First record of Larsonella pumilus (Teleostei: Gobiidae) from Japan, with phylogenetic placement of the genus Larsonella

During a survey of deep-sea fauna, using a Remotely Operated Vehicle, a single specimen (21.6 mm in standard length) of Larsonella pumilus (Larson & Hoese, 1980) was collected at a depth of 214 m off the coast of Okinawa Island, Japan. It represents the first record of this species from Japan. The collection site was far deeper than previous reports for this species. This suggests that the main habitat of L. pumilus is deeper than previously recognized and it may explain the paucity of records of this species. As the previously available morphological description of L. pumilus was based on only a single specimen (holotype), this new specimen is described herein. Its morphology corresponds closely to the original description of the holotype, except that faint melanophores are arranged radially around the eyes and scattered on the trunk and the fins. Mitochondrial genome sequences of L. pumilus and 19 related species demonstrate close relationships between L. pumilus and the genus Priolepis . These data also indicate that the genus Priolepis is not monophyletic. expressed as a percentage of standard length (SL). Measurements and counts follow Nakabo (2002), with the following modifications: body depth was measured at the pelvic- and anal-fin origins; length of first dorsal-fin base was measured from the origin of first dorsal fin to the base of the spine of the second dorsal fin. Scales and cephalic sensory organs were observed after staining with cyanine-blue solution. Teeth and osteological features were observed using computed tomography (CT) data. In order to observe nonde-structively, the specimen was placed in a plastic pack containing water and scanned with X-ray microcomputed tomography (micro-CT) R_mCT2 (Rigaku Co., Tokyo, Japan) at an X-ray setting of 90 kV and 200 mA. Micro-CT images were reconstructed from CT-slices (0.02 mm interval) using DICOM editing software AZE VirtualPlace (AZE Ltd., Tokyo, Japan). Teeth were also observed with a stereomicroscope. The dorsal-fin pterygiophore formula follows Birdsong et al . (1988). Color in life was described from a photograph taken when it was alive (Fig. 1A). Symbolic codes used to represent collections and institutions follow Sabaj (2016), except OCF (Okinawa Churashima Foundation Research Center).


Introduction
The order Gobiiformes (= suborder Gobioidei in Nelson 2006) is a very diverse fish taxon, including more than 2,000 goby species, belonging to more than 300 genera (Nelson et al. 2016). Gobies are distributed worldwide and occur in fresh, brackish, and marine waters. In the sea, they occupy nearshore environments, including rocky and sandy bottoms, mudflats, tidepools, coral reefs, and caves (Patzner et al. 2011). Although the deep sea may seem to be atypical goby habitat, more than ten genera (Antilligobius, Karsten, Lepidogobius, Lesueurigobius, Obliquogobius, Palatogobius, Pinnichthys, Priolepis, Sufflogobius, Suruga, Thorogobius, and Varicus) have been reported from >200 m depth (Eschmeyer & Herald 1983;Goren 1992;Goren & Baranes 1995;Bianchi et al. 1999;Shinohara et al. 2001;Greenfield 2002;Murdy 2002;Mytilineou et al. 2005;Shibukawa & Aonuma 2007;Tornabene et al. 2016;Baldwin et al. 2018;Sauberer et al. 2018). However, information about deep-sea gobies is extremely limited. Probably due to their small body sizes (usually <10 cm) and their cryptic natures (often dwelling in substrata or abandoned invertebrate exoskeletons), gobies are not easily detected. Most deep-sea gobies recorded were collected by trawling or dredging. We collected a small goby specimen identified as Larsonella pumilus from a depth of 214 m off the coast of Okinawa Island, Japan, using a Remotely Operated Vehicle (ROV) during a survey of deep-sea fauna.
Larsonella pumilus was originally described by Larson & Hoese (1980) as Lubricogobius pumilus. Subsequently, Randall & Senou (2001) reviewed the genus Lubricogobius and established a new genus Larsonella for this species on the basis of its depressed head, slenderer body, lack of a pelvic frenum, five rows of sensory papillae that radiate perpendicularly from the lower edge of the orbit, a snout longer than the eye, and the presence of scales posteriorly on the body. Currently L. pumilus is the only member of the genus Larsonella. The phylogenetic placement of Larsonella has not been studied, but Hoese & Larson (2010) suggested close relationships among Larson-ella, Lubricogobius, and Priolepis based on their morphological similarity. On the other hand, Thacker (2015) put Larsonella and Lubricogobius into the "Gobiodon lineage" sensu Agorreta et al. (2013) (= the "coral gobies" group  of Thacker & Roje 2011) with Gobiodon, Eviota, etc., rather than into the "Priolepis lineage" sensu Agorreta et al. (2013) (= the "tiny banded gobies" group of Thacker & Roje 2011).
In this paper, we report the first record of Larsonella pumilus from Japan. The morphology of this specimen is described because all morphological information about L. pumilus (see Hoese 1980 andSenou 2001) is based only on a single specimen (holotype) and our material had melanophores which are not described for the holotype. We also discuss the phylogenetic placement of Larsonella based on mitochondrial genome sequences of Larsonella pumilus and species in related genera.

Materials and methods
Sampling. A single specimen of Larsonella pumilus was collected with invertebrate specimens (see details in the "Habitat" section) on a muddy bottom at a depth of 209-220 m off East China Sea coast of Seragaki, Onna Village, Okinawa Island, Japan (26°31′N, 127°52′E), during a survey of deep-sea fauna. Sampling was conducted using a ROV (LEO, KOWA Corporation, Osaka, Japan) on 11 August 2017. The specimen was kept alive in a tank, and fixed in 10% formalin after it died on 22 November 2017, and then preserved in 70% ethanol. A piece of muscle was removed from a damaged part of the left side of the body prior to 10% formalin fixation and was preserved in 99.5% ethanol for mitochondrial DNA analysis.
Morphological observation. Measurements and counts were taken from the right side of the fish, because of damage to the left side. Measurements were made point-to-point to the nearest 0.1 mm, using a vernier caliper or a divider under a stereomicroscope, and were expressed as a percentage of standard length (SL). Measurements and counts follow Nakabo (2002), with the following modifications: body depth was measured at the pelvic-and analfin origins; length of first dorsal-fin base was measured from the origin of first dorsal fin to the base of the spine of the second dorsal fin. Scales and cephalic sensory organs were observed after staining with cyanine-blue solution. Teeth and osteological features were observed using computed tomography (CT) data. In order to observe nondestructively, the specimen was placed in a plastic pack containing water and scanned with X-ray microcomputed tomography (micro-CT) R_mCT2 (Rigaku Co., Tokyo, Japan) at an X-ray setting of 90 kV and 200 mA. Micro-CT images were reconstructed from CT-slices (0.02 mm interval) using DICOM editing software AZE VirtualPlace (AZE Ltd., Tokyo, Japan). Teeth were also observed with a stereomicroscope. The dorsal-fin pterygiophore formula follows Birdsong et al. (1988). Color in life was described from a photograph taken when it was alive (Fig. 1A). Symbolic codes used to represent collections and institutions follow Sabaj (2016), except OCF (Okinawa Churashima Foundation Research Center).
Mitochondrial DNA analysis. Total genomic DNA was extracted from muscle of the Larsonella pumilus specimen, and right pectoral fins, right eyes, or muscle piece of 19 related species preserved in 99.5 % ethanol, using Maxwell RSC Blood DNA Kit (Promega, Fitchburg, Wisconsin, USA) or DNeasy Blood & Tissue Kit (Quiagen, Hilden, Germany). Whole-genome shotgun sequencing libraries were prepared using a KAPA HyperPlus Kit, PCR-free (KAPA Biosystems, Wilmington, Massachusetts, USA). Extracted genomic DNA was enzymatically fragmented into pieces of 200-1000 bp. After repairing protruding ends and A-tailing, sequencing adaptors were ligated onto both ends of DNA fragments. Shotgun libraries were then sequenced on an Illumina HiSeq 2500 sequencer (Illumina, San Diego, California, USA) in Rapid Run mode version 2 using a HiSeq Rapid Cluster Kit v2 -Paired-End (Illumina) and a HiSeq Rapid SBS Kit v2 (Illumina) or an Illumina HiSeq 4000 sequencer (Illumina) using HiSeq 3000/4000 PE Cluster Kit (Illumina) following manufacturer's instructions.
Sequencing data from each library were assembled with the IDBA_UD assembler version 1.1.1 (Peng et al. 2012) with different kmer lengths (60, 80, 100). Identification of complete mitochondrial genomes from assembled contigs was performed by 1) comparing them with the complete Stiphodon alcedo mitochondrial genome (accession: AB613000.1) (BLASTN e-value ≤ 1e-100), and by 2) confirming that 100 bp of both head and tail DNA sequences of a contig were identical, indicating that the sequence was circular. Complete mitochondrial genomes were aligned using MAFFT v7.244 (Katoh & Standley 2013) and all positions with gaps were removed using tri-mAl (Capella-Gutierrez et al. 2009). Phylogenetic model selection for aligned whole mitochondrial genomes was performed using ModelTest-NG version 0.1.5 (Darriba et al. 2019). We performed molecular phylogenetic analyses FIRST RECORD OF LARSONELLA PUMILUS FROM JAPAN Zootaxa 4695 (4) © 2019 Magnolia Press · 369 of aligned mitochondrial genomes using the GTR+I+Gamma model suggested by the above evolutionary model selection. Maximum likelihood (ML) analysis was performed using RAxML version 8.2.3 (Stamatakis 2014) with 100 bootstrap replicates, and Bayesian inference analysis was performed using MrBayes version 3.2.7 (Ronquist et al. 2012) with one million generations. All sequenced raw data are available in the DDBJ Sequence Read Archive under BioProject accession number PRJDB5763. Assembled mitochondrial genome sequences with gene annotations are available in the DDBJ under accession numbers: AP019315-AP019347. Accession numbers for each individual are shown in Table 1.     Table 2. Head depressed, trunk nearly cylindrical, and tail compressed. Eyes located dorsolaterally. Mouth strongly oblique with angle to body axis about 60 degrees. Lower jaw protruding beyond upper jaw. Posterior end of upper jaw reaching below middle point between anterior margin of iris and anterior margin of pupil. Canine-like teeth aligned on edges of anterior halves of premaxilla and dentary; four and three teeth on one side of premaxilla and dentary, respectively; posterior teeth larger ( Fig. 2A). An inner row of conical teeth extending from anterior part to more posterior part of dentary than the outer canine-like teeth row (Fig. 2B). Additional small conical teeth observed on inner parts of premaxilla and dentary with a stereomicroscope, but no such small teeth with micro-CT.

Description. Counts and measurements are shown in
First dorsal fin with six spines. Second dorsal fin with one spine and nine soft rays. First and second dorsal fins connected by a low membrane behind last spine of the first dorsal fin. Anal fin with one spine and eight soft rays. Caudal fin rounded, with 17 segmented rays. Pectoral fin with 18 soft-rays. Pelvic fin with one spine and five soft rays. Posterior tips of pectoral fins reaching or exceeding position of anus, according to a photograph taken in life ( Fig. 1A), although they are broken in the preserved specimen. Pelvic fins without frenum. Vertebrae 10 + 16 = 26; dorsal-fin pterygiophore formula 3-22110; epural 1; anal-fin pterygiophores anterior to first haemal spine 2.
Head and body largely naked except for posterior part of caudal peduncle, involving three rows composed of 7-10 ctenoid scales along dorsal midline. A row of three ctenoid scales along lateral midline, and three rows composed of 6-8 ctenoid scales along ventral midline (Fig. 3). No sensory canals or associated pores on head. Cephalic sensory papillae patterns illustrated in Fig. 3. Infraorbital area with five transverse rows of sensory papillae.
Color in preservative (Fig. 1C): Background of head and tail white, trunk yellowish white. All fin membranes transparent. Melanophores scattered on dorsal half of trunk and on membranes of first and second dorsal, anal, and pelvic fins. Pectoral fin also with a few melanophores. Three rows of tiny melanophores arched between right and left eyes (Fig. 4A). Infraorbital area with two transverse rows of tiny melanophores (Fig. 4B) and another row of tiny melanophores behind eyes (Fig. 4B). These melanophore rows arranged radially around eyes.  Color in life (Fig. 1A): Body and all fins yellow or yellowish orange. Arrangement of melanophores same as after preservation, described above (Fig. 1A).
Habitat. We collected ten specimens of five invertebrate species from the muddy bottom at a depth of 209-220 m using the ROV, on 11 August 2017. These included five live comb jellies, Lyrocteis imperatoris, a sea cucumber, Holothuria dura, a starfish, Asterodiscides japonicus, a heart urchin, Pericosmus sp., and an empty shell of Xenophora chinensis (Fig. 5). After these animals were put into a tank on the boat, the L. pumilus specimen was found FIRST RECORD OF LARSONELLA PUMILUS FROM JAPAN Zootaxa 4695 (4) © 2019 Magnolia Press · 373 in the tank. Because Lubricogobius species, close relatives of Larsonella pumilus, often inhabit empty shells, sea urchin tests, tunicate siphons, bottles, etc. (Randall & Senou 2001;Allen & Erdmann 2016), we believe that the L. pumilus was inside the empty shell of X. chinensis (collected at a depth of 214 m) and was collected with it.

Discussion
Identification. The present specimen was identified as Larsonella pumilus (Larson & Hoese, 1980) as its morphological characters correspond almost exactly to those of the holotype described by Larson & Hoese (1980), although some characters, including fin morphology and nare shapes could not be confirmed due to their damaged condition. The only major difference found was the presence of melanophores. Larson & Hoese (1980) noted "no traces of dark pigment", while our specimen has melanophores arranged radially around eyes and scattered on trunk and fins. Because these melanophores were very small and invisible to the naked eye, we suppose that the melanophores were overlooked in the original description. It is also possible that melanophores vary individually or depending on body size and/or sex (we could not determine the sex of our specimen). Distribution and habitat. There have been few records of this species since the holotype of Lubricogobius pumilus was collected from the Indian Ocean off Somalia using an Agassiz trawl at a depth of 37-38 m (Larson & Hoese 1980). Other available records from the Indian Ocean are from La Digue in the Seychelles (4°23'S 55°49'E) from dredging at a depth of 30 m (Randall & van Egmond 1994;BPBM 35515 and ROM 66149;as L. pumilis) and from the northwest shelf of Australia (16°49'S to 19°52'S) (Hoese & Larson 2006). This species also appeared in a list of marine and brackish water goby species in the western central Pacific (Larson & Murdy 2001), but detailed locality information was not provided. According to the GBIF database (GBIF Secretariat 2017), L. pumilus was also collected in New Caledonia and Tonga (69 and 67 m in depth, respectively). The specimen reported in the present study was caught at a depth of 214 m off Okinawa Island. This is the first record of L. pumilus from Japan and the only report substantiated with a voucher from the Pacific Ocean. The depth is far greater than other records (214 m vs. 30-69 m). Thus, the main habitat of L. pumilus may be deeper sea than previously recognized. This may also explain the paucity of records since this area has been poorly explored. It also suggests that this species is distributed much more widely in the Indo-Pacific.
Phylogenetic placement. Among their hypothesized lineages based on molecular data, morphological characters, and literature review, Thacker (2015) placed the genus Larsonella into the "Gobiodon lineage (sensu Agorreta et al. 2013)" with the genera Bryaninops, Eviota, Gobiodon, etc. However, our results clearly demonstrate that L. pumilus belongs to the "Priolepis lineage (sensu Agorreta et al. 2013)" with Priolepis spp. and Trimma spp., not to the "Gobiodon lineage" (Fig. 6). Our tree also indicates that the genus Priolepis is not monophyletic, as L. pumilus nested within a clade composed of three species of Priolepis. But we could analyze only three of 35 species in Priolepis and no Lubricogobius species were involved in the present study, although L. pumilus is considered to be a close relative of Lubricogobius (Hoese & Larson 2010). Hoese & Larson (2010) doubted the validity of Larsonella based on the fact that a species of Lubricogobius actually has scales on its caudal peduncle, which is one of the major diagnostic characters to distinguish Larsonella from Lubricogobius. The mitochondrial genome sequence of L. pumilus included in the present study will help future research to review phylogenetic relationships among species in the genera Priolepis, Lubricogobius, and Larsonella.
Remarks. Larsonella pumilus is mentioned as "Larsonella pumila" in some online databases (e.g. Fricke et al. 2018;Froese & Pauly 2018), although no author spelled it this way. This species was originally described as a new species in Lubricogobius and the specific epithet is considered as a noun as the authors stated "Derivation of name: Latin -pumilus = dwarf fish" (Larson & Hoese 1980). Therefore, the original spelling of the specific epithet is to be retained with gender ending unchanged, even if gender of the generic name is changed. Although the specific epithet of this species is spelled "pumilis" in figure 536 of Larson & Hoese (1980) and Randall & van Egmond (1994), this is also a misspelling.