Morphological and molecular characterization of larval Echinocephalus sp. (Spirurida: Gnathostomatidae), a parasite of the greater lizard fish (Saurida undosquamis) and red porgy or common seabream (Pagrus pagrus).

Larvae of an unidentified Echinocephalus species were obtained from two fish species: red porgy or common seabream (Pagrus pagrus) and greater lizard fish (Saurida undosquamis) from the Red Sea. The prevalence of Echinocephalus sp. larvae in P. pagrus was 4.92% and 4.98% in S. undosquamis. The length, width, cephalic bulb, and spine shape and pattern of the larvae resembled Echinocephalus overstreeti. SSU gene sequences of larvae from P. pagrus and S. undosquamis were identical. Comparison of the SSU sequence to those available in GenBank showed that the larvae from P. pagrus and S. undosquamis are diagnosably distinct. Based on sequence similarity and published phylogenetic analysis, these larvae are most similar to an unknown species of Echinocephalus from an Australian sea snake (Hydrophis peronii). Despite morphological similarities of the Red Sea larvae to E. overstreeti, the SSU sequence differences show that they are not the same species.

PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

PHYLOGEOGRAPHY OF BAYLISASCARIS PROCYONIS (RACCOON ROUNDWORM) IN NORTH AMERICA.

Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 115 Baylisascaris procyonis individuals from 13 U.S. states and 1 Canadian province were obtained from 44 raccoon hosts to assess genetic variation and geographic structure. The maximum genetic distance between individuals was low (1.6%), consistent with a single species. Moderate COI haplotype (h = 0.60) and nucleotide (π = 0.0053) diversity were found overall. Low haplotype diversity was found among samples east of the Mississippi River (h = 0.036), suggesting that historical growth and expansion of raccoon populations in this region could be responsible for high parasite gene flow or a selective sweep of B. procyonis mtDNA. There was low genetic structure (average Φst = 0.07) for samples east of the continental divide, but samples from Colorado showed higher diversity and differentiation from midwestern and eastern samples. There was marked genetic structure between samples from east and west of the continental divide, with no haplotypes shared between these regions. There was no significant isolation by distance among any of these geographic samples. The phylogeographic patterns for B. procyonis are similar to genetic results reported for their raccoon definitive hosts. The phylogeographic divergence of B. procyonis from east and west of the continental divide may involve vicariance resulting from Pleistocene glaciation and associated climate variation.

Draft Genome Sequences of 28 Actinobacteria of the Family Microbacteriaceae Associated with Nematode-Infected Plants.

Draft genome sequences of 28 strains of Microbacteriaceae from plants infested by plant-parasitic nematodes were obtained using Illumina technology. The sequence data will provide useful baseline information for the development of comparative genomics and systematics of Microbacteriaceae and facilitate understanding of molecular mechanisms involved in interactions between plants and nematode-associated bacterial complexes.

Draft Genome Sequences of 13 Plant-Associated Actinobacteria of the Family Microbacteriaceae.

Draft genome sequences of 13 bacterial strains from the family Microbacteriaceae were generated using Illumina technology. The genome sizes varied from 3.0 to 4.8 Mb, and the DNA G+C content was 68.1 to 72.5%. The sequences obtained will contribute to the development of genome-based taxonomy and understanding of molecular interactions between bacteria and plants.

Host Population Expansion and the Genetic Architecture of the Pinniped Hookworm Uncinaria lucasi.

The long-term fidelity of pinniped hosts to their natal rookery site suggests the genetic architecture of their Uncinaria spp. hookworms should be strongly structured by host breeding biology. However, historical events affecting host populations may also shape parasite genetic structure. Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 86 Uncinaria lucasi individuals were obtained to assess genetic variation and structure of nematodes from 2 host species (68 hookworms from northern fur seals; 18 hookworms from Steller sea lions) and rookeries from 3 widely separated geographic regions: the western Bering Sea and Sea of Okhotsk, eastern Bering Sea, and the eastern Pacific Ocean. High COI haplotype (h = 0.96-0.98) and nucleotide (π = 0.014) diversity was found. The haplotype network showed a star-shaped pattern with a large number of haplotypes separated by few substitutions. The network did not show separation of U. lucasi by geographic region or host species. Fst values between U. lucasi individuals representing geographic regions showed no differentiation, consistent with the absence of genetic structure. At face value, this lack of genetic structure in U. lucasi suggests high gene flow but could also be explained by recent (post-glacial) population expansions of northern fur seals and their hookworms.

Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species.

Complete and draft genome sequences of 12 Rathayibacter strains were generated using Oxford Nanopore and Illumina technologies. The genome sizes of these strains are 3.21 to 4.61 Mb, with high G+C content (67.2% to 72.7%) genomic DNA. Genomic data will provide useful baseline information for natural taxonomy and comparative genomics of members of the genus Rathayibacter.

Vertical Transmission of a Nematode from Female Lizards to the Brains of Their Offspring.

Parasites have evolved a diversity of lifestyles that exploit the biology of their hosts. Some nematodes that parasitize mammals pass via the placenta or milk from one host to another. Similar cases of vertical transmission have never been reported in avian and nonavian reptiles, suggesting that egg laying may constrain the means of parasite transmission. However, here we report the first incidence of transovarial transmission of a previously undescribed nematode in an egg-laying amniote, the common wall lizard (Podarcis muralis). Nematodes enter the developing brain from the female ovary early in embryonic development. Infected lizard embryos develop normally and hatch with nematodes residing in their braincase. We present a morphological and molecular phylogenetic characterization of the nematode and suggest that particular features of lizard biology that are absent from birds and turtles facilitated the evolutionary origin of this novel life history.

Plagiorchis sp. in small mammals of Senegal and the potential emergence of a zoonotic trematodiasis.

Trematodes of the genus Plagiorchis have a wide geographical distribution and can exploit a variety of hosts. The occurrence and zoonotic potential of Plagiorchis spp. have been characterised across several countries in Asia; in contrast, information on Plagiorchis parasites in Africa remains anecdotal. We isolated a previously undescribed Plagiorchis species from the biliary tract and small intestine of 201 out of 427 small mammals collected in the region of Lake Guiers, Senegal, with local prevalence ranging from 38.6% to 77.0%. Conversely, Plagiorchis isolates were not observed in the 244 small mammals sampled in and around the town of Richard Toll, Senegal. Molecular phylogenetics of the internal transcribed spacer region, nuclear ribosomal DNA, and of the cytochrome c oxidase subunit 1 gene, mitochondrial DNA, supported the monophyly and multi-host spectrum of this newly discovered West African Plagiorchis species. Sequencing of individual cercariae shed by Radix natalensis (Gastropoda: Lymnaeidae) suggested that these freshwater snails may act as suitable first intermediate hosts. Phylogenetic analysis yielded a highly resolved topology indicating two different clades, one composed by Plagiorchis spp. infecting rodents, insectivores, and birds, while the other included parasites of bats. Our findings showed the low host specificity and high prevalence of the isolated Plagiorchis sp. in the Lake Guiers region, with Hubert's multimammate mice (Mastomys huberti) appearing to play a primary role in the epidemiology of this parasite. The results raise concern about the zoonotic potential of Plagiorchis sp. in local communities of the Lake Guiers region, and highlight food-borne trematodiases and their link to land-use change as a neglected public health issue in regions of West Africa.

Molecular phylogenetics and species-level systematics of Baylisascaris.

Nucleotide sequences representing nine genes and five presumptive genetic loci were used to infer phylogenetic relationships among seven Baylisascaris species, including one species with no previously available molecular data. These genes were used to test the species status of B. procyonis and B. columnaris using a coalescent approach. Phylogenetic analysis based on combined analysis of sequence data strongly supported monophyly of the genus and separated the species into two main clades. Clade 1 included B. procyonis, B. columnaris, and B. devosi, species hosted by musteloid carnivores. Clade 2 included B. transfuga and B. schroederi from ursids, B. ailuri, a species from the red panda (a musteloid), and B. tasmaniensis from a marsupial. Within clade 2, geographic isolates of B. transfuga, B. schroederi (from giant panda), and B. ailuri formed a strongly supported clade. In certain analyses (e.g., some single genes), B. tasmaniensis was sister to all other Baylisascaris species rather than sister to the species from ursids and red panda. Using one combination of priors corresponding to moderate population size and shallow genetic divergence, the multispecies coalescent analysis of B. procyonis and B. columnaris yielded moderate support (posterior probability 0.91) for these taxa as separate species. However, other prior combinations yielded weak or no support for delimiting these taxa as separate species. Similarly, tree topologies constrained to represent reciprocal monophyly of B. columnaris and B. procyonis individuals (topologies consistent with separate species) were significantly worse in some cases, but not others, depending on the dataset analyzed. An expanded analysis of SNPs and other genetic markers that were previously suggested to distinguish between individuals of B. procyonis and B. columnaris was made by characterization of additional individual nematodes. The results suggest that many of these SNPs do not represent fixed differences between nematodes derived from raccoon and skunk hosts.

Transuterine infection by Baylisascaris transfuga: Neurological migration and fatal debilitation in sibling moose calves (Alces alces gigas) from Alaska.

Larval Baylisascaris nematodes (L3), resulting from transuterine infection and neural migration, were discovered in the cerebrum of sibling moose calves (Alces alces gigas) near 1-3 days in age from Alaska. We provide the first definitive identification, linking morphology, biogeography, and molecular phylogenetics, of Baylisascaris transfuga in naturally infected ungulates. Life history and involvement of paratenic hosts across a broader assemblage of mammals, from rodents to ungulates, in the transmission of B. transfuga remains undefined. Neural infections, debilitating young moose, may seasonally predispose calves to predation by brown bears, facilitating transmission to definitive hosts. Discovery of fatal neurological infections by L3 of B. transfuga in mammalian hosts serves to demonstrate the potential for zoonotic infection, as widely established for B. procyonis, in other regions and where raccoon definitive hosts are abundant. In zones of sympatry for multi-species assemblages of Baylisascaris across the Holarctic region presumptive identification of B. procyonis in cases of neurological larval migrans must be considered with caution. Diagnostics in neural and somatic larval migrans involving species of Baylisascaris in mammalian and other vertebrate hosts should include molecular-based and authoritative identification established in a phylogenetic context.

Phylogenetic Relationships of Spiruromorph Nematodes (Spirurina: Spiruromorpha) In North American Freshwater Fishes.

Nematodes are common in the parasite communities of North American freshwater fishes, and the majority of them belong to 1 conventional order, Spirurida Chitwood, 1933. Within the Spirurida, the superfamilies Habronematoidea Chitwood and Wehr, 1932 and Thelazioidea Sobolev, 1949 have undergone considerable diversification. The dominant families of these 2 superfamilies, Cystidicolidae Skrjabin 1946 and Rhabdochonidae Railliet, 1916, respectively, are particularly common, widely distributed, and diverse, especially in North America, yet their phylogenetic relationships remain largely unexplored. In this study, we use near complete sequences of the 18S rRNA genes ( SSU rDNA) from species in 6 genera ( Capillospirura Skrjabin, 1924, Cystidicola Fischer, 1798, Salmonema Moravec, Santos and Brasil-Sato, 2008, Rhabdochona Railliet, 1916, Spinitectus Fourment, 1883, and a putative new cystidicolid in mooneyes, Hiodontidae), along with a species of Hedruris Nitzsch, 1812 from newts as a surrogate for the fish parasite Hedruris tiara VanCleave and Mueller, 1932, to explore their phylogenetic relationships. These sequences, together with available sequences from a range of other nematodes, including fish nematodes in other groups (Camallanoidea and 'Seuratoidea'), were analyzed using Bayesian inference and maximum likelihood. The results from both analyses indicate, for the first time, support for the close relationships of the sturgeon parasite Capillospirura with Ascarophis van Beneden, 1871 and Cystidicola; the relationship of the cystidicolid from Hiodontidae with Salmonema of salmonid fishes; the monophyly of the 2 dominant spiruridan genera of fishes, Rhabdochona and Spinitectus; and for previous relationships among Nearctic Spinitectus spp. The results also indicate a closer relationship of Rhabdochona and Spinitectus than is suggested by their conventional classification and reject the monophyly of Habronematoidea, Thelazioidea, and Cystidicolidae. Hedruridae appears to be an early branching lineage of spirurins. Finally, the pattern of association between the fish parasites in this study and their hosts indicates, with few exceptions, ecologically driven diversification events involving host shifting not related to the phylogenetic relationships of their hosts.

The bipartite mitochondrial genome of Ruizia karukerae (Rhigonematomorpha, Nematoda).

Mitochondrial genes and whole mitochondrial genome sequences are widely used as molecular markers in studying population genetics and resolving both deep and shallow nodes in phylogenetics. In animals the mitochondrial genome is generally composed of a single chromosome, but mystifying exceptions sometimes occur. We determined the complete mitochondrial genome of the millipede-parasitic nematode Ruizia karukerae and found its mitochondrial genome consists of two circular chromosomes, which is highly unusual in bilateral animals. Chromosome I is 7,659 bp and includes six protein-coding genes, two rRNA genes and nine tRNA genes. Chromosome II comprises 7,647 bp, with seven protein-coding genes and 16 tRNA genes. Interestingly, both chromosomes share a 1,010 bp sequence containing duplicate copies of cox2 and three tRNA genes (trnD, trnG and trnH), and the nucleotide sequences between the duplicated homologous gene copies are nearly identical, suggesting a possible recent genesis for this bipartite mitochondrial genome. Given that little is known about the formation, maintenance or evolution of abnormal mitochondrial genome structures, R. karukerae mtDNA may provide an important early glimpse into this process.

Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for Ascaridoid Nematodes.

Ascaridoids are among the commonest groups of zooparasitic nematodes (roundworms) and occur in the alimentary canal of all major vertebrate groups, including humans. They have an extremely high diversity and are of major socio-economic importance. However, their evolutionary history remains poorly known. Herein, we performed a comprehensive phylogenetic analysis of the Ascaridoidea. Our results divided the Ascaridoidea into six monophyletic major clades, i.e., the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, represents the sister clade to the remaining ascaridoids. The phylogeny was calibrated using an approach that involves time priors from fossils of the co-evolving hosts, and dates the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47-325.27 Ma). The divergence dates and ancestral host types indicated by our study suggest that members of the Ascaridoidea first parasitized terrestrial tetrapods, and subsequently, extended their host range to elasmobranchs and teleosts. We also propose that the fundamental terrestrial-aquatic switches of these nematodes were affected by changes in sea-level during the Triassic to the Early Cretaceous.

Similar yet different: co-analysis of the genetic diversity and structure of an invasive nematode parasite and its invasive mammalian host.

Animal parasitic nematodes can cause serious diseases and their emergence in new areas can be an issue of major concern for biodiversity conservation and human health. Their ability to adapt to new environments and hosts is likely to be affected by their degree of genetic diversity, with gene flow between distinct populations counteracting genetic drift and increasing effective population size. The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal parasite of the raccoon (Procyon lotor), has increased its global geographic range after being translocated with its host. The raccoon has been introduced multiple times to Germany, but not all its populations are infected with the parasite. While fewer introduced individuals may have led to reduced diversity in the parasite, admixture between different founder populations may have counteracted genetic drift and bottlenecks. Here, we analyse the population genetic structure of the roundworm and its raccoon host at the intersection of distinct raccoon populations infected with B. procyonis. We found evidence for two parasite clusters resulting from independent introductions. Both clusters exhibited an extremely low genetic diversity, suggesting small founding populations subjected to inbreeding and genetic drift with no, or very limited, genetic influx from population admixture. Comparison of the population genetic structures of both host and parasite suggested that the parasite spread to an uninfected raccoon founder population. On the other hand, an almost perfect match between cluster boundaries also suggested that the population genetic structure of B. procyonis has remained stable since its introduction, mirroring that of its raccoon host.

Phylogenetic analysis of two Plectus mitochondrial genomes (Nematoda: Plectida) supports a sister group relationship between Plectida and Rhabditida within Chromadorea.

Plectida is an important nematode order with species that occupy many different biological niches. The order includes free-living aquatic and soil-dwelling species, but its phylogenetic position has remained uncertain. We sequenced the complete mitochondrial genomes of two members of this order, Plectus acuminatus and Plectus aquatilis and compared them with those of other major nematode clades. The genome size and base composition of these species are similar to other nematodes; 14,831 and 14,372bp, respectively, with AT contents of 71.0% and 70.1%. Gene content was also similar to other nematodes, but gene order and coding direction of Plectus mtDNAs were dissimilar from other chromadorean species. P. acuminatus and P. aquatilis are the first chromadorean species found to contain a gene inversion. We reconstructed mitochondrial genome phylogenetic trees using nucleotide and amino acid datasets from 87 nematodes that represent major nematode clades, including the Plectus sequences. Trees from phylogenetic analyses using maximum likelihood and Bayesian methods depicted Plectida as the sister group to other sequenced chromadorean nematodes. This finding is consistent with several phylogenetic results based on SSU rDNA, but disagrees with a classification based on morphology. Mitogenomes representing other basal chromadorean groups (Araeolaimida, Monhysterida, Desmodorida, Chromadorida) are needed to confirm their phylogenetic relationships.

Mitochondrial Phylogenomics yields Strongly Supported Hypotheses for Ascaridomorph Nematodes.

Ascaridomorph nematodes threaten the health of humans and other animals worldwide. Despite their medical, veterinary and economic importance, the identification of species lineages and establishing their phylogenetic relationships have proved difficult in some cases. Many working hypotheses regarding the phylogeny of ascaridomorphs have been based on single-locus data, most typically nuclear ribosomal RNA. Such single-locus hypotheses lack independent corroboration, and for nuclear rRNA typically lack resolution for deep relationships. As an alternative approach, we analyzed the mitochondrial (mt) genomes of anisakids (~14 kb) from different fish hosts in multiple countries, in combination with those of other ascaridomorphs available in the GenBank database. The circular mt genomes range from 13,948-14,019 bp in size and encode 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNA genes. Our analysis showed that the Pseudoterranova decipiens complex consists of at least six cryptic species. In contrast, the hypothesis that Contracaecum ogmorhini represents a complex of cryptic species is not supported by mt genome data. Our analysis recovered several fundamental and uncontroversial ascaridomorph clades, including the monophyly of superfamilies and families, except for Ascaridiidae, which was consistent with the results based on nuclear rRNA analysis. In conclusion, mt genome analysis provided new insights into the phylogeny and taxonomy of ascaridomorph nematodes.

Five new species of the family Trischistomatidae (Nematoda: Enoplida) from North and Central America, with keys to the species of Trischistoma and Tripylina.

Three new species of Trischistoma and two new species of Tripylina (Trischistomatidae) are described. Trischistoma ripariana n. sp. was collected in the surface organic material and upper rhizosphere soil on a stream bank in Oakville, California, USA. It is characterized by a short, thin body, the vulva at 79-83%, a small index c (17-29) and a short tail, 34-57 µm. Trischistoma corticulensis n. sp. was found in moss on tree bark in a tropical forest at the La Mancha Ecological Institute, Veracruz State, México. The vulva is at 67-73% and the tail is elongate conoid (51-84 µm). Trischistoma helicoformis n. sp. was collected on lichen growing on tree bark near the Carretera Interamericana in Costa Rica. It is characterized by its spiral shape after fixation, the tail length (76-101 µm), a very small index c (10-14.5) and very small sclerotised pieces around the vagina. Tripylina rorkabanarum n. sp. was collected from moss on tree bark in a tropical forest at the La Mancha Ecological Institute. It is characterized by the presence of two cervical setae, the position of the subventral teeth posterior to the dorsal tooth, the absence of sclerotized pieces around the vagina and the distance of the dorsal tooth from the anterior, 10-15 µm. Tripylina iandrassyi n. sp. was collected from soil around a banana tree at the La Mancha Ecological Institute. It is characterized by the presence of a post-uterine sac, well-developed buccal lips, subventral teeth located posterior to the dorsal tooth, one cervical seta in females and two in males, and by spicules not completely surrounded by a muscular sheath.

Five new species of the genus Tripylella (Nematoda: Enoplida: Tripylidae).

Five new species of the genus Tripylella are described, two from México, one from Fátima, Portugal, one from Quito, Ecuador, and one from California, USA. Tripylella mexicana sp. n. is characterized by its short body (average 0.74 mm), short pharynx (average 161 µm), short tail (average 117 µm), presence of an excretory pore and small setae distributed sparsely along the body, the presence of body pores, the posterior position of the subventral teeth in relation to the small dorsal tooth with all teeth in contiguous stomal chambers, the finely-striated cuticle with many anastomoses, the non-protruding vulval lips, and the presence of sclerotized pieces in the vulval region. Tripylella muscusi sp. n. is characterized by its body length (average 0.94 mm), pharynx length (average 201 µm), tail length (average 140 µm), the anterior position of the subventral teeth in relation to the small dorsal tooth in a single stomal chamber, the presence of an excretory pore, the presence of body pores and sparse somatic setae, the finely-striated cuticle with sparse anastomoses, protruding vulval lips and sclerotized oval-shaped pieces present in the vulval region. Tripylella quitoensis sp. n. is characterized by the short body length (average 0.72 mm), the short outer labial setae, the short pharynx (average 175 µm), the location of the anterior subventral teeth and posterior dorsal tooth in the same stomal chamber, the short tail (average 98 µm), the apparent absence of an excretory pore, presence of body pores, presence of somatic setae, a finely-striated cuticle, non-protruding vulval lips, and very small oval sclerotized pieces in the vulva. Tripylella fatimaensis sp. n. is characterized by the short body, (average 0.74 mm) long, by the length of the pharynx (average 180 µm), the length of the tail (average 110 µm) and in the length of its reduced diameter portion, 45-58 µm, the presence of an excretory pore, body pores and three pairs of caudal setae (one pair each latero-ventral, latero-dorsal and ventral). Tripylella dentata sp. n. is characterized, and differs from all the species of the genus, by the presence of two adjacent stomal chambers, with two large teeth, one dorsal and one ventral, in the posterior stomal chamber and two subventral teeth in the anterior smaller chamber, short body (average 0.85 mm), pharynx length (average 209 µm), tail length (average 115 µm), the apparent absence of an excretory pore, the presence of two cervical setae in a lateral position, and by the presence of conspicuous pores along the body.

Pinworm diversity in free-ranging howler monkeys (Alouatta spp.) in Mexico: Morphological and molecular evidence for two new Trypanoxyuris species (Nematoda: Oxyuridae).

Two new species of Trypanoxyuris are described from the intestine of free-ranging howler monkeys in Mexico, Trypanoxyuris multilabiatus n. sp. from the mantled howler Alouatta palliata, and Trypanoxyuris pigrae n. sp. from the black howler Alouatta pigra. An integrative taxonomic approach is followed, where conspicuous morphological traits and phylogenetic trees based on DNA sequences are used to test the validity of the two new species. The mitochondrial cytochrome oxidase subunit 1 gene, and the nuclear ribosomal 18S and 28S rRNA genes were used for evolutionary analyses, with the concatenated dataset of all three genes used for maximum likelihood and Bayesian phylogenetic analyses. The two new species of pinworms from howler monkeys were morphologically distinct and formed reciprocally monophyletic lineages in molecular phylogenetic trees. The three species from howler monkeys, T. multilabiatus n. sp., T. pigrae n. sp., and Trypanoxyuris minutus, formed a monophyletic group with high bootstrap and posterior probability support values. Phylogenetic patterns inferred from sequence data support the hypothesis of a close evolutionary association between these primate hosts and their pinworm parasites. The results suggest that the diversity of pinworm parasites from Neotropical primates might be underestimated.