Research Article |
Corresponding author: Vladimir Lukhtanov ( lukhtanov@mail.ru ) Academic editor: Vladimir Gokhman
© 2014 Vladimir Lukhtanov, Nazar Shapoval, Alexander Dantchenko.
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 V, Shapoval N, Dantchenko A (2014) Taxonomic position of several enigmatic Polyommatus (Agrodiaetus) species (Lepidoptera, Lycaenidae) from Central and Eastern Iran: insights from molecular and chromosomal data. Comparative Cytogenetics 8(4): 313-322. https://doi.org/10.3897/CompCytogen.v8i4.8939
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The species-rich subgenus Polyommatus (Agrodiaetus) has become one of the best studied groups of Palearctic blue butterflies (Lepidoptera, Lycaenidae). However, the identity and phylogenetic position of some rare taxa from Iran have remained unclear. An enigmatic, recently described Central Iranian species P. (A.) shirkuhensis ten Hagen et Eckweiler, 2001 has been considered as a taxon closely related either to P. (A.) eckweileri ten Hagen, 1998 or to P. (A.) baltazardi (de Lesse, 1962). P. (A.) baltazardi, in its turn, was treated as a taxon close to Iranian-Pakistani P. (A.) bogra Evans, 1932. Here we used a combination of molecular and chromosomal markers to show that none of these hypotheses was true. Instead, P. (A.) shirkuhensis was recovered as a member of a species group close to P. (A.) cyaneus (Staudinger, 1899). From genetically closest species, P. (A.) kermansis (de Lesse, 1962), P. (A.) cyaneus and P. (A.) sennanensis (de Lesse, 1959), it differs by the wing coloration. From morphologically similar P. (A.) mofidii (de Lesse, 1963) and P. (A.) sorkhensis Eckweiler, 2003, it differs by its chromosome number, n=21. P. (A.) bogra and P. (A.) baltazardi were found to be members of two different species groups and, thus, are not closely related.
Agrodiaetus , chromosome number, COI , karyotype, Lycaenidae , Polyommatus
Agrodiaetus Hübner, 1822, a subgenus of the species-rich Palearctic genus Polyommatus Latreille, 1804 (
Here we use a combination of molecular mitochondrial (COI), molecular nuclear (ITS2) and nuclear chromosomal (karyotype) markers to analyze two recently described and little known taxa P. (A.) shirkuhensis ten Hagen et Eckweiler, 2001 (
The taxa P. (A.) shirkuhensis (Iran, Yazd Province, Shirkuh Mts., Deh-Bala village, 2900-3150 m, 12 July 2005, samples J299-1, J299-2 and J299-3, J302 and J304) and P. (A.) bogra birjandensis(Iran, South Khorasan Province, 26 km N of Birjand, 1900-2000 m, 14 July 2005, samples J305, J306, J307, J307-1, J307-2, J307-3, J307-4, J315, J318 and J319) were collected exactly in their type localities.
Fresh (not worn) adult males were used to investigate the karyotypes. After capturing a butterfly in the field, it was placed in a glassine envelope for 1-2 hours to keep it alive until we processed it. Testes were removed from the abdomen and placed into a small 0.5 ml vial with a freshly prepared fixative (ethanol and glacial acetic acid, 3:1). Then each wing was carefully removed from the body using forceps. The wingless body was placed into a plastic, 2 ml vial with pure 96% ethanol. The samples are kept in the Zoological Institute of the Russian Academy of Sciences.
Testes were stored in the fixative for 1-12 months at +4°C. Then the gonads were stained in 2% acetic orcein for 30-60 days at +18-20°C. Different stages of male meiosis were examined by using a light microscope Amplival, Carl Zeiss. We have used an original two-phase method of chromosome analysis (
A 643 bp fragment of mitochondrial gene cytochrome oxidase subunit I (COI) and 592 bp fragment of nuclear internal transcribed spacer 2 (ITS2) were used to analyze clustering of the specimens. Primers and the protocol of DNA amplification were given in our previous publication (
Sequences of the following additional representatives of the subgenus Agrodiaetus were found in GenBank (
Bayesian analysis was performed using the program MrBayes 3.2.2 (
Bayesian analysis of the gene COI resulted in a consensus phylogram which displayed a high level of posterior probability for the majority of the clades revealed. A fragment of this tree demonstrating the position of the target species P. (A.) shirkuhensis, P. (A.) eckweileri ten Hagen, 1998, P. (A.) baltazardi (de Lesse, 1962) and P. (A.) bogra birjandensis is shown on Fig.
Bayesian analysis of the sequence ITS2 resulted in a mostly unresolved consensus phylogram (Fig.
Fragment of consensus Bayesian tree of the subgenus Agrodiaetus inferred from COI sequences. Posterior probability values >50% are shown. Names of the target species are in bold. The complete tree is given online in the Suppl. material
P. (A.) shirkuhensis (Table
P. (A.) bogra birjandensis (Table
Male karyotype of Polyommatus (Agrodiaetus) shirkuhensis. a metaphase I, n = 21 b metaphase II, n = 21. Bar = 10µm.
Haploid chromosome number (n) of the taxa discussed and the species groups to which these taxa belong in classifications by
Taxon | n | Species group (classification after Eckweiler and Häuser) | Species group (classification after Kandul et al.) | Reference |
P. (A.) baltazardi | 45 | P. (A.) erschoffii | P. (A.) poseidon |
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P. (A.) bogra birjandensis | ca52–53 | P. (A.) erschoffii | P. (A.) erschoffii | This paper |
P. (A.) cyaneus | from 18 to 20 | P. (A.) damon | P. (A.) cyaneus |
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P. (A.) eckweileri | ca106 | unclear | P. (A.) erschoffii |
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P. (A.) kermansis | 22 | P. (A.) damon | P. (A.) cyaneus |
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P. (A.) mofidii | 35 | P. (A.) damon | P. (A.) cyaneus |
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P. (A.) sennanensis | 28–31 | P. (A.) dolus (Hübner, 1823) | P. (A.) cyaneus |
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P. (A.) shirkuhensis | 21 | unclear | P. (A.) cyaneus | This paper |
P. (A.) sorkhensis | 43 | P. (A.) damon | P. (A.) cyaneus |
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P. (A.) shirkuhensis is the only species of the subgenus Agrodiaetus known from Shirkuh Mts massif in Central Iran (province Yazd) (
However, analysis of COI clusters in the Bayesian tree (Fig.
P. (A.) baltazardi is found to be a member of P. (A.) poseidon (Herrich-Schäffer, [1851]) group and, thus, is not related to P. (A.) bogra The latter species has very isolated position within the P. erschoffii group. The karyotypes of P. (A.) baltazardi and P. (A.) bogra are also different (Table
Finally, our target species, P. (A.) shirkuhensis, is found to be a member of P. (A.) cyaneus (Staudinger, 1899) group and is especially close to P. (A.) kermansis (de Lesse, 1962), P. (A.) sennanensis (de Lesse, 1959) and P. (A.) cyaneus (Fig.
From P. (A.) kermansis, P. (A.) cyaneus and P. (A.) sennanensis, which possess closest COI haplotypes, P. (A.) shirkuhensis differs by blue upper side of wings in males (it is deep violet in P. (A.) kermansis, violet in P. (A.) cyaneus and whitish in P. (A.) sennanensis) (see figures in
To conclude, our study demonstrates that four allopatric taxa known from Central and East Iran, P. (A.) shirkuhensis, P. (A.) eckweileri, P. (A.) baltazardi and P. (A.) bogra birjandensis, which possess significant elements of morphological similarity, are not only specifically distinct from each other, but even belong to different distantly related groups of species within the subgenus Agrodiaetus.
The complete financial support for this study was provided by the grant from the Russian Science Foundation N 14-14-00541 to Zoological Institute of the Russian Academy of Sciences. Postdoctoral fellowship (N 1.50.1617.2013) was provided to A. Dantchenko from St. Petersburg State University.
Consensus Bayesian tree of the subgenus Polyommatus (Agrodiaetus) inferred from COI sequences
Data type: image
Explanation note: Consensus Bayesian tree of the subgenus Polyommatus (Agrodiaetus) inferred from COI sequences. Posterior probability values >50% are shown. Names of the target species are in bold.
Consensus Bayesian tree of the subgenus Polyommatus (Agrodiaetus) inferred from ITS2 sequences
Data type: image
Explanation note: Consensus Bayesian tree of the subgenus Polyommatus (Agrodiaetus) inferred from ITS2 sequences. Posterior probability values >50% are shown. Names of the target species are in bold.