A new species of Paracreptotrema (Digenea, Plagiorchiformes, Allocreadiidae) infecting two species of poeciliids in Río Malila of the Río Pánuco basin, Hidalgo, México, with a key to the species of the genus

Abstract Paracreptotrema rosenthali sp. n. was discovered in the intestine of Xiphophorus malinche and Pseudoxiphophorus jonesii, collected from the headwaters of Río Malila, tributary of Río Conzintla, in the Río Pánuco basin, Hidalgo, México, during 2008–2009. The new species differs from the five known species of Paracreptotrema Choudhury, Pérez-Ponce de León, Brooks & Daverdin, 2006 by having vitelline follicles that extend from a level anterior to the pharynx to mid-testes, the seminal vesicle which is more extensively folded, and a wider cirrus sac. The new species resembles Paracreptotrema heterandriae in the length of its ceca, which surpasses the posterior margin of the ovary but do not reach the testes. A key to the species of Paracreptotrema is provided.


Introduction
Despite an increase in our knowledge of the helminth parasites of the species of fish in México, Pérez-Ponce de León and Choudhury (2010) recently suggested that regions characterized by high biodiversity, such as the drainage basin of the Río Pánuco, need more intensive sampling. Their study indicated that the Poeciliidae, a family with many species endemic to México but with a limited range (Miller et al. 2005), could provide new information on the biodiversity of helminth parasites of freshwater fishes. Xiphophorus malinche Rauchenberger, Kallman & Morizot is such a poeciliid with a distribution restricted to the Río Pánuco basin. At present, it is known to inhabit only six isolated highland headwater streams (Culumber et al. 2011). Relatively little is known about the parasite communities of X. malinche; however, a recent study compared parasite communities between two populations of this species and reported differences in the helminth communities that the authors attributed to geographic isolation (Bautista-Hernández et al. 2014b). As part of that study, an undescribed species of Paracreptotrema Choudhury, Pérez-Ponce de León, Brooks & Daverdin, 2006 was recovered in one of these populations; it is described herein and a key to the known species is presented.

Materials and methods
Adult specimens of Xiphophorus malinche (60 individuals; May 2008 to July 2009) and Pseudoxiphophorus jonesii (Günther, 1874) (sensu Agorreta et al. 2013) (= Heterandria jonesii) (30 individuals; August 2012) were collected from the Río Malila, a tributary of the Río Conzintla, northeastern Hidalgo, México. Fish were collected using minnow traps, brought live to the laboratory of the Centro de Investigaciones Científicas de las Huastecas Aguazarca (CICHAZ) field station in Calnali, Hidalgo, and examined within 24 h after capture. Fish were fixed in ethyl alcohol (EtOH 96%) for confirmation of their identification. Trematodes were collected live, killed in warm water and fixed for 24 h in alcohol-formalin-acetic acid. Specimens were stained with Mayer's carmalum or Delafield's hematoxylin, mounted whole in Canada balsam, and examined using bright-field and differential interference contrast optics. Illustrations were made with a drawing tube attached to the microscope; measurements are given in micrometers (µm) and are expressed as the range of measurements followed by the mean ± standard deviation in parentheses. Comparisons of other members of the genus with the new species are made from the original descriptions, but full data on each species from all published works are given in Table 1; reported measurements are given exactly as in the original work because all of the original specimens were not available to be re-measured.  (Galicia et al. 2014).
Etymology. The species is named in honor of Gil G. Rosenthal, Department of Biology, Texas A&M University, College Station, Texas, and co-founder of the CICHAZ field station, for his friendship, contributions to the knowledge of species of Xiphophorus, and in recognition of his efforts to promote science in the Huasteca region of México.

Remarks
The genus Paracreptotrema includes four species: P. blancoi Choudhury, Pérez-Ponce de León, Brooks & Daverdin, 2006, P. mendezi (Sogandares-Bernal, 1955, P. profundulusi Salgado-Maldonado, Caspeta-Mandujano & Martínez-Ramírez, 2011, and P. heterandriae Salgado-Maldonado, Caspeta-Mandujano & Vázquez, 2012. The specimens of P. rosenthali sp. n. from X. malinche share the features established in the original concept of the genus (Choudhury et al. 2006). In general, there are five primary features that can be used to distinguish P. rosenthali sp. n. from the extant species: the shorter length of the ceca, the extent of the vitelline follicles, the extensive folding of the seminal vesicle, the width of the cirrus sac, and the extension (area occupied) of the uterus. Paracreptotrema rosenthali resembles P. mendezi, P. blancoi, and P. profundulusi in having a well-developed cirrus sac, but the new species stands out by having a seminal vesicle that is more extensively folded and the cirrus sac which is wider than those of the other three taxa. Paracreptotrema rosenthali sp. n. and P. heterandriae have ceca that extend past the ovary but not to the testes; however, in the latter species the body is longer and narrower than that of P. rosenthali sp. n. The vitellarium of the new species extends from a level anterior to the pharynx to the middle of the testes, and in some specimens reach but do not pass the posterior margin of the testes, and the follicular rows partially overlap the acetabulum dorsally. In P. mendezi, the vitellarum extends posteriorly from the oral sucker but does not pass the anterior margin of the testes. In P. blancoi it extends from the cecal bifurcation to the anterior edge of the testes. In P. profundulusi, the vitellarium extends to the postesticular area, and in P. heterandriae the vitelline follicles extend from the cecal bifurcation to the posterior margin of the testes. The extent of the uterus of P. rosenthali sp. n. is similar to the uterine distribution of P. blancoi and P. heterandriae in that the uterus extends to the posterior margin of the testes, often filling the post-testicular area; in P. profundulusi the uterus is mostly pretesticular. The mean size of the eggs of P. rosenthali sp. n. (52 long by 32 wide) is similar to that of P. blancoi (55 by 39) and P. profundulusi (57 × 31); the mean egg size of P. mendezi (46 × 37) is smaller and that of P. heterandriae (72 × 40) is larger. The number of eggs in the uterus ranged from 20-40 among the specimens of the new species; the specimens of P. blancoi that we examined had fewer than 10 eggs and those of the three other species that we observed had from 8-24 eggs in the uterus. . Four of the five species of Paracreptotrema, including the one described in this paper, are parasites of poeciliid fish. Choudhury et al. (2006) suggested that Paracreptotrema spp. might be parasites exclusive to poeciliids in the Neotropical region. However, Salgado-Maldonado et al. (2011) described P. profundulusi from and reported P. blancoi in species of the Profundulidae, arguing that this was evidence that Paracreptotrema spp. could have a closer relationship with freshwater members of the Profundulidae than with the Poeciliidae because of the restricted distribution of the latter family in Central America. The geographic distribution of Profundulus is restricted to hydrological basins of Central America, extending northward only to the Isthmus of Tehuantepec (southeastern México), so the co-occurrence of the two species of Paracreptotrema in those fish could be due to recent contact between dif-ferent host populations. The finding of the new species does not offer insights into the co-speciation of the members of the genus; i.e. the origin of each species and whether they originated in poeciliids or profundulids. For this reason, a phylogeny of the group is needed, ideally combined with a hypothesis regarding the taxa that host these species. Additionally, in cases where the localities of each species of helminth are widely separated, further studies are needed to verify the limits of the distribution of each.

Discussion
The distribution of X. malinche is restricted to the more northern Hidalgo anticline, separated from southern populations of fish by the barrier range of the Mexican plateau (Kallman and Kazianis 2006), so it is not clear how the population of P. rosenthali sp. n. is linked to those species of Central America. Consistent with hypotheses regarding the orogeny and isolation of headwater populations, Bautista-Hernández et al. (2014b) reported differences in parasite communities between two populations of X. malinche (Chicayotla and Malila) that are separated only by two mountain ridges. Specifically, the Malila population was infected with three species of helminth, whereas the Chicayotla population was infected with four species. Our finding a new species restricted to the Malila population further supports the importance of host biogeographic factors with regard to the structure of helminths communities. Although helminth diversity is affected by the restricted distribution of their host, further studies are needed to evaluate the familial host specificity of species of Paracreptotrema. Paracreptotrema mendezi was collected from fish living in a lake but all other known species are from stream-and river-dwelling populations of fish; whether or not this factor is important for our understanding of the ecological relationships of the members of the genus is still unknown.
The papillae on the oral sucker were difficult to discern on our specimens. Two papillae on the posterior margin of the sucker were visible on some specimens, but only some of the papillae along outer edge were visible on a few specimens; thus, no papillae were included in the figure. We could discern several papillae along the outer edge of the oral sucker in specimens of P. blancoi, P. profundulusi, and P. heterandriae, but the entire complement of papillae was not visible in any specimens we examined. Study of specimens using scanning electron microscopy will be necessary for a full assessment of the number of papillae present, but the number of specimens available at this time is not sufficient for such a study.
All known species of Paracreptotrema have an oral sucker that is wider than long ( Table 1). The new species is not different in this respect. However, one specimen we collected, the holotype (unfortunately), had an oral sucker longer than wide (Fig. 1). This specimen was processed differently to any of the others, and it was one of six specimens from single-worm infections, but it is the only one with the different sucker size ratio. Even with that worm removed from the comparison, the oral sucker of P. rosenthali sp. n. is the largest of the known species. Similarly, the average length of the acetabulum was greater than the width, but in some worms this was reversed.
The presence of Laurer's canal has been reported for the four previously known species. We were not able to discern the canal in specimens of the new species. The limited material precluded mounting of specimens in a more favorable position for Table 1. Comparison of morphological characteristics of the five species described as Paracreptotrema.

Data for P. blancoi
(México), P. blancoi (Costa Rica), P. mendezi and P. heterandriae taken from Choudhury et al. (2006) and Salgado-Maldonado et al. (2012). Note: measurements are given exactly as in the original work with the same precision as reported and presented as the range followed by the mean.    observations of this structure, and no specimens were available for histological study. The populations of fish from which the specimens were collected are limited in size and fragile, and this helminth has not been found in other populations of fish close to the locality (Bautista-Hernández et al. 2014a;Bautista-Hernández et al. 2014b), but the presence of Laurer's canal needs to be confirmed by future studies. Razo-Mendivil et al. (2014) provided molecular evidence that P. heterandriae is a member of the Allocreadiidae, affording strong support for the familial relationship previously suggested by Choudhury et al. (2006) and Salgado-Maldonado et al. (2012). A more inclusive molecular study of the new species would provide additional information on the relationships of this species with P. heterandriae and the other members of the genus. Molecular evidence would also provide confirmation of the specific identification of the putative species which have been identified to date. Morphological characters, some of which can vary intraspecifically, have been the primary features used to identify species; molecular techniques could verify or falsify the appropriateness of the morphological features that have been used. "El efecto de hibridización en la diversidad de helmintos parásitos de peces del género Xiphophorus" (Clave 0127310) to SM, as was a scholarship from CONACYT to CEB-H (217861) during her PhD research project.