Digeneans of cetaceanstaxonomy, evolutionary history and host specificity.

Resum

Parasitism is an extremely successful lifestyle among animals. In fact, every free-living organism is believed to harbour at least a parasite species, and cetaceans are not the exception. Host-parasite systems offer a suitable model for studying systematics, evolution, biogeography and ecology because the evolutionary fate of parasites is linked to that of their hosts. In particular, present-day associations between cetaceans and their parasites have been shaped by unique historical events. The parasites of the terrestrial ancestors of cetaceans likely faced an ecological barrier when their hosts colonized the marine environment; it is postulated that during the process terrestrial parasites went extinct, thus the current parasitic fauna of cetaceans was acquired through colonization events in the marine realm. Cetaceans harbour a trophically-transmitted helminth fauna that includes more than 100 species of acanthocephalans, cestodes, nematodes and digeneans. Records of helminths in cetaceans go back to the time when commercial whaling was allowed and material for scientific studies was available, and efforts for maintaining an updated list have been made. However, additional information from post-mortem examinations of stranded or by-caught animals, from rarely cetacean species, and from unexplored areas has become available over the last decades. In addition, new taxonomic rearrangements have been proposed and the use of molecular information has helped to unveil a significant portion of hidden biodiversity (i.e., cryptic species). Digeneans represent a highly diverse and relatively specific group of helminths infecting cetaceans. Records in cetaceans include four families of highly specific taxa, i.e., the monotypic Brauninidae with Braunina cordiformis as its only species, species of Ogmogaster (Notocotylidae), Pholeter gastrophilus (Heterophyidae), and species of the family Brachycladiidae. The systematic affiliation of these digeneans has been controversial with limited phylogenetic information. Only for the Brachycladiidae, available data indicates that the origin of this group, and its association with marine mammals, likely resulted from a host-switching event. However, in order to understand biogeographical and historical scenarios in which host and parasites become associated specific cophylogenetic studies for each family are also needed. The digeneans of cetaceans display sharp differences in host specificity, i.e., there are examples of highly specific species restricted to few host species (e.g., Synthesium pontoporiae, in the Franciscana dolphin, Pontoporia blainvillei), whereas other species are generalists and widely distributed among several host species (e.g., Pholeter gastrophilus, reported in at least 17 cetacean species). However, the number of hosts a parasite can exploit is not the only measure of host specificity, since hosts may differ in quality, and may not be equally compatible with each parasite species. There are examples of evident morphological and reproductive differences (i.e., dwarfism and impaired reproduction) when digeneans infect unsuitable cetacean species. Still, few studies have analysed how presumably equally suitable hosts and the environmental pressures where the parasite’s life cycle develops would affect the host compatibility of a generalist digenean species. The amount of gene flow between individuals likely contributes to the genetic cohesion of a species, and is highly determined by their capacity for dispersion, which in parasites depends on the most mobile host. Cetaceans are long-ranging animals with some highly structured populations. Currently, few studies have analysed the genetic structure of helminths in cetaceans, including the generalist nematode Anisakis typica, and the specific digenean Synthesium pontoporiae. No attempt has been made to study the genetic structure and gene flow in any generalist digenean species. This study addresses different aspects of the diversity, origin and diversification, and patterns of host specificity in digeneans of cetaceans, focusing on the following objectives: 1 – To update the systematic list of digeneans, and other helminths, infecting cetaceans. 2 – To describe a new digenean species found in cetaceans. 3 – To assess the taxonomic affiliation and to investigate the origin of the four families of digeneans found in cetaceans. 4 – To analyse the cophylogenetic relationship between the most diverse digenean family that parasitize cetaceans, i.e., the Brachycladiidae, and their marine mammal hosts. 5 – To investigate phylogeographical patterns of the most generalist species of digeneans infecting cetaceans, namely, Pholeter gastrophilus. 6 – To examine host specificity patterns in Pholeter gastrophilus, determining the extent to which the parasite’s performance varies according to host species, and how host’s habitat can influence the reproductive strategy of the parasite. The following results and conclusions were obtained from this study: Out of the 90 species currently recognised in the order Cetacea, 72 have been examined for helminths. The family Balaenopteridae among baleen whales and the family Delphinidae among toothed whales harbour the most diverse helminth fauna; this is partly accounted for by a higher sampling effort on these families. In contrast, the helminth fauna of the beaked whales (family Ziphiidae) is the least known since only 9 out of the 22 species in the family have been examined for parasites. Currently, there are 174 helminth species reported in cetaceans including nematodes (62 spp.), digeneans (54 spp.), cestodes (38 spp.) and acanthocephalans (20 spp.). However, 20 (11.5%) of these species represent accidental infections, which occur because cetaceans and other marine vertebrates (i.e., pinnipeds and marine birds) share a trophic guild. The families Anisakidae, Pseudaliidae and Tetrameridae (Nematoda), Brachycladiidae, Notocotylidae, Heterophyidae and Brauninidae (Digenea), Diphyllobothriidae, Phyllobothriidae and Tetrabothriidae (Cestoda), and Polymorphidae (Acanthocephala) are the most diverse. The update of the helminth diversity in cetaceans provides a baseline for future studies, stressing the biological importance of these organisms. A new species of a brachycladiid is described from the bile ducts of a Gervais’ beaked whale Mesoplodon europaeus (Ziphiidae) stranded on the North Atlantic coast of Florida. Brachycladium parvulum Fraija-Fernández, Aznar, Raga, Gibson et Fernández, 2014 differs from other species of the genus Brachycladium by the relative size of the oral and ventral suckers, the shape and size of the eggs and an extremely small body size. Four discriminating variables i.e., worm body area, oral sucker area, pharynx area and testes area were used in a canonical discriminant analysis between specimens of B. parvulum n. sp. (n = 17) and dwarf individuals of B. atlanticum (n = 16), which was considered the morphologically closest species. There were significant differences between the two samples, and a jack-knife classification showed that 96.2% of the specimens were correctly classified to their group. Phylogenetic relationships of the main groups of digeneans infecting cetaceans were also investigated. We obtained sequences of the small subunit rDNA (SSU), the large subunit rDNA (LSU) and the internal transcribed spacer 2 (ITS2) rDNA of Braunina cordiformis (Brauninidae), Ogmogaster antarcticus (Notocotylidae), Pholeter gastrophilus (Heterophyidae), and Campula oblonga, Nasitrema sp. and Oschmarinella rochebruni (Brachycladiidae). The phylogenetic position of taxa was estimated by Bayesian inference and maximum likelihood (ML) incorporating published sequences of 177 species of Digenea. In the case of P. gastrophilus, further Bayesian and ML analyses were performed with additional sequences of 14 species of the Heterophyidae and Opisthorchiidae. Species nominally assigned to the Brachycladiidae formed a clade that was embed among species of the Acanthocolpidae, thus making the relationship between these families paraphyletic. Braunina cordiformis formed a sister lineage to the Strigeidae and Diplostomidae, whereas O. antarcticus was placed within the Notocotylidae, in agreement with the previous taxonomy of this genus. Similarly, P. gastrophilus was placed within the Heterophyidae as originally described. However, our results suggest a paraphyletic relationship between the Heterophyidae and the Opisthorchiidae. The phylogenetic positions of these taxa indicate that the origin of digeneans in cetaceans occurred independently through host switching events from fish- eating birds and mammals (i.e. Strigeidae, Diplostomidae and Heterophyidae), parasites of marine fish (i.e. Acanthocolpidae) and other herbivorous aquatic birds and mammals (i.e. Notocotylidae). Only two clades, i.e., the Brachycladiidae and species of Ogmogaster (Notocotylidae) subsequently diversified exclusively among marine mammals. Specific cophylogenetic studies for each family are needed to understand the complete biogeographic and historical scenarios that account for the association between host and parasites. The association of the species from the largest family infecting cetaceans, the Brachycladiidae, and their hosts was studied under a cophylogenetic framework. Distance-based and event-based methods were used to explicitly test, for the first time, the coevolutionary events that have led to the current brachycladiid-marine mammal associations. The parasite phylogeny was constructed using sequences of the mtDNA NADH dehydrogenase, subunit 3 (ND3) of nine brachycladiid species, and host phylogeny using cytochrome b sequences of 104 mammalian species. A total of 50 host-parasite links were identified. Distance-based methods supported the hypothesis of a global non-random association of host and parasite phylogenies. Significant individual links were identified in 24 out of 50 associations, which were related to Campula oblonga, Nasitrema delphini, N. globicephalae and Brachycladium atlanticum and their associated taxa from the Delphinoidea. Regarding event-based methods, 54 schemes were explored using different combinations of costs for each potential coevolutionary event, i.e. duplication, loss, failure-to-diverge and host- switching. Three equally likely coevolutionary scenarios were identified across all schemes, and in all cases the number of loss events (87 – 156) was the most numerous, followed by failure-to- diverge (40), duplication, (3 – 6), host-switching (0 – 3) and cospeciation (0 – 2) events. Failure- to-diverge and colonization with or without subsequent diversification were thus identified as decisive events for the establishment of the associations between brachycladiids and marine mammals. Sequences from ribosomal (ITS2) and mitochondrial (COI and ND1) DNA were used to explore the phylogeography and historical demography of Pholeter gastrophilus. We used 68 specimens of P. gastrophilus collected from seven cetacean species in nine sampling localities from four regions, i.e., Mediterranean Sea, Central Eastern Atlantic, North Eastern Atlantic, and South Western Atlantic. Less than 1% of genetic variation in ribosomal and mitochondrial markers, and no reciprocal monophyly associated to geographic regions, suggest that P. gastrophilus is a single lineage in the Atlantic basin. The genetic cohesion of the species throughout the Atlantic Ocean and the Mediterranean Sea would be accounted for the ubiquity of P. gastrophilus in the food web, by infecting several species of invertebrates and vertebrates, which are used as intermediate hosts, and the extensive exploitation of long-ranging cetaceans as definitive hosts. Nevertheless, a significant pattern of isolation by distance associated to ecological barriers, and unique haplotypes for samples in the two most distant populations, i.e., South Atlantic Ocean and North Atlantic Ocean were found, indicating that the genetic structure of the population is accounted for samples from these areas. In fact, the results from AMOVA detected a significant population structure for P. gastrophilus (?ST = 0.40, p < 0.001), and a significant structure among regions (?CT = 0.37, p = 0.005). Most of the samples in the North Atlantic Ocean were collected from harbour porpoises, which is the only cetacean in this study restricted to coastal habitats. Thus, in this case the ecological isolation of hosts would be contributing to the genetic isolation of their parasites. Demographic analyses detected a recent population expansion for P. gastrophilus, which was estimated in 60,627 years ago. We raise the hypothesis that the population expansion in P. gastrophilus would be related to the origin of its association with cetaceans, which we postulate to have occurred via a host-switching event from fish-eating birds. Furthermore, the ubiquity of P. gastrophilus would be accounted for the extensive use of the food web through intermediate hosts and the exploitation of long-ranging cetaceans as definitive hosts. Finally, we investigated how the combined effect of cryptic host specificity and local selective pressures could shape key life-history traits of P. gastrophilus. In the western Mediterranean, P. gastrophilus infects the oceanic striped dolphin, Stenella coeruleoalba, but also the coastal bottlenose dolphin, Tursiops truncatus. The LSU rDNA, the ITS2 rDNA and the mtCOI indicated that worms collected from both dolphin species were conspecific. Also, no significant differences were observed in the infection parameters between both dolphin species. General Linear Mixed Models were used to analyse the influence of host species on four reproductive traits of P. gastrophilus: body size, maturity stage (non-gravid/gravid), egg size, and number of eggs in utero. AIC values were used to rank competing models, and p values to assess the effect of specific predictors. All worms collected were gravid and body size and egg size of individuals of P. gastrophilus were significantly larger in striped dolphins. The number of eggs in utero did not significantly differ between dolphin species but, for a given body size, worms in bottlenose dolphins harboured more eggs. A trade-off between egg size and egg number was found in worms from both dolphin species, with a higher slope in striped dolphins. Apparently, the striped dolphin is a more suitable host for P. gastrophilus, but reproductive investment seems to be adapted to the habitat where the life-cycle develops. Worms from striped dolphins likely face the problem of finding intermediate hosts in the oceanic realm and apparently investing into offspring size enhance the early survival of larvae and the potential to multiply asexually within the first intermediate host. The small-sized worms from bottlenose dolphins would be adapted to reproduce early because of higher adult mortality, generating smaller and numerous eggs in a coastal habitat where chances of transmission are presumably higher.