Operational criteria for cryptic species delimitation when evidence is limited, as exemplified by North American Entomobrya (Collembola: Entomobryidae

Evaluating species boundaries remains a significant challenge in rare, difficult to collect, and/or understudied groups because of a lack of available data. Uninformative morphology, unknown ecologies and/or geographical distributions , inadequate comparative sequence data, and limited sample sizes present substantial challenges when applying commonly used methods for species delimitation. Here we present an approach that overcomes the challenges previously mentioned by integrating phylogeny, genetic distances, and a fixed diagnostic character (i.e. colour pattern) into the species delimitation process. The genus Entomobrya (Collembola) includes many species marked by complex and variable colour patterns. Many Entomobrya species have been named based exclusively on colour pattern, but the value of this character as a species-level diagnostic marker has been challenged. To test the hypothesis that colour forms in Entomobrya represent independent evolutionary lineages, i.e. distinct species, we used phylogenetic methods to evaluate the association between colour pattern and molecular variation in the cytochrome c oxidase I gene (COI) in 11 species of North American Entomobrya. The comparative analysis focused on 13 colour forms distributed amongst the species Entomobrya assuta, Entomobrya clitellaria, Entomobrya ligata, and Entomobrya quadrilineata. Phylogenetic analysis and genetic divergences sorted the 13 colour forms into seven independent evolutionary lineages, including three morphologically cryptic lineages diagnosable by colour pattern. However, genetic divergence did not always correlate with colour pattern variation, indicating that the diagnostic utility of colour pattern is species dependent and requires individual evaluation for each species. We propose that incorporation of the explicit species delimitation criteria developed for this study will result in a substantial advance in the identification and description of species in understudied taxa.