Rosa Ana Sánchez-Guillén

Sexual and natural selection jointly contribute to the rapid evolution of mating-related traits, making hybridizing systems particularly informative for exploring how different classes of traits diverge during early speciation. The damselflies Ischnura elegans and Ischnura graellsii form two hybrid zones in Spain that differ in demographic structure and spatial genetic patterns, providing a natural framework to compare patterns of divergence in reproductive (male caudal appendages and the female prothorax) and non-reproductive (wings) traits. Using geometric morphometrics within a phylogenetic comparative framework, together with population genetic information, we compared evolutionary integration, rates of divergence, and evolutionary dynamics across traits and species. In I. elegans, both the shape and size of male caudal appendages and the female prothorax evolved in strong evolutionary correlation, whereas in I. graellsii this association was restricted to shape. Reproductive structures evolved at significantly higher rates than wings, indicating greater evolutionary lability in traits directly involved in mating. Evolutionary model comparisons revealed contrasting dynamics between shape and size, suggesting that multiple processes contribute to trait divergence rather than a single dominant mechanism. Estimates of isolation by distance and gene flow revealed contrasting demographic contexts between species, with I. elegans showing more restricted gene flow than I. graellsii. Overall, our findings suggest that reproductive and non-reproductive traits differ in their evolutionary integration, rates, and dynamics, highlighting how trait-specific evolutionary patterns and demographic context contribute to morphological divergence in recently diverged taxa.

Mechanical isolation is often viewed as a discrete species-level premating barrier arising from divergence in reproductive morphology. However, whether such isolation reflects discrete species-level incompatibility or emerges from continuous variation in morphological matching among interacting individuals remains unclear. We investigated how reinforcement and sexual selection shape morphological compatibility between male grasping appendages and the corresponding female contact structures in the damselflies Ischnura elegans and Ischnura graellsii, and how this compatibility constrains heterospecific tandem formation. Using geometric morphometrics across allopatric and hybrid-zone populations, we quantified sex-specific evolutionary trajectories of reproductive traits. Females of I. elegans exhibited greater phenotypic change consistent with asymmetric reproductive character displacement. Female trajectories converged in morphospace during late secondary contact, whereas male trajectories showed weaker and less directional phenotypic change. Partial least squares analyses revealed asymmetric covariance between heterospecific male and female reproductive traits during late secondary contact, indicating that, despite overall divergence, some male–female trait combinations allow functional matching. Linking the major axis of heterospecific covariance to tandem success showed that the probability of tandem formation success depends asymmetrically on the degree of size mismatch between partners, and that this relationship differs between reciprocal crosses. Our results suggest that reinforcement reshapes species-level trait distributions, but the outcome of heterospecific interaction depends on morphological matching rather than between individual males and females.

Female colour polymorphism is a widespread trait in Ischnura and has been linked to mating strategies, sexual conflict, and evolutionary diversification. In the Colombian Andes, Ischnura cyane and Ischnura capreolus occur in sympatry, and localities contain admixed/intermediate individuals. Here, we characterize female colour polymorphism, ontogenetic colour change, and pruinescence in I. cyane and individuals of admixed/intermediate morphology, within a comparative framework including I. capreolus. We evaluate mating-associated patterns using colour traits, operational sex ratio, and mating frequency observations, and we evaluate reproductive morphology. Our results show that both taxa are dimorphic, with androchrome and gynochrome females. In I. cyane, mating-associated traits were consistent with polyandry, whereas admixed/intermediate individuals exhibited patterns associated with monandry. Females of I. cyane and admixed/intermediate individuals cannot be distinguished based on colour traits or ontogenetic colour change, but they differ from I. capreolus. In contrast, males of the three populations are differentiated by caudal appendage morphology. No relationship was detected between male cercus size and female mesostigmal plate size among mating pairs. The coexistence of overlapping colour traits, asymmetric sexual differentiation, and contrasting mating-associated patterns supports the interpretation of an admixed system involving I. cyane and I. capreolus. Female colour polymorphism nevertheless remains conserved across contrasting mating-associated patterns in Andean Ischnura.

Hybridization outcomes often vary across space and time, yet the relative roles of demographic history and chromosomal differences in shaping introgression remain unclear. Here, we investigate three replicated hybrid zones between the damselflies Ischnura elegans and Ischnura graellsii across Spain to test whether genomic introgression patterns are repeatable across independently formed zones. Using genome-wide data, we combined demographic modeling, genomic cline, and functional annotation of introgressed loci. Demographic inference supported three independent secondary contact events of different ages: The South-east zone forming first (~207 years ago), followed by the North-west (~73.5 years) and North-central (~33 years) hybrid zones. Despite similar cline steepness across autosomes, asymmetric gene flow from I. graellsii into I. elegans was observed, with a low overlap of introgressed loci between zones. These loci were mainly associated with broad physiological and regulatory functions. In contrast, the X chromosome showed steeper clines, suggesting strong intrinsic genomic constraints. Together, demographic history explains geographic heterogeneity in introgression, whereas chromosome architecture imposes consistent constraints. These findings highlight how replicated hybrid zones can disentangle contingent versus repeatable genomic responses during early stages of speciation with gene flow.

Ischnura cruzi is an endemic damselfly of the Colombian Andes associated with high‐elevation wetlands, for which information on adult population dynamics at the local scale has been lacking. Here, we present the first integrated assessment of its population status, colour polymorphism and reproductive biology to provide baseline information relevant for conservation. Using mark–recapture data, we assessed population status through relative abundance, survival and adult life expectancy. In addition, we described female colour polymorphism and ontogenetic colour changes in both sexes and assessed mating patterns based on sex ratios, copulation frequency and behavioural observations in the field. Ischnura cruzi exhibited an extremely restricted distribution within the surveyed localities (2/22–9% of surveyed sites) and co‐occurred with another odonate species classified as Endangered on the International Union for Conservation of Nature Red List criteria (IUCN) Red List. The species showed female colour dimorphism (androchrome and aurantiaca morphs), pronounced ontogenetic colour changes in both sexes, and behavioural patterns consistent with apparent monandry. The species' narrow distribution, distinctive reproductive traits and reliance on high‐Andean wetlands suggests high sensitivity to habitat loss and potential vulnerability consistent with traits commonly associated with IUCN Red list criteria for threatened categories. Our results provide a biological and ecological baseline for I. cruzi and highlight the importance of integrating demographic and life‐history information into conservation assessments of endemic freshwater insects in montane ecosystems.

Hybridisation outcomes often vary across space and time, yet the relative roles of demographic history and genomic architecture in shaping introgression remain unclear. Here, we investigate three replicated hybrid zones between the damselflies Ischnura elegans and Ischnura graellsii across Spain to test whether genomic introgression patterns are repeatable across independently formed zones. Using genome-wide data, we combined demographic modelling, genomic cline, and functional annotation of introgressed loci. Demographic inference supported three independent secondary contact events of different ages: The South-east zone forming first (∼207 years ago), followed by the North-west (∼73.5 years) and North-central (∼33 years) hybrid zones. Despite similar cline steepness across autosomes, asymmetric gene flow from I. graellsii into I. elegans was observed, with a low overlap of introgressed loci between zones. These loci were mainly associated with broad regulatory and transport-related functions in both hybrid zones, indicating repeatability at the level of gene function rather than gene identity. In contrast, the X chromosome showed steeper clines, suggesting strong intrinsic genomic constraints. Together, demographic history explains geographic heterogeneity in introgression, whereas chromosome architecture imposes consistent constraints. These findings highlight how replicated hybrid zones can disentangle contingent versus repeatable genomic responses during early stages of speciation with gene flow.

Theoretical and empirical studies of reinforcement have advanced our understanding of speciation, yet its role in polymorphic species remains understudied. Because morphs differ in behavior, morphology, and reproductive strategies, reinforcement may act unevenly among them, generating asymmetric reproductive isolation. We tested this prediction in the polymorphic damselflies Ischnura elegans and Ischnura graellsii, in which female morphs adopt alternative reproductive strategies. These species form two independent hybrid zones where reinforcement has strengthened mechanical isolation and driven reproductive character displacement in mating-related structures. We quantified five reproductive barriers across female morphs to evaluate how color polymorphism interacts with reinforcement. We found clear asymmetry between morphs: gynochrome females of both species showed reinforced mechanical isolation, whereas androchromes did not, consistent with their contrasting reproductive strategies. Additionally, gametic barriers evolved in opposite directions between species. Fertility isolation was reinforced in I. elegans, while oviposition and fertility barriers relaxed in I. graellsii, but symmetrically between female morphs, likely reflecting gene flow and purging of incompatibilities. Reinforcement strengthened reproductive isolation in a morph-specific manner, as pre-existing differences between female morphs influenced the likelihood of heterospecific mating and therefore the strength of selection against hybridization. Future work should examine whether these asymmetric dynamics generate cascading effects within species and contribute to morph-level diversification.

Reproductive Character Displacement (RCD) often occurs when species with mating-related polymorphism come into secondary contact, leading to divergence in reproductive traits. Ischnura elegans and Ischnura graellsii have formed two independent hybrid zones in Spain where reinforcement has strengthened a mechanical barrier, and RCD has shaped mating-related structures, although reinforcement is asymmetric only in gynochrome females. This study examines the link between asymmetric reinforcement and asymmetric RCD. Using geometric morphometrics, we analyze prothorax shape and size in both female morphs and males, and male caudal appendages, to assess morphological divergence, determine whether gynochrome females show stronger divergence, and test for morphological covariation between male traits involved in the tandem position. Our results reveal consistent patterns of size and shape variation across species and zones: in I. elegans , androchromes are larger and resemble males in size, with clear shape differentiation between female morphs that diminishes in hybrid zones. In contrast, I. graellsii shows less consistent size differences between males and morphs, and weaker shape differentiation. Our results confirm RCD in prothorax shape in I. elegans females from both hybrid zones, but reveal that RCD in prothorax size is asymmetric, occurring only in gynochrome females from the NC hybrid zone. We also detected RCD in the prothorax shape of I. elegans males from the NC hybrid zone, extending previous evidence of RCD in male caudal appendages, while morphological covariation between male cerci and the prothorax was limited to size in I. elegans . Together, these findings illustrate how hybridization may generate morph-specific patterns of reproductive divergence.

Pair of species with multiple hybrid zones provide powerful systems to examine how reproductive isolation and reinforcement may depend on ecological and demographic contexts. We conducted a comparative analysis of two hybrid zones between Ischnura elegans and Ischnura graellsii in North-central (NC) and North-western (NW) Spain to evaluate how differences in reproductive barriers shape patterns of hybridization and admixture. First, we quantified five prezygotic barriers in the NC hybrid zone—where no barrier estimates previously existed—and compared them with published data from the NW hybrid zone. Second, we conducted genomic analyses using 5,702 SNPs to assess genetic structure, differentiation, and diversity between species; and 381 SNPs with species-specific alleles to characterize hybrid classes and introgression across both regions. Reproductive isolation was strongly context dependent. Mechanical reinforcement acted in the same cross direction in both hybrid zones, but the NC hybrid zone also showed stronger gametic (fertility) isolation. Consequently, total prezygotic isolation was higher in the NC hybrid zone. These differences were also observed genomically: the NC hybrid zone exhibited limited and largely unidirectional introgression, whereas the NW hybrid zone showed more extensive admixture, bidirectional gene flow, and reduced interspecific differentiation, consistent with a longer or more sustained history of hybridization. Our results demonstrate that hybridization outcomes can differ markedly between hybrid zones involving the same pair of species, underscoring the importance of evaluating spatial variation in reproductive barriers and introgression to assess the presence, asymmetry, and repeatability of reinforcement, as well as the microevolutionary processes shaping hybrid zone structure.