Gene flow increases phylogenetic structure and inflates cryptic species estimations: a case study on widespread Philippine puddle frogs (Occidozyga laevis)

In cryptic amphibian complexes, there is a growing trend to equate high
levels of genetic structure with hidden cryptic species diversity.
Typically, phylogenetic structure and distance-based approaches are used
to demonstrate the distinctness of clades and justify the recognition of
new cryptic species. However, this approach does not account for gene
flow, spatial, and environmental processes that can obfuscate phylogenetic
inference and bias species delimitation. As a case study, we sequenced
genome-wide exons and introns to evince the processes that underlie the
diversification of Philippine Puddle Frogs—a group that is widespread,
phenotypically conserved, and exhibits high levels of geographically-based
genetic structure. We showed that widely adopted tree- and distance-based
approaches inferred up to 20 species, compared to genomic analyses that
inferred an optimal number of five distinct genetic groups. Using a suite
of clustering, admixture, and phylogenetic network analyses, we
demonstrate extensive admixture among the five groups and elucidate two
specific ways in which gene flow can cause overestimations of species
diversity: (1) admixed populations can be inferred as distinct lineages
characterized by long branches in phylograms; and (2) admixed lineages can
appear to be genetically divergent, even from their parental populations
when simple measures of genetic distance are used. We demonstrate that the
relationship between mitochondrial and genome-wide nuclear p-distances is
decoupled in admixed clades, leading to erroneous estimates of genetic
distances and, consequently, species diversity. Additionally, genetic
distance was also biased by spatial and environmental processes. Overall,
we showed that high levels of genetic diversity in Philippine Puddle Frogs
predominantly comprise metapopulation lineages that arose through complex
patterns of admixture, isolation-by-distance, and isolation-by-environment
as opposed to species divergence. Our findings suggest that speciation may
not be the major process underlying the high levels of hidden diversity
observed in many taxonomic groups and that widely-adopted tree- and
distance-based methods overestimate species diversity in the presence of
gene flow.