American Journal of Botany 91(6): 9971001. 2004.

AMBORELLA

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DOUGLAS E. SOLTIS2
3

SO FAST1

PAMELA S. SOLTIS3

2 Department of Botany, University of Florida, Gainesville, Florida 32611 USA; and Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611 USA

The sequence of the plastid genome of Amborella trichopoda, the putative sister to all other extant angiosperms, was recently reported (Molecular Biology and Evolution 20: 14991505). Goremykin et al. used sequence data for 61 plastid genes from Amborella and 12 other embryophytes in phylogenetic analyses and concluded that Amborella is not the sister to the remaining owering plants; the monocots instead occupy this position. The authors attributed their results, which differ substantially from all recent phylogenetic analyses of angiosperms, to the increased character sampling (30 017 nucleotides in their aligned matrix) in their analysis relative to published studies that included fewer genes but more taxa. We hypothesized that the difference in topology is not due to limited character sampling in previous studies but to limited taxon sampling in the analysis by Goremykin et al. To test this, we conducted a series of phylogenetic analyses using a three-gene, 12 (or more)-taxon data set to evaluate the topological effects of (i) including three vs. 61 genes for (nearly) the same set of taxa, (ii) analyzing different codon positions, (iii) substituting representatives of other basal lineages for Amborella, (iv) replacing the grasses used to represent the monocots with other monocots, selected either for their phylogenetic position or randomly, and (v) adding other basal taxaNymphaea, Austrobaileya, magnoliids, and monocotsto the 12taxon data set. Our results demonstrate that the monocots basal topology obtained by Goremykin et al. is not due to increased character sampling of the plastid genome; their topology was obtained using only two plastid genes or two plastid genes and one nuclear gene. This topology was also retained when either Nymphaea or Austrobaileya was substituted for Amborella, demonstrating that any of the three basal lineages will attach to Calycanthus for lack of any other close branch. Furthermore, the monocots basal topology is not robust to changes in sampling of monocots. Simply adding Oncidium, for example, places Amborella sister to the other angiosperms. Thus, limited taxon sampling, focusing on organisms with complete genome sequences, can lead to artifactual results. Key words: Amborella; angiosperm phylogeny; basal angiosperms; taxon sampling; plastid DNA.

Goremykin et al. (2003) provide the complete sequence of the plastid genome of Amborella trichopoda (Amborellaceae), the putative sister to all other extant angiosperms (e.g., Mathews and Donoghue, 1999; Parkinson et al., 1999; Qiu et al., 1999; P. Soltis et al., 1999; D. Soltis et al., 2000; Savolainen et al., 2000; Barkman et al., 2000; Graham and Olmstead, 2000; Magallon and Sanderson, 2002; Zanis et al., 2002, 2003; Borsch et al., 2003; Hilu et al., 2003; Nickerson and Drouin, 2004). This plastid sequence, 162 686 bases in length, is one of fewer than 20 complete plastid sequences reported for land plants (including only 13 angiosperms; GenBank, 9/03) and is therefore an important contribution. However, Goremykin et al. then state that phylogenetic analyses of their data and other sequences obtained from databases indicate that Amborella is not a basal angiosperm, a conclusion that contrasts sharply with the results of recent molecular phylogenetic analyses (see references above). Although Goremykin et al. indicate a strong preference for the Amborella not basal topology, they do acknowledge that further studies are needed to understand the causes inuencing the position of Amborella obtained. . . Goremykin et al. (2003) used only 13 taxa in their analysis, including three non-angiosperm outgroups and Amborella, Calycanthus, and three monocots (all from Poaceae, a derived lineage of monocots) as the only non-eudicot angiosperms. Adequate taxon sampling is crucial in phylogeny reconstruction (e.g., Chase et al., 1993; Graybeal, 1998; Hillis, 1998; Pollock et al., 2002; Zwickl and Hillis, 2002). Furthermore,
Manuscript received 7 October 2003; revision accepted 10 February 2004. We thank Yin Long Qiu and Chuck Bell for helpful comments on earlier drafts of the manuscript. This work was supported in part by DEB-0090283 and PGR-0115684.
1

the constraint imposed by available complete plastid sequences precluded inclusion of other basal lineages that occupy important phylogenetic positions. That is, in addition to Amborella, Nymphaeaceae sensu APG II (2003) and Austrobaileyales (see APG II, 2003; Trimeniaceae, Schisandraceae, and Austrobaileyaceae) are successive sisters to all other extant owering plants, but neither of these lineages is represented in the analysis of Goremykin et al. Supporting our argument that the topology of Goremykin et al. is the result of limited taxon sampling are similar odd topologies recovered by ourselves and others in early analyses of plastid and nuclear genes when few taxa were included. For example, in our initial analyses of small data sets of 18S rDNA sequences, we obtained topologies in which monocots, either as a clade or as a single taxon, appeared as sister to all other extant angiosperms (D. Soltis et al., 1997). We recognized that the monocots sampled had long branches (see later), and we therefore continued to add taxa, ultimately obtaining stable trees in which Amborella, Austrobaileyales, and Nymphaeaceae were sisters to all other owering plants (D. Soltis et al., 1997). Chase et al. (1993) similarly pointed out the errors in the topologies recovered for angiosperms when small subsets of taxa were employed rather than large data sets. Likewise, Naylor and Brown (1998) obtained incorrect relationships in animals with a small sampling of complete mitochondrial DNA sequences. The topology of Goremykin et al. for angiosperms differs substantially from the consensus we obtained using three (P. Soltis et al., 1999; D. Soltis et al., 2000), ve (Qiu et al., 1999), six (Zanis et al., 2003), or 11 (Zanis et al., 2002) genes and from those reported by Mathews and Donoghue (1999), 997

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Parkinson et al. (1999), Barkman et al. (2000), Graham and Olmstead (2000), Magallon and Sanderson (2002), Borsch et al. (2003), Hilu et al. (2003), and Nickerson and Drouin (2004). Goremykin et al. contend that their results are due to the extensive plastid DNA data set (61 genes) they analyzed. However, we hypothesize that their topology has less to do with extensive character sampling than with limited taxon sampling. Goremykin et al. found Amborella and Calycanthus to be sisters, with 100% bootstrap support. However, we hypothesize that, given the taxon sampling by Goremykin et al., Amborella had nowhere to go other than with Calycanthus, the only other basal angiosperm included in their study other than the three grasses. If this hypothesis is correct, then a single species of either of the other two basalmost branches of angiospermsNymphaeaceae and Austrobaileyalesshould behave as Amborella did in the analysis by Goremykin et al. Furthermore, the placement of the monocots as the sister to all other angiosperms in the tree by Goremykin et al. and in those obtained when additional samples of basal lineages and magnoliids were added (see later), contra to all large-scale multi-gene analyses (e.g., P. Soltis et al., 1999; D. Soltis et al., 2000; Qiu et al., 1999; Zanis et al., 2002, 2003; Mathews and Donoghue, 1999; Parkinson et al., 1999; Barkman et al., 2000), suggested that this result was due to the selection of two grasses (three in Goremykin et al., 2003) as the representative monocots. Grasses are derived monocots and have long been known to have accelerated rates of cpDNA evolution (e.g., Gaut et al., 1992, 1996). Thus, the branch leading to the grasses is quite long, given both the phylogenetic position of the grasses and their rapid rate of cpDNA evolution. We therefore hypothesize that the selection of monocots other than grasses, or the inclusion of additional monocots to shorten the branch to the grasses, may alter the monocots basal topology. Character sampling: 61 vs. three genesTo test the effect of 61 vs. three genes, we constructed a 12-taxon data set using the same (or nearly the same) taxa used by Goremykin et al. and data from three genes: the plastid genes rbcL and atpB and nuclear 18S rDNA (angiosperms from D. Soltis et al., 2000; non-angiosperms from Pryer et al., 2001). Our pruned data set contained the same outgroups (Marchantia, Psilotum, and Pinus) used by Goremykin et al. (2003) and nine angiosperms, rather than 10 as in Goremykin et al. (2003); Triticum was not included in our analysis because it was not present in the D. Soltis et al. (2000) data set. Brassica (also of Brassicaceae) was substituted for Arabidopsis; Pisum (also of Fabaceae) was substituted for Lotus; and Epilobium ( Chamerion; also of Onagraceae) was substituted for Oenothera. Parsimony analysis (100 random taxon addition replicates, tree bisection-reconnection [TBR] branch swapping, saving all most parsimonious trees; using PAUP* 4.0 [Swofford, 2003]) was conducted using this three-gene, 12-taxon data set. Bootstrap analyses (100 replicates) were conducted with 100 random taxon addition replicates, using TBR branch swapping and saving all most parsimonious trees. Parsimony analysis found a single shortest tree (Fig. 1) with exactly the same topology as that reported by Goremykin et al.: Oryza Zea were sister to the rest of the angiosperms, Amborella Calycanthus were sisters, and all other relationships were identical to those in the tree of Goremykin et al. However, constraining Amborella to be sister to all other an-

Fig. 1. Single most parsimonious tree (length 2770 steps; CI 0.644; RI 0.469) obtained in a phylogenetic analysis of three genes (rbcL, atpB, 18S rDNA; data from D. Soltis et al. 2000). Bootstrap values are given above the branches. Branches without values received 50% bootstrap support.

giosperms, the position it occupies in the many studies cited earlier, lengthened the tree by only seven steps (length 2777 steps), an increase in length of only 0.25% over the unconstrained tree (2770 steps). The monocots basal result was obtained in analyses that included (i) all nucleotide positions of the three genes, (ii) all codon positions of the two plastid genes, and (iii) the rst and second codon positions of the two plastid genes. Because our analyses used only three instead of 61 genes and all analyses, regardless of codon positions included, yielded this same result, this topology is not due to the increased character sampling used by Goremykin et al. or to the exclusion of third positions. Instead, we argue that it must result from differences in taxon sampling. Taxon samplingTo test the hypothesis that representatives of either of the other two basalmost lineages of angiosperms would attach exactly as Amborella did, we used the 12-taxon, three-gene data set described earlier. We replaced Amborella rst with Nymphaea (of Nymphaeaceae) and then with Austrobaileya (of Austrobaileyales) and conducted parsimony analyses as described. Further analyses to evaluate the effects of additional basal lineages were conducted, including one or more of the following magnoliids: Magnolia, Myristica, Cinnamomum, Asarum, and Drimys. In analyses in which either

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TABLE 1. Randomly selected pairs of monocots (of the 102 monocots included in D. Soltis et al., 2000) used in replacement analyses to test the effects of monocot exemplars on the placement of Amborella vs. the monocots in 12-taxon, three-gene analyses.
Monocot Pair Sister to Rest of Angiosperms

Zostera and Glomeropitcairnia Bomarea and Chamaedorea Convallaria and Oncidium Blandfordia and Monocostus Ixiolirion and Nolina Behnia and Zingiber Aristea and Pleea Restio and Trillium Juncus and Tacca Chlorophytum and Dasypogon

Zostera Amborella Amborella Amborella Amborella Amborella Amborella Amborella Amborella Amborella

Nymphaea or Austrobaileya was substituted for Amborella, the substituted taxon was sister to Calycanthus, with no other changes to the topology: Nymphaea Calycanthus were sisters (but with 50% bootstrap support), and Austrobaileya Calycanthus received 100% bootstrap support (tree not shown). Therefore, the Amborella Calycanthus sister group reported by Goremykin et al. is not due to the close relationship of these taxa (per authors of the 19th and 20th centuries, as claimed by Goremykin et al.) but results from limited taxon sampling: a single representative of any of the three basal branches of angiosperms forms a sister group with Calycanthus. The addition of multiple representatives of these basal branches (e.g., Amborella, Nymphaea, Austrobaileya) plus additional magnoliids (Magnolia, Myristica, Cinnamomum, Drimys, Asarum) broke up the Amborella Calycanthus sister group, forming a clade in which Amborella, Nymphaea, and Austrobaileya are successive sisters to a clade of magnoliids, which includes Calycanthus. However, the monocots Oryza Zea remained sister to all other angiosperms, even when all of these taxa were added (trees not shown). To test the effect of the selection of two grasses as exemplars for monocots on the overall topology, we conducted a series of analyses that included alternative monocot exemplars. We (1) replaced the grasses with two other monocots (replacement analyses) and (2) added other monocots to the grasses to increase the diversity of monocot lineages represented and to break up the branch leading from the base of the monocots to the grasses (addition analyses). Two types of replacement analyses were conducted. First, we substituted Acorus (the sister group to all other monocots; e.g., P. Soltis et al., 1999; D. Soltis et al., 2000; Qiu et al., 1999; Zanis et al., 2002, 2003; Chase et al., 2000) and Spathiphyllum (of Alismatales, a basal clade of monocots) for Zea and Oryza. Although both Acorus and Spathiphyllum themselves have long branches (144 steps from the common ancestor of monocots to Acorus and 205 steps from the common ancestor of monocots to Spathiphyllum on one of the threegene, 567-taxon trees of D. Soltis et al., 2000), they are not as long as that leading to the grasses (447 steps from the common ancestor of the monocots to the common ancestor of the grasses; D. Soltis et al., 2000). Second, we randomly selected two monocots from the 102 monocots included in the threegene analysis of D. Soltis et al. (2000) and substituted them for Zea and Oryza; this was repeated 10 times (Table 1). If the position of the monocots as sister to all other angiosperms in the Goremykin et al. analysis (and our 12-taxon,

three-gene analysis described earlier) is an artifact of the long branch leading to grasses rather than the result of real phylogenetic signal, then the position of the monocots should shift to its more conventional location as the monocot branch is shortened by the addition of taxa. We included one to six additional monocots selected for their phylogenetic placements to test the robustness of the monocots basal topology to changes in taxon sampling. Further analyses included two to six additional monocots, along with Nymphaea, Austrobaileya, and additional representatives of magnoliids (Cinnamomum, Magnolia, Myristica, Drimys, and Asarum). In the replacement analyses in which Acorus and Spathiphyllum were substituted for the grasses Oryza and Zea, Amborella is the sister to all other angiosperms (tree not shown). However, Acorus and Spathiphyllum do not form a clade as expected, presumably due to limited sampling of monocots (see later), but are the successive sister groups to Calycanthus the eudicots (although bootstrap support for the Spathiphyllum (magnoliids eudicot clade) is 50%). However, when Nymphaea and Austrobaileya are also added to the analysis, Acorus and Spathiphyllum form a clade that is sister to the eudicots (tree not shown). The same result was obtained when additional magnoliids (Cinnamomum, Magnolia, Myristica, Asarum, and Drimys) were included. When randomly selected monocots were chosen to replace Oryza and Zea, the topology shifted from monocots basal to Amborella basal. In all cases but one, Amborella was the sister to the rest of the angiosperms, and the monocot pair was either sister to Calycanthus (one of the nine remaining analyses), sister to Calycanthus the eudicots (six), sister to the eudicots (one), or in a trichotomy with Calycanthus the eudicots (one). In the single case in which Amborella was not sister to all other angiosperms, the randomly selected monocots, Zostera (of Alismatales) and Glomeropitcairnia (of Bromeliaceae, the sister group of grasses), were successive sisters to all other angiosperms; Amborella was not paired with Calycanthus, however, as it was when the grasses were the monocot exemplars, but was sister to Calycanthus the eudicots. In only two of 14 analyses in which other monocot taxa were added to the grasses was the monocots basal topology obtained; in the other 12, Amborella was sister to all other angiosperms. The monocots basal result occurred when (1) Acorus and Spathiphyllum were added to Oryza and Zea as monocot exemplars and (2) Acorus and Puya were added to Oryza and Zea. Both analyses also included Nymphaea, Austrobaileya, Cinnamomum, Magnolia, Myristica, Drimys, and Asarum. However, in all other analyses, regardless of monocot sampling and whether other basal taxa were added, Amborella remained sister to all other angiosperms. For example, the addition of (1) the orchid Oncidium alone to Oryza and Zea (Fig. 2), (2) Puya alone, (3) Acorus and Oncidium, (4) Acorus and Puya, (5) Acorus, Oncidium, and Puya, (6) Acorus and Spathiphyllum, and (7) Acorus, Spathiphyllum, and Puya to the 12-taxon data set produced trees with the Amborella basal topology. Likewise, the other ve analyses with combinations of additional monocots and additional magnoliids found the Amborella basal topology (Appendix, see Supplemental Data accompanying online version of this article). In only one of 13 replacement analyses and two of 14 addition analyses is the monocots basal topology retained; in all other cases, the Amborella basal tree is obtained. Thus, the contention of Goremykin et al. that monocots form the sister group to all other angiosperms cannot be supported.

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Fig. 2. Single most parsimonious tree (length 2949 steps; CI 0.623; RI 0.459) obtained when the orchid Oncidium is included as an additional monocot exemplar to break up the long branch leading to the grasses. Bootstrap values are given above the branches. Branches without values received 50% bootstrap support.

More thorough phylogenetic sampling of monocots, representing basal lineages and the sister group of the grasses, for example, all yield the Amborella basal tree. The only exceptions occurred when the basal monocots Acorus and Spathiphyllum or Acorus and Puya, respectively, were added to data sets containing Oryza, Zea, Nymphaea, Austrobaileya, and additional magnoliids. In these two replacement cases, the long branches of grasses, Acorus, Spathiphyllum, and Puya grasses (e.g., D. Soltis et al., 2000) presumably distort relationships at the base of the tree; the addition of Puya, of Bromeliaceae, the sister group to all other Poales, does not break up the long branch to the grasses. Furthermore, even analyses that included randomly selected pairs of monocots found the Amborella basal tree in nine of 10 replicates. ConclusionsThe topology obtained by Goremykin et al., with the monocots sister to all other angiosperms and Amborella sister to Calycanthus, is not due to the increased character sampling in their study (61 genes vs. a maximum in previous analyses of 17; Graham and Olmstead, 2000), but rather to limited taxon sampling. Evidence supporting this conclusion is (1) analysis of three genes for the same (or nearly the same) 12 taxa (omitting Triticum) yielded the Goremykin et al. tree; (2) replacement of Amborella with either Nymphaea or Austrobaileya yielded trees in which these alternatives were sister to Calycanthus, and addition of Nymphaea, Austrobaileya, and multiple representatives of magnoliids to the original matrix

broke up the Amborella Calycanthus sister group; and (3) sensitivity analyses of the monocots basal topology, based on the inclusion of grasses as the only monocot exemplars in the original matrix, demonstrated that this position of the monocots is not robust to alternative sampling of monocots. Stefanovic et al. (unpublished data, Indiana University) re cently added a nearly complete plastid sequence for the monocot Acorus to the data set of Goremykin et al. (2003). Paralleling our results, Stefanovic et al. also recovered Amborella as sister to other angiosperms. Thus, available data cannot refute the position of Amborella as sister to all other extant angiosperms. A series of studies involving all three genomes and reasonable taxon sampling has demonstrated that Amborella (or Amborella Nymphaeaceae; Barkman et al., 2000) is sister to all other owering plants (e.g., D. Soltis et al., 1997, 2000; P. Soltis et al., 1999; Qiu et al., 1999; Mathews and Donoghue, 1999; Parkinson et al., 1999; Graham and Olmstead, 2000; Zanis et al., 2002, 2003; Borsch et al., 2003; Hilu et al., 2003). These analyses have sampled many nucleotides and many taxa and have applied diverse approaches, including parsimony, neighbor-joining, maximum likelihood, Bayesian methods, and compartmentalization. For example, the bootstrap support for Amborella and Nymphaeaceae as successive sisters to all other angiosperms is 91 and 98%, respectively, and the posterior probabilities inferred from Bayesian analyses for these same two nodes are 0.99 and 1.00, respectively (Zanis et al., 2002). Two recent analyses of fast-evolving plastid regions (Borsch et al., 2003; Hilu et al., 2003) only strengthen the argument for Amborella as sister to all other angiosperms. In fact, matK alone provides 86% bootstrap support for these same two basal nodes. Recent phylogenetic analyses of B-class oral genes not only recover Amborella and Nymphaeaceae as sisters to all other angiosperms, but also reveal structural features that indicate that Amborella alone is sister to all other angiosperms (Kim et al., 2004). All of these studies have in common a reasonable sampling of taxa (mostly over 100 taxa), especially of basal lineages. This reminder of the importance of sufcient and appropriate taxon sampling is particularly timely as attempts to use whole organellar genomes for phylogeny reconstruction are underway in several labs. As exciting as the genomic data are, extensive character sampling cannot compensate for inadequate taxon sampling. LITERATURE CITED
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