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 species from Southern Europe (: 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 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 , 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 DC. and Dicentra Bernh. () taxa as larval host plants (Korshunov 2002; Michel et al. 2008; Condamine 2018). Most species in this group are known to

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BOLOTOV ET AL. occur in Northern 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 , and the , with a few records from Western (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 and 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

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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 (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. Second, the genitalia of each specimen were mounted on a glass slide with Histofluid® (Paul Marienfeld GmbH & Co., Germany), and its dorsal view was photographed. Images of genitalia and wing scales were taken using a Leica M165C stereo microscope

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(Leica Microsystems GmbH, Germany). Images of the genitalia structures were taken with an AXIO Zoom.V16 research microscope (Carl Zeiss, Germany). Images of specimens were taken with a Canon EOS 500D digital camera (Canon Inc., Tokyo, Japan). An image of the P. mnemosyne lectotype was obtained from the Linnaeus’s Butterfly Type Specimens database of the Natural History Museum, London, UK (Honey and Scoble 2001, 2001a).

Table 1. List of the COI sequences used in this study.

COI acc. Species Catalogue no. Region Reference no. In-group: P. nebrodensis Turati, 1907 stat. rev. RMBH G0327 France: Pyrenees, foothill KF444468 This study of Mt. Neouville P. nebrodensis Turati, 1907 stat. rev. RMBH G0328 France: Pyrenees, foothill MW460988 This study of Mt. Neouville P. nebrodensis Turati, 1907 stat. rev. RMBH G0329 France: Pyrenees, foothill MW460989 This study of Mt. Neouville P. nebrodensis Turati, 1907 stat. rev. RMBH G0330 France: Pyrenees, foothill MW460990 This study of Mt. Neouville P. nebrodensis Turati, 1907 stat. rev. RMBH G0331 France: Pyrenees, foothill MW460991 This study of Mt. Neouville P. nebrodensis Turati, 1907 stat. rev. RMBH G0534 : eastern Pyrenees KX130690 This study P. nebrodensis Turati, 1907 stat. rev. RVcoll.09-V684 Spain: Pyrenees, JF848003 GenBank Cerdanya P. nebrodensis Turati, 1907 stat. rev. RVcoll.09-T076 Spain: Pyrenees, Lleida, JF847984 GenBank Val d'Aran P. nebrodensis Turati, 1907 stat. rev. RVcoll.07-C024 France: Pyrenees, foothill GU669633 GenBank of Pic Petit de Segre P. nebrodensis Turati, 1907 stat. rev. RVcoll.13-T947 Italy: Chieti, La MN145197 Dapporto et al. (2019) Maielletta P. nebrodensis Turati, 1907 stat. rev. LEP-SS-00563 Italy: Calabria MN144406 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 11-I228 Italy: Calabria MN144237 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. RVcoll.11-I226 Italy: Calabria MN143408 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. RVcoll.11-I225 Italy: Calabria MN141405 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 14-V321 Italy: Avio, Trente MN141943 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. LEP-SS-00001 Italy: Calabria MN143318 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 15-C862 Italy: Calabria MN143005 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. LEP-SS-00002 Italy: Calabria MN142317 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 15-C867 Italy: Calabria MN141765 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. RVcoll.11-H952 Italy: Sicily MN145369 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 11-H953 Italy: Sicily MN140069 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. RVcoll.11-H737 Italy: Sicily MN142921 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. 11-H738 Italy: Sicily MN144890 Dapporto et al. (2019) P. nebrodensis Turati, 1907 stat. rev. Pmne-ITNEB01 Italy: Nebrodi Mts., Sicily GU947642 Todisco et al. (2010) P. nebrodensis Turati, 1907 stat. rev. GBIFCH-BOL Switzerland: Valais, MK186621 Litman et al. (2008) LEPAA_0273 Martigny P. nebrodensis Turati, 1907 stat. rev. GBIFCH-BOL Switzerland: Ticino, MK186623 Litman et al. (2008) LEPAA_0229 Airolo P. nebrodensis Turati, 1907 stat. rev. GBIFCH-BOL Switzerland: Graubunden, MK186622 Litman et al. (2008) LEPAA_0743 Panix P. nebrodensis Turati, 1907 stat. rev. Ch765 Switzerland EU092983 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr525 France EU092985 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr527 France EU092986 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr528 France EU092987 Gratton et al. (2008)

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COI acc. Species Catalogue no. Region Reference no. P. nebrodensis Turati, 1907 stat. rev. It639 Italy EU092988 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr908 France EU092989 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr911 France EU092990 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Fr913 France EU092991 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Sp916 Spain EU092992 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Sp917 Spain EU092993 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Sp921 Spain EU092994 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Sp923 Spain EU092995 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Sp924 Spain EU092996 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. It878 Italy EU093015 Gratton et al. (2008) P. nebrodensis Turati, 1907 stat. rev. Ch892 Switzerland EU093017 Gratton et al. (2008) P. mnemosyne (Linnaeus, 1758) s. RMBH G0004 Belarus: Grodno Region KF444471 This study str. P. mnemosyne (Linnaeus, 1758) s. RMBH G0221 Russia: Arkhangelsk KF444472 This study str. Region P. mnemosyne (Linnaeus, 1758) s. RMBH G0286 Russia: Arkhangelsk KF444473 This study str. Region P. mnemosyne (Linnaeus, 1758) s. RMBH G0280 Moldova KF444470 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-532-4 Uzbekistan KX130692 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-532-2 Uzbekistan KX130691 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-332 Slovakia KX130689 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-326 Russia: Middle Urals KX130688 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-310 Russia: Nizhny Novgorod KX130687 This study str. Region P. mnemosyne (Linnaeus, 1758) s. IEPN-321 Russia: Lipetsk Region KX130686 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-275 Russia: Karelia KX130685 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-274 Russia: Karelia KX130684 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-271 Russia: Karelia KX130683 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-266 Russia: Karelia KX130682 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-269 Russia: Karelia KX130681 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-537-2 Russia: Krasnodar Region KX130680 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-537-1 Russia: Krasnodar Region KX130679 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-285 Russia: Arkhangelsk KX130678 This study str. Region P. mnemosyne (Linnaeus, 1758) s. IEPN-282 Moldova KX130677 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-279 Moldova KX130676 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-540 Kyrgyzstan KX130675 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-653 Kyrgyzstan KX130674 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-535 Iran KX130673 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-538 KX130672 This study str.

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COI acc. Species Catalogue no. Region Reference no. P. mnemosyne (Linnaeus, 1758) s. IEPN-646 Armenia KX130671 This study str. P. mnemosyne (Linnaeus, 1758) s. IEPN-644 Armenia KX130670 This study str. P. mnemosyne (Linnaeus, 1758) s. Hu630 Hungary EU092970 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. BH721 Bosnia EU092972 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. At606 Austria EU092977 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. At618 Austria EU092978 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. At626 Austria EU092981 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. Pmne-ITGAR05 Italy EU836659 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. Pmne-GRKEL03 Greece EU836665 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. Pmne-GRTAY01 Greece EU836669 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. Pmne-LTKRE03 Lithuania EU836676 Gratton et al. (2008) str. P. mnemosyne (Linnaeus, 1758) s. Pmne-RUSAR01 Central European Russia EU836682 Gratton et al. (2008) str. P. sp. Middle East W335 Turkey AM231418 Michel et al. (2008) P. sp. Middle East W280 Iran AM231420 Michel et al. (2008) P. sp. Middle East W311 Iran AM231419 Michel et al. (2008) P. sp. Middle East Tu1000 Turkey EU093005 Konopinski (2008) P. phoebus (Fabricius, 1793) [=P. RMBH G0525 Russia: Altai Mts. KX130693 This study ariadne (Lederer, 1853)] P. phoebus (Fabricius, 1793) [=P. N/A N/A EU093023 Konopinski (2008) ariadne (Lederer, 1853)] P. phoebus (Fabricius, 1793) [=P. AC4-14 N/A EF473794 GenBank ariadne (Lederer, 1853)] P. phoebus (Fabricius, 1793) [=P. W237 Russia: Altai Mts. AM231429 Michel et al. (2008) ariadne (Lederer, 1853)] P. phoebus (Fabricius, 1793) [=P. Pari-KZTRB02 Kazakhstan: Tarbagatai GU947640 Todisco et al. (2010) ariadne (Lederer, 1853)] Mts. P. hoenei Schweitzer, 1912 RMBH G0003.1 Russia: Sakhalin Island KF444469 This study P. hoenei Schweitzer, 1912 RMBH G0003.3 Russia: Sakhalin Island MW460983 This study P. hoenei Schweitzer, 1912 RMBH G0003.4 Russia: Sakhalin Island MW460984 This study P. hoenei Schweitzer, 1912 RMBH G0003.5 Russia: Sakhalin Island MW460985 This study P. hoenei Schweitzer, 1912 RMBH G0003.6 Russia: Sakhalin Island MW460986 This study P. hoenei Schweitzer, 1912 RMBH G0003.7 Russia: Sakhalin Island MW460987 This study P. hoenei Schweitzer, 1912 RMBH G0629 Russia: Sakhalin Island MW460995 This study P. hoenei Schweitzer, 1912 RMBH G0630 Russia: Sakhalin Island MW460996 This study P. hoenei Schweitzer, 1912 RMBH G0631 Russia: Sakhalin Island MW460997 This study P. hoenei Schweitzer, 1912 RMBH G0632 Russia: Sakhalin Island MW460998 This study P. hoenei Schweitzer, 1912 RMBH G0633 Russia: Sakhalin Island MW460999 This study P. hoenei Schweitzer, 1912 RMBH G0626 Russia: Kunashir Island MW460992 This study P. hoenei Schweitzer, 1912 RMBH G0627 Russia: Kunashir Island MW460993 This study P. hoenei Schweitzer, 1912 RMBH G0628 Russia: Kunashir Island MW460994 This study P. hoenei Schweitzer, 1912 W218 Japan: Hokkaido AM231427 Michel et al. (2008) P. hoenei Schweitzer, 1912 AC20-16 N/A EF473801 GenBank

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COI acc. Species Catalogue no. Region Reference no. P. hoenei Schweitzer, 1912 N/A N/A EU093019 Konopinski (2008) P. stubbendorfii Menetries, 1849 RMBH G0634 Russia: Primorye MW461000 This study P. stubbendorfii Menetries, 1849 AC5-2 South Korea EF473800 GenBank P. stubbendorfii Menetries, 1849 N/A South Korea GU372549 Kim et al. (2010) P. stubbendorfii Menetries, 1849 SWCN09-5002 South Korea GU696036 GenBank P. stubbendorfii Menetries, 1849 005-LOWA-815 Russia: Altai FJ663912 Lukhtanov et al. (2009) P. stubbendorfii Menetries, 1849 2005-LOWA-153 Russia: Altai FJ663914 Lukhtanov et al. (2009) P. eversmanni Ménétriés, [1850] AC23-68 N/A EF473797 GenBank P. eversmanni Ménétriés, [1850] AC1-14 N/A EF473796 GenBank P. eversmanni Ménétriés, [1850] KWP_Ento_3722 USA: Alaska KU875779 Sikes et al. (2017) 2 P. eversmanni Ménétriés, [1850] 2005-LOWA-107 Russia: Altai FJ663893 Lukhtanov et al. (2009) P. eversmanni Ménétriés, [1850] W251 Russia: Amur Region AM231430 Michel et al. (2008) P. orleans Oberthür, 1890 W212 China AM231433 Michel et al. (2008) P. orleans Oberthür, 1890 AC10-5 N/A EF473802 GenBank P. orleans Oberthür, 1890 GGS01 China MH518694 Tao et al. (2020) P. orleans Oberthür, 1890 PO-2011-59CI China JQ922045 GenBank P. nordmanni Ménétries in AC20-5 N/A EF473799 GenBank Simashko, 1850 P. nordmanni Ménétries in W226 Russia: Krasnodar Region AM231432 Michel et al. (2008) Simashko, 1850 P. nordmanni Ménétries in Pnor-RUKRS01 Russia: Krasnodar Region GU947641 Todisco et al. (2010) Simashko, 1850 P. glacialis Butler, 1866 TS28 China MH518684 Tao et al. (2020) P. glacialis Butler, 1866 SS26 China MH518656 Tao et al. (2020) P. glacialis Butler, 1866 NTS01 China MH518546 Tao et al. (2020) P. glacialis Butler, 1866 LJS20 China MH518454 Tao et al. (2020) P. glacialis Butler, 1866 NTS29 China MH518574 Tao et al. (2020) P. clodius Ménétriés, 1857 N/A Western North America MK947428 Zaman et al. (2019) P. clodius Ménétriés, 1857 N/A Western North America MK947427 Zaman et al. (2019) P. clodius Ménétriés, 1857 N/A Western North America MK947426 Zaman et al. (2019) P. clodius Ménétriés, 1857 N/A Western North America MK947425 Zaman et al. (2019) P. clodius Ménétriés, 1857 AC4-5 N/A EF473795 GenBank Outgroup: P. simo Gray, [1853] AC4-12 N/A EF473815 GenBank P. simo Gray, [1853] AC13-4 N/A EF473813 GenBank

N/A – not available. Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for explanation).

Phylogenetic analyses and species delimitation The alignments of the COI and Wg sequence data sets were performed using the MUSCLE algorithm implemented in MEGA7 (Kumar et al. 2016). For the phylogenetic analyses, we used a dataset with 116 unique haplotypes selected from 135 COI sequences (Table 1) with an online fasta sequence toolbox (FaBox v. 1.5; Villesen 2007). The maximum likelihood phylogenetic analyses were carried out with IQ-TREE v. 1.6.12 (Nguyen et al. 2015) through an online web server (http://iqtree.cibiv.univie.ac.at) (Trifinopoulos et al. 2016). The best-fit evolutionary model was selected for each codon separately using Model Finder based

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BOLOTOV ET AL. on Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al. 2017). Bootstrap support (BS) values were estimated by means of an ultrafast bootstrap (UFBoot2) approach (Hoang et al. 2018). The Bayesian phylogeny was calculated with MrBayes v. 3.2.7a (Ronquist et al. 2012) in the San Diego Supercomputer Center, USA through CIPRES Science Gateway (Miller et al. 2010). The MrBayes analyses were carried out in two separate runs of 20,000,000 generations, each with four Markov chains, one cold and three heated (temperature set at 0.1). Trees were sampled every 1000th generation. The first 15% of trees were discarded as burn-in, and the majority rule consensus phylogeny was calculated from the remaining trees. The GTR+G substitution model was applied to each codon position. The consensus COI phylogeny inferred from the W-IQ-TREE analyses was used as an input tree for the mPTP species-delimitation model of Kapli et al. (2017) via online mPTP server (http://mptp.h-its.org). The mPTP approach seems to be the most accurate tool to delineate species-level clades within large phylogenies (Kapli et al. 2017). This model returns more general Molecular Operational Taxonomic Units (MOTUs) compared with other species-delimitation methods (Kapli et al. 2017; Aksenova et al. 2019). Uncorrected p-distances between COI sequences were calculated using MEGA7 software (Kumar et al. 2016).

Table 2. Nucleotide substitutions between Parnassius species (subgenus Driopa) based on the nuclear Wg gene fragment.

Nucleotide position

GenBank

Species Region acc. No.

20 33 47 50 56 77

120 146 161 173 218 230 233 275 290 329 338

*P. nebrodensis France: MW468409 A/T C C/T T A T A G G A C C C G/T A/C C T stat. rev. Pyrenees *P. nebrodensis France: MW468414 A/T C C/T T A T A G G A C C C G/T A/C C T stat. rev. Pyrenees *P. nebrodensis France: MW468410 A/T C C/T T A T A G G A C C C G/T A/C C T stat. rev. Pyrenees *P. mnemosyne s. Belarus: MW468412 A C T T A T A G G A C C C G C C T str. Grodno *P. mnemosyne s. Belarus: MW468413 A C T T A T A G G A C C C G C C T str. Grodno *P. mnemosyne s. Russia: MW468407 A C T T A T C G G A C T C G C C T str. Arkhangels k Region *P. mnemosyne s. Russia: MW468408 A C T T A T C G G A C T C G C C T str. Arkhangels k Region *P. phoebus Russia: MW468411 A T T T A T A A G A T C C G C T C Altai Mts. *P. hoenei Russia: MW468405 A C T T G T A G G G C C A/C G C C C Sakhalin Isl. *P. hoenei Russia: MW468406 A C T T G T A G G G C C A/C G C C C Sakhalin Isl. P. clodius USA FJ756879 A C T C G C A G A A C C C G C C C

*New sequences generated under this study. Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for explanation).

Range mapping The distribution maps of Parnassius mnemosyne species complex and P. nebrodensis stat. rev. were compiled using ESRI ArcGIS 10 based on published sources (Weiss 1999; Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Todisco et al. 2010; Kudrna et al. 2011; Bolotov et al. 2013; Condamine et al. 2018; Litman et al. 2018; Dapporto et al. 2019) and our data. The type localities of subspecies were digitized based on original descriptions and general works (Eisner 1968, 1971, 1974, 1978; Ackery 1975; Weiss 1999).

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Figure 2. Range of Parnassius nebrodensis Turati, 1907 stat. rev. and a narrow contact zone between allopatric ranges of this species and P. mnemosyne (Linnaeus, 1758) s. str. based on DNA sequence data. (1) Range of P. nebrodensis (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) Occurrences of P. nebrodensis: (2a) Pyrenees (sample used for morphological and molecular analyses in this study), and (2b) other areas (data: Gratton 2006; Gratton et al. 2008). (3) Type locality of P. nebrodensis in Italy [Sicily: “monti Nebrodi”] (Turati 1907). (4) Type localities of nominal taxa that were described as P. mnemosyne subspecies but are being considered as synonyms of P. nebrodensis under this study (see Table 5 for detail). (5) A southern fragment of P. mnemosyne s. str. range adjoining the contact zone with P. nebrodensis (data: Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011): (5a) confirmed range, and (5b) uncertain distribution. (6) A contact zone between P. nebrodensis and P. mnemosyne s. str. ranges. (Map: Mikhail Y. Gofarov).

Results and discussion

Phylogenetic divergence between Parnassius (Driopa) taxa Both COI maximum likelihood and Bayesian phylogenies returned similar topology with 11 MOTUs: P. nebrodensis Turati, 1907 stat. rev., P. mnemosyne (Linnaeus, 1758) s. str., P. sp. ‘Middle East’, P. phoebus (Fabricius, 1793) [=P. ariadne (Lederer, 1853)], P. stubbendorfii Menetries, 1849, P. hoenei Schweitzer, 1912, P. glacialis Butler, 1866, P. eversmanni Ménétriés, [1850], P. orleans Oberthür, 1890, P. nordmanni Ménétries in Simashko, 1850, and P. clodius Ménétriés, 1857 (Figs 1-3). These MOTUs were supported by mPTP species-delimitation model of Kapli et al. (2017). Interestingly, P. phoebus was found to be more closely related to P. mnemosyne s. str. compared with P. nebrodensis (Fig. 3), as it was shown previously (Condamine et al. 2018). Mean uncorrected COI p-distances among the delineated MOTUs ranged from 2.57% (between P. clodius and P. eversmanni) to 8.47% (between P. orleans and P. glacialis) (Table 3). In almost all the MOTUs, maximum intraspecific distances were lower than minimum interspecific sequence divergence, except for Parnassius sp. ‘Middle East’ (2.71% vs 3.80%, respectively) (Table 4). P. mnemosyne s. str. and P. nebrodensis were separated by a 3.94% mean distance that is larger than that between well-defined

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species such as P. clodius vs P. eversmanni (2.57%), and P. mnemosyne s. str. vs P. phoebus (3.84%) (Table 3). Furthermore, our comparison of the Wg gene sequences revealed that all the studied species share 2-4 unique nucleotide substitutions, although P. nebrodensis has double peaks at the four informative sites (Table 2). In summary, our novel results align with earlier studies that underscored P. nebrodensis [=P. mnemosyne sp.3] as a separate cryptic species based on the COI, NADH dehydrogenase 1 (ND1), and NADH dehydrogenase 5 (ND5) gene fragments (Gratton 2006; Michel et al. 2008; Condamine et al. 2018). Furthermore, Gratton (2006) showed that the nuclear elongation factor-1 alpha (EF-1a) gene indicates the complete isolation of the Western Mediterranean haplogroup (i.e. P. nebrodensis) and P. mnemosyne s. str. and that any evidence of nuclear admixture between these taxa does not occur.

Table 3. Mean uncorrected p-distances (%) between Parnassius species (subgenus Driopa) based on the mitochondrial COI gene fragment.

Species ParNeb ParMne ParMEs ParPho ParStu ParHoe ParGla ParEve ParOrl ParNor P. nebrodensis stat. rev. [ParNeb] P. mnemosyne s. 3.94 str. [ParMne] P. sp. Middle 4.10 2.71 East [ParMEs] P. phoebus 4.38 3.84 4.32 [ParPho] P. stubbendorfii 5.78 6.36 6.51 6.44 [ParStu] P. hoenei 4.83 4.78 5.19 4.72 3.60 [ParHoe] P. glacialis 8.46 8.33 8.08 8.41 5.63 6.78 [ParGla] P. eversmanni 5.05 4.98 5.68 4.96 4.50 4.27 6.92 [ParEve] P. orleans 6.92 7.09 7.59 6.68 6.34 5.20 8.47 4.67 [ParOrl] P. nordmanni 6.92 6.37 6.85 6.40 6.34 5.86 8.39 5.65 6.85 [ParNor] P. clodius 4.69 4.54 5.04 4.61 4.82 4.06 6.89 2.57 5.06 5.45 [ParClo]

Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for explanation).

Differential diagnosis of Parnassius nebrodensis P. nebrodensis and P. mnemosyne s. str. cannot clearly be separated based on markings pattern alone, although the first species is usually darker, with stronger black markings on the wings, and a broader black spraying along veins distally (Fig. 4). However, several morphological features, outlined below, can help to distinguish these taxa. (1) Forewing venation (Fig. 5). P. nebrodensis shares an acute angle between veins R4 and R5 (ca. 30-50º in both sexes), while this angle is more obtuse in P. mnemosyne s. str. (ca. 60-70º in both sexes). This difference is caused by the degree of proximal bending of the vein R4: weak in P. nebrodensis vs strong in P. mnemosyne s. str. (see Fig. 5). Additionally, the endpoints of veins between R3 and Cu2 are shifted to the forewing apex in both sexes of P. nebrodensis compared with P. mnemosyne. The veins R3 and R4 in P. nebrodensis male are almost parallel, while in P. mnemosyne s. str. male vein R4 is slightly curved, and is lopsided to the forewing tornus (see Fig. 5). Finally, the female’s forewing cell of P. nebrodensis shares an elongate median veinlet, crossing the discocellular veinlet, prolonging to the postdical area, and finishing at the middle of the vein M1. This median veinlet is clearly distinct in the forewing markings pattern (Figs. 5 and 6B). Contrastingly, in P. mnemosyne s. str. female this median veinlet is short and indistinct (Figs. 5, 6C, and 6D).

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Figure 3. Maximum likelihood phylogeny of Parnassius (subgenus Driopa) taxa based on the COI haplotype sequences (Table 1). The red stars indicate the species-level clades supported by mPTP species-delimitation model. The name of each sequence contains the following information: GenBank accession number | sampling region. An asterisk indicates Parnassius nebrodensis sequence from the type locality (Nebrodi, Sicily). The black numbers near branches indicate bootstrap support values of IQ-TREE/BPP of MrBayes. Outgroup is not shown.

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(2) Forewing scale patterns (Fig. 6). In P. nebrodensis female, black diamond-shaped scales in the discocellular black spot are similar by size and shape to those in the blackish postdiscal band (Fig. 6B). In P. nebrodensis male, similar pattern occurs in the black postdiscal spot (if present) and in the space between veins Sc and R2 (Fig. 6E-F). Contrastingly, black scales at the blackish postdiscal band in P. mnemosyne s. str. female, and those at the black postdiscal spot and space between veins Sc and R2 in this species’ male are considerably smaller, with elongate, piliform shape (Fig. 6C-D, 6G-H). (3) Male genitalia structure (Fig. 7). P. nebrodensis shares a smaller and weakly sclerotized uncus; the uncus arms are triangular and straight, with narrow and sharp apex and a broad base (Fig. 7A3, 7A5, 7B1, and 7B3). Apical process of the P. nebrodensis valva is rounded at the end (Fig. 7A6 and 7B4). Aedeagus is slightly shorter (1.4 times longer than the valva’s length) (Fig. 7A1, 7A2, 7A4, and 7B2) than that in P. mnemosyne s. str. In contrast, P. mnemosyne s. str. shares a larger and strongly sclerotized uncus; the uncus arms are narrower at the base, with broader and thicker apex (Fig. 7C3, 7C5, 7D1, and 7D3). Apical process of the valva is flattened at the end (Fig. 7C6 and 7D4). Aedeagus is slightly longer (1.5 times longer than the valva’s length) (Fig. 7C1, 7C2, 7C4, and 7D2) than that in P. nebrodensis.

Table 4. Inter- and intraspecific genetic divergence of Parnassius species (subgenus Driopa) based on the mitochondrial COI gene fragment.

Minimum Maximum Mean Number of interspecific intraspecific intraspecific Species sequences sequence sequence sequence divergence (%) divergence (%) divergence (%) P. nebrodensis stat. rev. 42 3.94 2.22 0.98 P. mnemosyne s. str. 36 2.71 1.86 0.60 P. sp. Middle East 4 2.71 3.80 2.29 P. phoebus [=P. ariadne] 5 3.84 0.53 0.25 P. stubbendorfii 6 3.60 0.88 0.63 P. hoenei 17 3.60 0.91 0.37 P. glacialis 5 5.63 0.77 0.44 P. eversmanni 5 2.57 0.48 0.20 P. orleans 4 4.67 0.48 0.49 P. nordmanni 3 5.45 0.00 0.00 P. clodius 5 2.57 1.78 1.19

Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for explanation).

A brief morphological re-description of Parnassius nebrodensis Male external morphology and markings (Figs 4A, 5A, 6E-F). Forewing length is 28–31 mm. Antenna dark- brown, club black, fusiform, with narrower terminal segments (apiculus). Eye brown, surrounded by greyish- black scales. Frons and top of the head with mixed grey and black hairs. Palpus with mixed grey and black hairs on each segment. Thorax black with long yellowish-grey hairs. Legs black with grey hairs. Abdomen black with greyish-white hairs dorsally, and with yellowish-grey hairs ventrally. Upperside of the forewing with milk-white ground color and black veins. The base of the forewing black. Cell with two rectangular (or oval) black spots. Apical and subapical areas darkened. Black diamond-shaped scales in the black spot at the end of the cell are similar in size and shape to the scales in the black postdiscal spot (if present) and in the space between veins Sc and R2. Hindwing with the same ground color and black veins as those on the forewing; basal area and dorsum black, with dense greyish-black hairs. Unclear black spot in the basal part of the space between veins M1 and M2. The distal ends of veins usually marked with black. Underside of both wings with the same markings pattern as that on the upperside. Male genitalia (Figs 7A1-A6, 7B1-B4). Typical Driopa pattern. Uncus deeply bifurcated and weakly sclerotized, its two arms are triangular and straight (running parallel to each other), with narrow and sharp apex. Apical process of the valva rounded distally, with a curved thick spur. Aedeagus slender, 1.4 times longer than the length of the valva, broadened at the base. Saccus cylindrical, with a rounded apex, short, as long as 1/5 of the general length of the genitalia.

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Figure 4. Dorsal view of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str. specimens [coll. RMBH]. (A) Male of P. nebrodensis [France: Pyrenees, foothill of Mt. Neouville]. (B) Female of P. nebrodensis [the same locality]. (С) Male of P. mnemosyne [Republic of Belarus: 5 km E from Ruda Yavorskaya village]. (D) Female of P. mnemosyne [the same locality]. (Photos: Artem A. Frolov).

Female external morphology and markings (Figs 4B, 5B, and 6B). Forewing length is 29–30 mm. Antenna, eye, frons, top of the head, palpus, thorax, legs, and abdomen with the same patterns as those in male. Upperside of the forewing with milk-white ground color and black veins. The base of the forewing blackish. Cell with two rectangular (or oval) black spots. The postdiscal area with a transverse blackish band and an unclear blackish spot. Marginal area of the forewing strongly darkened. Black diamond shape scales in the black spot at the end of the forewing’s cell are identical to those forming the blackish postdiscal band. Hindwing with the same ground color and black markings along veins as those on the forewing. Basal area and dorsum black, with dense greyish-black hairs. The basal part of the space between veins M1 and M2 with a large black spot, which merges with black markings in the basal area and along the dorsum of the hindwing. The costal margin with a rounded black spot. Underside of both wings with the same markings pattern as that on the upperside. Female genitalia (Fig. 7E). Typical Driopa pattern. Ductus bursae thick, with a small funnel-shaped extension at the connection with membranous corpus bursae.

Distribution and biology of Parnassius nebrodensis Species within the P. mnemosyne species complex share largely allopatric ranges (Fig. 1). In particular, P. nebrodensis is endemic to the Western Mediterranean mountain ranges, i.e. the Pyrenees, Western and Central Alps, Apennines, and Nebrodi and Madonie on Sicily (Fig. 2). Habitats of this species are situated within the altitudinal belt of 900 to 2200 m (Dannehl 1929; Eisner 1968; Napolitano and Descimon 1994; Descimon 1995, 2006; Weiss 1999; Gratton 2006). In Italy, P. nebrodensis mostly occurs at the edges between deciduous forest and meadows, and flies from late May to mid-July (Gratton 2006). Corydalis

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Figure 5. Forewing venation of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str. (A) Male of P. nebrodensis [France: Pyrenees, foothill of Mt. Neouville]. (B) Female of P. nebrodensis [the same locality]. (C) Male of P. mnemosyne [Moldova]. (D) Female of P. mnemosyne [Linnaeus’s lectotype, Finland; redrawn after Honey and Scoble 2001a]. Red arrows indicate diagnostic features of the two species (see differential diagnosis for detail). (Graphics: Ivan N. Bolotov).

Taxonomic issues related to Parnassius nebrodensis More than 40 P. mnemosyne subspecies were described from the West Mediterranean Region (Turati 1907; Verity 1907; Fruhstorfer 1908; Pagenstecher 1911; Dannehl 1929; Ackery 1973; Eisner 1958, 1968, 1971, 1974, 1976, 1978; Sala and Bollino 1992; Weiss 1999; Beccaloni et al. 2003) (Table 5). The oldest names introduced from this region are as follows: P. mnemosyne nebrodensis Turati, 1907 (Sicily, the type locality (TL): “monti Nebrodi”), P. m. pyraenaeca Turati, 1907 (France, TL: “Gèdre, negli alti Pirenei”), and P. m. pyrenaica Verity, 1907 (France, TL: “Pyrenees Orientalis”). The last two names were found to be primary homonyms of P. apollo pyrenaica Harcourt, 1896 (Fruhstorfer 1908). Fruhstorfer (1908) assumed that the work of Turati (1907) was published in 1908. However, the numbers of 1–3 of the “Naturalista Siciliano”’s volume 20 containing Turati’s “Nuove forme di Lepidotteri” were issued in 1907. Further revisions also fixed this year (Eisner 1978; Weiss 1999; Beccaloni et al. 2003). Hence, P. nebrodensis is considered here as a valid name for the Western Mediterranean lineage of P. mnemosyne species complex (=P. mnemosyne sp.3 sensu Condamine, 2018). Altogether 38 subspecies of P. mnemosyne described from this region are considered here to be synonyms of P. nebrodensis based on the

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Figure 6. Forewing scales of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str. [coll. RMBH]. (A) Position of the enlarged fragment with scale patterns at the forewing. (B) Female of P. nebrodensis [France]. (C) Female of P. mnemosyne [Moldova]. (D) Female of P. mnemosyne [Northern European Russia]. (E) Male of P. nebrodensis [France]. (F) Male of P. nebrodensis [France]. (G) Male of P. mnemosyne [Moldova]. (H) Male of P. mnemosyne [Northern European Russia]. Red and blue arrows indicate diagnostic features of each species for the female and male, respectively (see differential diagnosis for detail). (Graphics and photos: Ivan N. Bolotov).

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Figure 7. Male and female genitalia of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str. [coll. RMBH]. (A) Male genitalia of P. nebrodensis [France; slide RMBH G0328]: (A1) lateral view, (A2) ventral view, (A3) dorsal view, (A4) aedeagus, (A5) uncus, and (A6) apical process of the valva. (B) Male genitalia of P. nebrodensis [France; slide RMBH G0330]: (B1) dorsal view, (B2) aedeagus, (B3) uncus, and (B4) apical process of the valva. (C) Male genitalia of P. mnemosyne [Moldova; slide RMBH G0279]: (C1) lateral view, (C2) ventral view, (C3) dorsal view, (C4) aedeagus, (C5) uncus, and (C6) apical process of the valva. (D) Male genitalia of P. mnemosyne [Belarus; slide RMBH G0004]: (D1) dorsal view, (D2) aedeagus, (D3) uncus, and D4) apical process of the valva. (E) Female genitalia of P. nebrodensis [France; slide RMBH G0331]. (F) Female genitalia of P. mnemosyne [Moldova; slide RMBH G0280]. (G) Female genitalia of P. mnemosyne [Belarus; slide RMBH G0005]. Black arrows indicate diagnostic features of the two species (see differential diagnosis for detail). (Photos: Artem A. Frolov).

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PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE type localities and available molecular data (Table 5 and Fig. 2). We did not revise several nominal taxa that can also belong to P. nebrodensis but whose type localities are situated within a narrow contact zone between the first species and P. mnemosyne s. str. in the Alps. These taxa are as follows: P. mnemosyne adamellicus Kunz, 1922, P. mnemosyne ausonicus Bryk, 1912, P. mnemosyne benacensis Dürck, 1922, P. mnemosyne cuneifer Fruhstorfer, 1908, P. mnemosyne lessinicus Dannehl, 1933, P. mnemosyne venetus Wagner, 1910 (see Weiss 1999). Their status and taxonomic affinities should be determined in the future using newly collected topotypes. Ackery (1973) listed subspecies P. mnemosyne esperi Bryk, 1922 (TL: “Mt. Superga, Piémont, Italien”) but earlier Eisner (1966) showed that the type locality of this taxon is erroneous, as the type series was collected in Württemberg, Germany. The intraspecific of P. nebrodensis and P. mnemosyne s. str. needs further revision using morphological and molecular analyses of topotypes. As for P. nebrodensis, there are at least two more or less divergent COI subclades within this species: (1) Sicily and southern Italy (Calabria), and (2) the Pyrenees, and the Italian Peninsula with Central and Western Alps (Gratton 2006; this study: Fig. 3). At first glance, these haplotype groups can correspond to subspecies-level taxa. For instance, the Sicilian populations might have evolved in isolation during approximately 400 Ka, and could represent a specific evolutionary unit (Gratton 2006).

Table 5. Subspecies of Parnassius mnemosyne (Linnaeus, 1758) synonymized with P. nebrodensis Turati, 1907 stat. rev. in this study.

Coordinates of the type No Taxa** Country Type locality locality*** * Latitude Longitude 1 P. mnemosyne aldinae Nardelli & Italy Sicily: eastern Nebrodi Mts, Cesaro, 37°52' N 14°41' E Giandolfo, 1991 syn. nov. Villa Miraglia 2 P. mnemosyne arollaensis Eisner, 1938 syn. Switzerland Arolla, Wallis 46°01′ N 07°29′ E nov. 3 P. mnemosyne calabricus Turati, 1911 syn. Italy Calabria: Aspromonte 38°10′ N 16°00′ E nov. 4 P. mnemosyne cassiensis Stépi, 1909 syn. France Ste. Baume Mt., near Marseille 43°19′ N 05°45′ E nov. 5 P. mnemosyne cayollensis Dujardin, 1967 France Southern Alps: Cayolle Pass 44°15' N 06°45' E syn. nov. 6 P. mnemosyne ceuzensis Eisner, 1957 syn. France Céüse Pass, south of Grenoble 44°31′ N 05°56′ E nov. 7 P. mnemosyne constantinii Turati, 1919 Italy Apennines: Lago Santo, S. 44°24′ N 10°00′ E syn. nov. Pelligrino 8 P. mnemosyne cosenzaensis Eisner, 1978 Italy Lago di Ampollino, Provinz 39°12′ N 16°38′ E syn. nov. Cosenza 9 P. mnemosyne costarum Bryk, 1922 syn. Italy Roccaraso, Valle de Petrella, 41°51′ N 14°05′ E nov. Caserta 10 P. mnemosyne dinianus Fruhstorfer, 1908 France Digne 44°06′ N 06°14′ E syn. nov. 11 P. mnemosyne ekplektus Rütimeyer, 1968 Switzerland Bern: Bundalp, Kiental 46°32′ N 07°46′ E syn. nov. 12 P. mnemosyne euaquilensis Bryk & Eisner, Italy Abruzzi: Gran Sasso 42°28′ N 13°33′ E 1932 syn. nov. 13 P. mnemosyne eucomitis Bryk & Eisner, Italy Abruzzi: Maiella 42°03′ N 14°03′ E 1932 14 P. mnemosyne excelsus Verity, 1911 syn. France French Alps: Mt Cenis 45°15′ N 06°54′ E nov. 15 P. mnemosyne fruhstorferi Turati, 1909 syn. Italy Italia centr., Mt Autore 41°57′ N 13°12′ E nov. 16 P. mnemosyne gallicus Bryk & Eisner, 1930 France Savoyen: Bonnéval-sur-Arc 45°22′ N 07°03′ E syn. nov. 17 P. mnemosyne guccinii Sala & Bollino, Italy Apennines: Passo della Cisa 44°28′ N 09°56′ E 1992 syn. nov. 18 P. mnemosyne hunti Dujardin, 1968 syn. France Southern Alps: St. Barnabé 43°48′ N 07°02′ E nov. 19 P. mnemosyne matuta Bryk, 1922 syn. nov. France Mte. Authion bei Sospel 43°53′ N 07°27′ E 20 P. mnemosyne mixtus Fruhstorfer, 1922 Switzerland Binn (also syntypes from Berisal 46°22′ N 08°11′ E syn. nov. and Lötschental)

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Coordinates of the type No Taxa** Country Type locality locality*** * Latitude Longitude 21 P. mnemosyne montdorensis Kolar, 1943 France Northern Massif Central: Mont Dore 45°34′ N 02°48′ E syn. nov. (Puy-de-Dôme) 22 P. mnemosyne ozalensis Bustillo & de Spain Northern Spain: Selva de Oza, 42°51′ N 00°42′ W Aizpurua, 1977 syn. nov. Hecho (Huesca) 23 P. mnemosyne parmenides Fruhstorfer, France Alpes maritimes 43°50′ N 07°10′ E 1908 syn. nov. 24 P. mnemosyne phaiohyalinus Rütimeyer, Switzerland Bern: Unteres, Urbachtal, südlich 46°26′ N 08°13′ E 1968 syn. nov. von Innertkirchen 25 P. mnemosyne puschlavensis Eisner, 1758 Switzerland Le Prese, Puschlav 46°18′ N 10°05′ E syn. nov. 26 P. mnemosyne rencurelensis Vergely & France Mt. Noir, Vercors 43°27′ N 02°20′ E Willien, 1972 syn. nov. 27 P. mnemosyne republicanus Peebles & Spain Hispania, Maladetta, Val de l'Esera 42°13′ N 00°21′ E Bryk, 1931 syn. nov. 28 P. mnemosyne rogervarleti Eisner & Switzerland Val Lavizarra, Ticino 46°26′ N 08°40′ E Epstein, 1968 syn. nov. 29 P. mnemosyne romanus Garavaglia, 1940 Italy Abruzzi: Terminillo 42°29′ N 13°00′ E syn. nov. 30 P. mnemosyne sbordonii Eisner & Racheli, Italy Mt Vulture 40°57′ N 15°38′ E 1971 syn. nov. 31 P. mnemosyne schawerdae Bryk, 1922 syn. Italy Apennines: Mti Sibillini, Mt 43°51′ N 11°45′ E nov. Nerone, Passo la Calla 32 P. mnemosyne symphorus Fruhstorfer, 1910 Italy Macugnaga 45°58′ N 07°58′ E syn. nov. 33 P. mnemosyne temora Fruhstorfer, 1922 Switzerland Glarus: Lake Talalpsee 47°06′ N 09°08′ E syn. nov. 34 P. mnemosyne tergestus Fruhstorfer, 1910 Switzerland Kanton Uri, Umgebung von Erstfeld 46°53′ N 08°38′ E syn. nov. 35 P. mnemosyne thebaida Fruhstorfer, 1922 Switzerland Val Maggina am Nordfuss des Mt. 46°08′ N 09°04′ E syn. nov. Camogh 36 P. mnemosyne turatii Fruhstorfer, 1908 syn. France Central Pyrénées: Gedre, Cauterets 42°53′ N 00°07′ W nov. 37 P. mnemosyne vernetanus Fruhstorfer, 1908 France Pyrénées Orientales 42°30′ N 02°45′ E syn. nov. 38 P. mnemosyne vivaricus Bernardi & Viette, France Southern Massif Central: Chambon 45°56′ N 00°41′ E 1961 syn. nov. Forest

*The numbers of localities are correspond to their numbers on the map (Fig. 2). **Taxonomic names and localities are given based on Weiss (1999). ***The majority of coordinates are rather approximate (±5 km or even more) due to the vague type localities in old references, being linked to a vast region.

Conclusion

Recent multi-locus phylogenetic studies revealed that P. mnemosyne sensu lato represents a complex of cryptic species that contains P. mnemosyne s. str. and two additional species-level taxa – P. mnemosyne sp.2 from the Middle East and P. mnemosyne sp.3 from Southern Europe (Condamine 2018; Condamine et al. 2018). In respect to the high levels of genetic divergence and weak morphological differences, the P. mnemosyne species complex is similar to a group of cryptic taxa discovered within the genus Leptidea Billberg, 1820 (Pieridae) (Dincă et al. 2011). The latter complex contains three species: Leptidea sinapis (Linnaeus, 1758), L. reali (Reissinger, 1989), and L. juvernica (Williams, 1946). These morphologically cryptic white-wood butterfly taxa can reliably be identified by means of either DNA sequences or karyological data (Dincă et al. 2011; Lehtonen et al. 2017; Talla et al. 2019). Additional remarkable examples of cryptic species were recently discovered within temperate and tropical butterflies such as (Ochsenheimer, 1808) (Hesperiidae), Lasiommata spp. (Nymphalidae), Polyommatus valiabadi (Rose & Schurian, 1977), and Rhamma spp. (Lycaenidae) (Lukhtanov et al. 2015; Prieto et al. 2019; Platania et al. 2020b; Hinojosa et al. 2021). Here, we show that P. mnemosyne sp.3 sensu Condamine (2018) and Condamine et al. (2018) belongs to P. nebrodensis. It is a distinct species and can be distinguished from P. mnemosyne s. str. based

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PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE on a combination of morphological features such as the forewing venation, forewing scales pattern, and the male genitalia structure. Phylogenetically, it is the most distant taxon among the P. mnemosyne species complex. This species complex contains not less than three additional species: P. mnemosyne s. str., P. phoebus [=P. ariadne], and P. sp. ‘Middle East’ (Omoto et al. 2004; Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Condamine 2018; Condamine et al. 2018). The status of Parnassius sp. ‘Middle East’ (=P. mnemosyne sp.2) from southern Anatolia and Iran (Gratton 2006; Gratton et al. 2008; Condamine 2018; Condamine et al. 2018) remains unclear, although it may belong to a separate cryptic species. However, the discussion on the taxonomic placement of this taxon is beyond the scope of this study, and will be published elsewhere. The P. stubbendorfii species complex represents another example of a clade combining allopatric cryptic taxa among the subgenus Driopa, and it contains P. stubbendorfii, P. hoenei, and P. glacialis (e.g. Yagi et al. 2011; Condamine et al. 2018; this study). It should be noted that subspecies-level taxa in Parnassius usually share much lower genetic distances (e.g. Yakovlev et al. 2020). Although Gratton (2006) assumed that the wing pattern’s differentiation could trigger a pre copula mechanism of reproductive isolation between P. nebrodensis and P. mnemosyne s. str., we show that these taxa share clear differences in the male genitalia structure, most likely precluding their interbreeding at the contact zonas of secondary sympatry (e.g. in northeastern Italy: see Figs 1-2). Michel et al. (2008) suggested a possibility of interspecific hybridization events between these clades, while Gratton (2006) revealed the lack of nuclear exchange between them using the EF-1a gene fragment. The latter conclusion was supported by our research based on the the Wg gene. In summary, the species status of P. nebrodensis is confirmed on the basis of five genes: COI, ND1, ND5, Wg, and EF-1a (Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Condamine 2018; Condamine et al. 2018; this study). P. nebrodensis is known to occur disjunctively throughout the Western Mediterranean mountain ranges (Spain, France, Andorra, Italy, and southeastern Switzerland). This region houses a plethora of regional endemic species, and shares the maximum level of butterfly endemism in Europe (Kudrna et al. 2011). Among papilionids, cassandra (Geyer, [1830]) and Z. rumina (Linnaeus, 1767) are characteristic examples of taxa, the local ranges of which are confined to certain parts of the Western Mediterranean Region (Kudrna et al. 2011; Zinetti et al. 2013; Camerini et al. 2018). Interestingly, P. nebrodensis reveals the high levels of intraspecific genetic variability in the COI gene. This unusual genetic pattern could be driven by a strong metapopulation structure at the regional scale, with a number of local populations having a larger or smaller degree of isolation from others, e.g. in southern France (Napolitano et al. 1988; Napolitano and Descimon 1994; Descimon 1995, 1996) and Italy (Gratton 2006). It should be noted that the distribution of P. nebrodensis is restricted compared with that of P. mnemosyne s. str. It occurs within a narrow range of mid- and high-altitude habitats, with several populations being threatened or declining due to human activities (Descimon 1995, 2006; Gratton 2006; Mérit and Mérit 2006). Moreover, rapid climate warming may reduce the altitudinal belt suitable to Parnassius butterflies at the southern edge of Europe (Descimon 2006). Hence, P. nebrodensis may be considered an endangered species due to its limited distribution (Figs 1-2), narrow range of habitats, and continuing population decline, although this tentative assessment must be clarified in the future. This species contains a variety of more or less isolated mountain populations, and special management and conservation efforts are urgently needed to prevent further fragmentation of its disjunctive range.

Acknowledgements This study was partly supported by the Ministry of Science and Higher Education of the Russian Federation (projects АААА-А17-117033010132-2 to Y.S.K. and V.M.S., and АААА-А18-118012390161-9 to M.Y.G.), and Russian Foundation for Basic Research (project 18-44-292001 to I.N.B. and A.V.K.). We are grateful to Dr. Oleg S. Pokrovsky and Dr. Lyudmila S. Shirokova (Toulouse, France), and to Artem A. Frolov (Moscow, Russia) for their help during this study.

References

Ackery, P. R. (1973) A list of the type specimens of Parnassius (Lepidoptera: Papilionidae) in the British Museum (Natural History). Bulletin of the British Museum of Natural History (Entomology), 29, 1– 35.

158

BOLOTOV ET AL.

Ackery, P. R. (1975) A guide to the genera and species of (Lepidoptera: Papilionidae). Bulletin of the British Museum of Natural History (Entomology), 31(4), 71–105. Aksenova, O. V., Bolotov, I. N., Gofarov, M. Y., Kondakov, A. V., Vinarski, M. V., Bespalaya, Y. V., Kolosova, Y. S., Palatov, D. M., Sokolova, S. E., Spitsyn, V. M., Tomilova, A. A., Travina, O. V. & Vikhrev, I. V. (2018) Species richness, molecular taxonomy and biogeography of the radicine pond snails (Gastropoda: Lymnaeidae) in the Old World. Scientific Reports, 8, 11199. https://doi.org/10.1038/s41598-018-29451-1 Beccaloni, G., Scoble, M., Kitching, I., Simonsen, T., Robinson, G., Pitkin, B., Hine, A. & Lyal, C. (Eds.) (2003) The Global Lepidoptera Names Index (LepIndex). The Natural History Museum, London, UK. Available from: https://www.nhm.ac.uk/our-science/data/lepindex/lepindex/ (accessed 15 January 2020). Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K., Meier, R., Winker, K., Ingram, K. K. & Das, I. (2007) Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution, 22(3), 148–155. https://doi.org/10.1016/j.tree.2006.11.004 Bolotov, I. N., Gofarov, M. Y., Rykov, A. M., Frolov, A. A. & Kogut, Y. I. (2013) Northern boundary of the range of the Clouded Apollo butterfly Parnassius mnemosyne (L.) (Papilionidae): climate influence or degradation of larval host plants? Nota lepidopterologica, 36 (1), 19–33. Bolotov, I. N., Kondakov, A. V. & Spitsyn, V. M. (2018) A review of tiger moths (Lepidoptera: Erebidae: Arctiinae: Arctiini) from Flores Island, Lesser Sunda Archipelago, with description of a new species and new subspecies. Ecologica Montenegrina, 16, 1–15. https://doi.org/10.37828/em.2018.16.1 Brower, A. V. Z. & DeSalle, R. (1998) Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference. Molecular Biology, 7(1), 73–82. https://doi.org/10.1046/j.1365-2583.1998.71052.x Camerini, G., Groppali, R. & Minerbi, T. (2018) Observations on the ecology of the endangered butterfly Zerynthia cassandra in a protected area of Northern Italy. Journal of Insect Conservation, 22, 41–49. https://doi.org/10.1007/s10841-017-0036-6 Condamine, F. L. (2018) Limited by the roof of the World: Mountain radiations of Apollo swallowtails controlled by diversity-dependence processes. Biology Letters, 14(3), 20170622. https://doi.org/10.1098/rsbl.2017.0622 Condamine, F. L., Rolland, J., Höhna, S., Sperling, F. A. & Sanmartín, I. (2018) Testing the role of the Red Queen and Court Jester as drivers of the macroevolution of Apollo butterflies. Systematic Biology, 67(6), 940–964. https://doi.org/10.1093/sysbio/syy009 Dannehl, F. (1929) Neue Formen und geographische Rassen aus meinen Ausbeuten und Erwerbungen der letzten Jahre. Mitteilungen der Münchner Entomologischen Gesellschaft, 19 (5–9), 97–116. Dapporto, L., Cini, A., Vodă, R., Dincă, V., Wiemers, M., Menchetti, M., Magini, G., Talavera, G., Shreeve, T., Bonelli, S., Casacci, L. P., Balletto, E., Scalercio, S. & Vila, R. (2019) Integrating three comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and paleogeographic events in European butterflies. Molecular Ecology Resources, 19(6), 1623–1636. https://doi.org/10.1111/1755-0998.13059 Descimon, H. (1995) La conservation des Parnassius en France: aspects zoogéographiques, écologiques, démographiques et génétiques. Rapport d'études de l'OPIE, 1, 1–54. http://www.insectes.xyz/re- parnass.htm Descimon, H. (2006). La conservation des Parnassius de France. Situation en 1995 et situation en 2006, 11 ans après. Bulletin des Lépidoptéristes Parisiens, 15 (33), 34–55. Dincă, V., Lukhtanov, V., Talavera, G. & Vila, R. (2011) Unexpected layers of cryptic diversity in wood white Leptidea butterflies. Nature Communications, 2, 324. https://doi.org/10.1038/ncomms1329 Dincă, V., Montagud, S., Talavera, G., Hernández-Roldán, J., Munguira, M. L., García-Barros, E., Hebert, P. D. N. & Vila, R. (2015) DNA barcode reference library for Iberian butterflies enables a continental- scale preview of potential cryptic diversity. Scientific Reports, 5, 12395. https://doi.org/10.1038/srep12395 Eisner, C. (1958) Parnassiana Nova XVIII. Varia. Zoologische Mededelingen, 34(1), 1–3. Eisner, C. (1966) Parnassiidae-Typen in der Sammlung J. C. Eisner. Zoologische Verhandelingen, 81(1), 1– 81. Eisner, C. (1968) Parnassiana Nova XLIII. Nachträgliche Betrachtungen zu der Revision der Subfamilie Parnassiinae (Fortsetzung 16). Zoologische Mededelingen, 43(2), 9–17.

Ecologica Montenegrina, 40, 2021, 140-163 159

PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE

Eisner, C. (1971) Parnassiana Nova XLVI. Nachträgliche Betrachtungen zu der Revision der Subfamilie Parnassiinae (Fortsetzung 19). Zoologische Mededelingen, 45(6), 87–90. Eisner, C. (1974) Parnassiana Nova XLIX. Die Arten und Unterarten der Baroniidae, Teinopalpidae und Parnassiidae (Erster Teil) (Lepidoptera). Zoologische Verhandelingen, 135, 3–96. Eisner, C. (1976) Parnassiana Nova XLIX. Die Arten und Unterarten der Baroniidae, Teinopalpidae und Parnassiidae (Zweiter Teil) (Lepidoptera). Zoologische Verhandelingen, 146, 99–259. Eisner, C. (1978) Parnassiana Nova LIV. Dr. S. Wagener's Bemerkungen zu den Parnassus-Formendes Apennin aus Geografisch-Ökologischer Sicht. Zoologische Mededelingen, 53(21), 237–243. Fruhstorfer, H. (1908) Neue Parnassier aus der mnemosyne-Gruppe. Internationale Entomologische Zeitschrift, 3, 17–18. Gratton, P. (2006) Phylogeography and conservation genetics of Parnassius mnemosyne L., 1758 (Lepidoptera, Papilionidae). Doctoral dissertation. Tor Vergata University of Rome, Italy, 102 pp. Gratton, P., Konopiński, M. K. & Sbordoni, V. (2008) Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a ‘time-dependent’ mitochondrial substitution rate. Molecular Ecology, 19, 4248–4262. https://doi.org/10.1111/j.1365- 294X.2008.03901.x Hall, T. A. (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98. Hanus J. & Theye M.-L. (2010) (Fabricius, 1793), a misidentified species (Lepidoptera: Papilionidae). Nachrichten des Entomologischen Vereins Apollo, 31(1–2), 7–84. Hinojosa, J. C., Dapporto, L., Brockmann, E., Dincă, V., Tikhonov, V., Grishin, N., Lukhtanov, V. A. & Vila, R. (2021) Overlooked cryptic diversity in Muschampia (Lepidoptera: Hesperiidae) adds two species to the European butterfly fauna. Zoological Journal of the Linnean Society, zlaa171. https://doi.org/10.1093/zoolinnean/zlaa171 Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. (2017) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35(2), 518–522. https://doi.org/10.1093/molbev/msx281 Honey, M. R. & Scoble, M. J. (2001) Linnaeus's Butterfly Type Specimens. The Natural History Museum, London, UK. Available from: http://www.nhm.ac.uk/research-curation/research/projects/linntypes (accessed 06 July 2013) Honey, M. R. & Scoble, M. J. (2001a) Linnaeus's butterflies (Lepidoptera: Papilionoidea and Hesperioidea). Zoological Journal of the Linnaean Society, 132, 277–399. https://doi.org/10.1006/zjls.2001.0265 Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587–589. https://doi.org/10.1038/nmeth.4285 Kapli, P., Lutteropp, S., Zhang, J., Kobert, K., Pavlidis, P., Stamatakis, A. & Flouri, T. (2017) Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo. Bioinformatics, 33(11), 1630–1638. https://doi.org/10.1093/bioinformatics/btx025 Kim, M., Wan, X., Kim, M. J., Jeong, H. C., Ahn, N.-H., Kim, K.-G., Han, Y. S. & Kim, I. (2010) Phylogenetic relationships of true butterflies (Lepidoptera: Papilionoidea) inferred from COI, 16S rRNA and EF-1α sequences. Molecules and Cells, 30, 409–425. https://doi.org/10.1007/s10059-010- 0141-9 Konopinski, M. K. (2008) A set of primers conserved in genus Parnassius (Lepidoptera, Papilionidae) for amplification and sequencing of 1016 bp fragment of cytochrome oxidase subunit I from museum specimens. Molecular Ecology Resources, 8, 675–677. https://doi.org/10.1111/j.1471- 8286.2007.02045.x Korshunov, Y. P. (2002) Butterflies of the Northern Asia [In Russian]. KMK Scientific Press Ltd., Moscow, 424 pp. Kudrna, O., Harpke, A., Lux, K., Pennerstorfer, J., Schweiger, O., Settele, J. & Wiemers, M. (2011) Distribution Atlas of butterflies in the Europe. Gesellschaft fur Schmetterlingschutz, Halle, 576 pp. Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870–1874. https://doi.org/10.1093/molbev/msw054

160

BOLOTOV ET AL.

Lehtonen, S., Lehtonen, I., Teräs, A., Varrela, J., Virta, P. & Vesterinen, E. J. (2017) DNA barcoding reveals widespread occurrence of Leptidea juvernica (Lepidoptera: Pieridae) in southern Finland. Entomologisk Tidskrift, 138, 151–159. Leraut, P. J. A. (1997) Liste systématique et synonymique des Lépidoptères de France, Belgique et Corse (deuxième édition). Alexanor Supplément, Paris, 526 pp. Litman, J., Chittaro, Y., Birrer, S., Praz, C., Wermeille, E., Fluri, M., Stalling, T., Schmid, S., Wyler, S. & Gonseth, Y. (2018) A DNA barcode reference library for Swiss butterflies and forester moths as a tool for species identification, systematics and conservation. PLoS ONE, 13(12), e0208639. https://doi.org/10.1371/journal.pone.0208639 Lukhtanov, V. A., Dantchenko, A. V., Vishnevskaya, M. S. & Saifitdinova, A. F. (2015) Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society, 116(2), 468–485. https://doi.org/10.1111/bij.12596 Lukhtanov, V. A., Sourakov, A., Zakharov, E. V. & Hebert, P. D. (2009) DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification. Molecular Ecology Resources, 9(5), 1302–1310. https://doi.org/10.1111/j.1755- 0998.2009.02577.x Luoto, M., Kuussaari, M., Rita, H., Salminen, J. & von Bonsdorff, T. (2001) Determinants of distribution and abundance in the Clouded Apollo butterfly: a landscape ecological approach. Ecography, 24, 601–617. https://doi.org/10.1111/j.1600-0587.2001.tb00494.x Mérit, X. & Mérit, V. (2006) Contribution à la connaissance des sous-espèces françaises de Parnassius (Driopa) mnemosyne (Linnaeus, 1758) (Lepidoptera, Papilionidae). Systématique, biologie et implications pour la conservation de l’espèce. Bulletin des Lépidoptéristes Parisiens, 15 (33), 2–7. Michel, F., Rebourg, C., Cosson, E. & Descimon, H. (2008) Molecular phylogeny of Parnassiinae butterflies (Lepidoptera: Papilionidae) based on the sequences of four mitochondrial DNA segments. Annales de la Société entomologique de France (N.S.), 44(1), 1–36. https://doi.org/10.1080/00379271.2008.10697541 Miller, M. A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, 1–8. Müller, A. (1973) Die mnemosyne-Gruppe der Gattung Parnassius Latreille unter Berücksichtigung neuer Schuppenmerkmale ihrer Arten (Lep. Parnassiidae). Deutsche Entomologische Zeitschrift, 20, (1-3), 211–276. Napolitano, M. & Descimon, H. (1994) Genetic structure of French populations of the mountain butterfly Parnassius mnemosyne L. (Lepidoptera: Papilionidae). Biological Journal of the Linnean Society, 53, 325–344. https://doi.org/10.1111/j.1095-8312.1994.tb01016.x Napolitano, M., Geiger, H. J. & Descimon, H. (1988) Structure démographique et génétique de quatre populations provençales de Parnassius mnemosyne (L.) (Lepidoptera Papilionidae): isolement et polymorphisme dans des populations “menaces”. Genetics Selection Evolution, 20, 51–62. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution, 32, 268–274. https://doi.org/10.1093/molbev/msu300 Omoto, K., Katoh, T., Chichvarkhin, A. & Yagi, T. (2004) Molecular systematics and evolution of the “Apollo” butterflies of the genus Parnassius (Lepidoptera: Papilionidae) based on mitochondrial DNA sequence data. Gene, 326, 141–147. https://doi.org/10.1016/j.gene.2003.10.020 Omoto, K., Yonezawa T. & Shinkawa T. (2009) Molecular systematics and evolution of the recently discovered “Parnassian” butterfly (Parnassius davydovi Churkin, 2006) and its allied species (Lepidoptera, Papilionidae). Gene, 441, 80–88. https://doi.org/10.1016/j.gene.2008.10.030 Pagenstecher A. (1911) Über die Geschichte, das Vorkommen und die Erscheinungsweise von Parnassius mnemosyne L. Jahrbücher des Nassauischen Vereins für Naturkunde, 64, 261–310. Platania, L., Menchetti, M., Dincă, V., Corbella, C., Kay‐Lavelle, I., Vila, R., Wiemers, M., Schweiger, O. & Dapporto, L. (2020a) Assigning occurrence data to cryptic taxa improves climatic niche assessments: Biodecrypt, a new tool tested on European butterflies. Global Ecology and Biogeography, 29(10), 1852–1865. https://doi.org/10.1111/geb.13154

Ecologica Montenegrina, 40, 2021, 140-163 161

PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE

Platania, L., Vodă, R., Dincă, V., Talavera, G., Vila, R., & Dapporto, L. (2020b) Integrative analyses on Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species. Journal of Zoological Systematics and Evolutionary Research. https://doi.org/10.1111/jzs.12356 Prieto, C., Nuñez, R., & Hausmann, A. (2019). Molecular species delimitation in the genus Rhamma Johnson, 1992 (Lepidoptera: Lycaenidae, Theclinae). Mitochondrial DNA Part A, 30(1), 101–117. https://doi.org/10.1080/24701394.2018.1462348 Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A. & Huelsenbeck, J. P. (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Systematic Biology, 61(3), 539–542. https://doi.org/10.1093/sysbio/sys029 Sala, G. & Bollino, M. (1992) A new subspecies of Parnassius mnemosyne L. from Tosco-Emilian Apennines and considerations about populations of the same range (Lepidoptera, Papilionidae). Atalanta, 23 (1–2), 123–126. Sikes, D. S., Bowser, M., Morton, J. M., Bickford, C., Meierotto, S. & Hildebrandt, K. (2017) Building a DNA barcode library of Alaska’s non-marine . Genome, 60(3), 248–259. https://doi.org/10.1139/gen-2015-0203 Talla, V., Johansson, A., Dincă, V., Vila, R., Friberg, M., Wiklund, C. & Backström, N. (2019) Lack of gene flow: narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species. Molecular Ecology, 28(16), 3756–3770. https://doi.org/10.1111/mec.15188 Tao, R., Xu, C., Wang, Y., Sun, X., Li, C., Ma, J., Hao, J. & Yang, Q. (2020) Spatiotemporal differentiation of Alpine butterfly (Papilionidae: Parnassiinae) in China: Evidence from mitochondrial DNA and nuclear single nucleotide polymorphisms. Genes, 11(2), 188. https://doi.org/10.3390/genes11020188 Todisco, V., Gratton, P., Cesaroni, D. & Sbordoni, V. (2010) Phylogeography of Parnassius apollo: hints on taxonomy and conservation of a vulnerable glacial butterfly invader. Biological Journal of the Linnean Society, 101(1), 169–183. https://doi.org/10.1111/j.1095-8312.2010.01476.x Trifinopoulos, J., Nguyen, L. T., von Haeseler, A. & Minh, B. Q. (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44 (W1), W232–W235. https://doi.org/10.1093/nar/gkw256 Turati, C. E. (1907) Nuove forme di Lepidotteri. Naturalista Siciliano, 20 (1–3), 1–48. Väisänen, R. & Somerma, P. (1985) The status of Parnassius mnemosyne (Lepidoptera: Papilionidae) in Finland. Notulae Entomologicae, 65, 109–118. Van Swaay, C. A. M. & Warren, M. S. (1999) Red Data Book of European Butterflies (Rhopalocera). Nature and Environment, No. 99. Council of Europe Publishing, Strasbourg, 259 pp. Van Swaay, C. A. M., Cuttelod, A., Collins, S., Maes, D., Munguira, M. L., Šašić, M., Settele, J., Verovnik, R., Verstrael, T., Warren, M., Wiemers, M. & Wynhoff, I. (2010) European Red List of Butterflies. Publications Office of the European Union, Luxembourg, 47 pp. Van Swaay, C., Collins , S., Dušej, G., Maes, D., Munguira M. L., Rakosy, L., Ryrholm , N., Šašić, M., Settele, J. , Thomas , J. A., Verovnik , R., Verstrael , T., Warren, M., Wiemers, M. & Wynhoff, I. (2012) Dos and Don'ts for butterflies of the Habitats Directive of the European Union. Nature Conservation, 1, 73–153. Verity, R. (1907) Rhopalocera Palaearctica, 1, 77–124. Villesen P. (2007) FaBox: an online toolbox for fasta sequences. Molecular Ecology Notes, 7, 965–968. https://doi.org/10.1111/j.1471-8286.2007.01821.x Weiss, J. C. (1999) The Parnassiinae of the World. Part 3. Hillside Books, Canterbury, pp. 137–235. Wiemers, M., Balletto, E., Dincă, V., Fric, Z. F., Lamas, G., Lukhtanov, V., Munguira, M. L., van Swaay, C. A. M., Vila, R., Vliegenthart, A., Wahlberg, N. & Verovnik, R. (2018) An updated checklist of the European butterflies (Lepidoptera, Papilionoidea). ZooKeys, 811, 9–45. https://doi.org/10.3897/zookeys.811.28712 Yagi, T., Katoh, T., Chichvarkhin, A., Shinkawa, T. & Omoto, K. (2001) Molecular phylogeny of butterflies Parnassius glacialis and P. stubbendorfii at various localities in East Asia. Genes & Genetic Systems, 76(4), 229–234. https://doi.org/10.1266/ggs.76.229 Yakovlev, R. V., Shapoval, N. A., Bakhaev, Y. I., Kuftina, G. N. & Khramov, B. A. (2020) A new subspecies of Parnassius arcticus (Eisner, 1968) from the Momsky Range (Yakutia, Russia). Acta Biologica Sibirica, 6, 93–105. https://doi.org/10.3897/abs.6.e55925

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BOLOTOV ET AL.

Zaman, K., Hubert, M. K. & Schoville, S. D. (2019) Testing the role of ecological selection on colour pattern variation in the butterfly . Molecular Ecology, 28(23), 5086–5102. https://doi.org/10.1111/mec.15279 Zinetti, F., Dapporto, L., Vovlas, A., Chelazzi, G., Bonelli, S., Balletto, E. & Ciofi, C. (2013) When the rule becomes the exception. No evidence of gene flow between two Zerynthia cryptic butterflies suggests the emergence of a new model group. PLoS ONE, 8(6), e65746. https://doi.org/10.1371/journal.pone.0065746

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