Molecular data reveal a new species of Rhopalias Stiles & Hassall, 1898 (Digenea, Echinostomatidae) in the Common opossum, Didelphismarsupialis L. (Mammalia, Didelphidae) in the Yucatán Peninsula, Mexico

Abstract A new species of Rhopalias Stiles & Hassall, 1898 is described from the small intestine of the Common opossum, Didelphismarsupialis Linnaeus from the Yucatán Peninsula, Mexico. Rhopaliasoochisp. nov. is morphologically very similar to the type species of the genus, Rhopaliascoronatus (Rudolphi, 1819) Stiles & Hassall 1898, a species widely distributed in opossums across Mexico. A molecular phylogenetic analysis using a mitochondrial gene (cox1), and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2), of specimens of R.coronatus collected in several localities of Mexico revealed that those from the Yucatán Peninsula, originally recorded on morphological grounds as R.coronatus actually represented an independent genetic lineage. Maximum Likelihood and Bayesian Inference analyses were performed for each data set independently, and for the concatenated data set (ITS1-5.8S-ITS2 + cox1). All phylogenetic analyses showed that the specimens from Yucatán represented a monophyletic lineage, with high bootstrap support and Bayesian posterior probabilities. In addition, the genetic divergence estimated between R.oochisp. nov. and two species of Rhopalias, R.coronatus, and R.macracanthus Chandler, 1932 that also occur in Mexican marsupials ranged between 7–8% and 16–17%, for cox1, and between 0.1–0.2% and 7% for the ITS region, respectively. The molecular evidence gathered in this study (reciprocal monophyly in both phylogenetic analyses, and estimated genetic divergence) suggested that the specimens found in the intestine of D.marsupialis originally reported as R.coronatus from Yucatán, actually represent a new species. Morphological evidence was found through light and scanning electron microscopy to support the species distinction based on molecular data.


Introduction
The genus Rhopalias Stiles & Hassall, 1898 includes six species of digenetic trematodes that infect the small intestine of didelphimorph marsupials of the New World (Haverkost and Gardner 2008). Members of this genus of echinostomatid trematodes (see Tkach et al. 2016) are distinguished by having two anterior tentacles armed with spines, which can be invaginated into a muscular pouch, one on each side of oral sucker (Kostadinova 2005). In a taxonomic review of the species of the genus Rhopalias, Haverkost and Gardner (2008) discussed the morphological characters that could be used for distinguishing among congeneric species, concluding that the number and size of tentacle spines, the presence or absence of oral and/or flanking spines, and the length of the muscular pouches are the most reliable characters. Nevertheless, they cautioned that a more extensive sampling of each species of Rhopalias was necessary to support the use of these characters for the species delimitation. Furthermore, with the exception of the sequence of the 28S rRNA gene from an individual of R. macracanthus Chandler, 1934, a parasite of the Virginia opossum, D. virginiana Kerr from the U.S. (Tkach et al. 2016), and genetic information about the other species of Rhopalias is lacking. The main objective of this study was to explore the genetic diversity among specimens of R. coronatus collected throughout a geographical range across southern Mexico, following a molecular prospecting approach in the search for cryptic species (sensu Blouin 2002). Molecular data were used in combination with a morphological study of newly sampled specimens of R. coronatus, and those deposited at the Colección Nacional de Helmintos (CNHE) to describe a new species of Rhopalias. Specimens originally recorded as R. coronatus from the Yucatán Peninsula by Acosta-Virgen et al. (2015) corresponded with an undescribed species. We describe the new species herein.

Specimen sampling
In total, 44 specimens of opossums were collected between August 2011 and November 2013, in seven localities across southeastern Mexico (Fig. 1, Table 1). Hosts were collected under the collecting permit FAUT-0057 issued to GPPL by the Secretaría del  Table 1. Medio Ambiente y Recursos Naturales. The mammals were sacrificed with an overdose of pentobarbital sodium, necropsied, and all organs were separated in Petri dishes with 0.85% saline, and examined under a stereomicroscope. A small piece of host tissue was taken from each individual and saved for further DNA studies. These tissues are available upon request. Trematodes were removed from the intestine of their hosts, and washed in saline for 3-5 min. Some specimens were fixed by sudden immersion in hot 4% formaldehyde and stored in 70% ethanol for morphological analyses. For molecular study, eight specimens were washed with saline solution, preserved in 100% ethanol, and stored at -20 °C. These specimens were used for DNA extraction, including four specimens that were cut in half and the posterior half processed for morphology (hologenophores, sensu Pleijel et al. 2008), and four complete specimens. The four hologenophores and remaining seven specimens (paragenophores) were stained in Mayer's paracarmine and mounted as permanent slides in Canada balsam. Specimens of Rhopalias were morphologically identified either as R. coronatus or R. macracanthus according with the description and morphometrical traits reported by Haverkost and Gardner (2008).

Amplification and sequencing of DNA
Individual worms fixed in 100% ethanol (or the posterior portion in some cases) were placed in tubes and digested overnight at 56 °C in a solution containing 10 mM Tris-HCl (pH 7.6), 20 mM NaCl, 100 mM Na 2 EDTA (pH 8.0), 1% Sarkosyl, and 0.1 mg/mL proteinase K. Following digestion, DNA was extracted from the supernatant using DNAzol reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer's instructions. A fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1), and ITS1-5.8S-ITS2 were amplified by PCR, using the primers shown in Table 2.
All PCRs were performed at a final volume of 25 μl consisted of 2.5 μl of 10× PCR buffer, 2.5 μl of 10 mM of dNTPs mixture (200 μl each), 1.25 μl MgCl 2 (50 mM), 1.0 μl each primer (10 pmol), 2 μl DNA template, and 1 unit of Taq DNA polymerase (Biogenica, Mexico City), and the remaining volume of sterilized distilled water. The amplification program for cox1 consisted of: initial denaturation at 96 °C for 3 min,  (Maddison and Maddison 2011), and trematode mitochondrial genetic code. All the cox1 and ITS1-5.8S-ITS2 sequences generated in this study were deposited in the GenBank (Table 1).

Phylogenetic analyses
DNA fragments of the cox1 and ITS region were aligned separately using the software Clustal W2 (Thompson et al. 1994) with a final manual adjusting in Mesquite v.2.75. The concatenated data set was aligned using the same software. Sequences of cox1 and ITS region of other species included within superfamily Echinostomatoidea Looss, 1899 available in the GenBank were used as outgroups. Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were performed for each data set, and for the concatenated data set (ITS1-5.8S-ITS2 + cox1) partitioned by gene. The program jModeltest v.3.0 (Posada and Crandall 1998) was used for inferring the best model of evolution for each data set using the Akaike information criterion. The TVM + I + G and TPMuf + G substitution model were the best models for cox1 and ITS region, respectively. The ML trees were inferred using RAxML v.7.0.4 (Stamatakis 2006  heating parameter value of 0.2 and a 'burn-in' of 10%. A 50% majority-rule consensus tree representing the posterior probability distribution of clades was produced of the sampled trees. Phylogenetic trees were displayed with the program FigTree v.1.4.2 (Rambaut 2006). Finally, genetic divergence (p-value) was calculated for each data set using MEGA v.6.0 (Tamura et al. 2013).

Morphological analyses
Representative specimens of the species of Rhopalias were stained with Mayer's paracarmine or Gomori's trichrome, dehydrated through a graded ethanol series (70%, 80%, 90% [twice], and 100%), cleared in methyl salicylate, and mounted in Canada balsam. The specimens were observed using an Olympus BX81 light microscope. Some worms were drawn with the aid of a drawing tube attached to an Olympus BX53 light microscope. Likewise, in order to obtain a complete digital record of the morphological traits, specimens were observed through the Differential Interference Contrast method (DIC), using an Olympus Provis AX70 microscope. Photomicrographs of the specimens were obtained with a digital camera Evolution 5.0 MP. All specimens were measured using the software Image Pro-Plus v.7.0. Measurements are presented in micrometres (μm) unless otherwise stated. For scanning electron microscope studies (SEM), the specimens were dehydrated in a graded series of alcohol solutions and then critical point dried with carbon dioxide. Specimens were mounted on metal stubs with carbon adhesive, and then gold coated and examined at 15kV in a Hitachi Stereoscan Model SU1510 SEM (Hitachi Ltd., Tokyo, Japan). Digital images of these specimens were obtained using digital imaging software attached to a computer. Specimens of the new species of Rhopalias were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City (Table 1).

Phylogenetic analysis
Cox1. One hundred twelve sequences of cox1 were obtained in the present study, including 101 of R. coronatus, three of R. macracanthus, and eight of the new species. The final alignment included seven sequences from GenBank (as outgroups), and consisted of 119 sequences with 666 bp. Phylogenetic trees reconstructed by ML and BI yielded similar topologies with high bootstrap support and Bayesian posterior probabilities, respectively (see Suppl. material 1: Figure S1). The tree obtained with both reconstruction methods shows three major clades, the same ones that were obtained in the concatenated data set (Fig. 2). Clade I, corresponding to R. coronatus according to their morphology, included 101 sequences from six localities: 26 isolates from Los Tuxtlas (LT), 16 isolates from Tlacotalpan (TL), 17 from Soyoltepec (SO), 39 from Agua Fría  Table 1), and host species where each isolate was collected.
(AF), two isolates from Cunduacán (CU) and one from Teapa (TE). Clade II included eight sequences from a single locality: Rancho Hobonil, Tzucacab (TZ), representing the new species. Finally, Clade III was composed by three isolates from Tlacotalpan (TL), which were morphologically determined as R. macracanthus. This last clade was recovered as the sister group of clades I and II (see Suppl. material 1: Fig. S1).

ITS.
A subsample of 30 sequences of ITS1-5.8S-ITS2 region were obtained in this study from some specimens corresponding with each of the three genetic lineages of Rhopalias from the cox1 analysis; samples included 25 individuals of R. coronatus, one of R. macracanthus, and four sequences of the new species. In addition, ITS sequences of other species included in the superfamily Echinostomatoidea were downloaded from GenBank and used as outgroups. The final alignment consisted of 32 sequences with 1093 bp. Phylogenetic analyses by ML and BI yielded the same topology (see Suppl. material 2: Figure 2). Both topologies showed two major clades (and not three as the cox1 tree), and relationships are supported by high bootstrap and posterior probability values. Clade I included 29 sequences from six localities: two isolates from Los Tuxtlas (LT), two isolates from Tlacotalpan (TL), nine from Soyoltepec (SO), eleven from Agua Fría (AF), one from Teapa (TE) and four isolates from Tzucacab (TZ). However, these four sequences formed a small subclade within Clade I, corresponding with the isolates of the new species; Clade II was composed by only one sequence of R. macracanthus from Los Tuxtlas (LT).
Concatenated data set. This data set consisted of ITS1-5.8S-ITS2 plus the cox1 gene sequences. The final alignment included 32 sequences with 1759 bp. Phylogenetic analyses of this concatenated data set were also conducted using ML and IB methods, yielding the same topologies. The ML tree is shown in Figure 2. The tree yielded three major clades well supported by bootstrap and Bayesian posterior probability values. The first one included all samples of R. coronatus: two specimens from Los Tuxtlas (LT), two specimens from Tlacotalpan (TL), nine specimens from Soyoltepec (SO), eleven specimens from Agua Fría (AF), and one from Teapa (TE). The second clade included four isolates from Rancho Hobonil, Tzucacab (TZ), representing the new species. Clade III included only one sequence from Tlacotalpan (TL), corresponding with R. macracanthus. All the individuals sequenced from each of these three Rhopalias species, sampled in most of their geographic distribution in Mexico, formed monophyletic groups, with R. coronatus and the new species grouped as sister species (Fig. 2).
Genetic divergence. The genetic divergence estimated among populations of the new species with respect to R. coronatus and R. macracanthus ranged between 7-8% and 16-17%, for cox1, respectively; for the internal transcribed spacers, interspecific divergence between the new species and the other two species of Rhopalias varied 0.1-0.2% and 7%, respectively. The intraspecific divergence among isolates of the three species of Rhopalias ranged from 0-1% for cox1, was null for ITS.   Etymology. The specific epithet refers to the common name of the host where the new species was found. In the Mayan language, "ooch" means opossum.
Description. Based on 11 adult specimens (including 4 hologenophores). Measurements are given in Table 3. Trematodes with a long body, forebody concave, wider than hindbody, with a pair of armed retractile tentacles with 4-7 spines (Fig. 3A, B, C). Tegument covered with spines reaching posterior end of body (Fig. 4A). Tegument spines U-shaped, with distal tip pectinated (Fig. 4D, E). Oral and flanking spines present (Figs 3C, 5A, 5D). Muscular sacs long, reaching far beyond posterior margin of pharynx, and may or may not reach the anterior margin of ventral sucker (Fig. 3A, B). Oral sucker subterminal, rounded, well-developed, short prepharynx, pharynx muscular, and relatively long oesophagus; caecal bifurcation at short distance anterior to genital pore (Fig. 3B); long caeca extending to the posterior end of body (Fig. 3A). Ventral sucker muscular and subspherical, in the first third of body, larger than oral sucker (Figs 3A, B, 4A). Testes two, elongated, in tandem, contiguous, no overlapped, located in mid-body; anterior testis shorter than posterior testis (Fig. 3B). Cirrus sac long, claviform, containing a welldeveloped prostate complex and seminal vesicle, extending beyond ventral sucker and terminating near anterior border of ovary. Genital pore between ventral sucker and caecal bifurcation. Ovary slightly oval, postacetabular, pretesticular. Uterus intercaecal, between ovary and genital pore. Metraterm long (Fig 4D). Vitelline follicles in lateral fields, beginning at mid-level between ventral sucker and ovary, ending at posterior end. Gravid specimens with few eggs, oval-shaped, operculated; embryonated eggs with thin shell.
Remarks. The genus Rhopalias currently contains six species as parasites of New World marsupials (Haverkost and Gardner 2008), i.e., Rhopalias coronatus, the type species; Rhopalias horridus (Diesing, 1850) Stiles and Hassall 1898; Rhopalias baculifer Braun, 1901;Rhopalias macracanthus Chandler, 1932; Rhopalias caballeroi Kifune & Uyema, 1982; and Rhopalias caucensis Rivillas, Caro, Carvajal & Vélez, 2004. Rhopalias oochi sp. nov. represents the seventh described species and is readily distinguished from five of the congeneric species, excepting R. coronatus by having tentacle sacs extending far beyond the posterior margin of pharynx. In their detailed morphological revision of the genus Rhopalias, Haverkost and Gardner (2008) provided a key to species of the genus and found that this character is reliable to distinguish between two groups of species. In R. horridus, the tentacle sacs surpass the posterior margin of pharynx to reach the mid-level of oesophagus; however, they never extend beyond the caecal bifurcation. In R. oochi sp. nov. the tentacle sacs extend to reach the anterior margin of ventral sucker. Rhopalias horridus further differ from the new species, and the remaining congeners by the large number of tentacle spines (> 30) (see key in Haverkost and Gardner 2008). The new species most closely resembles the type species, R. coronatus; in fact the specimens upon which the description of the new species is based were originally recorded as R. coronatus (see Acosta-Virgen et al. 2015). Genetic data revealed that they might actually represent a different species. In R. coronatus as in the new species tentacle sacs extend far beyond the posterior border of pharynx; in R. coronatus, both sacs usually surpass the anterior border of ventral sucker (although our observation of numerous voucher specimens indicate that the extension is variable among individuals); in the new species, the tentacle sacs consistently extend to almost reach the anterior border of ventral sucker, but never surpass it. Additionally, R. coronatus differ from the new species by having larger oral spines (at least four times larger), and by the distribution of vitelline follicles. In R. coronatus follicles extend between the posterior end of body and the posterior border of ventral sucker; instead, in R. oochi sp. nov. vitelline follicles do not reach the posterior end of body, and in the forebody, they reach the mid-level of cirrus sac. Morphometrically, most measurements are clearly overlapped between the new species and R. coronatus (except in oral spines size). We observed and measured 21 individuals of R. coronatus deposited at the CNHE. Our measurements, and those reported in other studies (see Table 3) indicate that R. coronatus is morphometrically quite variable. Furthermore, SEM study of the ultrastructure of the body surface provided additional evidence to distinguish the new species from R. coronatus and R. macracanthus; for these two species we collected specimens and some were fixed for a comparative SEM study (Fig. 4). In the three species body spines are denser in the forebody; however, the extension, size, and shape of spines clearly distinguish the three species. In R. oochi sp. nov., spines are robust and pectinate and they extend along the body almost to the posterior end (Fig. 4A, D, E); meanwhile, in R. coronatus spines are distributed to the mid-level of hindbody and are entire and tongue-shaped (Fig. 4B, F, G) and in R. macracanthus, even though spines extend more posteriorly, they are not as robust as in the new species; spines in R. macracanthus are pectinate and arrow-shaped (Fig. 4C, H, I).

Discussion
Specimens of Rhopalias studied in the present study were identified morphologically either as R. coronatus or R. macracanthus following the descriptions by Haverkost and Gardner (2008). Identification was based on the length of the muscular sacs. The species R. coronatus possess muscular sacs almost reaching the anterior end of ventral sucker; meanwhile in R. macracanthus muscular sacs are short and slightly overpass the pharynx level. Additionally, spines of the retractile tentacles are very large in R. macracanthus (see Suppl. material 3: Figure S3C) in comparison with those of R. coronatus. Morphometrically, specimens of the three species are also different (see Table  3). Our study followed a molecular prospecting approach by considering R. coronatus as a trematode species that infects three species of marsupials distributed across a wide geographical range in southeastern Mexico (Blouin 2002;Criscione et al. 2005;Vilas et al. 2005). Our analyses showed that the specimens from the Yucatán Peninsula actually represented a separate species; phylogenetic trees showed all isolates from  that locality as a reciprocally monophyletic assemblage, separated from isolates of R. coronatus, and this two as the sister taxa of R. macracanthus. Even though the internal transcribed spacers (ITS1-5.8S-ITS2) exhibited lower resolution, the mitochondrial gene (cox1) revealed relatively high genetic divergence values (7-8%) supporting the distinction of the new species. This level of genetic divergence has been found in studies with other members of the superfamily Echinostomatoidea. For instance, Saijuntha et al. (2011) reported divergence levels of 8-16% between two species of Echinostoma Rudolphi, 1809. Even though the pertinence of using a genetic yardstick to distinguish parasite species has been questioned (Nadler and Pérez-Ponce de León 2011), the species delimitation criteria followed in our study is also based on a hypothesis-testing framework (see Adams 2002;Nadler 2002).
A closer look at the morphology of the specimens from Yucatán using light and scanning electron microscopy corroborated the molecular results, and the new species was described as a parasite of the Common opossum, D. marsupialis. Rhopalias oochi sp. nov. represents the seventh described species in the genus, and the 5 th in marsupials distributed in Mexico. With the exception of R. macracanthus, a species described by Chandler (1932) in the Nearctic biogeographical region, from the Virginia opossum, in the U.S.A., all the other species included in the genus Rhopalias were originally described in marsupials from the Neotropical region. However, R. macracanthus was later found in South American marsupials, particularly in the Department of Santa Cruz, Bolivia (Haverkost and Gardner 2008). The type species, R. coronatus was described from the Common opossum, D. marsupialis in Brazil; R. horridus from the Water opossum, Chironectes minimus (Zimmerman) also in Brazil; R. baculifer from D. marsupialis in Brazil; R. caballeroi from D. marsupialis and from the Grey four-eyed opossum, Philander opossum (Linnaeus) in Brazil; and R. caucensis from P. opossum in Colombia (see Haverkost and Gardner 2008). According to García-Prieto et al. (2012) four species of Rhopalias have been recorded in Mexico, R. baculifer and R. caballeroi in restricted localities in the tropical rain forest of Los Tuxtlas, Veracruz, and other two species more widely distributed parasitizing three species of marsupials (P. opossum, D. virginiana and D. marsupialis), R. coronatus, and R. macracanthus in nine and 12 localities, respectively. A few additional records were more recently provided by Acosta-Virgen et al. (2015). Interestingly, in the region of Los Tuxtlas, Veracruz, where the three species of opossums are found in sympatry (see Cervantes et al. 2010), the four species of Rhopalias have been recorded. In our study, even though we sampled five individuals of D. marsupialis, one of D. virginiana and two of P. opossum from the same locality, we only collected specimens of R. coronatus and R. macracanthus.
Marsupials occurring across Mexico are heavily parasitized by helminths; at least 16 helminth taxa have been recorded for D. marsupialis, 30 for D. virginiana, and 17 for P. opossum across their distributional ranges in Mexico (Acosta-Virgen et al. 2015). The only study where DNA sequences were used to establish a more robust species delimitation for the helminth parasite fauna of marsupials is that of López-Caballero et al. (2015). These authors uncovered three genetic lineages for the acanthocephalan Oligacanthorhynchus microcephalus (Rudolphi, 1819) Schmidt, 1972, a species allegedly with a distribution from Brazil, where it was originally described, extended to the USA The fact that we detected another new species of a marsupial parasite through the use of DNA sequences clearly indicated that future studies should consider the use of molecular tools that greatly enhance our ability to delimit species, and this will increase our understanding of the species diversity of marsupial parasites.
Finally, we consider that the use of SEM is fundamental in determining reliable characters that distinguish among echinostomid species because the presence of a wide array of spines along the body and around the oral sucker. In this case, SEM was very important in showing that the species R. macracanthus do possess oral spines, although they are not completely visible using light microscopy (see Fig. 5C-F). For instance, Figure 5C shows the presence of 16 tongue-shaped oral spines in R. macracanthus, and these spines contrast in size and shape with flanking spines which are more visible. Haverkost and Gardner (2008) taxonomic key pointed out the lack of oral spines in R. macracanthus. However, our study demonstrated that these spines are present. Overall, the integrative taxonomy approach, where several sources of information are used to establish more robust species delimitation criteria, is highly recommended for a complete understanding of parasite diversity. to families Medina-Castillo in Agua Fría, Chiapas, and Hernández in Finca Brasil, Tapachula, Chiapas. We are grateful with Berenit Mendoza Garfias for her technical support taking the SEM pictures and Ana Isabel Bieler Antolin for assitance taking DIC microphotographs; David Hernández-Mena for obtaining of ITS sequences and Laura Márquez and Nelly López, LaNaBio, for obtaining sequences with the automatic sequencer. JLC also thanks CONACyT for the scholarship received to complete his PhD studies within Posgrado en Ciencias Biológicas, UNAM. This study was partly founded by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM) IN202617 and 220113 to GPPL and RML, respectively.