Speciation genetics of recently diverged species :

Species differentiation can be a consequence of evolutionary forces including natural selection and random genetic drift. Patterns of genomic differentiation vary across the tree of life. This variation seems to be dependent on, for example, differences in genomic architecture and molecular mechanisms. However, the knowledge we currently possess, both regarding the processes driving speciation and the resulting genomic signatures, is from a very small subset of the overall biodiversity that resides on the planet. Therefore, characterization of the architecture of genomic divergence from more organism groups will be important to understand the effects of molecular mechanisms and evolutionary forces driving divergence between lineages. Hence it has not been possible to come to a consensus on the relative importance of genetic drift and natural selection on divergence processes in general. In this thesis, I use genomic approaches to investigate the forces underlying species and population differentiation in the European cryptic wood white butterflies (Leptidea sinapis, L. reali and L. juvernica) and two closely related bird species, the chiffchaff (Phylloscopus collybita abietinus) and the Siberian chiffchaff (P. tristis). Both these groups contain recently diverged species, a prerequisite for investigating initial differentiation processes. However, the study systems also differ in several respects, allowing for applying distinct approaches to understand the divergence process in each system. In summary, by applying a suite of genomic approaches, my thesis work gives novel insights into the speciation history of wood whites and chiffchaff. I identify candidate genes for local adaptation in both systems and concludes that genome differentiation in wood white butterflies have been driven by a combination of random genetic drift and week directional selection in allopatry. In the chiffchaff, the general differentiation landscape seems to have been shaped by recurrent background selection (and potentially selective sweeps), likely as a consequence of regional variation in the recombination rate which has also been observed in other genome-scans in birds. Potentially, some of the highly differentiated regions contain barriers to gene-flow as these regions are still present in sympatry, where species exchange genetic material at a high rate

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 and there was a negative association between the magnitude of codon usage bias and GC-content in third codon positions. In contrast, 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

Heterogeneous Patterns of Genetic Diversity and Differentiation in European and Siberian Chiffchaff (Phylloscopus collybita abietinus/P. tristis)

Identification of candidate genes for trait variation in diverging lineages and characterization of mechanistic underpinnings of genome differentiation are key steps toward understanding the processes underlying the formation of new species. Hybrid zones provide a valuable resource for such investigations, since they allow us to study how genomes evolve as species exchange genetic material and to associate particular genetic regions with phenotypic traits of interest. Here, we use whole-genome resequencing of both allopatric and hybridizing populations of the European (Phylloscopus collybita abietinus) and the Siberian chiffchaff (P. tristis)-two recently diverged species which differ in morphology, plumage, song, habitat, and migration-to quantify the regional variation in genome-wide genetic diversity and differentiation, and to identify candidate regions for trait variation. We find that the levels of diversity, differentiation, and divergence are highly heterogeneous, with significantly reduced global differentiation, and more pronounced differentiation peaks in sympatry than in allopatry. This pattern is consistent with regional differences in effective population size and recurrent background selection or selective sweeps reducing the genetic diversity in specific regions prior to lineage divergence, but the data also suggest that post-divergence selection has resulted in increased differentiation and fixed differences in specific regions. We find that hybridization and backcrossing is common in sympatry, and that phenotype is a poor predictor of the genomic composition of sympatric birds. The combination of a differentiation scan approach with identification of fixed differences pinpoint a handful of candidate regions that might be important for trait variation between the two species

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 (F) regions generally had low genetic diversity (θ), but increased absolute divergence (D) 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.This work was supported by a junior research grant from the Swedish Research Council (VR) to NB. The authors acknowledge support from the National Genomics Infrastructure in Stockholm and Uppsala funded by the Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. RV was supported by project CGL2016‐76322‐P (AEI/FEDER, UE).Peer reviewe

Migratory behaviour is positively associated with genetic diversity in butterflies

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost¿benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2 -20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a "compensatory" demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales.This work was funded by the National Geographic Society (grant WW1-300R-18) and by the grant PID2020-117739GA-I00 from MCIN/AEI/10.13039/501100011033 to G.T., by fellowship FPU19/01593 to A.G.-B., by project LINKA20399 from the CSIC i-LINK-2021 programme to G.T., N.E.P., R.V. and N.B., by grants from the Putnam Expeditionary Fund of the Museum of Comparative Zoology to G.T. and N.E.P., by projects PID2019-107078GB-I00/MCIN/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to R.V. and G.T., by the University of Malaya (grant H50001-A-000027) to K.G.C., by the Swedish Collegium for Advanced Science (SCAS; Natural Sciences Programme, Knut and Alice Wallenberg Foundation, Postdoc funding) to D.S., by NSF grant DEB-1541560 to N.E.P., and by the Swedish Research Council FORMAS (grant 2019-00670) to N.B., G.T. and R.V.1 INTRODUCTION 2 MATERIALS AND METHODS 2.1 Heterozygosity estimates 2.2 Behavioural scoring 2.3 Comparative analyses 2.4 Vanessa cardui sampling and sequencing 2.5 Vanessa cardui reference-based assemblies 2.6 Vanessa cardui demographic inference 3 RESULTS 3.1 Heterozygosity rates in Lepidoptera 3.2 Vanessa cardui demographic history 4 DISCUSSION 4.1 Genomic heterozygosity, effective population size and migration 4.2 Short-term migratory cost but long-term demographic stability 4.3 Delimiting populations in migratory insect species AUTHOR CONTRIBUTIONS ACKNOWLEDGEMENTS CONFLICT OF INTERES

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 (643Mb) 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 72Mb/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

Dissecting the effects of selection and mutation on genetic diversity in three wood white (Leptidea) butterfly species

Abstract 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