為二氧化矽而非其他物質所堆積而成。可見於碎米蕨支的戟葉黑心蕨 (Doryopteris
鳳尾蕨科的一般表皮細胞依據其細胞長軸的有無、細胞軸向以及分支狀態可 屬 (Actiniopteris)、粉葉蕨屬 (Pityrogramma) 以及部分的書帶蕨 (vittarioid)；該類 型與矽異形細胞之形態相似。
(三) 脈上表皮細胞形態 (Tables 8, 9; Figure 20; Appendix 4) brunoniana)、翠蕨 (Anogramma leptophylla) 以及部分之金粉蕨 (Onychium)，則呈 現小羽片中肋之資料。
由於脈上表皮細胞之變化性相對較一般表皮細胞小，若脈上細胞之形態與一般 表皮細胞形態具有明顯不同，則必為具有單一明顯之主軸，且較一般表皮細胞長、
寬度窄且邊緣突起相對不明顯之細胞，因此在此僅將脈上表皮細胞概略分成三 型。(1) 短，與一般表皮細胞形態相同 (short, non-differentiate)；(2) 長，與一般表 皮細胞形態明顯不同 (long, differentiate)；(3) 長，與一般表皮細胞形態相同 (long, non-differentiate)
蕨屬、部分的鐵線蕨屬以及書帶蕨類群的物種之中，而該特徵與散布葉肉間的脈 間矽異形細胞具有相當的關連性。
(四) 特徵演化歷史重建
從利用 Schuettpelz et al. (2007) 六十七種鳳尾蕨科植物之 rbcL、atpB 以及 atpA 三段質體基因所建構之親緣關係樹，重建之表皮二氧化矽堆積形式與脈上表 皮細胞形態之演化歷史 (Figure 21) 中可見，鳳尾蕨科內具有矽異形細胞的類群集 在中鐵線蕨支以及鳳尾蕨支之中，然而，此二分類群之矽異形細胞應為不同起源 之構造，且這兩支內之矽異形細胞均可能具有多次起源；而若將表皮二氧化矽堆 積的特徵則視為具次序性 (ordered) 的，則鐵線蕨支之矽異形細胞為一次起源，而 鳳尾蕨支之矽異形細胞仍可能為多次起源。脈上表皮細胞形態之演化歷史則顯示 在碎米蕨支以及水蕨支之中，脈上表皮細胞分別變成了短且與一般表皮細胞形態 相同之類型，而這兩支則沒有矽異形細胞出現。相對的在鐵線蕨支以及鳳尾蕨支 之中維持了長形的脈上表皮細胞的模式，而這兩支中多數的物種均具有脈上矽異 形細胞；而脈間表皮細胞則常伴隨著長且與一般表皮細胞形態相同的脈上表皮細 胞出現。
Table 6. Four epidermal silica deposition types in Pteridaceae
Character Criteria Taxa
Silica deposition
Type I 1. No silica deposit or not consistently deposit at a specific region, no spicular cell
2. If there have silica deposition, it often deposit in outer tengential wall of epidermal cells, stomata, or wounded region
CR clade, CE clade, and most of CH clade species Anogramma and Taenitis in PT cldae
Type II 1. No spicular cell
2. Silica consistently deposit at tips of margin lobes of epidermal cells
3. Silica might also deposit in outer tengential wall of epidermal cells, stomata, or wounded region
Some species in CH clade
Some species in genus Pteris in PT clade
Type III 1. No interveinal spicular cell (ISC)
2. Have veinal spicular cells (VSC) or marginal spicular cells (MSC) or both 3. Some species also have silica deposits at tips of margin lobes of epidermal cells
4. Silica might also deposit in outer tengential wall of epidermal cells, stomata, or wounded region
Onychium and Pityrogramma in PT clade
Most of species species in Adiantum in AD clade and Pteris in PT clade
Type IV 1. Have interveinal spicular cells (ISC) 2. Have veinal spicular cells (ISC)
3. Some have marginal spicular cells (MSC) or have silica deposits at tips of margin lobes of epidermal cells
4. Silica might also deposit in outer tangential wall of epidermal cells, stomata, or wounded region
Vittarioids in AD clade and Actiniopteris in PT clade Some species in Adiantum in AD clade and Pteris in PT clade
Table 7. Epidermal silica deposition types of different taxa in Pteridaceae
Clade / Genus / Species Species sampled Type I Type II Type III Type IV Silica deposition type Cryptogrammoids (CR clade) 珠蕨支
Coniogramme Fée 鳳ㄚ蕨屬 3 3 - - - I
Cryptogramma R. Brown 珠蕨屬 2 2 - - - I
Ceratopteridoids (CE clade) 水蕨支
Acrostichum L. 鹵蕨屬 1 1 - - - I
Cheilanthoids (CH clade) 碎米蕨支
Aleuritopteris Fée 粉背蕨屬 3 3 - - - I
Characters in () only found in a few species in that genus
Figure 16. Epidermal silica deposition types in Pteridaceae (1): Type I & II. These two types do not have spicular cells. (A-C) Type I, no silica deposit. (A) Cryptogramma stelleri in CR clade. Bar = 100 μm. (B) Ceratopteris thalictroides in CE clade. Bar = 100 μm. (C) Cheilanthes chusana in CH clade. Bar = 100 μm. (D-F) Type II, silica deposit at tips of cell margin lobes. (D) Pteris semipinnata in PT clade. Bar = 100 μm. (E) Doryopteris ludens in CH clade. Bar = 100 μm. (F) Mildella henryi in CH clade. Bar = 100 μm.
Figure 17. Epidermal silica deposition types in Pteridaceae (2): Type III and IV. These two types have spicular cells.
(A-C) Type III, have veinal spicular cells (VSC) but no interveinal spicular cells (ISC). (A) Pityrogramma calomelanos in PT clade. Bar = 500 μm. (B) Pteris biaurita in PT clade. Bar = 500 μm. (C) Adiantum formosanum in AD clade. Bar = 500 μm. (D-F) Type IV, have ISC. (D) Actiniopteris radiata in PT clade. Bar = 100 μm. (E) Adiantum caudatum in AD clade. Both of the VSC and ISC present in this figure. Bar = 500 μm. (F) Haplopteris zosterifolia in AD clade. Bar = 500 μm.
Figure 18. EDX element analysis of some species with special silica deposition pattern. (A-F) Mildella henryi. Bars
= 50 μm. (A-C) Silicon and oxygen distribution of in epidermis. (A) Secondary electron image. The concave regions are cell margins. (B) Silicon mapping. The silicon signals are showed in bright dots, which aggregated at cell margin.
(C) Oxygen mapping. The oxygen signals are showed in bright dots. (D) Backscattered electron image (BEI).
Spectrum 5 and spectrum 6 are the dots sampled for element analysis. Spectrum 5 is at cell corner (white regions in
Spectron 5 Spectron 5 – Spectron 6
Spectron 9
Si
S Ca
Spectron 8 – Spectron 9
Table 8. Types of ordinary epidermal cell and veinal epidermal cell morphology in Pteridaceae
Character Criteria Taxa
Ordinary epidermal cell morphology
Stellate (S) 1. No significant main axes 2. No specific orientation
3. Cell margin often deeply lobed, but there have no ornamentations on the lobes
Acrostichum in CE clade
Most of species species in CH clade
Curve (C) 1. Have significant main axes, but often curved 2. No specific orientation
3. Often have lobate cell margin
Some species scattered in CE, CR, and CH clade
Simple elongated (E) 1. Have significant main axes
2. Long axes parallel to the lateral veins or midrib 3. Cell margin smoothly
Actiniopteris and Pityrogramma in PT clade Some vittarioid species in AD clade
Elongate sinuate (ES) 1. Have significant main axes
2. Long axes parallel to the lateral veins or midrib 3. Cell margin sinuate
Commonly found in CR clade and AD clade Some species in CH clade
Elongate crenate (EC) 1. Have significant main axes
2. Long axes parallel to the lateral veins or midrib 3. Cell margin crenate
Commonly found in PT clade and AD clade
Branched (B) 1. Except the main axes, lateral branches (deep marginal incisions with ornamentations) present
2. Cell margin often crenate
Pteris and Taenits in PT clade
Veinal epidermal cell morphology
Short, nondifferentiate (SN) 1. Cell short (length : width < 3 : 1)
2. Cell morphology have no significant different from ordinary epidermal cells
Most species in CE and CH clade
Long, differentiate (LD) 1. Cell long (length : width > 3 : 1)
2. Cell morphology have significantly different from ordinary epidermal cells
Coniogramma in CR clade, Pteris in PT clade, Adiantum in AD clade and some other species
Long, nondifferentiate (LN) 1. Cell long (length : width > 3 : 1)
2. Cell morphology have no significant different from ordinary epidermal cells
Cryptogramma in CR clade, Actiniopteris and Pityrogramma in PT clade, vittarioids in AD clade and some other species
Table 9. Epidermal characters of different taxa in Pteridaceae
Clade / Genus / Species Species sampled S C E B Epidermal cell type SN SD LD LN Vein epidermis type Stomata complex type Trichome type Cryptogrammoids (CR clade) 珠蕨支
Coniogramme Fée 鳳ㄚ蕨屬 3 - - 3 3* E - - 3 - LD anomocytic, polocytic clavellate
Cryptogramma R. Brown 珠蕨屬 2 - 1 1 2* E, C - - - 2 LN anomocytic clavellate
Ceratopteridoids (CE clade) 水蕨支
Acrostichum L. 鹵蕨屬 1 1 - - 1* S 1 - - - SN anomocytic clavellate
Cheilanthoids (CH clade) 碎米蕨支
Aleuritopteris Fée 粉背蕨屬 3 3 - - - S 3 - - - SN anomocytic glandular
Cheilanthes Swartz 碎米蕨屬 2 - - 2 - E 2 - - - SN anomocytic clavellate, (wolly)
Cheilanthes viridis (Forssk.) Prantl 1 - - 1 - E - - 1 - LD anomocytic clavellate
Doryopteris J. Smith 黑心蕨屬 1 1 - - - S 1 - - - SN anomocytic clavellate
Doryopteris ludens (Wall. ex. Hook.) J. Smith 戟葉黑心蕨 1 1 - - - S 1 - - - SN anomocytic clavellate
Figure 19. Types of ordinary epidermal cell shapes in Pteridaceae. (A) Stellate (S). Aleuritopteris argentea. Bar = 100 μm. (B) Curved (C). Cryptogramma stelleri. Bar = 100 μm. (C) Simple elongated (E). Haplopteris zosterifolia.
Bar = 100 μm. (D) Elongate sinuate (ES). Cheilanthes chusana. Bar = 100 μm. (E) Elongate crenate (EC).
Adiantum wangii. Bar = 100 μm. (F) Branched (B). Pteris longipes. Bar = 100 μm.
Figure 20. Veinal epidermal cell morphology and some other epidermal characteristics in Pteridaceae. (A) Short, not significant different from ordinary epidermal cells (SN). Aleuritopteris krameri. Bar = 100 μm. (B) Long, significant different from ordinary epidermal cells (LD). Coniogramme intermedia. Bar = 100 μm. (C) Long, not significant different from ordinary epidermal cells (LN). Actiniopteris semiflabellata. Bar = 100 μm. (D) Sunken stomata. Adiantum edgeworthii. Bar = 100 μm. (E) In Adiantum, veinal spicular cells (VSC) often interrupted by a
Figure 21. Phylogenetic relationships of taxa with different epidermal silica deposition type and veinal epidermal cell morphology in Pteridaceae. The maximum parsimony character evolution history was reconstructed by unordered model of Mesquite Version 2.6. (Maddison and Maddison, 2009). Tree modified from Schuettpelz et al.
(2007), based on plastid rbcL for 147 species. Only the branches indicate good posterior probability ( 0.95) ≧ or maxium likelihood ( 70) ≧ support was showed and most of the branches indicate good support from both of these two measurements. VSC: veinal spicular cell; ISC: interveinal spicular cell.
肆、討論
4% 之氫氧化鈉分別溶出葉綠素以及其它的胞內物質，通常浸泡至材料呈現透明狀 即可，依材料不同需歷時數天至數週不等，於光學顯微鏡下可清楚觀察到葉片內 各層細胞。
經透明法處理過的材料，可利用偏光顯微鏡進行觀察。在過去的研究中，以 特定的半偏光角度，能得到具有最佳顏色對比之影像 (Parry and Smithson, 1958;
Dayanandan, 1983)。在本研究中，於翅柄鳳尾蕨的觀察即相當成功，其矽異形細胞 為單矽酸 (monosilicic acid, Si(OH)4) 經聚合化 (polymerized) 所形成的非晶質二 氧化矽水合物 (hydrated amorphous silica) 所構成，基本上，其不具晶型，而僅有 矽酸體表層較緊密排列的部份具有偏光特性。在本研究預先測試的物種中，並非 所有種類皆能穩定的表現出偏光的特性。此外，利用濕式灰化法萃取矽酸體時，
偏光的特性亦會隨酸處理的時間加長而消退。因此，本研究沒有選取此法觀察鳳 尾蕨科內各分類群之矽異形細胞。
透明法的材料亦可利用染色的方式觀察其矽酸體，前人研究曾應用的染劑有 鹼性洋紅 (basic fuchsin)、孔雀石綠 (malachite green)、酚類 (phenol) (Molisch, 1923)、龍膽紫 (gentian violet)、亞甲基藍 (methylene blue) 以及番紅 (safranin) (Netolitzky, 1929)；上述的染劑雖多少有區別矽酸體之能力，然而專一性不佳。因 此，Dayanandan 等 (1983) 建議使用對矽醇基 (silanol group, SiOH) 具有專一性的 染劑 SAC (Silver-Ammine Chromate)、CVL (Crystal Violet Lactone) 以及 MR (Methyl Red)。然而，此三種染劑於本研究的應用上分別面臨了各自的問題。
CVL 以及 MR 應用於矽酸體染色，快速且美觀，但應用於透明法的材料時，
卻有染劑難以滲透之問題。在初步的測試中，需要染色數天才能呈色至拍照後能 夠辨識之程度。若依據發表文獻 (Dayanandan et al., 1983) 中利用硫酸進行前處 理，則不同材料的處理時間難以掌握且非專一性染色增加。因此，組織染色法雖 成的骨架。此外，乾式灰化法 (dry ashing or spodogram) (Molisch, 1923) 是利用高 溫燒去矽酸體以外有機的部份而取得矽酸體，然而，此法可能造成矽酸體形變 常用之研究法，而類似的方法也不斷的創新，如冷式灰化法 (cold plasma ashing,
積的材料並判斷矽酸體之分布。此外，利用背向散射電子 (backscattered electron) (Brandenburg et al., 1985) ，因材料具不同平均原子量而會接收到不同強度訊號之 特性區分矽異形細胞與一般表皮細胞。
Table 10. Comparison of different spicular cell studying methods used in this study
Method Cost Sample
preparation time Equipment
requirement Sensitivity Specificity Major advantages / Main usages in this study Stereomicroscope no no low - - A conventional observing method, fast and cheap
Epidermis impression very low short low - - A less harmful method to plant, used on tracing spicular cell development Paraffin section low long middle - - Observing internal structures of leaves
Clearing low middle low - - Cheap, easily manipulate, capable storage for long time, and compatible to other microscopic techniques or staining methods.
Clearing + Polarization low middle middle fine fine Cheap, easily manipulate, used on observing the distribution of spicular cells in epidermis and used as a supplement data for cell morphology observations Clearing + Histochemical staining low middle low bad good Double check the presence of silica in spicular cells
Wet oxidation low middle low - - Cheap, capable manipulating mass of samples at the same time and can store for long time. A conventional method in archeological studies. Good for observing silica body morphology
Wet oxidation + Histochemical staining
low middle low good good As above
Wet oxidation + SEM high middle high - - Good for observing the details and 3D structure of silica bodies
Ordinary SEM high long high - - Observing the epidermal cells morphology and epidermis topology, high resolution SEM + EDS very high long very high fine good One of the conventional methods in checking the component of an unknown structure
or the distribution of some specific elements; A reliable method for check the element