Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes☆
Graphical abstract
Introduction
Digenetic trematode larvae are often found in the eyes of fish. Metacercariae in species of Diplostomum, Tylodelphys and Austrodiplostomum (Digenea: Diplostomidae) may cause mortality or pathological changes (e.g., blindness) in freshwater fish (reviewed by Chappell et al., 1994 and Overstreet and Curran, 2004), with effects that vary among closely related parasite species (Larsen et al., 2005). Aside from their importance in fish health, eye-dwelling diplostomids are frequent subjects of evolutionary and ecological studies (e.g., Ballabeni and Ward, 1993, Kalbe and Kurtz, 2006). However, diplostomid metacercariae are difficult to identify morphologically and molecular methods are often used to distinguish species (Locke et al., 2010a, Locke et al., 2010b, Rellstab et al., 2011, Cavaleiro et al., 2012, Behrmann-Godel, 2013, Chibwana et al., 2013, Désilets et al., 2013, Georgieva et al., 2013, Ndeda et al., 2013, Blasco-Costa et al., 2014, Pérez-del-Olmo et al., 2014, Kuhn et al., 2015, García-Varela et al., 2015, Mateos-Gonzalez et al., 2015, Otachi et al., 2015). Consequently, DNA sequences from diplostomids are accumulating from all over the world, particularly from Diplostomum spp. Sequences can clarify whether morphological heterogeneity represents interspecific (Blasco-Costa et al., 2014, Faltýnková et al., 2014, Pérez-del-Olmo et al., 2014) or intraspecific variation among metacercariae (Cavaleiro et al., 2012, Otachi et al., 2015). Molecular links to adults from avian definitive hosts are usually needed to identify, rather than merely discriminate, species of metacercariae (Galazzo et al., 2002, Locke et al., 2010a, Locke et al., 2011, Georgieva et al., 2013, Pérez-del-Olmo et al., 2014). However, even when sequences are obtained from adult diplostomids, often not all species detected in surveys of metacercariae are resolved (Galazzo et al., 2002, Georgieva et al., 2013, Pérez-del-Olmo et al., 2014). The result is a sequence database dominated by unidentified, provisional species, many of which have been distinguished primarily through DNA sequence analysis.
This lack of links with available species names is undesirable but it may be unavoidable. Unrecognised diversity is routinely encountered in molecular surveys of digeneans (Poulin, 2011) and some lag can be expected between the genetic detection and identification, description or characterisation of putative species with other methods. Furthermore, provisional species delineations can be valuable. For example, provisional, molecular delineations of diplostomid species have revealed compositional differences (Rellstab et al., 2011) and evidence of interspecific interactions among metacercarial communities in fish eyes (Désilets et al., 2013). In other ecological field studies, diplostomids in fish eyes are often pooled into genera (e.g., Marcogliese et al., 2006), even when local species are known from prior molecular taxonomic work (Galazzo et al., 2002). Pooling metacercariae in this manner can change the factors implicated in, and reduce variance explained by, ecological analysis of parasite communities (Locke et al., 2013).
Besides increasing the resolution of ecological field data, provisional delineations of diplostomid species have also shed light on the biogeography and evolutionary relationships among species. For example, Diplostomum mashonensis was reallocated to Tylodelphys based on molecular phylogenetic analysis (Chibwana et al., 2013) that included a number of unidentified, putative species. In her review of the extensive pre-molecular literature on Diplostomum, Niewiadomska (1996) grouped similar species by the infection site of their metacercaria (eye lenses or other tissues), a distinction confirmed with recent molecular data in two ways. First, the fidelity of sequenced metacercariae of Diplostomum spp. for either lens or non-lens tissues (e.g., Locke et al., 2010a, Georgieva et al., 2013, Blasco-Costa et al., 2014) supports the diagnostic utility of the infection site. Second, molecular phylogenetic analysis shows lens and non-lens species each form a clade of closely related Diplostomum spp. (Blasco-Costa et al., 2014), which provides an evolutionary explanation for morphological similarity within these groups. Contrary to what Gibson (1996) expected based on the mobility and distribution of their avian hosts, none of the genetically distinguished Diplostomum spp. are common to both the Nearctic and Palearctic (Galazzo et al., 2002, Locke et al., 2010a, Cavaleiro et al., 2012, Georgieva et al., 2013). Intriguingly, it appears this biogeographic partition results mainly from two waves of Nearctic to Palearctic colonisation, one in the lens clade, the other in the clade of non-lens species (Blasco-Costa et al., 2014). Thus, while description or identification of species detected in sequencing surveys is urgently needed, insights are still possible in the interim. Moreover, prematurely naming genetically distinguished species may lead to misidentifications. For example, although the unreliability of many records of Diplostomum spathaceum was recognised before any diplostomid DNA was sequenced (Gibson, 1996, Niewiadomska, 1996), ignoring the probable commonness of this species can lead to questionable interpretations of molecular data (see Georgieva et al., 2013 for discussion).
Several results emerging from recent molecular studies are in need of further testing. Lester and Huizinga (1977) were among the first to propose a connection between metacercarial infection site and host specificity, which was later supported with molecular data from Diplostomum from the Great Lakes – St. Lawrence River basin, Canada (GLSLRB). The distribution of Diplostomum spp. distinguished by sequencing suggested that lower immune activity in the lens of the eye protects metacercariae, allowing resident species access to a wider variety of hosts than those inhabiting other tissues (Locke et al., 2010a). However, five of 12 Diplostomum spp. were encountered only once by Locke et al. (2010a), thus preventing comparison of host specificity in nearly half the species detected in the GLSLRB. Here we re-evaluate this phenomenon with additional data from recent studies and our own sampling. This could reveal the previously observed link between host specificity and the lens to be an artefact of sampling a small number of species in a single ecosystem (Poulin et al., 2011). A parallel goal was to expand the DNA barcode database in terms of specimens, host species and biogeographic regions, and to integrate past and present molecular species delineations in one framework. We employed distance-based analyses of sequences of cytochrome c oxidase 1 (CO1) mitochondrial DNA and internal transcribed spacers 1 and 2 and 5.8S nuclear ribosomal DNA (ITS). Our aim was not to test these phenetic methods, which produce results largely consistent with alpha taxonomy in diverse groups (Puillandre et al., 2012, Ratnasingham and Hebert, 2013). Here too, consistency with prior studies of diplostomids was the main determinant of the utility of these methods, which were also chosen due to their computational scalability, a consideration given the rapid growth of sequence databases (Collins and Cruickshank. 2014, Ondrejicka et al., 2014). Nonetheless, although generally reliable, these methods do not take into account potential artefacts of sampling effort or spatial scale. Thus, with the same forward-looking outlook, we explore the continued usefulness of these approaches as more data accumulate, by examining effects of sampling effort on key genetic distance metrics in diplostomids.
Section snippets
Data collection
Diplostomids were collected during necropsies of fish (mainly), amphibians, birds and molluscs in Eurasia, North and South America, including collections described by Moszczynska et al. (2009), Locke et al., 2010a, Locke et al., 2010b, Locke et al., 2011 and Désilets et al. (2013) (Fig. 1). Total DNA was extracted from individual parasites, except in a few cases where cercariae from individual snails were pooled. The barcode region of CO1 was amplified and sequenced at the Canadian Centre for
Species discrimination with CO1
Sequences of CO1 were obtained from 523 specimens. These were analysed with 1167 sequences generated by Moszczynska et al., 2009, Locke et al., 2010a, Désilets et al., 2013, which are published herein, and 336 sequences published by Moszczynska et al., 2009, Locke et al., 2010a, Behrmann-Godel, 2013, Chibwana et al., 2013, Georgieva et al., 2013, Blasco-Costa et al., 2014, O’Hear et al., 2014, Pérez-del-Olmo et al., 2014, Kuhn et al., 2015 and Otachi et al. (2015); details are in Supplementary
Discussion
Using distances between DNA sequences to delineate potential species as well as supporting evidence, we surveyed diplostomid diversity on a wide scale. Unsurprisingly, a metric reflecting a basic premise underlying genetic distance-based species delineation (NN ratio) was correlated with sampling effort (Fig. 5). This might seem to pose more of a problem for distance-based methods of delineating species than for those based on characters, approaches with important logical distinctions (DeSalle
Note added in proof
The species referred to herein as "Diplostomum mergi 1" is Diplostomum parviventosum (Selbach et al., 2015).
Acknowledgements
This research was made possible by funding from the Canadian Federal Government’s Genomics Research Development Initiative to DJM, by Discovery Grant A6979 from the National Sciences and Engineering Research Council (NSERC), Canada to JDM, by National Geographic, USA grant 9330-13 to SAL and by support from Paul D.N. Hebert (Center for Biodiversity Genomics, University of Guelph, Canada) from NSERC, Genome Canada, the Ontario Genomics Institute, Canada and the International Barcode of Life
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Note: Representative nucleotide sequences reported in this paper are available in GenBank under accession numbers KR271025–KR271523, KT186784–KT186806 and all data are on the BOLD database (see dx.doi.org/10.5883/DS-DIPLOIJP).