Inheritance Of Chloroplast DNA Research Articles

Identification of glacial refugia in south‐eastern North America by phylogeographical analyses of a forest understorey plant, Trillium cuneatum

AbstractAim We examine several hypotheses emerging from biogeographical and fossil records regarding glacial refugia of a southern thermophilic plant species. Specifically, we investigated the glacial history and post‐glacial colonization of a forest understorey species, Trillium cuneatum. We focused on the following questions: (1) Did T. cuneatum survive the Last Glacial Maximum (LGM) in multiple refugia, and (if so) where were they located, and is the modern genetic structure congruent with the fossil record‐based reconstruction of refugia for mesic deciduous forests? (2) What are the post‐glacial colonization patterns in the present geographical range?Location South‐eastern North America.Methods We sampled 45 populations of T. cuneatum throughout its current range. We conducted phylogeographical analyses based on maternally inherited chloroplast DNA (cpDNA haplotypes) and used TCS software to reconstruct intraspecific phylogeny.Results We detected six cpDNA haplotypes, geographically highly structured into non‐overlapping areas. With one exception, none of the populations had mixed haplotype composition. TCS analysis resulted in two intraspecific cpDNA lineages, with one clade subdivided further by shallower diversification.Main conclusions Our investigation revealed that T. cuneatum survived the LGM in multiple refugia, belonging to two (western, eastern) genealogical lineages geographically structured across south‐eastern North America. The western clade is confined to the south‐western corner of T. cuneatum’s modern range along the Lower Mississippi Valley, where fossil records document a major refugium of mesic deciduous forest. For the eastern clade, modern patterns of cpDNA haplotype distribution suggest cryptic vicariance, in the form of forest contractions and subsequent expansions associated with Pleistocene glacial cycles, rather than simple southern survival and subsequent northward colonization. The north–south partitioning of cpDNA haplotypes was unexpected, suggesting that populations of this rather southern thermophilic species may have survived in more northern locations than initially expected based on LGM climate reconstruction, and that the Appalachian Mountains functioned as a barrier to the dispersal of propagules originating in more southern refugia. Furthermore, our results reveal south‐west to north‐east directionality in historical migration through the Valley and Ridge region of north‐west Georgia.

Regeneration and molecular characterisation of two interspecific somatic hybrids of Citrus for potential rootstock improvement

SummaryTo produce allotetraploid somatic hybrid citrus rootstocks, protoplasts isolated from ‘Dancy’ tangerine or ‘Kinnow’ mandarin (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts of ‘Goutou’ sour orange (C. aurantium L.). Three and six plants were regenerated from these two fusions, respectively. They were confirmed to be tetraploids and diploids by flow cytometry. Nuclear DNA simple sequence repeats (SSR) analysis verified that they were true hybrids, with novel bands present. Chloroplast DNA SSR analysis showed random inheritance of chloroplast DNA (cpDNA) in the ‘Dancy’ + ‘Goutou’ hybrids, while all the cpDNA of the ‘Kinnow’ + ‘Goutou’ hybrids was derived from ‘Kinnow’, the callus parent. Cleaved amplified polymorphic sequence (CAPS) analysis using mitochondria-specific DNA (mtDNA) primer pairs, followed by restriction endonuclease digestion, showed that all the mtDNA of the ‘Dancy’ + ‘Goutou’ somatic hybrids was from ‘Dancy’, the callus parent; while that of the ‘Kinnow’ + ‘Goutou’ diploid hybrids was not distinguishable using various primer/endonuclease combinations. The potential of these two somatic hybrids for the improvement of Citrus rootstocks is discussed.

Mitochondrial and chloroplast phylogeography of Picea crassifolia Kom. (Pinaceae) in the Qinghai‐Tibetan Plateau and adjacent highlands

The disjunct distribution of forests in the Qinghai-Tibetan Plateau (QTP) and adjacent Helan Shan and Daqing Shan highlands provides an excellent model to examine vegetation shifts, glacial refugia and gene flow of key species in this complex landscape region in response to past climatic oscillations and human disturbance. In this study, we examined maternally inherited mitochondrial DNA (nad1 intron b/c and nad5 intron 1) and paternally inherited chloroplast DNA (trnC-trnD) sequence variation within a dominant forest species, Picea crassifolia Kom. We recovered nine mitotypes and two chlorotypes in a survey of 442 individuals from 32 populations sampled throughout the species' range. Significant mitochondrial DNA population subdivision was detected (G(ST) = 0.512; N(ST) = 0.679), suggesting low levels of recurrent gene flow through seeds among populations and significant phylogeographical structure (N(ST) > GST, P < 0.05). Plateau haplotypes differed in sequence from those in the adjacent highlands, suggesting a long period of allopatric fragmentation between the species in the two regions and the presence of independent refugia in each region during Quaternary glaciations. On the QTP platform, all but one of the disjunct populations surveyed were fixed for the same mitotype, while most populations at the plateau edge contained more than one haplotype with the mitotype that was fixed in plateau platform populations always present at high frequency. This distribution pattern suggests that present-day disjunct populations on the QTP platform experienced a common recolonization history. The same phylogeographical pattern, however, was not detected for paternally inherited chloroplast DNA haplotypes. Two chlorotypes were distributed throughout the range of the species with little geographical population differentiation (G(ST) = N(ST) = 0.093). This provides evidence for highly efficient pollen-mediated gene flow among isolated forest patches, both within and between the QTP and adjacent highland populations. A lack of isolation to pollen-mediated gene flow between forests on the QTP and adjacent highlands is surprising given that the Tengger Desert has been a geographical barrier between these two regions for approximately the last 1.8 million years.

Phylogeography of a northeast Asian spruce, Picea jezoensis, inferred from genetic variation observed in organelle DNA markers

Range-wide genetic variation of the widespread cold-temperate spruce Picea jezoensis was studied throughout northeast Asia using maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA markers. This study assessed 33 natural populations including three varieties of the species in Japan, Russia, China, and South Korea. We depicted sharp suture zones in straits around Japan in the geographical distribution pattern of mitochondrial haplotypes (GST=0.901; NST=0.934). In contrast, we detected possible extensive pollen flow without seed flow across the straits around Japan during the past population history in the distribution pattern of chloroplast haplotypes (GST=0.233; NST=0.333). The analysis of isolation by distance of the species implied that by acting as a barrier for the movement of seeds and pollen, the sharp suture zones contributed considerably to the level of genetic differentiation between populations. Constructed networks of mitochondrial haplotypes allowed inference of the phylogeographical history of the species. We deduced that the disjunction with Kamchatka populations reflects range expansion and contraction to the north of the current distribution. Within Japan, we detected phylogeographically different types of P. jezoensis between Hokkaido and Honshu islands; P. jezoensis in Honshu Island may have colonized this region from the Asian continent via the Korean peninsula and the species in Hokkaido Island is likely to have spread from the Asian continent via Sakhalin through land bridges. Japanese endemism of mitochondrial haplotypes in Hokkaido and Honshu islands might have been promoted by separation of these islands from each other and from the Asian continent by the straits during the late Quaternary.

Occurrence of Ploidy Variation in Camellia ×vernalis

Maternal inheritance of chloroplast DNA (cpDNA) in the genus Camellia was confirmed using the polymorphism of atpH‐atpI region. CpDNAs of all C.×vernalis cultivars showed the same type as those of C. sasanqua, and all the progenies from C.×vernalis, either open pollinated or crossed, had the same cpDNA type as their maternal plants. Flow cytometry subjected to estimate the ploidy level of C. japonica, C. sasanqua and C.×vernalis revealed that there is a very strong positive linear correlation (r 2 ＝0.981) between fluorescent intensity and ploidy level, suggesting that the method is useful to investigate the ploidy level in Camellia. Natural occurrence of ploidy variation was shown in the progenies of tetraploid C.×vernalis cultivars, and zygote patterns of the progenies are discussed.

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

AbstractSomatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.

Molecules and migration: biogeographical studies in cruciferous plants

In the past two decades our understanding of plant biogeography has been improved substantially by the introduction of various molecular marker systems. Especially within the angiosperms, maternally inherited chloroplast DNA based data sets have elucidated not only genetic relatedness but also geographic structuring of genetic variation. These findings were based on the observation that DNA molecules might mutate during migration, which consequently found its manifestation in the term phylogeography introduced in the late 80s by John Avise.

Rare recent natural hybridization in Hieracium s. str. – evidence from morphology, allozymes and chloroplast DNA

The first proven data on natural hybridization in the genus Hieracium s. str. are presented. Plants with intermediate morphological characters between the diploids H. alpinum and H. transsilvanicum were found in the Muntii Rodnei (Romanian Eastern Carpathians) in 2001 and in the Chornohora Mts (Ukrainian Eastern Carpathians) in 2003. While plants of intermediate morphology between usually so called basic species are usually tri- or tetraploid in Hieracium s. str., these plants were diploid (2n=18) like both parental species in this region. The Romanian plant did not produce fertile achenes in free pollination and in control backcrosses with H. transsilvanicum, two hybrids from Ukraine were completly seed sterile in free pollination and reciprocal crosses. Pollen stainability as an indirect measure of male fertility was quite high in the studied Ukrainian hybrid plants and similar to the parental taxa. Evidence from allozyme analysis also confirmed the hybrid origin of the studied plants. Sequencing and PCR-RFLP analyses of the trnT-trnL intergenic spacer revealed that all hybrid plants had the H. transsilvanicum chloroplast DNA haplotype. Maternal inheritance of chloroplast DNA in this particular cross was proved with artificial hybrids from reciprocal experimental crosses between H. alpinum and H. transsilvanicum. In both localities, the natural hybrid plants were found in disturbed habitats, exceptionally allowing contact of the otherwise ecologically vicariate parental species. Morphologically, the hybrid plants belong to H. × krasani Wol.

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids ( Dactylorhiza maculata group)

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or “spotted marsh-Orchids”. These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.

Chloroplast DNA Inheritance in the Orchid Anacamptis palustris Using Single-Seed Polymerase Chain Reaction

The modality of chloroplast inheritance in orchids has been investigated only in a few species due to the difficulties associated with the analysis of large progeny numbers from experimental crosses. To test chloroplast DNA inheritance in the orchid Anacamptis palustris, we took advantage of the presence of a highly variable minisatellite repeat located in the tRNA(LEU) intron in the chloroplast genome. Seed progeny obtained from experimental crosses between parental individuals carrying different chloroplast DNA (cpDNA) minisatellite repeat numbers were analyzed using a single-seed polymerase chain reaction (PCR) protocol. All examined seeds displayed the maternal cpDNA haplotypes, indicating that cpDNA inheritance is strictly maternal in this Mediterranean orchid species. No evidence for paternal leakage was found. This finding concurs with results obtained from PCR amplifications of pollen massulae that exclude the presence of chloroplast DNA in the pollen tetrads.

A Genetically Confirmed Intersubgeneric Hybrid in Nymphaea L. (Nymphaeaceae Salisb.)

A synthetic F1 water-lily hybrid has been obtained for the first time using parental species originating from different subgenera of Nymphaea. The cross was accomplished using Nymphaea gigantea Hook. `Andre Leu' (subgenus Anecphya Casp.) as the maternal parent and a white-flowered variant of Nymphaea colorata Peter (subgenus Brachyceras Casp.) as the paternal parent. Morphologically, the hybrid possesses some characteristics of both parents, some intermediate features, and some unique traits. The cross was confirmed using DNA sequencing and molecular cloning techniques to compare biparentally inherited nuclear genetic markers in the parents and hybrid plant. Each parent possessed distinct alleles that were found to combine in the hybrid. Maternally inherited chloroplast DNA sequences confirmed N. gigantea as the maternal parent of the cross.

Maternal inheritance of chloroplast DNA in intergeneric sexual hybrids of “true citrus fruit trees” revealed by PCR-RFLP analysis

SummaryPCR-RFLP analysis was employed to clarify the inheritance pattern of chloroplast DNA (cpDNA) in six kinds of intergeneric sexual hybrids among five genera, i.e. Fortunella, Eremocitrus, Poncirus, Microcitrus and Citrus within the “true citrus fruit trees” group of the subtribe Citrinae (Rutaceae). Two cpDNA gene regions were amplified from each material via PCR as following: one region of tRNA (trnD-trnT) and a variable region bounded by the conserved sequences in ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) and open reading frame 106 (ORF106). Twelve restriction endonucleases (MspI, XbaI, RsaI, AluI, HinfI, ScrFI, StyI, HaeIII, Sau3AI, NdeII, HhaI and TaqI) were used for RFLP analysis of the amplified regions of cpDNA. Polymorphisms were detected with five (HinfI, RsaI, NdeII, ScrFI and XbaI) and four (HinfI, RsaI, HaeIII and Sau3AI) enzymes for rbcL-ORFl06 and trnD-trnT regions, respectively. All progeny examined in each kind of the intergeneric sexual hybrid exhibited maternal inheritance of cpDNA. Also, cpDNA analysis for determining the maternal parent of a natural hybrid, Microcitrus virgata (Sydney hybrid) indicated that M. australis (Planch.) Swing. was the female parent of M. virgata.

Spatial patterns of maternal lineages and clones of Galium odoratum in a large ancient woodland: inferences about seedling recruitment

Summary We investigated spatial genetic patterns in the clonal herbaceous plant species Galium odoratum L., which has previously been shown to be significantly associated with ancient woodlands. A maternally inherited chloroplast DNA marker was developed for G. odoratum. Seven chloroplast haplotypes were detected in the study area and mapped at different spatial scales. Their spatial patterns suggest that both local and non‐local gene flow occurs via seeds. Fingerprints from DIG‐labelled AFLP technology were validated as markers for G. odoratum clone identification. A single primer–enzyme combination was sufficient to distinguish between clones. A typical patch of G. odoratum is composed of numerous genets, mainly occurring in separate clusters of ramets, which may be several meters in diameter. Pairwise similarities, calculated from the AFLP banding patterns, were used for spatial autocorrelation analysis. The distinct spatial genetic structure at the patch level may be due to the genetic similarity of neighbouring genets or to clonal growth. The spatial patterns obtained using the two DNA markers, suggest that a mixture of local and non‐local gene flow via both seeds and pollen is occurring. Repeated seedling recruitment (RSR) would be consistent with the life history of G. odoratum in ancient woodlands with long‐term spatial continuity.

Evidences for multiple maternal lineages of Caryocar brasiliense populations in the Brazilian Cerrado based on the analysis of chloroplast DNA sequences and microsatellite haplotype variation.

In this work we report on the phylogeography of the endangered tree species Caryocar brasiliense based on variability in two classes of maternally inherited chloroplast DNA sequences with different rates of molecular evolution. Eleven sequence haplotypes of a noncoding region between the genes trnT and trnF and 21 distinct 10-locus microsatellite haplotypes could be identified in a total of 160 individuals, collected in 10 widespread populations of C. brasiliense. An amova indicated that most of the variation can be attributed to differences among populations, both for DNA sequence (87.51%) and microsatellites (84.38%). Phylogeography based on a median-joining network analysis of the noncoding region showed a sharp difference from the analysis of microsatellite haplotypes. Nevertheless, both analyses indicated that multiple lineages may have contributed to the origin of C. brasiliense populations in Brazilian Cerrado. Incongruences in the microsatellite haplotypes network suggest that homoplasy, which emerged from recurrent and independent mutations, greatly influenced the evolution of the C. brasiliense chloroplast genome. We hypothesize that our results may show the outcome of the restriction of ancient relic populations to moist refugias during extended droughts coinciding with glaciation in the northern hemisphere. The subsequent spread to favourable areas throughout Central Brazil may have caused contact between different lineages during the interglacial periods. The extinction of megafauna dispersers in the last glaciation may have caused a restriction in seed movement and currently, gene flow has been occurring mainly by pollen movement.

Hybrid bridges to gene flow: a case study in milkweeds (Asclepias).

Natural hybridization occurs throughout areas of sympatry for the North American milkweeds Asclepias exaltata and A. syriaca (Asclepiadaceae), even though the formation of F1 hybrid seed is a rare event. For introgressive hybridization to proceed, F1 and advanced hybrids must be released from reproductive barriers and successfully mate with one or both parental species. I investigated the mating system of natural hybrids between A. exaltata and A. syriaca in three populations in Shenandoah National Park, Virginia. Allozyme data and a maximum-likelihood procedure were used to estimate the frequency of six genotypic classes (parentals, F1, F2, and backcrosses) of the hybridizing populations, the pollinia received by hybrid plants, and the paternal parents of seeds produced by hybrids. F1 hybrids, backcross A. syriaca, and parental A. syriaca individuals were common in three hybrid populations. Even though self-pollinations and interhybrid pollinations were common, F2 seed production and the occurrence of F2 individuals were rare in hybrid populations. Hybrid plants received more pollen from A. syriaca than A. exaltata, which resulted in the production of more backcross-A. syriaca seed than backcross-A. exaltata seed. Asclepias exaltata was rare in the hybrid populations, but A. exaltata pollinia were received by hybrids and this species sired between 15% and 36% of the seeds produced on hybrids. The potential for introgression with A. exaltata populations is lower because this species is unsuccessful as the maternal parent in interspecific and backcross hand-pollinations. The asymetry of hybridization with A. syriaca as the maternal parent is further supported by the incorporation of maternally inherited chloroplast DNA markers in hybrids. Hybrid milkweeds frequently backcross with both parental species and may be released from the reproductive barriers that limit the formation of F1 hybrids in natural populations. The direction of interspecific gene flow and introgression in milkweeds is influenced by the reproductive biology of hybrids, the constituency of the surrounding population, and failure of some crosses to produce seeds. Finally, introgressive hybridization remains an important evolutionary force even when the initial formation of F1 hybrids in natural populations is rare.

A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in Chlamydomonas maternal gametes.

Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt(+)) gametes is heavily methylated whereas that of paternal (mating type minus; mt(-)) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt(+) gametes, and not in mt(-) gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt(+) and mt(-) vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt(+) but not mt(-) cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt(+) gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt(-) mutant, mat-1, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii.

Reticulation or divergence: the origin of a rare serpentine endemic assessed with chloroplast, nuclear and RAPD markers

Species of Armeria from the southern part of the Iberian Peninsula have been postulated to undergo extensive reticulate evolution based on ITS sequences as well as morphometric, eco-geographical evidence and crossing experiments. The hypothesis that a rare serpentine endemic (A. villosa subsp. carratracensis) originated from a cross between another serpentine tolerant endemic (A. colorata) and a widespread limestone species (A. villosa subsp. longiaristata) is tested with molecular markers. Sequences from two chloroplast regions [trnL (UAA)-trnF (GAA) and trnD (GUC)-trnT (GGU)], from nuclear ribosomal DNA (ITS-1 + 5.8S + ITS-2) as well as RAPD data are used. Variation in chloroplast and nuclear sequence data shows a clear geographical structure irrespective of taxonomic ascription. The putative hybrid taxon shares locally restricted haplotypes and ribotypes both with A. colorata and populations of A. villosa subsp. longiaristata. Species specific markers for the progenitors have not been found and thus a divergent scenario cannot be conclusively excluded. A principal coordinate analysis as well as a UPGMA analysis of the RAPD data based on the Dice coefficient are consistent with the hybrid origin. A joint examination of the various markers together with the previous biological evidence and the geographical structure of the chloroplast and ITS markers raises the likelihood of the reticulate hypothesis. Reasons for inconclusiveness are discussed. Using PCR-RFLPs of artificially synthesized hybrids and backcrosses, evidence for a maternal inheritance of chloroplast DNA in Armeria is provided.

White oaks phylogeography in the Iberian Peninsula

The geographic distribution of maternally inherited chloroplast DNA polymorphisms was studied to determine the phylogeographic structure of white oaks in the Iberian Peninsula. Almost 1000 mature trees from nearly 200 populations were sampled in the distribution areas of the six white oak species that are recognised in Iberia. The analysed trees roughly represent the species distribution and their respective abundance. The PCR–RFLP procedure used by all project partners revealed up to 14 cpDNA haplotypes. Seven of them had not been previously described and have not been found elsewhere in Europe. Phylogenetic relationships among the haplotypes support the existence of four maternal lineages, one of them being restricted in Europe to the Mediterranean Spain and Corsica. Genetic diversity analysis confirmed the previously described extensive sharing of haplotypes among species, but also detected some species effect in intra- and inter-specific cytoplasmic gene flow. Geographic structuration of genotypes was studied by means of autocorrelation analysis. Autocorrelation was significant for ordered and unordered alleles both when considering all populations and when analysis was restricted to lineage B. However, in both instances there was not significant autocorrelation for the contribution of the genetic divergence between haplotypes to the total coefficient of differentiation. Autocorrelograms reflect the existence of a patchy distribution of haplotypes at different scales when all lineages are considered and when only lineage B is taken into account. Finally, discussion centres on the possible role played by the Iberian Peninsula as the European westernmost refugia for the white oaks during the last glacial period. We present several lines of evidence that make us to favour the persistence of white oaks in small northern refugia during the full glacial period.

Is there a correlation between chloroplastic and nuclear divergence, or what are the roles of history and selection on genetic diversity in European oaks?

The aim of this work was to investigate whether a correlation exists between maternal lineage, assessed by variation in maternally inherited chloroplast DNA (cpDNA) and nuclear controlled variation (phenotypic traits and gene markers). Variation in cpDNA and nuclear controlled traits (62 phenotypic traits, eight isozyme and 31 RAPD loci) was studied in deciduous oak trees (mostly Q. petraea) growing in 16 provenance tests. Results from two nuclear diversity studies were also included. The test for correlation was performed using two methods by: (1) comparing provenance mean values (or allele frequencies) among different lineages using ANOVA, (2) making pairwise comparisons of chloroplastic genetic distances (CGDs) with phenotypic differentiation index (DI) (or nuclear genetic distances (GD)) among all provenances using the Mantel test. Among the 62 phenotypic traits, only seven exhibited significant associations with maternal lineages when tested using ANOVA (six using Mantel test). This number decreased to two once correction for geographic distance was introduced in the calculation for Mantel test. There were stronger correlations between maternal lineage and nuclear gene markers. The existence of cytonuclear disequilibrium was shown by the significant differences in allozyme frequencies between the four maternal lineages (at least one allele for each locus). These associations were confirmed by significant correlations between CGD and GD. Finally associations were also found between levels of diversity in nuclear markers and maternal lineages. These results are discussed in the context of the glacial and postglacial history of oak populations in Europe. The analysis suggests that the processes which led to the current structure of chloroplastic diversity and variation for phenotypic traits can be subdivided into four major phases. (1) During the last long glacial period, deciduous oaks were probably confined to three major refugia which were genetically differentiated for chloroplastic and nuclear genes. (2) At the end of the glacial period, oaks migrated northwards and established a spatial pattern of distribution for chloroplastic genes which remains largely intact to this day. (3) As oaks progressively occupied the mid and northern parts of Europe, pollen flow established communication between stands originating from eastern and western refugia. This resulted in the gradual erosion of the original nuclear differentiation while conserving the chloroplastic variation installed during colonization. (4) Local selection pressures acting on the established populations eventually caused their genetic differentiation gradually to increase with time. New patterns of differentiation now exist which are totally different from those in place immediately following colonization. During this time, chloroplastic divergence remained unchanged. These processes led to the loss of any association between chloroplastic divergence and phenotypic traits, although some association with gene markers which are less affected by selection has been retained.

Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.