Research Article |
Corresponding author: Vladimir A. Lukhtanov ( lukhtanov@mail.ru ) Academic editor: Ilya Gavrilov-Zimin
© 2016 Elena A. Pazhenkova, Vladimir A. Lukhtanov.
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:
Pazhenkova EA, Lukhtanov VA (2016) Chromosomal and mitochondrial diversity in Melitaea didyma complex (Lepidoptera, Nymphalidae): eleven deeply diverged DNA barcode groups in one non-monophyletic species? Comparative Cytogenetics 10(4): 697-717. https://doi.org/10.3897/CompCytogen.v10i4.11069
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It is generally accepted that cases of species’ polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, and the detected cases reflect misidentifications or/and methodological errors. Here we studied the problem of species’ non-monophyly through chromosomal and molecular analysis of butterfly taxa close to Melitaea didyma (Esper, 1779) (Lepidoptera, Nymphalidae). We found absence or low interspecific chromosome number variation and presence of intraspecific variation, therefore we conclude that in this group, chromosome numbers have relatively low value as taxonomic markers. Despite low karyotype variability, the group was found to have unexpectedly high mitochondrial haplotype diversity. These haplotypes were clustered in 23 highly diverged haplogroups. Twelve of these haplogroups are associated with nine traditionally recognized and morphologically distinct species M. chitralensis Moore, 1901, M. deserticola Oberthür, 1909, M. didymoides Eversmann, 1847, M. gina Higgins, 1941, M. interrupta Colenati, 1846, M. latonigena Eversmann, 1847, M. mixta Evans, 1912, M. saxatilis Christoph, 1873 and M. sutschana Staudinger, 1892. The rest of the haplogroups (11 lineages) belong to a well-known west-palaearctic species M. didyma. The last species is particularly unusual in the haplotypes we obtained. First, it is clearly polyphyletic with respect to COI gene. Second, the differentiation in COI gene between these mostly allopatric (but in few cases sympatric) eleven lineages is extremely high (up to 7.4%), i.e. much deeper than the “standard” DNA barcode species threshold (2.7–3%). This level of divergence normally could correspond not even to different species, but to different genera. Despite this divergence, the bearers of these haplogroups were found to be morphologically indistinguishable and, most importantly, to share absolutely the same ecological niches, i.e. demonstrating the pattern which is hardly compatible with hypothesis of multiple cryptic species. Most likely such a profound irregularity in barcodes is caused by reasons other than speciation and represents an extraordinary example of intra-species barcode variability. Given the deep level of genetic differentiation between the lineages, we assume that there was a long period (up to 5.0 My) of allopatric differentiation when the lineages were separated by geographic or/and ecological barriers and evolved in late Pliocene and Pleistocene refugia of north Africa, the Iberian and Balkan Peninsulas, the Middle East and Central Asia. We discuss the refugia-within-refugia concept as a mechanism explaining the presence of additional diverged minor haplogroups within the areas of the major haplogroups. We also provide the first record of M. gina in Azerbaijan and the record of M. didyma turkestanica as a new taxon for Russia and Europe.
Biodiversity, butterflies, COI , chromosome, karyotype, mitochondrial DNA, monophyly, non-monopyletic species, Nymphalidae , phylogeography, Pleistocene refugium, taxonomy
The Melitaea didyma (Esper, 1779) species complex, a group of taxa close to M. didyma (
The significant reviews of this complex were published by
Combination of molecular and cytogenetic methods is a useful tool for detecting cryptic species (
In the present study we use a combination of molecular and chromosomal markers to analyse additional material collected in Armenia, Bulgaria, Georgia, Greece, Iran, Israel, Kazakhstan, Kyrgyzstan, Russia, Slovenia, Syria and Turkey, in order to reveal taxonomic and phylogeographic structure within the M. didyma species complex. In our opinion, this group includes the following species: M. didyma Esper, 1779, M. chitralensis Moore, 1901, M. deserticola Oberthür, 1909, M. didymoides Eversmann, 1847, M. gina Higgins, 1941, M. interrupta Colenati, 1846, M. latonigena Eversmann, 1847, M. mixta Evans, 1912, M. saxatilis Christoph, 1873 and M. sutschana Staudinger, 1892. This complex does not include the taxa of the M. persea complex (M. persea Kollar, 1849, M. casta Kollar, 1849, M. eberti Koçak, 1980 and M. higginsi Sakai, 1978) and the taxa of the M. ala complex (M. ala Staudinger, 1881, M. bundeli Kolesnichenko, 1999, M. kotshubeji Sheljuzhko, 1929, M. acraeina Staudinger, 1886, M. enarea Frühstorfer, 1917, M. ninae Sheljuzhko, 1935 and M. didymina Staudinger, 1895) which were shown to be strongly diverged with respect to genitalia structure (
We studied standard COI barcodes (658-bp 5’ segment of mitochondrial cytochrome oxidase subunit I). We obtained COI sequences from 121 specimens collected in Armenia, Bulgaria, Georgia, Greece, Iran, Israel, Kazakhstan, Kyrgyzstan, Russia, Slovenia, Syria and Turkey. DNA was extracted from a single leg removed from each voucher specimen.
Legs from 21 specimens were processed at Department of Karyosystematics of Zoological Institute of the Russian Academy of Sciences. Primers and PCR protocol are given in our previous publications (
The analysis involved 265 COI sequences (including outgroup) (Suppl. material
Within the studied samples, we are not completely sure of the identity of M. chitralensis specimens (their barcodes were obtained from GenBank) because we were not able to check these vouchers and used the identification of these samples accepted in
Sequences were aligned using BioEdit software (
Karyotypes were obtained from fresh adult males and processed as previously described (
The haploid chromosome number n=28 was found in prometaphase I, MI and MII cells of seven studied individuals (Table
Karyotypes in male meiosis of Melitaea gina from Iran. a sample Q183, prometaphase I, n = 28 b sample Q153, late prometaphase I, n = 28 c sample Q183, MI, n = 28 d sample Q155, M I, n = 28. Scale bar corresponds to 10µ in all figures.
Chromosome number and localities of Melitaea gina samples collected in Iran (province West Azerbaijan) (Collectors: V. Lukhtanov, E. Pazhenkova and N. Shapoval).
Sample | Karyotype | Haplotype | Locality | Altitude | Date |
---|---|---|---|---|---|
Q153 | n=28 | M18 | 25 km E of Mahabad (vic. Darman): N36°45'00"; E45°51'37" | 1900–2000 m | 10 August 2016 |
Q155 | n=28 | 25 km E of Mahabad (vic. Darman): N36°45'00"; E45°51'37" | 1900–2000 m | 10 August 2016 | |
Q156 | n=28 | M14 | 25 km E of Mahabad (vic. Darman): N36°45'00"; E45°51'37" | 1900–2000 m | 10 August 2016 |
Q157 | n=28 | M15 | 25 km E of Mahabad (vic. Darman): N36°45'00,30"; E45°51'36,60" | 1900–2000 m | 10 August 2016 |
Q182 | n=28 | 25 km E of Mahabad (vic. Darman): N36°45'00"; E45°51'37" | 1900–2000 m | 10 August 2016 | |
Q183 | n=28 | 25 km E of Mahabad (vic. Darman): N36°45'00"; E45°51'37" | 1900–2000 m | 10 August 2016 | |
Q211 | n=28 | 3 km W of Khalifen: N36°44'35"; E45°32'13" | 2100–2200 m | 11 August 2016 |
Bayesian analysis of the barcode region recovered the M. didyma complex as a monophyletic clade (Fig.
The Bayesian tree of Melitaea based on analysis of the cytochrome oxidase subunit I (COI) gene. Numbers at nodes indicate Bayesian posterior probability.
Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups neera and liliputana) based on analysis of COI gene. Numbers at nodes indicate Bayesian posterior probability.
Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups interrupta, occidentalis, saxatilis, lathonigena, didymoides, sutschana, sutschana 2, sutschana 3) based on analysis of COI gene. Numbers at nodes indicate Bayesian posterior probability.
Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups turkestanica, turkestanica 2, didyma) based on analysis of COI gene. Numbers at nodes indicate Bayesian posterior probability.
Fragment of the Bayesian tree of Melitaea didyma complex (haplogroups mixta, chitralensis, mauretanica, protaeoccidentis, neera2, gina and deserticola) based on analysis of the COI gene. Numbers at nodes indicate Bayesian posterior probability.
The uncorrected mean p-distances between the haplogroups were high (up to 9.1% between turkestanica4 and deserticola) (Table
Mean uncorrected COI p-distances between 23 haplogroups of the M. didyma species complex (%).
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1. chitralensis | ||||||||||||||||||||||
2. deserticola | 8.4 | |||||||||||||||||||||
3. didyma | 4.2 | 6.7 | ||||||||||||||||||||
4. didymoides | 6.3 | 6.9 | 4.7 | |||||||||||||||||||
5. gina | 5.5 | 7.5 | 4.2 | 5.3 | ||||||||||||||||||
6. gina 2 | 6.4 | 9.5 | 6.5 | 7.2 | 6.5 | |||||||||||||||||
7. interrupta | 4.9 | 6.2 | 2.7 | 3.1 | 4.4 | 6.1 | ||||||||||||||||
8. latonigena | 5.0 | 6.9 | 3.1 | 4.1 | 4.7 | 6.5 | 3.5 | |||||||||||||||
9. liliputana | 4.7 | 7.1 | 3.1 | 4.8 | 5.2 | 6.8 | 3.4 | 3.7 | ||||||||||||||
10. mauretanica | 4.1 | 6.3 | 2.1 | 4.2 | 4.8 | 7.1 | 2.2 | 3.1 | 3.5 | |||||||||||||
11. mixta | 2.4 | 6.9 | 3.5 | 5.0 | 5.2 | 6.9 | 4.1 | 4.6 | 4.3 | 3.6 | ||||||||||||
12. neera | 3.7 | 6.8 | 2.4 | 4.3 | 4.9 | 7.1 | 2.3 | 2.9 | 2.0 | 2.0 | 3.3 | |||||||||||
13. neera 2 | 3.2 | 6.7 | 1.9 | 4.7 | 4.5 | 6.4 | 2.6 | 3.0 | 2.8 | 2.0 | 2.7 | 1.7 | ||||||||||
14. occidentalis | 4.9 | 6.9 | 2.9 | 3.9 | 4.6 | 6.4 | 1.8 | 3.8 | 3.9 | 2.4 | 4.0 | 2.8 | 2.4 | |||||||||
15. protaeoccidentis | 3.3 | 5.6 | 2.1 | 4.1 | 4.2 | 6.7 | 2.7 | 2.7 | 3.0 | 2.1 | 2.9 | 2.1 | 2.0 | 3.0 | ||||||||
16. saxatilis | 5.0 | 7.9 | 4.0 | 4.7 | 5.4 | 7.4 | 3.5 | 4.5 | 4.3 | 3.3 | 5.0 | 3.7 | 3.9 | 3.9 | 3.8 | |||||||
17. sutschana | 5.6 | 6.9 | 3.4 | 3.5 | 4.5 | 6.7 | 3.1 | 2.4 | 3.7 | 3.5 | 4.6 | 2.6 | 3.3 | 3.1 | 3.2 | 3.9 | ||||||
18. sutschana 2 | 5.9 | 7.6 | 4.0 | 4.1 | 5.7 | 7.7 | 3.9 | 3.0 | 4.3 | 4.1 | 5.2 | 3.2 | 3.9 | 4.3 | 3.8 | 4.5 | 1.8 | |||||
19. sutschana 3 | 4.7 | 6.9 | 2.5 | 3.4 | 4.5 | 7.1 | 2.6 | 2.4 | 3.4 | 2.6 | 4.0 | 2.3 | 2.4 | 3.0 | 2.6 | 2.7 | 1.5 | 2.1 | ||||
20. turkestanica | 3.4 | 7.0 | 2.3 | 4.4 | 4.3 | 7.0 | 3.0 | 3.4 | 3.1 | 2.4 | 2.7 | 2.1 | 1.6 | 3.1 | 2.3 | 3.7 | 3.6 | 4.3 | 2.8 | |||
21. turkestanica 2 | 4.8 | 7.5 | 1.1 | 5.7 | 5.1 | 6.6 | 3.7 | 4.1 | 4.1 | 3.1 | 4.1 | 3.4 | 2.9 | 3.9 | 3.0 | 5.0 | 4.4 | 4.4 | 3.5 | 3.2 | ||
22. turkestanica 3 | 7.0 | 8.9 | 5.8 | 8.9 | 6.4 | 6.4 | 4.8 | 6.1 | 7.1 | 7.0 | 6.3 | 7.9 | 6.9 | 5.8 | 6.0 | 6.2 | 7.0 | 6.9 | 7.0 | 6.6 | 6.4 | |
23. turkestanica 4 | 7.0 | 9.1 | 6.5 | 7.4 | 7.0 | 4.3 | 6.1 | 7.3 | 6.7 | 7.3 | 7.4 | 7.2 | 6.4 | 6.6 | 6.5 | 7.5 | 7.2 | 8.0 | 7.2 | 7.4 | 7.0 | 4.4 |
Most of the haplogroups were found to be allopatric. However, in some cases barcodes’ clusters did not correspond to the simple allopatric geographical distribution. The sample Melitaea gina M22 (haplogroup gina2) was found in sympatry with the haplogroup gina in north-west Iran. The distance between gina and gina2 was 6.5%. Haplogroups turkestanica4, turkestanica3 and turkestanica2 were highly diverged (up to 7.4%) as compared with the haplogroup turkestanica and were found in sympatry with the haplogroup turkestanica (Fig.
Localization of neera and turkestanica haplogroups (yellow circles – neera, black – turkestanica, green – turkestanica2, red – turkestanica3, blue – turkestanica4)
Two samples with the turkestanica haplotypes (haplogroup turkestanica), one from Aktobe (Kazakhstan) and one from Samara (Russia) were found in sympatry with M. dimyma neera haplotypes (haplogroup neera). In Karabiryuk (Kazakhstan), two samples with the neera haplotypes (haplogroup neera) were found in sympatry with M. didyma turkestanica haplotypes (haplogroup turkestanica and turkestanica4).
The genus Melitaea is known to be characterized by relatively low interspecific chromosome number variation. The representatives of basal clades (see phylogeny in
The younger lineages, the M. fergana Staudinger, 1882 and M. didyma species groups, were found to possess lower chromosome numbers varying from n=27 to n=29-30. Within the M. fergana species group, M. athene Staudinger, 1881, the only karyologically studied species, was found to have n=29 (with n=30 as a rare intra-individual variation) (
Taxon | Chromosome number | Country | Locality | Reference |
---|---|---|---|---|
M. didyma ssp. | n=28 | Italy | Abruzzi |
|
M. didyma neera | n=28 | Kazakhstan | Altai |
|
M. didyma neera | n=27 | Russia | N Caucasus, Pyatigorsk | Lukhtanov and Kuznetsova 1988 |
M. interrupta | n=29 | Turkey |
|
|
M. interrupta | n=29 | Azerbaijan, Nakhichevan | Zangezur Mts |
|
M. latonigena | n=29–30 | Kazakhstan | Altai |
|
M. deserticola | n=29 | Lebanon |
|
|
M. gina | n=28 | Iran | W Azerbaijan | This study |
Together with M. deserticola (n=29,
Despite low level of chromosome number variability, the M. didyma complex was found to have unexpectedly high level of mitochondrial haplotype diversity. These haplotypes were clustered in 23 highly diverged haplogroups (Fig.
The rest of the haplogroups belong to the well-known west-palearctic species M. didyma. Despite intrapopulation and seasonal variability, this species is very homogenous with respect to morphology, including the structure of genitalia, a character which is most useful for species separation in Melitaea (
If we follow the opinion of experts in Melitaea taxonomy (
There are two theoretically possible explanations for this pattern. First, M. didyma sensu auctorum can be a mix of multiple species that mostly have allopatric distribution ranges, but some of them are sympatric. Second, the recovered haplogroups (at least the allopatric ones) can represent highly diverged intraspecific lineages. Of course, a combination of the first and the second hypotheses is possible, and a part of the haplogroups could represent different species, and another part of the haplogroups could represent intraspecific variations.
In our opinion, the second hypothesis seems to be more plausible. There are the following arguments for the second scenario. First, no morphological differences between the bearers of these haplogroups are known (except for lighter, more yellowish wing colour in the three M. didyma turkestanica haplogroups as compared with other haplogroups). The second (and the most convincing) argument is based on our field obseravtion of butterfly habitats and ecological preferences. In ecology the competitive exclusion principle, also known as Gause’s law is one of the most important rule (
M. didyma neera and M. didyma turkestanica are differentiated ecologically (
Interestingly, the haplogroup turkestanica2 is not related to the haplogroup turkestanica and is a derivative from West-European haplogroup didyma. This pattern can be treated as a result of ancient introgression. Generally, footprints of ancient and more recent introgression are both an evidence for transparency of boundaries between M. didyma populations.
The mega-analysis of species-level para- and polyphyly in DNA barcode gene trees was recently conducted by using a huge data set (4977 species and 41,583 specimens of European Lepidoptera) (
The M. didyma complex consists of at least 23 COI haplogroups, the majority of which demonstrated a strict attachment to particular geographic ranges: chitralensis (north Pakistan); deserticola (north Africa, Israel, Jordan, Lebanon, Syria); didyma (west Europe); didymoides (Asian Russia, Mongolia, North China); gina (W Iran, Azerbaijan); interrupta (Caucasus, NE Turkey); latonigena (Asian Russia, north-east Kazakhstan, Mongolia, north-west China); liliputana (Armenia, Turkey, Syria, Lebanon, Israel); mauretanica (south Spain); mixta (Tajikistan, Kyrgyzstan, Uzbekistan, Pakistan, Afghanistan); neera (east Europe, north Caucasus, west Siberia, north Kazakhstan); occidentalis (Spain); protaeoccidentis (north Africa); saxatilis (north Iran); sutschana (Russian Far East, Korea, north-east China) and turkestanica (Kazakhstan, Kyrgyzstan, Uzbekistan, Tajikistan, west China). With few exceptions (e.g. deserticola/protaeoccidentis, deserticola/liliputana), the ranges of these haplogroups do not overlap substantially (Fig.
We tentatively suggest interpreting the main clusters discovered within M. didyma sensu stricto (M. didyma didyma, M. didyma mauretanica, M. didyma occidentalis, M. didyma protaeoccidentis, M. didyma liliputana, M. didyma neera and M. didyma turkestanica) as subspecies because each of them has its own distribution range and is distinct with respect to mtDNA (i.e. represents by a monophyletic lineage or a combination of two or three monophyletic lineages). As a result we propose the following classification:
M. didyma (Esper, [1779])
M. didyma didyma (Esper, [1779])
M. didyma mauretanica Oberthür, 1909
M. didyma occidentalis Staudinger, 1961
M. didyma protaeoccidentis Verity, 1929
M. didyma liliputana Oberthür, 1909
M. didyma neera Fischer de Waldheim, 1840
M. didyma turkestanica Sheljuzhko, 1929
M. didymoides Eversmann, 1847
M. sutschana Staudinger, 1892
M. latonigena Eversmann, 1847
M. interrupta Colenati, 1846
M. mixta Evans, 1912
M. chitralensis Moore, 1901
M. deserticola Oberthür, 1909
M. saxatilis Christoph, 1873
M. gina Higgins, 1941
We provide the first record of M. gina in Azerbaijan (sample BPAL1697-12, Azerbaijan, Shamkir, 27 June 2011, collector V. Tikhonov).
We also record M. didyma turkestanica as a new taxon for Russia and Europe (samples BPAL3168-16, BPAL3169-16, BPAL3170-16, BPAL3173-16 Russia, Astrakhanskaya oblast, Bogdinsko-Baskunchaksky zapovednik, 24 May 2008, collector S. Nedoshivina).
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 S. Nedoshivina for samples from Astrakhanskaya oblast, V.V. Tikhonov for samples from Caucasus and to N.A. Shapoval for samples from Khakassia (Russia). We thank A. Novikova for help in collecting material in Israel and N.A. Shapoval for help in collecting material in Iran. The work was partially performed using equipment of the ‘Chromas’ Core Facility and Centre for Molecular and Cell Technologies of St. Petersburg State University.
Table S1
Data type: Microsoft Office Excel file
Explanation note: List of Melitaea samples used in this study.