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1 Global parasite trafficking: Asian Gyrodactylus (Monogenea) arrived to the U.S.A. via 1 2 2 invasive fish Misgurnus anguillicaudatus as a threat to amphibians 3 4 5 3 6 7 4 Florian B. Reyda 1 8 9 10 11 5 [email protected] 12 13 14 6 Scott M. Wells 2 15 16 17 18 7 Alexey V. Ermolenko 3 19 20 21 8 Marek S. Ziętara 4 22 23 24 25 9 Jaakko I. Lumme 5 26 27 2810 1. Biology Department and Biological Field Station, State University of New York at 29 30 3111 Oneonta, College at Oneonta, Oneonta, NY, U.S.A.; 32 3312 2. New York State State Department of Environmental Conservation, Region 4 Bureau of 34 3513 Fisheries Stamford, NY, U.S.A.; 36 37 3814 3. Institute of Biology and Soil Science 159, Prospekt Stoletiya, 690022 Vladivostok, Russia; 39 4015 4. Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, Wita 41 42 4316 Stwosza St., 59, PL-80-308 Gdańsk, Poland; 44 4517 5. Ecology and Genetics , University of Oulu, Oulu, Finland. 46 47 4818 Short title: Intercontinental transfer of parasites by invasive fish 49 50 5119 Running head/short title: Parasite transfer by invasive fish 52 53 5420 Keywords: Intercontinental parasite dispersal, molecular systematics, aquatic invasive species, 55 5621 chytrid fungus. 57 5822 59 60 61 62 63 64 1 65 23 Abstract 1 224 A monogenean flatworm Gyrodactylus jennyae Paetow, Cone, Huyse, McLaughlin & 3 4 525 Marcogliese, 2009 was previously described as a pathogen on bullfrog Lithobates 6 726 catesbeianus Shaw, 1802, in a Canadian captive population originating in Missouri, U.S.A. 8 9 1027 The ITS barcoding of G. jennyae showed relatedness to Asian Gyrodactylus macracanthus 11 1228 Hukuda 1940, a parasite of the Asian loach Misgurnus anguillicaudatus Cantor, 1842. The 13 1429 resulting suggestion that the globally invasive pet-trade of fish may be a mechanism for 15 16 1730 arrival of Gyrodactylus species to North America provided the framework for the current 18 1931 study. The present study was undertaken following the discovery of two other species of 20 21 2232 Gyrodactylus in a population of illegally introduced M. anguillicaudatus in New York State. 23 2433 Here the invasion hypothesis was tested via DNA sequencing of the ITS of the two 25 26 2734 Gyrodactylus species obtained from M. anguillicaudatus from New York, termed 28 2935 Gyrodactylus sp. A and Gyrodactylus sp. B. Both Gyrodactylus sp. A and Gyrodactylus sp. B 30 3136 were closely related to G. jennyae and G. macracanthus, and all belong to a molecularly well- 32 33 3437 supported monophyletic Asian freshwater group. In conclusion, this invasive fish has 35 3638 trafficked at least three parasite species to the U.S.A., one of them also found on frog. This 37 38 3939 route from the Asian wetlands to other continents is similar to that of amphibian chytrid fungi 40 4140 of genus Batrachochytrium Longcore, Pessier & Nichols, 1999. 42 43 4441 45 4642 Keywords 47 48 4943 Intercontinental parasite dispersal 50 5144 Molecular systematics 52 5345 Aquatic invasive species 54 55 5646 Chytrid fungus 57 5847 59 60 61 62 63 64 2 65 48 Introduction 1 2 349 Introduced and invasive species are increasingly recognized as a threat to local ecosystems. 4 550 The interactions of the invaders and the native populations are often simplified as resource 6 7 851 competition of the organisms at the same trophic level. Perhaps biological invasions are more 9 1052 complicated, and the harmful effects caused by new intruders surpass simple competition. 11 1253 Introduced organisms may shed their native parasites and diseases and gain a competitive 13 14 1554 advantage (Enemy release hypothesis, Torchin et al. 2003) in new ecosystems. The invaders 16 1755 may also introduce diseases which are new to the target ecosystem, and therefore harm the 18 19 2056 native populations of interacting species (Verneau et al. 2011). Disturbed ecosystems may in 21 2257 turn spread pathogens to humans and livestock and increase the number of zoonoses (EIDs or 23 24 2558 emerging infectious diseases; Daszak et al. 2000). One drastic example of harmful invasion in 26 2759 line with the focus of this paper was the transport of Baltic strains of Atlantic salmon (Salmo 28 2960 salar L.) to Norway. With clinically healthy (tolerant) Baltic fish, the monogenean flatworm 30 31 3261 parasite Gyrodactylus salaris Malmberg, 1957 arrived in Norway in 1975 and destroyed 45 33 3462 Atlantic salmon river populations in just a few decades (Johnsen and Jensen 1991; Bakke et 35 36 3763 al. 2002, 2007). In contrast, the long-isolated Baltic salmon (Lumme et al. 2015) is tolerant 38 3964 and co-adapted with G. salaris, so that the spatially and genetically differentiated host- 40 41 4265 parasite combinations remain stable despite the physical mobility (Lumme et al. 2016a). The 43 4466 parasite introduced to the susceptible Atlantic salmon populations spread rapidly, with fatal 45 46 4767 consequences. 48 4968 Gurevitch and Padilla (2004) asked whether species invasions are a major cause of 50 5169 extinctions, and claimed that data supporting this widely-accepted view often are anecdotal, 52 53 5470 speculative, and based on limited observation. A problem common with such observations 55 5671 about "change" is that many global changes occur in the same time axis and are temporally 57 58 5972 associated. This leads to skepticism against invoking explanations of "change". Pimentel et al. 60 61 62 63 64 3 65 73 (2005) estimated that there are 50,000 foreign plant or animal species in the United States 1 274 alone, and that 42% of endemic species are endangered or threatened because of aliens. The 3 4 575 authors concluded that the total economic damages and losses caused by invasive species add 6 776 up to an estimated $120 billion per year in the U.S.A. alone. 8 9 1077 This study maps a complex case of an introduced and rapidly spreading Asian fish in 11 1278 North America, accompanied by East Asian parasites. The starting point of this narrative was 13 1479 as follows. A monogenean flatworm species Gyrodactylus jennyae Paetow, Cone, Huyse, 15 16 1780 McLaughlin and Marcogliese 2009 was described as a new pathogenic parasite on the 18 1981 American bullfrog Lithobates catesbeianus Shaw, 1802 in North America. The bullfrog itself 20 21 2282 is widely trafficked (Mata-López et al. 2010). Paetow et al. (2013) also demonstrated that G. 23 2483 jennyae infection increased the mortality of the tadpoles associated with fungal disease 25 26 2784 Batrachochytrium dendrobatidis Longcore, Pessier & Nichols, 1999, making matters worse. 28 2985 This fungal disease is considered one of the main reasons for the contemporary decline of 30 3186 global amphibian populations (Collins and Crump 2009). The question was raised about the 32 33 3487 origin of this newly described Gyrodactylus species, which are monogenean ectoparasites of 35 3688 fish, seldom found on amphibians. Records on amphibians were reviewed in Paetow et al. 37 38 3989 2009. 40 4190 Fortunately, the formal description of G. jennyae was accompanied with the DNA 42 43 4491 sequence of complete ribosomal Internal Transcribed Spacer segments (ITS1-5.8S rDNA– 45 4692 ITS2), hereafter ITS. This fragment of DNA is the most popular in Gyrodactylus studies (e.g., 47 48 4993 Paetow et al. 2009), and consequently the most useful DNA segment for “barcoding” in the 50 5194 genus (Mendoza-Palmero et al., 2019). The parasites of genus Gyrodactylus are very small 52 5395 (<0.5 mm), living on the surface or gills of the fish, and most often clinically more or less 54 55 5696 asymptomatic. In fact, they are seldom detected without targeted search. Emphasis of research 57 5897 has been on fish farms, especially those of salmonids (Paladini 2012). A minimum number of 59 60 61 62 63 64 4 65 98 Gyrodactylus species diversity has been estimated at 20,000 (Bakke et al. 2002), with some 1 299 417 formally described (Harris et al. 2004), and only 200 barcoded by ITS. 3 4 1005 Paetow et al. (2009) conducted a BLAST search of G. jennyae among the published 6 1017 ITS sequences of Gyrodactylus spp., and confirmed closest matches of ITS1 and ITS2 8 9 10102 segments to species of the nominal subgenus G. (Gyrodactylus) which are mainly Eurasian 11 12103 parasites on cyprinids. The genetic distance of G. jennyae to G. neili LeBlanc, Hansen, Burt 13 14 104 & Cone 2006, a North American member of the subgenus G. (Gyrodactylus) is dK2P = 0.320. 15 16 17105 However, in a small unpublished collection of ITS sequences from Far East Asia maintained 18 19 106 by the authors, a much closer match dK2P = 0.085 was found between G. jennyae and G. 20 21 22107 macracanthus Hukuda, 1940, described in the Han River system, Korea. G. macracanthus is a 23 24108 parasite of the Asian pond loach (Misgurnus anguillicaudatus Cantor, 1842). 25 26 27109 Because M. anguillicaudatus is an invasive freshwater species trafficked by the 28 29110 aquarium pet trade (Chang et al. 2009) with established non-native populations in Australia, 30 31111 North and South America, and Europe (Belle et al. 2017 and references therein), we 32 33 34112 hypothesized that it might be a mechanism for transport of East Asian parasites to North 35 36113 America. A focused search was conducted in New York State (Wells 2014). 37 38 39114 40 41115 Methods 42 43 44116 The Far East and U.S.A. samples of Gyrodactylus 45 46117 The new ITS sequences used here were of Gyrodactylus macracanthus taken from M. 47 48 49118 anguillicaudatus collected from the wild near Vladivostok, Russia (43.25º N, 131.98º E) by 50 51119 hand net. The ITS samples from Topmouth gudgeon (a.k.a. Stone moroko) Pseudorasbora 52 53120 parva (Cyprinidae) were from the same geographical area, from the River Bol’shaya Ussurka 54 55 56121 (45.98º N, 134.03º E), and were included in this study because of the relatedness of the ITS. 57 58 59 60 61 62 63 64 5 65 122 The Russian fish were killed immediately by a blow to head and the fins and gills were 1 1232 removed and stored in 96 % ethanol. 3 4 1245 In central New York State, U.S.A., specimens of M. anguillicaudatus were collected 6 1257 by fish traps baited with dog food in the Manor Kill watershed near Conesville, New York 8 9 10126 (42.402 º N, -74.30 º W) on 21 October 2013 and 28 November 2015. 11 12127 The North American fish were maintained in aquaria for up to one month until 13 14128 examination for parasites. Fish were killed by cervical transection following submersion in 15 16 17129 Tricaine-S (Tricaine methanesulfonate, Western Chemical, Inc., Ferndale, Washington) in 18 19130 accordance with the guidelines of SUNY Oneonta (State University of New York College at 20 21 22131 Oneonta) IACUC protocol 201303. Gills were subsequently examined for monogeneans. 23 24132 Monogeneans encountered were preserved in 95–100% ethyl alcohol and examined with a 25 26 27133 light microscope and/or subjected to DNA sequencing (see below). 28 29134 30 31135 Morphological recording of the parasites 32 33 34136 A subset of the monogeneans encountered in New York State were photographed with a Leica 35 36137 DM2500 equipped with a DFC295 digital camera (Leica Microsystems, Buffalo Grove, 37 38 39138 Illinois). The photos of complete live specimens are provided here to serve as photo vouchers 40 41139 (Supplementary material). 42 43 44140 The monogenean specimens chosen for analysis of ITS sequence were stored in 45 46141 ethanol and cut in two parts. The opisthaptor was placed on a microscope slide for 47 48 49142 morphological inspection. When the ethanol was evaporated, a drop of proteinase K digestion 50 51143 mix (see below) was added and the digestion was followed under a microscope (100x phase 52 53144 contrast, Zeiss Axiolab). When the hard parts of the haptor were cleared, the sample was 54 55 56145 embedded in saturated ammonium picrate in glycerol under a coverslip, and fixed with nail 57 58146 polish. The samples were photographed during and after the digestion process with a Nikon 59 60 61 62 63 64 6 65 147 Coolpix 995 digital camera at 100x and 200x magnifications with phase contrast optics. 1 1482 Calibration for measurements was done with photos of a 0.01 mm scale (Graticules Ltd, 3 4 1495 Tonbridge, Kent, England). The microscope slides were deposited as vouchers or 6 1507 hologenophores (sensu Pleijel et al., 2008) in the Finnish National collection in Helsinki 8 9 10151 (MZH118018-19 for Gyrodactylus sp. A, #8 and #4, and MZH118018-20 for Gyrodactylus 11 12152 sp. B, #11). 13 14153 15 16 17154 DNA sequencing of the ITS segment 18 19155 The isolation of DNA, primary PCR and sequencing of the ITS of the anterior portions of 20 21 22156 ethanol fixed worms was conducted as described in previous Gyrodactylus studies (Ziętara 23 24157 and Lumme, 2002, 2003; Ziętara et al. 2006, 2008). The ethanol was evaporated. DNA was 25 26 27158 released by digesting single parasite halves in 10 μl of lysis solution consisting of 1 × PCR 28 29159 buffer, 0.45% (v/v) Tween 20, 0.45% (v/v) NP 40 and 60 µg/ml proteinase K. The samples 30 31160 were incubated at 65 °C for 25 minutes to allow proteinase K digestion, then at 95 °C for 10 32 33 34161 minutes to denature the proteinase and cooled down to 4 °C. 35 36162 Aliquots of 2 µl of this lysate were used as templates for PCR amplification. The 10 μl 37 38 39163 PCR mix consisted of 2 μl 5x Phusion HP buffer (7.5 mM MgCl2), 1 μl 2.0 mM dNTP, 0.5 1 40 41164 μl of each primer stock (20 μM), 0.2 U of Phusion DNA polymerase and 2 μl of digested 42 43 44165 parasite. The cycling profile was as follows: 98 °C for 30 sec, 35 cycles of 98 °C for 10 s, 45 46166 50 °C for 20 s and 72 °C 40 s, and a final extension at 72 °C for 5 min, and cooling down to 47 48 49167 4 °C. For primary PCR of whole ITS segment, the primers (Oligomer Oy, Helsinki, Finland) 50 51168 were ITS1F 5'- GTTTC CGTAG GTGAA CCT -3', and ITS2R 5'- GGTAA TCACG CTTGA 52 53169 ATC -3'. 54 55 56 57 58 59 60 61 62 63 64 7 65 170 The PCR product was checked in agarose gel. The product was purified by ExoSAP 1 1712 procedure: 0.8 μl Fast AP buffer, 0.5 μl Fast AP, 0.1 μl Exo I, 6.6 μl H2O, and 2 μl PCR 3 4 1725 product. 6 1737 The sequencing mix was 1.65 μl BigDye Terminator v 3.1 in 5x sequencing buffer, 8 9 10174 0.7 μl Big dye terminator v.3.1 Cycle in Sequencing RR-100, 0.65 μl of ITS1F, ITS2R, or the 11 12175 additional ITS2F or ITS1R primers, and 2 μl of Exosap purified PCR product. The internal 13 14176 sequencing primers were ITS2F 5'- TGGTG GATCA CTCGG CTCA -3' and ITS1R 5'- 15 16 17177 ATTTG CGTTC GAGAG AGACC -3'. Altogether, the coverage was threefold to fourfold. 18 19178 The sequencing reactions were purified by the Sephadex method and introduced to an 20 21 22179 ABI3730 automatic DNA reader. The ABI reads were decoded, edited and aligned by Codon 23 24180 Code Aligner, and transferred to MEGA7 for further analysis. 25 26 27181 28 29182 Sequence comparisons, alignment and phylogenetic tree construction 30 31183 The ITS sequences vary so much within Gyrodactylus that only 5.8S RNA and the terminal 32 33 34184 parts of the ITS1 and ITS2 sequences can be reliably aligned genus-wide. We selected the set 35 36185 of species to be compared with G. jennyae by phylogenetic hypothesis by internal BLAST 37 38 39186 search, confirmed by secondary structure comparison of the ITS2 (Zuker 2013). We utilized 40 41187 the MUSCLE (Edgar 2004) program as implemented in MEGA7. The hypervariable segment 42 43 44188 in ITS1 containing a variable number of TAAAAA repeats was hand-edited. 45 46189 The distance method K2P (Kimura's two parameter) was chosen because it is the most 47 48 49190 simple model that can be used for this type of DNA sequence data. The sequence evolution of 50 51191 rDNA is strictly constrained for maintaining the secondary structure stems and hairpins, 52 53192 which in ITS are needed for correct splicing of the segments to be removed. The phylogenetic 54 55 56193 reconstructions were made in MEGA7 by Neighbor Joining and Maximum Likelihood 57 58 59 60 61 62 63 64 8 65 194 methods, both tested by 500 rounds of bootstrapping. All methods produced a robust 1 1952 separation between the species and branches, with the same topology. 3 4 1965 The new ITS sequences from Far East Asia and North America were deposited in 6 1977 GenBank with accession numbers MH667459-MH667466 (Fig. 1). 8 9 10198 11 12199 Results 13 14200 In New York State, M. anguillicaudatus dominated the catch in the infested headwaters in the 15 16 17201 Manor Kill watershed, living together with native amphibians, the spotted salamander 18 19202 (Ambystoma maculatum Shaw, 1802) and red spotted newt (Notophthalmus viridescens 20 21 22203 Rafinesque, 1820), as described by Wells (2014). 23 24 25204 A total of 48 M. anguillicaudatus specimens were caught and the gills examined for 26 27 28205 monogeneans, including 30 from the October 2013 sample and 18 from the November 2015 29 30206 sample. Eight of the 48 fish examined were found to have Gyrodactylus specimens, for an 31 32 33207 overall prevalence of 16.7%. The maximum number of Gyrodactylus specimens found on a 34 35208 single fish was six. The ITS DNA sequence was obtained from a total of seven North 36 37209 American specimens of Gyrodactylus, and included in the phylogeny (Fig. 1). 38 39 40 41210 Molecular phylogenetic position of Gyrodactylus intruders in North America 42 43211 The species of Gyrodactylus to be compared with G. jennyae were selected from available 44 45 46212 GenBank data by BLAST search and sequence comparison. The group is monophyletic also 47 48213 in the partial global phylogeny by Mendoza-Palmero et al. (2019), supported by Posterior 49 50 51214 probability/ Bootstrap values 0.93/61. An apomorphic indel influencing the secondary 52 53215 structure folding in ITS2 confirmed the monophyly of the group in Fig. 1. 54 55 56216 A phylogenetic hypothesis containing a total of 29 published and new ITS1-5.8S-ITS2 57 58217 sequences was constructed for placing G. jennyae in a plausible systematic position in a 59 60218 monophyletic clade supported by Posterior probability/ Bootstrap values 0.93/61 in the partial 61 62 63 64 9 65 219 global phylogeny by Mendoza-Palmero et al. (2019), a study which helped us guide our 1 2202 sampling representation here. The phylogenetic hypothesis strongly supports three sister 3 4 2215 subclades presented in Fig 1: first one from Africa on Siluriformes (Přikrylová et al. 2012), 6 2227 second mostly on European cyprinids described as subgenus G. (Gyrodactylus) (Malmberg 8 9 10223 1970), and the third group in focus in this study, containing the Far East Asian species on 11 12224 Cobitidae and P. parva, along with the three found as intruders in North America. This cluster 13 14225 was characterized by a 15 16 17226 The branch from Far East Asia in Fig. 1 appears to be a sister clade to the 18 19227 predominantly European subgenus G. (Gyrodactylus). In the tree of Mendoza-Palmero et al. 20 21 22228 (2019), G. jennyae was paired with G. granoei in this clade, which now contains eight 23 24229 species. The two previously unknown Gyrodactylus parasites found following examination of 25 26 27230 M. anguillicaudatus from New York State clustered with G. jennyae and G. macracanthus, 28 29231 but they are clearly separate species differing from one another by a dK2P = 0.085. Not 30 31232 presented in the tree is G. misgurni Ling, 1962, because only ITS1 sequence (AJ407887) is 32 33 34233 available. The hypervariable ITS1 of G. misgurni is closer to G. macracanthus (dK2P = 0.084) 35 36 234 than to G. jennyae (dK2P = 0.145). Thus, it belongs to the subclade with the American G. sp. B 37 38 39235 and G. macracanthus. G. misgurni has been named and described from Misgurnus 40 41236 anguillicaudatus from China, but the ITS1 sequence is from European Misgurnus fossilis 42 43 44237 (Matĕjusová et al. 2001). 45 46238 Thus, the monophyletic sub-cluster including G. jennyae contains at least five parasite 47 48 49239 species from Misgurnus species. This strongly supports a fish origin of G. jennyae. The Far 50 51240 East Asia origin of G. jennyae and the two undescribed species A and B from New York is 52 53241 supported by relatedness of the sub-clade containing G. granoei on cobitids and the three 54 55 56242 unnamed species on the cyprinid Pseudorasbora parva. The combination of the two 57 58243 subclades is 100/100% supported and forms a novel Asian freshwater group of Gyrodactylus 59 60 61 62 63 64 10 65 244 species. Comparing it with the subgenus G. (Gyrodactylus) may suggest a subgeneric status. 1 2452 However, Malmberg's five subgenera are based on the osmoregulatory system, studied in 3 4 2465 living worms. Constructing new subgenera without this information seems unjustified. 6 2477 The two most basal species likely belonging to the subgenus G. (Gyrodactylus) are G. 8 9 10248 neili from chain pickerel (Esox niger Lesueur, 1818) from New Brunswick, Canada (LeBlanc 11 12249 et al. 2006), and G. laevisoides King, Cone, Mackley & Bentzen, 2013 from Northern 13 14250 redbelly dace (Chrosomus eos Cope, 1861) in Nova Scotia (King et al. 2013). Interestingly, 15 16 17251 these two are the only molecularly-identified North American members of subgenus G. 18 19252 (Gyrodactylus), and G. neili is the only species of this subgenus known from a non-cyprinid 20 21 22253 host. The phylogenetic analysis suggested that the inclusion of G. jennyae in subgenus G. 23 24254 (Gyrodactylus) is not supported. 25 26 27255 28 29256 Morphological comparisons 30 31257 The morphological taxonomy of Gyrodactylus is based on the form and size of the sclerotized 32 33 34258 parts of the haptor (opisthaptor) organ on the posterior of the animal, containing two major 35 36259 hooks (hamuli) and sixteen marginal hooks. The species descriptions available in Pugachev et 37 38 39260 al. (2009) and in GyroBase (http://www.gyrodb.net/) cover the present taxonomical 40 41261 knowledge of Gyrodactylus species in Far East Asia. The haptoral morphology of the two 42 43 44262 potentially new North American species, Gyrodactylus sp. A and Gyrodactylus sp. B, is 45 46263 presented in Fig. 2. 47 48 49264 The marginal hook morphology of Gyrodactylus sp. A and Gyrodactylus sp. B is 50 51265 characteristic for the molecularly confirmed morpho-group including the three American 52 53266 species, the far East Russian G. macracanthus, and the European specimen of G. misgurni. 54 55 56267 The base is hoof-like and looks hollow in light microscopy, but this is not visible in the all- 57 58268 black or outline profile drawings in the literature. The external part of the base is much 59 60 61 62 63 64 11 65 269 enlarged. The hooklet (sickle) is about the same length as the base, and rather straight until 1 2702 the curved tip, versus tightly curved from halfway as in G. granoei (You et al. 2010) and the 3 4 2715 unnamed species hosted by P. parva in Fig. 1. Five molecularly tagged taxa belong to this 6 2727 morpho-group parasitizing Cobitidae: G. jennyae, G. macracanthus and G. misgurni (ITS1 8 9 10273 only) and the two species G. sp. A and G. sp. B depicted and sequenced here. The following 11 12274 unsequenced species also belong to this morpho-group, characterized by specific marginal 13 14275 hooks, on the basis of drawings compiled in Pugachev et al. (2009) and in GyroBase 15 16 17276 (http://www.gyrodb.net/): G. micracanthus Hukuda, 1940 on M. anguillicaudatus (Korean 18 19277 Peninsula) and/or on Cobitis granoei Rendahl, 1935, (Russian Far East), G. molnari Ergens, 20 21 22278 1978, on Cobitis taenia L. (Hungary), G. yukhimenkoi Ergens, 1978, on Cobitis sibirica 23 24279 Gladkov, 1935 (Far East Asia), and G. latus Bychowsky, 1933, on Cobitis taenia (Europe). 25 26 27280 The G. macracanthus from Australian M. anguillicaudatus is also a member in this morpho- 28 29281 group, but it is tagged not with ITS, but by 28S ribosomal RNA, Histone 3 and Elongation 30 31282 factor 1 α sequences (Perkins et al. 2009). 32 33 34283 Total length of the hamulus (main hook) in G. sp. A was 53.2 - 54.5 µm, within the 35 36284 published range of G. yukhimenkoi (53-57 µm) and G. latus (50-57 µm). In G. sp. B, the 37 38 39285 hamulus was smaller, 48.4 µm, within the range reported for G. misgurni (42-51 µm). Hamuli 40 41286 of both G. sp. A and G. sp. B are clearly smaller than the hamulus of G. jennyae (62 µm) or 42 43 44287 G. molnari (63-65 µm). Both G. sp. A and G. sp. B have longer central hooks than the 45 46288 original Korean G. macracanthus (41-47 µm). The G. sp. B is close to the Australian sample 47 48 49289 named as G. macracanthus by Dove and Ernst (49 µm). The above species have not been 50 51290 molecularly confirmed, and thus may or may not be conspecific with any of the species 52 53291 mentioned in the phylogeny in Fig. 1. 54 55 56292 In the description of G. granoei (You et al. 2010) the marginal hooks of several of the 57 58293 above species were compared in figures 4-7, and they are clearly different: the hooklet of G. 59 60 61 62 63 64 12 65 294 granoei is much more curved. The marginal hooks of the parasites on Pseudorasbora parva 1 2952 (data not shown) confirm that they are morphologically related to G. granoei, but not to the 3 4 2965 group of G. jennyae. The parasites on P. parva presented in Fig. 1 are most probably all 6 2977 undescribed species. 8 9 10298 11 12299 Discussion 13 14300 The method of this report is molecular phylogeography (Avise 2000). The species 15 16 17301 names are less important than the DNA sequences and phylogenetic hypothesis constructed 18 19302 and overlaid on the continents. We have decided not to describe and name the few supposedly 20 21 22303 undescribed species in this study, owing to lack of adequate material (specimens), and 23 24304 because doing so is beyond the scope of this study. We report here two species related to G. 25 26 27305 jennyae that were found in North America, but they evidently originated from Far East Asia, 28 29306 together with their invasive host fish Misgurnus anguillicaudatus. There are several other 30 31307 morphologically related species described in Eurasia, but without DNA barcodes they cannot 32 33 34308 be reliably compared. In addition, three undescribed species on Pseudorasbora. parva 35 36309 barcoded here are surely novel, because the only species described on this host (G. parvae 37 38 39310 You, Easy & Cone, 2008, EF450249) belongs to subgenus G. (Limnonephrotus). However, 40 41311 they help in anchoring the clade to Far East Asia. 42 43 44312 The strong phylogenetic association of the barcoding ITS sequence of the amphibian 45 46313 parasite G. jennyae with G. macracanthus on the originally Asian invasive host fish lead to a 47 48 49314 hypothesis of intercontinental parasite trafficking. Autonomous dispersal is excluded as an 50 51315 explanation for a freshwater host and parasite. M. anguillicaudatus belongs to the family 52 53316 Cobitidae (~ 29 genera, ~ 110 species) which is native to freshwaters in Eurasia (> 100 54 55 56317 species) and Morocco, North Africa (only 2 species) (Kottelat 2012). Consequently, we 57 58318 predicted that M. anguillicaudatus may have also carried G. jennyae and perhaps other Asian 59 60 61 62 63 64 13 65 319 Gyrodactylus species into North America. If G. jennyae or other relative(s) of G. 1 3202 macracanthus could be found in established immigrant fish, they could be taken as direct 3 4 3215 evidence that the Asian loach carried them to a new continent. It was with this beginning 6 3227 hypothesis that we set out to investigate Gyrodactylus parasites on Oriental weatherfish in one 8 9 10323 of many feral populations now occurring in New York State (Wells 2014). 11 12324 Despite some gaps in the dataset, a global molecular systematic scaffold for 13 14325 Gyrodactylus has developed, supporting and complementing the morphological systematics of 15 16 17326 the genus based mostly on Eurasian material (Malmberg 1970; Ergens 1985). In a very 18 19327 important study, Boeger et al. (2003) developed a hypothesis that the viviparous genus 20 21 22328 Gyrodactylus originated in South America from an egg-laying predecessor, and later occupied 23 24329 the marine environments and expanded to other continents. Molecular phylogenetic 25 26 27330 investigations may support this hypothesis. Accumulating data from Europe (Matĕjusová et 28 29331 al. 2003; Ziętara and Lumme, 2004), North America (Gilmore et al. 2012) and Mexico 30 31332 (Garcia-Vasquez et al. 2015; Mendoza-Palmiero et al., 2019) help to test and further define 32 33 34333 such hypotheses. Among the marine (coastal) parasite groups, surprisingly distant samples 35 36334 show genetic relatedness. In the large dataset of ITS2 in GenBank, the overall genetic 37 38 39335 distance dITS2K2P = 0.449, even while deflated via comparisons within clades. Two Antarctic 40 41336 (Rokicka et al. 2009) and one Chilean (Ziętara et al. 2012) marine species of Gyrodactylus 42 43 44337 were phylogenetically connected with small clusters of respective Northern species groups. 45 46338 Representative species pairs are the Antarctic G. coriicepsi Rokicka, Lumme & Zietara. 2009, 47 48 49339 vs.G. groenlandicus Levinsen, 1881, from British Columbia (dITS2K2P = 0.046) and G. 50 51340 chileani Zietara, Lebedeva, Muňoz & Lumme, 2012 vs. Mediterranean G. orecchiae Paladini, 52 53 341 Cable, Fioravanti, Faria, Cave & Shinn, 2009 (dITS2K2P = 0.158) A surprising coincidence 54 55 56342 was that two species collected on Mexican freshwater poecilids, G. takoke Garcia-Vásquez, 57 58343 Razo-Mendivil & Rubio-Godoy, 2015 (KM514457; dITS2K2P = 0.031) and G. xalapensis 59 60 61 62 63 64 14 65 344 Rubio-Godoy, Paladini, Garcia-Vásquez & Shinn, 2010 (KJ621985; dITS2K2P = 0.018) were 1 3452 the nearest relatives of G. arcuatus Bychowsky, 1933 (AF328865), a circumpolar parasite on 3 4 3465 three-spined stickleback Gasterosteus aculeatus L. (Lumme et al., 2016b). At this scale, the 6 3477 differences between G. jennyae and its invasion partners are moderate (G. sp. A; 8 9 10348 dITS2K2P = 0.035; G. sp. B; dITS2K2P = 0.039). 11 12349 Monogenean parasites are only dispersed by the fish host. They have no known means 13 14350 of independent long-distance dispersal, and they are not known to use vectors; hence such 15 16 17351 cases of long-distance transfer are assumed to be anthropogenic. The invasive fishes may 18 19352 transport their own parasites to new areas, as shown here for M. anguillicaudatus. Some other 20 21 22353 examples are Gyrodactylus perccotti Ergens, Yukhimenko & Yukhimenko, 1973 on Amur 23 24354 sleeper Perccottus glennii Dybowski, 1877 from Russia to Eastern Europe (Ondračková et al. 25 26 27355 2012); Gyrodactylus proterohini Ergens, 1967 on Western tubenose goby Proterorhinus 28 29356 semilunaris (Heckel, 1837) from the Ponto-Caspian region to Western Europe, Belgium 30 31357 (Huyse et al. 2015) and G. salmonis (Yin & Sproston, 1948) on rainbow trout from boreal 32 33 34358 North America to Mexico (Rubio-Godoy et al. 2012). In these cases, subsequent spread of 35 36359 Gyrodactylus to any new fish (or amphibian) species of the new homeland was not reported, 37 38 39360 either due to limited examination or because the parasite failed to relocate. In another example 40 41361 of parasite trafficking, the topmouth gudgeon (P. parva) has introduced a protistan disease 42 43 44362 rosette agent (Sphaerothecum destruens) to the U.S.A., U.K. and the Netherlands (Ragan et 45 46363 al. 1996; Pinder et al. 2005; Spikmans et al. 2013), but no Gyrodactylus, as far as we know. 47 48 49364 Conversely, invasive fishes can adopt local parasites in their new localities, e.g. the 50 51365 unnamed Gyrodactylus (Limnonephrotus) sp. on round goby Neogobius melanostomus 52 53366 (Pallas, 1814) in Europe (Huyse et al. 2015). The rainbow trout (Oncorhynchus mykiss 54 55 56367 [Walbaum, 1792]), a global resident now (Fausch 2007) has adopted numerous European 57 58368 parasites, often via hybridization of two strains of Gyrodactylus (Lindenstrøm et al. 2003; 59 60 61 62 63 64 15 65 369 Rokicka et al. 2007, Kuusela et al. 2008, Ziętara et al. 2010, Ieshko et al. 2015). As a widely- 1 3702 cultured species, rainbow trout serves as an optimal platform to receive and subsequently 3 4 3715 serve as a vector for the introduction of opportunistic parasites to new locations. 6 3727 Of course, both scenarios can be combined. Introduced and local parasites may breed 8 9 10373 and produce novel combinations and perhaps transfer further, into new hosts and localities. 11 12374 Human trafficking has replaced the geological perturbations as a source of novelty by 13 14375 recombination. 15 16 17376 The conclusion based on the phylogenetic comparison in this work is that the clade of 18 19377 parasites related to G. jennyae on Pseudorasbora and Misgurnus appears to be a 20 21 22378 monophyletic branch originating from Asia. All native loach in the family Cobitidae are 23 24379 freshwater forms in Eurasia and in Morocco, Africa (Kottelat 2012). Consequently, the 25 26 27380 monogenean parasite species of this evolutionary clade are not expected to be endemic to any 28 29381 North American freshwater fishes, let alone frogs. 30 31382 32 33 34383 Similar invasion histories of Misgurnus, Gyrodactylus and chytridiomycosis. 35 36384 In the amphibian facet of the story, G. jennyae forms an intriguing coincidence with the 37 38 39385 fungal disease chytridiomycosis, caused by pathogenic fungi of genus Batrachochytrium, 40 41386 which have apparently invaded global amphibian populations. The global decline of 42 43 44387 amphibian populations is a major concern for scientists (Storfer 2003). The causes of this 45 46388 decline are diverse, complex, and still under much study and debate (e.g., Collins and Crump 47 48 49389 2009 vs. Pounds and Masters 2009). 50 51390 The enzymatic digestion and the hooked haptors of Gyrodactylus damage the frog’s 52 53391 epidermis (Nieto et al. 2007; Paetow et al. 2009, 2013). Epidermal damage on tadpole lips 54 55 56392 caused by G. jennyae (figs. 4A and 1A in Paetow et al. 2009 and 2013, respectively) appears 57 58393 similar to erosion caused by fungus Batrachochytrium dendrobatidis (fig. 6.10 in Collins and 59 60 61 62 63 64 16 65 394 Crump 2009). Such epidermal damage on frogs may facilitate opportunistic fungal infections 1 3952 which can result in osmoregulation failure and even death via heart failure (Voyles et al. 3 4 3965 2009; Paetow et al. 2013). 6 3977 Recently, a molecular phylogenetic framework for the chytrid fungi killing 8 9 10398 amphibians was generated, first in The Netherlands (Martel et al. 2013), describing a novel 11 12399 species, Batrachochytrium salamandrivorans Martel et al., 2013. The globally more widely 13 14400 spread B. dendrobatidis was extensively studied (Fisher et al. 2018; O'Hanlon et al. 2018). It 15 16 17401 was demonstrated that the worldwide pathogenic strains of the fungus originated in the 18 19402 Korean Peninsula and were subsequently spread most probably by the general pet trade, since 20 21 22403 1975. The pet trade routinely traffics both amphibians and fishes from areas where the 23 24404 collecting might be an important cottage industry. The worldwide industry is largely 25 26 27405 unregulated and lacks a central database to assess its impact upon the environment (Lee 28 29406 2014). The distribution history of M. anguillicaudatus demonstrates that it is commonly 30 31407 shipped live worldwide and may be included among the potential vectors of aquatic diseases. 32 33 34408 Here we raise the possibility that in their new environment, invasive parasites can 35 36409 move from invading fish to native amphibian hosts during the aquatic phases of their life 37 38 39410 cycle. By extension, we suggest that such novel/unknown parasites like Gyrodactylus spp. 40 41411 may be contributing to the global decline of amphibians. 42 43 44412 45 46413 Conclusions 47 48 49414 The phylogenetic position of North American bullfrog parasite G. jennyae and two 50 51415 additional Gyrodactylus species demonstrate that they are not native to North America but 52 53416 instead likely originated from Asia, by invasive fish. 54 55 56417 M. anguillicaudatus is a recent invader to several continents, and continues to spread 57 58418 into new/adjacent fresh waters. This Asian loach species carried G. jennyae from Asia to 59 60 61 62 63 64 17 65 419 North America and introduced it to bullfrog. We predict that M. anguillicaudatus has likely 1 4202 spread several other Gyrodactylus species or strains worldwide. 3 4 4215 We support previously stated concerns (Paetow et al. 2013) that M. anguillicaudatus 6 4227 and its Gyrodactylus parasites may contribute to the global spread of chytrid fungi, as vectors 8 9 10423 via both legal and illegal trafficking routes. 11 12424 Molecular phylogeography of Gyrodactylus (and Misgurnus) might well detect the 13 14425 routes of the insane trafficking of aquatic biota, and help to control it. 15 16 17426 18 19427 Acknowledgements 20 21 22428 Late Professor Ian Whittington informed us of the Australian situation. The early 23 24429 molecular work in Oulu University was supported by the Academy of Finland (Grants 63787, 25 26 27430 134592). Laura Törmälä conducted the DNA work in Oulu. The authors thank Timothy 28 29431 Pokorny (New York State Dept. of Environmental Conservation) for assisting with fish 30 31432 collections. We also acknowledge the students of the SUNY Oneonta fall 2013 parasitology 32 33 34433 class and other SUNY Oneonta students: Tara Aprill, Austin Borden, Jill Darpino, Margaret 35 36434 Doolin, Kathryn Forti, Kaylee Herzog, Elise Iwanyckyj, Sisina Macchiarelli, Zachary Piper, 37 38 39435 Sam VanDemark, and Craig Wert for their assistance with dissections of M. anguillicaudatus. 40 41436 This study was funded in part by a National Science Foundation Field Stations and Marine 42 43 44437 Laboratories grant to W. Harman (NSF DBI 1034744). The comments of two anonymous 45 46438 reviewers contributed to the development of this manuscript. 47 48 49 50439 51 52440 Literature Cited 53 54441 55 56 57 58 59 60 61 62 63 64 18 65 442 Arkush, KD, Mendoza L, Adkison MA, Hedrick RP (2003) Observations on the life stages of 1 4432 Sphaerothecum destruens n. g., n. sp., a Mesomycetozoean fish pathogen formally referred to 3 4 4445 as the Rosette Agent. 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Mfold web server for nucleic acid folding and hybridization prediction. 44 45627 Nucleic Acids Res. 31: 3406-15 46 47 48 49628 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 27 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59629 60 61 62 63 64 28 65 630 1 2 6313 Figure. 1. The phylogenetic hypothesis of G. jennyae and the Gyrodactylus spp. on 4 5 6326 Misgurnus anguillicaudatus and Pseudorasbora parva, compared with subgenus G. 7 6338 (Gyrodactylus) sensu Malmberg (1970) and rooted with a sister clade of three species from 9 10 11634 East Africa (Přikrylová et al. 2012). Respective Bootstrap values (500 replicates) are 12 13635 indicated for both Neighbor-Joining and Maximum Likelihood. The evolutionary distances 14 15636 were computed using the Kimura 2-parameter method and are in the units of the number of 16 17 18637 base substitutions per site. The analysis involved 29 nucleotide sequences varying in length 19 20638 from 881-896 bp. All gaps were removed, leaving a total of 665 positions in the final dataset. 21 22 23639 Evolutionary analyses were conducted in MEGA7. 24 25 26640 27 28 29 30641 31 32 33642 34 35 36 37643 38 39 40644 41 42 43 44645 45 46 47646 48 49 50 51647 52 53 54648 55 56 57 58649 59 60 61 62 63 64 29 65 650 1 2 6513 4 5 6 6527 8 9 10653 11 12 13654 14 15 16655 17 18 19 20656 21 22 23657 24 25 26658 27 28 29659 30 31 32 33660 34 35 36661 37 38 39662 40 41 42663 43 44 45664 46 47 48 49665 Figure. 2. Hamuli and marginal hooks of the Gyrodactylus sp. A (Figs. 2A,B; GenBank 50 51666 MH667465) and G. sp. B (Figs. 2C,D; GenBank MH667466) on Misgurnus anguillicaudatus in 52 53 54667 New York State, U.S.A. The hoof-like morphology of the base of marginal hooks is group-specific 55 56668 and is indistinguishable from G. jennyae, but separates the group clearly from the other frog 57 58 59669 parasites mentioned by Paetow et al. (2009). 60 61 62 63 64 30 65 Figure 1 Click here to access/download;Figure;Figure 1.pdf

. G. sp. B #2 Misgurnus anguillicaudatus U.S.A. 2015 100/100 G. sp. B #6 67/64 G. sp. B #11 G. sp. B M. anguillicaudatus U.S.A. 2013 MH667466 100/99 G. macracanthus M. anguillicaudatus FAR EAST MH667459 G. jennyae Lithobates catesbeianus U.S.A. EU678357 G. sp. A #4 97/96 G. sp. A #8 100/100 100 G. sp. A #15 M. anguillicaudatus U.S.A. 2015 MH667465 G. sp. 2 P. parva MH667461 100/100 G. sp. 2 P. parva MH667462 G. sp. 1 Pseudorasbora parva FAR EAST RUSSIA MH667460

100/98 G. granoei Cobitis granoei CHINA HM185817 G. sp. 3 P. parva MH667463 G. sp. 3 P. parva MH667464 100/99 G. sp. 3 P. parva G. sp. 3 P. parva 100/100 G. neili AY881175 NORTH AMERICA G. laevisoides KF263527 NORTH AMERICA 99/82 G. magnificus AY278035 EUROPE 65/84 100/100 G. phoxini AY278037 EUROPE Subgenus G. perccotti JN603638 EUROPE G. (Gyrodactylus) 97/65 G. laevis AY278036 EUROPE sensu G. alburnensis Malmberg 1970 42/44 AY278032 EUROPE 100/99 G. elegans AY278034 EUROPE 99/96 G. prostae AY278038 EUROPE G. rysavyi FR850679 EAST AFRICA 100/100 G. nigritae FR850686 EAST AFRICA 100/100 G. synodonti FR850684 EAST AFRICA

0.020 Figure 2 Click here to access/download;Figure;Fig 2.tif Supplementary Material

Click here to access/download Supplementary Material Loach parasites supplemental.pdf