Ecologica Montenegrina 40: 140-163 (2021) This journal is available online at: www.biotaxa.org/em http://dx.doi.org/10.37828/em.2021.40.13 https://zoobank.org/urn:lsid:zoobank.org:pub:6EDA9875-78A1-4D77-B840-42FF417CFA80 Parnassius nebrodensis: A threatened but neglected Apollo butterfly species from Southern Europe (Lepidoptera: Papilionidae) IVAN N. BOLOTOV1, MIKHAIL Y. GOFAROV1, VYACHESLAV V. GORBACH2, YULIA S. KOLOSOVA1, ALISA A. ZHELUDKOVA1, ALEXANDER V. KONDAKOV1 & VITALY M. SPITSYN1,* 1N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Northern Dvina Emb. 23, 163000 Arkhangelsk, Russia 2Petrozavodsk State University, Lenina Av. 20, 185035 Petrozavodsk, Russia *Corresponding author: [email protected] Received 22 January 2021 │ Accepted by V. Pešić: 22 March 2021 │ Published online 23 March 2021. Abstract Recent multi-locus phylogenetic studies repeatedly showed that what was thought to be the Clouded Apollo butterfly Parnassius mnemosyne (Linnaeus, 1758) represents a cryptic species complex. This complex contains at least three distant species-level phylogenetic lineages. Here, we compile a set of morphology- and DNA-based evidences supporting the distinctiveness of two species in this group, i.e. P. mnemosyne s. str. and P. nebrodensis Turati, 1907 stat. rev. These species can be distinguished from each other based on a combination of diagnostic characters in the male genitalia structure, wing scale patterns, and the forewing venation. The species status of P. nebrodensis is supported based on unique nucleotide substitutions in the mitochondrial (COI, ND1, and ND5) and nuclear (Wg and EF- 1a) genes. P. nebrodensis is endemic to the Western Mediterranean Region. This species shares a disjunctive range through the Pyrenees, Western and Central Alps, Apennines, and the Nebrodi and Madonie mountains on Sicily. Altogether 38 nominal taxa initially described as P. mnemosyne subspecies are considered here to be junior synonyms of P. nebrodensis. At first glance, P. nebrodensis can be assessed as an endangered species due to its restricted distribution, narrow range of habitats, and ongoing population decline. Isolated populations of this species scattered through mountain ranges need special management and conservation efforts. Key words: Apollo butterflies, cryptic species, phylogeny, Western Mediterranean Region, endangered species, conservation. Introduction The Clouded Apollo Parnassius mnemosyne (Linnaeus, 1758) belongs to the subgenus Driopa Korshunov, 1988 [=the Mnemosyne species group] (Müller 1973; Ackery 1975; Weiss 1999; Korshunov 2002; Omoto et al. 2004, 2009). All Driopa butterflies use Corydalis DC. and Dicentra Bernh. (Papaveraceae) taxa as larval host plants (Korshunov 2002; Michel et al. 2008; Condamine 2018). Most species in this group are known to Ecologica Montenegrina, 40, 2021, 140-163 BOLOTOV ET AL. occur in Northern Asia, Central Asia, and North America (Weiss 1999; Korshunov 2002). In contrast, the Clouded Apollo was thought to be a species widely distributed throughout Europe, the Urals, the Caucasus, eastern Kazakhstan, and the Middle East, with a few records from Western Siberia (Weiss 1999; Korshunov 2002; Kudrna et al. 2011; Wiemers et al. 2018). The northernmost populations of this butterfly were discovered in karst boreal landscapes of Northern European Russia between 65° and 66°N (Bolotov et al. 2013), while the southern boundary of the species’ range is situated in the Western Mediterranean Region and the Middle East (Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011). The Clouded Apollo was considered an endangered species in Europe (Van Swaay and Warren 1999; Van Swaay et al. 2010, 2012). It was shown that its population decline is largely associated with the cessation of traditional management practices such as grazing and mowing at semi-natural grasslands and coppicing in woodlands (Väisänen and Somerma 1985; Luoto et al. 2001; Descimon 2006; Van Swaay et al. 2012). A variety of P. mnemosyne morphological forms was described as intraspecific taxa from various parts of its broad range, including approximately 200 subspecies that were introduced based on minute differences in wing markings pattern and size (Weiss 1999). Available taxonomic reviews of intraspecific names linked to P. mnemosyne were based on morphological features alone (Leraut 1997; Weiss 1999; Mérit and Mérit 2006). Weiss (1999) subdivided all the subspecies into two categories as follows: (1) ‘strong’ subspecies (i.e. the taxa that can clearly be distinguished from others using morphological features), and (2) ‘weak’ subspecies (i.e. those displaying less clear diagnostic features). It was shown that the wing markings pattern in P. mnemosyne and related species is highly variable (Eisner 1968, 1971, 1974, 1976, 1978; Müller 1973; Weiss 1999). Ackery (1975) noted that the markings pattern could be applied as diagnostic features for the species groups within the genus Parnassius Latreille, 1804 but cannot be used to separate species- and subspecies-level taxa. It was found that the subspecies of P. mnemosyne are poorly correspond to the population genetic structure (Descimon 1995) and to phylogeographic and phylogenetic patterns (Gratton 2006; Gratton et al. 2008; Michel et al. 2008). For example, the subspecies from Northern Europe are generally less marked compared with those from southern regions (Weiss 1999). A long-term intraspecific morphological variability of P. mnemosyne within a single locality can be linked to climatic fluctuations (Eisner 1974). A growing body of DNA-based research revealed that the Clouded Apollo shares a deep phylogeographic structure, with three highly divergent lineages that could represent cryptic species (Gratton 2006; Gratton et al. 2008; Michel et al. 2008). In particular, the samples of P. mnemosyne collected in the Western Mediterranean Region and Southern Anatolia and Iran were found to be distant from those sampled through Northern and Eastern Europe and Central Asia (Figs 1-2). Recently, the existence of these species- level clades was confirmed using multi-locus time-calibrated phylogenies and species delimitation modeling (Condamine 2018; Condamine et al. 2018). It was shown that the Western Mediterranean lineage (=P. mnemosyne sp.3 sensu Condamine, 2018) was isolated from other populations of P. mnemosyne since the mid-Pliocene (Condamine 2018). Although the evaluation of cryptic species is a task of great importance to resolve a broad array of scientific, conservation, and management issues (Bickford et al. 2007; Dincă et al. 2011, 2015; Platania et al. 2020a), these Clouded Apollo lineages are yet to be studied by means of morphological and taxonomic approaches. This paper (1) presents a taxonomic evaluation and morphological diagnosis for the Western Mediterranean lineage of Parnassius mnemosyne species complex (=P. nebrodensis Turati, 1907 stat. rev.; =P. mnemosyne sp.3 sensu Condamine, 2018); (2) illustrates the distribution of this species; and (3) revises a number of P. mnemosyne subspecies, the type localities of which are situated within the range of P. nebrodensis. Materials and methods Data sampling, and DNA amplification and sequencing Samples of Parnassius species were studied in the collection of the Russian Museum of Biodiversity Hotspots, N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia. The new mitochondrial cytochrome c oxidase subunit I (COI) gene sequences were generated from a single leg of 48 specimens (Table 1) using the approaches of Konopinski (2008) and Gratton (2006). The PCR mix contained approximately 200 ng of genomic DNA, 10 pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 20 mmol MgCl2), 0.8 units Taq Ecologica Montenegrina, 40, 2021, 140-163 141 PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE DNA polymerase (SibEnzyme Ltd., Russia), and H2O was added for a final volume of 25 μl. The following PCR conditions were used in the amplifications: 95°C (4 min), 32 cycles of 95°C (45 sec), 52°C (40 sec), 72°C (50 sec) and a final extension at 72°C (5 min). Additionally, 76 COI sequences were obtained from NCBI’s GenBank, including two sequences of Parnassius simo Gray, 1853, a close relative of the subgenus Driopa (see Omoto et al. 2004, 2009; Condamine et al. 2018), as outgroup (Table 1). The nuclear wingless (Wg) gene fragment was amplified and sequenced from 10 specimens, including 3 specimens of Parnassius nebrodensis and 4 specimens of P. mnemosyne s. str. (Table 2) The primers Lepwg1 and Lepwg2 were applied for amplification (Brower and DeSalle 1998). The following PCR conditions were used in the amplifications: 95 °C (5 min), 36 cycles of 95 °C (50 sec), 50 °C (50 sec), 72 °C (50 sec) and a final extension at 72 °C (5 min). The resulting COI and Wg sequences were checked manually using Bioedit 7.1.9 (Hall 1999). Figure 1. Ranges of taxa within the Parnassius mnemosyne species complex. (1) Range of P. nebrodensis stat. rev. (data: Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Todisco et al. 2010; Condamine et al. 2018; Litman et al. 2018; Dapporto et al. 2019; this study). (2) Approximate range of P. sp. ‘Middle East’ (data: Gratton 2006; Gratton et al. 2008). (3) Range of P. mnemosyne s. str. (data: Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011; Bolotov et al. 2013): (3a) confirmed range, and (3b) uncertain distribution. (4) Approximate boundary of P. mnemosyne s. str. and P. sp. ‘Middle East’ ranges (data: Weiss 1999; Gratton 2006; Gratton et al. 2008; Kudrna et al. 2011; Bolotov et al. 2013). (5) Type locality of P. nebrodensis stat. rev. in Italy [Sicily: “monti Nebrodi”] (Turati 1907). (6) Type locality of P. mnemosyne s. str. in Finland (Honey and Scoble 2001, 2001a). (Map: Mikhail Y. Gofarov). Morphological analyses The dissection of the genitalia was performed using the standard methods for Lepidoptera (Bolotov et al. 2018). Each abdomen was macerated in a heated 10% KOH solution for 30 minutes. First, images of ventral and lateral views of unmounted genitalia were obtained.