Genetic variation at allozyme and RAPD markers in Pinus longaeva (Pinaceae) of the White Mountains, California

We compared genetic diversity estimated from allozymes and from random amplified polymorphic DNA (RAPDs) in a sample of 210 Great Basin bristlecone pines (Pinus longaeva Bailey) from three groves in the White Mountains, California, USA. The White Mountains are the most westerly extension of bristlecone pine and home to the oldest known living trees. We assayed two forks of each tree to determine whether they originated from multiple seed caches of the Clark's nutcracker. Despite the limited and fragmented distribution of bristlecone pine, its level of genetic diversity was comparable to that of other pines, but lower than that reported for eastern populations of Great Basin bristlecone pine. Twenty-six of 36 allozymes were polymorphic (p(95) = 38.9%; p = 63.0%), with observed heterozygosity (H(o)) of 0.122 and expected heterozygosity (H(e)) of 0.134. The proportion of the total variation among populations (G(ST)) was only 0.011. The high proportion of trees with multiple stems was not due to germination in seed caches; only six of 210 forked trees had multiple allozyme genotypes. Of the 42 RAPD loci scored, 27 were monomorphic. Genetic diversity for RAPDs was nearly the same as that for allozymes (p(95) = 34.1%, H(e) = 0.130). However, the estimates of diversity and differentiation were much higher (H(e) = 0.321, G(ST) = 0.039) after excluding monomorphic loci.     

A morphometric study of the geographic variation in Pinus contorta (Pinaceae)

This morphometric study of the geographic variation in Pinus contorta is based on 93 provenances cultivated in northern Jutland, Hjardemål Klitplantage, 57'04"08'48'E (Arboretum trial no. F275). The numerical methods employed were common principal components analysis (CPCA), non-metric multidimensional scaling (MDSCALE), minimum spanning trees (MST), and the neighbour-joining method (NJOIN). Although it is not always possible to draw a very clear line between the coastal var. contorta and the inland var. latifolia of P. contorta subsp. contorta, these varieties are distinct in their extreme forms. Critical provenances of both varieties seem to originate from the transition zone between the two taxa. The Californian or Sierra Nevada provenances of f contorta subsp. murrayana are very distinct from the Oregon provenances. Apparently most of the inland Oregon provenances of P. contorta are more or less intermediate between var. latifolia and subsp. murmyana s.str. The NJOIN method of phylogenetic reconstruction supports the division of P. contorta into two major groups or subspecies: 1. subsp. contorta, divided into var. contorta and var. latifolia, and 2. subsp. murrayana.     

Genetic diversity and phylogenetic relationships among five endemic Pinus taxa (Pinaceae) of China as revealed by SRAP markers

The genetic diversity and phylogenetic relationships among five endemic Pinus taxa of China (Pinus tabulaeformis, P. tabulaeformis var. mukdensis, P. tabulaeformis f. shekanensis, Pinus massoniana and Pinus henryi) were studied by SRAP markers. Using 10 SRAP primer pairs, 247 bands were generated. The percent of polymorphic bands (94.8%), Nei's genetic diversity (0.2134), and Shannon's information index (0.3426) revealed a high level of genetic diversity at the genus-level. At the taxon level, P. tabulaeformis f. shekanensis and P. henryi showed a higher genetic diversity than the others. The coefficient of genetic differentiation among taxa (0.3332) indicated a higher level of genetic diversity within taxon, rather than among taxa. An estimate of gene flow among taxa was 1.0004 and implied a certain amount of gene exchange among taxa. The results of neighbor-joining cluster analysis and principal co-ordinate analysis revealed that P. tabulaeformis, P. tabulaeformis var. mukdensis and P. tabulaeformis f. shekanensis were conspecific, which was in agreement with the traditional classification. Phylogenetic relationships analysis also indicated that P. henryi might be a distinct species closely related to P. tabulaeformis.     

Chemical Composition of Pinus sibirica (Pinaceae)

Siberian pine (Pinus sibirica), also known as Siberian cedar pine and Siberian cedar, is an important plant that has been long used as a source of natural compounds and materials (wood, needles, soft resin, turpentine, colophony). Its chemical composition has been studied well enough; however, to our surprise, no articles that compile the phytochemical data have been published so far. Presumably, this is due to the fact that most of the studies were published in journals difficult to access and not indexed by search systems. This review, for the first time, presents a systematic compilation of available data of secondary metabolites occurring in the needles, shoots, bark, wood, seeds, and oleoresin of Pinus sibirica.     

High population differentiation and genetic variation in the endangered Mexican pine Pinus Rzedowskii (Pinaceae)

Pinus rzedowskii is an endangered pine species from Michoaca´n (central Me´xico), which has been previously reported from only three localities. Classified within the subgenus Strobus, it exhibits intermediate morphological characters between subgenera Strobus and Pinus. We analyzed genetic aspects that could shed light on the evolution and conservation of this species. The genetic structure of nine populations was examined using 14 isozyme loci. Pinus rzedowskii has a relatively high level of genetic variation with 46.8% of the loci assayed being polymorphic, a total of 35 alleles, and a mean heterozygosity per population of 0.219. We calculated Wright's F_ST statistic to estimate gene flow indirectly and to evaluate whether or not there was genetic structuring among populations. We found a marked differentiation among populations (F_ST = 0.175) and significant inbreeding (F_IS = 0.247). No pattern of isolation by distance was found. We also constructed a dendrogram based on a genetic distance matrix to obtain an overview of the possible historical relationships among populations. Finally, we found a convex relationship between the genetic distance among populations and the number of ancestral lineages, suggesting that demographically this species has not been at risk recently. Although endangered, with small and fragmented populations, P. rzedowskii shows higher levels of genetic variation than other conifer species with larger populations or similar conservation status.     

Genetic diversity and spatial genetic structure of Pinus strobus (Pinaceae) across an island landscape inferred from allozyme and cpDNA markers

The Beaver Island Archipelago (BIA) provides a model system to address the impact of long-term isolation on genetic diversity and gene flow. Low lake levels are assumed to have caused the BIA to be attached to mainland Michigan for at least 4000 years (10000 yr B.P.- 6000 yr B.P.), eventually, rising lake levels would have kept the islands isolated since 6000 yr B.P. If the island populations of a plant species in the BIA were indeed once continuous with the mainland of Michigan, then we would expect similar levels of genetic diversity in populations of such a species on the islands vs. the mainland. We compared levels of allozyme genetic diversity of 20 plots of Pinus strobus in the BIA with two mainland populations in northern Michigan. In addition, if pollen is a primary agent of gene flow across islands, a low degree of allozyme differentiation among the island populations of P. strobus in the BIA would be evident. Furthermore, since seed dispersal is more limited than pollen dispersal in P. strobus, a more pronounced spatial genetic structure (SGS) is expected in allozymes than in cpDNA markers. To gain insights on the pattern of seed and pollen dispersal among the 20 plots, we further analyzed spatial autocorrelation using Moran's I-statistics for both data sets [biparentally inherited, allozymes and paternally inherited, cpDNA microsatellites (cpDNA SSR)]. We found a similar level of allozyme variability in both the BIA (mean H _e = 0.080) and the two mainland populations (mean H _e = 0.078). As predicted, we observed a low but significant degree of genetic divergence among populations for allozymes (mean F _ST = 0.033 across 20 plots). Our allozyme-based SGS analysis revealed significant evidence of SGS (i.e. isolation-by-distance; slope β = ?0.194 from regression analysis of observed averaged Moran's I values against the logarithm of the upper bound of six distance classes). In contrast, little evidence of SGS was found in cpDNA SSR data across the BIA (β = 0.013). These results suggest that although gene flow via seed dispersal is somewhat limited, pollen flow has been sufficient to maintain genetic diversity and prevent differentiation across the island landscape over several thousand years of isolation.     

Morphological evaluation of the Pinus kesiya complex (Pinaceae)

The main aim of this study is to compare all taxa from the Pinus kesiya complex and related P. tabuliformis using statistical, ordination and discrimination techniques focusing on ten most discriminating morphological traits. Special emphasis was placed on the recently described taxa Pinus densata subsp. tibetica and P. × naxiorum (=P. yunnanensis × P. densata). Population comparisons of the particular main geographic distribution areas of Pinus kesiya, P. densata subsp. tibetica and P. yunnanensis were also conducted separately within each mentioned taxon using the same data. Pinus densata subsp. tibetica proved to be sufficiently morphologically differentiated from subsp. densata as well as from other species of the P. kesiya complex and P. tabuliformis. The recently originating hybrid P. × naxiorum appears to be intermediate between its parents. The unique shrubby taxon P. densata var. pygmaea is clearly closer to P. densata than to P. yunnanensis, to which it has been classified in Chinese floras. Populations of four distant Pinus kesiya geographic distribution areas lack substantial differences that would support the recognition of infraspecific taxa such as subsp. insularis or var. langbianensis. Pinus yunnanensis is very similar to P. kesiya, with only one trait, leaf length, being significantly different.     

Dispersal limitation and spatial scale affect model based projections of Pinus uncinata response to climate change in the Pyrenees

Species Distribution Models (SDMs) were employed to assess the potential impact of climate change on the distribution of Pinus uncinata in the Pyrenees, where it is the dominant tree species in subalpine forest and alpine tree lines. Predicting forest response to climate change is a challenging task in mountain regions but also a conservation priority. We examined the potential impact of spatial scale on SDM projections by conducting all analyses at four spatial resolutions. We further examined the potential effect of dispersal constraints by applying a threshold distance of maximal advancement derived from a spatially explicit, individual‐based simulation model of tree line dynamics. Under current conditions, SDMs including climatic factors related to stress or growth limitation performed best. These models were then employed to project P. uncinata distribution under two emission scenarios, using data generated from several regional climate models. At the end of this century, P. uncinata is expected to migrate northward and upward, occupying habitat currently inhabited by alpine plant species. However, consideration of dispersal limitation and/or changing the spatial resolution of the analysis modified the assessment of climate change impact on mountain ecosystems, especially in the case of estimates of colonization and extinction at the regional scale. Our study highlights the need to improve the characterization of biological processes within SDMs, as well as to consider simultaneously different scales when assessing potential habitat loss under future climate conditions.     

Partial cambial mortality in high-elevation Pinus aristata (Pinaceae)

Partial cambial mortality is a growth form that is characteristic of Pinus aristata trees. To better elucidate their cambial death pattern, tree size and aspect of cambial death data were gathered from three Pinus aristata forests in central Colorado, USA. Stripping frequency tended to be higher for larger diameter classes. Partial cambial mortality exhibits significant directionality within each stand. Furthermore, cambial death was measured to be most frequent on the wind-exposed side of stripped trees in two of the three study sites and appeared to be at the third. Data presented here support the hypothesis that wind plays a role in the occurrence of partial cambial mortality in Pinus aristata. The mechanisms by which wind causes cambial mortality remain unclear.     

Genetic diversity and the mating system of a rare Mexican pinon, Pinus pinceana, and a comparison with Pinus maximartinezii (Pinaceae)

Weeping pi?on (Pinus pinceana) has a restricted and fragmented range, trees are widely scattered within populations, and reproduction is limited. Nevertheless, genetic diversity was high; based on 27 isozyme loci in 18 enzyme systems, unbiased expected heterozygosity averaged 0.174. Differentiation also was high (F(ST) = 0.152), reflecting isolation between southern, central, and northern fragments of the range. Among populations in the northern fragment, F(ST) was only 0.056, and the number of migrants per generation (Nm) was 4.21, which should preclude fixation. Nm between central and southern populations or between them and populations in the northern fragment was lower, 0.99-1.66, indicating a degree of genetic isolation. Multilocus outcrossing rates (t(m)) ranged from 0.836 in the south to 0.897 in the north. Therefore, selfing is low but statistically significant. The equilibrium inbreeding coefficient (F(e)) calculated from t(m) was in good agreement with observed inbreeding coefficients, suggesting that weeping pi?on may be near equilibrium with respect to inbreeding and selection against selfed trees. Weeping pi?on was variable at all loci polymorphic in maxipi?on (Pinus maximartinezii) and, therefore, qualifies as a possible progenitor of maxipi?on. Because of the high level of diversity, reasonable levels of gene flow within the northern fragment of weeping pi?on's range, high rates of outcrossing, and, perhaps, only weak selection against inbred trees, protection in reserves would be a viable option for conservation.     

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.     

Development of microsatellite loci for Pinus koraiensis (Pinaceae)

? Premise of the study: Microsatellite markers were developed for Pinus koraiensis to characterize its genetic diversity and understand its population structure. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 20 primer sets were developed in Chinese populations of P. koraiensis. Three of the markers showed polymorphism with two alleles per locus when assessed in a sample of two populations of P. koraiensis from the Changbai Mountain in the Jilin Province of China. Five and three loci were successfully amplified in P. taiwanensis and P. massoniana, respectively. The amplification size of these loci matches those in P. koraiensis. ? Conclusions: These markers may be useful for further investigation of population genetics of P. koraiensis.     

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.     

Spatial pattern of allozyme variation in a contact zone of Pinus Ponderosa and P. arizonica (Pinaceae)

The spatial distribution of genotypes for nine polymorphic allozyme loci was examined in a contact zone between Pinus ponderosa var. scopulorum and another tree regarded as either a separate species, Pinus arizonica, or variety, Pinus ponderosa var. arizonica, in southern Arizona. Previous work had identified a steep elevational cline for a key taxonomic trait, number of leaf-needles per fascicle, on the south slope of Mt. Lemmon. The present results indicate that the taxa are not fully interbreeding in this contact zone, because allozyme genotypes are considerably more spatially structured than expected for the dispersal characteristics of pines. The amount of spatial differentiation is also much less than that observed for needle number. It appears that this is due to the lack of differentiation for allozyme gene frequencies for the two types of trees, which is further evidenced by analysis of samples from two other populations away from the contact zone. It is likely that if the two taxa were isolated in the past, it was not for long enough nor complete enough to allow mutation-drift to create substantial differentiation between them. Another possible explanation is that introgression after recontact is so advanced that any differences have been erased throughout the Santa Catalina mountain range.     

Biodiversity of Pinus (Pinaceae) in Mexico: speciation and palaeo-endemism

Mexico, with 43 species of Pinus recognized in the most recent revision for Flora Neotropica, is the richest centre of diversity for the genus. Other centres are California and south-east U.S.A. In comparison, Eurasian pine regions are less diverse. This biogeography, combined with the virtual absence of fossil evidence in Mexico, has led to the consensus that Pinus is a relative newcomer to the region and that 'recent' speciation accounts for its diversity. Phylogenetic analysis indicates that several of the oldest and most primitive members of the genus may have originated in this part of the continent and coexist with the species resulting from more recent radiation.     

Nuclear DNA content variation in seeds from 22 half-sib families of jack pine (Pinus banksiana,Pinaceae)

In order to understand the amount of DNA content variation and its potential roles, both absolute DNA amount and cell cycle phases in 22 half-sib families of jack pine were examined using flow cytometry. When the variability due to differences in speed of germination was taken into account, embryos from superior families (classified on the basis of height growth during field trials) had significantly higher levels of all nuclei classes greater than 4C. Mean DNA contents per nucleus were significantly lower in embryos from superior families compared to inferior ones. Analysis of megagametophyte tissue showed that the mother trees of these embryos expressed a similar pattern. Absolute DNA values were also established on the emerging radicle and the hypocotyl + cotyledons region (HC) separately in five of the families. Nuclei isolated from the emerging radicles had significantly lower levels of DNA than those isolated from the HC region. For three of these families, absolute DNA values from nuclei of the hypocotyl + cotyledons region were established on individual embryos with varying cotyledon numbers. In all three families total DNA amount per nucleus decreased with increasing cotyledon number. A better understanding of differences observed in DNA content during germination, as well as in total DNA content per nuclei among different half-sib families of jack pine, may help in the identification of factors that influence growth and adaptation of this species.     

Microsatellite markers for the endangered fir Abies guatemalensis (Pinaceae)

Eight newly developed polymorphic microsatellite markers are presented for Abies guatemalensis Rehder, an endangered tree endemic to Mexico and Central America. These microsatellite markers are useful for evaluating the population genetics of the species which is threatened by greenery poaching in natural populations. Novel conservation plans are needed to secure a more sustainable use strategy and to preserve the genetic diversity of the species.     

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

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

Association genetics of the loblolly pine (Pinus taeda, Pinaceae) metabolome

The metabolome of a plant comprises all small molecule metabolites, which are produced during cellular processes. The genetic basis for metabolites in nonmodel plants is unknown, despite frequently observed correlations between metabolite concentrations and stress responses. A quantitative genetic analysis of metabolites in a nonmodel plant species is thus warranted. Here, we use standard association genetic methods to correlate 3563 single nucleotide polymorphisms (SNPs) to concentrations of 292 metabolites measured in a single loblolly pine (Pinus taeda) association population. A total of 28 single locus associations were detected, representing 24 and 20 unique SNPs and metabolites, respectively. Multilocus Bayesian mixed linear models identified 2998 additional associations for a total of 1617 unique SNPs associated to 255 metabolites. These SNPs explained sizeable fractions of metabolite heritabilities when considered jointly (56.6% on average) and had lower minor allele frequencies and magnitudes of population structure as compared with random SNPs. Modest sets of SNPs (n = 1-23) explained sizeable portions of genetic effects for many metabolites, thus highlighting the importance of multi-SNP models to association mapping, and exhibited patterns of polymorphism consistent with being linked to targets of natural selection. The implications for association mapping in forest trees are discussed.     

Genetic variation (protein markers and microsatellites) in endangered Catalonian donkeys

Genetic variation of the endangered Catalonian donkey breed (Equus asinus) has been analysed at 19 loci including seven protein loci and 12 microsatellite loci isolated from the domestic horse, in 98 individuals of both sexes. Only four protein markers and three microsatellites were polymorphic. Allele frequencies of the analysed loci showed close agreement with Hardy–Weinberg proportions, with the exception of the MPZ002 locus (P<0.01). The within-population inbreeding estimate was not significantly different from zero (as measured by F_IS-statistic). The cumulative-exclusion probability for all polymorphic loci was 82.9%, this value still being very low so that these markers could efficiently be utilised for verification of parentage.