Chromosomal and DNA barcode analysis of the Melitaea ala Staudinger, 1881 species complex (Lepidoptera, Nymphalidae).

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. Vladimir A. Lukhtanov, Anastasia V. Gagarina, Elena A. Pazhenkova.

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 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 (Esper, 1779) (Pazhenkova et al. 2015; Pazhenkova and Lukhtanov 2016) and Kollar, 1849 (Lukhtanov 2017) species complexes. The aim of this work is to study a complex of species close to 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: Staudinger, 1881, Sheljuzhko, 1929, Sheljuzhko, 1935, Moore, 1901, and Fruhstorfer, 1917. According to van Oorschot and Coutsis (2014), this complex consists of the following species: Staudinger, 1881, Sheljuzhko, 1935, Staudinger, 1881, Staudinger, 1895, Moore, 1901, Fruhstorfer, 1917, Kolesnichenko, 1999, Sheljuzhko, 1929, Staudinger, 1881 and Matsumura, 1927 (the latter taxon is usually considered a subspecies of , e.g. see Higgins, 1941).

Molecular phylogenetic analysis (Leneveu et al. 2009) demonstrated that and (cited in the article as Higgins, 1941) are sister species, and is a phylogenetically distant species which is a sister to the lineage ( + ). was found as a member of the species complex which is a sister to the lineage (( + ( + )) (Leneveu et al. 2009). In our study, we focused on the analysis of the lineage. We did not include , and 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 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 species group and outgroups ( Fruhstorfer, 1908 and Staudinger, 1881). Legs were used as a source for DNA isolation

Legs from 6 specimens ( 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 spp. were processed in the the Canadian Centre for DNA Barcoding (, 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 (), 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 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 (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 .

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

DNA barcode analysis

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

Figure 2.

The Bayesian 50% majority rule consensus tree of the analyzed samples of inferred from COI sequences. and 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. is , clade 1. is , clade 2. is is . is 1 is the species complex 2 is 3 is the species complex. 4 is . 5 is the species complex. I is species group. II is species group.

Within the clade, five supported (Bayesian posterior probabilities ranged from 0.9 to 1.0), relatively weakly differentiated subclades were found. These are (1) , (2) , (3) , (4) (clade b1) and (5) (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 .

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%

Table 4.

Uncorrected COI p-distances between the groups revealed within .

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%

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

Discussion

Chromosome number variation

The genus (Fabricius, 1807) has relatively low interspecific chromosome number variation. The representatives of basal clades (see phylogeny in Leneveu et al. 2009), the taxa of (Linnaeus, 1758), (Lang, 1789), (Rottemburg, 1775), ([Denis et Schiffermüller], 1775) and ([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 , but also for the family and for the order 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 .

The species group is one of the younger lineages of (Leneveu et al. 2009). This group is found to have lower chromosome numbers varying from n=27 to n=29–30 (Table 5). 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 species complex, only one species () is karyotyped (n=29). In the species complex, n=27 is found in two species. In the species complex, n=29 is found in two species studied.

Table 5.

Chromosome nmbers of taxa close to .

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 group. Thus, for the species of the complex n=29 is a symplesiomorphy.

Intraspecific taxonomy of the species group

The five identified clades within the species 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 and . Therefore, we are inclined, especially taking into account the geographical proximity of these lineages, to consider them as a single taxonomic unit, (= ).

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, .

Thus, within the studied populations, three subspecies can be distinguished. These are , and .

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 , and 1 type-locality of (Kazakhstan, Zyryanovsk district, Oktyabrsk, ) 2 type-locality of (Kazakhstan, Kurtchumski Mts, ) 3 (Kazakhstan, Dzhungarian Alatau, Kyzylagash and Kopal) 4 (clade b1) (China, Kyrgyzstan) 5 (clade b2) (Kyrgyzstan, Kara-Bura Pass; Kazakhstan, Kirgizski Mts) 6 (Tajikistan, Peter the Great Mts) 7 (Tajikistan, border with Kyrgyzstan, Alai Mts, ) 8 (Tajikistan).

Figure 4.

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

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 and .

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

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.

(Fig. 4h, i) was described as subspecies of (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 (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 ) that should be assigned to . 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 Bryk, 1940 has the wing pattern with clear characters of (Fig. 5a, b), but not of the subspecies (Fig. 4c) as supposed by Tuzov and Churkin (2000). Thus, should be synonymized with as suggested by Kolesnichenko (1999). We agree with Kolesnichenko (1999) that the label data of the syntype of (Fig. 5c) are probably wrong.

Figure 5.

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

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

The taxon from “Fu-Shu-Shi” (China) described by Bryk (1940) as 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 complex

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