Evolutionary History of the Complex Polymorphic Dobsonfly Genus

Systematic Entomology (2018), 43, 568–595 DOI: 10.1111/syen.12287

Evolutionary history of the complex polymorphic dobsonﬂy genus Neoneuromus (Megaloptera: Corydalidae)

, , , FAN YANG1 *, WENCHENG CHANG1 2 *, FUMIO HAYASHI3, JESSICA GILLUNG4,5, YUNLAN JIANG1, DING YANG1 andXINGYUE LIU1

1Department of Entomology, China Agricultural University, Beijing, China, 2Shanghai Agricultural Technology Extension and Service Center, Shanghai, China, 3Department of Biology, Tokyo Metropolitan University, Tokyo, Japan, 4California State Collection of Arthropods, Sacramento, CA, U.S.A. and 5Bohart Museum of Entomology, University of California, Davis, CA, U.S.A.

Abstract. The dobsonfly genus Neoneuromus van der Weele is endemic to the Oriental region. Species possess highly variable body and wing colouration and markings, not only among species but also among conspecific individuals of certain species. This stark polymorphism hinders accurate species identification, and obscures an undocumented species diversity as well as our understanding of the evolutionary history of this genus. We used multiple methods of molecular identification combined with morphological evidence to delimitate species, circumscribing a total of 13 species in Neoneuromus.Five new species are herein described: Neoneuromus indistinctus Liu, Hayashi & Yang, sp.n., Neoneuromus maculatus Liu, Hayashi & Yang, sp.n., Neoneuromus niger Liu, Hayashi & Yang, sp.n., Neoneuromus similis Liu, Hayashi & Yang, sp.n. and Neoneuromus vanderweelei Liu, Hayashi & Yang, sp.n. The dated phylogeny with reconstructed ancestral areas indicates an initial divergence of Neoneuromus during the mid-Eocene. A broad area including northeastern India and northern Indochina could be a centre for early divergence of the genus, while complex dispersal and vicariance events dating from the late Eocene to the Pliocene probably shaped the present diversity and distribution of the genus. Our ancestral character state reconstruction suggests that the pale and dark colour forms among different species, or conspecifics, could evolve rapidly and that changes in colouration could be driven by species-specific mate recognition.

Introduction the broadly subquadrate head with acute postocular spines, the male mandibles shorter than the head, the presence of four Neoneuromus van der Weele (Megaloptera: Corydalidae: Cory- or more forewing radial crossveins, the presence of accessory dalinae) is an endemic Oriental dobsonfly genus, currently crossveins among forewing CuA branches, and the diagnostic with nine described species (Liu & Yang, 2004; Yang & male genitalia with an internal fossa on tergum 9, an attenuate Liu, 2010). Individuals are relatively large-sized insects, with sternum 9, and a sclerite around the anus. Many Neoneuromus wingspan ranging from 90 to 150 mm (Fig. 1); they are some species are frequently confused with females of the giant dob- of the most commonly encountered megalopterans in Asia, and sonfly genus Acanthacorydalis van der Weele due to their large often have distinctive colouration and conspicuous mandibles. body size and their spectacularly sharp mandibles, although Morphologically, adults of Neoneuromus are characterized by Neoneuromus can be distinguished from Acanthacorydalis Correspondence: Xingyue Liu, Department of Entomology, by the absence of additional spines on the vertex and the China Agricultural University, Beijing 100193, China. E-mail: mandibles slightly shorter than head. The sister-group relation- [email protected] ship between Neoneuromus and Nevromus Rambur has been repeatedly recovered in previous phylogenetic studies of Cory- ∗ Co-first author. dalinae based on morphological and molecular data (Glorioso,

1981; Contreras-Ramos, 1998, 2011; Liu et al., 2015a; Jiang identified based on morphology (Table S1). Neoneuromus et al., 2016). territans (Needham,) is herein considered a junior synonym of ThelarvaeofNeoneuromus inhabit streams or riverlets in N. fenestralis (McLachlan,) and thus was not included in this hilly or mountainous areas. As the larvae are large and can be study, while Neoneuromus coomani Lestageishereconsidered collected in large numbers (e.g. Neoneuromus ignobilis Navás), a nominum dubia due to the poor species diagnosis and figure they are often used as food and medicine in southwestern China provided in the original description by Lestage (1927), in addi- (Cao, 2014). tion to the possible loss of the primary type after our intensive Despite their large body size, the identification of species of searching in many European collections. For the characteri- Neoneuromus is relatively challenging compared with its sister zation of the intraspecific variation as well as the detection of genus Nevromus. In general, corydaline male genitalia display cryptic species, more than one individual per species is needed; a set of significant characters useful for species identification therefore at least two specimens per species and, if possible, (Liu et al., 2016). However, the genitalia of Neoneuromus are specimens from different collecting sites were analysed, with conserved in morphogy with only a few rather inconspicuous the exception of Neoneuromus niger sp.n., for which only one diagnostic characters for each species. In contrast, patterns of specimen was available for the molecular study. All primary body colouration and wing markings are distinct among some types of the seven described species were examined. The iden- species and are considered important diagnostic characters for tifications of all sampled specimens were made by XYL based identification (Liu & Yang, 2004; Yang & Liu, 2010). In general, on the morphological comparisons with the type specimens, some species are very dark-coloured with black or brown wing original descriptions, figures, as well as the identification key markings [e.g. Neoneuromus fenestralis (McLachlan) (Fig. 1A), provided by Yang & Liu (2010). Our sampling for the phyloge- Neoneuromus maclachlani (van der Weele) (Fig. 1B)], while netic analysis is representative of the main distribution areas of some species are yellow with almost immaculate wings [e.g. Neoneuromus, including China, India, Laos and Vietnam. Neoneuromus sikkimmensis (van der Weele) (Fig. 1E)]. How- Eight species of Megaloptera were selected as outgroups, ever, with the considerably increasing number of examined spec- including Sialis sibirica McLachlan (Sialidae), Neochauliodes imens of all Neoneuromus species (c. 1000 specimens included punctatolosus Liu & Yang (Corydalidae: Chauliodinae), Acan- in this paper), the body colouration and the wing marking pat- thacorydalis fruhstorferi van der Weele, (Corydalidae: Cory- terns turn out to be highly variable both inter- and intraspecif- dalinae), Protohermes lii Liu, Hayashi & Yang (Corydalidae: ically (Figs 5–11). Even more confusingly, in some cases the Corydalinae), Nevromus aspoeck Liu, Hayashi & Yang, Nevro- colour form of one species resembles that of another species. mus exterior (Navás), Nevromus gloriosoi Liu, Hayashi & Yang, For example, the pale-coloured form of Neoneuromus tonkinen- and Nevromus intimus (McLachlan) (Corydalidae: Corydali- sis (van der Weele) (Fig. 5D) is similar to the typical form of nae). Among them, Nevromus is suggested to be the sister group both N. ignobilis (Fig. 6A) and Neoneuromus orientalis Liu & of Neoneuromus (Contreras-Ramos, 2011; Liu et al., 2015a). Yang, (Fig. 5E), although the male genitalia is very different The specimens used in this study are deposited in 36 institu- between the former species and the two latter species, namely tions (see Table S1 for the information of voucher specimens by the absence/presence of gonostyli 10. Herein, we found that used in the molecular phylogenetic study, and Table S8 for the seven species of Neoneuromus have extensive intraspecific vari- deposition of other specimens herein examined). ation in colouration. Unfortunately, this polymorphism in body In the taxonomic section, the label data of the name-bearing colour and wing markings in species of Neoneuromus hinders types are presented within quotation marks (‘ … ’). Added accurate species identification and obscures the estimation of the information that expands the often cryptic text of the type real species diversity within the genus. labels and provides the geographic coordinates is placed in Here we present the first phylogeny of Neoneuromus,based square brackets ([ … ]). Genitalia preparations were made on three mitochondrial genes. We used an integrative approach by clearing the apex of the abdomen in a cold, saturated to identify species, including morphology and multiple methods potassium hydroxide (KOH) solution for 8–10 h. After rinsing for molecular identification, which resulted in the discovery of with acetic acid and water, the apex of the abdomen was five new species. We also estimated the temporal and spatial transferred to glycerin for further dissection and examination. divergence of species of Neoneuromus and performed ancestral The terminology of the genitalia follows Liu et al. (2016). state reconstruction of the evolution of pale and dark colour forms. A systematic revision and identification key to species of Neoneuromus are provided under the present phylogenetic DNA extraction, amplification and sequencing framework. DNA was extracted and purified from the mesothoracic muscle using the TIANamp Genomic DNA Kit (TIANGEN, China). Material and methods Partial sequences of three mitochondrial genes (COI, ND2 and 16S rRNA) were amplified and sequenced. Two fragments of Specimen sampling COI were sequenced and are termed here as COI-1 (frequently used in previous molecular phylogenetic studies of Megaloptera; We sampled 77 specimens of seven described species and Chang et al., 2013; Yue et al., 2015; Liu et al., 2015a), and two new species of Neoneuromus; new species were primarily COI-2 (i.e. the standard DNA barcode of insects, following

Fig. 1. Adults of Neoneuromus spp. (A) Neoneuromus fenestralis (McLachlan), from western Yunnan (photograph by Chao Wu); (B) Neoneuromus maclachlani (van der Weele), from Chongqing (photograph by Yuansheng Li); (C) Neoneuromus tonkinensis (van der Weele) (colour form I), from northern Vietnam (photograph by Xingyue Liu); (D) Neoneuromus coomani Lestage, from southern Sichuan (photograph by Chengquan Cao); (E) Neoneuromus sikkimmensis (McLachlan), from western Yunnan (photograph by Chao Wu); (F) Neoneuromus ignobilis Navás (colour form I), from Guangxi (photograph by Xingyue Liu); (G) Neoneuromus ignobilis Navás (colour form II), from southern Yunnan (photograph by Xingyue Liu); (H) Neoneuromus orientalis Liu & Yang (form II), from southern Guangxi (photograph by Xingyue Liu).

Folmer et al., 1994 and Hebert et al., 2003). Primer sequences Gap Discovery (ABGD) and the Poisson tree processes (PTP) are presented in Table S2. All PCRs were performed using NEB method, to identify the molecular operational taxonomic unit Long Taq DNA polymerase (New England Biolabs, Ipswich, (MOTU), which has a similar extent as traditional species to MA, U.S.A.) under the following amplification conditions: 95∘C define the identity of conserved sequences (Blaxter, 2004). for 30 s, 40 cycles of denaturation at 95∘C for 10 s, annealing Afterwards, we linked the morphological species to the MOTUs at 43–55∘C (depending on the primer pair used) for 50 s, identified by the two methods above, and made further morpho- elongation at 65∘C for 1 kb/min (depending on the size of logical examinations of these MOTUs in order to obtain final amplicon), and the final elongation step at 65∘C for 10 min. The species delimitations. quality of PCR products was assessed using electrophoresis in a The ABGD is an iterative method to sort sequences into 1% agarose gel and staining with Gold View. genetic clusters or candidate species based solely on pairwise All PCR products were sequenced in both directions using the distances without an a priori species hypothesis (Puillandre BigDye Terminator Sequencing Kit (Applied Bio Systems, San et al., 2012). The analyses were conducted on the ABGD web Francisco, CA, U.S.A.) and the ABI 3730XL Genetic Analyzer interface (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb (PE Applied Biosystems, San Francisco, CA, U.S.A.) with two .html). Prior maximum divergence of intraspecific diver- vector-specific primers and internal primers for primer walking. sity P was investigated under K2P (Kimura, 1980). The parameters we set are as follows: Pmin = 0.001, Pmax = 0.1, Steps = 50, X (relative gap width) = 0.5, Nb bins (for distance Sequence alignment and phylogenetic analysis distribution) = 20. Sequence assembly was done using contigexpress. For the PTP analysis, we used the trees generated with The fragments of COI and ND2 were aligned based on raxml as the input data. The calculations were conducted on the amino acid translation using clustalw in mega 5.0 the bPTP websever (http://species.h-its.org/ptp/), with 500 000 (Tamura et al., 2011). The 16S rRNA sequences were MCMC generations, thinning set to 100 and burn-in at 25%. adjusted using g-blocks server (http:// molevol.cmima.csic The probability of clusters representing putative species was .es/castresana/Gblocks_server.html). Individual genes were calculated in two ways: (i) the Bayesian solution (PTP_sh), concatenated using sequencematrix v1.7.8 (Vaidya et al., which considers the frequency of the nodes across the sampling; 2010). and (ii) the maximum likelihood solution (PTP_ML), which Phylogeny estimation was performed using neighbour join- considers the most likely solution among the sampling (Zhang ing (NJ) (Saitou & Nei, 1987), maximum likelihood (ML) et al., 2013). and Bayesian inference (BI) methods. The NJ analysis was performed in mega 5.0 under the Kimura-2-Parameter (K2P) model. Bootstrapping with 1000 pseudo-replicates was used to Divergence time estimation examine the robustness of clades in the resulting tree (Felsen- stein, 1985). The genetic distance values of inter- and intraspe- Estimation of divergence times was performed using beast cific variations were calculated under the K2P model forthe 2.4.7 (Bouckaert et al., 2014). We performed analyses using COI-2 dataset. partitionfinder2 (Lanfear et al., 2012) was fossils. For the analysis with fossil calibrations, two calibration used to determine the optimum partitioning scheme and the densities were used: (i) the minimum age of Corydalidae + best-fitting model for each partition, using the Bayesian infor- Sialidae was set to 185 Ma based on the oldest known fossil mation criterion. We input the partition file containing ten par- of Sialidae (Dobbertinia reticulata Handlirsch, from the Lower titions, in which the protein-coding genes were divided into Jurassic of Dobbertin, Germany, ∼185 Ma) (Ansorge, 2001), an codon positions for each fragment. The ML analysis was per- exponential prior was imposed on the age of the node that ranged formed in raxml (Stamatakis et al., 2008), with 1000 bootstrap from 185.1 to 188.0 Ma; (ii) the minimum age of Chauliodinae replicates using the rapid bootstrap feature (random seed value + Corydalinae was set to 165 Ma based on the minimum age of set to 12 345). The BI analysis was performed using mrbayes an adult fishfly (Jurochauliodes ponomarenkoi Wang & Zhang, 3.2.2 (Ronquist & Huelsenbeck, 2003), with two simultaneous from the Middle Jurassic of Inner Mongolia, China, ∼165 Ma) Markov chain Monte Carlo (MCMC) runs of 2 million genera- (Liu et al., 2012b), an exponential prior was imposed on the age tions conducted for the dataset; tree samples were output every of the node that ranged from 165.1 to 168.0 Ma. The partitioning 1000 generations with a burn-in of 25%. Analyses terminated as scheme and nucleotide substitution model were as described the standard deviation of clade frequencies had reached 0.002; for the phylogenetic analysis , with a birth–death process of when it was below 0.01, we considered that stationarity had been speciation as tree prior. Site and clock models were unlinked and reached and stopped the run. Trees were visualized and edited all partitions were analysed using the uncorrelated exponential with figtree v1.3.1 (Rambaut, 2009). relaxed clock model. Two independent MCMC runs were performed for five million generations and sampled every 1000 generations. tracer v1.5 Species delimitation (Rambaut & Drummond, 2007) was used to check when the MCMCs had reached a stationary distribution by visual inspec- Based on the molecular data, we used two independent tion of plotted posterior estimates and by checking that the species delimitation approaches, the Automatic Barcoding effective sample size (ESS) was greater than 200 for most

© 2018 The Royal Entomological Society, Systematic Entomology, 43, 568–595 572 F. Yang et al. important parameters (e.g. posterior and tree likelihood). We Results combined the tree files of both runs using logcombiner 1.5.3, with the first 25% of the generations from each run discarded as Species delimitation burn-in. Finally, we used treeannotator 1.5.3 (Drummond & Rambaut, 2007) to calculate the consensus tree and annotate the Results of multi-locus species delimitation from the ABGD divergence times based on the combined tree file. The chrono- and PTP approaches were summarized in Fig. 2. For the ABGD gram was viewed and edited using figtree 1.3.1 (Rambaut, analysis, setting the default value for relative gap width (X = 1.5) 2009). did not produce any results for our dataset, so we used the smallest value that could be applied (X = 0.5). Both initial and recursive partitioning analyses clustered Neoneuromus taxa into 18 putative species. In contrast, the PTP analyses resulted in a Ancestral state reconstruction total of 27 putative species under both the best-fit ML search (PTP_ML) and simple heuristic search (PTP_sh). The support To infer the evolutionary history of different body colour values across PTP recognized entities are high, ranging from forms in Neoneuromus, we conducted an ancestral character 1.000 to 0.79 to 0.481. state reconstruction (ACSR). We defined the body colour form in The specific MOTUs resulting from the ABGD andPTP two states: dark-coloured form, and yellow-coloured form. The analyses are congruent for N. ignobilis, N. latratus, N. mac- ACSR analyses were performed under parsimony in mesquite ulatus sp.n., N. orientalis, and partial N. sikkimmensis.The v3.31 and under BI in the Multistate module of bayestraits inconsistent MOTUs from these two analyses are N. fenestralis, v2.0 (Pagel et al., 2004). We treated states as unordered, assum- N. tonkinensis, N. maclachlani, and remaining N. sikkimmensis. ing equal transition rates between states, and allowed the rates Surpringly, the PTP analysis indicated that the two specimens of of change between states to vary over each transition. Recon- N. fenestralis represent different putative species. However, N. structions were based on 5001 post-burn-in trees from the beast fenestralis is the most easily distinguishable species in Neoneu- analysis. We ran the MCMC implementation of MultiState for romus based on the lack of male gonostyli 10. The unchanged 200 million generations with a burn-in of two million genera- characteristics of male genitalia among individuals from differ- tions, sampling every 1000 generations, and with an exponential ent distributed areas indicate that there is no subdivision within distribution on character transition rates with a mean of 1.0. Con- N. fenestralis. The PTP analysis also separated two specimens vergence was assessed using tracer v1.5 to ensure that analyses (CAU00180 and CAU00184) of N. maclachlani into two differ- had reached stationarity and that ESS values for all parameters ent putative species. This result is problematic because these two were > 200. We re-ran the analyses under the prior to ensure specimens are almost identical in terms of morphology and have that there were no unexpected interactions causing erroneous all the typical diagnostic characters of N. maclachlani;also, induced prior distributions. these two specimens were even collected in the same locality and at the same date. Therefore, the results from the PTP analysis probably overestimated the species number of Neoneuromus. The morphological diagnosis of N. fenestralis, N. latratus, N. Ancestral area reconstruction maculatus sp.n.,andN. ignobilis is congruent with the result from the ABGD analysis, in which no subdivision within each Five endemic areas for Neoneuromus were defined for the of the species is found. Species with internal subdivisions as ancestral area reconstruction analyses: (i) northeastern India and inferred from the ABGD analysis include N. tonkinensis, N. its adjacent areas, including southeastern Xizang, western Yun- sikkimmensis, N. maclachlani and N. orientalis. nan and northern Myanmar; (ii) northern Indochina and the The suggested species subdivision within N. tonkinensis western part of South China; (iii) southeastern Indochina; (iv) resulting from the ABGD analysis indicates that the specimen the western part of Central China and adjacent areas, includ- (CAU00230) from Guangdong (southern China) represents a ing southern Sichuan and southeastern Guangxi; and (v) the putative separate species when compared with the other three eastern part of Central and South China (Fig. 4). The divi- individuals from Guangxi (southern China) and northern Viet- sion of Central and South China is based on the zoogeograph- nam. The genetic divergence between CAU00230 and the other ical regions proposed in Zhang (1999). We removed outgroup three N. tonkinensis specimens is relatively high (average of taxa that would preclude meaningful results. Ancestral distribu- 0.066). The wings of CAU00230 are much paler than those of tions were reconstructed using Bayes-Lagrange (or the S-DEC the other specimens in the group, these showing typical wing model) (Ree et al., 2005; Ree & Smith, 2008) approach. The colouration of N. tonkinensis (i.e. forewing with distal half dark analyses were performed in rasp (Reconstruct Ancestral State and with a dark spot on 1mp-cua). Nevertheless, such colour in Phylogenies) v3.2, build 20151028 (Yu et al., 2014). To form with very pale wings is found in Hong Kong, which is account for phylogenetic uncertainty, 5001 post-burn-in trees close to the collecting site of CAU00230 (Yiu, 2008), and the resulting from the beast analysis were integrated for infer- specimen is confirmed to be N. tonkinensis via examination of ence. The maximum number of ancestral areas was set to five, male genitalia. Therefore, as no differentiation in male genitalia as certain Neoneuromus species, such as N. ignobilis,canbe can be found between these two MOTUs, we herein consider widespread. the individual CAU00230 to be N. tonkinensis, together with the

Fig. 2. Phylogenetic tree of Neoneuromus based on the concatenated dataset (COI-1 + COI-2 + ND2 + 16S rRNA). Numbers at nodes are Bayesian posterior probabilities (left) and maximum likelihood bootstrap values (right). Results of molecular species delimitation based on Automatic Barcoding Gap Discovery (ABGD), Poisson tree process maximum likelihood (PTP_ML) and Bayesian (PTP_sh) methods are annotated on the right side of the tree.

© 2018 The Royal Entomological Society, Systematic Entomology, 43, 568–595 574 F. Yang et al. other three putative N. tonkinensis specimens. However, consid- cryptic species: Neoneuromus similis sp.n. and Neoneuromus ering the distribution gap between CAU00230 (Guangdong) and vanderweelei sp.n. The fourth MOTU (comprising the individ- the other three N. tonkinensis specimens (Guangxi and adjacent uals CORY013, CORY041, CORY047, and CORY048 from region in northern Vietnam), geographical variation and possible western Guangxi and northern Vietnam) is genetically distinct allopatric subspecies separation should be further investigated and is closely related to the MOTU identified as N. vanderweelei when more samples are available. sp.n. from western Yunnan. Regarding the external morphology, A similar case is also found in N. orientalis,whichis the body colour is either reddish brown (Fig. 11A) or blackish subdivided into two MOTUs based on the ABGD analysis. The brown (Fig. 11B) in these two MOTUs, but their genital char- clade from Guangdong, Fujian, Guizhou, and northern Guangxi acters are identical. Therefore, we consider these two MOTUs is characterized by large black cephalic markings (Fig. 5E), to be conspecific, with possible geographical variation. while the clade from Vietnam, Yunnan and southern Guangxi The single standard barcode data (herein named as COI-2) exhibits black cephalic markings that are greatly reduced in size worked well to distinguish the above identified species. In the (Fig. 5F). However, there is no morphological difference in the NJ tree based on the COI barcode data (Fig. S1), 15 groups were genitalia between these two clades. Hence, we regard these two recovered, including the 13 species described earlier and two clades as one single species, but possibly with geographical additional clades separated from typical N. orientalis and N. variation. vanderweelei sp.n. The same groupings were also obtained in Neoneuromus sikkimmensis here includes five MOTUs as sug- the NJ trees based on COI-1, ND2 and 16S rRNA (Fig. S1), suggested by the ABGD analysis. Moreover, these MOTUs rep- gesting that each of these genes is equally sufficient for species resent two independent monophyletic groups. The individuals delimitation in Corydalidae. The genetic divergences (Table S4) C831 (from northeastern India) and CORY004 (from southwest- within each group ranged from 1.04% to 5.81%. In particular, ern Yunnan), each assigned as a separate MOTU by the ABGD the genetic divergence within N. fesnestralis, N. maclachlani, analysis, are recovered as sister groups. However, there are no N. vanderweelei sp.n.,andN. sikkimmensis is slightly greater morphological differences between these two clades, and their than 2%, and that of N. tonkinensis is greater than 2%. It is geographical distribution is included within the distribution area evident that all conspecific specimens with distinct genetic of the typical N. sikkimmensis, whose type locality is Sikkim, divergence are from different distribution areas, suggesting northeastern India. Therefore, we consider these two MOTUs as possible population differentiation correlated with potential one single species, namely N. sikkimmensis. The remaining two geographical isolations, as mentioned earlier in the paper. MOTUs, which were recovered as closely related to N. sikkimmensis, can be easily distinguished from typical N. sikkimmensis by the strongly narrowed apex of male sternum 9 and the more Phylogenetic analysis inflated apex of male ectoprocts. However, there are no differences in the genitalia between these two MOTUs, although the For the ML and BI analyses, four mitochondrial gene frag- MOTU represented only by the individual H909 from central ments (COI-1, 726 bp; COI-2, 663 bp; ND2, 429 bp; 16S rRNA, Vietnam has much paler forewing marking patterns than the 488 bp) were aligned, and the best-fitting partitioning scheme MOTU from northern Vietnam, southern Yunnan and Sichuan. for the phylogenetic analyses and the selected nucleotide substi- Thus, we consider that these two MOTUs belong to the same tution models are summarized in Table S3. species, which is different from typical N. sikkimmensis and pos- The ML and BI analyses generated phylogenetic trees with sibly includes some intraspecific geographical variation. Here the same topology and overall high nodal supports (Fig. 2). we tentatively identify this species as Neoneuromus coomani Neoneuromus fenestralis was recovered as the sister group to the Lestage, which is a doubtful species due to the poor original monophyletic group including all other Neoneuromus species. description and loss of the primary type. The type locality of N. Within this monophyletic group, N. sikkimmensis + N. coomani coomani is from northern Vietnam, and this species is originally was found to be the sister group to the monophyletic subgroup noted to be very similar to N. sikkimmensis (Lestage, 1927). The comprising the remaining species. The sister-group relationship fifth MOTU apparently represents a separate species from N. between N. tonkinensis and the other species was recovered sikkimmensis, and herein is identified as a new cryptic species, with relatively low support [posterior probability (PP) = 0.66, namely Neoneuromus indistinctus sp.n. bootstrap percentage (BP) = 43]. Subsequently, the new cryptic Neoneuromus maclachlani comprises four MOTUs as indi- species, N. vanderweelei sp.n., was recovered at the base of cated by the ABGD analysis. After careful morphological a clade including all of the other species in the genus, with examinations, we found that the MOTU from southeastern N. maclachlani recovered as the sister species to N. maculatus China and the MOTU from western Yunnan are both slightly sp.n.; these two species together formed a clade including different, in terms of body colour and wing marking pat- N. similis sp.n., which is another new cryptic species herein terns, from the typical N. maclachlani from Sichuan, Guizhou separated from N. maclachlani. Neoneuromus orientalis was and northern Guangxi. There are also slight – but consis- recovered as the sister of the clade including N. ignobilis, N. tent – morphological differences on male ectoprocts between indistinctus sp.n., N. latratus and N. niger sp.n., but with low typical N. maclachlani and these two different putative species, support (PP = 0.87, BP = 43). Neoneuromus latratus, N. niger including the position and developmental extent of the distal sp.n. and N. indistinctus sp.n. form a monophylum, which is inflation. Hence, we consider these two MOTUs as two new the sister group of N. ignobilis.

Divergence time estimation also evolved from the dark-coloured form (see nodes 8 and 13 in Fig. 3). Similarly, the scenario reconstructed by parsi- The chronogram in Fig. 4 represents the divergence time mony shows bidirectional evolution between these two colour estimates (as common ancestor heights) for Neoneuromus.The forms, and the dark-coloured form could have evolved from the 95% highest posterior density (HPD) values for each node pale-coloured form at least three or four times (see nodes 9, 12, are given in Table S5. The time intervals of some deep nodes 14 and 19). are rather broad. Nevertheless, by comparing the estimated times with previous hypothesis on the temporal divergence of other Asian corydalids (Liu et al., 2010, 2012a, 2015b; Ancestral area reconstruction Yang et al., 2017), the mean nodal ages of these nodes seem more plausible and are considered for the following discus- The reconstructed ranges for the nodes resulting from sion. Our estimates indicate a Palaeocene origin for the genus Bayes–Lagrange (S-DEC) are shown in the chronogram of Neoneuromus at c. 64 Ma (HPD, 110.78–36.04 Ma), and we Neoneuromus in Fig. 4. Accordingly, the ancestral range of hypothesize that the beginning of their diversification occurred all Neoneuromus species was estimated to be northeastern during the mid-Eocene, at 47.10 Ma (HPD, 80.63–25.06 Ma). India and northern Indochina with their adjacent regions, with Neoneuromus indistinctus sp.n. and N. niger sp.n. are the the exclusion of the eastern part of Central and South China. youngest species in the clade and diverged during the Pliocene Northern Indochina and its northern adjacent region were recon- at 3.49 Ma (HPD, 6.42–0.91 Ma). Our results suggest that the structed as the ancestral range for several deep-level nodes, time interval ranging from the Late Eocene to the Mid Miocene such as nodes 19, 23, 51 and 61 (Fig. 4). (34.45–11.79 Ma) was an important period for the massive diversification of Neoneuromus, with the majority of extant Neoneuromus species diverging during that time. Discussion

Morphological characters for species delimitation Ancestral state reconstruction of colour forms The present integrated approach based on both DNA sequence The results of the parsimony and Bayesian reconstruction of data and morphology provides a convincing delimitation of colour forms are summarized in Fig. 3. The mean probabilities species of Neoneuromus. Even though the remarkable poly- for each state for the nodes reconstructed using MCMC are morphism in body and wing colouration can cause confusion provided in Table S6. Overall, the parsimony and Bayesian in species identification, some external morphological charac- approaches resulted in similar ancestral state reconstructions. ters are useful to distinguish species after careful comparison. However, parsimony reconstructed the common ancestor Notably, the dark/pale body and wing colour can be used for of all Neoneuromus species as being a pale-coloured form, distinguishing sympatrically distributed species. In other words, while the Bayesian reconstruction estimated this node as the species present in the same distribution area having either either dark-coloured or pale-coloured, with the probability dark or pale body and wing colour are different species in most of the former state being slightly higher than that of the lat- cases, except for N. tonkinensis, which exhibits great intraspe- ter. Moreover, the node representing the common ancestor cific variation in the colouration of the head and prothorax. of most species, except N. fenestralis, N. sikkimmensis and Also, the presence/absence of dark markings on the 1mp-cua N. coomani, was reconstructed as pale-coloured under parsi- crossvein in the forewing is an important – but previously over- mony and dark-coloured under Bayesian parameters. Parsimony looked – character to distinguish similar species. For example, also did not clearly reconstruct the ancestral state for nodes N. maclachlani and N. similis sp.n., both being dark-coloured 11 and 13 (see Fig. 3). Overall, results suggest a complex species with very similar genital characters, can be distinguished evolutionary history of colour form within Neoneuromus.The by this feature. common ancestor of all recent Neoneuromus species proba- The male genitalia, despite the generally conserved morphol- bly had pale body and wing colouration, being likely similar ogy among species of Neuneuromus, contain a number of novel to Nevromus, the sister group of Neoneuromus.However,a diagnostic characters of species. For instance, the configura- dark-coloured common ancestor of Neoneuromus is also pos- tion of the apex of sternum 9 (e.g. the truncate apex) (File sible, based on our analysis (see Fig. 3: node 3). Presumably, S2: Fig. 2B), the slightly incised apex with obtuse posterolat- the common ancestor of most Neoneuromus species except for eral projections (Fig. 10D), and the distinctly incised apex with N. fenestralis is likely to be pale-coloured (see Fig. 3: node digitiform posterolateral projections (Fig. 7C) are all examples 5), which is either a plesiomorphic condition or a derived of useful characters for species identification. In addition, the condition from a dark-coloured ancestor of all Neoneuromus strongly narrowed apex of sternum 9 (File S2: Fig. 1B) can be species. Subsequently, based on the scenario inferred from used to distinguish N. coomani from other similar species. Also, Bayesian reconstruction, the dark-coloured form evolved from the developmental degree of the distal inflation of ectoprocts the pale-coloured form four independent times (see nodes 7, is a previously neglected character to distinguish some species, 9, 14 and 19 in Fig. 3). It is noteworthy, however, that the including N. maclachlani versus N. similis sp.n. versus N. van- pale-coloured form in the common ancestor of some species derweelei sp.n.,andN. sikkimmensis versus N. coomani (File

Fig. 3. Summary of parsimony and Bayesian ancestral state reconstructions for the different colour forms of Neoneuromus species. The tree shown is the maximum clade credibility tree from the soft-bound beast analysis, with all outgroup taxa removed. The branch colour shows the ancestral state reconstructed through the parsimony approach; the pie charts at key nodes illustrate the mean posterior probability values for ancestral state reconstructed using BayesMultistate module in bayestraits. Numbers for key nodes are consistent with those in Table S6. Yellow indicates the pale-coloured form, black indicates the dark-coloured form. Habitus photos of Neoneuromus species with different colour forms are presented beside the tree and indicated by different coloured arrows.

S2: Fig. 1). Similarly, the relative length of the lateral arm of body (usually yellow to yellowish brown) could be a plesiomor- fused gonocoxites 10 can be used to differentiate N. indistinc- phic condition as it is present in the majority of species in tus sp.n. from N. sikkimmensis (File S2: Fig. 7C, Fig. 7D), and most donsonfly genera, including Chloroniella, the sister genus to distinguish N. ignobilis from N. orientalis (File S2: Fig. 3C, to all other Corydalinae, while only Neurhermes lacks such File S2: Fig. 6C). Lastly, the developmental extent of lateral pale-coloured species (Liu et al., 2015b). Likewise, the pale projections and gonostyli 10 on the fused gonocoxites 10 is an wing is probably also plesiomorphic in Corydalinae, while the important character to distinguish many species, including N. distinctly darkened wing could be apomorphic and only present ignobilis versus N. similis sp.n. (Fig. 10E, File S2: Fig. 3C), N. in a few species of Neoneuromus, Neurhermes and Protohermes tonkinensis versus N. orientalis (File S2: Figs 6C, 8C), N. indis- (Yang & Liu, 2010). In Neurhermes, the spectacular appearance tinctus sp.n. versus N. sikkimmensis (File S2: Fig. 7C, Fig. 7D), of the dark wing combined with the creamy white markings and and N. fenestralis versus N. maclachlani versus N. maculatus the blackish body with yellow prothorax was predicted to be sp.n. (File S2: Figs 2C, 5C, Fig. 8D). a kind of mimesis of diurnal toxic zygaenid moths (Hayashi, 1995). In Neoneuromus, the species having a dark body and wings include N. fenestralis, N. maclachlani, N. maculatus Evolution of body and wing colouration in Neoneuromus sp.n., N. tonkinensis, N. vanderweelei sp.n., N. similis sp.n. and N. niger sp.n. (Figs 5A, B, C, 8A, 9A, 10A-B, 11A-B). Most The greatly varied body and wing colouration among species possess transparent patches on their blackish brown forewings, as well as among conspecifics in Neoneuromus is remarkable and some species, such as N. maculatus sp.n. and N. tonkinen- within Megaloptera. In Corydalinae, the generally pale-coloured sis, have yellow markings on the head or abdomen. Interestingly,

Fig. 4. Chronogram with estimated divergence times amongst Neoneuromus species, with ancestral area reconstructed from Bayes–Lagrange (S-DEC) approaches in rasp. Letters at nodes, pie charts and in parenthenses ahead of sampling code correspond to the legend in the map at the bottom left. Nodes with dispersal events and those with vicariant events are indicated by a red star and a blue circle, respectively. Numbers at nodes are consistent with those in Tables S7.

Fig. 5. Habitus photos of Neoneuromus spp. (A) Neoneuromus fenestralis (McLachlan), male, from western Yunnan; (B) Neoneuromus maclachlani (van der Weele), holotype male, from Sichuan; (C) Neoneuromus tonkinensis (van der Weele) (colour form I), male, from northern Vietnam; (D) Neoneuromus tonkinensis (van der Weele) (colour form II), lectotype male, from northern Vietnam; (E) Neoneuromus orientalis Liu & Yang, male, from Guizhou; (F) Neoneuromus orientalis Liu & Yang, male, from northern Vietnam. Scale bar: 5.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com]. many of these species, including N. fenestralis, N. tonkinen- and N. maclachlani (dark-coloured). In Tongbiguan Nature sis and N. vanderweelei sp.n., sometimes co-occur with some Reserve in western Yunnan, China, there are four co-occuring Neurhermes species. Neoneuromus species: N. fenestralis (dark-coloured), N. orien- It has been suggested that the dark-coloured form in Neoneu- talis (pale-coloured), N. sikkimmensis (pale-coloured) and N. romus represents an example of mimesis of the colouration vanderweelei sp.n. (dark-coloured). The genital morphology exhibited by Neurhermes, possibly driven by predator selec- is relatively conserved in species of Neoneuromus, and genital tion. However, putative Neurhermes mimics of poisonous moths characters do not differ greatly, as in some other dobsonfly are separated temporally, as the moths are active diurnal fliers genera, such as Protohermes. Thus, body and wing colouration (Hayashi, 1995), while most species of Neoneuromus are noc- might play an important role for conspecific mates to be able to turnal. Furthermore, our ancestral state reconstruction suggests recognize each other during courtship. However, this hypoth- that the pale-coloured form in some Neoneuromus species could esis of different colour forms among sympatrically distributed also be a derived condition and might have evolved from the species evolving due to assortative mating needs further testing. dark-coloured form. Thus, this life-history difference between Neoneuromus and Neurhermes as well as our ancestral state reconstruction casts doubt on the hypothesis that the evolution Temporal diversification and historical biogeography of colour forms in Neoneuromus is driven by mimesis. of Neoneuromus Interestingly, different Neoneuromus species can be sympatrically distributed, with their larvae inhabiting the same The diversification patterns that gave origin to the incredibly stream. Moreover, based on all well-investigated areas with diversified fauna of Corydalidae in the Oriental region areof more than one Neoneuromus species known so far, it is evi- great interest, yet are still poorly understood. Previous studies dent that both pale-coloured and dark-coloured species are that attempted to elucidate the diversification of dobsonflies present. For example, there are two species distributed in Mt utilized morphology-based phylogenies to infer their evolution Emeishan, Sichuan, China, namely N. ignobilis (pale-coloured) and historical biogeography (Yang & Liu, 2010; Liu et al.,

Fig. 6. Habitus photos of Neoneuromus spp. (A) Neoneuromus ignobilis Navás, male, from Guangxi; (B) Neoneuromus ignobilis Navás, male, from southern Vietnam; (C) Neoneuromus latratus (McLachlan), male, from northeastern India; (D) Neoneuromus sikkimmensis (McLachlan), holotype male, from Sikkim; (E) Neoneuromus coomani Lestage, neotype male, from northern Vietnam; (F) Neoneuromus coomani Lestage, male, from northern Vietnam. Scale bar: 5.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

2008, 2010, 2012b, 2015b). These studies indicated an Indian northeastern India eastward to Indochina and the western part origin for the Oriental Corydalidae, with northeastern India and of Central and South China) is proposed at the basalmost node adjacent areas, specifically, being the diversification centre for (node 9 in Fig. 4) in this analysis. This result implies that the this group. Moreover, the common ancestors of some genera, Indian-origin hypothesis cannot be refuted for Neoneuromus or such as Nevromus and Neurhermes, are thought to have greatly for other Oriental corydalids. However, the ancestral species expanded their ranges due to dispersal in the southern part of might not have had to be introduced from Gondwana with Eurasia during the Eocene (Liu et al., 2012a, 2015b). the northward drifting of the Indian subcontinent, which was Our Neoneuromus chronogram based on molecular data pro- assumed to be the origin of the common ancestor of the Asian vides insight to further understand the divergence pattern and Corydalidae (Penny, 1993), as many Asian endemic corydalid historical biogeography of this genus. Our study also allows genera are estimated to have diverged during the Early Creta- further testing of the aforementioned hypothesis on the bio- ceous when the Indian subcontinent had not yet collided with geography of Oriental Corydalidae. First, our results indicate Eurasia (Yang et al., 2017). that the common ancestor of Neoneuromus was widely dis- Moreover, the time interval ranging from the late Eocene to tributed in northeastern India and northern Indochina (with adja- the mid-Miocene is demonstrated here to be an important period cent areas northwards), which is consistent with the presence for the diversification of Neoneuromus. Remarkably, northern of widespread ancestral species of some other Oriental dobson- Indochina and its adjacent areas to the north (i.e. area B in Fig. 4) fly genera (Liu et al., 2012a, 2015b). Moreover, the diversifica- was reconstructed as the ancestral area for many deep-level tion offset of Neoneuromus during the mid-Eocene, as indicated nodes, and this area could potentially have been a diversification by our results, also coincides well with the estimated time of centre for most species of Neoneuromus. It is also noteworthy the early divergence of other Oriental dobsonflies and fishflies that dispersal across many areas, as indicated in this study, might (Liu et al., 2010, 2012b, 2015b). Superficially, it appears that have taken place more frequently during the early divergence the Indian origin of Neoneuromus is not corroborated. How- of Neuneuromus than previously thought. Indeed, a dispersal ever, a dispersal event from A to BC or BCD (see Fig. 4) (from route from area B to E (Fig. 4) (from northern Indochina to

Fig. 7. Neoneuromus indistinctus sp.n. (A) Habitus photo, holotype male, from southeastern Yunnan; (B) male genitalia, dorsal view; (C) male genitalia, ventral view; (D) male gonocoxites + gonostyli 10, ventral view; (E) female genitalia, lateral view; (F) distribution map. as, anal sclerite; c, callus cercus; e, ectoproct; gx, gonocoxite; gst, gonostylus; if, internal fossa; T, tergum; S, sternum. Scale bar: (A) 5.0 mm; (B–E) 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 8. Neoneuromus maculatus sp.n. (A) Habitus photo, holotype male, from central Vietnam; (B) male genitalia, dorsal view; (C) male sternum 9, ventral view; (D) male gonocoxites + gonostyli 10, ventral view; (E) female genitalia, lateral view; (F) distribution map. Scale bar: 5.0 mm (A), 1.00 mm (B–E). [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 9. Neoneuromus niger sp.n. (A) Habitus photo, holotype male, from central Vietnam; (B) male genitalia, dorsal view; (C) male genitalia, ventral view; (D) male gonocoxites + gonostyli 10, ventral view; (E) distribution map. Scale bar: (A) 5.0 mm; (B–D) 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 10. Neoneuromus similis sp.n. (A) Habitus photo, holotype male, from Zhejiang; (B) habitus photo, paratype male, from Guangdong; (C) male genitalia, dorsal view; (D) male genitalia, ventral view; (E) male gonocoxites + gonostyli 10, ventral view; (F) female genitalia, lateral view; (G) distribution map. Scale bar: (A–B) 5.0 mm; (C–F) 1.0 mm . [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 11. Neoneuromus vanderweelei sp.n. (A) Habitus photo, holotype male, from western Yunnan; (B) habitus photo, paratype male, from northern Vietnam; (C) male genitalia, dorsal view; (D) male genitalia, ventral view; (E) male gonocoxites + gonostyli 10, ventral view; (F) female genitalia, lateral view; (G) distribution map. Scale bar: (A–B) 5.0 mm; (C–F) 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

© 2018 The Royal Entomological Society, Systematic Entomology, 43, 568–595 Neoneuromus systematics and phylogeny 585 the eastern part of Central and South China) is repeatedly Conclusions recovered. In addition, dispersal events from northern Indochina to southeastern Indochina (area C), the western part of Central Here we present the first DNA sequence-based phylogeny of the and South China (area D), and even to northeastern India and its Oriental endemic dobsonfly genus Neoneuromus, a remarkable adjacent regions (area A) were recovered in this study, and these group of Megaloptera with extremely polymorphic body and might be involved with certain speciation events. Extant species wing colouration patterns. Using an integrative approach, the of Neoneuromus are preferentialy found inhabiting relatively species diversity of Neoneuromus is thoroughly explored, and low-elevation regions, which may have enabled more frequent we identify 13 species in the clade, almost half of which are long-distance dispersals, resulting in the wide distributions of described here as new. The diversified body and wing coloura- many species, such as N. ignobilis and N. orientalis, among tion patterns among different species, as well as within species, others. This habitat preference might also have been present could be rapidly evolved, and they are thought to be associ- in ancestral species of Neoneuromus, which may explain the ated with the recognition of conspecific mates during courtship aforementioned ancient dispersal events. in the presence of other sympatrically distributed congeneric Additionaly, vicariance events are hypothesized to be the cause of the divergence of eight species, including the split of species. Finally, our chronogram and reconstructed ancestral N. coomani and N. sikkimmensis, the split of N. similis sp.n., areas suggest that the initial divergence of Neoneuromus took N. maclachlani and N. maculatus sp.n., and the split of N. place in a broad area including northeastern India and northern latratus, N. indistinctus sp.n. and N. niger sp.n.. Within these Indochina during the mid-Eocene, followed by a major diversi- species, N. sikkimmensis and N. latratus are mainly distributed fication series from the late Eocene to the mid-Miocene. Finally, in northeastern India (area A, Fig. 4), while N. maculatus sp.n. we demonstrate that complex dispersal and vicariance events and N. niger sp.n. are confined to southeastern Indochina from southeastern Asia shaped the present diversity and distri- (area C). Considering such distribution patterns, the geographic bution patterns of Neoneuromus. isolations causing the specific divisions of these species and their close relatives are probably correlated to the formation of some mountain ranges. More specifically, the Arakan Mountains Supporting Information or the paralleled distributed mountains in nearby northeastern Additional Supporting Information may be found in the online India and northwestern Myanmar could have been involved in the vicariant speciation of N. sikkimmensis and N. latratus. version of this article under the DOI reference: In southeastern Indochina, the southern part of the Annamite 10.1111/syen.12287 Range, which is the main mountain range in Indochina, is Figure S1. Neighbour-joining tree of Neoneuromus based likely to account for the isolation of N. maculatus sp.n. and on DNA sequences of each molecular marker, i.e. COI-1, N. niger sp.n.. Notably, these geographic barriers are found COI-2, ND2, and 16S rRNA. Numbers at the nodes are to be the causal factor, not only of the interspecific divisions bootstrap values. Red lines indicate the 15 groups (species discussed earlier, but also of some intraspecific divisions, such or putative species) as shown in Fig. 2. Numbers beside red as the subdivisions within N. coomani, N. vanderweelei sp.n., lines: 1, N. fenestralis;2,N. ignobilis;3,N. latratus;4,N. N. orientalis and N. ignobilis. maclachlani A; 5, N. maclachlani B; 6, N. maclachlani C; 7, Lastly, the diversification leading to the present fauna and distribution patterns of Neoneuromus apparently underwent com- N. maclachlani D; 8, N. maculatus sp.n.;9,N. niger sp.n.; plex processes featured by multiple dispersal and vicariance 10, N. orientalis A; 11, N. orientalis B; 12, N. sikkimmensis events, mainly in the southeastern part of Asia. An overall east- A; 13, N. sikkimmensis B; 14, N. sikkimmensis C; 15. N. ward and northward dispersal from northeastern India and north- tonkinensis. ern Indochina may account for the present distribution range Table S1. List of species sampled in phylogenetic analysis of the whole genus. Moreover, vicariance might have played and GenBank accession number. an important role driving the speciation of many Neoneuro- mus species. The orogeny caused by the collision of the Indian Table S2. Primers used for PCR and sequencing. subcontinent into Eurasia probably generated some geographic Table S3. The partitioning schemes and selected nucleotide barriers, such as the Arakan Mountains, causing speciation in Neoneuromus. There must have been many other geographic substitution models for phylogenetic analysis. barriers associated with the vicariant speciation of Neoneuro- Table S4. Genetic divergence among and within the 15 mus, but they remain undetected due to the rather limited knowl- groups (i.e. species or putative species) based on the edge of the geological history of the region where Neoneuromus sequences of COI-2. species occur. Apart from the geological processes, climatic conditions, such as the Pleistocene glaciations, could also have been Table S5. Divergence time estimates for nodes with fossil a factor shaping the present distribution patterns of Neoneu- calibrations in chronogram (Fig. 4), showing median node romus. These climatic events should be further investigated in heights and the 95% high posterior density (HPD) interval. future studies. Nodal age unit is in Ma.

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weapons and nuptial gifts? Proceedings of the Royal Society B: van der Weele, H.W. (1909) New genera and species of Megaloptera Biological Sciences, 282, 20150247. Latr. Notes from the Leyden Museum, 30, 249–264. Liu, X.Y., Hayashi, F. & Yang, D. (2015b) Systematics and bio- van der Weele, H.W. (1910) Megaloptera monographic revision. Collec- geography of the dobsonfly genus Neurhermes Navás (Megalopera: tions Zoologiques du Baron Edm. de Selys Longchamps, 5, 1–93. Corydalidae: Corydalinae). Arthropod Systematics & Phylogeny, 73, Yang, D. & Liu, X.Y. (2010) Fauna Sinica, Insecta, Megaloptara, Vol. 41–63. 51. Science Press, Beijing. Liu, X.Y., Lü, Y.N., Aspöck, H., Yang, D. & Aspöck, U. (2016) Homol- Yang, F., Jiang, Y.L., Yang, D. & Liu, X.Y. (2017) Mitochondrial ogy of the genital sclerites of Megaloptera (Insecta: Neuropterida) genomes of two Australian fishflies with an evolutionary timescale and their phylogenetic relevance. Systematic Entomology, 41, of Chauliodinae. Scientific Reports, 7, 4481. 256–286. Yiu, V. (2008) Brief notes on dobsonflies and fishflies (Megaloptera) of McLachlan, R. (1869) Chauliodes and its allies with notes and descrip- Hong Kong. Hong Kong Discovery, 44, 39–44. tions. The Annals and Magazine of Natural History, 4, 35–46. Yu, Y.,Harris, A.J. & He, X.J. (2014) RASP (Reconstruct Ancestral State Navás, L. (1929 (1928)) Comunicaciones entomológicas. 10. Insectos de in Phylogenies) 2.1b. URL http://mnh.scu.edu.cn/soft/blog/RASP. la India. 1.a serie. Revista de la [Real] Academia de Ciencias Exactas [accessed on 2014]. Fisico-Quimicas y Naturales de Zaragoza, 12, 177–197. Yue, L., Liu, X.Y., Hayashi, F., Wang, M.Q. & Yang, D. (2015) Navás, L. (1932) Decadas de insectos nuevos. Brotéria (Zoológica), 1, Molecular systematics of the fishfly genus Anachauliodes Kimmins, 145–155. 1954 (Megaloptera: Corydalidae: Chauliodinae). Zootaxa, 3941, Needham, J.G. (1909) Notes on the Neuroptera in the collection of the 91–103. Indian Museum. Records of the Indian Museum, 3, 185–210. Zhang, Y.Z. (1999) Zoogeography of China. Science Press, Beijing. Ôuchi, Y. (1939) Note on a supposed female of Corydalis orientalis Zhang, J.J., Kapli, P., Pavlidis, P. & Stamatakis, A. (2013) A general MacLachlan and a new species description belongs to Gen. Corydalis, species delimitation method with applications to phylogenetic place- Corydalidae, Megaloptera. The Journal of the Shanghai Science ments. Bioinformatics, 29, 2869–2876. Institute, 4, 227–232. Pagel, M., Meade, A. & Barker, D. (2004) Bayesian estimation of Accepted 5 February 2018 ancestral character states on phylogenies. Systematic Biology, 53, 673–684. Penny, N.D. (1993) The phylogenetic position of Chloroniella peringueyi (Megaloptera: Corydalidae) and its zoogeographic Appendix: Taxonomy of Neoneuromus significance. Entomological News, 104, 17–30. Puillandre, N., Lambert, A., Brouillet, S. & Achaz, G. (2012) ABGD, Genus Neoneuromus van der Weele Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21, 1864–1877. Neoneuromus van der Weele, 1909: 252. Type species: Neuro- Rambaut, A. (2009) FigTree Version 1.3. 1. Computer program dis- mus fenestralis McLachlan, 1869: 42, subsequently designated tributed by the author. [WWW document]. URL http://tree bio ed ac by van der Weele, 1910: 24. uk/software/figtree/. [accessed on 4 January 2011]. Rambaut, A. & Drummond, A.J. (2007) Tracer v1.4. URL http://beast .bio.ed.ac.uk/Tracer. [accessed on 2007]. Diagnosis. Large-sized (forewing length 43–68 mm). Body Ree, R.H. & Smith, S.A. (2008) Maximum likelihood inference of geo- yellow, yellowish brown, or reddish brown, usually with black graphic range evolution by dispersal, local extinction, and cladogen- markings on head and pronotum, but sometimes entirely black- esis. Systematic Biology, 57, 4–14. ish brown. Head subqudrate, flattened, with acutely pointed Ree, R.H., Moore, B.R., Webb, C.O. & Donoghue, M.J. (2005) A likelihood framework for inferring the evolution of geographic range postocular spines; clypeal margin slightly incised medially. on phylogenetic trees. Evolution, 59, 2299–2311. Antennae filiform, nearly as long as head plus prothorax. Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phy- Labrum ovoid, with anterior margin sparsely setose. Wings logengtic inference under mixed models. Bioinformatics, 19, with various dark marking patterns. RP nine to 12-branched; 1572–1574. MA bifurcated; four to seven crossveins between RA and Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new RP; anterior branch of MP three- to eight-branched, posterior method for reconstructing phylogenetic trees. Molecular Biology and branch of MP two-branched; A1 two-branched. Male tergum Evolution, 4, 406–425. 9 subtrapezoidal, anteriorly with a median internal fossa, pos- Stamatakis, A., Hoover, P. & Rougemont, J. (2008) A rapid bootstrap algorithm for the RAxML Web servers. Systematic Biology, 57, terolaterally produced into short arm, which bears unguiform 758–771. gonostylus 9; sternum 9 attenuate with tip more or less incised Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & (an important character for distinguishing this genus from Nevr- Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis mus which is the most closely related genus of Neoneuromus); using maximum likelihood, evolutionary distance, and maxi- ectoproct clavate with posterior half moderately or strongly mum parsimony methods. Molecular Biology and Evolution, 28, inflated; fused gonocoxites 10 present as a broad plate, medially 2731–2739. produced posteriad, usually with a pair of digitiform gonostyli Vaidya, G., Lohman, D.J. & Meier, R. (2010) SequenceMatrix: 10; a sclerite present around anus. Female fused gonocox- concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics, 27, ites 8 broad, with concaved posterior margin; gonocoxite 9 171–180. valvate with a cuitellate lateral sclerite; gonostylus 9 mostly van der Weele, H.W. (1907) Notizen uber Sialiden und Beschreibung fused with tip of gonocoxite 9, proximally lacking articulate einiger neuen Arten. Notes from the Leyden Museum, 28, 227–264. connection.

Distribution. China, India, Laos, Myanmar, Thailand, interrupted anteriorly; China (southern Sichuan and Yunnan), Vietnam. Laos, Thailand and Vietnam ...... N. coomani Lestage – Head yellowish brown, usually with broad black markings laterally, but sometimes black markings greatly reduced (Fig. 5E, F); pronotum with lateral black vittae not interrupted anteriorly; Key to species of the genus Neoneuromus southern China and northern Vietnam ...... N. orientalis Liu&Yang. 1. Head entirely reddish brown to blackish brown (if head 10. Head yellow to yellowish brown, only with narrow black yellowish brown, large black markings present on vertex, and postocular margins (Fig. 6D); pronotum with lateral black vittae forewing with distal half distinctly darkened); a distinct dark usually interrupted anteriorly; forewing with distal half feebly marking present on forewing 1mp-cua crossvein (Fig. 5A) . . . . darkened...... 11 ...... 2 – Head yellow to reddish brown, usually with broad black – Specieslackingoneorallofabovecharacters...... 6 markings laterally (if black markings reduced, forewing with 2. Forewing with distal half not entirely darkened, but having a distal half entirely brown) (Fig. 6A); pronotum with lateral black number of transparent patches (Fig. 8A); central Vietnam .... vittae usually not interrupted anteriorly (if lateral black vittae ...... N. maculatus Liu, Hayashi & Yang, sp.n. anteriorly interrupted, forewing with distal half entirely brown); – Forewing with distal half almost entirely darkened (Fig. 5B) forewing usually with distal half distinctly darkened (if forewing ...... 3 with distal half relatively pale, head laterally with broad black 3. Clypeus largely yellowish brown (Fig. 5C); male sternum 9 markings or longitudinal black bands) ...... 12 posterolaterally with a pair of short projections but medially 11. Male sternum 9 with apex nearly as wide as anal sclerite truncate at tip (File S2: Fig. 8B); southern China and northern (File S2: Fig. 7A); male ectoproct with apex distinctly inflated Vietnam ...... N. tonkinensis (van der Weele). (File S2: Fig. 7A); China (Yunnan), northeastern India, and – Clypeus reddish brown to blackish brown; tip of male northern Myanmar ...... N. sikkimmensis (van der Weele). sternum 9 medially concaved, or medially truncate but without – Male sternum 9 with apex slightly narrower than anal sclerite posterolateralprojections...... 4 (Fig. 7B); male ectoproct with apex not inflated (Fig. 7B); 4. Male sternum 9 truncate at tip, without posterolateral pro- China (Yunnan), Laos, and northern Vietnam ...... jections (File S2: Fig. 2B); male gonostyli 10 absent (File S2: ...... N. indistinctus Liu, Hayashi & Yang, sp.n. Fig. 2C); southwestern China (western Yunnan and south- 12. Head yellow to yellowish brown, laterally with broad eastern Tibet), northeastern India and northern Myanmar black markings or longitudinal black bands, occasionally black ...... N. fenestralis (McLachlan) markings absent (Fig. 6A, B); male sternum 9 distinctly incised – Male sternum 9 medially concaved at tip; male gonostyli 10 medially at tip, leaving a pair of short digitiform projections present...... 5 (File S2: Fig. 3B); male gonostylus 10 present on a relatively 5. Male ectoproct swollened about from proximal 1/4 (File long, digitiform projection (File S2: Fig. 3C); central and S2: Fig. 5A); China (Chongqing, Guangxi, Guizhou, Sichuan southern China, Laos and Vietnam ...... N. ignobilis Navás and northeastern Yunnan) ...... – Head yellowish brown, laterally with black markings reduced, ...... N. maclachlani (van der Weele) or reddish brown, laterally with broad black markings (Fig. 10A, – Male ectoproct swollen from about midpoint (Fig. 11C); B); male sternum 9 feebly incised medially at tip, leaving a pair China (southeastern and southwestern Yunnan) and north- of broad and rounded projections (Fig. 10D); male gonostylus ern Vietnam ……N. vanderweelei Liu, Hayashi & Yang, sp.n. 10 present on a short subtriangular projection (Fig. 10E); cen- 6. Head and pronotum mostly blackish brown; abdomen entirely tral and southern China ...... yellowish brown (Fig. 9A); central Vietnam ...... N. similis sp.n...... N. niger Liu, Hayashi & Yang, sp.n...... Liu, Hayashi & Yang, – Head and pronotum yellowish brown to reddish brown, usually with black markings laterally, and abdomen pale brown Neoneuromus coomani Lestage to blackish brown, or head only with narrow black lateral (File S1, File S2: Fig. 1, File S3: Fig. 1) margins, and abdomen yellowish brown ...... 7 Neoneuromus coomani Lestage, 1927: 111. Type locality: 7. Forewing with a distinct dark marking on 1mp-cua crossvein Vietnam...... 8 – Forewing without dark marking on 1mp-cua crossvein ...... 10 Diagnosis. Head yellow to pale reddish brown, with postoc- 8. Forewing with distal half dark (Fig. 6C); China (western ular margins and spines black. Pronotum yellow to pale reddish Yunnan) and northeastern India ...... brown, with a pair of short anterolateral black markings and ...... N. latratus (McLachlan). a pair of long lateral black vittae, sometimes the anterolateral – Forewing with distal half not distinctly darkened, but with marking fused with the posterolateral one. Forewing with distal darkspotsonmanycrossveins...... 9 half feebly darkened, dark marking on 1mp-cua usually present. 9. Head yellowish brown, only with narrow black postocular Male sternum 9 with apex strongly narrowed and feebly incised margins (Fig. 6E); pronotum with lateral black vittae usually medially at tip, forming a pair of obtuse projections; male

© 2018 The Royal Entomological Society, Systematic Entomology, 43, 568–595 Neoneuromus systematics and phylogeny 589 ectoproct strongly inflated distad; male gonocoxites 10 with Distribution. China (Yunnan); Myanmar (Chin); India short subtriangular lateral projections and short gonostyli 10. (Arunachal Pradesh, Sikkim, West Bengal).

Distribution. China (Sichuan, Yunnan); Laos (Attapeu, Remarks. In appearance, this species is similar to N. maclach- Champasak, Bokeo, Hua Phan, Savannakhet, Xieng Khouang); lani by the dark body colour and forewing marking patterns. Thailand (Chiang Mai); Vietnam (Hoa Binh, Lao Cai, Quang The latter species was originally described as a subspecies of Nam, Son La, Vinh Phuc). N. fenestralis (van der Weele, 1907). However, based on the male genitalia, N. fenestralis can be easily distinguished from all other Neoneuromus species by the sinuate inner margin of Remarks. This species was described by Lestage (1927) the ectoproct, the sternum 9 with truncate tip, and the absence based on a single specimen from northern Vietnam. Unfortu- of gonostyli 10. We found a few specimens of N. fenestralis nately, Lestage (1927) did not provide any figure of this species from Myanmar to be slightly darker on head and pronotum than in the original description, and the type which was reported the other specimens examined, while such variation should be to be deposited in the Lestage’s personal collection cannot intraspecific variation as the genital characters of all examined be found. Based on the original description (Lestage, 1927) specimens are the same. of N. coomani, the head is mostly immaculate except for the Corydalis territans was originally described by Needham black postocular margin, the pronotum bears two pairs of black (1909) based on a single female specimen from Sikkim, and markings (a small dot and a longitudinal stripe), the forewing it was placed into the genus Neoneuromus by Lestage (1927) is not distinctly darkened on the distal half but with dark sports according to the morphological characters and the distribution on some crossveins, and the abdomen is generally dark. The in Asia. The Needham’s type has not been found by far. combination of these characters can be found in all specimens However, by the original description of Needham (1909), the we examined and tentatively identified as N. coomani. Probably, morphological features of this species are quite similar to N. it is unlikely to find the primary type of N. coomani that might fenestralis. Ghosh (1991) reported a male of C. territans and have been lost or damaged, so here we designate a neotype that made a description on its genitalia, which is indeed identical to was also from northern Vietnam for this species. This species the male genitalia of N. fenestralis. Therefore, C. territans is resembles N. latratus and N. sikkimmensis, which was also probably conspecific with N. fenestralis, and herein we treated mentioned by Lestage (1927), by having similar head with it as a junior synonym of N. fenestralis. We also synomized reduced black markings and wings feebly darkened distad. Neoneuromus fenestralis var. zurbitui Navás which is distributed However, N. coomani can be distinguished from N. latratus from northeastern India because we did not find any specific and N. sikkimmensis by the male sternum 9 with strongly morphological difference to support it separated as a form or narrowed apex. Moreover, this species appears also similar to subspecies from N. fenestralis. N. indistinctus sp.n., but it can be distinguished from the latter species by the presence of dark marking on forewing 1mp-cua, the feebly incised apex of male sternum 9, and the strongly Neoneuromus ignobilis Navás inflated apex of male ectoprocts. It is notable that the specimens (File S1, File S2: Fig. 3, File S3: Fig. 3) of N. coomani from central Vietnam and southern Laos lack the Neonuromus ignobilis Navás, 1932: 147. Type locality: China dark marking on forewing 1mp-cua, and our molecular data also (Sichuan: Emeishan). suggest distinct genetic divergence of these specimens from the Corydalis huangshanensis Ôuchi, 1939: 230. Type locality: others. However, considering the identical genital characters China (Anhui: Huangshan). between these varied individuals, we herein identified them to be same species, but probably with geographical variation. Diagnosis. Head and prothorax yellow to yellowish brown, laterally with a pair of black markings on vertex (occasionally Neoneuromus fenestralis (McLachlan) absent) and a pair of black vittae on pronotum. Legs blackish (File S1, File S2: Fig. 2, File S3: Fig. 2) brown, with ventral portion of femora yellow. Wings with distal Neuromus fenestralis McLachlan, 1869: 42. Type locality: India half smoky brown, but forewing with a subquadrate hyaline (West Bengal: Darjeeling). area near proximal border of distal dark area; dark marking Corydalis territans Needham, 1909: 193. Type locality: India on forewing 1mp-cua absent. Male sternum 9 distinctly incised (Sikkim). syn.n. medially at tip, leaving a pair of short digitiform projections. Neoneuromus fenestralis var. zurbitui Navás, [1929] 1928: 194. Male fused gonocoxites 10 with long lateral arms and rather Type locality: India (Himalayas: Kurseong). syn.n. narrow apex; lateral projections narrow, long; gonostyli 10 present. Diagnosis. Body colouration pale reddish brown or blackish brown. Forewing with dark brown markings, among which two Distribution. China (Anhui, Chongqing, Gansu, Guangxi, broad hyaline areas are present; marking on 1mp-cua present. Guizhou, Hubei, Hunan, Jiangxi, Shaanxi, Sichuan, Yunnan, Male ectoproct with sinuate inner margin, sternum 9 with Zhejiang); Laos (Bokeo, Champasak, Hua Phan, Savannakhet, truncate tip, and gonostyli 10 absent. Xieng Khouang); Vietnam (Cao Bang, Ha Noi, Ha Tinh, Kon

Tum, Lai Chau, Lam Dong, Lang Son, Lao Cai, Nghe An, Son and suboval internal fossa, posterior margin truncate. Sternum 9 La, Thua Thien Hue). narrow and attenuate, with a pair of narrow longitudinal internal ridges, apex much narrower than anal sclerite, distinctly incised Remarks. This is one of the most common dobsonfly species medially at tip, leaving a pair of short digitiform projections. in Asia. Generally, there are two colour forms in this species. Gonostylus 9 unguiform. Ectoproct clavate and barely inflated The form I includes the individuals having broad vertexal black distad, with tip slightly curved ventrad. Fused gonocoxites 10 markings and forewing with distal half distinctly darkened strongly sclerotized, with long lateral arms and broad, truncate (Fig. 6A), and the form II includes those having narrow or apex; lateral projections subtrapezoidal; gonostyli 10 slenderly reduced vertexal black markings and forewing with distal half digitiform. slightly darkened (Fig. 6B). The distribution areas of the two Female. Body length 56–60 mm; forewing length forms also vary as the form I is from central and southeastern 59–65 mm, hindwing length 53–59 mm. China but the form II is from Indochina and its northern Fused gonocoxites 8 subquadrate, laterally with a pair of nar- bordering regions (e.g. Yunnan and southern Guangxi). row but strongly sclerotized areas, posterior margin shallowly and arcuately incised. Gonocoxite 9 broadly subtrapezoidal; gonostylus 9 rather small, slenderly digitiform. Ectoproct with Neoneuromus indistinctus Liu, Hayashi & Yang, sp.n. thick digitiform dorsal and relatively shorter, ovoid ventral lobe. http://zoobank.org/urn:lsid:zoobank.org:act:2F01013B-B3F7- 4D3B-A5A5-309D4ACBD4ED (Fig. 7) Materials examined. Holotype ♂, ‘China, Yunnan, Lvchun, Huanglianshan, Qimaba, 9.vi.2013, J. Zhang’ (CAU). Diagnosis. Head yellow, with only postocular margins and Paratypes: CHINA: Yunnan:1♀, Mangshi, Mukang, postocular spines black; pronotum yellow, laterally with a pair 14.vii.2013, W. Zhang (CAU); 1♂, Lvchun, Huanglianshan, of black vittae that are sometimes interrupted anteriorly, and Qimaba, 11.vi.2013, J. Wang & J. Yang (CAU); 1♀, Lvchun, anterolaterally with a pair of small black spots aside black vittae. Huanglianshan, Qimaba, 9.vi.2013, J. Zhang (CAU); 1♂2♀, Legs blackish brown, with femora mostly yellow. Forewing Lvchun, Huanglianshan, Qimaba, 24/25.vii.2013, M. Tan slightly smoky brown on distal half, lacking dark marking on (CAU). CHINA: Yunnan:1♂, [no locality data, but proba- 1mp-cua. Male sternum 9 with apex much narrower than anal bly collected from Lvchun, Yunnan] (CAU); 1♀, Menglun, sclerite, distinctly incised medially at tip, leaving a pair of short Zhiwuyuan, 26.ix.2006, H. Wang (CAU); 1♂7♀, Mengla, digitiform projections. Male fused gonocoxites 10 with long Shangyong, Longmen, 10.viii.2010, W. Li (CAU); 2♀, Mengla, lateral arms and narrow apex, lateral projections subtrapezoidal, Yaoqu, 6.viii.2010, H. Yu (CAU); 1♂2♀, Yunjinghong, Menglun gonostyli 10 present. Tropical Botanical Garden, Diaoqiao, 6.x.2014, C. Wu (CAU); LAOS: Hua Phan:1♂, Sam Neua, 970 m, 4.vii.2007, Y.Nakase Description. Male. Body length 47–51 mm; forewing length (HFC). 46–54 mm, hindwing length 42–48 mm. Head yellow, with postocular margins and spines black. Distribution. China (Yunnan); Laos (Hua Phan). Occiput respectively with a pair of black lateral markings dorsad and ventrad. Compound eyes brown, ocelli yellow, medially Etymology. The specific epithet ‘indistinctus’referstothe margined black. Antenna black; scape and pedicel brown with inconspicuous characteristics of the new species in comparison base and tip yellow. Mouthparts yellow; mandible black with to its similar species, such as N. sikkimmensis and N. latratus. distal half reddish brown; maxillary and labial palpi with distal two or three segments black. Prothorax yellow, pronotum laterally with a pair of black vit- Remarks. This species is quite similar to N. latratus, N. tae that are sometimes interrupted anteriorly, and with a pair of coomani and N. sikkimmensis in appearance, although it is anterolateral black spots beside black vittae. Meso- and metatho- assigned to be the sister species of N. niger sp.n. in the present rax yellow. Legs blackish brown, except for major part of femora phylogenetic analysis. It can be distinguished from N. latratus and lateral part of tibiae yellow; tarsal claws reddish brown. and N. coomani by the absence of dark marking on forewing Forewing hyaline, slightly smoky brown on distal half, medially 1mp-cua and the barely inflated apex of male ectoprocts. It with a subquadrate hyaline area proximally bordered by brown- differs from N. sikkimmensis by the strongly narrowed apex of ish marking on crossveins between MP and CuA, no dark spot male sternum 9, the barely inflated apex of male ectoprocts, on 1mp-cua crossvein, but some veins darkened, including ra-rp, and the male gonocoxites 10 with distinct lateral projections cua-cup, cup-1a, bases of CuA branches and stem of CuP. and long lateral arms. Finally, the new species can be easily Hindwing much paler than forewing, with tip slightly smoky separated from N. niger sp.n. by the yellow head and pronotum brown. Veins yellowish brown except for aforementioned dark- as well as the pale wings. ened veins. RP nine- or ten-branched; MA bifurcated; five or six crossveins between RA and RP; anterior branch of MP five- or Neoneuromus latratus (McLachlan) six-branched, posterior branch of MP two-branched. (File S1, File S2: Fig. 4, File S3: Fig. 4) Abdomen yellowish brown, slightly darker laterally. Tergum Neuromus latratus McLachlan, 1869: 43. Type locality: India 9 subtrapezoidal, anterior margin with deeply arched incision (India orientali).

Diagnosis. Head yellow to reddish brown, with a pair of small Description. Male. Body length 40–44 mm; forewing length black spots near antennal fossae, and usually with postocular 39–45 mm, hindwing length 36–40 mm. margins and postocular spines black; pronotum yellow to red- Head mostly black; clypeus medially with an orange subtrian- dish brown, laterally with a pair of black vittae, and anterolat- gular marking. Compound eyes greyish brown, ocelli dark red- erally with a pair of small black spots aside black vittae. Legs dish brown. Antenna black throughout. Mouthparts dark orange; blackish brown, with femora mostly yellow to reddish brown. mandible black with tips reddish brown; maxillary and labial Forewing slightly smoky brown on distal half, a dark marking palpi respectively with distal three segments black. present on 1mp-cua. Male sternum 9 distinctly incised medially Thorax mostly black, ventrally somewhat yellowish on the at tip, leaving a pair of short digitiform projections. Male fused joints among mesothorax, metathorax, wing bases and legs. gonocoxites 10 with long lateral arms and broad apex, lateral Legs black, with dense brownish setae; tarsal claws reddish projections subtrapezoidal, gonostyli 10 present. brown. Wings hyaline, with many blackish markings. Forewing with narrow stripes on proximal costal crossveins, and with Distribution. India (Arunachal Pradesh, Manipur); Myanmar numerous subquadrate markings on other crossveins, forming (Kachin). two transverse bands, which are bordered by three hyaline areas; wing apex blackish brown; proximal anal area hyaline. Hindwing with proximal half hyaline, medially with a few Remarks. This species is currently known only from north- markings on crossveins, and distally blackish brown. Veins eastern India and northern Myanmar. In appearance, this species blackish brown, with veins on proximal half of hindwings much looks similar to N. sikkimmensis, but can be distinguished from paler. RP nine- to 11-branched; MA bifurcated; five to seven the latter species by the presence of dark marking on forewing crossveins between RA and RP; anterior branch of MP four- to 1mp-cu and the male ectoproct strongly inflated on distal half. In six-branched, posterior branch of MP two-branched. N. sikkimmensis the forewing lacks the dark marking on 1mp-cu Abdomen blackish brown with venter yellowish brown, but and the male ectoproct is feebly inflated on distal half. The bright yellow on joint between pregenital segments. Tergum 9 present molecular phylogeny suggests its close relationships to subquadrate, with arched anterior incision and ovoid internal N. indistinctus sp.n. and N. niger sp.n. (see Remarks of N. indis- fossa. Sternum 9 attenuate, with a pair of narrow longitudi- tinctus sp.n.). nal internal ridges, distally with arched incision, leaving a pair of short digitiform projections. Gonostylus 9 unguiform. Ecto- Neoneuromus maclachlani (van der Weele) proct clavate, distal half distinctly inflated, with apex dorsally (File S1, File S2: Fig. 5, File S3: Fig. 5) depressed and curved ventrad. Fused gonocoxites 10 arched; Neuromus fenestralis maclachlani van der Weele, 1907: 241. median plate distinctly produced with truncate tip, laterally Type locality: China (Sichuan: Emei-shan). without projection; gonostyli 10 slenderly digitiform. Female. Body length 42–47 mm; forewing length 47–49 mm, hindwing length 45–48 mm. Diagnosis. Body reddish brown or blackish brown. Wings Fused gonocoxites 8 in lateral view subquadrate, with medial with distal half brown; forewing with a dark marking on portion slightly narrowed; posterior margin medially incised. 1mp-cua. Male ectoproct relatively short, strongly inflated from Gonocoxite 9 valvate and subtriangular, distally with small proximal 1/4, distally with an ovoid depression. Male fused gonostylus 9. Ectoproct short, posteriorly incised, leaving thick gonocoxites 10 with a pair of short subtriangular lateral projec- digitiform dorsal and suboval ventral lobes. tions, gonostyli 10 present.

Materials examined. ♂ VIETNAM Distribution. China (Chongqing, Guangxi, Guizhou, Holotype ,‘ : Thua Thien Hue, Bach Ma, 9.v.2003’ (CAU). Paratypes: VIETNAM: Thua Sichuan, Yunnan). Thien Hue:2♂3♀, Bach Ma, 5–9.v.2003 (CAU & HFC); 1♂, Bach Ma Nature Reserve, surr. Hotel Morin, 1350–1400 m., Remarks. See Remarks of N. fenestralis. 23–28.v.2014, L. Bartolozzi, G. Chelazzi, A. Bandinelli, S. Bambi & F. Fabiano (MSNF); Kon Tum:1♀, Ngoc Linh Mt., Neoneuromus maculatus Liu, Hayashi & Yang, sp.n. 1700 m, vi.2016, T. Le (LDPC); 1♀, Ko-Tich [untraceable local- http://zoobank.org/urn:lsid:zoobank.org:act:02B970F5-6FBA- ity of Vietnam], viii.1936, S. Masseyeff (NHM). VIETNAM: 4A91-93FB-E0306EF055D1 (Fig. 8) Da Nang:1♂1♀, Mt. Ba Na, 850 m, iii.2014 (HFC); Thua Thien Hue:1♂, Bach Ma, 1300 m, 8/11.vi.2002, M. Owada (NSM). Diagnosis. Body mostly black, head with an orange triangu- lar marking on clypeus. Forewing with many blackish brown markings which are bordered by three hyaline areas, dark Distribution. Vietnam (Da Nang, Kon Tum, Thua marking on 1mp-cua present; hindwing with blackish brown Thien Hue). apex and some blackish brown markings on crossveins of distal half. Male fused gonocoxites 10 with lateral projections reduced, Etymology. The specific epithet ‘maculatus’ refers to the gonostyli 10 present. remarkable wing marking pattern of ths new species.

Remarks. The new species can be distinguished from all the Distribution. Vietnam (Kon Tum, Thua Thien Hue). other Neoneuromus species by the generally black body and remarkable wing marking pattern, which has a series of hyaline Etymology. The specific epithet ‘niger’ refers to the black areas near forewing distal margin and a few scattered blackish head and pronotum as well as the thoroughly dark wings of the brown markings on distal half of hindwing. new species.

Neoneuromus niger Liu, Hayashi & Yang, sp.n. Remarks. The new species can be easily distinguished from http://zoobank.org/urn:lsid:zoobank.org:act:132CF144-BCB6- all other Neoneuromus species based on its mostly black head 420B-A017-F812DFA636ED (Fig. 9) and prothorax followed by the remaining yellowish brown body parts, and the immaculate but entirely dark wings. Diagnosis. Head and prothorax mostly black, but abdomen yellowish brown. Wings smoky brown throughout, without dis- Neoneuromus orientalis Liu&Yang tinct dark markings. Male ectoproct feebly inflated distad; fused (File S1, File S2: Fig. 6, File S3: Fig. 6) gonocoxites 10 with a pair of subquadrate lateral projections, Neoneuromus orientalis Liu & Yang, 2004: 154. Type locality: gonostyli 10 present. China (Guizhou: Liping).

Description. Male. Body length 47–48 mm; forewing length 43–45 mm, hindwing length 39–41 mm. Diagnosis. Head yellowish brown, laterally with black mark- Head black, slightly paler at middle of vertex, with a ings, but sometimes head with black markings reduced or absent. pair of subquadrate orange markings near inner margin Pronotum yellowish brown, laterally with a pair of black vittae. of compound eyes; clypeus reddish brown with yellowish Forewing with scattered brownish markings on most crossveins. margin. Compound eyes pale brown; ocelli yellow, medi- Male fused gonocoxites 10 subtrapezoid, with very short lateral ally margined black. Antenna black; scape and pedicel arms and with small but distinctly prominent lateral projections; orange. Mouthparts yellow; mandible black with tip reddish gonostyli 10 present. brown; maxillary and labial palpi with distal three segments black. Distribution. China (Anhui, Fujian, Guangdong, Guangxi, Prothorax black, pronotum posteriorly with subtriangular Guizhou, Hubei, Hunan, Jiangxi, Sichuan, Yunnan, Zhejiang); yellow marking medially. Meso- and metathorax yellow, each Vietnam (Bac Kan, Cao Bang, Hoa Binh, Lao Cai, Phu Tho, notum slightly brownish laterally. Legs yellow, with dense short Vinh Phuc). yellowish setae; tibia blackish brown except for yellow base; tarsus blackish brown; tarsal claw reddish brown. Both fore- Remarks. There are two colour forms in this species. The and hindwings smoky brown throughout, proximally slightly form I includes the individuals having broad vertexal black paler, without distinct dark markings. Veins brown, with costal markings (Fig. 5E), and the form II includes those having crossveins and crossveins on proximal half of forewing blackish reduced vertexal black markings (Fig. 5F). Nevertheless, the brown. RP 10 or 11-branched; MA bifurcated; 5 crossveins wing marking patterns and genitalia between these two forms are between RA and RP; anterior branch of MP 5 or 6-branched, the same. The distribution areas of the two forms also vary as the posterior branch of MP 2-branched. form I is from central and southeastern China but the form II is Abdomen yellow, slightly brownish dorsad. Tergum 9 sub- from northern Indochina and its northern bordering regions (e.g. trapezoidal, anterior margin with arched incision and suboval Yunnan and southern Guangxi). Our molecular data suggests internal fossa, posterior margin truncate. Sternum 9 narrow and that these two colour forms are genetically distinct from each attenuate, with a pair of narrow longitudinal internal ridges, apex other. However, considering the identical genital characters, we distinctly incised medially at tip, leaving a pair of short and still identified them to be the same species, but probably with obtuse projections. Gonostylus 9 unguiform. Ectoproct clavate, geographical variation. not inflated distad, with tip strongly curved ventrad. Fused gonocoxites 10 strongly sclerotized, with long lateral arms and concaved apex; lateral projections present, short, subquadrate; Neoneuromus sikkimmensis (van der Weele) gonostyli 10 slenderly digitiform. (File S1. File S2: Fig. 7, File S3: Fig. 7) Female. Unknown. Neuromus sikkimmensis van der Weele, 1907: 237. Type locality: India (Sikkim). Materials examined. Holotype ♂,‘VIETNAM: Thua Thien Hue Prov., Bach Ma National Park, 8.vi.2002, H. Karube’ Diagnosis. Head yellow to pale yellowish brown, with pos- (HFC). Paratypes: VIETNAM: Thua Thien Hue:1♂, Bach tocular margins and spines black. Pronotum yellow to pale yel- Ma National Park, 1200 m, 26.vii-7.viii.1996, V. Siniaev & E. lowish brown, with a pair of short anterolateral black markings Afonin (LDPC); Kon Tum:1♂, W border of Ngoc Linh Natural and a pair of long lateral black vittae. Forewing with distal Park, 20 km N Dak Glei, 900 m., 30.v.2014, L. Bartolozzi, G. half feebly darkened, dark marking on 1mp-cua absent. Male Chelazzi, A. Bandinelli, S. Bambi & F. Fabiano (MSNF). sternum 9 with apex nearly as wide as anal sclerite, feebly

© 2018 The Royal Entomological Society, Systematic Entomology, 43, 568–595 Neoneuromus systematics and phylogeny 593 incised medially at tip, forming a pair of obtuse projections; posterior margin truncate. Sternum 9 narrow and attenuate, with male ectoproct moderately inflated distad; male gonocoxites 10 a pair of narrow longitudinal internal ridges, apex shallowly with obtuse lateral projections and short gonostyli 10. incised at tip, leaving a pair of short, obtuse projections. Gonostylus 9 unguiform. Ectoproct clavate, strongly inflated Distribution. China (Yunnan); India (Arunachal Pradesh, distad from midpoint, distally with an ovoid depression and Assam, Meghalaya, Sikkim). distinctly curved ventrad. Fused gonocoxites 10 with a pair of short subtriangular lateral projections; gonostyli 10 digitiform. Female. Body length 42–63 mm; forewing length Remarks. This species in appearance resembles N. latratus 58–65 mm, hindwing length 52–59 mm. based on the head with a pair of black stripes along the postocu- Fused gonocoxites 8 subtrapezoidal in lateral view. Gonocox- lar margins and the wings with relatively indistinct markings. ite 9 valvate and subtriangular, distally with obtuse gonostylus 9. However, the overall body colouration of N. sikkimmensis is Ectoproct with dorsal lobe subtriangular, and with ventral lobe slightly paler than that of N. latratus, and the pronotal black lat- relatively longer and ovoid. eral marking is usually interrupted anteriorly in N. sikkimmensis but continuous in N. latratus. This species is also similar to N. coomani and N. indistinctus sp.n., and the comparison between Materials examined. Holotype ♂, ‘China, Zhejiang, Taishun, these species can be found in the Remarks of the latter two Wuyanling, 3.viii.2007, Yajun Zhu’ (CAU). Paratypes: CHINA: species. Anhui:1♀, Huangshan, Yungusi, 19.vii.1977, F. Li (CAU); Fujian:2♂6♀, Wuyishan, Xianfengling, 14.viii.2004, D. Zhou (CAU); Guangdong:1♂, Yingde, Shimentai, 29.viii.2005, J. Neoneuromus similis Liu, Hayashi & Yang, sp.n. Zhang (CAU); Zhejiang:1♀, Taishun, Wuyanling, 3.viii.2007, http://zoobank.org/urn:lsid:zoobank.org:act:DC287455-2D8E- Y. Z hu ( C AU) . CHINA: Anhui:2♂, Yixian, Hongcun, vi.2013 4083-B3AF-900312A48339 (Fig. 10) (CAU); Fujian:1♀, Kuatun, 2300 m, 13.vii.1938, J. Klop- perich (RMNH); 1♂1♀, Foochow, San Chiang, 1927, C. Pope Diagnosis. Head yellowish brown, posterolaterally with (NHM); 1♂1♀, Wuyishan, Xianfengling, 13.viii.2004, D. Zhou small black markings, or dark reddish brown, laterally with (NMNH); 33♂14♀, Wuyishan, Sangang, 16/20.viii.2006, H. broad black markings. Pronotum yellowish brown to dark Dong (CAU); 1♂, Wuyishan, Miaowan, 18.viii.2006, Q. Liu reddish brown, laterally with a pair of black vittae. Wings (CAU); 1♂, Wuyishan, Masu, 1250 m, 17.viii.2006, H. Dong with distal half brown. Male ectoproct strongly inflated from (CAU); 2♂, Wuyishan, Guadun, 13.viii.2006, H. Dong (CAU); midpoint, distally with an ovoid depression. Male fused gono- 16♂15♀, Wuyishan, 2009 (CAU); 14♂10♀, Wuyishan, Sangang, coxites 10 with a pair of short subtriangular lateral projections, 735 m, 9.vii.2009, L. Cao, L. Shi, X. Liu & X. Yang (CAU); 1♂, gonostyli 10 present. Wuyishan, 740–1160 m, 16/22.vii.2010, X. Liu (CAU); 6♂3♀, Wuyishan, 735 m, 12.vii.2009, L. Shi & X. Liu (CAU); 10♂10♀, Description. Male. Body length 47–54 mm; forewing length Dehua, Daiyunshan, 850 m, 11/13.vii.2010, X. Liu (CAU); 50–55 mm, hindwing length 48–50 mm. 7♂4♀, Wuyishan, Miaowan, 18.viii.2006, H. Dong (CAU); Head yellowish brown, posterolaterally with small black 3♂, Longyan, Meihuashan, 1200 m, 2/14.vi.2007, H. Huang markings, or dark reddish brown, laterally with broad black (CAU); 4♂1♀, Wuyishan, Kekaozhan, 735 m, 27.vi.2009, L. markings. Compound eyes brown; ocelli yellow, medially Shi & X. Liu (CAU); 1♂, Wuyishan, Sangang, 6.viii.1986, margined black. Antenna black; scape and pedicel with yellow- M. Xie (CAU); 1♀, Fujian, Wuyishan, 750 m, 20–30.vii.1983 ish brown tips. Labrum yellowish brown; mandible black with (CAU); 1♀, Dehua, Leifeng, Shuangfang, 3.vii.2011, X. Li apex and teeth reddish brown; maxilla yellow, except for dis- (CAU); 2♀, Dehua, Daiyuanshan, 3.vii.2011, X. Li (CAU); 2♀, tal three segments of maxillary palpus black; labium yellowish Dehua, Daiyunshan, 3.vii.2011, X. Li (CAU); Guangdong:1♀, brown except for distal three segments of labial palpus black. Yaoshan, 3.vii (MFN); 8♂1♀, Ruyuan, Nanling, 23/25.viii.2005 Prothorax yellowish brown to dark reddish brown; pronotum (CAU); 3♀, Nanling, 600–1400 m, 1–6.vi.2006, M. Takakuwa with black anterior margin and a pair of black vittae near (HFC); Jiangsu:1♀, Soochow, N. Gee (NMNH); Jiangxi:1♀, lateral margins. Meso- and metathorax brown to blackish brown, Jiulianshan, 390 m, 18.vii.1995, J. Wang (JXAU); 1♂1♀, Wuy- with lateral portions and scutellum much darker. Legs blackish ishan, 1.ix.1994, P. Zhu (JXAU); 12♂8♀, Shangrao, Wuyishan, brown, with dense reddish brown setae; coxae, trochanters, and 21.vii.2011, G. Wang (CAU); 15♂15♀, Shangrao, Wuyishan, ventral surface of femora slightly paler; tarsal claws reddish 22.vii.2011, G. Wang (CAU); 1♀, Jiangxi, Jingan, Daqishan, brown. Forewing hyaline, but dark brown on distal half, medially 16.vii.2014, K. Wang (CAU); Shaanxi:1♂, Y. Zhou (CAU); with a subquadrate hyaline area; dark marking on 1mp-cua Zhejiang:1♂, Mokanshan, 1918, N. Gee (NMNH); 1♂, Anji, absent. Hindwing brown on distal half. Veins pale brown to Longwangshan, 18.vii.1995, H. Wu (CAU); 1♂, Anji, Long- blackish brown. RP ten-branched; MA bifurcated; five to six wangshan, 18.vii.1996, Z. Wang (CAU); 1♂, Anji, Longwang- crossveins between RA and RP; anterior branch of MP six- to shan, 27.vii.1996, H. Wu (CAU); 1♀, Lin-an, Tianmushan, eight-branched, posterior branch of MP two-branched. Qianmutian, 30.vii.2011, T. Zhang (CAU); 50♂8♀,Lin-an, Abdomen blackish brown. Tergum 9 subtrapezoidal, anterior Tianmushan, Sanmuping, 25.vii.2011, T. Zhang (CAU); 1♀, margin with deeply arched incision and suboval internal fossa, Lin-an, Tianmushan, Zhonglieci, 26.vii.2011, X. Pan & X. Luo

(CAU); 1♀, Lin-an, Tianmushan, Xianrending, 27.vii.2011, X. Neoneuromus vanderweelei Liu, Hayashi & Yang, sp.n. Pan (CAU); 1♂, Longquan, Fengyangshan, 3.viii.2008, J. Liu http://zoobank.org/urn:lsid:zoobank.org:act:99DDA6CE- (CAU). CACD-4E40-9B3B-3E9F8B641208 (Fig. 11)

Distribution. China (Anhui, Fujian, Guangdong, Jiangsu, Diagnosis. Head and pronotum entirely reddish brown or Jiangxi, Shaanxi, Zhejiang). blackish brown, pronotum if reddish brown laterally with two pairs of black markings (a small anterior dot and a posterior longitudinal stripe). Wings with distal half brown; forewing Etymology. The specific epithet ‘similis’ refers to the undis- with a dark marking on 1mp-cua. Male ectoproct strongly tinguishable appearance of this cryptic new species. inflated on distal half, distally with an ovoid depression. Male fused gonocoxites 10 with a pair of short subtriangular lateral Remarks. The new species was previously thought to be projections, gonostyli 10 present. N. maclachlani in Liu & Yang (2004). The male genitalia between these two species are indeed very similar. Moreover, Description. Male. Body length 40–50 mm; forewing length the dark-coloured individuals of N. similis sp.n. are also quite 45–52 mm, hindwing length 40–47 mm. similar to N. maclachlani. However, the new species can be Head entirely reddish brown or blackish brown. Compound distinguished from N. maclachlani by the absence of dark eyes brown; ocelli yellow, medially margined black. Antenna marking on forewing 1mp-cua and the male ectoproct with black; scape and pedicel with yellowish tips. Labrum yellowish inflated apex slightly tapering distad. Besides, our molecular brown; mandible black with apex and teeth reddish brown; data also suggest a clear difference of N. similis sp.n. from maxilla yellow, except for distal three segments of maxillary N. maclachlani. Within this new species, some individuals palpus black; labium yellowish brown except for distal three are distinctly darker than the other examined specimens (see segments of labial palpus black. Fig. 10A, B). However, we did not find any correlation of such Prothorax reddish brown or blackish brown; pronotum, if variation with their geographical distributions. reddish brown, laterally with two pairs of black markings (a small anterior dot and a posterior longitudinal stripe). Meso- and metathorax brown to blackish brown, with lateral portions and Neoneuromus tonkinensis (van der Weele) scutellum much darker. Legs blackish brown, with dense reddish (File S1, File S2: Fig. 8, File S3: Fig. 8) brown setae; coxae, trochanters, and femora slightly paler; tarsal Neuromus latratus tonkinensis van der Weele, 1907: 239. Type claws reddish brown. Forewing hyaline, but dark brown on distal locality: Vietnam (Tuyen Quang: Chiem-Hoa). half, with brown markings on costal crossveins, ra-rp crossveins, and crossveins on proximal half, forming a broad hyaline area Diagnosis. Head blackish brown with clypeus almost entirely medially and some small hyaline areas around large hyaline yellowish brown, or head yellowish brown with a black frontal region. Hindwing slightly paler than forewing, but brown on marking and a pair of black vertexal markings. Pronotum distal half. Veins pale brown, except for veins in dark markings blackish brown, or yellowish brown with a pair of black lateral blackish brown. RP nine- to 12-branched; MA bifurcated; five to vittae. Forewing with distal half dark brown; dark marking on six crossveins between RA and RP; anterior branch of MP four- 1mp-cua present. Male sternum 9 posterolaterally with a pair of to five-branched, posterior branch of MP two-branched. short projections but medially truncate at tip; male gonostyli 10 Abdomen blackish brown. Tergum 9 subtrapezoidal, anterior absent. margin with deeply arched incision and suboval internal fossa, posterior margin slightly produced medially. Sternum 9 narrow and attenuate, with a pair of narrow longitudinal internal ridges, Distribution. China (Guangdong, Guangxi); Vietnam (Ha apex shallowly incised at tip, leaving a pair of short, obtuse pro- Tay, Hoa Binh, Lang Son, Lao Cai, Quang Ninh, Tuyen Quang, jections. Gonostylus 9 unguiform. Ectoproct clavate, strongly Vinh Phuc). inflated on distal half, distally with an ovoid depression and distinctly curved ventrad. Fused gonocoxites 10 with a pair of short Remarks. This species is sometimes confused with N. orien- subtriangular lateral projections; gonostyli 10 slenderly digiti- talis by the similarly conspicuous intraspecific variations, rang- form. ing from a blackish brown (colour form I) to yellowish brown Female. Body length 55–58 mm; forewing length (colour form II) species with similar wing marking patterns (see 65–67 mm, hindwing length 58–60 mm. Fig. 5C, D). Nevertheless, the forewing apex is largely dark in Fused gonocoxites 8 subquadrate in lateral view. Gonocoxite N. tonkinensis, whereas it is transparent with scattered small 9 valvate and suboval, distally with obtuse gonostylus 9. Ecto- dark spots in N. orientalis. Moreover, the male genitalia of N. proct with digitiform dorsal lobe and slightly shorter, obtuse tonkinensis is very distinguishable due to the male sternum 9 ventral lobe. with truncate apex and the male fused gonocoxites 10 lacking gonostyli. In N. orientalis the apex of male sternum 9 is medially Materials examined. Holotype ♂, ‘China, Yunnan, Yingjiang, incised, and the male gonostyli 10 are present. 1660 m, 29.vii.1979, Y. Hu’ (CAU). Paratypes: CHINA:

Guangxi:5♂3♀, Baise, Cenwanglaoshan, Dalongping, 1300 m, (MSNF); 7♂1♀, Tam Dao, 25–30.vii.2011, J. Constant & J. 28.vii.2013, F. Liang (CAU); 1♂, Tianlin, Cenwanglaoshan, Bresseel (IRSNB); 1♂, Tam Dao, 800–900 m, 12.vi.1994, Dalongping, 21.viii.2014 (CAU); Yunnan:2♂2♀,Baoshan, A. Baranov (ZIN); 1♂, Tam Dao, 800–900 m, 3.vii.1994, A. Baihualing, 31.vii-2.viii.2014, H. Han (CAU); 1♀, Yingjiang, Monastyrskyi (ZIN); 2♂1♀, Tam Dao, 800–900 m, 17.v.1995, Tongbiguan, 10.vii.2013, W. Zhang (CAU); 3♂, Lvchun, Huan- A.V. Gorokhov (ZIN); 1♀, Tam Dao, 9.vi.1997, S.A. Ryabov glianshan, Qimaba, 11.vi.2013, J. Wang & J. Yang (CAU); (ZIN); Yen Bai:2♀, Mucangchai, 1700 m, v.2016, T. Le 1♂, Lvchun, Huanglianshan, Qimaba, 9.vi.2013, J. Zhang (LDPC). (CAU); 1♀, Luxi, Etoushan, 23.vii.1979 (CAU); 1♂, Cangyuan, Banhong, 1100 m, 11.vi.1980 (CAU); 2♂, Xinping, Tszin- Distribution. China (Guangxi, Yunnan); Vietnam (Bac dun, 26–28.v.1956, A.K. Zagulyaev (ZIN); VIETNAM: Bac Giang, Cao Bang, Ha Noi, Lao Cai, Vinh Phuc, Yen Bai). Giang:1♀, Vietnam, Tay Yen Tu Nat. Res., 7–11.vii.2013, J. Constant & J. Bresseel (IRSNB); Cao Bang:1♀, Mt. Pia Oac, Etymology. The new species is dedicated to H.W. van der 22–28.v.1998, K. Matsumoto (HFC); 1♂, Phia Den, 8.viii.2010, Weele for his tremendous contribution on the taxonomy of J. Constant & P. Limbourg (IRSNB); Ha Noi:5♂1♀, Hanoi Megaloptera. (IRSNB); Lao Cai:1♂, Chapa, viii.1936, S. Masseyeff (NHM); 1♂2♀, N. Sa Pa, Deo O Quy Ho, 1750 m, 1/3.vii.1999, M. Remarks. This species looks quite similar to N. maclachlani Owada (NSM); 1♂, W. Sapa, 1100–1700 m, 29.v-11.vi.1996, in having the dark body and wing colouration, but can be K. Anton (ZMA); 1♂, Sa Pa, Mt. Fan-si-pan, 1500–1800 m, distinguished from the latter species by the male ectoproct that 30.vi-12.vii.1994, Brechlin & Schintimeister (ZMA); 1♂, is strongly inflated on distal half. In N. maclachlani the male Hoang Lien N.P., 1–5.vii.2013, J. Constant & J. Bresseel ectoproct is relatively short and inflated from about the proximal (IRSNB); 1♂1♀, Van Ban Nature Reserve, 23–26.v.2011, L. 1/4. Within this new species, the individuals from Yunnan are Bartolozzi (MSNF); Vinh Phuc:2♀, Tam Dao, 22/23.vi.2011, slightly paler than those from northern Vietnam and Guangxi G. Wang (CAU); 1♀, Tam Dao, 19.v.1995, S. Ueno (NSM); 1♂, (see Fig. 11A, B). Our molecular data also suggest that these two Tam Dao, 24.v.1997 (ONHM); 7♂, Tam Dao, 27.v-2.vi.1986, J. colour forms are genetically distinct from each other. However, Macek (NMPC); 1♂, Tam Dao, 18–21.v.1993, K. Matsumoto we could not find any difference of genital characters. Thus, (HFC); 1♂, Tam Dao, v.1993, H. Karube (HFC); 2♂,Tam we still consider these two colour forms to belong to the same Dao National Park, 5–12.vi.2010, L. Bartolozzi & S. Bambi species but might represent geographical variations.