Species delimitation in trematodes using DNA sequences: Middle-American Clinostomum as a case study

Abstract

The recent development of genetic methods allows the delineation of species boundaries, especially in organisms where morphological characters are not reliable to differentiate species. However, few empirical studies have used these tools to delineate species among parasitic metazoans. Here we investigate the species boundaries of Clinostomum, a cosmopolitan trematode genus with complex life cycle. We sequenced a mitochondrial [cytochrome c oxidase subunit I (COI)] gene for multiple individuals (adults and metacercariae) from Middle-America. Bayesian phylogenetic analysis of the COI uncovered five reciprocally monophyletic clades. COI sequences were then explored using the Automatic Barcode Gap Discovery to identify putative species; this species delimitation method recognized six species. A subsample was sequenced for a nuclear gene (ITS1, 5·8S, ITS2), and a concatenated phylogenetic analysis was performed through Bayesian inference. The species delimitation of Middle-American Clinostomum was finally validated using a multispecies coalescent analysis (species tree). In total, five putative species are recognized among our samples. Mapping the second intermediate hosts (fish) onto the species tree suggests that metacercariae of these five species exhibit some level of host specificity towards their fish intermediate host (at the family level), irrespective of geographical distribution.

Clinostomum poteae n. sp. (Digenea: Clinostomidae), in the trachea of a double-crested cormorant Phalacrocorax auritus Lesson, 1831 and molecular data linking the life-cycle stages of Clinostomum album Rosser, Alberson, Woodyard, Cunningham, Pote & Griffin, 2017 in Mississippi, USA

“…For almost 200 years researchers have debated the systematics of clinostomids. The advent of molecular techniques and the ability to supplement morphological data with nucleotide sequence data has identified the clinostomids as a truly diverse group of trematodes, with new species described regularly Sereno-Uribe et al, 2013;Pérez-Ponce de León et al, 2016;. Currently, six Clinostomum spp.…”

“…Currently, six Clinostomum spp. are recognised in North America based on detailed morphological descriptions of stained and mounted adults recovered from avian hosts (Bravo-Hollis, 1947;Hutton & Sogandares-Bernal, 1960;Pérez-Ponce de León et al, 2016;. Of these, few Clinostomum spp.…”

“…In the absence of experimental challenges, putative life histories and the other aspects of parasite ecology can be determined by linking molecular sequence data obtained from morphologically ambiguous larval stages to gene sequences collected from adult specimens identified by morphologically distinct characters (Criscione et al, 2005). Studies investigating the global and regional diversity of Clinostomum species have demonstrated an unexpected number of putative species identified by molecular and phylogenetic analyses of nuclear and mitochondrial genes (namely the internal transcribed spacer regions and cytochrome c oxidase subunit 1 gene) Pérez-Ponce de León et al, 2016). In North America, six distinct Clinostomum spp.…”

“…At least nine additional Clinostomum spp. are proposed, based solely on mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and ribosomal internal transcribed spacer regions (ITS) sequences obtained from larval and some adult stages (Pérez-Ponce de León et al, 2016), although corresponding morphological data for these putative species are limited. Further efforts to elucidate the species boundaries within the genus Clinostomum should require detailed morphometric analysis, in combination with molecular analyses of multiple gene targets, and when available, the inclusion of ecological data.…”

The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam

“…This is particularly so when phylogenetically divergent species are being compared and suggest that this region is not suitable for deep-level phylogenies [17]. At the level of genus and species, however, alignments of ITS sequences have proved valuable for phylogenetic studies and molecular taxonomy [17, 41, 42]. …”