Accepted Manuscript Deep phylogenetic incongruence in the angiosperm clade Rosidae Miao Sun, Douglas E. Soltis, Pamela S. Soltis, Xinyu Zhu, J. Gordon Burleigh, Zhiduan Chen PII: DOI: Reference:

S1055-7903(14)00387-X http://dx.doi.org/10.1016/j.ympev.2014.11.003 YMPEV 5067

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

10 June 2014 1 November 2014 5 November 2014

Please cite this article as: Sun, M., Soltis, D.E., Soltis, P.S., Zhu, X., Gordon Burleigh, J., Chen, Z., Deep phylogenetic incongruence in the angiosperm clade Rosidae, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/j.ympev.2014.11.003

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Deep phylogenetic incongruence in the angiosperm clade Rosidae

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Miao Suna, b, Douglas E. Soltis*, c, d, e, Pamela S. Soltisd, e, Xinyu Zhuf, J. Gordon

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Burleighc, e, Zhiduan Chen*, a

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a

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the Chinese Academy of Sciences, Beijing 100093, China;

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b

Graduate University of the Chinese Academy of Sciences, Beijing 100039, China;

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c

Department of Biology, University of Florida, Gainesville, FL 32611, USA;

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d

Florida Museum of Natural History, University of Florida, Gainesville, FL 32611,

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USA;

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e

University of Florida Genetics Institute

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f

School of Life Science, Nantong University, Nantong 226007, China;

State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany,

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*Corresponding authors:

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Zhiduan Chen: [email protected], 8610-62836090

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Douglas E. Soltis: [email protected], (1)-352-273-1963

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Running title: Deep phylogenetic incongruence in Rosidae

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Data Archival Location: Dryad (XXXX)

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Abstract

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Analysis of large data sets can help resolve difficult nodes in the tree of life and also

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reveal complex evolutionary histories. The placement of the

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Celastrales-Oxalidales-Malpighiales (COM) clade within Rosidae remains one of the

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most confounding phylogenetic questions in angiosperms, with previous analyses

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placing it with either Fabidae or Malvidae. To elucidate the position of COM, we

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assembled multi-gene matrices of chloroplast, mitochondrial, and nuclear sequences,

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as well as large single- and multi-copy nuclear gene data sets. Analyses of multi-gene

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data sets demonstrate conflict between the chloroplast and both nuclear and

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mitochondrial data sets, and the results are robust to various character-coding and

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data-exclusion treatments. Analyses of single- and multi-copy nuclear loci indicate

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that most loci support the placement of COM with Malvidae, fewer loci support COM

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with Fabidae, and almost no loci support COM outside a clade of Fabidae and

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Malvidae. Although incomplete lineage sorting and ancient introgressive

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hybridization remain as plausible explanations for the conflict among loci, more

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complete sampling is necessary to evaluate these hypotheses fully. Our results

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emphasize the importance of genomic data sets for revealing deep incongruence and

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complex patterns of evolution.

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Keywords

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Hybridization; introgression; incomplete lineage sorting; COM clade; incongruence;

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phylogenomics

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1. Introduction

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Genome-scale data can provide the power to resolve some of the most perplexing

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parts of the tree of life (e.g., Dunn et al., 2008; Lee et al., 2011; Simon et al., 2012;

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Smith et al., 2011; Yoder et al., 2013). Furthermore, estimates from numerous

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independent loci also can reveal phylogenetic incongruence caused by different

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evolutionary processes, such as gene duplication and loss, recombination,

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hybridization, lateral gene transfer, or incomplete lineage sorting (e.g., Cui et al., 2013;

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Degnan and Rosenberg, 2009; Doyle, 1992; Goodman et al., 1979; Hudson, 1983;

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Maddison, 1997; Oliver, 2013). Molecular phylogenetic analyses have resolved much

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of the backbone angiosperm phylogeny (e.g., Ruhfel et al., 2014; Soltis et al., 2009,

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2011) and clarified long-standing questions regarding relationships within major

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clades such as monocots (Monocotyledoneae; Chase et al., 2000; Givnish et al., 2006,

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2010; Graham et al., 2006; Jerrold et al., 2004; Saarela et al., 2008), asterids

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(Asteridae; Albach et al., 2001; Bremer et al., 2001, 2004; Hilu et al., 2003; Moore et

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al., 2011; Olmstead et al., 2000), and rosids (Rosidae; Hilu et al., 2003; Jansen et al.,

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2007; Moore et al., 2010; Qiu et al., 2010; Soltis et al., 2007, 2011; Wang et al., 2009).

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Yet much of this work is based either largely or exclusively on chloroplast sequence

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data, which represent a single, linked, and usually maternally inherited locus. New

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sequencing technologies make it feasible to obtain data sets of numerous independent

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nuclear loci, which can be used to evaluate results from analyses of chloroplast gene

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sequence data and reveal phylogenetic conflict among loci (e.g., Burleigh et al., 2011; 3 / 40

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Duarte et al., 2010; Lee et al., 2011; Xi et al., 2014; Zeng et al., 2014). Introgressive hybridization has played an important role in plant evolution, and

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incomplete lineage sorting also likely occurred during some rapid radiations.

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Consequently, there are numerous examples of discordance between chloroplast and

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nuclear gene trees in plants (e.g., Acosta and Premoli, 2010; Okuyama et al., 2005;

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Rieseberg and Soltis, 1991; Rieseberg and Wendel, 1993; Rieseberg et al., 1995,

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1996a; Soltis and Kuzoff, 1995; Soltis and Soltis, 2009; Tsitrone et al., 2003; Wendel

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et al., 1995; Xi et al., 2014). Although phylogenetic analyses of angiosperm backbone

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relationships based on nuclear, mitochondrial, and chloroplast loci have largely agreed,

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one major point of conflict is the placement of COM

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(Celastrales-Oxalidales-Malpighiales; Endress and Matthews, 2006; Zhu et al., 2007)

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within the large Rosidae clade.

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Rosidae comprise approximately one quarter of all angiosperm species, which

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are morphologically diverse, exhibit extraordinary heterogeneity in habit, habitat, and

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life form, and include most temperate and tropical forest trees (Wang et al., 2009).

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Some members possess novel biochemical pathways (e.g., production of glucosinolate,

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and cyanogenic glycosides for defense), and many are important crops (e.g., Fabaceae

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and Rosaceae). Symbioses with nitrogen-fixing bacteria are largely confined to this

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clade as well. Resolving relationships within Rosidae has been difficult (e.g., Hilu et

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al., 2003; Jansen et al., 2007; Lee et al., 2011; Moore et al., 2010, 2011; Qiu et al.,

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2010; Ruhfel et al., 2014; Soltis et al., 2005, 2007, 2011; Wang et al., 2009; Zhu et al.,

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2007) due to a series of rapid radiations (Wang et al., 2009). However, multi-gene 4 / 40

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studies have recovered two major, well-supported clades — the Fabidae (i.e., eurosids

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I, fabids) and Malvidae (i.e., eurosids II, malvids) (Hilu et al., 2003; Judd and

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Olmstead, 2004; Moore et al., 2010, 2011; Soltis et al., 1999, 2000, 2005, 2007, 2011;

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Wang et al., 2009; Xi et al., 2014).

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COM contains approximately one third of all Rosidae, 870 genera and ~19,000

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species (APG III, 2009). Molecular analyses, largely dominated by chloroplast genes,

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have usually placed COM with Fabidae (Table 1; e.g., Burleigh et al., 2009; Hilu et

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al., 2003; Jansen et al., 2007; Moore et al., 2010, 2011; Soltis et al., 2005, 2007, 2011;

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Wang et al., 2009). Analyses of the mitochondrial gene matR first suggested the

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placement of COM with Malvidae (Zhu et al., 2007), and subsequent studies based on

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nuclear or mitochondrial genes supported this placement, although typically with

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limited taxon sampling (Table 1; Burleigh et al., 2011; Duarte et al., 2010; Finet et al.,

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2010; Lee et al., 2011; Morton, 2011; Qiu et al., 2010; Shulaev et al., 2010; Xi et al.,

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2014; Zhang et al., 2012). Several floral characters also appear to link COM with

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Malvidae. For example, in COM and Malvidae species, the inner integument of the

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ovule is thicker than the outer integument at the time of fertilization, a feature that is

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extremely rare in Fabidae and other eudicots. Additionally, contorted petals and a

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tendency towards polystemony and polycarpy also suggest a placement of COM

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members with Malvidae rather than with Fabidae (Endress and Matthews, 2006;

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Endress et al., 2013).

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Although analyses of chloroplast gene sequence data generally appear to conflict with analyses of mitochondrial and nuclear gene sequence data, these studies often 5 / 40

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differ greatly in taxon sampling and analytical methods (Table 1; but see Xi et al.,

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2014). Thus, it is unclear whether the different placements of COM are due to errors

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in the analyses or biological incongruence among loci. The level of incongruence

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within the nuclear genome also is unknown. We use COM as an exemplar to

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investigate phylogenetic incongruence at deep levels in angiosperm phylogeny.

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Specifically, we first compare phylogenetic results from chloroplast, mitochondrial,

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and nuclear data sets having similar taxon sampling and examine whether the results

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are robust to various character-coding and data-exclusion protocols. We also survey

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large-scale nuclear data sets of both single-copy and multi-copy genes to investigate

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the patterns of phylogenetic discordance within the nuclear genome and then discuss

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whether these patterns are consistent with incomplete lineage sorting (i.e., deep

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coalescence) (Maddison, 1997; Maddison and Knowles, 2006; Page and Charleston,

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1998) or ancient hybridization and introgression (Chang et al., 2011; Cui et al., 2013;

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Linder and Rieseberg, 2004; Tsitrone et al., 2003; Zhang et al., 2014).

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2. Materials and methods

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Throughout this paper, to facilitate discussion, we treat COM, Fabidae, and Malvidae

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as three separate groups, despite current classifications that consider COM to be part

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of Fabidae (APG III, 2009; Cantino et al., 2007).

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2.1 Phylogenetic analyses of chloroplast, mitochondrial, and nuclear data

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To compare the placement of COM in analyses of chloroplast, mitochondrial, and

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nuclear gene data sets, we assembled published matrices with similar taxon sampling. 6 / 40

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For the chloroplast gene sequence data, we pruned 82 seed plant taxa from the

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78-gene chloroplast data set of Ruhfel et al. (2014). We also used the 92-taxon, 5-gene

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nuclear data set of Zhang et al. (2012), and the 79-taxon, 4-gene mitochondrial matrix

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of Qiu et al. (2010). The taxon sampling in all of these studies was designed to

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reconstruct relationships across angiosperms using representative sampling of major

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clades, including COM. We used the nuclear gene sequence data set of Zhang et al.

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(2012) to guide our assembly of chloroplast and mitochondrial gene data sets,

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attempting to ensure as much as possible that the taxa employed from these data sets

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are from the same species or genus.

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We performed a series of Maximum Likelihood (ML) phylogenetic analyses on

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each of the three data sets using RAxML v.7.2.8 (Stamatakis, 2008). For all analyses,

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we estimated the optimal ML tree and performed 100 nonparametric bootstrap (BS)

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replicates. First, we analyzed the full nucleotide alignments using an unpartitioned

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GTRCAT model. We also examined the variation of COM placement in single-gene

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topologies inferred from these three data sets using RAxML with the GTRCAT model.

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For the three multi-gene data sets, we also analyzed the amino acid (AA) alignment

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using the PROTCATJTT model (Jones et al., 1992). RY coding, which recodes the

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nucleotides as binary characters, either purines (A or G = R) or pyrimidines (C or T =

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Y), has been used to ameliorate biases caused by saturation, rate heterogeneity, and

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base composition (Delsuc et al., 2005; Gibson et al., 2005; Harrison et al., 2004;

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Phillips et al., 2004; Phillips and Penny, 2003). Thus, we also transformed the three

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full nucleotide matrices to RY coding and ran a ML analysis using the GTRCAT 7 / 40

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model. The elimination of potentially misleading sites from an alignment is a common

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practice in phylogenetic analysis (e.g., Delsuc et al., 2005; Philippe et al., 2005; Rajan,

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2013; Regier and Zwick, 2011). We used two methods to remove highly variable sites

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as a further means of exploring the data that may contribute to the discordant

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placements of COM. First, following Goremykin et al. (2010), we organized the

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nucleotide sites in each alignment in order of rate based on the observed variability

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(OV) criterion. For each sorted alignment, we then removed the most variable 5%,

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10%, 20%, 30%, 40%, and 50% of the sites. After each removal, we performed a ML

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analysis on the remaining sites in each alignment using the GTRCAT model. Second,

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we excluded the third codon positions and analyzed the alignments of the first and

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second codon positions only using RAxML with the GTRCAT model.

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2.2 Single-copy nuclear gene analysis

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The largest nuclear gene data set used to resolve the backbone of angiosperm

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relationships comprises 22,833 groups of orthologs (Lee et al., 2011). Although this

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data set includes only seven species of Malpighiales representing COM (no

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Celastrales or Oxalidales species were included), it provides estimates from by far the

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greatest number of presumably independent nuclear loci for the placement of COM.

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We examined the individual gene trees from this data set to look for variation in the

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placement of COM. First, we divided the full, concatenated nucleotide alignment

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from Lee et al. (2011; available on the BIGPLANT website: http://nybg.bio.nyu.edu/)

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into separate alignments, each representing a set of putative orthologs. Next we 8 / 40

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identified the ortholog sets that were potentially informative regarding the placement

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of COM; these alignments contained at least one COM species, one Malvidae, one

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Fabidae, and one other species not in any of these groups. In all, 8,445 of the ortholog

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sets were potentially informative regarding the placement of COM. For each

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potentially informative ortholog set alignment, we performed 100 ML bootstrap

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replicates using RAxML v.7.2.8 with the GTRCAT model (Stamatakis, 2008), and we

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counted how many bootstrap replicates support a clade of COM and Fabidae species,

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how many support a clade of COM and Malvidae species, and how many support

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COM outside a clade of Fabidae and Malvidae. The analysis of the support for the

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COM placement was automated using Perl scripts and Newick utilities (Junier and

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Zdobnov, 2010).

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2.3 Multi-copy nuclear gene analysis

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We also examined support for the placement of COM using multi-copy nuclear gene

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families, i.e., gene families that may have multiple sequences from one or more taxa.

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Unlike single-copy sets of orthologs, interpreting a phylogenetic tree supported by

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multi-copy gene families is not always straightforward. For example, in a multi-copy

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gene tree, a species from COM could have one sequence that groups with Fabidae

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and one sequence that groups with Malvidae. To solve this problem, we estimated the

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reconciliation cost of each gene family tree given a topology with COM sister to

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Fabidae, COM sister to Malvidae, and COM sister to a Fabidae + Malvidae clade.

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We used three different reconciliation costs, each implying a different evolutionary

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scenario: 1) the minimum number of implied gene duplications; 2) the minimum 9 / 40

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number of implied duplications and losses; and 3) the minimum number of implied

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deep coalescence events (e.g., Maddison, 1997). We used a parsimony criterion to

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distinguish among the three species tree topologies; the topology with the lowest

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reconciliation cost, i.e., the topology that implies the fewest evolutionary events, is

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the topology that is supported by the gene family. If two or three of the topologies

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have equal reconciliation costs, the gene family is considered uninformative regarding

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the placement of COM.

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We assembled a collection of 3,748 gene family alignments obtained from the

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genome sequences of 22 plant taxa with OrthoMCL (Chen et al., 2006) and aligned

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with MAFFT (Katoh et al., 2005). Included are Selaginella moellendorffii,

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Physcomitrella patens, and 20 angiosperm species, including one species representing

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COM, Populus trichocarpa. Although the taxon sampling is sparse, using only

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sequences from completely sequenced genomes may enable more accurate estimates

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of processes such as gene loss than incomplete transcriptome data sets.

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For each multi-copy gene alignment, we performed 100 bootstrap replicates

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using RAxML v.7.2.8 with the GTRCAT model (Stamatakis, 2008). For each of the

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resulting bootstrap trees, we calculated the reconciliation cost under the three different

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cost models (duplications, duplications and losses, and deep coalescence) using a

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species tree in which Populus (COM clade) was sister to Fabidae (Fragaria vesca,

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Medicago trunculata, and Glycine max), one in which Populus was sister to Malvidae

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(Arabidopsis thaliana, Thellungiella parvula, Carica papaya, and Theobroma cacao),

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and one in which Populus was sister to Fabidae + Malvidae. The rooting of gene trees 10 / 40

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can greatly affect the estimates of the reconciliation cost, and it is often difficult to

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infer the root of a multi-copy gene tree. Thus, for each gene tree, we used a rooting

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that minimized the reconciliation cost. We calculated the reconciliation costs for each

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gene tree bootstrap replicate under three possible species trees using the program

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OptRoot, written by Andre Wehe and available at http://www.wehe.us/optroot.html.

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All data sets and results are available on Dryad (XXX; www.datadryad.org).

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3. Results

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3.1 Chloroplast, mitochondrial, and nuclear data sets

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ML analyses of the chloroplast, mitochondrial, and nuclear multi-gene alignments

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with similar taxon sampling recover different placements of COM (Figures 1–3). We

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focus on the relationships among members of Rosidae, but all the trees generated in

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our analyses in the present study are available as supplemental data and on Dryad

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(XXXX).

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The phylogeny based on the 82-taxon, 78-gene chloroplast data set largely agrees

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with conclusions from previous chloroplast-dominated studies (APG III, 2009; Moore

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et al., 2010; Ruhfel et al., 2014; Soltis et al., 2011; Wang et al., 2009), supporting the

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placement of COM with Fabidae (Figure 1). COM received 100% BS support, as did

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a clade of all COM and Fabidae species, but the precise placement of COM was

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uncertain. There was 52% BS support for a sister relationship of COM and all

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Fabidae except Bulnesia (Zygophyllales; Figure 1), which was sister to COM and

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other Fabidae species. Although most chloroplast genes support a placement of COM 11 / 40

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with Fabidae, albeit generally with low BS support, no chloroplast genes provide

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even 50% BS support for COM with Malvidae (Table S1). The analysis of the full

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chloroplast alignment with RY coding indicates 100% BS support for a clade of COM

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and Fabidae species (Figure S1). AA coding indicates 47% BS support for a clade of

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the COM species and all Fabidae except Bulnesia, which is placed in Malvidae,

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although with low support (Figure S2). Removing the highly variable nucleotide sites

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from the chloroplast alignment quickly erodes support for COM with Fabidae.

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Bootstrap support for COM with Fabidae drops from 98% to 48% to 1% with the

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removal of the 5%, 10%, and 20% most variable sites, respectively, with no support

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after removing more sites. However, none of the site removal analyses indicates any

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support for a clade of COM and Malvidae or COM outside of Fabidae + Malvidae.

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Removing the third codon positions from the 78-gene chloroplast data set reduced the

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BS support for a clade of COM and Fabidae species to 90%, with Bulnesia initially

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sister to COM (Figure S3).

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Trees from analyses of the 79-taxon, 4-gene mitochondrial data set generally

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indicate a close relationship of COM with species from Malvidae (Figure 2). In the

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ML analysis of the full nucleotide data set, there is 94% BS support for a clade of

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COM species and all Malvidae except Stachyurus and Oenothera (Figure 2).

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Additionally, Guaiacum (Zygophyllales, Fabidae) is sister to Stachyurus

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(Crossosomatales, Malvidae) in agreement with Qiu et al. (2010). However, the

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placement of Guaiacum differs from those obtained in studies based largely on

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chloroplast genes (see APG III, 2009; Soltis et al., 2011). Analyses of the four 12 / 40

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individual mitochondrial genes either show weak (< 60%) BS support linking COM

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with Malvidae or yield trees that are unresolved, with little, if any, support for the

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monophyly of either clade or Rosidae (Table S1). RY coding greatly reduces support

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for relationships within Rosidae, with no support even for the monophyly of COM

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(not shown). AA coding indicates 74% BS support for a clade of COM species and all

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Malvidae except Stachyurus and Oenothera (Figure S4). Removing the most variable

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5% of sites yields 100% BS support for a clade of COM species and all Malvidae

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except Stachyurus and Oenothera. However, removing more variable sites greatly

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reduces support for relationships throughout the tree; after removing the 10% most

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variable sites, BS support for COM drops to 23%. After removing the third codon

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position from the 4-gene mitochondrial data set, there is 31% BS support for a clade

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of COM species and all Malvidae except Stachyurus and Oenothera (Figure S5).

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The results from analyses of the 92-taxon, 5-gene nuclear data set provide 100%

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BS support for a clade that includes COM species and all species of Malvidae except

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Pelargonium, Oenothera, and Lagerstroemia (Figure 3), as do the results from ML

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analyses of the RY and AA matrices (Figures S6, S7). This placement of COM with

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Malvidae is also in agreement with the ML analyses of the five individual nuclear

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genes, although with different levels of support (Table S1). Likewise, the ML analyses

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of nucleotides after removing 5% and 10% of the most variable sites yield 100% BS

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support for a clade that includes all of the COM species and Malvidae species, except

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Pelargonium, Oenothera, and Lagerstroemia. Removing 20% or 30% of the most

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variable sites reduces the support for this clade to 96% and 94%, respectively, but 13 / 40

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removing more sites greatly reduces support for relationships within Rosidae in

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general, including support for the monophyly of COM. Removing the third codon

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position from the 5-gene nuclear data set still resulted in 100% BS support for a clade

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of COM and all Malvidae species except Pelargonium, Oenothera, and Lagerstroemia

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(Figure S8).

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3.2 Single-copy nuclear gene analysis

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Although most of the orthologous gene sets from the Lee et al. (2011) analysis were

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not informative regarding the placement of COM, among those genes that do support

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one of the three possible placements (COM with Fabidae, COM with Malvidae, or

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COM outside of Fabidae + Malvidae), 62–75% support a clade of COM with

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Malvidae, 25-38% support a clade of COM with Fabidae, and none of the

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orthologous gene sets support COM outside of Fabidae + Malvidae (Table 2). While

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increasing the minimum bootstrap support cutoff reduces the number of orthologous

295

gene sets supporting each hypothesis, it has relatively little effect on the percentage of

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informative genes supporting COM with Malvidae versus COM with Fabidae (Table

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2).

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3.3 Multi-copy nuclear gene analysis

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Similar to the single-copy nuclear gene analysis, most of the multi-copy gene trees

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were not informative regarding the placement of COM, but the majority of

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informative genes support the placement of COM with Malvidae. Between 71–98% of

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the informative genes support a clade of COM and Malvidae species, depending on

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the model of reconciliation and the minimum bootstrap support level (Table 3). The 14 / 40

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duplication-only model provides the strongest support for a clade of COM with

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Malvidae (≥ 91%; Table 3). The maximum percentage of informative genes

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supporting COM with Fabidae is 27%, which is based on the deep coalescence

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reconciliation model (Table 3). A clade of COM outside of Fabidae + Malvidae is

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recovered by 0–6% of the genes in these analyses (Table 3).

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4. Discussion

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4.1 Conflict among multi-locus phylogenetic analyses

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In spite of much recent progress resolving the angiosperm tree of life, the

312

phylogenetic placement of COM remains uncertain. Most previous efforts to place

313

COM have used a variety of data sources, taxon sampling strategies, and phylogenetic

314

methods (but see Xi et al., 2014). Therefore, it is difficult to determine if the

315

conflicting placements of COM are due to errors or actual biological conflict among

316

loci (Table 1). Our ML analyses of multi-gene chloroplast, mitochondrial, and nuclear

317

data sets with similar taxon sampling reinforce the observation that analyses of

318

chloroplast loci yield a topology that differs from analyses of mitochondrial and most

319

nuclear loci (Table 1; Figures 1–3). The isolated placements of Lagerstroemia,

320

Oenothera, Pelargonium, and Stachyurus make Malvidae non-monophyletic in some

321

of our analyses of mitochondrial and nuclear data sets; however, their positions in our

322

trees are consistent with those of Qiu et al. (2010) and Zhang et al. (2012),

323

respectively. Furthermore, these four genera are respectively from Myrtales

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(Lagerstroemia, Lythraceae; Oenothera, Onagraceae), Geraniales (Pelargonium, 15 / 40

325

Geraniaceae), and Crossosomatales (Stachyurus, Stachyuraceae), the exact placement

326

of which within Rosidae varies among chloroplast, mitochondrial, and nuclear trees

327

(e.g., Morton, 2011; Qiu et al., 2010; Soltis et al., 2011; Xi et al. 2014; Zhang et al.,

328

2012; Zhu et al., 2007).

329

Our analyses of the chloroplast, mitochondrial, and nuclear data sets are robust to

330

different character-coding strategies, which are often used to detect heterogeneous

331

phylogenetic signals or error. AA matrices and RY coding are used to ameliorate

332

nucleotide saturation and composition biases (Delsuc et al., 2005; Gibson et al., 2005;

333

Harrison et al., 2004; Hashimoto et al., 1995; Phillips and Penny, 2003), and removal

334

of highly variable sites has been proposed to reduce long-branch attraction or

335

model-fitting error (see Philippe et al., 2005). Some of these experiments erode

336

phylogenetic signal for the placement of COM, but none support an alternative

337

placement of COM. Although we failed to find obvious signs of major systematic or

338

sampling biases, it is difficult to demonstrate the absence of error. In fact, the (weakly

339

supported) variation in single-gene topologies of linked chloroplast genes suggests

340

that some level of error may be present in chloroplast gene sequence analyses (Table

341

S1). Nonetheless, the consistency of the incongruence suggests that there may be an

342

underlying biological basis to the conflict among chloroplast, nuclear, and

343

mitochondrial loci.

344

4.2 Evolutionary patterns suggested by conflict among nuclear loci

345

If the conflict among chloroplast, nuclear, and mitochondrial gene sequence data is

346

due to evolutionary events such as ancient hybridization or incomplete lineage sorting, 16 / 40

347

we would also expect to see conflict among independent nuclear loci. Indeed, within

348

the single-copy nuclear gene data set from Lee et al. (2011), on average 66% of the

349

informative genes support a placement of COM with Malvidae, while on average 34%

350

weakly support the placement of COM with Fabidae (Table 2). The multi-copy genes

351

reveal similar levels of incongruence, with at least 71% of the informative genes

352

supporting COM with Malvidae, with far less support for COM with Fabidae and

353

very little support for COM outside a clade of Malvidae + Fabidae (Table 3). This

354

predominant placement of COM with Malvidae within multi-copy gene trees is

355

consistent with previous gene tree parsimony analyses (Burleigh et al., 2011; Górecki

356

et al., 2012). The placement of COM from multi-copy genes is robust to the model of

357

gene reconciliation (Table 3). Furthermore, in both the single- and multi-copy gene

358

results, the overall percentage of informative genes supporting each of the three

359

hypotheses is relatively stable no matter the bootstrap cutoff we use (Tables 2, 3).

360

If the differences in the position of COM among nuclear loci are not due to

361

errors, they may reflect biological processes such as ancient hybridization and/or

362

incomplete lineage sorting. Distinguishing between incomplete lineage sorting and

363

hybridization can be challenging (e.g., Buckley et al., 2006; Joly et al., 2009; Holder

364

et al., 2001; Holland et al., 2008; Sang and Zhong, 2000), and the sparse and

365

incomplete taxon sampling within our nuclear gene data sets, as well as the ancient

366

divergence time of the major rosid lineages (Bell et al., 2010; Wang et al., 2009),

367

make it especially difficult to differentiate between the two. Although the effects of

368

incomplete lineage sorting typically are studied on recent radiations, they can also 17 / 40

369

obscure the resolution of ancient radiations (Oliver, 2013; Whitfield and Lockhart,

370

2007), such as the deep relationships among mammals (McCormack et al., 2012;

371

Song et al., 2012). If we consider the placement of COM as a rooted 3-taxon (COM,

372

Fabidae, and Malvidae) phylogenetic problem, a process of incomplete lineage

373

sorting should yield approximately equal numbers of nuclear genes supporting the

374

two possible non-species tree topologies (Huson et al., 2005). Instead, we see that the

375

majority of genes supports COM with Malvidae, with far less support for COM with

376

Fabidae, and almost no support for COM outside of Fabidae + Malvidae (Tables 2, 3).

377

The differences in levels of support from nuclear loci suggest that incomplete lineage

378

sorting does not explain the phylogenetic discordance among genes. However, since

379

both Malvidae and Fabidae are not clades in some analyses (Figures 1–3), it is

380

possible that this 3-taxon case does not apply, and the expected patterns of support

381

from lineage sorting with more than three species are more complex (e.g., Rosenberg

382

and Nordberg, 2002; Degnan and Rosenberg, 2006).

383

Many plant lineages have experienced hybridization and introgression

384

throughout their evolutionary histories (e.g., Okuyama et al., 2005), and there are

385

more than a hundred records of interspecific hybridization among rosid taxa alone

386

(Rieseberg and Soltis, 1991; Rieseberg et al., 1996a). An ancient introgressive

387

hybridization event would likely produce conflict among independent loci (Wendel

388

and Doyle, 1998). The different placement of COM in trees constructed from

389

mitochondrial and chloroplast gene sequence data suggests that the evolutionary

390

histories of these two subcellular compartments are unlinked, with the chloroplast 18 / 40

391

genome derived from the Fabidae lineage and the mitochondrial genome from the

392

Malvidae lineage. This result is unexpected given that the chloroplast and

393

mitochondrial genomes typically are both maternally inherited in angiosperms (Birky,

394

1995, 2001; Corriveau and Coleman, 1988; Mogensen, 1996). However, there are

395

documented cases of biparental inheritance of organellar genomes (e.g., Fauré et al.,

396

1994; Havey et al., 1998; Testolin and Cipriani, 1997; Yang et al., 2000), and paternal

397

inheritance of chloroplast genomes has been documented in COM species (Turnera

398

ulmifolia; Shore et al., 1994; Shore and Triassi, 1998) and Fabidae (Medicago sativa;

399

Masoud et al., 1990; Schumann and Hancock, 1989; Larrea; Yang et al., 2000).

400

Additionally, empirical studies suggest that progeny from a hybridization event may

401

exhibit strong paternal chloroplast inheritance, while mitochondrial inheritance

402

remained exclusively maternal (Schumann and Hancock, 1989; Masoud et al., 1990;

403

Shore et al., 1994; Xu, 2005). Thus, it is conceivable that an ancient hybridization

404

event resulted in different evolutionary histories for the chloroplast and mitochondrial

405

genomes.

406

In this putative ancient hybridization scenario, an early member of Fabidae or its

407

immediate ancestor acted as the paternal parent and crossed with the maternal lineage

408

of a member of Malvidae, with accompanying paternal transmission of the chloroplast

409

to the ancestor of COM (F1). This event could have created conflicting histories in the

410

chloroplast and mitochondrial genomes and conflict among nuclear loci with half of

411

the alleles in the F1 contributed by each parent (Figures 1, 2, 4). Repeated selfing or

412

crossing of the hybrid derivatives would not explain the high percentage of nuclear 19 / 40

413

loci supporting the relationship of COM with Malvidae (Tables 2, 3), suggesting the

414

possibility of subsequent backcrosses of the early hybrids to the maternal Malvidae,

415

and reducing the number of nuclear loci supporting the placement of COM with

416

Fabidae (Figure 4). Considering the difficulty of producing fertile hybrids from

417

crosses of distantly related lineages, this proposed ancient hybridization event should

418

be viewed with caution.

419

5. Concluding remarks

420

Numerous plant systematics studies have demonstrated the promise of genomic

421

data to resolve angiosperm relationships that were not evident in analyses with a few

422

genes (Burleigh et al., 2011; Finet et al., 2010; Lee et al., 2011; Moore et al., 2010,

423

2011; Zeng et al., 2014). We demonstrate here that analyses of data sets with many

424

unlinked loci can highlight the ambiguity and discordance in phylogenetic

425

relationships and potentially reveal the complexity of angiosperm evolution. Most, but

426

not all, single- and multi-copy nuclear loci, as well as mitochondrial genes, support

427

the placement of COM with Malvidae. This placement is also consistent with patterns

428

of morphological evolution (Endress and Matthews, 2006), but it contradicts the

429

strongly supported analyses of chloroplast sequence data sets (Figures 1–4; Jansen et

430

al., 2007; Moore et al., 2010, 2011; Ruhfel et al., 2014). While analyses involving a

431

single data source, such as the chloroplast genome, seek a single phylogeny, it may be

432

more informative to appreciate the potentially chimeric origins of COM rather than to

433

force its placement in a binary species tree. Although with current sampling we cannot 20 / 40

434

conclusively infer the processes that caused the conflicting placement of COM, our

435

analyses emphasize the importance of phylogenomic data for highlighting

436

phylogenetic incongruence and directing future studies.

437

Acknowledgements

438

We thank Yin-Long Qiu, who contributed to the early design of this project, and Ning

439

Zhang, who graciously provided us with the 92-taxon, 5-gene nuDNA alignment used

440

in this study. This work was supported by the National Natural Science Foundation of

441

China (NNSF 31270268), National Basic Research Program of China (No.

442

2014CB954101), Chinese Academy of Sciences Visiting Professorship for Senior

443

International Scientists (grant number 2011T1S24), State Key Laboratory of

444

Systematic and Evolutionary Botany (grant number LSEB2011-10), and the US

445

National Science Foundation (DEB-1301828).

446

The authors declare no conflict of interest.

447

21 / 40

448

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810

30 / 40

811

Figure Legends

812

Figure 1: Majority-rule consensus tree of Maximum Likelihood (ML) bootstrap

813

analysis of 78-gene chloroplast matrix. These data place COM with Fabidae. To

814

highlight the position of COM, only the Fabidae, COM, and Malvidae clades were

815

labeled, and COM is isolated from the circumscription of Fabidae as previously

816

defined (Cantino et al., 2007). All the familial and ordinal names of the sampled taxa

817

follow APG III (2009). Numbers above branches are bootstrap percentages (BS).

818

Figure 2: Majority-rule consensus tree of Maximum Likelihood (ML) bootstrap

819

analysis of 4-gene mitochondrial matrix. These data place COM with Malvidae.

820

Figure 3: Majority-rule consensus tree of Maximum Likelihood (ML) bootstrap

821

analysis of 5-gene nuclear matrix. These data place COM with Malvidae.

822

Figure 4: Hypothetical reticulation scenario for the origin of COM from the ancestral

823

Fabidae and Malvidae lineages. Large circles reflect the plant lineages; the small

824

circles represent their nuclear DNA types (the red circle represents the Fabidae

825

nuclear DNA and the blue one the Malvidae nuclear DNA), the ovals represent the

826

chloroplast (the green ovals represent the Fabidae chloroplast, and the gray oval

827

represents the chloroplast from the Malvidae ancestor), and the diamonds represent

828

the mitochondria (the gray diamond represents the Fabidae mitochondrion and the

829

orange diamond the Malvidae mitochondrion); dashed arrows represent multiple

830

generations of backcrossing. During hybridization, the mitochondrion is maternally

831

inherited from the Malvidae ancestor, and the chloroplast is paternally inherited from 31 / 40

832

the Fabidae ancestor. After subsequent F1 backcrosses to the Malvidae, the resulting

833

generations contain chloroplasts from Fabidae, mitochondria from Malvidae, and a

834

majority of the nuclear genes from Malvidae, with a smaller number from Fabidae

835

(roughly 25%). The reticulate phylogeny at the bottom illustrates this hypothetical

836

introgressive hybridization scenario and shows the phylogenetic incongruence among

837

the three genomes with respect to COM.

838

32 / 40

839

Table 1. Summary of the placement of COM in previous phylogenetic studies.

Genome type

a

Relationship

Method of analysis/Support

Nr

Character-state weighting/–

COM + Fabidae COM + Fabidae COM + Fabidae

b

Parsimony/52% JK; BI/1.0 PP Parsimony jackknifing/77% JK; BI/1.0 PP ML/100% BS; MP/79% BS; BI/1.0 PP

Chloroplast

499

–

Chase et al., 1993

matK

374

16

Hilu et al., 2003

rbcL, atpB, 18S rDNA

560

64

81 cp

64

3

Jansen et al., 2007

567

59

Burleigh et al., 2009

rbcL, atpB, matK,

Soltis et al., 2000; Soltis et al.,2007

COM + Fabidae

ML/100% BS

10 cp, 2 nu

117

33

Wang et al., 2009

COM + Fabidae

ML/53% BS

83 cp

86

5

Moore et al., 2010

IR

244

14

Moore et al., 2011

11 cp, 2 nu, 4 mt

640

154

Soltis et al., 2011

78 cp

360

9

Ruhfel et al., 2014

COM + Fabida

33 / 40

rbcL

References

ML/89% BS

COM + Fabidae

Nuclear

COM sampling

c

COM + Fabidae

COM + Fabidae

Mitochondrial

Taxa Number

Marker

ML/99% BS (244 taxa); ML/89% BS (87 taxa) ML/57% BS ML/81% BS, 70% BS, 82% BS, 69% BS (ntAll, ntNo3rd, RY, AA)

18S rDNA, 26S rDNA

Malpighiales-F

ML/100% BS; BI/1.0 PP

45 cp

41

3

Xi et al., 2014

COM + Malvidae

ML/54% BS; MP/–

matR

174

21

Zhu et al., 2007

COM + Malvidae

ML/99% BS

atp1, matR, nad5, rps3

380

26

Qiu et al., 2010

Nr

–

18S rDNA

233

/

Oxalidales-M

ML/55% BS

Xdh

247

19

Morton 2011

COM + Malvidae

ML/>95% BS; BI/1.0 PP

SMC1, SMC2, MCM5,

94

5

Zhang et al., 2012

Soltis et al., 1997

MLH1, MSH1 Malpighiales-M COM + Malvidae Malpighiales-M

840

Note:

841

a

GTP-ML/18% BS (136 taxa); GTP-ML/75% BS (54 taxa) ML/>95% BS; MP/≤65% BS STAR/100% BP; MP-EST/100% BP; ML/100% BP; PhyloBayes/1.0 PP

18,896 gene trees

136

15

Burleigh et al., 2010

nuclear genome

101

7

Lee at al. 2011

310 nu

46

3

Xi et al., 2014

Nr = not resolved; COM + Fabidae = COM clade was placed in Fabidae; Malpighiales-F = only one member of COM Malpighiales, was

842

sampled and placed in Fabidae; COM + Malvidae = COM clade sister to Malvidae; Oxalidales-M = only Oxalidales sister to Malvidae;

843

Malpighiales-M = only Malpighiales sister to Malvidae.

844

b

JK = Jackknife value; BI = Bayesian inference; BS = Bootstrap value; BP = Bootstrap percentage; PP = Posterior probabilities; GTP = Gene

845

tree parsimony; STAR = a coalescent method: Species Tree Estimation using Average Ranks of Coalescence (Liu et al., 2009); MP-EST = a

846

coalescent method: Maximum Pseudo-likelihood for Estimating Species Trees (Liu et al., 2010); PhyloBayes = a Bayesian Monte Carlo

847

Markov Chain (MCMC) sampler for phylogenetic reconstruction using infinite mixtures

848

(http://megasun.bch.umontreal.ca/People/lartillot/www/downloadmpi.html).

849

c

81 cp = 81 chloroplast genes (Jansen et al., 2007); 10 cp, 2 nu = 10 chloroplast genes, rbcL, atpB, matK, psbBTNH region (4 genes), rpoC2,

34 / 40

850

ndhF, and rps4, and two nuclear genes, 18S rDNA and 26S rDNA; 83 cp = 83 chloroplast genes (Moore et al., 2010); IR means 25,000-bp

851

plastid Inverted Repeat region; 11 cp, 2 nu, 4 mt = 11 chloroplast genes, rbcL, atpB, matK, psbBTNH region (4 genes), rpoC2, ndhF, rps4

852

and rps16, and two nuclear genes, 18S rDNA and 26S rDNA, and four mitochondrial genes, atp1, matR, nad5, and rps3; ntAll, ntNo3rd, RY,

853

AA = four different character-coding matrix; ntAll = all nucleotide positions analysis; ntNo3rd = the first and second codon positions

854

analysis; RY = RY-coded analysis; AA = the amino acid analysis; 78 cp = chloroplast genes (Ruhfel et al., 2014); 45 cp = chloroplast genes

855

selected from plastid genome (See Supplementary Table S2 in Xi et al., 2014); 310 nu = low-copy nuclear genes selected from nuclear

856

genome and transcripts (See Supplementary Table S1 in Xi et al., 2014).

35 / 40

857

Table 2. Results from the single-copy nuclear gene analysis based on the ortholog

858

alignments from Lee et al. (2011). % BS

Fabidae

Malvidae

Fabidae + Malvidae

50

115 (36%) 208 (64%)

0

60

68 (34%)

131 (66%)

0

70

49 (36%)

89 (64%)

0

80

29 (38%)

48 (62%)

0

90

15 (35%)

28 (65%)

0

100

3 (25%)

9 (75%)

0

859

Note:

860

The columns list the number of orthologs (out of 8,445) and the percentage (in

861

parentheses) of all the informative genes that support a ‗COM + Fabidae‘ topology

862

(―Fabidae‖ column), a ‗COM + Malvidae‘ topology (―Malvidae‖ column), or COM

863

outside Fabidae + Malvidae (―Fabidae + Malvidae‖ column) with at least 50%

864

bootstrap (BS) support.

865

36 / 40

866

Table 3. Results from the multi-copy gene tree analysis based on genome

867

sequences of 22 land plant taxa.

868

Gene Duplications % BS Fabidae

869

38 (3%) 973 (91%)

62 (6%)

60

17 (2%) 723 (94%)

29 (4%)

70

12 (2%) 515 (96%)

11 (2%)

80

4 (1%)

308 (98%)

3 (1%)

90

2 (1%)

155 (98%)

1 (1%)

100

1 (6%)

15 (94%)

0 (0%)

Gene Duplications and Losses Fabidae

Malvidae

Fabidae + Malvidae

50

446 (20%) 1718 (76%)

82 (4%)

60

267 (16%) 1390 (81%)

47 (3%)

70

149 (12%) 1049 (86%)

26 (2%)

80

72 (9%)

715 (90%)

11 (1%)

90

30 (8%)

371 (91%)

5 (1%)

100

7 (11%)

54 (89%)

0 (0%)

Malvidae

Fabidae + Malvidae

Deep Coalescence % BS

871

Fabidae + Malvidae

50

% BS

870

Malvidae

Fabidae

50

547 (27%) 1468 (71%)

40 (2%)

60

358 (23%) 1160 (75%)

25 (2%)

70

229 (20%)

884 (79%)

13 (1%)

80

114 (16%)

598 (83%)

8 (1%)

90

58 (16%)

299 (83%)

4 (1%)

100

7 (13%)

44 (85%)

1 (2%)

Note: 37 / 40

872

We calculated the reconciliation cost based on the minimum number of implied gene

873

duplications, duplications + losses, and deep coalescence events, for 100 ML

874

bootstrap gene trees made from 3,784 multi-copy gene alignments. In the table, we

875

list the number and percentage (in parentheses) of genes (out of 3,784) that have at

876

least 50% bootstrap (BS) support for the three possible topologies based on the

877

reconciliation cost, respectively.

878

38 / 40

879

Supplementary materials

880

Table S1. Individual gene analyses for COM clade from 78-gene chloroplast, 4-gene

881

mitochondrial and 5-gene nuclear data sets.

882

Figure S1. RY coding tree from Maximum Likelihood (ML) bootstrap analysis of

883

78-gene chloroplast data set.

884

Figure S2. AA tree from Maximum Likelihood (ML) bootstrap analysis of 78-gene

885

chloroplast data set.

886

Figure S3. Best tree from Maximum Likelihood (ML) bootstrap analysis based on the

887

first and second codon positions only of 78-gene chloroplast data set.

888

Figure S4. AA tree from Maximum Likelihood (ML) bootstrap analysis of 4-gene

889

mitochondrial data set.

890

Figure S5. Best tree from Maximum Likelihood (ML) bootstrap analysis based on the

891

first and second codon positions only of 4-gene mitochondrial data set.

892

Figure S6. RY coding tree from Maximum Likelihood (ML) bootstrap analysis of

893

5-gene nuclear data set.

894

Figure S7. AA tree from Maximum Likelihood (ML) bootstrap analysis of 5-gene

895

nuclear data set.

39 / 40

896

Figure S8. Best tree from Maximum Likelihood (ML) bootstrap analysis based on the

897

first and second codon positions only of 5-gene nuclear data set.

40 / 40

100

64

100 100

100

52 81 100

100 100

100

100

100

100 100

99 100 100

95 73

100 100 100 100

100

100 62

100

100

100

59 100

100

100

100

100 100 100 100

100

100

100 100

100

100 100 100

55

67

100

100

100 100 100

98 72

100

100

95 59

100

100

100 100

100 100

100

100

98 92

53 100 100 100 100

100

Malvidae

100

88

COM

Rosidae

Bulnesia arborea Quercus nigra Glycine max Cucumis melo Ficus sp Morus indica Pentactina rupicola Euonymus americanus Oxalis latifolia Populus alba Passiflora biflora Jatropha curcas Ricinus communis Manihot esculenta Arabidopsis thaliana Carica papaya Gossypium arboreum Citrus sinensis Staphylea colchica Eucalyptus globulus Oenothera parviflora Pelargonium x Vitis vinifera Liquidambar styraciflua Heuchera sanguinea Sesamum indicum Epifagus virginiana Olea woodiana Solanum lycopersicum Ipomoea purpurea Coffea arabica Nerium oleander Aucuba japonica Lonicera japonica Daucus carota Eleutherococcus senticosus Panax ginseng Lactuca sativa Ilex cornuta Arbutus unedo Rhododendron simsii Franklinia alatamaha Cornus florida Davidia involucrata Silene vulgaris Anredera baselloides Fagopyrum esculentum Gunnera manicata Buxus microphylla Platanus occidentalis Meliosma aff cuneifolia Ranunculus macranthus Megaleranthis saniculifolia Nandina domestica Ceratophyllum demersum Saccharum hybrid Sorghum bicolor Oryza nivara Brachypodium distachyon Renealmia alpinia Musa acuminata Phoenix dactylifera Yucca schidigera Asparagus officinalis Iris virginica Lilium superbum Dioscorea elephantipes Colocasia esculenta Lemna minor Acorus americanus Piper cenocladum Calycanthus floridus Magnolia kwangsiensis Liriodendron tulipifera Chloranthus spicatus Illicium oligandrum Nuphar advena Nymphaea alba Amborella trichopoda Picea morrisonicola Pinus taeda Cycas taitungensis

Fabidae

64

27 63 97

71 33 73 83 94

100 99

53

94 97 100

86

55

61

36 28

100

13 86

96 100

100

47 100

100 55

27

95

38 60 83

22

77

100

100 68

98

34

90

100

92

71 100 100 100

59

98 67 45

100 61 60 27

99

31 74

53 88 100 54

88

100

44 22 100

25 100

96 100 100 100 100

Malvidae

Rosidae

Quercus Cucurbita Medicago Zelkova Morus Spiraea Euonymus Oxalis Euphorbia Hypericum Arabidopsis Carica Gossypium Citrus Schinus Cupaniopsis Stachyurus Guaiacum Oenothera Phytolacca Polygonum Vitis Corylopsis Cercidiphyllum Paeonia Lamium Scrophularia Syringa Nerium Galium Nicotiana Ipomoea Ilex Garrya Lonicera Helianthus Apium Hedera Pittosporum Hydrangea Cornus Actinidia Arbutus Diospyros Gunnera Pachysandra Buxus Meliosma Platanus Ranunculus Glaucidium Nandina Dicentra Ceratophyllum Chloranthus Oryza Maranta Strelitzia Chamaedorea Asparagus Iris Dioscorea Lilium Alisma Spathiphyllum Laurus Calycanthus Magnolia Liriodendron Houttuynia Thottea Aristolochia Schisandra Illicium Nuphar Brasenia Amborella Pinus Zamia

Fabidae COM

32

100

94 80

47

100

100 71 100

Rosidae

100

100

100

90 68 100

81

96

100

62 100 37 100

64

100 100

100

96 100

100

47 63

100

100

100

100

85

74

100 100 73

100 100

50

100 100

100

100

100 99

100

100 100 76

100

100

100

100 100 100 77

76 79 100

100

60 50 100

100

100

100 100

85

97

100 63

99 100

87 47

74

100 58

100

100 100 100

88

Malvidae

100

Cyclobalanopsis glauca Glycine max Morus alba Ulmus macrocarpa Photinia serrulata Cucumis sativus Euonymus carnosus Hypericum chinense Populus tricocarpa Ricinus communis Manihot esculenta Arabidopsis thaliana Arabidopsis lyrata Carica papaya Hibiscus syriacus Poncirus trifoliata Tetradium ruticarpum Rhus chinensis Sapindus mukorossi Stachyurus yunnanensis Oenothera erythrosepala Lagerstroemia limii Pelargonium hortorum Stellaria media Phytolacca americana Polygonum runcinatum Distylium buxifolium Cercidiphyllum japonicum Paeonia lactiflora Vitis vinifera Mimulus guttatus Callicarpa bodinieri Jassminum nudiflorum Solanum lycopersicum Pharbitis nil Nerium oleander Vinca major Galium aparine Ilex purpurea Aucuba japonica Lactuca sativa Lonicera japonica Hedera nepalensis Ligusticum chuanxiong Pittosporum tobira Rhododendron pulchrum Actinidia arguta Diospyros kaki Philadelphus incanus Cornus officinalis Cornus wisoniana Gunnera manicata Buxus sinica Pachysandra terminalis Platanus acerifolia Meliosma parviflora Ranunculus muricatus Aquilegia coerulea Nandina domestica Dicentra spectabilis Sorghum bicolor Setaria italica Brachypodium distachyon Oryza sativa Canna indica Musa basjoo Trachycarpus fortunei Dioscorea opposita Yucca filamentosa Asparagus officinalis Iris japonica Lilium brownii Alisma plantago-aquatica Pinellia ternata Acorus calamus Cinnamomum camphora Chimonanthus praecox Magnolia denudata Liriodendron Houttuynia cordata Asarum heterotropoides Aristolochia fimbriata Ceratophyllum taobao Chloranthus Schisandra Illicium henryi Brasenia schreber Nuphar advena Amborella trichopoda Pinus Picea Zamia fischeri

Fabidae COM

78 86

♀ Malvidae ancestor

Fabidae ancestor ♂

× × COM ancestor

Fabidae

COM

Malvidae

Nuclear gene tree Chloroplast gene tree Mitochondrial gene tree

Highlights • Report phylogenetic conflict of COM in chloroplast, mitochondrial, and nuclear data. • Results of multi-gene and genomic data show strong evidence for deep incongruence. • We provide an example for examination of other deep nodes of the tree of life. • Genomic datasets highlight patterns of deep incongruence in angiosperm phylogeny. • Stress the complexity of angiosperm evolution, which may be masked by a few genes.

Fabidae ancestor ♂

×

Fabidae chloroplast DNA Fabidae nuclear DNA Malvidae nuclear DNA Malvidae mitochodrial DNA

Fabidae

×

COM

Malvidae

Nuclear gene tree

COM ancestor

Chloroplast gene tree Mitochondrial gene tree

Malvidae ancestor ♀