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      Karyosystematics and molecular taxonomy of the anomalous blue butterflies (Lepidoptera, Lycaenidae) from the Balkan Peninsula

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          Abstract

          Abstract

          The Balkan Peninsula represents one of the hottest biodiversity spots in Europe. However, the invertebrate fauna of this region is still insufficiently investigated, even in respect of such well-studied organisms as Lepidoptera . Here we use a combination of chromosomal, molecular and morphological markers to rearrange the group of so-called anomalous blue butterflies (also known as ‘brown complex’ of the subgenus Agrodiaetus Hübner, [1822] and as the Polyommatus ( Agrodiaetus) admetus (Esper, 1783) species group) and to reveal its cryptic taxonomic structure. We demonstrate that Polyommatus aroaniensis (Brown, 1976) is not as widespread in the Balkans as was previously thought. In fact, it has a dot-like distribution range restricted to the Peloponnese Peninsula in South Greece. Polyommatus orphicus Kolev, 2005 is not as closely related to the Turkish species Polyommatus dantchenkoi (Lukhtanov & Wiemers, 2003) as was supposed earlier. Instead, it is a Balkan endemic represented by two subspecies: Polyommatus orphicus orphicus (Bulgaria) and Polyommatus orphicus eleniae Coutsis & De Prins, 2005 (Northern Greece). Polyommatus ripartii (Freyer, 1830) is represented in the Balkans by an endemic subspecies Polyommatus ripartii pelopi . The traditionally recognized Polyommatus admetus (Esper, 1783) is shown to be a heterogeneous complex and is divided into Polyommatus admetus sensu stricto (the Balkans and west Turkey) and Polyommatus yeranyani (Dantchenko & Lukhtanov, 2005) (east Turkey, Armenia, Azerbaijan and Iran). Polyommatus nephohiptamenos (Brown & Coutsis, 1978) is confirmed to be a species with a dot-like distribution range in Northern Greece. Finally, from Central Greece (Timfristos and Parnassos mountains) we describe Polyommatus timfristos Lukhtanov, Vishnevskaya & Shapoval, sp. n. which differs by its haploid chromosome number (n=38) from the closely related and morphologically similar Polyommatus aroaniensis (n=47-48) and Polyommatus orphicus (n=41-42). We provide chromosomal evidence for three separate south Balkan Pleistocene refugia (Peloponnesse, Central Greece and Northern Greece/South Bulgaria) and stress the biogeographic importance of Central Greece as a place of diversification. Then we argue that the data obtained have direct implications for butterfly karyology, taxonomy, biogeography and conservation.

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          Purification of nucleic acids by extraction with phenol:chloroform.

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            Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae)

            Background DNA barcoding, i.e. the use of a 648 bp section of the mitochondrial gene cytochrome c oxidase I, has recently been promoted as useful for the rapid identification and discovery of species. Its success is dependent either on the strength of the claim that interspecific variation exceeds intraspecific variation by one order of magnitude, thus establishing a "barcoding gap", or on the reciprocal monophyly of species. Results We present an analysis of intra- and interspecific variation in the butterfly family Lycaenidae which includes a well-sampled clade (genus Agrodiaetus) with a peculiar characteristic: most of its members are karyologically differentiated from each other which facilitates the recognition of species as reproductively isolated units even in allopatric populations. The analysis shows that there is an 18% overlap in the range of intra- and interspecific COI sequence divergence due to low interspecific divergence between many closely related species. In a Neighbour-Joining tree profile approach which does not depend on a barcoding gap, but on comprehensive sampling of taxa and the reciprocal monophyly of species, at least 16% of specimens with conspecific sequences in the profile were misidentified. This is due to paraphyly or polyphyly of conspecific DNA sequences probably caused by incomplete lineage sorting. Conclusion Our results indicate that the "barcoding gap" is an artifact of insufficient sampling across taxa. Although DNA barcodes can help to identify and distinguish species, we advocate using them in combination with other data, since otherwise there would be a high probability that sequences are misidentified. Although high differences in DNA sequences can help to identify cryptic species, a high percentage of well-differentiated species has similar or even identical COI sequences and would be overlooked in an isolated DNA barcoding approach.
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              Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies.

              The reinforcement model of evolution argues that natural selection enhances pre-zygotic isolation between divergent populations or species by selecting against unfit hybrids or costly interspecific matings. Reinforcement is distinguished from other models that consider the formation of reproductive isolation to be a by-product of divergent evolution. Although theory has shown that reinforcement is a possible mechanism that can lead to speciation, empirical evidence has been sufficiently scarce to raise doubts about the importance of reinforcement in nature. Agrodiaetus butterflies (Lepidoptera: Lycaenidae) exhibit unusual variability in chromosome number. Whereas their genitalia and other morphological characteristics are largely uniform, different species vary considerably in male wing colour, and provide a model system to study the role of reinforcement in speciation. Using comparative phylogenetic methods, we show that the sympatric distribution of 15 relatively young sister taxa of Agrodiaetus strongly correlates with differences in male wing colour, and that this pattern is most likely the result of reinforcement. We find little evidence supporting sympatric speciation: rather, in Agrodiaetus, karyotypic changes accumulate gradually in allopatry, prompting reinforcement when karyotypically divergent races come into contact.
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                Author and article information

                Journal
                Comp Cytogenet
                Comp Cytogenet
                CompCytogen
                Comparative Cytogenetics
                Pensoft Publishers
                1993-0771
                1993-078X
                2016
                20 December 2016
                : 10
                : 5
                : 1-85
                Affiliations
                [1 ]Department of Karyosystematics, Zoological Institute of Russian Academy of Sciences, Universitetskaya nab. 1, 199034 St. Petersburg, Russia
                [2 ]Department of Entomology, St Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
                [3 ]Department of Cytology and Histology, St Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
                Author notes
                Corresponding author: Vladimir A. Lukhtanov ( lukhtanov@ 123456mail.ru )

                Academic editor: V. Kuznetsova

                Article
                10.3897/CompCytogen.v10i5.10944
                5220643
                6194b5c7-9aa5-40f7-be1d-22c407827b7d
                Maria S. Vishnevskaya, Alsu F. Saifitdinova, 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.

                History
                : 27 October 2016
                : 29 November 2016
                Funding
                Funded by: Russian Science Foundation 501100006769 http://doi.org/10.13039/501100006769
                Categories
                Monograph

                agrodiaetus,biodiversity,chromosome,coi,conservation,cryptic species,dna barcode,its2,karyotype,mitochondrial marker,polyommatus timfristos sp. n.,protected species,red list,animalia,lepidoptera,lycaenidae

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