Zoologica Scripta A large-scale molecular survey of Clinostomum (Digenea, Clinostomidae) SEAN A. LOCKE,MONICA CAFFARA,DAVID J. MARCOGLIESE &MARIA L. FIORAVANTI Submitted: 21 July 2014 Locke S.A., Caffara M., Marcogliese D.J., Fioravanti M.L. (2015). A large-scale molecular Accepted: 9 November 2014 survey of Clinostomum (Digenea, Clinostomidae). —Zoologica Scripta, 44, 203–217. doi:10.1111/zsc.12096 Members of the genus Clinostomum Leidy, 1856 are parasites that mature in birds, with occasional reports in humans. Because morphological characters for reliable discrimination of species are lacking, the number of species considered valid has varied by an order of magnitude. In this study, sequences from the DNA barcode region of cytochrome c oxidase I (CO1) and/or internal transcribed spacer (ITS) from specimens from Mexico, Bolivia, Peru, Brazil, Kenya, China and Thailand were analysed together with published sequences from Europe, Africa, Indonesia and North America. Although ITS and CO1 distances among specimens were strongly correlated, distance-based analysis of each marker yielded different groups. Putative species indicated by CO1 distances were consistent with available morphological identifications, while those indicated by ITS conflicted with morphological identifications in three cases. There was little overlap in sequence variation within and between species, particularly for CO1. Although ITS and CO1 distances tended to increase in specimens that were further apart geographically, this did not impair distance-based spe- cies delineation. Phylogenetic analysis suggests a deep division between clades of Clinosto- mum inhabiting the New World and Old World, which parallels the distribution of their principal definitive hosts, the Ardeidae. Corresponding author: Sean A. Locke, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada. E-mail: [email protected] Sean A. Locke, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada Monica Caffara, Department of Veterinary Medical Sciences, Alma Mater Studiorum, University of Bologna, Via Tolara di Sopra 50, Ozzano Emilia, 40064, Italy. E-mail: [email protected] David J. Marcogliese, Aquatic Biodiversity Section, Watershed Hydrology and Ecology Research Division, Water Science and Technology Directorate, St. Lawrence Centre, Environment Canada, 105 McGill Street, 7th floor, Montreal, QC, H2Y 2E7, Canada. E-mail: david.marcogliese@ ec.gc.ca Maria L. Fioravanti, Department of Veterinary Medical Sciences, Alma Mater Studiorum, University of Bologna, Via Tolara di Sopra 50, Ozzano Emilia, 40064, Italy. E-mail: marialeti. fi[email protected] Sean A. Locke and Monica Caffara contributed equally to this study. Introduction Mirzoeva 1983), and there is also no reliable way to distin- Clinostomum Leidy, 1856 (Digenea: Clinostomidae) is a cos- guish species based on host species, because host associa- mopolitan genus of digenetic trematodes with complex, tions of all developmental stages are broad or poorly parasitic life cycles. Larval stages infect first gastropods and known (Lo et al. 1982; Matthews & Cribb 1998; Hoffman then fish or amphibians. Adults inhabit the oral cavity and 1999; Bullard & Overstreet 2008). oesophagus of vertebrates, mainly birds, which ingest sec- Molecular delineation of species has become routine in ond intermediate hosts. Humans are occasionally infected such situations, and DNA sequences are accumulating from after eating undercooked fish, leading to halzoun syndrome Clinostomum species from different biogeographic regions. (reviewed by Sutili et al. 2014; see also Table 1). Few mor- Variation in mitochondrial or ribosomal DNA now supports phological characters differ reliably among species of the validity of five species (C. complanatum, C. marginatum, Clinostomum (Ukoli 1966; Yamaguti 1971; Feizullaev & C. tataxumui, C. cutaneum and C. phalacrocoracis) and ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 Reproduced with the permission of the Minister of the Environment. 203 Molecular survey of Clinostomum spp. S. A. Locke et al. Table 1 Definitive hosts of Clinostomum spp. (excluding experimen- useful when estimating whether genetic variation is intra- tal infections, compiled from Gibson et al. 2005) specific or interspecific. Variation in the DNA barcode region of cytochrome c Number oxidase 1 (CO1) is effective for discriminating species in Order Family of records Clinostomum (Caffara et al. 2011, 2013, 2014; Sereno-Uribe Pelecaniformes Ardeidae 113 et al. 2013; Pinto et al. 2014), but the effect of geographic Suliformes Phalacroracidae 17 variation has not been assessed in these species, which show Primates Hominidae 13 both sympatric and allopatric distributions. Empirical stud- Pelecaniformes Threskiornithidae 5 Suliformes Anhingidae 4 ies of the effect of spatial scale on DNA barcoding are lim- Charadriiformes Laridae 3 ited to relatively well-described taxa (Hebert et al. 2004; Ciconiiformes Ciconiidae 3 Lukhtanov et al. 2009; Bergsten et al. 2012; Mutanen et al. Podicipediformes Podicipedidae 3 2012; Geiger et al. 2014). Herein, the effect of spatial scale Falconiformes Accipitridae 2 on species delineation is explored in two markers (CO1 and Charadriiformes Rynchopidae 1 Charadriiformes Sternidae 1 ITS) commonly employed in Clinostomum and other digen- Gruiformes Raillidae 1 eans. The use of multiple markers is often suggested to be Strigiformes Strigidae 1 preferable to single-locus species delimitation (e.g. Dupuis et al. 2012), but, to our knowledge, whether this adds clarity in wide sampling has not been quantitatively assessed. morphological differences between these species have also been observed (Dzikowski et al. 2004; Gustinelli et al. 2010; Materials and methods Caffara et al. 2011, 2013, 2014; Sereno-Uribe et al. 2013). Source of Clinostomum specimens and sequences Sequences indicate these species are restricted to single con- Specimens were collected from Canada, Mexico, Bolivia, tinents, which lends support to discrimination of Clinosto- Peru, Brazil, Kenya, China and Thailand (Table 2). Data mum species based on geographical distributions, a common were combined with those from collections in the Nearctic, but controversial practice in the premolecular era (Braun Neotropic, Palearctic, Indomalayan and Afrotropic zones 1901; Baer 1933; Dowsett & Lubinsky 1980). However, by Gustinelli et al. (2010), Caffara et al. (2011, 2013, 2014), molecular studies of Clinostomum have until now included Bonett et al. (2011), Riauwaty et al. (2012), Sereno-Uribe data from at most two continents and, given the vagility of et al. (2013) and Pinto et al. (2014). Sequences of CO1 avian hosts, intercontinental distributions of Clinostomum from Clinostomum phalacrocoracis and Clinostomum cutaneum species remain plausible. In this study, both newly obtained are from the specimens studied by Gustinelli et al. (2010). samples and existing data are used to expand the spatial scale Data from one specimen studied by Caffara et al. (2011) of sequence analysis in Clinostomum, with the primary aim of and one studied by Bonett et al. (2011), which had intra- better characterizing the species diversity and distribution of individual variation in mitochondrial or nuclear sequences, this parasite. were not included. The total data set includes sequences Increasing the scale of sampling presents a challenge, from Clinostomum from 54 host species, including six birds, however, because whether based on molecules or morphol- 41 fishes, six amphibians, and one gastropod. Sequences ogy, alpha taxonomy becomes more complex when species from this study are deposited on BOLD and in Gen- are sampled broadly (Thorpe 1987; Kuo & Avise 2005). In Bank (CO1 accessions KM518245-54, KP110515-63, widely dispersed samples, a complete lack of variation in KP150305-6; ITS accessions KM518255-59, KP110564-97, markers such as internal transcribed spacer rDNA (ITS) KP150307). Accessions for sequences used from other stud- can indicate a digenetic trematode species has a large geo- ies are found in Data S1. graphic range (e.g. Chambers & Cribb 2006). However, some level of genetic variation is expected within species Molecular methods, species discrimination and phylogenetic when sampled at large scales (Lo et al. 2001; Aiken et al. analysis 2007; Locke et al. 2012; Brant et al. 2013; McNamara et al. Total DNA was extracted from individual specimens, 2014). In samples collected at the same locality, high amplified and sequenced at one of two different laborato- genetic divergence is evidence of reproductive isolation, ries. In some cases (C. attenuatum, C. detruncatum, C. phi- and together syntopy and deep divergence constitute good lippinensis, Clinostomum sp. 8), specimens were stained, evidence of separate species even in the absence of other mounted on slides and studied morphologically after a distinguishing features (Padial et al. 2010; Kekkonen & small portion was removed for molecular analysis. Hebert 2014; McNamara et al. 2014). Thus, the spatial Sequences of internal transcribed spacers (ITS1, 5.8S, scale and magnitude of genetic differentiation are both ITS2) of rDNA and 600 bp from the barcode region of 204 ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 Zoologica Scripta ª 2015 Her Majesty the Queen in