Cryptic hybrids between Pinus uncinata and P. sylvestris

We tested the performance of molecular markers and biometric traits in the identification of hybrids between closely related mountain pine (Pinus uncinata) and Scots pine (Pinus sylvestris). A plastid DNA marker and a set of morphological and anatomical needle traits were applied in analyses of individuals from several sympatric stands of the species and a single‐species' population from southern Europe, used as a reference. A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) marker from the plastid trnL–trnF region and morphological and anatomical traits clearly discriminated between the pure species. Significant differences were found between P. uncinata and P. sylvestris, mostly in the shape of epidermal cells and the number of stomata. Four putative hybrids with P. sylvestris morphology, but with P. uncinata plastid DNA haplotypes, were found in a population from Sierra de Gúdar near Valdelinares, the southernmost locality of the latter species in eastern Spain. Discrimination analyses between and within populations placed these individuals on the edge of an agglomeration of P. sylvestris individuals. The results suggest that hybridization between the species is rare, but can result in cryptic hybrids morphologically similar to the maternal species. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 473–485.     

Ontogenetic differentiation between Mediterranean and Eurasian pines (sect. Pinus) at the seedling stage

Heteroblastic development in pine seedlings includes extreme morphological changes with still unclear adaptive and evolutionary significance. In particular, Mediterranean and Eurasian pines (section Pinus) living in the Mediterranean basin seem to follow quite distinct developmental trajectories at the seedling stage. Aiming to confirm this ontogenetic differentiation we performed a nursery experiment with seedlings of five Mediterranean pines (Pinus pinaster, P. brutia, P. halepensis, P. pinea and P. canariensis) and three Eurasian pines (P. sylvestris, P. uncinata, and P. nigra subsp. salzmannii), also including P. radiata as an outgroup. After destructive analyses at two harvest times (9 and 32 weeks), we found sharp differentiation between Mediterranean and Eurasian pines in a combination of traits linked to shoot heteroblasty. In particular, Mediterranean pines showed a marked delay in the proportion of adult needles to total needles in the shoot compared to Eurasian species, especially at the second harvest. However, two Mediterranean pines, P. pinaster and P. brutia showed a slightly higher proportion of secondary needles, and a higher rate of budset at a more advanced stage (68 weeks) compared to the other three Mediterranean species. Meaningfully, the outgroup P. radiata was the only species combining a high proportion of adult foliage since the first harvest with a delayed formation of the first terminal bud. We discussed the adaptive significance of these findings at the light of species’ climatic niches and life histories.     

Pines and oaks in the restoration of Mediterranean landscapes of Spain: New perspectives for an old practice — a review

Pines have been extensively used for land restoration in the Mediterranean basin and in other parts of the world, since the late 19^th century. The theoretical basis supporting pine utilisation was its stress-tolerant and pioneer features, and their attributed role of facilitating the development of late-successional hardwoods in the long-term. In the present work, the use of pines and hardwoods in forest restoration is discussed in the frame of the current disturbance regime and social demands for Mediterranean forests. Large pine plantations have recently disappeared because of their sensitivity to fire (e.g., Pinus nigra) or because of the short fire-intervals (e.g., Pinus halepensis). Combined pine and oak plantations are proposed for degraded land restoration on the basis of the complementary features of both groups of species. Seeding and containerised seedling plantation, soil amendments and plantation techniques to reduce transplant shock are evaluated for reforestation under water-stressing conditions, on the basis of several experiments performed in eastern Spain. Both P. halepensis and Quercus ilex are tested.     

Differentiating manool and 13-epimanool with NMR chiral shift reagents

Use of NMR chiral shift reagents showed that a compound isolated from Pinus contorta bark and Tsuga heterophylla wood was 13-epimanool. Southern pine (Pinus spp.) tall oil contained a mixture of manool and 13-epimanool.     

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.     

Phylogenetic Relationships and Species Delimitation in Pinus Section Trifoliae Inferrred from Plastid DNA

Recent diversification followed by secondary contact and hybridization may explain complex patterns of intra- and interspecific morphological and genetic variation in the North American hard pines (Pinus section Trifoliae), a group of approximately 49 tree species distributed in North and Central America and the Caribbean islands. We concatenated five plastid DNA markers for an average of 3.9 individuals per putative species and assessed the suitability of the five regions as DNA bar codes for species identification, species delimitation, and phylogenetic reconstruction. The ycf1 gene accounted for the greatest proportion of the alignment (46.9%), the greatest proportion of variable sites (74.9%), and the most unique sequences (75 haplotypes). Phylogenetic analysis recovered clades corresponding to subsections Australes, Contortae, and Ponderosae. Sequences for 23 of the 49 species were monophyletic and sequences for another 9 species were paraphyletic. Morphologically similar species within subsections usually grouped together, but there were exceptions consistent with incomplete lineage sorting or introgression. Bayesian relaxed molecular clock analyses indicated that all three subsections diversified relatively recently during the Miocene. The general mixed Yule-coalescent method gave a mixed model estimate of only 22 or 23 evolutionary entities for the plastid sequences, which corresponds to less than half the 49 species recognized based on morphological species assignments. Including more unique haplotypes per species may result in higher estimates, but low mutation rates, recent diversification, and large effective population sizes may limit the effectiveness of this method to detect evolutionary entities.     

Phylogeography of Pinus armandii and Its Relatives: Heterogeneous Contributions of Geography and Climate Changes to the Genetic Differentiation and Diversification of Chinese White Pines

Geographic barriers and Quaternary climate changes are two major forces driving the evolution, speciation, and genetic structuring of extant organisms. In this study, we used Pinus armandii and eleven other Asian white pines (subsection Strobus, subgenus Pinus) to explore the influences of geographic factors and Pleistocene climatic oscillations on species in South China, a region known to be centers of plant endemism and biodiversity hotspots. Range-wide patterns of genetic variation were investigated using chloroplast and mitochondrial DNA markers, with extensive sampling throughout the entire range of P. armandii. Both cpDNA and mtDNA revealed that P. armandii exhibits high levels of genetic diversity and significant population differentiation. Three geographically distinct subdivisions corresponding to the Qinling-Daba Mountains (QDM), Himalaya-Hengduan Mountains (HHM) and Yungui Plateau (YGP) were revealed in mainland China by cpDNA. Their break zone was located in the southeastern margin of the Qinghai-Tibetan Plateau (QTP). A series of massive mountains, induced by the QTP uplift, imposed significant geographic barriers to genetic exchange. The disjunct distribution patterns of ancestral haplotypes suggest that a large continuous population of the white pines may have existed from southwest to subtropical China. Repeated range shifts in response to the Pleistocene glaciations led to the isolation and diversification of the subtropical species. The two Taiwanese white pines share a common ancestor with the species in mainland China and obtain their chloroplasts via long-distance pollen dispersal from North Asian pines. Distinct genetic patterns were detected in populations from the Qinling-Daba Mountains, Yungui Plateau, Himalaya-Hengduan Mountains, and subtropical China, indicating significant contributions of geographic factors to the genetic differentiation in white pines. Our study depicts a clear picture of the evolutionary history of Chinese white pines and highlights the heterogeneous contributions of geography and Pleistocene climatic fluctuations to the extremely high plant species diversity and endemism in South China.     

Breeding performance and longevity of Tomicus destruens on Mediterranean and continental pine species

The pine shoot beetle Tomicus destruens (Wollaston) (Coleoptera: Curculionidae: Scolytinae) is one of the main pests of Mediterranean forests, where it is oligophagous on Mediterranean pines. However, possible global warming may make the insect move to higher latitudes and altitudes, allowing it to attack new pine species. In this respect, the aim of the present article was to assess both the acceptance and performance of T. destruens offered host and non‐host pine species. A no‐choice breeding experiment was set up under laboratory conditions, using logs of three Mediterranean (Pinus pinea L., Pinus pinaster Miller, and Pinus halepensis Aiton) and two continental (Pinus nigra Arnold and Pinus sylvestris L.) pine species. Log debarking at the end of adult emergence assessed parent fecundity, egg, and larval mortality. The quality of callow adults emerging from each tested pine was evaluated on the basis of their longevity on a semiartificial diet. Tomicus destruens colonised all tested pine species, but did not reproduce in Scots pine, taking about 79 days to complete development with no differences among pines tested. The best breeding performance, evaluated as female fecundity and adult production, was observed in P. halepensis, and the lowest in P. pinaster. On average, adults emerging from P. pinea survived longer (83 days) than from other pines, and adult longevity was the lowest in males emerging from P. nigra. Austrian pine, which under natural conditions is usually not a host of T. destruens, allowed insect development and adult production similar to P. pinea and P. pinaster.     

Taxonomy and systematics of the genus Pinus based on morphological, biogeographical and biochemical characters

For a long time, systematists have subdivided the genus Pinus into Diploxylon and Haploxylon according to morpho-anatomy and the number of needles. Nevertheless, divergent views remain regarding the structure of these two subgenera, mainly at the section and subsection levels. We propose to clarify some of these uncertainties by studying 45 Pinus taxa of different origins. Our results, based on morphometric and biochemical (flavonoids) parameters, complement those obtained from classical anatomical and morphological studies, and also modern macro-molecular markers (proteins, DNA). We confirm the subdivision of the genus into Pinus = Diploxylon versus Strobus = Haploxylon and the further sectioning of the first subgenus into sections Pinus and Trifoliae. Moreover, we specify the different subsections, whereby the contents of the methylated flavonol isorhamnetin coupled with needle morphometry play a significant role (subsections Pinus vs. Pinaster in section Pinus, Australes + Ponderosae vs. Contortae in section Trifoliae). Given that isorhamnetin proceeds from quercetin by the irreversible action of an O-methyl-transferase, this methylated flavonol becomes a dynamic marker in such way that the taxa rich in isorhamnetin can be considered as more “derived = evolved”. In addition, there exists a highly significant negative correlation between methylation index and number of needles. Consequently, the pines from the Holarctic Strobus group (with five needles and low isorhamnetin contents) can be considered as “ancestral”, in reference to a Laurasian origin of the genus. In the subgenus Pinus, the Nearctic group (=section Trifoliae) remains near the ancestral base. On the other hand, the Holarctic subset “densiflorae” is connected to the other members (mainly European) of the polyphyletic subsection Pinus, in particular with series “sylvestres”. Because of their very high contents of isorhamnetin, the Mediterranean pines result from an accentuation of this evolutionary trend (=subsection Pinaster). In fact, the pines growing under hot and dry climates (Mediterranean region) are highly evolved compared to those from cold and/or wet regions (Eurasia and North America but also, to a lesser extent, the south-eastern USA and East Asia). Our dynamic propositions based on plant phenolics data complete those from more modern macromolecular (DNA, proteins) studies.     

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.     

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.     

Pine invasion decreases density and changes native tree communities in woodland Cerrado

ABSTRACT

Background: Invasive plants can negatively impact native communities, but the majority of the effects of these invasions have been demonstrated only for temperate ecosystems. Tropical ecosystems, including the Cerrado, a biodiversity hotspot, are known to be invaded by numerous non-native species, but studies of their impacts are largely lacking.

Aims: Our research aimed at quantifying how Pinus spp. presence and density affected Cerrado plant communities.

Methods: We sampled areas invaded and non-invaded by Pinus spp. to determine if pine invasion affected native tree richness, diversity, evenness, and density. We also evaluated if community composition differed between invaded and non-invaded sites.

Results: We found invaded plots had lower native tree densities than non-invaded plots and that Pinus spp. invasions changed native tree communities by reducing native species abundances.

Conclusion: Invasive pines had negative impacts on the native Cerrado tree community by reducing native plant density and changing species abundances. Reduced density and abundance at early invasion stages can result in reduction in biodiversity in the long term.     

Spatial pattern of Quercus ilex and Quercus pubescens recruitment in Pinus halepensis dominated woodlands

Abstract. European Mediterranean landscapes have undergone changes in structure in recent years as a result of widespread agricultural land abandonment and cessation of silvicultural regimes. Studies concerning the regeneration dynamics of dominant forest species have become critical to the prediction of future landscape trends in these changing forest stands. Quercus ilex (holm oak) and Q. pubescens (downy oak) are considered to be the terminal point of secondary succession in extensive areas of the Mediterranean region. Recent studies, however, have suggested the existence of recruitment bottlenecks in oak genet populations as a result of current management regimes. In this study, we present evidence of the successful establishment of Q. ilex and Q. pubescens in Pinus halepensis (Aleppo pine) woodlands. We investigate the distribution patterns and spatial relationships among oak recruits and resident pines. Established P. halepensis is randomly distributed throughout the study area. Oak seedlings are positively associated with pine trees, suggesting that P. halepensis individuals provide safe sites for oak genet recruitment. We show that spatial patterns of recruitment are in agreement with the general model of spatial segregation described for other Mediterranean plant communities, with seeder species colonizing large openings after disturbance, followed by a more aggregated recruitment of resprouter species.     

Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments

RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. arguta×A. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomikta×A. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5′-trnL exon (a-b spacer DNA) and the trnL 3′ exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented.     

Assessing biodiversity and conservation value of forest patches secondarily fragmented by urbanisation in semiarid southeastern Spain

Semiarid Mediterranean areas patchily forested with Pinus halepensis Mill. are often affected by urbanisation plans, which influence the conservation of biodiversity in the remaining fragments. We assessed forest biodiversity in eleven Pinus halepensis patches of the municipality of Murcia (SE Spain), on the basis of the abundance, richness and conservation value of birds and woody flora. Under the hypothesis that biodiversity is conditioned by pine density, modulated by other patch features (age, physical substrate, surrounding land uses), mixed regression models were used to relate biodiversity indicators with environmental gradients, detected through principal component analyses (PCAs) performed on three sets of variables: landscape (L); habitat structure (H); and, stand development (S). PCA-L related patch size and connectivity to the abundance of typical woodland birds and to floral and bird species richness. PCA-H related shrub cover associated with Sylvia melanocephala Gmelin., to rock and dry grassland cover, a feature of moderately disturbed areas preferred by Lanius senator L. PCA-S associated taller trees and better physical conditions with canopy dwelling and soil probing bird species, respectively. The bird or plant conservation value of patches did not generally match their protection status, which was confirmed by the analysis of a larger sample of 36 natural patches. A negative relationship between floristic value and pine density suggests that reducing the density of reforestations can lead to more diverse and self-sustaining wooded formations. In contrast, conservation value for birds did not decrease with reforestation, stressing the need to use ornithological and floristic indexes as complementary assessment tools. The results seem useful for building a municipal network of reserves connecting higher status protected areas.     

Ectomycorrhizal fungal diversity decreases in Mediterranean pine forests adapted to recurrent fires

Fire is a major disturbance linked to the evolutionary history and climate of Mediterranean ecosystems, where the vegetation has evolved fire‐adaptive traits (e.g., serotiny in pines). In Mediterranean forests, mutualistic feedbacks between trees and ectomycorrhizal (ECM) fungi, essential for ecosystem dynamics, might be shaped by recurrent fires. We tested how the structure and function of ECM fungal communities of Pinus pinaster and Pinus halepensis vary among populations subjected to high and low fire recurrence in Mediterranean ecosystems, and analysed the relative contribution of environmental (climate, soil properties) and tree‐mediated (serotiny) factors. For both pines, local and regional ECM fungal diversity were lower in areas of high than low fire recurrence, although certain fungal species were favoured in the former. A general decline of ECM root‐tip enzymatic activity for P. pinaster was associated with high fire recurrence, but not for P. halepensis. Fire recurrence and fire‐related factors such as climate, soil properties or tree phenotype explained these results. In addition to the main influence of climate, the tree fire‐adaptive trait serotiny recovered a great portion of the variation in structure and function of ECM fungal communities associated with fire recurrence. Edaphic conditions (especially pH, tightly linked to bedrock type) were an important driver shaping ECM fungal communities, but mainly at the local scale and probably independently of the fire recurrence. Our results show that ECM fungal community shifts are associated with fire recurrence in fire‐prone dry Mediterranean forests, and reveal complex feedbacks among trees, mutualistic fungi and the surrounding environment in these ecosystems.     

Hydraulic properties of Pinus halepensis,Pinus pinea and Tetraclinis articulata in a dune ecosystem of Eastern Spain

The hydraulic properties of Pinus pinea, Pinus halepensis and Tetraclinis articulata were studied in a coastal dune area from Eastern Spain. The measured variables include vulnerability to xylem embolism (vulnerability curves), hydraulic conductivity and carbon isotopic discrimination in leaves. Leaf water potentials were also monitored in the three studied populations during an extremely dry period. Our results showed that roots had always wider vessels and higher hydraulic conductivity than branches. Roots were also more vulnerable to xylem embolism and operated closer to their hydraulic limit (i.e., with narrower safety margins). Although it was not quantified, extensive root mortality was observed in the two pines during the study period, in agreement with the high values of xylem embolism (> 75%) predicted from vulnerability curves and the water potentials measured in the field. T. articulata was much more resistant to embolism than P. pinea and P. halepensis. Since T. articulata experienced also lower water potentials, safety margins from hydraulic failure were only slightly wider in this species than in the pines. Combining species and tissues, high resistance to xylem embolism was associated with low hydraulic conductivity and with high wood density. Both relationships imply a cost of having a resistant xylem. The study outlined very different water-use strategies for T. articulata and the pines. Whereas T. articulata had a conservative strategy that relied on the low vulnerability of its conducting system to drought-induced xylem embolism, the two pines showed regulatory mechanisms at different levels (i.e., embolism, root demography) that constrained the absorption of water when it became scarce. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity and differentiation of two Mediterranean pines (Pinus halepensis Mill. and Pinus pinaster Ait.) along a latitudinal cline using chloroplast microsatellite markers

Several studies have reported glacial refugia and migration pathways for different pine species in the Iberian Peninsula, all of them based on a single‐species approach. In this paper, chloroplast microsatellites (cpSSRs) are used to compare population genetic structure and diversity estimates for interspecific pairs of populations located along a cline from southwestern (latitude 36°32′ N, longitude 5°17′ W) to northeastern Spain (latitude 42°14′ N, longitude 2°47′ E) in two widely distributed Mediterranean pines, Pinus halepensis Mill. and Pinus pinaster Ait. Some cpSSRs were shared between species, facilitating comparison of levels of gene diversity at the species level and inferences about within and among species differentiation. P. pinaster showed a much higher number of variants (29) and haplotypes (69) than P. halepensis (20 and 21, respectively). Moreover, genetic diversity estimates for interspecific pairs of populations along the cline were negatively correlated. Three main causes may explain the differences between species in the present‐day distribution of genetic diversity: (1) the distribution of genetic variability before the Quaternary glaciations, with an earlier presence of P. pinaster in the Iberian Peninsula and a late spread of P. halepensis from eastern and central Europe, (2) the location of the Holocene glacial refugia and the migration pathways from these refugia to the present‐day range (from northeast to southwest in P. halepensis and from southwest to northeast and northwest in P. pinaster) and (3) the interactions between species during the postglacial spread.     

Paternal chloroplast inheritance patterns in pine hybrids detected with trnL-trnF intergenic region polymorphism

The inheritance patterns of the chloroplast genomes of shortleaf pine (Pinus echinata Mill.), loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were investigated through the trnL-trnF intergenic spacer polymorphism analysis. The DNA sequences of this spacer differ among these three closely related Pinus species. A modified 'cold' PCR-SSCP (single-strand conformation polymorphism) analysis of this spacer shows that the artificial hybrids (F1) from the shortleaf pine (seed parent) 2 loblolly pine (pollen parent) cross, exhibit the loblolly pine profile. Additionally, nine putative hybrids between shortleaf pine and loblolly pine, previously identified by the IDH (Isocitrate dehydrogenase) allozyme marker, presented the shortleaf pine profile indicating that shortleaf pine, not loblolly pine, sired all of the putative hybrids. Nondenatured polyacrylamide-gel electrophoresis of the trnL-trnF intergenic spacer demonstrated that the artificial hybrids (F1) from the cross, slash pine (seed parent) 2 shortleaf pine (pollen parent), present the shortleaf pine profile. Those results confirmed that the chloroplast genome is paternally inherited in these three species of the genus Pinus. The significance of the trnL-trnF intergenic region polymorphism and our modified 'cold' SSCP protocol for population genetic studies is discussed.