Abstract
The blue-spotted maskray from Guadalcanal Island (Solomon archipelago) is morphologically distinct from Neotrygon kuhlii with which it was previously confused and it is genetically distinct from all other species in the genus Neotrygon. It is here described as a new species, Neotrygon vali sp. nov., on the basis of its nucleotide sequence at the cytochrome oxidase 1 (CO1) gene locus. It is unique in the genus Neotrygon by the possession of nucleotide T at nucleotide site 420 and G at nucleotide site 522 of the CO1 gene.
1 Introduction
Genetic studies of the dasyatid genus Neotrygon Castelnau, 1873 [1] or maskrays have pointed to the possible occurrence of several species complexes [2-7]. This genus currently comprises 10 nominal species: N. annotata (Last, 1987) [8], N. australiae Last, White and Séret, 2016 [9], N. caeruleopunctata Last, White and Séret, 2016 [9], N. kuhlii (Müller and Henle, 1841) [10], N. leylandi (Last, 1987) [8], N. ningalooensis Last, White and Puckridge, 2010 [11], N. orientale Last, White and Séret, 2016 [9], N. picta (Last, 1987) [8], N. trigonoides (Castelnau, 1873) [1] and N. varidens (Garman, 1885) [12]. The blue-spotted maskray, previously N. kuhlii, consists of up to eleven lineages representing separate species [4, 6, 7, 13] of which four (N. australiae, N. caeruleopunctata, N. orientale, N. varidens) have so far been formally described. One of the paratypes of N. kuhlii, a specimen from Vanikoro in the Santa Cruz archipelago, has been recently designated as lectotype [9], although the pigmentation patterns of the Vanikoro maskray, thus now the typical N. kuhlii, do not fit those of the original description of the species by J. Müller and F.G.J. Henle [10, 14]. In their re-description of N. kuhlii, P.R. Last and co-authors [9] hastily included a fresh specimen collected from Guadalcanal Island in the Solomon archipelago, over 800 km away from Vanikoro, the type-locality. Pigmentation patterns clearly distinguish the Guadalcanal maskray from N. kuhlii from Vanikoro [14], but not from other species previously under N. kuhlii except N. varidens [12].
Mitochondrial DNA sequence information contributes valuable diagnostic characters to the taxonomic description of species and is fundamental to the description of cryptic species [15]. The taxonomic value of mitochondrial DNA sequences has been demonstrated in morphologically intractable species complexes in Elasmobranchs such as Himantura uarnak [3, 16-18] or N. kuhlii [3-6, 13]. Based on the partial sequence of the CO1 gene, the objectives of the present paper are (1) to identify diagnostic characters that distinguish the Guadalcanal maskray from other species in the genus Neotrygon; and (2) to describe it as a new maskray species, a necessary step towards clarifying the intricate taxonomy of species in this genus.
2 Methods
Notice: the present article in portable document (.pdf) format is a published work in the sense of the International Code of Zoological Nomenclature [30] or Code and hence the new names contained herein are effectively published under the Code. This published work and the nomenclatural acts it contains have been registered in ZooBank (http://zoobank.org/), the online registration system for the International Commission on Zoological Nomenclature. The ZooBank life science identifier (LSID) for this publication is urn:lsid:zoobank.org:pub:69E3F1C8-1137-4EF9-B61A-5B56667477A3. The online version of this work is archived and available from the bioRxiv (http://biorxiv.org/) and from the Hal-IRD (http://www.hal.ird.fr/) repositories.
Because N. kuhlii from Vanikoro, the type-locality, has not yet been analyzed genetically, pigmentation patterns were used to distinguish it from the Guadalcanal maskray, following [5]. Two specimens were examined including specimen no. CSIRO H 7723-01 (p. 539 of [9]) and two live specimens photographed underwater, one by J.E. Randall ([19]: p. 18) and the other one by M.A. Rosenstein (Fig. 1). The diameter of ocellated blue spots on the dorsal side of the disk was measured on the photographs, relative to disk width. Ocellated blue spots were qualified as “small” when their maximum diameter was ≤ 2% disk width (DW), “medium” when ≤ 4% DW and “large” when > 4% DW [5]. On Randall’s [19] picture and on Fig. 1, DW was deduced from disk length (DL; measured from tip of snout to rear tip of pelvic fin) from the relationship DW = 1.13 DL, obtained from measurements on specimen no. CSIRO H 7723-01, a good photograph of which is available from [9]. Dark speckles (≤ 1% DW) and dark spots (> 1% DW) were also counted on the dorsal surface of the disk [5]. The counts did not include those speckles and spots located within the dark band around eyes that forms the mask [5]. The presence or absence of a scapular blotch was checked [5].
Guadalcanal maskray Neotrygon vali sp. nov. showing the pigmentation patterns that differentiate it from N. kuhlii from Vanikoro [14]. Photographed by M.A. Rosenstein near Mbike Wreck (09°06’S 160°11E), November 2014.
The Guadalcanal maskray was compared to other species in the genus Neotrygon based on nucleotide sequences of the CO1 gene. A total of 205 complete or partial CO1 gene sequences were found in the literature [2, 4-6, 9, 20, 21] and compiled into a single FASTA file which was edited under BIOEDIT [22]. The recently-described N. australiae and N. caeruleopunctata correspond to, respectively, clades V and VI of [4]. Clade IV of [4] included a distinct sub-clade that corresponds to N. varidens. All other haplotypes of clade IV of [4], together with GenBank no. JN184065 [21] correspond to N. orientale, except a distinct haplotype (GenBank no. AB485685) [20] here referred to as the Ryukyu maskray. Two haplotypes from the Indian Ocean (GenBank nos. JX263421 and KC249906) belonging to Haplogroup I of [4, 7] are here referred to as the Indian Ocean maskray. Sample sizes were: N = 8 for N. annotata; N = 11 for N. australiae; N = 12 for N. caeruleopunctata; N = 7 for N. leylandi; N = 1 for N. ningalooensis; N = 68 for N. orientale; N = 5 for N. picta; N = 18 for N. trigonoides; N = 1 for N. vali sp. nov. (holotype); N = 11 for N. varidens; N = 19 for clade II of [4]; N = 17 for clade III of [4]; N = 14 for clade VII of [4]; N = 10 for clade VIII of [4]; N = 1 for the Guadalcanal maskray; N = 2 for the Indian Ocean maskray; and N = 1 for the Ryukyu maskray. GenBank accession numbers for all the foregoing sequences are provided in Supplementary Table S1.
Average nucleotide divergences between pairs of sequences within a lineage and net nucleotide divergences between lineages were estimated according to the Tamura-3 parameter substitution model [23], the most likely model as inferred from the Bayesian information criterion using MEGA6 [24]. Variable nucleotide sites were determined automatically using MEGA6. Diagnostic nucleotide sites at the CO1 gene locus that distinguish the Guadalcanal maskray from all other lineages in the genus Neotrygon were then selected visually on the EXCEL (Microsoft Corporation, Redmond WA) file generated by MEGA6.
3 Results and Discussion
Pigmentation patterns on the dorsal side of each pectoral fin consisted of a variable number (N = 4-21) of small ocellated blue spots, a small number (N = 1-6) of medium-sized ocellated blue spots, 3-7 dark speckles and no scapular blotch (Table 1). The three Guadalcanal maskray specimens available for the present study thus lacked the dark spots and the scapular blotch that are present in N. kuhlii [14]. Given the relevance of pigmentation patterns in diagnosing species in the genus Neotrygon [5, 11, 25] and more generally in stingrays [16, 18], this observation alone suffices to reject the hypothesis that the Guadalcanal maskray might be synonymous with N. kuhlii as assumed previously by [9].
Pigmentation patterns on left or right dorsal side of disk in Guadalcanal maskray Neotrygon vali sp. nov. including numbers of ocellated blue spots, number of dark speckles or spots and presence or absence of a scapular blotch. Ocellated blue spots qualified as small when diameter ≤ 2% disk width (DW); medium when > 2% DW and ≤ 4% DW and large when > 4% DW; dark speckles ≤ 1% DW; dark spots > 1% DW [5]. N: number of speckles or spots.
The maximum-likelihood tree of CO1 haplotypes (Fig. 2) confirmed the monophyly of species in the genus Neotrygon, except N. picta which was paraphyletic with N. leylandi. Also, no distinction was evident between haplotypes of N. annotata and those previously assigned to a related undescribed lineage provisionally referred to as “Neotrygon cf. annotata” [6]. Estimates of nucleotide divergence at the CO1 locus among species and deep lineages (cryptic species remaining undescribed [13]) in the genus Neotrygon ranged from 0.015 to 0.301 (Table 2). They ranged from 0.015 to 0.038 among the four already-described blue-spotted maskray species (N. australiae, N. caeruleopunctata, N. orientale, N. varidens) previously under N. kuhlii (Table 2). Nucleotide divergence between the Guadalcanal maskray and other species in the genus Neotrygon was ≥ 0.049 (Table 2). Meanwhile, nucleotide divergence estimates within lineages ranged from 0 in N. caeruleopunctata to 0.011 in N. orientale and in clade II of [4] (Table 2), thus systematically lower than inter-specific estimates. The Guadalcanal maskray possessed nucleotides at two nucleotide sites at the CO1 locus, that were absent in N. annotata, N. australiae, N. caeruleopunctata, N. leylandi, N. ningalooensis, N. orientale, N. picta, N. trigonoides, N. varidens, and in 6 yet-undescribed blue-spotted maskray species sampled from the Indian Ocean, the Andaman Sea, the Banda Sea, the Ryukyu archipelago and West Papua [4, 7, 13] (Supplementary Table S1). Nucleotide sequences at the CO1 locus therefore provided diagnostic characters for the Guadalcanal maskray, relative to all other species in the genus Neotrygon. The Guadalcanal maskray is here considered to represent a distinct species, based on its distinct phylogenetic placement, its level of nucleotide distance with other species in the genus Neotrygon, and its unique nucleotide composition at the CO1 locus. No name being available for the Guadalcanal maskray [26], it is here described as a new species.
Neotrygon spp. Maximum-likelihood tree (Tamura 3-parameter model; Mega6 [24]) of nucleotide sequences at the CO1 locus (N = 205), compiled from several sources [2, 4-6, 9, 20, 21] showing the phylogenetic placement of the Guadalcanal maskray Neotrygon vali sp. nov. Numbers at nodes are bootstrap scores (500 bootstrap resampling runs under Mega6). Dotted vertical line: blue-spotted maskrays previously under N. kuhlii [7].
Neotrygon spp. Estimates of net nucleotide divergence (Tamura-3 parameter model; MEGA6 [24]) between lineages. Clades II, III, VII and VIII were defined by [4]. N sample size; ns number of base substitutions per site from averaging over all sequence pairs within each lineage (Tamura-3 parameter model; Mega6).
4 Taxonomy
Maskrays, genus Neotrygon Castelnau, 1873 belong to family Dasyatidae Jordan, 1888 [27]. The type species of the genus is N. trigonoides [1] previously resurrected from synonymy with N. kuhlii [5].
Neotrygon vali sp. nov. http://zoobank.org/A5BE7B5D-64A3-40C2-AD44-63ECAE060FF6. Previously referred to as: Guadalcanal maskray [13, 14]; erroneously placed under Neotrygon kuhlii by [9]. Specimen CSIRO H 7723-01, a female 295 mm DW, is here chosen as the holotype of Neotrygon vali sp. nov. This specimen was obtained on 7 May 2015 by [9] from the Plaza fish market, Honiara, Guadalcanal Island. The type locality is Guadalcanal Island in the Solomon archipelago.
The morphological description of the holotype of Neotrygon vali sp. nov. has been published previously (pp. 535-541 of [9]). This includes 11 meristic counts and 41 measurements made on the body (table 1 of [9]). Pigmentation patterns on the dorsal side of disk consist of a variable number of small ocellated blue spots and a moderate number of medium-sized ocellated blue spots, few dark speckles and no scapular blotch (present work). The CO1 gene sequence of Neotrygon vali sp. nov. is unique among species in the genus Neotrygon as it clusters with no one of its homologues in congeneric species (Fig. 2). The partial CO1 gene sequence of the holotype, comprised between homologous nucleotide sites nos. 95 and 696 of the CO1 gene in N. orientale (GenBank no. JN184065; [21]) is 5’- C T G G C C T C A G T T T A C T T A T C C G A A C A G A A C T A A G C C A A C C A G G C G C T T T A C T G G G T G A T G A T C A G A T T T A T A A T G T A A T C G T T A C T G C C C A C G C C T T C G T A A T A A T C T T C T T T A T A G T A A T A C C A A T T A T A A T C G G T G G G T T T G G T A A C T G A C T A G T G C C C C T G A T G A T T G G A G C T C C G G A C A T A G C C T T T C C A C G A A T A A A C A A C A T A A G T T T C T G A C T T C T G C C T C C C T C C T T C C T A T T A C T G C T A G C C T C A G C A G G A G T A G A A G C C G G A G C C G G A A C A G G T T G A A C A G T T T A T C C T C C A T T A G C T G G T A A T C T A G C A C A T G C T G G A G C T T C T G T G G A C C T T A C A A T C T T C T C T C T T C A C C T A G C A G G T G T T T C C T C T A T T C T G G C A T C C A T C A A C T T T A T C A C A A C A A T T A T T A A T A T A A A A C C G C C T G C A A T C T C C C A A T A T C A A A C C C C A T T A T T C G T C T G A T C C A T C C T T G T T A C A A C T G T G C T T C T C C T G C T A T C C C T A C C A G T C C T A G C A G C T G G C A T T A C T A T A C T C C T C A C A G A C C G A A A T C T T A A T A C A A C T T T C T T T G A T C C A G C T G G A G G A G G A G A T C C T A T T C T T T A C −3’ [9].
Based on Supplementary Table S1, Neotrygon vali sp. nov. is distinguished from all other species in the genus Neotrygon except N. kuhlii for which no genetic information is available yet, by the possession of nucleotide T at nucleotide site 420 and G at nucleotide site 522 of the CO1 gene. The Guadalcanal maskray is distinct from N. kuhlii by the lack of dark spots (> 1% DW) and by the lack of a pair of scapular blotches on the dorsal side.
Apart from the type locality (Honiara on the northern coast of Guadalcanal Island in the Solomon archipelago), the distribution of Neotrygon vali sp. nov. is likely to be confined within the part of Melanesia east of Cenderawasih Bay in West Papua, where the lineage present is Neotrygon clade VIII [4] and west of the Santa Cruz archipelago, where the species present is N. kuhlii.
Etymology: “vali” is the word for stingray in Gela, one of the languages spoken in Guadalcanal [28]. Epithet vali is intended to refer to the common name of the species among Guadalcanal fishers and it is a noun in apposition [29]. Proposed vernacular names: Guadalcanal maskray (English); vali Guadalcanal (Gela); pastenague masquée à points bleus de Guadalcanal (French).
Supplementary Table S1 Variable nucleotide sites at the CO1 locus that distinguish Neotrygon vali sp. nov. from congeneric species.
Variable nucleotide sites at the CO1 locus that distinguish Neotrygon vali sp. nov. from congeneric species. Nucleotides diagnostic of N. vali sp. nov. are highlighted in blue. Nucleotide sites numerotated starting from the origin of the CO1 gene in N. orientale, GenBank accession no. JN184065. The fragment used in this alignment is 519 bp long, spanning nucleotide sites 106-624.
Acknowledgements
I am grateful to Philippe Béarez (MNHN, Paris), Nicolas Hubert (IRD, Cibinong) and Bob Ward (CSIRO, Hobart) for stimulating discussions, to Yolande Yates (Tulagi Dive, Honiara) for helpful information and to Mark Rosenstein (ActWin, Cambridge MA) for kindly allowing me to use his underwater photograph of Guadalcanal maskray. Nineteenth-century books and articles were consulted online from the Biodiversity Heritage Library website (http://www.biodiversitylibrary.org/). This is a contribution of the PARI project, a cooperative research project by IRD, France and LIPI, Indonesia. I declare no conflict of interest and no specific funding for the writing of this paper, of which I am entirely responsible.
References
