Reassessing species demarcation criteria in viroid taxonomy by pairwise identity matrices.

With a small, circular and non-protein coding RNA genome, viroids are the smallest infectious agents. They invade plants, which in turn may develop symptoms. Since their discovery about 50 years ago, more than thirty viroids have been reported and classified using as species demarcation less than 90 per cent sequence identity on the overall genome and evidence of biological divergence with respect to the closest related viroids. In the last few years, new viroids have been identified that infect latently their (frequently) woody hosts and have a narrow experimental hosts range, complicating and slowing down studies on their biology. As a consequence, several viroids are still waiting for classification. Moreover, the number of new viroids is expected to increase in the next years due to the use of high-throughput sequencing technologies with diagnostics purposes. Therefore, establishment of reliable species demarcation criteria mainly based on molecular features of viroids is needed. Here, viroid classification is reassessed and a scheme based on pairwise sequence identity matrices is developed. After identifying a threshold pairwise identity score (PWIS) for each viroid genus, to be used as a species demarcation criterion, we show that most of those yet unclassified viroids can be assigned to a known or to a new species, thus limiting the need for additional biological evidence to only a few more complex situations. The advantages of this PWIS-based method are that the proposed identity thresholds for species demarcations are not arbitrarily established and evidence for biological divergence is not mandatory. Importantly, the current classification is not essentially modified. A protocol for a tentative fast classification of new viroids according to the proposed approach is also provided.