New Faces in an Old Genus: Museum Collections and New Materials Reveal New Diversity of Alaria and an Updated Key to Species

Alaria is a small genus of diplostomids that infect the intestines of mammalian definitive hosts. The life cycles of Alaria spp. require amphibian second intermediate hosts. However, snakes and small mammals often serve as paratenic hosts, and humans may be accidental dead-end paratenic hosts. Recently, the genus was expanded to include members of the former genus Pharyngostomoides, which primarily parasitizes common raccoon Procyon lotor. In the present study, we collected representatives of several Alaria spp. from amphibians, reptiles, and mammals in Georgia, Louisiana, North Dakota, and Wisconsin. Newly collected and previously available materials were used for morphological and molecular study. Additional specimens, including type series, were morphologically examined from various museums, teaching, and personal collections in North and South America. These archival specimens were critical in helping evaluate species diversity and assess potential evolutionary patterns of the genus. We provide descriptions of 3 new Alaria spp., 2 of them are based on new and museum specimens from common raccoons in Georgia and Minnesota, and the third new species is described based on museum specimens only from a mountain lion Puma concolor in Paraguay. The morphology of a species-level genetic lineage likely representing a new species is described based on specimens from a fisher Pekania pennanti in Wisconsin. Descriptions of Alaria procyonis (new material), Alaria marcianae (previously sequenced museum specimens), and Alaria nasuae (museum material; first description from wild-collected host) are provided. A redescription of Alaria adenocephala is provided based on type materials. Fragments of 3 genetic loci (28S + ITS region rDNA and COI mtDNA) were sequenced for the new materials (4 species; 1 species-level lineage). The name Alaria nattereri Lutz, 1933 is considered a nomen nudum. New sequences (ITS region and COI) of several previously studied Alaria isolates (5 species; 2 species-level lineages) were generated, along with the mitochondrial genome of Alaria ovalis, one of the species from raccoons. These DNA sequences were used to infer the phylogenetic relationships among Alaria spp. In total, our study increases the number of nominal Alaria spp. known from 10 to 14, of which 12 are limited to the Americas. A new key to Alaria spp. based on adult morphology is provided.