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

Siti N. Othman; Yucheol Shin; Hyun Tae Kim; Ming Feng Chuang; Yoonhyuk Bae; Jennifer Hoti; Yong Zhang; Yikweon Jang; Amaël Borzée

doi:10.1016/j.gecco.2022.e02253

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

Siti N. Othman, Yucheol Shin, Hyun Tae Kim, Ming Feng Chuang, Yoonhyuk Bae, Jennifer Hoti, Yong Zhang, Yikweon Jang, Amaël Borzée

Divison of EcoScience

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

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.

Original language	English
Article number	e02253
Journal	Global Ecology and Conservation
Volume	38
DOIs	https://doi.org/10.1016/j.gecco.2022.e02253
State	Published - Oct 2022

Bibliographical note

Funding Information:
The authors thank Hyunjung Kim for providing a photo for Fig. 5. The manuscript benefitted from reconstructive comments from Dr. Mi-Sook Min (Professor, Seoul National University, Republic of Korea). This project was financially supported by Korea Environmental Industry and Technology Institute (KEITI) of Republic of Korea under a project entitled ‘Development of behavioural ecological methods for population regulation of invasive amphibians and reptiles’ (Step 2) with grant number KEITI 2021002270001 to YJ, and the Foreign Youth Talent Program (QN2021014013L) from the Ministry of Science and Technology of the People's Republic of China to AB. SNO and AB conceived and designed the analysis. YS, HTK, MFC, YB and AB collected the data. YJ and ZY verified the data and supported the conceptual framework of the project. SNO performed the analysis, and YS, MFC and JH contributed to the data analysis tools. SNO wrote the manuscript and AB helped the interpretation of data. All authors involved in the editing of manuscript. All DNA sequences isolated in this study were deposited to Genbank (https://www.ncbi.nlm.nih.gov/nuccore) under the accession numbers: MZ349886-MZ519854 (16S rRNA gene sequences; see Table S2 in supplemental information). List of references used for reviewing the sensitivity of species assignment methods and all original codes, xml inputs and coding data generated during the analyses are available in following public database Othman, S. N. (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”, Mendeley Data, V2, doi:10.17632/mtd4yszp3d.2, https://data.mendeley.com/datasets/mtd4yszp3d/2. Any additional information required to reanalyse the data reported in this paper is available from the corresponding author upon request.

Funding Information:
The authors thank Hyunjung Kim for providing a photo for Fig. 5 . The manuscript benefitted from reconstructive comments from Dr. Mi-Sook Min (Professor, Seoul National University, Republic of Korea). This project was financially supported by Korea Environmental Industry and Technology Institute (KEITI) of Republic of Korea under a project entitled ‘Development of behavioural ecological methods for population regulation of invasive amphibians and reptiles ’ (Step 2) with grant number KEITI 2021002270001 to YJ, and the Foreign Youth Talent Program ( QN2021014013L ) from the Ministry of Science and Technology of the People’s Republic of China to AB.

Publisher Copyright:
© 2022

Keywords

Agricultural trade
Alien-invasive
Cryptic
Northeast Asia
Rana
Species delimitation

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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, Article e02253. https://doi.org/10.1016/j.gecco.2022.e02253

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abstract = "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.",

keywords = "Agricultural trade, Alien-invasive, Cryptic, Northeast Asia, Rana, Species delimitation",

author = "Othman, {Siti N.} and Yucheol Shin and Kim, {Hyun Tae} and Chuang, {Ming Feng} and Yoonhyuk Bae and Jennifer Hoti and Yong Zhang and Yikweon Jang and Ama{\"e}l Borz{\'e}e",

note = "Funding Information: The authors thank Hyunjung Kim for providing a photo for Fig. 5. The manuscript benefitted from reconstructive comments from Dr. Mi-Sook Min (Professor, Seoul National University, Republic of Korea). This project was financially supported by Korea Environmental Industry and Technology Institute (KEITI) of Republic of Korea under a project entitled {\textquoteleft}Development of behavioural ecological methods for population regulation of invasive amphibians and reptiles{\textquoteright} (Step 2) with grant number KEITI 2021002270001 to YJ, and the Foreign Youth Talent Program (QN2021014013L) from the Ministry of Science and Technology of the People's Republic of China to AB. SNO and AB conceived and designed the analysis. YS, HTK, MFC, YB and AB collected the data. YJ and ZY verified the data and supported the conceptual framework of the project. SNO performed the analysis, and YS, MFC and JH contributed to the data analysis tools. SNO wrote the manuscript and AB helped the interpretation of data. All authors involved in the editing of manuscript. All DNA sequences isolated in this study were deposited to Genbank (https://www.ncbi.nlm.nih.gov/nuccore) under the accession numbers: MZ349886-MZ519854 (16S rRNA gene sequences; see Table S2 in supplemental information). List of references used for reviewing the sensitivity of species assignment methods and all original codes, xml inputs and coding data generated during the analyses are available in following public database Othman, S. N. (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”, Mendeley Data, V2, doi:10.17632/mtd4yszp3d.2, https://data.mendeley.com/datasets/mtd4yszp3d/2. Any additional information required to reanalyse the data reported in this paper is available from the corresponding author upon request. Funding Information: The authors thank Hyunjung Kim for providing a photo for Fig. 5 . The manuscript benefitted from reconstructive comments from Dr. Mi-Sook Min (Professor, Seoul National University, Republic of Korea). This project was financially supported by Korea Environmental Industry and Technology Institute (KEITI) of Republic of Korea under a project entitled {\textquoteleft}Development of behavioural ecological methods for population regulation of invasive amphibians and reptiles {\textquoteright} (Step 2) with grant number KEITI 2021002270001 to YJ, and the Foreign Youth Talent Program ( QN2021014013L ) from the Ministry of Science and Technology of the People{\textquoteright}s Republic of China to AB. Publisher Copyright: {\textcopyright} 2022",

year = "2022",

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doi = "10.1016/j.gecco.2022.e02253",

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Othman, SN, Shin, Y, Kim, HT, Chuang, MF, 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, vol. 38, e02253. https://doi.org/10.1016/j.gecco.2022.e02253

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. / Othman, Siti N.; Shin, Yucheol; Kim, Hyun Tae et al.
In: Global Ecology and Conservation, Vol. 38, e02253, 10.2022.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - 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

AU - Othman, Siti N.

AU - Shin, Yucheol

AU - Kim, Hyun Tae

AU - Chuang, Ming Feng

AU - Bae, Yoonhyuk

AU - Hoti, Jennifer

AU - Zhang, Yong

AU - Jang, Yikweon

AU - Borzée, Amaël

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KW - Cryptic

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KW - Species delimitation

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