SnoRNA gene/gene cluster variation between and within

Seneciospecies

Analyses of most datasets for each primer pair separated distantly related species and sometimes more closely related species from each other, according to snoRNA fragment variation exhibited. However, most single pair primer datasets contained relatively few fragments which can often cause the generation of unstable and star like trees based on individual variation (Hollingsworth & Ennos, 2004). The topology of such trees might change markedly by variation in a single fragment and, thus, errors could greatly affect interpretation. Therefore, single primer pair datasets were combined for further analysis to improve the resolution of species relationships based on variation across several different genomic regions. Thus, the following discussion is based on the results from the analysis of combined datasets.

The initial primer-trial analysis, which employed radioactively labelled primer pairs, showed that samples of S. vulgaris did not form a distinct clade, but instead were grouped with samples ofS. aethnensis,S. chrysanthemifolius,S. squalidus,S. cambrensis

and S. teneriffae. Although some substructure was obtained within this group of taxa,

species were often intermixed. The subsequent more detailed analysis of snoRNA variation, employing fluorescence labelled primers, focussed particularly on relationships between species of this group and surveyed a much higher number of samples of S.

aethnensis, S. chrysanthemifolius, S. squalidus, S. vulgarisandS. cambrensis,and also of

S. madagascariensis. The application of fluorescence labelled primers increased the

sensitivity of fragment analysis by yielding a higher number of fragments which most likely accounted for the better resolution obtained. Analysis of the data from this analysis of combined data sets tended to separate species into three distinct groups - S.

madagascariensis, S. vulgaris/S. cambrensis, and S. aethnensis/S. chrysanthemifolius/S.

squalidus. The difference between the latter two groups of taxa was reflected by high

bootstrap support of the S. vulgaris/S. cambrensis group and high ΦST values between

them. Within these two groups, species were separated from each other, albeit with some degree of overlap. In particular, some S. vulgaris samples were intermingled with S.

cambrensisas was also found to be the case in a previous study of AFLP variation within

Chapter 4 Discussion indicated thatS. chrysanthemifolius was genetically more similar toS. squalidus than to

S. aethnensis.

In contrast to the situation in the other species examined, a high amount of variation (40%) was shown to be present within S. cambrensis, which reflects the fact that two different independently originated lineages (Welsh and the Edinburgh lineages) of this species (Abbott, 1992; Harris & Ingram, 1992a) were included in the survey.

In the NJ trees of the primer-trial analysis (Figure 4.5) S. mohavensis and S.

glaucuswere found close to S. flavuswhich appears not surprising as the former species

originated by hybridisation of the latter two species (Liston & Kadereit, 1995; Comes & Abbott, 2001; Colemanet al., 2003; Kadereit et al., 2006). Interestingly,S. flavus andS.

glaucus are very distantly related. In various ITS phylogenies S. mohavensis and S.

glaucus were placed within a poorly resolved clade containing also S. squalidus, S.

chrysanthemifolius, S. aethnensis and other species (i.e. S. squalidus clade (ITS

phylogeny in Chapter 1); also called Mediterranean complex (Comes & Abbott, 2001), Groundsel clade III (Coleman et al., 2003) and clade A (Pelser et al., 2007)), while S.

flavus was placed together with S. engleranus in a most distant position relative to this

clade. These two species, S. flavus and S. engleranus, may not even be part of Senecio

sensu stricto, but most closely related to the genus (Pelser et al., 2007; Milton, 2009).

Interestingly, RAPD analysis of 10 selected species of the Mediterranean complex, including S. mohavensis, S. glaucus and S. flavus, placed these three species within the same clade (clade A) and, thus, suggests a much closer relationship between these species (Comes & Abbott, 2001). Although a different set of species were used in the study presented here, the results were similar to the RAPD analysis and, therefore, support the findings of Comes & Abbott (2001).

S. glaucus was also found close to the group of species containing S. aethnensis,

S. chrysanthemifoliusandS. squalidus in the NJ trees of the primer-trial analysis. The S.

glaucus samples placed next to S. flavus were collected in Israel, whereas the one

phylogenetically close to S. squalidus group was sampled in Morocco. Samples from these locations differed considerably in their ITS sequences, showed high variation in their cpDNA haplotype (Comes & Abbott, 2001) and intraspecific geographical structure was shown by alloenzyme data (Comes & Abbott, 1999). Therefore, the high variation

Chapter 4 Discussion within S. glaucus found in the study presented here is in accordance with previous examinations and might be explained by strong geographical barriers and selection between different populations (Comes & Abbott, 1999).

Although samples of S. flavus were also collected from very distant sites, low within species variation was obtained. This was also evident in their ITS sequences and might be explained by relatively recently colonisation due to Pleistocene migration and/or long distance dispersal mediated by birds (Coleman et al., 2003). Less intraspecific variation was obtained forS. mohavensis, but it was possible to distinguish between the two disjunct subspecies mohavensis and breviflorus, respectively. The lack of ITS sequence variation is thought to be a result of the recent origin and disjunction (mediated by long distance dispersal from southwest Asia to North America) ofS. mohavensis ssp.

mohavensis (Coleman et al., 2003). Both, S. flavus and S. mohavensis are self-fertile

which is a great advantage in long distance dispersal due to the possibility of single colony establishment, decreased inbreeding depression and low pollinator dependence. However, self-fertilisation can result in a reduced amount of genetic variation and might, therefore, contribute to the low within species variation observed.

Both morphological and isoenzyme data suggested a considerable amount of variation within the S. madagascariensis complex (Radford et al., 2000) and ITS data showed some degree of intraspecific sequence differentiation (Le Roux et al., 2006). Thus, it is not surprising that relatively high intraspecific variation was obtained for S.

madagascariensis in this study in both the initial primer trial and the more detailed

analysis.