Phylogeny and Evolution of the Gobiid Genus <I>Coryphopterus</I>

BULLETIN OF MARINE SCIENCE, 70(3): 837–850, 2002

PHYLOGENY AND EVOLUTION OF THE GOBIID GENUS CORYPHOPTERUS

Christine E. Thacker and Kathleen S. Cole

ABSTRACT We use morphological and molecular data to resolve the relationships among species of the gobiid genus Coryphopterus, and between Coryphopterus and putative sister taxa Lophogobius and Fusigobius. Characters of the external morphology and the mitochondrial ND2 gene were combined in a total evidence cladistic analysis. The single most parsimonious topology that resulted indicates that Coryphopterus is not monophyletic, and we advocate the removal of C. nicholsii from Coryphopterus and resurrection of the name Rhinogobiops nicholsii. Our phylogeny agrees in many respects with an earlier hypothesis advanced by Smith and Tyler (1977); interpretations of the evolution of morphology and ecology on the two topologies are compared. The phylogeny also indicates that Coryphopterus is not closely related to Fusigobius, and so the synonomy of Randall (1995) should not be used. Instead, Coryphopterus is more closely related to the transisthmian Lophogobius than to any Indo-Pacific species examined, a finding that provides insight into the biogeography of the group. An ancillary result of our study is the suggestion that Fusigobius is not monophyletic, however, a comprehensive revision and phylogenetic analysis of Fusigobius is beyond the scope of this paper.

The goby genus Coryphopterus Gill includes nine western Atlantic and two eastern Pacific species. Among gobies, Coryphopterus species are unusual in that they occupy a range of habitats and have a sequential (protogynous) hermaphroditic life history. Spe- cies of Coryphopterus are generally small (11 to 50 mm SL) and vary in their general morphology. Most are pale, with few black, yellow or blue markings; the hover gobies (C. personatus (Jordan and Thompson) and C. hyalinus Böhlke and Robins) and the peppermint goby (C. lipernes Böhlke and Robins) are the smallest Coryphopterus, and are orange to gold with white markings. As is typical for gobies, most Coryphopterus species are benthic, inhabiting rocky or sandy substrates near reefs. The hover gobies and peppermint goby are exceptions to these patterns: hover gobies are found in large schooling aggregations above coral reefs, and the peppermint goby perches on corals; these three species are also found at deeper depths (up to 30 m) than the other Coryphopterus species. Previous work on Coryphopterus has mostly been taxonomic rather than phylogenetic. In the revision of Böhlke and Robins (1960), the two Pacific (Coryphopterus nicholsii (Bean) and C. urospilus Ginsberg) and six western Atlantic (C. alloides, C. dicrus, C. thrix, C. eidolon all new species, C. glaucofrenum Gill and C. punctipectophorus Springer) species are discussed and a key is provided. Böhlke and Robins (1960) provide 21 characters that are present in Coryphopterus, including the presence of six dorsal spines, with the sixth widely separated from the fifth; seventeen segmented caudal rays; no teeth on vomer or palatine; jaw teeth in several rows, the inner and outer rows enlarged; second dorsal and anal fins relatively short (13–15 second dorsal and 12–13 anal elements in C. nicholsii; 9–11 second dorsal and 12–13 anal elements in the other species); pelvic fin counts of I,5/I,5 with disc complete or deeply incised, frenum present or absent; enlarged neural and hemal spines on the vertebra preceding the hypural plate; body scaled, but

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cheek and opercle unscaled; and a fleshy ridge, variously developed, from dorsal fin origin to a point between and just behind the eyes. None of these characters is unique to Coryphopterus species, and most are fairly widespread among gobies. A further three western Atlantic species, Coryphopterus personatus (formerly Eviota personata), C. hyalinus and C. lipernes, were described in 1962 (Böhlke and Robins, 1962). Coryphopterus tortugae was discussed as a pallid form of C. glaucofrenum by Böhlke and Robins (1960); based on collections in the Colombian Caribbean, Garzon-Ferreira and Acero (1990) redescribe C. tortugae as a distinct species, and Greenfield and Johnson (1999) also recognize C. tortugae. The primary character used to differentiate the species is the pigment on the caudal peduncle: either two separate blotches (C. glaucofrenum) or a single bar (C. tortugae). Garzon-Ferreira and Acero (1990) also report that their C. tortugae specimens are overall more pallid and less robust than C. glaucofrenum. The coloration differences were attributed by Böhlke and Robins (1960) to differences in habitat where the fish were caught: clear, deeper water for C. tortugae and murkier, in- shore water for C. glaucofrenum. Böhlke and Robins (1960) also report that a full range of intermediates was observed. Garzon-Ferreira and Acero (1990) claim that no intergra- dation exists in the caudal pigmentation. The C. glaucofrenum examined for this study were collected from localities ranging from Panama, throughout the Greater and Lesser Antilles, to Venezuela, and all but one lot from Costa Rica (LACM 2250) exhibited two distinct blotches, without intervening pigment, on the caudal peduncle. The four fish in the Costa Rican lot were darker than the other fishes examined, and there was some dark pigment between the caudal peduncle blotches; however, the two blotches were clearly distinguishable and no other notable differences were observed. Because of the variation found in C. glaucofrenum and the presence of intermediates observed by Böhlke and Robins (1960), C. tortugae is not treated as a separate species in this study. Smith (1997) recognizes another species similar to C. glaucofrenum, C. venezulae. Originally described as a subspecies of C. glaucofrenum (Cervigón, 1966), it differs from C. glaucofrenum slightly in pigmentation and has one additional dorsal ray; we did not examine this species to determine its validity, and do not consider it further. Coryphopterus is currently placed in the Priolepis group of the gobiid subfamily Gobiinae (Birdsong et al., 1988), a large (54 genera) phenetically distinct subdivision of Gobioidei that includes most of the gobiids inhabiting the Indo-Pacific and Pacific Plate, as well as a significant portion of the western Atlantic gobiid fauna. Members of the Priolepis group share a dorsal-fin pterygiophore formula of 3-22110, 10 precaudal and 16 caudal vertebrae, one epural and two anal-fin pterygiophores anterior to the first hemal spine. More precise interrelationships between the Priolepis group genera, including the relationships of Coryphopterus, are not known. Two noteworthy candidates for the sister taxon to Coryphopterus have been proposed, both members of the Priolepis group. The first, Lophogobius Gill, includes three species, two western Atlantic (L. cyprinoides (Pallas) and L. androsensis Breder) and one eastern Pacific (L. cristulatus Ginsburg). Lophogobius shares several similarities with Coryphopterus, including gonad structure (which varies considerably among gobies) and a protogynous hermaphroditic sexual pattern (Cole, 1988). Lophogobius cyprinoides also has a conspicuous fleshy head crest similar to the fleshy ridge seen among Coryphopterus species. The development of this crest varies among species of Lophogobius: in L. cyprinoides the crest is prominent, and in the illustration of the holotype of L. androsensis (Breder, 1932) a large crest is figured. THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 839

Lophogobius cristulatus is described as differing from L. cyprinoides in that the crest is present, but markedly smaller (Ginsburg, 1939). An additional candidate that has been proposed as a close relative of Coryphopterus is Fusigobius Whitley. Fusigobius is an Indo-Pacific genus which also has a gonad structure similar to that of Coryphopterus and also exhibits protogynous hermaphroditism (Cole, 1988). There are six described Fusigobius species: F. neophytus (Günther, 1877), the type species, and F. l ongispinus Goren, 1978; F. duospilus Hoese and Reader, 1985; F. signipinnis Hoese and Obika, 1988; F. inframaculatus (Randall, 1994); and F. aureus Chen and Shao, 1997. Several species of Fusigobius are also similar in coloration and external morphology to those species of Coryphopterus that are associated with benthic reef substrate. This similarity led Randall (1995) to place all Indo-Pacific species of Fusigobius in the genus Coryphopterus, although other authors (Chen and Shao, 1997) advocate recognition of a Fusigobius distinct from Coryphopterus until a revision of Fusigobius is available. Several species of Fusigobius remain undescribed; the described species have only slight meristic differences, but vary in the degree to which their pelvic fins are joined, from separate to a complete pelvic disk. They are distinguished from one another primarily on the basis of color pattern. Determination of which (if either) of these genera is the sister taxon to Coryphopterus will inform hypotheses about the biogeography of the genera. If Fusigobius is the sister genus (or the same genus), the phylogeny would be consistent with the hypothesis that Coryphopterus originated in the Pacific and subsequently radiated into the Caribbean prior to the last closing of the land bridge betweeen South and Central America. Alterna- tively, if Lophogobius is the sister group to Coryphopterus, origin of Coryphopterus in the eastern Pacific or western Atlantic is hypothesized. Additionally, the monophyly of Coryphopterus is questionable: the species C. nicholsii was included by Böhlke and Rob- ins (1960), but they expressed doubts about this placement, and these doubts were echoed by Randall (1995). C. nicholsii is the largest Coryphopterus (up to 150 mm), and also the only temperate Coryphopterus species. Of the Pacific species, C. urospilus is found from the Pacific coast of Panama northward to the Gulf of California; C. nicholsii is distributed from Baja California to British Columbia. C. nicholsii also differs from the other Coryphopterus in fin ray counts and extent of squamation (Böhlke and Robins, 1960). A tree of relationships among the nine western Atlantic Coryphopterus species was presented by Smith and Tyler (1977). Their tree was constructed based on morphological characters including the conformation of the pelvic and dorsal fins, pigmentation, habitat and behavior; they did not hypothesize the sister taxon of Coryphopterus. In this study, we reexamine and augment Smith and Tyler’s (1977) morphological characters, add DNA sequence data, and use cladistic parsimony methods to hypothesize a total evidence phylogeny for species of Coryphopterus and outgroups including Lophogobius and Fusigobius. With this hypothesis, we evaluate the monophyly of Coryphopterus, specifically with respect to C. nicholsii, examine previous hypotheses of the sister taxon to Coryphopterus, and determine whether or not the synonomization of Coryphopterus and Fusigobius (Randall, 1995) is justified. We also use the phylogeny to interpret morphological and ecological character evolution among Coryphopterus species. 840 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

MATERIALS AND METHODS

Adults of all 11 Coryphopterus species, as well as L. cyprinoides, F. neophytus and outgroup species Bathygobius curacao (Metzelaar), Bathygobius lineatus (Jenyns), Gobiodon histrio (Valenciennes), G. citrinus (Rüppell) and Gnatholepis thompsoni Jordan were examined for external and internal morphology. The outgroups were selected based on the small amount of informa- tion available on gobioid relationships: Gobiodon is a member of Birdsong et al.’s (1988) Priolepis group, the group that also includes Coryphopterus, Fusigobius and Lophogobius. The Priolepis group is included in the subfamily Gobiinae. Bathygobius is also classified in Gobiinae, but in the Bathygobius group of Birdsong et al. (1988). The Bathygobius group differs from the Priolepis group in vertebral number: Bathygobius group genera have counts of 10+17=27, and Priolepis group genera are characterized by a count of 10+16 = 26. Gnatholepis thompsoni, used as the distal outgroup, is a member of the subfamily Gobionellinae (Pezold, 1993), and is characterized by different dorsal fin pterygiophore and head pore patterns, as well as several skeletal differences as compared to the gobiines. Most of the species were cleared and double stained for bone (alizarin red) and cartilage (alcian blue) by the method of Pothoff (1984) and dissected by a modification of the method for small teleosts outlined in Weitzman (1974); when not enough specimens were available to clear and stain, radiographs were prepared using a Hewlett-Packard Faxitron cabinet X-ray system with Kodak Ortho M film. For Bathygobius and Gobiodon, different species were sequenced than were cleared, stained and examined for internal morphology: B. lineatus rather than B. curacao, and G. citrinus rather than G. histrio. Morphological characters of Smith and Tyler (1977) were reexamined, and selected characters (those that were not autapomorphic or plesiomorphic) were recoded and used in this analysis. Additional characters of the external anatomy were added. Mor- phological characters for Fusigobius signipinnis were coded from the descriptions of Hoese and Obika (1988) and Chen and Shao (1997). Ethanol-preserved goby tissues for DNA sequencing were obtained from several sources. Coryphopterus dicrus, C. punctipectophorus, C. personatus, C. hyalinus, C. lipernes and C. eidolon were collected by K. Cole using SCUBA, quinaldine and hand nets on reefs near Carrie Bow Cay, Belize. K. Cole also collected specimens of L. cyprinoides in Florida. F. signpinnis was provided by Mark Westneat from collections at Santa Cruz Island, Solomon Islands. Coryphopterus urospilus was provided by Karen Crow from collections at Bahia Los Angeles. G. histrio was provided by Rob Reavis from a captive stock. Coryphopterus glaucofrenum and B. curacao were collected by C. Thacker using SCUBA, quinaldine and hand nets, at Carrie Bow Cay, Belize; C. nicholsii was collected with similar gear at Catalina Island, California. Two specimens of the species C. hyalinus, C. dicrus, C. punctipectophorus, C. nicholsii, L. cyprinoides and F. signipinnis were sequenced; for the other species, one specimen was used. Muscle tissue from each species was used for total genomic DNA extraction, performed with the QIAamp Tissue Kit (QIAGEN Inc., Chatsworth CA). Hotstart XL PCR was performed using primers L3827 (5'-GCA ATC CAG GTC GGT TTC TAT C- 3') to H6313 (5'-CTC TTA TTT AAG GCT TTG AAG GC-3') and Taq rTth XL polymerase with AmpliWax PCR Gems (Perkin-Elmer, Foster City, CA). The primers used in this study were designed by Sorenson et al. (1999) for use in amplification and sequencing of a variety of amniote taxa. The L3827/H6313 primer set amplifies a fragment from the 3' end of the 16s rDNA gene to the tryptophan tRNA that flanks the 3' end of the ND2 gene; the fragment includes approximately 200 base pairs of 16s, followed by tLeu, ND1, tIle, tGln, tMet, ND2 and tTrp. The PCR was performed with a profile of 94˚ for 5 min, followed by 16 cycles of 94˚ per 30 s denaturation, 50–53˚ per 20 s annealing and 70˚ per 4 min extension, then 21 cycles of the same profile but with 30 additional seconds of extension added at each step. These long (~2500 bp) fragments were used as template for a short PCR reaction using the primer pair L5758 (5'-GGC TGA ATR GGM CTN AAY CAR AC-3')/ H6313, which amplifies the 5' half of the ND2 gene. These amplifications were performed with AmpliTaq or AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA), with a profile of 94˚ for 3 min, followed by 35 cycles of 94˚ for 15 s denaturation, 50–55˚ for 20 s annealing and 70˚ for 1 min extension For some samples, these primers did not successfully am- THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 841

plify and a goby-specific primer in the middle of the ND2 gene was designed to replace L5758 (L5460: 5'-GGG GGC TGA GGG GGC TTA-3'). L5460 and H6313 were used to amplify the fragment directly from genomic DNA extraction, using the same PCR profile as the L5758-H6313 amplifications, and the enzyme AmpliTaq Gold DNA polymerase or Platinum Taq High Fidelity DNA Polymerase (Life Technologies, Rockville, MD). Using the same primers (1 µM rather than 10 µM) the short PCR fragments were cycle sequenced using Perkin-Elmer’s rhodamine dye termi- nator/Taq FS ready reaction kit and run on an ABI 377 automated sequencer. The heavy and light strands were sequenced separately. The resultant chromatograms for the heavy and light strands were reconciled in Perkin-Elmer’s Sequence Navigator to check basecalling, translated to amino acid sequence using the ‘universal mtDNA’ code, and aligned by eye. Aligned nucleotide sequences were exported from Sequence Navigator as text files and converted into a PAUP-ready NEXUS file using Sequence Compiler (Sorenson, 1996). The NEXUS file containing the DNA sequence data was opened in MacClade 3.0 (Maddison and Maddison, 1992), character codes were changed from A, C, G and T to numerical codes, and the morphological characters were added. The resultant matrix included 545 characters, 531 molecular and 14 morphological, for the eleven ingroup and six outgroup taxa. For two species (C. alloides and C. thrix), molecular data were not available, so only the morphological characters were coded and the ? entries in the matrix were treated as missing data. Phylogenetic analyses were performed using PAUP*, version 4.0b4a. One thousand replications of a heuristic search were performed, using TBR branch swapping, and Gnatholepis thompsoni was designated as the outgroup taxon. Decay indices (Bremer, 1994) were calculated with the aid of TreeRot version 2 (Sorenson, 1999).

RESULTS

The morphological characters are numbered in accordance with the character matrix, shown in Table 1. Examination of internal anatomy from cleared and stained specimens or radiographs did not reveal any characters useful for resolving relationships among most of the species. Although slight variations were present in the robustness of bones, squamation and dentition, no significant differences were observed. The exceptions were B. curacao, which has one more vertebra than the Priolepis group genera, and Gnatholepis thompsoni, which differed from the other species in dorsal fin ray formula (3-1221 rather than 3-2211), number of epurals (two rather than one), and some specializations of the suspensorium (similar to those described for G. anjerensis by Harrison, 1989). The head sensory pore pattern described by Böhlke and Robins (1960) for Coryphopterus species, and for F. s i gnipinnis by Hoese and Obika (1988), was present in all species examined except C. hyalinus and the outgroup G. thompsoni. Coryphopterus hyalinus has an autapomorphic variation of the pattern, with a pair of interorbital pores rather than a single pore. The lateral canal pore pattern in F. neophytus is variable within the species; some individuals exhibit the condition found in other Fusigobius and in Coryphopterus species, others have an extra lateral canal pore dorsal to the preopercular canal (two pores are present instead of the single pore G; Hoese and Reader, 1985; Hoese and Obika, 1988; Thacker and Hoese, pers. observ.). The head pore pattern in Gnatholepis thompsoni differs from the other species, instead resembling that figured for G. caurensis by Takagi (1989). 1. Pelvic frenum. (0) present; (1) absent. The pelvic fins may be joined anteriad by a frenum that connects the pelvic spines. The frenum is absent (state one) in Coryphopterus personatus, C. hyalinus, C. lipernes, C. alloides, C. dicrus, and F. signipinnis. 842 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

Table 1. Taxon by character matrix for morphological characters in Coryphopterus species and outgroups.

Traxon Characte 12 34567890111213141 Coryphopterus personatus 11111111100000 Coryphopterus hyalinus 11111111100000 Coryphopterus lipernes 11111101100000 Coryphopterus eidolon 00 110000011000 Coryphopterus alloides 11110000001000 Coryphopterus thrix 00010110011000 Coryphopterus glaucofrenum 00010000010110 Coryphopterus dicrus 10 110000010111 Coryphopterus punctipectophorus 00010000010111 Coryphopterus urospilus 00010000010100 Coryphopterus nicholsii 00010000000000 Lophogobius cyprinoides 00010000000000 Fusigobius neophytus 00000000000000 Fusigobius signipinnis 11100000000000 Bathygobius curacao 00000000000000 Gobiodon histrio 00000000000000 Gnatholepis thompsoni 00000000000000

2. Posterior pelvic membrane. (0) present; (1) absent. The pelvic fins may also be joined posteriad with a membrane. If the fins are joined both anteriad and posteriad (state zero for both characters one and two), the pelvic disc configuration common among gobies results. The posterior membrane is absent (state one) in Coryphopterus personatus, C. hyalinus, C. lipernes, C. alloides, and F. signipinnis. The range of variation seen among Coryphopterus species in pelvic frena and membranes is shown in Böhlke and Robins, (1960: fig. 3). 3. Fifth (innermost) pelvic ray shortened relative to fourth ray. (0) ray not short; (1) ray short. In the species C. personatus, C. hyalinus, C. lipernes, C. alloides, C. eidolon, C. dicrus, and F. signipinnis the fifth pelvic ray is as little as half the length of the fourth pelvic ray. In C. dicrus and C. eidolon this results in a pelvic disc which is emarginate posteriad. In the other species the pelvic fins are completely separate. 4. Crest or ridge on head. (0) absent; (1) present. A thin, fleshy crest or ridge may be present, extending from just anteriad to the first dorsal spine to between the eyes. The crest may be very large, as in Lophogobius cyprinoides, or smaller, as in Coryphopterus species. No crest or ridge is present in Fusigobius species examined or in other outgroup taxa (Fig. 1). 5. Pigmented periproct. (0) absent; (1) present. In three species (C. personatus, C. hyalinus and C. lipernes), a prominent black ring of pigment is found around the anus (Fig. 1). In other species this ring is absent. 6. Filamentous second dorsal-fin spine in males. (0) absent; (1) present. An elongate second dorsal-fin spine, sometimes reaching as far as the posterior border of the second dorsal fin, is seen in males of C. personatus, C. hyalinus, C. lipernes and C. thrix. 7. Filamentous second dorsal-fin spine in females. (0) absent; (1) present. The condition described in character 5 is present in the females of C. personatus, C. hyalinus, and THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 843

Figure 1: Left lateral view of (A) Fusigobius neophytus (LACM 31617-25; 44.3 mm SL), (B) Lophogobius cyprinoides (LACM 5970; 36.1 mm SL), (C) Coryphopterus nicholsii (LACM 32149− 8; 57.7 mm SL), (D) C. dicrus (LACM 2549; 31.4 mm SL), (E) C. eidolon (LACM 54113−004; 41.6 mm SL), and (F) C. personatus (UMMZ 176549; 24.6 mm SL). Numbered arrows indicate characters as described in the Results section; only the derived character states are highlighted. Illustration prepared by Steven Melendrez. 844 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

C. thrix only (Fig. 1). The elongate dorsal-fin spines have been reported as being present in both sexes in C. eidolon but only in young specimens “questionably referred to this species” (Böhlke and Robins, 1960). None of the C. eidolon examined for this study, several of which were between 15 and 22 mm SL, showed evidence of the filamentous dorsal spines, and so characters 5 and 6 were coded as absent in C. eidolon. 8. Orange or gold coloration on body. (0) absent; (1) present. This is found in three species, C. personatus, C. hyalinus and C. lipernes. These species also have hovering or coral perching habits and reduced body size. 9. Black pigmented area on face, around eyes and slightly posteriad, giving a masked appearance. (0) absent; (1) present. Present in three species, C. personatus (Fig. 1), C. hyalinus and C. lipernes, the hover and peppermint gobies. 10. Three horizontal lines lateral on head, one terminating anteriad at the center of orbit, one just ventral orbit, and one more ventral to orbit on cheek. (0) absent; (1) present. The stripes are present in Coryphopterus glaucofrenum, C. dicrus (Fig. 1), C. urospilus, C. punctipectophorus, C. eidolon (Fig. 1), and C. thrix. 11. Caudal peduncle pigment: black horizontal bar edging the most posterior portion of the peduncle, at base of caudal fin rays. (0) absent; (1) present. The caudal peduncle bar is present in Coryphopterus thrix, C. alloides and C. eidolon (Fig. 1). This pigment blotch is a distinctly different configuration than that seen in Fusigobius species, which is trian- gular or oblong and oriented vertically in the center of the peduncle. 12. Caudal peduncle pigment: blotch at edge of fin rays, in lower half of peduncle. (0) absent; (1) present. The blotch is present in C. glaucofrenum, C. dicrus (Fig. 1), C. urospilus, and C. punctipectophorus. 13. Caudal peduncle pigment: blotch at edge of fin rays, in upper half of peduncle. (0) absent; (1) present. The blotch is present in C. glaucofrenum, C. dicrus (Fig. 1), and C. punctipectophorus. 14. Pigment spot at base of pectoral fin. (0) absent; (1) present. The spot is present in C. dicrus and C. punctipectophorus. Of the 531 base pairs of the L5878/H6313 fragment, 410 were parsimony-informative (sequences available in Genbank under accession numbers AF322978-82; AF322984- 98; and AF386101). All morphological characters were informative, for a total of 424 informative characters in the combined data set. Parsimony analysis of the combined molecular and morphological data set resulted in a single phylogenetic hypothesis with length 1695, consistency index 0.599, retention index 0.698 and rescaled consistency index of 0.419 (Fig. 2). If the data are analyzed without the two species for which molecular data is lacking (C. alloides and C. thrix), the topology that results is the same as for the combined analysis, but with C. alloides and C. thrix pruned out. This phylogeny agrees in many, but not all, respects with the only other phylogenetic hypothesis proposed for Coryphopterus, that of Smith and Tyler (1977) (Fig. 3), and the implications of our hypothesis for interpretation of their morphological characters are discussed below.

DISCUSSION

The results of our phylogenetic hypothesis indicate the presence of three lineages within Coryphopterus: a basal clade including C. eidolon and C. thrix, sister to a clade compris- ing C. urospilus, C. glaucofrenum, C. dicrus and C. punctipectophorus, and a second clade that includes C. alloides, C. lipernes, C. personatus and C. hyalinus (Figure 2). Our THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 845

Figure 2: Phylogeny of Coryphopterus species and outgroups, generated by parsimony analysis of both morphological (14 characters) and molecular (531 characters; 408 informative) data. This hypothesis is a single most parsimonious topology, and has a consistency index of 0.599, retention index of 0.698 and rescaled consistency index of 0.419. Numbers on the nodes are decay indices.

hypothesis differs in a few respects from that of Smith and Tyler (1977); they hypothesize a basal polytomy of the species C. glaucofrenum, C. punctipectophorus and the pair C. eidolon and C. thrix, species for which our hypothesis is resolved, and they hypothesize a different placement of C. dicrus (Figure 3). Our hypotheses agree on the relationships of C. alloides, C. lipernes, C. personatus, and C. hyalinus. The differences between our phylogenetic hypothesis and the hypothesis of Smith and Tyler indicate different evolu- tionary scenarios for their habitat, pelvic and dorsal fin characters. The most complex of the non-autapomorphic characters used by Smith and Tyler (1977) concerns the configuration of the pelvic fins. They hypothesize that the conformation of the pelvic fins found in C. glaucofrenum and C. punctipectophorus, the typical gobiid round fused disc, is primitive (this configuration is also seen in C. urospilus, a species which they did not examine). In all remaining Coryphopterus species, the pelvic fins form an emarginate disc or are separate; in either case the medial pelvic rays are shorter than the lateral ones (Smith and Tyler (1977) indicate that C. thrix has an emarginate disc, but the disc is actually complete, although the membranes are very delicate). Fur- ther specialization is seen in C. dicrus, C. alloides, C. lipernes, C. personatus and C. hyalinus; the frenum found between the pelvic spines in the anterior portion of the pel- 846 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

Figure 3: Previous hypothesis of Coryphopterus phylogeny, redrawn from Smith and Tyler (1977). Their hypothesis includes only the western Atlantic Coryphopterus species and does not address the question of Coryphopterus outgroups. The hypothesis is a graphical summary of 18 morphological characters, not the result of a phylogenetic analysis.

vic disc is absent. Finally, in C. alloides, C. lipernes, C. personatus and C. hyalinus the pelvic fins are compeletely separate, lacking both the anterior frenum and the posterior membrane between the pelvic fin rays. Thus, they hypothesize a gradual reduction in the pelvic disc, from the typical gobiid round condition, to an emarginate condition, to a condition in which the anterior frenum and posterior membrane are lost, resulting in separate pelvic fins. Our phylogeny does not indicate a single reductive trend in pelvic fin morphology. In the clade (C. urospilus (C. glaucofrenum (C. punctipectophorus, C. dicrus))), only C. dicrus has a reduced pelvic fin morphology, with emarginate fins and no anterior frenum. Sister to this clade is the clade (C. alloides (C. lipernes (C. personatus, C. hyalinus))), all with separate pelvic fins, lacking both the anterior frenum and posterior membrane. C. eidolon and C. thrix, with emarginate and normal pelvic fins respectively, are the primitive sister taxa to the rest of Coryphopterus. Thus, the emarginate condition is inferred to have arisen twice, separately in C. eidolon and C. dicrus, and C. dicrus has additionally lost the anterior pelvic frenum. Coryphopterus eidolon, C. dicrus, C. alloides, C. lipernes, C. personatus and C. hyalinus all share the presence of shortened fifth pelvic rays. THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 847

In both our and Smith and Tyler’s (1977) hypotheses, the relationships of the species C. alloides, C. lipernes, C. personatus and C. hyalinus concur. These species are united by reduction in the pelvic disc resulting in completely separate pelvic fins. Coryphopterus lipernes, C. personatus and C. hyalinus also share the presence of a pigmented ring around the anus, orange or gold coloration on the body, a black pigment mask on the face, and an elongate, filamentous second dorsal-fin spine (found in males of C. lipernes and both sexes of C. personatus and C. hyalinus). In Smith and Tyler’s (1977) hypothesis, the elongate second dorsal-fin spine character is independently derived in C. thrix and C. eidolon; our hypothesis also indicates that C. thrix and C. eidolon are sister taxa, but the elongate second dorsal fin spine is not present in the specimens of C. eidolon examined, and therefore is hypothesized to be independently derived only in C. thrix. Our analysis provides evidence for the resolution of relationships among C. glaucofrenum, C. punctipectophorus and C. dicrus that differs from Smith and Tyler (1977), and we additionally include the eastern Pacific C. urospilus. All four of these species are united by the presence of a pigment blotch on the lower portion of the caudal peduncle, at the base of the caudal-fin rays. Three of the species (C. glaucofrenum, C. dicrus and C. punctipectophorus) additionally share the presence of an upper pigment blotch, resulting in a paired spot on the caudal peduncle. Finally, C. dicrus and C. punctipectophorus share the presence of a blotch of pigment at the base of the pectoral fin. This condition differs from the pigmented blotch present in C. thrix in that the blotch in C. dicrus and C. punctipectophorus is present at the base of the pectoral fin, and the blotch in C. thrix is dorsal to the fin, on the shoulder. We also code a character of the lateral head pigment: the presence of three vertical lines, shared by C. glaucofrenum, C. dicrus, C. urospilus, C. punctipectophorus, C. eidolon, and C. thrix. The ecological characters used by Smith and Tyler (1977) are analogous to the pelvic fin characters in that they indicate a trend from primitive hyaline coloration and benthic, sand-dwelling habits (in C. glaucofrenum, C. thrix, C. eidolon and C. punctipectophorus), through rock-dwelling in C. dicrus and C. alloides, association with live corals in C. lipernes, and hovering in schools above corals in C. personatus and C. hyalinus. Our hypothesis concurs with theirs in that the hyaline, benthic sand-dwelling ecology is primitive, but differs in indicating that the shift to rock-dwelling in C. dicrus is independently derived from the shift to rock-dwelling in C. alloides, and from the association with live corals (C. lipernes) and hovering (C. personatus and C. hyalinus). Our analysis also indicates that Coryphopterus, as presently defined, is not monophyletic. The species C. nicholsii is sister to a clade containing L. cyprinoides as well as the remainder of Coryphopterus, and the two Fusigobius species are, separately, sister taxa to this clade along with B. curacao and G. histrio. As has been previously suggested (Böhlke and Robins, 1960; Randall, 1995), C. nicholsii is not part of Coryphopterus. In accordance with the phylogeny, we remove C. nicholsii from Coryphopterus, assign it to the previously used name Rhinogobiops nicholsii Hubbs, 1926, and recognize a restricted Coryphopterus excluding that species. The genus Rhinogobiops is diagnosed by a combi- nation of characters: six dorsal spines, the sixth distant from the fifth; seventeen segmented caudal rays; no teeth on vomer or palatine; jaw teeth in several rows, the inner and outer rows enlarged; second dorsal with 13–15 elements; anal fin with 12–13 elements; pelvic disc complete, with counts of I,5/I,5; enlarged neural and hemal spines on the vertebra preceding the hypural plate; a low fleshy ridge from dorsal fin origin to a point between and just behind the eyes; posterior margin of spinous dorsal fin black 848 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

(Hubbs, 1926; Böhlke and Robins, 1960). Characters used to diagnose Coryphopterus including C. nicholsii by Böhlke and Robins (1960) still diagnose Coryphopterus s.s., and no additional diagnostic characters for Coryphopterus were identified. Keys to the species of Coryphopterus are given in Böhlke and Robins (1960 and 1962). Additional conclusions of our analysis are that the synonomization of Coryphopterus with Fusigobius by Randall (1995) was premature. Our data indicate Lophogobius is more closely related to Coryphopterus (sensu stricto) than either of the Fusigobius species included in this analysis, a placement supported not only by the molecular data, but by the morphological character of a fleshy crest or ridge at the cranial midline that ex- tends anteriad from the origin of the first dorsal fin to near or between the eyes. This character is present in all Coryphopterus species, R. nicholsii, and L. cyprinoides, but in no Fusigobius species. An ancillary conclusion of our analysis is that Fusigobius is not monophyletic: F. s i gnipinnis and F. neophytus do not group together. However, we sus- pend judgement on the relationships of Fusigobius (except to say that it is not the sister taxon to Coryphopterus) until a revision of Fusigobius is completed and the many undescribed species described. The phylogenetic conclusions presented here also provide insight into the biogeographic history of these taxa. Coryphopterus s.s. is most closely related to the transisthmian Lophogobius, not to the Indo-Pacific Fusigobius. Within Coryphopterus, the only eastern Pacific species (C. urospilus) is basal in its clade, but not the most basal taxon. The species pair C. eidolon and C. thrix form the most basal lineage of Coryphopterus; C. eidolon is widespread throughout the Greater and Lesser Antilles, whereas C. thrix is known from the Bahamas and the Gulf of Honduras (Böhlke and Robins, 1960; Greenfield and Johnson, 1999). Although a complete analysis of the biogeography of these taxa is beyond the scope of this paper, the phylogeny indicates that the ancestor of C. eidolon and C. thrix diverged from the ancestors of two other clades within Coryphopterus early in the history of the group. We surmise that this most likely occurred prior to the closure of the isthmus of Panama. The two clades began to diversify, perhaps including the spe- ciation of C. urospilus, followed by closure of the isthmus and isolation of C. urospilus in the eastern Pacific. The lineages of C. eidolon/C. thrix, C. alloides/C. lipernes/C. hyalinus/ C. personatus, and C. glaucofrenum/C. punctipectophorus/C. dicrus then diversified separately but in some cases sympatrically in the Caribbean and Western Atlantic. Species of Lophogobius are also distributed on both sides of the Isthmus of Panama, and Rhinogobiops nicholsii is found only in the eastern Pacific. This larger group of three genera, including Coryphopterus, Lophogobius and Rhinogobiops, all share the presence of a crest or fleshy ridge on the head, and appear to have diversified together in the transisthmian seaway area, before, during and after the closure of the Isthmus of Panama.

ACKNOWLEDGMENTS

We are grateful to M. Westneat, J. Williams, R. Reavis, C. St. Mary and K. Crow for providing tissue samples; M. McGrouther, S. Jewett and D. Nelson for the loan of specimens; and to J. Ast for invaluable assistance with PCR troubleshooting. D. Hoese aided greatly in the interpretation of characters of Fusigobius and Lophogobius. S. Melendrez prepared Figure 1. D. Mindell hosted part of this work in his lab at the University of Michigan Museum of Zoology, and part was performed at the Molecular Systematics Lab at the Natural History Museum, made possible by the support of THACKER AND COLE: CORYPHOPTERUS PHYLOGENY 849 the W. M. Keck Foundation. This is contribution number 2 of the W. M. Keck Foundation Program in Molecular Systematics and Evolution at the Natural History Museum of Los Angeles County.

LITERATURE CITED

Birdsong, R. S., E. O. Murdy and F. L. Pezold. 1988. A study of the vertebral column and median fin osteology in gobioid fishes with comments on gobioid relationships. Bull. Mar. Sci. 42: 174–214. Böhlke, J. E. and C. R. Robins. 1960. A revision of the gobioid fish genus Coryphopterus. Proc. Acad. Nat’l. Sci. Phila. 112(5): 103–128. ______and ______. 1962. The taxonomic position of the west Atlantic goby Eviota personata, with descriptions of two new related species. Proc. Acad. Nat’l. Sci. Phila. 114(5): 175–189. Breder, C. M. 1932. An annotated list of fishes from Lake Forsyth, Andros Island, Bahamas, with the descriptions of three new forms. Amer. Mus. Novit. 551: 1–8. Bremer, K. 1994. Branch support and tree stability. Cladistics 10: 295–304. Cervigón, F. 1966. Los peces marinos de Venezuela (2 vols.). Estacion de. Investigaciones Marihas de Margarita, Caracas. Chen, I.-S., and K.-T. Shao. 1997. Fusigobius aureus, a new species of gobiid fish (Perciformes, Gobiidae) from Flores Island, Indonesia. Acta Zool. Taiwanica 8(2): 87–92. Cole, K. S. 1988. Patterns of gonad structure in hermaphroditic gobies (Teleostei: Gobiidae). Env. Biol. Fish. 28: 125–142. Garzon-Ferreira, J. and A. Acero. 1990. Redescription of Coryphopterus tortugae (Jordan) (Osteichthyes: Gobiidae), a valid species of goby from the western Atlantic. Northeast Gulf Sci. 11(2): 105–112. Ginsburg, I. 1939. Twenty one new American gobies. J. Wash. Acad. Sci. 29(2): 51–63. Greenfield, D. W. and R. K. Johnson. 1999. Assemblage structure and habitat associations of western Caribbean gobies (Teleostei: Gobiidae). Copeia 1999: 251–266. Harrison, I. J. 1989. Specialization of the gobioid palatopterygoquadrate complex and its relevance to gobioid systematics. J. Nat. Hist. 23: 325–353. Hoese, D. F. and S. Reader. 1985. A new gobiid fish, Fusigobius duospilus, from the tropical Indo- Pacific. J. L. B. Smith Inst. Ichthyol. Spec. Pub. 36: 1–9. ______and Y. Obika. 1988. A new gobiid fish, Fusigobius signipinnis, from the Western Tropical Pacific. Japn. J. Ichthyol. 35(3): 282–288. Leviton, A. E., R. H. Gibbs, Jr., E. Heal and C. E. Dawson. 1985. Standards in ichthyology and herpetology: Part I. Standard symbolic codes for institutional resource collections in herpetology and ichthyology. Copeia 1985: 802–832. Maddison, W. P. and D. R. Maddison. 1992. MacClade: analysis of parsimony and character evolution. Ver. 3.01. Sinauer Assoc., Inc., Sunderland, Massachusetts. Pezold, F. 1993. Evidence for a monophyletic Gobiinae. Copeia 1993: 634–643. Pothoff, T. 1984. Clearing and staining techniques. Pages 35–37 in H. G. Moser, ed. Ontogeny and systematics of fishes. Spec. Pub. Amer. Soc. Ichthy. Herp. No. 1. Allen Press, Lawrence, Kan- sas. Randall, J. E. 1995. Fusigobius Whitley, a junior synonym of the gobiid fish genus Coryphopterus Gill. Bull. Mar. Sci. 56: 795–798. Smith, C. L. 1997. National Audobon Society field guide to tropical marine fishes of the Caribbean, Gulf of Mexico, Florida, the Bahamas and Bermuda. Alfred A. Knopf, New York. ______and J. C. Tyler. 1977. Redescription of the gobiid fish Coryphopterus lipernes Böhlke and Robins, with notes on its habits and relationships. Amer. Mus. Novit. 2616: 1–10. Sorenson, M. D. 1996. Sequence Compiler. Univ. Michigan, Ann Arbor. Supplied by author: [email protected]. 850 BULLETIN OF MARINE SCIENCE, VOL. 70, NO. 3, 2002

______. 1999. TreeRot, ver. 2. Boston Univ., Boston, Massachusetts. ______, J. C. Ast, D. E. Dimcheff, T. Yuri, and D. P. Mindell. 1999. Primers for a PCR- based approach to mitochondrial genome sequencing in birds and other vertebrates. Mole. Phylogen. Evol. 12: 105–114. Takagi, K. 1989. Cephalic sensory canal system of the gobioid fishes of Japan: comparative morphology with special reference to phylogenetic significance. J. Tokyo Univ. Fish. 75(2): 499– 568. Weitzman, S. H. 1974. Osteology and relationships of the Sternoptychidae, with a new classifica- tion of the stomiatoid fishes. Bull. Am. Mus. Nat. Hist. 153: 329–478.

ADDRESSES: (C.E.T.) Section of Vertebrates - Ichthyology, Natural History Museum of Los Angeles County, Los Angeles, California 90007; (K.S.C.) Department of Biology, University of Louisiana at Lafayette, P.O. Box 42451, Lafayette Louisiana 70504.

APPENDIX

MATERIAL EXAMINED.—Institutional acronyms follow Leviton et al. (1985). Taxa are listed alphabetically. The number of specimens in each lot is indicated in parentheses. An asterix after a lot number indicates the lot was cleared and stained, and if only part of a lot was cleared and stained, the number following the asterix indicates the number of cleared and stained specimens. An −‘x’ following a lot number indicates the lot was x-rayed.

Bathygobius curacao: LACM 1447(2). Bathygobius lineatus: LACM 43609−27(107)*−3. Coryphopterus alloides: USNM 267843(1)x. Coryphopterus dicrus: LACM 2480(9); LACM 2549(31)*−4; LACM 5977(3); LACM 6488(1); LACM 6489(2); LACM 6704−6(2); LACM 8942−8(1); LACM 36220(1). Coryphopterus eidolon: LACM 6727−10(1); LACM 7715(4); LACM 7756(6)*−3; LACM 8938− 30(3); LACM 8940−20(2); LACM 8941−25(5); LACM 54098.013(2); LACM 54113.004(4); USNM 317144(3); USNM 318815(4). Coryphopterus glaucofrenum: LACM 2250(4); LACM 5975(1); LACM 6837−13(25); LACM 7765(23)*−3; LACM 8938−29(1); LACM 8941−26(1); LACM 22153(2); LACM 22154(1); LACM 22155(1); LACM 22156(3); LACM 22157(3); LACM31573−13(3). Coryphopterus hyalinus: LACM 6727−17(4)x. Coryphopterus lipernes: LACM 54092.002(4)*−1; UMMZ 179634(1). Coryphopterus nicholsii: LACM 23117(10); LACM 31210−1(11); LACM 31223−1(9); LACM 31224−1(11); LACM 31228−1(19); LACM 32149−8(129)*−4. Coryphopterus personatus: LACM 6727−16(1); LACM 7828(5); LACM 8938-22(1); LACM 8939− 21(1); LACM 8940−14(2); LACM 8941−13(1); UMMZ 176549(6)*−2. Coryphopterus punctipectophorus: USNM 315530(1)x. Coryphopterus thrix: LACM 8939-33(1); USNM 317130(14)x; USNM 31731(8). Coryphopterus urospilus: LACM 6966−14(17); LACM 6973−11(79)*−4; LACM 31768-21(7); LACM 31774−37(12); LACM 31775−36(18); LACM 31781−14(20); LACM 32574(92). Fusigobius neophytus: LACM 31617−25(3); LACM 32819−2(1); LACM 33723-29(1); LACM 39986-7(1); LACM 54117.001(4)*−2; LACM 54118.004(3); LACM 54122.001(1); LACM 54125.001(1). Gnatholepis thompsoni: LACM 20636(2)*−1; LACM 54098.016(1); LACM 54113.005(6). Gobiodon citrinus: LACM 37393−8(3); LACM 37420−13(1); LACM 42491−69(5)*−2. Gobiodon histrio: LACM 42485−49(3); LACM 42456−70(1); LACM 42464−35(5). Lophogobius bleekeri: AMS 21258001(18); AMS 22055003(38). Lophogobius cyprinoides: LACM 1451(3); LACM 5970(20); LACM 7847(85)*−3.

Phylogeny and Evolution of the Gobiid Genus &lt;I&gt;Coryphopterus&lt;/I&gt;

Phylogeny and Evolution of the Gobiid Genus <I>Coryphopterus</I>