Family ties: molecular phylogenetics, evolution and radiation of flatworm parasites (Monogenea: capsalidae).

Abstract

The Capsalidae is a diverse family of ectoparasites of marine fish (Platyhelminthes: Monogenea). It is a large family with approximately 180 described species and many more yet to be discovered. Capsalids have a global distribution and parasitise a diversity of hosts from the Chondrichthyes to bony fishes. A morphological classification exists for the family based on a few key characters such as testes number, their arrangement and morphology of the posterior attachment organ (haptor). Phylogenetic relationships within the family and its position within the Monogenea are largely unexplored. I have used various molecular phylogenetic techniques to resolve relationships and explore the evolution and radiation of this family. Specimens from the Capsalidae and other monogenean families (outgroups) were obtained through fresh collections and generous donations by other parasitologists. Specimens were stored in 95% undenatured ethanol. Three unlinked nuclear genes (28S ribosomal RNA, Histone 3 and Elongation Factor 1 α) and two mitochondrial genes (Cytochrome Oxidase 3 and Cytochrome B) were amplified for 78 capsalid taxa in 28 genera representing all nine subfamilies and 30 outgroup taxa (eight Polyopisthocotylea and 22 Monopisthocotylea). Analyses showed the Capsalidae is monophyletic, with the sister group remaining unresolved. Some analyses supported Gyrodactylidae and Udonellidae as the sister group but in other analyses, it was unresolved with the Monocotylidae and Microbothriidae also possible sister groups. The Capsalinae, Encotyllabinae and Nitzschiinae are monophyletic, but analyses did not support monophyly for the Benedeniinae, Entobdellinae and Trochopodinae. Monophyly was supported for Capsala, Capsaloides, Encotyllabe, Entobdella, Listrocephalos, Neobenedenia, Nitzschia and Tristoma, but Metabenedeniella is paraphyletic and Benedenia and Neoentobdella are polyphyletic. Comparisons of the distribution of character states for the small number of morphological characters on a molecular phylogeny show a high frequency of apparent homoplasy. Consequently the current morphological classification for the Capsalidae shows little correspondence with the phylogenetic hypotheses I present. I also sequenced the first complete mitochondrial (mt) genome for a capsalid species, Benedenia seriolae. The mt genome of B. seriolae shows some tRNA rearrangements in comparison to three Gyrodactylus spp. (Gyrodactylidae), the only other complete monopisthocotylean mt genomes sequenced. It also lacks the duplicated, conserved non-coding regions present in Gyrodactylus spp. making the genome smaller in size. I combined this genome with other available platyhelminth mt genomes to investigate the monophyly of Monogenea and the evolution of diet across the Neodermata. Results confirm paraphyly for the Monogenea and also suggest paraphyly for the epidermal feeding Monopisthocotylea. I hypothesise that the Monopisthocotylea represent the first shift to parasitism in the Neodermata from a free living ancestor and following this, there was a dietary shift to blood feeding (Polyopisthocotylea). The Digenea and Cestoda independently evolved dietary specialisations to suit their diverse microhabitats and broad range of vertebrate final hosts. Using Elongation Factor 1 α I have estimated molecular divergence dates for the Neodermata, Monogenea and Capsalidae. Molecular clock analyses estimate the Neodermata diverged from the free living platyhelminths 513 million years ago (mya) (95% HPD [highest posterior density]: 473–605) before the appearance of vertebrates in the fossil record. The Monogenea diverged from the other neodermatan classes 441 mya (95% HPD: 420–547) coinciding with the appearance of fish in the fossil record. The most recent common ancestor of the Capsalidae arose approximately 235 million years ago (95% HPD: 200–274) following the Permian/Triassic extinction event and coinciding with the diversification of bony fishes in the marine environment. I compared the capsalid phylogeny to a phylogeny of fishes that I generated using eight nuclear and three mt genes to explore the evolution and the radiation of the Capsalidae across fishes. There is no significant global signal of coevolution with only five parasite-host associations that may be a result of coevolution. Estimated molecular divergence dates are also compared and do not reveal temporal congruence. Radiation of the family may have been driven by host switching constrained by shared host ecology, biology, behaviour and plasticity in morphological adaptations by the parasites. This study advances our understanding of the evolution of this monogenean family and provides insights into the evolution of the Neodermata and the complicated realities of reconstructing the evolutionary history of parasitic groups.