Yann J. K. Bertrand

Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data

Summary 1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

Phylogenetic properties of 50 nuclear loci in Medicago (Leguminosae) generated using multiplexed sequence capture and next-generation sequencing.

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.

First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Assignment of Homoeologs to Parental Genomes in Allopolyploids for Species Tree Inference, with an Example from Fumaria (Papaveraceae)

There is a rising awareness that species trees are best inferred from multiple loci while taking into account processes affecting individual gene trees, such as substitution model error (failure of the model to account for the complexity of the data) and coalescent stochasticity (presence of incomplete lineage sorting [ILS]). Although most studies have been carried out in the context of dichotomous species trees, these processes operate also in more complex evolutionary histories involving multiple hybridizations and polyploidy. Recently, methods have been developed that accurately handle ILS in allopolyploids, but they are thus far restricted to networks of diploids and tetraploids. We propose a procedure that improves on this limitation by designing a workflow that assigns homoeologs to hypothetical diploid ancestral genomes prior to genome tree construction. Conflicting assignment hypotheses are evaluated against substitution model error and coalescent stochasticity. Incongruence that cannot be explained by stochastic mechanisms needs to be explained by other processes (e.g., homoploid hybridization or paralogy). The data can then be filtered to build multilabeled genome phylogenies using inference methods that can recover species trees, either in the face of substitution model error and coalescent stochasticity alone, or while simultaneously accounting for hybridization. Methods are already available for folding the resulting multilabeled genome phylogeny into a network. We apply the workflow to the reconstruction of the reticulate phylogeny of the plant genus Fumaria (Papaveraceae) with ploidal levels ranging from 2[Formula: see text] to 14[Formula: see text]. We describe the challenges in recovering nuclear NRPB2 homoeologs in high ploidy species while combining in vivo cloning and direct sequencing techniques. Using parametric bootstrapping simulations we assign nuclear homoeologs and chloroplast sequences (four concatenated loci) to their common hypothetical diploid ancestral genomes. As these assignments hinge on effective population size assumptions, we investigate how varying these assumptions impacts the recovered multilabeled genome phylogeny.

Patterns of phylogenetic incongruence in Medicago found among six loci

The species phylogeny of Medicago L. (Leguminosae) remains unresolved, as there is significant incongruence between the published gene phylogenies. Here, we compare six of these gene phylogenies of Medicago, inferred from unlinked loci from the nuclear, chloroplast and mitochondrial genomes. Data from all loci were re-analysed, including gap-coding of initial data sets, and dated phylogenies were produced. The patterns of species relationships observed in the six dated phylogenies are compatible with several different biological processes, such as incomplete lineage sorting and hybridisation. A subset of the original sampling that included 29 taxa was also analysed using coalescent-based tree distance comparisons. The observed topological distances suggest that differences between gene phylogenies cannot be solely attributed to incomplete lineage sorting. Hybridisation is strongly suspected to have occurred in the history of many taxa in the genus, because of overlapping divergence times between suspected hybrids and each parental lineage, confirming earlier results based on only two genes. An attempt to reconcile the conflicting histories in a multispecies coalescent analysis, using multiple labels for taxa with hybrid histories, did not produce satisfactory results and may be fatally limited. We conclude that although the currently available data are not sufficient to clarify relationships in Medicago, many cases of hybridisation are probable. The phylogenetic history of the genus is therefore better understood as a network and not a single tree. This raises concerns over previous studies that have used single gene trees as summaries of the history of species relationships.

Molecular phylogeny of Acanthophyllum (Caryophyllaceae: Caryophylleae), with emphasis on infrageneric classification

Despite being one of the larger genera of Caryophyllaceae with about 60 cushion-forming subshrubby species, Acanthophyllum is represented poorly in previous molecular phylogenetic studies. The genus is an important component of the subalpine steppe flora in Central to Southwest Asia. Although the placement of Acanthophyllum in the tribe Caryophylleae and a close relationship to Allochrusa has already been suggested, the monophyly of the genus and its subgeneric taxa, as well as its relation to other closely related genera, have not been addressed. We have assembled datasets of nuclear ribosomal internal transcribed spacer (ITS) sequences and intron sequences of the chloroplast gene rps16 for 47 Acanthophyllum species and 63 species of 12 additional genera from Caryophylleae. Phylogenetic analyses were performed using maximum parsimony, maximum likelihood and Bayesian methods. Our analysis suggests that Allochrusa, Diaphanoptera, Ochotonophila and Scleranthopsis are nested within Acanthophyllum but that the traditionally recognized sections of Acanthophyllum are monophyletic after reassignment of a few species. Emarginate petals may be a synapomorphy for one of the two basal clades of Acanthophyllum. Moreover, non-monophyly of the genera Gypsophila and Diaphanoptera is suggested by the present study. The age of the crown clade of Acanthophyllum s.l. is estimated to be 11.1 Ma by *BEAST species tree analysis.

Using Genomic Location and Coalescent Simulation to Investigate Gene Tree Discordance in Medicago L.

Abstract.— Several well‐documented evolutionary processes are known to cause conflict between species‐level phylogenies and gene‐level phylogenies. Three of the most challenging processes for species tree inference are incomplete lineage sorting, hybridization and gene duplication, which may result in unwarranted comparisons of paralogous genes. Several existing methods have dealt with these processes but none has yet been able to untangle all three at once. Here, we propose a stepwise method by which these processes can be discerned using information on genomic location coupled with coalescent simulations. In the first step, highly discordant genes within genomic blocks (putative paralogs) are identified and excluded from the data set and, in the second step, blocks of linked genes are grouped according to their hybrid history. Existing multispecies coalescent software can then be applied to recover the principal tree(s) that make up the species tree/network without violating the underlying model. The potential of the approach is evaluated on simulated data derived from a species network composed of nine species, of which one is of hybrid origin, and displaying a single‐gene duplication that leads to paralogous comparisons. We apply our method to an empirical set of 12 genes from 7 species sampled in the plant genus Medicago that display phylogenetic discordance. We identify the causes of the discordance and demonstrate that the Medicago orbicularis lineage experienced an episode of ancient hybridization. Our results show promise as a new way to explore phylogenetic sequence data that can significantly improve species tree inference in presence of hybridization and undetected paralogy or other causes leading to extremely discordant gene trees. [Coalescent simulation; gene tree; genomic location; hybridization; incomplete lineage sorting; paralogy; phylogenetic incongruence; principal tree; species tree.]

A cryptic species produced by autopolyploidy and subsequent introgression involving Medicago prostrata (Fabaceae)

Although hybridisation through genome duplication is well known, hybridisation without genome duplication (homoploid hybrid speciation, HHS) is not. Few well-documented cases have been reported. A possible instance of HHS in Medicago prostrata Jacq. was suggested previously, based on only two genes and one individual. We tested whether this species was formed through HHS by sampling eight nuclear loci and 22 individuals, with additional individuals from related species, using gene capture and Illumina sequencing. Phylogenetic inference and coalescent simulations were performed to infer the causes of gene tree incongruence. We found no evidence that phylogenetic differences among M. prostrata individuals were the result of HHS. Instead, an autopolyploid origin of tetraploids with introgression from tetraploids of the M. sativa complex is likely. We argue that tetraploid M. prostrata individuals constitute a new species, characterised by a partially non-overlapping distribution and distinctive alleles (from the M. sativa complex). No gene flow from tetraploid to diploid M. prostrata is apparent, suggesting partial reproductive isolation. Thus, speciation via autopolyploidy appears to have been reinforced by introgression. This raises the intriguing possibility that introgressed alleles may be responsible for the increased range exploited by tetraploid M. prostrata with respect to that of the diploids.

Revisiting Recombination Signal in the Tick-Borne Encephalitis Virus: A Simulation Approach.

The hypothesis of wide spread reticulate evolution in Tick-Borne Encephalitis virus (TBEV) has recently gained momentum with several publications describing past recombination events involving various TBEV clades. Despite a large body of work, no consensus has yet emerged on TBEV evolutionary dynamics. Understanding the occurrence and frequency of recombination in TBEV bears significant impact on epidemiology, evolution, and vaccination with live vaccines. In this study, we investigated the possibility of detecting recombination events in TBEV by simulating recombinations at several locations on the virus’ phylogenetic tree and for different lengths of recombining fragments. We derived estimations of rates of true and false positive for the detection of past recombination events for seven recombination detection algorithms. Our analytical framework can be applied to any investigation dealing with the difficult task of distinguishing genuine recombination signal from background noise. Our results suggest that the problem of false positives associated with low detection P-values in TBEV, is more insidious than generally acknowledged. We reappraised the recombination signals present in the empirical data, and showed that reliable signals could only be obtained in a few cases when highly genetically divergent strains were involved, whereas false positives were common among genetically similar strains. We thus conclude that recombination among wild-type TBEV strains may occur, which has potential implications for vaccination with live vaccines, but that these events are surprisingly rare.

Robustness of RADseq for evolutionary network reconstruction from gene trees

Although hybridization has played an important role in the evolution of many species, phylogenetic reconstructions that include hybridizing lineages have been historically constrained by the available models and data. Recently, the combined development of high-throughput sequencing and evolutionary network models offer new opportunities for phylogenetic inference under complex patterns of hybridization in the context of incomplete lineage sorting. Restriction site associated DNA sequencing (RADseq) has been a popular sequencing technique for evolutionary reconstructions of close relatives in the Next Generation Sequencing (NGS) era. However, the utility of RADseq data for the reconstruction of complex evolutionary networks has not been thoroughly discussed. Here, we used new molecular data collected from diploid perennial Medicago species using single-digest RADseq to reconstruct evolutionary networks from gene trees, an approach that is computationally tractable with datasets that include several species and complex patterns of hybridization. Our analyses revealed that complex network reconstructions from RADseq-derived gene trees were not robust under variations of the assembly parameters and filters. Filters to exclusively select loci with high phylogenetic information created datasets that retrieved the most anomalous topologies. Conversely, alternative clustering thresholds or filters on the number of samples per locus affected the level of missing data but had a lower impact on networks. When most anomalous networks were discarded, all remaining network analyses consistently supported a hybrid origin for M. carstiensis and M. cretacea.