Molecular Phylogeny and Phylogeography of Reed Warblers and Allies (Aves: Acrocephalidae)

Reed warblers and allies (family Acrocephalidae) present a long history of classification and successive revisions. Due to a remarkable similarity in morphology and rapid radiation, the previous studies failed to solve all phylogenetic relationships between and within the genera. In this study DNA sequences from eight mitochondrial and nuclear loci (total, 6280 nt) as well as genomic ISSR fingerprinting were implemented to reconstruct the phylogenetic relationships among 35 species of reed warblers. The results are congruent with previous molecular analyses (Fregin et al. 2009; Helbig & Seibold 1999; Leisler et al. 1997) but support some of the sister-group relationships more strongly. Based on the analyses, the major clades of Acrocephalus, Iduna (except I. aedon), Hippolais, Nesillas and Calamonastides were recovered. The current taxonomic position of Calamonastides gracilirostris as a monotypic genus in the tree was supported. Although the inclusion of Chloropeta natalensis and Chloropeta similis in genus Iduna was not robustly supported, their close relationship could not be rejected. No support was found for inclusion of aedon in genus Iduna as suggested by Fregin et al. (2009), hence the resurrection of genus Phragamaticola for this species is proposed. A molecular clock analysis confirmed the hypothesis of the rapid radiation of the family from middle Miocene (12.5 MYA). This short evolutionary time causes low nodal support at the base of the some clades. Furthermore, phylogeographic patterns and evolutionary history of the Eurasian reed warbler (Acrocephalus scirpaceus) and marsh warbler (Acrocephalus palustris) were investigated. Two mitochondrial loci (cyt b and COI) and a combination of phylogeographic tools, molecular dating, and population genetic methods were employed to address several questions regarding the genetic diversity of these two long-distance migratory reed warblers across their widespread geographic distribution range. Investigation of mtDNA diversity among more than 400 individuals of A. scirpaceus from 20 sampling sites recovered three lineages. They correspond to three subspecies and split about 0.4 million years ago: one spanning Asia (A. s. fuscus); one encompassing Europe and Northern Africa (A. s. scirpaceus); and a third, including Eastern Africa and South-western Asia (A. s. avicenniae). Results from BEAST further suggested that the third clade has the basal position and diverged from its sister species African reed warbler A. baeticatus ca. 0.6 million years ago. This primitive subspecies (avicenniae) may have survived in one of the African refugia, likely low forest refugium of the Ethiopian Highlands during the Last Glacial Maximum. Moreover, DNA analysis could detect high number of identification errors between reed warblers in the field. In total, 6.8% of Eurasian reed warblers were misidentified as the wrong species. A. palustris was the most misidentified species; and at the subspecies level, it increased to 72.7% of the specimens. Based on the molecular analysis we could also report a first evidence of the Asian subspecies A. s. fuscus in Central Europe (Treysa, Germany). In contrast to the results of A. scirpaceus, the phylogeographic structure in ca. 230 individuals from 10 breeding, migrating and wintering populations of A. palustris presented a shallow divergence. The analyses based on both mtDNA loci failed to detect any population subdivision and indicate occupation of a single glacial refugium during the Pleistocene glaciation. High levels of gene flow among breeding populations (Nm = 13.69) is another factor which leads to high degree of admixture. Low nucleotide diversity, shallow phylogenetic tree, star-like haplotype network, unimodal mismatch distribution and the time to the most recent common ancestor (TMRCA = 0.45 MYA) all point to their recent origin during the last glacial periods.