Genome-wide transcriptome signatures of ant-farmed Squamellaria epiphytes reveal key functions in a unique symbiosis

The agricultural symbiosis between Squamellaria (Rubiaceae) epiphytic plants and Philidris nagasau ants in Fiji involves habitual planting, fertilization, defence, and harvest of the plant 'crop' by farmer ants. All Squamellaria individuals form a large tuber (domatium) with self-formed entrance holes and complex cavity networks that provide the ants with nesting and breeding areas. Key to this symbiosis is the differentiated inner domatium wall structure: There are smooth-surfaced cavity sections where ants nest and rear their brood and warty-surfaced sections were farming ants fertilize their crop by defecation. Here we use RNA sequencing (RNAseq) to investigate the genes expressed in the two wall types and in normal tuber tissue in the ant-farmed Squamellaria imberbis. Because the wall structure differentiation occurred in the most recent common ancestor of all farmed species of Squamellaria, we aimed to identify genetic pathways co-opted during the evolution of obligate agriculture in Rubiaceae. Most differentially expressed genes were upregulated in warty surfaces compared to smooth walls or tuber tissues and are involved in nutrient uptake and defence responses, indicating the role of warts as an interface between the plant and the external environment. Fifty-eight genes up-regulated in warty surface were involved in auxin transport and response, root development and nitrogen transport, pinpointing that warts are functionally and developmentally analogous to adventitious roots. Sixty-four genes linked to defence against pathogens and response to various external stimulus were upregulated in warts, unveiling the need to protect these permeable surfaces, which are the sites of nutrient transfer. In smooth surfaces, four genes functioning in suberin and wax biosynthesis were upregulated, contributing to the formation of sturdy and clean impermeable ant-nesting area in the domatium. This study throws light on a number of functional characteristics of farming symbioses.