由5个DNA片段推断极度濒危植物五针白皮松的系统位置

五针白皮松(Pinus squamata X．W．Li)是上世纪90年代描述的一种中国特有松树,目前野外只有立木32株,处于极度濒危状态。前人认为这个种可能是白松亚属(subgen．Strobus)的白皮松(P．bungeana Zucc．ex Endl．)与松亚属(subgen．Pinus)的云南松(P．yunnanensis Franch．)的过渡类型,并将其归入白皮松组狐尾松亚组(subsect．Balfourianae)。本文试图在前人分子系统学工作的基础上,检测五针白皮松5个DNA片段,叶绿体基因rbcL、matK、rpl20-rps18间隔区和trn V内含子以及核糖体ITS,将五针白皮松放在整个松属中探讨其系统位置。4个叶绿体基因单独分析结果和它们的联合数据分析结果以及根据ITS得到的系统树均表明,五针白皮松是白皮松亚组(subsect．Gerardianae)的一个稳定成员,其可能的姐妹群是西藏白皮松(P．gerardiana Wall．)。对白皮松亚组的地理分布作了初步探讨。     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

SEM Observation of the Inner Surface Structure of Needle Cuticles in Pinus

Comparative investigation on the inner surfaces of needle cuticle of Pinus was made for 17 species and two varieties under SEM. It is shown that the differences in protrusions and depressions of the internal cuticle surfaces of needles in the genus are not remarkable. However, the features of intercellular flanges are rather distinct and three types can be distinguished. They are: (1) Subgen. Strobus (Sweet) Rehd (except Sect. Parrya) is of the Pinus koraiensis type; (2) Subgen. Pinus is of the P. tabulaeformis type; (3) Sect. Parrya Mayr of Subgen. Strobus (Sweet) Rehd is of the P. bungeana type. The character may provide taxonomy of the genus Pinus with a new piece of evidence. Based on the features mentioned above, together with many others, such as wood anatomy, warts of wood tracheids, bark structure, needle anatomy and cuticle structure as well as karyotypic analysis in Pinus, the author considers that division of Pinus into two subgeuera is natural and that separation of Sect. Parrya Mayr from Subgen. Strobus (Sweet) Rehd. and thesubsequent establishment of the subgenus Parrya of its own are also reasonable.     

Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL, MATK, RPL20-RPS18 spacer, and TRNV intron sequences

The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.     

Pinus baileyi (section Pinus, Pinaceae) from the Paleogene of Idaho, USA

Pinus baileyi from the Paleogene of Idaho was initially related to the bristlecone pine P. longaeva (subgen. Strobus, sect. Parrya, subsect. Balfourianae) from western North America. Unlike the centromucronate condition in P. longaeva, P. baileyi cones have raised umbos that are excentromucronate, i.e., the mucro positioned in the upper umbo field above the keel. Cone size and scale morphology shows that P. baileyi more closely resembles excentromucronate pines of subsects. Halepenses and Pinus sensu Gernandt et al. (2005, Taxon 54: 29-42), but is most similar to P. resinosa, P. kesiya, and P. massoniana of subsect. Pinus. Morphologically, P. baileyi resembles the fossil species P. princetonensis and P. arnoldii from the Eocene Princeton Chert, British Columbia, Canada. Pinus baileyi extends the western North American range of ovulate cones resembling subsect. Pinus from the middle Eocene of British Columbia, Canada and Washington, USA to the Oligocene of Idaho, USA. Pinus baileyi, and possibly P. princetonensis and P. arnoldii, indicates the presence of early populations of subsect. Pinus-type pines in the western cordillera of North America, raising the possibility that P. resinosa and P. tropicalis may have evolved from this group.     

裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中,5SrRNA基因一级结构是高度保守的,二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异,发现55％的核苷酸位点是可变的,这种变异有68％发生在干区（双链区）,其中一些变异,如双链的互补性核苷酸替代,GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守,这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外,首次报道了松属环E区核苷酸的变异性,这可能与其他区域的变异一样,是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系,但由于基因长度短,信息量小,其在近缘种系统分类的应用受到限制。     

基于matK基因的松属白皮松组分子系统发育分析

基于matK基因对松属(Pinus L.)白皮松组(sect.Parrya Myre)进行了分子系统发育分析.白皮松组为一个并系类群,因为白松组的成员与该组(包括越南的扁叶松(P.krempfii Lecomte))的亚洲成员形成一个强烈支持的分支(靴带值92%).在这个分支中,白松组的3个代表种形成一个稳定的单系,而白皮松组的亚洲成员之间系统发育关系不明确.扁叶松和西藏白皮松(P. gerardiana Wall.ex D.Don)聚在一起,但只有61%的支持率.虽然在以前4个cpDNA基因序列分析时五针白皮松(P.squamata X.W.Li)与白皮松(P.bungeana Zucc.ex Loud.)和西藏白皮松形成一个单系,但在本文的分析中三者的关系不明确.在邻接树和多数一致简约树上,北美的白皮松组成员形成一个支持率低的分支.北美的subsect.Balfourianae Engelm.亚组(包括P.aristata Engelm.)是一个单系,但支持率较低.美洲另外两个亚组subsect.Cembroides Englem.和subsect.Rzedowskianae Carv.的组间和组内关系不确定,它们在严格一致简约树上形成一个多歧分支.     

Molecular Phylogeny of Section Parrya of Pinus （Pinaceae） Based on Chloroplast matK Gene Sequence Data

The molecular phylogenetics of sect. Parrya Myre of Pinus L. was analyzed based onchloroplast matKgene sequence data. The section was resolved as paraphyletic because members of thesect. Strobus were nested within a clade composed by the Asian members of the section, including theVietnamese P. krempfii Lecomte, which was strongly supported with a bootstrap value of 92%. [n thistopology, the three sampled species of sect. Strobus formed a strongly supported monophyletic group,while their relationships of Asian species of sect. Parrya were not clear. P. krempfii was grouped with P.gerardiana Wall. ex D. Don with low bootstrap support. The relationships among the Asian members of thesect. Parrya, i.e.P, bungeana Zucc. ex Loud., P. gerardiana and the recently described endangered pine, P.squarnata X. W. Li, was not resolved, although the monophyly of the three pines was strongly supported inthe combined analysis of four cpDNA sequences. The topology of the neighbor joining tree revealed anassemblage of the American members of the section, which also appeared in the majority rule tree withweak bootstrap support. However, this assemblage was not resolved in the consensus tree of theparsimonious analysis. The American subsect. Ba/fourianae Engelm. formed a weakly supported groupincluding P. aristata Engelm., while the relationships among and within the other two American subsections,Cembroides Englem. and tTzedowskianae Carv., were not resolved, as the members of them formed apolytomy in the consensus tree of the parsimonious analysis. The biogeographical implications of theresults are also discussed in this paper.     

基于matK基因的松属白皮松组分子系统发育分析(英文)

基于matK基因对松属(Pinus L.)白皮松组(sect.Parrya Myre)进行了分子系统发育分析。白皮松组为一个并系类群,因为白松组的成员与该组(包括越南的扁叶松(P.krempfii Lecomte))的亚洲成员形成一个强烈支持的分支(靴带值92％)。在这个分支中,白松组的3个代表种形成一个稳定的单系,而白皮松组的亚洲成员之间系统发育关系不明确。扁叶松和西藏白皮松(P.gerardiana Wall.ex D.Don)聚在一起,但只有61％的支持率。虽然在以前4个cpDNA基因序列分析时五针白皮松(P.squamata X.W.Li)与白皮松(P.bungeana Zucc.ex Loud.)和西藏白皮松形成一个单系,但在本文的分析中三者的关系不明确。在邻接树和多数一致简约树上,北美的白皮松组成员形成一个支持率低的分支。北美的subsect.Balfourianae Engelm.亚组(包括P.aristata Engelm.)是一个单系,但支持率较低。美洲另外两个亚组subsect.Cembroides Englem.和subsect.Rzedowskianae Carv.的组间和组内关系不确定,它们在严格一致简约树上形成一个多歧分支。     

Late Embryogeny of Pinus bungeana

Except for Pseudolarix, which is endemic to China, the late embryogeny of ten genera of Pinaceae has been reported before in the diffferent degree. Among them the mature embryos of Keteleeria evelyniana are different from those of the others in having well-developed cotyledons and a very short hypocotyl. As far as information we have is concerned there are three types in structure of the mature embryos of Pinaceae. The first type occurs in Keteleeria and Cedrus, which have very well-developed cotyledons; the second one appears in Picea and Larix, in which the hypocotyl and the root cap are equal in length; the third one, to which Pinus bungeana belongs, includes all the other genera of Pinaceae. The last type is of a prominent hypocotyl in the mature embryos. It is interesting to note that the mature embryos of Torreya grandis and Keteleeria evelyniana are very similar in having specially developed cotyledons, while the proportions of the various tissues in the mature embryo in Fokienia of Cupressaceae as well as Taxus and Amentotaxus of Taxaceae are similar to those of Pinus bungeana. The pith and secretory cells are usually present in the mature embryos of Pinaceae. Although no pith is present in those of Metasequoia and Taiwania of Taxodiaceae, the secretory cells generally occur in their embryos in Fokienia of Cupressaceae and Taxus and Pseudotaxus of Taxaceae, neither pith nor secretory cells are present in their hypocotyl. From above, the structures of mature embryos among Pinaceae, Taxodiaceae and Cupressaceae are different from one another to some degree. The most outstanding feature of the matur embryos in Pinus bungeana is that the shoot apex is very well developed, with a high H/D ratio, about 0.83 on an average, even up to 0.96 in some case. Above-mentioned H/D ratio of Pinus bungeana is rare in the mature embryos of conifers. Gifford (1943) reports that the average H/D ratio of shoot apex of Ephedra altissima is from 0.44 to 0.68, while in 5-year-old branch apex of Pinus ponderosa, the average ratio is about 0.25, and that in l5-year-old branch apex is about 0.35. For the apex of the dormant short shoots of Pinus densiflora the average ratio is about 0.35, but that of shoot apex is about 0.52 when new buds have just formed. According to the present data about shoot apices of both branches and mature embryos, the average ratio of shoot apex of mature embryos in Pinus bungeana is the largest one. From the present investigation the shoot apex of mature embryos of Pinus bungeana exhibits four distinct tissue zones, i.e. the apical initials, the central mother cell zone, the peripheral tissue zone and the rib meristem. It is worthy of note that the shoot apex of Pinus strobus may be divided into five zones, including transition zone between central mother cells and rib meristem (Owston, 1968). Four zones are recognized in the shoot apex of Pinus lambertiana and P. ponderosa, without transition one (Sacher, 1954). From cytological zonation, the shoot apex of mature embryos in Pinus bungeana is rather similar to that of Pinuslambertiana and P. ponderosa.     

吕梁山南端白皮松的群落特征及其多样性的研究

选择白皮松群落分布较集中的黑虎沟和柳家山两个地段设计26个样地,用样方法进行调查,共作样方426个。分析了白皮松群落的种类组成,区系特点,群落外貌,垂直结构和水平结构等群落学特征,并对该群落的环境概况进行分析,计测了不同地段上群落的频盖度指标和不同层次上的ShannonWiener指数、Simpson指数、Pielou指数和种间相遇指数。结果表明:白皮松主要分布于海拔900~1400米之间,在吕梁山南端白皮松群落组成种类约70种,北温带分布属占优势。矮高位芽植物种类多,数量大,是白皮松群落的优势层片。柳家山乔木层为白皮松单优组成,H值为0.706;灌木层H值最高为2.351;草本层H值为1.257。黑虎沟乔木层为多优树种组成,H值为1.825;灌木层H值为2.308;草本层H值为2.400。本研究为白皮松可持续利用和保护提供了有益的参考。     

Induction and Differentiation of Callus from Female Gametohyte Explants of Pinus bungeana Zucc. and P. tabulaeformis Carr.

Immature endosperms (female gametophyte) of Pinus bungeana Zucc. and P. tabulaeformis Carr. were used as explants for establishing tissue cultures. Calli induction and differentiation were studied on a modified MS medium containing 3 % sucrose and various concentrations of auxins and cytokinins. Callus tissues of P. bungeana and P. tabulaeforms could be induced on media supplemented with 1--6 mg/L naphthoxyacetic acid and 0.5 mg/L 6-BAP. The highest induction frequency of calli was 250%. Histocytological observation revealed that the callus cell was haploidy with, N= 12. Differentiation of green buds occurred on the medium supplemented with 0. 1 mg/L ABA, whereas no plantlet was developed, however.     

白皮松天然群体种实性状表型多样性研究

在白皮松天然林分布区共抽取了13个群体,分别测量了5个种实性状。方差分析表明：白皮松种实性状在群体间和群体内存在广泛的变异,5个性状在群体间的F值为2.44～14.68,群体内的F值为7.48～44.53,差异均达显著或极显著水平。群体内的方差分量接近80%,是白皮松表型遗传变异的主要部分。群体间的表型分化系数V_ST=22.8%,与其他松类树种相比,分化水平处于中等偏上。各群体内变异系数和相对极差存在显著或极显著差异,根据二者的多重比较,可将13个群体按群体内表型变异程度分为4组。种子性状地理梯度变异明显, 呈东北—西南走向。种子3个性状（亲代）的表型变异系数与子代的2年生苗高和地径、1年生苗的全干重之间达到或接近显著相关。     

Cuticle Micromorphology of Pinus krempfii Lecomte (Pinaceae) and Additional Species from Southeast Asia

Cuticle micromorphology of the unusual Vietnamese pine, Pinus krempfii Lecomte, and three additional endemic southeast Asian species of Pinus L. (Pinaceae) is characterized for the first time. Taxa studied include (1) P. krempfii, typically placed in its own subgenus Ducampopinus (Chevalier) Ferré ex Little & Critchfield; (2) the endemic Vietnamese species Pinus dalatensis Ferré and (3) the southeast Asian species Pinus kwantungensis Chun ex Tsiang, both of subgenus Strobus; and (4) the widespread Asian species Pinus kesiya Royle ex Gordon of subgenus Pinus. The current and previous studies demonstrate that the genus Pinus and its subgenera are delimited by unique combinations of cuticular characters, although some of these characters may occur individually in other conifers. Cuticular micromorphology supports taxonomic assignment of P. krempfii to subgenus Strobus rather than to its own subgenus, a result that is also indicated by other anatomical studies and recent molecular studies. Sectional affinities of P. krempfii are usually with Parrya, subsection Krempfianae. An alternative classification of P. krempfii with subsection Gerardianae can be supported by micromorphological characters including broad cuticular bridges between stomata, details of the intercellular flanges of the epidermal cells, and usually an amphistomatic stomatal distribution. Features of other Asian species studied are consistent with their taxonomic assignments. The study demonstrates the utility of cuticle micromorphology to taxonomic delimitation within the family Pinaceae.     

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.     

吕梁山南端白皮松幼林种群结构的研究

在吕梁山南端乔家湾、黑虎沟、郭家洼3个地段分别设置了3个样地,用样方法对白皮松幼林种群进行了调查,并做样方272个。分析了白皮松的群落学特征、径级结构及其密度变化规律;绘制了存活曲线和死亡曲线,从多角度研究了白皮松幼林种群的动态变化规律。结果表明：乔家湾白皮松幼林种群生长最好,基本接近于增长型种群。存活曲线表现为5－7．5cm径级后存活曲线显著下降,该种群的变化趋势接近Deevey I型。死亡曲线在7．5－10cm径级之间存在一个死亡高峰。黑虎沟和郭家洼的种群属于下降型种群,幼年个体补充不足严重影响着该种群的生存和发展。     

油松、白皮松和青木千花粉营养成分的研究

通过对我国有巨大生产量和应用潜力的油松、白皮松和青杄花粉的18种氨基酸、蛋白质、维生素A、维生素E、维生素C、磷脂、葡萄糖氧化酶等重要营养成分含量的测定,发现这三种花粉的以上成分含量都很高。其中青杄花粉的维生素A含量达70 641.6 IU/100 g,氨基酸总量达24.443 mg/100 mg。从营养的角度来看,青杄花粉是较优秀的花粉。     

Fossil calibration of molecular divergence infers a moderate mutation rate and recent radiations for pinus

Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31x10(-9) and 0.22-0.42x10(-9).site-1.year-1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.