Morphological variations of lobate phytoliths from grasses in China and the south-eastern United States

Abstract. Phytolith analysis of grasses is a useful tool in palaeoenvironmental and archaeobotanical research. Lobate phytolith is one of the most important morphotypes of grass phytoliths. This study describes morphological variations of diagnostic lobate phytoliths and produces a tentative classification scheme based on 250 modern grass species from China and the south‐eastern U.S.A. Eighty‐five grass species were found to contain lobate phytoliths. They are derived mainly from Panicoideae, but also include the Chloridoideae, Oryzoideae and Arundinoideae subfamilies. Twenty‐five lobate morphological types were observed from different subfamilies, genera or tribes of grasses, based on two important parameters: (1) the length of the lobate shank and (2) the shape of the outer margin of the two lobes. The identification of grass tribe or even genus is possible based on the differences in lobate shape parameters or the composition of assemblages. However, not all of the lobate assemblages have a definite relationship with the genera that produce them, because grasses can only produce a limited range of lobate shapes that often overlap from one genus to another. Several C₃ grasses and Chloridoideae subfamily grasses also produce characteristic lobate phytoliths. The variations of lobate morphologies can be related to environmental factors, especially moisture. Typical hygrophytic grasses tend to yield lobate phytoliths with very short shank, whereas typical xerophytic grasses tend to produce lobate phytoliths with a very long shank. The potential link between phytolith morphology, grass taxonomy and environmental conditions opens the possibility that phytolith morphology may be used as a proxy in palaeoclimatic reconstruction.     

Phytoliths of Indian grasses and their potential use in identification

Phytoliths are amorphous silicon dioxide (SiO₂.nH₂O) inclusions abundant in leaves, in-ternodes and glumes in members of Poaceae. They may occur as inclusions filling the entire lumen of the silica cells, bulliform cells and trichomes or may be part of the outer epidermal cell walls. Since phytoliths are resistant to fungal or animal digestive juices, a large quantity of phytoliths accumulate in the soil where grasses grow. Compared with the pollen grains of grasses which tend to be uniform, phytoliths vary in sue and morphology and can be of value in identification at different taxonomic levels and in the dating of past vegetation. The size and shape of phytoliths of about 100 species of grasses from Tamil Nadu, India, have been determined. Silica bodies were observed either after isolation or in cleared leaf blades. Size and shape of phytoliths were determined under a microscope or from micrographs of the specimens. Size and shape can be used to assign the phytoliths to their respective subfamilies and to distinguish some of the grasses at the generic level. Drawings of silica cells and an identification key are provided for 80 species.     

Phytolith analysis of Coprinisphaera,unlocking dung beetle behaviour,herbivore diets and palaeoenvironments along the Middle Eocene–Early Miocene of Patagonia

The analysis of the total number of phytoliths, and the absolute frequencies of the different morphotypes, extracted from fossil dung beetle brood balls (Coprinisphaera) from the Middle Eocene–Early Miocene Sarmiento Formation (Patagonia, Argentina), revealed that this trace fossil represents a concentrated locus for phytolith sampling. Particularly, differences found in the total number of phytoliths among parts of that trace fossil and the bearing paleosol, allowed to infer that the infilling represents mostly a sample of the original surrounding soil, whereas in most cases the wall shows a significantly higher number of phytoliths than in the paleosol or infilling. Modern brood balls also revealed that in an environment with high density of grasses, the walls, composed of soil and dung fibres, showed a lower concentration of phytoliths than the soil. In contrast, in other environment with scarce grass coverage, the dung, which had a higher concentration of phytoliths than the soil, added to the wall produced an increase in the number of phytoliths in it. Accordingly, the larger number of phytoliths of the Coprinisphaera wall in comparison with that of the paleosol, would be reflecting the addition of dung fibres to the wall in palaeoenvironments with moderate to poor presence of phytolith-bearing plants. The absence of differences in the total number of phytoliths between the internal and the external layer of the brood ball wall, suggests that the dung fibres would have been uniformly distributed in most of the wall, due to the addition of dung fibres during the brood ball construction by the dung beetle. In contrast, the absence of differences among wall and paleosol or infilling, could be suggesting that no dung fibres were added to construct the wall, that those added had no phytoliths, that the Coprinisphaera involved could had been a brood ball of a necrophagous scarab, or that the soils were richer in phytoltihs than the dung. Those balls that showed evidence of increased numbers of phytoliths in the wall, likely caused by dung fibres added to it, enable the study of diet preferences of the herbivores that produced the dung. Differences found in the phytolith morphotype frequencies among the wall, and the other two samples (infilling and paleosol), allow to infer that some herbivores were more generalists among phytolith-bearing plants feeding on the most abundant grasses and palms, whereas others preferred more rare grasses and dicots.     

Phytoliths Analysis for the Discrimination of Foxtail Millet (Setaria italica) and Common Millet (Panicum miliaceum)

Foxtail millet (Setaria italica) and Common millet (Panicum miliaceum) are the oldest domesticated dry farming crops in Eurasia. Identifying these two millets in the archaeobotanical remains are still problematic, especially because the millet grains preserve only when charred. Phytoliths analysis provides a viable method for identifying this important crop. However, to date, the identification of millet phytoliths has been questionable, because very little study has been done on their morphometry and taxonomy. Particularly, no clear diagnostic feature has been used to distinguish between Foxtail millet and Common millet. Here we examined the anatomy and silicon structure patterns in the glumes, lemmas, and paleas from the inflorescence bracts in 27 modern plants of Foxtail millet, Common millet, and closely related grasses, using light microscopy with phase-contrast and microscopic interferometer. Our research shows that five key diagnostic characteristics in phytolith morphology can be used to distinguish Foxtail millet from Common millet based on the presence of cross-shaped type, regularly arranged papillae, Ω-undulated type, endings structures of epidermal long cell, and surface ridgy line sculpture in the former species. We have established identification criteria that, when used together, give the only reliable way of distinguishing between Foxtail millet and Common millet species based on their phytoliths characteristics, thus making a methodological contribution to phytolith research. Our findings also have important implications in the fields of plant taxonomy, agricultural archaeology, and the culture history of ancient civilizations.     

Reproductive morphology of the early-divergent grass Streptochaeta and its bearing on the homologies of the grass spikelet

Reproductive morphology and development are described in the Brazilian grass Streptochaeta spicata, in order to assess the homologies of the characteristic grass inflorescence, termed a spikelet, and other reproductive organs. Streptochaeta possesses some features that are commonly found in Poaceae, including a well-differentiated embryo. It also possesses some relatively unusual, presumably derived features, such as non-plumose stigmas, which indicate that it could be insect-pollinated. It shares some features with other early-divergent grasses, such as Pharus, which could represent plesiomorphic conditions for grasses. The inflorescence unit in Streptochaeta has been interpreted as a compound branching system or pseudospikelet. The present data suggest that it is a highly modified spikelet, with a modified flower borne either on a different axis to the basal bracts (glumes) or on the same axis as the basal bracts. The three bracts below the stamens are interpreted as homologous to the lodicules. The Streptochaeta spikelet could be considered as morphologically intermediate between the true spikelet of grasses and reproductive units of close grass relatives.     

Phylogenomics,molecular evolution,and estimated ages of lineages from the deep phylogeny of Poaceae

The deeply diverging subfamilies of grasses: Anomochlooideae, Pharoideae, and Puelioideae, today inhabit tropical forest floors as sparsely distributed depauperate lineages. The BEP/PACMAD grasses, which make up the majority of the family, are the result of a more recent radiation. Species in the deeply diverging subfamilies were here investigated to better understand molecular evolutionary processes and ages of divergence. Complete chloroplast genomes (plastomes) of Pharus latifolius L., P. lappulaceus Aubl., and Puelia olyriformis (Franch.) Clayton were determined. Four plastome loci from seven species of the deep subfamilies were also sequenced. Phylogenetic and mutation analyses and divergence estimations were conducted on all sequences together with homologous sequences from other Poaceae. Mutation analyses surveyed insertion/deletion mutations across the plastomes, clarified a trend in the molecular evolution of the rpoC2 locus, and indicated unique pseudogenizations in the plastomes of Pharus and Puelia. Phylogenetic analyses largely confirmed earlier multi-gene phylogenies. Phylogenomic and divergence analyses produced estimated origins of the crown nodes of Anomochlooideae at 65–104 Ma, Pharoideae at 44–71 Ma, and Puelioideae at 62–96 Ma. The upper ends of our estimated ranges are in general agreement with previous estimates. However, the lower ends of our ranges are considerably older than previous estimates, reflecting the influence of the less commonly used oldest fossil calibration point. The deeply diverging subfamilies exhibited the accumulation of numerous substitution and indel mutations consistent with a long evolutionary history that predated the radiation of the BEP/PACMAD grasses. We hypothesize that relatively rapid warming and drying in Africa at 55–56.5 Ma may have acted as selective forces stimulating adaptive radiations of grasses from the African tropical forests into diverse habitats.     

Silica and Ash in Native Plants of the Central and Southeastern Regions of the United States

Ash and silica contents and depositional patterns were determinedfor different tissues of 11 plants growing in the southeasternand central parts of the USA. Silica content was high in theleaves, sheaths and inflorescences of the grasses studied, especiallyso in the inflorescence of the C₃ grass, Stipa comata Trise.and Rupr. The ash content was especially high in leaves of Polymniauvedalia L., which are also high in calcium. Calcium depositionwas largely in trichomes and in veins of the leaf. Energy-dispersiveX-ray analysis showed that the distribution of the element siliconis closely related to certain epidermal structures such as ridges,cell walls, rows of irregularly-shaped structures lying lenghthwisealong the leaf, dumb-bell shaped structures and trichomes. Thesestructures also correspond to the phytoliths left behind afterdecay of the plant. The C₃ grasses differed from the C₄ in thatthey showed oval structures and produced correspondingly ovalphytoliths. Silicified trichomes (particularly in the C₃ grasses)and long, narrow, silica fibres were common in the inflorescencesof the grasses studied. These sharp particles could be irritatingto oesophageal and other tissues. Similar fibres in other grasseshave been implicated in certain cancers. High silicificationof the inflorescence structures might afford protection forthe seed, as reported for other grasses. C₃ and C₄ grasses, silica and ash content, scanning electron microscopy, energy-dispersive X-ray analysis, silicon distribution, spectra of elements in plants, trichomes, silica fibres, phytoliths     

Starch-accumulating (S-type) sieve-element plastids in Hydatellaceae: implications for plastid evolution in flowering plants

TEM investigation of sieve-element plastids in three species of Trithuria, the sole genus of the small aquatic family Hydatellaceae, show that P-type plastids are absent from this genus and only starch-accumulating (S-type) sieve-element plastids are present. This discovery is consistent with the recent transfer of Hydatellaceae from the highly derived monocot order Poales (grasses and their allies) to the early-divergent angiosperm order Nymphaeales (waterlilies) based on molecular phylogenetic data. Species of Poales consistently possess P2-subtype plastids, in common with other monocots, but only S-type plastids are present in Nymphaeales. The results confirm that Hydatellaceae do not belong in monocots. Optimisation of the two major types of sieve-element plastid onto a recent phylogeny of early-divergent angiosperms confirms that S-type is the primitive form and indicates that P-type sieve-element plastids have evolved more than once in angiosperms.     

Structure and Function of Silica Bodies in the Epidermal System of Grass Shoots

Silica (SiO₂.nH₂O) is deposited in large quantities in the shootsystems of grasses. In the leaf epidermal system, it is incorporatedinto the cell wall matrix, primarily of outer epidermal walls,and within the lumena of some types of epidermal cells. This biogenic silica can be stained specifically with methylred, crystal violet lactone, and silver amine chromate. At theultrastructural level, the silica in lumens of silica cells,bulliform cells and long epidermal cells is made up of rodsabout 2.5 µm in length and 0.4µm in width. Ultimateparticles in the rods range from 1 to 2 nm in diameter. In contrast,silica in the cell wall matrix of trichomes and outer wallsof long epidermal cells is not rod-shaped, but rather, formsroughly spherical masses. Detailed analyses are presented on the frequencies of occurrenceof the different types of epidermal cells that contain silicain the leaves of representative C₃ and C₄ grasses. The C₄ grasseshave higher frequencies of bulliform cell clusters, silica cells,and long epidermal cells, whereas the C₃ grasses have higherfrequencies of trichomes. No correlation was found in the frequencyof occurrence of silica bodies in bulliform cells for C₃ grassesas compared with C₄ grasses. Of all the grasses examined, Coix,Oryza, and Eleusine had the highest densities of such bodies,and some taxa had no silica bodies apparent in their bulliformcells. The idea that silica bodies in bulliform cells and silica cellsmight act as "windows’ and trichomes might function as‘light pipes’ to facilitate light transmission throughthe epidermal system to photosynthetic mesophyll tissue belowwas tested. The experimental data presented do not support eitherof these hypotheses. C₂ and C₄ grasses, biogenic silica, light pipes, window hypothesis, silica staining, silica ultrastructure     

The spread of grass-dominated habitats in Turkey and surrounding areas during the Cenozoic: Phytolith evidence

The arrival of hipparionine horses in the eastern Mediterranean region around 11 Ma was traditionally thought to mark the simultaneous westward expansion of savanna vegetation across Eurasia. However, recent paleoecological reconstructions based on tooth wear, carbon isotopes, and functional morphology indicate that grasses played a minor role in Late Miocene ecosystems of the eastern Mediterranean, which were more likely dry woodlands or forests. The scarcity of grass macrofossils and pollen in Miocene floras of Europe and Asia Minor has been used to support this interpretation. Based on the combined evidence, it has therefore been suggested that Late Miocene ungulate faunal change in the eastern Mediterranean signals increased aridity and landscape openness, but not necessarily the development of grass-dominated habitats.

To shed new light on the Miocene evolution of eastern Mediterranean ecosystems, we used phytolith assemblages preserved in direct association with faunas as a proxy for paleovegetation structure (grassland vs. forest). We extracted phytoliths and other biogenic silica from sediment samples from well-known Early to Late Miocene ( 20–7 Ma) faunal localities in Greece, Turkey, and Iran. In addition, a Middle Eocene sample from Turkey yielded phytoliths and served as a baseline comparison for vegetation inference.

Phytolith analysis showed that the Middle Eocene assemblage consists of abundant grass phytoliths (grass silica short cells) interpreted as deriving from bambusoid grasses, as well as diverse forest indicator phytoliths from dicotyledonous angiosperms and palms, pointing to the presence of a woodland or forest with abundant bamboos. In contrast, the Miocene assemblages are dominated by diverse silica short cells typical of pooid open-habitat grasses. Forest indicator phytoliths are also present, but are rare in the Late Miocene (9–7 Ma) assemblages. Our analysis of the Miocene grass community composition is consistent with evidence from stable carbon isotopes from paleosols and ungulate tooth enamel, showing that C₄ grasses were rare in the Mediterranean throughout the Miocene. These data indicate that relatively open habitats had become common in Turkey and surrounding areas by at least the Early Miocene ( 20 Ma), > 7 million years before hipparionine horses reached Europe and arid conditions ensued, as judged by faunal data.     

Desert grassland dynamics estimated from carbon isotopes in grass phytoliths and soil organic matter

Abstract. We document the potential for using carbon isotopes in both soil organic matter (SOM) and grass phytoliths in soil to increase the temporal and taxonomic resolutions of long term vegetation dynamics. Carbon isotope values from both SOM and phytoliths are expected to describe both the age of material through ¹⁴C dating, and the photosynthetic pathway of the source plant material through ratios of ¹²C/¹³C. Taxonomic resolution is increased because the phytoliths examined are specific to grasses, whereas the SOM reflects the contribution of all the vegetation. Temporal resolution is increased because phytoliths are less mobile in the soil profile than SOM, and can therefore provide older dates from the same soil depth. Our results, from a desert grassland site in southwestern North America, largely confirm these expectations, and show that C₄ species have dominated the grass composition for the last 8000 yr, C₃ non‐grass vegetation increased about 100–350 yrBP, and no significant C₃ grass or non‐grass vegetation existed between 350–2000 yr BP.     

The phylogeny of the BEP clade in grasses revisited: evidence from the whole-genome sequences of chloroplasts

Despite the considerable efforts to reconstruct the phylogeny of grasses, the relationships among the subfamilies Bambusoideae, Pooideae and Ehrhartoideae in the BEP clade remain unresolved. Here we completely sequenced three chloroplast genomes of representative species from Bambusoideae and Ehrhartoideae and obtained 19 additional chloroplast genome sequences of other grasses from GenBank. Using sequences of 76 chloroplast protein-coding genes from the 22 grass species, we fully resolved the phylogeny of the BEP clade. Our results strongly supported the (B,P)E hypothesis, i.e., Bambusoideae and Pooideae are more closely related than Ehrhartoideae. This result was not biased by systematic or sampling errors and was impervious to phylogenetic methods or model specification. The divergence time estimate suggests that the initial diversification of the BEP clade into three subfamilies happened within a short time period (≈ 4 MY). The presence of these short internal branches may explain the inability of previous studies to achieve a confident resolution of the BEP clade. The combination of the sequences of the entire chloroplast genomes provided sufficient phylogenetic information to resolve the BEP phylogeny fully. These results provide a valuable evolutionary framework for comparative and functional genomic studies using the grass family as a model system.     

Clonal analysis of epidermal patterning during maize leaf development

In plants, specialized epidermal cells are arranged in semiordered patterns. In grasses such as maize, stomata and other specialized cell types differentiate in linear patterns within the leaf epidermis. A variety of mechanisms have been proposed to direct patterns of epidermal cell differentiation. One class of models proposes that patterns of cellular differentiation depend on the lineage relationships among epidermal cells. Another class of models proposes that epidermal patterning depends on positional information rather than lineage relationships. In the dicot epidermis, cell lineage is an important factor in the patterning of stomata, but not trichomes. In this study, the role of cell lineage in the linear patterning of stomata and bulliform cells in the maize leaf epidermis is investigated. Clones of epidermal cells in juvenile leaves were marked by excision of dSpm from gl15-m and in adult leaves by excision of Ds2 from bz2-m. These clones were analyzed in relation to patterns of stomata and bulliform cells, testing specific predictions of clonal origin hypotheses for the patterning of these cell types. We found that the great majority of clones analyzed failed to satisfy these predictions. Our results clearly show that lineage does not account for the linear patterning of stomata and bulliform cells, implying that positional information must direct the differentiation patterns of these cell types in maize.     

Racemose inflorescences of monocots: structural and morphogenetic interaction at the flower/inflorescence level

Background

Understanding and modelling early events of floral meristem patterning and floral development requires consideration of positional information regarding the organs surrounding the floral meristem, such as the flower-subtending bracts (FSBs) and floral prophylls (bracteoles). In common with models of regulation of floral patterning, the simplest models of phyllotaxy consider only unbranched uniaxial systems. Racemose inflorescences and thyrses offer a useful model system for investigating morphogenetic interactions between organs belonging to different axes.

Scope

This review considers (1) racemose inflorescences of early-divergent and lilioid monocots and their possible relationship with other inflorescence types, (2) hypotheses on the morphogenetic significance of phyllomes surrounding developing flowers, (3) patterns of FSB reduction and (4) vascular patterns in the primary inflorescence axis and lateral pedicels.

Conclusions

Racemose (partial) inflorescences represent the plesiomorphic condition in monocots. The presence or absence of a terminal flower or flower-like structure is labile among early-divergent monocots. In some Alismatales, a few-flowered racemose inflorescence can be entirely transformed into a terminal ‘flower’. The presence or absence and position of additional phyllomes on the lateral pedicels represent important taxonomic markers and key features in regulation of flower patterning. Racemose inflorescences with a single floral prophyll are closely related to thyrses. Floral patterning is either unidirectional or simultaneous in species that lack a floral prophyll or possess a single adaxial floral prophyll and usually spiral in the outer perianth whorl in species with a transversely oriented floral prophyll. Inhibitory fields of surrounding phyllomes are relevant but insufficient to explain these patterns; other important factors are meristem space economy and/or the inhibitory activity of the primary inflorescence axis. Two patterns of FSB reduction exist in basal monocots: (1) complete FSB suppression (cryptic flower-subtending bract) and (2) formation of a ‘hybrid’ organ by overlap of the developmental programmes of the FSB and the first abaxial organ formed on the floral pedicel. FSB reduction affects patterns of interaction between the conductive systems of the flower and the primary inflorescence axis.     

The first herders in the upper Ebro basin at Los Husos II (Álava,Spain): microarchaeology applied to <Emphasis Type="Italic">fumier</Emphasis> deposits

Fumier deposits are the product of the recurrent use of caves and rock shelters for stabling livestock and the periodic burning of the resulting dung. Their chronologies in the Mediterranean area extend from Neolithic times up to the Bronze Age, but they are scarce in or absent from Iron Age sites. The study of these deposits has provided important information to better understand past livestock and husbandry practices. The archaeological site of Los Husos II, Álava, in the upper Ebro basin, Spain, dating to 6,990–6,770 cal bp, represents one of the earliest pieces of evidence for animal domestication in the Basque Country. The Ebro basin is particularly important, since this was the main route by which the Neolithic economic system spread from the Mediterranean coast to the northern Iberian Peninsula and the western Pyrenees. We present here the results of the study of the fumier deposits from the Neolithic Levels IV to IX of Los Husos II, through analyses of phytoliths, faecal spherulites and ash pseudomorphs. The main goal was to discover the ways in which Neolithic populations in this region adapted these new practices and carried out their activities. The results indicate a constant pattern of keeping animals throughout the study period. The principal component of the livestock diet consisted of wild grasses from the vicinity. The presence of grass inflorescences suggests a diet rich in summer grass. In addition to grasses, dicotyledonous plants were also indicated, both through phytoliths and ash pseudomorphs. Faecal spherulites from herbivores were also noted in the samples and together with phytoliths and ash pseudomorphs they give important information regarding the formation processes of the studied deposits.     

中国-中新世湖泊的古生态:根据山旺盆地植硅体的初步解释

山东山旺中新世山旺组湖相沉积中富含保存精美的古生物化石。对于湖相沉积中的昆虫、植物和哺乳动物化石,前人已经做了大量的研究工作。其中植物大化石和孢粉的研究结果表明,中新世山旺组在沉积期间,山旺湖周围的植被处于湿润的暖温带到亚热带的古生态环境下。哺乳动物蝙蝠和貘化石的研究结果也支持这个结论。然而,迄今为止,还没有关于草本植物的种类组成及含量的研究报道。本文首次对山旺组样品中的植硅体进行了研究,为山旺中新世植被的研究提供了新的证据。对从6块山旺组样品中分析出的植硅体进行研究的结果表明,山旺中新世植硅体组合含有丰富的保存极好的草本植物植硅体,代表森林成分的双子叶植物植硅体,以及偶尔出现的棕榈科植硅体。草本植物的植硅体主要来自于C_3/C_4禾本科的PACCAD类群和早熟禾亚科的植物,少数可能来自于在林下封闭生活环境中生长的草本植物。山旺中新世植硅体组合显示,在湖边森林生活环境下,伴生大量的适合沼生到中生环境的草本植物,在较干旱的地区生长着早熟禾(和PACCAD)的草本植物。本文的研究结果进一步支持山旺中新世气候比现在更加湿润而且变动较小的观点。山旺中新世植硅体组合与土耳其和北美大平原中新世湖相沉积中的植硅体组合不同,后者的植硅体组合主要是由多种C_3早熟禾亚科草本和不同种类的PACCAD类群草本植物组成。今后的研究工作将进一步揭示这种差异是否反映了草本植物群落在大尺度的生物地理上的差异,或者是由于地区性的局部气候不同所导致。     

Biogenic Opal in Three Species of Gramineae

Particulate biogenic opaline silica is concentrated in cellwalls, intercellular deposits and cell lumina of all portionsof the above-ground plant body of three species of PanicoidGramineae,Andropogon gerardi, Sorgastrum nutans and Panicumvirgatum. Morphologically distinct opal phytoliths form notonly in long cells, short cells, trichomes, stomatal elementsand bulliform cells of the epidermis but also within the cellularstructure of mesophyll, vascular, and sclerenchyma tissues.Roots and rhizomes contain measurable quantities of opalinesilica, and phytoliths develop in epidermal long cells, saddle-shapedshort cells, vascular cells, and intercellular deposits. A morphologicallyunique plate-phytolith, formed by silicification of the innertangential wall of the endodermis, is present in the roots ofall three species. Differences in the quantity of opaline silicaoccur between species and between parts of the same species.The amount of opal deposited in the soil annually by root systemsand above-ground parts is approximately equal in magnitud Andropogon gerardi, Sorgastrum nutans, Panicum virgatum, opaline silica deposition     

Intracellular Silica Deposition in Immature Leaves in Three Species of the Gramineae

The formation of solid, discrete deposits of opaline silicawithin the cell lumen of leaf tissues is reported in speciesrepresenting three subfamilies of the Gramineae; the preparationof a silica-minimal nutrient solution is discussed. Opal phytolithnumbers are related to tissue age and to two external silicaconcentrations for tiller leaves over a period of 32 days followingbud initiation. Variations in silica deposition patterns among the individualleaves of a homologous series on the shoot apex are relatedto differential growth-rates. During ontogeny, deposition occursin an ordered sequence of cell types related to the basipetalmaturation gradient within the leaf. Initial deposition wasdetected in silica cells (idioblasts) of imbricated, bud leavesof Sieglingia decumbens, 1 mm long; phytoliths are confinedto these cells in expanding, basal portions of the leaf. Phytolithcounts/sq mm of epidermis vary with the degree of long-cellexpansion. The error is reduced by expressing silicificationas the number of phytoliths/100 silica cells. Post-expansiondeposition was initiated in epidermal long cells of the leafblade tip. The higher silica concentration resulted in a morerapid utilization of available deposition sites and larger phytoliths(P = 0.001). A passive cell influx of silicic acid is discussedin relation to cytodifferentiation.