Rincón Baron Edgar Javier, Rolleri Cristina Hilda, Alzate Guarin Fernando, Dorado Gálvez Jacinta Mireya
Rev Biol Trop. 2014 Mar;62(1):273-98.
Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5 microm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5 microm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.
石松科生殖方面的研究在科学文献中并不丰富，但这些研究构成了支持该类群分类和系统发育关系的重要信息。在此，我们对孢子囊的个体发育和孢子发生进行了详细研究，并对孢子形成过程中产生的几种化合物进行了化学测定。分析在该科六个属的14个分类群中进行，这些属包括扁枝石松属、双扇石松属、石杉属（本文将石松属中的马尾杉属也归入该属）、小石松属、石松属和垂穗石松属。标本采自哥伦比亚安第斯山脉的三个省份，海拔在1454 - 3677米之间。个体发育研究使用的是长1厘米的小型球果和茎段，将其固定在戊二醛或FAA中，用酒精脱水，包埋在LR White中，切成0.2 - 0.5微米厚的切片，并用甲苯胺蓝（TBO）染色，甲苯胺蓝是一种异染性染料，在这些过程中可检测孢粉素和木质素或其前体。对于其他研究，用番红 - 固绿和阿尔辛蓝 - 苏木精对包埋在石蜡中的切片（3 - 5微米）进行染色。还使用阿尔辛蓝（用于检测粘多糖）、卢戈氏碘液（用于检测淀粉）、苏丹III（用于检测脂质）、间苯三酚（用于检测木质素）和地衣红（用于检测染色体）对处于不同成熟阶段的新鲜孢子囊切片进行化学测试。切片用配备了微分干涉对比（DIC）的光学显微镜和荧光显微镜（用于观察未染色的孢子和孢子囊壁）进行观察。球果和孢子囊用2,2 - 二甲氧基丙烷脱水，临界点干燥，然后镀金用于扫描电子显微镜（SEM）观察。我们的结果表明，孢子囊的个体发育和孢子发生与之前在短叶石杉中观察到的非常相似。角质化发生在孢子囊细胞壁发育的早期阶段，但在其最后阶段细胞壁会木质化。至于孢子壁的发育，外壁是形成的第一层，由孢粉素组成。内壁沉积为一层由纤维素、果胶和羧化多糖组成的薄内层。周壁（如果存在）最后沉积。在孢子母细胞表面发现了粘多糖，其在孢子囊腔内的丰度一直持续到未成熟四分体阶段，然后消失。脂质在孢子母细胞、四分体和孢子中含量丰富，是孢子的主要能量来源。相比之下，在孢子中未检测到淀粉，但在减数分裂前的孢子母细胞和未成熟四分体中含量丰富，这些是细胞代谢活动较高的发育阶段。固有荧光证实了孢子囊壁中存在木质素和角质，而孢粉素仅限于外壁。转输细胞和周壁并非总是存在。然而，在所研究的所有分类群中，个体发育和孢子发生过程极其相似，这表明它们代表了该科保守的特征，而非特定属的特征。
