Genetic integrity of four species of Leptidea (Pieridae, Lepidoptera) as sampled in sympatry in West Siberia

In southern West Siberia, as many as four Leptidea Billberg, 1820 species are present sympatrically: Leptidea amurensis (Ménétriés, 1859), Leptidea morsei (Ménétriés, 1859), Leptidea sinapis (Linnaeus, 1758) and Leptidea juvernica Williams, 1946. The two latter were recently recognised as nearly sibling species on morphological and molecular characters. Specimens intermediate as to their subtle diagnostic characters occurring in West Siberia and elsewhere were interpreted as resulted from limited introgression. This supposition was tested via populational morphological and molecular analysis of spring brood specimens of all the four species taken from a limited (4.5 × 0.2 km) area in the suburbs of Novosibirsk. The samples were analysed with respect to the genitalic morphology, external characters, three nuclear (CAD, H1 gene and ITS2) and one mitochondrial (COI) molecular markers, infection of the intracellular maternally inherited bacterial symbiont Wolbachia Hertig, 1836 and its wsp gene coding for a hypervariable surface protein. Interspecific variation of the nuclear CAD and ITS2 sequences and the mitochondrial COI gene in Leptidea sinapis and Leptidea juvernica turned out concordant. The absence of molecular evidence of introgression suggests genetic integrity of these two species and allows their reliable identification by molecular characters. The genitalic (lengths of the saccus and valva) and external characters (wing pattern) of males overlap in Leptidea sinapis and Leptidea juvernica, as identified by molecular markers and thus are not so helpful in actual species identification. Only the ductus bursae length showed no overlap and can be used for identification of females. The histone H1 gene appeared five times less variable over the four studied species than COI, and found to be identical in species Leptidea sinapis and Leptidea juvernica. Wolbachia infection was found in all studied species. We identified three wsp variants of Wolbachia: 1) wsp-10 allele in Leptidea amurensis, Leptidea sinapis, Leptidea juvernica; 2) a very similar wsp-687 allele in Leptidea sinapis; and 3) wsp-688, highly divergent to the previous ones, in Leptidea morsei.