Phylogeny, Species Delimitation and Biogeography of the Endemic

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Zoological Journal of the Linnean Society, 2021, XX, 1–17. With 4 figures.

Phylogeny, species delimitation and biogeography of the endemic Palaearctic tribe Tomarini (Lepidoptera: Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 Lycaenidae)

ANATOLY V. KRUPITSKY1,2,*, , NAZAR A. SHAPOVAL3, DMITRY M. SCHEPETOV4, IRINA A. EKIMOVA4, and VLADIMIR A. LUKHTANOV3,

1Department of Entomology, Biological Faculty, Lomonosov Moscow State University, Leninskie gory, GSP-1, korp. 12, Moscow 119991, Russia 2Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky prospect 33, Moscow 119071, Russia 3Department of Karyosystematics, Zoological Institute of the Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia 4Department of Invertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskie gory, GSP-1, korp. 12, Moscow 119991, Russia

Received 31 March 2021; revised 20 May 2021; accepted for publication 6 July 2021

The tribe Tomarini is represented by the sole genus Tomares, comprising about eight species distributed from the western Mediterranean to Central Asia. We carried out a multilocus phylogenetic and a biogeographical analysis to test the taxonomy of the genus by several molecular species delimitation methods and reveal patterns shaping the current distribution of Tomares. The phylogenetic analysis based on four molecular markers recovered the monophyly of the genus and recovered two deep-branching lineages: an African clade and an Asian clade. Species delimitation analyses suggested six or ten putative species depending on the method applied. The haplotype network analysis of the Tomares nogelii clade revealed no phylogeographical and taxonomic structure. We consider the taxon Tomares nesimachus (syn. nov.) a synonym of T. nogelii and reinstate Tomares callimachus dentata stat. rev. for populations from south-eastern Turkey. Tomares originated between the early Oligocene and the early Miocene, most probably in south-west Asia. The split of the most recent common ancestor of Tomares occurred between the middle-late Miocene and middle-late Pliocene, probably as a response to increasing aridification and habitat fragmentation. Differentiation of the Asian clade took place in south-west Asia during the Pliocene and Pleistocene and coincided temporally with the evolution of Tomares host plants of the genus Astragalus (Fabaceae).

ADDITIONAL KEYWORDS: evolution – molecular phylogeny – phylogeography – taxonomy.

INTRODUCTION territory of the modern south-western Palaearctic underwent numerous interconnected geological and A large biogeographical area covering the climatic changes (Steininger & Rögl, 1996; Zachos Mediterranean, south-west Asia, the Pontic- et al., 2001). From an evolutionary viewpoint, the Caspian region and Central Asia, also known as most crucial events were uplifts and formations of the south-western Palaearctic, or Tethyan (Ancient mountain systems and islands, the closure of the Mediterranean) Subkingdom of the Boreal Kingdom Tethys Ocean, the Messinian salinity crisis and sensu Takhtajan (1986), is united by geological history Quaternary geological changes, including series of (de Lattin, 1967; Takhtajan, 1986). The distribution glaciations and interglacial periods (Hewitt, 2004; of organisms in the Tethyan region has been strongly Schmitt, 2007; Hamon et al., 2013; Pirouz et al., 2017). influenced by historical factors. In particular, the These events created conditions for the dispersal of organisms and speciation and shaped the region in *Corresponding author. E-mail: [email protected] question as a biodiversity hotspot (Myers et al., 2000).

The south-western Palaearctic is an area containing Nekrutenko & Effendi, 1980, Tomares fedtschenkoi many endemic groups of butterflies, including (Erschoff, 1874) and Tomares mauritanicus (Lucas, remarkable genera of Papilionidae, such as Zerynthia 1849) are characterized by distinct morphological Ochsenheimer, 1816, Allancastria Bryk, 1932, Archon diagnostic traits and are easily recognizable, whereas Hübner, 1822 and Hypermnestra Ménétriés, 1848, the status of the taxa Tomares dobrogensis Caradja,

Pieridae genus Zegris Boisduval, 1836, and numerous 1895, Tomares nesimachus Oberthür, 1894, Tomares Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 genera and subgenera of Nymphalidae and Lycaenidae, nogelii Herrich-Schäfer, 1851, Tomares romanovi including the diverse subgenus Agrodiaetus Hübner, Christoph, 1882 and Tomares telemachus Zhdanko, 1822. This region is also a diversity centre for many 2000, constituting the T. nogelii complex sensu Nazari Holarctic butterfly genera. & Ten Hagen (2020), remains disputable in different Historical biogeography and evolution of the taxonomic works (Van Oorschot & Wagener, 2000; Palaearctic butterflies has been a focus of numerous Weidenhoffer & Bozano, 2007; Nazari & Ten Hagen, studies. Several main geological events have been 2020). The morphological investigations (Van Oorschot hypothesized to play a key role in the diversity and & Wagener, 2000) and integrative studies combining evolution of the south-western Palaearctic butterfly morphological and molecular phylogenetic analyses genera. Quaternary glaciations shaped distribution based on fragments of COI and EF1α genes recently in the case of Pyrgus Hübner, 1819 (Hernández- performed by Nazari & Ten Hagen (2020) have Roldán et al., 2011), Proterebia Roos & Arnscheid, revealed no clear differentiation between the species 1980 (Bartoňová et al., 2018), Maniola Schrank, 1801 of this group. Research by Nazari & Ten Hagen (2020) (Kreuzinger et al., 2015) and Melanargia Meigen, reduced the number of Tomares species to eight by 1828 (Habel et al., 2005). Orogenies and isolations of formally synonymizing T. telemachus with T. romanovi landmasses acted as drivers of diversification in the and reconsidering the status of T. dobrogensis as a case of Allancastria (Nazari & Sperling, 2007; Nazari subspecies of T. nogelii. et al., 2007) and Hypermnestra (Nazari & Sperling, The species of the genus Tomares inhabit warm, 2007). Formation of the Mediterranean Basin, the grassy biotopes, from a coastal zone to a subalpine Messinian salinity crisis and Quaternary glaciations mountain belt ≤ 3000 m a.s.l. (Larsen, 1974; induced radiation and dispersal in Zerynthia (Nazari Hesselbarth et al., 1995; Tuzov et al., 2000). Larval host & Sperling, 2007; Dapporto, 2010), Pararge Hübner, plants of the genus are different species of Fabaceae 1819 (Weingartner et al., 2006; Livraghi et al., 2018) (Higgins & Riley, 1970; Weidenhoffer & Vanek, 1977; and Pseudophilotes Beuret, 1958 (Todisco et al., 2018; Jordano et al., 1990; Hesselbarth et al., 1995; Tennent, Bartoňová et al., 2020). 1996; Tuzov et al., 2000; Muñoz Sariot, 2011). Some One of the butterfly genera endemic to the south- species are monophagous or oligophagous, using only western Palaearctic is Tomares Rambur, 1840, from one or several species of the legume genera Astragalus the family Lycaenidae, subfamily Theclinae, the sole and Astracantha (Zhdanko, 1997; Van Oorschot & member of the tribe Tomarini Eliot, 1973. This tribe Wagener, 2000; Tuzov et al., 2000). Most Tomares was erected on the basis of external morphological species are locally distributed and strongly connected characters (Eliot, 1973). Later, Kuznetsov & Stekolnikov with their host plants. As a consequence, they suffer (2001) confirmed this treatment based on study of the from overgrazing and destruction of habitats owing functional morphology of the male genitalia. In recent to human activities. Some of them are of conservation phylogenetic studies, Tomarini is recovered as a sister concern in several regions; for instance, T. nogelii was group to a clade comprising Eumaeini, Loxurini and listed as a vulnerable species in Europe (regionally Deudorigini (Espeland et al., 2018). extinct in the European Union) in the International According to the taxonomic reviews of the genus, Union for Conservation of Nature European Red Tomares comprises between eight and ten species List of Butterflies (Van Swaay et al., 2010) and the (Hesselbarth & Schurian, 1984; Hesselbarth et al., Red Book of Ukraine (Budashkin & Plyushch, 2009), 1995; Van Oorschot & Wagener, 2000; Tuzov et al., and critically endangered and locally extinct in the 2000; Weidenhoffer & Bozano, 2007; Nazari & Ten Romanian Red List of Butterflies (Rákosy, 2003). Hagen, 2020) distributed throughout the south- The distribution range of the genus Tomares is western Palaearctic. The centre of diversity for the broadly divided into two disjoint parts. The first genus lies in Anatolia (Turkey), where up to six one comprises the territory uniting North Africa species have been recorded, some of them living and the western Mediterranean of Europe from sympatrically in certain localities (Hesselbarth Portugal to France, hosting two species (T. ballus and et al., 1995; Van Oorschot & Wagener, 2000). Species T. mauritanicus). The second part of the range is a wide such as Tomares ballus (Fabricius, 1787), Tomares area uniting the Pontic-Caspian and Irano-Turanian callimachus (Eversmann, 1848), Tomares desinens regions, which is inhabited by the rest of the species.

Tomares is absent in the two large Mediterranean in detail the phylogeography of the taxonomically peninsulas, the Apennines and Balkans, despite the complicated T. nogelii species group; and (4) estimate apparent presence of suitable habitats and host plants diversification times and biogeographical patterns in these regions and their important role in diversity shaping the current distribution of Tomares. and evolution shown for different Palaearctic animal

species (Hewitt, 1996, 2000, 2011; Taberlet et al., Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 1998; Kryštufek, 2004; Schmitt, 2007; Bregović & Zagmajster, 2016), including butterflies (Habel et al., MATERIAL AND METHODS 2005; Dapporto, 2010; Vishnevskaya et al., 2016). Sampling Despite the remarkable distribution pattern, the phylogeny and historical biogeography of Tomares Our ingroup dataset included all currently recognized remain unclear, as are the ages of its main clades and taxa of Tomares except the probably extinct Tomares potential drivers of diversification shaping its modern ballus mareoticus Graves, 1918 from north-east distribution in the south-western Palaearctic. Africa and Israel and the recently described Tomares The first attempt to reconstruct the phylogeny of callimachus huertasae Tshikolovets & Pagès, 2016 Tomares was performed recently by Nazari & Ten from Pakistani Baluchistan. We sequenced 60 Hagen (2020). It was based on a limited number specimens of Tomares collected across the distribution of specimens (28) and two genes (mitochondrial range, and also included in the analysis sequences of 37 specimens from GenBank and bold. As outgroup COI and nuclear EF1α) and resulted in a tree with mostly unsupported basal nodes. Although Nazari & taxa, we selected five species of the main Palaearctic Ten Hagen (2020) solved some taxonomic issues [i.e. lineages of the tribe Eumaeini. Four of them were they proposed several synonyms and reinstated the sequenced before our research [Callophrys rubi subspecies T. callimachus hafis (Kollar, 1849)], the total (Linnaeus, 1758), Callophrys titanus Zhdanko, 1998, phylogeny of Tomares remains unresolved. Taxonomic Satyrium spini (Fabricius [Denis & Shiffermüller], uncertainty remains in the T. nogelii species group: 1775) and Neolycaena baidula Zhdanko, 2000], and different colour morphs were described as distinct one (Ahlbergia korea Johnson, 1992) was obtained from taxa across the range of the group, and their status GenBank. Thus, the final dataset of the phylogenetic needs further exploration. analysis included 102 specimens (for details, see According to the phylogenetic tree covering most Supporting Information, Table S1; for distribution of of the tribes of butterflies (Espeland et al., 2018), the the samples in our study, see Fig. 1). age of the ancestor of Tomarini and a clade uniting the tribes Deudorigini, Eumaeini and Loxurini is estimated at ~35 Myr. The time-calibrated analysis DNA extraction, polymerase chain reaction, of the European butterflies inferred by Wiemers et al. sequencing and partitioning (2020), based on calibration points from Chazot et al. Standard COI barcodes (partial sequences of the (2019), included all the species of Tomares occurring cytochrome c oxidase subunit I gene) were obtained in Europe (T. ballus, T. callimachus and T. nogelii). from a single leg of five T. ballus, four T. mauritanicus, According to this analysis, the most recent common two T. nogelii, 11 T. nesimachus and two T. callimachus ancestor (MRCA) of the Tomarini and Eumaeini samples at the Canadian Centre for DNA Barcoding diverged ~22.62 Mya (29.5–16.6 Mya). The split of (CCDB, Biodiversity Institute of Ontario, University the MRCA of Tomares occurred ~5.55 Mya (9.0–2.7 of Guelph, ON, Canada) using the standard high- Mya) and gave rise to T. ballus and the ancestor of throughput protocol described by deWaard et al. (2008). the clade comprising T. nogelii and T. callimachus. For the rest of the specimens, DNA was extracted The MRCA of T. nogelii and T. callimachus diverged from two legs using PALL AcroPrep 96-well purification ~3.0 Mya (4.9–1.1 Mya) (Wiemers et al., 2020). In plates supplied by PALL Corp., Port Washington, contrast, Nazari & Ten Hagen (2020) suggested New York, USA (Ivanova et al., 2006), following Pleistocene diversification of the genus based on the manufacturer’s protocol, at the Department of the relatively low interspecific divergence in DNA Invertebrate Zoology, Biological Faculty, Moscow State barcodes. University (Moscow, Russia). Extracted DNA was In this paper we: (1) expand the dataset to include used as a template for the amplification of partial all described species of Tomares and nearly all cytochrome c oxidase subunit I (COI), 28S rRNA (28S), subspecies from most of the distribution range and histone H3 (H3) and elongation factor 1-alpha (EF1α) four molecular markers and present a phylogenetic genes. Polymerase chain reaction amplifications were analysis resolving the relationships within the genus; carried out in a 20 μL reaction volume, which included (2) test the current taxonomy of the genus by several 4 μL of 5× Screen Mix (Eurogen, Russia), 0.5 μL of each molecular species delimitation methods; (3) investigate primer (10 μM stock), 1 μL of genomic DNA and 14 μL

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Figure 1. Map showing the origins of the Tomares samples used for phylogenetic analyses in this study.

of ddH2O. The primers and polymerase chain reaction chains were sampled at intervals of 500 generations. protocols are listed in the Supporting Information Two runs of ten million generations with four (Table S2). chains (one cold and three heated) were performed. Sequencing of double-stranded product was Maximum likelihood-based phylogeny inference carried out using the BigDye v.3.1 sequencing kit was performed in the HPC-PTHREADS-AVX (Applied Biosystems). Sequencing reactions were version of RAxML (Stamatakis, 2014) with ultrafast analysed using the ABI 3500 Genetic Analyzer bootstrapping (UFBoot approximation approach; (Applied Biosystems) at the Koltsov Institute Minh et al., 2013) in 1000 pseudoreplicates under of Developmental Biology (Moscow, Russia). We the GTRCAT model of nucleotide evolution. The removed ambiguous regions at the 5′ and 3′ ends of final phylogenetic tree images were rendered in each primer region; hence, our dataset comprised FigTree v.1.4.0 (Rambaut, 2012). We regarded tree a total of 1733 bp (658 bp of COI, 387 bp of 28S, nodes with ML bootstrap (BS) values ≥ 75% and BI 328 bp of H3 and 360 bp of EF1α). All sequences posterior probabilities (PPs) > 0.95 to be sufficiently obtained in the present study were deposited to resolved a priori. GenBank (http://www.ncbi.nlm.nih.gov/; Supporting To test the taxonomic status of recovered clades, we Information, Table S1). The sequences were aligned used two species delimitations methods, which are with GENEIOUS v.7.1.9 (Kearse et al., 2012). commonly used for detection of species-level clusters The data were partitioned by gene. Nucleotide in taxonomic studies of various groups of insects, substitution models for each dataset were estimated including butterflies (Talavera et al., 2013a; Dincă using PartitionFinder v.2.1.1 (Lanfear et al., 2012) et al., 2015): the automatic barcode gap discovery based on the Bayesian information criterion (BIC) (ABGD) method (Puillandre et al., 2012) and the for each partition. The best-fitting models were as generalized mixed Yule coalescent (GMYC) model follows: GTR+G+I for COI, K2 for 28S, K2+G for H3 (Pons et al., 2006; Fujisawa & Barraclough, 2013). and K2+G for EF1α. For the ABGD test, we used COI alignment from the phylogenetic analysis, excluding outgroups, that was trimmed to the length of the shortest sequence Phylogenetic analyses and species delimitation (610 bp) in MEGA7 (Kumar et al., 2016). The Phylogenetic analyses were conducted for each gene analysis was run in the online version of the program independently and for nuclear genes combined using (http://www.abi.snv.jussieu.fr/public/abgd/abgdweb. the Bayesian inference (BI) approach (Supporting html) with the following settings: Pmin = 0.001, Information, Fig. S1) and for all datasets combined Pmax = 0.05, Steps = 10; X = 1.0; Nb bins = 20; using both BI and maximum likelihood (ML) and three proposed models: Jukes–Cantor (JC69), approaches. The Bayesian estimation of posterior Kimura (K80) and simple distance (SD). The GMYC probability was performed in MrBayes v.3.2.5 analysis was performed using the SPLITS package (Ronquist & Huelsenbeck, 2003), applying the for R (Ezard et al., 2009), with default settings, based selected evolutionary models for partitions. Markov on the COI Bayesian tree converted to ultrametric.

Analysis of the phylogeographical structure (BAYAREALIKE). We allowed for a maximum of five of the T. nogelii clade possible areas. For the analysis of the phylogeographical structure of the T. nogelii group, we used additional 107 sequences of COI barcodes obtained from GenBank previously RESULTS attributed to T. nogelii (including its subspecies Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 dobrogensis), T. nesimachus and T. romanovi (including Phylogenetic analysis and species delimitation the previously synonymized taxon T. telemachus) The phylogenetic analysis reveals Tomares as a covering the entire distribution range of these taxa. strongly supported monophyletic group (Fig. 2). The alignment was trimmed to the length of the The trees obtained from BI and ML analyses show shortest sequence (655 bp). A median-joining haplotype congruent results. Two deep-branching lineages are network was built using PopART v.1.7 software (Leigh recovered with high support: the first clade comprising & Bryant, 2015). African T. mauritanicus and African and the western Mediterranean T. ballus (African clade; PP = 0.96, BS = 96) and the second comprising the south-west Estimation of diversification time and Asian and Pontic T. nogelii complex, south-west Asian, biogeography Pontic-Caspian and Central Asian T. callimachus, To infer a dated phylogeny, we used BEAST v.2.6.2 south-west Asian T. desinens and Central Asian software (Bouckaert et al., 2014) with an uncorrelated T. fedtschenkoi (Asian clade; PP = 0.99, BS = 98). relaxed clock model and the tree prior set to birth– Within the African clade, each species forms a death. All other priors were set as the default. In monophyletic group with high support (PP = 1, BS = 96 order to have a fixed topology, we turned off the for T. ballus; PP = 1, BS = 100 for T. mauritanicus). topology operators in BEAUTi, and we specified the Three samples of T. ballus from Tunisia and one from topology obtained with RAxML. We used a secondary Morocco show a basal polytomy. The rest of the T. ballus calibration point of the MRCA of the tribe Eumaeini specimens from Morocco, Algeria and Spain form a (~16.66 Mya) from the dated phylogeny of European moderately supported clade (PP = 0.95, BS = 96). One butterflies by Wiemers et al. (2020) based on a recent T. mauritanicus sample from Morocco is recovered large-scale divergence analysis of butterflies (Chazot as sister to the rest of the species diversity (PP = 1, et al., 2019). BS = 100). Those specimens form a monophyletic The analyses were run for 50 million generations, group that receives high support in ML (BS = 100) but sampled every 5000 generations and repeated three is unsupported in BI (PP = 0.86). Within this lineage, times. The parameters of all three runs were compared two monophyletic groups are recognized: (1) two in Tracer v.1.5 (Drummond & Rambaut, 2007), in specimens from the Middle Atlas Mountains; and (2) which we also checked the model convergence (effective two specimens from the Anti-Atlas Mountains. sample size > 200). Trees from all three runs were The Asian clade is divided into two main groups, combined by LogCombiner v.1.8.4 (Drummond et al., the first one comprising the T. nogelii complex and 2012), and 10% of trees were discarded as burn-in. the second including all the remaining species The maximum credibility tree was selected using (T. callimachus, T. desinens and T. fedtschenkoi). TreeAnnotator v.1.8.4 (Drummond et al., 2012). The T. nogelii complex is strongly supported in BI The final phylogenetic tree was rendered in FigTree (PP = 1) but receives low support (BS = 61) in the ML v.1.4.0 (Rambaut, 2012). analysis. Within this complex, none of the currently We used a set of trees and the maximum recognized species (T. nogelii, T. nesimachus and credibility tree from the BEAST analysis for the T. romanovi) is recovered as monophyletic. Four biogeographical analysis. Five distribution areas monophyletic units are recognized: two samples of covering the range of Tomares were selected: North T. nogelii dobrogensis from Zaporizhzhia (Ukraine), Africa, the western Mediterranean, south-west Asia, two samples of T. nogelii dobrogensis from Crimea, the Pontic-Caspian region and Central Asia. We used four samples of T. nogelii nogelii from Turkey RASP v.4.0 (Yu et al., 2015), which incorporates the (provinces Niğde, Çankırı and Nevşehir) and eight R package BioGeoBEARS (Matzke, 2012, 2013), to samples of T. romanovi from Azerbaijan, Georgia, compare three possible models of past geographical Armenia and Turkey. In the latter group, a clade range estimation based on the Akaike information comprising three samples from Azerbaijan and criterion and, for each of them, also a variant Georgia corresponding to T. romanovi cachetinus with a founder effect (parameter j): dispersal– Nekrutenko, 1978 is recovered. extinction–cladogenesis (DEC), dispersal–vicariance The three remaining species (T. callimachus, analysis (DIVALIKE) and BI for discrete areas T. desinens and T. fedtschenkoi) are monophyletic with

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Figure 2. Molecular phylogenetic tree and putative species boundaries, with selected male specimens of Tomares. The Bayesian inference phylogenetic hypothesis was obtained from the combined datasets of cytochrome c oxidase subunit I (COI), 28S rRNA (28S), histone 3 (H3) and elongation factor 1-alpha (EF1α). Numbers above branches indicate the posterior probabilities; numbers below branches indicate bootstrap values obtained from the maximum likelihood analysis. Methods of species delimitation are as follows: GMYC, generalized mixed Yule coalescent method; ABGD, authomatic barcode gap discovery and its models, JC69 (Jukes–Cantor), K80 (Kimura) and SD (uncorrected p-distance). Branch names of the tree follow Weidenhoffer & Bozano (2007) and Nazari & Ten Hagen (2020). An updated classification based on the results of our study is given near the tree. Specimens: 1, T. romanovi cachetinus, Azerbaijan, Alty-Agatch; 2, T. romanovi romanovi, Armenia, Noravank; 3, T. nogelii dobrogensis (phenotype monotona), Ukraine, Dnepropetrovsk (photograph by Vadim Tshikolovets); 4, T. nogelii nogelii, Lebanon, Ain Zhatta; 5, T. nogelii nogelii (phenotype nesimachus), Syria, north Damascus, voucher BPAL2357-14; 6, T. callimachus callimachus, Crimea, Ordzhonikidze, voucher TOM044; 7, T. callimachus dentata, Turkey, Van Province, Erek Mountain; 8, T. desinens desinens, Azerbaijan, Talysh, Zuvand (paratype); 9, T. fedtschenkoi fedtschenkoi, Tajikistan, Dushanbe, voucher TOM031; 10, T. ballus ballus, Morocco, Igherm; 11, T. mauritanicus mauritanicus, Morocco, Asni.

© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, XX, 1–17 PHYLOGENY AND BIOGEOGRAPHY OF TOMARES 7 high support in both analyses. These species cluster recognized based on morphology, except T. nogelii, together in a single clade that has moderate support in T. nesimachus and T. romanovi of the T. nogelii group, ML analysis (BS = 73), but in BI it receives low support which are identified as a single species. (PP = 0.67). Tomares callimachus is a sister species to The JC69 and K80 models in ABGD delineate ten a clade comprising T. desinens and T. fedtschenkoi, but MOTUs. As in the GMYC and SD models, T. ballus,

the latter clade is not supported in BI or ML analysis T. desinens, T. fedtschenkoi and the T. nogelii clade Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 (PP = 0.71, BS = 56). Three monophyletic groups are are identified as candidate species. Two MOTUs are recovered within the T. callimachus hafis (Kollar, delineated within T. mauritanicus: the basal lineage 1849) clade. The first clade includes two samples of from Morocco and the rest of the samples. Within T. callimachus hafis from Iran, provinces Markazi the T. callimachus clade, four MOTUs are identified, and East Azerbaijan (PP = 0.98, BS = 100). The which correspond to: (1) the nominotypical subspecies second group comprises five specimens of T. c. hafis from Crimea, south Russia and north Kazakhstan; (2) from East Turkey, provinces Van and Hakkâri, and the recently re-established subspecies T. c. hafis from Tehran Province in Iran (PP = 0.93, BS = 69). Within central, south and north-west Iran; (3) samples from this group, two specimens from Hakkâri subsequently Tehran Province in Iran and Van Province in Turkey; form a clade (PP = 1, BS = 61). Finally, the third and (4) samples from Hakkâri Province in Turkey. monophyletic group is composed of nine specimens A graphical summary of the species delimitation of the nominotypical T. callimachus from Krasnodar analyses is illustrated in Fig. 2. Krai and Volgograd Oblast in Russia, Crimea and Kazakhstan (Karatau Mountains) (PP = 1, BS = 100). The T. desinens clade shows a split, resulting in Phylogeographical analysis of the T. nogelii subclades corresponding to the currently recognized clade subspecies T. desinens alborzicus Weidenhoffer & The haplotype network analysis of 143 COI sequences Bozano, 2007 and T. desinens mebep Koçak & Kemal, of the T. nogelii clade reveals 25 haplotypes. Most of 2005. Specimens of T. fedtschenkoi from Tajikistan and them are broadly shared by different populations Uzbekistan demonstrate no genetic differentiation. and phenotypes and generally demonstrate no The GMYC and SD models in ABGD give similar phylogeographical structure (Fig. 3). The majority of results, in which six molecular operational taxonomic the specimens comprise three haplotypes. The most units (MOTUs), or candidate species, are identified. widespread one (H1) comprises 48 specimens of They correspond to all species of Tomares currently the taxa T. romanovi, T. nesimachus, T. nogelii and

Figure 3. A, result of the statistical parsimony network analysis illustrating relationships of the 25 cytochrome c oxidase subunit I haplotypes of the Tomares nogelii species group. Colours of circles correspond to origins of samples. B, result of the statistical parsimony network analysis illustrating relationships of the 25 cytochrome c oxidase subunit I haplotypes of the T. nogelii species group. Colours of circles correspond to species previously considered within the group (Weidenhoffer & Bozano, 2007).

T. telemachus from Turkey, Armenia, Azerbaijan, Iran, Mya) and ~3.98 Mya (6.2–2.2 Mya), respectively. Within Turkmenistan, Israel and Syria. The second (H2) the Asian clade, it was subsequently followed by differs from H1 in one nucleotide substitution. It unites a rapid diversification, resulting in an ancestor of 41 specimens of the taxa T. nesimachus, T. nogelii and T. callimachus and an ancestor of T. fedtschenkoi T. dobrogensis from Turkey and Israel. The third most and T. desinens, which occurred between the early

common haplotype (H10) unites 12 specimens of the Pliocene and early Pleistocene ~3.59 Mya (5.6–1.9. Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 taxa T. nesimachus and T. nogelii from Turkey and Mya), and the split of an ancestor of T. fedtschenkoi Israel. Other haplotypes comprise between one and and T. desinens, which occurred between the middle six specimens represented by one taxon from one Pliocene and middle Pleistocene ~2.64 Mya (4.4–1.2 country except for H20, which unites six specimens of Mya). Finally, all species of Tomares differentiated the taxa T. romanovi and T. nesimachus from Georgia, during the Pleistocene. Syria, Jordan, Iran and Azerbaijan, and H13, which The preferred biogeographical model according unites the taxa T. romanovi and T. nogelii from Turkey to the corrected Akaike information criterion is a and Iran. likelihood version of the dispersal–vicariance model Most specimens from Israel included in our analysis with founder effect (DIVALIKE+j) followed by the belong to the closely related widespread haplotypes dispersal–extinction–cladogenesis model with founder H1, H2 and H10, and one specimen constitutes the effect (DEC+j) and the Bayesian inference of historical haplotype H11, which differs from H10 by one nucleotide biogeography for discrete areas model with founder substitution. Two specimens from Syria constitute effect (BAYAREALIKE+j) (Supporting Information the haplotype H12, which differs by two nucleotide Table S4). These models give similar results but substitutions from haplotypes H1 and H11. In all differ in the origin of the ancestor of Tomares (Fig. cases, specimens from Israel share their haplotypes 3; Supporting Information, Fig. S2). According to with geographically distant specimens of T. nogelii DIVALIKE+j and DEC+j, the ancestral distribution and T. nesimachus from Turkey and, in the case of of Tomares covered North Africa and south-west Asia. the haplotype H1, with specimens of T. romanovi from A vicariance event that occurred ~6.68 Mya gave Turkey, Armenia, Iran and Turkmenistan (including rise to the African and Asian clades. On the contrary, the recently synonymized taxon, T. telemachus). BAYAREALIKE+j suggests an origin of the MRCA of In contrast, geographically closer specimens from Tomares in south-west Asia, with subsequent dispersal Jordan belong to the haplotype H20, which unites to Africa. The West Mediterranean region was colonized four specimens of T. romanovi from Georgia, Iran by T. ballus from Africa in relatively recent times, and Azerbaijan and the rest of the specimens of during the late Pleistocene. The evolution of the Asian T. nesimachus from Syria. Populations from Ukraine clade occurred mainly in south-west Asia according to and Crimea represented by the taxon T. dobrogensis all models. Pontic-Caspian region was colonized from are represented by five unique haplotypes (H8, H9, south-west Asia three times during the Pleistocene, H15, H16 and H17). twice by T. nogelii and once by T. callimachus. Central The majority of the T. romanovi specimens share Asia was colonized twice, first from south-west Asia haplotypes with T. nogelii (H1, H13 and H20). Five by the ancestor of T. fedtschenkoi in the late Pliocene– haplotypes from Iran, Georgia and Azerbaijan (H14, middle Pleistocene and the second time from the H18, H19, H21 and H22) are unique for the species, Pontic-Caspian region by T. callimachus in the late differing from the haplotype H1 in only one or two Pleistocene. nucleotide substitutions. For the distribution of the haplotypes among specimens and localities, see the Supporting DISCUSSION Information (Table S3). Our analysis demonstrates that the genus Tomares is a well-supported monophyletic taxon comprising Estimation of diversification time and two major clades. Our results contradict the previous biogeography suggestion of Nazari & Ten Hagen (2020) that the Our dating estimates indicate that the genus Tomares Central Asian species T. fedtschenkoi represents a originated between the early Oligocene and the early sister clade to the North African and Mediterranean Miocene, ~24.14 Mya (32.0–17.0 Mya) (Fig. 4). The split T. ballus and T. mauritanicus. The position of of the MRCA of Tomares, resulting in the African and the T. nogelii clade as sister to T. desinens, with Asian clades, occurred between the middle-late Miocene T. callimachus sister to both (Nazari & Ten Hagen, and middle-late Pliocene ~6.68 Mya (10.25–3.75 Mya). 2020) is also not supported by our analysis. Both African and Asian clades diverged between the The phylogenetic analysis combined with species late Miocene and early Pleistocene, ~4.95 Mya (8.1–2.4 delimitation methods revealed six candidate

© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, XX, 1–17 PHYLOGENY AND BIOGEOGRAPHY OF TOMARES 9 Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021

Figure 4. Historical biogeography of Tomares. The most supported ancestral area reconstruction model (DIVALIKE+j) was estimated within the R package BioGeoBEARS. Pie charts on each node depict the relative probabilities of ancestral ranges. A, North Africa. B, western Mediterranean. C, south-west Asia. D, Pontic-Caspian region. E, Central Asia; *, other regions. Classification follows Weidenhoffer & Bozano (2007) and Nazari & Ten Hagen (2020).

species [T. ballus, T. mauritanicus, the T. nogelii a number of subspecies. The revised classification complex (T. nogelii, T. nesimachus and T. romanovi), of Tomares according to our data is summarized in T. callimachus, T. desinens and T. fedtschenkoi] and Table 1.

Table 1. Revised classification of the genus Tomares based on the results of our study

Revised classification Previous classification (Nazari & Ten Hagen, 2020)

Tomares ballus (Fabricius, 1787) Tomares ballus (Fabricius, 1787) T. ballus mareoticus Graves, 1918 (? east Morocco, ? Tunisia, Synonym of the nominotypical subspecies Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 north Lybia, north Egypt, central Israel; probably extinct in the eastern part of the range) Tomares mauritanicus (Lucas, 1849) Tomares mauritanicus (Lucas, 1849) T. m. antonius Brevignon, 1985 (Middle Atlas Mountains, Morocco) Synonym of the nominotypical subspecies T. m. amelnorum Tarrier, 1997 (Anti-Atlas Mountains, Morocco) Synonym of the nominotypical subspecies Tomares callimachus (Eversmann, 1848) Tomares callimachus (Eversmann, 1848) T. c. hafis (Kollar, 1849) (partim: central and south Iran) T. c. hafis (Kollar, 1849) (Transcaucasia, Turkey, Iran) T. c. dentata (Staudinger, 1892), stat. rev. (south-east Turkey, ? T. c. hafis (Kollar, 1849) (partim) Transcaucasia, ? north Iran) T. c. huertasae Tshikolovets & Pagès, 2016 (Pakistani Baluchistan) T. c. huertasae Tshikolovets & Pagès, 2016 (Paki- stani Baluchistan) Tomares nogelii (Herrich-Schäffer, 1851) Tomares nogelii (Herrich-Schäffer, 1851) T. n. dobrogensis (Caradja, 1895) (from south-east Romania to T. n. dobrogensis (Caradja, 1895) (from south-east Crimea; non-monophyletic taxon, status needs clarification) Romania to Crimea) Synonym of Tomares nogelii Tomares nesimachus (Oberthür, 1893) Tomares romanovi (Christoph, 1882) Tomares romanovi (Christoph, 1882) (non-monophyletic taxon, status needs clarification) T. r. cachetinus Nekrutenko, 1978 (east Georgia, north Azerbaijan) Colour form of the nominotypical subspecies Tomares desinens Nekrutenko & Effendi, 1980 Tomares desinens Nekrutenko & Effendi, 1980 T. d. mebep Koçak & Kemal, 2005 (south-east Turkey, north-west Synonym of the nominotypical subspecies Iran) T. d. alborzicus Weidenhoffer & Bozano, 2007 (north Iran) Synonym of the nominotypical subspecies Tomares fedtschenkoi (Erschoff, 1874) Tomares fedtschenkoi (Erschoff, 1874)

Species and the taxon with revised status are marked in bold.

Phylogeny and systematics of T. ballus and Anti-Atlas Mountains. One haplotype from Morocco T. mauritanicus recovered in two species delimitation analyses as a Within the T. ballus clade, three samples of T. ballus candidate species needs further exploration; probably, from Tunisia and one from Morocco showing a basal it represents an undescribed taxon. polytomy are detected. Their taxonomic status will remain unclear until specimens of probably extinct populations of T. ballus from north-east Africa and the Phylogeny, systematics and phylogeography of Near East are analysed. They were widely known in the T. nogelii clade the literature as subspecies T. ballus cyrenaica Turati, Neither T. nogelii nor T. nesimachus is recovered 1924 (type locality: Benghazi, Lybia), but Tshikolovets as a monophyletic entity in our analysis. Species (2011) has pointed out that the name T. ballus delimitation analysis suggests the species status for mareoticus Graves, 1918 (type locality: Alexandria, the whole T. nogelii clade, and the haplotype network Egypt) has priority over T. b. cyrenaica and should be analysis reveals no phylogeographical structure attributed to disjoined populations previously known within the clade. from southern Tunisia, Lybia, Egypt and Israel. Summarizing the results of the previous research Other specimens from Morocco, Algeria and Spain (Hesselbarth & Schurian, 1984; Hesselbarth et al. demonstrate no genetic differentiation and obviously 1995; Van Oorschot & Wagener, 2000; Nazari & represent the nominotypical subspecies. Ten Hagen, 2020), we can conclude that T. nogelii is Two units from Morocco are detected within presented by three phenotypes, all with intermediates the T. mauritanicus clade. They correspond to the between them: T. nogelii monotona Schwingenschuss, subspecies T. mauritanicus antonius Brevignon, 1985 1939 (wings dark brown dorsally, without red patches), from the Middle Atlas Mountains and the subspecies T. nogelii nogelii (wings red, with wide, dark border T. mauritanicus amelnorum Tarrier, 1997 from the dorsally) and T. nogelii nesimachus (wings red, with

© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, XX, 1–17 PHYLOGENY AND BIOGEOGRAPHY OF TOMARES 11 narrow, dark marginal border dorsally). Intermediate and Romania to the analysis will refine the taxonomic specimens were described as T. nogelii obscura Rühl, status of these populations. Until then, we follow 1893, T. nogelii cesa Koçak, Kemal & Seven, 2000 Nazari & Ten Hagen (2020) and provisionally consider and T. nogelii aurantiaca Staudinger, 1871. These T. n. dobrogensis as a subspecies of T. nogelii from the phenotypes, or colour morphs, and intermediate northern Pontic region. The specimens from Turkey

specimens differ in distribution but may co-occur. It previously attributed to T. n. dobrogensis by Nazari Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 was noted that adults of dark phenotypes (T. n. nogelii & Ten Hagen (2020) share the second widespread and T. n. monotona) tend to appear ~1 month haplotype (H2) with T. nogelii and ‘nesimachus’. later than those of T. n. nesimachus and/or inhabit Tomares romanovi is also not recovered as a higher elevations (Larsen, 1974; Hesselbarth et al., species in species delimitation analyses, but it clearly 1995). According to the literature, the north of the differs from parapatrically distributed T. nogelii in T. nogelii range (southern Ukraine and Crimea) morphological characters, overlapping with a small is inhabited by the phenotypes T. n. nogelii and part of the range of the latter in its easternmost part. T. n. monotona (Tshikolovets, 2011), Turkey is Several populations morphologically intermediate inhabited by all three colour morphs (Hesselbarth between T. nogelii and T. romanovi, including et al., 1995), both T. n. nogelii and T. n. nesimachus T. n. obscura from eastern Turkey and populations from are known from Lebanon and Syria (Larsen, 1974), Georgia and Azerbaijan, known as T. r. cachetinus, and the southernmost part of the range, in Israel and specimens with shared haplotypes were detected and Jordan, is inhabited by T. n. nesimachus (Larsen in an overlapping zone of their ranges (Hesselbarth & Nakamura, 1983; Tshikolovets, 2011). Specimens et al. 1995; Van Oorschot & Wagener, 2000; Nazari & of T. n. nesimachus from Israel, Jordan and Syria Ten Hagen, 2020). Phylogenetic analysis reveals that differ from those in Turkey in their well-developed samples of T. romanovi from Armenia, Azerbaijan red discal field, narrow, dark marginal border on the and Georgia form a clade within the T. nogelii group dorsal side of the wings and large size. They could be corresponding to the nominotypical subspecies of considered a distinct subspecies, but such treatment is T. romanovi, but this clade is not united with specimens not supported by phylogenetic and haplotype network of T. romanovi from Turkey, Iran and Turkmenistan, analyses because these specimens do not form a clade. which appear on an unresolved branch comprising The results of our analysis do not support the samples of T. nogelii from Syria, Turkey and Jordan. opinion of Nazari & Ten Hagen (2020) that T. nogelii In the phylogeographical analysis, the majority and T. nesimachus are distinct sister species. Taking of the T. romanovi specimens share haplotypes with our results into account, in addition to the absence of T. nogelii (H1, H13 and H20), and five haplotypes morphological diagnostic characters delimiting these from Iran, Georgia and Azerbaijan (H14, H18, H19, taxa, broadly shared distribution ranges and absence of H21 and H22) are unique for the species, differing clear reproductive barriers (Van Oorschot & Wagener, from the haplotype H1 in only one or two nucleotide 2000; Nazari & Ten Hagen, 2020), considering all substitutions. The overall haplotype network structure mentioned phenotypes a single species T. nogelii is (Fig. 3) shows a common haplotype (H1) shared by most reasonable. We consider T. n. nesimachus (syn. many specimens from different localities and a star- nov.) a synonym of T. n. nogelii to obtain taxonomic like pattern of its sister haplotypes. This might indicate stability and solve nearly a century and a half of that the hypothetical ancestral populations underwent confusion and attempts to discriminate these two taxa. a significant reduction in effective population size Apart from the nominotypical subspecies of and that a bottleneck event occurred in the recent T. nogelii inhabiting Turkey, Syria, Lebanon, Israel, historical past. At the same time, the large number Jordan and, according to old, unconfirmed data, of closely related haplotypes combined into a cluster Azerbaijan (Tshikolovets & Nekrutenko, 2012), with a dominant haplotype indicates that T. romanovi separation of a northern subspecies, T. n. dobrogensis, populations are currently undergoing exponential from south-eastern Romania, Moldova, southern growth (Avise, 2000). Ukraine and Crimea, is reasonable, but the specimens The taxonomic status of the populations of T. nogelii of T. n. dobrogensis do not form a monophyletic entity and T. romanovi with shared haplotypes needs in our phylogenetic reconstruction. In the haplotype further examination. This pattern can be the result of network analysis, they are represented by five unique hybridization leading to mitochondrial introgression or haplotypes, three related ones, from Crimea (H15 and incomplete lineage sorting, which occurs occasionally H17) and Kherson Oblast in Ukraine (H16), and two in Lycaenidae (Talavera et al., 2013b; Lukhtanov unconnected haplotypes, from Donetsk Oblast (H8) et al., 2015a, b). Another possible explanation of the and Zaporizhzhia (H9) in Ukraine. At least some of shared haplotypes within the T. nogelii group is an them should be attributed to T. n. dobrogensis. We influence of Wolbachia Hertig, 1936, an intracellular hope that the addition of specimens from Moldova rickettsial bacterium that infects arthropods and

© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, XX, 1–17 12 KRUPITSKY ET AL. filarial nematodes. As an endosymbiont associated from the southern part of the T. callimachus range. with mitochondria and thus maternally inherited, Further studies are required to clarify the taxonomic Wolbachia can cause selective sweeps in mitochondrial status of these clades, their boundaries and precise haplotypes owing to genetic hitchhiking (Whitworth geographical distribution. We hope that the addition to et al., 2007). Wolbachia infection can lead to analysis more specimens from Turkey, and the recently

mitochondrial introgression and reduce mitochondrial described subspecies T. c. huertasae from Pakistani Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 diversity, but can also mimic the speciation process, Baluchistan (Tshikolovets & Pagès, 2016), will refine causing a deep divergence in mitochondrial phylogenies the phylogeny and taxonomy of T. callimachus. (Ritter et al., 2013). It is possible that both species, Distinct monophyletic groups revealed within the T. nogelii and T. romanovi, share the same Wolbachia T. desinens clade correspond to geographically isolated strain owing to hybridization in the common parts of subspecies T. d. mebep and T. d. alborzicus. On the their distribution ranges in Eastern Turkey, which contrary, T. fedtschenkoi demonstrates no genetic has resulted in the observed star-like structure of the differentiation despite its rather wide distribution in haplotype network obtained. In the Lycaenidae, a case the mountain systems of Central Asia. of Wolbachia and mitochondrial DNA interference Systematics of Tomares according to the results of was found recently in the Pseudophilotes baton our research are summarized in Table 1. (Bergsträsser, 1779) species complex (Bartoňová et al., 2021). Their phylogeographical analysis also resulted in a star-like network structure, but in contrast to Diversification and evolutionary history of our case, it mirrored the geographical pattern of the Tomares revealed haplotypes and Wolbachia infection. The results of our analysis indicate that the ancestor Given the distinct morphological characters of the tribe Tomarini evolved during the early (Weidenhoffer & Bozano, 2007; Nazari & Ten Hagen, Oligocene–early Miocene. Given that North Africa 2020) and differences in ecology and distribution and south-west Asia were disjointed before the (Hesselbarth et al. 1995; Van Oorschot & Wagener, middle Miocene (~14 Mya) by the Tethys seaway 2000), T. romanovi is best regarded as a distinct species (Rögl, 1999; Hamon et al., 2013), and that the common until additional molecular studies are conducted. ancestor of the clade (Theclini + part of Arhopalini) and the clade (Tomarini + (Eumaeini + (Deudorigini + part of Loxurini))) according to Espeland et al. Phylogeny and systematics of T. callimachus, (2018) apparently evolved in Asia, the ancestor of T. desinens and T. fedtschenkoi Tomares seems to have originated in south-west Within the T. callimachus clade, the nominotypical Asia. The same region is ancestral for a majority of subspecies from the Pontic-Caspian region and clades of the diverse lycaenid subgenus Agrodiaetus Central Asia forms a monophyletic group, but the (Wiemers et al., 2009; Eckweiler & Bozano, 2016) and samples of recently re-established subspecies T. c. hafis of the Parnassiinae genera Allancastria, Archon and sensu Nazari & Ten Hagen (2020), which comprises Hypermnestra (Nazari et al., 2007). After the isolation populations of T. callimachus distributed to the south of the Mediterranean Sea from the Indian Ocean in of the Great Caucasus, are scattered among several the middle Miocene, the ancestor of Tomares probably clades and do not constitute a monophyletic unit. The colonized Africa through the Arabian Peninsula. ABGD analysis performed by JC69 and K80 methods Dispersals between Asia and Africa through the suggests three putative species within the southern Arabian Peninsula have also been proposed for populations of T. callimachus. The first of them other species of butterflies (Kodandaramaiah & comprises populations from Iran corresponding to the Wahlberg, 2007; Aduse-Poku et al., 2015; Fric et al., taxon T. c. hafis, the second unites populations from Van 2019). The crown group of Tomares differentiated Province in south-east Turkey and Tehran Province in the late Miocene owing to vicariance, probably in in Iran, and the third is represented by a population response to the increasing aridification and habitat from Hakkâri Province in south-east Turkey. There fragmentation that occurred in this period, followed is one more ‘forgotten’ name within T. callimachus, by the Messinian salinity crisis (Agakhanjanz & the taxon T. c. dentata described from south-eastern Breckle, 1995; Zachos et al., 2001). Our results Turkey (Mardin Province) on the basis of the serrated disprove the hypothesis of Nazari & Ten Hagen submarginal area of the forewing (Staudinger, 1892). (2020) that Tomares probably arose in Pleistocene. We tentatively consider T. c. dentata as a subspecies The estimates obtained for the differentiation of the of T. callimachus from south-eastern Turkey and, crown group of Tomares are slightly older than those probably, northern Iran, Tomares callimachus dentata calculated by Wiemers et al. (2020), who estimated (Staudinger, 1892) stat. rev. Our analysis does not the age of this split at 5.55 Mya, but our dating fits resolve phylogenetic relationships of populations into the same geological period.

The split of the African clade probably occurred in endemic south-west-Asian species, T. desinens, and the early-middle Pliocene and resulted in two lineages the only species of Tomares endemic to Central Asia, with contrasting evolutionary history, strongly T. fedtschenkoi, also originated in south-west Asia. The differing in morphology, habitat and host plant hypothetical ancestor of the latter probably colonized preference and distribution, i.e. the more specialized Central Asia around the Pliocene–Pleistocene border.

mountain species, T. mauritanicus, inhabiting the Atlas The route of colonization via the Alborz Mountains and Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlab055/6358824 by guest on 01 October 2021 Mountains in north-west Africa, and the polyphagous then via the Kopet Dag seems plausible. T. ballus, which dispersed across North Africa and Tomares colonized eastern Europe during the late colonized the western Mediterranean region during Pleistocene, but did not reach the Apennine and the late Pleistocene, probably owing to the glacial/ the Balkan Peninsulas. This type of distribution is interglacial sea-level oscillations (Frigola et al., 2012). remarkable from a biogeographical point of view and The evolution of the Asian clade took place in the generally has no equivalents among other groups of mountains of south-west Asia during the Pliocene and Palaearctic butterflies. A somewhat similar distribution Pleistocene. Members of this clade are mainly Astragalus pattern, characterized by the disjunction between the feeders (Weidenhoffer & Vanek, 1977; Hesselbarth et al., western Mediterranean and western Asia, the Pontic- 1995; Zhdanko, 1997; Van Oorschot & Wagener, 2000). Caspian region and Central Asia, is known for the pierid According to the recent reconstructions, the legume genus Zegris (Back, 2012). Other groups of butterflies genus Astragalus L. underwent rapid diversification in colonized these regions earlier in the Plio-Pleistocene south-west Asia at the same time as Tomares, caused and used them as refugia during Pleistocene glaciations by geographical isolation, environmental variation and (Dapporto, 2010; Dapporto et al., 2019). Tomares nogelii changes in resource availability, which were induced dobrogensis was apparently much more widespread in the by the climate fluctuations known as the Pliocene– steppes of south-east Europe than nowadays, reaching Pleistocene transition (Azani et al., 2019). It is possible as far north as Poltava Oblast in central Ukraine at the that the early diversification of Asian Tomares was beginning of the 20th century (Lvovsky & Morgun, 2007). shaped by the evolution of this genus of flowering plants. We cannot exclude the possibility that populations of Within the Asian clade of Tomares, three successive this species reached the Balkans through the steppes splits probably occurred in the middle Pliocene, the of Ukraine and Dobrogea in the late Pleistocene and mid-late Pliocene and around the Pliocene–Pleistocene existed there in historical time. If so, they could have border. They resulted in the ancestors of T. callimachus, disappeared after the decline of steppes and conversion T. desinens and T. fedtschenkoi and an ancestor of to agriculture, as is known for other steppe specialist the T. nogelii clade. This clade diverged during the butterflies (Bartoňová et al., 2018, 2020). Pleistocene and dispersed across Anatolia, the eastern Mediterranean, Transcaucasia and the western Iranian Plateau. Colonization of the Pontic region by T. nogelii ACKNOWLEDGEMENTS most probably occurred owing to the formation of steppes during glacial periods in the late Pleistocene, We thank Dmitry Morgun (Moscow Children and Youth after the final disconnection of the two large basins of Center of Ecology, Local History and Tourism, Moscow, the eastern Parathetys, namely the Kujalnician basin Russia), Dmitry Shovkoon (Samara National Research and the Akchagilian basin, resulting in the Black Sea University, Samara, Russia), Sergei Sinev and Alexander and the Caspian Sea, respectively (Popov et al., 2004, Lvovsky (Zoological Institute of the Russian Academy 2006). The same scenario explains colonization of the of Sciences, St. Petersburg, Russia), who provided an Pontic-Caspian region followed by the colonization of opportunity to work with collections, Galina Kuftina Central Asia by the most widespread Tomares species, (Zoological Institute of the Russian Academy of Sciences, T. callimachus, in the late Pleistocene. By that time, St. Petersburg, Russia), Maria Stanovova and Miroslava T. callimachus had colonized south-eastern Anatolia, Cherneva (Biological Faculty, Moscow State University, Transcaucasia, the Zagros, Alborz and Kopet Dag Moscow, Russia) for technical support, Valentina Mountains. Unfortunately, the origin of the populations Tambovtseva (Koltsov Institute of Developmental of T. c. huertasae from Baluchistan in the extreme south- Biology of Russian Academy of Sciences, Moscow, Russia) east of south-west Asia and their colonization routes are for assistance with Sanger sequencing and Vadim obscured, but the presence of endemic taxa of butterflies Tshikolovets (Pardubice, Czech Republic) for providing apparently of Iranian origin, including Lycaenidae, in photographs of specimens. The study was supported by this region and the absence of Tomares in neighbouring the Russian Science Foundation, grant no. 19-14-00202 regions of Pakistan and Afghanistan are more likely to to the Zoological Institute of the Russian Academy of indicate colonization from the western part of south- Sciences, and the project AAAA-A19-119020790106-0 west Asia rather than from Central Asia. Finally, our (V.A.L. and N.A.S.). We thank two anonymous reviewers analysis indicates that the common ancestor of an for comments and suggestions.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Overview of samples of Tomares and outgroup species used for total phylogeny study, with GenBank accession numbers/bold sequence ID. Accession numbers of sequences obtained before the present research are in bold. Classification follows Weidenhoffer & Bozano (2007) and Nazari & Ten Hagen (2020). Table S2. Amplification and sequencing primers used for molecular phylogeny reconstruction of the genus Tomares and protocols of polymerase chain reactions. Table S3. Overview of samples used for Tomares nogelii group phylogeography pattern studies, with GenBank accession numbers/bold sequence ID. Table S4. Comparison of biogeography models by BioGeoBEARS based on the corrected Akaike information criterion (AICc), where the lowest value indicates the best suitable model (marked in bold). Abbreviations: d, dispersal parameter; e, extinction parameter; j, founder effect parameter. Figure S1. Phylogenetic analyses of independent genes using Bayesian inference. Numbers above branches indicate the posterior probabilities. A, cytochrome c oxidase subunit I (COI). B, 28S rRNA (28S). C, histone 3 (H3). D, elongation factor 1-alpha (EF1α). E, concatenated nuclear dataset. Figure S2. Alternative biogeographical models of the genus Tomares ancestral distribution, resulting from modelling in BioGeoBears. A, DEC+J. B, BAYAREALIKE+J. Classification follows Weidenhoffer & Bozano (2007) and Nazari & Ten Hagen (2020).