Evaluating the efficiency of popular species identification analytical methods, and integrative workflow using morphometry and barcoding bioinformatics for taxonomy and origin of traded cryptic brown frogs

The unregulated wildlife trade increases the risk of global biological invasions and, therefore, accurate taxonomic assignments and origin-tracing methods are critical. First, using a meta-analysis comparing studies from the last 10 years, we quantified the efficiency of popular analytical methods used for species assignments and confirmed the higher sensitivity of coalescent-based methods in isolating cryptic operational taxonomic units (OTUs). Second, we proposed a workflow for species identification of unidentified animals from the trade, in this case cryptic Brown frogs (Rana) imported into the Republic of Korea. This integrated workflow relies on the use of a single-locus 16S rRNA gene barcoding along with morphometry, phylogenetic trait, species delimitation modelling and phylogeography. Out of 171 samples, we identified three erroneously imported non-native species: R. chensinensis, R. amurensis and R. kukunoris. Bayes factor delimitation modelling most supported the presence of 12 OTUs from the trade, highlighting a hidden genetic diversity. Both molecular and morphological analyses converged towards a high phenotypic crypticity and similarity in genetic sequences between Korean R. huanrenensis and Chinese R. chensinensis. The combined model-based OTUs and 16S rRNA gene phylogeny of traded and control specimens (n = 230) recovered the trade pathways, and revealed the widespread and likely wild-harvested origins of traded Rana individuals. Our results also highlight the independent evolution of toe webbings in Rana for the last 12.0 Mya, a potential key trait for species identification of northeastern Asian Rana. With the workflow for large-scale species identification developed herein, we urge the development of trade monitoring and legislation on Rana species in northeast Asia.