Zootaxa 4526 (3): 251–302 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2018 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4526.3.1 http://zoobank.org/urn:lsid:zoobank.org:pub:4DF63CE5-4838-46CA-BB0E-2F91841D5CB1

Taxonomic revision of species of Haematoloechus Looss, 1899 (Digenea: Plagiorchioidea), with molecular phylogenetic analysis and the description of three new species from Mexico

VIRGINIA LEÓN-RÈGAGNON1, 2 & JANET TOPAN2 1Estación de Biología Chamela, Instituto de Biología, Universidad Nacional Autónoma de México, A.P. 21, San Patricio, Jalisco, México, CP 48980. E-mail: [email protected] 2Centre for Biodiversity Genomics, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

Abstract

Lung flukes of the genus Haematoloechus Looss, 1899 are common parasites of anurans worldwide, but the taxonomy of the group has been confusing. In this taxonomic revision, 89 species of Haematoloechus (= Pneumonoeces Looss, 1902, Ostioloides Odening, 1960, Ostiolum Pratt, 1903, Skrjabinoeces Sudarikov, 1950, Neohaematoloechus Odening, 1960, Metahaematoloechus Yamaguti, 1971) are listed. Of these, 70 are considered valid, three are species inquirendae (H. legrandi Mañé–Garzón & Gil, 1959, H. latoricensis Kozák, 1968 & H. vitelloconfluentum (Rai, 1962) Saeed, Al–Barwari & Al-Harmni, 2007), one is a nomen nudum H. sudarikovi Belouss, 1962, 14 are junior synonyms and one belongs to Osti- oloides. This publication also describes three new species, H. occidentalis n. sp., H. veracruzanus n. sp. and H. mexicanus n. sp., parasitizing species of Rana Linnaeus in Mexico and redescribes Haematoloechus caballeroi (Skrjabin & Antipin, 1962) Yamaguti, 1971. The phylogenetic hypotheses based on sequences of mitochondrial and ribosomal DNA of Hae- matoloechus spp. show that genera proposed on the basis of morphological characters are not supported. The host records for species of Haematoloechus, together with the phylogenetic hypothesis of the genus, suggest that this host-parasite as- sociation predates the ranid diversification in the Cretaceous.

Key words: 28S rDNA, Amphibia, Biogeography, Coevolution, COI, Haematoloechidae, Haematoloechus mexicanus n. sp., Haematoloechus occidentalis n. sp., Haematoloechus veracruzanus n. sp., host list, Mexico, Platyhelminthes, Rani- dae, Phylogram, species list

Introduction

Members of the genus Haematoloechus Looss, 1899 are parasites in the lungs of anurans, known from every continent except Antarctica. First reported in the early 19th Century, the type species was originally named Distomum variegatum Rudolphi, 1819 but was transferred to the newly erected genus Haematoloechus by Looss (1899), who also described two European species, H. similis Looss, 1899, and H. asper Looss, 1899. In 1902, the genus was renamed as Pneumonoeces Looss, 1902 because a hemipteran genus had previously been named Haematoloecha Stal. Although Harwood (1932) and Ingles (1932) independently reinstated Haematoloechus, some other authors continued to use Pneumonoeces (Skrjabin & Antipin 1962). Some current members of this genus were placed in other genera mostly based on the varying arrangement of the uterine loops. Ostiolum Pratt, 1903 was, for example, proposed for species lacking extracecal longitudinal uterine loops (Pratt 1903), Pneumobites Ward, 1917 for those with longitudinal uterine loops extending to the pre-acetabular region of body, with H. longiplexus as its type species (Ward 1917). Skrjabinoeces Sudarikov, 1950 was proposed for species with the vitelline follicles placed anteriorly to testes with H. similis as its type species (Sudarikov 1950). Odening (1958) recognized the genus Ostiolum and three subgenera within Haematoloechus: Haematoloechus, Anomolecithus Odening, 1958 and Skrjabinoeces Odening, 1958 based on the arrangement of the vitelline follicles, and later erected Neohaematoloechus Odening, 1960 for those species lacking ventral sucker, with H. neivai (Travassos & Artigas, 1927) Ingles, 1933 as its type species (Odening 1960). This author also erected the genus

Accepted by N. Dronen: 17 Oct. 2018; published: 30 Nov. 2018 251 Ostioloides Odening, 1960 to include Haematoloechus rappiae (Szidat, 1932) Yamaguti, 1958. Skrjabin & Antipin (1962) continued to use the name Pneumonoeces, and recognized Ostiolum, Skrjabinoeces and Neohaematoloechus as distinct genera. From the previous taxonomic proposals, Yamaguti (1971) only recognized Neohaematoloechus, Ostioloides and erected a new genus, Metahaematoloechus Yamaguti, 1971, for species with extracecal testes, with H. exoterorchis Rees, 1964 as the type species. He placed all other species within Haematoloechus, recognizing two subgenera: Haematoloechus and Skrjabinoeces. Based on extensive studies of the host use and geographic distribution of North American species of Haematoloechus, Kennedy (1980a; 1980b) concluded that most characters used to differentiate species were examples of intraspecific variation. As a result, he synonymized 9 of the 15 species known at that time from the U.S.A. and Canada (Kennedy 1981). Unfortunately, the limited detail in the original descriptions (Stafford 1902; Seely 1906), and the lack of type material for some species, has led to confusion in the identification and delineation of taxa. More recent research, using molecular characters to complement morphology, has revalidated species once relegated to synonymy. These studies aided the identification of morphological characters valuable in differentiating species, and revealed that some genera erected to include species of Haematoloechus are polyphyletic (León-Règagnon et al. 1999, 2001; Snyder & Tkach 2001; León-Règagnon & Paredes-Calderón 2002; León-Règagnon & Brooks 2003; Bolek & Janovy 2007a; León- Règagnon 2010; Zamparo et al. 2011). Molecular evidence has also shown that H. complexus (Seely, 1906) a species prevalent in the eastern U.S.A. (Bolek & Janovy 2007a) and previously thought to occur in Mexico (Caballero 1942b; León-Règagnon 1992; Pérez-Ponce de León et al. 2000) is actually a complex of sibling species whose conserved morphology makes their differentiation difficult (León-Règagnon et al. 1999; León-Règagnon 2003, 2010). The present study revises the taxonomy of the genus in the light of recent molecular and morphological studies, describes three new species of Haematoloechus that parasitize frogs in Mexico, and provides a list of the species historically assigned to Haematoloechus and related genera.

Materials and methods

Original descriptions and taxonomic literature related to the genus were reviewed while voucher and type material were examined from the following collections: Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México; Canadian Museum of Nature Parasite Collection (CMNPA); U. S. National Parasite Collection at the National Museum of Natural History (NMNH); Natural History Museum, UK (NHM); and the Colecão Helmintológica do Instituto Oswaldo Cruz, Brazil (IOC). Host nomenclature and higher classification of anurans was based on the Amphibian Species of the World 6.0 (http://research.amnh.org/vz/ herpetology/amphibia/) (Frost 2018). Classification and nomenclature of the Ranidae Batsch was based on Hillis & Wilcox (2005) and Bossuyt et al. (2006). Considering the length of this manuscript, the genera Haematoloechus and Rana are not spelled out for each species the first time it is presented, but rather the abbreviations H. and R. are used to avoid excessive repetition. From 1997 to 2017, specimens of Lake Lerma salamander Ambystoma lermaense (Taylor), Rio Grande leopard frog Rana berlandieri Baird, Brown´s leopard frog R. brownorum Sanders, American bullfrog R. catesbeiana Shaw, green frog R. clamitans Latreille, Patzcuaro leopard frog R. dunni Zweifel, Forrer´s leopard frog R. cf. forreri, big–footed leopard frog R. megapoda Taylor, Montezuma leopard frog R. montezumae Baird, Transverse Volcanic leopard frog R. neovolcanica Hillis & Frost, Amazon River frog R. palmipes Spix, showy leopard frog R. spectabilis Hillis & Frost, common marsh frog R. vaillanti Brocchi, and Zweifel´s frog R. zweifeli Hillis, Frost & Webb were collected from varied localities in Mexico and Canada under scientific collection permits FAUT0056 (Mexico) and AUP3762 (Canada) issued to VLR and Paul D. N. Hebert. Amphibians were captured manually or using dip nets and killed by an overdose of sodium pentobarbital or double pithing. Helminthological examination was completed within 24 hours of capture. Digeneans recovered from these frogs were initially placed in saline (0.65%) solution, examined for preliminary identification and separated by morphotypes. For detailed morphometric study they were fixed by sudden immersion in hot 4% formaldehyde, and then preserved in 70% ethanol. Specimens were stained with Mayer's paracarmine or Gomori´s trichrome, dehydrated, cleared in methyl salicylate, and mounted in Canada balsam. Some specimens were permanently mounted between cover slips and held in Cobb slides. Measurements are presented as the range with means in parentheses and expressed in micrometers, unless otherwise stated. Figures were drawn with the aid of a drawing tube.

252 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN Total DNA was extracted from whole digeneans using standard glass fibre methods (Ivanova et al. 2006) or standard phenol extraction (Hillis et al. 1996). After purification, 2 µL of DNA was added to a PCR reaction consisting of 6.25 µL of 10% D–(+)–trehalose dihydrate (Fluka Analytical), 2.00 µL of Hyclone ultra–pure water

(Thermo Scientific), 1.25 µL of 10X PlatinumTaq buffer (Invitrogen), 0.625 µL of 50 mM MgCl2 (Invitrogen), 0.125 µL of each primer or primer cocktail, 0.0625 µL of 10 mM dNTP (KAPA Biosystems) and 0.060 µL of 5 U/ µL PlatinumTaq DNA Polymerase (Invitrogen) for a total reaction volume of 12.5 µL. Amplification and sequencing were performed using the primers JB3 5'-TTT TTT GGG CAT CCT GAG GTT TAT-3'/JB4.5 5'-TAA AGA AAG AAC ATA ATG AAA ATG–3' (Bowles et al. 1995) for the partial COI mtDNA region and digl2 5'- AAG CAT ATC ACT AAG CGG-3'/5'-GCT ATC CTG AG(AG) GAA ACT TCG-3' (Tkach et al. 2000) for the 28S rDNA region. Thermal cycling conditions were 94 °C for 1 min, five cycles at 94°C for 40 s, 45 °C for 40 s, 72°C for 1 min, followed by 35 cycles at 94°C for 40 s, 51°C for 40 s, 72°C for 1 min and a final extension at 72°C for 5 min. The resulting amplicons were visualized on a 2% agarose E-gel 96 precast gel (Invitrogen) and bidirectionally sequenced. Cycle sequencing was performed using a modified BigDye 3.1 Terminator (Applied Biosystems) protocol (Hajibabaei et al. 2005). Cycle sequencing conditions were 96 °C for 1 min followed by 35 cycles at 96°C for 10 s, 55°C for 5 s, 60°C for 2.5 min and a final extension at 60°C for 5 min. Sequencing was performed on an ABI 3730XL capillary sequencer (at the CBG, U of Guelph) or an ABI Prism 310 (at the IBUNAM) (Applied Biosystems). Traces were assembled and edited using CodonCode v. 3.0.1 (CodonCode Corporation, Dedham, Massachusetts). Sequences of complementary strands were edited and reconciled using MEGA 5.2 (Tamura et al. 2011). Sequences have been deposited in BOLD (http://www.boldsystems.org) within the project entitled: Platyhelminthes of Amphibians and Reptiles I (PLARI) and in GenBank under the accession numbers in Table 1. Sequences of all species of Haematoloechus available in GenBank were included in this study (Table 1). Alignments were built with CLUSTAL W (Thompson et al. 1994), resulting in a 28S alignment of 880 bp and a COI alignment of 361 bp. Trees were constructed in PAUP 4.0a (build 159) (Swofford 2002). Unweighted parsimony analysis using heuristic searches with 100 replicates were performed considering character states unordered and gaps as missing data; nonparametric Bootstrap (Felsenstein 1985) with 100 pseudoreplicates was applied to evaluate the stability of nodes of the resulting topologies.

Results

According to this revision, 89 species have been included in Haematoloechus (=Pneumonoeces), Ostiolum, Ostioloides, Skrjabinoeces, Neohaematoloechus or Metahaematoloechus; the list of species historically included in any of those genera is shown in Appendix 1. Based on the phylogenetic evidence presented herein, Ostiolum, Neohaematoloechus and Metahaematoloechus are not considered valid. Ostioloides is considered valid based on morphological evidence (see appendix 1). Further molecular and phylogenetic evidence is needed for the validation of Skrjabinoeces. Seventy of the 89 species are considered valid in this revision, three are species inquirendae, one is nomen nudum, 14 species are junior synonyms and one species belongs to the genus Ostioloides. Morphological examination of specimens from the CNHE and molecular evidence of newly-collected specimens from Mexico, resulted in the validation of H. caballeroi Skrjabin & Antipin, 1962, and this species is redescribed. Also, we discovered three new species and phylogenetic analyses of available DNA sequences from species of Haematoloechus were performed. Phylograms obtained from COI, 28S sequences and the concatenated data sets resulted in similar topologies (Figs. 1–3). The African species H. micrurus Rees, 1964 and H. exoterorchis appear in a basal position, followed by a diversification of Eurasian and American species throughout the tree. All the phylograms show three well supported clades: Clade I (H. longiplexus Stafford, 1902, H. macrorchis Caballero, 1941, H. asper Looss, 1899 & H. sibiricus (Isajcikov, 1927)), Clade II (H. complexus, H. occidentalis n. sp., H. humboldtensis Zamparo, Ferrao, Brooks, Bettaso & Mata-López, 2011, H. pulcher Bravo–Hollis, 1943, H. veracruzanus n. sp., H. caballeroi Skrjabin & Antipin, 1962 & H. longicollum León-Règagnon & Romero–Mayén, 2017), and Clade III (H. medioplexus Stafford, 1902, H. floedae Harwood, 1932, H. meridionalis León-Règagnon, Brooks & Zelmer, 2001, H. nicolasi León‒Règagnon, 2017, H. illimis Caballero, 1942, H. lobatus Seno, 1907, H. breviplexus Stafford, 1902, H. mexicanus n. sp. & H. parviplexus (Irwin, 1929)) (Figs. 1–3). The trees differ in the internal arrangement of species within those clades, and also differ in the position of H. varioplexus Stafford, 1902, which appears as the sister species of Clade III in the COI analysis (although with low bootstrap support) and sister

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 253 species of Clade II in the 28S and concatenated analyses. They also differ in the position of H. danbrooksi León- Règagnon & Paredes-Calderón, 2002, which appears as the sister species of Clade II & (Clade III + H. varioplexus) in the COI analysis, while nested within Clade III in the 28S and concatenated analyses. Haematoloechus abbreviatus (Bychowsky, 1932) + H. variegatus (Rudolphi, 1819) appear as the sister group of H. varioplexus + Clade II in the 28S and concatenated analyses, but no COI sequences were available for them. Phylogenetic relationships among species within Clades I, II & III were better resolved and supported in the concatenated tree (Figs. 1–3).

FIGURE 1. One of 4 most parsimonious phylogenetic trees (consistency index = 0.449) of available COI sequences for species in the genus Haematoloechus. Majority-rule consensus values above branches; bootstrap values below branches; *originally recorded as H. coloradensis by León‒Règagnon (2010); †originally recorded as H. complexus by León-Règagnon (2010); §originally recorded as H. varioplexus by León-Règagnon et al. (2005); I = Clade I, H. longiplexus & relatives; II = Clade II, H. complexus & relatives; III = H. medioplexus & relatives.

Haematoloechus caballeroi Skrjabin & Antipin, 1962 (Figs. 4 & 5)

Type host: Tlaloc´s leopard frog Rana tlaloci Hillis & Frost recorded as Montezuma leopard frog Rana montezumae (Caballero, 1942b; Skrjabin & Antipin, 1962). Type locality: Xochimilco, Mexico City, Mexico (Caballero, 1942b; Skrjabin & Antipin, 1962). Site of infection: lungs. Neotype: CNHE 10457.

254 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN FIGURE 2. One of 829 most parsimonious phylogenetic trees (consistency index = 0.625) of available 28S sequences for species in the genus Haematoloechus. Majority-rule consensus values above branches; bootstrap values below branches; *originally recorded as H. coloradensis by León-Règagnon & Brooks (2003); †originally recorded as H. cf. complexus León- Règagnon & Brooks (2003); §originally recorded as H. varioplexus by León-Règagnon et al. (2005); I = Clade I, H. longiplexus & relatives; II = Clade II, H. complexus & relatives; III = H. medioplexus & relatives.

Paratypes: CNHE 1428, 1551, 1552, 3376–3379, 3395, 3397, 3399, 3794, 4661, 10458–10460. Other hosts and localities: Montezuma leopard frog R. montezumae, Zempoala, Morelos (CNHE 10464), Patzcuaro leopard frog R. dunni Zweifel, Pátzcuaro, Michoacán (CNHE 10465); R. dunni, Zacapu, Michoacán (CNHE 10463). Other records: Mexico: Tlaloc´s leopard frog R. tlaloci, Xochimilco, Mexico City (Caballero &Sokoloff 1934 as H. complexus); Montezuma leopard frog R. montezumae, Ciénaga de Lerma, Mexico State (León- Règagnon 1992 as H. complexus; Lamothe et al. 1997 as H. coloradensis Cort, 1915; León-Règagnon et al. 1999 as H. complexus; Pérez-Ponce de León et al. 2000 as H. coloradensis and H. complexus); big-footed leopard frog R. megapoda, Cointzio Springs, Michoacán (Pérez-Ponce de León et al. 2000 as H. complexus); Transverse Volcanic leopard frog R. neovolcanica, Cointzio Springs, Michoacán (Pérez-Ponce de León et al. 2000 as H. complexus); Patzcuaro leopard frog R. dunni, Pátzcuaro, Michoacán (García-Altamirano et al. 1993; Pulido–Flores 1994; Pérez-Ponce de León et al. 2000 as H. coloradensis); Lerma Lake salamander Ambystoma lermaense, Ciénaga de Lerma, Mexico State (Pérez-Ponce de León et al. 2000 as H. complexus; Mata-López et al. 2002 as H. complexus). Redescription: Based on 33 mature specimens. Body elongate, with slender anterior region; 2.8–6.6 (5.0) mm long, 0.82–1.88 (1.27) mm of maximum width at testicular region. Tegument covered with thin spines that are larger and more abundant in anterior region; spines easily lost during fixation and staining procedures; 8–16 (13) long. Oral sucker subterminal, round, 243–487 (355) long, 260–511 (387) wide. Pharynx oval, 146–398 (255) long,

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 255 130–300 (206) wide; oral sucker/pharynx ratio 1: 0.41–0.86 (0.66). Pharynx and anterior region of esophagus surrounded by gland cells. Esophagus 24–162 (65) long, sometimes obscured by uterus. Ceca bifurcated at 414– 844 (643) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 219–438 (313) long, 203–430 (311) wide, at 0.98–2.7 (1.9) mm (30%–47% (37.6%) of BL) from anterior extremity. Oral sucker: ventral sucker length ratio 1: 0.53–1.0 (0.81). Testes 2, oval, slightly lobed in some specimens, oblique, inmmediately posterior to ovary. Anterior testis opposite to ovary, 219–795 (477) long, 170–682 (360) wide. Posterior testis 203–852 (557) long, 203–771 (396) wide. Cirrus sac reach anterior border of ventral sucker, mostly obscured by ascending uterus; internal seminal vesicle, elongate, slightly coiled. Ejaculatory duct strongly muscular, 190–200 (195) long, surrounded by prostatic gland cells. Ovary oval, 243–665 (414) long, 203–763 (313) wide; at 1.1–2.9 (2.1) mm (33%–51% (42%) of BL) from anterior extremity. Seminal receptacle adjacent partially overlapped with ovary; 203–730 (412) long, 138–568 (300) wide. Mehlis gland dorsal to seminal receptacle. Laurer’s canal not observed. Vitellaria in clusters overlapped with each other, distributed laterally, dorsally invade space between ceca in their anterior limit and in post-testicular region. Anterior limit of distribution 390–1607 (1055) (6.5%–28% (21%) of BL) from anterior end. Follicles extend to halfway between posterior testis and posterior end; in some specimens they extend to level of posterior testis on ovarian side of body, and more posteriorly on side opposite to ovary. Descending part of uterus form transverse and diagonal loops on ovarian side of body, partially overlapped with testis, filling intra- and extracecal space towards posterior end of body. Ascending uterus form one or two short diagonal loops oriented anteriorly on each side of body, and continues with transverse or diagonal loops that occasionally invade both sides of body, not totally overlapped with testes or ovary, and fill with transverse loops entire preovarian region. Genital pore median, ventral to pharynx. Eggs dark brown, 31–40 (36) long, 17–23 (20) wide. Excretory vesicle not observed. Excretory pore terminal. Remarks: In their revision of the family Plagiorchiidae Lühe, 1901, Skrjabin & Antipin (1962) described Haematoloechus caballeroi based on a specimen that Caballero (1942b) collected from Tlaloc´s leopard frog R. tlaloci in Xochimilco, Mexico, and was originally identified as H. complexus. This description was based on a single specimen, and was not recognized by any later author; all subsequent records of that morphotype from Mexico were assigned to H. complexus or H. coloradensis. Recent molecular and morphological evidence has shown that H. complexus sensu stricto does not occur in Mexico, but that there is a complex of species closely related to H. complexus, most undescribed (León-Règagnon & Brooks 2003; Bolek & Janovy 2007a; León- Règagnon 2010; León-Règagnon & Romero–Mayén 2017). The present species is one of those (GenBank AF532138, León-Règagnon & Brooks 2003). Haematoloechus caballeroi differs from most other species in the genus, but resembles the following 19 species by lacking longitudinal uterine loops that reach the posterior testis: H. arequipensis Ibáñez & Córdoba, 1979, H. aubriae Bourgat, Roure & Kulo, 1996, H. coloradensis, H. complexus, H. confusus Ingles, 1932, H. danbrooksi, H. dollfusinum (Odening, 1958), H. elongatus Caballero & Sokoloff, 1934, H. fuelleborni (Travassos & Darriba, 1930), H. humboldtensis, H. illimis, H. kernensis Ingles, 1932, H. longicollum, H. medioplexus, H. meridionalis, H. oxyorchis Ingles, 1932, H. parcivitellarius Caballero, 1942, H. pukinensis Ibáñez & Córdoba, 1979, and H. pulcher. It differs from H. aubriae, H. danbrooksi, H. medioplexus, and H. meridionalis in the large size of the ventral sucker compared to the oral sucker, which is less than one third in those four species vs more than half in H. caballeroi (Table 2) (Stafford 1902; Bourgat et al. 1996; León-Règagnon et al. 2001; León- Règagnon & Paredes-Calderón 2002). Haematoloechus coloradensis, H. confusus, and H. oxyorchis differ from H. caballeroi in the arrangement of the uterine loops, which are strictly intercecal in those species (Cort 1915; Ingles 1932; Bolek & Janovy 2007a), while they invade the extracecal region in H. caballeroi. Haematoloechus caballeroi differs from H. arequipensis in having oval rather than lobed testes (Ibañez & Córdoba 1979). It differs from H. illimis and H. dollfusinum in the shape of the ovary, which is lobed in those species (Caballero 1942a) and oval in H. caballeroi. The presence of diagonal uterine loops directed anteriorly at the posterior end of the body differentiates H. caballeroi from H. humboldtensis, H. longicollum, H. parcivitellarius, and H. pulcher, in which the diagonal uterine loops are either absent or directed posteriorly (Caballero 1942b; Bravo–Hollis 1943; Zamparo et al. 2011; León-Règagnon & Romero–Mayén 2017). Haematoloechus caballeroi most closely resembles H. complexus, H. elongatus, H. fuelleborni, H. kernensis and H. pukinensis. It differs from H. fuelleborni, H. complexus and H. elongatus in having a larger pharynx and ventral sucker compared to the oral sucker (1:0.45, 1:0.56, 1:51 & 1:0.5, 1:0.71, 1:0.70 respectively vs 1:0.66 & 1:0.81 in H. caballeroi) (Travassos & Darriba 1930; Caballero & Sokoloff 1934; Bolek & Janovy 2007a). It also

256 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN differs from H. fuelleborni in the distribution of the vitellaria; while they are limited to two groups, one anterior to the ventral sucker and other posterior to the testes in the South American species, they are distributed continuously from the anterior region of the ventral sucker to the posterior region of the testes in H. caballeroi. It also differs from H. complexus in the distribution of the vitellaria which are distributed asymmetrically in that species, being more restricted to the ovarian side of the body (Bolek & Janovy 2007a), while in H. caballeroi they reach the posterior region of the testes on both sides of the body. In addition to the differing size of the ventral sucker and pharynx, H. elongatus differs from H. caballeroi in body size, which is much larger in that species (9.5 mm vs 5.1 mm) than in H. caballeroi. Haematoloechus caballeroi differs from H. kernensis in the size of the ventral sucker compared with the oral sucker, which is larger in that species (1:1, vs 1:0.82 in H. caballeroi). It also differs from H. kernensis in the distribution of the vitellaria, which do not invade the intercecal region in the post-testicular region in that species, and in the arrangement of the uterus, which forms a few diagonal loops in the pre-acetabular and post-testicular region in H. kernensis, while it is filled with transverse and diagonal loops in both areas in H. caballeroi (Ingles 1932). Haematoloechus caballeroi differs from H. pukinensis in the arrangement of the uterine loops; while in H. pukinensis the ascending part of the uterus forms a few transverse loops in the pre-acetabular region (Ibáñez & Córdoba 1979), in H. caballeroi the ascending uterus entirely fills the pre-acetabular region. The distribution of the vitellaria is also different in H. pukinensis, being more restricted in the ovarian side of the body in that species.

FIGURE 3. One of 1823 most parsimonious phylogenetic trees (consistency index = 0.546) of the concatenated matrix of available sequences COI + 28S of species of the genus Haematoloechus. Majority-rule consensus values above branches; bootstrap values below branches; *originally recorded as H. coloradensis by León-Règagnon (2010) and León-Règagnon & Brooks (2003); †originally recorded as H. cf. complexus by León-Règagnon (2010) and León-Règagnon & Brooks (2003); §originally recorded as H. varioplexus by Leon–Règagnon et al. (2005); I = Clade I, H. longiplexus & relatives; II = Clade II, H. complexus & relatives; III = H. medioplexus & relatives.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 257 FIGURE 4. Haematoloechus caballeroi Skrjabin & Antipin, 1962, from the Tlaloc´s leopard frog, Rana tlaloci Hillis & Frost (Ranidae), from Xochimilco, Mexico. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, IC = intestinal ceca, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, T = testes, VF = vitelline follicles, VS = ventral sucker. Scale bar = 1 mm

258 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN FIGURE 5. Haematoloechus caballeroi (Skrjabin & Antipin, 1962), from the Tlaloc´s leopard frog, Rana tlaloci Hillis & Frost (Ranidae), from Xochimilco, Mexico. Male terminal genitalia; female proximal genialia. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, ED = ejaculatory duct, MG = Mehlis gland, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, SV = seminal vesicle, VD = vitelline ducts, VS = ventral sucker. Scale bar = 0.5 mm.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 259 TABLE 1. Accession number of 28S rDNA and COI mtDNA sequences of Haematoloechus spp. and outgroups used in the phylogenetic analyses. Species (sample ID) Host Locality 28S COI H. abbreviatus Bombina variegata Zakarpatska Region, Ukraine AF184251 (T) H. asper Rana arvalis Ivano-Frankivsk Region, AF151934 (T) Ukraine H. breviplexus (Ha71) R. luteiventris Glacier Park, Montana, U.S.A. AF531856 (LB) H. caballeroi (Ha30) R. montezumae Lerma, Estado de Mexico, MG672412 Mexico H. caballeroi (Ha29) R. montezumae Lerma, Estado de Mexico, MG672411 Mexico H. caballeroi (Ha23)2 R. montezumae Lerma, Estado de Mexico, AF532138 (LB) Mexico H. complexus PLARI224 R. pipiens Carbondale, Illinois, U.S.A. MG672413 MG647784 H. complexus PLARI225 R. pipiens Carbondale, Illinois, U.S.A. MG672414 MG647785 H. danbrooksi (Ha48) R. vaillanti Los Tuxtlas, Veracruz, Mexico MG672415 HQ141699 (L) H. danbrooksi (PLARI047) R. vaillanti Los Tuxtlas, Veracruz, Mexico MG672416 MG647786 H. danbrooksi (Ha14) R. vaillanti Los Tuxtlas, Veracruz, Mexico AF479652 (LB) HQ141683 (L) H. exoterorchis (Ha47) Hoplobatrachus Sierra Leone AF531858 (LB) HQ141698 (L) occipitalis H. floedae (Ha61) Rana sp. Tuxtla Gutiérrez, Chiapas, MG672417 HQ141703 (L) Mexico H. floedae (PLARI009) R. cf. forreri Ticuizitan, Colima, Mexico MG672418 MG647787 H. floedae (Ha70) R. catesbeiana Fort Stewart, Georgia, U.S.A. AY672119 (L) H. humboldtensis 1 R. boylii Humboldt Co., California, GU191156 GU191160 U.S.A. H. humboldtensis 2 R. boylii Humboldt Co., California, GU191157 GU191161 U.S.A. H. illimis (Ha20) R. montezumae Lerma, Estado de Mexico, AF531860 (LB) AY672122 (L) Mexico H. illimis (Ha21) R. montezumae Lerma, Estado de Mexico, HQ141686 (L) Mexico H. lobatus R. catesbeiana Kanto, Japan AB818362 (H) AB818359 (H) H. longicollum (Ha62) R. psilonota Atenquique, Jalisco, Mexico MG672419 HQ141704 (L) H. longicollum (PLARI034) Lithobates sp. Tlalquitenango, Morelos, MG672420 MG647788 Mexico H. longicollum (PLARI084) R. zweifeli Tzitzio, Michoacán, Mexico MG672421 MG647789 H. longiplexus (Ha24) R. catesbeiana Neligh, Nebraska, U.S.A. AF531861 (LB) HQ141688 (L) H. longiplexus (PLARI133) R. catesbeiana Eel Lake, Ontario, Canada MG672422 MG647790 H. macrorchis (Ha22) R. montezumae Lerma, Estado de Mexico, AF531862 (LB) HQ141687 (L) Mexico H. medioplexus (Ha17) R. pipiens Neligh, Nebraska, U.S.A. AF531863 (LB) AY672123 (L) H. medioplexus PLARI218 R. pipiens Manitoba, Canada MG672423 MG647791 H. medioplexus PLARI219 R. pipiens Manitoba, Canada MG672424 MG647792 H. meridionalis (PLARI040) R. vaillanti Catemaco, Veracruz, Mexico MG672425 MG647793 ...... continued on the next page

260 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN TABLE 1. (Continued) Species (sample ID) Host Locality 28S COI H. meridionalis (PLARI048) R. vaillanti Catemaco, Veracruz, Mexico MG672426 MG647794 H. meridionalis (Ha10) R. vaillanti Guanacaste, Costa Rica AF531864 (LB) HQ141681 (L) H. mexicanus (Ha19)1 R. dunni Pátzcuaro, Michoacán, AF531867 (LB) HQ141685 (L) Mexico H. mexicanus (Ha31) R. montezumae Lerma, Estado de Mexico, MG672427 Mexico H. micrurus (Ha44) Hoplobatrachus Sierra Leone AF531865 (LB) HQ141696 (L) occipitalis H. nicolasi (PLARI017) Lithobates sp. Atoyac, Guerrero, Mexico MG672428 MG647795 H. nicolasi (PLARI020) R. zweifeli Tumbiscatio, Michoacán, MG672429 MG647796 Mexico H. nicolasi (Ha65) R. zweifeli Santiago Tamazola, Oaxaca, MG672430 HQ141707 (L) Mexico H. occidentalis (Ha63)2 R. cf. forreri Coquimatlán, Colima, Mexico MG672431 HQ141705 (L) H. occidentalis (Ha59) R. cf. forreri Juluapan, Colima, Mexico MG672432 H. parviplexus (Ha16) R. catesbeiana Humboldt, Nebraska, U.S.A. AF479653 (LB) AY672124 (L) H. parviplexus (PLARI222) R. pipiens Manitoba, Canada MG672433 MG647797 H. parviplexus (PLARI243) R. catesbeiana Puslinch, Ontario, Canada MG672434 MG647798 H. pulcher (Ha85) Ambystoma Huapango, Estado de Mexico, MG672435 granulosum Mexico H. pulcher (Ha86) A. granulosum Huapango, Estado de Mexico, MG672436 HQ141710 (L) Mexico H. pulcher (Ha13) R. montezumae Lerma, Estado de Mexico, AF531866 (LB) Mexico H. sibiricus Pelophylax Kokone, Japan AB818366 (H) AB818363 (H) nigromaculatus H. variegatus P. ridibunda Ivano-Frankivsk Region, AF151916 (T) Ukraine H. varioplexus PLARI220 R. pipiens Manitoba, Canada MG672437 MG647799 H. varioplexus PLARI221 R. pipiens Manitoba, Canada MG672438 MG647800 H. veracruzanus (Ha15)2 R. vaillanti Los Tuxtlas, Veracruz, Mexico AF531857 (LB) HQ141684 (L) H. veracruzanus (PLARI049) R. vaillanti Catemaco, Veracruz, Mexico MG647801 H. veracruzanus (PLARI050) R. vaillanti Catemaco, Veracruz, Mexico MG672439 MG647802 Haematoloechus sp. (Ha58)2 R. pipiens Fort Stewart, Georgia, U.S.A. MG672440 HQ141702 (L) Haematoloechus sp. (Ha12)3 R. blairi Genoa, Nebraska, U.S.A. AY672127 AY672125 (LGA) (LGA) Glypthelmins hyloreus Martin, Pseudacris triseriata Nebraska, USA AY278050 AY278059 1969 Wied–Neuwied (RLP) (RLP) Glypthelmins brownorumae Rana brownorum Tabasco, Mexico, Mexico AY875674 AY278055 Razo–Mendívil, León–Règagnon (RLP) (RLP) & Pérez–Ponce de León, 2004 Brachycoelium salamandrae Salamandra Zakarpatska Region, Ukraine AF151932 (T) (Frölich, 1789) Dujardin, 1845 salamandra Opisthioglyphe ranae (Frölich, Rana arvalis Ivano–Frankivsk Region, AF151935 (T) 1789) Looss, 1899 Ukraine ...... continued on the next page

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 261 TABLE 1. (Continued) Species (sample ID) Host Locality 28S COI Plagiorchis koreanus Ogata, Lymnaea stagnalis Danube, Slovakia KJ533394 (Z) KJ533418 (Z) 1938 Linnaeus Plagiorchis maculosus L. stagnalis Bohdanecsky, Czech Rep. KJ533396 (Z) KJ533428 (Z) (Rudolphi, 1802) Braun, 1902 H = Hasegawa et al., 2013. L = León-Règagnon 2010. LB = León-Règagnon & Brooks 2003. LGA = León-Règagnon et al. 2005. RLP = Razo-Mendívil et al. 2004, 2006. T = Tkach et al. 2000. Z = Zikmundova et al. 2014. 1 originally recorded as H. coloradensis. 2 originally recorded as H. cf. complexus. 3 originally recorded as H. varioplexus.

Haematoloechus occidentalis n. sp. (Figs. 6 & 7)

Type host: Rana sp. These host specimens belong to an undescribed species of leopard frog that is closely related to Forrer´s leopard frog Rana forreri Boulenger (Zaldívar–Riverón et al. 2004). Type locality: Coquimatlán, Colima, México. Site of infection: lungs. Holotype: CNHE 10497 Paratypes: CNHE 10498–10504, 10506, 10507, 4448 Other hosts and localities: Rio Grande leopard frog R. berlandieri, San Antonio Chimalapas, Oaxaca; Brown´s leopard frog R. brownorum, Rizo de Oro, Chiapas (Velazquez–Urrieta & León-Règagnon 2017); Forrer´s leopard frog R. cf. forreri, San Pedro las Playas, Guerrero (Cabrera–Guzmán et al. 2007 as H. coloradensis), Tehuantepec, Oaxaca (CNHE 10509), Santa María del Oro, Nayarit; Transverse Volcanic leopard frog R. neovolcanica, Ocotlán, Jalisco; showy leopard frog R. spectabilis, Mitla, Oaxaca (CNHE 10510); Rana sp., San Pablo Huitzo, Oaxaca (CNHE 10508), Nizanda, Oaxaca (CNHE 10511), San Fernando, Chiapas; cane toad Rhinella marina, Tres Palos, Guerrero. Etymology: The species name refers to its distribution in western Mexico. Description: Based on 21 mature specimens. Body elongate, with slender anterior region; 2.4–6.2 (4.5) mm long, 0.64–1.66 (1.24) mm of maximum width at testicular region. Tegument aspinose, even in live specimens. Oral sucker subterminal, round, 195–527 (392) long, 203–487 (385) wide. Pharynx oval, 114–252 (196) long, 122– 227 (187) wide; oral sucker: pharynx ratio 1: 0.43–0.65 (0.51). Pharynx and anterior region of esophagus surrounded by abundant glans cells. Esophagus 32–130 (84) long, sometimes obscured by uterus. Ceca bifurcated at 325–852 (621) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 162–365 (264) long, 162–365 (271) wide, at 0.96–2.5 (1.7) mm (26%–53% (38%) of BL) from anterior extremity. Sucker length ratio 1: 0.61–0.8 (0.70). Testes 2, oval, slightly lobed in some specimens, oblique, inmmediately posterior to ovary. Anterior testis opposite to ovary, 219–730 (477) long, 203–730 (466) wide. Posterior testis 259– 893 (553) long, 235–812 (482) wide. Cirrus sac reaches anterior border of ventral sucker, mostly obscured by ascending uterus; internal seminal vesicle, elongate, slightly coiled. Ejaculatory duct weakly muscular, 220–230 (225) long, surrounded by prostatic gland cells. Ovary oval, 252–536 (409) long, 187–446 (301) wide; at 1.1–2.8 (1.9) mm (33%–55% (42%) of BL) from anterior extremity. Seminal receptacle adjacent, partially overlaps with ovary; 244–730 (472) long, 162–511 (337) wide. Mehlis gland dorsal to seminal receptacle. Laurer’s canal not observed. Vitellaria in clusters overlapped with each other, distributed laterally, dorsally invade space between ceca in their anterior limit and in postesticular region. Anterior limit of distribution 487–1152 (778) (10%–25% (17%) of BL) from anterior end. Follicles extend asymmetrically, to level of anterior testis on ovarian side of body, and

262 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN FIGURE 6. Haematoloechus occidentalis n. sp., from Rana sp., from Colima, México. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, IC = intestinal ceca, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, T = testes, VF = vitelline follicles,‒= ventral sucker. Scale bar = 1 mm.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 263 FIGURE 7. Haematoloechus occidentalis n. sp., from Rana sp., Colima, México. Male terminal genitalia; female proximal genialia. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, ED = ejaculatory duct, MG = Mehlis gland, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, SV = seminal vesicle, VD = vitelline ducts, VS = ventral sucker. Scale bar = 0.5 mm.

264 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN halfway between posterior testis and posterior end on side opposite to ovary. Uterine loops fill intra- and extracecal space, partially overlapped with testes and ovary. Descending and ascending parts of uterus form two lateral fields of transverse or diagonal loops that occasionally bend anteriorly or posteriorly and form very short longitudinal extracecal loops. One or two diagonal uterine loops oriented anteriorly often present in posterior end of body. Distal uterus fills entire preovarian region with diagonal loops. Genital pore median, ventral to pharynx. Eggs dark brown, 30–39 (34) long, 16–21 (19) wide. Excretory vesicle not observed. Excretory pore terminal. Remarks: Haematoloechus occidentalis n. sp. belongs to the group of species that are similar to H. complexus, and as with H. caballeroi, it differs from many other species in the genus by its lack of longitudinal uterine loops at the posterior end of body reaching at least the posterior testis, by the large size of the ventral sucker which is more than half the size of the oral sucker, by having uterine loops invading the extracecal area and by having an oval ovary and testes (see H. caballeroi remarks). Considering the characters mentioned above, Haematoloechus occidentalis n. sp. resembles H. caballeroi, H. complexus, H. elongatus, H. fuelleborni, H. humboldtensis, H. kernensis, H. longicollum, H. parcivitellarius, H. pukinensis and H. pulcher. It differs from H. humboldtensis, H. longicollum, H. parcivitellarius, and H. pulcher by its presence of diagonal uterine loops directed anteriorly at the posterior end of the body, which are either absent or directed posteriorly in those species (Caballero 1942b; Bravo–Hollis 1943; Zamparo et al. 2011; León-Règagnon & Romero–Mayén 2017). Haematoloechus occidentalis n. sp. differs from H. kernensis, and H. pukinensis in having a smaller ventral sucker compared to the oral sucker (1:1 & 1:0.94 respectively, vs 1:0.70 in H. occidentalis n. sp.) (Ingles 1932; Ibáñez & Córdoba 1979). It also differs from those two species in the arrangement of the uterine loops; while in H. kernensis and H. pukinensis the ascending part of the uterus forms a few transverse loops in the post-testicular and in the pre-acetabular region (Ibáñez & Córdoba 1979), in H. occidentalis n. sp. the ascending uterus fills both regions with transverse or diagonal loops. It differs from H. caballeroi in the uterine loops being transverse or diagonal in that species, never bending to form short longitudinal loops as happens in H. occidentalis n. sp.; also, the ejaculatory duct in H. caballeroi is strongly muscular while it is weakly muscular in H. occidentalis n. sp. (Figs. 5 & 7). Haematoloechus occidentalis n. sp. differs from H. fuelleborni in the size of the ventral sucker compared to the oral sucker, which is larger in the new species (1:0.5 in H. fuelleborni vs 1:0.7 in H. occidentalis n. sp.). Also, the distribution of the vitellaria is different; in H. fuelleborni they are limited to two groups, one anterior to the ventral sucker and the other posterior to the testes, while they are distributed continuously extending asymmetrically, from the region anterior to the ventral sucker, to the level of anterior testis on the ovarian side of body, and halfway between the posterior testis and the posterior end on the side opposite to the ovary in H. occidentalis n. sp. Haematoloechus occidentalis n. sp. differs from H. elongatus in the distribution of the vitellaria, which are asymmetrical in H. occidentalis n. sp. while they reach the posterior region of testes on both sides of the body in H. elongatus. Also, that species is much larger in body size than H. occidentalis n. sp. (9.5 mm vs 4.5 mm) and in H. elongatus the uterine loops are transverse or diagonal, never bending to form short longitudinal loops as does in H. occidentalis n. sp. (Caballero & Sokoloff 1934). Haematoloechus complexus and H. occidentalis n. sp. differ in the arrangement of the uterine loops. While in H. complexus the uterine loops are transverse in the post-acetabular region, and diagonal, oriented anteriorly in the posterior end (Bolek & Janovy 2007a), in H. occidentalis n. sp. the uterine loops are transverse or diagonal often bending to form short longitudinal loops that can be oriented anteriorly or posteriorly. Haematoloechus occidentalis n. sp. is one of the species that were differentiated using COI sequences by León-Règagnon (2010) (Ha63, Genbank HQ141705) and are included in the "complexus group".

Haematoloechus veracruzanus n. sp. (Figs. 8 & 9)

Type host: common marsh frog Rana vaillanti. Type locality: Laguna Escondida, Los Tuxtlas, Veracruz, Mexico. Site of infection: lungs Holotype: CNHE 4087 Paratypes: CNHE 4086, 4089, 4090, 10515.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 265 FIGURE 8. Haematoloechus veracruzanus n. sp., from the common marsh frog, Rana vaillanti, from Veracruz, Mexico. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, IC = intestinal ceca, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, T = testes, VF = vitelline follicles, VS = ventral sucker. Scale bar = 1 mm.

266 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN FIGURE 9. Haematoloechus veracruzanus n. sp., from the common marsh frog, Rana vaillanti, from Veracruz, Mexico. Male terminal genitalia; female proximal genialia. Dorsal view; AU = ascending uterus, DU = descending uterus, GP = genital pore, ED = ejaculatory duct, MG = Mehlis gland, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, SV = seminal vesicle, VD = vitelline ducts, VS = ventral sucker. Scale bar = 0.5 mm.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 267 Other hosts and localities: common marsh frog R. vaillanti, Los Tuxtlas, Veracruz (Paredes-Calderón et al. 2004 as H. complexus); Catemaco, Veracruz (GenBank MG647801, MG647802, MG672439); Rio Grande leopard frog R. berlandieri, Laguna Higueras, Nuevo León (León-Règagnon et al. 2005, as H. complexus), Huauchinango, Puebla (León-Régagnon 2003, as H. complexus CNHE 10517), Rana sp., Tierra Quemada (CNHE 10518) and Rancho el Borbotón (CNHE 10519), San Luis Potosí, Mexico. Etymology: Species name refers to Veracruz, the state in Mexico where the type locality is located. Description: Based on 11 mature specimens. Body elongate, with slender anterior region; 3.5–5.2 (4.3) mm long, 0.80–1.64 (1.17) mm of maximum width at testicular region. Tegument aspinose, even in live specimens. Oral sucker subterminal, round, 203–365 (310) long, 252–409 (318) wide. Pharynx oval, 97–195 (154) long, 105– 170 (145) wide; oral sucker: pharynx ratio 1: 0.38–0.56 (0.48). Pharynx and anterior region of esophagus surrounded by abundant gland cells. Esophagus 65–122 (96) long, sometimes obscured by uterus. Ceca bifurcated at 381–649 (496) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 178–292 (245) long, 203–340 (265) wide, at 0.9–1.84 (1.4) mm (26%–38% (33%) of BL) from anterior extremity. Sucker length ratio 1: 0.64–0.94 (0.84). Testes 2, oval, oblique to almost tandem, inmmediately posterior to ovary. Anterior testis opposite to ovary, 308–771 (476) long, 292–674 (415) wide. Posterior testis 446–795 (604) long, 284–771 (433) wide. Cirrus sac reaches anterior border of ventral sucker, mostly obscured by ascending uterus; internal seminal vesicle, elongate, slightly coiled. Ejaculatory duct weakly muscular, 260–330 (295) long, surrounded by prostatic gland cells. Ovary oval, 349–576 (449) long, 227–608 (335) wide; at 0.92–2.1 (1.6) mm (26%–42% (37%) of BL) from anterior extremity. Seminal receptacle adjacent and partially overlaps ovary; 349– 771 (548) long, 341–649 (429) wide. Mehlis gland dorsal to seminal receptacle. Laurer’s canal not observed. Vitellaria in clusters overlapped with each other, distributed laterally, dorsally invade space between ceca in their anterior limit and in post-testicular region. Anterior limit of distribution 471–1000 (662) (10%–23% (15%) of BL) from anterior end. Follicles extend asymmetrically, to level of posterior testis on ovarian side of body, and halfway between posterior testis and posterior end on side opposite to ovary. Uterine loops fill intra- and extracecal space, partially overlapped with testes and ovary. Descending part of uterus form several diagonal loops that frequently bend anteriorly or posteriorly and form short longitudinal extracecal loops. Uterus forms two diagonal uterine loops oriented anteriorly on each side of posterior end of body, sometimes reaches halfway between posterior end and posterior testis. Ascending part of uterus form transverse or diagonal loops on side opposite to ovary, they frequently invade ovarian side and bend anteriorly or posteriorly to form short longitudinal extracecal loops. Distal uterus fills entire preovarian region with diagonal loops. Genital pore median, ventral to anterior region of pharynx. Eggs dark brown, 34–40 (37) long, 18–23 (20) wide. Excretory vesicle not observed. Excretory pore terminal. Remarks: Haematoloechus veracruzanus n. sp. resembles H. arequipensis, H. caballeroi, H. complexus, H. danbrooksi, H. dollfusinum, H. elongatus, H. fuelleborni, H. humboldtensis, H. illimis, H. kernensis, H. longicollum, H. medioplexus, H. meridionalis, H. occidentalis n. sp., H. parcivitellarius, H. pukinensis, and H. pulcher and differs from other American species in the genus by having uterine loops invading the extracecal area, and by lacking longitudinal uterine loops reaching at least the level of the posterior testis. It differs from H. danbrooksi, H. medioplexus, and H. meridionalis in the size of the ventral sucker compared to the oral sucker, which is less than one third in those four species vs more than half in H. veracruzanus n. sp. (Table 2) (Stafford 1902; León-Règagnon et al. 2001; León-Règagnon & Paredes-Calderón 2002). Haematoloechus veracruzanus n. sp. differs from H. arequipensis in having oval rather than lobed testes (Ibañez & Córdoba 1979), and differs from H. illimis and H. dollfusinum in the shape of the ovary, which is lobed in those species (Caballero 1942a) and oval in H. veracruzanus. The presence of diagonal uterine loops directed anteriorly at the posterior end of the body differentiates H. veracruzanus n. sp. from H. humboldtensis, H. longicollum, H. parcivitellarius, and H. pulcher, in which the diagonal uterine loops are either absent or directed posteriorly (Caballero 1942b; Bravo–Hollis 1943; Zamparo et al. 2011; León-Règagnon & Romero–Mayén 2017). Haematoloechus veracruzanus n. sp. differs from H. fuelleborni, in having a larger ventral sucker compared to the oral sucker (1:0.5 vs 1:0.84 in H. veracruzanus n. sp.), and in the distribution of the vitellaria; while they are limited to two groups, one anterior to the ventral sucker and other posterior to the testes in the South American species, they are distributed continuously extending asymmetrically, from the region anterior to the ventral sucker to the level of the posterior testis on the ovarian side of the body, and halfway between the posterior testis and the posterior end on the side opposite to the ovary in H. veracruzanus n. sp. (Travassos & Darriba 1930). It differs from H. complexus and H. elongatus in the arrangement of the uterine loops. While in H. complexus and H. elongatus the uterine loops are transverse in the post-acetabular

268 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN region (Caballero & Sokoloff 1934; Bolek & Janovy 2007a), in H. veracruzanus n. sp. the uterine loops are transverse or diagonal often bending to form short longitudinal loops that can be oriented anteriorly or posteriorly. Haematoloechus elongatus is also much larger in body size than H. veracruzanus n. sp. (9.5 mm vs 4.3 mm). Haematoloechus veracruzanus n. sp. differs from H. occidentalis n. sp. in the arrangement of the uterine loops; while the descending and ascending loops form two lateral fields in the post-testicular region in H. occidentalis n. sp., the ascending uterine loops frequently invade both sides of the body in H. veracruzanus n. sp. (Figs. 6 & 8). Haematoloechus veracruzanus n. sp. differs from H. kernensis in the arrangement of the uterus, which forms a few diagonal loops in the pre-acetabular and post-testicular region in that species, while fills with transverse or diagonal loops in both areas in H. veracruzanus n. sp., and in the distribution of the vitellaria, which do not invade the intercecal region in the post-testicular area in H. kernensis (Ingles 1932). The new species also differs from H. pukinensis in the arrangement of the uterine loops; while in H. pukinensis the ascending part of the uterus forms a few transverse loops in the pre-acetabular region (Ibáñez & Córdoba 1979), in H. veracruzanus n. sp. the ascending uterus entirely fills the pre-acetabular region. Haematoloechus veracruzanus n. sp. most closely resembles H. caballeroi, but the two species can be differentiated by the arrangement of the uterus. While in H. caballeroi, the descending and ascending uterus form transverse loops that sometimes become diagonal and are oriented anteriorly in the extracecal region, in H. veracruzanus n. sp. the uterine loops are transverse or diagonal, frequently bending to form short longitudinal loops in the extracecal region that can be oriented anteriorly or posteriorly; also, the ejaculatory duct in H. caballeroi is strongly muscular while it is weakly muscular in H. veracruzanus n. sp. (Figs. 5 & 9). Haematoloechus veracruzanus n. sp. is another species that was differentiated using DNA sequences (León- Règagnon & Brooks 2003, Genbank AF531857; León-Règagnon 2010, GenBank HQ141684 (Ha15), HQ141701 (Ha51)) and is included in the "complexus group".

Haematoloechus mexicanus n. sp. (Figs. 10 & 11)

Type host: Montezuma leopard frog Rana montezumae (=Northern leopard frog R. pipiens Schreber and R. montezumae of Caballero 1941). Type locality: Ciénaga de Lerma, Estado de Mexico, Mexico. Site of infection: Lungs Holotype: CNHE 10489 Paratypes: CNHE 10490, 10491, 10492. Etymology: Species name refers to Estado de Mexico, the province of the type locality. Other hosts and localities: Mexico: Montezuma leopard frog Rana montezumae, Xochimilco, Mexico City (Caballero, 1941, as H. medioplexus); Patzcuaro leopard frog R. dunni, Pátzcuaro, Michoacán (León-Règagnon et al. 1999, as H. coloradensis); Transverse Volcanic leopard frog R. neovolcanica, Cointzio, Michoacán (this study). Description: Based on 17 mature specimens: Body slender, with thinner anterior region; 4.8–8.3 (7.0) mm long, 0.7–1.3 (1.0) mm of maximum width at testicular region. Tegument covered with abundant thin spines, easily lost during fixation; 7.5–12.5 (9.8) long. Oral sucker subterminal, round, 203–350 (284) long, 180–380 (278) wide. Pharynx oval, 140–280 (206) long, 122–220 (183) wide; oral sucker/pharynx ratio 1: 0.74–0.90 (0.83). Anterior border of pharynx and esophagus surrounded by gland cells. Esophagus 41–200 (129) long. Ceca bifurcated at 390–770 (609) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker small, weakly developed, frequently obscured by uterus, 52–125 (82) long, 57–130 (84) wide, at 1.5–3.0 (2.4) mm (31%– 41% (37%) of BL) from anterior extremity. Sucker length ratio 1:0.28–0.38 (0.33). Testes 2, elliptical, elongate, oblique, posterior to ovary; distance between ovary and anterior testis 350–825 (548). Anterior testis opposite to ovary, 365–1,080 (778) long, 243–600 (451) wide. Posterior testis 422–1200 (863) long, 260–830 (495) wide. Cirrus sac reaches anterior border of ventral sucker, mostly obscured by ascending uterus; internal seminal vesicle, elongate, slightly coiled. Ejaculatory duct weakly muscular, 150–160 (155) long, surrounded by prostatic gland cells. Ovary kidney shaped, lobed, 320–840 (623) long, 162–500 (342) wide; at 1.8–4.3 (2.8) mm (35%–57% (40%) of BL) from anterior extremity. Seminal receptacle posterior, partially overlapped with ovary; 300–1000 (644) long, 250–590 (410) wide. Mehlis gland dorsal to seminal receptacle. Laurer’s canal not observed. Vitellaria

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 269 FIGURE 10. Haematoloechus mexicanus n. sp., from the Montezuma leopard frog, Rana montezumae from Estado de Mexico, Mexico. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, OS = oral sucker, IC = intestinal ceca, OV = ovary, PH = pharynx, SR = seminal receptacle, T = testes, VF = vitelline follicles, VS = ventral sucker. Scale bar = 1 mm.

270 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN FIGURE 11. Haematoloechus mexicanus n. sp., from the Montezuma leopard frog, Rana montezumae from Estado de Mexico, Mexico. Male terminal genitalia; female proximal genialia. Ventral view; AU = ascending uterus, DU = descending uterus, GP = genital pore, ED = ejaculatory duct, MG = Mehlis gland, OS = oral sucker, OV = ovary, PH = pharynx, SR = seminal receptacle, SV = seminal vesicle, VD = vitelline ducts, VS = ventral sucker. Scale bar = 0.5 mm.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 271 in clusters of oval, well defined follicles, distributed laterally, dorsally invade space between ceca in anterior region of ovary and sometimes in post-testicular region. Anterior limit of distribution 982–3200 (1766) (19%–49% (25%) of BL) from anterior end. Follicles extend asymmetrically, to anterior region of posterior testis on ovarian side of body, and halfway between posterior testis and posterior end of body on side opposite to ovary. Uterine loops fill intra- and extracecal space, partially overlap testes and ovary. Descending part of uterus form several diagonal loops that frequently bend anteriorly or posteriorly and form short longitudinal extracecal loops on ovarian side of body. Uterus forms two longitudinal uterine loops on each side of posterior end of body that reach halfway between posterior end and posterior testis; one loop is frequently shorter. Ascending part of uterus forms diagonal loops on side opposite to ovary, frequently bends anteriorly or posteriorly to form longitudinal extracecal loops. Descending and ascending parts of uterus in two lateral fields rarely invade each other. Distal uterus fills intracecal preovarian region with diagonal loops. Genital pore median, ventral to middle region of pharynx. Eggs dark brown, 22–26 (24) long, 14–20 (17) wide. Excretory vesicle not observed. Excretory pore terminal. Remarks: Haematoloechus mexicanus n. sp. resembles those species of the genus possessing short longitudinal or diagonal uterine loops not reaching the posterior testis, namely H. aubriae, H. caballeroi, H. danbrooksi, H. fuelleborni, H. humboldtensis, H. illimis, H. kernensis, H. occidentalis n. sp., H. pukinensis, and H. veracruzanus n. sp. It also resembles those species with a ventral sucker less than half the size of the oral sucker, namely H. combesi Batchvarov & Bourgat, 1974, H. danbrooksi, H. darcheni Combes & Knoepffler, 1967, H. floedae, H. leonensis (Williams & Coker, 1967), H. medioplexus, H. meridionalis, H. nicolasi, H. ocellati Gassmann, 1975 and H. parviplexus (Table 2). This new species differs from H. caballeroi, H. fuelleborni, H. humboldtensis, H. illimis, H. kernensis, H. occidentalis n. sp., H. pukinensis, and H. veracruzanus n. sp. in the size of the ventral sucker compared to the oral sucker, which is smaller in H. mexicanus n. sp. (1: 0.5–1.0 in the other species vs 1: 0.33 in H. mexicanus n. sp.), and it differs from H. aubriae in the presence of ventral sucker, which is absent in that species. It also differs from H. aubriae, H. caballeroi, H. fuelleborni, H. humboldtensis, H. kernensis, H. occidentalis n. sp., H. pukinensis, and H. veracruzanus n. sp. in the shape of ovary and testes, which are oval in those species, while in H. mexicanus n. sp. the ovary is lobed and testes are elliptical or elongate. Haematoloechus mexicanus n. sp. differs from H. combesi, H. darcheni, H. floedae, H. leonensis, H. medioplexus, H. meridionalis, H. nicolasi, H. ocellati and H. parviplexus in the arrangement of the uterine loops. Haematoloechus medioplexus and H. meridionalis lack uterine longitudinal loops (Stafford, 1902; León-Regagnon et al. 2001), in H. combesi, H. floedae and H. leonensis they reach the level of the ovary (Williams & Coker 1967; Batchvarov & Bourgat 1974; León-Règagnon et al. 2005), in H. darcheni and H. ocellati they reach the level of the anterior testis (Combes & Knoepffler 1967; Gassmann 1975), in H. nicolasi and H. parviplexus they reach the level of the posterior testis (Irwin 1929; León-Règagnon 2017), while in H. mexicanus n. sp. they reach halfway between the posterior testis and the posterior end. In this new species there are frequently several short longitudinal uterine loops in the post- testicular region and at the level of testes, which are absent in the other species. Haematoloechus mexicanus n. sp. most closely resembles H. danbrooksi in the size of the ventral sucker and the presence of short diagonal or longitudinal uterine loops in the posterior end of body, but differs from that species in the shape of the ovary, which is oval or slightly bi-lobed in some specimens (León-Règagnon & Paredes-Calderón 2002) and deeply lobed in H. mexicanus n. sp. The arrangement of the uterus also differentiates these two species; while in H. danbrooksi the descending and ascending uterine loops often invade both sides of the body, in H. mexicanus n. sp. descending and ascending uterine loops form two lateral fields and rarely invade one another. Finally, the longitudinal uterine loops in the posterior end of the body are shorter in H. danbrooksi.

Discussion

Recent studies that used molecular evidence to complement morphology have aided in the identification of characters that are useful for the differentiation of species of Haematoloechus (León-Règagnon et al. 1999, 2001; León-Règagnon & Paredes-Calderón 2002; León-Règagnon & Brooks 2003; León-Règagnon 2010; Zamparo et al. 2011). In this study, we corroborated that characters as the size of body, reproductive organs, or the presence or absence of tegumental spines are not reliable to differentiate species because they are too variable, or may be easily altered during the fixation process. It is very useful to make observations on live specimens, but this is not always possible. Characters as the suckers ratio, oral sucker/pharynx ratio, shape of ovary and testes, the arrangement of

272 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN the uterine loops, the distribution of the vitellaria, are valuable characters to differentiate species; nevertheless, the differentiation must be based on the combination of several characters, and molecular evidence support is of key importance, considering morphological intraspecific variation in this genus. Although these morphological characters may be useful to differentiate species, none of them appears to reflect the evolutionary history of the group.

TABLE 2. Comparison of some general morphological characters among Haematoloechus spp. Species BL BW VS/OS Ovary Testes Vit EUL LEUL Africa mean; range mean; range mean; range H. aubriae Bourgat, Roure & Kulo, 92.4AOO, D1dASDA 1996 H. combesi Batchvarov & Bourgat, 5.2–6.1 2.8–3.3 0.37 I, L O, L, D 1a P OA 1974 H. darcheni Combes & Knoepffler, 4.8–6.7 1.4–1.8 0.25 I, L L, I, D 1a P AT 1967 H. dollfusinus (Odening, 1958) 18–18.9 3–3.2 0.66 L O, D 1a P A Yamaguti, 1971 H. exoterorchis Rees, 1964 7.32; 6.5–8.5 1.4; 1.3–1.4 0.79 O E, Pa, 3PPT Ex H. johnsoni Bourgat, 1977 3.9–7 1–2 0.55; 0.52–0.62 I, L L, I, D 1a P PT H. leonensis (Williams & Coker, 8.8; 7.9–12.4 2.7; 2.1–3.4 0.17 K O, D 1a P OA 1967) n. comb. H. lobogonadus (Meskal, 1970) 12.9; 12–14 3.6; 3.2–4 ≤0.51, L L, I, D 1d P PT Gassmann, 1975 H. micrurus Rees, 1964 10.4; 8.5–13 2.3; 1.7–2.6 0.5 O O, D 1a P Ph H. ocellati Gassmann, 1975 3.5–4.3 1–1.1 0.28 I, L O, L, D 1a P AT America H. arequipensis Ibáñez & Córdoba, 7.9; 7–8.4 2; 1.8–2.4 1; 0.9–1.1 O L, I, T 1a P A 1979 H. breviplexus Stafford, 1902 9; 5.8–12 2.1; 2–2.7 0.5 I, L E, L, D 1a P PT H. buttensis Ingles, 1936 7.4; 3.2–10 1.3; 0.7–2.2 0.7; 0.6–0.8 K O, D 1b P PT H. caballeroi (Skrjabin & Antipin, 4.9; 2.8–6.6 1.3; 0.8–1.9 0.82; 0.53–1 O, L O, L, D 1a P SDA 1962) n. comb. H. coloradensis (Cort, 1915) Ingles, 5.7; 3.3–8.1 1.1; 0.5–1.5 0.9; 0.75–1.14 O O, D 1d A A 1932 H. complexus (Seely, 1906) Ingles, 4.9; 1.2–6.08 1; 0.3–1.1 0.71; 0.59–0.9 O O, D 1a P SDA 1932 H. confusus Ingles, 1932 3.9; 3.3–4.9 0.9 0.75; 0.55–0.8 L, I L, I 1b A A H. danbrooksi León–Règagnon & 3.9 0.9 0.32 O, L O, D 1b P SDAP Paredes–Calderón, 2002 H. elongatus Caballero & Sokoloff, 9.5; 9–10 1.6; 1.5–1.7 0.7; 0.64–0.75 O O, D 1a P SDA 1934 H. floedae Harwood, 1932 6.3; 2–11.1 1.7; 0.7–2.7 0.35; 0.3–0.41 I, L O, D 1c P OA H. freitasi Mañé–Garzon & Gil, 1959 13.5 2.5 1.16 E L, I, D 1a P PT H. fuelleborni (Travassos & Darriba, 10 2.3 0.5 O O, D 1a P SDA 1930) Yamaguti, 1958 H. humboldtensis Zamparo, Ferrao, 6.2; 5.6–6.7 1.4; 1.2–1.7 0.76; 0.72–0.78 O O, D 1b P SDP Brooks, Bettaso & Mata–López, 2011 H. illimis Caballero, 1942 6.2; 4.8–6.86 2.1; 1.5–2.75 0.69; 0.56–0.83 L, K L, D 1a P SDA ...... continued on the next page

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 273 TABLE 2. (Continued) Species BL BW VS/OS Ovary Testes Vit EUL LEUL H. iturbei (Cordero & Vogelsang, 16.8 1.5 A O E, D 1b P PT 1939) Yamaguti, 1958 H. kernensis Ingles, 1932 6.3; 5.5–7 1.4 1; 0.85–1.06 O O, D 1a P SDA H. longicollum León–Règagnon & 5.6; 3.6–7.2 1.2; 0.6–1.9 0.81; 0.68–0.96 O O, D 1a P SDP Romero–Mayén, 2017 H. longiplexus Stafford, 1902 6.5; 4.6–8.0 1.9; 1.2–2.55 0.43; 0.4–0.5 I, L E, Pa 1c P Ph H. lutzi Freitas & Lent, 1939 4–6.5 1.3–2 0.9 O E, D 1a P PT H. macrorchis Caballero, 1941 6; 5.6–6.37 1.6 0.45; 0.41–0.49 I, L E, L, D 1c P OA H. medioplexus Stafford, 1902 8.5 1.2 0.3 O O, D 1b A A H. meridionalis León–Règagnon, 8.25 1.7 0.2 O O, D 1c P A Brooks & Zelmer, 2001 H. mexicanus n. sp. 7; 4.8–8.3 1.1; 0.7–1.3 0.33; 0.28–0.38 L, K O, E, D 1c P SDA H. neivai (Travassos & Artigas, 3.7–6.7 1.3–2.3 A O O, L, Pa 1a P OA 1927) Ingles, 1933 H. nicolasi León–Règagnon, 2017 3.9; 2.5–6 1.2; 0.8–1.9 0.31; 0.23–0.36 L E, L, D 1c P PT H. occidentalis n. sp. 4.5; 2.4–6.2 1.2; 0.6–1.7 0.71; 0.61–0.8 O, L O, D 1b P SDA H. oxyorchis Ingles, 1932 5.8; 3–6.5 0.9 0.78; 0.62–0.85 O O, D 1b A A H. ozorioi Freitas & Lent, 1939 7 2.1 1.25 O, L O, D 1a P PT H. parcivitellarius Caballero, 1942 6.6; 5.4–7.7 2; 1.5–2.4 0.82; 0.75–0.88 I, L O, D 1a P A H. parviplexus (Irwin, 1929) Ingles, 6.1 1.2 0.25 O O, D 1b P PT 1932 H. pukinensis Ibáñez & Córdoba, 3.8; 3.5–4.2 0.8; 0.7–0.9 0.94; 0.87–1 O O, L, D 1a P SDA 1979 H. pulcher Bravo–Hollis, 1943 4.0; 3.35– 1.1; 1–1.2 0.73; 0.65–0.81 O, K O, L, D 1a P SDP 4.86 H. tumidus Ingles, 1932 8.5 2.5 1.11 O, K O, L, D 1a P PT H. varioplexus Stafford, 1902 9 2 0.86 O O, D 1a P AT H. veracruzanus n. sp. 4.3; 3.5–5.2 1.3; 0.8–1.6 0.84; 0.64–0.94 O O, D 1c P SDAP Australia H. australis (Johnston, 1912) 3.5; 2.5–4.9 1.5 0.5 L O, D 1a P AT Yamaguti, 1958 Eurasia H. abbreviatus (Bychowsky, 1932) 6 1.6–2 0.57 O O, D 1a P OA Prokopic & Krivanec, 1974 H. almorai (Pande, 1937) Yamaguti, 1.7–7 0.7–1.6 0.5 E, L E, D 1a P OA 1958 H. asper Looss, 1899 2–10 1–1.6 0.75 O O, D 2 P PT H. breviansa (Sudarikov, 1950) 4.6 1.3 0.54 O O, D 1e P PT Yamaguti, 1958 H. bychovskii Odening, 1958 2.2–2.7 1.0–1.1 0.88 O O, Pa 2 P OA H. capyristes (Klein, 1905) Odening, 6.0 1.4 0.5 O I, E, D 1a P OA 1958 H. carbonelli Lluch, Navarro & 4.2–4.5 1.2; 1–1.4 0.5–0.62 I, L E 1a P PT Pérez–Soler, 1991 H. donicus (Shevchenko, 1965) n. 4.3–4.4 1.5–1.7 0.6–0.8 O O, D 1e P PT comb...... continued on the next page

274 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN TABLE 2. (Continued) Species BL BW VS/OS Ovary Testes Vit EUL LEUL H. japonicus (Yamaguti, 1936) n. 3–11.9 1–2.5 1.16–2 I, L E, D 1a P OA comb. H. jeholensis (Fukui & Ogata, 1938) 5 1.4 1.16 K O, D 1a P OA n. comb. H. lipsiensis (Odening, 1958) n. 6.5 1 0.75 O O, D 2 P PT comb. H. lobatus Seno, 1907 7.5–11.7 0.9 0.33 – 0.4 I, L L, D 1a P PT H. nanchangensis Hsiung, 1934 1.4–3.8 1.6–2.5 0.86 O O, Pa 2 P OA H. pyrenaicus Combes, 1965 10.1; 7.6– 2.3; 1.8–3.2 0.66 I, L E, L, D 1b P PT 13.7 H. schulzei (Wundsch, 1911) 8–18 1.5–2 0.6 O O, D 1a P AT Prokopic & Krivanec, 1974 H. sibiricus (Isajcikov, 1927) 1.5–11.2 0.6–3.7 1 O, K E, D 1a P OA Yamaguti, 1958 H. similis (Looss, 1899) Yamaguti, 4.5–10 1–2 0.66–0.75 O O, D 1e P AT 1958 H. sindensis Khan & Mohiuddin, 4.9; 2.6–7.7 1.5; 0.7–2.7 0.5 O O, D 1a P OA 1982 H. singaporensis Yuen, 1962 5.0–8.5 1.2–2.3 0.66 I, L O, D 1a P OA H. tientsinensis Hsiung, 1934 1.7–6.6 0.6–1.4 1.0–1.16 O E, D 1a P OA H. travdarribus (Skrjabin & Antipin, 4.6 1.7 1.1 O O, Pa 1c P OA 1962) Yamaguti, 1971 H. variegatus (Rudolphi, 1819) 2.5–18 0.6–2.2 0.5–0.75 E, L O, E, D 1a P OA Looss, 1899 H. vojtkovae Prokopic & Krivanec, 3.7 1 0.76 O O, D 1e P PT 1974 H. volgensis (Sudarikov, 1950) 4.5 1.2 0.78 O O, D 1e P AT Yamaguti, 1958 A = absent. AT = extend to the level of anterior testis. BL = body length. BW = maximum body width. D = in diagonal. E = elongate. EUL = uterine loops invading extracecal space. Ex = extracecal. I = irregular. K = kidney–shaped. L = lobed. LEUL = longitudinal extracecal uterine loops. O = oval. OA = extend to ovary or acetabulum level. P = present. Pa = parallel. Ph = reaching the level of cecal bifurcation or pharynx. PT = extend to the level of posterior testis. SDA = short, diagonal, oriented anteriorly, not reaching the level of posterior testis. SDP = short, diagonal, oriented posteriorly. SDAP = short, diagonal, some oriented anteriorly and some posteriorly. T = in tandem. Vit = vitellaria.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 275 1a = follicles in clusters, extending from the posterior region of the cecal bifurcation to near end of ceca, invading intracecal space anterior to acetabulum and posterior to testes. 1b = follicles in clusters, extending from the posterior region of the cecal bifurcation to testes level on ovarian side and to near end of ceca on the opposite side of ovary, invading intracecal space only anterior to acetabulum. 1c = follicles in clusters, extending from the posterior region of the cecal bifurcation to testes level on ovarian side and to near end of ceca on the opposite side of ovary, invading intracecal space anterior to acetabulum and posterior to testes. 1d = follicles in clusters, extending from the posterior region of the cecal bifurcation to testes level on ovarian side and to near end of ceca on the opposite side of ovary, not invading intracecal space. 1e = follicles in clusters; extending from the posterior region of the cecal bifurcation to the level of anterior testis (as described for Skrjabinoeces spp. Sudarikov, 1950). 2 = follicles unclustered, extending from the posterior region of the cecal bifurcation to near end of ceca, invading intracecal space anterior to acetabulum and posterior to testes (as described for (H.) Anomolecithus spp. Odening, 1960). 3 = follicles in clusters; two preacetabular lateral branches, follicles invading intra and extracecal space and one postovarian central branch, invading intra and cecal space.

TABLE 3. Host and geographical distribution of Haematoloechus spp. (Host nomenclature based on Bossuyt et al. (2006) for Ranidae and Frost (2018) for other families). Species Region/Country Host Family : Subfamily Africa H. aubriae Bourgat, Roure & Kulo, 1996 Tropical (Central) Ranidae: Pyxicephalinae H. combesi Batchvarov & Bourgat, 1974 Tropical (West) Ranidae: Conrauinae H. darcheni Combes & Knoepffler, 1967 Tropical (Central) Ranidae: Conrauinae H. dollfusinus (Odening, 1958) Yamaguti, Tropical (Central) Ranidae: Ptychadeninae 1971 H. exoterorchis Rees, 1964 Tropical (West) Ranidae: Dicroglossinae H. johnsoni Bourgat, 1977 Tropical (West, Central) Ranidae: Dicroglossinae H. leonensis (Williams & Coker, 1967) n. Tropical (West) Odontobatrachidae comb. H. lobogonadus (Meskal, 1970) Gassmann, Tropical (East) Ranidae: Pyxicephalidae 1975 H. micrurus Rees, 1964 Tropical Ranidae: Dicroglossinae H. ocellati Gassmann, 1975 Tropical (Central) Hyperoliidae North America H. breviplexus Stafford, 1902 North America Ranidae: Raninae H. buttensis Ingles, 1936 U.S.A. Ranidae: Raninae H. caballeroi (Skrjabin & Antipin, 1962) n. Central Mexico Ranidae: Raninae; Ambystomatidae (Acc) comb. H. coloradensis (Cort, 1915) Ingles, 1932 U.S.A. Ranidae: Raninae; Bufonidae (Acc) H. complexus (Seely, 1906) Ingles, 1932 U.S.A. Ranidae: Raninae; Bufonidae (Acc); Hylidae: Hylinae (Acc) H. confusus Ingles, 1932 West U.S.A. Ranidae: Raninae H. danbrooksi León–Règagnon & Paredes– Southeast Mexico Ranidae: Raninae; Bufonidae (Acc) Calderón, 2002 H. elongatus Caballero & Sokoloff, 1934 Central Mexico Ranidae: Raninae H. floedae1 Harwood, 1932 U.S.A., Mexico, Costa Rica Ranidae: Raninae H. humboldtensis Zamparo, Ferrao, Brooks, West U.S.A. Ranidae: Raninae Bettaso & Mata–López, 2011 H. illimis Caballero, 1942 Central Mexico Ranidae: Raninae H. kernensis Ingles, 1932 West U.S.A. Ranidae: Raninae; Bufonidae (Acc) ...... continued on the next page

276 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN TABLE 3. (Continued) Species Region/Country Host Family : Subfamily H. longicollum León–Règagnon & Romero– West Mexico Ranidae: Raninae Mayén, 2017 H. longiplexus1 Stafford, 1902 Canada, U.S.A., Argentina Ranidae: Raninae; Ceratophryidae; Leptodactylidae: Leptodactylinae; Bufonidae H. macrorchis Caballero, 1941 Central Mexico Ranidae: Raninae H. medioplexus Stafford, 1902 Canada, U.S.A. Ranidae: Raninae; Bufonidae (Acc) H. mexicanus n. sp. Central Mexico Ranidae: Raninae H. nicolasi León–Règagnon, 2017 Southwest Mexico Ranidae: Raninae H. occidentalis n. sp. West Mexico Ranidae: Raninae; Bufonidae (Acc) H. oxyorchis West U.S.A. Ranidae: Raninae H. parcivitellarius Caballero, 1942 Central Mexico Ranidae: Raninae H. parviplexus (Irwin, 1929) Ingles, 1932 U.S.A Ranidae: Raninae; Bufonidae (Acc) H. pulcher Bravo–Hollis, 1943 Central Mexico Ambystomatidae; Ranidae: Raninae H. tumidus Ingles, 1932 West U.S.A. Ranidae: Raninae H. varioplexus Stafford, 1902 Canada U.S.A. Ranidae: Raninae; Bufonidae; Hylidae: Acridinae (Acc); Gekkonidae (Acc) H. veracruzanus n. sp. East Mexico Ranidae: Raninae South America H. arequipensis Ibáñez & Córdoba, 1979 Peru Telmatobiidae H. freitasi Mañé–Garzon & Gil, 1959 Uruguay Leptodactylidae: Leptodactylinae H. fuelleborni (Travassos & Darriba, 1930) Brazil Bufonidae; Leptodactylidae: Leptodactylinae Yamaguti, 1958 H. iturbei (Cordero & Vogelsang, 1939) Venezuela, Brazil Ranidae: Raninae Yamaguti, 1958 H. lutzi Freitas & Lent, 1939 Venezuela Ranidae: Raninae H. meridionalis1 León–Règagnon, Brooks & Costa Rica, Mexico Ranidae: Raninae Zelmer, 2001 H. neivai (Travassos & Artigas, 1927) Ingles, Brazil, Venezuela Leptodactylidae: Leptodactylinae; Ranidae: 1933 Raninae; Hylidae: Pseudinae (Acc) H. ozorioi Freitas & Lent, 1939 Uruguay Leptodactylidae: Leptodactylinae H. pukinensis Ibáñez & Córdoba, 1979 Peru Telmatobiidae Australia H. australis (Johnston, 1912) Yamaguti, 1958 Australia Pelodryadidae: Pelodryadinae; Limnodynastidae Eurasia H. abbreviatus (Bychowsky, 1932) Prokopic & Central and Oriental Europe Bombinatoridae Krivanec, 1974 H. almorai (Pande, 1937) Yamaguti, 1958 India Ranidae: Dicroglossinae H. asper Looss, 1899 Central and Oriental Europe Ranidae: Raninae; Bombinatoridae (Acc); Bufonidae (Acc) H. breviansa (Sudarikov, 1950) Yamaguti, Oriental Europe Ranidae: Raninae 1958 H. bychovskii Odening, 1958 Russia Ranidae: Raninae H. capyristes (Klein, 1905) Odening, 1958 India Ranidae: Dicroglossinae

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HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 277 TABLE 3. (Continued) Species Region/Country Host Family : Subfamily H. carbonelli Lluch, Navarro & Pérez–Soler, Spain Ranidae: Raninae; Bombinatoridae (Acc); 1991 Salamandridae: Salamandrinae (Acc) H. donicus (Shevchenko, 1965) n. comb. Ukraine Ranidae: Raninae H. japonicus (Yamaguti, 1936) n. comb. Far East Ranidae: Raninae H. jeholensis (Fukui & Ogata, 1938) n. comb. Far East Ranidae: Raninae H. lipsiensis (Odening, 1958) n. comb. Central Europe Ranidae: Raninae H. lobatus Seno, 1907 Far East Ranidae: Raninae H. nanchangensis Hsiung, 1934 Far East Ranidae: Raninae H. pyrenaicus Combes, 1965 Spain Ranidae: Raninae; Bufonidae (Acc) H. schulzei (Wundsch, 1911) Prokopic & Central and Oriental Europe Ranidae: Raninae Krivanec, 1974 H. sibiricus (Isajcikov, 1927) Yamaguti, 1958 Russia Ranidae: Raninae H. similis (Looss, 1899) Yamaguti, 1958 Central and Oriental Europe Ranidae: Raninae H. sindensis Khan & Mohiuddin, 1982 Pakistan Ranidae: Dicroglossinae H. singaporensis Yuen, 1962 Malaysia Ranidae: Dicroglossinae H. tientsinensis Hsiung, 1934 China Ranidae: Raninae H. travdarribus (Skrjabin & Antipin, 1962) Germany Ranidae: Raninae Yamaguti, 1971 H. variegatus (Rudolphi, 1819) Looss, 1899 Europe Ranidae: Raninae; Bufonidae (Acc) H. vojtkovae Prokopic & Krivanec, 1974 Czech Republic Bombinatoridae H. volgensis (Sudarikov, 1950) Yamaguti, Oriental Europe Ranidae: Raninae 1958 (Acc) = accidental infections. 1 = Species distributed in North and South America. For detailed host records, distribution and references see Appendix 1.

According to the phylogenetic hypotheses based on ribosomal and mitochondrial DNA presented herein, three main groups are consistently recovered (Figs. 1–3): H. longiplexus and relatives (Clade I), H. complexus and relatives (Clade II) and H. medioplexus and relatives (Clade III), with the African species H. exoterorchis and H. micrurus diverging early in the evolution of the group. Previous analyses show congruent grouping patterns, although less species were included (Snyder & Tkach, 2001; León-Règagnon & Brooks, 2003; León-Règagnon, 2010). Morphological evidence supports the inclusion of H. danbrooksi within Clade III, as sister species of H. medioplexus (both having a small ventral sucker and no longitudinal uterine loops) as shown in the 28S and concatenated analysis (Figs. 2 & 3). Mapping the morphological traits that lead to previous taxonomic arrangements into the tree, they appear scattered throughout the tree not supporting the monophyly of previously proposed groups (Fig. 12). Consequently, Ostiolum which was erected based on the absence of longitudinal uterine loops (Pratt 1903), Pneumobites which was erected based on the presence of longitudinal uterine loops extending to the pre-acetabular region of body (Ward 1917), and the subgenera Haematoloechus and Anomolecithus, erected based on either clustered or unclustered vitelline follicles (Odening 1958, 1960; Skrjabin & Antipin 1962), are invalid. Molecular evidence of species previously assigned to the subgenus Skrjabinoeces and their inclusion in a phylogenetic study is necessary to test if the distribution of the vitellaria limited to the pretesticular region of body is a character that reflects the evolution of the group. The phylogenetic position of H. exoterorchis, proposed to belong to Metahaematoloechus (Yamaguti 1971) is conflicting among the hypotheses obtained from ribosomal and mitochondrial DNA (Figs. 1 & 2); while it is placed as the sister species of all other species of Haematoloechus in the hypothesis based on COI mtDNA sequences, it is placed within the group in the hypothesis based on 28S rDNA sequences. Considering the low bootstrap support of those clades in either analysis, and that the only morphological character supporting its separation in a different genus is the position of the testes, we therefore include H. exoterorchis in Haematoloechus

278 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN until additional evidence is gathered. Sequences of H. neivai or H. iturbei (Cordero & Vogelsang, 1939), previously considered members of the genus Neohaematoloechus (Odening 1960), were not available and thus they were not included in the molecular analysis. Considering the extremely reduced size of the ventral sucker in H. medioplexus and H. meridionalis, and that they do not appear as closest relatives in the tree (Fig. 12), it is premature to separate H. aubriae, H. neivai and H. iturbei based solely on the absence of the ventral sucker.

FIGURE 12. Geographic distribution and morphological traits that lead to previous taxonomic assignments for species of Haematoloechus mapped on the phylogenetic hypothesis based on COI + 28S sequences. Outgroups: Brachycoelium salamandrae, Glypthelmins brownorumae, Opisthioglyphe ranae & Plagiorchis koreanus.

Of the 70 valid species in Haematoloechus, 27 occur in North America, 24 in Eurasia, 10 in Africa, 8 in South America, and 1 in Australia. Most species of Haematoloechus employ members of the Ranidae (sensu Bossuyt et al. 2006) as hosts, with accidental infections (few isolated records of species common in ranids) in the Bufonidae Gray, Ceratophydae Tschudi, Hylidae Rafinesque, and Salamandridae Goldfuss, and presumable colonization events to members of the families Ambystomatidae Gray (Mexico), Leptodactylidae Werner, Telmatobiidae Fitzinger (South America), Bombinatoridae Gray (Europe), Odontobatrachidae Barej, Schmitz, Günther, Loader, Mahlow & Rödel, Hyperoliidae Laurent (Africa), Limnodynastidae Lynch and Pelodryadidae Günther (Australia) (Table 3). According to the diversification hypothesis for the Ranidae proposed by Bossuyt et al. (2006), this family originated in the southern hemisphere, with colonization routes from Gondwana to Laurasia via India and the Australia-New Guinea plate, and a recent colonization of South America through North America. The host records for species of Haematoloechus (Table 3), and the partial phylogenetic hypothesis for the genus presented herein, suggest that this host-parasite association predates the ranid diversification in the Cretaceous (Bossuyt et al. 2006). Several African species of Haematoloechus (H. aubriae, H. combesi, H. darcheni, H. dollfusinum, and H. lobogonadus (Meskal, 1970)) parasitize members of the Conrauinae, Ptychadeninae and Pyxicephalinae, groups that originated early in the radiation of the Ranidae in Africa (Bossuyt et al. 2006). When ancestral ranids

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 279 colonized Europe (Rana and Pelophylax) and the New World (Rana) in the Oligocene or Miocene (Bossuyt et al. 2006), they must have been already associated with Haematoloechus. This is clearly reflected in the phylogenetic hypothesis for Haematoloechus presented herein, as the African species H. exoterorchis and H. micrurus appear to have diverged early in the evolution of the group, and European and American species are present in the three larger clades of the tree (Fig. 12). Although the diversity of Haematoloechus appears much greater in the Palearctic and Nearctic regions, this could be the result of poor sampling efforts in other continents. However, the low diversity of ranids in South America and Australia may be an important contributing factor. Haematoloechus has colonized other groups of anurans (Leptodactylidae, Telmatobiidae) in South America; we would expect that South American species of Haematoloechus appear in a deeply nested position in the tree when included in the analysis, considering the recent colonization of ranids in this region and the presumed colonization of Haematoloechus to other groups of anurans, as apparently occurred with H. pulcher, that colonized members of the family Ambystomatidae in Mexico (Fig. 12). The same would be expected for species that parasitize other groups of amphibians in Europe (Bombinatoridae and Bufonidae) and Africa (Odontobatrachidae and Hyperoliidae), when additional representatives of the genus from those areas are included in the analysis. In spite of the supposedly complex and specialized life cycle of species of Haematoloechus that involves a snail and an insect as intermediate hosts and an amphibian as definitive host, host-switching events are possible and have been documented at least in one occasion (León-Règagnon et al. 2005). These host-switching events are possible for supposedly "host specific" parasite species due to ecological fitting (Janzen 1985), which in this case, is the capacity of the parasite to track widespread, phylogenetically conserved traits in the host, rather than particular host species (Brooks et al. 2006; Agosta et al. 2010; Hernández-Orts et al. 2017). This would explain the multiple host-switching events to different groups of anurans during the evolution of this group of digeneans.

Acknowledgments

The authors are grateful to María Antonieta Arizmendi-Espinoza, María Angélica Najar-Pacheco, David Osorio- Sarabia, Gerardo Pérez-Ponce de León, Ulises Razo-Mendívil, Ángeles Romero–Mayén, María Guadalupe Velarde-Aguilar, Alejandro Zaldívar Riverón (Instituto de Biología, UNAM), Rosario Mata-López, Edmundo Pérez-Ramos, (Facultad de Ciencias, UNAM), Elisa Cabrera-Guzmán (Oklahoma State Univeristy), Luis Jorge García-Márquez (Universidad de Colima), Agustín Jiménez-Ruiz (Southern Illinois Univeristy), Sean Locke (Universidad de Puerto Rico), and Elizabeth Martínez-Salazar (Universidad Autónoma de Zacatecas), for their help in the collection of specimens; Laura Márquez-Valdelamar (IBUNAM) for her help in the sequencing of samples; Luis García-Prieto (CNHE), Anna Phillips (NMNH), Jean-Marc Gagnon and Yemisi Dare (CMNPA), Ann Price (NHM) and Dely Noronha and Luis Muniz (IOC) for the loan of specimens. Thanks to Martins Aisien, Robert Bourgat, Scott Gardner, Hideo Hasegawa, Monika Hamann, José Iannacone and Georgina Ortega–Leite for their help in obtaining copies of original descriptions and related literature and Paul Hebert for his revision and comments on early versions of the manuscript. Special thanks to the associate editor of the journal and two anonymous reviewers for their careful and thorough revision of the manuscript. This study was partially funded by PAPIIT-UNAM Proj. IN203911 and IN209414, CONACyT Proj. 54475, 220408 and PASPA-2016 to VLR and by an award (Food From Thought) from the Canada First Research Excellence Fund to Paul Hebert.

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APPENDIX 1. Species historically included in the genera Haematoloechus (=Pneumonoeces), Ostiolum, Ostioloides, Skrjabinoeces, Neohaematoloechus or Metahaematoloechus listed by geographical region.

Africa Haematoloechus aubriae Bourgat, Roure & Kulo, 1996 Type host and locality: Brown ball frog Aubria subsigillata (Duméril), Porto–Novo, Benin (Bourgat et al. 1996). Other records: Nigeria: A. subsigillata Gelegele (Aisien et al. 2009). Taxonomic notes: Valid species, nominally described.

Haematoloechus combesi Batchvarov & Bourgat, 1974 Type host and locality: Togo slippery frog Conraua derooi Hulselmans, Klouto, Togo (Batchvarov & Bourgat 1974). Taxonomic notes: Valid species, nominally described.

Haematoloechus darcheni Combes & Knoepffler, 1967 Type host and locality: Abo slippery frog Conraua crassipes (Buchholz & Peters), Makokou, Gabon (Combes & Knoepffler 1967). Other records: Cameroon: Abo slippery frog C. crassipes (Gassmann 1975). Taxonomic notes: Valid species, nominally described.

Haematoloechus dollfusinus (Odening, 1958) Yamaguti, 1971 Type host and locality: Mascarene grass frog Ptychadena mascareniensis (Duméril & Bibron), Democratic Republic of the Congo (Odening 1958). Taxonomic notes: Valid species, described as Haematoloechus (Ostiolum) dollfusinus from material recorded by Dollfus in 1950 as Pneumonoeces variegatus (Odening 1960).

Haematoloechus exoterorchis Rees, 1964 Type host and locality: crowned bullfrog Hoplobatrachus occipitalis (Günther), Achimota, Legon and Nungua, Ghana (Rees 1964). Other records: Cameroon: H. occipitalis (Gassmann 1975); Ghana: H. occipitalis (Fischthal & Thomas 1968); Nigeria: H. occipitalis (Aisien et al. 2001, 2004; Imasuen & Aisien 2012; Aisien et al. 2015); Sierra Leone: H. occipitalis (León- Régagnon & Brooks 2003). Taxonomic notes: Valid species, nominally described; Yamaguti (1971) proposed the new genus Metahaematoloechus for this species based on the extracecal position of the testes, a character that is unique to this species. Molecular evidence from rDNA and mtDNA is conflicting (Figs. 1 & 2). As a result, we consider this species a member of Haematoloechus until additional evidence is gathered.

Haematoloechus johnsoni Bourgat, 1977 Type host and locality: crowned bullfrog Hoplobatrachus occipitalis, Awagome and Klikame, Togolese Republic (Bourgat 1977). Other records: Benin: H. occipitalis (Aisien et al. 2011); Sudan: H. occipitalis (Pike 1979). Taxonomic notes: Valid species, nominally described.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 291 Haematoloechus leonensis (Williams & Coker, 1967) n. comb. Type host and locality: Sierra Leone water frog Odontobatrachus natator (Boulenger 1905), Mount Aureole, Sierra Leone (Williams & Coker 1967). Taxonomic notes: Valid species, described as Haematoloechus variegatus leonensis.

Haematoloechus lobogonadus (Meskal, 1970) Gassmann, 1975 Type host and locality: Angolan river frog Amietia angolensis, Dima River, Ethiopia (Meskal 1970). Taxonomic notes: Valid species, described as Haematoloechus (Skrjabinoeces) lobogonadus.

Haematoloechus micrurus Rees, 1964 Type host and locality: crowned bullfrog Hoplobatrachus occipitalis (Günther), Achimota, Legon and Nungua, Ghana (Rees 1964). Other records: Cameroon: H. occipitalis (Gassmann 1975); Ghana: H. occipitalis (Fischthal & Thomas 1968); Ivory Coast: H. occipitalis (Baer 1972; Maeder 1973); Nigeria: H. occipitalis (Aisien et al. 2004, 2009); Sierra Leone: H. occipitalis (León-Régagnon & Brooks 2003); Sudan: H. occipitalis (Pike, 1979); Zaire: H. occipitalis (Fischthal 1977). Taxonomic notes: Valid species, nominally described.

Haematoloechus ocellati Gassmann, 1975 Type host and locality: golden–eyed reed frog Hyperolius ocellatus Günther, Nomayos, Cameroon (Gassmann 1975). Taxonomic notes: Valid species, nominally described.

Ostioloides rappiae (Szidat, 1932) Odening, 1960 Synonym: Haematoloechus rappiae (Szidat 1932) Yamaguti, 1958 Type host and locality: variable reed frog Hyperolius concolor (Hallowell), Liberia Other records: Ghana, H. concolor, long–nosed reed frog H. nasutus (Günther), long reed frog H. igbettensis Schiotz, plain reed frog H. nitidulus Peters, fire–sided skink Trachylepis perrotetii (Duméril & Bibron) (Fischthal & Thomas 1968). Taxonomic notes: Valid species, described as Haplometroides rappiae Szidat 1932, transferred to Haematoloechus by Yamaguti (1958) and used as the type species to erect Ostioloides Odening 1960. The intestinal ceca extending only two thirds of body and the post-bifurcal position of the genital pore support the position of O. rappiae in an independent genus (Odening 1960); nevertheless, it is necessary to obtain molecular evidence to test this hypothesis.

North & South America Haematoloechus arequipensis Ibáñez & Córdoba, 1979 Type host and locality: Peru water frog Telmatobius peruvianus Weigmann; Río Yarabamba, Arequipa, Peru (Ibáñez & Córdoba 1979). Taxonomic notes: Valid species, nominally described.

Haematoloechus breviplexus Stafford, 1902 Type host and locality: bullfrog Rana catesbeiana, Quebec and Ontario, Canada (Stafford 1902). Other records: Canada: R. catesbeiana, British Columbia, Nova Scotia and Ontario (Kennedy 1981); Ontario (Stafford 1905); Quebec (Fantham & Porter 1948); Northern leopard frog R. pipiens, Nova Scotia (Stafford 1902) host recorded as R. virescens Cope; Mexico: Tarahumara frog R. tarahumarae Boulenger, Sonora (Bursey & Goldberg 2001); U.S.A.: bullfrog R. catesbeiana, Arkansas (Rosen & Manis 1976); Indiana (Cort 1915, Lank 1971; Whitehouse 2002); Kentucky (Whitehouse 2002); Massachusetts (Kennedy 1981); Mississippi (Clark & Longest 1969; Brooks 1979); Nevada (Babero & Golling 1974); New Mexico (Dronen 1977); Oklahoma (Cort 1915; Trowbridge & Hefley 1934); Texas (Knight et al. 1965; Slagle 1966; Hollis 1972; Underwood & Dronen 1977; Yoder & Gomez 2007); Virginia (Campbell 1968); green frog R. clamitans, Indiana (Cort 1915), Michigan (Najarian 1955), Virginia (Campbell 1968); pig frog R. grylio Stejneger, Louisiana (Kennedy 1981); Columbia spotted frog R. luteiventris Thompson, Montana (León-Règagnon & Brooks 2003); Northern leopard frog R. pipiens, New Mexico (Dronen 1977); Texas (Slagle 1966); mink frog R. septentrionalis Baird, Minnesota (Schotthoefer et al. 2009); Southern leopard frog R. sphenocephala Cope, Florida (Kennedy 1981) recorded as R. utricularia Harlan; Texas (Underwood & Dronen 1977). Taxonomic notes: Valid species, nominally described; treated as H. (H.) breviplexus breviplexus by Odening (1960). Record of Snyder & Tkach (2001) of this species corresponds to H. floedae (León-Règagnon & Brooks 2003). Specimens from Oklahoma identified by Cort (1915) as H. breviplexus were re–determined by León-Règagnon et al. (2005) as H. floedae.

Haematoloechus buttensis Ingles, 1936 Type host and locality: yellow–legged frog Rana boylii Baird, California, U. S. A. (Ingles 1936). Other records: bullfrog R. catesbeiana, U. S. A.: Nevada (Babero & Golling 1974); Oregon spotted frog R. pretiosa Baird & Girard, Canada: British Columbia (Kennedy 1980a, 1981). Taxonomic notes: Valid species, nominally described; treated as H. (H.) buttensis by Odening (1960). Kennedy (1981) considered this species to be a junior synonym of H. varioplexus Stafford, 1902 based on morphological variability. Recent

292 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN molecular evidence support the morphological characters used to differentiate this species indicating its validity (Zamparo et al. 2011); these authors suggest the specimens examined by Kennedy (1980a, 1981) from British Columbia, Canada, belong to an undescribed species.

Haematoloechus caballeroi (Skrjabin & Antipin, 1962) n. comb. Type host and locality: Tlaloc´s leopard frog Rana tlaloci Hillis & Frost (=Rana montezumae of Caballero, 1942b), Xochimilco, Mexico City, Mexico (Skrjabin & Antipin 1962). Other records: R. tlaloci, Xochimilco, Mexico City (Caballero & Sokoloff 1934); Montezuma leopard frog R. montezumae, Ciénaga de Lerma, Mexico State (León-Règagnon 1992; Lamothe et al. 1997; León-Règagnon et al. 1999; Pérez-Ponce de León et al. 2000); big–footed leopard frog R. megapoda, Cointzio Springs, Michoacán (Pérez-Ponce de León et al. 2000); Transverse Volcanic leopard frog R. neovolcanica, Cointzio Springs, Michoacán (Pérez-Ponce de León et al. 2000); Patzcuaro leopard frog R. dunni, Pátzcuaro, Michoacán (García-Altamirano et al. 1993; Pulido–Flores 1994; Pérez-Ponce de León et al. 2000); Lake Lerma salamander Ambystoma lermaense, Ciénaga de Lerma, Mexico State (Pérez-Ponce de León et al. 2000; Mata-López et al. 2002). Taxonomic notes: Valid species, described as Pneumonoeces caballeroi Skrjabin & Antipin, 1962 based on material recorded by Caballero (1942b) as Haematoloechus complexus (Seely 1906), and redescribed herein.

Haematoloechus coloradensis (Cort, 1915) Ingles, 1932 Type host and locality: Northern leopard frog Rana pipiens, Colorado, U.S.A. (Cort 1915). Other records: U.S.A.: Woodhouse´s toad Anaxyrus woodhousii (Girard), Nebraska (Conant & Collins 1998; Bolek & Janovy 2007a), Utah (Frandsen & Grundman 1960, Parry & Grundman 1965); Arizona toad A. microscaphus (Cope), Utah (Frandsen & Grundman 1960, Parry & Grundman 1965); Rio Grande leopard frog R. berlandieri, New Mexico (Dronen 1978); Plains leopard frog R. blairi, Nebraska (Brooks 1976); Northern leopard frog R. pipiens, Idaho (Bolek & Janovy 2007a), Montana (USNPC Coll. by McIntosh 1937), Nebraska (Brooks 1976; Bolek & Janovy 2007a), New Mexico (Dronen 1975); Utah (Frandsen & Grundman 1960; Parry & Grundman 1965). Taxonomic notes. Valid species, described as Pneumonoeces coloradensis (Cort, 1915); treated as Ostiolum coloradense by Odening (1960). Kennedy (1981) considered this species to be a junior synonym of H. complexus based on morphological variation; recent morphological evidence supports its validity (Bolek & Janovy 2007a). Records of this species in Montezuma leopard frog R. montezumae and Patzcuaro leopard frog R. dunni of Mexico (Caballero & Sokoloff 1934; García-Altamirano et al. 1993; Pulido–Flores 1994; Lamothe et al. 1997; Pérez-Ponce de León et al. 2000) correspond to H. caballeroi re–described herein. Records of H. coloradensis in Forrer´s leopard frog R. cf. forreri in Mexico (Cabrera– Guzmán et al. 2007; Velázquez & León-Règagnon 2017) correspond to H. occidentalis n. sp. described herein. Record of León-Règagnon et al. (1999) in Patzcuaro leopard frog R. dunni from Pátzcuaro, Michoacán, belongs to H. mexicanus n. sp. described herein.

Haematoloechus complexus (Seely, 1906) Ingles, 1932 Type host and locality: Northern leopard frog Rana pipiens, North Carolina, U.S.A. (Seely 1906). Other records: U.S.A.: Woodhouse´s toad Anaxyrus woodhousii, Nebraska (Brooks 1976; Bolek & Janovy 2007a); Cope´s gray treefrog Dryophytes chrysoscelis (Cope), Nebraska (Bolek & Janovy 2007a); Plains leopard frog R. blairi Mecham, Littlejohn, Oldham, Brown & Brown, Colorado, Iowa and Kansas (Goldberg et al. 2000), Nebraska (Brooks 1976; Goldberg et al. 2000; Snyder & Tkach 2001; Bolek & Janovy 2007a; Langford et al. 2013); Chiricahua leopard frog R. chiricahuensis Platz & Mecham, Arizona (Goldberg et al. 1998); green frog R. clamitans, North Carolina (Krull 1933), Indiana and Kentucky (Whitehouse 2002); Wisconsin (Bolek & Janovy 2007a); Northern leopard frog R. pipiens, Indiana and Kentucky (Whitehouse 2002), Iowa (Ulmer 1970), Nebraska (Brooks 1976), North Carolina (Cort 1915); Southern leopard frog R. sphenocephala, Indiana and Kentucky (Whitehouse 2002); wood frog R. sylvatica LeConte, Ohio (Catalano & White 1977); Yavapai leopard frog R. yavapaiensis Platz & Frost 1984, Arizona (Goldberg et al. 1998); spring piper Pseudacris crucifer (Wied–Neuwied), Ohio (Catalano & White 1977); Mexico: Northwest Mexico leopard frog R. magnaocularis Frost & Bagnara, Sinaloa (Goldberg & Bursey 2002a). Taxonomic notes. Valid species, described as Pneumonoeces complexus (Seely 1906); treated as Ostiolum complexus by Odening (1960). According to Bolek & Janovy (2007a), Nebraska appears to be the western geographic limit for H. complexus; records from Colorado and Arizona, U.S.A. and Sinaloa, Mexico (Goldberg et al. 1998, 2000; Goldberg & Bursey 2002a) might correspond to other species, nevertheless, there is no way of corroborating these identifications because no voucher specimens were deposited. Records of H. complexus or H. cf. complexus from Montezuma leopard frog R. montezumae and Lerma Lake salamander Ambystoma lermaensis in Mexico (Caballero 1942b; León-Règagnon 1992; Lamothe et al. 1997; León-Règagnon et al. 1999; Pérez-Ponce de León et al. 2000; Mata-López et al. 2002; León- Règagnon & Brooks 2003) correspond to H. caballeroi re–described herein. Records of H. complexus in common marsh frog R. vaillanti (Paredes-Calderón et al. 2004), Rio Grande leopard frog R. berlandieri (León-Règagnon 2003; León- Règagnon et al. 2005) correspond to H. veracruzanus n. sp. described herein. COI sequence of H. complexus reported by León-Règagnon (2010) does not belong to this species (Fig. 1). Specimens of H. complexus reported in Rio Grande leopard frog R. berlandieri in Nuevo León, México by Martínez (1969) were not available for examination.

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 293 Haematoloechus confusus Ingles, 1932 Type host and locality: Northern red–legged frog Rana aurora Baird & Girard, California, U.S.A. (Ingles 1932). Taxonomic notes: Valid species, nominally described. Odening (1960) viewed this species as a junior synonym of H. oxyorchis Ingles, 1932 while Kennedy (1981) considered it a synonym of H. complexus Seely, 1906 based on morphological variation. This species was described from a few specimens collected from a single host (Ingles 1932), and it has not been recorded afterwards. Nevertheless, some characters that have proven to be useful to differentiate species such as the sucker ratio, oral sucker/pharynx ratio, shape of gonads and arrangement of uterine loops (León-Règagnon et al. 1999; León- Règagnon & Brooks 2003; Bolek & Janovy 2007a, 2007b; Zamparo et al. 2011) differentiate this species from the other two.

Haematoloechus danbrooksi León-Règagnon & Paredes-Calderón, 2002 Type host and locality: common marsh frog Rana vaillanti, Veracruz, Mexico (León-Règagnon & Paredes-Calderón 2002). Other records: Mexico: Southern Gulf Coast toad Incilius valliceps (Weigmann), Rio Grande leopard frog R. berlandieri, common marsh frog R. vaillanti, cane toad Rhinella marina (Linnaeus), Veracruz (Guillén-Hernández et al. 2000); common marsh frog R. vaillanti, Veracruz, Mexico (León-Règagnon et al. 1999), both records originally as H. medioplexus; common marsh frog R. vaillanti, Catemaco, Veracruz (this study CNHE10404). Taxonomic notes: Valid species, nominally described. Validated using morphological and molecular evidence (León-Règagnon & Paredes-Calderón 2002; León-Règagnon & Brooks 2003; León-Règagnon 2010).

Haematoloechus elongatus Caballero & Sokoloff, 1934 Type host and locality: Montezuma leopard frog Rana montezumae, Estado de Mexico, Mexico (Caballero & Sokoloff 1934). Other records: Mexico: Tlaloc´s leopard frog Rana tlaloci Hillis & Frost originally recorded as Northern leopard frog R. pipiens, Mexico City (Caballero & Sokoloff 1934). Taxonomic notes: Valid species, nominally described; treated as Ostiolum elongatum by Odening (1960). Records of H. elongatus in Montezuma leopard frog R. montezumae in Texcoco Lake (Lamothe-Argumedo et al. 1997) correspond to an undescribed species (León-Règagnon pers. obs.); records in showy leopard frog R. spectabilis and Zweifel´s frog R. zweifeli (León-Règagnon 2003) correspond to H. longicollum León-Règagnon & Romero–Mayén 2017 (León-Règagnon & Romero–Mayén 2017).

Haematoloechus floedae Harwood, 1932 Synonym: Haematoloechus uniplexus Harwood 1932 (syn. fide Odening 1960) Type host and locality: bullfrog Rana catesbeiana, Texas, U.S.A. (Harwood 1932). Other records: Costa Rica: Forrer´s leopard frog R. cf. forreri, common marsh frog R. vaillanti, Guanacaste (León-Règagnon et al. 2005); Mexico: Brown´s leopard frog R. brownorum, Yucatán (León-Règagnon et al. 2005; Yáñez–Arenas & Guillén- Hernández 2010); common marsh frog R. vaillanti, Yucatán (León-Règagnon et al. 2005); Forrer´s leopard frog R. cf. forreri, Chiapas (Velázquez & León-Règagnon in press), Colima, Jalisco (this study CNHE 10471–10473); Rana sp. Michoacán, Oaxaca (this study CNHE 10474, 10475, 10520); U.S.A.: bullfrog R. catesbeiana, Arizona (Snyder & Tkach 2001) recorded as H. breviplexus, California (León-Règagnon et al. 2005), Florida (Manter 1938); Georgia (León- Règagnon & Brooks 2003), Illinois (Andrews et al. 1992), Nevada (Babero & Golling 1974); South Carolina (USNPC), Texas (Jacobs & Morrison 1966; León-Règagnon et al. 2005); green frog R. clamitans, Texas (Harwood 1932); Southern leopard frog R. sphenocephala, Texas (Harwood 1932 as H. uniplexus). Taxonomic notes: Valid species, nominally described; treated as H. (H.) breviplexus floedae by Odening (1960). Harwood (1932) described Haematoloechus uniplexus based on one single specimen, and he noted that the specimen might be an "example of H. floedae that is somewhat stunted and malformed by residence in an unsuitable host". Kennedy (1981) considered H. floedae to be a junior synonym of H. varioplexus based on morphological variation; nevertheless, a revision of museum specimens at the light of molecular evidence lead to the validation of this species and re–determination of specimens originally recorded as H. breviplexus (Cort 1915 in part; Snyder & Tkach 2001), as H. floedae (León-Règagnon & Brooks 2003; León-Règagnon et al. 2005).

Haematoloechus freitasi Mañé–Garzon & Gil, 1959 Type host and locality: butter thin–toed frog Leptodactylus latrans (Steffen) (recorded as common thin–toed frog R. ocellatus Girard), Canelones, Uruguay (Mañé–Garzón & Gil 1959). Taxonomic notes: Valid species, nominally described.

Haematoloechus fuelleborni (Travassos & Darriba, 1930) Yamaguti, 1958 Type host and locality: cane toad Rhinella marina, Sao Paulo, Brasil (Travassos & Darriba 1930). Other records: Brazil: oven thin–toed frog Leptodactylus latinasus Jiménez de la Espada, South–Eastern Brazil (Toledo et al. 2015); yellow Cururu toad Rhinella icterica (Spix), Alto Paraná (Lux-Hoppe et al. 2008). Taxonomic notes: Valid species, described as Pneumonoeces fuelleborni (Travassos & Darriba 1930); treated as H. (Anomolecithus) fuelleborni by Odening (1960).

294 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN Haematoloechus humboldtensis Zamparo, Ferrao, Brooks, Bettaso & Mata-López, 2011 Type host and locality: yellow–legged frog Rana boylii, California, U.S.A. (Zamparo et al. 2011). Taxonomic notes: Valid species, nominally described. Validated using morphological and molecular evidence (Zamparo et al. 2011).

Haematoloechus illimis Caballero, 1942 Type host and locality: Montezuma leopard frogs Rana montezumae, Lerma, Estado de Mexico, Mexico (Caballero 1942a). Other records: Mexico: R. montezumae, Lerma, Estado de Mexico (Caballero 1941, as H. varioplexus; Caballero 1942c; León- Règagnon et al. 1999; Pérez-Ponce de León et al. 2000); Texcoco, Estado de Mexico; Xochimilco, Mexico City (Lamothe- Argumedo et al. 1997, as H. varioplexus); Zempoala, Morelos (this study, CNHE 19476). Taxonomic notes: Valid species, nominally described; treated as H. (Ostiolum) illime by Odening (1960). Validated using molecular evidence (León-Règagnon 1999). Caballero (1942a) described this species based on one single specimen; examination of additional material deposited in the CNHE revealed that uterine longitudinal loops may almost reach the posterior testis.

Haematoloechus iturbei (Cordero & Vogelsang, 1939) Yamaguti, 1958 Type host and locality: Amazon River frog Rana palmipes, Maracay, Venezuela (Cordero & Vogelsang 1939). Other records: Brazil: R. palmipes, Pernambuco (Dobbin 1957). Taxonomic notes: Valid species, described as Pneumonoeces iturbei (Cordero & Vogelsang 1939); treated as Neohaematoloechus iturbei by Odening (1960). Uribe–Piedrahita (1948) reported H. medioplexus in R. palmipes in Colombia, but later Dobbin (1957) considered those specimens to be H. iturbei. According to León-Règagnon et al. (2001), they correspond to H. meridionalis based on the description and drawing made by Uribe–Piedrahita (1948). Record of H. iturbei in R. palmipes from Costa Rica (León-Règagnon et al. 1999), corresponds to H. meridionalis in R. vaillanti (León-Règagnon et al. 2001). Caballero & Díaz–Ungria (1958) synonymized H. iturbei with H. neivai without justification. Odening (1960) did not agree with this synonymization considering the consistent morphological differences between both species; we concur.

Haematoloechus kernensis Ingles, 1932 Type host and locality: Northern red–legged frog Rana aurora, California, U.S.A. (Ingles 1932). Other records: U.S.A.: R. aurora, California (Ingles 1936); yellow–legged frog R. boylii, California (Bursey et al. 2010); Western toad Anaxyrus boreas (Baird & Girard), California (Goldberg et al. 1999). Taxonomic notes: Valid species, nominally described; treated as H. (H.) kernensis by Odening (1960).

Haematoloechus legrandi Mañé–Garzón & Gil, 1959 Type host and locality: butter thin–toed frog Leptodactylus latrans (recorded as L. ocellatus), Canelones, Uruguay (Mañé– Garzón & Gil 1959). Taxonomic notes: Species inquirendae, described based on a single, incomplete specimen, no drawing provided in original description; specimen not available for examination.

Haematoloechus longicollum León-Règagnon & Romero–Mayén, 2017 Type host and locality: smooth–backed frogs Rana psilonota Webb, Jalisco, Mexico (León-Règagnon & Romero–Mayén 2017). Other records: Mexico: Rana sp., Colima; Zweifel´s frog R. zweifeli, Guerrero, Michoacán, Morelos, Oaxaca (León-Règagnon & Romero–Mayén 2017). Taxonomic notes: Valid species, nominally described. Validated using morphological and molecular evidence (León-Règagnon & Romero–Mayén 2017).

Haematoloechus longiplexus Stafford, 1902 Type host and locality: bullfrog Rana catesbeiana, Ontario, Canada (Stafford 1902) Other records: Argentina: Chacoan horned frog Ceratophrys cranwelli Barrio, Santa Fe (Hamann & Pérez 1999); Vizcacheras' white-lipped thin-toed frog Leptodactylus bufonius Boulenger, Corrientes (Hamann et al. 2012); Chaco thin–toed frog L. chaquensis Cei, Corrientes (Hamann & Pérez 1999; Schaefer et al. 2006; Hamann et al. 2006a, 2013a); oven thin–toed frog L. latinasus, Corrientes (Hamann et al. 2006b, 2013a); butter thin–toed frog L. latrans, Corrientes (Hamann et al. 2013a); Bolivian swamp frog Pseudopaludicola boliviana Parker, Corrientes (Hamann et al. 2013a); Bella Vista toad Rhinella fernandezae (Gallardo), Corrientes (Hamann & Pérez 1999; Hamann et al. 2013a, 2013b); Canada: bullfrog R. catesbeiana, Quebec, New Brunswick, Nova Scotia (Stafford 1902); bullfrog R. catesbeiana, green frog R. clamitas, Northern leopard frog R. pipiens, New Brunswick (McAlpine 1997); Cuba: bullfrog R. catesbeiana (Odening 1960); U.S.A.: Woodhouse´s toad Anaxyrus woodhousii, Nebraska (Brooks 1976); Plains leopard frog R. blairi, Nebraska, (Brooks 1976; Bolek and Janovy 2007b); bullfrog R. catesbeiana, Arizona (Goldberg et al. 1998), Arkansas (Rosen & Manis 1976); California (Shields 1987; Goldberg & Bursey 2002b); Connecticut (Brooks 1976); Idaho (Waltz 1961; Schell 1965); Illinois (Cort 1915), Indiana (Cort 1915; Lank 1971; Whitehouse 2002), Iowa (Ulmer 1970, Cain & French

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 295 1975); Kentucky (Whitehouse 2002); Louisiana (Bennet 1938), Michigan (Krull 1932; Najarian 1955; Muzall 1991), Mississippi (Clark & Longest 1969); Nebraska (Manter 1938; Brooks 1976; León-Règagnon et al. 1999; Snyder & Tkach 2001; Bolek & Janovy 2007a); Nevada (Babero & Golling 1974), North Carolina (Brandt 1936), Ohio (Ashton & Rabalais 1978; Bursey & DeWolf 1998); Oklahoma (Trowbridge & Hefley 1934; Brooks 1976), Texas (Harwood 1932), Washington (Schell 1965); green frog R. clamitans, Indiana and Kentucky (Whitehouse 2002); Maine (Bouchard 1951); Ohio (Bursey & DeWolf, 1998), Texas (Harwood, 1932); pig frog R. grylio, Florida (Manter 1938; Parker 1941); Northern leopard frog R. pipiens, Idaho and Washington (Schell 1965), Illinois (Cort 1915), Iowa (Ulmer 1970); Oregon spotted frog R. pretiosa, Idaho and Washington (Schell 1965); mink frog R. septentrionalis, Maine (Bouchard 1951). Taxonomic notes: Valid species, nominally described; treated as H. (H.) longiplexus longiplexus by Odening (1960). Records from Argentina are based solely on morphological evidence; Hamman & Pérez (1999) noticed some differences with H. longiplexus from North America, but they concluded they were intraspecific variation. Considering the hosts and geographic distribution, additional evidence is needed to corroborate if specimens from Argentina are conspecific with H. longiplexus.

Haematoloechus lutzi Freitas & Lent, 1939 Synonym: H. tejerae Cordero & Vogelsang, 1939 (syn. fide Dobbin 1957). Type host and locality: Amazon River frog Rana palmipes, Maracay, Venezuela (Freitas & Lent 1939). Other records: Venezuela: R. palmipes, Maracay (Cordero & Vogelsang 1939; Caballero et al. 1953). Taxonomic notes: Valid species, nominally described; treated as H. (Anomolecithus) lutzi by Odening (1960).

Haematoloechus macrorchis Caballero, 1941 Type host and locality: Montezuma leopard frog Rana montezumae, Estado de Mexico, Mexico (Caballero 1941). Other records: Mexico: R. montezumae, Estado de Mexico (Lamothe-Argumedo et al. 1997; León-Règagnon et al. 1999; Pérez-Ponce de León 2000); Tlaloc´s leopard frog R. tlaloci (originally recorded as R. pipiens), Mexico City (Caballero 1941). Taxonomic notes: Valid species, nominally described; treated as H. (H.) longiplexus macrorchis by Odening (1960). León- Règagnon et al. (1999) and Pérez-Ponce de León et al. (2000) considered H. macrorchis to be a junior synonym of H. longiplexus. Further molecular evidence supported the validity of this species (León-Règagnon & Brooks 2003).

Haematoloechus medioplexus Stafford, 1902 Synonym: H. formosum Pratt, 1903 (syn. fide Stafford 1905; Cort 1915) Type host and locality: Northern leopard frog Rana pipiens (originally recorded as Rana virescens), Ontario, Canada (Stafford 1902). Other records: Canada: Southern toad Anaxyrus terrestris (originally recorded as common toad Bufo lentiginosus Shaw), New Brunswick, Nova Scotia (Stafford 1902); Northern leopard frog R. pipiens, Quebec (Stafford 1902; King et al. 2007), New Brunswick (McApline 1997); U.S.A.: American toad Anaxyrus americanus, Iowa (Ulmer 1970), Michigan (Krull 1931); Plains leopard frog R. blairi, Nebraska (Brooks 1976); bullfrog R. catesbeiana, Iowa (Cain & French 1975); green frog R. clamitans, Massachusetts (Rankin 1945), Michigan (Fortner 1923); pickerel frog R. palustris (LeConte), Maine (Bouchard 1951), Massachusetts (Rankin 1945); Northern leopard frog R. pipiens, Connecticut (Brooks 1976), Idaho (Waltz 1961), Illinois (Cort 1915), Iowa (Ulmer 1970), Massachusetts (Rankin 1945), Michigan (Fortner 1923; Krull 1930, 1931), Minnesota (Hollis 1972), Nebraska (Brooks 1976; León-Règagnon et al. 1999), Wisconsin (Snyder & Tkach 2001); wood frog R. sylvatica, Wisconsin (Williams & Taft 1980). Taxonomic notes: Valid species, nominally described; treated as Ostiolum medioplexus (Stafford, 1902) Odening, 1960 by Odening (1960). Uribe–Piedrahita (1948) reported H. medioplexus in R. palmipes in Colombia; later Dobbin (1957) considered those specimens to be H. iturbei. According to León-Règagnon et al. (2001), they correspond to H. meridionalis based on the description and drawing made by Uribe–Piedrahita (1948). Records of H. medioplexus in Veracruz, Mexico (León-Règagnon et al. 1999; Guillén-Hernández et al. 2000; Pérez-Ponce de León et al. 2000) correspond to H. danbrooksi (León-Règagnon & Paredes-Calderón 2002). According to the morphology of the specimens examined by Caballero (1941) in Estado de México and Mexico City, Mexico, these records belong to H. mexicanus n. sp. described herein.

Haematoloechus meridionalis León-Règagnon, Brooks & Zelmer, 2001 Type host and locality: common marsh frog Rana vaillanti, Guanacaste, Costa Rica (León-Règagnon et al. 2001). Other records: Colombia: Amazon River frog R. palmipes, Cali (Uribe–Piedrahita 1948, originally reported as H. medioplexus); Costa Rica: common marsh frog R. vaillanti, Guanacaste; R. taylori, Cartago (Goldberg & Bursey 2007); Mexico: R. vaillanti, Veracruz (León-Règagnon 2010; Razo–Mendívil & Pérez-Ponce de León 2011; this study CNHE7596). Taxonomic notes: Valid species, nominally described. Validated using morphological and molecular evidence (León-Règagnon et al. 2001). Record of H. medioplexus in Amazon River frog R. palmipes in Colombia (Uribe–Piedrahita 1948) corresponds to H. meridionalis (León-Règagnon et al. 2001).

296 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN Haematoloechus mexicanus n. sp. Type host and locality: Montezuma leopard frog Rana montezumae, Lerma, Estado de Mexico. Other records: Mexico: Tlaloc´s leopard frog Rana tlaloci, Xochimilco, Mexico City, Patzcuaro leopard frog R. dunni, Pátzcuaro, Michoacán (this study). Taxonomic notes: Recorded as H. medioplexus by Caballero (1941); record of H. coloradensis of León-Règagnon et al. (1999) and León-Règagnon & Brooks (2003) corresponds to H. mexicanus n. sp.

Haematoloechus neivai (Travassos & Artigas, 1927) Ingles, 1933 Synonym: Pneumonoeces planorbinus Lutz, 1928; P. pseudis Lutz, 1928 (Syn. fide Travassos & Darriba 1930). Type host and locality: butter thin–toed frog Leptodactylus latrans (recorded as common thin–toed frog L. ocellatus), Rio de Janeiro, Brazil (Travassos & Artigas 1927). Other records: Brazil: butter thin–toed frog L. latrans (recorded as common thin–toed frog L. ocellatus), Sao Paulo (Travassos & Artigas 1927); swimming frog Pseudis paradoxa (Linnaeus), Mato Grosso (Travassos & Freitas 1940); bladder thin– toed frog L. pentadactylus (Laurenti), Bahia (Fahel 1952); Venezuela: Amazon River frog Rana palmipes, Caracas (Freitas & Lent 1939); swimming frog Pseudis paradoxa, recorded as Pneumonoeces planorbinus and Pneumonoeces pseudis (Lutz 1928). Taxonomic notes: Valid species, described as Pneumonoeces neivai (Travassos & Artigas 1927); used as type species to erect the genus Neohaematoloechus (Odening 1960).

Haematoloechus nicolasi León-Règagnon, 2017 Type host and locality: Zweifel´s frog Rana zweifeli, Oaxaca, México (León-Règagnon 2017). Other records: Mexico: R. zweifeli, Guerrero (León-Règagnon 2017), Michoacán (this study, CNHE10405); Rana sp., Chiapas (León-Règagnon 2017). Taxonomic notes: Valid species, nominally described. Validated using morphological and molecular evidence (León-Règagnon 2010, 2017).

Haematoloechus occidentalis n. sp. Type host and locality: Rana sp., Coquimatlán, Colima, México. Other records: Mexico: Rio Grande leopard frog R. berlandieri, San Antonio Chimalapas, Oaxaca (this study); Brown´s leopard frog R. brownorum, Rizo de Oro, Chiapas (Velazquez–Urrieta & León-Règagnon, 2018); Forrer´s leopard frog R. cf. forreri, San Pedro las Playas, Guerrero (Cabrera–Guzmán et al. 2007), Rizo de Oro, Chiapas (Velazquez–Urrieta & León-Règagnon 2018), Tehuantepec, Oaxaca, Santa María del Oro, Nayarit; Transverse Volcanic leopard frog R. neovolcanica, Ocotlán, Jalisco; showy leopard frog R. spectabilis, Mitla, Oaxaca; Rana sp., San Pablo Huitzo, Oaxaca, Nizanda, Oaxaca, San Fernando, Chiapas; cane toad Rhinella marina, Tres Palos, Guerrero (this study).

Haematoloechus oxyorchis Ingles, 1932 Type host and locality: Northern red–legged frog Rana aurora, California, U.S.A. (Ingles 1932) Taxonomic notes: Valid species, nominally described; treated as Ostiolum oxyorchis by Odening (1960). Kennedy (1981) considered this species to be a junior synonym of H. complexus based on morphological variability. We validate this species considering recent evidence supporting the value of the distribution of uterine loops as a diagnostic character and its geographical distribution, very different of H complexus (Bolek & Janovy 2007a).

Haematoloechus ozorioi Freitas & Lent, 1939 Type host and locality: butter thin–toed frog Leptodactylus latrans (recorded as common thin–toed frog L. ocellatus), Montevideo, Uruguay (Freitas & Lent 1939). Taxonomic notes: Valid species, nominally described; treated as H. (Anomolecithus) ozorioi by Odening (1960).

Haematoloechus parcivitellarius Caballero, 1942 Type host and locality: Montezuma leopard frog Rana montezumae, Estado de Mexico, Mexico (Caballero 1942b). Other records: Mexico: R. montezumae, Estado de Mexico (Caballero 1942c; Pérez-Ponce de León et al., 2000, CNHE 3377, 3378 in part); Morelos (this study CNHE 10513); Tlaloc´s leopard frog R. tlaloci, Xochimilco, Mexico City (this study CNHE 10512). Taxonomic notes: Valid species, nominally described; treated as Ostiolum parcivitellarium by Odening (1960). The material recorded by Bursey & Goldberg (2005), collected from museum preserved specimens of Forrer´s leopard frog R. cf. forreri from Costa Rica, was in poor condition (USNPC 94842); revision of this material showed that according to the sucker ratio (1:0.58–0.66), the arrangement of the uterine loops, the geographic distribution and host, these specimens more likely belong to H. occidentalis n. sp.

Haematoloechus parviplexus (Irwin, 1929) Ingles, 1932 Type host and locality: green frog Rana clamitans, Minnesota, U.S.A. (Irwin 1929) Other records: U.S.A.: Woodhouse´s toad Anaxyrus woodhousii, Nebraska (Brooks 1976); bullfrog R. catesbeiana,

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 297 Connecticut (Brooks 1976), Louisiana (Bennett 1938), Michigan (Muzzall 1991), Nebraska (Brooks 1976; León- Règagnon & Paredes-Calderón 2002; Bolek & Janovy 2007b); Nevada (Babero & Gollin 1974); green frog R. clamitans, Idaho (Waltz 1961), Louisiana (Bennett 1938), Michigan (Krull 1930, 1931; Najarian 1955; Muzzall 1991), Nebraska (Bolek & Janovy 2007a), New York, Wisconsin (Bolek and Janovy 2007b; Bolek and Coggins 2001 reported as H. varioplexus); Oregon spotted frog Rana pretiosa, Idaho (Waltz 1961); mink frog R. septentrionalis, Minnesota (Schotthoefer et al. 2009); woods frog R. sylvatica, Michigan (Muzzall & Peebles 1991); Rana pretiosa x Rana sylvatica hybrid, Idaho (Waltz 1961). Taxonomic notes: Valid species, described as Pneumonoeces parviplexus Irwin, 1929; treated as H. (H.) parviplexus by Odening (1960). Kennedy (1981) considered this species to be a junior synonym of H. varioplexus based on morphological variation. Detailed morphological revision and molecular evidence support the validity of this species (León-Règagnon & Brooks 2003; Bolek & Janovy 2007b). Bolek & Janovy (2007b) suggest this species is host specific to bullfrogs (R. catesbeiana) and green frogs (R. clamitans) and records in Northern leopard frogs (R. pipiens), wood frogs (R. sylvatica), Oregon spotted frogs (R. pretiosa) and the hybrids Rana pretiosa x Rana sylvatica correspond to H. varioplexus. These authors also suggest that specimens described as H. parviplexus by Irwin (1929) and specimens described as H. varioplexus by Stafford (1902) belong to the same species, but still consider H. parviplexus as a valid name. We agree in this view, to avoid any further confusion in the taxonomy of these species.

Haematoloechus pukinensis Ibáñez & Córdoba, 1979 Type host and locality: Peru water frog Telmatobius peruvianus Weigmann, Arequipa, Peru (Ibáñez & Córdoba 1979). Other records: Peru: Acanchoca water frog Telmatobius jelskii (Peters), Ayacucho, Huanjavelica and Junín (Chero et al. 2014). Taxonomic notes: Valid species, nominally described.

Haematoloechus pulcher Bravo–Hollis, 1943 Type host and locality: Tiger salamander Ambystoma tigrinum (Green), Mexico City, Mexico (Bravo–Hollis 1943). Other records: Mexico: Lerma Lake salamander A. lermaense, Estado de México (Mata-López et al. 2002); granular salamander A. granulosum Taylor, Estado de México (this study, CNHE 10514); Montezuma leopard frog Rana montezumae, Estado de México (León-Règagnon et al. 1999; Pérez-Ponce de León et al. 2000). Taxonomic notes: Valid species, nominally described; treated as Ostiolum pulchrum by Odening (1960). Molecular evidence supports the validity of this species (León-Règagnon et al. 1999; this study).

Haematoloechus tumidus Ingles, 1932 Type host and locality: Northern red–legged frog Rana aurora, California, U.S.A. (Ingles 1932). Other records: U.S.A.: R. aurora, California, U.S.A. (Ingles 1936). Taxonomic notes: Valid species, nominally described; treated as H. (Anomolecithus) tumidus by Odening (1960). Kennedy (1981) considered this species to be a junior synonym of H. kernensis based of morphological variation. Recent molecular evidence supports the value of morphological characters used to differentiate this species such as the arrangement of the uterine loops, indicating its validity (León-Règagnon & Brooks 2003; León-Règagnon 2010).

Haematoloechus varioplexus Stafford, 1902 Synonym: H. similiplexus Stafford, 1902 (syn. fide Travassos & Darriba 1930) Type host and locality: bullfrog Rana catesbeiana, Ontario, Canada (Stafford 1902). Other records: Canada: R. catesbeiana, New Brunswick (McAlpine 1997), Quebec (Stafford 1902); green frog R. clamitans, New Brunswick (McAlpine 1997); Northern leopard frog R. pipiens, New Brunswick (McAlpine 1997), Ontario, Quebec (Stafford 1902 as H. similiplexus, host recorded as Rana virescens; King et al. 2007); Southern toad Anaxyrus terrestris, Ontario, Quebec (Stafford 1902 as H. similiplexus, host originally recorded as Bufo lentiginosus); U.S.A.: American toad A. americanus, Iowa (Ulmer 1970), Wisconsin (Bolek & Coggins 2003); Woodhouse´s toad A. woodhousii, Nebraska (Brooks 1976;); Plains leopard frog R. blairi, Nebraska (Brooks 1976); bullfrog R. catesbeiana, Virginia (Campbell 1968); green frog R. clamitans, Indiana (Whitehouse 2002); Maine (Bouchard 1951 as H. similiplexus), Michigan (Fortner 1923 as H. similiplexus; Muzzall et al. 2001), Virginia (Campbell 1968), Wisconsin (Snyder & Tkach 2001); Northern leopard frog R. pipiens, Illinois (Cort 1915 as H. similiplexus), Iowa (Ulmer 1970); Michigan (Fortner 1923 as H. similiplexus), Nebraska (Brooks 1976), South Dakota (Bolek and Janovy 2007b), Wisconsin (Bolek & Coggins 2003); Oregon spotted frog R. pretiosa, Idaho (Russell & Wallace 1992); Wyoming (Turner 1958); mink frog R. septentrionalis, Maine (Bouchard 1951 as H. similiplexus); wood frog R. sylvatica, Michigan (Najarian 1955), Wisconsin (Yoder & Coggins 1996; Bolek and Janovy 2007b); spring peeper Pseudacris crucifer, Wisconsin (Yoder & Coggins 1996); Tukish gecko Hemidactylus turcicus (Linnaeus), Louisiana, accidental infection (Criscione & Font 2001). Taxonomic notes: Valid species, nominally described; treated as H. (H.) varioplexus by Odening (1960). Stafford (1902) incompletely described this species based on few specimens that were not deposited in a formal collection, consequently, Cort (1915) considered H. varioplexus as species inquirendae. Travassos & Darriba (1930) erroneously declared H. varioplexus and H. similiplexus as synonyms following the arguments of Cort (1915) about their similarity, and kept H. varioplexus as the valid species because it was described first in Stafford´s manuscript. The synonymy was recognized by other authors in revisions of the genus (Caballero & Sokoloff 1934; Manter 1938; Odening 1960; Kennedy 1981), and

298 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN most records of the morphotype described by Stafford (1902) as H. similiplexus have been assigned to H. varioplexus ever since. Bolek & Janovy (2007b) suggest that specimens described as H. varioplexus by Stafford (1902) and specimens described as H. parviplexus by Irwin (1929) belong to the same species, and we share their opinion. According to the International Code of Zoological Nomenclature, H. parviplexus should be considered as a junior synonym of H. varioplexus, and the species H. similiplexus should be reinstated. Nevertheless, this would make the taxonomic confusion of the group even worse. We consider it to be easier to continue to call H. varioplexus the morphotype that Stafford (1902) described as H. similiplexus, and H. parviplexus to be the morphotype that he described as H. varioplexus, and Irwin redescribed as H. parviplexus. Bolek & Janovy (2007b) suggest H. varioplexus is host specific to Plains leopard frogs (R. blairi), Northern leopard frog (R. pipiens), wood frogs (R. sylvatica), and Oregon spotted frogs (R. pretiosa), while records in bullfrogs (R. catesbeiana) and green frogs (R. clamitans) correspond to H. parviplexus. Specimens recorded by Caballero (1941) are not available for examination, but according to his description and drawing, those specimens appear to be H. illimis; specimens recorded by Lamothe-Argumedo et al. (1997) in Montezuma leopard frogs (R. montezumae) in Texcoco, Estado de Mexico and Xochimilco, Mexico City, correspond to H. illimis.

Haematoloechus veracruzanus n. sp. Type host and locality: common marsh frog Rana vaillanti, Laguna Escondida, Los Tuxtlas, Veracruz. Other records: Mexico: R. vaillanti, Catemaco, Veracruz (this study); Rio Grande leopard frog R. berlandieri, Laguna Higueras, Nuevo León (León-Règagnon et al. 2005, as H. complexus); Huauchinango, Puebla (León-Règagnon 2003, as H. complexus); Rana sp., Tierra Quemada and Rancho el Borbotón, San Luis Potosí, Mexico (this study). Taxonomic notes: Records of H. complexus (Paredes-Calderón et al. 2004), Haematoloechus sp. (isolate Tuxtlas2 in León- Règagnon et al. 1999), H. cf. complexus (isolate H. cf. complexus (T) in León-Règagnon & Brooks 2003) belong to H. veracruzanus n. sp.

Australia Haematoloechus australis (Johnston, 1912) Yamaguti, 1958 Type host and locality: green and golden tree frog Ranoidea aurea (Lesson), New South Wales, Australia (Johnston 1912). Other records: Australia: Peron´s tree frog Limnodynastes peronii (Duméril & Bibron), New South Wales (Johnston 1912). Taxonomic notes: Valid species; described as Pneumonoeces australis Johnston, 1912; treated by Odening (1960) as H. (H.) australis.

Eurasia Haematoloechus abbreviatus (Bychowsky, 1932) Prokopic & Krivanec, 1974 Synonyms: H. mazurmovici Boschkow, 1964, and H. ellipticus Eckstein, 1922 (syn. fide Prokopic & Krivanec 1974). Type host and locality: fire–bellied toad Bombina bombina Linnaeus, Kiev, Ukraine (Bychowsky 1932). Other records: Czech Republic: B. bombina, yellow–bellied toad B. variegata, Charkovsk (Shevchenko 1965), Moravia (Vojtková & Krivanec 1970), Kvetná, Ostrava, Brno, Velké Kralovice, Vigantgice, Vranivice, Trebon, Klec (Prokopic & Krivanec 1974); Germany: B. bombina (Eckstein 1922, as Distomum ellipticum (variegatum); Republic of Albania: B. variegata (Prokopic & Krivanec 1974); Bulgaria: B. variegata (Boschkow 1964, as H. mazurmovici); Slovakia: B. bombina, B. variegata Linnaeus, Belánad Cirochou, Námestovo, Nová Lesná, Poprad, Slovakia, Eastern Slovakia (Prokopic & Krivanec 1974); Ukraine: Severnyi Donets valley (Shevchenko 1965). Taxonomic notes: Valid species; described as Pneumonoeces variegatus abbreviatus (Bychowsky 1932); treated as H. (Haematoloechus) variegatus abbreviatus by Odening (1960). Prokopic & Krivanec (1974) declared H. mazurmovici and H. ellipticus as synonyms of H. abbreviatus based on morphological variabilty.

Haematoloechus almorai (Pande, 1937) Yamaguti, 1958 Type host and locality: Indian skipper frog Euphlyctis cyanophlyctis (Schneider), India: Bangalore (Pande 1937). Taxonomic notes: Valid species; described as Pneumonoeces almorai Pande, 1937 Treated by Odening (1958) as a subspecies of H. (H.) variegatus. According to Odening (1960), the geographical boundary between H. (H.) v. variegatus and H. (H.) v. almorai may coincide with the border between the Eurasian marsh frog Pelophylax ridibundus (Pallas) (Tadshikistan) and the Indian skipper frog Euphlyctis cyanophlictis (Kashmir).

Haematoloechus asper Looss, 1899 Type host and locality: edible frog Pelophylax esculentus (Linnaeus), Berlin, Germany (Looss 1899). Other records: Albania: Eurasian marsh frog P. ridibundus (Prokopic & Krivanec 1974); Austria: P. esculentus (Odening 1960); Georgia: P. ridibundus, Bufo bufo (Murvanidze et al. 2008); Germany: P. esculentus, P. ridibundus (Odening 1960); Hungary: P. esculentus (Sey 1964, 1968; Murai et al. 1983); Iraq: P. ridibundus (Saeed et al. 2007); Poland: P. esculentus (Odening 1960); Romania: fire–bellied toad B. bombina, common toad Bufo bufo Linnaeus, P. esculentus, P. ridibundus, European common frog Rana temporaria Linnaeus (Odening 1960); Russia: P. esculentus (Chikhlyaev et al. 2009); P. ridibundus (Odening 1960; Rezvantseva 2008); R. temporaria (Chikhlyaev & Ruchin 2014); Slovakia: Altai brown frog Rana arvalis Nilsson, P. esculentus, P. ridibundus, Bombina variegata (Prokopic & Krivanec 1974); United Kingdom: P. esculentus (Odening 1960); Ukraine: P. ridibundus (Odening 1960); R. arvalis (Tkach et al. 2000).

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 299 Taxonomic notes: Valid species, nominally described. Treated as H. (Anomolecithus) asper asper by Odening (1960).

Haematoloechus breviansa (Sudarikov, 1950) Yamaguti, 1958 Type host and locality: Eurasian marsh frog Pelophylax ridibundus, Volga Region, Russia (Sudarikov 1950). Other records: Hungary: P. ridibundus (Sey & Eöry 1992); Iran: P. ridibundus (Mashaii et al. 2000); Russia: P. ridibundus (Romanova & Matveeva 2010); Turkey: P. ridibundus (Yildirimhan et al. 1996, 2005; Düsen & Öz 2006, 2013; Saglam & Arikan 2006; Düsen & Oguz 2010; Koyun et al. 2015); Levant green frog P. bedriagae (Camerano) (Demir et al. 2015); Ukraine: P. ridibundus, P. esculentus (Kovalenko 2007). Taxonomic notes: Valid species, described as Skrjabinoeces breviansa (Sudarikov 1950). Treated as H. (Skrjabinoeces) breviansa by Odening (1960); this author considered the specimens reported by Bychowski (1932) as H. similis in Ukraine to belong to H. breviansa.

Haematoloechus bychovskii Odening, 1958 Type host and locality: Asiatic grass frog Rana chensinensis David, Ussuriysk, Russia (Odening 1958). Taxonomic notes: Valid species, described as H. (Anomolecithus) nanchangensis bychovskii based on material reported by Bychowsky (1932) as Pneumonoeces schulzei Wundsch, 1911.

Haematoloechus capyristes (Klein, 1905) Ingles, 1932 Type host and locality: Indian green frog Euphlyctis hexadactyla (Lesson), Southern India (Klein 1905). Taxonomic notes: Valid species, described as Pneumonoeces capyristes Klein, 1905. Travassos & Darriba (1930) mistakenly wrote the species name as P. campyristis. Treated by Odening (1958) as H. (H). variegatus capyristes.

Haematoloechus carbonelli Lluch, Navarro & Pérez–Soler, 1991 Type host and locality: Iberian frog Rana iberica Boulenger, Ávila, Spain (Lluch et al. 1991). Other records: Spain: Ávila, common toad Bufo bufo, common fire salamander Salamandra salamandra (Linnaeus) (Lluch et al. 1991). Taxonomic notes: Valid species, nominally described.

Haematoloechus donicus (Shevchenko, 1965) n. comb. Type host and locality: Eurasian marsh frog Pelophylax ridibundus, Severnyi Donets Valley, Ukraine (Shevchenko 1965). Taxonomic notes: Valid species, described as Skrjabinoeces donicus Shevchenko, 1965.

Haematoloechus japonicus (Yamaguti, 1936) n. comb. Type host and locality: black–spotted pond frog Pelophylax nigromaculatus, Kyoto, Japan (Yamaguti 1936). Other records: China: Eastern golden frog Pelophylax plancyi (Lataste), P. nigromaculatus (Hallowell) (Zhaozhi & Qingkui 1999); Japan: P. nigromaculatus, Honshu (Goldberg & Bursey 2002c); Korea: P. nigromaculatus (Kim et al. 1992). Taxonomic notes: Valid species, described as Pneumonoeces sibiricus japonicus Yamaguti, 1936; treated as H. (Haematoloechus) sibiricus japonicus by Odening (1960). According to Odening (1960), records of H. variegatus (Rudolphi 1819) and H. sibiricus (Isajcikov 1927) in Japan belong to this species; record of Goldberg & Bursey (2002c) as H. sibiricus. Detailed morphological & molecular revision is needed to ascertain the validity of this species.

Haematoloechus jeholensis (Fukui & Ogata, 1938) n. comb. Type host and locality: black–spotted pond frog Pelophylax nigromaculatus, Lingyuan, China (Fukui & Ogata 1938). Other records: Korea: P. nigromaculatus (Fukui & Ogata 1938). Taxonomic notes: Valid species, described as Pneumonoeces jeholensis; treated as H. (Haematoloechus) sibiricus jeholensis by Odening (1960). Detailed morphological and molecular revision is needed to test the validity of this species.

Haematoloechus latoricensis Kozák, 1968 Type host and locality: edible frog Pelophylax esculenta, River Tisa, Slovakia (Kozák 1968). Taxonomic notes: Species inquirendae; according to Prokopic & Krivanec (1974), this species does not belong to the genus Haematoloechus.

Haematoloechus lipsiensis (Odening, 1958) n. comb. Type host and locality: edible frog Pelophylax esculentus, Leipzig, Germany (Odening 1958). Other records: Albania: Vlora, Brutinta, Eurasian marsh frog P. ridibundus, Agile frog Rana dalmatina Fitzinger, 1839 (Prokopic & Krivanec 1974). Taxonomic notes: Valid species, described as H. (Anomolecithus) asper lipsinensis (Odening 1958).

Haematoloechus lobatus Seno, 1907 Synonym: Haematoloechus lobatus koreanus Kim, Rim & Yoon, 1992 Type host and locality: black–spotted pond frog Pelophylax nigromaculatus (Hallowell), Kyoto, Japan (Seno 1907).

300 · Zootaxa 4526 (3) © 2018 Magnolia Press LEÓN-RÈGAGNON & TOPAN Other records: Japan: bullfrog Rana catesbeiana (Uchida & Itagaki 1976; Hasegawa et al. 2013); Korea: bullfrog R. catesbeiana, black-spotted pond frog P. nigromaculatus (Kim et al. 1992). Taxonomic notes: Valid species, poorly described by Seno (1907); considered a junior synonym of H. nanchangensis by Odening (1960), but sucker ratio clearly differentiate these two species. Uchida & Itagaki (1976) redescribed the species based on specimens collected in Japan from the introduced bullfrog R. catesbeiana. Kim et al. (1992) described H. lobatus koreanus based on specimens collected from black–spotted pond frogs P. nigromaculatus, differentiating the subspecies solely by the shape of testes, which can vary within species.

Haematoloechus nanchangensis Hsiung, 1934 Synonym: Pneumonoeces nanchangensis major Yamaguti, 1936 (syn fide Odening 1960) Type host and locality: Eastern golden frog Pelophylax plancyi, Nantschang, China (Hsiung 1934). Other records: Japan: black–spotted pond frogs P. nigromaculatus (Yamaguti 1936); Korea: P. nigromaculatus (Kim et al. 1992). Taxonomic notes: Valid species, nominally described; treated as H. (Anomolecithus) nanchangensis nanchangensis by Odening (1960).

Haematoloechus pyrenaicus Combes, 1965 Type host and locality: European common frog Rana temporaria, Eastern Pyrenees, France (Combes 1965). Other records: France: common toad Bufo bufo, Eastern Pyrenees (Combes 1965). Taxonomic notes: Valid species, nominally described.

Haematoloechus schulzei (Wundsch, 1911) Prokopic & Krivanec, 1974 Synonym: Haematoloechus odeningi Boschkow, 1964 (syn. fide Prokopic & Krivanec 1974). Type host and locality: Eurasian marsh frog Pelophylax ridibundus, Berlin, Germany (Wundsch 1911). Other records: Austria: edible frog P. esculentus (Odening 1960); Bulgaria: P. ridibundus (Boschkow, 1964); Czech Republic: P. esculentus (Prokopic & Krivanec 1974); Serbia: P. esculentus (Bjelic-Cabrilo et al. 2009). Taxonomic notes: Valid species, described as Pneumonoeces schulzei; treated as H. (Haematoloechus) schulzei by Odening (1960); this author states that the specimens recorded by Travassos & Darriba (1930) in Germany do not belong to H. schulzei.

Haematoloechus sibiricus (Isajcikov, 1927) Yamaguti, 1958 Type host and locality: Altai brown frog Rana arvalis, Siberia, Russia (Isajcikov 1927 in Odening 1960). Other records: Russia: black–spotted pond frogs P. nigromaculatus (= Rana chinensis), Vladivostok (Odening 1960); Amur brown frog Rana amurensis Boulenger, Siberia (Odening 1960; Tarasovskaya & Ibrayeva 2011). Taxonomic notes: Valid species, described as Pneumonoeces sibiricus; treated as H. (Haematoloechus) sibiricus sibiricus by Odening (1960).

Haematoloechus similis Looss, 1899 Synonyms: Haematoloechus similigenus Stiles & Hassall, 1902 (syn. fide Odening 1960); H. minimus Shevchenko, 1965 (syn. fide Prokopic & Krivanec 1974). Type host and locality: edible frog Pelophylax esculentus, Germany (Looss 1899). Other records: Austria: P. esculentus, Viena (Odening 1960); Bulgaria: Eurasian marsh frog P. ridibundus (Odening 1960); Czech Republic: P. esculentus (Prokopic & Krivanec 1974); France: P. esculentus (Bailenger & Chanseau 1954; Odening 1960); Germany: P. esculentus , P. ridibundus, European common frog Rana temporaria (Odening 1960); Rana sp. (Travassos & Darriba 1930); Iraq: P. ridibundus (Saeed et al. 2007); Iran: P. ridibundus (Combes and Knoepffler, 1972); Poland: P. esculentus (Odening 1960); Russia: P. esculentus (Chikhlaev et al. 2009); P. ridibundus (Romanova & Matveeva 2010); R. temporaria (Chikhlyaev & Ruchin 2014); Serbia: P. esculentus (Bjelic-Cabrilo et al. 2009); Tajikistan: P. ridibundus (Odening 1960); Ukraine: P. esculentus, P. ridibundus, Altai brown frog R. arvalis (Odening 1960; Tkach et al. 2000); former USSR: European region, R. temporaria (Odening 1960) Taxonomic notes: Valid species, nominally described; treated as H. (Skrjabinoeces) similis similis by Odening (1960).

Haematoloechus sindensis Khan & Mohiuddin, 1982 Type host and locality: Indian skipper frog Euphlyctis cyanophlyctis, Sind, Pakistan (Khan & Mohiuddin 1982). Taxonomic notes: Valid species, nominally described.

Haematoloechus singaporensis Yuen, 1962 Type host and locality: Asian brackish frog Fejervarya cancrivora (Gravenhorst), Singapore, Malaysia (Yuen 1962). Taxonomic notes: Valid species, nominally described.

Haematoloechus sudarikovi Belouss, 1962 in Skrjabin & Antipin (1962) Type host and locality: European common frog Rana temporaria, former USSR (Skrjabin & Antipin 1962).

HAEMATOLOECHUS LOOSS FROM MEXICO Zootaxa 4526 (3) © 2018 Magnolia Press · 301 Taxonomic notes: Nomen nudum, species described in a thesis, but not formally published; mentioned in Skrjabin & Antipin (1962).

Haematoloechus tientsinensis Hsiung, 1934 Type host and locality: black–spotted pond frogs Pelophylax nigromaculatus, Tientsin, China (Hsiung 1934). Other records: China: P. nigromaculatus (Zhaozhi & Qingkui, 1999). Taxonomic notes: Valid species, nominally described.

Haematoloechus travdarribus (Skrjabin & Antipin, 1962) Yamaguti, 1971 Type host and locality: Pelophylax sp., Hamburg, Germany (Skrjabin & Antipin 1962). Taxonomic notes: Valid species, described as Pneumonoeces travdarribus, based on the specimens recorded by Travassos & Darriba (1930) as H. schulzei. Odening (1960) states that the difference in the size of the eggs in these specimens does not allow the allocation of this form to H. schulzei.

Haematoloechus variegatus (Rudolphi, 1819) Looss, 1899 Synonym: Haematoloechus dubininae Odening 1958 (syn. fide Prokopic & Krivanec 1974). Type host and locality: edible frog Pelophylax esculentus, Berlin, Germany (Rudolphi 1819). Other records: Albania: Eurasian marsh frog P. ridibundus; Bulgaria: frog (Buchvarov 1977); Czech Republic: P. esculentus, P. ridibundus (Prokopic & Krivanec 1974); Europe: P esculentus, P. ridibundus, Altai brown frog R. arvalis, European common frog R. temporaria (Odening 1960); France: P. esculentus (Bailenger & Chanseau 1954; Dollfus 1961); Georgia: common toad Bufo bufo, European green toad Bufotes viridis (Laurentis), P. ridibundus, banded frog Rana macrocnemis Boulenger (Murvanidze et al. 2008); Hungary: P. ridibundus, P. esculentus (Herczeg et al. 2016); Korea: black-spotted pond frogs P. nigromaculatus (Kim et al. 1992); Poland (Kuc & Sulgostowska 1988a, 1988b; Popiolek et al. 2011; Okulewicz et al. 2014); Portugal (Rodrigues et al. 1973); Russia: P. esculentus (Chikhlaev et al. 2009), P. ridibundus (Rezvantseva 2008; Romanova & Matveeva 2010), Amur brown frog Rana amurensis (Odnokurtsev & Sedalishchev 2008), R. temporaria (Chikhlyaev & Ruchin 2014); Serbia: P. esculentus (Bjelic-Cabrilo et al. 2009); Spain: Iberian frog R. iberica (Salvador 2015); Turkey: P. ridibundus (Yildirimhan et al. 1996; Saglam & Arikan 2006); United Kingdom (Cox 1971); Ukraine: R. arvalis (Tkach et al. 2000). Taxonomic notes: Valid species, described as Distoma variegatum Rudolphi, 1819, transfered to Haematoloechus by Looss (1899), treated as H. (Haematoloechus) variegatus variegatus by Odening (1960).

Haematoloechus vitelloconfluentum (Rai, 1962) Saeed, Al–Barwari & Al–Harmni, 2007 Type host and locality: Indian bullfrog Hoplobatrachus tigerinus (Daudin), Piparia, India (Rai 1962) Other records: Iraq: Eurasian marsh frog Pelophylax ridibundus (Saeed et al. 2007). Taxonomic notes: Species inquirendae. This species was described as Tremiorchis vitelloconfluentum from the intestine of H. tigerinus and transfered to Haematoloechus by Saeed et al. (2007) without justification, when they reported specimens in the lungs of P. ridibundus. The position of the genital pore, immediately anterior to the ventral sucker, indicates that this species does not belong to Haematoloechus.

Haematoloechus vojtkovae Prokopic & Krivanec, 1974 Type host and locality: fire-bellied toad Bombina bombina, Vrané & Vltavou, Czech Republic (Prokopic & Krivanec 1974). Taxonomic notes: Valid species, nominally described.

Haematoloechus volgensis (Sudarikov, 1950) Yamaguti, 1958 Type host and locality: edible frog Pelophylax esculentus, Gorki, Russia (Sudarikov 1950). Other records: Iraq: Eurasian marsh frog P. ridibundus (Saeed et al. 2007). Taxonomic notes: Valid species, described as Skrjabinoeces volgensis Sudarikov, 1950; treated as H. (Skrjabinoeces) similis volgensis by Odening (1960).

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