Replicated secondary contact zones can provide insights into the barriers to gene flow that are important during speciation and can reveal to which degree secondary contact may result in similar evolutionary outcomes. Here, we studied two secondary contact zones between highly differentiated Alpine butterflies of the genus Erebia using whole-genome resequencing data. We assessed the genomic relationships between populations and species and found hybridization to be rare, with no to little current or historical introgression in either contact zone. There are large similarities between contact zones, consistent with an allopatric origin of interspecific differentiation, with no indications for ongoing reinforcing selection. Consistent with expected reduced effective population size, we further find that scaffolds related to the Z-chromosome show increased differentiation compared to the already high levels across the entire genome, which could also hint towards a contribution of the Z chromosome to species divergence in this system. Finally, we detected the presence of the endosymbiont Wolbachia, which can cause reproductive isolation between its hosts, in all E. cassioides, while it appears to be fully or largely absent in contact zone populations of E. tyndarus. We discuss how this rare pattern may have arisen and how it may have affected the dynamics of speciation upon secondary contact.

Pollinator-driven evolution of floral traits is thought to be a major driver of angiosperm speciation and diversification. Ophrys orchids mimic female insects to lure male pollinators into pseudocopulation. This strategy, called sexual deception, is species-specific, thereby providing strong premating reproductive isolation. Identifying the genomic architecture underlying pollinator adaptation and speciation may shed light on the mechanisms of angiosperm diversification. Here, we report the 5.2 Gb chromosome-scale genome sequence of Ophrys sphegodes. We find evidence for transposable element expansion that preceded the radiation of the O. sphegodes group, and for gene duplication having contributed to the evolution of chemical mimicry. We report a highly differentiated genomic candidate region for pollinator-mediated evolution on chromosome 2. The Ophrys genome will prove useful for investigations into the repeated evolution of sexual deception, pollinator adaptation and the genomic architectures that facilitate evolutionary radiations.

Speciation is a fundamental evolutionary process but the genetic changes accompanying speciation are difficult to determine since true species do not produce viable and fertile offspring. Partially reproductively isolated incipient species are useful for assessing genetic changes that occur prior to speciation. Drosophila melanogaster from Zimbabwe, Africa are partially sexually isolated from other D. melanogaster populations whose males have poor mating success with Zimbabwe females. We used the North American D. melanogaster Genetic Reference Panel (DGRP) to show that there is significant genetic variation in mating success of DGRP males with Zimbabwe females, to map genetic variants and genes associated with variation in mating success and to determine whether mating success to Zimbabwe females is associated with other quantitative traits previously measured in the DGRP. Incipient sexual isolation is highly polygenic and associated with the common African inversion In(3R)K and the amount of the sex pheromone 5,9-heptacosadiene in DGRP females. We functionally validated the effect of eight candidate genes using RNA interference to provide testable hypotheses for future studies investigating the molecular genetic basis of incipient sexual isolation in D. melanogaster.

Reproductive isolation is one of the mechanisms of speciation. The two currently accepted subspecies of Parodia haselbergii (P. haselbergii subsp. haselbergii and P. haselbergii subsp. graessneri) were studied regarding flower traits, phenology, breeding systems and pollination. In addition, a principal component analysis with 18 floral characters and germination tests under controlled conditions were performed for both taxa. Pollination was studied in the field, in two localities of Southern Brazil. Pollinators were recorded through photos and film. Breeding system experiments were performed by applying controlled pollinations to plants excluded from pollinators. Both taxa mostly differ in asynchronous flowering periods, floral traits (including floral part measurements and nectar concentration) and pollinators. The flowers of both subspecies are functionally protogynous and perform remarkably long lifespans (≥ 15 days), both traits being novelties for Cactaceae. Whereas the reddish flowers of P. haselbergii subsp. haselbergii (nectar concentration: ca. 18%) are pollinated by hummingbirds of Thalurania glaucopis, the greenish flowers of P. haselbergii subsp. graessneri (nectar concentration: ca. 29%) are pollinated by Augochlora bees (Halictidae). Both subspecies are self-compatible, yet pollinator-dependent. The principal component analysis evidenced that both subspecies are separated, regarding flower traits. The seeds of both subspecies performed differently in the germination tests, but the best results were recovered at 20 °C and germination considerably decreased around 30 °C. In conclusion, all these results support that both taxa are in reproductive isolation, and can be treated as different species.

Trade-offs are crucial for species divergence and reproductive isolation. Trade-offs between investment in growth versus defense against herbivores are implicated in tropical forest diversity. Empirically exploring the role of growth-defense trade-offs in closely related species\' reproductive isolation can clarify the eco-evolutionary dynamics through which growth-defense trade-offs contribute to diversity. Costus villosissimus and C. allenii are recently diverged, interfertile, and partially sympatric neotropical understory plant species primarily isolated by divergent habitat adaptation. This divergent adaptation involves differences in growth rate, which may constrain investment in defense. Here, we investigate growth-defense trade-offs and how they relate to the divergent habitat adaptation that isolates these species. We characterize leaf toughness and chemistry, evaluate the feeding preferences of primary beetle herbivores in controlled trials and field-based experiments, and investigate natural herbivory patterns. We find clear trade-offs between growth and defense: slower-growing C. allenii has tougher leaves and higher defensive chemical concentrations than faster-growing C. villosissimus. Costus villosissimus has rapid growth-based drought avoidance, enabling growth in drier habitats with few specialist herbivores. Therefore, growth-defense trade-offs mediate synergistic biotic and abiotic selection, causing the divergent habitat adaptation that prevents most interspecific mating between C. villosissimus and C. allenii. Our findings advance understanding of ecological speciation by highlighting the interplay of biotic and abiotic selection that dictates the outcome of trade-offs.
Los compromisos son cruciales para la divergencia de especies y el aislamiento reproductivo. El compromiso entre crecimiento y defensa contra los herbívoros juega un papel central en la diversidad de los bosques tropicales. Explorar empíricamente el papel de las compromisos entre crecimiento y defensa en el aislamiento reproductivo de especies recientemente divergentes puede aclarar la dinámica ecoevolutiva a través de la cual los compromisos entre crecimiento y defensa contribuyen a la diversidad. Costus villosissimus y C. allenii son dos especies de plantas de sotobosque neotropical que divergieron recientemente, son interfértiles y parcialmente simpátricas, aisladas principalmente por adaptación a hábitates divergentes. Esta adaptación divergente implica diferencias en la tasa de crecimiento, lo que puede limitar la inversión en defensas. Aquí investigamos las compensaciones entre crecimiento y defensa y cómo se relacionan con la adaptación divergente del hábitat que aísla a estas especies. Caracterizamos la dureza y la química de las hojas, evaluamos las preferencias alimenticia de los escarabajos herbívoros en ensayos controlados y experimentos de campo, e investigamos los patrones de herbivoría natural. Encontramos compromisos entre crecimiento y defensa: C. allenii, es de crecimiento lento, tiene hojas más duras y concentraciones de defensas químicas más altas que en C. villosissimus, que presenta un crecimiento rápido. Costus villosissimus evita la sequía usando una estrategia basada en crecimiento rápido que permite el crecimiento en hábitats más secos y con pocos herbívoros especializados. Por lo tanto, las compromisos entre crecimiento y defensa median presiones de selección bióticas y abióticas sinérgicas que causan la adaptación divergente del hábitat que previene la mayoría de entrecruzamientos interespecíficos entre C. villosissimus y C. allenii. Nuestros hallazgos avanzan en la comprensión de la especiación ecológica al resaltar la interacción de la selección biótica y abiótica que dicta el resultado de las compromisos.

Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) are sibling fruit fly species that are sympatric over much of their ranges. Premating isolation of these close relatives is thought to be maintained in part by allochrony-mating activity in B. tryoni peaks at dusk, whereas in B. neohumeralis, it peaks earlier in the day. To ascertain whether differences in pheromone composition may also contribute to premating isolation between them, this study used solid-phase microextraction and gas chromatography-mass spectrometry to characterize the rectal gland volatiles of a recently collected and a more domesticated strain of each species. These glands are typical production sites and reservoirs of pheromones in bactrocerans. A total of 120 peaks were detected and 50 were identified. Differences were found in the composition of the rectal gland emissions between the sexes, species, and recently collected versus domesticated strains of each species. The compositional variation included several presence/absence and many quantitative differences. Species and strain differences in males included several relatively small alcohols, esters, and aliphatic amides. Species and strain differences in females also included some of the amides but additionally involved many fatty acid esters and 3 spiroacetals. While the strain differences indicate there is also heritable variation in rectal gland emissions within each species, the species differences imply that compositional differences in pheromones emitted from rectal glands could contribute to the premating isolation between B. tryoni and B. neohumeralis. The changes during domestication could also have significant implications for the efficacy of Sterile Insect Technique control programs.

Dingoes come from an ancient canid lineage that originated in East Asia around 8,000 to 11,000 years BP. As Australia\'s largest terrestrial predator, dingoes play an important ecological role. A small, protected population exists on a world heritage listed offshore island, K\'gari (formerly Fraser Island). Concern regarding the persistence of dingoes on K\'gari has risen due to their low genetic diversity and elevated inbreeding levels. However, whole-genome sequence data is lacking from this population. Here, we include five new whole-genome sequences of K\'gari dingoes. We analyze a total of 18 whole-genome sequences of dingoes sampled from mainland Australia and K\'gari to assess the genomic consequences of their demographic histories. Long (>1 Mb) runs of homozygosity (ROHs)-indicators of inbreeding-are elevated in all sampled dingoes. However, K\'gari dingoes showed significantly higher levels of very long ROH (>5 Mb), providing genomic evidence for small population size, isolation, inbreeding, and a strong founder effect. Our results suggest that, despite current levels of inbreeding, the K\'gari population is purging strongly deleterious mutations, which, in the absence of further reductions in population size, may facilitate the persistence of small populations despite low genetic diversity and isolation. However, there may be little to no purging of mildly deleterious alleles, which may have important long-term consequences, and should be considered by conservation and management programs.

Mate choice is a key trait that determines fitness for most sexually reproducing organisms, with females often being the choosy sex. Female preference often results in strong selection on male traits that can drive rapid divergence of traits and preferences between lineages, leading to reproductive isolation. Despite this fundamental property of female mate choice, very few loci have been identified that contribute to mate choice and reproductive isolation. We used a combination of population genetics, quantitative complementation tests, and behavioural assays to demonstrate that alan shepard and Neuroglian contribute to female mate choice, and could contribute to partial reproductive isolation between populations of Drosophila melanogaster. Our study is among the first to identify genes that contribute to female mate preference in this historically important system, where female preference is an active premating barrier to reproduction. The identification of loci that are primarily known for their roles in neurodevelopment provides intriguing questions of how female mate preference evolves in populations via changes in sensory system and higher learning brain centres.

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.

Structural variations (SVs) play a significant role in speciation and adaptation in many species, yet few studies have explored the prevalence and impact of different categories of SVs. We conducted a comparative analysis of long-read assembled reference genomes of closely related Eucalyptus species to identify candidate SVs potentially influencing speciation and adaptation. Interspecies SVs can be either fixed differences or polymorphic in one or both species. To describe SV patterns, we employed short-read whole-genome sequencing on over 600 individuals of Eucalyptus melliodora and Eucalyptus sideroxylon, along with recent high-quality genome assemblies. We aligned reads and genotyped interspecies SVs predicted between species reference genomes. Our results revealed that 49,756 of 58,025 and 39,536 of 47,064 interspecies SVs could be typed with short reads in E. melliodora and E. sideroxylon, respectively. Focusing on inversions and translocations, symmetric SVs that are readily genotyped within both populations, 24 were found to be structural divergences, 2,623 structural polymorphisms, and 928 shared structural polymorphisms. We assessed the functional significance of fixed interspecies SVs by examining differences in estimated recombination rates and genetic differentiation between species, revealing a complex history of natural selection. Shared structural polymorphisms displayed enrichment of potentially adaptive genes. Understanding how different classes of genetic mutations contribute to genetic diversity and reproductive barriers is essential for understanding how organisms enhance fitness, adapt to changing environments, and diversify. Our findings reveal the prevalence of interspecies SVs and elucidate their role in genetic differentiation, adaptive evolution, and species divergence within and between populations.