A new species of Boholina (Crustacea, Copepoda, Calanoida) and a first record for stygobiotic calanoid fauna from a cave in Thailand

Abstract A new species of Calanoida belonging to the genus Boholina Fosshagen & Iliffe, 1989 was found in a freshwater pool within a cave of the Satun province, South Thailand. It is the first record of the genus and of a stygobiotic representative of calanoid fauna in this country. The new species is most similar to B. crassicephala Fosshagen & Iliffe, 1989, based on position of genital pores, structures of P4 and P5 in both sexes, relative length of subapical spine vestige on the male right P5, and shape of the male left P5 endopods. However, this new species is distinguished from its known congeners by: (1) relatively longer distal outer spines on the male right P5 exopods, (2) smaller endopods of the male left P5 and (3) elongated apical spines on the distal exopodal segment of the female P4 and P5. Furthermore, the distinctive characteristic of the Thai Boholina is the presence of inner minute seta on the distal segment of the male right P5 exopod. Detailed descriptions of the new species and a key to all six known species of the genus Boholina is provided.

During the investigation of cave-dwelling Copepoda in the Satun province, South Thailand, a representative of Calanoida was collected from a freshwater pool within a cave. Based on the unique characteristic of a grasping organ on the males' left P5, the cuticular pointed projection on the caudal rami (Fosshagen and Iliffe 1989), and the presence of an additional element on the inner margin of the males' right P5 exopod, a new species of the genus Boholina Fosshagen & Iliffe, 1989 was identified representing the first record of the genus and of the stygobiotic representative of calanoid species in Thailand.
According to Fosshagen and Iliffe (1989), the family Boholinidae and the genus Boholina were established on the basis of two new species collected from a brackish pool in San Vicente cave on Bohol Island in the Philippines. To date, five species are recognised in this genus, only known from East and Southeast Asia (Fig. 1); they are B. crassicephala Fosshagen &Iliffe, 1989, andB. purgata Fosshagen &Iliffe, 1989, recorded from a pool in the San Vicente Cave on Bohol Island in the Philippines, B. munaensis Boxshall & Jaume, 2012 from a spring in Lawou Cave on Muna Island in Indonesia, B. parapurgata Boxshall & Jaume, 2012 from sinkholes on the coast of Muna Island, Indonesia, and B. ganghwaensis Moon & Soh, 2014 from the burrows of Cleistostoma dilatatum (De Haan, 1833) in the inter-tidal mudflat of Ganghwa Island, western Korea (Fosshagen and Iliffe 1989;Boxshall and Jaume 2012;Moon and Soh 2014). More recently, the families Boholinidae and Ridgewayiidae were synonymised in the family Pseudocyclopidae by the morphology-based phylogenetic work of Bradford- Grieve et al. (2014).
In Thailand, six specimens of Boholina were collected from a freshwater pool within a cave. This type locality is in an area that has recently been designated as a UN-ESCO global geopark.

Materials and methods
Samples were collected from a pool in Khay Cave of Satun province, South Thailand ( Fig.  2) by hand net with a mesh size of 60 µm. They were placed in a plastic bottle with a 4 % formaldehyde solution as a fixative. In the laboratory, specimens were sorted under a stereomicroscope and stored in 70 % ethanol. They were placed in a mixture of glycerol and 70 % ethanol (ratio ~1:10 v/v) for 30 minutes before the morphological examination. Examination of habitus was done on the male and female specimens, which were placed in a drop of glycerol between a pair of coverslips on slide. Specimens were then dissected and mounted on slides using glycerol as a mounting medium. The examination was made with a Nikon ECLIPSE E200 compound light microscope at a magnification of ×1000. Habitus and dissected body parts were drawn using a drawing tube at- tached to a compound microscope, and the final versions of illustrations were prepared by Adobe Illustrator CC 2017. Description follows Huys and Boxshall (1991 beyond distal margin of prosome. Apical spine on female P4 Exp-3 elongated, ca. 3 × as long as outer terminal spine. Apical spine on female P5 Exp-3 ca. 1.8 × as long as outer terminal spine. Male: The left P5 Exp-3 highly transformed, bearing three irregular lobes; Endp oval-shaped, much shorter than right P5 Endp, ca. 1.6 × as long as wide. The male right P5 Exp with minute inner spiniform seta; distal outer spine elongated, ca. 3.4 × as long as proximal outer one and ca. 2.7 × as long as apical spine; subapical spine vestige ca. 0.7 × as long as apical spine. Description of adult female. Body (Fig. 3A) with a total length of 0.68 and 0.73 mm (measured from anterior margin of cephalosome to tip of projection of caudal rami, mean: 0.71 mm; N = 2). Prosome 5-segmented, elliptical, ca. 70 % of body length and 2.5 × as long as urosome, with greatest width at posterior end of first pedigerous somite; greatest width ca. 43 % of prosome length. Cephalosome and first three pedigerous somites free; postero-lateral corners rounded. Fourth and fifth pedigerous somites completely fused (Fig. 3A); postero-lateral corners rounded, symmetrical. Naupliar eye not discernible. Urosome 4-segmented, comprising genital double-somite, two free abdominal somites and very short anal somite ( Fig. 3A-D). Genital double-somite barrel-shaped, ca. 45 % of urosome length, with greatest width at mid-length of double-somite, with hyaline membrane all around the posterior margin; hyaline membrane with large medial notch ventrally. Genital pores paired, located ventrolaterally ( Fig. 3C). First and second free abdominal somites subequal in length, bearing hyaline membrane; hyaline membrane of the first free abdominal somite with serrulate margin, that of the second expanded dorso-medially to form a trapezoidal double-pointed flap, representing a pseudoperculum. Anal somite very short, telescoped within the preceding urosomite ( Fig. 3B, D).
Caudal rami (Fig. 3D) subrectangular, ca. 1.8 × as long as wide (measured from base to level of insertion of setae V), with triangular pointed-projection on distal margin dorsally (Fig. 3B); projection 0.4 × as long as ramus length; caudal seta II to VII present, caudal seta I absent; seta II spiniform, with setules along inner margin; seta III plumose, approx. mid-length of seta IV; seta IV shorter than seta V, with breaking planes and plumose; seta V longest, with breaking plane and plumose, sub-equal to urosome length; seta VI slim and plumose. Seta VII inserted dorso-medially near insertion of seta V and seta VI (Fig. 3D). Length ratio of caudal setae to ramus length, from seta II to seta VII: 0.6 : 2.3 : 4.3 : 5.5 : 3.7 : 1.0. Length ratio of caudal setae from seta II to seta VII: 1.0 : 3.7 : 6.9 : 8.9 : 6.0 : 1.5. Rostrum (Fig. 3E) weakly developed and V-shaped; base broad, completely fused to anterior margin of cephalic shield and tapering to rounded tip between bases of antennules, with two sensillae at middle third of rostrum.
Maxilla ( Fig. 5B) 6-segmented, comprising praecoxa, coxa, basis and 4-segmented Endp. Praecoxa partly fused to coxa, proximal and distal praecoxal endites with five and three apical setae, respectively. Coxa with two endites, each armed with three apical setae. Basis with large basal endite, armed with four strong apical setae; one of which ornamented with spinule row at mid-length of seta. Endp 4-segmented, setal formula 2, 2, 2, 3; ultimate segment with two long and one short setae. Maxilliped ( Fig. 5C) 8-segmented, comprising syncoxa, basis, and 6-segmented Endp. Syncoxa with four syncoxal endites, setal formula 1, 2, 2, 3; seta on first endite spinulose, basal seta on second endite strong, spinulose; distal endite with one long seta and two short, slender setae. Basis with three medial setae, with row of spinules on anterior surface. Endp with setal formula 2, 4, 4, 3, 3+1, 4; basal seta on first endopodal segment spinulose. P1-P4 ( Fig. 6A-D) biramous, comprising coxa, basis, and 3-segmented rami. Intercoxal sclerite trapezoidal. Coxa rectangular, with seta on distomedial corner. Basis of all swimming legs with lateral seta but lacking in P2; lateral seta inserted on posterior surface. Basis of P1 with robust seta on distomedial corner, with finger-like process on posterior surface arising near base of Exp; process reaching distal margin of Endp-1. Outer distal corner of all endopodal segments drawn out into triangular projection; projection relatively large in P1 and P2. P1 Endp-1 without any outer seta. Outer distal corner of P1 Exp-2 drawn out into spoon-like process, ornamented with spinules along outer margin. Outer distal corner of Exp-1 and Exp-2 of P2-P4 extended, forming 2-pointed sclerotised expansion, distal pointed process larger than proximal one.    Outer spine of Exp-3 of all swimming legs relatively short. P4 Exp-3 ca. 3.2 × as long as wide, with elongated, smooth apical spine, as long as segment bearing it and ca. 3 × as long as outer terminal spine, with row of curved spinules at its tip. Armature of swimming legs as presented in Table 1. P5 (Fig. 6E) biramous, with 3-segmented Exp and 2-segmented Endp; armament as in Table 1. Coxa and basis as in P3 and P4. Exp-3 ca. twice as long as wide, with apical and outer terminal spines on its tip; apical spine elongated, ca. 1.8 × as long as outer terminal spine, ca. 1.2 × as long as Exp-3 length. Endp much shorter than Exp, reaching level of articulation of Exp-2; Endp-1 as long as wide, without pointed process on outer distal corner; Endp-2 ca.2.6 × as long as wide, with small, pointed process on outer distal corner.
Description of adult male. Body with a total length of 0.65 and 0.67 mm (measured from anterior margin of cephalosome to tip of the projection of caudal rami; mean: 0.66 mm; N = 2). Habitus smaller and slenderer than in female (Fig. 7A). Prosome 5-segmented, as in female, ca. 70 % of body length and 2.5 × as long as urosome, with greatest width at posterior end of first pedigerous somite; greatest width ca. 47 % of prosome length. Cephalosome and first three pedigerous somites similar to those in female. Naupliar eye not discernible. Urosome 5-segmented; comprising genital somite, three free abdominal somites and very short anal somite. Genital somite slightly asymmetrical, ca. 25 % of urosome length; posterior margin with hyaline membrane dorsally. First three free abdominal somites similar in length, each with hyaline membrane all around posterior margin; hyaline membrane on third free abdominal somite as in female. Anal somite very short, telescoped within the preceding somite, as in female (Fig. 7B).
Caudal rami (Fig. 7B) relatively shorter than in female, ca. 1.8 × as long as wide. Armament and ornamentation as in female.
Examination of the structure of the genital double-somite, of P4 and P5 in both males and females, the relative length of subapical spine vestige on the male right P5 and the shape of the male left P5 Endp revealed that the new species is most similar to B. crassicephala, which had previously been described from a pool in a cave of Bohol Island, the Philippines. Several characters are shared by both species, especially the structure of the male P5. However, there are also remarkable differences ( Table 2). The characteristics which obviously distinguish Boholina laorsriae sp. nov. from B. crassicephala are as follows: i) Apical spine of the female P5 Exp-3 is ca. 1.8 × as long as outer terminal spine in the new species, but it is sub-equal to the outer terminal spine found in B. crassicephala. ii) Exopodal segment of the male right P5 has medial minute seta in the new species; however, it is absent in B. crassicephala. iii) Distal outer spine on exopodal segment of the male right P5 is relatively long, and the distal outer spine is ca. 2.9 × as long as the proximal one; however, in B. crassicephala, the spine on exopodal segment of the male right P5 is relative shorter and the distal outer spine is ca. 1.9 × as long as the proximal one. iv) The male left P5 Endp is relatively smaller in the new species than that of B. crassicephala.
The Thai Boholina can be easily distinguished from B. purgata, B. parapurgata, and B. ganghwaensis by the characteristics of the widely separated genital pores, relatively longer subapical spine vestige on the male right P5 when compared to the length of apical spine, the higher length ratio of the apical spine to the outer terminal spine in the female P5 Exp-3 and the elongated apical spine of the female P4 Exp ( Table 2). The genital double-somite of the new species is barrel-shaped, while it is globular in B. munaensis. Additionally, the ii) characteristic is unique for the Thai Boholina. Based on

Female
Body length ( Table 2. Remarks. Only six specimens were collected from a pool and the new species was not encountered in the other eight caves visited in this research project. Freshwater Cyclopoida belonging to the genera Thermocyclops, Metacyclops, and Mesocyclops, as well as harpacticoids of the genus Schizopera and of the family Ectosomatidae were also collected from the type locality. Boxshall and Jaume (2012) provided key to adults of the genus based on the four described species. In this paper, two more species from Korea and Thailand are added in the key.  (Brancelj 2005;Boxshall and Jaume 2012;Tran and Brancelj 2017). Of these, only representatives of Boholina were described from anchialine caves. The genus Boholina clearly shows affinities to the families Ridgewayiidae and Pseudocyclopidae, by the presence of several plesiomorphic characters in the antennule, antenna, mouthparts, and swimming legs. Nearly all species of Boholina were collected from anchialine caves: this habitat is different from a benthic environment in shallow water where the two families have frequently been found (Fosshagen and Iliffe 1989). Recently, the families Boholinidae and Ridgewayiidae were synonymised in the family Pseudocyclopidae, and the genus Boholina is sister to the clade of the genera Ridgewayia, Stygoridgewayia, Hondurella, Placocalanus, and Pseudocyclops, based on a morphology-based cladistic analysis of Bradford-Grieve et al. (2014). From a geographical point of view, Boholina has been recorded only from East and Southeast Asian countries along the western coast of the Pacific Ocean (Fig. 1). This suggests an Asian origin of the genus. The assumption is supported by the fact that its most closely related genera (Ridgewayia, Stygoridgewayia, Placocalanus, and Pseudocyclops) can also be found in this region. The only genus that has yet to be found in Asia is Hondurella, which was only obtained from Utila Island of Honduras in the Caribbean basin (Suárez-Morales and Iliffe 2007).

Key to the adult of Boholina
The geographical distribution of the new species is slightly different from those of all other species of Boholina, as it was collected from a freshwater pool within a cave located very far from the sea (ca. 6.5 km) compared with those of B. crassicephala and B. purgata (200 m), B. parapurgata and B. munaensis (700 m), and B. ganghwaensis (inter-tidal mudflat). The occurrence of the new species in a cave located so far from the coast is the same as that of Stygoridgewayia, which were collected from bores/wells located up to 450 km inland from the coast in the Cape Range Peninsula and Pilbara region, Western Australia (Tang et al. 2008). Tang et al. (2008) hypothesised that the occurrence of Stygoridgewayia in subterranean waters is due to secondary colonisation of freshwater after the regression of the epicontinental sea which inundated a large part of the land. From this geographical viewpoint, we postulate that the ancestor of the present-day population of Thai Boholina could have penetrated the cave in either the Cretaceous or Miocene periods. The assumption was postulated as the geological evidence shows that the sedimentary rocks in the area of the present-day Satun province and north-western Malay Peninsula were formed under the ancient sea for a very long time, spanning the Late Cambrian and Triassic periods, before uplifting of the area in Cretaceous, and the Quaternary sediments in the area below 10 m a.s.l. were interpreted as deposits of the epicontinental sea in the Holocene (The Malaysian and Thai Working Groups 2009).
Based on structure (i.e., degree of modification) of mouthparts, especially with respect to the mandible, maxilla, and maxilliped, feeding habits were suggested for several taxa of the families Epacteriscidae, Ridgewayiidae, and Pseudocyclopidae (e.g., Fosshagen 1968;Fosshagen et al. 2001;Jaume and Humphreys 2001;Fosshagen and Iliffe 2003, 2004a, 2004bSuárez-Morales and Iliffe 2007). The absence of the conspicuous modification of the raptorial feeding habit in the mandible, maxilla, and maxilliped in Thai Boholina and its congeners suggests that they are particle feeders. The characters that indicate particle-feeding habits in Boholina and in several ridgewayiids, such as Ridgewayia, Brattstromia, Exumellina, and Stargatia are are as follows: 1) the mandible bears numerous small teeth on the cutting-edge of the gnathobase and the endopod is well-developed, with four and more than nine setae on proximal and distal segments, respectively, and 2) the maxilla and maxilliped are armed with normal plumose setae. In the raptorial feeders, like epacteriscids and some ridgewayiids, such as Exumella, Palmeriella, and Normancavia, the general modifications of these three appendages include: 1) an enlargement of the teeth, especially the ventralmost teeth, and the reduction of the endopod, and 2) the transformation of setae on the distal part of the maxilla and maxilliped to stout, elongate, spinous setae or the reduction of setae on both the maxilla and maxilliped.
Even if many calanoid taxa in the superfamily Pseudocyclopoidea Giesbrecht, 1893 were collected from anchialine caves, it is likely that there is no specific adaption/modification in relation to the cave habitat in this family. Such a morphological adaptation or modification generally corresponds to the zones of the water column in which the copepod lives. Based on the relative length of the antennule, we suggest that the genus Boholina is a hyperbenthic form, because it has relatively short antennules (not extending beyond prosome) as in most epacteriscids. In the genera Exumellina and Stargatia, which were collected in the water column of anchialine caves, the antennules extend beyond the prosome (Fosshagen andIliffe, 1998, 2003). In our opinion, only the reductions of the eyes and outer seta on P1 Endp are adaptations of Boholina corresponding with life in caves, as Brancelj and Dumont (2007) suggested for the freshwater stygobiotic Calanoida.