A new genus (Copepoda, Harpacticoida, Laophontidae) from Jeju Island of Korea

Abstract A survey on the harpacticoid copepods from an intertidal zone in Hyeopjae sandy beach, Jeju Island, Korea, resulted in the discovery of an unusual laophontid, Jejulaophonte hyeopjaeensis sp. n., which cannot be placed in any extant genus within the family. To accommodate the species, a new genus of the family Laophontidae T. Scott, 1905 is proposed and fully described here. The new species is closely related to the lineage of the five primitive genera, Carraroenia McCormack, 2006, Coullia Hamond, 1973, Hemilaophonte Jakubisiak 1933, Psammoplatypus Lee & Huys, 1999, and Robustunguis Fiers, 1992 (the CCHPR-lineage) by the reduced P2 endopod, ovate shape of the female P5 exopod and sexual dimorphism in the P3 endopod. However, it displays discrepancies from the species of the CCHPR-lineage in the presence of an inner seta on P3 and P4 exp-2, four setae on P4 enp-2, and an inner seta on P3 and P4 enp-2 in the female. Furthermore, no other species within the family Laophontidae has three setae on P2 exp-3 and a seta on P2 enp-2 at the same time. The new species has sexual dimorphism in the antennule, genital segmentation and the legs from P2 to P5. The terminal seta on the second endopodal segment of P2 in the male is longer than that in the female. The endopod of P3 is 3-segmented and displays a short inner apophysis on the second segment in the male. The outer setae on the exopod of P3 and P4 are distinctly thicker and stronger in the male than in the female. Mitochondrial cytochrome oxidase subunit I (mtCOI) sequencing of the new species has been realized in order to be used in future phylogenetic analysis.


Introduction
Laophontid harpacticoids inhabit various environments including deep sea (Willen 1996;Lee and Huys 1999;Huys and Lee 2000). However the family Laophontidae is found mainly clinging to epiphytal habitats (Boxshall and Halsey 2004). Furthermore, several genera have been found in association with various organisms, for example, Hemilaophonte janinae Jakubisiak, 1933, collected from washings of the spider crab, Maia squinado (Herbst, 1788). They have highly reduced and modified appendages as specific adaptations to their host.
The family Laophontidae T. Scott, 1905 is a large group of harpacticoid copepods, comprising over 262 species in 63 genera and two families: Esolinae and Laophontinae (Boxshall and Halsey 2004). The type species, Laophonte cornuta, was published by Philippi (1840) and T. Scott (1905) proposed the family name, however he did not define the characters of this taxon. Lang (1944) divided the family in three subfamilies: Laophontinae, Normanellinae, and Donsiellinae. Later, Huys and Willems (1989) upgraded the Normanellinae to family rank and Hicks (1988) revised the Donsiellinae, creating four new genera and removing it to the family Thalestridae. Although many genera and species have been moved to other families, because of the numerous new genera and species described since Lang's (1965) key there is no easy way to identify the genera of Laophontidae. Huys and Lee (2000) provided a key to genera of subfamily Esolinae which included eight genera, and Huys (2009) suggested a key to species of five genera having reduced P2 endopod.
In this study, a survey on the harpacticoid copepods from an intertidal zone in Jeju Island, Korea resulted in the discovery of an unusual laophontid, which could not be allocated to any extant genera in the family Laophontidae. Sandy sediments around Jeju Island originate from volcanic rock called basalt. The sediment type of studied area, Hyeopjae beach, is silvery sand that is mixed with sand and various shell dusts. In addition, there are a lot of marine algae that are washed ashore by waves. The family Laophontidae includes various organisms, which are adapted to a habitat style, namely their cylindrical body shape and a reduced segmentation of their swimming legs (Gheerardyn et al. 2007). These interstitial species including associates with other invertebrates or alga also present a reduced segmentation of the thoracic appendages. To accommodate the new species, a new genus of Laophontidae is proposed and fully described here. In addition the mitochondrial cytochrome oxidase subunit I (mtCOI) sequences are obtained for using as molecular barcode of the new species.

Materials and methods
Sediments were collected by a small shovel and acryl cores (diameter 5.4 cm) in a submerged area of Hyeopjae sandy beach, Jeju island, Korea (about 1 m depth). The sediment samples were fixed in 5% neutralized formalin for taxonomic study. Copepods are extracted from the sediment samples by using the Ludox method (Burgess 2001) and fixed in 70% ethanol. Specimens were dissected in lactic acid, and the dissected parts were mounted on slides in lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings have been prepared using a camera lucida on an Olympus BX51 differential interference contrast microscope.
For scanning electron microscopy copepods were prefixed in 70% ethanol, dehydrated through graded ethanol for Hitachi S-2380N in Hanyang University or acetone series for Philips XL-30 in the Natural History Museum London, critical point dried, mounted on stubs using double-sided tape, coated with gold, and then examined with a scanning electron microscope (Hitachi S-2380N, Philips XL-30).
Etymology. Specific name refers the type locality of new species, Hyeopjae beach, Jeju Island, Korea.
DNA-barcode (mt COI). Sequences and traces were submitted to GenBank (GenBank Accession numbers: KF857218, KF857219) Description of female. Total body (Fig. 1A, B) length from anterior margin of rostrum to posterior margin of caudal rami 477 µm (n = 6, mean = 472 µm). Maximum width 88 µm measured at midway of cephalothorax. Body (Fig. 1A, B). Cylindrical and not dorsoventrally depressed with minute sensilla dorsally. Small sensilla well developed on the distal margin of prosomites and urosomites.
Labrum (Fig. 8B) with spinular ornamentation around distal margin; dense pattern of fine spinules anteriorly and distal patch of overlapping scales.

Discussion
The new species has a unique character set including the seta formula of P2-P4, and the shape of P5. Based on the Wells' key (2007), there is no extant genus that can harbor the specific character combination of the present new species. Especially, within the family Laophontidae the new species has a unique combination of three setae on P2 exp-3 and only one seta on P2 enp-2. The new genus, Jejulaophonte is placed in the subfamily Laophontinae based on character sets including the sub-chirocer male antennule, the typically uniramous mandible, the syncoxa of maxilliped armed with maximum only two setae, the P1 enp-1 without inner seta, the reduced P2 enp-2 without outer spine, and the proximal outer setae of female P5 exopod with a distinctly separated insertion site. Jejulaophonte is closely related to five genera (Carraroenia McCormack, 2006, Coullia Hamond, 1973, Hemilaophonte Jakubisiak 1933, Psammoplatypus Lee & Huys, 1999, and Robustunguis Fiers, 1992, the CCHPR-lineage, based on the reduced P2 endopod (Laophontidae typically has P2 larger than P3), the ovate shape of female P5 exopod, and the sexual dimorphism in the P3 endopod (Gomez and Boyko 2006, Huys 2009, McCormack 2006. Fiers (1992a, b) claimed that the main reason for some species of the genera Robustunguis and Hemilaophonte having reduced appendages is to adapt to their host. Lee and Huys (1999) discussed the relationship between Psammoplatypus and related genera based on the reduced P2 endopod, the swimming leg sexual dimorphism and the ovate shape of female P5 exopod. Additionally, McCormack (2006) observed that the species Carraroenia ruthae McCormack, 2006 shares some characters with this lineage. Huys (2009) consequently suggested that Phycolaophonte and Eolaophonte should be sub-  sumed into the synonymy of Coullia and provided a key to genera which have reduced P2 including the five genera Carraroenia, Coullia, Hemilaophonte, Psammoplatypus and Robustunguis. Especially, he recognized the reduced P2 endopod, sexual dimorphism in P3 endopod of male, and ovate shape of P5 as the shared characters. Importantly, the new genus shares those characters with the CCHPR-lineages. While the new genus shares the reduced P2 endopod with the CCHPR-lineage, there are several conspicuous differences in the seta formula of appendages (Table 1). Jejulaophonte, Psammoplatypus, and Carraroenia differ from its congeners in the presence of an inner seta on P3-P4 exp-2, four setae on P4 enp-2 (instead of 2 or 3 setae in other genera), one inner seta on P3-P4 enp-2 (instead of 0) in the female. On the other hand, Jejulaophonte shares the primitive characters of five setae on P1 exp-2 with Coullia insularis (Pallares, 1975), C. tongariki (Gomez & Boyko, 2006), and Psammoplatypus discipes (Noodt, 1958), one or two inner setae on P4 exp-3 with C. insularis, and C. tongariki (Table 1). However, Carraroenia can be regarded as the most primitive genus in the lineage rather than Jejulaophonte and Psammoplatypus, by having two inner setae in P4 enp-2, the retention of a inner seta on P2 exp-2 and the primitive P5 armed with six setae on exopod and with five setae on baseoendopod (Mccormack 2006 Although the female of Psammoplatypus proprius (Lang, 1965) has not yet been described, we suppose that the seta formula in the distal segment of P2 and P3 exopod is common in both sexes as the other species in this group do not have sexual dimorphism in the seta formula on the distal segment of P2 and P3 exopod (Table 1).
While Jejulaophonte shares some primitive characters with the lineage, the new species can be distinguished from the species of CCHPR-lineage by the reduced P5 setation. Except for Robustunguis minor Fiers, 1992 and Psammoplatypus discipes, all species in the CCHPR-lineage share the characters of the reduced P2, and the six setae on the P5 exopod in the female. However, the new species possesses a reduced setation of five setae on the P5 exopod in the female and four setae in the male. Furthermore, endopodal lobe of the male has only one seta (the others of CCHPR-lineage have at least two setae except for Robustunguis minor).
The nuclear ribosomal genes are useful for phylogenetic study (Huys et al. 2006, Huys 2009), however the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was proposed as a 'barcode' (Hebert et al. 2003;Bradford et al. 2010). Until now, mtCOI sequences of 27 harpacticoid species including the new species and the unpublished species Leptocaris canariensis were updated on GenBank ( Table 2). The sequences of the new species are the first barcode in the family Laophontidae, and it would be a useful template for laophontid barcode study.