Neoniphon pencei, a new species of holocentrid (Teleostei: Beryciformes) from Rarotonga, Cook Islands

Abstract Neoniphon pencei, n. sp., is described from thirteen specimens, 132-197 mm standard length (SL) collected from mesophotic coral ecosystems (MCEs) at Rarotonga, Cook Islands by divers using mixed-gas closed-circuit rebreathers. It differs from all other species of the genus in number of lateral line scales, scales above and below lateral line, elements of life color, and in COI and cytochrome b DNA sequences. Of the five other known species of Neoniphon, it is most similar to the Indo-Pacific N. aurolineatus and the western Atlantic N. marianus both morphologically and genetically.

Introduction marianus (n=1) collected from the Commonwealth of the Bahamas; and N. argenteus (n=6) collected from the Republic of Kiritimati. Total genomic DNA was extracted from each sample using the 'HotSHOT' protocol (Meeker et al. 2007). A 577-bp fragment of the mtDNA cytochrome b (Cyt b>) region was amplified using modified primers from Song et al. (1998) (5'-TGAAGTTGTCGGGATCTCCT-3') and Taberlet et al. (1992) (5'-TGCCGTGACGTAAACTATGG-3'). Polymerase chain reaction (PCR) was performed in a 15 µl reaction containing 7.5 µl BioMix Red (Biolone Inc., Springfield, NJ, USA), 0.2 µM of each primer, 5-50 ng template DNA, and nanopure water (Thermo Scientific* Barnstead, Dubuque, IA, USA) to volume. PCR cycling parameters were as follows: initial 95°C denaturation for 10 min. followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, followed by a final extension of 72°C for 10 min. PCR products were visualized using a 1.5% agarose gel with GelStarTM (Cambrex Bio Science Rockland, Inc., Rockland MA, USA) and then cleaned by incubating with 0.75 units of Exonuclease and 0.5 units of Shrimp Akaline Phosphate (ExoSAP; USB, Cleveland, OH, USA) per 7.5 µl of PCR product for 30 min. at 37°C followed by 85°C for 15 min. Sequencing was conducted in the forward direction and reverse direction when needed using a genetic analyzer (ABI 3130XL, Applied Biosystems, Foster City, California) at the Hawai'i Institute of Marine Biology EPSCoR Sequencing Facility. The sequences were aligned, edited and trimmed to a common length using Geneious Pro (v.5.6.6) DNA analysis software (Drummond et al. 2012). Twelve representative Cyt b> haplotypes were deposited in GenBank (accession numbers KJ188431-188436 and KJ201921-201926). jModelTest v.2.1.4 (Darriba et al. 2012, Guindon andGascuel 2003) was used with an Akaike information criterion (AIC) test to determine the best nucleotide substitution model for the data. The GTR+G model with gamma parameter 0.1840 was identified to be the best suited model for phylogenetic inference. Maximum Likelihood, Neighbor-Joining, and Maximum Parsimony tree-building methods were implemented using Mega v.5.2.2 (Tamura et al. 2011). Sargocentron rubrum (Genbank accession number AP004432.1) was used to root a maximum likelihood phylogenetic reconstruction. Clade support was evaluated by bootstrapping 1,000 replicates in all cases (Felsenstein 1985).
Color in life (Figs 1, 2, 3): Body silvery white with an orange-red tint above lateral line. Scales above lateral line with orange-red borders. Approximately eleven red to orangered stripes following and sometimes bisecting scales of each horizontal scale row, width of stripes on body alternating between very narrow stripes and stripes over three times wider, except for two consecutive wide stripes, numbers six and seven counted ventrally from the thin dorsal-most stripe, the eleventh ventral-most stripe thin and barely visible on some specimens. Preopercle silvery white with a narrow orange-red posterior border, faint on some specimens. Opercle, nape and interorbital space orange-red. Prominent red bar of less than pupil width extending across nape to level of pectoral axil when viewed underwater. Pectoral axil orange-red. Dorsal fin spines and rays light orange red. Membranes of spinous portion of dorsal fin red with white tips and a white semicircular spot encompassing the middle vertical third of each membrane, its greatest length along the preceding anterior spine and not extending to the posterior spine. Some specimens without a white spot on the first membrane. Soft dorsal fin, pectoral fin, anal fin and pelvic fins with transparent membranes, except anal fin with translucent white membrane between longest spines. Pectoral fin and pelvic fin rays with faint pinkish tint. Anal fin spines white with a faint orange tint on some specimens. Anal fin rays orange-red. Caudal fin rays orange-red, faint on inner rays, membranes translucent white.
Color in alcohol: Body pale yellowish-white. Narrow orange-tan stripes bisecting scales of horizontal scale rows, except for lateral line scale row, the stripes above lateral line faint, barely visible on some specimens. Preopercle white with narrow yellow-tan border. Opercle, nape and interorbital space yellow-tan. Spinous dorsal fin membranes translucent with a white tint. Soft dorsal fin, anal fin pectoral fin and pelvic fin membranes transparent. Caudal fin with orange-tan blotch on upper and lower base, extending faintly on to upper and lower rays. Middle third of caudal fin rays and membranes transparent.

Etymology
Named for David F. Pence, Dive Safety Officer for the University of Hawai'i, a member of the deep diving team that discovered this species, in recognition of his efforts to collect the type specimens.

Distribution
All type specimens of N. pencei were collected at Rarotonga, Cook Islands. An individual Neoniphon closely matching the life colors of N. pencei (and different from all other known species) was captured on video by Robert K. Whitton at a depth of 90 m at Moorea, in February 2012 (Fig. 3). It is likely that the species is more broadly distributed throughout the southeastern tropical Pacific, but has escaped noticed due to insufficient collecting activities at mesophotic depths in this region.

Genetic results
After alignment and editing, a 377-bp partial sequence of Cyt b was obtained for all thirtyfive Neoniphon samples, resulting in twelve unique haplotypes. All three phylogenetic methods used resulted in congruent tree topologies and are presented as a Maximum Likelihood reconstruction (Fig. 4). Phylogenetic reconstruction recovered strong support for clades corresponding to known Neoniphon species. The species N. pencei showed strong clade support (100% bootstrap support for all three methods) for belonging to a single clade distinct from currently described Neoniphon species. There was not enough signal to resolve the sister relationship between some members within the genus Neoniphon; however, this description is not necessary for the goals of this study. Neoniphon pencei shows 9-12.5% uncorrected sequence divergence and 34-47 mutations between all other known Neoniphon species and posesses 8 diagnostic sites unique from all other species of Neoniphon within this this region of Cyt b. This is consistent with species level sequence divergence found in other fish taxa (Bellwood et al. 2004, Fessler and Westneat 2007, Randall and Rocha 2009, Rocha 2004, Rocha et al. 2008).

Discussion
Most recent authors who have reported on Neoniphon (e.g., Randall and Heemstra 1985, Randall and Heemstra 1986, Kotlyar 1996, Kotlyar 1998, Randall and Greenfield 1999, Greenfield 2003, Satapoomin 2009) consider it to be a valid genus (a senior synonym of Flammeo), distinct from other genera in the subfamily Holocentrinae (particularly Sargocentron; Fowler 1904), primarily on the basis of the position of the last dorsal-fin spine (relative to the penultimate dorsal-fin spine and first dorsal-fin ray), and the protruding lower jaw in species of Neoniphon (Randall and Heemstra 1985). A more recent phylogenetic analysis of holocentrids by Dornburg et al. (2012), however, reported evidence that Sargocentron and Neoniphon are paraphyletic. Specifically, they found that four of the five species of Neoniphon (they did not include N. aurolineatus in their analyses) cluster among several subclades that include nine of the seventeen species of Sargocentron they analyzed ( S. coruscum, S. diadema, S. inaequalis, S. ittodai, S. microstoma, S. punctatissimum, S. suborbitalis [=suborbitale], S. vexillarium and S. xantherythrum). The other eight species of Sargocentron they analyzed ( S. caudimaculatum, S. cornutum, S. melanospilos, S. praslin, S. rubrum, S. seychellense, S. spiniferum and S. tiere) form a separate clade (their "Sargocentron group 1"). They argue that the characters used to differentiate these species are ecologically plastic and therefore current relationships represent ecotypes rather than their evolutionary relationships. We acknowledge the results of this study and welcome a new comprehensive analysis of the entire Holocentrinae in light of new genetic evidence. However, in the absence of observed morphological characters that are consistent with the genetic results, we choose to retain these six species within the genus Neoniphon, to the exclusion of Sargocentron, thereby maintaining nomenclatural stability. Neoniphon pencei clearly differs from all species placed in the genus Sargocentron on the basis of a closer association of the last dorsal-fin spine with the first soft-ray rather than the penultimate spine and the strongly protruding lower jaw (Randall and Heemstra 1985) as well as life color.
Meristic data of the type specimens of Neoniphon pencei are included in Table 1, and proportional measurements are included in Table 2. Neoniphon pencei is distinctive from all other species of holocentrids, both morphologically and genetically. Table 3 summarizes morphological differences between N. pencei and other species in the genus. It differs most substantially from all other Neoniphon in number of lateral line scales (48-52, compared with 38-47 among all other species), number of scales above the lateral line to the origin of the dorsal fin (5, compared with 2.5-3.5) and number of scales below the lateral line to the origin of the anal fin (6-7, compared with 7-9). It also differs from N. aurolineatus, N. opercularis, and N. argenteus in proportional length of the upper-jaw (2.3-2.6 in head length, compared with 2.0-2.3), and proportional length of of the third and fourth anal spines (1.1-1.3 and 1.7-2.0, compared with 1.4-1.9 and 1.9-2.7, respectively). It is further distinguished from N. aurolineatus in total number of gill rakers (19-20, compared with 15-17); from N. opercularis in head length (2.6-2.9 in SL, compared with 2.9-3.1), orbit diameter (1.2-1.4 in head length, compared with 3.0-3.5), snout length (1.2-1.4 in orbit diameter, compared with 1.2-1.5), and interorbital width (1.7-1.9 in orbit diameter, compared with 1.2-1.5); from N. argenteus in number of pectoral rays (14, compared with 12-13), interorbital width (1.7-1.9 in orbit diameter, compared with 1.2-1.7), and first dorsalspine length (3.3-4.1 in head length, compared with 2.4-3.1); and from N. sammara in number of soft dorsal-fin rays (13, compared with 11-12), interorbital width (1.7-1.9 in orbit diameter, compared with 1.3-1.6), and first dorsal-spine length (3.3-4.1 in head length, compared with 2.2-3.0). In addition to these morphometric characters, N. pencei differs from all other species of Neoniphon in life color, particularly in the pattern of white spots on the dorsal fin and overall body color, and the lack of yellow coloration on the body (as in N. aurolineatus and N. marianus). Genetically, it differs in its Cyt b sequence from N. argenteus by 9.8%, N. aurolineatus by 9-9.6%, N. marianus by 11.7%, N. opercularis by 9.8%, and N. sammara by 12-12.5%.  Neoniphon pencei appears most similar to N. aurolineatus and N. marianus, based on having the the fewest number of differences in morphometrics, greatest genetic similarity, and most similar aspects of life coloration with these two species. It is also similar to N. aurolineatus in the depth and habitat it occupies. However, the differences between N. pencei and these two species as noted above clearly warrant recognition of N. pencei as a distinct species. A more comprehensive phylogenetic analysis of the species of Neoniphon and related genera based on both morphology and genetics (with verified voucher specimens) is beyond the scope of this work.  Table 3.
Characters that differ from N. pencei are shown in bold. 1 as a proportion of Standard Length; 2 as a a proportion of orbit diameter; 3 as a proportion of head length.