Comparison of chloroplast and nuclear phylogeny in the autogamous annualMicroseris douglasii (Asteraceae: Lactuceae)

Morphology suggests that the Californian annualMicroseris douglasii is a monophyletic sister group to the other three diploid annuals ofMicroseris. Phylogenetic analysis of 44 inbred strains ofM. douglasii derived from 23 populations with 72 RAPD markers in the nuclear DNA strongly supports this phylogeny. However, 13 chloroplast RFLPs divideM. douglasii into four distinct groups. Two of these each share one or more cpRFLPs withM. bigelovii andM. pygmaea. Several hypotheses can explain the incongruence between nuclear and chloroplast phylogeny: (1) random sorting out of chloroplasts during phylogeny from a polymorphic pool, (2) cytoplasmic introgression from the related annualM. bigelovii intoM. douglasii after hybridization followed by elimination of theM. bigelovii nuclear genome. We suggest cytoplasmic introgression as the most likely origin. Possible remnants of nuclear introgression have been found in two populations ofM. douglasii that are polymorphic for chloroplast types. In these populationsM. bigelovii type chloroplast DNA seems to be accompanied by nuclear genes for flower color and leaf shape.     

Chloroplast and nuclear DNA variation among homozygous plants in a population of the autogamous annualMicroseris douglasii (Asteraceae,Lactuceae)

The autogamous diploid annualMicroseris douglasii of California occurs in many isolated populations. The populations consist of one to many highly inbred biotypes. Morphological variation among populations usually is greater than within populations. In spite of the virtual absence of gene flow even within populations, genetically determined character differences are randomly distributed and associated throughout the range of the species. Recent evidence even suggests introgression of chloroplasts from the relatedM. bigelovii. Offspring families from 25 plants of a very variable population were raised and examined for segregation of morphological and molecular (RAPD) markers. All 25 original plants were completely homozygous for all markers, but each differed from all others at least in some markers. The population consisted of two genetically isolated groups of plants: a distinct inbred line (3 plants) and 22 plants with random associations of a common set of markers and characters, possibly recombinant inbreds from a past hybridization event. One of these 22 plants contained a chloroplast genome found inM. bigelovii, the other 24 plants a chloroplast genome found only inM. douglasii.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.     

Allopolyploid origin of highly invasive Centaurea stoebe s.l. (Asteraceae)

Spotted knapweed (Centaurea stoebe) occurs from Western Asia to Western Europe both as diploid and tetraploid cytotypes, predominantly in single-cytotype populations with higher frequency of diploid populations. Interestingly, only tetraploids have been recorded so far from its introduced range in North America where they became highly invasive. We performed phylogenetic and network analyses of more than 40 accessions of the C. stoebe and C. paniculata groups and other related taxa using cloned internal transcribed spacer (ITS) and sequences of the chloroplast trnT-trnL and atpBrbcL regions to (i) assess the evolutionary origin of tetraploid C. stoebe s.l., and (ii) uncover the phylogeny of the C. stoebe group. Both issues have not been studied so far and thus remained controversial. Cloned ITS sequences showed the presence of two slightly divergent ribotypes occurring in tetraploid cytotype, while only one major ribotype was present in diploid C. stoebe s.str. This pattern suggests an allopolyploid origin of tetraploids with contribution of the diploid C. stoebe s.str. genome. Although we were not able to detect the second parental taxon, we hypothesize that hybridization might have triggered important changes in morphology and life history traits, which in turn may explain the colonization success of the tetraploid taxon. Bayesian relaxed clock estimations indicate a relatively recent--Pleistocene origin of the tetraploid C. stoebe s.l. Furthermore, our analyses showed a deep split between the C. paniculata and C. stoebe groups, and a young diversification of the taxa within the C. stoebe group. In contrast to nrDNA analyses, the observed pattern based on two cpDNA regions was inconclusive with respect to the origin and phylogeny of the studied taxa, most likely due to shared ancient polymorphism and frequent homoplasies.     

The karyotypes ofMicroseris bigelovii,M. douglasii,andM. pygmaea (Asteraceae)

The karyotypes of the three annuals,Microseris bigelovii, M. douglasii andM. pygmaea, consist of 2n = 18, small, submetacentric chromosomes. Length, centromere position, C-banding pattern, silver staining of NOR's, and the use of base specific fluorochromes, allow the identification of four of the nine chromosome pairs. The banding pattern ofM. bigelovii andM. pygmaea is identical, but intraspecific differences are found between strains ofM. douglasii.     

Molecular evidence for multiple polyploidization and lineage recombination in the Chrysanthemum indicum polyploid complex (Asteraceae)

The Chrysanthemum indicum polyploid complex comprises morphologically differentiated diploids, tetraploids and hybrids between C. indicum and C. lavandulifolium. The relationships between species and cytotypes within this complex remain poorly understood. Random amplified polymorphic DNAs (RAPDs), intersimple sequence repeats (ISSRs) and chloroplast SSR markers were used to elucidate the genetic diversity and relationships of the C. indicum polyploid complex. Molecular analysis of three diploid and nine tetraploid populations provided strong evidence for recurrent origins and lineage recombination in the C. indicum polyploid complex. The high similarity in molecular marker profiles and cpDNA haplotypes between the diploids and tetraploids distributed in the Shen-Nong-Jia Mountain area of China suggested an autopolyploid origin of the tetraploids, while the tetraploids from other populations may have originated via allopolyploidization. Lineage recombination was revealed by the extensive sharing of chloroplast haplotypes and genetic markers among the tetraploid populations with different origins. Multiple differentiation and hybridization/polyploidization cycles have led to an evolutionary reticulation in the C. indicum polyploid complex, and resulted in the difficulties in systematic classification.     

Genetic analysis of aMicroseris douglasii (Asteraceae) population polymorphic for an alien chloroplast type

Recent evidence suggests chloroplast introgression fromMicroseris bigelovii intoM. douglasii. We have examined 23 plants from a population ofM. douglasii polymorphic forM. douglasii andM. bigelovii chloroplast types. All 23 plants were completely homozygous for morphological and RAPD markers, and inbred lines derived by selfing have been used for DNA analysis. Chloroplast RFLP analysis identified 16 plants withM. bigelovii chloroplasts and seven withM. douglasii chloroplasts. The nuclear genomes of the 16 plants withM. bigelovii chloroplasts were examined with 22 primers for RAPD amplification products shared exclusively withM. bigelovii. Five of 268 markers appeared to be shared betweenM. bigelovii and one or more of these 16 plants on the basis of their position in gels. Detailed examination of these five amplification products showed that none of them are nuclear DNA fromM. bigelovii. Very little, if any, nuclear DNA fromM. bigelovii can be present inM. douglasii plants with chloroplasts typical ofM. bigelovii. The study demonstrates the usefulness of the RAPD technique for screening large numbers of markers to select a few potentially informative ones for rigorous examination.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

粗茎秦艽与西藏秦艽(龙胆科)种间的形态学、染色体与分子界定

粗茎秦艽Gentiana crassicaulis Duthie ex Burk和西藏秦艽G. tibetica King ex Hook. f.的主要区别是前者花冠小、檐部蓝色, 后者花大、檐部紫褐色; 但二者在西藏东南部地区的形态界限不清楚, 具有较多的中间形态过渡类型。本文以该地区居群作为主要研究对象, 并以云南和西藏其他地区的典型形态类群为对照, 比较研究了两个物种间的形态学、染色体和分子标记界定。共研究了10个居群69个个体的trnS-G和ITS序列、6个居群26株植株的染色体倍性和54份标本的形态变异。结果表明, 花冠大小与染色体倍性密切相关。云南等地分布的粗茎秦艽均为二倍体(2n=26), 花冠较小; 西藏西部的西藏秦艽均为四倍体(2n=52), 花冠较大。而西藏东南部的形态过渡类型也为四倍体, 花冠较大, 且其中的部分植株花颜色变浅, 类似西藏秦艽。但母系与双亲遗传背景的分子标记都表明西藏东南部居群与典型形态特征上的粗茎秦艽聚为一支, 应属于粗茎秦艽; ITS序列上也未发现杂合位点。这些分子证据都不支持近期杂交渗入造成形态过渡性的假设。综合分析表明多倍化造成了粗茎秦艽种内形态特征的变异, 其四倍体个体与四倍体西藏秦艽在形态特征上的趋同, 最终导致了二者在西藏东南部地区形态界限的模糊。但是, 分子证据可以很好地区分这种多倍化造成的形态趋同。我们的研究结果还表明, 综合利用分子、染色体和广义形态证据来解决近缘种之间种范围的界定具有更大的优势。此外, 还讨论了粗茎秦艽不同染色体倍性的分布以及该高山物种可能的起源与扩散途径。     

Chloroplast DNA variation in a wild plant, tolmiea menziesii

Few studies of cpDNA have provided evolutionary and/or phylogenetic information at the intraspecific level. We analyzed restriction site variation using 19 endonucleases in 37 populations representing both diploid (2n = 14) and autotetraploid (2n = 28) Tolmiea menziesii. Seven restriction site mutations and five length mutations were observed. Although diploid and tetraploid Tolmiea have been intensively studied using nuclear markers, cpDNA variation provided additional evolutionary insights not revealed previously. The chloroplast genomes of diploid and tetraploid Tolmiea are as distinct as those of many pairs of congeneric species of angiosperms. Based on outgroup comparisons, the primitive chloroplast genome is present in tetraploid rather than diploid Tolmiea. These findings suggest that either: (1) diploid and tetraploid Tolmiea may have diverged since the origin of the autotetraploid, (2) the original diploid donor of the cytoplasm present in the tetraploid subsequently became extinct, or (3) the diploid was actually derived from the tetraploid via polyhaploidy. cpDNA variation also revealed that despite their close geographic proximity, diploid and tetraploid Tolmiea do not experience cytoplasmic gene flow. Last, three cytoplasmically distinct groups of diploid populations exist, two of which occupy distinct geographic areas. These findings demonstrate that, at least in some plant species, restriction fragment analysis of cpDNA can provide important evolutionary and phylogenetic information at low taxonomic levels.     

云贵高原多星韭二倍体—四倍体分布格局研究

采用染色体压片技术对云贵高原17个多星韭居群,共412株个体进行染色体数目和核型研究。结合已有的细胞学资料,对云贵高原多星韭多倍体分布格局进行了研究。9个二倍体居群、6个二倍体与四倍体混生居群分布于云南中部至西北部,12个四倍体居群分布于整个云贵高原。二倍体除香格里拉d和保山居群核型类型为3A型外,其余均为2A型,四倍体除会泽和赫章居群为2 B型外,其余均为2A型。云南西北部四倍体核型不对称指数低于其它分布区,推测该地区四倍体可能起源较早。多星韭二倍体与四倍体核型类型和形态并无明显差异,表明多星韭四倍体可能为同源多倍体。     

Nuclear ribosomal ITS sequences and phylogeny in East AsianAconitum subgenusAconitum (Ranunculaceae), with special reference to extensive polymorphism in individual plants

Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) were used to examine the phylogeny of East Asian aconites. Individual aconites were discovered to contain as many as eight different ITS sequences after cloning and PCR-SSCP (single-stranded conformational polymorphisms) analysis. We identified eight putative ITS pseudogenes from four taxa with low predicted secondary structure stability and high substitution rates. Maximum likelihood (ML) and neighbor-joining (NJ) methods were used for phylogenetic reconstruction. The ITS trees agree with the previous chloroplast DNA (cpDNA) tree for the vast majority of the taxa. We found two East Asian clades in the ITS trees: 1) a clade with the Chinese diploid,Aconitum volubile and East Asian tetraploids, and 2) a clade of East Asian diploids and Siberian tetraploids. In the former clade, most tetraploid taxa appear to be polyphyletic; sequences from individual plants did not correspond to recognized taxonomic units. This indicates a recent divergence of the East Asian tetraploids.     

Phylogeny of the Malus (apple tree) species,inferred from its morphological traits and molecular DNA analysis

Some debated issues of the genus Malus (apple) taxonomy were examined using a variety of species from the collection of the Maikop Experimental Station, Vavilon Research Institute of Plant Industry (Krasnodar krai). Phylogenetic relationships among these species were studied using traditional analysis of morphological traits, RAPD, and complete sequencing of the 5'-internal transcribed spacer (ITS1), 5.8S rRNA, 3'-internal transcribed spacer (ITS2) (constituting a cluster of the rRNA genes), and the terminal fragment of the matK gene encoding chloroplast maturase. The results showed that the Sorbomalus section was polyphyletic; the American apple M. fusca was closely related to the species contributing to the East Asian center of the genus origin, and the American species M. angustifolia, M. coronaria, and M. ioensis were closely related to the M. trilobata relict species, whose assignment to the genus Malus is debated by some authors. Molecular analysis of the species relationships showed that the Middle Asian apple M. sieversii is the species from which apple domestication started.     

Population genetic structure of diploid sexual and polyploid apomictic hawthorns (Crataegus; Rosaceae) in the Pacific Northwest

Polyploidy and gametophytic apomixis are two important and associated processes in plants. Many hawthorn species are polyploids and can reproduce both sexually and apomictically. However, the population genetic structure of these species is poorly understood. Crataegus douglasii is represented exclusively by self-compatible tetraploid pseudogamous apomicts across North America, whereas Crataegus suksdorfii found in the Pacific Northwest is known to include self-incompatible diploid sexuals as well as polyploid apomicts. We compare population structure and genetic variability in these two closely related taxa using microsatellite and chloroplast sequence markers. Using 13 microsatellite loci located on four linkage groups, 251 alleles were detected in 239 individuals sampled from 15 localities. Within-population multilocus genotypic variation and molecular diversity are greatest in diploid sexuals and lowest in triploid apomicts. Apart from the isolation of eastern North American populations of C. douglasii , there is little evidence of isolation by distance in this taxon. Genetic diversity in western populations of C. douglasii suggests that gene flow is frequent, and that colonization and establishment are often successful. In contrast, local populations of C. suksdorfii are more markedly differentiated. Gene flow appears to be limited primarily by distance in diploids and by apomixis and self-compatibility in polyploids. We infer that apomixis and reproductive barriers between cytotypes are factors that reduce the frequency of gene flow among populations, and may ultimately lead to allopatric speciation in C. suksdorfii . Our findings shed light on evolution in woody plants that show heterogeneous ploidy levels and reproductive systems.     

Phylogenetic relationships and genetic diversity of the Salicornieae (Chenopodiaceae) native to the Atlantic coasts of France

Phylogenetic relationships of members of the tribe Salicornieae, native to the Atlantic coasts of France, were assessed by three molecular markers: the nuclear ribosomal internal transcribed spacer (ITS), the chloroplast trnL-F and the chloroplast matK sequences. In parallel to the phylogenetic studies, a population genetic study was carried out based on randomly amplified polymorphic DNAs (RAPD). Neither the MP/ML analyses of the sequences, nor the AMOVA and NJ analyses of RAPD fingerprints confirmed the morphology-based classification at the specific level within Salicornia. Instead, our investigations are in favour of the species aggregate concept. Two sister groups were revealed in the genus: one is composed of the diploid taxa, while the other clusters the tetraploid taxa. Conflicting nuclear versus plastid phylogenetic positions of some tetraploid samples, referred to as S. fragilis, indicate that they most likely derive from a reticulate evolution.     

GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)

Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trn L (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trn L intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trn L intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi.     

Nuclear DNA markers of the Australian tetraploid Microseris scapigera and its North American diploid relatives

The allotetraploid lactucean Microseris scapigera of Australia and New Zealand has presumably arisen in western North America by hybridization between an annual and a perennial diploid species followed by polyploidization and long-distance dispersal. A phylogenetic tree of various North American diploids, based on RFLPs in the nuclear DNA, confirmed the division of the genus into a clade containing the diploid annuals and a clade containing the diploid perennials. Four RFLP markers were shared among all accessions of M. scapigera and all the diploid accessions. Twelve markers found in the outgroup (Uropappus lindleyi) were absent in all Microseris. A cladogram of plants from six populations of M. scapigera based on eight RFLP markers shows a progressive specialization of three clades of two populations each. Two populations without any markers differentiating them from the North American diploids form the basic clade. These consist of plants with an apparently derived morphology that are self-compatible (or agamospermic) and thereby differ from most M. scapigera. Few markers in M. scapigera could be attributed to one or the other parental genome. As yet, we have found only one ITS 1 sequence of the nuclear ribosomal cistrons in M. scapigera. This sequence has features of both parental sequences.     

Molecular phylogeny and reticulate origins of the polyploid Bromus species from section Genea (Poaceae)

The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.     

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.