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It was found that the majority (77%) of pattern type combinations were explained by combinations containing the four recognised chloroplast lineages (CC+, CT+, TC-, CC-) with the main nuclear pattern types 5, 15, 17, and 20, and their

154 variants (Tables 3.24-3.27). Of this variation over half (46%) was CC+ (Table 3.24) supporting this lineage as the most wide spread and potentially the oldest. The above results support the idea of free intermixing between out-crossing individuals, allowing chloroplast and nuclear pattern types to become recombined. This modifies the diversity in the populations in a way proportional to the initial level of diversity.

Combinations containing rare nuclear pattern types collectively accounted for 22% of observed combinations (Tables 3.24-3.27). The CC+ lineage occurred in 82% of these (Tables 3.24, 3.25-3.27).

These results seem to suggest that lineages CC+/15 and CC+/5 are among the oldest. The frequencies of these lineages suggest there is a mechanism for promoting their wide distribution. As these patterns predominated in the commercial populations, it is likely that commercial activity has been a major factor in their spread. This agricultural spread probably changed allele frequencies, swamped the local variation with CC+/15 and CC+/5 and created new combinations through intermixing and recombination.

155 4.3.2 Chloroplast & nuclear results do they agree: or disagree?

Figure 4.13 The approximate relative pattern combinations, shown as circles and coloured to show different combinations.

Note grey circles represent combinations with rare nuclear pattern types.

Grey stars (Portugal and Spain) represent the rare chloroplast lineage CC-.

156 Distribution of combined pattern types, diversity in the regions

Figure 4.13 showed that diversity in pattern types was greater in Zone 1, Portugal and Spain, than in Zone 2. Within Zone 2, two regions, Northern Europe and the UK had the greatest number of pattern combinations. This differed from the results observed for the individual nuclear and chloroplast data sets.

The regions of diversity Portugal and Spain, Zone 1

The nuclear and chloroplast results both suggest Portugal as an area of diverse pattern types but the variation observed in Spain is less prominent (Fig. 4.13). However, as nearly twice as many individuals were sampled for ITS from Portugal as from Spain further sampling may show equivalent or greater diversity in Spanish populations.

North Africa

In the nuclear data North Africa was one of the most diverse regions. Although the

absence of chloroplast variation in North Africa suggested singular or limited T. pallescens-like lineages within the region. The prominence of rare nuclear pattern

types supports the hypothesis of multiple ancestral paternal lineages. However as the sample size was small, the results are tentative.

Northern and North-Western Europe

Northern and North-Western European populations showed a limited amount of

chloroplast diversity, but included the novel nuclear ITS lineage 17. This geographically restricted lineage was observed with each of the chloroplast types CC+, CT+ and TC-, creating combinations not observed anywhere else. The combined results suggested that despite limited chloroplast diversity, the amount of variation in nuclear pattern types, even in absence of rare nuclear pattern types, indicates considerable diversity within the region (Figs. 4. 5 and 4.12).

UK

The UK showed multiple combinations based on chloroplast and nuclear variation. The high diversity within the Kent landrace populations could be explained by several

factors: natural mutation over time, introgression from nearby T. repens or T. occidentale populations.

157 4.3.3 Diversity and models of origin, from a combined perspective

In order to evaluate models of origin of white clover it is necessary to consider the picture gained from a combined perspective, of nuclear and chloroplast data.

The main problem with this is that the different genetic markers, not only give different parental perspectives, but also evolve at different rates time (Eidesn et al., 2007; Volkov et al., 2007; Poczai and Hyvonen, 2010). The chloroplast genetic regions are thought to be under stronger constraint so evolve slower. This means that putting the two different perspectives together gives a better analogy of the actual story of origin.

An eastern origin, western origin or both for white clover?

Reasonable sequence diversity was observed in the pure Israeli sample, entirely through variation in ITS sequences and mostly involving combinations that also occurred to the west. Additionally, the high chloroplast diversity in South-West Europe and relative lack of chloroplast diversity in Central and Eastern Europe is consistent with a western region of origin. An eastern origin or influence cannot be ruled out under a “Melting pot” scenario (Petit et al., 2003; Marhold and Lihoviá, 2006; Parisod and Besnard, 2007). Interaction between the progenitor species speciation could have resulted in speciation. T. pallescens is known to occur in sporadic and widely scattered alpine populations in the eastern Mediterranean mountain ranges (Macedonia, Greece) (Professor Warren Williams, pers. com 2010).

In this study the population sampling strongly emphasised the western Mediterranean and western Europe, because this is where T. occidentale now grows. However had T. occidentale previously had a wider distribution in the east, a hypothesis for an eastern origin could be invoked.

Further what of separate eastern and western origins for T. repens? Given the amount of diversity recently observed within T. pallescens populations between glacial valleys in the Austrian Alps (Raffl et al., 2008) potential for island populations and diversity gradients, (Hewitt and Ibrahim 2001; Widmer and Lexer, 2001; Gabriel et al 2007;

Parisod and Besnard, 2007), it is reasonable to assume that different populations of T. pallescens, could have contributed variation through separate speciation events to

158 It is assumed that was this, the case populations from the east and west should show significant genetic differences, due to different founding parental populations.

To test this possibility further it will be necessary to carry out more intensive sampling of white clover populations from the eastern Mediterranean region, as well as

characterisation of T. pallescens populations from that region. Currently none are available in seed banks world-wide and it will initially be necessary to analyse herbarium specimens(Professor Warren Williams, pers. com, 2010).

Discussion of Chloroplast and Nuclear Models in the light of combined data

Chloroplast Models

Model 1: (Fig. 4.2) suggested that each of the main chloroplast lineages was the product of glaciations bringing different T. pallescens populations into contact with T. occidentale populations, sequentially resulting in several speciation events.

Model 2: (Fig. 4.3) suggests that CC+ is the oldest lineage, with the three less frequent pattern types being derived from it (Fig. 4.4).

Nuclear Models

Model 1: Glaciations led to origins (Fig. 4.10), this model suggests that each of the three separate nuclear lineages 5, 15, and 17 resulted when glacial and interglacial movements brought T. pallescens into contact with different T. occidentale populations, bringing about separate events of speciation.

Model 2: an alternative hypothesis for origin (Fig. 4.11). This is the hypothesis that pattern type 15 was the ancestral nuclear sequence and that types 5 and 17 arose from it.

Model 3: The T. occidentale introgression model. The key point of this model is that variation observed in the nuclear data could be the result of more recent introgression

from different T. occidentale populations after the formation of T. repens. The evidence excludes any introgression from T. pallescens, (Fig. 4.12).

159 Commerce

A further modifying influence is commerce, the effect of which is important and influential, but hard to establish. The argument is that commercial varieties have been spread over the top of natural populations, potentially obscuring natural variation in native populations.

Around the time of domestication of clover in southern Spain about the year 1000 AD, clover was widespread throughout Europe occurring in natural populations (Kjaegaard, 2003). It is likely that across Europe clover remained in a natural variable state until cultivation and domestication reached Lombardy and the Netherlands in the 16th century (Kjaegaard, 2003). Influence from commercial clover will likely have resulted in the wide spread of largely uniform varieties adding to the natural genetic variation present.

Northern and North-Western Europe

The influences on clover in Northern and North-Western Europe are not well known beyond, climatic influences and the similarity in French and Northern European white clover populations cannot be blamed wholly on mans influence through farming practice. The presence of the class of novel pattern types (17, 17' and 18) in Northern European populations and the novel pattern combinations extending from these suggest that there has been sufficient time within this area to give rise to a lineage that is

geographically restricted. However, without adequate sampling of the surrounding countries it cannot be determined if this observed distribution is evidence of an old or recent origin for the lineage. Sampling white clover from Germany where it was established in 1645 (Kjaegaard 2003) will help establish if the CC+/15 and others were introduced to this area, or were part of the existing natural variation prior to commerce and where lineage 17 is most frequent and likely to have originated from.

Further, this sampling may show that Germany acts a transition zone between Denmark, the Netherlands and Belgium. By sampling populations along and across the border between France and Germany, an indication of the extent and direction of the spread of white clover in these countries can be gained.

160 Multiple origins for white clover?

The models outlined in the chloroplast and nuclear sections collectively show the complexity of the problem for deciphering the events that caused the level of intra species diversity observed in current populations of white clover.

The data clearly show the greater contribution to diversity observed from the T. occidentale populations relative to the chloroplast contribution. It is clear that the ITS sequences of T. pallescens and T. occidentale are quite different and only T. occidentale like sequences were found in all the individuals sampled in this study. The present evidence suggests that if multiple speciation events have occurred; then the maternal NOR regions must have been lost or suppressed every time. FISH results on T. repens individuals are consistent with this, repeatedly showing no detectable signal from the 18S gene of the NOR on T. pallescens like chromosomes, (Ansari et al., 1999). If multiple origins have occurred, then cross talk between the parental genomes has consistently resulted in loss of the same parental pair of NORs, as is observed in some other allopolyploids (Kato et al., 2005; Wendel et al., 2005; Volkov et al., 2007). .

Other allopolyploids

Alternatively, it would seem that the ground is set for a single origin for T. repens, with current distribution of diversity indicating this origin as within Zone 1 Portugal and Spain. Natural dispersal could then be responsible for the distribution of white clover throughout Zone 2 and, more recently, human influence has spread the species

throughout Zone 3.

Under this hypothesis all of the observed diversity in nuclear pattern types and multiple chloroplast lineages would be due to recent mutation, natural selection and

environmental influences. This hypothesis fits with the chloroplast model 2 in which all the chloroplast lineages are derived from CC+ and nuclear models 1 and 3 that suggest variation post speciation and the possibility of introgression for further changes in allelic frequencies and diversity of DNA sequence.

161