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Clark, Justine Ann.
Genetic variation in the chloroplast genome of a newly described Aster species, Chrysopsis delaneyi
h [electronic resource] /
by Justine Ann Clark.
[Tampa, Fla] :
b University of South Florida,
ABSTRACT: The genus Chrysopsis (Asteraceae) contains eleven species native to Florida, including the newly described species, Chrysopsis delaneyi. Populations of this endemic plant species inhabit the Lake Wales Ridge (LWR) and the Atlantic Ridge (AR) of the Florida peninsula. Differences in morphology have been demonstrated within C. delaneyi, based on their locations. My objective was to determine the relationships between the LWR and the AR populations by analysis of chloroplast sequence and nuclear sequence variation. Approximately 160 samples of C. delaneyi and its sister species C. scabrella have been collected from fifteen sites throughout Florida. Six single base differences were detected, one insertion, and one variable short duplication. A total of four haplotypes (i.e.: groups that have different combinations of polymorphisms) have been found. For the most part, one haplotype is found in LWR populations and is indistinguishable from that found in C. scabrella. Another haplotype is found primarily in AR populations and is more similar to haplotypes found in the more distantly related C. highlandsensis and C. floridana. One haplotype is found within populations of C. scabrella. The last haplotype in one AR population contains two polymorphic loci, one site is representative of the AR populations, and the other site is that of the LWR populations. Only one mixed population has been found, at the northern end of the AR range. These results are not consistent with taxonomic relationships inferred from morphological characteristics; hence the results suggest that chloroplast DNA (cpDNA) relationships may be the consequence of one or more instances of chloroplast capture.
Thesis (M.S.)--University of South Florida, 2006.
Includes bibliographical references.
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Adviser: Bruce Cochrane, Ph.D.
Evolutionarily significant unit.
t USF Electronic Theses and Dissertations.
Genetic Variation in the Chloroplast Genome of a Newly Described Aster Species, Chrysopsis delaneyi By Justine Ann Clark A thesis submitted in partial fulfillment of the requirement s for the degree of Master of Science Department of Biology College of Arts and Science University of South Florida Major Professor: Bruce Cochrane, Ph. D. James Garey, Ph. D. Richard Wunderlin, Ph. D. Date of Approval: November 13, 2006 Keywords: Florida, cpDNA, haplot ype, chloroplast capture, evolutionarily significant unit Copyright 2006, Justine Ann Clark
ACKNOWLEDGEMENTS I would like to thank my advisor, Bruce Cochrane, for his guidance, confidence, understanding, and his time, espe cially during this past year. I also want to thank my committee members, James Garey and Richard Wunderlin, for their expertise and time. They all have been a great help to me throughout my research and education. I would also like to express my grat itude to Stefi, Terry, Kim, Hayden, and Alan for all of their help with my s equencing reactions, test preparations, and general assistance. Finally, I would like to thank my mot her-in-law Ann, my brother Ray, and my niece Roz for their support; my si ster-in-law Dana for her help and support; and my husband Robert for always believing in me.
i TABLE OF CONTENTS LIST OF TABLES .................................................................................................iii LIST OF FIGURES ...............................................................................................iv ABSTRACT ..........................................................................................................v INTRODUCTION ..................................................................................................1 Florida Plants and Habitats ........................................................................8 Chrysopsis delaneyi ................................................................................10 Overview ..................................................................................................13 MATERIALS AND METHODS ............................................................................15 Population Sites .......................................................................................15 DNA Extractions ......................................................................................16 PCR Amplifications ..................................................................................17 Restriction Enzyme Digests .....................................................................20 Gel Extractions ........................................................................................21 Sequencing Reactions .............................................................................21 PCR Fragment Cloning ............................................................................22 Nucleotide Sequence Comparisons .........................................................23 RESULTS ...........................................................................................................24 Restriction Enzyme Digests .....................................................................24 Sequencing Reactions .............................................................................24
ii BLAST Results ........................................................................................29 DISCUSSION .....................................................................................................32 CONCLUSION ....................................................................................................43 FUTURE DIRECTION ........................................................................................46 REFERENCES CITED .......................................................................................48
iii LIST OF TABLES Table 1 Number of Site s and Plants Sampled...............................................15 Table 2 Geographic Coordinates and Abbreviated Codes of Collectio n Sites .............................................................................16 Table 3 Thermocycling Conditions for PCR Reac tions..................................19 Table 4 BLAST Sequences of ccmp3 R egion............................................... 23 Table 5 ccmp3 Insert ion Sequenc es.............................................................26 Table 6 cpDNA Variations Found with matK Primers at Four Base Po sitions (BP).............................................................28 Table 7 Actin 1 Sequence Alignment with AR and LWR Populations...........30 Table 8 ccmp3 Sequence Alignm ent with Outg roups....................................31 Table 9 Table of Haploty pes.........................................................................33
iv LIST OF FIGURES Figure 1 Distribution of Four Chrysopsis Species in Peninsular Florida........ 11 Figure 2 Examples of C. delaneyi Populations and Habitats..........................14 2-A UCF Campus in Orange Coun ty................................................14 2-B Jonathon Dickens on State Pa rk.................................................14 Figure 3 Electrophoresis of Alu 1 Di gest........................................................25 Figure 4 Electrophoresis of DpnII Di gest........................................................25 Figure 5 Electrophoresis of ccmp3 PCR.........................................................25 Figure 6 Phylogenetic Tree of Chrysopsis Based on Chloroplast Data..........34 Figure 7 Population Distribution Map of Chrysopsis Species.........................39
v Genetic Variation in the Chloroplast Genome of a Newly Described Aster Species, Chrysopsis delaneyi Justine Clark ABSTRACT The genus Chrysopsis (Asteraceae) contains eleven species native to Florida, including the newly described species, Chrysopsis delaneyi Populations of this endemic plant species inhabit the Lake Wales Ridge (LWR) and the Atlantic Ridge (AR) of the Florida peninsula. Differences in morphology have been demonstrated within C. delaneyi based on their locations. My objective was to determine the rela tionships between the LWR and the AR populations by analysis of chloroplast sequence and nuclear sequence variation. Approximately 160 samples of C. delaneyi and its sister species C. scabrella have been collected from fifteen sites th roughout Florida. Six single base differences were detected, one inserti on, and one variable short duplication. A total of four haplotypes (i.e.: groups that have different combinations of polymorphisms) have been found For the most part, one haplotype is found in LWR populations and is indistingui shable from that found in C. scabrella Another haplotype is found primarily in AR populatio ns and is more similar to haplotypes
vi found in the more distantly related C. highlandsensis and C. floridana One haplotype is found within populations of C. scabrella The last haplotype in one AR population contains two polymorphic loci, one site is representative of the AR populations, and the other site is that of the LWR populations. Only one mixed population has been found, at the northern end of the AR range. These results are not consistent with ta xonomic relationships infe rred from morphological characteristics; hence the results s uggest that chloroplast DNA (cpDNA) relationships may be the consequence of one or more instances of chloroplast capture.
1 INTRODUCTION A key component in any conservation program is the preservation of genetic diversity of a species. This parti cular element of a conservation plan for endangered plant taxa can be one of the most difficult aspects to address, as human encroachment on sensit ive areas often causes fragmentation of habitat, which can result in the isolation of populations. This fragmentation can lead to genetic drift in isolated populations, dec reasing genetic variability of populations, and eventually decreased viability. Understandi ng the causes of genetic variation patterns in natural plant populations is an essential facet in conservation biology (Powell et al., 1996B). Molecular markers are used to facilitate the determination of genetic variation because they are accurate and can quantify the degree of genetic diversity between, as well as within plant populations (Lakshmi et al., 1997). The genetic information collected by such molecular techniques can then be used to develop and implement conservation recovery plans for endangered species. The organelle genomes of mitochondria and chloroplasts have been used in animal and plant studies that include areas of evolutionary population biology such as migration patterns, historic events, and differentiation gradations of populations (Provan et al., 2001). The unique characteri stics of the chloroplast make it a useful tool for such studies. The circular structure of the chloroplast
2 DNA (cpDNA) in land plants is highly conserved, and the gene order is usually maintained. The cpDNA is divided into a large single copy region (LSC) and the small single copy region (SSC), with inverted repeats between the two regions. The LSC region is less conserved than the SSC region, making this region ideal for low taxonomic evaluations (Grivet et al., 2001). The average size of the angiosperm chloroplast genome is approximat ely 148 kilobases (kb), providing a model size for restriction site anal yses and direct sequencing comparisons (Olmstead et al., 1994). Several other features of the ch loroplast genome are uniparental inheritance, and nonrecombinat ion. Specifically, the chloroplast genome is inherited maternally in most angiosperms (Ferris et al. 1997). Thus, directionality of seed and/or pollen dispersa l can be followed, as well as their contributions to the overall genetic arrangement of plant populations (Provan et al., 2001). Additionally, nonrecombinat ion of the chloroplast genome demonstrates how the chloroplast is inherited as a unit, and is, fo r the most part, resp onsible for the lack of cpDNA variation in populations. Therefore, questions of gene introgression and sex-biased dispersal may be addr essed by organellar polymorphism comparisons within and between populations (Wills et al ., 2005). The protein coding regions of chloropl asts are essential for photosynthetic activity as well as catabolic and metabolic functions. Thus, the frequency rate of mutations in the chloroplast genome is low, resulting in a lack of variation within these regions between species (Small et al., 2005). However, noncoding regions of cpDNA, such as intergenic spacers and introns, are more likely to show a
3 greater amount of variation because they are less functional and more likely to mutate (Shaw et al., 2005). Additionally, evolutionary changes of cpDNA such as small insertions and deletions of 1 100 base pairs (bp) have been documented. From a conservation perspective, chloroplast markers have been the fundamental means used in previous phy logenetic studies, particularly those involving seed and pollen dispersal and thei r influence on the genetic structures of populations, establishment and factors of hybrid zones (McCauley, 1995), as well as tracing patterns of migrations (Huang et al., 2002). Amplification of cpDNA by polymerase chain reaction (PCR) followed by restriction digests of the PCR product are useful tools for i dentifying intraspecific chloroplast polymorphisms (Provan et al., 2001). These patterns of polymorphisms are more prevalent than previously thought, thus allowing the da ta to be used to evaluate the population level processes (McCauley, 1995). Different types of molecular ma rkers have been used to identify DNA polymorphisms. Restriction fragment l ength polymorphisms (RFLPs) are single or low copy probes that have been us ed to evaluate the amount of genetic variability in the chloroplast and nuclear genomes. Several drawbacks to this method include the use of large quantities of relatively pure DNA required for assay, as well as low levels of poly morphism detection in some plant species (Powell et al., 1996B). Another technique us es arbitrary sequence markers known as randomly amplified polymorphi c DNA markers (RAPD). This procedure involves amplification of genomic DNA at di stinct loci by using random nucleotide sequence primers. The amp licons are then used to identify polymorphisms.
4 These fingerprints have been used to help determine the phylogenetic relationships within and between species (Rout, 2006). Restriction site mapping has been used widely in phylogenetic resear ch as this straightforward method allows sampling of a number of sites for each enzyme for an indirect comparison of genetic variation. Additionally, variat ions of restriction sites located in noncoding regions render far more useful data pertaining to species phylogenies (Olmstead et al., 1994). Simple sequence repeats (SSRs), al so known as microsatellites, are repetitive sequences of DNA, usually 1 6 bp repeated a number of times within the genome. In the chlor oplast genome, the repeats are generally runs of T residues that vary in length, which resu lt in the variations found within species (Powell et al., 1996A). The motifs are generally mo re conserved in closely related taxa as compared to that of more distantly related taxa (Provan et al., 2004). Several assumptions of SSR markers incl ude selective neutralit y, co-dominance, an equal distribution throughout the genome, and, these markers are effective in producing PCR products (Arnold et al., 2002). In addition to detecting polymorphisms on loci, SSRs are used to test for new alleles. In comparison with RFLP methods, SSR only use small amounts of plant tissue and its use have uncovered more polymorphisms than previous ly thought existed in several plant species (Provan et al. 2001). Sequencing of the chloroplast genome is another valuable tool used in genetic variation studies because eac h nucleotide in the sequence can be compared. The amplified PCR products can be either sequenced directly, or
5 cloned first and then sequenced. Considerat ions for the use of sequencing for comparative studies should include sequen ce length of the fragment, a general understanding of the substitution rate withi n the sequence region (i.e.: is the sequence in a coding or noncoding region), and the ability of sequence alignment to other sequences (Olmstead et al., 1994). This method can be used to resolve both higher-level and lower-level phylog enies, based on the regions examined. Early phylogenetic studies on plant species based on cpDNA proteincoding regions had drawbacks. These regions are highly conserved, and have low mutation rates, limiting their use to high-level phylogenetic studies (DumolinLapegue et al., 1997). The non-coding regions however, have demonstrated higher mutation rates, thus providing a more useful tool for taxonomic studies at lower levels (Shaw et al. 2005). This finding, combined with the advances of more complete chloroplast genome sequencing and its conserved gene arrangement, allowed for the development of universal primers. These primers are targeted at the conserved flanking regions of the noncoding regions (Small et al. 2005). Once amplified, the respecti ve PCR products are generally small enough for direct sequencing or, if large enough, can be digested with restriction enzymes. Another aspect of molecular marker use in conservation biology is defining units of flora and fauna for conservation purposes (Fraser & Bernatchez, 2001). In 1966, the U.S. Endangered Species Act ( ESA) was initiated with the objective of protecting endangered fish or wildlife. However, the initial legislation was too restrictive, therefore requiri ng changes that were more suitable and more specific
6 for endangered species protection (Pennock & Dimmick, 1997). The concept of the evolutionarily signif icant unit (ESU) was introduced to acknowledge and implement a classification system for di stinct groups lower than the already accepted taxonomic groups in order to preserve genetic diversity (Fraser & Bernatchez, 2001). Waples (1991) defined an ESU in order to distinguish the uniqueness of populations and, if found to be significant, warrant protection under the ESA. The definition states that an ESU is a population (or group of populations) that is isolated from and unable to reproduce with other populations of the same species, and plays an import ant role in the ev olutionary heritage of the species. The catch with this definiti on is that even if a population can show divergence, either adaptive or genetic, it may not qualify as an ESU if it does not demonstrate phylogenetic uniqueness, and will not be protected under the ESA (Young, 2001). Other definitions of ESUs incl ude those of Ryder (1986); Dizon et al ., (1992); Avise (1994); Moritz (1994); V ogler & DeSalle (1994); and Crandall et al., (2000) (Fraser & Bernatchez, 2001). Ryders 1986 definition, which initiated the ESU, was set in place to characterize subdivisions of a broad group of a particular species, whose genetic feat ures are unique and noteworthy for the preservation of current, as well as future populations of species. This definition too, had a catch, because it did not in clude rules for implementation (Fraser & Bernatchez, 2001). Dizon et al. (1992) looked at allele frequencies for their definition, specifically, concentrating on the divergence of these frequencies between populations of spec ies. Other factors causing reproductive isolation
7 were considered which included localizat ion of populations, species behavior, morphology and selection. Avises (1994) definition states that ESUs should be grouped by similar gene phylogenies which constantly result in population distinction into subgroups based on genet ic characterization and geographical levels. Moritz (1994) states th at the primary rationale for defining ESUs is to acknowledge and maintain the evolutionary lineage of a unit in order for genetic diversity to be passed along. Moritz based his definition of an ESU on demonstrating that there actua lly is the existence of a particular type of genetic difference, rather than just looking at the quantity. Genet ic differences are illustrated by the distribution of nuclear, mitochondrial or chloroplast alleles within populations (DeWeerdt, 2002). Therefore, Moritz points out several characteristics that should be included when describing an ESU: Members of ESUs should not share a common ancestor with any other individuals of anot her population, a term known as reciprocal monophyly. Nuclear allelic frequencies should demonstrate great divergence. The time period for reciprocal monophyly to occur in populations that have been separated should be 4N generations. As a result of high substitution rates compared to nuclear genomes, organellar genomes are anticipated to reach this state at a significantly fast rate.
8 Nuclear allele frequencies must be examined in concert with organellar frequencies and demonstr ate a good proportion of divergence in order to determi ne correctly phylogenies based on both data sets. Vogler & DeSalle (1994) take an alternative approach to ESUs. They define a conservation unit by character f eatures that cluster groups together, a theory known as Phylogenetic Species Concept (PSC). This concept looks at the ancestral condition for discrete clusters in order to define it as a conservation unit. Additionally, PSC can be examin ed by population aggregation analysis (PAA) to recognize the orders of relat ed species, but specifically to include groups that are joined by fixed charac ter states (Fraser & Bernatchez, 2001). Crandall et al. (2000) view defining distinct populations as a complete process involving different degrees of gene flow resulting in a groups individual uniqueness from adaptation through events such as genetic drift and natural selection. This principle is based on a null hypothesis of a populations uniqueness, then, if applicable, the popul ation is categorized for protection (Fraser & Bernatchez, 2001). Florida Plants and Habitats There are 4,189 plant taxa currently list ed in the state of Florida, of which 230 taxa are endemic. This diversity of pl ant species is attributed to Floridas geographic location, as well as its size and shape. Starting from the Atlantic and
9 Gulf Coastal Plains, Florida stretc hes down into the Caribbean, and is surrounded on the east coast by the Atlantic Ocean and the Gulf of Mexico on the west coast. This allows temperate plant species to thrive in north and central Florida and sub-tropical and tropical plant species to grow in the southern part of Florida. Additionally, the mild climate of Florida allows non-native (exotic) species to adapt and become naturalized, which make s up 31% of the recognized taxa of its flora (Wunderlin & Hansen, 2000). Florida scrub habitats are composed of sandy soils that are nutrient-poor. These conditions are ideal for woody, xeric vegetation, and are pre-disposed to infrequent high-intensity fires, which limit the plant diversity of this environment (Myers & Ewel, 1990). There are numerous pl ant species that are prevalent to the scrub habitat and are not found in any other habitat. Fifty-five species are presently listed at the f ederal level as endangered or threatened, and 22% are on the State of Floridas list. Rare scrub s pecies are limited to the Lake Wales Ridge of Florida, possibly as re sult of the ancient landsc ape and previous island-type environment along the ridge tops (Myers & Ewel, 1990). There are several characteristic layers in scrub habitats. The shrub layer consists of six commonly occurring specie s, listed in order of their presence and abundance: myrtle oak or scrub oak ( Quercus myrtifolia Q. inopina ), saw palmetto (Serenoa repens ), sand live oak (Q. geminata ), Chapmans oak ( Q. chapmanii ), rusty lyonia ( Lyonia ferruginea ), and Florida rosemary ( Ceratiola ericoides ). The ground layer includes gopher apple ( Licania michauxii ), beak rush (Rhynchospora megalocarpa ), milk peas ( Galactia spp.), Andropogon
10 floridanum and Panicum patentifolium the lichens British soldier moss ( Cladonia leporina), C. prostrata Cladina evansii and C. subtenuis (Myers & Ewel, 1990). Chrysopsis floridana, the Florida golden aster, is restricted to a limited number of scrub habitats in Hillsborough, Manatee, Pinellas, and Hardee Counties. Other species of Chrysopsis include C. scabrella which is found throughout the state, and C. highlandsensis which is found primarily in the central interior sections of the state in Glades, Highlands, and Polk Counties (Figure 1). Chrysopsis delaneyi Chrysopsis delaneyi is a short-lived perennial herb found throughout several counties in the Lake Wales Ri dge (LWR), and the southeast Atlantic Ridge (AR). Populations on the LWR occu r in southern Lake, western Osceola, eastern Polk, and northwestern Highlands C ounties, specifically in turkey oak sandhills and longleaf pine environments. Extant populations are small and fragmented. The AR populations i nhabit sand pine and hickory scrub environments along southern Brevard, I ndian River, St. Lucie, Martin, Palm Beach, and Broward Counties. There are numerous populations found along US highway 1 from Jonathan Dickinson State Park north to Hobe Sound, mainly growing on open dunes. A few populat ions are found on the Orange County Uplands, some located at close to the University of Central Florida (DeLaney et al. 2003) (Figure 1).
Figure 1. Distribution of Four Chrysopsis Species In Peninsular Florida C. floridana C. scabrella C. highlandsensis C. delaneyi In addition to the variable habitats C. delaneyi occupies, populations also display differences in morphology (Figure 2). Initially, C. delaneyi was identified as C. scabrella but has since been found to differ from that species. Both species share similar morphology, such as yellow-green colored leaves, small capitula, short, sparse trichomes, thin linear leaves, and overall small plant size compared to the silver-green colored sericeous-tomentose leaves, large capitula, thick 11
12 linear leaves, and larger plant size of C. highlandsensis. However, some AR plants are somewhat larger than the LW R plants, and have thicker trichomes, thicker, more densely sericeous-tomentose linear leaves, and larger capitula, and are more robust than LWR populations. Bas ed on this observation, the possibility exists that this species may typify se veral races that have genetically adapted to certain environmental conditions. As a re sult of the natural landscape of the upland ridge system, ce rtain populations of C. delaneyi have become isolated, thus allowing them to their uni queness due to allotropy (Delaneyi et al 2003). Additionally, some populations ma y have adapted to environmental changes within their own habitats resulting in vari ation in the genetic structure within the species. Chrysopsis delaneyi has a woolly-pubescent basal rosette, with rosette leaves (7.0)10.0-16.0(18.0) cm long and (0 .8)1.5-2.7(3.5) cm wide. These leaves are broadly spatulate, obl anceolate, or narrowly lanceolate to nearly linear. Rosettes with stems can be up to 15 cm tall. These mostly grow for two or more years before flowering, and often branch into clusters of multiple rosettes after the first year of growth. T he stems are (0.8)1.0-1.2(1.8 ) cm in diameter near the base, (0.6)0.8-1.2(1.5) m tall, densely l eafy, stipitate-glandul ar or glandular hirsute. The flower head is corymbiform or paniculiform, compact to moderately open, moderately branched, m easuring 30-70(200), (2.4)4.2(4.6) cm in diameter (including ray straps). The branches are stout and densely viscid stipitateglandular. Chrysopsis delaneyi flowers from mid-November to early January,
13 except for Orange County populations, which begin flowering in October (Delaney et al 2003). Overview The objective of this study is to investigate the chloroplast and nuclear genome distributions wit hin populations of C. delaneyi to determine the amount of differentiation within local species. The morphological differenc es of this plant species based on population locations raise some questions about the evolutionary processes taking place. Speculation that C. delaneyi is a species composed of several ecotypes has been suggested by DeLaney et al (2003). Therefore, investigating the possibility that C. delaneyi is an evolutionarily significant unit is included in this study. Based on the chloroplast data, I have found that the cpDNA variation distribution of C. delaneyi is consistent with the existence of two ESUs. Most of the LWR populations display differentiation from the AR populations, but similar to C. scabrella Additionally, there is a mixed population group containing both LWR and AR haplotypes and a population t hat contains two polymorphic loci consistent with one of each of the AR and LWR populations. The relationships with other Chrysopsis species are not consist ent with morphological data. Implications with respect to species conservation and management will be discussed.
Figure 2: Examples of C. delaneyi Populations and Habitats A: UCF Campus in Orange County B: Jonathon Dickenson State Park 2-A 2-B 14
15 MATERIALS AND METHODS Population Sites In November, 2003, one hundred sixt y three leaf samples of both C. delaneyi and C. scabrella were collected from fifteen sites (Table 1). Leaf samples were collected by removing two leaves from each flowering plant and placing them into plastic bags containing silica gel desiccant. The bags were subsequently labeled with an abbreviated code and number corresponding to the collection site (Table 2). The bags were then stored at -20 C until the DNA could be extracted. Table 1: Number of Sites and Plants Sampled Taxon Number of Sites Plants Collected C. scabrella 4 34 C. delaneyi (LWR) 6 67 C. delaneyi (AR) 5 62
16 Table 2: Geographic Coordinates and Abbrevia ted Codes of Collection Sites Population Abbreviation Site Designation County Latitude Longitude APA Avon Park Highland s 27.61508N 81.51349W APP Avon Park Highland s 27.60191N 81.50578W 1-708 NW US1 708 Martin 27.06102N 80.1377W BSNPRK Babson Park Polk 27.83356N 81.52695W ESUSI East Side US1 hundreds Martin 27.00865N 80.10195W FL708 SE Bridge Rd Martin 27.0581N 80.14137W HGH Highlands Avenue Highlands 27.57053N 81.49587W HTCWAT NE Inters. Hatchineha & Watkins Polk 28.03448N 81.52817W IRCL Daytona Blvd. W of 3rd St Brevard 27.86725N 80.49953W RSLND Roseland Indian River 27.83025N 80.4791W ScTIT1 S. Side FL50 Brevard 28.55427N 80.8201W ScYH2 W. Side 441 N Yeehaw Osceola 27.71323N 80.91133W STLUS1 W. Side US1 St. Lucie 27.49442N 80.3447W UCF UCF W. Entrance Univ Blvd Orange 28.59788N 81.20537W EEE Triple E Lake 28.61647 N 81.71317 W EMRLD W side Emerald Driv e Hernando 28.50953 N 82.1814 W DNA Extractions DNA extractions were performed using a Plant DNA Isolation Kit by Roche Diagnostics Corp. following the manufacturers protocol, with exceptions to the amount of buffers added as follows: Buffer 1 from 150 l to 300 l; Buffer 2 from 10 l to 20 l; and Buffer 3 from 50 l to 100 l.
17 PCR Amplifications All cpDNA used for PCR amplifications were diluted to a 1:10 concentration. All PCR amplifications were carried out as a 50 l volume reaction containing 5 l of 10X magnesium-fr ee reaction butter (50mM potassium chloride, 10mM Tris-HCl, 0.1% Trit ion X-100) supplied by Promega, 5 g (0.25 l) bovine serum albumin (BSA), 0.25mM each dNTP, 50 pmol primer and 10-50 ng template DNA (1 l), 1U Taq DNA polymerase (Promega), and 3 l of 1.5mM of 1X magnesium chloride. The PCR thermocycling conditions and primer sequences for the various PCR reactions are found in Table 3. All agarose gels were run in 1x TBE Buffer (Tris, boric acid, EDTA, pH 8) and visualized with ethidium bromide staining. The initial chloroplast markers used in this study were universal primers specific for the trn L CD region. These markers were used because previous analysis had revealed the presence of two restriction site polymorphisms, detected by digestion with Alu1 and Dpn II (Walker and Cochrane, unpublished). A 2% agarose gel was used for electrophoresis. Consensus chloroplast microsatellite prim ers (ccmp) specific for the intron of the trn G gene (ccmp3) were tested and subs equently used in this study. These primers not only target SSRs, but also have been used effectively in cpDNA variation studies, particu larly in angiosperms (Weising et al., 1999). The number of poly (A) microsate llites found in the amplicon cause the variations in species. These residues, which ar e less common in the organellar genome
18 compared to nuclear genome, are generally 20 bp long (Weising et al 1999). A 3-1/2% agarose gel was used for electrophoresis. A noncoding region of the trn K gene in the chloroplast genome was examined using matK6f and matK5r prim ers. Phylogenetic studies for both interspecific and intraspecific have re lied on noncoding cpDNA regions focused in the LSC. The conserved nature of the genes flanking these regions, especially in angiosperms, allows for easy and effective primer design for lower-level taxonomic studies (Shaw et al., 2005). A 2% agarose gel was used for electrophoresis.
Table 3: Thermocycling Conditions for PCR Reactions Primer Sequence Size (bp) PCR Conditions trnC 5 CCA GTT CAA ATC TGG GTG TC 3 ~500 5 minute at 94 C, 30 cycles of trnD 5 GGG ATT GTA GTT CAA TTG GT 3 30 seconds at 94 C, 30 seconds at 50 C, 1 minute at72 C, 5 minutes at 72 C ccmp3f 5 CAG ACC AAA AGC TGA CAT AG 3 ~120 5 minute at 94 C, 30 cycles of ccmp3r 5 GTT TCA TTC GGC TCC TTT AT 3 1 minute at 94 C, 1 minute at 52 C, 1 minute at 72 C, 5 minutes at 72 C matK6f 5 TGG GTT GCT AAC TCA ATG G 3 ~1500 5 minutes at 95 C, 35 cycles of matK5r 5 GCA TAA ATA TAY TCC YGA AAR ATA AGT GG 3 1 minute at 95 C, 1 minute at 50 C (ramp of 0.3 C/second), 5 minutes at 65 C, 5 minutes at 65 C 19
20 To examine the nuclear genome, the in tron region of the Actin 1 gene was used (Slomba et al., 2004). PCR reactions were performed using Actin 1 forward primers (5 CCC GAA TTC CTT GTT TG C GAC AAT GGA AC 3) and Actin 1 reverse primers (5 CCC GAA TTC AC A ATT CCA TGC TCA AT 3) to produce a 316 bp fragment. The thermocycl ing protocol was 1 minute at 95 C, 35 cycles of 15 seconds at 95 C, 30 seconds at 48 C, and 90 seconds at 72 C, followed by 10 minutes at 72 C. PCR amplified products were run on a 2% agarose gel in 1X TBE and visualized with ethidium bromide staining. Restriction Enzyme Digests The amplified products of the trn L CD region, as described above, were subsequently digested with both Alu1 and DpnII enzymes in separate reactions. The first reaction combined 1 l of Alu 1, 1 l of Alu 1 buffer (10mM Tric-HCl, 50mM NaCl, 10mM MgCl 2 1mM Dithiothreitol, pH 7.9 at 25 C), and 8 l (2538ng/l) of PCR product at 37 C for 2 hours. The second reaction combined 1 l of DpnII, 1 l of DpnII buffer (100mM NaCl, 50mM Bis Tris-HCl, 10mM MgCl 2 1mM Dithiothreitol, pH 6.0 at 25 C), and 8 l (25-38ng/l) of PCR product at 37 C for 2 hours. The digested PCR pro ducts were then run on a 2-1/2% agarose gel for the Alu1 digests and a 3% agarose gel for the DpnII digests and visualized with ethidium bromide staining.
21 Gel Extractions DNA samples from the matK and Actin 1 PCR amplifications were extracted from 1% agarose gels and pur ified for subsequent direct sequencing and cloning reactions, respectively, usi ng a QIAquick Gel Extraction Kit (Qiagen Corp.) following manufacturers protoc ol. Approximately 45 l of each PCR product was loaded into the gel. For the Actin 1 samples, 1 gel volume of isopropanol was added to the sample tubes and mixed to increase the yield of DNA fragments. The DNA was then stored at -20 C. Sequencing Reactions The thermocycler protocol for direct sequencing of the matK amplifications was 30 cycles of 20 seconds at 96 C, 20 seconds at 50 C, and 4 minutes at 60C. Reactions were carried out in 10 l volumes containing 25-38 ng of DNA, 0.0 l or 1.5 l sterile water, 1.6 pmol of matK5r primer, and 4 l QuickStart Master Mix (supplied with kit). The s equencing reactions were followed by ethanol precipitation of products first by adding 4 l of stop solution (50% volume of 3M sodium acetate and 50% of 100 mM EDTA) and 1 l of glycogen solution (supplied with kit) to each reaction tube. This was followed by the addition of 60 l of -20 C 95% ethanol/water (v/v) to each tube, which was then mixed by pipetting. The tubes were then centrifuged at 14,000 rpm for 15 minutes. The
22 ethanol was removed from the tubes by pipetting. This was followed by 200 l of -20 C 70% ethanol/water (v/v) added to each tube and centrifuged at 14,000 rpm for 5 minutes. The ethanol was remov ed from the tubes by pipetting and the 70% ethanol/water (v/v) step was repeated one more time. The pellets were dried at room temperature for 20 minutes, then resuspended in 40 l of Sample Loading Solution (SLS) (supplied with kit). PCR Fragment Cloning Several single band products from ccmp3 and Actin 1 PCR products were cloned using the TA Cloning Kit for Sequencing (Invitrogen TM Corp.) following manufacturers protocol. 50 l from each transformation reaction were spread on pre-warmed LB medium plates, each containing 30 g/ml of kanamycin. The plates were incubated at 37 C overnight. Following manufacturers protocol, one colony of each was picked and cultur ed overnight in LB broth containing 30 g/ml of kanamycin. Plasmid minipreps were performed with Purelink Quick Plasmid MiniPrep kit (Invitrogen TM Corp.) following manufa cturers protocol. The plasmid DNA was first heated for 3 minutes at 96 C and then cooled to room temperature before adding the rest of the reagents. Sequencing reactions were carried out in 10 l volumes containing 17ng/ul of plasmid DNA, 1.6pmol of M13 primer supplied with kit (M13F: 5 CTG GCC GTC GTT TTA C 3; M13R: 5
23 CAG GAA ACA GCT ATG AC 3), and 4 ul QuickStart Master Mix (supplied with kit). This was followed by ethanol precipitation as described above. Nucleotide Sequence Comparisons Sequences derived from PCR amplif ications with ccmp3 primers were used to search for other plant species that contained similar sequences (Table 4). A Basic Local Alignment Search Tool (BLAST) found at the National Center for Biotechnology Information (NCBI) website was used. An alignment was created with these sequenc es, along with several C. delaneyi C. scabrella and C. highlandsensis sequences using ClustalW in the Molecular Evolutionary Genetics Analysis (MEGA) software program (Kumer, Tamura, Nei, 1993-2005). Table 4: BLAST Sequences of ccmp3 Region Accession # Family Genus Species AY871258.1 Rosaceae Prunus ilicifolia AY727221.1 Asteraceae Trilisa paniculata AY727220.1 Asteraceae Carphephorus corymbosus AY727509.1 Solanaceae Solanum physalifolium AY727222.1 Asteraceae Eupatorium rotundifolium AY727513.1 Caryophyllaceae Minuartia uniflora DQ352338.1 Altingiaceae Altingia obovata
24 RESULTS Restriction Enzyme Digests A total of 122 C. delaneyi and 30 C. scabrella samples were digested with both Alu 1 and Dpn II. The results show t hat the AR populations have restriction sites for both of these enzymes. Alu 1 digestion produced two bands in the AR samples, compared to one band in the LWR and C. scabrella samples when visualized on a 2-1/2% agarose gel. Dpn II digestion produced a smaller band in the AR samples, compared to the slightly larger bands in the LWR and C. scabrella samples (Figure 3 and Figure 4). Sequencing Reactions The PCR products from the ccmp3 am plifications were examined by electrophoresis, using a 3-1/2% agarose gel. The LWR and C. scabrella samples showed larger bands by approximatel y 20 bps when compared to the AR samples (Figure 5). Subsequently, 5 samples were first cloned and then sequenced. Two samples were from the IRCL mixed population, one sample was a C. scabrella and two were AR samples. The results showed one insertion and one variable short duplication in the C. scabrella sample, as well as in one of the
IRCL samples (Table 5). The duplication sequence contains six A residues compared to the seven just upstream of it. Figure 3: Electrophoresis of Alu 1 Digest. Lane 3, AR sample with double bands. The other lanes are all LWR samples with a single band. Figure 4: Electrophoresis of DpnII Digest. Lane 2 is AR sample with smaller band. Lanes 3 is LWR sample with a slightly larger band Figure 5: Electrophoresis of ccmp3 PCR. Lanes 2 and 3 are AR samples with small bands. Lanes 4 8 are LWR and C. scabrella samples with larger bands 25
Table 5: ccmp3 Insertion Sequences 105 145 AR #APA3F TATGGAAAAT GGATATAT-T GCTA--------TGTGAAC CAACTTACAA AAAAATGATA CCCA--------------AR #FL708-3F .......... ........-. ....--------....... .......... .......... ....--------------AR #IRCL49F .......... ........G. ....--------....... .......... .......... ....--------------AR #IRCL50F .......... ........-. ....GATTTA AGA....... .......... .......... ....ACTTAC AAAAAATGAT SC #SCCL35F .......... ........-. ....GATTTA AGA....... .......... .......... ....ACTTAC AAAAAATGAT AR #APA3F -----TAACA AR #FL708-3F -----..... AR #IRCL49F -----..... AR #IRCL50F ACCCA..... SC #SCCL35F ACCCA..... 26
27 PCR fragments amplified with the matK primers were sequenced to examine cpDNA variations. In tota l, 134 samples comprised of 109 C. delaneyi 21 C. scabrella and 4 C. highlandsensis were directly sequenced. Variations between LWR and AR populations, within C. scabrella populations, and within one AR population were discove red in four positions (Table 6). The first variation appears in the C. scabrella scYH2 populations, where there is a single base change of G compared to an A in the other populations of C. scabrella as well as in the AR, LWR, and C. highlandsensis populations. The second variation distinguishes not only the LWR and AR populations, but also the C. scabrella and the C. highlandsensis populations. Both the LWR and C. scabrella populations have a T base at this locus, while AR and C. highlandsensis have a C base. Additionally, this similarity occurs again with the fourth locus. The LWR and C. scabrella have a C base, and the AR and C. highlandsensis have a T base. The third variation tends toward the same pa ttern as the fourth, however, in the AR RSLND populations, rather than retaining the G bas e as with the other AR populations, there is a base change to a C. The IRCL populations demonstrate a mixture of both the AR and LWR haplotypes.
28 Table 6: cpDNA Variations Found with matK Primers at Four Base Positions (BP) Population BP 212 BP 270 BP 329 BP 332 LWR G T C C AR G C G T C. highlandsensis G C G T C. scabrella G T C C C. scabrella (YH2) A T C C AR (RSLND) G C C T IRCL (5 samples) G T C C IRCL (5 samples) G C G T Two samples that were cloned from the actin 1 PCR products, one from an AR population and one from a LWR population, were sequenced. A BLAST search of each of these sequences re sulted in actin gene coding sequences or partial coding sequences in a number of pl ant families. An alignment between the two sequences shows two regions that are suspect of an insertion/deletion event (Table 7).
29 BLAST Results The results of the ccmp3 BLAST found various plant families that had some similarities in their sequences with the C. delaneyi and C. scabrella sequences (Table 8). The a lignment of these sequence s aligned 4 bps of the outgroups with the 9 bps of the first insertion site of the LWR and C. scabrella samples, with the exception of the Rosaceae family. The 7 outgroups do not show a duplication of the bases at t he second site as seen in the LWR and C. scabrella samples, however they do contain 18 to 21 similar base pairs between them.
Table 7: Actin 1 Sequence Alignment with AR and LWR Populations Identical=. Missing=? Indel=-; 50 #AR_STLU68F CCCGAATTCC TTGTTTGCGA CAATGGAACT GGAATGGTTA AGGTACCGAA TAATAGAATC TCTGCACACA CATTGATCTA #LWR_UCF7F .......... .......... .......... .......... ......TC.--------.T .TGCA...TG ...CAG--.. #AR_STLU68F AAAGTTACGC CCCAAAGTTA AATGCTTGTC TATATATAAA TGATGTTAAT TTGCAGGCTG GATTTGCGGG TGATGATGCA #LWR_UCF7F G..A..CAAA --TG.TT.C. G...G.A... ..A.C-C..G .A.CT..TTG .......... .T.....T.. A........T #AR_STLU68F CCACGAGCTG TGTTCCCAAG TATTGTGGGT CGTCCACGCC ATACTGGTGT GATGGTTGGC ATGGGCCAAA AAGATGCATA #LWR_UCF7F ...A.G.... .......... C.....A..C ..A..T..T. .C.....A.. .........A .......... .......T.. #AR_STLU68F TGTTGGTGAT GAGGCTCAGT CCAAGAGAGG TATCTTGACA CTGAAGTACC CGATTGAGCA TGGAATTGTG AATTCGGGAA #LWR_UCF7F ......A..C .......... .......G.. ......A..T .....A.... .A........ .......... .......... 333 #AR_STLU68F GGGCGAATTC GT-----TTA AACCTGCA--GGACTAG----#LWR_UCF7F .......... .CGGCCGC.. ..TTCA..TT C.CC...TAG TGAG 30
Table 8: ccmp3 Sequence Alignment with Outgroups Identical=. Missing=? Indel=-; #AR_APA3F ATGGAAAATG GATATAT-TG CTA--------TGTGAACC AACTTA-CAA AAAAATGATA CCCA--------------#AR_FL708-3F .......... .......-.. ...--------........ ......-... .......... ....--------------#Mixed_IRCL49F(AR) .......... .......G.. ...--------........ ......-... .......... ....--------------#Mixed_IRCL50F(LWR) .......... .......-.. ...GATTTAA GA........ ......-... .......... ....ACTTAC AAAAAATGAT #C_scabrella_SCCL35F .......... .......-.. ...GATTTAA GA........ ......-... .......... ....ACTTAC AAAAAATGAT #Rosaceae_Prunus GGAA..G.AT TT.G.T.CCA .CGAGCTAAA ACAA.TTGT. G.TG.CT.T. GT...CC.A. GT..TTGTTT AATAGCTATT #Asteraceae_Trilisa ..A.TT..G. .G.C.T.CG. T.TGATT----CA.ATT.. G.T----A.. ...CT.T..T T.AT---TTA AAGGATTGAA #Asteraceae_Carphephorus ..A.TT..G. .G.C.T.CG. T.TGATT----CA.ATT.. G.T----A.. ...CT.T..T T.AT---TTA AAGGATTGAA #Solanaceae_Solanum ..A.TT..A. .G.CCT.CG. T.TGATT----C..ATT.. G.T----... ...CT.T..T T.-T---TAA AAGGATTAAA #Asteraceae_Eupatorium ..A.TT..G. .G.C.T.CG. T.TGATT----CA.ATT.. G.T----A.. ...CT.T..T T.AT---TTA AAGGATTGAA #Caryophyllaceae_Minuartia..A.TT..A. .G.GCCAAG. T.TGATT----AC.ATT.. ...----..T T..CT.T..T T.T.---AAT AAGGAATTAA #Altingiaceae_Altingia -.A.TT..G. .G.CCT.CG. T.TGATT----CA.ATT.. G.T----... ...CT.T..T T.-T---TAA AAGGATTTAA #AR_APA3F -----TAA-#AR_FL708-3F -----...-#Mixed_IRCL49F(AR) -----...-#Mixed_IRCL50F(LWR) ACCCA...-#C_scabrella_SCCL35F ACCCA...-#Rosaceae_Prunus TTGCT.C.AT #Asteraceae_Trilisa TCCTT.-.-#Asteraceae_Carphephorus TCCTT.-.-#Solanaceae_Solanum TCCTT.-T-#Asteraceae_Eupatorium TCCTT.T.-#Caryophyllaceae_MinuartiaTCCCT.---#Altingiaceae_Altingia TCCTT.-.-31
32 DISCUSSION The objective of this study was to examine selected chloroplast and nuclear genes in order to determine variations within populations of C. delaneyi because of the morphological diff erences demonstrated among these populations. The primary focus wa s to resolve the question that C. delaneyi may be composed of several ecotypes. The re sults of the chloroplast data show strong evidence to support this. Additio nally, direct sequencing of the cpDNA showed within species variation of C. scabrella. A total of four haplotypes have been discovered with this study (Table 9). The presence or absence of the restriction sites in C. delaneyi populations clearly delineate the 2 groups. The result s of the ccmp3 sequencing substantiate the newfound relationship between AR and C. highlandsensis and between LWR and C. scabrella. The most likely evolutionary event occurring is that of duplication of an upstream sequence and insertion in the LWR and C. scabrella species. Intraspecific variation detect ed by the matK sequencing isolated the scYH2 population, located at Yeehaw Junction, from the rest of the C. scabrella populations. This single base difference was the only intraspecies specific variation found in all of the groups tested. Although preliminary, the alignment of the actin intron sequences from a LWR and an AR sample does suggest that
33 there may be sufficient variation within this region to be informative with respect to phylogenetic relationships in the genus. The predicted relationship between populations of the AR and the LWR C. delaneyi has not been found in this study. In stead, the cpDNA distribution points to a relationship between LWR and C. scabrella, and then reveals an unexpected relationship between AR C. delaneyi and C. highlandsensis (Figure 6). A key to understanding how these different relationshi ps may have evolved is to look at the possible interactions between these species as well as spatial patterns of the various populations of Chrysopsis Table 9: Table of Haplotypes Haplotype Variable Site Found in Populations trnL trnL-trnF* ccmp3** trnK-matK 1 GGA + GT CC BSNPRK, EEE, EMRL, HTCWAT, IRCL, OK, scCL, scTI1, UCF 2 ACG + GC GT AP, ESUS1, FL708, IRCL, STLUS1 3 GGA + ATCC scYH2 4 ACG + GCCT RSLND trnL-trnF : + Presence of Restriction Site Absence of Restriction Site ** ccmp3 : + Presence of Inse rtion/Duplication Sequence Absence of In sertion/Duplication Sequence
Figure 6: Phylogenetic Tree of Chrysopsis Based on Chloroplast Data C. scabrella C. delaneyi (LWR) C. delaneyi (AR) C. highlandsensis C. floridana The theory of introgression within the C. delaneyi species has been suggested by Semple (personal communication). Possible introgression patterns include the chloroplasts of C. scabrella incorporating into LWR populations or C. highlandsensis chloroplasts integrating into AR populations, and through backcrossing, each population group maintaining their chloroplast genotype respectively. Cases of chloroplast capture have been documented in several plant families such as Saxifragaceae (Mitella) and Asteraceae (Helianthus, and Artemisia). An early study conducted by Rieseberg et al. (1990) examined the relationship between Helianthus annuus ssp. texanus and Helianthus debilis ssp. cucumerifolius using a combination of chloroplast and nuclear ribosomal DNA markers. They concluded that the most likely scenario was chloroplast capture of H. debilis ssp. cucumerfifolius by H. annuus ssp. texanus. Another study by Kornkven et al. (1999) looked at cpDNA restriction site variations to determine phylogenetic relationships between 11 species of a woody shrub, Artemisia sect. 34
35 Tridentatae They found that 2 unrelated species, A. californica and A. filifolia were grouped in the Tridentata clade as a result of chloroplast capture. Finally, Okuyama et al (2005) examined three regions of DNA by direct sequencing in order to explain discrepancies found in the nuclear and chloroplast phylogenies of Mitella The chloroplast data were derived from the noncoding region of the trnL-F gene and the matK gene, as well as the external transcribed spacer (ETS) and internal transcribed spacer (ITS) regi ons of the nuclear ribosome. Grading the patterns of introgression from these re gions found that the chloroplast region was the most widespread, followed by t he ITS region. The ETS region did not demonstrated any pattern of introgression. The conclusion for the differences in the ITS and ETS patterns of introgressi on was nonuniform conc erted evolution. Successful chloroplast capture is dependent on a number of different factors. The initial obstacle would be t he adaptability of the donated chloroplast to the host species. A m odel presented by Tsitrone et al. (2003) suggests that the chloroplast genes and the nuclear genes would be incompatible with each other, giving rise to cytoplasmic male sterilit y (CMS), either parti al or complete in the first generation. This response to in trogression would, in turn, increase the fitness of the female by allocating t he energy from pollen production to seed production. Thus, breeding systems such as random mating and partial-selfing must be taken into consideration with this model, along with several assumptions, which include a single diploid nuclear locus and a single cytoplasmic locus each with 2 alleles, mate rnal inheritance of the cytoplasm, an
36 infinite population size, no homopla sy, no overlapping of generations, and sufficient pollen to maintain the population. According to this model, conditions involved in chloroplast capture include: A higher female fitness in the genotype that has the invading cytoplasm with the resident nucle ar alleles compared to the genotype with both the resident cytoplasm and nuclear alleles. A lower fitness of the heterozyg otes with the resident cytoplasm compared to the fitness of t he resident homozygotes with the invader cytoplasm. A lower fitness of the heterozyg otes with the invading cytoplasm compared to the fitness of t he resident homozygotes with the invading cytoplasm. These conditions favor the production of the homozygotes of the resident nuclear alleles with the invading chloropl ast, a condition that indicates successful chloroplast capture. However, if a cert ain percentage of nuclear genes introgress along with the invading chloroplasts to the resident species, conditions would be less restrained. Theoretical introgression rates have been calculated to occur in about 1000 generations. Actual experimental data of introgression rates of H. annuus cytoplasm into H. petiolares has been documented to occur in less than 50 generations. In addition, selfing rates of popul ations can play an important role in introgression rates. Reduction of the se lfing rate as a result of genome incompatibilities is predicted to increase t he rate of chloroplas t capture. If, on the
37 other hand, the selfing ra te of the resident is not affected by these incompatibilities, the rate of chloroplast capture would be similar to a randomly mating population (Tsitrone et al ., 2003). In order to use this model as a basis for chloroplast capture events in populations of C. delaneyi the assumptions must be examined. First, the chloroplasts are maternally inherited in the species. Next, there appears to be an adequate supply of pollen to maintain natur al populations and, in addition, it has been estimated by comparison studies with similar species that approximately 510% of selfing may occur in this spec ies (Semple, personal communication). Furthermore, the prevalence of homopla sy in these different populations of C. delaneyi does not seem feasible because the probability of these closely related species undergoing unrelated mutations that result in the same character state as compared to inheriting the charac ter state appears unlikely. However, populations of C. delaneyi are not of infinite size and in particular, LWR populations are more reduced compared to AR populations. These plants are short-lived perennials, ther efore this may violat e the assumption of no overlapping of generations. Populations of C. scabrella are found throughout Florida. Samples used in this study come from populations loca ted in Hernando, Brevard, and Osceola Counties. The LWR samples were taken from Lake, Orange, and Polk Counties. The LWR populations are centrally located within the outlying C. scabrella groups. The exception is the Osceola County population of C. scabrella This particular group is situated closer to AR populations. Populations of C.
38 highlandsensis are found in Polk County as well, but also in central southern Highlands and Glades Counties. AR popu lations occupy counties along the eastern coastline, starting from Indian River south to Palm Beach (Figure 7). These spatial patterns demons trate the proximities of C. highlandsensis and AR populations, as well as LWR and C. scabrella
Figure 7: Population Distribution Map of Chrysopsis species 39
40 Introgression appears to be a plausible cause for the discrepancies found within the chloroplast genome of C. delaneyi populations. Although extant populations are allopatric to C. scabrella and C. highlandsensis populations speculation of historic spatial patterni ng could include more contiguous ranges. Groups that have a tendency to hybridiz e may eventually replace one of the species, however until replacement is completed, the population may demonstrate features of being mixed or parapatric (McKinnon et al., 2004). As in the case of C. delaneyi two such populations do ex ist. First is the mixed haplotype population at the IRCL location, and the second is at RSLND, a population located directly southeast of IRCL, which cont ain 2 distinct haplotypes, one from each of the AR and the LWR haplotype. Adaptive strategies to the different landscaping and environmental conditions of Florida may have influenced the morphological changes found within C. delaneyi species. AR populations, as previously in dicated, are larger plants with a more sturdy structure than ar e the LWR populations. Thes e populations, which are established in open, sandy areas, may have adapted both physically and genetically to be more conducive to su ch harsh conditions. Accordingly, adaptation to the shaded turkey oak sandhi lls and longleaf pine habitats may have contributed to the reduction in plant size of LWR populations. Alternative hypotheses to introgression have been described in several papers, including Comes et al. (1997), Tsitrone et al. (2003), McKinnon et al. (2004), and Okuyama et al. (2005). The first of these is lineage sorting. This process involves either the preser vation or elimination of ancestral
41 polymorphisms in the descendant groups This symplesiomorphic condition between AR and C. highlandsensis is not very apparent. A more convincing scenario for this hypothesis would be t hat the polymorphisms would be shared within AR and LWR C. delaneyi and possibly C. scabrella as these groups are more closely related to each other than t hey are to the more distantly related C. highlandsensis The next hypothesis is that of c onvergent evolution. This reflects a condition of homoplasy rather than identity by descent. Although the populations of C. highlandsensis and AR are geographically close to each other, the probability that both hav e undergone similar mutation processes as a result of adaptation to similar environmental condi tions resulting in the same shared polymorphisms appear to be coincidental. The same can be said for C. scabrella and LWR populations. Finally, recurrent hybridization has been suspect with inconsistencies found in gene trees. This process involves frequent hybridization events that would affect either the or ganellar or nuclear genomes, which in turn would be passed on in a directional pattern to a resident species. While these alternative hypotheses seem unlikely in C. delaneyi populations, further testing is needed to completely rule these out. The most likely status for the ances tral state, based on the chloroplast data, appears to be t hat found in the C. highlandsensis and AR C. delaneyi, and the LWR C. delaneyi and C. scabrella are derived from this character state. While the restriction enzyme digests, doc umented to be identical for restriction enzymes in closely related species (Olmstead et al., 1994), lend support to the unconventional relationships between thes e groups by either the loss or the
42 acquisition of both restriction sites, the most convincing evidence comes from the direct sequencing of the ccmp3 region, whic h uncovered 2 insertion sites in the latter two groups. In addition, the alignm ent from the BLAST search of 7 plant families, including 3 Asteraceae, was used to assist in the determination of the ancestral state. The 4 bps that aligned in the first insertion site were not clearly comparable to those of the sample s equences. The alignment of the 18 21 bases of the outgroups at the second insert ion site show no signs of a duplication event as was found in the study samples. Additionally, these bps are exact in the 3 Asteraceae families and similar with the exception of 1 bp difference in the Solanaceae and Altingiaceae groups. The Rosaceae and Caryophyllaceae groups are both distinct from all of t he others. Comparisons between and within these families contribute to the conclusion that C. highlandsensis and AR contain the ancestral state. Nuclear data will be required to verify the ancestral state because the nature of inher itance of the chloroplas t genome as a complete single unit may interfere with interpreti ng patterns of species divergence due to introgression events (Olmstead et al 1994).
43 CONCLUSION The relationship between the LWR and AR C. delaneyi populations has not been resolved to its fullest potentia l. Clearly a distinction between C. delaneyi species has been identified based on chloroplast data alone, which may be the result of chloroplast capture. The discove ry of the distinct haplotypes gives rise for the need to distinguish suitable c onservation management practices for each individual ecotype if these populations ar e to be maintained in the wild. The LWR populations seem to be more at ri sk of eventual extinction than the AR populations. Although these populations have not yet been listed as endangered, factors such as human encroachment and recent years of severe weather conditions have proven detrimental to existing populations. Unless plans are implemented soon, these plants are at severe risk. In order to carry out a sound conser vation program, proper identification and prioritization of species, knowledge of habitat requirements as well as genetic diversity in populations must be established (Partel et al., 2004; Lee et al. 2006). The viability of a populat ion is controlled by its vital rates, which in turn are affected by genetic and environmental pr ocesses, respectively. An increase in genetic diversity may be a key component in a populations ability to survive environmental changes, either natural or anthropogenic (Lee et al. 2006). With an increase of human influx into sensitive areas, habitat fragmentation is
44 increasing. Once plant populations bec ome isolated, the general trend is a decrease in genetic diversity and a decreas e in population fit ness. Therefore, factors that need to be considered w hen designing a conservation plan should include the size of the population and a method to st rengthen gene flow among populations (Gao, 2005). One possible solution to prevent the loss of genetic diversity would be to generate a seed bank and stock plant collect ion of the wild plant populations. These collections must be representative of the actual population in order to maintain the genetic integrit y of the wild populations if it becomes necessary to use the seeds or plant stocks for restor ation purposes. If care is not taken when establishing a seed bank and the seeds are used in the wild populations, the genetic structure will be alter ed. Other options of conservation may want to be considered first. However, if these populations are already severely isolated and lack genetic diversity they may require an in flux from other populations in order to increase their fitness (Segarra-Moragues et al., 2005). With regards to the C. delaneyi populations, it is clear that two separate management programs would be needed. The LWR populations are more fragmented and isolated than the AR populations. In addition, there appears to be little chloroplast divergence between the LWR and C. scabrella populations. The AR populations are, for the time being, more robust and less fragmented than the LWR populations. Ther efore, they may not ha ve been subjected to a loss of genetic diversity as a result of is olation, thus maintaining the ancestral state of genetic variation. Therefore, conservation management would want to
45 include these larger populations in their c onservation efforts in order to preserve the ancestral structure of the species. In concert with this logic, allopatric populations such as the LWR populations demonstrate the change of genetic structure of the species as a result of adaptation processes thus representing important components in t he evolutionary history of a species warranting protection as well. Within popula tion variation like those found in C. scabrella populations results when the absence of gene flow from other populations occur after events such as Founders Effect or genetic drift. Natural selection takes over, selecting the genotypes that are most fit for the conditions of the establishing species. Again, this proce ss warrants protection of species in order to maintain intraspecific variati on by maintaining the gene flow among populations (Gao, 2005). The use of chloroplast molecular mark ers has proven effective in detecting cpDNA variations in this study. Th ey have identified two haplotypes of C. delaneyi, one haplotype withi n a population of C. scabrella and one haplotype within an AR population. Nu clear data is needed to corroborate the results. The nuclear markers used in this study were not as effective, thus optimizing conditions for these reactions will be requi red in order to produce accurate data.
46 FUTURE DIRECTION As stated in the definition of an ESU by Moritz (1994), data from the nuclear genome must be examined along with the organellar genome and show a significant amount of divergence in order to accurately determine phylogenies of species, and determine an ESU. Ther efore, more progress will need to be made with the nuclear genome. Initial reactions conducted in this study that involved PCR amplification and sequencing of the intron region of the Actin 1 gene proved to be problematic. One solution would be to design primers for this region either from sequences retrie ved from successful reactions on the C. delaneyi samples or by searching for simila r sequences in the Asteraceae family using Genbank. Additionally, exploring other nuclear regions such as the glycerol-3-phosphate acyltransferase (GPAT) gene (Tank & Sang, 2001), the nitrate reductase intron (Howarth & Baum, 2002), and the glyceraldehyde 3phosphate dehydrogenase (G3pdh) gene (Strand et al., 1997) may prove more successful. The cpDNA variations in natural populations need to be monitored in order to evaluate the amount of genetic diversit y maintained in populations. Initial data collected serves as a baseline of the chloroplast genomic structure of these populations at the current time. This as pect is important for the mixed IRCL
47 populations and RSLND popul ations in order to determi ne current evolutionary and adaptive processes taking place. To investigate the introgression hypot hesis, experimental design of plant crosses would determine the female fitness requirements proposed by Tsitrone et al. (2003). For example, setting up crosses between C. highlandsensis and AR C. delaneyi, using ovules from C. highlandsensis and the pollen from a close relative of AR C. delaneyi that does not contain t he invading chloroplast. A cytoplasm substitution line would be cr eated by repeated backcrossings and the female fitness could be determined by seed production. Along with chloroplast markers, mitochondrial markers can be us ed to compare both of these gene trees together. Similarities among them w ould indicate that introgression of the chloroplast has occurred. If, however there are inconsistencies found, homoplasy may be involved.
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