Molecular data reveal diversity of Tylodelphys spp. (Trematoda: Diplostomidae) in India: with evidence of new lineages, morphology and statistical analysis.

Background: Diplostomidae is a family of widespread digenean parasites distributed globally. The diplostomid genus Tylodelphys is frequently distributed worldwide and most published data originated from larval stages. Morphology-based identification of these parasites is typical and hard to use to identify at the species level, therefore, molecular data is needed. We cannot name these two species of Tylodelphys metacercariae yet because the adults are unknown. ITS1-5.8S-ITS2 and COX1 sequences were used for phylogenetic inference to establish the position of Tylodelphys sp. IND_ACBR1 and Tylodelphys sp. IND_ACVH1 spp. within the Diplostomoidea and to study their interrelationships with other closely related congeners.

Results: Tylodelphys metacercariae, infecting Ailia coila in India were collected to identify the species based on the morphology, molecular and statistical analysis. For morphology, live and stained fixed specimens were used. For molecular analysis, we have generated ITS cluster sequences (ITS1-5.8S-ITS2) of nuclear ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 (COX1) genes of Tylodelphys metacercariae. Principal component analysis (PCA) was run using R- software. Our results demonstrate that two diplostomids from India, i.e., Tylodelphys sp. IND_ACBR1 and Tylodelphys sp. IND_ACVH1 are likely undescribed taxons that belong within Diplostomidae. The results of morphology and phylogenetic analysis of both genes confirmed the status of Tylodelphys sp. IND_ACBR1 and Tylodelphys sp. IND_ACVH1 as separate species from previously published Tylodelphys species from India. The results of the statistical analysis of Tylodelphys spp. supported two distinct species status.

Conclusions: The novel DNA data from this study make available chances for future appraisals of larval diplostomids, especially in India. The present study also improves our understanding of the diversity of diplostomid metacercariae larval form and offers an opportunity to study their phylogenies.