Akdeniz University (grant no: 2010.02.0121.028); The Scientific and Technical Research Council of Turkey (TUBITAK, grant no: 210T169); PID2021-123298NB-I00; TED2021-129328B-I00, Peer reviewed

In this study we employ a geometric morphometric approach to quantify morphological differences among taxa of the genus Oropodisma Uvarov, a complex of alpine grasshoppers that includes several narrow endemic species distributed across different mountain ranges of the Balkan Peninsula. The genus was described by Uvarov in 1942 and currently comprises ten recognized species, primarily distinguished by subtle differences in the shape of the phallus apex and furculae of males. Some populations from central Greece exhibit an uncertain taxonomic status, suggesting the possible existence of undescribed species. Quantitative morphological comparisons with specimens collected from the type localities of all currently recognized taxa revealed the presence of two new species in the Pindus Range, Greece: O. tzoumerkae sp. nov. from Mt. Tzoumerka and O. agrafae sp. nov. from Mt. Agrafa. Geometric morphometric analyses indicate that the taxa O. tymphrestosi and O. willemsei, distributed in adjacent mountain ranges from central Greece, do not show statistically significant differences in either of the two studied traits. This finding raises doubts about their taxonomic status but may also reflect the limitations of geometric morphometric approaches in capturing subtle differences in the complex structures that characterize internal male genitalia. Overall, our analyses highlight the importance of quantitative approaches in formally reassessing the taxonomic status of species complexes. The description of two new and geographically restricted species of Oropodisma further emphasizes the role of Mediterranean mountains as key centers of microgeographic speciation for alpine biotas., TED2021-129328B-I00; PID2021-123298NB-I00, Peer reviewed

Mountains contribute disproportionately to global biodiversity, yet the ecological and evolutionary processes that make them important centers of speciation remain poorly understood. Here, we use a complex of Gomphocerinae grasshoppers distributed across the Pyrenees as a model system to investigate the process of speciation in mountain regions. Clustering analyses revealed a marked genetic fragmentation of populations and divergence time estimates indicated that all lineages split during the Pleistocene (<0.9 Ma). Although all taxa currently exhibit allopatric distributions, fine-scale temporal distribution modeling suggested that their ranges likely overlapped across extensive areas during glacial periods. Despite ample opportunities for historical hybridization, introgression tests indicated that gene flow was restricted to peripheral populations and only one population exhibited a high degree of admixed ancestry. Accordingly, our analyses revealed abrupt genetic discontinuities corresponding to the distributional boundaries of each taxon. Although taxa have probably not achieved complete reproductive isolation, semi-permeable species boundaries do not appear to have compromised their evolutionary independence. These results emphasize the interplay between high rates of lineage formation and the rapid evolution of reproductive barriers in promoting microgeographic speciation in topographically complex landscapes, shedding light on the proximate processes underlying the high levels of neo-endemism observed across mountain ranges worldwide., TED2021-129328B-I00; PID2021-123298NB-I00; Viera y Clavijo postdoctoral fellowship, Peer reviewed

All versions are available at DOI 10.6084/m9.figshare.29046218, Mountains contribute disproportionately to global biodiversity, yet the ecological and evolutionary processes that make them important centers of speciation remain poorly understood. Here, we use a complex of Gomphocerinae grasshoppers distributed across the Pyrenees as a model system to investigate the process of speciation in mountain regions. Clustering analyses revealed a marked genetic fragmentation of populations and divergence time estimates indicated that all lineages split during the Pleistocene (<0.9 Ma). Although all taxa currently exhibit allopatric distributions, fine-scale temporal distribution modeling suggested that their ranges likely overlapped across extensive areas during glacial periods. Despite ample opportunities for historical hybridization, introgression tests indicated that gene flow was restricted to peripheral populations and only one population exhibited a high degree of admixed ancestry. Accordingly, our analyses revealed abrupt genetic discontinuities corresponding to the distributional boundaries of each taxon. Although taxa have probably not achieved complete reproductive isolation, semi-permeable species boundaries do not appear to have compromised their evolutionary independence. These results emphasize the interplay between high rates of lineage formation and the rapid evolution of reproductive barriers in promoting microgeographic speciation in topographically complex landscapes, shedding light on the proximate processes underlying the high levels of neo-endemism observed across mountain ranges worldwide., TED2021-129328B-I00; PID2021-123298NB-I00; Viera y Clavijo postdoctoral fellowship, Peer reviewed

All versions are available at DOI 10.6084/m9.figshare.31136593, Recent Pleistocene radiations often generate cryptic diversity and semi-permeable species boundaries, challenging species delimitation and the reconstruction of evolutionary histories. Here, we investigate these processes in a species complex of Chorthippus grasshoppers from Greece using genome-wide ddRAD-seq data combined with geometric morphometrics. Phylogenomic analyses indicate that diversification occurred during the Middle Pleistocene and identify two major geographic clades separated by the Corinthian Gulf, followed by finer-scale divergence within the Peloponnese Peninsula. Most taxa form well-supported, reciprocally monophyletic lineages despite recent divergence, although several populations at range margins show signatures of historical hybridization, revealing semi-permeable species boundaries shaped by secondary contact. Introgression is geographically restricted and strongly asymmetric, occurring primarily from long-winged into short-winged taxa, consistent with differences in dispersal capacity. Species delimitation analyses largely support current taxonomy but also identify three previously unrecognized evolutionary lineages corresponding to geographically restricted populations, uncovering additional cryptic diversity. Morphological analyses reveal pronounced stasis in shape-related traits across lineages, with wing length emerging as the main axis of differentiation. Phylogenetic reconstructions indicate multiple independent reductions in wing size, highlighting repeated evolutionary transitions toward reduced dispersal. In at least one lineage, a pronounced geographic cline in wing length is mirrored by shallow phylogenetic structure, supporting an incipient evolutionary shift in dispersal capacity rather than phenotypic plasticity. Admixed populations consistently retain short-winged phenotypes, suggesting strong selection on dispersal-related traits despite genomic introgression. Overall, our results demonstrate how topographic complexity, Pleistocene climatic oscillations, and dispersal-related morphology interact to shape diversification and maintain lineage integrity in a Mediterranean biodiversity hotspot., CLOSING THE GAP BETWEEN DEMOGRAPHY AND DIVERSIFICATION: PERSPECTIVES FROM AN EVOLUTIONARY RADIATION OF GRASSHOPPERS, State Research Agency (AEI), PID2021-123298NB-I00, EVOLUTIONARY, DEMOGRAPHIC AND EXTINCTION RISK ASSESSMENT OF ALPINE BIOTAS IN MEDITERRANEAN MOUNTAIN ISLAND ARCHIPELAGOES WITH EXTREME VULNERABILITY TO CLIMATE CHANGE, State Research Agency (AEI), TED2021-129328B-I00, Peer reviewed

All versions are available at DOI 10.6084/m9.figshare.31136593, Recent Pleistocene radiations often generate cryptic diversity and semi-permeable species boundaries, challenging species delimitation and the reconstruction of evolutionary histories. Here, we investigate these processes in a species complex of Chorthippus grasshoppers from Greece using genome-wide ddRAD-seq data combined with geometric morphometrics. Phylogenomic analyses indicate that diversification occurred during the Middle Pleistocene and identify two major geographic clades separated by the Corinthian Gulf, followed by finer-scale divergence within the Peloponnese Peninsula. Most taxa form well-supported, reciprocally monophyletic lineages despite recent divergence, although several populations at range margins show signatures of historical hybridization, revealing semi-permeable species boundaries shaped by secondary contact. Introgression is geographically restricted and strongly asymmetric, occurring primarily from long-winged into short-winged taxa, consistent with differences in dispersal capacity. Species delimitation analyses largely support current taxonomy but also identify three previously unrecognized evolutionary lineages corresponding to geographically restricted populations, uncovering additional cryptic diversity. Morphological analyses reveal pronounced stasis in shape-related traits across lineages, with wing length emerging as the main axis of differentiation. Phylogenetic reconstructions indicate multiple independent reductions in wing size, highlighting repeated evolutionary transitions toward reduced dispersal. In at least one lineage, a pronounced geographic cline in wing length is mirrored by shallow phylogenetic structure, supporting an incipient evolutionary shift in dispersal capacity rather than phenotypic plasticity. Admixed populations consistently retain short-winged phenotypes, suggesting strong selection on dispersal-related traits despite genomic introgression. Overall, our results demonstrate how topographic complexity, Pleistocene climatic oscillations, and dispersal-related morphology interact to shape diversification and maintain lineage integrity in a Mediterranean biodiversity hotspot., CLOSING THE GAP BETWEEN DEMOGRAPHY AND DIVERSIFICATION: PERSPECTIVES FROM AN EVOLUTIONARY RADIATION OF GRASSHOPPERS, State Research Agency (AEI), PID2021-123298NB-I00, EVOLUTIONARY, DEMOGRAPHIC AND EXTINCTION RISK ASSESSMENT OF ALPINE BIOTAS IN MEDITERRANEAN MOUNTAIN ISLAND ARCHIPELAGOES WITH EXTREME VULNERABILITY TO CLIMATE CHANGE, State Research Agency (AEI), TED2021-129328B-I00, Peer reviewed

Understanding the geological, ecological, and microevolutionary processes that shape range size in alpine organisms is key to explaining the high rates of local endemism that characterize mountain ecosystems. Here, we investigate the geography of speciation in Oropodisma, a radiation of alpine grasshoppers from the Balkan Peninsula that includes several narrow-endemic species distributed along a latitudinal gradient of > 600 km. Phylogenomic-based species delimitation clarified taxonomic uncertainties and revealed striking variation in range size, with the northernmost species occupying an area nearly as large as all other congeneric taxa combined. Phylogenetic network analyses and divergence dating indicate a Pleistocene origin for the group, with recent speciation events (<0.2 Ma) and limited post-divergence gene flow, suggesting rapid evolution of reproductive isolation and short speciation times. Distributional shifts inferred from environmental niche modelling show that all taxa underwent severe range contractions during interglacial periods, leading to population fragmentation and demographic declines. Despite the ubiquity of these processes across taxa, both demographic stability and genetic connectivity among conspecific populations decreased at lower latitudes relative to the genus’ distribution. This suggests that greater genetic connectivity at higher relative latitudes, likely driven by regional range shifts and a more continuous availability of suitable habitats through time, has limited opportunities for microgeographic speciation and maintained genetic cohesiveness among populations across broader distributional ranges. Collectively, our findings support a model of interglacial speciation and illustrate how latitudinal variation in the balance between lineage formation and fusion —shaped by Pleistocene climatic oscillations— determines the rates at which alpine microendemic species emerge and accumulate in temperate mountain regions., CLOSING THE GAP BETWEEN DEMOGRAPHY AND DIVERSIFICATION: PERSPECTIVES FROM AN EVOLUTIONARY RADIATION OF GRASSHOPPERS, State Research Agency (AEI), PID2021-123298NB-I00, EVOLUTIONARY, DEMOGRAPHIC AND EXTINCTION RISK ASSESSMENT OF ALPINE BIOTAS IN MEDITERRANEAN MOUNTAIN ISLAND ARCHIPELAGOES WITH EXTREME VULNERABILITY TO CLIMATE CHANGE, State Research Agency (AEI), TED2021-129328B-I00, Peer reviewed

[Aim] Pleistocene climatic oscillations profoundly shaped Mediterranean mountain biotas through recurrent cycles of isolation and connectivity. Yet, despite the Central Apennines being a major centre of local endemism, this region has received limited attention in genomic studies addressing biogeographic processes. Here, we examine how climatically driven distributional shifts have promoted microgeographic diversification and endemism in sky-island systems of this mountain region., [Location] Central Apennines, Italy., [Taxon] Italohippus (Orthoptera: Acrididae)., [Methods] We used genome-wide ddRAD-seq data to infer phylogenetic relationships, divergence times, species limits, population structure, demographic history and introgression. Morphological differentiation was quantified using geometric and linear morphometrics of forewings. Past range dynamics and spatial patterns of climatic suitability were reconstructed by projecting species-specific environmental niche models from the Last Glacial Maximum to the present., [Results] Phylogenomic analyses reveal a Middle–Late Pleistocene diversification of Italohippus structured along a continuum of evolutionary divergence across the Central Apennines. Northern populations, corresponding to the taxon I. modestus, form a distinct allopatric lineage likely originating under geographic isolation and the evolution of partial reproductive barriers. In contrast, populations at the southern rear edge of the genus range, corresponding to I. albicornis, show weak genomic and morphological differentiation from I. monticola, consistent with incipient but incomplete speciation. Paleodistribution models and demographic reconstructions indicate marked latitudinal contrasts in glacial impacts, with higher availability and continuity of climatically suitable habitats and introgression in northern and central sectors and more persistent isolation in the south., [Main Conclusions] Our results support a biogeographic model of intermittently connected sky-island refugia in the Central Apennines, generating spatially heterogeneous opportunities for divergence and persistence across latitudes. By integrating different sources of data, we show how climatically driven distributional shifts have shaped the emergence and maintenance of geographically structured evolutionary lineages in Mediterranean mountain systems, highlighting key processes underlying the origin and persistence of endemic alpine biodiversity in the face of rapid climate warming., EVOLUTIONARY, DEMOGRAPHIC AND EXTINCTION RISK ASSESSMENT OF ALPINE BIOTAS IN MEDITERRANEAN MOUNTAIN ISLAND ARCHIPELAGOES WITH EXTREME VULNERABILITY TO CLIMATE CHANGE, State Research Agency (AEI) TED2021-129328B-I00, CLOSING THE GAP BETWEEN DEMOGRAPHY AND DIVERSIFICATION: PERSPECTIVES FROM AN EVOLUTIONARY RADIATION OF GRASSHOPPERS, State Research Agency (AEI) PID2021-123298NB-I00, NBFC – National Biodiversity Future Center, funded by the Italian Ministry of University and Research, PNRR, Missione 4 Componente 2, “Dalla ricerca all’impresa”, Investimento 1.4, Project CN00000033, Departments of Excellence—L. 232/2016—art.1, commi 314–337 awarded to Dept. of Science—University Roma Tre—Rome—Italy for 2023-2027, Peer reviewed

[Aim] Island archipelagos serve as natural laboratories for understanding how geological history, climate change and dispersal limitation interact to shape diversification. Here, we examine how these forces influence patterns of genomic divergence, demographic dynamics and phenotypic variation in island-endemic taxa, with a particular focus on evaluating whether contrasting connectivity regimes among archipelagos are associated with distinct microevolutionary outcomes., [Location] Ionian and Aegean archipelagos, Greece., [Taxon] Brachypterous grasshoppers including the Ionian-endemic Chorthippus sangiorgii and the Aegean-endemic Chorthippus biroi (Orthoptera: Acrididae)., [Method] We generated genome-wide ddRAD-seq data to estimate divergence times, infer genetic structure, assess relative levels of genetic connectivity and reconstruct the demographic trajectories of populations. In addition, geometric morphometric analyses of forewing and pronotum-head shape were conducted to evaluate morphological differentiation among island populations. Genomic and phenotypic patterns were interpreted in the context of palaeogeographic reconstructions of island connectivity associated with Quaternary sea-level oscillations., [Results] Both species show strong genetic structuring within and among islands, consistent with extremely limited dispersal. Whereas C. sangiorgii exhibits relatively shallow divergence compatible with recent and recurrent connectivity among the Ionian Islands, C. biroi displays deeper genetic structure consistent with the long-term isolation of Crete and the Cyclades. Demographic reconstructions for both species indicate population expansions during the Last Glacial Maximum followed by contractions throughout the Holocene, suggesting broadly similar responses to Late Quaternary climatic oscillations. Geometric morphometric analyses detected modest but statistically significant phenotypic differentiation among some island pairs in both species., [Main Conclusions] Our findings indicate that the microevolutionary and demographic dynamics in Mediterranean island grasshoppers are shaped by the interplay between limited dispersal, historical isolation and palaeogeographic connectivity driven by Quaternary sea-level oscillations. Phenotypic differentiation appears more limited and heterogeneous than genomic divergence, suggesting a partial decoupling between patterns of genetic and phenotypic variation. This study highlights the value of integrating genomic and phenotypic data to better characterize the microevolutionary trajectories in low-dispersal taxa inhabiting complex island systems., This work was supported by grant TED2021-129328B-I00 funded by MCIN/AEI/http://doi.org/10.13039/501100011033 and the European Union NextGenerationEU/PRTR, and grant PID2021-123298NB-I00 funded by MCIN/AEI/http://doi.org/10.13039/501100011033/FEDER, EU., Peer reviewed