﻿A description of a new species of Mongolodiaptomus Kiefer, 1937 (Copepoda, Calanoida, Diaptomidae) from Thailand with an up-to-date key to the genus

﻿Abstract The genus Mongolodiaptomus is widely distributed in stagnant water bodies in Southeast Asia. During a comprehensive collection of freshwater copepods from different areas in Thailand, a previously unknown species of calanoid copepod, Mongolodiaptomusphutakaensissp. nov., was recorded. Representatives were found in a natural swamp located in the Kok Phutaka community forest in Khon Kaen Province, northeastern Thailand. The new species belongs to the “M.loeiensis species group” and most closely resembles M.loeiensis and M.mekongensis by having a distinct shape of the second exopodal segment of the male right P5, with enlarged proximal and distal parts of the outer margin as well as a bent and twisted principal lateral spine. The new species can be distinguished from its congeners by various characters in the males. The ventral surface of the right caudal ramus has two chitinous teeth and two knobs. The intercoxal plate is slightly produced distally and without any spine. The right P5 basis lacks a hyaline membrane on the inner margin but has a distinct spur-like chitinous process at the mid-distal margin on the posterior surface. The left P5 basis has a thin, longer hyaline lamella on the inner margin. The new species is rare, having been observed in only one out of approximately 5,000 surveyed locations in Thailand. A detailed morphological comparison and an up-to-date key to the Mongolodiaptomus species are presented. Their taxonomic characters, interspecies relationships, and biogeography are discussed.


Introduction
In 1937, Kiefer created the genus Mongolodiaptomus to include a group of Asian freshwater diaptomid copepods, with Mongolodiaptomus formosanus Kiefer, 1937 as the type taxon (Kiefer 1937;Walter and Boxshall 2024).Ranga Reddy et al. (2000) recommended to use the ornamentation of the right second exopod of the male P5 as an important character to distinguish between diaptomid copepods, especially those from the three closely related genera Neodiaptomus Kiefer, 1932, Allodiaptomus Kiefer, 1936, and Mongolodiaptomus Kiefer, ZooKeys 1209: 19-42 (2024), DOI: 10.3897/zookeys.1209.125838Laorsri Sanoamuang & Kamonwan Koompoot: Mongolodiaptomus phutakaensis sp.nov.from northeast Thailand 1937.Thus, the characters used to distinguish the males of Mongolodiaptomus from related genera is the presence of at least two lateral spines on the second exopod of the right P5, one principal spine in the middle of the segment, and one or two accessory spines proximally or distally on the outer margin.
At present, the genus Mongolodiaptomus consists of 11 valid species, distributed across Asia, with countries in the lower Mekong River Basin as the epicenter (Sanoamuang and Watiroyram 2018).However, this is not the final number, since there are two more species with doubtful identities.Ranga Reddy et al. (1998) observed some morphological variabilities in M. botulifer (Kiefer, 1974) from Thailand, leading to serious doubt about the validity of the closely allied M. malaindosinensis (Lai & Fernando, 1978).Despite the similarities in morphological features, Sanoamuang and Dabseepai (2021: 20) provide detailed explanations that distinguish the two congeners from each other.The validity was confirmed in the previously mentioned paper.Ranga Reddy et al. (1998) originally described M. rarus (Ranga Reddy, Sanoamuang & Dumont, 1998) as Allodiaptomus rarus, based on a single male specimen from Thailand.This species was later transferred to the genus Mongolodiaptomus by Sanoamuang (2001).Since there are no type specimens of M. malaindosinensis available and no description of the female morphology of M. rarus, it would be advisable to redescribe M. malaindosinensis and M. rarus, pending the collection of new specimens.
Thailand is the most species-rich country of the Mongolodiaptomus species, with nine already known taxa plus one new species reported (Ranga Reddy et al. 1998Reddy et al. , 2000;;Sanoamuang 1999Sanoamuang , 2001;;Watiroyram and Sanoamuang 2017;Sanoamuang and Watiroyram 2018;Sanoamuang and Dabseepai 2021;this study).Recently, Watiroyram and Sanoamuang (2017) provided a key to the identification of both sexes of the valid Mongolodiaptomus species.
During the study of copepod diversity in the forest area of the Plant Genetics Conservation Project in Khon Kaen province, northeast Thailand, we came across a new species of the genus Mongolodiaptomus.As a result, this paper deals with the following: i) an illustrated description of M. phutakaensis sp.nov.; ii) a review and detailed morphological characteristics comparison of the genus Mongolodiaptomus; iii) the interspecies relationships; iv) the biogeography of the genus; and v) the updated key to the genus.

Materials and methods
The study area, Kok Phutaka community forest, is located in Wiang Kao District, 78 kilometers from the center of Khon Kaen Province in northeastern Thailand.Her Royal Highness Princess Maha Chakri Sirindhorn of Thailand initiated the Plant Genetics Conservation Project in 1992, which encompasses an area of approximately 1,150 square kilometers.This protected area is a dry dipterocarp forest that provides a source for researchers to study the biodiversity and utilization of plants, animals, and microorganisms.There is one natural swamp and four small artificial ponds in the forest.
Monthly sampling campaigns were conducted from January to December 2007 in all the five above-mentioned habitats using a plankton net with a mesh size of 60 μm.All samples were preserved in 70% ethanol immediately after collection.Specimens were put in a mixture of glycerol and 70% ethanol (ratio 1:10 v/v) and pure glycerol, respectively, just before dissection.Specimens were dissected and mounted at 40-100× magnification under an Olympus SZ51 stereomicroscope.An Olympus compound microscope (CX31) was used to examine all appendages and body ornamentation at 1,000× magnification.All the drawings were created using an Olympus U-DA drawing tube and a compound microscope configured for 100× magnification.Final versions of the drawings were made using the CorelDRAW® 12.0 graphic program.Specimens for scanning electron microscopy (SEM) were dehydrated in an ethanol series (50%, 70%, 80%, 90%, 95%, 100%), for 15 min at each concentration.Specimens were dried in a critical point dryer and coated with gold in a sputter coater.The SEM photographs were taken using a scanning electron microscope (LEO, 1450VP).
Mandible (Fig. 3B): ~ 6 cuspidate teeth dorsally and one seta on coxal gnathobase dorsally.Basis with four bare setae: one proximally and three distally along inner margin.Enp-1 with four setae on inner distal corner.Enp-2 with nine setae apically; two oblique rows of spinules along outer margin.Exp-1-3 each with one seta on inner margin; Exp-4 with three setae apically; all setae bare.
Maxillule (Fig. 3C): praecoxal arthrite with nine strong setae laterally and four slender submarginal setae.Coxal endite with four setae; coxal epipodite with nine setae; two proximal-most setae smaller than others.Two basal endites fused to segment bearing them: proximal and distal endite, each with four setae apically; basal exopodite with one short seta.Enp-1 and Enp-2 each with four setae apically, proximal segment fused to basis.Exp with six bare setae apically.
P1-P4 (Fig. 4A-D): coxa with a pinnate seta at innermost distal corner.P1 and P2 basis without setae; a reduced bare seta on outer distal margin of P3 and P4.Exp longer than Enp; two-segmented Enp and three-segmented Exp on P1, three-segmented Enp and Exp on P2-P4.Armature formula of P1-P4 as in Table 3.
Etymology.The specific epithet is derived from Kok Phutaka, reflecting the name of the area in which the type locality is located.The name is an adjective in the nominative singular, gender feminine.

Discussion
Mongolodiaptomus phutakaensis sp.nov.exhibits the distinguishing features of the genus, as described in the updated generic traits outlined by Ranga Reddy et al. (2000).For the males, the right P5 Exp-2 carries the characteristic three lateral spines, one principal spine inserted at the middle of the segment on the outer margin, and two accessory spines located proximally and distally.A comparison with its congeners shows that M. phutakaensis sp.nov.resembles the two recently described taxa from the Mekong region: M. loeiensis Watiroyram &Sanoamuang, 2017 andM. mekongensis Sanoamuang &Watiroyram, 2018, respectively.These three closely related species can be distinguished from the other congeners by the unique shape of the male right P5 Exp-2; the inner margin is slightly incurved, the proximal and distal parts of the outer margin are enlarged, and the principal lateral spine is bent and twisted.The antepenultimate segment of the male right antennule of all three related species has a comb-like process.Other similarities among the three closely related species are the male right P5 has a spur-like or irregular process on the basis, the coxa has a strong spine, the Exp-1 has an acute process on the outer distal margin, the left P5 has a hyaline lamella on the inner margin of the basis, and the strong spinules along the inner margin of the Exp-2.The female of the new species shares similarities with M. loeiensis and M. mekongensis by having two-segmented P1 Enp.
The new species can be differentiated from M. mekongensis and M. loeiensis by the characteristics of the male caudal rami and P5 (Table 4): the right caudal ramus of M. phutakaensis sp.nov.has four ventral chitinous processes (two proximal spine-like processes and two distal semi-circular knobs), while M. mekongensis has one spine-like process and one semi-circular knob, and M. loeiensis has two spine-like processes and one semi-circular knob.The intercoxal plate of the new species is slightly produced distally and without any spine, but it is well-produced with one strong spine and two spines on its distal margin in M. mekongensis and M. loeiensis, respectively.The right P5 basis in males lacks a hyaline membrane on the inner margin in the new species, which is present in M. mekongensis and M. loeiensis.The male P5 has a distinct mid-distal spur-like chitinous process on the posterior surface in the new species versus a small chitinous prominence on the same position in M. mekongensis and M. loeiensis.The left P5 basis has a thin, longer hyaline lamella on the inner margin in the new species but is somewhat shorter in M. mekongensis and M. loeiensis.Only M. mekongensis has an obviously longitudinal chitinous ridge on the posterior side of this segment.
In the females, the left wing of Pdg 5 is longer than the right one in the new species and M. mekongensis, whereas both wings are symmetrical in M. loeiensis.The genital double-somite is only slightly asymmetrical in the new species but pronounced in M. mekongensis and M. loeiensis, with well-developed posterolateral outgrowth on the right side.The P5 Exp-2 has longitudinal grooves (conveyor canals) on the posterior view in the new species and M. mekongensis, but these grooves are absent in M. loeiensis.
A comparison of male and female morphological characters of the Mongolodiaptomus species is presented in Tables 5-6.The prominent morphological characteristics of this genus are reviewed briefly hereafter.

Antennule
While the setal armature of the female antennules remains conservative among species, the characteristics of the male grasping antennules serve to identify species.The degree of spine development on segments 8 and 15 is important at the species level; segment 16 bears a spinous projection in eight species but is absent in five species (Table 5).The spinous process on the antepenultimate segment of most species is comb-like, but it is long and slender in three species (M.birulai, M. botulifer, and M. malaindosinensis).However, the shape and size of the comb-like projections are different across the species (Table 5).

Lateral wings of fifth pediger
In the female, the shape and size of the lateral wings and the position of the inner (posterior) spine on either wing are of significant taxonomic value.In most species, both left and right wings are moderate in size and moderately asymmetrical, only four (M.calcarus, M. dumonti, M. loeiensis, and M. pectinidactylus) have symmetrical wings (Table 6).

Urosome
The relative lengths of urosomites and caudal rami, as well as the structural details of the female's genital double-somite, are highly diagnostic.The genital double-somite's relative length varies greatly between species.It is strikingly asymmetrical.In five species (M. birulai, M. botulifer, M. loeiensis, M. mekongensis, and M. uenoi), the right proximal region has a well-developed posterolat-eral process, while in the other five species (M. gladiolus, M. malaindosinensis, M. pectinidactylus, M. phutakaensis sp. nov., and M. rarus), it has a moderately developed posterolateral process.In the male, most species have ventral hairs on urosomites 2 and 3, but only M. mephistopheles and M. rarus do not have ventral hairs on those segments.In general, the male right caudal ramus of most species is armed with one or two chitinous structures and sometimes with two minute semicircular knobs ventrally; only M. gladiolus and M. pectinidactylus do not have such structures.

Male fifth leg (P5)
The interspecific differences in the male P5 are well pronounced in this genus.
On the right and left P5, the shape and structure of the Exp-2 and of the Enp are different in most species (Table 5).On the right P5, the inner coxal plate is uniquely produced in six species, particularly with one and two strong spines in M. mekongensis and M. loeiensis, respectively.The basis in most species has inner hyaline lamella, except in M. calcarus, M. dumonti, M. gladiolus, and M. phutakaensis sp.nov.Furthermore, another distinctive characteristic of the genus is the presence of a spur-like process at the mid-distal margin of the posterior surface of the basis in seven species (Table 5).In seven species, the Exp-1 of the right P5 has a pointed spinous process in the distal outer corner.The principal lateral spine of Exp-2 is located at or close to mid-length of the outer margin in all species except M. dumonti, where such a spine is located at 3/4 length of the outer margin.The principal lateral spine of Exp-2 is either straight, curved, or twisted.On the left P5, the basis has either inner hyaline lamella or knoblike outgrowth in all but M. gladiolus.The Enp is one-segmented except for M. botulifer, M. malaindosinensis, and M. mekongensis.

Female fifth leg
The seta on basis is longer than ½ length of Exp-1 in most species except  Watiroyram 2018; Chaicharoen and Sanoamuang 2022).Laos has four species, while Indonesia and Taiwan have three species each, whereas Singapore and the Philippines both have only one species each (Lopez et al. 2017;Sanoamuang and Watiroyram 2018).
Most species are currently restricted to Southeast Asia; only six species have also been recorded outside the area, including M. birulai, M. calcarus, M. gladiolus, M. mekongensis, M. pectinidactylus, andM. uenoi (Ranga Reddy et al. 1998, 2000;Sanoamuang 2001;Alekseev et al. 2013;Tran et al. 2016;Lopez et al. 2017).Therefore, the distribution of these six species extends from Southeast Asia to South China.M. birulai has the widest distribution, occurring from Vietnam upwards to North China, including Taiwan (Young and Shih 2011;Tran et al. 2016;Li et al. 2018).M. mekongensis is a common species in the Mekong region and has been found in Thailand, Laos, Cambodia, Vietnam, and South China (Table 4).M. botulifer and M. malaindosinensis have been found only in Southeast Asia (Boonmak et al. 2018).M. dumonti, and M. mephistopheles, are confined in distribution to the Mekong region, and Malay Archipelago, respectively.Three species (M.loeiensis, and M. phutakaensis sp.nov., and M. rarus) are currently endemic to Thailand; for more details see also Table 2 in Sanoamuang and Watiroyram (2018).
In Thailand, the most widespread Mongolodiaptomus species are M. botulifer and M. calcarus.Both species live in both temporary and permanent water bodies throughout the country and occur throughout the year.M. malaindosinensis is moderately common and has been recorded throughout the country.M. mekongensis is widely distributed in the Mun River Basin, a tributary of the Mekong River, and mostly occurs in temporary water bodies.M. dumonti, M. rarus, and M. uenoi, are uncommon.M. rarus has been found only in temporary water bodies.In contrast, M. pectinidactylus is rare and has been reported only at two temporary water bodies (Sanoamuang 2002).M. loeiensis and M. phutakaensis sp.nov.are extremely rare and, to date, have been found only in a single locality each (Watiroyram and Sanoamuang 2017;this study).

Figure 1 .
Figure 1.Mongolodiaptomus phutakaensis sp.nov., SEM photographs of male A habitus, dorsal view B rostrum C comblike process on the antepenultimate segment of the right antennule D genital somite, and urosomites 2 and 3 E right caudal ramus, ventral view (white arrows indicate proximal chitinous spine and distal knob) F right P5 coxa and basis, posterior view (white arrows point to the coxal spine and spur-like hyaline membrane) G P5 in posterior view H distal part of left P5, posterior view I P5, anterior view (without end claw, white arrow points to the distal accessory spine) J left P5, anterior view (white arrow points to the hyaline membrane) K right P5 Exp-1 and 2, posterior view (white arrows point to the proximal accessory spine and twisted principal lateral spine).

Figure 8 .
Figure 8. Mongolodiaptomus phutakaensis sp.nov., SEM photographs of female A habitus, dorsal view B rostrum C pediger 5 and urosome, dorsal view (white arrows point to spines) D P5 Exp-1-2, posterior view (white arrow indicates longitudinal ridges) E P5, posterior view F P5, anterior view G P5 Enp, anterior view (white arrow indicates the border of the two segments) H urosome, ventral view I caudal rami, ventral view.

Table 1 .
Armature formula of the left male antennule of Mongolodiaptomus phutakaensis sp.nov.The number of setae (Arabic numerals), aesthetascs (ae), and spines (sp) is given.The Roman numerals refer to segment numbers.

Table 2 .
Armature formula of the right male antennule of Mongolodiaptomus phutakaensis sp.nov.The number of setae (Arabic numerals), aesthetascs (ae), and spines (sp) is given.The Roman numerals refer to segment numbers.

Table 3 .
Armature formula of the swimming legs of Mongolodiaptomus phutakaensis sp.nov.The number of setae (Arabic numerals) and spines (Roman numerals) is given in the following sequence: outer-inner margin or outer-apical-inner margin.

Table 4 .
The morphological characteristics and distribution of the closely related Mongolodiaptomus species: M. loeiensis, M. mekongensis, and M. phutakaensis sp.nov.

Table 5 .
Comparison of male morphological characters of Mongolodiaptomus species (? means unknown or doubtful).

Table 6 .
Comparison of female morphological characters of Mongolodiaptomus species (? means unknown or doubtful).