It is now apparent that phylogeographic inferences based on a single, non-recombining marker can be misleading [48, 49]. Consequently, phylogeographic studies are increasingly using multiple genetic markers and/or palaeodistribution modelling to draw more reliable inferences on population history. The results of the paleodistribution modelling and the patterns of genetic variation revealed by the phylogeographic analyses suggest that both Orthilia secunda and Monotropa hypopitys persisted throughout the LGM in Europe in southern refugia. Although both species generally exhibited a "southern richness vs. northern purity" distribution of genetic variation , this was more pronounced in the temperate M. hypopitys, where the only populations that displayed any within-population genetic variation for both the chloroplast rps 2 and nuclear ITS regions were located closest to the modelled refugial areas. Northern populations of O. secunda were more diverse, but the signatures of refugial areas i.e. high diversity coupled with unique haplotypes  were restricted to southern populations.
Based on the weight of evidence across modelling and the different markers used, our findings indicate a possible refugial area for O. secunda in Europe located in the vicinity of the French Alps. A second area of high diversity and endemic haplotypes included the Austrian Alps and Slovakia, but these populations lie outside the suitable climate envelope indicated by the palaeodistribution model. Nevertheless, although the precise locations of putative refugia are difficult to identify accurately, it is clear that the majority of genetic diversity is contained in southern populations. The occurrence of a fixed endemic ITS haplotype in one of the Estonian populations (EENN) more likely represents a relatively recent mutation that has become fixed through genetic drift, rather than indicating an extreme northern refugium. For M. hypopitys, the modelling and genetic data both indicated a likely refugial area in southeastern Europe. The identification of two genetic clusters with a broadly northern/eastern vs. southern/western geographical distribution for both species based on microsatellite data could indicate isolation in separate refugia followed by differential recolonization after the retreat of the ice .
Many studies have used modelling approaches to determine the effects of present and future climate change on the distribution ranges of plant species (e.g. [50–52]). We can extend this approach to investigate the potential effects of such distribution changes on intraspecific genetic diversity. The future modelled distributions of both O. secunda and M. hypopitys indicate substantial changes in the ranges of both species. For M. hypopitys in particular, these changes could have a profound impact on the genetic diversity of the species in Europe. Previous studies have suggested that range contraction during previous phases of climate change was characterized by population extinction, rather than migration [6, 53]. Although the future model indicates a range expansion at the northern edge, it also suggests extensive loss of suitable habitat in southeastern Europe. Given that this area represents the centre of genetic diversity for the species, extinction of these populations would lead to massive loss of genetic diversity since more northerly populations are genetically depauperate relative to populations in the southeast. A northern expansion of the species' range would not counter this, because the leading edge colonization would be from these low-diversity northern populations. Northern populations of O. secunda, however, tended to be more genetically diverse than those of M. hypopitys. Consequently, the loss of southern and central European O. secunda populations indicated by the species distribution model would not have the same overall effect on total intraspecific genetic diversity across the continent. Nevertheless, although the populations from the species' centres of diversity in the French and Austrian Alps would still lie within the future modelled climate envelope, this would most likely be as a result of altitudinal migration, since the mountain ranges of southern and eastern Europe represent the only climatically suitable areas in the region. Whilst altitudinal migration offers some short-term potential for countering the effects of climate change [54–57], its scope is ultimately limited . The situation in Europe is somewhat different from that in North America, where the occurrence of northern refugia for both species means that a lower proportion of the total genetic diversity in the continent is concentrated in southern populations [, Beatty & Provan, unpublished results] and thus the impact of loss of rear-edge populations might not be as extreme. It should also be borne in mind that models of future (and, indeed, past) climate are not guaranteed to be 100% accurate, and that many other factors such as changes in species tolerances through adaptation and species-species interactions will also determine species current and future ranges. Nevertheless, at least in Europe, the adverse encroachment of human activity on the boreal and temperate woodlands that form the natural habitat for these species, coupled with the fact that climate is changing faster now than at any time in the past, means that the impacts on the gene pools and subsequent adaptive potential of these, and possibly many other species, are likely to be potentially serious.