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Glutamine synthetase as a biological marker for fish phylogenetics : some new insights Laberge Macdonald, Tammy 2003

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GLUT AMINE SYNTHETASE AS A BIOLOGICAL MARKER FOR FISH PHYLOGENETICS: SOME NEW INSIGHTS by TAMMY LABERGE MACDONALD B.Sc, Simon Fraser University, 1991 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 2003 © Tammy Laberge MacDonald 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 'zLoolOfj ^ The University of British Columbia Vancouver, Canada Date Je^Q-a-VXl c\^) SLOP 5^ , DE-6 (2/88) 11 Abstract Glutamine synthetase is a key enzyme for nitrogen metabolism. It occurs in all organisms and is one of the oldest functioning genes. Many vertebrates have only one functional copy of this gene, while many plants have been shown to be multicopy for this gene. Pseudogenes for glutamine synthetase have also been reported in mammals. Until recently only a single copy of glutamine synthetase had been described in fish. However, six copies of this gene are expressed in rainbow trout Oncorhynchus myklss and two copies of this gene are expressed in the gulf toadfish Opsanus beta. We investigated a variety of intertidal fishes from British Columbia, Canada using PCR amplification of genomic DNA product and reverse transcriptase PCR to explore the diversity of glutamine synthetase in fish. We recovered two isoforms of glutamine synthetase in fourteen out of twenty-one fish. We describe the partial sequences for the two copies of this gene that differed in nucleotide composition by 8 to 22 percent. Phylogenetic analysis was performed using the different glutamine synthetase isoforms to generate trees for intertidal fishes collected in this study. Fish from the following orders were represented in this study: Myxiniformes, Lepisosteiformes, Salmoniformes, Gasterosteiformes, Syngnathiformes, Scorpaeniformes, Perciformes and Pleuronectiformes. Most species adhered to the traditional taxonomic classification although some representative fish did not. Ill Table Of Contents Abstract ii Table Of Contents iiList Of Tables v List Of Figures v Acknowledgements vi Introduction 1 Materials and Methods: 4 Sample Collection and Preservation 4 Primer Design 5 Genomic DNA Extractions 6 RNA ExtractionsReverse Transcriptase Reactions 7 PCR and SequencingSequence Analysis and Phylogenetics 8 Results 10 DNA Amplification 1Isoform designation  1 Overall Analysis GS products 12 Phylogenetic Analysis GS products 3 Isoform analysis and phylogenetic analysis of fishes 15 Discussion 17 Glutamine synthetase gene duplication(s) 1Phylogenetic analysis of GS products 20 Conclusions and future work 24 Species Name 26 Common NameReferences 49 Appendix 1 .' 52 Appendix 2 60 Appendix 3 1 Appendix 4 9 IV List Of Tables Table 1. Glutamine synthetase sequences available in Genbank for all vertebrates prior to August 1999 26 Table 2. Primers used in the amplification and sequencing of glutamine synthetase product. 27 Table 3. Names of animals used in this study including common names, species names, family names, subfamily names and order names 28 Table 4. Primers used to amplify PCR product for glutamine synthetase and approximate size of fragment produced 30 Table 5. Base composition percentage statistics for amplified glutamine synthetase fragments 31 List Of Figures Figure 1. Summary of DNA fragments of glutamine synthetase amplified by PCR for fishes used in this study 32 Figure 2. Pairwise comparisons for % differences in GS isoforms for fish used in this study that produced two GS isoforms 33 Figure 3. Neighbor joining tree constructed for all fish that produced two isoforms of glutamine synthetase for this study 4 Figure 4. Amino acid translation of glutamine synthetase products for all GS isoforms 35 Figure 5. Neighbor joining tree constructed from 432 bp fragment of glutamine synthetase from all isoforms amplified in all fish used in this study 38 Figure 6, Maximum parsimony tree constructed from 432 bp fragment of glutamine synthetase from all isoforms amplified in all fish used in this study 39 Figure 7. Maximum likelihood tree constructed from 432 bp fragments of all isoforms of glutamine synthetase for all fish used in this study 40 Figure 8. Neighbor joining analysis of Isoforms A only of glutamine synthetase 41 Figure 9. Maximum parsimony tree constructed from A isoforms only of glutamine synthetase 42 Figure 10. Maximum likelihood tree constructed A isoforms only of glutamine synthetase. 43 Figure 1 1. Neighbor joining tree constructed from B Isoforms only of glutamine synthetase 44 Figure 12. Maximum parsimony tree constructed from B isoforms only of glutamine synthetase 5 Figure 13. Maximum likelihood tree constructed B isoforms only of glutamine synthetase. 46 Figure 14. Pairwise comparisons of % differences in GS sequences of Isoforms A and Isoforms B 47 Figure 15. The two competing hypotheses for the resolution of the Superorder Acanthoptyerygii 8 vi Acknowledgements There are always so many people to thank when writing a thesis. I would like to start with my labmates: Shannon, Steve, Amanda, Ally and Lance. Someone once told me that labmates are special people because of what you have shared and that you will always have a tie to them. I have to agree. Shannon, you gave me my first fishing lesson and made coming to school fun. Steve, you always kept things interesting and made me realize life has a lot of aspects yet to explore. Amanda, you are always full of new ideas and were able to teach me not only about science, but about different ways to look at things. Lance, you were helpful, not only in the lab, but also in discussing future possibilities. Ally you helped me believe I could finish. You gave me confidence when I needed it and you make the best cookies in town! I have also had the pleasure of having two supervisors so far in my life, Andy Beckenbach and Martin Adamson. Andy, I can't adequately describe the gratitude I have for you. You took a chance on me and let me try. When I first came to your lab I knew nothing about molecular biology. You let me learn and were very patient with me. You gave me the freedom to decide what I liked and never pressured me. I learned so much in the years I spent in your lab. It gave me the foundation so I could move on. I would like to say thank you for this, but it really doesn't seem like enough. Martin, you also took a chance on me. You also gave me freedom, freedom to choose a project that wasn't your main area of interest. Martin, I must say it has always been fun in your lab. I do not think I will find another supervisor that will go for coffee everyday with his students. I learned that education is a much larger lesson in life in your lab. I have been fortunate to have a few mentors along the way and I would like to thank them now. Karen Beckenbach, Don Nelson and Murray Gilbert, thank you for teaching me Vll molecular techniques. I would never have been able to do this project without your guidance. I would also like to thank Don McPhail for his thoughtful discussions relating to my fish and the trees that resulted from my data. It is always great to be able to go to an expert for advice. I must also thank Bob Devlin and Rick Taylor who were on my committee. Thank you for your input into my thesis and its direction. Bob, you were able to make me feel much better about my project when I had some doubts. There are many family and friends I would like to thank for their support. I would like to thank my Mom and Dad, my sister Penny and my brother Todd for putting up with me for all these years. I would also like to thank Garvin and Shirley MacDonald for their prayers and well wishes for me and for my thesis. Thank you to the friends from school that kept me sane: Manu and Phillip Gardner, Beth Zimmer, Jean Paul Danko, Scott Morgan, Karen Needham, Durrell Kapan and Dawn Cooper. Karmavores, you are a big part of my life, thanks for playing with me. It is so nice to have such a great loving and supportive group of friends. I have one person left to thank. Glenn, thank you for always believing in me. You are my biggest fan. You allow me to be myself and support me in all that I do. I am always grateful for all that you do for me. Thank you and I love you. 1 Introduction Glutamine synthetase (GS) is considered to be one of the oldest functioning genes (Kumada et al. 1993) and may have been present during the origin of life (Kumada et al. 1993; Tateno 1994). It is involved in nitrogen metabolism of all living organisms (Pesole et al. 1991; Kumada et al. 1993; Tateno 1994) where it converts glutamate to glutamine (Meister 1985; Eisenberg et al. 2000); in fish, GS removes toxic ammonia during this conversion (Meister 1985; Mommsen and Walsh 1992; Eisenberg et al. 2000) mediating the reaction is shown in Equation 1. Mg+2 Equation 1. Glutamate + NH3 + ATP <-> Glutamine + ADP + P. For teleosts this reaction occurs just prior to the Ornithine-Urea Cycle (O-UC) (For review of O-UC see Mommsen and Walsh 1992). Glutamine synthetase has been used to study phylogenetic relationships in all major groups of prokaryotes and eukaryotes (Pesole et al. 1991; Kumada et al. 1993; Tateno 1994; Pesole et al. 1995; Saccone et al. 1995). Glutamine synthetase evolves very slowly and is therefore used to look at older relationships of organisms (Tateno 1994). Pesole et al. (1991) report that GS evolves in a clock-like manner and GS follows the neutral evolution model (Kumada et al. 1993; Tateno 1994). Of the two types of GS known; glutamine synthetase I occurs only in prokaryotes and glutamine synthetase II occurs mainly in eukaryotes, although some prokaryotes have been found with GSII (Hill et al. 1989; Goodman and Woods 1993; Kumada et al. 1993; Eisenberg et al. 2000). Glutamine synthetase I molecule is a dodecamer while the glutamine 2 synthetase II molecule is proposed to have eight subuits (Eisenberg et al. 2000). The existence of two types of GS implies gene duplication prior to the Prokaryote-Eukaryote split (Pesoleetal. 1991; Kumada et al. 1993; Pesole et al. 1995; Saccone et al. 1995). Active sites of GSI and GSII are invariant indicating that their function is similar (Eisenberg et al. 2000). Glutamine synthetase is a multimeric enzyme, and occurs in multigene families in plants (Cullimore et al. 1984; Tingey et al. 1987; Li et al. 1993; Temple et al. 1995). Some researchers found GS to be single-copy in a few vertebrates (Kuo and Darnell 1989; Pu and Young 1989; Campbell and Smith 1992; Laud and Campbell 1994). Multiple copies of GS were recently found in fish (Murray 2002; Walsh et al. 2002). This study reports on the sequence structure of a portion of the GS gene in a variety fish; it presents evidence of gene amplification and assesses phylogenetic relationships of these fish using GS as a biological marker. Genomic DNA is examined for the presence GS sequence and revealed more than one GS-like sequence with different introns. Genomic DNA is comprised of functional genes with introns and pseudogenes and therefore complementary DNA (cDNA) is examined to determine which sequence was the functional GS gene since cDNA expresses only functional genes. Twenty-one fish are examined in this study representing eight orders: Myxiniformes, Lepisosteiformes, Salrnoniformes, Gasterosteiformes, Syngnathiformes, Scorpaeniformes, Perciformes and Pleuronectiformes. Myxiniformes is the most primitive of all of these orders containing the hagfish (Nelson 1994; Helfman et al. 1997) and the Lepisosteiformes containing gars is also fairly primitive (Nelson 1994; Helfman et al. 1997). The Salrnoniformes are within the superorder Protacanthopterygii and are a sistergroup to the 3 superorder Acanthopterygii (Nelson 1994). Seventeen of 21 fish used in this study are within the last five orders and all fall within the superorder Acanthopterygii in the Series Percomorpha. The relationship of fishes within Series Percomorpha is an area of fish taxonomy that is still in flux (Johnson and Patterson 1993; Nelson 1994). While the composition of the species within Acanthopterygii is agreed upon, the taxonomy within this superorder is still unresolved (Johnson and Patterson 1993; Nelson 1994). There are two main topics addressed in this thesis: 1. Is glutamine synthetase a multicopy gene in fishes? Is glutamine synthetase an appropriate marker for phylogenetic analysis? 2. Do the fish used in this study follow the traditional classification system for these types of fishes? Do the.phytogenies produced in this study help resolve the superorder Acanthopterygii? 4 Materials and Methods; Sample Collection and Preservation Fish were sampled opportunistically by pole seining, rock tipping and dip netting and were identified using keys in Pacific Fishes of Canada (Hart 1988) and Fishes of the World (Nelson 1994). Tidepool sculpin {Oligocottus maculosus Girard 1856) were collected from Popham Island, B.C. in February 2000. Tubesnout (Aulorhynchus flavidus Gill, 1861), bay pipefish {Syngnathus leptorhynchus Girard, 1854), cabezon (Scorpaenichthys marmoratus Girard, 1854), striped seaperch {Embiotoca lateralis Agassiz, 1854), shiner perch (Cymatogaster aggregata Gibbons, 1854), high cockscomb (Anoplarchus purpurescens Gill, 1861), penpoint gunnel (Apodichthys flavidus Girard, 1854), crescent gunnel {Pholis laeta (Cope, 1873)), speckled sanddab {Citharichthys stigmaeus Jordan and Gilbert, 1882), buttersole (Isopsetta isolepis (Lockington, 1880)), and starry flounder (Platichthys stellatus (Pallas, 1788)) were collected from the waters surrounding Stanley Park, Vancouver, B.C. in August 2000. Fish were euthanized with tricane methane sulfonate in seawater before being cut open from pectoral girdle to anus and placed in 95% ethanol. Three spined stickleback (Gasterosteus aculeatus Linnaeus, 1758), white spotted green ling {Hexagrammos stelleri Tilesius, 1810), buffalo sculpin {Enophrys bison (Girard, 1854)), Pacific sanddab {Citharichthys sordidus (Girard, 1854)), A. flavidus, S. leptorhynchus, E. lateralis, C. aggregata, A. flavidus, P. laeta, P. ornata, C. stigmaeus and /. isolepis were sampled while fishing in August 2001 in the waters around Stanley Park, Vancouver, B.C. These fish were euthanized, their carcasses were cut open and tissue samples were collected and immersed in liquid nitrogen and then stored at -80.0 °C. Danny Kent from the Vancouver Aquarium provided additional samples of S. marmoratus, and P. stellatus collected from the waters around Stanley Park, Vancouver, B.C. These samples were frozen in liquid nitrogen and 5 stored at -80.0 °C. Chum salmon (Oncorhynchus keta (Walbaum, 1792)) and coho salmon (Oncorhynchus kitsutch (Walbaum, 1792)) were provided by Dr. Robert Devlin of DFO West Vancouver Labs. These samples were stored at -20.0 °C. Three spined stickleback (Gasterosteus aculeatus Linnaeus, 1758) and a fin clip of mossy sculpin (Clinocottus embryum (Jordan and Starks, 1895)) were provided by Patrick Tamkee from UBC. These samples were caught off Wizard Islet near Bamfield , B.C. and were preserved in 95% ethanol. Pacific hagfish (Eptatretus stoutii (Lockington, 1878)) samples were provided by Doug Fudge from UBC and were frozen at -20.0 °C and -80.0 °C. These samples were caught in the waters of Barclay Sound near Bamfield, B.C. Alligator gar (Astractosteus spatula Lacepede, 1803)) was provided by Dr. Robert Blake from UBC and was caught in the Gulf of Mexico. This sample was stored at -20.0 °C. Primer Design DNA sequence data for glutamine synthetase genes were retrieved from Genbank for all vertebrate specimens sequenced to August 1999 (Table 1). Initial alignment of Genbank sequences was performed using Clustal W (Thompson et al. 1994), and later adjusted by eye using ESEE 3.2S sequencing editor (Cabot and Beckenbach 1989)(Appendix 1). Several primers for glutamine synthetase were designed using OLIGO 4.04 (Rychlik and Rhoads 1989) based on regions of high conservation in the aligned sequences with the objective of providing specificity of amplification for GS sequences. Primers were designed to aVoid primer-dimers and hairpins. Primers were designed to have a G-C content between 40 and 65% and have similar annealing temperatures. Primers were also designed to have the last six bases of the three prime end match at least seven animals in the aligned vertebrate sequences. Primers were constructed by the Nucleic Acid Protein Services unit of the University of British Columbia. Primers were initially tested only on Oncorhynchus keta, Oncorhynchus kitsutch, and Oligocottus maculosus. Primers that amplified glutamine synthetase in any of these fish and were used in the present study are 6 listed in Table 2. The alignment created during the primer design phase was also used to assess nucleotide composition of glutamine synthetase for all vertebrates in Genbank prior to August 1999 (Appendix 2). Genomic DNA Extractions Muscle, skin or liver tissue was used for DNA extractions. Using a UV sterilized scalpel, 30 to 40 mg of tissue was cut away from the fish carcass and placed in a 1.7 ml microcentrifuge tube. Ethanol-preserved specimens were soaked in 0.5 ml proteinase K buffer (0.05M EDTA, 0.5% SDS, 0,01 M Tris, 2.0 M NaCl) for 5 minutes to remove traces of ethanol. The buffer was replaced with 0.36 ml fresh proteinase K buffer and 0.04 ml proteinase K enzyme (28 mg/ml). Samples were incubated at 65.0 °C until the tissue was digested (usually within 8 to 12 hours). Digested samples were extracted twice with phenol:sevag, and once with sevag ( 24 Chloroform: 1 isoamyl alcohol). DNA was precipitated in cold 95% ethanol and left overnight at -20.0 °C. DNA was pelleted by centrifugation, washed twice with cold 70% ethanol and left to air dry overnight. DNA pellets were resuspended in 0.1 ml water and stored at -20.0 °C. RNA Extractions RNA extractions were performed on frozen tissue only. RNA was extracted using Qiagen RNeasy Midi extraction kit (Mississauga, Ontario). The protocol for isolation of total RNA from heart, muscle, and skin tissue with the following modifications to the protocol: tissue was ground in liquid nitrogen with a mortar and pestle before homogenizing it in homogenization buffer in a 10 ml Kontes tissue grinder; samples were centrifuged at 4500 rpm for twice the recommended time outlined in the protocol. The RNA product was taken up in 50 - 100 ul of RNase-free water, precipitated in 3 volumes cold 95% ethanol and left overnight at -20.0 °C. RNA was pelleted by centrifugation, washed twice with cold 80% 7 ethanol and dried by heating in a 37.0 °C heating block for 15 minutes. RNA pellets were resuspended in 0.1 ml water and stored at -20.0 °C. Reverse Transcriptase Reactions Single stranded cDNA was generated from RNA by reverse transcription (RT). RNA was prepared for RT by combining 50 pmol Oligo d(T)12.lg (Amersham Pharmacia Biotech) with 1 -2 ug of RNA, and denatured by heating to 95.0 °C for two minutes then put on ice. RT was performed in 25 ul reactions containing the denatured RNA, 1 x PCR buffer (20 mM Tris-HCl (pH 8.4) and 50 mM KC1) (Invitrogen, Carlsbad, California), 0.01 M DTT, 2.5 mM MgCl2, 0.4 mM each dNTP, 15 units RNAguard (Amersham Pharmacia Biotech), 200 units Superscript II RT (Invitrogen) and ddH20. The RT reaction was placed into a thermocycler and incubated at 42.0 °C for 50 minutes, 65.0 °C for 15 minutes and then cooled to 4.0 °C. The cDNA product was stored at -20.0 'C. PCR and Sequencing One to five microlitres of genomic DNA or cDNA were used for PCR. PCR reactions were performed in 25.0 ul volumes each containing lx PCR buffer, 0.2 mM each dNTP, 1.6 mM MgCl,, 1.25 units Taq polymerase (Invitrogen), 0.38 mM each primer, and water. The primers used for glutamine synthetase amplification are outlined above. PCR was carried out on a Perkin Elmer Geneamp® PCR system 2400. PCR conditions for amplification were as follows: One denaturation cycle of 95.0 °C for 3 minutes followed by three initial amplification cycles of 95.0 °C for 90 seconds, 48.0 °C for 45 seconds, 70.0 °C for 2:00 minutes, then thirty two regular amplification cycles of 95.0 °C for 1:00 minute, 50.0 °C for 30 seconds, 70.0 °C for 2:00 minutes followed by a 72.0 °C extension for 5:00 minutes, and finally a 4.0 °C soak file was activated. Five microlitres of PCR product was 8 electrophoresed on a 1.0% agarose gel stained with ethidium bromide (5.7 x 10"4 mg/ml) to confirm presence of fragment. PCR product was gel purified using a QIAquick gel extraction kit (QIAGEN). The concentration of the PCR product was determined and 30-90 ng of the purified dsDNA PCR product was used for sequencing reactions. The PCR product was sequenced from both the 5' and 3' direction. Automated sequencing reactions used AmpliTaq FS DyeDeoxy Terminator Cycle Sequence chemistry (Applied Biosystems (ABI)). Excess terminators were removed by running the sequencing reaction product through Centri-Sep Spin columns (emp Biotech GmbH). Sequencing reactions were sent to the UBC NAPS sequencing facility where they were run on an ABI Model 373 Stretch DNA sequencer or an ABI Prism 377 DNA Sequencer. Sequence printouts were visually inspected for any anomalies. Sequence Analysis and Phylogenetics Sequences were manually aligned with ESEE Version 3.2S (Cabot and Beckenbach 1989) or IMSEA (Beckenbach unpublished) sequencing editors and compared to published glutamine synthetase sequence of gulf toadfish (Opsanus beta). Sequences were also compared to GS sequences present in Genbank using BLAST (Altschul et al. 1997). Introns were identified for genomic DNA by determining intron spice sites in the sequence using the methods of S. Mount (1982). Only sequence data from coding regions of DNA was used for analysis. Sequence data was analyzed for a 432 bp fragment of glutamine synthetase. Base composition, parsimony informative sites by codon position and pairwise distances were determined using IMSEA (Beckenbach unpublished). Phylogenetic trees were generated using parsimony, distance and likelihood methods of the PAUP* Version 4.0b 10 (Swofford 2002) and of MEGA version 2.1 (Kumar et al. 2001). Trees were 9 created using a heuristic search with random addition (50 replicates for parsimony and 10 replicates for maximum likelihood), and TBR branch swapping algorithm for PAUP* or CNI for MEGA. Neighbor joining trees were generated using Kimura-2-parameter distance. All trees were bootstrapped (n=100). Trees were run unweighted and weighted (2:4:1 by codon position). Pacific hagfish was used as an outgroup. Trees for individual isoforms of glutamine synthetase were also assessed. Trees were compared with a morphological tree based on orders of fishes (Nelson 1994; Helfman et al. 1997). Common names, family names and orders offish used in this study are listed in Table 3. 10 Results DNA Amplification Primer sets involving eleven primers successfully amplified glutamine synthetase-like product in Oncorhynchus keta, Oncorhynchus kitsutch, and Oligocottus maculosus. These primers were then used on the remainder of the fish. Amplification was not successful for all primers on all fish, so multiple primers were used on some fish. Fragment size varied due to amplification of different types of DNA product, complementary DNA (cDNA) and genomic DNA (Table 4). Complementary DNA produced a smaller fragment size and did not vary because cDNA does not contain introns. Genomic DNA produced products of varying lengths due to variation in intron lengths between species. Five intron sites were identified between positions 230/231, 392/393, 539/540, 667/668, and 867/868 of the coding sequence (Figure 1). Introns had an average length of 108bp, 88bp, 90bp, 102bp, and 122bp respectively. No intron site data was generated for fish whose GS product was generated from cDNA only. Five primers were largely successful and therefore used extensively. Primer GS-237 or primer GS-232 used with primer GSR-911 produced ~ 700 bp product when amplifying cDNA, and ~1050bp to ~1150 bp product when amplifying genomic DNA. Primer GS-448 used with primer GSR-977 amplified -550 bp product with cDNA and -800 bp to 900 bp product with genomic DNA. Sequence from the region of overlap amplified from the two primer sets (corresponding to positions 467 through 899 of published GS sequence Xenopus laevis Genbank accession number D50062) in the cDNA of the above fish produced two different glutamine synthetase products for shiner perch, coho salmon, white spotted greenling, penpoint gunnel, Pacific sanddab, three spined stickleback, tubesnout, buttersole, cabezon and 11 crescent gunnel (Figure 1 and Appendix 2). The average difference between these two isoforms from within the same fish was -18 % with the largest difference being 22% and occurring in both speckled sanddab and Pacific sanddab and the smallest difference of 8% occurring in shiner perch (Figure 2). Two different products from cDNA indicate that glutamine synthetase has more than one transcript for GS which is therefore not a single copy gene but a multicopy gene in these animals. A second copy of glutamine synthetase product was also observed in the coding sequence of the genomic product for speckled sanddab, starry flounder, high cockscomb, and mossy sculpin. These sequences also produced different introns between the two different GS products (Figure 1). Only one copy of glutamine synthetase was observed for hagfish, alligator gar, striped seaperch, buffalo sculpin, tidepool sculpin, bay pipefish and chum salmon. Isoform designation Neighbor joining compares the distances or raw sequence similarity between sequences and was performed for the multiple cDNA products and genomic products (after removal of introns). This produced a tree (Figure 3) with a distinct clade (bootstrap value 99) for one isoform in 12 of the 14 fishes compared: cabezon, white spotted greenling, mossy sculpin, tubesnout, three spine stickleback, high cockscomb, penpoint gunnel, crescent gunnel, starry flounder, buttersole, Pacific sanddab and speckled sanddab. Also grouped within this clade were both isoforms from the shiner perch. The distances between the fish within this clade was < 15 % (Figure 2). Therefore for this paper, an isoform of glutamine synthetase is identified as the sequence from either cDNA or genomic DNA (less introns) amplification which is resolved into a distinct clade by neighbor joining, whose overall similarity to other sequences within the clade is 15 % or less. A recent differentiation of an isoform may 12 occur for a fish within a clade, but is labeled by numbers after the isoform designation indicating more than one copy of the isoform from a particular fish originates somewhere within that clade. The clade with 13 of the fish represented is herein referred to as the A clade, with the isoforms found in it designated as A for each species and with two distinct shiner perch isoforms referred to as A1 and A2, where the average distances within the A clade was smaller for Al than A2 of the shiner perch isoforms. Figure 3 also shows a separation of the second isoforms of the above listed fish in Clade A (except shiner perch), indicating that the second sequence products isolated from these fish did not all represent the same glutamine synthetase isoform. Eight fish were grouped into the same clade for the second isoform (bootstrap 99): white spotted greenling, three spine stickleback,.tubesnout, mossy sculpin, cabezon, high cockscomb, penpoint gunnel and crescent gunnel. The pairwise distances for this clade were < 8% (Figure 2). This clade is therefore labeled as B and all the fish within it have an isoform designation of B. Four fish were grouped into another clade for their second isoform (bootstrap 79): Pacific sanddab, speckled sanddab, starry flounder and buttersole. The pairwise distances within this clade was < 15 % (Figure 2). This clade is therefore referred to as clade C and all the fish within it have an isoform designation of C. Also observed within this tree (Figure 3) was the separation of the coho genes into their own clade (bootstrap 72), with the pairwise distance between the two isoforms being only 13 % (Figure 3) and were therefore labeled Dl and D2. Overall Analysis GS products All amplification products from all fish used in this study, including those that only produced one gene product, were analyzed. A region corresponding to positions 467 to 899 of the published sequence of Xenopus laevis (Genbank accession number D50062) was 13 used for analysis. Base composition for fish sequence is reported in Table 5. and is similar with that reported for GS of other vertebrates (Appendix 3). Isoforms did not vary in overall base composition. Amino acid translation shows sites that are conserved in GS sequences of all other organisms (Eisenberg et al. 2000) are also conserved in the fish used in this study. One exception however, occurred at position 135, where all organisms code for alanine, whereas fish with the B isoform coded for isoleucine. Active sites for this region of GS (Eisenberg et al. 2000) were also maintained (Figure 4). Amino acid composition also loosely supports the existence of multiple genes of GS. At position 86 of Figure 4, fish with isoform C code for isoleucine while the amino acid for this position varies for the other isoforms from either valine or alanine. Fish with the D isoform code for serine at position 33 whereas other isoforms code for alanine at this position. Also seen in the D isoform a methionine at position 55 but the B isoform codes for aspartic acid here. At position 102 isoforms A, C and D code for alanine whereas isoform B codes for valine; at position 135 isoform A codes for alanine whereas isoform B codes for isoleucine. Phylogenetic Analysis GS products Phylogenetic analysis using neighbor joining, regardless of software used, weighted or unweighted, supported trees with similar topology (Figure 5). In each case isoforms A, B, C and D occurred in separate clades. Shiner perch had two sequences which clustered within the A clade. Both coho salmon isoforms clustered within the D clade. The pipefish GS isoform appears to have arisen as a sistergroup to gene A for GS although this branch 14 was not well supported but was consistently outside of the gene A cluster. The alligator gar isoform always arose on its own branch. Analysis of sequence data using IMSEA (Beckenbach unpublished) revealed 239 fixed sites in the nucleotide data. There were 193 parsimony informative sites, 41 occurring at first codon positions, 22 occurring at second codon positions and 130 occurring at third codon positions. Parsimony analysis both weighted and unweighted produced trees with similar topologies separating the GS isoforms (Figure 6) but not completely identical topologies as with the neighbor joining tree above. Using weighted parsimony analysis, the C isoforms did not separated into a single clade with two branches, but were part of a ladder from which the B clade branched off (Appendix 4). Unweighted parsimony analysis resulted in the separation of the two isoforms of coho salmon onto different branches but the branch with coho DI has low bootstrap support (under 50% not shown) (Figure 6). Both shiner perch isoforms clustered within the A clade. The pipefish isoform showed the same pattern as seen in the neighbor joining tree (Figure 5) branching as a sistergroup to the A clade. Maximum likelihood analysis also separated isoforms A and B both with weighted and unweighted analysis but isoform C did not form its own clade in either likelihood analysis. Isoform D formed its own clade in the weighted analysis (Figure 7), but not for the unweighted analysis (data not shown). Again both shiner perch genes clustered within the A cluster, however the pipefish isoform was also within this cluster although the branch was weakly supported (under 50% not shown). Weighted and unweighted maximum likelihood trees had similar topologies, however the weighted tree had better resolution and higher bootstrap support (Figure 7). 15 Isoform analysis and phylogenetic analysis of fishes Isoform A analysis produced trees with similar topologies regardless of method of analysis or weighting method (Figures 8, 9 and 10). The only difference between isoform A trees was the resolution level. Isoform B analysis also gave similar tree topologies regardless of method used for analysis or weighting method (Figures 11,12 and 13). Isoform C trees were all the same (Data not shown), with one clade of the order Pleuronectiformes with two branches, each with 100% bootstrap support; one for the family Paralichthyidae which includes the Pacific sanddab and the speckled sanddab and one for the family Pleuronectidae which includes the starry flounder and the buttersole. Generally fish clustered within their families. Order separation was not evident except in Scorpaeniformes. Within the order Scorpaeniformes, white spotted greenling (family Hexagammidae) never arose within the Cottidae clade (sculpins and cabezon) but always arose near this familial group. In all trees Pacific sanddab and speckled sanddab were grouped within their family Pleuronectidae, however the A isoform trees did not join the order Pleuronectiformes together with its two represented families Pleuronectidae and Paralichthyidae. In all trees was a clade of the order Gasteriformes, which paired as sister taxa tubesnout with three spine stickleback. Common to both isoform A and isoform B trees was a clade of the family Pholidae (penpoint gunnel and crescent gunnel) as sister group to the family Stichaeidae (high cockscomb). These fish are within the order Perciformes but never clustered with Embiotocidae, the other family within this order (shiner perch and striped seaperch). Shiner perch isoform Al always paired with striped seaperch isoform A in all A isoform trees but not with the Pholidae/Stichaeidae clade. Shiner perch A2 isoform always appeared as a 16 sistergroup to the butterole isoform A which is in a completely different order than the shiner perch. Since more species produced isoform A and isoform B products, these isoforms were used for phylogenetic analysis of the taxa sampled for this study with the exception of isoform C's grouping of the order Pleuronectiformes. The 432 bp fragment of isoform A was amplified in all fish except alligator gar. For isoform A there was 272 conserved sites and 160 variable sites. Pairwise differences revealed that penpoint gunnel and crescent gunnel were the most genetically similar for isoform A (0.5% difference), while shiner perch A2 and Pacific hagfish were the least genetically similar (27.1 % difference) and speckled sanddab and mossy sculpin were the least similar of all the A isoforms (15.0 % difference)(Figure 14). Weighted analyses gave better resolution and higher bootstrap values than unweighted analyses for isoform A. Isoform B was only amplified in eight taxa: white spotted greenling, penpoint gunnel, three spine stickleback, tubesnout, mossy sculpin, high cockscomb, cabezon and cresent gunnel. Again weighted analyses gave better resolution and higher bootstrap values than unweighted analyses. For isoform B there was 370 fixed sites and 62 variable sites. Pairwise differences again showed that penpoint gunnel and crescent gunnel were the most genetically similar for isoform B (1.0 % difference) and tubesnout was most different from Pacific hagfish (27.6% difference for these distances) while within the B isoforms, cabezon and three spine stickleback were the least genetically similar ( 7.7 % difference)(Figure 14). 17 Discussion Glutamine synthetase gene duplication(s) DNA and cDNA roducts isolated in this study are indeed glutamine synthetase gene(s) since the functional sites as given in Eisenberg (2000) are conserved (Figure 4) and the products are similar to GS products in Genbank. Glutamine synthetase can no longer be considered a single copy gene in eukaryotes. Multiple isoforms of GS in cDNA indicates that this gene is a multicopy gene perhaps even part of a gene family. Multiple copies of glutamine synthetase are also found in kidney bean, peas, alfalfa, and corn (Cullimore et al. 1984; Tingey et al. 1987; Li et al. 1993; Temple et al. 1995). Different genes in plants are expressed in different plant tissues. Multiple copies of GS have also been found in fish not used in this study and showed differential tissue expression (Murray 2002; Walsh et al. 2002). In other teleosts three copies of GS have been isolated in diploid fish and six GS isoforms were isolated in fish with a polyploid ancestry (2002 Busby, Ellen, University of Victoria, pers. comm.). Pseudogenes have also been observed in human and mouse (Chakrabarti et al. 1995) which may imply multiple copies of glutamine synthetase were present and were subsequently lost by mutation. Glutamine synthetase has recently been shown to have differential expression in different developmental stages in two eukaryotes Zea mays (Li et al. 1993) and Oncorhynchus mykiss (2002 Wright, P. A., University of Guelph, pers. comm.), however, no developmental information was obtained for this study. More than two different isoforms of GS are represented in this study. The neighbor-joining analysis of all isoforms reflects this (Figure 5). Multiple clades are formed with a higher degree of divergence than the within clade divergence. Most fish fall have an isoform that falls within the A clade; exceptions are bay pipefish, coho salmon, chum salmon and alligator gar. These fish likely have the A isoform but it was not amplified by the methods utilized in this study. Lack of PCR product of the A isoform only implies that 18 the primers utilized in this study were unable to amplify the A gene for the tissue used for extraction. Bay pipefish did not group within any clade and instead formed its own branch. Both coho salmon isoforms were isolated in a clade, distinct from the A, B or C clade. Chum salmon grouped with coho salmon. Alligator gar formed its own branch. This might have been predicted because it is a more primitive fish than the other fish used in this study and could be used as an outgroup with the Pacific hagfish, but this branch may represent a paralogous gene since gars are polyploid (Schultz 1980). The separation of B and C isoforms into separate clades indicates that the second isoform isolated from most fish in this study did not represent the same gene. Isoforms A and B were isolated for white spotted greenling, penpoint gunnel, crescent gunnel, high cockscomb, mossy sculpin, cabezon, three spine stickleback and tubesnout. Isoforms A and C were isolated for the Pacific sanddab, speckled sanddab, starry flounder, and buttersole. At the beginning of this study, differential expression of GS was not known, and therefore care was not taken to isolate specific organs. In some cases all of the organs within the gut cavity were combined. The GS sequence determined in bay pipefish was isolated from RNA and therefore sensitive to tissue specificity. The visceral tissue of this animal is surrounded by a large block of muscle tissue, and it is likely that the RNA extracted from this animal came predominantly from muscle tissue and secondarily from visceral tissue. Most other fish had large enough visceral cavities, that the predominant tissue isolated was liver or intestinal tissue for RNA extractions. Tissue from shiner perch was extracted multiple times due to amplification problems and isoform A2 was predominantly from gill 19 tissue, not liver, intestinal or muscle tissue. Isoform A2 from shiner perch represents a paralogous gene within shiner perch which has only differentiated a small amount from the A isoform. Differential expression of GS genes may explain why sequences in these fish were different from the other dominant isoforms and showed up in unexpected locations for the overall phylogenetic analyses. Glutamine synthetase enzyme is thought to have undergone a duplication event. Bacterial GS I is differs from eukaryotic GS II (Kumada et al. 1993; Tateno 1994; Pesole et al. 1995) and another type of GSIII also occur in Bacteroides fragilis (Hill et al. 1989) and Butyrivibrio fibrisolvens (Goodman and Woods 1993). This duplication and subsequent divergence occurred prior to the divergence of prokaryotes and eukaryotes (Pesole et al. 1991; Kumada etal. 1993; Tateno 1994; Pesole et al. 1995; Saccone et al. 1995). Gene duplications arise by multiple methods and can produce multilple sized products. Duplications can arise within a gene or spanning a complete gene by unequal crossing over during recombination resulting in tandem repeats on a chromosome (Ohno 1970; Li and Graur 1991; Twyman 1998; Freeman and Herron 2001). Evidence for tandem duplication occurs in the vertebrate lineage of the globin gene family (Proudfoot and Maniatis 1980; Freeman and Herron 2001). Duplication can also occur on a larger scale where regional portions of chromosomes or entire chromosomes (aneuploidy) are duplicated (Li and Graur 1991) (Ohno 1970; AUendorf and Thorgaard 1984; Twyman 1998). Finally, gene duplications can arise from polyploidy (Ohno 1970; AUendorf and Thorgaard 1984; Li and Graur 1991; Twyman 1998; Freeman and Herron 2001). Polyploidization results from a multiplication of an organism's entire genome. Many eukaryotes are descendants of lineages that have undergone polyploidization (For review see Otto and Whitton, 2000). Multiple rounds of genome duplication occurred in fish and this could explain the multiple 20 copies of genes or even gene families found in fish (Ohno 1970; Holland et al. 1994; Wittbrodt et al. 1998; Meyer and Schartl 1999; Taylor et al. 2001). Only three fish used in this study are known polyploids: alligator gar (Schultz 1980), coho salmon (Schultz 1980; Allendorf and Thorgaard 1984), and chum salmon (Schultz 1980; Allendorf and Thorgaard 1984). The latter two are partial tetraploids - only a portion of their genome remains polyploid. Chromosome numbers in polyploid fish are generally higher than non-polyploid fish (Schultz 1980; Allendorf and Thorgaard 1984). There is no indication that any of the other fish used in this study are polyploid. Their chromosome numbers are similar to the average chromosome number for non-polyploid fish (Froese and Pauly 2000). In alligator gar, coho salmon and chum salmon, the GS isoform(s) expressed did not group with the GS isoforms expressed in the majority of the rest of the fish in this study. It is likely that the isoforms expressed in the polyploid fish represented different paralogous genes not found in the non-polyploid fish and is therefore reflected in the phylogenetic trees when all isoforms were analyzed (Figures 5, 6 and 7) by appearing as separate branches. Isoforms orthologous to either the A gene or the B gene in the polyploid fish were not amplified likely due to the differential expression of the GS isoforms. The method of gene duplication for glutamine synthetase cannot be determined in this study. Phylogenetic analysis of GS products With the exception of Pacific hagfish, alligator gar, coho salmon and chum salmon, fish used in this study are within the superorder Acanthoptyergii. This superorder is not phylogenetically resolved by morphology alone. There are two competing hypotheses for the resolution of this group that differ mostly in the composition of the Series Percomorpha (Figure 15). Johnson and Patterson (1993) combine the five orders Synbranchiformes, Elassomatidae, Gastereosteiformes, Mugiloidei and Atherinomorpha into a monophyletic 21 group within the Percomorpha series and refer to this group as Smegmamorpha. Dactylopteriformes, Scorpaeniformes, Perciformes, Pleuronectiformes and Tetradontiformes orders remain as unresolved in the Johnson and Patterson (1993) classification system. Nelson (1994) however, does not recognize the Smegmamorpha and defines the Percomorpha differently, although Nelson's overall classification of the superorder Acanthoptyerigii comprises the same orders and families as that of Johnson and Patterson (Nelson 1994). Fish used in this study, except those listed above, fall into the Percomorpha. Phylogenies generated by the data for GS isoform A does not support either Johnson and Patterson's classification or Nelson's classification system. For both GS isoforms A and GS isoforms B, Gasteriformes (three spined stickleback and tubesnouts) always appear as sister taxa to the branch containing Pholidae (penpoint gunnel and crescent gunnel) and Stichaeidae (high cockscomb) which are members of the order Perciformes (Figures 5, 6, 7, 8, 9, 10, 11, 12 and 13). Stichaidae always form as a sister group to Pholididae. The Perciformes also includes the family Embiotocidae (shiner perch and striped seaperch) which do not group with the other Perciform families the Pholids or Stichaeids (Figures 5, 6, 7, 8, 9 and 10) anywhere in this study. This is not completely surprising as the Perciform order is considered polyphyletic (Johnson 1993; Johnson and Patterson 1993; Nelson 1994) and has no synapomorphy to support it (Nelson 1994). Nelson expects this order to undergo a re-classification in the near future (Nelson 1994); therefore Pholidae and Stichaeidae may eventually be re-classified outside of the Perfiformes branch and closer to the Gasteriformes. Glutamine synthetase isoform A did not result in a monophyletic grouping of the Order Pleuronectiformes (Pacific sanddab, speckled sanddab, starry flounder and buttersole) as 22 was predicted by the morphological analysis of Chapleau (1993). Species within the two families Paralichthyidae (Pacific sandddab and speckled sanddab) and Pleuronectidae (starry flounder and buttersole) did group together for both GS isoform A and GS isoform C. GS isoform C grouped the flatfish together, but the only Pleuronectiformes produced isoform C. Therefore grouping Pleuronectiformes together may be artificial due to the presence of a common isoform and not actually a phylogenetic resolution of the order. The familial relationships of the Pleuronectiformes are still not determined and are likely to change in the future (Johnson 1993; Johnson and Patterson 1993). Isoform A analysis clearly does not support monophyly of this order perhaps reflecting the gene phylogeny and not the species phylogeny. Scorpaeniformes (cabezon, buffalo sculpin, tidepool sculpin, mossy sculpin and white spotted greenling) grouped together for GS isoform A on one branch (Figures 8, 9 and 10) but did not form one branch for GS isoform B (Figures 11,12 and 13). Bootstrap support for GS isoforms B was also lower than that for GS isoforms A. The lack of resolution for GS isoform B may just reflect the overall smaller sequence divergence in GS isoform B than in GS isoform A (Figure 14) and therefore less informative sites available to group the Scorpaeniformes into one order on one branch. The phylogeny of the Scorpaeniform fish generated in this study for GS isoform A agrees with classification of Scorpaeniformes presented by Imamura and Shinohara (1998) and the classification of Nelson 1994. Glutamine synthetase isoform A separated the orders Scorpaeniformes and Gasteriformes into their own clades, but did not support monophyly of Perciformes or Pleuronectiformes (Figure 8, 9 and 10). Glutamine synthetase isoform B separated the Perciformes and Gasteriformes, but did not support monophyly of Scorpaeniformes. These differences may reflect both the differences in type and number of species available for analysis of both 23 isoforms for this study. These differences my also be caused by different phylogenetic patterns for the genes studied. 24 Conclusions and future work Glutamine synthetase is a multicopy gene or part of a gene family in fish. Not all the fish in this study produced two copies of glutamine synthetase but all of these fish likely have at least three copies of GS. This study did not assess the question of multiple copies of GS systematically. Differential expression of GS within the tissues was not addressed. Tissues sampled for this study were mostly from visceral tissue and not from heart, brain, gill (except shiner perch) or skin. In future studies each organ within a fish should be sampled separately for RNA in order to determine the exact number of GS genes and where in the fish these new genes are active. It is also important to sample fish at different developmental stages to determine if there is any developmental pattern of gene activity. The primers used in this study were based on vertebrate sequence alignments from Genbank (Appendix 1) and therefore may have a bias favoring one GS gene so the primers may not be suitable to amplify all three (or even two) GS genes. If all copies within a fish are determined from multiple fish, it may be possible to design new primers that target specific isoforms of GS. The number of GS isoforms may also be identified using cDNA libraries, but all tissues would have to be represented and each species offish addressed separately. Glutamine synthetase may no longer be useful for phylogenetic analysis of fishes unless all isoforms of GS are isolated for each fish and compared for analysis. Most of the fish used in this project were frozen and therefore effort should be made to isolate the remaining isoforms from the different tissues of the fish. 25 As for the phylogeny of the fishes used in this study, there is still much work to be done. Although this study mostly agreed with classical fish taxonomy, there were exceptions. I would not suggest re-classifying the Pholidae (penpoint and crescent gunnels) and Stichaeidae (high cockscomb) out of the order Perciformes and into their own order based on the evidence of one gene, but the relationship of these species should be re-examined and more genes should be analyzed. More molecular work should be done to try and aid in the classification of these fish. Future studies should be carefully planned to address some of the classification questions still unresolved and should include additional representative species from each order. 26 Species Name Common Name Genbank Accession Number Cricetulus griseus Chinese hamster AF150961 Opsanus beta Gulftoadfish AF118103 Heterodontus francisci Hornshark AF118104 Gillichthys mirabilis Long-jawed mudsucker AF266200 Scyliorhinus torazame Cloudy catshark AF306642 Danio rerio Zebrafish AW076779 Ictalurus punctatus Channel catfish BE469571 Xenopus laevis African clawed frog D50062 Bos taurus Cow J03604 Galius gallus Chicken M29076 Rattus norvegicus Norway Rat M29579 Mus musculus House Mouse M60803 Squalus acanthias Spiny dogfish U04617 Cricetulus longicaudatus Long-tailed hamster X03495 Homo sapiens Human X59834 Sus scrofa Pig Z29636 Table 1. Glutamine synthetase sequences available in Genbank for all vertebrates prior to August 1999. 27 Primer Name Direction Position* Sequence (5' to 3') GS-101 Forward 101 - 119 GTGAAGAAGCAGTACATGG GS-232 Forward 232 -249 TCTACCTGAATGGAACTT GS-237 Forward 237 - 254 CAGAATGGAACTTTGATGG GS-300 Forward 300 -318 TCGTTCCTGCTGCCATGTT GS-448 Forward 448 -465 CCCTTGGTTTGGAATGGA GS-537 Forward 537 - 554 AAGGTCCCTATTACTGTG GSR-548 Reverse 548 - 565 TGCTCCAAATCCACAGTA GSR-802 Reverse 802 -819 CACCCAGCACCATTCCAG GSR-91 1 Reverse 911 -928 GTAGGCAAGGATGTGGTA GSR-977 Reverse 977 -994 GTTGATGTTGGAGGTTTC GSR-1069 Reverse 1069 - 1086 CGGCGGTCTTCAAAGTAG Table 2. Primers used in the amplification and sequencing of glutamine synthetase product. * indicates sequence position in X. laevis of Appendix 1. 28 Common Name Latin Name Family Subfamily Order Pacific hagfish Eptatretus stoutii (Lockington, 1878) Myxinidae Eptatretinae Myxiniformes Alligator gar Atractosteus spatula (Lacepede, 1803) Lepisosteidae N/A Lepisosteiformes Chum salmon Oncorhynchus keta (Walbaum, 1792) Salmonidae Salmoninae Salmoniformes Coho salmon Oncorhynchus kisutch (Walbaum, 1792) Salmonidae Salmoninae Salmoniformes Three spined stickleback Gasterosteus aculeatus Linnaeus, 1758 Gasterosteidae Gasterosteinae Gasterosteiformes Tubesnout Aulorhynchus flavidus Gill, 1861 Aulorhynchidae N/A Gasterosteiformes Bay pipefish Syngnathus leptorhynchus Girard, 1854 Syngnathidae Syngnathinae Syngnathiformes White spotted greenling Hexagrammos stelleri Tilesius, 1810 Hexagrammidae Hexagramminae Scorpaeniformes Cabezon Scorpaenichthys marmoratus Girard, 1854 Cottidae N/A Scorpaeniformes Buffalo sculpin Enophrys bison (Girard,' 1854) Cottidae N/A Scorpaeniformes Tidepool sculpin Oligocottus maculosus Girard, 1856 Cottidae N/A Scorpaeniformes Calico sculpin a.k.a. Mossy Sculpin Clinocottus embryum (Jordan & Starks, 1895) Cottidae N/A Scorpaeniformes Table 3. Names of animals used in this study including common names, species names, family names, subfamily names and order names. 29 Common Name Latin Name Family Subfamily Order Striped seaperch Embiotoca lateralis Agassiz, 1854 Embiotocidae N/A Perciformes Shiner perch Cymatogaster aggregata Gibbons, 1854 Embiotocidae N/A Perciformes High cockscomb Anoplarchus purpurescens Gill, 1861 Stichaeidae N/A Perciformes Penpoint gunnel Apodichthys flavidus Girard, 1854 Pholidae N/A Perciformes Crescent gunnel Pholis laeta (Cope, 1873) Pholidae N/A Perciformes Pacific sanddab Citharichthys sordidus (Girard, 1854) Paralichthyidae (Bothidae) N/A Pleuronectiformes Speckled sanddab Citharichthys stigmaeus Jordan & Gilbert, 1882 Paralichthyidae (Bothidae) N/A Pleuronectiformes Buttersole Isopsetta isolepis (Lockington, 1880) Pleuronectidae Pleuronectinae Pleuronectiformes Starry flounder Platichthys stellatus (Pallas, 1788) Pleuronectidae Pleuronectinae Pleuronectiformes Table 3. continued 30 Primer Pair Approximate Size of Approximate size of Genomic DNA Fragment cDNA Fragment GS-101 with GSR-548 660 bp 470 bp GS-101 with GSR-1069 1500 bp 990 bp GS-232 with GSR-911 1100 bp 700 bp GS-232 with GSR-977 1160 bp 760 bp GS-232 with GSR-1069 1250 bp 850 bp GS-237 with GSR-911 1100 bp 700 bp GS-237 with GSR-977 1160 bp 760 bp GS-237 with GSR-1069 1250 bp 850 bp GS-300 with GSR-802 800 bp 520 bp GS-448 with GSR-977 860 bp 550 bp GS-448 with GSR-1069 920 bp 640 bp GS-537 with GSR-977 680 bp 460 bp GS-537 with GSR-1069 780 bp 780 bp Table 4. Primers used to amplify PCR product for glutamine synthetase. Numbers associated with primers give positional information of the location of the primers relative to the published glutamine synthetase sequence for Xenopus laevis (Genbank accession number D50062). Approximate fragment size indicates the size of the fragment produced when the specific primer pair indicated is used for amplification. bp = base pairs. Name Length 6 A T C j Cabezon_A 432 30 32 23 38 21 76 24 54 0 White_spotted_greenling_ _A 432 30 56 22 45 22 92 24 07 0 Mossy_sculpin_A 432 30 09 23 15 23 61 23 15 0 Tubesnout_A 432 31 02 22 92 21 99 24 07 0 Three_spine_stickleback^ _A 432 32 18 21 76 20 83 25 23 0 High_cockscomb_A 432 30 56 23 38 21 76 24 31 0 Penpoint_Gunnel_A 432 30 56 23 15 22 92 23 38 0 Crescent_gunnel_A 432 30 32 23 38 22 69 23 61 0 Starry_flounder_A 432 28 47 24 07 23 61 23 84 0 Buttersole_A 432 28 70 24 54 24 07 22 69 0 Pacific_sanddab_A 432 29 17 24 07 19 91 26 85 0 Speckled_sanddab_A 432 29 40 23 84 20 37 26 39 0 Shiner_perch_Al 432 28 94 24 54 22 92 23 61 0 Shiner_perch_A2 432 29 40 23 38 24 07 23 15 0 Buf falo_sculpin_A 432 29 63 23 84 22 69 23 84 0 Striped_seaperch_A 432 28 94 24 54 23 15 23 38 0 Tidepool_sculpin_A 432 30 56 22 92 22 92 23 61 0 White_spotted_greenling_ _B 432 30 79 22 45 21 76 25 00 0 Three_spine_stickleback_ _B 432 31 71 21 53 19 68 27 08 0 Tubesnout_B 432 31 48 22 69 20 14 25 69 0 Mossy_sculpin_B 432 31 59 21 89 20 65 25 87 30 Cabezon_B 432 30 98 22 20 21 46 25 37 22 High_cockscomb_B 432 32 41 21 30 20 37 25 93 0 Penpoint_gunnel_B 432 32 18 21 06 20 60 26 16 0 Crescent_gunnel_B 432 31 94 21 06 20 37 26 62 0 Starry_flounder_C 432 31 48 23 15 22 45 22 92 0 Buttersole_C 432 31 46 23 17 21 95 23 41 22 Speckled_sanddab_C 432 30 79 21 99 21 53 25 69 0 Pac i f i c_sanddab_C 432 30 79 21 99 21 76 25 46 0 Coho_salmon_Dl 432 30 56 23 15 22 45 23 84 0 Coho_salmon_D2 432 30 09 23 38 22 92 23 61 0 Chum_salmon_D 432 30 09 22 69 22 92 24 31 0 Bay_pipefish_E 432 30 79 22 92 17 82 28 47 0 Alligator_gar_F 432 30 56 22 92 19 68 26 85 0 Hagfish 432 29 86 21 06 27 55 21 53 0 Table 5. Base composition percentage statistics for amplified glutamine synthetase fragments. Fragments correspond to 432 bp fragment positioned from 467 to 899 of Xenopus laevis published glutamine synthetase sequence (Genbank accession number D50062). Letters after fish name indicate putative isoform designation. 32 232-237" Pacific hagfish Buffalo sculpin A Buttersole A Cabezon A Crescent gunnel A high cockscomb A Mossy sculpin A Pacific sanddab A Penpoint gunnel A I ' Speckled sanddab A |"*^-Starry flounder A Striped seaperch A Three-spine stickleback A TubesnoutA Tidepool sculpin A White spotted greenling A Shiner perch A1 Shiner perch A2 Cabezon B f"-^'y Crescent gunnel B High cockscomb B Mossy sculpin B Penpoint gunnel B Three-spine stickleback B Tubesnout B y,.,,,,,,,. White spotted greenling B Buttersole C Pacific sanddab C Speckled sanddab C Starry flounder C Chum salmon D Coho salmon D1 Coho salmon 02 Bay pipefish E Alligator gar F cDNA Product DNA Product An intron site having the size N A primer where N indicates position Figure 1: Summary of DNA fragments of glutamine synthetase amplified by PCR for fishes used in this study. Top line gives positional information relative to published sequence of Xenopus laevis glutamine synthetase gene (Genbank Accession number D50062) from position lOObp to 1 lOObp. 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Tree is based on Kimura-2-parameter distance constructed with unweighted 432 bp fragment of glutamine synthetase product, using CNI branch swapping algorithm in MEGA version 2.1. Tree was bootstrapped 100 times. 35 Conserved 1 * * * * Consensus 1 QEYTILGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVEICGTN Sm A 1 Hs A 1 Ce A 1 D F. . Auf A 1 Gaa A 1 Ap A Apf A PI A 1 . D 1 . D 1 . D Ps A 1 . D Ii A 1 . D Cso A 1 . K . M Cst A 1 . K . M Ca Al 1 . Q Ca A2 1 . H Eb A 1 El A 1 . Q Om A 1 Hs B 1 . . . . L . D Gaa B 1 . . . . M. . . . D Auf B 1 . . . . M. . . . D Ce B 1 . . . . L . K . D Sm B 1 . . . . L ....V..s. . K . D Ap B Apf B PI B 1 A ....V.... . D 1 A . D 1 A ....V.... . D Ps C 1 ....V.... . K . Q Ii C 1 ....V.... . K . Q Cst C 1 . . . . L . . . . M. . . . M Cso C 1 . . . . L . . . . M. . . . M Oki Dl 1 S . . . . M Oki D2 1 N S . . . .M Oke D 1 N  . . . .M SI E 1 S . . . . Q As F 1 . Q Es 1 . . . .L. .V ....V..s. FS . .N Figure 4. Amino acid translation of glutamine synthetase products for all isoforms of GS. * - indicate site conserved in all organisms for GS (Eisenberg et al. 2000). ! - indicate active site of GS conserved in all organisms for GS (Eisenberg et al. 2000). Numbers correspond to amino acid number 135 to 279 of the published Xenopus laevis sequence (Genbank accession number D50062). Amino acid translation sites that support isoform designations shown at positions 102 and 135 for isoform A; Sites 55, 102, and 135 for isoform B; Sites 86 and 102 for isoform C; Sites 33, 55, and 102 for isoform D. 36 Conserved !**! * * * *** * i i j Consensus 61 AEVMPAQWEFQVGPCEGINMGDHLWVARFILHRVCEDFGWASFDPKPITGNWNGAGCHT 12 0 Sm A 61 P . Hs A 1 S S. Ce A 61  . Auf A 1  L Gaa A 61 S Ap A 1Apf A 61 S I PI A 1 I Ps A 61 Ii A 1 A Cso A 61 S I P . Cst A 1  P . Ca Al 61 A P. Ca A2 1  R Eb A 61  . El A 1 A P. Om A 61 S . Hs B 1 V Gaa B 61 S V Auf B 1Ce B 61Sm B 1Ap B 61Apf B 1 V S. PI B 61  S. Ps C 1 I AIi C 61  T A. Cst C 1 E I Cso C 61  I Oki Dl 61 A P. Oki D2 61 S AOke D 1 PSI E 61 D I P As F 1 S...D I P Es 61 S VD L. . . L I P Figure 4. continued. 37 Conserved * * Consensus 121 NFSTKEMREDGGLKAIEESIEKLG Sm A 121 . . . . D . . Hs A 121 . . . . D . . Ce A 121 . . . . D . . Auf A 121 . . . . D . . Gaa A 121 D Ap A Apf A 121 121 . V PI A 121 . V Ps A 121 Ii A 121 E . . Cso A 121 . . . .D. . Cst A 121 . . . . D . . Ca Al 121 . . . . D . . Ca A2 121 E . . . M . . Eb A 121 . . . . D . . El A 121 ....D.. Om A 121 . . . . D . . Hs B 121 E . . . I . . D . . E Gaa B 121 E . . .1 R . P Auf B 121 E . . .1 R.A Ce B ' 121 Sm B •' 121 P. . . . I . Ap B 121 .1 R . A Apf B 121 . I R . A PI B 121 . . T .1 R . A Ps C 121 . .V R. A IiC 121 L Cst C 121 . . . . Y . .V R.A Cso C 121 . . . . Y . .V R.A Oki Dl 121 ..G..D.. Oki D2 121 E . . R Oke D 121 E . . R SI E 121 D . . As F 121 EN . . . Y R. S Es 121 ...SLA..QA.. .QH..YA. ... A Figure 4. continued. 38 Pacific hagfish 100 82 76 Pacific sanddab A Speckled sanddab A 100 61 72 100 52 51 98 58 93 Striped seaperch A Shiner perch A1 Starry flounder A Buttersole A Shiner perch A2 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A 98 87 98 White spotted greenling A Tubesnout A Three spine stickleback A 100 100 High cockscomb A Penpoint gunnel A Crescent gunnel A 100 67 Bay pipefish E Coho salmon D2 Chum salmon D 100 55 Coho salmon D1 White spotted greenling B 54 98 58 57 Penpoint gunnel B Crescent gunnel B High cockscomb B 70 Mossy sculpin B Cabezon B 100 100 73 100 Three spine stickleback B Tubesnout B Pacific sanddab C Speckled sanddab C Starry flounder C Buttersole C Alligator gar F 50 changes Figure 5. Neighbor joining tree based on Kimura-2-parameter distances constructed from 432 bp fragment of glutamine synthetase from all isoforms amplified in all fish used in this study. I used TBR branch swapping algorithm and tree was bootstrapped 100 times. 39 100 100 52 81 95 88 96 53 65 57 93 89 96 96 100 66 100 77 99 53 53 100 98 71 52 100 100 Pacific hagfish Pacific sanddab A Speckled sanddab A Striped seaperch A Shiner perch A1 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A Tubesnout A Three spine stickleback A High cockscomb A Penpoint gunnel A Crescent gunnel A Starry flounder A Buttersole A Shiner perch A2 Bay pipefish E Coho salmon D2 Chum salmon D Coho salmon D1 White spotted greenling B Penpoint gunnel B Crescent gunnel B High cockscomb B Three spine stickleback B Tubesnout B Cabezon B Mossy sculpin B Pacific sanddab C Speckled sanddab C Starry flounder C Buttersole C Alligator gar F Figure 6. Maximum parsimony tree constructed from 432 bp fragment of glutamine synthetase from all isoforms amplified in all fish used in this study. Parsimony criterion was set to random addition, 50 replicates, using TBR branch swapping algorithm. Tree was bootstrapped 100 times. 40 100 100 65 96 76 87 98 87 87 79 90 74 98 56 70 92 59 98 59 100 98 Pacific hagfish Pacific sanddab A Speckled sanddab A Bay pipefish E Striped seaperch A Shiner perch A1 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A Tubesnout A Three spine stickleback A High cockscomb A Penpoint gunnel A Crescent gunnel A Starry flounder A Buttersole A Shiner perch A2 Coho salmon D2 Chum salmon D Coho salmon D1 White spotted greenling B Penpoint gunnel B Crescent gunnel B High cockscomb B Three spine stickleback B Tubesnout B Mossy sculpin B Cabezon B Pacific sanddab C Speckled sanddab C Starry flounder C Buttersole C Alligator gar F Figure 7. Maximum likelihood tree constructed from 432 bp fragments of all isoforms of glutamine synthetase for all fish used in this study. Likelihood criterion was set to random addition, 10 replicates, TBR branch swapping algorithm. I used HKY85 for the Likelihood model. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 41 Pacific hagfish 100 69 69 58 Pacific sanddab A Speckled sanddab A 100 95 98 99 100 50 substitutions/site 83 57 100 - Striped seaperch A - Shiner perch A1 Starry flounder A Buttersole A Shiner perch A2 Cabezon A 94 86 Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A • Tubesnout A • Three spine stickleback A . High cockscomb A Penpoint gunnel A Crescent gunnel A Figure 8. Neighbor joining analysis of isoforms A only of glutamine synthetase using Kimura-2- parameter distance. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 42 100 62 99 68 97 98 86 90 96 83 60 Pacific hagfish Pacific sanddab A Speckled sanddab A Striped seaperch A Shiner perch A1 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A Tubesnout A Three spine stickleback A High cockscomb A Penpoint gunnel A Crescent gunnel A Starry flounder A Buttersole A Shiner perch A2 Figure 9. Maximum parsimony tree constructed from A isoforms only of glutamine synthetase. Parsimony criterion was set to random addition, 50 replicates, TBR branch swapping algorithm. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. c 43 76 100 94 68 89 82 90 78 90 98 65 Pacific hagfish Pacific sanddab A Speckled sanddab A Striped seaperch A Shiner perch A1 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A Tubesnout A Three spine stickleback A High cockscomb A Penpoint gunnel A Crescent gunnel A Starry flounder A Buttersole A Shiner perch A2 Figure 10. Maximum likelihood tree constructed A isoforms only of glutamine synthetase. Likelihood criterion set to random addition, 10 replicates, TBR branch swapping algorithm. I used HKY85 for the Likelihood model. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 44 Pacific hagfish White spotted greenling B 80 100 79 Penpoint gunnel B Crescent gunnel B High cockscomb B 98 Three spine stickleback B Tubesnout B lossy sculpin B Cabezon B 50 substitutions/site Figure 11. Neighbor joining tree constructed from B isoforms only of glutamine synthetase based on Kimura-2-parameter distances. I used TBR branch swapping algorithm. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 45 Pacific hagfish White spotted greenling B 58 97 64 80 Penpoint gunnel B Crescent gunnel B High cockscomb B 96 Three spine stickleback B Tubesnout B 56 Mossy sculpin B Cabezon B Figure 12. Maximum parsimony tree constructed from B isoforms only of glutamine synthetase. Parsimony criterion was set to random addition, 50 replicates, TBR branch swapping algorithm. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 46 Pacific hagfish White spotted greenling B 70 95 74 51 Penpoint gunnel B Crescent gunnel B High cockscomb B 100 Three spine stickleback B Tubesnout B Mossy sculpin B Cabezon B Figure 13. Maximum likelihood tree constructed B isoforms only of glutamine synthetase. Likelihood criterion was set to random addition, 10 replicates, TBR branch swapping algorithm. I used HKY85 for the Likelihood model. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 2_ 8 > 31 o ere" > c? s -a 3 a. l § cr a. c D. l5 Cd 3 2§ CT. O I g ^ 3' 3 OQ — ro o-o D-ro 13 c O ^ 3 on -O 2^' 1 D-> OQ 3" O o o 7T on O o 3 fa on ro o o 3 02 —! on O 3 on o -h O-li on ° ^ ro ro 2 o > cr ro on ro 3 o ro on on O C? —5 — . 3 = oo 03 3 1/1 D_ °> OQ C 3 on I 0 q = n 3 o 1 3 on 03 OQ 3 ro ro O 3 on o =^ 1° O- 03 ro 03 r' I 00 3 3" i ro I ro O on 03 ~o rf 3' N ro O on 3 '— o' pv ro cr 03 o 2V 13 =i' L> 2 ro ^ P" ^ ro ro g £ I = CL 3 03' 3 o OQ S 3- o on —• 3 ^ § £. E. 3 m Ef ^ 3' °> Pi: I 0 ro 3 1 g s ? 3 Cd o -a —. &3 4^ -J I O ro 03 °'2. § 3 ° CL cr ry § 0 3 O to 00 VO VO bo Ln Lfi to JO 0 — O O 0 ;— 0 b UJ b bv bv 41 43i bv VO VO VO to to 0 — 0 VO VO — 0 0 UJ in to bo 431 UJ VO _ _ p — — to UJ to to — 43i UJ to to ON bv Ov j>> b — 43i b ~J UJ UJ UJ UJ UJ to Ufl to UJ 41 421 UJ to to to b VO Ln b VO — bv tO to to to UJ to 431 to UJ UJ 41 UJ UJ UJ j> bv ij\ 421 421 VO vo 00 0 UJ un — — to VO UJ UJ Ov 421 431 — — UJ ^1 VO VO 00 0 J> to un - -VO UJ UJ bv 421 VO UJ UJ UJ VO 0 0 0 to 0 to —J 421 42* bv b UJ UJ to VO VO VO 0 0 <] J> UJ Ov UJ 0 00 00 00 VO 4^ Ov to 1— —' •—' vo vo 00 VO VO VO — Ov un UJ b b UJ UJ b b ^1 00 00 ^1 00 to 431 VO 00 bo VO bo to VO 0 vo Ov -J to ^1 b VO 00 00 -Fi Ov Ov Ov b bv Ov b un to to p Ov Ov 43i 43i b b to to Ln UJ . -J 421 un Ov b b b io <1 b un U/l Ov UJ 421 un to to 'to to to to to to to to to to Ov Ov Ov ^1 Ov Ov VO Ov VO bv bv Ov 43-to to UJ UJ to 421 vo — b to VO to 48 i) Percomorpha Tetraodontiformes Pleuronectiformes Perciformes Scorpaeniformes Dactylopteriformes Atherinomorpha Mugiloidei Gasterosteiformes Elassomatidae Synbranchiformes Beryciformes Zeiformes (less caproids) Stephanoberyciformes oo 3 re yl r 3 o -i •a _r ii) Percomorpha Tetraodontiformes Pleuronectiformes Perciformes Scorpaeniformes Synbranchiformes Gasterosteiformes Caproidei Zeioidei Holocentroidei Berycoidei Trachichthyoidei Stephanoberyciformes Atherinomorpha Mugilomorpha Figure 15. The two competing hypotheses for the resolution of the Superorder Acanthoptyerygii. i) Phylogenetic relationships of the Acanthoptyerygii as presented by Johnson and Patterson (1993). ii) Phylogenetic relationships of the Acanthoptyerygii as presented by Nelson (1994). A small arrow on each cladogram shows where the author(s) believe the series Percomorpha begins. 49 References Allendorf, F. W. and G. H. Thorgaard (1984). Tetraploidy and the Evolution of Salmonid Fishes. Evolutionary genetics of fishes. B. J. Turner. New York, Plenum Press: 1-53. Altschul, S. F., T. L. Madden, et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17): 3389-402. Cabot, E. L. and A. T. Beckenbach (1989). Simultaneous editing of multiple nucleic acid and protein sequences with ESEE. Comput Appl Biosci 5(3): 233-4. Campbell, J. W. and D. D. Smith, Jr. (1992). Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase. Mol Biol Evol 9(5): 787-805. Chakrabarti, R., J. B. McCracken, Jr., et al. (1995). Detection of a functional promoter/enhancer in an intron-less human gene encoding a glutamine synthetase-like enzyme. Gene 153(2): 163-99. Chapleau, F. (1993). Pleuronectiform relationships: A cladistic reassessment. Bulletin of Marine Science 52(1): 526-540. Cullimore, J. V., C. Gebhardt, et al. (1984). Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family. J Mol Appl Genet 2(6): 589-99. Eisenberg, D., H. S. Gill, et al. (2000). Structure-function relationships of glutamine synthetases. Biochim Biophys Acta 1477(1-2): 122-45. Freeman, S. and J. C. Herron (2001). Evolutionary Analysis. Upper Saddle River, New Jersey, Prentice-Hall Inc. Froese, R. and D. Pauly (2000). FishBase 2000: concepts, design and data sources. Los Banos, Laguna, Philippines. 344 p., ICLARM. 2000: 344. Goodman, H. J. and D. R. Woods (1993). Cloning and nucleotide sequence of the Butyrivibrio fibrisolvens gene encoding a type III glutamine synthetase. J Gen Microbiol 139(Pt 7): 1487-93. Hart, J. L. (1988). Pacific fishes of Canada. Ottawa, Canada, Canadian Government Publishing Centre. Helfman, G. S., B. B. Collette, et al. (1997). The diversity of fishes. Maiden, Mass., Blackwell Science. Hill, R. T., J. R. Parker, et al. (1989). Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis. J Gen Microbiol 135(Pt 12): 3271-9. Holland, P. W., J. Garcia-Fernandez, et al. (1994). Gene duplications and the origins of vertebrate development. Dev Suppl: 125-33. Imamura, H. and G. Shinohara (1998). Scorpaeniform fish phylogeny: An overview. Bulletin of the National Science Museum, Tokyo, Series A (Zoology). 24(3): 85-212. Johnson, G. D. (1993). Percomorph phylogeny: Progress and Problems. 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Graur (1991). Fundamentals of molecular evolution. Sunderland, Mass., Sinauer Associates. Meister, A., Ed. (1985). Glutamate, Glutamine. Glutathione, and Related Compounds. Methods in Enzymology. Orlando, Florida, Academic Press, Inc. Meyer, A. and M. Schartl (1999). Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Curr Opin Cell Biol 11(6): 699-704. Mommsen, T. P. and P. J. Walsh (1992). Biochemical and environmental perspectives on nitrogen metabolism in fishes. Experientia 48: 583-593. Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res 10(2): 459-72. Murray, B. W., Busby,E., Mommsen,T. and Wright,P.A. (2002). Evolution of glutamine synthetase in vertebrates: Multiple glutamine synthetase genes expressed in rainbow (Oncorhynchus mykiss).Journal of Experimental Biology In Review Nelson, J. S. (1994). Fishes of the world. New York, J. Wiley. Ohno, S. (1970). 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CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. 51 Tingey, S. V., E. L. Walker, et al. (1987). Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. Embo J 6(1): 1-9. Twyman, R. M. (1998). Advanced Molecular Biology: A Concise Reference. New York, Springer-Verlag New York Inc. Walsh, P. J., G. D. Mayer, et al. (2002). A Second Glutamine Synthetase Gene with Expression in the Gills of the Gulf Toadfish (Opsanus beta). Journal of Experimental Biology In Review. Wittbrodt, J., A. Meyer, et al. (1998). More genes in fish? BioEssavs 20: 511-515. 52 GLFTDFISH 1 XENOPUS 1 DOGFISH 1 CATSHARK 0 HORNSHARK 0 CHICKEN 1 CATFISH 0 MOUSE 1 PIG 1 COW 0 RAT 0 LT-HAMSTER 0 C_HAMSTER 0 HUMAN 0 ZEBRAFISH 1 MUDSUCKER 0 60 60 60 1 GGAGCCCCGCGCCGAGCCCCTGCCCGCAGCCCAGCCCAGGACAGCCCTCGCCAGCTCCGC 60 3TTCTCGTGACCTGTTCACCCATCCATCATCCAGCTGGCCACTGTTCTGAACACCTTC 6 0 3ATTCTCGCTCTCGCGGCCTGCCCGCCCTGCCTCCTGCTCGCCGCCCAGAACACCGTC 6 0 GTCCACCCATCCATCATCCTGCCGGCCACCGCTCTGAACACCTTC GCTCGTGGCCCTGTCCACCCCGTCCATCATCCCGCCGGCCACCGCTCAGAGCACCTTC GCTTTACCCGCCCGCCTGCTCGGCGACCAGAACACCTTC TTCGTCCATCCAGGTGTGTTATAGTAGCGGT 45 58 39 60 31 GLFTDFISH 61 XENOPUS 61 DOGFISH 61 CATSHARK 0 HORNSHARK 0 CHICKEN 61 CATFISH 0 MOUSE 61 PIG 61 cow 0 RAT 46 LT-HAMSTER 59 C_HAMSTER 0 HUMAN 40 ZEBRAFISH 61 MUDSUCKER 32 ATGGCCACCTCAGCAAGTTCCCACTTGAACAAAGGCATCAAGCAAATGTACATGTC 120 120 120 120 120 120 105 118 56 99 120 91 GLFTDFISH 121 XENOPUS 121 DOGFISH 121 CATSHARK 0 HORNSHARK 0 CHICKEN 121 CATFISH 0 MOUSE 121 PIG 121 cow 0 RAT 106 LT-HAMSTER 119 C_HAMSTER 57 HUMAN 100 ZEBRAFISH 121 MUDSUCKER 92 ACTCCCTCAGGGGGATAAAGTCCAAGCTATGTACATTTGGATTGATGGAACAGGGGAGGG 180 ACTGCCCCAAGGAGAAAAGGTCCAGGTCACCTACGTGTGGATCGACGGCACCGGGGAAGG 180 GCTGCCCCAAGATGGCAAGGTGCAAGCGATGTACATCTGGATAGACGGCACAGGGGAGGC 18 0 CCCCAAGATGGCAAGGTGCAAGCTATGTATATCTGGATCGATGGCACAGGAGAGGC 5 6 GCTGCCGCAGGGTGAGAAGGTCCAAGCCATGTACATCTGGATCGACGGGACTGGGGAGCA 180 CCTGCCCCAGGGTGAGAAAATCCAAGCCATGTATATCTGGGTTGATGGTACCGGAGAACC 180 CCTGCCCCAGGGCGAGAAAGTCCAAGCTATGTACATCTGGATTGACGGTACGGGAGAGGG 180 CCTGCCCCAGGGCGAGAAGATCCAACTCATGTATATCTGGGTTGATGGTACCGGGGAAGG 165 CCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGG 17 8 CCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACCGGAGAAGG 116 CCTGCCTCAGGGTGAGAAAGTCCAGGCCATGTATATCTGGATCGATGGTACTGGAGAAGG 159 TCTCCCTCAGGGAGAAAAAGTTCAGGTCATGTACATCTGGATTGTNGGATCCGTAGAGGG 180 GCTGCCTCAGGGAGATCTGGTGCAGGCTATGTACATCTGGATCGACGGCACTGGAGAGGG 151 Appendix 1. Sequence alignment of all vertebrate sequences present in Genbank prior to 1999. Glftdfish - Opsanus beta, Xenopus = Xenopus laevis, dogfish = Squalus acanthias, catshark = Scyliorhinus torazame, hornshark = Heteroclontus francisci, chicken = Gallus gallus, catfish = Ictalurus punctatus, mouse = Mus musculus, pig = Sus scrofa, cow = Bos taurus, rat = Rattus norvegicus, Lt-hamster = Cricetulus longicaudatus, C-hamster = Cricetulus griseus, human = Homo sapiens, zebrafish = Danio rerio, mudsucker = Gillichthys mirabilis. Position 1 corresponds to position 1 of Xenopus laevis published glutamine synthetase sequence (Genbank accession number D50062). 53 GLFTDFISH 181 XENOPUS 181 DOGFISH 181 CATSHARK 0 HORNSHARK 57 CHICKEN 181 CATFISH 0 MOUSE 181 PIG 181 COW 0 RAT 166 LT-HAMSTER 179 C_HAMSTER 117 HUMAN 160 ZEBRAFISH 181 MUDSUCKER 152 GLFTDFISH 238 XENOPUS 238 DOGFISH 238 CATSHARK 0 HORNSHARK 114 CHICKEN 238 CATFISH 0 MOUSE 241 PIG 238 COW 0 RAT 223 LT-HAMSTER 236 C_HAMSTER 174 HUMAN 217 ZEBRAFISH 238 MUDSUCKER 209 GLFTDFISH 298 XENOPUS 298 DOGFISH 298 CATSHARK 0 HORNSHARK 174 CHICKEN 298 CATFISH 0 MOUSE 301 PIG 298 COW 0 RAT 283 LT-HAMSTER 296 C_HAMSTER 234 HUMAN 277 ZEBRAFISH 298 MUDSUCKER 269 ACTCAGATGTAAAACCAGA-AGTGAGGTGCAAAACCAGG-CGTCCGCTGCAAGACCAGA-AGTCCGCTGTAAAACCAAA-CCTCCGCTGCAAAACCCGC--ACGCTGGATTCTGAACCCAAAAGCATTGAAGATCTTCC -ACTCTGGATCAGGAACCCAAAACCATAGATGAAATCCC -ACCTTGGACAATGAGCCCAAGAGCATTGCCGAACTCCC -ACCTTGGACAAGGAGCCCAAGAACATTACTGACCTCCC -ACTCTGGACCACGAACCCAAGAGCCTGGAAGATCTCCC ACTGCGCTGCAAGACCTGTCGTACCCTGGACTGTGAGCCCAAGTGTGTGGAAGAGTTACC ACTGCGCTGCAAGACCCGG ACCCTGGATTCTGAGCCCAAGTGTATAGAAGAGTTGCC GCTACGCTGCAAGACCCGT-ACTGCGCTGCAAAACCCGC-ACTGCGCTGCAAAACCCGC-ACTGCGCTGCAAGACCCGG-ATTGAGATGCAAAACCAGG-GCTGCGCTGCAAAACCAGG--ACTCTGGACTGTGACCCCAAGTGTGTAGAAGAGTTACC -ACCCTGGACTGTGAGCCCAAGTGTGTAGAAGAGTTACC -ACCCTGGACTGTGAGCCCAAGTGTGTAGAAGAGTTACC -ACCCTGGACAGTGAGCCCAAGTGTGTGGAAGAGTTGCC -ACTCTAGACTCTGAACCTAAATCTGTTGAAGAACTTNC -ACACTAGACTCTGAACCCAAAAGCATTGAAGATCTGCC GGAATGGAACTTTGACGGTTCCAGCACGTACCAGGCTGAGGGCTCCAACAGCGACATGTA TGAATGGAACTTCGATGGATCCAGTACTCACCAAGCAGAAGGCTCAAACAGTGACATGTA AGAATGGAACTTCGATGGCTCAAGTACGTATCAGTCAGAGGGGTCCAACAGCGACATGTA AGAATGGAACTTTGATGGCTCAAGTACATATCAGTCAGAGGGGTCCAACAGCGACATGTA CGAGTGGAACTTTGATGGCTCCAGCACCTTCCAAGCCGAAGGCTCCAACAGCGACATGTA TGAGTGGAACTTTGATGGCTCCAGTACCTTTCAGTCTGAAGGCTCCAACAGCAACATGTA CGAGTGGAATTTCGATGGCTCTAGTACTTTTCAGTCTGAAGGCTCCAACAGTGACATGTA CGAGTGGAACTTTGATGGTTCTAGTACGTTTCAGTCTGAAGGCTCCAACAGCGACATGTA TGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGACATGTA TGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGAGCTCCAACAGTGACATGTA TGAGTGGAATTTCGATGGCTCTAGTACTTTACAGTCTGAGGGTTCCAACAGTGACATGTA TGAGTGGAACTTTGATGGTTCCAGCACATATCAGGCTGAGGGGTCCAACAGTGACATGTA AGAATGGAACTTTGATGGCTCCAGCACATATCAAGCAGAAGGTTCCAATAGTGACATGTA CTTGGTTCCCGCTGCCATGTTCCGTGATCCCTTTCGCGAAGATCCCAACAAGCTTGTCCT TCTCATCCCAGTCCAGATGTTCAGAGACCCATTCTGCCTGGACCCCAATAAACTGGTTAT CCTGGTTCCATCTGCCATGTTCCGGGATCCTTTCCGTAGGGATCCAAACAAGCTCGTCCT CCTCATCCCATCTGCCATGTTCCGGGATCCTTTCCGTAAGGATCCAAACAAGCTCATCCT CCTGCGACCTGCTGCCATGTTCCGGGACCCTTTTCGCAAGGATCCCAACAAATTAGTTCT TCTCCATCCTGTTGCCATGTTTAGAGACCCCTTCCGC AACAAGCTGGTGCT TCTTGTCCCTGCTGCCATGTTTCGGGACCCTTTCCGCAAGGACCCCAACAAGCTGGTGTT CCTCCATCCTGTGGCCATGTTTCGAGACCCCTTCCGCAGAGACCCCAACAAGCTGGTGTT TCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCCAACAAGCTGGTGTT TCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAAAGAGCCCAACAAGCTGGTGTT TCTCGTGCCTGCTGCCATGTTTCGGGACCCCTTCCGTAAGGACCCTAACAAGCTGGTGTT TTTGTTCCCTCAAGCCATGTTCAGAGACCCTTTCAGGAAAGACCCCAACAAACTGGTTCT TCTGGTCCCTGCTGCCATGTTCCGTGACCTTTCCGCAAGACCCAACNAACTGGTCCTGTG 237 237 237 113 237 240 237 222 235 173 216 237 208 297 297 297 173 297 300 297 282 295 233 276 297 268 357 357 357 233 357 351 357 342 355 293 336 357 328 Appendix 1. continued 54 GLFTDFISH XENOPUS DOGFISH CATSHARK HORNSHARK CHICKEN CATFISH MOUSE PIG COW RAT LT-HAMSTER C_HAMSTER HUMAN ZEBRAFISH MUDSUCKER GLFTDFISH XENOPUS DOGFISH CATSHARK HORNSHARK CHICKEN CATFISH MOUSE PIG COW RAT LT-HAMSTER C_HAMSTER HUMAN ZEBRAFISH MUDSUCKER GLFTDFISH XENOPUS DOGFISH CATSHARK HORNSHARK CHICKEN CATFISH MOUSE PIG COW RAT LT-HAMSTER C^HAMSTER HUMAN ZEBRAFISH MUDSUCKER 3 5 8 TTGTGAAGTGCTGAAGTACAACCGCAAACCATCAGAATCCAATCTTCGGTTGAACTGTAA 417 3 5 8 GTGTGAAGTCTTGAAATACAACCGCAAGTCTGCAGAGACCAACCTGAGACACACATGCAA 417 3 58 CTGTGAGGTCCTCAAGTATAACAGGAAGCCAGCAGAATCTAATCTTAGACACTCATGCCA 417 0 2 3 4 CTGTGAAGTCTTCAAGTACAACAGAAAGCCAGCAGAAACTAATCTTAGAAACTCATGCCA 2 9 3 3 58 CTGTGAGGTCTTCAAATACAACCGCCAGTCTGCAGACACAAATCTTCGGCACACCTGTAG 417 0 3 52 ATGTGAAGTTTTCAAGTATAACCGGAAGCCTGCAGAGACCAACTTGAGGCACATCTGTAA 411 3 58 CTGTGAGGTCTTCAAGTACAACCGAAAGCCTGCAGAGACCAACTTAAGGCACACCTGTAA 417 0 3 4 3 CTGCGAAGTATTCAAGTATAACCGGAAGCCCGCAGAGACCAACCTGAGGCACAGCTGTAA 4 02 3 56 CTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGAGACCAATTTAAGGCACTCGTGTAA 415 2 9 4 CTGTGAAGTCTTCAAGTACAACCAGAAGCCTGCAGAGACCAATTTAAGACACACGTGTAA 3 53 3 3 7 ATGTGAAGTTTTCAAGTACAATCGAAGGCCTGCAGAGACCAATTTGAGGCACACCTGTAA 3 9 6 3 5 8 GTGCGATGTTCTGAAATACAACCATAAACCTGCAGAAACCAATCTTCGTCAGTCCTGTAA 417 3 2 9 TGAAGTGCTCAAGTTCCACCGCCAGCCTGCAGAAACCAACCTGAAGATTACATGTT 3 84 418 CAAGGTGATGAACATGGTCAAGGACCAGCATCCTTGGTTTGGCATGGAGCAAGAGTACAC 4 7 7 418 GAAGATC ATGGAGATGGTGAATGACCACCGCCCGTGGTTTGGAATGGAGCAGGAATACAC 47 7 418 GAAAATCATGTCCATGATCGCAAATGAATATCCATGGTTTGGAATGGAACAAGAGTACAC 4 7 7 0 2 9 4 GAAAGTCATGTCCATGGTCGCAGGTGAACACCCATGGTTTGGAATGGAACAGGAATACAC 3 53 418 GCGGATTATGGATATGGTGTCCAACCAGCACCCCTGGTTTGGGATGGAGCAGGAGTACAC 4 7 7 0 GTTTGGCATGGAGCAGGAGTACAC 24 412 ACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAGCAGGAATATAC 4 71 418 ACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAGCAGGAATATAC 4 7 7 0 4 03 GCGTATAATGGACATGGTGAGCAGCCAGCACCCCTGGTTTGGAATGGAACAGGAGTATAC 462 416 ACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAACAGGAGTATAC 47 5 3 54 ACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAACAGGAGTATAC 413 3 97 ACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGCATGGAGCAGGAGTATAC 4 56 418 GAAGATTATGGATATGGTCCAGAACCAGCATCCTTGGTTTGGAATGGAACAGGAGTACAC 47 7 4 7 8 CATTCTTGGCACAGATGGACATCCTTTCGGCTGGCCATCTAATGGATTTCCCGGACCACA 53 7 4 7 8 CTTGCTGGGCATTAATGGGCACCCGTATGGCTGGCCAGAAAATGGTTTCCC AGGGCCAC A 53 7 4 7 8 TTTGCTGGGAACGGACGGTCATCCCTTTGGATGGCCTTCCAATTGCTTTCCTGGACCACA 53 7 0 GGACCGCA 8 3 5 4 TCTTCTGGGAACAGATGGACATCCCTTTGGATGGCCTTCCAATGGGTTTCCTGGACCACA 413 4 7 8 CCTTCTGGGAACAGATGGTCATCCGTTTGGCTGGCCTTCCAATTGCTTCCCTGGACCCCA 53 7 2 5 CATCCTGGGAACGGACGGTCACCCGTTCGGCTGGCCTTCCAACGGCTTTCCCGGTCCTCA 8 4 4 7 2 TCTCATGGGAACAGACGGCCACCCGTTTGGTTGGCCTTTCAATGGCTTCCCTGGACCCCA 5 31 4 7 8 TCTCATGGGCACAGATGGACACCCCTTTGGTTGGCCTTCCAATGGCTTCCCTGGGCCCCA 53 7 0 4 63 TCTCATGGGAACAGACGGCCACCCTTTCGGCTGGCCTTCTAATGGCTTCCCTGGACCCCA 52 2 4 7 6 TCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCA 53 5 414 TCTCTTGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCGATGGCTTCCCTGGGCCCCA 4 7 3 4 57 CCTCATGGGGACAGATGGGCACCCCTTTGGTTGGCCTTCCAACGGCTTCCCAGGGCCCCA 516 4 7 8 TCTTCTCGGCACAGATGGTCATCCTTTCGGTTGGCCCTCCAATGGCTTCCCTGGACCTCA 53 7 Appendix 1. continued 55 GLFTDFISH 538 XENOPUS 538 DOGFISH 538 CATSHARK 9 HORNSHARK 414 CHICKEN 538 CATFISH 85 MOUSE 532 PIG 538 COW 0 RAT 523 LT-HAMSTER 536 C_HAMSTER 474 HUMAN 517 ZEBRAFISH 538 MUDSUCKER GLFTDFISH 598 XENOPUS 598 DOGFISH 598 CATSHARK 69 HORNSHARK 474 CHICKEN 598 CATFISH 145 MOUSE 592 PIG 598 COW 0 RAT 583 LT-HAMSTER 596 C_HAMSTER 534 HUMAN 577 ZEBRAFISH 598 MUDSUCKER AGGTCCATATTACTGTGGTGTGGGAGCAGACAAGGCCTACGGCAGAGACATAGTGGAGGC 59 7 AGGTCCCTATTACTGCGGCGTTGGAGCGGACAAGGTGTATGGCCGGGATGTGGTAGAGTC 597 AGGGCCCTATTACTGTGGAGTTGGTGCAGACAAAGCTTACGGC AGAGATATTGTCGAGGC 5 97 GGGACCCTATTACTGTGGCGTTGGTGCAGATAAAGCTTATGGTCGGGATATTGTGGAGGC 6 8 AGGGCCCTATTACTGTGGAGTTGGTGCAGACAAAGCTTACGGTAGAGATATTGTGGAAGC 4 7 3 AGGTCCGTACTACTGCGGTGTAGGAGCTGACAAAGCCTATGGCAGAGACATTGTGGAGGC 597 GGGGCCTTACTACTGTGGAGTCGGAGCGGACAAGGCGTACGGCAGGGATATTGTGGAAGC 144 AGGCCCATATTACTGCGGTGTGGGAGCAGACAAAGCCTATGGCAGGGACATCGTGGAGGC 5 91 AGGTCCGTACTATTGTGGTGTTGGAGCAGACAAAGCCTATGGCAGGGACATTGTGGAGGC 59 7 AGGACCCTATTACTGCGGTGTGGGAGCTGACAAGGCTTATGGCCGAGATATCGTGGAGGC 582 AGGTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGGAGGC 5 9 5 AGGTCTGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATCGCAGGGATATCATGGAGGC 5 3 3 GGGTCCATATTACTGTGGTGTGGGAGCAGACAGAGCCTATGGCAGGGACATCGTGGAGGC 57 6 AGGTCCATATTACTGTGGTGTTGGAGCTGATAANGCCTATGGACGAGATGTTGTAGAAGC 5 9 7 CCATTACAGAGCCTGTCTCTATGCTGGAGTCCAGATTTGTGGCACAAATGCAGAAGTAAT 657 GCATTATAAGGCCTGTCTGTACGCTGGCATTAAAATCTGTGGCACCAACGCAGAAGTCAT 65 7 TCACTACCGGGCGTGTCTGTATGCTGGAATTGAACTCAGTGGAACCAATGCTGAAGTTAT 657 TCACTACCGAGCATGTCTATATGCTGGGATTCACTTGTCTGGTACCAATGCTGAAGTGAT 12 8 TCACTACCGGGCTTGTCTGTATGCTGGAATCCATCTCTCTGGC ACCAATGCTGAAGTGAT 53 3 CCACTACCGAGCGTGCCTGTATGCTGGTGTGAAAATTGGAGGAACCAACGCAGAAGTGAT 657 CC ACTACAGAGCGTGTCTGTACGCCGGCGTGAATATCTGCGGCACGAACGCTGAGGTC AT 204 TCACTACCGGGCCTGCTTGTATGCCGGAGTCAAGATCACGGGGACAAATGCGGAGGTTAT 6 51 TCACTACCGGGCCTGCTTGTATGCCGGCATCAAGATTGGGGGCACCAATGCCGAGGTCAT 65 7 TCACTACCGGGCCTGCTTGTATGCTGGAATCAAGATCACAGGGACAAATGCCGAGGTTAT 64 2 TCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCAT 6 55 TCACTACCGTGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACATATGCTGAGGTCAA 5 9 3 CCATTACCGGGCCTGCTTGTATGCTGGAGTCAAGATTGCGGGGACTAATGCCGAGGTCAT 6 3 6 ACATTATAGAGCCTGTCTGTATGCTGGGGTAAAATCTGTGGCACCAATGCTGAGTCATGC 6 5 7 GLFTDFISH XENOPUS DOGFISH CATSHARK HORNSHARK CHICKEN CATFISH MOUSE PIG COW RAT LT-HAMSTER C_HAMSTER HUMAN ZEBRAFISH MUDSUCKER 6 5 8 GCCTGCACAGTGGGAGTTTCAGGTAGGACCTTGTGAGGGTATCAACATGGGCGATCATTT 717 6 5 8 GCCCTCGCAGTGGGAGTTCCAAGTGGGTCCGTGCGAAGGTATCGACATGGGGGACCACCT 717 6 5 8 GGCTGCTCAGTGGGAATACCAAGTTGGACCTTGTGAAGGTATCCAGATGGGTGACCACTT 717 12 9 GGCTTCTCAGTGGGAGTACCAGGTTGGACCTTGCGAGGGCATCCATATGGGTGACCACTT 188 5 3 4 GGCTTCTCAGTGGGAGTACCAAGTTGGACCTTGTGAAGGTATCAAGGTGGGTGACCACTT 5 9 3 6 5 8 GCCAGCCCAGTGGGAGTTCCAGGTGGGACCGTGCGAAGGGATTGAGATGGGGGATCACCT 717 2 0 5 GCCAGCTCAGTGGGAGTTCCAGGTGGGGCCGTGCGAGGGTATCGAGATGGGAGATCACCT 2 64 6 52 GCCTGCCCAGTGGGAATTCCAGATAGGACCCTGTGAGGGGATCCAGATGGGAGATCATCT 711 65 8 GCCCGCCCAGTGGGAATTCCAGATCGGACCCTGTGAAGGAATCGACATGGGAGATCACCT 717 0 6 4 3 GCCTGCCCAGTGGGAATTCCAGATAGGACCCTGCGAAGGGATCCGCATGGGAGATCATCT 7 02 65 6 GCCTGCCCAGTGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCT 715 5 94 GCATGCCCAGTGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCT 653 6 3 7 GCCTGCCCAGTGGGAATTTCAGATTGGACCTTGTGAAGGAATCAGCATGGGAGATCATCT 6 9 6 65 8 CTGCACAGTGG 6 68 Appendix 1. continued 56 GLFTDFISH 718 XENOPUS 718 DOGFISH 718 CATSHARK 189 HORNSHARK 594 CHICKEN 718 CATFISH 265 MOUSE 712 PIG 718 COW 0 RAT 703 LT-HAMSTER 716 C_HAMSTER 654 HUMAN 697 ZEBRAFISH MUDSUCKER GLFTDFISH 778 XENOPUS 778 DOGFISH 778 CATSHARK 249 HORNSHARK 654 CHICKEN 778 CATFISH 325 MOUSE 772 PIG 778 COW 0 RAT 763 LT-HAMSTER 776 C_HAMSTER 714 HUMAN 757 ZEBRAFISH MUDSUCKER GLFTDFISH 838 XENOPUS 838 DOGFISH 838 CATSHARK 309 HORNSHARK 714 CHICKEN 838 CATFISH 385 MOUSE 832 PIG 838 COW 0 RAT 823 LT-HAMSTER 836 C_HAMSTER 774 HUMAN 817 ZEBRAFISH MUDSUCKER CTGGGCGGCACGTTTCATCCTGCACCGTGTCTGTGAGGATTTGGGCGTGGTCGCTTCATT 7 7 7 GTGGATGGCCAGGTTCATCCTTCATCGGGTCTGTGAAGACTTTGGGGTGGTGGCGACTCT 7 7 7 GTGGATTTCCAGGTTTATTCTGCACAGGGTGTGCGAGGACTTCGGTATCATTGCTAGCTT 7 7 7 ATGGATGTCGAGGTTTATTCTGCACCGCGTGTGTGAGGACTTTGGGATCATCGCTAGCTT 2 4 8 GTGGATTTCAAGGTTTATTCTGCACAGGGTGTGCGAGGACTTTGGTATCATTGCTAGCTT 6 53 CTGGATAGCACGTTTCATCCTCCACCGGGTGTGCGAAGACTTTGGTGTCATTGTGTCCTT 7 7 7 GTGGGTGGCTCGTTTCATCCTGCACAGGGTGTGTGAAGACTTCGGCATCGTCGCCTCGTT 32 4 TTGGATAGCCTGTTTTATCTTGCATCGGGTATGCGAAGACTTTGGGGTGATAGCAACCTT 7 71 CTGGGTGGCCCGATTCATCTTGCATCGTGTGTGCGAAGACTTCGGAGTGATCGCCACCTT 77 7 CTGGGTAGCCCGTTTTATCTTGCATCGGGTATGCGAAGACTTTGGGGTGATAGCAACCTT 7 62 CTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTT 7 7 5 CTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTAAAGACTTTGGAGTAATAGCAACCTT 713 CTGGGTGGCCCGTTTCATCTTGCATCGTGTGTGTGAAGACTTTGGAGTGATAGCAACCTT 7 56 TGACCCTAAGCCCATCCCCGGAAACTGGAACGGTGCTGGCTGCCATACAAACTTCAGCAC 837 GGACCCCAAACCCATGACCGGAAACTGGAACGGAGCCGGGTGCCACACCAACTACAGCAC 83 7 TGACCCTAAGCCCATTCCTGGCAACTGGAATGGTGCTGGGTGCCACACTAACTTTAGCAC 83 7 TGACCCGAAGCCTATTCCTGGGAACTGGAACGGTGCTGGATGTCATACCAACTTTAGCAC 3 08 TGACCCGAAGCCCATTCCTGGCAACTGGAATGGGGCAGGGTGCCACACCAACTTTAGCAC 713 CGATCCCAAACCCATCCCTGGGAACTGGAACGGTGCTGGCTGTCACACCAACTTCAGCAC 83 7 CGACCCCAAACCCATCCCTGGGAACTGGAACGGCGCGGGATGTCACACCAACTTCAGCAC 3 84 TGACCCCAAGCCCATTCCAGGGAACTGGGATGGTGCAGGCTGCCATACCAACTTCAGCAC 8 31 TGATCCTAAGCCCATTCCTGGGAACTGGAATGGTGCCGGCTGCCACACCAACTTTAGCAC 8 3 7 TGACCCCAAGCCCATTCCAGGGAACTGGAATGGGGCAGGCTGCCACACCAACTTTAGCAC 8 2 2 TGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCAC 83 5 TGACTCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGTAC 7 7 3 TGATCCTAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTCAGCAC 816 GAAAGAGATGAGGGAAGACGGCGGATTAAAAGCCATTGAAGATGCGATTGAGAAGCTCGG 8 97 GGAGAGCATGAGGGTGGAAGGAGGACTCAAACACATTGAAGATGCCATAGAGAAGCTGGG 8 9 7 CAAAGCC ATGCGGGATGATGGAGGGTTGAAGTACATTGAAGACTC AATTGAAAAACTGGG 89 7 AAAATCTATGCGGGATGAGGGCGGTTTGAAATTCATTGAAGAGTGTATTGAAAAACTGGG 3 68 C'AAATCCATGCGGGAAGAGGGAGGGCTGAAGTACATTGAAGACTCCATTGAAAAACTGGG 7 7 3 CAAGAACATGAGGGAAGATGGAGGTCTCAAGCACATCGAGGAGGCCATCGAGAAGCTGAG 897 TAAAGAGACGCGGGAAGAAGGCGGGCTCAAATGCATTGAGGAATGTATCGAGAAACTGGC 44 4 CAAGGCCATGCGGGAGGAGAATGGTCTGAAGTGCATTGAGGAGGCCATTGACAAACTGAG 891 CAAGGCCATGCGAGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATCGAGAAGCTAAG 8 97 GTCTGAAGTACATTGAGGAGGCCATTGAGAAGCTAAG 3 7 CAAGGCCATGCGGGAGGAGAATGGTCTGAGGTGCATTGAGGAGGCCATTGATAAACTGAG 882 CAAGGCCATGCGGGAGGAGAATGGTCTGAAGCACATCGAGGAGGCCATCGAGAAACTAAG 89 5 CAAGACCATGCGGGAGGAGAATGGTCTGAAGCACATCAAGGAGGCCATTGAGAAACTAAG 83 3 CAAGGCCATGCGGGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATTGAGAAACTAAG 87 6 Appendix 1. continued 57 GLFTDFISH 898 GAAGAGGCACCACTACCACATTCGTGCCTATGACCCCAAAGGGGGGCTGGACAACGCCCG 957 XENOPUS 898 GAAGAGACACGACTACCACATCTGCGTCTACGACCCGCGGGGAGGGAAAGACAACTCCCG 957 DOGFISH 898 CAAGAGGCATCAGTACCACATTCGTGCCTATGATCCTAAAGGAGGGTTGGACAATGCTAG 957 CATSHARK 3 6 9 CAAGAGGCACCAATACCACATTCGTGCCTATGATCCTAAA 408 HORNSHARK 774 CAAGAGGCATCAGTACCACATTCGTGCCTATGACCCTAAAGGAGGGTTGGACAATGCTAG 833 CHICKEN 898 CAAGCGCCACCAGTACCACATCCGTGCCTACGACCCCAAAGGAGGGCTGGACAACGCCCG 957 CATFISH 445 GAAGAGACACAACTACCACATCCGTGCCTACGATCCTAAAGGAGGCCTGGACAACGCTCG 504 MOUSE 892 CAAGAGGCACCAGTACCACATCCACACCTACGATCCCAAGGGGGGCCTGGACAACTCCCG 951 PIG 898 CAAGCGGCACCAGTACCACATCCGAGCCTACGATCCCAAGGGGGGCCTGGACAACACACG 957 COW 38 CAAGCGCCACCAGTACCACATCCGAGCCTACGATCCCAAGGGGGGCCTGGACAACGCCCG 97 RAT 883 CAAGAGGCACCAGTACCACATCCGTGCCTACGACCCCAAGGGGGGCCTGGACAACGCCCG 942 LT-HAMSTER 896 CAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAAGGGGGGCCTGGACAATGCCCG 955 C_HAMSTER 834 CAAGCGGCACCGGTACCATATTCGAGCCTACGATCCCAAGGGGGGGCTGGACAATGCCCG 893 HUMAN 877 CAAGCGGCACCAGTACCACATCCGTGCCTATGATCCCAAGGGAGGCCTGGACAATGCCCG 936 ZEBRAFISH MUDSUCKER GLFTDFISH 958 CCGTCTCACCGGCCACCACGAAACCTCAAACATCCACGAGTTCTCTGCAGGTGTGGCCAA 1017 XENOPUS 958 GAGACTCACCGGCCAACACGAGACGTCGAGTATTCACGAGTTCTCGGCCGGCGTGGCCAA 1017 DOGFISH 958 AGCTTTGACAGGCCACCATGAAACCTCAAATATCAATGAGTTCTCAGCTGGTGTTGCCAA 1017 CATSHARK HORNSHARK 834 GCGTTTGACAGGCCACCATGAAACCTCAAATATCAATGAGTTCTCAGCTGGCGTTGCCAA 893 CHICKEN 958 GCGCCTGACGGGCTTCCACGAGACGTCCAGCATCCACGAGTTCTCCGCCGGCGTGGCCAA 1017 CATFISH 505 CCGCCTGACTGGCCACCACGAGACCTCCAACATCCACGAGTTCTCTGCCGGCGTCC 560 MOUSE 952 GCGTCTGACTGGATTCCACGAAACCTCCAACATCAACGACTTTTCTGCCAGTGTTGCCAA 1011 PIG 958 GCGCCTAACTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGCCGGCGTGGCCAA 1017 COW 98 GCGCCTAACTGGGTTCCACGAAACCTCCAACATCAACGACTTCTCTGCCGGCGTGGCCAA 157 RAT 943 CCGTCTGACTGGATTCCACGAAACCTCCAACATCAACGACTTTTCCGCTGGCGTTGCCAA 1002 LT-HAMSTER 956 TGGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTCTGCTGGTGTCGCCAA 1015 C_HAMSTER 894 TCGTCTGACTGGGTTCCACAAAACGTCCAACATCAACGACTTTTCAGCTGGCGTCGCCGA 953 HUMAN 937 ACGTCTAACTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGCTGGTGTAGCCAA 996 ZEBRAFISH MUDSUCKER GLFTDFISH 1018 CCGCGGCGCCAGCATTCGCATTCCCCGTAGTGTCGGCCAGGAGAAGAAGGGCTACTTTGA 1077 XENOPUS 1018 CCGGGGCGCCAGTATCCGCATCCCGCGTCAGGTGGGCCAGGAAGGCTACGGCTACTTTGA 1077 DOGFISH 1018 TAGAGGAGCCAGCATCCGAATCCCTCGATCCGTTGGCCAGGACAAGAAAGGCTACTTTGA 1077 CATSHARK HORNSHARK 894 TAGAGGAGCTAGCATCCGAATCCCTCGATCTGTTGGCCAGGACAAGAAAGGCTACTTTGA 953 CHICKEN 1018 CCGCGGCGCCAGCATCCGCATCCCACGCAACGTGGGCCATGAGAAGAAAGGCTACTTCGA 1077 CATFISH MOUSE 1012 CCGCAGTGCCAGTATCCGCATTCCCTGGACTGTCGGCCAGGAGAAGAAGGGCTACTTTGA 1071 PIG 1018 CCGTGGCGCTAGCATCCGCATTCCCCGGACTGGGGGCCAGGAGAAGAAGGGTTACTTCGA 1077 COW 158 CCGTGGTGCTAGCATCCGCATCCCCCGGACTGTTGGCCAGGAGAAGAAGGGCTACTTCGA 217 RAT 1003 CCGCAGCGCCAGTATCCGCATTCCCCGGATTGTCGGCCAGGAGAAGAAGGGTTACTTTGA 1062 LT-HAMSTER 1016 TCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGA 1075 C_HAMSTER 954 TCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGA 1013 HUMAN 997 TCGTAGCGCCAGACTACGCATTCCCCGGACTGTTGGCCAGGAGAAGAAGGGTTACTTTGA 1056 ZEBRAFISH MUDSUCKER Appendix 1. continued 58 GLFTDFISH 1078 GGACCGCCGACCGTCTGCCAACTGTGACCCGTACGGCGTAACGGAGGCCCTGATCCGCAC 1137 XENOPUS 1078 AGACCGACGGCCGGCAGCCAACTGCGACCCCTACGCAGTAACCGAGGCGCTGGTCAGGAC 1137 DOGFISH 1078 AGACCGCCGTCCATCTGCTAATTGTGACCCTTATGCAGTCACAGAAGCATTGGTCCGCAC 1137 CATSHARK HORNSHARK 954 AGACCGCCGTCCCTCTGCTAATTGTGACCCTTATGCAGTCACAGAAGCATTGGTCCGCAC 1013 CHICKEN 1078 GGACCGCGGGCCTTCAGCCAACTGCGATCCCTACGCCGTGACGGAGGCCCTGGTCCGTAC 1137 CATFISH MOUSE 1072 AGACCGTCGGCCTTCTGCCAATTGTGACCCCTATGCGGTGACAGAAGCCATCGTCCGCAC 1131 PIG 1078 AGACCGTCGCCCTTCTGCCAACTGTGACCCCTTTGCGGTGACAGAAGCTCTCATCCGCAC 1137 COW 218 AGACCGTCGCCCATCTGCCAACTGTGACCCCTTCGCCGTGACCGAAGCCCTCATCCGCAC 277 RAT 1063 AGACCGTCGGCCTTCTGCCAATTGCGACCCCTATGCGGTGACGGAAGCCATCGTCCGCAC 1122 LT-HAMSTER 1076 AGACCGCCGCCCCTCTGCCAATTGTGACCCCTTTGCAGTGACAGAAGCCATCGTCCGCAC 1135 C_HAMSTER 1014 AGCCCGCTGCCCCTCTGCCAATTGTGACCCCTTTGCAGTGACAGAAGCCATCGTCCGCAC 1073 HUMAN 1057 AGATCGTCGCCCCTCTGCCAACTGCGAGCCCTTTTCGGTGACAGAAGCCCTCATCCGCAC 1116 ZEBRAFISH MUDSUCKER GLFTDFISH 1138 GTGTTTGCTGAGCGAGGAAGGAGATGAACCTTTAGCTTACTGAATCCCACTCCCCTCCTG 1197 XENOPUS 1138 CACCATCCTGAACGAAACCGGCAGCGAGACCAAAGACTATAAGAACGGAGCTGGATTCTC 1197 DOGFISH 1138 ATGCCTATTGGATGAGTCTGGGGACAAGCCTATTGAGTACAACAAAAATTAAGCAAAATA 1197 CATSHARK HORNSHARK 1014 ATGCCTATTGGATGAGTCTGGGGACAAGCCT 1044 CHICKEN 1138 GTGTCTCCTCAACGAAACCGGGGACGAGCCTTTTGAGTACAAGAACTAAGTGGACTCGTG 1197 CATFISH MOUSE 1132 GTGTCTCCTCAACGAAACAGGCGACGAACCCTTCCAATACAAGAACTAAGCAGACTAGAC 1191 PIG 1138 GTGTCTCCTCAACGAAACTGGCGACGAGCCCTTCCAGTACAAAAACTAAGTGGACTAGAC 1197 COW 278 ATGTCTTCTGAATGAAACTGGCGACGAGCCCTTCCAGTACAAGAACTAAGTGGACTAGAC 337 RAT 1123 GTGTCTCCTCAACGAAACTGGCGACGAGCCCTTCCAATACAAGAACTAAGCGGACTCGAC 1182 LT-HAMSTER 1136 ATGCCTTCTCAATGAGACTGGCGACGAGCCCTTCCAATACAAAAACTAATTAGACTTTGA 1195 C_HAMSTER 1074 ATGCCTTCTCAATGAGACTGGCGACCAGCCCTTCCAATACAAAAACTAA 1122 HUMAN 1117 GTGTCTTCTCAATGAAACCGGCGATGAGCCCTTCCAGTACAAAAATTAAGTGGACTAGAC 1176 ZEBRAFISH MUDSUCKER GLFTDFISH 1198 ACATTCTTTTCTTTAAACTAGTACATTGTTTCTGTTCTCCTACTGAGATGATTTAACCTG 1257 XENOPUS 1198 CCGGGCAATCGGTATGGCATCTCCCCGAGACGCCGCTGTGTTTTAACCCGTTAGTCTCCC 1257 DOGFISH 1198 ATGCACTAATGGACCTGGCATTTGTAGCAGTGATAGCTGTTGAAATGTGGGACCTTTGGG 1257 CATSHARK HORNSHARK CHICKEN 1198 CCCACAGACACCGCCTTCCCCCTCCCCCCACCCCCCCCGTGCTCCCCGTACCCCTAAACT 1257 CATFISH MOUSE 1192 TTCCAGTGATCCCTCTCCCAGCTCTTCCCTCTCCCAGTTGTCCCCACTGTAACTCAAAGG 1251 PIG 1198 GGGCAGCCATCAAAACCCCTCCAATTCTACACCGCCCCCCCCCCCCTCGCCCTCTCAACT 1257 COW 338 TTGCAGCCCTCGAAACCCCTCTTAATTCTACATCTTACTCCCACTCTCG 386 RAT 1183 TTCCAGTGATCTTGAGCCCTTCCTAGTTCACCCCACTCCCAACTGTTCCCTCTCCCACTG 1242 LT-HAMSTER 1196 GTGATCTTGAGCCTTTCCTAGTTCATCCCACCCCGCCCCAGCTGTCTCATTGTAACTCAA 1255 C_HAMSTER HUMAN 1177 CTCCAGCTGTTGAGCCCCTCCTAGTTCTTCATCCCTGACTCCAACTCTTCCCCCTCTCCC 1236 ZEBRAFISH MUDSUCKER Appendix 1. continued 59 GLFTDFISH 1258 XENOPUS 1258 DOGFISH 1258 CATSHARK HORNSHARK CHICKEN 1258 CATFISH MOUSE 1252 PIG 1258 COW RAT 1243 LT-HAMSTER 1256 C_HAMSTER HUMAN 1237 ZEBRAFISH MUDSUCKER GLFTDFISH 1318 XENOPUS 1318 DOGFISH 1318 CATSHARK HORNSHARK CHICKEN 1318 CATFISH MOUSE 1312 PIG COW RAT 1303 LT-HAMSTER 1316 A C_HAMSTER HUMAN 1297 ZEBRAFISH MUDSUCKER GLFTDFISH 1378 XENOPUS 1378 DOGFISH 1378 CATSHARK HORNSHARK CHICKEN 1378 CATFISH MOUSE 1372 PIG COW RAT 1363 LT-HAMSTER C_HAMSTER HUMAN 1357 ZEBRAFISH MUDSUCKER CATTTTAATGGTTTAAAAGTTGGCTGGTCAACTTAAAACAAGGCGGTCTTGTCCTTGGTA 1317 TCCCTTCTAGTTGTAATCCTGAGGGTACAAGATAACACCTTCGTGTCTCAGTAACTCTTG 1317 12 3 7 AGTTGTCCCGATTGTAACTCAAAGGGTGGAATATCAAGGTCGTTTTTTTCATTCCATGTG 12 9 6 TGTATTTCAGAACCTAATTTCTTCTGTTGTTATCTGGAAGGTGAGGAATGAGGCTTGCGA 1377 13 03 AATTTTTGCCTTTATTGGTCAGAATAGAGGGGTCAGGTTCTTAATCTCTACACACCCAAC 13 62  AGAATAGAGGAGTCAAGTTCTT 1337 12 97 CCCAGTTAATCTTGCTTTCTTTTGTTTGGCTGGGATAGAGGGGTCAAGTTATTAATTTCT 13 5 6 13 6 3 CCCTTCTTTCCTAGCTAGCTTTCCAGTGGGGAACGGGAGGGGGTGGGGAAGGGTAACCCA 1422 13 57 TCACACCTACCCTCCTTTTTTTCCCTATCACTGAAGCTTTTTAGTGCATTAGTGGGGAGG 1416 Appendix 1. continued 60 Name Length G A T Gulftoadfish 432 29 17 25 23 22 69 22 92 African clawed frog 432 32 41 24 54 19 44 23 61 Spiny dogfish 432 28 47 24 31 25 93 21 30 Cloudy catshark 369 30 35 23 04 27 37 19 24 Hornshark 432 28 70 24 54 25 23 21 53 Chicken 432 30 32 23 84 20 60 25 23 Channel catfish 432 32 87 21 76 18 98 26 39 House Mouse 432' 30 56 24 07 21 53 23 84 Pig 432 29 . 63 22 92 21 30 26 16 Norway Rat 432 30 . 09 23 61 21 53 24 77 Long-tailed hamster 432 29 63 24 31 22 45 23 61 Chinese hamster 432 28 .24 25 23 23 61 22 92 Human 432 31 . 02 22 92 22 69 23 38 Zebrafish 202 27 .36 21 89 27 36 23 38 Appendix 2. Base composition percentage statistics for vertebrate sequences available in Genbank prior to August 1999. The fragment length of 432 bp spanned positions 467 to 899 of Xenopus laevis sequence in Appendix 1. Gulftoadfish (Opsanus beta), African clawed frog (Xenopus laevis), spiny dogfish (Squalus acanthias), cloudy catshark (Scyliorhinus torazame), hornshark (Heterodontus francisci), chicken (Gallus gallus), channel catfish (Ictalums punctatus), house mouse (Mus musculus), pig (Sus scrofa), Norway rat (Rattus norvegicus), long-tailed hamster (Cricetulus longicaudatus), Chinese hamster (Cricetulus griseus), human (Homo sapiens), zebraf ish (Danio rerio). Note: the zebrafish fragment and the cloudy catshark fragment only spanned a portion of the 432bp fragment length. 61 Consensus 1 CAGGAGTACACCATCCTGGGCACAGACGGACACCCCTTTGGCTGGCCATCCAACGGCTTC 6 0 Sm A T G G T..C T..T..T... Hs A  T C. .T. .T. .T. . . Ce A  G T....A T..T..T... Auf A T G..T..T..T..Gaa A C C T..T... ApA  G T..T..Apf A T T T T..T... PI A  T  T..T..Ps A  T A . . . Ii A  A . T A... Cso A G..G..T A..C T..T..T..Cst A T T T..T..T... Ca Al  A. . . Ca A2 T A T A. . . Eb A  T G T T..T..T... El A  T . . . OmA T G T T..T..T... Hs B C G. .T T. .T C Gaa B G..T T C C Auf B T G..T T C Ce B C T T C C . . SmB C G..T T C C T ApB T G..T T C C Apf B  G..T T CPi B  T T C C G... PsC G T T C IiC  T T T C Cst C ..A TC.G..T T G..T C T Cso C A TC.G. .T T G..TOki DI A T. .G T Oki D2 ..A TT T..T A CAA Oke D A A T..T A CASI E G C A. . . As F A G C A C T Es ..A T..AC....T.. GGT ...T A..T..T..T Appendix 3. Sequence alignment for amplified glutamine synthtase product for fish used in this study. The 432 bp region of glutamine synthetase amplified corresponds to the region of 467 to 899 of the published GS sequence for Xenopus laevis (Genbank accession number D50062). A dot represents an identical nucleotide base to the base given in the concensus sequence. Numbers indicate positional information relative to position 467 (with 1 being 467 and 432 being position 899) of the GS gene for Xenopus laevis. Species are designated by initials for species name and isoform designation is indicated by A, B, C, D, E or F. Ap - high cockscomb, Apf - penpoint gunnel, As - alligator gar, Auf - tubesnout, Ca - shiner perch, Ce - mossy sculpin, Cso - Pacific sanddab, Cst - speckled sanddab, Eb -buffalo sculpin, El - striped seaperch, Es - Pacific hagfish, Gaa - three spine stickleback, Hs - white spotted greenling, Ii - buttersole, Oke - chum Salmon, Oki - coho salmon, Om - tidepool sculpin, PI - crescent gunnel, Ps - starry flounder, SI - bay pipefish, Sm -cabezon. Note: Es does not have an isoform designation. ? - indicate base at that position is unknown for that fish. 62 Consensus 61 CCTGGACCACAAGGTCCATATTACTGTGGAGTGGGAGCTGACAAGGCCTATGGCAGAGAC 120 Sm A G r r A r T Hs A G r r r A T Ce A r r r A r T Auf A C G r r G A T Gaa A O r G c r G r G Ap A G r r A r Apf A G c r A c PI A G r r A r Ps A r r r r A . T . T Ii A r T r r A . T . T Cso A T r T r A T Cst A T r T r A T Ca Al r r T A Ca A2 r r r A . T . T Eb A r r A r T El A r T A T Om A r c r A r T Hs B 0 T T T G r AC . T Gaa B T G T T G G r GC C . Auf B c G G T T A G c AC c. Ce B . . G . T G r A T G r . AC Sm B G T G T A T G r AC . T Ap B T G T r r A T G r AC c. Apf B T G T r r r T G r AC c. Pi B T G T r r r T G r AC c. Ps C A T G G T T c A A T G AC Ii C A T G G c T A A T A AC Cst C . .A. r T G r r r T T G c GC G. Cso C . . A . r T G r r r . T . T G r . GC Oki Dl T r r T A G T Oki D2 r r T r T T Oke D r r T T . T . T SI E G r T G G r r T T A A r G T As F c c G r G T T GC Es T T G T T T A G Appendix 3.continued. 63 Consensus 121 ATAGTGGAGGCCCATTACAGAGCCTGTCTGTATGCTGGAGTTGAGATCTGTGGCACAAAT Sm A . . . . T . . . . .T.... C . ....c Hs A . . . . T . . . . .T.... C. ....c Ce A . .C . . . . T . . . . .T.... . . T . . . C . Auf A . . . . T . . . . . T . . . . C G. . . . . c Gaa A . . . .T. .G. .G.... ...C.... ....c Ap A . . . . T . . . . . T . . . . .. .c.. . . . . . . c. . c Apf A . . . . T . . . . ,T.... ....c..c PI A ....T.... . T . . . . . . . . c. . c Ps A ....T.... . T . . . . c C . . T T. Ii A ....T.... . T . . . . c C. .T T. Cso A A. . . . AG . . . . . C . . . . c. . c. .... CAT C Cst A A. . . . AG . ... . c. ... c... . .... CAT . . . . C Ca Al C . T . . . . . . . . CC T . Ca A2 . . . . T . . . . . T . . . . C.C..T T. Eb A . . C . T . . . . c. ....C El A c T. . . . . . .CC Om A . .c . . . . T . . . . .T. . . . c. Hs B . .c ...c... . . G C .C. . . Gaa B . .c C . G . ....CA . . .C .G. .G C C. . . . ,C. . . Auf B . . C . . T A . . .C .G. . G C . C . . . Ce B G. A. . ...c.... . G C . . . ." C C Sm B G T.G. A. . ...c.... .G C . C. . . Ap B G G ... c... . .G C .C. . . Apf B G . ...c.. . . .G C .c.. . PI B G G. ...c.... .G C . c. . . Ps C G A..T. A. . .G...C G. . c. . . Ii C G A..T. .C....A.. . G. .GC r c Cst C . .C T. .c...c... A AT c c Cso C . . C T . .c...c... A AT r c Oki DI ..C..A..A.... .C..T.... . T . . . . A . G ... AT ... A .c. . . Oki D2 . .T A. . . . .C . . . .c. .G. .CAT A. .c.. . Oke D . . T A. . . . . C . . T . . . . .G. .CAT .c.. . SI E . .C A. . . . . T . . C . c .G...C A. As F ..T..A..A..T. c.. . ....c. ...c..c. C .c.. . ES G AT . G . CT. .G..C. CT.T. . . . T . . GA . C . . T G . Appendix 3. continued. > 64 Consensus 181 GCAGAAGTGATGCCTGCTCAGTGGGAGTTCCAGGTTGGGCCTTGTGAAGGGATCAACATG Sm A C. .A. .G.... .G.... Hs A C. .A. .G.C.. .G.... Ce A A. .G.... Auf A G . . C C. .A. . G . . . . ..G.... Gaa A G. .C G. .A. .G.... .G.... ..G.... Ap A Apf A .CA... C . . . . . . G . . . . PI A C. . . . Ps A C. . . . . C . . . . . T . Ii A C. Cso A C . . C c. . c. . C . . . . . . G. . . . Cst A C . . C c. . c. . C . . . . . . G . . . . Ca Al c. .A.... Ca A2 c. Eb A A. . G . . . . .G.... El A c. .A.... Om A A. .G.... .G.... Hs B . .T C A Gaa B . .T C A .CA... ....C. Auf B . . T C A C . . . .CA.C. .:..T. Ce B . .T T A . . . . T . Sm B . .T C A . . . .T. Ap B . . T C A . . . .C. Apf B . . T C A . . ..c. PI B . . T C A . . . . c. Ps C . .T C A .A.... . . . . T . Ii C . .T C A .A.... . . . . T . Cst C . . T . . G . . C A . : T . .G.... ....c. TG.A... Cso C ..T..G..C A T . .G.... . . . . c. TG.A... Oki Dl . . T C A .A.... . . . . T . Oki D2 . .T C c. . . . . c. . . G . . . . Oke D . . T A A T . ....c. SI E C G..C C . .G..C. ....A. .G As F . . T C A. .C . C . .CA... . . . .A. .G Es T . A . . . ' G. .A. .A.... .G..TG GG Appendix 3. continued. 65 Consensus 241 GGTGATCATCTGTGGGTGGCTCGCTTCATCCTGCACCGCGTCTGTGAGGATTTTGGCGTG Sm A r C . T . . . . . . . .C. . . . . C T Hs A r C. G. . . . T . . . . ....C..A. T Ce A C. G. . . . T. . . . .C..A..T Auf A r . . . . c. . c. A. . . . T . . . . . . . . C . . . . .G C Gaa A G . ... c.. c. A. . . . T . . T . A. c Ap A c. G . . . . . . . .C. .A. c Apf A ....c..c. G . . . . T . A. c PI' A ....c..c. G. . . . T. A. c Ps A . . . . c. . c. A. .T. T. A. . . . . T . . T Ii A c. ..C...A..T. G. A. ....T..T Cso A r ....c.. . . C . . T . .G A. c Cst A G . . . . c. . . . C . . T . .G A. c Ca Al . . CA T T . A . T Ca A2 . . C T . T . . . G A. . . . . T . . T Eb A r c. G.... A . . T . . . . . . . .C. . . . .C .T. .T El A . . CA T . A. T Om A r c. G. . . . T . . . . ....C.... . C T Hs B G . c T...A.A. .G A. . C Gaa B G . . . T T...A.A. . G Auf B G . . . T T...A.A. .G Ce B G .c . . .T T...A.A. . G Sm B G . c . . .T T...A.A. . G . c Ap B G T. . .A. A. .G . c Apf B G . . . T C...A.A. .G .c PI B . . G . . . .T C...A.A. .G . c Ps C G . c. . c. . . . . A . T T. T...A.G. . . . .C. . . . Ii C G .c..c.... . A . T T. T...A.G. ....C.... Cst C r .c..c..c. . A. C A.G. .G..C.... . c Cso C r . c. . c. . c. .A.C A.G. .G..C.... . c Oki DI .c..c..c. . . CT G . C G. ,G A. r . . . . T . . . Oki D2 . . . .C .T. ..CA...A.G. ....T..C G. .G r . . . . T . . . Oke D . ... c.. c. C G. .G r SI E r .C..C..T. A. . . . .T..C..A. r . . . . G. . . As F A r CT... .A.T..CA.G . . . . T A.G. r .C .A. .C Es .C. .C. . . . .CT G. .TC.T..A..T..T. .G A. r A. A Appendix 3. continued. 66 Consensus 3 01 GTGGCCTCATTTGACCCCAAGCCGATCACTGGGAACTGGAACGGTGCTGGCTGCCACACC Sm A . . T . . . . .... A. .c. . . .C G. r T. .A Hs 'A . .T. . . . .... A. .T. . . .C T. .T Ce A ..A.... .T. . . .C. .T G Auf A . . T . . T . ... C ... . . C T . . A Gaa A . . T . . T . . . . . C . . C r T. .A Ap A . . T . . . . .C ....T..T..A Apf A A.T. . . . ,C .T..T..T..A PI A A.T.... ... C ... . .c .T..T..T..A Ps A . . T . . . . .... A. . . . C . . T T . . . Ii A . .T . . . . .A. .A. . T T. . . Cso A A.T. . . . ...c.... .A..A. .C.C. .A T..C. A Cst A A.T.... ...c.... .A..A. .C.C. .A C. A Ca Al . . T . . . . T . .C.C. . T  . . . . r T . . A Ca A2 . . T . . . . .A..A. . . .C. . T r T . . . Eb A ..A.... ....A. . T . . . . C . . T A El A . . T . . . . T. .C.C. . T . . . . r T. .A Om A ..A.... . T . . . .C. .T G Hs B ....T.. c .A..A. .C C. r Gaa B . . . . T . . c .A. .A. . . . G . . C  . A Auf B ....T.. c .A. .A. . . .G. C. r A Ce B . . . . T . . c .A..A. . . . G . c. r Sm B . . . . T . . c .A..A. . . .G. c. r Ap B . . . . T . . c .A.... . . .G. c. r Apf B . . . . T . . c .A.... . T . G . c. r PI B . . . . T . . c .A.... . T . G . c. r Ps C G. c T . . . . C . . G . A . . . . . T T. . . . r Ii C GA c T . . . .T. . G . A . . . . . T T. . . . r Cst C A. c .A.... . . .A. T. .C. Cso C A. C T. .A.... . . .A. T. . C . Oki Dl ...C.... ....C. .C. . . ....T T..C. T . . . Oki D2 .. . . c. .C. . . .A C .A Oke D . . . . T . . C . . . .A .A SI E A.C.... T .C.C. .c c. c A As F .A. .C .C. A. Es AGC T . . . . . c. . C . . . . . . . T T. .A. . T . . T Appendix 3. continued. 67 Consensus 3 61 AACTTCAGCACAAAGGAGATGAGGGAAGACGGTGGATTGAAAGCCATTGAAGAGTCCATT 420 Sm A A A . T . . . . A . . . c . Hs A r G . . T . Ce A T A T A . . . C . Auf A T . . . . T . . A .T..C. A . . . T . Gaa A . . . . T . . A G ....C. G A . . .T. Ap A ....G..A Apf A . . .G ....G..A PI A . . .G . . . . G . . A Ps A . . . . T . . . ....C. A C li A . . . . T . . . .A..C. A C Cso A . . . . T . . . .T..A. r . . .C. C Cst A . . . . T . . . .T..A. r . . . c. C Ca Al A .T.... G. . . . . . . c. Ca A2 . . . . T . . . .A..C. G. . . . C Eb A A A . . .c. El A A . T . . . . G. . . . . . . c. Om A .T A T A . . .c. Hs B . . . . C . . . r A G .A..C. r .AT. . r G . . T . Gaa B . . . .C. . . C A G .A..C. r .AT. . r G. . . . C Auf B . . . . C . . . r A G .A..C. r .AT. . r G. . . . T C Ce B . . . . C . . . r A G ....C. r Sm B . . . . C . . . . CCA. G ....c. r .AT. . A Ap B ....C... r A G . . . . c. r .AT. . G . . . . G C Apf B . . . . C . . . r A G . . . . c. r .AT. . G . . . . G C PI B C...C... r A G . . . . c. c .AT. . r G. . . . C Ps C . . T . . . . . G . . . .A. r A G r G.T. . A. . . Ii C . .T. . . . . G . . . .A. r A G r G AT GA 7777777777 Cst C T.T .A. A G r . . T . . . . .A. A C Cso C T.T .A. A G r G . T . . . . .A. A C Oki DI . . . . C . . . A . T . . C . G.G. . . . . T . Oki D2 . . T . A .A.... G G. . . . G. . . . Oke D . . T . . . . . C . . A r .A.,C. G A G. . . . G . . . . SI E A G r r G. .C. C As F . . . . C . . . AAAC . .c. GTA r G. . . . G, ,C Es T.TCTT CA. .C AC G CT.... .c. AC GCAT GT . TG A. .C Appendix 3. continued. 68 Consensus 421 GAGAAGCTGGGG Sm A C. . . Hs A T. . . Ce A C. . . Auf A T. . . Gaa A T .. . Ap A C. . . Apf A C. . . PI A C. . . Ps A T. .C Ii A T. .C Cso A c. .c Cst A c. .c Ca Al T. .A Ca A2 ....T...T..C Eb A T . . . El A T . . A Om A T . . . Hs B AA Gaa B ....G....CCC Auf B ....GA....C. Ce B Sm B Ap B . . . . G C. Apf B . . . ,G C. PI B . . . . G C. Ps C . .A.G CA Ii C Cst C ..A.G CA Cso C . .A.G CA Oki DI T .... A Oki D2 ..A.G Oke D . . . . G SI E C As F ....G....A.C Es CA 432 Appendix 3. continued. 69 100 77 51 100 51 76 91 95 95 91 92 94 85 61 100 55 55 92 64 86 51 64 94 65 100 99 100 Pacific hagfish Pacific sanddab A Speckled sanddab A Striped seaperch A Shiner perch A1 Cabezon A Buffalo sculpin A Tidepool sculpin A Mossy sculpin A White spotted greenling A Tubesnout A Three spine stickleback A High cockscomb A Penpoint gunnel A Crescent gunnel A Starry flounder A Buttersole A Shiner perch A2 Bay pipefish E Coho salmon D2 Chum salmon D Coho salmon D1 White spotted greenling B Penpoint gunnel B Crescent gunnel B High cockscomb B Three spine stickleback B Tubesnout B Cabezon B Mossy sculpin B Starry flounder C Buttersole C Pacific sanddab C Speckled sanddab C Alligator gar F Appendix 4. Maximum parsimony tree constructed from 432 bp fragments of all isoforms of glutamine synthetase for all fish used in this study. Parsimony criterion was set to random addition, 50 replicates, TBR branch swapping algorithm. Sequence data was weighted 2:4:1 by codon position and tree was bootstrapped 100 times. 

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