Chapter 9 : Molecular Phylogenetic Analysis

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Phylogenetic analysis, in the strictest sense, is the process of testing hypotheses about the descent of species from a common ancestor. This chapter provides an overview of a task essential to all: obtaining a working hypothesis of the evolutionary relationships among a group of organisms, summarized as a phylogenetic tree. Molecular phylogenetic analysis became more powerful and more accessible with the advent of rapid, inexpensive DNA sequencing, eventually leading to a major revision in the understanding of the evolutionary relationships among all living organisms. In order to provide a statistically robust representation of the phylogeny, a phylogenetic marker needs to have a sufficient number of independently evolving positions so that at least several changes differentiate the most closely related taxa of interest. After a phylogenetic marker has been selected, one must obtain sequence data. The core of automated alignment algorithms is optimization of a scoring function. Points are added for identities and similarities at each position, points are subtracted for mismatches, and many points are subtracted for gaps. The chapter emphasizes the use of aligned sequence databases in preference to de novo alignment not only for the savings in time and effort but also for the higher quality. It discusses maximum parsimony and maximum likelihood, the most widely implemented character-based methods, as well as Bayesian phylogenetic inference, a relatively new method rapidly growing in popularity. Although it may be possible to identify an optimal topology, the reader is cautioned that an optimal tree does not guarantee the true phylogeny.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9

Key Concept Ranking

Bacteria and Archaea
Group II Chaperonins
16s rRNA Sequencing
Group I Chaperonins
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Image of FIGURE 1

A typical workflow for molecular phylogenetic analysis.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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Image of FIGURE 2

Orthologous, paralogous, and xenologous homology. Organisms and genomes are depicted as rectangles and ovals, respectively, with large versions for ancestors and small versions representing extant strains available for sampling. The white and black bars on the genome represent genes that illustrate the relationship corresponding to each term. The small shapes decorating some genes indicate that additional changes can occur, leading to recognizable younger clades, but are not meant to imply that other genes have remained identical to the ancestral sequence. (A) The white and black versions of the gene are orthologous homologs in clades 1 and 2, since the divergence of the clades is the event that separated the white and black gene lineages. Any comparison between a white and a black gene is expected to reveal the initial divergence equally well. (B) The white and black versions of the gene are paralogous, having arisen from a gene duplication within a single genome. The common ancestor of each clade carried both paralogs; the use of either the white or the black version as a phylogenetic marker would be expected to depict the organismal phylogeny equally well. However, one member of clade 1 has lost the white paralog; the unwitting substitution of the black paralog in its place would incorrectly identify that organism as having branched off prior to the split between clades 1 and 2. (C) The white version of the gene is a xenolog of the black version, since its presence in clade 1 is due to a horizontal gene transfer event rather than inheritance from the common ancestor of clade 1. The ancestral gene is shown in the common ancestor of clades 1 and 2, implying that it has been lost in the lineage leading to the ancestor of clade 1, but it might also have been absent in the common ancestor of both clades and originated in the lineage leading to clade 2. Although clade 1 is shown as having no homolog of the gene prior to the horizontal transfer, in some cases a xenologous gene may either displace or coexist with an orthologous homolog.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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Image of FIGURE 3

BLAST interface at the website of the National Center for Biotechnology Information (NCBI). (A) Screen shot of the blastp (protein blast) query submission page. Numbered arrows highlight the following features: 1, button to access extensive online help; 2, user-generated job title for keeping track of multiple BLAST queries; 3, restriction of the search to a particular taxonomic group; 4, restriction of the search using an Entrez query, in this case to search only sequences from cultivated organisms (the unusual syntax, with an initial term that has no effect, is required in this case because the query is not permitted to begin with the Boolean term “NOT”); 5, algorithm parameters that can be customized but are not initially displayed. Usually choosing between the named algorithms above offers sufficient flexibility, but users with extensive or challenging searches may benefit from understanding and using the detailed parameter settings. (B) Screen shot of blastp results highlighting the following features: 1, panels to customize online and downloaded results format; 2, selection between multiple query sequences submitted in a single batch; 3, summary of the phylogenetic and taxonomic distribution of the BLAST hits; 4, links to the standard GenBank records; 5, links to “value-added” databases such as whole-genome sequences.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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Image of FIGURE 4

Ribosomal Database Project website. (A) Screen shot of the initial page for the Classifier tool, which provides a taxonomic classification for the sequences selected by the user. Numbered arrows highlight the following features: 1, additional RDP tools including Seqmatch, which finds the sequence(s) in the database most similar to each submitted sequence, and Treebuilder, which interactively generates small trees online; 2, ability to upload new sequences into a private myRDP account for automated alignment and storage on the server; 3, extensive online help files; 4, ability to apply tools to private sequences as well as those in the RDP database. (B) Screen shot of Classifier results highlighting the following features: 1, user-adjustable stringency for classification; 2, ability to download all sequence classifications as a single file; 3, numbers indicate the distribution of the submitted sequences to various taxa: clicking on taxon names will show which sequences are classified into each taxon.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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Image of FIGURE 5

Pfam website. (A) Screen shot of results from a protein sequence search. Numbered arrows highlight the following features: 1, extensive online help files; 2, architecture (protein domain structure) found in the query sequence; 3, table with information about each domain found in the query; 4, alignment shown between one domain of the query sequence and the hidden Markov model which describes the sequence information conserved in the domain family; 5, link to complete information about that particular Pfam domain family. (B) Screen shot of the home page for a Pfam domain family, highlighting the following features: 1, different categories of information available about the domain; 2, listing of all known protein architectures containing the domain; 3, link to all protein sequences of a particular architecture.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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Image of FIGURE 6

Alignment. (A) The region shown for hypothetical Sequences 1 to 3 can be aligned unambiguously, although the comparison between Sequences 1 and 2 indicates a mismatch and the comparison between Sequences 1 and 3 indicates an indel (insertion-deletion event). The alignment is an assertion that the nucleotides in a single column are homologous, i.e., descended from a single ancestral nucleotide. However, Sequence 4 cannot be aligned unambiguously with the information given; two possible alternatives are shown. See the text for additional discussion. (B) Alignment software. A screen shot of the Arb sequence editor is shown, which is one component of the Arb phylogenetic software designed especially for 16S rRNA data. The following features are highlighted: 1, sequence coordinates corresponding to the cursor position; 2, search fields for sequence motifs; 3, 16S rRNA sequence for reference; 4, symbols indicating position-specific secondary and tertiary interactions predicted by a model of 16S rRNA structure; 5, a sequence mask, with plus signs indicating columns aligned with sufficient confidence to be included in subsequent analysis; 6, aligned columns of nucleotides, color coded to assist in manual editing. Symbols underlining some nucleotides are indications of the potential for that nucleotide to participate in an interaction predicted by the structural model.

Citation: Dethlefsen L, Lepp P, Relman D. 2011. Molecular Phylogenetic Analysis, p 145-165. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch9
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