Research Article |
Corresponding author: Vladimir A. Lukhtanov ( lukhtanov@mail.ru ) Academic editor: Nazar Shapoval
© 2021 Vladimir A. Lukhtanov, Anastasia V. Gagarina, Elena A. Pazhenkova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Lukhtanov VA, Gagarina AV, Pazhenkova EA (2021) Chromosomal and DNA barcode analysis of the Melitaea ala Staudinger, 1881 species complex (Lepidoptera, Nymphalidae). Comparative Cytogenetics 15(2): 199-216. https://doi.org/10.3897/CompCytogen.v15.i2.66121
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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.
chromosome, COI, DNA barcode, karyosystematics, Melitaea, taxonomy
This work is a continuation of a series of publications (
The species of this complex are distributed in Central Asia (
Molecular phylogenetic analysis (
Karyotypes of four samples of M. kotshubeji kotshubeji were studied as previously described (
Standard COI barcodes (658-bp 5’ fragment of mitochondrial cytochrome oxidase subunit I) were studied as previously described (
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
Legs from 28 specimens of Melitaea spp. were processed in the the Canadian Centre for DNA Barcoding (
We also used 30 published COI sequences for DNA barcode analysis (
Species and subspecies | Species name as found in GenBank | Field code or BOLD number | GenBank number | Country | Locality | Reference |
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M. acentria | M. acentria | BOLD:BPAL2191-13 | KY777529 | Israel | Hermon |
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M. acraeina | M. acraeina | BOLD:GBLN1879-09 | FJ462229 | Uzbekistan | Komsomolobad |
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M. ala ala | M. ala | BPALB179-16; |
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 |
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M. ala bicolor | M. ala bicolor | BOLD:LOWA355-06 | FJ663775 | Kyrgyzstan | Moldatoo Mts, 41.5°N, 74.62°E |
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M. ala bicolor | M. ala bicolor | BOLD:LOWA356-06 | FJ663774 | Kyrgyzstan | Moldatoo Mts, 41.5°N, 74.62°E |
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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; |
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; |
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 |
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M. ala zaisana | M. ala zaisana | BOLD:LOWA175-06 | FJ663776 | Kazakhstan | Kalgutynski Pass, 48.47°N 84.12°E |
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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 |
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M. alatauica | Mellicta alatauica | BOLD:LOWA274-06 | FJ663810 | Kazakhstan | Dshungarski Alatau, Koksu, 44.72°N, 79.0°E |
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M. casta | M. casta | BOLD:BPAL2306-14 | KY777552 | Iran | Lorestan |
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M. deserticola | M. deserticola | BOLD:BPAL3124-15 | KY086157 | Israel | Jerusalem |
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M. didyma | M. didyma | BOLD:BPAL2495-14 | KT874733 | Austria | Tirol |
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M. didymoides | M. didymoides | BOLD:BPAL3493-16 | KY086178 | Russia | Buryatia |
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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; |
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 |
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M. gina | M. gina | BOLD:BPAL3083-15 | KY086152 | Iran | 35.32°N, 47.15°E |
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M. higginsi | M. higginsi | BOLD:BPAL2469-14 | KY777548 | Afghanistan |
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M. interrupta | M. interrupta | BOLD:BPAL3019-15 | KY086139 | Georgia | Bakuriani |
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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; |
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 |
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M. mauretanica | M. didyma | NW107-5; BOLD:GBLN1855-09 | FJ462253 | Morocco |
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M. mixta | M. chitralensis | BOLD:MABUT253-11 | KC158427 | Pakistan | 35.8333°N, 71.7667°E |
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M. mixta | M. chitralensis | BOLD:MABUT254-11 | KC158426 | Pakistan | 35.8333°N, 71.7667 °E |
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M. mixta | M. didyma | BOLD:BPAL2509-14 | KT874722 | Tajikistan | Farob |
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M. neera | M. neera | BOLD:BPAL3482-16 | MW672084 | Kazakhstan | Zyryanovsk region, 49.62°N, 83.62°E | This study |
M. neera liliputana | M. didyma |
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KT874744 | Israel | Hermon | |
M. occidentalis | M. didyma | RVcoll.08-L832 | GU676247 | Spain | Comunidad_de_Madrid | GenBank |
M. persea | M. persea | BOLD:BPAL2349-14 | KY777522 | Iran | Tehran |
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M. persea paphlagonia | M. persea | BOLD:BPAL2959-15 | KY777526 | Iran | Shahrud |
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M. saxatilis | M. saxatilis | NW120-8; BOLD:GBLN1828-09 | FJ462281 | Iran | Tehran |
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M. sutschana | M. sutschana | BOLD:BPAL2543-14 | KT874696 | Russia | Chita |
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M. telona | M. ornata telona | BOLD:BPAL3126-15 | MW672062 | Israel | This study | |
M. turkestanica | M. didyma | BOLD:BPAL2770-15 | KY086115 | Kazakhstan | Saikan |
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Sequences were aligned using the BioEdit software (
The haploid chromosome number n=29 was found in prometaphase I, MI and MII cells of four studied individuals of M. kotshubeji kotshubeji (Table
Code number of the specimen | Chromosome number | Locality, date and collector | Number of cells checked |
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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 |
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.
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
Group | Minimum p-distance | Maximum p-distance |
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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.
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% |
The genus Melitaea (Fabricius, 1807) has relatively low interspecific chromosome number variation. The representatives of basal clades (see phylogeny in
The Melitaea didyma species group is one of the younger lineages of Melitaea (
Species complex | Taxon | Chromosome number | Country | Locality | Reference |
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Melitaea didyma species complex | M. didyma | n=28 | Italy | Abruzzi |
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M. didyma neera | n=28 | Kazakhstan | Altai |
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M. didyma neera | n=27 | Russia | N Caucasus, Pyatigorsk |
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M. interrupta | n=29 | Turkey |
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M. interrupta | n=29 | Azerbaijan, Nakhichevan | Zangezur Mts |
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M. latonigena | n=29–30 | Kazakhstan | Altai |
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M. gina | n=28 | Iran | W Azerbaijan |
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Melitaea deserticola species complex | M. deserticola | n=29 | Lebanon |
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Melitaea ala species complex | M. ala | n=29 | Kazakhstan |
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M. kotshubeji | n=29 | Tajikistan | This study | ||
Melitaea persea species complex | M. persea | n=27 | Iran |
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M. acentria | n=27 | Israel |
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Based on the distribution of the known chromosome numbers (Table
The five identified clades within the species M. ala have relatively high support (Fig.
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.
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.
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.
Butterflies of the Melitaea ala species complex a M. ala ala, male, BPALB179-16 (
With regards to DNA barcodes, M. ala zaisana Lukhtanov, 1999 (Fig.
Syntypes of the taxa of the Melitaea ala species complex, originally described by Felix
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 (
Melitaea kotshubeji bundeli (Fig.
In our work we do not consider the intraspecific structure of M. enarea (Fig.
The taxon described by
The taxa described by
The taxon from “Fu-Shu-Shi” (China) described by
The specimens identified as Melitaea ninae (sample NW113-10, FJ462269, Kyrgyzstan), M. enarea (sample NW113-15, FJ462256, Tajikistan) (
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).