Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa

Accepted Manuscript Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa

Rocío Callejón, Ali Halajian, Cristina Cutillas PII: DOI: Reference:

S1567-1348(17)30115-6 doi: 10.1016/j.meegid.2017.04.002 MEEGID 3112

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

15 November 2016 2 April 2017 4 April 2017

Please cite this article as: Rocío Callejón, Ali Halajian, Cristina Cutillas , Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2017), doi: 10.1016/ j.meegid.2017.04.002

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ACCEPTED MANUSCRIPT Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa.

Department of Microbiology and Parasitology. Faculty of Pharmacy. University of

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Rocío Callejón1, Ali Halajian2 and Cristina Cutillas1*

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Seville. Profesor García González 2, 41012 Seville, Spain.

Department of Biodiversity (Zoology). University of Limpopo. Private Bag X1106,

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Sovenga, 0727 South Africa.

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Running tittle: Trichuris ursinus a new species from Papio ursinus Note: Supplementary data associated with this article

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* Corresponding author:

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Dr. Cristina Cutillas

Department of Microbiology and Parasitology. Faculty of Pharmacy. University of Seville. Prof. García González 2, 41012 Sevilla, Spain. Phone: +34 954556773 e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract In the present work, we carried out a morphological, biometrical and molecular study of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae) parasitizing Papio ursinus Keer 1792 (Chacma baboon). Biometrical and molecular data suggest a new species of Trichuris parasitizing baboons. In addition of main morphological features

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(spicule, spicule sheath, spicule tube, proximal cloacal tube, distal cloacal tube, vulva,

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vagina), the mean values of individual variables between Trichuris colobae, Trichuris

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suis, Trichuris trichiura examined by Student’s t tests suggest that T. ursinus n. sp. constitutes a new species. The combined analysis of three markers (cox1, cob and ITS2)

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revealed a sister relationships between T. colobae and T. ursinus n. sp. Mitochondrial sequences revealed a higher inter-specific similarity between T. ursinus n. sp., T. suis

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and T. colobae. Phylogenetic hypotheses for both mitochondrial genes strongly supported distinct genetic lineages corresponding to different species of the genus

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Trichuris associated with certain hosts. Thus, T. suis, T. colobae and T. ursinus n. sp.

species of primates.

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appeared as a sister group and separated from Trichuris spp. from humans and other

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DNA.

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Keywords: Papio ursinus, Trichuris, Chacma baboon, Ribosomal DNA, Mitochondrial

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ACCEPTED MANUSCRIPT 1. Introduction Trichuris trichiura Linnaeus, 1771 is a soil-transmitted helminth causing a parasitism known as “trichuriasis” with a global distribution, which affects approximately 600 million people worldwide (Bethony et al., 2006; Hotez et al., 2007; Hotez et al., 2009).

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Traditional parasitological research on T. trichiura from humans and primates has focused on differentiating it from Trichuris suis found in pigs (Beer, 1976; Ooi et al.,

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1993; Cutillas et al., 2009; Nissen et al., 2012; Liu et al., 2012). Morphological studies

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of Trichuris isolated from primates and humans conclude that the species infecting these hosts is the same, despite slight morphological variations that are distinguishable

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using scanning electron microscopy (Ooi et al., 1993). Nevertheless, many studies have

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been carried out so far to discriminate parasite species from human and non-human primates (NHP) hosts suggesting Trichuris spp. in human and NHP represent several species that differ in host specificity (Ravasi et al., 2012; Liu et al., 2013; Hawash et al.,

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2015). The hypothesised existence of more species of Trichuris in primates opens the possibility to revise the zoonotic potential and host specificity of T. trichiura and other putative new species of whipworms (Doležalová et al., 2015). Different molecular

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studies have been carried out to discriminate Trichuris species from humans and NHP

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(see Table 1). This is an interesting theme because multi-host parasites are difficult to control because reservoir hosts may serve as sources of re-infection for other populations in which the parasite has been eliminated (Anderson et al., 1992; Gottstein et al., 2009). Baboons of the genus Papio Erxleben, 1777 are distributed over wide ranges of Africa and even colonized parts of the Arabian Peninsula (Zinner et al., 2009). Traditionally, five phenotypically distinct species are recognized: P. anubis Lesson, 1827 (Olive baboon), P. cynocephalus Linnaeus, 1766 (Yellow baboon), P. hamadryas Linnaeus, 3

ACCEPTED MANUSCRIPT 1758 (Hamadryas baboon), P. papio Desmarest, 1820 (Guinea baboon) and P. ursinus (Chacma baboon) (Zinner et al., 2009). Despite the fact that there seems to be a pattern of infection with different Trichuris species infecting particular host species, there are several issues that need to be further explored (Betson et al., 2015). Are the different Trichuris species in primates host

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specific, i.e. can Trichuris in one clade infect hosts of the other clades? Betson et al.

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(2015) just started to unravel some of these questions. They concluded that is a need for

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further studies applying multiple genetic markers to Trichuris collected from humans and NHPs from sympatric areas and worldwide locations. This will clarify parasite

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transmission routes between these primates and allow implementation of appropriate

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control and prevention measures.

Previous findings (Ravasi et al., 2012) suggest the need for morphological analysis of Trichuris sp. adult worms collected from P. ursinus (Chacma baboon) from South

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Africa to determine whether the genetic lineages are corresponding with different morphological species. In the present paper, we carried out for the first time a morphological and biometrical study of Trichuris sp. from P. ursinus from the Cape

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Peninsula, Western Cape Province (South Africa). Furthermore, a molecular analysis is

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carried out based on cytochrome c oxidase 1 (cox1) and cytochrome b (cob) mitochondrial DNA (mtDNA) datasets and the combined analysis of mitochondrial and nuclear markers (cox1, cob and ITS2).

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ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Collection of samples Chacma baboons (P. ursinus) from the Cape Peninsula, an area of 470 km2 at the southwestern tip of the African continent, which stretches from the city of Cape Town

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to the Cape of Good Hope were studied for parasites. These specimens were obtained through the assistance of colleagues (see Acknowledgements).

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2.2. Biometrical and morphological studies

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Thirty-four adult specimens were collected from the large intestine of seven adult P.

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ursinus, washed extensively in a saline solution of 0.9 % sodium chloride, and stored separately in 70 % ethanol. Species identification was performed according to previous

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studies (Oliveros et al., 2000; Cutillas et al., 2002, 2004, 2007). Thirty Trichuris specimens (15 females and 15 males) of Trichuris sp. were measured.

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Measurements of new species are presented as follows: holotype male or allotype

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female, and paratypes with mean and range in parentheses. The holotype (101YT), allotype female (102 YT) and 28 paratypes (103YT) have been studied. All

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measurements are in millimetres.

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Trichuris specimens were measured according to parameters reported by Spakulová and Lýsek (1981), Suriano and Navone (1994) and Robles et al. (2006) who summarize the parameters used in the last years. A comparative study of morpho-biometrical data of T. suis, T. trichiura and T. colobae obtained for Cutillas et al. (2009, 2014) was carried out. Descriptive univariate statistics (mean values, standard deviations, and range) for all parameters were determined for four populations: T. suis from Sus scrofa domestica Linnaeus, 1758, T. trichiura from Pan troglodytes, T. colobae from C. g. kikuyensis and Trichuris sp. from P. ursinus. 5

ACCEPTED MANUSCRIPT Student’s t test (P<0.001) was used to test the equality of means for each variable. Statistical analysis was performed using Microsoft Excel 5.0 (Feliú et al., 2000). For scanning electron microscopy (SEM), the individuals were fixed for 2 h at 4° C in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, followed by washing several times in 0.1 M cacodylate, pH 7.4. The second fixation was carried out for 1 h in 1 %

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(w/v) osmium tetraoxide (4 °C) and washed in 0.1 M cacodylate, pH 7.4, added with

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sucrose. The fixed worms were dehydrated in an ascending series of alcohol (30– 50–70

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%) and acetone (70–80–90–100 %), dried in a critical point dryer, and mounted with

2.3. PCR and sequencing of specimens

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gold.

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Genomic DNA from 4 individual worms was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer´s protocol. Quality of extractions

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was assessed using 0.8 % agarose gel electrophoresis and ethidium bromide staining.

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The mitochondrial DNA (mtDNA) cob partial gene was amplified by PCR using a Perkin Elmer and the primers DP769 and DP770 (Liu et al., 2012). Amplification

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reactions (25 µl) consisted of 0.5 mM of each primer, 200 mM deoxynucleoside triphosphates, 3 mM MgCl2 and 1 unit of AmpliTaq® polymerase. PCR cycling

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parameters included denaturation at 94º C for 4 min, followed by 36 cycles of 94º C for 30 sec, 50º C for 30 sec, and 72º C for 30 sec, followed by a post-amplification extension at 72º C for 5 min. For each set of PCR reactions, a negative (no DNA) control sample and a positive DNA control sample were included. Cob PCR products were used for sequencing following enzymatic treatment using exonuclease I and shrimp alkaline phosphatase.

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ACCEPTED MANUSCRIPT Mitochondrial DNA (mtDNA) cox1 partial gene was amplified by PCR using a Eppendorf AG thermocycler and conditions specified for isolates of genus Trichinella Railliet, 1895 by Nagano et al. (1999) and sequenced using the following primers: The forward PCR primer was modified from the reverse primer of Folmer et al. (1994): HC02198F 5´-TGATTTTTTGGTCACCCTGAAGTTTA-3´ and the reverse PCR

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primer was modified from Nagano et al. (1999) to correspond to more broadly

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conserved cox1 sequence: CORA 5´-ACYACATAGTAGGTRTCATG-3´. These two primers were used successfully for cox1 PCR amplification of all species of Trichuris

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studied. Amplification reactions consisted of 5 μl 10x PCR buffer, 2 μl 10 mM dNTP

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mixture (0.4 mM each), 3 μl 50 mM MgCl2, 5 μl primer mix (1mM each), 5 μl template DNA, 0.5 μl Taq DNA polymerase (2.5 units) and autoclaved distilled water to 50 μl.

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The following PCR conditions were applied: 94º C for 5 min (denaturing), 40 cycles at 94º C for 1 min (denaturing), 48º C for 1 min (annealing), 72º C for 1 min (primer

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extension), followed by a post-amplification extension at 72º C for 7 min.

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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

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and PCR Clean-Up System (Promega). The purified PCR products were concentrated,

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and directly sequenced by Stab Vida (Portugal). All sequences were completely double-stranded for verification using reactions primed from the PCR primers. 2.4. Analysis Sequences 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 proteincoding genes (cox1 and cob) were first translated “in silico” to confirm that they lacked

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ACCEPTED MANUSCRIPT internal stop codons and to verify (by BLAST match) that inferred amino acid sequences were characteristic of the predicted nematode protein. Additional sequences from the National Centre for Biotechnology Information (NCBI) GenBank database were incorporated into the alignments; the analysis included the ITS2 nuclear marker and cox1 and cob mtDNA (Table 2). Nucleotide sequence data

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reported in this paper are available in the GenBankTM database (Accession numbers in

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Table 3).

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Phylogenetic trees were inferred using nucleotide data and produced using three methods: Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian

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Inference (BI), using MEGA 5.0 program (Tamura et al., 2011) for MP and ML and

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MrBayes version 3.2.6 (Ronquist and Huelsenbeck, 2003) for BI. JMODELTEST (Posada, 2008) was employed to compute the best partitioning scheme, as well as the best nucleotide substitution models for each partition. Models of evolution were chosen

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for subsequent analysis according to the Akaike Information Criterion (Huelsenbeck and Rannala, 1997; Posada and Buckley, 2004). The concatenation of mitochondrial markers and nuclear and mitochondrial markers (cox1, cob and ITS2) was analysed by

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BI method. Thus, dataset was partitioned by gene and models for individual genes

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within partitions were those selected by jModeltest. For ML inference, best-fit nucleotide substitution models included general time-reversible model with gammadistributed rate variation, GTR+G (cox1), general time-reversible model with gammadistributed rate variation and a proportion of invariable sites GTR+I+G (cob) and general time-reversible model with a proportion of invariable sites GTR+I (ITS2). Support for the topology was examined using bootstrapping (heuristic option) (Felsenstein, 1985) over 1000 replications to assess the relative reliability of clades. Models selected by jModeltest for BI were nst=6 with gamma rates (cox1), nst=6 with 8

ACCEPTED MANUSCRIPT invgamma rates (cob) and nst=6 with inv rates (ITS2). For BI, the standard deviation of split frequencies was used to assess if the number of generations completed was sufficient; the chain was sampled every 500 generations and each dataset was run for 10 million generations. Burn-in was determined empirically by examination of the log likelihood values of the chains. The Bayesian Posterior Probabilities (BPP) is

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percentage converted. The phylogenetic analysis, based on cox1, cob mtDNA, ITS2

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rDNA sequences was carried out using our sequences, and those obtained from

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GenBank database (Table 2). Sequences from two outgroup taxa (Table 2) were included in several analysis (cox1, cob and combined mitochondrial genes) to root the

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phylogenetic trees: Priapulus caudatus Lamarck, 1816 (Priapulida, Priapulidae) and

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Lithobius forficatus Rilling, 1968 (Arthropoda, Lithobiomorpha).

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ACCEPTED MANUSCRIPT 3. Results 3.1. Morphological and biometrical results The morphological study was carried out both, at the photomicroscope and SEM. A new species of Trichuris was found in P. ursinus, which is described here.

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Trichuris ursinus n. sp.

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(Figs. 1, 2 and 3)

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Diagnosis: Anterior part of body long, narrow, tapered, and whip-like; posterior part of body broad, and handle-like. Cuticle with fine transversal striation. Bacillary band

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located laterally in anterior portion of body. Bacillary band 0.30 – 0.64 from anterior end of body, and extends to body width region of 1.14-2.29. Vulvar aperture non-

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protrusive and without ornamentation (Fig. 1A, B, 2A-C) and a long and straight vagina (Fig. 1A) with a few rounded papillae (Fig. 2 A). Ratio between anterior and posterior

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body length is 1:1.9 in males and 1:2.2 females. Stichosome with 1 row of stichocytes,

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and 1 pair of conspicuous cells at esophagus-intestinal junction level (Fig. 1A, B). Male with spicule tube. Proximal cloacal tube united laterally to distal cloacal tube (Fig. 1C).

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Cloaca subterminal with one pair of paracloacal papilla not ornamented (Fig. 1C, 3A, 3C). Spicule sheath cylindrical without distal bulb and with triangular spines distributed

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from proximal to distal portion (Fig. 1C-D, 3B); Testis ends near the union of ejaculator conduct and intestine (Fig. 1C). Female with non-protrusive vulva located at esophagusintestinal junction level (Fig. 1A-B, 2A-B). Anus subterminal, with long caudal end finished with terminal torsion (Fig. 1E). Male (15 specimens): Body length 37.0, 36.5 ± 0.33 (32-41). Anterior portion of body (total length of esophagus) 25, 24.3 ± 0.19 (21-27) long,> thick portion of body 12.0, 12.67 ± 0.13 (11.0-14.0) long (Fig. 1C). Anterior body width 0.19, 0.17 ± 0.01 (0.1510

ACCEPTED MANUSCRIPT 0.19), maximum posterior body width 0.67, 0.62 ± 0.07 (0.53-0.75), width at esophagus-intestinal junction level 0.38, 0.38 ± 0.03 (0.32-0.46). Spicule length 2.3, 2.10 ± 0.11 (1.88-2.29) (Fig. 1C-D). Spicule sheath densely spinose 0.51, 0.54 ± 0.14 (0.23-0.69) long (Fig. 1C-D, 3B). Proximal cloacal tube 1.29, 1.33 ± 0.10 (1.23-1.47) long. Distal cloacal tube 1.67, 1.75 ± 0.09 (1.61-1.89) long (Fig. 1C). Ratio between

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total body length and posterior portion length 3.08, 2.92 ± 0.13 (2.77-3.25). Ratio

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between total body length and spicule length 17.4, 17.6 ± 0.08 (16.6-18.9). Ratio

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between posterior portion length and spicule length 5.6, 6.1 ± 0.04 (5.5-6.8). Ratio between proximal cloacal tube length and distal cloacal tube length 0.75, 0.77 ± 0.09

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(0.65-0.91). Ratio between maximum posterior body width and posterior portion length 0.056, 0.049 ± 0.08 (0.041–0.063).

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Female (15 specimens): Body length 39.0, 38.00 ± 0.55 (30.0-49.0). Anterior portion of body (total length of esophagus) 26, 26 ± 0.50 (20.0-37.0) long, thick portion of body

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13, 12.1 ± 0.17 (10.0-15.0) long. Anterior body width 0.18, 0.17 ± 0.02 (0.15-0.19);

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maximum posterior body width 0.71, 0.68 ± 0.08 (0.54-0.89); width at esophagusintestinal junction 0.41, 0.40 ± 0.02 (0.35-0.45) (Fig. 1A-B). Muscular portion of

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esophagus 0.93, 0.99 ± 0.20 (0.77-1.32), stichosome 1.67, 2.02 ± 0.42 (1.49-2.67). Distance between esophagus-intestinal junction and vulva 0.38, 0.18 ± 0.1 (0.03-0.38).

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Eggs oval, with bipolar plugs, (n= 9) 0.023-0.026 x 0.009-0.01 (Fig. 1F). Ratio between total body length and posterior portion length 3.0, 3.16 ± 0.46 (2.73-4.27). Ratio between maximum posterior body width and posterior portion length 0.06, 0.06 ± 0.007 (0.05-0.07). Taxonomic summary Type host species: Papio ursinus Keer, 1792 (Cercopithecidae). Type locality: Cape Town, Western Cape Province (South Africa). 11

ACCEPTED MANUSCRIPT Site of infection: Caecum. Type specimens: Holotype male (101 YT), allotype female (102 YT) and paratypes 103YT deposited at the Helminthological Collection of Museum National d'Histoire Naturelle of Paris (France).

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Etymology: Species name is derived from the species of the type host. Recorded in URN as urn: lsid: zoobank.org: pub: EFC90F8B-FF3C-4E26-8BB0-

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669619F7E742

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3.2. Differential diagnosis

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Different morphometric features compared the Trichuris species from different species of primates and pig. T. ursinus n. sp. can be separated from T. colobae and T. trichiura

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from human, macaque (Macaca fuscata) and baboon (Papio papio) by lacking a typical subterminal pericloacal papillae associated to a cluster of small papillae.

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The new species has a non-protrusive vulva and vagina without spines, such as T. suis. However, T. ursinus n. sp. can be separated from T. colobae and T. trichiura from human, monkey (M. fuscata) and baboon (P. papio) by an everted vagina covered with

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sharply pointed spines. Furthermore, T. colobae present a protrusive vulva with a crater-

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like appearance.

The length of spicule, the length of proximal cloacal tube, the maximum wide of posterior region of body, and distance between posterior part of testis and tail end of body is highest in males of T. suis from swine than those from T. ursinus n. sp., while the length of vagina and diameter of vulva turned over the surface of body is significantly higher in females of T. ursinus n. sp. than those from T. suis. The maximum width of posterior region of the body (thickness) of males and females is higher in T. suis from swine than T. ursinus n. sp. 12

ACCEPTED MANUSCRIPT Males and females of T. ursinus n. sp. have width of oesophageal region of body and the body width in the place of junction of esophagus and the intestine higher than T. colobae, while the length of bacillary stripes of males and females is higher in T. colobae. Furthermore, the length of spicule and the width of proximal end of spicule is

vagina was significantly higher in females of T. ursinus n. sp.

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highest in males of T. ursinus n. sp. than those from T. colobae, while the length of

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T. ursinus n. sp. can be separated from T. trichiura from P. troglodytes by nine male

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variables differing significantly (P<0.001): total body length of adult worm, length of oesophageal region of body, maximum width of posterior region of body (thickness),

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body width in the place of junction of esophagus and the intestine, length of spicule, maximum length of spicule sheath, width of proximal end of spicule, maximum width

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of spicule sheath and the distance between posterior part of testis and tail end of body. Furthermore, 8 female variables differed significantly: Width of oesophageal region of

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body, maximum width of posterior region of body (thickness), body width in the place

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of junction of oesophagus and the intestine, length of bacillary stripes, length of vagina, distance of posterior loop of uterus from tail end of body, distance of tail end of body

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(Table 5).

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and posterior fold of seminal receptacle and length of muscular zone of the esophagus

Moreover, the new species has a shorter proximal cloacal tube than T. suis and T. colobae and higher than T. trichiura. In addition, T. ursinus n. sp. presents a higher distal cloacal tube than T. suis, T. trichiura and T. colobae. T. ursinus n. sp. can be separated from T. vulpis, T. ovis and T. globulosa by lacking atypical subterminal pericloacal papillae, while T. arvicolae, T. muris, T. skrjabini and T. discolor have them. T. arvicolae, T. muris and T. skrjabini have a spicule length lesser than 1.2 mm while T. discolor and T. ursinus n. sp. have a spicule length higher 13

ACCEPTED MANUSCRIPT than 1.9 mm. T. discolor has a spicule with rounded tip while T. ursinus n. sp. has a spicule with pointed tip. 3.3. Molecular study 3.3.1. Cox1 mtDNA partial gene

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In order to include the largest possible number of sequences available for Trichuris species, further molecular analysis were performed using GenBank sequences of cox1

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partial gene of Trichuris spp. isolated from human, pigs and primates (Table 2).

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A portion (~350 base pairs (bp)) of the cox1 mtDNA gene was amplified and sequenced

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from 4 individuals of T. ursinus n. sp. Cox1 sequences were 342 (bp) in length and their G+C content was 35.9 % (Table 3). The cox1 sequences of T. ursinus n. sp. were

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identical (100 % of intra-specific similarity) (Data not shown). The multiple alignment of 31 cox1 nucleotide sequences (including outgroups) yielded a

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dataset of 342 characters. JModeltest determined that the best-fit model for cox1

Bayesian analysis.

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mtDNA datasets was GTR +G +I, which was used for Maximum Likelihood and

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The comparative study between different cox1 mtDNA sequences for each species

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(alignment not shown) revealed an intra-specific similarity of 100 % in T. ursinus n. sp., 99.7 % in T. trichiura, 99.9 % in T. colobae and 90.9 % in T. suis (Data not shown). The inter-specific similarity of Trichuris spp. based on cox1 mtDNA sequences range from 69.9 % (T. suis and Trichuris sp. from Macaca fascicularis Raffles, 1821 (Trichuris sp. MF) to 96.5 % (Trichuris sp. from Theropithecus gelada Rüppell, 1835 and Trichuris sp. from P. anubis (both populations named Trichuris sp. TG-PA)) (Data not shown). T. ursinus n. sp. sequences shared the maximum inter-specific similarity

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ACCEPTED MANUSCRIPT with the cox1 sequences from T. suis (81.5-83.9 %) and T. colobae (81.2-81.5 %) (Data not shown). Phylogenetic trees constructed by MP, ML and BI methods gave similar results (Fig. S1 and Table 6). Three main clades of Trichuris showed moderate to high (83-100 %) Bayesian Posteriors probabilities (BPP 1.0). Two of these clades also received high (82-

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100 % and 93-97 % support by MP and ML bootstrap (BV) analysis, the exception with

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low support 70 % and 78 % MP and ML BV respectively) was Trichuris sp.from Homo

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sapiens-Papio hamadryas (HS-PH). These three main clades included (1) T. suis, T. colobae and T. ursinus n. sp.; (2) Trichuris sp. from Homo sapiens Linnæus, 1758 and

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P. hamadryas (Trichuris sp. HS-PH); (3) Trichuris sp. TG-PA (Fig. S1). Within Clade

BV).

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3.3.2. Cob mtDNA partial gene

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1, T. ursinus n. sp. appeared more closely related to T. suis (97 % BPP and 100 % ML

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The cob partial gene sequences (513 bp) revealed a G+C content ranged 28.4-28.8 % (Table 3).

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The range of intra-population similarity was 89.7-100 % with the maximum and the minimum value corresponding to T. suis populations isolated from different geographic

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origins (Data not shown). The highest interspecific similarity (78.6 %) was observed between T. ursinus n. sp. and T. colobae, whereas the lowest similarity (70 %) was observed between T. ursinus n. sp. and Trichuris sp. from Homo sapiens. Nevertheless, the similarity observed between T. ursinus n. sp. and T. colobae (78.2-78.4 %) overlapped the range of similarity observed between T. ursinus n. sp. and T. suis (Data not shown).

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ACCEPTED MANUSCRIPT Phylogenetic trees constructed by MP, ML and BI methods based on cob datasets gave similar results (Fig. S2, Table 6). The BI and ML consensus trees for cob datasets lacked resolution within the Trichuris sp. TG-PA group, whereas these groups were resolved in MP analysis. Two main clades of Trichuris were obtained by three methods; (1) T. suis, T. colobae and T. ursinus n. sp.; (2) Trichuris sp. HS-PH (Fig. S2). Clade 1

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received moderate to high support (93 % BPP, 85 % MP and 100 % ML BV). Clade 2

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received strong support by MP method (100 % BV) and low support by BI method (<

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67 % BPP and ML, BV). Within Clade 1, the analysis that included cob datasets yielded a polytomy with respect to relationships of T. ursinus n. sp., T. suis and T. colobae (Fig.

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S2, Table 6).

3.3.3 Phylogenetic relationship based on concatenated cox1 and cob mtDNA sequence

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The concatenated dataset of cox1 and cob sequences included 854 aligned sites and 21

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taxa, including outgroups. BI, MP and ML analysis of the combined mtDNA (cox1 and cob) datasets yielded a similar tree topology to that observed by partitioned mitochondrial genes (Fig. S3 and Table 6) respect to the sister-group relationships

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between Trichuris spp. from pigs, human and primates. Three robust clades included (1)

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T. suis, T. colobae and T. ursinus n. sp.; (2) Trichuris ovis Abildgaard, 1795, Trichuris discolor Linstow, 1906, Trichuris skrjabini Baskakov, 1924, Trichuris muris Schrank, 1788, Trichuris arvicolae Feliú, Spakulová, Casanova, Renauld, Morand, Hugot, Santalla, and Durand, 2000 and T. vulpis; (3) Trichuris sp. HS-PH. Within Clade 1, BI indicated T. ursinus n. sp. group were more closely related to T. colobae (76 % BPP) however, this sister-species relationships was poorly bootstrap resampling (<65 % MP and ML BV). Analysis of the two mitochondrial datasets resolved Clade 1 and Clade 2 as sister groups by ML with moderate support (78 % BV); however, this sister 16

ACCEPTED MANUSCRIPT relationships was not strongly supported (<65 % BPP and MP BV) (Fig. S3 and Table 6). Phylogenetic analysis of the concatenated dataset yielded a strongly supported tree, with exception of: (1) the sister group relationships among T. colobae, T. suis and T. ursinus n. sp. respect to T. ovis, T. discolor, T. skrjabini, T. muris, T. arvicolae and T. vulpis; (2) the sister group relationships among T. colobae and T. ursinus n. sp. These

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clades were the only areas of discordance between the three methods (Fig. S3 and Table

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6).

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3.3.4 Phylogenetic relationship based on concatenated cox1 and cob mtDNA and ITS2 nuclear sequence dataset

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The concatenated dataset of cox1, cob and ITS2 sequences included 1164 aligned sites

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and 12 taxa. This phylogenetic analysis revealed strong support (> 96 % BPP, MP and ML bootstrap) for the separation of Trichuris spp. into three distinct clades, and the trees produced using the three different methods were essentially the same in topology

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(Fig. 4 and Table 6). Thus, Clade 1 clustered T. ovis, T. discolor and T. skrjabini, Clade 2 clustered T. suis, T. colobae and T. ursinus n. sp., and Clade 3 clustered T. vulpis, T. muris and T. arvicolae (Fig. S3, Table 6). In addition, within Clade 2, T. ursinus n. sp.

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appeared more closely related to T. colobae (100 % BPP, 83 % and 94 % MP and ML

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BV respectively). This result is in agreement with the combined analysis of mitochondrial genes (cox1 and cob) (Table 6).

17

ACCEPTED MANUSCRIPT 4. Discussion We have carried out the first comparative study of Trichuris species derived from different host primates and pig. The genetic and evolutionary relationship between Trichuris from humans and NHP is

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poorly understood. The identification of closely related species of Trichuris is very difficult. This is due to the phenotypic plasticity of the organisms themselves; host-

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induced variation, the paucity of morphological features, and the overlapping in

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morphologic characteristics that occur among species (Knight, 1984; Spakulová, 1994; Robles and Navone, 2006; Robles, 2011). Thus, genus Trichuris is a likely candidate to

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contain cryptic species. Thus, it is important include other complementary methods of

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identification.

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Many authors have carried out controversial studies about the different morphometric

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criteria to discriminate T. suis from T. trichiura. Spakulová (1994) found that the combination of seven metric characters, distinguished the whipworms from pig to

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human, while Cutillas et al. (2014) proposed a new species, T. colobae based on different parameters that discriminated significantly T. suis and T. trichiura to T.

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colobae from C. g. kikuyensis. Morphological and morphometric study showed that T. ursinus n. sp. differed significantly T. trichiura (nine different characters) and T. colobae (six different characters). Furthermore, T. ursinus n. sp. shows features close to T. suis (only three different characters) (see differential diagnosis above).

18

ACCEPTED MANUSCRIPT Whipworms in humans are traditionally designated T. trichiura. However, recent publications have begun to address whether several species present in the human population and whether NHPs are the same ones (Betson et al., 2015). Furthermore, molecular studies based on the amplification of cox1 and cob mitochondrial DNA confirmed that T. ursinus n. sp., T. suis and T. colobae are

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genetically distinct by phylogenetic analysis of sequences data sets representing these

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species and those from other species of whipworms.

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The high AT content of the cox1 and cob sequences in Trichuris is consistent with reports based on complete mitochondrial genomes (Park et al., 2011; Liu et al., 2012).

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The cox1 sequences of the four specimens of T. ursinus n. sp. were identical to each

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other while the percentage of inter-specific similarity based on cox1 and cob mtDNA sequences far exceeded the intra-specific similarity. Considering the inter-specific similarity observed in the genus Trichuris based on cox1 partial gene (Callejón et al.,

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2013, 2016) and cob partial gene (unpublished data), the similarities observed between T. ursinus n. sp. and T. suis and T. colobae are within the range of inter-specific variation reported for the genus corroborating that Trichuris from Papio ursinus

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represent a new species.

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The phylogenetic trees based on cox1 sequences showed different clades clustering different species of Trichuris from primates while phylogenetic analysis of cob mtDNA sequences showed T. ursinus n. sp. appearing in polytomy respect to T. suis and T. colobae (Fig. S2). Further, the phylogenetic analysis of the concatenated mitochondrial sequences (cox1 and cob) revealed T. ursinus n. sp. group were more closely related to T. colobae and separated of T. suis (Fig. 3). These results confirm those based on ITS1-5.8S-ITS2 region ribosomal DNA by Ravasi et al. (2012). These authors observed this population of Trichuris isolated from P. 19

ACCEPTED MANUSCRIPT ursinus from Groot Olifantsbos (GOB, South Africa) clustered to Trichuris spp. from the gibbon Nomascus gabriellae Thomas, 1909 and C. g. kikuyensis. Furthermore, that clade was more related to T. suis, and separated of Trichuris sp. from human and other species of NHP. On the other hand, Hawash et al. (2015), based on complete mt genome analysis, suggested the existence of a Trichuris species complex in primates and pigs.

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Hence, the cox1 phylogeny suggested at least five potential Trichuris sp. infecting

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primates. Likewise, Ghai et al. (2014) identified a distinct group of worms found only in

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NHP, and these might be related to the “Trichuris sp. non-human primates” clade observed by Hawash et al. (2015). However, the Trichuris from a black-and-white

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colobus was not identified as a separate species by Ghai et al. (2014), suggesting that this host can also be infected with different Trichuris spp., or it may reflect the use of

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different genetic markers between studies (Callejón et al., 2013). Phylogenetic analysis of the combined mtDNA (cox1 and cob) and rDNA (ITS2)

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datasets showed T. ursinus n. sp. closely related to T. colobae and nearly to T. suis. This

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analysis revealed strong support for the separation of Trichuris species from different hosts into three distinct clades (Fig. S3 and Table 6). These results are in agreement

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with those reported in previous molecular phylogenies of Trichuris based on nuclear

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and mitochondrial genes (Callejón et al., 2013, 2015).,

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ACCEPTED MANUSCRIPT 5. Conclusions In conclusion, morphological, biometrical and molecular data suggest a new species of Trichuris parasitizing P. ursinus. We report a description of this new species, T. ursinus n. sp. isolated from this species host. Relationships among T. ursinus n. sp. and T. suis and T. colobae have been resolved by molecular sequence data in this study. Thus, the

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combined analysis of three markers (cox1, cob and ITS2) revealed a sister relationships

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between T. colobae and T. ursinus n. sp. appearing as a different genetic lineage related

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to T. suis and separated from the rest of Trichuris sp. from other primates species.

Acknowledgements

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Thanks to Prof Mannus J. O’Riain (University of Cape Town) for providing the animals for health study by AH. Thanks extended to Prof Wilmien J. Luus-Powell (Biodiversity

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Research Chair, University of Limpopo) for supporting AH and Mr David Kunutu for

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assisting in parasites survey. The present work was supported by a grant of the V Plan

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Propio de Investigación of the University of Seville, Spain.

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ACCEPTED MANUSCRIPT References Anderson, R.M., May, R.M., Anderson, B., 1992. Infectious diseases of humans: dynamics and control. Vol. 28. Oxford University Press, Oxford. Beer, R. J. 1976. The relationship between Trichuris trichiura (Linnaeus 1758) of man

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Callejón, R., Robles, M.D.R., Panei, C.J., Cutillas, C., 2016. Molecular diversification of Trichuris spp. from Sigmodontinae (Cricetidae) rodents from Argentina based on mitochondrial DNA sequences. Parasitol. Res., 115 (8): 2933-45. Cutillas, C., Callejón, R., De Rojas, M., Tewes, B., Úbeda, J.M., Ariza, C., Guevara, D.C., 2009. Trichuris suis and Trichuris trichiura are different nematode species. Act. Trop. 111 (3), 299-307.

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ACCEPTED MANUSCRIPT Ghai, R.R., Simons, N.D., Chapman, C.A., Omeja, P.A., Davies, T.J., Ting, N., Goldberg, T.L., 2014. Hidden population structure and cross-species transmission of whipworms (Trichuris sp.) in humans and non-human primates in Uganda. PLoS Neg. Trop. Dis. 8(10), e3256. Doi:10.1371/journal.pntd.0003256. Gottstein, B., Pozio, E., Nöckler, K., 2009. Epidemiology, diagnosis, treatment, and

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ACCEPTED MANUSCRIPT Liu, G.H., Gasser, R.B., Su, A., Nejsum, P., Peng, L., Lin, R.Q., Li M.W., Xu, M.J., Zhu, X. Q., 2012. Clear genetic distinctiveness between human-and pig-derived Trichuris based on analyses of mitochondrial datasets. PLoS Neg. Trop. Dis. 6(2), e1539. Liu G.H., Gasser, R.B., Nejsum, P., Wang, Y., Chen, Q., Song, H.Q., Zhu X. Q., 2013.

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ACCEPTED MANUSCRIPT Suriano, D.M., Navone, G.T., 1994. Three new species of the genus Trichuris Roederer, 1761 (Nematoda: Trichuridae) from Cricetidae and Octodontidae rodents in Argentina. Res. Rev. Parasitol. 54(3), 39-46. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, under mixed

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models. Bioinformatics 19, 1572-1574.

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Mitochondrial

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phylogeography of baboons (Papio spp.). Indication for introgressive hybridization?

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ACCEPTED MANUSCRIPT Legends to figures Fig. 1. Drawings of Trichuris ursinus n. sp. A : Female, esophagus-intestine junction and vulva, lateral view. B : Female, detail of vulva, lateral view. C : Male, posterior end, spiny spicular sheath, spicule, spicule tube and proximal and distal cloacal tube, lateral view. D: Male, detail of the posterior extremity, lateral view. E: Female,

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posterior end, lateral view. F: Egg.

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Fig. 2. A-C: Females of T. ursinus n. sp. A: Vagina and vulva (arrowed). B: Vulva with

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high magnification (arrowed). C: Scanning electron micrographs of vagina everted. DE: T. suis. D: Vagina and vulva (arrowed). E: Vulva. F: Scanning electron micrograph

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of vagina everted. G-I: Trichuris colobae. G: Vagina and vulva everted (arrowed). H:

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Crater-like vulva. I: Scanning electronic micrographs of vulvar area and everted vagina showing typical triangular sharply spines

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Fig. 3. A-C: Males of T. ursinus n. sp. A: Caudal end showing typical pericloacal

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papillae. B: Spicule sheath with spines. C: Scanning electron micrographs of papillae. D-E: T. suis. D: Caudal end showing typical pericloacal papillae (arrowed). E: Spicule

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sheath with spines (arrowed). F: Scanning electron micrographs of papillae. G-I: T. trichiura. G: Caudal end showing typical pericloacal papillae. H: Spicule sheath with

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spines. I: Scanning electron micrographs of papillae. J-L: T. colobae. J: Pericloacal papillae and cluster of papillae at the caudal end (arrowed). K: Spicule sheath with spines and cluster of papillae. L: Scanning electron micrographs of papillae. Fig. 4. Phylogenetic tree of Trichuris species based on combined analysis of mitochondrial DNA (cytochrome c oxidase 1 and cytochrome b and nuclear ribosomal DNA (Internal Transcribed Spacer 2) inferred using Bayesian method. Bayesian Posterior Probabilities of clades are listed first, followed by Maximum Parsimony and

28

ACCEPTED MANUSCRIPT Maximum Likelihood bootstrap values, respectively, for clade frequencies exceeding 65 %. Fig. S1. Phylogenetic tree of Trichuris species based on cytochrome c oxidase 1 mitochondrial DNA sequences inferred using Bayesian method. Bayesian Posterior Probabilities of clades are listed first, followed by Maximum Parsimony and Maximum

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Likelihood bootstrap values, respectively, for clade frequencies exceeding 65 %.

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gelada and Papio Anubis; MF: Macaca fascicularis.

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Species codes: HS-PH: Homo sapiens and Papio hamadryas; TG-PA: Theropithecus

Fig. S2. Phylogenetic tree of Trichuris species based on cytochrome b mitochondrial

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DNA sequences inferred using Bayesian method. Bayesian Posterior Probabilities of

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clades are listed first, followed by Maximum Parsimony and Maximum Likelihood bootstrap values, respectively, for clade frequencies exceeding 65 %. Species codes:

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HS-PH: Homo sapiens and Papio hamadryas.

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Fig. S3. Phylogenetic tree of Trichuris species based on combined analysis of mitochondrial DNA (cytochrome c oxidase 1 and cytochrome b) inferred using

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Bayesian method. Bayesian Posterior Probabilities of clades are listed first, followed by Maximum Parsimony and Maximum Likelihood bootstrap values, respectively, for

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clade frequencies exceeding 65 %. Species codes: TG-PA: Theropithecus gelada and Papio anubis.

29

ACCEPTED MANUSCRIPT Table 1. Key studies carried out to discriminate Trichuris species from human and non-human primates Trichuris sequences reported Trichuris sp. (T. colobae since Cutillas et al., 2014) Trichuris sp.

Host species

Nomascus gabriellae Papio ursinus

Homo sapiens

Trichuris sp.

Trachypithecus francoisi

Trichuris trichiura Trichuris sp.

Mitochondrial DNA

Author

Geographical Origin

Colobus guereza

Trichuris trichiura

Trichuris sp.

Nuclear rDNA

ITS15.8SITS2

Cutillas et al., 2009

ITS15.8SITS2 ITS2 βtubuline

Ravasi et al., 2012

ITS1, ITS2

Nissen et al., 2012

Colobus guereza

Homo sapiens Colobus guereza Cercopithecus ascanius Cercopithecus lhoesti Cercopithecus mitis Lophocebus albigena Pan troglodytes Papio anubis Procolobus rufomitratus

18S

Mitochondrial genome

Liu et al., 2013

D E

Cox 1

T P E

Fuengirola Zoo, Spain

Callejón et al., 2013

N A

Fuengirola Zoo, Spain

C C

A

ITS1, ITS2

Ghai et al., 2014

I R

Two dintinct Trichuris genotypes infect both humans and nonhumans primates. “Heterozygotes” confirming the identification of two distinct Trichuris genotypes in primates “Heterozygote” worms isolated from human. Trichuris trichiura might consist of several subspecies, some being found mainly in non-human primates The molecular data indicate that the monkey-derived whipworm is a separate species from that of humans. There is a possibility that Trichuris sp. from Trachypithecus francoisi is transmissible to other primates, including humans Phylogenetic analysis revealed a relationship between Trichuris sp. from Colobus guereza and Trichuris suis, providing a strong support for the monophyly of the sampled primate and suid Trichuris

C S U

Zoo China

M

T P

Trichuris sp. from primates and Trichuris suis are different species

Cape Peninsula National Park, South Africa Uganda

Molecular findings

Kibale National Park, Uganda

Trichuris taxon should be considered a multi-host pathogen that is capable of infecting wild primates and humans. Trichuris is among the 20 % of helminths capable of cross-infecting primates and humans

30

ACCEPTED MANUSCRIPT

Trichuris colobae n. sp.

Colobus guereza

Trichuris colobae

Colobus guereza

Trichuris colobae

Colobus guereza

Trichuris sp.

Chlorocebus aethiops

Cutillas et al., 2014

TPI

Cyt b

Most phylogenetic analysis strongly support a sister group relationship between Trichuris suis clade (which includes Trichuris colobae) plus Trichuris trichiura and related with undescribed species from primate

N A

D E

PT

E C

Papio anubis

C A

Papio hamadryas

M

Doležalová et al., 2015

ITS2, 18S

Nomascus gabriellae

Trichuris trichiura

Fuengirola Zoo, Spain

Fuengirola Zoo , Spain Rubondo National Park, Tanzania Rubondo National Park,Tanzania

Macaca silenus

Theropithecus gelada Homo sapiens

Description of a new species “Trichuris colobae n. sp.”

T P

I R

C S U

Chlorocebus sabaeus Macaca fascicularis Macaca mulatta

Pan troglodytes

Callejón et al., 2015

Fuengirola Zoo, Spain

Liberez Zoo, Czech Republic

Primate whipworms form two independent lineages Trichuris trichiura and Trichuris suis clades. The former isolates thus may represent of more species of Trichuris in primates including humans

Bratislava Zoo, Slovak Republic Liberec Zoo, Czech Republic Guinea Bissau Netherlands National Park,Tanzania Brno Zoo, Czech Republic Czech Republic

31

ACCEPTED MANUSCRIPT

Trichuris trichiura

Chlorocebus aethiops

Trichuris sp.

Macaca fuscata

ITS2

Cavallero et al., 2015

Multiple host species (Review)

Trichuris trichiura

Betson et al., 2015

Trichuris sp. Papio hamadryas

Hawash et al., 2015

D E

Chlorocebus sabaeus

T P E

Papio anubis

Trichuris sp.

Papio anubis/ Papio cynocephalus Papio anubis/Papio hamadryas

C C

Nad1 rrnL

In captivity Texas (USA) Copenhagen Zoo/Denmark) Saint Kitts

M

I R

In captivity Texas (USA) They found evidence for an African origin of Trichuris trichiura which were then transmitted with human ancestors to Asia and futher to South America. A host shift to pigs may have occurred in Asia from where Trichuris suis seems to have been transmitted globally by a combination of natural host dispersal and anthropogenic factors

Hawash et al., 2016

A

Papio hamadryas

This review examines knowledge on the taxonomy, genetics and phylogeography of human Trichuris and its relationship to whipworm parasites in other host species. There seems to be host specific patterns in infection with particular Trichuris species or subspecies. However, there is evidence of zoonotic transmission, especially regarding Trichuris trichiura infections in non-human primates and posibly also for Trichuris suis in pigs and Trichuris vulpis in dogs Pig- and human- derived Trichuris may represent different species with the potential differences in endemicity, which may have important implications for implementing effective control strategies. Trichuris infecting primates represents a complex of cryptic species with some species being able to infect both humans and non-humans primates

C S U

N A

Uganda Mitochondrial genome

T P

Multiple localities

Homo sapiens Papio anubis

Genetic heterogeneity and phylogeny within the genus Trichuris from captive Japanese macaque and grivet provide evidences for the existence of distinct clades following analysis of nuclear ribosomal marker (ITS).The results confirmed the existence of Trichuris spp. other than Trichuris trichiura infecting nonhuman living primates

Bioparco of Rome Zoo, Italy

Copenhagen Zoo, Denmark,Knut henborg Park

32

ACCEPTED MANUSCRIPT Trichuris trichiura

Homo sapiens

Uganda, China, Ecuador

T P

I R

C S U

N A

D E

M

T P E

C C

A

33

ACCEPTED MANUSCRIPT Table 2. Sequences of Trichuris spp. and outgroup species obtained from GenBank and used for phylogenetic analysis.

Species

Host species / Geographical origin

Marker

Trichuris colobae

Colobus guereza kikuyensis / Spain

Cox1

Trichuris sp.

Colobus guereza kikuyensis / Czech Republic Theropithecus gelada / Czech Republic Papio anubis / Czech Republic

Papio hamadryas / Denmark

PT

JF690965 JF690964

SC

Papio hamadryas / Czech Republic

JF690968

RI

Papio anubis / USA

NU

Macaca fascicularis / Czech Republic Homo sapiens / Czech Republic Homo sapiens / China

KT449826

Sus scrofa scrofa / Spain

Sus scrofa domestica / Denmark

HE653127 HE653128 HE653129 HE653125 HE653126 HE653124 KT449822

Sus scrofa domestica / Uganda

KT449823

D

PT E CE AC

Sus scrofa domestica / China

Trichuris trichiura

Trichuris suis

KT449824 KT449825 JF690967

GU385218

Sus scrofa domestica / Spain

Trichuris sp.

JF690963

Homo sapiens / Uganda Trichuris suis

Trichuris colobae

KT449825

JF690962

MA

Trichuris trichiura

Accession number HE653118 HE653117 HE653116

Colobus guereza kikuyensis / Spain

Cob

HQ204208 HQ204210 HQ204209 GU070737 LM994704

Papio anubis / USA

KT449825

Papio hamadryas / Czech Republic

LM994703

Papio hamadryas / Denmark Homo sapiens / China

KT449824 KT449824 GU385218

Homo sapiens / Uganda

KT449826

Sus scrofa domestica / China

GU070737

34

ACCEPTED MANUSCRIPT Sus scrofa domestica / Denmark

KT449822

Sus scrofa domestica / Uganda

KT449823

Sus scrofa domestica / Spain

LM994696

Trichuris arvicolae

Myodes glareolus / Spain

Trichuris discolor

Bos taurus / Spain

Trichuris ovis

Capra hircus / Spain

Trichuris skrjabini

Capra hircus / Spain

Cox1 Cob Cox1 Cob

NC008557 NC008557 AF309492.1 AF309492.1

PT

Mus domesticus / Spain

HE653135 LM994699 AM234616 HE653134 LM994701 FM955267 FR851288 LM994698 FN543185 HE653139 LM994707 FR870272 HE653142 LM994697 AJ238220.1 HE653121 LM994700 AJ489248

RI

Trichuris muris

Cox1 Cob ITS2 Cox1 Cob ITS2 Cox1 Cob ITS2 Cox1 Cob ITS2 Cox1 Cob ITS2 Cox1 Cob ITS2

SC

Canis lupus familiaris / Spain

NU

Trichuris vulpis

OUTGROUP -

MA

Priapulus caudatus

-

AC

CE

PT E

D

Lithobius forficatus

35

ACCEPTED MANUSCRIPT

Table 3. GenBank accession number of cox1 and cob partial sequences of 4 individuals of T. ursinus n. sp. isolated from Papio ursinus from South Africa.

Code sample

Cox1

1 2 3 4 1 2 3 4

G+C % 35.9 35.9 35.9 35.9 28.4 28.4 28.6 28.8

Accession number LT627353 LT627354 LT627355 LT627356 LT627357 LT627358 LT627359 LT627360

AC

CE

PT E

D

MA

NU

SC

RI

Cob

Number of base pairs 342 342 342 342 513 513 513 513

PT

Gene

36

ACCEPTED MANUSCRIPT

Table 4. Biometrical data of males of T. suis isolated from S. s. domestica, T. trichiura isolated from P. troglodytes, T. colobae from C. g. kikuyensis and T. ursinus n. sp. from P. ursinus. † Significant differences between T. suis (Cutillas et al., 2009), T. trichiura (Cutillas et al., 2009) and T. colobae (Cutillas et al., 2014) compared with T. ursinus n. sp. (P < 0.001). M1= total body length of adult worm; M2 = length of oesophageal region of body; M3 = width of oesophageal region of body; M4 =maximum width of posterior region of body

T P

(thickness); M5= body width in the place of junction of oesophagus and the intestine; M6= distance from the head end to beginning of bacillary stripes; M7= length of bacillary

I R

stripes; M8= length of spicule; M9=maximum length of spicule sheath; M10 = width of proximal end of spicule; M11 =width of spicule sheath at the tail end of body; M12 =maximum width of spicule sheath; M13 distance between posterior part of testis and tail end of body. M14 = length of proximal cloacal tube; M15: length of distal cloacal tube.

C S U

Б= standard deviation. X=arithmetic mean. Data of M1 and M2 in cm. rest in mm.

T. suis from S. s. domestica (Cutillas et al., 2009)

(Cutillas et al., 2009)

Min-Max

X

б

Min-Max

M1

3.50-5.00

4.04

0.42

3.20-3.60

M2

2.30-3.30

2.61

0.31

M3

0.16-0.23

0.20

0.02

M4

0.76-0.94

0.87†

0.06

M5

0.28-0.40

0.35

0.04

M6

0.50-0.88

M7

1.00-1.76

A

M8

T. ursinus n. sp. from

C. g. kikuyensis

P. ursinus

(Cutillas et al., 2014)

(Present authors)

M

3.35†

Min-Max

X

б

Min-Max

X

б

0.19

2.70-4.10

3.45

0.34

3.20-4.10

3.65

0.33

2.05†

0.21

2.10-3.50

2.64

0.35

2.10-2.70

2.43

0.19

0.09-0.31

0.15

0.02

0.07-0.10

0.08†

0.01

0.15-0.19

0.17

0.01

0.39-0.60

0.50†

0.08

0.23-0.52

0.40†

0.01

0.53-0.75

0.62

0.07

0.15-0.24

0.2†

0.04

0.13-0.26

0.20†

0.04

0.32-0.46

0.38

0.03

D E

T P E

C C

X

б

T. colobae from

N A

T. trichiura from P. troglodytes

1.80-2.30

0.66

0.12

-

-

-

0.601.80

0.99

0.4

0.30-0.64

0.43

0.09

1.43

0.27

-

-

-

2.33-3.95

3.36†

0.43

1.14-2.29

1.67

0.32

2.05-2.57

2.35†

0.15

1.61-1.61

1.90†

0.23

1.48-2.07

1.64†

0.17

1.88-2.29

2.10

0.11

M9

0.16-0.76

0.34

0.20

0.220.22

0.22†

-

0.26-0.65

0.49

0.09

0.23-0.69

0.54

0.14

M10

0.04-0.10

0.05

0.02

0.01-0.06

0.02†

0.02

0.02-0.05

0.04†

0.01

0.06-0.12

0.08

0.02

37

ACCEPTED MANUSCRIPT

M11

0.06-0.08

0.07

0.01

0.05-0.09

0.07

0.02

0.03-0.06

0.04

0.01

0.05-0.07

0.059

0.01

M12

0.06-0.23

0.13

0.06

0.09-0.11

0.09†

0.01

0.040.12

0.05

0.02

0.06-0.09

0.07

0.01

3.42-4.92

4.31†

0.56

1.79-1.95

1.88†

0.07

2.55-3.06

2.88

0.07

2.77-3.79

3.18

0.27

1.23-1.47

1.34

0.1

1.61-1.89

1.75

0.09

M13

T P

Present authors M14

1.33-2,05

1.67

0.19

1.07

-

-

1.35-1.41

1.38

M15

1.44-1.95

1.68

0.17

1.10

-

-

1.32-1.51

1.4

C S U

I R 0.03 0.1

N A

D E

M

T P E

C C

A

38

ACCEPTED MANUSCRIPT

Table 5. Biometrical data of females of T. suis isolated from S. s. domestica, T. trichiura isolated from (P. troglodytes), T. colobae from C. g. kikuyensis and T. ursinus n. sp. from P. ursinus. † Significant differences between T. suis (Cutillas et al., 2009), T. trichiura (Cutillas et al., 2009) and T. colobae (Cutillas et al., 2014) compared with T. ursinus n. sp. (P < 0.001). F1 = Total body length of adult worm; F2 = Length of oesophageal region of body; F3 = Width of esophageal region of body; F4 = Maximum width of posterior region of body

T P

(thickness); F5 = Body width in the place of junction of oesophagus and the intestine; F6 = Distance from the head end to beginning of bacillary stripes; F7 = Length of bacillary

I R

stripes; F8 = Length of vagina; F 9 = Diameter of vulva turned over the surface of body; F 10 = Distance of vulva from place of junction of oesophagus and the intestine; F 11 = Distance of posterior loop of uterus from tail end of body; F 12 = Distance of tail end of body and posterior fold of seminal receptacle; F 13 = Length of muscular zone of the

C S U

oesophagus. Б = Standard desviation. X = Arithmetic mean.

T. suis from S. s.

T. trichiura from

domestica

P.troglodytes

(Cutillas et al., 2009)

(Cutillas et al., 2009)

D E

N A

T. colobae from C. g kikuyensis

T. ursinus n. sp. from P. ursinus

M б

Min-Max

X

б

Min-Max

X

б

(Cutillas et al., 2014)

(Present authors)

Min-Max

X

б

Min-Max

X

F1

4.10-5.00

4.43

0.28

2.00-4.20

3.34

0.78

4.10-5.20

4.60

0.35

3.00-4.90

3.80

0.55

F2

3.00-3.50

3.12

0.16

1.30-3.30

2.53

0.68

3.00-3.80

3.38

0.30

2.00-3.70

2.60

0.50

F3

0.18-0.21

0.19

0.01

0.09-0.19

0.14†

0.04

0.08-0.15

0.11†

0.02

0.15-0.19

0.17

0.02

F4

0.84-1.05

0.93†

0.10

0.38-0.64

0.45†

0.08

0.46-0.77

0.60

0.09

0.54-0.89

0.68

0.08

F5

0.30-0.40

0.36

0.03

0.13-0.23

0.17†

0.03

0.16-0.31

0.23†

0.04

0.35-0.45

0.40

0.02

F6

0.44-0.71

0.58

0.09

0.48-0.64

0.56

0.11

0.50-1.03

0.79

0.04

0.42-0.62

0.52

0.07

F7

1.20-1.66

1.46

0.16

0.36-0.63

0.50†

0.19

2.90-4.92

3.45†

0.70

1.29-2.00

1.70

0.23

F8

0.96-1.46

1.26†

0.17

0.68-1.29

0.96†

0.20

0.95-1.65

1.29†

0.28

1.88-2.81

2.26

0.33

F9

0:04-0.09

0.07†

0.02

0.03-0.05

0.04

0.008

0.04-0.08

0.06

0.02

0.06-0.12

0.08

0.02

E C

PT

C A

39

ACCEPTED MANUSCRIPT

F10

0.09-0.29

0.23

0.08

0.11-0.24

0.15

0.05

0.21-0.39

0.29

0.05

0.03-0.38

0.18

0.10

F11

0.11-0.77

0.41

0.23

0.13-0.24

0.21†

0.04

0.20-0.45

0.34

0.11

0.1-0.75

0.47

0.22

F12

0.42-0.91

0.61

0.19

0.32-0.53

0.44†

0.11

0.93-1.90

1.49

0.29

0.50-1.71

1.04

0.35

F13

1.04-1.31

1.19

0.12

0.66-0.67

0.67†

0.07

0.50-1.20

0.84

0.23

0.77-1.32

T P

0.99

0.20

I R

C S U

N A

D E

M

T P E

C C

A

40

ACCEPTED MANUSCRIPT Table 6. Monophyly of different markers (cox1, cob and ITS2) selected group based on different combinations of datasets and inference methods. BPP: Bayesian Posterior Probability; MP: Maximum Parsimony bootstrap; ML: Maximum Likelihood bootstrap; HS-PH: Homo sapiens-Papio hamadryas; TG-PA: Theropithecus gelada-Papio anubis.

Cox1

Cob

Mitochondrial markers (cox1 + cob)

Mitochondrial and nuclear markers (cox1+cob+ITS2) 100/100/100 -

Trichuris sp. TG-PA

100/100/97

-

-

-

T. suis grouped with T. ursinus n. sp.

97/-/100

-

-

-

T. colobae grouped with T. ursinus n. sp.

-

-

76/-/-

100/83/94

T. suis sister to T. colobae sister to T. ursinus n. sp.

100/82/93

93/85/100

-/-/78

100/100/100

T. suis sister to T. colobae sister to T. ursinus n. sp. sister to Trichuris sp. HS-PH

97/-/67

100/-/100

100/-/100

-

AC

CE

PT E

D

SC

100/99/100 98/85/100 67/100/-

RI

100/100/100 84/100/100 83/100/-

NU

100/97/98 100/100/98 100/100/100 83/70/78

MA

T. suis T. colobae T. ursinus n. sp. Trichuris sp. HS-PH

PT

BPP/MP/ML

41

ACCEPTED MANUSCRIPT Figure 1

B

A

PT

100 µm

NU

SC

RI

50 µm

D

D

MA

C

100 µm

AC

CE

PT E

50 µm

E

F 25 µm

50 µm 42

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 2

43

CE AC

Figure 3

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

44

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Figure 4

45

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

46

ACCEPTED MANUSCRIPT Highlights  

Biometrical and molecular data suggest a new species of Trichuris parasitizing Papio ursinus A description of a new species from Papio ursinus is carried out.



Biometrical values of Trichuris ursinus n.sp. are close to Trichuris suis



Molecular analysis revealed a sister relationships of Trichuris colobae and

AC

CE

PT E

D

MA

NU

SC

RI

PT

Trichuris ursinus n. sp.

47