Date Published: January 13, 2017
Publisher: Public Library of Science
Author(s): Natalia Fraija-Fernández, Mercedes Fernández, Kristina Lehnert, Juan Antonio Raga, Ursula Siebert, Francisco Javier Aznar, Tzen-Yuh Chiang.
We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, Pholeter gastrophilus, exploring the effects of isolation by distance, ecological barriers and hosts’ dispersal ability on the population structure of this parasite. The ITS2 rDNA, and the mitochondrial COI and ND1 from 68 individual parasites were analysed. Worms were collected from seven oceanic and coastal cetacean species from the south western Atlantic (SWA), central eastern Atlantic, north eastern Atlantic (NEA), and Mediterranean Sea. Pholeter gastrophilus was considered a single lineage because reciprocal monophyly was not detected in the ML cladogram of all individuals, and sequence variability was <1% for mtDNA and 0% for ITS2. These results rule out a recent suggestion that P. gastrophilus would actually be a cryptic-species complex. The genetic cohesion of P. gastrophilus could rely on the extensive exploitation of wide-ranging and highly mobile cetaceans, with a putative secondary role, if any, of intermediate hosts. Unique haplotypes were detected in SWA and NEA, and an AMOVA revealed significant population structure associated to the genetic variation in these regions. The Equator possibly acts as a significant geographical barrier for cetacean movements, possibly limiting gene flow between northern and southern populations of P. gastrophilus. A partial Mantel tests revealed that the significant isolation of NEA populations resulted from geographic clustering. Apparently, the limited mobility of cetaceans used by P. gastrophilus as definitive hosts in this region, coupled with oceanographic barriers and a patchy distribution of potential intermediate hosts could contribute to significant ecological isolation of P. gastrophilus in NEA. Rather unexpectedly, no genetic differentiation was found in the Mediterranean samples of this parasite. Historical demographic analyses suggested a recent population expansion of P. gastrophilus in the Atlantic Ocean, perhaps linked to initial association and subsequent spreading in cetaceans.
Digeneans are parasitic organisms that have complex life cycles including free living and parasitic stages, and alternation of asexual and sexual reproduction. Free-living stages, i.e. the miracium and the cercaria, have a limited capacity for dispersal, whereas other stages use invertebrates (usually molluscs) as first intermediate hosts, other invertebrates or vertebrates as second intermediate hosts, and vertebrates as definitive hosts . Consequently, gene flow in digeneans is largely determined by the dispersal ability of the most mobile host, which usually is the definitive host . This is particularly true in the marine environment where few barriers for gene flow presumably exist  and, therefore, the dispersal role of hosts is particularly important. Although studies on phylogeography of marine parasites are still scarce , evidence suggests contrasting patterns of genetic structure depending on host mobility [4, 5].
Our results support the hypothesis that all populations of P. gastrophilus surveyed in this study represent a single species. No variation was found in the ITS2 rDNA sequences from all samples, and a maximum genetic difference of just 0.3% was detected in the mtND1 and mtCOI between worms from the two most distant regions (SWA vs. NEA). Previous studies have suggested that the maximum intraspecific variation in digeneans to recognise separate species would be ~1% for ITS2 rDNA  and ~5% for mtDNA , well above the observed values for P. gastrophilus. The use of this “genetic yardstick” has been criticised (see, e.g., ), and reciprocal monophyly has been considered as a suitable alternative method for species delimitation [93–95]. In our study, the absence of reciprocal monophyly confirms that P. gastrophilus is a single lineage, with no deep genetic differentiation associated to specific geographical and/or ecological factors. In any event, sample sizes of P. gastrophilus are modest in some localities and, therefore, more sequences from the South Atlantic, and new samples from the Pacific Ocean, would provide additional support for the hypothesis of a single species.