ABSTRACT: Species delimitation among closely related species is challenging because traditional phenotype-based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high-throughput sequencing, it has become increasingly cost-effective to acquire genome-scale data which can resolve previously ambiguous species boundaries. As the availability of genome-scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro-endemism in a lineage of lichen-forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single-locus and genome-scale data consisting of (a) single-locus species delimitation analysis using ASAP, (b) maximum likelihood-based phylogenetic tree inference, (c) genome-scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross-validate results between genome-scale and single-locus datasets, differently sampled subsets of genomic data and to control for population-level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro-endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.
