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PubMed2004

Evolution of 4-coumarate:coenzyme A ligase (4CL) gene and divergence of Larix (Pinaceae).

The evolutionary dynamics of the 4CL gene encoding 4-coumarate:coenzyme A ligase was investigated in the genus Larix (Pinaceae) by comparing copy number, GC content and codon usage, sequence divergence, and phylogenetic analysis. All 4CL clones of Larix formed a strongly supported monophyletic group, in which two robust clades (4clA and 4clB) derived from an ancient gene duplication event in the common ancestor of Larix were identified. Further gene duplication in the 4clA clade gave rise to two subclades 4clA(1) and 4clA(2). Frequent duplication/deletion appears to be a common evolutionary phenomenon in the 4CL gene family and paralogous genes differ greatly in their evolution rate. The existence of L. speciosa in subclades 4clA(1) and 4clA(2) suggests that this species may represent a primitive form of Larix or the closest relative of the common ancestor of the Eurasian Sect. Multiserialis. In addition, cpDNA and nrDNA ITS analyses support the hypothesis of an early separation of Larix into a North American and a Eurasian clade, which is congruent with the results of previous allozyme and very recent AFLP analyses. The unexpected close relationship between North American larches and the short-bracted species L. gmelinii in East Asia, based on the 4CL gene tree, may stem from lineage sorting.

Base Composition↗
PubMed1999

IFG, a gypsy-like retrotransposon in Pinus (Pinaceae), has an extensive history in pines.

A 1 kb EcoRI restriction fragment cloned from a band visible in an agarose gel of Pinus lambertiana (sugar pine) genomic DNA is present in both subgenera of Pinus with at least 10(4) copies/genome. A full-length copy of this repeated element recovered from a P. radiata (Monterey pine) genomic DNA library was found to possess all of the sequence features associated with gypsy-like retrotransposons. This report describes the biology and history of the IFG (Institute of Forest Genetics) family of retrotransposons. The characterized IFG7 is 5937 bp long. Immediately interior to its 5' and 3' long terminal repeats are sequences consistent with primer binding sites for reverse transcription of the RNA genome. Presumptive gene products associated with retrotransposition appear to be coded in a single reading frame and are in the same order as the gypsy-like retrotransposons and retroviruses. The 1.0 kb EcoRI fragment of IFG elements codes for the 3' half of IFG's reverse transcriptase and the entire RNase H domain. Southern blot analysis suggests IFG was present in Pinaceae before its division into its modern genera. Sequence analysis of IFG 1.0 kb RI fragments and southern analysis also suggest that IFG continued to evolve in Pinus with restriction fragment length polymorphism (RFLP) subfamilies appearing early in the history of each subgenus often correlating with subdivisions of Pinus. Features shared with other plant retrotransposons are also discussed.

Amino Acid Sequence↗
PubMed2002

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.

Blotting, Southern↗
PubMed1999

Microtubule organization in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae).

The organization of microtubules in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae) was examined using primarily confocal microscopy. Pollination in conifers differs from angiosperms in the number of mitotic divisions between the microspore and the sperm and in the growth rate of the pollen tube. These differences may be orchestrated by the cytoskeleton, and this study finds that there are important functional differences in microtubule organization within conifer pollen compared to the angiosperm model systems. Pollen from P. abies contains two degenerated prothallial cells, a body cell, a stalk cell, and a vegetative cell. The body cell produces the sperm. In the vegetative cell, microtubules form a continuous network from within the pollen grain, out through the aperture, and down the length of the tube to the elongating tip. Within the grain, this network extends from the pollen grain wall to the body and stalk cell complex. Microtubules within the body and stalk cells form a densely packed array that enmeshes amyloplasts and the nucleus. Microtubule bundles can be traced between the body and stalk cells from the cytoplasm of the body cell to the adjoining cell wall and into the cytoplasm of the stalk cell. Body and stalk cells are connected by plasmodesmata. The organization of microtubules and the presence of plasmodesmata suggest that microtubules form a path for intercellular communication by projecting from the cytoplasm to interconnecting plasmodesmata. Microtubules in the elongating tube form a net axial array that ensheathes the vegetative nucleus. Microtubules are enriched at the elongating tip, where they form an array beneath the plasma membrane that is perpendicular to the direction of tube growth. This enriched region extends back 20 μm from the tip. There is an abrupt transition from a net perpendicular to a net axial organization at the edge of the enriched region. In medial sections, microtubules are present in the core of the elongating tip. The organization of microtubules in the tip differs from that seen in angiosperm pollen tubes.

Journal Article↗
PubMed2005

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi.

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.

China↗
PubMed2005

Molecular characterization of a glutathione transferase from Pinus tabulaeformis (Pinaceae).

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification metabolism in plants. To date, studies on GSTs in higher plants have focused largely on agricultural plants. In contrast, there is virtually no information on the molecular characteristics of GSTs in gymnosperms. The present study reports for the first time the cloning, expression and characteristics of a GST gene (PtGSTU1) from a pine, Pinus tabulaeformis, which is widely distributed from northern to central China covering cold temperate and drought regions. The PtGSTU1 gene encodes a protein of 228 amino acid residues with a calculated molecular mass of 26.37 kDa. Reverse transcription PCR revealed that PtGSTU1 was expressed in different tissues, both above and below ground, of P. tabulaeformis. The over-expressed recombinant PtGSTU1 showed high activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a Km of 0.47 mM and Vmax of 169.1 micromol/min per mg of protein. The recombinant PtGSTU1 retained more than 60% of its maximum enzymatic activity from 15 degrees C to 45 degrees C with a broad optimum Tm range of 25 degrees C - 35 degrees C. The enzyme had a maximum activity at approximately pH 8.5 - 9.0. Site-directed mutagenesis revealed that Ser13 in the N-terminal domain is a critical catalytic residue, responsible for stabilisation of the thiolate anion of enzyme-bound glutathione. Based on comparative analyses of its amino acid sequence, phylogeny and predicted three-dimensional structure, the PtGSTU1 should be classified as a tau class GST.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Seed plant phylogeny: gnetophytes are derived conifers and a sister group to Pinaceae.

The phylogenetic position of gnetophytes has long been controversial. We sequenced parts of the genes coding for the largest subunit of nuclear RNA polymerase I, II, and III and combined these sequences with those of four chloroplast genes, two mitochondrial genes, and 18S rRNA genes to address this issue. Both maximum likelihood and maximum parsimony analyses of the sites not affected by high substitution levels strongly support a phylogeny where gymnosperms and angiosperms are monophyletic, where cycads are at the base of gymnosperm tree and are followed by ginkgos, and where gnetophytes are grouped within conifers as the sister group of pines. The evolution of several morphological and molecular characters of gnetophytes and conifers will therefore need to be reinterpreted.

Base Sequence↗
PubMed2006

Phylogeography of the endangered Cathaya argyrophylla (Pinaceae) inferred from sequence variation of mitochondrial and nuclear DNA.

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.

Cell Nucleus↗
PubMed1999

Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences.

A 650-bp portion of the nuclear ribosomal DNA internal transcribed spacer region was sequenced in 47 species of Pinus, representing all recognized subsections of the genus, and 2 species of Picea and Cathaya as outgroups. Parsimony analyses of these length variable sequences were conducted using a manual alignment, 13 different automated alignments, elision of the automated alignments, and exclusion of all alignment ambiguous sites. High and moderately supported clades were consistently resolved across the different analyses, while poorly supported clades were inconsistently recovered. Comparison of the topologies highlights taxa of particularly problematic placement including Pinus nelsonii and P. aristata. Within subgenus Pinus, there is moderate support for the monophyly of a narrowly circumscribed subsect. Pinus (=subsect. Sylvestres) and strong support for a clade of North and Central American hard pines. The Himalayan P. roxburghii may be sister species to these "New World hard pines," which have two well-supported subgroups, subsect. Ponderosae and a clade of the remaining five subsections. The position of subsect. Contortae conflicts with its placement in a chloroplast DNA restriction site study. Within subgenus Strobus there is consistent support for the monophyly of a broadly circumscribed subsect. Strobi (including P. krempfii and a polyphyletic subsect. Cembrae) derived from a paraphyletic grade of the remaining soft pines. Relationships among subsects. Gerardianae, Cembroides, and Balfourianae are poorly resolved. Support for the monophyly of subgenus Pinus and subgenus Strobus is not consistently obtained.

DNA, Plant↗
PubMed2000

Molecular phylogenetic position of Japanese Abies (Pinaceae) based on chloroplast DNA sequences.

The phylogenetic positions of Japanese Abies species (A. firma, A. homolepis, A. veitchii, A. sachalinensis, and A. mariesii) were revealed based on nucleotide sequences of chloroplast DNA. First, rbcL sequences of 24 Abies species worldwide were analyzed to clarify the phylogenetic position of the Japanese Abies within this genus. While 4 of the Japanese species formed a monophyletic group with almost no sequence divergence, A. mariesii was placed into a clearly different group with North American species. Second, to clarify the relationships among the species in the monophyletic group, sequences of rbcL, matK, and six spacer regions (total ca. 5 kb) were analyzed for 12 species of the group, with A. mariesii as an outgroup. Although a unique sequence and some species-specific sequences were detected in A. mariesii and in A. firma and A. homolepis, respectively, A. veitchii and A. sachalinensis gave identical sequences for all the sequenced regions.

Abies↗
PubMed2006

Phylogeny, historical biogeography, and patterns of diversification for Pinus (Pinaceae): phylogenetic tests of fossil-based hypotheses.

Pines comprise one of the largest coniferous genera, are distributed throughout the Northern Hemisphere, and have an abundant fossil record. Distributions of fossils have been used to derive a three-step hypothesis of early pine evolution, which postulates a Mesozoic origin for the genus, east-west expansions across Laurasia, and retraction into Eocene refugia. Here, we present phylogenetic tests of this hypothesis using chloroplast sequence data from four loci for 83 pine species. We used the fossil-based hypothesis to derive null expectations concerning monophyly of taxonomic groups, dates of cladogenesis, and patterns of diversification. Phylogenetic analyses using several algorithms subsequently provided rigorous tests of these expectations. Our inferred phylogenies illustrated broad congruence with taxonomic groups, but highlighted consistent problems within subgenus Strobus. Estimated minimum dates of divergence derived from relaxed clock methods were largely consistent with the fossil record and yielded a date for the ingroup node of Pinus of 128+/-4 mya, depending upon the calibration used for subgenus Pinus. Ancestral area reconstructions showed Pinus to have most likely originated in Eurasia. Major clades differed in biogeographic patterns, but were consistent with the fossil-based hypothesis. We found weak support, however, for a change in diversification rate in the Eocene as interpretations of fossil distributions would have predicted.

Fossils↗
PubMed2006

Molecular phylogeny and biogeography of Picea (Pinaceae): implications for phylogeographical studies using cytoplasmic haplotypes.

The center of diversity is not necessarily the place of origin, as has been established by many plant molecular phylogenies. Picea is a complicated but very important genus in coniferous forests of the Northern Hemisphere, with a high species diversity in Asia. Its phylogeny and biogeography were investigated here using sequence analysis of the paternally inherited chloroplast trnC-trnD and trnT-trnF regions and the maternally inherited mitochondrial nad5 intron 1. We found that the North American P. breweriana and P. sitchensis were basal to the other spruces that were further divided into three clades in the cpDNA phylogeny, and that the New World species harbored four of five mitotypes detected, including two ancestral ones and three endemics. These results, combined with biogeographic analyses using DIVA and MacClade and fossil evidence, suggest that Picea originated in North America, and that its present distribution could stem from two times of dispersal from North America to Asia by the Beringian land bridge, and then from Asia to Europe. Most of the northeastern Asian species and the European P. abies could arise from a recent radiation given the very low interspecific genetic differentiation and pure mitotype of them. Considering that the ancestral mtDNA polymorphism can be preserved in many descendant species, even distantly related ones, we suggest that more species, at least the closely related ones, should be sampled in the phylogeographical study using cytoplasmic haplotypes if possible. In addition, we also discussed the evolution and phylogenetic utility of morphological characters in Picea.

Base Sequence↗
PubMed2003

Conflicting phylogenies of Larix (Pinaceae) based on cytoplasmic and nuclear DNA.

The phylogeny of Larix species was studied using chloroplast DNA RFLPs and nuclear ITS sequences and AFLPs. The study resolved the sister relationships between the Eurasian and Asian species and the monophyletic origin of each major geographic group. It also provides strong evidence for an early splitting of American Larix from other species in the genus. The discrepancy between phylogenies based on chloroplast DNA and nuclear DNA, previously observed by Qian et al. [Can. J. For. Res. 25 (1995) 1197], is confirmed. Various explanations for the incongruence between chloroplast DNA and nuclear DNA are discussed, and a tentative reconstruction of Eurasian Larix phylogeography is proposed.

Cluster Analysis↗
PubMed2003

Genetic diversity of nuclear and mitochondrial genomes in Pinus parviflora Sieb. & Zucc. (Pinaceae) populations.

Genetic diversities of the nuclear and mitochondrial genomes in Pinus parviflora were studied in 16 populations, which were distributed across most of the species' range in Japan. Six mitochondrial DNA haplotypes were identified among the 16 populations. The intrapopulation diversity of allozymes was similar to that of other endemic woody species (H(S)=0.259). Although P. parviflora is distributed in discrete populations, differentiation between these was very low (G(ST)=0.044). In addition, the extent of genetic differentiation between two varieties (var. pentaphylla and var. parviflora) was extremely low (G(VT)=0.001). Intrapopulation diversity of mitochondrial DNA was also very low (H(S)=0.098), but population differentiation was high (G(ST)=0.863). Moreover, the distribution of haplotypes reflected the taxonomic differences between P. parviflora var. pentaphylla and var. parviflora. The populations of var. pentaphylla and var. parviflora contained different haplotypes. Differing modes of inheritance may account for the differences in nuclear and mitochondrial genetic diversity.

Autoradiography↗
PubMed2001

Fine-scale genetic structure in Pinus clausa (Pinaceae) populations: effects of disturbance history.

Spatial autocorrelation analyses of 12 allozyme loci were used to compare genetic structure within populations of two varieties of Pinus clausa. P. clausa var. immuginata populations tend to be uneven-aged, with continuous recruitment in small gaps created by wind damage, whereas P. clausa var. clausa populations are more even-aged, with recruitment postdating periodic canopy fires. Three var. immuginata populations and three matched pairs of var. clausa populations, including both a mature and a nearby recently burned population, were examined. Aggregation of multilocus genotypes at small distances was evident in all young var. clausa populations. Little inbreeding was apparent among juveniles or adults in these populations; their genetic structure is likely to have resulted from limited seed dispersal. Genotypes were not significantly spatially structured in nearby matched mature populations. Genetic structure was less evident in var. immuginata populations. Aggregated genotypes were only apparent in the population where patches included juveniles of similar ages; dense juvenile clumps in the other two var. immuginata populations comprised a variety of ages. Interannual variability in allele frequencies of surviving seedlings may account for the absence of genetic structure in these populations.

Alleles↗
PubMed2000

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers.

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.

Base Sequence↗
PubMed2002

Impact of sawfly defoliation on growth of Scots pine Pinus sylvestris (Pinaceae) and associated economic losses.

Needle defoliation by diprionid sawflies decreases the increment and timber yield of Scots pine Pinus sylvestris L. The aim of this study was to provide approximate estimates of this decrease and preliminary estimates of the economic value of growth losses and tree mortality. Growth loss after needle damage by Diprion pini (Linnaeus) was studied in western Finland. Increment cores were sampled for radial growth measurements from trees subjected to slight, moderate and heavy defoliation. A literature survey was carried out to estimate radial growth losses after defoliation by Neodiprion sertifer (Geoffroy). Calculations for economic consequences were based on previous estimates and on the ongoing 9th Finnish National Forest Inventory. Moderate defoliation by N. sertifer and D. pini reduced volume growth by 21% and 86% and heavy defoliation by 38% and 94%, respectively. Tree mortality in defoliated stands was recorded as approximately 4% after an outbreak of N. sertifer and 30% after an outbreak of D. pini. The estimated average economic value of the losses due to reduced growth and tree mortality reached $40 ha-1 after a single-year outbreak of N. sertifer and $310 ha-1 in the case of D. pini. These preliminary estimates of economic losses indicate a much higher impact of pine sawflies than those revealed by the few earlier studies in Europe.

Animals↗