Genetic diversity, color pattern, and cryptic species delimitation in North American Entomobrya (Collembola)

Molecular phylogenetic studies have revealed that cryptic species represent a substantial proportion of earth's biodiversity. However, evaluating species boundaries remain a significant challenge in rare, difficult to collect, and/or understudied groups due to a lack of available data. Here we present an integrative approach that incorporates phylogeny, genetic distances, and a fixed diagnostic character (e.g. color pattern), to delineating species boundaries for morphologically cryptic groups characterized by high molecular divergence with unknown ecologies and/or distributions using Collembola, a largely understudied group of hexapods. Recent studies show that Collembola diversity estimates based on morphological characters have been greatly underestimated. High levels of DNA sequence divergence among congeneric species suggest that circumscription of species based solely on traditional morphological and color pattern characters provide a very conservative estimate of actual species diversity.  Many described species in the genera Orchesella, Isotomurus and Lepidocyrtus have been shown to comprise complexes of several morphologically cryptic species diagnosable by color pattern.  The genus Entomobrya includes many species marked by distinct, often complex, and variable color patterns, but to date, no one has explored the possible presence of cryptic species using DNA sequencing within this group.  In order to evaluate the association between color pattern and molecular variation and test the hypothesis that color forms within Entomobrya morphospecies represent distinct species, a phylogenetic analysis of the complete cytochrome c oxidase I gene (COI) was performed for 11 species of North American Entomobrya, including 13 color forms representing 4 morphospecies with different levels of color pattern variation: Entomobrya assuta, E. clitellaria, E. ligata, and E. quadrilineata. The analysis reveals genetic divergences reaching 18.5% between different color forms of the same morphospecies, equivalent to distances observed between other Entomobrya sister species differentiated by morphology. However, genetic divergence did not always correlate with color pattern variation, suggesting that the diagnostic utility of color pattern is species dependent and requires individual evaluation for each species. Following an integrative framework for species delimitation based on explicit species criteria developed for this study, 3 morphologically cryptic lineages, each diagnosable by color pattern, are identified and supported by evidence obtained from phylogeny, genetic distances, geographic distribution, and color pattern.