Decision letter: Convergent evolution of small molecule pheromones in Pristionchus nematodes (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The small molecules that mediate chemical communication between nematodes—so-called 'nematode-derived-modular-metabolites' (NDMMs)—are of major interest because of their ability to regulate development, behavior, and life-history. Pristionchus pacificus nematodes produce an impressive diversity of structurally complex NDMMs, some of which act as primer pheromones that are capable of triggering irreversible developmental switches. Many of these NDMMs have only ever been found in P. pacificus but no attempts have been made to study their evolution by profiling closely related species. This study brings a comparative perspective to the biochemical study of NDMMs through the systematic MS/MS- and NMR-based analysis of exo-metabolomes from over 30 Pristionchus species. We identified 36 novel compounds and found evidence for the convergent evolution of complex NDMMs in separate branches of the Pristionchus phylogeny. Our results demonstrate that biochemical innovation is a recurrent process in Pristionchus nematodes, a pattern that is probably typical across the animal kingdom. Introduction Pheromonal signaling appears to be the most ancient and widespread form of communication used in the animal kingdom (Agosta, 1992; Krieger and Breer, 1999). Deciphering the chemical components used for pheromonal communication is key to understanding why animals release information into their social environment, and to determining how we might exploit these signaling systems for management purposes. A major challenge to achieving these aims is the simple fact that pheromones are exceptionally diverse. In terms of biochemical structure, any molecule that can be released and subsequently detected can act as a pheromone. Moreover, animals often release complex blends of compounds, and they do so in context-dependent ways that are sensitive to sex, age, and condition (Wyatt, 2003). Finally, natural selection may shape these pheromonal signals to serve a wide variety of biological tasks, including mate detection, territorial defense, and quorum sensing (Agosta, 1992; Schneider, 1992; Hansson, 1995; Rutherford and Bassler, 2012). Consequently, pheromonal communication systems represent promising systems for exploring the evolution of diversity at the interface of biochemistry, molecular physiology, and behavioral ecology (Symonds and Elgar, 2008; Missbach et al., 2014). However, not all animals are equally suited to such integrative research—effectively studying the how and why of pheromonal communication requires systems that are both experimentally tractable and amenable to unbiased biochemical characterization. The nematodes Caenorhabditis elegans and Pristionchus pacificus have emerged as powerful systems for studying the biochemistry and genetics of pheromonal signaling (Ludewig and Schroeder, 2013; Sommer, 2015). Nematodes have long been known to communicate via pheromones, producing both releaser pheromones (those that trigger short-term behavioral responses, for example mate attraction) and primer pheromones (those that trigger long-term developmental responses, such as the production of quiescent dauer larvae) (Bone and Shorey, 1978; Golden and Riddle, 1982). Subsequent efforts to characterize these pheromonal signals biochemically revealed that nematodes release and respond to a diverse suite of ascarosides. Simple ascarosides consist of a 3,6-dideoxy-L-ascarylose sugar coupled to a fatty-acid-derived side chain (Jeong et al., 2005; Butcher et al., 2007). From this basic framework, nematodes produce hundreds of distinct molecules that are collectively referred to as nematode-derived modular metabolites (NDMMs). This structural diversity of NDMMs results from variation in the sugar (some NDMMs replace ascarylose with the related sugars paratose or L-3,6-dideoxy-lyxo-hexose), the fatty acid (length, saturation, and attachment position can all vary), the presence or absence of additional moieties (including both amino acid and nucleoside-derived moieties), and whether the NDMM exists as a monomer or a dimer (Bose, 2012; von Reuss et al., 2012; Ludewig and Schroeder, 2013; Schroeder, 2015; von Reuss and Schroeder, 2015; Butcher, 2017a; Butcher, 2017b; von Reuss, 2018; Artyukhin et al., 2018; Butcher, 2019; Bergame et al., 2019). Several NDMMs have been confirmed to influence aspects of behavior (e.g., aggregation behavior in C. elegans) and development (e.g., control of the mouth-form polyphenism in P. pacificus) (Srinivasan et al., 2012; Bose, 2012). Moreover, genetic studies have begun to shed light on both the biochemical pathways responsible for NDMM production and the neurobiological and developmental pathways that underlie receiver response (Butcher et al., 2009; von Reuss et al., 2012; Zhang et al., 2015; Leighton and Sternberg, 2016; Zhang et al., 2016; Panda et al., 2017; Falcke et al., 2018; Zhang et al., 2018; Zhou et al., 2018; McGrath and Ruvinsky, 2019). However, we currently know very little about the evolution of NDMMs. Targeted screening for known compounds confirmed that NDMM production is widespread across the nematode phylum (Choe et al., 2012), and earlier work identified long-chain ascarosides in the eggshells of parasitic Ascaris nematodes (Bartley et al., 1996), but there remains a need for unbiased biochemical screens that speak to the diversity of NDMMs present both within and across species. This is in strong contrast to past research on plants and insects, where small molecules involved in signaling have been surveyed broadly and shown to undergo rapid evolutionary changes (Symonds and Elgar, 2008; Engl et al., 2018; Lombe et al., 2019). With densely sampled comparative data in hand, it should become possible to address key questions about NDMM evolution, such as does phylogeny reliably predict NDMM production, and how repeatable is biochemical innovation? Pristionchus nematodes are well suited for phylogeny-informed research on NDMM diversity. P. pacificus is a free-living soil nematode that is reliably found in association with scarab beetles (Herrmann et al., 2006a; Herrmann et al., 2006b; Herrmann et al., 2007), and that has been developed as a model system in developmental genetics and comparative evolutionary biology. Many studies in this species focused on two examples of phenotypic plasticity, and these efforts revealed that P. pacificus and C. elegans produce and use NDMMs in distinct ways. The first example involves the development of the buccal cavity, with individuals adopting either the stenostomatous (St) mouth form with a single tooth, resulting in strict bacterial feeding, or the alternative eurystomatous (Eu) form with two teeth, which allows for predation on other nematodes (Bento et al., 2010; Bumbarger et al., 2013; Sieriebriennikov et al., 2017; Sieriebriennikov and Sommer, 2018). This behavioral-morphological polyphenism is broadly conserved within the Pristionchus genus and the larger Diplogastridae family (Susoy et al., 2015), but is not found in non-Diplogastrids such as C. elegans. Second, P. pacificus worms, similar to C. elegans worms, can enter an arrested and long-lived dauer stage in response to harsh environmental conditions (Ogawa et al., 2009). Both mouth-form plasticity and dauer induction are sensitive to population density in P. pacificus, with NDMMs serving as chemical indicators of competitive environments in which predation or diapause represent adaptive survival strategies (Bento et al., 2010; Werner et al., 2018a). However, completely different NDMMs are involved in the regulation of these two plastic traits (Bose, 2012; Sommer and Mayer, 2015), and P. pacificus and C. elegans employ different NDMMs as molecular triggers for dauer entry (Bose, 2012; Schroeder, 2015). Together, these studies demonstrated evidence for divergence of NDMM signaling between species and across developmental pathways. Biochemical analysis of the P. pacificus exo-metabolome showed that this species produces a surprising diversity of complex NDMMs that are not found in C. elegans, including the dimeric NDMM dasc#1 and several highly modular compounds that are characterized by the presence of complex sidechains that incorporate intermediates from different primary metabolic pathways (the UBAS, PASC, and NPAR NDMMs) (Figure 1; Bose, 2012; Yim et al., 2015; Artyukhin et al., 2018; Falcke et al., 2018). However, the common ancestor of P. pacificus and C. elegans existed around 100 million years ago (Prabh et al., 2018; Werner et al., 2018b; Hong et al., 2019) and comparing just these two species can provide only limited insights into patterns of small molecule evolution in nematodes. By contrast, comparative analysis involving multiple species across a shallower phylogenetic scale has the potential to shed light on the origins of complex NDMMs and the divergence of pheromone profiles. The availability of many culturable species and knowledge about phylogenetic relationships are key prerequisites for such studies, both of which are met in Pristionchus. Many Diplogastrid species, covering multiple genera, can be cultured in the laboratory, thereby allowing for fine-scale comparative analysis (Susoy et al., 2015). This diversity includes more than 40 Pristionchus species that have been isolated from world-wide samplings of scarab beetles and other invertebrates (Kanzaki et al., 2012a; Kanzaki et al., 2012b; Kanzaki et al., 2012c; Yoshida et al., 2018). These Pristionchus species fall into four major clades—the pacificus, maupasi, entomophagus, and triformis clades—plus an outgroup (Figure 1—figure supplement 1), nearly all of which are culturable under laboratory conditions. Within these clades, most species show geographically restricted distribution patterns that are often correlated with major continents. For example, species of the pacificus-clade are largely restricted to Asia. Although the majority of Pristionchus species are gonochoristic (comprised of males and females), self-fertilizing hermaphroditism has evolved at least six times independently within the genus, and the model species P. pacificus reproduces in this way (Rödelsperger et al., 2018; Weadick and Sommer, 2016a). Figure 1 with 1 supplement see all Download asset Open asset Known NDMMs from P. pacificus. (A) Simple ascarosides, and the paratoside part#9 [par-C5]. (B) Modular NDMMs (DASC, NPAR, PASC, and UBAS chemicals) from P. pacificus. Here, we bring the comparative method of evolutionary biology (Harvey and Pagel, 1991) to the chemical analysis of NDMMs to obtain insight into their evolutionary dynamics. Exo-metabolomes derived from 32 Pristionchus species and six non-Pristionchus species from different Diplogastridae genera were prepared for a systematic analysis using HPLC-MS/MS and NMR. Through these efforts, we uncovered an unexpected structural diversity of simple and complex NDMMs, including 16 novel DASC chemicals, 12 novel UBAS chemicals, and a new family of UPAS chemicals. By analyzing these results in the light of known Pristionchus relationships, we found evidence that NDMM production is strongly correlated with phylogeny, with several structurally distinct NDMMs showing clade-specific distributions, and with more closely related species tending to possess more similar NDMM compositional profiles. That said, our analyses also revealed the convergent evolution of highly modular NDMMs in distantly related branches of the phylogeny, indicating that biochemical innovation is a repeatable process in Pristionchus nematodes. We speculate that such patterns are probably typical of pheromonal small molecules across the animal kingdom. Results Uncovering the chemical diversity of NDMMs in Pristionchus nematodes P. pacificus produces a diverse set of NDMMs that can be classified into five major groups: simple, dimeric (DASC), NPAR, PASC, and UBAS (Figure 1). Of these, only a few of the simple ascarosides (i.e. ascr#9 [asc-C5], ascr#12 [asc-C6], and ascr#1 [asc-C7]) have also been identified in C. elegans or other nematodes. However, because of limited sampling, it is not currently possible to say whether this reflects true species-specificity, or whether the various complex compounds are shared broadly across species. Obtaining a historical and functional understanding of NDMM evolutionary innovation therefore requires a comparative perspective. To investigate the dynamics of NDMM production in Pristionchus nematodes, we selected 32 culturable species (Figure 1—figure supplement 1), including both hermaphroditic and gonochoristic species from all four major phylogeographic clades (Figure 2), and produced exo-metabolomes for comparative LC-MS/MS analysis (see 'Materials and methods'). Given our sample collection and biochemical analysis protocols, we employed an ion intensity threshold of 1.0 × 103: samples detected at levels above this value could be reliably quantified whereas samples detected at levels below this value were considered to be present only at trace levels. Figure 2 Download asset Open asset Comparative analysis of the exo-metabolomes of 32 Pristionchus nematodes revealed evolutionary diversity of NDMMs. Three biological replicates for each species were grown in 100 ml S-medium for comparative LC-MS/MS analysis (see 'Materials and methods'). Simple NDMMs and complex NDMMs including DASC, NPAR, PASC, and UBAS chemicals (Supplementary file 3—Tables 1–3) are summarized; compounds found at trace levels (including the newly described UPAS class) are not included. Values were standardized within classes to help to visualize variation in relative production levels within rare NDMM classes, as overall production levels were dominated by simple NDMMs (see Figure 2—source data 1). NDMM abundances were standardized by dividing mean peak area for each NDMM by the total for the respective NDMM class then expressing the abundance as a percentage for each species; for clarity, monomeric and dimeric UBAS compounds were standardized separately. Black cells represent the apparent absence of the chemical. Black and red compound labels represent known and novel NDMMs, respectively. Figure 2—source data 1 Quantification of NDMMs in 32 Pristionchus nematodes. https://cdn.elifesciences.org/articles/55687/elife-55687-fig2-data1-v2.xlsx Download elife-55687-fig2-data1-v2.xlsx The simple NDMMs, which often act as biosynthetic precursors for more complex compounds, consist of a single sugar coupled to a fatty acid side chain. Consistent with previous studies, we identified four simple NDMMs in P. pacificus: the ascarosides ascr#9, ascr#12, and ascr#1, which form a homologous series with fatty acid side chains of differing length, and the paratoside part#9, which is similar to ascr#9 but which uses paratose as the sugar moiety in place of ascarylose. We found six additional simple NDMMs when examining other Pristionchus species: ascr#5 [asc-ωC3], ascr#11 [asc-C4], ascr#7 [asc-ΔC7], ascr#14 [asc-C8], ascr#10 [asc-C9] and ascr#18 [asc-C11] (Figures 2 and 3A; Figure 3—figure supplements 1 and 2; Supplementary file 3—Table 1). Most of these are similar to ascr#9, ascr#12, and ascr#1, possessing saturated fatty acids attached via the ω−1 carbon, but ascr#5 and ascr#7 are structurally distinct: ascr#5 possesses a C3 fatty acid side chain that attaches to the sugar via the terminal ω carbon (Butcher et al., 2008), whereas ascr#7 possesses an unsaturated C7 side chain (Pungaliya et al., 2009). ascr#1, ascr#9 and ascr#12 were produced by all species, consistent with an ancient origin of these simple NDMMs (Figure 2 and Figure 2—source data 1; Supplementary file 1a—Figures 1–9). By contrast, a few NDMMs, most notably ascr#7 (Figure 3—figure supplement 3; Supplementary file 1a—Figures 10–11) and part#9, were restricted to specific clades; ancestral reconstruction analyses suggest a single origin for ascr#7 along the branch that defines the entomophagus-clade, and two origins for part#9 within the pacificus-clade (Supplementary file 4—Figures 1–2). The remaining simple NDMMs were phylogenetically patchy, possibly indicating repeated silencing and reactivation of conserved biochemical pathways. For example, ascr#10, a known attractant of C. elegans hermaphrodites (Izrayelit et al., 2012), was absent from 6 of 7 hermaphroditic species but present in 23 of 25 gonochoristic species, and was often produced by males at particularly high levels (Supplementary file 1a—Figures 12–24), suggesting a broadly conserved role as a sex pheromone in gonochoristic species (Figures 2 and 3B; Figure 3—figure supplement 4A). The lack of ascr#10 in hermaphroditic species might simply reflect the culture conditions (in which males are generally rare as the result of self-fertilization), but we note that ascr#10 was not detected in separate assays focused exclusively on P. pacificus males (Figure 3C and Figure 3—figure supplement 4B), consistent with a total loss of ascr#10 biosynthesis in at least one hermaphroditic species. Quantitative variation in simple NDMM levels was also apparent; depending on species, the most abundant compound was either ascr#1, ascr#9, ascr#10, or ascr#7. Moreover, a food limitation (starvation) treatment altered NDMM levels in P. pacificus hermaphrodites, indicating that NDMM production rates are plastic (Figure 3C and Figure 3—figure supplement 4B). Combined, these results demonstrate high levels of species-specificity within the simple NDMM class and indicate that NDMM production by Pristionchus correlates with phylogeny in some respects but is also influenced by sex, the mode of reproduction, and the environment. Similar patterns emerge from comparative study of complex NDMMs, albeit with more pronounced evidence for lineage-specificity and clear signatures of convergent evolution, as shown below. Figure 3 with 4 supplements see all Download asset Open asset Simple ascarosides detected from Pristionchus nematodes. (A) Chemical structures of newly detected simple ascarosides. (B) Males of 10 gonochoristic species release large amounts of ascr#10 [asc-C9] in a sex-specific manner. Dashed gray line indicates that nothing was detected in the culture, probably because of contamination of the P. arcanus females. (C) Males and hermaphrodites of P. pacificus released ascr#1 [asc-C7] and pasc#9, respectively, but no ascr#10 [asc-C9]. M, male; H, hermaphrodite. Table 1 Mode of evolution of major Pristionchus NDMMs. NDMMClassConservation PatternfitMk modelSIMMAP changesDP (Ho: D = 0)P (Ho: D = 1)Phylogenetic interpretationascr#1Simple NDMMConstant–––––Strictly conservedascr#10Simple NDMMVariableARD17.500.96450.25240.8989Variableascr#11Simple NDMMVariableER15.71−0.32630.66970.0051Somewhat conserved (Brownian?)ascr#12Simple NDMMConstant–––––Strictly conservedascr#14Simple NDMMVariableER26.250.44240.61160.2027Variableascr#5Simple NDMMVariableER8.540.22760.86700.1933Variableascr#7Simple NDMMVariableER1.10−3.01850.08540.0000Highly conservedascr#9Simple NDMMConstant–––––Strictly conservedpart#9Simple NDMMVariableER2.72−1.75180.08540.0000Highly conserveddasc#1DASCVariableER15.770.97140.24870.8989Variabledasc#10DASCVariableARD24.711.02610.19280.9866Variabledasc#12DASCVariableER1.64−2.44390.23350.0035Somewhat conserved (Brownian?)dasc#14DASCVariableER1.17−3.05600.08540.0000Highly conserveddasc#15DASCVariableER1.16−3.08050.08540.0000Highly conserveddasc#16DASCVariableER1.25−2.95280.09930.0000Highly conserveddasc#17DASCVariableER1.08−3.07720.08540.0000Highly conserveddasc#2DASCVariableER2.500.88500.66970.8989Variabledasc#3DASCVariableER2.661.50250.40770.7440Variabledasc#4DASCVariableER18.900.13250.86700.0644Somewhat conserved (Brownian?)dasc#5DASCVariableARD6.83−1.61650.24870.0032Somewhat conserved (Brownian?)dasc#6DASCVariableER37.880.85520.24870.7392Variabledasc#9DASCVariableARD17.900.52670.61160.3702Variablenpar#1NPARVariableER1.17−2.23880.08540.0000Highly conservednpar#2NPARVariableER1.19−2.23180.08540.0000Highly conservednpar#3NPARVariableARD8.11−0.58350.64630.0644Somewhat conserved (Brownian?)pasa#9PASCVariableARD7.16−0.72730.61160.0254Somewhat conserved (Brownian?)pasc#1PASCVariableARD7.89−0.16430.86010.0644Somewhat conserved (Brownian?)pasc#12PASCVariableER2.52−1.69140.05130.0000Highly conservedpasc#9PASCVariableER2.52−1.69280.05130.0000Highly conservedpaso#9PASCVariableARD4.19−1.74830.11080.0000Somewhat conserved (Brownian?)ubas#1UBASVariableER5.94−1.04250.28250.0006Somewhat conserved (Brownian?)ubas#12UBASVariableER5.990.65700.61160.6005Variableubas#2UBASVariableARD11.46−0.01600.91720.1368Variableubas#26UBASVariableARD6.372.24100.05130.0993Over-dispersedubas#27UBASSingleton–––––Youngubas#28UBASSingleton–––––Youngubas#29UBASSingleton–––––Youngubas#3UBASVariableARD11.67−0.19310.82620.0255Somewhat conserved (Brownian?)ubas#30UBASVariableER2.661.69390.28250.6054Variableubas#32UBASSingleton–––––Youngubas#33UBASSingleton–––––Youngubas#34UBASSingleton–––––Youngubas#35UBASSingleton–––––Youngubas#36UBASSingleton–––––Youngubas#4UBASVariableER6.010.66060.61160.6005Variableubas#5UBASVariableER9.220.64130.60070.5546Variable Pristionchus nematodes produce diverse dimeric ascarosides P. pacificus produces a dimeric from two ascr#1 that can trigger development of the mouth-form (Figure Bose, 2012). In additional DASC compounds could be produced by the use of alternative simple ascarosides alternative attachment We detected dasc#1 from most Pristionchus species a of additional DASC compounds that were from different simple ascarosides attached at either the or of the ascarylose moiety (Figures 2 and Supplementary file 3—Table These new were across of the 32 species, including P. pacificus and of the species for which dasc#1 was not DASC compounds were identified in of 32 species, suggesting that this NDMM class to the of the Pristionchus genus (Supplementary file Most species produced multiple which ascr#9 ascr#1 [asc-C7] were the most abundant (Figure supplement This pattern is at least in to the fact that these two precursors are produced at high levels in most species (Figure 2 and Figure 2—source data 1). Figure 4 with supplements see all Download asset Open asset DASC, UBAS, and UPAS chemicals detected from Pristionchus species. (A) of new DASC chemicals identified from Pristionchus nematodes with at the or that the and were as at the or respectively. (B) Nematodes from the produced four novel DASC chemicals one or two of (C) of novel UBAS chemicals identified from Pristionchus nematodes. identified UPAS chemicals a acid at the of ascarylose. For on NDMM see 'Materials and To the chemical structures of newly identified a of and was we used to the terminal side chain to the of (Figure supplement and we used data to whether the involved the or For example, of detected in P. and P. (Figures 2 and an ion of with a loss of the side chain (Supplementary file 1), which to the first and the ascarosides as ascr#9 and ascr#11 [asc-C4], (see Figure supplement for on the of DASC Subsequent analysis of the of from P. these and demonstrated that ascr#9 was to the of ascr#11 (Supplementary file 1 and Supplementary file 3—Table we found that two ascr#9 can be at the or resulting in two (Figures and Supplementary file 3—Table and (Figures and Supplementary file 3—Table the of which a high to nematodes of the (Figure supplements 2 and These revealed a chemical diversity of DASC chemicals in Pristionchus nematodes. Figure with supplements see all Download asset Open asset of the structures of novel NDMMs. (A) (B) (C) and of each compound are of selected between and with indicates that a acid moiety The in and the the absence of a in For see Supplementary file and Supplementary file chemical analysis revealed several homologous series of DASC chemicals in Pristionchus nematodes. The first series includes and which incorporate different simple ascarosides at the of ascr#9 (Figure Supplementary file Supplementary file 3—Table homologous series of DASC chemicals includes and dasc#1 (Figure Supplementary file Supplementary file 3—Table which each have a simple attached to the of ascr#1 Finally, we found four new dimeric ascarosides at least one ascr#7 and (Figures 2 and 4B). These DASC compounds were exclusively detected in species from the the only in which the ascr#7 was found (Figure supplement Here, analysis a ion of of the terminal side indicating that ascr#7 is the first in (Supplementary file to the the probably two ascr#7 (Supplementary file To these and were employed to and from P. and P. respectively. analysis demonstrated the presence of one ascarylose one ascarylose a side chain and an unsaturated C7 side chain in suggesting that ascr#7 was to the of ascr#9 (Figure and Supplementary file 3—Table data for confirmed that two ascr#7 were at the (Figure and Supplementary file 3—Table analysis of the of an of and that ascr#7 was also to the of ascr#1 [asc-C7] in (Supplementary file these four all have a at the These results show how the production of novel simple NDMMs which is found only in the can underlie the production of related modular compounds, thereby innovation at multiple structural levels. P. pacificus and produce a diverse suite of structurally complex NDMMs In to simple and dimeric NDMMs, P. pacificus produces a variety of more structurally complex NPAR, and UBAS NDMMs (Figure of which are known to act as pheromonal of dauer and mouth-form development (Bose, 2012; et al., Yim et al., 2015). These classes are by the presence of additional structural components that are attached to the sugar and the fatty acid side chain. The additional components from several different metabolic from to amino acid the phylogenetic of these highly modular compounds is a key in the origins of NDMM NPAR compounds, which are by the presence of and amino sidechains and by the use of paratose as the sugar were in the