Advances in ecological genomics in forest trees and applications to genetic resources conservation and breeding

Forest trees are an unparalleled group of organisms in their combined ecological, economic and societal importance. With widespread distributions, predominantly random mating systems and large population sizes, most tree species harbour extensive genetic variation both within and among populations. At the same time, demographic processes associated with Pleistocene climate oscillations and land‐use change have affected contemporary range‐wide diversity and may impinge on the potential for future adaptation. Understanding how these adaptive and neutral processes have shaped the genomes of trees species is therefore central to their management and conservation. As for many other taxa, the advent of high‐throughput sequencing methods is expected to yield an understanding of the interplay between the genome and environment at a level of detail and depth not possible only a few years ago. An international conference entitled ‘Genomics and Forest Tree Genetics’ was held in May 2016, in Arcachon (France), and brought together forest geneticists with a wide range of research interests to disseminate recent efforts that leverage contemporary genomic tools to probe the population, quantitative and evolutionary genomics of trees. An important goal of the conference was to discuss how such data can be applied to both genome‐enabled breeding and the conservation of forest genetic resources under land use and climate change. Here, we report discoveries presented at the meeting and discuss how the ecological genomic toolkit can be used to address both basic and applied questions in tree biology.     

Multiple genome rearrangement: a general approach via the evolutionary genome graph

MOTIVATION: In spite of a well-known fact that genome rearrangements are supposed to be viewed in the light of the evolutionary relationships within and between the species involved, no formal underlying framework based on the evolutionary considerations for treating the questions arising in the area has been proposed. If such an underlying framework is provided, all the basic questions in the area can be posed in a biologically more appropriate and useful form: e.g., the similarity between two genomes can then be computed via the nearest ancestor, rather than 'directly', ignoring the evolutionary connections. RESULTS: We outline an evolution-based general framework for answering questions related to the multiple genome rearrangement. In the proposed model, the evolutionary genome graph (EG-graph) encapsulates an evolutionary history of a genome family. For a set of all EG-graphs, we introduce a family of similarity measures, each defined via a fixed set of genome transformations. Given a set of genomes and restricting ourselves to the transpositions, an algorithm for constructing an EG-graph is presented. We also present the experimental results in the form of an EG-graph for a set of concrete genomes (for several species). This EG-graph turns out to be very close to the corresponding known phylogenetic tree.     

Anchor-based whole genome phylogeny (ABWGP): a tool for inferring evolutionary relationship among closely related microorganisms [corrected

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.     

Primate phylogenomics uncovers multiple rapid radiations and ancient interspecific introgression

Our understanding of the evolutionary history of primates is undergoing continual revision due to ongoing genome sequencing efforts. Bolstered by growing fossil evidence, these data have led to increased acceptance of once controversial hypotheses regarding phylogenetic relationships, hybridization and introgression, and the biogeographical history of primate groups. Among these findings is a pattern of recent introgression between species within all major primate groups examined to date, though little is known about introgression deeper in time. To address this and other phylogenetic questions, here, we present new reference genome assemblies for 3 Old World monkey (OWM) species: Colobus angolensis ssp. palliatus (the black and white colobus), Macaca nemestrina (southern pig-tailed macaque), and Mandrillus leucophaeus (the drill). We combine these data with 23 additional primate genomes to estimate both the species tree and individual gene trees using thousands of loci. While our species tree is largely consistent with previous phylogenetic hypotheses, the gene trees reveal high levels of genealogical discordance associated with multiple primate radiations. We use strongly asymmetric patterns of gene tree discordance around specific branches to identify multiple instances of introgression between ancestral primate lineages. In addition, we exploit recent fossil evidence to perform fossil-calibrated molecular dating analyses across the tree. Taken together, our genome-wide data help to resolve multiple contentious sets of relationships among primates, while also providing insight into the biological processes and technical artifacts that led to the disagreements in the first place.

Combining three newly sequenced primate genomes with other published genomes, this study adapts a little-known method for detecting ancient introgression to genome-scale data, revealing multiple previously unknown examples of hybridization between primate species.     

Anchor-Based Whole Genome Phylogeny (ABWGP): A Tool for Inferring Evolutionary Relationship among Closely Related Microorganims

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.     

MultiMSOAR 2.0: an accurate tool to identify ortholog groups among multiple genomes

The identification of orthologous genes shared by multiple genomes plays an important role in evolutionary studies and gene functional analyses. Based on a recently developed accurate tool, called MSOAR 2.0, for ortholog assignment between a pair of closely related genomes based on genome rearrangement, we present a new system MultiMSOAR 2.0, to identify ortholog groups among multiple genomes in this paper. In the system, we construct gene families for all the genomes using sequence similarity search and clustering, run MSOAR 2.0 for all pairs of genomes to obtain the pairwise orthology relationship, and partition each gene family into a set of disjoint sets of orthologous genes (called super ortholog groups or SOGs) such that each SOG contains at most one gene from each genome. For each such SOG, we label the leaves of the species tree using 1 or 0 to indicate if the SOG contains a gene from the corresponding species or not. The resulting tree is called a tree of ortholog groups (or TOGs). We then label the internal nodes of each TOG based on the parsimony principle and some biological constraints. Ortholog groups are finally identified from each fully labeled TOG. In comparison with a popular tool MultiParanoid on simulated data, MultiMSOAR 2.0 shows significantly higher prediction accuracy. It also outperforms MultiParanoid, the Roundup multi-ortholog repository and the Ensembl ortholog database in real data experiments using gene symbols as a validation tool. In addition to ortholog group identification, MultiMSOAR 2.0 also provides information about gene births, duplications and losses in evolution, which may be of independent biological interest. Our experiments on simulated data demonstrate that MultiMSOAR 2.0 is able to infer these evolutionary events much more accurately than a well-known software tool Notung. The software MultiMSOAR 2.0 is available to the public for free.     

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.     

Assessing the performance of single-copy genes for recovering robust phylogenies

Phylogenies involving nonmodel species are based on a few genes, mostly chosen following historical or practical criteria. Because gene trees are sometimes incongruent with species trees, the resulting phylogenies may not accurately reflect the evolutionary relationships among species. The increase in availability of genome sequences now provides large numbers of genes that could be used for building phylogenies. However, for practical reasons only a few genes can be sequenced for a wide range of species. Here we asked whether we can identify a few genes, among the single-copy genes common to most fungal genomes, that are sufficient for recovering accurate and well-supported phylogenies. Fungi represent a model group for phylogenomics because many complete fungal genomes are available. An automated procedure was developed to extract single-copy orthologous genes from complete fungal genomes using a Markov Clustering Algorithm (Tribe-MCL). Using 21 complete, publicly available fungal genomes with reliable protein predictions, 246 single-copy orthologous gene clusters were identified. We inferred the maximum likelihood trees using the individual orthologous sequences and constructed a reference tree from concatenated protein alignments. The topologies of the individual gene trees were compared to that of the reference tree using three different methods. The performance of individual genes in recovering the reference tree was highly variable. Gene size and the number of variable sites were highly correlated and significantly affected the performance of the genes, but the average substitution rate did not. Two genes recovered exactly the same topology as the reference tree, and when concatenated provided high bootstrap values. The genes typically used for fungal phylogenies did not perform well, which suggests that current fungal phylogenies based on these genes may not accurately reflect the evolutionary relationships among species. Analyses on subsets of species showed that the phylogenetic performance did not seem to depend strongly on the sample. We expect that the best-performing genes identified here will be very useful for phylogenetic studies of fungi, at least at a large taxonomic scale. Furthermore, we compare the method developed here for finding genes for building robust phylogenies with previous ones and we advocate that our method could be applied to other groups of organisms when more complete genomes are available.     

Support Measures to Estimate the Reliability of Evolutionary Events Predicted by Reconciliation Methods

The genome content of extant species is derived from that of ancestral genomes, distorted by evolutionary events such as gene duplications, transfers and losses. Reconciliation methods aim at recovering such events and at localizing them in the species history, by comparing gene family trees to species trees. These methods play an important role in studying genome evolution as well as in inferring orthology relationships. A major issue with reconciliation methods is that the reliability of predicted evolutionary events may be questioned for various reasons: Firstly, there may be multiple equally optimal reconciliations for a given species tree–gene tree pair. Secondly, reconciliation methods can be misled by inaccurate gene or species trees. Thirdly, predicted events may fluctuate with method parameters such as the cost or rate of elementary events. For all of these reasons, confidence values for predicted evolutionary events are sorely needed. It was recently suggested that the frequency of each event in the set of all optimal reconciliations could be used as a support measure. We put this proposition to the test here and also consider a variant where the support measure is obtained by additionally accounting for suboptimal reconciliations. Experiments on simulated data show the relevance of event supports computed by both methods, while resorting to suboptimal sampling was shown to be more effective. Unfortunately, we also show that, unlike the majority-rule consensus tree for phylogenies, there is no guarantee that a single reconciliation can contain all events having above 50% support. In this paper, we detail how to rely on the reconciliation graph to efficiently identify the median reconciliation. Such median reconciliation can be found in polynomial time within the potentially exponential set of most parsimonious reconciliations.     

A simple method for phylogenomic inference using the information of gene content of genomes

Many studies have been contributed to the inferences of phylogenies. Some studies are based on a single-gene (family), and some are based on entire genome data. In this paper, we propose a total loss genome distance approach based on gene content information to inferring phylogenies. Through various simulations, we demonstrate and evaluate the proposed approach. We compare it with some other approaches built upon gene content or extended gene content. Overall, the proposed approach performs equally well as the other methods do and is more efficient than some of the methods. We apply our approach to 34 microbial complete genomes from COG. The reconstructed tree agrees with the results from other approaches and the tree supports the concept of universal trees.     

Getting a better picture of microbial evolution en route to a network of genomes

Most current thinking about evolution is couched in the concept of trees. The notion of a tree with recursively bifurcating branches representing recurrent divergence events is a plausible metaphor to describe the evolution of multicellular organisms like vertebrates or land plants. But if we try to force the tree metaphor onto the whole of the evolutionary process, things go badly awry, because the more closely we inspect microbial genomes through the looking glass of gene and genome sequence comparisons, the smaller the amount of the data that fits the concept of a bifurcating tree becomes. That is mainly because among microbes, endosymbiosis and lateral gene transfer are important, two mechanisms of natural variation that differ from the kind of natural variation that Darwin had in mind. For such reasons, when it comes to discussing the relationships among all living things, that is, including the microbes and all of their genes rather than just one or a select few, many biologists are now beginning to talk about networks rather than trees in the context of evolutionary relationships among microbial chromosomes. But talk is not enough. If we were to actually construct networks instead of trees to describe the evolutionary process, what would they look like? Here we consider endosymbiosis and an example of a network of genomes involving 181 sequenced prokaryotes and how that squares off with some ideas about early cell evolution.     

The Tree versus the Forest: The Fungal Tree of Life and the Topological Diversity within the Yeast Phylome

A recurrent topic in phylogenomics is the combination of various sequence alignments to reconstruct a tree that describes the evolutionary relationships within a group of species. However, such approach has been criticized for not being able to properly represent the topological diversity found among gene trees. To evaluate the representativeness of species trees based on concatenated alignments, we reconstruct several fungal species trees and compare them with the complete collection of phylogenies of genes encoded in the Saccharomyces cerevisiae genome. We found that, despite high levels of among-gene topological variation, the species trees do represent widely supported phylogenetic relationships. Most topological discrepancies between gene and species trees are concentrated in certain conflicting nodes. We propose to map such information on the species tree so that it accounts for the levels of congruence across the genome. We identified the lack of sufficient accuracy of current alignment and phylogenetic methods as an important source for the topological diversity encountered among gene trees. Finally, we discuss the implications of the high levels of topological variation for phylogeny-based orthology prediction strategies.     

i-ADHoRe 3.0--fast and sensitive detection of genomic homology in extremely large data sets

Comparative genomics is a powerful means to gain insight into the evolutionary processes that shape the genomes of related species. As the number of sequenced genomes increases, the development of software to perform accurate cross-species analyses becomes indispensable. However, many implementations that have the ability to compare multiple genomes exhibit unfavorable computational and memory requirements, limiting the number of genomes that can be analyzed in one run. Here, we present a software package to unveil genomic homology based on the identification of conservation of gene content and gene order (collinearity), i-ADHoRe 3.0, and its application to eukaryotic genomes. The use of efficient algorithms and support for parallel computing enable the analysis of large-scale data sets. Unlike other tools, i-ADHoRe can process the Ensembl data set, containing 49 species, in 1?h. Furthermore, the profile search is more sensitive to detect degenerate genomic homology than chaining pairwise collinearity information based on transitive homology. From ultra-conserved collinear regions between mammals and birds, by integrating coexpression information and protein-protein interactions, we identified more than 400 regions in the human genome showing significant functional coherence. The different algorithmical improvements ensure that i-ADHoRe 3.0 will remain a powerful tool to study genome evolution.     

Phylogenomics reveal a robust fungal tree of life

Our understanding of the tree of life (TOL) is still fragmentary. Until recently, molecular phylogeneticists have built trees based on ribosomal RNA sequences and selected protein sequences, which, however, usually suffered from lack of support for the deeper branches and inconsistencies probably due to limited subsampling of the entire genome. Now, phylogenetic hypotheses can be based on the analysis of full genomes. We used available complete genome data as well as the eukaryote orthologous group (KOG) proteins to reconstruct with confidence basal branches of the fungal TOL. Phylogenetic analysis of a core of 531 KOGs shared among 21 fungal genomes, three animal genomes and one plant genome showed a single tree with high support resulting from four different methods of phylogenetic reconstruction. The single tree that we inferred from our dataset showed excellent nodal support for each branch, suggesting that it reflects the true phylogenetic relationships of the species involved.     

Breakpoint medians and breakpoint phylogenies: a fixed-parameter approach

With breakpoint distance, the genome rearrangement field delivered one of the currently most popular measures in phylogenetic studies for related species. Here, BREAKPOINT MEDIAN, which is NP-complete already for three given species (whose genomes are represented as signed orderings), is the core basic problem. For the important special case of three species, approximation (ratio 7/6) and exact heuristic algorithms were developed. Here, we provide an exact, fixed-parameter algorithm with provable performance bounds. For instance, a breakpoint median for three signed orderings over nelements that causes at most d breakpoints can be computed in time O((2.15)(d).n). We show the algorithm's practical usefulness through experimental studies. In particular, we demonstrate that a simple implementation of our algorithm combined with a new tree construction heuristic allows for a new approach to breakpoint phylogeny, yielding evolutionary trees that are competitive in comparison with known results developed in a recent series of papers that use clever algorithm engineering methods.     

SHOT: a web server for the construction of genome phylogenies

With the increasing availability of genome sequences, new methods are being proposed that exploit information from complete genomes to classify species in a phylogeny. Here we present SHOT, a web server for the classification of genomes on the basis of shared gene content or the conservation of gene order that reflects the dominant, phylogenetic signal in these genomic properties. In general, the genome trees are consistent with classical gene-based phylogenies, although some interesting exceptions indicate massive horizontal gene transfer. SHOT is a useful tool for analysing the tree of life from a genomic point of view. It is available at http://www.Bork.EMBL-Heidelberg.de/SHOT.     

Species tree estimation for the late blight pathogen, Phytophthora infestans, and close relatives

To better understand the evolutionary history of a group of organisms, an accurate estimate of the species phylogeny must be known. Traditionally, gene trees have served as a proxy for the species tree, although it was acknowledged early on that these trees represented different evolutionary processes. Discordances among gene trees and between the gene trees and the species tree are also expected in closely related species that have rapidly diverged, due to processes such as the incomplete sorting of ancestral polymorphisms. Recently, methods have been developed for the explicit estimation of species trees, using information from multilocus gene trees while accommodating heterogeneity among them. Here we have used three distinct approaches to estimate the species tree for five Phytophthora pathogens, including P. infestans, the causal agent of late blight disease in potato and tomato. Our concatenation-based "supergene" approach was unable to resolve relationships even with data from both the nuclear and mitochondrial genomes, and from multiple isolates per species. Our multispecies coalescent approach using both Bayesian and maximum likelihood methods was able to estimate a moderately supported species tree showing a close relationship among P. infestans, P. andina, and P. ipomoeae. The topology of the species tree was also identical to the dominant phylogenetic history estimated in our third approach, Bayesian concordance analysis. Our results support previous suggestions that P. andina is a hybrid species, with P. infestans representing one parental lineage. The other parental lineage is not known, but represents an independent evolutionary lineage more closely related to P. ipomoeae. While all five species likely originated in the New World, further study is needed to determine when and under what conditions this hybridization event may have occurred.     

Comparative Genomics and the Gene Complement of a Minimal Cell

The concept of a minimal cell is discussed from the viewpoint of comparative genomics. Analysis of published DNA content values determined for 641 different archaeal and bacterial species by pulsed field gel electrophoresis has lead to a more precise definition of the genome size ranges of free-living and host-associated organisms. DNA content is not an indicator of phylogenetic position. However, the smallest genomes in our sample do not have a random distribution in rRNA-based evolutionary trees, and are found mostly in (a) the basal branches of the tree where thermophiles are located; and (b) in late clades, such as those of Gram positive bacteria. While the smallest-known genome size for an endosymbiont is only 450 kb, no free-living prokaryote has been described to have genomes < 1450 kb. Estimates of the size of minimal gene complement can provide important insights in the primary biological functions required for a sustainable, reproducing cell nowadays and throughout evolutionary times, but definitions of the minimum cell is dependent on specific environments.     

The quest for orthologs: finding the corresponding gene across genomes

Orthology is a key evolutionary concept in many areas of genomic research. It provides a framework for subjects as diverse as the evolution of genomes, gene functions, cellular networks and functional genome annotation. Although orthologous proteins usually perform equivalent functions in different species, establishing true orthologous relationships requires a phylogenetic approach, which combines both trees and graphs (networks) using reliable species phylogeny and available genomic data from more than two species, and an insight into the processes of molecular evolution. Here, we evaluate the available bioinformatics tools and provide a set of guidelines to aid researchers in choosing the most appropriate tool for any situation.