Geographic structuring of chloroplast DNA genotypes inTiarella trifoliata (Saxifragaceae)

Tiarella trifoliata comprises varietieslaciniata, trifoliata, andunifoliata, and is distributed from southeastern Alaska to northern California. We analyzed restriction site variation of chloroplast DNA (cpDNA) using 23 endonucleases in 76 populations representing the entire geographic range of the species and the three recognized varieties. We also employed comparative restriction site mapping of PCR-amplified chloroplast DNA fragments using 16 restriction endonucleases. This species exhibits low cpDNA restriction site variation. No differentiation is evident among varieties of this species based on cpDNA data; some plants of each variety were characterized by each of the two major cpDNA types detected. The two major cpDNA clades, which differ by only a single restriction site mutation, are geographically structured. A northern clade comprises populations from Alaska to central Oregon; most populations analyzed from southern Oregon and California form a southern clade. Populations that possess the typical northern cpDNA type also occur disjunctly to the south at high elevations in the Siskiyou—Klamath Mountain area of southern Oregon and northern California. Conversely, the southern cpDNA type is found disjunctly to the north in the Olympic Peninsula of Washington. Both geographic areas characterized by disjunct cytoplasms are considered glacial refugia.Tiarella trifoliata joins two other species,Tolmiea menziesii andTellima grandiflora, in having well-demarcated northern and southern cpDNA lineages. All three species have similar life-history traits and geographic distributions. We suggest that glaciation may have played a major role in the formation of the cpDNA discontinuities present in these three taxa. The pronounced relationship between cpDNA variation and geographic distribution suggests the potential applicability of intraspecific phylogeography to plants via the analysis of intraspecific cpDNA variation. These three examples also join a rapidly growing data base which indicates that cytoplasms are often geographically structured within species and species complexes.     

INTRASPECIFIC CHLOROPLAST DNA VARIATION AND BIOGEOGRAPHY OF NORTH AMERICAN LIRIODENDRON L. (MAGNOLIACEAE)

Restriction site variation in chloroplast DNA (cpDNA) was surveyed to analyze population dynamics in Liriodendron tulipifera L., a woody angiosperm found in eastern North America. Two cpDNA haplotypes, differing by the presence or absence of five restriction site changes (nucleotide sequence divergence estimated as approximately 0.15%) are geographically structured; 61 widespread populations possess the “northern” haplotype and three isolated populations of central Florida possess the “southern” haplotype. This geographic break in cpDNA distribution corresponds to patterns of geographic distribution revealed by a previous survey of allozyme variation, with the exception that analyses of allozyme data further divided the populations containing the northern cpDNA haplotype into two groups, a widespread upland group and a coastal intermediate group. Analyses of these two independent data sets together support the hypothesis that L. tulipifera survived the glacial advances of the Pleistocene in two distinct refugia, possibly as different taxa, and the intermediate coastal group was putatively formed from recent hybridizations between these entities.     

Ribosomal and chloroplast DNA restriction site mutations and the radiation ofRobinsonia (Asteraceae: Senecioneae) on the Juan Fernandez Islands

Restriction site mutations in the chloroplast (cpDNA) and ribosomal DNA (rDNA) were examined in 41 populations representing five of the seven recognized species of the genusRobinsonia, which is endemic to the Juan Fernandez Islands. No intraspecific variation was detected for cpDNA but one population of one of the species (R. evenia) had a restriction site mutation in rDNA not detected elsewhere. No restriction site mutations were unique to all species ofRobinsonia relative to the species ofSenecio used as outgroups. All 13 mutations (eight from cpDNA and five from rDNA) are restricted to single species, and thus provide no cladistically useful information within the genus. The distribution of mutations is concordant with the hypothesis of a rapid adaptive radiation ofRobinsonia subsequent to the dispersal of its ancestor to Masatierra.     

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.     

Phylogenetic reticulation in subtribe Helianthinae

Incongruence between phylogenetic estimates based on nuclear and chloroplast DNA (cpDNA) markers was used to infer that there have been at least two instances of chloroplast transfer, presumably through wide hybridization, in subtribe Helianthinae. One instance involved Simsia dombeyana, which exhibited a cpDNA restriction site phenotype that was markedly divergent from all of the other species of the genus that were surveyed but that matched the restriction site pattern previously reported for South American species of Viguiera. In contrast, analysis of sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed Simsia to be entirely monophyletic and placed samples of S. dombeyana as the sister group to the relatively derived S. foetida, a result concordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sister group to a member of V. subg. Amphilepis, which was consistent with cpDNA restriction site data. Samples of Tithonia formed a single monophyletic clade based on ITS sequence data, whereas they were split between two divergent clades based on cpDNA restriction site analysis. The results suggested that cpDNA transfer has occurred between taxa diverged to the level of morphologically distinct genera, and highlight the need for careful and complete assessment of molecular data as a source of phylogenetic information.     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

CHLOROPLAST DNA VARIATION AND THE PHYLOGENY OF HORDEUM (POACEAE)

Restriction site variation in the chloroplast genome (cpDNA) was surveyed among 37 taxa or cytotypes (40 accessions) of the genus Hordeum. Seventeen restriction enzymes were employed, and a total of 491 restriction sites were assayed. Of these, 120 were variable among the taxa, including 70 synapomorphies. The level of sequence divergence (p) among species of Hordeum varied from 0.0 to 0.017, indicating that Hordeum possesses an about-average level of cpDNA diversity as compared to most other genera of flowering plants for which data are available. Wagner and polymorphism parsimony phytogenies were constructed from the restriction site data. These analyses divided the genus into several distinct groups; 1) American taxa; 2) diploid H. marinum; 3) Asian taxa; 4) H. vulgare-H. bulbosum; and 5) the H. murinum complex. Bootstrap-based confidence limits provided statistical support for the monophylesis of the latter three groups. The cpDNA data showed remarkably good congruence with previously published isoenzymatic, molecular, cytological, and crossing data.     

Chloroplast DNA variation of Panax (Araliaceae) in Nepal and its taxonomic implications

The restriction site and size variation of five PCR amplified fragments of noncoding chloroplast DNA (cpDNA) was examined in material from 13 populations ofPanax from Nepal and China. Fourteen restriction endonucleases produced 81 restriction site and length variations from the large single-copy region of cpDNA, 27 of which are polymorphic. The cpDNA dataset suggests two distinct groups ofPanax from Nepal (clades I and II). Clade I consists of two populations ofP. pseudoginseng subsp.pseudoginseng, and clade II is composed of material referable toP. pseudogingeng subsp.himalaicus (vars.himalaicus, angustifolius, andbipinnatifidus). The three accessions ofP. pseudoginseng subsp.japonicus andP. ginseng studied from China had cpDNA characters that differed from the HimalayanPanax. The highly distinctive cpDNA profile and morphology ofP. pseudoginseng subsp.pseudoginseng sensu Hara (1970) from central Nepal support its status as a separate species, which has an extremely restricted distribution.     

Intraspecific sequence variation of chloroplast DNA among the component species of evergreen broad-leaved forests in Japan

For the purpose of phylogeographic study of lucidophyllous (evergreen broad-leaved) forests in Japan, we surveyed intraspecific chloroplast DNA (cpDNA) variation in 41 component species of such forests. Intraspecific cpDNA variations were detected in 14 species. In 15 species and one species group, 16 non-coding cpDNA regions were examined to find intraspecific sequence variation. The extent of variation in these regions was compared. The largest amount of intraspecific variation was detected in the rps16 region. A relatively large amount of intraspecific variation was detected in the petD-rpoA, rpl16, and trnL-F regions. It is suggested that these regions of cpDNA would be useful for detecting intraspecific variation in plant species, and could provide valuable information for various research purposes.     

Cytoplasmic DNA variation and biogeography of Larix Mill. in Northeast Asia

Range‐wide variation in 54 populations of Dahurian larch (Larix gmelinii) and related taxa in Northeast Asia was assessed with four mitochondrial PCR‐RFLP and five chloroplast SSR markers. Eleven mitotypes and 115 chlorotypes were detected. The highest diversity was observed in the southern Russian Far East where hybrids of L. gmelinii, L. olgensis and L. kamtschatica are distributed. In contrast, only two mitotypes occurred in L. cajanderi and L. gmelinii. The Japanese larch (L. kaempferi) was found to be closely related to populations of L. kamtschatica inhabiting the Kuril Islands and South Sakhalin, populations from the northern part of Sakhalin being more closely related to continental species. In general, both mitochondrial (G_ST = 0.786; N_ST = 0.823) and chloroplast (G_ST = 0.144; R_ST = 0.432) markers showed a strong phylogeographical structure and evidence of isolation‐by‐distance. Yet both markers did not allow a clear delineation of species borders. In particular, and contrary to expectations, cpDNA was not significantly better than mtDNA at delineating species borders. This lack of concordance between morphological species and molecular markers could reflect extensive ancestral haplotype sharing and past and ongoing introgression. Finally the distribution of mtDNA and cpDNA variation suggests the presence of several refugia during Pleistocene glacial intervals. In particular, mtDNA and cpDNA reveal weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories of these species.     

CHLOROPLAST DNA VARIATION WITHIN AND AMONG GENERA OF THE HEUCHERA GROUP (SAXIFRAGACEAE): EVIDENCE FOR CHLOROPLAST TRANSFER AND PARAPHYLY

The Heuchera group (Saxifragaceae) comprises Bensoniella, Conimitella, Elmera, Heuchera, Lithophragma, Mitella, Tellima, Tiarella, and Totmiea. Earlier studies employing morphology, karyology, and flavonoid chemistry indicated that these genera form a natural group, but failed to resolve relationships among them. Restriction site analysis of chloroplast DNA (cpDNA) suggests that Bensoniella, Tolmiea, and Lithophragma are close allies and form the sister group of a large clade containing the remaining six genera. Mitella and Heuchera are both paraphyletic based on cpDNA data. cpDNA data, in conjunction with morphological and allozyme data, suggest at least four examples of intersectional hybridization and subsequent chloroplast capture in Heuchera. Several of these events may be explained via a stepping stone model in which the chloroplast genome of a species was captured by a second species, and then ultimately by a third taxon. Two well-differentiated groups of Tellima populations were detected: one group has a unique chloroplast genome characterized by nine autapomorphies, and the second group has a chloroplast genome identical to that found in M. trifida and M. diversifolia. cpDNA and allozyme data suggest that some Tellima populations probably obtained their chloroplast genome via intergeneric hybridization with M. trifida, M. diversifolia, or the ancestor of these taxa. The occurrence of intergeneric chloroplast transfer in some populations of Tellima, as well as extensive intersectional chloroplast capture in Heuchera, not only suggests caution in the use of cpDNA restriction site data in phylogenetic reconstruction, but also demonstrates again the importance of adequate sampling of conspecific populations. If the intergeneric relationships in the Heuchera group suggested by cpDNA analysis are accurate, fundamental questions arise regarding the validity of certain morphological traits as good taxonomic characters in Saxifragaceae. Furthermore, significant taxonomic changes at the generic level would be necessary.     

Evidence for phylogeographic structure in Lolium species related to the spread of agriculture in Europe. A cpDNA study

In order to explain the present distribution area of natural populations of two forage grasses species (Lolium perenne and L. rigidum), we studied genetic variation for maternally inherited chloroplast DNA (cpDNA) in 447 individual plants from 51 natural populations sampled throughout Europe and the Middle East. The detection of polymorphism by restriction analysis of PCR-amplified cpDNA fragments resulted in the identification of 15 haplotypes. Hierarchical analysis of chloroplastic diversity showed a high level of within-population diversity while, for both species, we found that about 40% of the total diversity still remains among populations. The use of previous isozymes data enabled us to estimate the pollen to seed flow ratio: pollen flow appears to be 3.5 times greater than seed flow for L. perenne and 2.2 times higher for L. rigidum. A stepwise weighted genetic distance between pairs of populations was calculated using the haplotypes frequencies of populations. A hierarchical clustering of populations clearly divides the two species, while two main clusters of L. perenne populations show a strong geographical structure. Different scenario are proposed for explaining the distribution area of the two species. Finally, evidence attesting that these geographical structures are related to the spread of agriculture in Europe are presented and discussed. Received: 5 November 1999 / Accepted: 24 November 1999     

Heteroplasmy of the chloroplast genome of Medicago sativa L. cv ‘Regen S’' confirmed by sequence analysis

The heteroplasmy of chloroplast DNA (cpDNA) observed in Medicago sativa L., which involves the presence (type B) or absence (type A) of an Xba I restriction site, was examined using closed fragments covering the variable XbaI site from type-A and type-B cpDNA. The 6.2-kb PstI fragment of DNA from type-A cpDNA (–XbaI) and from type-B cpDNA (+XbaI) was cloned into pUC19 plasmids. EcoRI fragments bearing the variable XbaI site from the type-A and type-B 6.2-kb PstI fragments were subcloned into pUC19. DNA sequences of both types of the 696-bp EcoRI fragments were determined and computer-assisted analysis of the sequence data carried out. Type-A cpDNA was found to differ from type-B cpDNA by 1 base, a G to T conversion, which results in a non-recognition site for XbaI in the type-A cpDNA. The sequence difference was in a non-coding region. Cloning and sequencing of the fragments verified the individual identity of the type-A and type-B cpDNA.     

Molecular diversity and derivations of populations of Silene acaulis and Saxifraga oppositifolia from the high Arctic and more southerly latitudes

A survey of allozyme diversity within and between populations of Silene acaulis from Spitsbergen, Norway, Iceland and Scotland, showed that populations from the high Arctic (Spitsbergen, > 76°N) contained high levels of diversity and were genetically similar to populations from more southern locations. Indirect measures of gene flow (Nm), calculated from Wrigh's F indicated that there had been extensive gene flow between Spitsbergen and some Norwegian populations. A restriction site analysis of chloroplast DNA (cpDNA) in S. acaulis revealed that all populations contained a single identical cpDNA haplotype, except one population from Norway which also contained a second haplotype. In contrast, five different cpDNA haplotypes were distinguished in a more limited survey of cpDNA variation in Saxifraga oppositifolia, with all five haplotypes present in one of two Spitsbergen populations surveyed. The contrasting cpDNA results for the two species suggest that whereas high-Arctic populations of Silene acaulis have most likely been derived from immigrants which arrived from the south after the last glacial period, high-Arctic populations of Saxifraga oppositifolia may be derived, in part, from ancient northern stocks which survived the last glaciation in high-Arctic refugia.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

EVOLUTIONARY IMPLICATIONS OF INTRASPECIFIC CHLOROPLAST DNA VARIATION IN DWARF DANDELIONS (KRIGIA; ASTERACEAE)

Intraspecific chloroplast DNA polymorphisms were examined for 51 populations of seven species in the genus Krigia. A total of 1,100 restriction sites was surveyed and 46 of these were variable at the intraspecific level. Twenty-two of the variable sites were found within K. virginica, giving this species one of the highest levels of intraspecific chloroplast DNA divergence of any examined species. In contrast, no restriction site variation was detected within K. dandelion, K. wrightii, and K. occidentalis. Five polymorphisms were identified from the 16 populations of the K. cespitosa-gracilis complex, but no mutations distinguished the K. cespitosa and K. gracilis types. Krigia montana and K. biflora showed 11 and eight restriction site polymorphisms, respectively. The chloroplast genome of the hexaploid K. montana was derived from the diploid K. biflora rather than the tetraploid K. montana. High levels of polymorphism were found in species having different ploidy levels, such as K. virginica, K. biflora, and K. montana. Furthermore, most mutations found in these three species were recorded from the tetraploid lineages. As a result, evolutionary rates between different ploidy levels differ significantly. The chloroplast DNA restriction site data suggest that all surveyed populations of the autotetraploid K. virginica originated from a common ancestor. Our results also indicate that certain regions of the chloroplast genome have changed more rapidly than others and have the potential to resolve evolutionary questions at the population level.     

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure

Summary The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.     

Chloroplast DNA and isozyme analysis of the progenitor-derivative species relationship between Senecio nebrodensis and S. viscosus (Asteraceae)

A comparison of chloroplast DNA (cpDNA) and isozyme diversity in Senecio nebrodensis L., a species restricted to certain mountain ranges in Spain, and S. viscosus L., a widespread ruderal species in Europe, revealed that S. viscosus possessed the more common of two cpDNA haplotypes resolved in S. nebrodensis and contained only a small subset of the alleles found in S. nebrodensis at enzyme coding loci. The two species shared one restriction length mutation and one site mutation in their cpDNA, which distinguished them from other European Senecio species examined previously. Taken overall, these results support the hypothesis that S. nebrodensis and S. viscosus are related as a progenitor-derivative pair of species. The fact that no novel alleles were found in S. viscosus would suggest a relatively recent origin of the species, most probably in late glacial or postglacial times.     

Molecular phylogeny ofIva (Asteraceae,Heliantheae) based on chloroplast DNA restriction site variation

Iva s.str. (comprising ten species) was examined by cpDNA restriction site variation to determine phyletic relationships within the group. The results were compared with relationships proposed from other data. A total of 86 restriction site mutations was detected, 47 of which proved phylogenetically informative. A single most parsimonious tree was obtained using both Wagner and Dollo parsimony. The tree revealed three main lineages that are congruent with the three chromosome lineages (base numbers of x = 16, 17, 18). The monophyly of the x = 16 and 18 groups was supported strongly by molecular data, while the monophyly of x = 17 lineage was only supported moderately. Relationships among the three lineages indicate that the sect.Iva is paraphyletic because sect.Linearbractea is nested within it. Both morphological data and the secondary chemical data are in agreement with the proposed cpDNA phylogeny. Because of this agreement, sect.Iva is revised such that,I. axillaris was excluded and positioned within the newly proposed sect.Rhizoma. Patterns and rates of cpDNA evolution were also examined. The results indicated an uneven evolution in the chloroplast genome with different rates of cpDNA evolution in at least a few species ofIva. However, the evolutionary clock hypothesis can not be rejected within most of the lineages inIva.     

Inheritance of plastid DNA in the Turnera ulmifolia complex (Turneraceae)

We used PCR to amplify most of the rbcL gene and identified restriction fragment length polymorphisms to study the inheritance of chloroplast DNA (cpDNA) in the cross between two taxonomic varieties of the Turnera ulmifolia L. complex, vars. angustifolia and velutina. We identified an Alu I restriction site polymorphism that distinguished the parental plants. All 23 progeny from the cross var. angustifolia × var. velutina, where var. angustifolia was the maternal parent, possessed the paternal cpDNA. Results for the reciprocal cross were more varied, and the 16 progeny showed maternal, paternal, or biparental inheritance. We believe this represents the first study of plastid inheritance for any species in the Tumeraceae. The results are unusual and warrant further investigation using other species in this family.