Long-Distance Travellers: Phylogeography of a Generalist Parasite, <i>Pholeter gastrophilus</i>, from Cetaceans

<div><p>We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, <i>Pholeter gastrophilus</i>, 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. <i>Pholeter gastrophilus</i> 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 <i>P</i>. <i>gastrophilus</i> would actually be a cryptic-species complex. The genetic cohesion of <i>P</i>. <i>gastrophilus</i> 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 <i>P</i>. <i>gastrophilus</i>. A partial Mantel tests revealed that the significant isolation of NEA populations resulted from geographic clustering. Apparently, the limited mobility of cetaceans used by <i>P</i>. <i>gastrophilus</i> 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 <i>P</i>. <i>gastrophilus</i> 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 <i>P</i>. <i>gastrophilus</i> in the Atlantic Ocean, perhaps linked to initial association and subsequent spreading in cetaceans.</p></div

Information on the specimens of the digenean <i>Pholeter gastrophilus</i> used for this study.

<p>Information on the specimens of the digenean <i>Pholeter gastrophilus</i> used for this study.</p

Haplotype network.

<p>Minimum Spanning network for the 16 haplotypes identified for the digenean <i>Pholeter gastrophilus</i> in this study. Each haplotype is labelled according to corresponding numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170184#pone.0170184.t003" target="_blank">Table 3</a>. Lines located transversally to connecting branches represent the number of base pair changes between haplotypes. Size and colour of circles match the information at the bottom-left side of the figure.</p

Geographical sampling of the digenean <i>Pholeter gastrophilus</i>.

<p>Sampling sites of the digenean <i>Pholeter gastrophilus</i>, identified by regions and host species. The map has been modified from a NASA image made by Reto Stockli, NASA's Earth Observatory Team, using data provided by the MODIS Land Science Team.</p

GenBank accession numbers of haplotypes identified for the digenean <i>Pholeter gastrophilus</i>.

<p>GenBank accession numbers of haplotypes identified for the digenean <i>Pholeter gastrophilus</i>.</p

Isolation by distance analysis.

<p>Graphical representation of the isolation by distance analysis among populations of the digenean <i>Pholeter gastrophilus</i>. The relationship between pairwise genetic distance (Φ_ST) and geographical distance (nm) between sampling sites are shown. Triangles represent comparisons of samples from the south western Atlantic (SWA); squares represent comparison of samples with the Mediterranean Sea (Med) and the central eastern Atlantic (CEA); and circles represent comparison of samples with the north eastern Atlantic (NEA).</p

Reports of cetacean species infected by the digenean <i>Pholeter gastrophilus</i>.

<p>Reports of cetacean species infected by the digenean <i>Pholeter gastrophilus</i>.</p

Maximum Likelihood analysis.

<p>Analysis for the phylogenetic relationships inferred from the mtND1 and mtCOI sequences of 68 individuals of the digenean <i>Pholeter gastrophilus</i>, collected from seven host species and four geographical regions. Support values for each node were calculated as ML bootstrap after 1000 replicates. Sequences are labelled according to their sampling localities as follows: central eastern Atlantic, CEA (Canary Islands, CI; Galicia North-West Spain, Gal; Strait of Gibraltar, SoG); Mediterranean Sea, Med (Alboran Sea, AS; western Mediterranean, WM); north eastern Atlantic, NEA (Celtic Sea, CS; North Sea, NS); south western Atlantic, SWA.</p

Analysis of Molecular Variance (AMOVA) of sequences of mtND1 and mtCOI from 68 individuals of the digenean <i>Pholeter gastrophilus</i>.

<p>Analysis of Molecular Variance (AMOVA) of sequences of mtND1 and mtCOI from 68 individuals of the digenean <i>Pholeter gastrophilus</i>.</p

Histopathology and immunohistochemistry of bronchial, bronchiolar and bronchial glandular epithelium of a harbor seal (No. 13) naturally infected with Seal/H10N7.

<p>Histopathology showed necrotizing bronchitis with accumulation of cellular debris in the lumen (asterisk) (A; magnification 100x), bronchial adenitis with necrosis of glandular epithelial cells (arrows) and accumulation of cellular debris in the glandular lumen (asterisks) (B; magnification 200x) and bronchiolitis (C; magnification 400x) with accumulation of sloughed epithelial and inflammatory cells in the lumen (arrows). Immunohistochemistry revealed marked influenza A virus antigen in bronchial (arrowheads) (D; magnification 100x), bronchial glandular (arrowheads) (E; magnification 200x) and bronchiolar (F; magnification 400x) epithelial cells and intraluminal cells.</p