A cactus survives harsh heat and drought with proteins that may act like tiny gates: their main structure stays stable, while the entrances subtly change what can pass through. Cereus has a broad evolutionary history, but the diversification and structural variation of gene families linked to stress-related processes remain poorly understood.

These proteins have recognized roles in lipid binding, transport, and stress responses in other plant lineages, yet they have not been investigated in this cactus genus. The central question is whether sequence variation gathers around the predicted lipid-binding cavities while the overall structural scaffold remains conserved.

The proteins keep the typical overall fold, while most variation sits in loop regions surrounding the internal binding cavity. It is like a house whose frame stays firm while the shape of a doorway changes. The pattern suggests functional flexibility without compromising the stable scaffold.

The evolutionary picture also shows frequent duplication and loss, along with copies found only in particular species. The alignment shows eight conserved cysteines characteristic of plant lipid-transfer proteins. In contrast, residues between the cysteines display greater variability, particularly in loop segments and solvent-exposed surfaces.

All the proteins retain the characteristic water-repelling tunnel found in plant lipid-transfer proteins. But predicted binding energies vary with both the lipid and the local shape of the pocket, indicating that the cavities are not energetically equivalent across the protein variants.

Copies from different Cereus species are extensively intermingled in the evolutionary tree rather than forming separate species-specific groups. That pattern indicates that much of the family’s diversification predates the spread of the living Cereus species, with many duplicated copies retained across descendant lineages.

Other groups contain multiple copies from the same species, revealing more recent duplications added to this older pattern. Evidence of episodic positive selection appeared at eighteen sites, and nine reached the stated significance threshold. The pressure was not spread evenly across the whole gene; it appeared locally in particular branches or lineages.

These results identify candidate sites associated with sequence diversification, rather than definitive targets of adaptive evolution, and their functional consequences remain unknown. The results should therefore be treated as hypotheses about evolutionary diversification, not direct evidence of ecological adaptation or physiological specialization.

The observed sequence diversification occurred through multiple independent events after lineages separated, rather than through selection acting across all copies. The candidate sites provide a rational starting point for future biochemical and functional studies, because their biological significance cannot be determined from sequence analysis alone.

The Cereus proteins keep a sturdy shared framework but vary around their internal passageways. That points to possible differences in lipid handling, while leaving the real effects to future experiments.