Inferring the History of the Polyploid Silene aegaea (Caryophyllaceae) Using Plastid and Homoeologous Nuclear DNA Sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.     

The origin and number of introductions of the Hawaiian endemic Silene species (Caryophyllaceae)

The Hawaiian endemic Silene are a small group of woody or semiwoody representatives from a large, predominantly herbaceous, species-rich genus. We here investigated the origin and number of introductions of the endemic Hawaiian Silene based on phylogenetic relationships inferred from DNA sequences from both the plastid (the rps16 intron) and the nuclear (ribosomal internal transcribed sequences, ITS, and intron 23 of the RPB2 gene) genomes. Silene antirrhina, a widespread weedy American annual, is strongly supported as sister to a monophyletic group consisting of the Hawaiian Silene, indicating a single colonization event. There are no obvious morphological similarities between S. antirrhina and any of the species of Hawaiian Silene. Our results suggest an American origin for the Hawaiian endemics because that would require only a single trans-ocean dispersal. Two of the Hawaiian endemics (S. struthioloides and S. hawaiiensis) that form a subclade in the analyses have evolved woodiness after introduction to the Hawaiian Islands. Our results contribute to other recent results based on molecular phylogenetics that emphasize the American continent as a source area for the Hawaiian flora and support a striking morphological radiation and evolution of woodiness from a single introduction to the archipelago.     

Reticulate phylogenetics and phytogeographical structure of Heliosperma (Sileneae, Caryophyllaceae) inferred from chloroplast and nuclear DNA sequences

The Balkan Peninsula is known to be one of the most diverse and species-rich parts of Europe, but its biota has gained much less attention in phylogenetic and evolutionary studies compared to other southern European mountain systems. We used nuclear ribosomal internal transcribed spacer (ITS) sequences and intron sequences of the chloroplast gene rps16 to examine phylogenetic and biogeographical patterns within the genus Heliosperma (Sileneae, Caryophyllaceae). The ITS and rps16 intron sequences both support monophyly of Heliosperma, but the data are not conclusive with regard to its exact origin. Three strongly supported clades are found in both data sets, corresponding to Heliosperma alpestre, Heliosperma macranthum and the Heliosperma pusillum clade, including all other taxa. The interrelationships among these three differ between the nuclear and the plastid data sets. Hierarchical relationships within the H. pusillum clade are poorly resolved by the ITS data, but the rps16 intron sequences form two well-supported clades which are geographically, rather than taxonomically, correlated. A similar geographical structure is found in the ITS data, when analyzed with the NeighbourNet method. The apparent rate of change within Heliosperma is slightly higher for rps16 as compared to ITS. In contrast, in the Sileneae outgroup, ITS substitution rates are more than twice as high as those for rps16, a situation more in agreement with what has been found in other rate comparisons of noncoding cpDNA and ITS. Unlike most other Sileneae ITS sequences, the H. pusillum group sequences display extensive polymorphism. A possible explanation to these patterns is extensive hybridization and gene flow within Heliosperma, which together with concerted evolution may have eradicated the ancient divergence suggested by the rps16 data. The morphological differentiation into high elevation, mainly widely distributed taxa, and low elevation narrow endemics is not correlated with the molecular data, and is possibly a result of ecological differentiation.     

Molecular phylogenetics of subfamily Calamoideae (Palmae) based on nrDNA ITS and cpDNA rps16 intron sequence data

Phylogenetic relationships among the 22 genera of the palm subfamily Calamoideae were investigated using DNA sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region and the chloroplast rps16 intron. The rps16 intron displayed low levels of variation, corroborating previous reports that the chloroplast genome of palms is highly conserved. High levels of within-individual polymorphism were identified in the ITS region, indicating that concerted evolution is not effectively homogenizing the ITS repeats. In the majority of cases, multiple clones from individuals resolved as monophyletic. However, the high levels of homoplasy in the ITS dataset, along with generally poor jackknife support for many clades, led to concerns that topologies obtained from these data might be unreliable. Nevertheless, congruence between trees based on ITS data alone and those based on rps16 intron data was high. Simultaneous analyses of both datasets yielded well-resolved topologies with high levels of jackknife support. A number of exciting groups emerged from the analyses: the African rattan clade comprising the endemic African rattan genera Laccosperma, Eremospatha, and Oncocalamus; the Lepidocaryeae-Raphia clade comprising the fan-leaved New World tribe Lepidocaryeae and the African genus Raphia; and the Asian clade comprising all Asian genera except Eugeissona. The position of Eugeissona was variable, although it did not resolve inside any of the three major clades mentioned above.     

Evolution of a RNA polymerase gene family in Silene (Caryophyllaceae)-incomplete concerted evolution and topological congruence among paralogues

Four low-copy nuclear DNA intron regions from the second largest subunits of the RNA polymerase gene family (RPA2, RPB2, RPD2a, and RPD2b), the internal transcribed spacers (ITSs) from the nuclear ribosomal regions, and the rps16 intron from the chloroplast were sequenced and used in a phylogenetic analysis of 29 species from the tribe Sileneae (Caryophyllaceae). We used a low stringency nested polymerase chain reaction (PCR) approach to overcome the difficulties of constructing specific primers for amplification of the low copy nuclear DNA regions. Maximum parsimony analyses resulted in largely congruent phylogenetic trees for all regions. We tested overall model congruence in a likelihood context using the software PLATO and found that ITSs, RPA2, and RPB2 deviated from the maximum likelihood model for the combined data. The topology parameter was then isolated and topological congruence assessed by nonparametric bootstrapping. No strong topological incongruence was found. The analysis of the combined data sets resolves previously poorly known major relationships within Sileneae. Two paralogues of RPD2 were found, and several independent losses and incomplete concerted evolution were inferred. The among-site rate variation was significantly lower in the RNA polymerase introns than in the rps16 intron and ITSs, a property that is attractive in phylogenetic analyses.     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

Discovery of paralogous nuclear gene sequences coding for the second-largest subunit of RNA polymerase II (RPB2) and their phylogenetic utility in gentianales of the asterids

Paralogous sequences of the RPB2 gene are demonstrated in the angiosperm order Gentianales. Two different copies were found by using different PCR primer pairs targeting a region that corresponds to exons 22-24 in the Arabidopsis RPB2 gene. One of the copies (RPB2-d) lacks introns in this region, whereas the other has introns at locations corresponding to those of green plants previously investigated. When analyzed with other available RPB2 sequences from this region, all 28 RPB2-d sequences obtained from the Gentianales and the four sequences from the Lamiales form a monophyletic group, together with a previously published tomato cDNA sequence. The substitution patterns, relative rates of change, and nucleotide compositions of the two paralogous RPB2 exon regions are similar, and none of them shows any signs of being a pseudogene. Although multiple copies of similar, paralogous sequences can confound phylogenetic interpretations, the lack of introns in RPB2-d make a priori homology assessment easy. The phylogenetic utility of RPB2-d within the Gentianales is evaluated in comparison with the chloroplast genes ndhF and rbcL. The hierarchical information in the RPB2-d region sequenced is more incongruent with that of the plastid genes than the plastid genes are with each other as determined by incongruence length difference tests. In contrast to the plastid genes, parsimony-informative third codon positions of RPB2 have a significantly higher rate of change than first and second positions. Topologically, the trees from the three genes are similar, and the differences are usually only weakly supported. In terms of support, RPB2 gives the highest jackknife support per sequenced nucleotide, whereas ndhF gives the highest Bremer support per sequenced nucleotide. The RPB2-d locus has the potential to be a valuable nuclear marker for determination of phylogenetic relationships within the euasterid I group of plants.     

A three-gene phylogeny of the Mycena pura complex reveals 11 phylogenetic species and shows ITS to be unreliable for species identification

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance.While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (<50) support, and the partition homogeneity incongruence length difference (ILD) tests were not significant. ILD tests revealed significant discordances between ITS and the tEF and RPB1 markers in several lineages. And our analyses suggested recombination between ITS1 and ITS2, most pronounced in one phylospecies that was identical in tEF and RPB1. Based on the agreement between tEF and RPB1, we defined 11 mutually concordant terminal clades as phylospecies inside the M. pura morphospecies; most of them cryptic. While neither of the markers showed an unequivocal barcoding gap between inter- and intraspecific diversity, the overlap was most pronounced for ITS (intraspecific diversity 0-3.5 %, interspecific diversity 0.4 %-8.8 %). A clustering analysis on tEF separated at a 1.5 % level returned all phylogenetic species as Operational Taxonomic Units (OTUs), while ITS at both a 1.5 % level and at a 3 % threshold level not only underestimated diversity as found by the tEF and RPB1, but also identified an OTU which was not a phylogenetic species. Thus, our investigation does not support the universal suitability of ITS for species recognition in particular, and emphasises the general limitation of single gene analyses combined with single percentage separation values.     

The enigma of the gene coding for ribosomal protein S12 in the chloroplasts of Nicotiana.

A 2.9 kbp region from within the inverted repeat of Nicotiana chloroplast DNA hybridized with a chloroplast DNA fragment from Euglena containing the complete rps12 gene coding for ribosomal protein S12. Nucleotide sequencing within this region revealed the existance of two rps12 coding stretches interrupted by 540 bp having class II intron structure. Joining and decoding the exon regions produced a sequence of 85 amino acids colinear and 81% homologous to the S12 protein of Euglena chloroplasts and E. coli, starting from amino acid residue 38 to the stop codon. Immediately upstream of codon 38, conserved intron sequences were located. However, the 5' 37 codon of Nicotiana chloroplast rps12 could not be identified by electron microscopy of RNA-DNA hybrids within a DNA region extending 4000 bp upstream of codon 38, nor by computer search of a completely sequenced region extending for more than 9000 bp upstream of this codon. In E. coli, alteration in rps12 codons 42 or 87 causes streptomycin resistance. However, the nucleotide sequence of the identified rps12 exons in two Nicotiana chloroplast mutants resistant to streptomycin were found to be identical to that of wild type.     

南方灵芝ITS、EF1-α和RPB2序列多态性研究

内转录间隔区（ITS）、延伸因子1-α（EF1-α）与RNA聚合酶II第二大亚基（RPB2）是真菌系统学研究中常用的基因片段。已有研究发现,在同一个体内ITS也可能存在差异,但EF1-α和RPB2是否也存在同样的现象却鲜有报道。本研究通过克隆测序,分析了南方灵芝Ganoderma australe的ITS、EF1-α和RPB2在同一个体中的序列差异,并探讨这种序列差异是否会影响分子鉴定的准确性。结果表明,在南方灵芝供试样品中,ITS、EF1-α和RPB2序列都有可能存在个体内变异,但个体内变异程度不尽相同。基于供试样品和克隆测序结果发现,ITS序列在同一个体内的变异最高可达2.3%,而RPB2序列在同一个体内的差异仅0.5%,EF1-α序列在同一个体内序列差异也可高达1.8%。ITS和EF1-α序列在同一个体内的差异可能超过了灵芝属部分物种间的差异,对于部分灵芝属物种来说直接利用克隆序列进行分子鉴定或系统发育分析可能存在一些问题。     

Molecular data suggest a hybrid origin for the invasive Caulerpa racemosa (Caulerpales,Chlorophyta) in the Mediterranean Sea

Morphological data has provided a basis for the hypothesis that three taxa belonging to the Caulerpa racemosa complex occur in the Mediterranean Sea: var. turbinata–uvifera, var. lamourouxi, and the `invasive variety'. In order to test this hypothesis and to determine the origin of the `invasive variety', the transcribed spacer ITS1–ITS2 and an 18S ribosomal DNA (rDNA) intron were analysed from 16 isolates of Caulerpa racemosa. The `invasive variety' shows intraindividual polymorphism for both types of sequences. The ITS1–ITS2 data confirm that the three morphological varieties of C. racemosa from the Mediterranean Sea are distinct taxonomic units. The 18S intron data suggest that the new `invasive variety' could be a recent hybrid between var. turbinata–uvifera and an unknown tropical strain. Incongruence between the phylogenetic tree computed from ITS1–ITS2 regions and the 18S intron indicates that homogenization processes of concerted evolution have run at different rates.     

Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies

We sampled and analyzed approximately 2900bp across the three loci from 54 taxa belonging to a taxonomically difficult group of Cortinarius subgenus Phlegmacium. The combined analyses of ITS and variable regions of RPB1 and RPB2 greatly increase the resolution and nodal support for phylogenies of these closely related species belonging to clades that until now have proven very difficult to resolve with the ribosomal markers, nLSU and ITS. We present the first study of the utility of variable regions of the genes encoding the two largest subunits of RNA polymerase II (RPB1 and RPB2) for inferring the phylogeny of mushroom-forming fungi in combination with and compared to the widely used ribosomal marker ITS. The studied region of RPB1 contains an intron of the size and variability of ITS along with many variable positions in coding regions. Though almost entirely coding, the studied region of RPB2 is more variable than ITS. Both RNA polymerase II genes were alignable across all taxa. Our results indicate that several sections of Cortinarius need redefinition, and that several taxa treated at subspecific and varietal level should be treated at specific level. We suggest a new section for the two species, C. caesiocortinatus and C. prasinocyaneus, which constitute a well-supported separate lineage. We speculate that sequence information from RNA polymerase II genes have the potential for resolving phylogenetic problems at several levels of the diverse and taxonomically very challenging genus Cortinarius.     

A phylogenetic analysis of palm subtribe Archontophoenicinae (Arecaceae) based on 14 DNA regions

Subtribe Archontophoenicinae belongs to Areceae, the largest of all palm tribes. It includes 15 species distributed in five genera, all found in the south‐western Pacific Region. Archontophoenicinae are rather homogeneous in morphology, making phylogenetic relationships problematic to reconstruct using morphological characters. In this study we investigated phylogenetic relationships in Archontophoenicinae based on all 15 species of the subtribe, using a combination of nine plastid and five nuclear DNA sequence markers. The plastid regions used were the coding rbcL, matK, ndhF and rpoC1 (exon 2) and the non‐coding rps16 intron, atpF‐atpH, psbK‐psbI, trnL‐trnF and trnQ‐rps16. The nuclear regions used were AG1, BRSC, ITS2, PRK and RPB2, which have all proved useful in palm systematics. We compared the phylogenetic hypotheses resulting from the plastid versus nuclear datasets, and combined both datasets to retrieve as much phylogenetic information as possible. Our results strongly support a clade composed of all species of Archontophoenix, Actinokentia, Chambeyronia and Kentiopsis, but raise the question of whether Actinorhytis, the fifth genus, should remain in Archontophoenicinae. Interspecific relationships in ‘core Archontophoenicinae’ still remain incompletely resolved, despite the gene and taxon sampling being substantially greater than in previous studies, and question the monophyly of the New Caledonian genera Chambeyronia and Kentiopsis. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 469–481.     

GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)

Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trn L (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trn L intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trn L intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi.     

The phylogenetic relationships among some Randia (Rubiaceae) taxa

Phylogenetic relationships among some Randia (Rubiaceae, Gardenieae) taxa were estimated based on sequence variation in the nuclear ribosomal internal transcribed spacers (ITS) and rps 16 intron (cpDNA). During the investigation of rpsl6 intron of 9 studied Central American Randia species, two well supported subclades were separated. Analysis of ITS data of 16 Randia species shows 3 major clades. A group of mainly lowland, South American Randia species is moderate supported (75%). Species from Mexico form a strongly supported (97%) clade, but the Central American and Mexican Randia species are low supported (58%). However the last two groups are well supported together (95%). The molecular delimination is well in line with the size of leaves combined with the texture of exocarp.     

Phylogenetic relationships among species of <Emphasis Type="Italic">Leotia</Emphasis> (Leotiales) based on ITS and RPB2 sequences

Thirty-three collections of Leotia were used to investigate inter-and infra-specific relationships in the genus. Collections were obtained from various parts of the world and represent the ascomatal color forms typical in species of the genus. The ITS rDNA and a variable region of the RNA polymerase II (RPB2) gene were sequenced and analyzed using parsimony and maximum likelihood methods. Although ITS and RPB2 tree topologies differed in regard to the position of two clades of L. lubrica and L. atrovirens, no significant conflict between ITS and RPB2 data or trees was found as determined by the partition homogeneity test. RPB2 sequences in general gave results comparable to ITS; the RPB2 sequences were more easily aligned. Phylogenetic analysis of the sequence data indicates that L. viscosa, L. lubrica and L. atrovirens are polyphyletic species. This suggests that ascomatal color in fresh specimens is not a reliable character alone for determining species in this group. Four major well-supported groups were found; these do not fully correspond to the commonly recognized species. Stipe color, in both fresh and dry condition, seems to correlate with the major recognized groups but features of the ascospores, asci and paraphyses prove too variable to be informative. The most basal group of Leotia species, identified as L. atrovirens, differ from all others by having stipes without gel tissue in their outer layers.