Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae)

We have determined the complete chloroplast genome sequences of four early-diverging lineages of angiosperms, Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae), to examine the organization and evolution of plastid genomes and to estimate phylogenetic relationships among angiosperms. For the most part, the organization of these plastid genomes is quite similar to the ancestral angiosperm plastid genome with a few notable exceptions. Dioscorea has lost one protein-coding gene, rps16; this gene loss has also happened independently in four other land plant lineages, liverworts, conifers, Populus, and legumes. There has also been a small expansion of the inverted repeat (IR) in Dioscorea that has duplicated trnH-GUG. This event has also occurred multiple times in angiosperms, including in monocots, and in the two basal angiosperms Nuphar and Drimys. The Illicium chloroplast genome is unusual by having a 10 kb contraction of the IR. The four taxa sequenced represent key groups in resolving phylogenetic relationships among angiosperms. Illicium is one of the basal angiosperms in the Austrobaileyales, Chloranthus (Chloranthales) remains unplaced in angiosperm classifications, and Buxus and Dioscorea are early-diverging eudicots and monocots, respectively. We have used sequences for 61 shared protein-coding genes from these four genomes and combined them with sequences from 35 other genomes to estimate phylogenetic relationships using parsimony, likelihood, and Bayesian methods. There is strong congruence among the trees generated by the three methods, and most nodes have high levels of support. The results indicate that Amborella alone is sister to the remaining angiosperms; the Nymphaeales represent the next-diverging clade followed by Illicium; Chloranthus is sister to the magnoliids and together this group is sister to a large clade that includes eudicots and monocots; and Dioscorea represents an early-diverging lineage of monocots just internal to Acorus.     

Complete Sequence of the Duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis>) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.     

Was the ANITA rooting of the angiosperm phylogeny affected by long-branch attraction? Amborella, Nymphaeales, Illiciales, Trimeniaceae, and Austrobaileya

Five groups of basal angiosperms, Amborella, Nymphaeales, Illiciales, Trimeniaceae, and Austrobaileya (ANITA), were identified in several recent studies as representing a series of the earliest-diverging lineages of the angiosperm phylogeny. All of these studies except one employed a multigene analysis approach and used gymnosperms as the outgroup to determine the ingroup topology. The high level of divergence between gymnosperms and angiosperms, however, has long been implicated in the difficulty of reconstructing relationships at the base of angiosperm phylogeny using DNA sequences, for fear of long-branch attraction (LBA). In this study, we replaced the gymnosperm sequences from the five-gene matrix (mitochondrial atp1 and matR, plastid atpB and rbcL, and nuclear 18S rDNA) used in our earlier study with four categories of divergent sequences--random sequences with equal base frequencies or equally AT- and GC-rich contents, homopolymers and heteropolymers, misaligned gymnosperm sequences, and aligned lycopod and bryophyte sequences--to evaluate whether the gymnosperms were an appropriate outgroup to angiosperms in our earlier study that identified the ANITA rooting. All 24 analyses performed rooted the angiosperm phylogeny at either Acorus or Alisma (or Alisma-Triglochin-Potamogeton in one case due to use of a slightly different alignment) and placed the monocots as a basal grade, producing genuine LBA results. These analyses demonstrate that the identification of ANITA as the basalmost extant angiosperms was based on historical signals preserved in the gymnosperm sequences and that the gymnosperms were an appropriate outgroup with which to root the angiosperm phylogeny in the multigene sequence analysis. This strategy of evaluating the appropriateness of an outgroup using artificial sequences and a series of outgroups with increments of divergence levels can be applied to investigations of phylogenetic patterns at the bases of other major clades, such as land plants, animals, and eukaryotes.     

Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms

Recent contributions from DNA sequences have revolutionized our concept of systematic relationships in angiosperms. However, parts of the angiosperm tree remain unclear. Previous studies have been based on coding or rDNA regions of relatively conserved genes. A phylogeny for basal angiosperms based on noncoding, fast-evolving sequences of the chloroplast genome region trnT-trnF is presented. The recognition of simple direct repeats allowed a robust alignment. Mutational hot spots appear to be confined to certain sectors, as in two stem-loop regions of the trnL intron secondary structure. Our highly resolved and well-supported phylogeny depicts the New Caledonian Amborella as the sister to all other angiosperms, followed by Nymphaeaceae and an Austrobaileya-Illicium-Schisandra clade. Ceratophyllum is substantiated as a close relative of monocots, as is a monophyletic eumagnoliid clade consisting of Piperales plus Winterales sister to Laurales plus Magnoliales. Possible reasons for the striking congruence between the trnT-trnF based phylogeny and phylogenies generated from combined multi-gene, multi-genome data are discussed.     

Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms

Sequences from 14 slowly evolving chloroplast genes (including three highly conserved introns) were obtained for representative basal angiosperm and seed-plant taxa, using novel primers described here. These data were combined with published sequences from atpB, rbcL, and newly obtained sequences from ndhF. Combined data from these 17 genes permit sturdy, well-resolved inference of major aspects of basal angiosperm relationships, demonstrating that the new primers are valuable tools for sorting out the deepest events in flowering plant phylogeny. Sequences from the inverted repeat (IR) proved to be particularly reliable (low homoplasy, high retention index). Representatives of Cabomba and Illicium were the first two successive branches of the angiosperms in an initial sampling of 19 exemplar taxa. This result was strongly supported by bootstrap analysis and by two small insertion/deletion events in the slowly evolving introns. Several paleoherb groups (representatives of Piperales) formed a strongly supported clade with taxa representing core woody magnoliids (Laurales, Magnoliales, and Winteraceae). The monophyly of the sampled eudicots and monocots was also well supported. Analyses of three major partitions of the data showed many of the same clades and supported the rooting seen with all the data combined. While Amborella trichopoda was supported as the sister group of the remaining angiosperms when we added Amborella and Nymphaea odorata to the analysis, a strongly conflicting rooting was observed when Amborella alone was added.     

Angiosperm phylogeny based on matK sequence information

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.     

Floral variation and floral genetics in basal angiosperms

Recent advances in phylogeny reconstruction and floral genetics set the stage for new investigations of the origin and diversification of the flower. We review the current state of angiosperm phylogeny, with an emphasis on basal lineages. With the surprising inclusion of Hydatellaceae with Nymphaeales, recent studies support the topology of Amborella sister to all other extant angiosperms, with Nymphaeales and then Austrobaileyales as subsequent sisters to all remaining angiosperms. Notable modifications from most recent analyses are the sister relationships of Chloranthaceae with the magnoliids and of Ceratophyllaceae with eudicots. We review "trends" in floral morphology and contrast historical, intuitive interpretations with explicit character-state reconstructions using molecular-based trees, focusing on (1) the size, number, and organization of floral organs; (2) the evolution of the perianth; (3) floral symmetry; and (4) floral synorganization. We provide summaries of those genes known to affect floral features that contribute to much of floral diversity. Although most floral genes have not been investigated outside of a few model systems, sufficient information is emerging to identify candidate genes for testing specific hypotheses in nonmodel plants. We conclude with a set of evo-devo case studies in which floral genetics have been linked to variation in floral morphology.     

Recent progress in reconstructing angiosperm phylogeny

In the past year, the study of angiosperm phylogeny has moved from tentative inferences based on relatively small data matrices into an era of sophisticated, multigene analyses and significantly greater confidence. Recent studies provide both strong statistical support and mutual corroboration for crucial aspects of angiosperm phylogeny. These include identifying the earliest extant lineages of angiosperms, confirming Amborella as the sister of all other angiosperms, confirming some previously proposed lineages and redefining other groups consistent with their phylogeny. This phylogenetic framework enables the exploration of both genotypic and phenotypic diversification among angiosperms.     

Amborella not a "basal angiosperm"? Not so fast

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: 1499-1505). 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 flowering 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 taxa-Nymphaea, Austrobaileya, magnoliids, and monocots-to the 12-taxon 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.