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A new nematode species of the genus Phasmarhabditis was isolated from the body surface of a slug ( Philomycus bilineatus Benson, PB). Morphological and molecular analyses confirmed this nematode as a new species. The nematode was named Phasmarhabditis zhejiangensis sp. nov. (Nematoda: Rhabditidae) and is dioecious. In males, the open bursa with genital papillae is characterized by the formula 1-1-1-2-1-3, and the spicule length is 58μm. In female, the vulva is located approximately in the middle of the body. The nematode belongs to papillosa group because of its tail shape pointed with filiform tip. The phasmids are rod-shaped. The posterior anus is slightly swollen. P . zhejiangensis was further characterized by internal transcribed spacer (ITS), 18S rDNA and 28S rDNA sequences. After the sequencing results were compared with sequences available from the National Center for Biotechnology Information (NCBI), the maximum similarities of ITS, 18S and 28S sequences were 89.81%, 96.22% and 95.28%, respectively. Phylogenetic analyses placed Phasmarhabditis zhejiangensis sp. nov. in the genus Phasmarhabditis .

Background Heterorhabditis are important biological control agents in agriculture. Two Heterorhabditis populations, S8 and S10, were isolated from agricultural soils in the United States of America. Molecular analyses, based on mitochondrial and nuclear genes, showed that these populations are conspecific and represent a novel species of the “ Bacteriophora ” clade. This species was named Heterorhabditis americana n. sp. and is described in this study. Methods To describe H. americana n. sp., we carried out phylogenetic reconstructions using multiple genes, characterized their morphology, conducted self-crossing and cross-hybridization experiments, and isolated and identified their symbiotic bacteria. Results Heterorhabditis americana n. sp. is molecularly and morphologically similar to H. georgiana . Morphological differences between the males of H. americana n. sp. and H. georgiana include variations in the excretory pore position, the gubernaculum size, the gubernaculum-to-spicule length ratio, the tail length, and the body diameter. Infective juveniles (IJs) of H. americana n. sp. differ from H. georgiana IJs because H. americana n. sp. IJs have an invisible bacterial cell pouch posterior to the cardia and a small posterior phasmid, whereas H. georgiana IJs have a visible bacterial cell pouch and an inconspicuous phasmid. Hermaphrodites of H. americana n. sp. and H. georgiana are differentiated by the body length, the nerve ring distance from the anterior end, the excretory pore distance from the anterior end, the anal body diameter, and the c′ ratio. Females of H. americana n. sp. can be differentiated from H. georgiana females by the anal body diameter and the c′ ratio. Reproductive isolation was confirmed, as H. americana n. sp. does not produce viable offspring with any of the species of the “ Bacteriophora ” clade. Heterorhabditis americana n. sp. is associated with the symbiotic bacterium Photorhabdus kleinii . Conclusions Based on the observed morphological and morphometric differences, the distinct phylogenetic placement, and the reproductive isolation, the nematode isolates S8 and S10 represent a novel species, which we named Heterorhabditis americana n. sp. This study provides a detailed characterization of this novel species, contributing to enhancing our knowledge of species diversity and evolutionary relationships of the Heterorhabditis genus. Graphical Abstract

Species of the genus Rhabdias Stiles & Hassall, 1905 are common parasitic nematodes occurring in the lungs of amphibians and reptiles worldwide. In the present study, Rhabdias macrocephalum n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions, and mitochondrial cox1 , cox2 , and 12S genes) based on specimens collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China. The complete mitochondrial genome of R. macrocephalum n. sp. was sequenced and annotated: it is 14,819 bp in length, including 12 protein coding genes (missing atp 8), 22 tRNA genes, 2 rRNA genes and three non-coding regions. The gene arrangement of R. macrocephalum n. sp. is different from all of the currently available mitogenomes of nematodes and represents a novel type of mitochondrial gene arrangement reported in Nematoda. Molecular phylogenetic results based on the ITS + 28S data support the monophyly of Entomelas , Pneumonema , Serpentirhabdias , and Rhabdias , and showed R. macrocephalum n. sp. forming a most basal lineage in Rhabdias . Les espèces du genre Rhabdias Stiles & Hassall, 1905 sont des nématodes parasites courants présents dans les poumons des amphibiens et des reptiles du monde entier. Dans cette étude, Rhabdias macrocephalum n. sp. est décrit à l’aide de méthodes morphologiques intégrées (microscopie optique et électronique à balayage) et d’approches moléculaires (séquençage des régions nucléaires 28S et ITS et des gènes mitochondriaux cox1 , cox2 et 12S ) basées sur des spécimens collectés chez le lézard Diploderma splendidum (Barbour & Dunn) (Reptilia : Agamidae) de Chine. Le génome mitochondrial complet de R. macrocephalum n. sp. a été séquencé et annoté : il a une longueur de 14 819 pb, dont 12 gènes codants pour des protéines ( atp8 manquant), 22 gènes d’ARNt, 2 gènes d’ARNr et trois régions non codantes. L’arrangement génétique de R. macrocephalum n. sp. est différent de tous les mitogénomes de nématodes actuellement disponibles et représente un nouveau type d’arrangement de gènes mitochondriaux signalé chez les nématodes. Les résultats phylogénétiques moléculaires basés sur les données ITS + 28S ont soutenu la monophylie d’ Entomelas , Pneumonema , Serpentirhabdias et Rhabdias , et ont montré que R. macrocephalum n. sp. forme la lignée la plus basale chez Rhabdias .

The genus Phasmarhabditis is an economically important group of rhabditid nematodes, to which the well-known slug-parasite P. hermaphrodita belongs. Despite the commercial use of Phasmarhabditis species as an attractive and promising approach for pest control, the taxonomy and systematics of this group of rhabditids are poorly understood, largely because of the lack of diagnostic morphological features and DNA sequences for distinguishing species or inferring phylogenetic relationship. During a nematode sampling effort for identifying free-living relatives of Caenorhabditis elegans in Huizhou City, Guangdong, China, a novel species belonging to the genus Phasmarhabditis was isolated from rotting leaves. Detailed morphology of the gonochoristic P. huizhouensis sp. nov. was described and illustrated. The adult female has a robust body, a relatively short and wide buccal capsule conjoined by a rhabditiform pharynx. Females are characterized by a short cupola-shaped tail end bearing a slender pointed tip, with the junction flanked by a pair of 'rod-like' phasmids. Males have an open peloderan bursa that is supported by 9 pairs of genital papillae and 1 terminal pair of phasmids. P. huizhouensis sp. nov. is morphologically very similar to the type species Phasmarhabditis papillosa but is distinguishable by its male caudal traits. The new species is readily differentiated from other taxa in the genus by its female tail shape. Molecular phylogenetic inferences based on small subunit (SSU) and the D2-D3 domain of large subunit (LSU) ribosomal DNA genes reveal that P. huizhouensis sp. nov. forms a unique branch in both phylogenies which is genetically related to P. hermaphrodita and other parasites such as Angiostoma spp. The host associations of P. huizhouensis sp. nov. and its ability to parasitize slugs are unknown.

The genus Rhabdias Stiles & Hassal, 1905 includes about 83 species of nematodes parasitic in amphibians and reptiles worldwide. Herein, we describe Rhabdias glaurungi sp. nov. from the hylid frog Scinax gr. ruber (Laurenti, 1768) in the Gunma Ecological Park, Santa Bárbara municipality, state of Pará, Brazil. This species has six small lips, an inflated cuticle along the entire body and a cup-shaped buccal capsule with smooth internal surface of its anterior part and irregularly folded internal surface of its posterior part in apical view. From the 17 valid species recognized in the Neotropical realm, the new species can be distinguished by the number of lips, the morphology and size of its buccal capsule, as well as the extent and shape of its cuticular inflation; in addition, there are molecular differences. Sequences of the mitochondrial Cytochrome c Oxidase subunit I gene strongly support the status of this form as a separate species. Molecular phylogenetic analysis shows R. glaurungi sp. nov. nested within the R. pseudosphaerocephala Kuzmin, Tkach & Brooks, 2007 species complex. Rhabdias glaurungi sp. nov. is the second species of the genus described from hosts of the family Hylidae in the Neotropical realm. We conclude that the diversity of Rhabdias within the Neotropics is likely largely underestimated.

A new entomopathogenic nematode in the genus Heterorhabditis is described from South Africa, from two singular isolates found 1000 km from each other, from beneath a fig tree and in a citrus orchard, respectively. Morphological and molecular studies indicate both isolates to be the same and a new undescribed Heterorhabditis species. Comparison of sequences of the internal transcribed spacer (ITS) rDNA and the D2D3 region of the 28S rDNA gene with available sequences of other described species within the genus, indicate the two isolates as a new species. Phylogenetic analysis of the sequence data concerned placed the new species, H. noenieputensis n. sp., closest to H. indica and H. gerrardi in the indica-group. The new species, H. noenieputensis n. sp., is distinguished from other species in the genus by a combination of several morphological traits of the males and the infective juveniles (IJs). The new species differs from all other species previously described, as regards the body length of the IJs, except for H. indica and H. taysearae, in which the IJ is smaller. The IJ also differs from that of H. indica in the length of the oesophagus, the body diameter, the length of the tail and the E%. In addition, males of H. noenieputensis n. sp. differ from their closest relative, H. indica, in the position of the excretory pore, SW% and D%; and from H. gerrardi in the length of the oesophagus and SW%. The seventh pair of genital papillae of H. noenieputensis n. sp. are normally developed, while for H. indica they are often branched or swollen at the base, while 8 and 9 are usually absent in both species.

Serpentirhabdias mussuranae n. sp. is described from the lungs of the mussurana, Clelia clelia (Daudin, 1803), from vicinities of Lábrea, Amazonas State, Brazil. The species is characterized by the triangular oral opening, the presence of teeth (onchia) in the oesophastome, the excretory glands longer than the oesophagus and the tail abruptly narrowing in its anterior half and gradually tapering in posterior half. Among the Neotropical representatives of the genus, three species are known to possess the onchia in the oesophastome: S. atroxi , S. moi and S. viperidicus . Serpentirhabdias mussuranae n. sp. differs from S. atroxi and S. viperidicus by its triangular shape of the oral opening and the oesophastome in apical view, vs. round in the latter two congeners. Additionally, S. viperidicus has a larger oesophastome, 13–22 micrometers wide and 13–23 micrometers deep. The new species has relatively longer excretory glands than S. moi . The new species is morphologically and genetically close to S. atroxi , S. moi and S. viperidicus , all parasitic in Brazilian snakes, based on the presence of onchia and the comparison of nucleotide sequences of nuclear ribosomal DNA and mitochondrial cox1 gene (differences varied between 3.8% and 7.1%). Data on the life cycle of S. mussuranae n. sp. is provided, and the life cycle is typical of the genus Serpentirhabdias , with the combination of direct development and heterogony. Free-living larval stages and the adults of amphimictic free-living generation are described. The results of molecular phylogenetic analysis based on nuclear ribosomal internal transcribed spacer (ITS) + partial 28S region and partial mitochondrial cox1 gene are provided.

Abstract Entomopathogenic nematodes (EPN) belonging to the Heterorhabditidae family are lethal parasites of soil-dwelling insects. Two species were reported in Argentina: Heterorhabditis argentinensis and Heterorhabditis bacteriophora characterized mainly by morphometric features. In this work a comparative and phylogenetic study between five Heterorhabditis populations from Argentina was conducted to analyze the variability between strains and to evaluate the taxonomic position of Heterorhabditis argentinensis. The PCA analyses of morphometric characters separated the larger juvenile, female and male H. argentinensis from H. bacteriophora populations. The juvenile (IJs) stage provided the clearest separation of Heterorhabditis populations presenting the least variability between strains. The variable L and MBW were highly related to H. argentinensis IJs. Three groups were separated by this stage considering PC1 and PC2: one formed by H. bacteriophora OLI, RIV and RN strains, (isolates from Córdoba and Río Negro province), one for H. bacteriophora VELI strain (Buenos Aires province) and one for H. argentinensis (Santa Fe province). Heterorhabditis bacteriophora VELI and H. argentinensis isolated from regions with more rainfalls and humidity presented larger values for morphometric features. Molecular analyses showed the Argentinian populations (H. bacteriophora VELI strain and H. argentinensis), forming a same clade, with six other H. bacteriophora populations (not from Argentina) with a genetic similarity between them of 99%. Heterorhabditis argentinensis presented one unique nucleotide that was not present in any of the other species of the clade. Considering the results of this study H. argentinensis would be conspecific to H. bacteriophora, constituting a strain with a great morphometric variation where the host and climatic conditions could have influenced on the measurements. Resumo Nematóides entomopatogênicos (EPN) pertencentes à família Heterorhabditidae são parasitas letais de insetos que vivem no solo. Duas espécies foram relatados na Argentina: Heterorhabditis argentinensis e Heterorhabditis bacteriophora, caracterizada principalmente por características morfométricas. Neste trabalho um estudo comparativo e filogenética entre cinco populações do Heterorhabditis da Argentina foi conduzido para analisar a variabilidade entre as linhagens e avaliar a posição taxonômica das Heterorhabditis argentinensis. Características morfométricas de Heterorhabditis bacteriophora VELI e H. argentinensis isoladas de regiões com mais chuvas e umidade apresentaram dimensões maiores. Analisa o PCA de personagens morfométricas separou a maior juvenil, feminino e masculino H. argentinensis de H. bacteriophora populações. A fase juvenil (JIs) fornece a mais clara separação de populações Heterorhabditis apresentando a menor variação entre as cepas. A L variável e MBW foram altamente relacionada com H. argentinensis JIs. Três grupos foram separados por esta fase considerando PC1 e PC2: um formado por H. bacteriophora OLI, RIV e estirpes RN, (isolados de Córdoba e província de Rio Negro), um para a estirpe H. bacteriophora VELI (província de Buenos Aires) e um para H. argentinensis (província de Santa Fe). Heterorhabditis bacteriophora VELI e H. argentinensis isolado a partir de regiões com mais chuvas e umidade apresentaram maiores valores para as características morfométricas. A análise molecular mostrou as populações da Argentina (estirpe H. bacteriophora VELI e H. argentinensis), formando um mesmo subtipo, com seis outras populações H. bacteriophora (não da Argentina), com uma similaridade genética entre eles de 99%. Heterorhabditis argentinensis apresentado um único nucleótido que não estava presente em nenhum dos outros espécies do clado. Considerando os resultados deste estudo H. argentinensis seria conspecific a H. bacteriophora, constituindo uma estirpe com uma grande variação morfométrica onde o anfitrião e as condições climáticas podem ter influenciado nas medições.

Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) is described from the lungs of the ‘Brazilian lancehead’ Bothrops moojeni (Hoge, 1966) from the savannah in São Paulo State, Brazil. The new species is the eighth species of Serpentirhabdias described in the Neotropical region, and differs from other species mainly by a combination of characters: lips slightly notable, presence of fine striations at posterior ends, presence of two parallel lines with intercalated pores, a pore-shaped phasmid situated at the level of the anal aperture and another two in the posterior half of the tail. It is the first species of Serpentirhabdias reported in this snake host and the second species of this genus found parasitizing South American viperidian snakes. Molecular phylogenetic analysis using ribosomal (ITS and 28S partial) genes confirms Serpentirhabdias viperidicus n. sp. as a new species that clustered in the Serpentirhabdias clade, sister taxon to Serpentirhabdias fuscovenosa and Serpentirhabdias elaphe. This is the first description of Serpentirhabdias species from Brazil using molecular approaches and morphological characters to confirm the monophyly of this recent genus.

Angiostoma norvegicum n. sp. (Angiostomatidae) is described from the oesophagus, crop and the buccal mass of five species of slugs of the family Arionidae, Arion vulgaris (Moquin-Tandon), Arion ater (L.), Arion fasciatus (Nilsson), Arion fuscus (Müller) and Arion rufus/Arion ater hybrid), collected throughout Norway. Angiostoma norvegicum n. sp. was found parasitising arionids at seven of the 30 sample sites examined (23.3%), and 9.9% of all Arion spp. were infected with this nematode. The new species is characterised by its large size (4.0–8.6 mm long) and in having: lateral alae; 6 + 6 papillae at the cephalic end; a large circular mouth aperture; a spacious stoma; a pharyngeal basal bulb without valvular apparatus; an excretory pore near the base of bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1+2/3+3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses demonstrated that although Angiostoma spp. cluster together, A. norvegicum n. sp. forms a tight monophyletic clade with the milacid nematode parasites Angiostoma margaretae Ross, Malan & Ivanova, 2011 and Angiostoma milacis Ivanova & Wilson, 2009.