Spot the difference: Two cryptic species of Gyrodactylus von Nordmann, 1832 (Platyhelminthes: Monogenea) infecting Astyanax aeneus (Actinopterygii, Characidae) in Mexico.

Over the course of one year, undescribed specimens of Gyrodactylus were recovered from banded tetra, Astyanax aeneus collected in the La Antigua and Nautla river basins in central Veracruz, Mexico. Parasites were processed for morphometric and molecular analyses. Morphometrically, Gyrodactylus samples collected in the La Antigua river had slightly smaller haptoral structures than those collected from the Nautla river. During the 12month-collection of samples, however, water temperature varied considerably (ca. 20°C to 30°C), and this abiotic factor is known to affect the size of gyrodactylid attachment structures. Moreover, no clear discrimination was possible between individual parasites collected from the two rivers based on the morphology of the marginal hook, which is recognised as a very informative character to discriminate between species. The morphology of the ventral bar, however, differed between specimens from both rivers: worms from Nautla all had long, rounded processes on the ventral bar, which formed a relatively closed angle with the dorsal edge of the bar proper, while most - but not all - specimens from La Antigua had comparatively slender processes forming a more open angle with respect to the ventral bar. Phylogenetic analyses based on the sequences of the ITS1, 5.8S rRNA gene, and ITS2 of gyrodactylids indicated the existence of two distinct, well-supported lineages whose sequences differ by >4%, one of which was only found in the Nautla basin, while the other was collected in both river systems. A posteriori, principal component analysis (PCA) of the morphometric data of sequenced specimens indicated that features of the dorsal bar, the hamuli and the ventral bar enable discrimination between the two phylogenetic lineages. Based on these independent sources of information (morphometric and molecular data), two new species of Gyrodactylus are described: Gyrodactyluspakan n. sp. and Gyrodactylusteken n. sp. The phylogenetic relationships of both new species to other gyrodactylids infecting characiformes (for which molecular data are available) are presented, which suggests that their closest relative is Gyrodactylus carolinae, a parasite of Characidium lanei in Brazil.