Skip to main content
PMC home page
ZooKeys logoLink to ZooKeys
. 2015 Nov 19;(538):35–46. doi: 10.3897/zookeys.538.6605

DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera, Nymphalidae)

Elena A Pazhenkova 1,2, Evgeny V Zakharov 3, Vladimir A Lukhtanov 1,2,4
PMCID: PMC4722858  PMID: 26807035

Abstract Abstract

The complex of butterfly taxa close to Melitaea didyma includes the traditionally recognized species Melitaea didyma, Melitaea didymoides and Melitaea sutschana, the taxa that were recognized as species only relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) as well as numerous described subspecies and forms with unclear taxonomic status. Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is monophyletic group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Six of these haplogroups are shown to correspond to six of the above-mentioned species (Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta). It is hypothesized that each of the remaining six haplogroups also represents a distinct species (Melitaea mauretanica, Melitaea occidentalis, Melitaea didyma, Melitaea neera, Melitaea liliputana and Melitaea turkestanica), since merging these haplogroups would result in a polyphyletic assemblage and the genetic distances between them are comparable with those found between the other six previously recognized species.

Keywords: Biodiversity, butterflies, COI, cryptic species, mitochondrial DNA, Nymphalidae, phylogeography, taxonomy

Introduction

The complex of butterfly taxa close to Melitaea didyma (Esper, 1779) is widely distributed in the Palaearctic region. This complex includes the traditionally recognized species Melitaea didyma, Melitaea didymoides Eversmann, 1847 and Melitaea sutschana Staudinger, 1892, the taxa that were recognized as species only recently (Melitaea latonigena Eversmann, 1847, Melitaea interrupta Colenati, 1846, Melitaea chitralensis Moore, 1901 and Melitaea mixta Evans, 1912) as well as numerous described subspecies and forms with unclear taxonomic status (Higgins 1941, 1955, Hesselbarth et al. 1995, Kolesnichenko 1999, Kolesnichenko et al. 2011). All these taxa are similar in male and female wing pattern and genitalia structure (Higgins 1941). In our opinion, this complex does not include the species Melitaea deserticola Oberthür, 1909, Melitaea ala Staudinger, 1881, Melitaea enarea Frühstorfer, 1917 and Melitaea persea Kollar, 1849 which are similar to Melitaea didyma in wing color and pattern but were shown to be distinctly different with respect to genitalia structure (Higgins 1941). The first significant review of this complex was published by Higgins (1941, 1955) in frame of a complete revision of the genus Melitaea. Recently the genus Melitaea was revised by Oorschot and Coutsis (2014). The taxa within the Melitaea didyma complex have a strong morphological variation between individuals of different generations and indistinct clinal variability in wing size and color from north to south (Lvovsky and Morgun 2007). Available cytogenetic (Lukhtanov and Kuznetsova 1989), morphological (Lvovsky and Morgun 2007, Kolesnichenko et al. 2011, Oorschot and Coutsis 2014) and molecular (Wahlberg and Zimmermann 2000, Lukhtanov et al. 2009, Dincă et al. 2015) data show that the Melitaea didyma species complex requires a more detailed taxonomic revision.

Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is a natural (monophyletic) group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Then the taxonomy of the Melitaea didyma species complex is discussed.

Material and methods

Standard COI barcodes (658-bp 5’ segment of mitochondrial cytochrome oxidase subunit I) were studied. COI sequences were obtained from 85 specimens collected in Afghanistan, Armenia, Austria, Bulgaria, China, Israel, Kazakhstan, Kyrgyzstan, Mongolia, Morocco, Russia, Syria, Tajikistan, Turkey and Uzbekistan. Collection data of these samples are presented in the Suppl. material 1.

Legs from 24 specimens (KT792884–KT792908, see the Suppl. material 2) were processed at the Department of Karyosystematics of the Zoological Institute of the Russian Academy of Sciences. The set of voucher specimens of these butterflies is kept in the Zoological Institute of the Russian Academy of Science (St. Petersburg). DNA was extracted from a single leg removed from each voucher specimen. For DNA extraction we used the GeneJet Genomic DNA Purification Kit (Fermentas) in accordance with the manufacturer’s instructions. Extracted DNA samples were stored at -20 °C.

For DNA amplification we used primers LepF 5’- ATTCAACCAATCATAAAGATATTGG-3’ and LepR (5’-TAAACTTCTGGATGTCCAAAAAATCA-3’ (deWaard et al. 2008). The (PCR) was carried out in 25-mL reactions using a DNA Engine thermal cycler (Eppendorf Mastercycler personal), and typically contained 0.5 mM of each primer, 0.8 mM dNTPs, Fermentas PCR buffer with additional MgCl2 to a final concentration of 2 mM and 1.25 units Fermentas Taq DNA polymerase. All reactions were initially denatured at 94 °C for 2 min, and then subjected to 30 cycles of 60 s at 94 °C denaturation, 60 s at 47 °C and 90 s at 72 °C extension. After amplification, double-stranded DNA was purified using GeneJet PCR Purification Kit (Fermentas). Sequencing of double-stranded product was carried out at the Research Resource Center for Molecular and Cell Technologies.

Legs from 61 specimens of Melitaea (HM404715–HM404718, KT874693–KT874751, see the Suppl. material 2) were processed at the (CCDB, Biodiversity Institute of Ontario, University of Guelph) using standard high-throughput protocol described in deWaard et al. (2008). The set of voucher specimens of these butterflies is kept at the McGuire Center for Lepidoptera and Biodiversity (University of Florida), at the Zoological Institute of the Russian Academy of Science (St. Petersburg) and in Museum for Insects, Pyatigorsk, Russia (Suppl. material 1).

The analysis involved 148 COI sequences (including outgroup). Among them there were 63 published sequences (Wahlberg and Zimmermann 2000, Vila and Bjorklund 2004, Leneveu et al. 2009, Lukhtanov et al. 2009, Dincă et al. 2011, 2015, Hausmann et al. 2011, Ashfaq et al. 2013) collected from GenBank (Suppl. material 2). Sequences were aligned using BioEdit software (Hall 1999) and edited manually. Phylogenetic hypotheses were inferred using (BI), (ML) and (MP) analyses as described previously (Vershinina and Lukhtanov 2010, Talavera et al. 2013a). Briefly, Bayesian analyses were performed using the program MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) with default settings as suggested by Mesquite (Maddison and Maddison 2015): burn-in=0.25, nst=6 (GTR + I + G). Two runs of 10,000,000 generations with four chains (one cold and three heated) were performed. Chains were sampled every 10,000 generations. The average value of the (PSRF) was 1.002 and average standard deviation of split frequencies was 0.01492, to the end of the analysis indicating that convergence was achieved, and a good sample from the posterior probability distribution was obtained.

The ML trees were inferred by using MEGA6 (Tamura et al. 2013) with the nucleotide substitution model T92 (Tamura 1992) as suggested by jModelTest (Posada 2008).

MP analysis was performed using a heuristic search as implemented in MEGA6 (Tamura et al. 2013). A heuristic search was carried out using the close-neighbour-interchange algorithm with search level 3 (Nei and Kumar 2000) in which the initial trees were obtained with the random addition of sequences (100 replicates). We used non-parametric bootstrap values (Felsenstein 1985) to estimate branch support on the reconstructed ML and MP tree. Branch support was assessed using 1000 bootstrap replicates.

Results and discussion

This analysis recovered the Melitaea didyma group as a strongly supported monophyletic clade (Fig. 1). Within this group many clades were well supported, whereas some of the relationships were not fully resolved (Figs 2 and 3). Within the complex we identified 12 differentiated major COI haplogroups. All of them showed a strict attachment to the localities (Fig. 4). Therefore in order to designate these haplogroups, we chose the oldest available name that was described from the area of each haplogroup: Melitaea mauretanica Oberthür, 1909, Melitaea occidentalis Staudinger, 1861, Melitaea didyma Esper, 1779, Melitaea neera Fischer de Waldheim, 1840, Melitaea interrupta Colenati, 1846, Melitaea liliputana Oberthür, 1909, Melitaea turkestanica Sheljuzhko, 1929, Melitaea mixta Evans, 1912, Melitaea chitralensis Moore, 1901, Melitaea latonigena Eversmann, 1847, Melitaea didymoides Eversmann, 1847 and Melitaea sutschana Staudinger, 1892 (Figs 2 and 3). The name Melitaea liliputana was selected for the Middle East populations of the Melitaea didyma complex. These populations have been known under the name libanotica Belter, 1934 in the literature (Larsen 1974, Benyamini 2002, Tshikolovets 2011). However, the name liliputana was preferred since ICZN states priority of the oldest available name (article 23, Principle of Priority).

Figure 1.

Figure 1.

Open in a new tab

The Bayesian tree of Melitaea based on analysis of (COI) gene. Numbers at nodes indicate Bayesian posterior probability/ML bootstrap/MP bootstrap values. Scale bar = 0.1 substitutions per position.

Figure 2.

Figure 2.

Open in a new tab

Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups neera, liliputana, occidentalis, interrupta, latonigena, sutschana and didymoides) based on analysis of the (COI) gene. Numbers at nodes indicate Bayesian posterior probability/ML bootstrap/MP bootstrap values, with nonmatching clades using different analyses indicated by ‘-’. Scale bar = 0.1 substitutions per position.

Figure 3.

Figure 3.

Open in a new tab

Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups turkestanica, mixta, chitralensis, mauretanica and didyma) based on analysis of the (COI) gene. Numbers at nodes indicate Bayesian posterior probability/ML bootstrap/MP bootstrap values, with nonmatching clades using different analyses indicated by ‘-’. Scale bar = 0.1 substitutions per position.

Figure 4.

Figure 4.

Open in a new tab

Distribution ranges of haplogroups didyma (■), didymoides (∆), interrupta (●), latonigena (○), liliputana (◊), mauretanica (*), mixta (▼), neera (▲), occidentalis (#), sutschana (♦), turkestanica (□) and chitralensis (►).

The discovered haplogroups correspond to two traditionally recognized species (Melitaea didymoides and Melitaea sutschana) (Higgins 1941), to four taxa that were recognized as species relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) (Lukhtanov and Kuznetsova 1989, Hesselbarth et al. 1995, Kolesnichenko 1999, Kolesnichenko et al. 2011), to five recognized subspecies (Melitaea didyma occidentalis, Melitaea didyma didyma, Melitaea didyma neera, Melitaea didyma liliputana and Melitaea didyma turkestanica) (Higgins 1941, Larsen 1974, Benyamini 2002, Tshikolovets 2011) and to one form (Melitaea mauretanica) whose status (subspecies or individual variations) is unclear (Higgins 1941).

There is good evidence based on analysis of morphology and observations of taxa in sympatry that Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta represent true biological species (Higgins 1941, Lukhtanov and Kuznetsova 1989, Hesselbarth et al. 1995, Kolesnichenko 1999, Kolesnichenko et al. 2011). Theoretically, the remainder of the Melitaea didyma complex can be interpreted as a single species Melitaea didyma. However, such an interpretation meets two difficulties. Firstly, such a lumping would result in a polyphyletic assemblage. Monophyly is the basic principle of phylogenetics and taxonomy. The majority of taxonomists currently believe that monophyly, in the narrow sense used by Hennig (Hennig 1966, Envall 2008, Hörandl and Stuessy 2010) is mandatory. Thus avoiding non-monophyletic groups and focusing on monophyletic entities is the preferable option in practical terms (Talavera et al. 2013b). The COI barcodes alone can provide weak evidence for monophyly of taxa since trees inferred from single markers sometimes display relationships that reflect the evolutionary histories of individual genes rather than the species being studied. Mitochondrial introgression (Zakharov et al. 2009) and Wolbachia infection (Ritter et al. 2013) can lead to additional bias in inferring phylogenetic relationships. Despite these limitations, we argue that, until not falsified, clusters based on DNA barcode monophyly represent preferable primary taxonomic hypotheses than the clusters based on para- or polyphyletic DNA barcode assemblages.

Secondly, the uncorrected p-distances between these taxa are high (from 1.3% between neera and liliputana to 3.9% between liliputana and occidentalis). Although some of them are lower than the ‘standard’ 2.7–3.0% DNA-barcoding threshold usually used for allopatric taxa as an indicator for their species distinctness (Lambert et al. 2005, Lukhtanov et al. 2015), even the lowest distances are comparable with those found between other six well recognized species. For example, distances between interrrupta, latonigena and mixta and their sympatric/parapatric non-conspecifics are 1.6-1.9% (Table 1).

Table 1.

Minimal uncorrected COI p-distances between 12 major haplogroups of the Melitaea didyma species complex (%).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
1. neera
2. liliputana 1.3
3. occidentalis 2.7 3.9
4. interrupta 1.8 3 1.9
5. latonigena 1.9 3.2 3.6 3.26
6. sutschana 2.2 3.6 3 3.28 1.89
7. didymoides 3.8 4.8 4.4 3 3.6 3.29
8. turkestanica 1.6 2.7 2.4 2.43 2.16 2.73 3.89
9. mixta 2.7 3.6 3 3.2 3.86 3.87 4.77 1.89
10. chitralensis 4.3 4.7 4.6 4.1 4.3 4.3 5.2 3,2 2.4
11. mauretanica 1.6 2.9 2.16 1.9 2.16 3 3.88 1.6 2.18 3.8
12. didyma 1.9 3 2.73 2.4 2.44 3 4.48 1.6 3 3.3 1.61
Open in a new tab

Sympatry (or at least parapatry) (shown by green color) was demonstrated for the following taxa pairs: mixta and turkestanica (Kolesnichenko et al. 2011), mixta and chitralensis (Higgins 1941), didymoides and sutschana (Gorbunov 2001), didymoides and latonigena (Gorbunov 2001), sutschana and latonigena (Gorbunov 2001), latonigena and neera (Lukhtanov et al. 2007), interrupta and neera (parapatry in the North Caucasus, Tuzov and Churkin 2000) and interrupta and liliputana (parapatry in Armenia and Turkey, Hesselbarth et al. 1995).

Here we also report an observation of parapatry between neera and turkestanica in South Altai and Zaisan valley in East Kazakhstan (shown by green color). In this area the distribution ranges of these taxa overlap, however, the taxa are separated ecologically: Melitaea neera is associated with the steppe biotopes and Melitaea turkestanica is associated with deserts.

Sympatry was also found between haplogroups occidentalis and didyma sensu stricto in Spain (shown by yellow, Dincă et al. 2015). However, morphology and ecology of the bearers of these haplogroups were not analyzed in the contact zone. Therefore, evolutionary and taxonomic interpretation of this case of sympatry is difficult. It may represent sympatric distribution of two different species or may be a consequence of mitochondrial introgression between the allopatric pair occidentalis-didyma.

Finally, five of the six remaining haplogroups (occidentalis, didyma sensu stricto, neera, liliputana and turkestanica) are morphologically distinct and have been considered as separate taxonomic entities (subspecies) (Higgins 1941, Larsen 1974, Benyamini 2002, Tshikolovets 2011). Their monophyly with respect to the COI gene reinforces the conclusion that they represent independent lineages of evolution.

Therefore, we hypothesize that the Melitaea didyma complex is represented by the following 12 species that can be recognized by a phylogenetic species concept (Cracraft 1989, Coyne and Orr 2004) (taxa 1–5) and by both phylogenetic and biological species concepts (taxa 6–12):

  1. Melitaea liliputana Oberthür, 1909 (Armenia, Turkey, Syria, Israel)

  2. Melitaea occidentalis Staudinger, 1961 (Spain)

  3. Melitaea didyma Esper, 1779 (west Europe)

  4. Melitaea neera Fischer de Waldheim, 1840 (east Europe, north Caucasus, west Siberia, north Kazakhstan)

  5. Melitaea mauretanica Oberthür, 1909 (north Africa, south Spain)

  6. Melitaea interrupta Colenati, 1846 (Caucasus, Turkey, Iran)

  7. Melitaea turkestanica Sheljuzhko, 1929 (Kazakhstan, Kyrgyzstan, Uzbekistan, Tajikistan, west China)

  8. Melitaea mixta Evans, 1912 (Tajikistan, Pakistan, Afghanistan)

  9. Melitaea chitralensis Moore, 1901 (north Pakistan)

  10. Melitaea latonigena Eversmann, 1847 (Asian Russia, north-east Kazakhstan, Mongolia, north-west China)

  11. Melitaea didymoides Eversmann, 1847 (Asian Russia, Mongolia, North China)

  12. Melitaea sutschana Staudinger, 1892 (Far East Russia, Korea, North-East China)

Acknowledgements

The financial support for this study was provided by the grant N 14-14-00541 from the Russian Science Foundation to the Zoological Institute of the Russian Academy of Sciences. We thank Andrei Sourakov and Andrew Warren (University of Florida) for their help in work with Lepidoptera collection in McGuire Center for Lepidoptera and Biodiversity. We are grateful to V.V.Tikhonov for samples from Caucasus. We thank A.V.Novikova, N.A.Shapoval and A.O.Vershinina for help in collecting material in Israel. The work was partially performed using equipment of the ‘Chromas’ Core Facility and Centre for Molecular and Cell Technologies of St. Petersburg State University.

Citation

Pazhenkova EA, Zakharov EV, Lukhtanov VA (2015) DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera, Nymphalidae). In: Lukhtanov VA, Kuznetsova VG, Grozeva S, Golub NV (Eds) Genetic and cytogenetic structure of biological diversity in insects. ZooKeys 538: 35–46. doi: 10.3897/zookeys.538.6605

Supplementary materials

Supplementary material 1

Table S1

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Elena A. Pazhenkova, Evgeny V. Zakharov, Vladimir A. Lukhtanov

Data type: Excel table.

Explanation note: Collection data of the samples sequenced in this study.

zookeys-538-035-s001.xls (46.5KB, xls)
Supplementary material 2

Table S2

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Elena A. Pazhenkova, Evgeny V. Zakharov, Vladimir A. Lukhtanov

Data type: Excel table.

Explanation note: List of the samples used in this study

References

  1. Ashfaq M, Akhtar S, Khan AM, Adamowicz SJ, Hebert PDN. (2013) DNA barcode analysis of butterfly species from Pakistan points towards regional endemism. Molecular Ecology Resources 13: 832–843. doi: 10.1111/1755-0998.12131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benyamini D. (2002) A Field Guide to the Butterflies of Israel. Keter Publishing House, Jerusalem, Israel, 248 pp. [Google Scholar]
  3. Coyne JA, Orr HA. (2004) Speciation. Sinauer Associates, Sunderland, Ma, USA, 545 pp. [Google Scholar]
  4. Cracraft J. (1989) Speciation and its ontology: the empirical consequences of alternative species concepts for understanding patterns and processes of differentiation. In: Otte D, Endler JA. (Eds) Speciation and its Consequences. Sinauer Associates, Sunderland, Massachusetts, USA, 28‒59. [Google Scholar]
  5. deWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN. (2008) Assembling DNA barcodes: analytical protocols. In: Martin CC. (Ed.) Environmental Genomics, Methods in Molecular Biology. Humana Press, Totowa, New Jersey, 410: 275‒283. doi: 10.1007/978-1-59745-548-0_15 [DOI] [PubMed] [Google Scholar]
  6. Dincă V, Montagud S, Talavera G, Hernández-Roldán H, Munguira ML, García-Barros E, Hebert PDN, Vila R. (2015) DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity. Scientific Reports 5: . doi: 10.1038/srep12395 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Envall M. (2008) On the difference between mono-, holo-, and paraphyletic groups: a consistent distinction of process and pattern. Biological Journal of the Linnean Society 94: 217‒220. doi: 10.1111/j.1095-8312.2008.00984.x [Google Scholar]
  8. Felsenstein J. (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791. [DOI] [PubMed] [Google Scholar]
  9. Gorbunov P. (2001) The Butterflies of Russia: Classification, Genitalia, Keys for Identification (Lepidoptera: Hesperioidea and Papilionoidea). Thesis, Ekaterinburg, Russia, 320 pp. [Google Scholar]
  10. Hall TA. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95‒98. [Google Scholar]
  11. Hennig W. (1966) Phylogenetic Systematics. University of Illinois Press, Urbana, IL, 263 pp. [Google Scholar]
  12. Hesselbarth G, Oorchot H, Wagener S. (1995) Die Tagfalter der Türkei unter Berücksichtigung der angrenzenden Länder. Selbstverlag Siegbert Wagener, Bocholt, Vol. 1‒3, 1354 pp. [Google Scholar]
  13. Higgins LG. (1941) An illustrated catalogue of the Palearctic Melitaea (Lep. Rhopalocera). Transactions of the Royal Entomological Society of London 91: 175–365. doi: 10.1111/j.1365-2311.1941.tb01045.x [Google Scholar]
  14. Higgins LG. (1955) A descriptive catalogue of the genus Mellicta Billberg (Lepidoptera: Nymphalidae) and its species, with supplementary notes on the genera Melitaea and Euphydryas. Transactions of the Royal Entomological Society of London 106: 1‒131. doi: 10.1111/j.1365-2311.1955.tb01262.x [Google Scholar]
  15. Hörandl E, Stuessy TF. (2010) Paraphyletic groups as natural units of biological classification. Taxon 59: 1641–1653. [Google Scholar]
  16. Huelsenbeck JP, Ronquist F. (2001) MrBayes: Bayesian inference of phylogeny. Bioinformatics 17: 754–755. doi: 10.1093/bioinformatics/17.8.754 [DOI] [PubMed] [Google Scholar]
  17. Kolesnichenko KA. (1999) A review of the East Palaearctic taxa of the Melitaea didyma (Esper, [1779])-group. 1. The M. ala Staudinger, 1881 – M. chitralensis Moore, 1901 – complex. Atalanta 30: 87‒117, 388‒391. [Google Scholar]
  18. Kolesnichenko KA, Churkin SV, Berdiev RK. (2011) A review of the East Palaearctic taxa of the Melitaea didyma (Esper, [1779]) ‒ group. Part II. Neue Entomologische Nachrichten 67: 257–278. [Google Scholar]
  19. Lambert DM, Baker A, Huynen L, Haddrath O, Hebert PDN, Millar CD. (2005) Is a large-scale DNA-based inventory of ancient life possible? Journal of Heredity 96(3): 279–284. doi: 10.1093/jhered/esi035 [DOI] [PubMed] [Google Scholar]
  20. Larsen TB. (1974) Butterflies of Lebanon. Classey Publications, Beirut, Lebanon, 256 pp. [Google Scholar]
  21. Leneveu J, Chichvarkhin A, Wahlberg N. (2009) Varying rates of diversification in the genus Melitaea (Lepidoptera: Nymphalidae) during the past 20 million years. Biological Journal of the Linnean Society 97: 346–361. doi: 10.1111/j.1095-8312.2009.01208.x [Google Scholar]
  22. Lukhtanov VA, Kuznetsova VG. (1989) The analysis of the karyotype variability in the butterflies of the Melitaea didyma group along with the evidence of the species distinctness of M. latonigena (Lepidoptera, Nymphalidae). Zoologichesky Zhurnal 68(12): 38‒46. [In Russian with English summary] [Google Scholar]
  23. Lukhtanov VA, Sourakov A, Zakharov EV, Hebert PDN. (2009) DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification. Molecular Ecology Resources 9: 1302–1310. doi: 10.1111/j.1755-0998.2009.02577.x [DOI] [PubMed] [Google Scholar]
  24. Lukhtanov VA, Dantchenko AV, Vishnevskaya MS, Saifitdinova AF. (2015) Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society 116(2): 468‒485. doi: 10.1111/bij.12596 [Google Scholar]
  25. Lukhtanov VA, Vishnevskaya MS, Volynkin AV, Yakovlev RV. (2007) Butterflies (Lepidoptera, Rhopalocera) of West Altai. Entomologicheskoe Obozrenie 86(2): 347‒369. doi: 10.1134/s001387380705003x [In Russian with English summary] [Google Scholar]
  26. Lvovsky AL, Morgun DV. (2007) Butterflies of the Eastern Europe. KMK Scientific Press, Ltd., Moscow, Russia, 443 pp [In Russian] [Google Scholar]
  27. Maddison WP, Maddison DR. (2015) Mesquite: a modular system for evolutionary analysis. Version 3.04. http://mesquiteproject.org
  28. Nei M, Kumar S. (2000) Molecular Evolution and Phylogenetics. Oxford University Press, NY, USA, 333 pp. [Google Scholar]
  29. Oorschot H van, Coutsis J. (2014) The Genus Melitaea Fabricius, 1807 (Lepidoptera: Nymphalidae, Nymphalinae). Taxonomy and systematics with special reference to the male genitalia. Tshikolovets Publications, Pardubice, Czech Republic, 360 pp. [Google Scholar]
  30. Posada D. (2008) jModel Test: phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253‒1256. doi: 10.1093/molbev/msn083 [DOI] [PubMed] [Google Scholar]
  31. Ritter S, Michalski SG, Settele J, Wiemers M, Fric ZF, Sielezniew M, Šašić M, Rozier Y, Durka W. (2013) Wolbachia infections mimic cryptic speciation in two parasitic butterfly species, Phengaris teleius and P. nausithous (Lepidoptera: Lycaenidae). PLoS ONE 8(11): 1‒13. doi: 10.1371/journal.pone.0078107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Talavera G, Lukhtanov VA, Rieppel L, Pierce NE, Vila R. (2013a) In the shadow of phylogenetic uncertainty: the recent diversification of Lysandra butterflies through chromosomal change. Molecular Phylogenetics and Evolution 69: 469–478. doi: 10.1016/j.ympev.2013.08.004 [DOI] [PubMed] [Google Scholar]
  33. Talavera G, Lukhtanov VA, Pierce NE, Vila R. (2013b) Establishing criteria for higher-level classification using molecular data: the systematics of Polyommatus blue butterflies (Lepidoptera, Lycaenidae). Cladistics 29: 166–192. doi: 10.1111/j.1096-0031.2012.00421.x [DOI] [PubMed] [Google Scholar]
  34. Tamura K. (1992) Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C content biases. Molecular Biology and Evolution 9(4): 678–687. [DOI] [PubMed] [Google Scholar]
  35. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725‒2729. doi: 10.1093/molbev/mst197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tshikolovets VV. (2011) Butterflies of Europe and the Mediterranean Area. Tshikolovets Publications, Pardubice, Czech Republic, [i–v]+544 pp. [Google Scholar]
  37. Tuzov VK, Churkin S. (2000) Genus Melitaea. In: Tuzov VK. (Ed.) Guide to the Butterflies of Russia and Adjacent Territories (Lepidoptera, Rhopalocera). Volume 2, Pensoft Publishers, Sofia-Moscow, 59‒76. [Google Scholar]
  38. Vershinina AO, Lukhtanov VA. (2010) Geographical distribution of the cryptic species Agrodiaetus alcestis alcestis, A. alcestis karacetinae and A. demavendi (Lepidoptera, Lycaenidae) revealed by cytogenetic analysis. Comparative Cytogenetics 4(1): 1‒11. doi: 10.3897/compcytogen.v4i1.21 [Google Scholar]
  39. Vila R, Bjorklund M. (2004) The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta). Journal of Molecular Evolution 58(3): 280‒290. doi: 10.1007/s00239-003-2550-2 [DOI] [PubMed] [Google Scholar]
  40. Wahlberg N, Zimmermann M. (2000) Pattern of phylogenetic relationships among members of the tribe Melitaeini (Lepidoptera: Nymphalidae) inferred from mitochondrial DNA sequences. Cladistics 16: 347–363. doi: 10.1006/clad.2000.0136 [DOI] [PubMed] [Google Scholar]
  41. Zakharov EV, Lobo NF, Nowak C, Hellma JJ. (2009) Introgression as a likely cause of mtDNA paraphyly in two allopatric skippers (Lepidoptera: Hesperiidae). Heredity 102: 590–599. doi: 10.1038/hdy.2009.26 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1

Table S1

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Elena A. Pazhenkova, Evgeny V. Zakharov, Vladimir A. Lukhtanov

Data type: Excel table.

Explanation note: Collection data of the samples sequenced in this study.

zookeys-538-035-s001.xls (46.5KB, xls)
Supplementary material 2

Table S2

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Elena A. Pazhenkova, Evgeny V. Zakharov, Vladimir A. Lukhtanov

Data type: Excel table.

Explanation note: List of the samples used in this study

zookeys-538-035-s002.xls (38KB, xls)

Articles from ZooKeys are provided here courtesy of Pensoft Publishers

RESOURCES