A number of Populus species, especially in the aspen section (sect. Populus, e.g. P. alba, P. tremula, P. tremuloides), produce sucker shoots from their root systems. Suckering allows these species to ‘invade’ adjacent territory more efficiently than seedlings in some situations. This phenomenon is often involved in large-scale landscape-level succession, e.g. from grassland to forest. Management practices like coppicing — cutting all trees in a stand at a relatively young age to enable re-sprouting from rootstocks — also enables these species to ‘take over’ whole stands. Within the species, clones that out-compete others may then dominate a single stand.
The extent of clones in Populus trees is often obvious from observing budburst and fall colour in monotypic stands. Sizable patches of natural forest often consist of same-sex trees, and a particular resemblance among them in stem form and stem colour is often observed (Fig. 1). However, such observations have limits in precision, e.g. when trying to compare old and young members of the presumed clone. Both Mock et al. (2008) and Ally (2008) show that these may not be useful indicators, as multiple genets often share a patch.
Clonality is an important process at the ecosystem level, e.g. certain clones may be preferred by herbivores (Whitham et al. 2003). Implications of clonality can get complicated depending on the landscape scale (Mock et al. 2008), as comparisons among different regions have to take sampling density into account. In some environments, suckers establish easier than seedlings which depend on raw soils — clonality allows genotypes to overcome unfavourable periods until seedling recruitment is possible again (‘windows of opportunity’, Mock et al. 2008). However, because of the lack of recombination during asexual reproduction, genetic load cannot be efficiently purged and deleterious mutations accumulate. For instance, the most widely dispersed clones identified by Mock et al. (2008) had high frequencies of aneuploidy, which may reduce the ability of these clones to reproduce sexually.
Geneticists have tried to analyse the extent of clones ever since isozymes became available (Muhs 1977), and in general, a link was made between clone size and age. Two studies in this issue of molecular ecology address clone age more directly, using either mutation rate and cell-growth estimates (Ally et al. 2008) or the proportion of ramets to genets (i.e. the variation due to somatic mutation vs. recombination) at the landscape scale (Mock et al. 2008). For estimating mutation rates in microsatellites, Ally et al. (2008) had to rely on indirect estimates which could be improved in the future by direct estimates from clones of known age (e.g. in provenance trials). Nevertheless, they clearly show evidence that the size of a clone is not at all indicative of its age.
One of the values of knowing the age of clones is finding out the disturbance and reproduction history of clonal stands, i.e. having an assessment of the timing and severity of the last disturbance. For instance, forest management by coppicing often leads to re-sprouting, whereas severe disturbances like fires, flooding and landslides more likely put seedlings at an advantage. Clone sizes and ages together, thus, may provide insights into the ability to survive changing environments in time and space (Mock et al. 2008). Previous studies in clonal organisms have addressed demographic events in the past from different points of view. Reusch et al. (1998) used microsatellite data and annual rhizome growth rates to estimate size and age of clones in sea grass. In their case, many small clones were found as a result of frequent disturbances, causing clone fragmentation and subsequent seedling establishment. In forestry, one widespread assumption is that some clones would out-compete all the others after repeated cycles of coppicing. In such a Quercus pyrenaica oak forests in Spain, however, high diversity was found — many clones survived (Valbuena-Carabaña et al. 2008).
What are the challenges in using microsatellites for clone identification and age determination? First, mutation rates differ between loci and are rarely known. There are no estimates for taxon-specific mutation rates in Populus. Second, even if a taxon-specific microsatellite mutation rate were known, it would be of little use for estimating clone age, as mutation rates are expected to be much higher in meiosis than in mitosis. The innovative approaches by Ally et al. (2008) are to combine estimates of cell division and growth rates with average physical distances within clones, and to set upper and lower time boundaries based on time since glaciation and ages of the oldest trees in the stands. Their estimates for somatic mutation rates (per cell division, or per year) are, as expected, a few orders of magnitude lower than (per generation) estimates for most sexually reproducing species. Third, it is difficult to distinguish somatic mutations from allelic variation. Both studies consider a somatic mutation one where it affects only a few alleles. Ally et al. (2008) consider an allele a somatic mutation when an individual ramet in a clone differs by one allele at one locus but shares all other alleles with the most frequent genotype. Mock et al. (2008) draw frequency graphs and argue that they see a gap between what are somatic mutations (affecting only few alleles) and genetic differences between distinct clones. Finally, possible technical errors, especially in polymerase chain reaction due to slippage of the enzyme, complicate the matter (Clarke et al. 2001).
One of the limitations of the Ally et al. (2008) method might be that they do not have enough power in the data with only 29 somatic mutations. For the model tree Populus, it should be possible in the near future to increase the number of loci and the number of ramets per clone. This might be more difficult for other less well-studied species. In the study of Mock et al. (2008), the sampling scheme might have been the limiting factor for delineation of clonal boundaries and estimating age, but dedicated studies can build upon the experience in both studies.
What other methods would be available to calibrate age estimate analysis? Tree ring counts and radiocarbon (C14) dating can help in finding the oldest part, or the oldest living tree, in a cohort. Recently, the latter approach was employed to find what is possibly a surviving spruce (Picea abies) clone from early after the last ice age (Kullman 2008), by dating fossil wood underneath a bushy, living clone in Northern Sweden. However, it is difficult to imagine this approach being widely applied, as the availability of fossil wood (attached to a living clone) is an exception rather than the rule. And tree rings will likewise not extend clone history into the very far past very often, although they might assist in estimating species-specific mutation rates.
Both studies break the ground in providing new methods for clone size and age estimation. Clearly, the next step is to merge these approaches and evaluate them in further studies. For instance, the age of the large ‘Pando‘ clone of Mock et al. (2008) could be estimated with the method of Ally et al. (2008). These approaches could be very useful in cultivated species with a long history of vegetative propagation. For instance, in grapevine cultivars, some of which may have been in continuous use for several hundred years already, somatic mutations in microsatellites were found in some distinct clones within cultivars (Lefort & Roubelakis-Angelakis 2001; Vouillamoz et al. 2003). Another example is the ‘English elm’ clone, Ulmus procera (syn. U. minor var. vulgaris), essentially dating back to the time of the Romans. Gil et al. (2004) did not find any microsatellite mutations in representatives of this clone from places as far apart as Britain and Spain (although there were very few amplified fragment length polymorphism differences). In the light of the mutation rates estimated by Ally et al. (2008), dozens or even hundreds of microsatellite loci would be required to find even a few mutations, if mutation rates are similar in Ulmus and Populus. A few thousand microsatellite loci are already available for Populus trichocarpa (Tuskan et al. 2004). As Gil et al. (2004) could not analyse so many loci, their results make good sense. The discovery of somatic mutations in grapevine, on the other hand, implies that either mutation rates are much higher in Vitis, or that these cultivars are quite old. High throughput molecular genetic tools and the calculation methods available now promise us exciting studies of clonality in the near future.