A hyper-diverse genus of acanthocephalans revealed by tree-based and non-tree-based species delimitation methods: Ten cryptic species of Neoechinorhynchus in Middle American freshwater fishes.

The genus Neoechinorhynchus represents a hyper-diverse group of acanthocephalans, parasites of fresh and brackish water fish and freshwater turtles, with approximately 116 species described worldwide. Forty-nine species have been recorded in the Americas, nine of them in Middle America. Even though species delimitation methods using DNA sequences have been rarely used for parasitic helminths, the genetic library for species of Neoechinorhynchus has grown in the past few years, enhancing the possibility of using these methods for inferring evolutionary relationships and for establishing more robust species boundaries. In this study, we used non-tree-based and tree-based methods through a coalescent approach to explore the species limits of specimens of Neoechinorhynchus collected in 57 localities across Middle America. We sequenced a large number of individuals to build a comprehensive dataset for three genes: the mitochondrial cytochrome c oxidase subunit I (352 individuals), the internal transcribed spacers (330 individuals), and the D2 + D3 domains of the large subunit (278 individuals). Several species delimitation methods were implemented, i.e., Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent Model (GMYC), Bayesian species delimitation (BPP) and species tree (∗BEAST). Additionally, we conducted a detailed morphological study of the diagnostic traits associated with the proboscis of 184 males and 169 females. Overall, our analyses allowed us to validate nine nominal species of Neoechinorhynchus and to identify 10 additional genetic lineages herein regarded as candidate species. This unexpected genetic diversity and the lack of reliable morphological traits show that the genus Neoechinorhynchus includes a group of cryptic species, at least in Middle America.