| Summary: | Abstract Premise The gametophytes of different fern species collected in the field can be difficult to distinguish because of their morphological similarities. Nonetheless, emerging molecular ecology techniques are starting to be used to tackle such limitations. Here, using case studies and a detailed protocol, we demonstrate a convenient methodology, tissue‐direct PCR (TD‐PCR), that foregoes a traditional DNA extraction and facilitates the identification of fern gametophytes, as well as enabling the elucidation of their natural distribution. Methods Based on updated plastome information, we designed a universal primer set targeting the trnL‐L‐F region, which is effective across extant ferns. We used this primer set to perform TD‐PCR on the case‐studied populations of Taiwanese Lomariopsis gametophytes, using the generated sequences for their identification. In the case study concerning the microhabitat preference of Vaginularia junghuhnii, we designed and used a taxon‐specific primer set. Results Compared with approaches requiring DNA extraction, the use of TD‐PCR with either universal or taxon‐specific primers could save significant time, money, labor, and research materials in the genetic identification of fern gametophytes. Discussion The use of modern genetic tools can aid in the identification of fern gametophytes. An updated TD‐PCR strategy not only facilitates the DNA‐based identification of gametophytes, but also promotes new avenues of research for investigating these plants in the field.
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