At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species.
We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnSUGA-trnGUCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnSUGA-trnGUCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species.
Citation: Dong W, Liu J, Yu J, Wang L, Zhou S (2012) Highly Variable Chloroplast Markers for Evaluating Plant Phylogeny at Low Taxonomic Levels and for DNA Barcoding. PLoS ONE7(4): e35071. https://doi.org/10.1371/journal.pone.0035071
Editor: Ahmed Moustafa, American University in Cairo, Egypt
Received: June 25, 2011; Accepted: March 13, 2012; Published: April 12, 2012
Copyright: © 2012 Dong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by National Natural Science Foundation of China (30930010, 30872062) and the Research Fund for the Large-scale Scientific Facilities of the Chinese Academy of Sciences (Grant No. 2009-LSF-GBOWS-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
At present, techniques for studying the molecular phylogeny of plants rely heavily on chloroplast genome sequence data. This is because the chloroplast genome has a simple and stable genetic structure, it is haploid, there are no (or very rare) recombination, it is generally uniparentally transmitted, and universal primers can be used to amplify target sequences. Another important reason is the ease of PCR amplification and sequencing of chloroplast genes, despite some intrinsic problems similar to those encountered when using animal mitochondrial DNA . Many fragments of coding regions, introns, and intergenic spacers, including atpB, atpB-rbcL, matK, ndhF, rbcL, rpl16, rps4-trnS, rps16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels , , , , , . Unfortunately, these regions often lack variations in closely related species, especially those that have diverged recently in evolution. Therefore, a concatenation of many individual genes must be used to improve the resolution of the phylogenetic analysis, and to obtain reasonable results. Such extra investments could be avoided if more variable locations were identified and universal primers were available.
Some regions of the chloroplast genome, for example, atpF-H, matK, psbK-I, rbcL, rpoB, rpoC1 and trnH-psbA have been relied upon heavily for development of candidate markers for plant DNA barcoding , , , , . The aim of DNA barcoding is to solve species identification problems, but some regions such as rbcL, rpoB, and rpoC1 are useful for identification at the family rather than species level. Recently, candidate loci and some other loci frequently used in phylogenetic analyses were critically evaluated for several flowering plant groups, including Amomum , Carex , Meteoriaceae , Cycadales , Compsonuera , Panax , peach  and tree peonies . It seems that matK and trnH-psbA are the two most promising choices of chloroplast regions. The matK gene is one of the most versatile candidates so far, because it is useful for identification at family, genus, and even species levels. However, it is difficult to amplify and sequence this region from certain taxa, and additional universal primers and optimization of PCR reactions are necessary , . trnH-psbA is the most variable region in the chloroplast genome across a wide range of groups. However, there are some exceptions and long mononucleotide repeats (poly-structures or single nucleotide microsatellites) can cause sequencing problems. Another problem is the presence of inversions in the middle of the sequence, which can lead to incorrect alignments .
Most of the regions that are commonly used for phylogenetic analyses were first identified in the 1990s, before entire genome sequences were available. Shaw et al.  summarized and evaluated the most frequently used chloroplast regions in seed plants, which significantly helped beginning researchers. Currently, about 191 entire chloroplast genomes are available, and some genera have two or more completely sequenced chloroplast genomes. Therefore, it is timely to reevaluate the variability of chloroplast regions at low taxonomic levels. Identification of variable loci in chloroplast genomes will be extremely useful for molecular systematics and DNA barcoding. Many plant species evolved via adaptive radiations or explosive patterns of speciation, and have evolutionary histories of only a few million years. The very short evolutionary histories result in low sequence divergence. The limited sequence variation is usually harbored in a few hotspots, and most of the loci available to researchers based on previous research provide very few informative characters.
To solve phylogenetic problems at the species level, or to identify species using DNA sequences, we need to identify regions with very high evolutionary rates. Greater availability of such regions will increase our ability to resolve such identification problems. Utilization of a larger number of regions of genes or sequences can minimize the noise of the evolutionary heterogeneity of genes or parts of a gene. Therefore, searching for more regions with high evolutionary rates is very important for plant phylogenetic analyses and for DNA barcoding. Fortunately, there are now many complete chloroplast genome sequences available, even for different species in same genera. This information allows the identification of most variable regions between or among species. In this paper, we summarize the results of comparative studies on chloroplast genomes of congeners of flowering plants. Our aim was to find the most variable regions that are common across many genera. Such regions can be used to resolve phylogenies and for DNA barcoding of closely related flowering plant species.
Identification of most variable loci in chloroplast genomes
There are 14 genera of seed plants in which the chloroplast genome has been sequenced for more than one species. We excluded gymnosperms from analyses because only Pinus has several genomes available. Of the angiosperm genera, we excluded Cuscuta from our analyses because of drastic genome reorganization and large deletions in the chloroplast genome. We analyzed a total of 12 genera among which nine genera have chloroplast genomic data readily available from GenBank, and the chloroplast genomic data of the other three genera are to be released (Table 1). The maximum number of polymorphic sites (S) within 600 bp in the 12 genera varied from 3 (Acorus) to 49 (Aethionema) with an arithmetic mean of 22.8. When the regions were restricted by the number of polymorphic sites (S, >+2stdev), there were 47 highly variable loci present in at least one genus, 29 were shared by two or more genera, 23 by three or more genera, 11 by four or more genera, 10 by 5 or more genera, and only 5 by 6 or more genera. To provide reasonable choices, we further analyzed 23 loci (Table S1). Among them, ndhF, trnK (containing matK), ycf1-a, and ycf1-b are largely coding regions, clpP and ndhA are introns, and the other 17 are intergenic spacers (Fig. 1). The most variable locus was ycf1, a gene of unknown function. The ycf1 locus is several kilobase-pairs long. Two regions of ycf1 showed high variability in 9 of 11 genera, and the π values of the ycf1 locus in 6 genera were markedly higher than in the other genera. The rpl32-trnL and trnK (including matK) loci were variable in 8 genera, and rps16-trnQ and trnSUGA-trnGUCC loci were variable in 6 genera. Judging from the values of nucleotide diversity (π values), ycf1, trnH-psbA, rpl32-trnL, rps16-trnQ, and ndhC-trnV were the most variable loci with average π values of greater than 0.01 over 12 genera. The other loci showed average π values of greater than 0.0048. The loci ndhC-trnV, rps16-trnQ, trnSUGA-trnGGCC, trnSUGA-trnGUCC, and trnT-psbD were rich in indels. Indels are usually informative in phylogenetic reconstructions and diagnostic to plant taxa.
Large white boxes indicate coding areas, small white boxes indicate intergenic spacers, and small black boxes indicate introns. Figures above boxes indicate length (bp).
Universality of primers
Although there are primers available for some loci, e.g., rps16-trnQ, trnH-psbA, trnK, and trnSUGA-trnGUCC, to provide more choices we designed new primers for 21 loci (Fig. 1, Table 2) based on available chloroplast genome data from GenBank. The two regions of ycf1 were too long and variable to design universal primers. All new primers were tested using eight species covering basal angiosperms, monocots, eudicots, rosids, and asterids. The primer pairs for 15 loci showed complete amplification successes with the eight testing species (Table 2). For some other primer pair/species combinations we failed to amplify sequences as follows: rbcL-accD of Typha orientalis and Prunus persica, accD-psaI of Panax bipinnatifidus, ndhF of Chimonanthus praecox, rpl32-trnL of Paeonia suffruticosa, trnSGCU-trnGGCC of T. orientalis, and trnW-psaJ of P. suffruticosa (Fig. 2). To check the quality of amplified fragments, PCR products from Nelumbo nucifera, Prunus mira, and Panax bipinnatifidus were purified with PEG8000 and directly sequenced. We considered 600 bp to be an acceptable length of a read for the sequence of a given product. One hundred and ninety-eight reads of the 210 in total (∼94.3%) reached 600 bp in length and had quality values (ratios of bases with QV >20 to the total bases of a read) higher than 90% after trimming both ends (Table 2).
Numbers shown at top are sequential order of loci as in Table 2 and Fig. 1. Numbers on right are size markers (kbp). Letters on left indicate species as follows: Cp: Chimonanthus praecox (L.) Link; To: Typha orientalis Presl.; Nn: Nelumbo nucifera Gaertn.; Ps: Paeonia suffruticosa Andrews; Pp: Prunus persica (L.) Batsch.; and Pb: Panax bipinnatifidus Seem.
Variability of 21 loci across Nelumbo, Panax, and Prunus, and comparisons with other loci
To assess the variability of the 21 loci, we selected two genera, Panax and Prunus, which have been studied for DNA barcoding purposes , , and the family Nelumbonaceae, which contains only two species. The number of variable sites, the nucleotide diversity, and the number of indels plus inverses were used as indicators. Four loci that have been suggested as candidate barcodes (atpF-H, rbcL, ropB, and rpoC1) were used as controls. The 21 loci performed satisfactory although not all of them were better than the controls in all three genera (Fig. 3).
Performances of the 21 loci, a case study on peaches
Peaches are a natural group of five or six species belonging to Prunus L. sect. Persica (L.) S. L. Zhou & X. Quan  or Amygdalus subg. Persica . Nine chloroplast loci had been evaluated for DNA barcoding purpose . The bootstrap consensus trees constructed using maximum parsimony based on the 21 loci (Fig. S1) show moderate to high resolutions. A combination of psbM-trnD intergenic spacer and clpP intron can solve all six species (Fig. 4).
It is believed that there are mutationally (or substitutionally) active regions in genomes, and our genomic survey of 12 genera indicated that such regions exist in chloroplast genomes. The number of polymorphic sites (S values) varied from 3 to 49 (Table 1) with an arithmetic mean value of 22.8 in 600 bp, indicating great potential for finding variable regions carrying phylogenetic information. Some considerably higher sequence variability of introns and intergenic spacers in chloroplast genomes has been reported (e.g. , , ). Mutationally active regions in chloroplast genomes are frequently regarded as problematic for phylogenetic analyses at higher taxonomic levels because of recombination and sequence convergence, although there are conflicting opinions on the issue (e.g. ). However, at lower taxonomic levels of flowering plants, the problems are less serious because in most cases there is insufficient sequence variation in chloroplast genes, rather than the high homoplasy that results from supervariability, as is found in mitochondrial genes.
The most variable locus was ycf1, a gene of unknown function. It is more variable than the matK locus in the Orchidaceae . The ycf1 locus is several kb long. The region locates in the IRb region is conservative and two regions located in SSC region are extremely variable, and thus suitable for phylogenetic studies or DNA barcoding at low taxonomic levels. Unfortunately, because of the vast sequence differences at the ycf1 locus, we could not obtain universal primers currently. It is worthy of working out universal primers after examining more sequences or taxon-specific primers such as matK . The matK region alone or together with trnK introns has been extensively used in molecular systematics and suggested to be a barcode for plants , . However, the variability of matK region is not as remarkable as some other loci in the genera we examined, except for in Chimonanthus. Therefore, the matK locus could be helpful in separating angiosperm families or genera but very rarely species. The 3′ region of ndhF locus is more variable than the 5′ region and such a kind of variations is suitable for phylogenetic reconstruction for either old or recent groups . The 3′ region of ndhF exhibited relatively high variability in Chimonanthus, Eucalyptus, Populus and Prunus (Table S1, Fig. 3E). The locus rpl32-trnL showed considerable length variation across taxa and a high level of positional variability. If the sequences can be unambiguously aligned, the rpl32-trnL region will be suitable for species identification. Unfortunately, the rpl32-trnL region has rarely been examined in systematics, and more case studies would further clarify its suitability for such analyses. The trnH-psbA was suggested as a candidate DNA barcode early , but has not been bolstered in subsequent studies. The trnH-psbA locus is really variable in most cases but suffers short length and, therefore, may not provide enough informative characters. Moreover, inversions or mononucleotide repeats are likely to exist at the trnH-psbA locus, which may result incorrect alignments or bring sequencing difficulties. The trnS-trnG has been well analyzed by Shaw et al.  and primer sequences were provided. The trnSUGA-trnGUCC includes psbZ. So the trnSGCU-trnGGCC region is likely to be more variable than trnSUGA-trnGUCC. Unexpectedly the variability of the two regions is taxon-dependent. The major problems are polyT sequences in the trnSGCU-trnGGCC region and (AT)n elements in the trnSUGA-trnGUCC region. A large indel in the trnSUGA-trnGUCC region was found variable within species . Although the trnSUGA was considered to be of mitochondrial origin for majority of taxa , it is also present in chloroplast genomes and the intergenic spacer trnSUGA-trnGUCC should be unique to the chloroplast genomes. The petA-psbJ and rps16-trnQ was variable in such genera as Acorus, Paeonia and Oryza in which very few variable regions exist. Similar to trnH-psbA indels and inversions are likely to happen in petA-psbJ. The loci ndhC-trnV, ndhF, rpoB-trnC, psbE-petL and rbcL-accD have been tried occasionally with varying successes. Among them the rbcL-accD deserves more attention because the rbcL has been suggested to be a barcode together with matK . The discrimination power of rbcL is not as high as matK. Inclusion of rbcL-accD would compensate the insufficient variations of rbcL. The 10 other loci shown in Table S1 were variable in 3 genera. Some showed high π values in some genera, indicating that they could be useful for resolving phylogenetic relationships in those taxa.
Most of the loci identified in this study have been used frequently for phylogenetic reconstructions, and their evolutionary features have been discussed , , . Some of the loci, e.g., clpP, petB-petD , rbcL-accD , , and trnW-psaJ have only been used occasionally. The two introns of clpP have seldom been considered in phylogenetic studies. The first intron of clpP exhibits moderate variability in some taxa, and has some potential applications, however, it may be absent from some taxa , . The trnW-psaJ locus includes two intergenic spacers and a coding region (trnW-trnP-psaJ). The trnW-psaJ region has never been used independently and its variability should be evaluated. The trnW-psaJ region was the most variable region in Acorus, and was also one of the most variable regions in Eucalyptus (Table S1). A complete assessment of the loci presented in this study should be conducted before making final decisions about markers used for analyses.
In rapidly evolving regions of the chloroplast genome, evolutionary events that occur include the formation of secondary structures, multiple-hit sites, and intra-molecular recombination events. These problems seem less serious in phylogenetic analyses of closely related species. However, to be frank our aim to accurately solve phylogenetic relationships by using the loci identified in this study may not always be achieved because of other problems. For example, the loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are likely to be absent from some genera, which limits their applications. Minute inversions are often observed in rapidly evolving regions such as introns and intergenic spacers, e.g., trnH-psbA, petN-psbM , . If such structures were not recognized, the resulting alignments could be problematic. In addition, incorrect use of information regarding length variation could lead to misguided conclusions, because it can be very difficult to determine homologies among sequences that vary substantially in length. Both mononucleotide and multinucleotide repeats (microsatellites, such as in psbM-trnD, trnSGCU-trnGGCC and trnSUGA-trnGUCC) are sometimes present in rapidly evolving regions, and the exact numbers of repeats are difficult to determine by direct sequencing of PCR products. Several authors have suggested ways to interpret the informative but problematic characters , .
The variability of chloroplast genes differs markedly among genera (Fig. 3). There are intrinsic difficulties and/or taxonomic problems in finding variable regions in chloroplast genomes. The sequence divergence between the two species in Acorus is so small that the maximum number of substitutions every 600 bp was only 3, compared with 49 in Aethionema. Acorus americanus and A. calamus are well diverged morphologically. The very small sequence difference between A. americanus and A. calamus is perhaps due to short time of divergence. Oryza sativa subsp. indica and O. nivara are also very closely related. The cultivated O. sativa originated from O. rufipogon and/or O. nivara a few thousand years ago as a result of human selection . Therefore, the variation between O. sativa subsp. indica and O. nivara is more properly considered intraspecific variation and it is unlikely that regions with high π values will be found. For such taxa, a combination of multiple genes is necessary to uncover more informative characters. In our case of peaches, many loci resolve the species better than matK (trnK), rbcL or trnH-psbA (Fig. S1) and a combination of psbM-trnD and clpP intron can resolve all six species (Fig. 4).
Although candidate genes have been significantly narrowed by this study, the loci we suggest may not be applicable for all flowering plants; however, most of the identified loci are good initial candidates for further evaluations. The controversies regarding plant DNA barcodes will continue even after markers become mandatory. Taxon-specific markers will have to be used for some difficult-to-differentiate taxa. In addition, markers that worked well in one case study do not guarantee suitability for another. Thus, pilot studies are necessary for any untested taxa and further assessments are required to better determine which loci will be useful for any given taxonomic and/or DNA barcoding questions. Moreover, if none of the candidates listed in Table S1 proves satisfactory, we have shown that additional choices are available for potential exploration (Table S3).
Collection of congener genome data and identification of variable loci
We downloaded from GenBank all chloroplast genome sequences in genera with at least two different species (Table 1). In our analyses, we also included six newly determined chloroplast genome sequences, three from the Calycanthaceae and three from Paeoniaceae (to be published). The sequences were first aligned using ClustalX 2.0 , and then manually adjusted with Se-Al 2.0a11 . Inversions, if present, were separated to avoid exaggerated sequence differences. The variability of the aligned genomes was evaluated using the sliding window method in DNAsp ver. 4.5 . The window length was set to 600 base pairs (bp), the typical length of DNA barcodes. The step size was set to 50 for relatively accurate positioning of variable regions. We only considered regions with the number of polymorphic sites (S) >+2 stdev. The regions were identified according to the original annotations, then extracted and compared among the genera after precise alignments. Regions were excluded from further consideration if they were present in fewer than three genera.
Primer design, applicability tests, and variability assessment
The locations of highly variable regions were precisely identified, and the conserved sequences flanking the regions were used for primer design. Primers for amplifying highly variable regions were designed using Primer Premier v. 5.0 (Premier Biosoft International, CA, USA) and Oligo v. 6.71 (Molecular Biology Insights, CO, USA). The primer pairs were synthesized by Sangon Biotech (Shanghai) Co. Ltd. (Beijing, China).
We used eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids (Table S2) to test the applicability of the primers and the variability of the selected loci. Total DNA was extracted by the CTAB method  from silicon gel-dried materials (Table S2). Polymerase chain reactions (PCR) were carried out in 20 µL reaction mixtures. Each PCR mixture contained 2.0 µL 10×buffer, 2.0 µL dNTPs (2 µmol/L), 1.0 µL each primer (5 µmol/L), 1.0 µL total DNA (∼25 ng), 0.2 µL Taq polymerase (5 U/µL), and 11.8 µL ddH2O. The PCR program was as follows: 94°C for 3 min, followed by 34 cycles of 94°C for 30 s, 52°C (regardless of the Tm values) for 30 s, 72°C for 2 min, with final extension at 72°C for 5 min. PCR amplifications were carried out using a Veriti 96 Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Six species from three genera presenting basal eudicots, rosids and asterids were used to evaluate the variability of the loci and all of the resulting fragments were sequenced on an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), following the manufacturer's instructions. The sequences were assembled using Sequencer 4.7 (Gene Codes, Ann Arbor, MI, USA), aligned with ClustalX , and adjusted manually with Se-Al 2.0 . The number of polymorphic sites and nucleotide diversity per site (π) were computed using DnaSP ver. 5.10 .
Considering very low sequence variations within species , sequences of all 21 loci from all six species were used to test the resolutions of the loci. The species [vouchers in parenthesis, please refer to Quan and Zhou  for more detailed information] are P. davidiana (QX095), P. ferganensis (QX020), P. kansuensis (QX026), P. mira (QX138), P. persica (QX048), and P. potanini (SL4805–83), and P. armeniaca (SL4802–69) was used as an outgroup. Prunus ferganensis considered to be a distinct species instead of treating it a subspecies in P. persica as Quan and Zhou . The methods for obtaining and analyzing the sequences are the same as above. The resolution of each locus was judged by the maximum parsimonious trees built with PAUP*  with the same settings as Quan and Zhou .
The twenty-three most variable regions in chloroplast genomes of 12 genera with two or more species.
Samples used to test the of 23 chloroplast loci.
Twenty-four loci of high potentials. The values are nucleotide diversity per site (π).
We thank John W. Stiller for his revision of the manuscript and suggestions. We are also grateful to the two anonymous reviewers for their comments that help significantly to improve the quality of this manuscript.
Conceived and designed the experiments: SZ WD. Performed the experiments: WD JY JL. Analyzed the data: WD. Wrote the paper: SZ LW WD.
- 1. Hurst GDD, Jiggins FM (2005) Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. Proceedings of the Royal Society B: Biological Sciences 272: 1525.GDD HurstFM Jiggins2005Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts.Proceedings of the Royal Society B: Biological Sciences2721525
- 2. Gao X, Zhu YP, Wu BC, Zhao YM, Chen JQ, et al. (2008) Phylogeny of Dioscorea sect. Stenophora based on chloroplast matK, rbcL and trnL-F sequences. Journal of Systematics and Evolution 46: 315–321.X. GaoYP ZhuBC WuYM ZhaoJQ Chen2008Phylogeny of Dioscorea sect. Stenophora based on chloroplast matK, rbcL and trnL-F sequences.Journal of Systematics and Evolution46315321
- 3. Li JH (2008) Phylogeny of Catalpa (Bignoniaceae) inferred from sequences of chloroplast ndhF and nuclear ribosomal DNA. Journal of Systematics and Evolution 46: 341–348.JH Li2008Phylogeny of Catalpa (Bignoniaceae) inferred from sequences of chloroplast ndhF and nuclear ribosomal DNA.Journal of Systematics and Evolution46341348
- 4. Wilson CA (2009) Phylogenetic relationships among the recognized series in Iris Section Limniris. Systematic Botany 34: 277–284.CA Wilson2009Phylogenetic relationships among the recognized series in Iris Section Limniris.Systematic Botany34277284
- 5. Peterson PM, Romaschenko K, Johnson G (2010) A classification of the Chloridoideae (Poaceae) based on multi-gene phylogenetic trees. Molecular Phylogenetics and Evolution 55: 580–598.PM PetersonK. RomaschenkoG. Johnson2010A classification of the Chloridoideae (Poaceae) based on multi-gene phylogenetic trees.Molecular Phylogenetics and Evolution55580598
- 6. Hilu KW, Black C, Diouf D, Burleigh JG (2008) Phylogenetic signal in matK vs. trnK: A case study in early diverging eudicots (angiosperms). Molecular Phylogenetics and Evolution 48: 1120–1130.KW HiluC. BlackD. DioufJG Burleigh2008Phylogenetic signal in matK vs. trnK: A case study in early diverging eudicots (angiosperms).Molecular Phylogenetics and Evolution4811201130
- 7. Kim KJ, Jansen RK (1995) NdhF sequence evolution and the major clades in the sunflower Family. Proceedings of the National Academy of Sciences of the United States of America 92: 10379–10383.KJ KimRK Jansen1995NdhF sequence evolution and the major clades in the sunflower Family.Proceedings of the National Academy of Sciences of the United States of America921037910383
- 8. Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005) Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences of the United States of America 102: 8369–8374.WJ KressKJ WurdackEA ZimmerLA WeigtDH Janzen2005Use of DNA barcodes to identify flowering plants.Proceedings of the National Academy of Sciences of the United States of America10283698374
- 9. Chase MW, Cowan RS, Hollingsworth PM, van den Berg C, Madrinan S, et al. (2007) A proposal for a standardised protocol to barcode all land plants. Taxon 56: 295–299.MW ChaseRS CowanPM HollingsworthC. van den BergS. Madrinan2007A proposal for a standardised protocol to barcode all land plants.Taxon56295299
- 10. Newmaster SG, Fazekas AJ, Ragupathy S (2006) DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany-Revue Canadienne De Botanique 84: 335–341.SG NewmasterAJ FazekasS. Ragupathy2006DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach.Canadian Journal of Botany-Revue Canadienne De Botanique84335341
- 11. Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. Plos One 6: e19254.PM HollingsworthSW GrahamDP Little2011Choosing and using a plant DNA barcode.Plos One6e19254
- 12. Seberg O, Petersen G (2009) How many loci does it take to DNA barcode a Crocus? Plos One 4: e4598.O. SebergG. Petersen2009How many loci does it take to DNA barcode a Crocus?Plos One4e4598
- 13. Yang ZY, Zhang L (2010) Screening potential DNA barcode regions in Amomum (Zingiberaceae). Acta Botanica Yunnanica 32: 393–400.ZY YangL. Zhang2010Screening potential DNA barcode regions in Amomum (Zingiberaceae).Acta Botanica Yunnanica32393400
- 14. Starr JR, Naczi RFC, Chouinard BN (2009) Plant DNA barcodes and species resolution in sedges (Carex, Cyperaceae). Molecular Ecology Resources 9: 151–163.JR StarrRFC NacziBN Chouinard2009Plant DNA barcodes and species resolution in sedges (Carex, Cyperaceae).Molecular Ecology Resources9151163
- 15. Zhao LJ, Jia Y, Zhou SL, Du GS (2010) The preliminary study on DNA barcoding of mosses-A case of part of genera of Meteoriaceae. Acta Botanica Yunnanica 32: 239–249.LJ ZhaoY. JiaSL ZhouGS Du2010The preliminary study on DNA barcoding of mosses-A case of part of genera of Meteoriaceae.Acta Botanica Yunnanica32239249
- 16. Sass C, Little DP, Stevenson DW, Specht CD (2007) DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of Cycads. Plos One 2: e1154.C. SassDP LittleDW StevensonCD Specht2007DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of Cycads.Plos One2e1154
- 17. Newmaster SG, Fazekas AJ, Steeves RAD, Janovec J (2008) Testing candidate plant barcode regions in the Myristicaceae. Molecular Ecology Resources 8: 480–490.SG NewmasterAJ FazekasRAD SteevesJ. Janovec2008Testing candidate plant barcode regions in the Myristicaceae.Molecular Ecology Resources8480490
- 18. Zuo YJ, Chen ZJ, Kondo K, Funamoto T, Wen J, et al. (2011) DNA barcoding of Panax species. Planta Medica 77: 182–187.YJ ZuoZJ ChenK. KondoT. FunamotoJ. Wen2011DNA barcoding of Panax species.Planta Medica77182187
- 19. Quan X, Zhou SL (2011) Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants. Journal of Systematics and Evolution 49: 138–145.X. QuanSL Zhou2011Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants.Journal of Systematics and Evolution49138145
- 20. Zhang JM, Wang JX, Xia T, Zhou SL (2009) DNA barcoding: species delimitation in tree peonies. Science in China Series C-Life Sciences 52: 568–578.JM ZhangJX WangT. XiaSL Zhou2009DNA barcoding: species delimitation in tree peonies.Science in China Series C-Life Sciences52568578
- 21. Dunning LT, Savolainen V (2010) Broad-scale amplification of matK for DNA barcoding plants, a technical note. Botanical Journal of the Linnean Society 164: 1–9.LT DunningV. Savolainen2010Broad-scale amplification of matK for DNA barcoding plants, a technical note.Botanical Journal of the Linnean Society16419
- 22. Yu J, Xue JH, Zhou SL (2011) New universal matK primers for DNA barcoding angiosperms. Journal of Systematics and Evolution 49: 176–181.J. YuJH XueSL Zhou2011New universal matK primers for DNA barcoding angiosperms.Journal of Systematics and Evolution49176181
- 23. Whitlock BA, Hale AM, Groff PA (2010) Intraspecific inversions pose a challenge for the trnH-psbA plant DNA barcode. Plos One 5: e11533.BA WhitlockAM HalePA Groff2010Intraspecific inversions pose a challenge for the trnH-psbA plant DNA barcode.Plos One5e11533
- 24. Shaw J, Lickey EB, Beck JT, Farmer SB, Liu WS, et al. (2005) The tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany 92: 142–166.J. ShawEB LickeyJT BeckSB FarmerWS Liu2005The tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis.American Journal of Botany92142166
- 25. Lu LT (1986) Amygdalus L. In: Yu TT, editor. Rosaceae (3). Flora Reipublicae Popularis Sinica 38. Beijing: Science Press. LT Lu1986Amygdalus L.TT YuRosaceae (3). Flora Reipublicae Popularis Sinica 38BeijingScience Press
- 26. Shaw J, Lickey EB, Schilling EE, Small RL (2007) Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The tortoise and the hare III. American Journal of Botany 94: 275–288.J. ShawEB LickeyEE SchillingRL Small2007Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The tortoise and the hare III.American Journal of Botany94275288
- 27. Borsch T, Quandt D (2009) Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA. Plant Systematics and Evolution 282: 169–199.T. BorschD. Quandt2009Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA.Plant Systematics and Evolution282169199
- 28. Müller KF, Borsch T, Hilu KW (2006) Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: Contrasting matK, trnT-F, and rbcL in basal angiosperms. Molecular Phylogenetics and Evolution 41: 99–117.KF MüllerT. BorschKW Hilu2006Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: Contrasting matK, trnT-F, and rbcL in basal angiosperms.Molecular Phylogenetics and Evolution4199117
- 29. Neubig K, Whitten W, Carlsward B, Blanco M, Endara L, et al. (2009) Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK. Plant Systematics and Evolution 277: 75–84.K. NeubigW. WhittenB. CarlswardM. BlancoL. Endara2009Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK.Plant Systematics and Evolution2777584
- 30. Neubig KM, Abbott JR (2010) Primer development for the plastid region ycf1 in Annonaceae and Other Magnoliids. American Journal of Botany 97: E52–E55.KM NeubigJR Abbott2010Primer development for the plastid region ycf1 in Annonaceae and Other Magnoliids.American Journal of Botany97E52E55
- 31. Group CPW (2009) A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106: 12794–12797.Group CPW2009A DNA barcode for land plants.Proceedings of the National Academy of Sciences of the United States of America1061279412797
- 32. Alverson AJ, Rice DW, Dickinson S, Barry K, Palmer JD (2011) Origins and Recombination of the Bacterial-Sized Multichromosomal Mitochondrial Genome of Cucumber. Plant Cell 23: 2499–2513.AJ AlversonDW RiceS. DickinsonK. BarryJD Palmer2011Origins and Recombination of the Bacterial-Sized Multichromosomal Mitochondrial Genome of Cucumber.Plant Cell2324992513
- 33. Korall P, Conant DS, Metzgar JS, Schneider H, Pryer KM (2007) A molecular phylogeny of scaly tree ferns (Cyatheaceae). American Journal of Botany 94: 873–886.P. KorallDS ConantJS MetzgarH. SchneiderKM Pryer2007A molecular phylogeny of scaly tree ferns (Cyatheaceae).American Journal of Botany94873886
- 34. Matsuda1a Y, Yoshimura1a H, Kanamoto H, Ujihara T, Tomizawa K, et al. (2005) Sequence variation in the rbcL-accD region in the chloroplast genome of Moraceae. Plant Biotechnology 22: 231–233.Y. Matsuda1aH. Yoshimura1aH. KanamotoT. UjiharaK. Tomizawa2005Sequence variation in the rbcL-accD region in the chloroplast genome of Moraceae.Plant Biotechnology22231233
- 35. Lee HL, Jansen RK, Chumley TW, Kim KJ (2007) Gene relocations within chloroplast genomes of Jasminum and Menodora (Oleaceae) are due to multiple, overlapping inversions. Molecular Biology and Evolution 24: 1161–1180.HL LeeRK JansenTW ChumleyKJ Kim2007Gene relocations within chloroplast genomes of Jasminum and Menodora (Oleaceae) are due to multiple, overlapping inversions.Molecular Biology and Evolution2411611180
- 36. Erixon P, Oxelman B (2008) Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene. Plos One 3: e1386.P. ErixonB. Oxelman2008Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene.Plos One3e1386
- 37. Kim KJ, Lee HL (2005) Widespread occurrence of small inversions in the chloroplast genomes of land plants. Molecules and Cells 19: 104–113.KJ KimHL Lee2005Widespread occurrence of small inversions in the chloroplast genomes of land plants.Molecules and Cells19104113
- 38. Kelchner SA (2000) The evolution of non-coding chloroplast DNA and its application in plant systematics. Annals of the Missouri Botanical Garden 87: 482–498.SA Kelchner2000The evolution of non-coding chloroplast DNA and its application in plant systematics.Annals of the Missouri Botanical Garden87482498
- 39. Simmons MP, Ochoterena H (2000) Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 369–381.MP SimmonsH. Ochoterena2000Gaps as characters in sequence-based phylogenetic analyses.Systematic Biology49369381
- 40. Londo JP, Chiang YC, Hung KH, Chiang TY, Schaal BA (2006) Phylogeography of Asian wild rice, Oryza rufipogon, reveals multiple independent domestications of cultivated rice, Oryza sativa. Proceedings of the National Academy of Sciences of the United States of America 103: 9578–9583.JP LondoYC ChiangKH HungTY ChiangBA Schaal2006Phylogeography of Asian wild rice, Oryza rufipogon, reveals multiple independent domestications of cultivated rice, Oryza sativa.Proceedings of the National Academy of Sciences of the United States of America10395789583
- 41. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007) Clustal W and clustal X version 2.0. Bioinformatics 23: 2947–2948.MA LarkinG. BlackshieldsNP BrownR. ChennaPA McGettigan2007Clustal W and clustal X version 2.0.Bioinformatics2329472948
- 42. Rambaut A (1996) Se-Al: Sequence Alignment Editor. version 2.0. Oxford: University of Oxford, Department of Zoology. A. Rambaut1996Se-Al: Sequence Alignment Editor. version 2.0OxfordUniversity of Oxford, Department of Zoology
- 43. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19: 2496–2497.J. RozasJC Sanchez-DelBarrioX. MesseguerR. Rozas2003DnaSP, DNA polymorphism analyses by the coalescent and other methods.Bioinformatics1924962497
- 44. Doyle J, Doyle J (1987) A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.J. DoyleJ. Doyle1987A rapid DNA isolation procedure for small amounts of fresh leaf tissue.Phytochemical Bulletin191115
- 45. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.JD ThompsonTJ GibsonF. PlewniakF. JeanmouginDG Higgins1997The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Research2548764882
- 46. Swofford D (2002) PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version 4b10. Sinauer, Sunderland, Massachusetts, USA. D. Swofford2002PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version 4b10Sinauer, Sunderland, Massachusetts, USA