BACKGROUND
comparative studies of the genetic structure of populations, particularly those utilising molecular makers, have provided new opportunities for better understanding historical and/or contemporary factors for modelling speciation.
surveys using dna variation in rdna and other sequence data have revealed several examples of morphological fungus species that have diverged into reproductively and/or genetically isolated morphologically indistinguishable monophyletic subgroups or cryptic species see, e.g.
<cit> .
in particular, patterns of geographic distribution have been noted as one of the main factors shaping species diversification.
for instance, geographically isolated populations  appear to be one of the most plausible causes of speciation.
in addition, for some species complexes, sympatric speciation likely has been caused by changes in ecological  <cit>  and/or life history traits and reinforced by negative selection towards hybrids.
such causes seem to be less frequent in allopatric speciation, where the major barrier to gene flow is geographic.
gene genealogies from independent dna loci have been recommended as a means of identifying phylogenetic species  <cit> .
molecular markers are very helpful for this purpose, especially within fungus groups with comparatively simple body plans that seriously restrict the number of constant and recognisable characters.
this is the case in the sebacinales, where high genetic diversity has been reported from the analysis of rdna sequences of lineages with low levels of morphological variation, see e.g.
<cit> .
for example, considerable intraspecific genetic variation has been found within the genus sebacina .
while sebacina vermifera is not known to develop visible basidiomata, s. epigaea develops relatively thin waxy-gelatinous, pale grey basidiomata  <cit> , and s. incrustans forms thick resupinate-incrusting, coriaceous, cream to ochre basidiomata  <cit> .
sebacina epigaea and s. incrustans are both broadly distributed in europe and form mycorrhizal associations with forest trees  <cit>  and/or orchids  <cit> .
both species are relatively easy to recognise and distinguish from one another in the field providing an exceptional opportunity to study their intraspecific diversity in combination with an evaluation of their ecological and morphological characteristics.
to explore the possible factors that have caused the high genetic diversity observed in the sebacinales, we have focussed our molecular phylogenetic approach on population samples of two morphological species, sebacina epigaea and s. incrustans distributed over a regional geographic range .
by characterising the genetic variation, estimating the genetic structure, and comparing the independent nucdna  and mtdna  markers from s. epigaea and s. incrustans population samples, we have been able to address the following questions:  what is the intraspecific genetic variation within these two morphospecies, how many species are found within each morphospecies and are these morphospecies monophyletic?
are their intraspecific nuclear and mitochondrial genealogies concordant?
if significant genetic diversity is present, is there morphological, geographical and/or ecological segregation of the different major lineages?
RESULTS
dna amplification and phylogenetic placement
in general, it was easy to amplify the nuclear dna  from the majority of the population samples.
on the other hand, for many population samples of sebacina epigaea, we were unable to amplify the mitochondrial dna .
phylogenetic analysis of our own complete its sequences and those of sebacina basidiomata available from the genbank/unite databases placed some of our sequences in rather isolated positions , except for s. epigaea haplotype h <dig> was identical to udb <dig> and haplotype h <dig> was identical to udb <dig> both from estonia, respectively.
for s. incrustans, haplotypes h <dig>  h <dig> and h <dig> appeared to be identical to ef <dig> , udb <dig>  and ay <dig> ; haplotype h <dig> the same as udb <dig> ; and haplotype h <dig> to be identical to aj <dig>  aj <dig> and aj <dig> .
in addition, some d1/d <dig> sequences of both species appeared to be identical to sequences of basidiomata in genbank from germany: s. epigaea h <dig> to af291267; s. incrustans h <dig> to af <dig> and fj644513; and s. incrustans h <dig>  h <dig> and h <dig> to ay <dig> and dq <dig>  and from austria: s. epigaea h <dig> to ay <dig> .
major phylogenetic lineages
genealogy analyses of its +  <dig> s and/or d1/d <dig>  as well as rpb <dig> and atp <dig> did not support the morphological species sebacina epigaea and s. incrustans as monophyletic taxa.
phylogenetic analyses of datasets with and without recombination blocks split both into identical major lineages.
in s. epigaea, three highly divergent lineages  were inferred from the its +  <dig> s + d1/d <dig> dataset ; the monophyly of el <dig> was not supported in the rpb <dig> and atp <dig> datasets, except for rpb <dig> inclusive recombinant blocks .
in s. incrustans, all loci strongly supported three highly divergent lineages, il <dig>  il <dig> and il <dig> .
approximately 95% of the population samples represented the lineage el <dig> and 60% the lineage il <dig>  the ild and sh tests did not detect any significant incongruent phylogenetic signal within  or among genes.
genetic diversity and spatial distribution of lineages
percentages of its +  <dig> s + d1/d <dig> sequence divergence before and after removing recombination blocks were higher in sebacina epigaea than in s. incrustans .
most of the polymorphisms were localised at the third codon position for the rpb <dig> and atp <dig> loci.
the dn/ds ratios were below one, indicating that negative selection is the predominant force in rpb <dig> and atp <dig> evolution .
for both species, a high proportion of population samples  included one or more heterozygous positions.
on the contrary, the atp <dig> sequences lacked any heterozygous positions.
heterozygous positions ranged between  <dig> and  <dig> in s. epigaea, and between  <dig> and  <dig> in s. incrustans in the rpb <dig> datasets.
with regard to the its +  <dig> s + d1/d <dig> datasets, between  <dig> and  <dig> heterozygous positions were found in s. epigaea, and between  <dig> and  <dig> in s. incrustans .
the total number of recombination blocks for s. epigaea was  <dig> in its +  <dig> s + d1/d <dig>   <dig> in rpb <dig> and  <dig> in atp <dig>  whereas for s. incrustans,  <dig> were detected in its +  <dig> s + d1/d <dig>   <dig> in rpb <dig> and  <dig> in atp <dig>  after removing indels and recombination blocks,  <dig> its +  <dig> s + d1/d <dig> haplotypes were found among the  <dig> population samples from s. epigaea and eight haplotypes in the  <dig> population samples for s. incrustans.
table  <dig> and figure  <dig> summarise the statistics of nucleotide variation in the its +  <dig> s + d1/d <dig> regions for both morphospecies, where the its region  was identified as the most variable region.
the neutrality tests performed had non-significant values in most cases; therefore, the equilibrium model of neutral evolution could be not rejected.
only for the whole sample of s. incrustans was significant value detected for fu & li’s d* test, suggesting background selection.
population samples from bavarian alps  for both species yielded significant results for the fu & li’s d* and f* tests .
sampling areas are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe.
n = sample size, h = number of haplotypes, s = number of segregating sites, hd = haplotype diversity, k = average number of nucleotide pairwise differences, π = number of nucleotide differences per site, θω = watterson’s estimate of ϕ per site.
significant tests  are marked with *.
the distribution of population samples of s. epigaea indicates that el <dig> has a wider distribution, while el <dig> is restricted to hohenlohe and el <dig> to neckar valley .
within s. incrustans, il <dig> has a wider geographic distribution, il <dig> is restricted to bavarian alps and neckar valley, and il <dig> to hohenlohe .
demographic structure
within each morphospecies, both network estimation approaches found essentially similar structure patterns between the its +  <dig> s + d1/d <dig> , rpb <dig>  and atp <dig>  haplotypes.
the median-joining network revealed three frequent haplotypes  in sebacina epigaea, whereas haplotypes h <dig> and h <dig> were obtained from the same collection, and h <dig> represented a single population sample.
all haplotypes were restricted to one or two sample areas .
in the s. incrustans its +  <dig> s + d1/d <dig> dataset, h <dig> was the most frequent haplotype, whereas h <dig> and h <dig> occurred at low frequency.
h <dig> was scattered throughout bavarian alps, eastern swabian alb, western swabian alb, and neckar valley, and appeared to have an ancestral position to the other haplotypes .
statistical parsimony analysis for s. epigaea yielded three its +  <dig> s + d1/d <dig> networks comprising eight connected , two connected haplotypes  and one single haplotype .
h <dig> had an ancestral position to h1-h <dig> and h6-h <dig> .
rpb <dig> and atp <dig> genes produced similar network structures .
analysis of the its +  <dig> s + d1/d <dig> dataset for s. incrustans resulted in one network with five haplotypes , one with two haplotypes  and one with a single haplotype .
rpb <dig> and atp <dig> genes produced nearly identical network structures .
in general, analyses of rpb <dig> from both morphospecies showed a greater number of haplotypes in comparison with its +  <dig> s + d1/d <dig> dataset.
conversely, a smaller number of haplotypes was detected in atp <dig> dataset.
when we performed nested clade analysis using its +  <dig> s + d1/d <dig> datasets containing recombination blocks within s. epigaea, a total of nine unconnected networks were detected, whereas s. incrustans yielded six unconnected networks .
in s. epigaea, only el <dig> was significantly separated from el <dig> and el <dig>  whereas in s. incrustans, all pairs of lineages had significant pairwise fst values and exact tests .
most of the pairwise fst values and exact tests between pairs of sampling areas were significant in s. epigaea and a minor number of pairs of sampling areas proved to be significant for s. incrustans .
mantel tests comparing genetic differentiation  and geographical distance matrices were not significant .
amova indicated non-significant differentiation among groups for both scenarios analysed, whereas percentages of total variance were significant among populations within groups and within populations .
between pairs of lineages ,  between pairs of collection areas, that are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe.
significant exact tests and fst values are indicated with asterisks: * p <  <dig> , ** p <  <dig> , *** p <  <dig> .
two grouping scenarios were tested .
sampling areas are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe.
ag = percentage of total variance among groups, wg = percentage of total variance among populations within groups, wp = percentage of total variance within populations.
bolded values are significant ..
morphological and ecological traits
main morphological and ecological traits analysed from both morphospecies are summarized as follows :
i. habitat and habit of basidiomata.
population samples within sebacina epigaea showed low phenotypic variation, whereas populations in s. incrustans displayed high levels of basidiomata plasticity that often were linked with the substrate on which they were growing and their age.
basidiomata of s. epigaea growing on naked soil with vertical exposure and on litter produced thin to 2 mm thick basidiomata of gelatinous consistency, which are smooth or meruloid and which often became translucent.
basidiomata of s. incrustans growing on diverse plant substrates are resupinate, up to 3 mm thick, smooth and cartilaginous, whereas those on naked soil have a habit reminiscent to s. epigaea.
ii.
colour of fresh and dried basidiomata.
fresh basidiomata of s. epigaea are uniformly gray and opalescent.
old and dried basidiomata in s. epigaea become membranous and brownish or greyish, and were often hard to see on the substrate.
both fresh and dried basidiomata in s. incrustans that grow on herbaceous substrates are typically whitish to cream or ochraceous; but the basidiomata that grow directly on naked soil have a colour similar to s. epigaea, but become yellow after drying.
iii.
microscopic characteristics.
there was no variation in microscopic characteristics among populations within s. epigaea el <dig> .
in s. incrustans, however, there was some differentiation of certain microscopic features, for example, wall thickness of the trama hyphae and the size of the basidia and basidiospores.
iv.
ecology.
basidiomata of both morphological species were mainly collected from forests with trees in the families fagaceae  and pinaceae , but some in vegetation with betulaceae  and salicaceae .
within s. epigaea, el <dig> and el <dig> seem to be rather restricted to picea abies and el <dig> occurred in a forest composed of frondose trees .
additional aspects of morphology, ecology and phylogenetic diversity within s. epigaea and s. incrustans are described and discussed in additional file  <dig>
discussion
intraspecific genetic variability at the regional scale
although our sampling area was rather geographically restricted, we found high levels of genetic diversity within sebacina epigaea and s. incrustans morphospecies, in contrast with the findings of nilsson et al.
<cit> .
there were two major factors that can explain some of the genetic diversity:  recombination events and  high frequency of synonymous mutations.
the first factor was detected previously by vandenkoornhuyse et al.
<cit>  for arbuscular mycorrhizal fungi, and could explain the high sequence diversity found in the its regions of environmental samples in sebacinales, see e.g.
<cit> .
the high number of recombination events detected in this study, particularly between populations in s. epigaea, suggests that sexuality has an influence on the patterns of genetic divesity and speciation at a regional geographic scale.
however, because both morphological species are obligate mycorrhizal formers and cannot be grown in axenic culture, mating experiments are not possible, and therefore we cannot determine whether or not they represent the same or different biological species.
the second factor of dna variation comprised ~95% of the observed dna polymorphisms in rpb <dig> and ~60% in the atp <dig> sequences, and was driven by negative selection in which rare, deleterious alleles are removed from a population.
the evolutionary significance of this type of selection is still not well understood, but there is an indication that some synonymous polymorphisms are involved in functional roles  <cit> .
we hypothesise that intraspecific genetic variation provide sebacinales symbionts the opportunity to select genotypes that adapt more rapidly, e.g.
those that are physiologically adapted to environmental heterogeneity and/or host availability  <cit> .
the total genetic variation of our data, however, cannot be completely explained by the evolutionary processes discussed above.
gene genealogies revealed three genetic divergent lineages within each morphospecies; interestingly, some of them were detected within the same site separated by only a few centimeters.
we suggest that these patterns of geographical distribution are driven by environmental discontinuities and/or heterogeneity in resources, as suggested by gryta et al.
<cit> .
however, further study is necessary to determine if highly divergent co-occurring lineages within sebacina epigaea and s. incrustans have evolved in sympatry or whether current patterns of geographic distribution are rather the result of recent dispersal events.
the its +  <dig> s + d1/d <dig> regions contain significant polymorphisms that may resolve lower and higher relationships across the sebacinales, even after removing recombinant segments.
in particular, the its region was useful for discriminating among populations and we therefore propose it as a potentially useful dna segment for barcoding in the sebacinales.
concordance of nuclear and mitochondrial genealogies
unlike other fungus taxa that have been affected by natural recombination , topologies of the genealogies inferred from two nuclear regions  and one mitochondrial region  clearly identified three distinctive lineages in both the sebacina epigaea and s. incrustans morphospecies, and these were significantly concordant.
the concordance between nucdna and mtdna sequence data may indicate either complete lineage sorting or an ancient diversification  <cit> .
circumscribing major lineages
surprisingly, both morphological species displayed relatively similar lineage/haplotype structures: a frequent and more widely distributed lineage and two small lineages.
sebacina epigaea el <dig> included population samples from estonia and s. incrustans il <dig> included population samples from austria and denmark.
sampling bias is a possible explanation for the presence of small, distinct lineages .
lineages/cryptic species within s. epigaea seems to have involved large evolutionary divergence accompanied by only a few changes in morphology and microscopic characteristics.
the lack of morphological innovations among the populations analysed could suggest that diversification may be driven by ecological opportunities offered by the availability of new habitats and/or host plants.
host specialisation and host switches have been considered to be important drivers for speciation  <cit> .
however, in this study, no obvious indication of substrate and host specialisation could be detected in circumscribing the major lineages .
because most of the collection sites had more than one potential host tree for both morphospecies, further study focusing on the molecular analysis of ectomycorrhizal root tips should allow us to find a link between mycobionts and host tree.
in s. incrustans, populations exhibited enormous plasticity in basidioma habit, which was linked with the type of substrate upon which they were formed.
such variation was observed even within the same haplotype.
nuclear and mitochondrial data showed evidence of gene flow between populations within major lineages in s. epigaea and s. incrustans morphospecies.
we did not find evidence of an isolation-by-distance model for population structure.
the swabian alp does not seem to represent a geographical barrier for wind or animal dispersal.
in addition, anthropogenic dispersal of spores and/or mycelia contained in the soil and roots should not be underestimated as means of dispersal.
an important putative scenario could be that these mycobionts have acquired their current geographical distribution through extensive planting of picea abies across europe.
the type specimens of sebacina epigaea and s. incrustans are more than 150 years old and catalogued as historical collections; therefore it is not possible to study the material or extract dna from the basidiomata.
genealogies inferred from nucdna and mtdna genes strongly support divergent lineages within these morphospecies.
however, considering the low morphological differentiation within and between lineages, we suggest that sampling specifically from the type localities in france and great britain of these species would increase the probability of more accurately defining each lineage in respect to its type specimen.
CONCLUSIONS
our results indicate that the high intraspecific genetic diversity within the morphospecies sebacina epigaea and s. incrustans is mainly the outcome of natural recombination events and synonymous mutations.
phylogenetic and demographic inferences from nuclear and mitochondrial loci support the division of each morphological species into three distinct phylogenetic lineages, which seem to have evolved independently for a long time.
none of these putative cryptic species appears to be linked to geographical provenance, host preference or morphological innovations.
however, future studies correlating genetic diversity across lineages with differences in ecological niches may help us to better understand the factors shaping speciation in these morphological species.

 comparative studies of the genetic structure of populations, particularly those utilising molecular makers, have provided new opportunities for better understanding historical and/or contemporary factors for modelling speciation. surveys using dna variation in rdna and other sequence data have revealed several examples of morphological fungus species that have diverged into reproductively and/or genetically isolated morphologically indistinguishable monophyletic subgroups or cryptic species see, e.g.. in particular, patterns of geographic distribution have been noted as one of the main factors shaping species diversification. for instance, geographically isolated populations appear to be one of the most plausible causes of speciation. in addition, for some species complexes, sympatric speciation likely has been caused by changes in ecological and/or life history traits and reinforced by negative selection towards hybrids. such causes seem to be less frequent in allopatric speciation, where the major barrier to gene flow is geographic. gene genealogies from independent dna loci have been recommended as a means of identifying phylogenetic species. molecular markers are very helpful for this purpose, especially within fungus groups with comparatively simple body plans that seriously restrict the number of constant and recognisable characters. this is the case in the sebacinales, where high genetic diversity has been reported from the analysis of rdna sequences of lineages with low levels of morphological variation, see e.g.. for example, considerable intraspecific genetic variation has been found within the genus sebacina. while sebacina vermifera is not known to develop visible basidiomata, s. epigaea develops relatively thin waxy-gelatinous, pale grey basidiomata, and s. incrustans forms thick resupinate-incrusting, coriaceous, cream to ochre basidiomata. sebacina epigaea and s. incrustans are both broadly distributed in europe and form mycorrhizal associations with forest trees and/or orchids. both species are relatively easy to recognise and distinguish from one another in the field providing an exceptional opportunity to study their intraspecific diversity in combination with an evaluation of their ecological and morphological characteristics. to explore the possible factors that have caused the high genetic diversity observed in the sebacinales, we have focussed our molecular phylogenetic approach on population samples of two morphological species, sebacina epigaea and s. incrustans distributed over a regional geographic range. by characterising the genetic variation, estimating the genetic structure, and comparing the independent nucdna and mtdna markers from s. epigaea and s. incrustans population samples, we have been able to address the following questions: what is the intraspecific genetic variation within these two morphospecies, how many species are found within each morphospecies and are these morphospecies monophyletic? are their intraspecific nuclear and mitochondrial genealogies concordant? if significant genetic diversity is present, is there morphological, geographical and/or ecological segregation of the different major lineages?  dna amplification and phylogenetic placement in general, it was easy to amplify the nuclear dna from the majority of the population samples. on the other hand, for many population samples of sebacina epigaea, we were unable to amplify the mitochondrial dna. phylogenetic analysis of our own complete its sequences and those of sebacina basidiomata available from the genbank/unite databases placed some of our sequences in rather isolated positions, except for s. epigaea haplotype h was identical to udb and haplotype h was identical to udb both from estonia, respectively. for s. incrustans, haplotypes h h and h appeared to be identical to ef, udb and ay; haplotype h the same as udb; and haplotype h to be identical to aj aj and aj. in addition, some d1/d sequences of both species appeared to be identical to sequences of basidiomata in genbank from germany: s. epigaea h to af291267; s. incrustans h to af and fj644513; and s. incrustans h h and h to ay and dq and from austria: s. epigaea h to ay. major phylogenetic lineages genealogy analyses of its +  s and/or d1/d as well as rpb and atp did not support the morphological species sebacina epigaea and s. incrustans as monophyletic taxa. phylogenetic analyses of datasets with and without recombination blocks split both into identical major lineages. in s. epigaea, three highly divergent lineages were inferred from the its +  s + d1/d dataset; the monophyly of el was not supported in the rpb and atp datasets, except for rpb inclusive recombinant blocks. in s. incrustans, all loci strongly supported three highly divergent lineages, il il and il. approximately 95% of the population samples represented the lineage el and 60% the lineage il the ild and sh tests did not detect any significant incongruent phylogenetic signal within or among genes. genetic diversity and spatial distribution of lineages percentages of its +  s + d1/d sequence divergence before and after removing recombination blocks were higher in sebacina epigaea than in s. incrustans. most of the polymorphisms were localised at the third codon position for the rpb and atp loci. the dn/ds ratios were below one, indicating that negative selection is the predominant force in rpb and atp evolution. for both species, a high proportion of population samples included one or more heterozygous positions. on the contrary, the atp sequences lacked any heterozygous positions. heterozygous positions ranged between and in s. epigaea, and between and in s. incrustans in the rpb datasets. with regard to the its +  s + d1/d datasets, between and heterozygous positions were found in s. epigaea, and between and in s. incrustans. the total number of recombination blocks for s. epigaea was in its +  s + d1/d in rpb and in atp whereas for s. incrustans, were detected in its +  s + d1/d in rpb and in atp after removing indels and recombination blocks, its +  s + d1/d haplotypes were found among the population samples from s. epigaea and eight haplotypes in the population samples for s. incrustans. table  and figure  summarise the statistics of nucleotide variation in the its +  s + d1/d regions for both morphospecies, where the its region was identified as the most variable region. the neutrality tests performed had non-significant values in most cases; therefore, the equilibrium model of neutral evolution could be not rejected. only for the whole sample of s. incrustans was significant value detected for fu & li’s d* test, suggesting background selection. population samples from bavarian alps for both species yielded significant results for the fu & li’s d* and f* tests. sampling areas are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe. n = sample size, h = number of haplotypes, s = number of segregating sites, hd = haplotype diversity, k = average number of nucleotide pairwise differences, π = number of nucleotide differences per site, θω = watterson’s estimate of ϕ per site. significant tests are marked with *. the distribution of population samples of s. epigaea indicates that el has a wider distribution, while el is restricted to hohenlohe and el to neckar valley. within s. incrustans, il has a wider geographic distribution, il is restricted to bavarian alps and neckar valley, and il to hohenlohe. demographic structure within each morphospecies, both network estimation approaches found essentially similar structure patterns between the its +  s + d1/d, rpb and atp haplotypes. the median-joining network revealed three frequent haplotypes in sebacina epigaea, whereas haplotypes h and h were obtained from the same collection, and h represented a single population sample. all haplotypes were restricted to one or two sample areas. in the s. incrustans its +  s + d1/d dataset, h was the most frequent haplotype, whereas h and h occurred at low frequency. h was scattered throughout bavarian alps, eastern swabian alb, western swabian alb, and neckar valley, and appeared to have an ancestral position to the other haplotypes. statistical parsimony analysis for s. epigaea yielded three its +  s + d1/d networks comprising eight connected, two connected haplotypes and one single haplotype. h had an ancestral position to h1-h and h6-h. rpb and atp genes produced similar network structures. analysis of the its +  s + d1/d dataset for s. incrustans resulted in one network with five haplotypes, one with two haplotypes and one with a single haplotype. rpb and atp genes produced nearly identical network structures. in general, analyses of rpb from both morphospecies showed a greater number of haplotypes in comparison with its +  s + d1/d dataset. conversely, a smaller number of haplotypes was detected in atp dataset. when we performed nested clade analysis using its +  s + d1/d datasets containing recombination blocks within s. epigaea, a total of nine unconnected networks were detected, whereas s. incrustans yielded six unconnected networks. in s. epigaea, only el was significantly separated from el and el whereas in s. incrustans, all pairs of lineages had significant pairwise fst values and exact tests. most of the pairwise fst values and exact tests between pairs of sampling areas were significant in s. epigaea and a minor number of pairs of sampling areas proved to be significant for s. incrustans. mantel tests comparing genetic differentiation and geographical distance matrices were not significant. amova indicated non-significant differentiation among groups for both scenarios analysed, whereas percentages of total variance were significant among populations within groups and within populations. between pairs of lineages, between pairs of collection areas, that are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe. significant exact tests and fst values are indicated with asterisks: * p < , ** p < , *** p < . two grouping scenarios were tested. sampling areas are coded as follows: ba = bavarian alps, wa = western swabian alb, ea = eastern swabian alb, nv = neckar valley, ho = hohenlohe. ag = percentage of total variance among groups, wg = percentage of total variance among populations within groups, wp = percentage of total variance within populations. bolded values are significant.. morphological and ecological traits main morphological and ecological traits analysed from both morphospecies are summarized as follows: i. habitat and habit of basidiomata. population samples within sebacina epigaea showed low phenotypic variation, whereas populations in s. incrustans displayed high levels of basidiomata plasticity that often were linked with the substrate on which they were growing and their age. basidiomata of s. epigaea growing on naked soil with vertical exposure and on litter produced thin to 2 mm thick basidiomata of gelatinous consistency, which are smooth or meruloid and which often became translucent. basidiomata of s. incrustans growing on diverse plant substrates are resupinate, up to 3 mm thick, smooth and cartilaginous, whereas those on naked soil have a habit reminiscent to s. epigaea. ii. colour of fresh and dried basidiomata. fresh basidiomata of s. epigaea are uniformly gray and opalescent. old and dried basidiomata in s. epigaea become membranous and brownish or greyish, and were often hard to see on the substrate. both fresh and dried basidiomata in s. incrustans that grow on herbaceous substrates are typically whitish to cream or ochraceous; but the basidiomata that grow directly on naked soil have a colour similar to s. epigaea, but become yellow after drying. iii. microscopic characteristics. there was no variation in microscopic characteristics among populations within s. epigaea el. in s. incrustans, however, there was some differentiation of certain microscopic features, for example, wall thickness of the trama hyphae and the size of the basidia and basidiospores. iv. ecology. basidiomata of both morphological species were mainly collected from forests with trees in the families fagaceae and pinaceae, but some in vegetation with betulaceae and salicaceae. within s. epigaea, el and el seem to be rather restricted to picea abies and el occurred in a forest composed of frondose trees. additional aspects of morphology, ecology and phylogenetic diversity within s. epigaea and s. incrustans are described and discussed in additional file discussion intraspecific genetic variability at the regional scale although our sampling area was rather geographically restricted, we found high levels of genetic diversity within sebacina epigaea and s. incrustans morphospecies, in contrast with the findings of nilsson et al.. there were two major factors that can explain some of the genetic diversity: recombination events and high frequency of synonymous mutations. the first factor was detected previously by vandenkoornhuyse et al. for arbuscular mycorrhizal fungi, and could explain the high sequence diversity found in the its regions of environmental samples in sebacinales, see e.g.. the high number of recombination events detected in this study, particularly between populations in s. epigaea, suggests that sexuality has an influence on the patterns of genetic divesity and speciation at a regional geographic scale. however, because both morphological species are obligate mycorrhizal formers and cannot be grown in axenic culture, mating experiments are not possible, and therefore we cannot determine whether or not they represent the same or different biological species. the second factor of dna variation comprised ~95% of the observed dna polymorphisms in rpb and ~60% in the atp sequences, and was driven by negative selection in which rare, deleterious alleles are removed from a population. the evolutionary significance of this type of selection is still not well understood, but there is an indication that some synonymous polymorphisms are involved in functional roles. we hypothesise that intraspecific genetic variation provide sebacinales symbionts the opportunity to select genotypes that adapt more rapidly, e.g. those that are physiologically adapted to environmental heterogeneity and/or host availability. the total genetic variation of our data, however, cannot be completely explained by the evolutionary processes discussed above. gene genealogies revealed three genetic divergent lineages within each morphospecies; interestingly, some of them were detected within the same site separated by only a few centimeters. we suggest that these patterns of geographical distribution are driven by environmental discontinuities and/or heterogeneity in resources, as suggested by gryta et al.. however, further study is necessary to determine if highly divergent co-occurring lineages within sebacina epigaea and s. incrustans have evolved in sympatry or whether current patterns of geographic distribution are rather the result of recent dispersal events. the its +  s + d1/d regions contain significant polymorphisms that may resolve lower and higher relationships across the sebacinales, even after removing recombinant segments. in particular, the its region was useful for discriminating among populations and we therefore propose it as a potentially useful dna segment for barcoding in the sebacinales. concordance of nuclear and mitochondrial genealogies unlike other fungus taxa that have been affected by natural recombination, topologies of the genealogies inferred from two nuclear regions and one mitochondrial region clearly identified three distinctive lineages in both the sebacina epigaea and s. incrustans morphospecies, and these were significantly concordant. the concordance between nucdna and mtdna sequence data may indicate either complete lineage sorting or an ancient diversification. circumscribing major lineages surprisingly, both morphological species displayed relatively similar lineage/haplotype structures: a frequent and more widely distributed lineage and two small lineages. sebacina epigaea el included population samples from estonia and s. incrustans il included population samples from austria and denmark. sampling bias is a possible explanation for the presence of small, distinct lineages. lineages/cryptic species within s. epigaea seems to have involved large evolutionary divergence accompanied by only a few changes in morphology and microscopic characteristics. the lack of morphological innovations among the populations analysed could suggest that diversification may be driven by ecological opportunities offered by the availability of new habitats and/or host plants. host specialisation and host switches have been considered to be important drivers for speciation. however, in this study, no obvious indication of substrate and host specialisation could be detected in circumscribing the major lineages. because most of the collection sites had more than one potential host tree for both morphospecies, further study focusing on the molecular analysis of ectomycorrhizal root tips should allow us to find a link between mycobionts and host tree. in s. incrustans, populations exhibited enormous plasticity in basidioma habit, which was linked with the type of substrate upon which they were formed. such variation was observed even within the same haplotype. nuclear and mitochondrial data showed evidence of gene flow between populations within major lineages in s. epigaea and s. incrustans morphospecies. we did not find evidence of an isolation-by-distance model for population structure. the swabian alp does not seem to represent a geographical barrier for wind or animal dispersal. in addition, anthropogenic dispersal of spores and/or mycelia contained in the soil and roots should not be underestimated as means of dispersal. an important putative scenario could be that these mycobionts have acquired their current geographical distribution through extensive planting of picea abies across europe. the type specimens of sebacina epigaea and s. incrustans are more than 150 years old and catalogued as historical collections; therefore it is not possible to study the material or extract dna from the basidiomata. genealogies inferred from nucdna and mtdna genes strongly support divergent lineages within these morphospecies. however, considering the low morphological differentiation within and between lineages, we suggest that sampling specifically from the type localities in france and great britain of these species would increase the probability of more accurately defining each lineage in respect to its type specimen.  our results indicate that the high intraspecific genetic diversity within the morphospecies sebacina epigaea and s. incrustans is mainly the outcome of natural recombination events and synonymous mutations. phylogenetic and demographic inferences from nuclear and mitochondrial loci support the division of each morphological species into three distinct phylogenetic lineages, which seem to have evolved independently for a long time. none of these putative cryptic species appears to be linked to geographical provenance, host preference or morphological innovations. however, future studies correlating genetic diversity across lineages with differences in ecological niches may help us to better understand the factors shaping speciation in these morphological species. 
concordance between gene genealogies identified lineages/cryptic species that have evolved independently for a relatively long period of time. in this study, we analysed patterns of genetic variation within two morphological species, sebacina epigaea and s. incrustans, based on dna haplotype sequences of independent genetic markers from the nuclear and mitochondrial genomes for population samples. these putative species were not associated with geographic provenance, geographic barrier, host preference or distinct phenotypic innovations. phylogenetic studies, particularly those based on rdna sequences from plant roots and basidiomata, have revealed a strikingly high genetic diversity in the sebacinales. however, the factors determining this genetic diversity at higher and lower taxonomic levels within this order are still unknown. by characterising the genetic population structure within these species, we provide insights into species boundaries and the possible factors responsible for genetic diversity at a regional geographic scale. we found that recombination events are relatively common between natural populations within sebacina epigaea and s. incrustans, and play a significant role in generating intraspecific genetic diversity. our results suggest that recombination and negative selection have played significant roles in generating genetic diversity within these morphological species at small geographical scales. phylogenetic and demographic analyses based on nuclear and mitochondrial loci revealed three distinct phylogenetic lineages within of each of the morphospecies s. epigaea and s. incrustans: one major and widely distributed lineage, and two geographically restricted lineages, respectively.
 phylogenetic studies, particularly those based on rdna sequences from plant roots and basidiomata, have revealed a strikingly high genetic diversity in the sebacinales. however, the factors determining this genetic diversity at higher and lower taxonomic levels within this order are still unknown. in this study, we analysed patterns of genetic variation within two morphological species, sebacina epigaea and s. incrustans, based on dna haplotype sequences of independent genetic markers from the nuclear and mitochondrial genomes for population samples. by characterising the genetic population structure within these species, we provide insights into species boundaries and the possible factors responsible for genetic diversity at a regional geographic scale.  we found that recombination events are relatively common between natural populations within sebacina epigaea and s. incrustans, and play a significant role in generating intraspecific genetic diversity. furthermore, we also found that rpb and atp genes display higher levels of intraspecific synonymous polymorphism. phylogenetic and demographic analyses based on nuclear and mitochondrial loci revealed three distinct phylogenetic lineages within of each of the morphospecies s. epigaea and s. incrustans: one major and widely distributed lineage, and two geographically restricted lineages, respectively. we found almost no differential morphological or ecological characteristics that could be used to discriminate between these lineages.  our results suggest that recombination and negative selection have played significant roles in generating genetic diversity within these morphological species at small geographical scales. concordance between gene genealogies identified lineages/cryptic species that have evolved independently for a relatively long period of time. these putative species were not associated with geographic provenance, geographic barrier, host preference or distinct phenotypic innovations. cryptic speciesspeciationdiversitypopulation structuremultilocus genealogiesbasidiomycotasebacinalessympatrysynonymous polymorphismectomycorrhiza 