Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA

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.

Figure 1. Sampling of European Rhithrogena.

Filled circles represent the sampled localities.

Figure 2. Bayesian majority-rule consensus tree of the reduced cox1 data set obtained using MrBayes.

Lineages 1 to 4 correspond to four different Rhithrogena morphological species groups (1: alpestris; 2: loyolaea; 3: diaphana; 4: semicolorata). Lineage 5 includes clades belonging to the hercynia species group (arrows) and the hybrida species group. Triangles represent collapsed lineages, (width proportional to the number of haplotypes). Filled stars indicate posterior probabilities (PP)>0.95, open stars indicate PP>0.75.

Figure 3. Bayesian majority-rule consensus tree of the reduced PEPCK data set obtained using MrBayes.

Lineages 1 to 4 correspond to four different Rhithrogena morphological species groups (1: alpestris; 2: loyolaea; 3: diaphana; 4: semicolorata). Lineage 5 includes clades belonging to the hercynia species group (arrows) and the hybrida species group. Triangles represent collapsed lineages, (width proportional to the number of haplotypes). Filled stars indicate posterior probabilities (PP)>0.95, open stars indicate PP>0.75.

Figure 4. Clock-constrained Bayesian maximum clade credibility tree of the complete cox1 data set obtained using BEAST.

The tree (upper panel), obtained under a relaxed lognormal molecular clock, is presented with its corresponding lineage-through-time plot (lower panel). The broken vertical line indicates the point of maximum likelihood fit of the single-threshold GMYC model, i.e. the point of transition from interspecies (Yule) to intraspecies (coalescent) branching events. The grey shading corresponds to the confidence interval of the transition point. The bars indicate significant clusters (arrows: significant singletons) that are inferred to be species. The five species groups are specified on subtending branches. All GMYC clusters were well supported (PP≥0.99).

Figure 5. Bayesian majority-rule consensus tree of the complete PEPCK data set obtained using MrBayes.

Triangles represent collapsed lineages, where filled triangles indicate clades congruent with cox1 GMYC species (see Table 4) and open triangles indicate the clades containing a mixture of Rh. gratianopolitana individuals (GMYC species 16 and 17; see Fig. 4 and section 3.4). For each collapsed lineage, filled stars indicate PP values>0.95, open stars indicate PP>0.75. The five species groups are specified.