Skip to main content
NCBI home page
Biology Open logoLink to Biology Open
. 2025 Oct 16;14(10):bio062018. doi: 10.1242/bio.062018

Coincident evolution and functional adaptation of the taxonomically restricted genes ivph-3 and gon-14 in Caenorhabditis nematodes

Nikita Jhaveri 1,*, Bhagwati Gupta 1, Helen M Chamberlin 2,✉
PMCID: PMC12570147  PMID: 40995696

ABSTRACT

Poorly conserved or taxonomically restricted genes represent a sizable portion of most genomes. Many of these genes participate in essential processes and can contribute to evolutionary innovation in species. Here, we evaluate two of the Caenorhabditis-restricted class of LIN-15B-domain-encoding genes, ivph-3 and gon-14, and compare their in vivo functions in two species, C. elegans and C. briggsae. We show that within the Elegans supergroup, ivph-3 and gon-14 exhibit sequence constraints distinct from other family members, including maintenance of a one-to-one orthology and a higher degree of sequence conservation. Coincidentally, mutants for either of the genes exhibit strong phenotypic defects that are similar within species (C. elegans or C. briggsae), but with notable differences when comparing between species. These findings highlight the genetic and genomic features associated with the evolution of a taxonomically restricted gene family.

Keywords: Taxonomically restricted genes, Gene adaptation, Caenorhabditis nematodes

Summary: This work identifies genetic and genomic hallmarks of the taxonomically restricted genes gon-14 and ivph-3 in Caenorhabditis, and highlights changes that correlate when a new gene integrates into important biological processes.

INTRODUCTION

The principles of gene sequence conservation and orthology are fundamental to our understanding of biological systems and underlie the powerful tools we use to assign and infer function across genomes, proteins and phyla (Ashburner et al., 2000). While a high degree of gene sequence conservation across species suggests functional importance, it has long been clear that the obverse – that lack of sequence or phylogenetic conservation suggests functional unimportance – does not hold (Khalturin et al., 2009; Chen et al., 2013; Kondo et al., 2017; Baalsrud et al., 2018). Technical innovations and new data-mining methodologies provide one important approach to identifying gene families and prospective functions for ‘orphan’ genes and others that represent the genomic and proteomic ‘dark matter’, genes whose roles remain elusive due to limited conservation, unique evolutionary trajectories, or insufficient functional annotation (Toll-Riera et al., 2009; Durairaj et al., 2023; McKnight, 2024; Pereira et al., 2025). However, understanding the emergence, evolution, and function of poorly conserved genes remains an important unanswered question.

Here, we investigate the evolutionary origins and in vivo genetic characteristics of two Caenorhabditis-restricted genes, ivph-3 and gon-14. ivph-3 and gon-14 are both members of a gene family that encode proteins with the RNAse H-fold-containing LIN-15B domain, a domain that may facilitate a protein's association with DNA and transcriptional repression (Rechtsteiner et al., 2019; Gal et al., 2021). LIN-15B-encoding genes are apparent only in a subset of Caenorhabditis species. Within the Elegans supergroup, ivph-3 and gon-14 are distinct from other family members in that they exhibit a higher degree of sequence conservation and retain a one-to-one orthology, suggestive of coincident adaptation of these two genes. We show that genetic disruption of Cbr-ivph-3 results in widespread perturbation of gene transcripts, suggesting the gene extensively impacts both positive and negative gene regulation. Further, we show that disruption of either of these genes has strong and similar within-species phenotypic consequences in two species (C. elegans or C. briggsae), but that there are notable differences when comparing between species. Cross-species rescue experiments indicate that these changes include both gene-specific and species-specific differences. Altogether, this work reveals genetic and genomic hallmarks of new gene family emergence, and highlights changes that correlate as specific family members integrate into and support essential processes.

RESULTS AND DISCUSSION

RNA transcripts exhibit widespread dysregulation in Cbr-ivph-3 mutants

Functions for the ivph-3 (also known as ivp-3) gene were first understood in the nematode C. briggsae, as mutant alleles were recovered in genetic screens for animals with a Multivulva (Muv) phenotype (Sharanya et al., 2015; Chamberlin et al., 2020). While this phenotype is the most obvious in mutants, the animals exhibit several additional defects including alterations to mobility and sensitivity to oxidative stress (Fig. S1). To better understand the function of Cbr-ivph-3, we performed RNAseq experiments using RNA recovered from synchronized L3-stage larval animals (Tables S1-S5). Initial analysis revealed differential expression of 3852 genes, with a roughly equal distribution of upregulated (1898) and downregulated (1954) genes (Fig. 1A). We compared the Cbr-ivph-3 data to previously reported datasets derived from two other C. briggsae mutants that exhibit the Muv phenotype and encode proteins that act to repress transcription of genes in C. elegans (Cbr-spr-4(gu163) encoding a C2H2 zinc finger protein and Cbr-htz-1(gu167) encoding H2A.z histone; Lakowski et al., 2003; Latorre et al., 2015; Chamberlin et al., 2020) to ask whether mutants for all three genes exhibit common characteristics with respect to gene dysregulation. First, notably more genes exhibited differential expression (DE) in Cbr-ivph-3 mutants compared to the other two (3852 versus 1241 and 505, respectively). In addition, upregulated genes represent a greater fraction of DE genes in Cbr-spr-4 and Cbr-htz-1 mutants, significantly different from Cbr-ivph-3 (Fig. 1B,C). Previous experiments indicated that the Muv phenotype associated with Cbr-ivph-3 mutants and others of this class results from increased and ectopic expression of the Cbr-lin-3/EGF gene (Chamberlin et al., 2020), and this gene is indeed represented among transcripts that exhibit increased abundance in the overlap of Cbr-spr-4, Cbr-htz-1 and Cbr-ivph-3 mutant strains (Table S4). Taken together, the results indicate that while Cbr-ivph-3 shares some gene regulatory network characteristics with Cbr-spr-4 and Cbr-htz-1, it is functionally distinct, impacting the transcripts of a broader number of genes with both positive and negative effects.

Fig. 1.

Fig. 1.

Open in a new tab

RNA transcripts exhibit widespread dysregulation in Cbr-ivph-3 mutants. (A) Volcano plot showing the differentially expressed (DE) genes in Cbr-ivph-3(sy5216) mutant transcriptome compared to AF16 wild type. 1898 genes exhibit increased abundance (log2 fold change >+1) and 1954 genes exhibit decreased abundance (log2 fold change <−1), or 49% of DE genes are increased compared to wild type. This is in contrast with the data from the Muv mutants Cbr-spr-4(gu163) and Cbr-htz-1(gu167) (Chamberlin et al., 2020) where the corresponding values are 843 and 398 (68% increased) for Cbr-spr-4(gu163) and 329 and 176 (65% increased) for Cbr-htz-1(gu167), values that are statistically different from the Cbr-ivph-3(sy5216) data, but not each other (P<0.05, two-tailed proportional z-test). (B) Overlap of upregulated genes present in transcriptomic data sets of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivph-3(sy5216). Fifty-six genes exhibit increased abundance in all three mutants, including Cbr-lin-3/EGF that is confirmed previously for all three strains using RT-qPCR (Chamberlin et al., 2020). (C) Overlap of downregulated genes present in transcriptomic data sets of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivph-3(sy5216). Only three genes exhibit decreased abundance in all three mutants. Gene Ontology (GO) analysis of the DE genes common to all three strains identified positive regulation of mitotic nuclear division (GO:0045840) and positive regulation of nuclear division (GO:0051785). Data are in Tables S1-S5 (DE gene lists and overlaps) and S12-S15 (GO term analysis).

IVPH-3 and other LIN-15B-domain-containing proteins are encoded by a gene class prevalent in a subset of Caenorhabditis species

Cbr-ivph-3 has been identified to be part of a gene family that encodes proteins with the LIN-15B domain. This domain extends over 500 amino acids, and includes a sub-region with sequences similar to hAT transposases (Chesney et al., 2006; Chamberlin et al., 2020; Fig. S2). LIN-15B-domain-encoding genes are prevalent in the genomes of some Caenorhabditis species, with seven clear representatives in C. elegans and six in C. briggsae (Fig. 2). However, there are no clear LIN-15B-domain containing proteins made in organisms with other highly curated genomes, such as human, mouse, zebrafish, or Drosophila (using Blastp; Altschul et al., 1990). While lack of Blastp hits does not rule out the possibility for highly divergent family members produced by a genome, these analyses support the classification of LIN-15B-domain encoding genes as lineage-restricted within Caenorhabditis, or poorly conserved across species.

Fig. 2.

Fig. 2.

Open in a new tab

Multiple genes encoding LIN-15B domain proteins are present in the genomes of Caenorhabditis species of the Elegans-supergroup and related nematodes, but not apparent in more distantly related Caenorhabditis species. (A) A phylogenetic tree for 16 Caenorhabditis species, adapted from Sun et al., 2022. The number of genes encoding proteins with a LIN-15B domain for each genome is listed in parentheses, based on Blastp hits at Caenorhabditis.org using default parameters. (B) Maximum likelihood tree of proteins with a LIN-15B domain from the species in panel A. C. elegans and C. briggsae IVPH-3 and GON-14 are highlighted, along with the three additional C. elegans protein sequences included in the alignment of Fig. S2. Scale is substitution per site. LIN-15B, F19F10.11 and ETS-6 also contain one or more THAP or THAP-like domains, but these are distinct and separate from the LIN-15B domain (Clouaire et al., 2005). (C) Predicted structures for Cel-IVPH-3 (AF-Q86DD2-F1-v4), Cel-GON-14 (AF-Q4PIU4-F1-v4), and Hsa-ZBED4 (AF-O75132-F1-v4) from AlphaFold (Jumper et al., 2021), annotated with the color coding of Fig. S2 and orientation adjusted using EzMol (Reynolds et al., 2018).

To better understand the evolution of this gene family within nematodes, we evaluated sequence data from 16 Caenorhabditis species, distributed across the genus (Stevens et al., 2019, 2020; Sun et al., 2022). While multiple LIN-15B-domain-encoding genes are present in genomes derived from Elegans supergroup species and some related species, they are not detected in more basal Caenorhabditis, or in outgroup nematodes Diploscapter pachys, Diploscapter coronatus, or Ascaris suum (Fig. 2A,B). To ask whether structurally similar proteins may be encoded by other genomes but not apparent at the primary sequence level using Blastp, we used AlphaFold to evaluate predicted structural features (Jumper et al., 2021; Varadi et al., 2024). LIN-15B-domain-containing proteins all have many high confidence domains (pLDDT>90) including a canonical RNAse H fold (Fig. 2C; Fig. S2). Human proteins with similar predicted structures identified using Foldseek (AFDB50, van Kempen et al., 2024) include zinc finger BED-domain containing proteins (ZBED) (Fig. 2C; Table S17). We conclude that LIN-15B-domain containing proteins share structural features with proteins from other species, but they lack clear orthologs outside of a subset of Caenorhabditis species. While the origin of these Caenorhabditis genes is not clear, it is notable that genes encoding structurally similar proteins, including ZBED genes in vertebrates and SLEEPER genes in angiosperms, originated as transposons that subsequently were ‘domesticated’, with their products adapted to critical roles in a variety of important host cell processes (Sinzelle et al., 2009; Arensburger et al., 2011; Knip et al., 2012; Hayward et al., 2013).

ivph-3 and gon-14 exhibit distinct sequence constraints in Elegans supergroup species compared to other LIN-15B-domain-encoding genes

We constructed a phylogenetic tree of the LIN-15B-domain-containing proteins to better understand the emergence and relationships among the encoding genes in Caenorhabditis (Fig. 2B). Our analysis revealed that the genomes exhibit a high degree of tolerance to duplication, loss, and sequence change for most genes. For example, the C. elegans genome contains seven genes in this family, five of which are lin-15B and two partial duplicates of the lin-15B gene, as well as the duplicate genes Cel-ets-6 and Cel-F19F10.11 (Fig. 2B). In contrast, among genomes of the Elegans supergroup species, genes encoding IVPH-3- or GON-14-related proteins retain an apparent one-to-one orthology and a relatively high level of sequence conservation compared to other family members (Fig. 2B). We conclude that ivph-3 and gon-14 exhibit gene copy number and sequence constraints distinct from the other family members. We hypothesize that these features, when observed in a novel or poorly conserved gene family, correlate with an adaptive transition of gene function. For example, such selection modifications may occur when a gene's product embeds in a biological process where enzymatic functions or interactive partners place greater constraints on stoichiometry and sequence characteristics (Worth et al., 2009). The coincident behavior for two genes in the family (ivph-3 and gon-14) is suggestive that they adapted to a common process.

ivph-3 and gon-14 mutants exhibit similar within-species phenotypes, and coincident evolution of phenotype

To better understand the relationship between ivph-3 and gon-14 and how these genes function in vivo, we evaluated mutant phenotypes in C. elegans compared to C. briggsae (Fig. 3). Cel-gon-14 mutants were isolated in genetic screens for hermaphrodites with defects in specification of the somatic gonad distal tip cells (DTCs) which are essential for proper development of the gonad and the support of germ cell proliferation (Siegfried et al., 2004; Chesney et al., 2006). Consistent with this function, homozygous Cel-gon-14 mutant hermaphrodites are sterile, and frequently lack one or both gonad arms (Fig. 3B,D-E; Siegfried et al., 2004). Many ivph-3(gk3691) mutants are sterile, but most derived from heterozygous mothers retain both gonad arms (Fig. 3C-E). Any offspring from fertile ivph-3(gk3691) homozygous mutants are sterile (Maternal Effect Sterile, Mes, Fig. 3E), and all exhibit defects in gonad arm outgrowth (Fig. 3D). We conclude that fertility and gonad arm development defects are shared phenotypes associated with Cel-ivph-3 and Cel-gon-14 mutants. In contrast to the C. elegans mutants, Cbr-ivph-3 and Cbr-gon-14 mutants are generally fertile (Fig. 3E, Chamberlin et al., 2020). For both C. elegans and C. briggsae, ivph-3; gon-14 double mutants do not exhibit an enhanced phenotype compared to single mutants, a result consistent with the idea that in both species, the two genes participate in a common process. Homozygous Cbr-ivph-3 or Cbr-gon-14 mutants exhibit a strong Muv phenotype (Fig. 3F; Sharanya et al., 2015; Chamberlin et al., 2020). By contrast, Cel-ivph-3 and Cel-gon-14 mutants are generally nonMuv with a normal pattern of vulva development, and this phenotype is not enhanced in double mutants (Fig. 3F-G; Chesney et al., 2006; Chamberlin et al., 2020).

Fig. 3.

Fig. 3.

Open in a new tab

Cel-gon-14 and Cel-ivph-3 mutants exhibit similar defects in gonad development and fertility, and are phenotypically distinct from Cbr-gon-14 and Cbr-ivph-3 mutants. (A-D) Gonad arm outgrowth is defective in Cel-gon-14 and Cel-ivph-3 mutants. (A) In wild-type hermaphrodites, two gonad arms develop from the gonad primordium in the animal midbody. In L4 larvae, the arms have developed toward the animal ends, completed a U-turn, and migrated back toward the midbody along the dorsal side of the body cavity. Asterisk indicates the position of the developing vulva opening, and a single gonad arm (posterior) is shown. (B) Cel-gon-14 mutants derived from heterozygous mothers exhibit defects in distal tip cell (DTC) differentiation and subsequent outgrowth of the gonad arms (Siegfried et al., 2004), and the gonad cells fail to migrate and frequently form a single mass of cells in the center of the animal. (C) While the gonads of Cel-ivph-3 mutants derived from heterozygous mothers are generally normal in morphology (not shown), Cel-ivph-3 mutant offspring produced by mutant mothers (m-z-) likewise exhibit gonad arm defects. (D) Quantification of the gonad arm defect. Introduction of Cel-ivph-3(gk3691) into the Cel-gon-14(q12) background does not enhance the phenotype. Asterisk indicates statistically different from Cel wild type (P<0.05, Fisher-Freeman-Halton test with Bonferroni correction). n.s., not significantly different. Cel-ivph-3; Cel-gon-14 is also not significantly different from Cel-gon-14(q12) (using the same test). (E) Homozygous mutant Cel-gon-14 or Cel-ivph-3 animals fail to produce offspring (Ste) or produce some offspring that all fail to produce offspring (Mes). Double mutant animals exhibit a phenotype similar to the single mutants. Homozygous mutant Cbr-gon-14 or Cbr-ivph-3 animals are generally fertile, as are double mutants (Chamberlin et al., 2020). Asterisk indicates statistically different from the wild type (P<0.05, Fisher-Freeman-Halton test with Bonferroni correction). n.s., not significantly different. (F) Homozygous mutant Cbr-gon-14 or Cbr-ivph-3 mutants exhibit a strong Multivulva (Muv) phenotype, whereas Cel-gon-14 and Cel-ivph-3 mutants are generally nonMuv. Asterisk indicates statistically different from the wild type (P<0.05, Fisher's exact test with Bonferroni correction). n.s., not significantly different. (G) Animals that do exhibit ventral protrusions (scored as Muv) typically exhibit defects in vulva morphogenesis rather than inappropriate vulva cell proliferation, as only the wild-type of three vulva precursor cells (VPCs) typically divide to produce vulva cells in Cel-gon-14 and Cel-ivph-3 mutants. Cel-gon-14 single and Cel-ivph-3; Cel-gon-14 double mutants exhibit increased variation in the number of induced cells, with some animals producing two or four induced cells rather than three as in the wild type. Asterisk indicates statistically different from the wild type (P<0.05, Fisher-Freeman-Halton test with Bonferroni correction). n.s., not significantly different. Although the mutant strains have an increased frequency of non-wild-type cell patterns, the average number of induced cells (induction index) for single and double mutants is not different from wild type (t-test with Bonferroni correction). Average number of induced cells for these data: Cel wild type (3, s.d.=0), Cel-ivph-3(gk3691) (3, s.d.=0), Cel-gon-14(q12) (2.9, s.d.=0.416), Cel-ivph-3; Cel-gon-14 (2.8, s.d.=0.648). Full genotypes for double mutants: Cel-ivph-3(gk3691); Cel-gon-14(q12). Cbr-ivph-3(sy5216); Cbr-gon-14(gu102). Sample sizes are indicated at the base of each bar.

Species differences in function for ivph-3 and gon-14 reflect both context-specific and gene-specific changes

To identify the source of the phenotypic differences between the two species, we evaluated the capacity of each gene to rescue genetic mutants. We introduced wild-type genomic DNA PCR fragments as transgenes, and asked whether they can rescue the mutant phenotype (Fig. 4). Cbr-gon-14(+) DNA rescues gon-14 mutants of either species, whereas Cel-gon-14(+) DNA only rescues Cel-gon-14 mutants (Fig. 4B,D). By contrast, Cel-ivph-3(+) DNA rescues ivph-3 mutants of either species, whereas Cbr-ivph-3(+) DNA only rescues Cbr-ivph-3 mutants (Fig. 4B,D). Since Cel-ivph-3 and Cbr-ivph-3 differ in that Cel-ivph-3 can produce transcripts with upstream exons not present in Cbr-ivph-3 (Fig. S3; wormbase.org Davis et al., 2022), we hypothesized that this difference might reflect Cel-ivph-3-specific functions for the longer transcript. However, either a fragment including all Cel-ivph-3 exons (Cel-ivph-3(+)L) or one that lacks the two most 5′ exons (Cel-ivph-3(+)S) is sufficient to rescue the Ste phenotype associated with Cel-ivph-3(gk3691) (Fig. 4B). Altogether, we conclude that the Cel-gon-14(+) or Cbr-ivph-3(+) sequences sufficient for function within species are not sufficient to rescue across different species, whereas those of Cbr-gon-14(+) and Cel-ivph-3(+) are. These results identify structural and functional differences between gon-14 and ivph-3 in the two species. Additionally, cellular and genetic context plays a role in the ultimate phenotypic consequences, since loss of Cbr-gon-14 has a minor impact on fertility and loss of Cel-ivph-3 has a minor impact on vulval development, but the wild-type gene can fully restore these functions when introduced into cross-species mutants with the defect.

Fig. 4.

Fig. 4.

Open in a new tab

ivph-3 and gon-14 exhibit distinct capacities to compensate across species. (A) Gene models for Cel-ivph-3, Cbr-ivph-3, Cel-gon-14 and Cbr-gon-14 (from wormbase.org, Davis et al., 2022), annotated to indicate the extent of genomic sequence included in the PCR products used for transgenic rescue in panels B-E. A triangle indicates the swap point for the chimeric genes evaluated in C and E. (B,C) Transgenic rescue experiments for Cbr-ivph-3 and Cbr-gon-14 mutants. (B) Either long (L) or short (S) Cel-ivph-3(+) DNA rescues the Muv phenotype associated with Cbr-ivph-3 mutants, whereas Cel-gon-14(+) DNA does not rescue the phenotype in Cbr-gon-14 mutants. (C) Either Cbr_Cel-ivph-3(+) DNA (Cbr sequences upstream and Cel sequences downstream of the gene start codon) or Cel_Cbr-ivph-3(+) DNA can rescue the Muv phenotype associated Cbr-ivph-3 mutants. Neither Cbr_Cel-gon-14(+) DNA nor Cel_Cbr-gon-14(+) DNA restores gene activity as does the intact Cbr-gon-14(+) DNA in Cbr-gon-14 mutants. (D,E) Transgenic rescue experiments for Cel-ivph-3 and Cel-gon-14 mutants. (D) Either Cel-gon-14(+) or Cbr-gon-14(+) DNA rescues the Ste phenotype associated with Cel-gon-14 mutants. Either long (L) or short (S) Cel-ivph-3(+) DNA can rescue Cel-ivph-3 mutants but Cbr-ivph-3(+) DNA does not. (E) Cbr_Cel-ivph-3(+) DNA can restore fertility to Cel-ivph-3 mutants, whereas Cel_Cbr-ivph-3(+) DNA does not. Cel_Cbr-ivph-3(+) transgene-bearing animals are statistically different from control as more animals are Ste rather than Mes. Either the Cbr_Cel-gon-14(+) DNA or the Cel_Cbr-gon-14(+) DNA can restore fertility to Cel-gon-14 mutants. Asterisk indicates statistically different from the homozygous mutant without a transgene (P<0.05, Fisher’s exact test or Fisher-Freeman-Halton test, with Bonferroni correction). n.s., not significantly different. Sample sizes are indicated at the base of each bar.

One explanation for the cross-species gene rescue results could be that the genes capable of rescuing in both species are expressed and function in a broader set of cells compared to the genes with more restricted rescue ability. Single cell RNAseq data indicate that the expression pattern for each gene is relatively well conserved, and both genes have relatively broad expression in both species (Large et al., 2025; Toker et al., 2025). Indeed, a cell-type expression specificity measure (Tau) indicates that in both cases the gene with ability to rescue in both species exhibits a greater cell-restricted pattern of expression than its ortholog (Large et al., 2025; Table S19). We evaluated the rescue ability of chimeric genes composed of sequences upstream of the start codon from one species and downstream sequences from the other, and found that while either chimeric transgene can rescue the mutant phenotype for strains rescued by the genomic DNA of both species, Cbr-gon-14 mutants were poorly rescued by both, and only the Cbr_Cel-ivph-3(+) transgene (upstream sequences from Cbr-ivph-3 and downstream from Cel-ivph-3) restored fertility to the Cel-ivph-3 mutants (Fig. 4C,E). Altogether, these results indicate there may be multiple distinctions between the species for each gene, although future experiments will be necessary to identify their specific differences.

Taxonomically restricted genes are prevalent in all genomes and have a variety of evolutionary origins (Pereira et al., 2025). They are of evolutionary interest as a class due to their capacity to be recruited to essential biological processes de novo, and to contribute to novel biological adaptations (Khalturin et al., 2009). Here, we evaluate the characteristics for one taxonomically restricted gene class, those encoding LIN-15B-domain proteins in Caenorhabditis. The LIN-15B domain includes an RNAse H fold, an evolutionary ancient protein structure that is prevalent in transposases and other nucleases (Ma et al., 2008; Nowotny, 2009; Majorek et al., 2014). While we do not rule out the possibility that IVPH-3 and GON-14 function as nucleases, other RNAse H fold-containing proteins such as those in the ZBED family associate with specific DNA elements and act as transcriptional regulators (Markljung et al., 2009; Hayward et al., 2013; Somerville et al., 2020). In C. elegans, LIN-15B itself associates with specific sites in the genome, and impacts the transcription of target genes (Rechtsteiner et al., 2019; Gal et al., 2021), and alleles of Cel-gon-14 have been identified in genetic screens for altered transcriptional response to mitochondrial dysfunction (Xu et al., 2024). However, the mechanisms for how GON-14 and IVPH-3 function remain important work for the future.

Our results indicate that Caenorhabditis LIN-15B domain-encoding genes exhibit distinct selection characteristics that correlate with in vivo function. Two genes, ivph-3 and gon-14, each play an important role in development in two species, C. elegans and C. briggsae. Where tested in C. elegans, genetic disruption of others in the family do not exhibit overt phenotypes on their own under standard growth conditions, although several exhibit synthetic or redundant functions with other genes (Clark et al., 1994; Huang et al., 1994; Byrne et al., 2007; Sawyer et al., 2011), and Cel-lin-15B participates in a variety of gene regulatory processes (Wang et al., 2005; Petrella et al., 2011; Gal et al., 2021). Overall, our analysis provides a deeper understanding of how novel taxonomically restricted genes transition from redundant or auxiliary roles to essential functions, shedding light on their evolutionary significance and their adaptation to species-specific roles.

MATERIALS AND METHODS

Worm maintenance and genetics

Caenorhabditis strains were grown on NGM (or NG-Agar) plates seeded with Escherichia coli strain OP50 as a food source (Stiernagle, 2006). All experiments were carried out at 20°C unless otherwise indicated. The wild-type C. elegans used was N2 Bristol. The wild-type C. briggsae used was AF16. Specific strains and genotypes are listed in Table S6. Cel-gon-14(q12) is a G to A substitution that results in a premature termination codon at amino acid 598 (exon 9) (Chesney et al., 2006). Cel-ivph-3(gk3691) is an allele that deletes exon 4 and the first nine bp of exon 5 of the longest transcript (Fig. 4; exon 1 and 2 of Y67D8C.3a.1; Chamberlin et al., 2020). Cbr-gon-14(gu102), Cbr-gon-14(gu168), Cbr-ivph-3(sy5216) and Cbr-ivph-3(gu236) each introduce a premature stop codon into their respective genes (Chamberlin et al., 2020). Cbr-gon-14(gu102) is a G to A substitution that alters the 5′ splice recognition site of intron 8 (of CBG18977b.1), resulting in failure to remove the intron and introduction of an in-frame stop codon. Cbr-gon-14(gu168) is a C to T substitution that introduces a premature stop codon into exon 7. Cbr-ivph-3(sy5216) is a G to A substitution that introduces a premature stop codon into exon 7 (of CBG03376.1). Cbr-ivph-3(gu236) is a C to T substitution that introduces a premature stop codon into exon 4.

RNA preparation, sequencing, and analysis

C. briggsae animals (AF16 wild type or PS9217 Cbr-ivph-3(sy5216)) were developmentally synchronized using a standard bleaching method (Porta-de-la-Riva et al., 2012). Synchronized L1s were plated onto OP50 seeded plates and incubated at 20°C. Larval animals were staged based on VPC division using Nomarski optics (Yochem, 2006; Chamberlin et al., 2020), and harvested for RNA following the Trizol (Invitrogen) protocol. Three independent biological replicates were prepared for each genotype and sequenced using the Illumina NovaSeq 6000 system by Genome Quebec to generate paired-end reads. The average number of reads obtained per sample was 2.9 million (ranging from 2.5 million to 3.4 million), with an average read length of 200 bp. RNA sequencing reads that were generated by Genome Quebec were processed using the online program, Galaxy (Galaxy Community, 2024, https://usegalaxy.org/). The algorithm for each program is outlined in Table S11. Gene Ontology (GO) enrichment analysis was performed using the PANTHER overrepresentation test (version 20240807; Mi et al., 2021) (Tables S12-S15).

Evaluation of IVPH-3-related protein sequence

The amino acid sequence of Cel-IVPH-3 was obtained from wormbase.org (WS294) (Davis et al., 2022), and used in the protein blast function on Caenorhabditis.org against datasets for the 16 Caenorhabditis species listed in Fig. 2A. The resulting sequences were filtered to retain a single (the longest) isoform for each predicted protein hit (Table S9). The tree of Fig. 2B was produced using these sequences, aligned using Muscle and evaluated using the Jones Taylor-Thornton Maximum Likelihood method within MEGA11 (Tamura et al., 2021).

Four C. elegans proteins have both a full-length LIN-15B-domain and predicted structures represented in the AlphaFold database (Cel-IVPH-3, Cel-GON-14, Cel-LIN-15B, Cel-ETS-6). These all have an average predicted local distance difference test (pLDDT) value between 60-75, but with many high confidence domains (pLDDT>90). All four proteins are annotated for an RNAse H fold (TED classification 3.30.420, Nucleotidyltransferase; domain 5; Lau et al., 2024) followed by one or more unclassified domains that correspond to other elements of the LIN-15B domain (Fig. 2C; Fig. S2). Structurally related proteins for Cel-IVPH-3 (AF-Q86DD2-F1-v4) and Cel-GON-14 (AF-Q4PIU4-F1-v4) were identified using Foldseek and both the PDB and AFDB50 databases (van Kempen et al., 2024) as implemented on the Alphafold website (AlphaFold.ebi.ac.uk; Jumper et al., 2021). This identified structurally related proteins for Cel-IVPH-3 [22 in PDB and the maximum (1000) in AFDB50] and Cel-GON-14 (23 in PDB and 64 in AFDB50). Top hits for each outside of Caenorhabditis were proteins annotated as including a BED domain (sorted by lowest E-value). The full list of hits for each protein was interrogated with the same species or genera queried for Blast search [human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio), fruit fly (Drosophila), nematodes (Caenorhabditis, Ascaris and Diploscapter)]. Proteins from none of these species are represented in the Cel-GON-14 sets. Within the Cel-IVPH-3-related sets, a single hit (human DNA mismatch repair protein Msh3) was identified in PDB (Table S16). Hits in the AFDB50 database include one in human (Zinc finger BED domain-containing protein 4), two in zebrafish, and seven in Drosophila species (two in D. melanogaster, and one each in five other species), all identified either as BED domain-containing or uncharacterized proteins (Table S17). No hits in either database were identified for the two outgroup nematode genera, although the AFDB50 database included over 27,000 structural model predictions from Ascaris (primarily Ascaris lumbicoides) and over 32,000 from Diploscapter (primarily Diploscapter pachys). Within Caenorhabditis, only hits from Elegans supergroup species were identified (Table S18), although very few predicted protein models are included for species outside of the Elegans supergroup in AFDB50. Predicted structures for Cel-IVPH-3 (AF-Q86DD2-F1-v4), Cel-GON-14 (AF-Q4PIU4-F1-v4), and Hsa-ZBED4 (AF-O75132-F1-v4) were obtained from AlphaFold.ebi.ac.uk (Jumper et al., 2021; Varadi et al., 2024). These structures were annotated with color to highlight structural features and adjusted for orientation using EzMol (www.sbg.bio.ic.ac.uk/ezmol/) (Reynolds et al., 2018).

Molecular biology and transgenics

PCR was used to amplify products from wild type (C. briggsae AF16 or C. elegans N2) genomic DNA (primers in Table S8). These PCR products were incorporated into injection mixes (25 ng/μl PCR product, 15 ng/μl pDP#MM016 (Cel-unc-119(+)), 10-20 ng/μl pCFJ90 (myo-2::mCherry), and 50 ng/μl 1 kb ladder (NEB) as a carrier), and injected into the gonad of adult hermaphrodites following standard C. elegans protocols (Mello et al., 1991). Specific transgene details are in Table S7.

Phenotypic assays

Fertility

L4 hermaphrodites (P0) were selected individually onto plates and observed for offspring after 2 days and after 7 days. Animals with no apparent eggs or offspring on either day were scored as Sterile (Ste). Animals with apparent eggs and offspring on both days, with clear production of offspring from the F1 progeny, were scored as Fertile. Animals with apparent eggs or offspring on day 2, but no apparent progeny from the F1 animals were scored as Maternal Effect Sterile (Mes). All experiments in a given graph were conducted in parallel, in 2-4 batches.

Vulva development

Production of the Multivulva (Muv) phenotype was measured in young adult animals by selecting L4 hermaphrodites onto plates and observing their morphology on the next day (Lyu and Chamberlin, 2024). To measure the extent of vulva cell development (induction; Fig. 3G), early L4 larval animals were selected and evaluated for the number of vulval cells and inferred number of VPCs that had divided to produce them (Sternberg and Horvitz, 1986). All experiments in a given graph were conducted in parallel, in 2-4 batches.

Gonad morphology

Microscopic slides were prepared as described above, and gonad arm morphology was scored in L4 larvae. In wild-type C. elegans (and C. briggsae) hermaphrodites, the gonad develops with two arms that extend from the mid-body along the ventral of the animal, then migrate to the dorsal and back toward the mid-body. Gonad arms lacking a distal tip cell (DTC) fail to migrate. L4 animals were evaluated for the presence of gonad arms that had completed an extent of ventral migration, as well as migration to the dorsal. All experiments in a given graph were conducted in parallel, in 2-4 batches.

Animal movement

Young adult animals (day-1 adult) were obtained by separating well-fed L4 animals to a fresh plate and performing the analyses on the next day. Pharyngeal pumping was measured in day-1 adult hermaphrodites under a dissecting microscope, counting the number of times pharyngeal contraction was observed in 30 s. Body bends were measured by counting the number of sinusoidal waves the worm made in 1 min (one wave corresponds to one body bend). The reading was not counted if the worm stopped moving before 1 min time elapsed.

Oxidative stress

Methyl viologen (Paraquat, PQ) was used to assay oxidative stress. Young adult (day-1) worms were washed off a synchronized plate and added into 300 μl of 200 mM PQ in M9 in 24-well plates. 50–100 worms were added in each well, and two wells were used for each strain. Animals were evaluated once per hour over a 4 h period for their response to touch – if animals did not respond and had a rod-like appearance, they were counted as dead.

Sequence alignment

The LIN-15B domains of C. elegans LIN-15B-related proteins were recovered and cropped from the C. elegans sequences in Table S9 (based on the blast alignment), yielding the sequences listed in Table S10. These sequences were aligned using the EMBL-EBI Clustal Omega server (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) (Sievers et al., 2011), and shaded using BoxShade (https://junli.netlify.app/apps/boxshade/).

Cbr-ivph-3 cDNA

To define the transcripts made from the Cbr-ivph-3 locus and to validate the predicted gene model, we harvested RNA from day-1 wild-type animals (Trizol), synthesized first strand cDNA using LunaScript RT Supermix kit (NEB #E3010) that contained random hexamers and oligo-dT primers and used PCR to amplify possible products with a Cbr-ivph-3-specific reverse primer in predicted exon 7, and primers to recognize splice leaders (SL1 and SL2). Two products were obtained using the SL1-specific primer. These products were separated on an agarose gene, excised, and subject to Sanger sequencing, which identified two SL1-spliced isoforms which include the longer isoform represented in the gene model, as well as an isoform that begins with exon 3 of the longer isoform. Primers are listed in Table S8.

Supplementary Material

biolopen-14-062018_review_history.pdf (406.1KB, pdf)
Supplementary information
biolopen-14-062018-s1.pdf (1.7MB, pdf)
DOI: 10.1242/biolopen.062018_sup1
Table S1.

List of DE genes in Cbr-ivp-3(sy5216).

biolopen-14-062018-TableS1.xlsx (2.1MB, xlsx)
Table S2.

Cbr-ivp-3(sy5216) DE genes with significant changes compared to wild type.

biolopen-14-062018-TableS2.xlsx (467.7KB, xlsx)
Table S3.

Overlap of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivp-3(sy5216) DE genes

biolopen-14-062018-TableS3.xlsx (254.6KB, xlsx)
Table S4.

Overlap between up-regualted DE genes of Cbr-spr-4(gu163), Cbrhtz-1(gu167) and Cbr-ivp-3(sy5216)

biolopen-14-062018-TableS4.xlsx (210.2KB, xlsx)
Table S5.

Overlap between down-regualted DE genes of Cbr-spr-4(gu163), Cbrhtz-1(gu167) and Cbr-ivp-3(sy5216)

biolopen-14-062018-TableS5.xlsx (213.1KB, xlsx)
Table S6.

Nematode strains used in this study

biolopen-14-062018-TableS6.xlsx (73.8KB, xlsx)
Table S7.

Details for transgenes used in this study

biolopen-14-062018-TableS7.xlsx (73.5KB, xlsx)
Table S8.

Primers used in this study

biolopen-14-062018-TableS8.xlsx (75.6KB, xlsx)
Table S9.

Sequences used to produce tree in Fig. 2B

biolopen-14-062018-TableS9.xlsx (57.8KB, xlsx)
Table S10.

Sequences used in LIN-15B domain alignment (Fig. S2).

biolopen-14-062018-TableS10.xlsx (12.4KB, xlsx)
Table S11.

Steps used in analysis of RNA seq data using the Galaxy platform

biolopen-14-062018-TableS11.xlsx (11.7KB, xlsx)
Table S12.

GO term enrichment in Cbr-spr-4(gu163) RNA seq data.

biolopen-14-062018-TableS12.xlsx (9.5KB, xlsx)
Table S13.

GO term enrichment in Cbr-htz-1(gu167) RNA seq data.

biolopen-14-062018-TableS13.xlsx (8.7KB, xlsx)
Table S14.

GO term enrichment in Cbr-ivph-3(sy5216) RNA seq data.

biolopen-14-062018-TableS14.xlsx (29.9KB, xlsx)
Table S15.

GO term enrichment in in the overlap of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivph-3(sy5216) RNA seq data.

biolopen-14-062018-TableS15.xlsx (8.5KB, xlsx)
Table S16.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against PDB (human, mouse, zebrafish, Drosophila, Ascaris, Diploscapter)

biolopen-14-062018-TableS16.xlsx (9KB, xlsx)
Table S17.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against AFDB50 (human, mouse, zebrafish, Drosophila, Ascaris, Diploscapter)

biolopen-14-062018-TableS17.xlsx (14.5KB, xlsx)
Table S18.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against AFDB50 (Caenorhabditis)

biolopen-14-062018-TableS18.xlsx (47.2KB, xlsx)
Table S19.

Analyses from Large et al. (2025): Jensen-Shannon Distance and Tau

biolopen-14-062018-TableS19.xlsx (72.8KB, xlsx)
Dataset 1.
biolopen-14-062018-Dataset1.zip (53.4KB, zip)

Acknowledgements

We thank A.T. Dawes for comments on the manuscript, and H. Lyu for technical assistance. Some strains were supplied by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Sanger sequencing was performed by the OSUCCC Genomics Core Shared Resource, which is subsidized by an NIH Cancer Center Support Grant (P30CA016058).

Footnotes

Author contributions

Conceptualization: N.J., B.G., H.C.; Data curation: N.J., H.C.; Funding acquisition: B.G., H.C.; Investigation: N.J., H.C.; Methodology: N.J., H.C.; Supervision: B.G., H.C.; Writing – original draft: H.C.; Writing – review & editing: N.J., B.G., H.C.

Funding

This work was supported by a National Institutes of Health award to H.M.C. (R21OD030067), and Natural Sciences and Engineering Research Council of Canada Discover grant to B.P.G. Additional support was from the Mitcas Globalink Research Award, the Global Experience Award (McMaster University, Spring 2022), a Spring/Summer GSA Travel Assistance Award (Spring 2022), and McMaster SGS Grant in Aid of Travel Research and Field Study (Spring 2022), all awarded to N.J. Open Access funding provided by Ohio State University. Deposited in PMC for immediate release.

Data and resource availability

Strains and plasmids are available upon request. Raw input data (genomes and gene annotations) can be retrieved from WormBase (under WS279 release) and NCBI (under PRJNA10731 and GSE288297 for RNA sequencing data sets of Cbr-ivph-3). Sequences were analyzed using MEGA11 (https://www.megasoftware.net/) (Tamura et al, 2021). All other relevant data and details of resources can be found within the article and its supplementary information.

Peer review history

The peer review history is available online at https://journals.biologists.com/bio/lookup/doi/10.1242/bio.062018.reviewer-comments.pdf

References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]
  2. Arensburger, P., Hice, R. H., Zhou, L., Smith, R. C., Tom, A. C., Wright, J. A., Knapp, J., O'Brochta, D. A., Craig, N. L. and Atkinson, P. W. (2011). Phylogenetic and functional characterization of the hAT transposon superfamily. Genetics 188, 45-57. 10.1534/genetics.111.126813 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T.et al. (2000). Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25-29. 10.1038/75556 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baalsrud, H. T., Tørresen, O. K., Solbakken, M. H., Salzburger, W., Hanel, R., Jakobsen, K. S. and Jentoft, S. (2018). De Novo gene evolution of antifreeze glycoproteins in codfishes revealed by whole genome sequence data. Mol. Biol. Evol. 35, 593-606. 10.1093/molbev/msx311 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byrne, A. B., Weirauch, M. T., Wong, V., Koeva, M., Dixon, S. J., Stuart, J. M. and Roy, P. J. (2007). A global analysis of genetic interactions in Caenorhabditis elegans. J. Biol. 6, 8. 10.1186/jbiol58 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chamberlin, H. M., Jain, I. M., Corchado-Sonera, M., Kelley, L. H., Sharanya, D., Jama, A., Pabla, R., Dawes, A. T. and Gupta, B. P. (2020). Evolution of transcriptional repressors impacts Caenorhabditis vulval development. Mol. Biol. Evol. 37, 1350-1361. 10.1093/molbev/msaa009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen, S., Krinsky, B. H. and Long, M. (2013). New genes as drivers of phenotypic evolution. Nat. Rev. Genet. 14, 645-660. 10.1038/nrg3521 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chesney, M. A., Kidd, A. R. and Kimble, J. (2006). gon-14 functions with class B and class C synthetic multivulva genes to control larval growth in Caenorhabditis elegans. Genetics 172, 915-928. 10.1534/genetics.105.048751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark, S. G., Lu, X. and Horvitz, H. R. (1994). The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins. Genetics 137, 987-997. 10.1093/genetics/137.4.987 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clouaire, T., Roussigne, M., Ecochard, V., Mathe, C., Amalric, F. and Girard, J.-P. (2005). The THAP domain of THAP1 is a large C2CH module with zinc-dependent sequence-specific DNA-binding activity. Proc. Natl Acad. Sci. USA 102, 6907-6912. 10.1073/pnas.0406882102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis, P., Zarowiecki, M., Arnaboldi, V., Becerra, A., Cain, S., Chan, J., Chen, W. J., Cho, J., da Veiga Beltrame, E., Diamantakis, S.et al. (2022). WormBase in 2022-data, processes, and tools for analyzing Caenorhabditis elegans. Genetics 220, iyac003. 10.1093/genetics/iyac003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Durairaj, J., Waterhouse, A. M., Mets, T., Brodiazhenko, T., Abdullah, M., Studer, G., Tauriello, G., Akdel, M., Andreeva, A., Bateman, A.et al. (2023). Uncovering new families and folds in the natural protein universe. Nature 622, 646-653. 10.1038/s41586-023-06622-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gal, C., Carelli, F. N., Appert, A., Cerrato, C., Huang, N., Dong, Y., Murphy, J., Frapporti, A. and Ahringer, J. (2021). DREAM represses distinct targets by cooperating with different THAP domain proteins. Cell Rep. 37, 109835. 10.1016/j.celrep.2021.109835 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galaxy Community. (2024). The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update. Nucleic Acids Res. 52, W83-W94. 10.1093/nar/gkae410 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayward, A., Ghazal, A., Andersson, G., Andersson, L. and Jern, P. (2013). ZBED evolution: repeated utilization of DNA transposons as regulators of diverse host functions. PLoS ONE 8, e59940. 10.1371/journal.pone.0059940 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang, L. S., Tzou, P. and Sternberg, P. W. (1994). The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development. Mol. Biol. Cell 5, 395-411. 10.1091/mbc.5.4.395 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A.et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589. 10.1038/s41586-021-03819-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Khalturin, K., Hemmrich, G., Fraune, S., Augustin, R. and Bosch, T. C. G. (2009). More than just orphans: are taxonomically-restricted genes important in evolution? Trends Genet. 25, 404-413. 10.1016/j.tig.2009.07.006 [DOI] [PubMed] [Google Scholar]
  19. Knip, M., de Pater, S. and Hooykaas, P. J. J. (2012). The SLEEPER genes: a transposase-derived angiosperm-specific gene family. BMC Plant Biol. 12, 192. 10.1186/1471-2229-12-192 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kondo, S., Vedanayagam, J., Mohammed, J., Eizadshenass, S., Kan, L., Pang, N., Aradhya, R., Siepel, A., Steinhauer, J. and Lai, E. C. (2017). New genes often acquire male-specific functions but rarely become essential in Drosophila. Genes Dev. 31, 1841-1846. 10.1101/gad.303131.117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lakowski, B., Eimer, S., Göbel, C., Böttcher, A., Wagler, B. and Baumeister, R. (2003). Two suppressors of sel-12 encode C2H2 zinc-finger proteins that regulate presenilin transcription in Caenorhabditis elegans. Development 130, 2117-2128. 10.1242/dev.00429 [DOI] [PubMed] [Google Scholar]
  22. Large, C. R. L., Khanal, R., Hillier, L., Huynh, C., Kubo, C., Kim, J., Waterston, R. H. and Murray, J. I. (2025). Lineage-resolved analysis of embryonic gene expression evolution in C. elegans and C. briggsae. Science 388, eadu8249. 10.1126/science.adu8249 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Latorre, I., Chesney, M. A., Garrigues, J. M., Stempor, P., Appert, A., Francesconi, M., Strome, S. and Ahringer, J. (2015). The DREAM complex promotes gene body H2A.Z for target repression. Genes Dev. 29, 495-500. 10.1101/gad.255810.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lau, A. M., Bordin, N., Kandathil, S. M., Sillitoe, I., Waman, V. P., Wells, J., Orengo, C. A. and Jones, D. T. (2024). Exploring structural diversity across the protein universe with The Encyclopedia of Domains. Science 386, eadq4946. 10.1126/science.adq4946 [DOI] [PubMed] [Google Scholar]
  25. Lyu, H. and Chamberlin, H. M. (2024). Functional distinction in oncogenic Ras variant activity in Caenorhabditis elegans. Dis. Model. Mech. 17, dmm050577. 10.1242/dmm.050577 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ma, B.-G., Chen, L., Ji, H.-F., Chen, Z.-H., Yang, F.-R., Wang, L., Qu, G., Jiang, Y.-Y., Ji, C. and Zhang, H.-Y. (2008). Characters of very ancient proteins. Biochem. Biophys. Res. Commun. 366, 607-611. 10.1016/j.bbrc.2007.12.014 [DOI] [PubMed] [Google Scholar]
  27. Majorek, K. A., Dunin-Horkawicz, S., Steczkiewicz, K., Muszewska, A., Nowotny, M., Ginalski, K. and Bujnicki, J. M. (2014). The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification. Nucleic Acids Res. 42, 4160-4179. 10.1093/nar/gkt1414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Markljung, E., Jiang, L., Jaffe, J. D., Mikkelsen, T. S., Wallerman, O., Larhammar, M., Zhang, X., Wang, L., Saenz-Vash, V., Gnirke, A.et al. (2009). ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth. PLoS Biol. 7, e1000256. 10.1371/journal.pbio.1000256 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McKnight, S. L. (2024). Protein domains of low sequence complexity-dark matter of the proteome. Genes Dev. 38, 205-212. 10.1101/gad.351465.123 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970. 10.1002/j.1460-2075.1991.tb04966.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mi, H., Ebert, D., Muruganujan, A., Mills, C., Albou, L.-P., Mushayamaha, T. and Thomas, P. D. (2021). PANTHER version 16: a revised family classification, tree-based classification tool, enhancer regions and extensive API. Nucleic Acids Res. 49, D394-D403. 10.1093/nar/gkaa1106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nowotny, M. (2009). Retroviral integrase superfamily: the structural perspective. EMBO Rep. 10, 144-151. 10.1038/embor.2008.256 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pereira, A. B., Marano, M., Bathala, R., Zaragoza, R. A., Neira, A., Samano, A., Owoyemi, A. and Casola, C. (2025). Orphan genes are not a distinct biological entity. BioEssays 47, 2400146. 10.1002/bies.202400146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Petrella, L. N., Wang, W., Spike, C. A., Rechtsteiner, A., Reinke, V. and Strome, S. (2011). synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival. Development 138, 1069-1079. 10.1242/dev.059501 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A. and Cerón, J. (2012). Basic Caenorhabditis elegans methods: synchronization and observation. J. Vis. Exp. 64, 4019. 10.3791/4019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rechtsteiner, A., Costello, M. E., Egelhofer, T. A., Garrigues, J. M., Strome, S. and Petrella, L. N. (2019). Repression of germline genes in Caenorhabditis elegans somatic tissues by H3K9 dimethylation of their promoters. Genetics 212, 125-140. 10.1534/genetics.118.301878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reynolds, C. R., Islam, S. A. and Sternberg, M. J. E. (2018). EzMol: a web server wizard for the rapid visualization and image production of protein and nucleic acid structures. J. Mol. Biol. 430, 2244-2248. 10.1016/j.jmb.2018.01.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sawyer, J. M., Glass, S., Li, T., Shemer, G., White, N. D., Starostina, N. G., Kipreos, E. T., Jones, C. D. and Goldstein, B. (2011). Overcoming redundancy: an RNAi enhancer screen for morphogenesis genes in Caenorhabditis elegans. Genetics 188, 549-564. 10.1534/genetics.111.129486 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sharanya, D., Fillis, C. J., Kim, J., Zitnik, E. M., Ward, K. A., Gallagher, M. E., Chamberlin, H. M. and Gupta, B. P. (2015). Mutations in Caenorhabditis briggsae identify new genes important for limiting the response to EGF signaling during vulval development. Evol. Dev. 17, 34-48. 10.1111/ede.12105 [DOI] [PubMed] [Google Scholar]
  40. Siegfried, K. R., Kidd, A. R., Chesney, M. A. and Kimble, J. (2004). The sys-1 and sys-3 genes cooperate with Wnt signaling to establish the proximal-distal axis of the Caenorhabditis elegans gonad. Genetics 166, 171-186. 10.1534/genetics.166.1.171 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J.et al. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539. 10.1038/msb.2011.75 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sinzelle, L., Izsvák, Z. and Ivics, Z. (2009). Molecular domestication of transposable elements: from detrimental parasites to useful host genes. Cell. Mol. Life Sci. 66, 1073-1093. 10.1007/s00018-009-8376-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Somerville, T. D. D., Xu, Y., Wu, X. S., Maia-Silva, D., Hur, S. K., de Almeida, L. M. N., Preall, J. B., Koo, P. K. and Vakoc, C. R. (2020). ZBED2 is an antagonist of interferon regulatory factor 1 and modifies cell identity in pancreatic cancer. Proc. Natl. Acad. Sci. USA 117, 11471-11482. 10.1073/pnas.1921484117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sternberg, P. W. and Horvitz, H. R. (1986). Pattern formation during vulval development in C. elegans. Cell 44, 761-772. 10.1016/0092-8674(86)90842-1 [DOI] [PubMed] [Google Scholar]
  45. Stevens, L., Félix, M.-A., Beltran, T., Braendle, C., Caurcel, C., Fausett, S., Fitch, D., Frézal, L., Gosse, C., Kaur, T.et al. (2019). Comparative genomics of 10 new Caenorhabditis species. Evol. Lett. 3, 217-236. 10.1002/evl3.110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stevens, L., Rooke, S., Falzon, L. C., Machuka, E. M., Momanyi, K., Murungi, M. K., Njoroge, S. M., Odinga, C. O., Ogendo, A., Ogola, J.et al. (2020). The Genome of Caenorhabditis bovis. Curr. Biol. 30, 1023-1031.e4. 10.1016/j.cub.2020.01.074 [DOI] [PubMed] [Google Scholar]
  47. Stiernagle, T. (2006). Maintenance of C. elegans. In WormBook: The Online Review of C. Elegans Biology, ed. The C. elegans Research Community, pub. WormBook, pp. 1-11. 10.1895/wormbook.1.101.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sun, S., Kanzaki, N., Dayi, M., Maeda, Y., Yoshida, A., Tanaka, R. and Kikuchi, T. (2022). The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus. BMC Genomics 23, 765. 10.1186/s12864-022-09011-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tamura, K., Stecher, G. and Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38, 3022-3027. 10.1093/molbev/msab120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Toker, I. A., Ripoll-Sánchez, L., Geiger, L. T., Sussfeld, A., Saini, K. S., Beets, I., Vértes, P. E., Schafer, W. R., Ben-David, E. and Hobert, O. (2025). Divergence in neuronal signaling pathways despite conserved neuronal identity among Caenorhabditis species. Curr. Biol. 35, 2927-2945.e7. 10.1016/j.cub.2025.05.036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Toll-Riera, M., Bosch, N., Bellora, N., Castelo, R., Armengol, L., Estivill, X. and Albà, M. M. (2009). Origin of primate orphan genes: a comparative genomics approach. Mol. Biol. Evol. 26, 603-612. 10.1093/molbev/msn281 [DOI] [PubMed] [Google Scholar]
  52. van Kempen, M., Kim, S. S., Tumescheit, C., Mirdita, M., Lee, J., Gilchrist, C. L. M., Söding, J. and Steinegger, M. (2024). Fast and accurate protein structure search with Foldseek. Nat. Biotechnol. 42, 243-246. 10.1038/s41587-023-01773-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Varadi, M., Bertoni, D., Magana, P., Paramval, U., Pidruchna, I., Radhakrishnan, M., Tsenkov, M., Nair, S., Mirdita, M., Yeo, J.et al. (2024). AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. Nucleic Acids Res. 52, D368-D375. 10.1093/nar/gkad1011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang, D., Kennedy, S., Conte, D., Kim, J. K., Gabel, H. W., Kamath, R. S., Mello, C. C. and Ruvkun, G. (2005). Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants. Nature 436, 593-597. 10.1038/nature04010 [DOI] [PubMed] [Google Scholar]
  55. Worth, C. L., Gong, S. and Blundell, T. L. (2009). Structural and functional constraints in the evolution of protein families. Nat. Rev. Mol. Cell Biol. 10, 709-720. 10.1038/nrm2762 [DOI] [PubMed] [Google Scholar]
  56. Xu, W., Sun, Y., Breen, P., Ruvkun, G. and Mao, K. (2024). Caenorhabditis elegans inositol hexaphosphate pathways couple to RNA interference and pathogen defense. Proc. Natl. Acad. Sci. USA 121, e2416982121. 10.1073/pnas.2416982121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yochem, J. K. (2006). Nomarski images for learning the anatomy, with tips for mosaic analysis. In WormBook: The Online Review of C. Elegans Biology, ed. The C. elegans Research Community, pub. WormBook, pp. 1-47. 10.1895/wormbook.1.100.1 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

biolopen-14-062018_review_history.pdf (406.1KB, pdf)
Supplementary information
biolopen-14-062018-s1.pdf (1.7MB, pdf)
DOI: 10.1242/biolopen.062018_sup1
Table S1.

List of DE genes in Cbr-ivp-3(sy5216).

biolopen-14-062018-TableS1.xlsx (2.1MB, xlsx)
Table S2.

Cbr-ivp-3(sy5216) DE genes with significant changes compared to wild type.

biolopen-14-062018-TableS2.xlsx (467.7KB, xlsx)
Table S3.

Overlap of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivp-3(sy5216) DE genes

biolopen-14-062018-TableS3.xlsx (254.6KB, xlsx)
Table S4.

Overlap between up-regualted DE genes of Cbr-spr-4(gu163), Cbrhtz-1(gu167) and Cbr-ivp-3(sy5216)

biolopen-14-062018-TableS4.xlsx (210.2KB, xlsx)
Table S5.

Overlap between down-regualted DE genes of Cbr-spr-4(gu163), Cbrhtz-1(gu167) and Cbr-ivp-3(sy5216)

biolopen-14-062018-TableS5.xlsx (213.1KB, xlsx)
Table S6.

Nematode strains used in this study

biolopen-14-062018-TableS6.xlsx (73.8KB, xlsx)
Table S7.

Details for transgenes used in this study

biolopen-14-062018-TableS7.xlsx (73.5KB, xlsx)
Table S8.

Primers used in this study

biolopen-14-062018-TableS8.xlsx (75.6KB, xlsx)
Table S9.

Sequences used to produce tree in Fig. 2B

biolopen-14-062018-TableS9.xlsx (57.8KB, xlsx)
Table S10.

Sequences used in LIN-15B domain alignment (Fig. S2).

biolopen-14-062018-TableS10.xlsx (12.4KB, xlsx)
Table S11.

Steps used in analysis of RNA seq data using the Galaxy platform

biolopen-14-062018-TableS11.xlsx (11.7KB, xlsx)
Table S12.

GO term enrichment in Cbr-spr-4(gu163) RNA seq data.

biolopen-14-062018-TableS12.xlsx (9.5KB, xlsx)
Table S13.

GO term enrichment in Cbr-htz-1(gu167) RNA seq data.

biolopen-14-062018-TableS13.xlsx (8.7KB, xlsx)
Table S14.

GO term enrichment in Cbr-ivph-3(sy5216) RNA seq data.

biolopen-14-062018-TableS14.xlsx (29.9KB, xlsx)
Table S15.

GO term enrichment in in the overlap of Cbr-spr-4(gu163), Cbr-htz-1(gu167) and Cbr-ivph-3(sy5216) RNA seq data.

biolopen-14-062018-TableS15.xlsx (8.5KB, xlsx)
Table S16.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against PDB (human, mouse, zebrafish, Drosophila, Ascaris, Diploscapter)

biolopen-14-062018-TableS16.xlsx (9KB, xlsx)
Table S17.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against AFDB50 (human, mouse, zebrafish, Drosophila, Ascaris, Diploscapter)

biolopen-14-062018-TableS17.xlsx (14.5KB, xlsx)
Table S18.

Foldseek hit set for Cel-IVPH-3 (AF-Q86DD2-F1-v4) against AFDB50 (Caenorhabditis)

biolopen-14-062018-TableS18.xlsx (47.2KB, xlsx)
Table S19.

Analyses from Large et al. (2025): Jensen-Shannon Distance and Tau

biolopen-14-062018-TableS19.xlsx (72.8KB, xlsx)
Dataset 1.
biolopen-14-062018-Dataset1.zip (53.4KB, zip)

Articles from Biology Open are provided here courtesy of Company of Biologists

RESOURCES