Diversity of the genus Tropodiaptomus Kiefer, 1932 (Crustacea, Copepoda, Calanoida, Diaptomidae) in Thailand, with the description of two new species

Tropodiaptomus is a genus of diaptomid copepods with 10 species currently recorded in Thailand. A recent study on DNA taxonomy revealed putative new species among specimens collected from freshwater habitats throughout Thailand. This study examined the morphological characteristics and confirmed the taxonomic status of the two putative new species of Tropodiaptomus . Results showed that the two new taxa were different from other species in the genus Tropodiaptomus . These two new species, T. pedecrassum sp. nov. and T. longiprocessus sp. nov. , were described and illustrated based on material collected from a swamp in northern Thailand and a pond in western Thailand, respectively. They were distinguished from their congeners by the length of the spinous process on the antepenultimate segment of the adult male right antennule, the number of lobes and serration pattern on the inner margin of the adult male left P5, and the shape and supplementary process on the surface structures of basis and distal exopod segments of the adult male right P5. These discoveries increased the number of records of this genus in Thailand to 12 species. A pictorial key to all species is provided, and their ecological and biogeographical distributions are updated and discussed.


Introduction
To date, 170 copepod species have been recorded from freshwater habitats in Thailand (Saetang et al. 2020;Sanoamuang and Dabseepai 2021).The diaptomid family in the calanoid group is highly diverse, comprising 42 species in Thailand, with 14 new species added during the last 20 years.Mongolodiaptomus and Tropodiaptomus are the two most diverse genera, each containing 10 species (Saetang et al. 2020;Sanoamuang and Dabseepai 2021).Interestingly, Tropodiaptomus has the highest number of 63 species in the family Diaptomidae worldwide (Walter and Boxshall 2023).Most species in this genus are restricted to one or a few biogeographic regions, with 21 species distributed in Asia (Saetang et al. 2020;Walter and Boxshall 2023).In Thailand, undiscovered species are postulated to exist within specific habitats such as temporary ponds and this genus remains underexplored.Saetang et al. (2020) investigated 468 samples collected from 190 freshwater habitats throughout Thailand.Eight species were reported with one new to science.Later, Saetang (2021) and Saetang et al. (2022) conducted extensive morphological and DNA taxonomy studies on Tropodiaptomus, with results determining at least 12 species recorded in Thailand.These integrative methods indicated a high level of genetic diversity in some Tropodiaptomus species, with the possibility of cryptic species.Based on genetic data, three more putative new species await morphological study and description (Saetang et al. 2022).
Our study proposes Tropodiaptomus sp. 1 and Tropodiaptomus sp. 2 mentioned in Saetang et al. (2022) as new species with detailed descriptions and illustrations of both males and females.A pictorial key for practical use to identify members of this genus was presented and the present diversity and species distribution were updated.

Materials and methods
Samples were qualitatively collected from swamp in Dok Kham Tai District, Phayao Province, northern Thailand (19°13'57.6"N,100°02'56.5"E) in January 2018 and Thong Phaphum District, Kanchanaburi Province (14°39'09.1"N,98°33'27.5"E) in June 2019 using a plankton net of 60 µm mesh and immediately preserved in 70% ethanol.All adult males and females were sorted using an Olympus SZ40 stereo microscope, and each specimen was dissected and mounted on a slide in glycerine and then sealed using nail varnish.
All type specimens were deposited in the reference collection of the Princess Maha Chakri Sirindhorn National History Museum, Prince of Songkla University, Songkhla, Thailand (PSUNHM).
Description of the adult male.Body (Figs 1A, 2A).Total body length about 1,145 µm (1.1 mm) (measured from anterior margin of rostrum to posterior margin of caudal rami).Prosome length about 2 times as long as urosome (including caudal rami).Fourth and fifth pedigers separated by distinct septum.Fifth pediger produced into small asymmetrical posterolateral wings (left wing shorter than right wing), each distal end with spine.Urosome 5-segmented.Genital somite with dorsolateral sensilla on right and left side, fourth somite with expanded right corner.Anal somite with deep cleft, length about 0.8 times as long as wide.Caudal rami parallel, symmetrical, length about 1.8 times as long as wide, with setules on inner margin.Each ramus with six setae.
Rostrum (Fig. 3A).Two rostral elements on anterior margin with suture in the middle.
Coxal endite with three plumose setae, and coxal epipodite with seven plumose setae and two bipinnate spines.Basis with two endites; the proximal with four  plumose setae and the distal with eight plumose setae, and basal exite with one bipinnate spines.Exopod 1-segmented with six plumose setae, one longitudinal row of setules on inner margin of segment.Endopod 1-segmented with four plumose setae.
P5 (Figs 6A, 8A-D).Asymmetrical.Left leg (Figs 6A, 8A-D), reaching slightly beyond proximal margin of exp-2 of right P5. Coxa as long as wide, with spine inserted on outer lobe.Basis cylindrical, about 1.4 times as long as wide, with one distal outer smooth seta.Exopod flattened, about 1.7 times as long as wide, inner margin one lobe, the serration gradually decreases in size from the proximal to distal end (Fig. 8C, arrowhead).Apex of exopod with usual 'finger-and-thumb' combination, 'finger' slim, and set with radiant, hair-like 'thumb' sphere, ventral surface of exopod with 2 hairy pads.Endopod 2-segmented with incomplete separation of segments (Fig. 8D, arrowhead), conical, reaching beyond middle of exp-1, rounded distally with two parallel rows of setules.Right leg (Figs 6A,8A,B), coxa as long as wide, with spine inserted on outer lobe.Basis cylindrical, about 1.3 times as long as wide, four structures occurring on dorsal surface: (i) one round process on proximal third close to inner margin, (ii) one triangular process in middle close to inner margin, (iii) one longitudinal hyaline lamella inserted near inner margin, and (iv) one distal outer smooth seta.Exopod 2-segmented.Exp-1 small, about 0.6 times as long as wide, with triangular lobe on inner margin, distal outer corner produced into acute spinous process, nearly as long as its segment.Exp-2 rhomboid, about 2.7 times as long as wide, dorsal surface with one semicircular hyaline knob on proximal outer margin, one semicircular hyaline lamella on distal inner margin, and one triangular process in middle of segment (Figs 6A, 8A, B (arrowhead)), and distal end of segment with one round hyaline prominence inserted between end claw and lateral spine (Fig. 8A, B (arrowhead)).Lateral spine nearly straight, acutely pointed, about as long as exp-2, inserted on distal corner of exp-2, with spinules on its inner margin.End claw curved and gradually tapering to acuminate tip, about 2.6 times as long as exp-2, inner margin with spinules distally.Endopod 1-segmented, conical, reaching distal end of exp-1, distal end with two rows of setules.
Variability.Morphological variability has been observed in: (i) the total body length (except of caudal setae) which ranged from 1,145-1,380 µm (mean 1,283 µm, n = 6) in the adult males and 1,520-1,626 µm (mean 1,575 µm, n = 5) in the adult females; (ii) the length of the spinous process on antepenultimate segment of the adult male right A1 is 3/4 to equal of segment 21 (Fig. 7A-G; see Table 2 in Saetang et al. 2022); (iii) segment 13 of the adult male right antennule with one hyaline knob (Fig. 3E (arrowhead)); and (iv) the number of setae on segment 13 of the adult male left A1 has one seta (n = 10) or two setae (n = 2) (Fig. 1D (arrowhead); see Table 2 in Saetang et al. 2022) Etymology.The specific name 'pedecrassum' is derived from the chubby shape of the adult male P5 that is clearly different from the more rectangular shape in other species of the genus.
Co-occurring species.In our samples, the new taxon co-occurred with one other copepod species, Mongolodiaptomus botulifer (Kiefer, 1974).
Distribution and ecology.Tropodiaptomus pedecrassum sp.nov.was found only in its type locality so far.It was recorded in two out of 471 samples collected from 206 freshwater habitats throughout Thailand between September 2017 and July 2019.Water temperature 19.6 °C, conductivity 620 µs cm-1, salinity

Swimming legs
Coxa Basis Exp Enp   tioned by Lai andFernando (1979a, 1979b), Sanoamuang (2002), Saetang et al. (2020): (i) the process on the antepenultimate segment of the adult male right antennule is always smooth; (ii) the exopod of the adult male left P5 fused into a single flattened piece and its inner margin is denticulate or serrate; (iii) the inner margin on the basis of the adult male right P5 has a hyaline lobe; (iv) the urosome of the adult female comprises two somites; and (v) the endopod segment of the adult female P5 has slender setae at the distal end.
This species differs from the congeneric species by the following characters: (i) antepenultimate segment of the male right antennule with straight spinous process reaching 3/4 or equal of next segment; (ii) inner margin of exopodal segment of the male left P5 with single lobe and the serration gradually decreases in size from the proximal to distal end; (iii) basis of the male right P5 with two processes and one longitudinal hyaline lamella; (iv) exp-1 of the male right P5 with triangular lobe on inner margin, distal outer corner produced into acute spinous process, length about as long as its segment; and (v) exp-2 of the male right P5 with rhomboid shape, dorsal surface with one semicircular hyaline knob on proximal outer margin, one semicircular hyaline lamella on distal inner margin, and one triangular process in middle of segment.
According to the identification key given by Saetang et al. ( 2020), Tropodiaptomus pedecrassum sp.nov. is most similar to T. hebereri.However, it distinctly differs from T. hebereri in the following characters: (i) inner margin of the basis of the adult male right P5 has one round process, one semicircular process, and one longitudinal hyaline lamella; (ii) length of spinous process of the outer corner of exp-1 of the adult male right P5 is as long as exp-1; (iii) inner margin of the exopod of the adult male left P5 has one lobe, and the serration gradually decreases in size from the proximal to the distal end; (iv) dorsal surface of exp-2 of the adult male right P5 has one semicircular hyaline knob, one semi-circular hyaline lamella, and one triangular process; and (v) caudal rami of the adult female have outer and inner setules (Table 2).
Rostrum (Fig. 9C).Two rostral elements on anterior margin with suture in the middle.
P5 (Figs 14A,E,16A,E).Asymmetrical.Left leg, reaching beyond middle of exp-2 of right P5. Coxa as long as wide, with spine inserted on outer lobe.Basis cylindrical, about 1.9 times as long as wide, with one distal outer smooth seta.Exopod flattened, about 2.2 times as long as wide, inner margin two lobes with uniform serration (Figs 14A, 16A, E).Apex of exopod with usual 'fingerand-thumb' combination, 'finger' slim, and set with radiant, hair-like 'thumb' sphere, ventral surface of exopod with two hairy pads.Endopod 2-segmented (Fig. 16G), conical, reaching beyond middle of exp-1, rounded distally with two parallel rows of setules.Right leg, coxa as long as wide, with spine inserted on outer lobe.Basis cylindrical, about 1.7 times as long as wide, three structures occurring on dorsal surface: (i) one triangular process in proximal third close to inner margin, (ii) one longitudinal hyaline lamella inserted near inner margin in middle, and (iii) one laterodistal smooth seta.Exopod 2-segmented.Exp-1 small, about 0.5 times as long as wide, with triangular lobe on inner margin, laterodistal corner produced into acute spinous process.Exp-2 rectangular, about 1.5 times as long as wide, dorsal surface with two longitudinal hyaline lamellae in middle and distal inner margin and one large and long process in middle near outer margin.Lateral spine nearly straight, acutely pointed, about 1.3 times as long as exp-2, inserted in laterodistal corner of exp-2, with spinules on its inner margin.End claw curved and gradually tapering to acuminate tip, about 2.5 times  margin of caudal rami).Prosome length about 3.1 times as long as urosome (including caudal rami).Fourth and fifth pedigers completely fused, fusion being indicated by indentation on each side.Fifth pediger produced into asymmetrical posterolateral wings (right wing shorter than left wing), each distal end with posterior spine, and each wing with one dorsal spine on inner margin (right spine smaller than left spine) (Fig. 16K).Dorsal surface of fifth pediger without prominence (Fig. 16J).Urosome 2-segmented.Genital double-somite asymmetrical, about twice times as long as wide, right sides of anteripart swollen (Figs 15B (arrowhead), 16K), with two unequal dorsolateral spines (right spine smaller than left spine) in anterior third (Fig. 15B).Right distal corner of genital double-somite without lobe (Figs 15B, 16K (arrowhead)).Genital area on ventral surface shows opercular pad protecting gonopores, characterised by rectangular and semicircular expansions (Fig. 15C).Anal somite about 1.2 times as long as wide.Caudal rami parallel, symmetrical, about 1.5 times as long as wide, with setules on outer and inner margins (Fig. 15B).Each ramus with six setae.A1, A2, mandible, maxillule, maxilla, maxilliped, P1-P4 (not shown) and rostrum (Fig. 15D) same as male.P5 (Figs 14F,16L).Symmetrical.Coxa spine on posterior lobe on caudal surface.Basis with one smooth outer seta on distolateral margin.Exopod 3-segmented.Exp-1 cylindrical, length about 1.9 time as long as wide.Exp-2 tapering into long claw, each side with one row of spinules starting in middle of segment.Exp-3 fused with exp-2 (Figs 14F, 16L (arrowhead)), with two unequal spines, inner spine about 4.9 times as long as outer spine, and with short spine laterally.Endopod 1-segmented, cylindrical, length about 0.7 time as long as exp-1, two strong smooth spiniform setae distally (Figs 14F, 16L (arrowhead)), outer seta longer than inner seta, two parallel rows of spinules on distal end.
Variability.Morphological variability has been observed in: (i) the total body length (except of cau- dal setae) which ranged from 1,490 -1,545 µm (mean 1,510 µm, n = 4) in the adult males and 1,570-1,700 µm (mean 1,646 µm, n = 6) in the adult females; and (ii) shape and length of large and long process on exp-2 of the adult male right P5 (Figs 14B-D, 16B-D).Moreover, it needs to be noted that the inner margin of exopod of the adult male left P5 appeared single lobe in positions other than dorsal which can lead to misidentification (Fig. 16F).
Etymology.The specific name 'longiprocessus' is derived from the presence of a long process on exp-2 of the right P5 in the adult male.
Co-occurring species.There were no other diaptomid copepods in these samples.Distribution and ecology.Tropodiaptomus longiprocessus sp.nov.was found only in its type locality.It is rare because it was found only in two of 471 samples collected from 206 freshwater habitats throughout Thailand between September 2017 and July 2019.Water temperature 26.6-28.0°C, conductivity 623.3-672.7 µs cm-1, salinity 0.3 ppt, total dissolved solids 385-424 mg L-1, dissolved oxygen 2.5-6.0 mg L-1, pH 7.2-7.3,and water depth 0.2-0.3m, substrate with mud.However, in order to understand more in their habitat preference, the whole year samples are needed to be examined.2).
The diversity of Tropodiaptomus in Thailand is presented as a pictorial key in Fig. 17 to facilitate easy and quick identification.

Tropodiaptomus species diversity and distribution in Thai water bodies
Tropodiaptomus species have been recorded from various ponds, lakes, streams, roadside canals and rice field habitats in Thailand (Sanoamuang 2002;Saetang et al. 2020Saetang et al. , 2022)), with 12 species now recorded (Table 3) (Saetang et al. 2020(Saetang et al. , 2022;;Sanoamuang and Dabseepai 2021).However, the status of some species remains unclear.Molecular data have confirmed that T. cf.lanaonus and T. lanaonus s. str.are different species (Saetang et al. 2022) but thorough morphological comparisons of numerous specimens are still needed to confirm species-level separation of T. cf.lanaonus.The validity of T. doriai is also in serious doubt, with no further reports on this species in Thailand since it was first reported by Daday in 1906 from a freshwater habitat close to Bangkok (Lai and Fernando 1981;Sanoamuang 2002).
Each species of Tropodiaptomus can be found in various types of freshwater habitats, while most occur in both temporary and permanent water bodies with a limited geographical distribution range (Fig. 18).Only Tropodiaptomus vicinus is widely distributed throughout Thailand, except in the north, while T. oryzanus is common but restricted to the northeastern region and T. hebereri, T. megahyaline, Tropodiaptomus sp. 1 and Tropodiaptomus sp. 2 have restricted distributions in one region in a few localities.Most species can be found  during all seasons in Thailand.T. oryzanus and T. vicinus can be found all year round, in both rainy and dry seasons, including during low temperature periods in Northern Thailand.Some species are seasonal, with T. pedecrassum found only during low temperature periods in the north and T. longiprocessus and Tropodiaptomus sp.1 found only during the rainy season, with distribution restricted to temporary pools (Table 3).

Discussion
This study assessed the morphological characteristics and molecular data recorded by Saetang et al. (2020) and confirmed the species status of Tropodiaptomus pedecrassum sp.nov.and Tropodiaptomus longiprocessus sp.nov.These two species clearly differ from their congeners: (i) the inner margin of the adult male left P5 had a single lobe and serration gradually decreased in size from the proximal to distal end in Tropodiaptomus pedecrassum sp.nov.with two lobes and uniform serration in Tropodiaptomus longiprocessus sp.nov.and (ii) exp-2 of the adult male right P5 was rhomboid-shaped, with one triangular-shaped process on the frontal surface in Tropodiaptomus pedecrassum sp.nov., and a rectangular shaped large and long process in the middle third lateral in Tropodiaptomus longiprocessus sp.nov.
The discovery of these two new species increases the number of Tropodiaptomus species recorded in Thailand from 10 to 12, thus accounting for 19% of species diversity worldwide.Based on the large number of samples taken throughout Thailand, most Tropodiaptomus species have a limited distribution at one site or one region, with only T. vicinus having a wide distribution.Moreover, Tropodiaptomus is distributed in a specific habitat, temporary pools without vegetation and the occurrences of this genus are seasonal.This type of habitat is undersampled in SE Asia, however it is important for our understanding of the diversity of this genus.However, recent findings of new records in Southeast Asia (Defaye 2002;Ambedkar and Elia 2014;Saetang et al. 2020), including the present discovery of two new species, indicate that this genus may have a wider geographical distribution than recorded previously (Lai and Fernando 1979b;Lai and Fernando1981;Ambedkar and Elia 2014).Saetang et al. (2022) considered that more undiscovered species exist in this genus in SE Asia.Further intensive study is required, in terms of intensive sampling year-round as well as the use of efficient tools such as molecular systematics together with morphological examinations.This will clarify the taxonomic status of currently confusing data and more accurately estimate the species richness and ecological and biogeographical distribution of members of this genus in Thailand.

Figure 17 .
Figure 17.Pictorial key of the adult male P5 of Tropodiaptomus species found in Thailand.Right exp-2 of P5 of Tropodiaptomus lanaonus and T. hebereri were modified from Kiefer (1982).

Figure 18 .
Figure 18.Distribution of Tropodiaptomus found in Thailand (Tropodiaptomus sp. 2 is not included in this figure because no distribution data are available).

Table 3 .
(Saetang et al. 2022ion of Tropodiaptomus species in Thai water bodies and remarks on their status.This species does not agree with the original description of T. lanaonus in (i) the length of the spinous process in the antepenultimate segment of adult male right antennule is 1/2 to 3/4 of segment 21, and (ii) the ornamentation on the basis of adult male right P5 has one hyaline lamella and one apophysis or one hyaline lamella and no apophysis(Saetang et al. 2022)5.T. megahyalinePond and rice field in both dry (May) and rainy season (June) in northeastern Thailand