A new species of Acartia (Copepoda, Calanoida) from the Philippines, based on morphological and molecular analyses

Abstract A new species of Acartia (Odontacartia), A. (O.) edentatasp. n., was collected from Leyte Island in the Philippines. Morphologically, the new species resembles A. (O.) pacifica Steuer, 1915. The female of the new species differs from other species of the A. (O.) erythraea Giesbrecht, 1889 species group in the absence of a pair of sharp spines on the posterior border of the genital double-somite and absence of setules on the lateral margins of urosomites 1–3. Unlike other congeners of the species group, males of the new species lack fine setules along the posterior margin of the prosome. Comparison of the new species with A. (O.) pacifica by pairwise distance data for the 16S (282 bp) gene indicates that these species differ by 20–21%, while the COI gene (636 bp) indicates a difference of 16–17%. The new species seems to be a coastal, occurring in warm waters having a salinity of 33.5.

The centura and erythraea species groups have so far accommodated 7 and 5 species, respectively, with the unassigned species A. (O.) lilljeborgi Giesbrecht, 1889. The centrura species group now accommodates the following seven species (Steuer 1923;Ueda 1986) The common A. (O.) pacifica has a wide distribution on the coasts of the Indo-West Pacific and in East Asian continental waters. The population in the brackish Ariake Sea, western Japan, was identified as a different species, and, after detailed morphological and molecular analyses, was described as A. (O.) ohtsukai Ueda & Bucklin, 2006 and considered to be a continental relict (Ueda and Bucklin 2006).
During our copepod surveys in southeastern Asia, we found an undescribed species of A. (Odontacartia) from Leyte Island, the Philippines. It is closely related to A. (O.) pacifica, but unique in lacking paired posterodorsal pointed processes on the female genital double-somite. Following Ueda and Bucklin's (2006) methodologies, we are able to define the new species described herein. A key to species of the subgenus Acartia (Odontacartia) is also provided.
Jeol Ltd, Tokyo, Japan). Terminology follows Huys and Boxshall (1991). Specimens of the species of Acartia and A. (O.) pacifica examined in the present study are deposited in the Institute of Marine Science, Burapha University (BIMS-Zoo-0266).
The structure of female and male antennules follows a pattern of the basically uniramous 28 segments. The antennules of both sexes are similar except for the geniculate right antennule in calanoid copepods as in the copepodid I (CI) setation pattern (Boxshall and Huys 1998). We followed Ueda and Bucklin (2006) because the new species more closely resembles A. (O.) pacifica in having equal antennule segment numbers, a similar setation pattern in right antennules of females and males, except segment 5 (XIII), and three rows of spinules ventrolaterally on the second somite of males.
Mandible (Fig. 3A) gnathobase having two sharp cuspid teeth, one blunt tooth, and three small sharp teeth bordered by small spinules at the proximal end; basis with fine setules on medial outer marginal, single seta distally, and patch of small spinules on surface at midlength; first endopodal segment short with two short setae, second segment with seven setae; exopod 4-segmented, first to fourth with setation formula 1, 1, 2, 2; first segment with row of small spinules.
Maxilliped ( Fig. 2F) highly reduced; syncoxa with setation formula of two long, one medium and one short seta; basis with one short strong, one long setae and row of setules along inner margin; endopod 2-segmented, with four inner spines and terminal spiniform element.
Legs 1 to 4 ( Fig. 3C-F) biramous, each with 2-segmented endopod and 3-segmented exopod; coxa unarmed; second endopodal segments of leg 1 and 4 and third exopodal segment of leg 1 with row of small spinules anteriorly. Seta and spine formula as shown in Table 1.
Male. Body (Fig. 4A, B) similar to that of female; cephalosome anterior bluntly triangular in dorsal view; rostrum (Fig. 4E) with paired filaments (Figs 4E, 5E). Posterior prosome symmetrical with pair of short acute processes dorsolaterally and longer ventrolateral acute processes, with pair of small prominences between two dorsolateral processes. Posterior margin of prosome naked. Urosome composed of five somites, symmetrical in dorsal view; genital somite (= first urosomite) as long as wide, bearing 2 dorsolateral rows of small spinules; second urosomite with two pairs of strong posterior dorsolateral, processes (Figs 4A, B, 5 E), outer shorter than inner, and furnished with Table 1. Acartia (Odontacartia) edentata sp. n. armature formula for legs 1-4, with spines and setae indicated by Roman and Arabic numerals, respectively, following Huys and Boxshall (1991).
Leg 5 (Fig. 3H) uniramous, coxae unarmed and completely fused with intercoxal sclerite; each side of basis with outer plumose seta subterminally, left basis approximately 2.5 times as long as wide, with concave inner margin. Right exopod 3-segmented, first segment approximately 3 times as long as wide with single seta subterminally, second segment with small subterminal spine at mid-length of inner irregularly triangular knob, third segment curved inward with small terminal spine and small inner spine midway. Left exopod 2-segmented, first segment about 2.5 times as long as wide, second segment with long inner seta and small terminal spine.
SEM observation of Acartia (Odontacartia) edentata sp. n. The absence of paired dorsal processes on the female genital double-somite and the thick rostrum were confirmed with scanning electron microscopy ( Fig. 5 A, D). Paired genital slits are located at midlength and moderately separated (Fig. 5B, C). A row of setules is located along the anterior margin of each genital slit (Fig. 5C).
SEM observations of the male urosomite clearly showed fine ornamentation on the posterior border of first urosomite (Fig. 5E) and second segment furnished with three rows of minute spinules ventrolaterally (Fig. 5F). No fine setules were observed along the inner posterior margin of prosome, as described in the above descriptions of the type specimens.
Genetic diversity. We obtained sequence data from mitochondrial 16S and COI genes for 14 individual specimens at three sites. A 282 bp fragment of the 16S gene was analyzed for five adult female specimens from the Philippines      (Treeramaethee et al. 2013). However, we concluded that three species of Acartia appear to occupy water bodies differing in temperature and salinity of the tropical and subtropical zone of the Pacific and Indian oceans (Fig. 7).

Discussion
Prior to the current study, it was believed that A. (O.) pacifica was represented by a single species with a wide geographic range occupying the coastal brackish waters throughout the Western Pacific and Indian oceans (FrÜchtl 1923;Sewell 1933;Shen and Lee 1963;Chen and Zhang 1965;Tanaka 1965;Brodsky 1967;Wellershaus 1969;Pillai 1971;Pinkaew 2003;Mulyadi 2004;Ueda and Bucklin 2006;Shang et al. 2007;Gao et al. 2008;Lan et al. 2008;Moon et al. 2008;Phukham 2008;Kang 2011;Treeramaethee et al. 2013). From the findings of the present study, A. (O.) edentata sp. n. is described based on morphological features that permit its discrimination from A. (O.) pacifica in the West Pacific Ocean. Ueda and Bucklin (2006), suggested that the features provided strong evidence specific to the habitats occupied by species and could be used to discriminate A. (O.) pacifica, which occupies neritic waters, from A. (O.) ohtsukai, which occupies brackish waters.
Mitochondrial markers within the 16S and COI genes have proved to be of great utility in investigating the systematics of ecologically and geographically isolated popu-lations of calanoid copepods (Bucklin et al. 2003). The molecular-based analyses using 16S and COI sequences in the current study lend good support to the morphology-based findings. The findings suggest two major clades that reflect the geographic distribution of Acartia in the Indo-West Pacific, i.e. A. (O.) edentata sp. n. and A. (O.) pacifica from the Seto Inland Sea and from Korean waters, and A. (O.) ohtsukai from Ariake Bay, Japan. Our sequencing results agree with those of Ueda and Bucklin (2006) and emphasize the wide divergence between A. (O.) ohtsukai from A. (O.) pacifica. The high level of sequence divergence observed in this study indicates that the Philippines Islands serve as a barrier limiting the spread of A. (O.) pacifica populations into the Philippine Archipelago. This supports the allopatric speciation hypothesis of Carpenter and Springer (2005) that an ecological vicariant seems to have blocked the migration of marine organisms in the Pleistocene from the West Pacific Ocean to the Indian Ocean. Srinui and Ohtsuka (2015) showed that the distribution patterns of 11 species of Acartiella could be separated into those inhabiting the West Pacific and those in the Indian Ocean. In the case of A. (O.) pacifica s.l., more studies on interand intra-specific molecular and morphological variation found in specimens collected from Asian waters are needed to further understand the distribution and evolution of sibling species in the West Pacific region.

Key to species of the subgenus Acartia (Odontacartia)
Thirteen species of the subgenus Acartia (Odontacartia), including A. (O.) edentata sp. n., have been described from the Indo-West Pacific (Razouls et al. 2018;present study), and are divided into three groups: the centrura and erythraea species groups and A. (O.) lilljeborgi (Steuer 1923;Ueda 1986). Key to species is provided below for both sexes of the subgenus Acartia (Odontacartia).