Figure 1
Species of Gardenieae and floral characteristics. a, j, m. Tocoyena formosa. b-c. Cordiera concolor. d-e, k-o. Genipa americana. f-g. Randia calycina. h-i. R. heteromera a, j, m. PF. b, d, f, h, k, n. FSF. c, e, g, i, l, o. FPF. j, Detail of a PF showing the pollen deposited on the stigma. k,, Detail of a FSF showing the pollen deposited on the non-functional stigma; above, portion of a dehiscent anther with pollen still present. l, Detail of a FPF; above, portion of a staminode, note the aborted tissue inside the thecae (arrow). m, Longitudinal section (LS) of PF showing a nectary around the style base. n, LS of inferior ovary of a FPF (the style has been removed). o, LS of the rudimentary ovary of a FSF. Abbreviations: n= nectary; ns= non-functional stigma, ov= ovary, ro= rudimentary ovary, s= stamen, sd= staminode, sg= stigma, st= style. Scales: a, d-i= 1 cm; b-c, k-l, n-o= 5 mm; m= 4 mm.
Figure 2
Cross section of anthers from the microspore mother cell stage (mmc) to microspore stages. a-e j, Randia calycina. f, h, l, o, p, Cordiera concolor. g, Tocoyena formosa. I, m, r, Genipa americana. k, n, q, Randia heteromera a, Anther (FSF). b, Portion of the anther where the mmc and several parietal strata are distinguished (FPF). c, mmc stained with aniline blue and observed under a fluorescence microscope (FSF). d, Septum and stomium region (FPF). e, Detail of the vascular bundle and cells with druses in the connective tissue. f, Portion of the connective tissue showing stomata, druses, cells with tannin in the epidermis and parenchyma (FPF). g, Detail of the placentoid (parenchyma + tapetum), the pollen sac is divided into two regions (*) due to the invasion of the placentoid. h, Detail of the anther showing: epidermis with tannin; tapetal cells with a large vacuole and the central mmc (*) in meiosis (FSF). i, Detail of an anther wall (FPF), the nuclei of the tapetal cells have several nucleoli (arrows). j, Tapetal placentoid in FSF. k, Tapetal placentoid in FPF. l,o-p,, Cordiera concolor. n, q, Randia heteromera m, r, Genipa americana. l, Detail of the mmc in meiosis surrounded by uninucleated tapetum. m, Decused tetrad. n, Tetrahedral tetrad stained with aniline blue and observed with fluorescence microscope. o, Cross section of anther showing wall layers during stage 3. p, Free microspore; the nucleus is displaced by a large vacuole. q, Microspores joined together to form permanent tetrads. r, Anther wall with the cytoplasm of the tapetum cells contracted. Abbreviations: ca= calose; cn= connective tissue; dr= druse; en= endothecium; ep= epidermis, ml= middle layers; mmc= microspore mother cell; pl= placentoide; se= septum; sr= stomiun region; st= stomata; tn= tannin; tp= tapetum; vb= vascular bundle. Scales: a-k, o, r= 50 µm,l-n, p-q= 10 µm.
Figure 3
Cross section of anthers with pollen grains (a-i) and in a dehiscence stage (j-m), and cross section of anthers of FPF in developmental arrest stages. a, Randia heteromera (FSF). b-d, j, Tocoyena formosa (PF). e-g, l-m, Cordiera concolor (FSF). h, Genipa Americana (FSF). i, k, Randia calycina (FSF). a, Permanent tetrads, in one pollen grain the vegetative and generative cells can be observed. b, Pollen grain with oncus (arrows) that protrudes from each aperture. c, Multilayered endothecium and placentoid, both with fibrous thickenings. d, Fusion of pollen sacs by the disintegration of the innermost cells of the stomium region. e, Septum with fibrous thickenings, no fusion of locules. The cells of the stomium have collapsed. f, Anther wall showing orbicules in the tapetal membrane. g, Connective cells with starch grains (stained with Lugol) and tanniferous idioblastos (stained with ferric chloride). h, Septum with fibrous thickenings, no fusion of locules. i, The cells of the septum collapse and disintegrate; the pollen sacs fuse in a common locule. j-k, Dehiscent anthers, note in j the placentoid with fibrous thickenings (right portion with polarized light). l, Pollen grain released at the 2-cell stage. m, Pollen grain released at the 3-cell stage. n-o, Cordiera concolor. p-r, Genipa americana. s-u, Randia calycina. v-x, Randia heteromera n, One pollen sac with tetrads and collapsed tapetum, the middle layers have enlarged cells. n’, Detail of a collapsed tetrad. 0, Pollen sacs of one theca with aborted tissues, endothecium with thickening (right portion with polarized light: PL). p-p’, mmc at different stages of meiosis (*). q, One pollen sac with tapetum and mmc cells aborted, middle layers of greater volume. r, Endothecium with thickening, collapsed stomium and linear and closed pollen sacs (image on the right with PL). s, The tetrads formed in the pollen sac begin to collapse. s’, Tetrads surrounded by callose. t, Linear pollen sac with aborted tetrads, the tapetum and placentoid cells have contracted cytoplasm, the middle layers are still present and the endothecium cells are voluminous. u, Endothecium with fibrous thickenings, collapsed tapetum, and pollen sacs of one theca nearby (image on the right with PL). v, the mmc in the pollen sac begin to collapse, the cytoplasm of the tapetum begins to contract. w, The cells of the tapetum and the placentoid with completely contracted cytoplasm, compressed pollen sac, and aborted mmc. x, Completely collapsed theca; linear pollen sacs with aborted cells inside. Abbreviations: at= aborted tissue; en= endothecium; ge= generative cell; ml= middle layers; mmc= microspore mother cell; or= orbicules; pl= placentoid; sa= starch grains; se= septum; sp= sperm cell; sr= stomium region; te= tetrad; tp= tapetum. vb= vascular bundle; ve= vegetative cell. Scales: a-b, l-m, n’, p-p’, s’= 10 µm; c-k, n, o, q-s, t-x= 50 µm.
Figure 4
Detail of pollen sacs with placentoid, orbicules (of Cordiera concolor), and pollen grains. a-d’, v-x’, LM. e-u, SEM. a-d, j, o, t , Randia calycina. a’-d’, g, l, q, Tocoyena formosa. e-f, h, m, r, Cordiera concolor. i, n, s, Genipa americana. k, p, u, Randia heteromera a-d, asterisks points to the placentoid (tapetal). a’-d’, asterisks points to the placentoid (tapetal and parenchymatic). a, Young placentoid b, mature placentoid invading the pollen sac. c, Collapsed placentoid. d, Pollen sac of a mature anther without placentoid. a’, Young placentoid, both the tapetal and parenchymal portions are well developed. b’, Mature placentoid dividing the pollen sac in two. c’, The tapetal portion of placentoid is collapsed. d’. The parenchymatic portion of placentoid develops thickenings along with the endothecium. e, General view of the orbicules. f, Detail of orbicules forming aggregates. g-k, General view of pollen grains. l-p, Detail of the exine. q-u, Detail of the aperture. v-x’ Non-acetolized pollen grains with pollenkitt, v-x, Genipa americana, monads. v’-x’, Randia heteromera, permanent tetrads. v, v’, Fresh pollen grain with pollenkitt (arrow). w, w’, Pollen grain stained with Lugol; negative reaction of pollenkitt (arrow). x, x’ Pollen grains stained with Sudan III; positive reaction in pollenkitt (arrow). Heteromera Scales: a-d’=50 µm; e-f= 2 µm; g-u= 5 µm; v-x’=10 µm.
Figure 5
a-o, Mechanism of secondary pollen presentation. p, Hypothetical evolutionary tendency of losses of tissues of anthers in Rubiaceae taking into account an anther with normal development of its tissues versus abnormal anthers. a-c, Representative outlines of the stages of the presentation mechanism in lateral (left) and apical (right) views. a, Phase 1 (young bud). b, Phase 2 (pre-anthesis at beginning of the floral anthesis). c, Phase 3 (post anthesis). d-f, Pollen is presented on the stigma surface. d, Genipa americana, e, Randia calycina, f, Randia heteromera g-k, Cordiera concolor, pollen is presented on the style surface. h, Lateral view of the style with pollen (fixed material). i, Lateral view of the style without pollen, note the furrows on its surface (fixed material). j, Detail of the surface of the style with trichomes. k, Detail of the surface of style close to a furrow where the pollen grains are retained (SEM). l-o, Protandry in Tocoyena formosa. l-m, Phases 2 and 3 of secondary pollen presentation, stigma with pollen on the surface. n, The pollen was removed by the pollinators from the stigma surface. o, The branches of stigma unfold. p, At the left, the anthers of the FSF or PF with a normal development of their tissues and pollen are represented, while the abnormal anthers of the FPF are represented to the right. The top row corresponds to anthers when pollen development stops (stage X) and the bottom row corresponds to indehiscent anthers with collapsed pollen sacs and aborted tissue. Abbreviations: en= endothecium without thickenings; et= endothecium with thickenings; ml= middle layers; mmc= microspore mother cells; po= pollen; sg= stigma; st= style; tp= tapetum. Scales: d-g, 1 mm; h-i, 0.5 mm; j, 0.2 mm; k, 20 µm; l-o, 2 mm.
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