A macroevolutionary role for chromosomal fusion and fission in Erebia butterflies.

The impact of large-scale chromosomal rearrangements, such as fusions and fissions, on speciation is a long-standing conundrum. We assessed whether bursts of change in chromosome numbers resulting from chromosomal fusion or fission are related to increased speciation rates in Erebia, one of the most species-rich and karyotypically variable butterfly groups. We established a genome-based phylogeny and used state-dependent birth-death models to infer trajectories of karyotype evolution. We demonstrated that rates of anagenetic chromosomal changes (i.e., along phylogenetic branches) exceed cladogenetic changes (i.e., at speciation events), but, when cladogenetic changes occur, they are mostly associated with chromosomal fissions rather than fusions. We found that the relative importance of fusion and fission differs among Erebia clades of different ages and that especially in younger, more karyotypically diverse clades, speciation is more frequently associated with cladogenetic chromosomal changes. Overall, our results imply that chromosomal fusions and fissions have contrasting macroevolutionary roles and that large-scale chromosomal rearrangements are associated with bursts of species diversification.

Asynchronous life cycles contribute to reproductive isolation between two Alpine butterflies.

Geographic isolation often leads to the emergence of distinct genetic lineages that are at least partially reproductively isolated. Zones of secondary contact between such lineages are natural experiments that allow investigation of how reproductive isolation evolves and co-existence is maintained. While temporal isolation through allochrony has been suggested to promote reproductive isolation in sympatry, its potential for isolation upon secondary contact is far less understood. Sampling two contact zones of a pair of mainly allopatric Alpine butterflies over several years and taking advantage of museum samples, we show that the contact zones have remained geographically stable over several decades. Furthermore, they seem to be maintained by the asynchronous life cycles of the two butterflies, with one reaching adulthood primarily in even and the other primarily in odd years. Genomic inferences document that allochrony is leaky and that gene flow from allopatric sites scales with the degree of geographic isolation. Overall, we show that allochrony has the potential to contribute to the maintenance of secondary contact zones of lineages that diverged in allopatry.

Holocentric repeat landscapes: From micro-evolutionary patterns to macro-evolutionary associations with karyotype evolution.

Repetitive elements can cause large-scale chromosomal rearrangements, for example through ectopic recombination, potentially promoting reproductive isolation and speciation. Species with holocentric chromosomes, that lack a localized centromere, might be more likely to retain chromosomal rearrangements that lead to karyotype changes such as fusions and fissions. This is because chromosome segregation during cell division should be less affected than in organisms with a localized centromere. The relationships between repetitive elements and chromosomal rearrangements and how they may translate to patterns of speciation in holocentric organisms are though poorly understood. Here, we use a reference-free approach based on low-coverage short-read sequencing data to characterize the repeat landscape of two independently evolved holocentric groups: Erebia butterflies and Carex sedges. We consider both micro- and macro-evolutionary scales to investigate the repeat landscape differentiation between Erebia populations and the association between repeats and karyotype changes in a phylogenetic framework for both Erebia and Carex. At a micro-evolutionary scale, we found population differentiation in repeat landscape that increases with overall intraspecific genetic differentiation among four Erebia species. At a macro-evolutionary scale, we found indications for an association between repetitive elements and karyotype changes along both Erebia and Carex phylogenies. Altogether, our results suggest that repetitive elements are associated with the level of population differentiation and chromosomal rearrangements in holocentric clades and therefore likely play a role in adaptation and potentially species diversification.

The Impact of Chromosomal Rearrangements in Speciation: From Micro- to Macroevolution.

Chromosomal rearrangements (CRs) have been known since almost the beginning of genetics. While an important role for CRs in speciation has been suggested, evidence primarily stems from theoretical and empirical studies focusing on the microevolutionary level (i.e., on taxon pairs where speciation is often incomplete). Although the role of CRs in eukaryotic speciation at a macroevolutionary level has been supported by associations between species diversity and rates of evolution of CRs across phylogenies, these findings are limited to a restricted range of CRs and taxa. Now that more broadly applicable and precise CR detection approaches have become available, we address the challenges in filling some of the conceptual and empirical gaps between micro- and macroevolutionary studies on the role of CRs in speciation. We synthesize what is known about the macroevolutionary impact of CRs and suggest new research avenues to overcome the pitfalls of previous studies to gain a more comprehensive understanding of the evolutionary significance of CRs in speciation across the tree of life.

Genomic hotspots of chromosome rearrangements explain conserved synteny despite high rates of chromosome evolution in a holocentric lineage.

Holocentric organisms, unlike typical monocentric organisms, have kinetochore activity distributed along almost the whole length of the chromosome. Because of this, chromosome rearrangements through fission and fusion are more likely to become fixed in holocentric species, which may account for the extraordinary rates of chromosome evolution that many holocentric lineages exhibit. Long blocks of genome synteny have been reported in animals with holocentric chromosomes despite high rates of chromosome rearrangements. Nothing is known from plants, however, despite the fact that holocentricity appears to have played a key role in the diversification of one of the largest angiosperm genera, Carex (Cyperaceae). In the current study, we compared genomes of Carex species and a distantly related Cyperaceae species to characterize conserved and rearranged genome regions. Our analyses span divergence times ranging between 2 and 50 million years. We also compared a C. scoparia chromosome-level genome assembly with a linkage map of the same species to study rearrangements at a population level and suppression of recombination patterns. We found longer genome synteny blocks than expected under a null model of random rearrangement breakpoints, even between very distantly related species. We also found repetitive DNA to be non-randomly associated with holocentromeres and rearranged regions of the genome. The evidence of conserved synteny in sedges despite high rates of chromosome fission and fusion suggests that conserved genomic hotspots of chromosome evolution related to repetitive DNA shape the evolution of recombination, gene order and crossability in sedges. This finding may help explain why sedges are able to maintain species cohesion even in the face of high interspecific chromosome rearrangements.

Genomic evidence for three distinct species in the Erebia manto complex in Central Europe (Lepidoptera, Nymphalidae).

A problem to implement conservation strategies is that in many cases recognized taxa are in fact complexes of several cryptic species. Failure to properly delineate species may lead to misplaced priorities or to inadequate conservation measures. One such species complex is the yellow-spotted ringlet Erebia manto, which comprises several phenotypically distinct lineages, whose degree of genomic isolation has so far not been assessed. Some of these lineages are geographically restricted and thus possibly represent distinct units with conservation priorities. Using several thousand nuclear genomic markers, we evaluated to which degree the bubastis lineage from the Alps and the vogesiaca lineage from the Vosges, are genetically isolated from the widespread manto lineage. Our results suggest that both lineages are genetically as strongly differentiated from manto as other taxonomically well separated sibling species in this genus from each other, supporting a delineation of bubastis and vogesiaca as independent species. Given the restricted and isolated range of vogesiaca as well as the disjunct distribution of bubastis, our findings have significant implication for future conservation efforts on these formerly cryptic species and highlight the need to investigate the genomic identity within species complexes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-023-01501-w.

Cryptic population structure at the northern range margin of the service tree Sorbus domestica.

Climate change has aroused interest in planting warm- and drought-adapted trees in managed forests and urban areas. An option is to focus on tree species that occur naturally, but have centers of distribution in warmer and drier areas. However, in order to protect the species pool of genetic diversity, efforts of planting and promotion should be informed by knowledge on the local genetic diversity. Here, we studied the macro- and micro-scale population genetic structure of the rare European fruit tree Sorbus domestica at its northern range margin, in western Switzerland. New microsatellite data were combined with published data from across the European distribution of the species. Analyses revealed the presence of mainly one of two species-wide ancestral clusters, i.e., the western European cluster, with evidence that it consists of two cryptic sub-clusters. Average pairwise F ST of 0.118 was low across the range, and only allelic richness was reduced in the northern margin compared to more southern and southeastern areas of Europe. Based on our finding of considerable genetic diversity of the species in western and northern Switzerland, we suggest that a national propagation program should focus on collecting seeds from natural, high-density tree stands and propagate locally. More generally, our study shows that rare tree species in marginal areas of their distributions do not necessarily have low genetic diversity or heightened levels of inbreeding, and in those cases probably need no assisted migration in efforts to propagate them.

Recent speciation associated with range expansion and a shift to self-fertilization in North American Arabidopsis.

The main processes classically evoked for promoting reproductive isolation and speciation are geographic separation reducing gene flow among populations, divergent selection, and chance genomic change. In a case study, we present evidence that the additional factors of climate change, range expansion and a shift in mating towards inbreeding can initiate the processes leading to parapatric speciation. At the end of the last Pleistocene glaciation cycle, the North American plant Arabidopsis lyrata expanded its range and concomitantly lost its reproductive mode of outcrossing multiple times. We show that in one of the newly colonized areas, the self-fertilizing recolonization lineage of A. lyrata gave rise to selfing A. arenicola, which expanded its range to subarctic and arctic Canada and Greenland, while the parental species remained restricted to temperate North America. Despite the vast range expansion by the new species, mutational load did not increase, probably because of selfing and quasi-clonal selection. We conclude that such peripheral parapatric speciation combined with range expansion and inbreeding may be an important but so far overlooked mode of speciation.

The genome sequence of the scotch argus butterfly, Erebia aethiops (Esper, 1777).

We present a genome assembly from an individual female Erebia aethiops (the scotch argus; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 473 megabases in span. The complete assembly is scaffolded into 20 chromosomal pseudomolecules, with the W and Z sex chromosomes assembled. The complete mitochondrial genome was also assembled and is 15.2 kilobases in length.

Secondary contact rather than coexistence-Erebia butterflies in the Alps.

Secondary contact zones are ideal systems to study the processes that govern the evolution of reproductive barriers, especially at advanced stages of the speciation process. An increase in reproductive isolation resulting from selection against maladaptive hybrids is thought to contribute to reproductive barrier buildup in secondary contact zones. Although such processes have been invoked for many systems, it remains unclear to which extent they influence contact zone dynamics in nature. Here, we study a very narrow contact zone between the butterfly species Erebia cassioides and Erebia tyndarus in the Swiss Alps. We quantified phenotypic traits related to wing shape and reproduction as well as ecology to compare the degree of intra- and interspecific differentiation. Even though only very few first-generation hybrids occur, we find no strong indications for current reinforcing selection, suggesting that if reinforcement occurred in our system, it likely operated in the past. Additionally, we show that both species differ less in their ecological niche at the contact zone than elsewhere, which could explain why coexistence between these butterflies may currently not be possible.

A holocentric twist to chromosomal speciation?

Chromosomal rearrangements trigger speciation by acting as barriers to gene flow. However, the underlying theory was developed with monocentric chromosomes in mind. Holocentric chromosomes, lacking a centromeric region, have repeatedly evolved and account for a significant fraction of extant biodiversity. Because chromosomal rearrangements may be more likely retained in holocentric species, holocentricity could provide a twist to chromosomal speciation. Here, we discuss how the abundance of chromosome-scale genomes, combined with novel analytical tools, offer the opportunity to assess the impacts of chromosomal rearrangements on rates of speciation by outlining a phylogenetic framework that aligns with the two major lines of chromosomal speciation theory. We further highlight how holocentric species could help to test for causal roles of chromosomal rearrangements in speciation.

The genome sequence of the Arran brown, Erebia ligea (Linnaeus, 1758).

We present a genome assembly from an individual male Erebia ligea (Arran brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 506 megabases in span. The majority (99.92%) of the assembly is scaffolded into 29 chromosomal pseudomolecules, with the Z sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.2 kilobases in length.

Prevalence and relationship of endosymbiotic Wolbachia in the butterfly genus Erebia.

BACKGROUND: Wolbachia is an endosymbiont common to most invertebrates, which can have significant evolutionary implications for its host species by acting as a barrier to gene flow. Despite the importance of Wolbachia, still little is known about its prevalence and diversification pattern among closely related host species. Wolbachia strains may phylogenetically coevolve with their hosts, unless horizontal host-switches are particularly common. We address  these issues in the genus Erebia, one of the most diverse Palearctic butterfly genera. RESULTS: We sequenced the Wolbachia genome from a strain infecting Erebia cassioides and showed that it belongs to the Wolbachia supergroup B, capable of infecting arthropods from different taxonomic orders. The prevalence of Wolbachia across 13 closely related Erebia host species based on extensive population-level genetic data revealed that multiple Wolbachia strains jointly infect all investigated taxa, but with varying prevalence. Finally, the phylogenetic relationships of Wolbachia strains are in some cases significantly associated to that of their hosts, especially among the most closely related Erebia species, demonstrating mixed evidence for phylogenetic coevolution. CONCLUSIONS: Closely related host species can be infected by closely related Wolbachia strains, evidencing some phylogenetic coevolution, but the actual pattern of infection more often reflects historical or contemporary geographic proximity among host species. Multiple processes, including survival in distinct glacial refugia, recent host shifts in sympatry, and a loss of Wolbachia during postglacial range expansion seem to have jointly shaped the complex interactions between Wolbachia evolution and the diversification of its host among our studied Erebia species.

Drivers of linkage disequilibrium across a species' geographic range.

While linkage disequilibrium (LD) is an important parameter in genetics and evolutionary biology, the drivers of LD remain elusive. Using whole-genome sequences from across a species' range, we assessed the impact of demographic history and mating system on LD. Both range expansion and a shift from outcrossing to selfing in North American Arabidopsis lyrata were associated with increased average genome-wide LD. Our results indicate that range expansion increases short-distance LD at the farthest range edges by about the same amount as a shift to selfing. However, the extent over which LD in genic regions unfolds was shorter for range expansion compared to selfing. Linkage among putatively neutral variants and between neutral and deleterious variants increased to a similar degree with range expansion, providing support that genome-wide LD was positively associated with mutational load. As a consequence, LD combined with mutational load may decelerate range expansions and set range limits. Finally, a small number of genes were identified as LD outliers, suggesting that they experience selection by either of the two demographic processes. These included genes involved in flowering and photoperiod for range expansion, and the self-incompatibility locus for mating system.

Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers.

Speciation, that is, the evolution of reproductive barriers eventually leading to complete isolation, is a crucial process generating biodiversity. Recent work has contributed much to our understanding of how reproductive barriers begin to evolve, and how they are maintained in the face of gene flow. However, little is known about the transition from partial to strong reproductive isolation (RI) and the completion of speciation. We argue that the evolution of strong RI is likely to involve different processes, or new interactions among processes, compared with the evolution of the first reproductive barriers. Transition to strong RI may be brought about by changing external conditions, for example, following secondary contact. However, the increasing levels of RI themselves create opportunities for new barriers to evolve and, and interaction or coupling among barriers. These changing processes may depend on genomic architecture and leave detectable signals in the genome. We outline outstanding questions and suggest more theoretical and empirical work, considering both patterns and processes associated with strong RI, is needed to understand how speciation is completed. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Speciation through chromosomal fusion and fission in Lepidoptera.

Changes in chromosome numbers may strongly affect reproductive barriers, because individuals heterozygous for distinct karyotypes are typically expected to be at least partially sterile or to show reduced recombination. Therefore, several classic speciation models are based on chromosomal changes. One import mechanism generating variation in chromosome numbers is fusion and fission of existing chromosomes, which is particularly likely in species with holocentric chromosomes, i.e. chromosomes that lack a single centromere. Holocentric chromosomes evolved repeatedly across the tree of life, including in Lepidoptera. Although changes in chromosome numbers are hypothesized to be an important driver of the spectacular diversification of Lepidoptera, comparative studies across the order are lacking. We performed the first comprehensive literature survey of karyotypes for Lepidoptera species since the 1970s and tested if, and how, chromosomal variation might affect speciation. Even though a meta-analysis of karyological differences between closely related taxa did not reveal an effect on the degree of reproductive isolation, phylogenetic diversification rate analyses across the 16 best-covered genera indicated a strong, positive association of rates of chromosome number evolution and speciation. These findings suggest a macroevolutionary impact of varying chromosome numbers in Lepidoptera and likely apply to other taxonomic groups, especially to those with holocentric chromosomes. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Secondary contact zones of closely-related Erebia butterflies overlap with narrow phenotypic and parasitic clines.

Zones of secondary contact between closely related taxa are a common legacy of the Quaternary ice ages. Despite their abundance, the factors that keep species apart and prevent hybridization are often unknown. Here, we study a very narrow contact zone between three closely related butterfly species of the Erebia tyndarus species complex. Using genomic data, we first determined whether gene flow occurs and then assessed whether it might be hampered by differences in chromosome number between some species. We found interspecific gene flow between sibling species that differ in karyotype by one chromosome. Conversely, only F1 hybrids occurred between two species that have the same karyotype, forming a steep genomic cline. In a second step, we fitted clines to phenotypic, ecological and parasitic data to identify the factors associated with the genetic cline. We found clines for phenotypic data and the prevalence of the endosymbiont parasite Wolbachia to overlap with the genetic cline, suggesting that they might be drivers for separating the two species. Overall, our results highlight that some gene flow is possible between closely related species despite different chromosome numbers, but that other barriers restrict such gene flow.

Lineage-specific adaptation to climate involves flowering time in North American Arabidopsis lyrata.

Adaptation to local climatic conditions is commonly found within species, but whether it involves the same intraspecific genomic variants is unknown. We studied this question in North American Arabidopsis lyrata, whose current distribution is shaped by post-glacial range expansion from two refugia, resulting in two distinct genetic clusters covering comparable climatic gradients. Using pooled whole-genome sequence data of 41 outcrossing populations, we identified loci associated with three niche-determining climatic variables in the two clusters and compared these outliers. Little evidence was found for parallelism in climate adaptation for single nucleotide polymorphisms (SNPs) and for genes with an accumulation of outlier SNPs. Significantly increased selection coefficients supported them as candidates of climate adaptation. However, the fraction of gene ontology (GO) terms shared between clusters was higher compared to outlier SNPs and outlier genes, suggesting that selection acts on similar pathways but not necessarily the same genes. Enriched GO terms involved responses to abiotic and biotic stress, circadian rhythm and development, with flower development and reproduction being among the most frequently detected. In line with GO enrichment, regulators of flowering time were detected as outlier genes. Our results suggest that while adaptation to environmental gradients on the genomic level are lineage-specific in A. lyrata, similar biological processes seem to be involved. Differential loss of standing genetic variation, probably driven by genetic drift, can in part account for the lack of parallel evolution on the genomic level.

Admixture between old lineages facilitated contemporary ecological speciation in Lake Constance stickleback.

Ecological speciation can sometimes rapidly generate reproductively isolated populations coexisting in sympatry, but the origin of genetic variation permitting this is rarely known. We previously explored the genomics of very recent ecological speciation into lake and stream ecotypes in stickleback from Lake Constance. Here, we reconstruct the origin of alleles underlying ecological speciation by combining demographic modelling on genome-wide single nucleotide polymorphisms, phenotypic data and mitochondrial sequence data in the wider European biogeographical context. We find that parallel differentiation between lake and stream ecotypes across replicate lake-stream ecotones resulted from recent secondary contact and admixture between old East and West European lineages. Unexpectedly, West European alleles that introgressed across the hybrid zone at the western end of the lake, were recruited to genomic islands of differentiation between ecotypes at the eastern end of the lake. Our results highlight an overlooked outcome of secondary contact: ecological speciation facilitated by admixture variation.