A three-dimensional genomics view for speciation research.

Genomic information is folded in a three-dimensional (3D) structure, a rarely explored evolutionary driver of speciation. Technological advances now enable the study of 3D genome structures (3DGSs) across the Tree of Life. At the onset of 3D speciation genomics, we discuss the putative roles of 3DGSs in speciation.

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.

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.

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.

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.

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.

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.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

Postglacial ecotype formation under outcrossing and self-fertilization in Arabidopsis lyrata.

The formation of ecotypes has been invoked as an important driver of postglacial biodiversity, because many species colonized heterogeneous habitats and experienced divergent selection. Ecotype formation has been predominantly studied in outcrossing taxa, while far less attention has been paid to the implications of mating system shifts. Here, we addressed whether substrate-related ecotypes exist in selfing and outcrossing populations of Arabidopsis lyrata subsp. lyrata and whether the genomic footprint differs between mating systems. The North American subspecies colonized both rocky and sandy habitats during postglacial range expansion and shifted the mating system from predominantly outcrossing to predominantly selfing in a number of regions. We performed an association study on pooled whole-genome sequence data of 20 selfing or outcrossing populations, which suggested genes involved in adaptation to substrate. Motivated by enriched gene ontology terms, we compared root growth between plants from the two substrates in a common environment and found that plants originating from sand grew roots faster and produced more side roots, independent of mating system. Furthermore, single nucleotide polymorphisms associated with substrate-related ecotypes were more clustered among selfing populations. Our study provides evidence for substrate-related ecotypes in A. lyrata and divergence in the genomic footprint between mating systems. The latter is the likely result of selfing populations having experienced divergent selection on larger genomic regions due to higher genome-wide linkage disequilibrium.

The role of structural genomic variants in population differentiation and ecotype formation in Timema cristinae walking sticks.

Theory predicts that structural genomic variants such as inversions can promote adaptive diversification and speciation. Despite increasing empirical evidence that adaptive divergence can be triggered by one or a few large inversions, the degree to which widespread genomic regions under divergent selection are associated with structural variants remains unclear. Here we test for an association between structural variants and genomic regions that underlie parallel host-plant-associated ecotype formation in Timema cristinae stick insects. Using mate-pair resequencing of 20 new whole genomes we find that moderately sized structural variants such as inversions, deletions and duplications are widespread across the genome, being retained as standing variation within and among populations. Using 160 previously published, standard-orientation whole genome sequences we find little to no evidence that the DNA sequences within inversions exhibit accentuated differentiation between ecotypes. In contrast, a formerly described large region of reduced recombination that harbours genes controlling colour-pattern exhibits evidence for accentuated differentiation between ecotypes, which is consistent with differences in the frequency of colour-pattern morphs between host-associated ecotypes. Our results suggest that some types of structural variants (e.g., large inversions) are more likely to underlie adaptive divergence than others, and that structural variants are not required for subtle yet genome-wide genetic differentiation with gene flow.

Allopatric and sympatric diversification within roach (Rutilus rutilus) of large pre-alpine lakes.

Intraspecific differentiation in response to divergent natural selection between environments is a common phenomenon in some northern freshwater fishes, especially salmonids and stickleback. Understanding why these taxa diversify and undergo adaptive radiations while most other fish species in the same environments do not, remains an open question. The possibility for intraspecific diversification has rarely been evaluated for most northern freshwater fish species. Here, we assess the potential for intraspecific differentiation between and within lake populations of roach (Rutilus rutilus)-a widespread and abundant cyprinid species-in lakes in which salmonids have evolved endemic adaptive radiations. Based on more than 3,000 polymorphic RADseq markers, we detected low but significant genetic differentiation between roach populations of two ultraoligotrophic lakes and between these and populations from other lakes. This, together with differentiation in head morphology and stable isotope signatures, suggests evolutionary and ecological differentiation among some of our studied populations. Next, we tested for intralacustrine diversification of roach within Lake Brienz, the most pristine lake surveyed in this study. We found significant phenotypic evidence for ecological intralacustrine differentiation between roach caught over a muddy substrate and those caught over a rocky substrate. However, evidence for intralacustrine genetic differentiation is at best subtle and phenotypic changes may therefore be mostly plastic. Overall, our findings suggest roach can differ between ecologically distinct lakes, but the extent of intralacustrine ecological differentiation is weak, which contrasts with the strong differentiation among endemic species of whitefish in the same lakes.

Genomics of Rapid Incipient Speciation in Sympatric Threespine Stickleback.

Ecological speciation is the process by which reproductively isolated populations emerge as a consequence of divergent natural or ecologically-mediated sexual selection. Most genomic studies of ecological speciation have investigated allopatric populations, making it difficult to infer reproductive isolation. The few studies on sympatric ecotypes have focused on advanced stages of the speciation process after thousands of generations of divergence. As a consequence, we still do not know what genomic signatures of the early onset of ecological speciation look like. Here, we examined genomic differentiation among migratory lake and resident stream ecotypes of threespine stickleback reproducing in sympatry in one stream, and in parapatry in another stream. Importantly, these ecotypes started diverging less than 150 years ago. We obtained 34,756 SNPs with restriction-site associated DNA sequencing and identified genomic islands of differentiation using a Hidden Markov Model approach. Consistent with incipient ecological speciation, we found significant genomic differentiation between ecotypes both in sympatry and parapatry. Of 19 islands of differentiation resisting gene flow in sympatry, all were also differentiated in parapatry and were thus likely driven by divergent selection among habitats. These islands clustered in quantitative trait loci controlling divergent traits among the ecotypes, many of them concentrated in one region with low to intermediate recombination. Our findings suggest that adaptive genomic differentiation at many genetic loci can arise and persist in sympatry at the very early stage of ecotype divergence, and that the genomic architecture of adaptation may facilitate this.

Distinct colonization waves underlie the diversification of the freshwater sculpin (Cottus gobio) in the Central European Alpine region.

Ecological speciation and adaptive radiation are key processes shaping northern temperate freshwater fish diversity. Both often involve parapatric differentiation between stream and lake populations and less often, sympatric intralacustrine diversification into habitat- and resource-associated ecotypes. However, few taxa have been studied, calling for studies of others to investigate the generality of these processes. Here, we test for diversification within catchments in freshwater sculpins in a network of peri-Alpine lakes and streams. Using 8047 and 13 182 restriction site-associated (RADseq) SNPs, respectively, we identify three deeply divergent phylogeographic lineages associated with different major European drainages. Within the Aare catchment, we observe populations from geographically distant lakes to be genetically more similar to each other than to populations from nearby streams. This pattern is consistent with two distinct colonization waves, rather than by parapatric ecological speciation after a single colonization wave. We further find two distinct depth distribution modes in three lakes of the Aare catchment, one in very shallow and one in very deep water, and significant genomewide differentiation between these in one lake. Sculpins in the Aare catchment appear to represent an early-stage adaptive radiation involving the evolution of a lacustrine lineage distinct from parapatric stream sculpins and the repeated onset of depth-related intralacustrine differentiation.

Long-term balancing selection on chromosomal variants associated with crypsis in a stick insect.

How polymorphisms are maintained within populations over long periods of time remains debated, because genetic drift and various forms of selection are expected to reduce variation. Here, we study the genetic architecture and maintenance of phenotypic morphs that confer crypsis in Timema cristinae stick insects, combining phenotypic information and genotyping-by-sequencing data from 1,360 samples across 21 populations. We find two highly divergent chromosomal variants that span megabases of sequence and are associated with colour polymorphism. We show that these variants exhibit strongly reduced effective recombination, are geographically widespread and probably diverged millions of generations ago. We detect heterokaryotype excess and signs of balancing selection acting on these variants through the species' history. A third chromosomal variant in the same genomic region likely evolved more recently from one of the two colour variants and is associated with dorsal pattern polymorphism. Our results suggest that large-scale genetic variation associated with crypsis has been maintained for long periods of time by potentially complex processes of balancing selection.

Genomic landscape of early ecological speciation initiated by selection on nuptial colour.

Ecological speciation is the evolution of reproductive isolation as a consequence of direct divergent natural selection or ecologically mediated divergent sexual selection. While the genomic signature of the former has been extensively studied in recent years, only few examples exist for genomic differentiation where environment-dependent sexual selection has played an important role. Here, we describe a very young (~90 years old) population of threespine sticklebacks exhibiting phenotypic and genomic differentiation between two habitats within the same pond. We show that differentiation among habitats is limited to male throat colour and nest type, traits known to be subject to sexual selection. Divergence in these traits mirrors divergence in much older benthic and limnetic stickleback species pairs from North American west coast lakes, which also occur in sympatry but are strongly reproductively isolated from each other. We demonstrate that in our population, differences in throat colour and breeding have been stable over a decade, but in contrast to North American benthic and limnetic stickleback species, these mating trait differences are not accompanied by divergence in morphology related to feeding, predator defence or swimming performance. Using genomewide SNP data, we find multiple genomic islands with moderate differentiation spread across several chromosomes, whereas the rest of the genome is undifferentiated. The islands contain potential candidate genes involved in visual perception of colour. Our results suggest that phenotypic and multichromosome genomic divergence of these morphs was driven by environment-dependent sexual selection, demonstrating incipient speciation after only a few decades of divergence in sympatry.

Hybrid 'superswarm' leads to rapid divergence and establishment of populations during a biological invasion.

Understanding the genetic background of invading species can be crucial information clarifying why they become invasive. Intraspecific genetic admixture among lineages separated in the native ranges may promote the rate and extent of an invasion by substantially increasing standing genetic variation. Here, we examined the genetic relationships among threespine stickleback that recently colonized Switzerland. This invasion results from several distinct genetic lineages that colonized multiple locations and have since undergone range expansions, where they coexist and admix in parts of their range. Using 17 microsatellites genotyped for 634 individuals collected from 17 Swiss and two non-Swiss European sites, we reconstruct the invasion of stickleback and investigate the potential and extent of admixture and hybridization among the colonizing lineages from a population genetic perspective. Specifically, we test for an increase in standing genetic variation in populations where multiple lineages coexist. We find strong evidence of massive hybridization early on, followed by what appears to be recent increased genetic isolation and the formation of several new genetically distinguishable populations, consistent with a hybrid 'superswarm'. This massive hybridization and population formation event(s) occurred over approximately 140 years and likely fuelled the successful invasion of a diverse range of habitats. The implications are that multiple colonizations coupled with hybridization can lead to the formation of new stable genetic populations potentially kick-starting speciation and adaptive radiation over a very short timescale.

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.

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.

When phenotypes do not match genotypes--unexpected phenotypic diversity and potential environmental constraints in Icelandic stickleback.

Divergent lateral plate phenotypes in stickleback represent one of only a few cases known, where a single gene underlies the phenotype under divergent selection between different habitats. However, the selection pressures leading to the repeated loss of lateral plates in freshwater are still not well understood. By genotyping 838 individuals from 9 independently colonized lakes and 1 marine population in Iceland, we found 1) that only in some lakes are phenotypes associated with the expected genotype and 2) that the independent repeated occurrence of a rarely described plate phenotype is expressed in the absence of an allele that is usually associated with this phenotype. This suggests that either other genes such as modifiers might be under divergent selection between lakes or that lateral plate expression in these populations is restricted due to environmental constraints.