Figure 5.2 Diagram showing basic features of starch granule morphology

Fissuring at the hilum was also observed within 18 specimens in the comparative collection. There are a range of different types of fissuring that can occur within starch (ICSN 2011), and a small number of these were present within the collected specimens. The most common type was longitudinal fissuring, where the fissure extends along the long axis of the grain. Nine specimens had granules with longitudinal fissuring, but it was only common in three species, Amorphophallus paeoniifolius, Dioscorea rotundata and P. methysticum. Radial fissuring was also identified. This is defined as fissuring that originates at the hilum and spreads outwards to the margins (ICSN 2011; Reichert 1913), and was commonly recorded within two species, Ipomoea polpha and Piper methysticum. Transverse fissuring extends at a right angle to the long axis of the grain (ICSN 2011), and was observed in a total of five specimens (see Table 5.1). Both I. polpha and Spondias dulcis granules commonly feature this hilum type. Stellate fissuring is another common type recorded within the comparative collection. These star-shaped fissures were common in three species- I. polpha, P. methysticum and S. dulcis, and rare in

Artocarpus heterophyllus and Inocarpus fagifer.

Several other types of hilum fissuring were identified within a smaller range of species. Oblique fissures are simple fissures that do not follow any particular axis on the grain (ICSN 2011, Reichert 1913) and were found commonly on granules from Dioscorea rotundata but only very rarely on Solanum tuberosum. Irregular fissures are those that are uneven in geometry and were observed on the starch of three specimens; however, this type was only common in I. polpha and P. methysticum. The presence of a mesial cleft, a large, deep and variably ragged interior crack that runs parallel to the long axis of the grain (ICSN 2011), was only noted in the starch of P. methysticum. Likewise, branching fissures were only observed in S. dulcis. These are fissures that feature subdivisions from the main branch. The final type of fissuring within the comparative collection is actually classed as an ‘indentation’ at the hilum, and was observed frequently in A. paeoniifolius but only rarely in I. fagifer.

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Table 5.1 Hilum fissuring of reference species

Lamellae

Lamellae are the growth layers that begin forming at the hilum and disperse towards the margins of the granule. These were ‘distinct’ in many large starch morphotypes deriving from the Dioscorea genus including Dioscorea alata, Dioscorea bulbifera, Dioscorea nummularia, Dioscorea pentaphylla, and Dioscorea rotundata. Distinct lamellae were also visible on all recorded starch from Curcuma longa and most starch from Musa sp. 1. ‘Indistinct’ or less prominent lamellae were observed on larger starch morphotypes from the fruit of Artocarpus altilis, Horsfieldia palauensis, Ipomoea batatas, Ipomoea polpha and Musa sp.2 .Smaller starch morphotypes, such as those seen within Alocasia macrorrhiza, Colocasia esculenta and

Dioscorea esculenta may have lamellae, but the limitation of light microscopy inhibit the ability to view these features, and lamellae were also not often visible using SEM.

Three-dimensional shape

Two and three-dimensional shapes are commonly used to describe and discriminate between starch morphotypes. In this study only three-dimensional (3D) shape was used as the granules were rolled during light microscopy to optimise visualisation of all planes of the granules. Fifteen different 3D shapes were observed within the comparative collection. These included a variety of rounded shapes such as spherical, sub-spherical, ovate, ellipsoidal, sub-ovate, pyriform (pear-shaped), reniform (kidney-shaped) and shapes with single or multiple facets such as dome, hemispherical, prismatic, polyhedral, quadrangular, cylindrical, conical, and prismatic. The nomenclature used to describe these shapes follow the International Code for Starch Nomenclature (ICSN) compiled in 2011 with some modification.

Rounded granules were observed within 27 of the 29 specimens within the comparative collection (see Table 5.2). The two species that did not have rounded starch morphotypes were

D. esculenta and C. longa. The most commonly observed shape was spherical or “…a sphere in

Longitudinal Radial Oblique

Amorphophallus paeoniifolius Artocarpus heterophyllus Dioscorea rotundata

Artocarpus altilis seed Barringtonia asiatica fruit Solanumtuberosum

Dioscorea nummularia Cyrtosperma merkusii

Dioscorea rotundata Dioscorea alata Indentation

Inocarpus fagifer seed Dioscorea bulbifera Amorphophallus paeoniifolius

Musa sp. 2 Ipomoea polpha Inocarpus fagifer seed

Piper methysticum Piper methysticum Transverse

Tacca leontopetaloides Barringtonia asiatica fruit

Xanthosoma sagittifolium Stellate Dioscorea rotundata

Irregular Artocarpus heterophyllus Ipomoea batatas

Cyrtosperma merkusii I.fagifer seed Ipomoea polpha

Ipomoea polpha Ipomoea polpha Spondias dulcis

Piper methysticum Piper methysticum Branching

Mesial cleft Spondias dulcis Spondias dulcis

67

which all radii are equal length.” (Reichert 1913), with a total of 22 specimens containing starch that matched this description. Within these specimens were members of a range of different families, and so there does not appear to be any diagnostic value in this shape for taxonomic classification. Likewise, sub-spherical granules were present within 15 specimens from the Anacardiaceae, Araceae, Convolvulaceae, Dioscoreaceae, Lecythidaceae, Moraceae, Musaceae, and Solanaceae. These are spherical granules with some small degree of curvature or are scalene, where all three main diameters are of unequal length. Also present in a large percentage of the reference collection were elongated granules which tend to be high in amylase (Gott et al. 2006). Ovate (n=16), subovate (n=2), and ellipsoidal (n=9) granules were commonly observed within members of the Dioscoreaceae and Musaceae families. These are distinguished from one another based on the diameter of the proximal, mesial and distal ends of the grain. Ellipsoidal granules have both distal and proximal ends equal in size, while ovate granules tend to be more egg-shaped with one end wider than the other. Sub-ovate are elongated granules with the same variation as described for sub-spherical granules. Other variations to these rounded granules included reniform or kidney-shaped granules, and pyriform or pear-shaped granules. These differ from the sub-ovate and sub-spherical granules based on a larger degree of curvature (reniform) and scalar width variation (pyriform). Pyriform granules were only recorded in those specimens belonging to the genus Musa, while reniform granules were present in five different specimens representing four different families including Dioscoreacae, Convolvulaceae, Musaceae, and Solanaceae.

More angular starch shapes were also very common within the comparative collection. Twenty eight of the 29 specimens had at least one angular morphotype. The only species that did not was Dioscorea pentaphylla. These shapes vary in the nature and number of straight or rounded sides (facets) that define the margins and diameters of the granules. Angular granules have been formed in compound starch granules, where a number of granules are clustered together within the parenchymatous cells, creating pressure facets. Single faceted 3D shapes include dome and hemispherical granules, which are differentiated from one another based on elongation. A domed granule is half an oval, while a hemispherical granule is half a sphere. Domed granules were present in 12 specimens, while hemispherical granules were slightly more common, being recorded within 15 specimens. Four of the five aroids, and both members of the

Artocarpus genera within the comparative collection contained hemispherical granules. The remaining aroid, Xanthosoma sagittifolium,instead had dome-shaped granules. Some specimens contained both shapes, including Artocarpus heterophyllus, Amorphophallus paeoniifolius, the seeds of Barringtonia asiatica, Cyrtosperma merkusii, Morinda citrifolia and Piper methysticum.

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Table 5.2 Three-dimensional shapes of reference species

Multi-faceted angular shapes recorded included conical, cylindrical, polyhedral, quadrangular, and prismatic. A number of specimens contained starch that could be described as conical, having a flat circular base and a tapering top. These included Curcuma longa, most of the members of the Dioscorea genus apart from D. pentaphylla which has more ovate granules, both Musa spp., and Solanum tuberosum. Cylindrical granules fall part way between rounded and angular, being described as having “…a circular base and top, both of equivalent size.” (Reichert 1913). This shape was not commonly observed, and was present only in three specimens. Four specimens contained granules that had six sides, with four being elongated, and thus could be described as quadrangular. Within these four specimens, less than five of these granules were observed. The most common multi-faceted angular shape by a significant margin was polyhedral. Polyhedral granules are defined as “…having many faces that are not

necessarily of the same two dimensional shape.” (Reichert 1913). These were recorded in all of the aroids, several members of the Dioscoreaceae family, and a range of other Monocots and Eudicots totalling 15 specimens.

Dome Reniform

Amorphophallus paeoniifolius Ipomoea batatas Artocarpus heterophyllus Dioscorea nummularia Artocarpus altilis fruit Inocarpus fagifer seed Amorphophallus paeoniifolius Dioscorea pentaphylla Artocarpus altilis seed Ipomoea polpha Barringtonia asiatica fruit Ipomoea polpha Artocarpus heterophyllus Morinda citrifolia fruit Barringtonia asiatica seed Musa sp. 1 Barringtonia asiatica fruit Musa sp. 1 Cyrtosperma merkusii Solanum tuberosum Barringtonia asiatica seed Piper methysticum Horsfieldia palauensis

Colocasia esculenta Pteridium sp. Ipomoea polpha Sub-ovate

Cyrtosperma merkusii Spondias dulcis Morinda citrifolia fruit Musa sp. 1 Dioscorea nummularia Solanum tuberosum Musa sp. 1 Solanum tuberosum Dioscorea pentaphylla Tacca leontopetaloides Piper methysticum

Horsfieldia palauensis Xanthosoma sagittifolium Pteridium sp.

Xanthosoma sagittifolium

Alocasia macrorrhiza Dioscorea rotundata Alocasia macrorrhiza Inocarpus fagifer seed Amorphophallus paeoniifolius Horsfieldia palauensis Artocarpus altilis fruit Ipomoea polpha Artocarpus altilis fruit Musa sp. 1 Artocarpus heterophyllus Musa sp. 1 Artocarpus altilis seed Musa sp. 2 Barringtonia asiatica fruit Musa sp. 2 Dioscorea alata Piper methysticum Barringtonia asiatica seed Piper methysticum Dioscorea bulbifera Pteridium sp. Dioscorea bulbifera Spondias dulcis Dioscorea nummularia Spondias dulcis Dioscorea rotundata Solanum tuberosum Dioscorea pentaphylla Solanum tuberosum Ipomoea batatas

Alocasia macrorrhiza Dioscorea bulbifera Alocasia macrorrhiza Horsfieldia palauensis Amorphophallus paeoniifolius Ipomoea batatas Amorphophallus paeoniifolius Ipomoea batatas Artocarpus altilis fruit Inocarpus fagifer seed Artocarpus altilis fruit Inocarpus fagifer seed Artocarpus altilis seed Morinda citrifolia fruit Artocarpus altilis seed Morinda citrifolia fruit Artocarpus heterophyllus Piper methysticum Artocarpus heterophyllus Pteridium sp. Barringtonia asiatica seed Spondias dulcis Colocasia esculenta Tacca leontopetaloides Colocasia esculenta Tacca leontopetaloides Cyrtosperma merkusii Xanthosoma sagittifolium

Cyrtosperma merkusii Dioscorea esculenta

Ellipsoidal Conical Quadrangular Pyriform

Barringtonia asiatica fruit Curcuma longa Artocarpus altilis seed Musa sp. 1 Barringtonia asiatica seed Dioscorea alata Artocarpus heterophyllus Musa sp. 2 Dioscorea bulbifera Dioscorea bulbifera Dioscorea esculenta

Dioscorea nummularia Dioscorea nummularia Ipomoea polpha

Ipomoea polpha Dioscorea rotundata Cylindrical Prismatic

Musa sp. 1 Musa sp. 1 Musa sp. 2 Xanthosoma sagittifolium

Musa sp. 2 Musa sp. 2 Spondias dulcis Pteridium sp. Solanum tuberosum Tacca leontopetaloides

Spherical

Ovate Sub-spherical

69 Modifiers of shape

These three-dimensional shapes can be altered by several other morphological attributes. Faceting was discussed above in terms of the development of overall shapes when granules are formed in compound clusters. Sometimes these ‘pressure facets’ are minimal, and only create slight impressions on the sides of granules. These granules are usually rounded in shape but have one or two pressure facets on the margins. Additionally, some dome or hemispherical granules can have an additional one to two pressure facets where these granules have been formed in clusters of three granules. They still retain the shape of half a sphere or oval. These types of pressure faceting are common in Amorphophallus paeoniifolius, Barringtonia asiatica, Cyrtosperma merkusii, Inocarpus fagifer, Ipomoea batatas, Ipomoea polpha, and Piper methysticum.

A small number of specimens within the comparative collection contained granules that were ‘compressed’ so that the granules had a smaller dimension in one plane than the other. These granules had to be consistently rolled to view this modification of shape in profile. Three of the yams- Dioscorea alata, Dioscorea bulbifera, and Dioscorea rotundata, and a ginger-

Curcuma longa, all have conical or ovate starch granules that are frequently compressed. In addition, both Musa spp. within the comparative collection also have compressed forms, but these are less commonly observed than non-compressed granules. Equitorial grooves often coincide with compressed granules or those that have uneven dimensions in different planes, and can be seen as a fold that runs along the long axis of the granules when these are turned on edge (ICSN 2011). This feature was common in Dioscorea rotundata and noted in Artocarpus altilis, Amorphophallus paeoniifolius, Dioscorea alata and Piper methysticum.

Several shape modifications were restricted to only one or two particular specimens within the comparative collection, and so can be considered diagnostic attributes of starch morphotypes. The first of these were ‘projections’, which were defined as areas that extend beyond the main surface of the grain (Reichert 1913), and were present on all recorded granules from C. longa, and a small number of granules of Dioscorea pentaphylla. The conical starch granules deriving from these specimens also had sharply tapering tips that differentiated these from granules seen in other specimens. The starch of Morinda citrifolia were often noticeably ‘concave-convex’ where one side curves inward and the other curves outward (ICSN 2011), creating a hollowed appearance. Finally, the conical starch of Dioscorea bulbifera each had a distinctive ‘bend’ at the proximal end of the granule just below the hilum. This was sharper than a ‘curve’ which is defined as a smooth bend in the form of the granule (Reichert 1913).

Granule length and width

The maximum length of starch granules is used by many starch analysts to identify unknown archaeological starch as a univariate statistical technique within an assemblage-type approach (Field et al. 2009; Therin et al. 1999). Following these studies, the range of granule lengths

70 within the comparative collection were analysed and compared as assemblages of each species (see Figure 5.3). Most of the recorded starch within the comparative collection falls into the 8- 25µm range (86% of species), and due to the relatively narrow range there is a significant amount of overlap between specimens. Only a small number of specimens have ranges that are larger or smaller than this. Several of the yams, Dioscorea pentaphylla and Dioscorea rotundata, as well as both Musa spp., Curcuma longa,Ipomoea polpha and Solanum tuberosum

all have maximum lengths that could exceed 40µm. Of these, the Dioscorea spp. are the only specimens that have an average length above 40µm. Overall, Dioscorea pentaphylla has the largest starch within the comparative collection, with an average length of 90µm, and a maximum of 145µm and are therefore taxonomically distinctive. On the smaller end of the spectrum, several specimens have starch morphotypes that can range below 8µm. These include

Artocarpus altilis seed and fruit, Artocarpus heterophyllus, Alocasia macrorrhiza, Colocasia esculenta, Cyrtosperma merkusii, Dioscorea bulbifera, Dioscorea esculenta, Horsfieldia palauensis, Inocarpus fagifer, Ipomoea batatas, Ipomoea polpha, Morinda citrifolia, Pteridium

sp., Tacca leontopetaloides and Xanthosoma sagittifolium. The only specimen to have the entire range of maximum lengths below 8µm is C. esculenta, which has an average length of 4µm. Despite this, this species cannot be distinguished from others solely on this attribute, as a number of other specimens have overall ranges that overlap with Colocasia esculenta such as

Alocasia macrorrhiza and Artocarpus altilis.

A study of granule widths revealed that there is substantial variation in the comparative collection based on this metric attribute. The starch of most taxa had widths between 7-19µm (93% of species), which is marginally smaller than the range of starch lengths for most species. However, unlike starch lengths, there is a greater number of specimens that contain starch smaller or larger than this range (see Figure 5.4). Sixteen specimens have granules that range in width above 19µm. Dioscorea pentaphylla is once again distinguishable by having the largest starch granules in this second dimension. A total of 20 specimens contain starch morphotypes that can range smaller than 7µm, but only six of these specimens have average widths below this point. The smallest width range was recorded within the starch of Colocasia esculenta

which has a median of 4µm, and a total range of 2-7µm.

Clearly, there is a significant amount of overlap in these two variables in the comparative collection. Only one specimen can be confidently distinguished from others based on either of these variables. Dioscorea pentaphylla has starch that ranges larger in length and width that any other specimens. On the smaller end of the spectrums for both widths and lengths, C. esculenta consistently has the lowest medians for these attributes, but this species still overlaps with the total range of others within the comparative collection. Therefore it is not able to be distinguished solely on these attributes.

71 Other metric variables

In addition to maximum length and width, a number of other metric variables were measured on all starch granules sampled from each species within the comparative collection. As mentioned previously, these included the ‘length of the extinction cross’, ‘maximum distance between the arms of the cross’, ‘distance between the hilum and the end of the granule’, and the ‘maximum angle within the arms of the extinction cross’. Most of these measurements are not diagnostic of species on its own when compared through univariate analyses of the starch assemblages in the comparative collection. These are more relevant when considered in multivariate statistical analyses as these dimensions are related to the three-dimensional shape and surface texture of starch granules. Therefore several of these will not be explored here, but instead will be compared as variables in the multivariate morphometric analysis later in this chapter.

Hilum position can be compared between specimens but only when this is considered as a ratio between the ‘length’ and the ‘distance between the hilum and the end of the granule’ (see Figure 5.5). This ratio enables statistical differentiation between starch that is centric (end-on) or eccentric (side-on). Centric hila are those that have a ratio that falls between 0.5-0.6, meaning that the hilum is close to the centre of the granule. These granules have been viewed and recorded end-on. Eccentric hila are those that have a ratio falling in the range of 0.61-1, and indicate that the granule has been viewed side-on. This is particularly relevant for granules that vary in dimension across different planes such as ovate or conical granules. When the distribution of these ranges is compared between specimens, it is clear that most sampled granules from each specimen have been recorded in both centric and eccentric views. However a number of specimens were primarily viewed side-on, including C. longa, D. alata, D. bulbifera, D. nummularia, D. pentaphylla, D. rotundata, Musa spp., S. tuberosum and S. dulcis.

This is most likely a result of the ovate, sub-ovate or conical shape of these granules which makes them hard or impossible to view completely end-on. Another 18 specimens were primarily viewed end-on, and this is similarly a result of shape, but in this case granules are mostly spherical or polygonal and so are even in length across all dimensions. These hilum position ratios were used within the following multivariate statistical analysis to divide the starch within the comparative collection into comparable datasets.

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Figure 5.3 Box plot of starch granule lengths within reference collection