Cuticle micromorphology of leaves of Pinus (Pinaceae) from North America

Cuticle micromorphology of 31 taxa of Pinus from North America was studied with scanning electron microscopy. Twenty-eight characters, 21 from the inner cuticle and seven from the outer cuticle, were described in detail. These characters have value either for infrageneric classification or for identifying individual taxa. Ten characters were diagnostic for subgenera. Numbers 1−5 relate to the epidermal cells: (1) periclinal wall texture; (2) degree of development; (3) shape of anticlinal walls; (4) shape of apex of anticlinal walls; (5) cuticular continuity. Numbers 6 and 7 relate to the subsidiary cells: (6) lateral texture; (7) groove on the inner surface. Numbers 8−10 relate to the Florin ring: (8) degree of development of the ridge; (9) furrow; (10) groove. Pinus resinosa , belonging to Pinus section Pinus , was clearly distinguished from both the other species and subsection by the tapering and oblique end wall and markedly developed anticlinal walls of the epidermal cells. The cuticular features of P. albicaulis belonging to Parrya subsection Cembrae have a cuticular pattern typical of the species of subgenus Strobus . The subsectional delimitation based on cuticular features did not show a clear resolution even though the taxa might be individually distinct based on the combination of characters examined.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 144 , 303−320.     

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.     

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.     

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。     

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.     

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.     

Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes.

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.     

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.     

Anchored reference loci in loblolly pine (Pinus taeda L.) for integrating pine genomics

Anchored reference loci provide a framework for comparative mapping. They are landmarks to denote conserved chromosomal segments, allowing the synthesis of genetic maps from multiple sources. We evaluated 90 expressed sequence tag polymorphisms (ESTPs) from loblolly pine (Pinus taeda L.) for this function. Primer sets were assayed for amplification and polymorphism in six pedigrees, representing two subgenera of Pinus and a distant member of the Pinaceae, Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). On average, 89% of primer sets amplified in four species of subgenus Pinus, 49% in one species of subgenus Strobus, and 22% in Douglas-fir. Polymorphisms were detected for 37-61% of the ESTPs within each pedigree. Comparative mapping in loblolly and slash pine (P. elliottii Englm.) revealed that ESTPs mapped to the same location. Disrupted synteny or significant disruptions in colinearity were not detected. Thirty-five ESTPs met criteria established for anchor loci. The majority of those that did not meet these criteria were excluded when map location was known in only a single species. Anchor loci provide a unifying tool for the community, facilitating the creation of a "generic" pine map and serving as a foundation for studies on genome organization and evolution.     

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。     

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.     

Fossil records of subsection Pinus (genus Pinus,Pinaceae) from the Cenozoic in Japan

Extant pines of subsection Pinus (section Pinus, genus Pinus, Pinaceae) are predominantly distributed in Eastern Asia. However, the extent of diversification in the section has yet to be fully clarified. We reviewed fossil records of subsection Pinus from Japan and collected permineralized materials, in which anatomical details are preserved for better understanding of the diversification. Our results suggest that this subsection appeared in Japan no earlier than the Middle Eocene, with extant species (i.e., Pinus densiflora and Pinus thunbergii) appearing around the beginning of the Pleistocene. Pinus fujiii (Early Miocene to Early Pleistocene) is inferred to have a close affinity to P. thunbergii based on the medial arrangement of its leaf resin canals. Additionally, P. fujiii has a similar cone morphology to those of extant species living in China, bridging the morphological gap between P. thunbergii and Chinese relatives of P. thunbergii as inferred by molecular phylogenetic analyses. Our results also suggest that taxonomic revisions of Pinus miocenica and Pinus oligolepis are required among the Japanese fossil species reported to date.     

Fossil wood of <Emphasis Type="Italic">Pinus priamurensis</Emphasis> sp. nov. (Pinaceae) from the Miocene deposits of the Erkovetskii Brown Coal Field,Amur Region

New species of the pine fossil wood, Pinus priamurensis sp. nov. (Pinaceae) from the Sazanka Formation (upper Middle Miocene–Upper Miocene) of the Erkovetskii Brown Coal Field (Amur Region) is described. The new species shares some wood anatomical features with modern species of the subsection Pinus (section Pinus, subgenus Pinus). This is the first record of fossil wood of Pinus in the Amur Region.     

Rapid expansion of microsatellite sequences in pines

Microsatellite persistence time and evolutionary change was studied among five species of pines, which included a pair of closely related species (Pinus sylvestris and Pinus resinosa) in the subgenus Pinus, their relative Pinus radiata, and another closely related species pair (Pinus strobus and Pinus lambertiana) in the subgenus Strobus. The effective population sizes of these species are known to have ranged from the very small bottlenecks of P. resinosa to vast populations of P. sylvestris. This background allowed us to place the microsatellite evolution in a well-defined phylogenetic setting. Of 30 loci originating from P. strobus and P. radiata, we were able to consistently amplify 4 in most of the these pine species. These priming sites had been conserved for over 100 Myr. The four microsatellites were sequenced in the five species. Flanking sequences were compared to establish that the loci were orthologous. All microsatellites had persisted in these species, despite very different population sizes. We found a recent microsatellite duplication: a closely related pair of loci in P. strobus, where the other four species had just one locus. On two independent occasions, the repeat area of this same microsatellite (locus RPS 105a/b) had grown from a very low repeat number to 15 or 17 in the last 10-25 Myr. Other parts of the same compound microsatellite had remained virtually unchanged. Locus PR 4.6 is known to be polymorphic in both P. radiata and P. sylvestris, but the polymorphism in the two species is due to different motifs. The very large pine genomes are highly repetitive, and microsatellite loci also occur as gene families.     

Variation of cuticle micromorphology of Metasequoia glyptostroboides (Taxodiaceae)

Leaf cuticle micromorphology of Metasequoia glyptostroboides Hu & W. C. Cheng was studied with SEM using samples collected from its natural population in south-central China and cultivated trees in Nanjing City. The cuticle characters from both natural and cultivated trees living in different environments allowed us to re-evaluate taxonomic values of certain cuticular characters and to assess their relationships with environmental factors and the degree of tree maturity. External and internal cuticular features of both adaxial (upper) and abaxial (lower) leaf surfaces revealed the following: (1) Cuticle micromorphology of M. glyptostroboides is distinct among Taxodiaceae, but that variation does not exceed the range of this family. (2) Except for an isolated tree outside the Metasequoia valley, the cuticular features displayed by individual trees from the main Metasequoia groves demonstrate a high degree of uniformity, which is in congruence with previous observations on the low population variability at the gross morphology level. (3) Cuticular characters of grafted Metasequoia trees living in different environments are identical to those of their original trees, indicating that no cuticular character of this species could be regarded as an environmental indicator. (4) Recognition of some unique cuticular features in a Metasequoia tree in an isolated location may lead to a rare source for increasing the variation of this endangered species.     

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.     

The phylogenetic position of the endemic flat-needle pinePinus krempfii (Pinaceae) from Vietnam, based on PCR-RFLP analysis of chloroplast DNA

Pinus krempfii is morphologically very unique as compared to otherPinus species by having flat leaf-like needles. Its taxonomic position has been problematic ever since its discovery. In this study, an attempt was made to infer the taxonomic status ofP. krempfii through restriction fragment length polymorphism analysis of 12 PCR amplified chloroplast (cp) DNA regions. Phylogenetic analysis was conducted using 10 representatives of the twoPinus subgenera:Strobus andPinus. In addition, to infer the position ofP. krempfii in Pinaceae in relation with other genera, 14 representatives of eight additional genera were included in the analysis. Our cpDNA-based results indicate that: 1)P. krempfii clearly belongs to the genusPinus. This result does not favour the creation of a new genusDucampopinus in Pinaceae for this taxon. 2) Within the genusPinus, P. krempfii is more allied with species in subgenusStrobus and differs distinctly from species in subgenusPinus. 3) Despite the similarity in certain morphological and anatomical leaf and wood characters toKeteleeria andPseudolarix, the cpDNA data do not support the hypothesis for close relationship betweenP. krempfii and these two genera.     

Evolutionary significance of a flat-leaved Pinus in Vietnamese rainforest

Pines are generally absent from tropical rainforests. An important exception, Pinus krempfii, is a unique tree that bears flattened needles and competes with evergreen angiosperm trees in southern Vietnam. Here, the photosynthetic and hydraulic physiology of P. krempfii leaves were examined to determine whether this species departs from the widespread pattern of high-light-demanding photosynthetic physiology displayed in needle-leaved Pinus species. Maximum photosynthesis and light saturation of photosynthesis, as well as stem and leaf hydraulic efficiencies, were all very low in P. krempfii compared with other Pinus species. These characteristics were consistent with our observations of P. krempfii seedling regeneration under the forest canopy. By possessing shade tolerance coupled with the production of flattened leaves, P. krempfii has converged morphologically and physiologically with many genera of the southern hemisphere conifer family Podocarpaceae. This convergence extends to a key feature of leaf anatomy, the production of tubular sclereids in the leaf for radial transport of water from the vein to the margin. These observations suggest that few adaptive possibilities are open to conifers when moving into tropical rainforest, meaning that Pinus is forced into direct competition with southern hemisphere conifers for a narrow niche in the equatorial zone.     

Taxonomic revision of phlebotomine sand fly species in the series davisi and panamensis of the subgenus Psychodopygus Mangabeira, 1941 (Diptera: Psychodidae: Phlebotominae)

Several species of the subgenus Psychodopygus Mangabeira, 1941 are known to be leishmaniosis vectors in Brazil. Some of them are morphologically similar, which makes their identification quite difficult concerning epidemiological studies. The aim of the current work is to study the morphology of adult specimens of the subgenus Psychodopygus, in accordance with the morphological similarity and still taking into account the epidemiological importance of some species. Thus 11 species have been studied, including four subspecies of adult specimens deposited in the phlebotomine collection of Centro de Pesquisas René Rachou-Fiocruz. Morphological characters found in the literature and new features observed in this study were recorded in a taxonomic discussion format. These characters make it easy to separate such species. Four taxa, previously considered as subspecies, were raised to the category of species.