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Chromosomal and DNA barcode analysis of the Melitaea ala Staudinger, 1881 species complex (Lepidoptera, Nymphalidae)
expand article infoVladimir A. Lukhtanov, Anastasia V. Gagarina, Elena A. Pazhenkova§
‡ Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia
§ St. Petersburg State University, St. Petersburg, Russia
Open Access

Abstract

The species of the Melitaea ala Staudinger, 1881 complex are distributed in Central Asia. Here we show that this complex is a monophyletic group including the species, M. ala, M. kotshubeji Sheljuzhko, 1929 and M. enarea Fruhstorfer, 1917. The haploid chromosome number n=29 is found in M. ala and M. kotshubeji and is, most likely, a symplesiomorphy of the M. ala complex. We show that M. ala consists of four subspecies: M. ala zaisana Lukhtanov, 1999 (=M. ala irtyshica Lukhtanov, 1999, syn. nov.) (South Altai, Zaisan Lake valley), M. ala ala (Dzhungarian Alatau), M. ala bicolor Seitz, 1908 (North, East, Central and West Tian-Shan) and M. ala determinata Bryk, 1940 (described from “Fu-Shu-Shi”, China). We demonstrate that M. kotshubeji kotshubeji (Peter the Great Mts in Tajikistan) and M. kotshubeji bundeli Kolesnichenko, 1999 (Alai Mts in Tajikistan and Kyrgyzstan) are distinct taxa despite their geographic proximity in East Tajikistan. Melitaea enarea is widely distributed in the southern part of Central Asia and is sympatric with M. kotshubeji.

Keywords

chromosome, COI, DNA barcode, karyosystematics, Melitaea, taxonomy

Introduction

This work is a continuation of a series of publications (Lukhtanov and Kuznetsova 1989; Pazhenkova et al. 2015; Pazhenkova and Lukhtanov 2016; Lukhtanov 2017) devoted to the analysis of chromosomal and mitochondrial haplotype diversity and taxonomy of butterflies of the species-rich butterfly genus Melitaea Fabricius, 1807. The combination of molecular and cytogenetic methods is a useful tool for taxonomic studies (Lukhtanov et al. 2015; Pazhenkova and Lukhtanov 2019) and can be a good addition to morphological analysis of taxonomically complicated groups of species (Lukhtanov et al. 2016). In our previous papers, we applied analysis of the DNA barcodes and karyotypes to study the genetic and taxonomic structure of the M. didyma (Esper, 1779) (Pazhenkova et al. 2015; Pazhenkova and Lukhtanov 2016) and M. persea Kollar, 1849 (Lukhtanov 2017) species complexes. The aim of this work is to study a complex of species close to M. ala Staudinger, 1881.

The species of this complex are distributed in Central Asia (Kolesnichenko 1999). According to Kolesnichenko (1999), this complex consists of the following species: Melitaea ala Staudinger, 1881, M. kotshubeji Sheljuzhko, 1929, M. ninae Sheljuzhko, 1935, M. chitralensis Moore, 1901, and M. enarea Fruhstorfer, 1917. According to van Oorschot and Coutsis (2014), this complex consists of the following species: M. acraeina Staudinger, 1881, M. ninae Sheljuzhko, 1935, Melitaea ala Staudinger, 1881, M. didymina Staudinger, 1895, M. chitralensis Moore, 1901, M. enarea Fruhstorfer, 1917, M. bundeli Kolesnichenko, 1999, M. kotshubeji Sheljuzhko, 1929, M. sutschana Staudinger, 1881 and M. yagakuana Matsumura, 1927 (the latter taxon is usually considered a subspecies of M. sutschana, e.g. see Higgins, 1941).

Molecular phylogenetic analysis (Leneveu et al. 2009) demonstrated that M. ala and M. enarea (cited in the article as M. permuta Higgins, 1941) are sister species, and M. acraeina is a phylogenetically distant species which is a sister to the lineage (M. ala + M. enarea). Melitaea sutschana was found as a member of the M. didyma species complex which is a sister to the lineage ((M. acraeina + (M. ala + M. enarea)) (Leneveu et al. 2009). In our study, we focused on the analysis of the M. ala lineage. We did not include M. ninae, M. didymina and M. chitralensis in the analysis, since for these species there has been no material suitable for chromosomal and molecular studies.

Materials and methods

Chromosomal analysis

Karyotypes of four samples of M. kotshubeji kotshubeji were studied as previously described (Lukhtanov et al. 2014; Vishnevskaya et al. 2016). Briefly, gonads were removed from the adult male abdomen and placed into freshly prepared fixative (3:1; 96% ethanol and glacial acetic acid) directly after capturing the butterfly in the field. Testes were stored in the fixative for 3–36 months at +4 °C. Then the gonads were stained in 2% acetic orcein for 5–10 days at +18–20 °C. Different stages of male meiosis, including metaphase I (MI) and metaphase II (MII) were examined using an original two-phase method of chromosome analysis (Lukhtanov et al. 2006, 2008). Leica DM2500 light microscope equipped with HC PL APO 100×/1.44 Oil CORR CS lens and S1/1.4 oil condenser head was used for bright-field microscopy analysis. A Leica DM2500 light microscope equipped with HC PL APO 100×/1.40 OIL PH3 lens was used for phase-contrast microscopy analysis.

Molecular methods and DNA barcode analysis

Standard COI barcodes (658-bp 5’ fragment of mitochondrial cytochrome oxidase subunit I) were studied as previously described (Lukhtanov et al. 2014; Vishnevskaya et al. 2016). COI sequences were obtained from 34 specimens representing the M. ala species group and outgroups (M. telona Fruhstorfer, 1908 and M. alatauica Staudinger, 1881). Legs were used as a source for DNA isolation

Legs from 6 specimens (M. kotshubeji bundeli Kolesnichenko, 1999) were processed in the Department of Karyosystematics of Zoological Institute of the Russian Academy of Sciences using primers and protocols described by Shapoval et al. (2017). Sequencing was carried out at the Research Resource Center for Molecular and Cell Technologies of St. Petersburg State University.

Legs from 28 specimens of Melitaea spp. were processed in the the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) using their standard high-throughput protocol described by Hajibabaei et al. (2005), Ivanova et al. (2006) and deWaard et al. (2008). The set of voucher specimens of butterflies is kept in the Zoological Institute of the Russian Academy of Science (St. Petersburg) and in the McGuire Center for Lepidoptera and Biodiversity (MGCL), Florida Museum of Natural History, University of Florida, Gainesville, Florida, USA. Photographs of these specimens, as well as collecting data are available in the of Life Data System (BOLD), projects Butterflies of Palearctic (BPAL) and Butterflies of Palearctic Part B (BPALB) at http://www.boldsystems.org/.

We also used 30 published COI sequences for DNA barcode analysis (Leneveu et al. 2009; Lukhtanov et al. 2009; Ashfaq et al. 2013; Pazhenkova et al., 2015; Pazhenkova and Lukhtanov 2016; Lukhtanov 2017) (Table 1).

Table 1.

Specimens of Melitaea spp. used in the DNA barcode analysis.

Species and subspecies Species name as found in GenBank Field code or BOLD number GenBank number Country Locality Reference
M. acentria M. acentria BOLD:BPAL2191-13 KY777529 Israel Hermon Lukhtanov 2017
M. acraeina M. acraeina BOLD:GBLN1879-09 FJ462229 Uzbekistan Komsomolobad Leneveu et al. 2009
M. ala ala M. ala BPALB179-16; CCDB-25458_G12 MW672072 Kazakhstan Dzhungarian Mts, Kopal, 45.08°N, 79.07°E This study
M. ala ala M. ala BOLD:BPAL039-10 MW672074 Kazakhstan Taldy-Kurgan region, Kysylagash This study
M. ala ala M. ala BOLD:BPAL3407-16 MW672077 Kazakhstan Taldy-Kurgan region, Kyzylagash This study
M. ala bicolor M. ala BOLD:GBLN1877-09 FJ462231 China Tian-Shan Leneveu et al. 2009
M. ala bicolor M. ala bicolor BOLD:LOWA355-06 FJ663775 Kyrgyzstan Moldatoo Mts, 41.5°N, 74.62°E Lukhtanov et al. 2009
M. ala bicolor M. ala bicolor BOLD:LOWA356-06 FJ663774 Kyrgyzstan Moldatoo Mts, 41.5°N, 74.62°E Lukhtanov et al. 2009
M. ala bicolor M. ala bicolor BOLD:BPAL2288-14 MW672075 China Xinjiang, Kunges Valley This study
M. ala bicolor M. ala bicolor BOLD:BPAL2289-14 MW672076 China Xinjiang, Kunges Valley This study
M. ala bicolor M. ala bicolor BOLD:BPAL012-10 MW672079 Kazakhstan Kirgizsky Mts, Kaindy This study
M. ala bicolor M. ala bicolor BOLD:BPAL013-10 MW672080 Kazakhstan Kirgizsky Mts, Kaindy This study
M. ala bicolor M. ala bicolor BOLD:BPAL026-10 MW672081 Kyrgyzstan Talassky Mts, Kara-Bura Pass This study
M. ala bicolor M. ala bicolor BOLD:BPAL027-10; RPVL-00027 MW672082 Kyrgyzstan Talassky Mts, Kara-Bura Pass This study
M. ala bicolor M. ala bicolor BOLD:BPAL3499-16 MW672089 Kyrgyzstan Talassky Mts, Kara-Bura Pass This study
M. ala bicolor M. ala bicolor BOLD:BPAL3500-16 MW672090 Kyrgyzstan Talassky Mts, Kara-Bura Pass This study
M. ala bicolor M. ala bicolor BOLD:BPAL3501-16 MW672091 Kyrgyzstan Talassky Mts, Kara-Bura Pass This study
M. ala bicolor M. ala bicolor BOLD:BPAL009-10; CCDB-03024-RPVL-00009 MW672078 Kazakhstan Kirgizsky Mts, Merke River This study
M. ala irtyshica M. ala BOLD:BPALB181-16 MW672073 Kazakhstan Zyryanovsk region, 49.62°N, 83.62°E This study
M. ala irtyshica M. ala BOLD:BPAL3481-16 MW672083 Kazakhstan Zyryanovsk region, 49.62°N, 83.62°E This study
M. ala irtyshica M. ala BOLD:BPAL3483-16 MW672085 Kazakhstan Zyryanovsk region, 49.62°N, 83.62°E This study
M. ala irtyshica M. ala BOLD:BPAL3484-16; CCDB-25456_F04 MW672086 Kazakhstan Zyryanovsk region, 49.62°N 83.62°E This study
M. ala irtyshica M. ala BOLD:BPAL3485-16 MW672087 Kazakhstan Zyryanovsk region, 49.62°N, 83.62°E This study
M. ala irtyshica M. ala BOLD:BPAL3486-16 MW672088 Kazakhstan Zyryanovsk region, 49.62°N 83.62°E This study
M. ala zaisana M. ala zaisana BOLD:LOWA174-06 FJ663777 Kazakhstan Kurchumski Khrebet 48.47°N, 84.12°E Lukhtanov et al. 2009
M. ala zaisana M. ala zaisana BOLD:LOWA175-06 FJ663776 Kazakhstan Kalgutynski Pass, 48.47°N 84.12°E Lukhtanov et al. 2009
M. alatauica Mellicta alatauica BOLD:PALB177-16 MW672064 Kazakhstan Dzhungarian Mts, Kopal, 45.08°N, 79.07°E This study
M. alatauica Mellicta alatauica BOLD:LOWA273-06 FJ663811 Kazakhstan Dshungarski Alatau, Koksu, 44.72°N, 79.0°E Lukhtanov et al. 2009
M. alatauica Mellicta alatauica BOLD:LOWA274-06 FJ663810 Kazakhstan Dshungarski Alatau, Koksu, 44.72°N, 79.0°E Lukhtanov et al. 2009
M. casta M. casta BOLD:BPAL2306-14 KY777552 Iran Lorestan Lukhtanov 2017
M. deserticola M. deserticola BOLD:BPAL3124-15 KY086157 Israel Jerusalem Pazhenkova and Lukhtanov 2016
M. didyma M. didyma BOLD:BPAL2495-14 KT874733 Austria Tirol Pazhenkova et al. 2015
M. didymoides M. didymoides BOLD:BPAL3493-16 KY086178 Russia Buryatia Pazhenkova and Lukhtanov 2016
M. enarea M. enarea BOLD:BPAL2656-14 MW672065 Tajikistan Tabakchi, 37.85° N, 68.98°E, 1200 m This study
M. enarea M. enarea BOLD:BPAL2657-14 MW672066 Tajikistan Chaltau, 37.9550°N, 69.1403°E; 1041m This study
M. enarea M. enarea BOLD:BPAL2658-14 MW672067 Tajikistan Chaltau, 37.9550°N, 69.1403°E; 1041m This study
M. enarea M. enarea BOLD:BPAL2659-14; CCDB-17967_H10 MW672068 Tajikistan Chaltau, 37.9550°N, 69.1403°E; 1041m This study
M. enarea permuta M. enarea permuta BOLD:GBLN1837-09 FJ462272 Uzbekistan Ghissar Mts Leneveu et al. 2009
M. gina M. gina BOLD:BPAL3083-15 KY086152 Iran 35.32°N, 47.15°E Pazhenkova and Lukhtanov 2016
M. higginsi M. higginsi BOLD:BPAL2469-14 KY777548 Afghanistan Lukhtanov 2017
M. interrupta M. interrupta BOLD:BPAL3019-15 KY086139 Georgia Bakuriani Pazhenkova and Lukhtanov 2016
M. kotshubeji bundeli Melitaea ala bundeli GA161 MW672092 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji bundeli Melitaea ala bundeli GA162 MW672093 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji bundeli Melitaea ala bundeli GA163 MW672094 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji bundeli Melitaea ala bundeli GA164 MW672095 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji bundeli Melitaea ala bundeli GA165 MW672096 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji bundeli Melitaea ala bundeli GA166 MW672097 Tajikistan Alai Mts, 39.42°N, 71.62°E This study
M. kotshubeji kotshubeji M. ala kotshubeji BOLD:BPAL2308-14 MW672069 Tajikistan Peter I Range, Garm This study
M. kotshubeji kotshubeji M. ala kotshubeji BOLD:BPAL2484-14; CCDB-17966 B02 MW672070 Tajikistan Peter I Range, 7 km S Tajikobad This study
M. kotshubeji kotshubeji M. ala kotshubeji BOLD:BPAL2485-14 MW672071 Tajikistan Peter I Range, Garm This study
M. latonigena M. latonigena BOLD:BPAL3476-16 KY086170 Russia Altai Pazhenkova and Lukhtanov 2016
M. mauretanica M. didyma NW107-5; BOLD:GBLN1855-09 FJ462253 Morocco Leneveu et al. 2009
M. mixta M. chitralensis BOLD:MABUT253-11 KC158427 Pakistan 35.8333°N, 71.7667°E Ashfaq et al. 2013
M. mixta M. chitralensis BOLD:MABUT254-11 KC158426 Pakistan 35.8333°N, 71.7667 °E Ashfaq et al. 2013
M. mixta M. didyma BOLD:BPAL2509-14 KT874722 Tajikistan Farob Pazhenkova et al. 2015
M. neera M. neera BOLD:BPAL3482-16 MW672084 Kazakhstan Zyryanovsk region, 49.62°N, 83.62°E This study
M. neera liliputana M. didyma CCDB-17968 E10; BOLD:BPAL2718-14 KT874744 Israel Hermon Pazhenkova at al. 2015
M. occidentalis M. didyma RVcoll.08-L832 GU676247 Spain Comunidad_de_Madrid GenBank
M. persea M. persea BOLD:BPAL2349-14 KY777522 Iran Tehran Lukhtanov 2017
M. persea paphlagonia M. persea BOLD:BPAL2959-15 KY777526 Iran Shahrud Lukhtanov 2017
M. saxatilis M. saxatilis NW120-8; BOLD:GBLN1828-09 FJ462281 Iran Tehran Leneveu et al. 2009
M. sutschana M. sutschana BOLD:BPAL2543-14 KT874696 Russia Chita Pazhenkova et al. 2015
M. telona M. ornata telona BOLD:BPAL3126-15 MW672062 Israel This study
M. turkestanica M. didyma BOLD:BPAL2770-15 KY086115 Kazakhstan Saikan Pazhenkova and Lukhtanov 2016

Sequences were aligned using the BioEdit software (Hall 1999) and edited manually. Phylogenetic hypotheses were inferred using Bayesian inference as described previously (Vershinina and Lukhtanov 2010; Przybyłowicz et al. 2014; Lukhtanov et al. 2016). Briefly, the Bayesian analysis was performed using the program MrBayes 3.2 (Ronquist et al. 2012) with default settings. Two runs of 10,000,000 generations with four chains (one cold and three heated) were performed. We checked runs for convergence and proper sampling of parameters [effective sample size (ESS) > 200] using the program Tracer v1.7.1 (Rambaut et al. 2018). The first 25% of each run was discarded as burn-in. The consensus of the obtained trees was visualized using FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).

Results

Karyotype

The haploid chromosome number n=29 was found in prometaphase I, MI and MII cells of four studied individuals of M. kotshubeji kotshubeji (Table 2, Fig. 1). All chromosome elements formed a gradient size row. The karyotype contained no exceptionally large or small chromosomes.

Table 2.

Chromosome number in studied samples of Melitaea kotshubeji kotshubeji.

Code number of the specimen Chromosome number Locality, date and collector Number of cells checked
VLcoll.17-AB028 n=29 Tajikistan, Peter the Great Mts, Ganishou, 2200 m, 30.VI.2017, E. Pazhenkova leg. 5
VLcoll.17-AB080 n=29 Tajikistan, Peter the Great Mts, Muk, 2800 m, 25.VII.017, V. Lukhtanov leg. 7
VLcoll.17-AB086 n=29 Tajikistan, Peter the Great Mts, Muk, 2800 m, 26.VII.2017, V. Lukhtanov leg. 11
VLcoll.17-AB087 n=29 Tajikistan, Peter the Great Mts, Muk, 2800 m, 26.VII.2017, V. Lukhtanov leg. 14
Figure 1.

Karyotype of M. kotshubeji a general view of six MI cells in a spermatocyst b M. kotshubeji, AB080, MI, n=29. Scale bar: 10 μm.

DNA barcode analysis

DNA barcode analysis revealed M. ala, M. kotshubeji and M. enarea as highly supported monophyletic entities. Together, these three species formed a monophyletic lineage (the M. ala species complex) (1 in Fig. 2). In relation to the M. ala species complex, M. acraeina was found as a phylogenetically distant sister group (2 in Fig. 2). Taxa close to M. didyma (the M. didyma species complex) also formed a clade, but its support was relatively low (3 in Fig. 2). The species M. deserticola formed an independent lineage within the M. didyma species group (4 in Fig. 2). Together, these four lineages (M. ala complex + M. acraeina + M. didyma complex+ M. deserticola) formed the well-supported M. didyma species group (I in Fig. 2). The species of the M. persea group also formed a supported clade, sister to the M. didyma group (5 and II in Fig. 2).

Figure 2.

The Bayesian 50% majority rule consensus tree of the analyzed samples of Melitaea inferred from COI sequences. Melitaea alatauica and M. telona sequences are used to root the tree. Museum ID numbers, GenBank accession numbers, species and subspecies names, and localities are shown to the right of the branches. Bayesian posterior probabilities higher than 0.75 are shown next to the recovered branches. b1 is M. ala bicolor, clade 1. b2 is M. ala bicolor, clade 2. i is M. ala irtyshica. k is M. kotshubeji kotshubeji. z is M. ala zaisana 1 is the M. ala species complex 2 is M. acraeina 3 is the M. didyma species complex. 4 is M. deserticola. 5 is the M. persea species complex. I is M. didyma species group. II is M. persea species group.

Within the M. ala clade, five supported (Bayesian posterior probabilities ranged from 0.9 to 1.0), relatively weakly differentiated subclades were found. These are (1) M. ala ala, (2) M. ala irtyshica, (3) M. ala zaisana, (4) M. ala bicolor (clade b1) and (5) M. ala bicolor (clade b2). We also calculated the uncorrected COI p-distances within (Table 3) and between (Table 4) the revealed clades and groups.

Table 3.

Intragroup uncorrected COI p-distances revealed within M. ala.

Group Minimum p-distance Maximum p-distance
irtyshica 0% 0.2%
zaisana 0% 0%
(irtyshica+zaisana) 0% 0.5%
ala 0% 0%
bicolor1 0% 0.6%
bicolor2 0% 0.2%
(bicolor1+bicolor2) 0% 0.8%

Melitaea kotshubeji kotshubeji and M. kotshubeji bundeli were found to differ by four fixed nucleotide substitutions in the COI barcode region.

Table 4.

Uncorrected COI p-distances between the groups revealed within M. ala.

Group 1 Group 2 Minimum p-distance Maximum p-distance
irtyshica zaisana 0.3% 0.5%
(irtyshica+zaisana) ala 0.9% 1.5%
(irtyshica+zaisana) bicolor1 0.9% 1.5%
(irtyshica+zaisana) bicolor2 0.9% 1.5%
ala bicolor1 0.9% 1.3%
ala bicolor2 0.9% 1.5%
bicolor1 bicolor2 0.3% 0.8%
(irtyshica+zaisana) (bicolor1+bicolor2) 0.9% 1.5%
ala (bicolor1+bicolor2) 0.9% 1.5%

Discussion

Chromosome number variation

The genus Melitaea (Fabricius, 1807) has relatively low interspecific chromosome number variation. The representatives of basal clades (see phylogeny in Leneveu et al. 2009), the taxa of M. cinxia (Linnaeus, 1758), M. diamina (Lang, 1789), M. athalia (Rottemburg, 1775), M. trivia ([Denis et Schiffermüller], 1775) and M. phoebe ([Denis et Schiffermüller], 1775) species groups demonstrate n=30–31 (Federley 1938; de Lesse 1960; Robinson 1971; Larsen 1975; Hesselbarth et al. 1995). These haploid numbers are modal ones not only for Melitaea, but also for the family Nymphalidae and for the order Lepidoptera in whole (Robinson 1971; Lukhtanov 2000, 2014). Most likely, one of them (probably, n=31, see Lukhtanov 2014) represents an ancestral lepidopteran state preserved in the basal lineages of Melitaea.

The Melitaea didyma species group is one of the younger lineages of Melitaea (Leneveu et al. 2009). This group is found to have lower chromosome numbers varying from n=27 to n=29–30 (Table 5). Melitaea didyma species complex is characterized by chromosome numbers from n n=27 to n=30, with n=28 and n=29 as modal numbers. In the Melitaea deserticola species complex, only one species (M. deserticola) is karyotyped (n=29). In the Melitaea persea species complex, n=27 is found in two species. In the Melitaea ala species complex, n=29 is found in two species studied.

Table 5.

Chromosome nmbers of taxa close to M. didyma.

Species complex Taxon Chromosome number Country Locality Reference
Melitaea didyma species complex M. didyma n=28 Italy Abruzzi de Lesse 1960
M. didyma neera n=28 Kazakhstan Altai Lukhtanov and Kuznetsova 1989
M. didyma neera n=27 Russia N Caucasus, Pyatigorsk Lukhtanov and Kuznetsova 1989
M. interrupta n=29 Turkey de Lesse 1960
M. interrupta n=29 Azerbaijan, Nakhichevan Zangezur Mts Lukhtanov and Kuznetsova 1989
M. latonigena n=29–30 Kazakhstan Altai Lukhtanov and Kuznetsova 1989
M. gina n=28 Iran W Azerbaijan Pazhenkova and Lukhtanov 2016
Melitaea deserticola species complex M. deserticola n=29 Lebanon Larsen 1975
Melitaea ala species complex M. ala n=29 Kazakhstan Lukhtanov and Kuznetsova 1989
M. kotshubeji n=29 Tajikistan This study
Melitaea persea species complex M. persea n=27 Iran de Lesse 1960
M. acentria n=27 Israel Lukhtanov 2017

Based on the distribution of the known chromosome numbers (Table 3) relative to the phylogeny (Fig. 2) and on the frequency of their occurrence, we can assume that n=29 is an ancestral state for the species of the M. didyma group. Thus, for the species of the M. ala complex n=29 is a symplesiomorphy.

Intraspecific taxonomy of the M. ala species group

The five identified clades within the species M. ala have relatively high support (Fig. 2) and can be considered as taxa, at least from the standpoint of the phylogenetic species concept (Cracraft 1989; Coyne and Orr 2004), in which diagnosable entities can be classified as species regardless of whether there is reproductive isolation between them or not. To assess the possibility of interpreting these clades as species or subspecies, we compared the level of COI divergence between the clades with the level of variability within the clades (Tables 3, 4). We found that in all cases, the distances between these clades were lower than ‘standard’ DNA-barcode species threshold (3%) (Hebert et al. 2003).

An especially low level of differentiation (0.3–0.5%) was found between the clades M. ala zaisanica and M. ala irtyshica. Therefore, we are inclined, especially taking into account the geographical proximity of these lineages, to consider them as a single taxonomic unit, M. ala zaisanica (= M. ala irtyshica).

A slightly higher average level of differentiation (0.3–0.8%) was found between the b1 and b2 clades (Fig. 2, Table 4). However, in this case, a rather high level of intragroup variability was observed (Table 3), and the maximum values of intragroup variability exceeded the minimum intergroup differences. Therefore, taking into account the geographical proximity of these lineages, we decided to consider them as a single taxonomic unit, M. ala bicolor.

Thus, within the studied populations, three subspecies can be distinguished. These are M. ala ala, M. ala bicolor and M. ala zaisana.

Melitaea ala ala is distributed in the Dzhungarian Alatau in East Kazakhstan (Fig. 3). This subspecies is characterized by darkening of the veins on the underside of the hind wing. These darkened veins form clear cells in the region of the median band (Fig. 4a).

Figure 3.

Locations of the analyzed samples of M. ala, M. kotshubeji and M. enarea 1 type-locality of M. ala irtyshica (Kazakhstan, Zyryanovsk district, Oktyabrsk, 49.62°N, 83.62°E) 2 type-locality of M. ala zaisanica (Kazakhstan, Kurtchumski Mts, 48.47°N, 84.12°E) 3 M. ala ala (Kazakhstan, Dzhungarian Alatau, Kyzylagash and Kopal) 4 M. ala bicolor (clade b1) (China, Kyrgyzstan) 5 M. ala bicolor (clade b2) (Kyrgyzstan, Kara-Bura Pass; Kazakhstan, Kirgizski Mts) 6 M. kotshubeji kotshubeji (Tajikistan, Peter the Great Mts) 7 M. kotshubeji bundeli (Tajikistan, border with Kyrgyzstan, Alai Mts, 39.42°N, 71.62°E) 8 M. enarea (Tajikistan).

Melitaea ala bicolor Seitz, 1908 is distributed in the North, East, Central and West Tian-Shan in SE Kazakhstan, NW China and Kyrgyzstan (Fig. 3). In this subspecies the veins on the underside of the hind wing are not strongly darkened. The cells of the median band are not highlighted. They are only marked with dark brackets on the outside of the median band (Fig. 4b). The specimens from the Tyshkantau Mts (SE part of the Dzhungarian Alatau in Kazakhstan) (Tuzov and Churkin 2000) and the eastern most part of the Tian-Shan (Kolesnichenko 1999) are intermediate between M. ala ala and M. ala bicolor.

Figure 4.

Butterflies of the Melitaea ala species complex a M. ala ala, male, BPALB179-16 (CCDB-25458_G12), Kazakhstan, Dzhungarian Alatau, Kopal, 45.04°N, 79.06°E, 1800–1900 m, 13.VI.2016, V. Lukhtanov leg. b M. ala bicolor, clade b1, male, Kyrgyzstan, Moldatoo Mts, 41.5°N, 74.62°E, 2100 m, 29.VI.1996, V. Lukhtanov leg. c M. ala zaisana, male, LOWA174-06, Kazakhstan, Kurchumski Khrebet, Kalgutinski Pass, 600 m, 48.47°N, 84.12°E, 9.VI.1998, V. Lukhtanov leg. d M. ala irtyshica, male, BPAL3484-16 (CCDB-25456_F04), Kazakhstan, Zyryanovsk distr., Oktyabrsk, 49.6178°N, 83.6219°E, 420 m, 08.VI.1999, V. Lukhtanov leg. e M. ala bicolor, clade b2, male, CCDB-03024-RPVL-00009, Kazakhstan, Kirgizski Mts, Merke, 42.69°N, 73.25°E, 1500m, 13.VI.2000, V. Lukhtanov leg. f M. ala bicolor, clade b2, male, BPAL027-10 (RPVL-00027), Kyrgyzstan, Talassky Mts, Kara-Bura pass, 42.27°N, 71.57°E, 2000m, 30.VI.2000, V. Lukhtanov leg. g M. ala bicolor, clade b2, male, BPAL026-10 (RPVL-00026), Kyrgyzstan, Talassky Mts, Kara-Bura pass, 42.27°N, 71.57°E, 2000m, 30.VI.2000, V. Lukhtanov leg. h M. kotshubeji bundeli, male, GA161, Tajikistan, Alai Mts, Kichi-Karamuk, 39.4258°N, 71.6125°E; 3150 m, 03.VIII.2019, V. Lukhtanov leg. i M. kotshubeji bundeli, female, GA166, Tajikistan, Alai Mts, Kichi-Karamuk, 39.4258°N, 71.6125°E; 3150 m, 03.VIII.2019, V. Lukhtanov leg. j M. kotshubeji kotshubeji, male, BPAL2484-14 (CCDB-17966 B02), Tajikistan, Peter I Range, 7 km S Tajikobad, 14.VIII.2003 k M. enarea, male, BPAL2656-14 (CCDB-17967_H07), Tajikistan, Tabakchi Mts, 37.85°N, 68.98°E, 1150 m, 01.V.2014, V. Lukhtanov leg. l M. enarea, female, BPAL2659-14 (CCDB-17967_H10), Tajikistan, Chaltau Mts, 37.9550°N, 69.1403°E, 1041m, 02.V.2014, V. Lukhtanov leg. Scale bar: 10 mm

With regards to DNA barcodes, M. ala zaisana Lukhtanov, 1999 (Fig. 4c) is distinct from the geographically closest M. ala ala. With regards to the wing pattern, M. ala zaisana is more similar to M. ala bicolor than to M. ala ala. Interestingly, the northernmost population of M. ala from Oktyabrsk (Kazakhstan) (Fig. 3d) is intermediate in its appearance between M. ala ala and M. ala zaisana. This population was described as M. ala irtyshica Lukhtanov, 1999 (Lukhtanov 1999) and was later erroneously synonymized with M. latonigena Eversmann, 1847 (Lukhtanov et al. 2007). DNA barcode analysis demonstrates that this population is similar to M. ala zaisana.

Figure 5.

Syntypes of the taxa of the Melitaea ala species complex, originally described by Felix Bryk (1940) as subspecies of M. didyma. All specimens are deposited in Swedish Museum of Natural History (Naturhistoriska riksmuseet, NRM) a M. didyma allah, upperside b M. didyma allah, underside c M. didyma allah, labels d M. didyma determinata, upperside e M. didyma determinata, underside f M. didyma determinata, labels g M. didyma sheljuzhkoi, upperside h M. didyma sheljuzhkoi, underside i M. didyma sheljuzhkoi, labels j M. didyma strandi, upperside k M. didyma strandi, underside l M. didyma strandi, labels.

Currently, there is a tendency to consider as a species any group of populations with a minimum set of fixed differences. We are almost certain that, given this trend, the subspecies discussed above will be interpreted by some authors as species in the future. Nevertheless, in our opinion, in accordance with the subspecies criteria (Lukhtanov et al. 2016; De Queiroz, 2020), they should be treated as subspecies of the same species.

Melitaea kotshubeji bundeli (Fig. 4h, i) was described as subspecies of Melitaea kotshubeji (Fig. 4j) (Kolesnichenko 1999), but later was treated as a distinct species (van Oorschot and Coutsis 2014) or alternatively as a synonym (Tshikolovets 2003, 2005). Our study demonstrates that these two taxa are not only distinct in the wing pattern, but also differ by four fixed nucleotide substitutions in the DNA barcode region, indicating the relative long independent evolution of these two sublineages. Interestingly, the distribution areas of these two allopatric taxa are in close proximity to each other and are separated by a narrow valley of the Surkhob River (in Kyrgyzstan, this river is called the Kyzylsu).

In our work we do not consider the intraspecific structure of M. enarea (Fig. 4k, l) due to the lack of molecular data for the northern populations of this species.

The taxa described by Bryk (1940)

Bryk (1940) described four taxa (all as subspecies of M. didyma) that should be assigned to M. ala. The types of these taxa were studied by the first author of this article in 2007 during a visit to Swedish Museum of Natural History.

The taxon described by Bryk (1940) as M. didyma allah Bryk, 1940 has the wing pattern with clear characters of M. ala ala (Fig. 5a, b), but not of the subspecies M. ala zaisana (Fig. 4c) as supposed by Tuzov and Churkin (2000). Thus, M. didyma allah should be synonymized with M. ala ala as suggested by Kolesnichenko (1999). We agree with Kolesnichenko (1999) that the label data of the syntype of M. didyma allah (Fig. 5c) are probably wrong.

The taxa described by Bryk (1940) as M. didyma sheljuzhkoi Bryk, 1940 (Fig. 5g–i) and M. didyma strandi (Fig. 5j–l) have the wing pattern with characters of M. ala bicolor. Most likely, they represent synonyms of M. ala bicolor.

The taxon from “Fu-Shu-Shi” (China) described by Bryk (1940) as M. didyma determinata Bryk, 1940 is characterized by the well-developed black wing pattern on both wing upper- and underside (Fig. 5d–f). Most likely, it represents a distinct subspecies. Unfortunately, we do not have material for molecular study to test this hypothesis.

Probably erroneous species identifications in the M. ala complex

The specimens identified as Melitaea ninae (sample NW113-10, FJ462269, Kyrgyzstan), M. enarea (sample NW113-15, FJ462256, Tajikistan) (Leneveu et al. 2009; Long et al. 2014) and M. chitralensis (samples KC158426 and KC158427) (Ashfaq et al. 2013) were reported in the cited molecular phylogenetic analyses of the genus Melitaea. According to the DNA barcodes of these samples, they most likely belong to M. turkestanica Sheljuzhko, 1929 (NW113-10) and M. mixta Evans, 1912 (NW113-15, KC158426 and KC158427).

Acknowledgements

We thank Kirill Kolesnichenko, Anatoly Krupitsky, Nazar Shapoval and Martin Wiemers for critical comments and suggestions. We thank Sergei Sinev and Alexander Lvovsky (Zoological Institute of the Russian Academy of Sciences, St. Petersburg), and Andrei Sourakov and Andrew Warren (McGuire Center for Lepidoptera and Biodiversity, University of Florida) who provided an opportunity to work with the collections of their institutions. The work was partially performed using equipment of the Centre for Molecular and Cell Technologies of St. Petersburg State University. Elena Pazhenkova was supported by RFBR, project number 19-34-90007 (taxonomic studies). Vladimir Lukhtanov and Anastasia Gagarina were supported by grant 19-14-00202 from the Russian Science Foundation to the Zoological Institute of the Russian Academy of Sciences (molecular studies).

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