Phylogenetic analysis of western Atlantic grouper (Epinephelinae) and other serranids (Teleostei : Serranidae) by peptide mapping of homologs of LDH-Aâ‚„

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Phylogenetic analysis of western Atlantic grouper (Epinephelinae) and other serranids (Teleostei : Serranidae) by peptide mapping of homologs of LDH-Aâ‚„

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Title:
Phylogenetic analysis of western Atlantic grouper (Epinephelinae) and other serranids (Teleostei : Serranidae) by peptide mapping of homologs of LDH-Aâ‚„
Creator:
Stokes, Rodger T.
Place of Publication:
Tampa, Florida
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University of South Florida
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Language:
English
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xi, 108 leaves : ill. ; 29 cm

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Subjects / Keywords:
Serranidae ( lcsh )
Amino acid sequence ( lcsh )
Lactate dehydrogenase ( lcsh )
Cladistic analysis ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

Notes

General Note:
Thesis (M.S.)--University of South Florida, 1996. Includes bibliographical references (leaves 48-51).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
022477258 ( ALEPH )
35771525 ( OCLC )
F51-00128 ( USFLDC DOI )
f51.128 ( USFLDC Handle )

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PHYLOGENETIC ANALYSIS OF WESTERN ATLANTIC GROUPER (EPINEPHELINAE) AND OTHER SERRANIDS (TELEOSTEI: SERRANIDAE) BY PEPTIDE MAPPING OF HOMOLOGS OF LDH-A4 by RODGER T. STOKES A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida May 1996 Major Professor: Raymond R. Wilson, Jr., Ph.D.

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Graduate School University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master's Thesis of RODGER T. STOKES with a major in Biological Oceanography has been approved by the Examining Committee o n December 15, 199 5 as satisfactory for the thesis requirement for the Master of Science degree Examininq f dommi t tee. : A Wilson, Jr. Ph.D.

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DEDICATION To my Dad, Taylor and Mom, Kathy who always believe in me and taught me to strive for my dreams, and to my girlfriend Nina de Verteuil who gives me immense support.

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ACKNOWLEDGMENTS I wish to express my gratitude for the helpful scholarly advice of Dr. Raymond R. Wilson, without whose guidance this thesis would never have come to fruition. Special thanks are due Dr. Lisa Robbins, Department of Geology for help with the HPLC, Mr. Tom Young for laboratory help, guidance and comraderie, Mr. Lew Bullock, Mr. Ron Taylor and Mr. Ron MacConnahey for donating specimens, and Mr. Mike Tringali, Mr. Seifu Seyoum, and Mr Mike Swain for assistance with data analysis. I will be forever appreciative of the support of my Mom, Dad, and girlfriend Nina, who never let me get down and urged me forward. To all the many friends and colleagues who have helped me sometime throughout this project, I extend my sincere thanks.

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TABLE OF CONTENTS LIST OF TABLES ii LIST OF FIGURES iii LIST OF SYMBOLS AND ACRONYMS vii ABSTRACT ix 1. INTRODUCTION 1 2. MATERIALS AND METHODS 11 Specimen Collection 11 Protein Electrophoresis 12 Affinity Chromatography Column Construction 12 Lactate Dehydrogenase (LDH-A4 ) Protein Purification 13 Denaturation, Carboxymethylation, and Tryptic Digestion of Protein 16 Reversed Phase High Performance Liquid Chromatography 18 Data Analysis 19 3 RESULTS 25 4. DISCUSSION 38 5. REFERENCES 48 6 APPENDICES 52 APPENDIX 1. PEPTIDE MAPS OF SINGLE SPECIES 5 3 APPENDIX 2. PEPTIDE MAPS OF TWO SPECIES 71 i

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Table 1. Table 2. Table 3. Table 4. LIST OF TABLES Species Included in Study Peak Presence/Absence Data from Pairwise Comparisons of Peptide Maps (Chromatograms) Dissimilarity Matrix of Peptide Maps Among 16 Serranids and 1 Centropomid (d = 1 -S) Distance Matrix of Peptide Maps Among 16 Serranids and 1 Centropomid (d = -ln S ) ii 10 22 23 24

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LIST OF FIGURES Figure 1. Classification of American Groupers (Smith, 1971) 4 Figure 2. Conversion of Pyruvic Acid to Lactic Acid by LDH 7 Figure 3. Electrophoretic Mobilities of LDH of Western Atlantic Groupers 26 Figure 4 Consensus Tree Produced by Wagner Parsimony Analysis of Binary Data in Table 2 Using the PENNY Algorithm 29 Figure 5. Consensus Tree Produced by Dollo Parsimony Analysis of Binary Data in Table 2 Using the DOLPENNY Algorithm 30 Figure 6. Consensus Tree Produced by Branch and Bound Parsimony Analysis of Binary Data in Table 2 Using Bootstrapping (50% Majority Rule) 32 Figure 7. Tree Produced by Fitch-Margoliash Distance Method Analysis of Dissimilarity Data in Table 3. 33 Figure 8. Tree Produced by Fitch-Margoliash Distance Method Analysis of Distance Data in Table 4. 34 Figure 9. Tree Produced by Neighbor-Joining Distance Method Analysis of Dissimilarity Data in Table 3 35 Figure 10. Tree Produced by Neighbor-Joining Distance Method Analysis of Distance Data in Table 4. 36 Figure 11. Tree modified from Smith (1971) showing only species included in this study. 39 Figure 12. Peptide Map of Centropomus undecimalis 54 iii

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Figure 13. Peptide Map of Paralabrax clathratus 55 Figure 14. Peptide Map of Paralabrax maculatofasciatus 56 Figure 15. Peptide Map of Paralabrax nebulifer 57 Figure 16. Peptide Map of Epinephelus adscensionis 58 Figure 17. Peptide Map of Epinephelus cruentatus 59 Figure 18. Peptide Map of Epinephelus drummondhayi 60 Figure 19. Peptide Map of Epinephelus fulvus 61 Figure 20. Peptide Map of Epinephelus inermis 62 Figure 21. Peptide Map of Epinephelus itajara 63 Figure 22. Peptide Map of Epinephelus mario 64 Figure 23. Peptide Map of Mycteroperca bonaci 65 Figure 24. Peptide Map of Mycteroperca interstitialis 66 Figure 25. Peptide Map of Mycteroperca microlepis 67 Figure 26. Peptide Map of My cteroperca phenax 68 Figure 27. Peptide Map of Mycteroperca venenosa 69 Figure 28. Peptide Map o f Paranthias furcifer 70 Figure 29. Peptide Map of Centropomus undecimalis and Paralabrax clathratus 72 Figure 30. Peptide Map of Centropomus undecimalis and Paralabrax maculatofasciatus 73 Figure 31. Peptide Map of Centropomus undecimalis and Paralabrax nebulifer 74 Figure 32. Peptide Map of Centropomus undecimalis and Epinephelus adscensionis 75 Figure 33. Peptide Map of Centropomus undecimalis and Epinephelus cruentatus 76 Figure 34. Peptide Map of Centropomus undecimalis and Epinephelus drummondhayi 77 iv

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Figure 35. Peptide Map of Cen tropomus undecimalis and Epinephelus fulvus 78 Figure 36. Peptide Map of Centropomus undecimalis and Epinephelus inermis 79 Figure 37. Peptide Map of Centropomus undecimalis and Epinephelus i tajara 80 Figure 38. Peptide Map of Centropomus undecimalis and Epinephelus moria 81 Figure 39. Peptide Map of Cen tropomus undecimalis and Mycteroperca bonaci 82 Figure 40. Peptide Map of Centropomus undecimalis and Mycteroperca interstitialis 83 Figure 41. Peptide Map of Cen tropomus undecimalis and Mycteroperca microlepis 84 Figure 42. Peptide Map of Cen tropomus undecimalis and Mycteroperca phenax 85 Figure 43. Peptide Map of Centropomus undecimalis and Mycteroperca venenosa 86 Figure 44. Peptide Map of Cen tropomus undecimalis and Paranthias furcifer 87 Figure 45. Peptide Map of Paralabrax clathratus and Paralabrax maculatofasciatus 88 Figure 46. Peptide Map of Paralabrax clathratus and Paralabrax nebulifer 89 Figure 47. Peptide Map of Paralabrax nebulifer and Paralabrax maculatofasciatus 90 Figure 48. Peptide Map of Epinephelus mario and Paralabrax clathratus 91 Figure 49. Peptide Map of Epinephelus moria and Paralabrax maculatofasciatus 92 Figure 50. Peptide Map of Epinephelus mario and Paralabrax nebulifer 93 Figure 51. Peptide Map of Epinephelus mario and Epinephelus adscensionis 94 Figure 52. Peptide Map of Epinephelus mario and Epinephelus cruentatus 95 v

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Figure 53. Peptide Map of Epinephelus morio and Epinephelus drummondhayi 96 Figure 54. Peptide Map of Epinephelu s morio and Epinephelus fulvus 97 Figure 55. Peptide Map of Epinephelu s morio and Epinephelus inermis 98 Figure 56. Peptide Map of Epinephelus morio and Epinephelus i tajara 99 Figure 57. Peptide Map of Epinephelus morio and Mycteroperca bonaci 100 Figure 58. Peptide Map of Epinephelus morio and Mycteroperca microlepis 1 0 1 Figure 59. Peptide Map of Epinephelus morio and Mycteroperca phenax 102 Figure 60. Peptide Map of Epinephelus morio and Mycteroperca venenosa 103 Figure 61. Peptide Map of Epinephelus morio and Paranthias furcifer 104 Figure 62. Peptide Map of Mycteroperca microlepis and Epinephelus itajara 105 Figure 63. Peptide Map of Mycteroperca phenax and Mycteroperca interstitialis 106 Figure 64. Peptide Map of Paranthias furcifer and Epinephelus i tajara 107 Figure 65. Peptide Map of Paranthias furcifer and Mycteroperca venenosa 108 vi

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DNA DTT EDTA g HCl IAA KCl L LDH M mg ml mM MW NAD NADH nm RFLP RPHPLC LIST OF SYMBOLS AND ACRONYMS Spectrophotometric Absorbance at Wavelength 280nm Deoxyribonucleic Acid Dithiothreitol Ethyleneditetraaminic Acid Grams or Gravity (Force of) Hydrochloric Acid Iodoacetic Acid Potassium Chloride Potassium Phosphate Liters Lactate Dehydrogenase Molar concentration of a solution Milligrams Milliliter Millimolar concentration of a solution Molecular Weight Nicotinamide Adenine Dinucleotide Nicotinamide Adenine Dinucleotide, Reduced Form Nanometers Restriction Fragment Length Polymorphism Reversed-Phase High Performance Liquid Chromatography vii

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TFA p.g p.l p.M p.moles lOX Triflouroacetic Acid Micrograms Microliter Micromolar concentration of a solution Micromoles Ten Times Concentration Needed viii

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PHYLOGENETIC ANALYSIS OF WESTERN ATLANTIC GROUPERS (EPINEPHELINAE) I AND OTHER SERRANIDS (TELEOSTEI: SERRANIDAE) BY PEPTIDE MAPPING OF HOMOLOGS OF LDH-A4 by RODGER T. STOKES 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 May 1996 Major Professor: Raymond R. Wilson, Jr. Ph.D. lX

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Seventeen species of fishes including sixteen serranids and one centropomid were collected from various sources and used to test C.L. Smith's (1971) taxonomic hypothesis of the American grouper interrelationships by peptide mapping of LDH. Lactate dehydrogenase was extracted and purified from the white muscle of one fish of each species using affinity chromatography. After electrophoretic determination of the presence of a single subunit, the lactate dehydrogenase was denatured, carboxyrnethylated, and cut with trypsin. The resuling peptides were prepared and separated with high performance liquid chromatography (HPLC) resulting in chromatograms (peptide maps) for each species. Chromatograms were compared between species and peaks were numbered sequentially. Mixed runs consisting of the peptides of the two species were performed to help designate individual peaks among the chromatograms of different species. Peak data were coded into a binary (presence/absence) data set and analyzed with discrete character (PENNY, DOLPENNY, and Maximum Parsimony Bootstrapping) computer parsimony programs. The binary data were also transformed into dissimilarity and distance matrices and analyzed with Fitch-Margoliash and Neighbor-Joining distance programs. The resulting trees did not agree well with Smith's morphometric hypothesis, with many of the species he grouped falling into different clades. Possible reasons for disagreement include: possible taxonomic oversplitting of groupers, the strength of the X

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peptide data/ the taxonomic value of the lactate dehydrogenase molecule1 or the imperfect fit of the peptide data to the assumptions inherent in the methods of analysis. Peptide mapping was not a suitably strong method for the taxonomic clarification of the interrelationships of the groupers hypothesized by Smith (1971) Abstract Approved: Major Profesuor: Raymond R. Wilson1 Jr.1 Ph.D. Assistant Professor/ Department of Marine Science Date Xl

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1 1. INTRODUCTION The Percoidei is the largest and most diverse suborder of the teleostean fishes. This group, belonging to the order Perciformes with 73 families, 589 genera, and 3524 species, is probably the basal evolutionary group from which all other perciform fishes as well as the orders Pleuronectiformes and Tetraodontiformes are derived (Nelson, 1984). Regan (1913b) defined the Percoidei "by having the absenc e of special peculiarities which characterize the suborders of the Percomorphi [=Perciformes] ." No better definition has been put forth since. The Percoidei has thus become a "dumping ground" for all families that have retained the generalized perciform characters (Johnson, 1984) At the heart of this problem are the basal percoids. Without even a single synapomorphy to unite the group, these families are especially problematic. A prime example of this is the family of sea basses, Serranidae. Johnson (1983) states that this family "has historically served as a classificatory 'waste basket' within the percoids, providing a convenient pigeonhole for those generalized perchlike fishes whose relationships could not obviously be shown to

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2 lie with some other percoid family." In essence, the Serranidae serves the Percoidei much in the same capacity as the Percoidei serves the Perciformes. The Serranidae consists of about 62 genera and 449 species (Nelson, 1994) Gosline (1966) defined the Serranidae and Johnson redefined it cladistically as possessing four derived anatomical features: (1) Presence of three opercular spines, (2) absence of the procurrent spur, (3) absence of third preural radial cartilages, and (4) absence of a posterior uroneural. Within the family, both Johnson (1983) and Gosline (1966) recognize three subfamilies, the Anthiinae, Epinephelinae, and Serraninae. Johnson (1983) further delineated five tribes of the Epinephelinae as follows: Niphonini, Epinephelinae, Diploprionini, Liopropomini, and Grammistini. The groupers fall into the Epinephelini which includes the genera Epinephelus, Mycteroperca, and Paranthias among others. In the eastern Gulf of Mexico, the serranid fauna is diverse and includes at least 47 species (Bullock and Smith, 1991) Of special interest to many researchers are the groupers due to their commercial importance. C.L. Smith (1971) revised the American groupers morphologically and proposed a phylogenetic classification of the interrelationships of the genera mentioned above. He recognized the Gulf of Mexico and the West Indies as centers of speciation of these groups. His study included only the

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three genera mentioned above of which genus Epinephelus was subdivided into five subgenera: Alphestes, Promicrops, Epinephelus, Cephalopholis, and Dermatolepis. His classification extends even beyond subgenera hypothesizing species-groups based on their shared similarities (Figure 1) Since Smith, no one has attempted a phylogenetic classification of these species to either support or refute his conclusions, and much might be learned from a more rigorous analysis, using the tools of modern systematics. 3 Systematic studies involving adult morphology have the inherent problems of character definition and the environmental influence on them. Using larval characteristics is also problematic due to the lack of early life history information and developmental series of these fishes (but see Baldwin and Johnson, 1993). Biochemical or molecular techniques could circumvent these problems by examining a gene itself, or the molecular product of a gene. Electrophoretic studies of proteins can provide a great amount of data in a relatively short amount of time (Buth, 1984, Wilson et al., 1990, Wilson, 1994 and many others), but there are intrinsic problems with this technique as well. For example, Ayala (1982) reported that approximately only 33% of the substitutions of amino acids, or point mutations in a protein gene, result in a change in the net electric charge; thus, a large fraction of the genetic variation among proteins may be undetectable through

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4 fulvus c ruenlotus ......:::::;...---ponomens i s striatut guttotus niveotus _,..---flovolimbotllf nigritus oconthistillf =------:::::-my1tocinvs onoiOCJU1 --odscensionis ----------c::c::::: itojoro -----...,.--::::: ofer multiguttotus v enenoso .. bonoci jo
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electrophoresis as isomobility is the criterion for "sameness". Another problem is in the designation of allozyme characters. Coding of electrophoretic data into discrete characters involves determining what comprises a character, how many states are included in a certain character scheme, and their subsequent ordering. Several methods have been proposed including the "independent allele" model, the "shared allele" model, the "minimum allele turnover" model, the "relative mobility" model, and the "systematic" method. There is still as yet, no agreed upon method, although the problems have been recognized (Buth, 1984) 5 Peptide mapping stands as a potential alternative to these two classic approaches for determining differences among taxonomic groups. The approach employs the separation of proteolytically digested proteins through the use of Reversed-Phase High Performance Liquid Chromatography (RPHPLC) In doing so, the technique affords a much greater resolution of differences between protein molecules than in standard starch-gel electrophoresis as is illustrated by Rivier and McClintock (1983) where human and rabbit insulin were separated. The only difference between the two insulin molecules is the change of one amino acid, out of 55, which differed by only one methylene group. The ability to separate peptides using RPHPLC depends on the subtle differences in the hydrophobicity of the

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molecule which determines the interaction it has with the RPHPLC column surface. Hydrophobicity in turn depends upon the amino acid sequence of the protein. Thus it follows that very small changes or differences could be resolved using this technique. 6 To employ peptide mapping as a taxonomic research tool, a specific molecule must be chosen to study. Upon making this decision, it should be acknowledged that the molecule must be extremely pure for use in an RPHPLC study. This poses a certain limitation in that purification methods exist for only a few such proteins. Past studies in molecular systematics have used metabolic enzymes as their focus, specifically Lactate Dehydrogenase, LDH (Wilson et al. 1991). This seems to be an excellent molecule to use in such a study, for it can be purified using an oxamateaminohexyl Sepharose column with specific affinity for LDH. Purification of LDH with an affinity column must consider that vertebrates possess two dominant LDH isozymes coded from two different genes (Markert et al., 1975). LDH-A, or M type, predominates in white muscle tissue and under anaerobic conditions serves to reduce pyruvate to lactate (Stock and Whitt, 1992). LDH-B, or H type, is primarily found in heart muscle and functions under aerobic conditions where it oxidizes lactate to pyruvate (Stock and Whitt, 1992). A third isozyme, LDH-C is present in mammalian sperm, columbid birds (Wheat and Goldberg, 1983),

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and typically in the eye or liver of actinopterygiian (ray-finned) fishes (Markert et al., 1975), but sometimes is expressed in white skeletal muscle as well (Wilson, pers. com.). 0 C-OH LDH I HO-C-H I CH3 Pyruvic Acid (s)-(+)-Lactic Acid Figure 2. Conversion of Pyruvic Acid to Lactic Acid by LDH (Solomons, 1986) Figure 2 illustrates the reaction for which the oxamate-aminohexyl Sepharose column is designed. In the presence of an enzyme cofactor, the reaction is favored toward the right and the LDH binds to the pyruvate or oxamate, its analog on the column. Since the oxamate is attached to Sepharose in the column, under the conditions described above, the LDH is retained on the column upon loading of the raw extract. Once the unbound proteins are rinsed off, the LDH can be eluted with a buffer solution 7 containing which forces the reaction back to the left, causing the enzyme to release the oxamate. The LDH can then be prepared for RPHPLC. LDH is present in the form of tetramers, often composed of two different isozymes coded for at two different genes, making them heterotetramers as opposed to homotetramers when

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all four subunits are of the same isozyme. LDH in white muscle can have the forms A4 A3B1 A2B2 A1B3 and B4 This can be determined electrophoretically and the correct LDH can be isolated by collecting fractions off the column. Differing affinities of each subunit to the column allow this to occur (O'Carra et al., 1974). 8 Once purification is achieved, peptides are generated by digesting the protein with the enzyme trypsin, which cleaves (hydrolyzes) the peptide bonds of the amino acids lysine and arginine specifically (Solomons, 1986). The digested peptides can then be loaded onto a RPHPLC column which has an organic stationary phase and a mobile inorganic polar phase (a gradient of TFA in acetonitrile) The peptides adsorb onto the hydrophobic surface of the column and desorb only when the polarity of the mobile phase reaches a certain level. The passing of the peptides out of the column is recorded as absorbance peaks at 280 nm wavelength by the data acquisition program as they pass through the detector, resulting in a chromatogram or "peptide map. The peptide maps are then compared between species and the absorbance peaks are then enumerated, and scored as either present (1) or absent (0 ) Pairwise comparisons can be performed on the peptide maps using these data with phylogenetic computer programs. The purpose of the present study was undertaken to

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9 produce a phylogenetic hypothesis of the Atlantic American grouper s (Table 1) using the LDHA4 (EC 1.1.1.27; NAD : lactate oxidoreductase) homologs from wh ite muscle tissue as a character for comparison. It will be used to test the earlier phylogenetic hypotheses o f C.L. S mith (1971) at the generic, subgeneric, and species level. Two outgroups were selected for comparison to "root" the tree, Centropomus undecimalis and Paralabrax spp. based on their supposed phylogenetic position relativ e to the groupers. These genera are placed in a more taxonomically primitive position, with Centropomus undecimalis belonging to a very basal percoid family Centropomidae (Nelson, 1994) and Paralabrax spp. belonging to the Serranidae subfamily Serraninae (Baldwin and Johnson, 1993). All other species included in the present study are included in the Serranidae subfamily Epinephelinae. The use of these taxa as outgroups will serve to root the trees found in analysis, as well as to allow comparison to other studies using the same species.

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Table 1. Species Included in Study Centropomus undecimalis Paralabrax clathratus Paralabrax maculatofasciatus Paralabrax nebulifer Epinephelus (Cephalopholis) cruentatus Epinephelus (Cephalopholis) fulvus Epinephelus (Derma tol epi s ) inermis Epinephelus (Epinephelus) adscensionis Epinephelus (Epinephelus) drummondhayi Epinephelus ( Epinephel us) morio Epinephelus (Promicrops) i tajara Mycteroperca bonaci Mycteroperca interstitialis Mycteroperca microlepis Mycteroperca phenax Mycteroperca venenosa Paranthias furcifer 10

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11 2. MATERIALS AND METHODS Specimen Collection Epinephelines and Centropomus undecimalis were acquired between 1991 and 1994 from the Florida Keys and the western Gulf of Mexico by sampling charter boat catches (The Florida Fisherman II) by hook and line on biological research cruises, by spearfishing with SCUBA, and from scientists at the Florida Department of Environmental Protection. Specimens of Paralabrax spp. were acquired from Ron MacConnaughey at the Scripps Institution of Oceanography in La Jolla, California. All specimens except those of Paralabrax spp. were sight identified, bagged, labelled, and frozen as quickly as possible after collection. When a shipboard freezer was not available, fish were kept on ice until freezing. Paralabrax specimens were sight identified at Scripps and transported on dry ice to Dr. Raymond Wilson's laboratory at the University of South Florida, St. Petersburg. Samples were subsequently maintained at -87C until used.

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12 Protein Electrophoresis White muscle extracts from each species, prepared by homogenizing in equal volumes of cold 100 mM KP02 4 pH 7.0 (Selander et al, 1971), were used in horizontal 12% starch-gel electrophoresis (tris-citrate, pH 6.9 and 8.0) and histochemically stained (Shaw and Prasad, 1970). The lactate dehydrogenase homologues were compared among all species based on electrophoretic mobility. To ensure LDH is homotetrameric. Affinity Chromatography Column Construction Separation and purification of LDH is most easily accomplished through affinity chromatography. The procedure involves the adsorption of the protein of interest onto a solid support matrix. Preparation of the affinity column followed the procedure of Yancey and Somero (1978), and the laboratory notes that Dr. Wilson borrowed from Dr. Joe Siebenaller, Department of Zoology and Physiology, Louisiana State University as follows: 1. Wash pre-swelled w-amino hexyl-Sepharose 4B over a sintered glass filter with 3 liters of deionized water. 2. Dissolve 15 grams potassium oxalate in 35 ml deionized water (may need to heat)

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13 3. Dissolve 5 grams 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma # E7750) in 10 ml deionized water. 4. Mix solutions from steps 1 and 2 above and let sit for 5 minutes. 5. Add mixture from step 4 t o gel of Sepharose beads and shake gently for 26 hours. 6. Wash beads with 3 liters of deionized water. 7 Suspend beads in 0 5 M KCl, 50 mM KP04 2-, pH 6.8 8. Store in chromatography refrigerator at 4C until use. (a 0.02% sodium azide solution can be added to beads to control biological growth when not in use) The beads were then poured into a Kontes (Vineland, N.J.) chromaflex AQ glass column with a sintered glass filter at the bottom to keep the beads from escaping while allowing the buffer solution to flow more or less unimpeded. More buffer was added, the beads settled to the bottom and a small amount of buffer was allowed to accumulate above the bead gel. The buffer was maintained throughout the column use to keep the beads moist. Buffer was added through a small plastic tubing by way of gravity flow started by a siphon. Column flow rate was adjusted to approximately 125 ml per hour . Lactate Dehyrdogenase (LDH-A4), Protein Purification The lactate dehydrogenase A4 isozyme was isolated and

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14 purified from all fish species included in this study (see Introduction) Purification began with approximately 100 g of white muscle tissue filleted from the right side of each fish. Muscle tissue was homogenized in a blender (Penncrest) with 300 ml of 50 mM KP04 2-, 1 mM 2-mercaptoethanol, pH 6.8, for 1 2 minutes. Homogenate was transferred to a beaker and then stirred for 30 min in a chromatography refrigerator at 4C. The homogenate was then centrifuged in a Sorvall RC5C (DuPont) centrifuge at 9000 rpm (13,200 x g) for 30 minutes at 4C. The supernatant was collected and filtered through glass wool into a graduated cylinder. A 1-2 ml aliquot was removed and frozen for later reference. The volume of the supernatant was recorded and an appropriate amount of 2 M KCl, 50 mM KP04 2, pH 6.8 stock solution was added bringing the solution to 0.5M KCl to prevent non-specific electrostatic binding to the column beads (Scopes, 1987). The sample was then brought to 200 NADH by addition of reduced from disodium salt (Sigma # N-8129) The Oxamate-Sepharose affinity column was preequilibrated with 500 ml of buffer containing 50 mM KP04 2-, 0.5 M KCl, 200 NADH, pH 6.8. Following equilibration the sample was loaded onto the column and the column was f lushed with the same buffer as used in equilibration. This flushing washes all excess unbound proteins from the column leaving only ligand specific proteins on the column. Flushing

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continued until spectrophotometric absorbance at A280 equalled zero (on Milton Roy Spectronic 1201), after being zeroed against a blank of flushing buffer. Elution of LDH was accomplished by passing approximately 500 ml of a buffer of 50 mM KP04 2-, 0.5 KCl, 2.2 mM NAD+ (Sigma# N-7381) through the column. The NAD drives the reaction previously outlined in the opposite direction causing the enzyme (LDH) to release the ligand (oxamate) and thus eluting it from the bottom of the column. The column was stripped with 1 liter of 50 mM KP04 2 1M KCl, pH 6.8. This removes any protein still on the column by disrupting all nonhydrophobic interactions between ligand and proteins (Scopes, 1987). 15 After elution, the presence of LDH was confirmed via an activity assay. This was done by placing 3 ml of a solution of 80 mM imidazole-HCl, 2 mM sodium pyruvate, and 0.15 mM NADH, into a spectrophotometric quartz cuvette, adding 20-30 of purified LDH eluent and reading the absorbance at A340 As LDH catalyzes the reaction and NADH is converted to NAD+ absorbance at A3 4 0 where NADH absorbs, drops. This allows for the presence of LDH to be ascertained. Next the enzyme was concentrated into a volume of 5 to 7 ml by membrane filtration under pressurized nitrogen using an Amicon (Danvers, MA) PM30 membrane. The concentrate was dialyzed overnight versus 4 L of 0.1 M Tris-HCl, 0 5 M EDTA, pH 8.3. All dialysis was conducted using Spectrapor

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(Spectrum Med. Ind. Inc. Houston, TX) molecular porous membrane tubing. The LDH was precipitated in a solutio n saturated with ammonium sulfate and maintained in the dialysis tube at 4C until tryptic digests were performed. Denatu ration, Ca rboxymethylation, and Tryptic Digestion of Protein Protein was dialyzed against 8 liters of 0.1 M Tris, 0.5 mM EDTA, pH 8 .3, over two days to remove ammonium sulfate. Amount of protein present was estimated using the 16 spectrophotometric absorbance at A280 (Bell and Bell, 1988). Appropriate amounts ( 5 mg) of each sampl e were then removed for eventual digestion with trypsin; the remainder was reprecipitated in ammonium sulfate, and maintained in dialysis tubing at 4C. The reconstituted native LDH was denatured by addition of solid urea to a concentration of 8 M, followed by addition of two fold molar excess of dithiothreitol (DTT) (Sigma # D-9779) over the cysteine residues. This reduces the disulfide bonds of cysteine forming a stable cyclic disulfide greatl y displacing the reaction equilibrium to the right (Torchinskii, 1974) A sample calculation is as follows: 1. Molecular weight of LDH is 36,000 Daltons or 36,000 LDH = # protein to be used/(36,000 i.e. 5 mg or 5000 then (5000 = 0 .1389 LDH.

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2. Assuming 7 cysteine residues per subunit, of cysteine can be calculated, i.e. (0.1389 LDH) (7 cysteine residues/subunit) = 0.972 cysteine. 17 3. Doubling the amount of cysteine gives the amount of DTT needed for a two-fold excess, i.e. (0.972) (2) = 1.94 DTT. Molecular weight of DTT is 154 # of of DTT = DTT) (154 i.e. (1.94 (154 = 299 DTT. A lOX(lO solution of DTT is used due to weighing difficulty of powder, thus an addition of 2.99 of the lOX solution is needed. After addition of urea and DTT, solution was allowed to incubate at room temperature for two hours. Iodoacetic acid (IAA) (Sigma # I-2512) was then added at double the molar ratio of cysteine and DTT to carboxymethylate the sulfhydryl groups formed in disulfide bond cleavage with DTT, resulting in a stable preparation of the reduced protein (Wilkinson, 1986, Torchinskii, 1974, and Crestfield et al., 1963) The amount of IAA to add was determined by addition of of DTT to of cysteine and doubling. Appropriate amounts of a lOX solution were added as with DTT above (step 3 ) Solution was incubated for two hours in the dark to avoid side reactions connected with formation of iodine by oxidation in the light of iodide ions released (Torchinskii, 1974) Reaction was stopped with a 10 times molar excess of DTT. The DTT amount is based on the molar amount of IAA, easily calculated by: 10(# IAA) (molecular weight DTT in = # DTT.

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18 The solution was then dialyzed overnight at 4C against 4 L of 50 mM ammonium bicarbonate, pH 8.3, to remove excess reagents and to reach appropriate buffer conditions for the tryptic digest (Wilkinson, 1986) Trypsin (Sigma # T-8642) was added at a 1% weight:weight ratio to the original amount of protein used. The digestion reaction was allowed to incubate for 6.5-7 hours at 37C. Digestion was terminated by freezing the solution at -87C (Wilkinson, 1986) Samples were lyophyllized for two days at -50C resulting in a dry, purified protein. This powder was maintained at -87C until analysis on HPLC. Reversed Phase High Performance Liquid Chromatography (RPHPLC) Pepitde mapping was performed on each total tryptic digest using an E-Lab gradient controller and data acquisition system, high pressure pump, and an Isco V4 Deuterium lamp absorbance detector equipped with a flow-through cell. A Synchropak (Alltech, Inc. ) C18 RP-P column (250 x 4.6mm) was used to separate peptides. Peptides were detected at 219nm wavelength -the absorbance wavelength of the carbon peptide bonds. Buffer A was triple distilled ultrapure water with 0 .1% (w/v) triflouroacetic acid (HPLC/Spectro grade sequanal quality), pH 2.2 and Buffer B was Acetonitrile (Burdick and Jackson, Inc.),

PAGE 34

19 triflouroacetic acid 0.1% (w/v). Peptides were separated over the following hour long gradient from Wilson et al. (1991) The gradient increased from 0-30% Buffer B in 7 minutes, reacned 50% B in 29 minutes, stayed at 50% for 18 minutes and returned to 0% in 6 minutes. Flow rate was adjusted to 1 ml/min through the column and a 15 min column reequilibration time was allowed ( 100% A) between samples. Column was flushed with and stored in HPLC grade methanol between uses. Mapping of total tryptic digests was done individually for each species, after each species was chromatographed at least three times to ensure reproducibility. Mixing digests of selected problematic species and mapping them together was used to confirm peak assignments. Absorbance data were recorded on diskette and will be archived for later reference. Mixed digest maps were produced from the following combinations of species: C. undecimalis/all others, E .morio/all others, Paralabrax/all other Paralabrax spp. Paranthias furcifer/E. itajara, Paranthias/M. venenosa, E. itajara/M. microlepis, M. phenax/M. interstitialis. Data Analysis Numbering of peaks followed Wilson et al. (1991) A

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20 11reference species11 was designated (Centropomus undecimalis) and each peak was sequential l y numbered i n the order of its elution. Chromatogra ms of eac h addit i onal species were compared with the reference species and all other s to identify both homologous and u nique peaks. Peaks were then renumbered according to their elution times. Peptide data were then analyzed using the discrete character parsimony programs PENNY, (PHYLI P ver. 3 .52c) as in Wilson et al. (1991), DOLPENNY, ( PHYLIP ver. 3.52c) and the PAUP (ver. 3 1 ) maximum parsimony 11branch and bound11 bootstrap method o f Felsenstein (1985). PENNY is a branch and bound algorithm which was first used by Hendy and Penny (1982) This program carries out Wagner parsimony where it is assumed that the ancestral state of each character is unknown, different characters and lineages evolve independently, and reversibility of characters in either direction is allowed. DOLPENNY is a program which carries out the Dollo parsimony metho d of Farris, (197 7 ) This method assumes that the ancestral state is known, the characters and lineages evolve independently, and t hat the probability of a forward change is smaller t han that of a reversion to the ancestral state. The PAUP bootstrap method (Swofford, 1993) involves sampling the original data with replacement to construct a series o f bootstrap repli c a tes o f the same size as the original data set. These replicates are analyzed and the variatio n among these replicate

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21 estimates is taken to be an indication of the error involved in making estimates from the original data. It assumes that each character is identically and individually distributed, and the characters are independent of each other. Each chromatogram was scored according to its HPLC profile (peptide map) such that each of the peaks were scored as being present (1), or absent (0) on individual chromatograms. The result was a data matrix (Table 2). This peak scoring corresponds with that used for discrete, two state (binary) characters as described in Swofford and Olsen (1990) Peptide data were also analyzed using the distance matrix programs FITCH (Fitch-Margoliash distance method) and NEIGHBOR (Neighbor-Joining method) [PHYLIP ver. 3 .52]. Data were entered as a dissimilarity matrix, d = 1 S (Table 3), [dissimilarity = 1 -number of shared peaks between two chromatograms/total number o f distinct peaks in both] and as a distance matrix (Table 4), [distance = ln(S)] obtained through a pairwise comparison of all chromatograms (Swofford and Olsen, 1990) The two different formulas for converting similarities to distances were used to guard against possible underestimating of relationships. The simplicity of the dissimilarity equation makes it the least preferred option (Swofford and Olsen, 1990)

PAGE 37

Peak Number Species Centropomus P.clathratus P.maculatofasciatus P.nebulifer E.adscensionis E.cruentatus E. drummondhayi E.fulvus E.inermis E.itajara E.morio M.bonaci M.interstitialis M.microlepis M.phenax M.venenosa Paranthias 123456789111111111122222222223333333333444444 012345678901234567890123456789012345 101101011100011111111100101100101100001000100 101101010000010011111100101000110111000101000 101011010001010011110000101100100110010101000 101111010001011011111000101000110100010100100 101011010001011011111100101100100101010010000 101111110001011011111100001100101101000010000 111111011000011011110100101101100111100010000 111101110001010011111100101000111101100010000 101101011001011011111111101100100101100010010 101101011011111011111101101100100101110010110 101111111001111011111100101100111100100000101 101101010001011011111100101100110101100010000 101101010001011011111100101100100101100010110 101101011001011011111100111110100101110010110 101101010001011011111100101100101111100010010 101101011011011011111100101100100101100010110 101101011001011011111101101000100101110010110 Table 2. Peak Presence/Absence from Pairwise Comparisons of Peptide Maps (Chr9matograms) tv tv

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Centropomus P. clathratus .414 P maculatofasciatus .500 .360 P. nebulifer .379 .346 .320 E. adscensionis .414 .407 .320 .308 E cruentatu s .379 .429 .464 .429 .200 E drununondhayi .419 .412 .448 .467 .321 .345 E. fulvus .452 .333 .533 .414 .357 .231 .367 E. inermis .387 .433 .516 .452 .286 .310 .300 .333 E. i tajara .394 .485 .515 .406 .300 .375 .364 .394 .167 E. morio .323 .469 .500 .333 .387 .300 3 94 .323 .364 .324 M. bonaci .379 .308 .464 .333 .200 .231 .286 .192 .185 .267 .300 M. interstitialis .345 3 93 .483 .357 .231 .259 .310 .286 .148 .172 .323 .120 M microlepis .375 .469 .500 .387 .276 .355 .344 .375 .200 .161 .353 .241 .143 M. phf!max .367 .357 .448 .433 .259 .222 .276 .250 .179 .258 .344 .154 .115 M. venenosa .333 .433 .516 .400 .286 .310 .300 .333 .143 .103 313 .185 077 Paranthias .387 .433 .516 .345 .286 .367 .355 .333 .143 .103 .363 .250 .179 Table 3. Dissimilarity Matrix of Peptide Maps among 16 Serranids and one Centropomid (d 1-S) .233 .138 .179 .138 .241 143 1\.) w

PAGE 39

Cen tropomus P. clathratus .534 P. maculatofasciatus .693 446 P. nebulifer 477 .423 .386 E. adscensionis .534 .523 .386 368 E cruentatus .477 .560 .624 .560 .223 E. drununondhayi 544 .534 .595 .661 .388 .423 E. fulvus .601
PAGE 40

25 3. RESULTS Starch-gel protein electrophoresis on white muscle extracts of each species (Tris-citrate pH 6.9 and 8 .0), indicated LDH was homotetrameric for the A 4 isozyme. Electrophoretic mobilities were isomobile among species with the exceptions of E fulvus being faster, and M. microlepis being slower than all others (Fig 3). Purification of LDH A 4 from approximately 100 g of epaxial white muscle tissue ranged from about 15-100 mg. Chromatograms of the tryptic digests of LDH from all species (Table 1), were generated using RPHPLC. Triplicate RPHPLC runs performed on each species (aliqliots from same sample) resulted in identical chromatograms, thus satisfying reproducibility requirements. Peak enumeration covered the first 15-20 minutes of the chromatogram, after which the peaks became exceedingly ambiguous and very difficult or impossible to score with confidence. Based on the assumption that each absorbance peak on the chromatogram represents a single peptide, 45 distinct peptides were identified among the LDH-A4 homologs of the 17 species (Appendix 1, Figures 12-28). The number of peptides identified from an individual chromatogram ranged from 29 in E. itajara (Fig. 21) to 20 in Paralabrax maculatofasciatus

PAGE 41

-----------------ABCDEFGH JKLMNOPQ Figure 3. Electrophoretic Mobilities of LDH of Western Atlantic Groupers (pH 6.9, and 8 .0) [data compiled from numerous gels]. Lane designations: A= E cruentatus, B = E. fulvus, C = E adscensionis, D = E. drummondhayi, E = M microlepis, F = M. phenax, G = M. interstitialis, H = M. venenosa, I = M. bonaci, J = Paranthias furcifer, K = E morio, L = E. inermis, M = E itajara, N = Paralabrax clathratus, 0 = P nebulifer, P = P. maculatofasciatus, Q = Centropomus undecimalis. 1\) en

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27 (Fig. 14). The number of tryptic peptides found per species is approximately the number expected when compared to the average lysine and arginine content of other LDH homologs (Pesce et al., 1967, Taylor and Oxley, 1976) The number reported here is low due to the difficulty of resolving ambiguous peaks from 20 to 60 minutes elution time. Examination of the tryptic digest RPHPLC profiles indicated that 12 (27%) of the peaks were evidently identical among all species. 31, and 34. These peaks were 1, 3, 6, 8, 14, 17-20, 27, Many of these peaks were readily identified and served as "reference peaks" in helping to align all chromatograms for comparisons. RPHPLC profiles of mixed samples of two different species, although less clear, yielded support of homologous peaks between species as corresponding peaks aligned adding to each other resulting in a peak twice as large. C. undecimalis, Paralabrax clathratus, P maculatofasciatus, P. nebulifer and E. morio appeared to share peak 41 with all species until mixed with the others. Only after mixed RPHPLC runs with others and amongst themselves was it possible to determine that the C undecimalis, E morio and Paralabrax spp. possessed unique peaks (Appendix 2, Figures 29-65). These peaks were renumbered 39 (Figure 12), 45 (Figure 22), and 40 (Figures 13-15) respectively. Wagner parsimony analysis of the peptide data using the PENNY algorithm produced no individual most parsimonious

PAGE 43

28 tree. That is to say several trees were produced having the same number of evolutionary 11steps11, or they were of the same length. From the total of all most parsimonious trees a consensus tree (Figure 4) was generated using CONSENSE (PHYLIP ver. 3 .52). This consensus tree placed Epinephelus morio as a sister taxon to all other serranids in the study. Three clades were produced by the algorithm, one containing P. maculatofasciatus, P. nebulifer, and E. adscensionis, the second containing P. clathratus, E. fulvus, E. cruentatus, and M. bonaci, and the third consisting of M. interstitialis, M. venenosa, M. microlepis, M. inermis, E. itajara, and Paranthias. Dollo parsimony analysis of the binary data also resulted 1n more than one most parsimonious tree. The tree produced by the consensus program showed a different result (Figure 5) Although E morio was still a sister taxon, the others were grouped into four clades. The largest contained all three Paralabrax species as well as E. adscensionis and E. drummondhayi. The second grouped three Mycteroperca species (phenax, bonaci, and fulvus) with E. cruentatus. The third contained M. interstitialis, M. venenosa, E. inermis and E. itajara, while the last clade consisted of M. microlepis and Paranthias fucifer. Analysis of the binary (presence/absence) data with a maximum-parsimony bootstrapping program (PAUP) with 100 replicates and SO% majority rule constraint produced no

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Figure 4. Consensus Tree Produced by Wagner Parsimony Analysis of Binary Data in Table 2 Using the PENNY Algorithm mario Centropomus undecimalis --< P. maculatofasciatus P. nebulifer E. adscensionis E. fulvus P. clathratus M. bonaci E. cruentatus E drummondhayi M. phenax M interstitia/is M. venenosa M. microlepis E. inermis E. itajara Paranthias furcifer tv 1.0

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Centropomus undecimalis Figure 5 Consensus T ree Produced by Dollo Parsimony An alysis Data in T a ble 2 Usin g the DOLPENNY Algorithm /E. morio P. macu/atofasciatus P. clathratus P. nebulifer E adscensionis E drummondhayi E. cruentatus M phenax M. bonaci E. fulvus M. interstitia/is M ; venenosa E inennis E. itajara M microlepis Paranthias furcifer of Binary w 0

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31 clades, resulting in a tree in which the relationships between taxa remained unresolved (Figure 6) This means no single node appeared in at least 50% of the most parsimonious (shortest) trees generated. Distance analysis consisted of both data matrices (Tables 3,4) run on identical programs. Fitch-Margoliash analysis of the two data matrices resulted in different trees (Figures 7,8). The difference, not including relative branch lengths, was the addition of E adscensionis to the Paralabrax clade. This addition was to the outside of the clade and did not disturb the relationships of these taxa. Analysis of the two data matrices by the NeighborJoining distance algorithm resulted in identical trees although of different branch lengths (Figures 9,10). E. morio remained outside all other serranids as in the other analysis methods. This method places E. drummondhayi outside all Epinephelines and groups E. cruentatus and E. fulvus together. With the above exception, these trees are identical in branching pattern to the Fitch-Margoliash tree of Figure 7. In all of the methods of distance analysis some branching arrangements occurred repeatedly. These included the placement of the Paralabrax species, the placement of Paranthias and E. itajara together as well as grouping the previous two taxa with M. microlepis, M. venenosa and E. inermis, and placing E. morio outside all other serranids.

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Figure 6. Consensus Tree Produced b y Branc h and B ound Parsimony Analysis o f Binary Data in Table 2 Using Bootst rapping (50% Majority Ru l e ) Cen tropomus undecimalis P. clathratus P. maculatofasciatus P. nubulifer E adscensionis E. cruentatus E drummondhayi -E. E. tnermts E. itajara E. morio M. bonaci M interstitia/is M microlepis M phenax M venenosa Paranthias furcifer w N

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I I r-'-.----'-E. -Paralabrax clathratus ------P. maculatofas. P. nebulifer M. bonaci ......__ M phenax M. interstitia/is M. venen E. E. fulvu I= r-r11an#, E. Paranthias furcifer E. itajara M. microlepis osa nermis E drummondhayi s tus is Centropomus undecimalis .307 .230 .153 .077 .000 Genetic Distance Figure 7. Tree Produced by FitchMargoliash Distance Method Analysis of Dissimilarity Data in Table 3 w w

PAGE 49

..___ ,------.490 .123 E. 1: tulvus E cruentatus M bonaci M phenax ,.--M. interstitia/is Para nth E itaja. ...M. mit M venenosa E inermis E dru E. adscensionis as furcifer a rolepis mmondhayi -------P. maculatofasciatus I P.' nebulifer p .245 G enetic Distance Paralabrax c/athratus ecima/is .368 .000 Figure 8 Tree Produced by Fitch-Margoliash Distance M ethod Analysis o f Distance Data in Table 4 w

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'----I I '--'--r---'--.327 .246 E. Paralabrax clathratus I P. maculatofas. I P. nebulifer E. drummondha yi E. adscensionis E cruentatus E. fu/vus E inermis I E. itajara -I Paranthias fu1 cifer ;-M microlepis M uPnPnn<:a II M. bo, Centropomus undecimal!s .164 Genetic Distance .082 .000 Figure 9 Tree Produced by Neighbor-Joining Distance Method of Analysis o f Dissimilarity Data in Table 3 w lJl

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r-E. morio j ......----------Paralabrax clathratus I ..-----------------P. maculatofasciatus I I L-L. .----.478 .359 I f. E. adscens E. cruent E. f. E ine P. nebulifer drummondhayi on is at us lvus rmis ,_r-f E itajara aranthias furcifer M microlepis ,---.__ M UD' osa /'v M M. bon Centropomus undecimalis ......, .239 .120 .000 Genetic Distance Figure 10. Tree Produced by Neighbor-Joining Distance Method of Analysis of Distance Data in Table 4 w 0'1

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37 E drummondhayi was placed outside all remaining Epinephelines except in Figure 8, where E adscensionis was grouped in the Paralabrax clade. Comparison between the two types 6f analyses, discrete character and distance, shows some similarity of the results. The PENNY, DOLPENNY and all the distance trees (Figures 4,5,7-10) agreed on grouping M interstitalis, M. venenosa, M. microlepis, E. inermis, E. itajara, and Paranthias together. All trees excluding the bootstrapped parsimony one have E. mario as a sister taxon to the rest. The DOLPENNY tree of the parsimony methods (Figure 5) was the one most similar to the distance trees of the parsimony methods. It grouped Paralabrax spp. together, placed E. fulvus, E. cruentatus, M. phenax, and M. bonaci together, which are also relatively close in the Neighbor-Joining trees (Figures 9,10) and put E. adscensionis and E. drummondhayi as being the Epinephelines closest to the Paralabrax species. This was also seen in the NeighborJoining trees. The PENNY tree showed much less similarity to the distance trees. The Paralabrax clade was disturbed, and many of the taxa occupied different terminal positions on the tree.

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38 4. DISCUSSION The results obtained in this study generally do not agree with the phylogenetic hypothesis of Smith (1971) based on mophology, although his was not rigorously produced. The discrepancies can be illustrated through comparison of the trees produced by the Neighbor-Joining Distance Method (Figures 9,10), and a tree modified from Smith's (1971) dendrogram (Figure 11) The arrangement of taxa in these trees are very different and show almost no agreement. Smith's (1971) tree has the genera Epinephelus, Mycteroperca, and Paranthias each possessing an individual clade, whereas the Neighbor-Joining analyses recognize no such groupings. In fact E. morio is placed outside all other groupers, while Smith has it immersed within the Epinephelus clade. The other two species contained in his Epinephelus subgenus E. drummondhayi, and E. adscensionis do not group together either. Paranthias furcifer, hypothesized to be derived earliest by Smith, is found in this study to be among the most recently derived species. Additionally, the peptide data's placement of E. itajara and E. inermis closer to all the Mycteroperca species than any other Epinephelus species, further questions Smith's classification.

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r-.---.___ . I l Paranthias furcifer E. fulvus E. cruentatus E. morio -------------E. drummondhayi E. adscensionis E. itajara I I I I E. inermis M. venenosa M. bonaci M. microlepis M. interstitia/is M. phenax aralabrax spp. entropomus undecimalis Figure 11. Tree Modified from Smith (1971) showing only species include d in this study w 1.0

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40 Heemstra and Randall (1993) place the species included in this study in the same genera as Smith, with the exception of recognizing Cephalopholis and Dermatolepis as genera. Cephalopholis contains E. cruentatus and E. fulvus which are the only grouping that agrees between this study and Smith's hypothesis. This genus is defined by Heemstra and Randall (1993) as having only IX dorsal-fin spines where most other groupers have XI dorsal fin spines. Another character that separates Cephalopholis from Epinephelus is the presence of 3-6 trisegrnental dorsal-fin pterygiophores in Cephalopholis while Epinephelus has only bisegrnental pterygiophores. This classification is supported here in the grouping of cruentatus and fulvus in a separate clade together. Dermatolepis includes E. inermis and is separated by Heemstra and Randall from the other groupers based on the combination of the scales being distinct, lack of a strong antrorse spine on the preopercle, and their larvae having a smooth neurocranium. Smith (1971) has this group within Epinephelus while this study finds them more closely associated with Mycteroperca. In light of the above comparisons and results it is prudent to ask if the groupers are taxonomically oversplit. The results of this study may shed light on the possibility of the groupers being one large taxonomic group that is split on characters of questionable taxonomic value.

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41 Bpinephelus seems to be used as a "catch all" for any grouper that has no distinctive defining character. Smith (1971) may have been on the right track when he placed many of the present genera into the Epinephelus as subgenera. The results produced here disagree with the morphological hypotheses, suggesting possibly that many more genera should be included as subgenera of the Epinephelus, including Paranthias, Mycteroperca, and even possibly Paralabrax1 until a more definitive suite of characters is identified. Another possibility for the disagreement of the biochemical analyses presented here and the heretofore accepted morphological hypotheses is the strength of the peptide data. Lactate dehydrogenase is a biochemically relatively plastic molecule that can be functionally adapted or modified to many different environments. Functional adaptations to hydrostatic pressure (Hennessey and Siebenaller, 1985, 1987a,b, Siebenaller and Somera/ 1979, 19821 Siebenaller et al. 19821 Siebenaller, 1984) and temperature (Yancey and Somera, 1978) have been documented for such groups as macrourids and scorpaenids although determining the structural basis for this has been elusive. The above studies centered on species that inhabit very extreme differences in both pressure and temperature. The differences that Wilson (1991) found in macrourids could have been adaptations to the specific environment where the species are found. The species included in his study ranged

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in depth from 475-4815 meters, and in pressure from 48.5-482.5 atmospheres. In essence he possibly was mapping the ecophenotypic adaptation of the LDH instead of the true genetic variation as intended. 42 Groupers inhabit a variety of environments although not as extreme as the macrourids. The species in this study inhabit more similar temperature and pressure regimes since almost all inhabit reef or rocky habitats. This could explain the lack of strength of the data when subjected to the discrete character analyses (Figures 4-6). The lack of environmental pressure on the enzyme may help to explain the differences between the morphological and biochemical trees since morphological adaptation may occur more readily in species inhabiting different habitats. The lack of habitat differences in groupers and their relatively recent occurence can only contribute to the lack of synapomorphic characters, both morphologically and biochemically. The macrourids are believed to be a much older group which may lend to the stronger defining morphological and biochemical characters. If they are indeed much older, they were allowed to arise and differentiate over a much greater time period. Over this larger period the differences between the species may become much more distinct. It may be that the grouper lineages are too young for their respective lactate dehydrogenases to have become significantly divergent, thus causing the phylogenetic signal of this molecule to be too

PAGE 58

43 weak for this method. Morphological and molecular evolution rates can differ greatly allowing for one taxonomic method to succeed where another cannot. Peptide mapping of lactate dehydrogenase as a taxonomic method in fishes is useful, although possibly not when looking at intergeneric problems or higher, due to some of the reasons discussed above. It does allow us to look at differences not apparent with protein electrophoresis. M. phenax and M interstitialis are so similar in appearance that some researchers are led to doubt the actual presence of two species (Bullock, pers. comm ) Protein electrophoresis of LDH on these species shows no differences (Figure 3). Peptide mapping shows three differences between the two chromatograms; M. phenax is distinguished by peaks 33 and 35, while M interstitialis has peak 43 (Figures 24, 26). None of these peaks are shared by the other, possibly supporting the assumption of different species. Support for this method also comes from the arrangement of the outgroup clade of Paralabrax species (Figures 7-10) Each of the distance trees produced using the peptide mapping data agreed with results of Graves et al. (1990), which used mitochondrial DNA RFLP data and allozyme electrophoresis. P. nebulifer and P. maculatofasciatus were found to be the closer to each other than either was to P. clathratus in both studies. While there is much to be gained through peptide

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44 mapping, there are some caveats as well. The coding of each chromatographic peak as a discrete, two-state, taxonomic character may not conform to certain inherent assumptions of parsimony analysis (Swofford and Olsen, 1990) Peptide characters are not as solidly defined as typical morphological characters. This is due to each chromatographic peak (i.e. character) having to occur in at least one chromatogram for detection of its absence in another to be possible. Thus the character cannot be recognized independently of its state (present or absent) The assumption of character independence is also a potential source of problem, since it is fundamental to parsimony analysis. The disappearance of one peak on the chromatogram of some species may be related to the appearance of other peaks. This may occur when an amino acid substitution in a peptide which alters its hydrophobicity and retention time on the HPLC column results in the disappearance of o n e chromatographic peak and the appearance of a new peak. This chromatogram would be scored as having lost one peak present in other chromatograms, and gaining one not present in the others. Similarly the result of a loss o f an arginine or lysine, which is the cleavage site of the trypsin, would cause the disappearance of two peaks from the chromatogram and the appearance of a new one. Problems associated with parsimony analysis outlined above seem to be avoided by the distance methods of

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45 analysis. In these the entire chromatogram is treated as a unit and is entirely independent of all others. Even so, the assumptions implicit with the use of these methods cannot positively be met by the data derived from the dissimilarity and distance matrices (Felsenstein, 1984) It has often been argued that discrete character methods (i.e. parsimony) are more powerful than distance methods, due to the direct use of the raw data and that some information is lost in transforming the data into distance matrices. This is a valid point; however, the fact that parsimony methods do use the raw data, it only uses a fraction of the data available. Some characters are termed uninformative in parsimony analysis due to their similarity in which case they are of no taxonomic value. The possible result is that a large portion of the data is ignored. The data used in this study had 45 characters from 17 taxa of which approximately half were informativ e This leaves almost one half of the data unused! For this reason, this method is possibly not as efficient as the distance methods used in this study, and could possibly account for no relationships above the 50% majority rule to be found in the maximum-parsimony bootstrapping analysis. Distance methods use all available dat a in their analyses and therefore could be a more reliable source of information for inferring taxonomic relationships. The different distance methods seem to agree more than the

PAGE 61

46 parsimony methods even though they have some different approaches to data analysis (see methods) In spite of the problem of uninformative data the two types of data analysis still agreed on some points, lending support to the trees found in the distance methods. For instance the presence of Paranthias furcifer, E. itajara, and E. inermis amoungst the Mycteroperca is seen in all analyses except the bootstrap (Figures 4, 5, 8-11). The Cephalopholids of Heemstra and Randall (1993), E. cruentatus and E. fulvus, are always included in the same clade as well. Another example consistent throughout the trees is the separation of E. morio from all other groupers. Peptide mapping is a very interesting and somewhat informative technique, but perhaps another approach to the taxonomic morass of the groupers would be more prudent. Of the techniques presently used DNA sequencing may prove to be the most informative. Sequencing a gene such as Cytochrome B in the mitochondrial genome may clarify many of the obscure relationships of these fishes. If this region is found to be too conserved, then possibly the D-loop of the same molecule could be used. Another possibility for attacking the problem could be a RFLP study of the mitochondrial DNA such as the one in Graves (see above) This may particularly attractive since the data for the Paralabrax are completed. Still another possibly fruitful although more labor intensive technique is an allozyme

PAGE 62

47 electrophoresis study using many loci, and buffer systems. All of the above methods of investigation are open for exploration due to most of the taxonomic hypotheses of this group being reached through adult and larval morphological studies. Biochemical and molecular studies may be the avenue to take in resolving the interrelationships of the groupers. Overall, peptide mapping of the LDH molecule as a method for testing Smith's (1971) hypothesis proved not as strong as had been hoped due to reasons outlined in this discussion. It did shed light on many facets of the relations of the groupers, lending support for some of the proposed classifications, as well as providing evidence against others. Much morphological work has been done on this group without a clear picture taking focus. Completion of this study does not allow for strong criticism of any morphological standpoint as the strength of the results are debateable. Further biochemical and molecular studies need to be undertaken for the interrelations of the groupers and other lower percoids to become more clear.

PAGE 63

48 5. REFERENCES Ayala, F.J. (1982). Genetic variation in natural populations: Problems of electrophoretically cryptic alleles. Proc. Natl. Acad. Sci. USA. 79: 550-554. Baldwin, C.C. and G.D. Johnson. (1993). Phylogeny of the Epinephelinae (Teleostei: Serranidae). Bull. Mar. Sci. 52(1): 240-283. Bell, J.E. and E.T. Bell. (1988). Proteins and Enzymes. Prentice Hall, Inc. New Jersey. Bullock, L. and G.B. Smith. (1991). Seabasses (Pisces: Serranidae). Mem. Hourglass Cruises. VIII (II) 243 p Buth, D.G. (1984). The application of electrophoretic data in systematic studies. Ann. Rev. Ecol. Syst. 15: 501522. Crestfield, A.M., S. Moore, and W.H. Stein. (1963). The preparation and enzymatic hydrolysis of reduced and scarboxymethylated proteins. J. Biol. Chern. 238: 622 Farris, J.S. (1977). Phylogenetic analysis under Dolle's law. Syst. Zool. 26: 77-88. Felsenstein, J. (1984). Distance methods for inferring phylogenies: A justification. Evol. 38: 16-24. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evol. 39: 783-791. Felsenstein, J (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Gosline, W.A. (1966). The limits of the fish family Serranidae, with notes on other lower percoids. Proc. Calif. Acad. Sci. 33: 91-111. Graves, J.E., M.J. Fellows, P.A. Oeth, and R.S. Waples. (1991) Biochemical genetics of southern California basses of the genus Paralabrax and a method for the specific identification of fresh and ethanol-preserved individual eggs and early larvae. Fish. Bull.

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Hendy, M D. and D. Penny. (1982). Branch and b ound algorithms to determine minimal evolutionary trees. Math. Biosci. 59: 277-290. 49 Hennessey, J.P., Jr. and J.F. Siebenaller. (1985). Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilal deep-living fishes. J. C o mp. Physiol. B 155: 647-652. Hennessey, J.P., Jr. and J F Siebenaller. (1987a). Inactivation of NAD-dependent dehydrogenases fro m shallo w and deep-living fishes by hydrostatic pressure and proteolysis. Biochemica et Biophysica Acta 913: 285-291. Hennessey, J.P., Jr. and J F Siebenaller. (1987b) Pressure-adaptive differences in proteolytic inactivation of M4-lactate dehydrogenase homologues from marine fishes. J. Exp. Zool. 241: 9-15. Johnson, G.D. (1983) Niphon spinosus: A primitive epinepheline serranid, with c omments on the monophyly and intrarelationships of the Serranidae. Copeia 1983: 777-787. Johnson, G.D. (1984). Percoidei: development and and D.M. relationships. Pages 464-498 in: Ontogeny systematics o f fishes. H.G. Moser, W.J. Richards, Cohen, M.P. Fahay, Kendall, Jr. and S.L. Richardson. eds. Amer. Soc. Ich. and Herp. Spec. Pub. No 1. Markert, C.L., J B Shakley, and G.S. Whitt. (1975) Evolution of a gene. S cience 189: 102-114. Nelson, J S (1984) Fishes of the world (2nd Ed.) Wiley and Sons Inc., New York. 0 Carra, P S. Barry, and E. Corcoran, ( 1974) Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortiv e complex formation. FEBS Letters. 43(2): 163-168. Pesce, A T.P. Fondy, F. Stolzenbach, F. Castillo, and N.O. Kaplan. (1967). The comparitiv e enzymology of lactate dehydrogenases: I Properties of the H4 and M4 enzymes from a number of vertebrates. J. Biol. Chern 242: 2151-2167. Regan, C.T. (1913b). On the classification of the percoid fishes. Ann. Mag. Nat. Hist. Ser. 8, 12:111-145.

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so Rivier, J. and R. McClintock. (1983). Reversed-phase high performance liquid chromatography of insulins from different species. J. Chrom. 268: 112-119. Scopes, R.K. (1987). Protein Purification: Principles and Practice (2nd Ed.) Springer-Verlag, New York. Selander, R. K., M. H. Smith, S Y. Yang, W. E. Johnson, and J.B. Gentry. (1971). Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old field mouse (Peromyscus polionotus) Studies in Gentics VI: Univ. Texas Pub. 7103: 49-90. Shaw, C.R. and R. Prasad. (1970). Starch gel electrophoresis of enzymes-a compilation of recipes. Biochem. Genet. 4 : 297-330. Siebenaller, J.F. (1984). Structural comparison of lactate dehydrogenase homologs differing in sensitivity to hydrostatic pressure. Biochimica et Biophysica Acta 786: 161-169. Siebenaller, J.F. and G N. Somero. (1982). The maintenance of different enzyme levels in congeneric fishes living at different depths. Phyiol. Zool. 55(2): 171-179. Siebenaller, J.F. G.N. Somero, and R.L. Haedrich. (1982). Biochemical characteristics of the macrourid fishes differing in their depths of distribution. Biol. Bull. 163: 240-249. Smith, C.L. (1971). A revision of the American groupers: Epinephelus and allied genera. Bull. Am. Mus. Nat. Hist. 146: 69-241. Solomons, T.W.G. (1986). Fundamentals of Organic Chemistry (2nd Ed.). Wiley and Sons Inc., New York. Somero, G.N. and J.F. Siebenaller. (1979). Inefficient lactate dehydrogenases of deep-sea fishes. Nature 282: 100-102. Stock, D.W. and G S Whitt. (1992). Evolutionary implications of the eDNA sequence of the single lactate dehydrogenase of a lamprey. Proc. Natl. Acad. Sci. USA. 89: 1799-1803. Swofford, D .L. (1993). PAUP: Phylogenetic Analysis Using Parsimony, Version 3 1 Computer program distributed by the Illinois Natural History Survey, Champaign, Illinois.

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Swofford, D.L. and G.J. Olsen. (1990). Phylogeny Reconstruction, pp. 411-501. In D.M. Hillis and C. Moritz (eds.), Molecular Systematics. Sinauer Assoc. Inc. Taylor, S.S. and S .S. Oxley. (1976). Homologies in the active site regions of lactate dehydrogenases. Arch. Biochem. Biophys. 175: 373-383. 51 Torchinskii, Y.M. (1974). Sulfhydryl and Disulfide Groups of Proteins. Studies in Soviet Science. Trans. by H.B.F. Dixon. Consultants Bureau, New York. Wheat, T.E. and E. Goldberg. (1983). In: Isozymes: Current topics in biological and medical research. M.C. Rattazzi, J.G. Scandalios and G.S. Whitt. eds. (Liss, New York), Vol.7, pp.ll3-130. Wilkinson, J .M. (1986) Fragmentation of Polypeptides by Enzymatic Methods. In: Practical Protein Chemistry -A Handbook. Ed. by A. Darbre. John Wiley & Sons, Ltd. London. Wilson. R.R., Jr. (1994). Interrelationships of the subgenera of Coryphenoides (Gadiformes: Macrouridae): comparison of protein electrophoresis and peptide mapping. Copeia 1994(1): 42-50. Wilson, R.R., Jr. J.F. Siebenaller, and B J Davis. (1990). Comparison of LDH-B4 allozymes of Macrourids (Teleostei: Gadiformes) by electrophoresis and by peptide mapping. Biochem. Syst. Ecol. 18(7/8): 565-572. Wilson, R.R., Jr., J.F. Siebenaller, and B.J. Davis. (1991). Phylogenetic analysis of species of three subgenera of Coryphaenoides (Teleostei: Macrouridae) by peptide mapping of homologs of LDH-A4 Biochem. Syst. Ecol. 19 (4) : 277-287. Yancey, P.H. and G.N. Somero. (1978). Temperature dependence of intracellular pH: Its role in the conservation of pyruvate apparent values of vertebrate lactate dehydrogenase. J. Comp. Physiol. 125: 129-134.

PAGE 67

52 APPENDICES (In all figures: Time = Retention Time in minutes)

PAGE 68

53 APPENDIX 1. PEPTIDE MAPS OF SINGLE SPECIES

PAGE 69

61111 39 27 19 ltllll E H {/) 0 17 n tJ:J 21111 3 31 20 6 14 4 16 '"-I 11 TIME Figure 12. Peptide Map of Centropomus undecimalis 21 31 "0 "0 t:t:l s H I-' () 0 rt f-' g CD 0.. Ul "'"

PAGE 70

-------------------... 4 0 1111 11 2 21 19 H t 21111 (/) 0 () ,./ I tJj 8 3 1 I --fi TIME Figure 13. Peptide Map of Paralabrax clathratus 2 1 3 1 ):.1 ttl ttl tJ:j s H ><: ..... n 0 ::s rt 1-' ::s c (1) 0.. l1l l1l

PAGE 71

40 Jallll 27 19 :u t>l 31111 H (/) 0 6l () 21111111 17 t>l 11'0.. I I 42 6 a 14 3 11 21 TIME Figure 14. Peptide Map of Paralabrax maculatofasciatus II :t:>' tO "0 tij s H :>< f-' n 0 ::::1 rt 1-'::::1 c It> p. U1 0'\

PAGE 72

H tii 211111 {J) 0 Q 1111111 ------------------------------, 40 27 19 31 .' 11 21 31 TIME Figure 15. Peptide Map of Paralabrax nebulifer 'lj 'lj tt:l s H :X: ....... n 0 rt 1-' c CD 0.. Ul ..J

PAGE 73

--------... ---4 1 51111 2 7 IIIII I I t1l H 31111., 17 I 111 8 {/) 0 21111 t1l I I I 6 1 4 A 1 S 3 A l l uv '\Jv J 11111. 3 6 J 11 21 ----a. T IME Figure 16. P eptide Map of Epinephelus adscensionis tO tO I:I:l s H :><: I-' n 0 :::1 n...... :::1 c CD 0.. U1 co

PAGE 74

31111 H 21111 {J) 0 () trJ 11111 ------.. -------------------41 21 I II 1 7 I I I Rll r L ----1 11 TIME Figure 17. Peptide Map of Epinephelus c ruentatus 21 81 :x:.o '"d '"d [:Ij z t::J H :>< I-' n 0 ::J rt f-' ::J s:: CD 0.. Ul I.D

PAGE 75

------------I _. .... .. I I 27 II I 311&1 H (/) 0 21111.J 17 () t1:l I I 121 1922 14 I I I I 31 6 I (I l 11111 I --------11 21 31 TIME Figure 18. Peptide Map of Epinephelus drummondhayi ttl ttl til E3 H :>< 1-' () 0 :::1 rt ....... :::1 c 0.. 0'\ 0

PAGE 76

"IIIII 27 31111111 II II 37 H (ll 21111 0 13 I 1;711 &l I I 6 14 111111 II II 3 .. 12 }L. I 11 TIME Figure 19. Peptide Map of Epinephelus fulvus I I I 21 II ttl t'IJ H >< ...... -n 0 ::1 rt 1-'-::1 c p. 0) .....

PAGE 77

51111 ...... E 31111 {fj 0 2tltllt () [tl 41 27 17 8 31 I I 3 6 14 34 36 37 { _ _ ------- 11 21 31 TIME Figure 20. Peptide Map of Epinephelus inermis 'U 'U tij s H :>< t-' n 0 rt 1-' 8 (I) 0.. 0'1 !\.)

PAGE 78

Ulll H ;;] G; (/) I t1l 21111 --------------------------------. 3 6 4 8 27 17 8 2 14 n i2;022 5 11 41 31 36 TIME Figure 21. Peptide Map of Epinephelus itajara 21 31 ):>' 'U 'U tr:l s H :X: .... () 0 ::I rt ..... ::I c CD 0.. m w

PAGE 79

APPENDIX l (Continued) 64 I I I l o 1 I N I I 0 j f"i w 0 :E E H e--{J) ... ::s ...... Ill ..c: ... g. I:: ;;; f"i tfr N ... I 0 0.. !::: "' I ;! m N <1) '0 <0 ...... .w 0.. ... <1) 0.. I I I N I l I N I I <1) I I I 1-.l ::s tn ...... I ex. D Cl D 00 .. N RELATIVE ABSORBANCE

PAGE 80

41 27 I I ..... 1-i H 8 ..... J 17 1121 II! i JJU\ .U! 1'111 36 11 TIME Figure 23. Peptide Map of Mycteroperca bonaci I 21 II ))! t-el t-el tzl H >< ....... -() 0 ::s rt ..... ::s p. 0'1 V1

PAGE 81

,...----------------------61111 41 27 _. .. 1 H tii t (I) 17 21111 I 31 6 210 y IS 3 36 34 4 11 21 TIME Figure 24 Peptide Map of Mycteroperca interstitialis II I'd tzl s H >< ..... () 0 :::3 rt .... g (1) p, 0'\ 0'\

PAGE 82

41 51111i l 27 -IIIIJ I II J I H IIIII (/) I ltza 0 17 211111 Ill 31 J 6 14 9 I 11 TIME Figure 25. Peptide Map of Mycteroperca microlepis I I 21 31 ):.1 "' "' tij H >< t-O -() 0 :::1 rt ..... :::1 c @ 0. 0'\ -.1

PAGE 83

41 _.I ... II 27 II I I 5:: ;IIIII >i H 2 (I) 21111 I 17 I 128 l I 'I 31 14 IUI19 22 11111 ... II A I I 1 Jl 36 TIME Figure 26. Peptide Map of Mycteroperca phenax I I I 21 31 1-0 1-0 M H >< ...... ....... n 0 ::s rt IJ ::s ro p. 0'1 (X)

PAGE 84

611111 41 27 Ullll H tii (/) 0 17 ":ze I 31 I I ll I 211111 3 ,.. Jr.-:z.o 22 I I 11 21 31 TIME Figure 27. Peptide Map of Mycteroperc a venenosa !Jll tU tU trl s H >< ..... n 0 rt ..... C1> p. 0"1 \D

PAGE 85

..... r--41 27 _. ... E 31tlt (I) I () I:Jj 21111 17 31 43 .... 3 J\.' 20' 14 .... I 22 I 11 TIME Figure 28 Peptide Map of Paranthias furcifer 21 31 )"' I'd I'd trJ E3 H :>< ..... n 0 ::l rt 1-':::l c Cll 0.. ....J 0

PAGE 86

71 APPENDIX 2. PEPTIDE MAPS OF TWO SPECIES

PAGE 87

500000-l 400000 -l II \ H tii 300000 en 0 200000 () t'l:1 100000 r 0 5 l II \ r 10 II I I I 15 TIME 20 25 Figure 29. Peptide Map o f Centropomus undecimalis and Paralabrax clathratus I I 30 'tl 'tl tij E3 H :>< N ...... n 0 cT ..... g ro 0.. ....J N

PAGE 88

4000001 1\ I I 3000001 II I I H t;j &; (I) 200000 () ttl 100000 0 0 1 0 20 TIME Figure 30 Peptide Map of Centropomus undecimalis and Paralabrax maculatofasciatus I I 30 ):>' "d "d 1:1:1 s H :>< tv -() 0 ::s rt ..... ::s c ro 0.. -..] w

PAGE 89

8000001 II I \ I I H til 400000 \ \ \ G; (I) 0 () tzl 200000 0 1-----------,-. 0 5 10 15 TIME 20 25 Figure 31. Peptide Map o f Centropomus undecimalis and Paralabrax nebulifer I I 30 ):I tU tU til s H :>< tv -n 0 ::s rt 1-' ::s c (1) 0.. -..J

PAGE 90

600000-l soooooi I \ I 1 -l H \ \ \ 300000 {/) 0 200000 tzl 100000 0 0 5 T 10 r 15 20 25 TIME Figure 32 Peptide Map of Centropomus undecimalis and Epinephelus adscensionis I I 30 t'(j t'(j tr.1 s H >< tv () 0 ("t 1-' c ro 0.. -..J l1l

PAGE 91

500000 400000 300000 H 200000 () tJl 100000 0 J l 5 20 25 I 15 0 I 10 TIME Figure 33. Peptide Map of Centropomus undecimalis and Epinephelus cruentatus 30 :x:or ttl ttl tx:l s H tv n 0 ::l rt ..... ::l c Cl> 0.. -...] 0"1

PAGE 92

600000-l 400000i t;] (/) 0 () (1l 200000 0 0 I I I I 10 20 TIME Figure 34. Peptide Map of Centropomus undecimalis and Epinephelus drummondhayi 30 t'{j t'{j tr:l s H :>< tv n 0 ::l rt 1-' ::l c CD 0.. ...J ...J

PAGE 93

800000-l G; 300000 Cll 0 Q 200000 100000 0 L-. 0 I l II I I I I 10 ll I 15 TIME 20 25 Figure 35. Peptide Map of Centropomus undecimalis and Epinephelus fulvus I I 30 tU tU tt:1 a H >< 'tv () 0 ==' rt ..... ==' ro 0... -...] Q)

PAGE 94

700000; 600000-l 5oooooJ 6; 300000 () trJ 200000 100000 0 0 II II \ I 10 II 15 TIME 20 25 Figur e 36. Peptide Map of Centropomus undecimalis and Epinephelus inermis I I I 30 "0 "0 ttl s H :>< N () 0 ::I rt ...... ::I ct> p. -.J \0

PAGE 95

eooooo-l I I H G; {I) 0 6J 200000 [r] 0 0 5 II II I I 10 I\ 15 TIME 20 25 Figure 37. Peptide Map of Centropomus undecimalis and Epinephelus itajara I I 30. ):>I tU tU [lj s H tv -() 0 ::s n-..... ::s c (I) 0. OJ 0

PAGE 96

6000001 400000 I H G; {I) 0 Q 200000 0 r 0 l 5 II I \ I I 10 1111 l \ 15 TIME 20 25 Figure 38. Peptide Map of Centropomus and Epinephelus morio I l 30 ):>1 toO toO ttl s H :>< 10 n 0 ::l rt 1-'-::l c (1) 0. 00 1-'

PAGE 97

500000-l l I \ 1\ H \ \. I \ til &; (I) 0 200000 () tJ:l 100000 0 I I I I 0 5 10 15 20 25 TIME Figure 39. Peptide Map of Centropomus undecimalis and Mycteroperca bonaci I I 30 ):>! ttl ttl tr:l E3 H >< tv () 0 ::I rt 1-' ::I c (1) 0. (X) tv

PAGE 98

5000001 fl H tii 300000 (J) 0 200000 tJl 100000 0 0 I I I I \ \ \ \ TIME 2 0 30 25 Figure 40. Peptide Map of Centropomus undecimalis and Mycteroperca interstitialis )" ttJ ttJ til s H >< tv ........ n 0 :::1 rt ..... :::1 (l) 0. CD w

PAGE 99

eooooo-l 5000001 4000001 H I 300000 (I) 200000 tr:l 100000 0 0 5 I 11 I \ 10 A I 15 TIME 20 25 Figur 41. Peptide Map of Centropomus undecimalis and Mycteroperca microlepis I I 30 'U 'U ttJ s H :>< tv -n 0 ::s rt 1-' ::s s:; C1> 0.. (X)

PAGE 100

A 500000i t:l 40CXM>ll s: H til 300000 Cll 0 Gl 200000 tz:1 100000 0 T 0 5 \ 1 10 I I\ A I \ 15 20 25 TIME Figure 42. Peptide Map of Centropomus undecimalis and Mycteroperca phenax I 30 tU tU t%j s H :>< N () 0 p rt ...... p c CD p. ():) Ul

PAGE 101

600000 400000 H tii 6; (I) 0 Q 200000 0 ------------6 1 10 20 TIME Figure 43. Peptide Map of Centropomus undecimalis and Mycteroperca venenosa 30 )"' ttl ttl l:1:j s H >< tv n 0 :::1 rt 1-':::1 c ro 0.. 0'1

PAGE 102

500000-l 400000i II I H \ I &; (I) 0 200000 s: () tJ:I 0 10 20 TIME Figure 44. Peptide Map of Centropomus unqecimalis and Paranthias furcifer I I 30 ttl ttl l1:l s H >< N n 0 ::J rt ...... ::J c Cl> 0.. (X) ...,J

PAGE 103

til H til (/) 0 () til 600000-l 4000001 II II II 200000 0 0 10 20 TIME Figure 45 Peptide Map of Paralabrax clathratus and Paralabrax maculatofasciatus I I 30 ):>' "tt "tt tiJ s H :>< tv -() 0 rt ..... c ro 0. ():) ():)

PAGE 104

500000-l A 400000.1 1\ Ill I \ JOOOOOJ II II H r;i (J) 0 200000 () tz:l 100000 0 0 10 20 TIME Figure 46. Peptide Map of Paralabrax clathratus and Paralabrax nebulifer I I I 30 ):>' "CC "CC ti1 s H :>< N (') 0 rt 1-' c (I) 0.. (X) \0

PAGE 105

600000 400000 H ;j G; {J) 0 () 200000 ttl 0 M I 0 10 20 TIME Figure 47. Peptide Map of Paralabrax nebulifer and Paralabrax maculatofasciatus 30 ):I tt) tt) til H >< I\) n 0 ::s rt ....... ::s c (1) 0. \0 0

PAGE 106

800000-' 5000001 4000001 H 300000 (I) 0 () 200000 tJl 100000 0 0 I 5 I 10 I II \ 15 TIME 20 25 Figure 48. Peptide Map of Epinephelus morio and Paralabrax clathratus I I 30 )::>' ttl ttl [Jj s H :>< tv -() 0 ::s rt 1-' ::s c (I) 0.. \D .....

PAGE 107

5000001 ft oooooJ I I 1\ I \ H (/) 200000 () tz:l 100000 0 I 0 5 10 15 20 25 TIME Figure 49 Peptide Map of Epinephelus morio and Paralabrax maculatofasciatus I I I 30 ;po 'U 'U tz:J s H >: N -n 0 rt 1-' ::1 ro 0. I.D N

PAGE 108

600000 500000 <400000 H t;) 300000 ?; (I) I 200000 1 00000 0 r 0 5 10 15 TIME 20 25 Figure 50. Peptide Map of Epinephelus morio and Paralabrax nebulifer 30 ):>' ttl ttl tt:J s H :>< N () 0 ::;! rt ..... CD p.. \0 w

PAGE 109

4000001 .300000l E I H 200000 100000 0 0 5 10 n \ 15 TIME I \ \1 20 25 Figure 51. Peptide Map of Epinephelus mario and.Epinephelus adscensionis I I 30 )::.1 >t:l >t:l 1:11 s H :>< IV () 0 :::1 rt 1-' :::1 c ct> 0.. \D

PAGE 110

400000-l II I \ H rn 200000 {I) 0 Q 100000 0 0 5 10 15 20 25 30. TIME Figure 52. Peptide Map of Epinephelus morio and Epinephelus cruentatus :t>' '"0 '"0 l:tj s H :>c: N () 0 rt 1-' c (1) 0. \0 Ul

PAGE 111

I' 4000001 \ 1\ I I 3000001 A I \ I 1-1 200000 () tzl 100000 0 0 1 5 10 15 20 25 30 TIME Figure 53. Peptide Map of Epinephelus morio and Epinephelus drummondhayi ):>' ttl ttl tt:l E3 H >< 1\) . (') 0 :::1 rt 1-J :::1 c ro 0.. \D 0'1

PAGE 112

500000-l l I II H \ \ 6; (I) 0 200000 () tzl 100000 0 0 5 10 15 20 25 TIME Figure 54. Peptide Map of Epinephelus morio and Epinephelus fulvus I I 30 't1 't1 ti:l s H :>< N n 0 ::l rt 1-' ::l (D 0.. \0 .....]

PAGE 113

oooooi 3000001 H ;;i (() 0 200000 100000 0 0 I \ 5 10 I\ I I I v \ 15 TIME 20 25 Figure 55. Peptide Map of Epinephelus morio and Epinephelus.inermis I I I 30 ):>' 'U 'U tz:l s H >< N () 0 ==' rt ..... ==' (I) 0.. \0
PAGE 114

APPENDIX 2 (Continued) 0 0 0 0 0 0 0 0 0 0 "' .. RELATIVE 0 0 0 0 0 N ABSORBANCE 6 0 N 0 .... 0 99 ns ns .r"'\ ns r-t Ol ::s ,..., Ql ..c:: r-t rd s:: ro 0 r-t 0 H Ol E-< ::s ,..., Ql ..c:: r-t 4-1 0 0. cu ::8 Q) '0 r-1 .w 0. Q) AI 1.0 li) Q) .,... ::s tJ) r-1 [x.

PAGE 115

500000-l 300000 H (/) 200000 () ttl 100000 0 T 0 5 ly\ I \. I 10 T 15 TIME I\ I \ I \ 20 25 Figure 57 Peptide Map of Epinephelus morio and Mycteroperc a bonaci I I I 30 :t:>' l'tl l'tl ti:l s H :><: tv ......... 0 0 ::I rt ..... ::I s:: (J) 0. 0 0

PAGE 116

APPENDIX 2. (Continued) 101 0 M Ul ., 1'"'-i 0 t> ., !:; (1j t> Q) g. Q) .u 0 t> N '0 s:: "' 0 ., 0 ::E !:; H E-< Ul ::s 1'"'-i Q) ...c:: 0 .... 4-1 0 0.. It! ::E Q) '0 r-1 J_) 0.. Q) co U1 Q) H ::l O'l r-1 0 li. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 "' ... "' RELATIVE ABSORBANCE

PAGE 117

500000l 400000 1 E h H 300000 G; (/) 0 200000 Q 0 0 10 l I 1 I II 11 r 1 15 TIME \ I 20 25 Figure 59. Peptide Map of Epinephelus morio and Mycteroperca phenax I I 30 ttl ttl [Ij H :>< tv () 0 :;:! rt .... :;:! (I) 0.. ...... 0 tv

PAGE 118

600000-l 5000001 400000l H tij 300000 (I) 0 200000 til 100000 0 0 T 5 '-... \ 10 I I I \ 1 5 TIME v \ 20 25 Figure 6 0 Peptid e M a p of Epinep helus m orio and Mycteroperca venenosa I I 30 ):>1 ttl ttl tr:J H 1\.) () 0 ::s rt I-' ::s c (1) 0... 1-' 0 w

PAGE 119

500000--l II 400000--l I I I I II J 1\. I \I\ 300000 >-3 H iii (I) 200000 Q 100000 0 0 5 10 15 20 25 TIME Figure 61. Peptide Map of Epinephelus morio and Paranthias furcifer I I I 30 ;:J;:>' 'U 'U tr:l z 0 H :>< tv n 0 p rt 1-' p c ro p. I-' 0

PAGE 120

600000-l I 400 0 001 \ \ s H l tii en 0 Q 200000 0 0 10 2 0 JO. TIME Figure 6 2 Peptide Map of Mycteroperca microlepis and Epinephelus itajar a ;x:.. tO tO trJ H N () 0 ::l rt r' ::l c (1) 0. ...... 0 l11

PAGE 121

600000-l 400000-j H I I {J) 0 Q 200000 0 0 10 2 0 TIME Figure 63. Peptide Map of Mycteroperca phenax and Mycteroperca interstitialis I 30 ):I to to t:tJ z t:J H >< N n 0 ::J rt ..... ::J s:: (!> p. 1-' 0 C7'l

PAGE 122

II oooooJ II I I H (J) 0 200000-t1l 0 0 10 20 TIME Figure 64. Peptide map of Paranthias furcifer and Epinephelus itajara I I 30 tU tU tr:l H :>< N ....... () 0 ::l rt ..... CD 0, ...... 0 -..]

PAGE 123

600000-l II 400000i II I I H t;] G; en 0 () 200000 t'1 0 0 10 20 TIME Figure 65. Peptide Map of Paranthias furcifer and Mycteroperca venenosa I I 30 t'(j t'(j tx:J H :X N ....... () 0 :::1 rt 1-'-:::1 c (1) 0.. 1-' 0 00


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