Habitat selection by Asiatic black bear (Ursus thibetanus) in Siran and Kaghan Valleys, Pakistan / Seleção de habitat pelo urso-negro asiático (Ursus thibetanus) nos vales Siran e Kaghan, Paquistão

Asiatic black bear is present in variety of habitats like broad-leaves and coniferous forests, extending form sea level to 4300m elevation and change their habitat for food purpose seasonally. The present study was conducted at Kaghan and Siran Valleys, District Mansehra, Khyber Pakhtunkhwa, Pakistan to assess habitat of black bear. Line transect method was used for observation of bear signs. Twelve meter circular radius plots were selected for the concern vegetation’s i.e. (trees, shrubs and herbs) and three to six plots were placed in each transect. At the result of sign survey, thirteen different categories of bear signs were recorded and encounter rate was calculated for each sign. A total of 1858 signs were observed during field surveys. Total (81%) coniferous species were recoded among trees, with the highest appearance of Pinus wallichiana (34.22%) and Spruce spp (27.76%), similarly broad leaves trees (18.56%) were also recoded from habitat plots. Most of the signs were encountered in bushy areas, whereas high number of Viburnum Spp (60.29%) was present. It is indicated that black bear prefers blend of Coniferous Trees, Viburnum and Ferns Species; probably because these plants provide enough food, protection, and meticulous shelter because more than 80% of habitat composed of these three species. Currently habitat destruction and increase in human population are the up-growing issues for wild animals (especially Asiatic black bear), which is highly sensitive to such problems. High levels of conservation efforts are recommended for the protection of black bear habitat and to avoid human interference in their territory.

O urso-negro asiático está presente em uma variedade de habitats, como folhas largas e florestas de coníferas, estendendo-se do nível do mar até 4.300 m de altitude e mudando seu habitat para fins alimentares sazonalmente. O presente estudo foi conduzido nos vales Kaghan e Siran, distrito de Mansehra, Khyber Pakhtunkhwa, Paquistão, para avaliar o habitat do urso preto. O método de transecto linear foi usado para a observação de sinais do urso. Parcelas de raio circular de 12 m foram selecionadas para a vegetação de preocupação, ou seja, árvores, arbustos e ervas, e 3 a 6 parcelas foram colocadas em cada transecto. No resultado da pesquisa de sinais, 13 categorias diferentes de sinais de urso foram registradas e a taxa de encontro foi calculada para cada sinal. Um total de 1858 sinais foi observado durante os levantamentos de campo. O total (81%) de espécies de coníferas foi recodificado entre as árvores, com maior aparecimento de Pinus wallichiana (34,22%) e Spruce spp (27,76%); árvores de folhas largas (18,56%) também foram recodificadas em parcelas de habitat. A maioria dos sinais foi encontrada em áreas com arbustos, enquanto um alto número de Viburnum spp (60,29%) estava presente. É indicado que o urso-negro prefere a mistura de árvores coníferas, espécies de viburnos e samambaias; provavelmente porque essas plantas fornecem alimento suficiente, proteção e abrigo meticuloso porque mais de 80% do habitat é composto por essas três espécies. Atualmente, a destruição do habitat e o aumento da população humana são questões crescentes para os animais selvagens, especialmente o urso-negro asiático, que é altamente sensível a esses problemas. Altos níveis de esforços de conservação são recomendados para a proteção do habitat do urso-negro e para evitar a interferência humana em seu território.

Lignin concentrations in phloem and outer bark are not associated with resistance to mountain pine beetle among high elevation pines.

A key component in understanding plant-insect interactions is the nature of host defenses. Research on defense traits among Pinus species has focused on specialized metabolites and axial resin ducts, but the role of lignin in defense within diverse systems is unclear. We investigated lignin levels in the outer bark and phloem of P. longaeva, P. balfouriana, and P. flexilis; tree species growing at high elevations in the western United States known to differ in susceptibility to mountain pine beetle (Dendroctonus ponderosae; MPB). Pinus longaeva and P. balfouriana are attacked by MPB less frequently than P. flexilis, and MPB brood production in P. longaeva is limited. Because greater lignification of feeding tissues has been shown to provide defense against bark beetles in related genera, such as Picea, we hypothesized that P. longaeva and P. balfouriana would have greater lignin concentrations than P. flexilis. Contrary to expectations, we found that the more MPB-susceptible P. flexilis had greater phloem lignin levels than the less susceptible P. longaeva and P. balfouriana. No differences in outer bark lignin levels among the species were found. We conclude that lignification in Pinus phloem and outer bark is likely not adaptive as a physical defense against MPB.

Phylogenomic and ecological analyses reveal the spatiotemporal evolution of global pines.

How coniferous forests evolved in the Northern Hemisphere remains largely unknown. Unlike most groups of organisms that generally follow a latitudinal diversity gradient, most conifer species in the Northern Hemisphere are distributed in mountainous areas at middle latitudes. It is of great interest to know whether the midlatitude region has been an evolutionary cradle or museum for conifers and how evolutionary and ecological factors have driven their spatiotemporal evolution. Here, we investigated the macroevolution of Pinus, the largest conifer genus and characteristic of northern temperate coniferous forests, based on nearly complete species sampling. Using 1,662 genes from transcriptome sequences, we reconstructed a robust species phylogeny and reestimated divergence times of global pines. We found that ∼90% of extant pine species originated in the Miocene in sharp contrast to the ancient origin of Pinus, indicating a Neogene rediversification. Surprisingly, species at middle latitudes are much older than those at other latitudes. This finding, coupled with net diversification rate analysis, indicates that the midlatitude region has provided an evolutionary museum for global pines. Analyses of 31 environmental variables, together with a comparison of evolutionary rates of niche and phenotypic traits with a net diversification rate, found that topography played a primary role in pine diversification, and the aridity index was decisive for the niche rate shift. Moreover, fire has forced diversification and adaptive evolution of Pinus Our study highlights the importance of integrating phylogenomic and ecological approaches to address evolution of biological groups at the global scale.

Evolutionary history of the mediterranean Pinus halepensis-brutia species complex using gene-resequencing and transcriptomic approaches.

KEY MESSAGE: Complementary gene-resequencing and transcriptomic approaches reveal contrasted evolutionary histories in a species complex. Pinus halepensis and Pinus brutia are closely related species that can intercross, but occupy different geographical ranges and bioclimates. To study the evolution of this species complex and to provide genomic resources for further research, we produce and analyze two new complementary sets of genetic resources: (i) a set of 172 re-sequenced genomic target loci analyzed in 45 individuals, and (ii) a set of 11 transcriptome assemblies. These two datasets provide insights congruent with previous studies: P. brutia displays high level of genetic diversity and no genetic sub-structure, while P. halepensis shows three main genetic clusters, the western Mediterranean and North African clusters displaying much lower genetic diversity than the eastern Mediterranean cluster, the latter cluster having similar genetic diversity to P. brutia. In addition, these datasets provide new insights on the timing of the species-complex history: the two species would have split at the end of the tertiary, and the changing climatic conditions of the Mediterranean region at the end of the Tertiary-beginning of the Quaternary, together with the distinct species tolerance to harsh climatic conditions would have resulted in different geographic distributions, demographic histories and genetic patterns of the two pines. The multiple glacial-interglacial cycles during the Quaternary would have led to the expansion of P. brutia in the Middle East, while P. halepensis would have been through bottlenecks. The last glaciations, from 0.6 Mya on, would have affected further the Western genetic pool of P. halepensis.

Morphological and Chemical Differentiation between Tunisian Populations of Pinus halepensis, Pinus brutia, and Pinus pinaster.

The lipid fraction of seeds from different pine species and populations was studied regarding total lipid content, fatty acid profile and vitamin E composition. The investigated seeds contained a high percentage of lipid (13.6 to 31.5 %). Lipid fractions were found to be rich in vitamin E, which varied significantly among species and populations. P. halepensis (Ph-Hn) showed the highest content of vitamin E (256.3 mg/kg of seeds) and the uppermost content of α-tocopherol (44 mg/kg). However, P. halepensis (Ph-Kas) was the richest in γ-tocopherol (204.9 mg/kg). Lipid fractions had a low content of δ-tocopherol (1.2 to 3.6 mg/kg. The highest content of γ-tocotrienol (∼18 %) was determined for P. halepensis (Ph-Dc and Ph-Hn). Thirteen fatty acids were identified by GC-FID with significant variation between the investigated species. The linoleic acid was the major fatty acid followed by oleic acid and palmitic acid. The chemical differentiation among species for the composition of fatty acids and vitamin E was confirmed by PCA. Significant correlations were observed between the content of vitamin E and fatty acids and ecological parameters of P. halepensis populations.

Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization.

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Comparative Study of Pine Reference Genomes Reveals Transposable Element Interconnected Gene Networks.

Sequencing the giga-genomes of several pine species has enabled comparative genomic analyses of these outcrossing tree species. Previous studies have revealed the wide distribution and extraordinary diversity of transposable elements (TEs) that occupy the large intergenic spaces in conifer genomes. In this study, we analyzed the distribution of TEs in gene regions of the assembled genomes of Pinus taeda and Pinus lambertiana using high-performance computing resources. The quality of draft genomes and the genome annotation have significant consequences for the investigation of TEs and these aspects are discussed. Several TE families frequently inserted into genes or their flanks were identified in both species' genomes. Potentially important sequence motifs were identified in TEs that could bind additional regulatory factors, promoting gene network formation with faster or enhanced transcription initiation. Node genes that contain many TEs were observed in multiple potential transposable element-associated networks. This study demonstrated the increased accumulation of TEs in the introns of stress-responsive genes of pines and suggests the possibility of rewiring them into responsive networks and sub-networks interconnected with node genes containing multiple TEs. Many such regulatory influences could lead to the adaptive environmental response clines that are characteristic of naturally spread pine populations.

A transcriptomic variation map provides insights into the genetic basis of Pinus massoniana Lamb. evolution and the association with oleoresin yield.

BACKGROUND: Masson pine (Pinus massoniana Lamb.), the dominant native coniferous species in southern China, is commercially important for supplying timber and oleoresin. However, knowledge of the genetic variability of masson pine germplasm is still limited. In this study, the genetic diversity and population structure of masson pine germplasm were assessed using 204 wild accessions from 10 main distribution regions using 94,194 core single-nucleotide polymorphisms (SNPs) obtained from transcriptome sequencing data. RESULTS: The average expected heterozygosity was 0.2724, implying abundant genetic diversity within masson pine germplasm. Analysis of molecular variance (AMOVA) revealed that 3.29% of the variation was sourced from genetic differentiation. Structure analysis identified two geographically distinct groups. Discriminant analysis of principal components (DAPC) showed that one of those groups was further divided into two clusters. Sichuan and Chongqing provenance is the geographical origin, which diffused outward along two different lines. Oleoresin yield is reflected in the evolution of the two groups, and exhibits two different trends along the two lines of diffusion. The oleoresin yield may be associated with the genes of chitinase, CYP720B, cytochrome P450, ABC transporter, and AP2/ethylene-responsive transcription factor (ERF) based on SNPs and expression. CONCLUSIONS: SNP markers from transcriptome sequencing are highly capable of evaluating genetic diversity within different species, as well as the genetic control of objective traits. The functions of these genes will be verified in future studies, and those genes strongly associated with oleoresin yield will be used to improve yields by means of early genotype selection and genetic engineering.

Sheep feeding preference as a tool to control pine invasion in Patagonia: influence of foliar toughness, terpenoids and resin content.

Herbivores modulate the structure and composition of plant communities, including plant invasions. This is conditioned by plant palatability which can be reduced by its chemical or physical traits. The effects that ungulates browsing has on pine invasions are variable and the empirical evidence on the causes of this variability is scarce. We experimentally explored how sheep browsing preference varies between seedlings of pine species with different invasiveness; Pinus contorta (high invasiveness), P. ponderosa (medium invasiveness), P. radiata (medium invasiveness) and P. jeffreyi (low invasiveness). Secondly, we quantified anti-herbivory chemical compounds and physical traits of these species and related them with sheep preference observed. The browsing incidence of P. contorta was 68%, P. ponderosa 58%, P. radiata 29%, and P. jeffreyi 84%. Among anti-herbivory traits analyzed, α-pinene concentration had a negative effect on the probability of a terminal bud being browsed and on browsing intensity. Meanwhile, foliar toughness was negatively related to browsing intensity and water concentration was positively related to browsing intensity. Also, the most invasive species, P. contorta, was highly damaged. Thus, sheep herbivory could be slowing pine invasion rate; suggesting that could be considered a tool to control early invasions, especially for this particular species.

Lipophilic Metabolites from Five-Needle Pines, Pinus armandii and Pinus kwangtungensis, Exhibiting Antibacterial Activity.

Lipophilic extractive metabolites from needles and defoliated twigs of Pinus armandii and P. kwangtungensis were studied by GC/MS. Needles of P. armandii contained predominantly 15-O-functionalized labdane type acids (anticopalic acid), fatty acids, nonacosan-10-ol, sterols, nonacosan-10-ol and sterol saponifiable esters, and acylglycerols, while P. kwangtungensis needles contained no anticopalic acid, but more trinorlabdane (14,15,16-trinor-8(17)-labdene-13,19-dioic acid) and other labdane type acids, nonacosan-10-ol and its saponifiable esters. The major compounds in the P. armandii defoliated twig extract were abietane and isopimarane type acids, fatty acids, sterols, labdanoids (cis-abienol), cembranoids (isocembrol and 4-epi-isocembrol), saponifiable sterol esters, and acylglycerols. The same extract of P. kwangtungensis contained larger quantities of fatty acids, caryophyllene oxide, serratanoids, sterols, saponifiable sterol esters, and acylglycerols, but lesser amounts of abietane and isopimarane type acids, cis-abienol, and lacked cembranoids. Both twig and needle extracts of P. armandii and P. kwangtungensis, as well as the extracts' fractions, significantly inhibited the growth of Gram-negative bacteria Serratia marcescens with MIC of 0.1 mg ml-1 , while in most cases they slightly stimulated the growth of Gram-positive bacteria Bacillus subtilis at the same concentrations. Thus, lipophilic extractive compounds from the needles and defoliated twigs of both pines are prospective for the development of antiseptics against Gram-negative bacteria.

On the Evolution and Functional Diversity of Terpene Synthases in the Pinus Species: A Review.

In the biosynthesis of terpenoids, the ample catalytic versatility of terpene synthases (TPS) allows the formation of thousands of different molecules. A steadily increasing number of sequenced plant genomes invariably show that the TPS gene family is medium to large in size, comprising from 30 to 100 functional members. In conifers, TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, and diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. Such substances are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress. Oleoresin terpenoids can be also profitably used in a number of different fields, from traditional and modern medicine to fine chemicals, fragrances, and flavours, and, in the last years, in biorefinery too. In the present work, after summarizing the current views on the biosynthesis and biological functions of terpenoids, recent advances on the evolution and functional diversification of plant TPSs are reviewed, with a focus on gymnosperms. In such context, an extensive characterization and phylogeny of all the known TPSs from different Pinus species is reported, which, for such genus, can be seen as the first effort to explore the evolutionary history of the large family of TPS genes involved in specialized metabolism. Finally, an approach is described in which the phylogeny of TPSs in Pinus spp. has been exploited to isolate for the first time mono-TPS sequences from Pinus nigra subsp. laricio, an ecologically important endemic pine in the Mediterranean area.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence.

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

Core set construction and association analysis of Pinus massoniana from Guangdong province in southern China using SLAF-seq.

Germplasm resource collection and utilization are important in forestry species breeding. High-through sequencing technologies have been playing increasing roles in forestry breeding. In this study, specific-locus amplified fragment sequencing (SLAF-seq) was employed to analyze 149 masson pine (Pinus massoniana) accessions collected from Guangdong in China. A large number of 471,660 SNPs in the total collection were identified from 599,164 polymorphic SLAF tags. Population structure analysis showed that 149 masson pines could not be obviously divided into subpopulations. Two core sets, containing 29 masson pine accessions for increasing resin and wood yield respectively, were obtained from the total collection. Phenotypic analyses of five traits showed abundant variations, 25 suggestive and 9 significant SNPs were associated with the resin-yielding capacity (RYC') and volume of wood (VW) using EMMAX and FaST-LMM; 22 suggestive and 11 significant SNPs were associated with RYC' and VW using mrMLM and FASTmrMLM. Moreover, a large number of associated SNPs were detected in trait HT, DBH, RW and RYC using mrMLM, FASTmrMLM, FASTmrEMMA and ISIS EM-BLASSO. The core germplasm sets would be a valuable resource for masson pine improvement and breeding. In addition, the associated SNP markers would be meaningful for masson pine resource selection.

[Effects of species diversity and phylogenetic diversity on productivity of a mixed broadleaved-Korean pine forest.] / ç‰©ç§å¤šæ ·æ€§å’Œç³»ç»Ÿå‘è‚²å¤šæ ·æ€§å¯¹é˜”å¶çº¢æ¾æž—ç”Ÿäº§åŠ›çš„å½±å“.

The relationship between biodiversity and ecosystem function is one of the hot spot in ecological research. The driving effect of plant diversity on forest productivity has been widely focused, but its underlying mechanisms are still controversial. We explored the impacts of species diversity and phylogenetic diversity on forest productivity at different spatial scales based on a 9 hm2 forest dynamic monitoring plot of typical mixed broadleaved-Korean pine (Pinus koraiensis) forest in Liangshui National Nature Reserve, Heilongjiang Province, with the censuses data in 2005 and 2015, using linear regression and structural equation models. The results showed that species diversity and phylogenetic diversity were both positively correlated with productivity. The strength of species diversity on productivity gradually enhanced as spatial scales increased, while the effects of phylogenetic diversity on productivity showed opposite trend. On small scales, the effects of phylogenetic diversity on productivity was stronger than species diversity. Moreover, productivity was affected by abiotic factors. Soil factors were significantly positively correlated with productivity at different scales, which was gradually dominated as spatial scales increased. Our results indicated that evolutionary information should be considered in future ecosystem function studies, which might provide additional explanatory power for other diversity metrics. Meanwhile, the effects of spatial scales and abiotic factors should also be considered, which could provide a scientific basis for further understanding the mechanisms underlying the changes of forest productivity.

Anatomical changes with needle length are correlated with leaf structural and physiological traits across five Pinus species.

The genus Pinus has wide geographical range and includes species that are the most economically valued among forest trees worldwide. Pine needle length varies greatly among species, but the effects of needle length on anatomy, function, and coordination and trade-offs among traits are poorly understood. We examined variation in leaf morphological, anatomical, mechanical, chemical, and physiological characteristics among five southern pine species: Pinus echinata, Pinus elliottii, Pinus palustris, Pinus taeda, and Pinus virginiana. We found that increasing needle length contributed to a trade-off between the relative fractions of support versus photosynthetic tissue (mesophyll) across species. From the shortest (7 cm) to the longest (36 cm) needles, mechanical tissue fraction increased by 50%, whereas needle dry density decreased by 21%, revealing multiple adjustments to a greater need for mechanical support in longer needles. We also found a fourfold increase in leaf hydraulic conductance over the range of needle length across species, associated with weaker upward trends in stomatal conductance and photosynthetic capacity. Our results suggest that the leaf size strongly influences their anatomical traits, which, in turn, are reflected in leaf mechanical support and physiological capacity.

[Microscopic and molecular identification of pine needles].

OBJECTIVE: To identify pine needles from different plant origins by microscopic and molecular approaches. METHODS: The characteristics of pine needles of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were investigated via plant morphology and microscopic characteristics. ITS2 and rbcL were analyzed with PCR amplification and bi-directional sequencing. MEGA 6.0 was used to calculate the intra-and inter-specific Kimura-2-Parameter (K2P) distances, and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. RESULTS: There were significant differences in the number and length of pine needles, number of vascular bundles, distribution of stomatal lines, number and distribution of resin channels among three kinds of pine needles. The lengths of ITS2 sequences of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 470, 469 and 470 bp, respectively. The lengths of rbcL sequences in three kinds of pine needles were 553 bp. The intraspecific variation rates of ITS2 sequences in Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 0%, 0.2%, and 2.8%, respectively; and the intraspecific variation rates of rbcL sequences were 0%, 2.4%, and 1.1%, respectively. There was no significant barcoding gap in intraspecific and interspecific genetic distances of ITS2 sequences. The intraspecific and interspecific distances of rbcL sequences were clearly separated in the barcoding gap test. The NJ tree based on rbcL showed that the three pine needles clustered into three separate groups, indicating that rbcL DNA marker could distinguish the Pinus massoniana Lamb., Pinus thunbergii Parl., Pinus armandii Franch. and its close relative species. CONCLUSIONS: s The three types of pine needles can be distinguished accurately and rapidly by microscopic and molecular identification. The study provides methodology and experimental basis for the quality evaluation and classification of pine needles.

Phylogeny and evolutionary history of Pinaceae updated by transcriptomic analysis.

Pinaceae comprises 11 genera, and represents the largest family of conifers with an extensive wild distribution in the Northern Hemisphere. Intergeneric relationships of Pinaceae have been investigated using many morphological characters and molecular markers, but phylogenetic positions of four genera, including Cathaya, Cedrus, Nothotsuga and Pseudolarix, remain controversial or have not been completely resolved. To completely resolve the intergeneric relationships of Pinaceae, we conducted a comparative transcriptomic study of 14 species representing all Pinaceae genera. Multiple data sets, containing up to 6,369,681 sites across 4676 loci, were analyzed using concatenation and coalescent methods. Our study generated a robust topology, which divides Pinaceae into two clades, one (pinoid) including Cathaya, Larix, Picea, Pinus, and Pseudotsuga, and the other (abietoid) including Abies, Cedrus, Keteleeria, Nothotsuga, Pseudolarix, and Tsuga. Cathaya and Pinus form a clade sister to Picea; Cedrus is sister to the remaining abietoid genera, and the two genera Nothotsuga and Tsuga form a clade sister to Pseudolarix. The discordant positions of Cathaya, Cedrus and Pseudolarix in different gene trees could be explained by ancient radiation and/or molecular homoplastic evolution. The hybrid origin hypothesis of Nothotsuga is not supported. Based on molecular dating, extant Pinaceae genera diverged since about 206 Mya, earlier than the break-up of Pangea, and the divergence among the pinoid genera occurred earlier than the split among the abietoid genera. Moreover, our study indicates that two radiation events occurred in the evolution of Pinaceae genera, and some important morphological characters evolved multiple times based on ancestral state reconstruction.

Development and characterization of chloroplast microsatellite markers for Pinus massoniana and their application in Pinus (Pinaceae) species.

Pinus massoniana is one of the important afforestation and pioneer tree species, which is widely distribute in southern China. Chloroplast simple sequence repeat markers (cpSSRs) have been widely used in studies of tree genetics, phylogenetic and breeding. We sequenced the whole chloroplast genome sequences of P. massoniana using PCR and Sanger sequencing. A total of 71 cpSSRs were identified, among which mononucleotide repeats were predominant (70.42%). Seventeen primer pairs were developed and amplification tests were conducted with 15 P. massoniana individuals. Also, cross-species amplification tests were conducted among 15 individuals per Pinus species, including P. elliottii, P. bungeana, P. armandii, P. caribaea, P. tabulaeformis, P. taiwanensis and P. yunnanensis which revealed polymorphic information content ranging from 0.2 to 0.8 and average of haploid diversity (h) ranging from 0.29 to 0.63. In addition, the polymorphic cpSSRs were useful in distinguishing the sampled pine species, and could be powerful tool in phylogenetic studies.

Chemotaxonomic Considerations of the n-Alkane Composition in Pinus heldreichii, P. nigra, and P. peuce.

The n-alkane composition in the leaf cuticular waxes of natural populations of Bosnian pine (Pinus heldreichii), Austrian pine (P. nigra), and Macedonian pine (P. peuce) was compared for the first time. The range of n-alkanes was wider in P. nigra (C16  - C33 ) than in P. heldreichii and P. peuce (C18  - C33 ). Species also diverged in abundance and range of dominant n-alkanes (P. heldreichii: C23 , C27 , and C25 ; P. nigra: C25 , C27 , C29 , and C23 ; P. peuce: C29 , C25 , C27 , and C23 ). Multivariate statistical analyses (PCA, DA, and CA) generally pointed out separation of populations of P. nigra from populations of P. heldreichii and P. peuce (which were, to a greater or lesser extent, separated too). However, position of these species on the basis of n-alkane composition was in accordance neither with infrageneric classification nor with recent molecular and terpene investigations.

Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes.

PREMISE OF THE STUDY: Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. METHODS: We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. KEY RESULTS: Concatenation- and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. CONCLUSIONS: Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow.