Diversity and Distributions (2003) 9, 73 8 7

BIODIVERSITY RESEARCH

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

HOUYUAN LU 1,2 * and KAM-BIU LIU 2 1 Institute of Geology and Geophysics, Chinese Academy of Sciences, PO Box 9825, Beijing 100029, China, E-mail: houyuanlu@peoplemail.com.cn 2 Department of Geography and Anthropology, Louisiana State University, Baton Rouge, Louisiana 70803, U.S.A.

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 classication scheme based on 250 modern grass species from China and the south-eastern U.S.A. Eighty-ve grass species were found to contain lobate phytoliths. They are derived mainly from Panicoideae, but also include the Chloridoideae, Oryzoideae and Arundinoideae subfamilies. Twenty-ve 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 identication 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 denite 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. Key words. Dumbbell, grasses, palaeoenvironment, palynology, phytoliths, phytolith-lobate, silica bodies, taxonomy.

INTRODUCTION
Grasses (Family: Gramineae) are an important group of plants in a variety of environments (Gould & Shaw, 1983). They are often the dominant plants in steppes or prairies, tundra, coastal marshes, pioneer or early successional communities, disturbed sites and in certain aquatic communities. Many important crops are grasses, such as maize, rice, wheat and sugar cane. Thus, the identication and classication of
* Corresponding author.

grasses from fossil assemblages are of great signicance in palaeoecological reconstruction. Unfortunately, except for Zea mays (maize), the pollen of Gramineae cannot be identied below the family level (Fearn & Liu, 1997). Thus, the use of grass pollen in palaeoecological reconstruction is limited. Grass phytoliths, on the other hand, offer a promising means to differentiate grasses at subfamily levels and, accordingly, to infer subtle changes in palaeoenvironmental conditions (Piperno, 1988; Rapp & Mulholland, 1992; Fredlund & Tieszen, 1994, 1997; Alexandre et al., 1997; Runge, 1999). In this paper, we focus

2003 Blackwell Publishing Ltd. http://www.blackwellpublishing.com/journals/ddi

73

74

H. Lu and K.-b. Liu

grass species can produce different types of phytoliths (i.e. multiplicity), and many different species can produce the same shapes (i.e. redundancy) (Rovner, 1971). In order to use phytoliths as a tool in environmental reconstruction and taxonomy, it is necessary to recognize morphological variations in phytoliths in different species of grasses. Bilobate (dumbbell) phytolith, one of the most important morphotypes in grass phytoliths, has been identied consistently as a distinctive silica body (Twiss et al., 1969; Brown, 1984; Piperno, 1988; Kondo et al., 1994; Rapp & Mulholland, 1992; Wang & Lu, 1993). The term dumbbell was rst used by Metcalfe (1960) as a morphological term for the shape of some intercostal short cell phytoliths. It has gradually become a name given to a loosely dened group of phytoliths characterized by having two lobes joined by a shank. However, many subsequent researchers, including Brown (1984), Fredlund & Tieszen (1994, 1997) and Piperno & Pearsall (1998) avoided using this term and favoured the alternative term bilobate. As a morphological term, dumbbell draws its analogy from an exercising equipment and makes sense only in the English language, whereas bilobate is rooted in Latin and has a well-founded scientic meaning that is more comprehensible to non-English-speakers. For this reason, we follow the convention of these subsequent researchers and use the term lobate to describe the morphological class of phytoliths that has two or more lobes connected by a shank, whereas the term bilobate refers only to the largest subgroup, known formerly as dumbbell, that has two lobes connected by a shank. In this paper, we propose a classication system for all lobate grass phytoliths, although our research and discussion will focus on the bilobate types. For the taxonomy and nomenclature of grasses in China and the United States, we follow the Institute of Botany, Chinese Academy of Sciences (1977) and Gould & Shaw (1983), respectively.

on the predominant type of grass phytoliths the lobate phytoliths, based on an investigation of 250 species of grasses from China and the southeastern U.S.A. Our objectives are twofold: (1) to document the morphological variations of lobate phytoliths in relation to grass taxonomy and (2) to investigate the relationship between lobate phytolith shapes and environmental factors, which may be useful for palaeoenvironmental reconstruction.

Background
Phytoliths are opal-A particles that precipitate within cells or between cells of living plant tissues (Piperno, 1988). Although nonsilicic (calcareous) phytoliths do exist (Cummings, 1992), most researchers (including the authors of this paper) use the term phytoliths to denote only the opal or silicic plant-cell inclusions, as dened above. Thus, phytoliths are also called silica bodies in the anatomical literature (Ellis, 1979). Their sizes range from a few microns (m) to about 150 m. They are well preserved in various sediments, even in oxidized environments such as soils, loess and sand dunes (Kelly et al., 1991; Wang & Lu, 1993; Lu et al., 1996; Horrocks et al., 2000). Early phytolith researchers noted that the different subfamilies of grasses produce different phytolith shapes; for example, grasses of the subfamily Panicoideae produce dumbbell and cross-shaped phytoliths, whereas Pooideae (Festucoideae) grasses produce rondels and sinuous types, and Chloridoideae grasses produce saddleshaped phytoliths (Twiss et al ., 1969). All members of the grass subfamilies Panicoideae and Oryzoideae produce the bilobate (dumbbell) type of phytoliths. However, bilobate (dumbbell) phytoliths, thought previously to be the most diagnostic marker of the Panicoideae subfamily (Twiss et al ., 1969), are also present in the Chloridoideae and Arundinoideae subfamilies (Mulholland, 1989; Lu, 1998; Piperno & Pearsall, 1998; Lu & Liu, in prep.). Much confusion exists in the classication and descriptive terminology of grass phytoliths. Existing classication of grass phytoliths is based on the micromorphology of discrete silica bodies, which is independent of the orientation of the bodies in silica cells in the various vegetative parts of the individual grass plant (Twiss, 1992). Unfortunately for palaeoecologists, the same

MATERIALS AND METHODS Samples of modern grass plants for phytolith analysis
Leaves, culms and inorescences from 250 species of modern grass plants in China (tropical,

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Morphological variations of lobate phytolith

75

Table 1 List of modern grasses from the Atlantic and Gulf coasts of the United States used for the analysis of lobate phytoliths Sample no.* 17 20 62 10 72 16 18 41 19 37 83 84 85 31 64

Name Andropogon glomeratus (Walt.) BSP Andropogon ternaries Michx. Anthaenantia rufa (Nutt.) Schult. Aristida desmantha Trin. & Rupr. Cenchrus incertus M. Curtis Chasmanthium laxum (L.) Yates. Chasmanthium ornithorhynchum (Steud.) Yates Ctenium aromaticum (Walter) A.W. Wood Erianthus strictus Spreng. Leersia oryzoides (L.) Sw. Panicum amarum Elliott Panicum dichotomiorum Michx. Panicum hemitomon Schult. Panicum verrucosum Muhl. Panicum virgatum L.

Subfamily Panicoideae Panicoideae Panicoideae Chloridoideae Panicoideae Arundinoideae Arundinoideae Chloridoideae Panicoideae Oryzoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae

Ecology or distribution Generally on wet sites Edge of pine forest Pine forest Sandy soil along coast Dry sand Edge of forest Moist area in pine atwoods Edge of forest Edge of pine forest and disturbed areas Wet roadside ditches and edges of lakes, streams, and other wet areas Sandy soil along coast Disturbed areas, especially in moist regions, throughout United States Coastal marsh, wet areas and in the inland part of coastal sites Frequent; disturbed areas and edges of forests, mostly in the pine regions Frequent; edges of pine forests and remnant strips in prairie regions; cheniers and spoil banks in coastal freshwater marsh Cultivated in tropical regions of the world

Saccharum ofcinarum L. Setaria sp. Sorghastrum nutans (L.) Nash Sorghum halepense (L.) Pers. Zizaniopsis miliacea (Michx.) Doell & Aschers. * See the sample numbers in Fig. 3.

Panicoideae Panicoideae Panicoideae Panicoideae Oryzoideae

Edge of forest and disturbed areas in pine and prairie regions Widespread throughout the world 63 Edges of lakes, streams, wet roadside ditches 38

68 9 21

subtropical and temperate forests, grasslands) and the subtropical Atlantic and Gulf coasts of the south-eastern United States (salt marshes, freshwater marshes, sand dunes and forests) were collected for phytolith analysis. The rationale for including samples from both China and the United States in this study is to ensure that our observations and conclusions have wide application and are not limited to only one geographical region. These 250 grass species include the representatives of all six subfamilies (Pooideae, Panicoideae, Chloridoideae, Bambusoideae, Oryzoideae and Arundinoideae) according to the classications of the Institute of Botany, Chinese Academy of Sciences (1977) and Gould & Shaw

(1983). Among these, 85 species, belonging mainly to the subfamily Panicoideae, contain lobate phytoliths. Panicoideae has about 32 genera and 325 species in the United States, and 190 genera and over 900 species in China (Institute of Botany, Chinese Academy of Sciences, 1977; Gould & Shaw, 1983). Some of the lobate phytoliths presented in this study, however, come from the subfamilies Oryzoideae, Chloridoideae, and Arundinoideae (Tables 1 and 2). It should be pointed out that the classication and nomenclature of grass subfamilies have recently been revised (GPWG, 2001). More studies are needed in the future to adapt our phytolith classication to the new classication system of grass subfamilies.

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Table 2 List of modern grasses from China used for the analysis of lobate phytoliths Sample no.* 28 48 1 4 59, 60 82 30 61 76 75 40 44 55 54 14 49 15 2 79 80 66 27 26 58 3 74 22 39 53 50

76 H. Lu and K.-b. Liu

Name Apluda mutica L. Arthraxon hispidus Makino var. cryptatherus (Hack.) Honda Arundinella hirta (Thunb.) Tanaka Arundinella setosa Trin. Bothriochloa ischaemum (L.) Keng Brachiaria ramose (L.) Stapf Capillipedium assimile (Steud.) A. Camus Capillipedium parviorum (R.Br.) Stapf Coix lacryma var. ma-yuen (Roman.) Stapf Coix lacryma-jobi L. Cymbopogom goeringii Steud Cyrtococcum patens (L.) A. Camus Digitaria adscendens (H. B.K.) Henrard Digitaria sanguinalis (L.) Scop. Digitaria sanguinalis var. ciliaris (Retz.) Parl. Digitaria violascens Link Dimeria ornithopoda Trin Eccoilopus cotulifer (Thunb.) A. Camus Echinochloa colonum (L.) Link Echinochloa crusgalli (L.) Beauv. Echinochloa crusgalli var. mitis (Pursh) Peterm Eragrostis ferruginea (Thunb.) Beauv. Eragrostis japonica (Thunb.) Trin. Eriochloa villosa (Thunb.) Kunth Eulalia speciosa (Debeaux) Kuntze Hackelochloa granularia (L.) Kuntze Imperata cylindrical (L.) Beauv. Var. major (Nees) C.E. Hubb. Isachne dispar Trin. Ischaemum aristatum L. Ischaemum antephoroides (Steud.) Miq.
2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Subfamily Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Chloridoideae Chloridoideae Panicoideae Panicoideae Panicoideae Panicoideae Chloridoideae Panicoideae Panicoideae

Ecology or distribution Southern China, edge of woodlands, side of streams Temperate regions of China, hillside, moist area East China, frequent growing in valley, side of streams, wetter sites Southern China, hillside Temperate regions of the world, hillside, roadside Tropical regions of the world East China, hillside East China, hillside, roadside Temperate and subtropical regions of the world, wet areas Temperate and subtropical regions of the world, wet areas Hillside Southern China, edge of forest Field, throughout China Hillside, eld, throughout China Southern China Southern China, roadside, hillsideeld Southern China, wet areas, hillside Southern China, hillside, eld East China, wet areas, roadside Temperate regions of the world, swamp, rice paddy Temperate regions of the world, swamp, wet areas East China, roadside, eld Southern China, hillside, eld East China, wet areas East China, hillside Tropical regions of the world, edges of streams Throughout China, dry soils Southern China, hillside, forest and edges of streams East China, hillside, roadside, coast Southern China, hillside, eld, coast

Table 2 continued. Sample no.* 51 36 33 32 52 70 71 45 42 35 46 56

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Name Ischaemum indicum (Houtt.) Merr. Leersia hexandra Swartz Leptochloa chinensis (L.) Nees Microstegium vimineum (Trin.) A. Camus Microstegium vimineum (Trin.) A. Camus var. imberbe (Nees) Honda Miscanthus oridulus (Labill.) Warb Miscanthus sinensis Anderss Oplismenus compositus (L.) Beauv Oplismenus undulatifolius (Arduino) Roem. Et Schult Oryza sativa L. Panicum austro-asiaticum Ohwi Panicum bisulcatum Yhunb Panicum notatum Retz Panicum repens L. Paspalum dilatatum Porst Paspalum orbiculare G. Forst Pennisetum alopecuroides (L.) Spreng Pennisetum purpureum Schumach Pogonatherum crinitum (Thunb.) Kunth Rottboellia exaltata L. f. Saccharum arundinaceum Retz Saccharum sinensis Roxb Sacciolepis myosuroides (R.Br.) A. Camus Schizachyrium brevifolium (Sw.) Nees ex Buse Setaria faberi Herrm. Setaria glauca (L.) Beauv.

Subfamily Panicoideae Oryzoideae Chloridoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Oryzoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae

Ecology or distribution Southern China, hillside, roadside, coast Southern China, a perennial grass growing in standing water or very damp ground Southern China, a common annual of waste elds and paddy elds East China, moist area East China, moist area Southern China, hillside wet areas, forest Hillside, edges of streams Southern China, wet areas, shady places under trees East China, wet areas, edge of forest Cultivated worldwide Southern China, wet areas East China, wet areas, edges of lakes, streams, wet roadside ditches Southern China, edge of forest Tropical and subtropical zones, waterside, coast Wet areas Tropical and subtropical regions of the world, hillside, eld Disturbed areas throughout China Elephant grass, native to Africa, a robust perennial with culms 24 meters tall Southern China, banks, edges of streams, often in sandy places. Southern China, hillside, roadside Southern China, hillside, edges of streams Cultivated in tropical regions of the world Southern China, paddy eld East China, an annual grass of rocky places with poor soil, hillside China, north of Yangtze River Temperate and tropical zones, eld and roadside

Morphological variations of lobate phytolith

13 73 81 78 11 12 47 29 69 67 43 25 5 8

77

78

H. Lu and K.-b. Liu

Sample no.*

Laboratory procedure for phytolith analysis

7 6 65 57 23 24 77 34

Subfamily

Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Panicoideae Oryzoideae

All collected plant samples were cleaned with distilled water in a water bath to remove adhering particles. Leaves and culms of each species were placed in 20 mL of saturated nitric acid for over 12 h to oxidize organic materials completely. Some species, such as Sporobolus dianger (Retz.) Beauv., S. indicus (L.) R.Br. var. purpurea-suffusus (Ohwi) T. koyama and Cymbopogon goeringii Steud, were oxidized for 24 h because they contain more vegetable tallow. The solutions were centrifuged at 2000 r.p.m. for 10 min, decanted and rinsed twice with distilled water, and then rinsed with 95% ethanol until the supernatants were clear. The phytolith sediments were transferred to storage vials. The residual subsamples were mounted onto microscopic slides in Canada balsam medium for photomicrography and in liquid medium for counting and line drawing. Light photomicrography at 400 magnication was used to record types of phytoliths found in each plant sample. An average of 270 lobate grains was counted in each sample. The percentages of different lobate categories were calculated on the basis of a sum consisting of all lobate phytoliths.

Ecology or distribution

Southern China, valley, hillside Southern China, valley, wet areas Cultivated in temperate regions of the world Temperate regions of the world, hillside, forest Southern China, hillside grassplot, edges of streams East, China, dry hillside Cultivated worldwide Southern China, streams, marsh

Classication of lobate phytoliths

Lobate phytoliths are originated from the short cells of grasses with identiable shape characteristics. Metcalfe (1960) identied three types of dumbbell or bilobate phytoliths. The Panicoid division in Twisss classication is composed of 11 types of dumbbells and crosses (Twiss et al., 1969). Brown (1984) recognized bilobates, polylobates and crosses. Mulholland & Rapp (1992) proposed the lobate class to denote phytoliths with denite lobes, including the cross, sinuate and dumbbell types. Based on our observation and statistics of the lobate phytoliths from 85 species of modern grass plants, we found that two important parameters can be used to characterize the morphological variations in lobate phytoliths: (1) the shape of the outer margins of the two lobes and (2) the length of the shank in the lobate structure (Fig. 1). These two parameters are relatively stable among different Panicoideae plants. We develop a lobate classication matrix based on these two criteria (Fig. 2). Pearsall
2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Setaria palmifolia (Koen.) Stapf Setaria plicata (Lamk.) T. Cooke Sorghum vulgare Pers. Spodipogon sibiricus Trin. Themeda gigantean var. caudate (Nees) Keng Themeda triandra var. japonica (Willd.) Makino Zea mays L. Zizania caduciora (Turcz. Ex Trin.) Hand.-Mazz.

Table 2 continued.

* See the sample numbers in Fig. 3.

Name

Morphological variations of lobate phytolith

79

Fig. 1 Morphological phytolith.

components

of

lobate

The rst criterion is the shape or outline of the lobe. We divide lobate phytoliths into A, B, C, D and E types based on the characteristics of ridged lines, rounded, truncated, concave and branched outer margins, respectively (Fig. 2). Type A has ridged lines running longitudinally along the whole length of the phytolith. The lines may be radiating towards the distal ends of the lobes. The shank is often wide and sturdy, connecting two indistinct lobes. The outline of the lobes has obvious edges and corners. Types B is characterized by having smooth and generally round outlines on the two lobes. In type C, the distal ends of the two lobes are truncated, forming a generally straight edge. In type D, the distal ends of the lobes are slightly indented, forming a smooth, concave curve. In type E, the distal ends of the lobes are deeply indented or distinctly branched. Type F is characterized by having multiple lobes. The second criterion is the length of the shank (a) relative to the length of lobes (b) (Fig. 1). Four types are recognized: Type Type Type Type 1: 2: 3: 4: a < 1/3b 1/3b < a < b a b a >b

Fig. 2 Classication of lobate phytoliths according to two criteria: outline of the lobes (A F), and length ratio between shank and lobe (1 4). See text for explanation.

& Piperno (1990) used a somewhat different approach to distinguish domesticated corn phytoliths on the basis of cross-shaped phytolith size (Pearsall, 1978) and cross-shaped three-dimensional structures (Piperno, 1984).

We divide lobate phytoliths into 20 types according to the combination of these two criteria. In addition, for the shapes of two half-lobes, three lobes, three lobes with radiation lines, more than four lobes and beaded lobes, we group them under a special category, type F, which consists of ve subtypes (designated by lower-case letters a e, respectively) according to the number of lobes present. Thus type F encompasses the trilobate and polylobate types that have been recognized widely in previous studies (Brown, 1984; Piperno, 1988). Altogether, there are 25 morphological types of lobate phytoliths in our classication system (Fig. 2). The above classication is based on a twodimensional view of phytolith shape, assuming that a perfect lateral view of the phytolith can be observed and measured. It should be pointed out that phytolith, like pollen, is not a two-dimensional object. In reality, phytoliths may be tilted at an angle from the viewer while being observed under the microscope. Consequently, great care must be exercised in observing and describing

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

80

H. Lu and K.-b. Liu

Table 3 List of 25 lobate phytolith types and their representative grass taxa Lobate types Representative Taxa A1 A2 Isachne dispar Sacciolepis myosuroides, Cyrtococcum patens, Oplismenus compositus, Panicum austro-asiaticum, Pogonatherum crinitum, Arthraxon hispidus var. cryptatherus, Digitaria violascens, Ischaemum antephoroides, I. Indicum, I. aristatum, Microstegium vimineum var. imberb Digitaria sanguinalis, D. sanguinalis var. ciliaris, D. adscendens Sacciolepis myosuroides Arundinella hirta, Eccoilopus cotulifer, Eulalia speciosa, Arundinella setosa, Setaria faberi, Pennisetum alopecuroides, P. purpureum, Echinochloa. crusgalli, Paspalum dilatatum, Brachiaria ramosa Setaria glauca, Aristida desmantha, Echinochloa crusgalli var. mitis, Saccharum sinensis, Sorghum vulgare, Saccharum arundinaceum, Miscanthus oridulus, Miscanthus sinensis, Cenchrus incertus Setaria glauca, Aristida desmantha, Pennisetum alopecuroides, P. purpureum, Panicum notatum Arundinella setosa, Schizachyrium brevifolium, Digitaria sanguinalis Oryza sativa, Leersia hexandra, Zizaniopsis miliacea, Z. caduciora, Panicum amarum Panicum bisulcatum, P. virgatum, Spodipogon sibiricus, Eriochloa villosa, Bothriochloa ischaemum, Capillipedium parviorum, Anthaenantia rufa, Sorghum halepense, Saccharum ofcinarum, Chasmanthium laxum, Miscanthus Anthaenantia ruoridulus, M. sinensis Digitaria sanguinalis var. ciliaris, Dimeria ornithopoda, Chasmanthium laxum, C. ornithorhynchum, Andropogon glomeratus, A. ternaries, Erianthus strictus, Sorghastrum nutans, Imperata cylindrica, Themeda gigantea var. caudate, Panicum virgatum Schizachyrium brevifolium, Eragrostis japonica, Eragrostis ferruginea Leersia oryzoides, Zizaniopsis miliacea, Anthaenantia rufa Leersia oryzoides, Zizania caduciora, Microstegium vimineum var. imberbe, Leptochloa chinensis Arundinella setosa, Setaria faberi, S. plicata, S. palmifolia Cymbopogom goeringii, Ctenium aromaticum Coix lacryma-jobi, Zea mays, Paspalum orbiculare, P. dilatatum, Echinochloa crusgalli, Brachiaria ramosa, Themeda triandra var. japonica, Panicum dichotomiforom, Arundinella hirta, Cyrtococcum patens, Oplismenus compositus, Bothriochloa ischaemum, Echinochloa crusgalli var. mitis, Saccharum sinensis, Sorghum vulgare Coix lacryma-jobi, Coix lacryma var. ma-yuen, Zea mays Setaria palmifolia, Themeda triandra var. japonica, Panicum dichotomiforom ? Capillipedium assimile, Panicum verrucosum Eulalia speciosa, Rottboellia exaltata, Capillipedium assimile, Panicum verrucosum, Oplismenus undulatifolius Sacciolepis myosuroides Panicum bisulcatum, Spodipogon sibiricus Apluda mutica

A3 A4 B1 B2 B3 B4 C1 C2

C3

C4 D1 D2 D3 D4 E1

E2 E3 E4 Fa Fb Fc Fd Fe

phytolith shapes. Fortunately, because phytoliths are translucent or transparent, we can recognize the structure or shape of the reverse side without having to turn over the specimen under observation. However, in order to avoid misclassication due to imperfect angles of observation it is important to use liquid mount in making phytolith slides, which allows rotation of object and more accurate measurement of lobe shape and shank length.

RESULTS
Figure 3 shows the relative abundance of different lobate phytolith types found in each of the 85 grass species. The identied morphotypes are illustrated in Fig. 4. Table 3 lists the representative grass taxa of different lobate phytolith types from China and the south-eastern United States. Type A is a phytolith with ridged lines on its shank and lobes. A1 occurs exclusively in Isachne

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

 2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Morphological variations of lobate phytolith 81

Fig. 3 Percentages of lobate phytolith morphotypes calculated based on the sum of lobate phytolith counts for each grass samples. Sample numbers on the left axis: 1, Arundinella hirta 2, Eccoilopus cotulifer 3, Eulalia speciosa 4, Arundinella setosa 5, Setaria faberi 6, S. plicata 7, S. palmifolia 8, Setaria glauca 9, S. sp. 10, Aristida desmantha 11, Pennisetum alopecuroides 12, P. purpureum 13, Panicum notatum 14, Digitaria sanguinalis var. ciliaris 15, Dimeria ornithopoda 16, Chasmanthium laxum 17, Andropogon glomeratus 18, Chasmanthium ornithorhynchum 19, Erianthus strictus 20, Andropogon ternaries 21, Sorghastrum nutans 22, Imperata cylindrica 23, Themeda gigantea var. caudate 24, Themeda triandra var. japonica 25, Schizachyrium brevifolium 26, Eragrostis japonica 27, E. ferruginea 28, Apluda mutica 29, Rottboellia exaltata 30, Capillipedium assimile 31, Panicum verrucosum 32, Microstegium vimineum 33, Leptochloa chinensis 34, Zizania caduciora 35, Oryza sativa 36, Leersia hexandra 37, L. oryzoides 38, Zizaniopsis miliacea 39, Isachne dispar 40, Cymbopogom goeringii 41, Ctenium aromaticum 42, Oplismenus undulatifolius 43, Sacciolepis myosuroides 44, Cyrtococcum patens 45, Oplismenus compositus 46, Panicum austro-asiaticum 47, Pogonatherum crinitum 48, Arthraxon hispidus var. cryptatherus 49, Digitaria violascens 50, Ischaemum antephoroides 51, I. indicum 52, Microstegium vimineum var. imberbe 53, Ischaemum aristatum 54, Digitaria sanguinalis 55, Digitaria adscendens 56, Panicum bisulcatum 57, Spodipogon sibiricus 58, Eriochloa villosa 59, Bothriochloa ischaemum (Southern China) 60, B. ischaemum (Northern China) 61, Capillipedium parviorum 62, Anthaenantia rufa 63, Sorghum halepense 64, Panicum virgatum 65, Sorghum vulgare 66, Echinochloa crusgalli var. mitis 67, Saccharum sinensis 68, Saccharum ofcinarum 69, Saccharum arundinaceum 70, Miscanthus oridulus 71, M. sinensis 72, Cenchrus incertus 73, Panicum repens 74, Hackelochloa granularia 75, Coix lacryma-jobi 76, Coix lacryma var. ma-yuen 77, Zea mays 78, Paspalum orbiculare 79, Echinochloa colonum 80, E. crusgalli 81, Paspalum dilatatum 82, Brachiaria ramosa 83, Panicum amarum 84, Panicum dichotomiorum 85, P. hemitomon. The order of arrangement of different grass species is based on the results of cluster analysis.

82

H. Lu and K.-b. Liu

Fig. 4 Photographs of some representative lobate phytoliths from grass plants 1, Ischaemum antephoroides 2, Saccharum arundinaceum 3, 24, 31, Oryza sativa 4, Miscanthus oridulus 5, 6, 11, Zizania caduciora 7, Zea mays 8, Arthraxon hispidus var. cryptatherus 9, 10, Setaria faberi 12, 18, Themeda triandra var. japonica 13, Coix lacryma-jobi 14, Echinochloa crusgalli 15, Pennisetum purpureum 16, Schizachyrium brevifolium 17, Themeda gigantea var. caudate 19, Rottboellia exaltata 20, 25, Sacciolepis myosuroides 21, Eragrostis ferruginea 22, Dimeria ornithopoda 23, Eragrostis japonica 26, 27, Paspalum orbiculare 28, Oplismenus compositus 29, Apluda mutica 30, Oplismenus undulatifolius.

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Morphological variations of lobate phytolith

dispar. A2 is represented by a wide range of grass taxa that are distributed mainly in the hillsides, grasslands, coasts and wetlands of southern China up to the Yangtze River. A3 is represented mainly by several species of Digitaria. Most of these grasses occur in subtropical South China and are found commonly in wet areas such as lakeshores, stream channels and wet roadside ditches. A4 has few representative species ( Sacciolepis myosuroides) (Fig. 3, no. 43). Type B are phytoliths with smooth rounded lobes. B1 occurs in many species of grasses, but its abundance in each species is relatively low (< 35%) compared to that of B2 and B3. Representative species of B2 are Aristida desmantha (Fig. 3, no. 10) , Saccharum sinensis (sugar cane, Fig. 3, no. 43), Sorghum vulgare (kaoliang or sorghum, Fig. 3, no. 65), Saccharum arundinaceum, Miscanthus oridulus and Miscanthus sinensis (Fig. 3, nos 69, 70, 71). They grow typically in elds and roadside habitats or in warm and humid environments. Representatives of B3 are Setaria glauca (Fig. 3, no. 8), Aristida desmantha , Pennisetum alopecuroides , P. purpureum and Panicum notatum (Fig. 3, nos 10, 11, 12, 13). They grow mainly on the hillsides, roadsides and dry sand dunes of relatively dry environments. B4 is an uncommon type that occurs only at relatively low frequencies in a few taxa. Type C, a phytolith with truncated margins at both ends of its lobes, has the largest number of representative plants. To some extent, C1 may be considered to be diagnostic of the Oryzoideae subfamily, occurring abundantly in such species as Oryza sativa (paddy rice), Leersia hexandra (wild rice), Zizaniopsis miliacea and Z. caduciora (water bamboo) (Fig. 3, nos 3538). Among other grasses, only Panicum amarum (Fig. 3, no. 83) of the Panicoideae subfamily, a typical aquatic and hygrophytic species, produces this type of phytoliths in signicant abundance. C2 is well represented in a large number of grass taxa that notably include Panicum bisulcatum , P. virgatum, Sorghum halepense (Fig. 3, nos 56, 64, 63) Saccharum ofcinarum, Miscanthus oridulus and M. sinensis , among others. These grasses are distributed widely in East China; most are hygrophytes and mesophytes. C3 is found in a smaller range of grasses than C2, but it occurs in great abundance in several taxa including, for example, Digitaria sanguinalis var. ciliaris, Dimeria ornithopoda ,

83

Chasmanthium laxum , Andropogon glomeratus (Fig. 3, nos 14 17), Erianthus strictus , Sorghastrum nutans and Imperata cylindrica. These are mainly heliophytes and drought-enduring mesophytes and xerophytes, growing typically on mountain slopes in both southern and northern China, and on forest edges and disturbed areas of pine woodlands and prairie regions in the United States. The C4 group is basically diagnostic of Eragrostis japonica and E. ferruginea (Fig. 3, nos 26, 27), as well as Schizachyrium brevifolium (Fig. 3, no. 25). The genus Eragrostis, a member of Chloridoideae, is found typically in areas of warm and dry climatic conditions. Type D is phytoliths with concave margins at the end of the lobes. There are only a few representative plants of D1 (Leersia oryzoides, Zizaniopsis miliacea and Anthaenantia rufa (Fig. 3, no. 62) and D4 (Cymbopogom goeringii and Ctenium aromaticum (Fig. 3, nos 40, 41). The representatives of D2 are Leersia oryzoides, Zizania caduciora, Microstegium vimineum var. imberbe and Leptochloa chinensis, which grow mainly in roadside wet ditches, stream channels and other wet habitats such as lakeshores. D3 phytoliths occur most abundantly in Setaria (S. faberi, S. plicata and S. palmifolia; Fig. 3, nos 5, 6, 7) and Arundinella setosa (Fig. 3, no. 4), species found typically in warm and wet subtropical environments. Type E is phytoliths with branched outer margins at the end of the lobes. E1 is a typical crossshape, the size and shape of which is somewhat variable. This type of phytolith occurs predominantly in a number of species that include (although not restricted to) some important agricultural crops in the temperate and tropical regions of the world, such as Zea mays (maize), Coix lacryma-jobi (Jobs tears), Saccharum sinensis (sugar cane) and Sorghum vulgare (kaoliang or sorghum) (Fig. 3, nos 77, 75, 67, 65). E2 occurs abundantly only in Coix lacryma var. ma-yuen (Jobs tears), a crop cultivated commonly in China. Incidentally, Coix lacryma var. ma-yuen and other members of the Maydeae subfamily produce only the E2 and E1 phytoliths. E3 can be found in many plants, but by itself is diagnostic of none. No E4 phytoliths were found in our samples in this study, although this morphotype has been observed in fossil assemblages (H.Y. Lu, unpublished data).

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

84

H. Lu and K.-b. Liu

C3 grasses from both Oryzoideae and Panicoideae subfamilies in this study are adapted typically to moist or marshy environments and are distributed widely in tropical and subtropical regions of the world.

Type F has multiple lobes and can be found in many plants. The plants that can produce the F type include Capillipedium assimile , Panicum verrucosum (Fa), Eulalia speciosa, Rottboellia exaltata, Capillipedium assimile, Panicum verrucosum, Oplismenus undulatifolius (Fb), Sacciolepis myosuroides (Fc), Panicum bisulcatum, Spodipogon sibiricus (Fd) and Apluda mutica (Fe). The plants of Type F are distributed mainly in the southern parts of China. It is worth noting that lobate phytoliths are also found in a few Chloridoideae plants, for example, in Leptochloa chinensis (C2), Eragrostis japonica and E. ferruginea (C4). More studies are needed on these non-Panicoideae grasses that produce lobate phytoliths.

Morphological variations in lobate phytoliths from Chloridoideae

Lobate phytoliths from the Chloridoideae subfamily have been reported by Brown (1984) and Mulholland (1989). In this study, many variations in lobate phytoliths were observed in Chloridoideae (including Eragrostis japonica, E. ferruginea, Ctenium aromaticum, Aristida desmantha and Leptochloa chinensis). Eragrostis japonica, E. ferruginea and Ctenium aromaticum produce different phytolith assemblages in which 40 98% of the lobates are C4 and D4 types with very a long shank between the lobes (Fig. 3, nos 26, 27, 41). These species are distributed widely in warm and arid regions. B2 and B3 types are the dominant phytoliths of Aristida desmantha (Fig. 3, no. 10) that also belongs to the Chloridoideae subfamily (or the Arundinoideae subfamily according to the classication system of Watson et al., 1985). The sample of Aristida desmantha was collected from dry sand dunes on the Georgia coast of the United States. Leptochloa chinensis (Fig. 3, no. 33) yields only two phytolith types: D2 (83%) and D3 (17%).

DISCUSSION Morphological variations in lobate phytoliths from C3 grasses

Early phytolith researchers noted that the different subfamilies of grass plants produce different phytolith shapes: Panicoideae produces dumbbell and cross-shaped phytoliths; Festucoid forms rondels and sinuous types, and Chloridoideae yields saddles (Brown, 1984; Mulholland, 1989; Fearn, 1998). The shape of individual phytoliths from grasses can be used as an indication of C3 or C4 photosynthetic pathways. For example, saddle phytolith indicates C4 short-grass prairie species that ourish in warm, arid to semi-arid regions where the available soil moisture is very low, whereas dumbbell and cross phytoliths represent the grasses of the C4 tall-grass prairies, which have high to medium soil-moisture availability (Twiss, 1992). In this study, we found that several C3 grasses (Waller et al., 1979; Gould & Shaw, 1983; Lu & Wang, 1991) also produce characteristic lobate morphotypes, such as Zizania caduciora, Oryza sativa, Leersia hexandra, L. oryzoides and Zizaniopsis miliacea of the Oryzoideae subfamily, and Oplismenus compositus, O. compositus, Sacciolepis myosuroides and Isachne dispar of the Panicoideae subfamily. Lobate phytoliths in Oryzoideae are characterized by high proportions of C1 and, to a lesser extent, D1 types with very short shank between the lobes. Other C3 grasses from Panicoideae produce characteristic A1 and A2 types with ridged lines on its lobes and F type with multiple lobes.

Can we infer grass genera from lobate phytolith assemblages?

The classication of phytolith shapes has long been criticized due to the multiplicity and redundancy of many grass morphotypes a problem preventing the attribution of individual phytolith to species or genus (Rovner, 1971; Brown, 1984; Mulholland, 1989). Because the same shapes of phytoliths can occur in different grass taxa, a single phytolith morphotype cannot be ascribed to a specic grass species. However, a phytolith assemblage could, to some extent, allow us to infer the predominant subfamily constituting the grass associations. Recently, many researchers have paid particular attention to this specic taxonomic problem of redundancy for grass phytolith classication. They indicated that it would never be possible to eliminate all redundancies at

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Morphological variations of lobate phytolith

the subfamily level, much less for genus (Fredlund & Tieszen, 1994). In this study, we tried to nd a potential relationship between the morphological variations in lobate phytoliths and the grass genera that produce them (Fig. 3, Table 3). We found some interesting patterns in several cases, as follows. Three grass plants belonging to the genus Setaria (S. faberi, S. plicata, S. palmifolia) were dened by a high abundance of D3 type. The two species of Eragrostis (E. japonica, E. ferruginea; subfamily Chloridoideae) produce almost exclusively the C4 type of lobate phytoliths. Zizania caduciora, Oryza sativa, Leersia hexandra, L. oryzoides and Zizaniopsis miliacea , all belonging to the subfamily Oryzoideae, produce a signicant amount of C1 and, to some extent, D2 types of lobate phytoliths. Miscanthus yields primarily B2 and C2 types of phytoliths. Maydeae (Coix lacrymajobi, C. l. var. ma-yuen, Zea mays) yields only E1 and E2 (cross) types. Remarkably, the three species of Saccharum (S. sinensis, S. ofcinarum and S. arundinaceum) produces different lobate phytolith assemblages (Fig. 3). Saccharum sinensis (sugar cane), in particular, is characterized by the codominance of E1 (54%) and B2 (29%) types, whereas S. ofcinarum is characterized by the codominance of C2 (35%) and C3 (24%) types, and S. arundinaceum by the preponderance of B2 (51%) mixed with some E1 and B1. Unfortunately, not all lobate phytolith assemblages have a denite and consistent relationship with the grass genera that produce them, because grasses only produce a limited range of lobate phytoliths, which often overlap from genus to genus. Moreover, the relatively limited sample size in each genus used in this study prevents us from generalizing the characteristics of lobate assemblages for all grass genera.

85

Morphological changes in lobate phytoliths along an environmental gradient

Although different parts of plant body from one species often contribute different lobate phytoliths to an assemblage, many grasses do produce predominantly a specic phytolith type recognizable by distinct shape, sculpture or size that can be assigned to a given taxon at various levels. Morphological variations in phytoliths can be produced by both botanical and environmental

factors (Mulholland et al ., 1988). Thus, it is possible to discuss the morphological changes in lobate phytoliths along environmental gradients. As shown in Fig. 3, the representatives of C1 are Oryzoideae that typically consist of aquatic and hygrophytic grasses. The principal members of B1 type are the grasses frequently growing in wet areas and on lakeshores, such as Echinochloa crusgalli (Fig. 3, no. 80) and Paspalum dilatatum (Fig. 3, no. 81). Grasses from the Chloridoideae subfamily (e.g. Eragrostis ) and Panicoideae subfamily (e.g. Cymbopogom goeringii, Ctenium aromaticum; Fig. 3, nos 40, 41) that grow in dry conditions produce predominantly C4 and D4 phytoliths. Grasses of the Maydeae subfamily, found typically in warm and moist areas, yield predominantly the cross phytoliths of E1 and E2 types. The representative grasses of B3 and B4 types (e.g. Setaria glauca, Aristida desmantha , Pennisetum alopecuroides; Fig. 3, nos 8, 10, 11) grow mainly in drier habitats such as hillsides, road sides and sand dunes of arid regions. As a rst-order generalization, it seems that the progression from type 1 to type 4 represents an environmental gradient of decreasing moisture (i.e. from wet to dry). In other words, grasses growing in drier habitats or environments tend to produce phytoliths with a longer shank and vice versa. The environmental signicance of the progression from types AE is less clear at this point. What is the cause of morphological variability in lobate phytoliths? Is it environmental phenotype or genotype? Wang & Lu (1993) compared morphological changes in short cell phytoliths from the same species of grasses growing in different environmental conditions in China. They showed that phytolith shapes are relatively stable, but sizes can change slightly. Piperno (1988) suggested that phytoliths formed in the short cells of the grass epidermis are under a considerable degree of active genetic control. To date, no strong evidence can be found from our data to resolve the question of whether phytolith shapes are phenotype or genotype. Regardless of the cause of morphological variations, our study suggests that in some ways phytolith morphology could be linked to grass taxonomy (e.g. C1 is diagnostic of Oryzoideae). As many grass taxa tend to have certain typical environmental adaptations (e.g. Oryzoideae occurring typically in wet habitats), it may be

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

86

H. Lu and K.-b. Liu

ACKNOWLEDGMENTS
We thank Y.J. Wang, X.Y. Zhou, C.A. Reese and C.M. Shen for providing modern grass reference samples and for helpful discussion. We are grateful to S.C. Mulholland, G.G. Fredlund and I. Rovner for valuable comments on an earlier version of this manuscript. We also thank the two anonymous reviewers for their helpful reviews of our original manuscript. This work was supported by NSFC (40024202, 49971077 and 49894174), NKBRSF (G1998040810), the Risk Prediction Initiative (RPI) of the Bermuda Biological Station for Research (RPI-00-1-002) and the U.S. National Science Foundation (SES-9122058; BCS-0213884).

possible to use phytolith morphology as a proxy for environmental conditions. More work is needed to substantiate this point.

CONCLUSIONS
This study documents the variability of 25 diagnostic lobate phytolith shapes occurring among 85 modern grass species collected from a variety of environments in China and the south-eastern United States. We propose a classication that is based on two important morphological parameters: the length of the lobate shank and the shape of the outer margin of the two lobes. These two morphological characteristics are relatively stable among the 85 modern grass species belonging to the Panicoideae, Chloridoideae, Oryzoideae and Arundinoideae subfamilies. In some cases, the identication of tribe or even genus is possible based on the differences in lobate shape parameters or the composition of assemblages. However, we should point out that not all of the lobate assemblages have a consistent and denite relationship with the genera that produce them. This is because grasses can only produce a limited range of lobate shapes, and there is often considerable overlap from one genus to another. In this study, we found that several C3 grasses of the Oryzoideae subfamily produce characteristic lobate morphotypes, which are characterized by a high proportion of C1 and D1 types and by a very short shank between the lobes. Other C3 grasses from Panicoideae produce characteristic A1 and A2 types with ridged lines and F type with multiple lobes. The Chloridoideae subfamily produces lobate assemblages in which 40 98% of phytoliths are C4 and D4 types with a very long shank between the lobes. This group of grasses is widely distributed in warm and arid regions. We also found that the variations of lobate morphologies can be related to environmental factors, especially moisture. Typical hygrophytic grasses tend to yield lobate phytoliths with a very short shank, whereas typical xerophytic grasses tend to produce lobate phytoliths with very long shank. This relationship, if supported by additional studies of lobate phytoliths derived from more grass species and from a wider range of environmental conditions, offers potential for using phytoliths in palaeoclimatic reconstruction.

REFERENCES
Alexandre, A., Meunier, J.-D., Lezine, A.-M., Vincens, A. & Schwartz, D. (1997) Phytoliths: indicators of grassland dynamics during the late Holocene in intertropical Africa. Palaeogeography, Palaeoclimatology, Palaeoecology 136, 213 219. Brown, D.A. (1984) Prospects and limits of a phytolith key for grasses in the central United States. Journal of Archaeological Sciences 11, 345 368. Cummings, L.S. (1992) Illustrated phytoliths from assorted food plants. Phytolith Systematics (ed. by G. Rapp, Jr and S.C. Mulholland), pp. 175 192. Plenum Press, New York. Ellis, R.P. (1979) A procedure for standardizing comparative leaf anatomy in the Poaceae. II. The epidermis as seen in surface view. Bothalia 12, 641 671. Fearn, M.L. (1998) Phytolith in sediment as indicators of grass pollen source. Review of Palaeobotany and Palynology 103, 75 81. Fearn, M.L. & Liu, K.-B. (1997) Identication of maize pollen: reply to Eubanks. American Antiquity 62, 146 148. Fredlund, G.G. & Tieszen, L.L. (1994) Modern phytolith assemblages from the North American Great Plains. Journal of Biogeography 21, 321 335. Fredlund, G.G. & Tieszen, L.L. (1997) Calibrating grass phytolith assemblages in climatic terms: application to late Pleistocene assemblages from Kansas and Nebraska. Palaeogeography, Palaeoclimatology, Palaeoecology 136, 199 211. Gould, F.W. & Shaw, R.B. (1983) Grass Systematics. Collage Station. Texas A & M University Press, College Station. GPWG (2001) Phylogeny and subfamilial classication of the grasses (Poaceae). Annals of the Missouri Botanical Garden 88, 373 457.

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87

Morphological variations of lobate phytolith

Horrocks, M., Deng, Y., Ogden, J. & Sutton, D.G. (2000) A reconstruction of the history of Holocene sand dune on Great Barrier Island, northern New Zealand, using pollen and phytolith analyses. Journal of Biogeography 27, 1269 1277. Institute of Botany, Chinese Academy of Sciences (1977) Iconographia Cormophytorum Sinicorum. Tomus V. Science Press, Beijing [in Chinese]. Kelly, E.F., Amundson, R.G., Marino, B.D. & Deniro, M.J. (1991) Stable isotope ratios of carbon in phytoliths as a quantitative method of monitoring vegetation and climate change. Quaternary Research 35, 222 233. Kondo, R., Childs, C. & Atkinson, I. (1994) Opal phytoliths of New Zealand. Manaaki Whenua Press, Canterbury. Lu, H.Y. (1998) Quaternary environmental changes recorded by magnetic susceptibility and plant fossils: quantitative estimates of paleoclimates. PhD Thesis. Institute of Geology, Chinese Academy of Sciences, China [in Chinese, with English abstract]. Lu, H.Y. & Wang, Y.J. (1991) A study on phytoliths in loess prole and paleoenvironmental evolution at Heimugou in Luochuan, Shaanxi province since late Pleistocene. Quaternary Sciences 1, 72 84 [in Chinese]. Lu, H.Y., Wu, N.Q., Liu, D.S., Han, J.M., Qin, X.G., Sun, X.J. & Wang, Y.J. (1996) Seasonal climatic variation recorded by phytolith assemblages from the Baoji loess sequence in central China over the last 150 000 a. Science in China (Series D) 39, 629 639. Metcalfe, C.R. (1960) Anatomy of the Monocotyledons. I. Gramineae. Clarendon Press, Oxford. Mulholland, S.C. (1989) Phytolith shape frequencies in North Dakota grasses: a comparison to general patterns. Journal of Archaeological Science 16, 489 511. Mulholland, S.C. & Rapp, G. Jr (1992) A morphological classication of grass silica-bodies. Phytolith systematics: advances in archaeological and museum science (ed. by G. Rapp Jr and S.C. Mulholland), pp. 65 89. Plenum Press, New York and London. Mulholland, S.C., Rapp, G. Jr & Ollendorf, A.L. (1988) Variation in phytoliths from corn leaves. Canadian Journal of Botany 66, 2001 2008. Pearsall, D.M. (1978) Phytolith analysis of archeo-

87

logical soils: evidence for maize cultivation in Formative Ecuador. Science 199, 177 178. Pearsall, D.M. & Piperno, D.R. (1990) Antiquity of maize cultivation in Ecuador: summary and reevaluation of the evidence. American Antiquity 55, 324 337. Piperno, D.R. (1984) A comparison and differenatiation of phytolith from maize and wild grasses: use of morphological criteria. American Antiquity 49, 361 383. Piperno, D.R. (1988) Phytolith analysis: an archaeological and geological perspective. Academic Press, San Diego. Piperno, D.R. & Pearsall, D.M. (1998) The silica bodies of tropical American grasses: morphology, taxonomy, and implications for grass systematics and fossil phytolith identication. Annals of the Smithsonian Institution, 85, 1 40. Rapp, G. Jr & Mulholland, S.C. (1992) Phytolith systematics: advances in archaeological and museum science. Plenum Press, New York and London. Rovner, I. (1971) Potential of opal phytoliths for use in paleoecological reconstruction. Quaternary Research 1, 343 359. Runge, F. (1999) The opal phytolith ineventory of soils in central Africa quantities, shapes, classication, and spectra. Review of Palaeobotany and Palynology 107, 23 53. Twiss, P.C. (1992) Predicted world distribution of C3 and C4 grass phytoliths. Phytolith systematics: advances in archaeological and museum science (ed. by G. Rapp Jr and S.C. Mulholland), pp. 113 128. Plenum Press, New York and London. Twiss, P.C., Suess, E. & Smith, R.M. (1969) Division S-5 soil genesis, morphology, and classication (morphological classication of grass phytoliths). Soil Science Society of America, Proceedings 33, 109 115. Waller, S.S. & Lewis, J.K. (1979) Occurrence of C3 and C4 photosynthetic pathways in North American grasses. Journal of Range Management 32, 12 28. Wang, Y.J. & Lu, H.Y. (1993) The study of phytolith and its application. China Ocean Press, Beijing [in Chinese]. Watson, L., Clifford, H.T. & Dallwitz, M.J. (1985) The classication of Poaceae: subfamilies and supertribes. Australian Journal of Botany 33, 433 484.

2003 Blackwell Publishing Ltd, Diversity and Distributions, 9, 73 87