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, 568 © 2018 The Royal Entomological Society Neoneuromus systematics and phylogeny 569 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

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