Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species

The mitochondrial genome (mtDNA) of Metazoa is a good model system for evolutionary genomic studies and the availability of more than 1000 sequences provides an almost unique opportunity to decode the mechanisms of genome evolution over a large phylogenetic range. In this paper, we review several structural features of the metazoan mtDNA, such as gene content, genome size, genome architecture and the new parameter of gene strand asymmetry in a phylogenetic framework. The data reviewed here show that: (1) the plasticity of Metazoa mtDNA is higher than previously thought and mainly due to variation in number and location of tRNA genes; (2) an exceptional trend towards stabilization of genomic features occurred in deuterostomes and was exacerbated in vertebrates, where gene content, genome architecture and gene strand asymmetry are almost invariant. Only tunicates exhibit a very high degree of genome variability comparable to that found outside deuterostomes. In order to analyse the genomic evolutionary process at short evolutionary distances, we have also compared mtDNAs of species belonging to the same genus: the variability observed in congeneric species significantly recapitulates the evolutionary dynamics observed at higher taxonomic ranks, especially for taxa showing high levels of genome plasticity and/or fast nucleotide substitution rates. Thus, the analysis of congeneric species promises to be a valuable approach for the assessment of the mtDNA evolutionary trend in poorly or not yet sampled metazoan groups.     

A Primer to Molecular Phylogenetic Analysis in Plants

Reconstructing a tree of life by inferring evolutionary history is an important focus of evolutionary biology. Phylogenetic reconstructions also provide useful information for a range of scientific disciplines such as botany, zoology, phylogeography, archaeology and biological anthropology. Until the development of protein and DNA sequencing techniques in the 1960s and 1970s, phylogenetic reconstructions were based on fossil records and comparative morphological/physiological analyses. Since then, progress in molecular phylogenetics has compensated for some of the shortcomings of phenotype-based comparisons. Comparisons at the molecular level increase the accuracy of phylogenetic inference because there is no environmental influence on DNA/peptide sequences and evaluation of sequence similarity is not subjective. While the number of morphological/physiological characters that are sufficiently conserved for phylogenetic inference is limited, molecular data provide a large number of datapoints and enable comparisons from diverse taxa. Over the last 20 years, developments in molecular phylogenetics have greatly contributed to our understanding of plant evolutionary relationships. Regions in the plant nuclear and organellar genomes that are optimal for phylogenetic inference have been determined and recent advances in DNA sequencing techniques have enabled comparisons at the whole genome level. Sequences from the nuclear and organellar genomes of thousands of plant species are readily available in public databases, enabling researchers without access to molecular biology tools to investigate phylogenetic relationships by sequence comparisons using the appropriate nucleotide substitution models and tree building algorithms. In the present review, the statistical models and algorithms used to reconstruct phylogenetic trees are introduced and advances in the exploration and utilization of plant genomes for molecular phylogenetic analyses are discussed.     

GO4genome: A Prokaryotic Phylogeny Based on Genome Organization

Determining the phylogeny of closely related prokaryotes may fail in an analysis of rRNA or a small set of sequences. Whole-genome phylogeny utilizes the maximally available sample space. For a precise determination of genome similarity, two aspects have to be considered when developing an algorithm of whole-genome phylogeny: (1) gene order conservation is a more precise signal than gene content; and (2) when using sequence similarity, failures in identifying orthologues or the in situ replacement of genes via horizontal gene transfer may give misleading results. GO4genome is a new paradigm, which is based on a detailed analysis of gene function and the location of the respective genes. For characterization of genes, the algorithm uses gene ontology enabling a comparison of function independent of evolutionary relationship. After the identification of locally optimal series of gene functions, their length distribution is utilized to compute a phylogenetic distance. The outcome is a classification of genomes based on metabolic capabilities and their organization. Thus, the impact of effects on genome organization that are not covered by methods of molecular phylogeny can be studied. Genomes of strains belonging to Escherichia coli, Shigella, Streptococcus, Methanosarcina, and Yersinia were analyzed. Differences from the findings of classical methods are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

General properties and phylogenetic utilities of nuclear ribosomal DNA and mitochondrial DNA commonly used in molecular systematics

To choose one or more appropriate molecular markers or gene regions for resolving a particular systematic question among the organisms at a certain categorical level is still a very difficult process. The primary goal of this review, therefore, is to provide a theoretical information in choosing one or more molecular markers or gene regions by illustrating general properties and phylogenetic utilities of nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) that have been most commonly used for phylogenetic researches. The highly conserved molecular markers and/or gene regions are useful for investigating phylogenetic relationships at higher categorical levels (deep branches of evolutionary history). On the other hand, the hypervariable molecular markers and/or gene regions are useful for elucidating phylogenetic relationships at lower categorical levels (recently diverged branches). In summary, different selective forces have led to the evolution of various molecular markers or gene regions with varying degrees of sequence conservation. Thus, appropriate molecular markers or gene regions should be chosen with even greater caution to deduce true phylogenetic relationships over a broad taxonomic spectrum.     

Molecular phylogeny and evolution of the order Tribonematales (Heterokonta, Xanthophyceae) based on analysis of plastidial genes rbcL and psaA

Tribonematales is an order of filamentous algae in the class Xanthophyceae (Heterokonta). Few molecular studies, all with a limited taxon sampling, have previously investigated its evolutionary history and phylogenetic relationships. We sequenced the chloroplast-encoded rbcL and psaA genes of several tribonematalean species and of several coccoid and siphonous forms that previous studies revealed to be strictly related to Tribonematales. Multiple alignments included mostly new sequences obtained from 42 taxa. Phylogenetic reconstructions were performed using the maximum likelihood method. The rbcL and psaA data sets were analyzed independently and combined in a single multiple alignment. Neither rbcL nor psaA genes showed intraspecific sequence variation. The former proved to be a better diagnostic marker than the latter for characterization of species. We explored effects produced on phylogenetic outcomes by selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on the combined data set. There is strong statistical support for trees that show several currently recognized taxonomic groups to be polyphyletic. The siphonous orders Botrydiales and Vaucheriales do not form a clade. Botrydiales and Tribonematales are polyphyletic as are the families Botrydiaceae, Centritractaceae and Tribonemataceae and the genera Xanthonema and Bumilleriopsis. We tentatively define new boundaries of the Tribonematales to include both coccoid and filamentous species having a bipartite cell wall and also the siphonous members of the genus Botrydium. Also, our results support morphological convergence at all taxonomic ranks in the evolution of the Xanthophyceae.     

A SINGLE ORIGIN OF PLASTIDS REVISITED: CONVERGENT EVOLUTION IN ORGANELLAR GENOME CONTENT1

In recent years a consensus has emerged from molecular phylogenetic investigations favoring a common endosymbiotic ancestor for all chloroplasts. It is within this conceptual framework that most comparative analyses of eukaryotic biochemistry and genetics now are interpreted. One of the first and most influential sources of data leading to this consensus is the remarkable similarity in genome content among all major plastid lineages. Here we report statistical analyses of two sequence data sets, genes encoding ribosomal proteins and transfer RNAs, from representatives of the three primary plastid lineages and a mitochondrion. The latter almost certainly originated in an independent endosymbiotic association and serves as a control for similarity due to convergent evolution. When genes related to organelle‐specific function are factored out, plastid genomes appear to be no more similar to each other than they are to the mitochondrion. Total similarities in gene content, measured as deviations from the expectation from a process of random gene loss, are correlated with the extent of reduction in the two genomes compared. They do not appear to reflect putative evolutionary relationships among plastids. These analyses indicate that similarities in plastid genome content are better explained by convergent evolution due to constraint on gene loss than by a shared evolutionary history. A review of other data cited as support for a single plastid origin suggests that the alternative hypothesis of multiple origins is at least equally consistent in most cases.     

Use of quantitative measures of gene order similarity to phylogenetic reconstructions (exemplified by bacteria of the genus Rickettsia)

Data reflecting evolutionary changes in chromosomal gene order can be used for phylogenetic reconstructions along with the results of nucleotide sequence comparison. By the example of bacteria of the genus Rickettsia, we have shown that phylogenetic reconstructions based on quantitative estimates of the similarity and cladistic analysis of gene order data, may, in some cases, amend and fill up classical phylogenetic trees. When applied, these approaches enabled us to substantiate the hypothesis that Rickettsia felis species had split before the typhus (R. typhi, R. prowazekii) and spotted fever (R. connorii) group divergence and thus R. felis does not belong to the latter group. In general, rickettsias evolved towards increasing intracellular parasitic specialization. Five Rickettsia species whose genomes have been sequenced and annotated completely actually form an evolutionary series R. hellii-R. felis-R. connorii-R. prowazekii-R. typhi. Within this series, a reduction in genome size and rapid decrease of genome rearrangement rates (genome plasticity loss) gradually occur.     

Ribosomal RNA diversity predicts genome diversity in gut bacteria and their relatives

The mammalian gut is an attractive model for exploring the general question of how habitat impacts the evolution of gene content. Therefore, we have characterized the relationship between 16 S rRNA gene sequence similarity and overall levels of gene conservation in four groups of species: gut specialists and cosmopolitans, each of which can be divided into pathogens and non-pathogens. At short phylogenetic distances, specialist or cosmopolitan bacteria found in the gut share fewer genes than is typical for genomes that come from non-gut environments, but at longer phylogenetic distances gut bacteria are more similar to each other than are genomes at equivalent evolutionary distances from non-gut environments, suggesting a pattern of short-term specialization but long-term convergence. Moreover, this pattern is observed in both pathogens and non-pathogens, and can even be seen in the plasmids carried by gut bacteria. This observation is consistent with the finding that, despite considerable interpersonal variation in species content, there is surprising functional convergence in the microbiome of different humans. Finally, we observe that even within bacterial species or genera 16S rRNA divergence provides useful information about average conservation of gene content. The results described here should be useful for guiding strain selection to maximize novel gene discovery in large-scale genome sequencing projects, while the approach could be applied in studies seeking to understand the effects of habitat adaptation on genome evolution across other body habitats or environment types.     

Discordant phylogenies within the rrn loci of Rhizobia

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of alpha-Proteobacteria by sequencing analysis of three loci within their rrn operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera Bradyrhizobium, Mesorhizobium and Sinorhizobium have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.     

Phylogenetic inferences from molecular sequences: review and critique

Conflicting results often accompany phylogenetic analyses of RNA, DNA, or protein sequences across diverse species. Causes contributing to these conflicts relate to ambiguities in identifying homologous characters of alignments, sensitivity of tree-making methods to unequal evolutionary rates, biases in species sampling, unrecognized paralogy, functional differentiation, loss of phylogenetic informational content due to long branches or fast evolution, and difficulties with the assumptions and approximations used to infer phylogenetic relationships. Attempts to surmount these conflicts by averaging over many proteins are problematic due to inherent biases of selected families, lack of signal in others, and events of lateral transfer, fusion, and/or chimerism. The process of assessing reliability of the results using the bootstrap method is strewn with obstacles because of lack of independence and inhomogeneity in the molecular data. Problems inherent to the three major procedures for developing phylogenetic trees--parsimony, likelihood, distance--are reviewed. Special attention is given to the problem of inferring evolutionary distances from patterns of similarity among sequences. The difficulties encountered by methods of phylogenetic reconstructions based on the analysis of divergent sequence families make new methods based on the analysis of complete genomes reasonable alternatives. Several of these are considered, including the signature sequences of Gupta and associates, the study of genome profiles, and the genomic signature set forth by Karlin and colleagues.     

VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing

Background

The 16S rRNA gene-based amplicon sequencing analysis is widely used to determine the taxonomic composition of microbial communities. Once the taxonomic composition of each community is obtained, evolutionary relationships among taxa are inferred by a phylogenetic tree. Thus, the combined representation of taxonomic composition and phylogenetic relationships among taxa is a powerful method for understanding microbial community structure; however, applying phylogenetic tree-based representation with information on the abundance of thousands or more taxa in each community is a difficult task. For this purpose, we previously developed the tool VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community), which is based on the genome-sequenced microbes’ phylogenetic information. Here, we introduce VITCOMIC2, which incorporates substantive improvements over VITCOMIC that were necessary to address several issues associated with 16S rRNA gene-based analysis of microbial communities.

Results

We developed VITCOMIC2 to provide (i) sequence identity searches against broad reference taxa including uncultured taxa; (ii) normalization of 16S rRNA gene copy number differences among taxa; (iii) rapid sequence identity searches by applying the graphics processing unit-based sequence identity search tool CLAST; (iv) accurate taxonomic composition inference and nearly full-length 16S rRNA gene sequence reconstructions for metagenomic shotgun sequencing; and (v) an interactive user interface for simultaneous representation of the taxonomic composition of microbial communities and phylogenetic relationships among taxa. We validated the accuracy of processes (ii) and (iv) by using metagenomic shotgun sequencing data from a mock microbial community.

Conclusions

The improvements incorporated into VITCOMIC2 enable users to acquire an intuitive understanding of microbial community composition based on the 16S rRNA gene sequence data obtained from both metagenomic shotgun and amplicon sequencing.

     

Inferring phylogenies from RAD sequence data

Reduced-representation genome sequencing represents a new source of data for systematics, and its potential utility in interspecific phylogeny reconstruction has not yet been explored. One approach that seems especially promising is the use of inexpensive short-read technologies (e.g., Illumina, SOLiD) to sequence restriction-site associated DNA (RAD)--the regions of the genome that flank the recognition sites of restriction enzymes. In this study, we simulated the collection of RAD sequences from sequenced genomes of different taxa (Drosophila, mammals, and yeasts) and developed a proof-of-concept workflow to test whether informative data could be extracted and used to accurately reconstruct "known" phylogenies of species within each group. The workflow consists of three basic steps: first, sequences are clustered by similarity to estimate orthology; second, clusters are filtered by taxonomic coverage; and third, they are aligned and concatenated for "total evidence" phylogenetic analysis. We evaluated the performance of clustering and filtering parameters by comparing the resulting topologies with well-supported reference trees and we were able to identify conditions under which the reference tree was inferred with high support. For Drosophila, whole genome alignments allowed us to directly evaluate which parameters most consistently recovered orthologous sequences. For the parameter ranges explored, we recovered the best results at the low ends of sequence similarity and taxonomic representation of loci; these generated the largest supermatrices with the highest proportion of missing data. Applications of the method to mammals and yeasts were less successful, which we suggest may be due partly to their much deeper evolutionary divergence times compared to Drosophila (crown ages of approximately 100 and 300 versus 60 Mya, respectively). RAD sequences thus appear to hold promise for reconstructing phylogenetic relationships in younger clades in which sufficient numbers of orthologous restriction sites are retained across species.     

Evaluation of methods for determination of a reconstructed history of gene sequence evolution

With whole-genome sequences being completed at an increasing rate, it is important to develop and assess tools to analyze them. Following annotation of the protein content of a genome, one can compare sequences with previously characterized homologous genes to detect novel functions within specific proteins in the evolution of the newly sequenced genome. One common statistical method to detect such changes is to compare the ratios of nonsynonymous (K(a)) to synonymous (K(s)) nucleotide substitution rates. Here, the effects of several parameters that can influence this calculation (sequence reconstruction method, phylogenetic tree branch length weighting, GC content, and codon bias) are examined. Also, two new alternative measures of adaptive evolution, the point accepted mutations (PAM)/neutral evolutionary distance (NED) ratio and the sequence space assessment (SSA) statistic are presented. All of these methods are compared using two sequence families: the recent divergence of leptin orthologs in primates, and the more ancient divergence of the deoxyribonucleoside kinase family. The examination of these and other measures to detect changes of gene function along branches of a phylogenetic tree will become increasingly important in the postgenomic era.     

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea)

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of free-living and endocommensal species. However, their traditional morphology-based classification conflicts with 18S rRNA gene phylogenies indicating (1) a deep bifurcation of the Litostomatea into Rhynchostomatia and Haptoria+Trichostomatia, and (2) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea. To test whether 18S rRNA molecules provide a suitable proxy for litostomatean evolutionary history, we used eighteen new ITS1-5.8S rRNA-ITS2 region sequences from various free-living litostomatean orders. These single- and multiple-locus analyses are in agreement with previous 18S rRNA gene phylogenies, supporting that both 18S rRNA gene and ITS region sequences are effective tools for resolving phylogenetic relationships among the litostomateans. Despite insertions, deletions and mutational saturations in the ITS region, the present study shows that ITS1 and ITS2 molecules can be used to infer phylogenetic relationships not only at species level but also at higher taxonomic ranks when their secondary structure information is utilized to aid alignment.     

New proposals for naming lower-ranked taxa within the frame of the International Code of Zoological Nomenclature

The recent multiplication of cladistic hypotheses for many zoological groups poses a challenge to zoological nomenclature following the International Code of Zoological Nomenclature: in order to account for these hypotheses, we will need many more ranks than currently allowed in this system, especially in lower taxonomy (around the ranks genus and species). The current Code allows the use of as many ranks as necessary in the family-series of nomina (except above superfamily), but forbids the use of more than a few ranks in the genus and species-series. It is here argued that this limitation has no theoretical background, does not respect the freedom of taxonomic thoughts or actions, and is harmful to zoological taxonomy in two respects at least: (1) it does not allow to express in detail hypothesized cladistic relationships among taxa at lower taxonomic levels (genus and species); (2) it does not allow to point taxonomically to low-level differentiation between populations of the same species, although this would be useful in some cases for conservation biology purposes. It is here proposed to modify the rules of the Code in order to allow use by taxonomists of an indeterminate number of ranks in all nominal-series. Such an 'expanded nomenclatural system' would be highly flexible and likely to be easily adapted to any new finding or hypothesis regarding cladistic relationships between taxa, at genus and species level and below. This system could be useful for phylogeographic analysis and in conservation biology. In zoological nomenclature, whereas robustness of nomina is necessary, the same does not hold for nomenclatural ranks, as the latter are arbitrary and carry no special biological, evolutionary or other information, except concerning the mutual relationships between taxa in the taxonomic hierarchy. Compared to the Phylocode project, the new system is equally unambiguous within the frame of a given taxonomic frame, but it provides more explicit and informative nomina for non-specialist users, and is more economic in terms of number of nomina needed to account for a given hierarchy. These ideas are exemplified by a comparative study of three possible nomenclatures for the taxonomy recently proposed by Hillis and Wilcox (2005) for American frogs traditionally referred to the genus Rana.     

Reconciling gene and genome duplication events: using multiple nuclear gene families to infer the phylogeny of the aquatic plant family Pontederiaceae

Most plant phylogenetic inference has used DNA sequence data from the plastid genome. This genome represents a single genealogical sample with no recombination among genes, potentially limiting the resolution of evolutionary relationships in some contexts. In contrast, nuclear DNA is inherently more difficult to employ for phylogeny reconstruction because major mutational events in the genome, including polyploidization, gene duplication, and gene extinction can result in homologous gene copies that are difficult to identify as orthologs or paralogs. Gene tree parsimony (GTP) can be used to infer the rooted species tree by fitting gene genealogies to species trees while simultaneously minimizing the estimated number of duplications needed to reconcile conflicts among them. Here, we use GTP for five nuclear gene families and a previously published plastid data set to reconstruct the phylogenetic backbone of the aquatic plant family Pontederiaceae. Plastid-based phylogenetic studies strongly supported extensive paraphyly of Eichhornia (one of the four major genera) but also depicted considerable ambiguity concerning the true root placement for the family. Our results indicate that species trees inferred from the nuclear genes (alone and in combination with the plastid data) are highly congruent with gene trees inferred from plastid data alone. Consideration of optimal and suboptimal gene tree reconciliations place the root of the family at (or near) a branch leading to the rare and locally restricted E. meyeri. We also explore methods to incorporate uncertainty in individual gene trees during reconciliation by considering their individual bootstrap profiles and relate inferred excesses of gene duplication events on individual branches to whole-genome duplication events inferred for the same branches. Our study improves understanding of the phylogenetic history of Pontederiaceae and also demonstrates the utility of GTP for phylogenetic analysis.     

Roundup: a multi-genome repository of orthologs and evolutionary distances

SUMMARY: We have created a tool for ortholog and phylogenetic profile retrieval called Roundup. Roundup is backed by a massive repository of orthologs and associated evolutionary distances that was built using the reciprocal smallest distance algorithm, an approach that has been shown to improve upon alternative approaches of ortholog detection, such as reciprocal blast. Presently, the Roundup repository contains all possible pair-wise comparisons for over 250 genomes, including 32 Eukaryotes, more than doubling the coverage of any similar resource. The orthologs are accessible through an intuitive web interface that allows searches by genome or gene identifier, presenting results as phylogenetic profiles together with gene and molecular function annotations. Results may be downloaded as phylogenetic matrices for subsequent analysis, including the construction of whole-genome phylogenies based on gene-content data. AVAILABILITY: http://rodeo.med.harvard.edu/tools/roundup.     

Application of quantitative measures of gene order similarity to phylogenetic reconstructions (exemplified by bacteria of the genus <Emphasis Type="Italic">Rickettsia</Emphasis>)

Data reflecting evolutionary changes in chromosomal gene order can be used for phylogenetic reconstructions along with the results of nucleotide sequence comparison. By the example of bacteria of the genus Rickettsia, we have shown that phylogenetic reconstructions based on quantitative estimates of the similarity and cladistic analysis of gene order data, may, in some cases, amend and fill up classical phylogenetic trees. When applied, these approaches enabled us to substantiate the hypothesis that Rickettsia felis species had split before the typhus (R. typhi, R. prowazekii) and spotted fever (R. connorii) group divergence and thus R. felis does not belong to the latter group. In general, rickettsias evolved towards increasing intracellular parasitic specialization. Five Rickettsia species whose genomes have been sequenced and annotated completely actually form an evolutionary series R. bellii—R. felis—R. conorii—R. prowazekii—R. typhi. Within this series, a reduction in genome size and rapid decrease of genome rearrangement rates (genome plasticity loss) gradually occur.     

Phylogenetic diversification of the globin gene superfamily in chordates

Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this article is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this, we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges.