Abstract
Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.
Highlights
The accurate delimitation of species is an essential step in evolutionary biology, ecology, and conservation research [1,2,3]
While the few studies that compared mitochondrial DNA (mtDNA) groups with ribosomal DNA genotypes found the two markers to be largely congruent (e.g. [14]), it remains to be determined whether nuclear DNA forms sequence clusters that can be statistically identified with a coalescent approach and that are comparable to species
The resulting phosphoenolpyruvate carboxykinase (PEPCK) alignment length (419 bp) was shorter than cox1 (658 bp), and there were more PEPCK genotypes than cox1 haplotypes, the number and proportion of parsimony-informative sites was higher for cox1 (Table 2)
Summary
The accurate delimitation of species is an essential step in evolutionary biology, ecology, and conservation research [1,2,3]. The GMYC approach estimates species boundaries directly from branching rates in mixed populationphylogenetic trees without the need for any prior definition of populations or species. This makes it suitable for large-scale, multispecies studies of taxonomic groups for which few genetic markers are readily available. Most studies have relied on a single locus, often mitochondrial DNA (mtDNA), for GMYC analysis. While the few studies that compared mtDNA groups with ribosomal DNA (rDNA) genotypes found the two markers to be largely congruent [14]), it remains to be determined whether nuclear DNA (nDNA) forms sequence clusters that can be statistically identified with a coalescent approach and that are comparable to species While the few studies that compared mtDNA groups with ribosomal DNA (rDNA) genotypes found the two markers to be largely congruent (e.g. [14]), it remains to be determined whether nuclear DNA (nDNA) forms sequence clusters that can be statistically identified with a coalescent approach and that are comparable to species
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