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The identification of functional, sequestered, symbiotic chloroplasts in Elysia clarki

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Material Information

Title:
The identification of functional, sequestered, symbiotic chloroplasts in Elysia clarki a crucial step in the study of horizontally transferred, nuclear algal genes
Physical Description:
Book
Language:
English
Creator:
Curtis, Nicholas E
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Bryopsidales
Kleptoplasty
Sacoglossan
rbcL
Chloroplast symbiosis penicillus
Halimeda
Bryopsis
Derbesia
Dissertations, Academic -- Biology -- Doctoral -- USF
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: A comparison of Elysia (=Tridachia) crispata (Mörch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki. Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters the chloroplasts, which actively photosynthesize for up to 4 months. We have determined the algal source of the chloroplasts in adult E. clarki from 2 populations in the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that adult E. clark i sequester chloroplasts from 7 different species of algae, representing two genera, of which 5 were identified; Penicillus lamourouxii, P. capitatus, Halimeda incrassata, H. monile, and Bryopsis pennata. In addition, chloroplasts from more than 1 species of algae are sequestered in the same digestive cell simultaneously. Phylogenetic analysis of rbcL sequences from the order Bryopsidales showed that E. clarki feeding was restricted to calcareous members of the family Udoteaceae and the family Bryopsidaceae. Feeding experiments were conducted, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae. For the first 14 d post-metamorphosis, juveniles ate only the thin filamentous coenocytes, Bryopsis plumosa or Derbesia tenuissima. Electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other ^macroalga, including the four calcareous species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ~ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Nicholas E. Curtis.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 115 pages.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001910232
oclc - 173276963
usfldc doi - E14-SFE0001688
usfldc handle - e14.1688
System ID:
SFS0026006:00001


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T he I de nt i f i c a t i on of F unc t i ona l S e que s t e r e d, S ym bi ot i c C hl or opl a s t s i n E l y s i a c l ar k i : A C r uc i a l S t e p i n t he S t udy of H or i z ont a l l y T r a ns f e r r e d, N uc l e a r A l ga l G e ne s by N i c hol a s E C ur t i s A t he s i s s ubm i t t e d i n pa r t i a l f ul f i l l m e nt of t he r e qui r e m e nt s f or t he de gr e e o f D oc t or of P hi l os ophy D e pa r t m e nt of B i ol ogy C ol l e ge of A r t s a nd S c i e nc e s U ni ve r s i t y of S ou t h F l o r i da M a j or P r of e s s or : S i dne y K P i e r c e J r P h. D C l i nt on J D a w e s P h D K a t hl e e n M S c ot t P h D B r i a n T L i v i ngs t on, P h D D a t e of A ppr ova l : J une 15, 2006 K e yw or ds : B r yops i da l e s kl e pt opl a s t y, s a c ogl os s a n, r bc L c hl or opl a s t s ym bi os i s P e ni c i l l us H al i m e da B r y ops i s D e r be s i a C opyr i ght 2006 N i c hol a s E C ur t i s

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N ot e t o R e a de r T he or i gi na l of t hi s doc um e nt c ont a i ns c ol or t ha t i s ne c e s s a r y f or unde r s t a ndi ng t he da t a T he or i gi na l di s s e r t a t i on i s on f i l e w i t h t he U S F l i b r a r y i n T a m pa F l o r i da

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i T a bl e of C ont e nt s L i s t of T a bl e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v L i s t of F i gur e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi A bs t r a c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x P r e f a c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 C ha pt e r 1: A M or phol ogi c a l a nd M ol e c ul a r C om p a r i s on B e t w e e n E l y s i a c r i s pat a a nd a N e w S pe c i e s of K l e pt opl a s t i c S a c og l os s a n S e a S l ug ( G a s t r opoda : O pi s t hobr a nc hi a ) f r om t he F l or i da K e ys U S A . . . . . . . . . . . . . . . . . . . 5 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 A ni m a l s a nd H a bi t a t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 F l or i da K e ys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 V i r gi n I s l a nds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 R a dul a r P r e pa r a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 C hl or opl a s t L oc a l i z a t i on a nd M or phol ogy . . . . . . . . . . . . . . . . . . . . . . . . 9 C hl or opl a s t I de nt i f i c a t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 P C R a nd S e que nc i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 C om pa r i s on of M i t oc hondr i a l G e ne S e que nc e s . . . . . . . . . . . . . . . . . . . 12

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i i R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 T a xonom y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 E t ym ol ogy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 P a r a t ype s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 D i s t r i but i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 C om pa r i s on B e t w e e n E c l ar k i n s p. a nd E c r i s pat a . . . . . . . . . . . . . . . 16 E xt e r na l M or phol ogy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 D or s a l S ur f a c e V e na t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 C hl or opl a s t L oc a t i on a nd M or phol ogy . . . . . . . . . . . . . . . . . . . . 26 C hl or opl a s t I de nt i f i c a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 C ha pt e r 2: M ol e c ul a r P hyl oge ny of t he B r yops i d a l e s ( U l vophyc e a e C hl or ophyt a ) B a s e d on a P a r t i a l S e que nc e of t he C hl or opl a s t E nc ode d, L a r ge S ubuni t R i bul os e B i s phos pha t e C a r boxyl a s e / O xyge na s e G e ne . . . . . . . . . . . 40 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 S pe c i m e n S a m pl i ng a nd O ut gr oup S e l e c t i on . . . . . . . . . . . . . . . . . . . . . 43 D N A E xt r a c t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 P C R a nd S e que nc i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 P hyl oge ne t i c A na l ys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 E xha us t i ve M a xi m um P a r s i m ony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 B a ye s i a n A na l ys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

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i i i D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 C ha pt e r 3: T he S ym b i ot i c C hl or opl a s t s i n t he S a c ogl os s a ns E l y s i a c l ar k i a r e f r om S e ve r a l A l ga l S pe c i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 A ni m a l s a nd A l ga e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 D N A E xt r a c t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 P C R a nd S e que nc i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 E l e c t r on M i c r os c opy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 F e e di ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 r bc L S e que nc e I de nt i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 E l e c t r on M i c r os c opy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 C ha pt e r 4: N e w l y M e t a m or phos e d E l y s i a c l ar k i J u ve ni l e s F e e d on a nd S e qu e s t e r C hl or opl a s t s f r om A l ga l S pe c i e s D i f f e r e nt f r om T hos e U t i l i z e d by A dul t S l ugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 S e a S l ugs a nd A l ga e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 C ul t ur e of E l y s i a c l ar k i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 J uve ni l e F e e di ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 E l e c t r on M i c r os c opy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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i v D N A E xt r a c t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 P C R a nd S e que nc i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 J uve ni l e F e e di ng P r e f e r e nc e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 U l t r a s t r uc t ur e of S ym bi ot i c C hl or opl a s t s . . . . . . . . . . . . . . . . . . . . . . . . 87 M ol e c ul a r I de nt i f i c a t i on o f E l y s i a c l ar k i C hl or opl a s t s . . . . . . . . . . . . . . 94 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 C onc l us i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 R e f e r e nc e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 A bout t he A ut hor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E nd P a ge

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v L i s t of T a bl e s T a bl e 1 C om pa r i s on of t he l e ngt hs o f r a dul a r t e e t h f r om V i r gi n I s l a nds s l ugs w i l d K e ys s l ugs a nd l a bor a t or y r a i s e d K e ys s l ugs t ha t ha ve be e n f e d onl y B r y ops i s pl um os a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 T a bl e 2 L i s t of a l ga l s pe c i e s s a m pl e d i n t h i s s t udy. . . . . . . . . . . . . . . . . . . . . . . . 44 T a bl e 3 L i s t of a l ga l s pe c i e s pr e s e nt e d t o j uve ni l e E l y s i a c l ar k i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

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vi L i s t of F i gur e s F i gur e 1 A D D or s a l a nd ve nt r a l vi e w s of E l y s i a s pe c i e s A C E c r i s pat a B D E c l ar k i n s p. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 F i gur e 2 L i g ht m i c r ogr a ph o f a s qua s he d, unf i xe d a nd uns t a i ne d pa r a podi um of a n E l y s i a c l ar k i n s p. . . . . . . . . . . . . . . . . . . . 18 F i gur e 3 A B E l e c t r on m i c r ogr a phs of c r os s s e c t i ons of t he t w o t ype s of r ound c e l l s pr e s e nt i n t he ha e m oc oe l of t h e pa r a podi a of E l y s i a c r i s pat a ( s i m i l a r c e l l s a r e a l s o pr e s e nt i n t he s a m e l oc a t i on i n E c l ar k i ) . . . . . . . . . . . . . . . . . . . . . . . . 19 F i gur e 4 A B P hot ogr a phs of t he a nt e r i or dor s a l s ur f a c e o f E l y s i a s pe c i e s i n t he r e gi on w he r e t he pa r a podi a c r os s i n f r ont o f t he pe r i c a r di um ( P ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 F i gur e 5 A H S c a nni ng e l e c t r on m i c r og r a phs of t he r a dul a e o f f i e l d c ol l e c t e d E l y s i a c l ar k i n s p. ( A C E G ) a nd E c r i s pat a ( B D F H ) s how i ng di f f e r e nc e s i n s ha pe be t w e e n s pe c i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 F i gur e 6 A D L i ght m i c r ogr a phs of t he t e r m i ni o f t he d i ge s t i ve di ve r t i c ul a t ubul e s of E l y s i a s pe c i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 F i gur e 7 M ont a ge of e l e c t r on m i c r ogr a phs of a l ongi t udi na l s e c t i on t hr ough t he t e r m i nus of a di ge s t i ve t ubul e of E l y s i a c l ar k i n. s p. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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vi i F i gur e 8 A B E l e c t r on m i c r ogr a phs of s e c t i ons of c hl or opl a s t c ont a i ni ng di ge s t i ve c e l l s i n E c r i s pat a . . . . . . . . . . . . . . . . . . . . . . . . . 30 F i gur e 9 N e i ghbor J oi ni ng t r e e ( F 84 m ode l s e e m e t hods ) of pa r t i a l r bc L nuc l e ot i de s e que nc e s of V I r bc L 1 6 obt a i ne d by P C R f r om E c r i s pat a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 F i gur e 10 M a xi m um l i ke l i hood phyl oge ny us i ng a he ur i s t i c s e a r c h of s e que nc e s ba s e d on C O I a nd 16S m i t oc hondr i a l D N A f r om E l y s i a c l ar k i n. s p a nd E c r i s pat a i ndi vi dua l s a nd out gr oup t a xa . . . . . . . . . . . . . . . . . . . . . . . . . . 33 F i gur e 11 E xha us t i ve M a xi m um P a r s i m ony t r e e ba s e d on 12 t a xa ge ne r a t e d us i ng P a up V 4 10b . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 F i gur e 12 B a ye s i a n a na l ys i s ba s e d on r bc L s e que nc e s . . . . . . . . . . . . . . . . . . . . . . 50 F i gur e 13 E l e c t r on m i c r ogr a ph o f a po r t i on o f a H al i m e da i nc r as s at a s i phon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 F i gur e 14 E l e c t r on m i c r ogr a ph o f a po r t i on o f a P e ni c i l l us c api t at us s i phon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 F i gur e 15 E l e c t r on m i c r ogr a ph o f a c hl o r opl a s t i n a f i e l d c ol l e c t e d E c l ar k i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 F i gur e 16 A B E l y s i a c l ar k i t ha t ha s be e n s t a r ve d a nd r e f e d on P e ni c i l l us c ap i t at us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 F i gur e 17 E l e c t r on m i c r ogr a ph o f c hl or opl a s t s ( C ) i ns i de c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um o f a s t a r ve d E c l ar k i a nd s ubs e que nt l y f e d on P c api t at us . . . . . . . . . . . . . . . . . . . . . . 70

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vi i i F i gur e 18 A B C om pa r i s on of E l y s i a c l ar k i f e d on di f f e r e nt a l ga e . . . . . . . . . . . . . . . . . 87 F i gur e 19 L ongi t udi na l s e c t i on ne a r t he t i p of a s i phona c e ous f i l a m e nt of D t e nui s s i m a s how i ng s e ve r a l c hl or opl a s t s ( C ) i n t he c yt opl a s m a nd t he out e r c e l l w a l l ( W ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 F i gur e 20 S e c t i on t hr ough c hl or opl a s t ( C ) c ont a i ni ng r e gi on of a f i l a m e nt of B r y ops i s pl um os a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 F i gu r e 21 C r os s s e c t i on of di ge s t i ve t ubul e of a l a b r e a r e d j uve ni l e E l y s i a c l ar k i = ( 3 w k o l d) f e d onl y D e r be s i a t e nui s s i m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 F i gur e 22 L ongi t udi na l s e c t i on of di ge s t i ve t ubul e of E l y s i a c l ar k i f e d B r y ops i s pl um os a a nd t he n P e ni c i l l us c api t at us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 F i gur e 23 C r os s s e c t i on of pl a s t i d c ont a i ni ng c e l l s i n di ge s t i v e t ubul e of E l y s i a c l ar k i c ol l e c t e d f r om m oo r i ng c a n a l on V a c a K e y, F l o r i da w he r e B r y ops i s pe nnat a i s a bunda nt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 F i gur e 24 N e i ghbor J oi ni ng t r e e ( F 84 m ode l ) of pa r t i a l r bc L nuc l e ot i de s e que nc e s obt a i n e d by P C R f r om E l y s i a c l ar k i c ol l e c t e d f r om m oo r i ng c a na l on F l o r i da B a y s i de of V a c a K e y, F l or i da ( C a na l r bc L 1 3 t e xt i n bol d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

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i x T he I de nt i f i c a t i on of F unc t i ona l S e que s t e r e d, S ym bi ot i c C hl or opl a s t s i n E l y s i a c l ar k i : A C r uc i a l S t e p i n t he S t udy o f H or i z ont a l l y T r a ns f e r r e d, N uc l e a r A l ga l G e ne s N i c hol a s E C ur t i s A B S T R A C T A c om pa r i s on of E l y s i a ( = T r i dac hi a ) c r i s pat a ( M r c h, 1863) f r om t he V i r g i n I s l a nds w i t h e l ys i i d s l ugs f r om t he m a ng r ove s w a m ps a n d c a na l s i n t he F l o r i da K e ys t ha t ha ve pr e vi ous l y be e n i de nt i f i e d a s E c r i s pat a r e ve a l s m a ny di f f e r e nc e s i n ha bi t a t gr os s a nd m i c r os c opi c a na t om y, f ood p r e f e r e nc e s of j uve ni l e s s our c e s of s ym bi ot i c c hl or opl a s t s a nd t he i r l oc a l i z a t i on w i t hi n t he di ge s t i ve t ubul e s r a dul a r m o r phol ogy, a nd nuc l e i c a c i d s e que nc e s of t w o ge ne s T he di f f e r e nc e s be t w e e n t he t w o gr oups o f s l ugs a r e s uc h t ha t t he F l or i da K e ys a ni m a l s a r e c ons i de r e d t o r e pr e s e nt a ne w s pe c i e s E l y s i a c l ar k i E l y s i a c l ar k i f e e ds on s i phona c e ous a l ga e a nd i nt r a c e l l ul a r l y s e que s t e r s t he c hl or opl a s t s w hi c h a c t i ve l y phot os ynt he s i z e f or up t o 4 m ont hs W e ha ve de t e r m i ne d t he a l ga l s our c e of t he c hl or opl a s t s i n a dul t E c l ar k i f r om 2 popul a t i ons i n t he F l or i da K e ys us i ng m ol e c ul a r t e c hni que s f e e di ng e xpe r i m e nt s a nd e l e c t r on m i c r os c opy. O ur r e s ul t s c l e a r l y de m o ns t r a t e t ha t a dul t E c l ar k i s e que s t e r c hl or opl a s t s f r om 7 di f f e r e nt s pe c i e s of a l ga e r e pr e s e nt i ng t w o ge ne r a of w hi c h 5 w e r e i de nt i f i e d; P e ni c i l l us l am our oux i i P c api t a t us H al i m e da i nc r as s at a H m oni l e a nd

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x B r y ops i s pe nnat a I n a ddi t i on c hl or opl a s t s f r o m m or e t ha n 1 s pe c i e s of a l ga e a r e s e que s t e r e d i n t he s a m e di ge s t i ve c e l l s i m ul t a ne ous l y. P hyl oge ne t i c a na l ys i s of r bc L s e que nc e s f r om t he or de r B r yops i da l e s s how e d t ha t E c l ar k i f e e di ng w a s r e s t r i c t e d t o c a l c a r e ous m e m be r s of t he f a m i l y U dot e a c e a e a nd t he f a m i l y B r yops i da c e a e F e e di ng e xpe r i m e nt s w e r e c onduc t e d us i ng i ndi vi dua l s r a i s e d i n t he l a bor a t or y f r om e gg m a s s e s l a i d by E c l ar k i a dul t s w hi c h ha d be e n c ol l e c t e d f r om G r a s s y K e y, F l or i da U S A a nd 29 s pe c i e s of m a c r oa l ga e F or t he f i r s t 14 d pos t m e t a m or phos i s j uve ni l e s a t e onl y t he t hi n f i l a m e nt ous c oe noc yt e s B r y ops i s pl um os a or D e r be s i a t e nui s s i m a E l e c t r on m i c r os c opy s how e d t ha t t h e c hl or opl a s t s f r om bo t h a l ga e w e r e s e que s t e r e d i nt r a c e l l ul a r l y i n j uve ni l e s l ugs I nd i vi d ua l s of f e r e d a ny ot he r m a c r oa l ga i nc l udi ng t he f ou r c a l c a r e ous s pe c i e s f e d on by a dul t s di d not f e e d on o r i nc or por a t e a ny c hl or opl a s t s a nd s oon di e d. J uve ni l e s s w i t c he d f r om B pl um os a t o P c api t at us a t a l e ngt h of ~ 1. 0 c m a nd f i xe d f o r m i c r os c opy 14 da ys l a t e r ha d i nt a c t i nt r a c e l l ul a r c hl or opl a s t s f r om bot h a l ga e

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xi P r e f ac e T hi s m a nus c r i pt c ont a i ns a l a r ge r a nge o f da t a i nc l udi ng l i ght m i c r os c opy, t r a ns m i s s i on e l e c t r on m i c r os c opy, s c a nni ng e l e c t r o n m i c r os c opy, g r os s a na t om i c a l c om pa r i s ons m ol e c ul a r da t a a nd phyl oge ne t i c s T he da t a pr e s e nt e d i n c ha pt e r 1 w a s c ol l e c t e d a s pa r t of a c onc e r t e d e f f or t t o de t e r m i ne i f a g r oup of a ppa r e nt l y m or phol ogi c a l l y di s t i nc t s e a s l ugs r e gul a r l y c ol l e c t e d f r om t he F l o r i da K e ys w e r e E l y s i a c r i s pat a ( a s or i gi na l l y i de nt i f i e d) or w e r e i n f a c t a pr e vi ous l y unde s c r i be d s a c ogl os s a n s pe c i e s ( P i e r c e e t a l 2006 ) A l t hough I w or ke d on t he m a j or i t y of t he da t a a po r t i on w a s c om pl e t e d by ot he r s but s i nc e i t i s vi t a l t o t he c onc l us i ons a l l da t a on t he pr oj e c t i s pr e s e nt e d. T hus I w oul d l i ke t o a c know l e dge t he s i gni f i c a nt c ont r i but i ons of S i dne y P i e r c e S t e ve n M a s s e y, S t e ve n K a r l A nna B a s s a nd C ol i n F i nne y t o c ha pt e r 1. I n a ddi t i on, I w oul d l i ke t o a c know l e dge T i m M a uge l w ho a s s i s t e d w i t h f i gu r e 20 i n c h a pt e r 4. S pe c i m e ns w e r e c ol l e c t e d unde r pe r m i t s i s s ue d t o S i dne y K P i e r c e by t he S t a t e of F l or i da S l ugs f r om S t T hom a s w e r e c ol l e c t e d a n d s hi ppe d t o T a m pa unde r a pe r m i t i s s ue d t o C ol i n F i nne y by t he V i r g i n I s l a nds D e pa r t m e nt of N a t u r a l R e s our c e s T hi s w or k w a s s uppor t e d by N S F g r a nt #I B N 0315227 a nd # D E B 0321924 ( S t e ve n A K a r l ) a nd t he D e pa r t m e nt o f B i ol ogy a t t he U ni ve r s i t y of S out h F l or i da I t ha nk B e t t y L or a a m m a nd S us a n B oe r ne r f o r t he i r he l p w i t h e l e c t r on m i c r os c opy, K a t hl e e n H ot c hki s s f or a s s i s t a nc e w i t h t he f i gu r e s a nd R a y M a r t i ne z f or a s s i s t a nc e w i t h t he phot ogr a phy.

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xi i I a l s o t ha nk D r N a nc y V os s of t he I nve r t e br a t e M us e um a t t he R os e ns t i e l S c hool of M a r i ne a nd A t m os phe r i c S c i e nc e a t t he U ni ve r s i t y of M i a m i f or a c c e s s t o t he m us e um s c ol l e c t i on of E c r i s pat a s pe c i m e ns S pe c i a l t ha nks a r e due t o D r B i l l R udm a n of t he A us t r a l i a n M us e um f or s ubs t a nt i a l e di t or i a l he l p i n c ha pt e r 1, pa r t i c ul a r l y w i t h s ynonom y a nd a na t om i c a l i s s ue s a nd c l a s s i c s P r of e s s or J D N oona n of t he U S F W or l d L a ngua ge D e pa r t m e nt w ho a s s i s t e d w i t h L a t i n gr a m m a r

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1 I n t r od u c t i on S a c ogl os s a n s e a s l ug s ( O pi s t obr a nc hi a M ol l us c a ) f e e d on s i phona c e ous a l ga e a nd t he n c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um t a ke up a nd s e que s t e r a l ga l c hl or opl a s t s f r om t he gut l um e n ( T r e n c h e t a l 1969 ) I t ha s be e n s ug ge s t e d t ha t t hi s m e c ha ni s m a r os e a s a f or m o f c a m ouf l a ge t o c om pe ns a t e f or t he l os s of a pr ot e c t i ve s he l l ( R um pho e t a l 2000 ) I n s om e s a c ogl os s a ns t he s e s t ol e n c hl or opl a s t s a r e phot os ynt he t i c a l l y f unc t i ona l ( C l a r k e t a l 1990 ) a nd c a n be m a i nt a i ne d f r om j us t a f e w da ys i n s om e s pe c i e s t o m a ny m ont hs i n ot he r s ( C l a r k & B us a c c a 1978) i n a n a s s oc i a t i on know n a s c hl or opl a s t s ym bi os i s or kl e pt opl a s t y" ( W i l l i a m s & W a l ke r 1999) T he c hl or opl a s t s a r e not ve r t i c a l l y t r a ns m i t t e d be t w e e n ge ne r a t i ons s o ne w c hl or opl a s t s m us t be a c qui r e d by j uve ni l e s l ugs i n e a c h ge ne r a t i on ( T r e nc h e t a l 1969 ) F unc t i ona l s e que s t e r e d c hl or opl a s t s pr ovi de s a c ogl os s a ns w i t h a s t a bl e f ood s uppl y, w hi c h a l l o w s t he m t o be l e s s de pe nda nt on f ood a va i l a bi l i t y f or s ur vi va l t ha n s pe c i e s w i t hout s e que s t e r e d c hl or opl a s t s T he a ni m a l s onl y ne e d t o f e e d t o r e pl a c e c hl or opl a s t s a s t he y a r e br e a ki ng dow n. T hus i n e nvi r onm e nt s w he r e a l ga e a r e s c a r c e s e a s ona l or hi ghl y va r i a bl e l onge r kl e pt opl a s t i c a s s oc i a t i ons pr ovi de s l ugs w i t h a n a dva nt a ge ove r or ga ni s m s t h a t m us t c ont i nuous l y f e e d.

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2 T he s e a s l ug E l y s i a ( = T r i dac hi a ) c r i s pa t a ( M RCH 1863) ha s one of t he l onge s t kl e pt opl a s t i c a s s oc i a t i on s know n ( 3 4 m ont hs ) ( C l a r k & B us a c c a 1978) a nd i s f ound on c or a l r e e f s t hr oughou t t he C a r i bbe a n ( J e ns e n & C l a r k 1983, W e a ve r & C l a r k 1981, 2V e r r i l l 1900) T he s e que s t e r e d c hl or opl a s t s i n E c r i s pat a a r e f unc t i ona l a nd phot os ynt he t i c pr oduc t s a r e us e d by t he a ni m a l ( T r e nc h e t a l 1969 C l a r k & B us a c c a 1978, G r e e ne 1970 ) B e c a us e of i t s l ong a s s oc i a t i on w i t h s e que s t e r e d c hl or opl a s t s E c r i s pat a i s a n i de a l or ga ni s m f o r s t udyi ng how s t ol e n c hl or opl a s t s a r e a bl e t o r e m a i n f unc t i ona l i n t he c om pl e t e a bs e nc e of a n a l ga l nuc l e us ( e g. P i e r c e e t a l 2003 ) R e s e a r c h c onduc t e d by our l a b on w ha t w e f i r s t t h ought t o be E l y s i a c r i s pat a f r om a bor r ow pi t m oor i ng c a na l s a nd m a ngr ove s w a m ps i n t he F l or i da K e ys ha s de m ons t r a t e d t ha t nuc l e a r e nc ode d c hl or opl a s t p r ot e i ns v i t a l t o phot os ynt he s i s a r e s ynt he s i z e d i n t he s e s l ugs ( P i e r c e e t a l 2003) I n a ddi t i on, a hom ol og o f t he nuc l e a r ge ne e nc odi ng f uc oxa nt hi n c hl or ophyl l bi ndi ng pr ot e i n ( f c p ) ha s be e n de t e c t e d i n t ot a l D N A e xt r a c t e d f r om t he s e a ni m a l s by S out he r n B l ot t i ng ( P i e r c e e t a l 2003 ) T hi s i s t he s t r onge s t e vi de nc e s o f a r t ha t a n a l ga l ge ne ha s be e n hor i z ont a l l y t r a ns f e r r e d t o t he s e a s l ug ge nom e U nf or t una t e l y, f ur t he r w or k on pot e nt i a l hor i z ont a l ge ne t r a ns f e r s i n t he s e a ni m a l s ha s be e n ha m pe r e d by s e ve r a l pr ob l e m s F i r s t t he r e i s c ons i de r a bl e c onf us i on i n t he l i t e r a t ur e ove r E c r i s pat a f e e di ng p r e f e r e nc e s I n s om e s t udi e s C aul e r pa v e r t i c i l l at a w a s de s c r i be d a s t he pr i m a r y f ood s our c e ( J e ns e n 1980, J e ns e n & C l a r k 1983 ) but C v e r t i c i l l at a w a s not e a t e n by E c r i s pat a i n ot he r s ( C l a r k & B us a c c a 1978, T ho m ps on & Y a r m a n 1989) C aul e r pa s e r t ul ar i oi de s w a s de t e r m i ne d t o be t he f ood s our c e i n s t i l l ot he r s t udi e s ( R ol l e r & B i a nc hi 1995) but w a s f ou nd t o be t oxi c i n ye t ot he r s ( T hom ps on

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3 & Y a r m a n 1989) F e e di ng e xpe r i m e nt s c om bi ne d w i t h c ha nge s i n w hol e a ni m a l c hl or ophyl l c ont e nt s how e d t ha t E c r i s pat a a dul t s f e d on H al i m e da s p P e ni c i l l us s p. B r y ops i s pl um os a, a nd B at ophor a oe r s t e di i ( C l a r k & B us a c c a 1978) H ow e ve r c onf us i ngl y, C l a r k a nd B us a c c a ( 1978) a l s o s t a t e t ha t F l or i da E c r i s pat a a r e not c a pa bl e of f e e di ng on P e ni c i l l us c api t at us O t he r r e por t e d a l ga l f ood s our c e s f or E c r i s pat a ha ve be e n C r ac e m os a C hae t om or pha s p. a nd H al i m e da di s c oi de a ( T hom ps on & J a r m a n 1989) T he s e s t udi e s us e d a va r i e t y of m e t hods a n d none l ooke d f or m ol e c ul a r or m i c r os c opi c e vi de nc e of s ym bi ot i c c hl or op l a s t or i gi n, or a c t ua l l y de t e r m i ne d c hl or opl a s t s e que s t r a t i on. I n a ddi t i on w he n w e ha d oc c a s i on t o c om pa r e t he a ni m a l s c ol l e c t e d f r om m a ngr ove s w a m ps a nd m oor i ng c a na l s o f t he F l or i da K e ys w i t h E l y s i a c r i s pat a c ol l e c t e d on c or a l r e e f s o f f of S t T hom a s V i r gi n I s l a nds i t be c a m e a ppa r e nt t ha t t he r e w e r e num e r ous l a r ge m or phol ogi c a l di f f e r e nc e s be t w e e n t he t w o popu l a t i ons F ur t he r m or e w e f ound s l ugs w i t h V i r gi n I s l a nd t y pe m or phol og y i n c or a l r e e f t ype ha bi t a t s a dj a c e nt t o, but ne ve r c o m i ngl i ng w i t h t hos e i n t he m a ng r ove s or c a na l s E l y s i a c r i s pat a w a s or i gi na l l y de s c r i be d ba s e d on s pe c i m e ns c ol l e c t e d f r om S t C r oi x, ( M r c h 1863 ) a nd i n a l l s ubs e que nt di s c us s i on a bout t he t a xonom y o f t h i s s pe c i e s none of t he s pe c i m e ns e xa m i ne d w e r e c ol l e c t e d i n F l or i da ( E ngl e 1927; P r vot F ol 1946; W hi t e 1952; M a r c us & M a r c us 1960; G os l i ne r 1995 ) F r om t he or i gi n a l de s c r i pt i ons s ubs e que nt dr a w i ngs phot os a nd pe r s ona l obs e r va t i o ns i t i s c l e a r t ha t a l l t he e nc ount e r e d s pe c i m e ns of E c r i s pat a f r om e ve r yw he r e i n i t s r a nge i nc l udi ng s l ugs i n t he r e e f ha bi t a t s a dj a c e nt t o t he ne a r s hor e m a ngr ove dw e l l i ng K e ys s l ugs m a t c h t he gr os s m or phol ogy o f t he M r c h ( a nd s ubs e que nt a ut hor s ) de s c r i pt i on. H ow e ve r t h e s l ugs c ol l e c t e d f r om m a ngr ove

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4 s w a m ps a nd m oor i ng c a na l s i n t he F l or i da K e ys di f f e r s o m uc h f r o m t he E c r i s pat a de s c r i pt i on, t ha t w e f e l t i t w a s ne c e s s a r y t o e s t a bl i s h i f t he s e s pe c i m e ns a r e E c r i s pat a or a ne w s pe c i e s B e f or e a ny f ur t he r w o r k on kl e pt opl a s t y a nd pot e n t i a l hor i z ont a l ge ne t r a ns f e r s i n t he s e or ga ni s m s c a n c ont i nue t he f e e di ng a nd t a xo nom i c i s s ue s m us t be c l a r i f i e d. T hus t hos e i s s ue s w e r e w or ke d out by c om pl e t i ng f our i nt e r a c t i ng i nve s t i ga t i ons F i r s t E c r i s pat a f r om S t T hom a s w e r e c a r e f ul l y c om pa r e d w i t h s l ugs c ol l e c t e d f r om m a ng r ove s w a m ps a nd m oor i ng c a na l s i n t he F l or i da K e ys us i ng gr os s m or phol ogy r a dul a m or phol ogy, de ve l opm e nt a l pa t t e r ns di ge s t i ve m o r phol ogy w i t h r e s pe c t t o kl e pt opl a s t y, a nd m ol e c ul a r m a r ke r s t o e s t a bl i s h i f t he r e a r e e nough di f f e r e nc e s t o r e qui r e a ne w de s i gna t i on f or t he K e ys s l ugs S e c ond, phy l oge ne t i c a na l ys e s on t he U l vophyc e a e a nd i n pa r t i c ul a r t he B r yops i da l e s w e r e ne c e s s a r y t o a i d i n t he i de nt i f i c a t i on o f s e que s t e r e d c hl or opl a s t s T hi r d, t he i de nt i t y of s e que s t e r e d c hl or opl a s t s f r om a dul t K e ys s l ugs w a s de t e r m i ne d us i ng m ol e c ul a r m a r ke r s a nd t r a ns m i s s i on e l e c t r on m i c r os c opy ( T E M ) F i na l l y, t he f e e di ng be ha vi or of K e ys s l ugs w a s f ur t he r e l uc i da t e d w i t h j uve ni l e f e e di ng e xpe r i m e nt s a nd c hl or opl a s t s e que s t r a t i on w a s ve r i f i e d us i ng T E M a nd m o l e c ul a r m a r ke r s

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5 C h ap t e r 1: A m or p h ol ogi c al an d m ol e c u l ar c om p ar i s on b e t w e e n E l y s i a c r i s pat a an d a n e w s p e c i e s of k l e p t op l as t i c s ac ogl os s an s e a s l u g ( G as t r op od a : O p i s t h ob r an c h i a) f r om t h e F l or i d a K e ys U S A I n t r od u c t i on F or s e ve r a l ye a r s w e ha ve be e n s t udyi ng t he m ol e c ul a r m e c ha ni s m s of s ym bi ot i c c hl or opl a s t m a i nt e na nc e i n e l ys i i d s a c ogl os s a n opi s t hobr a nc hs O ne s pe c i e s i n pa r t i c ul a r t ha t w e ha ve be e n ut i l i z i ng i s f ound i n t he m a ngr o ve s w a m ps a nd m oor i ng c a na l s i n t he F l or i da K e ys a nd ha s be e n i de nt i f i e d a s t he l e t t uc e s l ug E l y s i a ( = T r i dac hi a ) c r i s pat a ( M RC H 1863) A l t hough a l l o f ou r w or k on t hi s s pe c i e s ha s be e n done w i t h s pe c i m e ns f r om t hos e K e ys ha bi t a t s w e ha ve r e c e nt l y c om pa r e d t he K e ys s l ugs w i t h s pe c i m e n s of E c r i s pat a c ol l e c t e d i n t he w a t e r s a r ound S t T hom a s V i r gi n I s l a nds A l t hough t he g r os s m or phol ogy w a s s i m i l a r be t w e e n i ndi vi dua l s l ugs f r om e a c h l oc a l i t y i t w a s qui t e di f f e r e nt be t w e e n t he t w o g r oups I n a ddi t i on w e ha ve not e d l e t t uc e s l ugs w i t h V i r gi n I s l a nd t ype m or phol ogy i n F l or i da f r om r e e f t ype ha bi t a t s a dj a c e nt t o, but ne ve r c o m i ngl i ng w i t h t hos e i n t he m a ngr ove s o r c a na l s A n a na l ys i s of t he l i t e r a t ur e i nd i c a t e s t ha t E c r i s pat a i s a r e l a t i v e l y l a r ge s i z e d, e l ys i i d s l ug, c om m onl y oc c ur r i ng i n c o r a l r e e f

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6 ha bi t a t s t hr oughout a ge og r a phi c r a nge t ha t e xt e nd s f r om t he nor t h c oa s t of S out h A m e r i c a t hr oughou t t he C a r i bbe a n ( e g M a r c us 1 980) i n t o t he F l or i da K e ys ( J e ns e n & C l a r k 1983) t he B a ha m a s ( W e a ve r & C l a r k 1981) a nd B e r m uda ( V e r r i l l 1900) T he s pe c i e s ha s a c onf us e d a nd c om pl i c a t e d nom e nc l a t ur a l hi s t or y w hi c h i s di s c us s e d l a t e r i n t hi s pa pe r ( E nge l 1927; P r uvot F ol 1946; W hi t e 1952; M a r c us & M a r c us 1960; G os l i ne r 1995) I t ha s us ua l l y be e n pl a c e d i n t he ge nus T r i dac hi a D e s ha ye s 1857 but t he va l i di t y o f t he ge nus ha s be e n a m a t t e r of c ont i nui ng de ba t e ( G os l i ne r 1995) M os t r e c e nt l y t he uni que ne s s of T r i dac hi a ha s be e n s uppor t e d by m o l e c ul a r e vi de nc e ( B a s s & K a r l 2006) F r o m t he or i gi na l d e s c r i pt i ons s ubs e que nt dr a w i ngs phot os a nd our ow n obs e r va t i ons i t i s c l e a r t ha t a l l publ i s he d a c c ount s of E c r i s pat a f r om e l s e w he r e i n i t s r a nge a nd s l ugs i n t he r e e f h a bi t a t s a dj a c e nt t o t he ne a r s hor e m a ngr ove dw e l l i ng K e ys s l ugs m a t c h t he gr os s m or phol ogy of t he M r c h de s c r i pt i on a nd t ha t of s ubs e que nt a ut hor s ( F i g 1A C ) A de t a i l e d c om pa r i s on w a s unde r t a ke n be t w e e n E c r i s pat a f r om S t T hom a s V i r gi n I s l a nds a nd a ni m a l s f r om our c ol l e c t i ng s i t e s i n t he F l or i da K e ys us i ng g r os s m or phol ogy, r a d ul a r m or pho l ogy, de ve l opm e nt a l pa t t e r ns di ge s t i ve m or phol ogy w i t h r e s pe c t t o kl e pt opl a s t y. U s i ng m ol e c ul a r m a r ke r s w e ha ve de t e r m i ne d t he a l ga l s our c e s of t he s ym bi ot i c pl a s t i ds a nd t he nuc l e i c a c i d s e que nc e s of t w o ge ne s W hi l e w e f ound s om e s i m i l a r i t i e s be t w e e n t he t w o, t he K e ys s l ugs di f f e r e d f r om t he C a r i bbe a n s pe c i m e ns i n a n um be r of i m por t a nt a s pe c t s

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7 M at e r i al an d M e t h od s A n i m al s an d H ab i t a t s F l or i d a K e ys : S l ugs w e r e m a i nl y c ol l e c t e d f r om t w o s i t e s a bor r ow pi t a nd i t s a s s oc i a t e d m a ngr ove s w a m p on G r a s s y K e y a nd a m a n m a de m oor i ng c a na l ne a r t he e a s t e nd of V a c a K e y. B ot h s i t e s w e r e on t he F l o r i da B a y s i de ( nor t h) of t he i s l a nds T he pi t a nd c a na l ha ve a l m os t ve r t i c a l s i de s c ons i s t i ng of c a r ve d out l i m e s t one c ove r e d by a w i de a r r a y of a l ga l s pe c i e s T he w a t e r i n t he pi t i s us ua l l y s l i ght l y t ur b i d, w hi l e t ha t i n t he c a na l i s qui t e c l e a r T he m a ngr ove s w a m p ope ns i n t o F l or i da B a y a nd t he bo t t om s e di m e nt s a r e a l s o c ove r e d w i t h a b r oa d r a nge o f a l ga l s pe c i e s T he w a t e r i n t he s w a m p i s s ha l l ow ( 1. 5 m or l e s s ) a nd us ua l l y qui t e c l e a r T he s l ugs a r e not o f t e n a s s oc i a t e d w i t h a ny pa r t i c ul a r a l ga e i n a ny o f t he l oc a t i ons e xc e pt f or P e ni c i l l us s p. i f p r e s e nt A l l of t he s e a r e a s a r e f a i r l y s he l t e r e d f r om w a ve a c t i on, e xpe r i e nc e br oa d f l uc t ua t i ons i n t e m pe r a t ur e s e a s ona l l y ( 12 35C J e ns e n & C l a r k 1983) a nd a r e e xpos e d t o r unof f f ol l ow i ng r a i ns C l a r k o f t e n not e d t ha t t he s l ugs a p pe a r e d onl y a t c e r t a i n t i m e s of t he da y ( s e e f or e xa m pl e W e a ve r & C l a r k 1981) bu t w e w e r e a l w a ys a bl e t o f i nd t he m i n l a r ge num be r s r e ga r dl e s s of t he t i m e of da y or s t a ge of t he t i de W e ha ve not m a de s ys t e m a t i c or e xt e ns i ve c ol l e c t i ng e f f o r t s i n a l l a r e a s i n t he F l or i da K e ys but i n s i x ye a r s o f c ol l e c t i ng, w e ha ve ne ve r f ound a K e ys t ype s l ug o f f s hor e on t he r e e f s o r on t he A t l a nt i c s i de of a ny of t he i s l a nds w he r e a s w e ha ve f ound t hous a nds of s pe c i m e ns i n t he ot he r a r e a s de s c r i be d a bove O n t he ot he r ha nd, w e ha ve oc c a s i ona l l y f ound s pe c i m e ns w i t h

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8 t he V i r gi n I s l a nds m or phol ogy o f f s hor e of t he K e ys but ne ve r i n t he m a ngr ov e a r e a s or m a n m a de c a na l s I n a ddi t i on, w e ha ve e xa m i ne d s pe c i m e ns i de nt i f i e d a s E c r i s pat a i n t he I nve r t e br a t e M us e um c ol l e c t i on a t t he U ni ve r s i t y of M i a m i s R os e ns t i e l S c hool of M a r i ne a nd A t m os phe r i c S c i e nc e T he s e pr e s e r ve d s l ugs w e r e c ol l e c t e d a t s e ve r a l l oc a t i ons i n t he C a r i bbe a n, i n t he K e ys a nd no r t h i nt o B i s c a yne B a y by s e ve r a l bi ol ogi s t s ove r a pe r i od of s e ve r a l de c a de s S l ugs w i t h K e ys a nd V i r gi n I s l a nds m or phol ogi e s w e r e pr e s e nt i n t he c ol l e c t i on I n f or m a t i on on t he e xa c t c ol l e c t i on s i t e of t he s e s pe c i m e ns w a s s om e t i m e s va gue but w he r e w e c oul d t e l l t he K e ys t ype s l ugs w e r e a l w a ys a s s oc i a t e d w i t h m a ngr ove a r e a s s i m i l a r t o ou r c ol l e c t i ng s i t e s a nd V i r gi n I s l a nds t ype s l ugs ha d be e n c ol l e c t e d onl y f r o m of f s hor e r e e f s i nc l udi ng t hos e f r om F l or i da V i r gi n I s l an d s : S l ugs w e r e c ol l e c t e d f r om a va r i e t y of s i t e s a r ound S t T ho m a s T he s e i nc l ude d bot h hi gh e ne r gy s i t e s on t he nor t h s i de o f t he i s l a nd ( B or de a ux B a y a nd S t um py B a y) w hi c h a r e e xpos e d t o s w e l l s ge ne r a t e d by t he p r e va i l i ng N E t r a de w i nd s a nd f r om m or e p r ot e c t e d s i t e s i n t he s out h s i de of t he i s l a nd ( B r e w e r s B a y a nd P e r s e ve r a nc e B a y) T he s ubs t r a t e i n t he s e ba ys i s t ypi c a l l y f r i ngi ng c or a l r e e f a nd c ol oni z e d ha r d bot t om dom i na t e d by l i ve a nd de a d c or a l m a c r oa l ga e a nd s e s s i l e i nve r t e br a t e s gi vi ng w a y t o a s a ndy bot t om on t he s e a w a r d s i de T he w a t e r i s ge ne r a l l y c l e a r i n t he no r t h s i de ba ys a nd s l ugs w e r e c ol l e c t e d f r om de pt hs of l e s s t ha n 1m t o a ppr oxi m a t e l y 5 m W a t e r c l a r i t y i s of t e n r e duc e d i n t he S out hs i de ba ys a nd he r e t he s l ugs w e r e f ound i n de pt hs r a ngi ng f r om 0. 25 m t o a ppr oxi m a t e l y 2 m T he onl y ha bi t a t i n S t T ho m a s r e m ot e l y c om pa r a bl e t o t he K e ys c ol l e c t i ng s i t e s i s t he M a ngr ove L a goon on t he S E s i de of t he i s l a nd. M uc h of t he s ubs t r a t e he r e i s ha r d pa ve m e nt do m i na t e d by

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9 P e ni c i l l us c api t at us L A M A RCK 1813 a nd H al i m e da opunt i a ( L IN N A E U S ) L A M O U RO U X 1816. A r e c e nt s ur ve y of t he M a ngr ove L a goon ( i n t e r t i da l t o 3 m de pt h) f ound no s l ugs R ad u l ar P r e p ar at i on T he ve r y s m a l l r a dul a e w e r e f ound by s qua s hi ng t he a nt e r i or e nds o f s i m i l a r l y s i z e d s l ugs s e pa r a t e l y be t w e e n t w o t hi n gl a s s pl a t e s T he s qua s he d t i s s ue w a s e xa m i ne d unde r a m i c r os c ope t o l oc a t e t he r a dul a i ns i de t he s e m i t r a ns pa r e nt buc c a l m a s s w hi c h w a s of t e n r e c ogni z a bl e by i t s br ow ni s h c ol o r a nd s t r i a t i ons O nc e t he r a dul a w a s l oc a t e d, t he gl a s s s l i de s w e r e ge nt l y s e pa r a t e d, t he buc c a l m a s s c ont a i ni ng t he r a dul a r e m ove d f r om t he r e s t of t he de br i s a nd pl a c e d i nt o 5N N a O H ove r ni gh t dur i ng w hi c h m os t of t he t i s s ue di s s ol ve d, l e a vi ng t he r a dul a be hi nd. A ny r e m a i ni n g t i s s ue w a s c a r e f ul l y r e m ove d w i t h m i c r odi s s e c t i ng t ool s a nd t he N a O H c a r e f ul l y w a s he d a w a y w i t h s e ve r a l r i ns e s of di s t i l l e d w a t e r F i na l l y, t he w a t e r w a s r e m ove d w i t h s e ve r a l w a s he s w i t h 100% e t ha nol w hi c h, i n t ur n, w a s a l l ow e d t o e va por a t e t o d r yne s s T he c l e a n, d r y r a dul a w a s t r a ns f e r r e d t o a nd s t uc k on a m e t a l s t ub, s put t e r c oa t e d w i t h pl a t i num a nd t he n vi e w e d a nd phot ogr a phe d w i t h a s c a nni ng e l e c t r on m i c r os c ope ( S E M ) ( J E O L m ode l J S M 35) C h l or op l as t L oc al i z at i on a n d M or p h ol ogy I n or de r t o c om pa r e t he l oc a t i on a nd s t r uc t ur e of t h e s ym bi ot i c pl a s t i ds w i t hi n t he di ge s t i ve t ubul e s of t he s l ugs f r om t he K e ys w i t h t hos e f r om t he V i r gi n I s l a nds s m a l l s pe c i m e ns or c ut pi e c e s of pa r a podi a w e r e f i xe d i n 2% gl u t a r a l de hyde i n 0 15 M c a c odyl a t e buf f e r ( pH 7. 5 1050 m os m a dj us t e d w i t h s uc r os e ) a t l e a s t ove r ni ght T hi s w a s f ol l ow e d by pos t f i xa t i on w i t h 2% os m i um t e t r oxi de i n t he s a m e buf f e r f o r 1 h, f ol l ow e d by a s t a i n i n 2 % u r a nyl a c e t a t e e n bl oc f o r 1 h D e hydr a t i on c ons i s t e d of 10

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10 m i nut e s ( m i n) w a s he s i n a n e t ha nol s e r i e s ( 35% 1 00% ) f ol l ow e d by t hr e e 10 m i n w a s he s i n pr opyl e ne oxi de a nd t he n i nf i l t r a t e d by t hr e e 60 m i n w a s he s i n pr opyl e ne oxi de : A r a l di t e E pon r e s i n ( 1: 1 1: 2 a nd 1: 3 i n s e que nc e ) ( A r a l di t e 502/ E m be d 812 E l e c t r on M i c r os c opy S c i e nc e s ) S a m pl e s w e r e t he n i m m e r s e d i n 100% A r a l di t e E pon r e s i n f or 60 m i n a nd e m be dde d i n f r e s h A r a l di t e E pon T hi n s e c t i ons ( 60 90 nm ) w e r e c ut on a n ul t r a m i c r ot om e ( M 2T B S or va l l P or t e r B l um ) a nd pl a c e d on c oppe r m e s h gr i ds S a m pl e s w e r e s t a i ne d 1 m i n w i t h 0 01M l e a d c i t r a t e c o unt e r s t a i ne d w i t h 2 % u r a nyl a c e t a t e f or 1 m i n, s t a i ne d a ga i n w i t h l e a d c i t r a t e f o r 1 m i n, a nd r i ns e d a f i na l t i m e w i t h doubl e di s t i l l e d w a t e r S e c t i ons w e r e v i e w e d a nd phot ogr a phe d us i ng a t r a ns m i s s i on e l e c t r on m i c r os c ope ( T E M ) ( F E I m ode l M or ga gni 268D ) C h l or op l as t I d e n t i f i c a t i on s D N A e xt r a c t i ons : A l l s l ugs w e r e s t a r ve d f or a t l e a s t one w e e k be f or e D N A e xt r a c t i on T he n, e a c h s e a s l ug w a s gr ound t o a pow de r i n l i q ui d ni t r oge n us i ng a ba ke d m o r t a r a nd pe s t l e A n e xt r a c t i on m e di um c ons i s t i ng of a 1 : 1 m i x t ur e of phe nol ( pH 8. 0 ) a nd buf f e r [ 200 m M T r i s ( pH 8 0) 1% S D S 0 01M E D T A a nd 2. 5 m g/ m l he pa r i n] w a s a dde d t o t he pow de r m i xe d ge nt l y f or 10 m i n a nd c e nt r i f uge d 4300 x g f or 5 m i n T he a que ous pha s e w a s r e m ove d a nd m i xe d w i t h a n e qua l vol um e of phe nol : c hl or of o r m ( 1: 1) f or 5 m i n, a nd t he n c e nt r i f uge d a ga i n a s a bove T h e a que ous pha s e w a s s a ve d, m i xe d w i t h a n e qua l vol um e of c hl or of o r m f o r 5 m i n a nd c e nt r i f uge d a s be f o r e D N A w a s e t ha nol pr e c i pi t a t e d f r om t he r e s ul t i ng a que ous ph a s e l yophi l i z e d, a nd r e s us pe nde d i n doubl e di s t i l l e d, m e m br a ne f i l t e r e d ( 0. 2 m ) a ut oc l a ve d w a t e r T he D N A s ol ut i on w a s t he n c e nt r i f uge d a t 115, 000 x g ( L 2 80 U l t r a c e nt r i f uge B e c km a n) f o r 48 hour s

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11 on a 0. 87 g/ m l c e s i um c hl or i de g r a di e nt a nd e xt r a c t e d w i t h i s opr opa nol ( R um ph o e t a l 1994) P C R an d S e q u e n c i n g D e ge ne r a t e pr i m e r s e que nc e s w e r e de s i gne d t o c ons e r ve d r e gi ons of t he l a r ge s ubuni t of t he r i bul os e 1, 5 bi s phos pha t e c a r boxyl a s e ( r bc L ) g e ne a s de t e r m i ne d by a l i gnm e nt s of 31 c hl or ophyt e r bc L s e que nc e s a va i l a bl e i n G e nba nk. T he f or w a r d pr i m e r s e que nc e w a s 5 A A A G C X G G K G T W A A A G A Y T A 3 ( r bc L 1 ) a nd t he r e ve r s e pr i m e r s e que nc e w a s 5 C C A A C G C A T A R A D G G T T G W G A 3 ( r bc L 2 ) P C R r e a c t i on m i xt ur e s ( 25 L ) w e r e m a de w i t h 1X E nz ypl us 2000 r e a c t i on buf f e r ( E nz ypol D e nve r C O ) [ 2m M T r i s H C l ( pH 8. 8) 1m M K C l 1 m M ( N H 4) 2 S O 4, 0. 01 % T r i t on X 100, a nd 0 01 m g/ m l B S A ] 12 5 nM dN T P s 0 25 m M r bc L 1 0 25 m M r bc L 2, 1 n g/ l D N A a nd 1 uni t pol ym e r a s e ( E nz ypl us 2000, E nz ypol D e nve r C O ) P C R t ouc hdow n r e a c t i ons w e r e pe r f o r m e d on a t he r m oc yc l e r ( m ode l G e ne A m p P C R 2400 A ppl i e d B i os ys t e m s ) w i t h a de na t ur i ng t e m pe r a t ur e of 94C f or 30 s a nne a l i ng t e m pe r a t u r e s t a r t i ng a t 45C f or 30 s a nd a n e xt e ns i on t e m pe r a t ur e of 72C f or 1 m i n f o r 35 c y c l e s w i t h t he a nne a l i ng t e m pe r a t ur e l ow e r e d by 0 5C i n e a c h c yc l e a f t e r t he f i r s t P C R pr oduc t s w e r e pu r i f i e d us i ng t he S t r a t a P r e p P C R pu r i f i c a t i on ki t ( S t r a t a ge ne ) a nd t h e n c l one d us i ng t he Z e r o bl un t T O P O P C R c l oni ng ki t ( I nvi t r oge n) a c c or di ng t o t he m a n uf a c t ur e r s i ns t r uc t i ons a nd c ol oni e s w e r e s c r e e ne d by P C R us i ng T 3 a nd T 7 pr i m e r s C ol oni e s pos s e s s i ng i ns e r t s c l os e i n s i z e t o t he P C R pr oduc t w e r e m i n i pr e ppe d us i ng t he N uc l e os pi n P l a s m i d ki t ( C l ont e c h) S e que nc i ng w a s c onduc t e d by a c om m e r c i a l ve ndo r [ N or t hw oods D N A I nc S ol w a y, M N ( w w w nw dna c om ) ] us i ng T 3 a nd T 7 pr i m e r s T he r bc L s e que nc e s de t e r m i ne d w e r e

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12 a l i gne d w i t h 16 u l vophyt e r bc L s e que nc e s obt a i ne d f r om G e nba nk, us i ng C l us t a l W ( T hom ps on e t a l 1994 ) A l i gnm e nt s a r e a va i l a bl e f r om one o f t he a ut hor s ( N E C ) A N e i ghbor J oi ni ng t r e e w a s c ons t r uc t e d f r om t he a l i gnm e nt us i ng P hyl i p 3. 63 ( F e l s e ns t e i n 2004) ut i l i z i ng t he F 84 m ode l ( T hor ne e t a l 1992) w i t h 100 boot s t r a p r e pl i c a t e s C om p ar i s on of M i t oc h on d r i al G e n e S e q u e n c e s T w e nt y K e ys a nd s i x V i r g i n I s l a nds s l ugs w e r e a n e s t he t i z e d w i t h 0. 3M M gC l 2 a nd pl a c e d i n 95% e t ha nol T ot a l D N A w a s e xt r a c t e d f r om a s m a l l po r t i on of t he pos t e r i or e nd of e a c h s l ug by a non bo i l i ng c he l e x m e t hod ( W a l s h e t a l 1991 ) A n a l i quot of t he c he l e x i s ol a t e d D N A w a s di l ut e d w i t h 1X T E ( 10 m M T r i s 1 m M E D T A ; pH 7 5) a nd s t o r e d a t 20C unt i l us e d i n P C R a m pl i f i c a t i ons S e gm e nt s of t he m i t oc hondr i a c yt oc hr om e c oxi da s e s ubuni t I ge ne ( C O I ; ~ 700 b p) w e r e P C R a m pl i f i e d f o l l ow i ng B a s s a nd K a r l ( 2006 ) S e gm e nt s f r om t he l a r ge s ubuni t R N A ( 16S ; ~ 450 bp) w e r e P C R a m pl i f i e d f r om D N A e xt r a c t e d f r o m s i ngl e i ndi vi d ua l s f r om bot h l oc a t i ons A m pl i c ons w e r e pur i f i e d us i ng doubl e di s t i l l e d H 2 0 a nd f i l t r a t i on ( 30, 000 M W c ut of f M i l l i por e I nc B e df or d M A ) T he m a s s of t he a m pl i c ons w a s de t e r m i ne d by c om pa r i ng E t B R s t a i ni ng i nt e ns i t y of 2. 0 5 0 m l of e a c h pur i f i e d r e a c t i on pr oduc t r e l a t i ve t o a s t a nda r d m a s s D N A l a dde r ( I nvi t r oge n L i f e T e c hnol ogi e s C a r l s ba d, C A ) C yc l e s e que nc i ng r e a c t i ons ( A m e r s ha m E T T e r m i na t o r K i t A m e r s ha m B i os c i e nc e s C or p. P i s c a t a w a y, N J ) w e r e c onduc t e d w i t h a pp r oxi m a t e l y 100 ng of pu r i f i e d P C R pr oduc t a c c or di ng t o m a nuf a c t ur e r s s pe c i f i c a t i ons e xc e pt t ha t r e a c t i on s i z e s w e r e r e duc e d t o 25% F l uor e s c e nt l y l a be l e d pr oduc t s w e r e s i z e s or t e d a nd vi s ua l i z e d ( A B I 310 G e ne t i c A na l yz e r A ppl i e d B i os ys t e m s I nc F os t e r C i t y C A ) T he s e que nc e s f r om bot h s t r a nds

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13 of t he a m pl i c ons w e r e c om pa r e d a nd e di t e d i f ne e de d, us i ng S e que nc he r ( v4. 1; G e ne C ode s C or p. A nn A r bor M I ) T he ope n r e a di ng f r a m e f or C O I w a s us e d t o a i d pr ope r a l i gnm e nt T he 16S ge ne r e gi on f r a gm e nt s w e r e a l i gn e d t o A pl y s i om or pha s e c onda r y s t r uc t ur e us i ng S e a vi e w ( G a ul t i e r e t a l 1996 ) A l i g nm e nt s a r e a va i l a bl e on r e que s t f r om one of t he a ut hor s ( A L B ) C O I a nd 16 S s e que nc e s w e r e de t e r m i ne d f r om one i ndi vi dua l f r om t he K e ys a nd f r o m t he V i r gi n I s l a nds a l ong w i t h one i ndi vi dua l f r o m e a c h of t he out gr oups T he s e que nc e da t a w e r e us e d i n a phyl o ge ne t i c a na l ys i s I nt r a popul a t i on di ve r ge nc e e s t i m a t e s w e r e ba s e d on r e s ul t s f r om t w e nt y i ndi vi dua l s f r om t he K e ys a nd s i x f r om t he V i r gi n I s l a nds us i ng C O I s e que nc e s onl y. T he phyl oge ne t i c r e l a t i ons hi ps a m ong E l y s i e l l a pus i l l a B E RG H 1872, E l y s i a t i m i d a R IS S O 1818 ( da t a f r om B a s s a nd K a r l 2006) a nd t he K e ys a nd V i r gi n I s l a nd s l ugs w e r e e va l ua t e d us i ng bot h C O I a nd 16S c ons e ns u s s e que nc e s us i ng m a xi m um l i ke l i hood a nd a he ur i s t i c s e a r c h w i t h 10 r e pl i c a t e s of r a ndom s t e pw i s e a ddi t i on ( G e nba nk s e que nc e da t a a c c e s s i on num be r s : 16S E pus i l l a D Q 480201, E t i m i da D Q 480166, K e ys D Q 48019 7, V i r gi n I s l a nds D Q 480198 a nd C O I E pus i l l a D Q 471236, E t i m i da D Q 471245, K e ys D Q 471225, V i r gi n I s l a nds Q 471224) P e r c e nt s e que nc e di ve r ge nc e f or t he c o nc a t e na t e d s e que nc e s w a s e s t i m a t e d by m a xi m um l i ke l i hood di ve r ge nc e s w i t h P A U P ( v4 0b10; S w o f f or d 1998) us i ng t he be s t f i t m ode l ( G T R + I ) a s s e l e c t e d by M ode l t e s t w i t h A ka i ke i nf or m a t i on c r i t e r i a ( v3. 06 ; P os a da a nd C r a nda l l 1998) I nt r a a nd i nt e r popu l a t i on m a xi m um l i ke l i hood di ve r ge nc e s f or C O I w e r e c a l c ul a t e d s i m i l a r l y us i ng a T r N + I m ode l of e vol ut i on i ndi c a t e d w i t h A ka i ke i nf or m a t i on c r i t e r i a by M ode l t e s t

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14 R e s u l t s T axon om y O r de r S a c ogl os s a V O N I H E RI N G 1876 S upe r f a m i l y P l a c obr a nc hi ode a G RA Y 1840 F a m i l y P l a c obr a nc hi da e G RA Y 1840 ( s e e B ouc he t & R oc r oi 2005 ) G e nus E l y s i a R IS S O 1818 E l y s i a c l ar k i n. s p. E t ym ol ogy: N a m e d f or t he l a t e P r of e s s or K e r r y B C l a r k of t he F l or i da I ns t i t ut e of T e c hnol ogy, s om e o f w hos e s t ude nt s i nt r oduc e d us t o t he c ol l e c t i ng s i t e s t ha t he us e d i n hi s e xt e ns i ve s t udi e s of e l ys i i d s l ugs i n t he F l or i da K e ys T ype s pe c i m e ns : H ol ot ype : A m e r i c a n M us e um of N a t ur a l H i s t or y, A M N H 318222, 3 2 c m l ong a l i ve P ar at yp e s : A m e r i c a n M us e um of N a t ur a l H i s t or y A M N H 318223, 5 s pe c i m e ns 2 8, 3 2, 3. 1, 3. 7 4 6 c m l ong a l i ve : I nve r t e br a t e M us e um R os e ns t i e l S c hool of M a r i ne a nd A t m os phe r i c S c i e nc e s U ni ve r s i t y of M i a m i U M M L 30. 12717 F r om t he s a m e c ol l e c t i on a s t he hol ot ype m a t e r i a l 5 s pe c i m e ns 3. 2, 3 1, 3. 2 3. 8 5 5 c m l ong a l i ve T ype l oc a l i t y: E a s t e r n e nd o f V a c a K e y, F l or i da K e ys U S A 24 N 81 W F l or i da B a y s i de i n 1 m J ul y 2005. D i s t r i b u t i on : C om m onl y f ound i n m a ngr ove s w a m ps s om e bor r ow pi t s a nd m oor i ng c a na l s on t he F l or i da B a y s i de of L ong K e y G r a s s y K e y, C r a w l K e y, F a t D e e r K e y a nd

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15 V a c a K e y, F l or i da K e ys U S A i n de pt hs of 0 2 5 m D e s c r i pt i on: L a r ge f or t hi s ge nus up t o 35 m m l ong 3 0 m m w i de t a pe r i ng t o poi nt p os t e r i or l y. P r om i ne nt pos t e r i or of he a d but not f us e d, t he r e be i ng a s m a l l ga p e ve n i n l a r ge s t s pe c i m e ns P r om i ne nt ne c k a nd he a d e xt e ndi ng be yond a nt e r i o r e dge o f pa r a p odi a B a c kgr ound c ol or of m os t of e xt e r na l s ur f a c e gr e e n, f r om pr e s e nc e of i nt r a c e l l ul a r a l ga l c hl or opl a s t s l oc a t e d i n c e l l s a t t e r m i na l e nds of t he di ge s t i ve di ve r t i c ul a N um e r o us w hi t e t r a ns l uc e nt s pot s m os t l y c om pr i s e d of w hi t e a nd s c a r c e t ur quoi s e i r i de s c e nt pi gm e nt c e l l s di s pe r s e d on bot h dor s a l a nd ve nt r a l s ur f a c e s ; dor s a l t r a ns l uc e nt s pot s c i r c ul a r w i t h l a r ge r s pot s ( < 0. 75 m m di a m e t e r ) i nt e r s pe r s e d be t w e e n m a ny s m a l l e r s pot s ; ve nt r a l s pot s of t e n ova l a nd a l i gne d l e ngt h w i s e V e nt r a l f oot t r a ns pa r e nt a nd bot h gr e e n c ol or e d t ubul e s a nd t r a ns l uc e nt s pot s vi s i bl e t hr ough i t N a r r ow ( 0 5 m m ) di s t a l e dge of pa r a podi a t r a ns l uc e nt not opa que a nd unde r l a i d by a s m a ny a s t h r e e c ol or b a nds ; a n or a nge ba nd ( 0 1 m m w i de ) c ont i nuous di s c ont i nuous or l a c ki ng j us t be ne a t h t r a ns pa r e nt m a r gi n, f ol l ow e d pr oxi m a l l y by c ont i nuous gr e e n t u r quoi s e ba nd ( 0 1 m m w i de ) a l s o l a c ki ng i n s o m e s pe c i m e ns f ol l ow e d by pr ox i m a l c ont i nuous gr e y gr e e n ba nd ( 1. 25 m m w i de ) P e r i c a r di a l pr om i ne nc e t hi nl y c ove r e d by t r a ns pa r e nt m a nt l e w i t h t w o m a j o r ve s s e l s e xi t i ng pos t e r i or l y a nd r unni ng t ow a r ds pos t e r i or t i p of body; m a j or ve s s e l s w i t h f e w r a ndom br a nc he s R hi nophor e s pr o m i ne nt e nr ol l e d, t r a ns l uc e nt w hi t e ne a r t i p t u r ni ng t o gr e e n a t ba s e B l a c k e ye s pot a t ba s e of e a c h r hi no phor e M out h b r oa d w i t h pr om i ne nt l i ps c e nt r a l l y c l e f t R a dul a m i c r os c opi c s i ngl e r o w of 14 t e e t h on a ve r a ge ( r a nge 13 17 ) N o di s c a r de d t e e t h pr e s e nt i n a s c us of a ny s pe c i m e n e xa m i ne d. A l l t e e t h s i m i l a r l y da gge r s ha pe d, w i t h de e p gr oove on out e r f a c e a nd s e r r a t e d m i d r i dge on unde r s i de A ve r a ge t oot h l e ngt h 129 m m

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16 C om p ar i s on b e t w e e n E c l ar ki n s p an d E c r i s pat a F i gu r e 1 D o r s a l a nd ve nt r a l vi e w s of E l y s i a s pe c i e s A C E c r i s pat a B D E c l ar k i n. s p.

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17 E xt e r n al M or p h ol ogy : T he r e a r e s e ve r a l obvi ou s e xt e r na l m o r phol ogi c a l di f f e r e nc e s be t w e e n E c l ar k i a nd E c r i s pat a. W e l l f e d E c l ar k i a r e ne a r l y u ni f or m l y g r e e n i nt e r s pe r s e d w i t h c om pa r a t i ve l y s m a l l r ound o r ov a l w hi t e s pot s T he gr e e n c ol o r i s pr e s e nt on m os t e xt e r na l s ur f a c e s i nc l udi ng m uc h of t he he a d, t he di s t a l e dge s of t he pa r a podi a a nd t he s ol e of t he f oot w hi c h a r e a l m os t c om pl e t e l y t r a ns pa r e nt ( s e e be l ow ) ( F i g. 1B ) V i e w e d unde r a di s s e c t i ng m i c r os c ope i t i s c l e a r t ha t t he gr e e n c ol or i s l oc a l i s e d i n a l a r ge num be r of t i ny s pot s j us t be l ow t he e xt e r na l body s ur f a c e w hi c h a r e t he bl i nd e nds of t he di ge s t i ve di ve r t i c ul a t ha t c ont a i n t he s ym bi ot i c p l a s t i ds ( s e e be l ow ) T he w hi t e s pot s a r e i r i de s c e nt c l us t e r s of c e l l s c ont a i ni ng w hi t e or bl ui s h w hi t e p i gm e nt of unknow n c om pos i t i on a nd f unc t i on T he gr e e n c ol or i n E c r i s pat a i s m uc h m o r e l oc a l i s e d i n c e r t a i n a r e a s of t he body ( F i g 1A ) t ha n i n E c l ar k i T he dor s a l s ur f a c e be t w e e n t he pa r a podi a of w e l l f e d E c r i s pat a i s ge ne r a l l y gr e e n, i nt e r s pe r s e d w i t h m uc h l a r ge r w hi t e s pot s t ha n t hos e i n E c l ar k i T he he a d of E c r i s pat a i s us ua l l y onl y l i ght l y pi gm e nt e d w i t h gr e e n a l ong t he do r s a l s ur f a c e s o m e t i m e s w i t h a di f f us e bl ue ba nd e xt e ndi ng be t w e e n t he r hi nopho r e s T he out e r s ur f a c e of t he body be t w e e n t he ba s e of t he pa r a podi a a nd t he f oot a l s o i s gr e e n w i t h l a r ge w hi t e s pot s i n w e l l f e d i ndi vi dua l s T he out e r a s pe c t of t he pa r a podi a i n E c r i s pat a of t e n ha s a de e p bl ue ba nd o f va r i a bl e t hi c kne s s T he bl ue pi gm e nt i s c ont a i ne d i n l a r ge r ound c e l l s w hi c h a r e l oc a t e d be l ow t he e pi de r m i s i n t he ha e m oc oe l W e ha ve ne ve r s e e n t he bl ue ba nd i n t hous a nds of E c l ar k i a l t hough s om e ha ve a ve r y f a i nt bl ue t i nge on t he out e r e dge of t he pa r a podi a a nd on t he e dge s of t he m out h B ot h s pe c i e s c a n ha ve a n or a nge ba nd, s om e t i m e s t e ndi ng t o r e d i n E c r i s pat a a l ong t he di s t a l m a r gi n of t he pa r a podi a T hi s ba nd m a y be c ont i nuous i nt e r m i t t e nt o r l a c ki ng e nt i r e l y. T h e pi gm e nt i s c on t a i ne d w i t hi n l a r ge r ound, una t t a c he d

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18 c e l l s of unknow n f unc t i on l oc a t e d i n t he ha e m oc oe l a t t he d i s t a l e dge of t he pa r a podi a ( F i g. 2) F i gu r e 2 L i gh t m i c r og r a ph o f a s qua s he d, unf i xe d a nd uns t a i ne d pa r a podi um o f a n E l y s i a c r i s pat a n. s p. T he c e nt r a l c l e a r a r e a i s t r a ve r s e d by s m a l l di ge s t i ve di ve r t i c ul a t ubul e s ( D ) w hi c h t e r m i na t e i n t he g r e e n c ol or e d c hl or opl a s t c ont a i ni ng c e l l s ( C ) M uc us s e c r e t i ng c e l l s ( M ) a r e a l s o pr e s e nt T w o t ype s of f r e e f l oa t i ng r ound c e l l s a r e s how n he r e S m a l l e r r e d c e l l s ( O ) ( w hi c h a r e ge ne r a l l y c onc e nt r a t e d i n a br oke n o r c ont i nuous ba nd a t t he out e r e dge o f t he pa r a podi a [ s e e F i g 1A ] ) S i m i l a r s i z e d c e l l s a r e pr e s e nt i n t he E c l ar k i but a r e ge ne r a l l y or a nge i n c ol or M uc h l a r ge r br ow n c e l l s ( B ) ( w hi c h a r e g r e e n i n E c l ar k i ) w hi c h Y onge a nd N i c hol a s ( 1940) i nc or r e c t l y de s c r i be d a s z ooxa nt he l l a e S c a l e ba r = 50 m m I n E c l ar k i a l a ye r of m uc h l a r ge r una t t a c he d, r o und gr e e n c e l l s o f t e n a ppe a r i ng a s a n i r i de s c e nt gr e e n ba nd pr oxi m a l t o t he or a nge c e l l b a nd, i s a l s o pr e s e nt i n t he ha e m oc oe l F r e e r ound c e l l s s i m i l a r i n s i z e t o t he gr e e n c e l l s a r e a l s o pr e s e nt i n E c r i s pat a but t he y ha ve a br ow n c ol or e d pi gm e nt B a s e d on l i ght m i c r os c opy of s e c t i ons f r om pr e s e r ve d s l ugs Y onge a nd N i c hol s ( 1940) i de nt i f i e d t he s e b r ow n c e l l s a s z ooxa nt he l l a e but t he y c l e a r l y a r e not a s t he y l a c k c hl or op l a s t s a nd c e l l w a l l s ( F i g. 3) M os t of t he i nt e r i or o f t he

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19 c e l l i s c om pos e d o f a n a m or phous g r a nul a r a ppe a r i ng s ubs t a nc e m os t l i ke l y t he pi gm e nt w hi c h c onf i ne s t he nuc l e us ot he r or ga ne l l e s a nd c yt opl a s m t o a t hi n l a ye r j us t unde r t he out e r c e l l m e m b r a ne F i gu r e 3 E l e c t r on m i c r og r a phs of c r os s s e c t i ons of t he t w o t ype s of r ound c e l l s p r e s e nt i n t he ha e m oc oe l of t he pa r a podi a of E l y s i a c r i s pat a ( s i m i l a r c e l l s a r e a l s o pr e s e nt i n t he s a m e l oc a t i on i n E c l ar k i ) A L a r ge r o f t he t w o c e l l s w hi c h w a s de s c r i be d a s z ooxa nt he l l a e by Y onge a nd N i c hol a s ( 1940) T he c e nt r e of t he c e l l i s f i l l e d w i t h a m or phous dr opl e t s p r oba bl y of pi gm e nt T he dr o pl e t s a r e s ur r ounde d by a n out e r c or t i c a l l a ye r c om pr i s e d of a m or e e l e c t r on de ns e s ubs t a nc e T he c yt opl a s m a nd or ga ne l l e s i nc l udi ng t he nuc l e us a r e s que e z e d i nt o t he out e r r e a c he s of t he c e l l be t w e e n t he pi gm e nt a nd ou t e r c e l l m e m br a ne T he r e a r e no s i gns of c hl or opl a s t s or ot he r pl a nt c ha r a c t e r i s t i c s i n t he s e c e l l s B S m a l l e r of t he t w o c e l l t ype s T he s e c e l l s c ont a i n a br i ght r e d or o r a nge pi gm e nt a nd c a n be c onc e nt r a t e d i n s uf f i c i e nt num be r s a t t he e dge of t he pa r a podi a t o f or m a c ont i nuous ba nd o f c ol or H ow e ve r t he ba nd c a n a l s o be di s c ont i nuous or t he c e l l s c a n be l oc a t e d e nt i r e l y a w a y f r om t he pa r a podi a l e dge T he pi gm e nt i s l oc a t e d i n s e ve r a l l a r ge dr o pl e t s T he c y t opl a s m a nd or ga ne l l e s a r e i nt e r s pe r s e d be t w e e n t he dr opl e t s U nl i ke t he l a r ge r r ound c e l l t he nuc l e us i n t he s m a l l e r c e l l i s us ua l l y l oc a t e d i n t he c e nt r e o f t he c e l l T he r e a r e no s i gns of a ny pl a nt c ha r a c t e r i s t i c s i n t he s e c e l l s S t a r va t i on e ve nt ua l l y r e s ul t s i n c hl or opl a s t l os s f r o m t he di ge s t i ve c e l l s ( C ur t i s e t a l 2006a ) a nd c onc om i t a nt l os s of t he gr e e n c ol or D e pe ndi ng on t he l e ngt h of s t a r va t i on

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20 s l ugs c a n be c ol or e d f r o m a da r k gr e e n t hr ough va r i ous s ha de s of l i ght gr e e n t o ye l l ow w i t h no gr e e n r e m a i ni ng i n s l ugs t ha t ha ve no t f e d f or m ont hs T he gr e e n c ol o r w i l l us ua l l y r e a ppe a r ove r a pe r i od o f w e e ks i f s t a r ve d s l ugs a r e pr ovi de d w i t h t he a ppr op r i a t e a l ga e ( C ur t i s e t a l 2006a ) V e nt r a l l y t he f oot of a n E c l ar k i i s ne a r l y i ndi s t i ngui s ha bl e f r om t he r e s t of t he e xt e r na l s ur f a c e a nd i s a l m os t c om pl e t e l y t r a ns pa r e nt ( F i g 1D ) T he l a t e r a l m a r gi ns o f t he f oot a r e di f f i c ul t t o di s t i ngui s h f r om t he a dj oi ni ng body w a l l w i t hout m a gni f i c a t i on B ot h t he c hl or opl a s t c ont a i ni ng di ge s t i ve t ubul e s a nd t he w hi t e c e l l pi gm e nt s pot s a r e vi s i bl e t hr ough t he s ur f a c e o f t he f oot I n c ont r a s t t he f oot of a n E c r i s p at a i s t hi c k, w hi t e opa que ha s de f i ni t e l a t e r a l m a r gi ns a nd no pl a s t i ds a r e vi s i bl e i n i t or w i t hi n t he ve nt r a l pa r t o f t he b ody c ove r e d by t he f oot ( F i g 1C ) S pe c i m e ns of E c r i s pat a t ypi c a l l y ha ve a na r r ow bl ue pi gm e nt ba nd r unni ng t he l e ngt h o f t he o ut s i de m a r gi n of t he f oo t w hi c h i s a bs e nt i n E c l ar k i F ur t he r m or e t he f oot of E c r i s pat a e nds i n a bl unt s qua r e d of f m a r g i n a t t he pos t e r i or e nd, w hi l e t ha t of E c l ar k i t e r m i na t e s i n a t a pe r e d poi nt ( F i gs 1A B ) B ot h s pe c i e s ha ve pr om i ne nt w a vy pa r a podi a t ha t f or m t he l a t e r a l m a r gi n of t he dor s a l s ur f a c e a nd t oge t he r w i t h t he g r e e n c ol or gi ve t he s l ugs t he c om m on na m e l e t t uc e s l ug. A ve r y obvi ous d i f f e r e nc e be t w e e n t he t w o s pe c i e s i s t ha t i n E c r i s pat a t he pa r a podi a a r e f us e d i nt o a c ont i nuo us s t r uc t ur e t ha t c r os s e s t he a nt e r i or m i dl i ne be t w e e n t he pe r i c a r d i um a nd t he he a d, but t he pa r a podi a r e m a i n s e pa r a t e a l t hough of t e n ove r l a ppi ng i n E c l ar k i ( F i g. 4)

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21 F i gu r e 4 P hot og r a phs of t he a nt e r i or dor s a l s ur f a c e of E l y s i a s pe c i e s i n t he r e gi on w he r e t he pa r a podi a c r os s i n f r ont of t he pe r i c a r di um ( P ) A E c l ar k i n s p. B E c r i s pat a I n E c r i s pat a t he pa r a podi a a r e e nt i r e l y f us e d, w hi l e i n E c l ar k i t he pa r a podi a a r e s e pa r a t e d by a ga p ( a r r ow l a be l l e d G ) i n t he m i d l i ne T he un l a be l l e d a r r ow s a r e poi nt i ng i n t he a nt e r i or di r e c t i on f a c e d by e a c h s pe c i m e n. I n a ddi t i on t he r e a r e m a j or di f f e r e nc e s i n t he g r os s s t r uc t ur e of t he pa r a podi a I n E c r i s pat a t he pa r a podi a ha ve a num be r of pr i m a r y f ol ds t ha t a r e of t e n s ym m e t r i c a l on e a c h s i de ( F i g. 1A ) T he e dge s of t he pa r a podi a a l s o ha ve m a ny s e c onda r y f ol ds m a ki ng t he m a ppe a r ve r y f r i l l y, r e l a t i ve t o t he pa r a podi a of E c l ar k i ( F i g. 1B ) W hi l e t he pa r a podi a of E c l ar k i ha ve pr i m a r y f ol d i ng, t he f o l ds a r e ne i t he r s ym m e t r i c a l nor f i xe d. M ove m e nt of t he pa r a podi a l e dge s of E c l ar k i a ppe a r s pe r i s t a l t i c l i ke w hi l e i n E c r i s pat a t he pe r i s t a l s i s oc c ur s onl y a l ong t he e dge w i t hi n e a c h pr i m a r y f ol d I nde e d, w he n unf ur l e d i n t he s unl i ght E c l ar k i c a n be c om e f l a t l i ke a l e a f w i t h a n a l m os t s m oot h di s t a l pa r a podi a l e dge w hi l e t he pa r a podi a of E c r i s pat a r e t a i n bot h t he pr i m a r y a nd s e c onda r y f ol di ng. A l s o, t he d i s t a nc e be t w e e n t he a nt e r i or l i m i t of t he pa r a podi a a nd t he

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22 he a d i s c om pa r a t i ve l y m uc h s hor t e r i n E c r i s pat a s o w hi l e E c l ar k i ha s a di s t i nc t ne c k r e gi on E c r i s pat a i s a l m os t ne c kl e s s w i t h t he a nt e r i or pa r a podi a of t e n ove r l a ppi ng t he he a d ( F i g. 1A B ) D or s al S u r f ac e V e n at i on : T he pa t t e r n of s upe r f i c i a l ve i ns on t he do r s a l s ur f a c e ha s be e n us e d a s a di a gnos t i c c ha r a c t e r i s t i c a m ong e l y s i i d s pe c i e s by s om e ( M a r c us 1980) a nd di s m i s s e d by ot he r s a s l e s s r e l i a bl e be c a us e of w i t hi n s pe c i e s va r i a bi l i t y ( J e ns e n 1980) A l t ho ugh t he l oc a t i on a nd a r r a nge m e nt of m i nor a nd s e c onda r y ve i ns c a n be qui t e va r i a bl e f r om s l ug t o s l ug w i t hi n a s pe c i e s ( s e e f or e xa m pl e G os l i ne r 1995) t he a r r a nge m e nt of t he m a j or dor s a l s ur f a c e ve s s e l s i s qui t e s i m i l a r w i t hi n e a c h s pe c i e s I n E c l ar k i t w o m a j or ve i ns l e a ve t he pos t e r i o r a s pe c t o f t he pe r i c a r di um a t a bout a 45 a ngl e f r om t he m i d l i ne a nd r un a l ong t he s ur f a c e t o t he pr o xi m a l ba s e of t he i nne r f a c e of e a c h pa r a podi um B ot h m a i n ve i ns t h e n r un pos t e r i or l y a l ong t he s ur f a c e f o r t he l e ngt h of t he s l ug, oc c a s i ona l l y gi vi ng o f f l a t e r a l br a nc he s t ha t a r e us ua l l y not s ym m e t r i c a l i n pos i t i on o r num be r T he l a t e r a l br a nc he s t r a ve r s e t he s ur f a c e of t he i nne r f a c e of t he pa r a podi a a l m os t t o t he e dge B ot h t he t w o m a j or ve s s e l s a nd l a t e r a l pa r a podi a l ve s s e l s a r e c ove r e d by a t hi n t r a ns pa r e nt l a ye r o f t he m a nt l e a nd a r e e a s i l y vi s i bl e I n c ont r a s t t he t w o m a j o r ve i ns o f E c r i s pat a s l ugs a r e c ove r e d by a t hi c k l a ye r of m a nt l e a nd of t e n a r e obs c ur e d by t he gr e e n di ge s t i ve t ubul e s a nd t he w hi t e m a nt l e s pot s A ppr oxi m a t e l y ha l f w a y be t w e e n t he pe r i c a r di um a nd t he t a i l t he m a j or ve s s e l s di s a ppe a r f r om vi e w i n E c r i s pat a U s ua l l y, no l a t e r a l br a nc he s a r e vi s i bl e a nyw he r e a l ong t he dor s a l s ur f a c e T he pe r i c a r di um i n E c r i s pa t a i s c ove r e d by a m o r e opa que m a nt l e l a ye r t ha n t ha t of E c l ar k i T ypi c a l l y t he v e nt r i c l e i s vi s i bl e t hr ough t he e xt e r na l s ur f a c e i n E c l ar k i but e n t i r e l y obs c ur e d by gr e e n t ubul e s a nd w hi t e m a nt l e s pot s i n E

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23 c r i s pat a D e ve l opm e nt a l pa t t e r n: B ot h s l ugs de pos i t e gg m a s s e s a nd ha ve l e c i t hot r ophi c l a r va l de ve l opm e nt E c l ar k i ha t c h a s f r e e s w i m m i ng ve l i ge r s 28 t o 35 da ys a f t e r de pos i t i on ( 20 C i n f i l t e r e d a r t i f i c i a l s e a w a t e r ) W i t hi n 5 da ys t he ve l i ge r s m e t a m or phos e i nt o j uve ni l e s l ugs w hi c h e xt e ns i ve f e e di ng e xpe r i m e nt s ( C ur t i s e t a l 2004) ha ve f ound w i l l e a t B r y ops i s pl um os a ( H U D S O N ) A G A R D H 1823 or D e r be s i a t e nui s s i m a ( M O R IS & D E N O T A RIS ) C R O U A N & C R O U A N 1867 [ but not C aul e r pa v e r t i c i l l at a A G A RD H 1847 a s r e por t e d e l s e w he r e ( J e ns e n 1980) ] T he l a r va e f r om E c r i s pat a e gg m a s s e s ha t c h a s f r e e s w i m m i ng ve l i g e r s f a s t e r ( 19 da ys a f t e r de pos i t i on 20C i n f i l t e r e d a r t i f i c i a l s e a w a t e r ) a nd a l s o m e t a m or phos e a f t e r f our t o f i ve da ys W hi l e t he i r f ood s our c e i s unknow n, f e e di ng e xpe r i m e nt s f ound t ha t E c r i s pat a j uve ni l e s do no t e a t B r y ops i s hy pnoi de s L A M O U R O U X 1809, B r y op s i s pe nnat a L A M O U RO U X 1809 or C v e r t i c i l l at a T he l e ngt h of t he l i f e c yc l e i s un know n, but w e ha ve r a i s e d E c l ar k i f r om e ggs i n t he l a bor a t or y t ha t a r e s t i l l a l i ve a f t e r 22 m ont hs a nd ha ve not ye t r e pr oduc e d. R a dul a : A s w i t h ot he r e l ys i i ds t he r a dul a e i n bot h s pe c i e s a r e m i c r os c opi c T he r a dul a e ( f r om s pe c i m e ns of s i m i l a r body s i z e ) c ons i s t of a s hor t r ow o f s i ngl e t e e t h ( E c r i s pat a 12 x 0 1. 0; E c l a r ki 14 17 x 0. 1 0) ( F i g. 5A B )

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24 F i gu r e 5. S c a nni ng e l e c t r on m i c r og r a phs of t he r a dul a e of f i e l d c ol l e c t e d E l y s i a c l ar k i n. s p. ( A C E G ) a nd E c r i s pat a ( B D F H ) s how i ng di f f e r e nc e s i n s ha pe be t w e e n s pe c i e s I n bot h E a nd F t he l i ga m e nt t ha t a t t a c he s t he t oot h t o t he r a dul a r m e m br a ne i s s t i l l pr e s e nt G ( E c l ar k i ) a nd H ( E c r i s pat a ) a r e l a t e r a l vi e w s of a r a dul a r t oot h i n i t s pos i t i on w i t hi n t he r a dul a T he a r r ow i n ( G ) poi nt s t o t he a t t a c hm e nt l i ga m e nt S c a l e ba r s = 100 m m ( A B ) 10 m m ( C H )

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25 I n ove r a l l a ppe a r a nc e t he t e e t h ha ve a t ypi c a l e l ys i i d s ha pe ( s e e G os l i ne r 1995) but t he r e a r e s om e s pe c i f i c di f f e r e nc e s i n t oot h s t r uc t ur e be t w e e n t he t w o s pe c i e s I n bot h c a s e s t he t e e t h a r e na r r ow a nd poi nt e d w i t h a l on gi t udi na l c e nt r a l gr oove on t he do r s a l [ f ol l ow i ng G os l i ne r s ( 1995) t e r m i nol ogy] a s pe c t i nt o w hi c h f i t s t he ne xt di s t a l t oot h ( F i g. 5C D ) T he dor s a l s i de of t he t oot h i n E c l ar k i ( F i g. 5C ) ha s a de e pe r b r o a de r g r oove t ha n i n E c r i s pat a ( F i g. 5D ) t he t oot h i s l onge r a n d w i de r i n E c l ar k i a nd t he ba s a l a r t i c ul a t i ons a r e f u r t he r a pa r t a nd m uc h m or e p r om i ne nt T he ve nt r a l s i de s of t he t e e t h a r e s i m i l a r i n bot h s pe c i e s ( F i g 5 E F ) but t he b r o a de r l onge r s ha pe of t he E c l ar k i t oot h ( F i g 5 E ) c om pa r e d t o E c r i s pat a ( F i g. 5F ) i s obvi ous T he ve nt r a l c ut t i ng f a c e of t he t oot h ha s a c e nt r a l s e r r a t e d r i dge t ha t r uns t he l e ngt h f r om t he out e r m os t poi nt t o t he a pode m e l i ke a t t a c hm e nt poi nt w he r e t he bi l a t e r a l t e ndon a t t a c he s t he t oot h t o t he r a dul a r m e m br a ne ( F i g 5 E F ) T he s e r r a t i ons e xt e nd l a t e r a l l y be yond t he c e nt r a l r i dge t o t he l a t e r a l e dge s of t he ve nt r a l s i de o f t he t oot h. B e yond t he s e s i m i l a r i t i e s m e a s ur e m e nt s m a de of s e ve r a l a s pe c t s on r a dul a r t e e t h f r om s e ve r a l s i m i l a r l y s i z e d s pe c i m e ns of bot h s l ug t ype s r e ve a l e d t ha t t he t e e t h of E c r i s pat a a r e s i gni f i c a nt l y s hor t e r by a bout 10 % ( p < 0. 05, t t e s t ) ( T a bl e 1) a nd a ppe a r b r oa de r t ha n t h os e of E c l ar k i a l t hough m e a s ur i ng t hi s a s pe c t e xa c t l y f r om m i c r ogr a phs ( F i g. 5) i s ve r y di f f i c ul t A l t hough t he r e i s s om e e vi de nc e t ha t r a dul a r t oo t h m o r phol ogy c a n c ha ng e w i t h di e t i n s om e s a c ogl os s a ns ( B l e a kne y 1990; J e ns e n 1993) t he t e e t h i n E c l ar k i r a i s e d f r om e gg m a s s e s a nd m a i nt a i ne d f or a ye a r i n our a qua r i a f e e di ng onl y o n B r y ops i s pl um os a w e r e i ndi s t i ngui s ha bl e f r om f i e l d c ol l e c t e d F l o r i da K e ys s pe c i m e ns t ha t ha d be e n f e e di ng f or

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26 a t l e a s t t hr e e t o f our m ont hs be f or e c a pt ur e pr i m a r i l y on P e ni c i l l us c api t at us ( T a bl e 1) ba s e d on D N A e vi de nc e T ab l e 1. C om pa r i s on o f t he l e ngt hs of r a dul a r t e e t h f r om V i r gi n I s l a nds s l ugs w i l d K e ys s l ugs a nd l a bor a t or y r a i s e d K e ys s l ugs t ha t ha ve be e n f e d onl y B r y ops i s pl um os a A l l s l ugs w e r e a ppr oxi m a t e l y 1 c m i n l e ngt h S l ug T ype N um be r of r a dul a t e e t h T oot h l e ng t h** V i r gi n I s l a nds # 12 ( 12) 114 4. 2 ( n= 12 ) K e ys ( F i e l d c ol l e c t e d) 14 ( 13 17 ) 129 4. 1 ( n= 3 ) K e ys ( L a b r a i s e d) 14 ( 14) 128 2. 5 ( n= 3 ) *A ve r a ge num be r of t e e t h i n f our r a dul a e ( r a nge ) ** m m ( c e nt r a l t oot h m e a s ur e d f r o m m i c r og r a ph) S D # S e pa r a t e m e a s ur e m e nt s m a de on t e e t h f r om bl ue gr e e n a nd w hi t e s pe c i m e ns w e r e m e r ge d w he n no s i gni f i c a nt di f f e r e nc e s w e r e f oun d. C h l or op l as t L oc at i on a n d M or p h ol ogy : T he l oc a t i on of t he s ym bi ot i c c hl or op l a s t s w i t hi n t he di ge s t i ve di ve r t i c ul a i s di f f e r e nt i n t he t w o s pe c i e s I n E c l ar k i t w o t ype s o f t e r m i na l t ubul e s a r e p r e s e nt ( F i g 6 ) O ne t ype i s s t r a i ght e ndi ng bl i ndl y w i t h t he pl a s t i ds l oc a t e d i n s e ve r a l c e l l s a l ong t he l e ngt h of t he t e r m i nus ( F i g. 6B )

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27 F i gu r e 6 L i gh t m i c r og r a phs of t he t e r m i n i of t he di ge s t i ve di ve r t i c ul a t ubul e s of E l y s i a s pe c i e s A B E c l ar k i n. s p. C D E c r i s pat a T he t ubul e s i n E c l ar k i a r e us ua l l y m uc h m or e hi ghl y l obe d a t t he t e r m i nus t ha n i n E c r i s pat a ( c om pa r e A w i t h C ) a nd ne ve r e nd i n a s i ngl e l obe ( c om p a r e B w i t h D ) T he s ym bi ot i c pl a s t i ds a r e l oc a t e d i n a s i ngl e c e l l a t t he t i p of t he t ubul e ( a r r ow s i n C a nd a r r ow l a be l l e d c i n D ) i n E c r i s pa t a T he t ubul e c e l l s pr oxi m a l t o t he t e r m i nus ( a r r ow l a be l l e d T i n D ) do not c ont a i n s ym bi ot i c pl a s t i ds I n c ont r a s t t he s ym bi ot i c pl a s t i ds a r e l oc a t e d i n c e l l s a l ong t he l e ngt h of t he t ubul e s ( a r r ow s i n B ) a s w e l l a s t he t e r m i na l c e l l i n E c l ar k i S c a l e ba r s = 75 m m ( A ) 40 m m ( B ) 80 m m ( C ) 50 m m

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28 I n t he s e c ond t ype o f t ube t he e nd br a nc he s i nt o s e ve r a l l o be s ( F i g 6A ) a nd t he pl a s t i ds a r e a l l c ont a i ne d i n a s i ngl e c e l l t ha t f o r m s a c ul de s a c a r ound e a c h t e r m i nus o f e a c h l obe ( F i g 7)

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29 F i gu r e 7. M ont a ge of e l e c t r on m i c r og r a phs of a l o ngi t udi na l s e c t i on t hr ough t he t e r m i nus of a di ge s t i ve t ubul e of E l y s i a c l ar k i n. s p M a ny s ym bi ot i c pl a s t i ds ( C ) a r e pr e s e nt i n t he c e l l t ha t w r a ps a r ound t he t e r m i nus ( a r r ow l a be l l e d D ) o f t he t ubul e i n a c ul de s a c T he pl a s t i ds i n t hi s c e l l a r e m os t l y f r om P c api t at us b ut a f e w ( unl a be l e d a r r ow ) ha ve a di f f e r e nt m o r ph ol ogy, i n di c a t i ng t ha t t he y a r e f r o m a di f f e r e nt a l ga l s pe c i e s T he l um e n o f t he di ve r t i c ul um t ubul e ( T ) i s s ur r ou nde d by a s i ngl e l a ye r of c e l l s w hi c h ha ve a de ns e l a ye r of m i c r ovi l l i ( M V ) on t he l um i n a l s i de L i p i d bodi e s ( L ) a r e p r e s e nt i n bot h t he pl a s t i d c ont a i ni ng c e l l a nd t he c e l l s f ur t he r pr oxi m a l i n t he t ubul e T he t ubul e i s s ur r ounde d by ha e m ol ym ph ( H ) C e l l nuc l e i a r e l a be l l e d ( N ) S c a l e ba r = 3 m m T hi s c e l l i s of t e n pa c ke d w i t h 20 or m or e pl a s t i ds A l l of t he gr e e n t ubu l e t i ps l i e j us t unde r t he e xt e r na l s ur f a c e of t he s l ug. I n c ont r a s t di ge s t i ve t ubul e s i n E c r i s pat a a r e ge ne r a l l y s i m i l a r t o t he f i r s t t ype of t ubul e de s c r i b e d a bove ; s t r a i ght a nd e ndi ng bl i ndl y ( F i g. 6D ) U s ua l l y, t he c hl or opl a s t s a r e l oc a t e d on l y i n t he c e l l a t t he t ubul e e nd i n E c r i s pat a ( F i g. 6C D ) unl i ke E c l ar k i ( F i g. 6B ) T he e nds of t he t ubul e s a r e oc c a s i ona l l y br a nc he d i nt o l obe s i n E c r i s pat a ( F i g 6C ) bu t no t i nt o t he m ul t i br a nc he d, m u l t i l obe d t ype s e e n i n E c l ar k i I nde e d, i t i s t he hi ghl y br a nc he d, m ul t i l obe d t e r m i ni t ha t gi ve t he m or e uni f o r m g r e e n s ur f a c e c ol or t o E c l ar k i c om pa r e d t o E c r i s pat a I n bot h s l ugs i t i s not unus ua l t o f i nd pl a s t i ds f r om m o r e t ha n one a l g a l s pe c i e s i n t he s a m e c e l l ( F i g 8 ) A l s o i n bot h s l ug t ype s t he m o r phol ogy o f m os t of t he pl a s t i ds i s c ons i s t e nt w i t h t ha t i n P e ni c i l l us s pp. but l a r ge r pl a s t i ds c ont a i ni ng s t a r c h gr a nul e s a nd pyr e noi d bodi e s a r e of t e n pr e s e nt us ua l l y i n t he s a m e c e l l w i t h P e ni c i l l us pl a s t i ds T he s m a l l e r c hl o r opl a s t s f r om di f f e r e nt s pe c i e s of P e ni c i l l us a r e i ndi s t i ngui s ha bl e f r om e a c h ot he r a t t he e l e c t r on m i c r os c ope l e ve l T he l a r ge r pl a s t i ds a r e m or phol o gi c a l l y s i m i l a r t o t hos e i n ot he r s pe c i e s of a l ga e w hi c h w e ha ve i de nt i f i e d ( s e e ne xt s e c t i o n) but a r e d i f f e r e nt be t w e e n t he t w o s pe c i e s of s l u gs

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30 F i gu r e 8. A B E l e c t r on m i c r ogr a phs o f s e c t i ons o f c hl or opl a s t c ont a i ni ng di ge s t i ve c e l l s i n E c r i s pat a M os t of t he s ym bi ot i c pl a s t i ds ha ve a n i de nt i c a l m or phol ogy t o P e ni c i l l us pl a s t i ds ( C ) H ow e ve r i n A a l a r ge pl a s t i d c ont a i ni ng a pyr e noi d body ( P ) i s pr e s e nt i n t he m i ds t of t he s m a l l e r P e ni c i l l us pl a s t i ds a nd i n B a l a r ge pl a s t i d c ont a i ni ng s t a r c h gr a nul e s ( S ) i s pr e s e nt i n t he s a m e c e l l a s t he P e ni c i l l us pl a s t i ds T he s e l a r ge r pl a s t i ds a r e f r om ot he r a l ga l s pe c i e s M V = m i c r ovi l l i i n t he t u bul e l um e n, N = nuc l e us of t he pl a s t i d c ont a i ni ng c e l l unl a be l l e d a r r ow i n B poi n t s t o a c i l i um i n t he t ubul e l um e n. S c a l e ba r s = 2 m m

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31 C h l or op l as t I d e n t i f i c a t i on : P C R w i t h r bc L p r i m e r s on E c r i s pat a D N A yi e l de d a 600 bp a m pl i c on. S e que nc i ng of 20 c l on e s i de nt i f i e d s i x di f f e r e nt r bc L s e que nc e s ( V I r bc L 1 6) 562 bp i n l e ngt h, a l l of w hi c h g r oup phyl oge ne t i c a l l y w i t hi n t he gr e e n a l ga l f a m i l y U dot e a c e a e ( F i g. 9) F i gu r e 9. N e i ghbor J oi ni ng t r e e ( F 84 m ode l s e e m e t hods ) of pa r t i a l r bc L nuc l e ot i de s e que nc e s of V I r bc L 1 6 obt a i ne d by P C R f r om E c r i s pat a U l v a l ac t uc a i s t he out gr ou p T he s c a l e s how n r e pr e s e nt s s ubs t i t ut i ons pe r s i t e a nd t he boot s t r a p va l ue s ( = 50% ) a r e i ndi c a t e d a t t he node s V I r bc L 1 6 a l l g r oup w i t h i n t he U dot e a c e a e c l a de w i t h V I r bc L 3 4, a nd 6 ha vi ng ove r 99% s i m i l a r i t y t o H i nc r as s at a P c api t at us a nd H opunt i a r e s pe c t i ve l y. G e nba nk a c c e s s i on num be r s f or t he s e que nc e da t a a r e l i s t e d a f t e r e a c h s pe c i e s

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32 T hi s gr oup c ons i s t s of s e ve r a l s pe c i e s of s i phona c e ous of t e n c a l c a r e ous m a c r oa l ga e t ha t oc c ur t hr oughout t he C a r i bbe a n ( e g. L i t t l e r & L i t t l e r 2000) W e w e r e a bl e t o i de nt i f y t he e xa c t a l ga l s pe c i e s of c hl or opl a s t or i gi n f o r t w o of t he r bc L s e que nc e s f r om E c r i s pat a V I r bc L 3 w a s i de nt i c a l t o H al i m e da i nc r as s at a ( E l l i s ) L A M O U R O U X 1816 r bc L s e que nc e a nd V I r bc L 4 i s 1 bp di f f e r e nt f r om t he P e ni c i l l us c api t at us s e que nc e I n a ddi t i on V I r bc L 6 w a s 99% s i m i l a r t o H opunt i a ( 4 bp di f f e r e nc e ) a nd V I r bc L 5 c l e a r l y g r oups w i t hi n P e ni c i l l us but doe s not m a t c h t he r bc L s e que nc e s ( da t a not s how n) de r i ve d f r om a ny of t he f our know n C a r i bbe a n s pe c i e s P e ni c i l l us py r i f or m i s G E P P & G E P P 1905, P e ni c i l l us dum e t os us ( L A M O U RO U X ) B L A IN V IL L E 1834, P e ni c i l l us l am our oux i i D E CA IS N E 1842, or P c api t at us ( F i g 9) V I r bc L 1 a nd 2 di d not gr oup s t r o ng l y w i t h a ny ge nus f or w hi c h t he r e a r e r bc L s e que nc e s i n t he G e nba nk da t a ba s e a l t hough t he y c l e a r l y f a l l w i t hi n t he U dot e a c e a e c l a de ( F i g 9) I n c om pa r i s on, m ol e c ul a r a nd m i c r os c opi c e vi de nc e i ndi c a t e s t ha t E c l ar k i f e e ds on a nd s e qu e s t e r s c hl or opl a s t s f r om P c api t a t us a nd H i nc r as s at a i n t he f i e l d a nd w i l l s ur vi ve f or a s l ong a s t w o ye a r s i n t he l a bor a t o r y on B pl um os a ( C ur t i s e t a l 2006b) M i t oc h on d r i al ge n e s e q u e n c e s : A t ot a l of 1, 164 n uc l e ot i de s ( 630 f r om C O I a nd 534 f r om 16 S ) o f t he m i t oc hondr i a l D N A ge nom e w e r e s e que nc e d a nd a na l yz e d f r om a s i ngl e i ndi vi dua l E c l ar k i a nd E c r i s pat a a nd us e d i n a p hyl oge ne t i c a na l ys i s T he t hi r d pos i t i on of C O I w a s de l e t e d due t o s a t ur a t i on T he m a xi m um l i ke l i hood di ve r ge nc e w a s e s t i m a t e d a t 2. 26% be t w e e n t he t w o s l ugs ba s e d o n c onc a nt e na t e d C O I a nd 16S s e que nc e s T he t hi r d pos i t i ons w e r e not de l e t e d t o e s t i m a t e m e a n di ve r ge nc e i n C O I c om pa r i s ons a m ong m ul t i pl e i ndi vi dua l s a s s a t ur a t i on i s not a n i s s ue a t t hi s l e ve l T he

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33 m e a n pe r c e nt di ve r ge nc e a m ong 20 E c l ar k i i ndi v i dua l s s e que nc e d a t t he C O I ge ne w a s 0. 50% ( 0. 26 % S D ) a nd r a nge d f r om 0. 15 % 1. 11% T he s i t ua t i on f or t he 6 E c r i s pat a i ndi vi dua l s w a s s i m i l a r w i t h a n a ve r a ge di ve r ge nc e of 0 30% ( 0. 001 S D ) T he m e a n di ve r ge nc e f or C O I be t w e e n t he t w o s pe c i e s w a s 7. 08% ( 0. 44 S D ) a nd r a nge d f r om 6 23% 7 75% T he phyl oge ne t i c r e l a t i ons hi p s ( F i g. 10) w e r e c ons i s t e nt w i t h t he br oa de r s ys t e m a t i c s t udy of B a s s a nd K a r l ( 2006) F i gu r e 10. M a xi m um l i ke l i hood phyl oge ny us i ng a he ur i s t i c s e a r c h of s e que nc e s ba s e d on C O I a nd 16S m i t oc hondr i a l D N A f r om E l y s i a c l ar k i n. s p. a nd E c r i s pat a i ndi vi dua l s a nd out gr oup t a xa E l y s i a t i m i da E l y s i e l l a pus i l l a E l y s i a c l ar k i E c r i s pat a 0 1 s ubs t i t ut i ons / s i t e

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34 D i s c u s s i on T he di f f e r e nc e s w e ha ve f ound be t w e e n E c l ar k i a nd E c r i s pat a a t e ve r y l e ve l ( g r os s m or phol ogy, l i ght m i c r os c opy, e l e c t r on m i c r os c opy, de ve l opm e nt a l f e e di ng, a nd m ol e c ul a r ) a l ong w i t h t he ha bi t a t d i f f e r e nc e pr ovi de c onc l us i ve e vi de nc e t ha t t he s e s l ugs a r e di f f e r e n t s pe c i e s F ur t he r m or e i n s pi t e of t he i r a bunda nc e t he e xi s t e nc e of E c l ar k i w a s unknow n t o t he 19t h c e nt ur y bi o l ogi s t s w ho de s c r i be d E c r i s pat a S ubs e que nt i nve s t i ga t or s i nc l udi ng C l a r k J e ns e n a nd Y onge a t l e a s t m a y ha ve c ol l e c t e d a nd w or ke d on E c l ar k i w i t hout r e c ogni z i ng i t a s a un i que s pe c i e s E l y s i a c r i s pat a ( = T r i dac hi a c r i s pat a ) ha s a c om pl i c a t e d a nd c onf us e d nom e nc l a t ur a l hi s t or y I t w a s f i r s t not e d by D e s ha ye s ( 1857) i n a r e por t on t he c ol l e c t i ons of M S c h r a m m f r om G ua de l oupe i n w hi c h he e r e c t e d t he ge nus T r i dac hi a A l t hough D e s ha ye s i s not c ons i de r e d t o ha ve na m e d a s pe c i e s i n t hi s pa pe r he s a ys : N ous pr op os ons pour l e ge nr e l e nom de T R I D A C H I A e t p our l e s p c e c e l ui de M S c hr a m m a uque l e n e s t d l a d c ouve r t e ( w e pr opos e f o r t h i s ge nus t he na m e T r i dac hi a a n d f or t he s pe c i e s t he na m e o f M S c hr a m m w ho di s c ove r e d i t ) E l s e w he r e i n t ha t p ubl i c a t i on, D e s ha ye s na m e s a not he r s pe c i e s A pl y s i a s c hr a m m i w hi c h s ugge s t s t ha t he w a s i m pl yi ng s c hr am m i f or t he ne w s pe c i e s of T r i dac hi a I n our op i ni on, t he ne xt r e vi s or w a s M r c h ( 1863) w ho de s c r i be d a ne w s pe c i e s T r i dac hi a c r i s pat a w hi c h he c ons i d e r e d di f f e r e nt f r om T s c hr am m i I m por t a nt l y, he t r e a t e d t he na m e T r i dac hi a s c hr am m i not a s ne w but a s a n a c c e pt e d na m e i nt r oduc e d by D e s ha ye s ( 1857) A l t hough t h e R ul e s of N om e nc l a t ur e w e r e s t i l l

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35 e vol vi ng t he n, t he r e s e e m s no doubt t ha t M r c h c o ns i de r e d T s c hr am m i t o ha ve da t e pr i or i t y ove r T c r i s pat a O t he r na m e s w hi c h m a y a ppl y t o T s c hr am m i or T c r i s pat a a r e t oo poor l y know n t o r e s ol v e V e r r i l l ( 1900) i de nt i f i e d a n a ni m a l f r om B e r m uda a s E l y s i a c r i s pa M r c h w hi c h P r uvot F ol ( 1946) de c i de d w a s not T c r i s pat a M r c h, a nd he r e na m e d i t E l y s i a v e r r i l l i L a t e r M a r c us ( 1957 ) no t e d t he hom onym y w i t h E v e r r i l l i ( T H IE L E 1931 ) a nd i nt r oduc e d a s e c ond r e pl a c e m e nt na m e E pr uv ot f ol ae S i nc e V e r r i l l w a s not na m i ng a ne w s pe c i e s ne i t he r r e pl a c e m e nt na m e w oul d s e e m ne c e s s a r y. S i m i l a r l y, W hi t e ( 1952) i de nt i f i e d a n a ni m a l f r om D r y T o r t uga s F l o r i da w i t h t he H a w a i i a n E l y s i a or nat a ( P E A S E 1860 ) T ha t i de nt i f i c a t i on i s a l m os t c e r t a i nl y i nc or r e c t a nd M a r c us ( 1957) f e l t obl i ge d t o r e na m e i t T r i dac hi a w hi t e ae i nt r oduc i ng a not he r unne c e s s a r y na m e t o t he l i t e r a t ur e C l e a r l y, t hi s i s not t he pl a c e t o r e s ol ve t he c onf us i ng hi s t or y of t he s e na m e s ( s e e M a r c u s 1957; M a r c us a nd M a r c us 1960; M a r c us a nd M a r c us 1967; M a r c us 1980; M a r c us 1982) H ow e ve r i t i s i m por t a nt t o de t e r m i ne w he t he r or not T c r i s pat a or T s c hr am m i c oul d be a n e a r l i e r na m e f or E c l ar k i O ne of t he m or e obvi ous e xt e r na l di f f e r e nc e s r e por t e d he r e i s t ha t t he pa r a podi a a r e f us e d i n t he a nt e r i or m i dl i ne of E c r i s pat a a nd a r e s e pa r a t e i n E c l ar k i D e s ha ye s ( 1857: 137 143) i n hi s de s c r i pt i on of t he na m e T r i dac hi a ba s e d on S c hr a m m s m a t e r i a l c l e a r l y s t a t e s t ha t t he pa r a podi a j oi n i n t he a nt e r i o r m i d l i ne o c e l l e s d un c t s e r uni s s e nt c e l l e s de l a ut r e w hi c h m e a ns t ha t E s c hr am m i by de f i ni t i on, ha s t he pa r a podi a j oi ni ng a nt e r i or l y a s w e l l S o r e ga r dl e s s of t he t r ue r e l a t i o ns hi p be t w e e n E c r i s pat a a nd E s c hr am m i t he y bot h di f f e r f r om E c l ar k i w he r e t he pa r a podi a r e m a i n s e pa r a t e i n t he a nt e r i or m i dl i ne T hus t he V i r gi n I s l a nds E l y s i a i s pr ope r l y r e f e r r e d t o a s E

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36 c r i s pat a l e a vi ng t he K e ys s pe c i e s unna m e d unt i l n ow T he phyl oge ne t i c a nd popul a t i on a na l ys e s i ndi c a t e t ha t E c r i s p at a a nd E c l ar k i a r e c l os e l y r e l a t e d, but ge ne t i c a l l y di s t i nc t W e ha ve ne ve r s e e n a ny i ndi c a t i on of a m i xt ur e of phe not ype s i n e i t he r gr oup, nor doe s E c r i s pat a oc c ur i n t he m a ngr ove ha bi t a t o f E c l ar k i or vi c e ve r s a F u r t he r m or e t he ge ne t i c a na l ys i s us i ng C O I i ndi c a t e s c l e a r l y t ha t t he t w o s pe c i e s a r e ge ne t i c a l l y di s t i nc t T he a ve r a ge s e que nc e di ve r ge nc e be t w e e n E c r i s pat a a nd E c l ar k i i ndi vi dua l s w a s a ppr oxi m a t e l y 14 t i m e s l a r ge r t ha n w i t hi n E c l ar k i a nd 23 t i m e s l a r ge r t ha n w i t hi n E c r i s pat a i ndi c a t i ng a c l e a r e vol ut i ona r y s e pa r a t i on be t w e e n t he t w o s pe c i e s T he a ve r a ge m a gni t ude of d i ve r ge nc e be t w e e n E c r i s pat a a nd E c l ar k i us i ng t he C O I a nd 16S da t a i s w i t hi n t he r a nge of i nt r a s pe c i f i c c om p a r i s ons of r e l a t e d t a xa ( 5. 8 % 4. 9 S D B a s s & K a r l 2006) a ga i n i ndi c a t i ng t ha t E c l ar k i a nd E c r i s pat a a r e c l os e l y r e l a t e d a nd ge ne t i c a l l y di s t i nc t F u r t he r m or e t he s i gni f i c a nt C O I di ve r ge nc e i ndi c a t e s t ha t ge ne f l ow i s l i m i t e d be t w e e n t he t w o s pe c i e s T hus ge ne t i c di s t i nc t i on c a n be a dde d t o t he m or phol ogi c a l a nd phys i ol ogi c a l va r i a t i ons w e ha ve de s c r i be d. B ot h ou r m ol e c ul a r r e s ul t s a nd m i c r os c opy c onf i r m t ha t E c r i s pat a a nd E c l ar k i not on l y e a t a va r i e t y o f a l ga l s pe c i e s but a l s o a r e a bl e t o e s t a bl i s h a c hl or o pl a s t s ym bi os i s w i t h s e ve r a l s pe c i e s I n bot h c a s e s e l e c t r on m i c r os c opy i ndi c a t e d t ha t t he pr e dom i na t e i nt r a c e l l ul a r pl a s t i d i n bot h s l ug s pe c i e s w a s f r om P e ni c i l l us s pp. F u r t he r m or e i t w a s not unus ua l t o f i nd pl a s t i ds f r om m o r e t ha n one a l ga l s pe c i e s w i t hi n t h e s a m e di ge s t i ve c e l l I n a ddi t i on, t he r e s ul t s i ndi c a t e di f f e r e nc e s a s w e l l a s s i m i l a r i t i e s i n a l ga l s pe c i e s e a t e n be t w e e n E c r i s pat a a nd E c l ar k i T he K e ys s l ugs e a t a nd s e que s t e r c hl or opl a s t s f r om B r y ops i s pl um os a a nd D e r be s i a t e nui s s i m a a s j uve ni l e s ( C u r t i s e t a l 2004) a nd a s a dul t s e a t a nd s e que s t e r c hl or opl a s t s f r om P c api t at us P l am ou r oux i i H i nc r as s at a a nd H m oni l e i n

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37 a ddi t i on t o B pl um os a ( C ur t i s e t a l 2006a b) T he V i r gi n I s l a nds s l ugs do not e a t e i t he r B hy pnoi de s o r B pe nnat a a s j uve ni l e s L i ke E c l a r k i E c r i s pat a c ont a i ne d s ym bi ot i c pl a s t i ds f r om H i nc r as s at a a nd P c api t at us but n ot f r om a ny ot he r s pe c i e s of H al i m e da or P e ni c i l l us p r e s e nt i n t he V i r gi n I s l a nds ha bi t a t s T he m o l e c ul a r r e s ul t s pr e s e nt e d he r e i ndi c a t e t he pr e s e nc e of pl a s t i ds f r om ot he r a l ga l s pe c i e s i n E c r i s pat a a s w e l l w hi c h w e c oul d not i de nt i f y e xa c t l y, pr oba bl y due t o l a c k of i n f or m a t i on i n t he da t a ba s e E ve n s o, t he y a l l c l e a r l y gr oup w i t hi n t he U dot e a c e a e a nd none of t he unknow ns w e r e f ound i n E c l ar k i T he l a r ge num be r of a l ga l c hl or op l a s t do nor s pe c i e s ut i l i z e d by E c r i s pat a i s s ur pr i s i ng s i nc e m a ny s a c ogl os s a n s ha ve be e n r e por t e d t o ha ve a s i ngl e or onl y a f e w a l ga l s pe c i e s a s t he c hl or opl a s t s our c e ( e g. J e ns e n 1980) O ne f i na l i s s ue i s t he us e of t he p r e s e nc e or a bs e nc e of s o c a l l e d z ooxa nt he l l a e a s a t a xonom i c c ha r a c t e r i s t i c i n t he s e s pe c i e s O ur r e s ul t s ha ve de m ons t r a t e d t ha t z ooxa nt he l l a e a r e not pr e s e nt i n e i t he r E c r i s pat a or i n E c l ar k i N e ve r t he l e s s f o r s e ve r a l ye a r s a f t e r Y onge a nd N i c hol a s ( 1940) r e p o r t e d z ooxa nt he l l a e i n E c r i s pat a t he i r pr e s e nc e or a bs e nc e w a s us e d a s a t a xonom i c c ha r a c t e r ( M a r c us & M a r c us 1960; W hi t e 1952) unt i l T r e nc h ( 1969) r e por t e d t ha t t he z ooxa nt he l l a e de s c r i be d by Y onge a nd N i c hol a s w e r e a c t ua l l y s ym bi ot i c c hl or opl a s t s H o w e ve r Y onge a nd N i c hol a s ( 1940) a nd T r e nc h ( 1969 a l s o T r e nc h e t a l 1969) w e r e l ooki n g a t t w o d i f f e r e nt t hi ngs T r e nc h s di s c ove r y of s ym bi ot i c pl a s t i ds i n t he di ge s t i ve t ub ul e e nds i n E c r i s pat a w a s i nde e d c or r e c t but Y onge a nd N i c hol a s ha d m i s s e d t he pl a s t i ds e nt i r e l y. I ns t e a d, on t he ba s i s of i r on ha e m a t oxyl i n s t a i ni ng Y onge a nd N i c hol a s ( 1 940) c onc l ude d, i nc or r e c t l y, t ha t t he di ge s t i ve t ubul e e nds w e r e f ul l of z ym oge n gr a nul e s I n a ddi t i on Y onge a nd N i c hol a s

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3 8 f ound t he l a r ge r ound b r ow n c e l l s o f w hi c h T r e nc h ( 1969) m a ke s no m e nt i on a nd i de nt i f i e d t he m a s z ooxa nt he l l a e O ur e l e c t r on m i c r os c opy r e ve a l e d t ha t t he b r ow n c e l l s a r e not z ooxa nt he l l a e but a r e m ol l us c a n pi gm e nt c e l l s T he s a m e i s t r ue of t he s m a l l e r r ound r e d c e l l s T he f unc t i on of t he s e c e l l t ype s i s not know n, but t he pi gm e nt s m a y ha ve t o do w i t h pr ot e c t i on of t he pl a s t i ds f r om t oo m uc h s unl i ght T he e xa c t l oc a t i on of t he r ound c e l l s va r i e s f r om s l ug t o s l ug, s om e t i m e s f or m i ng a s ol i d ba nd a t t he e dge of t he pa r a podi a s om e t i m e s a b r oke n ba nd, s om e t i m e s n ot a t t he pa r a podi a l e dge s a t a l l B ot h t ype s of r ound c e l l s e e m t o be f r e e i n t he ha e m oc oe l a nd i t i s not c l e a r w ha t gove r ns t he i r di s t r i but i on. E l y s i a c l ar k i ha s s i m i l a r t ype s of r oun d c e l l s a l t hough t he pi gm e nt c ol or s a r e di f f e r e nt I n a ny e ve nt ou r r e s ul t s i ndi c a t e t ha t t he pr e s e nc e of a s ol i d br oke n o r e ve n a bs e nc e of a l i ne of pi gm e nt a t t he pa r a podi a l t i ps i s pr oba bl y of s om e phys i ol ogi c a l s i gni f i c a nc e but not a r e l i a bl e t a xonom i c c ha r a c t e r A not he r s pe c i e s w hi c h ne e ds t o be c ons i de r e d i s E l y s i a di om e de a ( B e r gh, 1894 ) f r om t he P a c i f i c c oa s t o f C e nt r a l A m e r i c a ( M a r c us & M a r c us 1967) I t ha s l ong be e n c ons i de r e d r e l a t e d t o E c r i s pat a f i r s t be i ng de s c r i be d a s a s pe c i e s of T r i dac hi a be c a us e of i t s e xt r e m e l y f ol de d pa r a podi a a nd t he n pl a c e d i n a s ubge nus T r i dac hi e l l a M a c F a r l a nd, 1924 be c a us e of t he a nt e r i or ga p s e pa r a t i ng t he pa r a podi a T he pa r a podi a i n E d i om e de a a r e not j oi ne d a nt e r i o r l y a nd br a nc he s of t he di ge s t i ve gl a nd r a m i f y i nt o t he s ol e of t he f oot c a us i ng gr e e n pa t c he s s i m i l a r t o E c l ar k i H ow e ve r i t d i f f e r s c ons i de r a bl y f r om E c l ar k i i n c ol o r a nd i n t he e xt r e m e l y f ol de d e dge t o t he pa r a podi a a s i n E c r i s pat a F u r t he r m o r e w hi l e i n E c l ar k i t he gr e e n on t he s ol e o f t he f oot i s i nt e r s pe r s e d w i t h r ounde d a nd ova l w hi t i s h s pot s i n E di om e de a t he t r a ns l uc e nt w hi t e r e gi ons a r e br oke n l i ne s r unni ng pa r a l l e l t o t he l on g a xi s of t he body. E l y s i a di om e de a

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39 a l s o di f f e r s i n ha vi ng a c ha r a c t e r i s t i c pa t t e r n of bl a c k a nd w hi t e l i ne s r unni ng dow n t he r hi nophor e s ont o t he he a d. O t he r di f f e r e nc e s i n c ol or c om pa r e d t o E c l ar k i a r e t he s m a l l bl ue s pot s s c a t t e r e d ove r t he dor s um a nd i nne r w a l l s of t he pa r a podi a a nd a bor de r of bl a c k s pot s a nd s om e t i m e s r e d or or a nge s pot s on t he pa r a podi a l e dge ( R udm a n 2001)

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40 C h ap t e r 2: M ol e c u l ar P h y l oge n y of t h e B r yop s i d al e s ( U l vop h yc e ae C h l or op h yt a ) b as e d on a p ar t i al s e q u e n c e of t h e c h l or op l as t e n c od e d l ar ge s u b u n i t r i b u l os e b i s p h os p h at e c ar b oxyl as e / oxyge n as e ge n e I n t r od u c t i on T he ne w l y de s i gna t e d s a c ogl os s a n s pe c i e s E l y s i a c l ar k i ( P IE RCE E T A L 2006) f e e ds by pi e r c i ng a l ga l s i phon c e l l w a l l s w i t h t he i r r a dul a t e e t h, a nd t he n s uc ki ng t he c ont e nt s out of t he f i l a m e nt ( C ur t i s e t a l 2006b ) A s t hi s s pe c i e s w a s j us t r e c e nt l y na m e d, t he r e i s a l a c k of da t a on t he a l ga l f ood s our c e o f t he s e que s t e r e d c hl or opl a s t s T he m os t i de a l s a c ogl os s a n f ood s our c e s w oul d be t hos e t ha t y i e l d t he m os t c hl or op l a s t s w i t h t he l e a s t e ne r ge t i c i nve s t m e nt T hus s i phona c e ous a l ga e of t he or de r B r yops i da l e s l a c ki ng c r os s w a l l s a r e t he m os t l i ke l y s a c ogl os s a n f ood s our c e s a s t he y w oul d yi e l d t he l a r ge s t a m ount of c e l l s a p f or t he a ni m a l pe r r a dul a punc t u r e ( G r e e ne 1970 ) I n or de r t o pos i t i ve l y i de nt i f y t he a l ga l c hl or opl a s t s t ha t a r e s e que s t e r e d by E c l ar k i t he c hl or opl a s t e nc ode d ge ne of t he l a r ge s ubuni t f or r i bul os e bi s p hos pha t e c a r boxyl a s e / oxyge na s e ( r bc L ) w a s P C R a m pl i f i e d a nd s e que nc e d f r om t ot a l E c l ar k i D N A ( S e e c ha pt e r s 1, 3, a nd 4) U nf or t una t e l y, a m a j or obs t a c l e t o i de nt i f yi ng t he r bc L s e que nc e s obt a i ne d w a s a n

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41 ov e r a l l l a c k of a va i l a bl e a l ga l r bc L s e que nc e s f or c om pa r i s on. H ow e ve r by c om pa r i ng t he obt a i ne d r bc L s e que nc e s t o know n a l ga l s e que nc e s t he ge ne r a l c l a de c a n be i de nt i f i e d, s i gni f i c a nt l y na r r ow i ng t he po t e nt i a l a l g a l s our c e s w hi c h c a n t he n be c ol l e c t e d f r om t he f i e l d a nd t he i r r bc L s e que nc e d. A s pa r t o f t hi s pr o j e c t a l a r ge s e t of r bc L s e que nc e s f r om t he B r yops i da l e s a nd i n pa r t i c ul a r t he U dot e a c e a e w e r e c ol l e c t e d. A l t hough t he pr i m a r y pu r pos e of t he s e a l ga l r bc L s e que nc e s w a s t o pr ovi de a m a t c h t o t he r bc L s e que nc e s obt a i ne d f r om E c l ar k i D N A t he y a l s o a l l ow f o r phyl oge ne t i c a na l ys i s pr ovi di ng s e ve r a l i m po r t a nt c onc l us i ons on t he t a xonom y o f t he B r yops i da l e s T hi s c ha pt e r f oc us e s on t he t a xonom y of t he B r yo ps i da l e s a nd t he phyl oge ne t i c s of t he o r de r ba s e d on r bc L s e que nc e a l i gnm e nt s T he c l a s s U l vophyc e a e i nc l ude s a l l gr e e n a l ga e t ha t pos s e s s a c r uc i a t e f l a ge l l a r r oot s ys t e m c ont a i n m ot i l e c e l l s t ha t di s pl a y a c l oc kw i s e a bs ol ut e or i e nt a t i on of ba s a l bodi e s a nd t he a bs e nc e of a phyc opl a s t ( M a t t ox & S t e w a r t 1984 H i l l i s C ol i nva ux 1984) T he ul vophyt e s a r e p r i m a r i l y m a r i ne m a c r oa l ga e a nd r e pr e s e nt a w i de r a nge of m or phol ogi e s i nc l udi ng m ul t i c e l l ul a r c oe noc yt i c a nd uni nuc l e a t e f or m s ( G r a ha m & W i l c ox 2000) W i t hi n t he U l vophyc e a e a r e f i ve o r de r s c ont a i ni ng m a r i ne a l ga e : B r yops i da l e s ( = C a ul e r pa l e s ) D a s yc l a da l e s C l a do phor a l e s ( = S i phonc l a da l e s ) U l va l e s a nd t he U l ot r i c ha l e s ( G r a ha m & W i l c ox 2000, W y nne 2005) A na l ys i s of nuc l e a r e nc ode d r D N A s e que nc e s ha s s how n t he U l vophyc e a e t o be pol yphyl e t i c w i t h t he m ul t i nuc l e a t e d a nd s e pt a l e oi de C l a dophor a l e s t he s i phona c e ous B r yops i da l e s a nd t he uni nuc l e a t e d D a s yc l a da l e s f or m i ng a m onophyl e t i c c l a de t ha t i s m o r e c l os e l y r e l a t e d t o t he c l a s s e s C hl or ophyc e a e a nd P l e ur a s t r ophyc e a e t ha n t o t he o r de r s U l v a l e s a nd U l ot r i c ha l e s ba s e d on m ol e c ul a r da t a ( Z e c hm a n e t a l 1990)

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42 T he or de r B r yops i da l e s i s r e pr e s e nt e d e xc l us i ve l y by a l ga e c om pos e d of c oe noc yt i c s i phona c e ous f i l a m e nt s a nd c ont a i ns 5 m a r i ne f a m i l i e s s pl i t i nt o t w o s ubor de r s : t he B r yops i di ne a e ( O s t r e obi a c e a e B r y ops i da c e a e a nd C odi a c e a e ) a nd t he H a l i m e di ne a e ( C a ul e r pa c e a e a nd U dot e a c e a e ) ( H i l l i s C ol i nva ux 1984) S o m e ha ve a l s o pl a c e d t he f r e s hw a t e r s pe c i e s D i c hot om os i phon t u be r os u s ( t he onl y m e m be r o f t he ge nus ) i n t he m onot ypi c f a m i l y ( D i c hot o m os i phona c e a e ) w i t hi n t he B r yops i da l e s ( O 'K e l l ys & F l oyd 1984) T he r e i s a l a r ge a m ount of m or phol o gi c a l di ve r s i t y w i t hi n t he B r yops i da l e s r a ngi ng f r o m f i ne f i l a m e nt ous a l ga e ( B r yops i da c e a e ) t o c a l c i f i e d c om pl e x m or phol ogi e s ( U dot e a c e a e ) a nd ge ne r a a r e c l a s s i f i e d ba s e d on t he ve ge t a t i ve t r a i t s of a dul t t ha l l i ( V r oom e t a l 1998) C l a di s t i c a na l ys i s ba s e d on m or phol ogi c a l a na t om i c a l r e pr oduc t i ve c e l l ul a r a nd e c ol ogi c a l c ha r a c t e r s ha s s how n t he B r yops i da l e s t o be m onophyl e t i c a nd ha s s uppor t e d t he m on ophyl y o f U dot e a c e a e C a ul e r pa c e a e C odi a c e a e a nd B r yops i da c e a e ( V r oom e t a l 1998 ) H ow e ve r t hr e e ge ne r a w i t hi n t he U dot e a c e a e ( P e ni c i l l us U dot e a a nd C hl or ode s m i s ) a r e pol yphyl e t i c ba s e d on nuc l e a r r D N A ( K ooi s t r a 2002) T he pur pos e of t hi s a na l ys i s w a s t o he l p c l a r i f y t he phyl oge ne t i c s t r uc t ur e o f t he B r yops i da l e s us i ng r bc L s e que nc e s T he a na l ys i s f oc us e s on t he vi a bi l i t y o f t he s ubor de r s H a l i m i di ne a e a nd B r yops i di ne a e a nd t he s t r uc t ur e bot h w i t hi n a nd be t w e e n e s t a bl i s he d f a m i l i e s P a r t i c ul a r a t t e nt i on w a s f oc us e d on t he U dot e a c e a e a s pr i or a na l ys i s ba s e d on nuc l e a r r D N A ha s s how n ge ne r a i n t he gr oup t o be pol yphy l e t i c

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43 M at e r i al s an d M e t h od s S p e c i m e n S a m p l i n g an d O u t gr ou p S e l e c t i on A l ga l s pe c i e s w e r e c ol l e c t e d i n F l o r i da f r om a bor r ow pi t a nd i t s a s s oc i a t e d m a ngr ove s w a m p on t he F l or i da B a y s i de of G r a s s y K e y ( 24 o 45. 5' N 80 o 59 5'W ) a nd V a c a K e y ( 24 o 44 1'N 81 o 02 0' W ) or f r om S uns e t B e a c h i n T a r pon S pr i ngs F l o r i da ( 27 75 2' N 82 76. 3' W ) ( T a bl e 2) A l s o, B r y ops i s pl um os a w a s c ol l e c t e d f r om t he s out h e nd of t he ba y s i de of t he S uns hi ne S kyw a y B r i dge i n T a m pa F l o r i da ( 27 62 0' N 82 65 5' W ) w hi l e B oodl e ops i s pus i l l a w a s c ol l e c t e d f r o m C oc kr oa c h B a y ne a r R us ki n, F l or i da ( 27 40. 7 N 82 30. 2' W ) P e ni c i l l us py r i f or m i s a nd B r y op s i s pe nnat a w e r e c ol l e c t e d f r om r e e f ha bi t a t s ne a r S t T hom a s V i r gi n I s l a nds by D r C ol i n M F i nne y a nd U dot e a c y at hi f or m i s w a s pr ovi de d f r om T w i n C a ys B e l i z e by R a ndi R ot j a n. V ouc he r s pe c i m e ns w e r e i de nt i f i e d by N E C a nd D r C l i nt on D a w e s a n d ha ve be e n de pos i t e d a t t he U ni ve r s i t y of S ou t h F l o r i da H e r ba r i um T he s pe c i e s U l v a c ur v at a w a s s e l e c t e d a s t he out gr oup ba s e d on pr i o r 18s r D N A a na l ys i s w hi c h s how e d t he C l a dophor a l e s D a s yc l a da l e s a nd B r yops i da l e s f or m i ng a m onophyl e t i c gr oup, a nd t he U l va l e s gr oupi ng s e p a r a t e l y ( Z e c hm a n e t a l 1990)

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44 T ab l e 2: L i s t o f a l ga l s pe c i e s s a m pl e d i n t hi s s t udy. P h y l u m O r d e r F a m i l y A l g a l S p e c i e s S o u r c e A c c e s s i o n N u m b e r Ch l o ro p h y t a Bry o p s i d a l e s U d o t e a c e a e H a l i m e d a d i s c o i d e a D E CA ISN E G E N BA N K A B0 3 8 4 8 8 H a l i m e d a o p u n t i a (L IN N A E U S ) L A MO U RO U X G E N BA N K A B0 3 8 4 8 9 H a l i m e d a i n c r a s s a t a (E L L IS ) L A M O U R O U X T a rp o n S p ri n g s F L D Q 4 6 9 3 2 8 H a l i m e d a m o n i l e ( E L L IS & S O L A N D E R ) L A M O U R O U X T a rp o n S p ri n g s F L D Q 4 6 9 3 2 9 P e n i c i l l u s c a p i t a t u s ( L A M A RCK ) T a rp o n S p ri n g s F L D Q 4 6 9 3 3 2 P e n i c i l l u s l a m o u r o u x i i D E CA IS N E G ra s s y K e y F L D Q 4 6 9 3 3 3 P e n i c i l l u s d u m e t o s i s ( L A M O U RO U X ) B L A I N V I L L E G ra s s y K e y F L D Q 4 6 9 3 3 0 P e n i c i l l u s p y r i f o r m i s ( G E PP & G E PP ) S t T h o m a s V I D Q 4 6 9 3 3 1 A v r a i n v i l l e a n i g r i c a n s f. f l o r i d a n a L IT T L E R & L I T T L E R G ra s s y K e y F L D Q 4 6 9 3 2 5 A v r a i n v i l l e a m a z e i M U RR A Y & B O O D L E G ra s s y K e y F L D Q 4 6 9 3 2 6 Cl a d o c e p h a l u s l u t e o f u s c u s ( C RO U A N & C RO U A N ) B RG E S E N G ra s s y K e y F L D Q 4 6 9 3 2 4 U d o t e a f l a b e l l u m ( E L L IS & S O L A N D E R ) H O W E G ra s s y K e y F L D Q 4 6 9 3 3 4 U d o t e a c o n g l u t i n a t a ( E L L IS & S O L A N D E R ) L A M O U R O U X V a c a K e y F L D Q 4 6 9 3 2 2 U d o t e a c y a t h i f o r m i s D E CA ISN E T w i n Ca y s Be l i z e D Q 4 6 9 3 1 8 R h i p o c e p h a l u s p h o e n i x ( E L L IS & S O L A N D E R ) K T Z IN G V a c a K e y F L D Q 4 6 9 3 2 1 R h i p o c e p h a l u s o b l o n g u s ( D E CA ISN E ) K T Z IN G V a c a K e y F L D Q 4 6 9 3 1 9 B o o d l e o p s i s p u s i l l a ( C O L L I N S ) T A Y L O R J O L Y & B E RN A T O W ICZ Co c k ro a c h Ba y F L D Q 4 6 9 3 2 0 Bry o p s i d a c e a e B r y o p s i s p l u m o s a ( H U D SO N ) A G A R D H T a m p a Ba y F L D Q 4 6 9 3 2 7 B r y o p s i s p e n n a t a L A MO U R O U X L o n g K e y F L D Q 4 6 9 3 2 3 Co d i a c e a e Co d i u m f r a g i l e ( S U RI N G A R ) H A RIO T G E N BA N K A B1 0 3 0 2 1 Co d i u m l u c a s i i S E T C H E L L G E N BA N K A B0 3 8 4 8 1 Ca u l e rp a c e a e Ca u l e r p a b r a c h y o p u s H A RV E Y G E N BA N K A B0 3 8 4 8 3 Ca u l e r p a r a c e m o s a ( F O RSSK L ) A G A RD H G E N BA N K A B0 3 8 4 8 6 Ca u l e r p a o k a m u r a e W E BE R V A N B O SSE G E N BA N K O s t re o b i a c e a e O s t r e o b i u m q u e k e t t i i B O RN E T & F L A H A U L T G E N BA N K A Y 0 0 4 7 6 5 D a s y c l a d a l e s B a t o p h o r a o e r s t e d i i A G A R D H G E N BA N K A Y 1 7 7 7 4 8 Cl a d o p h o ra l e s D i c t y o s p h a e ri a c a v e rn o s a ( F O RSS K L ) B RG E S E N G ra s s y K e y F L D Q 4 6 9 3 3 5 U l v a l e s U l v a c u r v a t a L IN N A E U S G E N BA N K A F 1 8 9 0 7 1 D N A E xt r ac t i on A l ga e w e r e r i ns e d w i t h de i on i z e d w a t e r pa t t e d dr y w i t h K i m w i pe s pl a c e d i n l i qui d ni t r oge n, a nd gr ound t o a f i ne pow de r us i ng a m o r t a r a nd pe s t l e A l ga l pow de r w a s a dde d t o 2x C T A B bu f f e r ( D oyl e & D oyl e 1987 ) ( 0. 1g a l ga l pow de r / m L C T A B buf f e r )

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45 m i xe d t hor oughl y a nd i nc uba t e d f or 1 h r a t 65 o C i n a w a t e r ba t h A n e qua l vo l um e of c hl or of or m : i s oa m yl a l c ohol ( 24: 1 ) w a s a dde d, m i x e d ge nt l y f o r 5 m i n, a nd c e nt r i f uge d a t 16, 000 x g f o r 10 m i n. T he s upe r na t a nt w a s r e m o ve d, a nd t he D N A w a s pr e c i pi t a t e d f r om i t w i t h 100% e t ha nol r i ns e d 3x i n 70% e t ha nol l yophi l i z e d, a nd r e s us pe nde d i n di s t i l l e d, f i l t e r e d, a nd a ut oc l a ve d ( d f a ) w a t e r P C R an d S e q u e n c i n g D e ge ne r a t e c ons e ns us pr i m e r s e que nc e s w e r e de s i gne d t o c ons e r ve d r e gi ons of t he c hl or opl a s t e nc ode d ge ne f or r i bul os e bi s phos pha t e c a r boxyl a s e / oxe ge na s e ( r bc L ) a s de t e r m i ne d by a l i gnm e nt s o f 31 c hl or ophyt e r bc L s e que nc e s a va i l a bl e i n G E N B A N K T hi s ge ne w a s c hos e n be c a us e r bc L s e que nc e s a r e c ons e r ve d e nough t o de s i gn c ons e ns us pr i m e r s ( e g M a nha r t 1994, M c C our t e t a l 2000) but di s t i nc t e nough be t w e e n e ve n t he m os t c l os e l y r e l a t e d s pe c i e s t o di s t i ngui s h t he m ( C ha s e e t a l 2005 N e gr i s ol o e t a l 2004) T he f or w a r d pr i m e r s e que nc e w a s 5 A A A G C X G G K G T W A A A G A Y T A 3 ( r bc L 1) a nd t he r e ve r s e p r i m e r s e que nc e w a s 5 C C A A C G C A T A R A D G G T T G W G A 3 ( r bc L 2) R e a c t i on m i xt ur e s w e r e m a de w i t h E nz ypl us 2000 r e a c t i on buf f e r ( E nz ypol ) [ 2m M T r i s H C l ( pH 8. 8) 1m M K C l 1m M ( N H 4 ) 2 S O 4 0. 01 % T r i t on X 100, a nd 0 01 m g/ m l B S A ] 12. 5 nM dN T P 0. 25 m M r bc L 1 0 25 m M r bc L 2 1 ng/ l D N A a nd 0. 1 uni t s / l pol ym e r a s e ( E nz ypl us 2000, E nz ypol ) P C R t ouc h dow n r e a c t i ons w e r e pe r f o r m e d on a t he r m oc yc l e r ( G e ne A m p P C R 2400, A ppl i e d B i os ys t e m s ) w i t h a de na t ur i ng t e m pe r a t ur e of 94 o C f o r 30 s a nne a l i ng t e m pe r a t u r e s t a r t i ng a t 45 o C f or 30 s a nd a n e xt e ns i on t e m pe r a t ur e of 72 o C f or 1 m i n f o r 35 c yc l e s w i t h t he a nne a l i ng t e m pe r a t ur e l ow e r e d by 0. 5 o C on e a c h s ubs e que nt c yc l e

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46 P C R pr oduc t s w e r e pur i f i e d, po l i s he d, l i ga t e d a nd t r a ns f or m e d us i ng t he P C R S c r i pt A m p ( s k+ ) c l oni ng ki t ( S t r a t a g e ne ) a c c or di n g t o t he m a nuf a c t ur e r s i ns t r uc t i ons a nd c ol oni e s w e r e s c r e e ne d by P C R us i ng T 3 a nd T 7 pr i m e r s C ol oni e s pos s e s s i ng i ns e r t s c l os e i n s i z e t o t he P C R p r oduc t w e r e m i ni pr e ppe d us i ng t he N uc l e os pi n P l a s m i d ki t ( C l ont e c h) S e que nc i ng r e a c t i ons w e r e pe r f or m e d us i ng t he C D Q D C T S Q ui c k S t a r t K i t ( B e c km a n C oul t e r ) a nd t he i ns e r t s e que nc e s w e r e de t e r m i ne d ( Q S M 8000, B e c km a n C oul t e r ) o r s e que nc e d by c om m e r c i a l ve ndor s ( N or t hw oods D N A M i nne s ot a or M a c r oge n, S out h K or e a ) w i t h T 3 a nd T 7 pr i m e r s P h yl oge n e t i c A n a l ys i s S e que nc e s w e r e obt a i ne d f r om c ol l e c t e d s pe c i m e ns or f r om G E N B A N K ( T a bl e 2 ) S e ve r a l t a xa ( C odi um l uc as i i C f r agi l e a nd C aul e r pa ok am ur ae ) c ont a i n a n i nt r on but onl y t he c odi ng r e gi ons of t he s e que nc e s w e r e us e d. T he ge ne r bc L i s a pp r opr i a t e f or e l uc i da t i ng r e l a t i ons hi ps f or bot h ol de r a nd younge r t a xa be c a us e a l t hough i t i s hi gh l y c ons e r ve d, t he r e i s e nough va r i a t i on, e s pe c i a l l y a t t he t hi r d c odon pos i t i on t o pr ovi de phyl oge ne t i c i nf or m a t i on ( K l l e r s j e t a l 1999) W hi l e c ons i de r e d uni nf or m a t i ve by s om e due t o s a t ur a t i on ( S w of f o r d e t a l 1996) o t he r s ha ve s how n t ha t t he t hi r d c odon pos i t i on c ont a i ns m os t of t he phyl oge ne t i c s i gna l ( Z e c hm a n 2003, N i c kr e nt e t a l 2000 K l l e r s j e t a l 1998) T he r e f or e a l l c odon pos i t i o ns w e r e i nc l ud e d i n t he a na l ys i s F o r a l l obt a i ne d s e que nc e s t he 5' a nd 3' pr i m e r s e que nc e s w e r e di s c a r de d, a nd s e que nc e s obt a i ne d f r om G E N B A N K w e r e e di t e d t o c or r e s pond t o t he r e m a i ni ng r e gi on. A l ga l r bc L s e que nc e s w e r e a l i gne d us i ng C l us t a l W ( T h om ps on e t a l 1994) a nd c onve r t e d t o N E X U S f o r m a t us i ng M E G A v 3 1 ( K um a r e t a l 2 004)

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47 T he pr opor t i ons of c ons e r ve d, va r i a bl e a nd pa r s i m ony i nf or m a t i ve s i t e s w e r e de t e r m i ne d us i ng M e ga v. 3 1 ( K um a r e t a l 2004) T o e va l ua t e t he a m ount o f phyl oge ne t i c s i gna l i n t he da t a s e t ( H i l l i s & H ue l s e nbe c k 1992) un w e i ght e d m a xi m um pa r s i m ony w a s us e d t o e va l ua t e t he s ke w ne s s ( g 1 ) of 100, 000 r a ndom t r e e l e ngt hs w i t h P a up 4. 10b ( S w of f or d 2003) E xha us t i ve M a xi m um P a r s i m ony ( M P t r e e ) w a s p e r f or m e d us i ng P a up v. 4. 10b ( S w of f or d 2 003) S i nc e i t i s not pos s i bl e t o pe r f or m a n e xha us t i ve s e a r c h w i t h m o r e t ha n 12 t a xa t a xa w e r e s e l e c t e d i n a n e f f or t t o r e pr e s e nt a l l m a j or gr oups B a ye s i a n a na l ys i s ( B I t r e e ) w a s pe r f or m e d us i ng M r B a ye s v. 3 1 ( H ue l s e nbe c k & R onqui s t 2001, R onqui s t & H ue l s e nbe c k 2003) T he be s t m ode l w a s de t e r m i ne d us i ng M ode l t e s t v. 3. 7 ( P os a da & C r a nda l l 1998) a nd t h e s e t t i ngs us e d w e r e ns t = 6" w i t h r a t e s = i nvga m m a T w o r uns w e r e ge ne r a t e d us i ng 4 c ha i ns a nd 1, 000 000 ge ne r a t i ons w i t h a f r e que nc y of s a m pl i n g of 10 R e s u l t s T he r e s ul t i ng r bc L s e que nc e f or t he m a j or i t y of s pe c i e s w a s 562 bp. T w o s e que nc e s ( U l v a c ur v at a a nd D i c ht y os phae r i a c av e r nos a ) w e r e 559 bp due t o a s i ngl e a m i no a c i d de l e t i on. A m ong t he a l i gne d c ha r a c t e r s 317 ( 56 4 % ) w e r e c ons t a n t 245 ( 43. 6 % ) w e r e va r i a bl e a nd 209 ( 37. 2 % ) w e r e pa r s i m ony i n f or m a t i ve R a ndom t r e e l e ngt h di s t r i but i ons w e r e l e f t s ke w e d ( g 1 = 0. 51 ) i ndi c a t i ng t ha t t he da t a c ont a i n phyl oge ne t i c s t r uc t ur e

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48 E xh au s t i ve M axi m u m P ar s i m on y E xha us t i ve M a xi m um pa r s i m ony a na l ys i s on 12 t a xa r e s ul t e d i n a s i ngl e t r e e ( F i g. 11) T hi s t r e e r e ve a l s t he t ha t D a s yc l a da l e s i s a s i s t e r t a xa t o t he B r yops i da l e s T he U dot e a c e a e i s pol yphyl e t i c w i t h t he s i ngl e s pe c i e s of P e ni c i l l us a nd H al i m e da gr oupi ng c l os e r t o t he C a ul e r pa c e a e t ha n t o t hos e of A v r ai nv i l l e a a nd C l adoc e phal us O s t r e obi um que k e t t i i i s a s i s t e r t a xa t o C aul e r pa r ac e m os a w hi l e D i c hot om os i phon t ube r os us gr oups w i t h t he s i ngl e s pe c i e s i n t he A v r ai nv i l l e a a nd C l a doc e phal us c l a de C odi um l uc as i i a nd B r y ops i s pl um os a gr oup t oge t he r a s a s i s t e r c l a de t o t he r e s t o f t he B r yops i da l e s F i gu r e 11. E xha us t i ve M a xi m um P a r s i m ony t r e e ba s e d on 12 t a xa ge ne r a t e d us i ng P a up V 4. 10b

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49 B aye s i an A n al ys i s B a ye s i a n a na l ys i s ( B I t r e e ) ( F i g 12 ) s how s t he or d e r B r yops i da l e s t o be a m ono phyl e t i c c l a de w i t h t he D a s yc l a da l e s a s a s i s t e r gr oup. T he s ubor de r B r yops i di ne a e i s s uppor t e d w i t h t he C odi a c e a e a nd t he B r yops i da c e a e a s s i s t e r t a xa a nd t he O s t r e obi a c e a e gr oupi ng ba s a l l y. S uppor t f o r t he s ubor de r H a l i m e di ne a e i s w e a k ( pos t e r i or p r oba bi l i t y = 0 51) T he f a m i l y U dot e a c e a e i s s how n t o be pol yphyl e t i c w i t h t he m a j or i t y of s pe c i e s gr oupi ng t oge t he r a s a s i s t e r c l a de t o t he C a ul e r pa c e a e ( w hi c h a r e s how n t o be m onophyl e t i c ) a nd t he ge ne r a C l adoc e phal us a nd A v r ai nv i l l e a gr oupi ng w i t h t he f r e s hw a t e r s pe c i e s D i c hot om os i phon t ube r os u s out s i de of t hi s c l a de T he ge ne r a P e ni c i l l us U dot e a, R hi p oc e phal us a nd H al i m e da a r e a l l s how n t o be pol yphyl e t i c w i t h H al i m e da be i ng ba s a l t o t he r e s t of t he U dot e a c e a e ( e xc e pt f o r A v r ai nv i l l e a a nd C l a doc e phal us ) W he n B at ophor a oe r s t e di i i s us e d a s t he out gr oup ( da t a not s how n) t he t opol ogy of t he t r e e i s i de nt i c a l e xc e pt t ha t t he pl a c e m e nt of O s t r e obi um que k e t t i i i s not r e s ol ve d a nd f a l l s out s i de of t he r e s t of t he B r yops i da l e s

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50 F i gu r e 12. B a ye s i a n a na l ys i s ba s e d on r bc L s e que nc e s T he va l ue s a t t he node s a r e pos t e r or pr oba bi l i t i e s a nd t he s c a l e ba r r e pr e s e nt s s ubs t i t ut i ons / s i t e T r e e ge ne r a t e d us i ng M r B a ye s v. 3 1

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51 D i s c u s s i on T he m a xi m um pa r s i m ony ( M P ) a nd ba ye s i a n i nf e r e nc e ( B I ) s up por t s i m i l a r t opo l ogi e s w i t h t he onl y m a j or di s c r e pa nc y be i ng t he pl a c e m e nt of O s t r e obi um que k e t i i W he n t he s ubor de r s H a l i m e di ne a e a nd B r yops i di ne a e w e r e or i gi na l l y e s t a bl i s he d, t he p l a c e m e nt of t he O s t r e obi a c e a e i n t he B r yops i di ne a e w a s t e nt a t i ve ( H i l l i s C ol i nva ux 1984) T he O s t r e obi a c e a e l i ve i n c a l c a r e ous s ubs t r a t e s a nd a r e s om e t i m e s s e pa r a t e d f r om t he r e s t of t he B r yops i da l e s due t o t he p r e s e nc e of oc c a s i ona l s e pt a ( L uka s 1974, K o r nm a nn & S a hl i ng 1980, i n V r oom e t a l 1998) H ow e ve r B I t r e e a ppe a r s t o s uppor t t he B r yops i di ne a e a s a c l a de w hi l e t he M P t r e e a na l ys i s s how s t he O s t r e obi a c e a e t o be pa r t of t he B r yops i da l e s but a s a s i s t e r t a xa t o t he C a ul e r pa c e a e P hyl oge ne t i c a na l ys i s of D a s yc l a da l e s r bc L s ugge s t s t ha t t he B I t r e e a na l ys i s i s m or e r obus t t ha n t he M P t r e e due t o pot e nt i a l i nc ons i s t e nc i e s c a us e d by t hi r d c odon pos i t i on s a t ur a t i on, a nd t he B I t r e e w a s m or e c ongr ue nt w i t h f os s i l r e c or ds t ha n t he M P t r e e a na l ys i s ( Z e c hm a n 2003) T he r e f o r e w he r e di s c r e pa nc i e s a r i s e t he B I t r e e i s gi ve n pr e f e r e nc e ove r t he M P t r e e i n t hi s a na l ys i s T he r e i s s t r ong s uppor t ( pos t e r i or p r oba bi l i t y [ pp] 0. 99) t o s ugge s t t ha t m e m be r s of t he U dot e a c e a e a r e m o r e c l os e l y r e l a t e d t o t he C a ul e r pa c e a e t ha n t o t he A v r ai nv i l l e a C l adoc e phal us D i c hot om os i phon c l a de T he r e s ul t s de m ons t r a t e t he f i r s t m ol e c ul a r ba s e d phyl oge ny t o e s t a bl i s h t he ge ne r a A v r ai nv i l l e a a nd C l adoc e phal us out s i de of t he U dot e a c e a e A v r ai nv i l l e a a nd C l adoc e phal us ha ve l ong be e n pl a c e d i n t he U dot e a c e a e ba s e d on c l a di s t i c a na l ys i s of m or phol ogi c a l c ha r a c t e r s ( V r oom e t a l 1998, L i t t l e r & L i t t l e r 2000, W ynne 2005) H ow e ve r R ot h a nd F r i e dm a n ( 1980) s ugge s t e d A v r ai nv i l l e a a nd C l adoc e phal us s houl d be pl a c e d i n a s e pa r a t e c l a de ba s e d on t he m a c r os e gr e ga t i on of

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52 nuc l e ol i a nd t he pr e s e nc e of py r e noi d s i n t he c hl or opl a s t s t ha t a r e s ur r ounde d by s t a r c h pl a t e s w hi c h a r e e nc i r c l e d by t hyl a koi d l a m e l l a e ( R ot h & F r i e dm a nn 1987) T he s e m or phol ogi c a l c ha r a c t e r s a r e not f ound i n a ny ot he r m e m be r s of t he B r yops i da l e s B ot h t he M P a nd B I t r e e s s how t ha t w hi l e t he A v r ai nv i l l e a C l adoc e phal us D i c hot om os i phon c l a de m a y be l ong i n t he s ubor de r H a l i m e di ne a e i t m a y be m or e a ppr opr i a t e t o c ons i de r t hi s c l a de a s e pa r a t e f a m i l y, s i s t e r t o t he C a ul e r pa c e a e U dot e a c e a e gr oup. T he pl a c e m e nt of D i c hot om os i phon t ube r os us i n t he B r yops i da l e s ha s be e n dubi ous s i nc e i t i s a f r e s hw a t e r a l ga l a c ks t he pi g m e nt s i phona xa nt hi n, a nd ha s ooga m ous r e pr oduc t i on ( H i l l i s C ol i nva ux 1984) H ow e ve r bot h t he B I a nd M P a na l ys i s c l e a r l y s how s D t ube r os us be l ongs i n t he B r yops i da l e s gr oupi ng ve r y c l os e l y w i t hi n t he A v r ai nv i l l e a C l adoc e phal us c l a de D i c hot om os i phon t ube r os us ha s s phe r i c a l s t r uc t ur e s ne a r t he ba s a l e nd of a m yl opl a s t s w hi c h a r e s i m i l a r t o s t r uc t u r e s f ound on l y i n A v r ai nv i l l e a a nd C l adoc e phal us of t he B r yops i da l e s ( R ot h & F r i e dm a nn 1987) A ddi t i ona l l y, e l e c t r on de ns e bodi e s c ont a i ne d i n v e s i c l e s f r om s pe c i e s of A v r ai nv i l l e a a nd C l adoc e phal us a r e s i m i l a r i n s i z e a nd s ha pe t o s t r uc t ur e s f ound i n D i c hot om os i phon t ube r os us ( R ot h & F r i e dm a nn 1987 M oe s t r up & H of f m a n 1973) T he s e c ha r a c t e r s m a y be c ons i de r e d s yna pom or phi e s of t he A v r ai nv i l l e a C l adoc e phal us a nd D i c hot om os i phon c l a de H ow e ve r t he di s t i nc t di f f e r e nc e s be t w e e n D i c hot om os i phon t ube r os us a nd t he r e s t of t hi s c l a de a r e l i ke l y due t o i t now be i ng a n e xc l us i ve l y f r e s hw a t e r a l ga I t i s pos s i bl e t ha t D t ube r os us a r os e f r om a m a r i ne a l g a s i m i l a r t o s pe c i e s of A v r ai nv i l l e a a nd C l adoc e phal us H ow e ve r onc e m ovi ng f r om a m a r i ne e nvi r onm e nt t o a n e xc l us i ve l y f r e s hw a t e r e nvi r onm e nt a w hol e ne w s e t of e nvi r o nm e nt a l f a c t or s w oul d d i r e c t t he e vol ut i on of t hi s a l ga l e a di ng t o r a pi d phe not ypi c c ha nge s I t i s i nt e r e s t i ng t o not e t ha t

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53 D i c hot om os i phon t ube r os us w a s l ong pl a c e d w i t h t he he t e r okont i c a l ga l ge nus V auc he r i a w hi c h i t i s o f t e n c onf us e d w i t h i t due t o s i m i l a r m o r phol ogi e s s i m i l a r f or m s o f r e pr oduc t i on, a nd c o oc c ur e nc e i n t he s a m e ha bi t a t ( de l G r os s o 1988, M e s t r up & H of f m a n 1973) T he s e s i m i l a r i t i e s m us t be due t o c onve r ge nt e vol ut i on f r om t he e vol ut i ona r y pr e s s ur e s of t he s a m e f r e s hw a t e r ha bi t a t a s t he t w o ge ne r a a r e f r om di f f e r e nt phyl a T he pol yphyl y of t he ge ne r a P e ni c i l l us a nd U dot e a s uppor t s pr e vi ous da t a ba s e d on nuc l e a r e nc ode d r R N A ( K ooi s t r a 2002) P e ni c i l l us a nd U dot e a w e r e o r i gi na l l y de s i gna t e d a s di s t i nc t ge ne r a due t o t he br us he s of P e ni c i l l us a nd t he bl a de s of U dot e a ( W ynne 2005) H ow e ve r t he br us h of P e ni c i l l us m a y be c ons i de r e d s i m pl y a n unc or t i c a t e d U dot e a bl a de w hos e s i phons ha ve l os t a dhe r e nc e ( K ooi s t r a e t a l 2002) w hi l e t he bl a de s of R hi poc e phal us m a y be c ons i de r e d a t ype o f U dot e a bl a de t ha t i s s pl i t r e gul a r l y, t hus e xpl a i ni ng t he a ppa r e nt non m onop hyl y of t he t hr e e g r oups T he gr oupi ng of P e ni c i l l us l am our oux i i m or e c l os e l y w i t h P dum e t os i s a nd P py r i f or m i s t ha n P c api t at us w a s s ur pr i s i ng s i nc e P c api t at us a nd P l am our oux i i bot h ha ve bl u nt t r unc a t e d l a t e r a l s i phons o f t he s t i pe w hi l e t he s t i pe l a t e r a l s i phons of P dum e t os i s a n d P py r i f or m i s e nd i n l ong t a pe r e d a pi c e s ( L i t t l e r & L i t t l e r 2000) H ow e ve r U c ongl ut i nat a w hi c h gr oups ba s a l l y t o P l am our oux i i ha s l a t e r a l a ppe nda ge s of t he s t i pe m o r e s i m i l a r t o P dum e t os i s t ha n t o P l am our oux i i a s do s e ve r a l ot he r s pe c i e s of U dot e a ( L i t t l e r & L i t t l e r 2000) F ur t he r t he l a t e r a l a ppe nda ge s of P dum e t os i s a nd P py r i f or m i s a r e s o s i m i l a r t ha t t hi s t r a i t i s l i ke l y a s yna pom or phy o f t h e t w o s pe c i e s T he a ppa r e nt pol yphyl e t i c na t ur e o f t he ge nus H al i m e da r e ve a l e d by B I a na l ys i s i s i nt e r e s t i ng be c a us e i t i s us ua l l y vi e w e d a s a s t r o ng m onophyl e t i c c l a de ( K ooi s t r a &

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54 V e r br ugge n 2005, V e r br ugge n e t a l 2005, V e r br u gge n & K ooi s t r a 2004 H i l l i s e t a l 1998 ) O ur a na l ys i s c ont a i ns r e pr e s e nt a t i ve s of t hr e e of t he f i ve r e c ogni z e d H a l i m e da l i ne a ge s : s e c t i on H a l i m e da ( H di s c oi de a ) s e c t i on O punt i a ( H opunt i a ) a nd s e c t i on R hi ps a l i s ( H i nc r as s at a a nd H m oni l e ) ( V e r b r ugg e n & K ooi s t r a 2004 V e r b r ugge n e t a l 2005) P r i or a na l ys i s bot h m ol e c ul a r a nd m or pho l ogi c a l w a s m a de w i t h t he a s s um pt i on t ha t t he ge nus H al i m e da i s a m onophyl e t i c g r oup, a nd ot he r U dot e a c e a e t a xa l i ke P e ni c i l l us c api t at us a nd U dot e a f l abe l l um a r e us e d a s out gr oups ( V e r b r ugge n e t a l 2005 V e r br ugge n & K ooi s t r a 2004) B a s e d on our r e s ul t s how e ve r P e ni c i l l us s p. or U dot e a s p. m a y not be a n a ppr op r i a t e out g r oup s e l e c t i on s i nc e H al i m e da gr oups ba s a l l y t o t he r e s t of t he U dot e a c e a e W i t hout t he i nc l us i on o f m or e t a xa f r om t he U d ot e a c e a e t he ge nus H a l i m e da w oul d a ppe a r t o be a m onophyl e t i c c l a de B a s e d on t he f os s i l r e c or d i t i s e s t i m a t e d t ha t H i nc r as s at a a ppe a r e d dur i ng t he e a r l y c r e t a c e ous w hi l e H opunt i a doe s not a ppe a r i n t he f os s i l r e c or d unt i l t he l a t e c r e t a c e ous pe r i od ( D r a ga s t on & S o l i m on 2002) T hi s c or r e l a t e s w i t h t he B I a na l ys i s w hi c h s how s H i nc r as s at a t o be ba s a l t o a c l a de t ha t i nc l ude s H opun t i a T he f os s i l r e c or d o f H di s c oi de a i s l e s s c l e a r H di s c oi de a w oul d be e xpe c t e d t o p r e da t e t he m i n t h e f os s i l r e c or d be c a us e i t g r oups ba s a l l y t o t he ot he r H al i m e da s pe c i e s i n t hi s s t udy, unl e s s i t a r os e f r o m a now e xt i nc t s pe c i e s A l t hough not r e f l e c t e d i n t he f os s i l r e c or d i t i s pos s i bl e t ha t t he r e m a i nde r of t he U dot e a c e a e ( e xc l udi ng m e m be r s of A v r ai nv i l l e a a nd C l adoc e phal us ) i s de r i ve d f r om t he H al i m e da s e c t i on O punt i a U nf or t una t e l y ou r s t u dy onl y c ont a i ns 4 s pe c i e s of H al i m e da r e pr e s e nt i ng t hr e e l i ne a ge s T hus t he H al i m e da l i n e a ge t ha t t he U dot e a c e a e a r os e f r om c a nnot be di s c e r ne d, but i t a ppe a r s t ha t t he U dot e a c e a e a r os e of t he H al i m e da g r oup.

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55 C l e a r l y a phyl oge ny t ha t i nc l ude s a m or e c om pl e t e s e t of H al i m e da s pe c i e s s houl d pr ovi de m or e i ns i ght i nt o t he or i gi ns of t he U dot e a c e a e t ha n i s pr e s e nt l y a va i l a bl e B oodl e ops i s pus i l l a i s t he onl y non c a l c i f i e d m e m be r of a c l a de i n t hi s a na l ys i s t ha t i s ot he r w i s e r e pr e s e nt e d by e nt i r e l y by c a l c i f i e d a l ga e B oodl e ops i s pus i l l a ha s s i m i l a r m or phol ogi c a l c ha r a c t e r i s t i c s t o A v r ai nv i l l e a s pe c i e s ( m oni l i f or m f i l a m e nt s ) ( L i t t l e r & L i t t l e r 2000) a nd D i c hot om os i phon t ube r os us ( br a nc h di c hot om i e s ) a nd w a s or i gi na l l y t hought t o be i n t he ge nus D i c hot om os i phon ( T a yl o r e t a l 1953 ) T he r e f or e i t c oul d be e xpe c t e d t o g r oup w i t h t he A v r ai nv i l l e a C l adoc e phal us D i c hot om os i phon c l a de B ut B pus i l l a a l s o ha s s i m i l a r i t i e s t o t h e j uve ni l e pha s e s of P e ni c i l l us a nd s om e s pe c i e s of U dot e a ( T a yl or e t a l 1953) a nd t he r e f o r e i t s pl a c e m e nt i n t he U dot e a P e ni c i l l us R hi poc e phal us c l a de a ppe a r s a ppr opr i a t e A s B p us i l l a gr oups ba s a l l y t o P e ni c i l l us a nd U dot e a i t m a y be t ha t t ha t t he U dot e a P e ni c i l l us R hi poc e phal us c l a de a r os e f r om B oode l ops i s or a now e xt i nc t t a xa s i m i l a r t o i t T he l os s a nd r e a qui s i t i on o f c a l c i f i c a t i on w i t hi n t he B r yops i da l e s a nd pa r t i c ul a r l y t he U dot e a c e a e i s a n i nt e r e s t i ng i s s ue T he m os t pa r s i m oni ous e xpl a na t i on i s t ha t c a l c i f i c a t i on w a s l os t i n t he ge nus B oodl e ps i s a nd w a s m a i nt a i ne d i n t he r e s t o f t he c l a de Y e t a na l ys i s ba s e d on r D N A ha s a l s o s how n t he non c a l c i f i e d ge nus C hl or ode s m i s t o be pa r t of t hi s c l a de a nd pol yphyl e t i c w i t h U dot e a a nd P e ni c i l l us i ndi c a t i n g c a l c i f i c a t i on ha s be e n l os t o r r e a c qui r e d m ul t i pl e t i m e s w i t hi n t hi s c l a de ( K ooi s t r a 2002) T ha t t he s i s t e r or de r t o t he B r yops i da l e s ( t he D a s yc l a da l e s ) a l s o c ont a i ns s om e c a l c i f i e d a nd s om e non c a l c i f i e d ge ne r a pr ovi de a not he r pos s i bl e s c e na r i o of w he r e c a l c i f i c a t i on w a s l os t or a r os e I t i s pos s i bl e t ha t t he a nc e s t or of t he bot h t he B r yops i d a l e s a nd t he D a s yc l a da l e s m a y ha ve be e n c a l c i f i e d, a nd t h i s w a s s ubs e que nt l y l os t s e ve r a l t i m e s i n t he bot h or de r s H ow e ve r

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56 t he f os s i l r e c or d s how s m a ny now e xt i nc t c hl or o ph yt e ge ne r a a nd c a l c i f i c a t i on w h i c h a r os e l ong be f or e t he B r yops i da l e s a nd t he D a s yc l a da l e s ( W r a y 1977) T he r e f or e i t m a y be t ha t t he m e c ha ni s m f o r c a l c i f i c a t i on i s r e l a t i ve l y s i m pl e t o t u r n on a nd of f a nd t he r e f or e ha s c om m onl y a r i s e n a nd be e n l os t i n m a ny gr e e n a l ga l t a xa I n c onc l us i on, t he B I t r e e a nd t he M P t r e e a na l ys i s de m ons t r a t e t ha t t he D a s yc l a da l e s a nd t he B r yops i da l e s a r e s i s t e r t a xa W i t hi n t he B r yops i da l e s B I a na l ys i s s t r ongl y s uppor t s t he m onophyl y of t he s ubor de r B r yops i di ne a e w i t h O s t r e obi um que k e t t i gr oupi ng ba s a l l y, a nd t he r e i s w e a k s uppor t f o r t he m onophyl y o f t he s ubor de r H a l i m e di ne a e B ot h t he M P t r e e a nd t he B I t r e e s h ow e d t he f a m i l y U dot e a c e a e t o be pol yphyl e t i c w i t h t he non c a l c i f i e d ge ne r a A v r ai nv i l l e a a nd C l adoc e phal us gr oupi ng w i t h D i c hot om os i phon t ube r os us i n a s e pa r a t e f a m i l y. T he r e s t o f t he U dot e a c e a e a r e m or e c l os e l y r e l a t e d t o t he m onophyl e t i c C a ul e r pa c e a e w i t h t he onl y non c a l c i f i e d m e m be r of t hi s gr oup be i ng B oode l ops i s pus i l l a W i t hi n t h e U dot e a c e a e t he B I t r e e c onf i r m s t he pol yphyl y of t he ge ne r a P e ni c i l l us R hi poc e phal us a nd U dot e a w hi l e P l am our oux i i gr oups w i t h P dum e t os i s a nd P py r i f or m i s T he ge nus H al i m e da doe s not f or m a m onophyl e t i c c l a de i n t he B I t r e e a nd g r ou ps ba s a l l y w i t h t he r e s t o f t he c a l c i f i e d m e m be r s of t he U dot e a c e a e s ugge s t i ng t ha t t he U dot e a c e a e a r os e f r om a l i ne a ge o f H al i m e da

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57 C h ap t e r 3: T h e S y m b i o t i c C h l or op l as t s i n t h e S ac ogl os s an s E l y s i a c l ar ki ar e F r om S e ve r al A l gal S p e c i e s I n t r od u c t i on S a c ogl os s a n s f e e d on s i phona c e ous a l ga e ; c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um t a ke up a nd s e que s t e r a l ga l c hl or opl a s t s f r om t he g ut l um e n ( T r e nc h e t a l 1969) I n E l y s i a c l ar k i P IE RCE E T A L 2006 t he c a pt ur e d, i nt r a c e l l ul a r pl a s t i ds r e t a i n phot os ynt he t i c f unc t i on f o r 3 4 m ont hs one of t he m os t pe r s i s t e nt c hl or opl a s t s ym bi os e s know n ( C ur t i s e t a l 2004 ) B ot h pl a s t i d a nd nuc l e a r e nc ode d c hl o r opl a s t pr ot e i ns a r e s ynt he s i z e d w hi l e t he c hl or opl a s t s r e s i de i n c e l l s i n E c l ar k i ( P i e r c e e t a l 2003 ) T he r e f o r e t he ge ne s f or t he nuc l e a r e nc ode d c hl or opl a s t pr ot e i ns m us t be p r e s e nt i n t he s e a s l ugs A ho m ol og o f f uc oxa nt hi n c hl or ophyl l bi ndi ng p r ot e i n ( f c p) a nu c l e a r e nc ode d a l ga l ge ne ha s be e n de t e c t e d i n t ot a l D N A e xt r a c t e d f r o m s pe c i m e ns of E c l ar k i T hi s i s t he f i r s t e vi de nc e f or t he hor i z ont a l t r a ns f e r of a ge ne be t w e e n m u l t i c e l l ul a r or ga ni s m s ( P i e r c e e t a l 2003) U nf or t una t e l y, t he a l ga l s our c e o f t he pl a s t i ds i n E c l ar k i ha s not be e n i de nt i f i e d t o da t e m a ki ng f ur t he r s t udy o f hor i z ont a l ge ne t r a ns f e r di f f i c ul t

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58 E l y s i a c l ar k i w a s onl y r e c e nt l y de s c r i be d a nd w a s pr e vi ous l y c onf us e d by us a nd ot he r s w i t h a c l os e l y r e l a t e d s pe c i e s E l y s i a c r i s pat a ( P i e r c e e t a l 2006) P r i or t o t a xonom i c c l a r i f i c a t i on, our r e por t s on a s pe c t s of t he c hl or opl a s t s ym bi os i s a t t r i but e our r e s ul t s t o E c r i s pat a, but i n f a c t a l l of our s t udi e s w e r e done w i t h E c l ar k i ( s e e f or e xa m pl e P i e r c e e t a l 2003) I n a ddi t i o n, i t i s no t c l e a r w he t he r pr e vi ous a ut ho r s ha ve w or ke d w i t h E c r i s pat a or E c l ar k i e s pe c i a l l y i n F l or i da ( C l a r k & B us a c c a 1978; J e ns e n 1980; T hom ps on & J a r m a n 1989) e ve n t hough i n ve s t i ga t i ons on c hl or opl a s t s ym bi os i s i n E c r i s pat a da t e ba c k t o t he 1940 s ( Y onge & N i c h ol a s 1940) M a ny s t udi e s of c hl or opl a s t s ym bi os i s ha ve f oc us e d on s a c ogl os s a ns i n w hi c h t he s our c e of t he c hl o r opl a s t s i s r e s t r i c t e d t o a s i ngl e a l ga l s pe c i e s F or e xa m pl e t he c hl or opl a s t s i n E l y s i a v i r i di s a r e t a ke n f r om C odi u m f r agi l e ( H a w e s 1979) a dul t E l y s i a t i m i da f e e d on A c e t abul ar i a ac e t abul um ( M a r i n & R os 1993) a nd s pe c i m e ns of E l y s i a c hl or ot i c a f e e d on a nd s e que s t e r pl a s t i ds f r om V au c he r i a l i t or e a a l t hough i n t he l a b i t w i l l a l s o c ons um e V c om pac t a ( W e s t e t a l 1984) I n m a ny ot he r s a c ogl os s a n s pe c i e s t he s our c e of c hl or opl a s t s w a s de t e r m i ne d e i t he r by e l e c t r on m i c r os c opy or by e xt e ns i ve l a bor a t or y f e e di ng e xpe r i m e nt s ( J e ns e n 1980; J e ns e n 1981; B r a ndl e y 1984 ) I n s om e c a s e s t he a l ga l s our c e of t he s ym bi ot i c c hl or opl a s t i s c ont r a di c t or y F o r e xa m pl e e ve n be f or e E c l ar k i w a s r e c ogni z e d a s a di s t i nc t s pe c i e s f r om E c r i s pat a, t he r e w e r e di s c r e pa nc i e s a bout f e e di ng i n E c r i s pat a. I n s om e s t udi e s C aul e r pa v e r t i c i l l at a w a s de s c r i be d a s t he pr i m a r y f ood s our c e ( J e ns e n 1980, J e ns e n & C l a r k 1983 ) but i n ot he r s C v e r t i c i l l at a w a s not e a t e n by i ndi vi dua l s of E c r i s pat a ( C l a r k & B us a c c a 1978, T hom ps on & Y a r m a n 1989) C aul e r pa s e r t ul ar i oi de s w a s t he f ood s our c e i n s t i l l ot he r s t udi e s ( R ol l e r & B i a nc hi 1995 ) but w a s t oxi c i n ye t ot he r s ( T hom ps on & Y a r m a n 1989 )

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59 F e e di ng e xpe r i m e nt s c om bi ne d w i t h c ha nge s i n w hol e a ni m a l c hl or ophyl l c ont e nt s how t ha t a dul t s of E c r i s pat a f e e d on H al i m e da s p. P e ni c i l l us s p. B r y ops i s pl um os a, a nd B at ophor a oe r s t e di i ( C l a r k & B us a c c a 1978) H ow e ve r c onf us i ngl y, C l a r k & B us a c c a ( 1978) a l s o s t a t e t ha t F l or i da E c r i s pat a ( w hi c h m a y ha ve a c t ua l l y be e n E c l ar k i ) a r e not c a pa bl e of f e e di ng on P e ni c i l l us c api t a t us O t he r r e por t e d a l ga l f ood s our c e s f or E c r i s pat a a r e C r ac e m os a, C hae t om or pha s p. a nd H al i m e da di s c oi de a ( T hom ps on & J a r m a n 1989) A l l of t he s e s t udi e s us e va r i ous t ype s of f e e di ng e xpe r i m e nt s a nd no ne l ook f or m ol e c ul a r o r m i c r os c opi c e vi de nc e of s ym bi ot i c c hl or opl a s t or i g i n. A t l e a s t i n or de r t o c l a r i f y our ow n w or k w e ha ve e xa m i ne d t he s our c e of t he s ym bi ot i c pl a s t i ds i n E c l ar k i M at e r i al s an d M e t h od s A n i m al s an d A l gae S pe c i m e ns of E l y s i a c l ar k i a nd s e ve r a l s pe c i e s of a l ga e w e r e c ol l e c t e d f r om a bo r r ow p i t a nd i t s a s s oc i a t e d m a ngr ove s w a m p on t he F l o r i da B a y s i de of G r a s s y K e y, F l o r i da ( 24 o 45. 5' N 80 o 59. 5'W ) B ot h t he bor r ow pi t a nd m a n gr ove s w a m p c ont a i ne d a w i de r a nge of m a c r oa l ga e A ddi t i ona l s a m pl e s of a l ga l s pe c i e s w e r e c ol l e c t e d f r om S uns e t B e a c h i n T a r pon S p r i ngs F l or i da A l ga l s a m pl e s w e r e i de nt i f i e d by N E C us i ng t a xonom i c ke ys ( L i t t l e r & L i t t l e r 2000) A l l s pe c i m e ns w e r e m a i nt a i ne d i n t he l a bor a t or y i n a qua r i a f i l l e d w i t h a e r a t e d a r t i f i c i a l s e a w a t e r ( A S W ) ( I ns t a nt O c e a n, 1000 m os m ) on a 16 hr l i ght 8 hr da r k c yc l e w i t h f l uor e s c e nt ( c ool w hi t e ) t ube s a t r o om t e m p ( 26 C )

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60 D N A E xt r ac t i on s D N A e xt r a c t i ons w e r e pe r f o r m e d on s e ve r a l pool e d s a m pl e s of 3 5 s e a s l ugs a nd s e pa r a t e l y on a s i ngl e s e a s l ug. A l l s e a s l ugs w e r e s t a r ve d f or a t l e a s t a w e e k be f or e D N A e xt r a c t i ons T he s l ugs w e r e gr ound t o a pow de r i n l i qui d ni t r oge n w i t h a ba ke d m or t a r a nd pe s t l e A n e xt r a c t i on m e di um c ons i s t i ng of a 1 : 1 m i xt ur e of phe nol ( pH 8. 0 ) a nd buf f e r [ 200 m M T r i s ( pH 8 0) 0. 1 % S D S 0. 01M E D T A a nd 0 5 m g/ m l he pa r i n] w a s a dde d t o t he pow de r a nd m i xe d ge nt l y f o r 10 m i n T he a que ous pha s e w a s r e m ove d a nd m i xe d w i t h a n e qua l vol um e o f phe nol : c hl or o f or m ( 1: 1) f or 5 m i n a nd t he n c e nt r i f uge d a t 16, 000 x g f or 5 m i n T he a que ous pha s e w a s a ga i n s a ve d, a nd t he n m i xe d ge nt l y w i t h a n e qua l vol um e of c hl or o f or m f or 5 m i n a nd c e nt r i f uge d ( 16 000 x g) f o r 5 m i n. D N A w a s pr e c i pi t a t e d f r om t he r e s ul t i ng a que ous pha s e w i t h 100% e t ha nol l yoph i l i z e d, a nd r e s us pe nde d i n doubl e di s t i l l e d, f i l t e r e d ( 0 2 m ) a ut oc l a ve d ( df a ) w a t e r T he D N A s ol ut i on w a s c e nt r i f uge d a t 115 000 x g ( L 2 80 U l t r a c e nt r i f uge B e c km a n) f or 48 h r on a 0. 9 g/ m l c e s i um c hl or i de g r a di e nt T he D N A ba nd w a s vi s ua l i z e d us i ng a U V l a m p r e m ove d w i t h a w i de bor e s yr i nge a nd e xt r a c t e d w i t h i s opr opa nol ( R um pho e t a l 1994 ) P C R an d S e q u e n c i n g D e ge ne r a t e c ons e ns us pr i m e r s e que nc e s w e r e de s i gne d t o c ons e r ve d r e gi ons of t he c hl or opl a s t e nc ode d ge ne f or r i bul os e bi s phos pha t e c a r boxyl a s e / oxyge na s e ( r bc L ) a s de t e r m i ne d by a l i gnm e nt s o f 31 c hl or ophyt e r bc L s e que nc e s a va i l a bl e i n G E N B A N K T he r bc L ge ne i s i de a l f or i de nt i f y i ng t he c hl o r opl a s t s i n E c l ar k i be c a us e i t i s

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61 c hl or opl a s t e nc ode d a nd s i ngl e c opy ( P om be r t e t a l 2005) I n a ddi t i on r bc L s e que nc e s a r e c ons e r ve d e nough t o de s i gn c ons e ns us pr i m e r s ( e g. M a nha r t 1994 M c C our t e t a l 2000) but a r e d i f f e r e nt e nough be t w e e n e ve n t he m os t c l os e l y r e l a t e d s pe c i e s t o di s t i ngui s h t he m ( C ha s e e t a l 2005) T he f or w a r d pr i m e r s e que nc e w a s 5 A A A G C X G G K G T W A A A G A Y T A 3 ( r bc L 1 ) a nd t he r e ve r s e pr i m e r s e que nc e w a s 5 C C A A C G C A T A R A D G G T T G W G A 3 ( r bc L 2 ) R e a c t i on m i xt ur e s w e r e m a de w i t h E nz ypl us 2000 r e a c t i on bu f f e r [ ( N H 4 ) 2 S O 4 0 01% T r i t on X 100 a nd 0. 01 m g/ m l B S A ] 12. 5 nM dN T P 0. 25 m M r bc L 1 0 25 m M r bc L 2 1 ng/ l D N A a nd 0. 1 uni t s / l pol ym e r a s e ( E nz ypl us 2000, E nz ypol ) P C R t ouc h dow n r e a c t i ons w e r e pe r f o r m e d on a t he r m oc yc l e r ( G e ne A m p P C R 2400, A ppl i e d B i os ys t e m s ) w i t h a de na t ur i ng t e m pe r a t ur e of 94 C f o r 30 s a nne a l i ng t e m pe r a t ur e s t a r t i ng a t 45 C f or 30 s a nd a n e xt e ns i on t e m pe r a t ur e of 72 C f or 1 m i n f or 35 c yc l e s w i t h t he a nne a l i ng t e m pe r a t ur e l ow e r e d by 0. 5 C on e a c h s ubs e que nt c yc l e P C R pr oduc t s w e r e pur i f i e d, po l i s he d, l i ga t e d a nd t r a ns f or m e d us i ng t he P C R S c r i pt A m p ( s k+ ) c l oni ng ki t ( S t r a t a ge ne ) a c c or di n g t o t he m a nuf a c t ur e r s i ns t r uc t i ons a nd c ol oni e s w e r e s c r e e ne d by P C R us i ng T 3 a nd T 7 pr i m e r s C ol oni e s pos s e s s i ng i ns e r t s c l os e i n s i z e t o t he P C R p r oduc t w e r e m i ni pr e ppe d us i ng t he N uc l e os pi n P l a s m i d ki t ( C l ont e c h) S e que nc i ng r e a c t i ons w e r e pe r f o r m e d us i ng t he C D Q D C T S Q ui c k S t a r t K i t ( B e c km a n C oul t e r ) a nd t he i ns e r t s e que nc e s w e r e de t e r m i ne d ( Q S M 8000 B e c km a n C oul t e r ) A l ga l r bc L s e que nc e s f r om G E N B A N K a nd obt a i ne d s l ug r bc L s e que nc e s w e r e a l i gne d us i ng C l us t a l W ( T hom ps on e t a l 1994)

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62 E l e c t r on M i c r os c op y B a s e d on t he P C R i de nt i f i c a t i ons s a m pl e s of E l y s i a c l ar k i P e ni c i l l us c api t at us L A M A RK 1813 P e ni c i l l us l am our oux i i D E C A IS N E 1842 H al i m e da i nc r as s at a ( E L L IS ) L A M O U RO U X 1816, H al i m e da m oni l e ( E L L IS & S O L A N D E R ) L A M O U RO U X 1816 w e r e f i x e d i n 2% g l ut a r a l de hyde i n c a c odyl a t e buf f e r ( 0 15M c a c odyl a t e c ont a i ni ng 0. 58M s uc r os e pH 7. 5) ove r ni ght f ol l ow e d by t hr e e 10 m i n w a s he s i n t he c a c odyl a t e buf f e r T he c a l c a r e ous a l ga l s a m pl e s w e r e t he n de c a l c i f i e d ove r ni ght i n 3% E D T A i n c a c odyl a t e buf f e r A l l s a m pl e s w e r e pos t f i xe d i n 2% os m i um t e t r oxi de f or 60 m i n f ol l ow e d by t hr e e 10 m i n w a s he s i n di s t i l l e d w a t e r S a m pl e s w e r e s o a ke d i n 2% a que ous ur a nyl a c e t a t e f o r 60 m i n. D e hydr a t i on c ons i s t e d of 10 m i n w a s he s i n a n e t ha nol s e r i e s ( 35 % 100% ) f ol l o w e d by t h r e e 10 m i n w a s he s i n pr opyl e ne ox i de a nd t he n i nf i l t r a t e d by t hr e e 60 m i n w a s he s i n pr opyl e ne oxi de : A r a l di t e E pon r e s i n ( 1: 1, 1: 2 a nd 1 : 3 i n s e que nc e ) ( A r a l di t e 502/ E m be d 812, E l e c t r on M i c r os c opy S c i e nc e s ) S a m pl e s w e r e t he n i m m e r s e d i n 100% A r a l di t e E pon r e s i n f o r 60 m i n a nd e m be dde d i n f r e s h A r a l di t e E pon. T hi n s e c t i ons ( 60 90 n m ) w e r e c ut on a n ul t r a m i c r ot om e ( M 2T B S o r va l l P or t e r B l um ) a nd pl a c e d on 200 m c oppe r m e s h gr i ds S a m pl e s w e r e s t a i ne d 1 m i n w i t h 0 01M l e a d c i t r a t e c ount e r s t a i ne d w i t h 2% ur a nyl a c e t a t e f or 1 m i n s t a i ne d a ga i n w i t h l e a d c i t r a t e f o r 1 m i n, a nd r i ns e d a f i na l t i m e w i t h doubl e di s t i l l e d w a t e r S e c t i ons w e r e vi e w e d a nd phot ogr a phe d us i ng a t r a ns m i s s i on e l e c t r on m i c r os c ope ( T E M ) ( M or ga gni 268D F E I ) F e e d i n g F e e di ng e xpe r i m e nt s w e r e pe r f o r m e d t o c onf i r m t h a t s pe c i m e ns of E l y s i a c l ar k i f e d on t he s pe c i e s of a l ga e i de nt i f i e d by t he m o l e c ul a r m a r ke r de s c r i be d a bove S pe c i m e ns of E

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63 c l a r k i w e r e i s ol a t e d i n A S W f or 3 m ont hs unt i l t he body c ol or a t i on t ur ne d f r om gr e e n t o ye l l ow i ndi c a t i ng c hl or opl a s t l os s T he s t a r ve d s e a s l ugs w e r e phot ogr a phe d a nd t he n pl a c e d on a s i ngl e s pe c i e s of f r e s h a l ga e ( P e ni c i l l u s c api t at us P l am our oux i i or H al i m e da i nc r as s at a ) f or t w o w e e ks a f t e r w hi c h t he s pe c i m e ns w e r e a ga i n phot ogr a phe d f or c ol or c om pa r i s on, a nd a s a m pl e o f t i s s ue w a s t a ke n f or e l e c t r on m i c r os c opy a s de s c r i be d a bove R e s u l t s r bc L S e q u e n c e I d e n t i t y T he P C R w i t h r bc L pr i m e r s us i ng E l y s i a c l ar k i t ot a l D N A pr oduc e d a 562 bp a m pl i c on. A f t e r c l oni ng t he P C R pr oduc t t he n s c r e e ni ng a nd s e que nc i ng c l one s f our di f f e r e nt r bc L s e que nc e s ( E c l ar k i 1, 2, 3, a nd 4) w e r e obt a i ne d f r om t he bo r r ow pi t s pe c i m e ns bot h i n t he pool e d D N A s a m pl e s ( 75 c l one s s e que nc e d) a nd i n t he D N A s a m pl e e xt r a c t e d f r om t he s i ngl e s l ug ( 20 c l one s s e que nc e d) T he s e que nc e E l y s i a c l ar k i 1 ( G E N B A N K # D Q 537220) ha d a 100 % s e que nc e i de nt i t y w i t h P e ni c i l l us c api t at us r bc L ( G E N B A N K # D Q 469332) E c l ar k i 2 ( G E N B A N K # D Q 537221 ) ha d a 100 % i de nt i t y t o P e ni c i l l us l am our oux i i r bc L ( G E N B A N K # D Q 469328 ) E c l ar k i 3 ( G E N B A N K # D Q 537222) ha d a 100% i de nt i t y t o H al i m e da i nc r as s at a r bc L ( G E N B A N K # D Q 469328) a nd E c l ar k i 4 ( G E N B A N K # D Q 537223) ha d a 100 % s e que nc e i de nt i t y t o H al i m e da m oni l e r bc L s e que nc e ( G E N B A N K # D Q 469329) N o ot he r r bc L s e que nc e s w e r e de t e c t e d i n a ny of t he s pe c i m e ns E l e c t r on m i c r os c op y T he f i ne s t r uc t ur e of t he c hl o r opl a s t s of H al i m e da i nc r as s at a ( F i g. 13) a nd H al i m e da m oni l e ( not s how n) w e r e i ndi s t i ngui s ha bl e

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64 F i gu r e 13. E l e c t r on m i c r og r a ph of a po r t i on o f a H al i m e da i nc r as s at a s i phon. T he c hl or opl a s t s ( C ) c ont a i n a l a r ge c e nt r a l s t a r c h g r a nul e ( S ) a nd s e ve r a l pl a s t ogl obul i ( P ) N uc l e us ( N ) m i t oc hondr i on ( M ) a nd t he c e l l w a l l ( C W ) S c a l e 1 m

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65 S i m i l a r l y, t he m o r phol ogy o f P e ni c i l l us c api t at us ( F i g. 14 ) c hl or op l a s t s w a s t he s a m e a s t ha t of P e ni c i l l us l am our oux i i ( no t s how n) c hl o r op l a s t s F i gu r e 14. E l e c t r on m i c r og r a ph of a po r t i on o f a P e ni c i l l us c api t at us s i phon. T he c hl or opl a s t s ( C ) do not c ont a i n a s t a r c h gr a nul e bu t us ua l l y c ont a i n pl a s t ogl obul i ( P ) L i pi ds ( L ) m i t oc hondr i a ( M ) a nd t he c e l l w a l l ( C W ) S c a l e 1 m

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66 H ow e ve r t he c hl or op l a s t m or phol ogy of P e ni c i l l u s s p. w a s di f f e r e nt i n s om e a s pe c t s f r om t ha t of H al i m e da s p. c hl or opl a s t s I n a l l f our s pe c i e s of a l ga e t he ova l c hl or opl a s t s w e r e bound by a s m oot h, doub l e m e m br a ne a nd c ont a i ne d s t a c ke d t hyl a koi ds i n ba nds of 2 5 l a m e l l a e l ongi t udi na l l y s pa nni ng t he c hl or opl a s t f r om one e nd t o t he ot he r ( F i g 1 3 a nd 14 ) O c c a s i ona l l y, ne i ghb or i ng t hyl a koi d ba nds l a y s i de by s i de a ppe a r i ng a s 6 10 s t a c ke d l a m e l l a e R ound pl a s t og l obul i r a ngi ng i n di a m e t e r f r om 100 300 nm w e r e pr e s e nt i n t he pl a s t i ds of t he H al i m e d a s p. ( F i g. 13) a nd P e ni c i l l us s p ( F i g 14) T he H al i m e da s p c hl o r opl a s t s w e r e di s t i ngui s he d f r om t hos e i n P e ni c i l l us s p. by a c e nt r a l s t a r c h gr a nul e r a ngi ng up t o 1500 nm i n l e ngt h ( F i g 13) T he pr e s e nc e or a bs e nc e of s uc h s t a r c h gr a i ns i n c hl or opl a s t s i s of phyl oge n e t i c i m por t a nc e i n ul vophyt e s ( C a l ve r t e t a l 1976 ) S t a r c h gr a nul e s w e r e not p r e s e nt i n c hl or opl a s t s of P c api t a t us ( F i g 14 ) I n t hi s a l ga l s pe c i e s s t a r c h w a s de pos i t e d i n a m yl opl a s t s out s i de t he pl a s t i d ( T ur ne r & F r i e dm a n 1974) T he s ym bi ot i c c hl or opl a s t s i n E l y s i a c l ar k i w e r e p r e s e nt i ns i de a l a r ge c e l l t ha t s ur r ounde d t he bl i nd e nd of t he t ubu l e s of t he di ge s t i ve di ve r t i c ul um ( F i g. 7) a nd i n s e ve r a l c e l l s t ha t s ur r ounde d t he t ubul e j us t be l ow t he t e r m i nus ( P i e r c e e t a l 2005 ) T he t ubul e s e xt e nde d di s t a l l y f r om t he c e nt e r of t he a ni m a l t o j us t be l ow t he e xt e r na l body s ur f a c e T he t ubul e s r a m i f i e d t hr oughout t he body of t he e nt i r e a ni m a l gi vi ng r i s e t o i t s gr e e n a ppe a r a nc e T he s e que s t e r e d c hl or opl a s t s w e r e m uc h r ounde r t ha n t he obl ong s ha pe of t he pl a s t i ds i n t he a l ga l c e l l T he s ym bi ot i c c hl or opl a s t s w e r e s e pa r a t e d f r om t he c yt opl a s m of t he di ge s t i ve di ve r t i c ul um c e l l s by a m e m br a ne a nd w e r e i nt e r s pe r s e d a m ong m i t oc hondr i a t he nuc l e us a nd ot he r i nt r a c e l l ul a r i nc l us i ons ( F i g 7 ) I n a ddi t i on,

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67 t he out e r m e m b r a ne w a s w a vy a nd a ppe a r e d m o r e l oos e l y a s s oc i a t e d w i t h t he c hl or opl a s t ( F i g. 15) unl i ke t he a l ga l c e l l w he r e t he out e r m e m br a ne w a s s m oot h a nd t i ght l y out l i ne d t he c hl or opl a s t ( F i gs 13 a nd 14) ( s e e a l s o T u r ne r e t a l 1974) F i gu r e 15. E l e c t r on m i c r og r a ph of a c hl o r opl a s t i n a f i e l d c ol l e c t e d E c l ar k i T he t hyl a koi ds ( T H ) a r e a r r a nge d i n ba nds of 2 6 l a m e l l a e a l l r unni ng i n t he s a m e di r e c t i on S m a l l l i pi d ( L ) d r opl e t s a r e p r e s e nt w i t hi n t he c hl o r opl a s t a nd l a r ge r l i p i d dr op l e t s a r e pr e s e nt i n t he c yt opl a s m be t w e e n t he c hl or opl a s t s T he pl a s t i d i s s ur r ounde d by a l oos e w a vy out e r m e m br a ne ( O ) out s i de t he c hl or op l a s t e nve l ope ( E ) a nd a ve s i c l e i s t r a ppe d be t w e e n t he m ( unl a be l l e d a r r ow ) M i t oc hondr i on ( M ) S c a l e 300 nm

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68 T he c hl o r opl a s t t hyl a koi ds i n E c l ar k i w e r e s t a c ke d i n ba nd s of 2 6 l a m e l l a e a nd of t e n c ont a i ne d l i pi d dr opl e t s ( F i g. 15) w hi c h w e r e ne ve r f ound i n c hl o r opl a s t s of t he a l ga e T he s e l i pi ds i n t he s ym bi ot i c c hl o r opl a s t s w e r e s i m i l a r i n s i z e a nd s ha pe t o pl a s t ogl obul i but w e r e c l e a r l y d i f f e r e nt s i nc e t he y s t a i ne d a l i ght gr e y c ol or a s oppos e d t he da r k bl a c k s t a i ni ng of pl a s t ogl obul i i n t he pl a s t i ds s t i l l i n t he a l ga e L a r ge l i p i d bodi e s w e r e a l s o pr e s e nt i n t he c yt opl a s m of t he E c l ar k i di ge s t i ve c e l l s ( F i g. 7) T he s e l i pi d bodi e s w e r e f ound i n t he a l ga l c e l l a l s o bu t m uc h l e s s f r e que nt l y ( F i g 14) O c c a s i ona l l y, c hl or opl a s t s w i t h a l a r ge c e nt r a l s t a r c h gr a nul e s uc h a s t ha t c ha r a c t e r i s t i c of H al i m e da s p. w e r e p r e s e nt i n E c l ar k i c e l l s ( F i g. 14) L a r ge s t a r c h gr a nul e s w e r e f ound i n t he c hl o r opl a s t s of bot h s pe c i e s of H al i m e da bu t not i n P e ni c i l l us s p. s o t he pr e s e nc e of t he s e r a r e r c hl or opl a s t s s ugge s t e d t ha t i ndi vi dua l c e l l s of E c l ar k i s e que s t e r e d pl a s t i ds f r om m or e t ha n one s pe c i e s of a l ga s i m ul t a ne ous l y. Y e l l ow c ol or e d, s t a r ve d E c l ar k i ( F i g 16A ) t ha t w e r e s ubs e que nt l y f e d on P c api t at us ( F i g. 16B ) o r P l am our oux i i ( da t a no t s h ow n) t ur ne d da r k g r e e n a f t e r 30 da ys E l y s i a c l ar k i di d not f e e d on H i nc r as s at a i n l a bor a t or y f e e di ng e xpe r i m e nt s T he r e c ol or e d P e ni c i l l us c api t at us f e d E c l ar k i ha d i nc or por a t e d c hl or opl a s t s i nt o t he di ge s t i ve di ve r t i c ul um c e l l s ( F i g. 17) T he m or phol ogy of t h e s e pl a s t i ds i n t he r e c ol or e d E c l ar k i w a s uni f or m t h r oughout t he r e c ol o r e d a ni m a l a nd t he s e que s t e r e d c hl or opl a s t s ( F i g. 16) w e r e i de nt i c a l t o t he m a j or i t y of c h l or opl a s t s i n f i e l d c ol l e c t e d s pe c i m e ns ( F i g. 7 ) T hus m os t of t he s e que s t e r e d c hl or opl a s t s i n t he f i e l d c ol l e c t e d a ni m a l s w e r e f r om a s pe c i e s of P e ni c i l l us

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69 F i gu r e 16. A S pe c i m e n o f E c l ar k i t ha t ha s be e n s t a r ve d f or 4 m ont hs unt i l i t s pl a s t i ds ha ve be e n l os t a nd i t ha s c ha nge d c ol or f r om g r e e n t o ye l l ow due t o c hl o r opl a s t l os s B T he s a m e s pe c i m e n f e d on P c api t at us f o r 30 da ys a f t e r s t a r va t i on ha s r e ga i ne d i t s g r e e n c ol or a t i on.

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70 F i gu r e 17. E l e c t r on m i c r og r a ph of c hl or opl a s t s ( C ) i n s i de c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um of a s t a r ve d E c l ar k i a nd s ubs e que nt l y f e d on P c api t at us ( S e e f i gs 5 A a nd 5B ) T he l um e n ( L ) o f t he di ge s t i ve di ve r t i c ul um i s de ns e l y l i ne d w i t h m i c r ovi l l i ( M V ) a nd c i l i a ( C L ) L i pi ds ( L ) a nd m i t oc hond r i a ( M ) S c a l e 1 m

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71 D i s c u s s i on T he P C R r e s ul t s s how t ha t E l y s i a c l ar k i s e que s t e r s c hl or opl a s t s f r om 4 s pe c i e s of a l ga e a t t he s a m e t i m e : P e ni c i l l us l am our oux i i P e ni c i l l us c api t at us H al i m e da i nc r as s at a a nd H al i m e da m oni l e T hi s r e s ul t i s une xpe c t e d be c a us e m a ny s pe c i e s of E l y s i a a r e t hought t o hous e s ym bi ot i c c hl or opl a s t s f r om onl y a s i ngl e a l ga l s pe c i e s S om e m i c r os c opi c e vi de nc e s ugge s t s t ha t bot h c hl or ophy t e pl a s t i ds a nd r hodophyt e pl a s t i ds m a y c o oc c ur i n E l y s i a f ur v ac auda ( B r a ndl e y 198 4) S t i l l t he s pe c i f i c a l g a l s our c e s of t he pl a s t i ds i n E f ur v ac auda w e r e no t r e po r t e d, a nd i t i s no t c l e a r i f t he r hodophyt e c hl or opl a s t s w e r e a c t ua l l y s e que s t e r e d i n c e l l s or w e r e j us t l oc a t e d i n t he l um e n o f t he di ge s t i ve di ve r t i c ul um a s no o t he r c e l l ul a r s t r uc t ur e s a r e vi s i bl e i n t he m i c r ogr a phs N e ve r t he l e s s bot h our m o l e c ul a r a nd m i c r os c opi c r e s ul t s pr e s e nt e d he r e s how t he s i m ul t a ne ous pr e s e nc e of s ym bi ot i c pl a s t i ds f r om m or e t ha n one a l ga l s pe c i e s i n t he E c l ar k i di ge s t i ve c e l l s T he c hl or opl a s t s i n E c l ar k i c e l l s ha ve a m or e r ou nde d s ha pe t ha n t he i r ova l c ount e r pa r t s i n t he a l ga A s i m i l a r s ha pe di f f e r e nc e w a s a l s o pr e s e nt be t w e e n s ym bi ot i c pl a s t i ds i n E l y s i a c hl or ot i c a a nd t hos e i n i t s a l ga l f ood V auc he r i a l i t or e a ( M ondy & P i e r c e 2003) a nd be t w e e n s e que s t e r e d c hl or op l a s t s of E l y s i a v i r i di s a nd i t s a l ga l f ood s our c e C odi um f r agi l e ( T r e nc h e t a l 1973) M os t pr e vi ous w or k on s a c ogl os s a n f e e di ng pr e f e r e nc e s w a s done be f or e t he a dve nt of m ode r n m ol e c ul a r t e c hni que s s uc h a s P C R I ns t e a d, s e a s l ug f e e di ng pr e f e r e nc e s w e r e de t e r m i ne d us i ng a dul t f e e di ng e xpe r i m e nt s c om bi ne d w i t h e i t he r

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72 c ha nge s i n body w e i ght ( T hom ps on & J a r m a n 198 9) or c ha nge s i n c hl or ophyl l c ont e nt ( C l a r k & B us a c c a 1978) a nd, oc c a s i ona l l y, e l e c t r o n m i c r os c opy ( B r a ndl e y 1984 ) U nf or t una t e l y, s uc h f e e di ng e x pe r i m e nt s a l one c a n not de t e r m i ne i f c hl or op l a s t s a r e be i ng s e que s t e r e d a nd, a s m a ny s pe c i e s of a l ga e ha ve s i m i l a r c hl or opl a s t m o r phol ogy ( f o r e xa m pl e P c api t at us a nd P l am our oux i i c hl o r opl a s t s a r e s t r uc t ur a l l y i ndi s t i ngui s ha bl e ) i t i s not a l w a ys pos s i b l e t o de t e r m i ne t he s pe c i f i c o r i gi n of s ym bi ot i c c hl o r opl a s t s i n f i e l d c ol l e c t e d s pe c i m e ns by e l e c t r on m i c r os c opy a l one T hus m ol e c ul a r m e t hods s uc h a s t hos e us e d i n t hi s s t udy a r e t he m os t c onc l us i ve m e t hod a va i l a bl e f or de t e r m i ni ng t he s pe c i e s i de nt i t y of s e que s t e r e d c hl or opl a s t s i n s a c ogl os s a ns N ot onl y do our m ol e c ul a r r e s ul t s de m ons t r a t e t ha t E c l ar k i f e e ds on P l am our oux i i a nd P c api t at us but a l s o ou r f e e di ng e xpe r i m e nt s c l e a r l y s how t ha t pl a s t i ds f r om bot h a l ga l s pe c i e s w e r e t a ke n up a nd s e que s t e r e d i n t he di ge s t i ve c e l l s I t s e e m s t he m a i n s our c e of s e que s t e r e d pl a s t i ds f or E c l ar k i i n t he bor r ow pi t w he r e w e c ol l e c t e d t he m i s P e ni c i l l us s i nc e t he m or phol ogy of t he m a j or i t y of c hl or opl a s t s i n c e l l s o f E c l ar k i c ol l e c t e d f r om t he f i e l d w e r e i de nt i c a l t o t ha t i n s l ugs w hi c h ha d be e n f e d P c api t at us i n t he l a b I nde e d, bot h P l am our oux i i a nd P c api t at us a r e a bunda nt i n t he qua r r y f r om w hi c h our s pe c i m e ns w e r e c ol l e c t e d. I n c ont r a s t a l t hough f i e l d c ol l e c t e d E c l ar k i c ont a i ne d s om e s ym bi ot i c c hl or opl a s t s f r om H i nc r as s at a, s t a r ve d E c l ar k i s how e d no s i gns of f e e di ng on H i nc r as s at a i n our e xpe r i m e nt s U nl i ke t he f i l a m e nt s f r om P e ni c i l l us s p. m a t ur e H i nc r as s at a s i phons a r e he a vi l y c a l c i f i e d, s o t he y m a y be di f f i c ul t f or E c l ar k i t o pi e r c e w i t h t he i r r a dul a e N e w gr ow t h of f i l a m e nt s o f H i nc r as s at a i s not c a l c i f i e d i ni t i a l l y ( B or ow i t z ka & L a r kum 1977 ) s o t he s e f i l a m e nt s m a y be e a s i e r f or t he s l ugs t o f e e d on. F or e xa m pl e E l y s i a t uc a f e e ds on t he nonc a l c i f i e d

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73 ga m e t a ngi c t ha l l i of H i nc r as s at a but not on t he c a l c i f i e d ve ge t a t i ve s i phons ( C l a r k & D e f r e e s e 1987) I n our a qua r i a H i nc r as s at a gr ow s s l ow l y, a nd t he ne w gr ow t h a r e a w a s l i ke l y t oo l i t t l e f o r t he s l ugs t o f e e d on e f f e c t i ve l y, pe r ha ps e xpl a i ni ng t he ne ga t i ve r e s ul t H ow e ve r t he c onf l i c t be t w e e n t he r e s ul t s of ou r P C R a nd f e e di ng e xpe r i m e nt s w i t h H i nc r as s at a de m ons t r a t e s t ha t our m o l e c ul a r pr ot oc ol i s a m o r e r e l i a bl e m e a ns of de t e r m i ni ng t he s our c e o f s e que s t e r e d c hl or opl a s t s i n s a c ogl os s a ns t ha n f e e di ng e xpe r i m e nt s a l one R e c e nt l y, t he i m por t a nc e o f a c c ur a t e s a c ogl os s a n f e e di ng pr e f e r e nc e s ha s be e n hi ghl i ght e d by t he c ons i de r a t i on of us i ng s a c ogl os s a n s e a s l ugs a s bi o c ont r ol a ge nt s f or i nf e s t a t i ons of a n i nva s i ve s t r a i n o f C aul e r pa t ax i f ol i a i n t he M e di t e r r a ne a n S e a ( T hi ba ut e t a l 2001 ) T hi s s t r a i n of C t ax i f o l i a i s unus ua l l y l a r ge f a s t g r ow i ng, not know n t o oc c ur na t ur a l l y, a nd hi ghl y t oxi c ( M us ni e r e t a l 20 01) a nd m os t c he m i c a l a nd phys i c a l c ont r ol m e t hods ha ve f a i l e d O x y noe ol i v ac e a, L ob i ge r s e r r adi f al c i a nd E l y s i a s ubor nat a ha ve a l l be e n pr opos e d a s pos s i bl e bi o c ont r ol c a n di da t e s ( T hi ba ut & M e i ne s z 2000; Z ul j e vi c e t a l 2001 ) W hi l e us i ng s a c ogl os s a ns t o c ont r ol C aul e r pa i nf e s t a t i ons s e e m s l ogi s t i c a l l y i m pos s i bl e a ny a t t e m pt t o do s o s houl d be s ur e t ha t t he s l ug s pe c i e s c hos e n a c t ua l l y e a t s t he a l ga e O ur m e t hods w oul d a l l ow c e r t a i n i de nt i f i c a t i on of t he f ood s our c e s of a l l a l ga l f e e di ng, kl e pt op l a s t i c s a c ogl os s a ns s o t ha t t he be s t c a ndi da t e f or bi o c ont r ol c oul d be r e c ogni z e d c or r e c t l y F i na l l y, t he va s t m a j or i t y of c hl or opl a s t pr o t e i ns a r e nuc l e a r e nc ode d. F or e xa m pl e e s t i m a t e s f r om t he A r abi dops i s t hal i ana ge nom e s ugge s t t ha t up t o 3500 di f f e r e nt nuc l e a r e nc ode d pr ot e i ns a r e t a r ge t e d t o t he c hl or opl a s t w hi l e t he c hl or opl a s t i t s e l f onl y c ont a i ns a bout 150 g e ne s ( B r uc e 2001 ) I n a ddi t i on phot os ynt he s i s i s

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74 a c c om pl i s he d i n pa r t by l i ght ha r ve s t i ng c om p l e x ( L H C ) pr ot e i ns w hi c h a r e a l l nuc l e a r e nc ode d ( D e a nne e t a l 2 000) F ur t he r m or e t hyl a k oi d pr ot e i ns a r e r a pi dl y de g r a de d dur i ng phot os ynt he s i s a nd m us t be c o nt i nuous l y r e pl a c e d ( M a t t oo e t a l 1984; Z e r & O ha d 1995) B ot h our pr e vi ous m i c r os c opi c a l ( M ondy & P i e r c e 2003) a nd bi oc he m i c a l ( P i e r c e e t a l 2003; G r e e n e t a l 2000 ) da t a i ndi c a t e t ha t onc e s e que s t e r e d by t he s e a s l ug, a n a l ga l c hl or opl a s t i s no l onge r a s s oc i a t e d w i t h a c or r e s pondi ng a l ga l nuc l e us H ow e ve r w e ha ve s how n pr e vi ous l y t ha t nuc l e a r e nc ode d c hl or opl a s t pr ot e i ns a r e s ynt he s i z e d i n E l y s i a c r i s pat a ( a c t ua l l y E c l ar k i ) ( P i e r c e e t a l 20 03) T hus i t i s l i ke l y t ha t t he a bi l i t y of E c l ar k i t o m a i nt a i n phot os ynt he t i c a l l y f unc t i ona l c hl or opl a s t s f r om f our d i f f e r e nt a l ga l s pe c i e s i n t w o di f f e r e nt ge ne r a a nd t he e xt r e m e l o nge vi t y of t he s e c hl or op l a s t s m us t be due t o t he p r e s e nc e of a l ga l nuc l e a r e nc ode d c hl or opl a s t ge ne s i n t he a ni m a l ; a hor i z ont a l ge ne t r a ns f e r be t w e e n a l ga a nd s l ug T he i de nt i f i c a t i on of t he a l ga l s our c e s of s e que s t e r e d c hl or opl a s t s i s a c r i t i c a l s t e p i n t he s t udy of pot e nt i a l hor i z ont a l ge ne t r a ns f e r s i n E c l ar k i I nde e d, w e ha ve a l r e a dy de t e c t e d a hom ol og of t he nuc l e a r e nc ode d V auc he r i a l i t or e a ge ne f c p, w hi c h i s a L H C pr ot e i n ge ne a s s oc i a t e d w i t h phot os ys t e m I I ( A pt e t a l 1995) i n E c r i s pat a ( a c t ua l l y E c l ar k i ) t ot a l D N A ( P i e r c e e t a l 2003) O ur r e s ul t s ha ve i de nt i f i e d t he p r e s e nc e of f our t yp e s of s ym bi ot i c c hl or opl a s t s f r om di f f e r e nt a l ga e ( P l am our oux i i P c api t at us H i nc r as s at a a nd H m oni l e ) i n E c l ar k i us i ng m ol e c ul a r a nd m i c r os c opi c t e c hni que s T he s e f i ndi ngs w i l l be us e d i n f ut ur e r e s e a r c h on c hl or opl a s t s ym bi os i s s e a r c hi ng f o r n uc l e a r ge ne s t ha t ha ve be e n t r a ns f e r r e d f r om a n a l ga l nuc l e us i nt o t he E c l ar k i ge nom e

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75 C h ap t e r 4: N e w l y M e t am or p h os e d E l y s i a c l ar ki Ju ve n i l e s F e e d on an d S e q u e s t e r C h l or op l as t s f r om A l gal S p e c i e s D i f f e r e n t f r o m T h os e U t i l i z e d b y A d u l t S l u gs I n t r od u c t i on T he r e c e nt l y na m e d kl e pt opl a s t i c s e a s l ug, E l y s i a c l ar k i P IE RCE E T A L 2006 f e e ds on s i phona c e ous a l ga e a nd c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um t a ke up a nd s e que s t e r a l ga l c hl or opl a s t s f r om t he gut l um e n ( P i e r c e e t a l 2006 ) T he c a pt ur e d i nt r a c e l l ul a r pl a s t i ds r e t a i n phot os ynt he t i c f unc t i on i n E c l ar k i f or 3 4 m o, one o f t he l onge s t c hl or opl a s t s ym bi os e s know n ( C ur t i s e t a l 2004) B ot h pl a s t i d e nc ode d a nd nuc l e a r e nc ode d c hl or opl a s t pr ot e i ns a r e s ynt he s i z e d w hi l e t he p l a s t i ds r e s i de i n E c l ar k i c e l l s ( P i e r c e e t a l 2003) T he r e f or e t he ge ne s f or t he nuc l e a r e nc ode d c hl or opl a s t pr ot e i ns m us t be pr e s e nt i n t he s e a s l ugs A l r e a dy a hom o l og of t he ge ne e nc odi ng f uc oxa nt hi n c hl or ophyl l bi ndi ng pr ot e i n ( f c p ) a nuc l e a r e nc ode d a l ga l ge ne ha s be e n de t e c t e d i n t ot a l D N A e xt r a c t e d f r om E c l ar k i t he s t r onge s t e vi de n c e s o f a r f o r a hor i z ont a l t r a ns f e r o f a nuc l e a r ge ne be t w e e n m ul t i c e l l ul a r or ga ni s m s ( P i e r c e e t a l 2003)

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76 F ur t he r w or k on hor i z ont a l ge ne t r a ns f e r s be t w e e n E l y s i a c l ar k i a nd a l ga e ha s be e n de l a ye d by a l a c k o f know l e dge o f t hi s s pe c i e s l i f e hi s t or y I n pa r t i c ul a r t he s pe c i e s i de nt i t y of t he s e que s t e r e d a l ga l c hl or opl a s t s vi t a l f or de s i gni ng e f f e c t i ve s t r a t e gi e s t o i de nt i f y t r a ns f e r r e d ge ne s i n E c l ar k i D N A w a s u nknow n unt i l r e c e nt l y. M ol e c ul a r m a r ke r s a nd t r a ns m i s s i on e l e c t r on m i c r os c opy ( T E M ) s how e d t ha t di ge s t i ve c e l l s of f i e l d c ol l e c t e d, a dul t E c l ar k i c ont a i n c hl or opl a s t s f r om f our di f f e r e nt s i phona c e ous c oe noc yt i c a l ga l s our c e s of t he o r de r B r yops i da l e s : P e ni c i l l us c api t at us P l am our oux i i H al i m e da i nc r as s at a a nd H m oni l e ( C ur t i s e t a l 2 006a ) W hi l e t he s e r e s ul t s une qui voc a l l y i de nt i f i e d t he a l ga l or i g i ns of s e que s t e r e d c hl or opl a s t s i n E c l ar k i a dul t s i n t he f i e l d, t he p r e s e nt s t udy i nve s t i ga t e s t he pos s i bi l i t y t ha t ot he r s pe c i e s of a l ga e m i ght be us e d a s pl a s t i d donor s i n e a r l i e r s t a ge s of t he E c l a r k i l i f e c yc l e S i m pl y t e s t i ng E c l ar k i f or i nge s t i on, a s i s of t e n t h e onl y obs e r va t i on m a de i n s a c ogl os s a n f e e di ng e xpe r i m e nt s ( J e ns e n 1983, T r ow br i dge & T odd 2001 ) i s not s uf f i c i e nt f o r our pur pos e s S e ve r a l l e ve l s of kl e pt opl a s t i c c om pl e xi t y ha ve be e n de s c r i be d, f r om i nge s t i on onl y t o t he m os t hi ghl y d e ve l ope d be i ng t he a bi l i t y t o t a ke up a nd s e que s t e r t he c hl or opl a s t s by c e r t a i n c e l l s l i ni n g t he di ge s t i ve di ve r t i c ul um r e t e nt i on of t h e pl a s t i ds i n t hos e c e l l s f or l onge r t ha n a w e e k a nd ongoi ng phot os ynt he s i s ( C l a r k e t a l 1990) I n a s pe c i e s w i t h t he l ong t e r m r e t e nt i on of f unc t i ona l s ym bi ot i c p l a s t i ds s uc h a s E c l ar k i i t s e e m s unl i ke l y t ha t s om e a l ga l s pe c i e s a r e m e r e l y e a t e n w i t h out s ubs e que nt s e que s t r a t i on of t he c hl o r opl a s t s H ow e ve r i f t ha t w e r e t he c a s e t hos e non s e que s t e r e d c hl or opl a s t s w oul d ha ve no r e l e va nc e t o pot e nt i a l ho r i z ont a l ge ne t r a ns f e r s T hus i n t hi s s t udy, w e de t e r m i ne d a l ga l f ood s pe c i e s w hi c h pr ovi de d c hl or opl a s t s t ha t w e r e s e que s t e r e d by E c l ar k i di ge s t i ve c e l l s

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77 T he a l ga l s pe c i e s f or ou r f e e di ng e xpe r i m e nt s w e r e c hos e n t o pr ovi de a w i de t a xonom i c c ove r a ge w i t hi n t he B r yops i da l e s ( c l a s s U l vophyc e a e ) s i nc e t he s e s i phona c e ous c oe noc yt i c a l ga a r e t he t ype f e d on by t he a dul t s l ugs I n a ddi t i on w e w i s he d t o know i f E l y s i a c l ar k i w oul d f e e d on a ny ot he r U l vophyt e s out s i de of t he B r yops i da l e s s o s pe c i e s f r om t he or de r s D a s yc l a da l e s U l va l e s a nd C l a dophor a l e s w e r e a l s o i nc l ude d. V auc he r i a l i t or e a w a s a l s o us e d i n f e e di ng e xpe r i m e nt s be c a us e e ve n t hough i t i s i n a d i f f e r e nt phyl um ( H e t e r okont a ) t h a n m os t s a c ogl os s a n f ood s our c e s ( C hl or ophyt a ) i t i s a c oe noc yt i c s i phona c e ous a l g a a nd i s t he pr e f e r r e d f ood of E l y s i a c hl or ot i c a ( W e s t e t a l 1984) M at e r i al s an d M e t h od s S e a S l u gs an d A l gae S pe c i m e ns of a dul t E l y s i a c l ar k i ( P i e r c e e t a l 200 6) a nd s e ve r a l s pe c i e s of a l ga e ( T a bl e 3 ) w e r e c ol l e c t e d f r om a bor r ow pi t a nd i t s a s s oc i a t e d m a ngr ove s w a m p on t he F l o r i da B a y s i de of G r a s s y K e y, F l o r i da ( 24 o 45 5' N 80 o 59 5' W ) a nd a m a n m a de m oor i ng c a na l ne a r t he e a s t e nd of V a c a K e y, F l or i da ( 24 o 44. 1'N 81 o 02 0' W ) a l s o on t he F l or i da B a y s i de W hi l e no qua nt i t a t i ve s ur ve ys ha ve be e n pe r f or m e d a t e i t he r s i t e t he a l ga l s pe c i e s H al i m e da s pp. a nd P e ni c i l l us s pp ( T a bl e 3 ) a r e a l m os t a l w a ys pr e s e nt a t t he G r a s s y K e y s i t e but B r y ops i s s pp. a nd D e r be s i a s pp. ha ve ne ve r be e n f ound t he r e i n 6 ye a r s o f c ol l e c t i ng. I n c ont r a s t B r y ops i s pe nnat a i s s om e t i m e s f ound a t t he V a c a K e y c ol l e c t i ng s i t e w hi l e P e ni c i l l us s pp. a nd H al i m e da s pp. ha ve ne ve r be e n f ound t he r e A ddi t i ona l s a m pl e s of a l ga l s pe c i e s w e r e c ol l e c t e d f r om S uns e t B e a c h i n T a r pon S pr i ngs F l o r i da or

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78 w e r e pur c ha s e d f r om T he C ul t u r e C ol l e c t i on of A l ga e a t t he U ni ve r s i t y o f T e xa s a t A us t i n, T e xa s ( U T E X ) ( T a bl e 3) B r y ops i s pl um os a w a s c ol l e c t e d f r om t he s out h e nd of t he ba y s i de of t he S uns hi ne S kyw a y B r i dge i n T a m pa F l o r i da a nd B r y ops i s pe nnat a w a s c ol l e c t e d f r om hol di ng t a nks c ont a i ni ng c i r c ul a t i ng na t ur a l s e a w a t e r a t t he K e ys M a r i ne L a b on L ong K e y F l o r i da C odi um f r agi l e a nd E nt e r om or pha pr ol i f e r a w e r e c ol l e c t e d f r om s t one j e t t i e s i n W oods H ol e M a s s a c hus e t t s V auc he r i a l i t or e a w a s obt a i ne d f r om our ow n l a bor a t or y c ul t ur e w hi c h w a s or i gi na l l y c ol l e c t e d f r om a s a l t m a r s h ne a r M e ne m s ha on M a r t ha s V i ne ya r d M a s s a c hu s e t t s ( P i e r c e e t a l 1996) A l l ot he r s pe c i m e ns w e r e m a i nt a i ne d i n t he l a bor a t or y i n a qua r i a f i l l e d w i t h a e r a t e d A r t i f i c i a l S e a W a t e r ( A S W ) ( I ns t a nt O c e a n, 1000 m os m ) on a 16 h l i ght : 8 h da r k c yc l e w i t h f l uor e s c e nt ( c ool w hi t e ) t ube s a t r oom t e m pe r a t u r e A l ga l s c pe c i m e ns w e r e i de nt i f i e d by N E C us i ng t a xonom i c ke ys ( L i t t l e r & L i t t l e r 2000 ) V ouc he r s pe c i m e ns f or a l l a l ga l s pe c i e s w e r e de pos i t e d a t t he U ni ve r s i t y of S out h F l or i da H e r ba r i u m

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79 T ab l e 3. L i s t of a l ga l s pe c i e s pr e s e nt e d t o j uve ni l e E l y s i a c l ar k i A l l a l ga l s pe c i e s w e r e pr e s e nt e d t o t he j uve ni l e s i n bot h f e e di ng e xpe r i m e nt s w i t h i de nt i c a l r e s ul t s P h y l u m O r d e r F a m i l y A l g a l S p e c i e s S o u r c e P o r t i o n P r e s e n t e d t o J u v e n i l e F e d o n b y J u v e n i l e s C h l o r o p h y t a B r y o p s i d a l e s U d o t e a c e a e H a l i m e d a d i s c o i d e a T a r p o n S p r i n g s F L G r o w i n g t i p H a l i m e d a i n c r a s s a t a T a r p o n S p r i n g s F L G r o w i n g t i p H a l i m e d a m o n i l e T a r p o n S p r i n g s F L G r o w i n g t i p P e n i c i l l u s c a p i t a t u s T a r p o n S p r i n g s F L B r u s h f i l a m e n t s P e n i c i l l u s l a m o u r o u x i i G r a s s y K e y F L B r u s h f i l a m e n t s A v r a i n v i l l e a n i g r i c a n s G r a s s y K e y F L W h o l e a l g a e B o o d l e o p s i s p u s i l l a G r a s s y K e y F L W h o l e a l g a e B r y o p s i d a c e a e B r y o p s i s p l u m o s a T a m p a B a y F L W h o l e a l g a e X D e r b e s i a t e n u i s s i m a U T E X W h o l e a l g a e X C o d i a c e a e C o d i u m f r a g i l e s s p t o m e n t o s o i d e s W o o d s H o l e M A W h o l e a l g a e C a u l e r p a c e a e C a u l e r p a m e x i c a n a G r a s s y K e y F L W h o l e a l g a e C a u l e r p a p a s p a l o i d e s G r a s s y K e y F L W h o l e a l g a e C a u l e r p a r a c e m o s a G r a s s y K e y F L W h o l e a l g a e C a u l e r p a v e r t i c i l l a t a G r a s s y K e y F L W h o l e a l g a e D a s y c l a d a l e s A c e t a b u l a r i a c r e n u l a t a G r a s s y K e y F L W h o l e a l g a e B a t o p h o r a o e r s t e d i i G r a s s y K e y F L W h o l e a l g a e C y m o p o l i a b a r b a t a U T E X W h o l e a l g a e N e o m e r i s d u m e t o s a U T E X W h o l e a l g a e D a s y c l a d u s v e r m i c u l a r i s G r a s s y K e y F L W h o l e a l g a e C l a d o p h o r a l e s C h a e t o m o r p h a a e r e a U T E X W h o l e a l g a e B o o d l e a c o m p o s i t a U T E X W h o l e a l g a e C l a d o p h o r a a l b i d a U T E X W h o l e a l g a e C l a d o p h o r a v a g a b u n d a U T E X W h o l e a l g a e C l a d o p h o r o p s i s m e m b r a n a c e a U T E X W h o l e a l g a e V a l o n i a m a c r o p h y s a U T E X W h o l e a l g a e U l v a l e s U l v a l i n g u l a t a U T E X W h o l e a l g a e U l v a p r o l i f e r a W o o d s H o l e M A W h o l e a l g a e U l v a l a c t u c a T a m p a B a y F L W h o l e a l g a e H e t e r o k o n t a V a u c h e r i a l e s V a u c h e r i a l i t o r e a L a b C u l t u r e W h o l e a l g a e

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80 C u l t u r e of E l y s i a c l ar ki N a t ur a l s e a w a t e r ( N S W 1050 m os m ) c ol l e c t e d f r om S t P e t e r s bur g B e a c h, F l or i da w a s dos e d w i t h pe ni c i l l i n G a nd s t r e pt om yc i n ( 100 g m l 1 e a c h) f i l t e r e d ( 0 2 m por e f i l t e r ) a nd s t or e d a t 4 o C A qua r i a c ont a i ni ng a dul t E l y s i a c l ar k i c ol l e c t e d f r om G r a s s y K e y, F l or i da w e r e m oni t or e d da i l y f o r t he a ppe a r a nc e of e gg m a s s e s A n e gg m a s s w a s c ol l e c t e d w i t hi n 24 h r o f de pos i t i on. A s e c ond f r e s hl y de pos i t e d e gg m a s s w a s c ol l e c t e d a t t he G r a s s y K e y, F L s i t e on a m a s s of B ood l e ops i s pus i l l a i n t he vi c i ni t y of s e ve r a l a dul t E c l ar k i T he e ggs w e r e c on f i r m e d t o be f r om E c l ar k i by g r ow i ng t he m i nt o a dul t s B ot h e gg m a s s e s w e r e pl a c e d i nt o a s e pa r a t e c ove r e d c ul t u r e di s h c ont a i ni ng w e e k ol d N S W w i t h 5 g m l 1 r i f a m p i c i n ( W e s t e t a l 1984) i n a n i nc uba t or on a 12 h l i ght : 12 h da r k c yc l e a t 26 o C L a r va l de ve l opm e n t w a s obs e r ve d da i l y unde r a di s s e c t i ng m i c r os c ope E ve r y 48 h e a c h e gg m a s s w a s ge nt l y r e m ove d f r om t he di s h, r i ns e d w i t h A S W on a 48 m nyl on m e s h, a nd r e pl a c e d i n f r e s h f i l t e r e d N S W a nd r i f a m pi c i n. E l y s i a c l ar k i e xhi bi t l e c i t hot r ophi c l a r va l de ve l opm e nt ha t c hi ng a s f r e e s w i m m i ng ve l i ge r l a r va e 28 35 d a f t e r e gg de pos i t i on ( a t 26 o C ) a nd t he y m e t a m or phos e i nt o j uve ni l e s 5 d a f t e r ha t c hi ng w i t hout a ny a l ga l c ue s ( P i e r c e e t a l 2006) T he s w i m m i ng, pos t ha t c he d ve l i ge r s w e r e not f e d, r i ns e d w i t h A S W e ve r y 48 h on a 48 m m e s h nyl on f i l t e r a nd t he n r e t ur ne d t o f r e s h s e a w a t e r unt i l t he y m e t a m or phos e d i nt o j uve ni l e s i n t he a bs e nc e of a ny m a c r oa l ga l c ue s

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81 Ju ve n i l e F e e d i n g S a m pl e s of f i e l d c ol l e c t e d a l ga e w e r e os m ot i c a l l y s hoc ke d i n f r e s h w a t e r f or 1 m i n a nd t he n t hor oughl y r i ns e d w i t h A S W t o c l e a n t he e xt e r na l s ur f a c e s of c ont a m i na t i ng or ga ni s m s T w o f e e di ng t r i a l s w e r e pe r f or m e d on e us i ng j uve ni l e s l ugs f r om t he f i e l d c ol l e c t e d e gg m a s s a nd t he ot he r us i ng t he e gg m a s s l a i d i n our a qua r i a T he s a m e a l ga l s pe c i e s w e r e pr e s e nt e d t o j uve ni l e s i n bot h t r i a l s I n e a c h f e e di ng e xpe r i m e nt 20 j uve ni l e s l ugs f r om t he s a m e e gg m a s s 1 2 d a f t e r m e t a m or phos i s w e r e pl a c e d i nt o i nd i vi dua l di s he s c ont a i ni ng a s i ngl e a l ga l s pe c i e s ( T a bl e 3) a nd w e r e ke pt i n a n i nc uba t or a t 20 o C i n N S W w i t h 5 g m l 1 r i f a m pi c i n ( 12 / 12 l i ght / da r k c yc l e ) T he a l ga l f i l a m e nt s c ove r e d t he e nt i r e bot t om of t he di s h s o t ha t i t w a s i m pos s i bl e f or t he j uve ni l e s t o m ove a r ound w i t hout e nc ount e r i ng t he a l ga e T he j uve ni l e s w e r e obs e r ve d da i l y unt i l e i t he r t he y a t e t he a l ga e gr e w a nd t u r ne d g r e e n or unt i l t he y di e d ( a ppr oxi m a t e l y 2 w ks a f t e r m e t a m or phos i s ) I n a ddi t i on E c l ar k i j uve ni l e s t h a t f e d a nd g r e w on B r y ops i s pl um os a w e r e t r a ns f e r r e d t o c ul t ur e s w i t h P e ni c i l l us c api t a t us a f t e r r e a c hi ng l e ngt hs o f 0 5 c m or 1. 0 c m a nd m oni t or e d f or e vi de nc e o f a ddi t i ona l g r ow t h. E l e c t r on M i c r os c op y E l y s i a c l ar k i a dul t s j uve ni l e s t ha t ha d e a t e n s i ngl e s pe c i e s of a l ga e i n t he f e e di ng e xpe r i m e nt D e r be s i a t e nui s s i m a a nd j uve ni l e s t ha t w e r e f e d P e ni c i l l u s c api t at us a f t e r i ni t i a l l y f e e di ng on B r y ops i s pl um os a w e r e f i xe d i n 2% ( v/ v) gl ut a r a l de hyde i n c a c odyl a t e buf f e r ( 0 15M c a c odyl a t e c ont a i ni ng 0 58M s uc r os e pH 7. 5) ove r ni ght f ol l ow e d by t h r e e 10 m i n w a s he s i n t he c a c odyl a t e buf f e r S a m pl e s w e r e pos t f i xe d w i t h

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82 2% ( w / v ) os m i um t e t r oxi de i n t he s a m e buf f e r f or 1 h, f ol l ow e d by s t a i ni ng i n 2% ( w / v) ur a nyl a c e t a t e e n bl oc f or 1 h D e hydr a t i on c ons i s t e d of 10 m i n w a s he s i n a n e t ha nol s e r i e s ( 35% 100% ) f ol l ow e d by t h r e e 10 m i n w a s he s i n pr opyl e ne oxi de a nd t he n i nf i l t r a t i on by t h r e e 60 m i n w a s he s i n pr opyl e ne o xi de : A r a l di t e E pon r e s i n ( 1: 1, 1: 2, a nd 1: 3 i n s e que nc e ) ( A r a l d i t e 502/ E m be d 812 E l e c t r on M i c r os c opy S c i e nc e s ) S a m pl e s w e r e t he n i m m e r s e d i n 100% A r a l di t e E pon r e s i n f or 60 m i n a nd e m be dde d i n f r e s h A r a l di t e E pon B r y ops i s pl um os a f i l a m e nt s di d not f i x w e l l w i t h t he a bove pr ot oc ol I ns t e a d, B pl um os a f i l a m e nt s w e r e i m m e r s e d i n 2 % ( v/ v) gl u t a r a l de hyde ( 4 o C ) i n A S W ( pH 7 3 900 m os m ) a nd pl a c e d i n a m i c r ow a ve ove n ( 344 0, P e l c o) ope r a t e d a t 100% pow e r w i t h t e m pe r a t ur e r e s t r i c t i on s e t t o 37 o C w i t h t he m i c r o w a ve on f or 10 s e c o f f f or 20 s e c on f or 10 s e c T he f i l a m e nt s w e r e p l a c e d out s i de t he o ve n f or 5 m i n a nd t hi s r e gi m e n w a s r e pe a t e d. S a m pl e s w e r e r i ns e d f o r 5 m i n e a c h i n A S W of de c r e a s i ng os m ol a r i t y ( 900 m os m 560 m os m 300 m os m ) t he n pos t f i xe d i n 2 % os m i um t e t r oxi de i n A S W ( pH 7 3, 300 m os m ) us i ng t he s a m e m i c r ow a ve r e gi m e n a s a bove 2 t i m e s T he s a m pl e s w e r e r i ns e d i n doubl e di s t i l l e d w a t e r 3 t i m e s f o r 5 m i n e a c h. D e hydr a t i on i n a c e t one c ons i s t e d of 4 s t e ps ( 50 % 70% 95% a nd 100% ) i n t he m i c r ow a ve ( t e m pe r a t ur e r e s t r i c t i on s e t t o 45 o C ) f or 40 s e c t w i c e e a c h. S a m pl e s w e r e i n f i l t r a t e d w i t h t hr e e w a s he s of a c e t one : S pur r s r e s i n ( 1: 1 15 m i n i n t he m i c r ow a ve f ul l po w e r t e m p 50 o C ; 1: 2 60 m i n t o ove r ni ght on be nc ht op; 1: 3, 60 m i n t o ove r ni ght o n be nc ht op; 100 % 60 m i n t hr e e t i m e s ) F i na l l y, t he a l ga l f i l a m e nt s w e r e e m be dde d i n f r e s h S pur r s r e s i n, a nd i nc uba t e d 8 15 h a t 70 o C

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83 T hi n s e c t i ons ( 60 90 nm ) w e r e c ut on a n ul t r a m i c r ot om e ( M 2T B S o r va l l P or t e r B l um ) a nd pl a c e d on c oppe r m e s h gr i ds S a m pl e s w e r e s t a i ne d f or 1 m i n w i t h 0 01M l e a d c i t r a t e s t a i ne d w i t h 2% ur a nyl a c e t a t e f or 1 m i n, s t a i ne d a ga i n w i t h l e a d c i t r a t e f or 1 m i n, a nd r i ns e d a f i na l t i m e w i t h doubl e di s t i l l e d w a t e r S e c t i ons w e r e vi e w e d a nd phot ogr a phe d us i ng a t r a ns m i s s i on e l e c t r on m i c r os c ope ( T E M ) ( M or ga gni 268D F E I ) D N A E xt r ac t i on s E l y s i a c l ar k i c ol l e c t e d f r om V a c a K e y, F l o r i da w e r e s t a r ve d f or a t l e a s t one w e e k be f or e D N A e xt r a c t i ons t o c l e a r t he gu t l um e n a nd e ns ur e t ha t a ny pl a s t i d e xt r a c t e d D N A w a s f r om s e que s t e r e d c hl or opl a s t s onl y. A s t he s ym bi ot i c c hl or opl a s t s i n E c l ar k i a r e m a i nt a i ne d f or up t o 3 m ont hs ( C ur t i s e t a l 2006a ) t he w e e k l ong s t a r va t i on ha s l i t t l e i m pa c t on t he nu m be r o f s e que s t e r e d c hl or opl a s t s E a c h s e a s l ug w a s gr ound t o a pow de r i n l i qui d n i t r oge n us i ng a ba ke d m or t a r a nd pe s t l e A n e xt r a c t i on m e di um c ons i s t i ng of a 1: 1 m i xt ur e of phe nol ( pH 8. 0 ) a nd bu f f e r ( 200 m M T r i s [ pH 8. 0 ] 1% S D S 0. 01M E D T A a nd 2 5 m g m l 1 he pa r i n) w a s a dde d t o t he pow de r m i xe d ge nt l y f o r 10 m i n a nd c e nt r i f uge d ( 4300 g) f or 5 m i n. T he a que ous pha s e w a s r e m ove d a nd m i xe d w i t h a n e qua l vol um e of phe nol : c hl or of o r m ( 1: 1) f or 5 m i n a nd t he n c e nt r i f uge d a ga i n a s a bove T he a que ous pha s e w a s s a ve d, m i xe d w i t h a n e qua l vol um e of c hl or o f or m f or 5 m i n, a nd c e nt r i f uge d a s be f or e D N A w a s e t ha nol pr e c i pi t a t e d f r om t he r e s ul t i ng a que ous pha s e l yophi l i z e d, a nd r e s us pe nde d i n doubl e di s t i l l e d m e m br a ne f i l t e r e d ( 0. 2 m ) a ut oc l a ve d w a t e r T he D N A s ol ut i on w a s t he n c e nt r i f uge d a t 115, 000 x g ( L 2 80 U l t r a c e nt r i f uge B e c km a n) f or 48 h on a 0 87 g m l 1 c e s i um c hl or i d e gr a di e nt a nd e xt r a c t e d w i t h i s opr opa nol ( P i e r c e e t a l 2006)

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84 A l ga e ( B r y ops i s pl um os a a nd B pe nnat a ) w e r e r i ns e d w i t h de i oni z e d w a t e r pa t t e d dr y w i t h K i m w i pe s pl a c e d i n l i qui d ni t r oge n, a nd g r ound t o a f i ne pow de r us i ng a m or t a r a nd pe s t l e A l ga l pow de r w a s a dde d t o 2x C T A B buf f e r ( D oyl e & D oyl e 1987) ( 0. 1g a l ga l pow de r m l 1 C T A B buf f e r ) m i xe d t ho r o ughl y, a nd i nc uba t e d f or 1 h a t 65 o C i n a w a t e r ba t h A n e qua l v ol um e of c hl or of o r m : i s oa m yl a l c ohol ( 24: 1 ) w a s a dde d, m i xe d ge nt l y f o r 5 m i n a nd c e nt r i f uge d a t 16, 000 g f or 10 m i n T he s upe r na t a nt w a s r e m ove d, a nd t he D N A w a s pr e c i pi t a t e d f r om i t w i t h 100% e t ha nol w a s he d t hr e e t i m e s w i t h 70% e t ha nol l yoph i l i z e d, a nd r e s us pe nde d i n doubl e di s t i l l e d, f i l t e r e d ( 0 2 m ) a ut oc l a ve d w a t e r ( C ur t i s e t a l 2006a ) P C R an d S e q u e n c i n g T o de t e r m i ne t he i de nt i t y of s e que s t e r e d c hl or opl a s t s i n f i e l d c ol l e c t e d E l y s i a c l ar k i t he ge ne f or r i bul os e bi s phos pha t e c a r boxyl a s e / oxe ge n a s e ( r bc L ) w hi c h i s l oc a t e d i n t he c hl or opl a s t ge nom e i n U l vophyt e s ( P om be r t e t a l 2005) w a s a m pl i f i e d f r om s e a s l ug t ot a l D N A s e que nc e d, a nd c om pa r e d t o a l ga l r bc L s e que nc e s D e ge ne r a t e pr i m e r s e que nc e s w e r e de s i gne d t o c ons e r ve d r e gi ons of t he ge ne e nc odi ng r bc L a s de t e r m i ne d by a l i gnm e nt s of 22 c hl or ophyt e r bc L s e que nc e s a va i l a bl e i n G E N B A N K T he f or w a r d pr i m e r s e que nc e w a s 5 G A T T A T C G T T T A M C A T A Y T A 3 ( r bc L 1 ) a nd t he r e ve r s e pr i m e r s e que nc e w a s 5 C A T A A T T T T T A G N G A T A A A C C T A 3 ( r bc L 2) R e a c t i on m i xt ur e s w e r e m a de w i t h E nz ypl us 2000 r e a c t i on buf f e r ( E nz ypol ) ( 20m M T r i s H C l [ pH 8. 8] ) 10m M K C l 10m M [ N H 4 ] 2 S O 4 0. 1 % T r i t on X 100, a nd 0. 1 m g m l 1 B S A ) 12. 5 M dN T P s 0 25 m M r bc L 1, 0. 25 m M r bc L 2 1 n g l 1 D N A a nd 1 uni t pol y m e r a s e ( E nz ypl us 2000, E nz ypol ) P C R t ouc hdow n r e a c t i ons w e r e pe r f or m e d on a t he r m oc yc l e r

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85 ( G e ne A m p P C R 2400, A ppl i e d B i os ys t e m s ) w i t h a de na t ur i ng t e m pe r a t u r e of 94 o C f or 30 s a nne a l i ng t e m pe r a t u r e s t a r t i ng a t 45 o C f o r 30 s a nd a n e xt e ns i on t e m pe r a t ur e o f 72 o C f or 1 m i n f or 35 c yc l e s w i t h t he a nne a l i ng t e m p e r a t ur e l ow e r e d by 0. 5 o C i n e a c h c yc l e a f t e r t he f i r s t ( C ur t i s e t a l 2006a ) P C R pr oduc t s w e r e pur i f i e d a nd c l one d us i ng t he Z e r o bl unt T O P O P C R c l oni ng ki t ( I nv i t r oge n ) a c c or di ng t o t he m a nuf a c t ur e r s i n s t r uc t i ons a nd c ol oni e s w e r e s c r e e ne d by P C R us i ng T 3 a nd T 7 pr i m e r s C ol oni e s pos s e s s i ng i ns e r t s c l os e i n s i z e t o t he P C R pr oduc t w e r e m i n i pr e ppe d us i ng t he N uc l e os pi n P l a s m i d ki t ( C l ont e c h) S e que nc i ng w a s c onduc t e d by a c om m e r c i a l ve ndo r ( N o r t hw oods D N A I nc S ol w a y, M i nne s ot a [ w w w nw dna c om ] ) us i ng T 3 a nd T 7 pr i m e r s T he r bc L s e que nc e s de t e r m i ne d f r om t hi s s e que nc i ng w e r e a l i gne d w i t h 22 ul vophyt e r bc L s e que nc e s obt a i ne d f r om G e nba nk us i ng C l us t a l W ( T hom ps on e t a l 1994) A N e i ghbor J oi ni ng t r e e w a s c ons t r uc t e d f r om t he a l i gnm e nt us i ng P hyl i p 3. 63 ( F e l s e ns t e i n 2004) us i ng t he F 84 m ode l ( T hor ne e t a l 1992) w i t h 100 boot s t r a p r e pl i c a t e s

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86 R e s u l t s Ju ve n i l e F e e d i n g P r e f e r e n c e s A l t hough s om e s a c ogl os s a n s p e c i e s r e qui r e a m i c r oa l ga l f ood s our c e a s ve l i ge r s a nd a m a c r oa l ga l pr e s e nc e f or m e t a m o r phos i s ( W e s t e t a l 1984) E c l ar k i ve l i ge r l a r va e ha t c he d a f t e r 28 d w i t hout t he a ddi t i on of a ny f ood s our c e s a nd m e t a m or phos e d a f t e r 4 5 d i n t he a bs e nc e of a ny a l ga O f t he 29 a l ga l s pe c i e s t e s t e d i n bot h f e e di ng t r i a l s j uve ni l e E c l ar k i f e d on l y on B r y ops i s pl um os a a nd D e r be s i a t e nui s s i m a ( T a bl e 3) J uve ni l e s t ha t f e d on t hos e t w o a l ga l s pe c i e s be ga n t o t ur n g r e e n a f t e r 6 d a nd a f t e r 8 d pa r a podi a a nd r h i nop hor e s s t a r t e d t o a ppe a r A f t e r 14 d o f f e e di ng t he s e a s l ugs r e a c he d a l e ngt h of 0. 5 c m a nd s how e d t he e xt e r na l m or phol ogi c a l c ha r a c t e r i s t i c s of a dul t E c l ar k i O nc e t hos e s l ugs f e d on B pl um os a ha d r e a c he d a t l e a s t 1 c m i n l e ngt h ( ~ 21 d o f f e e di ng) t he y w e r e t r a ns f e r r e d t o P c api t at us a pr e f e r r e d f ood o f f i e l d c ol l e c t e d a dul t s T he s e a s l ugs c ont i nue d t o f e e d a nd gr e w t o 3. 0 c m ( ~ 14 d o f f e e di ng) a nd w e r e s ubs e que nt l y f i xe d f or T E M a na l ys i s S m a l l e r E c l ar k i ( 0. 5 c m l ong ) r e a r e d on B pl um os a s t oppe d f e e di ng a nd ul t i m a t e l y di e d i f t r a ns f e r r e d t o P c api t at us J uve ni l e s pl a c e d on a l l t he o t he r s pe c i e s of a l ga e i nc l ud i ng t hos e pr e vi ous l y s how n t o be f e d on by E c l ar k i a dul t s ( C ur t i s e t a l 2006a ) s how e d no e vi de nc e o f f e e di ng a nd di e d w i t h i n 14 d A dul t E c l ar k i f e d e xc l us i ve l y on B pl um os a ha ve a da r ke r g r e e n c ol or a t i on t ha n a dul t E c l ar k i f r om t he G r a s s y K e y, F l or i da c ol l e c t i ng s i t e ( F i g. 18)

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87 F i gu r e 18. A E l y s i a c l ar k i S pe c i m e n w a s c ol l e c t e d f r om a bor r ow pi t a t G r a s s y K e y, F l or i da a nd ha d s pe nt a bout 4 w k i n a n a qua r i um I t s s ym bi ot i c pl a s t i ds w e r e f r om P e ni c i l l us c api t at us B L a r ge r s pe c i m e n i s 1 yr o l d. I t w a s r a i s e d f r om a n e gg m a s s i n t he l a b on B r yops i s pl um os a S m a l l e r s pe c i m e n w a s r a i s e d on B pe nnat a t ha t w a s c ol l e c t e d n e a r t he K e ys M a r i ne L a b on L ong K e y, F l or i da D a r k gr e e n c ol or of B pl um os a f e d s l ugs ha s onl y be e n s e e n i n t he f i e l d i n a popul a t i o n f ound i n a m oo r i ng c a na l on V a c a K e y, w he r e B pe nnat a i s a bunda nt U l t r as t r u c t u r e of S ym b i o t i c C h l or op l as t s T he ova l c h l or o pl a s t s of D e r be s i a t e nui s s i m a ( F i g. 19) a nd B r y ops i s pl um os a ( F i g. 20) bot h c ont a i n c e nt r a l p yr e noi d bodi e s s ur r ounde d b y m ul t i pl e s t a r c h gr a nul e s T he t hyl a koi ds a r e i n s t a c ks of 3 6 l a m e l l a e O ne t o t w o t hyl a koi ds t r a ve r s e t he m i ddl e of t he pyr e noi d body ( F i gs 19 a nd 20 )

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88 F i gu r e 19 L ongi t udi na l s e c t i on ne a r t he t i p o f a s i phona c e ous f i l a m e nt of D t e nui s s i m a s how i ng s e ve r a l c hl or opl a s t s ( C ) i n t he c yt opl a s m a nd t he out e r c e l l w a l l ( W ) T hyl a koi ds ( bl a c k a r r ow l a be l e d T ) r un t he e nt i r e l e ngt h of t he l ong a xi s o f t he ova l pl a s t i d, i n pa r a l l e l ba nds of 3 l a m e l l a e S e ve r a l s t a r c h gr a nul e s ( S ) a r e a l w a ys pr e s e nt i n e a c h pl a s t i d, i nt e r s pe r s e d be t w e e n t he t hyl a koi ds s ur r oundi ng a c e nt r a l py r e noi d body ( Y ) w hi c h s ur r ounds i n t u r n, a c e nt r a l t hyl a koi d ( w hi t e a r r ow l a be l e d T ) M a ny m i t oc hondr i a ( a r r ow l a be l e d M ) a l s o pr e s e nt S c a l e ba r = 1 m

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89 F i gu r e 20. S e c t i on t hr ough c hl o r opl a s t ( C ) c ont a i ni ng r e gi on o f a f i l a m e nt o f B r y ops i s pl um os a T hyl a koi ds ( a r r ow l a be l e d T ) r un t he l e n gt h of t he ova l c hl o r opl a s t s i n pa r a l l e l ba nds of 3 6 l a m e l l a e S t a r c h gr a nul e s ( S ) s ur r oun d a l a r ge c e nt r a l pyr e noi d body ( Y ) w hi c h i s t r a ve r s e d by 1 2 c e nt r a l t hyl a koi d ba nds ( a r r ow l a be l e d T ) M i t oc hondr i a ( M ) a r e a l s o vi s i bl e S c a l e ba r = 1 m H ow e ve r D t e nui s s i m a c hl or opl a s t s a r e s m a l l e r ( 3 4 m l e ngt h) t ha n t hos e i n B pl um os a ( 6 9 m l e ngt h) I n a ddi t i on t he B pl um os a f i l a m e nt s w e r e e xt r e m e l y di f f i c ul t t o f i x ( s e e m e t hods ) i m pl yi ng f ur t he r s t r uc t ur a l di f f e r e nc e s t ha t a r e not a ppa r e nt a t t he m i c r os c opi c l e ve l A f t e r i nge s t i on, t he c hl or op l a s t s of D e r be s i a t e nui s s i m a a r e s e que s t e r e d i nt o c e l l s l i ni ng t he l um e n of t he di ge s t i ve t ubul e of j uve ni l e E l y s i a c l ar k i ( F i g. 21)

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90 F i gu r e 21 C r os s s e c t i on of di ge s t i ve t ubul e o f a l a b r e a r e d j uve ni l e E l y s i a c l ar k i = ( 3 w k ol d) f e d onl y D e r be s i a t e nui s s i m a P a t e nt t ub ul e l um e n ( D ) i s l i ne d w i t h m i c r ovi l l i ( V ) a nd a l s o c ont a i ns a f e w c i l i a e xt e ndi ng i nt o t he c e nt e r C hl or opl a s t s ( C ) ha ve a l r e a dy be e n s e que s t e r e d i n t ubul e c e l l s of t h i s ve r y young s e a s l ug. P l a s t i ds ha ve a c e nt r a l pyr e noi d body ( Y ) s ur r ounde d by s t a r c h gr a nul e s ( S ) T hyl a koi ds ( a r r ow l a be l e d T ) r un i n bundl e s t he l e ngt h of t he pl a s t i d. S ha pe o f t he p l a s t i ds i s c i r c ul a r i ns i de di ge s t i ve c e l l s i ns t e a d of t he ova l e ugl e noi d s ha pe of t he p l a s t i ds i ns i de a l ga l f i l a m e nt T hi s s ha pe di f f e r e nc e oc c ur s a l s o w i t h t he P c api t a t us pl a s t i ds i ns i de t he a dul t di ge s t i ve c e l l s ( C ur t i s e t a l 2006a ) a nd, i n a ddi t i on, w i t h V auc he r i a l i t or e a pl a s t i ds i ns i de E c hl or ot i c a c e l l s ( M ondy & P i e r c e 2003) a nd i s m os t l i ke l y due t o a t i ght l y f i t t i ng s ur r oundi ng a ni m a l c e l l m e m br a ne M i t oc hondr i a ( a r r ow l a be l e d M ) a nd a c e l l nuc l e us ( N ) a r e pr e s e nt T ubul e i s s ur r ounde d by he m ol ym ph ( H ) S c a l e b a r = 1 m

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91 A l s o, t he j uve ni l e s f e d i ni t i a l l y on B pl um os a f or 21 d t he n l a t e r t r a ns f e r r e d t o P c api t at us f or 14 d ha d di ge s t i ve c e l l s c ont a i ni ng s e que s t e r e d c hl or opl a s t s w i t h t he m or phol ogi e s of bo t h B p l um os a a nd P c api t a t us pl a s t i ds ( F i g. 22 ) F i gu r e 22. L ongi t udi na l s e c t i on of di ge s t i ve t ubul e of E l y s i a c l ar k i f e d B r y ops i s pl um os a a nd t he n P e ni c i l l us c api t at us P l a s t i ds f r om bot h s pe c i e s of a l ga e a r e pr e s e nt i n t he s a m e t ubul e c e l l s T he P c api t at us c hl or opl a s t s ( a r r ow l a be l e d P ) a r e m uc h s m a l l e r ( 3. 5 m l ong) t ha n t he B pl um os a ( B ) c hl o r opl a s t s ( 5. 5 9 m l ong) L a t t e r c ont a i n s t a r c h gr a nul e s ( S ) a nd a c e nt r a l pyr e noi d body ( Y ) w hi c h a r e ne v e r pr e s e nt i n P c api t at us pl a s t i ds L um e n of di ge s t i ve t ubul e ( D ) i s f ul l of m i c r ovi l l i ( V ) N uc l e i ( N ) a nd l i pi d dr op l e t s ( L ) pr e s e nt i n t he c e l l s T ubul e i s s ur r ounde d by he m ol ym ph ( H ) S c a l e ba r = 3 m

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92 T he B pl um os a c hl o r opl a s t s a r e e a s i l y di s t i ngui s he d by t he c e nt r a l pyr e noi d bodi e s a nd m ul t i pl e s t a r c h gr a nul e s ne i t he r o f w hi c h i s pr e s e nt i n t he P c api t at us o r ga ne l l e s a s w e l l a s t he l a r ge r w i dt h ( 5 5 9 0 m di a m e t e r a nd l e ngt h) of t he B pl um os a t ha n P c api t at us c hl or opl a s t s ( 1. 5 4 0 m di a m e t e r a nd l e ngt h) ( C ur t i s e t a l 2006a ) A l l of t he s e que s t e r e d c hl or opl a s t s a r e r ound ( F i gs 21 a nd 2 2) unl i ke t he i r ova l s ha pe i n t he a l ga e ( F i gs 19 a nd 20) T hi s di f f e r e nc e i n t he m or phol o gy of a l ga l a nd s e que s t e r e d c hl or opl a s t s ha s be e n de m ons t r a t e d pr e vi ous l y ( T r e nc h e t a l 1973, M ondy & P i e r c e 2003, C ur t i s e t a l 2006a ) T he di ge s t i ve c e l l s of t he E l y s i a c l ar k i c ol l e c t e d i n t he m oor i ng c a na l i n t he pr e s e nc e of B r y ops i s pe nnat a c l e a r l y c ont a i ne d s e que s t e r e d c hl or opl a s t s f r om m or e t ha n one a l ga l s pe c i e s ( F i g. 23) A l t hough t h e s pe c i e s i de nt i t i e s of t he s e c hl or opl a s t s c a nnot be de t e r m i ne d f r om T E M a na l ys i s a l one c hl or opl a s t s w i t h pyr e noi d bodi e s a nd di m e ns i ons ( 5. 5 9. 0 m di a m e t e r a nd l e ngt h) s i m i l a r t o t hos e of B r y ops i s pl um os a a l ong w i t h ot he r s m a l l e r ( 2 5 4. 0 m d i a m e t e r a nd l e ngt h) c hl or opl a s t s r e s e m bl i ng P e ni c i l l us c api t at us w e r e bot h p r e s e nt

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93 F i gu r e 23. C r os s s e c t i on of pl a s t i d c ont a i ni ng c e l l s i n di ge s t i ve t ubul e o f E l y s i a c l ar k i c ol l e c t e d f r om m oor i ng c a na l on V a c a K e y, F l or i d a w he r e B r y ops i s pe nnat a i s a bunda nt L a r ge r c hl or opl a s t s ( B ) c ont a i n py r e noi ds ( Y ) a nd a r e s i m i l a r i n s i z e ( 5. 5 10 m l e ngt h) t o B pl um os a c hl or op l a s t s S m a l l e r c hl or opl a s t s a r e c l os e i n s i z e ( 2 5 4 m l e ngt h) t o P e ni c i l l us c api t at us ( P ) c hl or op l a s t s a nd s i m i l a r m or phol ogi c a l l y. N uc l e i ( N ) m i t oc hondr i a ( M ) a nd l i pi ds ( L ) a l s o pr e s e nt i n c e l l a nd t ubul e i s s ur r ounde d by he m ol ym ph ( H ) S c a l e ba r = 3 m

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94 M ol e c u l ar I d e n t i f i c at i on of E l y s i a c l ar ki C h l or o p l as t s T he P C R w i t h r bc L pr i m e r s of t o t a l D N A e xt r a c t e d f r om a dul t E c l ar k i c ol l e c t e d f r o m t he V a c a K e y m oor i ng c a na l yi e l de d a 500 bp a m p l i c on. S e que nc i ng of 20 c l one s yi e l de d 3 r bc L s e que nc e s 497 bp i n l e ngt h, i ndi c a t i ng t he pr e s e nc e of t hr e e di f f e r e nt a l ga l c hl or opl a s t s ( C a na l r bc L 1 3) T he phy l oge ne t i c a na l ys i s i ndi c a t e s t ha t onl y C a na l r bc L 1 ( D Q 537224) m a t c he d a know n s e que nc e w i t h 9 9% s i m i l a r i t y t o r bc L f r om B r y ops i s pe nnat a ( G E N B A N K # D Q 537227 ) c ol l e c t e d f r o m t he F l o r i da K e ys ( F i g 24) C a na l r bc L 2 a nd C a na l r bc L 3 di d not m a t c h a ny r bc L s e que nc e s a va i l a bl e i n G E N B A N K but phyl oge ne t i c a na l ys i s s how s t ha t C a na l r bc L 2 ( G E N B A N K # D Q 537226) g r oups w i t h a not he r s pe c i e s of D e r be s i a a nd, a l t hough i t doe s n ot gr oup w i t h a pa r t i c ul a r ge nus C a na l r bc L 3 ( G E N B A N K # D Q 537225) g r oups w i t hi n t he U dot e a c e a e c l a de ( F i g 24 )

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95 F i gu r e 24. N e i ghbor J oi ni ng t r e e ( F 84 m ode l ) o f p a r t i a l r bc L nuc l e ot i de s e que nc e s obt a i ne d by P C R f r om E l y s i a c l ar k i c ol l e c t e d f r o m m oor i ng c a na l on F l o r i da B a y s i de o f V a c a K e y, F l or i da ( C a na l r bc L 1 3 t e xt i n bol d) C hl am y dom onas r e i nha r dt i i w a s t he out gr oup. S c a l e s how n s ubs t i t ut i ons pe r s i t e a nd boot s t r a p va l ue s ( 50% ) a r e i nd i c a t e d a t node s C a na l r bc L 1 ha s 99 % s i m i l a r i t y t o t he r bc L s e que nc e f r om B r y ops i s pe nnat a c ol l e c t e d f r om F l or i da K e ys C a na l r bc L 2 gr oups w i t h D e r be s i a m ar i na a nd C a na l r bc L 3 gr oups w i t hi n t he f a m i l y U dot e a c e a e but not s t r ongl y w i t h a ny ge nus of a l ga P r e vi ous e xpe r i m e nt s ha ve de t e r m i ne d t ha t a dul t E c l ar k i s e que s t e r c hl or opl a s t s f r om t he a l ga l s pe c i e s P e ni c i l l us c api t at us P l am our oux i i H al i m e da i nc r as s at a a nd H m oni l e ( C ur t i s e t a l 2006a )

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96 D i s c u s s i on A l t hough E l y s i a c l ar k i a dul t s c ol l e c t e d f r om G r a s s y K e y c ont a i ne d s ym bi ot i c c hl or opl a s t s f r om P e ni c i l l us c api t at us P l am our o ux i i H al i m e da i nc r as s at a, a nd H m oni l e ( C ur t i s e t a l 2006a ) t he c ur r e nt s t udy f oun d t ha t E c l ar k i j uve ni l e s f r om 2 di f f e r e nt e gg m a s s e s ( one c ol l e c t e d f r om t he f i e l d a nd one c ol l e c t e d f r o m our a qua r i a ) di d not f e e d on t ho s e a l ga l s pe c i e s but i ns t e a d a t e a nd s e que s t e r e d c hl or opl a s t s f r om a l ga l s pe c i e s w i t h m uc h f i ne r f i l a m e nt s B r y ops i s pl um os a a nd D e r be s i a t e nui s s i m a I n a ddi t i on, E c l ar k i a dul t s f r om t he f i e l d or r a i s e d i n t he l a b, w i l l c ont i nue t o f e e d on B pl um os a, i f i t i s a va i l a bl e t o t he m f o r a t l e a s t 1. 5 y e a r s F ur t he r m o r e w hi l e j uve ni l e f e e di ng pr e f e r e nc e m a y be i nf l ue nc e d by t he f e e di ng pr e f e r e nc e s of t he pa r e nt s i n s om e s a c ogl os s a ns ( T r ow br i dge & T odd 2001) t he f e e di ng hi s t or y of a dul t E c l ar k i doe s not i n f l ue nc e t he of f s pr i ng. T he r bc L s e que nc e s f r om t he da r k g r e e n E l y s i a c l a r k i c ol l e c t e d f r om t he c a na l on V a c a K e y r e ve a l t ha t i ndi vi dua l s f r om t hi s popul a t i on s e que s t e r e d c hl or opl a s t s f r om t h r e e a l ga l s pe c i e s t ha t a r e di f f e r e nt f r om t hos e s e que s t e r e d by G r a s s y K e y A dul t s T he pr e s e nc e of a B r y ops i s pe nnat a s e que nc e ( C a na l r bc L 1) de m ons t r a t e s t ha t a dul t s l ugs f e e d on t hi s a l ga i n t he f i e l d, i f i t i s a va i l a bl e I n a ddi t i on, C a na l r bc L 2 a nd 3 r e pr e s e nt t w o m or e a l ga l s pe c i e s f r om w hi c h E c l ar k i s e que s t e r s c hl or opl a s t s W hi l e not m a t c hi ng a ny know n r bc L s e que nc e s e xa c t l y, t he phyl oge ne t i c r e s ul t s i ndi c a t e t ha t t he a l ga l s our c e s f a l l w i t hi n t he s a m e f a m i l y c l a de s a s t he i d e nt i f i e d a l ga l c hl or op l a s t s s e que s t e r e d i n E c l ar k i I n a ddi t i on, a l t hough c hl or opl a s t f i ne s t r uc t ur e doe s not a l l ow f or a n e xa c t

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97 s pe c i e s i de nt i f i c a t i on of t he a l ga l s our c e t he t w o m or phol ogi c a l l y di s t i nc t t ype s o f c hl or opl a s t s i n t he c e l l s of t he V a c a K e y c ol l e c t e d E c l ar k i a r e s i m i l a r t o B r y ops i s D e r be s i a s pp c hl or opl a s t s a nd i n t he s a m e c e l l s t he s m a l l e r c hl or opl a s t s r e s e m bl e t hos e of P e ni c i l l us H al i m e da s pp. H ow e ve r s i nc e no P e ni c i l l us s pp or H al i m e da s pp r bc L s e que nc e s w e r e f ound, t he s e s m a l l e r c hl or opl a s t s m a y be f r o m t he un i de nt i f i e d p l a s t i d s our c e of C a na l r bc L 3 O ur o r i gi na l i de nt i f i c a t i on o f t he a l ga l s our c e s of t he s e que s t e r e d c hl or opl a s t s i n G r a s s y K e y E l y s i a c l ar k i di d not f i nd B r y ops i s s pp. or D e r be s i a s pp. c hl or opl a s t s e i t he r t hr ough m i c r os c opy o r m ol e c ul a r t e c hni que s ( C ur t i s e t a l 2006a ) H ow e ve r a dul t E c l ar k i f r om G r a s s y K e y, w hi c h c ont a i ne d onl y s e que s t e r e d c hl or opl a s t s f r om H al i m e da s pp. a nd P e ni c i l l us s pp ( C ur t i s e t a l 2006a ) w i l l r e a di l y f e e d on B pl um os a i n l a bor a t or y e xpe r i m e nt s a nd E c l ar k i f r om V a c a K e y w i l l f e e d on P c api t at us i f pr e s e nt e d w i t h i t ( da t a not s how n) T hus i t i s l i ke l y t ha t B r y ops i s s pp w a s s i m pl y not a va i l a bl e t o t he E c l ar k i a t t he G r a s s y K e y s i t e w hi l e P e ni c i l l us c api t at us P l am our oux i i H al i m e da i nc r as s at a, a nd H m oni l e w e r e not a va i l a bl e t o t he V a c a K e y s l ugs C l e a r l y, l oc a l a l ga l s pe c i e s c om pos i t i on de t e r m i ne s w hi c h c hl or opl a s t s a r e pr e s e nt i n a pa r t i c ul a r E c l ar k i I n t he l a bo r a t or y, e ve n t hough t he s e a s l ugs c oul d s w i t c h f ood s our c e s s e que s t e r e d c hl or opl a s t s f r om t he or i g i na l f ood s our c e B r y ops i s pl um os a, pe r s i s t e d i n t he di ge s t i ve c e l l s a l ong s i de pl a s t i ds f r om P e ni c i l l us c api t at us T hus c hl o r opl a s t s f r om di f f e r e nt a l ga l s pe c i e s c a n be s e que s t e r e d i n t he s a m e di ge s t i ve c e l l s i m ul t a ne ous l y, a nd a r e not t u r ne d ove r w he n ne w c hl or opl a s t s a r e s e q ue s t e r e d. T he r e a s on t ha t j uve ni l e E l y s i a c l ar k i onl y f e e d on a f e w of t he a l ga l s pe c i e s ut i l i z e d by a dul t s i s unknow n but

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98 m e c ha ni c a l i s s ue s a r e a n obvi ous pos s i bi l i t y. I n o ur s t udy, a l l of t he a l ga l s pe c i e s e a t e n by E l y s i a c l ar k i a r e c oe noc yt i c H ow e ve r P e ni c i l l us c api t at us P l am our oux i i H al i m e da i nc r as s at a, a nd H m oni l e a r e a l l c a l c a r e ous a l ga e w hi c h m a y c a us e di f f i c ul t i e s f o r t he t e e t h of t he j uve ni l e r a dul a i n pi e r c i ng t he c e l l w a l l s o f t he f i l a m e nt s I n a ddi t i on t he s e f our a l ga e ha ve f a i r l y b r oa d f i l a m e n t s I n c ont r a s t B pl um os a a nd D t e nui s s i m a a r e not c a l c i f i e d a nd ha ve ve r y f i ne f i l a m e nt s w hi c h m a y be e a s i e r f or t he j uve ni l e s l ug t o g r a s p a nd pe r f o r a t e i n o r de r t o s uc k out t he c e l l ul a r c ont e nt s H ow e ve r j uve ni l e s di d not f e e d on V auc he r i a l i t or e a w hi c h i s ve r y s i m i l a r m or phol ogi c a l l y t o B pl um os a ( f i ne f i l a m e nt ous a nd c oe noc yt i c ) but i s a c hr om ophyt e T hus f i l a m e nt s i z e a nd s ha pe a l one doe s not de t e r m i ne f e e di ng a bi l i t y i n E c l ar k i T he r e i s a c l os e c or r e l a t i on be t w e e n t he s i z e a nd s ha pe of r a dul a r t e e t h i n ot he r s a c ogl os s a n s a nd t he di a m e t e r of t he f i l a m e nt s o f f ood a l ga e ( J e ns e n 19 81, J e ns e n 1993) A l s o, a l ga l c e l l w a l l c om pos i t i on m a y be a f a c t or a f f e c t i ng s a c ogl os s a n f e e di ng a bi l i t y ( J e ns e n 1981) O ur r e s ul t s s how t ha t unl i ke s om e e l ys i i ds s uc h a s E l y s i a c hl or ot i c a ( P i e r c e e t a l 2003) w hi c h r e l y on a s i ngl e s pe c i e s of a l ga e f or s ym bi ot i c pl a s t i ds E l y s i a c l ar k i f e e ds on a nd s e que s t e r s f unc t i ona l c hl or opl a s t s f r om a t l e a s t ni ne di f f e r e nt s pe c i e s of a l ga e r e pr e s e nt i ng f our ge ne r a du r i ng i t s l i f e c yc l e O t h e r s a c ogl os s a n s ha ve be e n r e por t e d t o f e e d on m ul t i p l e a l ga l s our c e s ( e g. E l y s i a v i r i di s [ T r ow br i dge 2000] a nd E l y s i a c f f ur v ac auda [ B r a ndl e y 1984] ) a l t hough f e e di ng s t udi e s do not of t e n pr ovi de e vi de nc e of c hl or opl a s t s e que s t r a t i on. E l y s i a c l ar k i c l e a r l y s e que s t e r s c hl or opl a s t s f r om a l l t he a l ga l s pe c i e s t ha t w e ha ve de m ons t r a t e d i t f e e ds on, a nd pl a s t i ds f r om m o r e t ha n one a l ga l s pe c i e s c o e xi s t i n t he s a m e di ge s t i ve c e l l T hi s pl a s t i d di ve r s i t y, t oge t he r w i t h t he m a i nt e n a nc e of t he a s s oc i a t i on f or m a ny m ont hs i ndi c a t e s t ha t t he pl a s t i d c ont a i ni ng

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99 c e l l s l i ni ng t he di ge s t i ve di ve r t i c ul um a r e w e l l s ui t e d t o c hl or opl a s t m a i nt e na nc e S i nc e m a ny c hl or opl a s t pr ot e i ns e s pe c i a l l y t hos e a s s oc i a t e d w i t h phot os ynt he s i s r e qui r e t ur nove r f o r pe r s i s t e nt f unc t i on a nd a r e a l s o nuc l e a r e nc ode d, E c l ar k i s a bi l i t y t o m a i nt a i n s uc h a w i de r a nge of c hl o r opl a s t s m us t b e due t he pr e s e nc e of a l ga l nuc l e a r e nc ode d c hl or opl a s t ge ne s i n t he a ni m a l I n c onc l us i on, t he r e s ul t s of t hi s s t udy s h ow t ha t E l y s i a c l ar k i f e e ds on a nd s e que s t e r s c hl or opl a s t s f r om a t l e a s t 9 di f f e r e nt s pe c i e s of gr e e n a l ga e f r om 4 ge ne r a a l l f r om t he or de r B r yops i da l e s N one o f t he j uve ni l e E c l ar k i f e d on t he a l ga e pr e f e r r e d by a dul t s ( P e ni c i l l us c api t at us P l am ou r oux i i H al i m e da i nc r as s at a, a nd H m oni l e ) I ns t e a d t he j uve ni l e s c hos e B r y ops i s pl um os a a nd D e r be s i a t e nui s s i m a a nd s e que s t e r e d c hl or opl a s t s f r om t hos e a l ga l s pe c i e s A dul t s i n t he f i e l d a nd i n t he l a b w i l l f e e d on B r y ops i s s pp i f i t i s a va i l a bl e a nd s e que s t e r pl a s t i ds A c om pl e t e unde r s t a ndi ng of t he f e e di ng pr e f e r e nc e s of E c l ar k i i s a c r i t i c a l s t e p i n t he s t udy of ho r i z ont a l t r a ns f e r of nuc l e a r ge ne s be t w e e n a l ga a nd s e a s l ug.

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100 C on c l u s i on s T he c om pa r i s ons be t w e e n V i r gi n I s l a nd c ol l e c t e d E l y s i a c r i s pat a a nd K e ys c ol l e c t e d s l ugs r e ve a l e nough c l e a r di f f e r e nc e s i n g r os s m or phol ogy, r a dul a r m or phol ogy de ve l opm e nt a l pa t t e r ns di ge s t i ve m or pho l ogy w i t h r e s pe c t t o kl e pt opl a s t y, a nd m ol e c ul a r s e que nc e s of t w o ge ne s t o r e qui r e a ne w s pe c i e s de s i gn a t i on f or t he K e ys s l ugs w hi c h w e ha ve na m e d E l y s i a c l ar k i ( P IE RCE E T A L 2006) A l t hough w e s ol ve d t ha t e s s e nt i a l i s s u e a l l of our pr e vi ous w or k on t he s e a ni m a l s us e d E c l ar k i a nd on E c r i s pat a ( P i e r c e e t a l 2003) H ow e ve r s i nc e t ha t w or k ha s s h ow n E c l ar k i ha s one of t he l onge s t know n kl e pt opl a s t i c a s s oc i a t i ons t ha t nuc l e a r e nc ode d, c hl or opl a s t p r ot e i ns a r e s ynt he s i z e d i n E c l ar k i a nd t ha t a ho m ol og of t he nuc l e a r e nc ode d a l ga l ge ne f c p, i s pr e s e nt i n s l ug t ot a l D N A ( P i e r c e e t a l 2003) i n s pi t e of t he t a xonom i c c onf us i on i t i s a good s ys t e m t o e xa m i ne c hl or opl a s t s ym bi os i s a n d i n pa r t i c ul a r pot e nt i a l ho r i z ont a l l y t r a ns f e r r e d nuc l e a r e nc ode d a l ga l ge ne s U nf or t una t e l y, pr i or t o t hi s s t udy, t he l i f e hi s t or y o f E c l ar k i w a s unknow n. T h e da t a c ol l e c t e d i n t hi s s t udy a r e e s s e nt i a l t o f u r t he r w or k on kl e pt opl a s t y i n t he s e s l ugs T he s our c e of s e que s t e r e d a l ga l c hl or opl a s t s i n E c l ar k i m us t be know n f o r a va r i e t y of r e a s ons i nc l udi ng pr ovi di ng t he c or r e c t e xpe r i m e nt a l c ont r ol s a c c ur a t e p r i m e r de s i gn, a nd s e que nc e c om pa r i s ons I n a ddi t i on, t hi s i s t he f i r s t s t udy t o i de nt i f y t he a l ga l s our c e of s e que s t e r e d c hl or opl a s t s i n a ny s a c ogl os s a n us i ng a m ol e c ul a r m a r ke r t he c hl o r opl a s t e nc ode d ge ne r bc L O ne of t he a dva nt a ge s of us i n g r bc L s e que nc e s t o i de nt i f y t he

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101 s e que s t e r e d c hl or opl a s t s i s t ha t i t a l l ow s f or t he e x a c t a l ga l s pe c i e s t o be de t e r m i ne d i f t he c or r e s pondi ng a l ga l r bc L s e que nc e i s a va i l a bl e E l y s i a c l ar k i w i l l f e e d on a t l e a s t 7 di f f e r e nt a l ga l s pe c i e s a s a dul t s i n t he f i e l d o f w h i c h w e w e r e a bl e t o pos i t i ve l y i de nt i f y P e ni c i l l us c api t at us P e ni c i l l us l am our oux i i H al i m e da i nc r as s at a, H al i m e da m oni l e a nd B r y ops i s pe nnat a T he t w o ot he r r bc L s e que nc e s di d not m a t c h a nyt hi ng i n t he da t a ba s e but one w a s c l os e l y r e l a t e d t o t he ge nus D e r be s i a a nd t he ot he r gr oupe d w i t hi n t he c a l c a r e ous m e m be r s of t he U dot e a c e a e T he phyl oge ne t i c a na l ys i s of t he a l ga l r bc L s e que n c e s a l s o a l l ow s f or s e ve r a l c onc l us i ons t o be dr a w n a bout E l y s i a c l ar k i f e e di n g. F i r s t a l l r bc L s e que nc e s obt a i ne d f r om E c l ar k i a r e w i t hi n t he m onophy l e t i c or de r B r yops i da l e s f r om t he m onophyl e t i c s ubor de r s B r yops i di ne a e a nd t he m onophyl e t i c H a l i m e di ne a e ( a l t hough pos t e r i or pr oba bi l i t y s uppor t f or t hi s c l a de w a s w e a k) F u r t he r m or e w i t hi n t he H a l i m e di ne a e t he f e e di ng a pp e a r s t o be r e s t r i c t e d t o c a l c i f i e d s pe c i e s of t he U dot e a c e a e N o r bc L s e que nc e s w e r e f ound f r om t he non c a l c i f i e d m e m be r s of t he C a ul e r pa c e a e or t he A v r ai nv i l l e a C l adoc e phal us D i c hot om ps i phon c l a de C l adoc e phal us l ut e of us c us a nd s e ve r a l s pe c i e s of C aul e r pa a nd A v r ai nv i l l e a a r e p r e s e nt i n t he bo r r ow pi t i n c l os e pr oxi m i t y t o c ol l e c t e d a dul t E l y s i a c l ar k i a nd ye t no r bc L s e que nc e s f r om t he s e a l ga e ha ve e ve r be e n f ound i n ou r a na l ys i s T hus w hi l e a dul t E c l ar k i w i l l e a t c a l c i f i e d a l ga e t he r e m us t be ot h e r f a c t or s p r e ve nt i ng t he m f r om f e e di ng on t he r e s t of t he non c a l c i f i e d H a l i m e di ne a e J uve ni l e E c l ar k i how e ve r do not f e e d on t he c a l c i f i e d a l ga l s pe c i e s ut i l i z e d by t he a dul t s I ns t e a d t he y e a t a nd r e t a i n pl a s t i ds f r o m t he f i ne f i l a m e nt ous s pe c i e s B r y ops i s pl um os a a nd D e r be s i a t e nui s s i m a T he r e a s on f o r t he i na bi l i t y o f j uve ni l e s t o f e e d on

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102 P e ni c i l l us s p. a nd H al i m e da s p. m a y be m e c ha ni c a l i s s ue s j uve ni l e E c l ar k i r a dul a r t e e t h a r e pr oba bl y t oo s m a l l t o pi e r c e t he l a r ge r c a l c i f i e d f i l a m e nt s w h i l e t he f i ne r un c a l c i f i e d f i l a m e nt s of B r y ops i s s p. a nd D e r be s i a s p. a r e e a s i e r t o pe ne t r a t e W he n t he j uve ni l e s ha ve gr ow n i nt o a dul t s a nd r e a c h a l e ngt h of a pp r oxi m a t e l y 1 c m t he y c a n s w i t c h t o P c api t at us i f p r e s e nt e d w i t h i t a l t hough t he y c a n c on t i nue t o gr ow nor m a l l y on B pl um os a O ur r e s ul t s a r e a c r i t i c a l s t e p i n t he s t udy o f kl e pt o pl a s t y i n E l y s i a c l ar k i O ur m e t hods a r e t he f i r s t de ve l ope d t ha t a l l ow pos i t i ve l y i de nt i f y s e que s t e r e d c hl or opl a s t s f r om f i e l d c ol l e c t e d a dul t s a t t he s pe c i e s l e ve l de p e ndi ng on t he a va i l a bi l i t y of c or r e s pondi ng a l ga l r bc L s e que nc e s S i nc e a l ga l s our c e s of s e que s t e r e d c hl or opl a s t s a r e now know n, t he c or r e c t a l ga l s pe c i e s c a n be c ol l e c t e d a nd c ul t ur e d t o pr ov i de c ont r ol s f or a l l w or k pe r f or m e d on t he s e a s l ugs

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103 R e f e r e n c e s A pt K E C l e nde nne n S K P ow e r s D & G r os s m a n A R ( 1995) T he ge ne f a m i l y e nc odi ng t he f uc oxa nt hi n c hl or ophy l l p r ot e i ns f r om t he b r o w n a l ga M ac r oc y s t i s py r i f e r a. M ol G e n. G e ne t 246: 455 464 B a s s A L & K a r l S A ( 2006 ) M ol e c ul a r ph yl oge ne t i c a na l ys i s of t he ge ne r a i n t he f a m i l y P l a kobr a nc hi da e ( M ol l us c a : O pi s t hobr a nc hi a : S a c ogl os s a ) P r oc S y m p. B i ol S ys t O pi s t h. ( I n pr e s s ) B l e a kne y J S ( 1990) I ndi r e c t e vi de nc e of a m o r ph ol ogi c a l r e s pons e i n t he r a dul a of P l ac i da de ndr i t i c a ( A l de r & H a nc oc k, 1843) ( O pi s t hobr a nc hi a : A s c ogl os s a / S a c ogl os s a ) t o di f f e r e nt a l ga l p r e y. V e l i ge r 33 : 111 115 B or ow i t z ka M A a nd L a r kum A W D ( 1977 ) C a l c i f i c a t i on i n t he g r e e n a l ga H al i m e da I A n ul t r a s t r uc t ur e s t udy of t ha l l us de ve l opm e nt J P hyc ol 13: 6 16. B ouc he t P & R oc r oi J P ( 2005 ) C l a s s i f i c a t i on a nd n om e nc l a t ur e of ga s t r opod f a m i l i e s M a l a c ol ogi a 47: 1 397. B r a ndl e y B ( 1984) A s pe c t s of t he e c ol ogy a nd phy s i ol ogy of E l y s i a c f f ur v ac auda ( M ol l us c a : S a c ogl os s a ) B ul l M a r S c i 34: 207 21 9. B r uc e B D ( 2001) T he pa r a dox of pl a s t i d t r a ns i t pe pt i de s : c ons e r va t i on of f unc t i on de s pi t e di ve r ge nc e i n pr i m a r y s t r uc t ur e B i oc hi m B i ophys A c t a 1541 : 2 21

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104 C a l ve r t H E D a w e s C J & B or ow i t z ka M A ( 1976) P hyl oge ne t i c r e l a t i ons hi ps of C aul e r pa ( C hl or ophyt a ) ba s e d on c om pa r a t i ve c hl or opl a s t ul t r a s t r uc t ur e ; J P hyc ol 12: 149 162 C ha s e M W S a l a m i n N W i l ki ns on M D unw e l l J M K e s a na kur t hi R P H a i da r N & S a vol a i ne n V ( 2005) L a nd pl a nt s a nd D N A ba r c ode s : S hor t t e r m a nd l ong t e r m goa l s P hi l os T r a ns R S oc L ond S e r B 360: 18 89 1895. C l a r k K B & B us a c c a M ( 1978) F e e di ng s pe c i f i c i t y a nd c hl or opl a s t r e t e nt i on i n f our t r opi c a l a s c ogl os s a w i t h a di s c us s i on of t he e xt e nt of c hl or opl a s t s ym bi os i s a nd t he e vol ut i on of t he o r de r J M ol l S t ud 44: 272 2 82. C l a r k K B & D e F r e e s e D ( 1987) P opul a t i on e c ol og y of C a r i bbe a n A s c ogl os s a ( M ol l us c a : O pi s t hobr a nc hi a ) : a s t udy of s pe c i a l i z e d a l ga l he r b i vor e s A m M a l a c ol B ul l 5: 259 280. C l a r k K B J e ns e n K R & S t i r t s H M ( 1990) S ur ve y f or f unc t i ona l kl e pt opl a s t y a m ong w e s t A t l a nt i c a s c ogl os s a ( = s a c ogl os s a ; M ol l us c a : O pi s t hobr a nc hi a ) V e l i ge r 33: 339 345 C ur t i s N E M a s s e y S E & P i e r c e S K ( 2006a ) T he s ym bi ot i c c hl or opl a s t s i n t he s a c ogl os s a n E l y s i a c l ar k i a r e f r om s e ve r a l a l ga l s p e c i e s I nve r t e br B i ol ( I n P r e s s ) C ur t i s N E P i e r c e S K M a s s e y S E S c hw a r t z J A & M a uge l T K ( 2006b) N e w l y m e t a m or phos e d E l y s i a c l ar k i j uve ni l e s f e e d on a nd s e que s t e r c hl or opl a s t s f r om a l ga l s pe c i e s di f f e r e nt f r om t hos e ut i l i z e d by a dul t s l ugs M a r B i ol ( I n pr e s s )

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105 C ur t i s N C M a s s e y S E S c hw a r t z J A T a gh i of H & P i e r c e S K ( 2004) T he i nt r a c e l l ul a r f unc t i ona l c hl or opl a s t s i n a dul t E c r i s pat a c om e f r om s e ve r a l a l ga l s pe c i e s a nd a r e di f f e r e nt f r om t hos e i n j uve ni l e s l ugs I nt e g C om p. B i ol 44: 686. D e a ne J A F r a unhol z M S u V M a i e r U G M a r t i n W D ur nf o r d D G & M c F a dde n G I ( 2000) E vi de nc e f or nuc l e om or ph t o hos t nuc l e us ge ne t r a ns f e r : l i ght ha r ve s t i ng c om pl e x pr ot e i ns f r o m c r ypt o m ona ds a nd c hl or a r a c hni ophyt e s P r ot i s t 151: 239 252. de l G r os s o F ( 1988 ) O n t he f o r m a t i on o f ga l l s i n D i c hot om os i phon t ube r os us ( A B r ) E r ns t ( C hl or ophyc e a e D i c hot om os i phona l e s ) pr o duc e d by r ot i f e r P r oal e s w e r ne c k i ( E hr e nbe r g ) H uds on e t G os s e N ova H e dw i gi a 46 ( 1 2) : 157 164 D e s ha ye s G P ( 1857) N ot e s ur di f f e r e nt s m ol l us qu e s de l a G ua de l oupe E nvoye s pa r M S c hr a m m J our n a l de C onc hyl i ol ogi e 6: 137 143 D oyl e J J & D oyl e J L ( 1987) A r a pi d D N A i s ol a t i o n pr oc e dur e f o r s m a l l qua nt i t i e s of f r e s h l e a f t i s s ue P hy t oc h. B ul l 19: 11 15. D r a ga s t a n O N & S ol i m a n H A ( 2002 ) P a l e oge ne c a l c a r e ous a l ga e f r om E gypt M i c r opa l e ont ol ogy 48: 1 30 E ngl e H ( 1927) W e s t i ndi s c he O pi s t hobr a nc hi a t e M ol l us ke n I I B i j dr a ge n t ot de D i e r kunde 25: 83 122 F e l s e ns t e i n J ( 2004) P H Y L I P ( P hyl oge ny I nf e r e nc e P a c ka ge ) ve r s i on 3. 6 D i s t r i but e d by t he a ut hor D e pa r t m e nt o f G e nom e S c i e nc e s U ni v e r s i t y of W a s hi ngt on, S e a t t l e G a ul t i e r N G ouy, M & G a ut i e r C ( 1996) S e a V i e w a nd P hyl o_w i n, t w o g r a phi c t ool s f or s e que nc e a l i gnm e nt a nd m ol e c ul a r phyl oge ny. C om put e r A ppl i c a t i ons i n t he B i os c i e nc e s 12, 543 548.

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106 G os l i ne r T M ( 1995 ) T he ge nus T hur i di l l a ( O pi s t h obr a nc hi a : E l ys i i da e ) f r om t he t r opi c a l I ndo P a c i f i c w i t h a r e vi s i on of t he phyl og e ny a nd s ys t e m a t i c s of t he E l ys i i da e P r oc C a l i f A c a d. S c i 49: 1 54. G r a ha m L E & W i l c ox L W ( 2000) A l gae P r e nt i c e H a l l 650 pp. G r e e n B J L i W Y M a nha r dt J R F ox T C S um m e r E L K e nne dy R A P i e r c e S K & R um pho M E ( 2000) M ol l us c a l ga l c hl or opl a s t e n dos ym bi os i s P hot os ynt he s i s t hyl a koi d pr ot e i n m a i nt e na nc e a nd c hl or opl a s t ge n e e xpr e s s i on c ont i nue f or m a ny m ont hs i n t he a bs e nc e of t he a l ga l nuc l e us P l a nt P hys i ol 124: 331 3 42 G r e e ne R W ( 1970) S ym bi os i s i n s a c ogl os s a n opi s t hobr a nc hs : f unc t i ona l c a pa c i t y of s ym bi ot i c c hl or opl a s t s M a r i ne B i ol 7: 138 142. H a w e s C R ( 1979) U l t r a s t r uc t ur a l a s pe c t s of t he s ym bi os i s be t w e e n a l ga l c hl or opl a s t s a nd E l y s i a v i r i di s N e w P hyt ol 83: 4 45 450. H i l l i s D M & H ue l s e nbe c k J P ( 1992) S i gna l noi s e a nd r e l i a bi l i t y i n m ol e c ul a r phyl oge ne t i c a na l ys i s J H e r d. 83: 189 195 H i l l i s L W E ngm a n J A & K ooi s t r a W H C E ( 1998 ) M or phol ogi c a l a nd m ol e c ul a r phyl oge ni e s of H a l i m e da ( C hl or ophy t a B r yops i da l e s ) i d e nt i f y t h r e e e vol ut i ona r y l i ne a ge s J P hyc ol 34 : 669 681 H i l l i s C ol i nva ux L ( 1984) S ys t e m a t i c s of t he S i ph ona l e s I n: S ys t e m a t i c s of t he g r e e n a l ga e ( E d. by D E G I r vi ne & D J ohn) A c a de m i c P r e s s L ondon. 271 296. H ue l s e nbe c k J P & R onqui s t F ( 2001 ) M R B A Y E S : B a ye s i a n i nf e r e nc e of phyl oge ny B i oi nf or m a t i c s 17: 754 755 J e ns e n K R ( 1980) A r e vi e w o f s a c ogl os s a n di e t s w i t h c om pa r a t i ve not e s on r a dul a r a nd buc c a l a na t om y. M a l a c ol R e v. 13: 55 77.

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109 M a r c us E & M a r c us E ( 1960) O pi s t hobr a nc hs f r o m A m e r i c a n A t l a nt i c w a r m w a t e r s B ul l M a r S c i G ul f C a r i bb. 10: 129 20 3. M a r c us E & M a r c us E ( 1967) A m e r i c a n opi s t hobr a nc h m ol l us ks P a r t 1, T r opi c a l A m e r i c a n opi s t hobr a nc hs S t udi e s T r o pi c a l O c e a n ogr a phy, M i a m i 6: 1 137. M a r i n A & R os J ( 1993 ) U l t r a s t r uc t ur a l a nd e c ol ogi c a l a s pe c t s of t he de ve l opm e nt of c hl or opl a s t r e t e nt i on i n t he s a c ogl os s a n ga s t r opod E l y s i a t i m i da J of M ol l S t ud. 59 : 95 104. M a t t oo A K H of f m a n F a l k H M a r de r J B & E de l m a n M ( 1984 ) R e gul a t i on o f p r ot e i n m e t a bol i s m : c oupl i ng of phot os ynt he t i c e l e c t r on t r a ns por t t o i n vi vo de gr a da t i on of t he r a pi dl y m e t a bol i z e d 32 ki l oda l t on pr ot e i n o f t he c hl or opl a s t m e m b r a ne s P r oc N a t l A c a d. S c i U S A 81 : 1380 1384 M a t t ox K R & S t e w a r t K D ( 1984 ) C l a s s i f i c a t i on of t he gr e e n a l ga e : A c onc e pt ba s e d on c om pa r a t i ve c yt ol ogy. I n I r vi ne D E G a nd J ohn, D M ( e ds ) Sy s t e m at i c s of t he G r e e n A l gae A c a de m i c P r e s s L ondon a nd O r l a nd o. pp 29 72 M c C our t R M K a r ol K G B e l l J H e l m B yc how s ki K M G r a j e w s ka A W oj c i e c how s ki M F & H os ha w R W ( 2000) J P hyc ol 36: 747 75 8. M e us ni e r I O l s e n J L S t a m W T D e s t om be C & V a l e r o M ( 2001) P hyl oge ne t i c a na l ys e s of C aul e r pa t ax i f ol i a ( C hl or ophyt a ) a nd of i t s a s s oc i a t e d ba c t e r i a l m i c r of l o r a pr ovi de c l u e s t o t he or i gi n of t he M e di t e r r a ne a n i nt r oduc t i on. M ol E c ol 10 : 931 946. M oe s t r up O & H of f m a n L R ( 1973) U l t r a s t r uc t ur e of t he ge e n a l ga D i c hot om os i phon t ube r os us w i t h s pe c i a l r e f e r e nc e t o t he oc c ur r e nc e of s t r i a t e d t ubul e s i n t he c hl or opl a s t J P hyc ol 11( 2) : 225 235

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110 M ondy W L & P i e r c e S K ( 2003) A popt ot i c l i ke m or phol ogy i s a s s oc i a t e d w i t h a nnua l s ync hr oni z e d de a t h i n kl e pt opl a s t i c s e a s l ugs ( E l y s i a c r i s pat a ) I nve r t e br B i ol 122: 126 137 M r c h O A L ( 1863 ) C ont r i bu t i ons a l a f a une m a l a c ol ogi que de s A nt i l l e s da noi s e s J our na l de C onc hyl i ol ogi e 11: 21 43. N e gr i s ol o E M a i s t r o S I nc a r bone M M or o I D a l l a V a l l e L B r oa dy P A & A ndr e ol i C ( 2004) M or phol ogi c a l c onve r ge nc e c ha r a c t e r i z e s t he e vol ut i on of X a nt hophyc e a e ( H e t e r okont ophyt a ) : e vi de nc e f r om n uc l e a r S S U r D N A a nd pl a s t i di a l r bc L ge ne s M ol e c ul a r P hyl oge ne t i c s a nd E vol ut i on 33: 156 1 70. N i c kr e nt D L P a r ki ns on C L P a l m e r J D & D uf f R J ( 2000) M ul t i ge ne phyl oge ny of l a nd pl a nt s w i t h s pe c i a l r e f e r e nc e t o br yophyt e s a nd t he e a r l i e s t l a nd pl a nt s M ol B i ol E vol 17: 1885 1895. O 'K e l l y C J & F l oyd G L ( 1984 ) C or r e l a t i ons a m on g pa t t e r ns of s por a ngi a l s t r uc t ur e a nd de ve l opm e nt l i f e hi s t or i e s a nd ul t r a s t r uc t ur a l f e a t ur e s i n t he U l vophyc e a e I n Sy s t e m at i c s of t he G r e e n A l gae T he S ys t e m a t i c s A s s oc i a t i on s p e c i a l vol um e no. 27. E d i t e d by D E G I r vi ne & D M J ohn. L ondo n: A c a de m i c P r e s s 121 156. P i e r c e S K C ur t i s N E M a s s e y S E B a s s A L K a r l S A & F i nne y C M ( 2006 ) A m or phol ogi c a l a nd m ol e c ul a r c om pa r i s on be t w e e n E l y s i a c r i s pat a a nd a ne w s pe c i e s of kl e pt opl a s t i c s a c ogl os s a n s e a s l ug ( G a s t r opoda : O pi s t hobr a nc hi a ) f r om t he F l or i da K e ys U S A M ol l us c a n R e s 26: 23 38 P i e r c e S K M a s s e y S E H a nt e n, J J & C ur t i s N E ( 2 003) H or i z ont a l t r a ns f e r o f f unc t i ona l nuc l e a r ge ne s be t w e e n m ul t i c e l l ul a r or ga ni s m s B i ol B u l l 204: 237 2 40.

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111 P i e r c e S K B i r on R W & R um pho M E ( 1996) E ndo s ym bi ot i c c hl or opl a s t s i n m ol l us c a n c e l l s c ont a i n pr ot e i ns s ynt he s i z e d a f t e r pl a s t i d c a pt ur e J E xp B i ol 199: 2323 2330 P os a da D & C r a nda l l K A ( 1998) M O D E L T E S T : t e s t i ng t he m ode l of D N A s ubs t i t ut i on. B i oi nf or m a t i c s 14: 817 818 P om be r t J F O t i s C L e m i e ux C & T u r m e l M ( 20 05) T he C hl or opl a s t G e nom e S e que nc e of t he G r e e n A l ga P s e ude ndoc l oni um a ki ne t um ( U l vophyc e a e ) R e ve a l s U nus ua l S t r uc t ur a l F e a t ur e s a nd N e w I ns i ght s i nt o t he B r a n c hi ng O r de r o f C h l or ophyt e L i ne a ge s M ol B i ol E vol 22: 1903 1918 P os a da D & C r a nda l l K A ( 1998) M O D E L T E S T : t e s t i ng t he m ode l of D N A s ubs t i t ut i on. B i oi nf or m a t i c s 14: 817 818 P r uvot F ol A ( 1946) R vi s i on c r i t i que de l a f a m i l l e de s E l ys i a da e J our na l de C onc hyl i ol ogi e 87: 29 44. R ol l e r R A & B i a nc hi T S ( 1995 ) H P L C a na l ys i s of c hl or opl a s t pi gm e nt s f r om t he m a r i ne a s c ogl os s a n T r i dac hi a c r i s pat a ( M r c h, 1863) ( M ol l us c a : O pi s t hobr a nc hi a ) A m M a l a c ol B ul l 11: 139 143 R onqui s t F & H ue l s e nbe c k J P ( 2003 ) M R B A Y E S 3: B a ye s i a n phyl oge ne t i c i nf e r e nc e unde r m i xe d m ode l s B i oi nf or m a t i c s 19: 1572 157 4. R ot h W C & F r i e dm a nn E I ( 1980 ) T a xonom i c s i gn i f i c a nc e of nuc l e us m i c r obody a s s oc i a t i on s S e gr e ga t e d nuc l e ol i a nd ot he r nuc l e a r f e a t ur e s i n s i phonous gr e e n a l ga e J P hyc ol 16: 449 46 4 R ot h W C & F r i e dm a nn E I ( 1987 ) U l t r a s t r uc t ur e o f t he s i phonous gr e e n a l ga e A vr a i nvi l l e a a nd C l a doc e pha l us P hyc ol ogi a 26 ( 1) : 70 81

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115 W i l l i a m s S I & W a l ke r D I ( 1999 ) M e s ohe r bi vor e m a c r oa l ga l i nt e r a c t i ons : F e e di ng e c ol ogy of s a c ogl os s a n s e a s l ugs ( M ol l us c a O pi s t hobr a nc hi a ) a nd t he i r e f f e c t s on t he i r f ood a l ga e O c e a nogr M a r B i ol A nnu. R e v. 37 : 87 128. W r a y J L ( 1977 ) C a l c a r e ous a l ga e D e ve l opm e nt s i n pa l a e ont ol ogy a nd s t r a t i g r a phy. N e w Y or k: E l s e vi e r 185 pp W ynne M J ( 2005) A c he c kl i s t o f be nt hi c m a r i ne a l ga e of t he t r opi c a l a nd s out h t r opi c a l W e s t e r n A t l a nt i c : S e c ond r e vi s i on. N ova H e dw i gi a B e i he f t 129: 1 152 Y onge C M & N i c hol a s H M ( 1940 ) S t r uc t ur e a nd f unc t i on of t he gut a nd s ym bi os i s w i t h z ooxa nt he l l a e i n T r i dac hi a c r i s pat a ( O e r s t ) B gh. P a p. T o r t uga s L a b 32: 287 301. Z e c hm a n F W ( 2003 ) P hy l oge ny of t he D a s yc l a da l e s ( C hl or ophyt a U l vophyc e a e ) ba s e d on a na l ys e s of r ubi s c o l a r ge s ubuni t ( r bc l ) ge ne s e que nc e s J P hyc ol 39: 819 827. Z e c hm a n F W T he r i ot E C Z i m m e r E A & C ha pm a n R L ( 1990) P hyl oge ny of t he U l vophyc e a e ( C hl or ophyt a ) : C l a di s t i c a na l ys i s of nuc l e a r e nc ode d r R N A s e que nc e da t a J P hyc ol 26: 700 710 Z e r H & O ha d I ( 1995) P hot oi na c t i va t i on o f phot o s ys t e m I I i nduc e s c ha nge s i n t he phot oc he m i c a l r e a c t i on c e nt e r I I a bol i s hi ng t he r e g ul a t or y r ol e of t he Q B s i t e i n t he D 1 pr ot e i n de gr a da t i on. E u r J B i oc he m 231: 448 453. Z ul j e vi c A T hi ba ut T E l l ouka l H & M e i ne s z A ( 2001) S e a s l ug di s pe r s e s t he i nva s i ve C aul e r pa t ax i f ol i a J M a r B i ol A s s oc U K 81: 343 344.

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A b ou t t h e A u t h or N i c hol a s C ur t i s r e c e i ve d a B a c he l or 's of S c i e nc e w i t h a dua l m a j or i n M a r i ne S c i e nc e / B i ol ogy f r om t he U ni ve r s i t y o f M i a m i i n t he S pr i ng o f 2000 H e s t a r t e d a D oc t or a l pr ogr a m i n B i ol ogy a t t he U ni ve r s i t y of S out h F l or i da i n t he f a l l of t ha t ye a r I n 2004 he r e c e i ve d t he N a noS pe c t i ve A w a r d f o r E xc e l l e nc e i n M i c r os c opy, a nd r e c e i ve d t he S e c ond P l a c e D i a t om e A w a r d i n 2005


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Curtis, Nicholas E.
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The identification of functional, sequestered, symbiotic chloroplasts in Elysia clarki :
b a crucial step in the study of horizontally transferred, nuclear algal genes
h [electronic resource] /
by Nicholas E. Curtis.
260
[Tampa, Fla] :
University of South Florida,
2006.
502
Dissertation (Ph.D.)--University of South Florida, 2006.
504
Includes bibliographical references.
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Text (Electronic dissertation or thesis) in PDF format.
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System requirements: World Wide Web browser and PDF reader.
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ABSTRACT: A comparison of Elysia (=Tridachia) crispata (Mrch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki. Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters the chloroplasts, which actively photosynthesize for up to 4 months. We have determined the algal source of the chloroplasts in adult E. clarki from 2 populations in the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that adult E. clark i sequester chloroplasts from 7 different species of algae, representing two genera, of which 5 were identified; Penicillus lamourouxii, P. capitatus, Halimeda incrassata, H. monile, and Bryopsis pennata. In addition, chloroplasts from more than 1 species of algae are sequestered in the same digestive cell simultaneously. Phylogenetic analysis of rbcL sequences from the order Bryopsidales showed that E. clarki feeding was restricted to calcareous members of the family Udoteaceae and the family Bryopsidaceae. Feeding experiments were conducted, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae. For the first 14 d post-metamorphosis, juveniles ate only the thin filamentous coenocytes, Bryopsis plumosa or Derbesia tenuissima. Electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other ^macroalga, including the four calcareous species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ~ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae.
590
Adviser: Sidney K. Pierce, Ph.D.
653
Bryopsidales.
Kleptoplasty.
Sacoglossan.
rbcL.
Chloroplast symbiosis penicillus.
Halimeda.
Bryopsis.
Derbesia.
690
Dissertations, Academic
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Doctoral.
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