A new species of Lipogramma from deep reefs of Roatan, Honduras (Teleostei, Grammatidae)

Abstract A new species of Lipogramma is described from submersible collections at 122–165 m depth off the coast of Roatan, Honduras, in the western Caribbean. The new species is distinguished from all other species in the genus by its bright blue coloration on the head, nape, and dorsal portion of the trunk beneath the spinous dorsal fin, a prominent round black blotch below the origin of the spinous dorsal fin, and a high number of gill rakers. A molecular phylogeny based on mitochondrial and nuclear genes shows that the new species belongs to a clade containing L.levinsoni, L.regia, and L.anabantoides. At Roatan, submersible observations of this and other Lipogramma species indicate clear, interspecific habitat partitioning by depth and substrate.


Introduction
Manned submersibles have proven to be highly effective for collecting fishes from deep-reef habitats (Gilmore 2016), particularly in the rariphotic zone (below ~130 m; Baldwin et al. 2018), where divers using closed-circuit rebreathers, which are lim-ited to depths less than ~ 150 m, are incapable of sampling for extended periods of time. This is especially true for cryptobenthic fishes such as grammatids and gobiids, many of which are associated with structurally complex reef and rocky habitats and are unlikely to be sampled using trawls or dredges. Partially because they are difficult to sample, cryptobenthic reef fishes as a whole are an understudied group, and recent studies suggest that they comprise nearly half of all fish diversity on coral reefs and possess a large number of undescribed species (Brandl et al. 2018). In recent years, researchers from the Smithsonian Deep Reef Observation Project (DROP) have used the manned submersible Curasub, located on the island of Curaçao in the southern Caribbean and capable of descending to 300 m, to collect and describe a cache of new species of reef fishes, including many species of cryptobenthic fishes (e.g. gobiids, Baldwin and Robertson 2015, Tornabene et al. 2016a, 2016b, Tornabene and Baldwin 2017labrisomids, Baldwin and Robertson 2013;andgrammatids, Baldwin et al. 2016, 2018).
In 2017, DROP operations expanded to Roatan, Honduras, where the Idabel manned submersible is located. On the first dive capturing specimens with Idabel off Halfmoon Bay, West End, Roatan, the authors collected a specimen of an undescribed species of Lipogramma (Grammatidae) at 165 m depth. Subsequent dives around this depth revealed that the species is relatively common, despite never having been collected before nor observed in more than 150 submersible dives throughout the Caribbean by us or others using Curasub, Idabel, or the Johnson Sea-Link subs. Four additional specimens, including one juvenile, were subsequently collected on later dives off Roatan.
The description of the new species from Roatan brings the total number of species in Lipogramma to 13, all of which occur in the tropical western Atlantic Ocean. Lipogramma and Gramma are currently classified in the family Grammatidae based on a single synapomorphy in the arrangement of cheek musculature (Gill and Mooi 1993). This relationship is not supported by molecular data, although the relationships between genera in the diverse Ovalentaria have proven to be difficult to resolve with traditional molecular markers, a combination of molecular markers and morphological characters, and phylogenomic data (Betancur-R et al. 2013;Mirande 2016;Eytan et al. 2015). Nearly all species of Lipogramma occur on deep reefs. Lipogramma trilineata and L. anabantoides are the only two species known to routinely occur above 50 m, which is approximately the limit of recreational scuba diving. Including the new species described here, five species of Lipogramma have been described from specimens collected using Curasub and Idabel. Several species are rare in collections, owing to the difficulties of collecting fishes from structurally complex deep-reef habitats. Currently L. haberorum, L. barrettorum, L. schrieri, L. robinsi, L. flavescens, L. regia, L. rosea, and the new species described here are each known from fewer than 10 specimens, although this may not accurately reflect an actual rarity in the wild. Increased sampling from Roatan and other localities across the Caribbean are certain to uncover additional undescribed species of Lipogramma and other cryptobenthic fishes.

Materials and methods
Specimens were collected using the Idabel submersible. The Idabel can accommodate a pilot and two scientists and is capable of diving to ~700 m. This sub was recently outfitted with a fish-catching system capable of delivering an anesthetic solution (5% quinaldine sulphate in seawater) and capturing specimens with a suction system powered by one of the submersible's vertical thrusters (Fig. 1). Four of the five type specimens were brought to the surface alive where they were photographed prior to euthanasia in MS222 and preservation. Tissue samples were taken and stored in 95% ethanol, and voucher specimens were fixed in 10% formalin and later transferred to 70-75% ethanol. Measurements were made weeks to months after preservation, and were taken to the nearest 0.1 mm with digital calipers. Counts and measurements follow Hubbs and Lagler (1947). Specimens were x-rayed with a digital radiography system. Type specimens were deposited at the University of Washington (UW), the National Museum of Natural History, Smithsonian Institution (USNM), the Florida Natural History Museum (UF) and the National History Museum of the National Autonomous University of Honduras in the Sula Valley (MUVS-V). Cephalic pores were viewed and photographed using a Zeiss Discovery V20 SteREO microscope with an attached Axiocam 503 digital camera. In addition to comparing our morphological data to those from original species descriptions, our comparative material examined here included several specimens (including types) of L. evides, L. levinsoni, L. barrettorum, L. schrieri, L. haberorum, as well as the voucher specimens from our phylogenetic analysis. Catalog numbers of these specimens are listed in the appendices of Baldwin et al. (2016) and Baldwin et al. (2018).
DNA was extracted from tissue samples using a Qiagen DNeasy Blood and Tissue kit. Four loci were sequenced for three specimens of the new species for phylogenetic analysis. A partial segment of the mitochondrial gene cytochrome c oxidase subunit I (COI), and three nuclear genes (TMO-4C4, Rag1, Rhodopsin) were amplified via PCR and sequenced using primers and PCR conditions from Weigt et al. (2012) and Hastings (2011, 2013). GenSeq nomenclature (Chakrabarty et al. 2013) and GenBank accession numbers are listed in Appendix 1: Table A1. The sequence data was aligned from the Roatan samples with data from our past studies (Baldwin et al. 2016(Baldwin et al. , 2018. The align ments were concatenated and phylogeny was inferred using Bayesian Inference (BI), partitioning by gene. For the BI analysis, the substitution models and the partitioning scheme were chosen using PartitionFinder (Lanfear et al. 2012) according to Bayesian Information Criterion scores. The BI phylogeny was inferred in the program MrBayes v. 3.2 (Ronquist et al. 2012) using two Metropolis-coupled Markov Chain Monte Carlo (MCMC) runs, each with four chains. The analyses ran for 10 million generations sampling trees and parameters every 1000 generations. Burn-in, convergence and mixing were assessed using Tracer (Rambaut and Drummond 2007) and by visually inspecting consensus trees from both runs. The ML analysis was done in the program RAxML v.8.2.9 (Stamatakis 2014), using 20 initial random searches, Figure 1. Idabel submersible outfitted with fish-catching system. A acrylic holding tank B housing for HD video camera C quinaldine sulphate delivery hose D suction for the system is powered by a PVC hose connecting to one of the submersible's vertical thrusters E two-way valve to allow for differential suction/ blowing of water and or anesthetic F carbon-fiber compensator holding up to 2.5 gallons of anesthetic solution, powered by pressurized air from a SCUBA cylinder (not shown, lower tank in image is oxygen for life support systems) G housing for solenoid valve, enabling scientists to control the flow of anesthetic from a switch inside the submersible. and topological support was assessed using 1000 bootstrap replicates. Outgroups for the phylo genetic analysis included two species of Gramma and several other genera from the Ovalentaria sensu Wainwright et al. (2012), i.e. Acanthemblemaria (Chaenopsidae), Blenniella (Blenniidae), and Tomicodon (Gobiesocidae).

Description. Counts and measurements of type specimens given in
Spinous and soft sections of dorsal fin confluent, several soft rays in posterior portion of fin forming slightly elevated lobe that extends posteriorly beyond base of caudal fin. Pelvic fin, when depressed, extending at least to origin of first soft ray of anal fin, and to origin of penultimate anal-fin ray in holotype. Dorsal profile from snout to origin of dorsal fin convex. Diameter of eye contained 2.8-3.0 times in head length. Pupil tear-shaped, with small aphakic space anteriorly. Scales extending anteriorly onto top of head, ending at a vertical just behind posterior margin of eye. Scales present on cheeks, operculum, and isthmus, absent on snout, jaws, and branchiostegals. Scales large and deciduous, missing on anterodorsal flank of several specimens. Approximately 22-25 lateral scales between shoulder and base of caudal fin (24 in holotype), 4-6 cheek rows, 11 rows across body from above anal-fin origin. Scales on nape and along dorsal midline with reduced or absent cteni, those on cheek and opercula lacking cteni. Fins naked except base of posteriormost dorsal-fin rays, which possess 1 or 2 small embedded scales. No modified lateral-line scales present on body, but faint indication of a lateral line present superficially in fresh photographs.
Margins of bones of opercular series smooth, opercle without spines. Single row of teeth on premaxilla posteriorly, broadening to 2 or 3 rows anteriorly, teeth in innermost row smallest, some teeth in outer row enlarged into small canines. Dentary similar with 3 or 4 enlarged teeth in outer row near symphysis. Vomer with chevronshaped patch of teeth that extends posterolaterally nearly length of premaxilla, palatine with long series of small teeth.
Cephalic head pores arranged as in Fig. 2, with conspicuous pores present in infraorbital canal (2), dentary canal (2), supraorbital canal (2 pores above each eye plus one median coronal pore), preopercular canal (7), posttemporal canal (3), plus a single pore in canal above post-temporal canal in line with preopercle. Anterior naris in elongate tube above upper lip, posterior naris a wide opening with slightly raised rim immediately anterior to supraorbital canal.
Coloration in fresh specimens (Fig. 3): Head: top of snout, top of head, and nape bright blue; lower part of head yellow brown, with faint blue overtone; eye with bright blue iris, black pupil; top and bottom lips with pale blue overtone; yellow oblique bar,  bordered with blue, extending from tip of snout to mid orbit and continuing below orbit towards lower corner of preopercle; opercle with lavender hue from bright red gills and blood vessels visible through gill cover. Trunk: yellowish to yellow-brown, paler and sometimes with faint bluish cast on isthmus and abdomen; large, eye-size round black blotch on upper back under the origin of dorsal fin, surrounded by thin bright blue ring; blue coloration on nape extends along the upper back to end of spinous dorsal. Spinous dorsal fin: bright blue anteriorly, fading to blue-grey posteriorly on basal half; outer edge blue-grey, with thin submarginal yellow stripe formed by series of close-set, horizontally elongate yellow spots; row of yellow spots (each on and just behind a spine), beginning at base of the first 2-3 spines, then continuing as row along other 1/3 of the fin. Soft dorsal fin: rays blue-grey anteriorly, with the last several posterior rays blue; upper margin whitish-blue, with a submarginal row of vertically elongate, inter-radial yellow spots, and 3-4 rows of vertically oval, interradial yellow spots, those at rear forming thin, yellow lines along the membranes between last 2 or 3 rays; pupil-sized, round, yellow-brown blotch containing darker scales ringed with bright blue at rear insertion of fin, with half of blotch covering base of last 6 or 7 rays and half on upper back. Anal fin: bluish grey, with brownish cast on scaled base of fin; 3 or 4 irregular rows of yellow spots along fin elements, central spots oval, basal and outer spots forming streaks along fin elements; outer margin of fin whitish-blue. Caudal fin: base translucent yellow; center of fin with rows of yellow spots along fin rays; outer part of fin translucent, with thin whitish-blue rear margin; vertical rows of pale blue spots on rays in center of fin. Pectoral fins: pectoral rays tinged with yellow, membranes translucent; base of fin paler than adjacent body. Pelvic fins: pale bluish grey, with elongate yellowish spots along fin rays, that yellow coloration strongest at base of fin. Juvenile coloration: Coloration of the single small juvenile is essentially the same as in the adult, except that the posterior portion of the body is more noticeably yellow, the entire center of the anal fin, and, apparently, much of the soft dorsal and caudal fins are yellow, and the dark center of the ocellus at the lower rear corner of the dorsal fin is solid black.
Comments about live coloration: As can be seen in the video of the holotype being captured (https://doi.org/10.5281/zenodo.1334518, or https://zenodo.org/record/1334518#. W89WsSPMyFU), live fish have the upper third of the head and body bright blue, the lower 2/3 yellow, and a prominent large round black blotch on the shoulder.
Color in preservation (Fig. 4): Overall pigmentation pattern largely similar to that of fresh individuals, except iridescent blue coloration replaced by dark brown pigment; oblique bar on head below eye pale in preservation versus yellow in life; dark ocelli below spinous and soft dorsal fins less distinct, with no clearly defined ring or margin; background body coloration darkest on head and below origin of spinous dorsal fin, gradually fading from brownish-tan anteriorly to pale-yellow posteriorly.
Habitat. The species was frequently observed in the mid-to-upper rariphotic zone between 122-165 m depth, in or around small rock crevices, rock piles, or caves situ-  Etymology. The specific epithet idabeli refers to the Idabel submersible, which was used to collect the type series, and recognizes of the efforts of its owner-designer and pilot Karl Stanley and engineer Thomas Trudel, who made these and other collections of fishes possible by constructing a fish-catching system that converted Idabel from an observation-only vessel to one capable of collecting scientific specimens. The name idabeli is to be treated as a patronym (adjective) formed from the female name Idabel. The generic name Lipogramma is feminine and is formed from lipo (without) and gramma (a line of text, feminine), referring to the absence of a well-developed lateral line. The common name Cabrilleta de Dorso Azul (Blue-backed Basslet in English) refers to its distinctive coloration.
Comparisons. Lipogramma idabeli is easily distinguished from all other species of Lipogramma by the bright blue coloration on the head and eye, and by the pair of blue-margined ocelli below the anterior origin of the dorsal fin and at the posterior insertion of the dorsal fin. There are only two other species of Lipogramma in which the head is a markedly different color than the body; L. klayi and L. anabantoides both have rose, pink, or purple heads with tan or yellow bodies. No other known species has bright blue coloration on the dorsal portion of the head, nape, and dorsal portion of the trunk. All species of Lipogramma except L. klayi, L. rosea, and L. trilineata possess an ocellus on the posterior portion of the dorsal fin, but the pattern of barring and shading on the head and body differ among all of those species (Fig. 6). In addition to the differences, none have the ocellus with a blue margin except L. idabeli. Furthermore, in L. idabeli, the ocellus varies from having a dark center as a juvenile to a yellow or dusky center as an adult, whereas in the other species, the ocellus is always black. The absence of prominent vertical barring on the body distinguishes L. idabeli from L. robinsi, L. barretorum, L. haberorum, L. evides, L. levinsoni, L. schrieri, and L. regia (Fig. 6). Lipogramma idabeli has more total gill rakers (18-20) than all other species except L. evides (19-22), L. klayi (19), and L. levinsoni (17-20); all other species have 16 or fewer. The combination of XII, 9 dorsal-fin elements and III, 8 anal-fin elements present in L. idabeli is shared among most species in the genus, except L. rosea (XI, 6 and III, 6), L. trilineata (XII, 10 and III, 7), and L. anabantoides (XIII,8 and III,8).
Phylogenetics and eco-evolutionary relationships. Coloration unambiguously diagnoses L. idabeli and supports its recognition as a distinct species. Molecular data from the ten species of Lipogramma for which tissue samples were available also support this distinction. The molecular phylogenies from the Bayesian and Maximum Likelihood analyses ( Fig. 7; Suppl. material 1: Figure S1), which were identical in topology to that from the BP&P coalescent species-tree analysis, show that the three specimens of L. idabeli form a monophyletic group with strong support (1.0 posterior probability; 100 bootstrap). The BP&P species delimitation analysis had overwhelming support for a 10-species model (posterior probability 0.997) versus models with fewer or more species, indicating perfect congruence between morphological and molecular delimitation approaches.
Our analyses show L. idabeli as part of a well-supported clade containing L. regia, L. levinsoni, and L. anabantoides, with the relationships within this clade being less resolved. All of these species occur from the mid-to-upper rariphotic zone and shallower (20-165 m). Similar to our observations of species of Lipogramma occurring off Curacao and other localities in the Caribbean (Baldwin et al. 2016(Baldwin et al. , 2018, species off Roatan appear to be partitioning the reef by depth and microhabitat association. The known depth range for L. idabeli off Roatan, based on collected specimens and visual observations, is 122-165 m. At Roatan, its depth range broadly overlaps with that of L. levinsoni, but the two species occupy very different microhabitats. Lipogramma levinsoni is typically found hovering around and above limestone rubble and small cobble habitats on gradual slopes, whereas L. idabeli is found around larger rocks, caves, and outcroppings on steeper slopes and vertical walls. In addition to L. idabeli and L. levinsoni, other nominal species of Lipogramma observed at Roatan include L. klayi, L. evides, and L. flavescens. Like L. idabeli, L. klayi also occurs around steep walls but at depths considerably shallower than L. idabeli (~65-120 m versus 122-165 m), where the reef wall is generally covered with more extensive growth of Halimeda, encrusting sponges, gorgonians, and other sessile habitat-forming organisms. Both L. flavescens and L. evides were observed deeper than L. idabeli at 213-250 m, with L. evides usually occurring on gradual rocky slopes with a heavy layer of cobbles (similar to the habitat of L. levinsoni), and L. flavescens found out in the open on bottoms of coarse sand with small, low, scattered piles of rock and rubble, far from the wall.  DRR, and funds from the University of Washington School of Aquatic and Fishery Sciences to LT. Rob Robins at UF, and Diane Pitassy and Jeff Williams at USNM assisted with cataloging specimens. We are indebted to Claudia Lardizabal and Alex Vallejo at Universidad Nacional Autónoma de Honduras -Valle de Sula for helping with research permits and deposition of specimens in the MUVS collection. This is Ocean Heritage Foundation/ Curacao Sea Aquarium/Substation Curacao (OHF/SCA/SC) contribution #35.