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Starch grain morphology of the seagrasses Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum
Aquatic Botany 96 (2012) 63–66
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
Starch grain morphology of the seagrasses Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum Stephanie Peek ∗ , Mark T. Clementz Department of Geology and Geophysics, Dept. #3006, 1000 E. University Ave, University of Wyoming, Laramie, WY 82071, USA
a r t i c l e
i n f o
Article history: Received 22 July 2011 Received in revised form 1 October 2011 Accepted 4 October 2011 Available online 12 October 2011 Keywords: Starch grain Morphology Seagrass Halodule wrightii Ruppia maritima Syringodium filiforme Thalassia testudinum
a b s t r a c t Starch grains are a ubiquitous component of plants that have been used in tandem with phytoliths, pollen, and macrofossils to reconstruct past floral diversity. This tool has yet to be fully explored for aquatic plants, specifically seagrasses, which lack phytoliths and are rarely preserved as macrofossils or pollen. If starch grains in seagrasses are morphologically distinct, this method has the potential to improve seagrass identification in the fossil record in such cases where its starch is preserved (e.g. scratches and occlusal surfaces of tooth enamel from seagrass consumers). The goals of this study were twofold: (1) to determine if starch is present in seagrass material and (2) to assess how starch grain morphology differs between different seagrasses. This study focused on four abundant and ecologically distinct seagrasses from the Caribbean: Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum. Starch grains were observed in all species except S. filiforme. Grains from H. wrightii are typically observed in side-on orientation, are subround to angular, and are fairly small (3-19 m, end-on). Grains of R. maritima are small spherical grains (4–8 m) that have a centric hilum and a straight extinction cross with a median angle between the arms of 90◦ . Grains from T. testudinum are large (9–31 m, end-on), conical in side-on and round/sub-round in end-on orientation, have a slightly eccentric hilum with an obvious particle, and prominent lamellae. Visual assessment and comparative statistics demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii are significantly different. With more extensive research, there is potential for the positive identification of starch grains from an unknown seagrass. The ability to identify seagrass from starch grains could facilitate the identification of seagrasses in the fossil record and supply information on seagrass evolution and distribution, climate effects on seagrass distribution, and the diets of seagrass consumers. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fossil evidence of seagrasses is known since the late Cretaceous but fossil localities are limited to only a handful of sites (Brasier, 1975; Lumbert et al., 1984), as plant remains of seagrasses are rare and nearly impossible to identify without the preservation of reproductive parts (Brasier, 1975). Additionally, seagrasses do not have phytoliths and their pollen is not preserved because it lacks exine (Brasier, 1975; Domning, 1982). For these reasons, the identification of fossil seagrasses primarily relies on associated fauna (e.g. foraminifera, mollusks, echinoids, crustaceans, and sirenians) and distinctive sedimentary features of seagrass communities (Brasier, 1975). However these identification methods have limitations, some of which could be overcome if starch grains from
∗ Corresponding author. Tel.: +1 307 766 6048; fax: +1 307 766 6679. E-mail addresses: [email protected] (S. Peek), [email protected] (M.T. Clementz). 0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.10.001
different species of seagrasses are distinct and are preserved in the fossil record. Starch is the energy source of a plant and, while present in all plant parts, it is most heavily concentrated in storage organs (i.e. roots, tubers, rhizomes, fruits, and seeds) (Gott et al., 2006). Starch grains occur in a variety of characteristic forms that can be used to identify plants to family and genus level, sometimes even species (Reichert, 1913). Starch is also highly resistant to alteration, which has enabled it to be preserved in a variety of climate regions ranging from arid to tropical and recovered from several substrates, including fecal material (Barton and Matthews, 2006); cracks, pits, and crevices in pottery, millstones, or other grinding tools and associated soils recovered from archaeological sites (Samuel, 1996; Piperno and Holst, 1998; Lentfer et al., 2002; Piperno et al., 2004; Barton and Matthews, 2006; Perry et al., 2007); and pyritized starch grains have even been found in rocks of Eocene age (Wilkinson, 1983). Unaltered starch grains could be preserved in the fossil record if they are protected from destructive elements such as microorganisms, soil moisture, soil pH, and oxygen following rapid
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burial (Barton and Matthews, 2006). One potential location for preservation could be in the cracks or occlusal surfaces of teeth from fossil seagrass consumers (i.e. manatees and dugongs) which could be similar to extracting starch from the grinding surfaces of tools. Most of the studies that have characterized and described starch grain morphology have been descriptive studies working with a variety of terrestrial, aquatic, and estuarine vegetation (Reichert, 1913) or have focused on food crops used by early human cultures (Ugent et al., 1987; Piperno and Holst, 1998; Lentfer et al., 2002; Piperno et al., 2004; Perry et al., 2007). However, starch grains of seagrasses have not been described in detail except for the analysis of seeds and pollen from Ruppia maritima, seeds from Zostera marina, and rhizomes of Z. noltii, formerly Z. nana (Reichert, 1913). Leaves and rhizomes are the most common part of the plant to be eaten by marine consumers, so additional research on starch from these parts of the plants is necessary. This study focused on four abundant and ecologically distinct tropical seagrasses from the Caribbean: Halodule wrightii, R. maritima, Syringodium filiforme, and Thalassia testudinum. The objectives of this study were to: (1) determine if starch grains are present in seagrasses and (2) determine if these starch grains are morphologically distinct between species, potentially enabling the identification of seagrass species from starch grains of unclear origin. With more rigorous study, this method could be used to collect important dietary information for seagrass consumers and aid in identifying the distribution of seagrasses in the fossil record. 2. Methods
2.3. Starch grain analysis Slides were analyzed using a polarizing light microscope in both plane-polarized light (PPL) with one polarizing filter in place and cross-polarized light (XPL) with two polarizing filters set at 90◦ to one another. Photographs were taken in rapid succession to minimize the effect of starch grain movement and taken so that at least 30 starch grains in end-on orientation were available for measurement. Additional digital photomicrographs were obtained at the Paleo Research Institute in white light, partial-polarized light, and XPL. Starch grain identifications were based on a set of the most commonly used morphological characters (Piperno et al., 2004; Torrence et al., 2004; Holst et al., 2007; Field, 2008; Fullagar et al., 2008). Grain type, size, shape, and presence or absence of lamellae, hilum, particles, and fissures were determined under PPL whereas measurements of extinction cross size and shape, angle between arms, and strength of birefringence were made under XPL. While the shape, size, and relative position of the arms of the extinction cross can be important diagnostic features of modern starch grains (Reichert, 1913; Torrence et al., 2004), ancient starches often lose their birefringent properties and as such, extinction cross features will not always be preserved in ancient starch. Statistical analyses were run using PAST (Hammer et al., 2001) with a significance level of ˛ = 0.05. Results from the Shapiro–Wilk test for normality indicated that only four of the 18 numerical variables resulted in normally distributed datasets. As such, the nonparametric Mann–Whitney–Wilcoxon rank-sum test was used to compare the difference in the sample medians.
2.1. Sample selection 3. Results Seagrass samples were collected from the Indian River Lagoon (IRL) in Florida, USA in 2003 and 2004. The IRL is located on the east coast of Florida between approximately 28◦ 24 N and 27◦ 11 N. Samples of whole plants (i.e. leaves and rhizomes) were taken for study from seagrass beds of H. wrightii, R. maritima, S. filiforme, and T. testudinum. Samples were rinsed in deionized water (DIW) to remove epiphytes, sediments, and salts and then frozen and stored before preparation for starch grain analysis. 2.2. Sample preparation Methods were adapted from Perry et al. (2007). For this study approximately 1 g of seagrass material (leaves and rhizomes together) was combined with 200 mL of DIW and then ground until fine using an immersion blender (approximately 5–10 min). For H. wrightii, R. maritima, and S. filiforme 5–10 individual plants were ground together; however given its larger size, only 2–3 plants of T. testudinum were used. Material was then passed through a stacked series of three sieves (250, 75, and 45 m) to isolate starch grains from the bulk plant matter. Initial analyses demonstrated no starch grains larger than 45 m were present and so this size was the smallest sieve used to remove other plant materials that would have obscured the view of the starch grains. This mixture was allowed to sit overnight and was then centrifuged for 5 min at 5000 rpm and the supernatant discarded. A few drops of 100% ethanol were added to the mixture to prevent molding. Starch grains were mounted in water; one drop of the <45 m fraction/DIW mixture was placed on a slide for analysis with a polarizing light microscope and the edges of the coverslip sealed with clear nail polish. To prevent contamination between samples, sieves were cleaned by sonication with DIW heated to 68 ◦ C for 30 min, which gelatinized and destroyed any starch grains adhering to the sieve.
Starch grains were observed in three of the four species of seagrass analyzed. Table 1 lists the summary statistics (i.e. median and range) and comparative statistics of measurements of starch grains in end-on orientation. Statistical comparisons were limited to endon orientation because starch grains of R. maritima did not occur in side-on orientation.
3.1. H. wrightii Starch grains of H. wrightii (Fig. 1a and b) were relatively abundant and typically observed in side-on orientation. In end-on orientation starch grains were simple, ranged from 3 to 19 m in diameter, were angular to sub-round, had an equal distribution of grains with a centric versus eccentric hilum, had the occasional particle and/or fissures, and occasionally had weak lamellae. The extinction cross was straight to rounded with median angle between the arms of 106◦ and strong birefringence. Grain characteristics did not change much in side-on orientation, although the grains typically had a greater length, which ranged from 8 to 24 m in diameter.
3.2. R. maritima Starch grains of R. maritima (Fig. 1a and b) were abundant; they were simple grains, roughly spherical and therefore only occurred in end-on orientation. The grains were small (4–8 m diameter), round to sub-round, with a centric hilum that typically lacked a particle, and occasionally had weak lamellae. The extinction cross was straight with a median angle between the arms of 90◦ and strong birefringence.
S. Peek, M.T. Clementz / Aquatic Botany 96 (2012) 63–66
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Table 1 Measurements of quantitative starch grain characteristics in plane-polarized light (PPL) and cross-polarized light (XPL) reported as: median (range). Statistical significance of the Mann–Whitney–Wilcoxon rank-sum test is designated with superscript letters. Halodule wrightii Grain size (PPL) Length (m) Width (m) Ratio (L:W) Extinction cross size (XPL) Length (m) Width (m) Largest angle
6.0A 5.8A 1.18A 6.0A 5.0A 106◦ A
Ruppia maritima (4.0–19.0) (3.0–13.0) (1.00–1.46) (3.0–15.0) (3.0–14.0) (90◦ –136◦ )
3.3. S. filiforme No starch grains were observed in any of the S. filiforme wholeplant samples from three different locations within the IRL. 3.4. T. testudinum Starch grains were very abundant in all whole-plant samples of T. testudinum (Fig. 1a and b). In end-on orientation the grains were simple, large (9–31 m diameter), round to sub-round, had a slightly eccentric hilum with a particle, and had prominent lamellae. The extinction cross was straight, rounded, or wavy with a median angle between the arms of 101◦ and strong birefringence. In side-on orientation, the grains were primarily conical in shape and the length ranged from 8 to 42 m in diameter and was greater than the width.
6.4A 6.4A 1.06B 5.0A 5.0A 90◦ B
Thalassia testudinum (4.5–8.2) (4.3–7.3) (1.00–1.30) (3.6–8.2) (3.6–7.3) (90◦ –125◦ )
20.0B 19.3B 1.04B 16.3B 14.8B 101 ◦ C
(9.6–31.1) (8.9–28.1) (1.00–1.21) (6.7–31.9) (5.9–30.4) (90◦ –129◦ )
when compared to R. maritima and to T. testudinum, but the shape of R. maritima and T. testudinum was not significantly different in endon orientation when compared to one another (Table 1). There were obvious differences in side-on orientation; starch grains from R. maritima were spherical and all views were end-on, whereas grains of T. testudinum were predominantly conical in side-on orientation and were larger. Extinction crosses of T. testudinum were significantly larger than those of R. maritima and H. wrightii whereas the size of the extinction crosses of R. maritima and H. wrightii were not significantly different (Table 1 and Fig. 1b). The median value of the largest angle between two arms of the extinction cross was significantly different among T. testudinum, R. maritima, and H. wrightii (Table 1). Extinction cross shape differed between all three species and was highly variable. Birefringence was not visually different among grains of the three seagrasses.
3.5. Comparisons 4. Discussion Measured in PPL, starch grains of T. testudinum were significantly larger in both median length and width than the starch grains from either R. maritima or H. wrightii (Table 1 and Fig. 1a). The starch grains of R. maritima and H. wrightii did not have significantly different median lengths or widths (Table 1 and Fig. 1a). In end-on orientation, grains of T. testudinum and R. maritima were predominantly round to sub-round whereas grains of H. wrightii were predominantly angular and round to sub-round. Starch grain shape is limited by being a visual qualitative classification (e.g. angular versus rounded) so we used the ratio of grain length to grain width (L:W) as measured from grains in end-on orientation as a proxy to separate grains classified as round/sub-round (L:W < 1.15) from all other grain shapes (e.g. ovate/sub-ovate, angular). Using L:W as a grain shape proxy, H. wrightii had a significantly different shape
The first objective of this study was to determine if starch is present in tropical seagrasses. We found abundant starch in H. wrightii, R. maritima, and T. testudinum but did not observe starch in S. filiforme. There are several possible factors that could explain the lack of observed starch grains in S. filiforme. In the Caribbean, S. filiforme is a pioneer species that rapidly colonizes, has a relatively short lifespan, and is more sensitive to environmental disturbances than the other three species (Creed et al., 2003). These factors suggest that S. filiforme might not store large amounts of starch in its rhizomes or leaves. Of the three species with observed starch, H. wrightii is most closely related to S. filiforme, however the former is much hardier and can tolerate a larger range of salinities, which may explain the presence of starch and the lack of observed starch
Fig. 1. Grain length versus width (a) measured on end-on grains in plane-polarized light; the grey shaded region represents a L:W ratio of 1–1.15 (the round/sub-round category). Extinction cross length versus width (b) measured on end-on grains in cross-polarized light. Images represent starch grains in PPL (a) and XPL (b). The scale bar denotes 10 m in all images. Symbols: Halodule wrightii (dark grey triangles); Ruppia maritima (grey diamonds); Thalassia testudinum (white circles).
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in S. filiforme. Alternatively, starch grains could be present in S. filiforme but may be too small to be detected by our instrumentation (e.g. very small transient starch <1 m). The second objective of the study was to determine if starch grains from these seagrasses are morphologically distinct. Our results have shown that traditional visual assessment and basic statistics are sufficient to demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii, are significantly different. These visual and statistical analyses highlight a number of important morphological differences among the starch grains that can be used to distinguish the three species. The first major difference is size: grains of T. testudinum are significantly larger than grains of H. wrightii or R. maritima. The second major difference is grain shape: grains of H. wrightii are more angular than grains of either R. maritima or T. testudinum with a significantly different L:W ratio in end-on orientation. Grains of R. maritima are spherical and only appear in end-on orientation whereas grains of T. testudinum are predominantly conical in side-on orientation. The third and fourth major differences are the extinction cross shape and the angle between the arms: grains of R. maritima have a high proportion of straight extinction crosses and a median angle between the arms of 90◦ . While extinction cross shape helps to further separate the morphologies of grains, it is not necessary for identification; size and shape are sufficient for distinguishing starch grains of these species. These results are the first to characterize differences in starch grains from three species of tropical seagrass from the IRL and suggest that further study into starch grain morphology from a wider variety of seagrass species is warranted. Because this study only looked at bulk plant samples from a handful of different locations on the IRL, future research should characterize starch from multiple plant parts, including seeds, pollen, leaves, and rhizomes. These studies should also assess spatial and temporal effects on starch grain morphology. Sampling should continue on a broader taxonomic range in order to expand potential taxonomic resolution. Additional research should be conducted into the preservation potential of starch in marine settings. In the future, this method could be used as an important tool in paleoecological research to help understand the diversity, distribution, and significance of past seagrass communities. Acknowledgements We thank the Smithsonian Marine Station and Kennedy Space Center for the collection of seagrass samples. Funding was provided by the Wyoming NASA Space Grant Consortium (NASA Grant # NNG05G165H), the Summer Research Apprenticeship Program (Wyoming NSF EPSCoR), and NSF (NSF-EAR # 0847413). We thank
C. Yost and L.S. Cummings at the Paleo Research Institute for help with digital photomicrographs, R. Frost at the University of Wyoming for access to microscopes and photographic equipment, and SRAP student A. Lopez for help with starch grain preparation and photography. References Barton, H., Matthews, P.J., 2006. Taphonomy. In: Torrence, R., Barton, H. (Eds.), Ancient Starch Research. Left Coast Press Inc., Walnut Creek, CA, p. 256. Brasier, M.D., 1975. An outline history of seagrass communities. Palaeontology 18, 681–702. Creed, J., Phillips, R., Van Tussenbroek, B., 2003. The seagrasses of the Caribbean. In: Green, E.P., Short, F.T. (Eds.), World Atlas of Seagrasses. University of California Press, Berkeley, p. 298. Domning, D.P., 1982. Evolution of manatees: a speculative history. J. Paleontol. 56, 599–619. Field, J., 2008. Analysis of botanical remains: starch grain analysis. In: Pearsall, D. (Ed.), Encyclopaedia of Archaeology. Elsevier, Oxford, pp. 2078–2082. Fullagar, R., Field, J., Kealhofer, L., 2008. Grinding stones and seeds of change: starch and phytoliths as evidence of plant food processing. In: Rowan, Y.M., Ebeling, J.R. (Eds.), New Approaches to Old Stones: Recent Studies of Ground Stone Artifacts. Equinox Press, London, pp. 159–172. Gott, B., Barton, H., Samuel, D., Torrence, R., 2006. Biology of starch. In: Torrence, R., Barton, H. (Eds.), Ancient Starch Research. Left Coast Press Inc., Walnut Creek, CA, p. 256. Hammer, Ø., Harper, D., Ryan, P., 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 9. Holst, I., Moreno, J.E., Piperno, D.R., 2007. Identification of teosinte, maize, and Tripsacum in Mesoamerica by using pollen, starch grains, and phytoliths. Proc. Natl. Acad. Sci. U.S.A. 104, 17608–17613. Lentfer, C., Therin, M., Torrence, R., 2002. Starch grains and environmental reconstruction: a modern test case from West New Britain, Papua New Guinea. J. Archaeol. Sci. 29, 687–698. Lumbert, S.H., Den Hartog, C., Phillips, R.C., Olsen, F.S., 1984. The occurrence of fossil seagrasses in the Avon Park Formation (late middle Eocene), Levy County Florida (U.S.A.). Aquat. Bot. 20, 121–129. Perry, L., Dickau, R., Zarillo, S., Holst, I., Peasall, D.M., Piperno, D.R., Berman, M.J., Cooke, R.G., Rademaker, K., Ranere, A.J., Raymond, J.S., Sandweiss, D.H., Scaramelli, F., Tarble, K., Zeidler, J.A., 2007. Starch fossils and the domestication and dispersal of chili peppers (Capsicum spp. L.). Science 315, 986–988. Piperno, D.R., Holst, I., 1998. The presence of starch grains on prehistoric stone tools from the humid neotropics: indications of early tuber use and agriculture in Panama. J. Archaeol. Sci. 25, 765–776. Piperno, D.R., Weiss, E., Holst, I., Nadel, D., 2004. Processing of wild cereal grains in the Upper Palaeolithic revealed by starch grain analysis. Nature 430, 670–673. Reichert, E.T., 1913. The Differentiation and Specificity of Starches in Relation to Genera, Species, etc.: Stereochemistry Applied to Protoplasmic Processes and Products and as a Strictly Scientific Basis for the Classification of Plants and Animals. Carnegie Institution of Washington, Washington, DC. Samuel, D., 1996. Investigation of ancient Egyptian baking and brewing methods by correlative microscopy. Science 273, 488–490. Torrence, R., Wright, R., Conway, R., 2004. Identification of starch granules using image analysis and multivariate techniques. J. Archaeol. Sci. 31, 519–532. Ugent, D., Dillehay, T., Ramirez, C., 1987. Potato remains from a Late Pleistocene settlement in Southcentral Chile. Econ. Bot. 41, 17–27. Wilkinson, H., 1983. Starch grain casts and moulds in Eocene (Tertiary) fossil mangrove hypocotyls. Ann. Bot. (Lond.) 51, 39–45.
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
Starch grain morphology of the seagrasses Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum Stephanie Peek ∗ , Mark T. Clementz Department of Geology and Geophysics, Dept. #3006, 1000 E. University Ave, University of Wyoming, Laramie, WY 82071, USA
a r t i c l e
i n f o
Article history: Received 22 July 2011 Received in revised form 1 October 2011 Accepted 4 October 2011 Available online 12 October 2011 Keywords: Starch grain Morphology Seagrass Halodule wrightii Ruppia maritima Syringodium filiforme Thalassia testudinum
a b s t r a c t Starch grains are a ubiquitous component of plants that have been used in tandem with phytoliths, pollen, and macrofossils to reconstruct past floral diversity. This tool has yet to be fully explored for aquatic plants, specifically seagrasses, which lack phytoliths and are rarely preserved as macrofossils or pollen. If starch grains in seagrasses are morphologically distinct, this method has the potential to improve seagrass identification in the fossil record in such cases where its starch is preserved (e.g. scratches and occlusal surfaces of tooth enamel from seagrass consumers). The goals of this study were twofold: (1) to determine if starch is present in seagrass material and (2) to assess how starch grain morphology differs between different seagrasses. This study focused on four abundant and ecologically distinct seagrasses from the Caribbean: Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum. Starch grains were observed in all species except S. filiforme. Grains from H. wrightii are typically observed in side-on orientation, are subround to angular, and are fairly small (3-19 m, end-on). Grains of R. maritima are small spherical grains (4–8 m) that have a centric hilum and a straight extinction cross with a median angle between the arms of 90◦ . Grains from T. testudinum are large (9–31 m, end-on), conical in side-on and round/sub-round in end-on orientation, have a slightly eccentric hilum with an obvious particle, and prominent lamellae. Visual assessment and comparative statistics demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii are significantly different. With more extensive research, there is potential for the positive identification of starch grains from an unknown seagrass. The ability to identify seagrass from starch grains could facilitate the identification of seagrasses in the fossil record and supply information on seagrass evolution and distribution, climate effects on seagrass distribution, and the diets of seagrass consumers. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fossil evidence of seagrasses is known since the late Cretaceous but fossil localities are limited to only a handful of sites (Brasier, 1975; Lumbert et al., 1984), as plant remains of seagrasses are rare and nearly impossible to identify without the preservation of reproductive parts (Brasier, 1975). Additionally, seagrasses do not have phytoliths and their pollen is not preserved because it lacks exine (Brasier, 1975; Domning, 1982). For these reasons, the identification of fossil seagrasses primarily relies on associated fauna (e.g. foraminifera, mollusks, echinoids, crustaceans, and sirenians) and distinctive sedimentary features of seagrass communities (Brasier, 1975). However these identification methods have limitations, some of which could be overcome if starch grains from
∗ Corresponding author. Tel.: +1 307 766 6048; fax: +1 307 766 6679. E-mail addresses: [email protected] (S. Peek), [email protected] (M.T. Clementz). 0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.10.001
different species of seagrasses are distinct and are preserved in the fossil record. Starch is the energy source of a plant and, while present in all plant parts, it is most heavily concentrated in storage organs (i.e. roots, tubers, rhizomes, fruits, and seeds) (Gott et al., 2006). Starch grains occur in a variety of characteristic forms that can be used to identify plants to family and genus level, sometimes even species (Reichert, 1913). Starch is also highly resistant to alteration, which has enabled it to be preserved in a variety of climate regions ranging from arid to tropical and recovered from several substrates, including fecal material (Barton and Matthews, 2006); cracks, pits, and crevices in pottery, millstones, or other grinding tools and associated soils recovered from archaeological sites (Samuel, 1996; Piperno and Holst, 1998; Lentfer et al., 2002; Piperno et al., 2004; Barton and Matthews, 2006; Perry et al., 2007); and pyritized starch grains have even been found in rocks of Eocene age (Wilkinson, 1983). Unaltered starch grains could be preserved in the fossil record if they are protected from destructive elements such as microorganisms, soil moisture, soil pH, and oxygen following rapid
64
S. Peek, M.T. Clementz / Aquatic Botany 96 (2012) 63–66
burial (Barton and Matthews, 2006). One potential location for preservation could be in the cracks or occlusal surfaces of teeth from fossil seagrass consumers (i.e. manatees and dugongs) which could be similar to extracting starch from the grinding surfaces of tools. Most of the studies that have characterized and described starch grain morphology have been descriptive studies working with a variety of terrestrial, aquatic, and estuarine vegetation (Reichert, 1913) or have focused on food crops used by early human cultures (Ugent et al., 1987; Piperno and Holst, 1998; Lentfer et al., 2002; Piperno et al., 2004; Perry et al., 2007). However, starch grains of seagrasses have not been described in detail except for the analysis of seeds and pollen from Ruppia maritima, seeds from Zostera marina, and rhizomes of Z. noltii, formerly Z. nana (Reichert, 1913). Leaves and rhizomes are the most common part of the plant to be eaten by marine consumers, so additional research on starch from these parts of the plants is necessary. This study focused on four abundant and ecologically distinct tropical seagrasses from the Caribbean: Halodule wrightii, R. maritima, Syringodium filiforme, and Thalassia testudinum. The objectives of this study were to: (1) determine if starch grains are present in seagrasses and (2) determine if these starch grains are morphologically distinct between species, potentially enabling the identification of seagrass species from starch grains of unclear origin. With more rigorous study, this method could be used to collect important dietary information for seagrass consumers and aid in identifying the distribution of seagrasses in the fossil record. 2. Methods
2.3. Starch grain analysis Slides were analyzed using a polarizing light microscope in both plane-polarized light (PPL) with one polarizing filter in place and cross-polarized light (XPL) with two polarizing filters set at 90◦ to one another. Photographs were taken in rapid succession to minimize the effect of starch grain movement and taken so that at least 30 starch grains in end-on orientation were available for measurement. Additional digital photomicrographs were obtained at the Paleo Research Institute in white light, partial-polarized light, and XPL. Starch grain identifications were based on a set of the most commonly used morphological characters (Piperno et al., 2004; Torrence et al., 2004; Holst et al., 2007; Field, 2008; Fullagar et al., 2008). Grain type, size, shape, and presence or absence of lamellae, hilum, particles, and fissures were determined under PPL whereas measurements of extinction cross size and shape, angle between arms, and strength of birefringence were made under XPL. While the shape, size, and relative position of the arms of the extinction cross can be important diagnostic features of modern starch grains (Reichert, 1913; Torrence et al., 2004), ancient starches often lose their birefringent properties and as such, extinction cross features will not always be preserved in ancient starch. Statistical analyses were run using PAST (Hammer et al., 2001) with a significance level of ˛ = 0.05. Results from the Shapiro–Wilk test for normality indicated that only four of the 18 numerical variables resulted in normally distributed datasets. As such, the nonparametric Mann–Whitney–Wilcoxon rank-sum test was used to compare the difference in the sample medians.
2.1. Sample selection 3. Results Seagrass samples were collected from the Indian River Lagoon (IRL) in Florida, USA in 2003 and 2004. The IRL is located on the east coast of Florida between approximately 28◦ 24 N and 27◦ 11 N. Samples of whole plants (i.e. leaves and rhizomes) were taken for study from seagrass beds of H. wrightii, R. maritima, S. filiforme, and T. testudinum. Samples were rinsed in deionized water (DIW) to remove epiphytes, sediments, and salts and then frozen and stored before preparation for starch grain analysis. 2.2. Sample preparation Methods were adapted from Perry et al. (2007). For this study approximately 1 g of seagrass material (leaves and rhizomes together) was combined with 200 mL of DIW and then ground until fine using an immersion blender (approximately 5–10 min). For H. wrightii, R. maritima, and S. filiforme 5–10 individual plants were ground together; however given its larger size, only 2–3 plants of T. testudinum were used. Material was then passed through a stacked series of three sieves (250, 75, and 45 m) to isolate starch grains from the bulk plant matter. Initial analyses demonstrated no starch grains larger than 45 m were present and so this size was the smallest sieve used to remove other plant materials that would have obscured the view of the starch grains. This mixture was allowed to sit overnight and was then centrifuged for 5 min at 5000 rpm and the supernatant discarded. A few drops of 100% ethanol were added to the mixture to prevent molding. Starch grains were mounted in water; one drop of the <45 m fraction/DIW mixture was placed on a slide for analysis with a polarizing light microscope and the edges of the coverslip sealed with clear nail polish. To prevent contamination between samples, sieves were cleaned by sonication with DIW heated to 68 ◦ C for 30 min, which gelatinized and destroyed any starch grains adhering to the sieve.
Starch grains were observed in three of the four species of seagrass analyzed. Table 1 lists the summary statistics (i.e. median and range) and comparative statistics of measurements of starch grains in end-on orientation. Statistical comparisons were limited to endon orientation because starch grains of R. maritima did not occur in side-on orientation.
3.1. H. wrightii Starch grains of H. wrightii (Fig. 1a and b) were relatively abundant and typically observed in side-on orientation. In end-on orientation starch grains were simple, ranged from 3 to 19 m in diameter, were angular to sub-round, had an equal distribution of grains with a centric versus eccentric hilum, had the occasional particle and/or fissures, and occasionally had weak lamellae. The extinction cross was straight to rounded with median angle between the arms of 106◦ and strong birefringence. Grain characteristics did not change much in side-on orientation, although the grains typically had a greater length, which ranged from 8 to 24 m in diameter.
3.2. R. maritima Starch grains of R. maritima (Fig. 1a and b) were abundant; they were simple grains, roughly spherical and therefore only occurred in end-on orientation. The grains were small (4–8 m diameter), round to sub-round, with a centric hilum that typically lacked a particle, and occasionally had weak lamellae. The extinction cross was straight with a median angle between the arms of 90◦ and strong birefringence.
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Table 1 Measurements of quantitative starch grain characteristics in plane-polarized light (PPL) and cross-polarized light (XPL) reported as: median (range). Statistical significance of the Mann–Whitney–Wilcoxon rank-sum test is designated with superscript letters. Halodule wrightii Grain size (PPL) Length (m) Width (m) Ratio (L:W) Extinction cross size (XPL) Length (m) Width (m) Largest angle
6.0A 5.8A 1.18A 6.0A 5.0A 106◦ A
Ruppia maritima (4.0–19.0) (3.0–13.0) (1.00–1.46) (3.0–15.0) (3.0–14.0) (90◦ –136◦ )
3.3. S. filiforme No starch grains were observed in any of the S. filiforme wholeplant samples from three different locations within the IRL. 3.4. T. testudinum Starch grains were very abundant in all whole-plant samples of T. testudinum (Fig. 1a and b). In end-on orientation the grains were simple, large (9–31 m diameter), round to sub-round, had a slightly eccentric hilum with a particle, and had prominent lamellae. The extinction cross was straight, rounded, or wavy with a median angle between the arms of 101◦ and strong birefringence. In side-on orientation, the grains were primarily conical in shape and the length ranged from 8 to 42 m in diameter and was greater than the width.
6.4A 6.4A 1.06B 5.0A 5.0A 90◦ B
Thalassia testudinum (4.5–8.2) (4.3–7.3) (1.00–1.30) (3.6–8.2) (3.6–7.3) (90◦ –125◦ )
20.0B 19.3B 1.04B 16.3B 14.8B 101 ◦ C
(9.6–31.1) (8.9–28.1) (1.00–1.21) (6.7–31.9) (5.9–30.4) (90◦ –129◦ )
when compared to R. maritima and to T. testudinum, but the shape of R. maritima and T. testudinum was not significantly different in endon orientation when compared to one another (Table 1). There were obvious differences in side-on orientation; starch grains from R. maritima were spherical and all views were end-on, whereas grains of T. testudinum were predominantly conical in side-on orientation and were larger. Extinction crosses of T. testudinum were significantly larger than those of R. maritima and H. wrightii whereas the size of the extinction crosses of R. maritima and H. wrightii were not significantly different (Table 1 and Fig. 1b). The median value of the largest angle between two arms of the extinction cross was significantly different among T. testudinum, R. maritima, and H. wrightii (Table 1). Extinction cross shape differed between all three species and was highly variable. Birefringence was not visually different among grains of the three seagrasses.
3.5. Comparisons 4. Discussion Measured in PPL, starch grains of T. testudinum were significantly larger in both median length and width than the starch grains from either R. maritima or H. wrightii (Table 1 and Fig. 1a). The starch grains of R. maritima and H. wrightii did not have significantly different median lengths or widths (Table 1 and Fig. 1a). In end-on orientation, grains of T. testudinum and R. maritima were predominantly round to sub-round whereas grains of H. wrightii were predominantly angular and round to sub-round. Starch grain shape is limited by being a visual qualitative classification (e.g. angular versus rounded) so we used the ratio of grain length to grain width (L:W) as measured from grains in end-on orientation as a proxy to separate grains classified as round/sub-round (L:W < 1.15) from all other grain shapes (e.g. ovate/sub-ovate, angular). Using L:W as a grain shape proxy, H. wrightii had a significantly different shape
The first objective of this study was to determine if starch is present in tropical seagrasses. We found abundant starch in H. wrightii, R. maritima, and T. testudinum but did not observe starch in S. filiforme. There are several possible factors that could explain the lack of observed starch grains in S. filiforme. In the Caribbean, S. filiforme is a pioneer species that rapidly colonizes, has a relatively short lifespan, and is more sensitive to environmental disturbances than the other three species (Creed et al., 2003). These factors suggest that S. filiforme might not store large amounts of starch in its rhizomes or leaves. Of the three species with observed starch, H. wrightii is most closely related to S. filiforme, however the former is much hardier and can tolerate a larger range of salinities, which may explain the presence of starch and the lack of observed starch
Fig. 1. Grain length versus width (a) measured on end-on grains in plane-polarized light; the grey shaded region represents a L:W ratio of 1–1.15 (the round/sub-round category). Extinction cross length versus width (b) measured on end-on grains in cross-polarized light. Images represent starch grains in PPL (a) and XPL (b). The scale bar denotes 10 m in all images. Symbols: Halodule wrightii (dark grey triangles); Ruppia maritima (grey diamonds); Thalassia testudinum (white circles).
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in S. filiforme. Alternatively, starch grains could be present in S. filiforme but may be too small to be detected by our instrumentation (e.g. very small transient starch <1 m). The second objective of the study was to determine if starch grains from these seagrasses are morphologically distinct. Our results have shown that traditional visual assessment and basic statistics are sufficient to demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii, are significantly different. These visual and statistical analyses highlight a number of important morphological differences among the starch grains that can be used to distinguish the three species. The first major difference is size: grains of T. testudinum are significantly larger than grains of H. wrightii or R. maritima. The second major difference is grain shape: grains of H. wrightii are more angular than grains of either R. maritima or T. testudinum with a significantly different L:W ratio in end-on orientation. Grains of R. maritima are spherical and only appear in end-on orientation whereas grains of T. testudinum are predominantly conical in side-on orientation. The third and fourth major differences are the extinction cross shape and the angle between the arms: grains of R. maritima have a high proportion of straight extinction crosses and a median angle between the arms of 90◦ . While extinction cross shape helps to further separate the morphologies of grains, it is not necessary for identification; size and shape are sufficient for distinguishing starch grains of these species. These results are the first to characterize differences in starch grains from three species of tropical seagrass from the IRL and suggest that further study into starch grain morphology from a wider variety of seagrass species is warranted. Because this study only looked at bulk plant samples from a handful of different locations on the IRL, future research should characterize starch from multiple plant parts, including seeds, pollen, leaves, and rhizomes. These studies should also assess spatial and temporal effects on starch grain morphology. Sampling should continue on a broader taxonomic range in order to expand potential taxonomic resolution. Additional research should be conducted into the preservation potential of starch in marine settings. In the future, this method could be used as an important tool in paleoecological research to help understand the diversity, distribution, and significance of past seagrass communities. Acknowledgements We thank the Smithsonian Marine Station and Kennedy Space Center for the collection of seagrass samples. Funding was provided by the Wyoming NASA Space Grant Consortium (NASA Grant # NNG05G165H), the Summer Research Apprenticeship Program (Wyoming NSF EPSCoR), and NSF (NSF-EAR # 0847413). We thank
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