Evolutionary association of stomatal traits with leaf vein density in Paphiopedilum, Orchidaceae.

BACKGROUND: Both leaf attributes and stomatal traits are linked to water economy in land plants. However, it is unclear whether these two components are associated evolutionarily. METHODOLOGY/PRINCIPAL FINDINGS: In characterizing the possible effect of phylogeny on leaf attributes and stomatal traits, we hypothesized that a correlated evolution exists between the two. Using a phylogenetic comparative method, we analyzed 14 leaf attributes and stomatal traits for 17 species in Paphiopedilum. Stomatal length (SL), stomatal area (SA), upper cuticular thickness (UCT), and total cuticular thickness (TCT) showed strong phylogenetic conservatism whereas stomatal density (SD) and stomatal index (SI) were significantly convergent. Leaf vein density was correlated with SL and SD whether or not phylogeny was considered. The lower epidermal thickness (LET) was correlated positively with SL, SA, and stomatal width but negatively with SD when phylogeny was not considered. When this phylogenetic influence was factored in, only the significant correlation between SL and LET remained. CONCLUSION/SIGNIFICANCE: Our results support the hypothesis for correlated evolution between stomatal traits and vein density in Paphiopedilum. However, they do not provide evidence for an evolutionary association between stomata and leaf thickness. These findings lend insight into the evolution of traits related to water economy for orchids under natural selection.

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy.

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m⁻² s⁻¹, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.

Leaf anatomical structures of Paphiopedilum and Cypripedium and their adaptive significance.

Paphiopedilum and Cypripedium are closely related in phylogeny, but have contrasting leaf traits and habitats. To understand the divergence in leaf traits of Paphiopedilum and Cypripedium and their adaptive significance, we analyzed the leaf anatomical structures, leaf dry mass per area (LMA), leaf lifespan (LL), leaf nitrogen concentration (N (mass)), leaf phosphorus concentration (P (mass)), mass-based light-saturated photosynthetic rate (A (mass)), water use efficiency (WUE), photosynthetic nitrogen use efficiency (PNUE) and leaf construction cost (CC) for six species. Compared with Cypripedium, Paphiopedilum was characterized by drought tolerance derived from its leaf anatomical structures, including fleshy leaves, thick surface cuticles, huge adaxial epidermis cells, lower total stoma area, and sunken stomata. The special leaf structures of Paphiopedilum were accompanied by longer LL; higher LMA, WUE, and CC; and lower N (mass), P (mass), A (mass), and PNUE compared with Cypripedium. Leaf traits in Paphiopedilum helped it adapt to arid and nutrient-poor karst habitats. However, the leaf traits of Cypripedium reflect adaptations to an environment characterized by rich soil, abundant soil water, and significant seasonal fluctuations in temperature and precipitation. The present results contribute to our understanding of the divergent adaptation of leaf traits in slipper orchids, which is beneficial for the conservation of endangered orchids.