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).     

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.     

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).     

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.     

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.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Population structure,gene diversity,and differentiation in natural populations of Cedar pines (Pinus subsect.Cembrae,Pinaceae) in the USSR

Twelve natural populations of four cedar pine species,Pinus sibirica, P. cembra, P. pumila, andP. koraiensis, occurring in the Soviet Union were investigated by starch-gel electrophoresis. Frequencies of 55 alleles at 19 loci were determined. Interpopulation genetic diversity inP. sibirica andP. pumila was only 2–4 per cent of the total genetic diversity. Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation among conspecific populations and among species. Dn values among populations ranged from 0.006 to 0.038. A dendrogram constructed using Dn values divided cedar pines species into 2 clusters:sibirica-cembra (Dn = 0.030) andpumila-koraiensis (Dn = 0.143). Nei's distance between these clusters was 0.232. On the basis of the data obtained it was possible to draw the following conclusion:P. sibirica, P. pumila, andP. koraiensis are distinct species, whileP. cembra should apparently be regarded as geographicalP. sibirica race.     

Chloroplast DNA typing by PCR-SSCP in thePinus pumila-P. parviflora var.pentaphylla complex (Pinaceae)

Pinus hakkodensis has been considered as a hybrid betweenP. pumila andP. parviflora var.pentaphylla. Chloroplast DNA typing of this putative hybrid and hypothesized parental species in the Tanigawa Mountains, Japan, was conducted by PCR and single-strand conformation polymorphism (SSCP) of the intergenic spacer betweentrnL(UAA)3′ exon andtrnF(GAA) of cpDNA. Each of the hypothesized parental species collected from other mountain regions displayed a diagnostic SSCP pattern, whereas all morphological intermediates sampled in the Tanigawa Mountains had the SSCP pattern ofP. parviflora var.pentaphylla. Furthermore, some individuals classified on the basis of needle morphology as belonging toP. pumila in this mountain region showed the SSCP pattern ofP. parviflora var.pentaphylla. This may suggest that pollen-mediated uni-directional introgression fromP. parviflora var.pentaphylla toP. pumila occurs in the Tanigawa Mountains.     

Chloroplast DNA variation in the genusSecale (Poaceae)

The chloroplast genomes of 44 accessions ofSecale were surveyed for restriction site polymorphisms. The accessions were chosen to represent the geographic as well as taxonomic range of the genus. Using 12 restriction enzymes a total of 348 sites were detected. Twenty-nine mutation sites were phylogenetically informative and used in a cladistic analysis. Further, a 0.1 kb insertion separatedSecale from the outgroup species. Only the annual speciesS. sylvestre was distinct from the rest of the taxa. Cultivated rye together with both wild annual and wild perennial accessions were mixed among each other. Sequence divergence (p) among taxa ofSecale was low, varying from 0.000 to 0.005, suggesting a rather recent origin of the genus.     

Pinus georginae (Pinaceae), a new species from western Jalisco, Mexico

Pinus georginae is described and illustrated as a new endemic species from the valleys in the southern basin of Río Ameca, Jalisco, western Mexico. It is closely related to Pinus praetermissa; however, after studying the cones and anatomy of the leaves in detail, characteristics were revealed that identify P. georginae as a distinct species.

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Resumen  Se describe e ilustra Pinus georginae como una especie nueva, endémica de algunos valles intermontanos al sur del Río Ameca, Jalisco, en el Occidente de México. Los árboles se parecen a Pinus praetermissa; sin embargo, al estudiar con detalle los conos y la anatomía de las acículas lo ubican a Pinus georginae como una entidad biológica con categoría específica independiente.

     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

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

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

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.     

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

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.     

Cytotaxonomy and phylogeny in certain species ofTaxodiaceae

Various species ofTaxodiaceae were selected for chromosome studies to indicate cytotaxonomic and phylogenetic relationships. Point dispersal patterns of diagrammatic presentations of the species' karyotypes, rather than marker chromosomes, were found to be the most significant cytotaxonomic characteristic in indicating phylogenetic relationships. Karyotypic evolution inTaxodiaceae appears to occur by unequal reciprocal translocations followed by pericentric and paracentric inversions. Cytotaxonomic relationships among species generally correspond to the phylogenetic relationships withinTaxodiaceae indicated by classical taxonomic classification. Presence and types of marker chromosomes may have the potential to indicate relationships between different coniferous families.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

Clone identification and clinal allozyme variation in populations ofAbies alba from the Eastern Alps (Austria)

Allelic frequencies at the polymorphic enzyme loci IDH-1, IDH-2, LAP-1, and 6-PGDH-1 were determined in population samples ofAbies alba from 5 different areas in Austria. The results reveal clear genetic differentiation between the eastern and the western provenances. Gradual frequency clines are observed with all four loci. The amount of variation, measured as average heterozygosity and genotypic diversity, is significantly higher in the west. The multilocus genotypes proved to be useful markers for the identification of clones. Functional differences with respect to substrate specifity were found for the two allozymes coded by the coexisting alleles of the IDH-2 locus.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.     

Chloroplast DNA diversity among wild and cultivated members of Cucurbita (Cucurbitaceae)

Summary Cladistic analysis of 86 chloroplast DNA restriction-site mutations among 30 samples representing 15 species of Cucurbita indicates that annual species of the genus are derived from perennials. The Malabar Gourd, C. ficifolia, is placed as a basal, sister taxon relative to other domesticated species and allied wild-types. The pattern of variation supports three species groups as monophyletic: (1) C. fraterna, C. pepo, and C. texana, (2) C. lundelliana, C. martinezii, C. mixta, C. moschata and C. sororia, and (3) C. foetidissima and C. pedatifolia. Domesticated samples representing subspecies of C. pepo are divided into two concordant groups, one of which is allied to wild-types referable to C. texana and C. fraterna. The data failed to resolve relationships among cultivars of C. moschata and C. mixta and their association to the wild C. sororia. The South American domesticate, C. maxima, and its companion weed, C. andreana, show close affinity and alliance to C. equadorensis.