Non-coding Chloroplast DNA Research Articles

Phylogeny and biogeography of Chinese Heracleum (Apiaceae tribe Tordylieae) with comments on their fruit morphology

The Sino-Himalayan Mountain Region is a major center of diversity of Heracleum (Apiaceae, Tordylieae, Tordyliinae), yet the phylogenetic relationships among its rich diversity of endemic taxa are largely unknown. In this study, we (1) investigate the monophyly of Heracleum and ascertain the phylogenetic placements of those species of Heracleum from China that fall outside of the group; (2) estimate phylogenetic relationships within Heracleumsensu stricto, particularly among those species occurring in the Hengduan Mountain Region of southwestern China; and (3) reconstruct the biogeographic history of Heracleum sensu stricto. To achieve these objectives, Bayesian inference and maximum-parsimony analyses of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region and five noncoding chloroplast DNA (cpDNA) loci (rps16-trnK, rps16 intron, trnQ-rps16, rpl32-trnL, and psbA-trnH), separately and in combination, were carried out. Fruit morphology of the Chinese Heracleum species was examined. Statistical dispersal-vicariance analysis (S-DIVA) was used to reconstruct the biogeographic history. In total, 114 accessions were considered, which included 88 species representing eight genera of subtribe Tordyliinae, 28 of the 29 species of Heracleum occurring in China, and 4 species of Angelica as outgroups. The genus Heracleum is monophyletic upon the exclusion of nine species of Heracleum from China and the inclusion of the Caucasian monotypic genera Mandenovia and Symphyoloma. These nine excluded species show affinities to Tetrataenium, Semenovia, and Angelica. The species of Heracleum endemic to southwestern China form a moderately to strongly supported clade; however, major discordance between the ITS- and cpDNA-derived phylogenies, lack of resolution in the trees, and polyphyly of several species preclude unequivocal hypotheses of phylogenetic relationship. The distinctly clavate vittae, shorter than the length of the mericarp, is a diagnostic feature for Heracleumsensu stricto. Heracleum originated in the West Palearctic, with three possible migration routes leading to its present-day distribution. Major topological conflicts between the ITS and cpDNA trees may be due to interspecific hybridization and/or incomplete lineage sorting. The taxonomic limits of many Chinese Heracleum species remain unclear, possibly because of recent radiation in the Hengduan Mountains.

Phylogenetic relationships of Brassicaceae in China: Insights from a non-coding chloroplast, mitochondrial, and nuclear DNA data set

Abstract Understanding of the systematics and evolution of the Brassicaceae has advanced greatly in recent years. In particular, molecular techniques offer a way to address issues of homoplasy that had limited morphology-based research. In this study, we used sequence data from chloroplast, mitochondrial, and nuclear genomes to construct the phylogeny of Brassicaceae. We adopted maximum parsimony, maximum likelihood, and Bayesian methods to illustrate the relationships of 71 species belonging to 51 genera. Results from trnS (GCU) -trnG (UUC) , nad 7 second intron, and pistillata ( PI ) first intron analyses supported the delimitation of Lineages I–III and agreed with previous findings from phyA , ITS, and ndhF analyses. The results indicated that Pachypterygium should be placed in the tribe Isatideae. The position of Leiospora agreed with speculation based on morphology. Furthermore, Conringia planisiliqua together with Orychophragmus violaceus can be recognized as a new tribe as proposed by previous studies. Finally, this study also supported the recently recognized new tribes Dontostemoneae and Erysimeae.

Discordant patterns of morphological and genetic divergence in the closely related species Schizanthus hookeri and S. grahamii (Solanaceae)

In this study I have examined the patterns of morphological and genetic differentiation between two species of the Andean genus Schizanthus that differ in their pollination and mating systems. Schizanthus hookeri has a bee pollination syndrome and is strongly dependent on pollinators for seed set. In contrast, S. grahamii has a hummingbird pollination syndrome and exhibits late autonomous selfing. Southern populations of the latter species have red flowers (reddish morph), while northern populations have yellow (yellowish morph) or pink flowers (pinkish morph). I used two noncoding chloroplast DNA (cpDNA) regions to investigate the genetic affinities between S. hookeri and the three morphs of S. grahamii. I also performed intra- and interspecific crosses to assess whether gene flow between species was possible. Phylogenetic analyses supported the existence of two differentiated clades that did not match currently accepted taxonomic classification. Accordingly, genetic distance did not correlate significantly with morphological distance. No fruits were produced from interspecific crosses, and there were no individuals with intermediate morphology that could indicate current and frequent hybridization events between species. I propose that the discordance between cpDNA data and conventional taxonomy could be explained by parallel evolution, or alternatively, by a very sporadic hybridization.

Genetic differentiation in the genus Lithops L. (Ruschioideae, Aizoaceae) reveals a high level of convergent evolution and reflects geographic distribution

Southern Africa is one of the hot spots for plant biodiversity, with ca. 80% of species endemic to this area. Rapid and recent radiations in Southern African plant genera were triggered by fine-scale differences in climate, topography and geology. The genus Lithops (Ruschioideae, Aizoaceae) contains 37 species and is widely distributed in Southern Africa. Species delimitation within the genus is challenging because the limited number of morphological characters in these reduced succulents varies intensely between populations, presumably as adaptations to local geological environments. We analysed phylogenetic relationships within Lithops using non-coding chloroplast DNA (trnS-trnG intergenic spacer), nuclear ribosomal internal transcribed spacer (nrITS) sequences and AFLP data. Genetic variability of the sequence data was very low, but AFLP data detected nine clades within Lithops that do not fit current morphology-based taxonomy. Two of these clades are separated by their distribution on the northern and eastern border of the distribution area, and four clades are found in the Gariep Centre in the estuary of the Orange River. Morphological similarities, especially colour of leaves, evolved repeatedly within the clades, thus we hypothesise that closely related species became adapted to different soil types in a mosaic-like geological environment. One-third of the species are found in the Gariep Centre, characterised by extremely diverse edaphic habitats.

Phylogenetic network of Linum species as revealed by non-coding chloroplast DNA sequences

Crop wild relatives are invaluable sources of novel genes for crop improvement and adaptation to changing environments. We assessed phylogenetic relationships among 29 Linum accessions representing 16 species, including cultivated flax and its progenitor pale flax, based on four non-coding regions of chloroplast DNA sequences. We obtained a cpDNA network showing that these 16 Linum species are appropriately connected based on previously defined taxonomic sections; these connections reflect the same evolutionary pathways as determined from earlier morphological and cytological data. These relationships also support an earlier hypothesis that cultivated flax is probably descended from a single domestication of pale flax plants, apparently for oil usage. The detailed species network not only is significant for understanding evolutionary relationships of Linum species, but also useful for classifying exotic gene pools of cultivated flax as a part of the ongoing exploration of new genetic diversity for flax improvement.

Erratum to: Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Erratum to: Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Gene pools in wild Lima bean (Phaseolus lunatus L.) from the Americas: Evidences for an Andean origin and past migrations

The aims of this research were to assess the genetic structure of wild Phaseolus lunatus L. in the Americas and the hypothesis of a relatively recent Andean origin of the species. For this purpose, nuclear and non-coding chloroplast DNA markers were analyzed in a collection of 59 wild Lima bean accessions and six allied species. Twenty-three chloroplast and 28 nuclear DNA haplotypes were identified and shown to be geographically structured. Three highly divergent wild Lima bean gene pools, AI, MI, and MII, with mostly non-overlapping geographic ranges, are proposed. The results support an Andean origin of wild Lima beans during Pleistocene times and an early divergence of the three gene pools at an age that is posterior to completion of the Isthmus of Panama and major Andean orogeny. Gene pools would have evolved and reached their current geographic distribution mainly in isolation and therefore are of high priority for conservation and breeding programs.

Phylogenetics of Eurasian plums, Prunus L. section Prunus (Rosaceae), according to coding and non-coding chloroplast DNA sequences

The genus Prunus contains the subgenus Prunus incorporating the European plums (section Prunus), the North American plums (section Prunocerasus) and the apricots (section Armeniaca). In section Prunus, there are approximately 20 species, which occur in three levels of ploidy, diploid \( \left( {2n = 2x = 16} \right) \), tetraploid \( \left( {2n = 4x = 32} \right) \) and hexaploid \( \left( {2n = 6x = 48} \right) \). Despite a clear distinction between section Prunus and the other sections, phylogenetic relationships between species within the section are unclear. We performed a phylogenetic analysis on members of the section Prunus and three outgroup species using sequence data from four single-copy phylogenetically informative chloroplast DNA regions (atpB-rbcL, matK, rpl16, and trnL-trnF). After alignment, the analysed regions totalled 4,696 bp of sequence, containing 68 parsimony-informative sites and 14 parsimony-informative indels. Data were analysed using both maximum parsimony and Bayesian likelihood and phylogenetic trees were reconstructed. The analyses recovered trees with congruent topologies and similar levels of statistical support for relationships between taxa. They confirmed that species belonging to section Prunus form a monophyletic clade within Prunus. The section is resolved into four well-supported clades, which correspond to the geographical distribution of the species. The hexaploid species could not be resolved into distinct species clades but formed a well-supported group separate from the tetraploid species, highlighting the distinct evolutionary origins of the different polyploid groups. The close relationship between the hexaploids and Prunus divaricata, Prunus cerasifera and Prunus ursina indicates the former may have derived from an ancestor of P. cerasifera and its allies.

Biogeographic history of the threatened species Araucaria araucana (Molina) K. Koch and implications for conservation: a case study with organelle DNA markers

Fragmentation of the habitat due to glaciations, fires and human activities affected the distribution range of Araucaria araucana in southern South America. On the borders of the Argentinean Patagonian steppe, the species is restricted to isolated patches without natural regeneration. Our objective is to test the hypothesis that these populations are relicts of pre-Pleistocene origin. A total of 224 individuals from 16 populations were sampled. Twenty chloroplast microsatellites, 19 non-coding chloroplast DNA regions and eight mitochondrial DNA fragments were screened for polymorphisms. A low transferability rate of universal primers from Pinaceae and also a low variation were detected for this ancient species. Only one non-coding region of the chloroplast DNA showed polymorphism allowing the identification of five haplotypes. A low genetic differentiation (G ST = 0.11; G′ ST = 0.267) and lack of geographic structure was found. Allelic richness was lower and genetic differentiation higher among the eastern isolated populations, suggesting a long lasting persistence. Conservation guidelines are given for these relictual populations, which are located outside the limits of the National Parks.

Molecular phylogeny of Ceropegia (Asclepiadoideae, Apocynaceae) from Indian Western Ghats

Ceropegia includes more than 200 species distributed in the Old World ranging from the Canary Islands to Australia. In India, there are about 50 species described on a morphological basis as belonging to Ceropegia, and most of them are endemic to the Western Ghats. To investigate evolutionary relationships among Indian Ceropegia taxa and their allies, a phylogenetic analysis was conducted to include 31 Indian taxa of Ceropegia and Brachystelma and their congeners from other geographical regions using nuclear ribosomal internal transcribed spacer (ITS) and three noncoding chloroplast DNA (cpDNA) sequences, including intergenic spacers trnT-L and trnL-F, and trnL intron. The Western Ghats Ceropegia species were found to be most closely related to Indian Brachystelma, with the two genera being placed sister to each other in the ITS phylogeny or with the Brachystelma clade nested within one of the two subclades of Indian Ceropegia in the cpDNA phylogeny. In contrast, Ceropegia species from other regions and African Brachystelma all formed separate clades basal to the Indian Ceropegia-Brachystelma clade. Thus, it can be concluded that the classical morphology-based delineation of the two genera needs revision to reflect their phylogenetic relationships, which are more in accordance with their geographical origin than with morphology.

Chloroplast simple sequence repeat markers for evolutionary studies in the sexually deceptive orchid genus Chiloglottis

The orchids in the genus Chiloglottis are pollinated exclusively by sexual deception. Extensive sequencing (>19.5kb) of noncoding chloroplast DNA revealed that simple sequence repeats (cpSSRs) were abundant, enabling a set of 41 cpSSR markers to be developed. All markers were polymorphic across the genus. Polymorphism reflected variation at both mononucleotide repeats and indels. For a subset of four taxa with 40 samples each, locus polymorphism varied from 46 to 81%, while the number of haplotypes ranged from seven to 21 per taxon. Extensive differentiation among the taxa was detected. These cpSSRs markers will enable novel insights into the evolution of this unique genus.

A new set of universal de novo sequencing primers for extensive coverage of noncoding chloroplast DNA: new opportunities for phylogenetic studies and cpSSR discovery

We present a new set of universal de novo sequencing primers targeting noncoding chloroplast DNA. The set of 107 polymerase chain reaction (PCR) primers span approximately 86% of the noncoding nucleotides in the large single copy region of Nicotiana tabacum, Oryza sativa and the orchid Phalaenopsis aphrodite. PCR tests confirmed the primers are effective in a wide range of monocots and dicots. More than 19.5kb of cpDNA sequence was obtained across representative orchid genera with up to 82 chloroplast simple sequence repeats (cpSSRs) detected per genus. This primer set will facilitate both phylogenetic studies and rapid discovery of cpSSRs for plants, such as orchids, where there are limited genomic resources.

Phylogenetic relationships of the Aurantioideae (Rutaceae) based on the nuclear ribosomal DNA ITS region and three noncoding chloroplast DNA regions, atpB-rbcL spacer, rps16, and trnL-trnF

The tribes and subtribes of Aurantioideae, an economically important subfamily of the Rutaceae, have a controversial taxonomic history because a phylogenetic framework has been lacking. In order to construct an evolutionary history and evaluate the most recent classification system [Swingle and Reece 1967. The botany of Citrus and its wild relatives, in: The Citrus Industry, vol. 1, History, World Distribution, Botany, and Varieties. University of California, Berkeley, pp. 190–430], one nuclear and three noncoding chloroplast genes were sequenced and analyzed phylogenetically along with selected non-molecular characters. Taxa representing tribes Citreae and Clauseneae and their six subtribes were sampled. In all analyses Aurantioideae is monophyletic. The majority-rule consensus tree from the combined analysis indicates that the two tribes are not monophyletic. The combined topology is not congruent with the widely used classification of Aurantioideae by Swingle and Reece (1967). The tribes and subtribes are in need of revision.

Estatus taxonómico de Oenocarpus bataua (Euterpeae, Arecaceae) inferido por secuencias del ADN cloroplástico

Las relaciones evolutivas entre la especie Oenocarpus bataua (anteriormente denominada Jessenia bataua) y otras especies del género Oenocarpus fueron estudiadas mediante un análisis filogenético de la tribu Euterpeae (Arecaceae) en base a 3,6 kb de secuencias de espaciadores intergénicos del cloroplasto. Los cladogramas recuperados sugieren: (1) la monofilia de la tribu Euterpeae, (2) la inclusión de los individuos de O. bataua dentro del clado de Oenocarpus, y (3) la divergencia molecular entre O. bataua var. bataua y var. oligocarpus, sugiriendo que estos taxa podrían representar especies distintas, así como fueron descritos originalmente. Sin embargo, los patrones filogenéticos mencionados tuvieron un bajo soporte estadístico, por lo cual las conclusiones derivadas de estos permanecen tentativas.

Diversification of Lupinus (Leguminosae) in the western New World: Derived evolution of perennial life history and colonization of montane habitats

Previous phylogenetic studies of Lupinus (Leguminosae) based on nuclear DNA have shown that the western New World taxa form a monophyletic group representing the majority of species in the genus, with evidence for high rates of recent diversification in South America following final uplift of the Andes 2–4 million years ago (Mya). For this study, three regions of rapidly evolving non-coding chloroplast DNA ( trnL intron, trnS– trnG, and trnT– trnL) were examined to estimate the timing and rates of diversification in the western New World, and to infer ancestral states for geographic range, life history, and maximum elevation. The western New World species (5.0–9.3 Mya, 0.6–1.1 spp./My) comprise a basally branching assemblage of annual plants endemic to the lower elevations of western North America, from which two species-rich clades are recently derived: (i) the western North American perennials from the Rocky Mountains, Great Basin, and Pacific Slope (0.7–2.1 Mya, 2.0–5.9 spp./My) and (ii) the predominantly perennial species from the Andes Mountains of South America and highlands of Mexico (0.8–3.4 Mya, 1.4–5.7 spp./My). Bayesian posterior predictive tests for association between life history and maximum elevation demonstrate that perennials are positively correlated with higher elevations. These results are consistent with a series of one or more recent radiations in the western New World, and indicate that rapid diversification of Lupinus coincides with the derived evolution of perennial life history, colonization of montane habitats, and range expansion from North America to South America.

Utility of Selected Non-coding Chloroplast DNA Sequences for Lineage Assessment of Musa Interspecific Hybrids

Single-copy chloroplast loci are used widely to infer phylogenetic relationship at different taxonomic levels among various groups of plants. To test the utility of chloroplast loci and to provide additional data applicable to hybrid evolution in Musa, we sequenced two introns, rpl16 and ndhA, and two intergenic spacers, psaA-ycf3 and petA-psbJ-psbL-psbF and combined these data. Using these four regions, Musa acuminata Colla (A)- and M. balbisiana Colla (B)-containing genomes were clearly distinguished. Some triploid interspecific hybrids contain A-type chloroplasts (the AAB/ABB) while others contain B-type chloroplasts (the BBA/BBB). The chloroplasts of all cultivars in 'Namwa' (BBA) group came from the same wild maternal origin, but the specific parents are still unrevealed. Though, average sequence divergences in each region were little (less than 2%), we propose that petA-psbJ intergenic spacer could be developed for diversity assessment within each genome. This segment contains three single nucleotide polymorphisms (SNPs) and two indels which could distinguish diversity within A genome whereas this same region also contains one SNP and an indel which could categorize B genome. However, an inverted repeat region which could form hairpin structure was detected in this spacer and thus was omitted from the analyses due to their incongruence to other regions. Until thoroughly identified in other members of Musaceae and Zingiberales clade, utility of this inverted repeat as phylogenetic marker in these taxa are cautioned.

Phylogenetic Relationships in Tribe Cariceae (Cyperaceae) Based on Nested Analyses of Four Molecular Data Sets

Phylogenetic reconstruction for Carex and relatives in tribe Cariceae is complicated by species richness and nearly cosmopolitan distribution. In this investigation, our main objective was to estimate evolutionary relationships in tribe Cariceae using DNA sequence data from two spacer regions in nuclear ribosomal genes (ITS and ETS-1f) combined with noncoding chloroplast DNA (trnL intron, trnL–trnF intergenic spacer, and trnE–trnD intergenic spacers). Parsimony analyses of separate and combined data and Bayesian analysis of the combined data matrix revealed strong support for monophyly of tribe Cariceae and for monophyly of two major lineages, one comprising principally Carex subgen. Carex and Vigneastra, and the other representing subgen. Vignea. A third clade with representatives from Kobresia and Uncinia, along with Cymophyllus fraserianus, Carex curvula, and several unispicate Carex received weak-to-moderate support. A small clade comprising Schoenoxiphium and two unispicate carices was placed as sister to the clades comprising multispicate Carex species in the parsimony analysis, but sister to the clade of Kobresia, Uncinia, and unispicate Carex in the Bayesian analysis. Two large widespread groups within subgen. Carex, sect. Hymenochlaenae and sect. Physocarpae s.l. (‘‘bladder sedges"), were highly polyphyletic, while ten clades that grouped species from two or more sections were each strongly supported as monophyletic. Within subgen. Vignea, three sections were strongly supported as monophyletic while sects. Phaestoglochin and Vulpinae were polyphyletic. Adding the variable ETS-1f region improved resolution and bootstrap support values over previous studies, but many of the characters supporting major branches came from the trnL region.

Systematics of Bromelioideae (Bromeliaceae)—Evidence from Molecular and Anatomical Studies

A reconstruction of the phylogeny of Bromeliaceae based on sequence data from three noncoding chloroplast DNA markers (trnL intron, trnT–trnL, and trnT–trnF intergenic spacer [IGS]) is presented, including 26 genera and 33 species. Relationships of Bromelioideae and phylogeny within this subfamily were analyzed in more detail on the basis of two of these markers (trnL intron and trnL–trnF IGS) using a set of 37 genera/74 species of Bromeliaceae, including 28 genera/60 species of Bromelioideae. Sister group relationships of Bromelioideae were not resolved with sufficient reliability, but the most likely candidates are the genera Fosterella and Puya. The basal phylogeny of Bromelioideae also was not resolved. Greigia, Ochagavia/Fascicularia/Fernseea, Deinacanthon, Bromelia, and a ‘‘core group’’ of the remaining Bromelioideae formed a basal polytomy. Within Bromelioideae, the AFLP technique was applied to assess relationships among selected groups of genera. In the Ochagavia/Fascicularia group (5 species and subspecies/16 accessions), AFLP data fully confirmed the systematic relationships based on morphological and anatomical characters. Investigation of 30 Aechmea species (33 accessions), including all subgenera and one species each from the related genera Ursulaea, Portea, Chevaliera, and Streptocalyx produced no resolution for several of the species. Clades that received good bootstrap support generally did not correspond with the delimitation of subgenera of Aechmea. Additionally, leaf blade anatomy of these species was investigated. The results corresponded partly with those of the AFLP analysis. Generic rank for Ursulaea and Portea was not supported.

Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana

The level of variation and the mutation rate were investigated in an empirical study of 244 chloroplast microsatellites in 15 accessions of Arabidopsis thaliana. In contrast to SNP variation, microsatellite variation in the chloroplast was found to be common, although less common than microsatellite variation in the nucleus. No microsatellite variation was found in coding regions of the chloroplast. To evaluate different models of microsatellite evolution as possible explanations for the observed pattern of variation, the length distribution of microsatellites in the published DNA sequence of the A. thaliana chloroplast was subsequently used. By combining information from these two analyses we found that the mode of evolution of the chloroplast mononucleotide microsatellites was best described by a linear relation between repeat length and mutation rate, when the repeat lengths exceeded about 7 bp. This model can readily predict the variation observed in non-coding chloroplast DNA. It was found that the number of uninterrupted repeat units had a large impact on the level of chloroplast microsatellite variation. No other factors investigated--such as the position of a locus within the chromosome, or imperfect repeats--appeared to affect the variability of chloroplast microsatellites. By fitting the slippage models to the Genbank sequence of chromosome 1, we show that the difference between microsatellite variation in the nucleus and the chloroplast is largely due to differences in slippage rate.

Amplification of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a significant source of characters for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte and monilophyte species to determine the potential utility of PCR amplification primers for 18 noncoding chloroplast DNA regions that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of five cpDNA regions for resolving relationships in Botrychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnL UAA– trnF GAA intergenic spacer in this group. To these published data we added sequences of the trnS GCU– trnG UUC intergenic spacer + the trnG UUC intron region, the trnS GGA– rpS4 intergenic spacer + rpS4 gene, and the rpL16 intron. Both the trnS GCU– trnG UUC and rpL16 regions are highly variable in angiosperms and the trnS GGA– rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the five sampled regions the trnS GCU– trnG UUC spacer + trnG UUC intron region provided the strongest support for the inferred phylogeny.