Cryptic variation in an ecological indicator organism: mitochondrial and nuclear DNA sequence data confirm distinct lineages of Baetis harrisoni Barnard (Ephemeroptera: Baetidae) in southern Africa

Baetis harrisoni Barnard is a mayfly frequently encountered in river studies across Africa, but the external morphological features used for identifying nymphs have been observed to vary subtly between different geographic locations. It has been associated with a wide range of ecological conditions, including pH extremes of pH 2.9-10.0 in polluted waters. We present a molecular study of the genetic variation within B. harrisoni across 21 rivers in its distribution range in southern Africa. Four gene regions were examined, two mitochondrial (cytochrome c oxidase subunit I [COI] and small subunit ribosomal 16S rDNA [16S]) and two nuclear (elongation factor 1 alpha [EF1α] and phosphoenolpyruvate carboxykinase [PEPCK]). Bayesian and parsimony approaches to phylogeny reconstruction resulted in five well-supported major lineages, which were confirmed using a general mixed Yule-coalescent (GMYC) model. Results from the EF1α gene were significantly incongruent with both mitochondrial and nuclear (PEPCK) results, possibly due to incomplete lineage sorting of the EF1α gene. Mean between-clade distance estimated using the COI and PEPCK data was found to be an order of magnitude greater than the within-clade distance and comparable to that previously reported for other recognised Baetis species. Analysis of the Isolation by Distance (IBD) between all samples showed a small but significant effect of IBD. Within each lineage the contribution of IBD was minimal. Tentative dating analyses using an uncorrelated log-normal relaxed clock and two published estimates of COI mutation rates suggest that diversification within the group occurred throughout the Pliocene and mid-Miocene (~2.4-11.5 mya). The distinct lineages of B. harrisoni correspond to categorical environmental variation, with two lineages comprising samples from streams that flow through acidic Table Mountain Sandstone and three lineages with samples from neutral-to-alkaline streams found within eastern South Africa, Malawi and Zambia. The results of this study suggest that B. harrisoni as it is currently recognised is not a single species with a wide geographic range and pH-tolerance, but may comprise up to five species under the phylogenetic species concept, each with limited pH-tolerances, and that the B. harrisoni species group is thus in need of taxonomic review.