Scale Molecular Survey of Clinostomum (Digenea, Clinostomidae)

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 species 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 olgc Scripta Zoologica ª

05HrMjsyteQeni ih fCanada of Right in Queen the Majesty Her 2015 Table 2 Specimens, sequences, origins and status of species of Clinostomum

N specimens and sequences

ª C. complanatum Species Cercariae Metacercariae Adults CO1 ITS Hosts Localities Status of putative species 05RylSeihAaeyo Sciences, of Academy Swedish Royal 2015 C. attenuatum 3a +13a +12a +1 Lithobates clamitans Quebec, Canada; Lithobates pipiens unknown state, USA 14a+10b 1a 10a+10b 12a+5b Barbus barbus Emilia Romagna, Barbus meridionalis Tuscany, Italy Lepomis gibbosus C. cutaneum Lissotriton vulgaris Phalacrocoraxsp. C. detruncatum Squalius cephalus Triturus carnifex c c c

44 1 1 22Ardea cinerea Sagana, Kenya

,Mrh21,p 203 pp 2015, March 2, , Oreochromis niloticus niloticus 331Synbranchus marmoratus Parana State, Brazil C. marginatum 1 + 23a +21d+23e 3a+10e 26a +21d+7e 15a+33e Ardea alba Ontario, Quebec, Canada; Ardea herodias Oaxaca, Chihuahua, Bufo boreas Durango, Veracruz, Catostomus nebuliferus Mexico

– Characodon lateralis 217 Cyprinodon meeki Eurycea tynerensis Lepomis macrochirus Notropissp. Micropterus dolomieu

C. Micropterus salmoides Locke A. S. Perca ﬂavescens Notemigonus crysoleucas Etheostoma nigrum f f j cf. marginatum 1 1 P. ﬂavescens Quebec, Canada CCS : Distinct CO1 (differs by ≥8.2% from C. marginatum al. et and ≥4.8% from C. attenuatum ) and sympatric with both

C. marginatum and C. attenuatum .

C. phalacrocoracis 1c+8g 2c 2 + 8g 1 + 2c+8g Ardea cinerea Sagana, Kenya of survey Molecular C. tataxumui Oreochromis niloticus niloticus Northern district, Israel Cichlidae gen. sp. C. philippinensis 1 + 1g 11+ 1g Trichogaster microlepis Thailand; Riau, Indonesia Trichogaster trychopterus 20 + 43e 23e 20 + 47e 10 + 63e Arde alba Guerrero, Jalisco, Oaxaca, Ardea herodias Veracruz, Tabasco, Mexico

Dormitator latifrons Clinostomum Dormitator maculatus Cichlasoma trimaculatum Eleotrix picta spp.

205 Gobiomorus maculatus 206 of survey Molecular

Table 2 Continued

N specimens and sequences

Species Cercariae Metacercariae Adults CO1 ITS Hosts Localities Status of putative species Clinostomum Clinostomum Petenia splendida Clinostomum Profundulus sp. Tigrisoma mexicanum sp. 1 1 1 1 Rhamdia guatamensis Yucatan, Mexico SCSj: Distinct CO1, but no support in ABGD analysis of ITS spp. (grouped with Clinostomum sp. 2, from which CO1 differs

by 2.5%, ITS by 0.27%). Locke A. S. Clinostomum sp. 2 5 5 1 Sicydium salvini Oaxaca, Mexico CCS: Distinct CO1 and ITS, sympatric with C. tataxumui , C. marginatum. sp. 31 11Poecilia mexicana Veracruz, Mexico CCS: Distinct CO1 and ITS, sympatric with C. tataxumui .

Clinostomum sp. 41 11Apistogramma sp. Iquitos, Peru SCS: Distinct CO1, but no support in ABGD analysis of ITS al. et (grouped with Clinostomum sp. 1, from which CO1 differs by ≥10.6%, ITS by 0.5% plus a 2-bp deletion). Clinostomum sp. 52 22Cichlasoma boliviense Santa Cruz, Bolivia CCS: Distinct CO1 and ITS, sympatric with Clinostomum spp. 6 and 7. Clinostomum sp. 61 11Cichlasoma boliviense Santa Cruz, Bolivia CCS: Distinct CO1 and ITS, sympatric with Clinostomum spp. 5 and 7. olgc Scripta Zoologica Clinostomum sp. 71h 1 + 5h 6h 1 + 4h Biomphalaria sp. Gymnotus carapo Minas Gerais, Brazil; CCS: Distinct CO1 and ITS, sympatric with Clinostomum Poecilia reticulata (experimental) anta Cruz, Bolivia spp. 5 and 6. Clinostomum sp. 89 99Ctenopharyngodon idella Hubei, China CCS: Distinct CO1, but no support in ABGD analysis of ITS Carassius auratus (grouped with C. complanatum , from which CO1 ª differs ≥3.5%, ITS by mean 0.1% (range 0–0.23%, 05RylSeihAaeyo Sciences, of Academy Swedish Royal 2015 plus a 6-bp insertion). Morphologically distinct from C. complanatum . Clinostomum sp. 94i 4i Anabas testudineus Yogyarkarta, Indonesia UCSj: Distinct ITS.

a data from Moszczynska et al. 2009; Caffara et al. 2011. b data from Caffara et al. 2013.

ª c data from Gustinelli et al. 2010. 05HrMjsyteQeni ih fCanada of Right in Queen the Majesty Her 2015 d data from Bonett et al. 2011. e data from Sereno-Uribe et al. 2013. f data from Van Steenkiste et al. 2014. g data from Caffara et al. 2014. 44 h ,Mrh21,p 203 pp 2015, March 2, , data from Pinto et al. 2014. i data transcribed from Riauwaty et al. 2012, nb., sequences are not published on Genbank. j CCS = confirmed candidate species, SCS = semi-confirmed candidate species, UCS = unconfirmed candidates species, Padial et al. 2010. – 217 S. A. Locke et al. Molecular survey of Clinostomum spp. cytochrome c oxidase I (CO1) were obtained from C. mar- Fast Fourier Transform (MAFFT, Katoh & Toh 2008), ginatum, as described by Moszczynska et al. (2009) and analysed in MEGA 5 (Tamura et al. 2011) and grouped Caffara et al. (2011) at the Canadian Centre for DNA Bar- into putative species using Automated Barcode Gap Dis- coding (CCDB) and, in one specimen, by Van Steenkiste covery (ABGD, Puillandre et al. 2012). For ABGD analysis et al. (2014). Most specimens of other species were pro- of CO1, default parameters were used to distinguish puta- cessed at the fish pathology laboratory, Department of Vet- tive species based on recursive partitions of genetic dis- erinary Medical Sciences (DIMEVET), where total DNA tances into those occurring within and between groups. was extracted with PureLinkTM Genomic DNA Kits (Invi- For ITS, we report results with the minimum limit of trogen) following the manufacturer’s protocol, and the intraspecific (Pmin) variation set to 0.01%, but other start- primers and protocols in Moszczynska et al. (2009) and ing conditions were also explored. Maximum-likelihood Caffara et al. (2011) were used to obtain sequences of the (ML) trees were constructed to determine whether the same region of CO1, and those of Gustinelli et al. (2010) phenetic clusters identified using the foregoing methods were used to obtain sequences of ITS. At both CCDB and were monophyletic. Diagnostic characters (nucleotides StarSEQ GmbH (Mainz, Germany), PCR products were shared by all specimens in a species, and no members of sequenced with an ABI 3730 DNA Analyzer. Chromato- other species) were assessed by inspecting alignments. A grams were assembled with Vector NTI AdvanceTM 11 subset of specimens were identified to species morphologi- software (Invitrogen, Carlsbad, CA, USA) or Geneious cally (in the present and other studies), and these identifi- (Biomatters, New Zealand). cations were extended to sequenced specimens with The main purpose of this study, to test existing species matching or similar DNA. hypotheses and propose new ones within Clinostomum,isa In tree-based species delineation using separate markers, goal that can now be achieved using over a dozen platforms the degree of mutual support for putative species can be for delineating species using DNA sequences (Jorger€ et al. compared using the topological congruence of trees, or 2012; Carstens et al. 2013; Prevot et al. 2013). Comparison assessed in trees made with concatenated markers. In the of these methods is better achieved in well-sampled taxa distance-based context of this study, agreement in ITS- (Lim et al. 2012) in which species boundaries can be veri- and CO1-distance matrices was assessed in a nonparametric fied independently and beyond the scope of this work. In Mantel test (10 000 permutations in RELATE, Clarke & this study, putative species are distinguished using two dis- Warwick 2001), which determined whether pairs of speci- tance-based algorithms (Puillandre et al. 2012; Ratnasing- mens with divergent ITS sequences also tend to have ham & Hebert 2013) that remove sources of potential bias divergent CO1 sequences. A similar approach was used to (in particular, the need for a pre-determined gap in varia- test for a relationship between genetic and geographic dis- tion within and between species). In datasets comprising a tances. Geographic distances between specimens were cal- combined total of 23 800 specimens from 4231 species culated from the latitude and longitude of collection or, (insects, crustaceans and vertebrates), these methods clus- when unavailable, an approximation based on place names tered barcode sequences into groups consistent with recog- (using PASSaGE, Rosenberg & Anderson 2011). nized taxonomy in 80–90% of specimens and species and Estimates of phylogenetic relationships among species one of the methods (Ratnasingham & Hebert 2013) pro- were obtained with separate ML analysis of non-redundant duced results consistent with tree-based species delineation. sequences (filtered using Cd-Hit, Li & Godzik 2006) of Where data permit, species delineated with these algo- CO1 and ITS. A Bayesian inference (BI) analysis of concat- rithms are assessed with an integrative approach similar to enated sequences was also conducted using MrBayes that used by Kekkonen & Hebert (2014). Conclusions are (Ronquist et al. 2012). The BI analysis was based on single, based on congruence of results between algorithms and strict majority consensus sequences (i.e. fewest ambiguities) markers, consistency with prior studies, morphology, created for each species, using Geneious. This generated monophyly, the presence of diagnostic nucleotides and co- longer sequences per species, thereby allowing more occurrence in host species and sampling localities and exhaustive analysis based on a larger number of characters, regions. and avoided fragmentation due to lack of overlap in Putative species were distinguished using the algorithm sequences of different lengths from different specimens. of Ratnasingham & Hebert (2013), which assigns Barcode Sequences from representatives of two species in the Dip- Index Numbers (BINs) to operational taxonomic units lostomidae (data from Galazzo et al. 2002; Locke et al. based on CO1 sequence cluster quality after alignment on 2010, 2011), which are closely related to Clinostomum BOLD (Ratnasingham & Hebert 2007). The BIN algo- (Olson et al. 2003) and from Euclinostomum (Clinostomidae) rithm is an online resource only implemented for CO1 (data from Senapin et al. 2014) were included as outgroups. sequences. Sequences were also separately aligned using Bayesian inference was based on five unlinked partitions of

ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 207 Molecular survey of Clinostomum spp. S. A. Locke et al. the data in which nucleotide substitution models and rates species, minimum interspecific variation in CO1 was were expected to differ: ITS sequence, gaps in the ITS greater than the maximum intraspecific variation (Fig. 4). alignment (coded using FastGap v1.2, Borchsenius 2009) All five species supported by prior studies combining mor- and one data set for each codon position in CO1, with phological and molecular identifications (Gustinelli et al. Diplostomum baeri (Diplostomidae) designated as out-group. 2010; Caffara et al. 2011, 2013, 2014; Sereno-Uribe et al. Nucleotide substitution models were selected with the 2013) were supported in this outcome, as were three addi- Bayesian information criterion in MEGA (Tamura et al. tional species identified in this study, and all species were 2011), with ITS modelled with Kimura 2-parameter and reciprocally monophyletic in ML analysis (Fig. S1). gamma shape parameter (nst = 2 and rates = gamma in Clinostomum detrunctaum was identified based on its tae- MrBayes), gaps in ITS by coding = variable, CO1 position noidean uterus, position of the testes in the posterior third 1 by Tamura-Nei 93 with a proportion of invariant sites of the body, and the cirrus pouch enwrapping the anterior (nst = 2, rates = propinv), CO1 position 2 Hasegawa- testis. Clinostomum philippinensis was identified based on its Kishino-Yano (HKY) (nst = 2, rates = equal) and CO1 consistency with the original description by Velasquez position 3 by HKY and gamma shape parameter (nst = 2, (1959) with some slight differences such as the position of rates = gamma). The BI analysis employed three chains of testes between middle and posterior part of the body, and a four Monte Carlo Markov chain tree searches, with four non-diverticulate uterus reaching the ventral sucker. Clino- random perturbations of trees, and chain sampling stomum attenuatum was identified based on its slender body occurred every 500 generations. Search chains were consid- shape, thick cuticular spines, proximity of the ventral sucker ered to have converged on equally likely trees when the to the anterior extremity, apparent specificity for Lithobates, average standard deviation of split frequencies of groups infection site (body cavity, rather than musculature), and fell below 0.01. provenance from the same region of north-eastern North America where Cort (1913) obtained the specimens on Results which he based the description of this species. Species discrimination and specimen identification Species delineations in ABGD analysis of ITS sequences Sequences of CO1 (n = 51) and/or ITS (n = 35) were were in 50–75% agreement with the results obtained from obtained from 49 metacercariae and three adults of Clino- CO1 data, and 0–3 of these species were not reciprocally stomum (Table 2). Combined with data from Moszczynska monophyletic in separate ITS ML trees (Table S1). Agree- et al. (2009), Bonett et al. (2011), Caffara et al. (2011, 2013, ment with CO1 results and the number of non-monophy- 2014), Riauwaty et al. (2012), Sereno-Uribe et al. (2013), letic species varied depending on the strategy employed to Pinto et al. (2014), this yielded 188 sequences of CO1 and cope with non-overlapping sequences in the alignment 182 of ITS from 206 metacercariae, 40 adults and one (Table S1). The group of ITS solutions closest to the out- sample of cercariae. Data from both markers were available come of CO1 analysis yielded 12 of 16 species indicated by in 124 specimens, and ITS only was obtained from 58, CO1 sequences (Fig. S2). Discrepancies with the CO1 CO1 only from 65. The ITS rDNA sequences were 580 to results were that C. marginatum was split into two groups, 1076 bp in length with 195 variable sites. The CO1 one of which also included C. attenuatum; C. complanatum sequences were 329 to 619 bp long, with 252 variable sites. was grouped with Clinostomum sp. 8; and Clinostomum spp. Overall divergence ranged from 0–21.05% in 188 CO1 1 and 2 were grouped. sequences and 0–8.8% in 182 ITS sequences (uncorrected In addition to not displaying reciprocal monophyly in p-distance) (Table 3, Fig. 1). Specimens were assigned to separate ML analysis, some groups distinguished in ABGD the same 16 putative species in analysis of CO1 sequences analysis of ITS distances conflicted with conclusions in the by the BIN algorithm (Ratnasingham & Hebert 2013) and present and prior studies using other evidence. Firstly, ITS ABGD (Fig. S1). In this scenario, there was little overlap sequences from adult specimens identified morphologically in genetic distances within and between species overall (see by Sereno-Uribe et al. (2013) as C. marginatum were split. histograms along edges of Figs 2 and 3) and, in any given Secondly, C. marginatum and C. attenuatum have identical

Table 3 Genetic distances (uncorrected CO1 (n = 188 sequences, 17 species) ITS (n = 182 sequences, p-distance expressed as per cent) within 17 species) Nucleotides Amino acids and between species in internal transcribed spacer and cytochrome c oxidase 1 Mean intraspeciﬁc (range) 0.173 (0–1.278) 0.538 (0–5.696) 0.135 (0–3.185) Mean interspeciﬁc (range) 5.108 (0–8.770) 16.563 (2.530–21.053) 5.669 (0–16.667)

208 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 S. A. Locke et al. Molecular survey of Clinostomum spp.

Fig. 1 Neighbor-joining phenogram of Kimura 2-parameter distances in sequences of cytochrome oxidase I (CO1, left panel) and internal transcribed spacer (ITS, right panel) in Clinostomum (pairwise deletion of gaps). White squares represent specimens from which both CO1 and ITS were obtained; ﬁlled squares are specimens in which only one marker was sequenced. For example, the ten white squares in the C. phalacrocoracis clusters in CO1 and ITS trees correspond to sequences from the same ten specimens, and single black square in the C. phalacrocoracis cluster in the ITS tree indicates a specimen from which no CO1 sequence was obtained.

ITS sequences and were not distinguished in ABGD. The complanatum, the testes span the middle and posterior distance-based analysis of ITS sequences does not take in- thirds of the body, and the anterior testis is left-displaced dels into account, and a 6-bp deletion in ITS2 also distin- by the cirrus pouch. guishes C. complanatum from Clinostomum sp. 8, which are Unidentified groups delineated by ABGD and BIN separated by mean 3.9% in CO1 (uncorrected p-distance, analysis of CO1 sequences were therefore considered can- range 3.5–5.2%). In addition, in Clinostomum sp. 8, the tes- didate species associated with varying degrees of confi- tes occur in the middle part of the body and the cirrus dence, following Padial et al. (2010), with ITS and other pouch is dextral to the medial anterior testis, while in C. data adduced as supporting evidence. Confirmed candidate

ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 209 Molecular survey of Clinostomum spp. S. A. Locke et al.

Fig. 2 Uncorrected distances in internal transcribed spacer plotted against uncorrected distances in cytochrome oxidase I in 118 specimens of Clinostomum. Symbols indicate interspecific (black points, n = 5317) and intraspecific (grey triangles, n = 1586) distances. Histograms along borders show relative frequency of distances on opposite axes (black = interspecific, grey = intraspecific). species are those supported by morphological characters, mum. Semi-confirmed candidate species are CO1-based ABGD-distinguished clustering in ITS, or occurring in clusters supported by interspecific variation in ITS that sympatry with other valid or candidate species of Clinosto- does not result in a separate group in ABGD analysis of

Fig. 3 Uncorrected distance in cytochrome oxidase I (upper panel) and internal transcribed spacer (lower panel) plotted against the geographic distance between specimens (CO1 n = 164 specimens, ITS n = 182 specimens). Symbols distinguish distances occurring between (black points, CO1 n = 13629 distances, ITS n = 12466) and within species (grey triangles, CO1 n = 3949, ITS n = 4005). Histograms on right show relative frequency of genetic distances along y axis (black = interspeciﬁc, grey = intraspeciﬁc).

210 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 S. A. Locke et al. Molecular survey of Clinostomum spp.

and ITS sequences consistent with morphologically defined species (Figs S1 and S2). Specimens within these species diverged by as much as 1.3% in ITS and 5.7% in CO1 and the latter variation was reflected in amino acid translations (Table 3). In any given species, the minimum CO1 distance between heterospecifics was greater than the maximum distance within the species, but some heterospecifics had identical ITS, and in some species, intraspecific ITS variation was relatively high (Fig. 4). The highest levels of intraspecific divergence in both markers, including in amino acid translations of CO1, occur in previously published sequences from C. marginatum. For example, CO1 sequences JX630993 and JX630995 (Sereno-Uribe et al. 2013) differ by 5.7% and, collectively, several CO1 sequences from Moszczynska et al. (2009), Bonett et al. (2011), Caffara et al. (2011) and Sereno-Uribe et al. 2013 vary by ≥4%. A number of ITS sequences from C. marginatum vary by 1–1.3% (e.g. JF718635-6, JX631049, JX631074-87 from Caffara et al. (2011) and Sereno-Uribe et al. (2013)). In species in which at least 3 specimens were sequenced, diagnostic characters were present in 9/9 species for CO1 (mean 4.3, range 1–19 diagnostic sites) and 3/ 8 species for ITS (Data files S2 and S3). Among 118 specimens comprising between one and 51 representatives of 16 species, uncorrected distance in ITS was strongly associated with that in CO1 (Rho = 0.803, P = 0.0001, Fig. 2). This relationship was also strong among distances between species (Rho = 0.593, P = 0.0001) but not significant among intraspecific distances (Rho = 0.11, P = 0.1344). Divergence in both markers increased with geographic distance between specimens (CO1 Rho = 0.743, n = 188 sequences; ITS Rho = 0.821, n = 182 sequences, both P = 0.0001; Fig. 3). Geographic distance was also associated with increasing genetic distances between specimens Fig. 4 Minimum uncorrected interspecific distance plotted against in different species (CO1 Rho = 0.596; ITS Rho = 0.748, the maximum intraspecific distance in cytochrome oxidase 1 (upper both P = 0.0001). Its effect on divergence within species panel) and internal transcribed spacer (lower panel). White data was also positive but substantially weaker (CO1 Rho=0.226, fi points indicate species that have been identi ed morphologically in P = 0.0003; ITS Rho = 0.174, P = 0.0001). fi the present and other studies, and lled data points are candidate The minimum genetic distance between specimens in species. Points are labelled with the number of specimens different species was regressed against the geographic dis- sequenced. The diagonals show the 1:1 line, such that points lying above it correspond to species in which the minimum distance tance between the specimens, as well as the minimum and between heterospecifics is greater than the maximum distance average geographic distances between the two species within the species, while this gap in variation does not occur in implicated, but there was no relationship in either marker species represented below the line. (P ≥ 0.47; Fig. S3). The maximum genetic variation within species in which ≥2 individuals were sequenced was compared to the mean and maximum spatial distances among ITS. Unconfirmed candidate species are based solely on specimens in that species, as well as the number of speci- allopatric CO1 or ITS clustering in ABGD and BIN analy- mens sequenced. Maximum intraspecific variation in CO1 = = = sis. Using these criteria, of the ten candidate species, seven increased with mean (R 0.669, P(2-tailed) 0.024, N 11) were confirmed, three semi-confirmed and one uncon- and maximum spatial distance between specimens fi = = = rmed; all form reciprocally monophyletic groups of CO1 (R 0.751, P(2-tailed) 0.008, N 11) and, with marginal

ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 211 Molecular survey of Clinostomum spp. S. A. Locke et al. significance, with the number of specimens sequenced with the exception of the unresolved placement of Clinosto- = = = (R 0.591, P(2-tailed) 0.055, N 11). Maximum intraspe- mum sp. 7 in the analysis of CO1. cific variation in ITS showed marginal associations with the same variables (mean spatial distance between speci- Discussion = = mens, R 0.552, P(2-tailed) 0.078; maximum spatial dis- In revisions of the genus Clinostomum Leidy, 1856, authors = = tance between specimens, R 0.587, P(2-tailed) 0.058; have recognized as few as one (Feizullaev & Mirzoeva = number of specimens sequenced R 0.514, P(2- 1983) or as many as 24 species (Vianna et al. 2003). In one = = fl tailed) 0.105, N 11). However, after Bonferroni correc- in uential treatment, Ukoli (1966) synonymized 20 species tion to account for multiple comparisons, only the relation- with C. complanatum and considered 13 to be valid. With ship between maximum intraspecific CO1 variation and the present study, molecular data now support the distinct maximum spatial distance between specimens remained sig- status of eight species, including C. attenuatum, C. margina- nificant at a = 0.05. Sampling effort and maximum intra- tum, C. complanatum, C. tataxumui, C. cutaneum, C. phala- specific variation can be compared in Fig. 4, where, if this crocoracis, C. philippinensis, C. detruncatum and ten additional were an important factor, better sampled species would unnamed species. Considering the distribution of candidate appear further to right (see also Fig. S3). species (Tables 2 and 4), it is likely that some of the unidentified diversity is not referable to species recognized Phylogenetic analysis by Ukoli (1966). For example, four of seven species known All genealogies (Fig. 5, Figs S4 and S5) indicated species of in the New World have been sequenced, but eight addi- Clinostomum form a monophyletic group, and most show a tional candidate species remain to be identified or well-supported, deep division between a New World clade described. of Nearctic and Neotropic species and an Old World clade The distinct status of a North American species in which of Palearctic, Afrotropic and Indomalayan species. The metacercariae occur primarily in fish, C. marginatum, has exception was the ML analysis of CO1 sequences, in which now been supported with morphological and molecular the separation of New World species was not strongly sup- analysis by Dzikowski et al. (2004), Caffara et al. (2011) ported, particularly in the unresolved placement of Clinosto- and Sereno-Uribe et al. (2013). We did not encounter this mum sp. 7 (Fig. S4). Old World species form a well- species in South America, which is significant because supported clade in all analyses, with C. complanatum and C. marginatum was described by Rudolphi in 1819 from Clinostomum sp. 8 consistently emerging as sister species, specimens collected in Brazil. This raises the possibility and C. philippinensis, C. phalacrocoracis and C. cutaneum that the North American species infecting fish is not placed in a separate clade. Relationships among New C. marginatum, but a similar but distinct species to be World species in separate ML analyses of ITS and CO1 renamed. The basis of Caffara et al.’s (2011) identification (Figs S4 and S5) were largely the same as those in Fig. 5, of C. marginatum was not only molecular, but morphologi-

Fig. 5 Consensus tree from Bayesian inference of concatenated consensus sequences of 532 nucleotides of cytochrome oxidase I partitioned by codon position, 616 nucleotides of internal transcribed spacer (ITS) and 16 gaps in the ITS alignment. Posterior probabilities at nodes (based on 1539 topologies) are indicated with ● = 1.0, = 0.979–0.995, ○ = 0.931. Three search chains converged on equally likely trees after 341000 generations. As indicated by images, the upper Clinostomum clade contains sequences originating from the New World, the lower clade sequences originating from the Old World.

212 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 S. A. Locke et al. Molecular survey of Clinostomum spp.

Table 4 Valid species of Clinostomum Leidy, 1856 according to increase in CO1 variation (note differences in scale of x- Ukoli (1966), Matthews & Cribb (1998) and Sereno-Uribe et al. and y-axes in Fig. 2). Biologically, it likely corresponds to a (2013) slower rate of mutation in rDNA. In Clinostomum, interspecific differences appear to plateau at 15–20% in CO1 but Country of in ITS a limit is not apparent (Figs 2 and 3). The clusters Species Type host type locality of CO1 sequences distinguished with BINs and by ABGD C. attenuatum Cort, 1913 Lithobates pipiens United States were consistent with prior taxonomic work and morpholog- C. australiense Johnson, 1917 Anhinga novaehollandiae Australia ical and ecological data, and therefore, they were inter- C. complanatum Rudolphi, 1814 Ardea cinerea Germany C. cutaneum Paperna, 1964 Ardea cinerea Kenya preted as likely to represent species even in cases when C. detruncatum Braun, 1899 Ciconia americana Brazil these supporting data were not available, or in cases of Mycteria americana conflict with ITS results. Species delineation with ITS may C. heluans Braun, 1899 Ardea coerulea Brazil have been impaired by the exclusion of alignment gaps in Nycticorax gardeni distance calculations. However, phylogenetic analysis of C. hornum Nicoll, 1914 Nycticorax caledonicus Australia C. intermedialis Lamont, 1920 Phalacrocorax vigua Venezuela alignment gaps is problematic (Pons & Vogler 2006) and it C. kassimovi Vaidova & Ardea cinerea Russia is unclear how gaps can be systematically implemented in Feizullaev, 1958 distance-based species delineation. Overall, the results sug- C. marginatum Rudolphi, 1819 Ardea sp. Brazil gest that taxonomic conclusions based solely on ITS data C. phalacrocoracis Dubois, 1930 Phalacrocorax levaillanti Angola should be made cautiously in Clinostomum, because intra- C. philippinensis Velasquez, 1959 Nycticorax spp. Philippines fi fi C. tataxumui Ardea alba Mexico speci c variation may be high and interspeci c variation Sereno-Uribe et al. 2013 can be absent. For example, only ITS is available from C. tilapiae Ukoli, 1966 Bubulcus ibis (experimental) Ghana Clinostomum collected from three-spot gourami (Trichogas- C. wilsoni Matthews & Cribb, 1998 Egretta intermedia Australia ter trychopterus) in Indonesia (Riauwaty et al. 2012). This sequence differs by one transition and two indels from that cal analysis consistent with Braun’s (1901) redescription of of C. philippinensis from moonlight gourami (T. microlepis) the type specimens, notably in terms of distance between from Thailand. Similarly, five transitions distinguish the the suckers and position of the genital pore. Moreover, ITS sequences of a single specimen collected in Bolivia adults and metacercariae from naturally infected hosts col- from the material sequenced by Pinto et al. (2014). In lected in Brazil by Pinto & Melo (2012) and Pinto et al. accordance with ABGD analysis of ITS, and in the absence (2013) are morphologically consistent with the description of any other data, both of these cases are provisionally trea- of North American C. marginatum by Caffara et al. (2011). ted as single species, although it is recognized this variation However, Brazilian isolates of Clinostomum sp. 7 also (0.25–0.5%) may well be interspecific (Table 3). While resemble C. marginatum as described by Caffara et al. there is also slight overlap between the absolute values of (2011), but are genetically distinct from the latter (Fig. 1; CO1 variation within and between all species (Figs 2 and Pinto et al. 2014). Because only a small number of 3), this was not the case in any given species (Fig. 4). sequences from South American Clinostomum have been These patterns are not unexpected, as the levels and overall obtained, it is still premature to speculate whether addi- pattern of divergence in both markers were comparable to tional molecular sampling is likely to reveal the presence in that observed in diverse platyhelminths (Vilas et al. 2005), South America of C. marginatum sensu Dzikowski et al. although variation is remarkably high in C. marginatum. (2004), Caffara et al. (2011) and Sereno-Uribe et al. (2013). Despite the uneven number of specimens sequenced across This question would be best addressed with sequences and species (a common problem, Lim et al. 2012), and large over- morphological data from topotypic specimens from hosts all spatial scale, there was an incomplete but clear gap in vari- in the type genus (Ardea). ation in DNA sequences within and between species (Figs 2– Despite strong correlation between ITS and CO1 varia- 4). This gap was less clear in ITS distances, but there was no tion, distance-based analyses of CO1 and ITS sequences indication that spatial scale was responsible, as intraspecific led to different sets of species. The shape of the curve in and interspecific distances both increased with spatial scale. Fig. 2 is remarkably similar to the relationship between There was no relationship between the minimum distances CO1 and ITS differences in Hurleytrematoides (Digenea: between heterospecifics and spatial scale, contrary to what Monorchiidae) observed by McNamara et al. (2014). Repli- Bergsten et al. (2012) observed in aquatic beetles (see also cation of this pattern in distantly related Clinostomum,ata Geiger et al. 2014). Spatial distance between specimens had larger spatial scale, suggests that it may be common. In a remarkably similar effects on variation in both markers, both statistical sense, the overall pattern reflects a tendency for a overall and within and between species. Given the (mostly small increase in ITS variation to be associated with a large marginal) tendency for intraspecific variation to increase with

ª 2015 Her Majesty the Queen in Right of Canada Zoologica Scripta ª 2015 Royal Swedish Academy of Sciences, 44, 2, March 2015, pp 203–217 213 Molecular survey of Clinostomum spp. S. A. Locke et al. sampling effort and spatial extent, it is notable that even in Whether the division between New and Old World spe- species that were intensively or widely sampled (C. tata- cies of Clinostomum will be supported in molecular data xumui, C. marginatum, C. phalacrocoracis), CO1 distances from additional samples is an open question. A single speci- between heterospecifics exceed those within species. men sequenced by Caffara et al. (2011) showed genetic Together these results contrast with empirical and simulated affinity with both Nearctic and Palearctic species, which is studies suggesting species discrimination using the barcode difficult to reconcile with the observed deep evolutionary region of CO1 may present increasingly ambiguous patterns divergence except given a recent hybridization event. Such in larger samples obtained at large spatial scales (Meyer & a hybrid lineage could be connected with the recent expan- Paulay 2005; Zhang et al. 2010; Bergsten et al. 2012; Geiger sion from Africa to the Americas of the cattle egret (Bubul- et al. 2014). However, expected problems may be mitigated cus ibis), which is a competent host for New World and with even a single sample from distant regions or populations Old World Clinostomum species (Ukoli 1966; Stuart et al. (Lou & Golding 2012) or, presumably, in species with high 1972) that has introduced at least one other parasite to the dispersal capabilities. Clinostomum is undoubtedly widely dis- New World (Barre et al. 1995). Nonetheless, the biogeo- persed by avian hosts, but effects of allopatric distributions graphic pattern observed here offers a fruitful way of would still be expected to emerge given the spatial scale of directing future collections in Clinostomum and potentially, the present study. other parasites from Ardeidae, or other hosts with geo- There was strong support for an evolutionary separation graphically structured distributions. of Clinostomum species in the New and Old World. This emerged from most phylogenetic analysis and was also Acknowledgements apparent in the distribution of AT repeats towards the 3’ This research was made possible by the Italian Ministry of end of ITS1, which have been recorded in a number of Health (RF-2010-2311360) to MC and MLF, a Natural digeneans (e.g. Morgan & Blair 1995; van Herwerden et al. Sciences and Engineering Research Council of Canada 1999; Tkach et al. 2000). These repeats occurred, in vary- (NSERC) Visiting Fellowship in Canadian Government ing numbers, only and in all specimens of the New World. Laboratories to SAL, Canadian Genomic Research and The apparent separation of the two Clinostomum clades Development Initiative funding to DJM, and a grant from could be related to the long isolation of avian definitive the Committee for Research and Exploration at National hosts by oceanic barriers, which have contributed strongly Geographic (9330-13) to SAL, and by support from Paul to global patterns of avian diversity and migration (Dar- D.N. Hebert (Center for Biodiversity Genomics, Univer- lington 1957; Faaborg 2005). Clinostomum spp. occur in sity of Guelph, Canada) from NSERC, Genome Canada, diverse birds, but are reported most frequently in Ardeidae the Ontario Genomics Institute, and the International Bar- (Table 1), in which 58 of 62 species occur in either the code of Life project. We are grateful to Tomas Scholz and New or Old Word, but not both (Kushlan & Hancock Roman Kuchta (Institute of Parasitology, ASCR, Czech 2005). Moreover, high genetic divergence within the few Republic), Thanapon Yooyen (Department of Biology, ardeids thought to be cosmopolitan indicates trans-oceanic Chiang Mai University, Thailand), Aline Acosta (UNESP contact has long been limited, or possibly separate species Instituto de Biociências, Departamento de Parasitologia, (Sasikala et al. 2012). Similar broad congruence in parasite Botucatu, S~ao Paulo, Brasil), Hudson A. Pinto (Departamen- evolutionary history and modern host distributions has to de Parasitologia, Instituto de Ciências Biologicas, Univer- been recorded before (Hoberg & Klassen 2002) but not, to sidade Federal de Minas Gerais, Belo Horizonte, Minas our knowledge, using molecular data. The clear separation Gerais, Brazil), Wenxiang Li (Institute of Hydrobiology, of Clinostomum species contrasts with phylogenetic relation- Chinese Academy of Sciences, China), Elizabeth Martınez ships within Diplostomum, a digenean with similar life cycle. Salazar (Guerrero samples; Unidad Academica de Ciencias For example, in a clade of Diplostomum species that mature Biologicas, Universidad Autonoma de Zacatecas), who gen- in the Laridae, Blasco-Costa et al. (2014) noted multiple erously provided Clinostomum specimens, to Heather Braid vicariance and recolonization events among European and (Auckland University of Technology, New Zealand), who North American species. The discrepancy with Clinostomum helped identify two fish species, and to two anonymous is suggestive, as Palearctic and Nearctic larids have overlap- reviewers, whose comments improved the manuscript. ping breeding areas in the Arctic (Liebers et al. 2004), where they may presumably exchange parasites, in contrast References to ardeid faunas isolated in the Old and New Worlds. Aiken, H. M., Bott, N. J., Mladineo, I., Montero, F. E., Nowak, B. Thus, in both Clinostomum and Diplostomum, the current F. & Hayward, C. J. (2007). Molecular evidence for cosmopoli- distributions of definitive hosts appear to be mirrored in tan distribution of platyhelminth parasites of tunas (Thunnus spp.). Fish and Fisheries, 8, 167–180. deep evolutionary history of their parasites.

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