Phylogeography of the Savi's pipistrelle (Vespertilionidae, chiroptera) complex based on whole mitochondrial genome analysis

Abstract

Understanding the phylogeography of species provides insight into the historical processes that lead the formation of their current geographic distributions and also how they evolve and adapt to variable environments over time. Intraspecific and interspecific genetic variations stand as a great indicator for elucidation of the evolutionary history of organisms in diverged phylogeographical patterns. Bats represent an immensely diverged group among mammals. They inhabit a wide variety of ecosystems, with the tropical belt hosting the highest number of bat species, similar to many other life forms. While the oldest known fossil of Chiroptera is approximately 52 million years old, they are hypothesized to be evolved around the Cretaceous-Tertiary boundary. During their evolution, they settled to the nocturnal niche. Echolocation and flight capabilities have affected the phylogenetic classification in the era of morphological and physiological taxonomy. However, the taxonomic classification of Chiroptera is a more complex phenomenon. The developments of modern genetic techniques transformed the historical perception of taxonomy. The introduction of high-throughput sequencing, which enables the sequencing of an organism's whole genetic material, has made genomic studies increasingly popular in biodiversity research. This powerful technique has revealed that organisms can exhibit significant genetic variation, even when their phenotypic characteristics do not reflect this diversity. Cryptic species, which arise from the discordance between morphological similarity and genetic divergence, exemplify this phenomenon. Resolving cryptic diversity is cruicial for identying evolutionary significant units and provides a new dimension for investigating the ecological dynamics of species. Furthermore, it is a significant concept in biodiversity assessment and monitoring, essential for inforimg conservation actions. Past studies have showned that the Palearctic Region hosts a rich fauna with signifacnt cryptic diversity, including among bats. Savi's Pipistrelle, Hypsugo savii, is a small-sized, vespertilionid bat species with a broad distribution range across various ecoregions in Europe, Asia, and North Africa. The accumulation of studies showing intraspecific variation within the species has drawn attention to the investigation of possible divergence within the taxon. Several studies idenfied deeply diverged mitochondrial lineages of H. savii. Two of such lineages have been proposed as distinct species statuses, H. darwinii and H. stubbei, based on their significant divergence from H.savii. Hypsugo savii hosts possibly further cryptic diversity. It is composed of deeply diverged clades, with sympatric occurences in Northwestern Africa, the Iberian Peninsula, Italy, and some Mediterranean islands (Sardinia, Sicily, and Malta). These studies, however, are based on limited sampling from a very broad geographical range. Furthermore, they utilize realtively short mitochondrial markers and marker selection is not usually consistent among studies. This study aims to invesitage the whole mitogemones of the previously identified H. savii lineages and the recently suggested related species. There are three main objectives: 1) establishing a reproducible workflow to de novo assemble mitogenome from whole genome sequencing data; 2) de novo assembling complete mitogenome of H. savii and the related species; and 3) reconstructing their phylogenetic relationships based on whole mitogenome sequences. Thirty samples from various regions including Central Asia, Sinai Peninsula, North Africa, continental Europea, and Mediterranean islands were analysed. High-throughput shotgun sequencing data was used for the analysis. The analysis workflow covers data filtering, de novo assembling of complete mitogenomes, and annotating the obtained mitogenomes. Complete mitochondrial genomes were succesfully de novo assembled for thirty samples, representing all of the previously identified lineages, as well as the closely related species, H. stubbei and H. alaschanicus. Mitochondrial genes were annotated on the assembled genomes. The read pool of each sample was mapped to the assembled mitogenomes to assess their coverage and also to edit possible misallignments and gaps. The samples which had uncircular genomes were manual edited and circularized. The tRNA profiles were analyzed with tRNAscan-SE. Phylogenetic relations of the analysed samples were investigated reconstructing phylogenetic trees. The sequences were aligned and the pairwise distance of the sequences were calculated with MEGA11. The phylogenies were obtained with Maximum-Likelihood model with IQ-TREE and RAxML tools. The trees were visualized with FigTree abd iTOL WebServer. The alignments were analysed with PopArt for a haplotype network analysis. The highly variable non-coding D-loop regions were removed from the sequences for all these analysis . All thirty-seven mitochondrial genes were annotated: two of them were ribosomal RNAs; twenty-two were transfer RNAs; and thirteen were protein-coding genes. D-loop regions of mitogenomes were also annotated. The secondary stuctures of the tRNA profiles were calculated and illustrated. The tRNA for serine amino acid was lacking the D- arm in the secondary stucture, which had no significant impact on its functionality. Phylogenetic analysis revealed that there are three main H. savii lineages in the Western Palearctic region. The related species, H. ariel, H.alaschanicus, and H. stubbei, also formed three distinct clusters. The latter was identified in Kyrgyzstan, which is outside of its known range. The distribution of Hypsugo savii lineages were in three main regions: Eastern Mediterranean, Western Mediterranean, and North African-Southwestern Mediterranean. In Sardinia, the latter of these three lineages were found together. The pairwise genetic distance between the clades in Europe were between 8 and 10%, close to the difference observed between H. alaschanicus and the other species. Similar levels of divergences were also found between newly proposed species, H. stubbei, and the European clades. These observed high levels of mitogenomic differences suggest that the H. savii complex probably harbours further cryptic diversity.