Parasitic insect herbivores
Cycads’ unique chemistries are also important for understanding cycads’ associations with parasitic insects. Cycads produce potent lineage-specific phytotoxins such as methylazoxymethanol (MAM) glycosides, β-methylamino-L-alanine (BMAA), and lesser studied compounds such as steryl glycosides. Yet cycad-feeding has been documented in at least six insect orders, with most cycad herbivores belonging to the orders Lepidoptera (moths and butterflies), Coleoptera (beetles and weevils), and Hemiptera (true bugs). A previous review concluded that cycad-feeding has evolved independently among Lepidoptera multiple times, and that insect defensive traits may be especially important in determining whether cycad-feeding lineages diversify (Whitaker & Salzman, 2020). A systematic review of cycad-feeding Coleoptera is currently underway to investigate the ecology and evolution of their incredible diversity of feeding ecologies, species interactions, and defensive traits.
Many cycad-associated insects are obligate cycad specialists and must possess adaptations to contend with cycads’ chemical defenses. In general, herbivorous insects can excrete, sequester, or detoxify phytotoxins, but it is not clear which strategies most cycad herbivores utilize. There is some evidence that pollinating weevils excrete BMAA in their frass and pupal casings, and some Lepidoptera have been shown to sequester MAM for protection from natural enemies (reviewed in Whitaker & Salzman, 2020). Some cycad-feeding Aulacoscelinae beetles reflexively bleed MAM-glycosides – presumably sequestered from their foodplants – when disturbed or threatened (Prado et al., 2011), and at least one lepidopteran has been shown to be capable of detoxifying MAM (Teas, 1967). Recently, the obligate cycad herbivore Eumaeus atalabutterfly was found to accumulate BMAA into their larval and adult tissues (Whitaker et al ., 2023), although the defensive value of sequestering BMAA remains questionable given its latent toxicity (Whitaker et al., 2022). Genomic evidence does suggest, however, that toxin tolerance is a key adaptation in the radiation ofEumaeus butterflies, a wholly cycadivorous neotropical genus of six species (Robbins et al., 2021). It has also been suggested that insects’ gut microbiomes may contribute to degrading cycad toxins (Salzman et al ., 2018), though this remains to be experimentally demonstrated.
Specialized cycad herbivores must also possess adaptations to locate and select feeding and oviposition sites, but very little is known about the chemical, thermal, and visual cues used in host selection. A more mechanistic view of host selection would help assess the potential for host switching and pest management as increasing cultivation of non-native cycads introduces opportunities for emerging pest dynamics (Rensburg et al ., 2023). Invasive pests such as the cycad scale represent major threats to cycad conservation (Marler, Lindström & Watson, 2021), and even native herbivores may pose a threat in some circumstances: recent host expansions have been documented among cycad-feeding lepidoptera where native and non-native cycads are co-cultivated, with potentially dire effects for cycad conservation (Marler, Lindström & Terry, 2012; Normark et al., 2017; Whitakeret al ., 2020). Cycad-herbivore interactions span from mutualistic to parasitic and provide the opportunity for investigating mechanisms of host selection, specialization, and toxin tolerance, a better understanding of which will improve species management and advance research on coevolution and plant-insect interactions.
Novel insights into the evolution of coralloid roots symbiosis
One of the first observations of cycad root symbionts is from the 19th century, in which various taxa were described by Schneider (1894), including fungi, bacteria and algae. Subsequent research however, focused mostly on Nostocales cyanobacteria as the key taxon in coralloid roots (Suárez-Moo et al ., 2019; Bell-Doyon et al ., 2020). These cyanobionts colonize a morphologically distinct cycad organ called the coralloid root (Fig. 1h). CyanobacterialNostocales in the coralloid root fix nitrogen in exchange for plant carbohydrates, but only a few species from the generaNostoc, Desmonostoc, Calothrix and Aulosira have been found inside the coralloid root forming three distinct phylogenetic clades (Bustos-Diaz et al ., in review; Fig. 3). While no “symbiotic genes” have been identified in cyanobiont genomes, experiments using cyanobionts isolated from cycads to infect other plants found multiple genes crucial for the establishment of the symbiosis (Alvarez et al. , 2022; Wong & Meeks, 2002), suggesting that while specific traits are required for the symbiosis, gene specificity, if any, is not apparent with current data (Bustos-Diazet al. , in review).
The mechanism of initiating symbiosis and the necessary morphological changes in both plant and symbiont is not well described, although recent evidence shows that multiple molecular mechanisms and signal recognition systems in both the cyanobiont and the host underlie the symbiosis, including the hormogonium–inducing factor produced by precoralloid roots (Hashidoko et al. , 2019; Figs. 1g & 3a). Based on Cycas panzhihuaensis coralloid roots transcriptomes (Liuet al ., 2022), additionally, terpenoids have been implicated with establishment of the symbiosis. The bacterial community has also been shown to produce various metabolites which might influence the symbiosis (Fig. 3a; Dehm et al. , 2019; Freitas et al ., 2022). Amongst these metabolites, BMAA has been linked to impaired cyanobacterial nitrogen fixation (Berntzon et al ., 2013) and may be produced by both the cyanobiont and host (Marler, Snyder & Shaw, 2010). The main challenge here is analytically distinguishing amongst the vast number of BMAA isomers and deciphering their interactions with other toxic cycad metabolites.
We are beginning to explore the role of gene expression in symbiosis . The recently sequenced cycad genome (see below) recovered a two-fold upregulation of 10 genes from the common symbiotic pathway of legume-rhizobial and plant-mycorrhizal symbiosis (Delaux & Schornack, 2021) (RAD1 , DHY , SymRK , EPP1 , VAPYRIN , CASTOR/POLLUX , NFP , CYTB561 ,GRAS , HEP ) in precoralloid roots compared to mature coralloid roots colonized by cyanobacteria. In contrast, five genes were highly expressed in colonized coralloid roots (CCaMK ,CYCLOPS , LIN , SYN and TAU ), while a large number of genes are preferentially up and down regulated in both root types (Fig. 3b). In addition, 24 conserved genes were found shared among plants with cyanobacterial symbiosis (Table 1). The specific role of all of these plant genes for symbiosis remains to be experimentally elucidated. Comparative genomics provides the necessary groundwork for an understanding of the evolutionary mechanisms of plant-cyanobacterial symbiosis.