Intra- and interspecific genetic differentiation in closely related pines fromPinus subsectionSylvestres (Pinaceae) in the former Soviet Union

Thirty-seven natural populations of four closely related species ofPinus subsect.Sylvestres, P. mugo, P. funebris, P. pallasiana, andP. sylvestris, occurring in the former Soviet Union were investigated by starch-gel electrophoresis. In the populations assayed 127 allelic variants at 25 loci were revealed.Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation amongP. sylvestris races and among closely related species. A dendrogram constructed using Dn values shows that of the fiveP. sylvestris races analyzed only the geographically isolated var.hamata exhibited sufficient differences at theDia-2 locus (a mean Dn value relative to the other four races is 0.025) to recognize it as a distinct taxon. The remaining races, sylvestris, cretacea, lapponica, and sibirica, have a similar gene pool (Dn values are not greater than 0.010), and they should be regarded as a single taxon,P. sylvestris var.sylvestris. Interspecific comparisons revealed thatP. sylvestris andP. mugo have the closest genetic affinities to each other withNei's genetic distance of 0.108. The dendrogram demonstrates thatP. funebris is closer toP. sylvestris andP. mugo thanP. pallasiana. The available paleontologic data allowed us to conclude thatNei's (1975) time scale estimate for the time of divergence of the taxa was more accurate thanNei's (1971) time scale estimate.     

A taxonomic analysis of seed proteins inPinus spp.(Pinaceae)

Seed storage proteins have proved to be a powerful biochemical marker for taxonomic research, but they have not been extensively employed in forest tree studies. In order to improve the understanding of the taxonomy of the genusPinus, total seed proteins of 12 pine species have been analyzed by means of SDS-PAGE (Sodium dodecylsulphate-polyacrylamide gel electrophoresis). The results showed the presence, in the genusPinus, of two main sub-taxa, corresponding to the subgeneraHaploxylon andDiploxylon. Differences and affinities between Mediterranean pine species were found in agreement with classification ofKlaus (1989).     

Chloroplast DNA-based phylogeny of AsianPinus species (Pinaceae)

The genusPinus includes over 90 species with approximately 24 species native to Asia. We have analyzed the chloroplast (cp) DNA variation of 18Pinus species, including 15 Asian, two Eurasian, and one European species using seven restriction enzymes and ten non-overlapping probes and inferred their phylogenetic relationships. Results of phenetic and cladistic approaches to phylogeny reconstruction were largely in agreement, suggesting two major lineages within the genus and confirmed the ancient character of haploxylon and diploxylon subgenera. Species from sectionParrya appear to have diverged earliest from the hypothesized phylogenetic centre for the haploxylon pines, withP. bungeana andP. gerardiana forming two basal, monotypic lineages. The range of estimated pairwise nucleotide substitutions per site ( ) was higher among haploxylon pines than among diploxylon species. CpDNA divergence was found to be low within the sectionSylvestres, relative to the divergence among haploxylon species, suggesting that the radiation of this group of taxa from its common ancestor occurred after the diversification of other groups. The low cpDNA divergence in this subsection corroborated earlier evidence for its phylogenetic cohesiveness and existence as a monophyletic group.     

Taxonomic evaluations of the genusPinus (Pinaceae) based on electrophoretic data of salt soluble and insoluble seed storage proteins

Electrophoretic studies on seed storage proteins of various gymnosperm species showed that both salt soluble and insoluble protein fractions give taxonomic information. Among species of the genusPinus evident differences are found between the subgeneraHaploxylon (Strobus) andDiploxylon (Pinus). In sect.Strobus the two subsectt.Strobi andCembrae are readily distinguished fromP. bungeana (sect.Parrya) which appears to fall in an intermediate position between haploxyl and diploxyl pines. Among the species of subg.Diploxylon, mountain pines show very similar protein patterns in agreement with their recent speciation (Quaternary).     

Population structure and interspecific differentiation of the peat moss sister speciesSphagnum rubellum andS. capillifolium (Sphagnaceae) in northern Europe

Isozyme electrophoresis was used to study the morphologically similar sister speciesSphagnum rubellum andS. capillifolium from a sample of 1313 plants representing 37 populations from Scandinavia, Great Britain and S Germany. The mean pairwise genetic identities (I) among conspecific populations were 0.976 forS. rubellum and 0.969 forS. capillifolium, versus 0.627 between populations of the two species. Interspecific gene flow was indicated by the observation of occasional plants in sympatric populations with alleles otherwise unique to the other species. Populations of bisexualS. capillifolium were significantly more variable than populations of unisexualS. rubellum. Alpine populations ofS. rubellum andS. capillifolium were dominated by few genotypes, and differentiation among populations was pronounced, indicating a low level of sexual recombination. InS. rubellum, maximum variability was found in western areas with high annual precipitation. Distribution of alleles inS. rubellum indicated restricted gene flow between Great Britain and Scandinavia. Postglacial migration from separate refugia may explain large-scale variation inS. rubellum.     

Structure and organization of the 5S rRNA genes (5S DNA) inPinus radiata (Pinaceae)

A 5S rRNA gene (5S DNA) from the coniferPinus radiata D. Don has been cloned and characterized at the nucleotide, genomic and chromosomal levels. Sequencing revealed a repeat unit of 524 base pairs which is present in approximately 3000 copies per diploid genome. Two-dimensional gel electrophoresis indicated that these copies are organized in tandem arrays of various length. Using in situ hybridization techniques, the tandem arrays appear to be present on all of the chromosomes. This complexity of chromosomal organization contrasts markedly with the few sites of uniform length found in angiosperm plants such as wheat, pea, and maize.     

Genetic diversity, gene flow and drift in Bavarian red deer populations (Cervus elaphus)

The red deer (Cervus elaphus) populationof Bavaria in Southern Germany was severelyreduced during the 19th century due toover-hunting. The species has since recoveredwithin designated areas. Subsequent habitatfragmentation presumably has changed thegenetic structure of Bavarian red deer.In order to assess the genetic diversity, weanalysed samples obtained from nine differentBavarian and two adjacent (Thueringen andCzech-Republic) red deer populations,genotyping 19 microsatellite loci. Our analysesrevealed moderate and significant differencesin diversity. Referring to assignment tests,the genetic differentiation of Bavarian reddeer was sufficient to assign an individual'sorigin to the correct population at an averageof 91.6%. The correlation of genetic andgeographic distance matrices revealed noevidence for isolation by distance. Thecoalescent model analysis suggests that thegenetic structure used to be characterized by adrift–gene flow equilibrium and is nowinfluenced by drift and disruption of the geneflow. Only three of the examined populationsshowed a probability of less than 10% that twogenes within these populations share a commonancestor (F _IS-value). Twopopulations had high F _IS values,indicating the influence of drift.Additionally, the intrapopulation indicesrevealed a low variability in thesepopulations. The estimated effective populationsizes (N _e) were generally in thesame range as the actual population sizes. Theinbreeding rates, based on the estimated N _e, and the inbreeding coefficientssuggested that the Bavarian red deerpopulations are in a stable state.     

The geographical distribution of genetic variation of silver fir (Abies alba,Pinaceae) in relation to its migration history

Silver fir presently occurs in many mountainous regions of eastern, western, southern and Central Europe. In order to elucidate the biogeographic history of fir populations in different European areas, the distribution of area-specific alleles at eight enzyme loci and the allele frequency distributions at seven polymorphic enzyme loci were investigated in seed or bud samples from 48 provenances comprising 98 single stands. Due to great genetic divergence among different populations located near the ancient glacial retreats, it was concluded that silver fir survived in five refugia during the last glaciation but migrated from only three refugia to its present natural range. The occurrence of unique alleles in particular fir areas indicated its migratory routes to the north and the existence of a few introgression zones where silver fir from different refugia has met during its expansion.     

Allozyme differentiation in the Mediterranean firs (Abies, Pinaceae). A first comparative study with phylogenetic implications

Nineteen natural Mediterranean fir populations, belonging to eight species and to one natural hybrid (A. ×borisii-regis), were investigated by starch and polyacrylamide gel electrophoresis. A total of 31 alleles was scored at eight loci (IDH-B, ACP-A, PER-B, 6PGD-A, 6PGD-B, MNR-B, PGI-B, PGM-A. Great variation was observed in the heterozygosity among the population studied and ranged from 0.010 (A. pinsapo) to 0.328 (A. cephalonica). The interpopulation genetic diversity was about 26% of the total genetic diversity. From the dendrogram, new phylogenetic relationships were revealed. High affinity was observed between the Calabrian fir population and the one from north-west Greece as well as betweenA. equi-trojani grown in Asia Minor and the southern Greek populations. Species specific alleles were found inA. cilicica. From the findings of the present work, a new hypothesis concerning the taxonomy, distribution and evolution ofAbies species in the Balkan Peninsula is supported.     

Contrasting patterns of genetic diversity in two tropical pines: Pinus kesiya (Royle ex Gordon) and P. merkusii (Jungh et De Vriese)

We studied allozyme and chloroplast (cp) DNA variation in natural populations of Pinus kesiya and P. merkusii from Thailand and Vietnam. The results showed striking differences between the two species in the amount and distribution of allozyme variation. P. kesiya harboured considerable allozyme variation and showed weak interpopulational differentiation. In contrast, P. merkmii had very low intrapopulational variability but a high level of interpopulational differentiation. The average Nei's genetic distance separating the two species was exceptionally high (0.701) taking into account their close taxonomic placement in the same subsection Sylvestres. The constructed phylogenetic trees revealed very early divergence of P. kesiya and P. merkusii. The present analysis of cpDNA variation also confirmed the dissimilar character of these two species and was compatible with other evidence indicating the outstanding position of P. merkusii as compared to other Asian members of the subsection Sylvestres. Analysis of cpDNA variation in sympatric populations of P. kesiya and P. merkusii revealed that they are pure representatives of the species in question. This result indicates that despite an overlapping distribution P. kesiya and P. merkusii do not hybridise in nature. We suggest that the distinctive character of P. merkusii is a result of an early separation from other Eurasian pines. Despite spatial proximity, P. kesiya and P. merkusii are kept apart by strong reproductive barriers. The low genetic variability of P. merkusii may be explained by previous bottlenecks, reduced gene flow among populations, and an inbreeding due to small population size and asynchronous flowering.     

Genetic diversity and genetic structure of populations of Ranunculus japonicus Thunb. (Ranunculaceae)

Abstract In order to clarify the genetic diversity and population structure of Ranunculus japonicus , allozymic analysis was conducted on 60 populations in southwestern Japan. Considerable genetic variati ons were detected among the populations of R. japonicus . The genetic diversities within species ( H _es = 0.215) and within populations ( H _ep = 0.172) were slightly higher than those of other perennial herbs with widespread distribution and outcrossing plants. Significantly higher values of fixation index were detected in some populations, which might have arisen from restricted mating partners. The majority of genetic variation (approx. 80%) resided within a population and a moderate level of genetic differentiation ( G _ST = 0.203) was observed among populations. The F _ST value (0.203) suggests the existence of a substantial population structure in this species. The highly significant correlation between geographic distance and F _ST values indicates that isolation by distance has played an important role in the construction of the genetic structure of this species.     

Mediterranean pines and their history

The assembly of Mediterranean pines in the sense ofMirov is inhomogeneous in respect to morphological, geographical and evolutionary affinities. Considering new or neglected characters (vegetative and particularly reproductive, cone scales, apophyses, mucros, seeds: Figs. 1–3) in extant populations and fossils, three groups are recognized. The group of coast and island pines extends from the Canary islands to the Himalaya region and is closely related to Caribbean and C. American taxa. This complex evidently has originated from haploxyl ancestors of sect.Parrya during the Mesozoic (Upper Jurassic/Lower Cretaceous) in the NW. Tethys area (Fig. 13).P. rzedowskii can be regarded as an extant survivor of this first phase of differentiation. The extremely variableP. canariensis (together withP. roxburghii in sect.Sula) marks a transitional phase towards the more advanced diploxyl species of sect.Pinea (withP. pinea, P. halepensis &P. brutia, and subsect.Oocarpae) and sect.Pinaster (withP. pinaster, etc.) (Figs. 4–12).—The second group consists of diploxyl mountain pines from the areas surrounding the Mediterranean. They are classified as members of the Eurasiatic sect.Pinus subsect.Sylvestres, have differentiated along the northern Parathetys area, and exhibit close links with E. Asiatic taxa. The third group includes the haploxyl mountain pinesP. cembra andP. peuce which can be regarded as western outposts of the circumpacific centred sect.Strobus with a pre-Tertiary origin.—As an appendix, an improved classification scheme is presented for the pine groups discussed.     

Genetic diversity of natural and cultivated populations ofOenanthe javanica in Korea

Enzyme electrophoresis was used to estimate genetic diversity and population structure in natural and cultivatedOenanthe javanica (Blume) DC. In the six natural populations, 8 of the 22 loci showed polymorphisms. Cultivated populations had fewer alleles per locus (1.84 vs. 1.91), fewer effective alleles per locus (1.47 vs. 1.52), a lower percentage of polymorphic loci (42.3 vs. 50.0), and lower diversity (0.210 vs. 0.228) than did natural populations. These parameters of genetic diversity indicate that the cultivated populations are genetically depauperate relative to their presumptive progenitor, and that the domestication process has partly eroded the level of genetic variation of this species. Nevertheless, the diversity of this species has higher-than-average values compared with other species having similar life-history traits. We propose that the mix-mating system; perennial, high gene flow; and large population sizes are possible factors contributing to this high diversity, which seemed to increase with distance from the coastlines.     

Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.     

Phylogenetic relationships amongPinus species (Pinaceae) inferred from different numbers of 6PGDH loci

Electrophoretic examination of variousPinus species from both subgenera revealed that several taxa differ in the number of loci that control the enzyme system 6-phosphogluconate dehydrogenase (6PGDH). Based on inheritance analyses and published data, it was established that all species of subg.Pinus possess only two 6PGDH loci, whereas all stone pines of subg.Strobus exhibit four controlling loci. In order to trace the phylogenetic links at which one or two gene duplications occurred during pine evolution, several species of subsect.Strobi (sectionStrobus) and two species of sect.Parrya were additionally investigated. Based on conclusions about the uniqueness of gene duplications and the different numbers of 6PGDH loci, a phylogenetic tree of the pine taxa was constructed. This tree shows some new features not recognized in earlier studies and supports several novel assignments postulated in very recent pine classifications.     

Population Genetic structure ofPotentilla fragariodes var.major (Rosaceae) in Korea

The genetic diversity and population structure of eighteenPotentilla fragariodes var.major (Rosaceae) populations in Korea were determined using genetic variations at 22 allozyme loci. The percent of polymorphic loci within the enzymes was 66.7%. Genetic diversity at the species level and at the population level was high (Hes = 0.203; Hep = 0.185, respectively), whereas the extent of the population divergence was relatively low (G_ST = 0.069). F_IS, a measure of the deviation from random mating within the 18 populations, was 0.075. An indirect estimate of the number of migrants per generation (Nm = 3.36) indicated that gene flow was high among Korean populations of the species. In addition, analysis of fixation indices revealed a slight heterozygote deficiency in some populations and at some loci. Wide geographic ranges, perennial herbaceous nature and the persistence of multiple generations are associated with the high level of genetic variation. AlthoughP. fragariodes var.major usually propagated by asexually-produced ramets, we could not rule out the possibility that sexual reproduction occurred at a low rate because each ramet may produce terminal flowers. Mean genetic identity between populations was 0.983. It is highly probable that directional movement toward genetic uniformity in a relatively homogeneous habitat operates among Korean populations ofP. fragariodes var.major.     

A genetic contribution to the taxonomy ofOenothera sect.Oenothera (including subsectionsEuoenothera,Emersonia, Raimannia andMunzia)

Based on an analysis of results from experimental hybridization, the plants assigned byMunz toOenothera subg.Oenothera and subg.Raimannia, now divided into approximately 76 species, are referred to a single section,Oenothera. This section is in turn divided into five subsections:Euoenothera, Munzia, Raimannia, Emersonia, and an undescribed group of three species related toOenothera pubescens. Euoenothera is maintained in the traditional sense, and includes about 14 species of North America, widely naturalized elsewhere.Munzia consists of 45 species, comprising three series, and native to South America.Raimannia is restricted to a group of approximately 11 North American species.Emersonia comprises four rather heterogenous species of northern Mexico and southern New Mexico, of whichOenothera macrosceles, O. maysillesii, andO. organensis have been described. Within these four subsections, interspecific hybrids can be made in general, although plastid differentiation often leads to incompatibilities. With varying degrees of difficulty, hybrids were produced in all intersectional combinations involvingEuoenothera, Emersonia, Munzia, andRaimannia, the most difficult being those betweenEmersonia andRaimannia. Based on their habit and distribution,Emersonia species, and especiallyOenothera maysillesii, appear to resemble most closely the common ancestor of the section,Euoenothera andMunzia to have been derived from it or its common ancestor, andRaimannia perhaps to be more closely related to the phylogenetic branch that leads toEuoenothera.     

Chromosome analysis and DNA homology in threePicea species,P. mariana,P. rubens,andP. glauca (Pinaceae)

A detailed karyotype analysis was made on the somatic complement ofPicea rubens andP. glauca. B-chromosomes were observed in someP. glauca populations. The karyotypes are generally asymmetrical with most of the chromosomes having median to median-submedian centromeres.Picea glauca chromosomes 2, 3, 7, and 8 have secondary constriction on their short arm and chromosome 10 has a secondary constriction on the long arm. Chromosome 3 was the most easily identifiable, as it has two secondary constrictions located on the short arm. InP. rubens, all the chromosomes but chromosomes 8 and 9 have one to four distinctive secondary constrictions. In general, the diagrammatic comparisons show a high degree of similarity amongP. mariana, P. rubens, andP. glauca. GenomicP. mariana probe strongly hybridized to dots of genomic DNA fromP. rubens andP. glauca indicating that there is a high sequence homology among these three species. The synchronizing agent, hydroxyurea was used at different concentrations to enhance the mitotic index of cell suspensions derived from embryogenic cultures. Hydroxyurea at 1.25 mM increased significantly the mitotic index. An increase of hydroxyurea from 1.25 mM to 5 mM and 10 mM resulted in a steady decrease of mitotic index.     

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.