Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta)

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine (Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion (”big-bang”) followed by recent, local population differentiation (”galaxy formation”). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10^–3. Received: 7 December 2000 / Accepted: 17 May 2001     

Construction of a framework map in Pinus contorta subsp. latifolia using random amplified polymorphic DNA markers.

We report on the construction of the first random amplified polymorphic DNA (RAPD) framework map in Pinus contorta subsp. latifolia. Genomic DNA of haploid megagametophytes from 90 open-pollinated seeds originating from a single tree were amplified with 840 random decamer oligonucleotide primers by the polymerase chain reaction. Three-hundred twenty-eight RAPD markers with fragment sizes that ranged between 260 and 3080 base pairs were found segregating at 110 random decamer oligonucleotide primers. Of these 328 RAPD markers, 148 were mapped to 16 framework linkage groups and 77 were mapped as accessory markers onto the framework linkage groups, on a support interval of minimal LOD score of 3. The 16 framework maps cover a distance of 2287 cM. The estimate of genome size was 2407 cM with a 95% confidence interval of 2304-2459 cM.     

Genetic diversity among cultivars of spring barley revealed by random amplified polymorphic DNA (RAPD)

RAPD (random amplified polymorphic DNA) polymorphism was studied in 23 malting and non-malting spring barley cultivars included in the official list of Polish cultivated varieties. Twenty-four 10-mer primers were tested in each cultivar, giving altogether 149 amplification products, 45% of which were polymorphic. The number of polymorphic bands revealed by one primer ranged from 1 to 6, with an average of 2.8. Genetic distance for all pairs of compared varieties was estimated and a dendrogram was constructed using unweighted pair group method of arithmetic means. The genetic distance between cultivars ranged from 0.11 for cvs. Apex and Bryl to 0.62 for cvs. Orthega and Madonna. Of the seven malting cultivars only two (Brenda and Stratus) formed one group at D = 0.25. The genetic distance between cvs. Brenda and Scarlett, especially recommended for brewery, was equal to 0.34. The detected polymorphism appeared to be sufficient for assessing genetic distances between cultivars, but on the basis of this polymorphism groups of malting and non-malting cultivars were not clearly distinguished.     

Random amplified polymorphic DNA diversity of marginal and central populations in Pinus contorta subsp. latifolia.

Fifteen populations of lodgepole pine (Pinus contorta subsp. latifolia) were surveyed for diversity across 52 random amplified polymorphic DNAs (RAPDs). The objective was to compare single-locus and multilocus structures in four marginal, three intermediate, and eight central populations. Single-locus estimates indicated average observed and expected heterozygosity to be 0.19 and 0.17, respectively. When these estimates were split into population categories, a clear trend of increasing diversity was detected in the direction of marginal to central populations. F-statistics indicated an excess of heterozygotes, with F(IS) ranging from -0.08 for marginal populations to -0.15 for central populations and averaging -0.12 over 15 populations. The estimates of F(ST) decreased towards the margins of the species range, indicating increased population differentiation. Forty-nine of 52 RAPDs tested neutral in the Ewens-Watterson analysis. Multilocus analysis showed significant two-locus and high-order gametic disequilibria in all 15 populations. The most prominent components of the two-locus analysis were the variance of disequilibrium (VD, 46.2%) and the multilocus Wahlund effect (31.9%). This high value for VD indicated that founder effects could explain much of the observed multilocus associations. When analyzed by population categories, the VD showed a decreasing trend indicating that variation due to founder effects was more prominent in marginal populations. The two-locus Wahlund effect (WC) that is characteristic of strong population subdivision was highest in the central populations. This indicated significant levels of gene flow between populations with different allelic combinations.     

Nuclear DNA content variation in seeds from 22 half-sib families of jack pine (Pinus banksiana,Pinaceae)

In order to understand the amount of DNA content variation and its potential roles, both absolute DNA amount and cell cycle phases in 22 half-sib families of jack pine were examined using flow cytometry. When the variability due to differences in speed of germination was taken into account, embryos from superior families (classified on the basis of height growth during field trials) had significantly higher levels of all nuclei classes greater than 4C. Mean DNA contents per nucleus were significantly lower in embryos from superior families compared to inferior ones. Analysis of megagametophyte tissue showed that the mother trees of these embryos expressed a similar pattern. Absolute DNA values were also established on the emerging radicle and the hypocotyl + cotyledons region (HC) separately in five of the families. Nuclei isolated from the emerging radicles had significantly lower levels of DNA than those isolated from the HC region. For three of these families, absolute DNA values from nuclei of the hypocotyl + cotyledons region were established on individual embryos with varying cotyledon numbers. In all three families total DNA amount per nucleus decreased with increasing cotyledon number. A better understanding of differences observed in DNA content during germination, as well as in total DNA content per nuclei among different half-sib families of jack pine, may help in the identification of factors that influence growth and adaptation of this species.     

Limitations to net photosynthesis as affected by nitrogen status in jack pine (Pinus banksiana Lamb.) seedlings

Relative limitations of nitrogen (N) status on the processescontributing to photosynthetic rate (A) were investigated. Jackpine {Pinus banksiana Lamb.) seedlings from seeds grown in sandculture were supplied with four different N treatments for 6weeks, which resulted in a needle N content ranging from 50–85mmol m^–2 (14–32 mg g^–1 dry weight). Leaf gasexchange at varying CO₂ levels was measured and limitationson A₃₅₀ (A at ambient CO₂ level) caused by finite, limitingcarboxylation efficiency (c.e.), maximum A (A_max)and stomatalconductance were estimated from an analysis of the responseof A to internal CO₂ concentration. Although c.e. and A_max decreasedlinearly with the decline in needle N, the magnitudes of theirchanges relative to A₃₅₀ differed. A_max varied with A₃₅₀ andalways exceeded A₃₅₀ by 37–38% c.e., however, declinedfaster than A₃₅₀, as needle N level decreased. Consequently,relative limitation on A₃₅₀ caused by inefficient A_max remainedconstant, but limitations caused by c.e. increased by 10–15%at low N levels. In contrast, the limitation by stomatal conductancedeclined initially, but remained stable when N content droppedbelow 75 mmol m^–2. The results suggest: (1) a decreasein biochemical capacity, but not stomatal conductance, contributedto the reduction of A₃₅₀ induced by N-deficiency in jack pineseedlings; and (2) the capacity of carboxylation appeared tobe impaired more than that of electron transport and/or photophosphorylationand its reduction may be the major reason for the reductionin A₃₅₀. Key words: A–C_i analysis, carboxylation efficiency, electron transport, nitrogen deficiency, stomatal conductance     

Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers

AIMS: To assess genotypic diversity within Ralstonia solanacearum isolates of a single field. METHODS AND RESULTS: A total of 44 field isolates and 22 in vitro generated clones of R. solanacearum were studied for genotypic diversity by random amplified polymorphic DNA (RAPD) technique. Genomic DNA of these isolates and clones was extracted by proteinase-K-SDS lysis mini-prep method. RAPD analysis was done with 30 decamer primers. The data were analysed using NTSYSpc 2.02h software. Forty-two out of 44 field isolates and all the clonal isolates were identified as distinct genotypes at 70% similarity level. CONCLUSION: Very high level of genome variability was observed within the field and clonal isolates of R. solanacearum. This might be a reason for the wide host range of this bacterium and for quick breakdown of wilt resistance in host plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it would be difficult to design specific diagnostic protocol for R. solanacearum even for a localized population and to breed cultivars with broad-spectrum resistance.     

Relationships between Bambusa species (Poaceae,Bambusoideae) revealed by random amplified polymorphic DNA

RAPD (random amplified polymorphic DNA) molecular markers were used to investigate relationships between a sample of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus by different authors. On the resultant neighbor-joining tree, a thorny core Bambusa cluster was distinguished, as was a Lingnania group, and a cluster of Dendrocalamus species with more capitate inflorescences. However, Leleba was found to be a polyphyletic group in the present study.     

Soil solution chemistry in lodgepole pine (Pinus contorta ssp. latifolia) ecosystems,southeastern Wyoming,USA

Concentrations of the principal inorganic and organic solutes in the soil root-zone were measured in six contrasting lodgepole pine (Pinus contorta ssp.latifolia) forest ecosystems for five years (1979–1983). Consistent temporal changes in the principal inorganic solutes (Ca, Mg, K, Na, SO₄, Cl) were observed in all the forest stands and years of study: high concentrations at the initiation of snowmelt in the spring were followed by rapid declines to rather constant values in the mid to late stages of snowmelt. Except for K, concentrations of these solutes differed significantly between sites and between years. Sulfate was the principal mobile anion in the root-zone soil solutions, but contributions of bicarbonate and organic anions also were important.The pH of root-zone solutions was relatively high (6.0), did not change significantly as snowmelt proceeded, and was significantly lower in high-clay soils. No consistent trends in bicarbonate alkalinity were observed and soil atmosphere CO₂ concentrations were only about 10 to 20 times above atmospheric levels, peaking at the end of the snowmelt interval. Concurrent changes in the concentrations of dissolved organic carbon, non-volatile acid-neutralizing capacity, and total Al and Fe indicated that these soil-forming metals were transported vertically in the soil as organic complexes. Precipitation of these complexes was more rapid and more complete in the soils with high clay content than in the coarser soils. Moreover, organic anions comprised up to 30% of the total anionic charge in the coarse-textured soils but less than 10% in the fine soils.Little seasonal or spatial variation of inorganic N and P concentrations was observed in root-zone solutions, probably as a result of high biotic demand for these limiting nutrients. Flux of N and P in these ecosystems was predominately via organic forms so that losses of these nutrients was strongly linked to the mobility of dissolved organic carbon. However, a two-fold increase in the organic N:P and C:P ratios was observed during passage of melt water from the forest floor to mineral soil, evidence of more rapid mineralization of organic P.     

A mitochondrial DNA minisatellite reveals the postglacial history of jack pine (Pinus banksiana), a broad-range North American conifer

Jack pine (Pinus banksiana Lamb.) is a broadly distributed North American conifer and its current range was covered by the Laurentian ice sheet during the last glacial maximum. To infer about the history and postglacial colonization of this boreal species, range-wide genetic variation was assessed using a new and highly variable minisatellite-like marker of the mitochondrial genome. Among the 543 trees analysed, 14 distinct haplotypes were detected, which corresponded to different repeat numbers of the 32-nucleotide minisatellite-like motif. Several haplotypes were rare with limited distribution, suggesting recent mutation events during the Holocene. At the population level, an average of 2.6 haplotypes and a mean haplotype diversity (H) of 0.328 were estimated. Population subdivision of genetic diversity was quite high with G(ST) and R(ST) values of 0.569 and 0.472, respectively. Spatial analyses identified three relatively homogeneous groups of populations presumably representative of genetically distinct glacial populations, one west and one east of the Appalachian Mountains in the United States and a third one presumably on the unglaciated northeastern coastal area in Canada. These results indicate the significant role of the northern part of the US Appalachian Mountains as a factor of vicariance during the ice age. A fourth distinct group of populations was observed in central Québec where the continental glacier retreated last. It included populations harbouring haplotypes present into the three previous groups, and it had higher level of haplotype diversity per population (H = 0.548) and lower population differentiation (G(ST) = 0.265), which indicates a zone of suture or secondary contact between the migration fronts of the three glacial populations. Introgression from Pinus contorta Dougl. var. latifolia Engelm. was apparent in one western population from Alberta. Altogether, these results indicate that the mitochondrial DNA variation of jack pine is geographically highly structured and it correlates well with large-scale patterns emerging from recent phylogeographical studies of other tree boreal species in North America.     

Genetic relationship of 32 cell lines of the euplotes octocarinatus species complex revealed by random amplified polymorphic DNA (RAPD) fingerprinting

Random amplified polymorphic DNA (RAPD) fingerprinting was used in this study to determine the genetic relationship of different cell lines of the hypotrichous ciliate Euplotes octocarinatus. Stocks isolated from different habitats in the USA, and from a group of genetically recombined laboratory strains, were characterized. Band-sharing indices (D) for all possible pairwise comparisons revealed a remarkable genetic diversity between the different cell lines. Investigation of the genetic structure in natural populations found diversity--although to a different extent--in all populations investigated. No clonal structure could be observed, as proposed for several protozoa and recently shown for E. daidaleos. These findings suggest frequent conjugation in the population of E. octocarinatus. No correlation between the genetic relationship of cell lines from different habitats and the distance between the corresponding sampling locations was found. Once separated geographically, the exchange of genetic material between populations appears to be nearly impossible. Therefore, these groups tend to separate into sibling species. The data generally support the occurrence of different syngens in the E. octocarinatus species complex. This finding is in accordance with our observation that the morphological 'species' of E. octocarinatus consists of several syngens or sibling species, similar to findings for the Paramecium aurelia-, Tetrahymena pyriformis- and E. vannus- species complexes.     

NaCl and Na2SO4 alter responses of jack pine (Pinus banksiana) seedlings to boron

Using sand culture, we examined the responses of 6-month-old jack pine (Pinus banksiana Lamb.) seedlings to boron and salinity (sodium chloride and sodium sulfate) treatments. During 4 weeks of treatments, 60 mM NaCl and 60 mM Na₂SO₄ significantly decreased survival, new shoot length, number of new roots, shoot to root dry weight ratio and transpiration rates. When applied in absence of the salts, B had little effect on the measured variables. However, when applied together with salts, B decreased seedling survival, increased needle injury and altered tissue elemental concentrations in jack pine seedlings. In 2 mM B treatment, B concentration was higher in the shoots than in the roots. However, when 2 mM B was present in NaCl and Na₂SO₄ treatments, shoot boron concentration declined and greater proportion of B accumulated in the roots. This shift corresponded to a decline in transpiration rates. In plants treated with NaCl, Na accumulated primarily in the shoots, while in Na₂SO₄-treated plants Na accumulated mostly in the roots. Based on the electrolyte leakage and needle necrosis data, Cl^– appears to be the major factor contributing to seedling injury and B aggravates the injurious effects of NaCl. We suggest that Cl^– may contribute to Na and B toxicity in jack pine by altering cell membrane permeability leading to increased Na concentration in the shoots.     

Genomic diversity within Taxus cuspidata var. nana revealed by random amplified polymorphic DNA markers

Taxus cuspidata var. nana is a cultivated variety of Taxus cuspidata and contains taxol, a valuable secondary metabolite of medical importance, both in their stems and leaves. In this paper, random amplified polymorphic DNA (RAPD) markers were used to assess the genomic diversity of individual plants within T. cuspidata population. Seventy-four randomly selected plants were analyzed by 29 selected primers among which 25 primers produced polymorphic banding patterns. The coefficient of similarity among the plants ranged from 0.30 to 1.00 with a mean of 0.605. Our results showed that a surprisingly high level of genomic diversity existed within T. cuspidata, and RAPD markers were effective in revealing the diversity. Cluster analysis divided the plants into two groups. This data, when taken together with earlier findings showing variation in taxol content within a natural population of T. cuspidata, suggests a tantalizing possibility for selecting genomically homogeneous T. cuspidata plant lines with elevated and stable taxol content. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 771–776. The text was submitted by the authors in English.     

Phylogeographic structure of jack pine (Pinus banksiana; Pinaceae) supports the existence of a coastal glacial refugium in northeastern North America

? Premise of the study: The genetic structure of jack pine (Pinus banksiana Lamb.), a North American boreal conifer with large longitudinal distribution, was investigated to test for the possible existence of a genetically distinct lineage in the Maritimes region in northeastern North America, which could be indicative of a mid-latitude coastal refuge during the last glaciation. ? Methods: One maternally inherited mitochondrial DNA (mtDNA) minisatellite marker and four paternally inherited chloroplast DNA (cpDNA) microsatellite markers were used to assess the range-wide geographical structure of jack pine populations with particular focus on northeastern North America. ? Key results: The populations from the Maritimes region presented a unique mtDNA background characterized by very low diversity and the preponderance of a distinctive mitotype. The distribution of cpDNA diversity was not spatially structured, though three chlorotypes were restricted to the east. ? Conclusions: MtDNA data suggest that populations from the Maritimes region derive from a genetically depauperated north-coastal refugium. Contrastingly, the much higher geographical uniformity observed for cpDNA variation indicates that gene flow by pollen had been much more effective than seed gene flow at homogenizing population structure.     

Effects of litter addition on ectomycorrhizal associates of a lodgepole pine (Pinus contorta) stand in Yellowstone National Park

Increasing soil nutrients through litter manipulation, pollution, or fertilization can adversely affect ectomycorrhizal (EM) communities by inhibiting fungal growth. In this study, we used molecular genetic methods to determine the effects of litter addition on the EM community of a Pinus contorta stand in Yellowstone National Park that regenerated after a stand-replacing fire. Two controls were used; in unmodified control plots nothing was added to the soil, and in perlite plots perlite, a chemically neutral substance, was added to maintain soil moisture and temperature at levels similar to those under litter. We found that (i) species richness did not change significantly following perlite addition (2.6 +/- 0.3 species/core in control plots, compared with 2.3 +/- 0.3 species/core in perlite plots) but decreased significantly (P < 0.05) following litter addition (1.8 +/- 0.3 species/core); (ii) EM infection was not affected by the addition of perlite but increased significantly (P < 0.001) in response to litter addition, and the increase occurred only in the upper soil layer, directly adjacent to the added litter; and (iii) Suillus granulatus, Wilcoxina mikolae, and agaricoid DD were the dominant organisms in controls, but the levels of W. mikolae and agaricoid DD decreased significantly in response to both perlite and litter addition. The relative levels of S. granulatus and a fourth fungus, Cortinariaceae species 2, increased significantly (P < 0.01 and P < 0.05, respectively) following litter addition. Thus, litter addition resulted in some negative effects that may be attributable to moisture-temperature relationships rather than to the increased nutrients associated with litter. Some species respond positively to litter addition, indicating that there are differences in their physiologies. Hence, changes in the EM community induced by litter accumulation also may affect ecosystem function.     

Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA (RAPD) markers

Hippophae rhamnoides is an outcrossing pioneer plant species with a severely fragmented distribution. Random amplified polymorphic DNA (RAPD) marker variation was analysed in 10 populations of ssp. rhamnoides and in one population of ssp. mongolica to estimate the amount and distribution of genetic variability. No less than 89.7% of the scorable markers were polymorphic, but few of these were fixed and populations consequently differed mainly by frequency variation of individual markers. Within-population gene diversity was somewhat low for an outcrossing plant species: 0.192 or 0.159 for ssp. rhamnoides , depending on whether it was based on all 156 polymorphic RAPDs or on only those 63 RAPDs that fulfilled the 3/ N criterion. Analysis of molecular variance applied to the ssp. rhamnoides showed only 15% between-population variability, indicating a relatively restricted population differentiation as expected in outcrossing species and shown in several other AMOVA studies. The tendency for island populations to be somewhat more differentiated, and to have less within-population diversity than mainland populations, may indicate an effect of population fragmentation. Genetic distance estimates among populations, obtained with and without pruning of RAPD loci on the basis of the 3/ N criterion, were generally in very good agreement. Cluster analyses and principal coordinate analyses showed populations of ssp. rhamnoides to be rather close, but quite isolated from the single ssp. mongolica population. Genetic and geographical distances between the ssp. rhamnoides populations were not associated, indicating that large-scale geographical and ecotypic differentiation was not reflected in the RAPD profiles.     

Features of DNA fragments obtained by random amplified polymorphic DNA (RAPD) assays

Random amplified polymorphic DNA (RAPD) fragments were prepared from samples of Calonectris diomedea (Cory's shearwater, Aves) and Haemonchus contortus (Nematoda) DNA by polymerase chain reaction (PCR) using decamers containing two restriction enzyme sites as primers. Six of 19 studied RAPD fragments probably originated from traces of commensal microorganisms. Many rearranged fragments, absent in the original genomic DNA, were synthesized and amplified during the processing of all the DNA samples, indicating that interactions occur within and between strands during the annealing step of PCR. The model of interactions between molecular species during DNA amplification with a single arbitrary oligonucleotide primer was modified to include nested primer annealing and interactions within and between strands. The presence of these artefacts in the final RAPD have a major effect on the interpretation of polymorphism studies.     

Comparison of homology and linkage of random amplified polymorphic DNA (RAPD) markers between individual trees of Scots pine (Pinus sylvestris L.)

Segregation, homology and linkage of 33 random amplified polymorphic DNA (RAPD) markers were studied in three trees [P304, E1101 and F1 (cross P315 × E1101)] of Scots pine (Pinus sylvestris L.). Markers were selected on the basis of a RAPD map constructed earlier for F1. Markers from three linkage groups were studied in P304 and E1101. In both P304 and E1101, 14 markers (42%) were heterozygous. Segregation deviating from 1:1 was found for three markers in P304 and one marker in E1101. With the restriction pattern test, two out of the 33 loci studied (6%) were found to be not homologous to the corresponding fragment in F1. Both of these loci were heterozygous, and they were not linked to any markers, which confirmed their nonhomology. Homologous heterozygous markers segregating 1:1 were mostly linked with similar order in P304 and E1101 as in F1. The results suggest that it might be necessary to verify the homology of comigrating fragments before using them even across individuals. It will often be desirable to use other codominant markers together with RAPDs.