First record of the genus Boholina (Copepoda, Calanoida, Pseudocyclopidae) in Vietnam, with description of a new species from an anchialine cave in Tra Ban Island

Abstract A new species, Boholina reductasp. nov., was found in a brackish pool within an anchialine cave in Tra Ban Island in Bai Tu Long Bay, north Vietnam. The new species is clearly distinguished from all the six species currently known in the genus Boholina by the following unique characteristics: reduction of the septum between gonopores; narrow and pointed rostrum; basal segment of mandibular palp armed with three setae; maxillule without seta on the basal exite, and exopod with 11 setae; second and third endopodal segments of the maxilliped bearing three setae each; exopod of male right leg 5 2-segmented, with two strong and one vestigial spines on the outer margin of the distal segment; and last exopodal segment of female leg 5 bearing only one spine on the outer margin. We provide a description of the new species, along with detailed illustrations and scanning electron microscopy photographs. The identification key to Boholina species is updated as well. This is the first record of the genus Boholina from Vietnam.


Introduction
The family Boholinidae was established for a single genus Boholina by Fosshagen and Iliffe (1989) on the basis of a combination of morphological characteristics: well-developed mouthparts; 3-segmented rami on P1-P4; female P5 with 3-segmented exopod and slightly reduced 2-segmented endopod; right antennule of male geniculated; and male P5 with a highly complex grasping organ. Fosshagen and Iliffe (1989) argued that it differed from the families Pseudocyclopidae Giesbrecht, 1893 andRidgewayiidae Wilson, 1958 by the modified terminal spine on the distal exopodal segment of P4 and the inner seta on the coxal segment of female P5 (Fosshagen and Iliffe 1989).
In this paper, we describe a new species of Boholina, based on specimens from an anchialine habitat of a karstic cave in Tra Ban Island, north Vietnam, along with detailed illustrations drawn under a differential interference microscope and by scanning electron microscopy. We also discuss its morphological relationships with congeners.

Materials and methods
Nha Tro Cave (or Hang Cam Cave) is located in Vietnam, on Tra Ban Island in Bai Tu Long Bay. The island is 20 km from Cam Pha City in Quang Ninh Province, and about 12 km from the mainland; it has an area of about 76.37 km² (Fig. 1A). The main geological composition of the island is stratified limestone, silicate and claystone (Uong et al. 2013).
The cave has a large entrance at 17 m above sea level (20°57'31.0"N, 107°29'12.1"E); a few meters from the entrance there is a larger downward-sloping hall (Fig. 1B). On the left of the cave is a steeply climbing branch, terminating after a few meters. On the right is the main gallery along the length of the cave with enormous boulders due to rockfalls and large concretions.
The floor is composed of pools and clay deposits; concretions are abundant with several discs, the largest one reaching three meters in diameter. The dimension of the cave is about 350 m in length, 10 m high and 15 m in depth (Donatis et al. 2010).
Copepods were collected from a pool inside the Nha Tro Cave (Fig. 1B). The pool is in a permanently dark section about 200 m from the cave exit. Physicochemical characteristics of the pool on 9 May 2018 are as follows: water temperature 19.8 °C; pH 7.82; dissolved oxygen 0.76 mg/L; water hardness (CaCO 3 ) 154 mg/L; electrical conductivity 1.12 mS/cm; salinity 0.18‰. Copepods were taken from the pool in knee-deep water, with a hand net with mesh size of 80 µm. They were fixed in about 80% ethanol in the field, and later stored in about 70% ethanol. Specimens were dissected and mounted in glycerol or lactophenol. The mounted specimens were observed under a differential interference contrast microscope with Nomarski optics (Nikon Eclipse Ni-U). All drawings were made with the aid of a camera lucida.
Material used for scanning electron microscopy (SEM) was fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2-7.4) for 2 hours, followed by fixation in 1% cold osmium tetroxide (at about 5°C) in the same buffer for 12 hours. After dehydration through a graded series of ethanol (70, 80, 90, 95 and 100%) for 30 minutes each, the material was critical point dried, coated with gold/palladium, and then examined with a scanning electron microscope Hitachi TM3000 TableTop operated at 15 KV. The following abbreviations are used, where required, throughout the text and figures: Endp = endopod; Exp = exopod; P1-P5 = swimming legs 1-5. General terminology for the description follows Huys and Boxshall (1991), including analysis of caudal setae (I-VII) and antennule segmentation (evident segments labeled with Arabic numerals, and ancestral segments with Roman numerals), and the terminology and homology for maxillary and maxilliped structures by Ivanenko (2001, 2008) is adopted herein.
Etymology. The proposed name refers to reduction of the terminal spine on the distal exopod segment of P5 in the male as well as the proximal outer spine on the distal exopodal segment of P5 in the female, which are the most remarkable characteristics differentiating this new species from all the congeneric species of Boholina.
Diagnosis. Boholinid form in both sexes. Postero-lateral corners of second and third pedigerous somites rounded, fourth and fifth pedigerous somites completely fused. Rostrum represented by a narrow chitinized projection with pointed tip. Medial lobe of distal segment A2 endopod with nine setae. Mandibular palp basis with three setae; distal segment of endopod with 11 setae; seta on first segment of exopod present. Maxillule exopod with 11 setae and seta on basal exite absent. Second and third segments of maxilliped endopod with three setae each. Terminal spine on exopod of leg 4 modified with row of large spinules on mid-inner margin. Female: Gonopores on double-somite located close together on mid-ventral surface, septum between gonopores reduced to vestige deep inside genital opening. P5 Exp-3 with only one spine on outer margin and four setae on inner margin; distal segment of P5 endopod with one seta on outer margin. Male: Process at antepenultimate segment of right antennule absent. Right P5 exopod 2-segmented; distal segment with three spines, including a vestigial one on outer margin, while terminal and inner spine absent.
Urosome 4-segmented, comprising genital double-somite, two free abdominal somites and anal somite. Genital double-somite slightly asymmetrical, widest about at mid-length; posterior margin ornamented with smooth hyaline membrane dorsally and small dentate hyaline frill ventrally, about as long as wide; paired gonopores equal in size, located close together on mid-ventral surface, the septum between gonopores reduced to vestige, deep inside genital opening; gonoporal plates small, and gonoporal slits large; two pairs of sensilla present (Figs 2D, 9C, D, arrows), one pair positioned adjacent to posterior margin of gonopores and second pair located ventrolaterally near posterior margin of double-somite. Third and fourth abdominal somites cylindrical, subequal in length (Fig. 2D); third with finely serrated hyaline membrane all around posterior margin, fourth with posterior margin hyaline membrane expanded mid-dorsally to four large spines functioning as pseudoperculum concealing anal opening and mid-ventrally with finely serrated hyaline membrane on posterior margin. Anal somite extremely short, posterior margin smooth, concealed within posterior rim and hyaline membrane of second free abdominal somite.
Caudal rami ( Fig. 2A, D) short, about 1.5 times longer than wide, with pointed dorsal process in middle of distal margin; distal inner margin with a row of setules; ventral surface with a small pore near inner distal edge; ornamented six caudal setae; seta I lacking, seta II spiniform, about 1.2 time as long as caudal ramus; setae III-VI plumose, ratio of setae V:IV:VI:III:II as 5.8:4.3:4.1:2.4:1.0; dorsal seta VII short, naked, about 0.5 times as long as seta II.
Rostral filaments absent, rostrum represented by a narrow chitinized projection with pointed tip (Figs 2B, C, 9B, arrows); pair of long sensilla present in proximal part of rostrum.
Basis of P1 with distally pointed digitiform process on anterior; inner basal seta crooked, bilaterally spinulate, reaching to distal end of second endopodal segment; second exopodal segment with conspicuous spinulate process distally in outer distal corner of segment. Outer proximal spine on third exopodal segment of P1 flagellate, other outer spines on P2-P4 with serrate marginal membrane(s) as figured. Terminal spine on exopod of P1 with naked outer margin and plumose internally; on P2 and P3 with serrate membrane externally and plumose internally; that on P4 modified with row of large spinules on mid-inner margin and armed slender spinules on outer margin and distal part of inner margin (Figs 5D, 9A).
P5 (Figs 5E, F, 8F) biramous, with 3-segmented exopod and 2-segmented endopod, intercoxal sclerite smooth and unornamented. Basis small, 1.4 times as wide as long, with acute process on posterior surface near base of exopod. Exopod longer than   endopod: tip of endopod only reaching to proximal inner seta on third exopodal segment. Distal endopodal segment 2.4 times as long as wide, armed with three inner, two apical and one outer naked setae. First and second exopodal segments each ornamented with a small pore on anterior surface at origin of outer spine. Distal exopodal segment 2.1 times as long as wide, bearing lateral spine (about 38-41 µm), subapical and apical spines of same length (about 51-54 µm); inner margin with four naked setae.
Urosome 5-segmented ( Fig. 6A-C), comprising genital somite, three free abdominal somites and anal somite. Genital somite slightly asymmetrical, distal part of right margin protuberant, slightly more expanded than left margin (Fig. 6C); both lateral margins smooth; four sensilla along dorsoposterior margin; posterior margin with finely serrated hyaline membrane. Second to fourth free abdominal somites cylindrical, subequal in size; second and third somites with finely serrated hyaline membrane on posterior margin; fourth with posterior margin hyaline membrane expanded middorsally to four large spines functioning as pseudoperculum concealing anal opening and mid-ventrally with finely serrated hyaline membrane on posterior margin. Anal somite short, ring-like, with posterior margin smooth.
Caudal rami symmetrical, 1.5-1.6 times as long as wide (mean 1.57, N = 10), bearing distal spinous process dorsally and row of small setules on distal inner margin; ventral surface with a small pore near inner distal edge; ornamented six caudal setae, included to caudal setae II-VII and absent seta I.
Remarks. The new species agrees well with the generic diagnosis of Boholina given by Fosshagen and Iliffe (1989), Boxshall and Jaume (2012), Moon and Soh (2014), and Boonyanusith et al. (2020): fourth and fifth pedigerous somites completely fused; urosome 4-segmented in the female and 5-segmented in the male with very short anal somite, telescoped within the preceding free abdominal somites in both sexes; genital openings paired, located ventromedially or ventrolaterally of genital double-somite; caudal rami produced into a pointed dorsal process in the middle of the distal margin; female antennule 24-segmented, with segments 8 and 9 partly fused or completely separated; P1 with 3-segmented endopod, each segment with a pointed outer distal corner, distal segment without any outer seta; P4 with slightly modified distomedial spine on the distal segment of the exopod; P5 with 2-segmented endopod in the female; and in the male P5 with a complex grasping organ and a highly modified exopod, reduced 1-segmented endopod on both sides.
Among six congeneric species currently recognized in Boholina, B. reducta sp. nov. shares the paired gonopores located either side of the ventral midline with B. ganghwaensis, B. parapurgata and B. purgata, and shares rounded postero-lateral corners of the second and third free pedigerous somites with B. munaensis, B. crassicephala and B. laorsriae. The new species is similar to B. laorsriae by the medial lobe of the distal segment of the antennary endopod having nine setae (while other congeners have eight setae). Boholina reducta sp. nov. resembles B. munaensis in bearing the single seta on the outer margin of the female P5 Endp-2 (against two in the other congeners). The new species is also similar to B. ganghwaensis in having the distal segment of the mandibular palp endopod with 11 setae (versus ten setae in the other congeners) (Table 2).
However, B. reducta sp. nov. is distinguished from all six congeners by the unique characteristics as follows (see Table 2): (1) a pair of gonopores are located close together on the mid-ventral surface of the genital double somites, and the septum between gonopores is only visible in the inner part of the genital opening. In Boholina, there are three species (B. ganghwaensis, B. parapurgata and B. purgata) with gonopores   No. of setae on medial lobe of antennary Enpd-2   (Fosshagen and Iliffe 1989;Boxshall and Jaume 2012;Moon and Soh 2014).
In other species of Boholina, the gonopores are widely separated (Fosshagen andIliffe 1989, Boonyanusith et al. 2020), (2) the rostrum has a narrow finger-like process with pointed tip. The shape of rostrum of new species is unique in the Pseudocyclopidae, (3) the basis of mandibular palp has three setae in the new species, while there are four setae in all the species of Boholina, (4) there is no outer seta on basal exite of maxillule and there are 11 setae on exopod of maxillule, while there are only ten setae in other species of Boholina, (5) the second and third endopodal segments of maxilliped have three setae each, (6) the distomedial spine of P4 Exp-3 is modified with a row of spinules inserted in the middle of inner margin of the spine, (7) the female P5 Exp-3 has only one spine on outer margin, and the proximal outer spine is missing. In Boholina, the outer margin of female P5 Exp-3 generally has two spines, (8)