Hather (1991, 2000) discriminates between the vegetative storage parenchyma that is found in roots and tubers, and all other parenchymatous organs such as fruits, seeds, and nuts. He makes this distinction because the function of these organs is to store reserve starch and carbohydrates during photosynthesis for plants, and thus these organs share many similar morphological traits. It is this storage function, and subsequent calorific and nutritional value, that led to the development of many prehistoric subsistence strategies based upon the collection or cultivation of these starchy organs (Barton and Paz 2007; Denham 2007b; Golson 2007; Holden, Hather and Watson 1995).
In the comparative collection of modern parenchyma developed for this study, a number of fruits and seeds were also included. The justification for these inclusions was that these organs also often produce starch and other vitamins and minerals, and have been recorded in the ethnographic record for Tonga (Beaglehole and Beaglehole 1941; Gifford 1929; Whistler 2009) to supplement diets either on a regular basis or during times of famine. Fruits such as breadfruit (Artocarpus altilis), the tropical almond (Inocarpus fagifer), and bananas (Musa spp.) were grown bordering plantations of yams (Dioscorea spp.), aroids (Colocasia esculenta, Alocasia macrorrhiza, Cyrtosperma merkusii), sweet potato (Ipomoea batatas), kava (Piper methysticum) and arrowroot (Tacca leontopetaloides). Under the premise that these crops were being processed and cooked in the same areas as the roots and tubers, it is possible that some macro-remains of these organs may end up charred in hearths and were preserved in archaeological contexts. This has been documented elsewhere in Polynesia by the recent find of breadfruit in Tahiti (Kahn and Ragone 2013).
The organisation of tissues within fruits does differ from that of stem and root-derived organs, and therefore they were described separately. The basic cell morphology and types of boundary, ground and some vascular tissues were recorded for each sample of non-vegetative storage parenchyma. Some similarities emerged such as the nature of the ground tissues and the arrangement of vascular bundles where these were present within the organ. Most of the fruits included in this study are Dicotyledons or Eudicotyledons, and thus have different anatomical features from Monocotyledons which form the bulk of the species within the comparative collection. This data thus provided a means of comparison with the stem and root-derived organs. Here, fresh samples will be analysed using univariate and multivariate statistical analyses to study parenchyma morphology, with a small study upon the changes that occur during charring. These studies summarise the descriptions provided in Appendix A.
83 Ground tissue morphology
Some of the distinguishing features of parenchyma ground tissue are best described through comparison of these tissues with hardwood. Some differences to wood charcoal include consistent cell shapes that are usually rounded or angular; the presence of distinctive vascular bundles or tissues, and there are usually very few rays dividing these tissues. When comparing vegetative and non-vegetative storage parenchyma from within roots, tubers, fruits and seeds with each other these morphological features need to be teased apart to allow identification of charred material where possible.
As described above, 40 cells from each specimen were recorded to assess the morphology of parenchymatous ground tissues within roots, tubers, fruits and seeds. The length and width of each cell was recorded, along with cell shape (rounded, angular or rounded- irregular), and cell dimensions (isodiametric or elongated). On top of these attributes of the individual cells, some overall descriptions were made about the ground tissue. These included the overall presence or absence of inter-cellular spaces, and the variety of cell contents that had been preserved in the histological thin sections.
Most species contained ground tissue that were consistently either rounded (60%, n=26), rounded-irregular (12%, n=5), or angular (21%, n=9); however a small percentage (7%, n=3) had both rounded and angular cell shapes within the same specimen. These included
Artocarpus altilis, Colocasia esculenta, and Dioscorea alata, and interestingly these species do not share any phenotypical or genotypical traits with one another aside from the fact that all three specimens are types of starchy organs. These specimens represent a range of different storage organs including a fruit (A. altilis), a corm (C. esculenta) and a tuber (D. alata). The yams (Dioscoreaceae) are traditionally classified as root tubers; however the anatomy of these organs is much closer to stem-derived tissue than true roots. Onwueme (1978) argues that the yam tuber is “probably neither a root nor a stem structure. Rather it is a structure that
originates from the hypocotyls- the transition zone between the stem and the root”. With this in mind, the attributes for describing stem anatomy have been used here for yams rather than root anatomy, especially with regard to the arrangement of vascular tissues and overall tissue organisation.
The ground tissues of the remaining yams (Dioscorea bulbifera, Dioscorea esculenta, and Dioscorea nummularia) and the Polynesian arrowroot (Tacca leontopetaloides), which also belongs to the Dioscoreaceae family, are all rounded in shape. Similarly, the remaining aroids (Alocasia macrorrhiza, Cyrtosperma merkusii, and Xanthosoma sagittifolium) have rounded cells within the ground tissues. Other starchy crops such as the bananas (Musa spp.), kava (P. methysticum) and a type of ginger (Zingiberaceae sp.) contain more irregularly rounded cells within the ground tissue. The majority of the Pteriodophytes or ferns have more angular shaped cells, including Asplenium sp., Pteridium sp., and Todea sp. Similarly many of the fruits such as
Barringtonia asiatica, Barringtonia racemosa, Ficus tinctorius, Spondias dulcis, and Syzygium malaccense have angular ground tissue cells.
Table 5.6 Ground tissue cell shapes of taxa in reference collection
The majority of specimens included in this analysis had ground tissue parenchyma cells that were a mixture of elongated (greater in length than width) and broadly isodiametric (roughly even in length and width) in dimension. These specimens with mixed dimensions totalled 37 out of the full 43 specimens, equating to 86%. The remaining 14% (n=6) of specimens had consistently broadly isodiametric cell dimensions. These included Barringtonia asiatica fruit, Barringtonia racemosa seed, Dioscorea nummularia, Ficus copiosa, Saccharum officinarum, and Xanthosoma sagittifolium, and represent a range of different plant families and organ types. There does not appear to be any patterning that may suggest why this particular range of specimens has solely isodiametric cells within the recorded random sample of ground tissue recorded.
Table 5.7 Ground tissue cell dimensions of taxa in the reference collection
Rounded- irregular Angular
Artocarpus altilis seed Epipremnum pinnatum Musa sp. 1 Asplenium sp.
Alocasia macrorrhiza Ficus copiosa Musa sp. 2 Barringtonia asiatica fruit Angiopteris sp. Inocarpus fagifer Piper methysticum Barringtonia racemosa seed Asplenium sp. Morinda citrifolia Solanum tuberosum Ficus tinctorius
Araceae sp. Pangium edule Zingiberaceae sp. Ipomoea batatas
seed Pueraria lobata Pteridium sp.
fruit Saccharum officinarum Spondias dulcis
Cordyline fruiticosa Pandanus tectorius Mixed Syzygium malaccense
Cyrtosperma merkusii Tabernaemontana
aurantiaca Artocarpus altilis fruit Todea sp. Dioscorea bulbifera Tacca leontopetaloides Colocasia esculenta
Dioscorea esculenta Xanthosoma sagittifolium Dioscorea alata Dioscorea nummularia Zingiberaceae sp.
Barringtonia asiatica fruit Artocarpus altilis seed Ipomoea batatas Barringtonia racemosa seed Artocarpus altilis fruit Inocarpus fagifer Dioscorea nummularia Alocasia macrorrhiza Morinda citrifolia
Ficus copiosa Angiopteris sp. Musa sp. 1
Saccharum officinarum Asplenium sp. Musa sp. 2 Xanthosoma sagittifolium Araceae sp. Pangium edule
Barringtonia asiatica seed Pueraria lobata Barringtonia racemosa fruit Piper methysticum Colocasia esculenta Pandanus tectorius Cordyline fruiticosa Pteridium sp. Cyrtosperma merkusii Spondias dulcis Dioscorea alata Syzygium malaccense Dioscorea bulbifera Solanum tuberosum
Dioscorea esculenta Tabernaemontana aurantiaca Dioscorea nummularia Tacca leontopetaloides Epipremnum pinnatum Todea sp.
Ficus tinctorius Zingiberaceae sp. Mixed
85 The statistical analysis of cell lengths and widths points to a large degree of overlap in these particular attributes of ground tissue morphology. Box plots were created using the PAST statistical software to visually display the range of cell lengths and widths for each of the specimens included in the modern comparative collection. Outliers were indicated within the ranges of almost every species and organ, indicating that the random sample of 40 cells recorded in this study may not fully represent the degree of variation within the ground tissue of each specimen.
Despite this, the box plots demonstrate that most cell lengths within the comparative collection fall into a range larger than 40µm and smaller than 160µm. A number of species have cell lengths that could exceed 160µm, including Alocasia macrorrhiza, Angiopteris sp.,
Asplenium sp., Dioscorea alata, Dioscorea esculenta, Dioscorea nummularia, Ficus copiosa,
Ficus tinctorius, Ipomoea batatas, Musa sp. 2, Spondias dulcis, Solanum tuberosum, Tacca leontopetaloides and Todea sp.The overall ranges of many of these species are very similar, and therefore the diagnostic value of this attribute of ground tissue morphology is reduced. To narrow this down further it may be more useful to consider the smaller number of species that have cell lengths that can exceed 240µm. These include Angiopteris sp., Asplenium sp., D. esculenta, D. nummularia and S. dulcis. The largest cells recorded within the comparative collection are in the ground tissues of Asplenium sp. and D. esculenta, which both can exceed 320µm in length. On the smaller end of the spectrum, some specimens have cell lengths that can range below 40µm such as the fruit of Artocarpus altilis and the stem of Saccharum officinarum. The two samples of the fruit phalange of Pandanus tectorius, and the root tuber of
D. nummularia indicate that there can be some intra-species diversity in cell lengths, and therefore this attribute of ground tissue morphology should be considered mostly undiagnostic on its own.
All taxa within the reference collection have cell widths within the ground tissues that can range between 30-120µm. However, just over a third of the specimens have cell width ranges where the minimum widths are smaller than 30µm (37%, n=16). These include A. altilis
fruit, Araceae sp., B. asiatica fruit, B. racemosa seed, C. merkusii, F. copiosa, F. tinctorius, I. fagifer, M. citrifolia, Musa sp. 1., Pangium edule, S. dulcis, Syzygium malaccense, S. officinarum, and both Zingiber spp. The specimens that have ground tissue cell widths that range above 120µm (35%, n=15) include Alocasia macrorrhiza, Angiopteris sp., Asplenium sp.,
D. alata, D. bulbifera, D. esculenta, D. nummularia, F. tinctorius, Musa sp.2, S. dulcis, S. malaccense, S. tuberosum, T. leontopetaloides and Todea sp.. The largest cell widths by quite a significant margin are those of D. esculenta, which range up to 230µm and so is a diagnostic attribute of parenchyma from this species.
86 Considering the degree of overlap between specimens in the univariate analysis of each of the recorded metric attributes, it was prudent to carry out a multivariate statistical study that could assess the relationships between sets of attributes in the modern comparative collection. A dataset was created in the PAST program that included the data of four attributes: cell lengths, widths, shapes and dimension of 40 recorded ground tissue cells from each specimen. Cell shape and dimensions were turned into nominal variables, with a number from 1-3 representing each shape (rounded, rounded-irregular or angular) and 1-2 representing each type of dimension (isodiametric or elongate).
DFA of the parenchyma attributes was carried out in the same way that the starch samples were analysed. A confusion matrix was created, with a total of only 21.8% of the recorded cells being correctly re-classified back into the original species. This figure is statistically very low, indicating that the majority of modern specimens could not be easily disciminated from one another based on these four attributes. Despite this, a small number of specimens were able to be separated from the remaining groupings reasonably well. The most easily discriminated was Musa sp. 1,which had a total of 78% correct reclassifications into original species. Closely following this was Dioscorea esculenta with 73%. Several species including the seed of Barringtonia racemosa (60%), and the fruit of Ficus copiosa (58%) had over half of all the cells correctly reclassified. The outputs of the DFA complemented the outcome of the univariate analysis, suggesting that there is substantial overlap within the morphology of ground tissues of many of the specimens in the modern comparative collection. Clearly some species will be more easily identified during the sorting of archaeological samples based on the morphology of ground tissue, such as those that have cells larger than 160µm or smaller than 40µm, those that have mixed cell shapes or consistently isodiametric cell dimensions. The majority of cells do not fall within these ranges, and so other non-metric attributes need to also be considered.
Figure 5.12 Plot showing classification of parenchyma within reference collection using DFA
Other features of the ground tissue can include the presence of inter-cellular spaces, sclerenchyma, collenchyma, fibre bundles, duct cavities and cell contents. These can also be used to differentiate some species from one another. For example, both Musa sp. 1 and Musa sp 2. have duct cavities three to four cells long throughout the ground tissue that separate rows of parenchymatous cells. These species also contain fibre bundles which are also common throughout Zingiber spp., Asplenium sp., Cordyline fruticosa, Epipremnum pinnatum, and
Pandanus tectorius. Regions of sclerenchyma can be observed within the pith of the fruit of
Artocarpus altilis, Pangium edule, and Piper methysticum; while collenchmya forms the pith of
Angiopteris sp. Inter-cellular spaces are present in most ground tissues; however it is absent in
Dioscorea bulbifera, Pteridium sp., Spondias dulcis, Saccharum officinarum, and Solanum tuberosum. Cell contents can include also raphides, starch granules, druses and other types of crystals. Raphides are most commonly seen in aroids, but are also noticeable in the ground tissue of Tacca leontopetaloides.
Vascular tissue morphology
The overall tissue organisation within the parenchyma of the modern specimens varies between the root and stem-derived organs, and the fruits and seeds, alongside the typical anatomical differences between Monocots and Dicots. The vascular tissues within these types of organs therefore also vary. Bundles of vascular tissues are present in stem-derived organs, where the phloem and xylem are either encircling or abutting one another. These bundles can be organised within the organ in one of three ways: 1) Dictyostele, where the bundle is formed as a chamber surrounding the pith and can be broken into segments; 2) Eustele, where bundles are arranged concentrically as either primary or secondary tissues radiating from the pith; or 3) Atactostele, where the bundles are organised seemingly haphazardly within the ground tissue of the organ.
90 The arrangement of the xylem and the phloem within the bundles can also be categorised into morphological types. These include:
Amphivasal concentric— xylem completely encircles the phloem;
Amphivasal open ends— xylem almost completely encircles phloem apart from each end of the bundle;
Amphivasal u-shaped-—xylem partially surrounds the phloem in a u-shape formation;
Amphicribal concentric— phloem completely encircles the xylem;
Amphicribal open ends— phloem almost completely encircles xylem apart from each end of the bundle;
Amphicribal u-shaped— phloem partially surrounds the xylem in a u-shape formation
Open collateral- bundles of phloem and xylem abut one another with a region of cambium between the two types of tissue;
Closed collateral- bundles of phloem and xylem abut one another with no region of cambium between the two types of tissue;
Bicollateral—bundles of phloem and xylem abut one another with a region of cambium between the two types of tissue, and another additional bundle of phloem or xylem is present below this arrangement.
Figure 5.13 Description of vascular bundle arrangements within vegetative parenchyma (from Hather 2000)
When considering the stem-derived organs such as corms, rhizomes and stem tubers, it is clear that the majority of vascular tissues are organised within an atactotstele morphology (58%, n=11). Much smaller numbers are organised within a eustele (16%, n=3), or dictyostele arrangement (26%, n=5). Those specimens that contain an atactostele arrangement of vascular tissues tend to be corms and stem tubers of Monocots, while the dictyostele arrangement is most
91 common in the Pteriodophytes or fern rhizomes, and the eustele arrangement is commonly seen in Dicots. When the arrangements of vascular tissues within these bundles are broken down into their respective categories, further patterning becomes apparent. Some specimens did not have visible or clear vascular tissues and so were not included in the below table (see Table 5.8).
All of the aroids (Araceae) assessed within this study have vascular bundles within an atactostele pattern of stele organisation (as these are Monocots), and the vascular tissues are of an amphivasal arrangement where the phloem is surrounded by the xylem. Colocasia esculenta
is the only aroid to contain two different types of amphivasal bundling, having both amphivasal concentric and u-shaped. Both Alocasia macrorrhiza and Cyrtosperma merkusii have amphivasal open-ended bundles, while Xanthosoma sagittifolium has bundles of amphivasal concentric arrangement. Another family belonging to the Monocots are the yams (Dioscoreaceae) and therefore have vascular bundles within an atactostele organisation; however these tissues differ from those belonging to the aroids as they are all of collateral bundling arrangement. Dioscorea alata and both samples of Dioscorea nummularia have bundles of open collateral arrangement with a region of cambium between the vascular tissues, while Dioscorea esculenta has closed collateral bundles without the layer of cambium. The only species from Dioscoreaceae to have a bicollateral arrangement is Tacca leontopetaloides, but outside this family Solanum tuberosum and Saccharum officinarum also contain bundles of this arrangement. The two specimens belonging to the Zingiberaceae family both have vascular tissues of amphicribal concentric arrangement where the xylem is surrounded the phloem.
Table 5.8 Vascular tissue arrangements of taxa in reference collection
Collateral Amphivasal Amphicribal
Dioscorea alata Alocasia macrorrhiza Zingiberaceae sp.
Dioscorea bulbifera Colocasia esculenta
Dioscorea esculenta Cordyline fruticosa Stem- Dictyostele
Dioscorea nummularia Cyrtosperma merkusii Angiopteris sp.
Tacca leontopetaloides Xanthosoma sagittifolium Asplenium sp.
Solanum tuberosum Epipremnum pinnatum Pteridium sp.
Saccharum officinarum Todea sp.
Collateral Amphicribal Unknown
Ficus copiosa Artocarpus altilis Pangium edule
Ficus copiosa Morinda citrifolia
Amphivasal Inocarpus fagifer Ficus tinctorius
Barringtonia asiatica Musa sp. 1
Barringtonia racemosa Musa sp. 2 Root
Pandanus tectorius Spondias dulcis Ipomoea batatas
Syzygium malaccense Pueraria lobata
Tabernaemontana aurantiaca Piper methysticum
92 The arrangement of vascular tissues within the fruits can vary significantly between samples. Where vascular bundles are present, these are not organised within an atactostele, eustele or dictyostele arrangements, but depend on other anatomical features of the organs. The arrangement of the tissues in the bundles is described, but not the overall organisation within the fruit as these do not fit any of the morphological categories used previously. The main purpose of including these samples is the description of basic cell and vascular morphology, in order to explore some differences between these organs and vegetative storage parenchyma. Almost half of the fruit specimens included within the comparative collection have vascular bundles of amphicribal arrangement (47%, n=8). Within these are a number of specimens with amphicribal concentric bundles including both Musa spp. and Tabernaemontana aurantiaca, and one example of a specimen with amphicribal u-shaped in Artocarpus altilis. A different version of