Abstract Background and Aims In the plant kingdom, gene flow occurs through pollen and seed dispersal, shaping both within-population spatial genetic structure and among-population genetic differentiation. Anthropogenic land-use change can affect levels of gene flow by reducing pollinator abundance and altering pollen and seed dispersal pathways. Yet, how environmental features shape gene flow events in common herbaceous plants – a fundamental building block of many ecosystems – remains poorly understood. We address this gap by investigating how environmental context influences population genetic structure at regional and local scales in the red campion ( Silene dioica ). Methods By sampling 1,005 individuals from 29 populations across habitats ranging from semi-natural to strongly human-altered, we assessed whether population size and landscape composition influenced within-population genetic diversity and population genetic differentiation. At the local scale, we examined whether landscape composition affected fine-scale spatial genetic structure and pollen dispersal distances in a subset of six populations representing the two extremes of an anthropogenic gradient. Key Results No effect of either demographic or landscape factors was found on levels of genetic diversity. We detected moderate levels of genetic differentiation among populations that matched an isolation-by-distance pattern, with high levels of population admixture, while landscape composition did not explain variation in population genetic differentiation. At the local scale, five of the six studied populations exhibited significant spatial genetic structuring, indicating distinct neighbourhoods at spatial scales less than 10 m. Paternity analyses based on 4,800 offspring further revealed predominantly short-distance pollen dispersal together with substantial immigration from external sources. Neither fine-scale genetic structure nor pollen dispersal distances differed between habitat types. Conclusions Altogether, our results demonstrate a multimodal pattern of gene flow and a remarkable resilience of this common herbaceous species to anthropogenic habitat change, as substantial genetic connectivity is maintained despite insect-mediated pollen dispersal and gravity-driven seed dispersal.

In most angiosperms, pollinators mediate access to sexual partners, making attractive floral traits key targets of sexual selection. Theory predicts stronger selection through the male function, due to greater dependence of male reproductive success on mate acquisition. However, habitat degradation may alter plant–pollinator interactions and disrupt sex-specific selection. In particular, reduced pollinator density may increase the dependency of female reproductive success on mate acquisition and strengthen selection on floral traits in both sexes. We tested this hypothesis in the dioecious species Silene dioica by measuring pollinator visitation, pollination service, and phenotypic selection on floral traits in both sexes across six populations located in either forest or anthropogenic habitats. Despite lower visitation in anthropogenic sites, pollination service was comparable between habitats with consistent selection on fertility-related traits in females. In contrast, we found stronger selection on a male trait likely promoting efficient pollen transfer in anthropogenic habitats, indicating a sex-specific response to land use change. Finally, male—but not female—reproductive success increased with the number of sexual partners in both habitats, supporting stronger sexual selection in males. More broadly, our results emphasize the need for a sex-specific perspective on floral trait evolution and demonstrate the persistence of male-biased sexual selection under environmental change.

Highway construction often leads to habitat fragmentation, altering the genetic features of wild populations. While compensatory measures are commonly implemented to mitigate their negative demographic and genetic impacts, their effectiveness remains species-dependent and difficult to assess. We analysed the short-term impact of a newly built highway in eastern France and the associated restoration measures on population genetic structure in the protected southern damselfly (Coenagrion mercuriale). We genotyped 46 populations collected after the highway construction using microsatellite and SNP markers over two successive years to detect potential genetic discontinuities across the highway. We also evaluated the effectiveness of watercourse restoration and identified the likely origin of recolonising individuals. We found no significant barrier effect of the highway construction on gene flow. However, these encouraging results should be interpreted with caution, considering the likely time-lag in populations’ responses to genetic drift effects. We observed a gradual recolonisation of restored watercourses that had undergone compensatory measures, suggesting positive effects of reproductive habitat recovery. Recolonisation involved nearby populations but also long-distance dispersal events occurring overland. Detected migrant individuals may come from geographically distant populations belonging to a different watercourse. This mirrors a migrant-pool model of colonisation that leaves high levels of genetic diversity and no evident spatial patterns in populations located close to the highway. Altogether, our study highlights both the potential and limitations of genetic tools to assess the impacts of linear infrastructure on gene flow and for understanding watercourse recolonisation processes after ecological management restoration.

Although attractive scents play a crucial role in reproduction in insect-pollinated plants, the degree of variation of this signal within and among populations remains understudied. Depending on the specifics of the reproductive system of the plant under scrutiny, it is possible to formulate predictions regarding this variation. In plants with separate sexes (dioecious species) and with highly specific pollination, one would predict (i) males to emit more scent than females, owing to sexual selection, (ii) scent bouquets to have a strictly similar composition between sexes, to guarantee efficient pollen transfer and (iii) variation of scent bouquet among populations that should mirror neutral genetic divergence. These hypotheses were tested in the European fan palm, Chamaerops humilis, by collecting scent in eight populations from three regions, Spain, Sardinia and Sicily, quantifying densities of pollinators-Derelomus sp. and Meligethinus pallidulus-and genotyping the plants on a set of neutral markers. Males emitted more scent than females. We detected some differences in bouquet composition between sexes, showing an imperfect inter-sex mimicry in some populations. We also found a strong geographical effect, with individuals sampled in Sicily emitting a strikingly different scent bouquet, which contained high proportions of a volatile compound that was never detected in the other two regions. Geographical variation of scent composition did not mirror neutral genetic structure: Sardinian and Spanish populations emitted similar scent bouquets but displayed very high levels of genetic differentiation. On the reverse, Sicilian populations showed both strong scent differences and appeared clearly genetically differentiated from populations found elsewhere, without any depletion in neutral genetic diversity. Synthesis: Our study confirmed higher scent emission rates in males compared to females, consistent with expectations of sexual selection. However, we also discovered significant variation in the composition of the floral bouquet, which was unexpected given the highly specific pollination context. Together with observations of spatial genetic structure, these findings suggest a shift in plant-pollinator interactions within the species across different regions.

Plant species with mixed pollination systems are under pollinator-mediated selection by both diurnal and nocturnal pollinator species. This could impact the strength and potentially direction of selection on floral traits, as different pollinators are not necessarily attracted by the same traits. In this study, we investigated how selection gradients on floral traits in Silene dioica were affected by (i) the pollinator community the plants were exposed to (diurnal versus nocturnal pollination) and (ii) the level of emission of a volatile organic compound typically linked to pollinator attraction (natural versus enhanced phenylacetylaldehyd (PAA) emission) in a fully crossed design. Female plants in all treatments achieved full seed set, suggesting no differences in pollination efficiency between diurnal and nocturnal pollinator communities in S. dioica. Nocturnal pollination resulted in stronger selection on corolla width and flower number in males, but not in females. We further found that increased PAA emission modified selection on attractive traits both in plants exposed to diurnal and nocturnal pollinators, with a stronger effect in males. This stronger response of selection patterns to pollinator community and scent emission could suggest that males are more dependent on pollinator attraction than females in their reproductive success.

Genomic markers are essential tools for studying species of conservation concern, yet nonmodel species often lack a reference genome. Here we describe a methodology for identifying and genotyping thousands of SNP loci in the southern damselfly (Coenagrion mercuriale), a bioindicator of freshwater stream quality classified as near-threatened, with locally declining populations. We used a hybrid approach combining reduced representation sequencing and target enrichment. First, we identified putative SNP loci using ddRADseq and de novo assembly. Then, single primer enrichment technology targeted 6000 of these SNPs across 1920 individuals. Challenges encountered included sequence recapture failure, coverage depth discrepancies, and aberrant FIS values. We provide recommendations to address such issues. After multiple filtering steps, 2092 SNPs were retained and used to analyze the genetic structure of 131 individuals belonging to 11 populations in France, comparing central and marginal populations. Genetic differentiation was lower among central populations, with no sign of inbreeding. As compared with microsatellite loci, SNPs exhibited greater resolution in detecting fine-scaled genetic structure, and identifying putative hybrids in adjacent populations. In this study, we emphasize the difficulties of large-scale SNP genotyping in nonmodel species via a hybrid method that ultimately did not offer the expected cost and time-saving compared with classical ddRAD approaches. However, SNPs showed greater power than previously available markers in identifying conservation units or admixture events, and the panel of reusable probes we describe here offers the potential to improve conservation efforts through future diachronic studies or finer estimations of key parameters like effective population size.

Premise of the study Paternal diversity and its distribution within maternal plants is governed by various factors, including frequency of pollinator visits, patterns of pollinator movement and pollen carryover. Floral display size may have opposite effects on fruit and plant-level paternal diversity, because plants with large displays should receive more pollinator visits but also more sequential probes per visit. Methods To investigate the effect of floral display size on the distribution of paternal diversity in Silene dioica , we genotyped and assigned paternity for 1320 seeds sampled in several fruits per plant in control and manipulated females, whose flower number was artificially increased. We estimated within-fruit paternal diversity and sire profile dissimilarity among fruits and compared these metrics between control and manipulated plants. We then studied pollen carryover using controlled pollinator visits and pollen counts. Key results Most fruits were multiply sired, but we found no effect of flower supplementation on within-fruit paternal diversity. Sire profiles were more similar between fruits located on the same than on different females, but sire profile similarity was not higher within manipulated than within control plants. The pollen deposition curve was steep, with an increasing carryover fraction throughout the visitation sequence. Conclusions Our results suggest that the effect of floral display size on pollinator foraging behaviour is too weak to affect paternal diversity and its distribution in S. dioica , or that other processes, such as limited pollen carryover and spatially restricted pollen dispersal, blur the effect of pollinator visit frequency and movements on sire profiles. ### Competing Interest Statement The authors have declared no competing interest. Data will be made available on Dryad (). Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-JCJC- EvoPoD (ANR-21-CE02-0011)

Genomic markers are essential tools for studying species of conservation concern, yet non-model species often lack a genome reference. Here we describe a methodology for identifying and genotyping thousands of SNP loci in the southern damselfly ( Coenagrion mercuriale ), a bioindicator of freshwater stream quality classified as near-threatened, with locally declining populations. We used a hybrid approach combining reduced representation sequencing and target enrichment. First, we identified putative SNP loci using ddRADseq and de novo assembly. Then, single primer enrichment technology targeted 6,000 of these SNPs across 1,920 individuals. Challenges encountered included sequence recapture failure, coverage depth discrepancies, and aberrant F IS values. We provide recommendations to address such issues. After multiple filtering, we retained 2,092 SNPs. We used them to characterise rear-edge populations of the southern damselfly in Northern France, a region where populations are sparsely distributed. Previous surveys utilising microsatellite markers allowed comparison of genetic diversity and differentiation estimates. Consistent with prior findings, genetic diversity estimates were similar across the studied populations that showed no sign of inbreeding. SNP markers exhibited greater resolution in detecting fine-scaled genetic structure, identifying two putative hybrids in adjacent populations, a feat unattainable with microsatellite loci. Altogether, this study highlighted the ongoing challenge of large-scale SNP genotyping using target sequencing techniques in non-model species to set conservation guidelines. Nonetheless, these new markers showed greater statistical power in identifying conservation units and offered the promise of greater precision in the identification of admixture events or the estimation of key population parameters such as effective population size. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND AND AIMS:Organelle genomes are usually maternally inherited in angiosperms. However, biparental inheritance has been observed, especially in hybrids resulting from crosses between divergent genetic lineages. When it concerns the plastid genome, this exceptional mode of inheritance might rescue inter-lineage hybrids suffering from plastid-nuclear incompatibilities. Genetically differentiated lineages of Silene nutans exhibit strong postzygotic isolation owing to plastid-nuclear incompatibilities, highlighted by inter-lineage hybrid chlorosis and mortality. Surviving hybrids can exhibit variegated leaves, which might indicate paternal leakage of the plastid genome. We tested whether the surviving hybrids inherited the paternal plastid genome and survived thanks to paternal leakage. METHODS:We characterized the leaf phenotype (fully green, variegated or white) of 504 surviving inter-lineage hybrids obtained from a reciprocal cross experiment among populations of four genetic lineages (W1, W2, W3 and E1) of S. nutans from Western Europe and genotyped 560 leaf samples (both green and white leaves for variegated hybrids) using six lineage-specific plastid single nucleotide polymorphisms. KEY RESULTS:A high proportion of the surviving hybrids (≤98 %) inherited the paternal plastid genome, indicating paternal leakage. The level of paternal leakage depended on cross type and cross direction. The E1 and W2 lineages as maternal lineages led to the highest hybrid mortality and to the highest paternal leakage from W1 and W3 lineages in the few surviving hybrids. This was consistent with E1 and W2 lineages, which contained the most divergent plastid genomes. When W3 was the mother, more hybrids survived, and no paternal leakage was detected. CONCLUSIONS:By providing a plastid genome potentially more compatible with the hybrid nuclear background, paternal leakage has the potential to rescue inter-lineage hybrids from plastid-nuclear incompatibilities. This phenomenon might slow down the speciation process, provided hybrid survival and reproduction can occur in the wild.

The effective population size (N e) is a key parameter in conservation and evolutionary biology, reflecting the strength of genetic drift and inbreeding. Although demographic estimations of N e are logistically and time-consuming, genetic methods have become more widely used due to increasing data availability. Nonetheless, accurately estimating N e remains challenging, with few studies comparing N e estimates across molecular markers types and estimators such as single-sample methods based on linkage disequilibrium or sibship analyses versus methods based on temporal variance in allele frequencies. This study aims at bridging this gap by analysing single-sample and temporally spaced populations in the southern damselfly (Coenagrion mercuriale), a bioindicator Odonata species of conservation concern found in southwestern Europe's freshwater stream networks. A total of 77 local populations were sampled from a semi-urbanised area located in eastern France near Strasbourg city, yielding 2842 individuals that were genotyped with microsatellites and 958 of which were also genotyped for 2092 SNPs. Spatial genetic structure was stable over time, suggesting porosity between alternate-year cohorts. When accounting for spatial genetic structure, single-sample and temporal estimations of N e were consistent for each set of molecular markers. Biologically meaningful results were obtained when the effect of migration was minimising by considering metapopulation N e estimates based on the level of genetic differentiation and population boundaries. In terms of applied conservation and management, most depicted metapopulations displayed large N e, indicating no immediate need for conservation measures to mitigate anthropogenic pressures, provided that a continuous suitable freshwater network is maintained. However, urbanisation negatively impacted N e levels in populations close to Strasbourg city. Because N e is used to inform conservation decisions, caution is crucial in interpreting N e estimates, especially in continuously distributed populations undergoing migration. Altogether, our study highlights the challenge of obtaining robust N e estimates and the necessity of careful interpretation to set relevant conservation guidelines.

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.

Aim: Human-induced environmental changes result in habitat loss and fragmentation, impacting wildlife population genetic structure and evolution. Urbanised and geographically peripheral areas often represent unfavourable environments, reducing connectivity among populations and causing higher population genetic differentiation and lower intra-population genetic diversity. We examined how geographic peripherality and anthropogenic pressures affect genetic diversity and genetic differentiation in the protected southern damselfly (Coenagrion mercuriale, Odonata), which has low dispersal capabilities and specific habitat requirements and whose populations are declining. Location: We studied two areas: one in semi-natural habitats at the periphery of the species geographic range (northern France) and the other more central to the species' range, in an urbanised area surrounding the city of Strasbourg (Alsace, eastern France). Methods: We genotyped 2743 individuals from 128 populations using 11 microsatellite loci. We analysed the spatial distribution of neutral genetic diversity (allelic richness, heterozygosity, levels of inbreeding and genetic relatedness), the extent of genetic differentiation and population affiliations (sPCAs) within the two areas. We also examined fine-scale patterns of gene flow in the urbanised area of Alsace by investigating patterns of isolation by distance and estimating effective migration surfaces (EEMS) method. Results: Northern peripheral populations showed lower levels of genetic diversity and higher levels of genetic differentiation than central Alsacian populations. Although located in anthropised habitats, geographically central Alsacian populations showed high levels of gene flow, with dispersal events mainly occurring overland and not restricted to watercourses. However, the highly urbanised city of Strasbourg negatively impacted nearby populations by reducing levels of genetic diversity and increasing population genetic differentiation. Main Conclusions: These results showed the need for management action by restoring breeding sites and creating migratory corridors for peripheral southern damselfly populations. However, our results also highlighted the resilience of southern damselfly in central range populations facing strong urbanisation pressures.

Abstract Helichrysum arenarium (L.) Moench (Asteraceae) is a self‐compatible, insect‐pollinated herb occurring in sand grasslands, and is declining and endangered in many parts of its European distribution range. A recovery plan of H. arenarium has been conducted in southern Belgium, involving plant translocations. We developed multiplex genotyping protocol for nine microsatellite markers previously published for Helichrysum italicum and two newly developed microsatellite markers for H. arenarium. Eleven polymorphic loci were associated (pooled) in two multiplex panels, to assess the genetic status of the only small remaining population in Belgium and of three large German populations used as seed source for propagating transplants. The small Belgian population was characterized by high clonality, with only two, however heterozygous, genets detected. The three large German populations showed high genetic diversity (He ranging from 0.635 to 0.670) and no significant inbreeding coefficient values, despite expectations of geitonogamous selfing. Management practices (grazing livestock) increasing seed dispersal distances, inbreeding depression at early stages of development, and mechanisms preventing or delaying selfing might be hypothesized to explain the observed patterns. The two Belgian genotypes remained within genetic variation range of German populations so that the high genetic differentiation between Belgian and German populations (FST values ranging from 0.186 to 0.206) likely resulted from genetic drift effects and small sample size. Transplants obtained from seeds sampled from the three large source populations from Germany constitute a highly diverse, noninbred gene pool, and are thus of high genetic quality for plant translocations.

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.

There is growing evidence that cytonuclear incompatibilities (i.e. disruption of cytonuclear coadaptation) might contribute to the speciation process. In a former study, we described the possible involvement of plastid-nuclear incompatibilities in the reproductive isolation between four lineages of Silene nutans (Caryophyllaceae). Because organellar genomes are usually cotransmitted, we assessed whether the mitochondrial genome could also be involved in the speciation process, knowing that the gynodioecious breeding system of S. nutans is expected to impact the evolutionary dynamics of this genome. Using hybrid capture and high-throughput DNA sequencing, we analyzed diversity patterns in the genic content of the organellar genomes in the four S. nutans lineages. Contrary to the plastid genome, which exhibited a large number of fixed substitutions between lineages, extensive sharing of polymorphisms between lineages was found in the mitochondrial genome. In addition, numerous recombination-like events were detected in the mitochondrial genome, loosening the linkage disequilibrium between the organellar genomes and leading to decoupled evolution. These results suggest that gynodioecy shaped mitochondrial diversity through balancing selection, maintaining ancestral polymorphism and, thus, limiting the involvement of the mitochondrial genome in evolution of hybrid inviability between S. nutans lineages.

Plant translocations represent a solution for rescuing nonviable populations and recreating population networks. An essential criterion for translocation success is genetic diversity enhancement or maintenance in post-translocation generations. Genetic mixing, i.e. individual relatedness and spatial structure, also needs to be considered. We investigated the processes involved in the spatial patterns of genetic diversity in the first post-translocation generation (recruits and seed progeny) of translocated populations of the insect-pollinated, self-incompatible herb Campanula glomerata. We combined direct and indirect estimates of contemporary mating and gene dispersal using 15 microsatellite loci to estimate clonal extent, and genetic diversity and structure, to reconstruct sibship and parentage and to estimate pollen and seed dispersal distances. Genetic diversity in the first post-translocation generation is representative of the natural seed-source populations. Combining sources enhanced the effective number of breeders (Ne = 102-175). Clonal propagation was only marginal. Pollen and seed dispersal occurred at short distances within populations (up to 18 m), favouring fine-scale spatial genetic structure (SGS). This means that the enhanced genetic diversity, random mating and high admixture levels are ascribed to the mixed spatial arrangement of transplants. Stronger SGS might lead to biparental inbreeding and reduced seed set due to a lack of compatible neighbours, and impact the long-term population genetic sustainability. Increasing seed-dispersal distances within and among sites might be mediated by grazing livestock used for grassland management. Some long-distance pollen flow events (0.68-2.35 km) between populations were also detected, suggesting the potential of creating large translocated populations to restore connectivity by gene flow.

Assisted gene flow interventions such as plant translocations are valuable complementary techniques to habitat restoration. Bringing new genetic variants can contribute to increasing genetic diversity and evolutionary resilience, counteract inbreeding depression and improve plant fitness through heterosis. Large, highly genetically variable populations are usually recommended as sources for translocation. Unfortunately, many critically endangered species only occur as small populations, which are expected to show low genetic variation, high inbreeding level, paucity of compatible mates in self-incompatible species, and increased genetic divergence. Therefore, assessment of population genetic status is required for an appropriate choice of the source populations. In this paper, we exemplify the different analyses relevant for genetic evaluation of populations combining both molecular (plastid and nuclear) markers and fitness-related quantitative traits. We assessed the genetic status of the adult generation and their seed progeny (the potential translocation founders) of small populations of Campanula glomerata (Campanulaceae), a self-incompatible insect-pollinated herbaceous species critically endangered in Belgium. Only a few small populations remain, so that the species has been part of a restoration project of calcareous grasslands implementing plant translocations. In particular, we estimated genetic diversity, inbreeding levels, genetic structure in adults and their seed progeny, recent bottlenecks, clonal extent in adults, contemporary gene flow, effective population size ( N e ), and parentage, sibship and seed progeny fitness variation. Small populations of C. glomerata presented high genetic diversity, and extensive contemporary pollen flow within populations, with multiple parentage among seed progenies, and so could be good seed source candidates for translocations. As populations are differentiated from each other, mixing the sources will not only optimize the number of variants and of compatible mates in translocated populations, but also representativeness of species regional genetic diversity. Genetic diversity is no immediate threat to population persistence, but small N e , restricted among-population gene flow, and evidence of processes leading to genetic erosion, inbreeding and inbreeding depression in the seed progeny require management measures to counteract these trends and stochastic vulnerability. Habitat restoration facilitating recruitment, flowering and pollination, reconnecting populations by biological corridors or stepping stones, and creating new populations through translocations in protected areas are particularly recommended.

Early stages of speciation in plants might involve genetic incompatibilities between plastid and nuclear genomes, leading to inter-lineage hybrid breakdown due to the disruption between co-adapted plastid and nuclear genes encoding subunits of the same plastid protein complexes. We tested this hypothesis in Silene nutans, a gynodioecious Caryophyllaceae, where four distinct genetic lineages exhibited strong reproductive isolation among each other, resulting in chlorotic or variegated hybrids. By sequencing the whole gene content of the four plastomes through gene capture, and a large part of the nuclear genes encoding plastid subunits from RNAseq data, we searched for non-synonymous substitutions fixed in each lineage on both genomes. Lineages of S. nutans exhibited a high level of dN/dS ratios for plastid and nuclear genes encoding most plastid complexes, with a strong pattern of coevolution for genes encoding the subunits of ribosome and cytochrome b6/f that could explain the chlorosis of hybrids. Overall, relaxation of selection due to past bottlenecks and positive selection have driven the diversity pattern observed in S. nutans plastid complexes, leading to plastid-nuclear incompatibilities. We discuss the possible role of gynodioecy in the evolutionary dynamics of the plastomes through linked selection.

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.