Two new species of Metacyclops Kiefer, 1927 (Copepoda, Cyclopoida) from Thailand and an up-to-date key to the species recorded in Asia

Metacyclops sakaeratensis sp. nov. and M. brancelji sp. nov. are described as being present in the mountainous areas of Northeastern and Southern Thailand, respectively. Two new species resemble M. woni in both sexes, but they are easily distinguished from their Cambodian relative by having: 1) transverse suture on the dorsal surface of the genital double-somite, 2) serrated hyaline frill on the posterior margin of third pedigerous somite, 3) diff erent length / width ratio of caudal ramus, and 4) a row of spinule on caudal surface of intercoxal sclerite of the third swimming legs. The signifi cant diff erences between M. sakaeratensis sp. nov. and M. brancelji sp. nov. are present in both sexes, including the body size, integumental ornamentation of the body surface, length / width ratio of caudal ramus, armature of the fi fth swimming leg, and the male sixth swimming leg. In addition, an up-to-date key to the female of all fi fteen species of Metacyclops recorded in Asia is provided.


Introduction
is the third largest genus of the subfamily Cyclopinae Kiefer, 1927, represented by 62 species and subspecies (Dussart & Defaye 2006). Representatives of the genus are known to be present in tropical and temperate regions worldwide, particularly in Europe, South America, and Africa (Reid 1991). Its members have been considered polyphyletic, and more recently they have been separated into several different genera after the revision of several species (Fiers 2001;Karanovic 2004a;Karanovic et al. 2011). Based on either fine-detailed re-examination or morphology-based cladistic analysis of the Australian and New Zealand species of Metacyclops, three new genera have been established, including Meridiecyclops Fiers, 2001, Fierscyclops Karanovic, 2004, and Pescecyclops Karanovic, Eberhard & Murdoch, 2011(Fiers 2001Karanovic 2004a;Karanovic et al. 2011).
In this paper, two new representatives of Metacyclops are proposed. One was discovered from a headwater stream in Sakaerat Environmental Research Station (SERS) located at the Sankhampaeng Mountain Range of Nakhon Ratchasima Province, Northeast Thailand. The second was collected from three caves of the Nakhon Sri Thammarat Mountain Range in Satun and Songkhla Provinces, South Thailand.

Material and methods
A plankton net with 60 µm mesh size was used to collect samples in headwater stream in SERS, Nakhon Ratchasima Province, and a hand net with 60 µm mesh size was used to collect samples in temporary pools, which are found in the vadose zone of the caves of Satun and Songkhla Provinces (Fig. 1). Samples were subsequently fixed in 4% formaldehyde solution. In the laboratory, copepods were sorted and stored in 70% ethanol. Before the morphological examination, adult of both female and male were placed in a mixture of glycerol and 70% ethanol (ratio ~1:10 v/v) for 30 minutes and later placed in a drop of glycerol between a pair of coverslips on a glass slide, one by one. Habitus was then examined using a Nikon ECLIPSE E200 compound light microscope at a magnification of 1000 ×. Each specimen was later dissected and mounted on slides by using glycerol as a mounting medium. Subsequently, morphological examination was done using 400 × and 1000 × magnifications. Habitus and dissected body parts were drawn using a drawing tube (Nikon Y-IDT) attached to a compound microscope and the Adobe Illustrator CC 2020 was adopted to prepare the final versions of illustrations. For scanning electron microscopy (SEM), additional material was dehydrated by a series of ethanol concentrations: 70%, 80%, 90% 95% and 100%, and then by the critical point drying process. Afterwards, specimens were mounted on stubs, coated with gold, and photographs were taken by a scanning electron microscope (FIB-FESEM).

Diagnosis
Female. Body size moderate (0.73-0.75 mm; n = 3), with shallow integumental pits. Posterior margin of second pedigerous somite undulated; those of third and fifth pedigerous somites with serrated hyaline frill. Genital double-somite dorsally with two sensilla and transverse suture, representing the remnant of ancestral articulation of the sixth thoracic somite and the first abdominal somite. Anal operculum developed, reaching insertion of caudal ramus; free margin smooth and straight. Caudal rami ca 2.1-2.3 × as long as wide, with few spinules at anterior third length on lateral surface and at base of seta II, combined with a row of strong spinules latero-ventrally at base of seta III. Seta VI slightly shorter than seta III. Setal and spine formulae of exp-2 of P1- P4 5.5.5.5 and 3.4.4.3, respectively. P4 exp-2 with single apical spine; spine slightly shorter than segment. Inner spine on free segment of P5 as long as segment; outer seta on P5 ca 4.5 × as long as inner spine.

Etymology
The species name is a noun. The specific epithet was raised after the ʻSakaeratʼ Subdistrict, where the new species was encountered.
Allotype THAILAND • ♂ (completely dissected and mounted on one slide in glycerol and sealed with nail polish); same collection data as for holotype; ZMB 34230 slide No. 5121.

Additional material examined
THAILAND • 1 ♀, 1 ♂ (stored in a mixture of glycerol and 70% ethanol (ratio ~1:10 v/v)); same collection data as for holotype; collection of the third author (CB) • 2 ♀♀ (processed for taking photographs by SEM); same collection data as for holotype; collection of the third author (CB).

Type locality
The new species was collected in a headwater stream in SERS, Sakaerat Subdistrict, Nakhon Ratchasima Province, Northeast Thailand ( Fig. 1A-C). The locality is located in a dry evergreen forest and has been known under the name of "Tham Ngu Jong Ang" (King cobra Cave) in Thai language. The name refers to a rock shelter, which is the characteristic of the stream bank in the type locality. The stream reach is about 30 m long and about 4-5 m wide, streambed with bedrock. During dry season, the water does not encompass the channel entirely. The new species was collected in pool-formed area, where the water flows slowly. The mean water temperature was 25.3°C, pH 6.98, conductivity 64.7 µS cm -1 , and dissolved oxygen 9.1 mg L -1 .

Adult female
Total body length, measured from tip of rostrum to posterior margin of caudal rami, 0.73-0.75 mm (mean = 0.75 mm; n = 3; holotype = 0.74 mm) ( Fig. 2A). Naupliar eye not discernible. Rostrum V-shaped in frontal view, completely fused to cephalothorax, with two sensilla laterally and rounded tip (Fig. 2B). Prosome ca 64% of body length and ca 1.75 × as long as length of urosome. Cephalothorax anteriorly oval, ca 33% of body length and ca 1.15 × as long as wide, with greatest width at posterior margin; posterior margin smooth. Posterior margin of second pedigerous somite undulated, third pedigerous somite with serrated hyaline frill on posterior margin, posterior margin of fourth pedigerous somite smooth ( Fig. 2A, C). Fifth pedigerous somite with two transversal rows of spinules located between proximal seta and free segment of P5 (Fig. 3B, D), with two sensilla dorsally; posterior margin with serrated hyaline frill (Fig. 2D). Genital somite and first abdominal somite fused, forming genital double-somite. Genital double-somite symmetrical, ca 0.84 × as long as wide, tapering posteriorly; dorsally with two sensilla and transverse suture, representing the remnant of ancestral articulation (Figs 2D, 4B); posterior margin with serrated hyaline frill (Figs 2D, 3A). Seminal receptacle with clear distinction between anterior and posterior lobes; anterior lobe short and wide; posterior lobe globular, narrower than anterior one (Fig. 3A). Second and third abdominal somites narrower than genital double-somite, ca 56% of double-somite width, with serrated hyaline frill on posterior margin (Fig. 2D). Anal somite with a row of minute spinules latero-ventrally on posterior margin and two sensilla dorsally at base of anal operculum (Fig. 2D). Anal operculum developed, trapezoidal, reaching insertion of caudal ramus; free margin smooth and straight (Fig. 2D). Body with numerous integumental pits; pits shallow, hard to observe and less developed on anal somite (Figs 2C, 4B, E).
Caudal rami (Figs 2D, 3C, 4C). Relatively short, ca 2.25 × as long as wide, with six setae; all setae pinnate. Seta I absent. Seta II inserted at ⅓ of caudal ramus length. Seta III spiniform, inserted at posterior outer corner of ramus. Seta IV and seta V with breaking planes. Seta V, the longest, ca 0.34 × as long as body length. Seta VI slender, slightly shorter than seta III. Seta VII inserted dorso-medially at ⅕ of ramus length. Length ratio of caudal setae to ramus length, from seta II to seta VII: 0.37: 0.91: 4.23: 5.57: 0.86: 1.21. Lateral surface ornamented with few minute spinules located at anterior ⅓ of ramus length, few minute spinules at base of seta II, and a row of strong spinules at base of seta III.
Antennule (Fig. 5A) 11-[8]. Fifth segment with short spine on posterior outer corner. Aesthetasc on eighth and tenth segments slender, inserted near outer seta, as long as outer seta. Eleventh segment with acrothek sub-apically.     . Four-segmented, comprising coxobasis and three-segmented enp; setal formula 3.1.9.7. Coxobasis robust, with three transversal rows of spinules on caudal surface; two smooth setae on inner distal corner; seta representing exp spinulose, inserted on outer distal corner and reaching tip of enp-3. Enp-1 ca 1.5 × as long as wide, with smooth seta on medial margin. Enp-2 ca 1.5 × as long as wide, with longitudinal row of minute spinules on outer margin and nine setae; seven setae inserted along medial margin and two setae inserted apically. Enp-3 ca 2.0 × as long as wide, with two longitudinal rows of minute spinules along outer margin and seven smooth setae apically; outermost seta shortest. Labrum (Fig. 5D). Trapezoidal in frontal view, with two rows of hairs; cutting edge with 12 teeth medially between two obtuse lateral teeth.
Mandible ( Fig. 6A-B). Gnathobase with strongly chitinized teeth on cutting edge and spinulose seta dorsally; seta completely fused to segment. Palp reduced, one-segmented, with one short, slender seta and two long, bipinnate setae; two long setae subequal in length, ca 10 × as long as shorter one.
Maxillule (Fig. 6C). Three-segmented, composed of robust praecoxa and two-segmented maxillulary palp, representing coxobasis and enp. Arthrite of praecoxa with three strong claw-like extensions apically and one spinulose seta sub-apically. Praecoxa with seven elements along medial margin; proximalmost seta minute, sub-proximal seta robust and spinulose, three middle setae slender and smooth, sub-distal seta robust and smooth, distalmost seta robust and spinulose. Basal segment of palp with three elements apically; outer apical seta robust and armed with long spinules on outer margin; inner apical and subapical ones smooth. Exp reduced, represented by spinulose seta near lateral segment of palp. Enp represented by lateral segment of palp, with two setae apically and one seta sub-apically; all setae spinulose, subequal in length. Fig. 6D). Five-segmented. Praecoxa and coxa partly fused frontally. Praecoxal endite prominent, inserted medially, with one smooth and one spinulose seta apically. Coxa with two endites; proximal endite with one smooth seta apically; distal endite rectangular, movable, with two spinulose setae apically; spinules on proximal seta on distal endite relatively long, those of distal one minute. Basis with claw-like endite and two setae at base of claw; longest seta strong, inserted ventrally to claw; shorter one slender, inserted on caudal surface above the longest seta; concave margin of claw with oblique row of spinules, spinules fused to basis and increased in size from frontal spinule to caudal one. Enp two-segmented; enp-1 with two robust setae; enp-2 with strong seta apically and two smooth, slender setae sub-apically. Maxilliped (Fig. 6E). Four-segmented, composed of syncoxa, basis and two-segmented enp. Syncoxa with two endites and ornamented with a row of spinules on outer margin; proximal endite with two subequal spinulose setae apically; distal endite with one spinulose seta. Basis with one seta on caudal surface; basal endite with one spinulose seta apically. Enp-1 with strong spinulose seta. Enp-2 with three setae; apical seta strong, two other ones slender and smooth. P1-P4 (Fig. 7). Two-segmented enp and exp. Intercoxal sclerite with minute spinules on distal prominences. Coxa with one seta on distal inner corner. Basis with one seta laterally and hairy medially. Setal and spine formulae of exp-2 of P1-P4: 5.5.5.5 and 3.4.4.3, respectively. Armature of swimming leg as in Table 1. P1 (Fig. 7A). Frontal and caudal surfaces of intercoxal sclerite bare, with 2-3 minute spinules on distal prominences. Lateral seta on basis ca 4 × as long as those of P2-P4; medial seta pinnate, reaching mid of enp-2. Exp-1 with outer spine and inner seta. Exp-2 as long as wide, with three spines and five setae; apical spine ca 0.7 × as long as segment. Enp-1 with inner seta. Enp-2 ca 1.5 × as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine and seta, inner margin with three setae; outer seta ca 1.2 × as long as length of apical spine; apical spine strong, slightly curved, as long as segment. P2 (Fig. 7B). Intercoxal sclerite as in P1, yet distal prominences with 3-4 minute spinules. Basis with two hook-like expansions: outer expansion located between insertions of exp and enp; inner one smaller, located at the same place where the medial seta of basis of P1 inserted. Lateral seta on basis ca 0.25 × as long as that of P1. Exp-1 with outer spine and inner seta. Exp-2 ca 1.3 × as long as wide, with four spines and five setae; apical spine slightly shorter than segment. Enp-1 with inner seta. Enp-2 ca 1.5 × as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine and seta, inner margin with four setae; outer seta inserted between claw-like extensions, ca 1.3 × as long as length of apical spine; apical spine strong and straight, as long as segment.
P4 (Figs 4D, 7E). Intercoxal sclerite similar to that of P3. Coxa with rows of spinules on caudal surface. Basis similar to those of P2 and P3. Exp-1 with outer spine. Exp-2 ca 2.0 × as long as wide, with three spines and five setae; spines smaller than those of P1-P3, apical spine ca 0.5 × as long as segment. Enp-1 with inner seta. Enp-2 ca 2.0 × as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine and seta, inner margin with four setae; outer seta ca 1.3 × as long as length of apical spine; apical spine ca 0.8 × as long as segment.
P5 (Figs 3A-B, D-E, 4E). One-segmented, inserted on postero-lateral corner of fifth pedigerous somite. Proximal segment completely fused to somite, represented by lateral seta. Distal segment free, subquadrate, ca 1.1 × as long as wide, apically with one slender outer seta and one inner spine; inner spine as long as segment and outer seta ca 4.5 × as long as inner spine.

Adult male
Total body length, excluding caudal seta, 0.62-0.67 mm (mean = 0.65 mm; n = 3; allotype = 0.65 mm). Habitus smaller and slenderer than in female (Fig. 8A). Naupliar eye and rostrum as in female. Prosome ca 62% of body length and ca 1.62 × as long as length of urosome. Cephalothorax anteriorly oval, representing ca 33% of body length and ca 1.16 × as long as wide. Posterior margins of cephalothorax and two subsequent pedigerous somites (Fig. 4A) similar to those of female; that of fourth pedigerous somite smooth. Fifth pedigerous somite as that of female. Genital somite swollen on mediolateral margin ( Fig. 8A-B), ca 25% length of urosome and ca 0.62 × as long as wide, with hyaline frill latero-dorsally. First to third abdominal somites narrower than genital somite, representing ca 60% of genital somite width, with serrated hyaline frill on posterior margin. Anal somite and operculum similar to those of female.
P4. Frontal surface of intercoxal sclerite bare, caudal one with transversal row of minute spinules and distal prominences with 3-5 minute spinules (Fig. 9D). Coxa, basis, enp and exp similar to that of female; apical spine on enp-2 ca 0.8 × as long as segment. P5 (Fig. 9E-F). Similar to that of female, yet outer seta on free segment short, ca 2.3 × as long as inner spine.
P6 (Fig. 9G). Reduced to cuticular plate with two elements, inner (ventral) one spine and outer (dorsal) one seta; inner spine ca 0.5 × as long as outer seta.

Variability
The body length of the female specimens varies in a range of 0.73-0.75 mm (n = 3) and that of the male specimens is 0.62-0.67 mm (n = 3). The variations in the length / width ratio of caudal ramus in the female, along with the length of spine on the female P5 and the male P6 are shown in Table 2.

Distribution
Metacyclops sakaeratensis sp. nov. has been known only from the type-locality.

Diagnosis
Female. Body size moderate (0.89-0.96 mm; n = 6), without integumental pits. Posterior margin of second pedigerous somite undulated, those of third and fifth pedigerous somite with serrated hyaline frill. Genital double-somite dorsally with two sensilla and transverse suture, representing the remnant of ancestral articulation of the sixth thoracic somite and the first abdominal somite. Anal operculum developed, reaching insertion of caudal ramus; free margin smooth and concave. Caudal rami ca 2.6-2.9 × as long as wide, ornamented with 3-4 spinules at anterior third length on lateral surface and few spinules at base of seta II, combined with a row of strong spinules latero-ventrally at base of seta III. Seta VI slightly shorter than seta III. Setal and spine formulae of exp-2 of P1-P4 5.5.5.5 and 3.4.4.3, respectively. P4 exp-2 with single apical spine; spine slightly shorter than segment. Inner spine of free segment of P5 longer than segment, ca 1.5 × as long as segment; outer seta on P5 ca 2.5 × as long as inner spine.

Etymology
The name is a masculine noun in genitive singular, raised after Professor Dr Anton Brancelj (National Institute of Biology, Ljubljana, Slovenia) in honor of his great contribution to the diversity of subterranean Copepoda in Thailand.  (Shen & Tai 1964) Paratypes THAILAND • 1 ♀, 1 ♂ (each completely dissected and mounted on a slide in glycerol and sealed with nail polish); same collection data as for holotype; ZMB 34231 slide No. 5126-5127 • 1 ♀ (stored in a mixture of glycerol and 70% ethanol (ratio ~1:10 v/v)); same collection data as for holotype; ZMB 34231a.

Type locality
The new species was collected from a pool in Phupha Phet Cave, Manang District, Satun Province, southern Thailand (Fig. 1A, D). The cave is located in a limestone hill of the Nakhon Sri Thammarat Mountain range. Beyond the entrance there is a very large cave tunnel, separated to several rooms and decorated well with stalactites, stalagmites, flowstones and rimstones, with a wooden bridge throughout the cave and installed lights. The collecting point is about 100 meters from the entrance, being a large temporary pool in the room, named "Dok Boa Khwam" (Upside-down lotus flower). Water comes primarily from the epikarst zone of the cave, and the depth varies according to season, ranging from 5 cm in dry season to 40 cm in rainy season. In collecting date, the water was about 40 cm deep, covering an area of about 30 m 2 , transparent and colorless.

Adult female
Total body length, measured from tip of rostrum to posterior margin of caudal rami, 0.89-0.96 mm (mean = 0.94 mm; n = 6; holotype = 0.89 mm) (Fig. 10A). Body without integumental pits (Fig. 11A-B). Naupliar eye not discernible. Rostrum V-shaped in frontal view, with rounded tip, completely fused to cephalothorax, with two sensilla laterally. Prosome ca 65% of body length and ca 1.87 × as long as length of urosome. Cephalothorax anteriorly oval, ca 32% of body length and ca 1.08 × as long as wide, with greatest width at posterior end; posterior margin smooth. Posterior margin of second pedigerous somite undulated; third pedigerous somite with serrated hyaline frill on posterior margin; posterior margin of fourth pedigerous somite smooth (Figs 10A, 11A). Fifth pedigerous somite with two transversal rows of spinules located between proximal seta and free segment of P5, with two sensilla dorsally; posterior margin with serrated hyaline frill. Genital double-somite symmetrical, ca 0.82 × as long as wide, tapering posteriorly, dorsally with two sensilla and transverse suture, representing the remnant of ancestral articulation; posterior margin with serrated hyaline frill (Figs 10A-B, 11B). Seminal receptacle with clear distinction between anterior and posterior lobes; anterior lobe short and wide; posterior lobe globular, narrower than anterior one. Second and third abdominal somites narrower than genital doublesomite, ca 56% of double-somite width, with serrated hyaline frill on posterior margin (Figs 10A, 11B). Anal somite with row of minute spinules latero-ventrally on posterior margin and two sensilla dorsally at base of anal operculum (Fig. 10C). Anal operculum developed, trapezoidal, reaching insertion of caudal ramus; free margin smooth and concave (Fig. 10C).
Caudal rami (Figs 10C-D, 11C). Relative short, ca 2.7 × as long as wide, with six setae; all setae pinnate. Seta I absent. Seta II inserted at ⅓ of caudal ramus length. Seta III spiniform, inserted at posterior outer corner of ramus. Seta IV and seta V with breaking planes. Seta V, the longest, ca 0.33 × as long as body length. Seta VI slender, slightly shorter than seta III. Seta VII inserted dorso-medially at ⅕ of ramus length. Length ratio of caudal setae to ramus length, from seta II to seta VII: 0.27: 0.84: 3.82: 4.98: 0.70: 0.82. Lateral surface ornamented with 4-5 minute spinules located at anterior ⅓ of ramus length, with few minute spinules at base of seta II and a row of strong spinules latero-ventrally at base of seta III.
Antenna (Fig. 12B). Four-segmented, comprising coxobasis and three-segmented enp; setal formula 3.1.9.7. Coxobasis robust, with three transversal rows of spinules on caudal surface and two smooth setae on inner distal corner; seta representing exp spinulose, inserted on outer distal corner and reaching tip of enp-3. Enp-1 ca 1.5 × as long as wide, with smooth seta on medial margin. Enp-2 ca 1.5 × as long as wide, with longitudinal row of minute spinules on outer margin and nine setae; seven setae inserted along medial margin and two setae inserted apically. Enp-3 ca 2.0 × as long as wide, with two longitudinal rows of minute spinules along outer margin and seven smooth setae apically; outermost seta shortest. Mandible (Fig. 12C). Gnathobase with strongly chitinized teeth on cutting edge and spinulose seta dorsally; seta completely fused to segment. Palp reduced, one-segmented, with one short, slender seta and two long, bipinnate setae; two long setae subequal in length, ca 10 × as long as shorter one. Maxillule (Fig. 12D). Three-segmented, composed of robust praecoxa and two-segmented maxillulary palp, representing coxobasis and enp. Arthrite of praecoxa with three strong claw-like extensions apically and one spinulose seta sub-apically. Praecoxa with seven elements along medial margin; proximalmost seta minute, sub-proximal seta robust and spinulose, three middle setae slender and smooth, sub-distal seta robust and smooth, distalmost seta robust and spinulose. Basal segment of palp with three elements apically; outer apical seta robust and armed with long spinules on outer margin; inner apical and subapical ones smooth. Exp reduced, represented by spinulose seta near lateral segment of palp. Enp represented by lateral segment of palp, with two setae apically and one seta sub-apically; all setae spinulose, subequal in length. (Fig. 12E). Five-segmented. Praecoxa and coxa partly fused frontally. Praecoxal endite prominent, inserted medially, with one smooth and one spinulose setae apically. Coxa with two endites; proximal endite with one smooth seta apically; distal endite rectangular, movable, with two spinulose setae apically; spinules on proximal seta of distal endite relatively long, those of distal one minute. Basis with claw-like endite and two setae at base of claw; longest seta strong, inserted ventrally to claw; shorter one slender, inserted on caudal surface above the longest seta; concave margin of claw with oblique row of spinules; spinules fused to basis and increased in size from frontal spinule to caudal one. Enp twosegmented; enp-1 with two robust setae; enp-2 with strong seta apically and two smooth, slender setae sub-apically.

Maxilla
Maxilliped (Fig. 12F). Four-segmented, composed of syncoxa, basis and two-segmented enp. Syncoxa with two endites and ornamented with arch row of spinules on outer margin; proximal endite with two subequal spinulose setae apically; distal endite with one spinulose seta. Basis with one seta on caudal surface; basal endite with one spinulose seta apically, with a row of long spinules on frontal surface. Enp-1 with strong spinulose seta. Enp-2 with three setae; apical seta strong, two other ones slender and smooth. P1-P4 (Fig. 13). Two-segmented enp and exp. Intercoxal sclerite of P1 with few spinules on distal prominences, those of P2-P4 with 3-6 smaller spinules. Coxa with one seta on distal inner corner. Basis with one seta laterally and hairy medially. Setal and spine formulae of exp-2 of P1-P4: 5.5.5.5 and 3.4.4.3, respectively. Armature of swimming leg as in Table 1. P1 (Fig. 13A). Frontal and caudal surfaces of intercoxal sclerite bare; distal prominence with 2-3 spinules. Lateral seta on basis ca 4 × as long as those of P2-P4; inner seta pinnate, reaching mid of enp-2. Exp-1 with outer spine and inner seta. Exp-2 as long as wide, with three spines and five setae; apical spine ca 0.71 × as long as segment. Enp-1 with inner seta. Enp-2 ca 1.5 × as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine and seta, inner margin with three setae; outer seta ca 1.3 × as long as length of apical spine; apical spine strong, slightly curved, as long as segment.   P2 (Fig. 13B). Intercoxal sclerite as in P1, with 4-5 spinules on distal prominence. Basis with two hooklike expansions: outer expansion located between insertions of exp and enp; inner one smaller, located at the same place where the medial seta of basis of P1 inserted. Lateral seta on basis ca 0.25 × as long as that of P1. Exp-1 with outer spine and inner seta. Exp-2 ca 1.5 × as long as wide, with four spines and five setae; apical spine slightly shorter than segment. Enp-1 with inner seta. Enp-2 ca 1.7 × as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine and seta, inner margin with four setae; outer seta ca 1.3 × as long as length of apical spine; apical spine strong and straight, as long as segment.
P3. Frontal surface of intercoxal sclerite bare; caudal surface with transversal row of minute spinules and distal prominences with 4-6 minute spinules (Fig. 13C). Basis, exp, and enp similar to those of P2. P4 (Fig. 13D). Intercoxal sclerite similar to that of P3; yet distal prominences with 3-4 spinules. Distal margin of coxa with rows of spinules on caudal surface. Basis similar to those of P2 and P3. Exp-1 with outer spine. Exp-2 ca 1.4 × as long as wide, with three spines and five setae; spines relatively smaller than those of P1-P3, apical spine ca 0.5 × as long as segment. Enp-1 with inner seta. Enp-2 ca 1.7 as long as wide, with outer seta inserted between claw-like expansion, apically with robust spine, inner margin with four setae; outer seta ca 1.3 × as long as length of apical spine; apical spine ca 0.9 × as long as segment.
P5 (Fig. 13E-F). One-segmented, inserted on postero-lateral corner of fifth pedigerous somite. Proximal segment completely fused to somite, represented by lateral seta. Distal segment free, subquadrate, ca 1.1 × as long as wide, with one slender outer seta and one inner spine apically; inner spine ca 1.5 × as long as segment and outer seta ca 2.5 × as long as inner spine.

Adult male
Total body length, excluding caudal seta, 0.71-0.76 mm (mean 0.74 mm; n = 3; allotype = 0.71 mm) (Fig. 14A). Habitus smaller and slenderer than in female. Naupliar eye and rostrum as in female. Prosome ca 62% of body length and ca 1.57 × as long as length of urosome. Cephalothorax anteriorly oval, representing ca 32% of body length and ca 1.12 × as long as wide. Second to fifth pedigerous somites similar to those of female. Genital somite swollen on mediolateral margin ( Fig. 14A-B); ca 25% length of urosome and ca 0.6 × as long as wide, with hyaline frill latero-dorsally. First abdominal somite and two subsequent somites narrower than genital somite, ca 60% of genital somite width, with serrated hyaline frill on posterior margin. Anal somite and operculum similar to those of female.
Caudal rami. As long as that in female, ca 2.7 × as long as wide. Armament and ornamentation similar to those of female. Length ratio of caudal setae to ramus length slightly different to that of female; ratio of caudal setae to ramus length from seta II to seta VII: 0.29: 0.82: 3.94: 5.50: 0.70: 0.97. Antennule (Fig. 14C-E P1. Frontal and caudal surfaces of intercoxal sclerite bare and distal prominences with 2-3 minute spinules (Fig. 15A). Coxa, basis, enp and exp similar to those of female. P2. Intercoxal sclerite as in P1, distal prominences with 4-5 minute spinules (Fig. 15B). Coxa, basis, enp and exp similar to those of female.
P3. Frontal surface of intercoxal sclerite bare, caudal one with transversal row of minute spinules and distal prominences with 4-5 minute spinules (Fig. 15C). Coxa, basis, enp and exp similar to those of female.
P4. Frontal surface of intercoxal sclerite bare, caudal one with transverse row of minute spinules and distal prominences with 3-4 minute spinules (Fig. 15D). Coxa, basis, enp and exp similar to that of female; apical spine on enp-2 ca 0.8 × as long as segment.
P5 (Figs 11D, 14B, 15E-F). Similar to that of female. Free segment ca 1.1 × as long as wide, with one slender outer seta and one inner spine apically; inner spine strong, ca 1.5 × as long as segment bearing it; outer seta ca 2.5 × as long as inner spine. P6 (Figs 11E, 14B, 15G). Reduced to cuticular plate with two elements; inner spine strong, slightly shorter than outer seta.

Variability
The female specimens collected in Phupha Phet Cave are slightly smaller (0.89 mm each, n = 2) than those of Rakhang Thong Cave (0.95-0.96 mm, n = 2) and Khao Nui Cave (0.95-0.96 mm, n = 2). Similarly, the male specimens collected in Phupha Phet Cave (0.71 mm, n = 1) are smaller than those of Rakhang Thong Cave (0.75-0.76 mm, n = 2). Unfortunately, because the male has not yet been encountered from Khao Nui Cave, the length of the male is doubtful for this cave. Minor variation in the length / width ratio of the caudal rami, the length of spines of the female P5 and the male P6 are shown in Table 2.

Habitat
Metacyclops brancelji sp. nov. was collected during the rainy season from temporary water bodies which are seasonally filled primarily by water from the epikarst zone of the cave. Specimens were collected by hand net from both a large pool (n = 53) and a rimstone (n = 10) (Fig. 1E-F), about 100 meters from the entrance of Phupha Phet Cave. The type locality is in the dark zone where the temperature is relatively constant. Based on four sampling occasions from December 2014 to December 2015, the temperature in the dark zone of Phupha Phet Cave varied from 24.4°C in July 2015 to 27.8°C in April 2015. In Khao Nui Cave, the new species was collected in December 2014 from a water body filled by dripping water (n = 4) at the deepest part of the twilight zone, about 20 meters from the entrance (Fig. 1H). In Rakhang Thong Cave, the new species was collected in July 2015 from a rimstone (n = 33) at the cave entrance. The entrance of Rakhang Thong Cave is located at the base of the hill. The area in front of the entrance is a rock shelter which has been modified for religious propose. Near the rimstone is a Buddha statue and the gutter was created to collect water from the top of the shelter above the rimstone (Fig. 1G). These let us hypothesize that the discovery of M. brancelji sp. nov. in the rimstone of Rakhang Thong Cave is accidental and the new species could be encountered in water bodies around the hill and in the cave, like in Phupha Phet Cave and Khao Nui Cave. Furthermore, if compared to the regional distribution of the three latest described copepod species in Satun Province, including Onychocamptus satunensis Boonyanusith, Saetang, Wongkamhaeng & Maiphae, 2018, Boholina laorsriae Boonyanusith, Wongkamhaeng & Athibai, 2020 and Rangabradya (Siamorangabradya) wongkamhaengae Boonyanusith & Athibai, 2021, the distribution of M. brancelji sp. nov. is wider since it was collected in three separate caves located along the south of the mountain range, while the three above-mentioned species were only encountered in a single cave. The characteristics mentioned above suggest a stygophylic nature of M. brancelji sp. nov. rather than stygophilic, as suggested by many authors that stygophile exhibits an adaptation to spend their whole life and reproduce in subterranean habitats and can live in epigean environments (Galassi 2001;Sket 2008;Brancelj 2015).
The armature of P5 and P6 are additional characters, separating the species (Table 2). In M. sakaeratensis sp. nov. and M. woni, the inner spine of P5 is minute, as long as the free segment, whereas it is ca 1.5 × as long as the segment in M. brancelji sp. nov. Conversely, the inner spine of male P6 is subequal in length with the outer seta in M. brancelji sp. nov. and M. woni, while it is much shorter in M. sakaeratensis sp. nov. Additionally, in M. sakaeratensis sp. nov. there are 2-3 spinules in the row of spinules on the anterior third of the lateral surface of the caudal ramus, while in M. brancelji sp. nov. and M. woni the row of spinules is composed of 4-5 spinules. Other differences between the two new species and M. woni are listed in Table 2.
Previously,  argued that the ornamentation of the spinule on the anterior third of the lateral surface of the caudal ramus is very characteristic for M. woni, but we realized that it has Kinnecaris iulianae Bruno & Cottarelli, 2015(Alekseev & Sanoamuang 2006Cottarelli et al. 2010;Bruno & Cottarelli 2015). Yet due to the differences of local conditions (e.g., substrate type, water velocity) within a headwater stream there are an enormous array of habitats and the geographical isolation of such streams support genetically isolated species which contribute to the diversity of aquatic fauna at the catchment-scale (Gomi et al. 2002;Meyer et al. 2007). Therefore, research on zooplankton diversity in headwater streams should be encouraged to fill the gap of knowledge on copepod diversity not only in Thailand but also in Southeast Asia.