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Abstract

This protocol presents a field-tested example workflow for the RFLP assay described in Cohn et al. (2026), used for quickly and cost-effectively identifying morphologically cryptic Pocillopora spp. in French Polynesia. The method is highly modular, and therefore flexible, to suit a variety of needs regarding project design, timelines, and equipment availability. See image above (and attached below) for a visual representation of the workflow. This protocol was designed for use in a field laboratory with limited access to full-size equipment (e.g., PCR thermalcyclers) but would require only minimal modifications for use in a standard molecular laboratory facility. Note that analysis can be performed after the completion of lab work or in parallel with lab work, depending on logistical needs. Analysis performed in parallel with lab work increases processing time but can greatly reduce cost and use of reagents, depending on the species present, because redundant steps can be omitted. Cohn et al. (2026) Abstract: Pocillopora is a widespread, dominant reef-building coral genus in the Indo-Pacific that exhibits high morphological similarity and plasticity. Given this, genetic tools are needed to robustly identify Pocillopora individuals to the species level. Quick and accurate identification approaches for Pocillopora species are critical to estimating biodiversity patterns under current and future environmental challenges. In recent years, the mitochondrial open reading frame (mtORF) and a histone region (PocHistone) have been validated using genome-wide data to become the most widely used species-level markers for Pocillopora. However, Sanger sequencing of a large number of samples can be prohibitively expensive and sequencing facilities are not always readily available. Therefore, we present restriction fragment length polymorphism (RFLP) digests here that identify the six species of Pocillopora (P. acuta, P. cf. effusa, P. grandis, P. meandrina, P. tuahiniensis, and P. verrucosa) found in French Polynesia, without sequencing. In uninformed validation tests (in silico and in vitro), our protocol identified each Pocillopora species with 100% accuracy. Given their cost-effective, rapid nature, the tailoring of additional RFLP digest protocols to identify cryptic coral species in reef regions around the world will support foundational reef science, conservation and restoration initiatives. Cohn, F. M., Johnston, E., Burgess, S. C., Sims, J. A., Layagala, K., Harnay, P., Putnam, H. M., & Correa, A. M. (2026). A Genetic Method for Distinguishing Cryptic Pocillopora Species in French Polynesia without Sequencing. Ecology & Evolution, 16(10):e74409. https://doi.org/10.1002/ece3.74409