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.

     

Allozyme Polymorphism of Swiss Stone Pine <Emphasis Type="Italic">Pinus cembra</Emphasis> L. in Mountain 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-Frankivs'ka oblast and one sample from Zakarpats'ka oblast of Ukraine). The allele frequencies of 30 isozyme loci, coding for enzymes ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SDH, SKDH, SOD, were analyzed using cluster analysis and Principal Component Analysis. 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 deficiency 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.     

Disentangling the effects of geographic peripherality and habitat suitability on neutral and adaptive genetic variation in Swiss stone pine

It is generally accepted that the spatial distribution of neutral genetic diversity within a species’ native range mostly depends on effective population size, demographic history, and geographic position. However, it is unclear how genetic diversity at adaptive loci correlates with geographic peripherality or with habitat suitability within the ecological niche. Using exome‐wide genomic data and distribution maps of the Alpine range, we first tested whether geographic peripherality correlates with four measures of population genetic diversity at > 17,000 SNP loci in 24 Alpine populations (480 individuals) of Swiss stone pine (Pinus cembra) from Switzerland. To distinguish between neutral and adaptive SNP sets, we used four approaches (two gene diversity estimates, F_ST outlier test, and environmental association analysis) that search for signatures of selection. Second, we established ecological niche models for P. cembra in the study range and investigated how habitat suitability correlates with genetic diversity at neutral and adaptive loci. All estimates of neutral genetic diversity decreased with geographic peripherality, but were uncorrelated with habitat suitability. However, heterozygosity (H_e) at adaptive loci based on Tajima's D declined significantly with increasingly suitable conditions. No other diversity estimates at adaptive loci were correlated with habitat suitability. Our findings suggest that populations at the edge of a species' geographic distribution harbour limited neutral genetic diversity due to demographic properties. Moreover, we argue that populations from suitable habitats went through strong selection processes, are thus well adapted to local conditions, and therefore exhibit reduced genetic diversity at adaptive loci compared to populations at niche margins.     

Variation in the chloroplast DNA of Swiss stone pine (Pinus cembra L.) reflects contrasting post-glacial history of populations from the Carpathians and the Alps

Aim To characterize the genetic structure and diversity of Pinus cembra L. populations native to two disjunct geographical areas, the Alps and the Carpathians, and to evaluate the rate of genetic differentiation among populations. Location The Swiss Alps and the Carpathians. Methods We screened 28 populations at three paternally inherited chloroplast simple sequence repeats (cpSSRs) for length variation in their mononucleotide repeats. Statistical analysis assessed haplotypic variation and fixation indices. Hierarchical analysis of molecular variance (AMOVA), Mantel test, spatial analysis of molecular variance (SAMOVA) and barrier analyses were applied to evaluate the geographical partitioning of genetic diversity across the species’ range. Results Haplotypic diversity was generally high throughout the natural range of P. cembra, with the mean value substantially higher in the Carpathians (H = 0.53) than in the Alps (H = 0.35). The isolated Carpathian populations showed the highest haplotype diversity among the populations originating from the High Tatras (Velka Studena Dolina) and South Carpathians (Retezat Mountains). AMOVA revealed that only 3% of the total genetic variation derived from genetic differentiation between the two mountain ranges. Differentiation among Carpathian populations was higher (F_ST = 0.19) than among Alpine populations (F_ST = 0.04). Low, but significant, correlation was found between the geographical and genetic distances among pairs of populations (r = 0.286, P < 0.001). SAMOVA results revealed no evident geographical structure of populations. barrier analysis showed the strongest differentiation in the eastern part of the species’ range, i.e. in the Carpathians. Main conclusions The populations of P. cembra within the two parts of the species’ range still share many cpDNA haplotypes, suggesting a common gene pool conserved from a previously large, continuous distribution range. Carpathian populations have maintained high haplotypic variation, even higher than Alpine populations, despite their small population sizes and spatial isolation. Based on our results, we emphasize the importance of the Carpathian populations of Swiss stone pine for conservation. These populations comprise private haplotypes and they may represent a particular legacy of the species’ evolutionary history.     

Speciation history of three closely related pines Pinus mugo (T.), P. uliginosa (N.) and P. sylvestris (L.)

Nucleotide polymorphisms at genomic regions including 17 nuclear loci, two chloroplast and one mitochondrial DNA fragments were used to study the speciation history of three pine species: dwarf mountain pine (Pinus mugo), peat‐bog pine (P. uliginosa) and Scots pine (P. sylvestris). We set out to investigate three specific speciation scenarios: (I) P. uliginosa is a homoploid hybrid between the other two, (II) the species have evolved without gene flow after divergence and (III) there has been substantial gene flow between the species since their divergence. Overall, the genetic data suggest that P. mugo and P. uliginosa share the same gene pool (average net divergence of 0.0001) and that the phenotypic differences (e.g. growth form) are most likely due to very limited areas of the genome. P. mugo and P. uliginosa are more diverged from P. sylvestris than from each other (average net divergence of 0.0027 and 0.0026, respectively). The nucleotide patterns can best be explained by the divergence with migration speciation scenario, although the hybrid speciation scenario with small genomic contribution from P. sylvestris cannot be completely ruled out. We suggest that the large amount of shared polymorphisms between the pine taxa and the lack of monophyly at all loci studied between P. sylvestris and P. mugo–P. uliginosa can largely be explained by relatively recent speciation history and large effective population sizes but also by interspecific gene flow. These closely related pine taxa form an excellent system for searching for loci involved in adaptive variation as they are differentiated in phenotype and ecology but have very similar genetic background.     

Population structure,gene diversity,and differentiation in natural populations of Cedar pines (Pinus subsect.Cembrae,Pinaceae) in the USSR

Twelve natural populations of four cedar pine species,Pinus sibirica, P. cembra, P. pumila, andP. koraiensis, occurring in the Soviet Union were investigated by starch-gel electrophoresis. Frequencies of 55 alleles at 19 loci were determined. Interpopulation genetic diversity inP. sibirica andP. pumila was only 2–4 per cent of the total genetic diversity. Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation among conspecific populations and among species. Dn values among populations ranged from 0.006 to 0.038. A dendrogram constructed using Dn values divided cedar pines species into 2 clusters:sibirica-cembra (Dn = 0.030) andpumila-koraiensis (Dn = 0.143). Nei's distance between these clusters was 0.232. On the basis of the data obtained it was possible to draw the following conclusion:P. sibirica, P. pumila, andP. koraiensis are distinct species, whileP. cembra should apparently be regarded as geographicalP. sibirica race.     

Influence of host log age and refuge from natural enemies on colonization and survival of Phoracantha semipunctata

The pine cone weevil, Pissodes validirostris Gyll. (Coleoptera: Curculionidae), attacks seed cones of most Eurasian pine species, except these of Swiss stone pine (Pinus cembra L.). Behavioural responses of adult weevils to cone volatile emissions of Swiss stone pine and to those of a common host, mountain pine (Pinus uncinata Ram.), were compared in an olfactometer. Weevils were significantly attracted by the volatile blend emitted by mountain pine, but Swiss stone pine volatiles elicited an inverse response, with most weevils moving in the opposite direction to the odour source. However, the majority of second instar weevil larvae that were extracted from mountain pine cones and transferred into Swiss stone pine cones were capable of developing to the adult stage. This suggests that Swiss stone pine cones do not contain strong feeding deterrents that could prevent larval development. The possible factors involved in the absence of colonization of Swiss stone pine cones by cone weevils are discussed.     

Comparative mapping in loblolly and radiata pine using RFLP and microsatellite markers

Genetic linkage maps were constructed for loblolly pine (Pinus taeda L.) and radiata pine (P. radiata D. Don) using a common set of RFLP and microsatellite markers. The map for loblolly pine combined data from two full-sib families and consisted of 20 linkage groups covering 1281 cM. The map for radiata pine had 14 linkage groups and covered 1223 cM. All of the RFLP probes readily hybridise between loblolly and radiata pine often producing similar hybridisation patterns. There were in total 60 homologous RFLP loci mapped in both species which could be used for comparative purposes. A set of 20 microsatellite markers derived from radiata pine were also assayed; however, only 9 amplified and revealed polymorphic loci in both species. Single-locus RFLP and microsatellite markers were used to match up linkage groups and compare order between species. Twelve syntenic groups were obtained each consisting of from 3 to 9 homologous loci. The order of homologous loci was colinear in most cases, suggesting no major chromosomal rearrangements in the evolution of these species. Comparative mapping between loblolly and radiata pine should facilitate genetic research in both species and provide a framework for mapping in other pine species. Received: 25 November 1998 / /Accepted: 19 December 1998     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

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.     

Genetic structure in Pinus cembra from the Carpathian Mountains inferred from nuclear and chloroplast microsatellites confirms post-glacial range contraction and identifies introduced individuals

Genetic differentiation of scattered populations at neutral loci is characterized by genetic drift counteracted by the remaining gene flow. Populations of Pinus cembra in the Carpathian Mountains are isolated and restricted to island-like stands at high-elevation mountain ranges. In contrast, paleobotanical data suggest an extended early Holocene distribution of P. cembra in the Carpathians and its surrounding areas, which has contracted to the currently disjunct occurrences. We analyzed the genetic variation of 11 Carpathian populations of P. cembra at chloroplast and, in part newly developed, nuclear microsatellites. Both marker types revealed low levels of genetic differentiation and a lack of isolation by distance, reflecting the post-glacial retraction of the species to its current distribution. Stronger effects of genetic drift were implied by the higher genetic differentiation found for haploid chloroplast than for diploid nuclear markers. Moreover, we found no association between the values of population genetic differentiation for the two marker types. Several populations indicated recent genetic bottlenecks and inbreeding as a consequence of decline in population sizes. Moreover, we found individuals in two populations from the Rodnei Mountains that strikingly differed in assignment probabilities from the remaining specimens, suggesting that they had been introduced from a provenance outside the studied populations. Comparison with Eastern Alpine P. cembra and individuals of the closely related Pinus sibirica suggests that these individuals presumably are P. sibirica. Our study highlights the importance of the maintenance of sufficiently large local population sizes for conservation due to low connectivity between local occurrences.     

Responses to recent climatewarming of Pinus sylvestris and Pinus cembra within their montane transition zone in the Swiss Alps

Abstract. Altitudinal and latitudinal distribution limits of trees are mainly controlled by temperature. Therefore climate warming is expected to induce upslope or poleward migrations. In the Swiss Central Alps, summers in the period 1982-1991 were on average 0.8 °C warmer than those of the period 30 yr before. We investigated whether populations of conifers at the montane Pinus sylvestris-Pinus cembra ecocline exhibit demographic trends in response to that warming. We found no evidence for this. Young seedlings of Pinus sylvestris, the species which is expected to expand its range upward in a warmer climate, were virtually absent from all sites, whereas large fractions of Pinus cembra populations were observed in the seedling and juvenile categories even below the present lower distribution limit of adult trees. This suggests that there are no major altitudinal shifts in response to the recent sequence of warmer summers. Germination and seedling survival trials with Pinus sylvestris suggest that temperature per se would not exclude this species even from establishing at the current treeline in the Swiss Central Alps. Similar results were found at the polar treeline. Phytotron tests of seedling survival showed much less drought resistance in Pinus sylvestris than in Pinus cembra which is in contrast to their phytogeographic distributions. Thus, the montane pine ecocline in the Swiss Central Alps seems to be stabilized by species interactions and may not be directly responsive to moderate climatic change, which needs to be taken into account in predictive attempts.     

Microsatellite DNA markers for the snow vole (Chionomys nivalis)

A total of 14 dinucleotide microsatellite loci were characterized in the snow vole (Chionomys nivalis). Allelic polymorphism across all loci and 28 individuals representing a single population in the Swiss Alps was high (mean = 10.1 alleles). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These loci will be useful for describing mating systems and population structure and to investigate the genetic consequences of a species living in a highly fragmented habitat.     

Development and characterization of nine polymorphic microsatellite markers in the sand goby Pomatoschistus minutus (Gobiidae)

A microsatellite‐enriched genomic library was constructed for the sand goby, Pomatoschistus minutus (Pallas 1770), and nine polymorphic DNA microsatellite markers of high quality were successfully optimized. Characterization of 96 individuals from the Vaccarès lagoon (France) showed moderate to high levels of polymorphism (two to 54 alleles). All the markers conformed to Hardy–Weinberg equilibrium and showed no evidence of null alleles, large allele dropout, stuttering and linkage disequilibrium between pairs of loci. These markers successfully amplify in three closely related species and can be employed to investigate population genetic structure and to clarify paternity in Pomatoschistus species.     

Characterization of five microsatellite loci in the pine shoot beetle Tomicus piniperda (Coleoptera: Scolytidae)

Tomicus piniperda is a bark beetle that causes damage to various pine species across a wide geographical range. We developed five microsatellite polymorphic markers using an enrichment protocol. All loci could be successfully amplified with no evidence of null alleles and will be useful for population genetic studies. Cross‐species amplifications show that at least some of the markers could be useful in four other Tomicus species.     

Comparative genome and QTL mapping between maritime and loblolly pines

Genetic markers developed from expressed sequence tags (ESTs) were used as orthologous loci for comparative genome studies in the genus Pinus. A total of 309 ESTs derived from conifer gene sequences were tested for amplification and polymorphism in maritime pine (Pinus pinaster Ait.). Electrophoresis-based techniques made it possible to map 50 expressed sequence tag polymorphisms (ESTPs). The map positions of 32 markers were compared to putative orthologous loci on the loblolly pine (Pinus taeda L.) linkage map, which is the reference map of the conifer genetic mapping community. Overall, synteny was maintained between the two species. This report agrees with other pairwise genome comparisons in pine and supports the cytogenetic evidence that chromosome evolution in the genus is conservative. The alignment of homologous linkage groups allowed, for the first time in conifers, the comparison of QTL location. The position of two QTLs controlling wood density and cell wall components were found to be conserved between the two species.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Development and characterization of microsatellite markers for Psychotria tenuinervis (Rubiaceae), a shrub species from the Atlantic forest, and primers transferability from Coffea

The shrub species Psychotria tenuinervis (Rubiaceae) is native to the Brazilian Atlantic forest and is largely found within natural and disturbed forest fragments. Aiming to develop studies on population genetic structure of forest fragment species, eigth microsatellite markers were developed for P. tenuinervis. Also, 15 loci already developed for Coffea (Rubiaceae) were tested for transferability to this species. We utilized 45 individuals from natural populations of three different fragments-anthropic edge, interior fragment and natural edge, within the Brazilian Atlantic forest. The average number of alleles per locus was 2.5 (two–four alleles/locus). These loci will be useful for future population genetic studies aiming to the conservation and management of this species.     

马尾松和黄山松两个核基因位点的群体遗传多样性和种间分化

利用两个核基因座位C3H和GI, 对重叠分布于中国东南部的两个松属(Pinus)物种马尾松(P. massoniana)和黄山松(P. hwangshanensis)的22个群体88个个体进行了遗传多样性和种间分化模式研究。在这两个核基因座位上, 两种植物都表现出较低的核苷酸多样性水平(马尾松π_sil = 0.001 71; 黄山松π_sil = 0.003 40), 但是马尾松要显著低于黄山松; 在种内分化水平上, 马尾松的种内遗传分化也明显低于黄山松(马尾松F_ST = 0.059; 黄山松F_ST = 0.339)。这可能是由于黄山松的海拔分布高于马尾松, 而高海拔分布使黄山松的分布区域更加片段化, 促使其形成较高的种内遗传多样性和遗传分化。分子变异分析(AMOVA)发现, 两物种基于两个核基因座位的种间差异为48.86%, 而GI基因座位上的种间差异明显高于C3H座位(GI: 77.24%, C3H: 20.48%), 同时, 基因谱系显示两物种的共享单倍型仅在C3H座位上存在。结合这两个基因的功能, 推测GI基因可能在物种形成过程中受到了一定的选择压力, 因为GI基因参与调控植物的开花时间, 而C3H与木质素表达水平的调控有关。不同的选择压力使得GI的进化速度相对较快, 从而加速了黄山松和马尾松的物种分化。