﻿New and little-known stonefly species of the Rhopalopsolevietnamica ‘western assemblage’ group (Plecoptera, Leuctridae) from China

﻿Abstract We examined Chinese stonefly specimens of the Rhopalopsolevietnamica ‘western assemblage’ group. A new species from Hainan Province, R.bawanglinga Li, Li & Yang, sp. nov. is described and illustrated from male and female adults, and it is compared to closely related taxa. The hitherto unknown female of R.hainana Li & Yang, 2010 is described. Morphological evidence is presented for the identity of R.dentiloba Wu, 1973, on the basis of topotypes from Yunnan Province, southwestern China.


Material and methods
The specimens were collected by hand and preserved in 75% ethanol.Most of the studied materials are deposited in the Entomological Museum of China Agricultural University, Beijing, China (CAU).Additional specimens are deposited in the Insect Collection of Henan Institute of Science and Technology, Xinxiang, Henan Province, China (HIST), as indicated in the text.The color illustrations were made using an Imaging Source CCD attached to a Leica M205FA dissecting microscope.The morphological terminology follows that of Sivec et al. (2008).The association of sexes of Rhopalopsole hainana is based on the type locality and barcoding data.
Total genomic DNA was extracted from thorax muscle using the TIANamp Genomic DNA kit (Tiangen Biotechnology, Beijing, China) following the manufacturer's protocol.The universal primers LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify the target COI gene region.Polymerase chain reaction (PCR) program was as follows: initial denaturation at 95 °C for 60 s, followed by 40 cycles of 30 s at 95 °C, 50 s at 40-60 °C, and 60 s at 65 °C, and a final extension phase of 65 °C for 10 min.The PCR products were confirmed by 1% agarose gel electrophoresis and then sent to Sangon Biotechnology Co. Ltd. (Shanghai, China) for DNA sequencing.The primary sequences were assembled using Contigexpress software (Vector NTI Informax).Genetic distances between sequences were calculated in MEGA v.5.2 and bootstrap analysis was conducted using 1000 replicates.The GenBank accession numbers for the three specimens are in Table 2. Diagnosis.Male adult of this species is characterized by the lateral projections of the tergum 10 being bifurcate with the upper spine longer than the lower one in lateral view.The cercus bears a stout dorsoapical spine, and the epiproct is subtrapezoid with deep, circularly incised anterior margin in dorsal view.Females are diagnosed by the posterior margin of sternum 7 forming a narrowly produced pregenital plate, posteromedial portion slightly bilobed.
Description.Adult habitus.Forewing length is 6.8 mm in the male, 7.0-7.4mm in the females.Head brown to dark brown, slightly wider than pronotum; compound eyes black; antennae and mouthparts brownish to brown.Pronotum brown with dark rugosities; wings subhyaline with darker veins; legs brown.Abdomen brown.
Male (Figs 1, 3).Tergum 9 (Figs 1A, 3A) weakly sclerotized except anterior margin and lateral portions, medial 1/3 with a large trapezoidal membranous area surrounded by lateral sclerotized portion and ornamented posteromedial margin which terminates in a thin, produced sclerite with tiny granules.Sternum 9 (Figs 1B, 3B) longer than wide, distal portion with distinct trapezoidal subgenital plate, the plate about as long as wide, with a subapical constriction; vesicle dark brown, ovum-shaped in ventral view, tongue-like in lateral view, covered with dense hairs.Tergum 10 (Figs 1A, C, 3A, C) with well sclerotized, bifurcate lateral projections, terminating in two acute, parallel-sided points in dorsal aspect, the upper spine is longer than lower spine in lateral view.Central plate (Figs 1A, D, 3A, D) sclerotized and trilobed, lateral lobes subtriangular, together with a circular anteromedial incision of the medial lobe resemble flying wings; medial lobe darkly pigmented, with a small knob-like projection.Transverse bars slightly elevated medially, triangular, posterior margin and inner portion sclerotized.Cercus barely 3× longer than wide, curved dorsally, with a stout subapical spine.Epiproct (Figs 1A, E, 3A, E) strongly sclerotized and darkly  Etymology.The specific name refers to the Bawangling National Forest Park.

Distribution. China (Hainan).
Remarks.The new species is a typical member of the R. vietnamica group, western assemblage sensu Sivec et al. (2008).The male of the new species seems closely related to R. hainana from Hainan and R. cestroidea Li & Yang, 2017(in: Li et al. 2017) from Guangxi, as they share a similar wide epiproct.However, it can be differentiated from both by bifurcate lateral projections on tergum 10, with the upper spine being longer than the lower one in lateral view, and the presence of a concave anterior margin of the epiproct.In R. hainana and R. cestroidea, the terminal bifurcation of lateral projections on tergum 10 is of equal length in lateral view, and the epiproct has a truncate or convex anterior margin.Additionally, the cercus of R. cestroidea lacks a spine, and that of R. hainana has a sharp subapical spine, whereas the cercal spine in the new species is stout.The female is distinctive due to the bilobed distal tip of the pregenital plate.

Rhopalopsole hainana Li & Yang, 2010
Fig. 4 Rhopalopsole hainana Li & Yang, 2010: 59 (original description).Remarks.Genetic distance refers to the genetic divergence between species and can be used to compare the genetic similarity between difference species (Wang et al. 2013); generally, more than 98% of congeneric species or sister species pairs have greater than 0.02 sequence divergence (Johns and Avise 1998;Hebert et al. 2003;Park et al. 2011).In our study, pairwise distances of the Rhopalopsole species were 0-0.9%; lower than the 2% threshold considered for a rough differentiation between intraspecific and interspecific distances (Zhou et al. 2009).The genetic distance of COI between J66 and J61 is 0 and between J66 and J64 is 0.9% (Table 3).The combination of morphology and molecular data suggest the female is R. hainana.
We compared the holotype with the specimens from Baisha County, which originated from near the type locality.The hitherto unknown female resembles the female of R. bawanglinga, but the tip of the pregenital plate is not bilobed, and the transverse band on sternum 8 is more pronounced.Wu, 1973 Figs 5, 6 Rhopalopsole dentiloba Wu, 1973: 105 (original description).

Rhopalopsole dentiloba
Material examined.4 males, 1 female (CAU), China: Yunnan Province, Xishuangbanna, Meng shimron botanical garden, 22.IV.2007,21°55'9.12"N,101°16'6.96"E,550 m, leg.D. Hui. 1 male (HIST), China: Yunnan Province, Remarks.We examined several specimens from Xishuangbanna, Yunnan Province, which is the same location where R. dentiloba was described.The original description and illustrations did not provide sufficient details for identification.Therefore, we discuss herein the distinctive morphological characters of our specimens.This species is a typical member of the R. vietnamica group, western assemblage sensu Sivec et al. (2008).The male tergum 9 (Fig. 5A, C, D) has a small triangular, upraised sclerotized process on hind margin.Tergum 10 (Fig. 5A, B) bears a small bifurcate spine at each posterolateral corner in dorsal and lateral aspects, but the inner/lower spine is not easily observed.In lateral view (Fig. 5C, D), unbranched, sclerotized lateral projections are seen.The epiproct is small, hook-like.The minute teeth of the subanal lobe described for the holotype are possibly the distinct furrows seen in the males observed in this study.The identity of these specimens is primarily based on a similar female and their proximity to the type locality (Mengsong town or Mengsong village in this town).The female (Fig. 6A, B) is identical to the original illustration.However, doubts may arise about association of the original female and the male holotype, as tropical Yunnan is well known for its high biodiversity, where more Rhopalopsole species may co-occur.

Conclusion
Thirteen species were previously recorded in the R. vietnamica 'western assemblage' group, as specified in Table 1.Rhopalopsole dentiloba was included in the R. shaanxiensis species group but is now transferred to the R. vietnamica 'western assemblage' group due to its great similarities to R. sinensis Yang & Yang, 1993.Considering the geographical (Fig. 7) differences between populations of R. sinensis, molecular methods should be applied to confirm the status of R. dentiloba in the future.With the description of the new taxon and the inclusion of R. dentiloba, the number of species in this group in China is now up to eleven, and more new species may await discovery.

Figure 7 .
Figure 7. Distribution of species of the Rhopalopsole vietnamica 'western assemblage' group of China.City records shaded in grey.

Table 1 .
World list of species belonging to the Rhopalopsole vietnamica 'western assemblage' group.Abbreviation: M, male; F, female.

Table 2 .
China, Hainan Province specimens used for COI based identification, genetic differentiation, and circumscription analyses of three Rhopalopsole species.