Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

Base Composition, Codon Usage, and Patterns of Gene Sequence Evolution in Butterflies.

Coding sequence evolution is influenced by both natural selection and neutral evolutionary forces. In many species, the effects of mutation bias, codon usage, and GC-biased gene conversion (gBGC) on gene sequence evolution have not been detailed. Quantification of how these forces shape substitution patterns is therefore necessary to understand the strength and direction of natural selection. Here, we used comparative genomics to investigate the association between base composition and codon usage bias on gene sequence evolution in butterflies and moths (Lepidoptera), including an in-depth analysis of underlying patterns and processes in one species, Leptidea sinapis. The data revealed significant G/C to A/T substitution bias at third codon position with some variation in the strength among different butterfly lineages. However, the substitution bias was lower than expected from previously estimated mutation rate ratios, partly due to the influence of gBGC. We found that A/T-ending codons were overrepresented in most species, but there was a positive association between the magnitude of codon usage bias and GC-content in third codon positions. In addition, the tRNA-gene population in L. sinapis showed higher GC-content at third codon positions compared to coding sequences in general and less overrepresentation of A/T-ending codons. There was an inverse relationship between synonymous substitutions and codon usage bias indicating selection on synonymous sites. We conclude that the evolutionary rate in Lepidoptera is affected by a complex interaction between underlying G/C -> A/T mutation bias and partly counteracting fixation biases, predominantly conferred by overall purifying selection, gBGC, and selection on codon usage.

The genome sequence of the wood white butterfly, Leptidea sinapis (Linnaeus, 1758).

We present a genome assembly from an individual male Leptidea sinapis (the wood white; Arthropoda; Insecta; Lepidoptera; Pieridae). The genome sequence is 686 megabases in span. The majority (99.99%) of the assembly is scaffolded into 48 chromosomal pseudomolecules, with three Z sex chromosomes assembled. Gene annotation of this assembly on Ensembl has identified 14,800 protein coding genes.

Dissecting the Effects of Selection and Mutation on Genetic Diversity in Three Wood White (Leptidea) Butterfly Species.

The relative role of natural selection and genetic drift in evolution is a major topic of debate in evolutionary biology. Most knowledge spring from a small group of organisms and originate from before it was possible to generate genome-wide data on genetic variation. Hence, it is necessary to extend to a larger number of taxonomic groups, descriptive and hypothesis-based research aiming at understanding the proximate and ultimate mechanisms underlying both levels of genetic polymorphism and the efficiency of natural selection. In this study, we used data from 60 whole-genome resequenced individuals of three cryptic butterfly species (Leptidea sp.), together with novel gene annotation information and population recombination data. We characterized the overall prevalence of natural selection and investigated the effects of mutation and linked selection on regional variation in nucleotide diversity. Our analyses showed that genome-wide diversity and rate of adaptive substitutions were comparatively low, whereas nonsynonymous to synonymous polymorphism and substitution levels were comparatively high in Leptidea, suggesting small long-term effective population sizes. Still, negative selection on linked sites (background selection) has resulted in reduced nucleotide diversity in regions with relatively high gene density and low recombination rate. We also found a significant effect of mutation rate variation on levels of polymorphism. Finally, there were considerable population differences in levels of genetic diversity and pervasiveness of selection against slightly deleterious alleles, in line with expectations from differences in estimated effective population sizes.

Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species.

Genome scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large-scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence-whether radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here, we used whole-genome resequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships, and there was no evidence for postdivergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis)-a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic, and outlier regions were narrow and dispersed. Highly differentiated (FST ) regions generally had low genetic diversity (θπ ), but increased absolute divergence (DXY ) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression.

Rapid Increase in Genome Size as a Consequence of Transposable Element Hyperactivity in Wood-White (Leptidea) Butterflies.

Characterizing and quantifying genome size variation among organisms and understanding if genome size evolves as a consequence of adaptive or stochastic processes have been long-standing goals in evolutionary biology. Here, we investigate genome size variation and association with transposable elements (TEs) across lepidopteran lineages using a novel genome assembly of the common wood-white (Leptidea sinapis) and population re-sequencing data from both L. sinapis and the closely related L. reali and L. juvernica together with 12 previously available lepidopteran genome assemblies. A phylogenetic analysis confirms established relationships among species, but identifies previously unknown intraspecific structure within Leptidea lineages. The genome assembly of L. sinapis is one of the largest of any lepidopteran taxon so far (643 Mb) and genome size is correlated with abundance of TEs, both in Lepidoptera in general and within Leptidea where L. juvernica from Kazakhstan has considerably larger genome size than any other Leptidea population. Specific TE subclasses have been active in different Lepidoptera lineages with a pronounced expansion of predominantly LINEs, DNA elements, and unclassified TEs in the Leptidea lineage after the split from other Pieridae. The rate of genome expansion in Leptidea in general has been in the range of four Mb/Million year (My), with an increase in a particular L. juvernica population to 72 Mb/My. The considerable differences in accumulation rates of specific TE classes in different lineages indicate that TE activity plays a major role in genome size evolution in butterflies and moths.

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

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.

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: Leptideaamurensis (Ménétriés, 1859), Leptideamorsei (Ménétriés, 1859), Leptideasinapis (Linnaeus, 1758) and Leptideajuvernica 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 Leptideasinapis and Leptideajuvernica 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 Leptideasinapis and Leptideajuvernica, 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 Leptideasinapis and Leptideajuvernica. Wolbachia infection was found in all studied species. We identified three wsp variants of Wolbachia: 1) wsp-10 allele in Leptideaamurensis, Leptideasinapis, Leptideajuvernica; 2) a very similar wsp-687 allele in Leptideasinapis; and 3) wsp-688, highly divergent to the previous ones, in Leptideamorsei.

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex.

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter- and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.

Separation possibilities and genital measurement variations in two cryptic species of European pierid butterflies, Leptidea juvernica Williams, 1946 and L. sinapis (Linnaeus, 1758).

Of the three recently separated cryptic butterfly species of the Leptidea sinapis complex, the two species L. sinapis and L. juvernica occur sympatrically and syntopically in central Europe. As the separation of these species requires genital or genetic characters, their correct identification is a fundamental problem to be solved prior to any biological and distributional studies. In the present study a morphometric approach was applied to test for separation possibilities and to examine genital measurement variations based on large population samples of the two species (347 females and 636 males). Butterflies were collected at 456 localities distributed across Poland. Specimens of both sexes could be separated using either the shape or the length of the antrum bursae (females), phallus and saccus (males). Intraspecific seasonal differences in these measurements may affect the discrimination of males only. Genital characters were significantly larger in butterflies of the spring brood than in those of the second brood. In females, antrum bursae length ranges were separated by an interval, allowing for the exact identification of each specimen. In males, no such intervals were found between phallus and saccus length ranges, which slightly overlapped. Discriminant analysis resulted in 100% of males correctly classified. The present study confirmed the validity of interspecific differences in the shape of phallus and saccus for species identification without measurements. The length of these two characters discriminates the two species when separation is limited to specimens from the same generations. As an alternative for discriminant analysis, either phallus length and vinculum width or phallus length and the ratio of the ventral edge of genital capsule length and saccus length (VEL/SL ratio) is recommended for the practical separation of the two species. The differences in the shape and size of the copulatory organs of the two species seem to indicate reproductive isolation due to mechanical incompatibility of their genitals.