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13.2.1 Flowering Phenology and Production

Hellenge Hill and Woodborough ecotypes began flowering significantly earlier than the other ecotypes (Figure 55), this may be a reflection of these two sites having the highest altitudes in the study (160m and 190m respectively) (Table 7) as flowering time variation has been found over an altitudal gradient in a clinal study (Suter et al., 2014).

Two clear flowering peaks were seen in the first year (April and July) but different patterns occurred in the second, some ecotypes having just one peak and all finishing earlier (which could explain the lower seed pod count for the second year). Springate and Kover (2014) found elevated temperatures accelerated flowering time and Meineri et al. (2014) found it to increase flowering production, both of which could be the case here, as Figure 26 shows higher monthly temperatures in this year [to the previous year] for June to October. However, in contrast, Frei et al. (2014) found elevated

temperature did not affect flower number of Trifolium montanum L., Ranunculus bulbosus L. and Briza media L. in their study and Reisch & Poschlod (2009) instead found genetic differentiation of flowering phenology was stronger between land managements than it was between geographic regions. Looking again at the flowering pattern, only cut and calcareous loam ecotypes still had two distinct peaks (in April and June) again during the second year (Figure 56). As all calcareous loam ecotypes were either from grazed or cut sites, this could be an adaptation to the timing of seed pod loss

through biomass removal at home-sites. Research on Scabiosa columbaria L. (Reisch & Poschlod, 2009) found genetic variation of floral display was clearly linked with land-use, shown by populations of mown sites flowering

significantly earlier than those from grazed, and Warren & Billington (2005) also found hay meadows flowered earlier than other grasslands.

The calcareous loam ecotype had produced significantly more flowers by harvest and there was significant positive interaction between the sand ecotype in matching soil type, and unmanaged ecotype in unmanaged treatment. This could indicate more productive ecotypes and/or a matching soil type or management advantage (and adaptation). Indeed, Stephenson (1984) concluded that L.corniculatus flower number was regulated by soil resource availability, Ollerton & Lack (1998) determined larger plant size to be correlated with a longer flowering period in L.corniculatus and Forrest (2014) stated that in many plant populations early flowering and improved fecundity are positively correlated with larger plant size which [size] could also be indicating greater fitness here, potentially from factors such soil resources. Treatments led to significant differences in all flower parameters; plants grown in calcareous loam soil had significantly shorter time to first flowering,

supporting Ollerton and Lack’s (1998) and Forrest’s (2014) theory of larger plants flowering earlier. Plants grown in neutral loam treatment soil produced significantly more flowers at harvest and plants of unmanaged treatment had signifiantly stronger flower scent in both pre-harvest flower scent and bagged flower scent tests. Such differences in flower phenology should cause

negligible implication for herbivory as this plasticity would presumably be constant at the herbivore home-site due to the treatments being the cause rather than the ecotypes.

13.2.2 Growth Habit and Hirsuteness

There was no management treatment difference within hirsuteness though calcareous loam treatment soil produced significantly more glabrous plants (P<0.001) (Table 29). Ecotypic variation was also seen for hirsuteness with the three sand ecotypes (Berrow Dunes, Woolacombe Warren and Dawlish Warren) significantly more hirsute than the Cockey Down standard (P<0.001).

These differences in hirsuteness could be another adaptation of the sand ecotypes to a dry environment as leaf surface hairs can reduce moisture loss (Maun, 2009). When grouped together, ecotypes from neutral loam sites and those from cut sites were significantly more glabrous than the standard. This could have fitness implications in terms of plant longevity, in a mismatched receptor site, a glabrous plant from a loamy meadow translocated to a dry, coastal site with high desiccation may exhibit signs of drought stress. Hirsuteness could also reduce feeding ability, if animals at the receptor site are not adapted to digesting trichomes, often found to be a form of structural defence (Hanley et al., 2007). However it should be noted that mean results (for ecotypes and treatments) each rounded to 2 ‘slightly hirsute’.

Contrasting to previous research (Kelman et al., 1997), only treatment

differences were shown to be significant for growth habit with neutral loam soil and unmanaged treatments producing more erect profiles. Panagiota et al. (2014) found L. corniculatus had a more prostrate growth habit when heavily grazed and Kelman noticed associations between prostrate growth habits of L. corniculatus and condensed tannin levels. However, growth habit here was considered to be phenotypic plasticity only, due to lack of differentiation between ecotypes.

13.2.3 Biomass

Treatments had the greatest effect on biomass, with higher grazed treatment dry clippings (Figure 63) and harvest biomass (Table 30) found for plants grown in calcareous loam treatment soil (P<0.001 for both). In other studies, nitrogen has been shown to be the main limiting factor in plant biomass production (Prine & Burton, 1956; McLeod & Murphy, 1983; McNaughton et al., 1983; Zhang et al., 2015), which may be the case here as sand and

neutral loam treatment soils had significantly less nitrate than calcareous loam (P<0.050) (Table 11).

Unsurprisingly, unmanaged treatment plants had significantly higher dry harvest weight (mean 3.7g compared to 1.8g for grazed treatment P<0.001), likely due to no previous vegetation removal. However, grazed treatment plants had significantly higher relative moisture content (mean 71.2%

compared to 68.5% in unmanaged, P<0.001), a result also found in previous research of defoliation effects on a sedge (Kyllinga nervosa Steud)

(McNaughton et al., 1983), where it was considered plant material lost [through grazing] conserved soil-water and increased relative moisture content of the plant, both of which contributed to increased plant growth after grazing. It could be that harvesting fresh-growth regularly (through the grazed treatment) reduced dry material that would have instead become thickened and lignified from maturity (Engels & Jung, 1998).

The only significant variation between ecotypes for biomass (Figure 62, Table 30) was Woodborough which had significantly lower grazed treatment

clippings biomass (P=0.035) (Figure 62). As this was the only site differing it could be this ecotype is just a poorer biomass producer than the others.

13.2.4 Chemical Properties

For leaf-nitrogen it was treatment that resulted in greatest variation (Table 31, Table 32). Dawlish Warren was the only ecotype to show significant

difference (in the model) with lower leaf-nitrogen than the standard in unmanaged treatment. Dawlish Warren’s lower leaf-nitrogen could reflect a reduced capacity to retain this element where the ecotype has become

adapted to low amounts at the home-site (Chapin, 1980; IPNI, 2013) [Dawlish Warren had the lowest nitrate content of all ecotype soils at 6.12ppm] (Table 3), this possible adaptive feature is reflected in the treatment differences. It has also been found that low plant nitrate content (and therefore low

nutritional value) can be used as a form of herbivore defence when low dietary nitrogen cannot be compensated for by herbivores by infinite eating (Mattson, 1980). However, nitrogen is also needed for production of nitrogen based chemical defences such as HCN (Hermes & Mattson, 1992).

The management treatment was shown to be the most influential factor in the model, with significantly higher leaf-nitrogen shown in the grazed treatment plants (mean 0.71%). Such increased leaf-nitrogen is thought to be at least partly due to induced reaction of secondary metabolite production after herbivory (Bardgett et al., 1998). The sand treatment soil produced

be assumed that this low leaf-nitrogen reflects that available from the soil (Aerts, 1996; Cornelissen et al., 2003). This effect in the sand treatment could be due to leaching of nitrate from the soil, nitrate is a mobile element (IPNI, 2013) and lack of soil organic matter and clay (mean in sand soil = 1.71%) (Figure 20) in this substrate would have created a faster rate of loss and a lower reservoir (normally held in the organic matter) (IPNI, 2013).

Hydrogen cyanide (HCN) was found to be significantly lower in Salisbury Plain plants (mean 0.73 degrees of colour) (Table 33, Figure 65), this could be a factor of the unmanaged condition of this home-site, as HCN is thought to be used as herbivore defence (Morant et al., 2008; Pentzold et al., 2014).

Although historically the Salisbury Plain site would have been grazed by rabbits and sheep it is now one of the sites with the longest sward height of all ecotypes sampled (Appendix V). This site also contained the least cyanogenic plants (1), which contrasts with Ellis (1977) who suggested only highly

exposed coastal sites would contain predominantly acyanogenic plants.

The three significantly higher leaf-HCN ecotypes (Southstoke, Woodborough, and Folly Farm) (means respectively 35.67, 25.40, 37.53 degrees of colour) which also contained the least acyanogenic plants of all, were from cut sites. This was supported in the ecotype management grouping model which

showed cut ecotypes as having significantly higher leaf-HCN compared to the grazed standard, and higher still than the unmanaged ecotypes. This result suggests an environmental adaptation to a stressed home-site management, corresponding with previous work in which predominantly acyanogenic

L.corniculatus often occurs in environmental conditions where cost of producing the chemical is outweighed by other factors (Bloom et al., 1985; Till-Bottraud & Gouyon, 1992).

Ecotype soil groupings (Table 33) also showed variation with those from calcareous loam sites having significantly more leaf-HCN (mean 53.07 degrees of colour) compared to the sand ecotypes (mean 16.6 degrees of colour). This may be from adaptation to homesite conditions where decreased nutrient uptake of an easily leaching sand soil (Aerts, 1996; Cornelissen et al.,

2003; IPNI, 2013) prioritises nitrogen availability towards plant growth, whereas excess nitrogen (to that needed in growth) in the loam soils have been allocated to HCN production (Herms & Mattson, 1992), as HCN is found to be higher in plants when nitrogen is in high supply (Gleadow & Møller 2014). This result could also be due to the sand ecotypes close proximity to the sea, and therefore greater exposure to wind-borne salt, where numbers of selective herbivores are found to be lower (Ellis et al., 1977). However, in contrast to Ellis et al. (1997) Berrow Dunes contained 52% cyanogenic plants, and Dawlish Warren 69% rather than predominantly acyanogenic plants. Ellis et al. (1977)’s thoeory of coastal exposure could however, explain differences between the sand ecotypes, as Dawlish Warren is located on the south coast as apposed to the north (more exposed coast) of the other two sand ecotypes this could be a contributing factor to its higher amount of cyanogenic plants. Although there was no correlation between leaf-nitrogen and leaf-HCN, it is interesting to see Dawlish Warren had significantly higher leaf-HCN, yet significantly lower nitrogen (compared to the model standard), suggesting a resource allocation trade-off between the two with nitrogen being used in HCN production as priority over plant growth (Hermes & Mattson, 1992). It may also suggest this site has had long historical grazing of wild and/or domestic

herbivores (Morant et al., 2008).

There were no management treatment differences for leaf-HCN, this result was surprising as previous research has pointed towards herbivory (and therefore tissue damage) as activating chemical defences (Briggs, 1991; Morant et al., 2008; Pentzold et al., 2014). However, it has also been found that this interaction is more likely to happen in nectar rather than leaves (Alder et al., 2006). There were both cyanogenic and acyanogenic forms within each treatment and each ecotype, as found in previous research (Ellis et al., 1977). Although leaf-HCN levels did not differ significantly in response to soil or management treatments, significant differences were recorded between ecotypes. Such ecotype differences are thought more likely to be genetic adaptations to home-sites rather than phenotypic plasticity.