PHYLOGEOGRAPHY OF BAYLISASCARIS PROCYONIS (RACCOON ROUNDWORM) IN NORTH AMERICA.

Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 115 Baylisascaris procyonis individuals from 13 U.S. states and 1 Canadian province were obtained from 44 raccoon hosts to assess genetic variation and geographic structure. The maximum genetic distance between individuals was low (1.6%), consistent with a single species. Moderate COI haplotype (h = 0.60) and nucleotide (π = 0.0053) diversity were found overall. Low haplotype diversity was found among samples east of the Mississippi River (h = 0.036), suggesting that historical growth and expansion of raccoon populations in this region could be responsible for high parasite gene flow or a selective sweep of B. procyonis mtDNA. There was low genetic structure (average Φst = 0.07) for samples east of the continental divide, but samples from Colorado showed higher diversity and differentiation from midwestern and eastern samples. There was marked genetic structure between samples from east and west of the continental divide, with no haplotypes shared between these regions. There was no significant isolation by distance among any of these geographic samples. The phylogeographic patterns for B. procyonis are similar to genetic results reported for their raccoon definitive hosts. The phylogeographic divergence of B. procyonis from east and west of the continental divide may involve vicariance resulting from Pleistocene glaciation and associated climate variation.

Molecular phylogenetics and species-level systematics of Baylisascaris.

Nucleotide sequences representing nine genes and five presumptive genetic loci were used to infer phylogenetic relationships among seven Baylisascaris species, including one species with no previously available molecular data. These genes were used to test the species status of B. procyonis and B. columnaris using a coalescent approach. Phylogenetic analysis based on combined analysis of sequence data strongly supported monophyly of the genus and separated the species into two main clades. Clade 1 included B. procyonis, B. columnaris, and B. devosi, species hosted by musteloid carnivores. Clade 2 included B. transfuga and B. schroederi from ursids, B. ailuri, a species from the red panda (a musteloid), and B. tasmaniensis from a marsupial. Within clade 2, geographic isolates of B. transfuga, B. schroederi (from giant panda), and B. ailuri formed a strongly supported clade. In certain analyses (e.g., some single genes), B. tasmaniensis was sister to all other Baylisascaris species rather than sister to the species from ursids and red panda. Using one combination of priors corresponding to moderate population size and shallow genetic divergence, the multispecies coalescent analysis of B. procyonis and B. columnaris yielded moderate support (posterior probability 0.91) for these taxa as separate species. However, other prior combinations yielded weak or no support for delimiting these taxa as separate species. Similarly, tree topologies constrained to represent reciprocal monophyly of B. columnaris and B. procyonis individuals (topologies consistent with separate species) were significantly worse in some cases, but not others, depending on the dataset analyzed. An expanded analysis of SNPs and other genetic markers that were previously suggested to distinguish between individuals of B. procyonis and B. columnaris was made by characterization of additional individual nematodes. The results suggest that many of these SNPs do not represent fixed differences between nematodes derived from raccoon and skunk hosts.

Morphological variability and molecular identification of Uncinaria spp. (Nematoda: Ancylostomatidae) from grizzly and black bears: new species or phenotypic plasticity?

The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis ( Wolfgang, 1956 ) were formally described from grizzly ( Ursus arctos horribilis) and black bears ( Ursus americanus ) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada and isolated Uncinaria specimens with anatomical traits never previously documented. We applied morphological and molecular techniques to investigate the taxonomy and phylogeny of these Uncinaria parasites. The morphological analysis supported polymorphism at the vulvar region for females of both U. rauschi and U. yukonensis. The hypothesis of morphological plasticity for U. rauschi and U. yukonensis was confirmed by genetic analysis of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. Two distinct genotypes were identified, differing at 5 fixed sites for ITS-1 (432 base pairs [bp]) and 7 for ITS-2 (274 bp). Morphometric data for U. rauschi revealed host-related size differences: adult U. rauschi were significantly larger in black bears than in grizzly bears. Interpretation of these results, considering the historical biogeography of North American bears, suggests a relatively recent host-switching event of U. rauschi from black bears to grizzly bears which likely occurred after the end of the Wisconsin glaciation. Phylogenetic maximum parsimony (MP) and maximum likelihood (ML) analyses of the concatenated ITS-1 and ITS-2 datasets strongly supported monophyly of U. rauschi and U. yukonensis and their close relationship with Uncinaria stenocephala (Railliet, 1884), the latter a parasite primarily of canids and felids. Relationships among species within this group, although resolved by ML, were unsupported by MP and bootstrap resampling. The clade of U. rauschi, U. yukonensis, and U. stenocephala was recovered as sister to the clade represented by Uncinaria spp. from otariid pinnipeds. These results support the absence of strict host-parasite co-phylogeny for Uncinaria spp. and their carnivore hosts. Phylogenetic relationships among Uncinaria spp. provided a framework to develop the hypothesis of similar transmission patterns for the closely related U. rauschi, U. yukonensis, and U. stenocephala.

Molecular systematics of pinniped hookworms (Nematoda: Uncinaria): species delimitation, host associations and host-induced morphometric variation.

Hookworms of the genus Uncinaria have been widely reported from juvenile pinnipeds, however investigations of their systematics has been limited, with only two species described, Uncinaria lucasi from northern fur seals (Callorhinus ursinus) and Uncinaria hamiltoni from South American sea lions (Otaria flavescens). Hookworms were sampled from these hosts and seven additional species including Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), South American fur seals (Arctocephalus australis), Australian fur seals (Arctocephalus pusillus), New Zealand sea lions (Phocarctos hookeri), southern elephant seals (Mirounga leonina), and the Mediterranean monk seal (Monachus monachus). One hundred and thirteen individual hookworms, including an outgroup species, were sequenced for four genes representing two loci (nuclear ribosomal DNA and mitochondrial DNA). Phylogenetic analyses of these sequences recovered seven independent evolutionary lineages or species, including the described species and five undescribed species. The molecular evidence shows that U. lucasi parasitises both C. ursinus and E. jubatus, whereas U. hamiltoni parasitises O. flavescens and A. australis. The five undescribed hookworm species were each associated with single host species (Z. californianus, A. pusillus, P. hookeri, M. leonina and M. monachus). For parasites of otarids, patterns of Uncinaria host-sharing and phylogenetic relationships had a strong biogeographic component with separate clades of parasites from northern versus southern hemisphere hosts. Comparison of phylogenies for these hookworms and their hosts suggests that the association of U. lucasi with northern fur seals results from a host-switch from Steller sea lions. Morphometric data for U. lucasi shows marked host-associated size differences for both sexes, with U. lucasi individuals from E. jubatus significantly larger. This result suggests that adult growth of U. lucasi is reduced within the host species representing the more recent host-parasite association. Intraspecific host-induced size differences are inconsistent with the exclusive use of morphometrics to delimit and diagnose species of Uncinaria from pinnipeds.

Structural restoration of nematodes and acanthocephalans fixed in high percentage alcohol using DESS solution and rehydration.

Ninety-five percent ethanol is the most widely used field and laboratory preservative for nematodes and other helminth specimens intended for use in molecular systematics. Preservation of nematodes in high-concentration alcohols results in structural dehydration artifacts, including shrinkage and body surface distortions sufficient to obscure features required for morphological identification and analysis, thereby compromising precise morphometrics. However, treating dehydrated nematodes using a solution of DMSO, disodium EDTA, and NaCl, followed by rehydration in water produces marked improvements in specimen shape and surface features, resulting from diffusion of water into the tissues and pseudocoelom as the internal salt concentration is reduced. Following rehydration, tissue samples can be obtained for molecular research and individuals can be fixed for morphological examination. This treatment method is demonstrated for species of 3 nematode genera that vary substantially in body size ( Baylisascaris , Uncinaria , and Bidigiticauda ). The method also works on nematodes that have been cut in half, provided the individuals are large enough to be folded and clamped during treatment. This method appears promising for other helminths, although for an acanthocephalan, the treatment restored the body surface but failed to reverse the retracted proboscis.