Fungal endophytes of native Gossypium species in Australia

Fungal endophytes of 17 genera were found in stems of four native Gossypium species (G. australe, G. bickii, G. nelsonii, G. sturtianum) collected from inland areas in Queensland, the Northern Territory, and South Australia in 2001. Phoma, Alternaria, Fusarium, Botryosphaeria, Dichomera, and Phomopsis were common, accounting for 58, 18, 11, 3, 1, and 1 % of the 281 recovered isolates, respectively, and occurring in 47, 29, 19, 5, 5, and 4 % of the 79 sampled populations. Among the four Gossypium species in Queensland and the Northern Territory, Alternaria spp. and Fusarium spp. had the greatest recovery frequency in G. bickii stems. The recovery frequencies of Phoma spp. and Alternaria spp. were significantly greater in the G. sturtianum stems collected from South Australia than in those from Queensland and the Northern Territory. Pathogenicity of 42 representative isolates was tested on cultivated cotton (G. hirsutum). All isolates caused some localized discoloration in stem tissue when inoculation was conducted with the stem puncturing method, but none of the isolates could induce any foliar symptoms during the five-week experimental period by either inoculation method (root dipping or stem puncturing), suggesting that the endophytic fungi of native Gossypium species are unlikely sources of cotton pathogens.     

THE CYTOLOGY AND PHYLOGENETICS OF THE DIPLOID SPECIES OF GOSSYPIUM

Meiotic chromosome behavior of 11 inter-genomic hybrids of Gossypium (2n = 26) were investigated. Per cell univalent frequencies at meiotic metaphase I in these hybrids were: A genome × Cgenome—G. herbaceum × sturtianum, 10.53; G. herbaceum × australe, 18.05. A genome × E genome—G. smnalense × arboreum, 21.82. B genome × C genome—G. anomalum × sturtianum, 9.23; G. anomalum × australe, 13.11. B genome × D genome—G. anomalum × klotzschianum, 17.45; G. anomalum × raimondii, 18.83. C genome × D genome—G. robinsonii × davidsonii, 12.77; G. sturtianum (armourianum × thurberi), 8.63. C genome × E genome—G. somalense × australe, 23.78; G. somalense × bickii, 25.58. Trivalent and quadrivalent frequencies were relatively high for those hybrids involving a C genome species, indicating that a reciprocal translocation differentiates the C genome from the A, B, D, and E genomes. The results of this study and the data of similar studies cited from the literature on Gossypium cytogenetics are discussed relative to the phylogenetics and evolution of the major (genome) groups of Gossypium and their constituent taxa.     

EVOLUTION OF POPULUS NIGRA (SECT. AIGEIROS):INTROGRESSIVE HYBRIDIZATION AND THE CHLOROPLAST CONTRIBUTION OF POPULUS ALBA (SECT. POPULUS)

Restriction site variation in chloroplast DNA and nuclear ribosomal DNA was examined in 16 accessions from the Salicaceae comprising ten species of Populus and one outgroup species of Salix. Forty-nine restriction site mutations in the chloroplast DNAs were used to generate one most parsimonious phylogenetic tree. This tree indicates that all varieties of P. nigra (black poplars of sect. Aigeiros) have a chloroplast genome, maternally inherited, derived from the clade including the white poplars (P. alba and segregate species of sect. Populus) and divergent from the American cottonwoods of their own section. Twenty-one restriction site mutations in the nuclear ribosomal DNAs generated a single most parsimonious phylogenetic tree that indicates that the nuclear genome ofP. nigra is distinct from both the white poplars and American cottonwoods. The incongruity of these independent molecular phylogenies provides evidence for an unusual origin of the black poplars. Populus alba or its immediate ancestor acted as the maternal parent in a hybridization event with the paternal lineage of P. nigra. Subsequent backcrosses to the paternal species gave rise to the extant P. nigra with a chloroplast genome of P. alba and the nuclear genome of the paternal species. These hybridization and introgression events must have pre-dated the divergence of the black poplar varieties. The biphyletic nature of the P. nigra genomes suggests that dependency on one class of molecular or morphological markers or the merging of the two kinds of data sets to derive accurate estimates of true phylogenies could be misleading in plants.     

Restriction fragment length polymorphisms reveal considerable nuclear divergence within a well-supported maternal clade in allium section Cepa (Alliaceae)

Maternal phylogenies estimated by restriction fragment length polymorphisms (RFLPs) in the chloroplast DNA (cpDNA) delineated a well-supported clade containing Allium altaicum, A. cepa (bulb onion), A. fistulosum (Japanese bunching onion), A. galanthum, and A. vavilovii. Few polymorphic restriction-enzyme sites were detected among the wild and cultivated species within this clade, and relationships could not be confidently estimated. Random nuclear RFLPs revealed considerable variation among these species and three distinct groups were identified (Altaicum, Cepa, and Galanthum). Relationships were estimated using principal components and cluster analyses of the Jaccard's similarity matrix. For five out of six analyses, nuclear phylogenies estimated by UPGMA and neighbor joining of the Jaccard's similarity matrix produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, and A. galanthum, disagreeing with maternal phylogenies that produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, A. cepa, and A. vavilovii. Allium oschaninii was closely related to A. galanthum and these two species may represent the progenitor types. Overall, restriction-enzyme analyses of the nuclear and cpDNA produced few synapomorphies among closely related species in Allium section Cepa.     

Chloroplast DNA as an evolutionary marker in thePhaseolus vulgaris complex

We have analyzed the changes occurring in the chloroplast DNA (cpDNA) of taxa belonging to thePhaseolus vulgaris complex to help clarify relationships among species of this complex. Two restriction maps for 11 restriction enzymes comprising the whole chloroplast genome from a wildP. vulgaris and a wildP. coccineus accession were constructed. These maps allowed us to compare a total of 330 restriction sites between the two genomes in order to identify polymorphisms, assess the type of mutations detected, and identify regions of high variability. A region, located in the large single-copy region near the borders with the inverted repeats, accounted for a large portion of the variation. Most of the mutations detected were due to restriction sites gains or losses. Variable and conserved regions were then evaluated in 30 accessions belonging to taxa of theP. vulgaris complex. Phylogenetic analyses were made using parsimony methods. Conclusions obtained from such analyses were the following: (1) there was high cpDNA variability withinP. coccineus but not inP. vulgaris. (2)P. coccineus subsp.glabellus showed a very distinct cpDNA type that strongly suggests that it actually belongs to a different but as yet undetermined section of the genus. Our cpDNA observations are supported by distinctive morphological traits and reproductive biology of this taxon. (3) InP. coccineus subsp.darwinianus (also classified asP. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers and suggested a reticulated origin by hybridization betweenP. coccineus as the male parent and an ancestralP. polyanthus type, closely allied toP. vulgaris, as the seed parent. This initial cross was presumably followed by repeated backcrossing toP. coccineus. Our cpDNA studies illustrate the importance of molecular markers in elucidating phylogenetic relationships. They also indicate that accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes.     

Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome

The present work describes the plant regeneration via somatic embryogenesis in two wild cotton species belonging to G genome: Gossypium nelsonii Fryx and Gossypium australe F Muell. The role of plant hormones and carbohydrates was also evaluated for somatic embryogenesis and somatic embryo development. Normal plants were obtained from G. nelsonii Fryx; abnormal plants and somatic embryos were obtained from G. australe F Muell. The best medium for callus induction for these G genome wild cotton species was MSB₅ supplemented with 0.1 mg L⁻¹ KT and 0.1 mg L⁻¹ 2,4-D. For embryogenic callus proliferation, the best medium used was MSB₅ supplemented with 0.2 mg L⁻¹ KT and 0.5 mg L⁻¹ IBA. The medium MSB₅ supplemented with 0.15 mg L⁻¹ KT and 0.5 mg L⁻¹ NAA was used successfully for root initiation and plant growth. In addition, adding CuSO₄ and AgNO₃ in the callus-inducing and proliferation medium resulted in a number of somatic embryos. Glucose and maltose, the carbon sources in somatic culture, were used for callus induction, but maltose worked even better than glucose for proliferation of embryogenic callus and development of somatic embryos.     

Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii

Summary Alloplasmic male sterile (cms) and restoration-of-fertility (Rf) lines of the AD allotetraploid Gossypium hirsutum were earlier derived from the presumed introgression of the cytoplasm of the D species G. harknessii. To confirm that this happened and address its significance, cytoplasms of the maternal progenitor, backcross intermediates, derived breeding lines, related A, D, and F species, and a synthetic AD tetraploid were examined by agarose and polyacrylamide gel electrophoresis of 140 restriction enzyme fragments of chloroplast DNA. Length mutations of 10–50 nucleotides predominate over site loss/gain mutations. Chloroplast DNA is maternally inherited and that of G. harknessii has been maintained in the cms lines for at least 13 successive generations without detectable alteration. Chloroplast DNA divergence is consistent with current nuclear genome classification and shows that the A progenitor was the maternal parent of the AD tetraploids. As predicted from incompatability models of cms, the degree of male sterility in alloplasmic Gossypium tetraploids is correlated with the extent of evolutionary divergence of their cytoplasms. It is suggested that the A genome in the AD tetraploids dominates those nuclear-cytoplasm interactions reflected by male fertility.     

PHYLOGEOGRAPHY OF GRACILARIA TIKVAHIAE (GRACILARIACEAE,RHODOPHYTA): A STUDY OF GENETIC DISCONTINUITY IN A CONTINUOUSLY DISTRIBUTED SPECIES BASED ON MOLECULAR EVIDENCE1

Gracilaria tikvahiae, a highly morphologically variable red alga, is one of the most common species of Gracilariaceae inhabiting Atlantic estuarine environments and the Intracoastal Waterway of eastern North America. Populations of G. tikvahiae at the extremes of their geographic range (Canada and southern Mexico) are subjected to very different environmental regimes. In this study, we used two types of genetic markers, the chloroplast‐encoded rbcL and the nuclear internal transcribed spacer (ITS) region, to examine the genetic variability within G. tikvahiae, for inferring the taxonomic and phylogenetic relationships between geographically isolated populations, and to discuss its distributional information in a phylogeographic framework. Based on rbcL and ITS phylogenies, specimens from populations collected at the extreme distributional ranges reported for G. tikvahiae are indeed part of the same species; however, rbcL‐ but not ITS‐based phylogenies detected phylogenetic structure among the ten G. tikvahiae different haplotypes found in this study. The four distinct rbcL lineages were identified as 1) a Canadian–northeast U.S. lineage, 2) a southeast Florida lineage, 3) an eastern Gulf of Mexico lineage, and 4) a western Gulf of Mexico lineage. We found no evidence for the occurrence of G. tikvahiae in the Caribbean Sea. Observed phylogeographic patterns match patterns of genetic structures reported for marine animal taxa with continuous and quasicontinuous geographic distribution along the same geographic ranges.     

The Phylogeny of Triticum L. and Aegilops L. Inferred from Comparative Analysis of Nucleotide Sequences in rDNA Promoter Regions

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat and aegilops species from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum–aestivumand timopheevi,diploid Aegilops speltoides acted as a maternal form. In addition to cytoplasmic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilops species confirmed the accepted wheat phylogenies.     

Chloroplast DNA study of the generaAmbrosia s.l. andHymenoclea (Asteraceae): Systematic implications

The relationship ofAmbrosia (ragweed) andFranseria has long been debated. Their treatment as separate genera has been repeatedly challenged. In this study, chloroplast DNA restriction site variation was examined for species from bothAmbrosia andFranseria as well as taxa from the closely related genusHymenoclea. The chloroplast genomes of members of these three genera were examined using 21 restriction endonucleases and the restriction mutations were used to construct phylogenetic trees. Wagner and Dollo parsimony as well as weighted parsimony were employed to compare the different phylogenies. The results support a close relationship betweenAmbrosia andFranseria, but indicate that the two groups are well separated. Compared toFranseria, Ambrosia is a much more strongly supported group, and the results indicate thatHymenoclea is closer toFranseria than toAmbrosia. The cpDNA phylogeny was used as a framework to examine evolutionary trends in morphology and secondary chemistry.     

Subgenome analysis of two southern hemisphere allotriploid species in Sphagnum (Sphagnaceae)

Sphagnum ×australe s.l. and S. ×falcatulum s.l. are both cryptic species complexes of gametophytically allodiploid and allotriploid cytotypes, with the allodiploid cytotype being one parent of the respective allotriploid cytotype. Phylogenetic analyses of S. ×australe s.l. and S. ×falcatulum s.l. were undertaken using sequences from two plastid loci and cloned sequences from three nuclear loci. Subgenomes from three subgenera were detected in allotriploid S. ×australe. The haploid parent of allotriploid S. ×australe was S. fimbriatum (subg. Acutifolia). One of the two subgenomes contributed by allodiploid S. australe was associated with S. strictum (subg. Rigida); the parental species associated with the other subgenome could not be identified, either at the species level or in terms of subgenus association. It may represent an early diverging lineage of subg. Sphagnum or a previously undetected subgenus. Allotriploid S. ×falcatulum has subgenomes from two subgenera. This study confirms prior findings that S. cuspidatum (subg. Cuspidata) was the haploid parent of allotriploid S. ×falcatulum. One of the two subgenomes contributed by allodiploid S. ×falcatulum was associated with subg. Cuspidata (species unidentified) and the second was associated with subg. Subsecunda (species unidentified). Recurrent allopolyploidy appears to have been associated with the complex evolutionary processes (inter-subgeneric hybridization, interploidal hybridization, double allopolyploidy) which resulted in each of these two allotriploids. The highest level of divergence detected among the subgenomes in each of these Sphagnum allotriploids is comparable to the average genetic divergence reported for angiosperm allopolyploids.     

ASSESSMENT OF EVOLUTIONARY AFFINITIES IN GOSSYPIUM BY PROTEIN ELECTROPHORESIS

Protein band patterns from 25 species of Gossypium were obtained by electrophoresis of crude seed extracts on polyacrylamide gel. Band homologies between species were verified by electrophoresis of a mixture of their extracts. The patterns were found to be largely consistent with the conventional classification of the diploids into 6 genomic groups, A–F. However, G. triphyllum and G. bickii showed unique patterns differing respectively from those of the B and C groups, and G. australe showed closer affinity with the Arabian E- than with the Australian C-genome species. Affinities among the D-genome species were different from those implied by their former grouping into taxonomic sections but remarkably similar to those indicated in the most recent taxonomic revision of the genus. They were classifiable into two subgroups, β and ɛ. The clustering pattern of the diploids based on correlation coefficients calculated from densitometer curves of the electrophoretic spectra suggested that the genomic groups were derived from an African progenitor type, and that the American β and ɛ subgroups, most closely related to the African B- and the Arabian E-genome groups respectively, evolved under comparative mutual isolation, possibly separated by the Tertiary Amazonas basin.     

IRIS NELSONII (IRIDACEAE): ORIGIN AND GENETIC COMPOSITION OF A HOMOPLOID HYBRID SPECIES

Nuclear and chloroplast DNA variation was assayed for two populations of Louisiana irises (Bayou Teche and Young's Coulee) that demonstrated extreme morphological variation and for a sample of the putative hybrid species, Iris nelsonii. The genetic markers examined in this analysis were diagnostic for either Iris fulva. Iris hexagona, or Iris brevicaulis. These data demonstrated that the two morphologically variable populations were hybrid associations involving all three of these species and that all three of these species were involved in the origin of I. nelsonii. The distribution of genetic variation in I. nelsonii was significantly different from that present in either of the two hybrid populations. I. nelsonii demonstrated significantly fewer foreign markers than the two hybrid populations. This finding is in accord with the prediction that I. nelsonii is a hybrid species that has undergone stabilization with regard to genetic recombination and segregation. Although the genotypic makeup of I. nelsonii was significantly different from other parental and hybrid populations, individual plants from this species cannot be unequivocally differentiated from either I. fulva or certain hybrid genotypes. This reflects the paradoxical nature of genotypic variation in hybrid species. Thus, a hybrid species may include genotypes that overlap with both parental and contemporary hybrid populations. In the case of I. nelsonii it is necessary to utilize additional information (morphological, chromosomal, ecological) to identify plants belonging to this taxon. One hybrid population (Young's Coulee) is suggested as a paradigm for the progenitor population that gave rise to I. nelsonii.     

Phylogeny and infrageneric classification of Correa Andrews (Rutaceae) on the basis of nuclear and chloroplast DNA

This paper presents phylogenies of the small but ecologically and horticulturally important Australian genus Correa (Rutaceae). Consensus phylogenies generated using parsimony were congruent with their counterparts generated by Bayesian analysis, although usually less well resolved. The phylogeny generated from the second internal transcribed spacer region of the nuclear ribosomal DNA supported the monophyly of Correa and identified two well supported clades (one comprising C. lawrenceana and C. baeuerlenii and the other containing all other species of the genus). Phylogenetic reconstructions based on the combined trnL-trnF spacer and the trnK intron (including the matK gene) regions of chloroplast DNA also supported the monophyly of Correa and of the C. lawrenceana/C. baeuerlenii clade, but the topology among the other species differed markedly from that in the ITS-based phylogeny. The major clades identified in the chloroplast phylogenies seemed to follow geographic patterns rather than species boundaries, with different samples of C. glabra bearing chloroplast genotypes from different clades. These patterns are likely to be because of independent evolution of the chloroplast and nuclear genomes, and are typical of cases of introgressive hybridisation among species or incomplete lineage sorting of chloroplast genomes leading to incongruence between chloroplast and nuclear phylogenies. Thus, the phylogenies based on nuclear DNA should reflect species relations better than the chloroplast phylogeny in Correa, and we propose a new subgeneric classification of the genus on the basis of the ITS-based phylogeny and morphology. Correa subgenus Persistens Othman, Duretto and G.J. Jord., containing C. lawrenceana and C. baeuerlenii, is formally described.     

Phylogenomics and multigene phylogenies decipher two new cryptic marine algae from California,Gelidium gabrielsonii and G. kathyanniae (Gelidiales,Rhodophyta)

Molecular surveys are leading to the discovery of many new cryptic species of marine algae. This is particularly true for red algal intertidal species, which exhibit a high degree of morphological convergence. DNA sequencing of recent collections of Gelidium along the coast of California, USA, identified two morphologically similar entities that differed in DNA sequence from existing species. To characterize the two new species of Gelidium and to determine their evolutionary relationships to other known taxa, phylogenomic, multigene analyses, and morphological observations were performed. Three complete mitogenomes and five plastid genomes were deciphered, including those from the new species candidates and the type materials of two closely related congeners. The mitogenomes contained 45 genes and had similar lengths (24,963–24,964 bp). The plastid genomes contained 232 genes and were roughly similar in size (175,499–177,099 bp). The organellar genomes showed a high level of gene synteny. The two Gelidium species are diminutive, turf‐forming, and superficially resemble several long established species from the Pacific Ocean. The phylogenomic analysis, multigene phylogeny, and morphological evidence confirms the recognition and naming of two new species, describe herein as G. gabrielsonii and G. kathyanniae. On the basis of the monophyly of G. coulteri, G. gabrielsonii, G. galapagense, and G. kathyanniae, we suggest that this lineage likely evolved in California. Organellar genomes provide a powerful tool for discovering cryptic intertidal species and they continue to improve our understanding of the evolutionary biology of red algae and the systematics of the Gelidiales.     

RECONSTRUCTING THE COMPLEX EVOLUTIONARY ORIGIN OF WILD ALLOPOLYPLOID TOBACCOS (NICOTIANA SECTION SUAVEOLENTES)

Nicotiana (Solanaceae) provides an ideal system for understanding polyploidization, a pervasive and powerful evolutionary force in plants, as this genus contains several groups of allotetraploids that formed at different times from different diploid progenitors. However, the parental lineages of the largest group of allotetraploids, Nicotiana section Suaveolentes, have been problematic to identify. Using data from four regions of three low‐copy nuclear genes, nuclear ribosomal DNA, and regions of the plastid genome, we have reconstructed the evolutionary origin of sect. Suaveolentes and identified the most likely diploid progenitors by using a combination of gene trees and network approaches to uncover the most strongly supported evidence of species relationships. Our analyses best support a scenario where a member of the sect. Sylvestres lineage acted as the paternal progenitor and a member of either sect. Petunioides or sect. Noctiflorae that also contained introgressed DNA from the other, or a hypothetical hybrid species between these two sections, was the maternal progenitor. Nicotiana exemplifies many of the factors that can complicate the reconstruction of polyploid evolutionary history and highlights how reticulate evolution at the diploid level can add even greater complexity to allopolyploid genomes.     

Variation in the nrDNA ITS of Pinus Subsection Cembroides: Implications for Molecular Systematic Studies of Pine Species Complexes

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.     

Introgression of the low-gossypol seed & high-gossypol plant trait in upland cotton: Analysis of [(Gossypium hirsutum?×?G. raimondii)2?×?G. sturtianum] trispecific hybrid and selected derivatives using mapped SSRs

In order to select genotypes of Gossypium hirsutum genetically balanced and expressing the low-gossypol seed & high-gossypol plant trait introgressed from the Australian wild diploid species G. sturtianum, the [(G. hirsutum × G. raimondii)2 × G. sturtianum] triple hybrid was backcrossed to G. hirsutum and autopollinated to produce backcross and selfed progenies. Two hundred and six mapped SSR markers of G. hirsutum were used to monitor the introgression of SSR alleles specific to G. sturtianum and G. raimondii in the selected progenies. A high level of heterozygosity, varying from 25 to 100%, was observed for all G. sturtianum-specific SSR markers conserved in the most advanced progenies. These results indicate the existence of segregation distortion factors that are associated with the genes controlling the researched trait. This study represents a starting point to map the genes involved in the expression of the trait and better understand its genetic determinism.