Evolution of extrafloral nectaries in Passifloraceae

Passiflora (passionflowers) represent one of the most morphologically diverse groups of angiosperms. Among the many unique features of this group is the extraordinary diversity of extrafloral nectaries (EFNs)—glands located outside of flowers that secrete sugar-rich nectar to attract mutualistic insects, especially ants, which in turn help protect the plant from herbivores. The genus Passiflora contains more species with EFNs than any other genus of vascular plants, making it an ideal system for studying both the developmental and evolutionary origins of these structures.

Extrafloral nectary diversity in the salicoid lineage. A-F: Salicaceae. A. Banara nitida petiole EFN; B. Xylosma venosa petiole and leaf teeth EFNs with ants; C. Hasseltia floribunda laminar EFNs with ants; D. Banara guianensis leaf teeth EFNs; E. Neosprucea montana leaf teeth EFNs; F. Populus sp. leaf teeth EFNs with longhorn beetle. G-H: Turneraceae. G. Turnera ulmifolia petiole EFNs with ants; H. Tricliceras lobatum petiole EFNs with ant. I-R: Passifloraceae. I. Adenia sp. laminar EFNs; J. Passiflora parritae petiole and leaf teeth EFNs; K. P. morifolia petiole EFNs; L. P. auriculata petiole egg mimic EFNs; M. P. alnifolia laminar EFNs with caterpillar; N. P. tatei laminar EFNs; O. P. boenderi laminar egg mimic EFNs; P. P. mathewsii stipule teeth EFNs; Q. P. vitifolia floral bract teeth EFNs; R. P. incarnata floral bract margin EFNs with assassin bug.

Our research examines the anatomical diversity, developmental origins, genetic control, and evolutionary history of EFNs across Passiflora and its relatives. This project integrates approaches from systematics, developmental anatomy, comparative transcriptomics, and plant-insect ecology to understand how these highly diverse structures evolve and function.

Research Questions

Our ongoing work addresses multiple interrelated questions:

• How have EFNs evolved within Passiflora and the broader Passifloraceae?
• Are EFNs in different species homologous structures or the result of repeated evolutionary recruitment of different developmental pathways?
• How are EFNs developmentally and evolutionarily related to leaf teeth and other glandular structures?
• What molecular and genetic pathways control the initiation and differentiation of EFNs across divergent species?
• How do EFNs influence plant-insect interactions, particularly with ants and herbivorous butterflies?
• How have EFN modifications contributed to evolutionary innovations such as egg mimicry and herbivore deterrence?

Developmental and Anatomical Diversity

Across Passiflora, EFNs exhibit remarkable variation in position, structure, and anatomy:

• EFNs may occur on petioles, leaf laminae (adaxial and abaxial surfaces), stipules, floral bracts, or combinations of these structures.
• The anatomical complexity of EFNs ranges from simple epidermal domes to multi-layered secretory complexes with specialized vascularization, parenchyma, and secretory pores.
• In many species, EFNs appear to be serially homologous to leaf teeth, reflecting shared developmental origins but diversification into distinct morphologies.

Anatomical studies using paraffin sections, histological staining, and scanning electron microscopy have documented these structures across diverse species and subgenera of Passiflora, revealing both conserved and highly derived forms of EFN development.

SEM images of Passiflora matthewsii extrafloral nectary/leaf teeth development from the petiole upwards onto the leaf margins.

Evolutionary Context and Homology

• EFNs are distributed across nearly all major clades within Passiflora, with multiple transitions between different EFN types.
• Evidence suggests that some EFNs are developmentally derived from modified leaf teeth, consistent with broader patterns observed across Malpighiales and the “Salicoid” clade.
• Certain EFNs have likely arisen multiple times independently through convergent evolution, while others may represent deeply homologous structures conserved across evolutionary time.
• Our group is developing hypotheses of EFN homology that incorporate both ontogenetic data and phylogenetic distribution across the genus.

Egg Mimicry and Defensive Roles

In addition to recruiting ants through EFN secretion, some Passiflora species have evolved highly specialized egg mimic structures that serve to deter oviposition by butterflies (e.g. Heliconius spp.):

• These structures often mimic the size, shape, and color of butterfly eggs, discouraging further egg laying and reducing herbivory.
• Egg mimics are sometimes derived from aborted floral buds, modified stipules, petiolar structures, or laminar EFNs themselves.
• Anatomical studies show that these mimetic structures frequently contain secretory cells, carbohydrate-rich tissues, and calcium oxalate crystals, linking their formation to EFN-related developmental pathways.

The close integration of EFNs and egg mimics reflects the highly adaptive nature of Passiflora defense strategies and the evolutionary flexibility of these glandular structures.

Molecular and Genomic Approaches

To complement anatomical and developmental studies, our research incorporates molecular tools aimed at identifying the underlying genetic mechanisms controlling EFN formation:

• Comparative transcriptomics (RNA-seq) is being used to compare gene expression between tissues with and without EFNs across multiple species.
• Early results suggest a combination of conserved genetic pathways shared across lineages, as well as lineage-specific gene recruitment that contributes to the morphological diversity of EFNs.
• These data will ultimately allow us to test models of developmental homology, gene network evolution, and adaptive innovation in EFN development.

Collaborators

Our research group collaborates closely with:

• Dr. David Hearn (Towson University) — Comparative genomics, EFN development, and phylogenetic context.
• Dr. Bikash Shrestha (JGI) — Transcriptomics and bioinformatics support.
• Dr. John MacDougal (Missouri Botanical Garden) — morphological variation and phylogenetic context.

Broader Significance

This research contributes broadly to multiple areas of plant biology:

• Evolutionary Developmental Biology (Evo-Devo): Understanding how novel structures arise through shifts in gene networks.
• Plant-Insect Interactions: EFNs mediate complex mutualistic and defensive relationships that affect plant fitness and survival.
• Comparative Morphology: The tremendous diversity of EFNs in Passiflora provides a model for studying serial homology, modularity, and evolutionary convergence.
• Systematics and Phylogenetics: Documenting EFN evolution informs broader questions of character evolution within Malpighiales.