The sibling species Leptidea juvernica and L. sinapis (Lepidoptera, Pieridae) in the Balkan Peninsula: ecology, genetic structure, and morphological variation
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
References (45)
Separation possibilities and genital measurement variations in two cryptic species of European pierid butterflies, Leptidea juvernica Williams, 1946 and L. sinapis (Linnaeus, 1758)
Zoology
(2013)An Atlas of the Distribution of the Butterflies in Bulgaria (Lepidoptera: Hesperioidea & Papilionoidea)
Pensoft Publishers
(2001)- Amiet, J.L., 2004. Séparation des niches écologiques chez deux espèces jumelles sympatriques de Leptidea (Lepidoptera,...
- et al.
Do the sibling species of small whites, Leptidea sinapis and L. reali (Lepidoptera, Pieridae) differ in habitat preferences?
Biologia (Bratisl.)
(2003) - et al.
Testing for phylogenetic signal in comparative data: behavioral traits are more labile
Evolution
(2003) New subspecies of Leptidea reali Reissinger, 1989 (Lepidoptera: Pieridae) from the mountain regions of Middle Asia
Eversmannia
(2006)- et al.
TCS: a computer program to estimate gene genealogies
Mol. Ecol.
(2000) Leptidea sinapis and Leptidea reali (Lepidoptera: Pieridae): at what point does the first one of the two end, and the other one begin?
Phegea
(2013)- et al.
Leptidea sinapis and Leptidea reali (Lepidoptera: Pieridae) in the Netherlands
Entomol. Berichten
(2002) - et al.
Unexpected layers of cryptic diversity in wood white Leptidea butterflies
Nat. Commun.
(2011)