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.
16S matrix
The mitochondrial 16S alignment in fasta format
16S.fasta
cox1 matrix
The mitochondrial cox1 alignment in fasta format
cox1.fasta
EF-1α matrix
The nuclear EF-1α alignment in fasta format
EF-1alpha.fasta
PEPCK matrix
The nuclear PEPCK alignment in fasta format
PEPCK.fasta
wg matrix
The nuclear wg alignment in fasta format
wg.fasta
Species tree *BEAST input file
The species tree *BEAST input file in xml format
sp_tree_STARBEAST_input.xml
16S *BEAST tree file
The mitochondrial 16S *BEAST output tree
16S_STARBEAST.tree
cox1 pos1-2_*BEAST tree file
The mitochondrial cox1 *BEAST output tree for codon positions 1 and 2
cox1_pos1-2_STARBEAST.tree
cox1 pos3 *BEAST tree file
The mitochondrial cox1 *BEAST output tree for codon position 3
cox1_pos3_STARBEAST.tree
EF-1α *BEAST tree file
The nuclear EF-1α *BEAST output tree
EF-1alpha_STARBEAST.tree
PEPCK pos1-2 *BEAST tree file
The nuclear PEPCK *BEAST output tree for codon positions 1 and 2
PEPCK_pos1-2_STARBEAST.tree
PEPCK pos3-intron *BEAST tree file
The nuclear PEPCK *BEAST output tree for codon position 3 and intron
PEPCK_pos3-intron_STARBEAST.tree
wg *BEAST tree file
The nuclear wg *BEAST output tree
wg_STARBEAST.tree
species tree *BEAST output file
The *BEAST output species tree including the five genes (cox1 + 16S + PEPCK + EF-1α + wg)
species_tree_STARBEAST.tree
mitochondrial MrBayes input file
The concatenated mitochondrial (cox1 + 16S) MrBayes input file in nexus format
mt_MrBayes_input.nex
nuclear MrBayes input file
The concatenated nuclear (PEPCK + EF-1α + wg) MrBayes input file in nexus format
nuc_MrBayes_input.nex
concatenated MrBayes input file
The concatenated (cox1 + 16S + PEPCK + EF-1α + wg) Mrbayes input file in nexus format
combined_MrBayes_input.nex
cox1 MrBayes tree file
The mitochondrial cox1 MrBayes output tree
cox1_MrBayes_tree.nex
16S MrBayes tree flle
The mitochondrial 16S MrBayes output tree
16S_MrBayes_tree.nex
PEPCK MrBayes treee file
The nuclear PEPCK MrBayes output tree
PEPCK_MrBayes_treee.nex
EF-1α MrBayes tree file
The nuclear EF-1α MrBayes output tree
EF-1alpha_MrBayes_tree.nex
wg MrBayes tree file
The nuclear wg MrBayes output tree
wg_MrBayes_tree.nex
mitochondrial MrBayes tree file
The concatenated mitochondrial (cox1 + 16S) MrBayes output tree
mt_MrBayes_tree.nex
nuclear MrBayes tree file
The concatenated nuclear (PEPCK + EF-1α + wg) MrBayes output tree
nuc_MrBayes_tree.nex
concatenated MrBayes tree file
The concatenated (cox1 + 16S + PEPCK + EF-1α + wg) MrBayes output tree
combined_MrBayes_tree.nex
mitochondrial Garli tree file
The concatenated mitochondrial (cox1 + 16S) Garli output tree
mt_Garli_tree.tre
nuclear Garli tree file
The concatenated nuclear (PEPCK + EF-1α + wg) Garli output tree
nuc_Garli_tree.tre
concatenated Garli tree file
The concatenated (cox1 + 16S + PEPCK + EF-1α + wg) Garli output tree
combined_Garli_tree.tre
mitochondrial BEAST input file
The mitochondrial (cox1 + 16S) Beast input file (dating analysis) in xml format
mt_BEAST_input.xml
mitochondrial BEAST tree file
The mitochondrial (cox1 + 16S) Beast (dating analysis) output tree
mt_BEAST.tree
CorSiM R script
the R script of the CorSiM analyses for simulation of missing species
CorSiM_script.R