Morphological and molecular characterization of a new trichuris species (nematoda-Trichuridae), and phylogenetic relationships of Trichuris species of cricetid rodents from Argentina

Morphological and Molecular Characterization of a New Trichuris Species (Nematoda- Trichuridae), and Phylogenetic Relationships of Trichuris Species of Cricetid Rodents from Argentina Marı ´a del Rosario Robles 1,4 *, Cristina Cutillas 2 , Carlos Javier Panei 3,4 , Rocı ´o Callejo ´n 2 1Centro de Estudios Parasitolo ´gicos y de Vectores (CEPAVE), CCTCONICET- La Plata/Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina, 2Departmento de Microbiologı ´a y Parasitologı ´a, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Espan ˜a, 3Virologı ´a, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina, 4Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (CONICET), Buenos Aires, Argentina Abstract Populations of Trichuris spp. isolated from six species of sigmodontine rodents from Argentina were analyzed based on morphological characteristics and ITS2 (rDNA) region sequences. Molecular data provided an opportunity to discuss the phylogenetic relationships among the Trichuris spp. from Noth and South America (mainly from Argentina). Trichuris specimens were identified morphologically as Trichuris pardinasi,T. navonae,Trichuris sp. and Trichuris new species, described in this paper. Sequences analyzed by Maximum Parsimony, Maximum Likelihood and Bayesian inference methods showed four main clades corresponding with the four different species regardless of geographical origin and host species. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages observed among Trichuris species from sigmodontine rodents which supported the proposal of a new species. Moreover, host distribution showed correspondence with the different tribes within the subfamily Sigmodontinae. Citation: Robles MdR, Cutillas C, Panei CJ, Callejo ´n R (2014) Morphological and Molecular Characterization of a New Trichuris Species (Nematoda- Trichuridae), and Phylogenetic Relationships of Trichuris Species of Cricetid Rodents from Argentina. PLoS ONE 9(11): e112069. doi:10.1371/journal.pone.0112069 Editor: Philippe Castagnone-Sereno, INRA, France Received August 24, 2014; Accepted October 9, 2014; Published November 13, 2014 Copyright: ß2014 Robles et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: The authors received no specific funding for this work. However, some grants contributed to the cost of sampling and laboratory supplies: Agencia Nacional de Promocio ´n Cientı ´fica y Tecnolo ´gica (PICT 2010-0924); Proyecto de incentivos de la Universidad Nacional de La Plata (UNLP N 627 and travel grant). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]u.ar Introduction Species of Trichuris Roederer, 1761 (Nematoda: Trichuridae) have a cosmopolitan distribution and parasitize a broad range of mammalian hosts, such as ruminants, marsupials, rodents, and primates, including humans [1,2]. The presence of Trichuris species among host species is probably related to a combination of factors, such as the immunologic status and the behavior of the host species, and characteristics of the environment where the host lives [2,3]. To discriminate between the alternative hypotheses of co-speciation (host-parasite) versus geographical differentiation, it is necessary to integrate studies of both morphological and molecular analysis [4]. Several features, such as the presence/absence of the spicular tube, the shape and distribution of the spines of the spicular sheath, length of the spicule and the cloacal tube, the shape of the proximal and distal cloacal tube, and the vulvar morphology, along with classic morphometric characteristics have been used as characteristics with high discriminatory value to differenciate the species of Trichuris i.e. [5–10]. Moreover, scanning electron microscopy (SEM) has been used as a diagnostic tool in some studies [10–15]. Trichuris species have been described with a narrow range of anatomic and biometric characteristics; and they have been insufficiently compared with their congeneric species i.e. [5,16–19]. Consequently, different populations with overlapping morphometrical features [8] result in taxonomic and nomenclatorial problems, e.g. individuals of the same species recognized as different species (under different names: synonyms), and different species referred as the same species (sibling species) [20,21]. To date, 24 Trichuris species have been described from 10 families of North and South American rodents [22]. Among these, three species are parasites of Cricetidae in North America: T. opaca Barker and Noyes, 1915 from Arvicolinae, T. neotomae Chandler, 1945 and T. peromysci Chandler, 1946 from Neotominae; and five parasites of Cricetidae in South America: T. chilensis Babero, Cattan and Cabello, 1976, T. travassosi Correa Gomes, Lanfredi, Pinto and Souza, 1992, T. laevitestis Suriano and Navone, 1994; T. pardinasi Robles, Navone and Notarnicola, 2006, and T. navonae Robles, all from Sigmodontinae. The last three species were found from Argentina [7,10,22]. Of the Trichuris from Cricetidae, 66% share a general morphological pattern, including the absence of a spicular tube, spicular sheath with spines (most with a cylindrical shape), and a PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e112069 non-protrusive vulva, thus these can be separated mainly by morphometric characters with high discriminatory value [6,10,22– 25]. However, this also exemplifies the difficulty in finding morphological differences among species in this genus. For this reason, some studies have used isoenzymatic patterns and molecular studies to identify these nematodes [25–29]. The genes encoding the rRNA subunits are particularly useful in phylogenetic studies. In fact, recent studies have demonstrated that internal transcribed spacers (ITS1-5.8S-ITS2) of nuclear ribosomal DNA (rDNA) provide genetic markers for the accurate identification of closely related nematode species [30]. Sequence data of the ribosomal ITS2 have also been shown to be a valuable tool in species identification; first because they are highly speciesspecific and, second because they are flanked by conservative regions of the rDNA that allows the use of universal primers that bind to the 5.8S and 28S rDNA genes of many helminths e.g. [31– 37]. Since, the ITS2 region is much more variable than ITS1, it allows a better discrimination at the species level. For example, ITS2 have been used for the unequivocal delineation of morphologically well defined adults such as Trichuris ovis, Trichuris leporis and Trichuris suis with high levels (32.8– 58.64%) of interspecific variation e.g. [21,27]. Also, ITS2 provides significant phylogenetic insights [38,39]. Studies on the comparative phylogeny of taxa strongly linked by an ecological factor, such as parasitism, have shown that the degree of phylogenetic congruence increases with the forced character of the host– parasite relationship [40]. The purpose of this paper was to study different populations of Trichuris isolated from six species of Sigmodontinae rodents from Argentina, based on morphological characteristics and ITS2 of nuclear rDNA region sequences. Also, the description of a new species of Trichuris is provided in this study, and the level of variation among the ITS2 sequences of studied populations was determined. Molecular data are also used to analyze and discuss the phylogenetic relationships among the Trichuris spp from the Americas, and mainly from Argentina. Material and Methods Cricetid rodents were trapped during different field studies between 2009 and 2012 (see collectors in acknowledgements). A total of 81 adult specimens of Trichuris were studied from six species of Sigmodontinae rodents (Cricetidae) from eight/seven localities respectively: 61 for morphological analyses and 20 for molecular characterization (see Table 1). Ethics Statement The research has been conducted according to Argentine laws. Sample collection was carried out during fieldwork under oficial permits granted by Ministerio de Asuntos Agrarios de la provincia de Buenos Aires (expedient 22500-7981-2010-0) and Organismo Provincial para el Desarrollo Sustentable (OPDS) (expedient 2145- 6077/10), Ministerio de Produccio´n y Ambiente de la Provincia de Formosa (authorization s/n; Guı ´a de Tra´nsito: 004076), Ministerio de Ecologı ´a, RNR y Turismo, Provincia de Misiones (authorization #27, Guı ´a Tra´nsito 000316). This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The specimens obtained with methods for live capture were studied and humanely sacrificed (euthanasia by thoracic compression under ether anesthesia), following the procedures and protocols approved by national laws (Animal Protection National law 14.346 and references in the provincial permits) and Ethics Committee for Research on Laboratory Animals, Farm and Obtained from Nature of National Council of Scientific and Technical Research (CONICET) (Resolution 1047, section 2, annex II), and subsequently by National Agency for the Promotion of Science and Technology of Argentina (ANPCYT) (PICT 2010-0924). No endangered species were involved in this study. Morphological analysis Nematodes were preserved in 70% ethanol, and cleared in lactophenol, and studied using a light microscope. Morphological identification was performed using characteristics listed by Robles et al. [10] and Robles [22]. Drawings of specimens of Trichuris from Sooretamys angouya were made with the aid of a drawing tube. Four specimens of this population were dehydrated in an ethanol series (75%, 80%, 85%, 90%, 96%, 100%), dried using the critical point method, and examined with the aid of a scanning electron microscope (Jeol 6360 LVLV, Tokyo, Japan). Measurements of new species are presented as follows: holotype male or allotype female, and paratypes with mean, standard deviations, and range in parentheses. We tested for statistical differences for some variables. When data met parametric requirements, Student’s t-test was used for pairwise comparisons; otherwise a non-parametric Mann–Whitney U-test was used. For all calculations, we tested significance at the a= 0.05 level. Statistical analysis was performed using Past 3.01 (Paleontological Statistics, free software). All measurements are given in millimeters (mm). The scales of figures are given in micrometers (mm). Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ‘‘http://zoobank.org/’’. The LSID for this publication is: urn:lsid:- zoobank.org:pub: A22989C7-144F-4F9F-A3E8-0D13023B0413. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS. Specimens of nematodes were deposited in the Helminthological Collection of Museo de La Plata (MLP), La Plata, Buenos Aires, and hosts in Mastozoological Collections of the Centro Nacional Patago´nico (CNP), Puerto Madryn, Chubut, and Mastozoological Collections of Museo de La Plata (MLP) La Plata, Buenos Aires, Argentina. Molecular analysis The specimens previously identified were washed extensively in 0.9% saline solution and stored in 70% ethanol until used for PCR and sequencing. PCR and sequencing of specimens. Genomic DNA from individual worms was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. Quality of extractions was assessed using 0.8% agarose gel electrophoresis and ethidium bromide staining. The Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA (rDNA) region was amplified by PCR using a Perkin Elmer thermocycler and the following PCR mix: 10 ml106PCR buffer, 2 ml 10 mM dNTP mixture (0.2 mM each), 3 ml 50 mM MgCl 2 ,5ml primer mix (0.5 mM each), 5 ml template DNA, 0.5 mlTaq DNA polymerase Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 2 November 2014 | Volume 9 | Issue 11 | e112069 (2.5 units) and autoclaved distilled water to 100 ml. The following conditions were applied: 94uC for 3 min (denaturing), 35 cycles at 94uC for 1 min (denaturing), 50uC for 1 min (annealing), 72uC for 1 min (primer extension), followed by 10 min at 72uC. DNA sequences of the forward primer 5.8S F (59-GTAGGT- GAACCTGCGGAAGGATCATT-39) and reverse primer ITS2R (59-TTAGTTTCTTTTCCTCCGCT-39) corresponded to the conserved 39-59ends of the ITS1-5.8S-ITS2 flanking the 5.8S and 28S gene regions. Thus, DNA sequence of the reverse primer was cited by Gasser et al. [41], while forward primer was designed by us. For each set of PCR reactions and extraction of the DNA, samples without DNA (negative) and a known (positive) control DNA samples were also included. The PCR products were checked on ethidium bromide-stained 2% Tris-Borate-EDTA (TBE) agarose gels. Bands were eluted from the agarose by using the Wizard SV Gel and PCR Clean-Up System (Promega). The purificated PCR products were concentrated, and directly sequenced by Stab Vida (Portugal). The rDNA intra-individual similarity was determined by sequencing between three to five clones of one individual per population of Trichuris species. Thus, the isolated DNA was cloned into Escherichia coli DH5ausing pGEM-T Easy vector system (Promega). Transformed cells were selected by overnight incubation at 37uC on LBB/Amp/X-gal/ IPTG plates. In order to check for successful cloning and to study the intra-individual variation, at least ten single recombinants (clones) were screened for the DNA insert and sequenced. The ten clones containing the correct insert were used to inoculate 5 ml of LBB/Amp broth and incubated, shaked at 37uC for 12 h. Plasmid were purified using a Wizard Plus SV (Promega)and sequenced by Stab Vida (Portugal) with an universal primer (M13). The intraspecific similarity was determined for the rDNA by sequencing, at least, three individuals of each locality and host. Furthermore, all the sequences were aligned and compared with each other using the CLUSTAL W program. Alignments were manually adjusted. Restriction maps of the different ITS2 sequences were determined by using the ‘‘Map’’ program available on Gen Bank. Sequence analysis. Phylogenetic trees based on ITS2 rDNA were rooted by including five outgroups representing members of the genus Trichuris from Murinae and Arvicolinae rodents isolated from Europe, Africa and North America (Table 2). Phylogenetic trees were produced using three methods: Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI), using the PhyML package [42], MEGA 5.0 program [43] and MrBayes version 3.1.2 [44], respectively. jModeltest version 0.1.1 [45] was used to choose a best-fit model of sequence evolution [45]. For the Bayesian analysis, we ran three independent runs of four Markov chains for 10 million generations, sampling every 500 generations. For ML inference, the rapid Table 1. Trichuris specimens studied from different rodent species of Argentina. Trichuris spp. Number of studied specimens Host species Locality/Province Code Geographical point Morphological Molecular 83Phyllotis bonariensis Cerro Bahı ´a Blanca, Parque Provincial Ernesto Tornquist, Sierra de la Ventana, Partido de Tornquist, Buenos Aires province SV 38u04947.990S, 62u00922.480W 10 2 Phyllotis xanthopygus Cerro Los Linderos, Departamento Calamuchita, Co ´rdoba province SC 32u00917.820S, 64u56901.510W Robles, 2011; Robles and Navone, 2014 4Akodon montensis Refugio Mocona ´, Departamento San Pedro, Misiones province RM 27u89S, 53u559W 10 3 Reserva de Vida Silvestre Urugua-ı ´, Fundacio ´n Vida Silvestre, Departamento General Manuel Belgrano, Misiones province UR 25u59908.190S, 54u06936.150W 6 1 Campo Anexo M. Belgrano, INTA, San Antonio, Departamento General Manuel Belgrano, Misiones province SA 26u02952.600S, 53u469210W 21Thaptomys nigrita Reserva de Vida Silvestre Urugua-ı ´, Fundacio ´n Vida Silvestre, Departamento General Manuel Belgrano, Misiones province UR 25u58932.290S, 54u07900.080W 3 2 Campo Anexo M. Belgrano, INTA, San Antonio, Departamento General Manuel Belgrano, Misiones province SA 26u02954.210S, 53u46932.400W 51Necromys obscurus Estacio ´n Experimental Agropecuaria Balcarce, INTA, Partido de Balcarce, Buenos Aires province BA 37u42959.530S, 58u16923.120W 12 2 Sooretamys angouya Refugio Mocona ´, Departamento San Pedro, Misiones province RM 27u89S, 53u559W 3 1 Estacio ´n de Animales Silvestres Guaycolec, Departmento Formosa, Formosa Province GU 25u589510S, 58u99520W 2 0 Reserva de Usos Mu ´ltiples Guaranı ´, Departamento Guaranı ´, Misiones province RG 26u569S, 54u139W doi:10.1371/journal.pone.0112069.t001 Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 3 November 2014 | Volume 9 | Issue 11 | e112069 bootstrap algorithm (with GTRCAT) was used (1000 replicates) to assess the relative reliability of clades, whereas the best ML tree was found using the GTRGAMMA model and a more thorough optimization. Results Morphological analysis Specimens of Trichuris from eight populations were identified based on morphological characteristics (Table 3). The specimens found in Phyllotis bonariensis Crespo, 1964 and P. xanthopygus Waterhouse, 1837 were identified as Trichuris pardinasi; and those found in Akodon montensis Thomas, 1913 and Thaptomys nigrita (Lichtenstein, 1829) as T. navonae. Although the Trichuris specimens from Necromys obscurus (Waterhouse, 1837) were studied in detail, species identification was not possible since only one male was present. A new species of Trichuris was found in Sooretamys angouya (Fischer, 1814), which is described here. Moreover, Cerro Los Linderos (SC), Co´rdoba province; Reserva de Vida Silvestre Urugua-ı ´(UR) and Campo Anexo M. Belgrano, INTA, San Antonio (SA), Misiones province; Estacio´n Experimental Agropecuaria Balcarce, INTA (BA), Buenos Aires province, and Estacio´n de Animales Silvestres Guaycolec (GU), Formosa Province constitute new geographical records for the genus Trichuris (Table 3). Description. Trichuris bainae n. sp. urn: lsid: zoobank.org: act: C53665A4-4DAE-4A2C-988A-59754EBA54A7. (Figs. 1, 2) Diagnosis: Cuticle with fine transversal striation (Figs. 1C, 2K). Anterior part of body long, narrow, tapered, and whip-like; posterior part of body broad, and handle-like (Fig. 1A). Ratio between anterior and posterior body length is 1:1.8 in males and 1:1.4 females. Stichosome with 1 row of stichocytes, and 1 pair of conspicuous cells at esophagus-intestinal junction level (Figs. 1B, 1D, 1G). Male with spicular tube absent. Proximal cloacal tube, united laterally to distal cloacal tube (Fig. 1E). Spicular sheath cylindrical with spines distributed from proximal to distal portion; distal spines very sharpened and joined together (Figs. 1F, 2L, 2M, 2N). Testis ends near final third of distal cloacal tube, showing different degree of convolutions (Fig. 1E). Cloaca subterminal with 1 pair of paracloacal papillae, not ornamented (Figs. 1F, 2M). Female with ornamentated protrusive vulva located at esophagusintestinal junction level (Figs. 1G, 1H). Anus subterminal, with long caudal end finished with terminal torsion (Fig. 1I). Bacillary band located laterally in anterior portion of body (Figs. 1C, 2K). Bacillary band 0.05–0.09 from anterior end of body, and extends to body width region of 0.18–0.24. With SEM, cuticular inflations appear bordering bacillary band from 0.2–0.37 to 0.6–0.87 in the anterior end of body, forming low rings of thick walls and very reduced interior cavity. These structures limit laterally to abundant and visible bacillary glands with conspicuous pore (Fig. 1C, 2K). Cuticle around vulvar aperture with transversally striated pattern (Fig. 1H). Male (6 specimens): Body length 13.9, 13.660.58 (12.97–14.4). Anterior portion of body 8.67, 8.9861.04 (7.4–9.92) long and thick portion of body 5.25, 4.9660.83 (4.15–6.05) long. Anterior body width 0.75, 0.06860.01 (0.050–0.075), maximum posterior body width 0.25, 0.3560.061 (0.27–0.42), width at esophagusintestinal junction level 0.17, 0.2260.035 (0.17–0.25) (Figs. 1B, 1D). Total length of esophagus 8.65, 8.8960.89 (7.3–9.9), Table 2. Trichuris species included in the phylogenetic analysis based on the ITS2 rDNA. Species Host species Host family/subfamily Geographical Origin Code Accession Number Trichuris sp. Microtus townsendii Cricetidae/Arvicolinae Oregon, USA OR FR849676 Trichuris arvicolae Myodes glareolus Cricetidae/Arvicolinae Montseny, Spain MO FR849660 Trichuris muris Mus domesticus Muridae/Murinae Calafel, Spain CA FN543175 Trichuris carlieri Gerbilliscus vicinus Muridae/Murinae Maguha, Tanzania MA JX683522 Trichuris mastomysi Mastomys natalensis Muridae/Murinae Berega Tanzania BE JX683517 doi:10.1371/journal.pone.0112069.t002 Table 3. Trichuris species found from different rodent species from Argentina (see Table 1), with new localities records and molecular data. Species Host species Localities Base pairs G + C% GenBank Accession number T. pardinasi Phyllotis bonariensis SV 429 430 431 60.3 60.7 61.7 HG934448 HG934445 HG934449 Phyllotis xanthopygus SC (new locality) 429 433 60.4 60.1 HG934447 HG934446 T. navonae Akodon montensis RM 427 427 427 427 59.4 59.7 60.2 60.1 HG934435 HG934436 HG934437 HG934438 UR (new locality) 428 435 427 59.6 59.5 59.7 HG934443 HG934444 HG934441 SA (new locality) 427 59.7 HG934434 T. navonae Thaptomys nigrita UR (new locality) 427 59.7 HG934439 SA (new locality) 427 427 59.5 60.4 HG934440 Trichuris sp. Necromys obscurus BA (new locality) 388 59.3 HG934450 Trichuris bainae n. sp Sooretamys angouya RM 441 441 59.9 59.7 HG934431 HG934432 GU (new locality) 441 60.6 HG934433 doi:10.1371/journal.pone.0112069.t003 Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 4 November 2014 | Volume 9 | Issue 11 | e112069 Figure 1. Drawings of Trichuris bainae n. sp. (A) Complete female specimen. (B) Esophagus, muscular and stichosome portions. (C) Esophagus, muscular and stichosome portions, with bacillary band and cuticular inflations view. (D) Male, esophagus-intestine junction and proximal portion of testis, with bacillary band view. (E) Male, posterior end, spiny spicular sheath, spicule and proximal and distal cloacal tube, lateral view. (F) Male, detail of the posterior extremity, lateral view. (G) Female, esophagus-intestine junction and vulva, lateral view. (H) Female, detail of vulva, lateral view. (I) Female, posterior end, lateral view. (J) Egg. doi:10.1371/journal.pone.0112069.g001 Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 5 November 2014 | Volume 9 | Issue 11 | e112069 muscular portion 0.45, 0.3660.16 (0.2–0.47) long, stichosome portion 8.2, 8.761.1 (7–9.8) long. Spicule length 1.94, 2.1660.13 (1.93–2.3) (Fig. 1E). Spicular sheath densely spinose 1.7, 1.861.1 (1.5–2) long (Figs. 1F, 2L). Proximal cloacal tube 1.2, 1.5760.35 (1.19–2.11) long, distal cloacal tube 1.79, 1.3660.31 (1.15–1.9) long (Fig. 1E). Ratio between total body length and posterior portion length 2.64, 2.8960.45 (2.22–3.23). Ratio between total body length and spicule length 7.16, 6.3360.53 (5.87–6.97). Ratio between posterior portion length and spicule length 2.7, 2.3260.52 (1.89–3.13). Ratio beween proximal cloacal tube length and distal cloacal tube length 0.67, 1.1560.17 (1.01– 1.43). Ratio between maximum posterior body width and posterior portion length 0.04, 0.0760.01 (0.06–0.095). Female (2 specimens): Body length 22.18, 23.3. Anterior portion of body 12.66, 14.2 long and thick portion of body 9.52, 9.1 long (Fig. 1A). Anterior body width 0.05, 0.075, maximum posterior body width 0.5, 0.5; width at esophagus-intestinal junction 0.2, 0.22 (Fig. 1G). Total length of esophagus 12.65, 13.7, muscular portion 0.38, 0.35 long, stichosome portion 12.27, 13.66 long. Distance between esophagus-intestinal junction and vulva 0.22, 0.20. Eggs oval, with bipolar plugs, (n = 10) 0.020–0.02560.045– 0.05 (Fig. 1J). Ratio between total body length and posterior portion length 2.33, 2.56. Ratio between maximum posterior body width and posterior portion length 0.052, 0.055. Taxonomic summary. Type host species:Sooretamys angouya (Fischer, 1814) (Sigmodontinae: Oryzomyini). Symbiotype: Female CNP 1998. Other hosts housed: CNP 2529 and CNP 3634. Type locality: Refugio Mocona´ (27u89S, 53u559W), Guaranı ´ Department, Misiones province, Argentina. Other localities: Estacio´n de Animales Silvestres Guaycolec (25u989S, 58u169W), Formosa Department, Formosa Province and Reserva de Usos Mu´ltiples Guaranı ´(26u569S, 54u139W), Guaranı ´Department, Misiones province. Site of infection: Caecum. Type specimens: Holotype male MLP-He 6760, allotype female MLP-He 6761, 6 paratypes MLP-He 6762 deposited at the Helminthological Collection of the Museo de La Plata. Etymology: Dedicated to the memory of Odile Bain, a widely recognized parasitologist from Paris, France; who contributed valuable knowledge on trichurid nematodes from many host groups and different parts of the world. Differential diagnosis. The Trichuris species from North and South American rodents were compared by different morphometric features [5–7,9,10,19,22–24,46–54]. Trichuris bainae n. sp. resembles T. travassosi and T. navonae in their similar general size, cloacal tubes and distance from anterior end to vulva. However, the new species differs from T. travassosi by the unequal distribution of the spines on the spicular sheath, and differs from both species by the presence of a vulva ornamented with spines as well as morphometric features. Trichuris bainae n. sp. can be separated from eight of the species that parasitize American rodents, i.e., T. citelli,T. perognathi,T. neotomae,T peromysci,T. madisonensis,T. dipodomys,T. fulvi and T. laevitestis by the absence of a spicular tube (the spicule lies entirely within the distal cloacal tube). The new species differs from T. opaca,T. fossor,T. citelli,T. neotomae,T. dipodomys, and T. bursacaudata by lacking a spicular sheath with a spiny distal spherical bulge or a spiny campanuliform shape. Among those species with a cylindric spicular sheath, the new species can be separated from T. travassosi and T. pampeana by the distribution of the spines. The new species has a shorter spicule than T. myocastoris, T. bradleyi, T. chilensis, T. fulvi, T. robusti, T. laevitestis, T. bursacaudata, T. pampeana and T. pardinasi and longer than T. opaca, T. fossor, T. perognathi, T. neotomae, T peromysci, T. madisonensis, T. dipodomys, and T. elatoris. Although the ranges of spicule length among T. bainae n. sp., T. travassosi and T. Figure 2. Scanning electron micrographs of Trichuris bainae n. sp. - SEM. (K) Bacillary band, with detail of bacillary glands. (L) Male, posterior end, ventral view. (M) Male, detail of the proximal portion of spiny spicular sheath. (N) Male, detail of the distal portion of spiny spicular sheath. doi:10.1371/journal.pone.0112069.g002 Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 6 November 2014 | Volume 9 | Issue 11 | e112069 navonae overlap in part, the means are differents (2.16, 1.63 and 2.3, respectively). Moreover, T. bainae n. sp. has a shorter distal cloacal tube than T. bradleyi, T. chilensis, T. robusti, T. bursacaudata, T. pampeana, and T. pardinasi and longer than T. perognathi, T. neotomae, T. peromysci, T. dipodomys and T. fulvi. The most similar biometrical features were found between T. bainae n. sp. and T. navonae. However, some statistical differences were found in characters such as spicule length (T-test, t= 6.61, p,0.001), proximal cloacal tube (T-test, t= 3.94, p,0.001), and the ratio between posterior portion length and spicule length (U- test, w = 12, p= 0.037). The new species has an ornamentated protrusive vulva, which is absent in the rest of the species from American rodent hosts. In addition, T. bainae n. sp. has a smaller distance to vulva from the anterior end than T. gracilis, T. opaca, T. myocastoris, T. citelli,T. perognathi, T. neotomae, T. peromysci, T. madisonensis, T. dolichotis,T. dipodomys, T. bradleyi,T. bursacaudata, T. pampeana, and T. pardinasi. Since the males of T. gracilis and T. dolichotis have not been described, these species were not included in the preceding comparison. However, the females of these species can be separated from the new species by the dimensions of the body length, and the lengths of the anterior and posterior portions of the body. Molecular analysis Internal Transcribed Spacer 2 (ITS2) rDNA sequences of the specimens of Trichuris from seven populations of four species were obtained and analyzed. These sequences ranged from 388 to 441 base pairs (bp) (exclusive of the primers) and their G+C content ranged from 59.3%–61.7% (Table 3). Multiple alignment and sequence model selection. The alignment of 25 ITS2 sequences of Trichuris species from rodents from Africa, North America, Europe (Table 2) and South America yield a dataset of 450 characters. jModelTest determined that the best-fit model for ITS2 rDNA datasets was GTR+I+G, which was used for Maximum Likelihood and Bayesian inference. Intra-individual, intra- and inter-specific similarities of Trichuris spp. from Argentina based on ITS2 Rdna. The intra-individual similarity, observed for 3 to 5 clones of one individual per population, ranged from 97.4% to 100% (data not shown). The highest value corresponded to all the individuals of Trichuris populations from Argentina; nevertheless, the minimum value was observed in T. navonae individuals. The range of intra-specific similarity of Trichuris species based on ITS2 rDNA ranged from 95.6% to100%. Trichuris pardinasi from Co´rdoba showed the minimum value (Table 4). In order to analyze inter-specific similarities, different species of Trichuris isolated from Sigmodontine rodents from Argentina were compared. All the ITS2 rDNA sequences obtained for each species were included in the analysis (alignment not shown). The highest similarity was found between Trichuris navonae and T. bainae n. sp. both from Misiones (95.5%) and between T. navonae and T. bainae n. sp. (94.5%), from Misiones and Formosa, respectively. The lowest similarities were found between Trichuris pardinasi from Buenos Aires and T. bainae n. sp. from Misiones (88.3%), and Trichuris sp. from Buenos Aires and T. navonae and T. bainae n. sp. from Misiones (88.8%) (Table 4). Relationships of Trichuris based on ITS2 rDNA sequences. Phylogenetic trees (Fig. 3) provided robust phylogenetic resolution among most Trichuris taxa regardless of the inference method. The topology among Trichuris species showed that all the species of Trichuris from Argentina are separated from those belonging to Trichuris isolated from rodents of Africa, North America and Europe (Bootstrap Values (BP), 100% and 100% in ML and BI methods) (Fig. 3). Furthermore, Trichuris muris from Europe and South Africa clustered together and separated of T. arvicolae from Europe and North America with high BP (81%, 98% and 100% in ML, MP, and BI respectively) (Fig. 3). Phylogenetic trees based on ITS2 sequences of Trichuris species from Argentina showed four main clades by ML, MP and BI methods (Fig. 3). These four main clades included: Clade 1 clustered T. navonae from different hosts from different localities of Misiones region (BP 100%, 98% and 98% in ML, MP and BI methods). Within clade 1, we observed a polytomy of populations of T. navonae regardless of the geographical origin and host species. Clade 2 clustered Trichuris bainae n. sp., from Formosa and Misiones provinces with high BP values (93%, 100% and 100% in ML, MP and BI). Clade 3 included Trichuris sp. from N. obscurus from Buenos Aires. Finally, Clade 4 clustered T. pardinasi populations from Buenos Aires and Co´rdoba. Based on the ITS2 sequences, restriction mapping identified many endonucleases that could be used to delineate different species of Trichuris from sigmodontine rodents. Thus, BsePI, BssHII,HinfI,SacII and SstII sites were present in the sequences of T. bainae n. sp., but not in T. navonae. Otherwise, AgeI,BshTI, HindIII and Sspl sites were present in the sequences of T. navonae but not in those of T. bainae n. sp. Interestingly, HinfI site was only present in T. bainae n. sp., but not in T. navonae, T. pardinasi and Trichuris sp, therefore this endonuclease is specific for the determination of this new species of Trichuris. Discussion The four species of Trichuris studied showed the same general morphological pattern in the male reproductive system. In fact, identification of closely related species is very difficult. This is due in part to the phenotypic plasticity of the organisms themselves, host-induced variation, the paucity of morphological features, and the extensive overlap in morphometric characteristics that occur among species e.g. [8,18,15,22]. However, isoenzymatic patterns and molecular studies for identification of these nematodes have been used successfully [25–29]. The specimens of Trichuris collected from Sooretamys angouya in Misiones province belong to a new species with clear morphobiometrical differences in respect to the rest of the species of Trichuris. Molecular data corroborated these results. Although Trichuris specimens from Necromys obscurus were morphologically studied, species identification was not attained. However, molecular characterization was achieved for those individuals as well as T. pardinasi and T. navonae. This is the first study that provides the molecular characterization of Trichuris species of Sigmodontinae rodents. Also, it is confirmed that Thaptomys nigrita is a host of T. navonae, since this was previously characterized as Trichuris cf. navonae [3]. In addition, five new localities for the four species of Trichuris studied were recorded. The internal transcribed spacers (ITS1 and ITS2) located in the ribosomal DNA are considered appropriate molecular markers to resolve relationships at the species level [30]. It has been demonstrated that there is little, if any, intraspecific variation in the sequence of ITS2 and, further, that closely related species show unequivocal differences in these sequences [31–35,55,56]. In this context, for example, ITS rDNA and 5.8S sequences data have been used to test the existence of two species: T. muris and T. arvicolae in Muridae and Arvicolidae hosts, respectively and a phylogenetic analysis based on combined 5.8S and ITS2 sequences was carried out [27]. The results obtained clearly Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 7 November 2014 | Volume 9 | Issue 11 | e112069 indicated that the ITS+region of rDNA provides genetic markers for whipworm species. Previously, the results of the analysis of the ITS1-5.8S-ITS2 sequence of the ribosomal DNA had confirmed the presence of DNA polymorphisms among T. muris isolates from Europe, suggesting the presence of different lineages/species [57]. The percentage of interspecific variation observed among the four species far exceeded the intraindividual and intraspecific variation which is, in general, slight in the ITS2 sequences. These Table 4. Intra-specific (*) and inter-specific similarity observed in ITS2 sequences in Trichuris populations isolated from different rodent species. Species T. pardinasi (Buenos Aires) T. pardinasi (Co ´rdoba) T. navonae (Misiones) Trichuris sp. (Buenos Aires) T. bainae n. sp. (Misiones) T. bainae n. sp. (Formosa) T. pardinasi (Buenos Aires) 95.6–97.3% * T. pardinasi (Co ´rdoba) 97.6% * 98% * T. navonae (Misiones) 89.3% 90.6% 96.3%–100% * Trichuris sp. (Buenos Aires) 92.1% 92.7% 88.8% 100% * T. bainae n. sp. (Misiones) 88.3% 89.8% 95.5% 88.8% 97.4%–100% * T. bainae n. sp. (Formosa) 89.7% 90.1% 94.5% 89% 99.2% * 98.1% * doi:10.1371/journal.pone.0112069.t004 Figure 3. Phylogenetic tree of of Trichuris species from rodents of Sigmodontinae, Arvicolinae and Murinae of different geographical origins (see Table 1 and 2) based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA inferred using Maximum Composite Likelihood. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown on the branches (Maximum Composite Likelihood/Maximum Parsimony/Bayesian Inference). Bootstrap values lower than 65% are not shown. doi:10.1371/journal.pone.0112069.g003 Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 8 November 2014 | Volume 9 | Issue 11 | e112069 results indicate that T. pardinasi,T. navonae,Trichuris sp. and T. bainae n. sp. must be considered different species. Also, based on ITS2 sequences, some specific recognition sites for endonucleases were detected. Trichuris navonae and T. bainae n. sp. from Misiones and T. navonae from Misiones and T. bainae n. sp., from Formosa were the populations with the most similar sequences, but the HinfI restriction site was only present in T. bainae n. sp., being a useful endonuclease for the determination of this new species. Phylogenetic studies that includes ecological and host geographical distribution data allows a better interpretation of possible processes that determine the geographical distribution of parasites, subdivisions of populations, speciation events or ecological adaptation [58]. The phylogeographic study of Trichuris populations isolated from Cricetid rodents by Callejo´n et al. [4] used the ITS1-5.8S-ITS2 fragment of the ribosomal DNA and the first subunit of the cytochrome coxidase (cox1) region of mitochondrial DNA. This study confirmed the presence of DNA polimorphism among Trichuris arvicolae and Trichuris sp. isolated from the Western Nearctic and the Western half of the Palearctic region. Also, this survey indicated that there might be a second species of Trichuris in arvicoline rodents. The analysis of ITS2 sequences of Trichuris species from Argentina showed four main clades by ML, MP and BI methods corresponding with four different species regardless of the geographical origin and host species: Trichuris navonae,T. pardinasi,Trichuris sp. and T. bainae n. sp. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages were found among Trichuris species from sigmodontine rodents, which supported the proposal of a new species. The host distribution of the studied species showed correspondence with different tribes included in Sigmodontinae rodents such as, Clade 1-Akodontini; Clade 2- Oryzomyini, Clade 4-Phyllotini. Trichuris navonae (Clade 1) is a parasite of Akodon montensis, one of the most abundant host species present in a wide geographical distribution from the Atlantic forest and cerrado in Brazil, Paraguay and Argentina [59–63]. Also, T. navonae is present in another sympatric host species, Thaptomys nigrita, which is considered rare (not easy to capture) and not abundant (not caught in numbers) [64,65]. Both rodents occur in the same microhabitat, living in primary and secondary forests [66]. The new species Trichuris bainae n. sp. (Clade 2) is a parasite of Sooretamys angouya, a rodent with sympatric distribution with the two akodontines mentioned above, although this species uses trees and ground more frequently [64,67–69]. This rodent is considered common (easy to capture) but not abundant (not caught in numbers) [64]. Trichuris pardinasi (Clade 3) is a parasite of Phyllotis bonariensis and P. xanthopygus, both abundant species which are found in a wide variety of habitats, but these are restricted mainly to rocky outcrops [70,71]. Phyllotis bonariensis is distributed only in Sierra de la Ventana, southeast of Buenos Aires province (Argentina) [72,73] while P. xanthopygus has a wide distribution, along the Andes from west central Peru to Santa Cruz Province (Argentina) and the adjacent Magellan Region of Chile [74,75]. The population of P. bonariensis from Sierra de la Ventana was originally cited as an endemic species by Crespo [72], Reig [76], Galliari et al. [77], and Musser and Carleton [74]. Later, the populations of P. xanthopygus were considered as stated in Pardin˜as et al. [78]. Currently, there is no solid evidence available to justify this second proposal and specific status [79]. In this paper we follow the first taxonomic proposal. However, Sierras de Co´rdoba and Sierra de la Ventana are areas considered faunistic islands that share a considerable number of species and subspecies, i.e., molluscs, insects, amphibians [80], as well as T. pardinasi. In this study, three clades showed different levels of host specificity. Clade 1 and 2 indicated the presence of different species of Trichuris in the same biome, but with each species associated with a different host tribe and ecological habits. In addition, these species of whipworm follow their hosts along their geographical distribution (e.g. T. bainae in S. angouya from Misiones and Formosa provinces). Clade 4 indicated the presence of the same species of Trichuris in two congener host species in two disjunct areas; showing a probably specificity at the generic level of host (or specific level if futures studies confirm that P. bonariensis and P. xanthopygus are conspecific). Although other closely related species of Trichuris spp. from Sigmodontinae, such as T. laevitestis from Akodon azarae (Fischer, 1829), Scapteromys aquaticus Thomas, 1920 and Necromys lasiurus (Lund, 1840) [3]; and T. travassosi from Oligoryzomys nigripes (Olfers, 1818) [6] were not studied here, it is possible that the correspondence between parasite-tribe host is maintained, even though probably the level of host specificity could be different in agree with the ecology of the species host and the history of the areas where the hosts lives [81]. The understanding of the phylogeography of these nematodes would be improved by the study of a larger number of specimens and integrating biogeographic information from potential hosts. For example, Trichuris spp. in Clade 3 consisted of only one specimen and it was not possible to provide any hypothesis about its host and geographical distribution. This study highlights the importance of an integrated study of Trichuris spp., allowing a more complete understanding of the taxonomy, host and geographical distribution, and biology of whipworms. Acknowledgments We thank Graciela T. Navone for her support in the laboratory and important advice with nematodes; Carlos Galliari, Ulyses Pardin˜as, Marcela Lareschi, Guillermo Panisse, Natalia Guerreiro Martins, and Jualiana Notarnicola for their help during host collections; Carlos Galliari and Ulyses Pardin˜as for identification of the hosts; Marı ´a Cristina Estivariz for the drawings; Patricia Sarmiento for the SEM photographs. Luciana Riccialdelli for the revision of the English version, and Mike Kinsella for the valuable critical reading of the manuscript and for the revision of the English version. Author Contributions Conceived and designed the experiments: MdRR CC CJP RC. Performed the experiments: MdRR CC CJP RC. Analyzed the data: MdRR CC CJP RC. Contributed reagents/materials/analysis tools: MdRR CC CJP RC. Wrote the paper: MdRR CC CJP RC. References 1. Cafrune MM, Aguirre DH, Rickard LG (1999) Recovery of Trichuris tenuis Chandler, 1930, from camelids (Lama glama and Vicugna vicugna)in Argentina. Journal of Parasitology 85: 961–962. 2. Anderson RC (2000) Nematode parasites of vertebrates. Their Development and Transmission. 2 nd ed. CAB International ed. Wallingford Oxon UK. 650 p. 3. Robles MdR, Navone GT (2014) New host records and geographic distribution of species of Trichuris (Nematoda: Trichuriidae) in rodents from Argentina with an updated summary of records from Ame´rica. Mastozoologı ´a Neotropical 21: 67–78. 4. Callejo´n R, Halajian A, de Rojas M, Marrugal A, Guevara DC, et al. (2012) 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal Trichuris Species, Morphological and Molecular Characterization PLOS ONE | www.plosone.org 9 November 2014 | Volume 9 | Issue 11 | e112069