A priori and a posteriori methods in comparative evolutionary studies of host–parasite associations

Brooks parsimony analysis (BPA) and reconciliation methods in studies of host–parasite associations differ fundamentally, despite using the same null hypothesis. Reconciliation methods may eliminate or modify input data to maximize fit of single parasite clades to a null hypothesis of cospeciation, by invoking different a priori assumptions, including a known host phylogeny. By examining the degree of phylogenetic congruence among multiple parasite clades, using hosts as analogs of taxa but not presuming a host phylogeny or any degree of cospeciation a priori, BPA modifies the null hypothesis of cospeciation if necessary to maintain the integrity of the input data. Two exemplars illustrate critical empirical differences between reconciliation methods and BPA: (1) reconciliation methods rather than BPA may select the incorrect general host cladogram for a set of data from different clades of parasites, (2) BPA rather than reconciliation methods provides the most parsimonious interpretation of all available data, and (3) secondary BPA, proposed in 1990, when applied to data sets in which host‐switching produces hosts with reticulate histories, provides the most parsimonious and biologically realistic interpretations of general host cladograms. The extent to which these general host cladograms, based on cospeciation among different parasite clades inhabiting the same hosts, correspond to host phylogeny can be tested, a posteriori, by comparison with a host phylogeny generated from nonparasite data. These observations lead to the conclusion that BPA and reconciliation methods are designed to implement different research programs based on different epistemologies. BPA is an a posteriori method that is designed to assess the host context of parasite speciation events, whereas reconciliation methods are a priori methods that are designed to fit parasite phylogenies to a host phylogeny. Host‐switching events are essential for explaining complex histories of host–parasite associations. BPA assumes coevolutionary complexity (historical contingency), relying on parsimony as an a posteriori explanatory tool to summarize complex results, whereas reconciliation methods, which embody formalized assumptions of maximum cospeciation, are based on a priori conceptual parsimony. Modifications of basic reconciliation methods, embodied in TreeMap 1.0 and TreeMap 2.02, represent the addition of weighting schemes in which the researcher specifies allowed departures from cospeciation a priori, with the result that TreeMap results more closely agree with BPA results than do reconciled tree analysis results.     

Phylogenetic relationships and ecological speciation in the mistletoe Tristerix (Loranthaceae): the influence of pollinators, dispersers, and hosts

Phylogenies can provide valuable information on biotic and abiotic factors associated with speciation. We examined species relationships in Tristerix (Loranthaceae), a genus of 11 species with an Andean distribution from Colombia to Chile. A previous classification divided Tristerix into subgenera Tristerix (two species) and Metastachys (nine species). We tested this classification by generating a molecular phylogeny of the genus using nuclear ribosomal DNA ITS and chloroplast atpB-rbcL intergenic spacer and trnL-F regions. All partitions generally gave congruent trees, thus a combined analysis was conducted. Tristerix was composed of a northern clade (six species) and a southern clade (four species). Tristerix verticillatus and T. penduliflorus (Metastachys) were strongly supported as members of the (southern) subgenus Tristerix clade. Speciation appears to be correlated with the emergence of matorral and cloud forest biomes and is driven by interactions with pollinators and seed dispersers. Tristerix aphyllus is sister to T. corymbosus of the matorral, not to neighboring temperate forest populations, thus rendering the latter species paraphyletic. This ecological speciation event may have occurred in sympatry. Tristerix provides excellent examples of how, during the orography of the Andes, many dynamic and interacting ecological factors have influenced their speciation.     

Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.     

Phylogeography of two European newt species--discordance between mtDNA and morphology

The newts Triturus vulgaris and Triturus montandoni are sister species that exhibit contrasting levels of intraspecific morphological variation. Triturus vulgaris has a broad Eurasiatic distribution encompassing both formerly glaciated and unglaciated areas and shows substantial morphological differentiation in the southern part of its range, while T. montandoni, confined to the Carpathians, is morphologically uniform. We analysed sequence variation of two mtDNA fragments of the total length of c. 1850 bp in 285 individuals of both species collected from 103 localities. Phylogenetic analysis of 200 unique haplotypes defined 12 major clades, their age estimated at c. 4.5-1.0 million years (Myr). Most of the older clades were found in the southern part of the range, and also in central Europe, mainly in Romania. The distribution of mtDNA clades points to the existence of several glacial refugia, located in the Caucasus region, Anatolia, the Balkan Peninsula, Italy, and more to the north in central Europe. The concordance between mtDNA based phylogeny and the distribution of T. vulgaris subspecies was weak. Triturus montandoni haplotypes did not form a monophyletic group. Instead they were found in six clades, in five of them mixed with T. vulgaris haplotypes, most likely as a result of past or ongoing hybridization and multiple introgression of mtDNA from T. vulgaris to T. montandoni. Patterns of sequence variation within clades suggested long-term demographic stability in the southern groups, moderate and relatively old demographic growth in the populations inhabiting central Europe, and high growth in some of the groups that colonized northern parts of Europe after the last glacial maximum.     

Phylogeography and freshwater basins in central Mexico: recent history as revealed by the fish parasite Rhabdochona lichtenfelsi (Nematoda)

Aim The phylogeography of Rhabdochona lichtenfelsi, a nematode parasite specific to endemic goodeids in Mexico, is used to infer the biogeographical history of fragmentation and recent evolution of the Mesa Central drainages. Geological history of the west‐central region of Mexico suggests that extant freshwater basins are the result of different vicariant events that fragmented ancient watercourses and lakes within the Mesa Central. Location Major freshwater river basins of the Mesa Central, Mexico: Ameca, Cotija, Lerma, Rio Verde, Panuco, and lakes Cuitzeo and Zacapu. Methods Haplotype diversity and phylogeographical structure of 10 populations of R. lichtenfelsi, sampled from the complete range of this species, were analysed with partial sequences of cytochrome c oxidase subunit I (456 bp). Analyses performed included phylogenetic tree estimation methods (neighbour‐joining, maximum parsimony and maximum likelihood), genetic diversity, distance and structure estimates, and nested clade analysis. Results High overall haplotype diversity, unique haplotypes, and strongly structured populations were found in the basins sampled. Three phylogenetically and demographically identifiable clades were recovered. These clades fit an isolation‐by‐distance model. Significant population expansion was observed for two clades and for the entire population. Time of divergence was estimated as 1.0 and 0.84 Ma for the different clades. Main conclusions The distribution of R. lichtenfelsi haplotypes does not correspond to the present distribution of the basins of Mesa Central, but instead reflects the distribution of those basins during a recent geological period (Pleistocene). While our current knowledge of the evolution and geographical relationships of the Mesa Central basins comes from studies of freshwater fish encompassing a more ancient history, our results suggest that, during the past million years, old basins and connections existed where today isolated freshwater bodies stand, thus unravelling a novel biogeographical history for the Mesa Central of Mexico.     

Sequence variation at cpDNA regions of watermelon and related wild species: implications for the evolution of Citrullus haplotypes

Sequencing analysis of one coding and four noncoding cpDNA regions was conducted to infer biogeographic and evolutionary relationships in the genus Citrullus. Eighteen taxa from diverse geographical areas were included. A low number of parsimony informative characters (1.1%) was observed at the ～4 kb section of cpDNA. Variability within Citrullus was detected primarily at noncoding regions of high A + T content. Substitution rates varied from 0-0.48% for ndhF with A + T content of 68.4% to 0.39- 1.69% for the intergenic region of atpA with A + T content of 82.8%, mainly resulting in indels and transversions. Indels at several regions acted as valuable parsimony informative markers. Citrullus lanatus var. lanatus, the cultivated watermelon, and C. ecirrhosus and C. rehmii from Namibia, lacked molecular variability. The genus Citrullus is supported monophyletically and shows two main clades, one of which contains C. colocynthis. In the other clade, C. rehmii is sister to a clade containing C. ecirrhosus and C. lanatus. Two clades were recovered within C. lanatus, consisting of domesticated watermelon and wild citron, var. citroides. Five haplotypes within C. colocynthis were used to deduce colonization routes of the species. Biogeographic patterns point to separate colonization events into Africa and the Far East.     

Mitochondrial DNA variation and biogeography of eastern gorillas

Mitochondrial DNA variation in 109 individuals from four populations of wild living gorillas in East Africa was ascertained by sequencing the first hypervariable segment of the control region, or 'd-loop', amplified from noninvasively collected hair and faeces. D-loop haplotypes from eastern gorillas fell into two distinct clades, each with low levels of genetic diversity; most observed haplotypes within each clade differing by only one or two mutations. Both clades show evidence of population bottlenecks in the recent past, perhaps concomitant with the tropical forest reduction and fragmentation brought on by global cooling and drying associated with the last glacial maximum.     

Population genetic studies of Alouatta caraya (Alouattinae, Primates): inferences on geographic distribution and ecology

Cytochrome b DNA sequence data (ca. 1,140 bp) of 44 Alouatta caraya, including 42 specimens from three localities of Brazil and two from Bolivia, were used for phylogenetic reconstructions and population studies. Seventeen haplotypes were identified, eight of which were present in more than one individual. Seven of these eight haplotypes were shared by individuals from a same locality and one by individuals from two localities. We found 26 variable sites along the entire gene, consisting of 18 transitions and eight transversions; most replacements occurring at the third codon position (65.39%) in contrast to first and second positions (26.92 and 7.69%, respectively). In the sample collected at Chapada dos Guimarães (Brazil), nucleotide and haplotype diversity estimates were π=0.002325 and h=0.8772, respectively. Maximum parsimony analysis grouped all haplotypes in two clades, separating Bolivian haplotypes from Brazilian haplotypes, the grouping of which did not show a straightforward correspondence with geographic distribution. Median‐joining and TCS network pointed to haplotypes 11 or 12 as the most likely ancestral ones. Mismatch distribution and the goodness‐of‐fit test (SSD estimate=0.0027; P=0.6999) indicated that the population from Chapada dos Guimarães experienced a demographic expansion, in agreement with the median‐joining star‐like pattern, although this finding could not be confirmed by Fu's F_s test. Am. J. Primatol. 69:1093–1104, 2007. © 2007 Wiley‐Liss, Inc.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

Widespread and structured distributions of blood parasite haplotypes across a migratory divide of the Swainson's thrush (Catharus ustulatus)

We examined the phylogenetic distribution of cytochrome b haplotypes of the avian blood parasite genera Haemoproteus and Plasmodium across the migratory divide of the Swainson's thrush (Catharus ustulatus) in British Columbia, Canada. From 87 host individuals, we identified 8 parasite haplotypes; 4 of Plasmodium and 4 of Haemoproteus. Six haplotypes were novel; 1 Haemoproteus haplotype was identical to H. majoris found in the blue tit (Parus caeruleus) in Sweden, and another halotype was identical to a Plasmodium haplotype found in the white-crowned sparrow (Zonotrichia leucophrys) in Oregon. The 2 most abundant parasite haplotypes were widely distributed across the contact zone, whereas 2 other parasite haplotypes seem to have structured distributions. Compared with 74 Plasmodium and Haemoproteus haplotypes published in GenBank, haplotypes recovered from Swainson's thrushes do not form monophyletic groups, and they are closely related to haplotypes from a variety of other hosts and localities. In addition, we recovered 2 Swainson's thrush Plasmodium haplotypes from the nonmigratory orange-billed nightingale thrush (Catharus aurantiirostris) in Costa Rica. This study is the first to elucidate avian blood parasite transmission, distribution, and phylogenetic relationships in an avian contact zone in North America.     

Evolution after the flood: phylogeography of the desert fish Utah Chub

The Bonneville Basin and upper Snake River drainage of western North America underwent extensive hydrological changes during the late Pleistocene, potentially influencing the geographic distribution and evolutionary trajectories of aquatic species that occupied this region. To test this hypothesis, I reconstructed the phylogeographic history of the desert fish Utah chub (Gila atraria) by examining 16 populations that span the natural distribution of this species across the Bonneville Basin and upper Snake River. I compared mitochondrial control region sequences (934 bp) among 77 individuals revealing 24 unique haplotypes. Geographic and phylogenetic relationships among haplotypes were explored using parsimony, maximum likelihood, nested clade analysis, and analysis of molecular variance. I found that G. atraria is composed of two distinct clades that represent an early Pleistocene split between the upper Snake River and Bonneville Basin. Within each of these clades, geographic structuring was highly concordant with the hydrological history of late Pleistocene Lake Bonneville and the upper Snake River, suggesting that glacial-induced shifts in climate and unpredictable geological events have played a major role in shaping genetic subdivision among populations. To examine the effects of vicariant events on phenotypic divergence among Utah chub populations, I mapped chub life histories to the control region haplotype network. I found a nonrandom association between haplotypes and life-history phenotypes. These results suggest that historical events responsible for population fragmentation may have also contributed to phenotypic shifts in life histories, both indirectly by limiting gene flow among populations and directly by altering the selective environments where populations persisted.     

Molecular evidence for host–parasite co-speciation between lizards and Schellackia parasites

Current and past parasite transmission may depend on the overlap of host distributions, potentially affecting parasite specificity and co-evolutionary processes. Nonetheless, parasite diversification may take place in sympatry when parasites are transmitted by vectors with low mobility. Here, we test the co-speciation hypothesis between lizard final hosts of the Family Lacertidae, and blood parasites of the genus Schellackia, which are potentially transmitted by haematophagous mites. The effects of current distributional overlap of host species on parasite specificity are also investigated. We sampled 27 localities on the Iberian Peninsula and three in northern Africa, and collected blood samples from 981 individual lizards of seven genera and 18 species. The overall prevalence of infection by parasites of the genus Schellackia was ～35%. We detected 16 Schellackia haplotypes of the 18S rRNA gene, revealing that the genus Schellackia is more diverse than previously thought. Phylogenetic analyses showed that Schellackia haplotypes grouped into two main monophyletic clades, the first including those detected in host species endemic to the Mediterranean region and the second those detected in host genera Acanthodactylus, Zootoca and Takydromus. All but one of the Schellackia haplotypes exhibited a high degree of host specificity at the generic level and 78.5% of them exclusively infected single host species. Some host species within the genera Podarcis (six species) and Iberolacerta (two species) were infected by three non-specific haplotypes of Schellackia, suggesting that host switching might have positively influenced past diversification of the genus. However, the results supported the idea that current host switching is rare because there existed a significant positive correlation between the number of exclusive parasite haplotypes and the number of host species with current sympatric distribution. This result, together with significant support for host–parasite molecular co-speciation, suggests that parasites of the genus Schellackia co-evolved with their lizard hosts.     

Phylogenetic divergence in leatherside chub (Gila copei) inferred from mitochondrial cytochrome b sequences

We examined intra-specific phylogenetic relationships in leatherside chub, Gila copei. The complete mitochondrial (mt) cytochrome b gene (1140 bp) was sequenced for 30 individuals from 10 populations that span the geographical distribution of this species. Traditional phylogenetic analyses revealed two deeply divergent and evolutionarily distinct mtDNA clades that are geographically separated in northern and southern drainage basins. Interpopulation sequence variation between clades ranged from 7.7 to 8.1%. The northern clade was genetically more similar and phylogenetically more closely related to the selected out-group Lepidomeda m. mollispinus than to the southern clade, suggesting that the taxonomy of this species may require revision. Sequence variation among populations within clades ranged from 0 to 0.3% in the north and from 0 to 0.7% in the south. Statistical parsimony was used to construct phylogenetic networks of haplotypes within clades. Nested clade analysis revealed that geographical fragmentation has played an important role in genetic structuring within northern and southern clades.     

Phylogeography of Japanese water crowfoot based on chloroplast DNA haplotypes

Water crowfoot, Ranunculus subgenus Batrachium, are submerged macrophytes in the Ranunculaceae. We aimed to infer the Quaternary history of these aquatic macrophytes in the Japanese archipelago. We studied 212 individuals of three perennial and one annual species from 46 populations covering the entire geographic range and found eight haplotypes based on approximately 1800 bp of four spacers in chloroplast DNA. The relationships among haplotypes were resolved using maximum parsimony and parsimony network analyses. To identify the zones of clear genetic boundaries, Monmonier's algorithm was used. The eight haplotypes were distinguished from adjacent haplotypes by one substitution or indel. Each of the 46 populations was fixed for a single haplotype. Inconsistency between cpDNA haplotypes and Batrachium taxa was found, except in one annual species. The distribution of the haplotypes in perennial species was highly geographically structured. An abrupt genetic change was detected between the Tohoku region and more southerly regions. Since the perennial Batrachium are cold-adapted, this genetic differentiation may be due to historical changes in their distributions caused by Quaternary climatic oscillations: interglacial retreats in colder refugia and glacial range expansions. The single haplotype composition of each population may have been shaped by founder effects during colonization and/or by genetic drift. The interglacial refugial populations must have been small enough to deplete haplotype diversity. Inconsistency between cpDNA haplotypes and Batrachium taxa may be due to incomplete lineage sorting of ancestral polymorphic haplotypes.     

Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds

A fragment of the mitochondrial cytochrome b gene of avian malaria (genera Haemoproteus and Plasmodium) was amplified from blood samples of 12 species of passerine birds from the genera Acrocephalus, Phylloscopus and Parus. By sequencing 478 nucleotides of the obtained fragments, we found 17 different mitochondrial haplotypes of Haemoproteus or Plasmodium among the 12 bird species investigated. Only one out of the 17 haplotypes was found in more than one host species, this exception being a haplotype detected in both blue tits (Parus caeruleus) and great tits (Parus major). The phylogenetic tree which was constructed grouped the sequences into two clades, most probably representing Haemoproteus and Plasmodium, respectively. We found two to four different parasite mitochondrial DNA (mtDNA) haplotypes in four bird species. The phylogenetic tree obtained from the mtDNA of the parasites matched the phylogenetic tree of the bird hosts poorly. For example, the two tit species and the willow warbler (Phylloscopus trochilus) carried parasites differing by only 0.6% sequence divergence, suggesting that Haemoproteus shift both between species within the same genus and also between species in different families. Hence, host shifts seem to have occurred repeatedly in this parasite host system. We discuss this in terms of the possible evolutionary consequences for these bird species.     

线粒体DNA序列变异显示鸭绿江茴鱼为黑龙江茴鱼同物异名

经测序获得北极茴鱼鸭绿江亚种Thymallus arcticus yaluensis和黑龙江茴鱼T.grubii共26尾的线粒体DNA控制区基因全序列,以下游黑龙江茴鱼T.tugarinae、北极茴鱼指名亚种T.a.arcticus和欧洲茴鱼T.thymallus作外群,构建了茴鱼属部分种分子系统发育树,以探讨北极茴鱼鸭绿江亚种分类地位的有效性。结果显示,鸭绿江茴鱼与黑龙江茴鱼的4个地理种群(呼玛河、甘河、牡丹江、松花江)间的Kimura 2-parameter遗传距离仅为0·007~0·015,处于黑龙江茴鱼不同地理种群间的变异范围之内(0·004~0·015),明显小于属内种间的遗传距离(0·046~0·062)。以邻接法(NJ)和最大简约法(MP)构建的茴鱼属部分种系统发育树表明,鸭绿江茴鱼未构成独立的单系群,也未与北极茴鱼T.a.arcticus形成亚种关系,而是聚到黑龙江茴鱼种群内部,共同构为一个单系群。研究表明,鸭绿江茴鱼T.a.yaluensis并非有效种,应为黑龙江茴鱼T.grubii的同物异名,可视为黑龙江茴鱼的一个地理生态种群。     

Genetic variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of multiple introductions

To analyze the genetic relatedness and phylogeographic structure of Aedes aegypti, we collected samples from 36 localities throughout the Americas (Brazil, Peru, Venezuela, Guatemala, US), three from Africa (Guinea, Senegal, Uganda), and three from Asia (Singapore, Cambodia, Tahiti). Amplification and sequencing of a fragment of the mitochondrial NADH dehydrogenase subunit 4 gene identified 20 distinct haplotypes, of which 14 are exclusive to the Americas, four to African/Asian countries, one is common to the Americas and Africa, and one to the Americas and Asia. Nested clade analysis (NCA), pairwise distribution, statistical parsimony, and maximum parsimony analyses were used to infer evolutionary and historic processes, and to estimate phylogenetic relationships among haplotypes. Two clusters were found in all the analyses. Haplotypes clustered in the two clades were separated by eight mutational steps. Phylogeographic structure detected by the NCA was consistent with distant colonization within one clade and fragmentation followed by range expansion via long distance dispersal in the other. Three percent of nucleotide divergence between these two clades is suggestive of a gene pool division that may support the hypothesis of occurrence of two subspecies of Ae. aegypti in the Americas.     

Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences

A portion of the mitochondrial control region (494 bp) was sequenced in 106 great reed warblers sampled in six breeding populations in Europe and one wintering population in Africa. In total, 33 different haplotypes were found. There was little evidence of divergence between populations in northern and western Europe whereas the sample from Greece differed significantly from the other European breeding populations. The lowest haplotypc diversity was found near the distribution range limit in Sweden and in The Netherlands suggesting recent effects of bottlenecks/founder events in these areas. A neighbour-joining analysis of the different haplotypes placed the haplotypes into two distinctive clades, A and B. The divergence of the two clades was on average 1.29%. Accounting for the within cladc variation suggested a divergence time between these lines approximately 70 000 years BP. The frequency of the two clades changed longitudinally across Europe with the A haplotypc in the west and the B haplotypc in the east. All birds from Kenya carried the B haplotypc suggesting an origin of these birds east of Latvia/Greece. The long-term female effective population size was estimated to be 20 000 individuals, which is approximately 2% of current population size.     

The phylogeography of European Atlantic salmon (Salmo salar L.) based on RFLP analysis of the NDl/16sRNA region of the mtDNA

The diversity and distribution of mtDNA haplotypes in Atlantic salmon (Salmo salar L.) from 13 river systems across the species' European range was investigated. Salmon were screened by agarose electrophoresis for variation in a 1400 base pair fragment spanning the ND-1 and 16SrRNA genes. The fragment was amplified by PCR and digested using the restriction endonucleases Avail, Dral, Haelll, Hinfl and Rsal. Nine haplotypes were identified and resolved by parsimony analysis into two major clades. Clade I was ubiquitous and predominated in all samples while Clade II was restricted to eight out of 94 individuals in two of the 13 rivers. The first clade shows two sublineages whose frequency distribution is strongly associated with geography. One sublineage dominated in river systems draining into the Baltic sea and in Iceland, and the other in the river systems elsewhere in Europe. No geographical patterns were apparent within these regions but haplotype frequencies among samples, both within and outside the Baltic region, were significantly heterogeneous. Approximately 8% of haplotype frequency variation occurred among samples, 44% between Baltic and non-Baltic samples and 48% within samples. Baltic samples had a significantly lower haplotype and nucleotide diversity than non-Baltic samples. Current and historical factors potentially responsible for the observed levels and distribution of the haplotype variation are discussed.     

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta,Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.