﻿Phyllodiaptomusparachristineae, a new species of copepod (Copepoda, Calanoida, Diaptomidae) from the floodplain of the lower Mekong River Basin in Thailand and Cambodia

﻿Abstract Phyllodiaptomus (Phyllodiaptomus) parachristineaesp. nov., a new diaptomid copepod, was collected from 30 sites in the lower Mekong River Basin floodplain in northeastern Thailand and nine sites in Cambodia. The new species is the 13th species of the genus to have been recorded across Asia. It has a wide range of habitats, occurring in small to large, temporary to permanent water bodies, and it can be found at any time of the year. The morphology of the new species is most similar to that of the males of P. (P.) christineae, having a comb on the antepenultimate segment on the right antennule, symmetrical caudal rami, a narrow hyaline lamella on the left P5 basis, a distal accessory spine on the right P5Exp-2, and a single-lobed Enp on the right P5. On the other hand, the males of the new species have second and third urosomites without hair-like setae on the ventral margin, a rounded distal margin of the P5 intersclerite plate, a semi-circular lamella on the right P5 basis, a strong principal lateral spine inserted at the proximal 1/3 of the right P5Exp-2, and a two-segmented left P5Enp. Morphological differences among species in the subgenus Phyllodiaptomus (Phyllodiaptomus) as well as the distribution and habitats of the 13 species and two subspecies of Phyllodiaptomus in Asia are discussed.


Introduction
The Mekong River is Southeast Asia's longest and the 12 th longest river in the world. It flows 4,350 kilometers from the Tibetan Plateau to the Mekong Delta and into the South China Sea, making it one of Asia's major river systems (Sanoamuang and Watiroyram 2018). Approximately 75% of the Mekong's drainage area is located in Laos, Thailand, Cambodia, and Vietnam. The Mekong River catchment area is divided into two major sections. The upper part of the Mekong flows 1,955 kilometers through southwestern China, accounting for approximately 25% of the total land area. The lower part of the Mekong is a 2,390 km-long stretch that drains the Khorat Plateau of northeastern Thailand, the western slopes of the Annamese Cordillera in Laos and Vietnam, and the (1) P. blanci (Guerne & Richard, 1896) from Uzbekistan; (2) P. tunguidus Shen & Tai, 1964 from China, Laos and Vietnam; (3) P. longipes Kiefer, 1965 from Indonesia; (4) P. irakiensis Khalaf, 2008 from Iraq; (5) P. christineae Dumont, Ranga Reddy & Sanoamuang, 1996 from Thailand; (6) P. surinensis Sanoamuang & Yindee, 2001 from Thailand; (7) P. thailandicus Sanoamuang & Teeramaethee, 2006 from Thailand; (8) P. roietensis Sanoamuang & Watiroyram, 2020 from Thailand and Cambodia; and (9) Phyllodiaptomus sp. from Thailand (Sanoamuang & Dabseepai, 2021) and Cambodia (Chaicharoen & Sanoamuang, 2022).
Here, we describe the previously called Phyllodiaptomus sp. from Thailand and Cambodia as Phyllodiaptomus parachristineae sp. nov. The new species have been found at several sites in the floodplains of the Mekong River tributaries, the Mun River in Thailand, and the Tonle Sap Lake-River Complex in Cambodia. It is the sixth species in Thailand, the third in Cambodia, and the 13 th in the genus.

Materials and methods
In Thailand, samples were qualitatively taken from the Mun River Basin in Surin Province (December 1998, August 1999, and April 1999 and Ubon Ratchatani Province (June 2002 andOctober 2002) (Fig. 1). In total, 379 samples were obtained from 224 sites (temporary and permanent waters), including rice fields, roadside canals, ponds, and reservoirs. In Cambodia, samples were collected from 252 sites in seven provinces (Banteay Meanchey, Battambang, Siem Reap, Kampong Thom, Pursat, Kratie, and Stung Treng) from February to October 2007.
Samples were collected using a 60-µm-mesh plankton net and preserved immediately in 4% formalin. Adult specimens were sorted under an Olympus SZ51 stereomicroscope at 40-x magnification and placed in a mixture of glycerol and 70% ethanol (ratio of 1:10 v/v). The animals were then transferred to a small drop of pure glycerol. They were dissected and prepared on a glycerin-mounted slide under a stereomicroscope at 40-100× magnification. All appendages and body ornamentation were examined at 1000× magnification under an Olympus CX31 compound microscope. The drawings were made using an Olympus U-DA drawing tube mounted on a compound microscope. The final versions of the drawings were made using the CorelDRAW 12.0 graphic program.
The dissected specimens were prepared for semi-permanent slides by mounting them in pure glycerine and sealing them with transparent nail varnish. All un-dissected specimens were stored in 70% ethanol in 1.5 mL microtubes. Type specimens are deposited at the Thailand Natural History Museum, Pathum Thani, Thailand (THNHM), and the Applied Taxonomic Research Center, Khon Kaen University, Khon Kaen, Thailand (KKU).
Female. Pedigerous somites 4 and 5 completely fused. Pedigerous somite 5 with asymmetrical wings: right wing round with two strong spines, left wing triangular with one strong spine. Genital double-somite incompletely fused dorsolaterally, with right dorsolateral spine. Exp-2 without lateral spine.
Maxillule (Fig. 4C): 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 exite with one short seta. Enp-1 and Enp-2 each with four setae apically, proximal segment fused to basis. Exp with six setae apically.

Coxa Basis Exp Enp
P5 (Figs 5E, F, 6A): intercoxal sclerite fused to coxa, produced into a rounded lobe distally (Fig. 5F1). Right P5: coxa with an acute, strong spine on posterior surface. Basis subrectangular, larger than that on the left side; semi-circular lamella at the middle of inner margin; short longitudinal ridge at proximal half of posterior surface; short, thin seta on the distal outer margin. Enp one-segmented, distal half tapering, tipped with tiny spinules; reaching beyond 1/3 of Exp-2, close to insertion of its principal lateral spine on outer margin. Exp-1 shorter than wide, with a bifid knob at inner margin; distolateral margin with a small acute process. Exp-2 oval with two lateral spines. Principal lateral spine articulated, located at 1/3 length of Exp-2 outer margin, thick, S-shaped, with a sharp curved tip, reaching the distal margin of segment. Accessory lateral spine minute, close to insertion of end-claw. End-claw sickle-shaped, with a blunt tip; ~ 1.5× as long as Exp-2.
Left P5 (Figs 5E, F, 6A): coxa with thin seta on posterior lobe near distal inner corner; longer, slender than spine on right coxal segment. Basis with narrow hyaline lamella along 3/4 distal length of inner margin; with a small seta at distal outer margin. Exp-1 triangular, tapering towards distal end, medial margin concave with a field of long setules. Exp-2 smaller than Exp-1, bulbous, with a strong pinnate seta at mid-length of medial margin, with a field of long  setules proximally, and a field of tiny spinules distally. Apical hyaline process, thumb-like, with blunt tip. Enp two-segmented, shorter than Exp-1; Enp-1 unarmed. Enp-2 longer than Enp-1, slightly tapering distal end, tipped with a row of tiny spinules.
Antennule symmetrical; left antennule, antenna, mouthparts, and P1-P4 as in male. P5 (Fig. 8) asymmetrical. Coxa with a blunt, stout spine on distal outer margin. Basis with a thin, bare seta on outer margin, reaching almost distal end of Exp-1. Exp-1 sub-rectangular, more than twice as long as wide, longer than Enp. Exp-2 triangular, with a row of strong spinules along both margins; right side stouter than left one (Fig. 8A); left side with a longitudinal groove (i.e., a conveyor canal) on anterior view (Fig. 8B). Exp-3 reduced, represented by a small segment on proximal outer margin of Exp-2, with one short spine and a longer medial spiniform seta apically. Enp one-segmented, conical, with a circular row of spinules apically.
Etymology. The specific name parachristineae is a combination of the Greek prefix para-, meaning to resemble, and the specific name christineae, referring to the fact that the male P5 of the new species resembles Phyllodiaptomus (P.) christineae Dumont, Ranga Reddy & Sanoamuang, 1996 (Fig. 6B).  Distribution. At present, the new species have been found throughout the year in both temporary and permanent water bodies, like ponds, roadside canals, irrigation canals, puddles in rice fields, and reservoirs. The new species was prominently found at 30 sites in the Mun River Basin, which is one of the largest tributaries of the Mekong River in Thailand (Fig. 1). In Cambodia, the new species has been found in habitats similar to those in Thailand. However, it was recorded at only nine of the 252 sampled sites in the floodplain of Tonle Sap Lake and the Mekong River Basin (Fig. 1).
Phyllodiaptomus (P.) parachristineae sp. nov. can be easily distinguished from the three other species of the subgenus Phyllodiaptomus (Phyllodiaptomus) in Thailand by morphological differences in the males. The new species has an unproduced distal margin of the intersclerite plate, whereas P. (P.) thailandicus has a distinct, bilobed one. The new species has a moderate spine on the right coxa, versus a large spine in P. (P.) roietensis and P. (P.) surinensis, but a reduced spine in P. (P.) thailandicus. The new species has no chitinous process on the right basis, but it is present in these three species. The right P5 of the new species has a single-lobed Enp, but a bilobed Enp in P. (P.) roietensis and P. (P.) surinensis. The principal lateral spine of new species is inserted on the proximal half of its Exp-2 outer margin, but such a spine is inserted on the middle length of Exp-2 in P. (P.) thailandicus and on the distal half in P. (P.) roietensis and P. (P.) surinensis. The new species has symmetrical caudal rami, but they are transformed in P. (P.) roietensis and P. (P.) surinensis. Furthermore, the new species lacks hairs on urosomites 2-3 ventrally, whereas P. (P.) thailandicus has hairy urosomites 2 and 3 (for more information, see Table 4).  Kiefer (1965) has drawn the distal inner margin of the intercoxal sclerite with a roughly triangular lobe rather than a conical lobe in the view of Dumont and Ranga Reddy (1993). 2 = Khalaf (2008) described the left P5 basis of a male with a small hyaline lobe projecting from the distal inner corner, but such a hyaline did not figure on their leg. 3 = Dumont et al. (1996) mentioned that the conveyor canal in the SEM figure is not clear.
The male of the new species is also different from the males of the four other Asian species in that the distal edge of the P5 interslerite plate is rounded instead of bilobed, conical, or triangular, as it is in P. (P.) irakiensis, P. (P.) tunguidus, P. (P.) blanci, and P. (P.) longipes. The right P5 Exp-2 of the new species has a principal lateral spine on the proximal half of the outer margin, but on the middle length in P. (P.) irakiensis and on the distal half in P. (P.) blanci and P. (P.) tunguidus. The new species' left P5 has a two-segmented Enp but an unsegmented Enp in P. (P.) blanci and P. (P.) longipes. The new species has untransformed caudal rami, versus transformed ones in P. (P.) tunguidus. The urosomites 2 and 3 of the new species lack hairs, but urosomite 2 is hairy in P. (P.) blanci, and both somites are hairy in P. (P.) longipes. Segment XX of the right antennule of the new species has a serrated process versus a smooth one in P. (P.) longipes. The female of the new species has only one right spine on the genital double-somite, versus a pair of spines in other species. Moreover, the new species lacks spines on the P5 Exp-2 but has outstanding spines in its congeners (see Table 4).
The new species (Fig. 6A) is most similar to P. (P.) christineae (Fig. 6B), particularly the morphology of the male right P5. The male P5 of the new species has a moderate spine on the right coxa, a long and narrow lamella on the left basis, the right Enp is unsegmented, and there are two spines on the Exp-2 outer margin, whereas the female P5 has an asymmetrical leg and a two-segmented Enp. Other shared characteristics are the right antennule with a serrated spine on segment 20, and the caudal rami are not transformed in males.
The new species was easily misidentified as P. (P.) christineae (see discussion below). After re-examination of several samples collected from the Mun River Basin, many major morphological differences are defined in both sexes. The male urosomite of the new species has no ornamentation, versus the hairy urosomite 2 in P. (P.) christineae. There are many different traits in the male P5.
The new species has a distal margin of the intersclerite plate that produces a rounded lobe versus the mostly triangular-lobed P. (P.) christineae. The new species has a semi-circular lamella on the right basis, but a long, narrow lamella in P. (P.) christineae; the right Exp-2 is strongly oval in the new species versus round or slightly oval in P. (P.) christineae; the new species has a reduced distal accessory spine on the right Exp-2, which is obviously smaller than those of P. christineae; and the new species has a sturdy principal lateral spine inserted at the proximal half of the right Exp-2 outer margin versus slender and presented at the middle of the right outer margin in P. (P.) christineae.
Other differences in the male left P5 are: (1) the new species has two-segmented Enp but is unsegmented in P. (P.) christineae; (2) the new species has no hyaline lamella on Enp but is presented in P. (P.) christineae. However, such hyaline lamellae are hardly observed under a light microscope in some populations of P. (P.) christineae, too (personal observation).
The female of the new species differs from P. (P.) christineae by: (1) having only one spine on the left wing of pediger 5, whereas P. (P.) christineae has a spine on both wings; (2) the new species has only one right spine on the genital double-somite, whereas P. (P.) christineae has a pair of spines; (3) the new species has a conveyor canal along the anterior view of the left P5 Exp-2, but this canal is absent in P. (P.) christineae; (4) the female legs of the new species show an apomorphies character for the genus by the Exp-2 without a distal lateral spine, versus P. (P.) christineae, which has a normal form like its congeners by having this spine.
The presence of a spine on the P5 Exp-2 in female diaptomid copepods in Thailand is found in the genera Tropodiaptomus Kiefer, 1932;Heliodiaptomus Kiefer, 1932;Neodiaptomus Kiefer, 1932;Allodiaptomus Kiefer, 1936;and Mongolodiaptomus Kiefer, 1937; but it is the opposite scenario in Eodiaptomus Kiefer, 1932, including E. draconisignivomi Brehm, 1952E. phuphanensis Sanoamuang, 2001a;E. phuvongi Sanoamuang & Sivongxay, 2004;and E. sanuamuangae Ranga Reddy & Dumont, 1998. The genus Eodiaptomus has only two species with such a spine on P5 Exp-2, i.e., E. japonicus (Burckhardt, 1913) from Japan and E. sinensis (Burckhardt, 1913) from China, which perhaps use the reduced form or absence of this lateral spine as a generic character (Ranga Reddy and Dumont 1998). In addition, the genus Dentodiaptomus Shen & Tai, 1964, also shared this character with the new species, which is found in the recent species described from Thailand, D. orientalis Sanoamuang & Watiroyram, 2021. The sharing of synapomorphies among different genera probably reveals their evolutionary relationships, or, in other words, 'the relationship is based on the synapomorphies possession' (Boxshall 1986;Sanoamuang and Watiroyram 2021).
In Thailand, the most widespread diaptomid species is P. (C.) praedictus praedictus. It lives in both temporary and permanent water bodies. During the rainy season, it was also discovered in seeping pools in caves as a result of drift from the surface (Watiroyram 2021). After P. (C.) praedictus praedictus, P. (P.) christineae is the most often encountered species, followed by P. (P.) thailandicus, which is likewise found in both ephemeral and permanent water bodies (Sanoamuang 2002). Conversely, P. (P.) roietensis and P. (P.) surinensis are very rare and restricted to temporary water bodies (Sanoamuang 2002;Sanoamuang and Watiroyram 2020;Sanoamuang and Dabseepai 2021). Phyllodiaptomus (P.) parachristineae sp. nov. was present in both temporary and permanent water bodies from the Mun River basin in the northeastern part of Thailand and was found more often than P. (P.) roietensis and P. (P.) surinensis, respectively. The distribution of P. (P.) christineae and P. (P.) parachristineae sp. nov. is puzzling when reviewing unpublished documents from previous studies. Prior authors probably identified the new species as its closet species, P. (P.) christineae, especially the specimens collected from the Chi River and Mun River basins. Based on the available information, P. (P.) christineae lives mostly in large water bodies, like lakes, rivers, and reservoirs, in the north, center, and east of Thailand, and it is rare in the Songkhram River basin. P. (P.) parachristineae sp. nov., on the other hand, lives in small water bodies rather than large ones in the Mun River basin from lower northeastern Thailand to Cambodia. However, more sampling and specimen examinations are required to explain their distribution in the northeast of Thailand.
The distribution and habitats of the 13 species and two subspecies of Phyllodiaptomus in Asia are represented in Table 5. The common species live in a wide variety of habitats from small to large water bodies, such as P. (P.) blanci, which has been reported in Iran, Iraq, Israel, India, Nepal, Uzbekistan, Kazakhstan, Turkmenistan, and Tajikistan. Although P. (C.) praedictus praedictus and P. (P.) parachristineae sp. nov. have been found in both temporary and permanent water bodies, they have so far been known only from Thailand and Cambodia. Species that live in large water bodies (i.e., ponds, lakes, or rivers) also show a widespread distribution, such as P. (C.) annae (Sri Lanka, India, Bangladesh), P. (C.) praedictus sulawesensis (Indonesia, Philippines), and P. (P.) christineae (Thailand, Laos), except P. (P.) irakiensis and P. (P.) longipes, which are exclusively found only in Iraq  (2022); Zhang et al. (2021Zhang et al. ( , 2023 and Indonesia, respectively. The small water bodies or temporary waters are separated from other waters, and then it is a naturally occurring barrier for species distribution, such as in the cases of rare species or endemic species like P. (C.) sasikumari (India), P. (P.) roietensis (Thailand, Cambodia), and P. (P.) surinensis (Thailand) Sanoamuang 1999;Khalaf 2008;Alekseev et al. 2013;Marrone et al. 2014;Kulkarni and Pai 2016;Guinto et al. 2018;Sanoamuang and Watiroyram 2020; Dabseepai 2021) (see Table 5).