BACKGROUND
fossil analogs of modern fagaceae family are well represented in the northern hemisphere, indicating long-term presence and differential patterns of species diversification throughout the tertiary and quaternary period  <cit> .
in italy during the tyrrhenian, the last interglacial period which started around  <dig>  years ago, palaeontological and palynological data support the existence in the veneto-po plain of a mixed forest of colchic type dominated by fir-trees  spach) and pine-trees, together with the broad-leaved trees zelkova spach, quercus l., ulmus l., tilia l., carpinus l., corylus l., fagus l., betula l. and castanea miller  <cit> .
although a complete agreement has not been reached, most authors indicate that the fagaceae family includes nine genera: fagus l., castanea l., castanopsis spach., chrysolepis hjelmquist, colombobalanus  nixon & crepet, formanodendron  nixon & crepet, lithocarpus bl., quercus l., and trigonobalanus forman  <cit> .
most genera are in temperate forests of the northern hemisphere, and they show a hot-spot of diversity in tropical zones of the southern-east asia  <cit> .
the fagaceae family has been extensively studied at the genetic  <cit> , biogeographic and phylogeographic  <cit>  level.
thanks to macrofossils richness several taxonomic and macroevolutionary studies have been performed as well  <cit> .
despite this, the taxonomic subdivision in the fagaceae family is still controversial, due to the small number of prominent characteristics of the fruit and floral  <cit> .
the traditional classification of beech in western eurasia recognizes one species, f. sylvatica with two subspecies f. s. sylvatica and f. s. orientalis, and two intermediary types, f. moesiaca and f. taurica  <cit> .
fagus sylvatica l. is distributed in western, central and southern europe with marginal and isolated occurrences in england, and scandinavia.
the natural range of f. orientalis is asia minor, the amanus mts.
, the elburz mts.
, and caucasus.
isolated occurrences of this taxa outside the natural range were found in eastern serbia, in macedonia, in banate, in moldova, in dobrudja, and in central bulgaria.
contact zones between the natural ranges of both taxa are reported in northern greece and bulgaria  <cit> .
authors disagree about the taxonomic position of fagus taurica popl.
and fagus moesiaca czecz.
.
f. taurica is considered by some authors as the intermediate form between f. sylvatica and f. orientalis, and by others as an independent species.
similar doubts regard the taxonomy of f. moesiaca considered an independent species or, most frequently, the subspecies of f. sylvatica  <cit> .
the description of these taxa has been mainly based on the morphological traits of leaves  <cit> .
standing the difficulties in discriminating closely related taxa and the overlapping of their natural range, the molecular data by manos and stanford  <cit>  provided the most reliable phylogenetic reconstruction of fagaceae family.
however, they presented limited data from european taxa, which have been traditionally included in various morphologically distinct groups.
also denk and colleagues  <cit>  tried to combine morphological and molecular data to resolve the controversy within the western eurasiatic taxa without succeeding.
from these statements it is clear that is still controversial whether the two taxa  are really separated, and whether the intermediates are two different taxonomic units .
analysing populations of the western eurasiatic taxa f. sylvatica , f. moesiaca , f. taurica  and f. orientalis , using the trnl-trnf chloroplast dna  sequence , that has been previously proved efficient in discriminating quercus entities  <cit> , we aim at:  inferring relationships among the species within the fagaceae family;  identifying the western eurasiatic taxa;  combining, for the first time, phylogeny analyses on present fagus spp.
with molecular analyses of pollen from italian fossil specimens as old as about  <dig>  yrs before present.
in addition f. crenata , f. grandifolia  f. japonica , f. hayatae , quercus spp.
and castanea spp.
were analysed to better resolve the phylogeny inference .
RESULTS
identification of fagaceae family and species
the phylogenetic relationship among and within quercus, castanea and fagus genera belonging to fagaceae family have been inferred analysing the trnl-trnf cpdna region in several species and in several individuals .
the entire trnl-trnf cpdna ranged from  <dig> bp in c. crenata sieb.
to  <dig> bp in f. crenata blume.
the multiple alignment of the trnl-trnf region among the different species resulted in a sequence  <dig> bp long; the trnl intron had a length of  <dig> bp, the second trnl exon had a length of  <dig> bp, and the trnl-trnf partial intergenic spacer had a length of  <dig> bp .
the trnl intron belonging to intron i group is interesting for rna secondary structure that permits to conserve active form for "autosplicing".
these introns have different nucleotide primary structure, but the same secondary structure is conserved from cyanobacteria, to algae, and to plant.
the conserved nucleotide regions are short four sequences named p, q, r, and s  <cit> .
these sequences are always present in the same order within intron, but the distance among contiguous elements range from few to hundred nucleotides  <cit> .
the nucleotide conserved sequences in trnl intron were identified in quercus genus by paffetti et al  <cit> : p , q , r  and s .
in the present work the same sequences have been found in castanea and fagus genera.
the intron and exon of trnl show few nucleotides changes within fagaceae family, particularly among the castanea and quercus genera according to the low rate of chloroplast dna variation within this family, as previously shown for other cpdna regions by several authors  <cit> .
the fagus genus is divergent from the other genera due to an insertion of  <dig> nucleotides within the intron region .
the kimura- <dig> model  <cit>  with a proportion of invariant sites  and a rate variation among sites  was selected as best-fit model of sequence evolution and then employed in maximum likelihood  and bayesian analyses .
since trees recovered by the three different methods, maximum parsimony , ml and bi, substantially display the same topology, we show only the ml tree .
the different fagus species were grouped in the same unresolved cluster, providing no information about their phylogenetic relationships.
high bootstrap and bayesian posterior probability values are present only in the part of the tree involving the separation between castanea and quercus genera and inside the cluster of castanea species.
the phylogenetic reconstruction shows a subdivision of the fagaceae family in fagoideae, quercoideae and castanoideae subfamily according to traditional systematic and to data obtained by manos and steele  <cit>  analysing mitochondrial matk gene, but in contrast with the results obtained by nixon and crepet  <cit>  with macrofossil data.
moreover, the results confirm a recent monophyletic origin of the fagoideae subfamily and common origin  of the quercoideae and castanoideae confirming data obtained by manos and steele  <cit> .
the region in the alignment ranging from position  <dig> to  <dig> can distinguish the genera .
particularly, the fagus genus is distinguished from quercus and castanea genera by four transversion  and by one transition at site  <dig>  moreover, the castanea genus is distinguished from quercus genus by one transversion at site  <dig>
a sequence comparative analysis of the trnl-trnf cpdna region has been done among all the fagus spp.
considered .
the entire region ranged from  <dig> bp in f. sylvatica and in f. grandifolia ehrh., to  <dig> bp in f. crenata.
the trnl intron harbour some informative sites that allow to discriminate the different species inside the genus, with the intergenic region showing a higher variability at the intraspecific level.
these data confirmed the results of previous studies on the genus quercus, highlighting the usefulness of this sequence for taxa identification  <cit> .
these are the most informative sites:  a  <dig> bp insertion  in the intron, and a  <dig> bp deletion  in the intergenic region characterise the genus fagus;  two transversions  in the intron identify f. sylvatica;  two transitions  in the intron identify f. grandifolia;  one transition  in the intron identifies f. japonica bl.
;  one transversion  in the intron identifies f. moesiaca; and  two transitions  in the intergenic identify f. taurica .
the transversion  at site  <dig> is particularly interesting as it discriminates the western eurasiatic taxa from the others.
it is to notice that all the european taxa have an a, and the asiatic species have a c. as also the other genus quercus and genus castanea have a c in the site  <dig>  this is indicating that this base was present in the common ancestor of the genus fagaceae, and that its mutation to a occurred in european fagus more recently.
therefore, the asiatic species are more ancient than the european ones.
moreover, f. orientalis seems to be originated early from the other european groups, as it presents an identical sequence to the asiatic ones except for the site  <dig>
it is possible to distinguish the f. grandifolia species from asiatic and western eurasiatic taxa, and the asiatic species from western eurasiatic taxa; among the latter f. orientalis, f. moesiaca, f. taurica and f. sylvatica can be identified, and f. orientalis can be regarded as the ancestral species in european beech.
in comparison to previous works, the region analysed in this study is able to discriminate the different fagus species as some mutation are fixed and specific for each fagus species.
nevertheless, we also find that within fagus spp.
the differences are very few suggesting that speciation process within this group might be still in progress  <cit> .
identification of ancient dna
it is well known that dna degradation processes pose serious limits to the length of the fragments that can be retrieved from ancient specimens  <cit> : only stretches of dna less than  <dig> bp can be confidently analysed.
therefore, considering the age of our sample, we decided to amplify from ancient samples the trnl-trnf region ranging from site  <dig> to site  <dig>  namely the region that proved useful to distinguish among the different genera  and also among the main taxa  inside the fagus genus.
this region was first amplified by real-time pcr  to estimate the copy number of cpdna in ancient sample for the assessment of its authenticity.
a post-amplification melting curve analysis was carried out to verify that artefactual products  were not present within each reaction.
a unique melting curve characteristic of our dna fragment  was obtained indicating that the reaction was problem free.
we determined that the initial amount of genuine chloroplast molecules in the fossil sample is about  <dig>  molecules/μl.
one pollen grain contains about  <dig>  cpdna molecules, and five fagus pollen grains are present in  <dig> μl of our sample , so the estimated copy number is of  <dig>  genomes/μl; a value very similar to the estimation made by qpcr.
therefore, we can say that our result is reliable enough for a study based on ancient dna amplification  <cit> .
the sequences of  <dig> bp obtained from fossil material, dated older than  <dig>  years before present  and collected near bocca di malamocco  , belong to fagus genus, since they display the informative transversions at sites  <dig> and  <dig>  all the  <dig> clones analysed showed the same nucleotide sequences.
the mutations present in these sequences are characteristic of the western eurasiatic group due to the presence of the transversion at site  <dig> position.
moreover, these sequences do not belong to f. sylvatica since they show transversions  at both sites  <dig> and  <dig> .
therefore, the results show that f. orientalis complex  and not f. sylvatica was present in val padana   <dig>  years bp.
these data confirm the palynological data obtained by calderoni and colleagues  <cit> , who was not able to discriminate among the fagus spp.
for the limitation of the methods.
therefore, our molecular data add new crucial information to the palynological data.
euroasiatic fagus taxa and ancient pollen grouping
the same 37– <dig> cpdna region was used to construct a statistical parsimony network containing both ancient and modern samples of f. sylvatica, f. moesiaca, f. taurica and f. orientalis .
only  <dig> different haplotypes were recovered out of the  <dig> specimens analysed  .
the network clearly shows that two major groups, separated by at least three mutational steps, can be identified.
the cluster a, comprising haplotypes from  <dig> to  <dig>  is represented by the f. orientalis, f. moesiaca, f. taurica samples, plus the ancient specimens and some f. sylvatica samples.
the cluster b, with haplotypes from  <dig> to  <dig>  is formed by samples referred only to f. sylvatica taxa.
it is evident that the cluster b is homogeneous confirming previously data on f. sylvatica in western europe  <cit> .
the heterogeneity of cluster a is due to the presence of a mix of taxa and it can be explained, at a first attempt, by the length of the sequence used in the construction of the network, which does not contain the informative sites necessary to distinguish f. taurica and f. moesiaca from f. orientalis.
another source of apparent incongruence is the presence, in this cluster a, of some populations, morphologically described as f. sylvatica.
in effect, the sequence analysis showed that these populations have the typical sequence of the f. orientalis.
nonetheless, some authors already pointed out that some central-south italian populations look like the balkans peninsula ones  <cit> .
this seems to be the result of an ancient connection, during the pliocene, between the italian and balkan peninsulas, as confirmed by previous isoenzyme analyses.
this connection might have favoured gene flow and genetic admixture between the gene pool originated from the two peninsulas  <cit> .
considering that fossil material analysed in this study resulted belonging to the f. orientalis complex, another hypothesis can be put forward: these taxa were present in the po valley  <dig>  years bp.
therefore, we can hypothesize that during the interglacial period the f. orientalis complex was the dominant forest vegetation in italian peninsula, and that the actual f. sylvatica populations  can be considered remains of ancient populations.
previous studies already showed that these populations are divergent from other f. sylvatica populations.
in fact, the presence of unique haplotypes in central-southern populations was previously demonstrated  <cit> .
magri  <cit>  suggested that beech populations might have survived during the last glacial period at different locations in the italian peninsula, so that no clear large-scale migration trends can be recognised in southern and central italy.
the presence of populations displaying high divergence in central-southern italy may be associated to the fact that beech populations persisted in these regions since the middle pleistocene, as also suggested by follieri and colleagues  <cit> .
during the saint germain i , beech represented a major component of the vegetation in central italy.
pollen data revealed that the italian peninsula might have been an important area for the survival of beech not only during the last glaciation, but also during previous interglacial periods  <cit> .
our results seem to confirm these hypotheses, adding the new information that the f. orientalis complex was already present.
the sporadic occurrence of f. orientalis in greek and romania populations reported in previous work  <cit>  can explain the presence of some of these populations in the cluster a.
accordingly to the results of the network, we subdivided our modern samples in two groups, f. orientalis-complex  and f. sylvatica , comprising respectively  <dig> and  <dig> samples.
using the entire cpdna sequence of fagus spp.,  <dig> bp long, we submitted these groups to specific population genetics analyses.
the genetic differentiation between f. orientalis-complex  and f. sylvatica  resulted very strong with a Φst value equal to  <dig>  .
the f. orientalis-complex  is characterised by a value of allelic richness as three times as greater than that of f. sylvatica  .
the greater genetic diversity within f. orientalis-complex  is confirmed by another measure not strictly dependent on the number of samples being compared, such as the mean number of pairwise differences: we see that the f. orientalis-complex cluster has a value as twice as higher than that of f. sylvatica.
we applied two statistical tests for neutrality, tajima's d and fu's fs.
both tests gave statistically significant negative values only for the f. orientalis-complex .
assuming neutrality selection at the locus under study, these results can be interpreted as a signature of a demographic expansion.
standard and molecular indexes of the f. sylvatica cluster a and f. orientalis complex as determined by the network of haplotypes .
symbols used: n, sample size; k, number of different haplotypes; h, genetic diversity; ar, allelic richness calculated with a rarefaction size of 20; π, nucleotide diversity; mpd, mean number of pairwise difference; ns, not significant.
all these results can reinforce the hypothesis according to which f. orientalis is the ancestral species in european beech; as also previously hypothesize by the sequence analysis.
in fact, if we consider f. sylvatica a species derived from the ancestral f. orientalis, this can partially explain its reduction in genetic variability  <cit> .
one general observation within fagaceae is that sequence divergence among and within genera is fairly low.
this is based on a variety of sources, including the cpdna genes, rbcl, trnk and matk  <cit> , and the its region  <cit> .
the various noncoding regions of cpdna sequenced in this study also reveal appreciably low levels of sequence divergence between disjoint species of most of the fagus species.
estimating divergence times between species with low sequence divergence can be problematic because of low statistical power of relative rate tests and high levels of standard error  <cit> .
calculation of divergence times using the highest estimate of the rate of synonymous changes for rbcl  in fagaceae  <cit> , calibrated using fossil data for the minimum divergence time of castanea and quercus , provides a rough guide to estimate divergence times based on the noncoding cpdna sequences examined here.
we adopted a coalescent-based method specifically devised for recent divergence events  <cit> , which takes also into account the contribution of gene flow subsequent to the divergence event.
for fagus, where roughly equal amounts of nucleotide variation were obtained for cpdna noncoding regions, we calculated divergence times using the same substitution rate across the total amount of sequence.
values are derived from two different mutation rates, slow and fast which refers to the overall substitution rate  and the synonymous rate  of frascaria et al  <cit> , respectively.
this suggests minimum and maximum divergence between f. orientalis and f. sylvatica of about  <dig>  million and  <dig>  million years bp .
estimated divergence times, t, between the group denoted as 'orientalis' and 'sylvatica' as defined on the basis of genetic information .
tmrca is the estimated time to the most recent common ancestor of the two groups.
values are derived from two different mutation rates, slow and fast which refers to the overall substitution rate  and the synonymous rate  of frascaria et al.
<cit> , respectively.
even using the slowest mutation rate, it turns out that the two species diverged recently.
the method we adopted allowed us to exclude that the relative low amount of divergence can be attributed to an older event of splitting followed by moderate gene flow.
CONCLUSIONS
we can conclude that at the end of tertiary period the ancestral f. orientalis complex was present in italy, and that only at the beginning of the pliocene f. sylvatica became differentiated from f. orientalis, slowly turning to a predominant role among the fagus species in west europe.
the analysis of dna from samples dated before the last glacial maximum, allowed us to provide a direct demonstration that this event dramatically affected the distribution of forest trees such as the beech.
to better understand how and why the f. sylvatica became predominant in comparison to f. orientalis in western europe more extensive molecular study on fossils remains from different geographic area and from different ages is required, combining the results with palynological and paleoclimatic data.

 fossil analogs of modern fagaceae family are well represented in the northern hemisphere, indicating long-term presence and differential patterns of species diversification throughout the tertiary and quaternary period. in italy during the tyrrhenian, the last interglacial period which started around years ago, palaeontological and palynological data support the existence in the veneto-po plain of a mixed forest of colchic type dominated by fir-trees spach) and pine-trees, together with the broad-leaved trees zelkova spach, quercus l., ulmus l., tilia l., carpinus l., corylus l., fagus l., betula l. and castanea miller. although a complete agreement has not been reached, most authors indicate that the fagaceae family includes nine genera: fagus l., castanea l., castanopsis spach., chrysolepis hjelmquist, colombobalanus nixon & crepet, formanodendron nixon & crepet, lithocarpus bl., quercus l., and trigonobalanus forman. most genera are in temperate forests of the northern hemisphere, and they show a hot-spot of diversity in tropical zones of the southern-east asia. the fagaceae family has been extensively studied at the genetic, biogeographic and phylogeographic level. thanks to macrofossils richness several taxonomic and macroevolutionary studies have been performed as well. despite this, the taxonomic subdivision in the fagaceae family is still controversial, due to the small number of prominent characteristics of the fruit and floral. the traditional classification of beech in western eurasia recognizes one species, f. sylvatica with two subspecies f. s. sylvatica and f. s. orientalis, and two intermediary types, f. moesiaca and f. taurica. fagus sylvatica l. is distributed in western, central and southern europe with marginal and isolated occurrences in england, and scandinavia. the natural range of f. orientalis is asia minor, the amanus mts., the elburz mts., and caucasus. isolated occurrences of this taxa outside the natural range were found in eastern serbia, in macedonia, in banate, in moldova, in dobrudja, and in central bulgaria. contact zones between the natural ranges of both taxa are reported in northern greece and bulgaria. authors disagree about the taxonomic position of fagus taurica popl. and fagus moesiaca czecz.. f. taurica is considered by some authors as the intermediate form between f. sylvatica and f. orientalis, and by others as an independent species. similar doubts regard the taxonomy of f. moesiaca considered an independent species or, most frequently, the subspecies of f. sylvatica. the description of these taxa has been mainly based on the morphological traits of leaves. standing the difficulties in discriminating closely related taxa and the overlapping of their natural range, the molecular data by manos and stanford provided the most reliable phylogenetic reconstruction of fagaceae family. however, they presented limited data from european taxa, which have been traditionally included in various morphologically distinct groups. also denk and colleagues tried to combine morphological and molecular data to resolve the controversy within the western eurasiatic taxa without succeeding. from these statements it is clear that is still controversial whether the two taxa are really separated, and whether the intermediates are two different taxonomic units. analysing populations of the western eurasiatic taxa f. sylvatica, f. moesiaca, f. taurica and f. orientalis, using the trnl-trnf chloroplast dna sequence, that has been previously proved efficient in discriminating quercus entities, we aim at: inferring relationships among the species within the fagaceae family; identifying the western eurasiatic taxa; combining, for the first time, phylogeny analyses on present fagus spp. with molecular analyses of pollen from italian fossil specimens as old as about yrs before present. in addition f. crenata, f. grandifolia f. japonica, f. hayatae, quercus spp. and castanea spp. were analysed to better resolve the phylogeny inference.  identification of fagaceae family and species the phylogenetic relationship among and within quercus, castanea and fagus genera belonging to fagaceae family have been inferred analysing the trnl-trnf cpdna region in several species and in several individuals. the entire trnl-trnf cpdna ranged from bp in c. crenata sieb. to bp in f. crenata blume. the multiple alignment of the trnl-trnf region among the different species resulted in a sequence bp long; the trnl intron had a length of bp, the second trnl exon had a length of bp, and the trnl-trnf partial intergenic spacer had a length of bp. the trnl intron belonging to intron i group is interesting for rna secondary structure that permits to conserve active form for "autosplicing". these introns have different nucleotide primary structure, but the same secondary structure is conserved from cyanobacteria, to algae, and to plant. the conserved nucleotide regions are short four sequences named p, q, r, and s. these sequences are always present in the same order within intron, but the distance among contiguous elements range from few to hundred nucleotides. the nucleotide conserved sequences in trnl intron were identified in quercus genus by paffetti et al: p, q, r and s. in the present work the same sequences have been found in castanea and fagus genera. the intron and exon of trnl show few nucleotides changes within fagaceae family, particularly among the castanea and quercus genera according to the low rate of chloroplast dna variation within this family, as previously shown for other cpdna regions by several authors. the fagus genus is divergent from the other genera due to an insertion of nucleotides within the intron region. the kimura- model with a proportion of invariant sites and a rate variation among sites was selected as best-fit model of sequence evolution and then employed in maximum likelihood and bayesian analyses. since trees recovered by the three different methods, maximum parsimony, ml and bi, substantially display the same topology, we show only the ml tree. the different fagus species were grouped in the same unresolved cluster, providing no information about their phylogenetic relationships. high bootstrap and bayesian posterior probability values are present only in the part of the tree involving the separation between castanea and quercus genera and inside the cluster of castanea species. the phylogenetic reconstruction shows a subdivision of the fagaceae family in fagoideae, quercoideae and castanoideae subfamily according to traditional systematic and to data obtained by manos and steele analysing mitochondrial matk gene, but in contrast with the results obtained by nixon and crepet with macrofossil data. moreover, the results confirm a recent monophyletic origin of the fagoideae subfamily and common origin of the quercoideae and castanoideae confirming data obtained by manos and steele. the region in the alignment ranging from position to can distinguish the genera. particularly, the fagus genus is distinguished from quercus and castanea genera by four transversion and by one transition at site moreover, the castanea genus is distinguished from quercus genus by one transversion at site a sequence comparative analysis of the trnl-trnf cpdna region has been done among all the fagus spp. considered. the entire region ranged from bp in f. sylvatica and in f. grandifolia ehrh., to bp in f. crenata. the trnl intron harbour some informative sites that allow to discriminate the different species inside the genus, with the intergenic region showing a higher variability at the intraspecific level. these data confirmed the results of previous studies on the genus quercus, highlighting the usefulness of this sequence for taxa identification. these are the most informative sites: a bp insertion in the intron, and a bp deletion in the intergenic region characterise the genus fagus; two transversions in the intron identify f. sylvatica; two transitions in the intron identify f. grandifolia; one transition in the intron identifies f. japonica bl.; one transversion in the intron identifies f. moesiaca; and two transitions in the intergenic identify f. taurica. the transversion at site is particularly interesting as it discriminates the western eurasiatic taxa from the others. it is to notice that all the european taxa have an a, and the asiatic species have a c. as also the other genus quercus and genus castanea have a c in the site this is indicating that this base was present in the common ancestor of the genus fagaceae, and that its mutation to a occurred in european fagus more recently. therefore, the asiatic species are more ancient than the european ones. moreover, f. orientalis seems to be originated early from the other european groups, as it presents an identical sequence to the asiatic ones except for the site it is possible to distinguish the f. grandifolia species from asiatic and western eurasiatic taxa, and the asiatic species from western eurasiatic taxa; among the latter f. orientalis, f. moesiaca, f. taurica and f. sylvatica can be identified, and f. orientalis can be regarded as the ancestral species in european beech. in comparison to previous works, the region analysed in this study is able to discriminate the different fagus species as some mutation are fixed and specific for each fagus species. nevertheless, we also find that within fagus spp. the differences are very few suggesting that speciation process within this group might be still in progress. identification of ancient dna it is well known that dna degradation processes pose serious limits to the length of the fragments that can be retrieved from ancient specimens: only stretches of dna less than bp can be confidently analysed. therefore, considering the age of our sample, we decided to amplify from ancient samples the trnl-trnf region ranging from site to site namely the region that proved useful to distinguish among the different genera and also among the main taxa inside the fagus genus. this region was first amplified by real-time pcr to estimate the copy number of cpdna in ancient sample for the assessment of its authenticity. a post-amplification melting curve analysis was carried out to verify that artefactual products were not present within each reaction. a unique melting curve characteristic of our dna fragment was obtained indicating that the reaction was problem free. we determined that the initial amount of genuine chloroplast molecules in the fossil sample is about molecules/μl. one pollen grain contains about cpdna molecules, and five fagus pollen grains are present in μl of our sample, so the estimated copy number is of genomes/μl; a value very similar to the estimation made by qpcr. therefore, we can say that our result is reliable enough for a study based on ancient dna amplification. the sequences of bp obtained from fossil material, dated older than years before present and collected near bocca di malamocco, belong to fagus genus, since they display the informative transversions at sites and all the clones analysed showed the same nucleotide sequences. the mutations present in these sequences are characteristic of the western eurasiatic group due to the presence of the transversion at site position. moreover, these sequences do not belong to f. sylvatica since they show transversions at both sites and. therefore, the results show that f. orientalis complex and not f. sylvatica was present in val padana years bp. these data confirm the palynological data obtained by calderoni and colleagues, who was not able to discriminate among the fagus spp. for the limitation of the methods. therefore, our molecular data add new crucial information to the palynological data. euroasiatic fagus taxa and ancient pollen grouping the same 37– cpdna region was used to construct a statistical parsimony network containing both ancient and modern samples of f. sylvatica, f. moesiaca, f. taurica and f. orientalis. only different haplotypes were recovered out of the specimens analysed. the network clearly shows that two major groups, separated by at least three mutational steps, can be identified. the cluster a, comprising haplotypes from to is represented by the f. orientalis, f. moesiaca, f. taurica samples, plus the ancient specimens and some f. sylvatica samples. the cluster b, with haplotypes from to is formed by samples referred only to f. sylvatica taxa. it is evident that the cluster b is homogeneous confirming previously data on f. sylvatica in western europe. the heterogeneity of cluster a is due to the presence of a mix of taxa and it can be explained, at a first attempt, by the length of the sequence used in the construction of the network, which does not contain the informative sites necessary to distinguish f. taurica and f. moesiaca from f. orientalis. another source of apparent incongruence is the presence, in this cluster a, of some populations, morphologically described as f. sylvatica. in effect, the sequence analysis showed that these populations have the typical sequence of the f. orientalis. nonetheless, some authors already pointed out that some central-south italian populations look like the balkans peninsula ones. this seems to be the result of an ancient connection, during the pliocene, between the italian and balkan peninsulas, as confirmed by previous isoenzyme analyses. this connection might have favoured gene flow and genetic admixture between the gene pool originated from the two peninsulas. considering that fossil material analysed in this study resulted belonging to the f. orientalis complex, another hypothesis can be put forward: these taxa were present in the po valley years bp. therefore, we can hypothesize that during the interglacial period the f. orientalis complex was the dominant forest vegetation in italian peninsula, and that the actual f. sylvatica populations can be considered remains of ancient populations. previous studies already showed that these populations are divergent from other f. sylvatica populations. in fact, the presence of unique haplotypes in central-southern populations was previously demonstrated. magri suggested that beech populations might have survived during the last glacial period at different locations in the italian peninsula, so that no clear large-scale migration trends can be recognised in southern and central italy. the presence of populations displaying high divergence in central-southern italy may be associated to the fact that beech populations persisted in these regions since the middle pleistocene, as also suggested by follieri and colleagues. during the saint germain i, beech represented a major component of the vegetation in central italy. pollen data revealed that the italian peninsula might have been an important area for the survival of beech not only during the last glaciation, but also during previous interglacial periods. our results seem to confirm these hypotheses, adding the new information that the f. orientalis complex was already present. the sporadic occurrence of f. orientalis in greek and romania populations reported in previous work can explain the presence of some of these populations in the cluster a. accordingly to the results of the network, we subdivided our modern samples in two groups, f. orientalis-complex and f. sylvatica, comprising respectively and samples. using the entire cpdna sequence of fagus spp., bp long, we submitted these groups to specific population genetics analyses. the genetic differentiation between f. orientalis-complex and f. sylvatica resulted very strong with a Φst value equal to. the f. orientalis-complex is characterised by a value of allelic richness as three times as greater than that of f. sylvatica. the greater genetic diversity within f. orientalis-complex is confirmed by another measure not strictly dependent on the number of samples being compared, such as the mean number of pairwise differences: we see that the f. orientalis-complex cluster has a value as twice as higher than that of f. sylvatica. we applied two statistical tests for neutrality, tajima's d and fu's fs. both tests gave statistically significant negative values only for the f. orientalis-complex. assuming neutrality selection at the locus under study, these results can be interpreted as a signature of a demographic expansion. standard and molecular indexes of the f. sylvatica cluster a and f. orientalis complex as determined by the network of haplotypes. symbols used: n, sample size; k, number of different haplotypes; h, genetic diversity; ar, allelic richness calculated with a rarefaction size of 20; π, nucleotide diversity; mpd, mean number of pairwise difference; ns, not significant. all these results can reinforce the hypothesis according to which f. orientalis is the ancestral species in european beech; as also previously hypothesize by the sequence analysis. in fact, if we consider f. sylvatica a species derived from the ancestral f. orientalis, this can partially explain its reduction in genetic variability. one general observation within fagaceae is that sequence divergence among and within genera is fairly low. this is based on a variety of sources, including the cpdna genes, rbcl, trnk and matk, and the its region. the various noncoding regions of cpdna sequenced in this study also reveal appreciably low levels of sequence divergence between disjoint species of most of the fagus species. estimating divergence times between species with low sequence divergence can be problematic because of low statistical power of relative rate tests and high levels of standard error. calculation of divergence times using the highest estimate of the rate of synonymous changes for rbcl in fagaceae, calibrated using fossil data for the minimum divergence time of castanea and quercus, provides a rough guide to estimate divergence times based on the noncoding cpdna sequences examined here. we adopted a coalescent-based method specifically devised for recent divergence events, which takes also into account the contribution of gene flow subsequent to the divergence event. for fagus, where roughly equal amounts of nucleotide variation were obtained for cpdna noncoding regions, we calculated divergence times using the same substitution rate across the total amount of sequence. values are derived from two different mutation rates, slow and fast which refers to the overall substitution rate and the synonymous rate of frascaria et al, respectively. this suggests minimum and maximum divergence between f. orientalis and f. sylvatica of about million and million years bp. estimated divergence times, t, between the group denoted as'orientalis' and'sylvatica' as defined on the basis of genetic information. tmrca is the estimated time to the most recent common ancestor of the two groups. values are derived from two different mutation rates, slow and fast which refers to the overall substitution rate and the synonymous rate of frascaria et al., respectively. even using the slowest mutation rate, it turns out that the two species diverged recently. the method we adopted allowed us to exclude that the relative low amount of divergence can be attributed to an older event of splitting followed by moderate gene flow.  we can conclude that at the end of tertiary period the ancestral f. orientalis complex was present in italy, and that only at the beginning of the pliocene f. sylvatica became differentiated from f. orientalis, slowly turning to a predominant role among the fagus species in west europe. the analysis of dna from samples dated before the last glacial maximum, allowed us to provide a direct demonstration that this event dramatically affected the distribution of forest trees such as the beech. to better understand how and why the f. sylvatica became predominant in comparison to f. orientalis in western europe more extensive molecular study on fossils remains from different geographic area and from different ages is required, combining the results with palynological and paleoclimatic data. 
in this work we aim at testing if the trnl-trnf chloroplast dna region, that has been previously proved efficient in discriminating different quercus taxa, can be employed in distinguishing the fagus species and in identifying the ancient pollen. in particular, few specific and fixed mutations were able to discriminate and identify all the different fagus species. the ancient dna sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the f. orientalis complex was already present during the tyrrhenian period in what is now the venice lagoon. considering a short fragment of base pairs within the trnl intron, transversions were found able in distinguishing the f. orientalis complex taxa from the remaining fagus spp. populations from western euroasistic taxa were sampled, and sequenced for the trnl-trnf region to verify the efficiency of this cpdna region in identifying the fagus spp.. furthermore, fagus crenata, fagus grandifolia, fagus japonica, fagus hayatae, quercus species and castanea species were analysed to better resolve the phylogenetic inference. our results show that this cpdna region harbour some informative sites that allow to infer relationships among the species within the fagaceae family. phylogeographic analyses on the western euroasiatic fagus taxa is available, however, the subdivision of fagus spp. for the first time molecular analyses of ancient pollen, dated at least years ago, were used in combination with the phylogeny analysis on current species, to identify the fagus spp. this permits to analyse this fragment also in ancient samples, where dna is usually highly degraded. the sequences data indicate that the dna recovered from ancient pollen belongs to the f. orientalis complex since it displays the informative sites characteristic of this complex. this is a new and important insight considering that nowadays west europe is not the natural area of fagus orientalis complex, and up to now nobody has hypothesized the presence during the last interglacial period of f. orientalis complex in italy. present during the last interglacial period in italy.
 phylogeographic analyses on the western euroasiatic fagus taxa is available, however, the subdivision of fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification of fagus eurasiatic taxa. for the first time molecular analyses of ancient pollen, dated at least years ago, were used in combination with the phylogeny analysis on current species, to identify the fagus spp. present during the last interglacial period in italy. in this work we aim at testing if the trnl-trnf chloroplast dna region, that has been previously proved efficient in discriminating different quercus taxa, can be employed in distinguishing the fagus species and in identifying the ancient pollen.  populations from western euroasistic taxa were sampled, and sequenced for the trnl-trnf region to verify the efficiency of this cpdna region in identifying the fagus spp.. furthermore, fagus crenata, fagus grandifolia, fagus japonica, fagus hayatae, quercus species and castanea species were analysed to better resolve the phylogenetic inference. our results show that this cpdna region harbour some informative sites that allow to infer relationships among the species within the fagaceae family. in particular, few specific and fixed mutations were able to discriminate and identify all the different fagus species. considering a short fragment of base pairs within the trnl intron, transversions were found able in distinguishing the f. orientalis complex taxa from the remaining fagus spp.. this permits to analyse this fragment also in ancient samples, where dna is usually highly degraded. the sequences data indicate that the dna recovered from ancient pollen belongs to the f. orientalis complex since it displays the informative sites characteristic of this complex.  the ancient dna sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the f. orientalis complex was already present during the tyrrhenian period in what is now the venice lagoon. this is a new and important insight considering that nowadays west europe is not the natural area of fagus orientalis complex, and up to now nobody has hypothesized the presence during the last interglacial period of f. orientalis complex in italy. 4– september second congress of italian evolutionary biologists florence, italy 