NSF Proposal - 7. Phylogenetic Analysis

Principles of OTU and character selection. Due to the integrative nature of the proposed analyses, in which data from many sources will be considered, the concepts of "OTU" and character will vary within and among datasets. Data at this level are always compiled from study of different organisms considered to represent the same OTU. Thus OTUs are always composites in practice; their composition varying depending on the scale of analysis. Likewise, what counts as a useful character changes depending on the scale of analysis. The columns in a data matrix are already refined hypotheses of phylogenetic homology. There is also a clear reciprocal relationship between OTUs and characters. An OTU can best be defined as a set of individual samples that are homogeneous for characters currently known, while a character can be defined as a potential marker for shared history of some subset of the known OTUs. This means that OTUs and characters emerge during a process of "reciprocal illumination." To a large extent their definitions are interlinked, so how do we proceed empirically in a way that avoids circularity" We will take great care to examine both concepts of character and OTU in the proposed research. We will use the relatively advanced state of knowledge of characters and phylogenetic structure in the green plants as a model system for testing alternative approaches to analysis in a systematic manner. The overarching goal is to develop ways to scale OTU composition and character definition up and down the many fractally-nested levels making up the tree of life.

Character analysis. Phylogenetic analysis can be broken down into two discrete phases: character analysis and cladistic analysis. In the former phase, a data matrix is assembled as discussed above. Potential characters are evaluated by rules of character analysis, an evaluation of evidence for:(1) homology and heritability of a character across the taxa being studied, (2) independent evolution of different characters, and (3) presence in each character of a system of at least two discrete states. These criteria will be applied here to data matrices assembled at several scales of analysis. The deepest scale will be a matrix of the ca. 50 exemplar green plants plus outgroups (see section 4), with much of the data newly generated from this proposal. These OTUs will be thoroughly studied for the morphological characters covered above, and have completely sequenced mitochondrial and chloroplast genomes. We will also have nuclear BAC libraries constructed for most exemplars, which will facilitate discovering gene translocations between the organellar genome and the nuclear genome, and sequencing of new candidate nuclear genes. Characters will be evaluated in the following categories:

1. Genomic characters. Structural genomic differences resulting from inversions, translocations, gene losses, duplications, and insertion/deletion of introns will be identified within and between the three genomes and likely homologies established (e.g, examining the ends of breakpoints to see whether a single event is likely to have occurred).
2. Morphological characters. All features that can be compared across this deep level of analysis will be evaluated for independence and discrete states. The literature will be used, but wherever possible original material will be reexamined.
3. DNA sequence data. To compare DNA sequence characters with genomic and morphological characters, we will also align all genes available in the three genomes, We will do this two ways (in order to compare results): a liberal alignment using as much sequence as possible, and a conservative alignment using only regions that are unambiguously alignable. Both amino acid and nucleotide alignments will be analyzed where appropriate for protein coding genes.

Matrices will be developed for local clades using data appropriate at that level. These data will come almost entirely from other research groups and collaborators as discussed in the management plan section.

Cladistic analysis. The second phase of phylogenetic analysis involves turning data matrices into a recontructions of a phylogenetic tree. We will explore the full spectrum of approaches to building phylogenetic trees from data matrices and how to concatenate the results from the different scales of phylogenetic analysis to be undertaken here. We will use only character-based methods of phylogenetic analysis, and mainly work within a maximum parsimony framework (given the very heterogeneous set of characters). However, we will compare and contrast equal and differentially-weighted parsimony and maximum likelihood methods as applied to DNA sequence data. The first task will be to analyse the data matrix of the ca. 50 exemplar taxa, and the mixture of genomic, morphological, and DNA sequence characters discussed above, to produce a "backbone" phylogeny of basal green plants. The next task will be to use this sparsely-sampled, but extremely character-rich, global phylogeny to connect up all the many local phylogenetic data sets available from other research groups. These local data sets sample many more taxa (thousands taken all together), but with considerably less character data available.

For this second task we will assemble all published phylogenetic trees on the relevant chlorophyte and streptophyte lineages (e.g., references above), and will closely coordinate our efforts with ongoing phylogenetic projects of direct relevance to ours (see "Management Plan"). We will insure that all relevant phylogenetic studies are entered into TreeBASE (www.treebase.org), thereby providing ready access to project members (and the entire scientific community) to phylogenetic knowledge on green plant lineages.

Concatenation analyses. This assembly of individual phylogenetic trees and data sets will be critical to the construction of large-scale concatenated trees. In collaboration with M. Sanderson (UC Davis) we will use green plant phylogenies to explore a variety of algorithms for producing supermatrices and supertrees, such as Matrix Representation Parsimony (e.g., [133-135]) and methods that can take branch lengths into account [136]. This will allow direct comparisons to be made with other approaches, such as simultaneous analysis of concatenated data matrices and compartmentalization methods (references above; also [137]. There is a full spectrum of approaches for concatenating analyses at different scales:

At the left end of this spectrum, the approach is to include all possible OTUs and potential characters in one matrix. Generally this is not actually done, because the sheer amount of data (millions of possible OTUs) makes thorough phylogenetic analysis computationally impossible. The most common approach is to select a few representatives of a large, clearly monophyletic group (the exemplar method). Care is sometimes taken to select representatives that are "basal" OTUs within the group to be represented; however, this still does not avoid two important problems: (i) within-group variation is not fully represented in the analysis, and (ii) an increase both in terminal branch lengths and in asymmetry between lengths of different branches is introduced. These problems can lead to erroneous branch attractions in global analyses.

At the right end of the spectrum, local analyses are simply grafted together at the place where shared taxa occur, without reference back to the original data. There are many ways to do this in detail (as reviewed by Sanderson), but the important thing is that the analyses on real character data are only done locally, and the concatenation is based on the combination of local topologies rather than a combination of local data sets into a global data set.

We will explore both of these approaches even though both seem too extreme, one too global, the other too local. Thus we will also explore a promising synthetic approach called compartmentalization (by analogy to a water-tight compartment on a ship -- homoplasy is not allowed in or out) that represents diverse yet clearly monophyletic clades by their inferred ancestral states in larger-scale cladistic analyses. A well-supported local topology is sought first, then an inferred "archetype" or hypothetical ancestor (HTU) for the group is inserted into a more inclusive analysis. In more detail, the procedure we will use is to: (1) perform global analyses, determine the best supported clades (these become the compartments); (2) perform local analyses within compartments, including more taxa and characters (more characters can be used within compartments due to improved homology assessments among closely related organims); (3) return to a global analyses, in one of two ways, either (a) with compartments represented by single HTUs (the archetypes), or (b) with compartments constrained to the topology found in local analyses (for smaller data sets, this approach is better because it allows character optimizations within each compartment).

The compartmentalization approach differs from the exemplar approach in that the representative character-states coded for the archetype are based on all the taxa in the compartment, thus the reconstructed HTU is likely to be quite different from any real OTU. As an estimate of the states of the most recent common ancestor of all the local OTUs, the HTU is likely to have a much shorter terminal branch with respect to the global analysis, which in turn can have the beneficial global effect of reducing long-branch attraction. In addition to these advantages of compartmentalization at the global level, the local analyses will be better because one can: (1) include all local OTUs for which data are available; (2) incorporate more (and better justified) characters, by adding in those characters for which homology could not be determined (aligned) globally; (3) avoid spurious homoplasy that can change the local topology due to long-branch attractions with distant outgroups. The effects of compartmentalization are thus to cut large data sets down to manageable size, suppress the impact of spurious homoplasy, and allow the use of more information in analyses. This approach is self-reinforcing; as better understanding of phylogeny is gained, the support for compartments will be improved, leading in turn to refined understanding of appropriate characters and OTUs.

Phylogenetic database enhancements. As our data sets develop, we plan to assist larger efforts to develop a new generation of phylogenetic data-bases, including TreeBase and a pending ITR proposal for a national resource in phyloinformatics (see management plan). The next generation of data resources needs to be much more flexible than existing data bases (e.g., GenBank, which is essentially "flat" with respect to phylogeny), and sensitive to scale and the fractal nature of phylogenies (with their many hierachically nested scales).

The exploration of the basic nature of phylogenetic data described above will be applied to data-base research through modeling studies. We will address fundamental questions about the nature of data before, during, and after phylogenetic analysis. Biologists in this project will work with collaborating computer scientists to model: (1) How are elements of the data matrix (OTUs, characters, and states) defined and recognized in any particular study? (2) How can heterogeneous data types (e.g., DNA sequences, genomic rearrangements, morphology) be compared/combined? (3) How can data sets and analyses at very different scales be concatenated (e.g. supertree, compartmentalization, or global approaches as discussed above)? (4) How can data sets at these different concatenated scales, where OTUs are nested inside larger ones and character definitions (e.g., alignments) change as you move up and down the scale, be presented to the user community?

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