Phyllodiaptomus (Phyllodiaptomus) roietensis, a new diaptomid copepod (Copepoda, Calanoida) from temporary waters in Thailand and Cambodia, with a key to the species

Abstract Phyllodiaptomus (Phyllodiaptomus) roietensissp. nov. was collected from temporary water bodies in Roi Et and Nakhon Ratchasima provinces in northeastern Thailand and Kampong Thom Province in central Cambodia. The new species is closely related to Phyllodiaptomus (P.) surinensis Sanoamuang & Yindee, 2001 in that it shares common morphological characters in the males: urosomites 2–3, P5 intercoxal sclerite, right P5 Exp-2, and left P5 Exp. Minor differences on the right antennule, right caudal ramus, P5 basis and Enp exist. The females differ in their Pdg 5, genital double-somite, and P5. An updated key to the species of the genus Phyllodiaptomus Kiefer, 1936 is provided.

During seasonal sampling collections of freshwater copepods from several localities in Thailand and Cambodia, we encountered another hitherto unknown species of Phyllodiaptomus. In this paper, we describe Phyllodiaptomus (P.) roietensis sp. nov. from two localities in Roi Et and Nakhon Ratchasima provinces, northeast Thailand, and two localities in Kampong Thom Province in central Cambodia (Fig. 1).

Materials and methods
Samples were collected using a plankton net with a mesh size of 60 µm and preserved immediately in 70% ethanol. Adult copepods were selected under an Olympus SZ51 stereomicroscope at 40-x magnification and placed in a mixture of glycerol and 70% ethanol (ratio ~ 1:10 v/v). After 10 min the animals were transferred to pure glycerol. The animals were dissected and prepared in a glycerin-mounted slide under a stereomicroscope at 40-100-x magnifications. The dissected specimens were mounted in pure glycerin on a glass slide and sealed under a cover glass with transparent nail varnish. All un-dissected specimens were stored in 70% ethanol in 1.5 mL microtubes.
All appendages and body ornamentation were examined at 1000-x 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.
Left antennule, antenna, mouthparts, and P1-P4 as in female. P5 (Figs 6D, 8A, G) intercoxal sclerite with rounded lobe on free margin. Right P5: coxa with acute, stout spine on posterior lobe. Basis (Fig. 8B, G) with large proximomedial triangular lamella at one-fourth length of inner margin; with large three-lobed chitinous medial prominence on posterior surface; distal outer margin with long, thin seta, slightly extending beyond Exp-1. Enp (Fig. 8B, H, G) with bi-lobed distal margin, tipped with spinules and hyaline lamella on inner and outer lobes, respectively; reaching downward to approximately one-third of Exp-2. Exp-1 (Fig. 8A, B, H) with semi-circular knob on distomedial margin; distolateral margin with small acute process. Exp-2 (Fig. 8C, H) elliptic, with three accessory lateral spines, one proximal, middle, and distal on lateral margin. Principal lateral spine articulated, located at two-third length of Exp-2, flat, thick, digitiform, with sharp tip; long, with approximately half of segment bearing it; slightly twisted in posterolateral direction. End claw (Figs 6D, 8A) medially sickle-shaped, slender towards tip, more than 1.5 times as long as Exp-2; medial margin serrated with row of tiny spinules.
Left P5 (Figs 6D, 8D): coxa with moderate strong seta inserted on posterior lobe at distal inner corner, slightly shorter than distal margin of basis. Basis with flap of longitudinal hyaline lamella at medial margin; with long, thin seta at posterolateral margin, extending to approximately half of Exp-1. Exp-1 (Fig. 8F) tapering towards posterior margin, medial margin concave, with field of setules and tiny spinules. Exp-2 smaller than Exp-1, conical; with large seta at mid-length of medial margin, as long as Exp-2 and apical process combined; with few setules proximally and widespread with spinules distally along inner margin, thickness of spinules increased from proximal to distal; apical process stout, bare, and blunt-tip. Enp (Fig. 8F, J) bi-segmented, longer than Exp-1, Enp-2 tipped with row of spinules distally. Differential diagnosis. Phyllodiaptomus (P.) roietensis sp. nov. with the male P5 Exp-2 displays an affinity to the subgenus Phyllodiaptomus sensu Dumont et al. (1996): the lateral side of the right Exp-2, medially concave in posterior view, principal lateral spine inserted on distal to mid-outer margin and three accessary spines arranged from proximal, middle, and distal, respectively; the left Exp-2 with patch of strong spinules along medial margin.
The male of the new species has serrated outgrowth on the antepenultimate segment of the right antennule. The right caudal ramus with small chitinous spine near distal margin on ventral side and triangular prominence along proximal one-third length of outer margin. The P5 intercoxal sclerite produced, with convex distal margin. The right P5 with (1) short, strong spine on posterior lobe of coxa, (2) triangular hyaline lamella on proximal inner margin and large chitinous outgrowth on posterior surface of basis, (3) acute distal outer corner of Exp-1 (4) Exp-2 oval and concave, with strong, flat, curved principal spine and three accessary spines, and (5) bi-lobed Enp. The left P5 with long and narrow hyaline lamella along inner margin, Exp-2 with patch of strong spinules along medial margin, and bi-segmented Enp.  Female with asymmetrical Pdg 5 wings, left wing more elongated in posterio-lateral direction; posterior and dorsal spines short and strong. Genital double-somite with posterolateral directed process on right side. One pair of genital spines on lateral side slightly symmetrical and strong. P5 Exp-2 with conveyor canal on anterior surface. P5 with bi-segmented Enp.
Etymology. The specific name roietensis is taken after the type locality, Roi Et Province. The name with the Latin suffix "-ensis" is the adjective for a location.
The right antennule is mainly used as a clasping organ in all males of the family Diaptomidae, and it normally bears spines or spinous processes on segments 8, 10-16, and 20 (Kulkarni et al. 2018). However, P.  fig. 6D). The males of these species may manage to mate more easily with females using the unique ornamentation of antennule and caudal ramus. In females, Pdg 5 wings and genital double-somites are probably important for species recognition and mating behavior of their males (Ohtsuka and Huys 2001;Ali et al. 2014). Although the male morphological features of the two parapatric Phyllodiaptomus are different, they are able to differentiate their conspecific females during mating, as the females of the new species can be distinguished from its congeners by the presence of posterolateral process on both sides of genital double-somite, which are absent in other congeners except P. (P.) thailandicus. However, the characteristic that differentiates the new diaptomid from P. (P.) thailandicus is the presence of a single process on each side of the genital doublesomite; P. (P.) thailandicus has two processes only on the right side (Figs 2C-E, 3C-F; Sanoamuang and Teeramathee 2006: figs 1, 24). In contrast to their males, the new species and P. (P.) surinensis have unique females which can be easily differentiated. The female P5 Exp-2 of the new species is obviously asymmetrical compared with that of P. (P.) surinensis which has a slightly asymmetrical P5 Exp-2. Dumont and Ranga Reddy (1993) observed that the conveyor canal on the P5 Exp-2 in females is species-specific and unique to the genus Phyllodiaptomus: the new species has two longitudinal ridges on the anterior surface versus multi-longitudinal ridges in P. With regard to the comparative morphology above, the male of the new species is most similar to those of P. (P.) surinensis. However, there are three major differences among the males, i.e. the right caudal ramus, left P5 basis, and left P5 Enp as described above. The fine detail on its inner hyaline lamella on the right P5 basis is also different: triangular in the new species versus oval bi-lobed in P. (P.) surinensis.
Ranga  provided the first key to species and included six species of Phyllodiaptomus (P. (P.) tunguidus, P. (P.) blanci, P. (P.) longipes, P. (C.) annae, P. (C.) wellekensae, and P. (C.) sasikumari); he also gave morphological descriptions of these six species. In this study, the key is updated as follows: Keys to worldwide species of Phyllodiaptomus Kiefer, 1936 Males: