Allozyme polymorphism of Swiss stone pine Pinus cembra L. in mountainous populations of the Alps and the Eastern Carpathians

Swiss stone pine Pinus cembra L. is a species with fragmented range, occurring in the Alpine-East Carpathian mountain system. Seeds of P. cembra are dispersed by nutcrackers, which offers potential possibilities for gene exchange among populations. Using isozyme analysis, we have examined five samples from two parts of the Swiss stone pine range: the Alps (Switzerland and Austria) and the Carpathians (two samples from the northern macroslope of the Gorgany Ridge, Eastern Carpathians, Ivano-Frankovsk oblast and one sample from Trans-Carpathian oblast of Ukraine). The allele frequencies of 30 isozyme loci, coding for enzymes ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6PGD, PGI, PGM, SDH, SKDH, SOD, were analyzed using cluster analysis and methods of principal components. Two clusters, corresponding to the isolated Alpine and Carpathian parts of the range, were found. The main contribution to these differences were made by loci Adh-1, Adh-2, Fest-2, Lap-3, Mdh-4, and Sod-4. The interpopulation differentiation proved to be somewhat higher than that typical for pines (F(ST) = 7.4%), but within the limits characteristic for taxonomically close species. Thus, isolation of the populations did not lead to their marked differentiation, which may be explained by gene flow and balancing selection, which equalizes gene frequencies across the fragmented species area. Interlocus F(ST) heterogeneity (from 0.003 to 0.173) suggests adaptive significance of some of the allozyme polymorphisms or linkage of some loci with adaptive genes. The Carpathian populations were shown to have higher gene diversity than the Alpine ones (expected heterozygosities 0.095-0.114 and 0.060-0.080, respectively). A deficit of heterozygotes (as compared to the Hardy-Weinberg proportions), observed in the embryo sample, was probably explained by inbreeding. The reduction in the area of Carpathian pine forests in Holocene, caused by the global climatic changes and the anthropogenic impact, is hazardous for the gene pool of the species. The maintenance of genetic uniqueness of both Carpathian populations of P. cembra in general, and individual stands in particular, requires special measures for protection of Swiss stone pine in the Eastern Carpathians.     

Pinus cembra L. (arolla pine), a common tree in the inner French Alps since the early Holocene and above the present tree line: a synthesis based on charcoal data from soils and travertines

Aim In this study, charcoal‐based data for Pinus cembra L. (arolla pine) were gathered from soil and travertine sequences in order to reconstruct its historical biogeography at the landscape level in the inner western Alps during the Holocene. Location The study sites are located between 1700 and 2990 m a.s.l., in the southern (Queyras Massif and Ubaye Valley) and the northern (Maurienne Valley) parts of the inner French Alps. Methods Charcoal fragments were extracted from sediments by water sieving, using meshes of 5, 2, 0.8 and 0.4 mm. The charcoal mass of P. cembra was determined in each charcoal assemblage. Accelerator mass spectrometry and conventional ¹⁴C measurements were used to date the fragments. Results Supported by 40 ¹⁴C datings, the fragments show that, over 2000 m a.s.l., P. cembra accounts for around 40% (mean value) of identified fragments. Data reveal that arolla pine once extended between 260 and 375 m above the present‐day local tree lines. It was established in the southern and the northern French Alps from at least c. 9000 and 6000 cal yr bp , respectively. Main conclusions While present‐day populations of P. cembra are very fragmented in the inner French Alps, charcoal records indicate large past occurrences of this tree since the early Holocene. Human disturbance since the Neolithic seems to be the main reason for the regression of the arolla pine woodlands. On the south‐facing slopes of the study sites, currently deforested, this species extended up to 2800 m a.s.l. In the northern areas, charcoal records of the P. cembra expansion are consistent with the regional pollen archives, but in the southern massifs charcoal records indicate its presence c. 2600 years earlier than other palaeobotanical studies suggest. This discrepancy highlights the necessity to crosscheck data using several different proxies in order to assess the validity of conclusions regarding tree development in space and time.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Chloroplast microsatellites and mitochondrial nad1 intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila)

We studied the phylogenetic relationships among the three stone pine species, Pinus cembra, P. sibirica, and P. pumila, using chloroplast microsatellites and mitochondrial nad1 intron 2 sequences. The three chloroplast microsatellite loci combined into a total of 18 haplotypes. Fourteen haplotypes were detected in 15 populations of P. cembra and one population of P. sibirica, five of which were shared between the two species, and the two populations of P. pumila comprised four species-specific haplotypes. Mitochondrial intron sequences confirmed this grouping of species. Sequences of P. cembra and P. sibirica were identical, but P. pumila differed by several nucleotide substitutions and insertions/deletions. A repeat region found in the former two species showed no intraspecific variation. These results indicate a relatively recent evolutionary separation of P. cembra and P. sibirica, despite their currently disjunct distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help to trace the hybridization in their overlapping distribution area and to identify fossil remains with respect to the still unresolved postglacial re-colonization history of these two species.     

Variation in chloroplast single-sequence repeats in Portuguese maritime pine (Pinus pinaster Ait.)

Genetic variation in 12 Pinus pinaster (maritime pine) populations spanning most of the distribution range of the species in Portugal was evaluated using six polymorphic chloroplast microsatellite (cpSSR) loci. Thirty-two haplotypes were found. There were indications of very weak differentiation among populations (Weir’s θcoefficient, 0.023), and the R _ST value, derived from the stepwise mutation model (SMM), was not significantly different from zero. The pattern, in which similarities in allele size, in base pairs, do not contribute to the genetic structure, may be due to the recent mixing of genetic material from different stands through plantations. Overall, a high level of haplotypic variation within populations was detected. Using the SMM estimator (mean genetic distance of individuals within populations, D ² _sh –––) we divided the populations into two groups, with above and below average values. The first group contained 5 populations, mainly from the central part of the country, which possess, in general, high levels of haplotypic diversity. Among them, 2 populations were divergent from the others based on the pair-wise Nei’s distance. The results indicate that there is no discernible geographic genetic pattern for the Portuguese populations of P. pinaster investigated. The history of expansion of the species range in Portugal during the twentieth century (mainly due to human activity) and extensive gene flow among populations associated with the expansion could explain this finding. Received: 15 February 2000 / Accepted: 14 April 2000     

DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis)

Uniparentally inherited mitochondrial (mt)DNA and chloroplast (cp)DNA microsatellites (cpSSRs) were used to examine population genetic structure and biogeographic patterns of bird-dispersed seed and wind-disseminated pollen of whitebark pine (Pinus albicaulis Engelm.). Sampling was conducted from 41 populations throughout the range of the species. Analyses provide evidence for an ancestral haplotype and two derived mtDNA haplotypes with distinct regional distributions. An abrupt contact zone between mtDNA haplotypes in the Cascade Range suggests postglacial biogeographic movements. Among three cpSSR loci, 42 haplotypes were detected within 28 cpSSR sample populations that were aggregated into six regions. Analysis of molecular variance (amova) was used to determine the hierarchical genetic structure of cpSSRs. amova and population pairwise comparisons (FST ) of cpSSR, and geographical distribution of mtDNA haplotypes provide insights into historical changes in biogeography. The genetic data suggest that whitebark pine has been intimately tied to climatic change and associated glaciation, which has led to range movements facilitated by seed dispersal by Clark's nutcracker (Nucifraga columbiana Wilson). The two hypotheses proposed to explain the genetic structure are: (i) a northward expansion into Canada and the northern Cascades in the early Holocene; and (ii) historical gene flow between Idaho and the Oregon Cascades when more continuous habitat existed in Central Oregon during the late Pleistocene. Genetic structure and insights gained from historical seed movements provide a basis on which to develop recovery plans for a species that is at risk from multiple threats.     

Phylogenetic analysis of the hard pines (Pinus subgenus Pinus,Pinaceae) from chloroplast DNA restriction site analysis

Phylogenetic analysis of plastid DNA restriction site and rearrangement mutations suggests a number of major revisions to taxonomy and phylogenetic concepts in the hard pines. Total genomic DNA from 18 species that sampled all nine subsections was digested with 19 restriction enzymes, blotted, and probed with 70% of the Douglas-fir (Pseudotsuga menziesii) chloroplast genome, or, with clones encompassing the entire chloroplast genome of Pinus contorta. A total of 204 site mutations and five rearrangement mutations were generated, of which 126 were phylogenetically informative. Wagner parsimony analyses revealed 11 clades that were strongly supported by bootstrap and decay index analyses. All North American species except P. resinosa formed a distinct monophyletic group that was strongly separated from the Eurasian species. Within the Eurasian clade subsect. Sylvestres was polyphyletic; its Mediterranean species were closely allied with members of sect. Pinea. Sect. Pinea appeared polyphyletic as well; both species of its subsect. Leiophyllae showed a close affinity to Mesoamerican pines of subsect. Oocarpae in sect. Pinus. Within the North American pines subsects. Ponderosae and Oocarpae were polyphyletic. Despite its shallow fossil record, subsect. Contortae emerged as a sister group to all of the North American pines apart from P. resinosa, which was allied with Eurasian species of subsect. Sylvestres. The remaining North American subsections formed two groups: a poorly resolved clade with subsects. Ponderosae and Sabinianae, and sequentially nested clades represented by: P. radiata; P. taeda; representatives of subsects. Oocarpae and Ponderosae from Mesoamerica, and subsect. Leiophyllae. We present estimates of divergence times for each of these major clades based on molecular clocks calibrated using two hard pine fossil observations.     

Evaluation of the non-climatic (age-related) trends of stable oxygen and carbon isotopes in Swiss stone pine (Pinus cembra L.) tree rings from the Eastern Carpathians,Romania

The presence, magnitude, and duration of age and/or size-related trends in the stable isotopes in tree-ring cellulose time series has been a subject of scientific debate. Where present, their evaluation and removal are key to the development of robust climate calibrations and reconstructions, especially in the low-frequency domain and where sample replication levels are low. Where reported, results suggest that the age/size/height-related trend of stable oxygen (δ¹⁸O) and carbon (δ¹³C) isotope compositions in tree-ring cellulose may vary according to the tree species and the individual tree location. For this reason, it is important when developing long palaeoclimate reconstructions for a new species, geographical region or ecological setting to perform studies to investigate non-climatic (age-related) trends. This study evaluates the ontogenetic pattern in the δ¹⁸O and δ¹³C data in Swiss stone pine trees from the Eastern Carpathians, Romania. For this, we used a new multi-centennial dataset consisting of 5 living and 10 relict Swiss stone pine samples collected from the Eastern Carpathians, which were annually resolved for δ¹⁸O and δ¹³C composition. Age-related trends were evaluated using the conventional “slope of the mean” approach as well as the “mean of the slope” method. The results suggest that neither the traditional “slope-of-the mean” nor the “mean-of-the-slope” approach indicates a persistent linear trend in the Swiss stone pine δ¹⁸O data, while for the δ¹³C data a systematic enrichment in ¹³C was observed over a < 40 year-long period after germination. Despite the limited sample size of this developing dataset these findings help to inform more detailed analyses and future sampling strategies in the Eastern Carpathian Swiss stone pine stands.     

Comparison of genetic diversity estimates within and among populations of maritime pine using chloroplast simple-sequence repeat and amplified fragment length polymorphism data

We compared the genetic variation of Pinus pinaster populations using amplified fragment length polymorphism (AFLP) and chloroplast simple-sequence repeat (cpSSR) loci. Populations' levels of diversity within groups were found to be similar with AFLPs, but not with cpSSRs. The high interlocus variance associated with the AFLP loci could account for the lack of differences in the former. Although AFLPs revealed much lower genetic diversity than cpSSRs, the levels of among-population differentiation found with the two types of marker were similar, provided that loci showing fewer than four null-homozygotes, in any population, were pruned from the AFLP data. Moreover, the French and Portuguese populations were clearly differentiated from each other, with both markers. The Mantel test showed that the genetic distance matrix calculated using the AFLP data was correlated with the matrix derived from the cpSSRs. Because of the concordance found between markers we conclude that gene flow was indeed the predominant force shaping nuclear and chloroplastic genetic variation of the populations within regions, at the geographical scale studied.     

Genetic structure of Hypochaeris uniflora (Asteraceae) suggests vicariance in the Carpathians and rapid post-glacial colonization of the Alps from an eastern Alpine refugium

Aim The range of the subalpine species Hypochaeris uniflora covers the Alps, Carpathians and Sudetes Mountains. Whilst the genetic structure and post‐glacial history of many high‐mountain plant taxa of the Alps is relatively well documented, the Carpathian populations have often been neglected in phylogeographical studies. The aim of the present study is to compare the genetic variation of the species in two major European mountain systems – the Alps and the Carpathians. Location Alps and Carpathians. Methods The genetic variation of 77 populations, each consisting of three plants, was studied using amplified fragment length polymorphism (AFLP). Results Neighbour joining and principal coordinate analyses revealed three well‐supported phylogeographical groups of populations corresponding to three disjunct geographical regions – the Alps and the western and south‐eastern Carpathians. Moreover, two further clusters could be distinguished within the latter mountain range, one consisting of populations from the eastern Carpathians and the second consisting of populations from the southern Carpathians. Populations from the Apuseni Mountains had an intermediate position between the eastern and southern Carpathians. The genetic clustering of populations into four groups was also supported by an analysis of molecular variance, which showed that most genetic variation (almost 46%) was found among these four groups. By far the highest within‐population variation was found in the eastern Carpathians, followed by populations from the southern and western Carpathians. Generally, the populations from the Alps were considerably less variable and displayed substantially fewer region‐diagnostic markers than those from the south‐eastern Carpathians. Although no clear geographical structure was found within the Alps, based on neighbour joining or principal coordinate analyses, some trends were obvious: populations from the easternmost part were genetically more variable and, together with those from the south‐western part, exhibited a higher proportion of rare AFLP fragments than populations in other areas. Moreover, the total number of AFLP fragments per population, the percentage of polymorphic loci and the proportion of rare AFLP fragments significantly decreased from east to west. Main conclusions Deep infraspecific phylogeographical gaps between the populations from the Alps and the western and south‐eastern Carpathians suggest the survival of H. uniflora in three separate refugia during the last glaciation. Our AFLP data provide molecular evidence for a long‐term geographical disjunction between the eastern and western Carpathians, previously suggested from the floristic composition at the end of 19th century. It is likely that Alpine populations survived the Last Glacial in the eastern part of the Alps, from where they rapidly colonized the rest of the Alps after the ice sheet retreated. Multiple founder effects may explain a gradual loss of genetic variation during westward colonization of the Alps.     

Gradual decline in genetic diversity in Swiss stone pine populations (Pinus cembra) across Switzerland suggests postglacial re-colonization into the Alps from a common eastern glacial refugium

Molecular genetic markers may reveal informative patterns of population processes such as historical migration, which may substantiate inference on postglacial re-colonization inferred, e.g., from fossil records. Palynological records of Swiss stone pine (Pinus cembra) suggest that the species has re-colonized the central Alps from a southeastern Alpine refugium after the last glacial maximum. Such a migration pathway likely resulted in a gradual decrease in genetic diversity with increasing distance to the glacial refugium, owing to founder events at the leading range edge. The present distribution of P. cembra in Switzerland consists of two rather distinct ranges, namely the inner-alpine parts of the Grisons and Valais, respectively, and additional disjunct occurrences in the northern and southern periphery of the Alps as well as between the two main ranges. We screened chloroplast microsatellite loci on 39 Swiss P. cembra populations and show that the genetic structure detected was congruent with a common ancestry from a single glacial refugium, likely located at the (south-)eastern periphery of the Alps. In contrast, our data rejected the alternative hypothesis of a distinct genetic separation of the two main ranges of Swiss stone pine in Switzerland. We further show that low genetic diversity within and high differentiation among peripheral populations in the northern Alps as well as the genetic differentiation between core and peripheral populations reflect genetic drift as a consequence of colonization history and limited gene flow by pollen and seed.

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Zusammenfassung  Molekulargenetische Marker enthalten wertvolle Information über Populationsprozesse wie historische Wanderungen, wodurch Annahmen zur postglazialen Wiederbesiedelung, beispielsweise abgeleitet von Fossilfunden, unterstützt werden k?nnen. Palynologische Funde von Arve (Pinus cembra) lassen vermuten, dass diese Art nach dem letzten glazialen Maximum von einem Refugium in den süd?stlichen Alpen wieder in die zentralen Alpen eingewandert ist. Ein solcher Rückwanderungsweg dürfte aufgrund von Gründereffekten an der Wanderungsfront eine graduelle Verringerung der genetischen Variation mit zunehmender Distanz zum Glazialrefugium bewirkt haben. Die heutige Verbreitung von P. cembra in der Schweiz weist zwei deutlich getrennte Gebiete auf, n?mlich inneralpine Bereiche der Kantone Graubünden und Wallis, mit jeweils isolierten Vorkommen in den n?rdlichen und südlichen Randalpen. Wir untersuchten Chloroplasten-Mikrosatelliten in 39 Schweizer Populationen von P. cembra und zeigen, dass die gefundene genetische Struktur übereinstimmt mit der Annahme einer gemeinsamen Abstammung aus einem einzigen Glazialrefugium, welches vermutlich am (süd-)?stlichen Rand der Alpen lag. Im Gegensatz dazu widerlegen unsere Daten die alternative Hypothese einer deutlichen genetischen Trennung der zwei Hauptvorkommen der Arve in der Schweiz. Im Weiteren zeigen unsere Resultate eine geringe genetische Variation innerhalb und einen hohen Differenzierungsgrad zwischen n?rdlichen Randalpenvorkommen, sowie eine genetische Differenzierung zwischen zentralen und peripheren Populationen. Dies weist auf genetische Drift hin, welche die Besiedlungsgeschichte und einen beschr?nkten Genfluss durch Pollen und Samen widerspiegelt.

     

Inferring long-distance dispersal and topographic barriers during post-glacial colonization from the genetic structure of red maple (Acer rubrum L.) in New England

Aim  This study aims to assess the role of long-distance seed dispersal and topographic barriers in the post-glacial colonization of red maple ( Acer rubrum L.) using chloroplast DNA (cpDNA) variation, and to understand whether this explains the relatively higher northern diversity found in eastern North American tree species compared with that in Europe.
Location  North-eastern United States.
Methods  The distribution of intraspecific cpDNA variation in temperate tree populations has been used to identify aspects of post-glacial population spread, including topographic barriers to population expansion and spread by long-distance seed dispersal. We sequenced c.  370 cpDNA base pairs from 221 individuals in 100 populations throughout the north-eastern United States, and analysed spatial patterns of diversity and differentiation.
Results  Red maple has high genetic diversity near its northern range limit, but this diversity is not partitioned by topographic barriers, suggesting that the northern Appalachian Mountains were not a barrier to the colonization of red maple. We also found no evidence of the patchy genetic structure that has been associated with spread by rare long-distance seed dispersal in previous studies.
Main conclusions  Constraints on post-glacial colonization in eastern North America seem to have been less stringent than those in northern Europe, where bottlenecks arising from long-distance colonization and topographic barriers appear to have strongly reduced genetic diversity. In eastern North America, high northern genetic diversity may have been maintained by a combination of frequent long-distance dispersal, minor topographic obstacles and diffuse northern refugia near the ice sheet.     

No reduction in genetic diversity of Swiss stone pine (Pinus cembra L.) in Tatra Mountains despite high fragmentation and small population size

In Europe, most of the alpine timberline ecotone has been altered by human activities and climate change. Hence, mountain forests are of the highest conservation interest. Here, we screened 25 populations of Swiss stone pine (Pinus cembra L.) from the Carpathians and the Alps, using a set of ten microsatellite primers to assess the relative conservation value of populations sampled in Polish and Slovak Tatra National Parks, where potential extinction risk is the highest within the Carpathian range. Although endangered, with small and fragmented populations, P. cembra in the Tatra Mts. shows high levels of allelic richness (AR = 5.0) and observed heterozygosity (H _o  = 0.554). Our results suggest that anthropogenic habitat fragmentation has had little impact on DNA variation of Swiss stone pine in the Tatra Mts. However, the effects of changing conditions on the genetic structure may occur with a substantial time delay due to the long life span of P. cembra. Moreover, inbreeding depression may occur in the next generations, since we found inbreeding (F _IS  = 0.063) and elevated coancestry coefficient (θ = 0.062) in all populations. Also a shallow pattern of genetic differentiation between populations was found, indicating recent fragmentation of a common gene pool that formerly occupied a larger range. Therefore, the Tatra Mts. can be considered as a single conservation unit. Based on our results, we suggest possible conservation activities for Swiss stone pine both in Poland and Slovakia.     

The distribution of genetic variation in Norway spruce (Picea abies Karst.) populations in the western Alps

Aim  In order to look for a possible centre of survival for the Norway spruce ( Picea abies Karst.) in the south-western Alps, six natural populations of this area were investigated by means of genetic markers in order to assess the degree and the distribution of genetic diversity within the species.
Location  Western and South-western Alps.
Methods  Populations were genotyped using seven simple sequence repeat (SSR) markers. Basic population genetics parameters were estimated and the amount of genetic differentiation calculated.
Results  A large amount of variability was found (0.59 <  H _e < 0.67); genetic differentiation as measured by F _ST was 0.05, close to other similar studies; no isolation by distance was detected by a Mantel test. Analysis of molecular variance confirmed a high degree of variability within populations and a low degree of variability among populations. Finally, the number of populations from which those observed could have arisen was estimated by Bayesian analysis.
Main conclusions  The results presented here suggest that the present populations derive their genetic make-up from three inferred clusters. The possible existence in this area of a relict/refuge population during the last glaciation is discussed.     

Genetic diversity within and among populations of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf pine (n = 93) and loblolly pine (n = 112) trees representing 22 seed sources or 16 physiographic populations were sampled from Southwide Southern Pine Seed Source Study plantings located in Oklahoma, Arkansas, and Mississippi. The sampled trees were grown from shortleaf pine and loblolly pine seeds formed in 1951 and 1952, prior to the start of intensive forest management across their native ranges. Amplification fragment length polymorphism (AFLP) markers were developed and used to study genetic diversity and its structure in these pine species. After screening 48 primer pairs, 17 and 21 pairs were selected that produced 794 and 647 AFLPs in shortleaf pine and loblolly pine, respectively. High-AFLP-based genetic diversity exists within shortleaf pine and loblolly pine, and most (84.73% in shortleaf pine; 87.69% in loblolly pine) of this diversity is maintained within physiographic populations. The high value of unbiased measures of genetic identity and low value of genetic distance for all pairwise comparisons indicates that the populations have similar genetic structures. For shortleaf pine, there was no significant correlation between geographic distance and genetic distance (r = 0.28), while for loblolly pine there was a weak but significant correlation (r = 0.51).     

Geography determines genetic relationships between species of mountain pine (Pinus mugo complex) in western Europe

Aim Our aims were to test whether morphological species of mountain pines were genetically supported in the western part of the distribution range of the Pinus mugo species complex (Pinus mugo Turra sensu lato), to resolve genetically homogeneous clusters of populations, to determine historical demographic processes, and to assess the potential hybridization of mountain pines with Scots pine, Pinus sylvestris L. Location Populations were sampled in the Iberian System, the Pyrenees, the French Mont Ventoux, Vosges and Jura mountains, the German Black Forest and throughout the Alps. This corresponded to a range‐wide sampling for mountain pine sensu stricto (Pinus uncinata Ram.) and to a sampling of the western parts of the ranges of dwarf mountain pine (Pinus mugo Turra sensu stricto) and bog pine/peatbog pine [Pinus rotundata Link/Pinus × pseudopumilio (Willk.) Beck]. Methods In total, 786 individuals of P. mugo sensu lato from 29 natural populations, and 85 individuals of P. sylvestris from four natural populations were genotyped at three chloroplast microsatellites (cpSSRs). Populations were characterized for standard genetic diversity statistics and signs of demographic expansion. Genetic structure was explored using analysis of molecular variance, differentiation statistics and Bayesian analysis of population structure (BAPS). Results One hundred haplotypes were identified in P. mugo sensu lato. There was a stronger differentiation between geographical regions than between morphologically identified taxa (P. mugo sensu stricto, P. uncinata and P. rotundata/P. ×pseudopumilio). Overall genetic differentiation was weak (G_ST = 0.070) and displayed a clear phylogeographic structure [N_ST = 0.263, N_ST > N_ST (permuted), P < 0.001]. BAPS identified a Pyrenean and an Alpine gene pool, along with several smaller genetic clusters corresponding to peripheral populations. Main conclusions The core regions of the Pyrenees and Alps were probably recolonized, respectively by P. uncinata and P. uncinata/P. mugo sensu stricto, from multiple glacial refugia that were well connected by pollen flow within the mountain chains. Pinus rotundata/P. × pseudopumilio populations from the Black Forest, Vosges and Jura mountains were probably recolonized from various glacial populations that kept their genetic distinctiveness despite late glacial and early Holocene expansion. Marginal P. uncinata populations from the Iberian System are compatible with elevational shifts and long‐term isolation. The causes of haplotype sharing between P. mugo sensu lato and P. sylvestris require further research.     

A little luxury doesn’t hurt: Swiss stone pine (Pinus cembra L.) - an unexpected item in the diet of central European Mesolithic hunter-gatherers

Our knowledge of the plant diet of the last European hunter-gatherers is hindered by the difficulties of recording poorly durable plant tissues. One exception is the hazelnut fruit, which preserves well at dry archaeological sites, although usually only in a charred state. Here we give the first evidence for the prehistoric (Mesolithic) use of seeds of the Swiss stone pine, specifically for the time period 8239???7943 cal bc, when this edible seed-bearing tree was an important element of local boreal forests in northern Bohemia, Czech Republic. This local population of Swiss stone pine became extinct at the end of the Early Holocene without leaving modern offspring in the lowlands and middle elevations of central Europe.

     

Distribution of genetic variability in southern populations of Scots pine (Pinus sylvestris L.) from the Alps to the Apennines

Alpine and Northern Apennine populations of Pinus sylvestris collected from eight different Italian sites were analyzed by mitochondrial nadI intron and InterSimple Sequence Repeat (ISSR) markers, in order to describe the natural level of genetic variability and to clarify their genetic relationships. The small Northern Apennine populations are the southernmost populations of this conifer in Italy. All the analyzed populations were spontaneous and reforested areas were excluded. The analysis of the polymorphisms in the nad 1 intron sequence confirmed that the Italian P. sylvestris populations have the same mitotype (mitotype a) as the Central European ones. In the genomic ISSR analysis the proportion of shared alleles between the individuals showed the highest degree of differentiation between French and Italian populations and a divergence between the Alpine and Apennine populations. Alpine populations showed a higher genetic variability (GD 0.310±0.0252) than Apennine samples (GD 0.217±0.019). In addition, the individuals from the Apennines did not show a clear population structure, suggesting a common genetic constitution of Apennine P. sylvestris. It is likely that this constitution is the result of a progressive genetic isolation between the Alpine and the Northern Apennine populations from the early Holocene. The genetic constitution of the Northern Apennine populations suggests the opportunity of a management where in situ conservation of such small populations could be coupled to their use as sources of suitable local reforesting materials.     

Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

Aim The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post‐glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. Methods As our model system we used Pulsatilla vernalis, a widely distributed European herbaceous plant occurring both in the high‐mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution‐wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment‐length polymorphisms (PCR–RFLPs) (trnH–trnK, trnK–trnK, trnC–trnD, psbC–trnS, psaA–trnS, trnL–trnF) and further sequencing of trnL–trnF and trnH–psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus‐scale cpDNA variation. Results Eleven PCR–RFLP polymorphisms were detected in P. vernalis, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section Pulsatilla was low, but P. vernalis did not share haplotypes with other species. Main conclusions The genetic distinctiveness of P. vernalis populations from the south‐western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high‐mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post‐glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post‐glacial colonization of these two areas, with possible founder effects.     

Morphometric and chemical fruit variability of selected stone pine trees (Pinus pinea L.) grown in non-native environments

Few studies have been conducted on Stone pine (Pinus pinea) morphology, cone to kernel yield (KY) and composition. To study the species variability, a morphometric and chemical characterization of pine nuts was performed across the species distribution in Chile. Seventy-six highly productive trees were selected in three macrozones. Ten cones per tree were harvested, and cone weight, in-shell pine nut number cone^?1 and size and weight of in-shell pine nuts and kernels were measured; KY was determined. Chemical and nutritional characterization of pine nuts was performed in 38 trees. Differences among macrozones were tested with mixed linear models. Partial correlation was used to test for correlation between cone and pine nut morphometric data, chemical features, and climate. Canonical correlation coefficients between groups were tested. Selected trees differed in cone and in-shell pine nut size, kernel morphometry, and KY, with lowest trait values being generally found in the coast. KY was higher than in Europe. Chemical composition was similar to values reported in producer countries, homogeneous among macrozones, with the exception of oleic acid and potassium. Detected differences would respond to the environment and to the interaction genotype?×?environment given the restricted origins of the genetic material, confirming the species adaptability.