Complete mitochondrial genome of trichuris trichiura from macaca sylvanus and papio papio

life Article Complete Mitochondrial Genome of Trichuris trichiura from Macaca sylvanus and Papio papio Julia Rivero , Rocío Callejón and Cristina Cutillas *   Citation: Rivero, J.; Callejón, R.; Cutillas, C. Complete Mitochondrial Genome of Trichuris trichiura from Macaca sylvanus and Papio papio.Life 2021,11, 126. https://doi.org/ 10.3390/life11020126 Academic Editor: Angela Anna Messina Received: 9 January 2021 Accepted: 3 February 2021 Published: 6 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Calle San Fernando, 4, 41004 Sevilla, Spain; [email protected] (J.R.); [email protected] (R.C.) *Correspondence: [email protected] Abstract: 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. Keywords: complete mitochondrial genome; Trichuris trichiura; phylogeny; whipworms 1. 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 [4–9]. 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 [6,10–12]. 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. https://doi.org/10.3390/life11020126 https://www.mdpi.com/journal/life 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]. Nematode mitogenomes are like those of other animals in many respects, but have a few unusual features including high variation in conservation of gene order across major branches and the occasional presence of multiple chromosomes [ 13 ]. The nematode mitogenome is usually a single, circular molecule ranging in size from 12 to 22 kb and containing 36 (sometimes 37) genes—12 (or 13) protein coding genes (PCGs) (cox1–cox3, cytb, nad1-nad6, nad4L, atp6, and rarely atp8), two ribosomal RNA (rRNA) (rrnL and rrnS), and 22 transfer RNA genes (tRNAs). The atp8 gene, which is found in most other metazoan mitogenomes (except the parasitic Platyhelminthes clade Neodermata) [ 14 ], is usually absent in nematode mitogenomes, although it does appear in the order Trichinellida (Trichinella spp. and Trichuris spp.) [ 13 , 15 – 20 ]. Furthermore, within class Enoplea, members of order Trichinellida (Trichinella spp., Trichuris spp.) show a substantial gene rearrangement even among closely related species, while members of Chromadorea show far less rearrangement in theirs mitogenomes [21]. 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. 2. Materials and Methods 2.1. 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. 2.2. 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) Life 2021,11, 126 3 of 15 2.3. 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. 2.4. 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]. 2.5. 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). Life 2021,11, 126 4 of 15 Table 1. Sequences analyzed in the first data set for phylogenetic analyses. Species Host Species/Geographical Origin GenBank Accession Number Trichuris trichiura Macaca sylvanus/Spain MW448470 Trichuris sp. Macaca sylvanus/Spain MW448471 Trichuris trichiura Papio papio/Spain MW448472 Trichuris trichiura Homo sapiens/(unknown geographical location) AP017704 Trichuris trichiura Homo sapiens/China NC_017750 Trichuris trichiura Homo sapiens/China GU385218 Trichuris trichiura Homo sapiens/Uganda KT449826 Trichuris sp. Papio anubis/USA KT449825 Trichuris sp. Papio hamadryas/Denmark KT449824 Trichuris sp. Trachypithecus francoisi/China KC461179 1Trichinella pseudospiralis Coragypus atratus/USA KM357411 1Used as an outgroup. 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]. Table 2. Sequences analyzed in the second data set for phylogenetic analyses. Species Host Species/ Geographical Origin Order GenBank Accession Number Trichuris trichiura Macaca sylvanus/Spain Trichinellida MW448470 Trichuris sp. Macaca sylvanus/Spain Trichinellida MW448471 Trichuris trichiura Papio papio/Spain Trichinellida MW448472 Trichuris trichiura Homo sapiens/(unknown geographical location) Trichinellida AP017704 Trichuris trichiura Homo sapiens/China Trichinellida NC_017750 Trichuris trichiura Homo sapiens/China Trichinellida GU385218 Trichuris trichiura Homo sapiens/Uganda Trichinellida KT449826 Trichuris sp. Papio anubis/USA Trichinellida KT449825 Trichuris sp. Papio hamadryas/Denmark Trichinellida KT449824 Trichuris sp. Trachypithecus francoisi/China Trichinellida KC461179 Trichuris rhinopiptheroxella Rhinopithecus roxellana/China Trichinellida MG189593 Trichuris ovis Addax nasomaculatus/China Trichinellida NC_018597 Trichuris discolor Bos grunniens mutus/China Trichinellida NC_018596 Trichuris muris - /United Kingdom Trichinellida LC050561 Trichuris suis Sus scrofa/Uganda Trichinellida KT449823 Trichuris suis Sus scrofa/China Trichinellida GU070737 Trichinella pseudospiralis Coragypus atratus/USA Trichinellida KM357411 Xiphinema americanum Plant ectoparasite Dorylaimida NC_005928 Hexamermis agrotis - Mermithida NC_008828 Agamermis sp. - Mermithida NC_008231 Romanomermis culicivorax - Mermithida NC_008640 Life 2021,11, 126 5 of 15 Table 2. Cont. Species Host Species/Geographical Origin Order GenBank Accession Number Romanomermis iyengari - Mermithida NC_008693 Romanomermis nielseni - Mermithida NC_008692 Strelkovimermis spiculatus - Mermithida NC_008047 Thaumamermis cosgrovei - Mermithida NC_008046 1Brugia malayi - Rhabditida NC_004298 1Ascaris suum - Rhabditida HQ704901 1Used as an outgroup. 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. 3. Results 3.1. Annotation and Features of Mitochondrial Genomes The complete mtDNA sequences of the primate worms TMF31, TMM5 and TPM1 were 14,091, 14,047 and 14,089 bp in length, respectively (GenBank accession nos. MW448470-2) (Figure 1). The mt genomes contained 37 genes—13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS) (Table 3). All genes are transcribed from the heavy strand, except four PCGs (nad2, nad4, nad4L and nad5) and 10 tRNA (tRNA-Met, tRNA-Phe, tRNA-His, tRNA-Arg, tRNA-Pro, tRNA-Trp, tRNA-Ile, tRNA-Gly, tRNA-Cys, and tRNA-Tyr) that are transcribed from the light strand. 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. Life 2021,11, 126 6 of 15 Life 2021, 11, x FOR PEER REVIEW 6 of 16 Figure 1. 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. Table 3. Mitochondrial genomes of Trichuris from Macaca sylvanus (TMF31, TMM5) and Papio papio (TPM1). Protein coding, transfer RNA (tRNA), and ribosomal RNA (rRNA) genes with lengths in nucleotides (nt) are given. The lengths are not identical, and differences are given in parentheses (TMM5/TPM1), likewise for the initiation and termination codons. Genes Positions Lengths Codons Strand TMF31 TMM5 TPM1 nt Initiation Termination cox1 1–1545 1–1545 1–1545 1545 ATG TAA (TAG) + cox2 1558–2232 1556–2230 1558–2232 675 ATG TAG (TAA) + tRNA-leu (L2) 2255–2317 2252–2311 2255–2317 63 (60) + tRNA-glu (E) 2324–2384 2320–2377 2324–2384 61 (58) + nad1 2406–3305 2401–3300 2406–3305 900 ATA (ATG) TAG (TAA) + Non-coding region (NCR-L) 3306–3435 3303–3442 3306–3430 tRNA-lys (K) 3436–3501 3441–3502 3431–3496 66 (62) + Figure 1. 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. Table 3. Mitochondrial genomes of Trichuris from Macaca sylvanus (TMF31, TMM5) and Papio papio (TPM1). Protein coding, transfer RNA (tRNA), and ribosomal RNA (rRNA) genes with lengths in nucleotides (nt) are given. The lengths are not identical, and differences are given in parentheses (TMM5/TPM1), likewise for the initiation and termination codons. Genes Positions Lengths Codons Strand TMF31 TMM5 TPM1 nt Initiation Termination cox1 1–1545 1–1545 1–1545 1545 ATG TAA (TAG) + cox2 1558–2232 1556–2230 1558–2232 675 ATG TAG (TAA) + tRNA-leu (L2) 2255–2317 2252–2311 2255–2317 63 (60) + tRNA-glu (E) 2324–2384 2320–2377 2324–2384 61 (58) + nad1 2406–3305 2401–3300 2406–3305 900 ATA (ATG) TAG (TAA) + Non-coding region (NCR-L) 3306–3435 3303–3442 3306–3430 tRNA-lys (K) 3436–3501 3441–3502 3431–3496 66 (62) + nad2 3499–4395 3505–4401 3494–4390 897 ATA TAA (TAG) − tRNA-met (M) 4396–4456 4402–4462 4391–4451 61 − tRNA-phe (F) 4451–4507 4457–4513 4446–4502 57 − nad5 4499–6055 4520–6067 4494–6050 1557 (1553) ATA TAG (TAA) − tRNA-his (H) 6049–6106 6065–6118 6044–6101 58 (54) − tRNA-arg (R) 6108–6171 6115–6181 6103–6166 64 (67) − nad4 6176–7396 6183–7394 6171–7391 1221 (1212) ATG (ATA) TAA − nad4L 7419–7631 7425–7673 7414–7626 213 (249) ATA TAA (TAG) − tRNA-thr (T) 7672–7729 7679–7736 7667–7724 58 + tRNA-pro (P) 7729–7787 7736–7795 7724–7782 59 (60) − nad6 7780–8256 7788–8264 7775–8251 477 ATT TAA (TAG) + cob 8263–9369 8272–9378 8258–9364 1107 ATG TAG + tRNA-ser (S1) 9368–9420 9377–9426 9363–9415 53 (50) + rrnS 9413–10116 9419–10112 9408–10,111 704 (694) + tRNA-val (V) 10,118–10,174 10,114–10,170 10,113–10,169 57 + rrnL 10,176–11,184 10,170–11,180 10,171–11,179 1009 (1011) + atp6 11,155–11,967 11,151–11,990 11,150–11,962 813 (840) ATG TAA + cox3 11,973–12,746 11,965–12,738 11,968–12,741 774 ATG TAA (TAG) + tRNA-trp (W) 12,759–12,821 12,743–12,805 12,754–12,816 63 − tRNA-gln (Q) 12,825–12,880 12,807–12,862 12,820–12,875 56 + tRNA-Ile (I) 12,883–12,943 12,864–12,925 12,878–12,938 61 (62) − tRNA-gly (G) 12,957–13,013 12,934–12,989 12,952–13,008 57 (56) − tRNA-asp (D) 13,020–13,077 12,996–13,060 13,015–13,072 58 (65) + atp8 13,066–13,233 13,042–13,209 13,061–13,228 168 ATT (ATA) TAG + nad3 13,243–13,584 13,219–13,560 13,238–13,579 342 ATT TAA + Non-coding region NCR-S) 13,585–13,676 13,561–13,659 13,580–13,672 tRNA-ser (S2) 13,677–13,726 13,660–13,709 13,673–13,723 50 (50/51) + tRNA-asn (N) 13,727–13,781 13,710–13,763 13,724–13,778 55 (54) + tRNA-leu (L1) 13,789–13,848 13,770–13,835 13,786–13,845 60 (66) + tRNA-ala (A) 13,860–13,917 13,842–13,897 13,857–13,914 58 (56) + tRNA-cys (C) 13,960–14,013 13,924–13,976 13,957–14,010 54 (53) − tRNA-tyr (Y) 14,014–14,074 13,977–14,038 14,011–14,071 61 (62) − Total length 14,091 14,047 14,089 3.2. 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. Life 2021,11, 126 8 of 15 Table 4. Pairwise genetic and protein distances between the different complete mt genomes for different Trichuris trichiura and Trichuris sp. in different primates and human hosts at different countries. The nucleotide distances are given below the diagonal and the amino acid distances above the diagonal. TMF31 TPM1 TMM5 AP017704 T. trichiura H. sapiens (Unknown Geographical Location) NC_017750 T. trichiura H. sapiens China GU385218 T. trichiura H. sapiens China KT2449826 T. trichiura H. sapiens Uganda KT449825 Trichuris sp. TTB2 P. anubis USA KT449824 Trichuris sp. P. hamadryas Denmark KC461179 Trichuris sp. GHL T. francoisi China TMF31 0.41 14.5 14.7 14.8 14.8 0.6 14.6 0.49 26.9 TPM1 0.25 14.6 14.8 14.9 14.9 0.7 14.6 0.34 26.8 TMM5 18.7 18.7 10.9 11.1 11.1 14.5 10.3 14.6 28.2 AP017704 T. trichiura H. sapiens (unknown geographical location) 18.7 18.7 15.7 4.68 4.68 14.7 3.47 14.9 28.3 NC_017750 T. trichiura H. sapiens China 18.6 18.6 15.9 6.51 0 14.8 4.66 14.9 27.8 GU385218 T. trichiura H. sapiens China 18.6 18.6 15.9 6.51 0 14.8 4.66 14.9 27.8 KT2449826 T. trichiura H. sapiens Uganda 0.4 0.47 18.8 18.7 18.6 18.6 14.5 0.78 26.8 KT449825 Trichuris sp. TTB2 P. anubis USA 18.8 18.8 15.8 4.7 6.48 6.48 18.8 14.7 28.2 KT449824 Trichuris sp. P. hamadryas Denmark 0.34 0.28 18.8 18.7 18.6 18.6 0.55 18.8 26.8 KC461179 Trichuris sp. GHL T. francoisi China 27.1 27.1 28.4 28.3 28 28 27.1 28.6 27.2 Life 2021,11, 126 9 of 15 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. Life 2021, 11, x FOR PEER REVIEW 8 of 16 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 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. 3.2. 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. Figure 2. Nucleotide diversity (π) for all protein-coding genes (PCGs) and ribosomal rRNA (rrnS and rrnL) measured using a sliding window of 100 bp with 25 bp steps. The aligned dataset for Trichuris in primates (baboons, humans and Francois’ leaf monkey). Figure 2. Nucleotide diversity ( π ) for all protein-coding genes (PCGs) and ribosomal rRNA (rrnS and rrnL) measured using a sliding window of 100 bp with 25 bp steps. The aligned dataset for Trichuris in primates (baboons, humans and Francois’ leaf monkey). 3.3. 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.