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.

Spatial and temporal heterogeneity alter the cost of plasticity in Pristionchus pacificus.

Phenotypic plasticity, the ability of a single genotype to produce distinct phenotypes under different environmental conditions, has become a leading concept in ecology and evolutionary biology, with the most extreme examples being the formation of alternative phenotypes (polyphenisms). However, several aspects associated with phenotypic plasticity remain controversial, such as the existence of associated costs. While already predicted by some of the pioneers of plasticity research, i.e. Schmalhausen and Bradshaw, experimental and theoretical approaches have provided limited support for the costs of plasticity. In experimental studies, one common restriction is the measurement of all relevant parameters over long time periods. Similarly, theoretical studies rarely use modelling approaches that incorporate specific experimentally-derived fitness parameters. Therefore, the existence of the costs of plasticity remains disputed. Here, we provide an integrative approach to understand the cost of adaptive plasticity and its ecological ramifications, by combining laboratory data from the nematode plasticity model system Pristionchus pacificus with a stage-structured population model. Taking advantage of measurements of two isogenic strains grown on two distinct diets, we illustrate how spatial and temporal heterogeneity with regard to the distribution of resources on a metapopulation can alter the outcome of the competition and alleviate the realized cost of plasticity.

Chitin contributes to the formation of a feeding structure in a predatory nematode.

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

A New Hope: A Hermaphroditic Nematode Enables Analysis of a Recent Whole Genome Duplication Event.

Whole genome duplication (WGD) is often considered a major driver of evolution that leads to phenotypic novelties. However, the importance of WGD for evolution is still controversial because most documented WGD events occurred anciently and few experimental systems amenable to genetic analysis are available. Here, we report a recent WGD event in the hermaphroditic nematode Allodiplogaster sudhausi and present a comparison with a gonochoristic (male/female) sister species that did not undergo WGD. Self-fertilizing reproduction of A. sudhausi makes it amenable to functional analysis and an ideal system to study WGD events. We document WGD in A. sudhausi through karyotype analysis and whole genome sequencing, the latter of which allowed us to 1) identify functional bias in retention of protein domains and metabolic pathways, 2) show most duplicate genes are under evolutionary constraint, 3) show a link between sequence and expression divergence, and 4) characterize differentially expressed duplicates. We additionally show WGD is associated with increased body size and an abundance of repeat elements (36% of the genome), including a recent expansion of the DNA-hAT/Ac transposon family. Finally, we demonstrate the use of CRISPR/Cas9 to generate mutant knockouts, whereby two WGD-derived duplicate genes display functional redundancy in that they both need to be knocked out to generate a phenotype. Together, we present a novel experimental system that is convenient for examining and characterizing WGD-derived genes both computationally and functionally.

Multidimensional competition of nematodes affects plastic traits in a beetle ecosystem.

Resource competition has driven the evolution of novel polyphenisms in numerous organisms, enhancing fitness in constantly changing environmental conditions. In natural communities, the myriad interactions among diverse species are difficult to disentangle, but the multidimensional microscopic environment of a decaying insect teeming with bacteria and fighting nematodes provides pliable systems to investigate. Necromenic nematodes of the family Diplogastridae live on beetles worldwide, innocuously waiting for their hosts' deaths to feast on the blooming bacteria. Often, more than one worm species either affiliates with the insect or joins the microbial meal; thus, competition over limited food ensues, and phenotypic plasticity provides perks for species capable of employing polyphenisms. The recently established system of cockchafer Gymnogaster bupthalma and its occasional co-infestation of Pristionchus mayeri and Acrostichus spp. has revealed that these worms will simultaneously utilize two polyphenisms to thrive in a competitive environment. While both genera maintain plastic capacities in mouth form (strictly bacterial-feeding and omnivorous predation) and developmental pathway (direct and arrested development, dauer), P. mayeri employs both when faced with competition from Acrostichus. Here, we took advantage of the malleable system and added a third competitor, model nematode Pristionchus pacificus. Intriguingly, with a third competitor, P. mayeri is quicker to exit dauer and devour available food, while Acrostichus hides in dauer, waiting for the two Pristionchus species to leave the immediate environment before resuming development. Thus, experimental manipulation of short-lived ecosystems can be used to study the roles of polyphenisms in organismal interactions and their potential significance for evolution.

Sex or cannibalism: Polyphenism and kin recognition control social action strategies in nematodes.

Resource polyphenisms, where single genotypes produce alternative feeding strategies in response to changing environments, are thought to be facilitators of evolutionary novelty. However, understanding the interplay between environment, morphology, and behavior and its significance is complex. We explore a radiation of Pristionchus nematodes with discrete polyphenic mouth forms and associated microbivorous versus cannibalistic traits. Notably, comparing 29 Pristionchus species reveals that reproductive mode strongly correlates with mouth-form plasticity. Male-female species exhibit the microbivorous morph and avoid parent-offspring conflict as indicated by genetic hybrids. In contrast, hermaphroditic species display cannibalistic morphs encouraging competition. Testing predation between 36 co-occurring strains of the hermaphrodite P. pacificus showed that killing inversely correlates with genomic relatedness. These empirical data together with theory reveal that polyphenism (plasticity), kin recognition, and relatedness are three major factors that shape cannibalistic behaviors. Thus, developmental plasticity influences cooperative versus competitive social action strategies in diverse animals.

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.

Phenotypic Plasticity: From Theory and Genetics to Current and Future Challenges.

Phenotypic plasticity is defined as the property of organisms to produce distinct phenotypes in response to environmental variation. While for more than a century, biologists have proposed this organismal feature to play an important role in evolution and the origin of novelty, the idea has remained contentious. Plasticity is found in all domains of life, but only recently has there been an increase in empirical studies. This contribution is intended as a fresh view and will discuss current and future challenges of plasticity research, and the need to identify associated molecular mechanisms. After a brief summary of conceptual, theoretical, and historical aspects, some of which were responsible for confusion and contention, I will formulate three major research directions and predictions for the role of plasticity as a facilitator of novelty. These predictions result in a four-step model that, when properly filled with molecular mechanisms, will reveal plasticity as a major factor of evolution. Such mechanistic insight must be complemented with comparative investigations to show that plasticity has indeed created novelty and innovation. Together, such studies will help develop a true developmental evolutionary biology.

Small peptide-mediated self-recognition prevents cannibalism in predatory nematodes.

Self-recognition is observed abundantly throughout the natural world, regulating diverse biological processes. Although ubiquitous, often little is known of the associated molecular machinery, and so far, organismal self-recognition has never been described in nematodes. We investigated the predatory nematode Pristionchus pacificus and, through interactions with its prey, revealed a self-recognition mechanism acting on the nematode surface, capable of distinguishing self-progeny from closely related strains. We identified the small peptide SELF-1, which is composed of an invariant domain and a hypervariable C terminus, as a key component of self-recognition. Modifications to the hypervariable region, including single-amino acid substitutions, are sufficient to eliminate self-recognition. Thus, the P. pacificus self-recognition system enables this nematode to avoid cannibalism while promoting the killing of competing nematodes.

Developmental Plasticity and Robustness of a Nematode Mouth-Form Polyphenism.

In the last decade, case studies in plants and animals provided increasing insight into the molecular mechanisms of developmental plasticity. When complemented with evolutionary and ecological analyses, these studies suggest that plasticity represents a mechanism facilitating adaptive change, increasing diversity and fostering the evolution of novelty. Here, we summarize genetic, molecular and evolutionary studies on developmental plasticity of feeding structures in nematodes, focusing on the model organism Pristionchus pacificus and its relatives. Like its famous cousin Caenorhabditis elegans, P. pacificus reproduces as a self-fertilizing hermaphrodite and can be cultured in the laboratory on E. coli indefinitely with a four-day generation time. However, in contrast to C. elegans, Pristionchus worms show more complex feeding structures in adaptation to their life history. Pristionchus nematodes live in the soil and are reliably found in association with scarab beetles, but only reproduce after the insects' death. Insect carcasses usually exist only for a short time period and their turnover is partially unpredictable. Strikingly, Pristionchus worms can have two alternative mouth-forms; animals are either stenostomatous (St) with a single tooth resulting in strict bacterial feeding, or alternatively, they are eurystomatous (Eu) with two teeth allowing facultative predation. Laboratory-based studies revealed a regulatory network that controls the irreversible decision of individual worms to adopt the St or Eu form. These studies revealed that a developmental switch controls the mouth-form decision, confirming long-standing theory about the role of switch genes in developmental plasticity. Here, we describe the current understanding of P. pacificus mouth-form regulation. In contrast to plasticity, robustness describes the property of organisms to produce unchanged phenotypes despite environmental perturbations. While largely opposite in principle, the relationship between developmental plasticity and robustness has only rarely been tested in particular study systems. Based on a study of the Hsp90 chaperones in nematodes, we suggest that robustness and plasticity are indeed complementary concepts. Genetic switch networks regulating plasticity require robustness to produce reproducible responses to the multitude of environmental inputs and the phenotypic output requires robustness because the range of possible phenotypic outcomes is constrained. Thus, plasticity and robustness are actually not mutually exclusive, but rather complementary concepts.

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.

The Nuclear Hormone Receptor NHR-40 Acts Downstream of the Sulfatase EUD-1 as Part of a Developmental Plasticity Switch in Pristionchus.

Developmental plasticity, the ability of one genotype to produce distinct phenotypes in different environments, has been suggested to facilitate phenotypic diversification, and several examples in plants and animals support its macroevolutionary potential [1-8]. However, little is known about associated molecular mechanisms, because environmental effects on development are difficult to study by laboratory approaches. One promising system is the mouth dimorphism of the nematode Pristionchus pacificus [9-12]. Following an irreversible decision in larval development, these nematodes form moveable teeth that occur in either of two discrete morphs. The "eurystomatous" (Eu) form has a wide mouth and two teeth, allowing predatory feeding on other nematodes. In contrast, the alternative ("stenostomatous"; St) form has diminutive mouthparts that largely constrain its diet to microbes. The sulfatase EUD-1 was previously discovered to execute a polyphenism switch based on dosage of functional alleles [13] and confirmed a prediction of evolutionary theory about how developmental switches control plasticity [1, 3]. However, the genetic context of this single gene, and hence the molecular complexity of switch mechanisms, was previously unknown. Here we use a suppressor screen to identify factors downstream of eud-1 in mouth-form regulation. We isolated three dominant, X-linked mutants in the nuclear hormone receptor gene nhr-40 that are haploinsufficient. Both eud-1 nhr-40 double and nhr-40 single mutants are all Eu, whereas transgenic overexpression of nhr-40 does not restore the wild-type phenotype but instead results in nearly all-St lines. Thus, NHR-40 is part of a developmental switch, suggesting that switch mechanisms controlling plasticity consist of multi-component hormonal signaling systems.

Large-scale diversification without genetic isolation in nematode symbionts of figs.

Diversification is commonly understood to be the divergence of phenotypes accompanying that of lineages. In contrast, alternative phenotypes arising from a single genotype are almost exclusively limited to dimorphism in nature. We report a remarkable case of macroevolutionary-scale diversification without genetic divergence. Upon colonizing the island-like microecosystem of individual figs, symbiotic nematodes of the genus Pristionchus accumulated a polyphenism with up to five discrete adult morphotypes per species. By integrating laboratory and field experiments with extensive genotyping of individuals, including the analysis of 49 genomes from a single species, we show that rapid filling of potential ecological niches is possible without diversifying selection on genotypes. This uncoupling of morphological diversification and speciation in fig-associated nematodes has resulted from a remarkable expansion of discontinuous developmental plasticity.

Rapid diversification associated with a macroevolutionary pulse of developmental plasticity.

Developmental plasticity has been proposed to facilitate phenotypic diversification in plants and animals, but the macroevolutionary potential of plastic traits remains to be objectively tested. We studied the evolution of feeding structures in a group of 90 nematodes, including Caenorhabditis elegans, some species of which have evolved a mouthpart polyphenism, moveable teeth, and predatory feeding. Comparative analyses of shape and form, using geometric morphometrics, and of structural complexity revealed a rapid process of diversification associated with developmental plasticity. First, dimorphism was associated with a sharp increase in complexity and elevated evolutionary rates, represented by a radiation of feeding-forms with structural novelties. Second, the subsequent assimilation of a single phenotype coincided with a decrease in mouthpart complexity but an even stronger increase in evolutionary rates. Our results suggest that a macroevolutionary 'pulse' of plasticity promotes novelties and, even after the secondary fixation of phenotypes, permits sustained rapid exploration of morphospace.

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.

Two new and two recharacterized species from a radiation of pristionchus (nematoda: diplogastridae) in europe.

Two new species of Pristionchus, P. lucani n. sp. and P. bulgaricus n. sp., are described from France and Bulgaria, respectively. Additionally, new morphological and morphometric data are provided for two previously described species from Europe, P. brevicauda (Kotlán, 1928) Paramonov, 1952 and P. clavus (von Linstow, 1901) Sudhaus and Fürst von Lieven, 2003. A phylogeny including these four species was inferred from a dataset including 26 ribosomal protein-coding genes, sequences of which are original for P. bulgaricus n. sp. and P. clavus. Relationships support a radiation of all sequenced European Pristionchus species from a single, gonochoristic common ancestor, and current knowledge of species ranges supports "western" and "eastern" clades. Similar diagnostic morphologies reflect the close relationships among the new and recharacterized species, especially P. bulgaricus n. sp., P. brevicauda, and P. clavus, although mating tests as well as genetic and phylogenetic separation support their identities as unique species. Our results show that Pristionchus species in Europe are more diverse than typological characters suggest, and thus biological and molecular profiling will be essential for future delimitation of Pristionchus species from the region.

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.