Molecular phylogeny of Gyrodactylus (Monogenea) parasitizing fishes in fresh water, estuarine, and marine habitats in Canada

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1 776 Molecular phylogeny of Gyrodactylus (Monogenea) parasitizing fishes in fresh water, estuarine, and marine habitats in Canada S.R. Gilmore, D.K. Cone, G. Lowe, S.K. King, S.R.M. Jones, and C.L. Abbott Introduction Abstract: Gyrodactylus Nordmann, 1832 is a genus of monogenean flatworms that is well-studied in Europe and best known for the destructive pathogen of Atlantic salmon, Gyrodactylus salaris Malmberg, However, there is a paucity of information on species of Gyrodactylus occurring elsewhere despite that the genus is thought to be extremely speciose. Here we present the first major molecular phylogeny of Gyrodactylus using samples from host fishes in North America. Our aims were to characterize species and molecular diversity of North American Gyrodactylus to allow a determination of their evolutionary relationship with congenerics sampled from Europe. Specimens belonging to 25 species of Gyrodactylus and closely related species from Canada and the northern United States of America were identified using morphology and sequenced at 18S rdna and ITS2 rdna (totaling ca bp). Significant molecular groups in North American Gyrodactylus were found to correspond to a major division in the genus based on the structure of the male copulatory organ. Results corroborate earlier findings suggesting that the genus Fundulotrema Kritsky and Thatcher, 1977, traditionally thought to be distinct from Gyrodactylus owing to the presence of a peduncular bar, falls within Gyrodactylus. The usefulness and application of the broadly conserved 18S primers as a standard tool for Gyrodactylus taxonomy is discussed and advocated. Key words: Gyrodactylus, phylogeny, Monogenea, Gyrodactylus salaris, 18S rdna. Résumé : Gyrodactylus Nordmann, 1832 est un genre de vers plats monogénéens bien étudié en Europe et dont le représentant le plus connu est Gyrodactylus salaris Malmberg, 1957, un pathogène destructif du saumon atlantique. Peu d information est toutefois disponible sur les espèces de Gyrodactylus présentes en d autres endroits et ce, malgré le fait que le genre soit vraisemblablement d une très grande richesse spécifique. Nous présentons la première grande phylogénie moléculaire de Gyrodactylus établie à partir d échantillons prélevés de poissons hôtes en Amérique du Nord. L étude avait pour but la caractérisation de la diversité spécifique et moléculaire des Gyrodactylus nord-américains afin d en déterminer la relation évolutive avec leurs congénères européens. Des spécimens appartenant à 25 espèces de Gyrodactylus et à des espèces étroitement reliées du Canada et du nord des États-Unis ont été identifiés sur la base de leur morphologie, et leurs ADNr 18S et ADNr ITS2 ont été séquencés (environ 1430 pb au total). D importants groupes moléculaires de Gyrodactylus nordaméricains se sont avérés correspondre à une grande division du genre à la lumière de la structure de l organe copulateur mâle. Les résultats corroborent des constatations antérieures voulant que le genre Fundulotrema Kritsky et Thatcher, 1977, traditionnellement vu comme étant distinct de Gyrodactylus en raison de la présence d une tige pédonculaire, ferait plutôt partie de ce genre. L utilité et l application d amorces 18S très conservées comme outil normalisé pour établir la taxinomie de Gyrodactylus sont abordées et mises de l avant. Mots clés : Gyrodactylus, phylogénie, monogénéens, Gyrodactylus salaris, ADNr 18S. [Traduit par la Rédaction] Gyrodactylus Nordmann, 1832 is a large, globally distributed genus of monogenean parasites with 400 named species (Harris et al. 2004); however, true diversity within the genus is postulated at up to species (Bakke et al. 2002). The genus is best known in Europe where the salmonid pathogen Gyrodactylus salaris has caused exorbitant economic losses and drawn significant attention to the group, thus prompting scientific research. Many factors make it reasonable to expect that an abundance of discoveries of new species of Gyrodactylus may be on the horizon, including (i) the high rate of comprehensive new species descriptions currently emerging from European studies (e.g., Kuusela et al. 2008; Paladini et al. 2009; Paladini et al. 2010); (ii) the relatively poor understanding of the diversity of Gyrodactylus occurring Received 19 August Accepted 23 March Published at on 30 May S.R. Gilmore Andrea Crescent, Lantzville, BC V0R 2H0, Canada. D.K. Cone. Department of Biology, Saint Mary s University, Halifax, NS B3H 3C3, Canada. G. Lowe, S.R.M. Jones, and C.L. Abbott. Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, BC V9T 6N7, Canada. S.K. King. Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada. Corresponding author: Cathryn Abbott ( cathryn.abbott@dfo-mpo.gc.ca). Can. J. Zool. 90: (2012) doi: /z

2 Gilmore et al. 777 elsewhere; and (iii) the ability of contemporary molecular tools to facilitate species level designations given that Gyrodactylus species are typically morphologically very similar. Little attention has been paid to Gyrodactylus in North America and extant species diversity is poorly characterized. Hoffman (1999) lists 102 species of Gyrodactylus from North America, predominantly found on freshwater fish. In Canada specifically, 55 named Gyrodactylus species have been recorded from across 52 host finfish species (Margolis and Arthur 1979; Beverley-Burton 1984; McDonald and Margolis 1995; LeBlanc et al. 2006; Cone et al. 2007), two of which are now placed in the closely related genus Fundulotrema Kritsky et Thatcher, A further 47 finfish host species, many of which do not have a named Gyrodactylus associated with them, have had unidentified species of Gyrodactylus recorded infecting them (Margolis and Arthur 1979; McDonald and Margolis 1995; Cone et al. 2007). There is good reason to think this is the tip of the iceberg. Gyrodactylus has been confirmed on several Canadian fish hosts (Poulin 1992), and the majority of these (60%, which is likely an underestimate because misidentifications of species based on morphology is a common problem) exhibit high host specificity having been reported from only one host species (Harris et al. 2004). Furthermore, with 1100 fish species present in Canada (Froese and Pauly 2000), many more Gyrodactylus species are expected to exist in Canada than are currently known. Taxonomy of these parasites traditionally is based on morphology, particularly the shape and size of hard parts within the haptor, the organ of attachment to the host fish (Malmberg 1970). Drawbacks of this method include that these hard parts show little morphological diversity among species and can vary ecophenotypically depending on parasite age, season, geographic distribution, location on host, and species of host (e.g., Appleby 1996; Geets et al. 1999; Olstad et al and references therein). The challenge of traditional Gyrodactylus identification is further exacerbated by the existence of so many species (Bakke et al. 2002; Ziętara and Lumme 2003). Molecular phylogenetic reconstruction allows evolutionary relationships among species to be viewed independently of morphological, geographical, temporal, or host similarities. Previous molecular phylogenies constructed for Gyrodactylus have examined within-genus phylogenetic relationships using various sections of the ribosomal DNA (rdna) cistron: 18S (specifically, the variable V4 region ), first internal transcribed spacer (ITS1), and second internal transcribed spacer (ITS2) (see references in Bakke et al. 2007). In particular, 18S sequence data have shown that Gyrodactylus is a monophyletic group (Matějusová et al. 2003). More variable regions have been targeted for within-species analyses, such as the mitochondrial cytochrome oxidase (cox1) gene and the rdna intergenic spacer (IGS) (Collins and Cunningham 2000; Meinilä et al. 2002; Cunningham et al. 2003; Hansen et al. 2003; Meinilä et al. 2004; Hansen et al. 2006; Gilmore et al. 2010). The bulk of Gyrodactylus genetic work has been completed on European species (Bakke et al and references therein); to date, with the exception of a recent paper (Gilmore et al. 2010), no named North American Gyrodactylus has been included in a molecular phylogeny. Only an ITS2 sequence from Fundulotrema stableri (Kritsky and Boeger 2003) and other unnamed Gyrodactylus samples (Kritsky and Boeger 2003; Ziętara and Lumme 2004) from North America have been included in phylogenetic analyses. Our aim is to use molecular methods to complement the traditional morphological approach, the goal being to enhance our understanding of the diversity of Gyrodactylus in North America and establish their relationship to better known European taxa. We do this by generating novel DNA sequence data for 25 taxa collected from both coasts of North America using newly developed rdna primers to complement traditional primers. In addition, this study provides a basis for naming of new taxa in North America by encouraging that all new species be sequenced at standard molecular markers as suggested in the literature (Ziętara et al. 2002; Ziętara and Lumme 2003) and discussed herein. Materials and methods Sample collection and morphological identification Gyrodactylus specimens were collected from their finfish hosts at fish farms, hatcheries, and natural marine and freshwater environments across Canada and, in a few cases, from the east and west coasts of the USA (Table 1). Either fishes were necropsied separately where the body surfaces, fins and gills were examined microscopically for the parasites, or livecaught host fish were immersed in a 1:4000 solution of formalin in water for min and then placed in a container of ambient water to recover before being released. The solution was poured through a 120 µm sieve and material on each sieve was rinsed into a separate scintillation vial with 95% ethanol, which were then stored at room temperature until examined microscopically. For vials found to contain Gyrodactylus, a variety of strategies were used to obtain morphological identifications: (i) single specimens were identified microscopically by making temporary wet mounts in distilled water, removing the cover slip, and returning the specimens to ethanol for subsequent DNA extraction; (ii) haptors were excised from single worms and mounted on 50% glycerine wet mounts for identification while the body of the worm was returned to ethanol for DNA extraction; or (iii) when multiple worms were collected from the same fish, one was used for identification by microscopy and another was used for DNA extraction. In this last scenario, results of morphological identifications and DNA sequence analysis were compared to determine whether there were any discordance that may be explained by the two worms analyzed having been taxonomically different. European specimens were obtained from Kurt Buchmann (G. salaris) and Catherine Collins (Gyrodactylus derjavinoides and Gyrodactylus truttae). rdna sequencing and analysis DNA extraction, polymerase chain reaction (PCR), and sequencing followed the methods described in Gilmore et al. (2010). At the time of this study, the only PCR primers available for amplifying partial 18S rdna in Gyrodactylus were for the V4 region (Cunningham et al. 1995), which is known to exhibit interspecific variation in Gyrodactylus (Cunningham et al. 1995; Matějusová et al. 2001). However, most North American samples failed to amplify at this region using primers V4F and V4R (Cunningham et al. 1995), so an alignment of com-

3 Table 1. Species, number of individuals sequenced here (N), host and host family, sampling location, and GenBank sequence accession information for Gyrodactylus and related genera included in phylogenetic analyses (Figs. 1 and 2). GenBank accession no. Species N Host Host family Sampling location 18S1 18S2 ITS Gyrodactylus aideni Mullen, Cone, Easy and Burt, 2010 Winter flounder, Pseudopleuronectes americanus (Walbaum, 1792) Pleuronectidae New Brunswick HM Gyrodactylus aeglefini (Bychowsky and Polyansky, 1953) 3 Haddock, Melanogrammus aeglefinus (L., 1758) Gadidae New Brunswick JF JF JF Malmberg, 1970 Gyrodactylus alexanderi Mizelle and Kritsky, Three-spined stickleback, Gasterosteus aculeatus L., 1758 Gasterosteidae British Columbia JF JF JF Gyrodactylus anguillae Ergens, 1960 American eel, Anguilla rostrata (LeSueur, 1817) Anguillidae Maryland, USA AB Gyrodactylus arcuatus Bychowsky, Three-spined stickleback Gasterosteidae Nova Scotia E JF JF JF Three-spined stickleback Gasterosteidae British Columbia* E JF JF JF Gyrodactylus cameroni Hanek and Threlfall, Ninespine stickleback, Pungitius pungitius (L., 1758) Gasterosteidae Nova Scotia JF JF JF Fourspine stickleback, Apeltes quadracus (Mitchill, 1815) Gasterosteidae Nova Scotia JF JF JF Gyrodactylus colemanensis Mizelle and Kritsky, Brook trout, Salvelinus fontinalis (Mitchill, 1814) Salmonidae Nova Scotia* JF JF JF Gyrodactylus emembranatus Malmberg, Atlantic cod, Gadus morhua L., 1758 Gadidae Nova Scotia E GU JF JF Gyrodactylus eos Mayes, Northern redbelly dace, Phoxinus eos Cope, 1861 Cyprinidae Ontario JF JF JF Gyrodactylus groenlandicus Levinsen, Shorthorn sculpin, Myoxocephalus scorpius (L., 1758) Cottidae Nova Scotia E JF JF Gyrodactylus harengi Malmberg, Atlantic herring, Clupea harengus L., 1758 Clupeidae New Brunswick E JF JF Gyrodactylus jennyae Paetow, Cone, Huyse, McLaughlin American Bullfrog, Rana catesbeiana Shaw, 1802 Ranidae Canada* EU and Marcogliese, 2009 Gyrodactylus marinus Bychowsky and Polyansky, Atlantic cod Gadidae Nova Scotia E JF JF JF Gyrodactylus neili LeBlanc, Hansen, Burt and Cone, 2006 Chain pickerel, Esox niger LeSueur, 1818 Esocidae New Brunswick AY Gyrodactylus notatae King, Forest and Cone, Atlantic silverside, Menidia menidia (L., 1766) Atherinopsidae Nova Scotia JF JF FJ Gyrodactylus pharyngicus Malmberg, Atlantic cod Gadidae New Brunswick E JF JF JF Atlantic cod Gadidae Nova Scotia E JF JF JF Gyrodactylus pleuronecti Cone, 1981 Winter flounder Pleuronectidae New Brunswick HM Gyrodactylus salmonis Yin and Sproston, Atlantic salmon, Salmo salar L., 1758 Salmonidae Nova Scotia* JF JF GQ Brook trout Salmonidae Maine, USA* JF JF GQ Rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) Salmonidae Washington, USA JF JF GQ Rainbow trout Salmonidae British Columbia JF JF GQ Sockeye salmon, Oncorhynchus nerka (Walbaum, 1792) Salmonidae British Columbia JF JF GQ Brook trout Salmonidae Nova Scotia JF JF GQ Gyrodactylus spathulatus Müller, White sucker, Catostomus commersoni (Lacepède, 1803) Catostomidae Nova Scotia JF JF JF Gyrodactylus stephanus Müller, Common mummichog, Fundulus heteroclitus (L., 1766) Fundulidae Nova Scotia JF JF FJ Gyrodactylus sp. 1 Tomcod, Microgadus tomcod (Walbaum, 1792) Gadidae New Brunswick JF JF JF Pearl dace, Margariscus margarita (Cope, 1867) Cyprinidae Ontario JF JF JF Golden shiner, Notemigonus crysoleucas (Mitchill, 1814) Cyprinidae Ontario JF JF JF Prickly sculpin, Cottus asper Richardson, 1836 Cottidae British Columbia JF JF JF Bay pipefish, Syngnathus leptorhynchus Girard, 1854 Syngnathinae British Columbia JF JF JF Koi carp, Cyprinus carpio carpio L., 1758 Cyprinidae British Columbia* JF JF JF Brown bullhead, Ameiurus nebulosus (LeSueur, 1819) Ictaluridae Ontario JF JF Speckled dace, Rhinichthys osculus (Girard, 1856) Cyprinidae Idaho, USA AY Fathead minnow, Pimephales promelas Rafinesque, 1820 Cyprinidae Idaho, USA AY Northern plains killifish, Fundulus kansae Garman, 1895 Fundulidae Colorado, USA AY Fundulotrema foxi (Rawson, 1973) Common mummichog Fundulidae Nova Scotia GQ Fundulotrema porterensis King and Cone, Common mummichog Fundulidae Nova Scotia JF JF FJ Fundulotrema prolongis Hargis, Common mummichog Fundulidae Nova Scotia JF JF GQ Fundulotrema stableri (Hathaway and Herlevich, 1973) Northern plains killifish Fundulidae Colorado, USA AY Gyrocerviceanseris passamaquoddyensis Cone, Abbott, 2 Silver hake, Merluccius bilinearis (Mitchill, 1814) Merlucciidae New Brunswick GU JF Gilmore and Burt, 2010 Gyrodactylus brachymystacis Ergens, Manchurian trout, Brachymystax lenok (Pallas, 1773) Salmonidae China JF JF GQ Can. J. Zool. Vol. 90, 2012

4 Gilmore et al. 779 Table 1 (concluded). GenBank accession no. Species N Host Host family Sampling location 18S1 18S2 ITS 2 Atlantic salmon Salmonidae Scotland JF JF GQ Rainbow trout Salmonidae Scotland JF JF GQ Brook trout Salmonidae Scotland JF JF GQ Gyrodactylus derjavinoides Malmberg, Collins, Cunningham and Behiar, 2007 Gyrodactylus salaris Malmberg, Atlantic salmon Salmonidae Denmark JF JF GQ Gyrodactylus truttae Gläser, Brook trout Salmonidae Scotland JF JF GQ Gyrodactylus rutilensis Gläser, 1974 Roach, Rutilus rutilus (L., 1758) Cyprinidae Czech Republic AJ AJ Gyrodactylus rhodei Žitňan, 1964 Bitterling, Rhodeus sericeus (Pallas, 1776) Cyprinidae Czech Republic AJ AJ Gyrodactylus gobiensis Gläser, 1974 Gudgeon, Gobio gobio (L., 1758) Cyprinidae Czech Republic AJ AJ Gyrodactylus carassi Malmberg, 1957 Bleak, Alburnus alburnus (L., 1758) Cyprinidae Czech Republic AJ AJ Gyrodactylus sedelnikovi Gvozdev, 1950 Stone loach, Barbatula barbatula (L., 1758) Balitoridae Czech Republic AJ AJ Macrogyrodactylus polypteri Malmberg, 1957 na na na AJ AJ Gyrodactyloides bychowskii Albova, 1948 Atlantic salmon Salmonidae Scotland AJ AJ Haliotrema pratasensis Sun, Kritsky and Yang, 2007 na na na EU EU Note: All species sampled outside of North America are not currently known to co-occur there; species sampled in North America that are known also to occur in Europe are marked with a superscript E by their sampling location. Asterisks denote locations where sampled fish were captive. New data generated in this study have GenBank accession numbers starting with JF. na, not available. plete 18S sequences in GenBank from Gyrodactylus and related taxa was assembled. The alignment was done in BioEdit (Hall 1999) using the Clustal W multiple alignment option and contained 8 taxa (Gyrodactyloidesbychowskii, AJ566379; Macrogyrodactylus polypteri, AJ567671; Gyrodactylus gobiensis, AJ566375; Gyrodactylus rhodei, AJ567670; Gyrodactylus rutilensis, AJ566376; Gyrodactylus carassii, AJ566377; Gyrodactylus sedelnikovi, AJ566378; G. salaris, Z26942). Visual inspection of the 1980 bp alignment showed three conserved and two variable regions within 18S, and that the V4 primers matched well with G. salaris but poorly with most other Gyrodactylus. New primers were designed in conserved flanking regions of the two variable regions (see Results) as follows: primers PBS18SF and PBS18SR (Cone et al. 2010) amplify an approximately 485 bp variable region located between base pairs 440 and 924 of G. gobiensis (AJ566375) and that contains the V4 region and additional variable sites. Primers PBS1325F (5 -GACGGAAGGGCACCACCAGGAGT-3 ) and PBS1863R (5 -CAAAGGGCAGGGACGTATTCAGCACA-3 ) amplify a different, approximately 537 bp variable region located between base pairs 1267 and 1800 of G. gobiensis (AJ566375). Cycling conditions for primer pair PBS18SF/PBS18SR are described by Cone et al. (2010); those for PBS1325F/PBS1863R deviated from these in the following ways only: the 5 touchdown cycles were from 70 to 62 C (less 2 C per cycle), and the next 30 cycles used an annealing temperature of 60 C. Sequence data for ITS2 were also generated because this molecular region is better represented in GenBank for Gyrodactylus species. Whenever possible, ITS2 was amplified using variable forward primers ITS1 (Cunningham 1997) or ITS1A (Matějusová et al. 2001) with the reverse primer ITS2 (Cunningham 1997), as this allowed the whole ITS1 5.8S ITS2 array to be sequenced. However, those forward primers failed in several species, so in these cases, we used forward primer ITS4.5 (Matějusová et al. 2001) with reverse primer ITS2 to amplify ITS2 only. All rdna PCR products described here were sequenced using their PCR primers only, with the exception that ITS1/ITS2 or ITS1A/ITS2 products were also sequenced using ITS4.5 and ITSR3A (Matějusová et al. 2001). GenBank was searched for 18S and ITS sequences from other Gyrodactylus and related genera (the latter for use as the outgroup). All available Gyrodactylus sequences that covered the same regions within 18S as were amplified here were included in the analysis. For ITS2, only those samples collected from North America were included in the analysis because including the large number of samples collected from Europe is outside the scope of this paper. Haliotrema pratasensis was chosen as an outgroup because a sequence was available that covered the 18S regions amplified in this study; it was sufficiently far from Gyrodactylus to help resolve the question of monophyly of the genus; and it aligned readily with Gyrodactylus sequences. One sequence from each Gyrodactylus species was used in phylogenetic analyses (refer to Table 1). Separate alignments of 18S and ITS were done using BioEdit (Hall 1999) using the Clustal W multiple alignment option with default settings and were checked by eye to confirm similarity among samples. Phylogenetic analyses were performed for the two 18S regions separately, as well as combined, and for the ITS region using MEGA version 4 (Tamura et al. 2007) as described by Gilmore et al.

5 780 Can. J. Zool. Vol. 90, 2012 (2010). This software also was used to calculate sequence divergence (Gilmore et al. 2010). Results A total of 53 gyrodactylid specimens were collected from their finfish hosts sampled from across Canada and parts of the USA, representing 22 species of Gyrodactylus, 2 species of Fundulotrema, and Gyrocerviceanseris passamaquoddyensis (Table 1). An additional seven specimens obtained from overseas were also included (Table 1). 18S alignment and phylogeny Primers PBS18SF and PBS18SR amplified a single, clean product in all species tested that ranged in length (excluding primers) from 432 bp (Gyrodactylus sp. from brown bullhead, Ameiurus nebulosus) to 449 bp (Gyrodactylus sp. on golden shiner, Notemigonus crysoleucas). Primers PBS1325F and PBS1863R amplified a single, clean product in all species tested that ranged in length from 482 bp (both Fundulotrema) to 488 bp (G. salmonis and related species). Noteworthy is that the GenBank sequence for G. salaris (Z26942) had one deletion, one insertion, and a double substitution compared with the G. salaris sequence generated here and all other ingroup and outgroup sequences, alerting to the possibility that these are errors in this published sequence. Incidentally, for the V4F primer, Cunningham et al. (1995) incorporated the deletion and this may be why it was prone to failure in our tests. As such, only the G. salaris sequences generated here were used in analyses. A total of around 60 sequences per fragment was generated across 29 taxa. Sample sizes within single taxa were low (up to 10 samples obtained for G. salmonis but typically one or two sequences per taxon) and no notable within-species differences were found, resulting in one sequence per taxon being retained in final analyses. Each final 18S alignment included: (i) newly generated Gyrodactylus (n = 25) and Fundulotrema (n = 2) sequences from North America (n = 23 species), Europe (n = 3 species), and China (1 species); (ii) six Gyrodactylus sequences from GenBank or previously published samples from our laboratory along with Gyrocerviceanseris passamaquoddyensis; and (iii) three GenBank sequences of related taxa for use as outgroups (Table 1). Sequence similarity matrix Using the combined data set, pairwise sequence divergence for these 18S regions between North American Gyrodactylus varied from 0.1% (G. marinus G. aeglefini; one change across 926 bp) to 16.4% (G. emembranatus to Gyrodactylus sp. (on koi carp, Cyprinus carpio carpio), 136 changes across 931 bp). Variation between ingroup and outgroup taxa was between 29.9% and 9.1%, suggesting some outgroup taxa were in fact closely related to or part of the ingroup (see phylogenetic results below). When analysed separately, the first 18S segment was more variable than the second (minimum 0% for both, maximum 23% vs. 12.8%, and mean 14.5% vs. 6.5%). Phylogenetic analysis Minimum evolution (ME), neighbour-joining (NJ), and maximum parsimony (MP) analyses of the combined 18S sequences gave the same basic tree structure (Fig. 1). Bootstrap support values at individual nodes obtained across the three methods were generally concordant, with some exceptions (Fig. 1). Results indicate that Gyrodactylus is not monophyletic because Gyrocerviceanseris and Fundulotrema fall significantly within the bounds of Gyrodactylus. The placement of Gyrodactyloides and Macrogyrodactylus compared with Gyrodactylus is less certain with low bootstrap values and would require analysis with a deeper gene region to resolve relatedness (Fig. 1). Some significant structure is seen in these analyses (significance seen as 70% bootstrap following Hillis and Bull 1993); in particular, three major groups are identified (labeled group A, B, and C in Fig. 1). Group A consists of the European G. sedelnikowi and G. carassii, the only two known representatives of the subgenus Gyrodactylus (Gyrodactylus) in this study (Malmberg 1970). Closely related to them is an unnamed freshwater sample collected from golden shiner in central Canada. Also within this group are Gyrocerviceanseris passamaquoddyensis and Gyrodactylus emembranatus. Together groups B and C form a significant clade (or group) that is distinctly different from group A. Taxa in group B consist of European and Asian Gyrodactylus known to infect salmonid and cyprinid fishes with the only North American species being G. salmonis (the phylogeny of which was presented by Gilmore et al. 2010), an unnamed sample from koi carp (an introduced fish), and an unnamed sample from bay pipefish (Syngnathus leptorhynchus) whose sequence sits at the edge of the group and may represent another distinct group (Fig. 1). The rest of the North American samples, and the vast majority, are found in group C. This group has only some significant internal structuring and has samples from nine different fish families (Fig. 1). The morphologically distinct genus Fundulotrema is found within this group. Only one sample of Gyrodactylus does not place significantly with one of the three groups; it is an unnamed sample collected from brown bullhead in Ontario and falls between groups B and C and may represent another distinct lineage. Analyses of the separate 18S fragments gave very similar results (not shown). Variation was found in significance levels of some nodes. Group B always had significant bootstrap support and group A had significant support in all but one occasion (MP second fragment). The support for group C showed the greatest variation with significant bootstrap values for the first 18S region for two of the three methods (ME/NJ/MP = 87/91/68) but only with NJ methods using the second 18S region ( /71/ ). Sequence length variation Along with bootstrap support for the three groups, sequence lengths of the 18S fragments varied among groups. Sequence lengths for the first 18S segment were 447 or 449 bp for species in group A, 441 or 442 bp for species in group B, and bp for species in group C. This sequence for Gyrodactylus sp. (brown bullhead), which stayed ungrouped, was 432 bp long. Sequence lengths for the second 18S segment varied minimally; it ranged from 486 to 488 bp in all taxa except the two Fundulotrema species (both 482 bp) and G. arcuatus G. cameroni G. stephanus (all 484 bp). When combined, the two 18S segments had

6 Gilmore et al. 781 Fig. 1. Minimum evolution (ME) phylogenetic tree for 951 bp alignment of combined 18S segments of North American species of Gyrodactylus and related genera. Bootstrap values obtained from 1000 resamplings using ME, neighbour-joining, and maximum parsimony methods are shown in the same order as just described and separated by a solidus (/). Asterisks denote bootstrap values below 50%. (Parsimony tree statistics: consistency index (CI) = 0.46; retention index(ri) = 0.72; six most parsimonious trees, length = 865.) See Table 1 for full sample details. Groups labeled A, B, and C represent deep divisions in the genus (see text).

7 782 Can. J. Zool. Vol. 90, 2012 nonoverlapping length ranges for the three main Gyrodactylus groups (group A: bp; group B: bp: group C: bp). ITS2 alignment and phylogeny A total of 34 taxa were included in analyses using ITS2. They included 16 newly collected North American taxa: 14 North American taxa from GenBank (5 of which were recently sequenced in our laboratory), 3 European samples and 1 Chinese sample sequenced in our laboratory and previously published in GenBank (Table 1). Four species sequenced at 18S could not be amplified with any ITS primers tested (G. groenlandicus, G. harengi, Gyrodactylus sp. from brown bullhead, and Gyrocerviceanseris passamaquoddyensis). The ITS region of rdna analysed here included 51 bp of 5.8S, complete ITS2, and 42 bp of 28S. The length of the analyzed sequences ranged from 443 bp in G. neili to 555 bp in Fundulotrema porterensis. Our sequence data could not be aligned with sufficient confidence to construct a genus-wide phylogeny. Default alignments, although possibly correct, produced differences up to 51.5% among sequences. This difficulty is known for ITS2 in Gyrodactylus despite common use of this region for within-group phylogenies but is scarcely discussed in the literature (see Bakke et al. 2007). Rather than lose data by removing ambiguous sites, we used separate alignments that could be assembled with confidence. When looking at a series of unaligned sequences, these groups are immediately apparent and they are also identical to the three groups found in the 18S phylogenetic analyses. Pairwise sequence divergence varied between 18.5% and 35.8% in the four samples in group A. The topology of group A ITS2 phylogenies cannot be compared with the 18S group A phylogenies because the analyses share only two taxa. The sequence divergence suggests no close species grouping in this small analysis (Fig. 2a). In group B, pairwise sequence divergence ranged from as little as 1.6% to a high of 30.1%. The tree structure and support of group B based on ITS2 concords well with those generated using 18S, the only exception being that G. salaris and Gyrodactylus brachymystacis are not significantly grouping in the former. Gyrodactylus anguillae from American eel (Anguilla rostrata) is significantly related to Gyrodactylus sp. from bay pipefish at the edge of the group (Fig. 2b). Pairwise sequence divergence in group C taxa varied from 0% to 32.0%. Phylogenetic analysis of ITS2 gave the same basic groupings as did 18S but with some changes in the relationships among the groups. Four Fundulotrema species group significantly together but, unlike the 18S results, are no longer closer to the G. arcuatus group than to the rest of the taxa. Most notably, G. notatae groups closer to the G. arcuatus G. stephanus group along with an unnamed Gyrodactylus also from Fundulus than it did in the 18S data set (Fig. 2c). Discussion This phylogenetic study presents the first broad geographic scale evolutionary analysis of the genus Gyrodactylus with specimens sampled from North America. Named species of Gyrodactylus collected from North America had hitherto never been included in a phylogenetic analysis of the genus (but see Gilmore et al. 2010). By examining two segments of 18S rdna (one encompassing the V4 region) and the ITS2 region, evolutionary relationships among species from North America are assessed. With the majority of prior molecular analysis in this genera coming from European samples (Bakke et al. 2007), this study provides data for a northern hemisphere approach to studying this complicated and diverse genus. Paraphyly of Gyrodactylus Results presented here imply that Gyrodactylus is not monophyletic: two of the three major clades identified contained multiple genera. This agrees with a previous molecular analysis that suggested the genus was paraphyletic (Kritsky and Boeger 2003). It disagrees with Matějusová et al. (2003), whose analysis of complete 18S and partial ITS sequences found Gyrodactylus to be monophyletic; however, it is now clear that this result was driven by biased taxon sampling. It would seem that to definitively resolve the relationship of outgroups like Macrogyrodactylus and Gyrodactyloides with Gyrodactylus will require improved sampling of species just outside of Gyrodactylus. A multigenic phylogenetic approach is also likely needed; the inclusion of different genes, portions of genes, and samples across different studies has clearly influenced results and conclusions drawn. However, irrespective of the placement of Macrogyrodactylus and Gyrodactyloides, our finding that Fundulotrema and the recently named Gyrocerviceanseris (Cone et al. 2010) both fall definitively within Gyrodactylus substantially strengthens available evidence that Gyrodactylus is paraphyletic. Kritsky and Boeger (2003) found that a species of Fundulotrema, which is defined by the presence of a peduncular bar (Cone and Odense 1988), fell within the bounds of Gyrodactylus when examining ITS2, but they also found these two genera to be distinct from a morphological analysis. Bakke et al. (2007) suggested that the genus Fundulotrema is not valid given that, from a morphological perspective, it appears to have derived from certain Gyrodactylus species that infect fish belonging to the genus Fundulus. Phylogenetic analysis of 18S and ITS2 presented here do not include the similar Gyrodactylus species listed by Bakke et al. (2007) (Gyrodactylus funduli Hargis, 1955 and Gyrodactylus stegurus Müller, 1937) but clearly show that the genetic diversity among Fundulotrema species is high with long branches and that the genus is not closely related to other Gyrodactylus samples collected from Fundulus (Figs. 1 and 2c). Gyrodactylus stephanus (collected from common mummichog, Fundulus heteroclitus) is nearly morphologically identical to G. stegurus and likely they are closely related sister taxa. In the molecular analysis using 18S, G. stephanus is closely related to G. arcuatus and G. cameroni, which all together are grouped with the Fundulotrema clade. This grouping may suggest a shared evolutionary history with an ancient radiation as parasites of cyprinodontids. Morphological similarity between Fundulotrema and certain Gyrodactylus species that infect Fundulus are likely due to a deep shared ancestry. Numerous differences exist between Fundulotrema and Gyrodactylus infecting Fundulus with Fundulotrema having wide morphological variation among species, again suggestive of ancient beginnings to the genus, but consistent characteristics

8 Gilmore et al. 783 Fig. 2. Phylogenies of second internal transcribed spacer (ITS2) region of rdna for groups A, B, and C as defined using 18S analysis (Fig. 1). Bootstrap values obtained from 1000 resamplings using minimum evolution (ME), neighbour-joining, and maximum parsimony methods are shown in the same order as just described and separated by a solidus (/). Asterisks denote bootstrap values below 50%. See Table 1 for full sample details. that separate them from Gyrodactylus, namely the presence of a peduncular bar, distinct marginal hook arrangement, and the presence of marginal hook ligaments. In addition, there has been a reduction in the length of the 18S region in Fundulotrema compared with the closest Gyrodactylus relatives. Relationships within Gyrodactylus and evaluation of subgenera We resolved three main clades within Gyrodactylus using 18S sequence data and a broader representation of species than has been used in previous studies. Even so, these three clades (referred to here as groups A, B, and C) are repeatedly seen in the literature; they correspond to length variation and sequence differences in ITS1, 5.8S, and ITS2 (Cable et al. 1999; Matějusová et al. 2003; Ziętara et al. 2002; Ziętara and Lumme 2004). Here we reinforce results of previous data sets by showing that these clades also emerge when using 18S sequence data and that there is length variation associated with them in this gene region. From the small representation of species for which 18S sequences are available, it appears that the length of the first 18S region alone may be sufficient to classify species into one of the three groups. The one species sampled here that did not fall phylogenetically into one of the three main clades was positioned between groups B and C, and had a shorter length than any other species (Gyrodactylus sp. on brown bullhead; length 432 bp). It is important to note that we refer to these clades as groups A, B, and C for ease of discussion only; we do not intend to suggest that these are the only major clades within Gyrodactylus. Given the very high species diversity in this genus and that most of it remains unsampled, we predict that as more species are discovered more deep divisions within the genus will also be resolved. Indeed, there is already the indication of this occurring based on our current data set. Gyrodactylus sp. on bay pipefish is separated from all other

9 784 Can. J. Zool. Vol. 90, 2012 taxa in clade B by very high bootstrap support, and a preliminary phylogenetic analysis using limited sequence data and a small taxon set indicates that Gyrodactylus species parasitizing syngnathids may form a distinct clade (Paladini et al. 2010). Interestingly, the rather deep division between group A and groups B and C combined corresponds to a distinct morphological difference that has previously gone unrecognized. Members of group A display significant variability in the form of the male copulatory organ (MCO), with a common feature being multiple rows of small spines, whereas members of groups B and C have a MCO that has a single large spine and varying numbers of small spines all in a single terminal row. This distinction in MCO types is a fundamental difference and may prove to be taxonomically informative. This molecular distinction together with the difference in morphology of the MCO may delineate a major division in the genus. From an ecological perspective, species in groups A and C are found on both marine and freshwater hosts, whereas group B is restricted mostly to freshwater hosts. The hosts in group A are mostly freshwater ostariophysid fishes; two, however, (G. emembranatus and Gyrocervicianseris) parasitize marine coastal fishes, suggesting there may have been an early offshoot of ancestral viviparous gyrodactylids that diversified among marine paracanthopterygians, fishes that arose shortly after the ostariophysids. Group B consists mostly of the Gyrodactylus wageneri Malmberg, 1956 species group that radiated successfully among salmonid fishes of the northern hemisphere and various small-bodied cyprinids that are found living sympatrically with salmonids and, not surprisingly, includes North American and Eurasian species from related hosts. Hosts affected by Gyrodactylus species in group C include freshwater, estuarine, and marine fishes. Terminal clades within this group lead us to speculate that species may have either radiated within a host lineage (e.g., the G. marinus group within the gadoids, or G. groenlandicus and Gyrodactylus sp. from sculpins that occur on opposite coasts suggesting a widespread marine distribution) or habitat (e.g., the Fundulotrema from fundulids and the G. arcuatus types from sticklebacks, both of which occupy shallow coastal marine bays). An extensive study of the excretory system of many Gyrodactylus species led to subgeneric classification of different groups within the genus by Malmberg (1970). Previous molecular studies have generated discrepant results with respect to the monophyletic nature of these groups (Cable et al. 1999; Ziętara et al. 2002; Matějusová et al. 2003; Ziętara and Lumme 2004). All analytical methods used here support the grouping of sampled Gyrodactylus species into three distinct groups, with group A being more distantly related to groups B and C than the latter are to each other. Sample sizes used here are too low to truly test the phylogenetic status of all four sampled subgenera; however, analyses presented here support them weakly at best. The two subgenera Gyrodactylus (Metanephrotus) and Gyrodactylus (Mesonephrotus) are not monophyletic but rather are dispersed throughout groups A and C, and group C, respectively, and hence are likely not evolutionarily accurate. Although Gyrodactylus (Limnonephrotus) does form a distinct monophyletic clade within group B, this may be an artefact of biased species representation because Matějusová et al. (2003) found Gyrodactylus (Limnonephrotus) to be paraphyletic. Although still true that improved sampling of all subgenera from a broader geographic and host range would enable a thorough assessment of how useful these excretory characters are for subgeneric classification, our results support the suggestion by Matějusová et al. (2003) that they are not conserved enough for subgeneric classification and hence should not be considered to be indicators of phylogeny. It now seems apparent that molecular-based phylogenies will be more accurate at defining evolutionary relationships within Gyrodactylus than ones based on traditionally used morphological characters. Ideally, they will aid in the identification of synapomorphic morphological characters within the genus that are taxonomically informative. 18S for future Gyrodactylus phylogenies and molecular taxonomy The 18S data set contained sufficient similarity among sequences to allow easy alignment across all ingroup and outgroup taxa, which is known to be problematic for other markers, but also allowed good species-level resolution to be achieved. As such, this molecular marker allows a fast, easy comparison to aid in taxonomic placement of newly discovered or unidentified Gyrodactylus collected from this continent. The 18S regions were easily amplified and aligned in all species tested, with sequence variation up to 16.4% among them. These characteristics and that these primers also work outside the genus in closely related monogenean genera (e.g., Discocotyle Diesing, 1850, Haliotrema Johnson and Tiegs, 1922, Anoncohaptor Müller, 1938, Dactylogyrus Diesing, 1850) and the digenean genus Axina Kirby, 1818 (results not shown) may make them superior to ITS for this purpose. No two species shared both sequences and with the exception of the close clustering of G. arcuatus G. cameroni and G. marinus G. aeglefini Gyrodactylus sp. (on tomcod, Microgadus tomcod), species differentiation was easily done with either fragment. Matějusová et al. (2001) found that sequence variation in the V4 region, which is within the 18S region analyzed here, varied up to 40%. However, the most distant species in that analysis, Gyrodactylus markakulensis Gvosdev, 1950, was not included in our data set; when it is excluded, the diversity of the V4 region ranges up to 34%. Sequence variation seen here for 18S of up to 16.4% is between that found in the V4 region by Matějusová et al. (2001) and the much lower level of up to 6.4% found within Gyrodactylus at 5.8S (Ziętara et al. 2002). Ziętara and Lumme (2003) suggested the inclusion of DNA sequence data to modern species descriptions of Gyrodactylus, something that very few North American species had when we started this study. By presenting the first genus-wide phylogeny of Gyrodactylus that includes North American samples, this paper provides baseline data against which future collections of North American Gyrodactylus can be compared. Molecular data have the advantage of independently confirming morphological changes as consistent and related to speciation instead of being due to external influences. In addition, molecular data can suggest a requirement for more detailed morphological differentiation between divergent genetic lineages. In addition to ITS1 and ITS2, we suggest that the regions of 18S defined here be in-

10 Gilmore et al. 785 cluded in such studies and sequenced in as many species as possible. Bakke et al. (2007) states that establishing large and robust gyrodactylid phylogenies has been difficult because markers inappropriate for the purpose have been used. The ribosomal 5.8S and V4 region of 18S show good levels of variation and phylogenetic signal but are too short for analyses involving large sample sizes (Ziętara and Lumme 2004). ITS2 has shown its validity in determining species-level differences, with variation in the complete ITS1 5.8S ITS2 rdna cistron above 1% suggested as being outside of species bounds (Ziętara and Lumme 2003). It does not, however, correspond to a molecular region that is easily aligned and therefore useful to resolve higher level differences in Gyrodactylus as a whole. The 18S rdna data used in this study are easily aligned and enable resolution of distinct phylogenetic groups, making it seem the superior marker for genus-wide phylogenies of Gyrodactylus. However, they are not without limitations: the data are composed of two segments that are each too short to resolve relationships among large numbers of species when used alone (although the two could be amplified as one segment if needed), and they provide poor support for within group relationships. Further isolation of independent genetically variable regions is required to derive well-resolved Gyrodactylus-wide phylogenies to facilitate demarcation of new generic boundaries should they be required. Taxonomic revision to the genus is clearly needed owing to its paraphyletic nature at present. This will require thorough worldwide representation of taxa within the genus and morphologically similar species and would be strongest with the use of multigenic molecular and morphological approaches. Acknowledgements We thank J. Wade for sample collections in British Columbia, C. Collins for providing samples of G. derjavinoides and G. truttae, and K. Buchmann for providing a sample of G. salaris. This research was funded by a Centre of Expertise for Aquatic Animal Health Research and Development (Fisheries and Oceans Canada) grant to C.L.A. References Appleby, C Variability of the opisthaptoral hard parts of Gyrodactylus callariatis Malmberg, 1957 (Monogenea: Gyrodactylidae) from Atlantic cod Gadus morhua L. in the Oslo Fjord, Norway. Syst. Parasitol. 33(3): doi: / BF Bakke, T.A., Harris, P.D., and Cable, J Host specificity dynamics: observations on gyrodactylid monogeneans. Int. J. Parasitol. 32(3): doi: /s (01) PMID: Bakke, T.A., Cable, J., and Harris, P.D The biology of gyrodactylid monogeneans: the Russian-doll killers. In Advances in parasitology. Edited by J.R. Baker, R. Muller, and D. Rollinson. Academic Press, London, U.K. pp Beverley-Burton, M Monogenea and Turbellaria. 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