Lactate dehydrogenase isozyme patterns of Paracanthopterygians (Osteichthyes : Teleostei) : evaluation of a putative Gadiform synapomorphy, with comments on implications for sister group relationships and phylogenetic position of Gobiesociformes

Lactate dehydrogenase isozyme patterns of Paracanthopterygians (Osteichthyes : Teleostei) : evaluation of a putative Gadiform synapomorphy, with comments on implications for sister group relationships and phylogenetic position of Gobiesociformes

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Lactate dehydrogenase isozyme patterns of Paracanthopterygians (Osteichthyes : Teleostei) : evaluation of a putative Gadiform synapomorphy, with comments on implications for sister group relationships and phylogenetic position of Gobiesociformes
Stengard, Fredrik John
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Tampa, Florida
University of South Florida
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ix, 132 leaves : col. ill. ; 29 cm.


Subjects / Keywords:
Gadiforms -- Genetics ( lcsh )
Clingfishes ( lcsh )
Lactate dehydrogenase ( lcsh )
Dissertations, Academic -- Marine science -- Masters -- USF ( FTS )


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Thesis (M.S.)--University of South Florida, 1998. Includes bibliographical references (leaves 101-122).

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University of South Florida
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Universtity of South Florida
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025980263 ( ALEPH )
41978902 ( OCLC )
F51-00141 ( USFLDC DOI )
f51.141 ( USFLDC Handle )

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LACTATE DEHYDROGENASE ISO.lYME PATTERNS OF PARACANTHOPTERYGIANS (OSTEICHfHYES: TELEOSTEI) : EVALUATION OF A PUTATIVE GADIFORM SYNAPOMORPHY, WITH COMMENTS ON IMPLICATIONS FOR SISTER GROUP RELATIONSHIPS AND PHYLOGENETIC POSffiON OF GOBIESOCIFORMES b y / FREDRIK JOHN STENGARD A thesis submitt e d in partial fulfillm e nt of the requirements for the degree of Master of Science Department of Marine Science University of South Florida Decemb e r 199 8 Major Professor : Raymond R. Wilson, Jr., Ph. D


Graduate School University of South Florida Tampa Florida CERTIFICATE OF APPROVAL Master s Thesis This is to certify that the Master's Thesis of FREDRIK JOHN STENGARD with a major in Marine Science has been approved by the Examining Committee on November 23, 1998 as satisfactory for the thesis requirement for the Master of Science degree Examining Committee: R. Wilson.zJL Ph O Memter: Pamela Hallock-M1dler, Ph.D: Member: Ted VanVleet Ph. D


ACKNOWLEDGEMENTS During a seminar course on fish phylogenetics, Dr. Raymond Wilson introduced me to the collection of papers presented at the International Workshop on Gadiform Systematics held in Los Angeles 1986, and in particular to the ideas ofMarkle (1989) ; with the hunt for the tadpole cod, Raniceps rani nus, in order to answer a question therein, this project began. I wish to express my appreciation to Dr. Wilson and my other committee members, Dr. Ted VanVleet and Dr. Pamela Hallock-Muller as well as the Department ofMarine Science at the University of South Florida without whose support and assistance this work would have been impossible I also thank my bosses at the Florida Marine Research Institute (FMRI) Dr. Roy Crabtree, and later Dr. Rich McBride, for allowing me the flexibility of irregular hours as I made good use of the walkway connecting the two institutions Special thanks are due to all those who assisted in collecting specimens for this study, especially the captains of the Friendly Fishermen (Ed Thompson) and the Reel Time (Will Ward), Paul Thurman and others of the Fisheries Independent Monitoring section ofFMRI in St. Petersburg as well as the field labs in Tequesta, Cedar Key, and the Keys regional lab in Marathon, Dr. Behzad Mahmoudi's Baitfish group, Sandra Levett and Phil Steele of Shrimp by-catch, and Dr. Rich McBride ofGamefish (all of FMRI, St. Petersburg, FL), Dr. Frank Maturo (University of Florida Gainesville FL), Dr. M J Allen (Southern California Coastal Waters Research Project Westminster, CA) Andrew Sexton (Woods Hole Biological Laboratory), Matz Berggren (Kristineberg Marine Laboratory, Fiskebackskil Sweden) and Beverly Dickson (Portobello Marine Lab, Dunedin, New Zealand) Additionally, I wish to thank my lab mates Kim Donaldson, Rod Stokes, Andrea Johnson and Jennifer Jarrell (USF), and Dan Merryman, Connie Stevens Heather Patterson, and David Harshany (FMRI) without whom these years would have been a lot less educational, and a lot less fun Roy helped morale by allowing Dan and I to whoop the boss in tennis each Friday and Dave, proprietor of Caddy's on the Water, across the street on Sunset Beach, freed up time for my thesis work by removing the pool table Finally I wish to thank my parents Bertil and Birgitta, as well as Gigi for their (un)wavering support and belief in me, my three younger siblings, Annika Katarina and Petter (who now whoops me in tennis) for providing inspiration by all starting college later and finishing earlier, and especially to Annelie whose love renews me each day


TABLE OF CONTENTS LIST OF TABLES 111 LIST OF FIGURES IV ABSTRACT Vll 1. INTRODUCTION 1 The Cods ofFaro 1 The Codfishes (Order Gadiformes) 2 Gadiform Taxonomy 6 Paracanthopterygian Taxonomy 14 Protein Electrophoresis 18 The Lactate Dehydrogenase Enzyme System 20 Summary ofResearch Aims 24 2 :METHODS 25 Specimen Collection 25 Tissue/Specimen Handling and Storage 26 Specimen Identification 33 Chemicals 34 Preparation of Tissue Extracts 35 Electrophoresis 36 Thermo lability 37 Otolith Preparation 38 Documentation 39 3 RESULTS 40 LDH-C Expression of Acanthopterygians 40 LDH-C Expression of Raniceps raninus 43 LDH-C Expression of Other Gadiforms 48


Other Paracanthopterygians : LDH-C Expression ofPercopsiformes 49 LDH-C Expression of Ophidiiformes (Superorder Paracanthopterygii) 51 LDH-C Expression of the Lophiiformes, Suborder Lophioidei (Paracanthopterygii) 53 LDH-C Expression of the Lophiiformes, Suborder Antennarioidei (Paracanthopterygii) 55 LDH-C Expression of the Lophiiformes, Suborder Chaunacoidei (Paracanthopterygii) 57 LDH-C Expression of the Lophiiformes, Suborders Ogcocephaloidei + Ceratioidei (Paracanthopterygii) 57 LDH-C Expression of the Batrachoidiformes (Paracanthopterygii) 57 LDH-C Expression of the Suborder Gobiesocoidei (Superorder Acanthopterygii sensu Nelson 1994) 66 LDH-C Expression of the Suborder Callionymoidei (Superorder Acanthopterygii) 68 Analysis ofDual Expression : Test for "Nothing Dehydrogenases" 72 Thermolability Analysis 75 Search for LDH-C Heterozygotes 78 Otolith Examinations 79 Age Determination of Raniceps 80 4. DISCUSSION 84 LDH Pattern ofExpression as a Synapomorphy for the Gadiformes 84 Dual expression of Slow Liver-predominant and Fast Eye-predominant LDH-C Forms in the Same Fish 85 Implications ofLDH Patterns of Expression for Gadiform Sister Group Analysis 90 JncertaeSedis Past Members and Proposed Additions 92 Gobiesociformes 96 Conclusions 99 REFERENCES 101 APPENDICES 123 APPENDIX 1. CHEMICALS 124 APPENDIX 2. RECIPES 125 APPENDIX 3. SLICING AND STAINING PROTOCOL 128 ADDENDUM 130 ])


Table 1. Table 2. Table 3. LIST OF TABLES Species Invest i gated Arranged in Taxonomic Order Summary of Specimen Collections, 1995-1998: Numbers Collected, SL, Capture Locations. Lactate Dehydrogenase Pattern of Expression of Selected Taxa Ill 27 30 70


LIST OF FIGURES Figure 1 Codfish Split and Hung Out to Dry in a Norwegian Fishing Village (Ommanney 1969). 3 Figure 2 The "Sacred Cod" Wood Carving of the State House, Boston, Massachusetts (Vosburgh 1969) 4 Figure 3. Variation ofTaxonomic Posit i on of the Monotypic Famil y Ranicipitidae in Dendrograms Presented at WOGADS 11 Figure 4. Toadfish Batrachoidiformes) and Tadpo l e cod Gadiformes). 16 Figure 5 Acanthopterygian Pattern -LDH Isozymes of Scorpaeniformes 41 Figure 6 Acanthopterygian Pattern -LDH Isozymes ofPerciformes 42 Figure 7 LDH Isozymes of Three Specimens of the Tadpo l e Cod, Rani c eps raninus 44 Figure 8 Slice from Raniceps Gel as in Fig. 3 After 90 Minutes Staining Time 45 Figure 9. Rani c eps Isozyme Visualization after Three Hours Staining Time. 46 Figure 10 Second Slice of Raniceps Gel, Allowed to Stain for 18 Hours at Room Temperature 47 Figure 11. LDH Isozyme Patterns of Representative Gadiforms : Coelorhynchus sp ., Nezumia bairdi, and Urophycis floridana 49 Figure 12 Dual e x pression of the Two Forms ofLDH-C in the Pirate Perch, Aphredoderus sayanus 50 I V


Figure 13. Lactate Dehydrogenase Isozyme Patterns of the Ophidiiforms Ophidion holbrooki and Lepophidium cervinum 52 Figure 14 Lactate Dehydrogenase Pattern of the Goosefish, Lophius americanus 53 Figure 15 LDH Pattern of the Blackfin Goosefish, Lophius gastrophysus 54 Figure 16 LDH Pattern of the Antennarioids Antennarius striatus A biocel/atus and Histrio histrio 56 Figure 17 LDH Pattern for Three Individuals of the Chaunacoid Chaunax suttkusi 58 Figure 18 Lactate Dehydrogenase Isozyme Pattern of the Polka-dot batfish, Ogcocephalus radiatus (Ogcocephaloidei) 59 Figure 19 LDH Pattern of Two Batrachiodiforms of the Subfamily Batrachoidinae the Leopard Toadfish (Opsanus pardus) and the Oyster Toadfish (0. tau) 60 Figure 20. Lactate Dehydrogenase Pattern of the GulfToadfish (Opsanus beta) and the "Orange Toadfish" (0. sp ) Subfamily Batrachoidinae. 61 Figure 21. Electrophoretic Results of the Batrachoidiform Porichthys myriaster Subfamily Porichthyinae 62 Figure 22 LDH Patterns of Further Porichthys myriaster Specimens ; Liver -Eye Extract Comparisons 63 Figure 23. Intentionally Over-stained Gel of Porichthys myriast e r 64 Figure 24 Electrophoretic Results of the Batrachoidiform Porichthys notatus Subfamily Porichthyinae 65 Figure 25 Lactate Dehydrogenase Patterns of Three Specimens of the Skilletfish Gobiesox strumosus (Gobiesocoidei) 67 Figure 26 LDH Patterns of Pterosyn c hiropus picturatus, P. splendidus and N e osynchiropus ace/latus (Suborder Callionymoidei) 69 Figure 27A. Test for "Nothing Dehydrogenases" ; Substrate Added 73 v


Figure 27B Test for "Nothing Dehydrogenases"; Substrate Omitted 74 Figure 28A. Comparison ofLDH Allozyme Thermo lability of Opsanus beta. 76 Figure 28B. Comparison ofLDH Allozyme Thermolability of Opsanus beta; Back Slice. 77 Figure 29. Sagittae of the Skilletfish, Gobiesox strumosus 81 Figure 30. Sagitta of the Tadpole Cod, Raniceps raninus 82 Figure 31. Ageing of Raniceps raninus. 83 Figure 32. Opsanus beta with Orange Toadfish (0. sp.?) and Color "Hybrid" 131 VI


LACTATE DEHYDROGENASE ISOZY'ME PATTERNS OF P ARACANTHOPTERYGIANS (OSTEICHTHYES: TELEOSTEI) : EVALUATION OF A PUTATIVE GADIFORM SYNAPOMORPHY, WITH COMMENTS ON IMPLICATIONS FOR SISTER GROUP RELATIONSIDPS AND PHYLOGENETIC POSmON OF GOBIESOCIFORMES by FREDRIK JOHN STENGARD An Abstract Of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida December 1998 Major Professor : Raymond R. Wilson, Jr., Ph. D Vll


Gadiforms comprise over one quarter of the world's commercial marine catch (Nelson 1994) and have a long taxonomic history (Cohen 1984). Desp ite this man y aspects of their systematics as well as of the superorder to which they belong the Paracanthopterygii are still not well understood (Cohen 1989 ). Good characters uni t ing these taxa are limited The few morpholog i cal characters that have been proposed to define the group (synapomorph i es) all suffer from putative member species where the character either is lacking, or is indeterminable (Patterson & Rosen 1989) A molecular character, the lactate deh y drogenase LDH-C* locus pattern of expression has unique tissue specificities and electrophoretic mobil i ties in gadiforms studied to date (Markert et al. 1975 Shaklee & Whitt 1981) Prior to this investigation, however LDH had not been adequately studied in key taxa of the order Gadiformes and superorder Paracanthopterygii to answer the following questions : 1) Would the putative LDH-C synapomorphy hold for the primitive sister group of all the other gadiforms (sensu Markle 1989) Raniceps raninus? 2) Is this pattern truly restricted to the Gadiformes ; i.e Is it a synapomorphic character for the order or some wider taxon? 3) Can the LDH pattern of expression among paracanthopterygians or among incerta e sedis past members illuminate gadiform sister group relationships intra relationships of the group or other group membership questions? The work contributes the following four find i ngs : 1 The LDH-C pattern of expression listed as a putative synapomorphy for the Gadiformes holds for its most basal member Raniceps (but see 4 below) Vlll


2 An unexpected result, dual expression offast eye-predominant and slow liver-predominant LDHC, is documented and will have implications for how we view the lactate dehydrogenase enzyme system as well as for how we view electrophoretic data (based on mobility differences of enzymes) as a whole 3 Many groups at several levels within the Paracanthopterygii express both eye and liver LDH-C forms, and this characteristic may hold some utility in explaining intrarelationships or in defining the group itself Dual expression appears to be the pleisiomorphic state for the taxon 4 Gobiesocoidei (sensu Nelson 1994), presently a suborder within the Perciformes, express the gadiform LDH-C pattern. Given its tenuous affinities to perciforms its final removal from the Perciformes and inclusion in the Paracanthopterygii (as Gobiesociformes) is recommended It should possibly be accorded a position as sister group to the Gadiformes, in which case the lack of eye type LDH-C and expression of l iver type LDH-C will no longer be tenable as a gadiform synapomorphy. Abstract Approved : -.....---=t-r-----------+r ....... ----' __, .......... <'---Major Professor : Raymond R. Wilson, Jr., Ph. D. Associate Professor, Department of Marine Science Date Approved : IX


1. INTRODUCTION Salt fish were stacked on the wharves looking like corded wood, maple and yellow birch with the bark left on. I mistook them for this at first, and such in one sense they were,-fuel to maintain our vital fires-an eastern wood which grew on the Grand Banks. The Cods of Faro -Henry David Thoreau, Cape Cod, 1851 At about the midpoint between the peninsulas ofLanghammar and Norsholmen, the old fisherman stopped rowing and carefully placed the oars inside the wooden gunnels He motioned to his friend to lower the anchor and announced it was time to catch torsk. Jigging heavy chrome-plated metal spoons on handlines resulted in strikes as 1 soon as they hit bottom We hauled up one fish after another until our arms ached In fact, catching the fish was so easy that we competed in seeing who could keep their spoon near the bottom the longest without hooking a fish That day, we stopped fishing when the little boat could not safely hold more cod. This may sound like a tall tale, but it is not. The island of Faro lies just north of Gotland in the Baltic Sea. Most of the land is dry, windswept, and rocky ; geologically it is an ancient coral reef which thrived in the warmer waters of the Tethys Sea The inhabitants are predominantly farmers and fishermen That summer I was nine the year was 1970, and cod were plentiful. Such fishing trips were repeated several times over the


early years of the decade By the late 1970 s however fishermen on the island were talking about the decline in cod stocks of the Baltic and it was reflected in the numbers I caught. In the summer of 1982 a trip to the same area yielded not a cod, not a bite and I haven't caught one there since In the span of my youth I had seen the cod of Faro disappear The Codfishes (Order Gadiformes) 2 The codfish (cod haddock, hake, grenadiers, and related forms) are largely cold water bottom dwellers of the continental shelves and slopes though one Holarctic species the burbot (Lata lata) is completely confined to freshwater (Curry-Lindahl1985 & Fichter 1976 Robins et al. 1986 Vosburgh 1969) Plentiful easily captured and of superior food quality, cod became integral to the economic and cultural structure of the peoples ofEurope before the beginnings of written history ( e g Jensen 1972) By the ninth century, the Scandinavians had perfected the art of preserving cod by hanging and drying (F i g 1), and protein stores provided by dried cod made possible the Vikings' voyages to Greenland and North America (Kurlansky 1997) In fact the Scandinavian word for cod tarsk is a shortening of"tarr fisk" meaning literally "dry fish" (Muus & Dahlstrom 1985) Codfishing has been a large industry since the Middle Ages (Lancaster-Brown et al. 1976) and with the discovery of the great resources of the Grand Banks, was


3 paramount to the economic development of colonial New England as well as Nova Scotia and Newfoundland By the eighteenth century, cod had made New England an international commercial power ; those whose fortunes were won in this trade became known as the "codfish aristocracy" British meddling in this valuable enterprise helped solidify support for the Revolution and rights to the fishery figured prominently in the negotiations for the treaty that ended the American War of Independence (Jensen 1972 Figure 1. Codfish Split and Hung Out to Dry in a Norwegian Fishing Village (Ommanney 1969) Copyright Time Inc


4 Kudansky 1997). Cape Cod is today perhaps one of the most recognized place names in the Americas The importance of cod to the New England area is evidenced by the use of a codfish in the State Seal ofMassachusetts (Migdalski & Fichter 1976); the gilded carving of the "Sacred Cod" (Fig 2) has hung in the State House (Boston) since 1784 (Moyle & Cech 1996). Global commercial fisheries for gadiforms constitute over one quarter of the world's marine catch (Nelson 1994), second only to clupeiforms (anchovies and sardines ; Gross 1987) In Iceland, codfishing generates nearly 50% of the gross domestic product (Hannesson 1996). Due to this heavy dependence, and in an effort to curtail competition Figure 2 The "Sacred Cod" Wood Carving of the State House, Boston, Massachusetts (Vosburgh 1969) Copyright National Geographic Society


5 from foreign fleets fishing around Iceland, the island nation unilaterally declared an extension oftheir exclusive fishing rights, first out to 4 nautical miles (1952), then 12 (1958), 50 (1972), and finally 200 (1975). Conflicts with the British Navy over these actions became known as the Cod Wars, but Iceland persevered and the U.N. Conference on the Law of the Sea (UNCLOS) eventually adopted as an international standard the 200nm exclusive economic zone (EEZ; Hannesson 1996 Hickling & Lancaster-Brown 1981, Kurlansky 1997). Despite their tremendous value, however, the west Atlantic stock crashed in the early 1990's, and a complete moratorium on fishing has been imposed in this area (Hannesson 1996). Cooperative management regionally under the European Community has not prevented declines in other cod stocks (Norse 1993), and a council ofBaltic States is urging a complete moratorium for that body of water also starting next year (though in the case of the Baltic, natural cycles in the influx of salty north Atlantic water across the Kattegatt sill between Sweden and Denmark may be as much to blame) Their management, as a whole, is perhaps the classic case ofHardin' s (1968) tragedy of the commons" The codfish, therefore, have implications (economic, scientific, political legal, literary, historical, and cultural) which perhaps no other fishes can match; their study is not only exciting but has substantial importance beyond the theoretical and academic In this context, phylogenetic investigations of gadiforms and the evolutionary histories these phylogenies help explain are all the more relevant. Placed in the larger picture of vertebrate evolution, fish phylogenetics trace the path of animal development from the


6 most advanced invertebrates the hemichordates urochordates and cephalochordates (including Amphioxus) and through the coelacanths and lungfishes, to the earliest amphibians (Ichthyostega, Acanthost e ga) and other terrestrial tetrapods (c f Barnes 1987 Long 1995, Nelson 1994). Gadiform Taxonomy Despite their commercial and ecological importance (detailed above, Moyle & Cech 1996), as well as their long taxonomic history (Cohen 1984), there are many aspects ofgadiform systematics that are still not resolved (Cohen 1989, Fahay & Markle 1984) In the most recent attempt to systematically define the group Patterson & Rosen (1989) at the International Workshop on Gadiform Systematics (WOGADS) Los Angeles CA (1986) stated : . accepting the current consensus including macrouroids and excluding ophidioids (=Ophidiiformes) and zoarcids ..... [We] find Gadiformes to be mono-phyletic [and] characterized by three or four synapomorphies These putative synapomorphies (shared deri v ed character states ; as opposed to ples i omorphic or ancestral ; cf Hennig 1966, Maddison et al. 1984 Richter & Meier 1994) uniting the gadiforms and defin i ng them as a monophyletic group (Patterson &


7 Rosen 1989 Fig 16 p 33) are 1) X andY bones in the caudal skeleton ; 2) no epipleurals on the first two vertebrae ; 3) scapular foramen between scapula and coracoid; and 4) lactate dehydrogenase electrophoretic pattern (i.e ., LDH-C slow or cathodal, and compartmentalized predominantly in the liver ; see Protein Electrophoresis and The Lactate Dehydrogenase Enzyme System below). These first three characters are problematic. The details of gadiform osteology will not be focused on in this study, but the taxonomic distribution of the characters are discussed here merely to illustrate their somewhat tenuous applicability. The first X and Y bones (Monod 1968, Fahay & Markle 1984), are sometimes absent in Raniceps only present occasionally in the lotid genera Lota and Molva absent in Melanonus and in all gadids (sensu Patterson & Rosen [but see Dunn 1989]); in Euclichthy s, X is present but Y is sometimes absent while inMuraenolepis the dorsal and anal fins run together with the caudal making recognition of X and Y bones impossible (Patterson & Rosen 1989). Furthermore, many gadiforms lack caudal fins altogether (e.g and can therefore not be evaluated with respect to the character. Markle (1982) and Cohen (1984) see the absence of X andY bones as a derived gadiform character; Patterson & Rosen (1989) conclude that, "in any case we learn from this [review of character distribution] that a decision on whether or not X and Y bones are a gadiform character depends on a decision about gadiform phylogeny or relationships, which in tum must depend on prior decisions about gadiform characters. It seems to follow that X and Y bones cannot be cited a priori as gadiform characters Nonetheless, it remains a character restricted to the gadiforms.


Of the second putative synapomorphy, no epipleurals on first two vertebrae, Patterson & Rosen, in the text (p 27) elaborate : 8 "The third character [of eight potential synapomorphies listed earlier (p. 19)] is no ribs or epipleurals on vertebrae 1 or 2 .... pediculates [i.e ., Batrachoidiformes + Lophiiformes] lack ribs entirely so they satisfy this character in part As to epipleurals, lophiiforms have none ; at least some batrachoids have them forward to the first ... or second ... neural arch At least some ophidiiforms ... have epipleurals on the first two vertebrae, but others lack them . and all appear to lack ribs there ... Thus this character should be split into two: no ribs on vertebrae 1 and 2 characterizes [the] node [linking the acanthopterygians and "beryciforms" with the paracanthopterygians] or, more probably, some lower node; epipleurals on those vertebrae are incongruent because of either multiple loss (in some ophidiiforms, in lophiiforms, and in gadiforms) or secondary extension forwards in some batrachoidiforms They repeat this conclusion (p. 30), "Thus out of seven [sic] putative gadiform characters three ... [among them no ribs or epipleurals on vertebrae 1 or 2] fit at lower nodes on the cladogram"; nonetheless this character is rendered on their cladogram (i.e., Fig. 16) as a gadiform synapomorphy though they note in the legend that the character is


homoplast i c in also being pos s essed by some bythitoids and all lophi i forms Patterson & Johnson (1995) find "one unattached bone on one side ofV1 in our Raniceps". The third putative gadiform synapomorphy scapular foramen between scapula and coracoid (rather than within the scapula) was first observed by Gosline (1968) as distinguishing the gadiforms from the ophidiiforms This condition is lacking in some macrourids and in Bromse but does not occur in any of the other paracanthopterygians (Patterson & Rosen 1989) It is with the fourth putative synapomorphy la c tate dehydrogenas e pattern, that this work is concerned. From the discussion above, it is clear that the gadiforms are poorly characterized The point is that the three suggested osteological synapomorphies are in many gadiforms either i ndeterminable secondarily lost or homoplastic LDH holds the promise of being readily determinable in all members The question is "Does the specialized lactate dehydrogenase pattern, with a slow liver-predominant LDH-C (as opposed to the common pattern of a highly-anodal eye-predominant LDHC of other higher teleosts) hold up for the entire group ? And out of this inquiry a secondary one becomes paramount : "Is this specialized pattern expressed by any members of putative sister taxa (i.e if the syapomorphy holds for the gadiforms does it also hold for a wider assemblage of species outside ofthe Gadiformes)"? An alternative way of looking at this second question is : "Can LDHC patterns in this wider group (the Paracanthopterygii s e nsu Nelson 1994 ; see Paracanthopterygian Taxonomy) as a whole shed light on the gad i form sister group question? Addressing these questions with specific reference to L DH-C expression is the primary goal of this research 9


10 Though many investigators have studied distributions of various character states and interand intrarelationships of selected taxa within the Gadiformes (e.g Dunn 1989, Dunn & Matarese 1984, Fahay 1989, Fedotov & Bannikov 1989, Houde 1984a, Howes 1987, 1988, 1989, Inada 1989, Iwamoto 1989, Marshall & Iwamoto 1973, Merrett 1989, Okamura 1989, Paulin 1989, Renaud 1989), Markle (1989) was the first to attempt a rigorous systematic analysis of intrarelationships for the group as a whole. This work derived from attempts to identify developmental patterns, on a broad scale, in gadiform fishes which led him to incongruencies between relationships evidenced by developmental features and accepted relationships within the group based on works by Svetovidov (1948), Marshall (1966), Gosline (1968), Marshall & Cohen (1973), and others It is with a study such as Markle's, which defines order within the group of interest, that one must start when assessing the value of proposed synapomorphies. In his paper, which was "strongly influenced by upper gill arch morphology", Markle (1989) concludes that the primitive sister group of all other gadiforms is the family Ranicipitidae, with Raniceps raninus as its sole member (Fig. 3A). There has been disagreement on this point. Howes (1989), in a study with conclusions based on cranial myology and arthrology, nests the Ranicipitidae within the suborder Gadoidei, with the families Muraenolepididae/Lotidae + Phycidae as a sister group; on his dendrogram (p 126) at the 9th branchpoint of maximally 11 (Fig. 3B). He writes (p 125), "The genus Raniceps, considered by some to be a 'primitive' or 'basal' gadid (Dunn & Martrese 1984) has been referred to its own subfamily


------------------------------------------------------Ranicipitidae ------------------------------------------Melanonidae [ ------Moridae ------Steindachneriidae ------------------------------Macrouronidae [-----Bregmacerotidae ------Muraenolepididae --------------Phycidae --------------Merlucciidae '----i ------Lotidae A Markle 1989 ------Gadidae .-----------------------fC ____ Macrourinae --j :----------------1.-----Jl_ _____ Ga domus --------------Moridae ------Steindachneriidae ------Melanonidae B. Howes 1989 ---------------------------------------Euclichthyidae -? ---------------------------------------Bregmacerotidae Merluciidae r-------------1 -------Phycidae* ------Gadidae r----------------Ranicipitidae .._ __ L_ ------Lotidae [ L_ ______ Phycidae* --------------Muraenolepididae Figure 3. Variation ofTaxonomic Position of the Monotypic Family Ranicipitidae in Dendrograms Presented at WOGADS. A As the sister group to all other gadiforms (Markle 1989) ; B As an advanced gadiform (Howes 1989) ; C Intermediate position (Nolf & Steurbaut 1989c). Continued on next page paraphyletic


--------------------------------------------------------------Muraenolepididae ----------------------------------------------------------Bregmacerotidae ----------------------------------------------Euclichthyidae -------------------Bathygadinae 1-------------i C{----Macrourinae -----Macrouroidinae -----Trachyrhincinae --------------------------------------------------------Melanonidae ---------------------------------------------Moridae 12 r------------Lotinae (Raniceps)* [-------Steindachneriinae "---------Merlucciinae r-----------------------------------------II Gadiderum II ------------------------------Phycinae r-------------------------Gadini C Nolf & Steurbaut 1989c ----------------"Gadidarum" -------------------Trisopterus [------Merlangius ------Micromesistius ------------Gadiculus '---1 I -----Boreogadus ------Arctogadus Figure 3 (Continued). *possibly paraphyletic (Gaemers 1976). However, myologically, Raniceps displays several derived features (Howes 1988) and in my scheme ofrelationships would be ranked at the family level." He considers Ranicipitidae as among the more advanced, or "higher gadoids (p 127)


Intermediate to the d iffe ring v iews of Markle and Howes is that ofNolf & Steurbaut (1989c) who based on otolith morphology place Ranic eps within the subfamily Lotinae in Gadidae though they offer no explanation for this placement. It is then in a position in which its consubfamilials of the Lotinae have as a sister group the subfamilies Steindachneriinae + Merlucciinae They question the Lotinae, constituted thusly, as possibly paraphyletic however and write (p 98), "The most interesting genus among the Lotinae is undoubtedly Raniceps ; its otoliths show very clearly all of the generalized gadid features ... In their resulting dendrogram (p 1 07) Raniceps ends up at the 4th branchpoint of maximally eight (Fig 3C) 13 Thus, Raniceps is placed either basally among the gadiforms (i.e as the primitive sister group to all other gadiforms) at an intermediate level or as an advanced member of the taxon Nolf & Steurbaut s (1989c) data could easily be reinterpreted with Raniceps in a basal position, and though Howes (1989) gives only one character apomorphy separating Ranicipitidae from Gadidae (rectus communis tendinously attached to head of urohyal), Markle details 11 non homoplastic apomorphies between the two taxa and at least 6 separating Rani cipitidae from the more inclus ive suborder Gadoide i I therefore favor the basal position for Rani ceps.


14 If this position as the sister group to all other gadiforrns is correct then confirming putative gadiform synapomorphies on this species takes on further importance. According to Markle (1989), the three morphological gadiforrn synapomorphies listed above are all possessed by ranicipitids, but "their LDH-C pattern is unknown As mentioned above, this putative synapomorphy is perhaps the most important of those proposed since Raniceps and all other gadiforms will likely possess some version of the character ; i.e it is unaffected by body form differences mode of swimming, feeding, etc. which often confound morphological characters It was therefore the first goal of this research to identify that pattern in Raniceps and determine if it corresponds to that generally found in Gadiforrnes Regarding the larger question of gadiform sister groups and paracanthoperygian intra-relationships as determinable from C4 isozyme expression, a survey of representative species in the various taxa is required. It is necessary to do this in order to further confirm (or refute) the synapomorphic status of liver-predominant expression in Gadiforrnes Paracanthopterygian Taxonomy In the words of Greenwood et al. (1966), who first defined the Paracanthopterygii "The fishes grouped together in this superorder represent a spiny-finned radiation more or less comparable morphologically with that of the Acanthopterygii." That is among the


15 most advanced of the bony fishes the Paracanthopterygii are considered a sort of side branch (para meaning by the side of, alongside) of equal taxonomic rank, to the more speciose Acanthopterygii (an assemblage which includes the familiar perch-like fishes and related groups). As first constituted by Greenwood et al. (1966), the Paracanthopterygii included the Percopsiformes (trout-perches, pirate perch cavefishes), Gadiformes Batrachoidiformes (toadfishes midshipmen) Gobiesociformes ( clingfishes) and Lophi i formes (anglerfishes frogfishes, batfishes) Since then, various autho r s have proposed additions or deletions (for example, the reasons for exclud i ng the Zoarcidae from Gadiformes and the Paracanthopterygii [Gosline 1971 Anderson & Hubbs 1981, Anderson 1984] have been generally accepted; see Patterson & Rosen 1989 for a detailed review of these cases), but the core has remained essentially the same The group as currently understood is thought to include five orders : Percopsiformes Ophidiiformes cusk-eels) Gadiformes Batrachoidiformes and Lophiiformes (Johnson & Patterson 1993 Nelson 1994), though acceptance ofthis combination of members is not universal (e g Carroll, 1988 Eschmeyer 1990, 1998 who retain Gobiesociformes [including Cheilobranchidae (Springer & Fraser 1976)] next to the Batrachoidiformes and Lophiiformes) Perhaps the first clue to gadiform sister group relationships was unwittingl y provided by Linnaeus when he named the first toadfish discovered Gadus tau (Greenwood et al 1966; now known as Opsanus tau) The error of including a toadfish within the cods is understandable when one compares a toadfish to Raniceps, a fish common on the west coast ofLinnaeus' homeland of Sweden and described by h i m in


16 1758 (Fig 4) Not only are their appearances, preferred habitats, diets, and sociobiology very similar (cf Bohlke & Chaplin 1993 Curry-Lindahl1985 Muus & Dahlstrom 1985 Robins et al. 1986), but Markle (1989) lists six deri v ed shared states between the two A. B Figure 4 Toadfish (Opsanus; Batrachoidiformes) and Tadpole cod (Raniceps ; Gadiformes). A. 0. beta ; Bohlke & Chaplin 1993 cop y right Uni v Texas Press B Curry Lindahll985, copyright P .A. Norstedt & Soners Forlag


17 orders and considers them sister groups He writes of Raniceps that they .. are plesiomorphic, so much so that they are 'batrachoidiform' in many characters .. This similarity of appearance is reflected in the common names given for example, tadpole cod in English (Nelson 1994) and paddtorsk (literally, toadcod) in Swedish The genus name derives from the Latin for "frog head" (Markle 1989) Slightly different is the analysis by Patterson & Rosen (1989) who see the Batrachoidiformes together with the Lophiiformes (as a named taxon they call the Pediculati, this assemblage being traditionally accepted ; cf Valenciennes 1837a b Gill 1872, Jordan & Sindo 1902 Regan 1912, Monod 1960, Greenwood et al. 1966, Rosen & Patterson 1969, Pietsch & Grobecker 1987) as constituting the sister group to the gadiforms Others hold that there is a more or less close relationship between gadiforms and the "primitive" order of the Paracanthopterygii, the Percopsiformes (e g Greenwood et al. 1966, Lauder & Liem 1983) Those who retain Gobiesociformes within the Paracanthopterygii generally favor placing this order nearer the lophiiforms and batrachoidiforms than to the gadiforms (Greenwood et al. 1966 Fraser 1972, Lauder & Liem 1983, Nelson 1984) That the Paracanthopterygii constitute a natural group is far from certain either It has been widely recognized that the superorder is woefully short on defining characteristics (i e., apomorphies; e g Rosen 1985, Patterson & Rosen 1989, Johnson 1992, Roberts 1993) yet it survives since few characters are evident to tear it asunder (cf Johnson & Patterson 1993). The questions of affinities with i n the groups, as well as membership both of the Gadiformes and wider Paracanthopterygii then, are as old as


Linnaeus and have been the subject of much controversy since the erection ofthe superorder in 1966. A more interesting group to study would be hard to find. 18 Regarding the applicability ofLDH patterns of expression to resolve these, an example has already been set. Shaklee & Whitt (1981), working under the assumption that the Gadiformes as constituted in Nelson (1976), based on the works of Greenwood et al. (1966) and Rosen & Patterson (1969), including the suborders Ophidioidei and Zoarcoidei, found that all 19 species they tested belonging to the suborders Gadoidei and Macrouroidei possessed the cathodally-migrating liver-predominant LDH-C pattern of expression while all 16 representatives of the Ophidioidei and Zoarcoidei they tested expressed the highly anodal, eye-predominant LDH-C type. Based on this dichotomy, they successfully proposed a splitting of the Gadiformes, removing ophidioids and zoarchoids from them This conclusion had already been reached independently by other investigators working with morphological features (e g., Gosline 1971; cf Patterson & Rosen 1989). Protein Electrophoresis Proteins are composed of amino acids arranged in a sequence determined by the underlying genetic code (e.g Richardson et al. 1986). Each of the 20 common amino acids used to synthesize enzymes and other proteins have unique side chains, and five of these are charged (Avise 1994; Murphy et al. 1990). Lysine, arginine, and histidine are


19 basic and positively charged (NH3) while aspartic acid and glutamic acid are acid i c and negatively charged (COO} The technique analyses mobility differences among enzymes subjected to an electric field The combination of starch gel electrophoresis (Smithies 1955) with histochemical staining techniques (Hunter & Markert 1957) allowed visualization of functionally s i milar versions of a given enzyme (iso-enzymes = isozymes) and allelic variants of a particular enzyme locus (allozymes) due to mobility dist i nctions accounted for by net charge differences (May 1992) Enzyme shape also has significant consequences for electrophoresis Its three dimensional (tertiary) structure determines the orientat i on of am i no acids and therefore impacts relative mobility. Additionally many enzymes are composed of more than one copy of the polypeptide chain ( dimers trimers, tetrameres) and its quaternary structure (i.e ., size and shape) then impacts to some degree mobility through the gel matrix, though less so than net charge Quaternary structure also has implications for banding patterns since the gene products of loci encoding multimeric enzymes usually combine randomly (following a b i nomial distribution) to produce the active form of the enzyme (May 1992) These polypeptide chains may even combine between distinct loci of the same enzyme. A tetramer composed of identical peptide chains derived from just one locus is termed a homotetramer (or homomer) while one composed of different subunits is termed a heterotetramer (Murphy et al. 1990). Subscripts are often employed to denote these relationships ; thus ADH-A 2 would i nd icate the alcohol dehydrogenase-A homodimer (i e ., two -A subunits forming the enzyme) while LDH-A2B2 would ind icate the lactate dehydrogenase-AlB heterotetramer (i e two -A subunits joined with two -B


subunits ; see Shaklee et al. 1990 for conventions on gene nomenclature and Goldberg 1966, Salthe et al. 1965 for lactate dehydroganase heterotetramer hybridization) 20 A key advantage of electrophoretic data with respect to systematics is its versatility Information obtained from protein electrophoresis has been successfully applied to phylogenetic questions ofvery different scales-from population(e g Ayala et al. 1972, Buth & Mayden 1981, Salini & Shaklee 1988) species(e g. Boileau & Hebert 1988, Shaklee & Tamaru 1981) and subgenus/genus-level (e g. Buth 1979a, b 1980, Buth et al. 1980, Dimmick & Page 1992, Leibel 1995) to higher (e g ord i nal) level problems (Shaklee & Whitt 1981 Buth 1984) Additionally, much systematically-useful information can be obtained from analysis of just a few individuals (cf A vise 1975 Hedges 1987 Nei 1978, Shaklee et al. 1982 ; though see also Archie et al. 1989) The Lactate Dehydrogenase Enzyme System The lactate dehydrogenase family ofisozymes (E.C.; Shaklee et al. 1990) is one of the most extensively studied and informative multilocus enzyme systems known (cf Shaklee et al. 1973 Whittet al. 1973, Dalziel 1975, Holbrook et al 1975, Rossman et al. 1975) This enzyme functions chiefly in the reduction of pyruvate to lactate in the glycolytic pathway during periods of transient anaerob iosis (Shaklee & Whitt 1981) with this conversion concomitantly producing oxidized NAD+ from a co-


21 factor, NADH, allowing glycolysis, and therefore, ATP (energy) production to continue (Shaklee 1972, Whittet al. 1973; see Everse et al. for differential locus functioning) Lactate dehydrogenase is of particular importance and utility in the study of fish systematics since it exists in multiple forms (i.e ., isozymes) and is often tissue-restricted in remarkably predictable patterns (e g Chatterjee & Dhar 1985, Kettler &nWhitt 1986, Markert & Faulhaber 1965 Markert et al. 1975, Nakano & Whiteley 1965 Sensabaugh & Kaplan 1972, Whitt 1968, 1969, 1970a, b, 1987 Whittet al. 1971; such tissueand subcellular-level restrictions have been found for other enzyme systems as well : e g. creatine kinase [Fisher & Whitt 1978], phosphoglucose isomerase [Al-Hassan & Ahmed 1985], enzymes of carbamoyl phosphate and urea synthesis [Anderson & Walsh], xanthine dehydrogenase [Padhi & Khuda-Bukhsh 1990], multiple systems [Kettler et al. 1986] ; cf Somera & Hand 1990, Somera et al. 1991). In lactate dehydrogenase these forms (isozymes) are encoded by three separate gene loci known as -A* B and -C* (letters ra t her than numbers are employed to designate these genes/loci since orthology with lactate dehydrogenase isozymes of other taxa has been clearly established ; cf Bailey & Wilson 1968, Gorman et al. 1971 Nadal Ginard & Markert 1975 Pesce et al. 1967, Salthe 1975, Shaklee et al 1990 Syner & Goodman 1966, Tsuji et al. 1994 Wilson et al. 1964 and details below. Much work has been carried out elucidating the genealogies of these loci ; see Masters & Holmes 1974Matson 1989 Powers 1991, Quattro et al. 1993 Stock & Whitt 1992, Whittet al 1975 and for examples of isozyme duplication in fish see Engel et al. 1973 Klose et al.


22 1969) The LDH-C4 homotetramer has proven to be of exceptional taxonomic va lue for just these reasons (e g Shaklee & Whitt 1981 ). It has long been known that the C4 isozyme of mammals and birds (e.g., Blanco et al. Goldberg Zinkham et al. 1964) is also widespread among fishes (Morrison & Wright 1966 Markert et al 1975, Shaklee et al. 1973, Whitt 1975, Whitt & Maeda 1970) In most fishes that have been studied, LDH-C exh ibits a highly anodal mobility under standard electrophoretic conditions and is predominantly compartmentalized in neural tissues, particularly the eye and brain (e.g., Holmes & Markert 1969, Horowitz & Whitt 1972); first to report this "fast eye" isozyme of the -C locus were Nakano & Whiteley (1965) and Markert & Faulhaber (1965). Other groups of fishes exhibit an alternative isozyme ofLDH-C, one in which the enzyme is predominantly compartmentalized in the liver and which exhibits low anodal or even cathodal mobility under standard electrophoretic conditions (e.g., Odense et al 1969, Numachi 1972, Sensabaugh & Kaplan 1972, Shaklee 1972) Of particular interest, the gadiforms examined to date possess this latter LDH-C pattern of expression ( Markert & Faulhaber 1965, Sensabaugh & Kaplan 1972 Shaklee & Whitt 1981) along with several other distantly related fish taxa: Cyprinifonnes (Kepes & Whitt 1972, Shaklee et al. 1973) the pallid sturgeon, Scaphirhynchus a/bus (Acipenseriformes) and Xenomystus nigri Markert et al 1975 Shaklee 1972) Despite their widely differing electrophoretic mobilities and tissue specificities, these C 4 's are nonetheless regarded as distinct tetrameric LDH isozymes of the same presumptive LDH-C* gene locus Many lines of evidence indicating their synonymy have


23 been marshalled to support this, including immunochemical, genetic physical and phylogenetic (Markert et al. 1975). One ofthe cornerstones of this view is that despite extensive surveys of the bony fishes (over 150 species; de Panepucci et al. 1984 Lush et al. 1969, Markert et al. 1975, Page & Whitt 1973, Shaklee 1972, Shaklee & Whitt 1981) all that express LDH-C activity demonstrate either the liver-predominant or the eye predominant isozymes but never both (but see Holt & Leibel 1987) Shaklee (1972) wrote (p 217), "Although the presence of retinal-specific (Markert & Faulhaber 1965 ; Whitt 1970a) and liver-specific (Odense et al. 1969, Lush 1970 Sensabaugh & Kaplan 1972) LDH isozymes led some authors to suggest the existence of additional LDH genes (Odense et al1969, Sensabaugh & Kaplan 1972) the present study demonstrates that the retinal-specific (eye-band) and liver specific isozymes are merely alternative expressions of the LDH-C locus. The definitive observation allowing this generalization is that all the fish species invest i gated exhibited either an eye-band or a liver-band (or kidney band). Species were never observed with both forms and every species examined had one or the other."


24 Summary of Research Aims The methodology (i.e ., investigation ofLDH-C patterns of expression) it follows is applicable to the level of questions asked and has proven capable of resolving problems within the superorder Paracanthopterygii To explicitly restate the questions t o be addressed by the research herein, 1 Is the LDH pattern of expression seen in many gadiforms shared in the most primitive species of the order Ran i ceps rani nus ? 2. Is this pattern truly restricted to the Gadiformes, or does it extend beyond the "boundaries" of this group? That is, Is it a synapomorphic character for the order ? 3. Can the LDH pattern of expression among paracanthopterygians or among in c erta e s e dis past members illuminate gadiform sister group relationships intra relationships of the group or other group membership questions ?


25 2.METHODS Specimen Collection Specimens for this study were collected between 1995-1998 using a variety of methods These included 100 foot seine (Opsanus beta, Gobiesox strumosus), shrimp trawl (0. beta 0 sp., Chilomycterus schopft, and Urophycisjloridana), modified otter trawl [FDEP permit# 97S-372 (0. beta Ogcocephalus radiatus)], coastal and deep-sea otter trawls deployed from research vessels in the Gulf of Mexico, Florida Straits, and coastal Southern California ( Ophidion ho/brooki, Chaetodipterus faber, Lepophidium cervinum, Scorpaena brazi/iensis, Anclyopsetta quadroce//ata, Prionotus scitu/us, Coe/orhynchus sp., Nezumia bairdii, Porichthys myriaster Porichthys notatus, Chaunax suttkusi, and Lophius gastrophysus), SCUBA (0. beta), hook and line (Opsanus pardus, 0 beta, Seriola zonata, Rachycentron canadum, Echeneis naucrates, Corypha.ena hippurus Orthopristis chrysoptera, and Lagodon rhomboides), fish traps (Raniceps raninus), and purse sein (Scomberomorus maculatus). Additional specimens were acquired from biological supply companies: Woods Hole Marine Biological Laboratory, Woods Hole, MA (Lophius americanus, Opsanus tau), Gulf Specimen Co., Panacea, FL (G. strumosus); from reputable local aquarium fish dealers specializing in direct air freight import of live specimens: Marine Warehouse, St Petersburg FL (Histrio histrio, Antennarius biocellatus, Antennarius striatus, Opsanus


26 sp ., and Neosynchiropus ocellatus) Aquatic Wonders, St. Petersburg, FL (Pterosynchiropus picturatus, Pterosynchiropus splendidus), and Fish and Other Ichthy Stuff, Oldsmar, FL (Gobiesox sp ); or from private collectors : John Brill Livingston, NJ (Aphredoderus sayanus) Species investigated, in taxonomic order and with species abbreviations, are listed in Table I ; specimen lots with numbers of individuals examined and collection locations are listed in Table 2. Tissue/Specimen Handling and Storage Specimens collected in the field were either immediately stored on ice until arrival at the laboratory, or were frozen in a shipboard freezer at -20 C Tissues (liver, white skeletal muscle, heart, eye, and brain) and/or whole specimens were subsequently stored in a laboratory at the University of South Florida in a -87 C freezer until needed for electrophoresis Tissue homogenates and extracts not used up during electrophoresis were immediately re-frozen at -87 C for possible later use Specimens shipped from other locations to this laboratory were frozen prior to shipping packed on dry ice, and express delivered overnight by FedEx (P. notatus, P myriast e r), or hand carried by the author in the same manner (R. raninus) These were subsequently stored as above Whenever possible specimens were kept live until immediately before electrophoresis in order to ensure maximum enzyme activity. These were brought back


Table 1 Species Investigated Arranged in Taxonomic Order. Sensu Nelson 1994, with modifications as in Markle 1989 Pietsch 1981, 1984, Pietsch & Grobecker 1987. Abbreviations used follow in parentheses Taxon Species Superorder Paracanthopterygii Order Percopsiformes Suborder Percopsoidei Suborder Aphredoderoidei Aphredoderus sayan us Order Ophidiiformes Suborder Ophidioidei Lepophidium cervinum Ophidion holbrooki Suborder Bythitoidei Order Gadiformes Suborder Ranicipitoidei Raniceps raninus Suborder Melanonoidei Suborder Macrouroidei Coe/orhynchus sp. Nezumia bairdi Suborder Gadoidei Urophycis floridana Order Batrachoidiformes Opsanus beta Opsanus pardus Opsanus sp. Opsanus tau Porichthys myriaster Porichthys notatus Order Lophiiformes Suborder Lophioidei Lophius americanus Lophius gastrophysus continued on next page Abbrev (As) (Lc) (Oh) (Rr) (C) (Nb) (Uf) (Ob) (Opp) (Osp) (Ot) (Pm) (Pn) (La) (Lg) 27


Table 1 (Continued) Taxon Species Suborder Antennarioidei Antennarius biocel/atus Antennarius striatus Histrio histrio Suborder Chaunacoidei Chaunax suttkusi Suborder Ogcocephaloidei + Ceratioidei Ogcocephalus radiatus (= cubifrons; Bradbury 1980) Superorder Acanthopterygii Order Scorpaeniformes Suborder Scorpaenoidei Abbrev (Ab) (Ans) (Hh) (Cs) (Or) Scorpaena braziliensis (Sb) Prionotus scitu/us (Psc) Order Perciformes Suborder Percoidei Chaetodipterus faber (Cf) Coryphaena hippurus (Ch) Echeneis naucrates (En) Lagodon rhomboides (Lr) Orthopristis chrysoptera (Oc) Rachycentron canadum (Rc) Serio/a zonata (Sz) Suborder Gobiesocoidei Gobiesox sp. ( Gsp) Gobiesox strumosus (Gs) Callionymoide i Neosynchiropus ocellatus (= Synchiropus ; (No) Fricke 1982, 1983) Pterosynchiropus picturatus (= Synchiropus; (Pp) Fricke 1982 1983) Pterosynchiropus splendidus (= Synchiropus ; (Ps) Fricke 1982, 1983) continued on next page 28


Table 1 (Continued). Taxon Species Suborder Scombroidei Scomberomorus maculatus Order Pleuronectiformes Suborder Pleuronectoidei Anclyopsetta quadrocel/ata Order T etraodontiformes Suborder T etraodontoidei Chi/omycterus schoepfi 29 Abbrev (Sm) (Aq) (Cs)


30 Table 2 Summary of Specimen Collections 1995-1998 : Numbers Collected, SL, Capture Locations Tissue(s) Specimen ld's n sampled SL (mm) Location Uf970302001 1 LMHEB 175 Tampa Bay, FL Uf970302002-003 2 L 102-127 Tampa Bay, FL Lc960921001 1 L 187 G of M (off Naples) Lc960921002 1 LMHEB 186 G of M (off Naples) Oh960920001 1 L 224 G of M (off Ft. Myers) Oh960920002 1 LMHEB 188 G. of M (off Ft. Myers) Oh960920003 1 L 193 G. of M. (off Ft. Myers) Oh981 01 0004 1 LMHEB 166 Port Richey, FL Ob960917001 1 LMHEB 114 Key Largo, FL Ob960917002 1 L 64 Key Largo FL Ob970124003 1 L 226 Pass-a-Grille, FL Ob97031 0004-005 2 LMHEB 72 76 Tampa Bay, FL Ob970618006-008 3 LMHEB 112-131 Terra Ceia, FL Ob980907009 1 LMHEB N/A Pass-a Grille, FL Ob98090701 0-011 2 LMHEB N/A Madeira Bch FL Ob981 010012-050 39 LE 91-222 Port Richey, FL Rr960721 001-003 3 LMHEB 152-239 KML, Sweden c 001002 2 LMHEB 189 196 N/A Nb951005001 1 LMHEB 121 S of Marq Keys, FL Or970417001-002 2 LMHEB 65-95 Tampa Bay FL Or970417003 1 LMHEB 92 Tampa Bay FL Or980830004-006 3 LMHEB 172-201 Cedar Keys, FL Or980830007 -008 2 LMHEB 147-156 Cedar Keys, FL Or981010009 1 LMHEB 140 Port Richey, FL Opp970507001 1 LMHEB 173 off Madeira Bch., FL Opp970507002-003 2 LMHEB 245-265 off Madeira Bch FL continued on next page


31 Table 2. (Cont i nued) Spec i men ld's n Tissue(s) SL (mm) Location sampled Opp980828004 1 LMHEB 308 off Madeira Bch. FL Opp980831005 1 LMHEB 299 off Madeira Bch FL Sm970227003 1 LMEB Egmont Key FL Sm970329008 2 LMEB Egmont Key FL Sz00 1 -010 10 LMEB off Madeira Bch., FL La970924001 1 LMHEB 625 Woods Hole MA Cs980825001-003 3 LMHEB 93-144 Gulf of Me x ico Gs980821001 1 whole body 27 Bradenton, FL (pooled) Gs980827002-004 3 LMHEB 62-69 Panacea, FL Gsp981002001 1 LMHEB 54 N/A Lg980825001-002 2 LMHEB 171-242 Gulf of Mexico Lg980825003 1 LMHEB 261 Gulf of Mexico Pm980811 001-003 3 LMHEB 168-326 Hun ti ngton Bch CA Pm98081 0004 1 LMHEB 278 Balsa Chica Lgn CA Pm98081 0005 1 LE 145 Balsa Chica Lgn CA Pm98081 0006-012 7 LE 128-202 Los Angeles CA Pn980819001-004 4 LMHEB 164-172 Santa Monica Bay CA Pn980819005-052 48 LE 72-161 Santa Monica Bay CA As980829001-002 2 LMHEB 55-59 Ocean Co NJ Ot970923001 1 LMHEB 217 Woods Hole, MA Ans980901001 1 LMHEB 57 Philippean I slands Sp980901001 1 LMHEB 56 Phil i ppean Is l ands continued on next page


32 Table 2 (Continued) Specimen ld's n Tissue(s) SL (mm) Locat i on sampled Ps980901001 1 LMHEB 51 Philippean Islands No980901001 1 LMHEB 64 Philippean Islands Osp980907001 1 LMHEB 96 Dunedin FL Rc001-005 5 LMEB 715-1206 Anclote Key FL En001-005 5 LMEB 219-559 Gulf of Mexico coastal FL Ch001-005 5 LMEB 438-768 Miami, FL Sb981 010001-002 2 LMHEB 74-89 Port Richey, FL Hh970801001 1 LMHEB 83 Philippean Islands Ab970801001 1 LMHEB 76 Philippean Islands Psc0422001-002 2 LMHEB 165-170 Gulf of Mex ico Lr981 027001 1 LMHEB 194 St. Pete FL Oc981027001 1 LMHEB 203 St. Pete FL Cf960422001 1 LMHEB 131 Gulf of Mexico Aq960415001 1 LMHEB 228 Gulf of Mexico Cs981104001 1 LMHEB 63 Port Richey FL


33 to the laboratory in vessels containing sea water (e .g. 0. beta L. rhomboides 0 chrysoptera), maintained in fresh water (A. sayanus) or salt water aquaria (H. h i strio A biocellatus, A striatus, G stumosus, G sp, P picturatus P splendidus, N ace/latus, and 0. sp ) or kept in 500 gallon tanks in the recirculating sea water wet lab at the Univers i ty of South Florida Voucher specimens of all species except Coelorhyn c hus sp R canadum A quadrocellata and C hippurus are housed in the ichthyological collection of the Florida Marine Research Institute (Florida Department of Environmental Protection) St. Petersburg, FL. Specimen Identification Specimens were either sight identified at the time of collect ion or were keyed out using recognized primary and secondary literature before or after tissue samples had been taken (Bohlke & Chaplin 1993, Bradbury 1978 1980 1988 Briggs 1955, 1963, 1969, Burgess et al. 1988 Caruso 1978, 1985, 1989, Cohen 1978 Cohen & Nielsen 1978, Collette 1966, 1973, 1974 1983, Collette & Russo 1981, Curry-Lindahl 1985, Eddy & Underhill1978 Fricke 1982 1983 Gould 1965 Greenfield & Greenfield 1973 Roese & Moore 1977 Humann 1994, Hutchins 1976 1981, Johnson & Greenfield 1983, McClane 1978 Myers 1991 Nakabo 1982 1983a Page & Burr 1991 Randall 1968, Robins et al.


34 1986 Schultz & Reid 1937, Smith 1952 Tinker 1991, Walker & Rosenblatt 1988 Wall s 1975 Walters & Robins 1961) The point at which identifications were determined varied since at times the keying-out of specimens was difficult and therefore, tissue samples were first taken and frozen in order to preserve enzyme activity In other cases particularly when specimens were quite small (e .g., callionymids, gobiesocids) or when diagnostic features would be destroyed from tissue extraction (e g the illium/esca of antennarids or the neuromast patterns of chaunacids) identification was effected first. Chemicals All chemicals used for this study were of reagent grade or higher and purchased from Sigma Chemical Co., St. Louis Missouri unless specified otherwise (see Appendix 1 for a complete list of specific chemicals used and Appendix 2 for recipes to electrophoretic buffer systems grinding buffer tracking dye histochemical staining solutions and buffer, starch gels, gel fixer etc.)


35 Preparation of Tissue Extracts Tissues (liver, muscle, heart eye and brain) were dissected from specimens, placed in separate plastic centrifugation tubes, and immediately frozen. Muscle tissue was white skeletal (epaxial) removed from the back region (left side) near the dorsal fin ; eye tissue consisted of the whole eye for smaller specim e ns and the retinal (colored) tissue onl y if the specimen s size allo w ed dissection of the eyeball and recovery of retinal tissue It has been shown that the time of first expression ofLDH isozymes in tissues corresponds to the time of functional differentiat ion of the tissue (Whitt 1984). Thus for example, the time of first LDH-C appearance in developing bonefish (Albula vulpes ) corresponds to the time of differentiation of the retina (Pfeiler & Vrijenhoek 1988). Since all specimens used in this study were past the time of tissue differentiation, it was assumed that age of ind i viduals had no bearing on isozyme expression In preparation for electrophoresis, tissues were manually homogenized in the centrifugation tubes by mashing with a spatula in an approx i mately 1 : I v/v ratio of chilled 0.1 M potassium phosphate (pH 7.0) grinding buffer (Wilson et al. 1990) Extracts were centrifuged at 4 C for about five minutes at 10,000 x g in an RC-SC automatic superspeed refrigerated centrifuge (Sorvall Instruments) supernatants recovered and then either kept at 4 C for immediate electrophoresis or refrozen at 87 C for later use (Wilson 1994) With small specimens the quantity oftissue recovered was so limited so as to render centrifugation and collection of supernatant an unuseful exercise ; with these


the raw homogenized extracts were used immediately in order to obtain sufficient enzyme activity Electrophoresis 36 Horizontal starch gel electrophoresis of tissue extracts was performed on 12% Sigma starch gels and buffers, carried out at 4-6 C in a chromatography refrigerator for 4-10 hours, depending on the buffer system, according to established procedures (e.g. Murphy et al. 1990). Three buffer systems were used for these experiments : discontinuous EDT A-boric acid-Tris, pH 8 6 (EBT; with modifications from Boyer et al. 1963, Shaklee et al. 1973, Shaklee & Keenan 1986, Murphy et al. 1990); discontinuous Tris-citric acid, pH 6 9 (TC 6.9 ; with modifications from Whitt 1970a, Rainboth & Whitt 1974); and Tris-citric acid, pH 8 0 (TC 8 .0; with modifications from Shaw & Prasad 1970, Selander et al. 1971) After electrophoresis was complete, gels were sliced, and stained histochemicall y using established recipes (Shaw & Prasad 1970, Selander et al. 1971, Buth & Murphy 1990 ; see Appendix 3 for protocol). These were incubated in the dark at 37 C (e.g., Shaklee et al. 1973) in a Tempcon Oven (American Scientific Products) for various lengths of time (5 minutes to 2 hours) depending on strength of resulting bands (i e., level of exhibited enzyme activity), and sometimes left to continue staining for longer periods (e g., 12-36 hours) in the dark at room temperature (in lab drawers). In order to v erif y the


37 specificity of the staining reaction, control slices were stained in the usual manner but without the addition of the substrate (i e to control for nonspecific reductases or "nothing dehydrogenases"; cf Markert & Faulhaber 1965, Shaklee et al. 1973, Shaklee & Whitt 1981, Shaw & Koen 1965) When bands were sufficiently resolved the reaction was terminated by decanting off the staining solution, washing the gel gently with DI H20, and treating the gel in fixing solution (Murphy et al. 1990) for 24 hours Finally, gels were dried off and wrapped in Cellophane plastic for storage Thermolability Thermolability of the various isozymes ofLDH were investigated according to the paradigm of Whitt (1970a) In order to obtain enough activity of the tissues (of eye in particular) for the analysis, several individuals of the species to be investigated were run in the regular manner to look at mobilities ofthe isozymes coded by the -A*, -B*, andC* loci. If these were found to be identical, tissues could be pooled For the analysis, eye, brain, liver, and heart were pooled. These were homogenized manually in a centrifuge tube in approximately 1:1 v/v of grinding buffer (circa 1 ml) This was then spun down at 10,000 x g for 5 minutes at 4 C The resulting supernatant was pipetted off (ca 0 7 ml), and from this, 45 111 aliquots were added to centrifugation tubes marked corresponding to their times to be incubated (0 s, 15 s, 30 s, 45 s, 1 min, 2, 3 4, 5, 10, 15, 20, 25, and 30 min .).


38 The aliquots so marked were then incubated at 70 C in a Model 183 water bath (Precision Scientific) for these lengths oftime, removed, and stored on ice until the last aliquot had been treated From these aliquots a 5 111 of sample was added directl y to a wick of filter paper lying flat on a paper towel and subsequentl y loaded directly onto a gel for electrophoresis on EBT 8 6 for 4 hours In order to mainta i n as nearly as possible the identical volume of extract on each wick for electrophoresis these were not blotted on a paper towel to remove excess extract as is routinely done otherwise Prior to pooling of the tissues wicks dipped in the extracts of liver muscle heart eye and brain of one individual is loaded onto the gel in the first five slots in order to help in determin ing ident i ty of the allozymes after staining Otolith Preparation For R raninus (typical gadoid) and all gobiesocids one of each pair of otoliths the sagitta (saccular) asteriscus (utricular), and lapillus (utricular) were removed from specimens to be examined using a dissecting microscope Subsequently, only sagittae were used due to lack of comparative material for the other two pairs (cf Nolf 1985 Nolf & Steurbaut 1989a b) Otoliths were then cleaned for 30 seconds in bleach (5 25% sodium hypochlorite) and rinsed first in water and then 95% ethanol (Crabtree et al. 1996).


39 For otolith morphology (shape) comparisons dried whole otoliths were examined without further preparation For age determinations three or four thin sections (approximately 0 5 mm) were cut in order that one encompass the otolith core using a Buehler Isomet low-speed saw with a diamond blade These sections were then mounted onto a microscope slide with Histomount for examination and annuli counting using transmitted light and a compound microscope (1 Ox power) Documentation Electrophoresis gels were initially photographed using a 35 mm Minolta camera and a wide-angle lens with Kodachrome 200 color film The developed pictures were later scanned into computer files using a Hewlett Packard Photosmart photograph scanner. Later, gels were scanned directly with Deskscan ll using a Hewlett Packard ScanJet Tic. Images of sectioned and whole otoliths were acquired using a CCD Video Camera (CV-M50) mounted on a Zeiss dissecting microscope and using Optimas 5 2 software. These images were then printed using either a Hewlett Packard DeskJet 660C color printer or a Sony Color Video Printer.


40 3.RESULTS LDH-C Expression of Acanthopterygians Acanthopterygians have been reported to express the fast eye-predominant form ofLDH-C4 (e.g., Shaklee 1972, Markert et al. 1975) This pattern is demonstrated clearly by selected scorpaeniforms (Fig 5) and perciforms (Fig. 6). Muscle, heart, eye, and brain display (predominant in muscle) and LDH-B 4 (predominant in heart) to varying degrees, while liver exhibits no LDH-C activity, and little LDH activity overalL Thus, the pattern ofhomotetramer isozyme mobilities (relative anodal mobility, RAM, of Shaklee 1972, Shaklee et al. 1973, Markert et al. 1975) can be expressed as C>B>A (i.e., LDH-C fastest/most anodal). [In reading gel scans, note that the anode (positive pole) is always at the top of the gel, the cathode at the bottom. The origin is where the filter paper wicks containing extracts are loaded. In Fig. 5 as in all gels, the tissues are loaded in the order : liver, muscle, heart, eye, and brain unless otherwise noted (see labeling) Arrows indicate, from top to bottom, the fast eye-predominant LDH-C4 homotetramer, the -B homotetramer, the A2B2 heterotetramer, and closest to the origin the homotetramer. Note also that in the brain column, though not labeled, is a -BC heterotetramere (this band is probably B 2 C 2 ; usually charges on the polypeptide chains are additive, resulting in evenly spaced heterotetramers, but post-translational events may occasionally affect


41 Figure 5 Acanthopterygian patternLDH Isozymes of Scorpaeniformes. Specimens are of Scorpaena braziliensis (slots 1-5, 6-10 ; two individuals) and Prionotus scitulus (slots 11-15) ; tissue extracts for each specimen were loaded in the order liver, muscle, heart eye and brain Note the distinct fast LDH-C present in the eye slots of each individual (somewhat weaker in brain) and the absence of any cathodal or slow liver bands


42 u u 0 Figure 6. Acanthopterygian Pattern-LDH Isozymes ofPerciformes. Specimens are of Lagodon rhomboides (slots 1-5), Orthopristis chrysoptera (slots 6-10), and Chae todipterus faber (slots 11-15); tissue extra cts for each specimen were loaded in the order liver, muscle, heart eye, and brain. As with scorpaeniforms, note fast LDH-C present in eye (and brain) and the absence of slow/cathodal l iver bands


43 relative band position [Richardson et al. 1986]) The first two groups of extracts are of Scorpaena braziliensis while the third group is of Prionotus scitulus; note that the mobilities of the various isozymes are somewhat different between the two species All loci will not be labeled on the gel scans following this one, but see accompanying text for clarifications.] LDH-C Expression of Raniceps raninus Raniceps clearly expresses the typical gadiform pattern (Fig 7) LDH-C is compartmentalized predominantly in liver; the other tissues (muscle, heart eye, and brain) display LDH-A (predominant in muscle; most anodal) and LDH-B (predominant in heart; intermediate mobility). There is no evidence of a highly anodal, "eye-specific" LDH-C in any of the three specimens. Thus, Raniceps' pattern ofhomotetramer isozyme mobilities can be expressed as A>B>C. Lactate dehydrogenase activity in many tissues is evidently weak Staining times (i e ., the time the substrate and histological staining solution is allowed to interact with the enzyme of interest on the gel) impacts dramatically the visualization of bands where enzyme activity is weak (Figs. 8-10) These images, along with Fig 7, provide a staining time-line: these are 30 minutes, 90 minutes, 3 hours, and 18 hours, respectively After 90 minutes, slight LDH-C activity can be seen also in heart tissue ofRr002 (Fig. 8) and is very evident in heart (all individuals) and eye (Rr001, 003) after 18 hours (Fig 10) In


Figure 7 LDH Isozymes of Three Specimens of the Tadpo le Cod Raniceps raninus Electrophoresis was for 4 hours 20 minutes on EBT 8 .6. Tissue extracts for each specimen (in order: RrOOl-003) were loaded in the order liver muscle heart, eye, and brain Note the distinct slow LDH-C present in the liver slo ts of each individual and the absence of any highly anodal eye bands. 44


Figure 8. Slice fromRaniceps Gel as in Fig. 7, after 90 Minutes Staining Time. Note slight LDH-C activity visualized in heart column ofRr002. 45


46 Figure 9. Raniceps Isozyme Visualization after Three Hours Stain i ng Time. Same s l ice as above (Figs. 7, 8); note the clear visualization of the A2B2 heterotetramers


Figure 10. Second Slice of Raniceps Gel, Allowed to Stain for 18 Hours at Room Temperature. See methods. Note the clear visualization ofLDH-C activity in heart and eye tissues in addition to liver. Also note the limited visualization of the A-B heterotetramers 47


48 this latter picture, it is also evident that there exists substantial LDH-B, and somewhat less LDH-A acti vi t y in eye extract a fact no t readily discernable from an initial analysis of the gel after 30 minutes Furthermore, heterotetrameres of -A and -B (A2B2 ) also become more clearly visualized with sta i ning time (e g., Fig. 9), but there exists an upper limit when vis ualization deteriorates (Fig 10 ) LDH-C Expression of Other Gadiforms Other gadiforms investigated (Co e lorhyn c hus sp., Nezumia bairdii, and Urophycisfloridana) show the typical pattern for this group of fishes (Fig 11) These representatives clearly demonstrate the slow LDH-C compartmentalized predominan tly in the liver but present to lesser degrees in other tissues (here, visualized most clearly in the heart) and absolutely no indication of a highly anodal eye band (as has been shown by Shaklee 1972, Markert et al. 1975, and Shaklee & Whitt 1981 for a wide array of gadiforms) Activity of -A and -B is characteristically weak in th is latter t issue For Coelorhynchus and Urophycis RAM pattern is B > A > C ; for Nezumia it is A > B > C (Fig 11) As with Ranic eps, A2B2 heterotetrameres are visualized clearly


4 9 A. B. Figure 11. LDH Isozyme Patterns ofRepresentative Gadiforms : Coelor h y n chus sp ., N e zumia bairdii and Urophyci s floridana Slots 1-5 6-10 and 111 5 re spectiv e ly; ti s sue order as above : liver muscle, heart, eye and b rain Specimens w e r e run o n E B T 8 6 for 4 hours 15 minutes A slice one ; B back slice of same st ained s o m e wha t longer (presented here flipped horizontally with imaging program in ord e r t o m a i n tain c onstan t tissue order for simplicity) Note slow (in Coelorhyn c hus and U roph y ci s, ca t h o dal ) l iv er predominant LDH-C and especially (in Fig 11B) the -C acti vity a l so manife sti n g in heart of each individual all different in mobilit y fr om e ac h oth e r y e t o f iden ti c al mobility to that visualized in the liver of each indi v idual. Other Paracanthopterygians: LDH-C Expression of Percopsiformes The lactate deh y drogenase pattern of a percopsif o rm Aphre d o d e ru s say an u s is s hown i n Fig 12. This species expresses both f orms (isoz y me s) ofLDH-C ( i .e., du a l express e s)


50 u u Figure 12. Dual expression (co-expression) of the Two Forms ofLDH-C4 in the Pirate Perch, Aphredoderus sayanus. AsOOl, 002 ; tissue order: liv er, muscle he a rt, eye brain. Electropporesis w as with EBT 8 6 for 4 hours 40 minutes Note the nearl y equal levels of C activity expressed in the liver and eye and the lack of any slow-C" a c tivity in the eye


51 very clearly, with the e v ident activity level approximately equal in liver and eye tissue No "slow-C' is evident in any other tissue, though fast-C" is also present in brain. LDH A and-B are very nearly isomobile with -A being slightly more negatively charged under these conditions (i .e., pH 8.6) and running somewhat faster towards the anode Liver also displays some -B activity but no A In muscle, only -A is expressed. Heterotetramer formation between -A and -B cannot be evaluated since the two homomers are so close together. The spacing of those consisting partly of -C subunits is not readily interpretable as being from combinations with strictly either -A or -B. The RAM pattern for Aphredoderus is thus C > A > B > C LDH-C Expression of Ophidiiformes (Superorder Paracanthopterygii) Two species of ophidiiforms Ophidion holbrooki and Lepophidium cervinum, were investigated and their patterns are rendered i n Fig 13A, B These two representatives of the suborder Ophidioidei show the pattern of lactate dehydrogenase common in higher fishes; namely, fast LDH-C compartmentalized in the neural tissues eye and brain In liver there is only min i mal LDH activity this being LDH-A ; there is no i ndication in either species of the slow form of -C. For Ophidion the -A homomer is substantially more negative than -B which is nearly isoelectric (barely off the origin i n the anodal direction) at pH 8 6 All heterotetramers expected are plainly visible in brain


52 tissue. Lepophidium displays much less difference in mobility between these same homomeres, but the same relative mobilities; their RAM pattern is the same : C > A>B. Figure 13. Lactate Dehydrogenase Isozyme Patterns of the Ophidioids Ophidion holbroold and Lepophidium cervinum. A. Slots 1-5 and 6 10, are 0. holbroold and L. cervinum, respectively; tissue order as before (L,M,H,E,B). Electrophoresis was on EBT 8.6 for 4 hours, 15 minutes. B. 0. holbroold; electrophoresis on EBT 8.6 for 4 hours 10 minutes Note clearly expressed fast LDH-C in eye extracts of both representatives of this order, also expressed in brain, and the complete absence of slow LDH-C in all tissues For Ophidion, the -B4 isozyme is almost isoelectric at this pH and barely moves off the origin.


53 LDH-C Expression of the Lophiiformes, Suborder Lophioidei (Paracanthopterygii) Though Lophius americanus proved ambiguous (Fig. 14), the L. gastrophysus allowed the general pattern to be discerned (Fig. 15). These two representatives of the suborder share a pattern of A faster than B and A-B heterotetramer activity equal to, or greater than, B4 homo mer activity. In L. gastrophysus, none of the three specimens display the A1B3 heteropolymer, thus leading to a fourbanded pattern This is also seen in L. americanus, though it is impossible to say that it is due to the lack ofthe AtBJ heterotetramer since those that do show activity are equidistant. For L. americanus, there is no indication of slow liver or fast eye LDH -C activity, but for L. gastrophysus there is Figure 14 Lactate Dehydrogenase Pattern of the Goosefish Lophius americanus Run on EBT 8 6 for 4 tissue order : L,M,H,E,B. Note the absence of any LDH-C activity in either liver or eye, and the unusual four-banded heterotetramer pattern.


54 0 Figure 15. LDH Pattern o f the Blackfin Goosefish, Lophius gastrophysus. Run on EBT 8 6 for 5 hours 45 minutes ; (Lg 001-003 ; tissue order : L M,H,E ,B). Note the A-C heterotetramer bands in eye ofLg003 and the total absence of any s l ow LDHC i n any tissues of any specimens Note also the absence of the A1B3 hetero t etramer i n all specimens, resulting in the unusual four banded pattern seen also i n L. americ anus abo v e


55 Although the gel was run somewhat too long (5 hours 45 minutes) it is clear from Lg003 that A-C heteromers are present in the eye tissue ofthat individual and that the RAM pattern for these fish can be represented as C>A>B. LDH-C Expression for Lophiiformes, Suborder Antennarioidei (Paracanthopterygii) Three species from two antennarioid genera were examined and their LDH allozyme pattern is shown in Fig. 16 As with the Aphredoderioidei (Percopsiformes), these fish show dual express the two types ofLDH-C clearly, and their relative activity levels appear to be similar. Slow -C is present predominantly in the liver but is also present in heart tissue of all three representatives Fast-Cis expressed in both the eye and brain extracts. In no single tissue are both forms ofLDH-C co-expressed together Interestingly, Antennarius striatus differs from the other two antennarids in expressing little B4 activity in the heart is expressed there while eye and brain show clear B4 bands) A. biocellatus and Histrio histrio, on the other hand, show normal NB tissue distributions The general RAM pattern of this suborder would be represented by C>B>A>C


56 u co<( 0 Figure 16. LDH of the Antennarioids Antennarius striatus, A. biocellatus, and Histrio histrio Slots 1-5, 6-10, and 11-15, respectively; tissue order: L M,H,E,B Electrophoresis was carried out on EBT 8 6 for 4 hours Note the clear dual expression of liver-predominant slow LDH-C, and the fast eye-predominant LDH-C Slow-C a l so expressed in heart; fast C also expressed in brain


57 LDH-C Expression in Lophiiformes, Suborder Chaunacoidei (Paracanthopterygii) As with L. americanus above, the pattern ofLDH expression for Chaunax suttkusi is difficult to decipher (Fig 17) It is atypical in that Chaunax does not display fast eye predominant LDH-C. Nor is there any evidence of a slow liver locus either If the slow locus is B, this taxon would have a pattern quite similar to Lophius, with the exception of the heteropolymers Without visualization of the C-locus, its RAM pattern is unknown LDH-C Expression in Lophiiformes, Suborders Ogcocephaloidei + Ceratioidei (Paracanthopterygii) Batfish were chosen as a representative of this group and run on two separate gels (Figs 18A, B). Ogcocephaloids show a characteristic fast eye-predominant LDH-C and no indication of the slow -C. and B4 homotetramers are very nearly isomobile, with A slightly anodal ofB. The pattern ofRAM for this taxon, therefore, is C > A >B. LDH-C Expression of the Batrachoidiformes (Paracanthopterygii) Five established species ( Opsanus beta, 0 pardus 0 tau Porichthys myriaster P notatus) and one taxon whose species status remains undetermined (0. sp .) in the


58 Figure 17. LDH Pattern for Three Indiv iduals of the Chaunacoid Chaunax suttkusi. Cs001-003 in slots 1-5, 6-10, and 11-15, respectively ; electroph o resed on EBT 8 6 for 4 hours 3 5 minutes Note lack of e v idence for either fast or slow LDH-C b ands Rela tive positions of A and B difficult to detennine also


59 A. B c Figure 18. Lactate Dehydrogenase Isozyme Pattern of the Polka-dot batfish, Ogcocephalus radiatus (Ogcocephaloidei) A 0. radiatus (Or0 0 3) run for 4 hours on EBT 8 6; B 0. radiatus (Or009) run for 4 hours 10 minutes Note fast LDH-C present in eye, and no slow -C expressed in any tissue. monofamilial Batrachoidiformes were investigated All representati v es ofthe subfamil y Batrachoidinae (Opsanus) very clearly co-expressed both forms ofLDH-C with the fast form in eye and brain, the slow in liver (predominantly) but some activity also in heart (Figs 19, 20) The RAM pattern of these fish is C > A > B > C The two representatives of the subfamily Porichthyinae also showed dual expression but much less clearly so (Figs 21-24) In these fish, activit y of the slow, liver dominant LDH-C has been almost entirely lost (though B is expressed) Cis expressed cathodally, but so faintly so as to be barely vis i ble (a third ge l was run and intentionall y


0 0 Figure 19. LDH Pattern of Two Batrachiodiforms of the Subfamily Batrachoidinae, the Leopard Toadfish (Opsanus pardus) and the Oyster Toadfish (0. tau). Electrophoresis was for 4 hours 45 minutes on EBT 8.6 (slots 1-5 Opp004, 6-10 Opp005, 11-15 Ot001; tissue order L,M,H,E,B) Note the fast LDH-C expressed in eye and brain as well as the slow -C expressed predominantly in liver but also visualized weakly in heart (especially Opp004, 005). 60


0 <( co 0 Figure 20 Lactate Dehydrogenase Pattern of the GulfToadfish (Opsanus beta) and the "Orange Toadfish" (0. sp ) Subfamily Batrachoidinae Gel was run on EBT 8 6 buffer s y stem for 4 hours (slots 1-5 Ob009 6-10 Ob010 11-15 Ob011 16-20 Osp001 ; tissue order : L M,H,E,B) Note same pattern ofLDH expression as with other species of Opsanus in Fig. 19 (fast LDH-C in eye brain ; slow -C in liver possibly also heart of Ob009) 61


62 Figure 21. Electrophoretic Results of the Batrachoidiform Porichthys myriaster, Subfamily Porichthyinae Pm001-004 ; tissue order : L M,H,E B ; run on EBT 8.6 for 5 hours, 15 minutes Note very faint slow LDH-C bands (cathodally) under liver and heart columns, and clear expression of fast eye (and brain) -C (ran to anode).


63 Figure 22. LDH Patterns of Further Porichthys myriaster Liver Eye Extract Comparisons Run on EBT 8 6 for 7 hours 45 minutes The first five columns represent L M,H,E, and B the remaining columns (6-21) are liver (first) and eye (second) alternating for remainingP myriaster specimens (Pm 005 012) Note faint l ive r slow LDH-C bands especially evident in columns 8, 10, 12, 14, and 16. Fast eye LDHC stains sharply under these same conditions due to many-fold greater activity leve l s (ran to anode)


64 Figure 23 Intentionally Over stained Gel of Porichthys myriaster Gel run shorter ( 4 hours) and intentionally over-stained in order to establish presence of slow liver dominant LDH-C (slots 1-5 are Pm003, 6-10 Pm006, 11-15 Pm007 and 16-20 Pm012 ; tissue order: L,M,H,E,B). Note distinct cathodal LDH-C bands vis ualized for Pm006,007, and 012. Gel buffer system: EBT 8 6


65 Figure 24. Electrophoretic Results of the Batrachoidiform Pori c hthys notatus, Subfamil y Porichthyinae Pn001-004 ; tissue order : L M,H,E,B; run on EBT 8.6 for 4 hours, 15 minutes As with P myriaster, note very faint bands cathodally for slow LDHC, especially evident under liver column for Pn002 (column 6), and liver and heart for 004 (column 16 and 18) Note also anomalous A-fastC heterotetramer pattern for Pn002 (four-band pattern as seen in Lophioids for A-B heteromers ).


66 over-stained in order to establish with certainty the presence of these bands; see Fig. 23) On the other hand fast eye-dominant LDH-C expresses very clearly with high activity levels in all specimens examined The RAM pattern for this taxon is the same as for the Batrachoidinae; i.e., C>A > B>C LDH-C Expression of the Suborder Gobiesocoidei (Superorder Acanthopterygii sensu Nelson 1994) Three individuals of a representative species of the Gobiesocoidei, Gobiesox strumoStis, were run on EBT 8 .6. These fish displayed the classic gadiform pattern of LDH-C allozyme (Fig 25) with prominent slow, liver-dominant LDH-C, and no indication of any fast, eye-dominant -C fonn in either eye or brain extracts. Additionally, something visualized cathodally of the locus in the eye column of Gs004 though well anodal ofLDH-C (this was not seen in the other specimens and remains unexplained) A second species Gobiesox sp. demonstrated the same pattern (not shown) The RAM pattern of Gobiesox, then, is B> A>C.


67 co <( u Figure 25. Lactate Dehydrogenase Patterns of Three Specimens of the Skilletfish Gobiesox strumosus (Gobiesocoidei). Run on EBT 8 6 for 4 hours 35 minutes (Gs002004 ; tissue order: L,M,H,E,B) Note the clear slow LDH-C in liver, and the complete absence of any fast -C activity in any tissue Note also the unexplained band just cathodal in eye column ofGs004.


68 LDH-C Expression of the Suborder Callionymoidei (Superorder Acanthopterygii) Three species of callionymoids were investigated to see if their pattern of lactate dehydrogenase expression would correlate with Gobiesox or with the standard pattern of Perciformes and higher teleosts (i e ., acanthopterygians) The three representatives of this taxon Pterosynchiropus picturatus P splendidus and Neosynchiropus ocellatus (sometimes all placed in the common genus Synchiropus ; cf Fricke 1982 1983) clearly displayed the common pattern of higher teleosts with no indication of slow, liver dominant LDH-C but rather the very negatively-charged fast LDH-C variant (Fig. 26). The and B4 homomers are similar in their mobilities, with A moving somewhat more anodally Interestingly P p i cturatus demonstrates no LDH activity at all in the heart, and no LDH-B activity in any tissue, a pattern distinctly different from the other two species whose patterns are similar except for a different allele at the fast LDH-C locus As with the pattern seen for Antennarius striatus (Fig 16), these latter two species exhibit only LDH-A in the heart, while -B allozyme act i vity is restricted to the eye and brain (neural) tissues (Antennarius is different from these taxa, however, by co-expressing the two LDH-Cs and having reverse A-B mobilities) These observations may indicate a closer relationship between P splendidus and Neosynchiropus than either has to P picturatus The RAM for the group as indicated by these representatives is : C > A>B RAM patterns for all investigated taxa are summarized in Table 3


69 u Figure 26. LDH Patterns of Pterosynchiropus picturatus, P. splendidus, and Neosynchiropus ocellatus (Suborder Callionymoidei) Slots 1 -5 SpOOl 6-10 PsOOl, 11-15 order : run on EBT 8 6 for 4 hours. These individuals display the common pattern of perciforms and other higher teleosts with no slow LDH-C acti v ity in liver but prominent fast eye-dominant -C (also evident in brain) Note also divergent pattern of expression between P. picturatus on the one hand and the other two taxa on the other


70 Table 3. L actate Dehydro g enase Pattern o f E x press i on of Selected Taxa Expression of fast eye predom i nant slow live r -predom i nant LDH C forms dua l express i on and re l ative anodal movement (RAM) pattern of LDH-A, 8 and C homotetramers Taxonom i c order as i n Table 1 (i.e., sensu Ne l son 1994 w i th mod i f i cations but see Gobiesociformes i n discussion LDH-C Expression Pattern Species RAM li ver form eye for m Superorde r Paracanthopterygii O r der Percops i formes Aphredoderus sayan us C>A>B>C X X O r der Oph i d ii formes Lepophidium cervinum C>A>B X Ophidion holbrooki C>A>B X Order Gadiformes Suborder Ranicipitoidei Raniceps raninus A>B>C X Coelorhynchus sp. B>A>C X Nezumia bairdi A>B>C X Urophycis floridana B>A>C X Order Batrachoid i fo r mes Opsanus beta C>A>B>C X X Opsanus pardus C>A>B>C X X Opsanussp. C>A>B>C X X Opsanus tau C>A>B>C X X Porichthys myriaster C>A>B>C X* X Porichthys notatus C>A>B>C X* X Order Lophiiformes Suborder Lophio i de i Lophius gastrophysus C>A>B X Suborder Antennario i dei Antennarius biocellatus C>B>A > C X X Antennarius striatus C>B>A>C X X Histrio histrio C > B>A>C X X Suborder C h aunaco i de i Chaunax suttkusi ? ? ? Suborder Ogcocephalo i dei Ogcocepha/us radiatus C>A>B X cont i nued on next page


71 Table 3 (Continued) LDH-C Expression Pattern Species RAM liver form eye form Superorder Acanthopterygii Order Scorpaeniformes Scorpaena braziliensis C>B>A X Prionotus scitulus C>B>A X Order Perciformes Suborder Perco i dei Lagodon rhomboides C>B>A X Orthopristis chrysoptera C>B>A X Chaetodipterus faber C>B>A X Suborder Gobiesocoidei Gobiesox sp. B>A>C X Gobiesox strumosus B>A>C X Suborder Callionymoidei Neosynchiropus ace/latus C>A>B X Pterosynchiropus picturatus C>A>B X Pterosynchiropus C>A>B X sp/endidus Suborder Scombroidei Scomberomorus maculatus C>B>A X Order Pleuronectiformes Suborder Pleuronectoidei Anclyopsetta quadrocelata C>B=A? X Order T etraodontiformes Suborder T etraodontoidei Chiolomycterus schoepfi C>B=A? X very weakly expressed in Porichthys


72 Analysis of Dual Expression: Test for "Nothing Dehydrogenases" Since dual expression of "fast-eye"and "slow-liver" -predominant forms of LDH-C4 isozymes has essentially not been reported before (but see Holt & Leibel 1987) and was completely unexpected the question therefore arises as to whether either band is the result of non-specific staining Control for nonspecific reducta s es or "nothing dehydrogenases" revealed that the LDH bands visualized were not the product of alcohol dehydrogenases which may sometimes stain to some degree even in the absence of alcohol substrate (cf Markert & Faulhaber 1965 ; Shaklee & Whitt 1981) or the result of other enzyme systems (Figs 27A, B) While the top slice had all the components of the staining mixture added, the bottom (back) slice had everything but the lactic acid (1.0 M Lithium lactate pH 8 0 ; see Appendix 2 for recipes) added and was incubated under the same conditions and for the same length of time as the top slice (note that the first two fish are lophiiforms ofFig. 17B and do not show dual expression while the next three are Opsanus beta specimensOb001 004 005 not shown elsewherewhich dual express both forms ofLDH-C). This was particularly important to establish for the dual expressing slow liver-dominant and fast eye-dominant LDH-C bands of the enzyme system Though these bands do not visualize without the substrate they can be m a de to visualize subsequently by "back-adding" substrate and i n cubating further These controls were carried out on several g e ls of different taxa throughout the study with the same (negative) results When incubated for an extreme period oftime (2 hours+), certain


Figure 27 A. Test for "Nothing Dehydrogenases" ; Substrate Added. Gel with two lophiiforms (OpOOl, 002) and the batrachoidiforms (ObOOl, 004 005) stained for LDH in the normal way (see Appendix 3) Specimens electrophoresed on EBT 8.6 for 6 hours 55 minutes. Note dual expression of slow and fast LDH-C bands in Opsanus (00 1, 004 005; fast -C to anode) 73


Figure 27B Test for "Nothing Substrate Omitted Top (back) slice of gel in Fig. 27A, stained the same way, except for the omission of the substrate (lactate) ; incubated in the same manner and for the same length of time as p r evious slice Note the complete absence of activity of all bands including the dual expressed LDH-Cs. 74


75 other faint bands could be produced ; these were demonstrated to be identical to those produced on the same gel when stained for ADH (not shown) These bands did not display identical mobilities to LDH bands observed, and were quite faint. They were not visualized during normal incubation! staining regimens (see Appendix 3). Thermolability Analysis If the dual expressing bands are indeed real, a second possibility exists; namely, that they are not "dual" expressions of one locus (i.e LDH-C*) at all but rather the products of two separate loci, one of which may be LDH-C* and the other a new locus (this was indeed suggested by Holt & Leibel [1987] to account for observations with their cichlids) Comparing thermolability profiles of enzymes has been one way in which these types of questions have been investigated (e.g. Whitt 1970a, Shaklee 1972) Since the mobilities ofLDH-A, -B, and -C isozymes for Ob009, 010, and 011 were identical for all loci (see Fig. 20), eye, liver, brain, and heart from these fish were pooled for the purposes of this procedure. When pooled tissue homogenates incubated for differing lengths oftime were electrophoresed, d i fferences in the isozymes of lactate dehydrogenase with respect to temperature sensitivity became apparent (Figs 28A, B). With regard to the LDH gene products it can be seen from the gel that:


Figure 28A. Comparison ofLDH Allozyme Thermo lability of Opsanus beta Run on EBT 8.6 for 4 hours 5 minutes. Tissue order : Ob011 L,M,H, E,B (slots 1-5 ) pooled extract incubated 0 sec 15 s, 30 s, 45 s, 1 min., 2 3 4 5 10,15, 20,25 and 30 min (slots 6-19) See text for conclusions. A. Bottom slice, B. Top (back) slice, flipped (mirror image) in order to preserve tissue order next page) Both slices are presented since proximity of superoxide dismutase bands (non-staining white bands ) and glare make reading final several fast LDH-C bands difficult. 76


77 Figure 28B. Comparison ofLDH Allozyme Thermo lability of Opsanus beta ; Back Slice See previous page (Fig 28A) for details


--Fast eye-predominant LDH-C is the most stable, still displaying some activity at 30 minutes. --At is almost as stable as fast C4; there is no activity in slot 12 (after 20 minutes). --B4 is quite thermolabile, denaturing and showing no activity after just 2-3 minutes 78 -Slow liver-predominant-Cis also thermolabile, denaturing as B 4 at approximately 2-3 minutes. Thus fast-Cis similar to A, and slow-Cis similar to B with respect to thermolability Search For LDH-C Heterozygotes Perhaps the most direct way of determining the identity or distinctness of these isozyme forms is to find a heterozygote for the locus (i.e LDH-C*) If the visualized bands are products of one locus then the heterozygote type banding pattern (i e., a five banded pattern resulting from random association of four polypeptide chains according to the binomial distribution) should be demonstrated by both the slow form in liver extract and the fast form in eye extract. Conversely, if these forms are encoded by two different loci, a heterozygous condition at one locus would presumably have no bearing on the banding pattern at the other. Either way identification of an individual heterozygous at LDH-C* would answer the question (theoretically, of course, there is the possibility that if these indeed were products of two distinct loci, an individual may be found who happens by chance to be heterozygous at both loci; the odds of this are low, but


79 observation of such a result [i. e demonstration of the heterozygous 5-banded pattern for both the liverand eye-predominant forms] would therefore not eliminate the possibility of two distinct loci encoding for the two forms) The problem with this line of invest i gation i s that heterozygotes are rare Liver and eye extracts for 50 0 beta specimens were run but no individuals heterozygous for either form were identified Additionally 54 P notatus specimens were investigated but as was mentioned under LDH-C Expression of the Batrachoidiformes, liver predominant expression was so weak as to make meaningful evaluation of homozygotes / heterozygotes impossible ; there were no heterozygotes of the eye-predominant form observed Otolith Examinations Sagittae of three specimens of Gobiesox strumosus were removed and cleaned i n order that they might be compared to the general i zed gadiform type described b y Nolf & Steurbaut (1989a) and developed from a wide survey of otolith forms (e g. Nolf 1978a, b c 1993). This was done since it was a another angle from which to evaluate gobiesocid relationships given the results ofLDH-C expression detailed above According to Nolf & Steurbaut ( 1989a) the otoliths of gob iesocids are small and are not useful in determining relationships due to lack of diagnostic features This conclusion in tum, was based on Nolfs (1985) analysis of just one gobiesocid species Dipl ec o g aster bim a culata Nolf &


80 Steurbaut's conclusions seem congruent with those evident from the sagittae obtained from the three specimens examined here (Fig. 29). By comparison, an otolith from Raniceps (Fig 30) displays many of the generalized gadiform features (Nolf & Steurbaut 1989c) -cauda well developed in both the ostium and cauda (on the ostial side extending to the rim, but not opening to it), pince-nez-shaped sulcus, and a central collicular crest just above the ostium-cauda junction of the crista inferior ( cf Nolf & Steurbaut 1989a) Age Determination of Raniceps This specimen (Rr002) displayed well-defined and easily readable annuli (Fig 31 ) The seventh annual increment is on the margin, thus the individual is 7 years old. It was captured on the 12th of April near Kristineberg, Sweden and measured 207 mm SL. Thus, tadpole cod seem to be moderately long-lived, slow-growing fishes


Figure 29 Sagittae of the Skilletfish, Gobi esox strumosus. Upper left= Gs004 (right sagitta) ; upper right= Gs002 (right sagitta) ; lower= Gs003 (left sagitta) Note lack of generalized gadiform features (cf Raniceps otolith Fig 30) 81


Figure 30 Sagitta of the Tadpole Cod, Rani ceps raninus Rr002 ; displaying many of the generalized gadiform features (cf Nolf & Steurbaut 1989a, c). 82


83 Figure 31. Ageing of Raniceps raninus Rr002 : a seven year old, 207 mm SL tadpole cod captured on the 12th of April near Kristineberg Sweden.


84 4. DISCUSSION LDH Pattern of Expression as a Synapomorphy for the Gadiformes Markle (1989) placed Raniceps as the primitive sister group to all the other gadiforms Of the four putative synapomorphies identified for the Gadiformes by he and Patterson & Rosen (I 989), LDH expression was the only one which had not been evaluated for this critical species Raniceps clearly demonstrates the typical gadiform pattern as it has previously been reported for other members of the taxon (e g., Shaklee 1972, Sensabaugh & Kaplan 1972) Although Markle points out (p 85) that corroborating evidence for the position afforded Raniceps in his dendrogram can be found in the fossil otolith record as reported by Nolf & Steurbaut (1989b) others have suggested Muraenolepis as a candidate for this spot (e.g. Nolf & Steurbaut 1989b). Cohen (1984) says ofthe genus (p 262), "Muraenolepis is not obviously related to any other gadiform and appears to represent an ancient lineage." Unlike Raniceps, the X andY bones of the caudal skeleton are not present in Muraenolepis (or in many other gadiforms; see Introduction and Patterson & Rosen 1989) It would therefore be interesting to examine the LDH pattern of expression in this genus Unfortunately, I was unable to obtain a specimen as was Shaklee & Whitt (1981) for their overview of gadiform fishes. This taxon has a circum-Antarctic distribution and


85 representatives are hard to come by. Bev erly Dickson of the Portobello Marine Lab, New Zealand whose laboratory collects icefishes (Notothenioids) for the research ofDr. DeVries and many others, has yet to be successful in this endeavor ; it will necessarily have to be left for future investigations Until a species from this genus is tested LDH pattern cannot be absolutely upheld as a synapomorphy of the Gadiformes (and see also discussion under Gobiesociformes). Dual Expression of Slow Liver-predominant and Fast Eye-predominant LDH-C Forms in the Same Fish This phenomenon of dual expression of slow and fast forms ofLDHC was unexpected consider ing the numbers of species that have been screened for lactate dehydrogenase activity by other investigators (e g Shaklee 1972 Markert et al. 1975, Shaklee & Whitt 1981, etc. ) Possible explanations include 1) one or both of the -C bands visualized are artifacts of the staining process ; 2) one or the other of the bands represents a fourth heretofore undiscovered LDH locus or 3) this truly is dual expression of two very d i fferent forms of the same locus modified in one or the other by some post transcriptional or post translational agent(s). In order to investigate the first possibility, controls for "nothing dehydrogenases" and non-specific reductases were run treating gels the same way as was usually done, but omitting the specific substrate (in this case lactate) As stated in the results section, it


86 could not be demonstrated that the bands visualized were artifactual. The investigations suggest that they are indeed real and the result of the substrate being reduced from lactate to pyruvate The second possibility (no 2 above) would confront massive evidence to the contrary Shaklee (1972) demonstrated convincingly that the eye-predominant LDH-C although often highly anodal in its mobility actually is quite variable. It ranges on a continuum from highly anodal in perciforms to intermediate in an aulopiform to only slightly anodal in an osmeriform (Shaklee 1972) He also showed that both the "eye" and "liver" forms were in fact present in a wide range of tissues in addition to those where activity was greatest (that is, the slow "liver" form when present, was also found in many taxa to be expressed in a number of other tissue extracts, though usually not at the same level of activity; likewise with the fast eye "; never, however, were they found to co occur) This makes much more plausible the possibility that they are indeed products of the same locus. Several investigators have shown through immunochemical and catalytic comparisons that both the "eye" LDH-C and the "liver" LDH-C resemble the B4 homopolymer more closely than either does the ( cf Sensabaugh & Kaplan 1972 Whitt 1970a, Shaklee 1972) Other lines of evidence indicating their identity include thermolability profiles (e g Shaklee et al. 1973), and kinetic and physical likenesses (Markert et al. 1975)


87 Therefore, I choose to accept the most parsimonious alternative (no 3) and conclude that these are indeed dual e x pressions of products encoded by the same gene locus but modified differently in their respective tissues One can envision a host of mechanisms by which the identical gene product could exhibit great differences in a v ariety of physical and chemical properties The effec t s oftemperature on proteins have recei v ed substantial attention (Coppes & Somero 1990, Dahlhoff et al. 1990 Dietz & Somero 1993 Hofmann & Somero 1996 Holland et al. 1997 Somero 1995) and much work has been carried out lately on temperature effects on isozyme production and express ion (e g ., Segal & Crawford 1994, Lin & Somero 1995a b). Temperature induced alterations of isozyme patterns (i.e mobility differences) in acclimated fish have been known for some time (e g Baldwin & Hochachka 1970 Hochachka & Lewis 1970 Moon & Hochach.ka 1972a, b ; though this is not universal ; cf. Wilson et al. 1975). Temperature has been shown to produce differential folding shapes or "conformers of the same proteins which in tum impact a variety of chemical (e g protein surface hydrophobicity) and physical (e g protein stab i lity) characteristics (Koti k & Zuber 1992 ) and lactate dehydrogenase folding i ntermediates have recently been v i sualized by 19F NMR spectroscopy (Sun et al 1996). Chaperones or chaperonins are a class of protein molecules that direct the folding process assembly, and/or transport of other proteins or enz y mes during and immed i ately after synthesis (e g Badcoe et al 1991 Voet & Voet 1995) Of the 20 amino acids available as building blocks of proteins five have sidechains which i mpart pos i tive (lysine, arginine, and histidine) or negative (aspartic acid and glutamic acid) charge to the

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molecule This is the basis for differential movement through an electrophoretic gel (Murphy et al. 1990) 88 It has been shown that a substitution of just one charged amino acid for another of a different charge is enough to produce electrophoretically-detectable mobility differences It is easy to see, therefore, how a chaperone protein effect i ng a differential tertiary folding shape in an enzyme such as lactat e deh y drogenase folding some charged sidechains in toward the middle of the molecule or out toward its periphery could easily produce great changes in isozyme mobility completely independent of the underlying genetic make-up of the gene Fields & Somero (1997) recently demonstrated differences in thermolability and kinetics from two closely related gobies found to have identical amino acid sequences These differing conformers of otherwise identical enzymes were not due to temperature-dependent folding since they were aclimated to the same temperature regimes. Instead, they hypothesize the involvement of either low-molecular-mass costituents bind i ng to nascent LDH-A polypeptides to effect folding or of molecular chaperones (Fields & Somero 1997) If mechanisms like these can cause mobility differences in isozymes as seems very likely, our presumption when scoring or evaluating bands generated from electrophoresisthat mobility differences signify underlying genomic differences may not be valid. The implications of this for allozyme studies should not be overlooked Many studies comparing dendrograms generated from allozyme/isozyme electrophoresis to those generated from mitochondrial DNA or from anatomical characters show general congruence (e g. Stepi e n 1992 Stepien et al. 1993

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89 Wilson 1994, Wilson et al. 1990) so it wou l d seem that this is not a common occurrence. It is however potentially a confounding, and unquant i fied factor which must be taken into consideration (much like hidden heterogeneity "null" alleles etc. must be borne in mind ; cf Johnson 1977, Engel et al. 1973, Ferguson et al. 1988) Dual expression of distinct eye and l i ver-predominant LDH-C isozymes in the same individuals has only been reported one other time to my knowledge Holt & Leibel ( 1987) report finding such express i on in several taxa of cichlid fishes (order Perc i formes, superorder Acanthopterygii ; see also Oppenheimer et al. 1989) They argued, based on differences between the eye-C and liver-C in thermolability elution behavior on oxamate-Sepharose and Blue Dextran Sepharose affinity columns, that these might represent products from a fourth locus due to a third gene duplication event. They pointed out, however that possible tissue spec i fic post-transcr i ptual or post-translational modifications cannot be eliminated with the information in hand It is easy to see how different conformers of the same enzyme could account for the observed differences in thermolability Results from thermolability runs performed in the course of this study show that eye"-C is most like A, and "liver"-C is most like B, but I am unsure of what that means in the light of Fields & Somero's work. Since shape changes of an otherwise identical molecule can greatly affect characteristics such as kinetics and thermo labil ity, pointing up differences between liverand eye-predominant forms ofLDH-C tell us little about the nature of the underlying gene encoding the enzyme Molecular probing/sequencing must be employed to answer these qurstions

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90 Implications of LDH Patterns of Expression for Gadiform Sister Group Analysis The survey of representatives from the main groups of the superorder Paracanthopterygii (which might a priori be thought to offer the best candidates for a gadiform sister group) demonstrate that dual expression is evidently widespread within the superorder The percopsiform Aphredoderus expressed the two forms very clearly, with approximately equal levels of activity manifested in eye and liver for the two forms ofLDH-C. This does not agree with what appears to be stated in Markert et al. (1975), but in that paper, the legend to their Table 1 (p 1 04) states, marginal presence; undetectable; blank, tissue not examined Therefore, it seems to indicate, by their own explanation that a blank space under a tissue might mean either that -C was undetectable or that the tissue was not examined Liver for A sayanus in their Table 1 is blank, so it remains unclear if they missed it or never looked at it. This possible misrepresentation was then manifested in Patterson & Rosen's (1989) adaption of this information into their Table 3 (p 16) where they represent A sayanus liver expression with a dash, indicating absent. The Ophidiiformes examined show the typical "higher teleost" pattern previously reported for this group of fishes by Shaklee & Whitt (1981). The Lophiiformes demonstrate both this same pattern (i.e ., the typical"higher teleost") as well as clear co-

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91 expression of both LDH-C forms in Antennarioidei with Chaunacoidei indeterminable The Batrachoidiformes all dual express very clearly, and as in Percopsiformes display approximately equal activity levels in the two tissues Finally Gadiformes (including Raniceps) show their distinctive pattern of slow liver -Conly (cf. Table 3) From this distribution, one could speculate that dual expression is the ancestral (plesiomorphic) state, retained in some groups while specialization/ compartmentalization into either eye or liver is derived Alternatively, one can see the problem from the point of view that what allows the mobility changes is some sort of chaperone protein, modifying either the "regularly-folded" liver -C into eye -C or the reverse With this scenario, the determination of what visualizes where depends on two factors in combination : presence/absence of the -C isozyme and presence/absence of the chaperone protein in a given tissue Fast eye-C and/or slow liver-C then pops in and out" throughout the taxon wherever these combine What seems safe to say is that the presence of slow liver -C in general, and the manifestation of co-expression in particular seem to be common within the superorder, and might be a characteristic of many members of the group A final note regarding current members of the Paracanthopterygii needs to be made. Of the five orders currently recognized in the superorder, only the Ophidiiformes lack any member which demonstrates the slow, liver-predominant LDH-C form of expression (see also Shaklee & Whitt 1981) Though many researchers have pointed out similarities between ophidiiforms and gadiforms (e g. Greenwood et al. 1966 McAllister 1968, Freihofer 1970 1978), Gosline (1968 1971) argues that these are the result of

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92 convergent evolution based on shared feeding habits ( cf Fraser 1972, Marshall & Cohen 1973) The question ofophidiiform affinities remains an open one (cf Gordon et al. 1984). Incertae Sedis Past Members and Proposed Additions As mentioned in the introduction, there has been some tinkering with the membership of the Paracanthopterygii since its inception by Greenwood et al in 1966 Patterson & Rosen (1989) reviewed in some detail proposed additions and deletions and they are commented on here since they have the potential to bear on sister group relationships of the Gadiformes Freihofer (1970) suggested that Gobioidei (as Gobiiformes) should be added to the Paracanthopterygii based on similarities of nerve patterns in particular the ramus lateralis accessorius-to the percopsiforms Others however, have argued convincingly that the basal gobioid Rhyacichthys (and therefore the group as a whole) has perciform affinities (Miller 1973, Springer 1983 ; cf Rosen & Patterson 1969 Roese 1984) Bannister (1970) argued that!ndostomus (as Indostomiiformes) should be added due to their many primitive paracanthopteryg i an features and anatomic similarities with batrachoidiforms and gobiecociforms Fraser (1972) rejects this argument and their placement within the Acanthopterygii i s now accepted (e. g Johnson 1993 Johnson &

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Patterson 1993, Nelson 199 4) Winterbottom (1993) explores the possibility of a gob iesoc i d-callionymoid lineage be i ng the gobioid sister group 93 The zoarcids have been all i ed with gadiforms and ophidiiforms by several investigators (e. g Rosen 1962, Freihofer 1963, Greenwood et al. 1966 Rosen & Patterson 1969) but the i r removal from the paracanthoppterygii (Gosline 1970, Anderson & Hubbs 1981 Anderson 1984) and placement in the acanthopterygii has also been generally accepted (e g Patterson & Rosen 1989 Nelson 1994) The i r LDH-C pattern of expression has been demonstrated to be that of the "higher teleosts" ; i e ., with a h i ghly anodal (=fast) eye-predominant LDH-C and no indication of a slow liver-predominant form (Shaklee & Whitt 1981, Simonsen & Christiansen 1985) Polymixoide i has likewise been removed but in the oppos ite direction Fraser (1972) po i nts to characters the taxon shares with berycoids and Stiassny (1986) places Polymixia as the sister group to the Paracanthopterygii Acanthopterygii based on two upper jaw ligament characters. Others have reached s i milar conclusions on the position of Polym ix ia (e g Rosen 1985 Stiassny & Moore 1992 Johnson & Patterson 1993 Nelson 1994) but see Rosen (1973) Parenti (1993) A final group bears mentioning in this regard Scott et al. (1986) found glycopeptide antifreezes in the Notothen i oide i that were "essentially identical" to those of the Gadiformes and postulated that they ha v e a shared evolutionary history recommending that the notothenioids should be re-aligned w i th i n the Paracanthopterygii They observe that, "to our knowledge, no lactate dehydrogenase analysis has been performed on the nototheniids ...... We conjecture that lactate dehydrogenase analysis of

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94 nototheniids would indicate their alliance with the more primitive Gadiformes and support the argument that these two groups diverged approximately 30-40 million yr ago after their glycopeptide antifreeze development had been established." [Fitch (1988, 1989) has in fact characterized LDH kinetics and electrophoretic patterns ofNotothenia neglecta but only for -A and -B; he did not investigate liver or eye tissues and makes no mention ofLDH-C patterns ] They hypothesize that though gadiforms have generally been thought to have evolved in the Northern Hemisphere, their origins may in fact be the Southern Hemispere Support for this idea was provided by Eastman & Grande's (1991) find on Seymour Island, Antarctic Peninsula, of what appeared to them to be gadiform remains from the Late Eocene (40 million ybp) Jerzmanska & Swidnicki (1992) found further remains in the same formation and these are almost of comparable age to the earliest gadiforms of the Northern Hemisphere (cf Patterson 1993a, b). Eastman (1993) however, does not agree with the hypothesis of Scott et al. He states that, "Antifreeze glycopeptides were probably derived by mutation and amplification of a gene having a wide phyletic distribution in higher teleosts. I believe that AFGPs evolved independently in gadiforms and notothenioids and that they should not be used as a basis for inferring common ancestry A subsequent re-examination by Balushkin (1994) of the Seymour Island fossil finds resulted in the determination that they were in fact early notothenioids and not gadiforms. Analysis of representatives of some of the aforementioned taxa for lactate dehydrogenase isozyme pattern is des i rable, but will be left for further investigations.

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Notothenioids are relevant to these lines of investigation all the more so since they are hypothesized to have given rise to the gobiesocoid-callionymoid lineage by those who favor placement of the gobiesocids within the acanthopterygians (e g Gosline 1970, Nelson 1994) 95 An intriguing variation of this relationship is suggested by Parenti & Song (1996). Based on a detailed re-analysis of the bones of the pectoral-pelvic fin association of gobiesocids, 11misinterpreted for over a century" and hinted at by GUnther (1861) in conjunction with innervation patterns, they note .. that if the posteriorly attenuate anterior pelvic bones of gobiesocids are homologous with the posteriorly attenuate pelvic bones of the trachinioid (notothenioid of Gosline, 1970) Cheimarrichthys, these two taxa are more closely related to each other than either is to callionymoids represented in our study by Synchiropus, which lack posteriory attenuate pelvic bones 11 If either hypothesized relationship is indeed correct (i e., a notothenio i d-gobiesocoid-callionymoid or a notothenioid-gobiesocid lineage) and given the Gobiesox pattern ofLDH-C expression (see discussion below) then ascertaining the pattern expressed by nototheniods becomes very useful. These fishes are an important component of the Antarctic fish fauna and of commercial fisheries ofthe area (e g McKenna 1991 1993) and should not be impossible to obtain Efforts to do so continue as of the writing of this thesis and will await later investigation

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96 Gobiesociformes Gobiesox displays the generalized gadiform LDH pattern as mentioned earlier ( cf Fig. 25). The implications of this observation can be viewed in either of three ways : 1 Gobiesocids are essentially gadiforms since they share the putative gadiform LDH-C synapomorphy 2 Gobiesocids and gadiforms are sister taxa, united by their synapomorphy, the LDH-C pattern ; this pattern i s thus no longer tenable as a gadiform synapomorphy (but rather is a synplesiomorphic character of the gobiesocid + gadiform group) 3 Gobiesocids are correctly placed as they currently are (sensu Nelson 1994) near the Callionymidae (Perciformes: superorder Acanthopterygi i) and their atypical LDH pattern (for acanthopterygians), presumably not due to common ancestry has no bearing on the evaluation of the gad i forms much like the patterns of the Cypriniformes and the pallid sturgeon (cf Markert et al. 1975) far removed from the Paracanthopterygii have no evident bearing In order to help clarify the cho i ces between these alternatives three callionymids were investigated and these taxa were found not to share the gobiesocid pattern, but rather to display that typical of"higher teleosts" (see Fig 25) [As mentioned above attempts to obtain notothenio i ds have not yet been successful, so their pattern remains unknown.] As originally constituted by Greenwood et al. (1966) gobiesocids (as

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Gobiesociformes) were members of the Paracanthopterygii They were subsequently removed by Gosline (1970), however and placed near the Callionymidae and Draconettidae, and a review by Patterson & Rosen (1989) found no evidence to align them with the Paracanthopterygii 97 The case for affinity with the callionymoids is not exceptionally strong either however. As early as 1905, Starks evaluated this possibility; he wrote, "The families Batrachididae [i.e., Batrachoididae] and Callionymidae offer some slight indications of relationship to the Gobiesocidae, and the weight of evidence is thrown towards the former family by the young of some or all of them having a ventral sucking disk just behind the base ofthe pectorals." Hayashi et al. (1986) listed many differences between the two groups and there appears to be little similarity between developmental patterns of gobiesocids and callionymoids (Allen 1984, Houde 1984b) Rosen & Patterson (1990) with regards to a survey of dorsal gill arches wrote, "although clingfishes (gobiesocids) might not belong with the paracanthopterygians (Patterson & Rosen, 1989), neither do they belong with the dragonets [ callionymids] Clingfishes have four short basibranchials converging on a short basibranchial copula without ossifications as do some uranoscopids, but dragonets have a primitive ventral gill arch configuration with well developed ossified basibranchials" (see also Nakabo 1983b) Recently sperm ultrastructure (biflagellarity and/or spiral nucleus) of the gobiesocids lead some investigators to align them as a sister-group to the Batrachoidiformes (Jamieson 1991, Mattei 1991) Lauder & Liem (1983) list several synapomorphies for their batrachoidiforrn lophiiform gobiesociforrn clade : skull roof

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98 flattened ; parasphenoid and frontal bones either approaching each other or sutured to each other ; large sphenot i cs flaring forward and laterally ; and progressive reduction in the ossification ofthe palatopterygoid. Eschmeier (1990 1998) retains the Gobiesocids in a position next to the Batrachoidiformes and Lophiiformes Other than the gadiforms no other fish above the level of Ostariophysi has been reported to possess a slow, liver-predominant LDH-C while simultaneously exhibiting no fast eye-predominant LDH-C until now Regardless of the difficulties with deciphering the implications of dual expression at various le v els within the Paracanthopterygii, this synapomorphy" between the gobiesocids and the gadiforms would appear to have real phylogenetic meaning. This information, in conjunction with old doubts about both its removal from the Paracanthopterygii as well as its weak affinities with the higher perciforms and new evidence of its spermatozoological ties to the batrachoidiforms leads me to suggest their final removal from the Perciformes and inclusion once again among the Paracanthopterygii as Gobiesociformes

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99 Conclusions This work has as its main contributions the following four findings: 1. The LDH pattern of expression listed as a putative synapomorphy for the Gadiformes by Patterson & Rosen (1989) holds for its suggested most basal member, Raniceps (sensu Markle) but see below. 2 Dual expression of fast eye-predominant and slow liver-predominant LDH-C is documented and will have implications for how we view the lactate dehydrogenase enzyme system, as well as perhaps how we view electrophoretic data, based on mobility differences of enzymes, as a whole. 3 Many groups at several levels within the Paracanthopterygii express both eye and liver LDH-C forms, and this characteristic may hold some utility in elucidating intra relationships or in defining the group itself. Dual expression appears to be the pleisiomorphic state for the taxon 4 Gobiesocoidei (sensu Nelson 1994), presently a suborder within the Perciformes express the gadiform LDH-C pattern Given its tenuous affinities to perciforms, its final removal from the Perciformes and inclusion in the Paracanthopterygii (as Gobiesociformes) is recommended It possibly should be accorded a position as sister

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100 group to the Gadiformes in which case the lack of eye type LDH and expression of liver type LDH will no longer be tenable as a gadiform synapomorphy

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APPENDIX 1. CHEMICALS Sigma Chemical Co.(company designation in parentheses) : DL Lactic acid Trizma base Potato starch EDTA f3Nicotinamide adenine dinucleotide [NAD] Nitro blue tetrazolium [NBT] Phenazine methosulfate [PMS] Fisher Scientific company designat i on in parentheses) : Boric acid MeOH Glacial acetic acid Potasium phosphate monobasic Potasium phosphate dibasic (L-1500) (T-1503) (S-4501) (E-5134) (N-7381) (N-6876) (P-9625) (A73-3) (A452 4) (A38-212 (P285-500) (P288-500) 124

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APPENDIX 2 RECIPES 1 0 M Lithium lactate pH 8. 0 : mix lactic acid with stain buffer 4 80 g to 50ml buffer K2HPOJKH2P04 potassium phosphate grinding buffer, 0.1 M ; pH 6 95-7 .00: For 250 ml : 0 351 g K2HP04 0 .131 gKH2P04 250 mlDIH20 Tris-Borate EDTA (EBT) pH 8.6 2 slice gels 23. 5 g starch 10. 5 ml Stock sol. 199.5 ml DI continued on next page 125

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Appendix 2. (Continued) electrode / reservo i r 1:5 dilution of stock sol. For 1 L total, 166 7 ml Stock sol., 833.3 ml DI EBT, pH 8 6 Stock sol. For 3 0 L: 0 5 M boric acid [FW=61.83] 0 02 MEDTA [FW=372.2] 0 9 M Tris [FW=121.1] Stain recipe (LDH-A, -B -C) : 0 2 M Tris-HCI, pH 8 0 (stain buffer) 1 0 M Lithium lactate 10 mg/ml NAD 5 mg/ml NBT 5 mg/ml PMS continued on next page =92 7 g =22 3 g =327 g 50 0 ml 8 0 ml 1.0 ml 1.0 ml 1.0 ml 126

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127 Appendix 2 (Continued) Stain buffer (0.2 M Tris-HCI, pH 8 0) : For 2 0 L, 48.44 g Trizma base adjust pH down with cone HCl Fixing solution: 5 : 5 : 1 mix ofMeOH:DI: Glacial acetic acid

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APPENDIX 3 GEL SLICING AND STAINING PROTOCOL 1 After the gel has been removed from the electrophoretic chamber, remove cellophane wrap and straw at cathodal end of gel. 128 2 Using scalpel and straight-edge cut away top and bottom of gel where sponges have overlain (1-2 em), and excess gel on left and right (where no wicks/extracts were loaded) 3 On left side of remaining gel, cut a small V in gel at origin (or other similar mark) in order to denote orientation of subsequent slices. 4 Remove wicks from area between gel halves 5 Position gel halves on slice tray and, using "bow slicer" cut a slice of thickness determined by the slice tray height. Repeat according to thickness of gel run The final slice (top) is also used (if desired) ; this is the "back" slice and is stained upside down since top is glossy and resistant to penetrat ion of staining chemicals (and somewhat opaque) 6 Place slices to be stained in staining boxes 7 Add stain chemicals: 50 0 ml Tris-HCI staining buffer 8.0 mllithium lactate contnued on next page

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Appendix 3 (Continued) 1.0 ml NAD 1.0 ml NBT 1 0 mlPMS 129 8 Incubate slices at 37 C in the dark in oven (usually for 15-30 nimutes) If further staining of weak bands is desired, or as controls for presence of bands not readily visualized, slices were allowed to sit in the dark at room temperature (in a drawer) overnight. 9. When the staining has proceeded to the desired point, the staining process is haled by decanting the staining solution out of the staining box, rinsing a few times with DI water, and adding fixing solution 10. After 12-36 hours in fixing solution, gel slices are ready for wrapping in cellophane for storage, and/or documentation (as described in Methods).

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130 ADDENDUM During the course of this study, I came across a toad fish which I referred to as Opsanus sp. in the text (see LDH Expression of the Batrachoidiformes (Paracanthopterygii)). This form is bright orange red, and markedly different from the color of Opsanus beta which it resembles in the roseatte patterns of the head and medial sides I have examined >20 of these individuals and have found nothing other than their unique color expression to differentiate them from 0. beta (e g comparing counts as by Schultz & Reid 1937) Certain individuals diplay a "hybrid11 color scheme with deep orange blotches confined primarily to the head region (Fig 32). I have obtained specimens of this form through the aquarium/tropical fish trade where they are sold under the name orange toadfish A local diver/dealer who collects from the Dunedin, FL area had a holding tank with about 30 of them as of September (1998) Through field work with Phil Steele's shrimp by-catch reduction research (at the Florida Marine Research Institute) I have encountered them in this area myself (off of Port Richey FL) A night's catch which results in approximately 1000 regular 0 beta's will on average produce 2-3 orange forms and 0-1 "hybrids11 Dr. Ed Matheson (FMRI) in the course of his extensive sampling of the seagrass communities ofFlorida Bay, has come across the "hybrid11 form a few times i n the upper bay (though never the solid orange form) indicating their range may be more widespread Batrachoids are rather sessile fish, inhabiting excavated burrows and holes in seagrass beds rock piles, oysterbeds, and similar habitats as adults and lacking a

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131 Figure 32. Opsanus beta with Orange Toadfish (0. sp. ? ) and Color "Hybrid" All three specimens were taken sympatrically in Thallassia beds off Port Riche y FL in November 1998

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132 dispersive stage in the reproductive cycle as the eggs are demersal and brooded by the males in their burrows (Avise et al. 1987, Robins et al. 1986) Several batrachoids are known to have rather narrow geographic ranges. The coral toad, Sanopus splendidus is known only from Cozumel Island, Mexico (Collette 1974), Opsanus phobetron might be a glacial relict only just holding on around Bimini in the Bahamas ( cf Bohlke & Chaplin 1993), and Sanopus greenjie/dorum and S. astrijer is known only from areas ofBelize and offshore atolls (Collette 1983, Humann 1994) Collette (1983) described S greenjieldorum and destinguished it from the very similar S. astrifer on the basis of two specimens of the former, four of the latter Other than color differences (black and white in both but with white lines on a dark background in greenjie/dorum and white dots on a dark background in astrifer), no apparent features separates the two forms [Collette states that in addition, the eye is slightly smaller in greenfieldorum but his own data shows the orbital diameter to be 41 thousands of SL for green.fie/dorum and 41-50 for astrifer based on n=6]. Batrachoids seem to offer many opportunities for genetic and zoogeographic lines of inquiry. The New World is clearly the center of diversity for this family (Collette 1983) and cases of incipient speciation as well as decline towards potential extinction (in the case of 0 phobetron) may be awaiting the researcher who takes the challenge.


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