Chloroplast rbcL Gene Research Articles

Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations.

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML.

A taxonomic revision of cleistocarpous species of Weissia (Pottiaceae, Bryophyta) in Japan

Four species, including one newly described, of Japanese cleistocarpous species of Weissia (Pottiaceae): W. exserta, W. japonica, W. kiiensis and W. parajaponica sp. nov. are recognized based on molecular phylogenetic inference and morphological reassessment. For each species, typification, description, distribution, illustrations and photographs are presented, and a key to the species is included. Rapid sporophyte modifications in Weissia and monophyletic positions of these four species are supported by the analysis using concatenated chloroplast rbcL and rps4 gene sequences. A lectotype is designated for W. controversa which is the type species of the genus, and a new combination, W. loncophylla is proposed for Trachycarpidium lonchophyllum.

Laurencia majuscula var. elegans (Rhodophyta, Rhodomelaceae) is reinstated to specific rank as L. elegans

SUMMARYLaurencia majuscula has recently been determined to be conspecific with Laurencia dendroidea. To determine the correct taxonomic placement of its only non‐typical variety, L. majuscula var. elegans, mitochondrial COI‐5P and chloroplast rbcL gene sequences were analyzed and its phylogenetic position was inferred. Laurencia majuscula var. elegans consistently formed a well‐supported clade distinctly separate from the L. dendroidea clade. Molecular analyses revealed that L. majuscula var. elegans generally shows two distinct forms; a long‐branched form, and a compact form. Often the compact form has been identified as a different species based on morphology alone. Included in these analyses were topotype sequences of two species with which L. majuscula var. elegans is most often misidentified; Laurencia nidifica and Laurencia mcdermidiae. Laurencia nidifica formed a well‐supported clade separate from L. majuscula var. elegans. Laurencia mcdermidiae, however, formed a well‐defined clade sister to the L. majuscula var. elegans clade. Morphological comparisons were also made. It is concluded that L. majuscula var. elegans is morphologically distinct from L. dendroidea, L. nidifica, and L. mcdermidiae. It is proposed that L. majuscula var. elegans be reinstated to species level as L. elegans.

Contribution to Globularia phylogeny based on nuclear ribosomal spacer and two chloroplast DNA regions

Background and Purpose: Molecular approach has a major impact on phylogenetic studies of plants, considering that it gives useful information about evolutionary events and relations on all taxonomic levels. The sequence data of the nuclear ITS and of two chloroplast regions, trnL-trnF spacer and rbcL gene, obtained from thirteen Globularia L. taxa, including five Anatolian endemics, representing six sections altogether, were analyzed in order to determine the relations between the European and the Anatolian species and get a better insight into the phylogeny of several closely related Globularia taxa. Materials and Methods: Total cellular DNA was extracted from fresh or frozen leaf tissue of thirteen Globularia samples. The ITS regions of nuclear DNA and two chloroplast DNA regions were amplified and sequenced. Obtained nuclear and combined plastid data matrices were subjected to Maximum Parsimony analyses. Results and Conclusions: Molecular data that were obtained in this study indicate the existence of separate centers of diversification for the European and the Anatolian Globularia. The results provide support for relationships among the studied Anatolian endemic species and indications for a redefinition of affinities of some of the European species. The results presented herein are discussed along with available morphological, karyological, phytogeographical and molecular data. Keywords: Globularia, ITS, trnL-trnF, rbcL, Maximum Parsimony, Quaternary

Adaptive evolution of the rbcL gene in the genus Rheum (Polygonaceae)

ABSTRACTRapid putative radiations of Rheum might be caused by the recent uplifts of the Qinghai–Tibetan Plateau and the quaternary climate oscillations. To better understand the molecular adaptation associated with Rheum radiation, in this study, the adaptive evolution of the chloroplast rbcL gene was analysed using the Phylogenetic Analysis Program. The results showed that two amino acid residues (75F, 203I) were under positive selection. The spatial analysis indicated that the site (75F) was located in the β-sheet of the N-terminal loops involved in subunit interactions in the L8S8 molecule, and the site (203I) was in the α/β-barrel active centre located on the C-terminal domain of the large subunit of Rubisco. These results suggest that potential positive selection in rbcL might have played an important role in the adaption of Rheum species to the extreme environments in Qinghai–Tibetan Plateau regions, and different species lineages might have been subjected to different selective pressures.

Evolutionary divergence in Chenopodium and validation of SNPs in chloroplast rbcL and matk genes by allele-specific PCR for development of Chenopodium quinoa-specific markers

The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbcL and matK gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825, which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2Mya. Whereas multiple alignments of rbcL gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68% interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the matK gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbcL and matK regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR, designed from rbcL sequences, that could differentiate quinoa from 64 genera including 13 species of the genus Chenopodium. With an estimated age of 10.5–4.1million years (Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.

Cladosiphon takenoensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

SUMMARYThe new brown algal species Cladosiphon takenoensis H. Kawai (Chordariaceae, Ectocarpales s.l.) is described from Takeno, Hyogo, Japan based on morphology and DNA sequences. The species is a spring annual, growing on subtidal rocks at more or less exposed sites. It resembles C. umezakii in its gross morphology, and the two often grow together, but is distinguishable from C. umezakii in having a more hairy appearance. Cladosiphon takenoensis has a slimy, cylindrical, multiaxial and sympodial erect thallus, branching once to twice, and is provided with long assimilatory filaments (up to 1.8 mm long, composed of up to 100 cells). Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most related to C. umezakii and has the same basic thallus structures, but differs from C. umezakii and other Cladosiphon species in lacking phaeophycean hairs and plurilocular zoidangia of the assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and the nuclear rDNA ITS2 region support the distinctness of the species. The genus Cladosiphon was paraphyletic in our analyses because the clades of C. okamuranus/C. zosterae and C. takenoensis/C. umezakii were split by Mesogloia vermiculata. However, since the genus‐level taxonomy of Chordariaceae needs considerable revision, we suspend the genus‐level taxonomy of the new species, and tentatively describe it as C. takenoensis.

Phylogeny of the Rosidae: A dense taxon sampling analysis

AbstractRosidae, a clade of approximately 90 000 species of angiosperms, exhibits remarkable morphological diversity and extraordinary heterogeneity in habitats and life forms. Resolving phylogenetic relationships within Rosidae has been difficult, in large part due to nested radiations and the enormous size of the clade. Current estimates of phylogeny contain areas of poor resolution and/or support, and there have been few attempts to synthesize the available data into a comprehensive view of Rosidae phylogeny. We aim to improve understanding of the phylogeny of Rosidae with a dense sampling scheme using both newly generated sequences and data from GenBank of the chloroplast rbcL, atpB, and matK genes and the mitochondrial matR gene. We combined sequences from 9300 species, representing 2775 genera, 138 families, and 17 orders into a supermatrix. Although 59.26% of the cells in the supermatrix have no data, our results generally agree with previous estimates of Rosidae phylogeny and provide greater resolution and support in several areas of the topology. Several noteworthy phylogenetic relationships are recovered, including some novel relationships. Two families (Euphorbiaceae and Salvadoraceae) and 467 genera are recovered as non‐monophyletic in our sampling, suggesting the need for future systematic studies of these groups. Our study shows the value of a botanically informed bioinformatics approach and dense taxonomic sampling for resolving rosid relationships. The resulting tree provides a starting point for large‐scale analyses of the evolutionary patterns within Rosidae.

Phylogenetic relationships of New Zealand Lycopodiaceae

We examine the global relationships of all New Zealand’s Lycopodiaceae species through DNA sequencing of the chloroplast rbcL gene and phylogeny reconstruction. The molecular phylogeny largely agreed with recent taxonomic schemes based on morphology. However, the grouping of Lycopodiella serpentina with Lycopodiella caroliniana, as either Lycopodiella sect. Caroliniana or Pseudolycopodiella was not supported in the phylogeny, indicating that these classification schemes need adjustment. Several New Zealand species showed intraspecific genetic variation within New Zealand and/or compared to conspecific samples from overseas. The chloroplast sequences could not distinguish Lycopodiella diffusa and Lycopodiella lateralis, nor the different morphologies of Phlegmariurus varius characteristic of high and low altitudes.

Foeniculum sanguineum Triano y A. Pujadas (Apiaceae) nueva especie del suroeste de la Región Mediterránea

Se describe Foeniculum sanguineum Triano & A. Pujadas, sp. nov., del suroeste de la Región Mediterránea (España y Marruecos). Para su caracterización se ha realizado el análisis morfológico, citológico, fitoquímico y molecular. Se ha llevado a cabo el studio comparativo con Foeniculum vulgare Mill. La nueva especie F. sanguineum se distingue principalmente por sus pétalos rojos, polen rosado en fresco y por su estilopodio rojo. Es una especie diploide (2n= 22). Se ha encontrado una alta proporción de óxido de limoneno y piperitenona (ausente en F. vulgare) en la composición de aceite esencial de los frutos secos de F. sanguineum y una elevada cantidad (aproximadamente 50%) de α-felandreno en sus raíces y tallos. El análisis filogenético se realizó utilizando las secuencias del espaciador transcrito interno de ADN ribosomal nuclear (ITS) y las secuencias del gen cloroplástico rbcL. El análisis ITS apoya la existencia de la nueva especie, al tiempo que revela la divergencia de secuencias tanto a nivel intraespecífico como a nivel interespecífico. La divergencia de secuencia encontrada en el gen cloroplástico, aunque reducida a un nucleótido, proporcionó apoyo adicional para la caracterización de la nueva especie, para la que se propone el nombre de Foeniculum sanguineum.

Morphological and genetic variability of assemblages of Cyclotella ocellata Pantocsek/C. comensis Grunow complex (Bacillariophyta, Thalassiosirales)

Centric diatom taxa in the Cyclotella ocellata and C. comensis complexes show high morphological variability and often apparently continuous morphological transitions. In this study, we investigated natural assemblages of the C. ocellata/C. comensis complex from Hungarian and Croatian lakes and from Turkish streams using morphological and molecular methods. The studied assemblages contained cells with morphologies resembling C. ocellata as well as other, closely related, species: C. comensis, C. pseudocomensis, C. costei, and C. trichnoidea. The goal of our paper was to assess whether the observed morphological differences were due to intraspecific variability or suggest the existence of several, putatively distinct species.Ten morphometric characters were measured, which, either individually, or in pairs, did not differentiate the nominal taxa in our assemblages. However, multivariate discriminant analysis has revealed a group including C. ocellata and C. trichonidea morphologies could be separated from another containing C. comensis, C. pseudocomensis and C. costei.A nuclear (18S rDNA) and a chloroplast (rbcL) gene were amplified and partially sequenced from environmental DNA or from isolated cells. The sequences showed little variability among the assemblages and nominal species. Although general congruence of molecular and morphometric separation supports the species level separation of C. ocellata/trichonidea from the probably conspecific C. comensis/pseudocomensis/costei, sequence divergences between the groups are in the same range as within them, so that a conspecificity of all four taxa cannot be unequivocally excluded.

Identification and Individualization of Lophophora using DNA Analysis of the trnL/trnF Region and rbcL Gene.

Lophophora williamsii (peyote) is a small, spineless, greenish-blue cactus found in Mexico and the southwestern United States. Ingestion of the cactus can result in hallucinations due to its content of mescaline. In the United States, L.williamsii is classified as a Schedule I controlled substance. In this study, we use DNA analysis of the chloroplast trnL/trnF region and chloroplast rbcL gene to identify the individuals of Lophophora. Using the rbcL gene, Lophophora specimens could be distinguished from outgroups, but species within the genus could not be distinguished. The trnL/trnF region split the Lophophora genus into several groups based on the length and substructure of an AT-rich segment of the sequence. Our results indicate that the genetic variability at the trnL/trnF locus is greater than previously recognized. Although DNA structures at the trnL/trnF region and rbcL gene do not align with the classification of Lophophora species, they can be used to aid in forensic analysis.

Phylogenetic analysis of the genus Pseudoroegneria and the Triticeae tribe using the rbcL gene

The Pseudoroegneria species are perennial grasses in the Triticeae tribe, whose St genome has been linked to several important polyploid species. Due to frequent hybridization and complex genetic mechanism, the relationships within Pseudoroegneria, and within the Triticeae have been heavily disputed. Using the chloroplast rbcL gene we estimated the nucleotide diversity of 8 Pseudoroegneria species. We also examined the phylogenetic relationships within Pseudoroegneria and of Pseudoroegneria within the Triticeae. The estimates of nucleotide diversity indicated that Pseudoroegneria tauri and Pseudoroegneria spicata species had the highest diversity, while Pseudoroegneria gracillima had the lowest diversity. The phylogenetic analysis of Pseudoroegneria placed all P. spicata species into a clade separate from the other Pseudoroegneria species, while the relationship of the other Pseudoroegneria species could not be determined. Due to the groupings of Pseudoroegneria with the polyploid Elymus, our results strongly supported Pseudoroegneria as the maternal genome donor to Elymus. There was also weak support that P. spicata may be the maternal donor to the StH Elymus species.

Morphology and molecular phylogeny of Chaetoceros dayaensis sp. nov. (Bacillariophyceae), characterized by two 90° rotations of the resting spore during maturation.

Recent studies suggest that a high species diversity of Chaetoceros exists in tropical waters. Based on plankton samples from Chinese tropical waters, Daya Bay of the South China Sea, a new species, Chaetoceros dayaensis sp. nov., is described. Vegetative cells and resting spores were examined by light microscopy as well as transmission and scanning electron microscopy. The nuclear rDNA molecular markers SSU, ITS and D1-D3 of LSU, and the chloroplast rbcL gene were sequenced for information on phylogenetic relationships. The species is characterized by the presence of two 90° rotations of the resting spore during maturation. First a rotation in the apical plane of the mother cell resulting in the valvar plane of the resting spore turning from parallel to perpendicular to that of the mother cell, and a second 90° rotation in the valvar plane of the mother cell resulting in the valve faces of the resting spore turning from facing narrow girdle view to broad girdle view of the mother cell. It is the first report of two 90° rotations of resting spores during maturation in Chaetoceros. Based on this, the maturation rotation of the resting spore of C.rotosporus was reevaluated. C.dayaensis belongs to the subgenus Hyalochaete Gran and we suggest placing it in the section Laciniosa, although a new subdivision of Chaetoceros is needed. The description of C.dayaensis was based on analyses of the phylogenetic relationships combined with morphological comparisons with other similar species.

Sequencing of historic and modern specimens reveals cryptic diversity in Nothogenia (Scinaiaceae, Rhodophyta)

:Nothogenia fastigiata has been reported to exhibit great morphological variability and has been considered to be widely distributed in the Southern Hemisphere. To test its current circumscription, sequences from type material of N. fastigiata and other species currently synonymized with it were compared to those from recent collections of this and other species in the genus. Eight distinct species previously subsumed under the name N. fastigiata were identified. Multiple specimens from southern Chile and a single specimen from Campbell Island (subantarctic New Zealand) were conspecific with type material of N. fastigiata from the Falkland Islands. For other species, molecular analyses of recent collections using the nuclear ITS1-5.8S-ITS2 region of the ribosomal cistron, the chloroplast rbcL and psbA genes and the mitochondrial COI gene indicated a strong geographic pattern to species relationships. Other specimens identified as N. fastigiata from Chile represented up to five species, including N. chilensis and N. fragilis, based on sequences of type material; these Chilean species occurred on a monophyletic branch. We also recognized N. lingula comb. nov. from Tasmania, which is closely related to N. fastigiata, based on sequences of type material. Specimens from mainland New Zealand identified as N. fastigiata fell into a distinct clade with New Zealand N. pulvinata and represented a previously undescribed species, described here as N. neilliae sp. nov. Another New Zealand species, N. pseudosaccata, was distantly related to N. variolosa from Auckland Island and other subantarctic islands south of New Zealand. The New Zealand species were more closely related to South African N. erinacea and N. ovalis than to species of Nothogenia from Chile, including N. fastigiata, although bootstrap support for this interpretation was weak. These genetic data demonstrate that matching DNA sequences from archival Nothogenia material to modern specimens can be used to identify and define new and old cryptic species.

On the systematic position of the genus Timmiella (Dicranidae, Bryopsida) and its allied genera, with the description of a new family Timmiellaceae

Based on our molecular phylogenetic analysis of haplolepideous mosses with concatenated sequences of chloroplast rps4 and rbcL genes, a new family Timmiellaceae is erected to accommodate the genera Timmiella and Luisierella, both of which have been formerly included in the family Pottiaceae. The family Timmiellaceae is resolved as a second-branching clade together with Distichium (Distichiaceae) within the Dicranidae (haplolepideous moss) lineages and phylogenetically distinct from the Pottiaceae. Reassessment of morphological characters suggests that a combination of the characters: 1) adaxially bulging and abaxially flat leaf surfaces, 2) sinistrorse or straight peristomes, when present, and 3) sinistrorsely arranged operculum cells is unique to Timmiellaceae and discriminates it from other haplolepideous moss families.

Biogeography and Phylogenetic Position of a Warm-stenotherm Centric Diatom, Skeletonema potamos (C.I. Weber) Hasle and its Long-term Dynamics in the River Danube

Skeletonema potamos is a poorly known freshwater species in the ancestrally and predominantly marine genus Skeletonema. With phylogenetic analysis of two nuclear {partial SSU (18S) and partial LSU (28S) rDNA)} and two chloroplast (rbcL and psbC) genes, we verified its placement within the genus Skeletonema and identified the mostly brackish species, Skeletonema subsalsum, as its closest known relative. Comparisons of SSU and LSU rRNA genes from S. potamos populations from Europe and North America revealed no intraspecific variation. Skeletonema potamos can be a dominant element of the phytoplankton community in various ecosystems, including the River Danube. We tracked phytoplankton composition in the River Danube weekly from 1979 to 2 012, and throughout this period, S. potamos exhibited a strong increase in proportion of total phytoplankton abundance and biomass - an increase that was correlated with increasing water temperature over the same time period. Current records indicate a temperate distribution of S. potamos, but ecological data predict possible expansion of its geographic range and increase in seasonal duration within existing habitats in response to the warming of surface waters.

The evolution of rbcL: A methodology to follow the evolution patterns of Medicago and Sulla (Fabaceae) genera

The chloroplast rbcL gene is selected to be a standard barcode for the phylogenetic analyses of several land plants. Using universal primers pair, the 3’ frame was used to assess the phylogeny of the two Legume genera Medicago and Sulla and the evolution of the translated protein. The multiple alignment exhibited high conservation rates (97.56% and 96.43% for Medicago and Sulla respectively), which might be a brake for the phylogenetic assessment. Nevertheless, the topologies of the cladograms drawn using Maximum Likelihood method, showed a low intraspecific divergence and a slightly high interspecific variation, which confirms the efficiency of rbcL for such characterization’ analyses. Besides, the composition and the patterns of the Amino-Acid sequence and the deduced Codon Usage, highlighted a non-aleatoire distribution of the codons. Thus, the non-synonymous substitutions rates traduced a positive selection occurring in the rbcL of the Medicago and Sulla species.

Annonaceae substitution rates: a codon model perspective

The Annonaceae includes cultivated species of economic interest and represents an important source of information for better understanding the evolution of tropical rainforests. In phylogenetic analyses of DNA sequence data that are used to address evolutionary questions, it is imperative to use appropriate statistical models. Annonaceae are cases in point: Two sister clades, the subfamilies Annonoideae and Malmeoideae, contain the majority of Annonaceae species diversity. The Annonoideae generally show a greater degree of sequence divergence compared to the Malmeoideae, resulting in stark differences in branch lengths in phylogenetic trees. Uncertainty in how to interpret and analyse these differences has led to inconsistent results when estimating the ages of clades in Annonaceae using molecular dating techniques. We ask whether these differences may be attributed to inappropriate modelling assumptions in the phylogenetic analyses. Specifically, we test for (clade-specific) differences in rates of non-synonymous and synonymous substitutions. A high ratio of nonsynonymous to synonymous substitutions may lead to similarity of DNA sequences due to convergence instead of common ancestry, and as a result confound phylogenetic analyses. We use a dataset of three chloroplast genes (rbcL, matK, ndhF) for 129 species representative of the family. We find that differences in branch lengths between major clades are not attributable to different rates of non-synonymous and synonymous substitutions. The differences in evolutionary rate between the major clades of Annonaceae pose a challenge for current molecular dating techniques that should be seen as a warning for the interpretation of such results in other organisms.

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes.

We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yellow etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex-traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using proteinase K, and precipitation of polysaccharides in the presence of a high salt concentration. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calculated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproducible, and suitable for PCR-based markers.