TCS analysis of combined pattern types, with alignment order, ITS-CD IRB21NE.

This analysis based on 200 individuals of both commercial and non commercial accessions from throughout the sampled areas was performed under the setting gaps as missing data. The data set contained the two previously stated points of variation in IRB21NE noted in section 3.4.1, a C/T presence in place of the presence or absence of the indentified insert in IRB21NE, and an A/T presence at the end of IRB21NE

sequence (464 in the Huia reference).

The presentation of the data by the TCS software shows that the relationships within the data based on DNA patterns can be broken down into 13 groupings of two or more individuals. Clear separation of chloroplast and ITS pattern combinations is observable. Of these the group 1H is the largest and contains a collection of individuals with

commonality in ITS type 15, and variants on this pattern type, caused by the presence of ambiguous positions and rare variants. The disproportionally large size of this group is attributable to the large number of commercial varieties present in the group and

114 indicates the effect of commerce on the spread of type 15, (Appendix F). Individuals in this group contain both chloroplast types CC+ and CT+ although prominence of CC+ is observed suggesting a high contribution of this lineage to commercial varieties,

(Appendix G). Two groups (40 and 81), show a connection of ITS type 5 with CC+ and related variant ITS types in numerous individuals.

A significant separation of Portuguese and Spanish individuals was observed as

separation of group 24, and the number of individual green circles. Two further groups 65 and 51, identified connections between Portugal, Spain and the landrace Kent. Further Portuguese and Spanish individuals were observed as part of the smaller groups 40 and 140 suggestive of the diversity observable in these countries, and their potential as source populations for wide spread diversity. This was further supported by their presence in group 81 which contained a mix of Kent, Spanish, Portuguese, Barbian, Morso, Regal, Lodi, Dusi, Crusader and Algerian individuals.

A small grouping of individuals from both assessed Algerian accessions showed a placement separate from the majority of the data set, through multiple changes,

indicating a potentially separate origin or a different evolutionary trajectory for Algerian germplasm. The closeness of this Algerian group to Portuguese individuals also

supports the prominence of one dominant ITS sequence type, type 5, in both countries (Figs: 3.13, 3.14, 3.29, 3.30. 4.4 and Tables: 3.14, 3.23).The similarity in prevalence of type 15 as observed in the combined and ITS results (Figs: 3.15, 3.16, 3.29, 3.30, 4.4 and Tables: 3.15, 3.23, 3.29, 3.36) for Morocco and Spain for was not obvious in this analysis.

In this analysis a large number of individuals were observed with DNA pattern types separate from the main groupings. The minor groupings 167, 300, 342, and 309 show chloroplast pattern types with CC+ or CT+ in connection with rarer ITS patterns types. These ambiguities could not be resolved through resequencing and are treated by TCS as missing data hence; these groupings may not be correct.

However, what was most noticeable about the individuals in this analysis was the spatial arrangement of; Spanish individuals with their close proximity to Portuguese individuals that were more closely placed with European individuals. Portuguese

115 individuals are wider spread throughout the diagram, and showed close placement to individuals and groupings of European and/or Algerian individuals. Derived positions for individuals from France, (207) C7690, Bouches du Rhône, Entressen, in the Cote d'Azur South East France, Crau (4A) South France, and Crusader (444), support a suggestive role of France in the region of origin of white clover, (Figs: 3.17, 3.18, 4.44 and Table 3.1.

116

Figure 3.37 Combined pattern types TCS analysis with C/T and A/T, with the alignment ordered ITS-trn L- IRB21NE and gaps treated as missing data. Groupings: raspberry pink- Huia Reference, Spain, Portugal, France, Crau, Aran, Crusader, Italy, Tammisto, Tamar, Barbian, Morso, Kent, Regal, Blanca, Hafia, S. Africa- Dusi, and Morocco, dark green- Portugal and Spain, turquoise- Portugal, Spain and Kent, grey/blue- Portugal, Tammisto, Barbian, Morso and Blanca, purple- Lodi and Regal, maroon- Portugal and Italy, navy blue- Morocco and Blanca, orange-red- Portugal, Spain, Kent, Barbian, Morso, Regal, Italy, Algeria, and Dusi, brown- Algeria C6452 and C1751. Individuals: lime green- Spain- (11A (N), 72 (NW), 161 (NW), 189 (N), 206 (W)), medium green- Portugal- (47 (SW), 89 (NW), 134 (C), 153/157 (SW), 188 (SW), 4-4 (NE), 8-1 (NW), 9-1, 9-2, 9-4, 9-5 (NE)), medium blue- Tammisto, pale blue- Kent, raspberry pink- Crau (France), medium pink- Crusader (France/Syria), dark purple- Lodi (Italy), dark grey- Tamar (Israel), brown- (Algeria).Note: the numbers in the brackets refer to the number of individuals sampled.

117

Figure 3.38 Combined pattern types TCS analysis with C/T, but with A/T treated as non-existent, with the alignment ordered ITS-trn L- IRB21NE and gaps treated as missing data. Groupings: raspberry pink- Huia Reference, Spain, Portugal, France, Crau, Aran, Crusader, Italy, Tammisto, Tamar, Barbian, Morso, Kent, Regal, Blanca, Hafia, S. Africa- Dusi, and Morocco, dark green- Portugal/ Spain, turquoise- Portugal, Spain and Kent, grey/blue- Portugal, Tammisto, Barbian, Morso and Blanca, purple- Lodi and Regal, maroon- Portugal and Italy, navy blue- Morocco and Blanca, orange-red- Portugal, Spain, Kent, Barbian, Morso, Regal, Italy, Algeria, and Dusi, brown- Algeria 6452 and 1751. Individuals: lime green- Spain- (11A (N), 72 (NW), 161 (NW), 189 (N), 206 (W)), medium green- Portugal- (89 (NW), 96 (NW), 134 (C), 153/157 (SW), 188 (SW), 203 (S), 4-4 (NE), 8-1 (NW), 9- 1, 9-2, 9-4, 9-5, 9-6 (NE)), medium blue- Tammisto, pale blue- Kent, raspberry pink- Crau (France), medium pink- Crusader (France/Syria), dark purple- Lodi (Italy), dark grey- Tamar (Israel), brown- (Algeria). Note: the numbers in the brackets refer to the number of individuals sampled.

118

Figure 3.38 analysis of combined pattern types with absence of the T insert in