Two new species of Parapharyngodon parasites of Sceloporus pyrocephalus, with a key to the species found in Mexico (Nematoda, Pharyngodonidae)

Abstract Two new species of Parapharyngodon collected from the intestine of the Mexican boulder spiny lizard Sceloporus pyrocephalus are described. This study increases to 49 the number of valid species assigned to Parapharyngodon worldwide, 11 of them distributed in Mexico. Males of the two new species share the presence of four pairs of caudal papillae, an anterior echinate cloacal lip and the presence of lateral alae; however, both differ from each other in lateral alae extension and echinate cloacal anterior lip morphology. Females of both species have a prebulbar uterus and eggs shell punctuate with pores, characteristics shared with few other species of Parapharyngodon. Both new species differ from other congeneric species in the papillar arrangement, the anterior cloacal lip morphology, the lateral alae extension and total length/spicule ratio. A taxonomic key for the species of Parapharyngodon distributed in Mexico is provided.


Introduction
Mexico has a species-rich reptile fauna, with 864 species (8.7% of the worldwide total); 57% of them are endemic (Flores-Villela and García-Vázquez 2014). Although Mexico has a long tradition of helminthological and herpetological research, there are large gaps in the knowledge of the species diversity of helminths associated with these vertebrates (Pérez-Ponce de León et al. 2011). Sceloporus Wiegmann is a reptile genus distributed in the Americas; it inhabits a wide range of environments, and it is the most representative lizard taxon of the Mexican herpetofauna with 92 of 97 species that composed the genus, with the majority of them endemic (Flores-Villela 1993). Sceloporus pyrocephalus Cope is an oviparous endemic lizard to Mexico. It is associated with streams and rivers within tropical deciduous and semi-deciduous forest, and it is distributed from the Southwestern Pacific coast of Jalisco and Colima to Michoacán, Guerrero, South-western Mexico State, and Southern Morelos (Uetz and Hošek 2013). There are scarce studies on this phrynosomatid lizard; the majority of these are focused in taxonomy, conservation, and reproductive research (Ramírez-Bautista and Olvera-Becerril 2004, Calisi et al. 2007, Leaché 2010.
Recently, the validity of species of Parapharyngodon Chatterji, 1933was reviewed (Velarde-Aguilar et al. 2015, Bursey and Goldberg 2015. In accordance with these authors, of the 78 Parapharyngodon species assigned to this genus, only 47 have been properly described, and recognized as valid species. Nine of these species are distributed in the Panamanian realm, seven in Oriental, seven in Palearctic, six in Neotropical, five in Afrotropical, five in Nearctic, five in Saharo-Arabian, one Australian, one in Sino-Japanese realm, and one species in Madagascan region. In this paper, two new species of Parapharyngodon parasites of S. pyrocephalus collected from Michoacán and Guerrero states, Mexico, are described, increasing the basic knowledge about helminths of Mexican lizards.

Materials and methods
During the breeding season months of June-July in 2003, 2004 and 2005, 16 S. pyrocephalus were captured (under the collection permit SEMARNAT FAUT-0056 issued to VLR) by noosing or hand in ten different locations (seven from Michoacán and three from Guerrero states, Mexico, Table 1). Hosts were killed by an intraperitoneal injection of sodium pentobarbital overdose. The mouth, peritoneal cavity and all internal organs were examined for helminths with the use of stereoscope. Nematodes found were counted, fixed in hot 4% formaldehyde solution and stored in alcohol 70%. For morphological study, specimens were cleared in glycerin-alcohol 70% solution at 1:1 ratio, and mounted on temporary slides for examination under a light microscopy. Original drawings were made with an Olympus BX53 microscope equipped with camera lucida. For scanning electron microscopy (SEM), worms were dehydrated through ethanol series, dried with a K850 Critical Point Drier (Emitech, Ashford, England), coated with gold using a Q150R Modular Coatin System (Quórum, Ashford, England), and examined in a Hitachi S-2460N (Hitachi, Tokyo, Japan) and SU1015 SEM (Hitachi) SEM. Measurements are provided in millimeters, including the range, followed by average and standard deviation, and the sample size. Host specimens collected were deposited in the Herpethology Collection of the Museo de Zoología, Facultad de Ciencias (MZFC), UNAM, and helminths were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, UNAM. Etymology. The species is named in honor of the 43 missing students from "Escuela Normal Rural Raúl Isidro Burgos" in Ayotzinapa, Guerrero, Mexico; in solidarity with their families and the Mexican people.

Family
Diagnosis. Robust, small and white fusiform nematodes, males smaller than females. Cuticle with prominent transverse striations along the whole body except tail. Triangular oral opening surrounded by three simple lips in males and bilobed in females, in both sexes ventrolateral lips have an amphid each one, in females it is located on the dorsal lobe. Within buccal cavity, both sexes have three transverse plates, bilobed in males and complete in females. Esophageal bulb with sclerotized apparatus. Excretory pore evident, it is located at level of posterior edge of esophageal bulb. A vesicular body surrounds excretory duct. Males with lateral alae covering the last third of body, females lacking lateral alae. Males without caudal alae. Four pairs of caudal papillae. Caudal filament sub-terminal and directed dorsally in males. Females with a conical posterior end. Vulva located at middle region. Eggs no shown alae, with a punctuated shell and subpolar operculum, embryo in early stage of cleavage.
Biology. Nematode species parasite of the intestine of Sceloporus pyrocephalus Cope, collected on July 6, 2005.
Diagnosis. Fusiform and robust nematodes, males smaller than females. Cuticle with thin transverse striations constant in width along the whole body except tail. Triangular oral opening surrounded by three lips simple in males and bilobed in females, in both sexes ventrolateral lips with an amphid each one. Within buccal cavity, both sexes have three transverse plates, bilobed in males and complete in females. Esophageal bulb with sclerotized apparatus. Excretory pore evident, it is located posterior to esophageal bulb-intestine junction. A vesicular body surrounds excretory duct. Males with lateral alae covering almost of body length, females lacking lateral alae. Males without caudal alae. Four pairs of caudal papillae. Caudal filament subterminal and directed dorsally in males. Females with a conical posterior end. Vulva located at middle region. Eggs without alae with punctuated shell and a subpolar operculum, embryo in early stage of cleavage.
Biology. Nematode species parasite of the intestine of Sceloporus pyrocephalus Cope, collected on June 21, 2004.

Remarks
Parapharyngodon tikuinii sp. n. is the 80 th species assigned to Parapharyngodon and the 49 th valid species of the genus. It is characterized by the presence of a cuticular outgrowth at base of posterior cloacal lip. In addition, the following composition of traits allow us to differentiate the new species described herein: four pairs of caudal papillae, an echinate anterior cloacal lip, lateral alae covering almost the length of the body, spicule length 3.287% of total body length, prebulbar ovaries coiling around prebulbar esophagus and eggs shell punctuate. Nineteen of the 48 valid species described before (including Parapharyngodon ayotzinapaensis), share with P. tikuinii the arrangement of caudal papillae (four pairs of caudal papillae: one precloacal, one paracloacal, one at postcloacal lip and one at caudal filament). Thirteen of these species have echinate precloacal lip and lateral alae as the second new species described herein; of these, only P. grenadaensis, P. colonensis and P. ayotzinapaensis share the presence of prebulbar ovaries and a punctuate egg shell with P. tikuinii. Nonetheless, P. tikuinii differs from P. grenadaensis, P. colonensis and P. ayotzinapaensis in the lateral alae extension (which start at level of nerve ring and end at level of precloacal papillae in these three species, whereas in P. tikuinii lateral alae cover exclusively the last portion of the body); in addition, spicule length-total body length ratio is greater in P. grenadaensis (4.488%), P. colonensis (3.765%) and P. ayotzinapaensis (3.614%) than in P. tikuinii (3.287%) (Bursey and Goldberg 2005, Bursey et al. 2013, Velarde-Aguilar et al. 2015, Bursey and Goldberg 2015. Finally, males of P. grenadaensis and P. colonensis have three bilobed lips which are simple in P. tikuinii. Consequently, P. tikuinii is proposed as new species for the genus and the 11th recorded in Mexico.

Discussion
Parapharyngodon includes species parasitizing ectothermic vertebrates (mainly reptiles), few species of amphibians [Rhinella marina Linnaeus (Anura: Bufonidae) parasitized by P. grenadaensis, Phrynohyas venulosa Laurenti (Anura: Hylidae) parasitized by P. duniae, Onychodactylus japonicus Houttuyn (Caudata: Hynobiidae) parasitized by P. japonicus, Triprion petasatus Cope (Anura: Hylidae) parasitized by P. hylidae, and Diaglena spatulata Günther (Anura: Hylidae) parasitized by P. chamelensis] and one ancestral mammal species [Tachyglossus aculeatus Shaw (Monotremata: Tachyglossidae) (Irwin and Raharison 2009)]. Only two species of Parapharyngodon have been described as parasites of phrynosomatid lizards: P. grismeri and P. iguanae in Petrosaurus repens Van Denburgh and Petrosaurus mearnsi Stejneger, respectively (Paredes-León et al. 2008, Velarde-Aguilar et al. 2015. In this study, we describe two additional species infecting phrynosomatid lizards: P. ayotzinapaenis and P. tikuinii. Some authors had emphasized the relationship between food habits and composition of helminths richness in reptiles , Pereira et al. 2013, Roca 1999. In this sense, Petter (1966) and Petter and Quentin (1976) recognized an evolutionary trend within Pharyngodonidae and distinguished two groups of genera: 1) Parasites of herbivorous iguanids and testudines, and 2) Parasites of omnivorous and insectivorous reptiles. In Mexico, nine pharyngodonid genera have been registered, five in herbivorous iguanids and testudines (Ozolaimus Dujardin, 1845;Tachygonetria Wedl, 1862;Alaeuris Thapar, 1925;Thaparia Ortlepp, 1933;Gopheruris Petter & Douglas, 1976), and four in omnivorous and insectivorous reptiles (Parapharyngodon; Pharyngodon, Diesing, 1861;Spauligodon Skrjabin, Schikhobalova &Lagodovskaja, 1960, andSkrjabinodon Inglis, 1968). In this sense, the presence of Parapharyngodon species infecting S. pyrocephalus confirm indirectly the feeding habits observed in this lizard, which is considered an omnivorous and insectivorous species by Alvarado-Diaz et al. (2009). On the other hand, there are scarce studies focused on evolutionary history of Pharyngodonidae and only include few genera from the same region (Jorge et al. 2011, Jorge et al. 2012) and some species parasitic in herbivores (Bouamer and Morand 2003). This information is not enough to attempt to relate the trend observed among pharyngodonids to parasitize hosts based on its different feeding habits with their evolutionary history. Robust analyses based on molecular and morphological information about the relationship of pharyngodonid species and their hosts will allow analysis of the patterns and process involved in their evolutionary history.