Long branch attraction, taxon sampling, and the earliest angiosperms: Amborella or monocots?
© Stefanović et al; licensee BioMed Central Ltd. 2004
Received: 06 August 2004
Accepted: 28 September 2004
Published: 28 September 2004
Numerous studies, using in aggregate some 28 genes, have achieved a consensus in recognizing three groups of plants, including Amborella, as comprising the basal-most grade of all other angiosperms. A major exception is the recent study by Goremykin et al. (2003; Mol. Biol. Evol. 20:1499–1505), whose analyses of 61 genes from 13 sequenced chloroplast genomes of land plants nearly always found 100% support for monocots as the deepest angiosperms relative to Amborella, Calycanthus, and eudicots. We hypothesized that this conflict reflects a misrooting of angiosperms resulting from inadequate taxon sampling, inappropriate phylogenetic methodology, and rapid evolution in the grass lineage used to represent monocots.
We used two main approaches to test this hypothesis. First, we sequenced a large number of chloroplast genes from the monocot Acorus and added these plus previously sequenced Acorus genes to the Goremykin et al. (2003) dataset in order to explore the effects of altered monocot sampling under the same analytical conditions used in their study. With Acorus alone representing monocots, strongly supported Amborella-sister trees were obtained in all maximum likelihood and parsimony analyses, and in some distance-based analyses. Trees with both Acorus and grasses gave either a well-supported Amborella-sister topology or else a highly unlikely topology with 100% support for grasses-sister and paraphyly of monocots (i.e., Acorus sister to "dicots" rather than to grasses). Second, we reanalyzed the Goremykin et al. (2003) dataset focusing on methods designed to account for rate heterogeneity. These analyses supported an Amborella-sister hypothesis, with bootstrap support values often conflicting strongly with cognate analyses performed without allowing for rate heterogeneity. In addition, we carried out a limited set of analyses that included the chloroplast genome of Nymphaea, whose position as a basal angiosperm was also, and very recently, challenged.
These analyses show that Amborella (or Amborella plus Nymphaea), but not monocots, is the sister group of all other angiosperms among this limited set of taxa and that the grasses-sister topology is a long-branch-attraction artifact leading to incorrect rooting of angiosperms. These results highlight the danger of having lots of characters but too few and, especially, molecularly divergent taxa, a situation long recognized as potentially producing strongly misleading molecular trees. They also emphasize the importance in phylogenetic analysis of using appropriate evolutionary models.
Comparison of recent studiesa that identify the sister lineages of angiosperms.
No. of genes (genomesb)
No. of angiosperms
No. of nucleotides
Amborella sister to the rest of angiospermsc
Basal vs. core angiospermsc
Monophyly of monocotsc
5 (c, m, n)
5 (c, m, n)
3 (c, n)
6 (c, m, n)
In sharp contrast stands the study of Goremykin et al. , in which the Amborella chloroplast genome was sequenced and in which 61 protein genes shared among 13 land plants (including 10 angiosperms) were analyzed. In 31 of 33 phylogenetic analyses this study found that "Amborella is not the basal angiosperm and not even the deepest branching among dicots" ( Abstract). Instead, these results indicate, with 100% BS in most analyses, that the first split within angiosperm evolution occurred between monocots and dicots. Goremykin et al.  imply that the earlier studies are in error with respect to the placement of Amborella because these "studies were based on a limited number of characters derived from only a few genes" and used "unmasked sequences of chloroplast genes [i.e., with all three codon positions included] with high substitution rates at their synonymous sites" (p. 1503).
Thus, we are faced with a major paradox. On the one hand, many different studies, employing in aggregate 28 different genes (19 chloroplast, five mitochondrial, and four nuclear; Table 1), consistently and strongly place the branch leading to Amborella deeper in angiosperm evolution than the branch leading to the monocots, whereas a study that employed twice as many genes found the opposite result, also with strong support. It is critical to resolve this paradox, for the groups and issues involved are such important ones in angiosperm phylogeny.
One notable difference between the two sets of studies concerns taxon sampling, which can be critical in phylogenetic analysis [20–24]. Even though sampling strategies in the Amborella-deep studies listed in Table 1 varied substantially, ranging from 18 to 553 species of angiosperms and from 2,208 to 14,244 nucleotides (NT) of aligned data, a commonality was their relatively broad taxon sampling. Most of these studies attempted to represent the diversity of living angiosperms by including critical species identified by prior morphological [25–28] and single-gene molecular analyses [29–31]. Even the listed study with the fewest taxa  was based on exemplar species, compiled by the Green Plant Phylogeny Research Coordination Group and chosen to represent most of the major putatively basal lineages suggested by a large body of previously accumulated results. In contrast, the Goremykin et al.  study included only 10 angiosperms. Five of these belong to a single derived group (eudicots) and three are grasses (the only monocots sampled), leaving Amborella and Calycanthus (the only sampled member of the other three lineages of core angiosperms) as the other two angiosperms sampled (Fig. 1). It is known that grasses have accelerated substitution rates in all three genomes [9, 32–35], especially the chloroplast genome, making them a poor representative for such a large and diverse group as monocots.
Relevant here is that the grasses-sister topology obtained by Goremykin et al.  (see their Fig. 3, which also corresponds to our Fig. 3A) shows one long branch, leading to grasses, connecting to another long branch, separating angiosperms from the outgroups. When the outgroups are removed and the Goremykin et al.  tree is taken as an unrooted network, it becomes apparent that there is no difference between their ingroup topology and those of studies that obtained the Amborella-sister rooting. In other words, given the taxonomic sampling of Goremykin et al. , their grasses-sister topology differs from the canonical Amborella-sister topology only with respect to where the outgroup branch attaches , either to grasses or to Amborella (see Discussion and Fig. 8 for an elaboration of this point).
These observations led us to suspect that the grasses-sister topology is an artifact stemming from long branch attraction (LBA), a phenomenon known [37–39] to give strongly supported, but spurious results under precisely the set of conditions operative in the Goremykin et al.  study. These are 1) inadequate taxon sampling, 2) large amounts of data per taxon, 3) two known long branches (the grass branch and the outgroup branch) separated by short internodes, and 4) phylogenetic analyses that do not account for rate heterogeneity.
The current study was undertaken to test whether the grasses-sister topology is indeed an LBA artifact. We hypothesize that, by analyzing the Goremykin et al.  dataset with a focus on rate heterogeneity and taxon sampling of monocots, the Amborella-sister topology will be recovered instead. In addition, we carried out a similar, but much more limited set of analyses in response to a follow-up paper by Goremykin et al.  that appeared while this manuscript was in the final stages of preparation and which similarly challenged the position of Nymphaea as a basal angiosperm.
Addition of Acorus
We gathered new sequence data for an additional monocot representative, Acorus, and added it to the 13 taxa, 61 gene first- and second-position alignment matrix of Goremykin et al.  to give a 14 taxa, 61 gene first- and second-position alignment matrix. Acorus was chosen for two reasons. First, it is well supported as the sister to all other monocots [41–43]. Thus, Acorus plus grasses represent monocot diversity about as well as any two groups of monocots. Second, unlike grasses, its chloroplast genome does not appear to have evolved at unusually high rates [9, 44]. The Acorus dataset consisted of 40 protein gene sequences, 22 newly determined in this study and 18 from preexisting databases. This corresponds to 65.6% (40/61) of the genes and 71.4% (32,072/44,937) of the nucleotide characters analyzed by Goremykin et al. .
A number of initial analyses were conducted in parallel on the "full" Acorus matrix, containing data for all 61 genes and including gaps where data for Acorus were not available, and a "truncated" matrix, containing only those 40 genes where Acorus sequences were available. Inspection of the resulting trees revealed no topological incongruences and no significant change in bootstrap support (BS) between the full and truncated analyses [see Additional files 1 and 2]. The results presented hereafter for Acorus are based on the full matrix dataset. This allows us to include all available relevant data, allowing the fullest and most direct comparisons to the Goremykin et al.  analyses.
Inclusion of both grasses and Acorus produced two very different topologies, depending on the method used. On the one hand, standard MP, with both nucleotides (Fig. 2C) and amino acids (Fig. 2F), gives a grasses-sister topology in which monocots are paraphyletic with 100% BS (i.e., there is 100% support for Acorus as the sister to "dicots" to the exclusion of grasses). On the other hand, equal-rates ML (Fig. 2I) and transversion parsimony (Fig. 2L) give an Amborella-sister topology, with moderate (79%) to strong (98%) support, in which monocots are monophyletic with equivalent support.
To make the results more directly comparable to the Goremykin et al. study  and to investigate the performance of various distance-based models, we tested many different neighbor joining (NJ) models. We did this also because, of all MP, ML and NJ methods initially investigated, the only approaches that failed to give the Amborella-sister topology when Acorus was substituted for grasses were the NJ methods without a ML model. When the PAUP*  distance is set to any of 12 settings (Mean, P, JC , F81 , TajNei , K2P , F84 , HKY85 , K3P , TamNei , GTR [54, 55] or LogDet [56, 57]), Amborella, Calycanthus, and Acorus form a monophyletic group with 100% BS. Importantly, however, this same grouping is obtained, with all 12 distance settings, even when grasses are included, such that, as in equal-weighted parsimony analyses (Figs. 2C and 2F), grasses are sister to all other angiosperms and monocots are not monophyletic (Fig. 3C and analyses not shown).
Finally, it should be noted that ML and NJ methods using models (see next section) that give Amborella-sister when only grasses represent monocots, continue to do so, but with higher BS, when Acorus is added, either with or without grasses [see Additional files 1 and 2].
Site-to-site rate heterogeneity
If the lineage leading to Amborella is sister to the rest of angiosperms, as the analyses with Acorus strongly indicate, why do so many of the Goremykin et al.  analyses support the grasses-sister topology? We explored this question by conducting analyses using a broad range of models and methods as applied to their data matrix (i.e., with only grasses representing monocots).
The 56 MODELTEST models and the grasses- or Amborella-sister topology that received the higher likelihood.
These results held when the parameters estimated on one topology (either Amborella- or grasses- sister) were used to calculate the likelihood of the other topology (the topology used had only a minor effect on the values of the parameter estimates). For both topologies, the model chosen by MODELTEST using either the hierarchical likelihood ratio tests or the Akaike information criterion was the 5-substitution-type-transversion (TVM) + I + G model, where the probability of going between A and G is equal to that of C and T. With this model, using parameter estimates from either topology, a heuristic search found the Amborella-sister topology with 98% BS, and the SH-test  showed the grasses-sister topology to be significantly worse at the 5% level (p = 0.04).
We performed a similar analysis with the proportion of invariant sites (Plnvar option in PAUP). Using the estimated PInvar = 0.58 without gamma-distributed rate categories, we obtained the Amborella-sister topology (Fig. 4C) with 97% BS. As with α, the Amborella-sister topology was stable over a wide range of PInvar [0.09 <= PInvar <= 0.995 (Fig. 5B)]. The BS and the SH-test p-value for Amborella-sister improve as PInvar increases (Fig. 5B). The SH-test for Amborella-sister is significant at the 5% level using the estimated value of PInvar and remains significant as PInvar increases.
The BS for a sister-group relationship of Amborella and Calycanthus is identical (within the variance expected for BS values) with that for grasses-sister across the entire range of both α and PInvar values, while both of these BS values always equal 100 minus the BS value for Amborella-sister (Figs. 5A and 5B). This is exactly as expected (see Discussion) if the only difference between the grasses-sister/Amborella+Calycanthus topology and the Amborella-sister topology is where the outgroup branch roots within angiosperms. Put another way, almost all of the BS replicates were one of these two topologies.
There are 20,071 (out of 30,017; 66.9%) constant sites in theGoremykin et al.  matrix. When these constant sites are removed, the highest HKY85 ML tree (using equal rates) places Amborella-sister with 98% BS and with p = 0.03 for the SH-test relative to grasses-sister [see Additional file 4, Fig. A]. Furthermore, NJ analysis with the equal-rate ML model also obtains Amborella-sister (with 100% BS) when constant sites are removed [see Additional file 4, Fig. B]. This is another way of allowing the rates to increase since the rates of the sites that are changing are not constrained by the constant sites. This allows the ML model to work with a more homogenous set of rates and reduces the need for using rate categories. Removing these constant sites allows the ML model to simulate the actual evolutionary process of sites that are changing more accurately than when imposing a proportion of invariant sites because there is no invariant site weighting of the sites that are changing. As a consequence of the faster rate with constant sites excluded, the branch lengths of the resulting trees are ~2.6 times longer than when constant sites are included.
We further explored the NJ method using ML models of evolution to compute distances and with constant sites included. We were able to precisely reproduce the grasses-sister result (Fig. 3 from Goremykin et al. ) with NJ and the K2P model(Fig. 3A). NJ using a distance matrix calculated based on ML and using parameters estimated with the HKY85 model with equal rates alsogives grasses-sister with 100% BS. However, distances calculated using the ML HKY85 model and estimated proportion of invariant sites puts Amborella- sister with low BS of 58% [see Additional file 5], while distances derived from the ML HKY85 model with four gamma-distributed rate categories estimated gives Amborella-sister with stronger support (89%; Fig. 3B).
Third codon positions
In order to most directly assess the Goremykin et al.  analyses, which used only first and second codon position, the above analyses were restricted to first and second codon positions. In addition, however, most of the above analyses were also carried out with a dataset that includes all three codon positions. The resulting trees provide similar if not higher support for Amborella-sister than those obtained with just first and second positions. For example, using all three positions, the gamma rates ML tree analogous to Fig. 4B gives 100% BS for Amborella-sister, and the ML distance based NJ tree analogous to Fig. 3B gives 99% BS for Amborella-sister (trees available upon request). The most noteworthy shift towards stronger support involves ML analysis with equal rates, where inclusion of third positions changes the topology, from grasses-sister (with 61% BS; Fig. 4A) to Amborella-sister (and with 100% support; Fig. 4D). We also conducted a few analyses of third positions only (again using the set of taxa analyzed by Goremykin et al. ). These too recovered Amborella-sister, with 100% BS using ML with either equal rates or gamma-distributed rates [see Additional file 6].
Individual gene analyses
The single gene trees also illustrate the effect of taxon sampling. When Acorus is added and all three positions are used in ML analyses with four rate categories, none of the gene trees find monocots sister, whereas exactly half of the 40 genes put Amborella sister [see Additional file 8, top figure]. When the third position is excluded, 12 genes put Amborella sister and BS levels drop significantly, while still no genes put monocots sister [see Additional file 8, bottom figure]. Very similar results are obtained when the grasses are removed [see Additional file 9]. In contrast to the parsimony results without Acorus (where grasses-sister is the favored topology; Fig. 6B), when Acorus is added and parsimony is used (with all three positions), only two genes put monocots sister (and both with low, 13 and 34%, BS), whereas 11 of 40 genes put Amborella sister [see Additional file 10, top figure]. With Acorus added and grasses removed, 21 genes place Amborella sister and 1 places Acorus sister [see Additional file 10, bottom figure].
Addition of Nymphaea
While this manuscript was in the final stages of preparation, the chloroplast genome sequence of Nymphaea alba became available (released to EMBL database on July 13, 2004). This sequence was generated as part of a very recent study, also by Goremykin et al. , in which it was added, as the only new sequence, to the same data matrix as analyzed in their earlier study  and subjected to a similar set of phylogenetic analyses. Under these conditions, the grasses-sister topology was again recovered (and with 100% support) in nearly all analyses, with Nymphaea and Amborella recovered as sister taxa (also with 100% support). In their abstract, Goremykin et al.  present these findings as supporting their prior conclusion  that monocots are sister to the rest of angiosperms. However, their Discussion presents a more nuanced treatment than before, concluding that "we may be some ways from being confident of identifying the most basal angiosperms. Clearly the sequencing of genomes for more closely related outgroups and putatively basal angiosperms will be important for overcoming potential problems of model misspecification and long-branch attraction."
The grasses-sister topology is an LBA artifact
That long branch attraction can be a serious problem in phylogenetic inference has long been known to the systematics community, ever since this phenomenon was first explored by Felsenstein . Felsenstein described conditions of unequal evolutionary rates under which phylogenetic inference will result not only in an incorrect topology, but will converge asymptotically to the wrong phylogeny with increasing confidence as more data are added, ultimately producing 100% support for the wrong tree (hence, be positively misleading). Hendy and Penny  showed that this phenomenon can occur for parsimony even under equal evolutionary rates if taxa are insufficiently sampled along a branch, while Lockhart et al.  showed that an ML equal-rates model can incorrectly join long branches when there is rate heterogeneity across sites. In the case of DNA sequence data, due to the limited number of character states, taxa with the greatest sequence divergence are expected to be "attracted" to each other by chance alone if long and short branches are sufficiently different in length. With large amounts of data, this can result in spurious, yet strongly supported, relationships.
The Amborella-sister topology is in agreement with the many diverse phylogenetic studies summarized in Table 1 and in Background, except for that of Goremykin et al. . With Acorus included (Figs. 2I and 2L), it also shows monocots as monophyletic, consistent with a large body of evidence [7, 35, 41–43, 63], and depicts faster chloroplast DNA evolution on the monocot lineage leading to grasses than in the Acorus lineage, also consistent with a substantial body of evidence (e.g. [9, 44]). Conversely, the grasses-sister topology (Figs. 2C and 2F) is consistent only with the Goremykin et al.  results, fails to recover monophyly of monocots [has them either paraphyletic (Figs. 2C and 2F) or even polyphyletic (Fig. 3C), and always with 100% support], and fails to portray the known rapid evolution of chloroplast DNA in the lineages leading to grasses. All this leads us to conclude that the grasses-sister topology is almost certainly an artifact, most likely due to LBA between the long branches leading to grasses and to angiosperms.
Second, we reanalyzed the same dataset used by Goremykin et al.  and found that methods that account for rate heterogeneity across sites [61, 64–67] put Amborella sister, usually with high BS (Figs. 2J, 3B, 4B, 4C, and 5; also see most Additional files). This was true for all 14 MODELTEST substitution models (Table 2) except for the simplest, JC model. When rates vary between sites, as with the chloroplast dataset under consideration, it is usually appropriate to model the evolutionary process to reflect this. The evolutionary models explored here point to LBA as the cause of the controversial grasses-sister topology and demonstrate that even with conservative corrections for rate heterogeneity, Amborella moves to the sister position within angiosperms (e.g., Figs. 5A and 5B).
In summary, our two principal approaches for reassessing the results and analyses of Goremykin et al.  lead to what we regard as compelling evidence for two major conclusions. First, Amborella, not grasses, is the sister angiosperm among this set of taxa. Second, any tendency for angiosperms to root on grasses is an LBA artifact stemming from the confluence of limited taxon sampling, rapid evolution in grasses, a long branch between the outgroups and angiosperms, and rate heterogeneity across sites. Furthermore, we point out that while our manuscript was nearly finished, two independent papers appeared [68, 69] that also challenged Goremykin et al.  and reached similar conclusions to our study. Both studies are complementary to ours, because instead of taking the Goremykin et al.  61-gene chloroplast dataset as the starting point, as we did, they used a 3-gene dataset (the same two chloroplast genes and one nuclear gene) plus the Goremykin et al.  set of taxa as the starting point for a variety of taxon-sampling experiments. In addition, an important forthcoming study  which added five new chloroplast genome sequences to the dataset of Goremykin et al. , found "strong support" for the Amborella-sister topology. That four entirely independent studies, using a variety of taxon sets, character sets, and analytical approaches, all lead to such similar results and conclusions makes it all the more likely that the grasses-sister topology is indeed a phylogenetic artifact.
Is Amborella or Amborella+Nymphaeaceae sister to the rest of angiosperms?
Although our results reject grasses/monocots as the sister to all other angiosperms, support for Amborella as the first branch of angiosperm evolution must necessarily be qualified given the very limited sampling of whole chloroplast genomes (besides Amborella, only monocots, Calycanthus, and eudicots; see Fig. 1). There is still uncertainty as to the exact placement of Amborella relative to the other two deepest lineages of angiosperms, especially Nymphaeaceae [8, 9], although the overall weight of published evidence currently favors Amborella as the deepest angiosperm (see [10, 12] and references in Table 1). This uncertainty is heightened by our limited analyses that included Nymphaea and used methods that account for rate heterogeneity. These analyses never recovered an Amborella-sister topology. Instead, they most commonly found a sister clade comprising both Amborella and Nymphaea (Figs. 7B,7C,7D), or even found Nymphaea alone to be the sister-most angiosperm (Fig. 7A). Likewise, in the one analysis reported by Goremykin et al.  in which Amborella and Nymphaea were found sister to the other angiosperms these two taxa clustered as sisters rather than forming a basal grade.
Clearly, then, the question of which group is sister to the rest of extant angiosperms should be regarded as unsettled and in need of further exploration, using much more data (such as whole chloroplast genomes from a large number of diverse angiosperms, as well as more mitochondrial and/or nuclear data) and better analytical methodologies as they become available. At the same time, we must face up to two serious limitations arising from extinction. First, Amborella trichopoda is the only known species in the entire Amborellaceae/Amborellales, i.e., it is the only taxon available whose DNA can be used to represent a lineage of ca. 150 million years in age arising at or near the base of angiosperms. Second, the stem branch leading to angiosperms is long in length and years [9, 62] (also approaching 150 million years) and thus represents a long-branch attractor, with the potential to spuriously attract other branches besides that leading to grasses. LBA between outgroup and ingroups is particularly insidious, because, as illustrated in Fig. 2 (C and F vs. I and L), it tends to mask the long nature of the ingroup branches. Amborella does not show any evidence of having a long branch in published analyses with more extensive taxon sampling. It is nonetheless difficult to rule out (but see ) the possibility that Amborella may be only near-sister among angiosperms (e.g., part of a Nymphaeaceae/Amborella clade that itself is the earliest branch of angiosperms; as suggested by Barkman et al.  and some of our analyses), with its generally sister position representing only a slight topological distortion (nearest neighbor interchange) caused by attraction to the long outgroup branch. For that matter, we point out (also see ) that the long branch leading to angiosperms also makes it difficult to rule out the possibility that the monophyletic-gymnosperm topologies recovered by multigene analyses (e.g., [35, 72–74]) might result from LBA between angiosperms and the outgroup branch leading to seed plants.
Many of our analyses, including all but one of the 61-gene concatenate analyses shown, included only first and second codon positions. This is because Goremykin et al.  chose to exclude third codon positions from their analyses, and because we wanted to make the most direct comparisons possible to their analyses. Third positions were excluded because most of the 61 chloroplast genes were claimed to be "very divergent" at synonymous sites (Ks for most genes between Pinus and angiosperms was between 0.50 and 1.50 substitutions/site), which they felt could lead to "misleading" phylogenetic results. However, because our analyses with all three positions or only third positions gave such similar results to those using only first and second positions, we believe that for this particular dataset third positions are not contributing "excessive" homoplasy and leading to spurious affiliations. This conclusion is consistent with a considerable body of literature dealing with the phylogenetic utility of third positions in organellar genes [75–80], while simulations have shown that "saturated" data can be very reliable, provided that taxon sampling is sufficiently high [21, 24]. Caution is nonetheless well advised in situations involving relatively sparse taxon sampling (some of which may be unavoidable, i.e., where extinction has been significant) and/or greater divergences than in this study. For example, chloroplast third positions are problematic in analyses across all of algal/plant evolution (e.g., ), and even appear to be problematic at the relatively shallow level of seed plant phylogeny [35, 73, 82].
Our findings, and those of others [68–70, 83], highlight the potential danger of phylogenetic analyses that employ lots of genes, but too few and/or the wrong taxa. Adequate taxon sampling is in a sense even more important here than with single or few-gene trees, because of the potential for even subtle systematic bias in a particular lineage's evolution to generate strongly supported misleading trees. Equally, if not more importantly, our results emphasize the crucial importance of using phylogenetic methods that best model the underlying molecular evolutionary processes, especially by accounting for site-to-site rate variation.
Sequencing chloroplast genes from Acorus
We used long PCR to generate full-length or partial sequences from Acorus gramineus Soland. (a voucher specimen is deposited at the IND herbarium) for 22 of the 61 chloroplast genes analyzed by Goremykin et al. . Long PCRs were conducted using the AccuTaq™ LA DNA Polymerase (Sigma, Atlanta, GA, USA), following instructions provided by the manufacturer. Initially, sets of primers designed by Graham and Olmstead , which cover a large portion of the chloroplast genome (psbC-D and psbE-J operons; from rpl2 to 3'-rps12 gene), as well as the primers described in [84–87] for the rbcL, atpB, trnL-F, and trnE-D region, respectively, were used for amplifications and/or sequencing. For the most part, however, based on the initial sequences, a number of sequencing primers were designed and used for chromosome walking with long PCR products. Primer sequences are available upon request from SS. PCR products were separated by electrophoresis using 0.8% agarose gels, visualized with ethidium-bromide, and cleaned using Qiagen columns (Valencia, CA, USA). Cleaned products were then directly sequenced using the BigDye™ Terminator cycle sequencing kit (PE Applied Biosystem, Foster City, CA, USA) on an ABI 3100 DNA automated sequencer (PE Applied Biosystem, Foster City, CA, USA). Sequence data were edited and assembled using Sequencher™ 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). The Acorus sequences for these 22 chloroplast genes (atpA, atpE, clpP, cemA, lhbA, 3'-petB, petD, petG, petL, psaB, psaI, rpl20, rpoA, rpoB, rpoC1, rpoC2, rps2, rps14, rps18, rps19, ycf3, ycf4) are deposited in GenBank (accession numbers AY757810-AY757831). These were combined for phylogenetic analyses with full-length or partial Acorus sequences already available in GenBank for 18 other chloroplast genes [AF123843 (psbB, psbT, psbN, psbH), AF123771 (rps7, 3'-rps12), AF123828 (psbE, psbF, psbL), AF123813 (psbD, psbC), AF123785 (rpl2), D28866 (rbcL), X84107 (rps4), U96631 (psbA), AB040155 (matK), AF197616 (atpB), and AJ344261 (psaA)]. The 40 Acorus genes used here come from two closely related species – A. calamus (14 genes) and A. gramineus (26 genes) – and correspond to 65.6% (40/61) of the genes and 71.4% (32,072/44,937) of the nucleotide characters analyzed by Goremykin et al. .
For all first and second codon position analyses, the data matrix provided by V. Goremykin was used without modification. For analyses that included Acorus, the Acorus genes were individually aligned with the individually extracted gene alignments from the Goremykin et al.  dataset using CLUSTALW , and the resulting gene alignments were concatenated to regenerate a matrix identical to the original except for the extra row containing Acorus. Using the same procedure, Acorus was also added to the amino acid matrix provided by V. Goremykin. The relevant 61 chloroplast genes of Nymphaea  were likewise added to both alignments.
We also constructed a new matrix consisting of all three codon positions by extracting genes from 13 sequenced chloroplast genomes of land plants (GenBank numbers: AP002983, AP000423, AJ271079, Z00044, AJ400848, AJ506156, AJ428413, X86563, AB042240, X15901, D17510, AP004638, X04465), aligning them, and hand editing apparent mistakes. The first and second position version of this matrix was nearly identical to the Goremykin et al.  matrix, except for a few minor differences (the overall length was slightly shorter due to removal of terminal extensions that either were created by single taxon indels or where multiple extending genes were nonhomologous). All phylogenetic trees resulting from this first and second position matrix and the Goremykin et al.  matrix were identical in topology and nearly identical in BS values. All alignments used in this study are available in Nexus format upon request of DWR.
Phylogenetic analyses were performed in PAUP* 4.0b10 . Unless specified, all nucleotide-based trees were built using only first- and second-codon positions. For ML analyses, parameters were initially estimated using an equal-weighted parsimony tree. A ML tree was then built, and parameters were re-estimated using this tree if it differed from the parsimony tree. This iteration was continued until the last two topologies converged (the final ML topology was almost always equal to the one in which the ML parameters were estimated from the parsimony topology). For all ML analyses we also calculated a NJ tree using distances calculated from the ML model being tested. For DNA and protein parsimony the default PAUP* 4.0b10  step matrices were used.
Bootstrap support  was estimated with 100 replicates using parameters estimated from the final topology. Thus the methodology cited for a particular tree refers to the model used for the bootstrap replicates. For parsimony and ML searches the heuristic algorithm was used with simple and as-is stepwise addition, respectively; tree bisection-reconnection swapping; and no limit on the number of trees saved in memory. Unless specified, the default PAUP* settings were used in all analyses. An automated script (available upon request from DWR) was used to run the analyses. Detailed log files and trees of each analysis were saved and are available upon request from DWR. Most analyses were performed on two 3 GHz Linux machines. Treetool  was used for viewing and printing trees.
The Shimodaira-Hasegawa (SH) test  was performed using the "lscores" command of PAUP* with the options SHTest = RELL and BootReps = 10000. ML parameters being tested were estimated on each topology to calculate its own log likelihood except where otherwise specified.
long branch attraction
proportion of invariant sites
We thank Ulfar Bergthorsson, Eric Knox, and Richard Olmstead for useful comments on earlier versions of the manuscript, and Vadim Goremykin for providing the 61-gene data matrices that were the starting point for this study. Funding for this work came from National Institutes of Health grant GM-35087 to J.D.P.
- Mathews S, Donoghue MJ: The root of angiosperm phylogeny inferred from duplicate phytochrome genes. Science. 1999, 286: 947-950. 10.1126/science.286.5441.947.View ArticlePubMedGoogle Scholar
- Mathews S, Donoghue MJ: Basal angiosperm phylogeny inferred from duplicate phytochromes A and C. Int J Plant Sci. 2000, 161: S41-S55. 10.1086/317582.View ArticleGoogle Scholar
- Parkinson CL, Adams KL, Palmer JD: Multigene analyses identify the three earliest lineages of extant flowering plants. Curr Biol. 1999, 9: 1485-1488. 10.1016/S0960-9822(00)80119-0.View ArticlePubMedGoogle Scholar
- Qiu Y-L JLee, Bernasconi-Quadroni F, Soltis DE, Soltis PS, Zanis MJ, Zimmer EA, Chen Z, Savolainen V, Chase MW: The earliest angiosperms: evidence from mitochondrial, plastid and nuclear genomes. Nature. 1999, 402: 404-407. 10.1038/46536.View ArticlePubMedGoogle Scholar
- Qiu Y-L JLee, Bernasconi-Quadroni F, Soltis DE, Soltis PS, Zanis MJ, Zimmer EA, Chen Z, Savolainen V, Chase MW: Phylogeny of basal angiosperms: analyses of five genes from three genomes. Int J Plant Sci. 2000, 161: S3-S27. 10.1086/317584.View ArticleGoogle Scholar
- Soltis PS, Soltis DE, Chase MW: Angiosperm phylogeny inferred from multiple genes as a research tool for comparative biology. Nature. 1999, 402: 402-404. 10.1038/46528.View ArticlePubMedGoogle Scholar
- Soltis PS, Soltis DE, Zanis MJ, Kim S: Basal lineages of angiosperms: relationships and implications for floral evolution. Am J Bot. 2000, 161: S97-S107. 10.1086/317581.Google Scholar
- Barkman TJ, Chenery G, McNeal JR, Lyons-Weiler J, Ellisens WJ, Moore G, Wolfe AD, dePamphilis CW: Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny. Proc Natl Acad Sci USA. 2000, 97 (24): 13166-13171. 10.1073/pnas.220427497.PubMed CentralView ArticlePubMedGoogle Scholar
- Graham SW, Olmstead RG: Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms. Am J Bot. 2000, 87: 1712-1730.View ArticlePubMedGoogle Scholar
- Qiu YL, Lee J, Whitlock BA, Bernasconi-Quadroni F, Dombrovska O: Was the ANITA rooting of the angiosperm phylogeny affected by long-branch attraction?. Mol Biol Evol. 2001, 18 (9): 1745-1753.View ArticlePubMedGoogle Scholar
- Borsch T, Hilu KW, Quandt D, Wilde V, Neinhuis C, Barthlott W: Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms. J Evol Biol. 2003, 16 (4): 558-576. 10.1046/j.1420-9101.2003.00577.x.View ArticlePubMedGoogle Scholar
- Zanis MJ, Soltis DE, Soltis PS, Mathews S, Donoghue MJ: The root of the angiosperms revisited. Proc Natl Acad Sci USA. 2002, 99: 6848-6853. 10.1073/pnas.092136399.PubMed CentralView ArticlePubMedGoogle Scholar
- Zanis MJ, Soltis PS, Qiu YL, Zimmer E, Soltis DE: Phylogenetic analyses and perianth evolution in basal angiosperms. Ann Mo Bot Gard. 2003, 90: 129-150.View ArticleGoogle Scholar
- Hilu KW, Borsch T, Muller K, Soltis DE, Soltis PS, Savolainen V, Chase MW, Powell MP, Alice LA, Evans R, Sauquet H, Neinhuis C, Slotta TAB, Rohwer JG, Campbell CS, Chatrou LW: Angiosperm phylogeny based on matK sequence information. Am J Bot. 2003, 90 (12): 1758-1776.View ArticlePubMedGoogle Scholar
- Nickerson J, Drouin G: The sequence of the largest subunit of RNA polymerase II is a useful marker for inferring seed plant phylogeny. Mol Phylogenet Evol. 2004, 31: 403-415. 10.1016/j.ympev.2003.08.013.View ArticlePubMedGoogle Scholar
- Stellari GM, Jaramillo MA, Kramer EM: Evolution of the APETALA3 and PISTILLATA lineages of MADS-box-containing genes in the basal angiosperms. Mol Biol Evol. 2004, 21 (3): 506-519. 10.1093/molbev/msh044.View ArticlePubMedGoogle Scholar
- Aoki S, Uehara K, Imafuku M, Hasebe M, Ito M: Phylogeny and divergence of basal angiosperms inferred from APETALA3- and PISTILLATA-like MADS-box genes. J Plant Res. 2004, 117 (3): 229-244. 10.1007/s10265-004-0153-7.View ArticlePubMedGoogle Scholar
- Judd Walter S., Campbell CS, Kellogg EA, Stevens PF, Donoghue MJ: Plant systematics : a phylogenetic approach. 2002, Sunderland, Mass., Sinauer Associates, xvi, 576-2ndGoogle Scholar
- Goremykin VV, Hirsch-Ernst KI, Wölfl S, Hellwig FH: Analysis of the Amborella trichopoda chloroplast genome sequence suggests that Amborella is not a basal angiosperm. Mol Biol Evol. 2003, 20: 1499-1505. 10.1093/molbev/msg159.View ArticlePubMedGoogle Scholar
- Hillis DM: Inferring complex phylogenies. Nature. 1996, 383: 130-131. 10.1038/383130a0.View ArticlePubMedGoogle Scholar
- Hillis DM: Taxonomic sampling, phylogenetic accuracy, and investigator bias. Syst Biol. 1998, 47: 3-8. 10.1080/106351598260987.View ArticlePubMedGoogle Scholar
- Graybeal A: Is it better to add taxa or characters to a difficult phylogenetic problem?. Syst Biol. 1998, 47: 9-17. 10.1080/106351598260996.View ArticlePubMedGoogle Scholar
- Pollock DD, Zwickl DJ, McGuire JA, Hillis DM: Increased taxon sampling is advantageous for phylogenetic inference. Syst Biol. 2002, 51: 664-671. 10.1080/10635150290102357.PubMed CentralView ArticlePubMedGoogle Scholar
- Zwickl DJ, Hillis DM: Increased taxon sampling greatly reduces phylogenetic error. Syst Biol. 2002, 51: 588-598. 10.1080/10635150290102339.View ArticlePubMedGoogle Scholar
- Dahlgren R, Bremer K: Major clades of angiosperms. Cladistics. 1985, 1: 349-368.View ArticleGoogle Scholar
- Doyle JA, Donoghue MJ: Seed plant phylogeny and the origin of angiosperms: an experimental cladistic approach. Bot Rev. 1986, 52: 321-431.View ArticleGoogle Scholar
- Loconte H, Stevenson DW: Cladistics of the Spermatophyta. Brittonia. 1990, 42: 197-211.View ArticleGoogle Scholar
- Nixon KC, Crepet WL, Stevenson D, Friis EM: A reevaluation of seed plant phylogeny. Ann Missouri Bot Garden. 1994, 81: 484-533.View ArticleGoogle Scholar
- Chase MW, Soltis DE, Olmstead RG, Morgan D, Les DH, Mishler BD, Duvall MR, Price RA, Hills HG, Qiu YL, Kron KA, Rettig JH, Conti E, Palmer JD, Manhart JR, Sytsma KJ, Michaels HJ, Kress WJ, Karol KG, Clark WD, Hedren M, Gaut BS, Jansen RK, Kim KJ, Wimpee CF, Smith JF, Furnier GR, Strauss SH, Xiang QY, Plunkett GM, Soltis PS, Swensen SM, Williams SE, Gadek PA, Quinn CJ, Eguiarte LE, Golenberg E, Learn GH, Graham SW, Barrett SCH, Dayanandan S, Albert VA: Phylogenetics of seed plants - an analysis of nucleotide-sequences from the plastid gene rbcL. Ann Mo Bot Gard. 1993, 80 (3): 528-580.View ArticleGoogle Scholar
- Soltis DE, Soltis PS, Nickrent DL, Johnson LA, Hahn WJ, Hoot SB, Sweere JA, Kuzoff RK, Kron KA, Chase MW, Swensen SM, Zimmer EA, Chaw SM, Gillespie LJ, Kress WJ, Sytsma KJ: Angiosperm phylogeny inferred from 18S ribosomal DNA sequences. Ann Mo Bot Gard. 1997, 84 (1): 1-49.View ArticleGoogle Scholar
- Savolainen V, Chase MW, Hoot SB, Morton CM, Soltis DE, Bayer C, Fay MF, deBruijn AY, Sullivan S, Qiu YL: Phylogenetics of flowering plants based upon a combined analysis of plastid atpB and rbcL gene sequences. Syst Biol. 2000, 49: 306-362. 10.1080/10635159950173861.View ArticlePubMedGoogle Scholar
- Gaut BS, Muse SV, Clark WD, Clegg MT: Relative rates of nucleotide substitution at the rbcL locus of monocotyledonous plants. J Mol Evol. 1992, 35: 292-303. 10.1007/BF00161167.View ArticlePubMedGoogle Scholar
- Zhang LQ, Pond SK, Gaut BS: A survey of the molecular evolutionary dynamics of twenty-five multigene families from four grass taxa. J Mol Evol. 2001, 52: 144-156.View ArticlePubMedGoogle Scholar
- Eyre-Walker A, Gaut BS, Felsenstein J: Correlated rates of synonymous site evolution across plant genomes. Mol Biol Evol. 1997, 14: 455-460.View ArticlePubMedGoogle Scholar
- Chaw SM, Parkinson CL, Cheng Y, Vincent TM, Palmer JD: Seed plant phylogeny inferred from all three plant genomes: monophyly of extant gymnosperms and origin of Gnetales from conifers. Proc Natl Acad Sci U S A. 2000, 97: 4086-4091. 10.1073/pnas.97.8.4086.PubMed CentralView ArticlePubMedGoogle Scholar
- Graham SW, Olmstead RG, Barrett SCH: Rooting phylogenetic trees with distant outgroups: A case study from the commelinoid monocots. Mol Biol Evol. 2002, 19 (10): 1769-1781.View ArticlePubMedGoogle Scholar
- Felsenstein J: Cases in which parsimony or compatibility methods will be positively misleading. Syst Zool. 1978, 27 (4): 401-410.View ArticleGoogle Scholar
- Felsenstein J: Parsimony in systematics: biological and statistical issues. Annu Rev Ecol Syst. 1983, 14: 313-333. 10.1146/annurev.es.14.110183.001525.View ArticleGoogle Scholar
- Hendy MD, Penny D: A framework for the quantitative study of evolutionary trees. Syst Zool. 1989, 38: 296-309.Google Scholar
- Goremykin VV, Hirsch-Ernst KI, Wölfl S, Hellwig FH: The chloroplast genome of Nymphaea alba: Whole-genome analyses and the problem of identifying the most basal angiosperm. Mol Biol Evol. 2004, 21: 1445-1454. 10.1093/molbev/msh147.View ArticlePubMedGoogle Scholar
- Duvall MR, Clegg MT, Chase MW, Clark WD, Kress WJ, Hills HG, Eguiarte LE, Smith JF, Gaut BS, Zimmer EA, Learn GH: Phylogenetic hypotheses for the monocotyledons constructed from rbcL sequence data. Ann Mo Bot Gard. 1993, 80 (3): 607-619.View ArticleGoogle Scholar
- Angiosperm Phylogeny Group (APG): An update of the Phylogeny Group classification for the orders and families of flowering plants: APGII. Bot J Linn Soc. 2003, 141: 399-436. 10.1046/j.1095-8339.2003.t01-1-00158.x.View ArticleGoogle Scholar
- Chase MW: Monocot relationships: an overview. Am J Bot. 2004, 91: 1645-1655.View ArticlePubMedGoogle Scholar
- Chase MW, Soltis DE, Soltis PS, Rudall PJ, Fay MF, Hahn WH, Sullivan S, Joseph J, Givinish TJ, Systma KJ, Pires JC: Higher-level systematics of the monocotyledons: an assessment of current knowledge and new classification. Monocots: systematics and evolution. Edited by: Wilson K L and Morrison D A. 2000, Collingwood, Australia, CSIRO Publishing, 3-16.Google Scholar
- Swofford DL: PAUP*: Phylogenetic analysis using parsimony (* and other methods). Version 4.0b10. 2003, Sunderland, Massachusetts, Sinauer AssociatesGoogle Scholar
- Jukes TH, Cantor CR: Evolution of protein molecules. Mammalian Protein Metabolism. Edited by: Munro H N. 1969, New York, Academic Press, 21-132.View ArticleGoogle Scholar
- Felsenstein J: Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 1981, 17 (6): 368-376.View ArticlePubMedGoogle Scholar
- Tajima F, Nei M: Estimation of evolutionary distance between nucleotide sequences. Mol Biol Evol. 1984, 1 (3): 269-285.PubMedGoogle Scholar
- Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980, 16 (2): 111-120.View ArticlePubMedGoogle Scholar
- Felsenstein J: Distance methods for inferring phylogenies - a justification. Evolution. 1984, 38 (1): 16-24.View ArticleGoogle Scholar
- Hasegawa M, Kishino H, Yano T: Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol. 1985, 22: 160-174.View ArticlePubMedGoogle Scholar
- Kimura M: Estimation of evolutionary distances between homologous nucleotide sequences. Proc Natl Acad Sci U S A. 1981, 78 (1): 454-458.PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Nei M: Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993, 10 (3): 512-526.PubMedGoogle Scholar
- Lanave C, Preparata G, Saccone C, Serio G: A new method for calculating evolutionary substitution rates. J Mol Evol. 1984, 20 (1): 86-93.View ArticlePubMedGoogle Scholar
- Rodriguez F, Oliver JL, Marin A, Medina JR: The general stochastic model of nucleotide substitution. J Theor Biol. 1990, 142 (4): 485-501.View ArticlePubMedGoogle Scholar
- Steel MA: Recovering a tree from the leaf colourations it generates under a Markov model. App Math Lett. 1994, 7 (2): 19-24. 10.1016/0893-9659(94)90024-8.View ArticleGoogle Scholar
- Lockhart PJ, Steel MA, Penny D, Hendy MD: Recovering evolutionary trees under a more realistic model of sequence evolution. Mol Biol Evol. 1994, 11 (4): 605-612.PubMedGoogle Scholar
- Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14 (9): 817-818. 10.1093/bioinformatics/14.9.817.View ArticlePubMedGoogle Scholar
- Shimodaira H, Hasegawa M: Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol. 1999, 16: 1114-1116.View ArticleGoogle Scholar
- Yang ZH: Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites - approximate methods. J MOL EVOL J MOL EVOL. 1994, 39 (3): 306-314. 10.1007/BF00160154.View ArticlePubMedGoogle Scholar
- Lockhart PJ, Larkum AWD, Steel MA, Waddell PJ, Penny D: Evolution of chlorophyll and bacteriochlorophyll: The problem of invariant sites in sequence analysis. Proc Natl Acad Sci USA. 1996, 93 (5): 1930-1934. 10.1073/pnas.93.5.1930.PubMed CentralView ArticlePubMedGoogle Scholar
- Qiu YL, Chase MW, Les DH, Parks CR: Molecular Phylogenetics of the Magnoliidae: Cladistic Analyses of Nucleotide Sequences of the Plastid Gene rbcL. Ann Mo Bot Gard. 1993, 80 (3): 587-606.View ArticleGoogle Scholar
- Stevenson DW, Loconte H: Cladistic analysis of monocot families. Monocotyledons: systematics and evolution. Edited by: Rudall P J, Cribb P J, Cutler D F and Humphries C J. 1995, Kew, UK, Royal Botanical GardensGoogle Scholar
- Yang Z: Among-site rate variation and its impact on phylogenetic analyses. Trends Ecol Evol. 1996, 11: 367-372. 10.1016/0169-5347(96)10041-0.View ArticlePubMedGoogle Scholar
- Felsenstein J: Taking variation of evolutionary rates between sites into account in inferring phylogenies. J Mol Evol. 2001, 53: 447-455. 10.1007/s002390010234.View ArticlePubMedGoogle Scholar
- Buckley TR, Simon C, Chambers GK: Exploring among-site rate variation models in a maximum likelihood framework using empirical data: Effects of model assumptions on estimates of topology, branch lengths, and bootstrap support. Syst Biol. 2001, 50 (1): 67-86. 10.1080/106351501750107495.View ArticlePubMedGoogle Scholar
- Rogers JS: Maximum likelihood estimation of phylogenetic trees is consistent when substitution rates vary according to the invariable sites plus gamma distribution. Syst Biol. 2001, 50: 713-722. 10.1080/106351501753328839.View ArticlePubMedGoogle Scholar
- Soltis DE, Soltis PS: Amborella not a "basal angiosperm"? Not so fast. Am J Bot. 2004, 91: 997-1001.View ArticlePubMedGoogle Scholar
- Degtjareva TH, Samigullin DD, Sokoloff CM, Valiejo-Roman CM: Gene sampling versus taxon sampling: Is Amborella (Amborellaceae) a sister group to all other extant angiosperms. Botanichesky Zhurnal. 2004, 6: 896-907.Google Scholar
- Jansen RK, dePamphilis CW, Raubeson L, Leebens-Mack J, McNeal JR, Cui L, Zhang Y, Wyman S, Boore J, Kuehl J: The utility of whole chloroplast genome sequencing for reconstructing deep nodes in plants with an example from basal angiosperms [abstract]. Botony 2004. 2004, Salt Lake City, Utah, Botanical Society of America, [http://www.2004.botanyconference.org/engine/search/index.php?func=detail&aid=308]Google Scholar
- Donoghue MJ, Doyle JA: Seed plant phylogeny: demise of the anthophyte hypothesis?. Curr Biol. 2000, 10: R106-R109. 10.1016/S0960-9822(00)00304-3.View ArticlePubMedGoogle Scholar
- Bowe LM, Coat G, dePamphilis CW: Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers. Proc Natl Acad Sci U S A. 2000, 97 (8): 4092-4097. 10.1073/pnas.97.8.4092.PubMed CentralView ArticlePubMedGoogle Scholar
- Soltis DE, Soltis PS, Zanis MJ: Phylogeny of seed plants based on evidence from eight genes. Am J Bot. 2002, 89: 1670-1681.View ArticlePubMedGoogle Scholar
- Burleigh JG, Mathews S: Phylogenetic signal in nucleotide data from seed plants: implications for resolving the seed plant tree of life. Am J Bot. 2004, 91: 1599-1613.View ArticlePubMedGoogle Scholar
- Yoder AD, Vilgalys R, Ruvolo M: Molecular evolutionary dynamics of cytochrome b in strepsirrhine primates: the phylogenetic significance of third-position transversions. Mol Biol Evol. 1996, 13: 1339-1350.View ArticlePubMedGoogle Scholar
- Olmstead RG, Reeves PA, Yen AC: Patterns of sequence evolution and implications for parsimony analysis of chloroplast DNA. Molecular systematics of plants II : DNA sequencing. Edited by: Soltis Douglas E, Soltis Pamela S and Doyle Jeff J. 1998, Boston, Kluwer Academic Publishers, 164-187.View ArticleGoogle Scholar
- Källersjö M, Farris JS, Chase MW, Bremer B, Fay MF, Humphries CJ, Peterson G, Seberg O, Bremer K: Simultaneous parsimony jackknife analysis of 2538 rbcL DNA sequences reveals support for major clades of green plants, land plants, seed plants and flowering plants. Plant Syst Evol. 1998, 213: 259-287.View ArticleGoogle Scholar
- Källersjö M, Albert VA, Farris JS: Homoplasy increases phylogenetic structure. Cladistics. 1999, 15: 91-93. 10.1006/clad.1999.0085.Google Scholar
- Savolainen V, Chase MW, Salamin N, Soltis DE, Soltis PS, López A, Fédrigo O, Naylor GJP: Phylogeny reconstruction and functional constraints in organellar genomes: plastid atpB and rbcL sequences versus animal mitochondrion. Syst Biol. 2002, 51: 638-647. 10.1080/10635150290102348.View ArticlePubMedGoogle Scholar
- Sanderson MJ, Wojciechowski MF, Hu JM, Khan TS, Brady SG: Error, bias, and long-branch attraction in data for two chloroplast photosystem genes in seed plants. Mol Biol Evol. 2000, 17 (5): 782-797.View ArticlePubMedGoogle Scholar
- Delwiche CF, Kuhsel M, Palmer JD: Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids. Mol Phylogenet Evol. 1995, 4: 110-128. 10.1006/mpev.1995.1012.View ArticlePubMedGoogle Scholar
- Magallón S, Sanderson MJ: Relationships among seed plants inferred from highly conserved genes: sorting conflicting phylogenetic signals among ancient lineages. Am J Bot. 2002, 89: 1991-2006.View ArticlePubMedGoogle Scholar
- Phillips MJ, Delsuc F, Penny D: Genome-scale phylogeny and the detection of systematic biases. Mol Biol Evol. 2004, 21: 1455-1458. 10.1093/molbev/msh137.View ArticlePubMedGoogle Scholar
- Taberlet P, Gielly L, Pautou G, Bouvet J: Universal primers for amplification of the non-coding regions of chloroplast DNA. Pl Mol Biol. 1991, 17: 1105-1109. 10.1007/BF00037152.View ArticleGoogle Scholar
- Olmstead RG, Michaels HJ, Scott KM, Palmer JD: Monophyly of the Asteridae and identification of their major lineages inferred from DNA sequences of rbcL. Ann Missouri Bot Garden. 1992, 79: 249-265.View ArticleGoogle Scholar
- Hoot SB, Culham A, Crane PR: The utility of atpB gene sequences in phylogenetic relationships: comparison with rbcL and 18S ribosomal DNA sequences in Lardizabalaceae. Ann Mo Bot Gard. 1995, 82: 194-207.View ArticleGoogle Scholar
- Yen AC-T: Molecular systematics of Cyperaceae tribe Cariceae and genus Carex. 1999, Seattle, Washington, University of WashingtonGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680.PubMed CentralView ArticlePubMedGoogle Scholar
- Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985, 39: 783-791.View ArticleGoogle Scholar
- Maciukenas M: TREETOOL: a phylogenetic data visualizer, editor, and tree formatter. 1994, [http://iubio.bio.indiana.edu/soft/molbiol/unix/treetool]Google Scholar
- Soltis PS, Soltis DE: The origin and diversification of angiosperms. Am J Bot. 2004, 91: 1614-1626.View ArticlePubMedGoogle Scholar
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