Ancestral maximum likelihood of evolutionary trees is hard

Maximum likelihood (ML) (Neyman, 1971) is an increasingly popular optimality criterion for selecting evolutionary trees. Finding optimal ML trees appears to be a very hard computational task--in particular, algorithms and heuristics for ML take longer to run than algorithms and heuristics for maximum parsimony (MP). However, while MP has been known to be NP-complete for over 20 years, no such hardness result has been obtained so far for ML. In this work we make a first step in this direction by proving that ancestral maximum likelihood (AML) is NP-complete. The input to this problem is a set of aligned sequences of equal length and the goal is to find a tree and an assignment of ancestral sequences for all of that tree's internal vertices such that the likelihood of generating both the ancestral and contemporary sequences is maximized. Our NP-hardness proof follows that for MP given in (Day, Johnson and Sankoff, 1986) in that we use the same reduction from Vertex Cover; however, the proof of correctness for this reduction relative to AML is different and substantially more involved.     

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.      

Does choice in model selection affect maximum likelihood analysis?

In order to have confidence in model-based phylogenetic analysis, the model of nucleotide substitution adopted must be selected in a statistically rigorous manner. Several model-selection methods are applicable to maximum likelihood (ML) analysis, including the hierarchical likelihood-ratio test (hLRT), Akaike information criterion (AIC), Bayesian information criterion (BIC), and decision theory (DT), but their performance relative to empirical data has not been investigated thoroughly. In this study, we use 250 phylogenetic data sets obtained from TreeBASE to examine the effects that choice in model selection has on ML estimation of phylogeny, with an emphasis on optimal topology, bootstrap support, and hypothesis testing. We show that the use of different methods leads to the selection of two or more models for approximately 80% of the data sets and that the AIC typically selects more complex models than alternative approaches. Although ML estimation with different best-fit models results in incongruent tree topologies approximately 50% of the time, these differences are primarily attributable to alternative resolutions of poorly supported nodes. Furthermore, topologies and bootstrap values estimated with ML using alternative statistically supported models are more similar to each other than to topologies and bootstrap values estimated with ML under the Kimura two-parameter (K2P) model or maximum parsimony (MP). In addition, Swofford-Olsen-Waddell-Hillis (SOWH) tests indicate that ML trees estimated with alternative best-fit models are usually not significantly different from each other when evaluated with the same model. However, ML trees estimated with statistically supported models are often significantly suboptimal to ML trees made with the K2P model when both are evaluated with K2P, indicating that not all models perform in an equivalent manner. Nevertheless, the use of alternative statistically supported models generally does not affect tests of monophyletic relationships under either the Shimodaira-Hasegawa (S-H) or SOWH methods. Our results suggest that although choice in model selection has a strong impact on optimal tree topology, it rarely affects evolutionary inferences drawn from the data because differences are mainly confined to poorly supported nodes. Moreover, since ML with alternative best-fit models tends to produce more similar estimates of phylogeny than ML under the K2P model or MP, the use of any statistically based model-selection method is vastly preferable to forgoing the model-selection process altogether.     

Phylogenetic Depth of the Bacterial Genera Aquifex and Thermotoga Inferred from Analysis of Ribosomal Protein, Elongation Factor, and RNA Polymerase Subunit Sequences

The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1α) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase β- and β′-type subunits encoded in the rpoBC operon (1130 aa positions). Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences. In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1α) and EF-G(2) data sets, which lack both resolution and statistical robustness. In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A. pyrophilus and T. maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A. pyrophilus–T. maritima cluster situated at the base of the bacterial clade. RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements. The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed. However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm. A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a ``long branch attraction' artifact. Received: 22 September 1999 / Accepted: 11 January 2000     

Molecular phylogeny of oribatid mites (Oribatida, Acari): evidence for multiple radiations of parthenogenetic lineages

Nucleotide sequences of the D3 expansion segment and its flanking regions of the 28S rDNA gene were used to evaluate phylogenetic relationships among representative sexual and asexual oribatid mites (Oribatida, Acariformes). The aim of this study was to investigate the hypothesis that oribatid mites consist of species-rich clusters of asexual species that may have radiated while being parthenogenetic. Furthermore, the systematic position of the astigmate mites (Astigmata, Acariformes) which have been hypothesised to represent a paedomorphic lineage within the oribatid mites, is investigated. This is the first phylogenetic tree for oribatid mites s.l. (incl. Astigmata) based on nucleotide sequences. Intraspecific genetic variation in the D3 region was very low, confirming the hypothesis that this region is a good species marker. Results from neighbour joining (NJ) and maximum parsimony (MP) algorithms indicate that several species-rich parthenogenetic groups like Camisiidae, Nanhermanniidae and Malaconothridae are monophyletic, consistent with the hypothesis that some oribatid mite groups diversified despite being parthenogenetic. The MP and maximum likelihood (ML) method indicated that the D3 region is a good tool for elucidating the relationship of oribatid mite species on a small scale(genera, families) but is not reliable for large-scale taxonomy, because branches from the NJ algorithm collapsed in the MP and ML tree. In all trees calculated by different algorithms the Astigmata clustered within the oribatid mites, as proposed earlier.     

Parsimony, likelihood, and the role of models in molecular phylogenetics

Methods such as maximum parsimony (MP) are frequently criticized as being statistically unsound and not being based on any "model." On the other hand, advocates of MP claim that maximum likelihood (ML) has some fundamental problems. Here, we explore the connection between the different versions of MP and ML methods, particularly in light of recent theoretical results. We describe links between the two methods--for example, we describe how MP can be regarded as an ML method when there is no common mechanism between sites (such as might occur with morphological data and certain forms of molecular data). In the process, we clarify certain historical points of disagreement between proponents of the two methodologies, including a discussion of several forms of the ML optimality criterion. We also describe some additional results that shed light on how much needs to be assumed about underlying models of sequence evolution in order to successfully reconstruct evolutionary trees.     

Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences

Phylogenetic relationships of mushrooms and their relatives within the order Agaricales were addressed by using nuclear large subunit ribosomal DNA sequences. Approximately 900 bases of the 5' end of the nucleus-encoded large subunit RNA gene were sequenced for 154 selected taxa representing most families within the Agaricales. Several phylogenetic methods were used, including weighted and equally weighted parsimony (MP), maximum likelihood (ML), and distance methods (NJ). The starting tree for branch swapping in the ML analyses was the tree with the highest ML score among previously produced MP and NJ trees. A high degree of consensus was observed between phylogenetic estimates obtained through MP and ML. NJ trees differed according to the distance model that was used; however, all NJ trees still supported most of the same terminal groupings as the MP and ML trees did. NJ trees were always significantly suboptimal when evaluated against the best MP and ML trees, by both parsimony and likelihood tests. Our analyses suggest that weighted MP and ML provide the best estimates of Agaricales phylogeny. Similar support was observed between bootstrapping and jackknifing methods for evaluation of tree robustness. Phylogenetic analyses revealed many groups of agaricoid fungi that are supported by moderate to high bootstrap or jackknife values or are consistent with morphology-based classification schemes. Analyses also support separate placement of the boletes and russules, which are basal to the main core group of gilled mushrooms (the Agaricineae of Singer). Examples of monophyletic groups include the families Amanitaceae, Coprinaceae (excluding Coprinus comatus and subfamily Panaeolideae), Agaricaceae (excluding the Cystodermateae), and Strophariaceae pro parte (Stropharia, Pholiota, and Hypholoma); the mycorrhizal species of Tricholoma (including Leucopaxillus, also mycorrhizal); Mycena and Resinomycena; Termitomyces, Podabrella, and Lyophyllum; and Pleurotus with Hohenbuehelia. Several groups revealed by these data to be nonmonophyletic include the families Tricholomataceae, Cortinariaceae, and Hygrophoraceae and the genera Clitocybe, Omphalina, and Marasmius. This study provides a framework for future systematics studies in the Agaricales and suggestions for analyzing large molecular data sets.     

Systematics of Fraxinus (Oleaceae) and evolution of dioecy

Phylogenetic relationships among 40 of the 43 recognized species of Fraxinus L. (Oleaceae) were estimated on the basis of 106 nuclear ribosomal ITS sequences. ITS trees resulting from maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) are congruent and identify six distinct lineages. These clades allow establishing sections with high molecular and morphological support. The basal resolution generally has low ML bootstrap and MP jackknife support, but the Bayesian posterior probabilities are high for certain relationships. An independent data set of combined sequences from the chloroplast rps16 and trnL-F regions contains few informative sites but corroborate most of the relationships in the ITS tree. The molecular phylogeny is discussed in the light of morphological and other data and a revised infrageneric classification with six sections are presented. The subgenera and subsections are abandoned and the section Pauciflorae is a new combination. Fraxinus quadrangulata and Fraxinus anomala are united with Fraxinus dipetala in the section Dipetalae and Fraxinus platypoda is transferred to the section Fraxinus. Fraxinus chiisanensis, Fraxinus spaethiana and Fraxinus cuspidata are treated as incertae sedis. A sectional key is given, together with a systematic list of the 43 recognized species, with common synonyms and distribution. Breeding system and other traits mapped on the phylogeny show that dioecy has three separate origins, and in each case followed after the transition from insect to wind pollination. In one instance dioecy evolved from hermaphroditism via androdioecy and twice via polygamy.     

Reanalysis of Murphy et al.'s data gives various mammalian phylogenies and suggests overcredibility of Bayesian trees

Murphy and colleagues reported that the mammalian phylogeny was resolved by Bayesian phylogenetics. However, the DNA sequences they used had many alignment gaps and undetermined nucleotide sites. We therefore reanalyzed their data by minimizing unshared nucleotide sites and retaining as many species as possible (13 species). In constructing phylogenetic trees, we used the Bayesian, maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ) methods with different substitution models. These trees were constructed by using both protein and DNA sequences. The results showed that the posterior probabilities for Bayesian trees were generally much higher than the bootstrap values for ML, MP, and NJ trees. Two different Bayesian topologies for the same set of species were sometimes supported by high posterior probabilities, implying that two different topologies can be judged to be correct by Bayesian phylogenetics. This suggests that the posterior probability in Bayesian analysis can be excessively high as an indication of statistical confidence and therefore Murphy et al.'s tree, which largely depends on Bayesian posterior probability, may not be correct.     

Heterotachy and long-branch attraction in phylogenetics

Background  

Probabilistic methods have progressively supplanted the Maximum Parsimony (MP) method for inferring phylogenetic trees. One of the major reasons for this shift was that MP is much more sensitive to the Long Branch Attraction (LBA) artefact than is Maximum Likelihood (ML). However, recent work by Kolaczkowski and Thornton suggested, on the basis of simulations, that MP is less sensitive than ML to tree reconstruction artefacts generated by heterotachy, a phenomenon that corresponds to shifts in site-specific evolutionary rates over time. These results led these authors to recommend that the results of ML and MP analyses should be both reported and interpreted with the same caution. This specific conclusion revived the debate on the choice of the most accurate phylogenetic method for analysing real data in which various types of heterogeneities occur. However, variation of evolutionary rates across species was not explicitly incorporated in the original study of Kolaczkowski and Thornton, and in most of the subsequent heterotachous simulations published to date, where all terminal branch lengths were kept equal, an assumption that is biologically unrealistic.     

Molecular Phylogeny of the Genus Paramesotriton (Caudata: Salamandridae)

To elucidate the phylogeny of the genus Paramesotriton (Caudata: Salamandridae), we investigated three mitochondrial DNA gene fragments (1207 bp in total) of cytochrome b, ND2, and ND4 for its six recognized species. The phylogenetic relationships within Paramesotriton were reconstructed by maximum parsimony (MP) and maximum likelihood (ML) methods. Phylogenetic trees (MP and ML trees) that were constructed from the combined data set of the three gene fragments indicated that all six species of Paramesotriton formed a monophyletic group, with P. caudopunctatus as basal to the other five species. This result suggests that P. fuzhongensis is a valid species in Paramesotriton.     

DNA barcodes of eight species in genus Sebastes

DNA barcode is effective for biological taxonomy and is able to identify species from any life-history stage. In the present study, eight species which belong to four different subgenera of genus Sebastes found in China sea waters were identified by cytochrome c oxidase I (COI) barcode. The results indicated that the intra-species variation in DNA barcode was less than inter-species variation. When the phylogenetic trees were reconstructed by neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods, all the species clustered in their groups distinguishable by high bootstrap values, which proved that COI barcode is a powerful means to differentiate species of Sebastes and supports their identification. When the molecular tree was compared to the morphological tree, only Sebastes trivittatus in subgenus Sebastocles settled in the different positions. It is suggested that S. trivittatus is one of the shallowest occurring species in the Northwest Pacific due to its life characters.     

丝瓜藓属(Pohlia Hedw.)的系统位置及属内种间关系探讨

丝瓜藓属的系统地位及属内种间关系存在争议。本研究首次利用核糖体DNA内转录间区(ITS)序列数据对丝瓜藓属及相关科属植物进行系统发育分析。最大简约法,最大似然法及贝叶斯推论法构建的系统树均表明,ITS序列数据不支持将丝瓜藓属划归提灯藓科。丝瓜藓属植物与缺齿藓属植物构成一个单系分支。Brotherus(1903),Ochi(1959),Shaw(1984)及Hill等(2006)等学者提出的丝瓜藓属属下分类均不是单系类群。     

Evolutionary Triplet Models of Structured RNA

The reconstruction and synthesis of ancestral RNAs is a feasible goal for paleogenetics. This will require new bioinformatics methods, including a robust statistical framework for reconstructing histories of substitutions, indels and structural changes. We describe a “transducer composition” algorithm for extending pairwise probabilistic models of RNA structural evolution to models of multiple sequences related by a phylogenetic tree. This algorithm draws on formal models of computational linguistics as well as the 1985 protosequence algorithm of David Sankoff. The output of the composition algorithm is a multiple-sequence stochastic context-free grammar. We describe dynamic programming algorithms, which are robust to null cycles and empty bifurcations, for parsing this grammar. Example applications include structural alignment of non-coding RNAs, propagation of structural information from an experimentally-characterized sequence to its homologs, and inference of the ancestral structure of a set of diverged RNAs. We implemented the above algorithms for a simple model of pairwise RNA structural evolution; in particular, the algorithms for maximum likelihood (ML) alignment of three known RNA structures and a known phylogeny and inference of the common ancestral structure. We compared this ML algorithm to a variety of related, but simpler, techniques, including ML alignment algorithms for simpler models that omitted various aspects of the full model and also a posterior-decoding alignment algorithm for one of the simpler models. In our tests, incorporation of basepair structure was the most important factor for accurate alignment inference; appropriate use of posterior-decoding was next; and fine details of the model were least important. Posterior-decoding heuristics can be substantially faster than exact phylogenetic inference, so this motivates the use of sum-over-pairs heuristics where possible (and approximate sum-over-pairs). For more exact probabilistic inference, we discuss the use of transducer composition for ML (or MCMC) inference on phylogenies, including possible ways to make the core operations tractable.     

Efficiencies of fast algorithms of phylogenetic inference under the criteria of maximum parsimony, minimum evolution, and maximum likelihood when a large number of sequences are used

In phylogenetic inference by maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods, it is customary to conduct extensive heuristic searches of MP, ME, and ML trees, examining a large number of different topologies. However, these extensive searches tend to give incorrect tree topologies. Here we show by extensive computer simulation that when the number of nucleotide sequences (m) is large and the number of nucleotides used (n) is relatively small, the simple MP or ML tree search algorithms such as the stepwise addition (SA) plus nearest neighbor interchange (NNI) search and the SA plus subtree pruning regrafting (SPR) search are as efficient as the extensive search algorithms such as the SA plus tree bisection-reconnection (TBR) search in inferring the true tree. In the case of ME methods, the simple neighbor-joining (NJ) algorithm is as efficient as or more efficient than the extensive NJ+TBR search. We show that when ME methods are used, the simple p distance generally gives better results in phylogenetic inference than more complicated distance measures such as the Hasegawa-Kishino-Yano (HKY) distance, even when nucleotide substitution follows the HKY model. When ML methods are used, the simple Jukes-Cantor (JC) model of phylogenetic inference generally shows a better performance than the HKY model even if the likelihood value for the HKY model is much higher than that for the JC model. This indicates that at least in the present case, selecting of a substitution model by using the likelihood ratio test or the AIC index is not appropriate. When n is small relative to m and the extent of sequence divergence is high, the NJ method with p distance often shows a better performance than ML methods with the JC model. However, when the level of sequence divergence is low, this is not the case.     

Reconstructing phylogeny by quadratically approximated maximum likelihood

Maximum likelihood (ML) for phylogenetic inference from sequence data remains a method of choice, but has computational limitations. In particular, it cannot be applied for a global search through all potential trees when the number of taxa is large, and hence a heuristic restriction in the search space is required. In this paper, we derive a quadratic approximation, QAML, to the likelihood function whose maximum is easily determined for a given tree. The derivation depends on Hadamard conjugation, and hence is limited to the simple symmetric models of Kimura and of Jukes and Cantor. Preliminary testing has demonstrated the accuracy of QAML is close to that of ML.     

基于COI基因序列的九种真蝽属昆虫的分子系统学研究(半翅目:蝽科)

通过对真蝽属Pentatoma 9种昆虫线粒体COI基因约798bp的序列进行分子进化分析,并以同蝽科宽铗同蝽Acanthosoma labiduroides为外群,采用最大简约法、最大似然法和邻接法构建了分子系统树,来探讨真蝽属的系统发育关系.研究结果支持褐真蝽群P. semiannulata-group的划分,绿角真蝽Pentatoma viridicornuta应划归到褐真蝽群P. Semiannulata-group;红足真蝽群中的角肩真蝽P. angulata与红足真蝽P. rufipes遗传距离较小,它们是否为1个物种值得关注;真蝽属各群之间的系统发育关系以及是否可分为3个属值得进一步研究.     

Phylogenetic place of mitochondrion-lacking protozoan, Giardia lamblia, inferred from amino acid sequences of elongation factor 2

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a mitochondrion-lacking protozoan, Giardia lamblia, were amplified by polymerase chain reaction, and their primary structures were analyzed. The deduced amino acid sequence was aligned with other eukaryotic and archaebacterial EF-2's, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) and the maximum parsimony (MP) methods. The ML analyses using six different models of amino acid substitutions and the MP analysis consistently suggest that among eukaryotic species being analyzed, G. lamblia is likely to have diverged from other higher eukaryotes on the early phase of eukaryotic evolution.      

Molecular systematics and biogeography of the southern South american freshwater "crabs" Aegla (decapoda: Anomura: Aeglidae) using multiple heuristic tree search approaches

Recently new heuristic genetic algorithms such as Treefinder and MetaGA have been developed to search for optimal trees in a maximum likelihood (ML) framework. In this study we combined these methods with other standard heuristic approaches such as ML and maximum parsimony hill-climbing searches and Bayesian inference coupled with Markov chain Monte Carlo techniques under homogeneous and mixed models of evolution to conduct an extensive phylogenetic analysis of the most abundant and widely distributed southern South American freshwater"crab,"the Aegla(Anomura: Aeglidae). A total of 167 samples representing 64 Aegla species and subspecies were sequenced for one nuclear (28S rDNA) and four mitochondrial (12S and 16S rDNA, COI, and COII) genes (5352 bp total). Additionally, six other anomuran species from the genera Munida,Pachycheles, and Uroptychus(Galatheoidea), Lithodes(Paguroidea), and Lomis(Lomisoidea) and the nuclear 18S rDNA gene (1964 bp) were included in preliminary analyses for rooting the Aegla tree. Nonsignificantly different phylogenetic hypotheses resulted from all the different heuristic methods used here, although the best scored topologies found under the ML hill-climbing, Bayesian, and MetaGA approaches showed considerably better likelihood scores (Delta> 54) than those found under the MP and Treefinder approaches. Our trees provided strong support for most of the recognized Aegla species except for A. cholchol,A. jarai,A. parana,A. marginata, A. platensis, and A. franciscana, which may actually represent multiple species. Geographically, the Aegla group was divided into a basal western clade (21 species and subspecies) composed of two subclades with overlapping distributions, and a more recent central-eastern clade (43 species) composed of three subclades with fairly well-recognized distributions. This result supports the Pacific-Origin Hypothesis postulated for the group; alternative hypotheses of Atlantic or multiple origins were significantly rejected by our analyses. Finally, we combined our phylogenetic results with previous hypotheses of South American paleodrainages since the Jurassic to propose a biogeographical framework of the Aegla radiation.     

ITS2 barcode for identifying the officinal rhubarb source plants from its adulterants

Officinal rhubarb, the dried root and rhizome of Rheum officinale Baill., Rh. palmatum L., and Rh. tanguticum (Maxim. ex Regel) Maxim. ex Balf. of Polygonaceae family, is one of the most well-known and important traditional Chinese medicines. The growing herbal market has led to the introduction of adulterants, and difficulties in morphological based discrimination has also resulted in concerns over consumer safety. ITS2 as a potential barcode was employed to discriminate officinal rhubarb sampled from the entire distribution range and its adulterants. The minimum K2P interspecific distance between officinal rhubarb source plants and the adulterants were higher than the maximum K2P intraspecific distance within the officinal rhubarb source plants. When the phylogenetic trees were constructed by neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods, all Rheum species were grouped into one clade, while Polygonum and Rumex species were clustered in another clade. In the Rheum clade, all the officinal rhubarb source plants constituted a distinct subclade, while other Rheum species formed other subclades. Our results suggested that the ITS2 region is a more suitable and accurate DNA barcode for distinguishing officinal rhubarb source plants from their adulterants.