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Urbanization is altering landscapes globally at an unprecedented rate. While ecological differences between urban and rural environments often promote phenotypic divergence among populations, it is unclear to what degree these trait differences arise from genetic divergence as opposed to phenotypic plasticity. Furthermore, little is known about how specific landscape elements, such as green corridors, impact genetic divergence in urban environments. We tested the hypotheses that: (1) urbanization, and (2) proximity to an urban green corridor influence genetic divergence in common milkweed (Asclepias syriaca) populations for phenotypic traits. Using seeds from 52 populations along three urban-to-rural subtransects in the Greater Toronto Area, Canada, one of which followed a green corridor, we grew ~ 1000 plants in a common garden setup and measured > 20 ecologically-important traits associated with plant defense/damage, reproduction, and growth over four years. We found significant heritable variation for nine traits within common milkweed populations and weak phenotypic divergence among populations. However, neither urbanization nor an urban green corridor influenced genetic divergence in individual traits or multivariate phenotype. These findings contrast with the expanding literature demonstrating that urbanization promotes rapid evolutionary change and offer preliminary insights into the eco-evolutionary role of green corridors in urban environments.

 

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.

 

A major predicted constraint on the evolution of anti-herbivore defense in plants is the nonindependent 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.

 

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. 

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 transgenicDrosophilathat 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.

 

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.