Long-Term Balancing Selection and the Genetic Load Linked to the Self-Incompatibility Locus in Arabidopsis halleri and A. lyrata.

Balancing selection is a form of natural selection maintaining diversity at the sites it targets and at linked nucleotide sites. Due to selection favoring heterozygosity, it has the potential to facilitate the accumulation of a "sheltered" load of tightly linked recessive deleterious mutations. However, precisely evaluating the extent of these effects has remained challenging. Taking advantage of plant self-incompatibility as one of the best-understood examples of long-term balancing selection, we provide a highly resolved picture of the genomic extent of balancing selection on the sheltered genetic load. We used targeted genome resequencing to reveal polymorphism of the genomic region flanking the self-incompatibility locus in three sample sets in each of the two closely related plant species Arabidopsis halleri and Arabidopsis lyrata, and used 100 control regions from throughout the genome to factor out differences in demographic histories and/or sample structure. Nucleotide polymorphism increased strongly around the S-locus in all sample sets, but only over a limited genomic region, as it became indistinguishable from the genomic background beyond the first 25-30 kb. Genes in this chromosomal interval exhibited no excess of mutations at 0-fold degenerated sites relative to putatively neutral sites, hence revealing no detectable weakening of the efficacy of purifying selection even for these most tightly linked genes. Overall, our results are consistent with the predictions of a narrow genomic influence of linkage to the S-locus and clarify how natural selection in one genomic region affects the evolution of the adjacent genomic regions.

Transition to Self-compatibility Associated With Dominant S-allele in a Diploid Siberian Progenitor of Allotetraploid Arabidopsis kamchatica Revealed by Arabidopsis lyrata Genomes.

A transition to selfing can be beneficial when mating partners are scarce, for example, due to ploidy changes or at species range edges. Here, we explain how self-compatibility evolved in diploid Siberian Arabidopsis lyrata, and how it contributed to the establishment of allotetraploid Arabidopsis kamchatica. First, we provide chromosome-level genome assemblies for two self-fertilizing diploid A. lyrata accessions, one from North America and one from Siberia, including a fully assembled S-locus for the latter. We then propose a sequence of events leading to the loss of self-incompatibility in Siberian A. lyrata, date this independent transition to ∼90 Kya, and infer evolutionary relationships between Siberian and North American A. lyrata, showing an independent transition to selfing in Siberia. Finally, we provide evidence that this selfing Siberian A. lyrata lineage contributed to the formation of the allotetraploid A. kamchatica and propose that the selfing of the latter is mediated by the loss-of-function mutation in a dominant S-allele inherited from A. lyrata.

Rapid Genetic Isolation Among Four Lineages Of Silene nutans.

Speciation is the process leading to the emergence of new species. While being usually progressive, it can sometimes be fast with rapid emergence of reproductive barriers leading to high level of reproductive isolation. Some reproductive barriers might leave signatures in the genome, through elevated level of genetic differentiation at specific loci. Similar signatures might also be the results of linked selection acting in low recombination regions. Nottingham catchfly (Silene nutans) is a Caryophyllaceae species composed of four genetically differentiated lineages for which strong and asymmetric levels of reproductive isolation have been identified. Using population transcriptomic data from several individuals of the four lineages, we inferred the best evo-demographic scenario leading to the current reproductive isolation of these four lineages. We also tested whether loci exhibiting high level of genetic differentiation represented barrier loci or were located in low recombination regions, evolving under strong influence of linked selection. Overall, the four lineages of S. nutans have diverged in strict isolation, likely during the different glacial period, through migration in distinct glacial refugia. Speciation between these four lineages appeared to be particularly fast, likely due to fast evolving plastid genome accelerating plastid-nuclear co-evolution and the probability of plastid-nuclear incompatibilities in inter-lineage hybrids.

Maintenance of Adaptive Dynamics and No Detectable Load in a Range-Edge Outcrossing Plant Population.

During range expansion, edge populations are expected to face increased genetic drift, which in turn can alter and potentially compromise adaptive dynamics, preventing the removal of deleterious mutations and slowing down adaptation. Here, we contrast populations of the European subspecies Arabidopsis lyrata ssp. petraea, which expanded its Northern range after the last glaciation. We document a sharp decline in effective population size in the range-edge population and observe that nonsynonymous variants segregate at higher frequencies. We detect a 4.9% excess of derived nonsynonymous variants per individual in the range-edge population, suggesting an increase of the genomic burden of deleterious mutations. Inference of the fitness effects of mutations and modeling of allele frequencies under the explicit demographic history of each population predicts a depletion of rare deleterious variants in the range-edge population, but an enrichment for fixed ones, consistent with the bottleneck effect. However, the demographic history of the range-edge population predicts a small net decrease in per-individual fitness. Consistent with this prediction, the range-edge population is not impaired in its growth and survival measured in a common garden experiment. We further observe that the allelic diversity at the self-incompatibility locus, which ensures strict outcrossing and evolves under negative frequency-dependent selection, has remained unchanged. Genomic footprints indicative of selective sweeps are broader in the Northern population but not less frequent. We conclude that the outcrossing species A. lyrata ssp. petraea shows a strong resilience to the effect of range expansion.

Genotyping and De Novo Discovery of Allelic Variants at the Brassicaceae Self-Incompatibility Locus from Short-Read Sequencing Data.

Plant self-incompatibility (SI) is a genetic system that prevents selfing and enforces outcrossing. Because of strong balancing selection, the genes encoding SI are predicted to maintain extraordinarily high levels of polymorphism, both in terms of the number of functionally distinct S-alleles that segregate in SI species and in terms of their nucleotide sequence divergence. However, because of these two combined features, documenting polymorphism of these genes also presents important methodological challenges that have so far largely prevented the comprehensive analysis of complete allelic series in natural populations, and also precluded the obtention of complete genic sequences for many S-alleles. Here, we develop a powerful methodological approach based on a computationally optimized comparison of short Illumina sequencing reads from genomic DNA to a database of known nucleotide sequences of the extracellular domain of SRK (eSRK). By examining mapping patterns along the reference sequences, we obtain highly reliable predictions of S-genotypes from individuals collected from natural populations of Arabidopsis halleri. Furthermore, using a de novo assembly approach of the filtered short reads, we obtain full-length sequences of eSRK even when the initial sequence in the database was only partial, and we discover putative new SRK alleles that were not initially present in the database. When including those new alleles in the reference database, we were able to resolve the complete diploid SI genotypes of all individuals. Beyond the specific case of Brassicaceae S-alleles, our approach can be readily applied to other polymorphic loci, given reference allelic sequences are available.

Whole-genome sequencing and genome regions of special interest: Lessons from major histocompatibility complex, sex determination, and plant self-incompatibility.

Whole-genome sequencing of non-model organisms is now widely accessible and has allowed a range of questions in the field of molecular ecology to be investigated with greater power. However, some genomic regions that are of high biological interest remain problematic for assembly and data-handling. Three such regions are the major histocompatibility complex (MHC), sex-determining regions (SDRs) and the plant self-incompatibility locus (S-locus). Using these as examples, we illustrate the challenges of both assembling and resequencing these highly polymorphic regions and how bioinformatic and technological developments are enabling new approaches to their study. Mapping short-read sequences against multiple alternative references improves genotyping comprehensiveness at the S-locus thereby contributing to more accurate assessments of allelic frequencies. Long-read sequencing, producing reads of several tens to hundreds of kilobase pairs in length, facilitates the assembly of such regions as single sequences can span the multiple duplicated gene copies of the MHC region, and sequence through repetitive stretches and translocations in SDRs and S-locus haplotypes. These advances are adding value to short-read genome resequencing approaches by allowing, for example, more accurate haplotype phasing across longer regions. Finally, we assessed further technical improvements, such as nanopore adaptive sequencing and bioinformatic tools using pangenomes, which have the potential to further expand our knowledge of a number of genomic regions that remain challenging to study with classical resequencing approaches.

Adaptive divergence in shell morphology in an ongoing gastropod radiation from Lake Malawi.

BACKGROUND: Ecological speciation is a prominent mechanism of diversification but in many evolutionary radiations, particularly in invertebrates, it remains unclear whether supposedly critical ecological traits drove or facilitated diversification. As a result, we lack accurate knowledge on the drivers of diversification for most evolutionary radiations along the tree of life. Freshwater mollusks present an enigmatic example: Putatively adaptive radiations are being described in various families, typically from long-lived lakes, whereas other taxa represent celebrated model systems in the study of ecophenotypic plasticity. Here we examine determinants of shell-shape variation in three nominal species of an ongoing ampullariid radiation in the Malawi Basin (Lanistes nyassanus, L. solidus and Lanistes sp. (ovum-like)) with a common garden experiment and semi-landmark morphometrics. RESULTS: We found significant differences in survival and fecundity among these species in contrasting habitats. Morphological differences observed in the wild persisted in our experiments for L. nyassanus versus L. solidus and L. sp. (ovum-like), but differences between L. solidus and L. sp. (ovum-like) disappeared and re-emerged in the F1 and F2 generations, respectively. These results indicate that plasticity occurred, but that it is not solely responsible for the observed differences. Our experiments provide the first unambiguous evidence for genetic divergence in shell morphology in an ongoing freshwater gastropod radiation in association with marked fitness differences among species under controlled habitat conditions. CONCLUSIONS: Our results indicate that differences in shell morphology among Lanistes species occupying different habitats have an adaptive value. These results also facilitate an accurate reinterpretation of morphological variation in fossil Lanistes radiations, and thus macroevolutionary dynamics. Finally, our work testifies that the shells of freshwater gastropods may retain signatures of adaptation at low taxonomic levels, beyond representing an evolutionary novelty responsible for much of the diversity and disparity in mollusks altogether.

Diversification dynamics of freshwater bivalves (Unionidae: Parreysiinae: Coelaturini) indicate historic hydrographic connections throughout the East African Rift System.

Invertebrates are exceptionally diverse, but many are in decline because of anthropogenic changes to their habitat. This situation is particularly problematic for taxa that are not well monitored or taxonomically poorly understood, because the lack of knowledge hampers conservation. Despite their important functional role in freshwater ecosystems, African bivalves of the family Unionidae remain poorly studied compared to their highly threatened relatives in Europe, the U.S.A. and Canada. To resolve relationships and to study diversification dynamics in space and time, we performed time-calibrated phylogenetic studies and biogeographical modeling on the unionids from the East African Rift System and surroundings, including representatives of all currently recognized Afrotropical genera except for Brazzaea (and Unio from southern Africa). Our analyses indicate that all sampled Afrotropical unionids belong to the tribe Coelaturini (subfamily Parreysiinae), as does the genus Moncetia from Lake Tanganyika, which is currently attributed to the family Iridinidae. Colonization of Africa from Eurasia by Parreysiinae occurred ~17 Ma ago, and the subsequent diversification of Coelaturini in Africa continued at a steady pace, although net diversification decreased over time as more niches and ecoregions became occupied. Clades in Coelaturini largely reflect drainage basins, with the oldest lineages and highest regional diversity occurring in Lake Tanganyika, followed by the Congo Basin watershed in general. The species assemblage of Lake Tanganyika reflects multiple independent events of colonization and intralacustrine diversification since the Late Miocene or Early Pliocene. The clades of other regions, including that containing the species from Lake Malawi, are comparatively young. Biogeographical analyses indicate that the colonization history was mainly driven by cladogenesis in sympatry, whereas few anagenetic events contributed to the modern distribution of Coelaturini. Ancestral range estimations demonstrate that Coelaturini originated in the Victoria and/or Tanganyika ecoregions, and that the Congo Basin played an essential role in the colonization of Africa by Coelaturini.

Patterns of Polymorphism at the Self-Incompatibility Locus in 1,083 Arabidopsis thaliana Genomes.

Although the transition to selfing in the model plant Arabidopsis thaliana involved the loss of the self-incompatibility (SI) system, it clearly did not occur due to the fixation of a single inactivating mutation at the locus determining the specificities of SI (the S-locus). At least three groups of divergent haplotypes (haplogroups), corresponding to ancient functional S-alleles, have been maintained at this locus, and extensive functional studies have shown that all three carry distinct inactivating mutations. However, the historical process of loss of SI is not well understood, in particular its relation with the last glaciation. Here, we took advantage of recently published genomic resequencing data in 1,083 Arabidopsis thaliana accessions that we combined with BAC sequencing to obtain polymorphism information for the whole S-locus region at a species-wide scale. The accessions differed by several major rearrangements including large deletions and interhaplogroup recombinations, forming a set of haplogroups that are widely distributed throughout the native range and largely overlap geographically. "Relict" A. thaliana accessions that directly derive from glacial refugia are polymorphic at the S-locus, suggesting that the three haplogroups were already present when glacial refugia from the last Ice Age became isolated. Interhaplogroup recombinant haplotypes were highly frequent, and detailed analysis of recombination breakpoints suggested multiple independent origins. These findings suggest that the complete loss of SI in A. thaliana involved independent self-compatible mutants that arose prior to the last Ice Age, and experienced further rearrangements during postglacial colonization.

The unusual S locus of Leavenworthia is composed of two sets of paralogous loci.

The Leavenworthia self-incompatibility locus (S locus) consists of paralogs (Lal2, SCRL) of the canonical Brassicaceae S locus genes (SRK, SCR), and is situated in a genomic position that differs from the ancestral one in the Brassicaceae. Unexpectedly, in a small number of Leavenworthia alabamica plants examined, sequences closely resembling exon 1 of SRK have been found, but the function of these has remained unclear. BAC cloning and expression analyses were employed to characterize these SRK-like sequences. An SRK-positive Bacterial Artificial Chromosome clone was found to contain complete SRK and SCR sequences located close by one another in the derived genomic position of the Leavenworthia S locus, and in place of the more typical Lal2 and SCRL sequences. These sequences are expressed in stigmas and anthers, respectively, and crossing data show that the SRK/SCR haplotype is functional in self-incompatibility. Population surveys indicate that < 5% of Leavenworthia S loci possess such alleles. An ancestral translocation or recombination event involving SRK/SCR and Lal2/SCRL likely occurred, together with neofunctionalization of Lal2/SCRL, and both haplotype groups now function as Leavenworthia S locus alleles. These findings suggest that S locus alleles can have distinctly different evolutionary origins.

Secondary evolution of a self-incompatibility locus in the Brassicaceae genus Leavenworthia.

Self-incompatibility (SI) is the flowering plant reproductive system in which self pollen tube growth is inhibited, thereby preventing self-fertilization. SI has evolved independently in several different flowering plant lineages. In all Brassicaceae species in which the molecular basis of SI has been investigated in detail, the product of the S-locus receptor kinase (SRK) gene functions as receptor in the initial step of the self pollen-rejection pathway, while that of the S-locus cysteine-rich (SCR) gene functions as ligand. Here we examine the hypothesis that the S locus in the Brassicaceae genus Leavenworthia is paralogous with the S locus previously characterized in other members of the family. We also test the hypothesis that self-compatibility in this group is based on disruption of the pollen ligand-producing gene. Sequence analysis of the S-locus genes in Leavenworthia, phylogeny of S alleles, gene expression patterns, and comparative genomics analyses provide support for both hypotheses. Of special interest are two genes located in a non-S locus genomic region of Arabidopsis lyrata that exhibit domain structures, sequences, and phylogenetic histories similar to those of the S-locus genes in Leavenworthia, and that also share synteny with these genes. These A. lyrata genes resemble those comprising the A. lyrata S locus, but they do not function in self-recognition. Moreover, they appear to belong to a lineage that diverged from the ancestral Brassicaceae S-locus genes before allelic diversification at the S locus. We hypothesize that there has been neo-functionalization of these S-locus-like genes in the Leavenworthia lineage, resulting in evolution of a separate ligand-receptor system of SI. Our results also provide support for theoretical models that predict that the least constrained pathway to the evolution of self-compatibility is one involving loss of pollen gene function.

Contrasted patterns of molecular evolution in dominant and recessive self-incompatibility haplotypes in Arabidopsis.

Self-incompatibility has been considered by geneticists a model system for reproductive biology and balancing selection, but our understanding of the genetic basis and evolution of this molecular lock-and-key system has remained limited by the extreme level of sequence divergence among haplotypes, resulting in a lack of appropriate genomic sequences. In this study, we report and analyze the full sequence of eleven distinct haplotypes of the self-incompatibility locus (S-locus) in two closely related Arabidopsis species, obtained from individual BAC libraries. We use this extensive dataset to highlight sharply contrasted patterns of molecular evolution of each of the two genes controlling self-incompatibility themselves, as well as of the genomic region surrounding them. We find strong collinearity of the flanking regions among haplotypes on each side of the S-locus together with high levels of sequence similarity. In contrast, the S-locus region itself shows spectacularly deep gene genealogies, high variability in size and gene organization, as well as complete absence of sequence similarity in intergenic sequences and striking accumulation of transposable elements. Of particular interest, we demonstrate that dominant and recessive S-haplotypes experience sharply contrasted patterns of molecular evolution. Indeed, dominant haplotypes exhibit larger size and a much higher density of transposable elements, being matched only by that in the centromere. Overall, these properties highlight that the S-locus presents many striking similarities with other regions involved in the determination of mating-types, such as sex chromosomes in animals or in plants, or the mating-type locus in fungi and green algae.

Recent and ancient signature of balancing selection around the S-locus in Arabidopsis halleri and A. lyrata.

Balancing selection can maintain different alleles over long evolutionary times. Beyond this direct effect on the molecular targets of selection, balancing selection is also expected to increase neutral polymorphism in linked genome regions, in inverse proportion to their genetic map distances from the selected sites. The genes controlling plant self-incompatibility are subject to one of the strongest forms of balancing selection, and they show clear signatures of balancing selection. The genome region containing those genes (the S-locus) is generally described as nonrecombining, and the physical size of the region with low recombination has recently been established in a few species. However, the size of the region showing the indirect footprints of selection due to linkage to the S-locus is only roughly known. Here, we improved estimates of this region by surveying synonymous polymorphism and estimating recombination rates at 12 flanking region loci at known physical distances from the S-locus region boundary, in two closely related self-incompatible plants Arabidopsis halleri and A. lyrata. In addition to studying more loci than previous studies and using known physical distances, we simulated an explicit demographic scenario for the divergence between the two species, to evaluate the extent of the genomic region whose diversity departs significantly from neutral expectations. At the closest flanking loci, we detected signatures of both recent and ancient indirect effects of selection on the S-locus flanking genes, finding ancestral polymorphisms shared by both species, as well as an excess of derived mutations private to either species. However, these effects are detected only in a physically small region, suggesting that recombination in the flanking regions is sufficient to quickly break up linkage disequilibrium with the S-locus. Our approach may be useful for distinguishing cases of ancient versus recently evolved balancing selection in other systems.

Trait transitions in explicit ecological and genomic contexts: plant mating systems as case studies.

Plants are astonishingly diverse in how they reproduce sexually, and the study of plant mating systems provides some of the most compelling cases of parallel and independent evolutionary transitions. In this chapter, we review how the massive amount of genomic data being produced is allowing long-standing predictions from ecological and evolutionary theory to be put to test. After a review of theoretical predictions about the importance of considering the genomic architecture of the mating system, we focus on a set of recent discoveries on how the mating system is controlled in a variety of model and non-model species. In parallel, genomic approaches have revealed the complex interaction between the evolution of genes controlling mating systems and genome evolution, both genome-wide and in the mating system control region. In several cases, major transitions in the mating system can be clearly associated with important ecological changes, hence illuminating an important interplay between ecological and genomic approaches. We also list a number of major unsolved questions that remain for the field, and highlight foreseeable conceptual developments that are likely to play a major role in our understanding of how plant mating systems evolve in Nature.

Dominance between self-incompatibility alleles determines the mating system of Capsella allopolyploids.

The shift from outcrossing to self-fertilization is one of the main evolutionary transitions in plants and has broad effects on evolutionary trajectories. In Brassicaceae, the ability to inhibit self-fertilization is controlled by 2 genes, SCR and SRK, tightly linked within the S-locus. A series of small non-coding RNAs also encoded within the S-locus regulates the transcriptional activity of SCR alleles, resulting in a linear dominance hierarchy between them. In Brassicaceae, natural allopolyploid species are often self-compatible (SC) even when one of the progenitor species is self-incompatible, but the reason why polyploid lineages tend to lose self-incompatibility (SI) and the timing of the loss of SI (immediately after ancestral hybridization between the progenitor species, or at a later stage after the formation of allopolyploid lineages) have generally remained elusive. We used a series of synthetic diploid and tetraploid hybrids obtained between self-fertilizing Capsella orientalis and outcrossing Capsella grandiflora to test whether the breakdown of SI could be observed immediately after hybridization, and whether the occurrence of SC phenotypes could be explained by the dominance interactions between S-haplotypes inherited from the parental lineages. We used RNA-sequencing data from young inflorescences to measure allele-specific expression of the SCR gene and infer dominance interactions in the synthetic hybrids. We then evaluated the seed set from autonomous self-pollination in the synthetic hybrids. Our results demonstrate that self-compatibility of the hybrids depends on the relative dominance between S-alleles inherited from the parental species, confirming that SI can be lost instantaneously upon formation of the ancestral allopolyploid lineage. They also confirm that the epigenetic regulation that controls dominance interactions between S-alleles can function between subgenomes in allopolyploids. Together, our results illustrate how a detailed knowledge of the mechanisms controlling SI can illuminate our understanding of the patterns of co-variation between the mating system and changes in ploidy.

The genetic architecture of the load linked to dominant and recessive self-incompatibility alleles in Arabidopsis halleri and Arabidopsis lyrata.

The long-term balancing selection acting on mating types or sex-determining genes is expected to lead to the accumulation of deleterious mutations in the tightly linked chromosomal segments that are locally 'sheltered' from purifying selection. However, the factors determining the extent of this accumulation are poorly understood. Here, we took advantage of variations in the intensity of balancing selection along a dominance hierarchy formed by alleles at the sporophytic self-incompatibility system of the Brassicaceae to compare the pace at which linked deleterious mutations accumulate among them. We first experimentally measured the phenotypic manifestation of the linked load at three different levels of the dominance hierarchy. We then sequenced and phased polymorphisms in the chromosomal regions linked to 126 distinct copies of S-alleles in two populations of Arabidopsis halleri and three populations of Arabidopsis lyrata. We find that linkage to the S-locus locally distorts phylogenies over about 10-30 kb along the chromosome. The more intense balancing selection on dominant S-alleles results in greater fixation of linked deleterious mutations, while recessive S-alleles accumulate more linked deleterious mutations that are segregating. Hence, the structure rather than the overall magnitude of the linked genetic load differs between dominant and recessive S-alleles. Our results have consequences for the long-term evolution of new S-alleles, the evolution of dominance modifiers between them, and raise the question of why the non-recombining regions of some sex and mating type chromosomes expand over evolutionary times while others, such as the S-locus of the Brassicaceae, remain restricted to small chromosomal regions.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

Inferring the Demographic History and Inheritance Mode of Tetraploid Species Using ABC.

Genomic patterns of diversity and divergence are impacted by certain life history traits, reproductive systems, and demographic history. The latter is characterized by fluctuations in population sizes over time, as well as by temporal patterns of introgression. For a given organism, identifying a demographic history that deviates from the standard neutral model allows a better understanding of its evolution but also helps to reduce the risk of false positives when screening for molecular targets of natural selection. Tetraploid organisms and beyond have demographic histories that are complicated by the mode of polyploidization, the mode of inheritance, and different scenarios of gene flow between sub-genomes and diploid parental species. Here we provide guidelines for experimenters wishing to address these issues through a flexible statistical framework: approximate Bayesian computation (ABC). The emphasis is on the general philosophy of the approach to encourage future users to exploit the enormous flexibility of ABC beyond the limitations imposed by generalist data analysis pipelines.

Target enrichment of long open reading frames and ultraconserved elements to link microevolution and macroevolution in non-model organisms.

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here, we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced substantial data sets. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites). Variant calling on ORFs and UCEs of Coelaturini from the Malawi Basin produced ~2000 SNPs per population pair. Estimates of nucleotide diversity and population differentiation were similar for ORFs and UCEs. They were low compared to previous estimates in molluscs, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming off-target sequence data from the same enriched libraries of Coelaturini from the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays the gene order inferred for the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for integrative genomic studies of microevolutionary and macroevolutionary dynamics in non-model organisms.