Description of a new deep-water dogfish shark from Hawaii, with comments on the Squalus mitsukurii species complex in the West Pacific

Toby S. Daly-Engel; Amber Koch; James M. Anderson; Charles F. Cotton; R. Dean Grubbs

doi:10.3897/zookeys.798.28375

Abstract

Dogfish sharks of the genus Squalus are small, deep-water sharks with a slow rate of molecular evolution that has led to their designation as a series of species complexes, with low between-species diversity relative to other taxa. The largest of these complexes is named for the Shortspine spurdog (Squalus mitsukurii Jordan & Snyder), a medium-sized dogfish shark common to warm upper slope and seamount habitats, with a putative circumglobal distribution that has come under investigation recently due to geographic variation in morphology and genetic diversity. The Hawaiian population of Squalus mitsukurii was examined using both morphological and molecular analyses, putting this group in an evolutionary context with animals from the type population in Japan and closely-related congeners. External morphology differs significantly between the Hawaiian and Japanese S. mitsukurii, especially in dorsal fin size and relative interdorsal length, and molecular analysis of 1,311 base pairs of the mitochondrial genes ND2 and COI show significant, species-level divergence on par with other taxonomic studies of this genus. The dogfish shark in Hawaii represents a new species in the genus, and the name Squalus hawaiiensis, the Hawaiian spurdog, is designated after the type location.

Keywords

Chondrichthyes , DNA barcoding, Elasmobranchii , morphology, Squalus hawaiiensis , taxonomy

Introduction

Deep-water sharks like the dogfish sharks (Squaliformes, Squalidae) and the gulper sharks (Squaliformes, Centrophoridae) have proven confounding groups for systematists to resolve due to their highly conserved morphology, wide ranges, and patchy, infrequently-sampled distributions (Veríssimo et al. 2014; Cotton and Grubbs 2015; Veríssimo et al. 2017; Daly-Engel et al. submitted). Recent years have shown that DNA sequencing in conjunction with morphological analyses is an effective approach for elucidating the alpha taxonomy of deep-water sharks (Avise 2004; Ward et al. 2005; Last et al. 2007c; Ebert et al. 2010; Veríssimo et al. 2014; Pfleger et al. 2018). Findings from several studies have shown a mix of low genetic distances between well-established morphological species, and deep genetic splits between animals identified as conspecifics (Daly-Engel et al. 2010; Veríssimo et al. 2017; Daly-Engel et al. 2018; Pfleger et al. 2018; Daly-Engel et al. submitted).

Taxonomic delineation that incorporates DNA analysis has often relied upon consistencies among within- and between-species divergences in the barcoding gene (COI), as measured by percent nucleotide sequence variation (Avise 2004; Ward et al. 2005; Naylor et al. 2012). DNA barcoding is an effective, widely-used molecular method among taxonomists because the cytochrome oxidase I gene (COI) records a low rate of mutation compared with other loci (Avise 2004; Ward et al. 2005). While a reliable metric among distantly-related groups in which mutations accumulate consistently, COI may fail to elucidate shallow divergences among taxa with low genetic diversity. Complicating identification issues is the fact that deep-water sharks, whose cold environment results in low metabolic rates relative to other elasmobranchs, may undergo an overall slower rate of molecular evolution compared with shallow coastal species (Martin et al. 1992; Martin and Palumbi 1993). As a result, a number of investigators have found the more-rapidly evolving ND2 gene to be an effective genetic marker for estimating both inter- and intraspecific variation in dogfish sharks (Veríssimo et al. 2010; Naylor et al. 2012; Veríssimo et al. 2017; Pfleger et al. 2018).

Much work among shark systematists has focused on clarifying species delineations in dogfishes of the genus Squalus, an abundant, speciose, globally-distributed group of morphologically-similar, small-bodied demersal sharks that primarily inhabit circumglobal shelf and slope habitats from 100 - 1000 m depth (Compagno et al. 2005; Last et al. 2007c). Within this genus, genetic and morphological examinations have revealed a series of species complexes characterized by relatively shallow evolutionary divergences among putative species (Last et al. 2007c; Gaither et al. 2016; Veríssimo et al. 2017). Such complexes are not unusual in nature, having been observed across a variety of phyla from insects (Perring 2001) and nematodes (Chilton et al. 1995) to bony fishes (Barluenga and Meyer 2004), and have been shown to harbor “cryptic” diversity not always apparent from morphology alone (Daly-Engel et al. Submitted). Taxonomic reevaluation of Squalus in the Indo-Pacific and elsewhere has revealed many undescribed species that were historically lumped together under a single name (Ward et al. 2007; Naylor et al. 2012; Viana et al. 2016; Pfleger et al. 2018): although the relatively shallow spiny dogfish Squalus acanthias Linnaeus comprises just one wide-ranging species apart from the North Pacific (Ebert et al. 2010; Veríssimo et al. 2010), we now know that both the Shortspine dogfish shark Squalus mitsukurii Jordan & Snyder and the shortnose dogfish shark Squalus megalops Macleay comprise global species complexes (Veríssimo et al. 2010; Naylor et al. 2012; Veríssimo et al. 2017; Pfleger et al. 2018; Daly-Engel et al. Submitted).

Recent taxonomic studies on Squalus have focused on Squalus mitsukurii, a putative circumglobal species found on continental and insular shelves and upper slopes and on seamounts between 100 and 950 m depth (Compagno et al. 2005). Re-examination of local S. mitsukurii stocks has revealed many new species, including four from the West Pacific alone: S. formosus White & Iglesias (2011), S. chloroculus Last, White, & Motomura (Last et al. 2007b), S. montalbani Whitley (Last et al. 2007b), and S. griffini Phillips (Duffy and Last 2007). Other revisions of S. mitsukurii have been done in the Atlantic using either genetic tools or morphological characters (Viana et al. 2016; Veríssimo et al. 2017), though not both (but see Pfleger et al. 2018).

Along the Hawaiian Archipelago in the Central Pacific, the Shortspine spurdog (Squalus cf. mitsukurii) is the only Squalus species known, aggregating in large numbers on or near the bottom at a depth of 100–950 m (Wilson and Seki 1994). Observable differences between specimens of S. mitsukurii in Hawaii and its conspecifics from the West Pacific first came to light during a genetic study (Daly-Engel et al. 2010), and subsequent research showed that growth (L_∞, k) and reproductive parameters (size-at-maturity) for Squalus cf. mitsukurii in Hawaii differed from published data (putatively as S. mitsukurii) from other regions (Cotton et al. 2011). Together with the relative geographic isolation of the Hawaiian Islands and the high levels of endemism observed there [25% of fish species in Hawaii are endemic, the most in the Indo-Pacific region (Roberts et al. 2002; Randall 2007; Briggs and Bowen 2011)] make this population a likely candidate for redescription.

We undertook a taxonomic evaluation of Squalus mitsukurii from the Hawaiian Islands using molecular and morphological data, couching these in the evolutionary context of closely-related, previously-recognized congeners from the West Pacific. DNA barcoding with COI can discriminate among species in the genus Squalus (Ward et al. 2007), but low resolution in this marker may fail to identify cryptic diversity. We therefore supplemented DNA sequences derived from the COI barcoding gene with data from ND2, a faster-evolving mitochondrial gene with the potential to distinguish evolutionary relationships with a high degree of resolution (Avise 2004; Naylor et al. 2012). In addition, morphological and meristic comparisons were made comparing S. cf. mitsukurii from Hawaii with measurements taken from the Japanese holotype, and reported by Last et al. (2007c) and in Viana et al. (2016).

Materials and methods

Tissue collections

Whole specimens and genetic samples of Squalus cf. mitsukurii were collected primarily during longline surveys conducted on the insular slope around the Hawaiian Island of Oahu. Survey methods are described in Daly-Engel et al. (2010) and Cotton et al. (2011). Additional specimens and samples were collected from bottom fish surveys from Maui to Lisianski Atoll, spanning nearly 2,000 km of the Hawaiian Archipelago. Because the 2010 genetic study showed extremely low diversity, we deemed 5–10 specimens adequate for taxonomic evaluation, a number on par with other revisions in this genus (Ward et al. 2007; Pfleger et al. 2018). To that end, five whole mature females and three whole mature male S. cf. mitsukurii from Oahu were retained as voucher specimens for morphometric and genetic analyses. Small (< 1cm³) samples of fin or muscle tissue were taken using scissors and stored in 2 mL vials containing 1.5 mL 20% dimethylsulfoxide (DMSO) saturated salt (NaCl) buffer (Seutin et al. 1991) or >70% ethanol (EtOH).

Genetic examination of 130 tissue samples from 25–30 Squalus dogfish species has shown that S. cf. mitsukurii from Hawaii clustered closely with S. nasutus Last, Marshall, & White from Australia, S. japonicus Ishikawa from Japan and elsewhere, and S. mitsukurii from Japan (Daly-Engel et al. Submitted), and well apart from other congeners in the region; hence the current analysis focuses on these species. Because it is impossible to extract undamaged DNA from the formalin-fixed S. mitsukurii holotype, we referenced DNA extracted from two specimens identified as S. mitsukurii by expert Japanese systematist Dr. Sho Tanaka (Tanaka et al. 1975; Yano and Tanaka 1984; Yano et al. 2017) collected from Suruga Bay in mainland Japan, which is approximately 100 kilometers from the type locality of Misaki. Tissue samples of S. nasutus (N = 2) and S. japonicus (N = 8) were obtained from Australia and Japan (Appendix 1).

Genetic analysis

DNA was extracted from fin clips using a DNeasy Blood & Tissue Kit from Qiagen (Germantown, MD). Primers were obtained from Integrated DNA Technologies, Inc. (Coralville, Iowa). PCR reactions consisting of 7 μL BioMix Red from Bioline (London, UK) at the recommended concentration, 1 μL (3 μg) template DNA, and 1 μL (1.0 μM) each primer (10 μL total PCR volume). PCR amplification on a C1000 Touch Thermal Cycler (Bio-Rad; Hercules, California) consisted of an initial denaturation at 95 °C for 4 minutes followed by 36 cycles of 1 min at 95 °C, followed by 30s at 58 °C, and 30s at 72 °C with a final extension at 72 °C for 20 minutes. DNA from two mitochondrial genes were sequenced for a total of 1,131 base pairs (bp; Table 1): Cytochrome Oxidase I (COI; 602 bp), and NADH dehydrogenase 2 (ND2; 529 bp). COI primers were standard barcoding primers by Folmer et al. (1994), and NADH 2 primers that were utilized were designed by Veríssimo et al. (2010; Table 1). PCR products were cleaned with ExoFAP (Thermo Fisher Scientific, Waltham, Massachusetts) and sequenced on an Applied Biosystems 3730XL DNA Analyzer at the University of Arizona Genetics Core.

DNA sequences were trimmed in Geneious v9.1.4 (Kearse et al. 2012) and aligned using Mafft (Katoh et al. 2002) implemented in Geneious. MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) was used to construct Bayesian inference phylogenetic trees: first, analyses of Markov Chain Monte Carlo (MCMC) chains were run for 10,000,000 generations while sampling one tree per 100 generations. Convergence between simultaneous runs was reached when the average standard deviation of split frequencies fell below 0.01 (Ronquist et al. 2005). Following a burn-in phase of 10,000 steps (10% of generations discarded), parameter values were averaged, and posterior clade probabilities were calculated and the likelihood scores for all the topologies averaged. PhyML v3.0 (Guindon et al. 2010) implemented in Geneious was used to construct a maximum likelihood (ML) tree with 10,000 bootstrap replicates, which was compared with the Bayesian topology (Figure 1). TCS networks (Clement et al. 2000) and gene diversity statistics were generated in PopART (Leigh et al. 2014), while mutational model was calculated for each gene independently using jModeltest (Table 1; Posada 2008). Genetic distance expressed as percent sequence divergence (Table 2) was calculated in MrBayes (Huelsenbeck and Ronquist 2001).

Figure 1.

Phylogenetic tree of concatenated ND2 and COI sequences. Bayesian phylogenetic tree of concatenated mitochondrial ND2 and COI sequences for Squalus species used in this study, which was concordant with maximum likelihood methods. Numbers at nodes represent maximum likelihood bootstrap support/Bayesian posterior probability.

Table 1.

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Details and diversities of genetic loci amplified in Squalus hawaiiensis sp. n. Abbreviations: COI = Cytochrome oxidase I; ND2 = ND2 dehydrogenase 2; N = number of individual specimens included in the analysis; T_a = annealing temperature; π = nucleotide diversity; S = segregating sites; I = informative sites; H = number of haplotypes; h = haplotype diversity.

Gene	F primer	R primer	# bp	N	π	S	I	H	model	Citation
COI	LCO	HCO	602	20	0.0038	8	8	6	HKY	(Folmer et al. 1994)
ND2	ND2F	ND2R	529	20	0.0069	15	10	9	TRN	(Veríssimo et al. 2010)

Table 2.

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Genetic distances expressed as a percent divergence between Squalus species. Lower wedge is average between-species divergence in concatenated ND2 and COI genes; upper wedge is between-species divergence in each gene expressed as ND2/COI. Shaded boxes show within-species variation in concatenated sequences (top) and ND2/COI (bottom).

	S. mitsukurii	S. japonicus	S. hawaiiensis sp nov	S. nasutus
S. mitsukurii (N = 2)	0.09	1.07/0.30	1.01/0.50	1.70/0.70
S. mitsukurii (N = 2)	0.20/0.00	1.07/0.30	1.01/0.50	1.70/0.70
S. japonicus (N = 8)	0.71	0.12	0.84/0.50	1.07/0.70
S. japonicus (N = 8)	0.71	0.13/0.18	0.84/0.50	1.07/0.70
S. hawaiiensis sp nov (N = 8)	0.73	1.90	0.00	1.01/0.80
S. hawaiiensis sp nov (N = 8)	0.73	1.90	0.00/0.00	1.01/0.80
S. nasutus (N = 2)	1.15	0.87	0.91	0.09
S. nasutus (N = 2)	1.15	0.87	0.91	0.20/0.00

Morphometrics and meristics

Morphological measurements were used to discriminate between Japanese S. mitsukurii, including the holotype as measured by Last et al. (2007c) and Viana et al. (2016), and S. cf. mitsukurii collected from Hawaii. Measurements were performed on fresh specimens in accordance with conventional techniques used for sharks (Compagno 1984), including taxon-specific adaptations (e.g. fin spine measurements) used in recent publications (Last et al. 2007c; Veríssimo et al. 2014). A suite of 82 morphological and meristic measurements were recorded for eight specimens. Measurements were taken by two readers for each individual, and the average measurement between the two readers is reported for three specimens to be designated as a holotype and two paratypes, along with the minimum and maximum values measured across the five remaining specimens. Vertebral meristic data were obtained for six specimens, including the three type specimens, using X-radiographs conducted at the Shepherd Spring Animal Hospital in Crawfordville, Florida. Dermal denticles from a male specimen (72.5 cm TL) were imaged by the Florida State University’s Biological Science Imaging Resource (BSIR) using a scanning electron microscope with Everhart-Thornley Detector (SEM ETD; FEI Nova 400 NanoSEM; BAL-TEC CPD030 Critical Point Dryer) at 15 kV, with a spot size of 3 at magnifications of 195–800×.

Results

Genetic analyses

Mitochondrial DNA sampled from four conspecific shark taxa in the genus Squalus from the Central and West Pacific (S. mitsukurii, S. nasutus, S. japonicus, and S. cf. mitsukurii) clustered into four genetically distinct genetic groups with a high degree of confidence using both Maximum Likelihood (89–98% bootstrap support) and Bayesian methodology (1.00 posterior probability), except for S. japonicus (80% bootstrap support, 0.80 posterior probability). COI and ND2 trees were concordant, though jModeltest showed slightly different best-fit models of molecular evolution for each (Table 1), and the concatenated tree is shown (Figure 1). As expected, ND2 showed roughly twofold-higher diversity (? = 0.0069) than COI (? = 0.0038), though the evolutionary patterns they describe are similar (Table 1). TCS networks illustrate distinct genetic separation between the four taxa, with some haplotypes being closely related, but none shared (Figure 2).

Figure 2.

Haplotype networks for 20 individuals from four putative Squalus species. TCS plots show two the mitochondrial genes (COI and ND2) sequenced this study. The size of the circles or wedges represents the number of samples within each haplotype, and uninterrupted branches represent single mutational steps.

Among the four species examined here, interspecific divergence across 1,131 bp of concatenated mtDNA ranged from 0.71% between S. mitsukurii and S. japonicus to 1.90% between S. japonicus and S. cf mitsukurii (average = 1.05±0.18%). Average pairwise genetic distance between S. cf mitsukurii and the three named species was 1.18%, greater than the average distance linking named pairs (0.91%). Intraspecific divergence ranged from 0.00% among eight S. cf. mitsukurii to 1.12% in the same number of S. japonicus. Such lack of diversity is consistent with a 2010 population genetic study of Squalus from Hawaii that recovered only eight CO1 haplotypes in 112 individuals, and only five haplotypes in the 91 sharks sampled from Oahu (Daly-Engel et al. 2010). Haplotype diversity was also low in S. nasutus and S. mitsukurii, likely because these were represented by just two samples each. Novel DNA sequences have been made publicly available via GenBank (Appendix 1).

Squalus hawaiiensis sp. n.

http://zoobank.org/105A6FF0-9FFD-4425-BE9C-85019A911B25

Diagnosis

A large species of Squalus of the ‘mitsukurii group’ with the following combination of characters: body relatively slender, trunk height 8.7–12.4% TL (mean 10.1% TL, n=8; Figure 3); snout is angular and short to moderate in length, mouth width 1.35–1.60 (1.48) times horizontal prenarial length and pre-oral length is 1.92–2.06 (1.97) times the prenarial length (Figure 4); pre-first dorsal length 30.3–31.5 (30.2)% TL; pre-second dorsal length 63.6–67.0 (65.5)% TL; interdorsal space 26.7–30.0 (28.6)% TL; pelvic-caudal space 25.2–29.3 (27.1)% TL; relatively small, upright dorsal fins; first dorsal fin length 11.4–12.8 (12.2)% TL, height 6.5–7.8 (7.3)% TL, inner margin length 4.9–5.7 (5.4)%% TL; second dorsal fin length 10.6–11.7 (11.1)% TL, height 4.0–4.6 (4.4)% TL, inner margin length 4.3–4.9 (4.6)% TL; first dorsal fin spine length 46.6–64.6 (55.6)% of first dorsal fin height; second dorsal spine length 104.5–114.5 (109.0)% of second dorsal fin height; caudal bar triangular, extending from the caudal fork nearly to the anterior edge of the lower caudal, distinct upper caudal blotch and fringe in juveniles, upper caudal blotch diffuse in adults but extending to the posterior margin of the upper caudal fin, upper and lower caudal fins white tipped; flank denticles tricuspid (Figure 5A–C); teeth are similar in appearance in the upper and lower jaw, with numbers ranging from 26–28 in the upper jaw and 23 in the lower jaw; 41–45 monospondylous centra, 85–89 precaudal centra, 112–116 total centra; adult maximum size at least 101 cm TL.

Figure 3.

Holotype. Lateral view of Squalus hawaiiensis sp. n. holotype (UF241161, female 750.5 mm TL).

Figure 4.

Holotype. Ventral view of Squalus hawaiiensis sp. n. holotype (UF241161, female 750.5 mm TL).

Figure 5.

SEM images of dermal denticles. Three views of dermal denticles from adult male (TL = 72.5 cm) Squalus hawaiiensis.

Description

Morphometric data are provided in Table 3. Squalus hawaiiensis sp. n. is a relatively large dogfish shark with a fusiform body, a relatively short snout, and small dorsal fins. The nape is modestly humped over the pectoral fins, particularly in large females. Head length is 21.4–23.9% TL. The snout is relatively short but angular and relatively pointed in dorsal view, with a pre-narial length that is 49–52% of the pre-oral length and 1.06–1.31 times eye length. Pre-oral length is 2.04–2.42 times the internarial space. Pre-vent length is 50.4–53.6% of the TL. Mouth width is 0.69–0.83 times the pre-oral length. Eye is large (3.9–4.9% of TL) and strongly notched posteriorly. Upper and lower labial furrows pronounced. Upper labial furrow length 1.9–2.5% TL, 24.9–33.0% of mouth width, and 19.3–24.7% of pre-oral length. Inner nostril labial furrow space is 1.89–2.27 times labial furrow length. Pre-first dorsal fin length is 30.3–31.5% of TL, pre-second dorsal space is 63.6–67.0% of TL and the interdorsal space ranges from 26.7% to 30.0% of TL. The first dorsal fin is rounded at the apex. First dorsal fin length measures 1.62–1.81 times first dorsal fin height. First dorsal fin length is 1.02–1.16 times second dorsal fin length and the height of the first dorsal fin is 1.57–1.80 times the height of the second dorsal fin. Second dorsal fin length 2.36–2.79 times the second dorsal fin height. Dorsal fin spines are stout, with the spine on the second dorsal fin typically longer (4.1–5.0%TL) than the spine on the first dorsal fin (3.6–4.6%TL). First dorsal spine length is 0.39–0.65 (mean: 0.53%) times the first dorsal fin height. Second dorsal spine length is 0.84–1.15 (mean: 1.04%) times the second dorsal fin height. The pectoral fins are well developed with an anterior margin that is 12.8–16.0% of the TL. The pectoral inner margin is 6.4–7.4% of total length and free rear tip is rounded (Figure 6A–C).

Figure 6.

Holotype. A First dorsal fin B second dorsal fin C pectoral fin of Squalus hawaiiensis holotype (UF241161, female 750.5 mm TL).

Squalus hawaiiensis is morphologically similar to other species in the “mitsukurii” group. It is distinguished morphologically by a very long inter-dorsal space which ranges from 26.7% to 30.0% of TL compared to 18.7–25.5% in Squalus mitsukurii (Last et al. 2007a) and 23.5–24.6% in Squalus formosus (White and Iglésias 2011), both from Taiwan and southern Japan, and to 23.5–25.6 in S. edmundsi, 20.6–23.8% in S. grahami (White et al. 2007), 21.7–25.9% in S. montalbani (Last et al. 2007b), all from Australia and 22.6–26.0% in S. griffini (Duffy and Last 2007) from New Zealand, but overlaps with S. chloroculus (23.7–27.5%) from Australia (Last et al. 2007b), S. nasutus (24.4–28.0%) from Australia, Indonesia, and the Philippines (Last et al. 2007a) and S. japonicus from Japan (28.0–29.5%TL) (Chen et al. 1979). Squalus hawaiiensis is further distinguished from S. mitsukurii by having smaller first and second dorsal fin lengths and anterior margins and a longer body or torso (longer pre-caudal and pre-second dorsal lengths but shorter dorsal caudal margin; Table 3). The longer torso is reflected in differences in the ranges of the following ratios between S. mitsukurii type specimens (reported in Last et al. 2007) and all S. mitsukurii measured here (N=8): pre-first dorsal length 1.45–1.73 vs. 1.01–1.16 times interdorsal space; prepectoral length 1.09–1.28 vs. 0.74–0.86 times interdorsal space; prepectoral length 1.02–1.07 vs. 0.78–0.89 times pelvic-caudal space. Based on data from Chen et al. (1979), S. mitsukurii has higher vertebral meristic counts (45–51 monospondylous centra, 87–93 precaudal centra, 118–127 total centra) than S. hawaiiensis (41–45 monospondylous centra, 85–89 precaudal centra, 112–116 total centra). Squalus chloroculus has a caudal bar that extends much higher on the upper caudal fin and lacks the upper caudal blotch characteristic of S. hawaiiensis (Figure 7A–C). Squalus chloroculus also has much shorter first dorsal fin spines (2.3–3.3%TL) and second dorsal fin spines (2.5–3.9%TL) than S. hawaiiensis. Squalus nasutus has a much longer snout with pre-narial lengths of 5.9–7.5%TL and pre-oral lengths of 11.1–12.7%TL compared to 4.8–5.4%TL and 9.6–10.4%TL respectively for S. hawaiiensis. Based on the morphometrics from Chen et al. (1979), the closely related S. japonicus differs from S. hawaiiensis in having a smaller mouth (6.4–6.9%TL compared to 7.0–8.1%TL) and shorter first and second dorsal fin lengths. First dorsal fin length in S. japonicus is 10.1–11.0%TL compared to 11.4–12.8%TL in S. hawaiiensis. Second dorsal fin length is 7.9–8.4%TL S. japonicus compared to 10.6–11.7%TL in S. hawaiiensis.

Figure 7.

Caudal fin of Squalus hawaiiensis sp. n. A Holotype (UF241161, female 750.5 mm TL) B fresh adult male C fresh adult female.

Color. In life (based on many captured specimens): dorsal surface uniformly dark gray to brown, light gray to white ventrally. Dorsal fins uniformly gray to brown with think black tips that narrow with age, free rear tips slightly paler. Caudal fin mostly dusky with a broken white trailing edge, dark caudal bar triangular, extending from the caudal fork nearly to the anterior edge of the lower caudal (Figure 8A–B). Upper caudal blotch diffuse in adults, extending to a short length of the posterior margin of the upper caudal fin, upper and lower caudal fins white tipped; pectoral and pelvic fins greyish dorsally, darker in the middle and with well-defined white posterior margin; Juveniles with much more pronounced fin markings; dorsal fins with black fringes, dark blotch in pectoral fins, caudal bar distinct on lower caudal from the fork to the anterior edge, well-defined and separated black upper caudal blotch and upper caudal fringe with upper caudal blotch not reaching the posterior margin of the upper caudal fin. In juvenile S. mitsukurii the upper caudal blotch is smaller and indistinct from the upper caudal fringe and the caudal bar is diagonal rather than triangular and does not reach the posterior edge of the lower caudal fin. In preservative: holotype similar, dark markings on fins faint but evident; caudal bar faint; broad, pale posterior margins on pectoral and pelvic fins well-defined. Eyes bright green in life (Figure 8C).

Figure 8.

Images of the Hawaiian spurdog, Squalus hawaiiensis. A Lateral view of adult female Squalus hawaiiensis, drawing by R. McPhie B embryonic Squalus hawaiiensis, lateral and dorsal views, drawings by R. McPhie C embryonic Squalus hawaiiensis, dorsal view. Photo by RDG.

Size. Based on 197 Hawaii specimens surveyed, 156 females and 41 males (Daly-Engel et al. 2010; Cotton et al. 2011), the maximum observed length of females and males was 101 cm TL and 78 cm TL respectively. Cotton et al. (2011) reported that females reach maturity at ~64 cm TL and males reach maturity at ~47 cm TL.

Etymology. Derived from the type locality in the Hawaiian Archipelago

Vernacular. Hawaiian Spurdog

Discussion

We found marked genetic variation across 1,311 base pairs of mitochondrial DNA separating Squalus hawaiiensis from Squalus mitsukurii specimens collected from the Japanese type locality, as well as closely-related congeners from elsewhere in the Pacific (Figures 1, 2). Patterns of relatedness and inter- and intraspecific genetic distances were comparable to other phylogenetic studies on Squalus, including species descriptions (Ward et al. 2005; Ward et al. 2007; Naylor et al. 2012). Our data show that S. mitsukurii from Japan and S. hawaiiensis are demonstrably distinct sister species (Figures 1, 2), most closely related to S. japonicus from Japan and S. nasutus from Australia. Morphological examination also distinguished S. hawaiiensis from these three species by a combination of differences in the trunk length (interdorsal distance), snout length, fin and fin spine lengths, and caudal coloration. We conclude that S. hawaiiensis represents a novel, previously-unidentified species. A holotype and paratypes a and b have been deposited into the Florida Museum of Natural History (FLMNH catalog numbers UF241161, UF241162, and UF241163; Table 3).

Table 3.

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Morphological data from Squalus hawaiiensis sp. n. and S. mitsukurii from Japan. Morphological data from type specimens of two Squalus species expressed as a percentage of total length (TL) in cm following the methods of Last et al. (2007b). Morphometrics from the holotype, two paratypes a and b, and the range of values from five additional specimens of Squalus hawaiiensis sp. n. are shown; type specimens are listed with FLMNH catalog number and genetic ID from Figure 1. Morphometrics for S. mitsukurii were taken directly from published studies, including two independent sets for the S. mitsukurii holotype: ¹Last et al. (2007b) and ²Viana et al. (2016). Also shown are the minimum and maximum values from Last et al. (2007b) for four S. mitsukurii paratypes from Japan; “Min” and “Max” represent a range of values for these paratypes, not including holotype values. Abbreviations: ♂ = male, ♀ = female, bolded numbers indicate non-overlapping size ranges between S. mitsukurii and S. hawaiiensis.

		S. hawaiiensis sp. n.					S. mitsukurii (Japan)
		Holotype (♀ UF241161, Sha116)	Paratype^a (♂, UF241162, Sha114)	Paratype^b (♂, UF241163, Sha117)	Min	Max	Holotype¹	Holotype²	Min	Max
STL	Stretched total length	774.5	628	502.5			–	–	–	–
TL	Total length	750.5	608	487.5	535	836	719	710	266	855
PCL	Precaudal length	81.3	82.6	80.3	80.3	83.1	76.6	77.5	78.2	79.0
FL	Fork Length	90.2	91.3	89.8	88.4	92.5	–	–	–	–
PD2	Pre-second dorsal length	65.8	65.6	64.6	63.6	67.0	59.8	61.0	58.6	61.2
PD1	Pre-first dorsal length	31.5	30.4	30.9	30.3	31.3	30.9	32.4	28.5	32.3
SVL	Pre-vent length	53.1	52.4	51.1	50.4	53.6	51.5	50.0	48.9	52.2
PP2	Prepelvic length	52.2	50.1	48.6	48.9	52.4	48.5	47.9	47.4	50.1
PP1	Prepectoral length	22.5	24.3	23.1	22.0	23.3	23.3	24.6	19.9	23.9
HDL	Head Length	21.8	23.9	22.9	21.4	22.4	23.4	24.2	20.9	23.5
PG1	Prebranchial length	18.3	19.2	19.1	17.9	20.7	19.5	20.4	18.0	20.1
PSP	Prespiracular length	12.2	12.9	13.2	12.0	12.9	12.8	12.8	12.1	13.3
POB	Preorbital length	7.6	7.8	7.8	7.4	7.8	7.5	7.3	7.3	7.9
PRN	Prenarial length	4.9	5.2	5.4	4.8	5.1	5.5	5.6	5.0	5.4
PINL	Pre-inner nostril	5.0	5.2	5.3	4.9	5.1	–	–	–	–
POR	Preoral length	9.9	10.1	10.4	9.6	10.2	10.8	10.3	9.4	10.6
INLF	Inner nostril-labial furrow space	4.3	4.7	4.8	4.3	4.7	4.4	4.3	4.2	4.7
MOW	Mouth width	7.7	7.6	7.2	7.0	8.1	6.2	8.6	6.3	7.5
ULA	Labial furrow length	1.9	2.5	2.1	1.9	2.3	2.4	2.5	2.1	2.5
INW	Internarial space	4.4	4.9	4.4	4.0	4.8	4.8	4.7	4.0	4.9
INO	Interorbital space	8.0	7.8	8.0	6.7	7.8	8.1	9.3	7.9	8.4
EYL	Eye length	4.3	4.9	4.5	3.9	4.7	3.4	3.6	3.8	4.7
EYH	Eye height	3.0	3.1	2.9	1.7	3.5	1.3	0.9	1.8	2.5
SPL	Spiracle length	1.2	1.4	1.7	1.2	1.6	1.2	1.3	1.2	1.5
GS1	First gill-slit height	1.7	1.6	1.4	1.5	1.9	1.9	1.7	1.6	1.7
GS5	Fifth gill-slit height	2.2	1.9	2.2	2.0	2.4	2.1	2.3	1.8	2.0
IDS	Interdorsal space	27.8	28.9	26.7	28.1	30.0	21.3	21.1	18.7	25.5
DCS	Dorsal-caudal space	10.2	12.1	11.4	10.9	11.6	9.8	10.6	9.9	11.2
PPS	Pectoral-pelvic space	26.2	22.9	22.8	23.6	27.7	22.5	21.8	21.3	24.5
PCA	Pelvic-caudal space	25.4	29.0	27.4	25.2	29.3	22.7	23.7	22.3	27.4
D1L	First dorsal length	12.5	11.4	11.9	11.6	12.8	14.5	13.6	12.5	15.7
D1A	First dorsal anterior margin	11.0	9.1	10.6	10.0	11.1	12.0	12.0	10.5	11.1
D1B	First dorsal base length	7.2	6.2	6.9	6.4	7.4	8.3	8.2	7.8	7.8
D1H	First dorsal height	7.7	6.5	7.8	6.9	7.7	8.5	9.8	4.5	8.3
D1I	First dorsal inner margin	5.5	5.2	5.4	4.9	5.7	6.3	6.2	4.9	6.4
D1P	First dorsal posterior margin	8.1	7.7	8.0	7.6	9.0	9.7	9.3	4.6	7.9
D1ES	First dorsal spine length	4.6	4.2	3.8	3.6	4.4	3.3	3.9	3.5	4.8
D1BS	First dorsal spine base width	0.9	0.8	0.9	0.7	1.0	0.8	1.0	0.6	0.8
D2L	Second dorsal length	9.4	9.7	9.7	9.2	9.9	–	–	–	–
D2L*	Second dorsal length (incl. cartilage)	11.5	11.1	10.8	10.6	11.7	12.7	12.3	12.0	13.9
D2A	Second dorsal anterior margin	6.9	6.5	7.3	6.7	7.4	–	–	–	–
D2A*	Second dorsal anterior margin (incl. cartilage)	9.5	8.1	8.6	8.3	9.2	10.2	10.2	10.4	10.7
D2B	Second dorsal base length	5.0	4.9	4.9	5.2	5.5	–	–	–	–
D2B*	Second dorsal base length (incl. cartilage)	6.8	6.4	6.3	5.9	6.9	7.2	7.2	8.0	9.2
D2H	Second dorsal height	4.3	4.1	4.6	4.0	4.6	4.5	6.8	3.0	4.6
D2I	Second dorsal inner margin	4.6	4.7	4.6	4.3	4.9	5.1	5.3	4.2	5.4
D2P	Second dorsal posterior margin	5.4	5.7	5.5	4.8	6.3	5.2	6.3	4.1	4.4
D2ES	Second dorsal spine length	4.1	4.7	5.0	4.1	4.6	3.8	4.2	3.8	5.0
D2BS	Second dorsal spine base width	0.8	0.8	0.9	0.7	0.9	0.7	0.9	0.7	0.9
P1A	Pectoral anterior margin	16.0	12.8	13.6	12.9	15.6	15.0	15.2	11.7	16.1
P1I	Pectoral inner margin	6.6	6.8	7.1	6.4	7.4	8.2	9.5	7.0	7.5
P1B	Pectoral base length	5.6	5.4	5.3	5.0	5.8	6.8	5.3	5.0	6.1
P1P	Pectoral posterior margin	12.2	9.9	9.9	10.1	12.3	11.0	11.7	7.6	11.4
P2L	Pelvic length	9.9	11.4	10.9	9.3	10.7	10.8	11.5	9.6	10.3
P2H	Pelvic height	3.9	3.4	3.1	3.0	5.2	5.6	–	4.0	4.9
P2I	Pelvic inner margin	4.6	5.7	5.9	3.9	6.0	5.8	6.3	2.0	3.1
CDM	Dorsal caudal margin	20.7	20.1	21.1	19.4	21.4	22.6	24.4	21.2	21.3
CPV	Preventral caudal margin	11.2	9.9	10.3	10.2	12.0	12.3	12.1	10.2	12.2
CPU	Upper postventral caudal margin	16.6	15.0	15.4	14.3	16.6	16.4	–	13.2	16.2
CPL	Lower postventral caudal margin	5.2	4.0	3.2	3.7	5.4	4.8	–	3.4	5.6
CF.W	Caudal fork width	6.7	6.7	6.6	6.5	7.2	6.7	7.0	5.9	6.7
CF.L	Caudal fork length	8.5	8.1	8.5	8.2	9.3	9.2	–	9.3	10.3
HANW	Head width at nostrils	7.2	7.7	7.6	6.5	7.3	7.7	7.3	7.6	7.7
HAMW	Head width at mouth	10.8	11.1	10.5	9.9	10.6	11.5	12.2	10.1	10.8
HDW	Head width	13.6	11.7	10.8	11.7	15.8	14.8	22.5	11.5	13.8
TRW	Trunk width	15.0	10.6	10.7	11.7	14.2	–	18.3	8.2	10.7
ABW	Abdomen width	15.1	10.9	9.6	10.0	14.4	–	15.5	6.4	9.6
TAW	Tail width	7.1	7.1	5.9	5.9	7.9	6.3	–	4.7	6.7
CPW	Caudal peduncle width	3.0	3.0	2.7	2.4	3.4	2.5	–	2.4	3.1
HDH	Head height	8.2	8.3	8.2	8.1	10.7	8.5	12.7	7.5	11.7
TRH	Trunk height	9.2	8.7	8.8	8.8	12.4	–	10.3	7.9	9.1
ABH	Abdomen height	11.1	9.3	9.2	8.6	14.2	–	7.7	7.7	8.4
TAH	Tail height	6.3	5.9	5.3	5.7	8.8	7.2	–	5.3	6.2
CPH	Caudal peduncle height	2.3	2.2	2.2	2.3	2.5	2.6	–	2.3	2.5
CLO	Clasper outer length		4.5	3.8	5.1	5.1	–	–	1.7	2.6
CLI	Clasper inner length		7.7	6.3	8.4	8.4	–	–	5.2	6.0
CLB	Clasper base width		1.7	1.2	1.6	1.6	–	–	0.9	1.1

The holotype of Squalus mitsukurii was first listed from Misaki, Japan by Jordan and Snyder (1901) before being officially described two years later by Jordan and Fowler (1903), though as in the 1901 paper, the accompanying drawing was of Squalus acanthias. Squalus mitsukurii from Hawaii was referenced by Jordan & Evermann shortly thereafter in the publication “Shore Fishes of the Hawaiian Islands” (1905), with a copy of the misattributed illustration from 1903. Little scientific investigation has been done on Squalus from the Central Pacific since then, with the exception of a 1994 account of the rapid depletion of the dogfish stock around Hancock Seamount in the Northwestern Hawaiian Islands as a result of bycatch in the armorhead fishery (Wilson and Seki 1994), and a more recent investigation of age, growth, and reproduction (Cotton et al. 2011). In 2010, the authors found that S. cf. mitsukurii from Hawaii has the lowest rate of both multiple paternity and genetic diversity estimated in a shark population to date, indicating that this species might have a particularly low rebound potential in the face of fishing pressure (Simpfendorfer and Kyne 2009; Daly-Engel et al. 2010).

Because taxonomic descriptions that incorporate molecular data may use different marker types, study taxa, and methods of estimating divergence, it can be difficult to directly compare genetic distances among studies, or define a genetic threshold for speciation. But a lack of shared haplotypes, plus variation between species that is generally an order of magnitude higher than variation within species, is a consistent pattern reported in many elasmobranch species descriptions (Spies et al. 2006; Ward et al. 2007; Veríssimo et al. 2014; Daly-Engel et al. 2018; Pfleger et al. 2018). Among the four closely-related species we studied, average concatenated sequence divergence between species (1.045±0.183%) was nearly fourteen times the average within-species divergence (0.075±0.026), and therefore consistent with species-level differences reported for other elasmobranchs, including Squalus (Ward et al. 2007; Ebert et al. 2010; Viana et al. 2016).

In addition to being taxonomically unresolved, members of genus Squalus are often subject to high fishing pressure as bycatch in commercial trawl fisheries, sometimes resulting in severe population depletion (Wilson and Seki 1994; Graham et al. 2001; Kyne and Simpfendorfer 2007; Dulvy et al. 2014; Pfleger et al. 2018). Furthermore, their long reproductive intervals (12–24 months) and slow growth results in a low rate of replacement (Musick 1999; Musick and Ellis 2005; Cotton et al. 2011; Cotton and Grubbs 2015), compounding the depleting effect of fishing pressure. In general, S. mitsukurii is classified by the International Union for Conservation of Nature (IUCN) as Data Deficient globally (Cavanagh et al. 2007), but life history parameters among Squalus species likely varies due to undiagnosed taxonomic variation. The combination of these variables may result in the extirpation or extinction of deep-water stocks and species before they are described by science, so taxonomic evaluation is of vital importance to ensure the survival of species that may not yet be managed as distinct evolutionary units. Further, though the barcoding gene, COI, has great utility for species identification, it may not provide sufficient resolution for diagnosing differences between organisms with low rates of molecular evolution, such as deep-water sharks. Because so many potential species remain unexamined, the name S. mitsukurii now represents a series of geographically distinct Evolutionary Significant Units (ESUs), each meriting its own taxonomic examination (Daly-Engel et al. Submitted).

Conclusions

Morphological and genetic differences indicate that the dogfish shark in Hawaii represents a novel species, designated here as Squalus hawaiiensis, the Hawaiian spurdog shark, named for the type location. Further, Squalus mitsukurii in Japan is subject to taxonomic confusion even among experts, and may comprise multiple distinct species, one of which likely includes the holotype. There, thorough morphological and genetic examination is warranted to elucidate the subtle differences between co-occurring populations that are morphologically indistinguishable but genetically unique. Given the number of previously-cryptic species identified in the S. mitsukurii complex alone, analysis of other populations will likely yield further identification of cryptic diversity within the genus Squalus.

Acknowledgements

The authors gratefully acknowledge R. McPhie for the wonderful illustrations. W. White, G. Kume, A. Yamaguchi, J. Kawauchi, S. Tanaka, and P. Reinthal helped with tissue collections. For laboratory and field support we thank we thank K. Holland, B. Bowen, J. Eble, J. Romine, M. Blake, D. Coffey, M. Royer, S. Macias, B. Freese, S. Boleyn, C. Hitchcock, E. Pereira, M. Pfleger, and the members of the Daly-Engel Shark Conservation Lab. We thank N. Griggs, J. Whited, and J. Burdette from Shepherd Spring Animal Hospital for their assistance on conducting X-radiographs. SEM images were provided by G. Platt, D. Trickey, and E. Lochner at the FSU Biological Science Imaging Resource. Funding came from the Florida Institute of Technology Department of Ocean Engineering and Marine Sciences, the University of West Florida Department of Biology, College of Science and Engineering, Research and Sponsored Programs, a SAHLS-Seifert Foundation Grant to A.K., and the Office of Undergraduate Research. Support was also provided by grant No. 2 K12 GM000708 to the PERT Program at the University of Arizona on behalf of T.D.E. from the National Institutes of Health. Publication of this article was funded in part by the Open Access Subvention Fund and the Florida Tech Libraries.

References

Avise JC (2004) Molecular markers, natural history, and evolution, second edition. Sinauer Associates, Inc., Sunderland, MA.

Barluenga M, Meyer A (2004) The Midas cichlid species complex: incipient sympatric speciation in Nicaraguan cichlid fishes? Molecular Ecology 13: 2061–2076. https://doi.org/10.1111/j.1365-294X.2004.02211.x

Briggs JC, Bowen BW (2011) A realignment of marine biogeographic provinces with particular reference to fish distributions. Journal of Biogeography 39: 12–30. https://doi.org/10.1111/j.1365-2699.2011.02613.x

Cavanagh RD, Lisney TJ, White W (2007) Squalus mitsukurii. The IUCN Red List of Threatened Species 2007: e.T41877A10583487. https://doi.org/10.2305/IUCN.UK.2007.RLTS.T41877A10583487.en

Chen C, Taniuchi T, Nose Y (1979) Blainville’s dogfish, Squalus blainville, from Japan, with notes on S. mitsukurii and S. japonicus. Japanese Journal of Ichthyology 26: 26–42.

Chilton NB, Gasser RB, Beveridge I (1995) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). International Journal for Parasitology 25: 647–651. https://doi.org/10.1016/0020-7519(94)00171-J

Clement M, Posada D, Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657–1660. https://doi.org/10.1046/j.1365-294x.2000.01020.x

Compagno LJV (1984) Sharks of the world: an annotated and illustrated catalogue of shark species known to date. Part 1: Hexanchiformes - Lamniformes. FAO Fisheries Synopsis, pp.

Compagno LJV, Dando M, Fowler SL (2005) Sharks of the World. Princeton University Press, Princeton, NJ.

Cotton CF, Grubbs RD (2015) Biology of deep-water chondrichthyans: Introduction. Deep Sea Research Part II: Topical Studies in Oceanography 115: 1–10. https://doi.org/10.1016/j.dsr2.2015.02.030

Cotton CF, Grubbs RD, Daly-Engel TS, Lynch PD, Musick JA (2011) Age, growth and reproduction of Squalus cf. mitsukurii from Hawaiian waters. Marine and Freshwater research 62: 811–822. https://doi.org/10.1071/MF10307

Daly-Engel TS, Baremore IE, Grubbs RD, Gulak SJB, Graham RT, Enzenauer MP (2018) Resurrection of the sixgill shark Hexanchus vitulus Springer & Waller, 1969 (Hexanchiformes, Hexanchidae), with comments on its distribution in the northwest Atlantic Ocean. Marine Biodiversity: 1–10. https://doi.org/10.1007/s12526-018-0849-x

Daly-Engel TS, Grubbs RD, Feldheim KA, Bowen BW, Toonen RJ (2010) Is multiple mating beneficial or unavoidable? Low multiple paternity and genetic diversity in the shortspine spurdog (Squalus mitsukurii). Marine Ecology Progress Series 403: 255–267. https://doi.org/10.3354/meps08417

Daly-Engel TS, Lockhart B, Pereira E, Reece JS (Submitted) Dispersal potential in deep-water dogfish sharks of the Genus Squalus (Squaliformes, Squalidae). PloS ONE.

Duffy CAJ, Last P (2007) Part 9 — Redescription of the Northern Spiny Dogfish Squalus griffini Phillips, 1931 from New Zealand. In: Last P, White WT, Pogonoski JJ (Eds) Descriptions of new dogfishes of the genus Squalus (Squaloidea: Squalidae).CSIRO Marine and Atmospheric Research, Hobart, Tasmania, 91–100.

Dulvy NK, Fowler SL, Musick JA, Cavanagh RD, Kyne PM, Harrison LR, Carlson JK, Davidson LN, Fordham SV, Francis MP, Pollock CM, Simpfendorfer CA, Burgess GH, Carpenter KE, Compagno LJ, Ebert DA, Gibson C, Heupel MR, Livingstone SR, Sanciangco JC, Stevens JD, Valenti S, White WT (2014) Extinction risk and conservation of the world’s sharks and rays. Elife 3: e00590. https://doi.org/10.7554/eLife.00590

Ebert DA, White WT, Goldman KJ, Compagno LJV, Daly-Engel TS, Ward RD (2010) Resurrection and redescription of Squalus suckleyi (Girard, 1854) from the North Pacific, with comments on the Squalus acanthias subgroup (Squaliformes: Squalidae). Zootaxa 2612: 22–40.

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Gaither MR, Bowen BW, Rocha LA, Briggs JC (2016) Fishes that rule the world: circumtropical distributions revisited. Fish and Fisheries 17: 664–679. https://doi.org/10.1111/faf.12136

Graham KJ, Andrew NL, Hodgson KE (2001) Changes in relative abundance of sharks and rays on Australian South East Fishery trawl grounds after twenty years of fishing. Marine and Freshwater research 52: 549–561. https://doi.org/10.1071/MF99174

Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. https://doi.org/10.1093/sysbio/syq010

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. https://doi.org/10.1093/bioinformatics/17.8.754

Jordan DS, Evermann BW (1905) Shore Fishes of the Hawaiian Islands, With a General Account of the Fish Fauna. United States Government Printing Office, Washington, DC, 45 pp.

Jordan DS, Fowler HW (1903) A Review of the Elasmobranchiate Fishes of Japan. In: Snyder M (Ed.) Proceedings of the United States National Museum.The Smithsonian Institution, Washington, 629–630.

Jordan DS, Snyder JO (1901) A preliminary check list of the fishes of Japan. Societatis Zoologicae Tokyonensis, 129 pp.

Katoh K, Misawa K, Kuma Ki, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059–3066. https://doi.org/10.1093/nar/gkf436

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649. https://doi.org/10.1093/bioinformatics/bts199

Kyne PM, Simpfendorfer CA (2007) A collation and summarization of available data on deepwater chondrichthyans: biodiversity, life history and fisheries. A report prepared by the IUCN SSC Shark Specialist Group for the Marine Conservation Biology Institute, 11 pp.

Last PR, Marshall LJ, White WT (2007a) Part 8—Squalus nasutus sp nov, a new long-snout spurdog of the ‘japonicus group’ from the Indian Ocean. In: Last PR, White WT, Pogonoski JJ (Eds) Descriptions of New Dogfishes of the Genus Squalus (Squaloidea: Squalidae) CSIRO Marine and Atmospheric Research, Hobart, Tasmania, 84–90.

Last PR, White WT, Motomura H (2007b) Part 6—A description of Squalus chloroculus sp. nov., a new spurdog from southern Australia, and the resurrection of S. montalbani Whitley. In: Last PR, White WT, Pogonoski JJ (Eds) Descriptions of New Dogfishes of the Genus Squalus (Squaloidea: Squalidae) CSIRO Marine and Atmospheric Research, Hobart, Tasmania, 55–69.

Last PR, White WT, Pogonoski JJ (2007c) Descriptions of new dogfishes of the genus ‘Squalus’ (Squaloidea: Squalidae). CSIRO Marine and Atmospheric Research, Hobart, Tasmania.

PopART (2018) (Population Analysis with Reticulate Trees). http://popart.otago.ac.nz [accessed]

Martin AP, Naylor GJP, Palumbi SR (1992) Rates of mitochondrial DNA evolution in sharks are slow compared with mammals. Nature 357: 153–155. https://doi.org/10.1038/357153a0

Martin AP, Palumbi SR (1993) Body size, metabolic rate, generation time, and the molecular clock. Proceedings of the National Academy of Sciences 90: 4087–4091. https://doi.org/10.1073/pnas.90.9.4087

Musick JA (1999) Ecology and conservation of long-lived marine animals. In: Musick JA (Ed.) Life in the Slow Lane: Ecology and Conservation of Long-Lived Marine Animals.American Fisheries Society Symposium 23, Bethesda, MD, 1–10.

Musick JA, Ellis JK (2005) Reproductive Evolution of Chondrichthyes. In: Hamlett WC (Ed.) Reproductive Biology and Phylogeny of Chondrichthyes: Sharks, Batoids, and Chimaeras.Science Publishers, Inc., Plymouth, UK, 45–79.

Naylor GJP, Caira JN, Jensen K, Rosana KAM, White WT, Last PR (2012) A DNA Sequence–Based Approach To the Identification of Shark and Ray Species and Its Implications for Global Elasmobranch Diversity and Parasitology. Bulletin of the American Museum of Natural History: 1–262. https://doi.org/10.1206/754.1

Perring TM (2001) The Bemisia tabaci species complex. Crop Protection 20: 725–737. https://doi.org/10.1016/S0261-2194(01)00109-0

Pfleger MO, Grubbs RD, Cotton CF, Daly-Engel TS (2018) Squalus clarkae sp. nov., a new dogfish shark from the Northwest Atlantic and Gulf of Mexico, with comments on the Squalus mitsukurii species complex. Zootaxa 4444: 101–119. https://doi.org/10.11646/zootaxa.4444.2.1

Posada D (2008) jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution 25: 1253–1256. https://doi.org/10.1093/molbev/msn083

Randall JE (2007) Reef and Shore Fishes of the Hawaiian Islands. University of Hawaii Press, Honolulu, HI.

Roberts CM, McClean CJ, Veron JEN, Hawkins JP, Allen GR, McAllister DE, Mittermeier CG, Schueler FW, Spalding M, Wells F (2002) Marine biodiversity hotspots and conservation priorities for tropical reefs. science 295: 1280–1284. https://doi.org/10.1126/science.1067728

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. https://doi.org/10.1093/bioinformatics/btg180

Ronquist F, Huelsenbeck JP, van der Mark P (2005) MrBayes 3.1 manual, 1–69.

Seutin G, White BN, Boag PT (1991) Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology 69: 82–90. https://doi.org/10.1139/z91-013

Simpfendorfer CA, Kyne PM (2009) Limited potential to recover from overfishing raises concerns for deep-sea sharks, rays and chimaeras. Environmental Conservation 36: 97–103. https://doi.org/10.1017/S0376892909990191

Spies IB, Gaichas S, Stevenson DE, Orr JW, Canino MF (2006) DNA‐based identification of Alaska skates (Amblyraja, Bathyraja and Raja: Rajidae) using cytochrome c oxidase subunit I (coI) variation. Journal of Fish Biology 69: 283–292. https://doi.org/10.1111/j.1095-8649.2006.01286.x

Tanaka S, Teshima K, Mizue K (1975) Studies on Sharks―X Morphological and Ecological Study on the Reproductive Organs in Male Heptranchias perlo. Bulletin of the Faculty of Fisheries, Nagasaki University 40: 15–22.

Veríssimo A, Cotton CF, Buch RH, Guallart J, Burgess GH (2014) Species diversity of the deep-water gulper sharks (Squaliformes: Centrophoridae: Centrophorus) in North Atlantic waters – current status and taxonomic issues. Zoological Journal of the Linnean Society 172: 803–830. https://doi.org/10.1111/zoj.12194

Veríssimo A, McDowell JR, Graves JE (2010) Global population structure of the spiny dogfish Squalus acanthias, a temperate shark with an antitropical distribution. Molecular Ecology 19: 1651–1662. https://doi.org/10.1111/j.1365-294X.2010.04598.x

Veríssimo A, Zaera‐Perez D, Leslie R, Iglésias SP, Séret B, Grigoriou P, Sterioti A, Gubili C, Barría C, Duffy C (2017) Molecular diversity and distribution of eastern Atlantic and Mediterranean dogfishes Squalus highlight taxonomic issues in the genus. Zoologica Scripta 46: 414–428. https://doi.org/10.1111/zsc.12224

Viana ST, Carvalho MR, Gomes UL (2016) Taxonomy and morphology of species of the genus Squalus Linnaeus, 1758 from the Southwestern Atlantic Ocean (Chondrichthyes: Squaliformes: Squalidae). Zootaxa 4133: 1–89. https://doi.org/10.11646/zootaxa.4133.1.1

Ward RD, Holmes BH, Zemlak TS, Smith PJ (2007) Part 12—DNA barcoding discriminates spurdogs of the genus Squalus. In: Last PR, White WT, Pogonoski JJ (Eds) Descriptions of New Dogfishes of the Genus Squalus (Squaloidea: Squalidae).CSIRO Marine and Atmospheric Research, Tasmania, Australia, 117–130.

Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society B: Biological Sciences 360: 1847–1857. https://doi.org/10.1098/rstb.2005.1716

White WT, Iglésias SP (2011) Squalus formosus, a new species of spurdog shark (Squaliformes: Squalidae), from the western North Pacific Ocean. Journal of Fish Biology 79: 954–968. https://doi.org/10.1111/j.1095-8649.2011.03068.x

White WT, Last PR, Stevens JD (2007) Part 7 — Two new species of Squalus of the ‘mitsukurii group’ from the Indo–Pacific. In: Last PR, White WT, Pogonoski JJ (Eds) Descriptions of new dogfishes of the genus Squalus (Squaloidea: Squalidae).CSIRO Marine and Atmospheric Research, Hobart, Tasmania, 71–81.

Wilson CD, Seki MP (1994) Biology and population characteristics of Squalus mitsukurii from a seamount in the central North Pacific Ocean. Fisheries Bulletin 92: 851–864.

Yano K, Tanaka S (1984) Review of the deep sea squaloid shark genus Scymnodon of Japan, with a description of a new species. Japanese Journal of Ichthyology 30: 341–360.

Yano T, Ohshimo S, Kanaiwa M, Hattori T, Fukuwaka Ma, Nagasawa T, Tanaka S (2017) Spatial distribution analysis of the North Pacific spiny dogfish, Squalus suckleyi, in the North Pacific using generalized additive models. Fisheries Oceanography 26: 668–679. https://doi.org/10.1111/fog.12225

Appendix 1

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Demographic data for specimens used in this study. ID numbers refer to those used in Figure 1; GenBank Accession numbers are listed by gene (ND2/CO1); CSIRO = Commonwealth Scientific and Industrial Research Organisation Marine & Atmospheric Research (Hobart, Tasmania).

ID #	Squalus species	GenBank Accession #s	Source	Collector/ identifier	Origin	Latitude / Longitude	Collection date	Depth (m)	Sex	TL (mm)
Sna021	nasutus	MG654959.1/ MK005141	CSIRO	P. Last & W. White	West Australia, W of Shark Bay	25.0633S, 112.1483E	23 Apr 2006	340
Sna022	nasutus	MG654960.1/ MK005140	CSIRO	W. White	West Australia, W of Leander Point	29.3100S, 113.9467E	06 Feb 1991	505
Sja049	japonicus	MG654922.1/ MK005129	CSIRO	W. White	Taiwan, Tashi fish market, near I-Lan (NE coast)		24 May 2005
Sja050		MG654923.1/ MK012557	CSIRO	W. White	Taiwan, Tashi fish market, near I-Lan (NE coast)		23 May 2005
Sja051		MG654924.1/MK005128	CSIRO	P. Last	Taiwan, Tashi fish market, near I-Lan (NE coast)		23 May 2005
Sja074		MG654925.1/MK005130	A. Veríssimo	N. Straube	Suruga Bay, Japan
Sja075		MG654926.1/MK005127	A. Veríssimo	N. Straube	Suruga Bay, Japan
Sja121		MG654927.1/MG792167.1	A. Yamaguchi	Sho Tanaka	Suruga Bay, Japan		May 5 2011		F	945
Sja123		MG654928.1/ MG792169.1	A. Yamaguchi	Sho Tanaka	Suruga Bay, Japan		21 Mar 2007		F	885
Sja124		MG654929.1/ MG792170.1	A. Yamaguchi	Sho Tanaka	Suruga Bay, Japan		24 Apr 2009		M	774
Smi120	mitsukurii	MG654933.1/ MG792166.1	A. Yamaguchi	Sho Tanaka	Suruga Bay, Japan		21 Mar 2007		M	778
Smi122	mitsukurii	MG654934.1/ MG792168.1	A. Yamaguchi	Sho Tanaka	Suruga Bay, Japan		10 May 2011		F	1096
Sha090	hawaiiensis	MG654906.1/MK005139	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4916N, 157.7525W	17 Aug 2015	360	F	58.4
Sha091		MG654907.1/MK005138	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4916N, 157.7525W	17 Aug 2015	360	F	64.8
Sha093		MG654908.1/MK005136	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4916N, 157.7525W	17 Aug 2015	360	F	63.5
Sha114		MG654909.1/MK005131	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4983N, 157.7310W	29 Jan 2016	305	M	60.8
Sha115		MG654910.1/MK005135	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4983N, 157.7310W	29 Jan 2016	305	M	57.5
Sha116		MG654911.1/MK005134	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4983N, 157.7310W	29 Jan 2016	305	F	75.1
Sha117		MG654912.1/MK005133	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4983N, 157.7310W	29 Jan 2016	305	M	48.8
Sha118		MG654913.1/MK005132	J.M. Anderson	J.M. Anderson	Kaneohe Bay, Oahu, Hawaii	21.4983N, 157.7310W	29 Jan 2016	305	M	55.5