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Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae).

We investigated the evolutionary dynamics of duplicated copies of the granule-bound starch synthase I gene (GBSSI or Waxy) within polyploid Spartina species. Molecular cloning, sequencing, and phylogenetic analyses revealed incongruences between the expected species phylogeny and the inferred gene trees. Some genes within species were more divergent than expected from ploidy level alone, suggesting the existence of paralogous sets of Waxy loci in Spartina. Phylogenetic analyses indicate that this paralogy originated from a duplication that occurred prior to the divergence of Spartina from other Chloridoideae. Gene tree topologies revealed three divergent homoeologous sequences in the hexaploid S. alterniflora that are consistent with the proposal of an allopolyploid origin of the hexaploid clade. Waxy sequences differ in insertion-deletion events in introns, which may be used to diagnose gene copies. Both paralogous and homoeologous coding regions appear to evolving under selective constraints.

Base Sequence↗

Exclusive localization of tandem repetitive sequences in subtelomeric heterochromatin regions of Leymus racemosus (Poaceae, Triticeae).

Two kinds of tandem repetitive sequences were isolated from Leymus racemosus (Lam.) Tzvelev. One of them was classified in the 350-bp family originally isolated from Secale. The other was a novel repetitive sequence family, named 'TaiI family', which consisted of a repeat unit of 570 bp. Fluorescence in-situ hybridization of the chromosomes of L. racemosus indicated that both families were located in subtelomeric heterochromatin and that the 350-bp family and TaiI family occupied different heterochromatin regions. In addition, even homologous chromosomes did not show the same patterns of TaiI and 350-bp families. The combination of these two families of repetitive sequences, together with Afa-family sequences and rDNAs, helps to identify the ten homologous chromosome pairs of L. racemosus. From these data, we proposed a karyotype of L. racemosus and compared it with other karyotypes already reported.

Base Sequence↗

Trigenomic origin of the hexaploid Psammopyrum athericum (Triticeae: Poaceae) revealed by in-situ hybridization.

The genomic constitution of the hexaploid Psammopyrum athericum was studied with in-situ DNA hybridization using both genomic DNA and isolated cloned sequences as probes. A genomic probe from Thinopyrum bessarabicum (E genome) hybridized successfully to 14 chromosomes of Ps. athericum and a probe from Festucopsis serpentinii (L genome) hybridized to another 14 chromosomes. The remaining chromosomes did not hybridize, apart from in the centromeric region, to any of the genomic probes used. It is thus proposed that Ps. athericum contains the genomes E, L and X where X stands for a so-far unknown genome. Psammopyrum athericum has three pairs of pTa71 sites and approximately 30 pSc119:2 sites. The origin of the third genome will be a matter for further research using genomic and genome-specific probes.

Biological Evolution↗

Integrated genomics and morphological approach reveals interspecific gene flow cases and decodes the origin of selected feathergrasses (Poaceae, Stipa).

Central Asia is a diversity hotspot of arid-adapted grasses from the genus Stipa, with approximately 100 taxa found in the region. Recent studies in the steppe areas of Kazakhstan revealed specimens displaying intermediate morphology, distinguishing them from other taxa that grow sympatrically. Using integrative taxonomy, we investigated whether these individuals resulted from natural speciation or hybridisation, and if so, we would like to know which species were involved in this process feathergrasses. Research conducted in steppes of central Kazakhstan (Kyzylorda region), revealed the existence of individuals morphologically intermediate between S. arabica and S. richteriana, suggesting that these are probably of hybrid origin. Morphology and SNP markers validated the specimens as F1 hybrid between the aforementioned species by cladding separately based on neighbor-joining phylogenetic tree. Moreover, genetic structure displayed a separate cluster and showed almost equal genetic admixture between S. arabica and S. richteriana. Additionally, fastStructure analysis detected two geographically separated cryptic genotypes within S. richteriana population and their involvement in the hybridisation resulted in occurrence of S. × heptapotamica, S. × czerepanovii and S. × korshinskyi which recently were suggested as hybrids. Based on these evidences, we described a new nothospecies S. × kyzylordensis, as F1 hybrid. Furthermore, morphologically, the nothospecies delimited with other hybrids in Kazakh steppe area, marking the first report of hybridisation between S. arabica and S. richteriana, along with molecular evidence for the origin of further species supposed to be hybrids. This finding is crucial to understanding species diversity and hybridisation process in morphologically and genetically distant Stipa species.

Poaceae↗

Genetic differentiation among populations of Sesleria albicans Kit. ex Schultes (Poaceae) from ecologically different habitats in central Europe.

As observed for many other plant species, the populations of Sesleria albicans in Central Europe are located in habitats, which differ to a high degree from each other with regard to ecological factors such as nutrients, light and water as well as in type of land use. The species colonizes limestone cliffs, pavements, screes, grazed and mown grasslands, heaths, fens and open woodlands. In this study, we used random amplified polymorphic DNA (RAPD) analysis to investigate the genetic differentiation among 25 populations of S. albicans from six different types of habitat (beech forests, alpine and lowland rocky ridges, lowland screes, fens, calcareous grasslands). With RAPD analysis, 344 fragments could be amplified, of which 95.9% were polymorphic. The level of polymorphism ranged from 29.7 to 56.7% polymorphic bands per population and was correlated with population size. In an analysis of molecular variance (AMOVA), used to detect variation among individuals within populations, among populations from the same habitat and among different habitats, most of the genetic variation was found within populations (62.06%) and among populations from the same habitat (33.36%). In contrast, only a very low level of differentiation could be observed among different habitats (4.58%). The results of our study give only little evidence for an ecotypic differentiation of Sesleria albicans. This differentiation is principally conceivable, but obviously not related to the investigated RAPD loci.

Analysis of Variance↗

Uniformity of the nuclear and chloroplast genomes of Spartina maritima (Poaceae), a salt-marsh species in decline along the Western European Coast.

Spartina maritima is a salt-marsh species from European and African Atlantic coasts. In the northern range of the species (including north-west France), a rapid decline of the populations has been observed during the 20th century. In this paper, the molecular diversity of 10 populations of S. maritima from France has been investigated using nuclear and chloroplast DNA markers: inter-simple sequence polymorphism (ISSR), randomly amplified polymorphic DNA (RAPD), inter-retrotransposon amplified polymorphism (IRAP), and PCR-RFLP of a 5 kb long portion of chloroplast DNA. The results reveal an extremely low level of genetic variation in this species: only one nuclear marker (out of 98) was polymorphic, with the presence of two genotypes randomly distributed among the populations. The lack of genetic diversity is interpreted in light of the almost exclusive vegetative propagation of the species in its northern range, the colonization history of the populations, and the origin of S. maritima (2n = 60) in the hexaploid lineage of the genus and in the context of the management of S. maritima populations.

DNA Primers↗

Haplotype variation of cpDNA in the agamic grass complex Pennisetum section Brevivalvula (Poaceae).

The Brevivalvula section of the grass polyploid complex Pennisetum shows various reproductive systems, apomixis being the most widespread. Haplotype variation of chloroplast DNA was studied in the six morphological taxa (species) of this section by using RFLP analysis in 54 plants corresponding to 14 elementary taxa, each characterized on the basis of morphology and ploidy level. Two additional species, Pennisetum glaucum and P. purpureum, which belong to another section of the same genus, were analysed for comparison. In Brevivalvula, chloroplast DNA size was estimated to range between 130 and 133 kb. Thirteen of 15 distinct haplotypes identified in the study were specific to the Brevivalvula section. They were unequally distributed among the morphotypes, the ploidy levels and sampling sites. Within the Brevivalvula section, plants of P. setosum, which are perennial and reproduce vegetatively or by agamospermy, possessed a single specific haplotype. This species differed clearly from the five other morphological species, which are known to be annual, to show either sexual or agamospermic reproduction and which shared most of the 12 other haplotypes observed in the section, suggesting the occurrence of multiple hybridization events between the taxa. Chloroplast DNA variation was highly geographically structured, suggesting low seed dispersal between sites, whereas the substantial haplotype diversity observed in the sites may indicate that agamic reproduction is responsible for the maintenance of distinct genetically isolated clones. Haplotype classification using Wagner's parsimony suggested the occurrence of bidirectional gene flow between the diploids and the polyploids, as reported already in other related apomictic complexes.

Africa, Western↗

Molecular investigations in populations of Spartina anglica C.E. Hubbard (Poaceae) invading coastal Brittany (France).

Spartina anglica is a classical example of recent alloploid speciation. It arose during the end of the nineteenth century in England by hybridization between the indigenous Spartina maritima and the introduced East-American Spartina alterniflora. Duplication of the hybrid genome (Spartina x townsendii) gave rise to a vigorous allopolyploid involved in natural and artificial invasions on different continents. Spartina anglica was first recorded in France in 1906, and since then, it has spread all along the western French coast. Earlier studies revealed that native British populations display consistent morphological plasticity and lack of isozyme variation. In this paper, we use different molecular markers (randomly amplified polymorphic DNA, intersimple sequence repeats and restriction patterns from nuclear and chloroplast DNA sequences) to analyse the genetic patterns of the French populations of S. anglica. Our results show that French populations are mainly composed of one "major" multilocus genotype. This genotype is identical to the first-generation hybrid S. x townsendii from England. Losses of few markers from this genotype are observed but are restricted to a few populations from Brittany; it is likely that they appeared independently, subsequent to their introduction. In southern Brittany, no hybrids between S. anglica and S. maritima have been found where the two species co-occur. All French populations of S. anglica display the same chloroplast DNA sequences as S. alterniflora, the maternal genome donor. These findings are consistent with a severe genetic bottleneck at the time of the species formation, as a consequence of a unique origin of the species. Both parental nuclear sequences are present in the allopolyploid populations, revealing that for the markers investigated, no extensive changes have occurred in this young species.

DNA, Plant↗

Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae).

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchard-grass (Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0.9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t-test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P=0.0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.

Chromosome Aberrations↗

Characterization of a genetic resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR.

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat markers were employed to characterize a genetic resource collection of Miscanthus, a grass under trial in Europe as a biomass crop. The 26 polymorphic markers produced by two ISSR fingerprinting primers were able to discriminate taxa and identify putative clones. AFLP fingerprints were fully reproducible and produced a larger number of markers for the three primer pairs tested, of which 998 were polymorphic (representing 79.3% of all bands). AFLP markers distinguished species, infra-specific taxa (varieties and cultivars) and putatively clonal material. They were also used to assess the inter-relationships of the taxa, to investigate the origin of important hybrid plants and to estimate the overall level of genetic variation in the collection. They were useful for assessing the species status of certain taxa such as M. transmorrisonensis, an endemic from Taiwan that was clearly distinct from M. sinensis; whereas other taxa of disputed species status, such as M. condensatus and M. yakushimanum were not genetically distinct from M. sinensis. The AFLP markers detected a high degree of infra-specific variation and allowed subdivisions of the genetic resource collection to be made, particularly within M. sinensis.

DNA Primers↗

Silica accumulation in long-lived leaves of Sasa veitchii (Carrière) Rehder (Poaceae-Bambusoideae).

Silica accumulation in long-lived leaves of Sasa veitchii was investigated using the molybdenum blue method. In general, silica accumulation was rapid during spring and summer, and slow during winter. The leaves continuously accumulate silica throughout their life. The significance of these observations is discussed in relation to two hypotheses for silica accumulation in plants: (1) that silica accumulation is a result of water consumption by the plant: and (2) that silica is actively accumulated to protect the plant. The results of the present study support the former hypothesis.

Plant Leaves↗

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution.

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.

DNA, Ribosomal Spacer↗

Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae).

Spartina x townsendii arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced Spartina alterniflora, native to the eastern seaboard of North America. Duplication of the hybrid genome gave rise to Spartina anglica, a vigorous allopolyploid involved in natural and artificial invasions on several continents. This system allows investigation of the early evolutionary changes that accompany stabilization of new allopolyploid species. Because allopolyploidy may be a genomic shock, eliciting retroelement insertional activity, we examined whether retrotransposons present in the parental species have been activated in the genome of S. anglica. For this purpose we used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposons-microsatellite amplified polymorphism (REMAP) markers, which are multilocus PCR-based methods detecting retrotransposon integration events in the genome. IRAP and REMAP allowed the screening of insertional polymorphisms in populations of S. anglica. The populations are composed mainly of one major multilocus genotype, identical to the first-generation hybrid S. x townsendii. Few new integration sites were encountered in the young allopolyploid genome. We also found strict additivity of the parental subgenomes in the allopolyploid. Both these findings indicate that the genome of S. anglica has not undergone extensive changes since its formation. This contrasts with previous results from the literature, which report rapid structural changes in experimentally resynthesized allopolyploids.

DNA, Plant↗

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae).

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.

Bacterial Proteins↗

Phylogenetic evidence for excision of Stowaway miniature inverted-repeat transposable elements in triticeae (Poaceae).

The mode of transposition of miniature inverted-repeat transposable elements (MITEs) is unknown, but it has been suggested that they are duplicated rather than excised at transposition. However, the present investigation demonstrates that a particular family of MITEs, Stowaway:, is excised. Mapped onto a gene tree based on partial sequences of disrupted meiotic cDNA1 (DMC1) from 30 species of the Triticeae grasses, it is evident that at least two excisions have occurred, leaving short footprints. These footprints may subsequently be reduced in length or deleted. Excision of Stowaway: elements lends strong support to the suggestion that MITEs are DNA transposons and should be classified as class II elements. The evolution of Stowaway: elements can also be traced by scrutiny of the gene tree. It appears that base substitutions are as frequent in the conserved terminal inverted repeats (TIRs) as in the core of the element. Neither substitutions nor deletions lead to compensatory changes; hence, the highly stable secondary structure of the elements may gradually be reduced.

Base Sequence↗

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae.

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

Amino Acid Sequence↗

Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae).

To study the consequences of hybridization and genome duplication on polyploid genome evolution and adaptation, we used independently formed hybrids (Spartina x townsendii and Spartina x neyrautii) that originated from natural crosses between Spartina alterniflora, an American introduced species, and the European native Spartina maritima. The hybrid from England, S. x townsendii, gave rise to the invasive allopolyploid, salt-marsh species, Spartina anglica. Recent studies indicated that allopolyploid speciation may be associated with rapid genetic and epigenetic changes. To assess this in Spartina, we performed AFLP (amplified fragment length polymorphism) and MSAP (methylation sensitive amplification polymorphism) on young hybrids and the allopolyploid. By comparing the subgenomes in the hybrids and the allopolyploid to the parental species, we inferred structural changes that arose repeatedly in the two independently formed hybrids. Surprisingly, 30% of the parental methylation patterns are altered in the hybrids and the allopolyploid. This high level of epigenetic regulation might explain the morphological plasticity of Spartina anglica and its larger ecological amplitude. Hybridization rather than genome doubling seems to have triggered most of the methylation changes observed in Spartina anglica.

DNA Methylation↗

Microsatellite variation within and among populations of Oryza officinalis (Poaceae), an endangered wild rice from China.

Oryza officinalis Wall. ex Watt. is an agriculturally important but seriously endangered species of wild rice. To obtain more accurate estimates of population structure for improved conservation planning of the species, genetic variability at 14 microsatellite DNA loci was examined in population samples covering most of the species' range in China. Considerable genetic variability (overall Na = 1.886, P = 62%, HO = 0.056, HE = 0.216, and HS = 0.277) was detected at the 14 loci in 442 individuals of the 18 natural populations. The evaluation of partitioning of genetic variability (FST = 0.442) suggested high genetic differentiation among the Chinese O. officinalis populations. An overall value of Nm = 0.316 suggested limited gene flow occurred among the sampled populations. Most of the populations showed heterozygote deficits in tests of Hardy-Weinberg equilibrium and significantly positive FIS values. This could be due to some inbreeding occurring in this predominantly outcrossing species. For effective in situ conservation and restoration genetics, maintenance of significant historical processes is particularly important, including high outbreeding, considerable gene flow, and large population effective sizes. The high FST values detected among populations in this study are instructive for adopting a conservation plan that includes representative populations with the greatest genetic variation for either in situ conservation management or germplasm collection expeditions.

China↗