Elsevier

Zoology

Volume 119, Issue 1, February 2016, Pages 11-20
Zoology

The sibling species Leptidea juvernica and L. sinapis (Lepidoptera, Pieridae) in the Balkan Peninsula: ecology, genetic structure, and morphological variation

https://doi.org/10.1016/j.zool.2015.12.003Get rights and content

Highlights

  • The Balkan populations of L. sinapis and L. juvernica are not genetically differentiated from other mainland populations.

  • In the Balkans, L. sinapis is a widespread habitat generalist; L. juvernica exhibits a transition from a habitat generalist in the west to a localised habitat specialist in the east.

  • Morphometric analysis of the male genitalia reveals a mosaic of regional morphological variation.

  • The morphological variation is not correlated to DNA and is likely a result of environmental and ecological factors.

  • A simple and efficient discriminant function is derived based on the genitalia scaling relations.

Abstract

The wood white butterfly Leptidea sinapis and its more recently discovered sibling species L. reali and L. juvernica have emerged as a model system for studying the speciation and evolution of cryptic species, as well as their ecological interactions in conditions of sympatry. Leptidea sinapis is widely distributed from Western Europe to Central Asia while the synmorphic L. juvernica and L. reali have allopatric distributions, both occurring in sympatry with L. sinapis and exhibiting an intricate, regionally variable ecological niche separation. Until now, the Balkan Peninsula remained one of the major unknowns in terms of distribution, genetic structure, and ecological preferences of the Leptidea triplet in Europe. We present new molecular and morphological data from a detailed survey of the region. Our DNA analysis suggests that the Balkan populations belong only to L. sinapis and L. juvernica, and that they are not genetically differentiated from other mainland populations. The distribution data reveal that L. sinapis is a widespread habitat generalist, while L. juvernica exhibits a transition from a habitat generalist in the western Balkans to a localised habitat specialist, confined to humid mountain habitats, in the east. The morphometric analysis of male genitalia and a comparison to data from other parts of the species' ranges suggest an interesting mosaic of regional morphological variation that is likely linked to environmental and ecological factors. We also demonstrate the usefulness of the genitalia scaling relations for analysing the morphological variation and discuss the implications for species identification.

Introduction

The wood white butterfly Leptidea sinapis (Linnaeus, 1758) and its two sibling species (Leptidea reali Reissinger, 1989 and Leptidea juvernica Williams, 1946) represent a fascinating example of the intricate relations developing within a cryptic species complex. Remarkably, in spite of more than two decades of extensive studies, the full taxonomic diversity of the complex was only recently brought to light. The existence of three well differentiated species was first revealed through molecular, karyological and morphological analyses (Dincă et al., 2011) and later reinforced by demonstrating reproductive isolation due to female mate choice (Dincă et al., 2013). The three species are virtually indistinguishable externally and two of them (L. reali and L. juvernica) also have extremely similar genitalia (Dincă et al., 2011). Because of its specific patterns of distribution and ecological interaction, the Leptidea triplet is an excellent model system for studying the origin and evolution of cryptic species, as well as their ecological interaction in conditions of sympatry (Dincă et al., 2013, Friberg et al., 2013). In addition, due to pronounced karyotype variability, this triplet (particularly L. sinapis) emerges as a promising model to study speciation linked to chromosomal rearrangements and sex determination systems (Dincă et al., 2011, Lukhtanov et al., 2011, Šíchová et al., 2015).

Of the three species, L. sinapis is the one with the widest distribution ranging from western Spain and Ireland to eastern Kazakhstan (Dincă et al., 2011, Dincă et al., 2013) and further east to lake Baikal (Sinev, 2008). The other two species have allopatric distributions, but both are known to occur in sympatry with L. sinapis in parts of their respective ranges (Dincă et al., 2013). L. reali occurs in Western Europe (Spain, southern France, and Italy) whereas L. juvernica extends from Ireland and France to eastern Kazakhstan (Dincă et al., 2011, Dincă et al., 2013), Tian Shan in Kyrgyzstan, northwestern China (Bolshakov, 2006) and the Republic of Tuva (Russia) (Sinev, 2008). Old (pre-2011) reports for L. reali from Central Europe eastwards therefore refer to L. juvernica, although the situation has not been fully clarified in the Balkans (see below).

Studies of the habitat preferences of Leptidea populations have revealed a variable niche specialization in different parts of the species' ranges. The same species can be a habitat generalist in some regions and a habitat specialist in others, where the sympatric sibling species may be a habitat generalist (Friberg et al., 2013). For example, in Sweden L. juvernica is a habitat specialist while L. sinapis is a generalist (Friberg et al., 2008); in Poland both species are widespread generalists (Sachanowicz et al., 2011); in the Czech Republic L. sinapis is the habitat specialist (Beneš et al., 2003); and in Slovenia both species are widespread with partial habitat segregation observed near the Adriatic Sea where L. juvernica is confined to humid habitats (Verovnik et al., 2012). Documenting this geographical variation can provide essential information for understanding the causes of niche specialization and their relation to the microevolutionary processes that eventually produce sibling species (Dincă et al., 2011, Friberg et al., 2013).

The Balkan Peninsula remains relatively poorly studied in terms of species composition, distribution, ecology, and genetic structure of the Leptidea triplet. Data from this region are scarce and sporadic. Some authors assumed that all unverified reports from the region belong to L. sinapis (Abadjiev, 2001, Pamperis, 2009). A series of other studies based on genital morphology or DNA data have been published, with data for Croatia (Lorković, 1993, Mazel and Leestmans, 1999, Dincă et al., 2011, Dincă et al., 2013), Bosnia-Herzegovina (Lelo, 2007, Lelo, 2010), Bulgaria (Kristal and Nässig, 1996, Mazel and Leestmans, 1999, Dincă et al., 2011, Dincă et al., 2013), Greece (Kristal and Nässig, 1996, Mazel and Leestmans, 1999, Dincă et al., 2011, Dincă et al., 2013, Coutsis, 2013), and Romania (Rákosy, 1996, Dincă et al., 2011, Dincă et al., 2013). L. reali sensu lato has also been reported from several localities in Serbia and Montenegro (Jakšić, 1999, Jakšić and Ristić, 1999); however, these records may represent erroneous identifications because the depicted male genitalia (Jakšić and Ristić, 1999) do not correspond to the L. reali-juvernica type. All these works reported isolated findings that were not the result of a systematic survey. Moreover, since L. reali and L. juvernica appear to have overlapping genital morphology, even studies using this character have limited resolution (being able to separate only L. sinapis) in the absence of DNA data. Notably, with the exception of the northwestern limit (Slovenia), all available DNA-based data from the Balkans refer to L. sinapis. It is thus necessary to clarify whether previous records of L. reali from this area actually refer to L. juvernica or not. The known distribution of L. reali is largely restricted to two of the three main European peninsulas (Iberia and Italy), and the possibility that populations of this species also occur in the Balkans cannot be discarded without verification.

The degree of intraspecific (including regional) vs. interspecific morphological variation of the Leptidea triplet presents another largely overlooked problem. Dincă et al. (2011) have noted the almost complete overlap in genital morphology of L. reali and L. juvernica, and the overlap in the size of genitalia features between L. juvernica and L. sinapis has been highlighted in other studies (Freese and Fiedler, 2004, Verovnik and Glogovčan, 2007, Coutsis, 2013), but has not been quantitatively studied. In addition, an overview of published data by Sachanowicz (2013) has indicated that the ranges of variation of genitalia measurements may differ significantly between regions. The extent to which this variation can affect the accuracy of discrimination among the species has not been investigated in detail, in spite of its potentially profound implications on our ability to use genital morphology to reliably identify the sympatric species pairs and trace their regional distribution.

The objective of the present study is to partially fill the existing gap in our knowledge of the Leptidea triplet by establishing what species occur in the Balkan Peninsula and by carrying out a systematic study of their distribution, ecology, genetic structure, and morphological variation. A detailed morphometric analysis combined with molecular data is used for identification, and the regional morphological and genetic variation of the Balkan populations is placed in a broader context by a comparison with published data from other parts of the species' ranges. Habitat preferences and conservation aspects are discussed in the context of the known variable niche specialization of Leptidea.

Section snippets

DNA analyses

We sequenced 655 bp of the mitochondrial cytochrome c oxidase subunit I gene (COI) for 16 Leptidea specimens, 13 of which originated from the Balkans (see supplementary Table S1 in the online Appendix). Total genomic DNA was extracted using Chelex 100 resin, 100–200 mesh, sodium form (Bio-Rad Laboratories, Hercules, CA, USA), under the following protocol: one leg was removed and introduced into 100 μl of Chelex 10% and 5 μl of proteinase K (20 mg/ml) were added. The samples were incubated overnight

DNA data

The DNA analyses (Fig. 2 and supplementary Table S1) indicated that 41 of the sequenced specimens from the Balkans belong to L. sinapis (Bosnia and Herzegovina, Bulgaria, Croatia, Greece, Macedonia, Romania, Serbia, and Slovenia), while 14 belong to L. juvernica (Slovenia, southwestern Bulgaria, and northern Greece). No L. reali were identified among the samples analysed. DNA-based identifications were always congruent with the morphology of the genitalia for the analysed specimens (see

DNA

Previous studies have suggested that L. juvernica and L. reali are synmorphic species that can be differentiated from L. sinapis through male or female genitalia examination, but can be separated from each other only by DNA data (Dincă et al., 2011). The available molecular data have shown that L. reali is confined to the northern half of the Iberian Peninsula, parts of southern France and at least central Italy, while L. juvernica replaces it in the rest of the Eurasian range (Dincă et al.,

Conclusion

Until now, the Balkan Peninsula remained one of the major unknowns in the distribution, genetic structure, and ecological preferences of the Leptidea triplet of sibling species in Europe. Our molecular data indicate that the Leptidea triplet is most likely represented in the Balkans only by L. sinapis and L. juvernica, while L. reali is confined to Iberia, southern France and Italy. We also show that the Balkan populations of L. sinapis and L. juvernica are not genetically differentiated from

Acknowledgements

We are grateful to Stoyan Beshkov for providing access and arranging specimen loan from the NMNH collection, and to Wolfgang Nässig, John Coutsis, Lazaros Pamperis, and Miloš Popović for the provided information and helpful discussions. We also thank Hristos Anastassiu, Artur Choch, Sylvain Cuvelier, Juha Jantunen, and Raluca Vodă for material sequenced in this study. This research was partially funded by Grant CGL2013-48277-P from the Spanish Ministerio de Economía y Competitividad to R.V. and

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