NSF Proposal - 4. "Deep" Problems in Green Plant Phylogeny

Current topology of the green plant tree (Fig. 1) arises from considerable morphological and ultrastructural data that have accumulated over the last three decades (e.g. [14-26]), and from the molecular tools that have been applied at many levels (e.g., [27-41]). Many recent advances reflect innovations in data gathering and analysis that have resulted from increased coordination of effort among different laboratories (see Management section). Despite these advances, several nodes in the "deep" phylogeny? remain unresolved (Fig. 1). We propose to resolve these problematic nodes.

Ambiguity in the origin of green plants. It is generally accepted that there are two principal clades of green plants: the streptophytes, consisting of the land plants and the green algae most closely related to them, and the chlorophytes, containing most of the remaining green algae. At the base of these two clades is a "residuum" consisting of unicellular "prasinophytes". These algae are regarded as ancestral within the green plants, on the basis of morphology and ultrastructure (e.g. [42]), nuclear gene sequences (e.g. [43, 32, 44]) and organelle genome features [45-48]. However, the relationships among the prasinophytes have been difficult to recover. One species only, Mesostigma viride, has been placed with some confidence at the base of the streptophytes [49, 50]. Most of the remaining prasinophytes have been placed at the base of the chlorophytes, but without clear affinities to any other chlorophyte lineage [44].

Which are the most ancestral prasinophytes? The "phycomate" prasinophytes (those with large, thick-walled floating stages, or "phycomata") are candidates. They have ultrastructural features common to both the chlorophyte and streptophyte lines [42], they are the only green algae with mixotrophy (nutrition by both photosynthesis and phagotrophy [51]), a presumptive precondition for endosymbiosis of chloroplasts, and they have a fossil record extending to the latest Precambrian and perhaps much earlier [52]. One extant genus, Tasmanites, has a fossil record dating back =600 million years, making it the oldest of all green plants [53, 54]. To test this result, we will incorporate a phycomate prasinophyte, Pterosperma, into our primary analysis, and attempt concatenation of our results with ongoing research on living (collaboration with Fawley) and fossil (collaboration with Knoll) prasinophytes. 0228655

Figure 1. Outline phylogeny of the green plants, indicating the currently understood phylogenetic position for 51 candidate exemplar organisms (represented by generic names; see Table 1) and the higher-level taxa to which they are thought to belong. All branches are subject to further testing, but the best- supported branches with current data are indicated with a thick line, branches with some support are indicated with a thin line, and areas of the tree that remain controversial are indicated by labelled ovals. See text for further explanation: 1. Base of green plants: outgroup relationships and basal branching among prasinophytes. 2. Relationships among major lineages of Chlorophyta (some groupings are more firmly established, e.g. Ulvales/Ulotrichales, Chlorophyceae/Trebouxiophyceae). 3. Relationships among the siphonous algae and their placement in the Chlorophyta. 4. Base of the land plants. 5. Base of the ferns (moniliforms).


To identify the ancestral prasinophyte, we need to define outgroups for green plants. Many recent gene-sequence trees indicate that the green plants are most closely related to the red algae and the glaucocystophytes, the other two groups of algae that appear to have gained their chloroplasts through a primary endosymbiosis with a cyanobacterium [55]. However, red algae and glaucocystophytes differ significantly from each other and from green plants in key morphological, reproductive and ultrastructural features, so much so that homologies are difficult to establish (e.g. [42]), and the molecular results have been questioned (e.g. [56]). The most ancient green algae have organellar genomes that may be more ancestral (more like eubacteria) than those in the red algae examined to date [57, 45-48]. We will test the idea that red and glaucocystophyte algae represent the proximal outgroups for green plants by incorporating published data from selected representatives of these outgroups into our analyses, and by interacting with colleagues who will be conducting research on potential outgroups from among both algae and protozoa (collaboration with Lang).

The Ulvophyceae. The Ulvophyceae is one of the 3 classes currently recognized in the chlorophyte lineage, the others being Chlorophyceae and Trebouxiophyceae (formerly Pleurastrophyceae; [58]). Most members are marine, and the majority of green "seaweeds", including well-known species of Ulva, Acetabularia and Caulerpa, are placed in this class. Conversely, the Chlorophyceae and Trebouxiophyceae, and the streptophytes, consist almost entirely of non-marine organisms. In classifications based on morphology and ultrastructure [59, 60, 42], the Ulvophyceae have been separated from other chlorophytes mostly on the basis of characters associated with mitosis, cytokinesis, and the flagellar apparati of zoospores and gametes. In molecular analyses, however, the relationships among these three classes are less clear [61]. Moreover, the "siphonous" orders of Ulvophyceae (Cladophorales, Dasycladales, Caulerpales) are difficult to resolve vis-à-vis each other and with other Ulvophyceae (orders Ulotrichales and Ulvales); phylogenetic trees based on single gene sequences reveal long branch lengths between "siphonous" sequences and those of other chlorophytes [59, 62] (O'Kelly unpublished, Friedl unpublished).

Are Ulvophyceae as conceived by Floyd and O'Kelly [64] monophyletic, and how are ulvophyte clade(s) related to other chlorophytes? We think it possible that the "siphonous" "ulvophyceae" represent a clade separate from, and basal to, the remaining chlorophytes (Chlorophyceae, Trebouxiophyceae and non-siphonous "Ulvophyceae"). We will test this idea by incorporating both siphonous and non-siphonous Ulvophyceae, together with representative Chlorophyceae and Trebouxiophyceae, into our large-scale analysis, and attempt concatenation of our results with those from other ongoing research on ulvophytes (O'Kelly ulvophyte grant). Some algae in our large-scale investigations, particularly Acrochaete, Blastophysa, Bolbocoleon, Halochlorococcum, Ignatius, Ostreobium, and Trentepohlia, we selected because very recent research (O'Kelly and Friedl, unpublished) suggests they may break up some of the long ulvophyte branches.

The "bryophytes" - early embryophyte radiation. There is no consensus as to the primary branching patterns at the base of the land plants. Molecular and morphological evidence [63-67, 25] suggests that either hornworts or liverworts are the oldest living lineage of land plants. In the ?hornworts- basal? hypothesis, a moss + liverwort clade is typically supported. In the ?liverworts-basal? hypothesis, all three main bryophyte lineages are paraphyletic, with either hornworts or mosses sister to the vascular plants. Fossil evidence supports the liverworts-basal hypothesis, as the first unambigous hornwort fossils date to the Cretaceous and liverwort fossils to the Devonian. However, ornamentation of Paleozoic spores from the Silurian (>410 Mya) are comparable to that of Anthoceros, raising the possibility that hornworts were the first bryophyte lineage to appear. Monophyly of the mosses is widely accepted, but the interrelationships among the four major moss lineages are unresolved. The hornworts are undoubtedly monophyletic but within-group phylogeny has not been fully explored. Liverworts are highly diverse and interrelationships are ambiguous to the extent that monophyly of the group is questionable. DNA sequence data have been equivocal, supporting a number of conflicting branching orders, in part due to poor taxon sampling or limited sequence lengths [68-70, 66, 35, 24, 39]. Our large-scale analysis is likely to resolve the bryophyte tangle.

The basal vascular plant radiation. Within the tracheophytes, the lycophytes are sister to all other tracheophytes (e.g., [71, 72]), a result supported by analyses of both morphological and DNA sequence data. A comprehensive analysis of morphological and molecular characters in basal tracheophytes [36] produced the topology: (lycophytes (((Psilotum + ophioglossoid ferns) + (Equisetum + marattioid ferns + leptosporangiate ferns)) + seed plants)). This novel topology unites horsetails together with all ferns as a monophyletic group that is sister to seed plants and refutes the earlier view that horsetails are transitional evolutionary grades between bryophytes and seed plants. This conclusion was supported consistently by data from morphology and spermatogenesis [73, 24, 25]. The sister relationship of Psilotum with ophioglossoid ferns, which was suggested previously (e.g., [74, 70, 75, 76]), is now strongly supported [36].

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