A new species of Harpacticella Sars, 1908 (Copepoda, Harpacticoida), from a tidal pool on Jeju Island, Korea

Abstract A new species of the genus Harpacticella Sars, 1908 is described from a tidal pool on Jeju Island, Korea. Harpacticella jejuensis sp. n. is closely related to Harpacticella itoi Chang & Kim, 1991, with regard to the structure of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the setal number of the P5 exopod in males. However, the new species is clearly distinguishable from Harpacticella itoi by the combined following characters: six setae on the P5 exopod in females, one naked seta on the inner margin of P1 exp-2, the short endopod of P1 compared to the exopod, and a naked long seta on the proximal inner margin of the P5 exopod of males. The mtCOI partial sequence is provided as a DNA barcode for the new species.


Introduction
Harpacticella Sars, 1908 is a genus of harpacticoid copepods, family Harpacticidae Dana, 1846. The genus has been reported from various habitats (fresh water, brackish water, and marine), mostly in Asian waters (Itô and Kikuchi 1977;Chang and Kim 1991), and has also been recorded from the Pacific Northwest, USA (Cordell et al. 2007).
The first Harpacticella species report was by G.O. Sars (1908) who examined sandy littoral sediment of Lake Baikal. He proposed a new genus for this species, based on a reduced number of antennule segments and a two-segmented antennary exopod. So far, six species in the genus Harpacticella have been reported (Wells 2007). Among these species, H. inopinata Sars, 1908, H. paradoxa (Brehm, 1924), and H. amurensis Borutzky, 1952 were described from freshwater, H. lacustris Sewell, 1924and H. itoi Chang & Kim, 1991from brackish water, and H. oceanica Itô, 1977 from the marine environment.
During a study of the harpacticoid community along the coast of Jeju Island of Korea, we collected a new species of Harpacticella from a tidal pool. Here, we describe the new species and provide a key to species in the genus Harpacticella. Partial mtCOI sequence was also obtained as a DNA barcode for the new species.

Materials and methods
Samples were collected by hand net (63 µm mesh-size) from a tidal pool on the coast of Jeju Island, Korea. Specimens were preserved in 99% ethanol. Specimens were dissected in lactic acid, and the dissected parts were mounted on slides with lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings were prepared using a drawing tube attached to an Olympus BX51 differential interference contrast microscope.
Caudal ramus (Figs 1A-B, 2D) wider than long; seta I inserted at half length of caudal ramus, ventrolaterally; lateral seta II longer than seta I, inserted close to distal   outer corner; seta III as long as lateral seta II; apical seta IV unipinnate, slightly longer than urosome; apical seta V bipinnate, as long as whole body; apical seta VI similar in length to seta III; dorsal seta VII bare and bi-articulate at its base.
Swimming legs 1-4 (Figs 4A-B, 5A-B) biramous, P1-P4 with 3-segmented exopod and 3-segmented endopod; spinules along inner and outer margins as illustrated. Intercoxal sclerites well developed. P1 (Fig. 4A): coxa with row of spinules along outer lateral margin. Basis shorter than wide, with strong outer seta; inner spine inserted near inner distal corner, with several spinules. Endopod shorter than exopod reaching about half of exp-2; end-1 with plumose seta at the middle of the inner margin; enp-2 small without ornamentation but a row of spinules along outer lateral margin; enp-3 longer than wide with pinnate claw distally and inner naked seta on distal inner margin. Exp-1 as long as exp-2, with spinules along outer margin and spine on distal outer corner; exp-2 with short naked seta on distal part of inner margin and pinnate spine at middle part of the outer margin; exp-3 short with 4 curved pinnate claws and bare seta. P2 (Fig. 4B): coxa with 2 rows of spinules along outer lateral margin. Basis shorter than wide, with slender outer seta and spinules on outer lateral margin. Endopod as long as exopod; row of spinules along outer margin of each segment. Exopodal segments with row of spinules along outer margin; exp-1 with 2 rows of spinules and pore on anterior surface; exp-2 with pore on anterior surface. P3 (Fig. 5A): coxa with 2 rows of spinules along outer lateral margin. Basis shorter than wide, with slender outer seta and spinules along outer lateral margin. Endopod reaching to middle of exp-3; row of spinules along outer margin of each segment. Exopodal segments with row of spinules along outer margin; exp-1 with 2 rows of spinules and pore on anterior surface; exp-2 with pore on anterior surface. P4 (Fig. 5B): coxa with 2 rows of spinules along outer lateral margin. Basis shorter than wide, with slender outer seta and spinules on outer lateral margin. Endopod shorter than exopod, reaching to proximal half of exp-3; row of spinules along outer margin of each segment. Exopodal segments with row of spinules along outer margin; exp-1 with 2 rows of spinules and pore on anterior surface; exp-2 with pore on anterior surface.
Armature formulae as follows:  (Fig. 3E): exopod and baseoendopod well separated. Baseoendopod with slender and bare outer lateral seta. Endopodal lobe larger than exopod and extended beyond distal margin of exopod; with 3 pinnate and 2 bare setae. Exopod oval, with 6 setae; rows of spinules along inner and outer margins.

Discussion
The new species clearly fits in the genus Harpacticella based on the combination of following character sets: a) 7-segmented antennule in the female, b) 2-segmented antennary exopod, c) 3-segmented P1 endopod and exopod, d) only one seta on the inner edge of P2 enp-2 and e) spiniform outer spine of exp-3 P3 and P4 (Table 1). Harpacticella jejuensis sp. n. is closely related to H. itoi Chang & Kim, 1991 based on the length of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the four setae on the P5 exopod of males. However, H. jejuensis can be distinguished from H. itoi by the following distinctive characters: (1) six setae on the P5 exopod of females (Fig. 2E) compared to five setae in H. itoi (see Fig. 3A; Chang and Kim 1991; this character is a unique character within the genus); (2) one bare seta on inner margin of P1 exp-2 (Fig. 4A), which is absent in H. itoi (see Fig. 1C; Chang and Kim 1991); (3) P1 endopod much shorter than exopod (ratio = 0.64:1), but in H. itoi it is as long as exopod (see Fig. 1C; Chang and Kim 1991); (4) naked seta on proximal inner margin of male P5 (Fig. 6D), but plumose-type in H. itoi (see Fig. 3E; Chang and Kim 1991); (5) naked seta on proximal inner margin of male's P5 is three times longer than length of male's P5 exopod (Fig. 6D), but it is almost two times longer in H. itoi (see Fig. 3E; Chang and Kim 1991).
Harpacticella species have a wide distribution ranging from freshwater to true marine environments, and have been found in Asian waters, American waters, and the Aldabra Atoll in the Indian Ocean (Fig. 8). All species in this genus except H. amurensis Borutzky, 1952 andH. inopinata Sars, 1908 have been recorded from at least two localities; the former two species have been recorded only in the type locality (Itô and Kikuchi 1977;Evstigneeva 1993). H. itoi Chang & Kim, 1991 has been found in several locations in the southeastern part of Korean peninsula and H. oceanica Itô, 1977 was documented in Korean and Japanese marine waters (Itô 1977;Chang and Kim 1991;Song and Chang 1993). H. lacustris Sewell, 1924 has a discontinuous distribution and has been recorded in India, China, and Japan (Sewell 1924;Wells and McKenzie 1973;Ishida 1989b). H. paradoxa (Brehm, 1924) is the most ubiquitous species; it has been recorded in China, Japan, and the northwest coast of the USA (Brehm 1924;Pesta 1930;Itô and Kikuchi 1977;Ishida 1987;Ishida 1989a;

Type locality
Amur River (Borutzky 1952) Lake Baikal (Sars 1908) Tamjin River, Korea (Chang and Kim 1991) Chilka Lake near Calcutta, India (Sewell 1924) Bonin Island, Japan (Itô 1977) Talifu Lake in Yunnan Province, China (Brehm 1924) Seogwipo in Jeju Island, Korea 2003; Ishida et al. 2004;Cordell et al. 2007). Cordell et al. (2007) suggested that the introduction of H. paradoxa may have been due to anthropogenic factors such as ballast waters. Small marine invertebrates have been shown to be introduced into new marine ecosystems via ballast water (Orsi and Ohtsuka 1999). Recently, molecular approaches have been used to determine the origin of these invasive organisms (Le Roux and Wieczorek 2009;Simon et al. 2011) DNA barcoding is an efficient tool to identify species, especially morphologically similar species (Floyd et al. 2002;Hebert et al. 2003;Guidetti et al. 2005;Bhadury et al. 2006). This barcode can also be used for biogeographical analysis of invasive or widely distributed species (Garrick et al. 2004). We obtained a 619-bp partial sequence of mtCOI (KM272559) for use in future studies; no sequences have been obtained from congeners to date, even though it would be interesting to determine the phylogenetic relationships among congeners based on analysis of mtCOI sequences.