Lack of trnL intron in the thermoacidophilic red alga Galdieria sulphuraria (Galdieri) Merola

Abstract

The plastid trnC‐trnL(UAA)‐ilvH region from Galdieria sulphuraria was cloned and sequenced with the aim of verifying the absence of the trnL intron. The sequence alignment shows both the absence of a trnL intron and the colinearity of the whole region of the plastidial DNA of G. sulphuraria with that of the other thermoacidophilic red algae.     

Evolution of the Euphrasia transmorrisonensis complex (Orobanchaceae) in alpine areas of Taiwan

Aims To unravel isolation and differentiation of the genetic structure of the Euphrasia transmorrisonensis complex, a showy herb, among alpine regions of mountain peaks in subtropical Taiwan and to infer its evolutionary history. Location Alpine ecosystems of high‐montane regions of Taiwan. Methods Phylogenetic analyses of the trnL intron and the trnL–trnF intergenic spacer of chloroplast (cp) DNA, and the intertranscribed spacer (ITS) of nuclear ribosomal (nr) DNA between 18S and 26S were carried out on 18 populations of the E. transmorrisonensis complex in Taiwan. Results In total, 10 haplotypes for cpDNA and 14 haplotypes for nrDNA were detected. Three population groups located in the northern, north‐eastern, and south‐central regions of the Central Mountain Range (CMR) were revealed according to the frequencies of haplotypes and haplotype lineages of nrDNA. Balancing selection might have played a role in the evolution of Euphrasia in Taiwan. Main conclusions By integrating the spatial‐genetic patterns of cpDNA and nrDNA, two possible evolutionary histories of Euphrasia in Taiwan were inferred. The favourable hypotheses for interpreting the data suggest at least three origins of the E. transmorrisonensis complex in Taiwan, corresponding to each nuclear lineage in the northern (II), northern/north‐eastern (I), and central/southern regions (III) with subsequent hybridization between lineages I and II and lineages II and III. These lineage boundaries are strengthened by the finding that haplotypes of C derived from cpDNA were found in the geographical region of lineage II of nrDNA, while haplotypes of A derived from cpDNA were found in the region of lineage III of nrDNA. Thus, the origin of chloroplasts exclusive to lineages II and III supports their long‐term isolation from one another.     

Phylogenetic relationships between disjunctly occurring groups of Tristicha trifaria (Podostemaceae)

Aim To reveal the phylogeographic relationship of disjunct specimens of Tristicha trifaria (Bory ex Willd.) Spreng., a member of the Podostemaceae river‐weed family, which is distributed exceptionally widely, but disjunctly, in Africa and the Americas. Location Brazil, Mexico, Ghana, Tanzania and Madagascar. Methods The chloroplast matK and rbcL genes, a trnK intron, the trnS‐trnG intergenic spacer (IGS), the two IGSs of trnT‐trnL‐trnF, a trnL intron, and nuclear ribosomal ITS regions were sequenced and analysed. Phylogenetic analyses were conducted using maximum likelihood and maximum parsimony methods. Results The T. trifaria samples analysed were separated into two groups in a rooted tree based on a combined matK/rbcL/ITS dataset; one contained the West African and all of the American samples, and the other contained the East African and Madagascan samples. An unrooted tree obtained from a combined analysis of all the chloroplast DNA and nuclear ITS data showed that a sample from West Africa was sister to an American T. trifaria group. Main conclusions The American and West African T. trifaria are closely related, despite the great distance between their locations. This observation, along with a tree of the whole Tristichoideae subfamily and estimated divergence times, suggests that an ancestor of T. trifaria migrated from Asia to Africa during the early Tertiary, and that this was followed by further westward migration to the Americas at the end of the Miocene or in the early Pliocene.     

Multiple autopolyploidizations and range expansion of Allium przewalskianum Regel. (Alliaceae) in the Qinghai‐Tibetan Plateau

We used the Allium przewalskianum diploid–tetraploid complex on the Qinghai‐Tibetan Plateau (QTP) as a model to examine how this complex responded to the Quaternary climatic oscillations, and whether multiple autopolyploidizations have occurred. We sequenced five chloroplast DNA (cpDNA) fragments (accD‐psaI, trnH‐psbA, trnL‐trnF, trnS‐trnG and rpl16‐intron) in 306 individuals (all of known ploidy level) from 48 populations across the distribution of this species complex. We identified a total of 32 haplotypes—11 in diploids only, 13 in tetraploids only, and 8 found in both cytotypes. This, plus network analyses, indicated that tetraploids have arisen independently from diploids at least eight times. Most populations in the eastern QTP contained multiple haplotypes, but only a single haplotype was found for 17 tetraploid populations on the western QTP, suggesting a recent colonization of the western QTP. We further found that this species complex underwent an earlier range expansion around 5–150 thousand years ago (kya), after the largest glacial period (800–170 kya) in the QTP. In addition, the high frequencies of tetraploids in the QTP suggested that the tetraploid A. przewalskianum cytotype has evolutionary advantages over diploids in colonizing and/or surviving the arid habitats of the QTP.     

Phylogeny of the Madagascan endemic family Didiereaceae

A molecular phylogeny of the Didiereaceae was produced through parsimony analysis of chloroplastrpl16 intron andtrnL-trnF andtrnT-trnL intergenic spacer sequences of all eleven species of the Didiereaceae and several outgroup taxa from the Portulacaceae. Results indicated that: 1) the Didiereaceae were embedded within the Portulacaceae, withCalyptrotheca as the sister group of the family; 2) present generic limits were supported; 3)Alluaudiopsis was the most basal lineage; 4) at least two separate episodes of polyploidization within the genusAlluaudia had occurred, and 5) unusually low amounts of variation were present in rapidly evolving noncoding plastid sequences.     

The complete chloroplast genome of Myriophyllum spicatum reveals a 4‐kb inversion and new insights regarding plastome evolution in Haloragaceae

Myriophyllum, among the most species‐rich genera of aquatic angiosperms with ca. 68 species, is an extensively distributed hydrophyte lineage in the cosmopolitan family Haloragaceae. The chloroplast (cp) genome is useful in the study of genetic evolution, phylogenetic analysis, and molecular dating of controversial taxa. Here, we sequenced and assembled the whole chloroplast genome of Myriophyllum spicatum L. and compared it to other species in the order Saxifragales. The complete chloroplast genome sequence of M. spicatum is 158,858 bp long and displays a quadripartite structure with two inverted repeats (IR) separating the large single copy (LSC) region from the small single copy (SSC) region. Based on sequence identification and the phylogenetic analysis, a 4‐kb phylogenetically informative inversion between trnE‐trnC in Myriophyllum was determined, and we have placed this inversion on a lineage specific to Myriophyllum and its close relatives. The divergence time estimation suggested that the trnE‐trnC inversion possibly occurred between the upper Cretaceous (72.54 MYA) and middle Eocene (47.28 MYA) before the divergence of Myriophyllum from its most recent common ancestor. The unique 4‐kb inversion might be caused by an occurrence of nonrandom recombination associated with climate changes around the K‐Pg boundary, making it interesting for future evolutionary investigations.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

DNA sequences of tobacco chloroplast genes for tRNASer (GGA), tRNAThr (UGU), tRNALeu (UAA), tRNAPhe (GAA): the tRNALeu gene contains a 503 bp intron

Summary The location and nucleotide sequence of tobacco chloroplast genes for tRNA^Ser (GGA), tRNA^Thr (UGU), tRNA^Leu (UAA) and tRNA^Phe (GAA) (trnS-GGA, trnT-UGU, trnL-UAA and trnF-GAA, respectively) have been determined. These genes are located in the 10 kbp BamHI fragment which lies in the middle of the large single-copy region of the chloroplast DNA. The gene order is trnS-trnT-trnL-trnF. The trnS, trnL and trnF are encoded on the same strand while the trnT on the opposite strand. The trnL contains a 503 bp intron like maize and broad bean trnL-UAAs.     

Autecological traits determined two evolutionary strategies in Mediterranean plants during the Quaternary: low differentiation and range expansion versus geographical speciation in Linaria

The evolutionary patterns of the Mediterranean flora during the Quaternary have been relatively well documented based on phylogenetic and biogeographic analyses, but few studies have addressed the evolutionary traits that determined diversification and range expansion success during this period. We analysed previously published and newly generated sequences of three plastid noncoding regions (rpl32‐trnL^UAG, trnS‐trnG and trnL‐trnF), the nuclear ribosomal internal transcribed spacer (ITS) and a low‐copy nuclear gene intron (AGT1) of Linaria sect. Supinae, a group of angiosperms that diversified in the Quaternary. The origin and recent colonization dynamics of closely related lineages were inferred by biogeographic reconstruction and phylogeographic analyses, while breeding system experiments coupled with ecological and morphological data were used to test association with range expansion and diversification. A combination of traits, including selfing, short lifespan and the ability to tolerate a wide variety of substrates, were key factors underlying range expansion after long‐distance dispersal throughout the Mediterranean basin. By contrast, self‐incompatibility may have promoted higher diversification rates in narrow ranges of the Iberian Peninsula. We argue that a few traits contributed to the adoption of two contrasting strategies that may have been predominant in the evolution of Mediterranean angiosperms.     

Phylogenetic relationships within Luzula DC. and Juncus L. (Juncaceae): A comparison of phylogenetic signals of trnL-trnF intergenic spacer, trnL intron and rbcL plastome sequence data

Juncus and Luzula are the largest, almost cosmopolitan, genera in the Juncaceae. Relationships within Juncus and Luzula and among other genera of Juncaceae (Distichia, Marsippospermum, Oxychloë, Patosia and Rostkovia) remain incompletely resolved. RbcL sequence data resolved a part of the supraspecific phylogeny, but many clades remain polytomic. For this reason, the non‐coding cpDNA regions, trnL intron and trnL‐trnF intergenic spacer, were sequenced. We intended to create hypotheses of relationships within Juncaceae and to test the classification of the sections, but a primary goal to this study was to assess the relationships within Juncus and Luzula and to test for monophyly of groups recognized from rbcL data (especially the monophyly of genus Luzula and the Southern Hemisphere Clade (SHC)). Furthermore, we tested the influence of different rooting and ingroup composition on the tree topology. The parsimony analyses revealed several well‐supported lineages. The traditionally distinguished genus Luzula is monophyletic and Juncus is non‐monophyletic. Two subgenera of Luzula (Pterodes and Luzula) are non‐monophyletic, while subg. Marlenia forms a sister group to the whole Luzula clade (trnL‐F data set). Within Juncus, both subgenus Juncus and subgenus Agathryon are non‐monophyletic. SHC is clustered not only with the South African J. lomatophylus and J. capensis, but also together with members of the section Juncus, Caespitosi and Graminifolii. These sections form a well‐separated sister group to the SHC. Within the genera Juncus and Luzula, monophyly is demonstrated for a number of groups (e.g., Juncus section Stygiopsis, Luzula section Luzula) but questioned for others (e.g., Juncus section Graminifolii). The unusual, separate positioning of Juncus trifidus and J. monanthos were clarified by trnL‐trnF sequence data, but vary within the tree topology depending on outgroup selection and also due to LBA phenomenon. © The Willi Hennig Society 2006.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.     

Amplicon pyrosequencing late Pleistocene permafrost: the removal of putative contaminant sequences and small‐scale reproducibility

DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small‐scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi‐squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present‐day Yukon.     

Clonal diversity and geographic structure in <Emphasis Type="Italic">Pleurochaete squarrosa</Emphasis> (Pottiaceae): different sampling scale approach

This paper describes our investigation of genetic variation and clonal structure of the Mediterranean moss Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae), using inter-simple sequence repeat (ISSR) molecular markers and trnL_UAA (intron of plastid gene for Leu tRNA) sequence, choosing different sampling strategies and scales on 16 European populations. Moreover, the intercontinental distribution of two trnL haplotypes, previously found over a large area and including 24 populations in three continents, was also investigated. Despite the prevalent asexual reproduction, P. squarrosa shows a high level of genetic diversity. Some site features seem to affect the clonal structure at the local scale, influencing the relocation of detached fragments and the level of intermingling, but they do not substantially affect genetic diversity. The peculiar vegetative reproduction coupled with somatic mutation could partly account for the genetic variation detected. Genetic distances highlight geographic isolation and limited gene flow among populations. We found only two trnL haplotypes in Europe due to length polymorphism, but, over an intercontinental scale, only non-delete trnL was found in Africa and the USA. ISSR analysis within each population detected a higher genetic distance between the samples with different trnL haplotypes, suggesting the presence of two different genetic lineages within this species, geographically overlapping in the Mediterranean Basin.     

TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.     

Using molecular data to test the monophyly of Lallemantia in the subtribe Nepetinae (Mentheae,Lamiaceae)

Phylogenetic relationships among the species of Lallemantia and its close allies (Lamiaceae, Mentheae) were investigated using nuclear (ITS) and plastid (trnL, trnL/F, trnS/G, rpl32, and rpl32-trnL) DNA sequences. Phylogenetic results from Bayesian and parsimony analyses show that (1) Lallemantia is monophyletic, (2) Hymenocrater is nested within Nepeta, and (3) Lallemantia is more closely related to Dracocephalum than other genera in Nepetinae. Based on the molecular results, the genus Lallemantia comprises two disparate lineages, with each lineage supported by distinct morphological characters (e.g. floral structures and pollen grains).     

Phylogeny of kangaroo apples (<Emphasis Type="Italic">Solanum</Emphasis> subg. <Emphasis Type="Italic">Archaesolanum</Emphasis>, Solanaceae)

Kangaroo apples, subgenus Archaesolanum, are a unique and still poorly known group within the genus Solanum. Here we aimed to reveal phylogeny, historical biogeography and age of diversification of Archaesolanum. We sampled all recognized species of the group and sequenced three chloroplast regions, the trnT-trnL spacer, trnL intron and trnL-trnF spacer to calibrate a molecular clock to estimate the age of the group. Distributional data were combined with the results of phylogenetic analysis to track the historical processes responsible for the current range of the group. Our analysis supported the monophyly of the kangaroo apples and the biogeographical disjunction between the two subclades within the group. Based on the divergence time estimates the most recent common ancestor of kangaroo apples is from the late Miocene age (~9 MYA). Based on the age estimate the common ancestors of the kangaroo apples are presumed to have arrived in Australia by long-distance dispersal. The two distinct lineages within the group have separated during the aridification of the continent and further speciated in the brief resurgence of rainforests during the Pliocene.     

'Missing link' species Capsella orientalis and Capsella thracica elucidate evolution of model plant genus Capsella (Brassicaceae)

To elucidate the evolutionary history of the genus Capsella, we included the hitherto poorly known species C. orientalis and C. thracica into our studies together with C. grandiflora, C. rubella and C. bursa‐pastoris. We sequenced the ITS and four loci of noncoding cpDNA regions (trnL – F, rps16, trnH –psbA and trnQ –rps16). Sequence data were evaluated with parsimony and Bayesian analyses. Divergence time estimates were carried out with the software package BEAST. We also performed isozyme, cytological, morphological and biogeographic studies. Capsella orientalis (self‐compatible, SC; 2n = 16) forms a clade (eastern lineage) with C. bursa‐pastoris (SC; 2n = 32), which is a sister clade (western lineage) to C. grandiflora (self‐incompatible, SI; 2n = 16) and C. rubella (SC; 2n = 16). Capsella bursa‐pastoris is an autopolyploid species of multiple origin, whereas the Bulgarian endemic C. thracica (SC; 2n = 32) is allopolyploid and emerged from interspecific hybridization between C. bursa‐pastoris and C. grandiflora. The common ancestor of the two lineages was diploid and SI, and its distribution ranged from eastern Europe to central Asia, predominantly confined to steppe‐like habitats. Biogeographic dynamics during the Pleistocene caused geographic and genetic subdivisions within the common ancestor giving rise to the two extant lineages.     

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

Pollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.     

Phylogenetics of Quiinaceae (Malpighiales): evidence from trnL-trnF sequence data and morphology

We present the first parsimony analyses of the Neotropical family Quiinaceae using nucleotide sequence data from the non-coding trnL intron and trnL-trnF intergenic spacer of the plastid genome, analysed separately as well as in combination with morphology. Both molecules and combined data recover Quiinaceae as a well-supported monophyletic group. Quiinaceae form a polytomy together with their potential sister groups, the monophyletic Ochnaceae s.str. and the monotypic Medusagynaceae from the Seychelles in the Indian Ocean. Froesia is resolved as sister to the rest of the family. Other members of the family, Lacunaria, Quiina, and Touroulia, are all recovered as monophyletic despite the inclusion of strikingly distinctive representatives (L. oppositifolia and Q. pteridophylla). Relationships among the last three genera, however, are yet uncertain. Optimising characters of breeding system onto the molecular phylogeny reveals that bisexual flowers (Froesia) are the ancestral state in Quiinaceae, whereas androdioecy (Quiina, Touroulia) and dioecy (Lacunaria) are derived breeding systems.     

DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep

Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle‐class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL‐F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL‐F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree‐based methods for nrITS in 67% of the tested tubers, 58% for trnL‐F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross‐border trade of Salep tubers.