Taxonomic revision of species of Haematoloechus Looss...

ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright 2018 Magnolia Press

Zootaxa 4526 (3): 251302 http://www.mapress.com/j/zt/

Article

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 LEN-RGAGNON1, 2 & JANET TOPAN21Estacin de Biologa Chamela, Instituto de Biologa, Universidad Nacional Autnoma de Mxico, A.P. 21, San Patricio, Jalisco,

Mxico, CP 48980. E-mail: [email protected] 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 MaGarzn & Gil, 1959, H. latoricensis Kozk, 1968 & H. vitelloconfluentum (Rai, 1962) Saeed, AlBarwari

& 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 (Len-Rgagnon et al. 1999, 2001; Snyder & Tkach 2001; Len-Rgagnon & Paredes-Caldern 2002; Len-Rgagnon & Brooks 2003; Bolek & Janovy 2007a; Len-Rgagnon 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; Len-Rgagnon 1992; Prez-Ponce de Len et al. 2000) is actually a complex of sibling species whose conserved morphology makes their differentiation difficult (Len-Rgagnon et al. 1999; Len-Rgagnon 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: Coleccin Nacional de Helmintos (CNHE), Instituto de Biologa, Universidad Nacional Autnoma de Mxico; 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 Coleco Helmintolgica 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 Haematoloechusand 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, Browns leopard frog R. brownorum Sanders, American bullfrog R. catesbeiana Shaw, green frog R. clamitans Latreille, Patzcuaro leopard frog R. dunni Zweifel, Forrers leopard frog R. cf. forreri, bigfooted 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 Zweifels 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 Gomoris 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.

LEN-RGAGNON & TOPAN252 Zootaxa 4526 (3) 2018 Magnolia Press

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 L of DNA was added to a PCR reaction consisting of 6.25 L of 10% D(+)trehalose dihydrate (Fluka Analytical), 2.00 L of Hyclone ultrapure water (Thermo Scientific), 1.25 L of 10X PlatinumTaq buffer (Invitrogen), 0.625 L of 50 mM MgCl

2 (Invitrogen),

0.125 L of each primer or primer cocktail, 0.0625 L of 10 mM dNTP (KAPA Biosystems) and 0.060 L of 5 U/L PlatinumTaq DNA Polymerase (Invitrogen) for a total reaction volume of 12.5 L. 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 ATG3' (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 94C for 40 s, 45 C for 40 s, 72C for 1 min, followed by 35 cycles at 94C for 40 s, 51C for 40 s, 72C for 1 min and a final extension at 72C 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 96C for 10 s, 55C for 5 s, 60C for 2.5 min and a final extension at 60C 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. 13). 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-Lpez, 2011, H. pulcher BravoHollis, 1943, H. veracruzanus n. sp., H. caballeroi Skrjabin & Antipin, 1962 & H. longicollum Len-Rgagnon & RomeroMayn, 2017), and Clade III (H. medioplexus Stafford, 1902, H. floedae Harwood, 1932, H. meridionalis Len-Rgagnon, Brooks & Zelmer, 2001, H. nicolasi LenRgagnon, 2017, H. illimis Caballero, 1942, H. lobatus Seno, 1907, H. breviplexus Stafford, 1902, H. mexicanus n. sp. & H. parviplexus (Irwin, 1929)) (Figs. 13). 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

Zootaxa 4526 (3) 2018 Magnolia Press 253HAEMATOLOECHUS LOOSS FROM MEXICO

species of Clade II in the 28S and concatenated analyses. They also differ in the position of H. danbrooksi Len-Rgagnon & Paredes-Caldern, 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. 13).

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 LenRgagnon (2010); originally recorded as H. complexus by Len-Rgagnon (2010); originally recorded as H. varioplexus by Len-Rgagnon 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: Tlalocs 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.


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 Len-Rgagnon & Brooks (2003); originally recorded as H. cf. complexus Len-Rgagnon & Brooks (2003); originally recorded as H. varioplexus by Len-Rgagnon et al. (2005); I = Clade I, H. longiplexus & relatives; II = Clade II, H. complexus & relatives; III = H. medioplexus & relatives.

Paratypes: CNHE 1428, 1551, 1552, 33763379, 3395, 3397, 3399, 3794, 4661, 1045810460.Other hosts and localities: Montezuma leopard frog R. montezumae, Zempoala, Morelos (CNHE 10464),

Patzcuaro leopard frog R. dunni Zweifel, Ptzcuaro, Michoacn (CNHE 10465); R. dunni, Zacapu, Michoacn (CNHE 10463).

Other records: Mexico: Tlalocs leopard frog R. tlaloci, Xochimilco, Mexico City (Caballero &Sokoloff 1934 as H. complexus); Montezuma leopard frog R. montezumae, Cinaga de Lerma, Mexico State (Len-Rgagnon 1992 as H. complexus; Lamothe et al. 1997 as H. coloradensis Cort, 1915; Len-Rgagnon et al. 1999 as H. complexus; Prez-Ponce de Len et al. 2000 as H. coloradensis and H. complexus); big-footed leopard frog R. megapoda, Cointzio Springs, Michoacn (Prez-Ponce de Len et al. 2000 as H. complexus); Transverse Volcanic leopard frog R. neovolcanica, Cointzio Springs, Michoacn (Prez-Ponce de Len et al. 2000 as H. complexus); Patzcuaro leopard frog R. dunni, Ptzcuaro, Michoacn (Garca-Altamirano et al. 1993; PulidoFlores 1994; Prez-Ponce de Len et al. 2000 as H. coloradensis); Lerma Lake salamander Ambystoma lermaense, Cinaga de Lerma, Mexico State (Prez-Ponce de Len et al. 2000 as H. complexus; Mata-Lpez et al. 2002 as H. complexus).

Redescription: Based on 33 mature specimens. Body elongate, with slender anterior region; 2.86.6 (5.0) mm long, 0.821.88 (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; 816 (13) long. Oral sucker subterminal, round, 243487 (355) long, 260511 (387) wide. Pharynx oval, 146398 (255) long,


130300 (206) wide; oral sucker/pharynx ratio 1: 0.410.86 (0.66). Pharynx and anterior region of esophagus surrounded by gland cells. Esophagus 24162 (65) long, sometimes obscured by uterus. Ceca bifurcated at 414844 (643) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 219438 (313) long, 203430 (311) wide, at 0.982.7 (1.9) mm (30%47% (37.6%) of BL) from anterior extremity. Oral sucker: ventral sucker length ratio 1: 0.531.0 (0.81). Testes 2, oval, slightly lobed in some specimens, oblique, inmmediately posterior to ovary. Anterior testis opposite to ovary, 219795 (477) long, 170682 (360) wide. Posterior testis 203852 (557) long, 203771 (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, 190200 (195) long, surrounded by prostatic gland cells. Ovary oval, 243665 (414) long, 203763 (313) wide; at 1.12.9 (2.1) mm (33%51% (42%) of BL) from anterior extremity. Seminal receptacle adjacent partially overlapped with ovary; 203730 (412) long, 138568 (300) wide. Mehlis gland dorsal to seminal receptacle. Laurers 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 3901607 (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, 3140 (36) long, 1723 (20) wide. Excretory vesicle not observed. Excretory pore terminal.

Remarks: In their revision of the family Plagiorchiidae Lhe, 1901, Skrjabin & Antipin (1962) described Haematoloechus caballeroi based on a specimen that Caballero (1942b) collected from Tlalocs 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 (Len-Rgagnon & Brooks 2003; Bolek & Janovy 2007a; Len-Rgagnon 2010; Len-Rgagnon & RomeroMayn 2017). The present species is one of those (GenBank AF532138, Len-Rgagnon & 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 Ibez & Crdoba, 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 Ibez & Crdoba, 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; Len-Rgagnon et al. 2001; Len-Rgagnon & Paredes-Caldern 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 (Ibaez & Crdoba 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; BravoHollis 1943; Zamparo et al. 2011; Len-Rgagnon & RomeroMayn 2017).

Haematoloechus caballeroi most closely resembles H. complexus, H. elongatus, H. fuelleborni, H. kernensisand 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


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 (Ibez & Crdoba 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 Len-Rgagnon (2010) and Len-Rgagnon & Brooks (2003); originally recorded as H. cf. complexus by Len-Rgagnon (2010) and Len-Rgagnon & Brooks (2003); originally recorded as H. varioplexus by LeonRgagnon et al. (2005); I = Clade I, H. longiplexus & relatives; II = Clade II, H. complexus & relatives; III = H. medioplexus & relatives.


FIGURE 4. Haematoloechus caballeroi Skrjabin & Antipin, 1962, from the Tlalocs 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


FIGURE 5. Haematoloechus caballeroi (Skrjabin & Antipin, 1962), from the Tlalocs 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.


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, Ukraine

AF151934 (T)

H. breviplexus (Ha71) R. luteiventris Glacier Park, Montana, U.S.A. AF531856 (LB)

H. caballeroi (Ha30) R. montezumae Lerma, Estado de Mexico, Mexico

MG672412

H. caballeroi (Ha29) R. montezumae Lerma, Estado de Mexico, Mexico

MG672411

H. caballeroi (Ha23)2 R. montezumae Lerma, Estado de Mexico, Mexico

AF532138 (LB)

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 occipitalis

Sierra Leone AF531858 (LB) HQ141698 (L)

H. floedae (Ha61) Rana sp. Tuxtla Gutirrez, Chiapas, Mexico

MG672417 HQ141703 (L)

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, U.S.A.

GU191156 GU191160

H. humboldtensis 2 R. boylii Humboldt Co., California, U.S.A.

GU191157 GU191161

H. illimis (Ha20) R. montezumae Lerma, Estado de Mexico, Mexico

AF531860 (LB) AY672122 (L)

H. illimis (Ha21) R. montezumae Lerma, Estado de Mexico, Mexico

HQ141686 (L)

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, Mexico

MG672420 MG647788

H. longicollum (PLARI084) R. zweifeli Tzitzio, Michoacn, 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, Mexico

AF531862 (LB) HQ141687 (L)

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


TABLE 1. (Continued)


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 Ptzcuaro, Michoacn, Mexico

AF531867 (LB) HQ141685 (L)

H. mexicanus (Ha31) R. montezumae Lerma, Estado de Mexico, Mexico

MG672427

H. micrurus (Ha44) Hoplobatrachus occipitalis

Sierra Leone AF531865 (LB) HQ141696 (L)

H. nicolasi (PLARI017) Lithobates sp. Atoyac, Guerrero, Mexico MG672428 MG647795

H. nicolasi (PLARI020) R. zweifeli Tumbiscatio, Michoacn, Mexico

MG672429 MG647796

H. nicolasi (Ha65) R. zweifeli Santiago Tamazola, Oaxaca, Mexico

MG672430 HQ141707 (L)

H. occidentalis (Ha63)2 R. cf. forreri Coquimatln, 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 granulosum

Huapango, Estado de Mexico, Mexico

MG672435

H. pulcher (Ha86) A. granulosum Huapango, Estado de Mexico, Mexico

MG672436 HQ141710 (L)

H. pulcher (Ha13) R. montezumae Lerma, Estado de Mexico, Mexico

AF531866 (LB)

H. sibiricus Pelophylax nigromaculatus

Kokone, Japan AB818366 (H) AB818363 (H)

H. variegatus P. ridibunda Ivano-Frankivsk Region, Ukraine

AF151916 (T)

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 (LGA)

AY672125 (LGA)

Glypthelmins hyloreus Martin, 1969

Pseudacris triseriata WiedNeuwied

Nebraska, USA AY278050 (RLP)

AY278059 (RLP)

Glypthelmins brownorumae RazoMendvil, LenRgagnon & PrezPonce de Len, 2004

Rana brownorum Tabasco, Mexico, Mexico AY875674 (RLP)

AY278055 (RLP)

Brachycoelium salamandrae (Frlich, 1789) Dujardin, 1845

Salamandra salamandra

Zakarpatska Region, Ukraine AF151932 (T)

Opisthioglyphe ranae (Frlich, 1789) Looss, 1899

Rana arvalis IvanoFrankivsk Region, Ukraine

AF151935 (T)



H = Hasegawa et al., 2013.

L = Len-Rgagnon 2010.

LB = Len-Rgagnon & Brooks 2003.

LGA = Len-Rgagnon et al. 2005.

RLP = Razo-Mendvil 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 Forrers leopard frog Rana forreri Boulenger (ZaldvarRivern et al. 2004).

Type locality: Coquimatln, Colima, Mxico. Site of infection: lungs.Holotype: CNHE 10497 Paratypes: CNHE 1049810504, 10506, 10507, 4448Other hosts and localities: Rio Grande leopard frog R. berlandieri, San Antonio Chimalapas, Oaxaca;

Browns leopard frog R. brownorum, Rizo de Oro, Chiapas (VelazquezUrrieta & Len-Rgagnon 2017); Forrers leopard frog R. cf. forreri, San Pedro las Playas, Guerrero (CabreraGuzmn et al. 2007 as H. coloradensis),Tehuantepec, Oaxaca (CNHE 10509), Santa Mara del Oro, Nayarit; Transverse Volcanic leopard frog R. neovolcanica, Ocotln, 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; 2.46.2 (4.5) mm

long, 0.641.66 (1.24) mm of maximum width at testicular region. Tegument aspinose, even in live specimens. Oral sucker subterminal, round, 195527 (392) long, 203487 (385) wide. Pharynx oval, 114252 (196) long, 122227 (187) wide; oral sucker: pharynx ratio 1: 0.430.65 (0.51). Pharynx and anterior region of esophagus surrounded by abundant glans cells. Esophagus 32130 (84) long, sometimes obscured by uterus. Ceca bifurcated at 325852 (621) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 162365 (264) long, 162365 (271) wide, at 0.962.5 (1.7) mm (26%53% (38%) of BL) from anterior extremity. Sucker length ratio 1: 0.610.8 (0.70). Testes 2, oval, slightly lobed in some specimens, oblique, inmmediately posterior to ovary. Anterior testis opposite to ovary, 219730 (477) long, 203730 (466) wide. Posterior testis 259893 (553) long, 235812 (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, 220230 (225) long, surrounded by prostatic gland cells. Ovary oval, 252536 (409) long, 187446 (301) wide; at 1.12.8 (1.9) mm (33%55% (42%) of BL) from anterior extremity. Seminal receptacle adjacent, partially overlaps with ovary; 244730 (472) long, 162511 (337) wide. Mehlis gland dorsal to seminal receptacle. Laurers 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 4871152 (778) (10%25% (17%) of BL) from anterior end. Follicles extend asymmetrically, to level of anterior testis on ovarian side of body, and



Plagiorchis koreanus Ogata, 1938

Lymnaea stagnalis Linnaeus

Danube, Slovakia KJ533394 (Z) KJ533418 (Z)

Plagiorchis maculosus (Rudolphi, 1802) Braun, 1902

L. stagnalis Bohdanecsky, Czech Rep. KJ533396 (Z) KJ533428 (Z)


FIGURE 6. Haematoloechus occidentalis n. sp., from Rana sp., from Colima, Mxico. 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.


FIGURE 7. Haematoloechus occidentalis n. sp., from Rana sp., Colima, Mxico. 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.


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, 3039 (34) long, 1621 (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; BravoHollis 1943; Zamparo et al. 2011; Len-Rgagnon & RomeroMayn 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. occidentalisn. sp.) (Ingles 1932; Ibez & Crdoba 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 (Ibez & Crdoba 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 Len-Rgagnon (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: lungsHolotype: CNHE 4087Paratypes: CNHE 4086, 4089, 4090, 10515.


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.


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.


Other hosts and localities: common marsh frog R. vaillanti, Los Tuxtlas, Veracruz (Paredes-Caldern et al. 2004 as H. complexus); Catemaco, Veracruz (GenBank MG647801, MG647802, MG672439); Rio Grande leopard frog R. berlandieri, Laguna Higueras, Nuevo Len (Len-Rgagnon et al. 2005, as H. complexus), Huauchinango, Puebla (Len-Rgagnon 2003, as H. complexus CNHE 10517), Rana sp., Tierra Quemada (CNHE 10518) and Rancho el Borbotn (CNHE 10519), San Luis Potos, 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; 3.55.2 (4.3) mm

long, 0.801.64 (1.17) mm of maximum width at testicular region. Tegument aspinose, even in live specimens. Oral sucker subterminal, round, 203365 (310) long, 252409 (318) wide. Pharynx oval, 97195 (154) long, 105170 (145) wide; oral sucker: pharynx ratio 1: 0.380.56 (0.48). Pharynx and anterior region of esophagus surrounded by abundant gland cells. Esophagus 65122 (96) long, sometimes obscured by uterus. Ceca bifurcated at 381649 (496) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker round, 178292 (245) long, 203340 (265) wide, at 0.91.84 (1.4) mm (26%38% (33%) of BL) from anterior extremity. Sucker length ratio 1: 0.640.94 (0.84). Testes 2, oval, oblique to almost tandem, inmmediately posterior to ovary. Anterior testis opposite to ovary, 308771 (476) long, 292674 (415) wide. Posterior testis 446795 (604) long, 284771 (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, 260330 (295) long, surrounded by prostatic gland cells. Ovary oval, 349576 (449) long, 227608 (335) wide; at 0.922.1 (1.6) mm (26%42% (37%) of BL) from anterior extremity. Seminal receptacle adjacent and partially overlaps ovary; 349771 (548) long, 341649 (429) wide. Mehlis gland dorsal to seminal receptacle. Laurers 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 4711000 (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, 3440 (37) long, 1823 (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; Len-Rgagnon et al. 2001; Len-Rgagnon & Paredes-Caldern 2002). Haematoloechus veracruzanus n. sp. differs from H. arequipensis in having oval rather than lobed testes (Ibaez & Crdoba 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; BravoHollis 1943; Zamparo et al. 2011; Len-Rgagnon & RomeroMayn 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


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 (Ibez & Crdoba 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 (Len-Rgagnon & Brooks 2003, Genbank AF531857; Len-Rgagnon 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: Cinaga de Lerma, Estado de Mexico, Mexico.Site of infection: LungsHolotype: CNHE 10489Paratypes: 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, Ptzcuaro, Michoacn (Len-Rgagnon et al. 1999, as H. coloradensis); Transverse Volcanic leopard frog R. neovolcanica, Cointzio, Michoacn (this study).

Description: Based on 17 mature specimens: Body slender, with thinner anterior region; 4.88.3 (7.0) mm long, 0.71.3 (1.0) mm of maximum width at testicular region. Tegument covered with abundant thin spines, easily lost during fixation; 7.512.5 (9.8) long. Oral sucker subterminal, round, 203350 (284) long, 180380 (278) wide. Pharynx oval, 140280 (206) long, 122220 (183) wide; oral sucker/pharynx ratio 1: 0.740.90 (0.83). Anterior border of pharynx and esophagus surrounded by gland cells. Esophagus 41200 (129) long. Ceca bifurcated at 390770 (609) from anterior extremity. Ceca terminate blindly near posterior extremity. Ventral sucker small, weakly developed, frequently obscured by uterus, 52125 (82) long, 57130 (84) wide, at 1.53.0 (2.4) mm (31%41% (37%) of BL) from anterior extremity. Sucker length ratio 1:0.280.38 (0.33). Testes 2, elliptical, elongate, oblique, posterior to ovary; distance between ovary and anterior testis 350825 (548). Anterior testis opposite to ovary, 3651,080 (778) long, 243600 (451) wide. Posterior testis 4221200 (863) long, 260830 (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, 150160 (155) long, surrounded by prostatic gland cells. Ovary kidney shaped, lobed, 320840 (623) long, 162500 (342) wide; at 1.84.3 (2.8) mm (35%57% (40%) of BL) from anterior extremity. Seminal receptacle posterior, partially overlapped with ovary; 3001000 (644) long, 250590 (410) wide. Mehlis gland dorsal to seminal receptacle. Laurers canal not observed. Vitellaria


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.


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.


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 9823200 (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, 2226 (24) long, 1420 (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: 0.51.0 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; Len-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; Len-Rgagnon 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; Len-Rgagnon 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 (Len-Rgagnon & Paredes-Caldern 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 (Len-Rgagnon et al. 1999, 2001; Len-Rgagnon & Paredes-Caldern 2002; Len-Rgagnon & Brooks 2003; Len-Rgagnon 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


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, 1996

9 2.4 A O O, D 1d A SDA

H. combesi Batchvarov & Bourgat, 1974

5.26.1 2.83.3 0.37 I, L O, L, D 1a P OA

H. darcheni Combes & Knoepffler, 1967

4.86.7 1.41.8 0.25 I, L L, I, D 1a P AT

H. dollfusinus (Odening, 1958) Yamaguti, 1971

1818.9 33.2 0.66 L O, D 1a P A

H. exoterorchis Rees, 1964 7.32; 6.58.5 1.4; 1.31.4 0.79 O E, Pa, Ex

3 P PT

H. johnsoni Bourgat, 1977 3.97 12 0.55; 0.520.62 I, L L, I, D 1a P PT

H. leonensis (Williams & Coker, 1967) n. comb.

8.8; 7.912.4 2.7; 2.13.4 0.17 K O, D 1a P OA

H. lobogonadus (Meskal, 1970) Gassmann, 1975

12.9; 1214 3.6; 3.24 0.51, L L, I, D 1d P PT

H. micrurus Rees, 1964 10.4; 8.513 2.3; 1.72.6 0.5 O O, D 1a P Ph

H. ocellati Gassmann, 1975 3.54.3 11.1 0.28 I, L O, L, D 1a P AT

America

H. arequipensis Ibez & Crdoba, 1979

7.9; 78.4 2; 1.82.4 1; 0.91.1 O L, I, T 1a P A

H. breviplexus Stafford, 1902 9; 5.812 2.1; 22.7 0.5 I, L E, L, D 1a P PT

H. buttensis Ingles, 1936 7.4; 3.210 1.3; 0.72.2 0.7; 0.60.8 K O, D 1b P PT

H. caballeroi (Skrjabin & Antipin, 1962) n. comb.

4.9; 2.86.6 1.3; 0.81.9 0.82; 0.531 O, L O, L, D 1a P SDA

H. coloradensis (Cort, 1915) Ingles, 1932

5.7; 3.38.1 1.1; 0.51.5 0.9; 0.751.14 O O, D 1d A A

H. complexus (Seely, 1906) Ingles, 1932

4.9; 1.26.08 1; 0.31.1 0.71; 0.590.9 O O, D 1a P SDA

H. confusus Ingles, 1932 3.9; 3.34.9 0.9 0.75; 0.550.8 L, I L, I 1b A A

H. danbrooksi LenRgagnon & ParedesCaldern, 2002

3.9 0.9 0.32 O, L O, D 1b P SDAP

H. elongatus Caballero & Sokoloff, 1934

9.5; 910 1.6; 1.51.7 0.7; 0.640.75 O O, D 1a P SDA

H. floedae Harwood, 1932 6.3; 211.1 1.7; 0.72.7 0.35; 0.30.41 I, L O, D 1c P OA

H. freitasi MaGarzon & Gil, 1959 13.5 2.5 1.16 E L, I, D 1a P PT

H. fuelleborni (Travassos & Darriba, 1930) Yamaguti, 1958

10 2.3 0.5 O O, D 1a P SDA

H. humboldtensis Zamparo, Ferrao, Brooks, Bettaso & MataLpez, 2011

6.2; 5.66.7 1.4; 1.21.7 0.76; 0.720.78 O O, D 1b P SDP

H. illimis Caballero, 1942 6.2; 4.86.86 2.1; 1.52.75 0.69; 0.560.83 L, K L, D 1a P SDA





H. iturbei (Cordero & Vogelsang, 1939) Yamaguti, 1958

16.8 1.5 A O E, D 1b P PT

H. kernensis Ingles, 1932 6.3; 5.57 1.4 1; 0.851.06 O O, D 1a P SDA

H. longicollum LenRgagnon & RomeroMayn, 2017

5.6; 3.67.2 1.2; 0.61.9 0.81; 0.680.96 O O, D 1a P SDP

H. longiplexus Stafford, 1902 6.5; 4.68.0 1.9; 1.22.55 0.43; 0.40.5 I, L E, Pa 1c P Ph

H. lutzi Freitas & Lent, 1939 46.5 1.32 0.9 O E, D 1a P PT

H. macrorchis Caballero, 1941 6; 5.66.37 1.6 0.45; 0.410.49 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 LenRgagnon, Brooks & Zelmer, 2001

8.25 1.7 0.2 O O, D 1c P A

H. mexicanus n. sp. 7; 4.88.3 1.1; 0.71.3 0.33; 0.280.38 L, K O, E, D 1c P SDA

H. neivai (Travassos & Artigas, 1927) Ingles, 1933

3.76.7 1.32.3 A O O, L, Pa 1a P OA

H. nicolasi LenRgagnon, 2017 3.9; 2.56 1.2; 0.81.9 0.31; 0.230.36 L E, L, D 1c P PT

H. occidentalis n. sp. 4.5; 2.46.2 1.2; 0.61.7 0.71; 0.610.8 O, L O, D 1b P SDA

H. oxyorchis Ingles, 1932 5.8; 36.5 0.9 0.78; 0.620.85 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; 5.47.7 2; 1.52.4 0.82; 0.750.88 I, L O, D 1a P A

H. parviplexus (Irwin, 1929) Ingles, 1932

6.1 1.2 0.25 O O, D 1b P PT

H. pukinensis Ibez & Crdoba, 1979

3.8; 3.54.2 0.8; 0.70.9 0.94; 0.871 O O, L, D 1a P SDA

H. pulcher BravoHollis, 1943 4.0; 3.354.86

1.1; 11.2 0.73; 0.650.81 O, K O, L, D 1a P SDP

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; 3.55.2 1.3; 0.81.6 0.84; 0.640.94 O O, D 1c P SDAP

Australia

H. australis (Johnston, 1912) Yamaguti, 1958

3.5; 2.54.9 1.5 0.5 L O, D 1a P AT

Eurasia

H. abbreviatus (Bychowsky, 1932) Prokopic & Krivanec, 1974

6 1.62 0.57 O O, D 1a P OA

H. almorai (Pande, 1937) Yamaguti, 1958

1.77 0.71.6 0.5 E, L E, D 1a P OA

H. asper Looss, 1899 210 11.6 0.75 O O, D 2 P PT

H. breviansa (Sudarikov, 1950) Yamaguti, 1958

4.6 1.3 0.54 O O, D 1e P PT

H. bychovskii Odening, 1958 2.22.7 1.01.1 0.88 O O, Pa 2 P OA

H. capyristes (Klein, 1905) Odening, 1958

6.0 1.4 0.5 O I, E, D 1a P OA

H. carbonelli Lluch, Navarro & PrezSoler, 1991

4.24.5 1.2; 11.4 0.50.62 I, L E 1a P PT

H. donicus (Shevchenko, 1965) n. comb.

4.34.4 1.51.7 0.60.8 O O, D 1e P PT



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 = kidneyshaped.

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.



H. japonicus (Yamaguti, 1936) n. comb.

311.9 12.5 1.162 I, L E, D 1a P OA

H. jeholensis (Fukui & Ogata, 1938) n. comb.

5 1.4 1.16 K O, D 1a P OA

H. lipsiensis (Odening, 1958) n. comb.

6.5 1 0.75 O O, D 2 P PT

H. lobatus Seno, 1907 7.511.7 0.9 0.33 0.4 I, L L, D 1a P PT

H. nanchangensis Hsiung, 1934 1.43.8 1.62.5 0.86 O O, Pa 2 P OA

H. pyrenaicus Combes, 1965 10.1; 7.613.7

2.3; 1.83.2 0.66 I, L E, L, D 1b P PT

H. schulzei (Wundsch, 1911) Prokopic & Krivanec, 1974

818 1.52 0.6 O O, D 1a P AT

H. sibiricus (Isajcikov, 1927) Yamaguti, 1958

1.511.2 0.63.7 1 O, K E, D 1a P OA

H. similis (Looss, 1899) Yamaguti, 1958

4.510 12 0.660.75 O O, D 1e P AT

H. sindensis Khan & Mohiuddin, 1982

4.9; 2.67.7 1.5; 0.72.7 0.5 O O, D 1a P OA

H. singaporensis Yuen, 1962 5.08.5 1.22.3 0.66 I, L O, D 1a P OA

H. tientsinensis Hsiung, 1934 1.76.6 0.61.4 1.01.16 O E, D 1a P OA

H. travdarribus (Skrjabin & Antipin, 1962) Yamaguti, 1971

4.6 1.7 1.1 O O, Pa 1c P OA

H. variegatus (Rudolphi, 1819) Looss, 1899

2.518 0.62.2 0.50.75 E, L O, E, D 1a P OA

H. vojtkovae Prokopic & Krivanec, 1974

3.7 1 0.76 O O, D 1e P PT

H. volgensis (Sudarikov, 1950) Yamaguti, 1958

4.5 1.2 0.78 O O, D 1e P AT


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, 1971

Tropical (Central) Ranidae: Ptychadeninae

H. exoterorchis Rees, 1964 Tropical (West) Ranidae: Dicroglossinae

H. johnsoni Bourgat, 1977 Tropical (West, Central) Ranidae: Dicroglossinae

H. leonensis (Williams & Coker, 1967) n. comb.

Tropical (West) Odontobatrachidae

H. lobogonadus (Meskal, 1970) Gassmann, 1975

Tropical (East) Ranidae: Pyxicephalidae

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. comb.

Central Mexico Ranidae: Raninae; Ambystomatidae (Acc)

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 LenRgagnon & ParedesCaldern, 2002

Southeast Mexico Ranidae: Raninae; Bufonidae (Acc)

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, Bettaso & MataLpez, 2011

West U.S.A. Ranidae: Raninae

H. illimis Caballero, 1942 Central Mexico Ranidae: Raninae

H. kernensis Ingles, 1932 West U.S.A. Ranidae: Raninae; Bufonidae (Acc)





H. longicollum LenRgagnon & RomeroMayn, 2017

West Mexico Ranidae: Raninae

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 LenRgagnon, 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 BravoHollis, 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 Ibez & Crdoba, 1979 Peru Telmatobiidae

H. freitasi MaGarzon & Gil, 1959 Uruguay Leptodactylidae: Leptodactylinae

H. fuelleborni (Travassos & Darriba, 1930) Yamaguti, 1958

Brazil Bufonidae; Leptodactylidae: Leptodactylinae

H. iturbei (Cordero & Vogelsang, 1939) Yamaguti, 1958

Venezuela, Brazil Ranidae: Raninae

H. lutzi Freitas & Lent, 1939 Venezuela Ranidae: Raninae

H. meridionalis1 LenRgagnon, Brooks & Zelmer, 2001

Costa Rica, Mexico Ranidae: Raninae

H. neivai (Travassos & Artigas, 1927) Ingles, 1933

Brazil, Venezuela Leptodactylidae: Leptodactylinae; Ranidae: Raninae; Hylidae: Pseudinae (Acc)

H. ozorioi Freitas & Lent, 1939 Uruguay Leptodactylidae: Leptodactylinae

H. pukinensis Ibez & Crdoba, 1979 Peru Telmatobiidae

Australia

H. australis (Johnston, 1912) Yamaguti, 1958 Australia Pelodryadidae: Pelodryadinae; Limnodynastidae

Eurasia

H. abbreviatus (Bychowsky, 1932) Prokopic & Krivanec, 1974

Central and Oriental Europe Bombinatoridae

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, 1958

Oriental Europe Ranidae: Raninae

H. bychovskii Odening, 1958 Russia Ranidae: Raninae

H. capyristes (Klein, 1905) Odening, 1958 India Ranidae: Dicroglossinae



(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. 13): 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; Len-Rgagnon & Brooks, 2003; Len-Rgagnon, 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



H. carbonelli Lluch, Navarro & PrezSoler, 1991

Spain Ranidae: Raninae; Bombinatoridae (Acc); 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 & Krivanec, 1974

Central and Oriental Europe Ranidae: Raninae

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) Yamaguti, 1971

Germany Ranidae: Raninae

H. variegatus (Rudolphi, 1819) Looss, 1899 Europe Ranidae: Raninae; Bufonidae (Acc)

H. vojtkovae Prokopic & Krivanec, 1974 Czech Republic Bombinatoridae

H. volgensis (Sudarikov, 1950) Yamaguti, 1958

Oriental Europe Ranidae: Raninae


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, Gnther, Loader, Mahlow & Rdel, Hyperoliidae Laurent (Africa), Limnodynastidae Lynch and Pelodryadidae Gnther (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


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 (Len-Rgagnon 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; Hernndez-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 Mara Antonieta Arizmendi-Espinoza, Mara Anglica Najar-Pacheco, David Osorio-Sarabia, Gerardo Prez-Ponce de Len, Ulises Razo-Mendvil, ngeles RomeroMayn, Mara Guadalupe Velarde-Aguilar, Alejandro Zaldvar Rivern (Instituto de Biologa, UNAM), Rosario Mata-Lpez, Edmundo Prez-Ramos, (Facultad de Ciencias, UNAM), Elisa Cabrera-Guzmn (Oklahoma State Univeristy), Luis Jorge Garca-Mrquez (Universidad de Colima), Agustn Jimnez-Ruiz (Southern Illinois Univeristy), Sean Locke (Universidad de Puerto Rico), and Elizabeth Martnez-Salazar (Universidad Autnoma de Zacatecas), for their help in the collection of specimens; Laura Mrquez-Valdelamar (IBUNAM) for her help in the sequencing of samples; Luis Garca-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, Jos Iannacone and Georgina OrtegaLeite 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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