Corresponding author: Gunilla Ståhls (
Academic editor: T. Backeljau
A majority of the known
The use of a standardized gene region, i.e. a 650 bp fragment of the 5’-region of the mitochondrial cytochrome
The butterfly genus
The Central Asian mountainous regions harbour nearly half of all
The first contribution to the species classification of
The aim of the present study was to test the usefulness of COI barcodes for species identification of a broad representation of Central Asian
This study includes material from the mountain regions of Kirgizistan, Tadzhikistan, northern Afghanistan, northern Pakistan and India (e.g. mountain ranges Tian Shan, Hindu Kush, Karakorum, Himalaya) and the mountain regions in the Chinese provinces Qinghai, Gansu, Sichuan, Yunnan and the autonomous regions Tibet and Xinjiang Uygur. The
The taxon sampling aimed to cover as many of the
List of specimens used for molecular analyses including GenBank accession numbers.
Species | Sex | Locality and date | Lab code | COI accession number | RpS2 accession number |
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male | Russia, SW Transbaikalia, Buryatia, Selenga river district, Gusinoye Ozero village env., steppe rivulet valley, 7.6.2003 | MZH_JL35 |
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male | Russia, SW Transbaikalia, Buryatia, Selenga river district, Gusinoye Ozero village env., steppe rivulet valley, 7.6.2003 | MZH_JL44 |
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male | China, Gansu, Xia-He, 3400 m, |
MZH_JL61 |
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female | Kyrgyzstan, Alai mts., 4 km SE Tengizbai pass, 3400 m, 3.7.2001 | MZH_JL37 |
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female | Kyrgyzstan, Alai mts., 4 km SE Tengizbai pass, 3400 m, 3.7.2001 | MZH_JL51 |
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male | China, S Tibet, Himalaya Mts., Lablungla pass, 4800 m, 18–22.7.2001 | MZH_JL48 |
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male | China, Tibet, Lhodak, 4600 m, 15.7.2002 | MZH_JL55 |
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female | Tadjikistan, Turkestanskyi Mts., Kumbel pass, 3000 m, July 2002 | MZH_JL45 |
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male | Kyrgyzstan, Alai Mts., W end of Tengizbai pass, 3700 m, 5–6.7.2001 | MZH_JL67 |
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male | Kyrgyzstan, Suusamyr Mt. r., Alabel pass, 3200 m, 10.7.2002 | MZH_JL43 |
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male | Tajikistan, E Pamir, Ak-Buura Mts., 4250 m, 14–15.7.2003 | MZH_JL34 |
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male | Kyrgyzstan, Sary Dzhaz riv. bas., Kaindy-Ketta mts., Tashkoro village, 3000 m 10.7.2003 | MZH_JL19 |
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male | Kyrgyzstan, W end of Tengizbai pass, 3700 m, 5–6.7.2001 | MZH_JL1 |
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male | Kyrgyzstan, W end of Tengizbai pass, 3700 m, 5–6.7.2001 | MZH_JL40 |
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male | China, Sichuan, Zhangia, 3000 m, |
MZH_JL50 |
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female | China, Gansu, Shin-Long-Shan, 2800 m, |
MZH_JL60 |
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female | China, Gansu, Altun Shan, road from Aksay to Danjing pass, 2500–2800 m, 22–23.7.2002 | MZH_JL54 |
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male | Russia, SW Transbaikalia, Buryatia, Selenga river district, Gusinoye Ozero village env., steppe rivulet valley, 1.7.2003 | MZH_JL39 |
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male | Russia, SW Transbaikalia, Buryatia, Selenga river district, Gusinoye Ozero village env., steppe rivulet valley, 1.7.2003 | MZH_JL46 |
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male | China, Sichuan, Maningano surr., |
MZH_JL7 |
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male | China, Sichuan, Maningano surr., |
MZH_JL27 |
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male | China, SW Tibet, Himalaya Mts., 100km W Paryang, 4650–5000 m, 13.6.2004 | MZH_JL4 |
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male | China, SW Tibet, Himalaya Mts., 100km W Paryang, 4650–5000 m, 13.6.2004 | MZH_JL57 |
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male | Tadjikistan, E Pamir, Dunkeldyk Lake, 4400 m, 25.7.2003 | MZH_JL30 |
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male | Tadjikistan, E Pamir, Dunkeldyk Lake, 4400 m, 25.7.2003 | MZH_JL33 |
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male | Tadjikistan, E Pamir, Dunkeldyk Lake, 4400 m, 25.7.2003 | MZH_JL41 |
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male | China, Sichuan, Maningano surr., |
MZH_JL59 |
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male | China, Sichuan, Maningano surr., |
MZH_JL9 |
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male | China, Sichuan, Maningano surr., |
MZH_JL24 |
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male | China, Sichuan, Maningano surr., |
MZH_JL26 |
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male | China, SW Tibet, Himalaya Mts., 60 km S Saga, 4600–5000 m, 7–8.6.2004 | MZH_JL53 |
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male | China, SW Tibet, Himalaya Mts., Lablongla pass, 4800 m, 5.6.2004 | MZH_JL58 |
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male | Kyrgyzstan, Kaindy-Ketta Mt. r., Kumar pass, 3200 m, 12.7.2003 | MZH_JL8 |
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male | Kyrgyzstan, Kaindy-Ketta Mt. r., Kumar pass, 3200 m, 12.7.2003 | MZH_JL42 |
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male | Kyrgyzstan, Alai mts., 4 km SE Tengizbai pass, 3400 m, 7–8.7.2001 | MZH_JL3 |
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male | Kyrgyzstan, Alai mts., 4 km SE Tengizbai pass, 3400 m, 7–8.7.2001 | MZH_JL47 |
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male | Tadjikistan, Peter I Mts., Ganishob, 2400 m, 17.6.2004 | MZH_JL70 |
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male | China, Gansu, Xia-He, 3400 m, |
MZH_JL11 |
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male | China, Gansu, Xia-He, 3400 m, |
MZH_JL64 |
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male | Kyrgyzstan, Zaalaisky (Transalai) Mts., Altyn Dara river, 3000 m, 25.7.2000 | MZH_JL2 |
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male | Kyrgyzstan, Zaalaisky (Transalai) Mts., Altyn Dara river, 3000 m, 25.7.2000 | MZH_JL13 |
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male | Kyrgyzstan, Zaalaisky (Transalai) Mts., Altyn Dara river, 3000 m, 25.7.2000 | MZH_JL23 |
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male | India, Jammu Kashmir, Ladakh Range, Markha Valley, Ganda Pass, 4600 m, 12.7.2001 | MZH_JL15 |
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male | Kyrgyzstan, Sary Dzhaz riv. bas., Kaindy-Ketta mts., Tashkoro village, 3000 m, 10.7.2003 | MZH_JL10 |
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female | Kyrgyzstan, Sary Dzhaz riv. bas., Kaindy-Ketta mts., Tashkoro village, 3000 m, 10.7.2003 | MZH_JL17 |
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female | Kyrgyzstan, Sary Dzhaz riv. bas., Kaindy-Ketta mts., Tashkoro village, 3000 m, 10.7.2003 | MZH_JL25 |
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male | China, W Tibet, Mandhata Mt., 4900 m, 15–16.7.2003 | MZH_JL14 |
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male | China, Tibet, Himalaya Mts., Nyalam, 4200 m, 8.7.2003 | MZH_JL6 |
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male | China, SW Tibet, Himalaya Mts., Nyalam, 3700–4200 m, 28–30.6.2004 | MZH_JL63 |
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male | China, Qinghai, 20km NW of Zhidoi City, 4700–5000 m, 16.7.2000 | MZH_JL66 |
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male | China, S. Tibet, Cona, 4500–4700 m, 24–25.6.2004 | MZH_JL69 |
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male | Uzbekistan, Chandalas Mts., Chakmksh village, 2600 m, 27.6.2004 | MZH_JL71 |
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male | Iran, Khorasan, 75km SE of Birjand, 2200 m, 18–20.5.2002 | MZH_JL68 |
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male | Kyrgyzstan, Alai mts., 4km SE Tengizbai pass, 3400 m, 3.7.2001 | MZH_JL65 |
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male | Russia, East Siberia, Lake Baikal, Khamar-Daban Mts., Slyudyanka river, taiga, 800 m, 14.6.2003 | MZH_JL5 |
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male | Russia, East Sayan, Buryatia, Mondy env., Huruma river, 1500 m, 6.6.2002 | MZH_JL12 |
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Total genomic DNA was extracted form 2-5 legs of dried, pinned butterfly specimens using NucleoSpin® Tissue Kit (Machery-Nagel), according to manufacturer’s protocols, and resuspended in 50 µl ultrapure water.
The primer pair LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994) was used to amplify a ca. 650 bp fragment of the mitochondrial COI gene. The polymerase chain reactions (PCR) were done under the following parameters: initial heating 95 °C for 2 min, following 30 cycles of 94 °C for 30 s, 49 °C for 30 s and 72 °C for 2 min, followed by a final extension of 72 °C for 7 min. The primer pair RpS2 nF (5’-ATCWCGYGGTGGYGATAGAG-3’) and RpS2 nR (5’-ATGRGGCTTKCCRATCTTGT-3’) (
We analysed and clustered our sequence data using parsimony and Neighbour-Joining (NJ) of K2P-distances. We used parsimony and NJ for our newly generated COI sequence dataset, NJ for RpS2 sequences, parsimony for the concatenated COI and RpS2 sequences, and, finally, NJ for the combined COI sequences generated in this study and those in GB. All trees were rooted using
Parsimony analysis was performed using NONA (
We obtained a 643 bp COI barcode for 56
Uncorrected pairwise divergences between ingroup taxa ranged between 1.09 and 4.09% (mean 2.77%) for COI and 0.0–1.7% (mean 1.0%) for RpS2. GenBank accession numbers are given in
The parsimony analysis of the new COI sequences yielded four equally parsimonious trees (CI = 0.59, RI = 0.75) the strict consensus tree of which is presented in
The majority of the species could be identified with COI alone, as no COI haplotypes were shared between species. Both parsimony and NJ trees recovered 25 (out of 28) species as monophyletic groups (
Strict consensus cladogram of
Neighbour-Joining tree using the K2P-model for the COI sequences obtained in this study.
Neighbour-Joining tree using the Tamura-Nei model with gamma distributed rates for the RpS2 sequences.
Species haplotypes for 17 variable positions of RpS2 for Central Asian
Haplotype | positions of RpS2 |
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MZH_JL35_hyale | TCCCCGGGTCCATTTTC |
MZH_JL44_hyale | TCCCCGGGTCCATTTTC |
MZH_JL02_staudingeri | TCCTCGAGTTCAAATCC |
MZH_JL13_staudingeri | TCCTCGAGTTCAAATCC |
MZH_JL23_staudingeri | TCCTCGAGTTCAAATCC |
MZH_JL43_cocandica_cocandica | TCCCCGAGTTCAAATCC |
MZH_JL41_marcopolo | TACCCGAGTTCAAAACC |
MZH_JL30_marcopolo | TACCCGAGTTCAAAACC |
MZH_JL07_lada | TCCCAAAAGTCGATTCC |
MZH_JL27_lada | TCCCAAAAGTCGATTCC |
MZH_JL25_thisoa | TCCCAAAAGTCGATTCC |
MZH_JL10_thisoa | TCCCAAAAGTCGATTCC |
MZH_JL17_thisoa | TCCCAAAAGTCGATTCC |
MZH_JL05_tyche | TCCCAAAAGTCGATTCC |
MZH_JL12_tyche | TCCCAAAAGTCGTTTCC |
MZH_JL39_heos | TCCCAAAAGTCGATTCC |
MZH_JL46_heos | TCCCAAAAGTCGATTCC |
MZH_JL53_nina | TCCCAAAAGTCGATTCC |
MZH_JL58_nina | CCCCCGAAGTCGATTCC |
MZH_JL11_sifanica | TCCCCGAGGTCGWTTCC |
MZH_JL64_sifanica | TCTCCGAGGTCGATTCC |
MZH_JL57_ladakensis | TCCCCGAGGTCGATTCC |
MZH_JL06_tibetana | TCCTCGAGGTTATTTCC |
MZH_JL09_nebulosa | TCCTCGAGGTTATTTCC |
MZH_JL26_nebulosa | TCCTCGAGGTTATTTCC |
MZH_JL14_thrasibulus | TCCTCGAGGTTATTTCC |
MZH_JL01_eogene | TCCTCGAGGTTATTTCT |
MZH_JL04_ladakensis | TCTCCGAGGTTATTTCC |
MZH_JL15_stoliczkana | TCTCCGAGGTTGTTTCT |
MZH_JL19_cocandica_pljushtchi | TCCTCGAGTTCATTTCC |
MZH_JL34_cocandica_hinducucia | TCCTCGAGTTCATTTCC |
MZH_JL03_romanovi | TCCTCGAGTTCATTTCC |
MZH_JL08_regia | TCCCCGAGTTCATTTCT |
MZH_JL42_regia | TCCCCGAGTTCATTTCT |
MZH_JL47_romanovi | CCCTCGAGTTCATTTCC |
MZH_JL51_alpherakii | TCCCCGAGTTCATTTCC |
MZH_JL37_alpherakii | CACCCGAGTTCATTTCC |
MZH_JL67_christophi_christophi | TCCTCGAGTTCATTTCC |
MZH_JL45_christophi_kali | TCCTCGAGTTCGTTTCC |
MZH_JL40_eogene | TCCTCGAGGTTGTTTCT |
MZH_JL24_nebulosa | TCCTCGAGGTCGTTTCC |
MZH_JL59_montium | CCCTCGAGGTTGTTTCC |
MZH_JL61_adelaidae | TCCTCGAGGTCGTTTCC |
MZH_JL60_fieldii | TCCTCGAGGTTATTTCC |
MZH_JL50_fieldii | TCCTCGAGGTTATTTCT |
MZH_JL33_marcopolo | TCCCCGAGGTCATTACT |
MZH_JL63_tibetana | TCCTCGAGGTTATWTCC |
MZH_JL48_berylla | TCCCCGAGGTCGAATCC |
MZH_JL55_berylla | TCCCCGAGGTCGAATCC |
The parsimony analysis of COI + RpS2 yielded nine trees of length 560 steps (CI = 0.63, RI = 0.72), the strict consensus tree of which is shown in
Strict consensus cladogram of the concatenated data set of COI + RpS2.
The strict consensus cladogram for all the available COI data resolved the taxa in the same positions as in the tree of the new COI sequences only. For ten species of the present study sequences were also available from GB. Sequences of most species clustered together as monophyletic entities, except for
Neither the Himalayan and south Tibetan adjacent mountain
Several COI haplotypes were noted for a few species, even among specimens obtained from the same locality (e.g.
The fact that the three
Even though
The utility of RpS2 as a species barcode for
The strict consensus tree was more resolved than either of the trees resulting from separate analyses of the gene regions (
Although the concatenated data did not resolve the phylogenetic relationships among all
The analyses did not support the monophyly of the subgenera
The parsimony (
Strict consensus cladogram of COI sequences for Palaearctic
Neighbour-Joining tree using the K2P-model of COI sequences for Palaearctc
Intraspecific variation is notable between some of the recognized subspecies, both among our own samples and those downloaded from GB. The intraspecific variation can partly be explained by morphologically clearly distinct subspecies, such as those of
The combined COI data of our sequences and sequences downloaded from GB include species belonging to one additional subgenus,
Our findings generally support COI as a species specific barcode for
JL thanks the Societas Entomologica Helsingforsiensis for support for the DNA work.