System-wide rewiring underlies behavioral differences in predatory and bacterial-feeding nematodes.

The relationship between neural circuit function and patterns of synaptic connectivity is poorly understood, in part due to a lack of comparative data for larger complete systems. We compare system-wide maps of synaptic connectivity generated from serial transmission electron microscopy for the pharyngeal nervous systems of two nematodes with divergent feeding behavior: the microbivore Caenorhabditis elegans and the predatory nematode Pristionchus pacificus. We uncover a massive rewiring in a complex system of identified neurons, all of which are homologous based on neurite anatomy and cell body position. Comparative graph theoretical analysis reveals a striking pattern of neuronal wiring with increased connectional complexity in the anterior pharynx correlating with tooth-like denticles, a morphological feature in the mouth of P. pacificus. We apply focused centrality methods to identify neurons I1 and I2 as candidates for regulating predatory feeding and predict substantial divergence in the function of pharyngeal glands.

A developmental switch coupled to the evolution of plasticity acts through a sulfatase.

Developmental plasticity has been suggested to facilitate phenotypic diversity, but the molecular mechanisms underlying this relationship are little understood. We analyzed a feeding dimorphism in Pristionchus nematodes whereby one of two alternative adult mouth forms is executed after an irreversible developmental decision. By integrating developmental genetics with functional tests in phenotypically divergent populations and species, we identified a regulator of plasticity, eud-1, that acts in a developmental switch. eud-1 mutations eliminate one mouth form, whereas overexpression of eud-1 fixes it. EUD-1 is a sulfatase that acts dosage dependently, is necessary and sufficient to control the sexual dimorphism of feeding forms, and has a conserved function in Pristionchus evolution. It is epistatic to known signaling cascades and results from lineage-specific gene duplications. EUD-1 thus executes a developmental switch for morphological plasticity in the adult stage, showing that regulatory pathways can evolve by terminal addition of new genes.

The role of plasticity and stochasticity in coexistence.

Species coexistence in ecological communities is a central feature of biodiversity. Different concepts, i.e., contemporary niche theory, modern coexistence theory, and the unified neutral theory, have identified many building blocks of such ecological assemblies. However, other factors, such as phenotypic plasticity and stochastic inter-individual variation, have received little attention, in particular in animals. For example, how resource polyphenisms resulting in predator-prey interactions affect coexistence is currently unknown. Here, we present an integrative theoretical-experimental framework using the nematode plasticity model Pristionchus pacificus with its well-studied mouth-form dimorphism resulting in cannibalism. We develop an individual-based model that relies upon synthetic data based on our empirical measurements of fecundity and polyphenism to preserve demographic heterogeneity. We demonstrate how the interplay between plasticity and individual stochasticity result in all-or-nothing outcomes at the local level. Coexistence is made possible when spatial structure is introduced.

Feeding-structure morphogenesis in "rhabditid" and diplogastrid nematodes is not controlled by a conserved genetic module.

Disentangling the evolution of the molecular processes and genetic networks that facilitate the emergence of morphological novelties is one of the main objectives in evolutionary developmental biology. Here, we investigated the evolutionary history of a gene regulatory network controlling the development of novel tooth-like feeding structures in diplogastrid nematodes. Focusing on NHR-1 and NHR-40, the two transcription factors that regulate the morphogenesis of these feeding structures in Pristionchus pacificus, we sought to determine whether they have a similar function in Caenorhabditis elegans, an outgroup species to the Diplogastridae which has typical "rhabditid" flaps instead of teeth. Contrary to our initial expectations, we found that they do not have a similar function. While both receptors are co-expressed in the tissues that produce the feeding structures in the two nematodes, genetic inactivation of either receptor had no impact on feeding-structure morphogenesis in C. elegans. Transcriptomic experiments revealed that NHR-1 and NHR-40 have highly species-specific regulatory targets. These results suggest two possible evolutionary scenarios: either the genetic module responsible for feeding-structure morphogenesis in Diplogastridae already existed in the last common ancestor of C. elegans and P. pacificus, and subsequently disintegrated in the former as NHR-1 and NHR-40 acquired new targets, or it evolved in conjunction with teeth in Diplogastridae. These findings indicate that feeding-structure morphogenesis is regulated by different genetic programs in P. pacificus and C. elegans, hinting at developmental systems drift during the flap-to-tooth transformation. Further research in other "rhabditid" species is needed to fully reconstruct the developmental genetic changes which facilitated the evolution of novel feeding structures in Diplogastridae.

Single worm transcriptomics identifies a developmental core network of oscillating genes with deep conservation across nematodes.

High-resolution spatial and temporal maps of gene expression have facilitated a comprehensive understanding of animal development and evolution. In nematodes, the small body size represented a major challenge for such studies, but recent advancements have helped overcome this limitation. Here, we have implemented single worm transcriptomics (SWT) in the nematode model organism Pristionchus pacificus to provide a high-resolution map of the developmental transcriptome. We selected 38 time points from hatching of the J2 larvae to young adults to perform transcriptome analysis over 60 h of postembryonic development. A mean sequencing depth of 4.5 million read pairs allowed the detection of more than 23,135 (80%) of all genes. Nearly 3000 (10%) genes showed oscillatory expression with discrete expression levels, phases, and amplitudes. Gene age analysis revealed an overrepresentation of ancient gene classes among oscillating genes, and around one-third of them have 1:1 orthologs in C. elegans One important gene family overrepresented among oscillating genes is collagens. Several of these collagen genes are regulated by the developmental switch gene eud-1, indicating a potential function in the regulation of mouth-form plasticity, a key developmental process in this facultative predatory nematode. Together, our analysis provides (1) an updated protocol for SWT in nematodes that is applicable to many microscopic species, (2) a 1- to 2-h high-resolution catalog of P. pacificus gene expression throughout postembryonic development, and (3) a comparative analysis of oscillatory gene expression between the two model organisms P. pacificus and C. elegans and associated evolutionary dynamics.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2, and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25, and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus.

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.

Nematode biphasic 'boom and bust' dynamics are dependent on host bacterial load while linking dauer and mouth-form polyphenisms.

Cross-kingdom interactions involve dynamic processes that shape terrestrial ecosystems and represent striking examples of co-evolution. The multifaceted relationships of entomopathogenic nematodes with their insect hosts and symbiotic bacteria are well-studied cases of co-evolution and pathogenicity. In contrast, microbial interactions in soil after the natural death of insects and other invertebrates are minimally understood. In particular, the turnover and succession of nematodes and bacteria during insect decay have not been well documented - although it represents a rich ecological niche with multiple species interactions. Here, we utilize developmentally plastic nematode Pristionchus pacificus and its associated scarab beetles as models. On La Réunion Island, we collected rhinoceros beetle Oryctes borbonicus, induced death, and placed carcasses in cages both on the island and in a mock-natural environment in the laboratory controlling for high spatial and temporal resolution. Investigating nematode population density and dispersal dynamics, we were able to connect two imperative plasticities, dauer and mouth form. We observed a biphasic 'boom and bust' dispersal dynamic of dauer larvae that corresponds to bacterial load on carcasses but not bacterial type. Strikingly, all post-dauer adults have the predatory mouth form, demonstrating novel intricate interactions on decaying insect hosts. Thus, ecologically relevant survival strategies incorporate critical plastic traits.

Two independent sulfation processes regulate mouth-form plasticity in the nematode Pristionchus pacificus.

Sulfation of biomolecules, like phosphorylation, is one of the most fundamental and ubiquitous biochemical modifications with important functions during detoxification. This process is reversible, involving two enzyme classes: a sulfotransferase, which adds a sulfo group to a substrate; and a sulfatase that removes the sulfo group. However, unlike phosphorylation, the role of sulfation in organismal development is poorly understood. In this study, we find that two independent sulfation events regulate the development of mouth morphology in the nematode Pristionchus pacificus. This nematode has the ability to form two alternative mouth morphologies depending on environmental cues, an example of phenotypic plasticity. We found that, in addition to a previously described sulfatase, a sulfotransferase is involved in regulating the mouth-form dimorphism in P. pacificus However, it is unlikely that both of these sulfation-associated enzymes act upon the same substrates, as they are expressed in different cell types. Furthermore, animals mutant in genes encoding both enzymes show condition-dependent epistatic interactions. Thus, our study highlights the role of sulfation-associated enzymes in phenotypic plasticity of mouth structures in Pristionchus.

Cilia drive developmental plasticity and are essential for efficient prey detection in predatory nematodes.

Cilia are complex organelles involved in a broad array of functions in eukaryotic organisms. Nematodes employ cilia for environmental sensing, which shapes developmental decisions and influences morphologically plastic traits and adaptive behaviours. Here, we assess the role of cilia in the nematode Pristionchus pacificus, and determine their importance in regulating the developmentally plastic mouth-form decision in addition to predatory feeding and self-recognition behaviours, all of which are not present in Caenorhabditis elegans. An analysis of a multitude of cilia-related mutants including representatives of the six protein subcomplexes required in intraflagellar transport (IFT) plus the regulatory factor X transcription factor daf-19 revealed that cilia are essential for processing the external cues influencing the mouth-form decision and for the efficient detection of prey. Surprisingly, we observed that loss-of-function mutations in the different IFT components resulted in contrasting mouth-form phenotypes and different degrees of predation deficiencies. This observation supports the idea that perturbing different IFT subcomplexes has different effects on signalling downstream of the cilium. Finally, self-recognition was maintained in the cilia deficient mutants tested, indicating that the mechanisms triggering self-recognition in P. pacificus may not require the presence of fully functional cilia.

Two new Species of Pristionchus (Nematoda: Diplogastridae) include the Gonochoristic Sister Species of P. fissidentatus.

The genus Pristionchus (Kreis, 1932) consists of more than 30 soil nematode species that are often found in association with scarab beetles. Three major radiations have resulted in the "maupasi species group" in America, the "pacificus species group" in Asia, and the "lheritieri species group," which contains species from Europe and Asia. Phylogenetic analysis indicates that a group of three species, including the gonochorists P. elegans and P. bucculentus and the hermaphrodite P. fissidentatus, is basal to the above-mentioned radiations. Two novel species are described here: Pristionchus paulseni sp. n. from Taiwan and P. yamagatae sp. n. from Japan by means of morphology, morphometrics and genome-wide transcriptome sequence analysis. Previous phylotranscriptomic analysis of the complete Pristionchus genus recognized P. paulseni sp. n. as the sister species of P. fissidentatus, and thus its importance for macro-evolutionary studies. Specifically, the gonochorist P. paulseni sp. n. and the hermaphrodite P. fissidentatus form a species pair that is the sister group to all other described Pristionchus species. P. paulseni sp. n. has two distinct mouth forms, supporting the notion that the mouth dimorphism is ancestral in the genus Pristionchus.The genus Pristionchus (Kreis, 1932) consists of more than 30 soil nematode species that are often found in association with scarab beetles. Three major radiations have resulted in the "maupasi species group" in America, the "pacificus species group" in Asia, and the "lheritieri species group," which contains species from Europe and Asia. Phylogenetic analysis indicates that a group of three species, including the gonochorists P. elegans and P. bucculentus and the hermaphrodite P. fissidentatus, is basal to the above-mentioned radiations. Two novel species are described here: Pristionchus paulseni sp. n. from Taiwan and P. yamagatae sp. n. from Japan by means of morphology, morphometrics and genome-wide transcriptome sequence analysis. Previous phylotranscriptomic analysis of the complete Pristionchus genus recognized P. paulseni sp. n. as the sister species of P. fissidentatus, and thus its importance for macro-evolutionary studies. Specifically, the gonochorist P. paulseni sp. n. and the hermaphrodite P. fissidentatus form a species pair that is the sister group to all other described Pristionchus species. P. paulseni sp. n. has two distinct mouth forms, supporting the notion that the mouth dimorphism is ancestral in the genus Pristionchus.

The Role of DAF-21/Hsp90 in Mouth-Form Plasticity in Pristionchus pacificus.

Phenotypic plasticity is increasingly recognized to facilitate adaptive change in plants and animals, including insects, nematodes, and vertebrates. Plasticity can occur as continuous or discrete (polyphenisms) variation. In social insects, for example, in ants, some species have workers of distinct size classes while in other closely related species variation in size may be continuous. Despite the abundance of examples in nature, how discrete morphs are specified remains currently unknown. In theory, polyphenisms might require robustness, whereby the distribution of morphologies would be limited by the same mechanisms that execute buffering from stochastic perturbations, a function attributed to heat-shock proteins of the Hsp90 family. However, this possibility has never been directly tested because plasticity and robustness are considered to represent opposite evolutionary principles. Here, we used a polyphenism of feeding structures in the nematode Pristionchus pacificus to test the relationship between robustness and plasticity using geometric morphometrics of 20 mouth-form landmarks. We show that reducing heat-shock protein activity, which reduces developmental robustness, increases the range of mouth-form morphologies. Specifically, elevated temperature led to a shift within morphospace, pharmacological inhibition of all Hsp90 genes using radicicol treatment increased shape variability in both mouth-forms, and CRISPR/Cas9-induced Ppa-daf-21/Hsp90 knockout had a combined effect. Thus, Hsp90 canalizes the morphologies of plastic traits resulting in discrete polyphenism of mouth-forms.

Co-option of the hormone-signalling module dafachronic acid-DAF-12 in nematode evolution.

Morphological novelties are lineage-specific traits that serve new functions. Developmental polyphenisms have been proposed to be facilitators of phenotypic evolution, but little is known about the interplay between the associated genetic and environmental factors. Here, we study two alternative morphologies in the mouth of the nematode Pristionchus pacificus and the formation of teeth-like structures that are associated with bacteriovorous feeding and predatory behaviour on fungi and other worms. These teeth-like denticles represent an evolutionary novelty, which is restricted to some members of the nematode family Diplogastridae but is absent from Caenorhabditis elegans and related nematodes. We show that the mouth dimorphism is a polyphenism that is controlled by starvation and the co-option of an endocrine switch mechanism. Mutations in the nuclear hormone receptor DAF-12 and application of its ligand, the sterol hormone dafachronic acid, strongly influence this switch mechanism. The dafachronic acid-DAF-12 module has been shown to control the formation of arrested dauer larvae in both C. elegans and P. pacificus, as well as related life-history decisions in distantly related nematodes. The comparison of dauer formation and mouth morphology switch reveals that different thresholds of dafachronic acid signalling provide specificity. This study shows how hormonal signalling acts by coupling environmental change and genetic regulation and identifies dafachronic acid as a key hormone in nematode evolution.

Predatory feeding behaviour in Pristionchus nematodes is dependent on phenotypic plasticity and induced by serotonin.

Behavioural innovation and morphological adaptation are intrinsically linked but their relationship is often poorly understood. In nematodes, a huge diversity of feeding morphologies and behaviours can be observed to meet their distinctive dietary and environmental demands. Pristionchus and their relatives show varied feeding activities, both consuming bacteria and also predating other nematodes. In addition, Pristionchus nematodes display dimorphic mouth structures triggered by an irreversible developmental switch, which generates a narrower mouthed form with a single tooth and a wider mouthed form with an additional tooth. However, little is known about the specific predatory adaptations of these mouth forms or the associated mechanisms and behaviours. Through a mechanistic analysis of predation behaviours, in particular in the model organism Pristionchus pacificus, we reveal multifaceted feeding modes characterised by dynamic rhythmic switching and tooth stimulation. This complex feeding mode switch is regulated by the neurotransmitter serotonin in a previously uncharacterised role, a process that appears conserved across several predatory nematode species. Furthermore, we investigated the effects of starvation, prey size and prey preference on P. pacificus predatory feeding kinetics, revealing predation to be a fundamental component of the P. pacificus feeding repertoire, thus providing an additional rich source of nutrition in addition to bacteria. Finally, we found that mouth form morphology also has a striking impact on predation, suppressing predatory behaviour in the narrow mouthed form. Our results therefore hint at the regulatory networks involved in controlling predatory feeding and underscore P. pacificus as a model for understanding the evolution of complex behaviours.

Pristionchus pacificus daf-16 is essential for dauer formation but dispensable for mouth form dimorphism.

The nematode Pristionchus pacificus shows two forms of phenotypic plasticity: dauer formation and dimorphism of mouth form morphologies. It can therefore serve as a model for studying the evolutionary mechanisms that underlie phenotypic plasticity. Formation of dauer larvae is observed in many other species and constitutes one of the most crucial survival strategies in nematodes, whereas the mouth form dimorphism is an evolutionary novelty observed only in P. pacificus and related nematodes. We have previously shown that the same environmental cues and steroid signaling control both dauer formation and mouth form dimorphism. Here, we examine by mutational analysis and whole-genome sequencing the function of P. pacificus (Ppa) daf-16, which encodes a forkhead transcription factor; in C. elegans, daf-16 is the target of insulin signaling and plays important roles in dauer formation. We found that mutations in Ppa-daf-16 cause strong dauer formation-defective phenotypes, suggesting that Ppa-daf-16 represents one of the evolutionarily conserved regulators of dauer formation. Upon strong dauer induction with lophenol, Ppa-daf-16 individuals formed arrested larvae that partially resemble wild-type dauer larvae, indicating that Ppa-daf-16 is also required for dauer morphogenesis. By contrast, regulation of mouth form dimorphism was unaffected by Ppa-daf-16 mutations and mutant animals responded normally to environmental cues. Our results suggest that mechanisms for dauer formation and mouth form regulation overlap partially, but not completely, and one of two key transcriptional regulators of the dauer regulatory network was either independently co-opted for, or subsequently lost by, the mouth form regulatory network.

Adaptive value of a predatory mouth-form in a dimorphic nematode.

Polyphenisms can be adaptations to environments that are heterogeneous in space and time, but to persist they require conditional-specific advantages. The nematode Pristionchus pacificus is a facultative predator that displays an evolutionarily conserved polyphenism of its mouthparts. During development, P. pacificus irreversibly executes either a eurystomatous (Eu) or stenostomatous (St) mouth-form, which differ in the shape and number of movable teeth. The Eu form, which has an additional tooth, is more complex than the St form and is thus more highly derived relative to species lacking teeth. Here, we investigate a putative fitness trade-off for the alternative feeding-structures of P. pacificus. We show that the complex Eu form confers a greater ability to kill prey. When adults were provided with a prey diet, Eu nematodes exhibited greater fitness than St nematodes by several measures, including longevity, offspring survival and fecundity when followed by bacterial feeding. However, the two mouth-forms had similar fecundity when fed ad libitum on bacteria, a condition that would confer benefit on the more rapidly developing St form. Thus, the two forms show conditional fitness advantages in different environments. This study provides, to our knowledge, the first functional context for dimorphism in a model for the genetics of plasticity.

Leptojacobus dorci n. gen., n. sp. (Nematoda: Diplogastridae), an Associate of Dorcus Stag Beetles (Coleoptera: Lucanidae).

A new species of diplogastrid nematode, Leptojacobus dorci n. gen., n. sp., was isolated from adults of the stag beetle Dorcus ritsemae (Coleoptera: Lucanidae) that were purchased from a pet shop in Japan. Leptojacobus n. gen. is circumscribed by a very thin, delicate body and by a small stoma with minute armature. A combination of other stomatal characters, namely the division of the cheilostom into adradial plates, the symmetry of the subventral stegostomatal sectors, and the presence of a thin, conical dorsal tooth, further distinguishes Leptojacobus n. gen. from other genera of Diplogastridae. Phylogenetic analysis of nearly full-length SSU rRNA sequences support the new species, together with an isolate identified previously as Koerneria luziae, to be excluded from a clade including all other molecularly characterized diplogastrids with teeth and stomatal dimorphism. Therefore, the new species will be of importance for reconstruction of ancestral character histories in Diplogastridae, a family circumscribed by a suite of feeding-related novelties.

Feeding plasticity in the nematode Pristionchus pacificus is influenced by sex and social context and is linked to developmental speed.

The increasing evidence for a role of developmental plasticity in evolution offers exciting prospects for testing interactions between ecological and developmental genetic processes. Recent advances with the model organism Pristionchus pacificus have provided inroads to a mechanistic understanding of a developmental plasticity. The developmental plasticity of P. pacificus comprises two discontinuous adult mouth-forms, a stenostomatous ("narrow mouthed") and a eurystomatous ("wide mouthed") form, the latter of which is structurally more complex and associated with predatory feeding. Both forms are consistently present in populations, but fundamental properties guiding fluctuations in their appearance have been poorly understood. Here, we provide a systematic characterization of the mouth plasticity in P. pacificus, quantifying a strong sexual dimorphism and revealing that, in an inbred genetic background, maternal phenotype is linked to that of male offspring. Furthermore, cues from conspecifics influenced the developmental decision in juvenile nematodes. Separating individuals from a population resulted in a lower eurystomatous frequency, which decreased incrementally with earlier isolation. Finally, the time to the reproductively mature stage was, in the presence of an abundant bacterial food supply, less for stenostomatous than for eurystomatous individuals, suggesting the potential for a fitness trade-off between developmental time and breadth of diet. This study provides a baseline understanding of the mouth dimorphism in P. pacificus as a necessary reference point for comparative analysis.

Two new species of Pristionchus (Nematoda: Diplogastridae) support the biogeographic importance of Japan for the evolution of the genus Pristionchus and the model system P. pacificus.

Pristionchus pacificus Sommer, Carta, Kim, and Sternberg, 1996 is an important model organism in evolutionary biology that integrates developmental biology with ecology and population genetics. This species is part of a sub-complex of the genus Pristionchus that is considered to have originated in East Asia. Here, we describe two new species of Pristionchus, P. maxplancki and P. quartusdecimus, which were isolated from beetles in Japan, supporting the hypothesis that a region including Japan is the origin of diversification of the P. pacificus species complex. Phytogeny inferred from a partial small subunit rRNA gene and 25 ribosomal protein genes shows P. maxplancki to be the closest known outgroup to a triad of sibling species, including P. pacificus. Pristionchus quartusdecimus is a putative outgroup to the P. pacificus species complex, supporting a more ancient origin of Pristionchus species in the region. Species diagnoses are based on morphological and molecular characters, in addition to reproductive isolation for P. maxplancki. Members of the P. pacificus species complex as well as P. quartusdecimus are distinguished by stegostomatal structures, male genital papilla arrangement, and gubernaculum shape. The discovery of a new member of the P. pacificus species complex allows greater precision in polarizing and reconstructing ancestral states in the comparative model system centering on P. pacificus. Together with previous reports, these findings support an important biogeographic role of Japan in the evolution of the genus Pristionchus and the P. pacificus species complex, especially the associated phenotypic evolution of mouth morphology.