ITS Polymorphisms Shed Light on Hybrid Evolution in Apomictic Plants: A Case Study on the Ranunculus auricomus Complex

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.     

Divergent and reticulate species relationships in Leucaena (Fabaceae) inferred from multiple data sources: insights into polyploid origins and nrDNA polymorphism

Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.     

Structural evolution of nrDNA ITS in Pinaceae and its phylogenetic implications

Nuclear ribosomal DNA (nrDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels. In comparison with its fast concerted evolution in angiosperms, nrDNA is symbolized by slow concerted evolution and substantial ITS region length variation in gymnosperms, particularly in Pinaceae. Here we studied structure characteristics, including subrepeat composition, size, GC content and secondary structure, of nrDNA ITS regions of all Pinaceae genera. The results showed that the ITS regions of all taxa studied contained subrepeat units, ranging from 2 to 9 in number, and these units could be divided into two types, longer subrepeat (LSR) without the motif (5'-GGCCACCCTAGTC) and shorter subrepeat (SSR) with the motif. Phylogenetic analyses indicate that the homology of some SSRs still can be recognized, providing important informations for the evolutionary history of nrDNA ITS and phylogeny of Pinaceae. In particular, the adjacent tandem SSRs are not more closely related to one another than they are to remote SSRs in some genera, which may imply that multiple structure variations such as recombination have occurred in the ITS1 region of these groups. This study also found that GC content in the ITS1 region is relevant to its sequence length and subrepeat number, and could provide some phylogenetic information, especially supporting the close relationships among Picea, Pinus, and Cathaya. Moreover, several characteristics of the secondary structure of Pinaceae ITS1 were found as follows: (1) the structure is dominated by several extended hairpins; (2) the configuration complexity is positively correlated with subrepeat number; (3) paired subrepeats often partially overlap at the conserved motif (5'-GGCCACCCTAGTC), and form a long stem, while other subrepeats fold onto itself, leaving part of the conserved motif exposed in hairpin loops.     

Comparative ITS and AFLP analysis of diploid Cardamine (Brassicaceae) taxa from closely related polyploid complexes

BACKGROUND AND AIMS: Diploid representatives from the related polyploid complexes of Cardamine amara, C. pratensis and C. raphanifolia (Brassicaceae), were studied to elucidate phylogenetic relationships among the complexes and among the individual taxa included. METHODS: Two independent molecular data sets were used: nucleotide sequences from the internal transcribed spacers (ITS) of nrDNA, and amplified fragment length polymorphism (AFLP) markers. Seventeen diploid taxa from the studied groups were sampled. KEY RESULTS: Both ITS and AFLP analyses provided congruent results in inferred relationships, and revealed two main lineages. While the C. amara group, consisting of C. wiedemanniana and four subspecies of C. amara, was resolved as a well-supported monophyletic group, taxa from the C. pratensis and C. tenera groups (the latter representing diploid taxa of the complex of C. raphanifolia) all appeared together in a single clade/cluster with no support for the recognition of either of the groups. Intra-individual polymorphisms and patterns of nucleotide variation in the ITS region in C. uliginosa and C. tenera, together with the distribution of AFLP bands, indicate ancient hybridization and introgression among these Caucasian diploids. CONCLUSIONS: The lack of supported hierarchical structure suggests that extensive reticulate evolution between these groups, even at the diploid level, has occurred (although an alternative explanation, namely ancestral polymorphism in ITS data, cannot be completely excluded). Several implications for the investigation of the polyploid complexes of concern are drawn. When tracing origins of polyploid taxa, a much more complex scenario should be expected, taking into account all relatives as potential parents, irrespective of the group in which they are classified.     

Allopolyploid origin of Cardamine asarifolia (Brassicaceae): incongruence between plastid and nuclear ribosomal DNA sequences solved by a single-copy nuclear gene

Interspecific hybridization and polyploidization have played central roles in plant diversification. However, technical difficulties in the analyses of low-copy genes have limited the study of the origins of hybrid and polyploid plants. Here, we present a phylogenetic analysis of the hexaploid Cardamine asarifolia, distributed in the southern European Alps and northern Apennines. Our study included all relevant taxa of the genus found in Europe. A marked discrepancy was revealed between the trnL-trnF region of cpDNA and internal transcribed spacer (nrDNA ITS) sequences. To solve the incongruence, we sequenced a single-copy nuclear CHS gene (chalcone synthase) using a novel method to design homoeologue-specific PCR primers to bypass artefacts caused by artificial recombination of homoeologues during PCR and/or cloning. Three homoeologues were isolated from C. asarifolia, providing evidence for its allopolyploid origin. One homoeologue, showing the same phylogenetic position as the ITS sequences, most likely originated from an extinct parent. Furthermore, we documented recurrent polytopic hybridizations between C. asarifolia and diploid C. amara. The allohexaploidization and the following hybridization with a diploid species exemplify the ongoing dynamic processes of speciation in the genus Cardamine.     

Phylogenetic and population genetic analyses of diploid Leucaena (Leguminosae; Mimosoideae) reveal cryptic species diversity and patterns of divergent allopatric speciation

? Premise of the study: Leucaena comprises 17 diploid species, five tetraploid species, and a complex series of hybrids whose evolutionary histories have been influenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled multilocus phylogenetic and population genetic approaches to address species delimitation, interspecific relationships, hybridization, and the predominant mode of speciation among diploids. ? Methods: Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci representing 42 populations and 424 individuals. ? Results: Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identified two cryptic species that are consistent with geography and morphology. ? Conclusions: Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation among diploid Leucaena. These findings contrast with the potential hybrid origin of several tetraploid species and highlight the importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized species (L. cruziana) and the elevation of another taxon (L. collinsii subsp. zacapana) to specific status (L. zacapana) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.     

Phacopsis — A lichenicolous genus of the family Parmeliaceae

SSU nrDNA studies of two representatives of the lichenicolous genus Phacopsis revealed that they belong to the family Parmeliaceae (Lecanorales) and therefore represent lichenicolous lichens with an endokapylic thallus. Because they are the only lichenicolous taxa within this family, it is suggested that their transition from a foliose and/or fruticose precursor lichen might have been due to a unique (or rare) evolutionary one step event(s). Phylogenetic analyses of the ITS nrDNA of the type species of Phacopsis (P. vulpina) and Nesolechia (N. oxyspora = P. oxyspora) could neither confirm nor reject a monophyletic origin of these two Parmeliacean genera, as previously implied by their synonymization. However, it is considered premature to draw nomenclatorial consequences.     

Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria

We present phylogenetic analyses of 37 taxa of Fritillaria (Liliaceae), 15 species of Lilium, and several outgroup taxa from Liliaceae s.s. to investigate the generic delimitation of Fritillaria in relation to Lilium as well as infrageneric relationships within Fritillaria. We used DNA sequences from the maturase-coding plastid matK gene and the trnK intron, the intron of the ribosomal protein-coding rpl16 plastid gene, and the nuclear ribosomal internal transcribed spacers (ITS). Phylogenetic analysis using maximum parsimony defined Fritillaria and Lilium (the latter including Nomocharis) as sister taxa. Fritillaria sections Fritillaria and Liliorhiza are supported in part, and some of the most enigmatic species usually included in Fritillaria (sections Petilium and Theresia and the monotypic genus Korolkowia) are closely related. The results support the new classification of Fritillaria proposed by Rix. We postulate independent origins of the underground bulbils found in Fritillaria davidii and the remainder of subgenus Liliorhiza.     

Hybridization between the threatened plant, <Emphasis Type="Italic">Lespedeza leptostachya</Emphasis> Englem. and its co-occurring congener <Emphasis Type="Italic">Lespedeza capitata</Emphasis> Michx.: morphological and molecular evidence

Natural hybridization is common in the genus Lespedeza. No hybrids between Lespedeza leptostachya Englem. and Lespedeza capitata Michx. are formally recognized in any of the current floras, however observations in the field suggest that hybridization might occur in many of their shared habitats. Putative hybrids were compared to L. leptostachya and L. capitata using morphological measurements and screened for the presence of species-specific trnL-F gene region (cpDNA) and the ITS gene region (nrDNA). A discriminate analysis of 10 morphological measurements identified the hybrids as intermediate to both parents with two PCA axes explaining 99% of the variation between taxa. The presence of hybrids was confirmed by genetic markers with individuals morphologically identified as hybrids having cpDNA trnL-F genotypes identical to L. leptostachya and the ITS (nrDNA) phenotypes in most cases contain the ITS genotype of both parents, however, some putative hybrid individuals contained the ITS genotype of only one parents. Those individuals with L. leptostachya ITS and trnL-F could be a case of misclassification, but the presence of both L. capitata ITS genotypes and L. leptostachya trnL-F genotypes suggest segregation has occurred, which may result from either selfing or backcrossing.     

Worldwide phylogeny and biogeography of Cardamine flexuosa (Brassicaceae) and its relatives

Phylogenetic relationships, biogeography, and taxonomy of a group of taxa putatively related to the tetraploid Cardamine flexuosa were explored using sequences of the internal transcribed spacer region of nrDNA (ITS) and the trnL-trnF region of cpDNA. Taxon sampling focused on eastern Asia, North America, and Europe, and included 19 taxa represented by 177 and 182 accessions for each data set, respectively. Our analyses provided unequivocal evidence that Asian weedy populations traditionally assigned to C. flexuosa form an independent evolutionary lineage and represent a distinct taxon from European C. flexuosa. The allopolyploid origin of this common weed in paddy fields, its origin, and/or spread associated with the establishment of suitable man-made habitats are suggested. It is also found as an introduced weed in Australia and North America. Phylogenetic relationships and the associated taxonomic implications are presented and discussed for the group as a whole. Contrasting patterns of genetic variation (particularly in cpDNA) among different species were revealed. While very little haplotype diversity was found in widespread C. hirsuta and C. flexuosa, greater variation, showing phylogeographic structure, was observed in the tetraploid C. scutata within a relatively small area of Japan.     

VARIATION IN CHROMATOGRAPHIC PATTERNS IN THE TRAGOPOGON DUBIUS-PRATENSIS-PORRIFOLIUS COMPLEX (COMPOSITAE)

Natural populations of the diploid species, Tragopogon dubius, T. pratensis and T. porrifolius, the F₁ hybrids of these species, and the two amphidiploids, T. mirus and T. miscellus, were obtained from the same localities used for previous morphological and cytological studies of the evolutionary relationships of this complex. Inter- and intra-populational comparisons were made utilizing 2-dimensional chromatograms of these taxa. Members of the complex shared a group of 18 flavonoid-type pattern components, no one of which was completely species specific. Therefore, species patterns were expressed for a population rather than an individual with pattern components being expressed as a frequency. Patterns for F₁ hybrids and amphidiploids, expressed in the same manner, were relatable to the parental species. Diploid species and F₁ hybrid patterns were the most variable in areas of greatest sympatry, indicating considerable genetic interchange between species. The distribution of specific components in certain populations was interpreted as chemical introgression. Evidence was presented for separate origins of T. mirus in two localities. The results confirm previous interpretations of the evolutionary relationships in this complex, and when compared to those obtained for Baptisia, they implicate the different breeding systems as important factors in establishing the kind of variation observed.     

Molecular phylogeny of selected Old World Astragalus (Fabaceae): incongruence among chloroplast trnL-F, ndhF and nuclear ribosomal DNA ITS sequences

This study reports maximum parsimony and Bayesian phylogenetic analyses of selected Old World Astragalus using two chloroplast fragments including trnL-F and ndhF and the nuclear ribosomal internal transcribed spacer (nrDNA ITS). A total of 52 taxa including 34 euploid Old World and New World Astragalus , one aneuploid species from the Neo-Astragalus clade as a representative and 14 other Astragalean taxa, plus Cheseneya astragalina and two species of Caragana as outgroups were analyzed for both trnL-F and nrDNA ITS regions. ndhF was analyzed in 30 taxa and the same number for the combination of these three datasets were examined. In general, the trnL-F dataset and the ndhF and nrDNA ITS datasets generated more or less the same clades within Astragalus . However, in the trnL-F and ndhF phylogenies, Astragalus species are not gathered in a single clade, the so-called Astragalus s.s., as indicated by the nrDNA ITS tree. Visual inspection of these three phylogenies revealed that they were inconsistent regarding the position and relationships of Astragalus hemsleyi , A. ophiocarpus , A. annularis–A. epiglottis / Astragalus pelecinus, A. echinatus and A. arizonicus . Incongruence length difference test suggested that the trnL-F , ndhF and nrDNA ITS datasets were incongruent. In spite of this, phylogenetic analyses of the combined datasets as one unit or as three partitions generated trees that were topologically similar as a mix of the cpDNA and the nrDNA ITS trees. However, the combined dataset provided more resolved and statistically supported clades. The recently described A. memoriosus appeared closely related to A. stocksii (both from sect. Caraganella ) based on both trnL-F and nrDNA ITS sequences.     

Hybridogenetic Reproduction and Maternal Ancestry of Polyploid Iberian Fish: The Tropidophoxinellus Alburnoides Complex

Iberian minnows collectively known as the Tropidophoxinellus alburnoides STEINDACHNER complex comprise diploid and polyploid forms with highly female biased sex ratios. Previous investigators suggested that all-female clonal reproduction and interspecific hybridization may occur in this complex. We examined nuclear (allozymes) and cytoplasmic genes (mtDNA) to assess the evolutionary origins, relationships, and reproductive modes of T. alburnoides from western Spain. The multi-locus allozyme data clearly revealed the hybrid nature of all polyploid forms of this fish and some diploid forms as well. Diagnostic markers identified fish from the genus Leuciscus as the paternal ancestor of hybrids in the Duero and Guadiana River Basins. Additionally, analysis of nuclear markers revealed that hybridogenetic reproduction occurs in the diploid and triploid hybrids. The hybrids fully express the paternal Leuciscus genome and then discard it during oogenesis. Hybridogenetic ova contain only maternal nuclear genes and mtDNA from a non-hybrid T. alburnoides ancestor. Apparently diploid and triploid hybrids of T. alburnoides persist as sperm parasites on males of a sexually reproducing Leuciscus host species.     

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

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

Karyotype variation, evolution and phylogeny in Borago (Boraginaceae), with emphasis on subgenus Buglossites in the Corso-Sardinian system

BACKGROUND AND AIMS: Karyological variation in the Mediterranean genus Borago and cytogeography of subgenus Buglossites in Corsica, Sardinia and the Tuscan Archipelago were investigated in combination with a molecular phylogenetic analysis aimed at elucidating relationships between subgenera and taxa with different chromosome features. METHODS: Karyotype analysis was performed on population samples of B. pygmaea, B. morisiana, B. trabutii and B. officinalis. Phylogenetic analyses were based on ITS1 nrDNA and matK cpDNA sequences. KEY RESULTS: Four base numbers were found, x = 6, 8, 9 and 15, and three ploidy levels based on x = 8. In subgenus Buglossites the Sardinian endemic B. morisiana is diploid with 2n = 18, while B. pygmaea includes three allopatric cytotypes with 2n = 30 (Sardinia), 2n = 32 (southern Corsica) and 2n = 48 (central northern Corsica and Capraia). In subgenus Borago, the Moroccan endemic B. trabutii and the widespread B. officinalis have 2n = 12 and 2n = 16, respectively. Molecular data support the monophyly of Borago, while relationships in subgenus Borago remain unclear. Borago trabutii appears as the earliest divergent lineage and is sister to a clade with B. officinalis, B. morisiana and B. pygmaea. Subgenus Buglossites is also monophyletic, but no correspondence between ITS1 phylogeny and B. pygmaea cytotypes occurs. CONCLUSIONS: Chromosome variation in Borago is wider than previously known. Two base numbers may represent the ancestral condition in this small genus, x = 6 or x = 8. An increase in chromosome number and karyotype asymmetry, a decrease in chromosome size and heterochromatin content, and the appearance of polyploidy are the most significant karyological changes associated with the divergence of the Buglossites clade. High ITS1 variation in the tetra- and hypotetraploid races of B. pygmaea suggests a multiple origin, while the lower polymorphism of the hexaploid race and its allopatric distribution in the northernmost part of the range is better explained with a single origin via union of unreduced and reduced gametes.     

A continental-wide perspective: the genepool of nuclear encoded ribosomal DNA and single-copy gene sequences in North American Boechera (Brassicaceae)

74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data.     

Potential phylogenetic utility of the low-copy nuclear gene pistillata in dicotyledonous plants: comparison to nrDNA ITS and trnL intron in Sphaerocardamum and other Brassicaceae.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6-30.8%, 284 characters) and ITS (0-24%, 94 characters) than in the chloroplast trnL intron (0-4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0-2.4%, 1 character) and nrDNA ITS (0-2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15-3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.     

Molecular phylogeny and evolution of Caricaceae based on rDNA internal transcribed spacers and chloroplast sequence data

This study focused on clarifying phylogenetic relationships and evolution within Caricaceae. Our phylogenetic analysis based on nucleotide sequences from the ITS of the ribosomal DNA and three chloroplast fragments (matK, trnL-trnF, and psbA-trnH) included 29 taxa belonging to five genera: the neotropical genera Carica, Vasconcellea, Jarilla, and Jacaratia and the equatorial African genus Cylicomorpha. Having a relatively low mutation rate, matK, and trnL-trnF were used for estimating relationships at the generic level, while intrageneric evolution within Vasconcellea was studied with the more variable ITS and psbA-trnH sequences. Gaps, coded as binary characters, were added to the sequence alignments before performing Maximum Parsimony and Maximum Likelihood analyses. Monophyly of Caricaceae as well as phylogenetic distance between Carica and Vasconcellea species, previously belonging to the same genus, and monophyly of the resurrected genus Vasconcellea were emphasized. Within Vasconcellea, the largest genus of this family, two well-confirmed evolutionary lineages could be discerned: (1) V.xheilbornii, V. weberbaueri, V. stipulata, and V. parviflora and (2) a clade holding all other taxa of the genus. Incongruence between nuclear ITS and chloroplast psbA-trnH datasets, shown to be significantly caused by some taxa of the genus Vasconcellea, indicated that reticulate events in this genus might be more frequent than previously suspected. Moreover, intra-individual ITS sequence heterogeneity provided further evidence for the hybrid or introgressed origin of different taxa and one presumed hybrid belonging to this genus.     

Ribosomal DNA variation,recombination and inheritance in the basidiomycete Trichaptum abietinum: implications for reticulate evolution

Two divergent nuclear ribosomal DNA (nrDNA) types, designated alpha and beta, were found distributed in 11 North European populations of the basidiomycete Trichaptum abietinum. These types differed by a 220 bp indel in the internal transcribed spacer 1 (ITS1) sequence and a number of linked substitutions and small indel motives in the internal transcribed and intergenic spacers (ITS1, ITS2, IGS1 and IGS2). The alpha and beta haplotypes co-occurred in heterozygous somatic individuals (dikaryons) and segregated in a Mendelian fashion in monokaryotic single spore progenies. This result suggests that the haplotypes are encoded in different nuclei of field-collected dikaryons and inherited as a single locus. No meiotic recombinants were observed among the sequenced monokaryons. Population genetic analyses by PCR-RFLP revealed that a low frequency of evolutionary intermediate nrDNA types also existed in natural populations, presumably as a result of meiotic recombination of alpha and beta nrDNA. The existence of divergent nrDNA types in T. abietinum could be a result of a former independent evolution followed by a hybridization event. Phylogenetic analyses of ITS sequences suggest that the sister taxon T. fusco-violaceum has been involved in the evolutionary history of T. abietinum. Sequence polymorphisms observed in the translation elongation factor 1alpha (efa) and glyceraldehyde-3-phosphate dehydrogenase (gpd) genes, did not reveal two well-defined types of these genes. The results are discussed in the light of other evolutionary mechanisms as well.