From the wild to our plates: crypticity in the northeast Asian trade of brown frogs – a muti-tool approach

New lab paper on the trade of Brown Frogs (Rana sp.) in northeast Asia, including the delineation of cryptic species, and morphological tools for species identification to act against the trade of mis-identified individuals.

The trade of amphibians increases the risk of invasions across the world, and northeast Asia. To understand the trade in the area, we developed an accurate species assignment and workflow guiding a broad-scale species identification and resolving the trade pathways. We tested our workflow on unidentified cryptic brown frogs imported into the Republic of Korea.

Comparative species delimitations determining the OTUs on a calibrated timetree and matching comparative morphometry. (A) Comparative species delimitation based on the 16S rRNA gene barcode marker for 171 individuals of Rana originated from the trade determined by Automatic Barcode Gap (ABGD), General Mixed Yule Coalescent (GMYC) and Bayesian-Poisson Tree Process (bPTP) schemes. The partitions of the OTUs for each scheme is indicated below the tree nodes with specific colour gradients. The principal component analysis (PCA) for the 15 morphometric variables analysed for 208 individuals originating from the trade and a museum collection adjusted for (B) polyphyletic clades, and (C) parapatric clades (statistical analyses in Supplementary Tables S7 and S9). Each Rana clade is colour coded, matching with the colour of its range on the distributional map. The placement of R. uenoi in clade 2 was adapted from the Bayesian Inference tree and marked in the time tree (dashed box). 

The workflow combined morphometry, phylogenetic trait, species delimitation modelling and phylogeography, and successfully identified three non-native species imported in Korea: Rana chensinensis, Rana amurensis and Rana kukunoris out of 171 specimens bought from the trade.

Using a machine-learning approach with Bayes Factor Delimitation method to evaluate the taxonomy models from four commonly used species delimitation tools (BLAST, ABGD, sGMYC and bPTP), and isolated 12 operational taxonomic units (OTUs) from the 171 individuals. We then combined the information from the 12 OTUs and combined it into a 16S phylogeny including traded and control specimens (n = 230).

The model-based OTUs and phylogeny revealed the widespread and likely wild-harvested origins of traded Rana individuals, cycled through farm in China to be integrated in the legal trade. Apart from the genetic, our morphometry also indicates extremely high phenotype crypticity between the endemic Korean species, especially R. huanrenensis and those brown frogs from the trade.

 However, the trait evolution results also highlight a pattern of independent evolution of toe webbings in Rana for the last 12.0 Mya, most likely a useful key-trait for Rana species identification across northeast Asian. With the workflow for broad-scale species identification developed herein, we urge the development of trade monitoring and legislation on Rana species in northeast Asia.

Phenotypic evolution of toe webbing in selected northeast Asian Rana since the Miocene. The ancestral range reconstruction from the calibrated species tree including 181 individuals includes six taxa from northeast Asian Rana (385 bp of 16S rRNA). The colour coding in the trait value indicates the rate of evolution in phenotypic trait related to the angle made by the webbing between webbed toes I and II (WTA). The species the closest to the ancestral character is Rana coreana and the derived character rose in R. kukunoris. The earliest and the most recent transition times for WTA character to evolve from the ancestral to the youngest clades mark on the tree time with straight dotted and solid arrows, respectively.

Othman S. N., Shin Y., Kim H.-T., Chuang M.-F., Bae Y., Hoti J., Zhang Y., Jang Y. & Borzée A. (2022).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.Global Ecology and Conservation. 38:e02253. DOI: 10.1016/j.gecco.2022.e02253

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