Stephan Nylinder

An open source chimera checker for the fungal ITS region

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit holds a central position in the pursuit of the taxonomic affiliation of fungi recovered through environmental sampling. Newly generated fungal ITS sequences are typically compared against the International Nucleotide Sequence Databases for a species or genus name using the sequence similarity software suite blast. Such searches are not without complications however, and one of them is the presence of chimeric entries among the query or reference sequences. Chimeras are artificial sequences, generated unintentionally during the polymerase chain reaction step, that feature sequence data from two (or possibly more) distinct species. Available software solutions for chimera control do not readily target the fungal ITS region, but the present study introduces a blast‐based open source software package (available at http://www.emerencia.org/chimerachecker.html) to examine newly generated fungal ITS sequences for the presence of potentially chimeric elements in batch mode. We used the software package on a random set of 12 300 environmental fungal ITS sequences in the public sequence databases and found 1.5% of the entries to be chimeric at the ordinal level after manual verification of the results. The proportion of chimeras in the sequence databases can be hypothesized to increase as emerging sequencing technologies drawing from pooled DNA samples are becoming important tools in molecular ecology research.

Estimating the Phanerozoic history of the Ascomycota lineages: Combining fossil and molecular data

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant 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.

Paleo-Drainage Basin Connectivity Predicts Evolutionary Relationships across Three Southeast Asian Biodiversity Hotspots

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earths land surface, more than half the worlds plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the regions dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

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.

Are Asteraceae 1.5 Billion Years Old? A Reply to Heads

Are Asteraceae 1.5 Billion Years Old? A Reply to Heads ULF SWENSON1,* , STEPHAN NYLINDER2 , AND STEVEN J. WAGSTAFF3 1Department of Phanerogamic Botany, Swedish Museum of Natural History, PO Box 50007, 104 05 Stockholm, Sweden; 2Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, 405 30 Gothenburg, Sweden; and 3Allan Herbarium, Landcare Research, PO Box 40, Lincoln 7640, New Zealand; ∗Correspondence to be sent to: Department of Phanerogamic Botany, Swedish Museum of Natural History, PO Box 50007, 104 05 Stockholm, Sweden; E-mail: ulf.swenson@nrm.se.

On the Biogeography of Centipeda: A Species-Tree Diffusion Approach

Reconstructing the biogeographic history of groups present in continuous arid landscapes is challenging due to the difficulties in defining discrete areas for analyses, and even more so when species largely overlap both in terms of geography and habitat preference. In this study, we use a novel approach to estimate ancestral areas for the small plant genus Centipeda. We apply continuous diffusion of geography by a relaxed random walk where each species is sampled from its extant distribution on an empirical distribution of time-calibrated species-trees. Using a distribution of previously published substitution rates of the internal transcribed spacer (ITS) for Asteraceae, we show how the evolution of Centipeda correlates with the temporal increase of aridity in the arid zone since the Pliocene. Geographic estimates of ancestral species show a consistent pattern of speciation of early lineages in the Lake Eyre region, with a division in more northerly and southerly groups since ∼840 ka. Summarizing the geographic slices of species-trees at the time of the latest speciation event (∼20 ka), indicates no presence of the genus in Australia west of the combined desert belt of the Nullabor Plain, the Great Victoria Desert, the Gibson Desert, and the Great Sandy Desert, or beyond the main continental shelf of Australia. The result indicates all western occurrences of the genus to be a result of recent dispersal rather than ancient vicariance. This study contributes to our understanding of the spatiotemporal processes shaping the flora of the arid zone, and offers a significant improvement in inference of ancestral areas for any organismal group distributed where it remains difficult to describe geography in terms of discrete areas.

Towards a natural classification of Sapotaceae subfamily Chrysophylloideae in Oceania and Southeast Asia based on nuclear sequence data

Generic limits within subfamily Chrysophylloideae (Sapotaceae) from Oceania and Southeast Asia are reconciled based on a molecular phylogeny. We analysed sequences of nuclear ribosomal DNA (ETS, IT ...

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)‐amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele‐specific primers. We tested two methods to enhance the specificity of allele‐specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele‐specific primer to 15–13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele‐specific amplification in regular PCR rather than in direct sequencing.

Species tree phylogeny and biogeography of the Neotropical genus Pradosia (Sapotaceae, Chrysophylloideae).

Recent phylogenetic studies in Sapotaceae have demonstrated that many genera need to be redefined to better correspond to natural groups. The Neotropical genus Pradosia is believed to be monophyletic and includes 26 recognized species. Here we reconstruct the generic phylogeny by a species-tree approach using (∗)BEAST, 21 recognized species (36 accessions), sequence data from three nuclear markers (ITS, ETS, and RPB2), a relaxed lognormal clock model, and a fossil calibration. We explore the evolution of five selected morphological characters, reconstruct the evolution of habitat (white-sand vs. clayish soils) preference, as well as space and time by using a recently developed continuous diffusion model in biogeography. We find Pradosia to be monophyletic in its current circumscription and to have originated in the Amazon basin at ∼47.5Ma. Selected morphological characters are useful to readily distinguish three clades. Preferences to white-sand and/or clay are somewhat important for the majority of species, but speciation has not been powered by habitat shifts. Pradosia brevipes is a relative young species (∼1.3Ma) that has evolved a unique geoxylic life strategy within Pradosia and is restricted to savannahs. Molecular dating and phylogenetic pattern indicate that Pradosia reached the Brazilian Atlantic coast at least three times: at 34.4Ma (P. longipedicellata), at 11.7Ma (P. kuhlmannii), and at 3.9Ma (weakly supported node within the red-flowered clade).