Heth longquani sp. n.(Rhigonematomorpha: Hethidae)obligate commensalistic nematode of camballoid millipede (Spirostreptida: Cambalopsidae) from Viet Nam.

A new nematode species, Heth longquani sp. n. (Hethidae, Ransonematoidea, Rhigonematomorpha, Rhabditida) is described from the camballoid millipede, Chonecambala crassicauda from Thanh Ha province, Viet Nam. The morphological features of this new species were studied with brightfield and scanning electron microscopies. Partial sequences of LSU rDNA and Cox1 mtDNA are provided. The phylogeny of the nematodes of the genus Heth Cobb, 1898 is discussed.

Redescription and molecular characterization of Panagrellus ceylonensis (Nematoda, Rhabditida, Panagrolaimidae) from India.

A species of genus Panagrellus was discovered from a wet season form of an oriental common evening brown butterfly Melanitis leda. In this study, a detailed description of Panagrellus ceylonensis is provided including the morphometry, light microscopy and molecular (18S and 28S rDNA genes) studies. Morphological studies on the species agree with original description and characterized by having 12501481m long body in females and 9491305m in males, lateral fields with four longitudinal incisures, lip region continuous and 811 m wide, six offset lips with protruding labial sensilla, neck 124173 m long, excretory pore at the level of basal bulb, vulva post-equatorial (V = 6871), vagina anteriorly orientated with heavily muscled vaginal walls, post-vulval uterine sac 111135 m long or 1.72.6 times as long as the corresponding body diameter, tail conical elongate with an acute terminus in both sexes, spicule 7191 m long, ventrally curved having hooked manubrium and bifurcated lamina tip, lamina ventrally curved with dorsal deflexion at about 60% of spicule length, gubernaculum 2631 m long and well developed. Morphologically, the Indian population of P. ceylonensis does not show a significant difference from the type material of P. ceylonensis in the original description. For molecular studies of this species, the sequence of 18S rDNA is obtained for the first time. Phylogenetic trees based on 18S and 28S rDNA sequences are provided in this study. Additionally, bionomics and global distribution of the species of Panagrellus genus are also discussed. In conclusion, our study provides a comprehensive morphological characterisation and molecular marker sequences of 18S, and 28S genes that can be used to support future taxonomical research on this species and emphasizes the importance of combining molecular data with morphological data to describe the species accurately.

Morphological and molecular profiling of an entomopathogenic nematode Steinernema feltiae: Unlocking its biocontrol potential against vegetable insect pests.

A population of entomopathogenic nematodes, belonging to the Feltiae-clade and labelled J13, was discovered in the agricultural soils of the hilly regions of the Union territory of Jammu and Kashmir, India. Based on morphological, morphometric, and molecular analyses, the nematodes were identified as Steinernema feltiae. The J13 nematode isolate was tested in a laboratory assay for its pathogenicity against six major pests of vegetable crops: Pieris brassicae, Plutella xylostella, Helicoverpa armigera, Agrotis iplison, Trichoplusia ni, and Exelastis atomosa. The morphology of the isolated nematode closely matched the original description, except for the adult females, which had prominent epiptygmata instead of the weakly developed, double-flapped epiptygmata described in the original report. Analysis of the internal transcribed spacer and large subunit rRNA data from the J13 nematodes showed 100% similarity to sequences of the type population, indicating that they are conspecific. The virulence assays revealed that the nematode caused 100% mortality in the tested insect pests within 4872 hours, even at the lowest concentration of 50 infective juveniles per insect. The calculated median lethal concentration varied among the pests, with the lowest number of infective juveniles needed to achieve 50% larval killing being 117 for P. xylostella, 181.74 for P. brassicae, 226.35 for H. armigera, and 202.07 for T. ni at 24 hours post-inoculation. These findings suggest that S. feltiae isolated during the present investigation, may be a viable option for the biocontrol of these insect pests in Kashmir valley, India.

First report of Caenorhabditis brenneri (Nematoda: Rhabditida) isolated from the cadaver of Philippinella moellendorffi (Stylommatophora: Ariophantidae), a terrestrial slug in the Philippines.

Gastropod-associated nematodes have been previously studied and documented worldwide, with some species forming host-specific association as obligate parasites of molluscs while others form intermediate and temporary association. Philippinella moellendorffi from Imelda, Zamboanga Sibugay, Philippines, were collected, washed and maintained in the laboratory until death. Cadavers were placed on nutrient agar to allow nematode proliferation. Nematode pure culture was obtained using one gravid female for propagation. Morphology and molecular analyses (18S ribosomal DNA (rDNA) and D2-D3 expansion segments of 28S rDNA) were employed as diagnostic tools in identifying the nematode species isolated from P. moellendorffi. The newly isolated nematode was identified as Caenorhabditis brenneri, thus designated as C. brenneri strain IZSP from the Philippines. This is the first record of C. brenneri isolated from the terrestrial slug P. moellendorffi.

TARANTOBELUS JEFFDANIELSI N. SP. (PANAGROLAIMOMORPHA; PANAGROLAIMIDAE), A NEMATODE PARASITE OF TARANTULAS.

Multiple tarantula deaths for a wholesale breeder were reported in 2018. The breeder noticed white discharge in the oral cavities of the tarantulas. Upon inspection, it was discovered that the white discharge was a large group of nematodes intertwined inside the tarantula's oral cavity. We examined the nematodes and propose a new species, Tarantobelus jeffdanielsi n. sp., in the currently monotypic genus Tarantobelus based on a combination of morphological and morphometrical data and unique nuclear rDNA 28S and 18S sequences. Based on phylogenetic analyses, the previously described Tarantobelus arachnicida was relocated, along with T. jeffdanielsi, into the family Panagrolaimidae. We also provide evidence of the ability of T. jeffdanielsi to parasitize Galleria mellonella larvae and the tarantula Grammostola pulchra. The life span and fecundity of the new species were also assessed, resulting in an 11.2-d average life span, and a total fertility rate of 158 nematodes/adult.

Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living.

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditis tufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus-specifically from non-extreme locales-establishes a comparative system for the study of extremophilic traits in this model.

Morphological, molecular and ecological characterization of a native isolate of Steinernema feltiae (Rhabditida: Steinernematidae) from southern Chile.

BACKGROUND: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches. METHODS: Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction. RESULTS: Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity. CONCLUSIONS: For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.

Description of Hemicycliophora cardamomi sp. n. (Nematoda: Hemicycliophoridae) associated with Amomum longiligulare T.L. Wu and a web-based key for the identification of Hemicycliophora spp.

During a survey of plant diseases in the Central Highlands of Vietnam, a new plant-parasitic nematode, Hemicycliophora cardamomi sp. n., was discovered in the growing areas of Amomum longiligulare, a valuable medicinal plant with high economical value. The new species is characterized by a cuticular sheath loosely fitting body; a labial region continuous to the body contour bearing 3 annuli; a lateral field frequently marked by a discontinuous single line with indistinct additional short lines in some parts along the body; a modified vulval lip with an anterior lip c. 4 annuli long; and a post-vulval body tapering to a pointed tail tip with an abrupt constriction at the last third of the post-vulval body. Morphology and molecular characterizations of D2-D3 expansion segment of 28S rRNA, ITS, and COI mtDNA gene regions clearly distinguish the new species from all other 133 known species. This study also provides a newly developed web-based key for the identification of Hemicycliophora spp. in order to accelerate the identification process, to minimize the increasing error load associated with larger datasets, and to avoid any dependence on a single starting entry. This key includes both an existing Bray-Curtis similarity measure and a newly developed similarity formula.

Structure, development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida).

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested "rhabditid" pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.

Conserved nuclear hormone receptors controlling a novel plastic trait target fast-evolving genes expressed in a single cell.

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.

Morphological characteristics of microfilariae in blood smears of the common treeshrew Tupaia glis (Mammalia: Scandentia) in Gemas, Negeri Sembilan, Malaysia.

Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.

Evolution of neuronal anatomy and circuitry in two highly divergent nematode species.

The nematodes C. elegans and P. pacificus populate diverse habitats and display distinct patterns of behavior. To understand how their nervous systems have diverged, we undertook a detailed examination of the neuroanatomy of the chemosensory system of P. pacificus. Using independent features such as cell body position, axon projections and lipophilic dye uptake, we have assigned homologies between the amphid neurons, their first-layer interneurons, and several internal receptor neurons of P. pacificus and C. elegans. We found that neuronal number and soma position are highly conserved. However, the morphological elaborations of several amphid cilia are different between them, most notably in the absence of 'winged' cilia morphology in P. pacificus. We established a synaptic wiring diagram of amphid sensory neurons and amphid interneurons in P. pacificus and found striking patterns of conservation and divergence in connectivity relative to C. elegans, but very little changes in relative neighborhood of neuronal processes. These findings demonstrate the existence of several constraints in patterning the nervous system and suggest that major substrates for evolutionary novelty lie in the alterations of dendritic structures and synaptic connectivity.

Two Ektaphelenchinae Paramonov, 1964 (Nematoda: Rhabditida) from Iran have tripartite stylet, with similar observations in other species.

Two ektaphelenchid nematodes representing one new and one known species are illustrated and characterized using morphological and molecular data. Ektaphelenchus kanzakii n. sp. is mainly characterized by its tripartite stylet having a well visible wide lumen, encompassing a sclerotized and acute anterior part (the conus), a short and slightly tapering middle part (the conophore) that is equally sclerotized but clearly separate from the conus, and a long posterior part that is cylindrical and only weakly sclerotized (the shaft) without basal knobs or swellings. It is further characterized by 863.5 (772-926) µm long females having 23.8 (21.2-27.0) µm long total stylet, distinctly annulated cuticle, three lines in lateral field, vulva at 76.6 (75.3-80.0)%, no rectum, vestigial anus in some individuals, conical posterior body end (tail) with narrow ventrally bent tip, common males in population with two pairs of caudal papillae (the single precloacal papilla and the third caudal pair lacking), spicules with dorsally bent tip and conical tail with sharp or blunt tip. The new species is morphologically compared with close species having conical posterior body end and stylet lacking basal knobs or swellings. Iranian population of Devibursaphelenchus teratospicularis, the second studied species, is characterized by 679.5 (620-709) µm long females having 18.6 (17.5-20.0) µm long total stylet with similar structure to the previous species, subcylindrical body end with widely rounded tip, and rare males with typical spicules of this species and a pair of precloacal and a pair of caudal papillae. Molecular phylogenetic studies of the two recovered species using small and large subunit ribosomal DNA (SSU and LSU rDNA) partial sequences revealed they have close phylogenetic affinities with Ektaphelenchus obtusus in both reconstructed trees. However, species of both genera Ektaphelenchus and Devibursaphelenchus don't form monophyletic groups in SSU and LSU trees. New observations on stylet structure of the two presently studied and some other ektaphelenchid species having available light microphotographs (LM) yielded on definition of a new term "conophore" for the middle part of the ektaphelenchid-type tripartite stylet.

Study of Steinernema hermaphroditum (Nematoda, Rhabditida), from the West Uttar Pradesh, India.

INTRODUCTION: The entomopathogenic nematodes have been reported from all continents (except Antarctica) and almost all regions of the world. Surveys of EPNs in India has resulted in the recovery of several isolates of Steinernema. Among one of them, isolate CS34 was identified as S. hermaphroditum Stock, Griffin & Chaerani, 2004. We investigated the identification and the pathogenicity of S. hermaphroditum in District Meerut of Western Uttar Pradesh, India. MATERIALS AND METHODS: The Steinernema was examined for its pathogenicity and accurate identification by the mean of morphological and molecular technique and its geographical distribution was mapped based on meta-analysis of the ITS GenBank records. RESULTS: The surveys of agricultural soils of district Meerut, India, resulted in the isolation of one strain from entomopathogenic nematode labelled CS34 through Galleria baiting technique. Morphological characters and morphometrical analysis indicated that the strain CS34 was closely related to the "glaseri" group of Steinernema spp. The Nblast results indicated that ITS rDNA sequence had no nucleotide differences in comparison with the S. hermaphroditum (JQ687355). However, one variation in the D2-D3 segment of 28S rDNA was observed in comparison with the AY598358. The phylogenetic analysis using ITS and 28S rDNA indicated that the Indian S. hermaphroditum could be placed together with other S. hermaphroditum, with strong posterior probability. Besides, the PCA analysis demonstrated some variability within the test populations. The distribution of S. hermaphroditum based on meta-analysis of the GenBank records showed its presence in the three Asian countries-India, Thailand and Indonesia. The Indian strain of S. hermaphroditum also tested positively for its virulence against three major pests, namely, Galleria mellonella, Helicoverpa armigera, and Spodoptera litura, with resultant which showed good efficacy on the mortalities. CONCLUSIONS: In conclusion, the economy of India is agriculture-based, but there are huge losses due to different insect pests infesting different crops. Steinernema hermaphroditum CS34 is an indigenous species to Indian subcontinent and efforts should be made to evaluate its virulence and pathogenicity against the other agricultural pests hampering productivity throughout the country. This may lead to incorporate S. hermaphroditum strain CS34 as a regular biological control agent against important lepidopteran pest in integrated pest management programs in the future.

Description of a New Species of Rhabdias Stiles and Hassall, 1905 (Nematoda, Rhabdiasidae) in Duttaphrynus melanostictus (Schneider, 1899) from West Bengal, India.

AIMS: The purpose of this article is to describe a new species of Rhabdias Stiles and Hassall, 1905 from West Bengal, India. Rhabdias bengalensis sp. nov. (Nematoda, Rhabdiasidae) recovered from the lungs of Asian common toad, Duttaphrynus melanostictus (Schneider, 1899), collected from Illambazar town, Birbhum district of West Bengal, India. METHODS: The collected nematode specimens were clean by shaking them in 0.67% NaCl solution and were killed and fixed in steaming 70% alcohol; lactophenol was used as a mounting medium for light microscopic examination. The lactophenol treated specimens after examination were directly transferred to 70% alcohol and could be stored satisfactorily. RESULTS: The new species is named as, R. bengalensis sp. nov. (Nematoda, Rhabdiasidae), in reference to its locality of collection, West Bengal, India. The worms are small [(2.84-2.96 mm) in length], anterior end somewhat truncated and posterior end tapering. R. bengalensis sp. nov. is characterised by rounded mouth without lips, inflated cuticle, cup-shaped buccal capsule, pre-equatorial vulva and a conical tail. CONCLUSION: The present nematodes, thus, appear to be new in the field of science and name R. bengalensis sp. nov. is proposed for them. R. bengalensis sp. nov. represents 17th species described from oriental region and the 10th species from India.

Characterization of Polish Steinernema silvaticum isolates (Nematoda: Steinernematidae) using morphological and molecular data.

Four strains of entomopathogenic nematodes were isolated with a live trap method in southern Poland. The isolates were identified as Steinernema silvaticum based on morphological, morphometric and molecular data. Infective juveniles of Polish S. silvaticum isolates differ in body length from S. weiseri (951 vs 740 µm, respectively), and in the hyaline tail portion from S. kraussei (48 vs 38%, respectively). First-generation males of S. silvaticum are longer than those of S. kraussei, S. weiseri and S. ichnusae (1829 vs 1400, 1180 and 1341 µm, respectively). Males of S. silvaticum and a sister species S. kraussei can be distinguished by the distance from the anterior end to the nerve ring (142 vs 105 µm), spicule (66 vs 49 µm) and gubernaculum length (45 vs 33 µm), and the presence of a mucron. The analysis of internal transcribed spacer (ITS), D2-D3 and cox1 sequences of the tested nematodes revealed differences of 3-5%, 3% and 12-13%, respectively, from S. kraussei strains. The phylogeny of both nuclear and mitochondrial genes indicated close relationships of the Polish S. silvaticum isolates with S. kraussei, S. oregonense and S. cholashanense. The reproductive isolation of the studied isolates was confirmed by hybridization tests with other European feltiae-kraussei group representatives. This study has supplemented the original description of S. silvaticum with morphological and morphometric characterization of the first-generation males and females. This is also the first molecular study of this species based on a multi-gene approach.

Redescription of Macrolaimus canadensis Sanwal, 1960 and M. ruehmi Andrássy, 1966 (Nematoda, Rhabditida, Chambersiellidae), and new data on M. crucis Maupas, 1900.

Three species belonging to the genus Macrolaimus, namely M. canadensis, M. crucis and M. ruehmi, have been obtained from areas of natural vegetation in Bosnia and Herzegovina, Corsica (France), the Czech Republic and Spain. These three species are characterized by the body length of males and females, stomatal proportion of the gymnostom and cheilostom, excretory pore position, postvulval uterine sac length, male and female tail length and morphology, and the length and morphology of the spicules and gubernacula. The occurrence of M. crucis in Spain has, as a result of this study, now also been expanded to a larger area of the southern Iberian Peninsula. Morphological and morphometrical analyses showed that M. canadensis and M. ruehmi are very similar, sharing apomorphic characters. In contrast, M. crucis has plesiomorphic characters. Description, measurements and illustrations are provided for these three species.

Steinernema khuongi n. sp. (Panagrolaimomorpha, Steinernematidae), a new entomopathogenic nematode species from Florida, USA.

In this study, molecular (ribosomal sequence data), morphological and cross-hybridization properties were used to identify a new Steinernema sp. from Florida, USA. Molecular and morphological data provided evidence for placing the novel species into Clade V, or the 'glaseri-group' of Steinernema spp. Within this clade, analysis of sequence data of the rDNA genes, 28S and internal transcribed spacer (ITS), depicted the novel species as a distinctive entity and closely related to S. glaseri and S. cubanum. Additionally, cross-hybridization assays showed that the new species is unable to interbreed with either of the latter two species, reinforcing its uniqueness from a biological species concept standpoint. Key morphological diagnostic characters for S. khuongi n. sp. include the mean morphometric features of the third-stage infective juveniles: total body length (average: 1066 µm), tail length (average: 65 µm), location of the excretory pore (average: 80.5 µm) and the values of c (average: 16.4), D% (average: 60.5), E% (average: 126) and H% (average: 46.6). Additionally, males can be differentiated from S. glaseri and S. cubanum by the values of several ratios: D% (average: 68), E% (average: 323) and SW% (average: 120). The natural distribution of this species in Florida encompasses both natural areas and citrus groves, primarily in shallow groundwater ecoregions designated as 'flatwoods'. The morphological, molecular, phylogenetic and ecological data associated with this nematode support its identity as a new species in the S. glaseri-group.

A sulfotransferase dosage-dependently regulates mouthpart polyphenism in the nematode Pristionchus pacificus.

Polyphenism, the extreme form of developmental plasticity, is the ability of a genotype to produce discrete morphologies matched to alternative environments. Because polyphenism is likely to be under switch-like molecular control, a comparative genetic approach could reveal the molecular targets of plasticity evolution. Here we report that the lineage-specific sulfotransferase SEUD-1, which responds to environmental cues, dosage-dependently regulates polyphenism of mouthparts in the nematode Pristionchus pacificus. SEUD-1 is expressed in cells producing dimorphic morphologies, thereby integrating an intercellular signalling mechanism at its ultimate target. Additionally, multiple alterations of seud-1 support it as a potential target for plasticity evolution. First, a recent duplication of seud-1 in a sister species reveals a direct correlation between genomic dosage and polyphenism threshold. Second, inbreeding to produce divergent polyphenism thresholds resulted in changes in transcriptional dosage of seud-1. Our study thus offers a genetic explanation for how plastic responses evolve.

Description of two three-gendered nematode species in the new genus Auanema (Rhabditina) that are models for reproductive mode evolution.

The co-existence of males, females and hermaphrodites, a rare mating system known as trioecy, has been considered as an evolutionarily transient state. In nematodes, androdioecy (males/hermaphrodites) as found in Caenorhabditis elegans, is thought to have evolved from dioecy (males/females) through a trioecious intermediate. Thus, trioecious species are good models to understand the steps and requirements for the evolution of new mating systems. Here we describe two new species of nematodes with trioecy, Auanema rhodensis and A. freiburgensis. Along with molecular barcodes, we provide a detailed analysis of the morphology of these species, and document it with drawings and light and SEM micrographs. Based on morphological data, these free-living nematodes were assigned to a new genus, Auanema, together with three other species described previously. Auanema species display convergent evolution in some features with parasitic nematodes with complex life cycles, such as the production of few males after outcrossing and the obligatory development of dauers into self-propagating adults.