Abstract
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. In the nematode Pristionchus pacificus, which form two alternative feeding-morphs, the polyphenism threshold is set by relative dosage of two lineage-specific enzymes that respond to morph-inducing cues. One enzyme, the sulfotransferase SEUD-1, integrates an intercellular signalling mechanism at its ultimate target, the cells producing dimorphic mouthparts. 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 the polyphenism threshold. Second, laboratory selection on the polyphenism threshold resulted in changes in relative transcriptional dosage. Our study thus offers a genetic explanation for how plastic responses evolve.
Developmental plasticity, the ability to produce multiple phenotypes from a single genotype, is a ubiquitous feature of multicellular life. At its extreme, plasticity takes the form of polyphenism, by which discrete alternative morphs develop, often with disparate ecological roles and sometimes exhibiting evolutionary novelties1,2. Because its binary outputs imply a limited number of factors needed for a switch mechanism, polyphenism offers a tractable model for studying how plasticity is controlled and responds to selective pressures. Genetic regulators of morphological polyphenism have indeed begun to be revealed3–6, and the selectable variation shown for polyphenisms7,8 suggests that genetic changes in the control of polyphenism may ultimately be identified. However, the genetic architecture of polyphenism is still not well understood, and it has remained elusive how such architecture should change across species with different plastic responses under different ecological pressures.
The omnivorous nematode Pristionchus pacificus has a polyphenism in its adult mouthparts, allowing individuals to take different diets in response to local environmental cues experienced as postembryonic larvae (Fig. 1). Specifically, the nematodes develop either into a “stenostomatous” (St) morph, which has a narrow mouth specialised for feeding on microbes, or into a “eurystomatous” (Eu) morph with opposing, moveable teeth that allow a broader diet, including predation on other nematodes9. Production of the Eu morph, which confers a fitness benefit over the St morph when offered a diet of only prey9, is influenced by the local availability of microbial food as sensed by starvation10 and local crowding by conspecifics11. How responsive (i.e., plastic) the polyphenism decision is to environmental changes varies across populations and species3,12 — for example, the laboratory reference strain of P. pacificus is mostly (90%) Eu even when well-fed under laboratory conditions — such that the “set point” of the polyphenism threshold can vary, potentially offering more or less sensitivity to given cues according to local environmental pressures. At a genetic level, this polyphenism is regulated by the lineage-specific, dosage-dependent sulfatase EUD-13. This completely penetrant regulator channels pheromone signalling13, endocrine (DAF-12-dafachronic acid) signalling10, and information from chromatin modifiers and antisense RNAs14 into a single switch. EUD-1 activity depends on the nuclear receptor NHR-40, which was proposed to be at the transcriptional terminus of the switch mechanism15. Yet despite an emerging genetic knowledge of this polyphenism and of plasticity in general, how genetic changes result in the evolution of plastic responses is little understood. Here, through the identification of a new polyphenism switch-gene, we show that a plastic response (i.e., morph ratio) is decided by the relative dosage of putative signal-modifying enzymes. Specifically, we have discovered a sulfotransferase with dosage-dependent epistasis over EUD-1 to set a threshold for a plasticity switch. Furthermore, we show that environmental, artificially selected, and interspecies changes in plasticity correlate with changes in the relative dosage of these genes. Our findings thus provide genetic insight into how the molecular regulation of polyphenism evolves to produce new plastic responses.
Results
Multiple recessive mutants suppress a polyphenism switch gene
As an unbiased approach to identify genes making up a polyphenism switch, we conducted a forward genetic screen for recessive suppressors of the mutant strain eud-1(tu445), which is completely St (i.e., Eud, eurystomatous-form-defective). We mutagenised eud-1(tu445) mothers, isolated their putatively heterozygous F1, and screened segregant F2 for mutants showing an Eu phenotype. From a screen of ~10,300 haploid genomes, we identified seven recessive mutants. These mutants fell into one of three complementation groups, suggesting that the polyphenism switch comprises, and can feasibly be described from, a finite number of factors with non-deleterious mutant phenotypes. One of the complementation groups consisted of two mutant alleles, sup(iub7) and sup(iub8), which were fully penetrant for their constitutive suppression of the Eud phenotype, even under well-fed conditions (Fig. 2A; Table S1). We named the gene represented by these alleles seud-1 (suppressor-of-eud-1).
A polyphenism-regulating sulfatase is suppressed by a sulfotransferase
To identify the locus of seud-1(iub7) and seud-1(iub8), we backcrossed each mutant and resequenced its genome. We scanned all annotated genes for non-identical and potentially harmful (nonsense, missense, and splice-site) mutations in both mutants. The entire genome yielded only one predicted gene, Contig20-aug8366.tl, with harmful lesions unique to each mutant. This gene, located on Chromosome I, encodes a cytosolic sulfotransferase and is one of several P. pacificus homologs of Caenorhabditis eiegans ssu-1. In C. elegans, SSU-1 functions in dauer diapause, another type of environmentally conditioned development, acting with the steroid hormone receptor DAF-12/VDR in ASJ amphid neurons to suppress formation of dauer downstream of insulin (DAF-2) signalling16,17. Mutant lesions in iub7 and iub8 were nonsense mutations in Exon 8 and Exon 1, respectively, of the gene as annotated from expressed RNAs (Fig. 2B). The iub8 lesion lies upstream of sequences encoding a proton acceptor (H164) for a substrate-binding domain, and iub7 lies within a sulfate-donor (3′-phosphoadenosine-5′-phosphosulfate, PAPS) domain (Table S2). Sequences for both functional domains are highly conserved across nematodes and the mammalian homolog SULT2A1 (Fig. S1), suggesting the conserved function of SEUD-1 as a cytosolic sulfotransferase in P. pacificus.
SEUD-1 is a genetic intermediary between EUD-1 and NHR-40
Given the likely function of SSU-1 as a sulfotransferase, we hypothesised that this enzyme may act, like the sulfatase EUD-1, on a substrate upstream of NHR-40. To test this idea, we investigated its epistasis with nhr-40, particularly using a line of the null mutant seud-1(iub7) that was outcrossed to restore wild-type alleles at eud-1 (Table S3). Because nhr-40 and seud-1 both promote the St form in wild-type animals, we performed epistasis tests by combining loss-of-function mutations in one gene with transgenic overexpression in the other. These tests specifically used an nhr-40 mutant allele (tu505) that, like all previously recovered P. pacificus nhr-40 mutants, incurred a non-synonymous mutation that is fully penetrant for its loss of St-promoting function15. First, we transgenically over-expressed a wild-type construct of nhr-40 in seud-1(iub7) mutants. This resulted in the all-Eu phenotype of seud-1(iub7) rather than the mostly-St phenotype of the over-expressing line Ex[nhr-40] (χ2 = 4.93, df = 1, P < 0.0001; Fig. 2C; Table S4), indicating that seud-1 either acts downstream of NHR-40 or, alternatively, is necessary to sulfate a signal that ultimately activates NHR-40-mediated transcription. In the former model, seud-1 overexpression should suppress the phenotype of nhr-40 mutants, whereas in the latter model the reverse should be the case. To distinguish between these possibilities, we transgenically overexpressed a wild-type genomic clone of seud-1, including its presumptive 5′ and 3′ regulatory elements, in nhr-40(tu505) mutants (Table S5). Although transgenic Ex[seud-1] animals wild-type for nhr-40 were mostly St, Ex[seud-1] animals with loss-of-function nhr-40 alleles exhibited the nhr-40 mutant phenotype (χ2 = 1.07, df = 1, P < 0.0001; Fig. 2C; Table S4). Therefore, reciprocal epistasis tests indicate that SEUD-1 acts between EUD-1 and NHR-40 and has an enzymatic function necessary for NHR-40 function.
SEUD-1 is expressed at the site of the polyphenism
Because expression of eud-1 and nhr-40 does not overlap, and because nhr-40 is pleiotropically expressed across several tissues15, the anatomical scope of the polyphenism switch was previously unknown. We therefore studied the expression of seud-1 to reconstruct the switch in space. To localise expression of seud-1, we generated a transcriptional, nucleus-localised, fluorescent (RFP) reporter using the same promoter sequence that was sufficient to over-express seud-1. This transgenic construct reported seud-1 expression in the cells making up the anterior body wall (face), stoma (mouth), and anterior pharynx, which have homologs in C. elegans18 and which together produce the feeding morphology that is dimorphic in P. pacificus (Fig. 3). We detected expression in these cells throughout postembryonic juvenile stages (J1-J4), dauer (a facultative, third-stage diapause larva), and young adults (Fig. S2). Expression was consistently most pervasive across cell classes at the J2 and J3 stages (Fig. 3A), thereafter tapering in the J4 and early adult stages (Fig. 3B), and indeed at the adult stage, when morphs could be distinguished, no obvious qualitative differences in expression between Eu and St morphs were observed. Additionally, although individuals of a given life-stage varied in which particular cells were expressed when the animals were caught for examination, all larval stages were collectively shown to express seud-1 in this set of cells. These findings suggest that this polyphenism switch factor is active in dimorphic tissue throughout postembryonic development, including diapause, until the polyphenism is expressed at the adult stage.
The specific cells expressing the reporter were those making up the anterior epidermal (hyp) syncytia, which together comprise the anterior body wall and secrete the outer lining of the mouth (cheilostom)19, the region of the mouth and head that is dimorphic in width; the anterior and posterior arcade cells, which join the mouth opening to the pharynx20; epithelial cells e2 and mc1, and myoepithelial cells pm1, pm3, and pm4, which together comprise the pharyngeal muscle and secrete the extracellular teeth21. In addition to cells in the mouth and anterior pharynx, strong expression was detected at early juvenile (J2 and J3) stages in the pharyngeal gland (g1) cell, the nuclei of which are in the pharyngeal basal bulb (Fig. 3A). This cell comprises a gland whose duct terminates in the dorsal tooth, the secretions of which have been hypothesized to be involved in predatory feeding and which is, compared with C. elegans, highly innervated by the pharyngeal nervous system22. Other cell classes reporting seud-1 were M1, I1, and possibly 12 (Fig. 3A), all of which innervate pm1, the cell actuating the dorsal tooth, and are thus hypothesized to reflect dimorphism in pharyngeal behaviour18. Because the cells reporting seud-1 overlap with several cells in which nhr-40 is expressed15, seud-1 and nhr-40 likely control the polyphenism decision at the site of the morphological dimorphism. Together, these results indicate that seud-1 is expressed at the terminal integration site of the polyphenism pathway in P. pacificus, whereas EUD-1 regulates signalling upstream of these target cells.
Polyphenism is determined by relative dosage of enzymes with opposing functions
Because transgenic over-expression of seud-1 showed the gene to be dosage-dependent, we hypothesised that the relative dosages of seud-1 and eud-1 may together establish the threshold for the polyphenism switch. To test this idea, we manipulated wild-type copy number, a feature shown to affect expressivity in eud-1 hemizygotes and heterozygous mutants3. Specifically, we used eud-1 and seud-1 mutants to generate all possible combinations of homozygotes, heterozygotes, and hemizygotes, from both cross directions, for both genes (Table S6). As a result, relative copy number of functional eud-1 and seud-1 alleles was sufficient to gradate the ratio of morphs among the F1 from all-Eu to all-St (Fig. 4A). All genotypes with one copy of each gene restored the phenotype of wild-type hermaphrodites (morphological females), which have two copies of each, indicating the relative dosage dependence of eud-1 and seud-1. Moreover, animals with one copy of seud-1 but two copies of eud-1 were almost all-Eu, as the Eu-promoting eud-1 is favoured, although a few animals were still St. Lastly, no number of copies of eud-1 could rescue Eu formation in the absence of wild-type seud-1. Together, these results indicate that, under a given environmental regimen, relative dosage of the two genes set the threshold for the mouth-morph ratio of P. pacificus.
Morph-inducing cues differentially regulate eud-1 and seud-1
Given the dependence of the morph ratio on relative genomic dosage of eud-1 and seud-1, we investigated whether transcriptional dosage of these genes reflected differences in morph ratio induced under different environments. Namely, we predicted that the different morph-inducing cues would result in higher eud-1:seud-1 transcript ratios in an Eu-inducing compared with a St-inducing environment. To test this prediction, we performed an analysis of RNA transcripts produced across multiple nutritive environments demonstrated to elicit different plastic responses in P. pacificus23. Because the only variable in this experiment was food source, we interpreted any differences in phenotype and gene expression to be the result of dietary cues. Results of this analysis showed that nematodes cultured on a relatively St-inducing diet of Cryptococcus albicus yeasts resulted in higher enrichment of seud-1 expression compared with a diet of E. coli OP50 (Table 1). In contrast, a relatively Eu-inducing diet of C. curvatus yeasts showed no difference in either phenotype or seud-1 expression. Further, eud-1 transcript levels were lower on a diet of C. albicus, and only slightly lower in C. curvatus, further shifting the ratio of eud-1 to seud-1 transcripts on the former diet. Our results thus indicate that, in a single genetic background, different morph-inducing environmental cues result in relative transcriptional dosage of opposing factors making up the polyphenism switch.
seud-1 homologs are dynamically radiating in Pristionchus nematodes
To reconstruct the evolutionary origin of seud-1, we performed a phylogenetic analysis of seud-1 and other identifiable homologs of C. elegans ssu-1 in Pristionchus and outgroups (Table S7). This analysis showed that three sibling species of Pristionchus each had at least four to seven homologs of ssu-1. Several of these genes having high inferred divergence and dubious orthology (Fig. 4B), despite the species’ being close enough to interbreed24. In contrast, the most recent common ancestor of Pristionchus and outgroups likely carried a single ssu-1 homolog. Therefore, a radiation of ssu-1 duplicates has been specific to a lineage including Pristionchus and coincides with the presence of polyphenism in evolutionary history12. A product of this radiation was seud-1, for which orthology could be established across Pristionchus species (Fig. 4B). However, even within this clade, a recent duplication of seud-1 was detected, specifically in P. exspectatus. Pairwise counts of dN/ds between the two Pex-seud-1 duplicates (0.027), as well between each copy and the reference allele for P. pacificus (0.012, 0.011), suggest that both are under strong purifying selection (i.e., dN/ds << 1.0) rather than harbouring an incipient pseudogene. Furthermore, the coding sequences of the two copies are >99% identical at the amino-acid level, suggesting a similar enzymatic function between duplicates.
Gene duplication correlates with new plasticity phenotypes across species
Because the P. pacificus polyphenism is sensitive to genomic dosage of switch genes, we hypothesised that the recent duplication of seud-1 in P. exspectatus has changed its plasticity phenotype. The sampled strain of this species, prior to its laboratory inbreeding to an all-St phenotype, produced more St animals than most strains of P. pacificus under laboratory conditions3. In principle, duplication could provide an instant change in a plasticity response given the standing trans-regulation of factors making up a polyphenism switch: in P. exspectatus, this would result in the St morph being more likely to develop in a given environment. We tested whether Pex-seud-1.1 and Pex-seud-1.2, which are both expressed (Table S8), had a combined influence on the polyphenism decision, using P. pacificus mutants to create interspecific hybrids with different relative copy numbers of eud-1 and seud-1 (Table S9). By examining F1 females from these crosses, we could standardise the regulatory genetic background of these genotypes, which has likely diverged in addition to gene dosage. As predicted, changing the ratio of copies of eud-1 to seud-1 (1:3,1:2, 2:3, and 2:2) in hybrids resulted in a gradation of plasticity phenotypes (Fig. 4C). In particular, hybrid phenotypes showed a difference between double “heterozygotes” (1:2) and double “homozygotes” (2:3) from either P. pacificus mothers (Z = 6.00, P < 0.0001) or P. exspectatus mothers (Z = 7.03, P < 0.0001), consistent with both Pex-seud-1 duplicates having an influence on the morph ratio in P. exspectatus. Taken together, these results show a clear correlation between the duplication of one of two opposing switch factors and a divergent plastic response (i.e., morph ratio) to a given environmental regimen.
Laboratory evolution of plasticity accompanies regulatory changes in opposing switch factors
Lastly, we examined whether changes in relative transcriptional dosage could be detected among haplotypes differing in their plastic responses. First, we quantified expression of eud-1 and seud-1 in three genetically distant P. pacificus isolates25,26 that vary in their morph ratios in a standardised environment3. While two St-biased strains (RS5200B, RS5410), as predicted, showed lower (P < 0.05) or a trend toward lower (P = 0.1) eud-1 expression than the California strain, seud-1 expression was also lower in these St-biased strains (P < 0.05), indicating that at least some variation must be attributed to regulatory differences beyond the simple scaling of eud-1 and seud-1 expression. Therefore, we next performed an experiment to test whether relative dosages could be artificially selected on a shorter time scale, specifically from a single, heterozygous laboratory population. To do this, we artificially inbred lines of P. exspectatus, a gonochoristic species harbouring higher levels of heterozygosity than P. pacificus26, possibly including for plasticity-regulating genes. Systematic inbreeding for 10 generations indeed resulted in lines being fixed for different polyphenism thresholds in standardised environments (Fig. 4D). Furthermore, when we measured the relative expression of eud-1 and total “seud-1” (seud-1.1 + seud-1.2) in these lines, we found that an increase in St animals correlated with increased relative transcription (P < 0.001) of combined seud-1 duplicates (Fig. 4E). Thus, natural variation in transcriptional regulation could be artificially selected, resulting both in divergent plastic responses and in divergent switch-gene transcription, implicating these genes as potential targets for plasticity evolution.
Discussion
In summary, we describe a genetic model for a signalling system regulating developmental plasticity, and we have identified molecular targets of plasticity evolution (Fig. 5). Our findings support two major insights about plasticity regulation and its capacity to change. First, we have detailed how a pair of enzymes can fine-tune a polyphenism threshold without requiring changes to signal production itself. Although hormones have been long known to mediate morphological polyphenism in animals27, the molecular mechanisms that integrate polyphenism have been poorly understood. In P. pacificus, a plasticity threshold is mediated by opposing factors regulating local signalling function, particularly through sulfation and desulfation. Further, sulfation by SEUD-1 is instructive, promoting the activity of NHR-40, possibly by locally providing a metabolic precursor or modifying transport of an active ligand28. Alternatively, SEUD-1 and EUD-1 may regulate NHR-40 itself, such as through its acetylation state29, although the absence of EUD-1 from polyphenic tissue makes this scenario less likely. Signal modification would also be consistent with the inferred activity of C. elegans ssu-1, for which mutants show altered levels of sulfated sterols14. We therefore propose that a signalling molecule, still to be identified, is alternately modified to regulate its local activity at the targets of the polyphenism pathway.
Secondly, our study gives a genetic explanation for how genetic changes to a polyphenism mechanism produce new plasticity responses in different lineages. Opposing signalling modifiers offer an alternative evolutionary target besides the production of signals themselves, which are often pleiotropic and likely to be constrained30,31. This target also differs from a signal’s ultimate receptor, to which mutations can have a dramatic impact on phenotype32 and also be hindered by pleiotropy, as observed for NHR-4033. Thus, the balance between competing enzymatic regulators can be shifted to change the threshold response required for a switch. In principle, changes to such a system may be fine-tuned by allowing selection on opposing factors that individually channel separate, upstream influences on polyphenism. It is also possible that gene duplications, as observed in P. exspectatus, could result from gene amplification in such a system. In such a case, fixation of duplicates and the specialisation of new developmental regulators would thereafter be possible34. Although functional assays have not yet revealed polyphenism function for paralogs of eud-135, the more extensive radiation of seud-1 homologs allows a better test of this idea. By revealing the precise factors controlling a polyphenism switch, it is now possible to reconstruct what changes to a switch have occurred across species that have diverged in their sensitivity to environmental pressures.
In conclusion, we have identified the genetic regulatory points that establish a threshold between plastic developmental phenotypes, and we have shown that modifications to these regulatory points correlate with divergent morph-ratios between species. Importantly, this model offers a foundation for identifying the genetic modifications that have occurred in other instances of polyphenism evolution, thereby revealing its generalizable features. The nematode family (Diplogastridae) that includes Pristionchus has many other species with this ancestral polyphenism, and among lineages there is a diversity of plasticity responses as well as plastic morphologies12,36. With our genetic description of a switch apparatus, it is possible to comparatively explore how evolutionary modifications to this switch ‒ in regulation, function, or downstream targets ‒ have facilitated a dramatic radiation of polyphenism-governed traits.
Methods
Nematode cultures
All Pristionchus strains were maintained on 6-cm Petri plates containing nematode growth medium (NGM) agar seeded with 300 μl of Escherichia coli grown in L-broth. All cultures were kept at 20-25 ºc.
Forward genetics screen
Mutagenesis of the P. pacificus null mutant eud-1(tu445) followed described methods37. Self-fertilizing hermaphrodites (P0) were mutagenised at the J4 (preadult) stage, transferred onto individual 10-cm NGM plates, and allowed to produce ~20 offspring each. These F1 self-fertilised to produce F2 on the same plates, and once most F2 had reached the adult stage, we screened for nematodes of the eurystomatous (Eu) morph, which suppressed the Eud (all-St) phenotype. Eu F2 were cloned into individual culture lines. Phenotypes of >100 F3 clones of each line were screened to confirm the mutant phenotype to be both homozygous and recessive. Pairwise complementation tests were performed for the seven isolated recessive mutants and grouped alleles iub7 and iub8. These two mutants were then backcrossed to the eud-1(tu445) strain four and six times, respectively, using the following strategy: for two rounds of backcrossing, mutant hermaphrodites were first crossed to eud-1(tu445) males, after which F1 were backcrossed to the strain pdl-2(tu463); eud-1(tu445), a strain in which the parental line had been marked with a recessive “dumpy” mutation15; F2 were then selfed to recover Eu F3, which were cloned and then backcrossed further as above.
Phenotype scoring
Mouth phenotypes were scored as previously described11. In short, the Eu phenotype was determined by the presence of (i) a claw-shaped dorsal tooth, (ii) a large, hooked subventral tooth, and (iii) a mouth wider than deep; the St phenotype was determined by (i) a narrow, dorsoventrally symmetrical dorsal tooth, (ii) absence of a subventral tooth, and (iii) a mouth narrower than deep. Rare (<0.5%) intermediates between the two morphs were excluded from phenotype counts. Phenotypes were scored using differential interference (DIC) microscopy using a Zeiss AxioScope, except in the F2 screen for mutants, for which a Zeiss Discovery V.20 stereoscope was used for higher throughput.At least 60 individuals, with one exception (n = 25), were scored for each genotype (Tables S4, S6, S9).
Genomic resequencing for mutant identification
Nematodes were prepared for DNA extraction as previously described3. Genomic DNA was extracted using a MasterPure DNA Purification Kit (Epicentre) and was quantified using an Agilent TapeStation 2200. DNA libraries for sequencing were prepared using a Nextera kit, diluted to a concentration of 0.45 pM in 0.1% EB-Tween, and pooled as 6-plex. The libraries were sequenced as 150-bp paired-end reads on an lllumina NextSeq 300 to a theoretical coverage of 24x. Raw sequencing data were mapped and variants called as previously described38.
Identification of mutant lesions
With the lists of potential variants for iub7 and iub8, we identified the causal lesions as follows. First, we excluded all variants from each list that were shared with the resequenced genome of the mutagenised eud-1(tu445) strain3 and with the dumpy pdl-2(tu463) strain (voucher EJR1018) used to create the pdl-2(tu463); eud-1(tu445) strain for backcrossing (unpubl. data). Variants in predicted coding sequences were categorised with respect to the “Hybridl” assembly and AUGUSTUS annotation for the genome of P. pacificus (www.pristionchus.org). From the intersecting lists of variants, we excluded SNPs manually determined to be artefactual using the Integrative Genomics Viewer39. Following the identification of the mutant locus as Contig20-aug8366.tl from resequenced genomes, mutant lesions were verified by Sanger sequencing (Table S3).
Outcrossing of the eud-1 mutation
To determine the phenotype of a null seud-1 mutant without a background eud-1 mutation, we outcrossed the mutant seud-1(iub7); eud-1(iub8) to the wild-type reference (“California,” PS312) strain of P. pacificus. For the P0 cross, we used males of the mutant strain and California hermaphrodites, so that F1 males would inherit the autosomal seud-1 mutation but not the X-linked eud-1 mutation. F1 males were then outcrossed again to California hermaphrodites, virgin (BC1) offspring of which were let to selfcross to re-segregate mutant seud-1 alleles from putative heterozygotes. Multiple BC1F2 lines were cloned and >200 individuals per line were screened to confirm their all-Eu phenotype. Finally, the presence of the identified seud-1(iub7) lesion and the absence of the eud-1(tu445) lesion were additionally confirmed by PCR and Sanger sequencing (Table S3).
Over-expression of seud-1 by genetic transformation
Transgenic P. pacificus nematodes were generated as previously described40. To rescue function of and over-express seud-1, ovaries of outcrossed seud-1(iub7) mutants were injected with an 11-kb genomic clone containing the putative 4.2-kb promoter region of wild-type seud-1 and a 6.8-kb coding sequence including its 60-bp 3′ regulatory region (10 ng/μl). This construct was delivered with the co-injection marker eg1-20promoter::TurboRFP (10 ng/μl) and digested genomic carrier DNA (60 ng/μl) from the recipient strain. To over-express nhr-40 in seud-1 mutants, an extrachromosomal array from a - nhr-40 rescue line generated previously15 was crossed into the outcrossed seud-1 (iub7) strain.
Crosses to study effects of relative gene dosage
Genetic crossing experiments were performed to test for haploinsufficiency and opposing dosages between eud-1 and seud-1. In our cross panel, all possible genotype combinations (i.e., homozygotes, heterozygotes, and hemizygotes) of wild-type (California) and mutant (tu445 and iub7) alleles of eud-1 and seud-1 were created. The backcrossed, double-mutant line seud-1(iub7); eud-1(tu445) (voucher EJR1029), the outcrossed line seud-1(iub7) (voucher EJR1039), the backcrossed (x6) parental line eud-1(tu445), and the California strain were used for creating these genotypes. To correctly identify F1 instead of self-offspring from these crosses, all polyphenism mutants were marked with the recessive dumpy marker pdl-2(tu463), such that cross-offspring could be easily distinguished by the rescue of a non-dumpy phenotype. In addition to using previously generated lines15, we created the double mutant seud-1(iub7); pdl-2(tu463) and triple mutant seud-1(iub7); pdl-2(tu463); eud-1(tu445). Homozygosity of mutations was confirmed by Sanger sequencing of all alleles in >10 clones per line (Table S3).
Localization of seud-1 expression
We localised expression of seud-1 in P. pacificus using a transcriptional reporter. To express this reporter, we transformed P. pacificus germ cells along with co-injection markers as described above. Because seud-1 is dosage-dependent, we predicted its expression in the California strain to be low, as shown previously for the St-promoting nhr-4015. Therefore, we used P. pacificus strain RS5200B for transformation, as this strain is a natural variant that is highly St in laboratory culture3 and was thus more likely than the California strain to express seud-1 at detectable levels. The construct used for genetic transformation was ligated from the following sequence fragments: (i) the 4.2-kb putative promoter region of seud-1, (ii) a coding sequence consisting of a start codon, a nuclear localization signal, and TurboRFP, and (iii) the 3′ untranslated region of the gene Ppa-rpi-23. The construct was cloned into pCR4 TOPO vector and maintained in a TOPIO E. coli clone, which served as its source for microinjection experiments. Two independent transgenic lines were generated, and 10 animals at each life-stage (for adults, 5 St and 5 Eu) were examined using a Zeiss Axiolmager.
Analysis of eud-1 and seud-1 expression under different culture conditions
Raw RNA reads, which were produced by nematodes raised on E. coii OP50, Cryptococcus albicans, and C. curvatus23, were downloaded from the NCBI database (accession number: SRP081198). Paired-end reads (2 × 150bp) from two biological replicates for each condition were aligned to the P. pacificus reference genome (version El Paco, www.pristionchus.org) using STAR (version 2.5.3)41 under default settings. Read counts were achieved using the function FeatureCounts in Rsubread Bioconductor package (version 1.24.2)42 and genes with a low number count (i.e., low-expressed) were filtered out. Reads mapped to seud-1 and eud-1 genes were examined manually in IGV viewer (version 2.3.77)39 to eliminate false mapping. Differential expression analysis was performed in edgeR43, with p-values adjusted according to the Benjamini Hochberg procedure to restrict false discovery rate.
Phylogenetic analysis
Evolutionary history of seud-1 homologs in Pristionchus and outgroups was inferred from predicted homologs mined from available nematode genomes using the criterion of reciprocal best BLAST similarity with C. elegans ssu-1. For reconstruction of the phylogenetic history of seud-1, predicted amino-acid sequences encoded by identified ssu-1 homologs were aligned using the E-INS-I algorithm and default settings in MAFFT (version 7.1)44. The gene tree of predicted SSU-1 amino-acid sequences was inferred under the maximum likelihood (ML) criterion, as implemented in RAxML (version 8)45. 50 independent analyses were performed. Analyses invoked a Whelan and Goldman model with a gammashaped distribution of rates across sites. Bootstrap support for the most likely tree among all runs was estimated by 500 pseudoreplicates.
Selection analyses of seud-1 duplicates
We tested for purifying selection among seud-1 alleles using a pairwise counting method of dN/ds46, as implemented in the program PAML47. We performed three comparisons: (i) Pex-seud-1.1 to Pex-seud-1.2; (ii) Pex-seud-1.1 to Ppa-seud-1(California); (iii) Pex-seud-1.2 to Ppa-seud-1(California).
Hybrid crosses to study effects of gene amplification
Hybrid crosses followed the same logic described for studying relative gene dosage in P. pacificus, with the following modifications. In crosses of P. pacificus males to P. exspectatus females, a phenotypic (e.g., dumpy) marker was not used or needed, as P. exspectatus requires sexual reproduction to produce offspring. Also, only female hybrids were used to score phenotypes, as male F1 from reciprocal crosses have different genetic backgrounds that were previously found to have asymmetrical epistatic effects on the mouth polyphenism3.
Quantification of seud-1 and eud-1 expression in natural polyphenism variants
To quantify transcripts from the California strain and two St-biased strains (RS5200B and RS5410), we used J3 and J4 nematodes from each of these populations. To collect nematodes mostly at these stages, cultures were synchronized by starting with eggs released by bleaching gravid mothers48, 48 hours after which nematodes were washed from plates, pooled, and prepared for RNA extraction following published protocols3. For RT-PCR, total RNA was extracted from the homogenized solutions of nematodes in TRIzol using the Zymo Direct-zol RNA Miniprep kit (Zymo Research). 1 μg of total RNA was used for first-strand cDNA synthesis with random hexamers in a 20-μL reaction using the superscript III Reverse Transcriptase kit (Invitrogen). cDNA was diluted five times, and 1.5 μl of the final volume was used for a 10 μl PCR. Real-time PCR to quantify expression was performed on a Roche LightCycler 96 system, using SYBR GREEN reaction mix and the manufacturer’s (Roche) software and with the genes encoding beta-tubulin (tbb-4) and an iron-binding protein (Y45F10D) as the reference genes49. Primers for RT-qPCR of P. pacificus seud-1 and eud-1 are listed in Table S10. The PCR cycle was: 10 min at 95 ºC, followed by 45 cycles of 10 sec at 95 ºc, 20 sec at 52 ºC, and 15 sec at 72 ºC, with a single fluorescence read at the end of each extension. Each PCR reaction was performed on two independent biological replicates and two technical replicates for each group. Melting-curve analysis and gel electrophoresis were performed to ensure the absence of non-specific products or primer dimerization, and PCR efficiency was identified with a 5-log titration of pooled cDNA. Relative expression levels were determined using the ΔΔCt method49, with California strain designated as the control group.
Genetic inbreeding of P. exspectatus polyphenism variants
For artificial selection, we started with a source population of P. exspectatus (RS5522) previously shown to harbour heterozygosity in genes affecting the mouth-morph ratio3. This population was recovered from a frozen voucher of a culture that, prior to freezing, had been established from multiple individuals collected from a vector beetle24 and had been kept in laboratory culture for about one year on large-diameter (13 cm) plates, with subcultures established through “chunking”50, whereby dozens of individuals were transferred. This voucher comprised worms from ~10 separate cultures maintained under the conditions above and frozen according to standard protocols37. This population was the parent source of the inbred strain RS5522B, in which a Eud phenotype had previously been fixed through inbreeding3. From the revived RS5522 strain, we systematically inbred two new lines for 10 generations each, using a single male-female pair at each generation. Because systematic inbreeding of a dioecious, or obligately outcrossing, species was likely to lead to negative fitness effects on the populations, 10 individual pairs were mated at each generation, with the subsequent generation being established from the most fecund pair; furthermore, all pairs from the previous four generations were kept stable at low temperature (6 ºC) as insurance in case lines from selected pairs suffered inbreeding generations later. All crosses were performed under standardised environmental conditions as described for experimental crosses above. As a result of this inbreeding method, we were able to establish lines, which were in turn frozen as vouchers that were both inbred and without obvious negative effects on brood size and generation time. Additionally, because the thawed voucher was highly St-biased, we promoted the retention of the polyphenism (i.e., as opposed to a fixed Eud phenotype), by artificially selecting the Eu morph for two generations.
Specifically, males and females selected for breeding during the first two generations were both Eu, whereas in subsequent generations inbreeding was random with respect to mouth phenotype. The variable morph-ratio phenotypes of the resulting inbred lines (EJR16 and EJR18) were then recorded under a standardised environment as described above.
Quantification of seud-1 and eud-1 expression in inbred polyphenism variants
To quantify transcripts from inbred P. exspectatus lines, mixed-stage nematodes were collected and prepared for quantitative real-time PCR (RT-qPCR) as previously described3. Because the coding sequences of Pex-seud-1.1 and Pex-seud-1.2 are highly similar, primers matching both genes were used to amplify the total number of Pex-seud-1 transcripts. Two non-overlapping pairs of primers were used, and transcripts of both Pex-seud-1.1 and Pex-seud-1.2 were confirmed by manual inspection of trace files of sequences for both RT-qPCR amplicons. RT-qPCR followed the procedure described above for the quantification of P. pacificus variants, with tbb-4 (encoding beta-tubulin) used as the reference gene49. Relative expression levels were determined with line EJR16 designated as the control group. To quantify seud-1 expression, two primer sets were used and all data from two separate runs with the set “seud-1A” (Table S8) set and one run with the set “seud-1B” were combined. PCR products were purified and submitted to Sanger Sequencing (Eurofins Genomics LLC) to confirm the presence of both duplicate genes. eud-1 expression was quantified from two separate runs using eud-1 primers (Table S8).
Statistical analyses
All statistical analyses were conducted in R (version 3.4.3)51. Polyphenism phenotypes were recorded as proportional data, specifically the percentage of Eu per total number of individuals screened. To determine whether there were differences in morph ratios, we used generalized linear models with binomial error and a logit link function, designating genotype as the explanatory variable. Significance was assessed for (i) epistatic interactions of nhr-40 and seud-1 (Fig. 2A); (ii) genotypes from hybrid crosses; (iii) differences between P. exspectatus inbred lines (Fig. 4D). Replicates within lines were included in each model, and significance of differences was determined using a χ2 test. Significant differences between specific contrasts (i.e., between results of hybrid crosses) in the final models were determined using post-hoc Tukey’s honest significant difference tests as implemented in the package Ismeans52. For graphical representation of phenotype data, mouth-morph frequencies were pooled across replicates within lines, with 95% confidence intervals estimated using a binomial test; these results are qualitatively identical to those derived from the generalized linear model.
Significance of differences in expression of eud-1 and seud-1 transcripts among wild isolates of P. pacificus and between inbred P. exspectatus lines was determined by a linear mixed model, which accounted for repeated measures within biological replicates, using the Ime function in the nlme package53. Log fold change was used as the response variable, strain as a fixed explanatory variable and biological replicate as a random variable. For tests involving P. exspectatus, this method also confirmed that there were no differences among experimental runs (i.e., different primer pairs) for quantification of seud-1 expression.
Author contributions
E.J.R. conceived and designed the study; L.T.B. and N.A.I. conducted the experiments; L.T.B., N.A.I., and E.J.R. analysed the data; L.T.B., N.A.I., and E.J.R. wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgements
We thank the Indiana University Center for Genomics and Bioinformatics for DNA library preparation and genomic resequencing, Susan Feldt and Ryan Mueller for their help with nematode crosses, R. Taylor Raborn for suggestions on transcriptomic analyses, and Meagan Pritchard for media preparation and strain keeping. We also thank Ralf Sommer and Christian Rödelsperger (Max Planck Institute for Developmental Biology) for sharing their unpublished genome sequence of P. arcanus. This work was funded by the National Science Foundation (IOS 1557873).