More new deep-reef basslets (Teleostei, Grammatidae, Lipogramma), with updates on the eco-evolutionary relationships within the genus

Abstract Two new Lipogramma basslets are described, L. barrettorum and L. schrieri, captured during submersible diving to 300 m depth off Curaçao, southern Caribbean. Superficially resembling L. robinsi in having 11–12 bars of pigment on the trunk, L. barrettorum is distinct from L. robinsi in having a stripe of blue-white pigment along the dorsal midline of the head (vs. a cap of yellow pigment), in patterns of pigment on the median fins, and in having 8–10 gill rakers on the lower limb of the first arch (vs. 11–12). Lipogramma schrieri is distinct from all congeners in having seven or eight dark bars of pigment on the trunk and broad, irregular, whitish blue markings on the dorsal portion of the head. The new species are genetically distinct from one another and from seven other Lipogramma species for which genetic data are available. A phylogenetic hypothesis derived from mitochondrial and nuclear genes suggests that the new species belong to a clade that also comprises L. evides and L. haberi. Collectively those four species are the deepest-living members of the genus, occurring at depths predominantly below 140 m. This study thus provides further evidence of eco-evolutionary correlations between depth and phylogeny in Caribbean reef fishes. Tropical deep reefs are globally underexplored ecosystems, and further investigation of Caribbean deep reefs undoubtedly will provide samples of species for which no genetic material currently exists and reveal more cryptic species diversity in the genus.


Introduction
described two new species of western Atlantic Lipogramma basslets collected during diving to 300 m by the Curasub submersible, as part of the ongoing Deep Reef Observation Project (DROP) in the southern Caribbean. That brought the total number of species in the genus to 10. However, they noted that their deep-reef collections included two additional putative new species that superficially resemble L. robinsi Gilmore 1997. Further investigation has confirmed that these two species represent additional cryptic diversity in the Grammatidae, a family of usually small, brightly colored fishes restricted to deep reefs of the tropical northwest Atlantic Ocean. Here we describe the two new species of Lipogramma based on integrated morphological and molecular data, provide a revised phylogenetic hypothesis of relationships within the genus that includes L. regia (which we recently caught using the Curasub off St. Eustatius), comment on the eco-evolutionary history of the group based on the phylogenetic pattern of species' depth distributions, and present a revised key to the species of the genus.

Methods
Collecting and morphology. Basslets were collected using Substation Curaçao's manned submersible Curasub (http://www.substation-curacao.com). The sub has two flexible, hy draulic arms, one of which is equipped with a quinaldine/ethanol-ejection system and the other with a suction hose. Anesthetized fish specimens were captured with the suc tion hose, which empties into a vented plexiglass cylinder attached to the outside of the sub. At the surface, the specimens were photographed, tissue sampled, and fixed in 10% formalin. Measurements were made weeks to months after fixation and subsequent preservation in 75% ethanol and were taken to the nearest 0.1 mm with dial calipers or an ocular micrometer fitted into a Wild stereomicroscope. Selected preserved specimens were later photographed to document preserved pigment pat tern and X-rayed with a digital radiography system. Images of parasitic cysts were made using a Zeiss Axiocam on a Zeiss Discovery V12 SteREO microscope. Counts and measurements follow Hubbs and Lagler (1947). Symbolism for configuration of supraneural bones, anterior neural spines, and anterior dorsal pterygiophores follows Ahlstrom et al. (1976): USNM = Smithsonian Institution, National Museum of Natural History; UF = Florida Museum of Natural History. Molecular analyses. Tissue samples for 98 specimens assignable to nine species of Lipogramma were used for molecular analyses (Appendix 1). Tissues of L. robinsi, margin of orbit, a pore present between one above mid orbit and one above posterodorsal corner of orbit; caudal fin rounded; body mostly yellow in life with 11 or 12 narrow brownish bars on trunk; posterior base of soft dorsal fin with large white-or blue-rimmed black ocellus; dorsal, anal and caudal fins yellow with blue/grey (brown in preservative) wavy bars or square-shaped spots. Pelvic fins blue/grey with scattered yellow-ringed dark spots. The new species is further differentiated genetically from congeners for which molecular data are available in mitochondrial COI and nuclear Histone 3, Rhodopsin, TMO-4C4, and RAG1.
Spinous and soft dorsal fins confluent, several soft rays at rear of fin forming slightly elevated lobe that extends posteriorly beyond base of caudal fin. Pelvic fin, when depressed, extending posteriorly to point between base of second or third anal-fin spine and posterior base of anal fin, first pelvic-fin ray elongate. Dorsal profile from snout to origin of dorsal fin convex. Diameter of eye of holotype contained three times in head length. Pupil slightly tear shaped, with small aphakic space anteriorly. Scales extending anteriorly onto posterior portion of head, ending short of coronal pore. Scales present on cheeks, opercle, preopercle, interopercle, and isthmus. Scales lacking on top of head, snout, jaws, and branchiostegals. Scales large and deciduous, too many scales missing in most specimens to make accurate scale counts. In one paratype (USNM 436460) approximately 21 lateral scales between shoulder and base of caudal fin, approximately 4 scale rows on cheek, and approximately 9 scale rows across body above anal-fin origin. Scales on head and nape without cteni, scales on rest of body ctenoid. Fins naked.
Margins of bones of opercular series smooth, opercle without spines. Single row of teeth on premaxilla posteriorly, broadening to 2-3 rows anteriorly, teeth in innermost row smallest, some teeth in outer row enlarged into small canines. Dentary similar, holotype with 6 enlarged teeth in outer row near symphysis. Vomer with chevronshaped patch of teeth. Palatine with long series of small teeth. Conspicuous pores present in infraorbital canal (2 pores), portion of supraorbital canal bordering dorsal portion of orbit (4), on top of head (1 median coronal pore), preopercle (at least 5), Table 1. Counts and measurements of type specimens of Lipogramma barrettorum sp. n. Measurements are in percent SL. "Other Caudal Rays" include "i" -a slender, flexible, non-spinous, and typically nonsegmented ray and "I" -a spinous procurrent ray.  XII,9 XII,9 XII,9 XII,9 XII,9 XII,9 XII, and lateral-line canal in the post-temporal region (3). The 4 supraorbital pores situated as illustrated by Baldwin et al. (2016: fig 4) for L. evides. Posterior nostril situated just ventral to anteriormost supraorbital pore, nostril a single large opening. Anterior nostril at apex of elongate narial tube and situated just posterior to upper lip. No lateral line present on body. Coloration: In life or deceased but prior to preservation ( Fig. 1): ground color of body brownish yellow, head darker than trunk, especially on underside. Head: dorsal midline of head with thin blue-white stripe beginning on lower lip, continuing on upper lip and over snout to nape; iris yellow, blue-white bars anterior and posterior to pupil; an indistinct yellow/brown bar from center of lower edge of iris to lower jaw, bar bordered anteriorly and posteriorly by smaller pale bars that extend up to top of eye. Trunk: 9-12 narrow dark bars between pectoral-fin base and caudal peduncle, bars about as wide as paler interspaces. Dorsal fin: yellow, with a thin blue-grey margin; series of straight, wavy, or irregular blue-grey bars on basal 2/3 of spinous dorsal fin; basal half of soft-dorsal with a large black ocellus complete ringed in blue-white pigment, ocellus extending onto trunk; distal half of fin with 2-3 rows blue-grey roundto square-shaped spots, these markings (here and on other unpaired fins) with pale centers and darker edges. Anal fin: yellow, with a thin blue-grey margin; 5-6 rows of blue-grey square-shaped spots between fin base and margin, some of which may fuse to form irregular lines. Caudal fin: yellow, with thin blue-grey posterior margin  and 6-7 bars formed by vertical rows of blue-grey square-shaped spots on inter-radial membranes, basal two rows palest. Pectoral fins: translucent, base as dark as trunk bars. Pelvic fins: opposite color scheme to that of other fins, i.e., mostly blue-grey with yel-low spots along inter-radial membranes; proximally, spots with tiny black center; distally, dark centers larger, some spots appearing completely dark. Juveniles: the 12-and 15-mm SL paratypes ( Fig. 2) with similar pigment pattern as adults. Comment regarding live coloration: photographed against a light background (Fig. 1A, C), "blue-grey" in description above = grey; photographed against a black background (Fig. 1B), "bluegrey" = blue. Preserved coloration (Fig. 3A): Head mostly brown, trunk mostly tan with darker tan to brown bars. Yellow portions of median fins in life clear in preservative, blue-grey markings on fins in life dark brown to black in preservative.
Distribution. Known only from specimens collected off Curaçao, southern Caribbean. Habitat. Lives in or immediately above elevated rocky habitat with ample cracks or holes into which the fish retreated upon approach of the submersible. The holotype was collected at 161 m, which is the only discrete depth recording for the species. Depth ranges for two specimens were recorded as 123-160 m and 132-141 m, thus providing a potential total depth range of 123-161 m. Depth ranges for two additional specimens of 90-249 m and 50-246 m reflect depths visited during an entire submersible dive and provide little information relevant to establishing this species' depth distribution.
Etymology. Named Lipogramma barrettorum in recognition of the support of Craig and Barbara Barrett for the Smithsonian's Deep Reef Observation Project (DROP).
Common name. We propose blue-spotted basslet in reference to the numerous blue/grey markings on the dorsal, anal, and caudal fins in life.
Genetic comparisons. Table 2 shows average inter-and intraspecific divergences in COI among species of Lipogramma analyzed genetically in this study. Average intraspecific divergence among the seven specimens of L. barrettorum is 0.003 substitutions per site, and interspecific divergences between it and the other species for which data are available range from 9.9% (L. evides) to 25.3% (L. klayi).
Comments. The holotype has two cysts, one at the base of the uppermost left pectoral-fin ray and one about mid-way along the length of the elongate first left pelvic-fin ray (Fig. 4). The cysts or galls are likely parasitic, but further analysis is needed. No other cysts were observed on the holotype or paratypes.  Diagnosis. A species of Lipogramma distinguishable from congeners by the following combination of characters: pectoral-fin rays 16-17 (modally 16), gill rakers 11-13 (modally 12, 8-9 rakers on lower limb); four supraorbital pores present along dorsal margin of orbit, a pore present between one above mid orbit and one above posterodorsal corner of orbit; caudal fin rounded; body mostly tan to brown in life with 7 or 8 narrow darker brown bars on trunk; head with broad, irregular, whitish blue markings along dorsal midline from lower lip across upper lip and snout to nape; dark bar through eye bordered anteriorly and posteriorly by bluish-white bars; posterior base of soft dorsal fin with large white-or blue-rimmed black ocellus; dorsal and anal fins blue-grey with yellow spots or bars. Caudal fin mostly yellow with wide blue-grey margin and several bars comprising blue-grey mostly square-shaped spots. Pelvic fins grey/blue with scattered yellow-ringed dark spots. Juveniles with irregular white blotches of pigment on trunk and two triangular white blotches on caudal-fin base. The new species is further differentiated genetically from congeners for which molecular data are available in mitochondrial COI and nuclear Histone 3, Rhodopsin, TMO-4C4, and RAG1.

Lipogramma schrieri
Description. Counts and measurements of type specimens given in Table 3. Seven specimens examined, 17.2-61.9 mm SL. Dorsal-fin rays XII, 9 (last ray composite); largest specimen (USNM 414911) with 9 pterygiophores in soft anal fin, but only 8 externally visible rays, the 8 th appearing to represent fusion of two rays; anal-fin rays III, 8 (last ray composite); pectoral-fin rays 16-17, modally 16, 16 on both sides in holotype; pelvic-fin rays I,5; total caudal-fin rays 25 (13 + 12), principal rays 17 (9 + 8), spinous procurrent rays 6 (III + III), and 2 additional rays (i + i) between principal and procurrent rays that are neither spinous nor typically segmented; vertebrae 25 (10 + 15); pattern of supraneural bones, anterior dorsal-fin pterygiophores, and dorsal-fin spines usually 0/0/0+2/1+1/1/, one paratype (USNM 435299) aberrant in having first two dorsal-fin spines supported in supernumerary association by separate pterygiophores Table 3. Counts and measurements of type specimens of Lipogramma schrieri sp. n. Measurements are in percent SL. "Other Caudal Rays" include "i" -a slender, flexible, non-spinous, and typically nonsegmented ray and "I" -a spinous procurrent ray. vs. a single pterygiophore -0/0/0+1+1/1+1/1/; ribs on vertebrae 3-10; epineural bones present on at least vertebrae 1-15 in holotype and two paratypes, difficult to assess in other specimens; gill rakers on first arch 11-13 (3-4 + 8-9), 11 (3 + 8) in holotype; upper-limb rakers and lowermost one or two rakers very small or present only as nubs, all other gill rakers elongate and slender with tooth-like secondary rakers as in L. evides (Baldwin et al. [2016: fig 3]); pseudobranchial filaments 7-11 (9 in holotype), filaments poorly or well developed (well developed in holotype); branchiostegals 6. Spinous and soft dorsal fins confluent, several soft rays in posterior portion of fin forming slightly elevated lobe that extends posteriorly beyond base of caudal fin. Pelvic fin extending posteriorly to base of third anal-fin spine in preserved holotype when depressed, to middle or posterior portion of anal fin in aquarium photos (e.g., Fig. 5D). Dorsal profile from snout to origin of dorsal fin convex. Diameter of eye of holotype contained 3.3 times in head length. Pupil slightly tear shaped with small aphakic space anteriorly. Scales extending anteriorly onto top of head, ending short of coronal pore. Scales present on cheeks, operculum, and isthmus. Scales lacking on frontal region, snout, jaws, and branchiostegals. Scales large and deciduous, too many missing in most preserved specimens to make counts, but counts made from photographs of specimens prior to preservation indicate approximately 25-27 lateral scales between shoulder and base of caudal fin (27 in holotype), 5 cheek rows, and 12 rows across body above anal-fin origin. Scales on head and nape without cteni, scales on rest of body ctenoid. Fins naked.
Margins of bones of opercular series smooth, opercle without spines. Premaxilla with band of small conical teeth, band widest at symphysis, outer row with largest teeth, 3 or 4 (4 in holotype) near symphysis enlarged. Dentary similar except 8 anterior teeth enlarge. Vomer with chevron-shaped patch of teeth, palatine with long series of small teeth. Conspicuous pores present in infraorbital canal (2 pores), portion of supraorbital canal bordering dorsal portion of orbit (4), on top of head (1 median coronal pore), preopercle (at least 5), and lateral-line canal in the posttemporal region (3). The 4 supraorbital pores situated as illustrated by Baldwin et al. (2016: fig 4) for L. evides. Posterior nostril situated just ventral to anteriormost supraorbital pore, nostril a single large opening. Anterior nostril at apex of elongate narial tube and situated just posterior to upper lip. No lateral line present on body.
Coloration: In life or deceased but prior to preservation (Fig. 5), ground color of body light brown. Head: dorsal midline of head with broad area of irregular, blue-white markings beginning on lower lip and continuing on upper lip and over snout to nape; a dark brown, pupil-width bar extending across orbit to lower jaw, this bar bordered on either side by thin whitish bar that runs from top of eye through front and rear of iris to lower jaw. Trunk: 7-8 narrow, dark-brown bars between posterior edge of operculum and caudal peduncle, bars narrower than paler interspaces. Dorsal fin: spinous dorsal and anterior portion of soft dorsal blue-grey with stripes comprising short yellow bars proximally and yellow spots distally; posterior portion of soft dorsal with large black ocellus ringed in blue-white pigment that extends onto trunk; several rows of yellow spots above ocellus. Anal fin: blue-grey, with yellow markings similar to those on spinous dorsal fin. Caudal fin: yellow, with vertical bars comprising blue-grey, square-shaped spots on interradial membranes on anterior 2/3 of fin; wide, blue-grey margin distally. Pectoral fins: most of fin translucent, base and anterior portion of fin dark. Pelvic fins: pale blue-grey to bright blue with yellow-ringed dark spots, spots mostly dark brown distally. Juveniles: An ontogenetic series from 17-33 mm SL is shown in Figure 6. The 17-mm SL specimen lacking body bars and with row of four large, irregular white blotches on or just below lateral midline of trunk, smaller white spots along back above that row, white spot at posterior base of anal fin, and two large, roughly triangular white blotches on caudalfin base. First four dark trunk bars evident anteriorly in 26-mm SL juvenile, which has smaller white markings (spots vs. blotches). In 33-mm SL specimen, all trunk bars present, and remnants of each white caudal-fin blotch present as small white spot before indistinct pale vertical bar. Comment regarding live coloration: photographed against a light background (Fig. 5A-C), "blue-grey" in description above = grey; photographed against a darker background (Fig. 5D, E), "blue-grey" = blue. Preserved coloration (Fig.  3B): Head and trunk tan with darker tan to brown bars. Yellow portions of median fins in life clear in preservative, blue-grey markings on fins in life dark brown in preservative.
Distribution. Known only from specimens collected off Curaçao, southern Caribbean.
Habitat. Elevated rocky habitat with ample cracks or holes into which the fish retreated upon approach of the submersible. The holotype was collected at 197 m, and three paratypes were collected at 173-207 m. The range of 156-290 m recorded for another paratype reflects all depths visited on the submersible dive during which the specimen was collected and provides little relevant depth information.  Etymology. Named Lipogramma schrieri in honor of Adriaan (Dutch) Schrier, owner of Substation Curaçao. Although the Curasub submersible was not built originally for scientific research, Dutch's enthusiastic support of research use of his sub has exponentially expanded our understanding of fish and invertebrate faunas of Caribbean mesophotic and deeper reefs.
Common name. We propose Maori Basslet, in reference to the similarity of the markings on the dorsal midline of the forehead to the beautiful facial tattoo of the Maoris, indigenous Polynesian people of New Zealand.
Genetic Comparisons. Table 2 shows average inter-and intraspecific divergences in COI among species of Lipogramma analyzed genetically in this study. Average intraspecific divergence among the seven specimens of L. schrieri is 0.002 substitutions per site, and interspecific divergences between it and the other species for which data are available range from 11.7% (L. barrettorum) to 25.3% (L. klayi).
Comments. A 52.5 mm SL Lipogramma (RGG uncataloged), collected at 296 m in 1997 by one of us (RGG) and Richard Robins off Cuba (Fig. 7), could be a specimen of L. schrieri. It has a similar color pattern, including seven dark body bars, the "maori" pattern of pigment on top of the head (based on examination of the preserved specimen), and similar pattern of fin pigment. However, this specimen has yellow pigment around the eye (vs. brown in L. schrieri), yellow pigment in a triangular-shaped subocular bar (vs. brown pigment in a rectangular-shaped bar), and a yellow pectoralfin base (vs. dark brown). Furthermore, the Cuban specimen has 15 pectoral-fin rays on each side vs. 16-17 (modally 16) in L. schrieri, and 14 total gill rakers vs. 11-13 in L. schrieri. Further study is needed to determine if this specimen represents a variant of L. schrieri or an additional cryptic species in the genus.

Discussion
Morphological comparisons. Lipogramma barrettorum, L. robinsi, and L. schrieri differ from all congeners in having at least seven dark trunk bars. A comparison of major morphological and pigmentation differences among those three species is provided in Table 4. They are easily separable from one another on the basis of live or fresh color patterns, most notably the following: (1) the presence of 7-8 dark brown trunk bars against a tan background in L. schrieri, 10-12 tan/yellow bars against a flesh to greenish background in L. robinsi, and 11-12 light brown bars against a yellow background in L. barrettorum (trunk bars also evident in preserved specimens); (2) the presence of a bright yellow nape in L. robinsi, a blue-white stripe from the tip of the lower jaw to the base of the dorsal fin in L. barrettorum, and irregular, broad, blue-white markings from the tip of the lower jaw to the nape in L. schrieri (stripe in L. barrettorum and irregular markings in L. schrieri also evident in preserved specimens); (3) the presence of a distinct dark bar through the eye in L. schrieri, no such bar in L. robinsi, and an indistinct bar in L. barrettorum (orbital bar, when present, also evident in preserved specimens); and (4) median fins transparent with yellow spots in L. robinsi, yellow with blue/grey spots in L. barrettorum, and blue/grey with yellow spots in L. schrieri. Lipogramma robinsi can further be distinguished from the other two species by having more lower-limb gill rakers on the first arch (11-12 vs. 8-9 in L. schrieri and 8-10 in L. barrettorum) and usually having fewer pectoral-fin rays (15 vs. modally 16 in L. schrieri and L. barrettorum). Based on available material, Lipogramma schrieri reaches a larger size (~62 mm SL) than L. robinsi (~22 mm SL) and L. barrettorum (~25 mm SL).
Species delimitation and phylogeny. Comparative morphological analysis supports the recognition of L. barrettorum and L. schrieri as distinct species, and combinations of diagnostic morphological features that distinguish them from all other Lipogramma species are provided in the species descriptions above. Molecular data for the nine Lipogramma species for which genetic data existed prior to this study (Baldwin et al. 2016) or was generated in this study (L. anabantoides, L. barrettorum, L. evides, L. haberi, L. klayi, L. levinsoni, L. regia, L. schrieri, L. trilineata) unequivocally support the presence of nine species (molecular data not available for L. flavescens, L. roseum and L. robinsi). The neighbor-joining network (Suppl. Material 1) derived from COI data shows nine distinct lineages with very high genetic distances between lineages (10-27%, mean = 19%), which are at least 20 times greater than variation in COI within lineages (range 0.1-0.5%, mean = 0.3%). The molecular phylogeny from the Bayesian analysis of the concatenated dataset (Fig. 8) was identical in topology to the BP&P coalescent-based species-tree analysis. The BP&P analysis also had overwhelming support for a nine-species model (posterior probability 0.996) versus models with fewer or more species.
Eco-evolutionary relationships. The two new species, L. barrettorum and L. schrieri, belong to a clade that includes L. evides and L. haberi. Collectively the members of this clade are the deepest-living species in our analysis, occurring at depths predominantly below 140 m (Figure 9). Sister to this clade is a group comprising L. levinsoni, L. anabantoides, and L. regia, species that inhabit depths predominantly shallower than 150 m, in  the zone traditionally referred to as mesophotic coral ecosystems (MCEs-~30-150 m; Kahng et al. 2010). A second shallow/MCE (< 150 m) clade that is sister to both those clades comprises L. trilineata and L. klayi. There is clear habitat partitioning by depth within both shallow clades, particularly between sister species: L. trilineata vs L. klayi and L levinsoni vs L. anabantoides. Based on available data, there is no clear depth partitioning evident within the more speciose L evides clade. We note, however, that these depth distributions are based on collections and observations of different species from different locations. Hence, we cannot rule out the possibility that some of the differences in depth distributions in Figure 9 represent location effects rather than depth partitioning. In addition to depth, we have observed distinct interspecific variation in the types of substrata with which some of these species associate and the nature of their associations with substrata. Off Curaçao, Lipogramma klayi is a very commonly observed species, especially on the upper level of vertical faces or slopes of ~30-60° that are heavily indented with small caves and overhangs and festooned with gorgonians and other growth. We have commonly seen it occupying the same habitat at Bonaire, Dominica, Roatan, and St. Eustatius. Its sister species, L. trilineata is much more cryptic than L. klayi. It is rarely observed, as it tends to stay close to ceilings of cavities, whether those are caves or small holes formed in large rock or coral heads. Within the clade comprising the two new species, L. evides and L. levinsoni are commonly associated with small patches of cobble scattered among rocky areas, whereas L. barrettorum and L. schrieri are associated with elevated rocky habitat with ample cracks or holes. Finally, two of us (LT and DRR) recently observed multiple instances of L. flavescens off Roatan sitting on coarse sand, meters away from shelter, in areas of sand and scattered small low patches of rock. Baldwin et al. (2016) noted that members of the large clade comprising all Lipogramma species except L. trilineata and L. klayi are characterized by a dark ocellus on the posterior base of the dorsal fin. Based on this character, a relatively shallow depth range, and a modal count of 17 rays in the pectoral fin, they hypothesized that L. regia (not sampled in that study) is most closely related to L. anabantoides and L. levinsoni. The present phylogenetic analysis includes a recently collected specimen of L. regia and supports this hypothesis. Baldwin et al. (2016) also hypothesized that L. flavescens may be part of the deep-dwelling clade comprising L. evides, L. haberi, L. barrettorum, and L. schrieri (the last two as "Lipogramma 'robinsi'" in their phylogeny) and, based on the deep depth-range of L. flavescens, a dark ocellus on the dorsal fin, bright yellow body coloration, a dark bar through the eye, and a low gill-raker count (15-16), possibly most closely related to L. haberi. Collection of fresh material of L. flavescens would provide the genetic material needed to test their hypotheses. We note that L. robinsi is likely part of this deep-dwelling clade as well, based on the presence of a dorsal-fin ocellus, a barring pattern on the body similar to that of L. schrieri and L. barrettorum, and its depth range (although the latter is based on very few specimens). Fresh material of L. robinsi for genetic analyses is also desirable. In addition, more information is needed on the depth distributions of all of the less common species, particularly to determine the extent of habitat partitioning within locations, as well as its consistency between locations.
Submersible exploration. Effective capture of fish specimens during deep-sea submersible dives has only been realized since 1982 with the Johnson Sea-Link submersibles and, much more recently, with the Curasub. Fresh and often living specimens are brought to the surface, providing quality material for color photography and genetic analyses to investigate phylogenetic relationships and evolutionary trends. Capture of cryptobenthic species, including gobiids (Baldwin and Robertson 2015;Tornabene et al. 2016aTornabene and Baldwin 2017), blennioids (Baldwin and Robertson 2013), grammatids (Gilmore and Jones 1988;Gilmore 1997;Baldwin et al. 2016b; this study), serranids Johnson 2014, Baldwin andRobertson 2014), and scorpaenids ) using manned submersibles is allowing unprecedented examination of microhabitat relationships, depth and temperature preferences, and biogeography, along with comparative morphology and molecular phylogenetic relationships in previously unknown or inaccessible species. We cannot overemphasize the value of these manned undersea operations to increasing our knowledge and understanding of tropical deep-reef fish assemblages.