Palms of the past: can morphometric phytolith analysis inform deep time evolution and palaeoecology of Arecaceae?

BACKGROUND AND AIMS: Palm fossils are often used as evidence for warm and wet palaeoenvironments, reflecting the affinities of most modern palms. However, several extant palm lineages tolerate cool and/or arid climates, making a clear understanding of the taxonomic composition of ancient palm communities important for reliable palaeoenvironmental inference. However, taxonomically identifiable palm fossils are rare and often confined to specific facies. Although the resolution of taxonomic information they provide remains unclear, phytoliths (microscopic silica bodies) provide a possible solution because of their high preservation potential under conditions where other plant fossils are scarce. We thus evaluate the taxonomic and palaeoenvironmental utility of palm phytoliths. METHODS: We quantified phytolith morphology of 97 modern palm and other monocot species. Using this dataset, we tested the ability of five common discriminant methods to identify nine major palm clades. We then compiled a dataset of species' climate preferences and tested if they were correlated with phytolith morphology using a phylogenetic comparative approach. Finally, we reconstructed palm communities and palaeoenvironmental conditions at six fossil sites. KEY RESULTS: Best-performing models correctly identified phytoliths to their clade of origin only 59 % of the time. Although palms were generally distinguished from non-palms, few palm clades were highly distinct, and phytolith morphology was weakly correlated with species' environmental preferences. Reconstructions at all fossil sites suggested that palm communities were dominated by Trachycarpeae and Areceae, with warm, equable climates and high, potentially seasonal rainfall. However, fossil site reconstructions had high uncertainty and often conflicted with other climate proxies. CONCLUSIONS: While phytolith morphology provides some distinction among palm clades, caution is warranted. Unlike prior spatially restricted studies, our geographically and phylogenetically broad study indicates phytolith morphology may not reliably differentiate most palm taxa in deep time. Nevertheless, it reveals distinct clades, including some likely to be palaeoenvironmentally informative.

Inter- and intra-observer variation in phytolith morphometry.

BACKGROUND: Archaeobotanists and palaeoecologists extensively use geometric morphometrics to identify plant opal phytoliths. Particularly when applied to assemblages of phytoliths from concentrations retrieved from closed contexts, morphometric data from archaeological phytoliths compared with similar data from reference material may allow taxonomic attribution. Observer variation is one aspect of phytolith morphometry that has received little attention but may be an important source of error, and hence cause of potential misidentification of plant remains. SCOPE: To investigate inter- and intra-observer variation in phytolith morphometry, eight researchers (observers) from different laboratories measured 50 samples each from three phytolith morphotypes, Bilobate, Bulliform flabellate and Elongate dendritic, three times, under the auspices of the International Committee for Phytolith Morphometrics (ICPM). METHODS: Data for 17 size and shape variables were collected for each phytolith by manually digitising a phytolith outline (mask) from a photograph, followed by measurement of the mask with open-source morphometric software. KEY RESULTS: Inter-observer variation ranged from 0 to 23% difference from the mean of all observers. Intra-observer variation ranged from 0 to 9% difference from the mean of individual observers per week. Inter- and intra-observer variation was generally higher among inexperienced researchers. CONCLUSIONS: Scaling errors were a major cause of variation and occurred more with less experienced researchers, which is likely related to familiarity with data collection. The results indicate that inter- and intra-observer variation can be substantially reduced by providing clear instructions for and training with the equipment, photo capturing, software, data collection and data cleaning. In this paper, the ICPM provides recommendations to minimise variation.Advances in automatic data collection may eventually reduce inter- and intra-observer variation, but until this is common practice, the ICPM recommends that phytolith morphometric analyses adhere to standardised guidelines to assure that measured phytolith variables are accurate, consistent and comparable between different researchers and laboratories.

Evapotranspiration-linked silica deposition in a basal tracheophyte plant (Lycopodiaceae: Lycopodiella alopecuroides): implications for the evolutionary origins of phytoliths.

Phytoliths, microscopic deposits of hydrated silica within plants, play a myriad of functional roles in extant tracheophytes - yet their evolutionary origins and the original selective pressures leading to their deposition remain poorly understood. To gain new insights into the ancestral condition of tracheophyte phytolith production and function, phytolith content was intensively assayed in a basal, morphologically conserved tracheophyte: the foxtail clubmoss Lycopodiella alopecuroides. Wet ashing was employed to perform phytolith extractions from every major anatomical region of L. alopecuroides. Phytolith occurrence was recorded, alongside abundance, morphometric information, and morphological descriptions. Phytoliths were recovered exclusively from the microphylls, which were apicodistally silicified into multiphytolith aggregates. Phytolith aggregates were larger and more numerous in anatomical regions engaging in greater evapotranspirational activity. The tissue distribution of L. alopecuroides phytoliths is inconsistent with the expectations of proposed adaptive hypotheses of phytolith evolutionary origin. Instead, it is hypothesized that phytoliths may have arisen incidentally in the L. alopecuroides-like ancestral plant, polymerizing from intraplant silicon accumulations arising via bulk flow and 'leaky' cellular micronutrient channels. This basal, nonadaptive phytolith formation model would provide the evolutionary 'raw material' for later modification into the useful, adaptative, phytolith deposits seen in later-diverging plant clades.

Seasonal drought events in tropical East Asia over the last 60,000 y.

The cause of seasonal hydrologic changes in tropical East Asia during interstadial/stadial oscillations of the last glaciation remains controversial. Here, we show seven seasonal drought events that occurred during the relatively warm interstadials by phytolith and pollen records. These events are significantly manifested as high percentages of bilobate phytoliths and are consistent with the large zonal sea-surface temperature (SST) gradient from the western to eastern tropical Pacific, suggesting that the reduction in seasonal precipitation could be interpreted by westward shifts of the western Pacific subtropical high triggered by changes of zonal SST gradient over the tropical Pacific and Hadley circulation in the Northern Hemisphere. Our findings highlight that both zonal and meridional ocean-atmosphere circulations, rather than solely the Intertropical Convergence Zone or El Niño-Southern Oscillation, controlled the hydrologic changes in tropical East Asia during the last glaciation.

High potential of stable carbon sequestration in phytoliths of China's grasslands.

Phytolith carbon (C) sequestration plays a key role in mitigating global climate change at a centennial to millennial time scale. However, previous estimates of phytolith-occluded carbon (PhytOC) storage and potential in China's grasslands have large uncertainties mainly due to multiple data sources. This contributes to the uncertainty in predicting long-term C sequestration in terrestrial ecosystems using Earth System Models. In this study, we carried out an intensive field investigation (79 sites, 237 soil profiles [0-100 cm], and 61 vegetation assessments) to quantify PhytOC storage in China's grasslands and to better explore the biogeographical patterns and influencing factors. Generally, PhytOC production flux and soil PhytOC density in both the Tibetan Plateau and the Inner Mongolian Plateau had a decreasing trend from the Northeast to the Southwest. The aboveground PhytOC production rate in China's grassland was 0.48 × 106 t CO2 a-1 , and the soil PhytOC storage was 383 × 106 t CO2 . About 45% of soil PhytOC was stored in the deep soil layers (50-100 cm), highlighting the importance of deep soil layers for C stock assessments. Importantly, the Tibetan Plateau had the greatest contribution (more than 70%) to the PhytOC storage in China's grasslands. The results of multiple regression analysis indicated that altitude and soil texture significantly influenced the spatial distribution of soil PhytOC, explaining 78.1% of the total variation. Soil phytolith turnover time in China's grasslands was mainly controlled by climatic conditions, with the turnover time on the Tibetan Plateau being significantly longer than that on the Inner Mongolian Plateau. Our results offer more accurate estimates of the potential for phytolith C sequestration from ecological restoration projects in degraded grassland ecosystems. These estimates are essential to parameterizing and validating global C models.

Thermal induced changes of rice straw phytolith in relation to arsenic release: A perspective of rice straw arsenic under open burning.

On-site open burning is a common practice for handling rice straw, but its negative impacts, e.g., biomass loss and air pollution, are largely debated worldwide. To address the negative effects of open burning, many efforts have been made to 'ignite' worldwide bans. However, these bans are likely based on a singular view in which some positive aspects of open burning are overlooked. In this study, we aimed to determine the thermal-induced changes of straw and straw arsenic (As) under open burning and heat-treatments (in the temperature range from 300 to 900 °C). It was found that silica phase in rice straw (so-called phytolith) can encapsulate As in its structure. Open burning or heat-treatment of straw resulted in a tighter association of As and phytolith, thereby reducing dissolution of As. We proposed an opinion that open burning causes air pollution, but it can increase the activity of phytolith in sequestrating As, enabling delayed As cycle in rice ecosystems. The combat of on-site open burning of rice straw to reduce air pollution will alter straw handling routines, thereby changing the cycle of straw phytolith and the route of straw As.

Rare earth elements, aluminium and silicon distribution in the fern Dicranopteris linearis revealed by µPIXE Maia analysis.

BACKGROUND: The fern Dicranopteris linearis is a hyperaccumulator of rare earth elements (REEs), aluminium (Al) and silicon (Si). However, the physiological mechanisms of tissue-level tolerance of high concentrations of REE and Al, and possible interactions with Si, are currently incompletely known. METHODS: A particle-induced X-ray emission (µPIXE) microprobe with the Maia detector, scanning electron microscopy with energy-dispersive spectroscopy and chemical speciation modelling were used to decipher the localization and biochemistry of REEs, Al and Si in D. linearis during uptake, translocation and sequestration processes. RESULTS: In the roots >80 % of REEs and Al were in apoplastic fractions, among which the REEs were most significantly co-localized with Si and phosphorus (P) in the epidermis. In the xylem sap, REEs were nearly 100 % present as REEH3SiO42+, without significant differences between the REEs, while 24-45 % of Al was present as Al-citrate and only 1.7-16 % Al was present as AlH3SiO42+. In the pinnules, REEs were mainly concentrated in necrotic lesions and in the epidermis, and REEs and Al were possibly co-deposited within phytoliths (SiO2). Different REEs had similar spatial localizations in the epidermis and exodermis of roots, the necrosis, veins and epidermis of pinnae of D. linearis. CONCLUSIONS: We posit that Si plays a critical role in REE and Al tolerance within the root apoplast, transport within the vascular bundle and sequestration within the blade of D. linearis.

Inter- and intraspecific variation in grass phytolith shape and size: a geometric morphometrics perspective.

BACKGROUND AND AIMS: The relative contributions of inter- and intraspecific variation to phytolith shape and size have only been investigated in a limited number of studies. However, a detailed understanding of phytolith variation patterns among populations or even within a single plant specimen is of key importance for the correct taxonomic identification of grass taxa in fossil samples and for the reconstruction of vegetation and environmental conditions in the past. In this study, we used geometric morphometric analysis for the quantification of different sources of phytolith shape and size variation. METHODS: We used landmark-based geometric morphometric methods for the analysis of phytolith shapes in two extant grass species (Brachypodium pinnatum and B. sylvaticum). For each species, 1200 phytoliths were analysed from 12 leaves originating from six plants growing in three populations. Phytolith shape and size data were subjected to multivariate Procrustes analysis of variance (ANOVA), multivariate regression, principal component analysis and linear discriminant analysis. KEY RESULTS: Interspecific variation largely outweighed intraspecific variation with respect to phytolith shape. Individual phytolith shapes were classified with 83 % accuracy into their respective species. Conversely, variation in phytolith shapes within species but among populations, possibly related to environmental heterogeneity, was comparatively low. CONCLUSIONS: Our results imply that phytolith shape relatively closely corresponds to the taxonomic identity of closely related grass species. Moreover, our methodological approach, applied here in phytolith analysis for the first time, enabled the quantification and separation of variation that is not related to species discrimination. Our findings strengthen the role of grass phytoliths in the reconstruction of past vegetation dynamics.

Aluminium-silicon interactions in higher plants: an update.

Aluminium (Al) and silicon (Si) are abundant in soils, but their availability for plant uptake is limited by low solubility. However, Al toxicity is a major problem in naturally occurring acid soils and in soils affected by acidic precipitation. When, in 1995, we reviewed this topic for the Journal of Experimental Botany, it was clear that under certain circumstances soluble Si could ameliorate the toxic effects of Al, an effect mirrored in organisms beyond the plant kingdom. In the 25 years since our review, it has become evident that the amelioration phenomenon occurs in the root apoplast, with the formation of hydroxyaluminosilicates being part of the mechanism. A much better knowledge of the molecular basis for Si and Al uptake by plants and of Al toxicity mechanisms has been developed. However, relating this work to amelioration by Si is at an early stage. It is now clear that co-deposition of Al and Si in phytoliths is a fairly common phenomenon in the plant kingdom, and this may be important in detoxification of Al. Relatively little work on Al-Si interactions in field situations has been done in the last 25 years, and this is a key area for future development.

Siliplant1 protein precipitates silica in sorghum silica cells.

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

High silicon concentrations in grasses are linked to environmental conditions and not associated with C4 photosynthesis.

The uptake and deposition of silicon (Si) as silica phytoliths is common among land plants and is associated with a variety of functions. Among these, herbivore defense has received significant attention, particularly with regard to grasses and grasslands. Grasses are well known for their high silica content, a trait which has important implications ranging from defense to global Si cycling. Here, we test the classic hypothesis that C4 grasses evolved stronger mechanical defenses than C3 grasses through increased phytolith deposition, in response to extensive ungulate herbivory ("C4 -grazer hypothesis"). Despite mixed support, this hypothesis has received broad attention, even outside the realm of plant biology. Because C3 and C4 grasses typically dominate in different climates, with the latter more abundant in hot, dry regions, we also investigated the effects of water availability and temperature on Si deposition. We compiled a large dataset of grasses grown under controlled environmental conditions. Using phylogenetically informed generalized linear mixed models and character evolution models, we evaluated whether photosynthetic pathway or growth condition influenced Si concentration. We found that C4 grasses did not show consistently elevated Si concentrations compared with C3 grasses. High temperature treatments were associated with increased concentration, especially in taxa adapted to warm regions. Although the effect was less pronounced, reduced water treatment also promoted silica deposition, with slightly stronger response in dry habitat species. The evidence presented here rejects the "C4 -grazer hypothesis." Instead, we propose that the tendency for C4 grasses to outcompete C3 species under hot, dry conditions explains previous observations supporting this hypothesis. These findings also suggest a mechanism via which anthropogenic climate change may influence silica deposition in grasses and, by extension, alter the important ecological and geochemical processes it affects.

Dating rice remains through phytolith carbon-14 study reveals domestication at the beginning of the Holocene.

Phytolith remains of rice (Oryza sativa L.) recovered from the Shangshan site in the Lower Yangtze of China have previously been recognized as the earliest examples of rice cultivation. However, because of the poor preservation of macroplant fossils, many radiocarbon dates were derived from undifferentiated organic materials in pottery sherds. These materials remain a source of debate because of potential contamination by old carbon. Direct dating of the rice remains might serve to clarify their age. Here, we first validate the reliability of phytolith dating in the study region through a comparison with dates obtained from other material from the same layer or context. Our phytolith data indicate that rice remains retrieved from early stages of the Shangshan and Hehuashan sites have ages of approximately 9,400 and 9,000 calibrated years before the present, respectively. The morphology of rice bulliform phytoliths indicates they are closer to modern domesticated species than to wild species, suggesting that rice domestication may have begun at Shangshan during the beginning of the Holocene.

Revealing a 5,000-y-old beer recipe in China.

The pottery vessels from the Mijiaya site reveal, to our knowledge, the first direct evidence of in situ beer making in China, based on the analyses of starch, phytolith, and chemical residues. Our data reveal a surprising beer recipe in which broomcorn millet (Panicum miliaceum), barley (Hordeum vulgare), Job's tears (Coix lacryma-jobi), and tubers were fermented together. The results indicate that people in China established advanced beer-brewing technology by using specialized tools and creating favorable fermentation conditions around 5,000 y ago. Our findings imply that early beer making may have motivated the initial translocation of barley from the Western Eurasia into the Central Plain of China before the crop became a part of agricultural subsistence in the region 3,000 y later.

Association of wheat chaff derived silica fiber and esophageal cancer in north China.

BACKGROUND: Despite decades of research and intervention programs, the epidemic of esophageal squamous cell carcinoma (ESCC) in the Taihang Mountain area of north China has not seen convincing explanation by any risk factor yet and the incidence has not seen a substantial decrease. Based on recently disclosed association of aridity and wheat consumption with esophageal cancer, we revisited the hypothesis of biogenic silica in esophageal cancer development. METHODS: From the archives of the Pathology Department of Heping Hospital, Changzhi Medical College, we selected three pairs of formalin-fixed samples, tumor tissues and distant normal tissues, of three patients operated for ESCC who had no history of workplace exposure to silica dust. Two pairs of dried tissue samples were used for phytolith (silica body) analysis and another pair for microanalysis with Transmission Electron Microscope (TEM). RESULTS: One of the phytoliths in ESCC tumor tissue was similar to the prickle hair on the surface of wheat bract. In the mineral particles detected in the tumor tissue the predominant elements were Si, Ca, and P, whereas Si signals were not obvious in the distant normal tissue. CONCLUSIONS: The preliminary findings on the detection of phytoliths and the higher than normal Si concentration in ESCC tumor tissue warrants further testing the role of biogenic silica in esophageal cancer.

Copper encapsulated in grass-derived phytoliths: Characterization, dissolution properties and the relation of content to soil properties.

The formation of phytoliths as a result of the precipitation of Si in many Si-rich plant species is known to encapsulate organic matter. This work aims to examine the possible encapsulation of Cu in grass phytoliths in an orange growing area, where Cu-rich fungicides have been excessively applied. Batch experiments, in combination with SEM-EDS and microscopy, were conducted for the grass-derived phytoliths and phytoliths separated from soil, thus revealing their dissolution properties, morphotypes and contents, in relation to soil properties. By measuring the Cu release accompanying the dissolution of phytoliths by different extractants, especially an Na2CO3/HNO3 solution, it was revealed that Cu was encapsulated within the silica body of the phytolith. This sink of Cu in the grass can be cycled to serve as a new Cu source in soils. Phytolith contents in the soil were up to 17.7 g kg-1 and tended to accumulate in soil depths from 0 to 20 cm. A positive correlation was found for soil phytolith and phytCu contents and may be indicative of the role of phytoliths as an enhancer of Cu accumulation in soil. It would be worth developing suitable techniques for the determination of phytCu, because common extraction/digestion methods are not suited for evaluating this Cu pool.

Four hundred million years of silica biomineralization in land plants.

Biomineralization plays a fundamental role in the global silicon cycle. Grasses are known to mobilize significant quantities of Si in the form of silica biominerals and dominate the terrestrial realm today, but they have relatively recent origins and only rose to taxonomic and ecological prominence within the Cenozoic Era. This raises questions regarding when and how the biological silica cycle evolved. To address these questions, we examined silica abundances of extant members of early-diverging land plant clades, which show that silica biomineralization is widespread across terrestrial plant linages. Particularly high silica abundances are observed in lycophytes and early-diverging ferns. However, silica biomineralization is rare within later-evolving gymnosperms, implying a complex evolutionary history within the seed plants. Electron microscopy and X-ray spectroscopy show that the most common silica-mineralized tissues include the vascular system, epidermal cells, and stomata, which is consistent with the hypothesis that biomineralization in plants is frequently coupled to transpiration. Furthermore, sequence, phylogenetic, and structural analysis of nodulin 26-like intrinsic proteins from diverse plant genomes points to a plastic and ancient capacity for silica accumulation within terrestrial plants. The integration of these two comparative biology approaches demonstrates that silica biomineralization has been an important process for land plants over the course of their >400 My evolutionary history.

Microscopic Chemical Characterization and Reactivity in Cementing Systems of Elephant Grass Leaf Ashes.

Many agrowastes are being used for energy production by combustion in power plants. This process generates huge amounts of ash, which has a potential pozzolanic activity for blending with Portland cement or hydrated lime. In this paper, the ash obtained from elephant grass (Pennisetum purpureum Schum var. purple) leaves (EGLs) was studied, including the silicon content and its distribution, the presence of other compounds, and in addition, the presence of silica bodies (phytoliths). Combustion temperatures of 450 and 650°C produced an unaltered inorganic skeleton (spodogram), whereas at 850°C, there is a sintering process because of high potassium content in the ash. Phytoliths and different types of hairs were identified, and they contained high percentages of silica. Magnesium (mainly as periclase) was distributed in the most porous parts in the interior of the leaves. The silica can react with calcium hydroxide (pozzolanic reaction) forming calcium silicate hydrates (observed by field-emission scanning electron microscopy and thermogravimetric analysis). Fixed lime percentages at 28 curing days (63%) indicated the high reactivity of EGL ashes in calcium hydroxide pastes due to the pozzolanic reaction. This study demonstrates the possibility of the reuse of ashes from EGLs for the production of environmental-friendly cements.

Effect of phytoliths for mitigating water stress in durum wheat.

The role of silicon (Si) in alleviating biotic and abiotic stresses in crops is well evidenced by empirical studies; however, the mechanisms by which it works are still poorly known. The aim of this study is to determine whether or not phytolith composition and distribution in wheat are affected by drought and, if so, why. Durum wheat was grown using hydroponics in the presence of polyethylene glycol (PEG)-6000 to perform a water-stress simulation. We developed an original method for in situ analysis of phytoliths in leaves via X-ray imaging. PEG was efficient in inhibiting water uptake by roots and creating stress, and prevented a small fraction of Si from being accumulated in the shoots. The application of Si with PEG maintained shoot and root fresh weights (FW) and relative water content at higher values than for plants without Si, especially at PEG 12%. Our data show that, under water stress in the presence of Si, accumulation of phytoliths over the veins provides better support to the leaf, thus allowing for a better development of the whole plant than in the absence of Si. The development of silicified trichomes in durum wheat depends primarily on the availability of Si in soil and is not an adaptation to water stress.

Microscopy Characterization of Silica-Rich Agrowastes to be used in Cement Binders: Bamboo and Sugarcane Leaves.

Agrowastes are produced worldwide in huge quantities and they contain interesting elements for producing inorganic cementing binders, especially silicon. Conversion of agrowastes into ash is an interesting way of yielding raw material used in the manufacture of low-CO2 binders. Silica-rich ashes are preferred for preparing inorganic binders. Sugarcane leaves (Saccharum officinarum, SL) and bamboo leaves (Bambusa vulgaris, BvL and Bambusa gigantea, BgL), and their corresponding ashes (SLA, BvLA, and BgLA), were chosen as case studies. These samples were analyzed by means of optical microscopy, Cryo-scanning electron microscopy (SEM), SEM, and field emission scanning electron microscopy. Spodograms were obtained for BvLA and BgLA, which have high proportions of silicon, but no spodogram was obtained for SLA because of the low silicon content. Different types of phytoliths (specific cells, reservoirs of silica in plants) in the studied leaves were observed. These phytoliths maintained their form after calcination at temperatures in the 350-850°C range. Owing to the chemical composition of these ashes, they are of interest for use in cements and concrete because of their possible pozzolanic reactivity. However, the presence of significant amounts of K and Cl in the prepared ashes implies a limitation of their applications.

Tooth length and incisal wear and growth in guinea pigs (Cavia porcellus) fed diets of different abrasiveness.

Dental diseases are among the most important reasons for presenting guinea pigs (Cavia porcellus) and other rodents to veterinary clinics, but the aetiopathology of this disease complex is unclear. Clinicians tend to believe that the ever-growing teeth of rabbits and rodents have a constant growth that needs to be worn down by the mastication of an appropriate diet. In this study, we tested the effect of four different pelleted diets of increasing abrasiveness [due to both internal (phytoliths) and external abrasives (sand)] or whole grass hay fed for 2 weeks each in random order to 16 guinea pigs on incisor growth and wear, and tooth length of incisors and cheek teeth. There was a positive correlation between wear and growth of incisors. Tooth lengths depended both on internal and external abrasives, but only upper incisors were additionally affected by the feeding of whole hay. Diet effects were most prominent in anterior cheek teeth, in particular M1 and m1. Cheek tooth angle did not become shallower with decreasing diet abrasiveness, suggesting that a lack of dietary abrasiveness does not cause the typical 'bridge formation' of anterior cheek teeth frequently observed in guinea pigs. The findings suggest that other factors than diet abrasiveness, such as mineral imbalances and in particular hereditary malocclusion, are more likely causes for dental problems observed in this species.