Attheyella (Canthosella) thailandica sp. nov. (Copepoda, Harpacticoida, Canthocamptidae) from caves in Thailand

During this sampling campaign, the canthocamptid Attheyella (Canthosella) thailandica sp. nov. was collected from various caves in Thailand. The new species is widely distributed in the country and favours habitats, such as phytotelmata and wet soil. Attheyella (Canthosella) thailandica sp. nov. is the second member of the genus to be found in Thailand, after Attheyella (Canthosella) vietnamica Borutzky (1967), which is most similar to it. Amongst Asian species, both A. (C.) thailandica sp. nov. and A. (C.) vietnamica have identical setal formulae, with a greater number of armatures on the distal endopods of legs 2–4. However, A. (C.) thailandica sp. nov. markedly differs from A. (C.) vietnamica in the insertion point of the dorsal seta and the presence of inner spinules on the caudal ramus. Additionally, the leg 4 endopod is two-segmented in A. (C.) thailandica sp. nov., but one-segmented in A. (C.) vietnamica.


Material and methods
This research mainly focused on cave-dwelling copepods in freshwater from the epikarst zone and related habitats -especially water dripping from rocks and plants at cave entrances (Fig. 1). Samples were collected using a filtering bottle with a mesh size of 60 µm and were preserved immediately in 70% ethanol. In the laboratory, samples were rinsed with tap water through a sieve with 60 µm mesh size. Adult specimens were sorted under an Olympus SZ51 stereomicroscope at 40× magnification and were placed in a mixture of glycerol and 70% ethanol (ratio ~ 1:10 v/v) to pure glycerol. Animals were dissected and prepared on a glycerine-mounted slide under a stereomicroscope at 40-100× magnification. The specimens were mounted in pure glycerine on a glass slide and were sealed under a cover glass with transparent nail varnish. Whole specimens were stored in 70% ethanol.
All appendages and body ornamentation were examined with 1000× magnification under an Olympus CX31 compound microscope. Drawings were made using an Olympus U-DA drawing tube mounted on the microscope. Final versions of the drawings were done using the CorelDRAW 12.0 graphic programme.
Specimens for scanning electron microscopy (SEM) were dehydrated in an ethanol series (50%, 70%, 80%, 90%, 95%, 100% and 100%) for 15 min at each concentration. Specimens were dried in a critical point dryer and mounted on stubs. Mounted specimens were coated with gold in a sputter-coater. SEM photographs were carried out using a LEO 1450VP scanning electron microscope.
The descriptive terminology follows Huys and Boxshall (1991).  Etymology. The specific name of the new species, 'thailandica', refers to Thailand, where the species was collected.
Type specimens. Holotype: one adult female dissected and mounted on one slide, NHMUK 2020.56; Allotype: one adult male dissected and mounted on one slide, NHMUK 2020.57; Paratypes: three adult females and three adult males not dissected and stored in a 1.5 ml microtube with 70% ethanol, NHMUK 2020.58-63; one adult female dissected and mounted on one slide, NPU 2020-003; one adult male dissected and mounted on one slide, NPU 2020-004; three adult females and three adult males not dissected and stored in a 1.5 ml microtube with 70% ethanol, NPU 2020-005.
Antenna ( Fig. 3B) with small, unarmed coxa. Allobasis elongated, with two spinules on abexopodal margin. Exp one-segmented, with two inner and two apical unipinnate setae. Enp one-segmented, with two outer spines, one apical spine and five apical setae (two normal and three geniculated setae); inner and outer margins ornamented with two rows of spinules.
Mandible ( Fig. 3C) with two large teeth, seven small teeth distally and one lateral seta on gnathobase, with a small knob on the disto-lateral margin. Palp one-segmented, with four apical setae.
Maxillule ( Fig. 3D) with five apical spines and one anterior seta on praecoxal arthrite. Coxal endite with one spine and one seta apically. Basis with one spine, two setae apically. Exp and Enp reduced, represented by four lateral setae on basis.
Maxilla ( Fig. 3E) with two endites on syncoxa, each endite with three apical setae. Basis elongated, drawn out into a claw, with one proximal accessory seta. Enp reduced and represented by two setae.
Maxilliped ( Fig. 3F) with unarmed coxa, ornamented with row of spinules on median surface. Basis with two groups of spinules at inner and outer margins. Enp one-segmented, transformed into claw-like segment, accompanied by one small seta inserted proximally.
P1-P4 with three-segmented Exp and two-segmented Enp. The armature formula is as follows (Arabic and Roman numerals indicate number of setae and spines, respectively; not including spinules): *the formula for the male P3Enp: Enp-1-3 is 0-0, 0-1, 0-2-0, respectively. P1 ( Fig. 5A) basis with short, strong outer spiniform spine and long, slender, smooth inner seta. Enp shorter than Exp; Enp-1 reaching to middle of Exp-2, with one inner unipinnate-tipped seta, ornamented with strong outer spinules and inner setules. Enp-2 with one smooth distal inner seta, one distal inner geniculated seta and one distal outer unipinnate seta; ornamented with strong outer spinules. Exp-1-2 with one distal outer spiniform spine; both segments ornamented with strong outer spinules. Exp-3 with one distal outer spiniform spine, two apical setae (inner seta geniculated, outer one unipinnate) and one distal inner geniculated seta; ornamented with few distal outer spinules. P2 (Fig. 5B) basis with short, strong outer spine. Enp as long as Exp-1; Enp-1 shorter than wide, unarmed. Enp-2 with two elements apically; outer spine slightly longer than segment; inner seta bipinnate, long, extending beyond Exp. Exp-1 with one enlarged distal outer spine; ornamented with outer spinules. Exp-2 with one distal outer spine and one smooth distal inner seta. Exp-3 with two distal outer spines, two apical setae (inner seta bipinnate, outer one unipinnate) and one smooth inner seta; ornamented with disto-outer spinules. P3 (Fig. 5C) basis with long, smooth, slender outer seta. Enp as long as Exp-1; Enp-1 shorter than wide, unarmed. Enp-2 with three elements apically; outer spine and innermost seta shorter than segment, subequal in length; middle seta bipinnate, reaching beyond Exp. Exp-1-2 with one distal outer spine, ornamented with spinules along disto-outer margin; Exp-2 with one additional smooth distal inner seta. Exp-3 with two distal outer spines, two apical setae (inner seta bipinnate, outer one unipinnate) and two inner setae (distal seta unipinnate, about 3.0× longer than proximal seta); ornamented with spinules along disto-outer margin. P4 (Fig. 5D) basis and Exp as in P3, but Exp-3 shorter. Enp as long as Exp-1; Enp-1 small, shorter than wide, unarmed. Enp-2 with two bipinnate setae apically, both longer than segment; inner seta shorter than outer seta. P5 (Fig. 5E) without ornamentation on surface. Basal seta smooth, slender. Baseoendopod separated from Exp, well-developed, exceeding Exp, with six spiniform setae; third inner seta longest apically; remaining setae decreasing in length to outer and inner margins of Enp. Exp sub-oval, with five setae, second inner seta longest; two innermost setae spiniform; three outer remaining setae smooth and decreasing in length to margin of Exp. P6 (Fig. 4A) reduced to a single bipinnate seta inserted on the small plate, anterior to the seminal receptacle on the first half of genital double-somite.
P5 (Figs 6C, 7E) separated from somite, baseoendopod of left and right sides fused medially. Basal seta long, slender and smooth. Baseoendopod separated from Exp, reaching one-half of Exp, with two spiniform setae; inner seta over 4.0× longer than outer seta. Exp with four setae; second inner seta longest, followed by second outer seta, innermost seta and outermost seta, respectively; two inner setae bipinnate, two outer setae smooth.
Variability. (a) The free distal margin of the anal operculum varies from six to ten spinules in females and six to eight spinules in males, a characteristic which is, perhaps, useless for differentiating amongst species, as mentioned by Gaviria and Defaye (2012). (b) The posterior margin of urosomite 3 in female has a continuous row of ventral spinules (Fig. 4A'; one of the other five examined specimens collected from the type locality). (c) The P2-P3 Enp-2 has a different number of spinules along the outer margin, with two spinules on P2 and three spinules on P3 in females (not shown in Figure; one of the other five female from the type locality) and with two spinules on P2 in males (Fig. 7B'; one of the other five examined specimens collected from the type locality). (d) The seta size of the P4 Enp-2 in both sexes, the outer seta about 2.0× longer than the inner seta in female and the inner seta longer than or equal in length to outer seta in male (Figs 5D', 7D'; two of the other five examined specimens collected from Pra Hor cave). (e) The female P5 in the southern population shows mostly shorter Exp and End setae (Fig. 5E'; two other females from Pra Hor cave).
Ecology. Attheyella (C.) thailandica sp. nov. is usually found in pools of water at cave entrances, where water seeps through soil and plants before flowing down into the cave (see Fig. 1B, D, G). In this research, some samples were found in a pool at the twilight zone of a cave (Fig. 1C) and in a pool in the dark zone of a cave, which was directly fed by the dripping water from stalactites (Fig. 1F). The favourite habitats of A. (C.) thailandica sp. nov. in the present study are likely to be phytotelmata or wet soils, as is already known from most species of Attheyella (subgenus Canthosella) (Reid 2001). At times, A. (C.) thailandica sp. nov. has been found together with Bryocyclop muscicola (Menzel, 1926); both are recognised as stygophile species and are widely distributed in Thailand (Watiroyram 2018;present study).

Discussion
Attheyella (C.) thailandica sp. nov. has been identified as belonging to the subgenus Canthosella Chappuis, 1931 because it shares the following characteristics with other members of the subgenus: posterior margin of somites smooth; caudal ramus longer than wide; antenna with one-segmented Enp; P1-P4 with two-segmented Enp (P1 Enp shorter than Exp, P4 Enp-1 much smaller than other legs of Enp-1); P2-P4 Enp-2 with at least two elements; male P3 with three-segmented Enp bearing an inner apophysis on Enp-2; male P4 Exp-3 without transformed spine; P2-P4 Exp-2-3 with at least one inner seta and one outer spine; female P5 baseoendopod well-developed (reaching beyond Exp) bearing six setae, while Exp bears five setae; male P5 with two setae on baseoendopod and four setae on Exp.
However, A. (C.) thailandica sp. nov. shows morphological differences from other species of the subgenus, related to the ornamentation of the caudal rami. Most species of Attheyella (Canthosella), except A. (C.) antillica and A. (C.) mervini, carry spinules on the inner margin of the caudal ramus (at least in the female). This margin is bare in both sexes of A. (C.) thailandica sp. nov.
At present, the Canthosella subgenus shows two lineages, which include ten American and six SEA species. They can easily be differentiated by the number of armatures on their P2-P4 legs. In contrast to SEA species, all American species, except A. (C.) acanthophora, show a higher number of setae on the P2-P4 Enp-2 in both sexes. Females of these American species have three to four, three to five or two elements on P2-P4, respectively -except for the P4 of A. (C.) antillica, which has one seta. Females of SEA species show one to two setae, one to three setae or one seta on P2-P4, respectively -except for the P4 of A. (C.) vietnamica and A. (C.) thailandica sp. nov., which carry two setae. Additionally, the male P3 Enp-3 of the American species shows two apical setae -except in A. (C.) aliena, which has only one seta. Males of the SEA species have only one seta on the male P3 Enp-3, except for A. (C.) thailandica sp. nov., which has two setae. However, a minute seta located close to a long distal seta on the male P3 Enp-3 could easily have been overlooked in previous descriptions (P.H.C. Corgosinho, personal communication).
Amongst the six SEA species, A. (C.) thailandica sp. nov. is most similar to A. (C.) vietnamica for the following reasons (Table 1) The P4 Enp in both sexes bears two apical setae, while it bears only one seta in all other individuals, except the male of A. (C.) silvicola, which has two setae. (c) The P2 Exp-1 in both sexes of these species has an enlarged outer spine, but this spine is normal in the other species; this characteristic is presumed to be a synapomorphy, which can be used to define closely-related species.
Nevertheless, A. (C.) thailandica sp. nov. also shows strong morphological differences from A. (C.) vietnamica in the following aspects. (a) The P4 Enp is two-segmented in both sexes of A. (C.) thailandica sp. nov. (Fig. 6B) (2007) holds that A. (C.) lacustris with three spines and setae, counting two apical setae and one inner seta (though the latter appears to be a spinule rather than a seta). However, Well (2007) does not count those similar elements on the inner margin of the P3 Enp-2 in A. (C.) fluviatilis. Therefore, the present study notes that A. (C.) lacustris has two apical elements. 2 = A. (C.) muscicola has one apical seta and one outer seta, in contrast to A. (C.) lacustris with two apical setae. 3 = Well (2007) holds that A. (C.) fluviatilis has three spines and setae, probably by counting one apical seta and two small inner setae as described in Chappuis (1931). However, Well (2007) does not generally count armatures (like spinules) on the inner margin of Attheyella species described in Chappuis (1931), and the male P2 Enp-2 usually has the same number of armatures on its segment (or fewer) than females. Therefore, the present study notes that A. (C.) fluviatilis has one apical seta. 4 = An original description notes, in the leg formula, that the male P4 of A. (C.) silvicola has a two-segmented Enp, but it is presented as one-segmented Enp in the related Figure. Well (2007) notes that the male has a one-segmented Enp.
dorsal seta located at three-quarters of the length of the caudal ramus, while this seta is located almost at the distal end in