Morphological and Molecular Data for a New

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Morphological and molecular data for a new species of Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in the Mexican redhorse Moxostoma austrinum Bean (Cyprinifo...

Article in Systematic Parasitology · November 2017 DOI: 10.1007/s11230-017-9756-y

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Martín García-Varela Berenit Mendoza-Garfias Universidad Nacional Autónoma de México Universidad Nacional Autónoma de México

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Anindo Choudhury Gerardo Pérez-Ponce de León St. Norbert College Escuela Nacional de Estudios Superiores unidad Mérida. UNAM

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The user has requested enhancement of the downloaded file. Morphological and molecular data for a new species of Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in the Mexican redhorse Moxostoma austrinum Bean (Cypriniformes: Catostomidae) in central Mexico Martín García-Varela, Berenit Mendoza- Garfias, Anindo Choudhury & Gerardo

Systematic Parasitology Pérez-PonceAn International Journal de León

ISSN 0165-5752 Volume 94 Number 9

Syst Parasitol (2017) 94:989-1006 DOI 10.1007/s11230-017-9756-y

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Syst Parasitol (2017) 94:989–1006 DOI 10.1007/s11230-017-9756-y

Morphological and molecular data for a new species of Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in the Mexican redhorse Moxostoma austrinum Bean (Cypriniformes: Catostomidae) in central Mexico

Martıń Garcıá-Varela . Berenit Mendoza-Garfias . Anindo Choudhury . Gerardo Pe´rez-Ponce de Leoń

Received: 25 April 2017 / Accepted: 5 August 2017 / Published online: 12 October 2017 Ó Springer Science+Business Media B.V. 2017

Abstract Pomphorhynchus purhepechus n. sp. is Pomphorhynchus bulbocolli Linkins in Van Cleave, described from the intestine of the Mexican redhorse 1919, a species widely distributed in several freshwa- Moxostoma austrinum Bean (Catostomidae) in central ter fish species across Canada, USA, and Mexico. The Mexico. The new species can be distinguished from genetic divergence estimated between the new species the other seven described species of Pomphorhynchus and the isolates of P. bulbocolli ranged between 13 Monticelli, 1905 in the Americas by a subspherical and 14% for cox1, and between 0.6 and 0.8% for LSU. proboscis and 14 longitudinal rows with 16–18 hooks Maximum likelihood and Bayesian inference analyses each; the third and the fourth row of hooks are of each dataset showed that the isolates of P. alternately longest. Sequences of the mitochondrial bulbocolli parasitising freshwater fishes from three cytochrome c oxidase subunit 1 (cox1) gene and the families, the Catostomidae, Cyprinidae and Centrar- large subunit (LSU) rDNA (including the domains D2- chidae, represent a separate lineage, and that the D3) were used to corroborate the morphological acanthocephalans collected from two localities in distinction between the new species and central Mexico comprise an independent lineage. In addition, our analysis of the genetic variation of P. bulbocolli demonstrates that individuals of this acanthocephalan from different host species are conspe- This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as 865ABA9F-4748-41CA-8685- cific. Finally, the distribution, host-association, and D72800D4B2AB. This article was published as an Online First phylogenetic relationship of the new species, when article on the online publication date shown on this page. The placed in the context of the region’s geological article should be cited by using the doi number. This is the history, suggest that both host and parasite underwent Version of Record. speciation after their ancestors became isolated in This article is part of the Topical Collection Acanthocephala. Central Mexico.

M. Garcıá-Varela (&) Á B. Mendoza-Garfias Á G. Pe´rez-Ponce de Leoń Introduction Departamento de Zoologıá, Instituto de Biologıá, Universidad Nacional Autońoma de Me´xico, Me´xico City, DF, Mexico Species of Pomphorhynchus Monticelli, 1905 are e-mail: [email protected] mainly endoparasites of freshwater fishes; four species parasitise marine or estuarine fishes and one occurs in A. Choudhury Division of Natural Sciences, St. Norbert College, 100 amphibians. In the most recent review of the genus, 29 Grant Street, DePere, Wisconsin 54115, USA species are recognised as valid (Amin, 2013). In the 123 Author's personal copy

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Americas, the genus Pomphorhynchus has an amphi- from several localities across Canada, USA and American distribution (see Choudhury et al., 2017), northern Mexico, led to a more detailed study using with eight recognised species: five species occur in morphological and molecular data, which indicated South America (Pomphorhynchus yamagutii Schmidt that this acanthocephalan from M. austrinum was new & Higgins, 1973; P. patagonicus Ortubay, Ubeda, to science. The main objectives of this paper are to Semenas & Kennedy, 1991; P. sphaericus Gil de describe the new species using integrative taxonomy, Pertierra, Spatz & Doma, 1996; P. moyanoi Olmos & to explore the genetic divergence of populations of P. Habit, 2007; and P. omarsegundoi Arredondo & Gil de bulbocolli across a wide host and geographic range in Pertierra, 2010) and only three species occur in North North America, and to discuss the biogeographical America (P. bulbocolli Linkins in Van Cleave, 1919; implications of our findings. P. rocci Cordonnier & Ward, 1967; and P. lucyi Williams & Rogers, 1984) (Oturbay et al., 1991; Gil de Pertierra et al., 1996; Olmos & Habit, 2007; Materials and methods Arredondo & Gil de Pertierra, 2010). Of the three North American species, Pom- Specimen collection phorhynchus bulbocolli appears to be the most widely Adult acanthocephalans were collected from the distributed. Although this species is primarily a intestine of the Mexican redhorse in two localities of parasite of catostomids, it has been reported from central Mexico, i.e. Pateo, and La Luz spring, hosts in five other fish families, the Acipenseridae, Michoacań (Table 1; Fig. 1). Additionally other areas Amiidae, Centrarchidae, Cyprinidae and Ictaluridae, across Canada, USA and northern Mexico, individuals across Canada, USA and Mexico (see Samuel et al., identified as P. bulbocolli were collected in nine 1976; Choudhury & Dick, 1998; Hoffman, 1999; localities (Table 1; Fig. 1). Fish were captured with Garcıá-Prieto et al., 2010; Martıńez-Aquino et al., seine nets and by electrofishing, kept alive, and 2011; Aguilar-Aguilar et al., 2014). The list of hosts transported to laboratory. Fish taxonomy follows may be inflated because it is not always clear if records Froese & Pauly (2017). Fishes were euthanised of P. bulbocolli in hosts distantly related to catosto- individually and examined immediately after death. mids refer to sexually mature or gravid worms, and Acanthocephalans were placed in distilled water for these reports may need to be verified. Pom- 10–12 h at 4°C to relax. Specimens were subsequently phorhynchus lucyi has a more limited distribution, in preserved in 100% ethanol, and stored at 4°C. For northern Florida and southern Alabama, but its hosts, morphological identification, some specimens were four fish species from seven families (Williams & stained with Mayer’s paracarmine, dehydrated in a Rogers, 1984), are also disparate. The third species, P. graded ethanol series, cleared with methyl salicylate, rocci, was described from the anadromous striped bass and mounted in Canada balsam. Specimens (par- Morone saxatilis (Walbaum) in North Carolina (Cor- agenophores sensu Pleijel et al., 2008) were deposited donnier & Ward, 1967) but was subsequently reported in the Coleccioń Nacional de Helmintos (CNHE), from the freshwater drum Aplodinotus grunniens Instituto de Biologıá, Universidad Nacional Autoń- Rafinesque and the rock bass Ambloplites rupestris oma de Mexico, Mexico City. A sub-sample from (Rafinesque) in Canada and USA (Hoffman, 1999). these isolates were also fixed in 95% or 100% ethanol During a recent survey of fish parasites in the Trans- for molecular work. Specimens collected from north- Mexican Volcanic Belt (TMVB) of Central Mexico, a ern Mexico were fixed in 100% ethanol after being critical transition zone between the Neotropical and relaxed in water for 12 h. Some specimens were Nearctic biogeographic regions (Domıńguez-Domıń- stained with Mayer’s paracarmine, dehydrated in a guez & Pe´rez-Pone de Leoń, 2009; Morrone, 2006), graded ethanol series, cleared with methyl salicylate, adult specimens of a species of Pomphorhynchus were and mounted on permanent slides in Canada balsam; collected from the intestine of the Mexican redhorse voucher material was also deposited at the CNHE. Moxostoma austrinum Bean (Catostomidae). Initial Acanthocephalans assigned to Pomphorhynchus were examination of these newly collected specimens of compared with the original descriptions and other Pomphorhynchus and a comparison with published published accounts. Measurements are given in descriptions and specimens of P. bulbocolli, collected micrometres unless otherwise stated, as the range 123 Author's personal copy

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Table 1 Species of Pomphorhynchus collected in this study, including collection sites (CS), localities, hosts species, geographical coordinates and GenBank accession numbers. The collection site number (CS) for each locality corresponds to the number in Fig. 1 CS Locality Host Coordinates GenBank ID Species NW cox 1 LSU

Canada Manitoba 1 Red River Catostomus commersonii 49°0500600 100°0300500 KY911296–97 P. bulbocolli (Lace´pe`de) 2 Delta Marsh Ictiobus cyprinellus 50°1105500 98°120700 KY911298–300 P. bulbocolli (Valenciennes) USA Wisconsin 3 Lake Lepomis gibbosus 44°4803600 88°3004700 KY911301 KY911338 P. bulbocolli Shawano (Linnaeus) Lepomis macrochirus KY911302–310 KY911339–346 P. bulbocolli Rafinesque Ambloplites rupestris KY911311–313 KY911347 P. bulbocolli (Rafinesque) Carpioides cyprinus KY911314 P. bulbocolli (Lesueur) 4 Lake Ictiobus cyprinellus 44°0101200 88°2501300 KY911315 KY911348 P. bulbocolli Winnebago (Valenciennes) 5 Nace Creek Ambloplites rupestris 44°5201800 89°2106300 KY911316 P. bulbocolli (Rafinesque) Luxilus cornutus KY911317–319 KY911349; P. bulbocolli (Mitchill) KY911350 6 Duck Creek Semotilus atromaculatus 44°3003500 88°1803600 KY911320–21; KY911351 P. bulbocolli (Mitchill); Luxilus KY911322 cornutus (Mitchill) 7 Wolf River Moxostoma erythrurum 44°1404300 88°5103600 KY911323 P. bulbocolli (Rafinesque) Michigan 8 Gull Lake Lepomis macrochirus 42°3808500 85°3903800 KY911294; P. bulbocolli Rafinesque KY911295; DQ089709 Mexico Durango 9 Barranca Catastomus nebuliferus 23°54027.600 12°330300 KY911293 KY911335 P. bulbocolli Garman Michoacań 10 La Luz Moxostoma austrinum 19°5600800 102°1800000 KY911281–286 KY911324–329 P. Bean purhepechus n. sp. 11 Pateo Moxostoma austrinum 19°5401700 100°1806.2900 KY911287–292 KY911330–334 P. Bean purhepechus n. sp.

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130°0'0"W 120°0'0"W 110°0'0"W 100°0'0"W 90°0'0"W 80°0'0"W 70°0'0"W 60°0'0"W

2 50°0'0"N 1 50°0'0"N

5 3 6 7 4 8 40°0'0"N 40°0'0"N

30°0'0"N 30°0'0"N

11 20°0'0"N 20°0'0"N 10*

130°0'0"W 120°0'0"W 110°0'0"W 100°0'0"W 90°0'0"W 80°0'0"W 70°0'0"W 60°0'0"W

Fig. 1 Map showing the sampling sites of the fish hosts across North America. The type-locality of Pomphorhynchus purhepechus n. sp. is indicated by an asterisk. Legend for localities: 1, Red River, Manitoba, Canada; 2, Delta Marsh, Manitoba, Canada; 3, Shawano Lake Wisconsin, USA; 4, Winnebago Lake Wisconsin, USA; 5, Nace Creek Wisconsin, USA; 6, Duck Creek Wisconsin, USA; 7, Wolf River, Wisconsin, USA; 8, Gull Lake, Michigan, USA; 9, Barranca, Durango, Mexico; 10, La luz, Michoacań, Mexico; 11, Pateo, Michoacań, Mexico

followed by the mean in parentheses. Measurements with a Hitachi Stereoscan Model SU1510 SEM and drawings of eggs were made from mature (Hitachi Ltd., Tokyo, Japan). specimens. Specimens were illustrated with the aid of a drawing tube attached to an Olympus BX-51 DNA extraction, amplification and sequencing compound microscope. Whole bodies of 43 acanthocephalans were individ- For scanning electron microscopy (SEM), two ually digested overnight at 56°C in a solution specimens of P. bulbocolli from the bluegill Lepomis containing 10 mM Tris-HCl (pH 7.6), 20 mM NaCl, macrochirus Rafinesque from Shawano Lake, Wis- 100 mM Na2 EDTA (pH 8Á0), 1% Sarkosyl and 10 mg/ consin, USA, one specimen each from the catostomids ml of proteinase K. Following digestion, DNA was Ictiobus cyprinellus (Valenciennes) from the Red extracted from the supernatant using the DNAzol River, Delta Marsh, Manitoba and Carpiodes cyprinus reagent (Molecular Research Center, Cincinnati, (Lesueur) from Lake Winnebago, Wisconsin, USA, Ohio, USA) according to the manufacturer’s instruc- and two specimens of the new species from La Luz, tions. A fragment of 655 bp of the cytochrome Michoacan, were dehydrated through a graded ethanol c oxidase subunit 1 (cox1) gene was amplified using series, critical-point dried with carbon dioxide. Spec- the polymerase chain reaction (PCR) with the forward imens were mounted on metal stubs with carbon primer 507 50-AGT TCT AAT CAT AA(R) GAT adhesive tabs, gold coated, and examined at 10 kV AT(Y) GG-30 and reverse primer 588 50-TAA ACT

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TCA GGG TGA CCA AAA AAT CA-30 (Folmer models inferred with the jModelTest program v0.1.1 et al., 1994). The domains D2-D3 of the large subunit (Posada, 2008) were GRT ? G for cox1 and HKY ? I (LSU) of the ribosomal DNA (rDNA) were amplified for LSU datasets. ML tree searches were performed using the forward primer 502 50-CAA GTA CCG TGA using 100 random taxon addition heuristic searches GGG AAA GTT GC-30 and the reverse primer 536 50- with branch and bound searches. Clade support was CAG CTA TCC TGA GGG AAA C-30 (Garcıá-Varela assessed by bootstrap resampling with 1,000 bootstrap & Nadler, 2005). PCR reactions (25 ll) consisted of 10 replicates using PAUP (Swofford, 2002). The best-fit lM of each primer, 2.5 llof109 PCR Rxn buffer, 1.5 model for each dataset was used for the Bayesian ll of 2 mM MgCl2, and 1 U of Taq DNA polymerase inference analyses (Ronquist et al., 2012). Settings (Platinum Taq, Invitrogen Corporation, Saõ Paulo, were two simultaneous runs of the Markov chain Brazil). PCR cycling parameters for rDNA amplifica- (MCMC) for 10 million generations, sampling every tions were as follows: denaturation at 94°C for 1 min, 1,000 generations, a heating parameter value of 0.2 followed by 35 cycles at 94°C for 1 min, annealing at and a ‘burn-in’ of 25%. Trees were drawn using 40 or 50°C for 1 min (optimised for each amplifica- FigTree version 1.3.1 (http://tree.bio.ed.ac.uk/ tion), extension at 72°C for 1 min, followed by a post- software/figtree/). The genetic divergence among amplification incubation at 72°C for 10 min. Sequenc- taxa was estimated using uncorrected p-distances with ing reactions were performed using ABI Big Dye the program MEGA version 6 (Tamura et al., 2011). (Applied Biosystems, Boston, Massachusetts, USA) terminator sequencing chemistry. The reactions were Family Pomphorhynchidae Yamaguti, 1939 read using an ABI 3730 capillary DNA sequencer. Genus Pomphorhynchus Monticelli, 1905 Contiguous sequences were assembled and base- calling differences resolved using Codoncode Aligner Pomphorhynchus purhepechus n. sp. version 5.0.2 (Codoncode Corporation, Dedham, Type-host: Moxostoma austrinum Bean (Osteichthyes: Massachusetts). Sequences obtained were deposited Catostomidae). in the GenBank database under the accession numbers Type-locality: La Luz (19°5600800N, 102°1800000W), KY911281–KY911335 (see Table 1 for details). Michoacań, Mexico. Other locality: Pateo (19°540 1700N, 100°1806.2900W) Alignments and phylogenetic analyses Michoacań, Mexico. LSU rDNA and cox1 sequences were aligned inde- Type-material: Holotype (CNHE 10059), allotype pendently. For cox1, the alignment included (CNHE 10060) and 14 paratypes (CNHE 10061, sequences of two other congeneric species, i.e. three 10062). individuals of Pomphorhynchus laevis (Zoega in Site in host: Intestine. Mu¨ller 1776), and a single Pomphorhynchus tereti- Prevalence: La Luz Michoacań: 100% (10 out of 10 collis (Rudolphi, 1809). The following taxa, members fish); Pateo Michoacań: 75% (6 out of 8 fish). of Illiosentidae, were used as the outgroup: Illiosentis Representative DNA sequences: See Table 1. sp. (GenBank: LSU AY829092; cox1 DQ089705); ZooBank registration: To comply with the regulations Koronacantha mexicana Monks & Pe´rez Ponce de set out in article 8.5 of the amended 2012 version of Leoń, 1996 (GenBank: LSU AY829095; cox1 the International Code of Zoological Nomenclature DQ089708), Koronacantha pectinaria (Van Cleave, (ICZN, 2012), details of the new species have been 1940) (GenBank: LSU AY829094; cox1 DQ092433) submitted to ZooBank. The Life Science Identifier and Pseudoleptorhynchoides lamothei Salgado-Mal- (LSID) for Pomphorhynchus purhepechus n. sp. is donado, 1976 (GenBank: LSU EU090951; cox1 urn:lsid:zoobank.org:act:EAC33876-7ADD-42F3- EU090949). Sequences were aligned with the soft- 852F-0DEC47588C9E. ware Clustal W (Thompson et al., 1994) with default Etymology: The species is named after the Purhe´pe- parameter settings and adjusted manually with the cha, an indigenous ethnic group whose main settle- Mesquite program (Maddison & Maddison, 2008). ment was in the state of Michoacań, where both The final alignments consisted of 52 sequences with localities are located. 509 nucleotides (cox1), and 32 sequences with 688 nucleotides (LSU). The best-fit nucleotide substitution 123 Author's personal copy

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Description (Figs. 2, 3) Length of hooks varying by position: first (anteriormost) 17–27 (20); second 17–35 (25); third 17–41 (29); General [Based on 16 specimens, 5 males and 11 fourth 20–54 (38); fifth 28–40 (29); sixth 24–26 (23); females.] Live individuals white. Body wall thick. seventh 21–28 (23); eighth 18–26 (23); ninth 18–24 Trunk spineless, elongate, cylindrical, with swollen (20); tenth 15–29 (22); eleventh 15–27 (21); twelfth anterior region (Fig. 2A). Proboscis comprised of 14–28 (20); thirteenth 14–27 (20); fourteenth 14–23 anterior bulbous half and posterior narrow region, (21); fifteenth 14–23 (19); sixteenth 14–29 (23); armed with 14 longitudinal rows, with 16–18 hooks seventeenth 14–31 (25); eighteenth 31–43 (38). Pro- per row (Fig. 3A–C). Viewed en face, hooks arranged boscis receptacle 2.75–3.62 (3.16) mm long. Lemnisci in offset circles with 7 hooks per circle, except basal 1.21–2.14 (1.53) mm long (Fig. 2F–H). Eggs fusiform, circle that has 14 hooks. Hooks with simple roots. 51–65 9 11–13 (58 9 11), with polar prolongation. Anterior hooks (first and second) short, third or fourth hooks alternately larger. Basal hooks largest. Cerebral Remarks ganglion oval, at base of proboscis receptacle. Bulb well-developed, spherical. Lemnisci claviform, sube- Pomphorhynchus purhepechus n. sp. can be distin- qual, extending beyond proboscis receptacle (Fig. 2A, guished from four of the five species of Pomphorhynchus B), gonopore subterminal. described from freshwater fishes from South America, i.e. P. yamagutii, P. patagonicus, P. moyanoi and P. Male [Based on five specimens.] Trunk 4.8–10.4 9 omarsegundoi by having a symmetrical bulb (see Olmos 0.58–1.20 (7.6 9 0.85) mm. Neck and proboscis & Habit, 2007; Arredondo & Gil de Pertierra, 2010). The 2.7–4.5 (3.28) mm in length. Proboscis 502–691 9 new species differs from P. sphaericus, another South 201– 303 (567 9 270) (widest point in anterior half), American species, by having 14 longitudinal rows with 158–311 (256) wide at base; subspherical bulb 16–18 hooks per row as opposed to 12 longitudinal rows 417–1,209 9 491–1,652 (794 9 867). Proboscis with with 14–16 hooks per row in P. sphaericus (see Gil de 14 longitudinal rows of 16 hooks. Length of hooks Pertierra et al., 1996). varying by position: first (anteriormost) 18–26 (22); The new species closely resembles the three North second 20–27 (23); third 34–43 (38); fourth 21–41 American species of Pomphorhynchus, i.e. P. bulbo- (31); fifth 16–21 (18); sixth 15–25 (20); seventh 15–24 colli, P. rocci, and P. lucyi. All three species possesses (19); eighth 14–24 (20); ninth 19–21 (20); tenth 18–20 a symmetrical bulb. However, P. purhepechus n. sp. (19); eleventh 19–21 (20); twelfth 20–21 (20); can be readily distinguished by the number of thirteenth 18–20 (20); fourteenth 18–20 (20); fifteenth longitudinal rows and the number of hooks per row 20–24 (21); sixteenth 27–30 (29) (Fig. 2D, E). and circle in the proboscis. The new species possesses Proboscis receptacle 1.2–3.1 (2.08) mm long. Lem- 14 longitudinal rows with 16–18 hooks per row nisci 1.2–2.14 (1.81) mm long (Fig. 2B). Testes ovoid, (Fig. 3A, B) with 7 hooks per circle in all but the symmetrical, posterior to proboscis receptacle at basal circle. In contrast, P. bulbocolli possesses 12 middle of trunk (Fig. 2C); anterior testis 558–1,370 longitudinal rows with 10–19 hooks with 6 hooks per 9 324–654 (1,010 9 481); posterior testis 455–1,440 circle in all but the basal circle (see figure 5A–G in 9 321–695 (934 9 564). Cement glands 6, pyriform, Van Cleave & Mueller, 1934; Huffman & Nickol, 519–941 9 140– 440 (639 9 313). Sa¨fftigen’s pouch 1978), P. rocci possesses 12 longitudinal rows with 438–459 (445). Copulatory bursa 204–491 9 327–631 15–18 hooks per row, and P. lucyi possesses 12 (347 9 479) (Fig. 2C). longitudinal rows with 20–23 hooks (Table 2). The male of P. purhepechus n. sp. can be easily distin- Female [Based on six gravid and five mature speci- guished from other three (P. bulbocolli, P. rocci, and mens.] Trunk 4.4–10.3 9 0.53–1.54 (6.5 9 0.97) mm P. lucyi) congeneric species from North America by (widest point). Neck and proboscis 2.26–5.56 (4.08) having larger cement glands. The female of the new mm in length. Proboscis 455–630 9 228– 327 (540 9 species possess smaller eggs in comparison to the 283), 170–344 (244) wide at base; subspherical bulb other three species as P. bulbocolli, P. rocci, and P. 576–1,280 9 507–1,470 (751 9 815). Proboscis with lucyi (Table 2). 14 longitudinal rows of hooks, with 18 hooks per row. 123 Author's personal copy

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A B CD

E F H G

Fig. 2 Pomphorhynchus purhepechus n. sp. ex Moxostoma austrinum. A, Adult male, total view (holotype); B, Anterior region; C, Posterior region; D, Proboscis; E, Row of hooks (holotype), lateral view; F, Adult female, (allotype), ventral view; G, Female reproductive system (allotype), ventral view; H, Egg. Scale-bars: a, b, c, f, 1.0 mm; d, e, g, 250 lm; h, 50 lm

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Fig. 3 Scanning electron micrographs of proboscis (A, B) and bulb (C) of adult male Pomphorhynchus purhepechus n. sp. ex Moxostoma austrinum. Scale-bars: A, C, 100 lm; B, 50 lm Pomphorhynchus bulbocolli Linkins in Van Cleave, Hooks with simple roots. First and second hooks on 1919 each longitudinal row smaller than third and fourth hook that are alternately larger in anterior part. Basal Hosts: Catostomus commersonii (Lace´pe`de), Ictiobus hooks largest. Cerebral ganglion oval, located at base cyprinellus (Valenciennes), Carpiodes cyprinus (Le- of receptacle. Bulb spherical. Lemnisci claviform, sueur), Catostomus nebuliferus Garman, Moxostoma slightly subequal, extending beyond proboscis recep- erythrurum (Rafinesque), Luxilus cornutus (Mitchill), tacle (Fig. 4A, B), gonopore terminal. Semotilus atromaculatus (Mitchill), Ambloplites rupestris (Rafinesque), Lepomis gibbosus (Linnaeus), Male [Based on four specimens.] Trunk 4.55–6.46 9 Lepomis macrochirus Rafinesque. 0.81–1.03 (5.3 9 0.97) mm. Neck and proboscis Localities: Red River, Delta Marsh, Manitoba, Canada; 3.2–3.6 (3.36) mm in length. Proboscis 610–657 9 Shawano Lake, Winnebago Lake, Nace Creek, Duck 248–291 (629 9 275) (at widest point), 198–257 (221) Creek, Wolf River, Wisconsin, USA; Gull Lake, wide at base. Subspherical bulb 1.38–1.86 9 Michigan, USA; Barranca, Durango, Mexico. 1.30–1.99 (1.65 9 1.63) mm. Proboscis with 12 Voucher material: Six specimens (CNHE 10063). longitudinal rows of hooks, each with 14 hooks (5A– Site in host: Intestine. G). Length of hooks varying by position: first (anteriormost) 30–35 (32); second 35–44 (38); third 38–51 Description (Figs. 4, 5) (45); fourth 40–45 (42); fifth 35–49 (39); sixth 34–42 (37); seventh 27–46 (38); eighth 28–44 (35); ninth General [Based on 6 specimens, 4 male and 2 female.] 29–42 (32); tenth 28–38 (30); eleventh 25–35 (27); Pomphorhynchidae Yamaguti, 1939, with character- twelfth 27–35 (30); thirteenth 26–33 (28); fourteenth, istics of genus. Trunk cylindrical with swollen anterior 41–46 (42). Proboscis receptacle 2.8–3.7 (3.21) mm. region (Fig. 4A, B). Proboscis cylindrical, armed with Lemnisci 315–1,270 (793) (Fig. 4A). Testes ovoid, 12 longitudinal rows of 14 hooks per row (Fig. 5A–G). symmetrical, in posterior half of body (Fig. 4A). Viewed en face, hooks arranged in offset circles with 6 Anterior testis 436–680 9 366–458 (567 9 423). hooks per circle, except basal circle that has 12 hooks. Posterior testis 448–701 9 351–439 (570 9 399).

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Table 2 Comparative metrical data for males and females of species of Pomphorhynchus from North America. Measurements in micrometres, unless otherwise indicated Species P. purhepechus P. rocci P. lucyi Williams P. bulbocolli Linkins in Van Cleave, 1919 n. sp. Cordonnier & Rogers, 1984 & Ward, 1967 Reference This study Cordonnier Williams Van Cleave Samuel et al. This study & Ward (1967) & Rogers (1984) (1919) (1976)

General No. of longitudinal rows 14 12 12 12 12 12 of hooks No. of hooks per row 16–18 15–18 20–23 12–14 12–18 (15) 14 Male Trunk length (mm) 4.8–10.4 (7.6) 12–17 (13) 5.0–6.6 (5.6) – 8.0–16.0 4.55–6.46 (11.0) (5.3) Trunk width (mm) 0.5–1.20 (0.8) 1.0 0.8–1.2 (1.0) – 0.8–1.2 0.8–1.03 (0.9) Neck and proboscis 2.7–4.5 (3.28) 5.0 4.6–7.2 (5.9) 4.0–6.0 2.3–4.4 (3.6) 3.2–3.6 (3.3) length (mm) Proboscis length 502–691 (567) 510–930 (800) 1,200–1,500 500–600 500–920 (650) 610–657 (1,400) (629) Proboscis width at 201–303 (270) 180–350 (260) 440–480 (464) 500–600 70–380 (260) 248–291 widest point (275) Proboscis width at base 158–311 (256) 130–210 (150) 364–400 (368) – – 198–257 (221) Proboscis receptacle 1.2–3.1 (2.08) 5.0 5.0–6.3 (5.6) – 3.1–5.1 (4.3) 2.8–3.7 length (mm) (3.21) Leminiscus length (mm) 1.2–2.1 (1.8) 0.91–1.56 0.8–1.3 – 0.58–2.25 0.31–1.27 (1.40) (0.79) Anterior testis length 0.55–1.3 (1.01) 0.83–1.56 0.41–0.71 (0.56) – 0.50–1.20 0.43–0.68 (mm) (0.88) (0.56) Anterior testis width 324–654 (481) 390–730 238–324 (274) – 290–470 (370) 366–458 (423) Posterior testis length 0.45–1.45 0.98–1.63 0.49–0.80 (0.60) – 0.54–1.20 0.44–0.70 (mm) (0.93) (0.98) (0.57) Posterior testis width 321–695 (564) 410–650 244–316 (284) – 260–500 (430) 351–439 (406) Cement gland length 519–941 (639) 570–700 138–182 – – 207–331 (259) Sa¨fftigen’s pouch 438-459 (445) – – – – 349–467 (406) Female Trunk length (mm) 4.4–10.3 (6.5) 11.0–19.0 (13) 5.4–6.3 (5.7) – 9.0–20.0 5.02–6.60 (13.0) (5.81) Trunk width (mm) 0.53–1.54 1.0–1.56 (1.3) 0.9–1.1 (1.0) – 1.0–1.7 (1.4) 0.94–1.26 (0.97) (1.10) Proboscis length 455–630 (540) – 1100–1300 – 498–718 (1200) (608) Proboscis width at 228–327 (283) – 324–356 (344) – – 266–342 (304) widest point Proboscis width at base 170–344 (244) – 256–324 (280) – – 177–263 (220)

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Neck and proboscis 2.26–5.56 (4.08) – 4.4–6.6 (5.7) – – 3.49–3.58 (3.53) length (mm) Proboscis receptacle 2.75–3.62 (3.16) – – – – 3.22–3.40 (3.31) length (mm) Leminiscus length mm 1.21–2.14 (1.53) – – – – 1.39–1.82 (1.60) Egg length 51–65 (58) 65–83 (78) 66–78 53–83 (63) 53–83 (66) 64–75 (66) Egg width 11–13 (11) 10–11 (11) 12–15 58–13 (10) 8–13 (10) 10–15 (12)

ninth 27–34 (30); tenth 29–32 (30); eleventh 26–27 A B (26); twelfth 27–30 (28); thirteenth 30–34 (32); fourteenth 30–34 (32). Proboscis 177–263 (220) wide at base. Subspherical bulb 1.52–1. 81 9 1.49–2.15 (1.66 9 1.82) mm. Proboscis receptacle 3.22–3.40 (3.31) mm. Neck and proboscis 3.49–3.58 (3.53) mm. Lemnisci 1.39–1.82 (1.60) mm. Eggs fusiform, 64–75 9 10–15 (66 9 12), with polar prolongation (Fig. 4B).

Remarks

Based on morphology and morphometrics, the specimens found in catostomids, centrarchids and cyprinids across Canada, USA, and northern Mexico were identified as P. bulbocolli. Our specimens conform to the original description (see Van Cleave, 1919)in several key features: a cylindrical trunk with a wider Fig. 4 Pomphorhynchus bulbocolli ex Lepomis macrochirus in anterior region, a cylindrical proboscis with 12 Shawano Lake (Locality 3 see Fig. 1 and Table 1). A, Adult male, whole worm, voucher (CNHE 10063), lateral view; B, longitudinal rows with 14 hooks per row (Fig. 5A– Adult female, whole worm, voucher (CNHE 10063), lateral G), with subspherical and symmetrical bulb in the view. Scale-bars: 1.0 mm anterior region, and a long neck (see Table 2). Males possess six pyriform cement glands and females Cement glands 6, pyriform, 207–331 9 118–166 (259 possess fusiform eggs with polar prolongations. 9 141). Sa¨fftigen’s pouch 349–467 (406) (Fig. 4A). However, the newly collected specimens provide new morphometric information not mentioned in the Female [Based on two gravid specimens]. Trunk original description (Van Cleave, 1919) such as the 5.02–6.60 9 0.95–1.26 (5.81 9 1.10) mm. Proboscis width of the proboscis at its base, length of the 498–718 9 266–342 (608 9 304) (at widest point). proboscis receptacle and lemnisci, testis length and Proboscis hooks with 12 longitudinal rows, each with width and length of the cement glands and Sa¨fftigen’s 14 hooks. Length of hooks varying by position: first pouch (see Table 2) (Fig. 4A, B). (anteriormost) 35–44 (39); second 36–53 (44); third 42–67 (54); fourth 48–57 (52); fifth 40–48 (44); sixth 34–50 (42); seventh 30–39 (34); eighth 31–38 (34);

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Fig. 5 Scanning electron micrographs of proboscis and bulb of adult male Pomphorhynchus bulbocolli. A–C, Specimen ex bluegill Lepomis macrochirus Raﬁnesque in Shawano Lake, Wisconsin, USA; D–E, Specimen ex catostomid Ictiobus cyprinellus in the Red River, Delta Marsh, Manitoba; F–G, Specimen ex Carpiodes cyprinus in Lake Winnebago, Wisconsin, USA. Scale-bars:A,D,F,100 lm; C, 500 lm; B, E, G, 50 lm

Genetic divergence and phylogenetic analyses characters, and yielded a tree with a -ln likelihood of 3273.86. The topology of the ML tree was identical to DNA sequences of the cox1 and LSU rDNA were the Bayesian inference (BI) consensus tree. Both trees ampliﬁed for individuals representing two species of showed four major clades corresponding to four the genus Pomphorhynchus, P. purhepechus n. sp. and species of Pomphorhynchus. Pomphorhynchus P. bulbocolli. PCR amplicons were 509 nt long for purhepechus n. sp. was the sister species of P. cox1, and 648–653 nt for the LSU rDNA fragments. bulbocolli (represented by specimens from Canada, The genetic divergence of the cox1 sequences between USA and northern Mexico), whereas P. laevis and P. P. purhepechus n. sp. and P. bulbocolli ranged tereticollis are two independent species that in the tree between 13–14%, whereas the intraspeciﬁc genetic received moderate bootstrap and Bayesian posterior divergence among specimens of the new species probability support values (Fig. 6), with a basal ranged between 0–5%, and among specimens of P. polytomy. The LSU rDNA dataset included 32 bulbocolli from Canada, USA and Mexico, between terminals, with 688 characters. Maximum likelihood 0–4%. The genetic divergence between P. purhep- analysis yielded a tree with a -ln likelihood of echus n. sp. and the other two congeneric species for 2524.3422. This tree was identical with the Bayesian which sequences of the cox1 mitochondrial gene are inference tree. Both trees showed two clades with available in the GenBank, i.e. P. laevis and P. short branches, one containing specimens of P. tereticollis from Europe, varied between 23–26%. In bulbocolli from Canada, USA and northern Mexico contrast, the genetic divergence estimated with the and the second containing specimens of P. purhep- LSU dataset was very low, ranging from 0.6 to 0.8% (7 echus n. sp. (Fig. 7). nt) between P. purhepechus n. sp. and P. bulbocolli. No sequences for LSU rDNA of other species of Pomphorhynchus are available for comparison. The cox1 dataset used for the Maximum Likelihood (ML) analysis included 52 terminals, with 509 aligned

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KY911281 78 / 0.9 KY911282 KY911283 KY911284 Centrarchidae KY911285 73/ 1 KY911286 Pomphorhynchus purhepechus n. sp. KY911287 KY911288 KY911289 KY911290 KY911291 Catostomidae KY911292

DQ089709 * KY911294 * KY911295 * KY911301 * KY911305 * KY911306 * Cyprinidae KY911308 * KY911311 * KY911313 * 66 / 0.9 KY911303 * KY911307 * KY911310 * KY911312 * KY911296 (CA) Pomphorhynchus bulbocolli KY911300 (CA) KY911302 * KY911304 * KY911309 * KY911317 * KY911319 * 100 / 1 KY911320 * KY911316 * KY911322 * KY911315 * KY911298 (CA) KY911318 * 100 / 1 KY911321 * KY911314 * KY911297 (CA) KY911323 * KY911299 (CA) KY911293 (Mex) AY423353 Pomphorhynchus tereticollis AY423350 75/ 0.9 AY423349 Pomphorhynchus laevis AY423348 Pseudoleptorhynchoides lamothei EU090949 Koronacantha mexicana DQ089708 Koronacantha pectinaria DQ089707 Illiosentis sp. DQ089705

0.4

Fig. 6 Maximum likelihood (ML) tree inferred from the cox1 dataset. Numbers near internal nodes show ML bootstrap clade frequencies and posterior probabilities (BI) as ML/BI. Accession numbers with an asterisk are for sequences based on material from the USA. Abbreviations: CA, Canada; Mex, Mexico

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Discussion relationship between the new species and P. bulbocolli. LSU rDNA has proved to be useful for discrim- Pomphorhynchus purhepechus n. sp. represents the inating among species of other acanthocephalans such eighth species of the genus described from the as Neoechinorhynchus and Floridosentis Ward, 1953 Americas and the fourth in North America. Thus far, (see Rosas-Valdez et al., 2012; Pinacho-Pinacho et al., only few species of Pomphorhynchus have been 2015). Vardic´ Smrzlic´ et al. (2015), found that other characterised using molecular methods. The only relatively conserved regions such as the ITS and 18S species in the Americas that has been characterised rDNA are not useful in studying genetic variability molecularly (for ITS) is P. lucyi (see Kra´l’ova´- among pomphorhynchids such as P. laevis.Itis Hromadova´ et al., 2003). In addition, genetic diver- possible that in this group of acanthocephalans only gence has been studied for congeners in Europe, i.e. P. mitochondrial genes or other more rapidly evolving laevis and P. tereticollis, using nuclear and/or mito- markers will allow interspecific differences to be chondrial genes (e.g. Perrot-Minot, 2004; O’Mahony recognised. et al., 2004;Sˇpakulova´ et al., 2011; Vardic´ Smrzlic´ Pomphorhynchus bulbocolli is a widely distributed et al., 2015). In our study, the phylogenetic trees species across the Nearctic region, and has been inferred with the mitochondrial cox1 gene, showed reported in freshwater fishes of at least six, often that the four species of Pomphorhynchus in the disparate, families of fishes, i.e. Acipenseridae, Ami- analysis, two from North America (P. purhepechus idae, Catostomidae, Centrarchidae, Cyprinidae and n. sp. and P. bulbocolli) and two from Europe (P. Ictaluridae (see Samuel et al., 1976; Choudhury & laevis and P. tereticollis) represent independent Dick, 1998; Hoffman, 1999; Garcıá-Prieto et al., 2010; lineages, supported by moderate bootstrap (ML) and Aguilar-Aguilar et al., 2014), although the species posterior probability (BI) values. The genetic diver- seems to prefer suckers (Catostomidae) (see Margolis gence between the new species and P. bulbocolli & Arthur, 1979; McDonald & Margolis, 1995; Hoff- (13–14%) is similar to the divergence observed among man, 1999 and references therein; Muzzall & Whelan, other species of acanthocephalans for the same 2011). Pomphorhynchus bulbocolli was first reported molecular marker, e.g. 20% between P. laevis and P. in Mexico from the marine fish Diapterus auratus tereticollis (see Perrot-Minot, 2004). In other groups Ranzani, obtained from a fish market in Mexico City, of acanthocephalans, such genetic variation is indica- and apparently collected in Campeche, on the Gulf of tive of distinct species. The genetic divergence ranged Mexico coast (Salgado-Maldonado, 1978), but the between 15–18% among three closely related species record is doubtful. We examined these specimens, of the polymorphid Pseudocorynosoma Aznar, Pe´rez- deposited at the CNHE (accession number 680); most Ponce de Leoń & Raga, 2006 (Garcıá-Varela et al., of them are immature and, as expected from speci- 2017), and between 20.3–24.9% among three species mens not collected fresh, in very poor condition. In our of Neoechinorhynchus Stiles & Hassall, 1905 (see opinion, these specimens cannot be assigned to any Pinacho-Pinacho et al., 2012). In contrast, the described species of Pomphorhynchus, and certainly intraspecific genetic divergence among isolates of do not correspond to P. bulbocolli, which is a both, P. purhepechus n. sp. and P. bulbocolli, was freshwater species, whereas the host in this case is comparatively very low (range 0–5%). Specimens of marine (Salgado-Maldonado, 1978). Similarly, spec- P. laevis from Salmo trutta Linnaeus from Ireland, imens identified as Pomphorhynchus cf. bulbocolli England and Scotland showed a mean sequence from Zoogoneticus purhepechus Domıńguez-Domıń- divergence of 0.7% and a mean of sequence diver- guez, Pe´rez-Rodrı´guez & Doadrio in central Mexico gence of 0.35% compared with samples of P. laevis (see Martıńez-Aquino et al., 2011) (CNHE 7795), are from Leuciscus cephalus (Linnaeus) and Cottus gobio immature and poorly preserved and it is not possible to Linnaeus (O’Mahony et al., 2004). Vardic´ Smrzlic´ assign them to any species of Pomphorhynchus either. et al. (2015) reported differences of 2.3–3.3% in Therefore the only two valid records of P. bulbocolli partial cox1 sequences of P. laevis in the River Sava in Mexico are specimens collected from the Nazas basin in Croatia. sucker Catostomus nebuliferus Garman and the Nazas The phylogenetic trees inferred with the LSU data chub Gila conspersa Garman (Pe´rez-Ponce de Leoń set yielded trees with very low resolution for the et al., 2009) in the Nazas River basin, Durango State, 123 Author's personal copy

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Illiosentis sp. AY829092

Koronacantha mexicana AY829095 100/ 1 Koronacantha pectinaria AY829094

Centrarchidae

KY911325 Catostomidae KY911326

KY911328

KY911327

KY911324 55/ 1 KY911329 Pomphorhynchus purhepechus n. sp. Cyprinidae KY911333

KY911330

KY911332

KY911331

KY911334

KY911340 *

KY911345 *

KY911347 * 100/ 0.6 KY911336 *

KY911349 *

KY911339 *

KY911338 *

KY911342 * Pomphorhynchus bulbocolli KY911344 *

KY911341* KY911337 *

KY911348

KY911346 *

0.2 KY911350 * AY829096 *

KY911343 *

KY911351*

KY911335 (Mex)

Fig. 7 Maximum likelihood tree inferred from the LSU dataset. Numbers near internal nodes show ML bootstrap clade frequencies and posterior probabilities (BI). Accession numbers with an asterisk are from the USA. Abbreviation: Mex, Mexico

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Syst Parasitol (2017) 94:989–1006 1003 and from the red shiner Cyprinella lutrensis (Baird & facilitated the speciation of P. purhepechus n. sp. The Girard), in Cuatro Cienegas, Coahuila State (Aguilar- TMVB is an area where Neotropical and Nearctic Aguilar et al., 2014). The identification of specimens biotas overlap and represents a zone of biotic from Durango was corroborated with sequences of the exchange that has favored the diversification of cox1 and LSU rRNA genes in the current study but no species and endemism in different taxa (see Morrone, specimens from Cuatro Cieńegas were preserved for 2006), including helminths (see Lira-Guerrero et al., molecular work. The phylogenetic trees inferred with 2008; Martıńez-Aquino et al., 2014). This study each gene placed specimens of P. bulbocolli from provides additional empirical evidence of speciation northern Mexico in a clade also containing specimens events across this biogeographically rich and diverse of P. bulbocolli from Canada and USA, with a very region, and further illustrates the usefulness of an low genetic divergence values among individuals. integrative taxonomy approach in the recognition of Therefore, we confirmed that the specimens of P. independent evolutionary lineages, species complexes bulbocolli (CNHE 6707) from northern Mexico (from or even the description of new species of parasites. Durango) actually represents the first record of the Such approach may considerably increase the recog- species in Mexico (Pe´rez-Ponce de Leoń et al., 2009). nised biodiversity of this transitional zone. Pomphorhynchus purhepechus n. sp. is only found Biogeographically and in terms of host association, in the catostomid M. austrinum, apparently the only the amphi-American genus Pomphorhynchus (see species of catostomid that inhabits the Lerma-Santi- Choudhury et al., 2017) seems to form two separate ago river system (Miller et al., 2005;Pe´rez-Rodrı´guez assemblages in the Americas. The South American et al., 2016). This fish species appears to be closely species occur in the austral portion of South America, related to an undescribed species of Moxostoma with two of them in Chile (P. yamagutii and P. (M. cf. austrinum Bean) from the River Santiago, moyanoi) and three in Argentina (P. patagonicus, P. and both are sister taxa to M. milleri Robins & Raney, sphaericus and P. omarsegundoi). No species of a species found in the River Mezquital located further Pomphorhynchus has been found in central and north, forming a clade with species occurring across northern areas of South America and the freshwater the Pacific slope drainages (Pe´rez-Rodrı´guez et al., acanthocephalan fauna of Middle-America is notably 2016). Other morphotypes of M. cf. austrinum occur in species poor (Pe´rez-Ponce de Leoń & Choudhury, river basins of northern Mexico, but they seem to 2005; Salgado-Maldonado, 2008; Choudhury et al., represent independent species, and all these species 2017), indicating the amphi-temperate nature of this are nested within a clade of seven other Moxostoma group of parasites. In fact, while the species in North spp. from the southwestern, southern and central America, including the new species described in this United States (Doosey et al., 2010). Interestingly, P. study, are parasites of typically Nearctic freshwater bulbocolli has been reported from some of these fishes such as catostomids, cyprinids, centrarchids and closely related species (see Hoffman, 1999) but a few others, no species of Pomphorhynchus is found isolates from these hosts were not available for this in the endemic freshwater fish fauna of Mexico such as study. The wide geographical distribution of P. goodeids and atherinomorphs. As mentioned before, bulbocolli attests to its high dispersal ability across a the record of P. cf. bulbocolli in an endemic goodeid in large area of North America. Thus, we may speculate central Mexico (Martıńez-Aquino et al., 2011) repre- that the ancestor of P. purhepechus n. sp. arguably sents an accidental infection; the acanthocephalans colonised central Mexico, and in particular the Lerma- were immature and were collected in La Luz, which is Santiago drainage, along with the ancestral popula- the same spring where we collected the new species tions of their hosts as a part of this wide dispersal route described in this study. (Harris et al., 2002;Pe´rez Rodrı´guez et al., 2016), and The absence of Pomphorhynchus in other, Neotrop- both, host and parasite evolved as separate species in ical, components of the Middle-American fish fauna is isolation. Although the parasite fauna of M. austrinum also remarkable. No species is found in cichlids, across its full geographical range is not known, we characids and poeciliids. In South America, species of may hypothesize that drainage isolation, as a result of Pomphorhynchus are mostly parasites of austral fish tectonic and volcanic activity in central Mexico since species. For instance, P. yamagutii was recorded as a the early Miocene (Ferrari et al., 2000), may have parasite of the percichthyid Percichthys melanops 123 Author's personal copy

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Girard, which is distributed in pre-andean streams of Tracey and Shane Hoffmann, St. Norbert College. AC also central Chile (Schmidt & Hugghins, 1973); P. patag- thanks Miki Oros, Slovak Academy of Sciences, Kosice, Slovakia, for help with necropsy work, and Kevin Campbell, onicus parasitises the patagonic atherinopsid Odon- University of Manitoba, for use of laboratory facilities. testhes hatcheri (Eigenmann), the galaxid Galaxias platei Steindachner, the percichthyid Percichthys Funding This research was partially funded by the Programa trucha (Valenciennes), and introduced salmonids de Apoyo a Proyectos de Investigacioń e Inovacioń Tecnolo´gica (PAPIIT No IN206716). AC acknowledges funding from the St. rainbow trout Oncorhynchus mykiss (Walbaum) and Norbert College Faculty Development Summer Grants brook charr/trout Salvelinus fontinalis (Mitchill) in Programme. Argentina; P. sphaericus is a parasite of the pimelodid catfishes Pimelodus maculatus Lace´pe`de, P. albicans (Valenciennes), Iheringichthys labrosus (Lu¨tken), Compliance with ethical standards Parapimelodus valenciennis (Lu¨tken) and Lu- Conflict of interest The authors declare that they have no ciopimelodus pati (Valenciennes) in Argentina; P. conflict of interest. moyanoi parasitises the percichthyid Percilia gillissi Girard in Chile; and finally, the last described species, Ethical approval All applicable institutional, national and international guidelines for the care and use of animals were P. omarsegundoi is a parasite of the gymnotid followed. Hosts were collected under the Cartilla Nacional de Gymnotus carapo Linnaeus in northern Argentina Colector Cientı´fico de Flora y Fauna Silvestre FAUT 0202 (Oturbay et al., 1991; Gil de Pertierra et al., 1996; issued by the Secretaria del Medio Ambiente y Recursos Nat- Olmos & Habit, 2007; Arredondo & Gil de Pertierra, urales (SEMARNAT), to MGV. Fish were collected in Wis- consin and Manitoba under collecting permits Nos. SCP-NER- 2010). Clearly, all these hosts, from the southern 130 (Wisconsin), and CP 44-13 and CP 30-14 (Manitoba). regions of South America, represent temperate freshwater fishes. The phylogenetic relationships of both amphi- References temperate members of Pomphorhynchus in the Amer- icas is pending a molecular phylogenetic analysis. But, Aguilar-Aguilar, R., Martıńez-Aquino, A., Espinosa Pe´rez, H., based on distribution and host associations, we posit &Pe´rez-Ponce de Leoń, G. (2014). Helminth parasites of that species in North and South America will form freshwater fishes from Cuatro Cieńegas, Coahuila, in the Chihuahua desert of Me´xico: Inventory and biogeograph- distinct monophyletic assemblages that will not be ical implications. Integrative Zoology, 9, 328–339. closely related to each other. Instead, we hypothesize Amin, O. M. (2013). Classification of the Acanthocephala. Folia that the North American species will nest with the Parasitologica, 60, 273–305. other species occurring in the Palaearctic, showing a Arredondo, N. J., & Gil de Pertierra, A. A. (2010). Pom- phorhynchus omarsegundoi sp. n. (Acanthocephala:Pom- Laurasian origin, while the South American species porhynchidae), parasite of the banded knifefish Gymnotus will show a separate but common Gondwanan origin. carapo (Gymnotiformes:Gymnotidae) from the Parana´ If corroborated, it will also imply that despite the River basin, Argentina. Folia Parasitologica, 57, 307–311. amphi-American distribution of Pomphoryhynchus, Choudhury, A., & Dick, T. A. (1998). The historical biogeography of sturgeons (Osteichthyes: Acipenseridae) a syn- its distribution is not the result of the faunal inter- thesis of phylogenetics, palaeontology and palaeography. change through the Great American Biotic Inter- Journal of Biogeography, 25, 623–640. change (see Choudhury et al., 2017). Choudhury, A., Garcıá-Varela, M., & Pe´rez-Ponce de Leoń, G. (2017). Parasites of freshwater fishes and the Great Acknowledgements We thank Hugo Mejıá-Madrid for his American Biotic Interchange: a bridge too far? Journal of help collecting acanthocephalans from La Luz, Michoacań. We Helminthology, 91, 174–196. are grateful to Liliana Cervantes for obtaining some of the Cordonnier, L. M., & Ward, H. L. (1967). Pomphorhynchus sequences of Pomphorhynchus, to Luis Garcıá Prieto for rocci sp. n. (Acanthocephala) from the rock bass, Roccus providing specimens of Pomphorhynchus from the CNHE, saxatilis. Journal of Parasitology, 53, 1295–1297. ´ ´ ´ ´ and Laura Marquez for her help with the use of the DNA Domınguez-Domınguez, O., & Perez-Ponce de Leon, G. (2009). ´ sequencer. AC acknowledges the help of the following people in >La mesa central de Mexico es una provincia bio- ´ ´ sampling fish: Doug Watkinson, Department of Fisheries and geografica? Analisis descriptivo basado en componentes ´ ´ Oceans and Patrick Nelson, North-South Consultants, bioticos dulceacuıcolas. Revista Mexicana de Biodiversi- Winnipeg, Manitoba, Canada, Ron Bruch, Ryan Koenigs and dad, 80, 835–852. Ryan Zernzach, Wisconsin Department of Natural Resources, Doosey, M. H., Bart, Henry L., Jr., Saitoh, K., & Miya, M. Oshkosh, Wisconsin, USA, and Joe Beuchel, Chris Levas, John (2010). Phylogenetic relationships of catostomid fishes

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