A new species of Mongolodiaptomus Kiefer, 1938 from northeast Thailand and a key to the species (Crustacea, Copepoda, Calanoida, Diaptomidae)

Abstract This study describes the new species Mongolodiaptomus loeiensis sp. n. collected from a temporary pond nearby a cave located in Loei Province, in northeastern Thailand. Mongolodiaptomus loeiensis sp. n. is similar to M. calcarus (Shen & Tai, 1965) in the male but can be distinguished from its congeners by the following unique characteristics in the males: (1) the right caudal ramus has 3 ventral chitinous prominences; (2) intercoxal plate of P5 is produced into 2 spine-like lobes on distal margin; (3) the basis of right P5 has a subglobular chitinous prominence on mid-distal caudal surface; and (4) the principal lateral spine on the right Exp-2 P5 is extremely bent at its tip. The occurrence of diaptomid copepods in the study area is discussed and an identification key to worldwide species of the genus Mongolodiaptomus Kiefer, 1938 is presented herein.


Introduction
The freshwater calanoid copepods have been intensively studied in Thailand especially in northeast Thailand. However, their study in the Loei Province in the upper northeast region has so far been largely neglected. According to Sanoamuang (2002), only three diaptomid species were previously recorded in Loei Province, namely Mongolodiaptomus botulifer (Kiefer, 1974), M. calcarus (Shen & Tai, 1965), and Phyllodiaptomus praedictus (Dumont & Reddy, 1994). During the years 2014-2016, the first author had led sampling surveys on planktonic and cave-dwelling copepods in the upper northeastern region in order to fill the gap of copepod richness and distribution in this region (Watiroyram et al. 2015(Watiroyram et al. , 2017. As results of this study, Mongolodiaptomus loeiensis sp. n. and other diaptomids were discovered in water bodies outside the caves. The genus Mongolodiaptomus Kiefer, 1938 was defined by Kiefer (1938) by several characters, especially in the male fifth leg. Nevertheless, some of these characters are not useful for separating the species because they are shared by certain species of Allodiaptomus Kiefer, 1936 andNeodiaptomus Kiefer, 1932. After the revision of Reddy et al. (2000), the solution on diagnosis of problematic species was well defined on generic characters. Based on this revision, Mongolodiaptomus is characterized by the second exopod of the right male P5 having 3 spines and processes on its outer margin; one principal spine somewhat on middle of segment, and 1-2 spinous processes proximally or/and distally. As a result, nine species of Allodiaptomus, Diaptomus and Neodiaptomus were transferred into the genus Mongolodiaptomus; namely M. birulai (Rylov, 1922), M. botulifer, M. calcarus, M. gladiolus (Shen & Lee, 1963), M. malaindosinensis (Lai & Fernando, 1978), M. mephistopheles (Brehm, 1933), M. pectinidactylus (Shen & Tai, 1964), M. rarus (Reddy, Sanoamuang & Dumont, 1998), M. uenoi (Kikuchi, 1936) (for more details see Kiefer 1939;Reddy et al. 1998Reddy et al. , 2000Sanoamuang, 1999Sanoamuang, , 2002Luong et al. 2016).
To date, 37 diaptomid species are known from inland waters of Thailand (Sanoamuang, 2002). Of these, seven species belong to the genus Mongolodiaptomus: M. malaindosinensis, M. botulifer, M. calcarus, M. dumonti Sanoamuang, 2001, M. pectinidactylus, M. rarus, and M. uenoi. Mongolodiaptomus loeiensis sp. n., the eighth species from Thailand, is illustrated and described herein together with a dichotomous key to the worldwide species of the genus Mongolodiaptomus. Additionally, the geographical distribution of the recorded diaptomids in Loei Province is briefly discussed.

Material and methods
Samples were collected using a plankton net with a mesh size of 60 µm. The copepod samples were transferred into 120 ml plastic bottles and preserved in 70% ethanol. In the laboratory, samples were selected for individual adults and placed in a mixture of glycerol and 70% ethanol (ratio ~ 1:10 v/v) under a stereomicroscope at 40× magnification.
Specimens were transferred to pure glycerol and dissected at 40-100-× magnification under an Olympus SZ51 stereomicroscope.
All appendages and body ornamentation were examined at1000-× magnification. All the drawings were made at the same magnification (1000-×), with a drawing tube mounted on an Olympus compound microscope (CX31). The final versions of the drawings were made using the CORELDRAW ® 12.0 graphic program. For permanent slides, all body parts were put in a drop of glycerol on a microscope slide, covered by a cover glass, and sealed with nail polish.
Specimens for a scanning electron microscopy (SEM) were dehydrated in an ethanol series (50%, 70%, 80%, 90%, 95%, 100%, and 100%) for 15 min each concentration. After dehydration, specimens were dried in a critical point dryer using liquid carbon dioxide as the exchange medium. Dried specimens were mounted on stubs using adhesive tape under a stereomicroscope. Then, specimens were coated with gold in a sputter-coater. The SEM photographs were carried out using a scanning electron microscope (FEI Helios NanoLab G3 CX).
The nomenclature and descriptive terminology follow Huys and Boxshall (1991), including analysis of caudal setae (I-VII). Specimens were deposited at the Natural History Museum, London, United Kingdom (NHMUK) and at the Nakhon Phanom University, Faculty of Science, Thailand (NPU).
Holotype. One adult male, NHMUK 2017.134, dissected and mounted in glycerol on one slide: collected on 5 August 2015 by S. Watiroyram. Allotype. One adult female, NHMUK 2017.135, dissected and mounted in glycerol on one slide: collected on same date by the same collector.
Differential diagnosis. The right P5 Exp-2 of male in Mongolodiaptomus loeiensis sp. n. with principal spine slightly posterior to mid-outer margin and one spinous process each proximally and distally, fits the diagnostic features of the genus Mongolodiaptomus sensu Reddy et al. (2000).
The male of new species is most similar to M. calcarus by the segment 20 of right antennule with serrated spine, and the armature of P5: on right P5 (1) the presence of strong and stout coxal spine, (2) Exp-2 with similar shape and size, and (3) Exp-2 with strong and bent principal lateral spine; on left P5 (1) basis with long and narrow hyaline membrane on inner margin, and (2) Exp-2 with row of strong spinules on inner margin at distal half of segment. However, M. loeiensis sp. n. differs from M. calcarus by following morphological characters: (1) the new species with three chitinous processes on ventral surface of the right caudal ramus while M. calcarus with only two chitinous processes, (2) intercoxal plate of the new species with outgrowth process into two-spine like lobes at distal margin while M. calcarus without any outgrowth process, (3) basis of right P5 in the new species with inner hyaline membrane and with sub-globular chitinous process on caudal surface while M. calcarus without hyaline membrane, and with spur-like chitinous process, (4) the new species with long and slender end claw while it is typical short and robust in M. calcarus. Additional differences occur in female characteristics as follows: (1) the genital somite of M. loeiensis sp. n. with bulges on proximal, sub-middle, and middle region at right margin while M. calcarus is slightly convex on those region, (2) the right side of genital somite with spine located on outgrowth process in M. loeiensis sp. n. but it is directly inserted on somite in M. calcarus, (3) the left side of genital somite with slightly proximal dilated and spine inserted on small prominence while M. calcarus with obviously proximal rounded lobe and spine directly inserted on its segment.
Description of adult female. Body length (Fig. 1A), measured from anterior margin of rostrum to posterior margin of caudal rami, 1.0-1.3 mm. Rostral spines (Figs 1B, 4B) with two teeth-like process in anterior margin. Prosome ( Fig. 1A) ovoid, with cephalosome and 5 pedigers; pediger 4 and 5 fused, partly separated at lateral side. Pediger 5 (Figs 1C, 4A) with symmetrical postero-lateral wings, reaching proximal part of genital somite; each wing with one inner and one posterior spine on posterolateral margins (former spine smaller than later one). Urosome (Fig. 1A, D) consisting of 3-somites including genital somite, urosomite 2 and anal somite, approximately 1/2 as long as prosome. Genital somite (Figs 1C-F, 4A) exceeding in length urosomite 2, anal somite and caudal ramus combined. Right side: with dilated laterally in three regions on proximal half of segment length; with postero-laterally directed outgrowth (sub-conical process), extended beyond half of somite; with a short and blunt posterior spine on respective outgrowth. Left side: with regular margin, with a spine on dorso-lateral surface at proximal part, larger and sharper than the right spine. A pair of gonopores located beneath a genital operculum on mid-ventrally proximal part. Urosomite 2 symmetrical, shorter than latter somite. Anal somite (Figs 1A, D, 4C) slightly expanded at distal end, almost as long as caudal rami; anal operculum small and slightly concave on its posterior margin.
Additional ornamentation of P1-P5 as in Figs 3A-F, 5F-I. Adult females with a single egg sac containing 8-10 eggs. Description of adult male. Body length (Fig. 6A) measured from anterior margin of rostrum to posterior margin of caudal rami, 0.9 -1.1 mm (mean = 1.0 mm, n = 5), smaller than female. Prosome as in female but lateral wings not well developed compared to those in female, pediger 5 without inner spine inserted on dorso-posterior margin of each wing. Urosome (Figs 4D, 6B-C) 5-segmented and asymmetrical, oriented downward to right side. Genital somite (Fig. 6B) dilated postero-laterally on right side accompanied with a small seta on distal corner. Urosomites 2-3 (Figs 4D, 6C) with a field of long hairs ventrally along middle of segment. Urosomite 4 (Figs 4D-E, 6B) with posterolateral dilated on right side; dorso-posterior margin expanded beyond anal operculum. Anal somite (Figs 4D-E, 6B-D) similar to female but asymmetrical, right side located at lower position than opposite side. Caudal rami (Figs 4D-E, 6B-D) similar to female in setation but having different shape and ornamentation on right ramus. Ramus asymmetrical and cylindrical shaped: right ramus with three ventral chitinous prominences: two teeth-like on large bulge at proximal region (outer one small and with sharped tip; inner one large and with rounded tip), and one semi-circular ridge located above insertion of caudal seta IV and V.
Left antennule, antenna, mouthparts, and P1-P4 similar to those in female. P5 (Figs 5A-E, 7) asymmetrical, right leg reaching beyond caudal setae. Intercoxal plate with two tooth-like lobes on distal margin, its tip bent forward to left leg. Right P5: coxa with strong and stout spine inserted on well-developed posterior lobe on caudal surface. Basis with long and narrow hyaline lamella at inner margin; small chitinous prominence (sub-globular in shape) located approximately mid-distal of segment on caudal surface; distal outer margin with short, thin seta on frontal surface. Exp 2-segmented: Exp-1 small and shorter than wide, approximately 0.6 times as long as wide; with two prominent knobs on caudal surface; distal outer corner produced. Exp-2 enlarged, approximately 2.0 times as long as wide; proximal and distal part enlarged in similar size, inner margin slightly convex and outer margin concave; with two minute knobs and one principal (lateral) spine at outer margin (knob-like projection located on proximal and distal region; lateral spine inserted slightly anterior to mid-outer margin of segment). Lateral spine strong, cylindrical, approximately 1/2 of segment length; its tip bent upward to posterolateral direction on caudal view. End claw sickle-shaped, strong and pointed tip; approximately 1.5 times as long as Exp-2, inner and outer margins smooth. Enp 1-segmented, conical, reaching to proximal expansion of Exp-2, with cluster of spinules at rounded tip. Left P5: coxa with thin seta inserted on posterior lobe at distal inner corner, exceeding basis, and posterolateral margin with semi-circular concave on caudal surface. Basis with long, narrow hyaline lamella at inner margin (small size than right P5); with long, thin seta at posterolateral margin on caudal surface. Exp 2-segmented: Exp-1 longer than wide, gradually tapering posteriorly, inner margin concave and outer one convex; with field of setules on inner margin at distal end. Exp-2 smaller than Exp-1, conical; with seta on inner margin at distal end on frontal surface, as long as segment; with a cluster of strong spinules along inner margin; apical process stout, bare, and short. Enp 1-segmented, longer than Exp-1, with a cluster of spinules at its tip.
Etymology. The specific name loeiensis refers to the place "Loei" where the new species was first recognized. The name with the Latin suffix "-ensis" is an adjective for place. Distribution and ecology of the new species and other diaptomids in the area of study. Mongolodiaptomus loeiensis sp. n. was found in a temporary pond with a mean water temperature of 25.5 °C, conductivity 259 µS/cm, and pH 7.6. The new species was a single calanoid copepod occurring in type locality. However, it was found co-occurring with other microcrustaceans, i.e. Diaphanosoma excisum Sars, 1885, Thermocyclops decipiens (Kiefer, 1929), and Mesocyclops thermocyclopoides Harada, 1931. In over 3,000 samples collected from Thailand (59 samples from Loei Province provided by the first author and about 3,000 samples from other provinces by the second author and her colleagues), the new species can by considered as a rare species because it was present only in the type locality from Loei Province (Sanoamuang 2002).
The occurrence of the new species is similar to that of Phyllodiaptomus thailandicus Sanoamuang & Teeramaethee, 2006 and Tropodiaptomus cf. ruttneri (Brehm, 1923), which have so far been known as rare species in Thailand and here each species was found in a single location or in about 2% of collected samples. P. thailandicus has hitherto been found in six provinces; Prachinburi, Chanthaburi, Sa Kaeo, Suphanburi, Kanchanaburi, and Chumphon Teeramaethee 2006, Koompoot andSanoamuang 2012) whereas Tropodiaptomus cf. ruttneri was only known from Phayao and Nan provinces (Sanoamuang 2002). The findings of P. thailandicus and Tropodiaptomus cf. ruttneri in this study are a new record to northeastern Thailand and provide more understanding on its distribution range in the country. Vietodiaptomus blachei (Brehm, 1951) was found in a few samples or approximately 9% of collected samples whereas P. praedictus, M. botulifer, and M. calcarus were frequently found in about 41, 39 and 6% of collected samples, respectively.