Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E. puniceoluteum (Orchidaceae).

The hypothesis of gene flow between species with large differences in chromosome numbers has rarely been tested in the wild, mainly because species of different ploidy are commonly assumed to be reproductively isolated from each other because of instantaneous and strong postzygotic barriers. In this study, a broad-scale survey of molecular variation was carried out between two orchid species with different ploidy levels: Epidendrum fulgens (2n = 2x = 24 chromosomes) and Epidendrum puniceoluteum (2n = 4x = 52 chromosomes). To test the strength of their reproductive barriers, we investigated the distribution of genetic variation in sympatric and allopatric populations of these two species and conducted crossing experiments. Nuclear and plastid microsatellite loci were used to genotype 463 individuals from eight populations across the geographical range of both species along the Brazilian coastal plain. All six sympatric populations analysed presented hybrid zones, indicating that hybridization between E. fulgens and E. puniceoluteum is a common phenomenon. Bayesian assignment analysis detected the presence of F(1) and F(2) individuals and also signs of introgression, demonstrating a high potential for interspecific gene flow. Introgression occurs preferentially from E. fulgens to E. puniceoluteum. Pure parental individuals of both species display strong genotype-habitat associations, indicating that environment-dependent selection could be acting in all hybrid zones. This study suggests that hybridization and introgression are evolutionary processes playing a role in the diversification of Epidendrum and indicates the importance of investigations of hybrid zones in understanding reproductive barriers and speciation processes in Neotropical orchid species.

Endogenous hormonal levels and growth of dark-incubated shoots of Catasetum fimbriatum.

Apical shoots and Lateral buds of the epiphytic orchid Catasetum fimbriatum give rise to rootless etiolated stolons, when cultured in the presence of light and then transferred to the dark. The stolons are characterized by fast and continuous apical longitudinal growth. Measurements of four endogenous cytokinin, indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in etiolated shoots and light-grown plants were low. However, after transfer of green plants to the dark, cytokinin Levels increased 3- and 7-fold by 10 and 30 days of incubation, respectively. IAA levels also increased significantly, but the increase was not as great as for cytokinins. A similar trend was observed in the roots. A close relationship seems to exist between both cytokinin accumulation and the formation of etiolated stolons. Variations in ABA levels were practically inconspicuous. The presence of paclobutrazol in the medium, a potent inhibitor of gibberellin synthesis, strongly inhibited etiolated and non-etiolated longitudinal shoot growth, although no apparent effect was observed on apical meristem activity.

Phylogeographic structure and outbreeding depression reveal early stages of reproductive isolation in the neotropical orchid Epidendrum denticulatum.

Phylogeographic studies provide an important framework for investigating the mechanisms operating during the earliest stages of speciation, as reproductive barriers can be examined among divergent lineages in a geographic context. We investigated the evolution of early stages of intrinsic postmating isolation among different populations and lineages of Epidendrum denticulatum, a Neotropical orchid distributed across different biomes in South America. We estimated genetic diversity and structure for both nuclear and plastid markers, using a haplotype network, differentiation tests, Bayesian assignment analysis, and divergence time estimates of the main lineages. Reproductive barriers among divergent lineages were examined by analyzing seed viability following reciprocal crossing experiments. Strong plastid phylogeographic structure was found, indicating that E. denticulatum was restricted to multiple refuges during South American forest expansion events. In contrast, significant phylogeographic structure was not found for nuclear markers, suggesting higher gene flow by pollen than by seeds. Large asymmetries in seed set were observed among different plastid genetic groups, suggesting the presence of polymorphic genic incompatibilities associated with cytonuclear interactions. Our results confirm the importance of phylogeographic studies associated with reproductive isolation experiments and suggest an important role for outbreeding depression during the early stages of lineage diversification.

Dark-induced hormone changes coincide with the resumption of light-inhibited shoot growth in Catasetum fimbriatum (Orchidaceae).

Catasetum fimbriatum plants cultivated in the absence of light exhibit continuous shoot growth leading to the formation of nodes and internodes. On the other hand, when these plants are incubated in the presence of light, shoot longitudinal growth is inhibited and pseudobulbs develop just below the shoot apical meristem. These facts provide evidence of a possible influence of light on mitotic cell division in the shoot apex as well as on pseudobulb initiation. The effects of light and dark on the interruption and/or maintenance of shoot apex mitotic activity and the subsequent formation of pseudobulbs in the sub-meristematic regions were investigated by means of histological and hormonal studies. The interruption of shoot apex development occurred around the 150th d of light incubation and seems to have resulted from the establishment of a strong storage sink in the region of the future pseudobulb, in detriment to the continuous activity of the shoot apical meristem. The reduced total cytokinins/IAA ratio in the apex, mainly due to high levels of IAA, could be a key factor in the interruption of cell divisions. Transfer to the dark brings about the resumption of shoot apex development of plants through the re-entrance of cells in the cell cycle which coincides with a significant increase in the total cytokinins/IAA ratio.