75 (3): 497– 521 20.12.2017 © Senckenberg Gesellschaft für Naturforschung, 2017. High cryptic diversity in aquatic insects: an integrative approach to study the enigmatic Leuctra inermis species group (Plecoptera) Simon Vitecek *, 1, 4, #, Gilles Vinçon 2, #, Wolfram Graf 3 & Steffen U. Pauls 4 1 Department for Limnology and Bio-Oceanography, Universität Wien, UZA I, Althanstrasse 14, 1090 Vienna, Austria; Simon Vitecek [simon. [email protected]] — 2 55 Bd Joseph Vallier, F 38100 Grenoble, France; Gilles Vinçon [[email protected]] — 3 Institute of Hydrobio- logy and Aquatic Ecology Management, University of Natural Resources and Life Sciences, Gregor-Mendel-Straße 33, 1180 Vienna, Austria; Wolfram Graf [[email protected]] — 4 Senckenberg Research Institute and Natural History Museum, Senckenberganlage 25, 60325 Frankfurt am Main, Germany; Steffen U. Pauls [[email protected]] — * Corresponding author; # Equally contributing authors Accepted 05.x.2017. Published online at www.senckenberg.de/arthropod-systematics on 11.xii.2017. Editors in charge: Gavin Svenson & Klaus-Dieter Klass Abstract Within the genus Leuctra (Plecoptera: Leuctridae) the L. inermis species group comprises 17 – 18 species in which males lack the char- acteristic tergal abdominal ornamentation of many Leuctra species, and females have an accessory receptacle in the dorsal portion of the vagina. Taxonomically the group is challenging, and congruence of existing morphological species concepts and phylogenetic relationships of taxa is hitherto not assessed. Here, we estimate phylogenetic relations of morphologically defined species by concatenated maximum likelihood (ML) and Bayesian combined species tree and species delimitation analysis. We aim to clarify the status of 15 European species of the L. inermis group and 2 potential new species. To this end, we infer relationships on a 2580 bp, 4 loci (mtCOI, mt12S, nuH3, nu28S) molecular sequence dataset. We further depict abdominal terminalia with their morphological characteristics for 15 L. inermis group spe- cies, and describe 2 micro-endemic species from the Southern Alps and the Apennine Mountains corroborated by distinct morphological and molecular characteristics. Our analyses support close relationships of the L. inermis group species and corroborate the a priori mor- phological definition of all included species. However, relationships among species are largely unresolved, indicative of a putatively recent diversification of the group. Phylogenetic inference suggests sister taxon relationships between morphologically similar species. Morpho- logical variation within single species is linked to geographical location of populations. Our results thus suggest that further study on the differentiation of geographically isolated populations as distinct lineages or species is warranted. Based on the high number of regional endemic lineages and species we highlight the necessity to protect high-altitude aquatic habitats. Key words Leuctridae, STACEY, integrative taxonomy, Leuctra fochettii, Leuctra grafi, new species, aquatic biodiversity. 1. Introduction Plecoptera are a group of highly niche-specific aquatic zoogeography (ILLIES 1965; HYNES 1976; ZWICK 2000; insects widely used as indicators of water quality (ROSEN­ GRAF et al. 2009, 2017; DEWALT et al. 2015). European BERG & RESH 1993; GRAF et al. 2002 – 2011; MOOG 2002 – Plecoptera exhibit high degrees of endemism, and more 2011; HERING et al. 2003, 2006; BIRK et al. 2012; DEWALT than half of the known species (264 of 514) are consid- et al. 2015). The group comprises many cold-stenotopic ered regional or micro-endemics (GRAF et al. 2009, 2017). taxa that contribute to its high diversity and interesting Population fragmentation and ensuing endemism in stone- ISSN 1863-7221 (print) | eISSN 1864-8312 (online) 497 Vitecek et al.: A taxonomists’ battle of Leuctra flies are often linked to the relatively poor dispersal ca- Ravizza, 1996, L. handlirschi Kempny, 1899, L. inermis pacity of both mature and immature stages (FOCHETTI Kempny, 1899, L. insubrica Aubert, 1949, L. kempnyi et al. 2011; ELBRECHT et al. 2014). Evolutionary history Mosely, 1932, L. metsovonica Aubert, 1966, L. pusilla and extant diversity patterns of Plecoptera are moreover Krno, 1985, L. quadrimaculata Kis, 1962, L. rauscheri shaped by allopatric diversification driven by historic Aubert, 1957, L. silana Aubert, 1953 (likely a synonym glaciation, orogenesis, and small-scale population frag- of L. inermis Kempny; G. Vinçon unpubl. data), L. sim­ mentation (ZWICK 2000; FOCHETTI & TIERNO DE FIGUEROA plex Zhiltzova, 1960, L. teriolensis Kempny, 1900, and 2008; FOCHETTI et al. 2009, 2011; WEISS et al. 2011; THEIS­ L. uncinata Martynov, 1928. SINGER et al. 2013). Ecologically, a slight majority of Based on scarce autecological data, European spe- Plecoptera taxa behave as detritivores, while other spe- cies of the group can be assumed to differ little in their cies (particularly systellognathan ones) are carnivores preferred ecological niche. No ecological data exist (HYNES 1976; SILVERI et al. 2008; GRAF et al. 2009, 2017; for the Caucasian L. simplex and L. uncinata. Larvae FENOGLIO et al. 2010; BOTTOVÁ et al. 2013; DEWALT et of L. inermis group species mostly occur in crenal to al. 2015). Larval habitats of Plecoptera comprise lotic rhithral stretches of cold-water streams in montane to and lentic waterbodies, and the majority of species prefer alpine habitats, and behave as detritivores (GRAF et al. cool to cold temperature conditions (HYNES 1976; GRAF 2009, 2017). To our current knowledge, adult emer- et al. 2009, 2017). gence peaks in spring to summer; we thus assume a uni- Among European Plecoptera, the family Leuctridae voltine annual life cycle (BO & FENOGLIO 2009; GRAF (Plecoptera, Arctoperlaria, Euholognatha) is second only et al. 2009, 2017). As in other Leuctridae, larvae of the to Nemouridae in number of described species (TIERNO group cannot be reliably identified based on their mor- DE FIGUEROA & FOCHETTI 2014; GRAF et al. 2017). In Eu- phology. rope, Leuctridae are represented by the genera Pachy­ Taxonomically, the L. inermis group could contain leuctra [3 species], Tyrrhenoleuctra [5 species], and high micro-endemic species-level or subspecies-level Leuctra [151 species] (TIERNO DE FIGUEROA & FOCHETTI diversity: they share ecological traits (e.g., habitat pre- 2014; GRAF et al. 2017). The overwhelming majority of ferences, altitudinal distribution, phenology) with other the taxa are mostly small, darkly coloured, and save a groups of aquatic insects comprising many endemic spe- few exceptions, are macropterous. Males of the genus cies such as, among others, Rhyacophilidae (Insecta, Leuctra exhibit diagnostic tergal ornamentation patterns Trichoptera), Drusinae (Trichoptera, Limnephilidae), on abdominal segments. Differences in the sclerotized Con sorophylax (Trichoptera, Limnephilidae, Stenophy- tergal processes of the males and form of external geni- la­­cini) or Pediciidae (Insecta, Diptera) (Previšić et al. talia in both sexes are the also most important characters 2014; DENÉS et al. 2015; IBRAHIMI et al. 2015, 2016; GRAF in Leuctridae taxonomy (e.g., RAVIZZA & VINÇON 1998). et al. 2015; VITECEK et al. 2015a,b; GRAF & VITECEK Differentiation of larval stages in Leuctridae is rarely 2016). Also, sexual selection via development of dis- achieved, and only few species can be reliably identified tinct drumming call dialects could enhance divergence of as larvae (ZWICK 2004; GRAF & SCHMIDT-KLOIBER 2010). populations (BOUMANS & JOHNSEN 2014). Consequently, Also, adults of some species groups exhibit little mor- other allopatric, micro-endemic taxa of the L. inermis phological differentiation and are difficult to distinguish group can be assumed to exist. (RAVIZZA 2002). Alternatively, different morphologically defined spe- A taxonomically particularly challenging species cies of the L. inermis group as previously recognized group of European Leuctra is the L. inermis group. Males could represent morphological variants of a few wide- exhibit reduced abdominal tergal sclerotization without spread species (cf. FOCHETTI et al. 2011). Intriguingly, raised dorsal processes; the female genital tract develops geographical ranges of many species of the L. inermis an accessory receptacle on the dorsal side of the vagina, group overlap, and result in syntopic occurrence of cer- and the external species-specific female genitalic charac- tain taxa (BOJKOVÁ & SOLDÁN 2011; PETROVIC et al. 2014; ters seem to oscillate between two major forms (AUBERT GRAF et al. 2005; GRAF 2010; GRAF & SCHMIDT-KLOIBER 1957; ZWICK 1973). The first comprehensive treatment of 2010; Kroča 2011). Indeed, these reported distribution the group was conducted by AUBERT (1957), who reco- patterns could be due to the existence of morphological gnized 10 species in 3 phyletic species groups based on variants within several currently unrecognized wide- similarities in sclerotization/pigmentation patterns in spread species (FOCHETTI et al. 2011). males and external structure of male and female genitalia. In this contribution, we test species boundaries of Five additional species included by Aubert due to their morphologically defined taxa in the enigmaticL . inermis similarity to the L. inermis group have since been reco- group. To this end, we infer phylogenetic relationships gnised as more closely related to other groups (BERTHÉLE­ between 15 currently accepted and 2 potentially new Eu- MY 1968;