While previous studies have highlighted the potential of DNA‐based methods for the biomonitoring of freshwater macroinvertebrates, a limited number have investigated homogenization of bulk samples that include debris, in order to reduce sample‐processing costs. This study explores the use of several DNA‐based survey methods for water quality and biodiversity assessment in South Africa, comparing morphological and molecular‐based identification of freshwater macroinvertebrates at the family level and the level of molecular operational taxonomic units (mOTUs). Seven sites were studied across three rivers with four different sample types collected per site: a standard SASS biomonitoring sample split into a picked sample (also used for morphological identification) and a leftover debris sample; a more intensive‐search comprehensive sample; and a filtered water eDNA sample. DNA‐based methods recovered higher diversity than morphology, but did not always recover the same taxa, even at the family level. Regardless of the differences in SASS taxon scores, most DNA‐based methods, except a few eDNA samples, returned the same water quality assessment category as the standard morphology‐based assessment. Homogenized comprehensive samples recovered more freshwater invertebrate diversity than all other methods, suggesting the standardized SASS method overlooks taxa. The eDNA samples recovered more diversity than any other method; however, 90% of the reads were nontarget and as a result eDNA recovered the lowest target (macroinvertebrate) diversity. However, eDNA did find some target taxa that all other methods failed to detect. This study shows that unsorted bulk samples have the potential to be used for water quality biomonitoring, providing higher diversity estimates for macroinvertebrates than either SASS picked or eDNA samples. These results also show the value of incorporating DNA‐based approaches into existing South African metrics, providing additional taxonomic resolution to develop more refined metrics for biodiversity management.

BackgroundBaetis 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.ResultsFour 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).ConclusionsThe 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.

Many studies have highlighted the potential of DNA-based methods for the biomonitoring of freshwater macroinvertebrates, however only a few studies have investigated homogenisation of bulk samples that include debris to reduce sample-processing time. In order to explore the use of DNA-based methods in water quality assessment in South Africa, this study compares morphological and molecular-based identification of freshwater macroinvertebrates at the mixed higher taxon and mOTU level while investigating abundance and comparing mOTU recovery with historical species records. From seven sites across three rivers in South Africa, we collected a biomonitoring sample, an intensive-search comprehensive sample and an eDNA sample per site. The biomonitoring sample was picked and scored according to standard protocols and the leftover debris and comprehensive samples were homogenised including all debris. DNA-based methods recovered higher diversity than morphology, but did not always recover the same taxa, even at the family level. Regardless of the differences in taxon scores, most DNA-based methods except some eDNA samples, returned the same water quality assessment category as the standard morphology-based assessment. Homogenised comprehensive samples recovered more freshwater invertebrate diversity than all other methods. The eDNA samples recovered 2 to 10 times more mOTUs than any other method, however 90% of reads were non-target and as a result eDNA recovered the lowest target diversity. However, eDNA did find some target taxa that the other methods failed to detect. This study shows that unsorted samples recover the same water quality scores as a morphology-based assessment and much higher diversity scores than both picked and eDNA samples. As a result, there is potential to integrate DNA-based approaches into existing metrics quickly while providing much more information for the development of more refined metrics at the species or mOTU level with distributional data which can be used for conservation and biodiversity management.