Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

Species relationships between antifungal chitinase and nuclear rDNA (internal transcribed spacer) sequences in the genus Hordeum.

The sequences of the chitinase gene (Chi-26) and the internal transcribed spacer of 18S - 5.8S - 26S rDNA (ITS1) were determined to analyze the phylogenetic relationships among species representing the four basic genomes of the genus Hordeum. Grouping analysis based on data for Chi-26 gene sequences placed Hordeum secalinum (H genome) near the Hordeum murinum complex (Xu genome), and Hordeum bulbosum distant from the other species that carried the I genome. ITS sequence data showed the expected grouping based on the genome classification of the species studied. Different sequences of ITS were detected even in the genomes of the diploid species. The results are interpreted in terms of defective or unfinished concerted evolution processes in each taxon.     

Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences

To estimate the phylogenetic relationship of polyploid Elymus in Triticeae, nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F sequences of 45 Elymus accessions containing various genomes were analysed with those of five Pseudoroegneria (St), two Hordeum (H), three Agropyron (P) and two Australopyrum (W) accessions. The ITS sequences revealed a close phylogenetic relationship between the polyploid Elymus and species from the other genera. The ITS and trnL-F trees indicated considerable differentiation of the StY genome species. The trnL-F sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. Both the ITS and trnL-F trees suggested multiple origins and recurrent hybridization of Elymus species. The results suggested that: the St, H, P, and W genomes in polyploid Elymus were donated by Pseudoroegneria, Hordeum, Agropyron and Australopyrum, respectively, and the St and Y genomes may have originated from the same ancestor; Pseudoroegneria was the maternal donor of the polyploid Elymus; and some Elymus species showed multiple origin and experienced recurrent hybridization.     

Phylogeny in the genus Hordeum based on nucleotide sequences closely linked to the vrs1 locus (row number of spikelets).

The phylogenetic relationship between four basic genomes designated H, I, Xa, and Xu in the genus Hordeum was studied using a nuclear DNA sequence. The sequence, cMWG699, is single copy in the H. vulgare genome, and tightly linked to the vrs1 locus which controls two- and six-rowed spikes. DNA fragments homologous to cMWG699 were amplified from diploid Hordeum species and the nucleotide sequences were determined. A phylogeny based on both base substitutions and an insertion-deletion event showed that the H- and Xa-genome groups are positioned in one monophyletic group indicating that the Xa-genome taxa should be included in the H-genome group. The large H-genome group is highly homogeneous. The I and Xu genomes are distinctly separated from H and Xa, and form sister groups. Another phylogeny pattern based on data excluding the insertion-deletion gave a result that the Xa genome forms a sister group to the H-genome group. The difference between the H and Xa genomes was affected only by a single base insertion-deletion event, thus the H and Xa genomes are likely to be closely related. The I and Xu genomes were again distinctly separated from the H and Xa genomes.     

Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences

Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.     

The molecular phylogeny of the genus Rhizopus based on rDNA sequences

In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate.     

Genome evolution in a Mediterranean species complex: phylogeny and cytogenetics of Helictotrichon (Poaceae) allopolyploids based on nuclear DNA sequences (rDNA,topoisomerase gene) and FISH

We examined the molecular phylogeny and chromosomal features of European Helictotrichon species to explore the relationships within the genus and to investigate the origin of several polyploids. Using both approaches, molecular and cytogenetic, revealed the strong impact of allopolyploidization on genome organization from chromosome structure to sequence level. Our research focused on Mediterranean and endemic species of the Alps. Altogether, the molecular phylogenetic analyses include a sample of 17 Helictotrichon species and subspecies, used DNA sequences from the nuclear ribosomal (nr) internal transcribed spacer region (ITS) and the single copy gene topoisomerase 6 (Topo6), and were analysed by maximum parsimony and Bayesian methods. Karyotype structures were investigated by fluorescence in situ hybridization (FISH) and fluorochrome banding. Cytogenetic characters were mapped on the combined phylogenetic tree. The absence or comparatively rare occurrence of different ITS sequence types in some (allo-) polyploid species of Helictotrichon suggests frequent intergenomic homogenization of ribosomal DNA (rDNA) loci due to the phenomenon of concerted evolution. This result implies that the ITS region is not an ideal marker to study polyploid evolution of these grasses. The phylogenetic analysis of the Topo6 region revealed three major clades that concur with three different copy types (termed SAR, SET, PAR), representing the major genome groups in Helictotrichon. A comparison of the molecular phylogenetic trees with the chromosome and karyotype structure supports allopolyploidy of several Helictotrichon species and identifies potential genome donors. A correlation between molecular phylogenetic/cytogenetic results and geographic distribution is expressed by a west-east disjunction, in the narrower or wider sense, of the analysed species. While SAR represents a geographically narrowly distributed southwest Mediterranean genome group, PAR and SET are very widespread (Mediterranean to Asia) and encompass several instances of west-east disjunctions.     

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.     

Phylogeographic congruence between mtDNA and rDNA ITS markers in brown trout

Variation in the internal transcribed spacer (ITS) of rDNA was examined throughout the range of the brown trout (Salmo trutta) to analyze the usefulness of this molecular marker for phylogeographic analysis. The results were compared with those previously obtained with mtDNA, a region exhaustively analyzed along the brown trout distribution. ITS2 was essentially conserved at all populations sampled, no informative characters being detected across the main lineages described in this species. Conversely, ITS1 showed a greater homogenization than other genetic markers at a microgeographic scale, with variation partitioning into several major phylogenetic groups. Phylogeographic patterns were partially congruent between both ITS1 and mtDNA. The main discrepancies were the detection of intra-individual variation and putative recombinant ITS1 sequences in hybridization areas between genetically different, yet historically overlapping, assemblages. Also, the existence of an ancient ITS1 sequence in the Mediterranean-southeastern area (rMEDA), not revealed by mtDNA analysis, was evidenced after rDNA ITS1 analysis.     

Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to other genomes in Elymus species

It has been hypothesized from isozymic and cytological studies of Elymus species that the Old and New World taxa may be of separate origin of the H genome in the StH genome species. To test this hypothesis, and estimate the phylogenetic relationships of polyploid Elymus species within the Triticeae, the second largest subunit of RNA polymerase II (RPB2) sequence of 36 Elymus accessions containing StH or StY genomes was analyzed with those of Pseudoroegneria (St), Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum(Ee), Thinopyrum(Eb) and Dasypyrum (V). Our data indicated that the H genome in Elymus species is differentiated in accordance with geographical origin, and that the Eurasian and American StH genome species have independent alloploid origins with different H-genome donors. Phylogenetic analysis of Y genome sequences with other genome donors (St, H, P, W) of Elymus revealed that W and P genomes are sister to Y genome with a 87% bootstrap support, and that StY and StH species group might have acquired their RPB2 St sequences from distinct Pseudoroegneria gene pools. Our data did not support the suggestion that the St and Y genomes have the same origin as put forward in a previous study using ITS data. Our result provides some insight on the origin of Y genome and its relationship to other genomes in Elymus.     

Phylogenetic Analysis of Leymus (Poaceae: Triticeae) Inferred from Nuclear rDNA ITS Sequences

To investigate the phylogenetic relationships of polyploid Leymus (Poaceae: Triticeae), sequences of the nuclear rDNA internal transcribed spacer region (ITS) were analyzed for 34 Leymus accessions representing 25 species, together with three Psathyrostachys species (Ns genome), two Pseudoroegneria (St genome) species, Lophopyrum elongatum (E(e) genome), and Thinopyrum bessarabicum (E(b) genome). The phylogenetic analyses (maximum likelihood and Bayesian inference) supported two major clades, one including 21 Leymus species and three Psathyrostachys species, the other with nine Leymus species and four diploid species. The ITS RNA secondary structure of the Leymus species was compared with that of their putative diploid donor. It is suggested that (1) the species from the same areas or neighboring geographic regions are closely related to each other; (2) L. coreanus, L. duthiei, L. duthiei var. longearistatus, and L. komarovii are closely related to other Leymus species, and it is reasonable to transfer these species from the genus Hystrix to Leymus; (3) the ITS sequences of Leymus are evolutionarily distinct; (4) the different Leymus species and different distribution of a species derived their Ns genome from different Psathyrostachys species; and (5) there is a close relationship among Leymus, Pseudoroegneria, Lophopyrum, and Thinopyrum, but it is difficult to presume that the St, E(e), and E(b) genome may be the Xm genome donor of the Leymus species.     

Progenitor-derivative relationships of Hordeum polyploids (Poaceae, Triticeae) inferred from sequences of TOPO6, a nuclear low-copy gene region

Polyploidization is a major mechanism of speciation in plants. Within the barley genus Hordeum, approximately half of the taxa are polyploids. While for diploid species a good hypothesis of phylogenetic relationships exists, there is little information available for the polyploids (4×, 6×) of Hordeum. Relationships among all 33 diploid and polyploid Hordeum species were analyzed with the low-copy nuclear marker region TOPO6 for 341 Hordeum individuals and eight outgroup species. PCR products were either directly sequenced or cloned and on average 12 clones per individual were included in phylogenetic analyses. In most diploid Hordeum species TOPO6 is probably a single-copy locus. Most sequences found in polyploid individuals phylogenetically cluster together with sequences derived from diploid species and thus allow the identification of parental taxa of polyploids. Four groups of sequences occurring only in polyploid taxa are interpreted as footprints of extinct diploid taxa, which contributed to allopolyploid evolution. Our analysis identifies three key species involved in the evolution of the American polyploids of the genus. (i) All but one of the American tetraploids have a TOPO6 copy originating from the Central Asian diploid H. roshevitzii, the second copy clustering with different American diploid species. (ii) All hexaploid species from the New World have a copy of an extinct close relative of H. californicum and (iii) possess the TOPO6 sequence pattern of tetraploid H. jubatum, each with an additional copy derived from different American diploids. Tetraploid H. bulbosum is an autopolyploid, while the assumed autopolyploid H. brevisubulatum (4×, 6×) was identified as allopolyploid throughout most of its distribution area. The use of a proof-reading DNA polymerase in PCR reduced the proportion of chimerical sequences in polyploids in comparison to Taq polymerase.     

Cytogenetic and phylogenetic studies of diploid and polyploid members of tribe Anemoninae (Ranunculaceae)

The ancestry, phylogenetic differentiation and systematic classification of the worldwide-distributed genus Anemone have been debated for many years. In this paper 11 Anemone, three Pulsatilla species and Hepatica nobilis were subjected to detailed karyotype analysis with the aim of obtaining new cytogenetic data that will contribute to karyotype evolutionary studies of the tribe Anemoninae. The results are interpreted in a phylogenetic context, established from the intergenic nontranscribed spacer (NTS) of 5S rDNA and internal transcribed spacer (ITS) of 35S rDNA. One to three 35S and one to three 5S rDNA loci are present in diploid and polyploid taxa. The 35S rDNA loci are located terminally on the short arm of acrocentric chromosomes, while for 5S rDNA there is no preferential chromosomal position as it exhibits terminal, subterminal, interstitial or pericentromeric positions, and is located either on acrocentric or metacentric chromosomes. The karyotype of hexaploid A. baldensis (2n = 6x = 48) is presented for the first time, and A. sylvestris is proposed as one of its putative parental species. Chromosome fusion/translocation is proposed as the key mechanism involved in reduction of the basic chromosome number from 8 in the Anemone subgenus to 7 in the Anemonidium subgenus. The cytogenetic data obtained are mainly supported by ITS and NTS phylogeny. Diversification of the genus Anemone was accompanied by a large reduction of heterochromatin, from the Mediterranean anemones that have large amounts of heterochromatin to the New World anemones without any detectable heterochromatic blocks.     

Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers

Cytologically, the species of Passiflora with known chromosome number can be divided into four groups: (1) 2n = 12, 24, 36; (2) 2n = 24; (3) 2n = 18, 72; and (4) 2n = 20. The base chromosome number proposed for the genus is x = 6, with x = 9, x = 10 and x = 12 being considered secondary base numbers. In the present study, variability of 5S and 45S rDNA sites was investigated in 20 species of these four groups to check the reliability of this hypothesis. In the group with x = 6, five diploid species (2n = 12) exhibit two 5S rDNA sites and two (P. capsularis, P. morifolia and P. rubra) or four (P. misera 2x and P. tricuspis) 45S rDNA sites. The hexaploid cytotype of P. misera had 12 45S rDNA sites and six 5S rDNA. A tetraploid species, P. suberosa, had ten 45S rDNA sites and four 5S rDNA sites, both in the same chromosomes as the 45S rDNA sites. In the group with x = 9, P. actinia, P. amethystina, P. edmundoi, P. elegans, P. galbana, P. glandulosa and P. mucronata displayed six 45S rDNA sites, whereas P. alata, P. cincinnata, P. edulis f. flavicarpa, P. edulis var. roxo and P. laurifolia had four sites. In this group, all species were diploid (2n = 18) and had only two 5S rDNA sites. Passiflora foetida, the only species with 2n = 20, had six 45S rDNA sites and four 5S rDNA sites. The species with x = 12 (2n = 24), P. haematostigma and P. pentagona, showed four 45S rDNA sites and two 5S rDNA. In general, the number and location of 5S and 45S rDNA sites were consistent with the hypothesis of x = 6 as the probable ancestral genome for the genus, while the groups of species with x = 9, x = 10 and x = 12 were considered to be of tetraploid origin with descending dysploidy and gene silencing of some redundant gene sites, mainly those of 5S rDNA.     

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.     

Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne

Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.     

紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and beta-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade (Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira-Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.     

A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization

Background and Aims

Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids.

Methods

Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids.

Key Results

In Australian/NZ A-clade Lepidium polyploids, GISH identified African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n = 11x = 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type.

Conclusions

The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be confirmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively.Key words: Lepidium, Brassicaceae, FISH, GISH, hybridization, polyploidy, long-distance dispersal, ITS, rDNA, Australia, New Zealand     

Phylogenetic significance of morphological characters in the taxonomy of Pestalotiopsis species

There has been considerable disagreement regarding the relationships among Pestalotiopsis species and their delimitations. A molecular phylogenetic analysis was conducted on 32 species of Pestalotiopsis in order to evaluate the utility of morphological characters currently used in their taxonomy. Phylogenetic relationships were inferred from nucleotide sequences in the ITS regions and 5.8S gene of the rDNA under four optimality criteria: maximum parsimony, weighted parsimony, maximum likelihood, and neighbor joining. Phylogenies estimated from all analyses yielded trees of essentially similar topology and revealed 3 major groups that correspond with morphology-based classification systems. Molecular data indicated that the genus contains two distinct lineages based on pigmentation of median cells and four distinct groupings based on morphology of apical appendages. The analyses did not support reliability of other phenotypic characters of this genus, such as spore dimensions. Characters with particular phylogenetic significance are discussed in relation to the taxonomy of Pestalotiopsis.