New species of Foleyellides (Nematoda: Onchocercidae: Waltonellinae), parasite of Lithobates brownorum (Amphibia: Ranidae) from South-eastern Mexico and genetic barcodes of the Mexican species of the genus

Specimens of Foleyellides were collected from the body cavity of frogs in different regions of Mexico; Lithobates brownorum from Yucatán, Quintana Roo and Campeche; L. megapoda from Jalisco and Rhinella marina, from Guerrero. Foleyellides calakmulesis n. sp. is described based on specimens found parasitizing L. brownorum. The new species is distinguished from the other members of the genus by the combination of the following male characters: four pairs of caudal papillae different in size and the presence of a preanal plaque. Partial DNA sequences of the mitochondrial Cytochrome Oxidase C, subunit I of the four known Mexican species of Foleyellides and two potentially new species collected in this study were generated and compared, validating the erection of the new species.

Mitochondrial DNA sequences, in particular partial COI sequences, known as barcodes, have shown to be useful to differentiate species of nematodes (Lima-Monteiro et al., 2018;Powers et al., 2018;Siddall et al., 2012), given the morphological conservatism in some groups and high phenotypic plasticity in others (Nadler, 2002;Powers et al., 2011).Nevertheless, the only barcodes representing Foleyellides available in GenBank belong to F. mayenae.The aim of this study is to describe a new species of Foleyellides from southeast Mexico based on morphological and molecular evidence, as well as to ameliorate the lack of molecular information for this group available in public repositories, since partial sequences of the mitochondrial COI gene were generated for the four known Mexican species and two potentially new species collected in this study.

Specimens collection
Specimens of Lithobates brownorum were collected in Yucata ´n, Quintana Roo and Campeche, Mexico, during June and July 2016 (table 1).Specimens were collected under the scientific collection permits FAUT0056 issued to VLR and SGPA/DGVS/ 02798/16 to AOF by Secretarı ´a del Medio Ambiente y Recursos Naturales (SEMARNAT).Amphibians were captured using dip nets and euthanized by an overdose of sodium pentobarbital, dissected and examined under stereomicroscope; worms were placed in saline solution (0.65%) for 4-8 min and examined in vivo for distinctive morphological traits.For morphological study, specimens were fixed in hot ethanol (96%) and preserved in ethanol (70%).For molecular analyses, worms were fixed and preserved in ethanol (100%).Nematodes previously collected and identified as F. striatus of L. megapoda from Jalisco (July 2012) and F. rhinellae of Rhinella marina from Guerrero (August 2010) were also processed for molecular analyses (table 1).

Morphological analyses
Specimens were cleared with glycerine for 24 h and mounted between coverslips; measurements are given in millimetres (unless otherwise indicated), with minimum and maximum, mean and standard deviation in parentheses.Drawings were made using a camera lucida attached to a microscope.Helminth specimens were deposited in the Coleccio ´n Nacional de Helmintos (CNHE), Instituto de Biologı ´a, Universidad Nacional Auto ´noma de Me ´xico, Mexico City.Host specimens were desposited in the Coleccio ´n Nacional de Anfibios y Reptiles (CNAR).For scanning electron microscopy (SEM), specimens were dehydrated through a graded ethanol series, critical point dried with K850 Critical Point Drier (Emitech, Ashford, England), sputter-coated with gold with Q150R Modular Coating System (QuoRum, Ashford, England), and examined with a Hitachi SU1510 SEM (Hitachi, Tokyo, Japan) at the Laboratorio Nacional de la Biodiversidad (LANABIO), Instituto de Biologı ´a, Universidad Nacional Auto ´noma de Me ´xico.

Molecular analyses
For molecular analyses, total DNA was extracted using the Jena Bioscience kit, following the protocol provided by the company (Jena Bioscience, Jena, Germany).Amplification and sequencing of partial DNA sequences of the mitochondrial Cytochrome Oxidase C subunit I (COI) locus were carried out using a cocktail of six primers: forward (C_NemF1t1: NemF1_t1?NemF2_t1?NemF3_t1), reverse (C_NemR1_t1: NemR1_t1?NemR2_t1 ?NemR3_t1) (Prosser et al., 2013).Thermal cycling conditions for amplification reactions were 94 C for 1 min, five cycles at 94 C for 45 s, 45 C for 40 s, 72 C for 1 min, followed by 35 cycles at 94 C for 40 s, 51 C for 40 s, 72 C for 1 min and a final extension at 72 C for 5 min.Sequencing reactions were accomplished using an ABI 3730xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at the LANABIO.
The phylogenetic analysis was performed through Bayesian inference (BI), using Markov Chain Monte Carlo (MCMC) in Mr. Bayes V 3.1.2(Ronquist et al., 2012).The appropriate model of evolution (GTR?1?C) was determined with jModeltest 0.1.1 (Posada, 2008).The chains run for 1,500,000 generations, sampling trees every 1,000 generations; the first 25% of the sampled trees were discarded according to Tracer V 1.5 (htt://beast.bio.ed.ac.uk/ tracer); consensus topology and posterior probability values were calculated from the remaining 75% of the trees.

Remarks
Foleyellides calakmulensis n. sp. is included in the genus based on morphological characters referred by Esslinger (1986) and Gibbons (2010), such as the presence of cuticularized parastomal structures, lateral and caudal alae in both sexes, and the lack of distinct cuticularized buccal capsule or annular bands of longitudinally oriented bosses on the cuticle of midbody region.
The new species differs from some other species of Foleyellides (F.americana, F. brachyoptera, F. confusa, F. flexicauda, F. malayensis, F. mayenae, F. ranae, and F. rhinellae) in the number of male caudal papillae, four pairs in the new species and more than four in the other species (Garcı ´a-Prieto et al., 2014;Romero-Maye ´n & Leo ´n-Re `gagnon, 2016;Schmidt & Kuntz, 1969;Wehr & Causey, 1939;Witenerg & Gerichter, 1944).The new species also differs from F. brachyoptera, F. confusa and F. mayenae in the presence of a distinctive cuticularized preanal plaque, which is absent in those species (Garcı ´a-Prieto et al., 2014;Romero-Maye ´n & Leo ´n-Re `gagnon, 2016;Schmidt & Kuntz, 1969).On the other hand, the new species resembles F. duboisi, F. dolichoptera and F. striatus, by having males with four pairs of caudal papillae (Esslinger, 1986;Wehr & Causey, 1939;Witenerg & Gerichter, 1944).However, in F. duboisi and F. dolichoptera the preanal plaque is absent, contrasting with F. calakmulensis.The new species most closely resembles F. striatus in the number of papillae and in the presence of a preanal plaque, but they differ in several features: 1) the size of females, which are smaller in the new species (38-74 in F. striatus vs 19-49 in the new species); 2) the difference in the size of left spicule, which is longer in F. striatus (336-465 vs 265 -375); 3) the size of papillae (post-anal papillae are the same size in F. striatus (Esslinger, 1986), while different in size in the new species).These combined characteristics distinguish F. calakmulensis n. sp.from F. striatus and from the other described species of the genus.

Genetic distances and phylogenetic analyses
Specimens of two potentially new species of Foleyellides were collected during this study in Yucatan and Quintana Roo, Mexico (table 1); nevertheless, only female specimens were found in spite of intensive collecting efforts in both Mexican states.Without the male characters, those species can not be described in this study, and only COI sequences are presented.Genetic distances of the mitochondrial COI sequences of specimens of F. calakmulensis n. sp.from the same individual host and type locality range from 0 to 0.05%, and 0 to 0.70% between different localities.Genetic distances between F. calakmulensis and F. striatus range from 12.6-13.1%;12.5-13.2from F. mayenae, 15.5% from F. rhinellae, 10.5-11.5% from Foleyellides sp. 1 and 14.3-14.5% from Foleyellides sp. 2. Phylogenetic analysis results are presented in figure 3.

Discussion
Eleven species of Foleyellides have been described in the world, the majority of which have been recorded in North America.Ten species are parasites of frogs of the family Ranidae and only F. rhinellae of toads (Rhinella marina) (Garcı ´a-Prieto et al., 2014); all of them inhabit the body cavity of the host, with exception of F. confusa which is subcutaneous (Schmidt & Kuntz, 1969).Taxonomy of the genus Foleyellides is mainly based on morphological characters (body size, number of caudal papillae, presence of cuticularized preanal plaque and size of the spicules); however, many of these characters are variable, and in some cases are difficult to distinguish between species.For example, males of F. calakmulensis n. sp. and F. striatus both have four pairs of papillae, and only with scanning electron microscopy it was possible to clearly corroborate that they are different in size (see Velarde-Aguilar, 2014).
In this sense, molecular tools are important for the differentiation and delimitation of species.We obtained COI sequences of F. calakmulensis n. sp., F. rhinellae, F. striatus, Foleyellides sp. 1 and Foleyellides sp. 2 (table 1), and compared them with sequences of F. mayenae, which were the only available sequences of the genus in GenBank, in order to corroborate the validity of the new species.We also included sequences of other species in the subfamily Waltonellinae: Neofoleyellides boerewors, N. steyni, N. martini (Kuzmin et al., 2021;Netherlands et al., 2020) and three unidentified samples of Ochoterenella (Lefoulon et al., 2015).In the phylogenetic analysis, F. calakmulensis n. sp.appears within a highly supported clade that includes species collected from Lithobates spp. in Jalisco (F.mayenae and F. striatus), Yucata ´n (Foleyellides sp. 1) and Quintana Roo (Foleyellides sp. 2) (fig.3).Foleyellides rhinellae, the only species of the genus described parasitizing toads, appears nested within samples of Ochoterenella, a genus that has been typically found in this group of hosts.Further morphological and molecular information would be needed to revise the phylogenetic position and taxonomy of F. rhinellae and to determine if this species should be transferred to Ochoterenella.All other species of Foleyellides included in the analysis are parasites of frogs in the genus Lithobates.It is interesting to note that species that share morphological traits (four pairs of papillae in F. striatus and F. calakmulensis n. sp.) or share geographical distribution and host species (Foleyellides calakmulensis n. sp., Foleyellides sp. 1 and Foleyellides sp. 2, distributed in the Yucatan peninsula) are not sister species to each other in the tree.Further investigation on the phylogenetic relationships among species of Waltonaellinae is needed.
The new species is distributed only in south-eastern Mexico (Campeche, Quintana Roo and Yucatan), although additional geographic sampling is needed in order to determine the geographical distribution of the species in this genus, and also sequences of additional genes are needed to elucidate the evolution of morphological traits and host specificity of Foleyellides species.

Fig. 1
Fig. 1 Foleyellides calakmulensis n. sp.Line drawing; male, ventral view of posterior end (a); male, lateral view of posterior end (b); apical view showing four pairs of papillae and parastomal structures (c); female, lateral view of anterior region showing position of vulva (d); male, ventral view of anterior end (e).

Fig. 2
Fig. 2 SEM of a male of Foleyellides calakmulensis n. sp.Lateral view of anterior region (a); apical view of anterior region showing four pairs of papillae (b); apical view showing parastomal structures (c); male, ventral view of caudal region, showing the distribution and size of papillae, preanal plaque and spicules (d).

Fig. 3
Fig. 3 Bayesian phylogenetic tree of Foleyellides spp.based on COI sequences, showing the phylogenetic position of Foleyellides calakmulensis n. sp.; numbers above branches indicate posterior probabilities.The scale bar indicates the expected number of substitutions per site.

Table 1
Hosts, localities and GenBank accession numbers of Foleyellides spp.and outgroups included in our analysis