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Climate is often considered to be an important, but indirect driver of speciation. Indeed, environmental factors may contribute to the formation of biodiversity, but to date this crucial relationship remains largely unexplored. Here we investigate the possible role of climate, geological factors, and biogeographical processes in the formation of a freshwater insect species group, the Chaetopteryx rugulosa species complex (Trichoptera) in the Western Balkans. We used multi-locus DNA sequence data to establish a dated phylogenetic hypothesis for the group. The comparison of the dated phylogeny with the geological history of the Western Balkans shows that lineage formation coincided with major past Earth surface and climatic events in the region. By reconstructing present-day habitat conditions (climate, bedrock geology), we show that the lineages of C. rugulosa species complex have distinct climatic but not bedrock geological niches. Without exception, all splits associated with Pliocene/Pleistocene transition led to independent, parallel split into ‘warm’ and ‘cold’ sister lineages. This indicates a non-random diversification on the C. rugulosa species complex associated with late Pliocene climate in the region. We interpreted the results as the diversification of the species complex were mainly driven by ecological diversification linked to past climate change, along with geographical isolation.

The Cankiri Basin, located in the northern part of the Central Anatolian Plateau, is a large Tertiary basin where thick Miocene to Quaternary continental sediments overlay the Cretaceous-Tertiary units. This investigation focuses on the Tuglu Formation, an Upper Miocene succession mainly composed of dark grey silty and organic rich clays. The type section of Tuglu has been sampled for an array of multidisciplinary analyses. The palaeontological proxies included ostracod, foraminifer, nannoplankton, pollen, molluscs, charophytes, small mammal assemblages, fish, and crab remains. The abiotic parameters studied were: palaeomagnetism and environmental magnetism, stable carbon and oxygen isotope ratios (on ostracods and bulk sediment samples), strontium isotope ratios (on ostracods and foraminifera), and major elemental composition of the sediments. All analysed proxies point to a continental setting characterised by permanent water bodies affected by strong salinity oscillations. A shallow saline lake developed in a permanent freshwater lake. Barren layers, potentially linked to a short subaerial exposure, mark the end of the saline lake and the transition to a fluvial environment. Geochemical analysis confirms aridity-humidity oscillations as recorded by the micropalaeontological proxies. Analysis of small mammal assemblages refined the chronology of the Tuglu Formation, with the onset of the deposition at the base of the mammal zone MN9 (around 11 Ma) and continuous deposition until the MN11 zone (around 8 Ma). The stable oxygen isotope records from the Tuglu section point to Miocene delta O-18 water values consistent with subdued topography where no prominent mountain belts were yet developed at the northern plateau margin. If correct, this suggests that at least until the end of the deposition of the Tuglu Formation, the Cankiri Basin did not yet experience rain shadow conditions, and the regional surface uplift of the area most likely occurred after 8 Ma.