The importance of setting the right genetic distance threshold for identification of clones using amplified fragment length polymorphism: a case study with five species in the tropical plant genus <I>Piper</I>

Abstract

Abstract Amplified fragment length polymorphism (AFLP) has been widely used for clone identification, but numerous studies have shown that clonemates do not always present identical AFLP fingerprints. Pairwise AFLP distances that distinguish known clones from nonclones have been used to identify a threshold genetic dissimilarity distance below which samples are considered to represent a single clone. Most studies to date have reported threshold values between 2% and 4%. Here, I determine the consistency of the clonal threshold across five species in the tropical plant genus Piper, and evaluate the sensitivity of genetic diversity indices and estimates of frequency of clonal reproduction to the threshold value selected. I sampled multiple ramets per individual from widely distributed plants for each of the five Piper species to set a threshold at the point where the error rate of clonal assignments was lowest. I then sampled all individuals of each shade-tolerant species in a 1-ha plot, and of each light-demanding species in 25 x 35-m plot, to estimate the frequency of asexual recruitment in natural populations using a series of different thresholds including the threshold set with the preliminary sampling. Clonal threshold values for the different species ranged from 0% to 5% AFLP genetic dissimilarity distance. To determine the sensitivity of estimates of clonal reproduction, I calculated several clonal diversity indexes for the natural populations of each of the five species guided by the range in clonal threshold values observed across the five Piper species. I show that small changes in the value of the clonal threshold can lead to very different conclusions regarding the level of clonal reproduction in natural populations.