Mechanisms of survival during the Pleistocene glaciation periods have been studied for more than a century. Until now, molecular studies that confirmed animal survival on Alpine nunataks, that is, ice-free summits surrounded by glaciers, were restricted to peripheral areas. Here, we search for molecular signatures of inner-Alpine survival of the narrow endemic and putatively parthenogenetic Alpine jumping bristletail Machilis pallida combining mitochondrial and AFLP data from its three known populations. The mitochondrial data indicate survival on both peripheral and central nunataks, the latter suggesting that refugia in the centre of the Alpine main ridge were more widespread than previously recognized. Incongruences between mitochondrial and AFLP patterns suggest a complex evolutionary history of the species and may be explained via parallel fixation of parthenogenesis of different origins during the last glacial maximum. We suggest that the inferred parthenogenesis may have been essential for central nunatak survival, but may pose a serious threat for M. pallida in consideration of the present climatic changes.

Endemic species play an important role in conservation ecology. However, knowledge of the real distribution and ecology is still scarce for many endemics. The aims of this study were to predict the distribution of the short-range endemic Alpine jumping bristletail Machilis pallida; to evaluate the actual level of endemism via ground validation using an iterative approach for testing the models in field trips and increasing the quality of the prediction step by step; and to test the potential of species distribution modelling for increasing the knowledge about the ecological niche. Based on seven known locations of M. pallida, we used species distribution modelling via Maxent. After a set of seven field trips a new model was built if new locations were found. Three such iterations were performed to increase model quality. We discovered four new locations of M. pallida, increasing the area of known distribution from 470 to 4,890 km 2 . The distribution of M. pallida is thus wider than formerly known, but our results support Eastern Alpine endemism of the species. The knowledge about the ecological niche could be increased due to the newly found locations. Our study showcases the potential of the iterative approach of modelling and ground validation to evaluate the actual level of endemism and the ecological niche in Alpine species and beyond.

Accurate species delimitation is fundamental to biology. Traditionally, species were delimited based on morphological characters, sometimes leading to taxonomic uncertainty in morphologically conserved taxa. Recently, multiple taxonomically challenging cases have benefited from integrative taxonomy—an approach that highlights congruence among different disciplines and invokes evolutionary explanations for incongruence, acknowledging that different methods can mirror different stages of the speciation continuum. Here, we used a cohesive protocol for integrative taxonomy to revise species limits in 20 nominal species and 4 morphospecies of an ancestrally wingless insect group, the jumping bristletail genus Machilis from the European Eastern Alps. Even though morphologically conserved, several small-scale endemic species have been described from the Eastern Alps based on variation in hypodermal pigmentation patterns—a highly questionable character. As valuable as these endemics are for conservation, they have never been verified by alternative methods. Using traditional morphometrics, mitochondrial DNA, ribosomal DNA, and amplified fragment-length polymorphism markers, we identify six nominal species as taxonomic junior synonyms (Machilis alpicola Janetschek, 1953 syn. n. under M. vagans Wygodzinsky, 1941; M. ladensis Janetschek, 1950 syn. n., M. robusta Wygodzinsky, 1941 syn. n., and M. vicina Wygodzinsky, 1941 syn. n. under M. inermis Wygodzinsky, 1941; M. aleamaculata Wygodzinsky, 1941 syn. n. under M. montana Wygodzinsky, 1941; M. pulchra Janetschek, 1950 syn. n. under M. helleri Verhoeff, 1910) and describe two new species (Machilis cryptoglacialis sp. n. and Machilis albida sp. n.), one uncovered from morphological crypsis and one never sampled before. Building on numerous cases of incongruence among data sources, we further shed light on complex evolutionary histories including hybrid speciation, historical and recent hybridization, and ongoing speciation. We hypothesize that an inherent affinity to hybridization, combined with parallel switches to parthenogenesis and repeated postglacial colonization events may have boosted endemicity in Eastern Alpine Machilis. We thus emphasize the importance of integrative taxonomy for rigorous species delimitation and its implication for evolutionary research and conservation in taxonomically challenging taxa.

Parthenogenesis in animals is often associated with polyploidy and restriction to extreme habitats or recently deglaciated areas. It has been hypothesized that benefits conferred by asexual reproduction and polyploidy are essential for colonizing these habitats. However, while evolutionary routes to parthenogenesis are manifold, study systems including polyploids are scarce in arthropods. The jumping-bristletail genus Machilis (Insecta: Archaeognatha) includes both sexual and parthenogenetic species, and recently, the occurrence of polyploidy has been postulated. Here, we applied flow cytometry, karyotyping, and mitochondrial DNA sequencing to three sexual and five putatively parthenogenetic Eastern-Alpine Machilis species to investigate whether (1) parthenogenesis originated once or multiply and (2) whether parthenogenesis is strictly associated with polyploidy. The mitochondrial phylogeny revealed that parthenogenesis evolved at least five times independently among Eastern-Alpine representatives of this genus. One parthenogenetic species was exclusively triploid, while a second consisted of both diploid and triploid populations. The three other parthenogenetic species and all sexual species were diploid. Our results thus indicate that polyploidy can co-occur with parthenogenesis, but that it was not mandatory for the emergence of parthenogenesis in Machilis. Overall, we found a weak negative correlation of monoploid genome size (Cx) and chromosome base number (x), and this connection is stronger among parthenogenetic species alone. Likewise, monoploid genome size decreased with elevation, and we therefore hypothesize that genome downsizing could have been crucial for the persistence of alpine Machilis species. Finally, we discuss the evolutionary consequences of intraspecific chromosomal rearrangements and the presence of B chromosomes. In doing so, we highlight the potential of Alpine Machilis species for research on chromosomal and genome-size alterations during speciation.

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