Molecular barcoding of orchids remains challenging and unreliable owing to the wide diversity within the family combined with poorly populated sequence databases. In highly diverse tropical ecosystems, identifying orchid taxa is challenging using morphological characteristics alone. This problem is compounded in seedlings, juvenile individuals and root samples that do not have reproductive structures. Current orchid barcoding research has utilized a multilocus approach, combining different barcode regions including nrITS, matK, trnH-psbA and trnL-F. To establish a reference database for a long term ecological study and to enable barcode based identification of orchids encountered in Central America, we collected up to five flowering individuals from each morpho-species from our study site in the Parque Nacional Tapantí in Costa Rica, which harbors more than 400 orchid species altogether. Each morpho-species was photographed, morphologically identified, and vouchered. Total genomic DNA was extracted from leaf tissues, the ITS region was amplified using 17SE/26SE while the matK region was amplified using a new primer combination 19F/1300R to obtain longer amplicons. Edited sequences were used for BLAST searches against GenBank/NCBI database to assess taxonomic identifications.
Twenty-six species representing 16 genera and five subtribes were morphologically identified in this survey. Sixteen of the 26 species were members of the highly diverse subtribe Pleurothallidinae. The primer pair 17SE and 26SE amplified the entire ITS region for each orchid species. With the primer combination 19F and 1300R, we were able to cover 1000 bases of the matK region. Only nine of our sequences had over 97% similarity with currently available sequences in GenBank, which highlights the of updated gene information from tropical orhids in publically available genomic databases. Our Tapanti orchid sequence reference database will be updated on a regular basis when new surveys are made throughout the year.