No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Functional evidence supports adaptive plant chemical defense along a geographical cline
Abstract
Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host’s range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed ( Asclepias syriaca ) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na ⁺ /K ⁺ -ATPase); there was little variation among compounds in inhibition of an unadapted Na ⁺ /K ⁺ -ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltu s. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.
... According to the criteria defined by Agrawal et al. [48], which classify molecules with IC 50 > 100 µM as nontoxic, 10 to 100 µM as moderately toxic, IC 50 < 10 µM as toxic and IC 50 < 1 µM as highly toxic (also refer [49]), we found that the coroglaucigenin cardenolides (glucopyranosyl frugoside and frugoside) are toxic (IC 50 < 10 µM) for black-headed grosbeak and highly toxic (IC 50 < 1 µM) for zebra finch. For the invertebrate predator, the giant Asian mantid, the monoglycosidic cardenolides (frugoside and gofruside) were highly toxic (IC 50 < 1 µM; figure 2; electronic supplementary material, table S5). ...
... For example, certain cardenolides can be detrimental to insect growth [72], as well as redox state [56]. Sequestration might also be constrained by transport and modification of specific compounds [48,73]. Testing the uptake, transport, modification and accumulation of the cardenolides that we found in similar concentrations in the body and defensive fluid (i.e. ...
... Testing the uptake, transport, modification and accumulation of the cardenolides that we found in similar concentrations in the body and defensive fluid (i.e. 16α-hydroxycalotropin), and comparing to those that were more abundant in the bugs than the seeds (frugoside and gofruside; [40]), would be useful for establishing which key metabolic process the bugs use when feeding on Asclepias (refer to Agrawal et al. [48]). ...
Aposematic animals rely on diverse secondary metabolites for defence. Various hypotheses, such as competition, life history and multifunctionality, have been posited to explain defence variability and diversity. We investigate the compound selectivity hypothesis using large milkweed bugs, Oncopeltus fasciatus, to determine if distinct cardenolides vary in toxicity to different predators. We quantify cardenolides in the bug’s defensive secretions and body tissues and test the individual compounds against predator target sites, the Na⁺/K⁺-ATPases, that are predicted to differ in sensitivity. Frugoside, gofruside, glucopyranosyl frugoside and glucopyranosyl gofruside were the dominant cardenolides in the body tissues of the insects, whereas the two monoglycosidic cardenolides—frugoside and gofruside—were the most abundant in the defensive fluid. These monoglycosidic cardenolides were highly toxic (IC50 < 1 μM) to an invertebrate and a sensitive vertebrate enzyme, in comparison to the glucosylated compounds. Gofruside was the weakest inhibitor for a putatively resistant vertebrate predator. Glucopyranosyl calotropin, found in only 60% of bugs, was also an effective inhibitor of sensitive vertebrate enzymes. Our results suggest that the compounds sequestered by O. fasciatus probably provide consistency in protection against a range of predators and underscore the need to consider predator communities in prey defence evolution.
... Therefore, population differentiation in plant defense expression may be contingent on the relative selection imposed by herbivores that feed on different plant parts, limits due to the genetic architecture of defense expression (Prasad et al., 2012;Züst et al., 2012), and gene flow. In the milkweed system (Asclepias spp.), cardiac glycosides (i.e., cardenolides) have an exclusive defensive function against their physiological target, the Na + /K + -ATPase (hereafter "sodium pump") of animals, including the community of specialized milkweed insect herbivores (Agrawal, 2005;Agrawal et al., 2012bAgrawal et al., , 2022. Cardenolide richness may reach 30 distinct compounds in individual plants, and both compound diversity and concentration are variable among tissues eaten by insects (Lopez-Goldar et al., 2022;Rasmann & Agrawal, 2011). ...
... Putative identity of individual cardenolides was assigned after comparing HPLC-UV output obtained from similar methods with LC-MS and NMR analyses following Agrawal et al. (2022). ...
... For instance, milkweed leaves are eaten by insects in at least four different taxonomic orders that also vary dramatically in their adaptive mechanisms to circumvent plant defenses (Agrawal et al., 2012b;Taverner et al., 2019). Recent work suggests that, despite their known negative impact against herbivores overall, independent evolution of specific cardenolides may be proceeding due to differences in their effectiveness against distinct herbivores varying in tolerance strategies (Agrawal & Hastings, 2023;Agrawal et al., 2022;Lopez-Goldar et al., 2022). On the other hand, seeds showed more abundant and positive genetic correlations among cardenolides compared with roots and leaves, particularly between more polar compounds. ...
A major predicted constraint on the evolution of anti-herbivore defense in plants is the non-independent expression of traits mediating resistance. Since herbivore attack can be highly variable across plant tissues, we hypothesized that correlations in toxin expression within and between plant tissues may limit population differentiation and, thus, plant adaptation. Using full-sib families from two nearby (<1 km) common milkweed (Asclepias syriaca) populations, we investigated genetic correlations among 28 distinct cardenolide toxins within and between roots, leaves and seeds, and examined signatures of tissue-specific divergent selection between populations by QST-FST comparisons. The prevalence, direction and strength of genetic correlations among cardenolides were tissue-specific, and concentrations of individual cardenolides were moderately correlated between tissues; nonetheless, the direction and strength of correlations were population-specific. Population divergence in the cardenolide chemistry was stronger in roots than in leaves and seeds. Divergent selection on individual cardenolides was tissue- and toxin-specific, except for a single highly toxic cardenolide (labriformin), that showed divergent selection across all plant tissues. Heterogeneous evolution of cardenolides within and between tissues across populations appears possible due to their highly independent expression. This independence may be common in nature, especially in specialized interactions in which distinct herbivores feed on different plant tissues.
... Recently, the interactions between particular toxic cardenolide heart poisons and insect target sites have begun to be identified (Petschenka et al., 2018;Agrawal et al., 2022;López-Goldar et al., 2022). Agrawal et al. (2022) found that the dominant seed cardenolide in Asclepias syriaca (glycosylated aspecioside) is more inhibitory to the target site of the seed herbivore than its sequestered conversion product (aspecioside A) and that the subdominant seed cardenolides-diglycosylated syriogenin and labriformin-were significantly more inhibitory than the more abundant compounds. ...
... Recently, the interactions between particular toxic cardenolide heart poisons and insect target sites have begun to be identified (Petschenka et al., 2018;Agrawal et al., 2022;López-Goldar et al., 2022). Agrawal et al. (2022) found that the dominant seed cardenolide in Asclepias syriaca (glycosylated aspecioside) is more inhibitory to the target site of the seed herbivore than its sequestered conversion product (aspecioside A) and that the subdominant seed cardenolides-diglycosylated syriogenin and labriformin-were significantly more inhibitory than the more abundant compounds. The differential accumulation of the compounds could be evidence that they are under selection, especially if this is correlated to geographical areas that vary in insect diversity and herbivory. ...
... . . Cardenolide toxicity tested by functional Na + /K + -ATPase assays Na + /K + -ATPase extractions from the cardenolide-resistant seed bug (Oncopeltus fasciatus; Bramer et al., 2015;Agrawal et al., 2022;López-Goldar et al., 2022) and one non-adapted insect (Drosophila melanogaster wild type Canton S) (Dalla and Dobler, 2016;Dobler et al., 2019) were prepared according to the methods described in Petschenka et al. (2013Petschenka et al. ( , 2018Petschenka et al. ( , 2022. Fruit flies were reared under controlled conditions, and O. fasciatus were reared in terrarium boxes as described in section Cardenolides from Asclepias curassavica seeds and sequestration except that for this analysis, O. fasciatus adults were reared on sunflower seeds (Helianthus annuus). ...
Milkweed–herbivore systems are characterized by cardenolide chemical defenses and specialized herbivore adaptations such as physiological target site insensitivity. Cardenolide defenses in milkweeds can vary in terms of the total concentration, differences in the polarity of individual cardenolides, and the substitution of the steroidal structures that can contribute to the molecule's reactivity. The variability in cardenolide defenses could represent the plant's response to natural selection and adaptation of resistant herbivores and is a characteristic of phenotype-matching between defensive and offensive traits resulting from coevolution. Here, we test the phenotypic match of the cardenolide composition of seeds of Asclepias curassavica and those sequestered by nymphs and adults of the specialized seed herbivore Oncopeltus fasciatus, combined with tests of the inhibitory capacity of a subset of seed cardenolides against the Na⁺/K⁺-ATPase of O. fasciatus and a non-adapted insect (Drosophila melanogaster). We compare this with the inhibitory capacity against the highly sensitive porcine Na⁺/K⁺-ATPase. Among the five most abundant cardenolides present in milkweed seeds, glucopyranosyl frugoside, glucopyranosyl gofruside, and glucopyranosyl calotropin were significantly more abundant in the seeds than in the adults and nymphs; the bugs contained higher concentrations of the deglucosylated compounds. The most abundant compound, glucopyranosyl frugoside, was also the most inhibitory for O. fasciatus, but O. fasciatus was significantly more tolerant to all compounds compared to D. melanogaster and the highly sensitive porcine enzyme. Our results add to the evidence that O. fasciatus sequesters specific individual cardenolides from its Asclepias host plants that are not directly linked to the concentration and inhibitory potency.
... Although leaf and root cardenolides show correlated evolution across milkweed species, plants tend to invest in higher concentrations of a few cardenolides in roots compared to a higher diversity of lower concentration compounds in leaves (20). Because the genetic and physiological basis of cardenolide tolerance in the insects' Na + /K + -ATPases is well studied (21)(22)(23), challenging different insects' Na + /K + -ATPases with distinct cardenolides can provide insight into coevolutionary interactions (10,19,24,25). Among the herbivores of cardenolide-containing plants, insect specialists feed on roots, leaves, seeds, phloem sap, and even pith tissue (26). ...
... Little is known about the mechanisms of sequestration or the location of stored cardenolides in Tetraopes, but beetles typically upconcentrate cardenolides at the level of whole insects (18,30). Although other milkweed specialist herbivores detoxify highly potent cardenolides and sequester less potent compounds (10,25), it is unclear whether Tetraopes does the same. We hypothesized that elytra (the hardened protective wing coverings) may be a safer place to store potent toxins than body tissue, while also providing a first line of defense against predators. ...
... Studies of the mechanistic basis of interaction traits and their phenotypic (often physiological) expression have recently yielded insight into ongoing coevolution. For example, we recently showed that some milkweeds produce unusual cardenolides in leaves and seeds that are highly potent against the most adapted specialist sequestering herbivores, although they have mediocre potency against sensitive insects who have not evolved in response to cardenolides (10,25). Conversely, leaf-and seed-feeding specialists do not sequester these compounds, but rather chemically alter them to reduce toxicity to themselves. ...
In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.
... Another possibility is that it is due to our holding the nutritional background constant, only varying the quantity of additional cardenolides, whereas in Blount et al. (2023) monarchs were reared on whole food plants which differ not only in phytochemical profile but also in other metabolic and physical parameters that could have contributed further to changes in redox state. We also found that O. fasciatus only sequestered ouabain, and there were no digitoxin metabolites (as was also reported in Pokharel et al., 2021), so the costs of sequestration may differ when milkweed bugs are feeding on plants with more complex mixtures of cardenolides that require metabolic transformation (see also Agrawal et al., 2022). O. fasciatus do experience oxidative damage in other contexts (López-Muñoz et al., 2019), so the stable levels of damage we measured here could be related to their higher cardenolide resistance rather than a general resistance to oxidative stress. ...
... While the overall tolerance of large milkweed bugs to cardenolides is well established (Bramer et al., 2015;Isman, 1977;Lohr et al., 2017;Scudder et al., 1986), our results add to the recent evidence that sequestration of cardenolides by O. fasciatus impacts life-history traits (Agrawal et al., 2022;Pokharel et al., 2021), and has molecular costs Dalla & Dobler, 2016). This is after what Agrawal et al. (2022) describe as "a long history of finding no costs of cardenolide exposure or sequestration" (Chaplin & Chaplin, 1981;Isman, 1977;Vaughan, 1979). ...
... While the overall tolerance of large milkweed bugs to cardenolides is well established (Bramer et al., 2015;Isman, 1977;Lohr et al., 2017;Scudder et al., 1986), our results add to the recent evidence that sequestration of cardenolides by O. fasciatus impacts life-history traits (Agrawal et al., 2022;Pokharel et al., 2021), and has molecular costs Dalla & Dobler, 2016). This is after what Agrawal et al. (2022) describe as "a long history of finding no costs of cardenolide exposure or sequestration" (Chaplin & Chaplin, 1981;Isman, 1977;Vaughan, 1979). Our results also add to the growing literature that the production and maintenance of warning signals and chemical defenses are affected and limited by resource availability (Barzaghi et al., 2022;Blount et al., 2009Blount et al., , 2012Blount et al., , 2023Burdfield-Steel et al., 2019;Lindstedt et al., 2010Lindstedt et al., , 2020Ottocento et al., 2023). ...
In some aposematic species the conspicuousness of an individual's warning signal and the concentration of its chemical defense are positively correlated. Several mechanisms have been proposed to explain this phenomenon, including resource allocation trade-offs where the same limiting resource is needed to produce both the warning signal and chemical defense. Here, the large milkweed bug (Oncopeltus fasciatus: Heteroptera, Lygaeinae) was used to test whether allocation of antioxidants, that can impart color, trade against their availability to prevent self-damage caused by toxin sequestration. We investigated if (i) the sequestration of cardenolides is associated with costs in the form of changes in oxidative state; and (ii) oxidative state can affect the capacity of individuals to produce warning signals. We reared milkweed bugs on artificial diets with increasing quantities of cardenolides and examined how this affected signal quality (brightness and chroma) across different instars. We then related the expression of warning colors to the quantity of sequestered cardenolides and indicators of oxidative state-oxidative lipid damage (malondialdehyde), and two antioxidants: total superoxide dismutase and total glutathione. Bugs that sequestered more cardenolides had significantly lower levels of the antioxidant glutathione, and bugs with less total glutathione had less luminant orange warning signals and reduced chroma of their black patches compared to bugs with more glutathione. Bugs that sequestered more cardenolides also had reduced red-green chroma of their black patches that was unrelated to oxidative state. Our results give tentative support for a physiological cost of sequestration in milkweed bugs and a mechanistic link between antioxidant availability, sequestration, and warning signals.
... The cardenolide profiles of the bugs frequently diverge from those of their host plants (Isman et al. 1977). This variance can be attributed to the metabolic conversion of certain cardenolides into unique compounds, mitigating self-toxicity and minimising the costs of sequestration (Agrawal et al. 2022). Rubiano-Buitrago et al. (2023a) have found higher concentrations of the cardenolides frugoside and gofruside in O. fasciatus than is available in the seeds on which they feed. ...
... Cardenolide toxicity to predators tested by functional Na + /K + -ATPase assay According to the criteria defined by Agrawal et al. (2022), we found that the coroglaucigenin cardenolides (glucopyranosyl frugoside and frugoside) are toxic (IC 50 <10 μM for black-headed grosbeak, and highly toxic (IC 50 <1 μM) for zebra finch. For the invertebrate predator, the giant Asian mantis, the monoglycosidic cardenolides (frugoside and gofruside) were highly toxic (IC 50 <1 μM, Figure 2, Table S5). ...
The defences of aposematic animals are characterised by diversity and variability of secondary metabolites. Here we examine the nature and function of chemical defence diversity in large milkweed bugs, Oncopeltus fasciatus, testing the hypothesis that different chemical defence compounds have evolved in response to different enemies. We profiled and quantified the cardenolides sequestered by large milkweed bugs in their defensive secretions and their bodies, and measured the inhibitory properties of a subset of isolated milkweed cardenolides in the insect’s defence against the Na+/K+—ATPase target site of vertebrate and invertebrate predators, using porcine Na+/K+—ATPase data as a reference. We show that highly concentrated coroglaucigenin cardenolides in the insect’s defence (glucopyranosyl frugoside and frugoside) are toxic for both resistant and sensitive predators, whereas corotoxigenin and calotropagenin cardenolides have varying degrees of enzyme inhibition among various predators. Overall, O. fasciatus is well defended against a range of enemies due to the differential effect of these compounds´ target sites. Our results suggest that the compounds the insect sequester have evolved in response to predation pressure.
... The total concentration (but not composition as measured by polarity and diversity) of plant cardenolides was induced proportionally to the extent of plant feeding by O. fasciatus, which may have impacts on O. fasciatus sequestration from milkweed plant tissue. It has been shown that the performance of O. fasciatus is largely insensitive to the concentration of cardenolides in its diet [21,25,36]. Accordingly, we speculate that such induction may be adaptive for the bug, as induced plants will have higher concentrations of defensive cardenolides for sequestration than uninduced plants. ...
... Despite large differences in the cardenolide profiles of milkweed plant tissue and seeds, cardenolides sequestered by bugs converged on an intermediate polarity and diversity relative to their two food sources, supporting previous findings that sequestration of cardenolides by O. fasciatus is selective and involves chemical modification [36]. This result is consistent with work on other milkweed herbivores; when fed on the leaves of multiple species of milkweeds, monarch butterflies (Danaus plexippus) converged in terms of the polarities of sequestered cardenolides [35,43]. ...
Plant toxicity shapes the dietary choices of herbivores. Especially when herbivores sequester plant toxins, they may experience a trade-off between gaining protection from natural enemies and avoiding toxicity. The availability of toxins for sequestration may additionally trade off with the nutritional quality of a potential food source for sequestering herbivores. We hypothesized that diet mixing might allow a sequestering herbivore to balance nutrition and defence (via sequestration of plant toxins). Accordingly, here we address diet mixing and sequestration of large milkweed bugs (Oncopeltus fasciatus) when they have differential access to toxins (cardenolides) in their diet. In the absence of toxins from a preferred food (milkweed seeds), large milkweed bugs fed on nutritionally adequate non-toxic seeds, but supplemented their diet by feeding on nutritionally poor, but cardenolide-rich milkweed leaf and stem tissues. This dietary shift corresponded to reduced insect growth but facilitated sequestration of defensive toxins. Plant production of cardenolides was also substantially induced by bug feeding on leaf and stem tissues, perhaps benefitting this cardenolide-resistant herbivore. Thus, sequestration appears to drive diet mixing in this toxic plant generalist, even at the cost of feeding on nutritionally poor plant tissue.
... For seed-eating milkweed bugs (Heteroptera: Lygaeidae, Lygaeinae), artificial diets have been used to address various physiological, ecological, and evolutionary questions (Ramoska & Todd, 1985;Jones et al., 1986;Cohen, 2000;Igarashi et al., 2013). One remarkable characteristic of the entire subfamily Lygaeinae is the ability to sequester plant toxins in defense against predators (Bramer et al., 2015) rendering them an important model system for the study of insect-plant coevolution (Agrawal et al., 2022;Petschenka et al., 2022). Milkweed bugs are primarily associated with plant species in the Apocynaceae, which commonly produce cardenolides (Bramer et al., 2015), potent inhibitors of the ubiquitous animal enzyme Na + /K + -ATPase (Agrawal et al., 2012). ...
... We demonstrated that O. fasciatus and S. saxatilis can sequester ouabain and colchicine included into our diet in a crystalline form. The accumulation of toxins above diet concentrations that our results seem to indicate in both cases has also been reported in the same model species (i.e., O. fasciatus feeding on Asclepias syriaca L. seeds (Agrawal et al., 2022) as well as in other sequestration systems such as Longitarsus sp. beetles sequestering pyrrolizidine alkaloids (Dobler et al., 2000) and the lepidopteran Zygaena filipendulae (L.) sequestering cyanogenic glucosides (Zagrobelny et al., 2014). ...
Insect artificial diets are not only an important tool for mass rearing, nutritional research, and maintaining laboratory colonies but also for studying insect‐plant interactions. For herbivorous insects able to sequester plant toxins, feeding and sequestration assays based on artificial diet allow for the investigation of physiological, ecological, and evolutionary questions which may be difficult to study using real plants representing complex chemical environments. We developed a simple artificial diet, consisting of sunflower meal pressed into pills, for the milkweed bugs Oncopeltus fasciatus (Dallas) and Spilostethus saxatilis (Scopoli) (Heteroptera: Lygaeidae), which are capable of sequestering cardenolides and colchicum alkaloids, respectively. We assessed insect performance, suitability of the diet for sequestration assays, and its shelf life. Compared to sunflower seeds which are widely used as a laboratory maintenance diet for milkweed bugs, no differences were found in terms of weight development, presence of deformities, speed of development, or mortality. Importantly, after feeding O. fasciatus and S. saxatilis sunflower pills enriched with crystalline ouabain (cardenolide) or colchicine (colchicum alkaloid), respectively, sequestration was observed in both species. Moreover, as a prerequisite to test ecological hypotheses, our method allows for adequate concentration control and homogenous distribution of toxins across the diet. Under relatively warm conditions (27 °C and 60% r.h.), the new diet was stable for up to 10 days when used for feeding assays with adult bugs. Therefore, studies focusing on the role of plant toxins in predator–prey interactions and plant defense, but also insecticide research could benefit from using this approach.
... The large milkweed bug, Oncopeltus fasciatus (Heteroptera, Lygaeinae), shares a long history of coevolution with cardiac glycoside containing host plants (Agrawal et al., 2022;Bramer et al., 2015). Like many other lygaeid bugs, the species tolerates the toxins in its food (Bramer et al., 2015;Scudder & Duffey, 1972) and even profits from their presence by sequestering them in specialized storage compartments from where defence fluids are released upon predator attack (Bramer et al., 2017;Duffey et al., 1978;Scudder et al., 1986). ...
... All α/β-combinations of O. fasciatus NKAs, including those containing αC, displayed a strikingly high resistance to inhibition by cardenolides. They even outperform the well-known cardiac glycoside resistance of the monarch butterfly (Danaus plexippus; see also Agrawal et al., 2022;Bramer et al., 2015). Nevertheless, we found that the individual α/β-combinations strongly differed in their resistance against the tested cardenolides. ...
Cardiac glycosides are chemical defence toxins known to fatally inhibit the Na,K-ATPase (NKA) throughout the animal kingdom. Several animals, however, have evolved target-site insensitivity through substitutions in the otherwise highly conserved cardiac glycoside binding pocket of the NKA. The large milkweed bug, Oncopeltus fasciatus, shares a long evolutionary history with cardiac glycoside containing plants that led to intricate adaptations. Most strikingly, several duplications of the bugs' NKA1α gene provided the opportunity for differential resistance-conferring substitutions and subsequent sub-functionalization of the enzymes. Here, we analysed cardiac glycoside resistance and ion pumping activity of nine functional NKA α/β-combinations of O. fasciatus expressed in cell culture. We tested the enzymes with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a standard cardiac glycoside. The identity and number of known resistance-conferring substitutions in the cardiac glycoside binding site significantly impacted activity and toxin resistance in the three α-subunits. The β-subunits also influenced the enzymes' characteristics, yet to a lesser extent. Enzymes containing the more ancient αC-subunit were inhibited by both compounds but much more strongly by the host plant toxin calotropin than by ouabain. The sensitivity to calotropin was diminished in enzymes containing the more derived αB and αA, which were only marginally inhibited by both cardiac glycosides. This trend culminated in αAβ1 having higher resistance against calotropin than against ouabain. These results support the coevolutionary escalation of plant defences and herbivore tolerance mechanisms. The possession of multiple paralogs additionally mitigates pleiotropic effects by compromising between ion pumping activity and resistance.
... syriaca and A. curassavica; n = 3 replicates per tissue type per plant species). We follow the protocol described in Agrawal et al. (2022). Briefly, we used reversed-phase chromatography in a Dionex 3000 LC coupled to an Orbitrap Q-Exactive mass spectrometer controlled by Xcalibur software (ThermoFisher Scientific). ...
Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na⁺/K⁺‐ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na⁺/K⁺‐ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenic Drosophila that were CRISPR‐edited with the monarch's Na⁺/K⁺‐ATPase. Thus, the monarch's selective sequestration appears to reduce self‐harm while maintaining protection from enemies.
... For example, chemical defences include secondary metabolites (e.g. tannins, phenolics, terpenes, alkaloids), which are stored in plant tissues and are detrimental to herbivores (Wink 1988;Agrawal 2007;Mithöfer and Boland 2012;Agrawal et al. 2022). Physical defences include leaf toughness, thorns and trichomes, which limit plant consumption or decrease plant digestibility (Ohata et al. 2010;Massad et al. 2011). ...
Plant strategies against herbivores are classically divided into chemical, physical, biotic defences. However, little is known
about the relative importance of each type of plant defence, especially in the same species. Using the myrmecophyte Triplaris
americana (both with and without ants), and the congeneric non-myrmecophyte T. gardneriana, we tested whether ant defence
is more efective than other defences of naturally ant-free myrmecophytes and the non-myrmecophyte congeneric species, all
spatially co-occurring. In addition, we investigated how plant traits vary among plant groups, and how these traits modulate
herbivory. We sampled data on leaf area loss and plant traits from these tree groups in the Brazilian Pantanal foodplain, and
found that herbivory is sixfold lower in plants with ants than in ant-free plants, supporting a major role of biotic defences
against herbivory. Whereas ant-free plants had more physical defences (sclerophylly and trichomes), they had little efect on
herbivory—only sclerophylly modulated herbivory, but with opposite efects depending on ants’ presence and species identity.
Despite little variation in the chemicals among plant groups, tannin concentrations and δ13C signatures negatively afected
herbivory in T. americana plants with ants and in T. gardneriana, respectively. We showed that ant defence in myrmecophytic
systems is the most efective against herbivory, as the studied plants could not fully compensate the lack of this biotic defence.
We highlight the importance of positive insect-plant interactions in limiting herbivory, and therefore potentially plant ftness.
... Insect adaptations to host-plant toxins involve mechanisms that metabolise toxins and prevent autotoxicity (Agrawal et al., 2022). In cardenolide-sequestering Aphis nerii, gene expression analysis (Birnbaum et al., 2017) showed the expression of a variety of classical detoxification gene families, such as cytochrome P450s, UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette transporters (Heckel, 2014), when the insects were fed on cardenolide-producing plants. ...
Plants produce chemicals (or plant specialised/secondary metabolites, PSM) to protect themselves against various biological antagonists. Herbivorous insects use plants in two ways: as a food source and as a defence source. Insects can detoxify and sequester PSMs in their bodies as a defence mechanism against predators and pathogens. Here, I review the literature on the cost of PSM detoxification and sequestration in insects. I argue that no-cost meals might not exist for insects feeding on toxic plants and suggest that potential costs could be detected in an ecophysiological framework.
... Recently, Agrawal et al. (2022) tested the inhibitory capacity of a subset of purified cardenolides from Asclepias syriaca on the Na + /K + -ATPase. There was little variation among compounds in inhibition of an unadapted Na + /K + -ATPase, but impacts on that of monarchs and Oncopeltus varied significantly [41]. Here, we focus instead on Asclepias curassavica which, although native to the Caribbean area, is cultivated widely as an ornamental plant, and it is now found in numerous semitropical areas [6]. A. curassavica is understudied in its interactions with specialist herbivores in comparison with other species, despite being a critical hostplant worldwide [19]. ...
Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias, cardenolides in milkweed plants have an established role in plant–herbivore and predator–prey interactions, based on their ability to inhibit the membrane-bound Na+/K+-ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica. We report the structures of new compounds: 3-O-β-allopyranosyl coroglaucigenin (1), 3-[4′-O-β-glucopyranosyl-β-allopyranosyl] coroglaucigenin (2), 3′-O-β-glucopyranosyl-15-β-hydroxycalotropin (3), and 3-O-β-glucopyranosyl-12-β-hydroxyl coroglaucigenin (4), as well as six previously reported cardenolides (5–10). We test the in vitro inhibition of these compounds on the sensitive porcine Na+/K+-ATPase. The least inhibitory compound was also the most abundant in the seeds—4′-O-β-glucopyranosyl frugoside (5). Gofruside (9) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant–herbivore interactions.
... Nonetheless, it has previously been shown that O. fasciatus chemically modifies some of the most potent compounds from common milkweed (A. syriaca) seeds into less toxic forms (Agrawal et al., 2022). ...
Many herbivores sequester plant toxins, and this occurs along a gradient of specialization including “toxic plant generalists” that also consume non-toxic hosts. We hypothesized that availability of toxins for sequestration may trade off with nutritional quality to shape dietary choices in a Lygaeid bug. In the absence of toxins (cardenolides) from a preferred food (milkweed seeds), bugs increased feeding on nutritionally poor, but cardenolide-rich milkweed plant tissue, corresponding to lower growth but greater sequestration. While bugs feeding on only milkweed plant tissue sequestered lower cardenolides than those feeding on seeds, they sequestered more diverse and less polar compounds. Cardenolide production was also induced by feeding on plant tissues, which may have downstream effects on future herbivory and sequestration. Accordingly, sequestration is a driver of diet choice in this toxic plant generalist, even at the cost of feeding on nutritionally poor plant tissue; reciprocally, plants defensively respond to such feeding choices.
... Wide variation in cardenolide solubility, stability, and binding site specificity may be necessary to protect plants against herbivores with different physiological characteristics. For instance, unique nitrogen-containing cardenolides in some milkweeds (e.g., voruscharin; Fig. 2) provide protection against monarch butterfly caterpillars and seed bugs, which are otherwise highly resistant to these compounds (Agrawal et al., 2021;Agrawal et al., 2022). Both in vivo toxicity and in vitro Na + /K + -ATPase inhibition assays show that not all cardiac glycosides function in the same manner, and the most abundant compounds are not necessarily the most efficacious against a particular herbivore (Petschenka et al., 2018;Tverskoi et al., 2021). ...
The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na⁺/K⁺-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na⁺/K⁺-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na⁺/K⁺-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na⁺/K⁺-ATPase activity by plant extracts, and (3) extraction of insect Na⁺/K⁺-ATPases for inhibition assays.
... In Agrawal's Inaugural Article (2), he explores the evolutionary interaction between common milkweeds and large milkweed bugs, using monarch butterflies as a point of comparison. He also explores geographical patterns in milkweed defense by sampling common milkweed populations across almost 13°of latitude, from Quebec City, Canada, to Bishop, North Carolina. ...
Considering both pollinator and herbivore pressures on plant reproductive and defensive traits is key to understanding patterns of selection for plants. However, phenotypic selection studies connecting floral traits and plant defenses with pollinator activity and herbivore damage remain rare. We used the common milkweed, Asclepias syriaca (Apocynaceae), to study phenotypic selection on attractive and defensive traits, and nectar rewards. We measured herbivore (leaf damage) and pollinator activity (pollinia movement) and quantified selection via female (pollinia insertions and fruit number) and male fitness (pollinia removals). We found selection to increase plant and inflorescence size and to decrease floral size (i.e. petal width) via female fitness. We also detected selection to increase floral but not leaf latex. The lack of selection on leaf latex was congruent with the low herbivory observed, however we also did not observe florivory in the population that would explain the advantage of more floral latex. Interestingly, we found selection on attractive traits differed via pollinia insertions and fruits initiated, suggesting that something other than pollinators was driving selection via fruit production. In contrast to female fitness, we did not find selection on any trait through male fitness, suggesting no sexual conflicting selection, at least through these proxies. Our findings reinforce the importance of the direct assessment of pollinator pressures in phenotypic selection studies before assuming pollinators as drivers of floral evolution by natural selection. Further work in southern populations closer to the centre of the species range, where herbivory and plant defense investment are higher, may help elucidate selection on attractive and defensive traits.
Aim
Adaptation to local environments has been considered a driving force of ecological speciation. Previous phylogenetic studies at higher taxonomic levels have strongly suggested that herbivore diversification occurs through local adaptation to host plants. However, whether similar mechanisms contribute to within‐species diversification remains poorly understood. We reconstructed species expansion processes using the phylogenetic relationships between two Luehdorfia butterflies and their host plants and tested the local adaptation hypothesis during species diversification.
Location
Japanese archipelago.
Taxa
Luehdorfia japonica and L . puziloi.
Methods
We analysed mitochondrial DNA and single‐nucleotide polymorphisms to trace the expansion processes of two species in the Japanese archipelago. We analysed data collected during trans‐host plant feeding experiments to test for local host plant adaptation in L. japonica .
Results
Luehdorfia japonica differentiated 17.0 million years ago (MYA) from a group of four Luehdorfia species in East Asia, and L . puziloi differentiated 13.3 MYA from the remaining three species. We observed several genetic groups in both species, reflecting geographical differences among populations. Some host plants had detrimental effects on the pupation rates of L. japonica , demonstrating the presence of genetic barriers between populations of this species.
Main Conclusions
Although these two sibling species were influenced by the geological formation of the Japanese archipelago, their present population structures differ greatly: L . japonica expanded its distribution and adapted to local host plants along at least four expansion routes independent of host plant phylogeny, and L . puziloi exhibited a more isolated population structure and was less strongly influenced by gene flow among populations. These results suggest that both isolation by distance and isolation by adaptation have been important drivers resulting in the present‐day population structure of L . japonica . Unidirectional gene flow through local adaptation to the host plant may have played a role in initial speciation.
Many herbivorous insects not only cope with plant toxins but also sequester them as a defense against predators and parasitoids. Sequestration is a product of the evolutionary arms race between plants and herbivorous insects and has been hypothesized to incur physiological costs due to specific adaptations required. Contradictory evidence about these costs exists for insects sequestering only one class of toxin, but very little is known about the physiological implications for species sequestering structurally different classes of compounds. Spilostethus saxatilis is a milkweed bug belonging to the cardenolide-sequestering heteropteran subfamily Lygaeinae (Heteroptera: Lygaeidae) that has shifted to the colchicine-containing plant Colchicum autumnale, a resource of chemically unrelated alkaloids. Using feeding-assays on artificial diet and chemical analysis, we assessed whether S. saxatilis is still able to sequester cardenolides apart from colchicine and related metabolites (colchicoids), and tested the effect of (1) either a natural cardenolide concentration (using ouabain as a model compound) or a natural colchicine concentration, (2) an increased concentration of both toxins, and (3) seeds of either Asclepias syriaca (cardenolides) or C. autumnale (colchicoids) on a set of life-history traits. For comparison, we assessed the same life-history traits in the milkweed bug Oncopeltus fasciatus exposed to cardenolides only. Although cardenolides and colchicoids have different physiological targets (Na+/K+-ATPase vs tubulin) and thus require different resistance traits, chronic exposure and sequestration of both isolated toxins caused no physiological costs such as reduced growth, increased mortality, lower fertility, or shorter adult life span in S. saxatilis. Indeed, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis when feeding on isolated ouabain and isolated colchicine, respectively. Positive effects were even more pronounced when insects were provided with natural toxic seeds (i.e. C. autumnale for S. saxatilis and A. syriaca for O. fasciatus), especially in O. fasciatus. Our findings suggest, that S. saxatilis can sequester two chemically unrelated classes of plant compounds at a cost-free level, and that colchicoids may even play a beneficial role in terms of fertility.
Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na⁺/K⁺-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na⁺/K⁺-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony’s lifespan.
In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here we address the demographic history of the monarch butterfly, Danaus plexippus , and its dominant host plant, the common milkweed Asclepias syriaca , using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using Approximate Bayesian Computation via Random Forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced concurrent range expansion during the most recent recession of North American glaciers ∼12,000 years ago. Our data identify an expansion of milkweed during the large-scale clearing of eastern forests (∼200 years ago) but was inconclusive as to expansion or contraction of the monarch butterfly population during this time. Finally, our results indicate that neither species experienced a population contraction over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.
Coevolution between plants and herbivores often involves escalation of defense‐offense strategies, but attack by multiple herbivores may obscure the match of plant defense to any one attacker. As herbivores often specialize on distinct plant parts, we hypothesized that defense‐offense interactions in coevolved systems may become physiologically and evolutionarily compartmentalized between plant tissues. We report that roots, leaves, flower buds and seeds of the tropical milkweed (Asclepias curassavica) show increasing concentrations of cardenolide toxins acropetally, with latex showing the highest concentration. In vitro assays of the physiological target of cardenolides, the Na+/K+–ATPase (hereafter ‘sodium pump’), of three specialized milkweed herbivores (root‐feeding Tetraopes tetrophthalmus, leaf‐feeding Danaus plexippus, and seed‐feeding Oncopeltus fasciatus) show that they are proportionally tolerant to the cardenolide concentrations of the tissues they eat. Indeed, molecular substitutions in the insects’ sodium pumps predicted their tolerance to toxins from their target tissues. Nonetheless, the relative inhibition of the sodium pumps of these specialists by the concentration vs. composition (inhibition controlled for concentration, what we term ‘potency’) of cardenolides from their target vs. non‐target plant tissues revealed different degrees of insect adaptation to tissue‐specific toxins. In addition, a trade‐off between toxin concentration and potency emerged across plant tissues, potentially reflecting coevolutionary history or plant physiological constraints. Our findings suggest that tissue‐specific coevolutionary dynamics may be proceeding between the plant and its specialized community of herbivores. This novel finding may be common in nature, contributing to ways in which coevolution proceeds in multi‐species communities.
Sequestration, that is, the accumulation of plant toxins into body tissues for defense, was predicted to incur physiological costs and may require resistance traits different from those of non-sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant-derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na⁺/K⁺-ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na⁺/K⁺-ATPases. Both adaptations, resistance and sequestration, are ancestral traits of the Lygaeinae. Using four milkweed bug species (Heteroptera: Lygaeidae: Lygaeinae) and the related European firebug (Heteroptera: Pyrrhocoridae: Pyrrhocoris apterus) showing different combinations of the traits “cardenolide resistance” and “cardenolide sequestration,” we tested how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na⁺/K⁺-ATPase nor sequestering cardenolides, growth was not affected in the non-sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na⁺/K⁺-ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na⁺/K⁺-ATPase. We furthermore assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, longevity of adults, and reproductive success in O. fasciatus. Unexpectedly, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide-containing diet for their entire lifetime but not when adults were transferred to non-toxic sunflower seeds. We speculate that the resistant Na⁺/K⁺-ATPase of milkweed bugs is selected for working optimally in a “toxic environment,” that is, when sequestered cardenolides are stored in the body.
Since its first release over a decade ago, the MetaboAnalyst web-based platform has become widely used for comprehensive metabolomics data analysis and interpretation. Here we introduce MetaboAnalyst version 5.0, aiming to narrow the gap from raw data to functional insights for global metabolomics based on high-resolution mass spectrometry (HRMS). Three modules have been developed to help achieve this goal, including: (i) a LC-MS Spectra Processing module which offers an easy-to-use pipeline that can perform automated parameter optimization and resumable analysis to significantly lower the barriers to LC-MS1 spectra processing; (ii) a Functional Analysis module which expands the previous MS Peaks to Pathways module to allow users to intuitively select any peak groups of interest and evaluate their enrichment of potential functions as defined by metabolic pathways and metabolite sets; (iii) a Functional Meta-Analysis module to combine multiple global metabolomics datasets obtained under complementary conditions or from similar studies to arrive at comprehensive functional insights. There are many other new functions including weighted joint-pathway analysis, data-driven network analysis, batch effect correction, merging technical replicates, improved compound name matching, etc. The web interface, graphics and underlying codebase have also been refactored to improve performance and user experience. At the end of an analysis session, users can now easily switch to other compatible modules for a more streamlined data analysis. MetaboAnalyst 5.0 is freely available at https://www.metaboanalyst.ca.
Despite long-standing interest in local adaptation of plants to their biotic and abiotic environment, existing theory, and many case studies, little work to date has addressed within-species evolution of concerted strategies and how these might contrast with patterns across species. Here we consider the interactions between pollinators, herbivores, and resource availability in shaping plant local adaptation, how these interactions impact plant phenotypes and gene flow, and the conditions where multiple traits align along major environmental gradients such as latitude and elevation. Continued work in emerging model systems will benefit from the melding of classic experimental approaches with novel population genetic analyses to reveal patterns and processes in plant local adaptation.
Significance
Interactions between plants and herbivores constitute a major pathway of energy transfer up the food chain. As a consequence, evolution by natural selection has honed the chemically mediated antagonistic interactions between these groups. Monarch butterflies and milkweeds serve as royal representatives in deciphering such coevolution, and our study takes a mechanistic and manipulative approach to understand how the tropical milkweed, Asclepias curassavica , defends itself against monarch butterflies, which would seem to be impervious feeders. By directly observing plant–herbivore interactions and coupling this with experiments on isolated toxins and the monarch’s neural sodium-potassium pump enzymes, we show that tropical milkweed produces a burdensome cardenolide toxin, and monarchs convert it to less toxic compounds, the latter sequestered for their own benefit.
Cardiac glycosides are known to fatally inhibit the Na,K-ATPase throughout the animal kingdom. Several animals, however, evolved target-site insensitivity by substitution in the otherwise highly conserved cardiac glycoside binding pocket located on the Na,K-ATPase α-subunit. The minimal functional enzyme consists of an α- and a β-subunit, considered mainly as a chaperone responsible for correct folding and membrane integration. We here analyze resistance to cardiac glycosides and kinetic properties of different Na,K-ATPase α-β-combinations of the large milkweed bug, Oncopeltus fasciatus . These insects have adapted to high concentrations of cardiac glycosides in their food plants via several rounds of ATPase α1 gene duplications followed by subfunctionalization of the enzymes.
To investigate their characteristics we expressed nine combinations of Na,K-ATPase α/β-sunbunits (three each) in Sf 9 cells and tested them with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a commonly used toxin. Differences in the number and identity of amino acid substitutions in the cardiac glycoside binding site resulted in large differences in activity and toxin resistance of the three α-subunits. The enzymes’ kinetics were also influenced by the β-subunits leading to increased activities (αCβ3) or altered resistances. Calotropin proved to be a much more potent inhibitor than ouabain. Our results show that the possession of multiple paralogs enables adaptation to plant toxins and mitigates pleiotropic effects by a compromise between ion pumping activity and resistance.
Summary statement
Activity and cardiac glycoside resistance of the Oncopeltus fasciatus ’ Na,K-ATPase is determined by amino acid substitutions in the α-subunit binding pocket, the α/β-subunit combination and the cardiac glycoside identity.
Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.
Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.
Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness¹. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2,3,4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6,7,8,9,10,11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na⁺/K⁺-ATPase)7,8,9,10,11, the physiological target of cardiac glycosides¹². Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR–Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides¹⁶, culminating in triple mutant ‘monarch flies’ that were as insensitive to cardiac glycosides as monarch butterflies. ‘Monarch flies’ retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17,18,19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20,21,22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6,7,8,9,10,11,15,17,18,19.
Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na+,K+-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.
Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na⁺/K⁺ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na⁺/K⁺ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed’s diverse community of specialist cardenolide-sequestering insect herbivores.
A central tenet of plant defense theory is that adaptation to the abiotic environment sets the template for defense strategies, imposing a trade-off between plant growth and defense. Yet this trade-off, commonly found among species occupying divergent resource environments, may not occur across populations of single species. We hypothesized that more favorable climates and higher levels of herbivory would lead to increases in growth and defense across plant populations. We evaluated whether plant growth and defense traits co-varied across 18 populations of showy milkweed (Asclepias speciosa) inhabiting an east-west climate gradient spanning 257 of longitude. A suite of traits impacting defense (e.g., latex, cardenolides), growth (e.g., size), or both (e.g., specific leaf area [SLA], trichomes) were measured in natural populations and in a common garden, allowing us to evaluate plastic and genetically based variation in these traits. In natural populations, herbivore pressure increased toward warmer sites with longer growing seasons. Growth and defense traits showed strong clinal patterns and were positively correlated. In a common garden, clines with climatic origin were recapitulated only for defense traits. Correlations between growth and defense traits were also weaker and more negative in the common garden than in the natural populations. Thus, our data suggest that climatically favorable sites likely facilitate the evolution of greater defense at minimal costs to growth, likely because of increased resource acquisition.
A major prediction of coevolutionary theory is that plants may target particular herbivores with secondary compounds that are selectively defensive. The highly specialized monarch butterfly (Danaus plexippus) copes well with cardiac glycosides (inhibitors of animal Na+/K+-ATPases) from its milkweed host plants, but selective inhibition of its Na+/K+-ATPase by different compounds has not been previously tested. We applied 17 cardiac glycosides to the D. plexippus-Na+/K+-ATPase and to the more susceptible Na+/K+-ATPases of two non-adapted insects (Euploea core and Schistocerca gregaria). Structural features (e.g., sugar residues) predicted in vitro inhibitory activity and comparison of insect Na+/K+-ATPases revealed that the monarch has evolved a highly resistant enzyme overall. Nonetheless, we found evidence for relative selectivity of individual cardiac glycosides reaching from four to 94-fold differences of inhibition between non-adapted Na+/K+-ATPase and D. plexippus- Na+/K+-ATPase. This toxin-receptor specificity suggests a mechanism how plants could target herbivores selectively and thus provides a strong basis for pairwise coevolutionary interactions between plants and herbivorous insects.
A major prediction of coevolutionary theory is that plants may target particular herbivores with secondary compounds that are selectively defensive. The highly specialized monarch butterfly (Danaus plexippus) copes well with cardiac glycosides (inhibitors of animal Na⁺/K⁺-ATPases) from its milkweed host plants, but selective inhibition of its Na⁺/K⁺-ATPase by different compounds has not been previously tested. We applied 17 cardiac glycosides to the D. plexippus-Na⁺/K⁺-ATPase and to the more susceptible Na⁺/K⁺-ATPases of two non-adapted insects (Euploea core and Schistocerca gregaria). Structural features (e.g., sugar residues) predicted in vitro inhibitory activity and comparison of insect Na⁺/K⁺-ATPases revealed that the monarch has evolved a highly resistant enzyme overall. Nonetheless, we found evidence for relative selectivity of individual cardiac glycosides reaching from 4- to 94-fold differences of inhibition between non-adapted Na⁺/K⁺-ATPase and D. plexippus-Na⁺/K⁺-ATPase. This toxin receptor specificity suggests a mechanism how plants could target herbivores selectively and thus provides a strong basis for pairwise coevolutionary interactions between plants and herbivorous insects.
Background:
The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell's resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C.
Results:
Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls).
Conclusions:
These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge.
The latitudinal herbivory defense hypothesis (LHDH) postulates that the prevalence of species interactions, including herbivory, is greater at lower latitudes, leading to selection for increased levels of plant defense. While latitudinal defense clines may be caused by spatial variation in herbivore pressure, optimal defense theory predicts that clines could also be caused by ecogeographic variation in the cost of defense. For instance, allocation of resources to defense may not increase plant fitness when growing seasons are short and plants must reproduce quickly. Here we use a common garden experiment to survey genetic variation for constitutive and induced phenylpropanoid glycoside (PPG) concentrations across 35 Mimulus guttatus populations over a ~13° latitudinal transect. Our sampling regime is unique among studies of the LHDH in that it allows us to disentangle the effects of growing season length from those of latitude, temperature and elevation. For five of the seven PPGs surveyed, we find associations between latitude and plant defense that are robust to population structure. However, contrary to the LHDH, only two PPGs were found at higher levels in low latitude populations, and total PPG concentrations were higher at higher latitudes. PPG levels are strongly correlated with growing season length, with higher levels of PPGs in plants from areas with longer growing seasons. Further, flowering time is positively correlated with the concentration of nearly all PPGs, suggesting that there may be a strong tradeoff between development time and defense production. Our results reveal that ecogeographic patterns in plant defense may reflect variation in the cost of producing defense compounds in addition to variation in herbivore pressure. Thus, the biogeographic pattern predicted by the LHDH may not be accurate because the underlying factors driving variation in defense, in this case, growing season length, are not always associated with latitude in the same manner. Given these results, we conclude that LHDH cannot be interpreted without considering life history, and we recommend that future work on the LHDH move beyond solely testing the core LHDH prediction and place greater emphasis on isolating agents of selection that generate spatial variation in defense and herbivore pressure. This article is protected by copyright. All rights reserved.
Greater plant defence is predicted to evolve at lower latitudes in response to increased herbivore pressure. However, recent studies question the generality of this pattern. In this study, we tested for genetically based latitudinal clines in resistance to herbivores and underlying defence traits of Oenothera biennis. We grew plants from 137 populations from across the entire native range of O. biennis. Populations from lower latitudes showed greater resistance to multiple specialist and generalist herbivores. These patterns were associated with an increase in total phenolics at lower latitudes. A significant proportion of the phenolics were driven by the concentrations of two major ellagitannins, which exhibited opposing latitudinal clines. Our analyses suggest that these findings are unlikely to be explained by local adaptation of herbivore populations or genetic variation in phenology. Rather greater herbivory at high latitudes can be explained by latitudinal clines in the evolution of plant defences.
Substantial research has addressed adaptation of nonnative biota to novel environments, yet surprisingly little work has integrated population genetic structure and the mechanisms underlying phenotypic differentiation in ecologically important traits. We report on studies of the common milkweed Asclepias syriaca, which was introduced from North America to Europe over the past 400 years and which lacks most of its specialized herbivores in the introduced range. Using 10 populations from each continent grown in a common environment, we identified several growth and defense traits that have diverged, despite low neutral genetic differentiation between continents. We next developed a Bayesian modeling approach to account for relationships between molecular and phenotypic differences, confirming that continental trait differentiation was greater than expected from neutral genetic differentiation. We found evidence that growth-related traits adaptively diverged within and between continents. Inducible defenses triggered by monarch butterfly herbivory were substantially reduced in European populations, and this reduction in inducibility was concordant with altered phytohormonal dynamics, reduced plant growth, and a trade-off with constitutive investment. Freedom from the community of native and specialized herbivores may have favored constitutive over induced defense. Our replicated analysis of plant growth and defense, including phenotypically plastic traits, suggests adaptive evolution following a continental introduction.
Broad‐scale geographical gradients in the abiotic environment and interspecific interactions should select for clinal adaptation. How trait clines evolve has recently received increased attention because of anticipated climate change and the importance of rapid evolution in invasive species. This issue is particularly relevant for clines in growth and defense of plants, because both sets of traits are closely tied to fitness and because such sessile organisms experience strong local selection. Yet despite widespread recognition that growth and defense traits are intertwined, the general issue of their joint clinal evolution is not well resolved.
To address heritable clinal variation and adaptation of growth and defense traits of common milkweed ( Asclepias syriaca ), we planted seed from 22 populations encompassing the species' latitudinal range in common gardens near the range center (New York) and toward the range edges (New Brunswick and North Carolina). Populations were differentiated in 13 traits, and six traits showed genetically based latitudinal clines. Higher‐latitude populations had earlier phenology, lower shoot biomass, more root buds and clonal growth, higher root‐to‐shoot ratio, and greater latex production. The cline in shoot biomass was consistent in all three locations. Selection on phenology was reversed in New Brunswick and North Carolina, with early genotypes favored in the north but not the south. We found no clines in foliar trichomes or toxic cardenolides. Annual precipitation of source populations explained variation in phenology, clonal growth, root‐to‐shoot ratio, and latex. Across four traits measured in New Brunswick and North Carolina, we found garden‐by‐latitude (and garden‐by‐precipitation) interactions, indicating plasticity in genetically based trait clines.
In the two gardens with substantial herbivory (New York and North Carolina), northern populations showed higher resistance to insects. Resistance to aphids was driven by trichomes and water content, while resistance to monarch caterpillars was driven by latex. However, surveys of natural populations indicated that leaf damage and insect diversity on milkweed are low at the geographical extremes (New Brunswick and North Carolina) and higher toward the range center. We speculate that milkweed plants evolved clines in growth traits in response to climate, and that this set the template for tolerance to herbivory, which subsequently shaped the evolution of defensive traits.
Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na(+)/K(+)-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na(+)/K(+)-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na(+)/K(+)-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.
© 2015 The Author(s) Published by the Royal Society. All rights reserved.
Monarch butterfly,Danaus plexippus (L.), larvae were collected during August 1983 from the common milkweed,Asclepias syriaca L., across its extensive North American range from North Dakota, east to Vermont, and south to Virginia. This confirms that the late summer distribution of breeding monarchs in eastern North America coincides with the range of this extremely abundant milkweed resource. Plant cardenolide concentrations, assayed by spectrophotometry in 158 samples from 27 collection sites, were biased towards plants with low cardenolide, and ranged from 4 to 229 μg/ 0.1 g dry weight, with a mean of 50 μg/0.1 g. Monarch larvae reared on these plants stored cardenolides logarithmically, and produced 158 adults with a normally distributed concentration range from 0 to 792 μg/0. l g dry butterfly, with a mean of 234 μg/0.1 g. Thus butterflies increased the mean plant cardenolide concentration by 4.7. The eastern plants and their resultant butterflies had higher cardenolide concentrations than those from the west, and in some areas monarchs sequestered more cardenolide from equivalent plants. Plants growing in small patches had higher cardenolide concentrations than those in larger patches, but this did not influence butterfly concentration. However, younger plants and those at habitat edges had higher cardenolide concentrations than either older, shaded, or open habitat plants, and this did influence butterfly storage. There were no apparent topographical differences reflected in the cardenolides of plants and butterflies. Twenty-eight cardenolides were recognized by thin-layer chromatography, with 27 in plants and 21 in butterflies. Butterflies stored cardenolides within the more polar 46% of the plantR
d range, these being sequestered in higher relative concentrations than they occurred in the plants. By comparison with published TLC cardenolide mobilities, spots 3, 4, 9, 16, 24 or 25, 26, and 27, may be the cardenolides syrioside, uzarin, syriobioside, syriogenin, uzarigenin, labriformidin, and labriformin, respectively. Cochromatography with cardenolide standards indicated that desglucosyrioside did not occur in the plants but did occur in 70% of the butterflies, and aspecioside was in 99% of the plants and 100% of the butterflies. The polar aspecioside was the single most concentrated and diagnostic cardenolide in both plants and butterflies. ButterflyR
d values were dependent on those of the plant, and both showed remarkable uniformity over the range of areas sampled. Thus contrary to previous reports,A. syriaca has a biogeographically consistent cardenolide fingerprint pattern. The ecological implications of this for understanding the monarch's annual migration cycle are significant.
Ecological processes that determine the abundance of species within ecological com-munities vary across space and time. These scale-dependent processes are especially important when they affect key members of a community, such as ecosystem engi-neers that create shelter and food resources for other species. Yet, few studies have examined the suite of processes that shape the abundance of ecosystem engineers. Here, we evaluated the relative influence of temporal variation, local processes, and latitude on the abundance of an engineering insect—a rosette-galling midge, Rhopalomyia solidaginis (Diptera: Cecidomyiidae). Over a period of 3–5 years, we studied the density and size of galls across a suite of local experiments that manip-ulated genetic variation, soil nutrient availability, and the removal of other insects from the host plant, Solidago altissima (tall goldenrod). We also surveyed gall density within a single growing season across a 2,300 km latitudinal transect of goldenrod populations in the eastern United States. At the local scale, we found that host-plant genotypic variation was the best predictor of rosette gall density and size within a single year. We found that the removal of other insect herbivores resulted in an increase in gall density and size. The amendment of soil nutrients for four years had no effect on gall density, but galls were smaller in carbon-added plots compared to control and nitrogen additions. Finally, we observed that gall density varied several fold across years. At the biogeographic scale, we observed that the density of rosette gallers peaked at mid-latitudes. Using meta-analytic approaches, we found that the effect size of time, followed by host-plant genetic variation and latitude were the best predictors of gall density. Taken together, our study provides a unique comparison of multiple factors across different spatial and temporal scales that govern engineering insect herbivore density.
Broad-scale geographical gradients in the abiotic environment and interspecific interactions should select for clinal adaptation. How trait clines evolve has recently received increased attention because of anticipated climate change and the importance of rapid evolution in invasive species. This issue is particularly relevant for clines in growth and defense of plants, because both sets of traits are closely tied to fitness and because such sessile organisms experience strong local selection. Yet despite widespread recognition that growth and defense traits are intertwined, the general issue of their joint clinal evolution is not well resolved. To address heritable clinal variation and adaptation of growth and defense traits of common milkweed (Asclepias syriaca), we planted seed from 22 populations encompassing the species' latitudinal range in common gardens near the range center (New York) and toward the range edges (New Brunswick and North Carolina). Populations were differentiated in 13 traits, and six traits showed genetically based latitudinal clines. Higher-latitude populations had earlier phenology, lower shoot biomass, more root buds and clonal growth, higher rootto-shoot ratio, and greater latex production. The cline in shoot biomass was consistent in all three locations. Selection on phenology was reversed in New Brunswick and North Carolina, with early genotypes favored in the north but not the south. We found no clines in foliar trichomes or toxic cardenolides. Annual precipitation of source populations explained variation in phenology, clonal growth, root-to-shoot ratio, and latex. Across four traits measured in New Brunswick and North Carolina, we found garden-by-latitude (and gardenby-precipitation) interactions, indicating plasticity in genetically based trait clines. In the two gardens with substantial herbivory (New York and North Carolina), northern populations showed higher resistance to insects. Resistance to aphids was driven by trichomes and water content, while resistance to monarch caterpillars was driven by latex. However, surveys of natural populations indicated that leaf damage and insect diversity on milkweed are low at the geographical extremes (New Brunswick and North Carolina) and higher toward the range center. We speculate that milkweed plants evolved clines in growth traits in response to climate, and that this set the template for tolerance to herbivory, which subsequently shaped the evolution of defensive traits.
The extent of convergent molecular evolution is largely unknown, yet is critical to understanding the genetics of adaptation. Target site insensitivity to cardenolides is a prime candidate for studying molecular convergence because herbivores in six orders of insects have specialized on these plant poisons, which gain their toxicity by blocking an essential transmembrane carrier, the sodium pump (Na,K-ATPase). We investigated gene sequences of the Na,K-ATPase α-subunit in 18 insects feeding on cardenolide-containing plants (spanning 15 genera and four orders) to screen for amino acid substitutions that might lower sensitivity to cardenolides. The replacement N122H that was previously shown to confer resistance in the monarch butterfly (Danaus plexippus) and Chrysochus leaf beetles was found in four additional species, Oncopeltus fasciatus and Lygaeus kalmii (Heteroptera, Lygaeidae), Labidomera clivicollis (Coleoptera, Chrysomelidae), and Liriomyza asclepiadis (Diptera, Agromyzidae). Thus, across 300 Myr of insect divergence, specialization on cardenolide-containing plants resulted in molecular convergence for an adaptation likely involved in coevolution. Our screen revealed a number of other substitutions connected to cardenolide binding in mammals. We confirmed that some of the particular substitutions provide resistance to cardenolides by introducing five distinct constructs of the Drosophila melanogaster gene into susceptible eucaryotic cells under an ouabain selection regime. These functional assays demonstrate that combined substitutions of Q(111) and N(122) are synergistic, with greater than twofold higher resistance than either substitution alone and >12-fold resistance over the wild type. Thus, even across deep phylogenetic branches, evolutionary degrees of freedom seem to be limited by physiological constraints, such that the same molecular substitutions confer adaptation.
The contribution of Miriam Rothschild to the monarch cardenolide story is reviewed in the light of the 1914 challenge by the evolutionary biologist, E.B. Poulton for North American chemists to explain the chemical basis of unpalatability in monarch butterflies and their milkweed host plants. This challenge had lain unaccepted for nearly 50 years until Miriam Rothschild took up the gauntlet and showed with the help of many able colleagues that monarchs are aposematically coloured because they sequester toxic cardenolides from milkweed host plants for use as a defence against predators. By virtue of Dr Rothschild's inspiration and industry, and subsequently that of Lincoln Brower and his colleagues, this tritrophic interaction has become a familiar paradigm for the evolution of chemical defences and warning colouration. We now know that the cardenolide contents of different milkweeds vary quantitatively, qualitatively and spatially, both within and among species and we are starting to appreciate the implications of such variation. However, as Dr Rothschild has pointed out in her publications, cardenolides have sometimes blinded us to reality and it is curious how little evidence there is for a defensive function to cardenolides in plants — especially against adapted specialists such as the monarch. Thus the review will conclude with a discussion of the significance of temporal variation and induction of cardenolide production in plants, the lethal plant defence paradox and an emphasis on the dynamics of the cardenolide-mediated interaction between milkweeds and monarch larvae.
Cardenolides are remarkable steroidal toxins that have become model systems, critical in the development of theories for chemical ecology and coevolution. Because cardenolides inhibit the ubiquitous and essential animal enzyme Na⁺/K⁺-ATPase, most insects that feed on cardenolide-containing plants are highly specialized. With a huge diversity of chemical forms, these secondary metabolites are sporadically distributed across 12 botanical families, but dominate the Apocynaceae where they are found in > 30 genera. Studies over the past decade have demonstrated patterns in the distribution of cardenolides among plant organs, including all tissue types, and across broad geographic gradients within and across species. Cardenolide production has a genetic basis and is subject to natural selection by herbivores. In addition, there is strong evidence for phenotypic plasticity, with the biotic and abiotic environment predictably impacting cardenolide production. Mounting evidence indicates a high degree of specificity in herbivore-induced cardenolides in Asclepias. While herbivores of cardenolide-containing plants often sequester the toxins, are aposematic, and possess several physiological adaptations (including target site insensitivity), there is strong evidence that these specialists are nonetheless negatively impacted by cardenolides. While reviewing both the mechanisms and evolutionary ecology of cardenolide-mediated interactions, we advance novel hypotheses and suggest directions for future work.
Mass spectrometry (MS) coupled with online separation methods is commonly applied for differential and quantitative profiling of biological samples in metabolomic as well as proteomic research. Such approaches are used for systems biology, functional genomics, and biomarker discovery, among others. An ongoing challenge of these molecular profiling approaches, however, is the development of better data processing methods. Here we introduce a new generation of a popular open-source data processing toolbox, MZmine 2.
A key concept of the MZmine 2 software design is the strict separation of core functionality and data processing modules, with emphasis on easy usability and support for high-resolution spectra processing. Data processing modules take advantage of embedded visualization tools, allowing for immediate previews of parameter settings. Newly introduced functionality includes the identification of peaks using online databases, MSn data support, improved isotope pattern support, scatter plot visualization, and a new method for peak list alignment based on the random sample consensus (RANSAC) algorithm. The performance of the RANSAC alignment was evaluated using synthetic datasets as well as actual experimental data, and the results were compared to those obtained using other alignment algorithms.
MZmine 2 is freely available under a GNU GPL license and can be obtained from the project website at: http://mzmine.sourceforge.net/. The current version of MZmine 2 is suitable for processing large batches of data and has been applied to both targeted and non-targeted metabolomic analyses.
Q(ST) is a standardized measure of the genetic differentiation of a quantitative trait among populations. The distribution of Q(ST)'s for neutral traits can be predicted from the F(ST) for neutral marker loci. To test for the neutral differentiation of a quantitative trait among populations, it is necessary to ask whether the Q(ST) of that trait is in the tail of the probability distribution of neutral traits. This neutral distribution can be estimated using the Lewontin-Krakauer distribution and the F(ST) from a relatively small number of marker loci. We develop a simulation method to test whether the Q(ST) of a given trait is consistent with the null hypothesis of selective neutrality over space. The method is most powerful with small mean F(ST), strong selection, and a large number (>10) of measured populations. The power and type I error rate of the new method are far superior to the traditional method of comparing Q(ST) and F(ST).
The parsnip webworm Depressaria pastinacella is restricted to two hostplant genera containing six structurally diverse furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole‐larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut‐specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin‐inducible. Computer docking and energy‐minimization of a CYP6AE89 model with all six furanocoumarins showed that 5‐methoxylated bergapten, and 8‐methoxylated xanthotoxin had the smallest distances from the heme to the proton‐donor residue in the catalytic I‐helix, and that the 5,8‐dimethoxylated isopimpinellin and bergapten had the smallest energy‐minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an E. coli system, and used it to detect high catalytic activity against only the two mono‐methoxylated linear furanocoumarins—bergapten, and xanthotoxin, and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is likely specialized for detoxifying only a subset of hostplant furanocoumarins. A maximum‐likelihood tree built with six representative lepidopterans with manually annotated P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure. This article is protected by copyright. All rights reserved.
Classic research on elevational gradients in plant-herbivore interactions holds that insect herbivore pressure is stronger under the warmer, less seasonal climates characteristic of low elevations, and that this in turn selects for increased defence in low- (relative to high-) elevation plants. However, recent work has questioned this paradigm, arguing that it overly simplifies the ecological complexity in which plant-insect herbivore interactions are embedded along elevational gradients. Numerous biotic and abiotic factors vary with elevation, and their simultaneous influences are the focus of current work on elevational gradients in insect herbivory and plant defences. The present review (i) synthesizes current knowledge on elevational gradients in plant-insect herbivore interactions; (ii) critically analyses research gaps and highlights recent advances that contribute to filling these gaps; and (iii) outlines new research opportunities to uncover underlying mechanisms and build towards a unified theory on elevational gradients. We conclude that the next generation of studies should embrace community complexity–including multi-trophic dynamics and the multivariate nature of plant defence–and to do so by combining observational data, manipulative experiments and emerging analytical tools.
A long-standing paradigm in ecology holds that herbivore pressure and thus plant defences increase towards lower latitudes. However, recent work has challenged this prediction where studies have found no relationship or opposite trends where herbivory or plant defences increase at higher latitudes. Here we tested for latitudinal variation in herbivory, chemical defences (phenolic compounds), and nutritional traits (phosphorus and nitrogen) in leaves of a long-lived tree species, the English oak Quercus robur. We further investigated the underlying climatic and soil factors associated with such variation. Across 38 populations of Q. robur distributed along an 18° latitudinal gradient, covering almost the entire latitudinal and climatic range of this species, we observed strong but divergent latitudinal gradients in leaf herbivory and leaf chemical defences and nutrients. As expected, there was a negative relationship between latitude and leaf herbivory where oak populations from lower latitudes exhibited higher levels of leaf herbivory. However, counter to predictions there was a positive relationship between leaf chemical defences and latitude where populations at higher latitudes were better defended. Similarly, leaf phosphorus and nitrogen increased with latitude. Path analysis indicated a significant (negative) effect of plant chemical defences (condensed tannins) on leaf herbivory, suggesting that the latitudinal gradient in leaf herbivory was driven by an inverse gradient in defensive investment. Leaf nutrients had no independent influence on herbivory. Further, we found significant indirect effects of precipitation and soil porosity on leaf herbivory, which were mediated by plant chemical defences. These findings suggest that abiotic factors shape latitudinal variation in plant defences and that these defences in turn underlie latitudinal variation in leaf herbivory. Overall, this study contributes to a better understanding of latitudinal variation in plant-herbivore interactions by determining the identity and modus operandi of abiotic factors concurrently shaping plant defences and herbivory.
Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase α-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, we expressed the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. Our results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier. This article is protected by copyright. All rights reserved
Both herbivory and plant defenses against herbivores have been predicted to increase toward tropical regions. Early tests of this latitudinal herbivory-defense hypothesis (LHDH) were supportive, but accumulating evidence has been mixed. We argue that the lack of clarity might be due to heterogeneity in methodology and problems with study design and interpretation. We suggest possible solutions. Latitudinal studies need to carefully consider spatial and phylogenetic scale, to link plant defense measurements to herbivore performance, and to incorporate additional concepts from plant defense theory such as tolerance and induced defense. In addition, we call for consistent measures of herbivory to standardize comparisons across biomes. Improving methodology in future studies of LHDH should resolve much of the current controversy.
This paper is the first in a series on cardenolide fingerprinting of the monarch butterfly. New methodologies are presented which allow both qualitative and quantitative descriptions of the constituent cardenolides which these insects derive in the wild from specificAsclepias foodplants. Analyses of thin-layer Chromatographic profiles ofAsclepias eriocarpa cardenolides in 85 individual plant-butterfly pairs collected at six widely separate localities in California indicate a relatively invariant pattern of 16–20 distinct cardenolides which we here define as theAsclepias eriocarpa cardenolide fingerprint profile. Cardenolide concentrations vary widely in the plant samples, but monarchs appear able to regulate total storage by increasing their concentrations relative to their larval host plant when reared on plants containing low concentrations, and vice versa. Forced-feeding of blue jays with powdered butterfly and plant material and with one of the constituent plant cardenolides, labriformin, established that theA. eriocarpa cardenolides are extremely emetic, and that monarchs which have fed on this plant contain an average of 16 emetic-dose fifty (ED50) units. The relatively nonpolar labriformin and labriformidin in the plant are not stored by the monarch but are metabolized in vivo to desglucosyrioside which the butterfly does store. This is chemically analogous to the way in which monarchs and grasshoppers metabolize another series of milkweed cardenolides, those found inA. curassavica. It appears that the sugar moiety through functionality at C-3′ determines which cardenolides are metabolized and which are stored. The monarch also appears able to store several lowR
f
cardenolides fromA. eriocarpa without altering them. Differences in the sequestering process in monarchs and milkweed bugs (Oncopeltus) may be less than emphasized in the literature. The monarch is seen as a central organism involved in a coevolutionary triad simultaneously affecting and affected by both its avian predators and the secondary chemistry of the milkweeds with which it is intimately involved.
The cardenolide content of the gut, wings, and fat body ofOncopeltus fasciatus was examined. The female fat body contained 4-5% of the total cardenolide content of the insect. The cardenolide content of male fat body, and gut and wings of both sexes was below the detection limit of the cardenolide assay. Thin-layer chromatography was used to determine the cardenolide array of various tissues and secretions ofO. fasciatus reared on seeds of a single species of milkweed (A. Speciosa) and adult extracts and dorsolateral space fluid ofO. fasciatus reared on seeds of two species of milkweed with different cardenolide arrays (A. speciosa andA. syriaca). Our results indicate that cardenolides are not sequestered in the insect simply on the basis of polarity and that metabolism and differential excretion of cardenolides are involved in the sequestration of cardenolides inO. fasciatus. The similarities in the cardenolide profiles ofO. fasciatus reared on different food sources, and tissues ofO. fasciatus reared on a single food source indicates that there is regulation of the cardenolide array inO. Fasciatus.
The morphological and physiological adaptations associated with sequestration of cardenolides by the lygaeidOncopeltus fasciatus are summarized and discussed. Cardenolides are efficiently accumulated inO. fasciatus; however, the insect does not appear to suffer any physiological cost as a result of handling large amounts of these plant toxins. Morphological adaptations of the insect include a modified integument composed of a double layered epidermis with an inner layer (the dorsolateral space) specialized for cardenolide storage. Special weak areas of the cuticle are found on both the thorax and abdomen, which rupture when the insect is squeezed, resulting in the cardenolide-rich contents of the inner epidermal layer being released onto the body surface in the form of discrete spherical droplets. Physiological adaptations include selective sequestration of food plant cardenolides, concentration of cardenolides in the dorsolateral space, passive uptake of cardenolides at the gut and dorsolateral space requiring little energy output, reabsorption of secreted cardenolides by the Malpighian tubules, high in vivo tolerance to cardenolides, and the presence of cardenolide-resistant Na,K-ATPases.
The majority (85%) of 394 monarch butterflies sampled from overwintering sites in Mexico contain the same epoxy cardenolide glycosides, including most conspicuously a novel polar glycoside with a single genin-sugar bridge (aspecioside), as occur in the milkweedsAsclepias speciosa andA. syriaca. This cardenolide commonality was established by isolating aspecioside and syriobioside from the wings of overwintering monarchs and the two plant species, and comparing Chromatographie and NMR spectrometric characteristics of the isolates. When combined with the migratory pattern of monarchs and the distribution of these two milkweed species, this chemical evidence lends strong support to the hypothesis thatA. syriaca is the major late summer food plant of monarchs in eastern North America. This finding may be of ecological importance, forA. syriaca contributes less cardenolide and cardenolides of lower emetic potency to monarchs than most milkweeds studied to date.
Asclepias eriocarpa and A. labriformis contain three new cardenolides, the structures of which have been partially assigned by their spectral properties and comparison with the known cardenolides of A. curassavica. They include labriformin (C31H39O10NS), labriformidin (C29H31O11) and eriocarpin.
Larvae of the milkweed bug Oncopeltus fasciatus were reared on the seeds of eight different species of milkweed (Asclepias), representing a wide range of cardenolide concentrations in the diet. There were few significant differences in larval developmental period, wet body weight of teneral adults, dry weight of adults, and pronotal width of adults reared on the different diets. However, the data indicate no significant correlations between cardenolide content, and body weight or size of the adult insects.There was no evidence in this study of a physiological cost or adverse effect on the larval growth and development of insects which sequestered and stored differing quantities of cardenolides. Instead, the data support a recently-proposed model of cardenolide sequestration which may be energy-independent.The validity of evidence supporting a physiological cost hypothesis for sequestration of cardenolides by the monarch butterfly is discussed in the light of these findings.
Oncopeltus fasciatus sequesters cardiac glycosides from seeds of Asclepias syriaca, and in the adult, concentrates these chemicals in the dorsolateral spaces of the thorax and abdomen; 60 to 95% of the cardenolide in such adults is stored in the dorsolateral complex. Very little cardenolide is detectable in the haemolymph of adults and larvae, but cardiac glycosides do occur in the adult ventral metathoracic gland secretions and in the larval middorsal abdominal gland fluid.O. fasciatus sequesters cardenolides throughout its life cycle. The complement of cardenolides acquired by feeding throughout the development is not related simply to increase in body weight. Unknown factors during late larval development are important in the accumulative uptake of the heart poisons.It is shown that O. fasciatus sequesters polar but not non-polar cardenolides into the dorsolateral complex in the adult. The accumulation of the polar cardenolides in both the larva and adult appears to be, at least in part, associated with the ability of the insect to metabolize heart poisons. The non-polar cardenolide digitoxin was converted to at least two more polar cardenolides. Adult O. fasciatus did not, however, metabolically alter the chromatographic behavior of ouabain that had been added to the food.
The effect of different host plant diets on the growth dynamics of the large milkweed bug, Oncopeltus fasciatus , was investigated by rearing this specialized seed feeder on the seeds of seven milkweed species occurring in central Missouri. Growth rate, growth efficiency, and ♂:♀ biomass ratio proved to be the most sensitive measures of growth in detecting significant differences between diets. Ascleptas syriaca L., A. verticillata L. and A. hirtella (Pennell) Woods, supported the most rapid and efficient growth, while A. incarnata L., A. purpurescens L., A. viridiflora Raf., and A. quadrifolia Jacq. were less suitable host species. In general, O. fasciatus grew best on the host species it utilizes most commonly in the field, thus supporting the proposed host familiarity hypothesis. The most commonly exploited plant species shared three characteristics of seed production and quality which accounted for 87%‐96% of the variability in growth dynamics of O. fasciatus . The most important of these was the mean number of pods produced per plant. Seed nitrogen content was the next best parameter in further accounting for variability in growth rate and ♂:♀ biomass ratio but seed cardenolide content was the second parameter for growth efficiency.
RÉSUMÉ
DYNAMIQUE DU DÉVELOPPEMENT D'UN CONSOMMATEUR SPÉCIALISTE DE GRAINES D'ASCLEPIADACÉES (ONCOPELTUS FASCIATUS), SUR DES GRAINES D' ASCLEPIAS HABITUELLEMENT OU NON CONSOMMÉES
L'élevage d' Oncopeltus fasciatus sur les graines de 7 espèces d' Asclepias de la région centrale du Missouri a permis d'examiner l'influence de l'alimentation sur différentes plantes hôtes sur la dynamique du développement de cet insecte.
La survie, la durée du développement, la biomasse des adultes et les calories de l'aliment consommé ou de la biomasse produite, présentent peu de différences significatives suivant les aliments.
Par contre, 3 autres paramètres: le taux de croissance, l'efficacité de la croissance, et le rapport des biomasses ♂/♀, sont plus sensibles pour la mise en évidence de différences. Asclepias syriaca, A. verticillata et A. hirtella permettent la croissance la plus rapide et la plus efficace, tandis que A. incarnata. A. purpurescens, A. viridiflora et A. quadrifolia sont des hôtes moins favorables. En général, O fasciatus se développe mieux sur la plante qu'il consomme le plus fréquemment dans la nature, ce que était l'hypothèse des hôtes familiers. 87% de la variabilité du développement sur les différents régimes peut être expliquée par 3 paramètres liés à la production el à la qualité des graines. Le nombre moyen de gousses par plante intervient le plus dans la détermination de la variabilité du taux de croissance. La teneur des graines en azote est le paramètre qui intervient ensuite pour le taux de croissance et le rapport de biomasses ♂/♀, mais la teneur des graines en cardenolide est le second paramètre pour l'efficacité de la croissance.
Aus den oberirdischen Teilen der Seidenpflanze, Asclepias syriacaL. (Asclepiadaceae) isolierten Masler et al. [2] [3] fünf krist. Cardenolide, u. a. Syriobiosid und Syriosid, denen sie die Formeln 5 und 6 zuschrieben. A. syriaca ist eine der Futterpflanzen, auf denen die Larven von Schmetterlingen leben, welche die Cardenolide der Nahrung zu speichern vermögen und dadurch von der Vertilgung durch insektenfressende Tiere (bes. Vögel) teilweise geschützt sind. Die Inhaltsstoffe der Pflanze variieren stark. Bei dem uns zur Verfügung stehenden Material enthielten Blätter und Stengel nur Spuren von Cardenoliden, relativ viel enthielten die Wurzeln. Aus solchen konnte krist. Syriosid sowie eine Spur krist. Syriobiosid direkt durch Chromatographie gewonnen werden, die Hauptmenge des letzteren wurde erst nach fermentativem Abbau mit β-Glucosidasen erhalten. Chemische und physikalische Methoden zeigten, dass die vorgeschlagenen Formeln 5 und 6 unrichtig sind. Syriobiosid besitzt vermutlich Formel 7 und Syriosid Formel 10. Letzteres liefert bei fermentativem Abbau mit β-Glucosidasen nicht Syriobiosid, wie die tschechischen Autoren glaubten, sondern einen um zwei H-Atome ärmeren Stoff, den wir Desglucosyriosid (12) nennen. Die Formeln sind gut begründet, aber nicht eindeutig bewiesen. Syriosid und Syriobiosid enthalten somit als Zuckerbaustein eine 4,6-Didesoxy-hexosulose (33), wie sie im Gomphosid (20) und den Calotropis-Cardenoliden (22, 24etc.) vorkommt, die ebenfalls von den Larven der genannten Schmetterlinge mit der Nahrung aufgenommen werden und als Abwehrstoffe wirksam sind.
Milkweed bugs,Oncopeltus fasciatus, were fed seeds of common milkweed,Asclepias syriaca, that contained differing concentrations of cardiac glycoside. Whole seeds had a mean cardiac glycoside concentration of 4.01 mg equivalents to digitoxin per g dry weight, and seed embryos had a mean concentration of 5.56 mg/g dry weight. Bugs fed these seeds concentrated cardiac glycoside: their mean concentration was 6.85 mg/g dry weight. Milkweed bugs fed seeds of lower cardiac glycoside content sequestered a greater percent of the available glycoside than bugs fed seeds of high glycoside content. The quantitative variation of cardiac glycoside content of the seeds of this single species did not significantly affect the growth of bugs. In a separate feeding preference experiment, bugs were offered seeds of both high (5.18 mg/g dry weight) and low (2.30 mg/g dry weight) cardiac glycoside content. The bugs showed no indication of selecting seeds of either high or low glycoside content.
According to the geographic mosaic theory of coevolution, selection intensity in interactions varies across a landscape, forming a selection mosaic; interaction traits match at coevolutionary hotspots where selection is reciprocal and mismatch at coldspots where reciprocity is not a factor. Chemical traits play an important role in the interaction between wild parsnip (Pastinaca sativa) and the parsnip webworm (Depressaria pastinacella). Furanocoumarins, produced as plant defenses, are detoxified by the webworms by cytochrome P450 monooxygenases; significant additive genetic variation exists for both furanocoumarin production in the plant and detoxification in the insect, making these traits available for selection. To test the hypothesis that differences in selection intensity affect the distribution of coevolutionary hotspots and coldspots in this interaction, we examined 20 populations of webworms and wild parsnips in Illinois and Wisconsin that varied in size, extent of infestation, proximity to woods (and potential vertebrate predators), and proximity to a chemically distinct alternate host plant, Heracleum lanatum (cow parsnip). Twelve of 20 populations displayed phenotype matching between plant defense and insect detoxification profiles. Of the eight mismatched populations, a logistic regression model related matching probability to two predictors: the presence of the alternate host and average content of xanthotoxin (one of the five furanocoumarins produced by P. sativa). The odds of mismatching were significantly increased by the presence of the alternate host (odds ratio = 15.4) and by increased xanthotoxin content (odds ratio = 6.053). Parsnips growing near cow parsnip displayed chemical phenotypes that were chemically intermediate between cow parsnip and parsnips growing in isolation. Rapid phenotype matching in this system is likely due in part to differential mortality every season; larvae transferred to a plant 30 m or more from the plant on which they developed tended to experience increased mortality over larvae transferred to another umbel on the same plant on which they had developed, and plant populations that mismatched in 2001 displayed a change in chemical phenotype distribution from the previous year. Trait mixing through gene flow is also a likely factor in determining mismatch frequency. Populations from which webworms were eradicated the previous year were all recolonized; in three of seven of these populations, infestation rates exceeded 90%. Our findings, consistent with the geographic mosaic theory, suggest that the presence of a chemically distinct alternate host plant can affect selection intensity in such a way as to reduce the likelihood of reciprocity in the coevolutionary interaction between wild parsnip and the parsnip webworm.
Local adaptation through natural selection can be inferred in case the additive genetic divergence in a quantitative trait across populations (Q(st)) exceeds the neutral expectation based on differentiation of neutral alleles across these populations (e.g. F(st)). As such, measuring Q(st) in relation to neutral differentiation presents a first-line investigation applicable in evolutionary biology (selection on functional genes) and conservation biology (identification of locally adapted coding genes). However, many species, especially those in need of conservation actions, are not amenable for the kind of breeding design required to estimate either narrow- or broad-sense Q(st). In such cases, Q(st) has been approximated by the phenotypic divergence in a trait across populations (P(st)). I here argue that the critical aspect for how well P(st) approximates Q(st) depends on the extent that additive genetic effects determine variation between populations relative to within populations. I review how the sensitivity of conclusions regarding local adaptation based on P(st) have been evaluated in the literature and find that many studies make a anticonservative null assumption in estimating P(st) and/or use a nonconservative approach to explore sensitivity of their conclusions. Data from two studies that have provided a second, independent assessment of selection in their system suggest that P(st)-F(st) comparisons should be interpreted very conservatively. I conclude with recommendations for improving the robustness of the inferences drawn from comparing P(st) with neutral differentiation.
J. M. Benson, J. N. Seiber, C. V. Bagley, R. F. Keeler, A. E. Johnson and S. Young. Effects on sheep of the milkweeds Asclepias eriocarpa and A. labriformis and of cardiac glycoside-containing derivative material. Toxicon17, 155–165, 1979.—Some milkweeds native to the western United States are extremely toxic to range animals. As little as 0·05 and 0·25% of an animal's weight of Asclepias labriformis and Asclepias eriocarpa, respectively, may be lethal (Kingsbury, 1964). Cardiac glycosides (cardenolides) in these species have been implicated as toxic principles. In experiments conducted to verify this toxicity, four preparations were tested on sheep: dried, ground A. labriformis and A. eriocarpa plants, a crude ethanolic A. eriocarpa extract, and a partially purified extract from which pigments and fats had been removed. In addition, labriformin (a purified cardenolide present in A. labriformis and A. eriocarpa), and digitoxin (a clinically important cardenolide) were administered. Toxic symptoms and gross and histopathological lesions were qualitatively similar regardless of whether plants, extracts, labriformin or digitoxin were administered. Results of these experiments strongly suggest that cardenolides found in A. labriformis and A. eriocarpa account for the effects of these plants on sheep.
Steroidal compounds in the roots of Asclepias tuberosa were investigated and 17alpha-hydroxyandrosta-4,6,15-trien-3-one 17-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside, termed ascandroside, was isolated from the CHCl3-soluble fraction. Among five doubly-linked cardenolide glycosides, two were identified as 3'-spiro-linked thiazolidinone (4) and S-oxythiazolidinone derivatives (5) of delta5-calotropin. The stereochemistry at the C-3' in these two cardenolides is discussed. 3'-O-beta-D-Glucopyranosyl-delta5-calotropin (3) was also isolated along with A5-calotropin and its 3'-acetate. Nine glycosides of uzarigenin, coroglaucigenin and corotoxigenin were identified.
According to the geographic mosaic theory of coevolution, selection intensity in interactions varies across a landscape, forming a selection mosaic; interaction traits match at coevolutionary hotspots where selection is reciprocal and mismatch at coldspots where reciprocity is not a factor. Chemical traits play an important role in the interaction between wild parsnip (Pastinaca sativa) and the parsnip webworm (Depressaria pastinacella). Furanocoumarins, produced as plant defenses, are detoxified by the webworms by cytochrome P450 monooxygenases; significant additive genetic variation exists for both furanocoumarin production in the plant and detoxification in the insect, making these traits available for selection. To test the hypothesis that differences in selection intensity affect the distribution of coevolutionary hotspots and coldspots in this interaction, we examined 20 populations of webworms and wild parsnips in Illinois and Wisconsin that varied in size, extent of infestation, proximity to woods (and potential vertebrate predators), and proximity to a chemically distinct alternate host plant, Heracleum lanatum (cow parsnip). Twelve of 20 populations displayed phenotype matching between plant defense and insect detoxification profiles. Of the eight mismatched populations, a logistic regression model related matching probability to two predictors: the presence of the alternate host and average content of xanthotoxin (one of the five furanocoumarins produced by P. sativa). The odds of mismatching were significantly increased by the presence of the alternate host (odds ratio = 15.4) and by increased xanthotoxin content (odds ratio = 6.053). Parsnips growing near cow parsnip displayed chemical phenotypes that were chemically intermediate between cow parsnip and parsnips growing in isolation. Rapid phenotype matching in this system is likely due in part to differential mortality every season; larvae transferred to a plant 30 m or more from the plant on which they developed tended to experience increased mortality over larvae transferred to another umbel on the same plant on which they had developed, and plant populations that mismatched in 2001 displayed a change in chemical phenotype distribution from the previous year. Trait mixing through gene flow is also a likely factor in determining mismatch frequency. Populations from which webworms were eradicated the previous year were all recolonized; in three of seven of these populations, infestation rates exceeded 90%. Our findings, consistent with the geographic mosaic theory, suggest that the presence of a chemically distinct alternate host plant can affect selection intensity in such a way as to reduce the likelihood of reciprocity in the coevolutionary interaction between wild parsnip and the parsnip webworm.
Diversification of cytochrome P450 monooxygenases (P450s) is thought to result from antagonistic interactions between plants and their herbivorous enemies. However, little direct evidence demonstrates the relationship between selection by plant toxins and adaptive changes in herbivore P450s. Here we show that the furanocoumarin-metabolic activity of CYP6B proteins in two species of swallowtail caterpillars is associated with the probability of encountering host plant furanocoumarins. Catalytic activity was compared in two closely related CYP6B4 and CYP6B17 groups in the polyphagous congeners Papilio glaucus and Papilio canadensis. Generally, P450s from P. glaucus, which feeds occasionally on furanocoumarin-containing host plants, display higher activities against furanocoumarins than those from P. canadensis, which normally does not encounter furanocoumarins. These P450s in turn catalyze a larger range of furanocoumarins at lower efficiency than CYP6B1, a P450 from Papilio polyxenes, which feeds exclusively on furanocoumarin-containing host plants. Reconstruction of the ancestral CYP6B sequences using maximum likelihood predictions and comparisons of the sequence and geometry of their active sites to those of contemporary CYP6B proteins indicate that host plant diversity is directly related to P450 activity and inversely related to substrate specificity. These predictions suggest that, along the lineage leading to Papilio P450s, the ancestral, highly versatile CYP6B protein presumed to exist in a polyphagous species evolved through time into a more efficient and specialized CYP6B1-like protein in Papilio species with continual exposure to furanocoumarins. Further diversification of Papilio CYP6Bs has likely involved interspersed events of positive selection in oligophagous species and relaxation of functional constraints in polyphagous species.