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Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity [1]

['Mohannad Dardiry', 'Max-Planck Institute For Biology Tübingen', 'Tübingen', 'Department Of Genetics', 'Faculty Of Agriculture', 'Cairo University', 'Giza', 'Gabi Eberhard', 'Hanh Witte', 'Christian Rödelsperger']

Date: 2023-08 

The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.

Funding: This work was funded by the Max-Planck Society through institutional funds to RJS, including the salaries of all co-authors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

( A ) P. pacificus mouth-form dimorphism. The predatory Eu form has a larger mouth opening and 2 teeth, in comparison to the St nonpredatory form with a narrow opening and a single tooth. ( B ) Killing behavior of an Eu adult biting C. elegans larval prey. ( C ) Phylogenetic relationship of a subset of the more than 300 P. pacificus isolates with strains from La Réunion Island indicated in green. Modified from [ 8 ]. Focal strains for subsequent analysis are RSA076 and RSC011 from clade B. ( D ) RSA076 and RSC011 are from neighboring high-altitude regions on La Réunion and were isolated from the endemic stag beetle Amneidus godefroyi, like all other clade B strains. The map is modified from [ 17 ]. Map figure made with GeoMapApp ( www.geomapapp.org ) / CC BY /CC BY [ 19 ]. ( E ) Crossing scheme of the 2 parental strains for RIL analysis. Eu, eurystomatous; QTL, quantitative-trait-locus; RIL, recombinant-inbred-line; St, stenostomatous.

Resource polyphenisms are a special form of adaptive developmental plasticity that facilitate the exploitation of distinct food sources across animals [ 1 ]. Recent studies have started to identify the gene regulatory networks (GRNs) that control resource polyphenisms and other forms of plasticity [ 2 , 3 ]. One example is mouth-form plasticity in the hermaphroditic nematode P. pacificus with its predatory “eurystomatous” (Eu) and nonpredatory “stenostomatous” (St) morphs ( Fig 1A and 1B ) [ 4 ]. Mouth-form plasticity is controlled by the sulfatase EUD-1 that acts as a developmental switch: Expression above a certain threshold will result in the execution of the Eu form, whereas in the absence of eud-1 expression, the St morph is formed [ 5 ]. eud-1 is located in a multigene locus and is part of a complex GRN controlling P. pacificus mouth-form plasticity [ 6 , 7 ]. Knowledge about this GRN also provides a framework for natural variation studies and allows the testing of the contribution of plasticity for evolution.

A single QTL regulates natural variation of mouth-form plasticity

To capture natural variation of mouth-form plasticity and the Eu versus St mouth-form ratio, we made use of a collection of around 1,500 P. pacificus isolates with more than 300 strains from La Réunion Island that were previously whole-genome sequenced (Fig 1C) [8]. We identified several pairs of closely related strains that differ in the preferential expression of mouth form when grown under standard laboratory conditions. For example, in P. pacificus clade B that is endemic to high-altitude locations on La Réunion, RSA076 from Nez du Boeuf (NB) is nearly exclusively Eu, whereas the closely related strain P. pacificus RSC011 from Coteau Kerveguen (CK) is preferentially St (80% St:20% Eu) (Fig 1D). Like all strains of clade B, RSA076 and RSC011 were isolated from the endemic stag beetle Amneidus godefroyi, which is restricted to high-altitude habitats like NB, CK, and neighboring regions (Fig 1D) [9].

We generated F1 hybrids between both strains and allowed F1 animals to self-fertilize for 12 generations to create 160 recombinant-inbred-lines (RILs) (Fig 1E). These RILs have different mouth-form ratios covering the complete range of 20% to 100% Eu, reflecting their mosaic homozygous genetic makeup (S1A Fig). We performed QTL analysis to statistically associate mouth-form ratios to genomic region(s) in the sequenced RILs (Fig 2A). This analysis initially identified 3 highly significant peaks across the genome. However, due to the genetic distance of RSA076 and RSC011 from the P. pacificus reference strain PS312, two of these signals were subsequently demonstrated to represent small X chromosome translocations [10] (S1B Fig). Thus, there is only 1 QTL, which spans a region of more than 200 kb. This QTL contains 35 predicted genes and, importantly, covers the previously described multigene switch locus including eud-1 (Fig 2B). Besides the sulfatase-encoding eud-1, this locus contains the eud-1 paralog sul.2.2.1, and 2 α-N-acetylglucosaminidase-encoding genes (nag-1 and nag-2), which result in all-Eu animals when mutated [6]. Thus, QTL analysis identified a single major locus regulating natural variation of mouth-form plasticity.

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TIFF original image Download: Fig 2. RIL and QTL analyses between RSA076 and RSC011. (A) QTL analysis reveals a single peak of around 200 kb at the left end of the X chromosome. (B) This region includes the multigene locus that contains the eud-1 developmental switch gene. The upstream region of eud-1 contains 5 SNPs (red) and 1 large CNV (yellow) with sequence differences indicated. An additional SNP exists in intron 1 (red). Arrows refer to the 32-bp repeated sequence, which contains the potential FBS (GTAAACAT). (C) RNAseq experiments indicate a 40% higher expression of eud-1 in RSA076 relative to RSC011, consistent with its preferential Eu mouth-form. (D) Mouth-form ratios of various CRISPR-induced mutants introducing RSC011 variants in the RSA076 genetic background result in the sequential appearance of the RSC011 mouth-form ratio. Parental phenotypes are indicated in purple (RSA076) and green (RSC011), respectively. CNV of a potential forkhead transcription factor binding site in the eud-1 promoter with RSA076 having 2 copies, whereas RSC011 has only a single copy (hexagonal shapes). Intron 1 has 1 single-nucleotide-polymorphism (G vs. A) between both strains. For detailed information, see S1 Data. CNV, copy number variation; FPKM, xxxx; LOD, xxxx; QTL, quantitative-trait-locus; RIL, recombinant-inbred-line; SNP, single nucleotide polymorphism. https://doi.org/10.1371/journal.pbio.3002270.g002

Polymorphisms in cis-regulatory regions include copy number variations in potential transcription factor binding sites Genetic variants at the multigene switch locus would be strong candidates to control mouth-form plasticity. We found a total of 41 single nucleotide polymorphisms (SNPs) within the 30-kb region spanning the multigene locus between RSA076 and RSC011. However, within the coding region of the four mouth-form associated genes, only a single nonsynonymous SNP was identified. This is found within nag-2 and causes a Phe415Ile change. Using CRISPR/Cas-9 engineering, we introduced the RSA076 parental nucleotide into the RSC011 genetic background. Two independent lines carrying this substitution (tu1489, tu1490) did not show any change in the highly St phenotype, dismissing a role for this substitution in controlling mouth-form variation (S2 Fig and S1 Table). All other SNPs between the parental strains either are in intergenic or intronic regions or represent synonymous changes in genes of the multigene locus. Therefore, we focused on potential cis-regulatory variation as numerous studies have shown the involvement of cis-regulatory elements in adaptive divergence, particularly in promoter and enhancer regions of developmental control genes [11–16]. The highest number of SNPs between RSA076 and RSC011 are in the upstream region and the first intron of eud-1 (S2 Table). Specifically, 5 SNPs in the upstream region and 1 SNP in intron 1 of eud-1 are shared between related strains of RSA076 and RSC011 (Figs 2B and S3). In addition, we detected a 32-bp element that contains sequence similarity to a potential Forkhead transcription factor binding site (hereafter, Forkhead binding site (FBS)) in the upstream region of eud-1 (Fig 2B). Interestingly, we observed copy number variation (CNV) of this 32-bp element between strains. RSA076 has 2 copies of this element, whereas RSC011 has only a single copy (Fig 2B). These SNPs and the CNV might be involved in the regulation of eud-1, as eud-1 expression in RSA076 is 40% higher, consistent with its role in the specification of the Eu morph (Fig 2C).

Cis-regulatory variants affect eud-1 expression Next, we used quantitative reverse transcription PCR (qRT-PCR) experiments to provide direct evidence that the engineered CRISPR lines shifting the mouth-form ratio from Eu to St are indeed affecting eud-1 expression. For this, we measured eud-1 expression relative to the normalized eud-1 expression of the parental RSA076 strain. We found that lines with the deletion of both FBS and the intronic swap (tu1870, tu1871) show strongly reduced eud-1 expression, similar to RSC011 (Fig 3C). This was further validated in the line with the 31-bp deletion in intron 1, which also resulted in a strong reduction of eud-1 expression (Fig 3C). These findings indicate that the cis-regulatory variants in natural isolates of P. pacificus affect eud-1 expression and thereby influence mouth-form execution.

Population-scale whole-genome sequencing suggests the Eu pattern to be ancestral Finally, we tested if the QTL shows any evidence for selection and wanted to determine the direction of evolutionary change in mouth-form ratio. For that, we employed available population-scale whole-genome sequencing data for the strains used in this study [8] to compare their genotype with mouth-form ratios (S8 Fig). Specifically, we compared all 10 available strains from CK, where RSC011 was isolated, with representative strains from NB, the origin of RSA076 (S8 Fig). Strikingly, we identified 4 of the 10 strains from CK that had a preferentially St mouth form and shared the same haplotype at the eud-1 locus with RSC011 (S8A Fig). In contrast, the remaining isolates exhibited either the RSA076-type variants at the eud-1 locus or a mixed pattern (RSC010, RSC173) and were all preferentially Eu (S8B Fig). No such variation was seen in the strains from NB with all strains being preferentially Eu and having the RSA076 haplotype (S8B Fig). These results indicate that the RSA076 Eu pattern is ancestral in P. pacificus clade B and that the RSC011 St phenotype has recently evolved. Moreover, the CK strains with the RSC011 haplotype at the QTL peak showed a remarkably low diversity suggesting that this haplotype was introduced very recently into the population (Figs 3D and S8C). To distinguish if these patterns are shaped by neutrality or natural selection will require future sampling efforts with a higher temporal resolution.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002270

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