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Molecular diversification of Trichuris spp. from Sigmodontinae (Cricetidae) rodents from Argentina based on mitochondrial DNA sequences

A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from mitochondrial cytochrome c oxidase 1 (cox1) and cytochrome b (cob). The taxa consisted of nine populations of whipworm from five species of Sigmodontinae rodents from Argentina. Bayesian Inference, Maximum Parsimony, and Maximum Likelihood methods were used to infer phylogenies for each gene separately but also for the combined mitochondrial data and the combined mitochondrial and nuclear dataset. Phylogenetic results based on cox1 and cob mitochondrial DNA (mtDNA) revealed three clades strongly resolved corresponding to three different species (Trichuris navonae, Trichuris bainae, and Trichuris pardinasi) showing phylogeographic variation, but relationships among Trichuris species were poorly resolved. Phylogenetic reconstruction based on concatenated sequences had greater phylogenetic resolution for delimiting species and populations intra-specific of Trichuris than those based on partitioned genes. Thus, populations of T. bainae and T. pardinasi could be affected by geographical factors and co-divergence parasite-host.


Introduction
The Old World mice and rats (Murinae-Muridae) are the largest mammalian subfamily, comprising over 500 species (Musser and Carleton 2005). Possibly related to the Murinae are the Gerbillinae, hopping species that live in African and Asian deserts. The second-largest mammalian subfamily is the Sigmodontinae (Cricetidae), endemic from American continent. In the same family, they are often grouped with the Cricetinae (Old World hamsters) and sometimes with Arvicolinae (voles, lemmings, and muskrats). The Arvicolinae are a diverse Holarctic group (>125 species) (Steppan et al. 2004). The importance of sigmodontines cannot be underestimated. Some species are reservoirs of the etiological agents of a number of human and animal diseases. In South America, all hantaviruses known to cause Hantavirus Pulmonary Syndrome are associated with species of sigmodontine rodents, including Oligoryzomys spp., Necromys benefactus (Thomas, 1919), Akodon azarae (Fischer, 1829), Calomys laucha (Fischer, 1814), and Sigmodon alstoni (Thomas, 1881) (D'Elía 2003). In addition, sigmodontine rodents are reservoirs of protozoa (Jansen and Roque 2010;Lanos-Cuentas et al. 1999;Lallo et al. 2009) and helminthes (Maldonado et al. 2012;Robles et al. 2008Robles et al. , 2016Cubillos et al. 1991;Miño et al. 2013;Kuhnen et al. 2012). In most of the cases, the infections in this host group may indicate the occurrence and level of environmental contamination Rocío Callejón and María Del Rosario Robles contributed equally to this paper and should be considered as co-first authors of different zoonotic parasites, affecting many domestic animals (Herrera et al. 2005;Reperant et al. 2009).
Trichuris species from Sigmodontinae rodents present an interesting evolutive history since these hosts are endemic from American rodents. These rodents live in different environment and geographical distributions, and have been included in diverse phylogenetic hypotheses (e.g., Steppan et al. 2004;Cox and Hautier 2015). Five out of six species of Trichuris from sigmodontine rodents present similar morphological features, including the absence of a spicular tube, spicular sheath with spines (mostly with a cylindrical shape), and a non-protrusive vulva (Tiner 1950;Babero et al. 1976;Correa-Gomes et al. 1992;Feliú et al. 2000;Robles et al. 2006;Robles 2011). Therefore, it is probable finding several cryptic species. In this context, although most studies of Trichuris molecular systematics have focused on delimiting species, accurately inferring relationships among species is essential for testing macroevolutionary hypotheses including co-phylogeny and biogeography, both in support of the phylogenetic relationships proposed for their hosts and those linked to the geographic areas of distribution.
Molecular markers used for species-level questions in Trichuris include the Internal Transcribed Spacer 1 and 2 nuclear regions (ITS1 and ITS2) (Oliveros et al. 2000;Cutillas et al. 2002Cutillas et al. , 2004Cutillas et al. , 2007Cutillas et al. , 2009Callejón et al. 2012b;Salaba et al. 2013;Robles et al. 2014;Doležalová et al. 2015), 18S nuclear ribosomal RNA gene (Callejón et al. 2013;Guardone et al. 2013;Doležalová et al. 2015), mitochondrial 16S ribosomal RNA gene (Callejón et al. 2012b), and protein-coding mitochondrial genes, including the 12 common genes obtained from mitochondrial genome sequences (Liu et al. 2012(Liu et al. , 2013, cytochrome c oxidase 1 (cox1) mitochondrial DNA partial gene (Callejón et al. 2013;Doležalová et al. 2015) and cytochrome b (cob) mitochondrial DNA partial gene . These genes have different attributes and shortcomings for inferring Trichuris phylogeny, including substantially different rates and patterns of evolution. Studies combining more than one locus, such as nuclear and mitochondrial genes, together with morphological and ontogenetic comparisons, provide many advantages over single-locus studies, particularly for estimates of species-level relationships (Ballard and Rand 2005). Thus, different authors evaluated phylogenetic hypotheses for Trichuris based on two independent loci (Callejón et al. 2013Liu et al. 2013;Doležalová et al. 2015).
The main objective of this manuscript is to develop a molecular phylogenetic hypothesis for whipworm species isolated from five species of Sigmodontinae rodents from Argentina using mitochondrial genes, including partial sequences of cox1 and cob mtDNA. A phylogenetic reconstruction based on combined analysis of mitochondrial genes (cox1 and cob) and mitochondrial and nuclear markers (cox1, cob, and ITS2) is also evaluated.

Sampling
Twenty adult Trichuris specimens were collected from different cricetid (Sigmodontinae) rodents and geographical origins (Argentina) ( Akodon montensis is widespread from eastern Paraguay, northeastern Argentina in gallery forests along rivers or wetlands in the provinces of Formosa, Chaco, Corrientes, and Misiones, and along the southern coast of Brazil. Moreover, this species is the dominant member of the small mammal assemblage in both secondary forest and other anthropogenic disturbed habitats in Argentina. Thaptomys nigrita is restricted to moist tropical forest and second-growth forest in southeastern Brazil, eastern Paraguay, and northeastern Argentina. Sooretamys angouya is widely distributed in eastern South America, from the Brazilian Atlantic rainforest to the humid forests of eastern Argentina and Paraguay. Phyllotis bonariensis lives in rocky places at about 400 m elevation in a range of hills north of Bahia Blanca, Buenos Aires province. Phyllotis xanthopygus lives in rocky microhabitats on arid Andean slopes from Junín in Peru to Magallanes in Chile and Santa Cruz in Argentina, where boulder fields, rock slides, cliffs, small shale outcroppings, stone walls, and stone huts are all satisfactory habitations. Also, this species is found at higher elevations in the Sierras of San Luis and Córdoba provinces in Argentina (D'Elia and Pardiñas 2015).
Species identification was performed according to previous studies (Robles et al. 2006(Robles et al. , 2014Robles 2011). Worms were washed extensively in 0.9 % saline solution and stored in 70 % ethanol until required for DNA extraction, PCR amplification, and posterior sequencing.

Sequences
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.
Mitochondrial cob partial gene was amplified and sequenced using primers designed from comparisons of complete mtDNA genome sequences of Trichuris discolor Linstow 1906 (NC_018596), Trichuris ovis Abildgaard 1795 (NC_018597), Trichuris suis Schrank 1788 (NC_017747), and Trichuris , and 72°C for 30 s (primer extension), followed by a post-amplification extension at 72°C for 7 min. The PCR products were checked on ethidium bromidestained 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 purified PCR products were concentrated and directly sequenced by Stab Vida (Portugal).
All sequences were completely double-stranded for verification using reactions primed from the PCR primers.

Analyses
Sequences based on mtDNA (cox1 and cob) were aligned using CLUSTAL X (Larkin et al. 2007) as described by Callejón et al. (2013). The nucleotide sequences of the protein-coding genes (cox1 and cob) were first translated Bin silico^to confirm that they lacked internal stop codons and to verify (by BLAST match) that inferred amino acid sequences were characteristic of the predicted nematode protein.
ITS2 sequences based on nuclear ribosomal DNA (rDNA) obtained from GenBank (Robles et al. 2014) were incorporated to a comparative analysis (Table 2). Nucleotide sequence data reported in this paper are available in the GenBank™ database (accession numbers in Table 3).
Sequences from two outgroup taxa ( Phylogenetic trees were inferred using nucleotide data and produced using three methods: Bayesian Inference (BI), Maximum Parsimony (MP), and Maximum Likelihood (ML) using the PhyML package (Guindon and Gascuel 2003), MEGA 5.0 program (Tamura et al. 2011), and MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003), respectively. jModelTest version 0.1.1 (Posada 2008) was used to compare the fit of nucleotide substitution models using the Akaike information criterion. For the study of the three concatenated datasets (cox1, cob, and ITS2), analyses were partitioned by gene. For Bayesian analysis, models for individual genes within partitions were those selected by jModelTest. For ML inference using PhyML, the rapid 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. Models selected by jModelTest for BI included nst = 6 with gamma rates (cox1 and cob), nst = 6 with invgamma rates (ITS2), and nst = mixed for concatenated analysis. For the Bayesian analysis, we ran three independent runs of four Markov chains for 10 million generations, sampling every 500 generations.

Results
Cox1 mtDNA partial gene A single cox1 PCR product was amplified from each Trichuris species included in this study. The partial sequences were 401 and 405 base pairs (bp) in length. The G + C content of the cox1 partial gene of Trichuris species was 35-38 %, the maximum values corresponding to three individuals of T. bainae and the minimum values to six individuals of T. navonae from Misiones (Table 3).
The multiple alignment of 20 cox1 nucleotide sequences (including outgroups) yielded a dataset of 341 characters. jModelTest determined that the best-fit model for cox1 mtDNA datasets was GTR + G, which was used for Maximum Likelihood and Bayesian analysis.
The range of intra-population similarity of Trichuris spp. based on cox1 mtDNA sequences was 97.7-100 % with the maximum value corresponding to T. bainae from Formosa and the minimum value corresponding to T. pardinasi from Buenos Aires. On the other hand, the inter-population similarity was analyzed in T. bainae isolated from Misiones and Formosa with the maximum value (99.6 %) and T. pardinasi isolated from Córdoba and Buenos Aires with the minimum value (97.0 %).
The comparative study between different cox1 mtDNA sequences obtained for each species (alignment not shown) revealed the highest similarity (90.4 %) between T. pardinasi from Córdoba and T. bainae from Misiones, whereas the lowest similarity (86.7 %) was observed between T. navonae from Misiones and T. pardinasi from Córdoba. The cox1 datasets provided moderate phylogenetic resolution among most Trichuris taxa regardless of inference method. The consensus tree showed three clear clades corresponding with three different Trichuris spp. with good resolution; nevertheless, within the Trichuris spp. group from Argentina, all relationships among clades were not resolved by the three methods (BI, MP, and ML) ( Table 4). The topology showed all species of Trichuris from South America (Argentina) separated from those belonging to Trichuris spp. from Europe (Spain) with absolute support (100 % Bayesian Posterior Probabilities (BPP) and 100 % ML Bootstrap Value (BV)). Furthermore, T. arvicolae from Europe appeared clustered together and separated of T. muris from Europe with high support (100 % BPP, 99 % MP, and 92 % ML BV) (unpublished data).
Trichuris spp. from Argentina showed three main clades by BI, MP, and ML methods (Table 4). Clade 1 clustered T. navonae from different hosts and localities from Misiones. Clade 2 clustered T. bainae from Misiones and Formosa. Finally, Clade 3 clustered T. pardinasi populations from Buenos Aires and Córdoba. In addition, a subclade 2a including T. bainae from Formosa was observed by the three methods with robust support (Table 4) separated of T. bainae from Misiones and a subclade 3a including T. pardinasi from Buenos Aires with moderate support which appeared separated from T. pardinasi from Córdoba.
Cob mtDNA partial gene A single cob PCR product was amplified from each Trichuris species included in this study. The partial sequences were 505 bp in length. The G + C content of the cob partial gene of Trichuris species ranged from 28 to 32 %, the maximum values corresponding to one individual of T. pardinasi from Córdoba and one individual of T. pardinasi from Buenos Aires and the minimum values to seven individuals of T. navonae from Misiones (Table 3).
The multiple alignment of 21 cob nucleotide sequences (including outgroups) yielded a dataset of 494 characters. jModelTest determined that the best-fit model for cob mtDNA datasets was GTR + G, which was used for Maximum Likelihood and Bayesian analysis.
The range of intra-population similarity of Trichuris spp. based on cob mtDNA sequences was 98.4-99.8 % with the maximum value corresponding to T. bainae from Formosa and Misiones and the minimum value corresponding to T. pardinasi from Córdoba. On the other hand, the interpopulation similarity was analyzed in T. bainae isolated from Buenos Aires and Formosa with the maximum value (99.6 %) and T. pardinasi isolated from Córdoba and Buenos Aires with the minimum value (98.0 %).
When the cob mtDNA sequences of the different species and host isolates of the genus Trichuris were compared, the highest similarity (88.9 %) was obtained between T. navonae from Misiones and T. bainae from Misiones and Formosa, respectively, whereas the lowest similarity (84.8 %) was observed between T. pardinasi compared with T. navonae and T. bainae populations. The cob datasets provided moderate  (Table 4). The topology showed that Clade 1 (T. navonae) related with Clade 2 (T. bainae). In addition, a subclade 2b clustered T. bainae from Misiones separated of T. bainae from Formosa. A subclade 3a including T. pardinasi from Buenos Aires with moderate support was recovered in cob data. In contrast to the cox1 mtDNA, a subclade 2a corresponding to T. bainae from Formosa was not revealed by cob mtDNA sequences (Table 4). Phylogenetic relationship based on concatenated cox1 and cob mtDNA sequence datasets The combined analysis of mitochondrial genes (cox1 and cob) revealed a similar topology than those obtained by separate analysis of the two genes (Fig. 1). Thus, three clades were observed according to three different species of Trichuris. The concatenated analysis of the mitochondrial genes showed that Clade 1 (T. navonae) related with Clade 2 (T. bainae) with moderate support (100 % BPP, 68 % MP, and 80 % ML BV).
Phylogenetic relationship based on concatenated mtDNA (cox1 and cob) and rDNA (ITS2) sequence datasets BI, MP, and ML analysis of the combined mtDNA (cox1 and cob) and rDNA (ITS2) datasets yielded a similar tree topology to that observed by partitioned mitochondrial and nuclear markers (Fig. 2). Phylogenetic trees revealed a strong support for Clade 1, Clade 2, and Clade 3 (>95 %). The combined analysis of the mtDNA and rDNA sequences revealed the sister-group relationship among Clade 1 and 2. Furthermore, in concordance with cox1 and cob data, a subclade 3a including T. pardinasi from Buenos Aires with high support was recovered ( Fig. 2 and Table 4).

Discussion
Comparative analyses of coding and non-coding regions of ribosomal DNA (rDNA) have become a useful tool for the construction of the phylogenetic trees of many organisms including nematodes (Subbotin et al. 2001). Internal Transcribed Spacer regions (ITS1, ITS2) of the rDNA are often useful for differentiating closely related nematode species (Xie et al. 1994). The ribosomal DNA segments ITS1 and ITS2 have been shown to be two of the best molecular markers for analyzing genetic relationships at species level in Trichuris. For example, sequences obtained by amplification of the ITSs of seven different Trichuris species (Trichuris leporis Frolich 1789, T. ovis, T. muris, T. arvicolae, T. suis, T. discolor, and T. trichiura) permit reliable diagnosis (Oliveros et al. 2000;Cutillas et al. 2002Cutillas et al. , 2004Cutillas et al. , 2007Cutillas et al. , 2009Callejón et al. 2010Callejón et al. , 2012a. Furthermore, a comparative phylogenetic study of ITS1 and ITS2 sequences from Trichuris species from different hosts revealed three different genetic lineages corresponding with host groups (Callejón et al. 2012a). Similarly, analyses of nuclear ribosomal RNA, including SSU (Callejón et al. 2013) and ITS sequences (Ravasi et al. 2012;Nissen et al. 2012), support the specific characterization of T. suis and the determination of a new species: Trichuris colobae Cutillas, De Rojas, Zurita, Oliveros and Callejón 2014, a parasite of the primate Colobus guereza kikuyensis Rúppell 1835 .
Nevertheless, previous studies have mainly relied on sequence analysis of the ITS regions (Cavallero et al. 2015;Ravasi et al. 2012;Ghai et al. 2014;Cutillas et al. 2009;Nissen et al. 2012), and as this region contains multiple repeats, the alignment of sequences even between closely related worms will include a number of gaps. This makes inference of the phylogenetic relationship between Trichuris species problematic, and it is therefore highly recommended to supplement such analysis with other genetic markers such as mitochondrial DNA genes (Callejón et al. 2013).  MtDNA has proved useful in molecular phylogeny due to its maternal inheritance, rapid rate of divergence, and lack of recombination (Arrivillaga et al. 2002). Mitochondrial genes data have been used for the characterization of Trichuris species and their relationships (Cutillas et al. 2009;2012bLiu et al. 2012;Hawash et al. 2015;Doležalová et al. 2015).
Phylogenetic investigations that include multiple loci have advantages over single-locus studies not only for estimates of species-level relationships (Ballard and Rand 2005) but for testing hypotheses of species or species delimitation (Pérez-Ponce de León and Nadler 2010; Nadler and Pérez-Ponce de León 2011). Recent progress has been made in understanding the phylogeny of the Trichuris genus. There seem to be host-specific patterns in infection with particular Trichuris species or subspecies (Betson et al. 2015). Future research priorities should include multiple genetic marker analysis of Trichuris sampled from different hosts and diverse geographical locations to provide insights into parasite transmission within and between host species.
In the present paper, we carried out a molecular study, based on multiple genetic marker analysis, of populations of Trichuris spp. isolated from five Sigmodontinae rodents' species from Argentina. Three species were identified: T. navonae, T. bainae, and T. pardinasi. The overall A + T content of the cox1 and cob sequences in these species is consistent with mitochondrial genomes of T. trichiura (68.1 %) and T. suis (71.5 %) (Liu et al. 2012).
The percentage of inter-specific similarity observed along the three species based on cox1 and cob mtDNA partial genes far exceeded the intra-population and inter-population similarity. The highest values of inter-specific similarity based on cox1 partial gene (90.4 %) were observed between T. bainae from Misiones and T. pardinasi from Córdoba, while the highest values of inter-specific similarity based on cob partial gene (88.9 %) was observed between T. navonae (Misiones) and T. bainae (Formosa). Although the clades were strongly supported, in the case of subclades they were moderate, suggesting that these individuals may represent the same species, although they could be on the way to diverge. Attending to the inter-specific similarity observed in the genus Trichuris based The phylogenetic analysis of the concatenated sequences (cox1 and cob) revealed three different clades: Clade 1 and Clade 2 (T. navonae and T. bainae, respectively) which appeared related but separated and a Clade 3 clustering both populations of T. pardinasi (Buenos Aires and Córdoba). These results are in agreement with those cited by Robles et al. (2014). The concatenation of these mitochondrial genes with nuclear sequences (ITS2) revealed phylogenetic results similar to those observed by individual gene analysis. Thus, these results suggest that resolving relationships representing the deepest nodes in the Trichuris phylogeny will require analysis of additional independent loci represented by nuclear genes.
Since the survival of Trichuris individuals in a host depends on many factors as host immunologic status (mainly associated with the taxonomy) or/and ecology aspects, the characteristics of its life cycle and environmental factors have been the main aspects used by different authors to explain the geographical distribution of Trichuris species (Bundy et al. 1988;Bundy and Cooper 1989;Grencis et al. 1993;Anderson 2000). Thus, the question is if the species of Trichuris could act as markers of their hosts and/or areas and if this could be observed as a co-divergence or captured process in co-phylogeny. In this context, the geographical and host distributions of Trichuris spp. have been poorly analyzed.
Rodent hosts could be used to understand the ecological and evolutionary factors that affect the geographical and host distribution of Trichuris spp. The Trichuris species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents. The host distribution of T. navonae, T. bainae, and T. pardinasi showed correspondence with different tribes included in Sigmodontinae rodents such as Clade 1-Akodontini, Clade 2-Oryzomyini, and Clade 3-Phyllotini. T. navonae (Clade 1) is a parasite of A. montensis, Likelihood bootstrap values, respectively, for clade frequencies exceeding 60 % one of the most abundant host species present in a wide geographical distribution from the Atlantic forest and Cerrado to Brazil, Paraguay, and Argentina (Pardiñas et al. 2006Cirignoli et al. 2011;De la Sancha 2014). Also, T. navonae is present in another sympatric host species, T. nigrita, that is not easy to capture and is less abundant (Bonvicino et al. 2002;Patton et al. 2008). Both rodents occur in the same microhabitat, living in primary and secondary forests .
T. bainae (Clade 2) is a parasite of S. angouya, a rodent with sympatric distribution with the two akodontines mentioned above, although this species lives more frequently in trees and ground (Bonvicino et al. 2002;Cirignoli et al. 2005;Teta et al. 2007;Percequillo et al. 2008). This rodent is considered common (easy to capture) but not abundant (Bonvicino et al. 2002).
In this study, the 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, such as T. bainae (Clade 2 and subclade 2a) in S. angouya from Misiones and Formosa provinces. Although the humid Chaco (Formosa) and tropical forests (Misiones) are very different in floristic composition, getting the Argentina area, these biomes are geographically closed (Mayle 2004;Pennington et al. 2000).
On the other hand, T. pardinasi (Clade 3) is a parasite of P. bonariensis and P. xanthopygus, both abundant species which are found in a wide variety of habitats, but these are restricted mainly to rocky outcrops (Polop 1989;Kramer et al. 1999). P. bonariensis is distributed only in Sierra de la Ventana, southeast of Buenos Aires province (Argentina) (Crespo 1964; Pardiñas and Jayat 2008), 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 (Musser and Carleton 2005). The population of P. bonariensis from Sierra de la Ventana was originally cited as an endemic species by Crespo (1964), Reig (1987), Galliari et al. (1996), and Musser and Carleton (2005). Later, this population was considered as P. xanthopygus as stated by Pardiñas et al. (2004). Currently, there is no solid evidence available to justify this second proposal and specific status (Steppan et al. 2007); for this reason, we follow the first taxonomic proposal. Clade 3 indicated the presence of the same species of Trichuris in two congener host species in two disjoint areas suggesting specificity at the host generic level (or specific level if futures studies confirm that P. bonariensis (subclade 3a) and P. xanthopygus are conspecific). Notably, the Sierras de Córdoba and Sierra de la Ventana are areas considered faunistic islands that share a considerable number of species and subspecies, i.e., mollusks, insects, and amphibians (Ringuelet 1961), as well as T. pardinasi.
Interestingly, Trichuris from different host origin (Muridae and Cricetidae) from the Old World generated a monophyletic group separated from Trichuris of Sigmodontinae (Cricetidae) from the New World ( Figs. 1 and 2). The question leads again to the lack of knowledge on the behavior of distribution of Trichuris: Are the geographical factors and processes that affect the distribution of Trichuris greater than those of phylogenetic origin?

Conclusion
Mitochondrial (cox1 and cob) markers, used in the present work, are useful tools to discriminate Trichuris spp. (T. navonae, T. bainae, and T. pardinasi) and different populations of T. bainae and T. pardinasi with different geographical origins and rodent hosts. The phylogenetic tree based on combined mitochondrial and nuclear sequences of Trichuris spp. from different rodent hosts from Argentina revealed three clades corresponding to these three Trichuris spp. showing different levels of host specificity. More comprehensive understanding of the co-divergence parasite-host will require increased taxa sampling of Trichuris species and the increased resolution provided by multigene molecular phylogenies.