Background
The emergence of DNA taxonomy sparked a paradigm shift in biodiversity assessments and revealed the existence of many morphologically cryptic species in all ecosystems worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, an increasing number of studies report patterns of mito-nuclear discordance originating for example from shared ancestral polymorphisms or introgression, leading to an over- or underestimation of true species diversity. Therefore, reports of mitochondrial lineages should be verified by other methods. Freshwater amphipods are ideal model organisms since putative cryptic species have been reported in many of the nominal taxa. In this study, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within the Mediterranean freshwater amphipod Echinogammarus sicilianus on Sicily. For validation, we used an integrative taxonomic approach combining DNA barcoding with behavioural data, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq).
Results
Within a relatively small sampling area (approx. 200 km2), we detected 12 divergent COI MOTUs, with genetic distances ranging from 1.8% to 20.3% and a mean distance of 11%. At 17 of the 25 sampling sites up to six MOTUs occurred in syntopy. The four most common MOTUs were analysed with high resolution morphometric data (49 measurements per specimen), but we found no clear evidence of morphological differentiation among them. We also observed a high level of MOTU intermixing (38%) in precopulatory pairs. Similarly, ten of the mitochondrial MOTUs were not supported by the genome-wide ddRAD-seq data. However, for the two remaining mitochondrial MOTUs found at sites outside of the main river stretch, ddRAD-seq and COI data were congruent, indicating that those MOTUs might indeed represent cryptic species.
Conclusions
Our study on the amphipod E. sicilianus reveals another prominent example of mito-nuclear discordance indicating that species delimitation based solely on mitochondrial markers can significantly overestimate species diversity. The study highlights that integrative taxonomy approaches are important for validating species status, especially in hyperdiverse species complexes, where many mitochondrial MOTUs occur in close proximity or even syntopy.