Description of the First Species of Polylepion (Teleostei: Labridae) from the Atlantic Ocean with Analysis of Evolutionary Relationships of the New Species

Submersible diving in the 1980/90s in the Bahamas and Cuba and 2013–2018 at Curaçao, Dominica, and Roatan resulted in the collection of a new species of Polylepion, a genus of wrasse previously known only from the Indian and Pacific Oceans. The new species, which inhabits the rariphotic reef-fish faunal zone at depths of 219–457 m, is another example of a deep-reef species belonging to a largely shallow-reef family, in this case the family Labridae. Here, we describe the new species and provide a phylogenetic placement for it by adding new sequence data from 12 genetic markers for the new species and one of its two congeners (P. cruentum from the eastern Pacific Ocean) to a previously published dataset comprising 336 species of wrasses that includes the other congener, P. russelli, from the Indian and West/Central Pacific Oceans. Our results resolve the phylogenetic history of the species of Polylepion and provide the first molecular support for the monophyly of the genus.

T HE marine-fish family Labridae comprises over 600 species in 82 genera displaying a broad array of anatomical and ecological adaptations across tropical and subtropical waters (e.g., Sanderson, 1990;Parenti and Randall, 2000;Tatom-Naecker and Westneat, 2018;Ghezelayagh et al., 2022). Although most wrasses inhabit shallow waters, a few species are limited to depths of~100 to 400 m depth, including those in Polylepion Gomon 1977. This genus is currently known from P. cruentum Gomon 1977 in the tropical eastern Pacific Ocean (Gomon, 1995;Britzke et al., 2021) and P. russelli (Gomon and Randall, 1975) in the Central Pacific (Gomon and Randall, 1975), West Pacific (Masuda et al., 1984;Hubert et al., 2011;GBIF, 2022;Rob Meyers, pers. comm.), and Indian (Béarez et al., 2013;Hobbs et al., 2014) Oceans. While exploring the fish fauna of deep reefs at Curaçao in 2013-2016 through human-occupied submersible diving as part of the Smithsonian Institution's Deep Reef Observation Project (DROP), several specimens of Polylepion were collected at depths of 219-272 m using the Curasub. The genus is easily distinguished from closely related wrasse genera by numbers of dorsal-and anal-fin rays (Gomon, 1977(Gomon, , 1997. Subsequent correspondence with Grant Gilmore, formerly of the Harbor Branch Oceanographic Institute, revealed that he and colleagues had previously collected the same species decades earlier to depths of 367 m in the Bahamas and Cuba aboard the Johnson Sea Link-I (JSL-I) submersible and that he had made precise color-pencil sketches of juveniles and adults of the species. Further DROP diving in Dominica (Curasub) in 2016 and Roatan Island (Idabel) in 2017-2018 resulted in the collection of additional specimens at depths of~300 m. ROV imagery of the Atlantic Polylepion was captured off the Florida Keys in 2011 and Puerto Rico in 2013 (see ''Distribution and Habitat'' below).
In his description of Polylepion, Gomon (1977) noted that, morphologically, the genus closely resembles Decodon, Pimelometopon, Semicossyphus, and Bodianus, the last a historically catch-all wrasse genus to which P. russelli was originally assigned (Gomon and Randall, 1975) and to which Semicossyphus (including Pimelometopon based on Gomon [1997]) was recently provisionally relegated (Santini et al., 2016). Molecular phylogenies of the Labridae revealed a sister-group relationship between Polylepion and Decodon (Aiello et al., 2017;Hughes et al., 2022;Larouche et al., 2022) and a paraphyletic Bodianus without the inclusion of Clepticus, Decodon, Polylepion, and Semicossyphus (Aiello et al., 2017) or without the inclusion of Clepticus and Semicossyphus (Hughes et al., 2022;Larouche et al., 2022). By combining the recently published time-calibrated phylogenomic tree for the Labridae (Hughes et al., 2022) with genetic sequences for over 300 species of wrasses (Aiello et al., 2017), we investigated the evolutionary history and timing of speciation events of the genus Polylepion, incorporating new genetic data for P. cruentum and the new species. Here we describe the new Polylepion as the first species of the genus in the Atlantic and report the results of the phylogenetic analysis.

MATERIALS AND METHODS
Specimen collection.-Specimens of the new species were collected from Caribbean and Bahamian deep reefs using human-occupied submersibles in Curaçao and Dominica (Curasub), Honduras (Idabel), and Bahamas (Johnson Sea Link-I-JSL-I). Both the Curasub and Idabel deployed a quinaldine fish-anesthetic solution to immobilize fishes prior to suction into a hose that transfers specimens into a collection bucket. Technology for collecting with the JSL-I was similar except that the fish toxicant rotenone was used. Most Caribbean specimens were photographed at the surface, and some in situ video was recorded at Curaçao and Roatan. Caribbean specimens were tissue sampled prior to fixation in formalin and then subsequently transferred to 75% ethanol. Bahamian specimens were collected prior to the onset of routine tissue sampling in the field and are not genetic vouchers; however, Richard G. Gilmore, Jr. made detailed color-pencil sketches of some specimens prior to preservation. Specimens were accessioned in fish collections at the Scripps Institution of Oceanography (SIO), Smithsonian Institution's National Museum of Natural History (USNM), University of Florida (UF), University of Washington (UW), and Museo Historia Natural at Universidad Nacional Autonoma de Honduras en el Valle de Sula (MUVS). Collecting at Curaçao, Dominica, and Roatan was done under ACUC permits 2014-13 and 2017-11 to Baldwin.
Morphological character examination.-Counts and measurements follow Gomon and Randall (1978). Most specimens were X-rayed with a digital radiography system at USNM. Molecular data and phylogenetic analyses.-Taxonomic sampling for genetic analyses comprised 336 species of wrasses from the most recent published dataset of the family Labridae (Aiello et al., 2017), including newly collected sequence data from P. cruentum and the new species (USNM 435298). For P. cruentum, we sequenced samples obtained from the Marine Vertebrate Collections at Scripps (SIO 10-18 and SIO 09-377). We follow Fricke et al. (2023, and references therein) in recognizing species of Clepticus and Semicossyphus as members of Bodianus.
DNA was extracted using the DNeasy tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Double-stranded DNA was synthesized via PCR using the same set of primers and PCR conditions described by Aiello et al. (2017) and Westneat and Alfaro (2005) for 12 genetic markers (12S, 16S, S7, CO1, CYTB, BMP4, DLX2, OTX1, RAG1, RAG2, TMO4C4, RHOD). The PCR products were electrophoresed in 1.5% low melting point agarose gels using a tris-acetate buffer for quality control and submitted for purification and sequencing in both directions at the Sequencing and Genomic Facilities of the University of Puerto Rico. Sequences were assembled, visualized, inspected, and concatenated in Geneious v10.0.9. All exon markers were aligned individually based on their underlying reading frame using the MAFFT aligner (Katoh and Standley, 2013). The 16S and 12S sequences were aligned with SATé using MAFFT and 25 iterations (Liu et al., 2009). The molecular dataset consisted of 6,209 sites for 336 species. All sequences produced for this study have been deposited in NCBI (Supplemental Table S1; see Data Accessibility).
Phylogenetic relationships among maximum likelihood (ML) searches were conducted in RAxML v8.2.10 using the GTRGAMMA model (Stamatakis, 2014). All gene fragments were partitioned by codon position and concatenated in a single super matrix. A total of 30 tree replicates were conducted to obtain the best-scoring tree across the different searches for the concatenated dataset and each gene fragment. Branch support was estimated using the rapid bootstrapping algorithm with 1,000 replicates under the GTRGAMMA model; the collection of bootstrapped trees was used to draw bipartition frequencies onto the optimal tree. Species-trees analyses were conducted using ASTRAL-2 (Mirarab and Warnow, 2015). This method addresses gene tree incongruence due to incomplete lineage sorting using unrooted gene trees as input to maximize the number of quartet trees shared between the gene tree and the species trees.
Divergence time estimation analyses used a ''congruification'' approach (Eastman et al., 2013) that matches compatible nodes between a reference and target phylogenetic tree. The reference tree consisted of the genome-wide timecalibrated tree of the family Labridae (Hughes et al., 2022) based on seven fossil calibrations, †Phyllopharyngodon longipinnis, †Trigonodon jugleri, †Tautoga sp., †Symphodus westneati, †Calotomus preisli, †Bolbometopon sp., and †Coris sigismundi. The target tree consisted of the Labridae tree based on nine genetic markers and 336 species (Aiello et al., 2017), including the newly generated data for P. cruentum and the new species of Polylepion. To interpole the dates for the same nodes as the reference in the target tree, an ML tree was estimated using a backbone constraint approach. Based on the tree assembled using nine genetic markers, 158 of the 336 species were placed on the backbone tree based on 1,105 exon markers (Hughes et al., 2022). The remaining 178 species were placed in the phylogenomic backbone tree using a constraint ML search in RAxML and by codon partitions. The resulting ML tree was calibrated in TreePL (Smith and O'Meara, 2012) using the secondary calibrations extracted from the reference backbone phylogeny and the function ''congruify'' implemented in the R package geiger (Harmon et al., 2008).  Tables 1-2, Supplemental Table T1 Polylepion species A, Curaçao, Robertson and Van Tassell (2016), https://biogeodb.stri.si.edu/caribbean/en/gallery/ genus/5244. Diagnosis.-A revised diagnosis of Polylepion is presented after the species description. Polylepion gilmorei, the only species of Polylepion in the Atlantic Ocean, is distinguished from other wrasses by the following combination of characters: dorsalfin rays usually XI, 11; anal-fin rays usually III, 11; pectoralfin rays usually ii17; lateral-line complete, 38-44 tubed scales; scales above lateral line 2-4; scales not extending onto bases of dorsal and anal fins; scales on forehead extending anteriorly beyond or nearly to level of anterior nostrils; in life, multiple orange-red bars of pigment on dorsal portion of the trunk against an otherwise whitish background, and a red (adults) or black (juveniles) circular blotch on dorsal portion of caudal-fin base; inhabiting depths of 219-457 m.
Body deepest around midbody, tapering posteriorly. Head pointed, profile mostly straight except for convexity between orbits. Scales on trunk not extending onto dorsal-and analfin bases. Lateral line continuous, smoothly curved. Head mostly scaled except snout, lips, gular region, and branchiostegals. Scales extending anteriorly on forehead in advance of or nearly to anterior nostrils.
Posterior end of maxilla reaching vertical through anterior edge of orbit. Upper and lower jaws each with two pairs of large canines anteriorly. Anterior canines in upper jaw of similar size, followed by a row of much smaller canines (7 in holotype) and one moderately large canine at rear corner of jaw. Medial canines of large anterior pair in lower jaw smaller than more lateral ones.
Dorsal fin continuous, originating just anterior to vertical through base of pectoral fin. Membrane between dorsal-and anal-fin spines deeply incised, membrane forming small flags of tissue at tips of spines. Dorsal and anal fins slightly pointed posteriorly, longest rays not reaching margin of hypural plate in adults. Posterior margin of caudal fin somewhat rounded in juveniles, more truncate in adults. Posterior extent of pelvic fin not reaching anus.
Adults pale in alcohol with no dark markings on head, trunk, or fins (Fig. 1A). Juveniles similar except with circular blotch of melanophores on dorsal base of caudal fin. In addition to that blotch, smallest juvenile (27.9 mm SL) with six indistinct black bars on trunk, a dark stripe extending from snout to operculum, some dark pigment on pectoral-fin base, and dark spots present on lower caudal-fin base. A 35.7 mm juvenile with remnants of these dark markings.
In life (Figs. 1B, 2C), adults with six orange-red bars of pigment on dorsal portion of trunk, anteriormost bars terminating ventrally slightly below midbody, last bar extending from dorsal to ventral margins of caudal peduncle, bar much paler ventrally. Large red circular blotch on dorsal base of caudal fin, irregular and paler red bar below this blotch. Upper half of head red, eye mostly yellow. Indistinct stripe of red pigment extending along center of entire dorsal fin, stripe narrow on spinous dorsal fin, usually broader on soft dorsal fin. Uppermost and lowermost caudal-fin rays red, forming the margins of a broad, indistinct bar of red pigment on central area of caudal fin. Oblong blotch or streak of red pigment on pectoral-fin base. Gilmore's color-pencil sketch of the holotype (Fig. 1B) shows a broad red stripe centrally on   Table 1. Counts and differences in head squamation of species of Polylepion. Data for P. russelli are from Gomon and Randall (1978) and Béarez et al. (2013), for P. cruentum from Gomon (1977)  soft portion of anal fin, but this pigment not apparent in photographs of freshly caught specimens, in which the extent of red pigmentation also varies on the dorsal fin (Fig.  2). Juveniles ( Fig. 2A, B) with the six orange-red trunk bars extending further ventrally than in adults, bars containing some melanophores mixed in with the red chromatophores in smallest juveniles. Spot on upper base of caudal fin relatively larger than in adults, black or a black/red mix and broadly rimmed with iridescent white. Head pigment similar to that of adults except red on upper portion of head pale, setting off a distinctive narrow red stripe running from tips of jaws through center of eye to upper rear corner of operculum. Other fin pigment similar to that of adults.   Tables 1 and 2. Currently the only known species of the genus in the Atlantic Ocean, P. gilmorei differs from P. cruentum in having fewer anal-fin rays (III, 10-11, almost always 11, vs. III, 12-13, usually 12 in P. cruentum). It differs from P. russelli in having fewer pectoralfin rays (ii, 16-17, usually 17, vs. ii, 18-19 in P. russelli) and fewer scales above the lateral line (2-4 vs. 5 in P. russelli). Polylepion gilmorei differs from both congeners in having fewer lateral-line scales (38-44 vs. 48-52 in P. russelli and 49-53 in P. cruentum) and scales below the lateral line (9-12 vs. 17-18 in P. russelli and 16-18 in P. cruentum). The new species further differs from its congeners in color pattern, especially in having multiple red bars of pigment on the dorsal portion of the trunk against an otherwise whitish background (vs. color pattern of trunk variable in P. cruentum and P. russelli, usually with at least some yellow stripes on the trunk against a pinkish background in small specimens, never with distinct red bars). The largest specimen of P. gilmorei is 114 mm SL, whereas that of P. cruentum is 199 mm SL and P. russelli 313 mm SL, which could indicate that P. gilmorei is a smaller species than its congeners.
Relationships.- (Fig. 3) Early morphological studies noted the resemblance among Bodianus, Decodon, and Polylepion and suggested a close relationship between species of Polylepion and Decodon (Gomon, 1997). The results of our phylogenomic analysis are congruent with those of Aiello et al. (2017), Hughes et al. (2022), and Larouche et al. (2022) in strongly supporting a sister-group relationship between the deep-reef genera Polylepion and Decodon (bootstrap support ¼ 97%) and with those studies and Santini et al. (2016) in Table 2. Morphometric data for type specimens of species of Polylepion. Standard length is in mm, other measurements in percentages of standard length. Data for P. russelli are from Gomon and Randall (1978), for P. cruentum from Gomon (1977). Measurements for P. gilmorei were taken to the nearest tenth of a millimeter, and percentages of standard length are represented by appropriate number of significant digits. 13.1 12.1-13.2 9.5 7.6-12.3 10 6.4-11 Orbit diameter 6.5 6.5-7.2 10.7 8.0-12.0 11 9.1-12 Bony interorbital width 6.3 6.3-8.1 5.6 4.5-6.0 9.1 7.9-12 Upper jaw length 11.2 11.2-11.9 11.1 9.7-14.2 9.9 7. revealing that Bodianus includes species formerly classified in the genera Clepticus and Semicossyphus (Supplemental Fig. S2; see Data Accessibility). Unlike the phylogeny of Aiello et al. (2017), in which Decodon and Polylepion were embedded in the Bodianus clade, our results agree with those of Hughes et al. (2022) and Larouche et al. (2022) in resolving Decodon and Polylepion as distinct from Bodianus. Further taxonomic study of Bodianus is needed to resolve the species composition of that genus. Our results provide the first molecular evidence for the monophyly of the genus Polylepion (bootstrap support ¼ 100%) and support the recognition of P. gilmorei as a distinct species. The phylogeny further suggests that the eastern Pacific P. cruentum is sister to the clade comprising the western Atlantic P. gilmorei and Indo-Central-West Pacific P. russelli.
Biogeography.-One possible biogeographic scenario for the current distribution of Polylepion based on the time-calibrated phylogeny (Fig. 3) involves an initial split of the eastern Pacific population approximately 10 mya, with westward migration that ultimately isolated P. russelli. That occurred at about the same time as the separation of the other Neotropical genus of deep-reef wrasses, Decodon, into eastern Pacific and western Atlantic populations, suggesting a linkage of these events in both genera to shoaling of the central American isthmus prior to its final closure~2 mya (Lessios, 2008;O'Dea et al., 2016). Since P. russelli is known as far west as Réunion Island in the southwest Indian Ocean, further westward migration around the tip of South Africa approximately 6 mya could have led to the establishment of an isolated Atlantic population. Colonization from the Indian Ocean directly into the western Atlantic has been hypothesized for some shallow-reef fishes, including Centropyge (Bowen et al., 2006, and references therein), which, like Polylepion, is not known from the tropical eastern Atlantic. Gordon (2003) noted that dispersal directly from the Indian Ocean into the western Atlantic could occur via the surfaceflowing South Equatorial Current, which runs below the equator from the eastern Atlantic to Brazil. However, given that the global tropical deep-reef fish fauna is poorly known, additional species of Polylepion may be discovered, and other biogeographic scenarios are possible.
Revised diagnosis of Polylepion.- Gomon (1977) proposed the generic name Polylepion (''many small scales'') in reference to large number of lateral-line scales (48-53) relative to those in the closely related genera Bodianus (30-48), Decodon (26-30), and Semicossyphus (42-45), although he noted that Pimelometopon has 53-57 (Gomon, 1977: table 1). Polylepion gilmorei has fewer lateral-line scales (38-44) than its congeners, a count that overlaps those of Bodianus and ''Semicossyphus.'' Discovery of the new Atlantic species necessitates minor changes in the diagnosis of Polylepion, and the new diagnosis follows with Gomon's (1977) diagnostic characters given in parentheses if different: dorsal-fin rays XI, 10-12, usually 11 (XI, 11); anal-fin rays III, [10][11][12][13]usually 11 or 12 (III,[11][12]; pectoral-fin rays ii, 16-19, usually 17 or 18 (ii, 17-19); lateral-line scales 38-53 (48-52); scales above lateral line 2-5 (2 1 / 2 -5); scales below lateral line 9-18 (16-19, usually 17 or 18); predorsal scales approximately 20-32 (25-32); scales on forehead reaching forward to a vertical between anterior edge of orbit and one anterior to anterior nostrils; color in alcohol pale with dusky spot on dorsal base of caudal fin in juveniles. Since neither P. gilmorei nor P. russelli has black interspinal membranes in the anterior portion of the dorsal fin, that character in Gomon's (1977) diagnosis is removed from the revised diagnosis. Baldwin et al. (2018) presented a classification of reef-fish faunal zones at Curaçao that comprises the altiphotic (0 tõ 40 m), mesophotic (~40 to~130 m), and rariphotic (~130 to at least 300 m). The new species joins a growing list of new Caribbean deep-reef fish species discovered at mesophotic and rariphotic depths and collected with the aid of robotic arms and fish anesthetics using human-occupied submersibles (Robertson et al., 2022). Including the new species of Polylepion, exploration of the Caribbean deep-reef fish fauna by the Smithsonian's Deep Reef Observation Project (DROP) has resulted in the description of one new genus and more than 25 new species, with additional new species yet to be described (e.g., Tornabene and Baldwin, 2019, and references therein;McFarland et al., 2020;Tornabene et al., 2023). While some of the new species inhabit mesophotic depths, P. gilmorei and several new species in the genera Birdsongichthys, Haptoclinus, Liopropoma, Lipogramma, Robinsichthys, and Varicus live in the rariphotic, below the mesophotic but above the deep aphotic zone (Baldwin et al., 2018;Tornabene et al., 2023). While the rariphotic reef-fish assemblage is taxonomically distinct from shallower faunas, the two faunas share strong evolutionary affinities, as most rariphotic species belong to reef-fish families inhabiting altiphotic and mesophotic reefs (Baldwin et al., 2018). At Curaçao, the seabass family Serranidae, with eight rariphotic species, Gobiidae (6), Grammatidae (4), and Labridae (3, including the new species of Polylepion) are the most common families of typical reef fishes with species that primarily inhabit the rariphotic zone (Baldwin et al., 2018;Tornabene et al., 2023). Tornabene et al. (2016Tornabene et al. ( , 2023 used time-calibrated phylogenies of some members of the Gobiidae to hypothesize multiple transitions from shallow-to deep-reef habitats, all of which occurred approximately 10-30 mya. Our time-calibrated phylogeny of the Labridae (Fig. 3) suggests that by 30 mya, a split had occurred between the deep-reef clade comprising Decodon þ Polylepion and the shallower-reef clade comprising Bodianus þ Clepticus þ Semicossyphus, with Decodon and Polylepion diverging slightly more than 20 mya. Future exploration of tropical deep reefs is likely to reveal additional new fish species and possibly new genera, which may alter our phylogenetic and biogeographic hypotheses. For now, the discovery of the new Polylepion provides another example of the value of continued exploratory expeditions to enhance our globally poor knowledge of tropical deep reefs.

DATA ACCESSIBILITY
Supplemental material is available at https://www. ichthyologyandherpetology.org/i2022075. Unless an alternative copyright or statement noting that a figure is reprinted from a previous source is noted in a figure caption, the published images and illustrations in this article are licensed by the American Society of Ichthyologists and Herpetologists for use if the use includes a citation to the original source (American Society of Ichthyologists and Herpetologists, the DOI of the Ichthyology & Herpetology article, and any individual image credits listed in the figure caption) in accordance with the Creative Commons Attribution CC BY License. ZooBank publication urn:lsid:zoobank.org:pub: 1FA0F7E4-3DF9-41EF-99B9-4BADF08E1AD5.

ACKNOWLEDGMENTS
We thank G. Gilmore for valuable specimens, data, artwork, and discussion related to the new species. B. Brandt, B. Brown, T. Christiaan, L. Schenk, A. ''Dutch'' Schrier, K. Stanley, T. Trudel, and B. van Bebber assisted in various ways with submersible operations; C. Castillo and T. Devine provided project management and DNA technician services; R. Manning provided assistance in the field; M. Westneat provided the original gene alignments published by Aiello et al. (2017); L. Hughes provided the time-calibrated phylogenomic tree of the Labridae; D. Pitassy and K. Maslenikov provided collection-management assistance; S. Raredon provided photographs and radiographs of type material; A. David (NOAA) provided the image in Supplemental Fig. 1 and information on depth and habitat of the fish in that image, and J. Reed was chief scientist on the NOAA expedition that secured the image; C. Lardizibal at MUVS helped facilitate collecting in Roatan and assisted with cataloging specimens. Funding for the Smithsonian Institution's Deep Reef Observation Project was provided by the Smithsonian's Consortium for Understanding and Sustaining a Biodiverse Planet to CCB, the Smithsonian's Competitive Grants for the Promotion of Science program to CCB and DRR, the Herbert R. and Evelyn Axelrod Endowment Fund for systematic ichthyology to CCB, the Prince Albert II of Monaco Foundation's grant and gift to CCB, STRI funds to DRR, and University of Washington School of Aquatic and Fishery Sciences funds to LT. Aspects of the work by CCB and DA were completed under National Science Foundation grants DEB-1541552, DEB-2015404, and DEB-2144325. This is Ocean Heritage Foundation/Curaçao Sea Aquarium/Substation Curaçao (OHF/SCA/SC) contribution number 52.