High Local Diversity of <i>Trypanosoma</i> in a Common Bat Species, and Implications for the Biogeography and Taxonomy of the <i>T. cruzi</i> Clade

<div><p>The <i>Trypanosoma cruzi</i> clade is a group of parasites that comprises <i>T. cruzi</i> sensu lato and its closest relatives. Although several species have been confirmed phylogenetically to belong to this clade, it is uncertain how many more species can be expected to belong into this group. Here, we present the results of a survey of trypanosome parasites of the bat <i>Artibeus jamaicensis</i> from the Panamá Canal Zone, an important seed disperser. Using a genealogical species delimitation approach, the Poisson tree processes (PTP), we tentatively identified five species of trypanosomes – all belonging to the <i>T. cruzi</i> clade. A small monophyletic group of three putative <i>Trypanosoma</i> species places at the base of the clade phylogeny, providing evidence for at least five independent colonization events of these parasites into the New World. <i>Artibeus jamaicensis</i> presents a high diversity of these blood parasites and is the vertebrate with the highest number of putative trypanosome species reported from a single locality. Our results emphasize the need for continued efforts to survey mammalian trypanosomes.</p></div

Five independent colonization events of <i>Trypanosoma cruzi</i> clade members to the New World.

<p>Cladogram of species belonging to the <i>Trypanosoma cruzi</i> clade, depicting in red are the branches that indicate dispersal events to the Americas with grey branches denoting the species that are not present in the New World. Note that <i>T. dionisii</i> 2 and <i>T. conorhini</i> are distributed in the New and Old Worlds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603-Hamilton3" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603-Hamilton4" target="_blank">[43]</a>. Branch lengths in the cladogram are not proportional to divergence; for branch length estimates refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone-0108603-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone-0108603-g002" target="_blank">2</a>.</p

Phylogeny of the <i>Trypanosoma cruzi</i> clade.

<p>Maximum likelihood tree constructed with concatenated 18S rRNA and gGAPDH genes. Numbers on the branches represent support values corresponding to bootstrap replicates (right) and Bayesian posterior probabilities (left). Clades highlighted with colored boxes correspond to trypanosomes detected in Panamá. Collapsed branches at the base of the phylogeny indicate low support for the placement of the three Panamian lineages. The symbol//on the branch to the outgroup indicates a shortened branch. GenBank accession numbers of the samples used in this phylogeny are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603.s001" target="_blank">Table S1</a>.</p

Putative species delimitation of members of the <i>Trypanosoma cruzi</i> clade.

<p>Maximum likelihood phylogeny with Bayesian support values presented for all 18 lineages recognized as species for the PTP analyses. Monophyletic groups in red indicate a single putative species as well as terminal branches in blue. Names of terminals indicate codes of the samples. Names in bold after a | symbol are taxonomic or geographic identifiers of the putative species. Arrows indicate the 5 putative species found in <i>Artibeus jamaicensis</i> in Panamá.</p

Bats, Trypanosomes, and Triatomines in Ecuador: New Insights into the Diversity, Transmission, and Origins of <i>Trypanosoma cruzi</i> and Chagas Disease

<div><p>The generalist parasite <i>Trypanosoma cruzi</i> has two phylogenetic lineages associated almost exclusively with bats—<i>Trypanosoma cruzi</i> Tcbat and the subspecies <i>T</i>. <i>c</i>. <i>marinkellei</i>. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time <i>T</i>. <i>cruzi</i> Tcbat and <i>T</i>. <i>c</i>. <i>marinkellei</i> in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines <i>Cavernicola pilosa</i> and <i>Triatoma dispar</i> sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for <i>T</i>. <i>c</i>. <i>marinkellei</i> than any of the <i>T</i>. <i>c</i>. <i>cruzi</i> genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of <i>T</i>. <i>c</i>. <i>marinkellei</i> supports a long evolutionary relationship between <i>T</i>. <i>cruzi</i> and bats, implying that bats are the original hosts of this important parasite.</p></div

Mitochondrial diversity of <i>Trypanosoma cruzi</i>.

<p>Nucleotide diversity (π) of mitochondrial lineages of <i>Trypanosoma cruzi</i> calculated for the haplotypes of the cytb gene. The subspecies <i>T</i>. <i>c</i>. <i>marinkellei</i> shows larger nucleotide diversity than the other examined lineages. Whiskers in each bar indicate the standard error.</p

Results of PCR screenings for <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i>.

<p>Results of PCR screenings for <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i>.</p

Constructions in Ecuador where <i>Cavernicola pilosa</i> and <i>Triatoma dispar</i> were found in association with bats.

<p><b>A.</b> House with cinderblock walls at Rancho Alegre, Zamora Chinchipe where <i>C</i>. <i>pilosa</i> (inset) was inhabiting a roost of <i>Myotis</i> sp. <b>B.</b> Adobe barn in Chinguilamaca, Loja where <i>T</i>. <i>dispar</i> (inset) was found associated with <i>Molossus molossus</i> and <i>Myotis</i> sp. Arrows indicate the entrances to the bat roosts where the insects were collected.</p

Network genealogy using partial 18S rRNA gene sequences

<p>from eight new trypanosomes characterised in this study (in bold) plus 70 other sequences from all DTUs (TcI-TcVI and Tcbat) of <i>T</i>. <i>cruzi</i> and 22 sequences from <i>T</i>. <i>c</i>. <i>marinkellei</i>. Network constructed with the NeighborNet algorithm excluding all conserved sites and with uncorrected p-distance. Numbers in nodes correspond to bootstrap support values using the same parameter optimized for network inferences.</p

Mitochondrial phylogeny of <i>Trypanosoma cruzi</i>.

<p>Maximum likelihood tree of a fragment of the cytb gene of <i>Trypanosoma cruzi</i> and <i>T</i>. <i>dionisii</i> as outgroup, representing 60 haplotypes from 362 sequences. The parentheses at the tip labels contain the number of identical sequences per each haplotype, and in the TcIII-TcVI group the DTU identity for each haplotype is indicated. Numbers on branches correspond to bootstrap support values. Stars indicate the haplotypes found in Ecuador.</p