Reproductive development and genetic structure of the mycoheterotrophic orchid Pogoniopsis schenckii Cogn

Abstract

Background: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations.

Results: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations.

Conclusions: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant.

Keywords: Autogamy; Embryology; Fungal hyphae; Genetic diversity; Mycoheterotrophy; Orchid.

Fig. 1

Longitudinal sections of Pogoniopsis schenckii fruits. A General view of the ovary, showing the fertile and sterile valves. B Detail of the fertile and sterile valve, where no dehiscence line is formed. C Median region of the ovary. D Median region of a young fruit. E Ripe fruit, with collapsed cells. Arrows indicate the cell collapse of the epicarp towards the endocarp. F Detail of the placental region of a ripe fruit with collapsed cells. cc = collapsed cells; ie = inner epiderms; ed = endocarp; ep = epicarp; fv = fertile valve; oe = outer epiderms; om = ovarian mesophyll; me = mesocarp; se = seed sv = sterile valve; vb = vascular bundle. Scale bars: 50 µm

Fig. 2

Longitudinal sections of the Pogoniopsis schenckii fruit. A Sterile valve with starch grains and protein granules evidenced by Toluinine Blue. B Placental region showing starch grains evidenced by Lugol. C Region placenta showing protein granules evidenced by Xylidine. Detail of the fruit showing protein granules. D-F Presence of fungal hyphae in the placental region evidenced by double staining with cotton blue and safranin. D Fungal hyphae between cells. E–F Fungal hyphae inside cells. fh = fungal hyphae; pg = protein granules; sc = starch grains. Scale bars: 50 µm

Fig. 3

Transmission microscopy electromicrograph in the mesocarp and placental region of Pogoniopsis shenckii. A Overview of fruit cells with high cytoplasmic content. B Detail of the dense cytoplasm. C Vesicles concentrated in the cytoplasm. D Vesicles scattered in the cell vacuole. E Dense cytoplasm, release of secretion near the cell wall. F-L Fungal hyphae. F Fungal hyphae in the cell of the placental region. G Fungal hyphae in the mesocarp cells. H Fungal hyphae penetrating the cell wall. I-J Detail of the fungal hyphae in the mesocarp cells. K Fungal hyphae in the mesocarp cells of the ripe fruit. L Fungal hyphae in the collapsed cells of the ripe fruit. c = cytoplasm; fh = fungal hyphae; cw = cell wall; v = vesicles. Scale bars: I,J = 500 nm, E,F = 1 µm, B,C = 2 µm, D,G,H,K = 5 µm, A = 20 µm, L = 500 µm

Fig. 4

Schematic model of the participation of fungus hyphae over the life cycle of Pogoniopsis schenckii. A Individual showing different interactions with fungi. In purple, fungal hyphae that interact with the floral stem. In green fungal hyphae that interact with the roots. B Flower ovary. Note that has not interactions with fungal hyphae. C Fruit in development. At this stage, it is possible to observe interaction between the cells of the fruit and fungal hyphae in the placental region. Fungal hyphae are represented in purple. D Ripe fruit showing fungal hyphae. Fungal hyphae are represented in purple. E Ripe fruit dropped on the ground. Fungal hyphae are represented in purple. F Seed released after the fruit disintegrates. At this moment, it is already involved with the fungi that will promote its germination. Fungal hyphae are represented in purple. df = developing fruit; f = flower; fh = fungal hyphae; fs = floral stem; mf = mature fruit; o = ovary; r = root; se = seed; so = soil

Fig. 5

Flowers of Pogoniopsis schenckii. A Population of P. schenckii. B Flower in anthesis. Scale bars: 1 cm