Evidence for opal phytolith preservation in the Langebaanweg ‘E’ Quarry Varswater Formation and its potential for palaeohabitat reconstruction

Introduction The distribution of savannas and grasslands are associated with a range of climatic parameters and provide a link to past climate changes. More specifically, grasses are potentially good indicators of past climates since the taxonomic composition of grass-dominated ecosystems indirectly reflect local climatic conditions. This is because grasses are likely to adapt relatively quickly to environmental changes, including variation in atmospheric carbon dioxide, temperature and moisture availability. During the Late Miocene to Early Pliocene (7–5 Myr), a major vegetational shift occurred when C4 grasslands expanded globally at the expense of tree-, shruband forb-dominated C3 ecosystems. The reason for this shift is not overtly clear. However, there is a suggestion that a drop in CO2 concentrations at the time would have favoured plants using the C4 photosynthetic pathway. The shift towards grass-dominated biomes may in turn have driven many of the evolutionary changes seen in terminal Miocene faunal communities around the globe. Faunal changes were particularly evident in Africa, where there was a proliferation in grazing ungulates and open-country adapted carnivores. Most significantly, some of the earliest known possible human ancestors also made their appearance. At a time when C4 grasses were spreading through most of Africa, C3 grasses may have been spreading along South Africa’s southwest coast. Stable isotope analyses of a variety of ungulate specimens from ‘E’ Quarry (32°58’S, 18°7’E), a well-known Late Miocene to Early Pliocene fossil locality near the town of Langebaanweg (Fig. 1), indicate that the local environment remained dominated by C3 taxa. 15 While this might indicate a closed environment dominated by trees, shrubs and forbs, the occurrence of several species of high-crowned (hypsodont) ungulates in the ‘E’ Quarry faunal assemblage, hint at the presence of a substantial grass component. Despite this evidence, very little is known about the evolution, nature and importance of grass in the Langebaanweg ‘E’ Quarry biome. As a first step towards answering these questions, we initiated a trial investigation to assess whether sediments at the site are conducive to the preservation of phytoliths, an important tool in palaeohabitat reconstruction. Here we present the results of this preliminary study and elaborate on how we plan to take the research further.


Introduction
The distribution of savannas and grasslands are associated with a range of climatic parameters and provide a link to past climate changes. 1 More specifically, grasses are potentially good indicators of past climates since the taxonomic composition of grass-dominated ecosystems indirectly reflect local climatic conditions. 2,3This is because grasses are likely to adapt relatively quickly to environmental changes, including variation in atmospheric carbon dioxide, temperature and moisture availability. 46][7][8] The reason for this shift is not overtly clear.0][11] The shift towards grass-dominated biomes may in turn have driven many of the evolutionary changes seen in terminal Miocene faunal communities around the globe. 7aunal changes were particularly evident in Africa, where there was a proliferation in grazing ungulates and open-country adapted carnivores.[14] At a time when C 4 grasses were spreading through most of Africa, C 3 grasses may have been spreading along South Africa's southwest coast.Stable isotope analyses of a variety of ungulate specimens from 'E' Quarry (32°58'S, 18°7'E), a well-known Late Miocene to Early Pliocene fossil locality near the town of Langebaanweg (Fig. 1), indicate that the local environment remained dominated by C 3 taxa. 15While this might indicate a closed environment dominated by trees, shrubs and forbs, the occurrence of several species of high-crowned (hypsodont) ungulates in the 'E' Quarry faunal assemblage, hint at the presence of a substantial grass component.Despite this evidence, very little is known about the evolution, nature and importance of grass in the Langebaanweg 'E' Quarry biome.As a first step towards answering these questions, we initiated a trial investigation to assess whether sediments at the site are conducive to the preservation of phytoliths, an important tool in palaeohabitat reconstruction.Here we present the results of this preliminary study and elaborate on how we plan to take the research further.

Langebaanweg fossil deposits and geological sequence
Fossil deposits were first discovered close to the town of Langebaanweg during phosphate mining operations which initially began at Baard's Quarry in 1943 and later moved to 'C' and 'E' Quarries (Fig. 1).However, the importance of these and other fossil localities in the area was only recognised in the late 1950s and 1960s, once scientists commenced with formal fossil descriptions.The prolific bone beds of 'E' Quarry in particular, soon established this locality as a fossil site of great importance.From the late 1950s until the late 1980s, 'E' Quarry was a major focus of research at the South African Museum (now Iziko South African Museum).][18][19][20][21] All the 'E' Quarry fossils originate from the phosphatic sediments of the Mio-Pliocene Varswater Formation. 17,21,22Four members are recognised in the most recent lithostratigraphic review of this formation-the Middle/Late Miocene Langeenheid Sandy Clay Member (LSCM) and Konings Vlei Gravel Member (KVGM), and the Late Miocene/Early Pliocene Langeberg Quartzose Sand Member (LQSM) and Muishond Fontein Pelletal Phosphate Member (MPPM) 22 (Fig. 2).The LQSM and the MPPM produced the majority of fossils (more than 230 vertebrate and invertebrate taxa).The former is construed as primarily a floodplain and salt marsh deposit, while the latter is interpreted as primarily a river channel deposit. 22,23Two river channels, beds 3aS and 3aN, have been identified.Although the temporal relationship between these two beds remains unclarified, bed 3aS appears to be the earlier of the two. 24,25In any event, both river channels reflect a progressive northward shift of the lower course of the proto-Berg River.

The 'E' Quarry palaeoenvironment
Pollen sequences from the LSCM point towards the presence of subtropical forests, palms and marshy vegetation in the region during the early Late Miocene.However, Podocarpus and grass pollens tend to dominate in subsequent members of the Varswater Formation. 26,27There is also an increase in taxa which are characteristic of fynbos shrublands. 28,29s previously mentioned, stable carbon isotope analyses of tooth enamel carbonate of several ungulates from LQSM and MPPM, some with hypsodont dentition, provided values that remain within the C 3 range.9][40] The presence of hypsodont ungulates with C 3 stable carbon isotope values suggests that C 3 grasses were the prevalent grass type and also implies that the modern wet winter/dry summer climate regime was established by Late Miocene to Early Pliocene times. 15Whereas fynbos vegetation today is characterised by an insignificant C 3 grass component, 41 the 'E' Quarry Late Miocene/Early Pliocene habitat may have included a substantial C 3 grass component along with riverine forests and fynbos shrublands. 42

Phytolith analysis
Opal phytoliths are particles of hydrated silica that are produced in various parts of living plants. 43erbaceous plants, especially monocotyledonous plants, are prolific producers of phytoliths, particularly in the epidermal tissue of stems and leaves. 44he morphology and rate of production of phytoliths are largely under genetic control and there is evidence for metabolic exclusion or preferential concentration of silica in some plants. 44][47] Phytoliths preserve well in oxidising environments and have the potential to yield a record of past vegetation cover where there is no other information available. 43,48The abundant production of opaline silica in grasses, for example, is reflected in post-depositional contexts, which facilitate quantitative applications of fossil grass phytolith assemblages. 49This suggests that a predominance of any particular phytolith type in the soil is evidently a reflection of a locally-dominant group of plants.The predictive power of grass phytoliths also yields additional potential because the distribution of grasses in southern Africa is primarily linked to growing season temperature, which also accounts for the geographic distribution of C 3 and C 4 grasses. 2[52]

Methods
Eleven soil samples were collected from three different localities in 'E' Quarry.Sampling of the LSCM, the top of the KVGM and the LQSM was conducted in an old test pit, approximately half a kilometre southwest of the current fossil exhibition tunnel.Three samples were collected from the MPPM-two from previously excavated areas in the exhibition tunnel and one from a newly excavated area in the adjacent tunnel, which currently is closed to the public.An additional two samples were collected from fine-grained sandy deposits of the LQSM, in the latter tunnel.
The procedure for recovering phytoliths from sediment samples is based on published techniques. 53,54Approximately 50 g of each sample was used for each phytolith extraction.Essential steps included deflocculation, removal of clays by means of sedimentation, and the elimination of carbonates using HCl in low concentration (10%).Phytolith extraction also involved mineral separation with a heavy liquid solution of sodium polytungstate (s.d.= 2.3).Fractions were mounted on microscope slides in glycerin jelly and scanned under a Nikon 50i polarising microscope at ×400 magnification.Morphological descriptions follow the guidelines provided by the International Code for Phytolith Nomenclature 1.0. 55A modern phytolith comparative collection aided the identification of fossil phytoliths.

Results
Preservational conditions for biogenic silica are generally favourable throughout the sequence despite comparatively low and more variable phytolith counts in the LQSM and MPPM samples (Table 1 and Fig. 3).The predominantly sandy matrix of the estuarine LQSM and fluviatile MPPM deposits most likely facilitated leaching of biogenic silica during waterlogged conditions, but more detailed analyses of phytoliths from the various depositional environments are needed to adequately interpret post-depositional processes.
Diagnostic grass phytolith morphotypes, as well as morphotypes produced by the monocot families Restionaceae (Cape reed grasses) (Figs 4e and 4r) and Arecaceae (palms) (Fig. 4f), were identified.Globular cavate phytoliths, analogous to silica bodies produced by the gymnospermous Encephalartoideae (cycad subfamily), were identified in the KVGM (Figs 4l  and 4m).In addition, pennate diatoms, with surface markings at right angles to the long axis, abundant in the LSCM (Fig. 4i).Four grass short-cell phytolith types were recognised, namely bilobates, saddles, rondels and trapeziform morphotypes 43,[46][47][48] (Figs 4a-d,  4k, 4o and 4p).Saddle-shaped grass phytoliths were absent in the KVGM, LQSM and MPPM samples.This is noteworthy and seems to support the suggestion that C 3 grasses prevailed during Late Miocene/Early Pliocene times at Langebaanweg.In contrast, saddle-shaped grass phytoliths were identified in the LSCM (Figs 4b and 4c), which is significant given that in the modern environment these phytoliths are almost exclusively found in the Chloridoideae, a grass subfamily that utilises the C 4 photosynthetic pathway. 3,56he results are very promising and show that fossil phytoliths are sufficiently preserved in terms of both quantity and variety (Table 2).The occurrence of palm and cycad-type phytoliths in the LSCM and KVGM supports previous work based on palynological indicators. 26Except for the LQSM, grasses are well represented in the Varswater Formation-more than 50% of the total phytolith count in the LSCM and about 30% of the total phytolith count in the KVGM and MPPM (Fig. 5).A comparatively large proportion of restoid phytoliths in the LQSM and MPPM compliments earlier and more recent reconstructions, suggesting that South Africa's southwest coast was becoming drier and more open during the Early Pliocene. 57,58The presence of chloridoid-type grass phytoliths in the LSCM and subsequent lack in the KVGM, LQSM and MPPM further suggest that radical changes in grassland composition may have occurred towards the end of the Miocene when climate changed from subtropical to more temperate. 27,59While intrigued, we are nevertheless reluctant to make firm inferences regarding the presence of C 3 or C 4 grasses in the 'E' Quarry Varswater sediments at this preliminary stage.Rather, we intend to proceed with a comprehensive quantitative study of grass phytoliths from the  Varswater Formation to ascertain of Mio-Pliocene grass expansion in the southwestern Cape.This study will involve referencing modern plant material from the region, systematic soil sampling of the geological succession and evaluation of the fossil phytolith data through standard laboratory and analytical techniques.
At the end of the Miocene epoch, C 4 grasslands began to expand at the expense of tree-, shrub-and forb-dominated C 3 ecosystems.While C 4 grasses were spreading throughout most regions of the world, C 3 grasses may have been spreading along South Africa's southwest coast.Stable isotope analyses of hypsodont fossil ungulates from 'E' Quarry, a well-known Late Miocene/Early Pliocene fossil locality near the town of Langebaanweg, suggest that the local environment might have included a substantial C 3 grass component.Besides this indirect evidence, little is known about the evolution, nature and importance of grass in the 'E' Quarry biome.As a preliminary step towards addressing these questions, we initiated a trial investigation to assess whether sediments at the site are conducive to the preservation of phytoliths, an important tool in the reconstruction of palaeohabitats.Results indicate that fossil phytoliths are sufficiently well preserved to allow a comprehensive analysis of the 'E' Quarry phytolith assemblage.

Fig. 2 .
Fig. 2. Division of the geological members in the Varswater Formation (modified from Roberts22 ).

HeritageFig. 5 .
Fig. 5. Representation of phytoliths in a 100% stacked column.The graph compares the percentage each value contributes to the total across categories.

Table 1 .
Total number of phytoliths counted per sample.

Table 2 .
Number of phytoliths counted per sample according to each category.