Host-related genetic differentiation in the anther smut fungus Microbotryum violaceum in sympatric, parapatric and allopatric populations of two host species Silene latifolia and S. dioica

We investigated genetic diversity in West European populations of the fungal pathogen Microbotryum violaceum in sympatric, parapatric and allopatric populations of the host species Silene latifolia and S. dioica, using four polymorphic microsatellite loci. In allopatric host populations, the fungus was highly differentiated by host species, exhibiting high values of F(ST) and R(ST), and revealed clear and distinct host races. In sympatric and parapatric populations we found significant population differentiation as well, except for one sympatric population in which the two host species grew truly intermingled. The mean number of alleles per locus for isolates from each of the host species was significantly higher in sympatric/parapatric than in allopatric populations. This suggests that either gene flow between host races in sympatry, or in case of less neutral loci, selection in a more heterogeneous host environment can increase the level of genetic variation in each of the demes. The observed pattern of host-related genetic differentiation among these geographically spread populations suggest a long-term divergence between these host races. In sympatric host populations, both host races presumably come in secondary contact, and host-specific alleles are exchanged depending on the amount of fungal gene flow.     

Intraspecific competition and mating between fungal strains of the anther smut Microbotryum violaceum from the host plants Silene latifolia and S. dioica

Abstract We studied intraspecific competition and assortative mating between strains of the anther smut fungus Microbotryum violaceum from two of its host species, Silene latifolia and S. dioica . Specifically, we investigated whether strains from allopatric host populations have higher competitive ability on their native host species and show positive assortative mating. In general, strains isolated from S. latifolia outcompeted strains isolated from S. dioica on both host species, but in female hosts, heterotypic dikaryons (i.e., dikaryons composed of a haploid strain originating from S. latifolia and a haploid strain originating from S. dioica ) were most successful in competition. Furthermore, the latency period was significantly shorter for heterokaryons that contained at least one strain originating from S. latifolia , compared to heterokaryons that only contained strains originating from S. dioica . The frequencies of conjugations between strains originating from S. latifolia were much higher than conjugation frequencies between strains originating from S. dioica . A significant positive correlation was detected between the relative success of strains in competition and in conjugation, suggesting that success of a strain in competition might be partly determined by its swiftness of mating. In addition, reciprocal differences within heterotypic crosses revealed a significant effect of fungal mating type, with mating type a₁ being the main determinant of mating pace. The observed differences in infection success, conjugation rate, and latency period in favor of strains from S. latifolia relative to strains from S. dioica on both host species are discussed in an evolutionary context of opportunities for the maintenance of differentiation between different formae speciales upon secondary contact.     

Specificity and seasonal prevalence of anther smut disease Microbotryum on sympatric Himalayan Silene species

Host sympatry provides opportunities for cross‐species disease transmission and compounded disease effects on host population and community structure. Using the Silene–Microbotryum interaction (the castrating anther smut disease), eleven Himalayan Silene species were assessed in regions of high host diversity to ascertain levels of pathogen specificity. We also investigated disease prevalence, seasonal dynamics of infection and flowering patterns in five co‐blooming Silene species. We identified several new Microbotryum lineages with varying degrees of specialization that is likely influenced by degrees of host divergence and ecological similarities (i.e. shared pollinator guilds). Affected species had 15%–40% of plants infected by anther smut. Flowering was seasonally overlapping among host species (except for the species pair S. asclepiadea and S. atrocastanea), but diseased flowering onset was earlier than healthy plants, leading to dramatic seasonal shifts in observed disease prevalence. Overlapping distributions and flowering provides opportunities for floral pathogen movement between host species, but host specialization may be constrained by the plant phylogenetic relatedness, adaptation to micro‐habitats and difference in pollinator/vector guilds.     

Seed fitness of hermaphrodites in areas with females and anther smut disease: Silene acaulis and Microbotryum violaceum

Sex-dependent infection rates could change the effective sex ratio of a population. Here, I tested whether females and hermaphrodites of Silene acaulis were equally likely to be infected by Microbotryum violaceum, a fungus that sterilizes the host, and whether sex allocation in hermaphrodites differed between low and high disease plots. Sex ratios of healthy and diseased plants were estimated in five natural plots. Fitness gained through seed production was estimated by measuring seed quantity and quality for each sex morph in eight plots for 2 yr; four plots had 1-5% disease frequency and four plots had 18-25% disease frequency. Sex ratios of healthy and diseased plants did not differ in five plots. The proportion of fitness hermaphrodites gained through ovules varied from 25 to 48%, indicating that this population is near the cosexual end of gynodioecy. Variation in functional gender of hermaphrodites was not explained by sex-dependent infection rates. Spatial heterogeneity in resources and microclimate seems to be important in explaining both disease frequency and variation in seed production by females and hermaphrodites.     

Glacial refugia in pathogens: European genetic structure of anther smut pathogens on Silene latifolia and Silene dioica

Climate warming is predicted to increase the frequency of invasions by pathogens and to cause the large-scale redistribution of native host species, with dramatic consequences on the health of domesticated and wild populations of plants and animals. The study of historic range shifts in response to climate change, such as during interglacial cycles, can help in the prediction of the routes and dynamics of infectious diseases during the impending ecosystem changes. Here we studied the population structure in Europe of two Microbotryum species causing anther smut disease on the plants Silene latifolia and Silene dioica. Clustering analyses revealed the existence of genetically distinct groups for the pathogen on S. latifolia, providing a clear-cut example of European phylogeography reflecting recolonization from southern refugia after glaciation. The pathogen genetic structure was congruent with the genetic structure of its host species S. latifolia, suggesting dependence of the migration pathway of the anther smut fungus on its host. The fungus, however, appeared to have persisted in more numerous and smaller refugia than its host and to have experienced fewer events of large-scale dispersal. The anther smut pathogen on S. dioica also showed a strong phylogeographic structure that might be related to more northern glacial refugia. Differences in host ecology probably played a role in these differences in the pathogen population structure. Very high selfing rates were inferred in both fungal species, explaining the low levels of admixture between the genetic clusters. The systems studied here indicate that migration patterns caused by climate change can be expected to include pathogen invasions that follow the redistribution of their host species at continental scales, but also that the recolonization by pathogens is not simply a mirror of their hosts, even for obligate biotrophs, and that the ecology of hosts and pathogen mating systems likely affects recolonization patterns.     

Host adaptation in the anther smut fungus Ustilago violacea (Microbotryum violaceum): infection success,spore production and alteration of floral traits on two host species and their F1-hybrid

It is often assumed that host specialization is promoted by trade-offs in the performance of parasites on different host species, but experimental evidence for such trade-offs is scant. We studied differences in performance among strains of the anther smut fungus Ustilago violacea from two closely related host plant species, Silene alba and S. dioica, on progeny of (1) the host species from which they originated, (2) the alternative host species, and (3) inter-specific hybrids. Significant intra-specific variation in the pathogen was found for both infection success on a range of host genotypes (virulence) and components of spore production per infected host (aggressiveness) (sensu Burdon 1987). Strains did not have overall higher virulence on conspecifics of their host of origin than on strains from the heterospecific host, but they did have a significantly (c. 3 times) higher spore production per infected male host. This finding suggests that host adaptation may have evolved with respect to aggressiveness rather than virulence. The higher aggressiveness of strains on conspecifics of their host of origin resulted both from higher spore production per infected flower (spores are produced in the anthers), and greater ability to stimulate flower production on infected hosts. The latter indicates the presence of adaptive intraspecific variation in the ability of host manipulation. As transmission of the fungus is mediated by insects that are both pollinators of the host and vectors of the disease, we also assessed the effect of strains on host floral traits. Infection resulted in a reduction of inflorescence height, flower size, and nectar production per flower. Strains did not differ in their effect on nectar production, but infection with strains from S. alba resulted in a stronger reduction of inflorescence height and petal size on both host species. Vectors may therefore in principle discriminate among hosts infected by different strains and affect their efficiency of transmission. Contrary to assumptions of recent hypotheses about the role of host hybrids in the evolution of parasites, hybrids were not generally more susceptible than parental hosts. It is therefore unlikely that the rate of evolution of the pathogen on the parental species is slowed down by selection for specialization on the hybrids.     

Distribution and population structure of the anther smut Microbotryum silenes‐acaulis parasitizing an arctic–alpine plant

Cold‐adapted organisms with current arctic–alpine distributions have persisted during the last glaciation in multiple ice‐free refugia, leaving footprints in their population structure that contrast with temperate plants and animals. However, pathogens that live within hosts having arctic–alpine distributions have been little studied. Here, we therefore investigated the geographical range and population structure of a fungus parasitizing an arctic–alpine plant. A total of 1437 herbarium specimens of the plant Silene acaulis were examined, and the anther smut pathogen Microbotryum silenes‐acaulis was present throughout the host's geographical range. There was significantly greater incidence of anther smut disease in more northern latitudes and where the host locations were less dense, indicating a major influence of environmental factors and/or host demographic structure on the pathogen distribution. Genetic analyses with seven microsatellite markers on recent collections of 195 M. silenes‐acaulis individuals revealed three main genetic clusters, in North America, northern Europe and southern Europe, likely corresponding to differentiation in distinct refugia during the last glaciation. The lower genetic diversity in northern Europe indicates postglacial recolonization northwards from southern refugia. This study combining herbarium surveys and population genetics thus uniquely reveals the effects of climate and environmental factors on a plant pathogen species with an arctic–alpine distribution.     

Colony structure of a slavemaking ant. II. Frequency of slave raids and impact on the host population

The parasite pressure exerted by the slavemaker ant Protomognathus americanus on its host species Leptothorax longispinosus was analyzed demographically and genetically. The origin of slaves found in colonies of the obligate slavemaker was examined with nuclear and mitochondrial DNA markers to make inferences about the frequency and severity of slave raids. Relatedness of enslaved L. longispinosus workers in the same nest was very low, and our data suggest that, on average, each slavemaker nest raids six host colonies per season. Therefore, the influence of slavemaker species on their hosts is much stronger than simple numerical ratios suggest. We also found that slave relatedness was higher in small than in large slavemaker nests; thus, larger nests wield a much stronger influence on the host. We estimated that in the study population, on average, a host nest has a 50% chance of being attacked by a slavemaker colony per year. Free-living Leptothorax colonies in the vicinity of slavemaker nests did not represent the source of slaves working in P. americanus colonies, which suggests that raided nests either do not survive or migrate after being raided. Colony composition and intranest relatedness of free-living L. longispinosus colonies differed markedly between areas with slavemakers and those that are parasite-free. In the presence of slavemakers, host colonies were less likely to be polygynous and had fewer workers and a higher relatedness among worker brood. Host nests with slavemaker neighbors allocated more resources into sexuals, possibly caused by these shifts in nest demography. Finally, enslaved Leptothorax workers in P. americanus nests appeared to be less efficient than their counterparts in free-living colonies. Thus, slavemakers exert a much stronger impact on their hosts than had previously been suspected and represent an unique system to study parasite-host coevolution.     

Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States

We used herbarium specimens of Silene virginica, S. caroliniana, S. rotundifolia, and S. latifolia to survey the incidence of anther-smut disease (caused by Microbotryum violaceum sensu lato) in the eastern USA. We found no evidence of a collector bias against diseased specimens. Diseased specimens were frequently found in collections of S. virginica and S. caroliniana, but not in those of S. rotundifolia or S. latifolia. Disease incidence in S. virginica and S. caroliniana increased significantly over the past century and was higher in marginal populations. The absence of disease in specimens of S. rotundifolia is consistent with field observations, but its presence in natural populations of S. latifolia (especially in Virginia) suggests that the disease is recently introduced. Changes in the host distributions were also evident. The relative abundance of S. caroliniana declined over time (especially further north), while the relative abundance of S. virginica increased. Silene latifolia was absent or rare south of Pennsylvania before ca. 1920, indicating that S. latifolia and its anther smut are likely to be recent introductions in Virginia. Methods are also presented that quantify the completeness of coverage provided by herbarium specimens.     

Substitution rates in the X- and Y-linked genes of the plants,Silene latifolia and S. dioica

Theory predicts that selection should be less effective in the nonrecombining genes of Y-chromosomes, relative to the situation for genes on the other chromosomes, and this should lead to the accumulation of deleterious nonsynonymous substitutions. In addition, synonymous substitution rates may differ between X- and Y-linked genes because of the male-driven evolution effect and also because of actual differences in per-replication mutation rates between the sex chromosomes. Here, we report the first study of synonymous and nonsynonymous substitution rates on plant sex chromosomes. We sequenced two pairs of sex-linked genes, SlX1-SlY1 and SlX4-SlY4, from dioecious Silene latifolia and S. dioica, and their non-sex-linked homologues from nondioecious S. vulgaris and Lychnis flos-jovis, respectively. The rate of nonsynonymous substitutions in the SlY4 gene is significantly higher than that in the SlX4 gene. Silent substitution rates are also significantly higher in both Y-linked genes, compared with their X-linked homologues. The higher nonsynonymous substitution rate in the SlY4 gene is therefore likely to be caused by a mutation rate difference between the sex chromosomes. The difference in silent substitution rates between the SlX4 and SlY4 genes is too great to be explained solely by a higher per-generation mutation rate in males than females. It is thus probably caused by a difference in per-replication mutation rates between the sex chromosomes. This suggests that the local mutation rate can change in a relatively short evolutionary time.     

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.     

麦瓶草种群中的花药黑粉菌感染：花的形态学变异、菌孢子散布式样及病原菌—传媒调节性选择

花药黑粉菌（Microbotryum violaceum)可系统侵染其寄主植物麦瓶草（Silene dioica),使其不育而代之以在植物花中布满菌孢子,这些菌孢子主要由花传媒昆虫带到健康植株,花传媒昆虫对花部特征变异的行为反应不仅可能影响花粉的输入/输出率,而且影响菌孢子的着落率和植株的发病与否,为研究S.dioica雌株花部性状特征与花粉传布,菌孢子着落及由此而导致的植株染病之间的相关性,用采自4个不同S.dioica种群的植物进行了移栽实验,讠4个种群均位于瑞典北部的Skeppsvik群岛,代表了从健康（岛1,种群1）,低度发病（岛2,种群2）到高度发病（岛3、4,种群3、4）的发病梯度,从上述4个不同种群中各采50个植株移栽至岛4的中部,来自健康种群的植株较来自高发病种群的植株具有较大的花,较长的花柱,研究发现,着落在花上的花粉粒数和菌孢子数呈强正相关,来自健康种群的植株每朵花上着落的花粉粒和菌孢子数分别是高发病种群（种群4）植株的4倍和9倍,导致来自4个不同种群的植株的发病率存在着显差异。种群1、2、3、4的发病率分别为37%,20%,18%,0。在涉及10个种群的田间调查研究中,发现种群中健康植株的平均花柱长度（与花冠大小,胚珠数目正相关）与植株的发病率显负相关,讨论了病原体-传媒调节的植物花部性状特征选择潜势及其导致的麦瓶草S.dioica种群音质 基因漂移。     

麦瓶草种群中的花药黑粉菌感染:花的形态学变异、菌孢子散布式样及病原菌-传媒调节性选择

花药黑粉菌(Microbotryum violaceum)可系统侵染其寄主植物麦瓶草(Silene dioica),使其不育而代之以在植物花中布满菌孢子.这些菌孢子主要由花传媒昆虫带到健康植株.花传媒昆虫对花部特征变异的行为反应不仅可能影响花粉的输入/输出率,而且影响菌孢子的着落率和植株的发病与否.为研究S.dioica雌株花部性状特征与花粉传布、菌孢子着落及由此而导致的植株染病之间的相关性,用采自4个不同S.dioica种群的植物进行了移栽实验.该4个种群均位于瑞典北部的Skeppsvik群岛,代表了从健康(岛1,种群1),低度发病(岛2,种群2)到高度发病(岛3、4,种群3、4)的发病梯度.从上述4个不同种群中各采50个植株移栽至岛4的中部.来自健康种群的植株较来自高发病种群的植株具有较大的花,较长的花柱.研究发现,着落在花上的花粉粒数和菌孢子数呈强正相关.来自健康种群的植株每朵花上着落的花粉粒和菌孢子数分别是高发病种群(种群4)植株的4倍和9倍,导致来自4个不同种群的植株的发病率存在着显著差异,种群1、2、3、4的发病率分别为37%、20%、18%、0.在涉及10个种群的田间调查研究中,发现种群中健康植株的平均花柱长度(与花冠大小,胚珠数目正相关)与植株的发病率显著负相关.讨论了病原体-传媒调节的植物花部性状特征选择潜势及由其导致的麦瓶草S. dioica种群间的基因漂移.     

Importance of Silene latifolia ssp. alba and S. dioica (Caryophyllaceae) as host plants of the parasitic pollinator Hadena bicruris (Lepidoptera, Noctuidae)

With regards to pollination there exist several mutualistic relationships between Hadena -species and Caryophyllaceae. As mutualists have both negative and positive effects on their partners, mutualism is often betoken as reciprocative exploitation which may shift to parasitism if the exploitation of one partner becomes prevalent.
Several Silene - and Saponaria -species are considered to be larval host plants of Hadena bicruris . Although Silene latifolia ssp. alba and S. dioica are frequently cited as hosts of the seed eating larvae, field and laboratory observations at Ulm were suggestive for only S. latifolia ssp. alba being a suitable host. Records of the oviposition behavior of H. bicruris made it evident that in fact a considerable number of eggs could be found in planted stands of both species. On the other hand, phenological data of the flowering periods and of the oviposition behavior of H. bicruris showed that S. latifolia ssp. alba is clearly preferred for oviposition if host selection is possible due to contemporaneous flowering of individuals of both plant species growing at close range. In addition, the flowering periods of S. latifolia ssp. alba and the periods of moth activity overlap to a large extent. This is not the case in S. dioica . Feeding experiences first indicated that the caterpillars may not prefer one of the species to the other, but comparison of the pupal weight of the animals reared on fruits of exclusively one of the species showed that the seeds of S. latifolia ssp. alba were more profitable for nutrition than those of S. dioica ; the pupal weight of animals reared on seeds of the former species significantly exceeded that of animals reared on seeds of the latter one. The question arises if the symbiosis of H. bicruris and its hosts constitutes a stable situation or if an evolutionary shift to mutualism or parasitism will take place.     

Colony and population genetic structure of the Formosan subterranean termite,Coptotermes formosanus,in Japan

Subterranean termites have unusual plasticity in their breeding systems. As a result of their cryptic foraging and nesting habits, detailed information on the numbers and types of reproductive individuals in colonies has been difficult to obtain. In this study, we used microsatellite markers to infer the major features of the breeding system of the Formosan subterranean termite, Coptotermes formosanus, in southern Japan, where it is believed to have been introduced from China. A total of 30 colonies was sampled from two islands (Kyushu and Fukue) located 100 km apart. Twenty workers from each colony were genotyped at six microsatellite loci. Analysis of worker genotypes within colonies indicated that 27 colonies (90%) were simple (Mendelian) families. The remaining three colonies, all from Kyushu, were consistent with being extended families having begun as simple families but being currently headed by multiple neotenic (secondary) reproductives descended from the original king and queen. Workers from simple families in both populations were significantly inbred (FIT = 0.10 for Kyushu and 0.46 for Fukue) and highly related to their nestmates (coefficient of relatedness, r = 0.59 for Kyushu and 0.77 for Fukue), suggesting that many simple-family colonies were headed by closely related reproductives, especially in the Fukue population. This conclusion is supported by the high coefficient of relatedness between nestmate reproductives in simple-family colonies (r = 0.23 for Kyushu and 0.61 for Fukue) based on genotypes inferred from their worker offspring. There was moderate genetic differentiation (FST = 0.12) between the two populations, suggesting rather restricted gene flow between them. There was no significant isolation by distance among colonies, as might be expected given the limited dispersal of reproductives, presumably because of the frequent movement of colonies by humans. Finally, there was no evidence of a recent bottleneck, a finding possibly consistent with the more than 300-year history of this species in Japan.     

X linkage of AP3A, a homolog of the Y-linked MADS-box gene AP3Y in Silene latifolia and S. dioica

Background

The duplication of autosomal genes onto the Y chromosome may be an important element in the evolution of sexual dimorphism.A previous cytological study reported on a putative example of such a duplication event in a dioecious tribe of Silene (Caryophyllaceae): it was inferred that the Y-linked MADS-box gene AP3Y originated from a duplication of the reportedly autosomal orthologAP3A. However, a recent study, also using cytological methods, indicated that AP3A is X-linked in Silenelatifolia.

Methodology/Principal Findings

In this study, we hybridized S. latifolia and S. dioicato investigate whether the pattern of X linkage is consistent among distinct populations, occurs in both species, and is robust to genetic methods. We found inheritance patterns indicative of X linkage of AP3A in widely distributed populations of both species.

Conclusions/Significance

X linkage ofAP3A and Y linkage of AP3Yin both species indicates that the genes'' ancestral progenitor resided on the autosomes that gave rise to the sex chromosomesand that neither gene has moved between chromosomes since species divergence.Consequently, our results do not support the contention that inter-chromosomal gene transfer occurred in the evolution of SlAP3Y from SlAP3A.     

Contrasting demographic history and population structure in Capsella rubella and Capsella grandiflora, two closely related species with different mating systems

Both mating system and population history can have large impacts on genetic diversity and population structure. Here, we use multilocus sequence data to investigate how these factors impact two closely related Brassicaceae species: the selfing Capsella rubella and the outcrossing C. grandiflora. To do this, we have sequenced 16 loci in approximately 70 individuals from 7 populations of each species. Patterns of population structure differ strongly between the two species. In C. grandiflora, we observe an isolation-by-distance pattern and identify three clearly delineated genetic groups. In C. rubella, where we estimate the selfing rate to be 0.90-0.94, the pattern is less clear with some sampling populations forming separate genetic clusters while others are highly mixed. The two species also have divergent histories. Our analysis gives support for a bottleneck approximately 73 kya (20-139 kya) in C. rubella, which most likely represents speciation from C. grandiflora. In C. grandiflora, there is moderate support for the standard neutral model in 2 of 3 genetic clusters, while the third cluster and the total data set show evidence of expansion. It is clear that mating system has an impact on these two species, for example affecting the level of genetic variation and the genetic structure. However, our results also clearly show that a combination of past and present processes, some of which are not affected by mating system, is needed to explain the differences between C. rubella and C. grandiflora.     

Geographically congruent large‐scale patterns of plastid haplotype variation in the European herbs Silene dioica and S. latifolia (Caryophyllaceae)

The closely related dioecious herbs Silene latifolia and Silene dioica are widespread and predominantly sympatric in Europe. The species are interfertile, but morphologically and ecologically distinct. A study of large‐scale patterns of plastid DNA (polymerase chain reaction–restriction fragment length polymorphism) haplotypes in a sample of 198 populations from most of the European ranges of both species revealed extensive interspecific haplotype sharing. Four of the 28 detected haplotypes were frequent (found in > 40 populations) and widespread. Three of these frequent haplotypes occurred in both species and the geographic distribution of each haplotype was broadly congruent in both species. Each of these three, shared and widespread haplotypes is likely to have colonized central and/or northern Europe after the last glaciation from one or more of refugial areas in southern Europe. Interspecific hybridization and plastid introgression within refugial regions and/or during the early stages of postglacial expansion is the most plausible explanation for the broadly similar distribution patterns of the shared, frequent chloroplast haplotypes in the two species. The fourth frequent, widespread haplotype was absent from S. latifolia and almost entirely restricted to Nordic S. dioica. It is most likely that this haplotype spread into the Nordic countries from a central or northern European source or from a refugial area in Russia. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 153–170.