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Interrelationships of the subgenera of Coryphaenoides (Teleostei: Gadiformes: Macrouridae): synthesis of allozyme, peptide mapping, and DNA sequence data
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 27 (2003) 343–347 www.elsevier.com/locate/ympev
Interrelationships of the subgenera of Coryphaenoides (Teleostei: Gadiformes: Macrouridae): synthesis of allozyme, peptide mapping, and DNA sequence data Raymond R. Wilson Jr.* and Phoebe Attia Department of Biological Sciences, California State University Long Beach, Long Beach, CA 90840, USA Received 12 June 2002; revised 15 October 2002
Abstract DNA sequences of the 12s rRNA mitochondrial gene from 12 species key to the question of the monophyly of the deep-sea fish genus Coryphaenoides (Macrouridae) were analyzed phylogenetically using maximum parsimony and maximum likelihood. The results were compared with those of three previous studies in which allozyme, peptide mapping, and DNA sequence data were similarly analyzed. The allozyme and DNA sequence data suggested that the largest subgenus (Coryphaenoides), which contained most of the species inhabiting continental slopes between approximately 600 and 2000 m depth, is monophyletic. Two of the three subgenera containing the species inhabiting abyssal ocean basins below approximately 2000 m together formed a sister group to subgenus Coryphaenoides. The macrourids of the abyssal basins and those of the continental slopes thus appear to have experienced separate radiations from a common ancestor. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction The macrourid genus Coryphaenoides contains 61 recognized species that are presently organized into five subgenera (Cohen et al., 1990; Iwamoto and Sazonov, 1988). Subgenus Coryphaenoides has 43 species, Chalinura 10, Nematonurus five, Lionurus two, and Bogoslovius one (Cohen et al., 1990). These Coryphaenoides (sensu lato) species inhabit the worldÕs continental slopes and deepest ocean basins but their greatest diversity is on continental slopes approximately between 600 and 2000 m depth where species of subgenus Coryphaenoides typically reside. The few truly abyssal species are among the subgenera Chalinura, Nematonurus, and Lionurus. Coryphaenoides (Chalinura) leptolepis ranges between ca. 2000 and 4000 m in the North Pacific Ocean (Stein and Pearcy, 1982) and between ca. 600 and 4700 m in the North Atlantic Ocean (Merrett et al., 1991). C. (Ch.) profundicolis, the deepest reported species of Chalinura, ranges in the eastern North Atlantic Ocean between ca. 3900 and 4800 m (Merrett et al., 1991). Coryphaenoides * Corresponding author. Fax: 1-562-985-8878. E-mail address: [email protected] (R.R. Wilson Jr.).
(Nematonurus) armatus ranges chiefly between ca. 2000 and 4300 m in the North Pacific Ocean (Endo and Okamura, 1992; Stein and Pearcy, 1982; Wilson and Waples, 1983), and between ca. 2100 and 4800 m in the North Atlantic Ocean (Merrett, 1992; Middleton and Musick, 1986). Its close congener C. (N.) yaquinae ranges in the Pacific Ocean basin from ca. 3400 to at least 6450 m (Endo and Okamura, 1992; Wilson and Waples, 1983) and is evidently the deepest living macrourid in the worldÕs oceans. The deepest reported species of Lionurus, C. (Lionurus) carapinus, ranges below 4200 m to as deep as 5600 m in the North Atlantic Ocean basin (Marshall, 1973). Iwamoto and Stein (1974) established the present organization of genus Coryphaenoides. At that time they placed Coryphaenoides pectoralis in the subgenus Nematonurus. Since then, the species has been reassigned to its own monotypic genus Albatrossia (Iwamoto and Sazonov, 1988). Wilson et al. (1991) and Wilson (1994) questioned whether the subgenera erected by Iwamoto and Stein (1974) each represented a natural (monophyletic) group. Peptide mapping of trypsin digests of LDH-A4 homologs from a collection of Atlantic and Pacific ocean species representing three of the four
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subgenera indicated a Nematonurus + Chalinura clade nested in subgenus Coryphaenoides, making Coryphaenoides (sensu stricto) appear paraphyletic (Wilson et al., 1991). Starch-gel electrophoresis of gene products from 24 presumptive allozyme loci (Wilson, 1994) also suggested a Nematonurus + Chalinura clade but the lack of tree rooting in that study precluded discussion of the issues pertaining to monophyly of the subgenera. It appeared, nevertheless, that C. (N.) pectoralis was in fact referable to subgenus Coryphaenoides rather than to Nematonurus or to Chalinura. Morita (1999) analyzed DNA sequences from portions of the 12s rRNA and COI mitochondrial genes for seven Coryphaenoides species representing the subgenera Bogoslovius, Coryphaenoides, and Nematonurus, plus two outgroup species that are now referred to the macrourid genus Caelorinchus, an upper slope group. MoritaÕs (1999) study included four Coryphaenoides species in common with Wilson et al. (1991) and three in common with Wilson (1994). MoritaÕs two abyssal species were C. (N.) armatus and C. (N.) yaquinae. Morita (1999) found that the two Nematonurus species formed a sister group to the other Coryphaenoides species, which appeared to represent a natural (monophyletic) group for subgenus Coryphaenoides upon a reassignment of C. longifilis and Albatrossia pectoralis to that subgenus. Morita (1999) suggested that the abyssal species had experienced a radiation separate from that of the slopedwelling species of Coryphaenoides, whereas Wilson (1994) suggested the abyssal species might have arisen from among the shallower slope-dwelling species of subgenus Coryphaenoides. A posteriori rooting of WilsonÕs (1994) trees in accordance with MoritaÕs (1999) phylogenetic hypothesis converged to essentially MoritaÕs result, thus supporting a similar interpretation of a possibly monophyletic subgenus Coryphaenoides and separate radiations for Coryphaenoides (s.s.) and the abyssal group of species. As mentioned above, however, MoritaÕs (1999) abyssal group was restricted to the North Pacific C. (N.) yaquinae and C. (N.) armatus. C. (N.) yaquinae is the evident ecological depth replacement of C. (N.) armatus in the North Pacific Ocean (Endo and Okamura, 1992; Wilson and Waples, 1983). The two species are long believed closely related (e.g., Iwamoto and Stein, 1974; Wilson and Waples, 1983) so a node uniting them would be expected. However, with the absence of C. (Ch.) leptolepis or, for example, C. (Ch.) profundicolis from MoritaÕs analysis, the two Nematonurus species alone do not represent the same abyssal group that Wilson (1994) and Wilson et al. (1991) studied. In addition, all of MoritaÕs (1999) species of subgenus Coryphaenoides inhabit proximal or coincident regions of the North Pacific Ocean as do his outgroup species; whereas, the species that Wilson (1994) and Wilson et al. (1991) studied included representatives from the Atlantic
Ocean and the Gulf of Mexico in addition to ones from the North Pacific Ocean. Thus, the separate studies are not fully comparable. Our purpose in this paper is to extend the research of previous authors (Morita, 1999; Wilson, 1994; Wilson et al., 1991) on the interrelationships of selected Coryphaenoides (s.l.) species by including additional species germane to the question of the phylogenetic origin of abyssal macrourids, as well as to that of the monophyly of subgenus Coryphaenoides. In doing so, we continued using 12s rRNA gene sequences because the phylogenetic hypothesis Morita (1999) discussed was obtainable solely from analysis of 12s rRNA sequences.
2. Materials and methods 2.1. Taxa and DNA extraction Specimens of C. (Ch.) leptolepis, C. (C.) filifer, C. (C.) rupestris, C. (C.) mexicanus, and C. (C.) zaniophorus were collected as described in Wilson (1994). The specimens have been stored continuously in an ultra-low freezer at )87 °C since 1987, except for brief transfer on dry ice to the present location at California State University, Long Beach. Vouchers representing each of those species were deposited in the Scripps Institution of Oceanography collection under museum Accession Nos. SIO93-36, SIO87-80, SIO93-35, and SIO93-38, respectively. Caelorinchus occa was collected in 1989 from the eastern Gulf of Mexico near 24°170 N, 82°350 W, ca. 400 m depth. DNA was extracted from ca. 25 mg of muscle tissue using DNeasy Tissue Kits (Qiagen, Tustin, CA; Catalog No. 69506). The extracts were stored in buffer at 4 °C until used. PCR amplification of the 12s rRNA gene was performed in 100 ll using the reaction mixture of Wilson et al. (1997). Cycling parameters were: 94 °C 4:10 min, 92 °C 50 s, 55 °C 40 s, and 72 °C 1:30 min for 35 cycles, then, 72 °C 2:00 min, 5 ll of template was used in each PCR. We used MoritaÕs (1999) primers (L640: 50 -AAGCTATTA TGATGGGCCCT-30 and H1110: 50 -GTTCGAGTGAA GTACCATCA-30 ) and/or the reverse complement of Kocher et al.Õs (1989) 12SAL primer (12SARH: 50 -TGGG GTATCTAATCCCAGT-30 ) with MoritaÕs (1999) L640 primer to amplify the left (upstream) portion of the 12s gene. To amplify the downstream portion, we used Kocher et al.Õs (1989) primers (12SAL and 16SAH: 50 -ATGT TTTGATAAACAGGCG-30 ) and a second reverse primer we designed from C. (C.) rupestris sequence (RD12SP: 50 -CCAAGTACACCTTCCGGTACA-30 ) together with Kocher et al.Õs (1989) 12SAL primer. Amplicons were purified directly from the PCR cocktail using the QIAquick PCR Purification Kit (Qiagen, Tustin, CA, Catalog No. 28106), then cycled– sequenced bi-directionally for 30 cycles in a single re-
R.R. Wilson Jr., P. Attia / Molecular Phylogenetics and Evolution 27 (2003) 343–347
action using the Sequi-Therm Excel II DNA kit (Epicentre Technologies, Catalog No. SE9101LC) and dye-labeled sequencing primers. The cycle sequencing parameters were: 92 °C 30 s, 57 °C 15 s, and 70 °C 15 s. Sequenced products were run on a 5.5% TBE–polyacrylamide gel as part a LiCOR Model 4200L autosequencing system. 2.2. Alignment and phylogenetic analysis The six new 12s rRNA sequences were aligned using Clustal W (web version) multiple alignment program (Thomson et al., 1994) with MoritaÕs (1999) nine 12s sequences (GenBank Accession Nos: AB018224 Coryphaenoides (N.) armatus; AB018225 C. (N.) yaquinae; AB018226 C. (C.) nasutus; AB018227 C. (B.) longifilis; AB018228 C. (C.) acrolepis; AB018229 C. (C.) cinereus; AB018230 C. (C.) pectoralis; AB018231 Ca. gilberti; and AB018232 A. macrochir). In addition, we aligned the corresponding portion of the 12s rRNA sequence of Ca. kishinouyei, Accession No. AP002929. Altogether 16 macrourid species (12 ingroup and four outgroup) were aligned and analyzed without modification of the Clustal W alignment. The six new sequences were deposited in GenBank, Accession Nos. AY161229–AY161234. PAUP (Swofford, 1998) was used for maximum parsimony (MP) analysis using the branch and bound search option where gaps were treated as a new state, all characters were weighted equally, and bootstrapping was conducted at 100 replicates. The three Caelorinchus species were used collectively as outgroups for rooting the MP trees. DNAML in PHYLIP (Felsenstein, 1995) was used for maximum likelihood (ML) analysis (Felsenstein, 1981, 1985). Ca. kishinouyei was the outgroup root in the ML analysis. Because the new sequences overlapped MoritaÕs (1999) sequences, four of them fully, we used his observations on the distribution of variable sites between stems and loops to set parameter options for the ML analysis. His ratio of stem/loop sites was ca. 434/ 395 ¼ 1.1. The substitution rate of stems was ca. 51/ 434 ¼ 0.12; the substitution rate for loops was 63/ 395 ¼ 0.16. Thus, we input two standardized rates, a stem rate of 1.0 and a loop rate of 1.3. The probability of a stem substitution was set to 0.52 and a loop substitution was set to 0.48. The transition/transversion ratio was set to 4.0.
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mtDNA sequence of Ca. kishinouyei. C. (Ch.) leptolepis, C. (C.) filifer, C. (C.) rupestris, and Ca. occa constituted a full alignment of 832 bases with MoritaÕs (1999) sequences. C. (C.) zaniophorus and C. (C.) mexicanus constituted partial alignments of 396 and 374 bases, respectively, from positions 434 and 456, respectively, to position 830. The undetermined nucleotides of the shorter sequences of C. (C.) zaniophorus and C. (C.) mexicanus were coded as missing data in the overall alignment that we analyzed. The overall base composition from the alignment was A: 31%, C: 25%, G: 22%, and T: 22%. MP found 15 most parsimonious trees of 233 steps from 108 parsimony-informative characters (consistency index ¼ 0.7124, retention index ¼ 0.7880, and rescaled consistency index ¼ 0.5614). In each of the 15 MP trees the abyssal species clade of [C. (Ch.) leptolepis + C. (N.) armatus + C. N.) yaquinae)] was sister to a monophyletic subgenus Coryphaenoides assuming reassignment of C. longifilis and A. pectoralis to that subgenus. The bootstrapped MP tree (Fig. 1) showed 96% support for the abyssal clade and 73% for the Coryphaenoides (s.s.) clade. In subgenus Coryphaenoides the North Pacific species [minus C. (C.) nasutus] formed a clade with 72% support. One of the 15 MP trees agreed with the maximum likelihood tree (ln likelihood ¼ )12,396) (Fig. 2). In subgenus Coryphaenoides, C. (C.) longifilis was sister to the North Pacific Ocean group of [(C. (C.) pectoralis + C. (C.) acrolepis)) + (C. (C.) filifer + C. (C.) cinereus)]. The North Pacific C. (C.) nasutus was sister to an Atlantic species clade of [C. (C.) rupestris + (C. (C.) mexicanus + C. (C.) zaniophorus)]. As in the MP trees, the abyssal species clade was sister to a monophyletic Coryphaenoides. The bootstrapped ML tree showed 93% support for the abyssal clade, and 65% for the Coryphaenoides (s.s.) clade. In subgenus Coryphaenoides, the
3. Results Our alignment data block contained 832 characters versus MoritaÕs (1999) 829 the difference being due to insertions. The alignment was from two positions before the start of the 12s rRNA gene to position number 830 as referenced to the 12s rRNA gene in the complete
Fig. 1. Bootstrapped maximum parsimony consensus tree created from DNA sequence data of the 12s rRNA mitochondrial gene, indicating a monophyletic Coryphaenoides (s.s.). C. longifilis and C. pectoralis are here reassigned to subgenus Coryphaenoides as explained in text, and so depicted.
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Fig. 2. Bootstrapped maximum likelihood consensus tree created from DNA sequence data of the 12s rRNA mitochondrial gene, indicating a monophyletic Coryphaenoides (s.s.). C. longifilis and C. pectoralis treated as stated above.
North Pacific species [minus C. (C.) nasutus] clade had only 39% support.
4. Discussion The ML tree constructed from the partial sequences of the mitochondrial 12s rRNA gene agreed with MoritaÕs (1999) phylogeny with respect to the species studied in common except for placement of C. (C.) longifilis (Fig. 2). The MP consensus tree (Fig. 1) did not contradict the ML tree, and one MP tree was the same as the ML tree. The ML tree agreed closely for species in common with WilsonÕs (1994) MP tree from peptidemapping data that had the shortest FREQPARS length (Swofford and Berlocher, 1987), if the latter were rooted accordingly (Fig. 3A). The only difference was the joining of C. (C.) filifer and C. (C.) acrolepis, instead of C. (C.) acrolepis and C. (C.) pectoralis, as sister taxa. The ML tree agreed equally well with WilsonÕs (1994) ML tree from allozyme data that had the longest FREQ-
PARS length, if the later were rooted accordingly (Fig. 3B). The only difference was the uniting of C. (C.) filifer and C. (C.) pectoralis, instead of C. (C.) acrolepis and C. (C.) pectoralis, as sister taxa. C. (C.) longifilis and C. (C.) pectoralis ( ¼ A. pectoralis) have clear phylogenetic affinity with subgenus Coryphaenoides and should be reassigned to that subgenus. Although retaining pectoralis in the genus Albatrossia might be tenable morphologically, it would not be with respect to molecular phylogeny or as a matter of reflecting its evolutionary history. Iwamoto and Stein (1974) reviewed the similarities among C. (C.) pectoralis, C. (C.) cinereus, C. (C.) filifer, and C. (C.) acrolepis. The two most similar species were C. (C.) cinereus and C. (C.) filifer; C. (C.) acrolepis was more similar to C. (C.) filifer than to C. (C.) cinereus, C. (C.) pectoralis seemed the most derived due to its flaccid muscle tissue, poorly ossified skull bones, reduced swim bladder, and large adult size. Those features among others were responsible for its reassignment to Albatrossia (Iwamoto and Sazonov, 1988). However, the features likely reflect its protracted early life history stage in mid-water (Iwamoto and Stein, 1974; Novikov, 1970) and its frequent foraging there (Drazen et al., 2001). A similar situation applies to C. longifilis, a species presently referred to the monotypic subgenus Bogoslovius (Cohen et al., 1990). Both MP and ML analysis clearly place it among the slope-dwelling Coryphaenoides (s.s.) clade. Wilson et al. (1991) used Ca. occa, a species in common with the present study, as the outgroup root for their MP trees derived from peptide-mapping data; however, subgenus Coryphaenoides appeared paraphyletic in that study. A recent exhaustive search for an MP tree using PAUP on Wilson et al.Õs (1991) data again suggested paraphyly with low bootstrap support for any node save Chalinura + Nematonurus. Thus, data from peptide-mapping by reversed-phase high-performance liquid chromatography of trypsin digests of a single protein is evidently not as powerful in phylogenetic
Fig. 3. A posteriori rooted trees of Wilson (1994) for eight macrourids; placement of the presumed root is in accordance with that indicated in Morita (1999). (A) Maximum parsimony tree created from peptide-mapping data from trypsin digests of LDH-A4 , and evaluated against allozyme data for total tree length using FREQPARS (WilsonÕs Fig. 2C). FREQPARS length ¼ 163.9; branch lengths have meaning. (B) Maximum likelihood tree created from allozyme data from 24 presumptive gene loci, and evaluated for tree length using FREQPARS (WilsonÕs Fig. 1A). FREQPARS length ¼ 162.0; branch lengths have meaning.
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studies as allozyme data involving multiple loci, or when peptide and allozyme data are combined (Wilson, 1994). Future attempts to employ peptide mapping should include more than one protein in combination with more than one protease. In conclusion, comparative phylogenetic analyses of allozyme and 12s rRNA gene sequence data from 16 macrourids from four ocean regions with eight of 12 Coryphaenoides species in common for both data types strongly suggest that subgenus Coryphaenoides is a natural (monophyletic) group within genus Coryphaenoides and that the abyssal macrourids of the subgenera Chalinura and Nematonurus have experienced a separate radiation from the common ancestor. The latter scenario has been suggested from comparative morphology (Iwamoto and Sazonov, 1988). The remaining questions on the organization of the genus are whether species of subgenus Lionurus will prove the sister group of the Chalinura + Nematonurus clade within the abyssal group, as also previously suggested (Iwamoto and Sazonov, 1988), and whether inclusion of yet more species of Coryphaenoides (s.s.) in future studies will further confirm monophyly of the subgenus.
Acknowledgments This research was supported in part by an award from the Minority Student Development (MSD) program of the NIH, Award #GM584882 to M. Lopez, Principal Investigator, by faculty start-up funds from the College of Natural Sciences and Mathematics, California State University, Long Beach, and by NSF award BSR86-00186 to R. Wilson. Specimen collections in the Gulf of Mexico were facilitated by ship time from the Florida Institute of Oceanography, St. Petersburg, Florida. E. Gallardo, CSULB, assisted with DNA sequencing. We especially thank G. Nagel, College of Natural Sciences and Mathematics, for his continuing financial support of student research.
References Cohen, D.M., Inada, T., Iwamoto, T., Scialabba, N., 1990. Gadiform fishes of the world (Order: Gadiformes). FAO Fisheries Synopsis, No. 125, vol. 10, 442 pp. Drazen, J.C., Buckley, T.W., Hoff, G.R., 2001. The feeding habits of slope dwelling macrourid fishes in the eastern North Pacific. DeepSea Res. 48, 909–935. Endo, H., Okamura, O., 1992. New records of the abyssal grenadiers Coryphaenoides armatus and C. yaquinae from the western North Pacific. Jpn. J. Ichyol. 38, 433–438. Felsenstein, J., 1981. Evolutionary trees from DNA sequences: a maximum-likelihood approach. J. Mol. Evol. 17, 363–376.
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Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Felsenstein, J., 1995. Phylogeny Inference Package (PHYLIP), Version 3.5c00 . Department of Genetics, SK-50, University of Washington, Seattle, WA. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 6196–6200. Iwamoto, T., Stein, D.L., 1974. A systematic review of the rattail fishes (Macrouridae: Gadiformes) from Oregon and adjacent waters. Occas. Pap. Calif. Acad. Sci. 111, 1–79. Iwamoto, I., Sazonov, Y.I., 1988. A review of the southeastern Pacific Coryphaenoides (sensu lato) (Pisces, Gadiformes, Macrouridae). Proc. Calif. Acad. Sci. USA 45, 35–82. Marshall, N.B., 1973. Subfamily Macrourinae. In: Cohen, D.M. (Ed.), Fishers of the western North Atlantic. Memoir. Sears Found. Mar. Res. Part 6, New Haven, Connecticut, p. 596. Merrett, N.R., Haedrich, R.L., Gordon, J.D.M., Stehmann, M., 1991. Deep demersal fish assemblage structure in the Porcupine Seabight (Eastern North Atlantic): results of single warp trawling at lower slope to abyssal soundings. J. Mar. Biol. Assoc. UK 71, 359–373. Merrett, N.R., 1992. Demersal ichthyofaunal distribution in the abyssal eastern North Atlantic, with special reference to Coryphaenoides (Nematonurus) armatus (Macrouridae). J. Mar. Biol. Assoc. UK 72, 5–24. Middleton, R.W., Musick, J.A., 1986. The abundance and distribution of the family Macrouridae (Pisces: Gadiformes) in the Norfolk Canyon area. Fish. Bull. 84, 35–62. Morita, T., 1999. Molecular phylogenetic relationships of the deep-sea fish genus Coryphaenoides (Gadiformes: Macrouridae) based on mitochondrial DNA. Mol. Phylogenet. Evol. 13, 447–454. Novikov, N.P., 1970. Biology of Chalinura pectoralis in the North Pacific. In: Moiseev, P.A. (Ed.), Soviet fisheries investigations in the northwestern Pacific. Proc. Sci. Res. Inst. Mar. Fish. Ocean (VINRO), vol. 70, pp. 304–331. Stein, D.L., Pearcy, W.G., 1982. Aspects of reproduction, early life history, and biology of macrourid fishes off Oregon, USA. DeepSea Res. 29, 1313–1329. Swofford, D.L., 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods), Version 4. Sinauer Associates, Sunderland, MA. Swofford, D.L., Berlocher, S.H., 1987. Inferring evolutionary trees from gene frequency data under the principle of maximum parsimony. Syst. Zool. 36, 293–325. Thomson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wilson Jr., R.R., 1994. Interrelationships of the subgenera of Coryphaenoides (Gadiformes: Macrouridae): comparison of protein electrophoresis and peptide mapping. Copeia 1994, 42–50. Wilson Jr., R.R., Donaldson, K.A., Frischer, M.E., Young, T.B., 1997. Mitochondrial DNA control region of common snook and its prospect for use as a genetic tag. Trans. Amer. Fish. Soc. 126, 594–606. Wilson Jr., R.R., Siebenaller, J.F., Davis, B.J., 1991. Phylogenetic analysis of species of three subgenera of Coryphaenoides (Teleostei: Macrouridae) by peptide mapping of homologs of LDH-A4 . Biochem. Syst. Ecol. 18, 565–572. Wilson Jr., R.R., Waples, R.S., 1983. Distribution, morphology, and biochemical genetics of Coryphaenoides armatus and C. yaquinae (Pisces: Macrouridae) in the central and eastern North Pacific. Deep-Sea Res. 30, 1127–1145.
Interrelationships of the subgenera of Coryphaenoides (Teleostei: Gadiformes: Macrouridae): synthesis of allozyme, peptide mapping, and DNA sequence data Raymond R. Wilson Jr.* and Phoebe Attia Department of Biological Sciences, California State University Long Beach, Long Beach, CA 90840, USA Received 12 June 2002; revised 15 October 2002
Abstract DNA sequences of the 12s rRNA mitochondrial gene from 12 species key to the question of the monophyly of the deep-sea fish genus Coryphaenoides (Macrouridae) were analyzed phylogenetically using maximum parsimony and maximum likelihood. The results were compared with those of three previous studies in which allozyme, peptide mapping, and DNA sequence data were similarly analyzed. The allozyme and DNA sequence data suggested that the largest subgenus (Coryphaenoides), which contained most of the species inhabiting continental slopes between approximately 600 and 2000 m depth, is monophyletic. Two of the three subgenera containing the species inhabiting abyssal ocean basins below approximately 2000 m together formed a sister group to subgenus Coryphaenoides. The macrourids of the abyssal basins and those of the continental slopes thus appear to have experienced separate radiations from a common ancestor. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction The macrourid genus Coryphaenoides contains 61 recognized species that are presently organized into five subgenera (Cohen et al., 1990; Iwamoto and Sazonov, 1988). Subgenus Coryphaenoides has 43 species, Chalinura 10, Nematonurus five, Lionurus two, and Bogoslovius one (Cohen et al., 1990). These Coryphaenoides (sensu lato) species inhabit the worldÕs continental slopes and deepest ocean basins but their greatest diversity is on continental slopes approximately between 600 and 2000 m depth where species of subgenus Coryphaenoides typically reside. The few truly abyssal species are among the subgenera Chalinura, Nematonurus, and Lionurus. Coryphaenoides (Chalinura) leptolepis ranges between ca. 2000 and 4000 m in the North Pacific Ocean (Stein and Pearcy, 1982) and between ca. 600 and 4700 m in the North Atlantic Ocean (Merrett et al., 1991). C. (Ch.) profundicolis, the deepest reported species of Chalinura, ranges in the eastern North Atlantic Ocean between ca. 3900 and 4800 m (Merrett et al., 1991). Coryphaenoides * Corresponding author. Fax: 1-562-985-8878. E-mail address: [email protected] (R.R. Wilson Jr.).
(Nematonurus) armatus ranges chiefly between ca. 2000 and 4300 m in the North Pacific Ocean (Endo and Okamura, 1992; Stein and Pearcy, 1982; Wilson and Waples, 1983), and between ca. 2100 and 4800 m in the North Atlantic Ocean (Merrett, 1992; Middleton and Musick, 1986). Its close congener C. (N.) yaquinae ranges in the Pacific Ocean basin from ca. 3400 to at least 6450 m (Endo and Okamura, 1992; Wilson and Waples, 1983) and is evidently the deepest living macrourid in the worldÕs oceans. The deepest reported species of Lionurus, C. (Lionurus) carapinus, ranges below 4200 m to as deep as 5600 m in the North Atlantic Ocean basin (Marshall, 1973). Iwamoto and Stein (1974) established the present organization of genus Coryphaenoides. At that time they placed Coryphaenoides pectoralis in the subgenus Nematonurus. Since then, the species has been reassigned to its own monotypic genus Albatrossia (Iwamoto and Sazonov, 1988). Wilson et al. (1991) and Wilson (1994) questioned whether the subgenera erected by Iwamoto and Stein (1974) each represented a natural (monophyletic) group. Peptide mapping of trypsin digests of LDH-A4 homologs from a collection of Atlantic and Pacific ocean species representing three of the four
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subgenera indicated a Nematonurus + Chalinura clade nested in subgenus Coryphaenoides, making Coryphaenoides (sensu stricto) appear paraphyletic (Wilson et al., 1991). Starch-gel electrophoresis of gene products from 24 presumptive allozyme loci (Wilson, 1994) also suggested a Nematonurus + Chalinura clade but the lack of tree rooting in that study precluded discussion of the issues pertaining to monophyly of the subgenera. It appeared, nevertheless, that C. (N.) pectoralis was in fact referable to subgenus Coryphaenoides rather than to Nematonurus or to Chalinura. Morita (1999) analyzed DNA sequences from portions of the 12s rRNA and COI mitochondrial genes for seven Coryphaenoides species representing the subgenera Bogoslovius, Coryphaenoides, and Nematonurus, plus two outgroup species that are now referred to the macrourid genus Caelorinchus, an upper slope group. MoritaÕs (1999) study included four Coryphaenoides species in common with Wilson et al. (1991) and three in common with Wilson (1994). MoritaÕs two abyssal species were C. (N.) armatus and C. (N.) yaquinae. Morita (1999) found that the two Nematonurus species formed a sister group to the other Coryphaenoides species, which appeared to represent a natural (monophyletic) group for subgenus Coryphaenoides upon a reassignment of C. longifilis and Albatrossia pectoralis to that subgenus. Morita (1999) suggested that the abyssal species had experienced a radiation separate from that of the slopedwelling species of Coryphaenoides, whereas Wilson (1994) suggested the abyssal species might have arisen from among the shallower slope-dwelling species of subgenus Coryphaenoides. A posteriori rooting of WilsonÕs (1994) trees in accordance with MoritaÕs (1999) phylogenetic hypothesis converged to essentially MoritaÕs result, thus supporting a similar interpretation of a possibly monophyletic subgenus Coryphaenoides and separate radiations for Coryphaenoides (s.s.) and the abyssal group of species. As mentioned above, however, MoritaÕs (1999) abyssal group was restricted to the North Pacific C. (N.) yaquinae and C. (N.) armatus. C. (N.) yaquinae is the evident ecological depth replacement of C. (N.) armatus in the North Pacific Ocean (Endo and Okamura, 1992; Wilson and Waples, 1983). The two species are long believed closely related (e.g., Iwamoto and Stein, 1974; Wilson and Waples, 1983) so a node uniting them would be expected. However, with the absence of C. (Ch.) leptolepis or, for example, C. (Ch.) profundicolis from MoritaÕs analysis, the two Nematonurus species alone do not represent the same abyssal group that Wilson (1994) and Wilson et al. (1991) studied. In addition, all of MoritaÕs (1999) species of subgenus Coryphaenoides inhabit proximal or coincident regions of the North Pacific Ocean as do his outgroup species; whereas, the species that Wilson (1994) and Wilson et al. (1991) studied included representatives from the Atlantic
Ocean and the Gulf of Mexico in addition to ones from the North Pacific Ocean. Thus, the separate studies are not fully comparable. Our purpose in this paper is to extend the research of previous authors (Morita, 1999; Wilson, 1994; Wilson et al., 1991) on the interrelationships of selected Coryphaenoides (s.l.) species by including additional species germane to the question of the phylogenetic origin of abyssal macrourids, as well as to that of the monophyly of subgenus Coryphaenoides. In doing so, we continued using 12s rRNA gene sequences because the phylogenetic hypothesis Morita (1999) discussed was obtainable solely from analysis of 12s rRNA sequences.
2. Materials and methods 2.1. Taxa and DNA extraction Specimens of C. (Ch.) leptolepis, C. (C.) filifer, C. (C.) rupestris, C. (C.) mexicanus, and C. (C.) zaniophorus were collected as described in Wilson (1994). The specimens have been stored continuously in an ultra-low freezer at )87 °C since 1987, except for brief transfer on dry ice to the present location at California State University, Long Beach. Vouchers representing each of those species were deposited in the Scripps Institution of Oceanography collection under museum Accession Nos. SIO93-36, SIO87-80, SIO93-35, and SIO93-38, respectively. Caelorinchus occa was collected in 1989 from the eastern Gulf of Mexico near 24°170 N, 82°350 W, ca. 400 m depth. DNA was extracted from ca. 25 mg of muscle tissue using DNeasy Tissue Kits (Qiagen, Tustin, CA; Catalog No. 69506). The extracts were stored in buffer at 4 °C until used. PCR amplification of the 12s rRNA gene was performed in 100 ll using the reaction mixture of Wilson et al. (1997). Cycling parameters were: 94 °C 4:10 min, 92 °C 50 s, 55 °C 40 s, and 72 °C 1:30 min for 35 cycles, then, 72 °C 2:00 min, 5 ll of template was used in each PCR. We used MoritaÕs (1999) primers (L640: 50 -AAGCTATTA TGATGGGCCCT-30 and H1110: 50 -GTTCGAGTGAA GTACCATCA-30 ) and/or the reverse complement of Kocher et al.Õs (1989) 12SAL primer (12SARH: 50 -TGGG GTATCTAATCCCAGT-30 ) with MoritaÕs (1999) L640 primer to amplify the left (upstream) portion of the 12s gene. To amplify the downstream portion, we used Kocher et al.Õs (1989) primers (12SAL and 16SAH: 50 -ATGT TTTGATAAACAGGCG-30 ) and a second reverse primer we designed from C. (C.) rupestris sequence (RD12SP: 50 -CCAAGTACACCTTCCGGTACA-30 ) together with Kocher et al.Õs (1989) 12SAL primer. Amplicons were purified directly from the PCR cocktail using the QIAquick PCR Purification Kit (Qiagen, Tustin, CA, Catalog No. 28106), then cycled– sequenced bi-directionally for 30 cycles in a single re-
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action using the Sequi-Therm Excel II DNA kit (Epicentre Technologies, Catalog No. SE9101LC) and dye-labeled sequencing primers. The cycle sequencing parameters were: 92 °C 30 s, 57 °C 15 s, and 70 °C 15 s. Sequenced products were run on a 5.5% TBE–polyacrylamide gel as part a LiCOR Model 4200L autosequencing system. 2.2. Alignment and phylogenetic analysis The six new 12s rRNA sequences were aligned using Clustal W (web version) multiple alignment program (Thomson et al., 1994) with MoritaÕs (1999) nine 12s sequences (GenBank Accession Nos: AB018224 Coryphaenoides (N.) armatus; AB018225 C. (N.) yaquinae; AB018226 C. (C.) nasutus; AB018227 C. (B.) longifilis; AB018228 C. (C.) acrolepis; AB018229 C. (C.) cinereus; AB018230 C. (C.) pectoralis; AB018231 Ca. gilberti; and AB018232 A. macrochir). In addition, we aligned the corresponding portion of the 12s rRNA sequence of Ca. kishinouyei, Accession No. AP002929. Altogether 16 macrourid species (12 ingroup and four outgroup) were aligned and analyzed without modification of the Clustal W alignment. The six new sequences were deposited in GenBank, Accession Nos. AY161229–AY161234. PAUP (Swofford, 1998) was used for maximum parsimony (MP) analysis using the branch and bound search option where gaps were treated as a new state, all characters were weighted equally, and bootstrapping was conducted at 100 replicates. The three Caelorinchus species were used collectively as outgroups for rooting the MP trees. DNAML in PHYLIP (Felsenstein, 1995) was used for maximum likelihood (ML) analysis (Felsenstein, 1981, 1985). Ca. kishinouyei was the outgroup root in the ML analysis. Because the new sequences overlapped MoritaÕs (1999) sequences, four of them fully, we used his observations on the distribution of variable sites between stems and loops to set parameter options for the ML analysis. His ratio of stem/loop sites was ca. 434/ 395 ¼ 1.1. The substitution rate of stems was ca. 51/ 434 ¼ 0.12; the substitution rate for loops was 63/ 395 ¼ 0.16. Thus, we input two standardized rates, a stem rate of 1.0 and a loop rate of 1.3. The probability of a stem substitution was set to 0.52 and a loop substitution was set to 0.48. The transition/transversion ratio was set to 4.0.
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mtDNA sequence of Ca. kishinouyei. C. (Ch.) leptolepis, C. (C.) filifer, C. (C.) rupestris, and Ca. occa constituted a full alignment of 832 bases with MoritaÕs (1999) sequences. C. (C.) zaniophorus and C. (C.) mexicanus constituted partial alignments of 396 and 374 bases, respectively, from positions 434 and 456, respectively, to position 830. The undetermined nucleotides of the shorter sequences of C. (C.) zaniophorus and C. (C.) mexicanus were coded as missing data in the overall alignment that we analyzed. The overall base composition from the alignment was A: 31%, C: 25%, G: 22%, and T: 22%. MP found 15 most parsimonious trees of 233 steps from 108 parsimony-informative characters (consistency index ¼ 0.7124, retention index ¼ 0.7880, and rescaled consistency index ¼ 0.5614). In each of the 15 MP trees the abyssal species clade of [C. (Ch.) leptolepis + C. (N.) armatus + C. N.) yaquinae)] was sister to a monophyletic subgenus Coryphaenoides assuming reassignment of C. longifilis and A. pectoralis to that subgenus. The bootstrapped MP tree (Fig. 1) showed 96% support for the abyssal clade and 73% for the Coryphaenoides (s.s.) clade. In subgenus Coryphaenoides the North Pacific species [minus C. (C.) nasutus] formed a clade with 72% support. One of the 15 MP trees agreed with the maximum likelihood tree (ln likelihood ¼ )12,396) (Fig. 2). In subgenus Coryphaenoides, C. (C.) longifilis was sister to the North Pacific Ocean group of [(C. (C.) pectoralis + C. (C.) acrolepis)) + (C. (C.) filifer + C. (C.) cinereus)]. The North Pacific C. (C.) nasutus was sister to an Atlantic species clade of [C. (C.) rupestris + (C. (C.) mexicanus + C. (C.) zaniophorus)]. As in the MP trees, the abyssal species clade was sister to a monophyletic Coryphaenoides. The bootstrapped ML tree showed 93% support for the abyssal clade, and 65% for the Coryphaenoides (s.s.) clade. In subgenus Coryphaenoides, the
3. Results Our alignment data block contained 832 characters versus MoritaÕs (1999) 829 the difference being due to insertions. The alignment was from two positions before the start of the 12s rRNA gene to position number 830 as referenced to the 12s rRNA gene in the complete
Fig. 1. Bootstrapped maximum parsimony consensus tree created from DNA sequence data of the 12s rRNA mitochondrial gene, indicating a monophyletic Coryphaenoides (s.s.). C. longifilis and C. pectoralis are here reassigned to subgenus Coryphaenoides as explained in text, and so depicted.
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Fig. 2. Bootstrapped maximum likelihood consensus tree created from DNA sequence data of the 12s rRNA mitochondrial gene, indicating a monophyletic Coryphaenoides (s.s.). C. longifilis and C. pectoralis treated as stated above.
North Pacific species [minus C. (C.) nasutus] clade had only 39% support.
4. Discussion The ML tree constructed from the partial sequences of the mitochondrial 12s rRNA gene agreed with MoritaÕs (1999) phylogeny with respect to the species studied in common except for placement of C. (C.) longifilis (Fig. 2). The MP consensus tree (Fig. 1) did not contradict the ML tree, and one MP tree was the same as the ML tree. The ML tree agreed closely for species in common with WilsonÕs (1994) MP tree from peptidemapping data that had the shortest FREQPARS length (Swofford and Berlocher, 1987), if the latter were rooted accordingly (Fig. 3A). The only difference was the joining of C. (C.) filifer and C. (C.) acrolepis, instead of C. (C.) acrolepis and C. (C.) pectoralis, as sister taxa. The ML tree agreed equally well with WilsonÕs (1994) ML tree from allozyme data that had the longest FREQ-
PARS length, if the later were rooted accordingly (Fig. 3B). The only difference was the uniting of C. (C.) filifer and C. (C.) pectoralis, instead of C. (C.) acrolepis and C. (C.) pectoralis, as sister taxa. C. (C.) longifilis and C. (C.) pectoralis ( ¼ A. pectoralis) have clear phylogenetic affinity with subgenus Coryphaenoides and should be reassigned to that subgenus. Although retaining pectoralis in the genus Albatrossia might be tenable morphologically, it would not be with respect to molecular phylogeny or as a matter of reflecting its evolutionary history. Iwamoto and Stein (1974) reviewed the similarities among C. (C.) pectoralis, C. (C.) cinereus, C. (C.) filifer, and C. (C.) acrolepis. The two most similar species were C. (C.) cinereus and C. (C.) filifer; C. (C.) acrolepis was more similar to C. (C.) filifer than to C. (C.) cinereus, C. (C.) pectoralis seemed the most derived due to its flaccid muscle tissue, poorly ossified skull bones, reduced swim bladder, and large adult size. Those features among others were responsible for its reassignment to Albatrossia (Iwamoto and Sazonov, 1988). However, the features likely reflect its protracted early life history stage in mid-water (Iwamoto and Stein, 1974; Novikov, 1970) and its frequent foraging there (Drazen et al., 2001). A similar situation applies to C. longifilis, a species presently referred to the monotypic subgenus Bogoslovius (Cohen et al., 1990). Both MP and ML analysis clearly place it among the slope-dwelling Coryphaenoides (s.s.) clade. Wilson et al. (1991) used Ca. occa, a species in common with the present study, as the outgroup root for their MP trees derived from peptide-mapping data; however, subgenus Coryphaenoides appeared paraphyletic in that study. A recent exhaustive search for an MP tree using PAUP on Wilson et al.Õs (1991) data again suggested paraphyly with low bootstrap support for any node save Chalinura + Nematonurus. Thus, data from peptide-mapping by reversed-phase high-performance liquid chromatography of trypsin digests of a single protein is evidently not as powerful in phylogenetic
Fig. 3. A posteriori rooted trees of Wilson (1994) for eight macrourids; placement of the presumed root is in accordance with that indicated in Morita (1999). (A) Maximum parsimony tree created from peptide-mapping data from trypsin digests of LDH-A4 , and evaluated against allozyme data for total tree length using FREQPARS (WilsonÕs Fig. 2C). FREQPARS length ¼ 163.9; branch lengths have meaning. (B) Maximum likelihood tree created from allozyme data from 24 presumptive gene loci, and evaluated for tree length using FREQPARS (WilsonÕs Fig. 1A). FREQPARS length ¼ 162.0; branch lengths have meaning.
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studies as allozyme data involving multiple loci, or when peptide and allozyme data are combined (Wilson, 1994). Future attempts to employ peptide mapping should include more than one protein in combination with more than one protease. In conclusion, comparative phylogenetic analyses of allozyme and 12s rRNA gene sequence data from 16 macrourids from four ocean regions with eight of 12 Coryphaenoides species in common for both data types strongly suggest that subgenus Coryphaenoides is a natural (monophyletic) group within genus Coryphaenoides and that the abyssal macrourids of the subgenera Chalinura and Nematonurus have experienced a separate radiation from the common ancestor. The latter scenario has been suggested from comparative morphology (Iwamoto and Sazonov, 1988). The remaining questions on the organization of the genus are whether species of subgenus Lionurus will prove the sister group of the Chalinura + Nematonurus clade within the abyssal group, as also previously suggested (Iwamoto and Sazonov, 1988), and whether inclusion of yet more species of Coryphaenoides (s.s.) in future studies will further confirm monophyly of the subgenus.
Acknowledgments This research was supported in part by an award from the Minority Student Development (MSD) program of the NIH, Award #GM584882 to M. Lopez, Principal Investigator, by faculty start-up funds from the College of Natural Sciences and Mathematics, California State University, Long Beach, and by NSF award BSR86-00186 to R. Wilson. Specimen collections in the Gulf of Mexico were facilitated by ship time from the Florida Institute of Oceanography, St. Petersburg, Florida. E. Gallardo, CSULB, assisted with DNA sequencing. We especially thank G. Nagel, College of Natural Sciences and Mathematics, for his continuing financial support of student research.
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