| 1. |
- Bengtsson-Palme, Johan, 1985, et al.
(author)
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Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data
- 2013
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In: Methods in Ecology and Evolution. - 2041-210X. ; 4:10, s. 914-919
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Journal article (peer-reviewed)abstract
- 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. 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. 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. 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.
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| 2. |
- Lopes de Abreu, Narjara, et al.
(author)
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The use of chloroplast genome sequences to solve phylogenetic incongruences in Polystachya Hook (Orchidaceae Juss)
- 2018
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In: Peerj. - : PeerJ. - 2167-8359. ; 6
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Journal article (peer-reviewed)abstract
- Background: Current evidence suggests that for more robust estimates of species tree and divergence times, several unlinked genes are required. However, most phylogenetic trees for non-model organisms are based on single sequences or just a few regions, using traditional sequencing methods. Techniques for massive parallel sequencing or next generation sequencing (NGS) are an alternative to traditional methods that allow access to hundreds of DNA regions. Here we use this approach to resolve the phylogenetic incongruence found in Polystachya Hook. (Orchidaceae), a genus that stands out due to several interesting aspects, including cytological (polyploid and diploid species), evolutionary (reticulate evolution) and biogeographical (species widely distributed in the tropics and high endemism in Brazil). The genus has a notoriously complicated taxonomy, with several sections that are widely used but probably not monophyletic. Methods: We generated the complete plastid genome of 40 individuals from one clade within the genus. The method consisted in construction of genomic libraries, hybridization to RNA probes designed from available sequences of a related species, and subsequent sequencing of the product. We also tested how well a smaller sample of the plastid genome would perform in phylogenetic inference in two ways: by duplicating a fast region and analyzing multiple copies of this dataset, and by sampling without replacement from all non-coding regions in our alignment. We further examined the phylogenetic implications of non-coding sequences that appear to have undergone hairpin inversions (reverse complemented sequences associated with small loops). Results: We retrieved 131,214 bp, including coding and non-coding regions of the plastid genome. The phylogeny was able to fully resolve the relationships among all species in the targeted clade with high support values. The first divergent species are represented by African accessions and the most recent ones are among Neotropical species. Discussion: Our results indicate that using the entire plastid genome is a better option than screening highly variable markers, especially when the expected tree is likely to contain many short branches. The phylogeny inferred is consistent with the proposed origin of the genus, showing a probable origin in Africa, with later dispersal into the Neotropics, as evidenced by a clade containing all Neotropical individuals. The multiple positions of Polystachya concreta (Jacq.) Garay & Sweet in the phylogeny are explained by allotetraploidy. Polystachya estrellensis Rchb.f. can be considered a genetically distinct species from P. concreta and P. foliosa (Lindl.) Rchb.f., but the delimitation of P. concreta remains uncertain. Our study shows that NGS provides a powerful tool for inferring relationships at low taxonomic levels, even in taxonomically challenging groups with short branches and intricate morphology.
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| 3. |
- Bertrand, Yann, et al.
(author)
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Assignment of homoeologues to parental genomes in allopolyploids for species tree inference, with an example from Fumaria (Papaveraceae)
- 2015
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In: Systematic Biology. - : Oxford University Press (OUP). - 1063-5157 .- 1076-836X. ; 64:3, s. 448-471
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Journal article (peer-reviewed)abstract
- 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). 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 incomplete lineage sorting 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 homoeologues 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 2x to 14x. We describe the challenges in recovering nuclear NRPB2 homoeologues in high ploidy species while combining in vivo cloning and direct sequencing techniques. Using parametric bootstrapping simulations we assign nuclear homoeologues 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.
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| 4. |
- Eriksson, Jonna S., 1989, et al.
(author)
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A cryptic species produced by autopolyploidy and subsequent introgression involving Medicago prostrata (Fabaceae)
- 2017
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In: Molecular Phylogenetics and Evolution. - : Elsevier BV. - 1055-7903 .- 1095-9513. ; 107, s. 367-381
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Eriksson, Jonna S., 1989, et al.
(author)
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Allele phasing is critical to revealing a shared allopolyploid origin of Medicago arborea and M. strasseri (Fabaceae)
- 2018
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In: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 18:1
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Journal article (peer-reviewed)abstract
- Background: Whole genome duplication plays a central role in plant evolution. There are two main classes of polyploid formation: autopolyploids which arise within one species by doubling of similar homologous genomes; in contrast, allopolyploidy (hybrid polyploidy) arise via hybridization and subsequent doubling of nonhomologous (homoeologous) genomes. The distinction between polyploid origins can be made using gene phylogenies, if alleles from each genome can be correctly retrieved. We examined whether two closely related tetraploid Mediterranean shrubs (Medicago arborea and M. strasseri) have an allopolyploid origin - a question that has remained unsolved despite substantial previous research. We sequenced and analyzed ten low-copy nuclear genes from these and related species, phasing all alleles. To test the efficacy of allele phasing on the ability to recover the evolutionary origin of polyploids, we compared these results to analyses using unphased sequences. Results: In eight of the gene trees the alleles inferred from the tetraploids formed two clades, in a non-sister relationship. Each of these clades was more closely related to alleles sampled from other species of Medicago, a pattern typical of allopolyploids. However, we also observed that alleles from one of the remaining genes formed two clades that were sister to one another, as is expected for autopolyploids. Trees inferred from unphased sequences were very different, with the tetraploids often placed in poorly supported and different positions compared to results obtained using phased alleles. Conclusions: The complex phylogenetic history of M. arborea and M. strasseri is explained predominantly by shared allotetraploidy. We also observed that an increase in woodiness is correlated with polyploidy in this group of species and present a new possibility that woodiness could be a transgressive phenotype. Correctly phased homoeologues are likely to be critical for inferring the hybrid origin of allopolyploid species, when most genes retain more than one homoeologue. Ignoring homoeologous variation by merging the homoeologues can obscure the signal of hybrid polyploid origins and produce inaccurate results. © 2018 The Author(s).
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| 6. |
- Nilsson, R. Henrik, 1976, et al.
(author)
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A comprehensive, automatically updated fungal ITS sequence dataset for reference-based chimera control in environmental sequencing efforts
- 2015
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In: Microbes and Environments. - 1342-6311 .- 1347-4405. ; 30:2, s. 145-150
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Journal article (peer-reviewed)abstract
- The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.
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| 7. |
- Nilsson, R. Henrik, 1976, et al.
(author)
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Improving ITS sequence data for identification of plant pathogenic fungi
- 2014
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In: Fungal Diversity. - : Springer Science and Business Media LLC. - 1560-2745 .- 1878-9129. ; 67:1, s. 11-19
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Journal article (peer-reviewed)abstract
- Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.
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| 8. |
- Sousa, Filipe de, 1982
(author)
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Next-generation Molecular Systematics and Evolution: insights into Medicago
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Evolutionary relationships among species have in recent years been inferred using DNA sequences from specific genes and represented by tree diagrams (phylogenies). However, the signal contained in different genes can produce conflicting sets of relationships – i.e., gene tree incongruence. Incongruence among gene trees in Medicago L. (Leguminosae) has been observed in several studies but not yet resolved. This thesis aims to re-examine the existing gene phylogenies, generate new data and apply new methods of analysis in order to achieve a deeper understanding of the causes of incongruence among species of Medicago. A comparison and reanalysis of six previously published gene phylogenies for Medicago indicates that different biological processes, such as incomplete lineage sorting, paralogy and hybridisation, intervene simultaneously to produce highly incongruent phylogenetic patterns in this plant genus. In order to discern between these different processes, more genomic data is required, and therefore a new approach, using recently developed sequence-capture techniques, is adopted, resulting in the largest, most widely sampled and comparable set of gene phylogenies generated to date for this genus. Identifying causes of incongruence also requires the development a theoretical framework that would allow for the sorting of these data according to different patterns. A model that takes genomic location into account and uses coalescent simulation to compare gene tree topologies is presented. This model is formulated as a flow of tests that culminate in the sorting of data for the inference of species trees, referred to as principal trees when hybridisation is present. The data produced and the methods developed are used to investigate the phylogeny of two sub-clades within Medicago, the Medicago murex clade and section Spirocarpos subsection Intertextae. Additionally, coalescent-based species delimitation methods are used to clarify species relationships. A species phylogeny is produced for the Medicago murex clade, confirming the separation between M. murex and M. lesinsii, two species with different karyotype. The inferred species relationships suggest a double event of chromosome speciation in this clade. Two hybridisation events are shown in subsect. Intertextae, both affecting lineages of Medicago ciliaris. The principal trees that represent these events are recovered through species tree inference analysis from data sorted by coalescent-based tests. A case of cryptic allopolyploid speciation is discovered in Medicago prostrata, a species with known diploid and polyploid karyotypes. Finally, a chloroplast phylogeny of Medicago is presented. This thesis shows that hybridisation has played an important role in the evolutionary history of Medicago, both at diploid and polyploid levels. It provides new insights into the evolutionary history of Medicago and shows the importance of hybridisation in the evolution of plant species.
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| 9. |
- Sousa, Filipe de, 1982, et al.
(author)
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Patterns of phylogenetic incongruence in Medicago found among six linkage groups
- 2016
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In: Plant Systematics and Evolution. - : Springer Science and Business Media LLC. - 0378-2697 .- 1615-6110 .- 2199-6881. ; 302:5, s. 493-513
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Sousa, Filipe de, 1982, et al.
(author)
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Phylogenetic properties of 50 nuclear loci in Medicago (Leguminosae) generated using multiplexed sequence capture and next-generation sequencing
- 2014
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:10
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Journal article (peer-reviewed)abstract
- 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.
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