title: Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).
abstract: BACKGROUND
  Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown.
  
  
  RESULTS
  Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups.
  
  
  CONCLUSIONS
  Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects.
authors.affiliation: Musée cantonal de zoologie, Palais de Rumine, Place de la Riponne 6, 1014, Lausanne, Switzerland. laurentvuataz@yahoo.fr.|Department of Ecology and Evolution, Biophore, University of Lausanne, 1015, Lausanne, Switzerland. laurentvuataz@yahoo.fr.
authors.forename: Laurent
authors.identifier: http://orcid.org/0000-0001-9193-8683
authors.initials: L
authors.lastname: Vuataz
authors.affiliation: Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), 12587, Berlin, Germany.|Berlin Center for Genomics in Biodiversity Research, 14195, Berlin, Germany.|Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310, Vigo, Spain.
authors.forename: Sereina
authors.initials: S
authors.lastname: Rutschmann
authors.affiliation: Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), 12587, Berlin, Germany.|Berlin Center for Genomics in Biodiversity Research, 14195, Berlin, Germany.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Monaghan
authors.affiliation: Musée cantonal de zoologie, Palais de Rumine, Place de la Riponne 6, 1014, Lausanne, Switzerland.|Department of Ecology and Evolution, Biophore, University of Lausanne, 1015, Lausanne, Switzerland.
authors.forename: Michel
authors.initials: M
authors.lastname: Sartori
keywords: Dated phylogeny; Diversification; Ephemeroptera; Freshwater insects; Headwater; Lineage-through-time; Mayfly; Pleistocene glaciations; Speciation