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Complete Mitochondrial Genome of Trichuris trichiura from Macaca sylvanus and Papio papio

Trichuriasis is among the most prevalent worldwide parasitism caused by helminths. For many years, Trichuris spp. have been described with a relatively narrow range of both morphological and biometrical features. The use of the complete mitochondrial genome (mitogenome) is an alternative and powerful molecular method for inferring phylogenies. Here, we present an overview of the contributions of mitogenome for Trichuris spp. from human and non-human primates. In addition, we carry out structural and phylogenetic comparative analyses with genomes of Trichuris species available in public datasets. The complete mt genomes of Trichuris trichiura and Trichuris sp. from Macaca sylvanus and T. trichiura from Papio papio are 14,091 bp, 14,047 bp and 14,089 bp in length, respectively. The three mt genomes are circular and consist of 37 genes—13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS). The molecular evidence presented here supports the hypothesis that T. trichiura de M. sylvanus (TMF31) and T. trichiura de P. papio (TPM1) were similar but genetically different with respect to Trichuris sp. from macaques (TMM5). The phylogenetic study also supported the evolution of the different Trichuris species. In conclusion, we suggest the existence of two cryptic species parasitizing M. sylvanus.


Introduction
Soil-transmitted helminths (STHs) (Ascaris lumbricoides (Ascariasis), Trichuris trichiura (Trichuriasis)) and hookworms (Ancylostomiasis/Necatoriasis) are widely distributed in tropical and subtropical areas, with the greatest numbers occurring in sub-Saharan Africa, the Americas, China, and East Asia [1]. In areas with favorable climatic and environmental conditions, poor access to potable water supply, sanitation, and hygiene resources, the transmission of these parasites is higher [2]. Recent data show that about 1.5 billion people are estimated to be affected by STHs worldwide [1].
Although Trichuriasis is a seriously neglected disease, fortunately, in recent years hereditary studies are increasing as well as related methodologies that provide new opportunities for the discovery of novel intervention strategies, with major implications for improving animal and human health and welfare globally [3]. In addition, the implications of genomic studies could also be very relevant in relation to the search for new treatments for immunopathological diseases in humans [4][5][6][7][8][9]. For example, it has been reported that infections of human patients suffering from immunological disorders (such as Crohn's disease) suppress clinical symptoms significantly with pig-Trichuris eggs [6,[10][11][12].
The nematode mitogenome (complete mitochondrial genome) has several practical strengths as a phylogenetic marker, and has yielded well-supported results for clades, which were not well resolved using other approaches [13]. The whole genome and transcriptome studies have contributed to our understanding of deep node nematode phylogeny but have been limited by low taxon sampling (which is also biased toward some Life 2021, 11, 126 2 of 15 parasitic groups) and by the technical challenges inherent in obtaining a large quantity of genetic information from a single nematode with a small body size. In such cases, the use of mitogenome sequences is one alternative that has been widely applied to many nematode branches where relationships were unclear [13].
Kern et al. [13] concluded that the mitochondrial genome is a useful tool for nematode phylogenetic because the diversity within nematode mitogenome architecture, its variable rate of gene rearrangement, and the representation of nearly every kind of lifestyle and habitat ecology within nematodes make this phylum an exciting area for addressing questions about mitogenome evolution.
Currently, the complete mt genome of several species of whipworms has been sequenced-Trichuris trichiura from humans and baboons [3,19]; Trichuris sp. from the Endangered François' Leaf-Monkey [17]; Trichuris rhinopiptheroxella from the endangered golden snub-nosed monkey [20]; Trichuris suis from pig [3,19]; Trichuris ovis from antelope [16]; Trichuris discolor from wild yak [16]; Trichuris skrjabini from sheep [22]; and Trichuris muris (LC050561, unpublished). This fact, and considering the hypothesis that Trichuris infecting primates represent a complex of cryptic species with some species capable of infecting both humans and non-human primates (NHPs), reflects the need to delve into the mt genomes of Trichuris spp. of NHPs compared to T. trichiura from humans.
Thus, the aim of the present study was to sequence the complete mt genome of Trichuris trichiura isolated from different NHPs. In addition, we carry out structural and phylogenetic comparative analyses with genomes of species of genus Trichuris available in public datasets.

Ethics Statement
This study does not require approval by an ethics committee. Macaca sylvanus, from which Trichuris specimens were collected from their caeca post-mortem. Trichuris specimens, recovered from the feces of one Papio papio after anthelmintic treatment, were handled and housed in a zoo in strict accordance with good animal practices.

Parasites, DNA Extraction and Genotyping of Worms
Specimen Trichuris worms were recollected from a Guinea baboon (P. papio) at Parque de la Naturaleza de Cabárceno (Cantabria, Spain), from a stool sample after anthelmintic treatment, and a Barbary macaque (M. sylvanus) in Castellar Zoo (Cádiz, Spain), from its caeca post-mortem. Adult Trichuris were recovered and washed in physiological saline, identified morphologically, and then, stored at −20 • C until use. Total genomic DNA was isolated from three individual worms according to the manufacturer's protocol the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Düsseldorf, Germany),used to extract the genomic DNA. Quality of extractions was assessed using 0.8% agarose gel electrophoresis infused with SYBR ® Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA)

Mitochondrial Genome Amplification and Sequencing
For long-range PCR amplification and next generation sequencing (NGS), different primate derived Trichuris specimens were chosen. One Guinea baboon worm (TPM1) and two Barbary macaque worms (TMF31 and TMM5) were chosen based on their distinct haplotypes identified in previous studies by sequencing the partial gene of mtDNA (cox1, cob and rrnL) and rDNA (ITS1 and ITS2) [23]. To obtain the complete mt genome, firstly, we used the primers designed by Hawash et al. [19] to amplify the Trichuris sp. from baboon (TTB1) and T. trichiura from humans from Uganda (TTHUG) genomes in two overlapping fragments (~8 and~6 kbp), Nevertheless, we only could amplify the second fragment (~6 kbp), from rrnL to nad1. Then, new primers (MS1F and MS1R) were designed to amplify the other fragment using Primer3 from nad1 to rrnL (http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 1 February 2021)). PCR mix, PCR conditions and PCR primers are summarized in the Supplementary materials (Table S1). The PCR products were checked on SYBR ® Safe stained 0.8% Tris-Borate-EDTA (TBE) agarose gels and bands were eluted and purified from the agarose gel using the QWizard SV Gel and PCR Clean Up System Kit (Promega, Madison, WI, USA). Once purified, the samples were concentrated and measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Stab Vida (Lisbon, Portugal) sequenced the mt genomes. The PCR products were used for library construction using Illumina Nextera XT library preparation kit (San Diego, CA, USA) and the generated DNA fragments were sequenced in the Illumina MiSeq platform (San Diego, CA, USA), using 300 bp paired-end sequencing reads.

Assembly, Annotation, and Genome Sequence Analyses
Sequences were assembled and analyzed using MacVector package v17.5.4 (Oxford Molecular Group, Waterbeach, Cambridge, UK). The identity of the sequences was made using BLAST by comparison with other sequences available in GenBank database. Genome annotation was performed using the pipeline MITOS web server (http://mitos.bioinf.unileipzig.de/index.py (accessed on 1 February 2021)) [24] using the mitochondrial invertebrate genetic code, and MacVector and protein-encoding genes of T. trichiura. The majority of the tRNA genes were identified using the ARWEN tool (http://130.235.244.92/ARWEN/ (accessed on 1 February 2021)) to get rRNA [25] and tRNAScan-SE web server (http: //trna.ucsc.edu/tRNAscan-SE/ (accessed on 1 February 2021)) [26], but the remaining tRNA genes were recognized manually by comparison.
The genomes were compared with T. trichiura and Trichuris sp. Sequences-T. trichiura from humans (unknown geographical location) (AP017704), T. trichiura from humans in China (NC_017750), T. trichiura from humans in China (GU385218), T. trichiura from humans in Uganda (KT449826), T. trichiura from P. anubis in USA (KT449825), T. trichiura from P. hamadryas in Denmark (KT449824) and Trichuris sp. from T. francoisi in China (KC461179). All protein-coding genes (PCGs) and ribosomal RNA genes (rRNAs) sequences were individually extracted, and a nucleotide data set was generated by concatenated sequences (all PCGs and rRNAs genes). This data set was used to estimate genetic distances using MEGA X v.10.1.8 (Penn State, PA, USA) [27]. The genomes of Trichuris from humans and NHP were used to calculate the < Nucleotide diversity (π) using a sliding window of 100 bp with 25 bp steps implemented in DnaSP v.6 [28].

Phylogenetic Analysis
For the phylogenetic analyses, two different data sets were generated. The first included nucleotides sequences, with the 13 PCGs (cox1-3, nad1-6, nad4L, atp6, atp8 and cob) and the two rRNAs (rrnL and rrnS). The data set was aligned using MEGA X v.10.1.8 [27] and concatenated for Trichuris sp. and T. trichiura species from baboons and humans. We used as an outgroup Trichinella pseudospiralis (Table 1). Another data set was generated using amino acid sequences inferred from the 12 PCGs. The sequences were aligned using MEGA X v.10.1.8 [27] and then concatenated excluding atp8 (because it is not present in the mitochondrial genome of all the species of nematodes except in Trichinella and Trichuris species). The data set, using the sequences previously used and all the sequences available of the complete mt genome of Trichuris spp. and with those of 9 other enoplid nematodes, using Brugia malayi and Ascaris suum as the outgroups, was generated ( Table 2). The ambiguous regions of the alignment were excluded using Gblocks Server v.0.91b (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks (accessed on 1 February 2021)) with the default settings being used to select the option of less strict conservation of flanking positions [29,30].  For phylogenetic re-constructions we used three methods-Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inferences (BI). The ML tree was generated using PHYML package [31,32], and for the MP tree we used MEGA X v.10.1.8 [27] and for BI we used MrBayes v.3.2.6. [33]. To resolve the best-fit substitution model for the nucleotide data set we employed jModelTest [34] and ProtTest 3.4 for the amino acid data set. Models of evolution were defined according to the Akaike Information Criterion [35,36]. For the nucleotide data set, GTR + I + G model, with rate variation along the length of the alignment (+ G) and allowing for a proportion of invariant sites (+ I) was selected, and for the amino acid data set, the MtArt + I + G + F model, with residue frequencies estimated from the data (+ F) was chosen. Support for the topology was examined using bootstrapping (heuristic option) [37] over 1000 replications to assess the relative reliability of clades. The commands used in MrBayes for BI were nst = mixed. The standard deviation of split frequencies was used to determine whether the number of generations completed was sufficient; the chain was sampled every 500 generations and each dataset was run for 10 million generations. Trees from the first million generations were discarded based on an assessment of convergence. Burn-in was determined empirically by examination of the log likelihood values of the chains. The Bayesian posterior probabilities (BPPs) comprise the percentage converted.
The mt genomes of Trichuris sp. contain an AT-rich region consisting of two noncoding regions (NCRs), including a long non-coding region (NCR-L) and short non-coding region (NCR-S). The nucleotide composition (%) is summarized in Table S3. The content of A + T is 69.4%, 68% and 69.3% for TMH31, TMM5 and TPM1, respectively.
Between TMF31 and TPM1 genomes there were slight differences. The sequences were identical in terms of all initiation and termination codons and gene lengths, except for tRNA-ser (S2), which presented one nucleotide more in TPM1. In addition, between TMF31 and TMM5, 22 genes of 37 were different in length. Genes were represented to standard nomenclature, but tRNAs were represented using one-letter amino acid codes, with numbers differentiating each of the two leucine-and serinespecifying tRNAs. NCR-L refers to large non-coding region and NCR-S to small non-coding region. . Mitochondrial genome structure of Trichuris trichiura from Barbary macaque (TMF31) and from Guinea baboon (TPM1), and Trichuris sp. from Barbary macaque (TMM5). Genes were represented to standard nomenclature, but tRNAs were represented using one-letter amino acid codes, with numbers differentiating each of the two leucine-and serinespecifying tRNAs. NCR-L refers to large non-coding region and NCR-S to small non-coding region.
The start/stop codons for some PCGs differed between TMM5 and the genomes previously cited (TMF31 and TPM1), which were similar. Codon usage analyses of mt genomes showed that three start and seven termination codons were different (Table 3). For instance, the starting codon for TMF31 and TPM1 is ATA for the nad1 gene, while it reads ATG in the TMM5 genome; for the nad4 gene, the starting codon is ATG in TMF31 and TPM1, while being ATA in TMM5, and in the atp8 gene, ATT is the start codon in TMF31 and TPM1, while it reads ATA in TMM5. In the cox1 gene, TAA is the termination codon in TMF31 and TPM1, whereas it is TAG in TMM5; in cox2 gene, TAG is the stop codon in TMF31 and TPM1, while being TAA in TMM5; in nad1, TAG is the stop codon in TMF31 and TPM1, but is TAA in TMM5; in nad2 gene, TAA is the stop codon in TMF31 and TPM1, and is TAG in TMM5; the stop codon in the nad5 gene is TAG for TMF31 and TPM1 and TAA for TMM5; in the nad4L and nad6 genes, TAA is the stop codon in TMF31 and TPM1 while it reads TAG in TMM5, and in cox3 gene TAA is the stop codon for TMF31 and Life 2021, 11, 126 7 of 15 TPM1 and TAG for TMM5. There are overlaps between rrnL and atp6 in TMF31, between rrnL, atp6 and cox3 in TMM5, and between rrnL and atp6 in TPM1.

Comparative Sequence Analyses
Genetic distances between worms for individual PCGs and rRNAs genes are found in Table S2. The genetic distances between the mt genomes of Trichuris spp. in primates and humans are given in Table 4. The gene with highest genetic variation was the atp8 gene, and the most conserved was the rrnS gene, however between all PCGs, cox1 was the most conserved gene. Among the mt genomes obtained in this study, nucleotide and amino acid differences between TMF31 and TPM1 were 0.25% and 0.41%, and between TMF31 and TMM5 they were 18.7% and 14.5%, respectively. Within all sequences of Trichuris spp. studied, the mt genome of T. francoisi was the most variable (with a nucleotide difference of 27.1-28.6% and an amino acid difference of 26.8-28.2%) relative to the other worms from baboons and humans. Among the Trichuris genome dataset, the nucleotide diversity was analyzed using the sliding window approach for the 13 PCGs and the two rRNA genes. The number of polymorphic sites was 5108 and the nucleotide diversity was 0.166 ( Figure 2). The genes with lowest nucleotide diversity were rRNAs (rrnL and rrnS) and cox1. most conserved gene. Among the mt genomes obtained in this study, nucleotide and amino acid differences between TMF31 and TPM1 were 0.25% and 0.41%, and between TMF31 and TMM5 they were 18.7% and 14.5%, respectively. Within all sequences of Trichuris spp. studied, the mt genome of T. francoisi was the most variable (with a nucleotide difference of 27.1-28.6% and an amino acid difference of 26.8-28.2%) relative to the other worms from baboons and humans.
Among the Trichuris genome dataset, the nucleotide diversity was analyzed using the sliding window approach for the 13 PCGs and the two rRNA genes. The number of polymorphic sites was 5108 and the nucleotide diversity was 0.166 ( Figure 2). The genes with lowest nucleotide diversity were rRNAs (rrnL and rrnS) and cox1.

Phylogenetic Analyses
The phylogenetics analyses of nucleotide sequence datasets with partial genome (the 13 PCGs (cox1-3, nad1-6, nad4L, atp6, atp8 and cytb) and the two rRNAs (rrnL and rrnS)) and with mt genome complete datasets reflecting similar tree topologies by the three methods studied (ML, MP and BI) (Figure 3). Within Trichuris populations from humans and NHPs, Trichuris sp. from T. francoisi appeared separated of the other sequences. Within this last group, there are three main clades. The first clade (clade 1) is composed of sequences of Trichuris sp. from H. sapiens and P. anubis from Asia, Japan and USA, the clade 2 is composed of a sequence of Trichuris sp. from M. sylvanus, and the clade 3 corresponded with T. trichiura from humans and NHPs (M. sylvanus, P. papio and P. hamadryas) from Europe and Africa.
Phylogenetic trees inferred from a concatenated amino acid sequence dataset of 12 PCGs among selected enoplid nematodes, using the chromadorean nematode, B. malayi (NC_004298) and A. suum (HQ704901) as outgroups, revealed congruence with phylogenetics analyses of nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS), and complete mt genomes. Thus, phylogenetics analyses of all 12 genes showed a general pattern with very strong support for clades near the terminals of the tree (Figure 4). Thus, clade 1, clade 2 and clade 3 for Trichuris spp. from humans and NHPs, and the monophyly of the genus Trichuris, are very strongly supported (100% ML BV, 100% MP BV and 100% BPP). There are no marked differences in support values between the 12 gene datasets analyzed using nucleotide versus amino acid sequences. and with mt genome complete datasets reflecting similar tree topologies by the three methods studied (ML, MP and BI) (Figure 3). Within Trichuris populations from humans and NHPs, Trichuris sp. from T. francoisi appeared separated of the other sequences. Within this last group, there are three main clades. The first clade (clade 1) is composed of sequences of Trichuris sp. from H. sapiens and P. anubis from Asia, Japan and USA, the clade 2 is composed of a sequence of Trichuris sp. from M. sylvanus, and the clade 3 corresponded with T. trichiura from humans and NHPs (M. sylvanus, P. papio and P. hamadryas) from Europe and Africa. Phylogenetic trees inferred from a concatenated amino acid sequence dataset of 12 PCGs among selected enoplid nematodes, using the chromadorean nematode, B. malayi (NC_004298) and A. suum (HQ704901) as outgroups, revealed congruence with phylogenetics analyses of nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS), and complete mt genomes. Thus, phylogenetics analyses of all 12 genes showed a general pattern with very strong support for clades near the terminals of the tree ( Figure  4). Thus, clade 1, clade 2 and clade 3 for Trichuris spp. from humans and NHPs, and the monophyly of the genus Trichuris, are very strongly supported (100% ML BV, 100% MP BV and 100% BPP). There are no marked differences in support values between the 12 gene datasets analyzed using nucleotide versus amino acid sequences. The phylogenetic analyses reflected the clear distinctiveness between T. trichiura and Trichuris sp. from humans and NHPs with respect to T. suis from suids, T. ovis and T. discolor from herbivorous and T. muris from rodents and grouping these members of Trichuris with T. pseudospiralis (order Trichinellida) with absolute support (100% ML BV, MP BV 100% and 100% BPP), but excluding the members of Dorylaimida and Mermithida (Figure 4).
In the present study, we resolved the complete mt genome sequences of two haplo- The phylogenetic analyses reflected the clear distinctiveness between T. trichiura and Trichuris sp. from humans and NHPs with respect to T. suis from suids, T. ovis and T. discolor from herbivorous and T. muris from rodents and grouping these members of Trichuris with T. pseudospiralis (order Trichinellida) with absolute support (100% ML BV, MP BV 100% and 100% BPP), but excluding the members of Dorylaimida and Mermithida (Figure 4).
In the present study, we resolved the complete mt genome sequences of two haplotypes of Trichuris isolated from M. sylvanus and one complete mt genome of Trichuris from P. papio. The size of the complete mt genomes were within the range reported for the Trichuridae family (ranging from 13,904 bp (T. discolor) to 14,521 bp (T. suis)) [3,16,17,19,22]. The complete mtDNA is a circular molecule and encodes 37 genes-13 PCGs (atp6, atp8, cox1-3, cob, nad1-6 and nad4L), two rRNAs (rrnS and rrnL), and 22 for tRNAs.
The complete mt genomes of one of the haplotypes of Trichuris from macaques (TMF31) and that from baboon (TPM1) were genetically similar, with a difference in nucleotide and amino acid sequence of 0.25% and 0.41%, and having 14,091 and 14,098 bp, respectively, and both genomes being similar with that of T. trichiura from humans from Uganda, which was 14,079 bp in length, and with TTB1 (13,984 bp in length) from Trichuris sp. from P. hamadryas [19], with a sequence variation of 0.28-0.47%. In contrast, the complete mt genome of the other haplotype from macaques (TMM5) was 14,047 bp, like human T. trichiura from China that was 14,046 bp in length [3], showing a nucleotide difference of 15.9%. Moreover, the mt genome of Trichuris sp. from P. anubis (USA) was 14,009 bp [19], with a nucleotide difference to TMM5 of 15.8%. A substantial level of nucleotide difference was detected between TMF31 (Clade 3) with respect to Trichuris sp. included in clades 1 and 2 (15.8-18.8%). Comparison of the nucleotide and amino acid sequences between TMF31 and TMM5 was 18.7% and 14.5%, respectively. The sequence variation detected in the 13 PCGs between TMF31 and TMM5 was 20.3% (nucleotide sequence) and 34.4% (amino acid sequence). The percentage of dissimilarity observed between the different sequences corresponded to previously cited ranges within different nematode species. Thus, Hawash et al. [19] suggested the presence of two Trichuris species with a difference in nucleotide and amino acid sequences of around 18.8% and 14.6%, respectively, of Trichuris from Ugandan humans and Chinese humans. Furthermore, T. suis and T. trichiura presented an amino acid difference of 33.3-39.2% [19], T. trichiura from human and Trichuris sp. from leaf monkey was 29.4% [17], T. ovis and T. discolor ranged from 11.0-33.9% [16], 11.7% between Wucheria bancrofti and B. malayi [42], 10.3% between Chabertia ovina and C. erschowi [43], 4-18% between different Trichinella spp. [18] and 4.12% between Ancylostoma duodenale and Ancylostoma caninum [44]. Blouin [45] indicated that the difference in mt genome sequences between closely related species was normally 10-20%. Thus, the molecular evidence presented in the present manuscript supports the hypothesis that the haplotypes TMF31 and TMM5 of Trichuris from M. sylvanus display differences in amino acid and nucleotide sequences that are within the range of those previously reported by different authors to consider them as different species.
On the other hand, the complete mt genomes of Trichuris spp. from different hosts were evaluated to clarify the evolutionary relationships. Considering that complete mt genome sequences are maternally inherited and mutate at a rapid rate related to nuclear genes, theses markers could prove useful for evolutionary and phylogenetic analyses [46].
Phylogenetics trees inferred from both datasets (sequences dataset of 12 PCGs among enoplid nematodes and nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS)) showed congruence with a high support for the differentiation between Trichuris spp. and the different clades within Trichuris populations parasitizing humans and NHPs (clade 1, clade 2 and clade 3). Furthermore, the monophyly of the genus Trichuris was strongly supported and grouping these species of Trichuris (traditionally named as Trichocephalida) with T. pseuospirallis (Trichinellida,) with the exclusion of the members of the Dorylaimida and Mermithida (Figure 4). Similar results were reported by Liu et al. [3,16] who characterized the complete mitochondrial genomes of several whipworms and compared them with other enoplid nematodes.
The atp8 gene is present in mitogenomes of the Order Trichinellida (Trichuris and Trichinella species), but it is usually absent in mitogenomes of members of the phylum Nematoda [3,13,[15][16][17][18][19]. These authors reported the lack of atp8 in the mtDNA of Chromadorean nematodes (traditionally named as Class Secernentea) and it could be derived, within that lineage, after the divergence of Chromadorean nematodes from other groups. Furthermore, phylogenetic inferences confirm the clear relationships between Trichinellida and Mermithida correlating both groups with animal parasites of nematodes separated of Dorylaimida, corresponding to free-living nematodes and plant parasitic nematodes [47].
The phylogenetic study also supported the evolution of the different Trichuris species showing T. trichiura (clade 3), to be closer to Trichuris spp. (clade 1 and clade 2) parasitizing humans and NHPs. As reported by previous authors [19,23,38,39,41,48], Trichuris populations parasitizing humans and NHPs showed a species complex in primates. In addition, the high difference in nucleotide and amino acid sequences between T. trichiura (clade 3) and the minority population of Trichuris sp. from M. sylvanus (clade 2) and Trichuris sp. from humans and NHPs from China, Japan, and USA (clade 1) was confirmed in the phylogenetic trees with strong support for the different clades. Thus, based on our results, we suggest the existence of two cryptic species parasitizing M. sylvanus. Trichuris genus is a likely candidate to containing cryptic species as it has a wide geographical distribution and infects several host species [49]. As revealed by recent studies, there is more than one taxon capable of infecting humans and other primates, including individuals in captivity, suggesting that T. trichiura should be considered a complex species that includes different cryptic units [19]. In addition, and based on morphobiometric and molecular parameters, new species of Trichuris have been described in primates, such as T. rhinopiptheroxella [20], that was found in the golden snub-nosed monkey (Rhinopithecus roxellana), Trichuris colobae from Colobus guereza kikuyensis [50], and Trichuris ursinus from Papio ursinus [51]. As a result, it has been confirmed that T. trichiura and other Trichuris species are present in humans and NHPs.
This fact has two main epidemiological repercussions-(i) the zoonotic potential of Trichuris spp. of NHPs for humans. This is particularly important when humans and NHPs are living in proximity, as is becoming increasingly common with human encroachment into habitats where NHPs accessing gardens and farms in search of food and has significant implications for both human health and wildlife conservation [52]. This implies that in communities where access to NHPs is common, simple public health measures should be encouraged, including thorough handwashing with soap (particularly for children) and rising and cooking of vegetables, and (ii) the presence of different cryptic species might also be very important for implementation of appropriate control strategies. Different control strategies for Opistorchirs viverrini have been identified due to the existence of different cryptic species based on the different fecundity as measured by eggs/g/worm [53].

Conclusions
In the present study, based on complete mt genome analyses, the molecular data suggested two distinct species in whipworms isolated from M. sylvanus. The whipworms Life 2021, 11, 126 13 of 15 from P. papio, TPM1, and TMF31 from M. sylvanus are T. trichiura since the sequences were within the majority clade, with sequences from Ugandan humans and P. hamadryas (Europe)-derived Trichuris. Further, we suggested the existence of two cryptic species parasitizing M. sylvanus. Moreover, a major source of mitochondrial markers is given and may be used as the basis of subsequent epidemiological research, to clarify appropriate control measures and transmission routes.
Supplementary Materials: The following are available online at https://www.mdpi.com/2075-1 729/11/2/126/s1, Table S1: PCR mix, primers and conditions used for each molecular marker sequenced in the present study, Table S2: Pairwise nucleotide and amino acid distances for the different 13 PCGs and RNAs (rrnS and rrnL) genes for Trichuris species from different primates and human hosts. Nucleotide genetics distances are given below the diagonal and amino acid genetic distances above the diagonal, Table S3: Nucleotide composition (%) of the mt genomes studied.
Funding: This research was financially supported by a grant from the Ministry of Economy, Industry and Competitiveness (CGL2017-83057), which included FEDER/Ministry of Science, Innovation and Universities -State Research Agency/ CGL2017-83057, the Junta de Andalucía (BIO-338) and a grant from the V and VI Plan Propio de Investigación of the University of Seville, Spain.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are openly available in GenBank database (GenBank accession nos. MW448470-2).