Abstract
Orchidaceae is among the largest plant families in the world; that is why it is notoriously difficult to identify species using traditional taxonomic methods. This problem is especially apparent in the Andean region of South America, a global hotspot for orchid diversity. Plant DNA barcoding has been proposed to help with orchid species discrimination. However, the utility of the rbcL and matK DNA barcode markers to discriminate between orchid species still needs investigating. The goal of this study is to determine the utility of the rbcL and matK DNA barcode markers to discriminate orchid species from a localized Andean montane forest in Ecuador. A total of 174 samples from the Siempre Verde Preserve, Ecuador, were sequenced, returning 51 unique rbcL and 81 unique matK barcode gene region sequences. Similar rates of sequencing success, rbcL (76%) and matK (75%), were found for both gene regions. Sequences were then queried to GenBank, where top matches were compared to the taxonomic identification of samples. Analysis of 132 unique orchid sequences indicated that, overall, the matK gene region was more robust for species (63%) and genus level (78%) discrimination compared to the rbcL gene region (species: 29%; genus: 36%). However, for 11 of 29 genera, rbcL and matK had similar success at genus level discrimination. Collectively, results emphasize the utility of the matK DNA barcode for the discrimination of orchid taxa and the importance of incorporating taxonomic and genetic methods when investigating the identity of plant species from complex groups.
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Availability of data and material
The orchid flora of Siempre Verde can be found in Online Resource 1. The GenBank accession numbers for all successfully sequenced rbcL and matK DNA barcodes can be found in Online Resource 2. Sequences are also publicly available on the Barcode of Life Data System (BOLD). Alignments used to generate the phylogenetic trees in Fig. 1, Online Resource 5, and Online Resource 6 can be found at https://doi.org/10.5281/zenodo.6377406.
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Acknowledgements
We wish to thank the Ministry of the Environment of Ecuador for granting the permit MAE-DNB-CM-2015-0031 to carry out this research. We thank the Lovett School, Department of Biology and Graduate School at Columbus State University, and Pontificia Universidad Católica del Ecuador for logistical support. We thank Daniela Cevallos and Nicolas Zapata for assistance with DNA voucher collections and Maria (Masha) Kuzmina at the Biodiversity Institute of Ontario for her invaluable assistance with DNA barcoding.
Funding
All fieldwork and laboratory work was supported by the Lovett School, the Herbario-QCA at Pontificia Universidad Católica del Ecuador, and the Department of Biology and Graduate School at Columbus State University. SJW and KB were supported by the Center for Global Engagement at Columbus State University, William S. Birkhead Fund for Biology, and Student Research and Creative Endeavors Grants.
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KB and KSB took part in conceptualization; KB, SJW, AR, ÁJP, KSB involved in methodology; KB, SJW, and EP involved in data curation; SJW, KB, AR, JC-S, ÁJP, and KSB participated in funding acquisition; SJW and KB took part in formal analysis; SJW involved in visualization; KSB took part in supervision; SJW, KB, and KSB involved in writing—original draft; all authors contributed to the writing, reviewing, and editing.
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Online Resource 1. The current orchid flora of Siempre Verde. Formal documentation of the flora began in 2013 and is ongoing.
Online Resource 2. Specimens for which samples were taken for DNA barcode sequencing.
Online Resource 3. Sequence variation between samples for the rbcL gene region determined using the dist.alignment function of the “seqinr” package (Charif and Lobry 2007) in R programming language (R Core Team 2019).
Online Resource 4. Sequence variation between samples for the matK gene region determined using the dist.alignment function of the “seqinr” package (Charif and Lobry 2007) in R programming language (R Core Team 2019).
Online Resource 5. Phylogenetic tree depicting genetic relationships among 132 orchid samples based on the rbcL gene region.
Online Resource 6. Phylogenetic tree depicting genetic relationships among 131 orchid samples based on the matK gene region.
Online Resource 7. Matches of sequences queried to GenBank using BLASTn analysis.
Online Resource 8. Matches of sequences queried to GenBank using BLASTn analysis for samples either not taxonomically identified to the species level or without a species level sequence on GenBank for the sequence to match when queried.
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Worthy, S.J., Bucalo, K., Perry, E. et al. Ability of rbcL and matK DNA barcodes to discriminate between montane forest orchids. Plant Syst Evol 308, 19 (2022). https://doi.org/10.1007/s00606-022-01809-z
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DOI: https://doi.org/10.1007/s00606-022-01809-z