Robin van Velzen

Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

DNA Barcoding of Recently Diverged Species: Relative Performance of Matching Methods

Recently diverged species are challenging for identification, yet they are frequently of special interest scientifically as well as from a regulatory perspective. DNA barcoding has proven instrumental in species identification, especially in insects and vertebrates, but for the identification of recently diverged species it has been reported to be problematic in some cases. Problems are mostly due to incomplete lineage sorting or simply lack of a ‘barcode gap’ and probably related to large effective population size and/or low mutation rate. Our objective was to compare six methods in their ability to correctly identify recently diverged species with DNA barcodes: neighbor joining and parsimony (both tree-based), nearest neighbor and BLAST (similarity-based), and the diagnostic methods DNA-BAR, and BLOG. We analyzed simulated data assuming three different effective population sizes as well as three selected empirical data sets from published studies. Results show, as expected, that success rates are significantly lower for recently diverged species (∼75%) than for older species (∼97%) (P<0.00001). Similarity-based and diagnostic methods significantly outperform tree-based methods, when applied to simulated DNA barcode data (P<0.00001). The diagnostic method BLOG had highest correct query identification rate based on simulated (86.2%) as well as empirical data (93.1%), indicating that it is a consistently better method overall. Another advantage of BLOG is that it offers species-level information that can be used outside the realm of DNA barcoding, for instance in species description or molecular detection assays. Even though we can confirm that identification success based on DNA barcoding is generally high in our data, recently diverged species remain difficult to identify. Nevertheless, our results contribute to improved solutions for their accurate identification.

BLOG 2.0: a software system for character-based species classification with DNA Barcode sequences. What it does, how to use it

BLOG (Barcoding with LOGic) is a diagnostic and character‐based DNA Barcode analysis method. Its aim is to classify specimens to species based on DNA Barcode sequences and on a supervised machine learning approach, using classification rules that compactly characterize species in terms of DNA Barcode locations of key diagnostic nucleotides. The BLOG 2.0 software, its fundamental modules, online/offline user interfaces and recent improvements are described. These improvements affect both methodology and software design, and lead to the availability of different releases on the website http://dmb.iasi.cnr.it/blog-downloads.php. Previous and new experimental tests show that BLOG 2.0 outperforms previous versions as well as other DNA Barcode analysis methods.

Allopatric origin of cryptic butterfly species that were discovered feeding on distinct host plants in sympatry

Surveys of tropical insects are increasingly uncovering cryptic species – morphologically similar yet reproductively isolated taxa once thought to comprise a single interbreeding entity. The vast majority of such species are described from a single location. This leaves us with little information on geographic range and intraspecific variation and limits our ability to infer the forces responsible for generating such diversity. For example, in herbivorous and parasitic insects, multiple specialists are often discovered within what were thought to be single more generalized species. Host shifts are likely to have contributed to speciation in these cases. But when and where did those shifts occur, and were they facilitated by geographic isolation? We attempted to answer these questions for two cryptic species within the butterfly Cymothoe egesta that were recently discovered on different host plants in central Cameroon. We first used mtDNA markers to separate individuals collected on the two hosts within Cameroon and then extended our analysis to incorporate individuals collected across the entire pan‐Afrotropical range of the original taxon. To our surprise, we found that the species are almost entirely allopatric, dividing the original range and overlapping only in the narrow zone of West‐Central Africa where they were first discovered in sympatry. This finding, combined with analyses of genetic variation within each butterfly species, strongly suggests that speciation occurred in allopatry, probably during the Pleistocene. We discuss the implications of our results for understanding speciation among other cryptic species recently discovered in the tropics and argue that more work is needed on geographic patterns and host usage in such taxa.

Molecular phylogenetics and character evolution of Cannabaceae

Cannabaceae includes ten genera that are widely distributed in tropical to temperate regions of the world. Because of limited taxon and character sampling in previous studies, intergeneric phylogenetic relationships within this family have been poorly resolved. We conducted a molecular phylogenetic study based on four plastid loci (atpB-rbcL, rbcL, rps16, trnL-trnF) from 36 ingroup taxa, representing all ten recognized Cannabaceae genera, and six related taxa as outgroups. The molecular results strongly supported this expanded family to be a monophyletic group. All genera were monophyletic except for Trema, which was paraphyletic with respect to Parasponia. The Aphananthe clade was sister to all other Cannabaceae, and the other genera formed a strongly supported clade further resolved into a Lozanella clade, a Gironniera clade, and a trichotomy formed by the remaining genera. Morphological ancestral state reconstructions indicated the complex evolution pattern of most analyzed morphological characters, and it is difficult to identify morphological synapomorphies for most clades within Cannabaceae.

Phylogenetics of African Rinorea (Violaceae): Elucidating Infrageneric Relationships using Plastid and Nuclear DNA Sequences

Abstract Rinorea is a pantropical genus of shrubs and small trees within the family Violaceae. The genus is particularly diverse in Africa where species are ecologically important as they are often abundant or even dominant in particular forest types and act as larval host plants for highly specialized Cymothoe butterflies. Despite their importance, species identification of African Rinorea is difficult and a taxonomic revision is needed. Previous phylogenetic studies have suggested that neotropical taxa are sister to a palaeotropical clade, with multiple independent dispersals to Madagascar, but these were based on plastid data only. We therefore present an updated phylogeny of Rinorea with increased sampling of African taxa, using plastid as well as nuclear DNA sequences. Phylogenetic relationships inferred from nuclear DNA data were generally congruent with those based on evidence from plastid haplotypes from earlier studies. Our increased taxonomic sampling also revealed previously undiscovered African Rinorea clades, some of which warrant further taxonomic study. Ancestral state reconstructions refute previous hypotheses about the evolution of morphological characters traditionally used for Rinorea infrageneric classification. In addition, some widespread species may comprise species complexes. It is clear that African Rinorea require comprehensive taxonomic revision; our contribution to understanding Rinorea infrageneric relationships will facilitate this task.

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legume Parasponia

Rhizobium nitrogen-fixing nodules are a well-known trait of legumes, but nodules also occur in other plant lineages either with rhizobium or the actinomycete Frankia as microsymbiont. The widely accepted hypothesis is that nodulation evolved independently multiple times, with only a few losses. However, insight in the evolutionary trajectory of nodulation is lacking. We conducted comparative studies using Parasponia (Cannabaceae), the only non-legume able to establish nitrogen fixing nodules with rhizobium. This revealed that Parasponia and legumes utilize a large set of orthologous symbiosis genes. Comparing genomes of Parasponia and its non-nodulating relative Trema did not reveal specific gene duplications that could explain a recent gain of nodulation in Parasponia. Rather, Trema and other non-nodulating species in the Order Rosales show evidence of pseudogenization or loss of key symbiosis genes. This demonstrates that these species have lost the potential to nodulate. This finding challenges a long-standing hypothesis on evolution of nitrogen-fixing symbioses, and has profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

Draft Genome Sequence of the Nitrogen-Fixing Rhizobium sullae Type Strain IS123T Focusing on the Key Genes for Symbiosis with its Host Hedysarum coronarium L.

The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments.

Annonaceae substitution rates: a codon model perspective

The Annonaceae includes cultivated species of economic interest and represents an important source of information for better understanding the evolution of tropical rainforests. In phylogenetic analyses of DNA sequence data that are used to address evolutionary questions, it is imperative to use appropriate statistical models. Annonaceae are cases in point: Two sister clades, the subfamilies Annonoideae and Malmeoideae, contain the majority of Annonaceae species diversity. The Annonoideae generally show a greater degree of sequence divergence compared to the Malmeoideae, resulting in stark differences in branch lengths in phylogenetic trees. Uncertainty in how to interpret and analyse these differences has led to inconsistent results when estimating the ages of clades in Annonaceae using molecular dating techniques. We ask whether these differences may be attributed to inappropriate modelling assumptions in the phylogenetic analyses. Specifically, we test for (clade-specific) differences in rates of non-synonymous and synonymous substitutions. A high ratio of nonsynonymous to synonymous substitutions may lead to similarity of DNA sequences due to convergence instead of common ancestry, and as a result confound phylogenetic analyses. We use a dataset of three chloroplast genes (rbcL, matK, ndhF) for 129 species representative of the family. We find that differences in branch lengths between major clades are not attributable to different rates of non-synonymous and synonymous substitutions. The differences in evolutionary rate between the major clades of Annonaceae pose a challenge for current molecular dating techniques that should be seen as a warning for the interpretation of such results in other organisms.

Comparative transcriptome analysis of Poncirus trifoliata identifies a core set of genes involved in arbuscular mycorrhizal symbiosis

A comparative transcriptome analysis of Poncirus trifoliata and four herbaceous model plants identifies a core set of arbuscular mycorrhiza-induced genes that are likely to play key roles in symbiosis.