Benjamin Stielow

Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In todays next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports.

Pathology to vertebrate hosts has emerged repeatedly in the order Ophiostomatales. Occasional infections have been observed in Sporothrix mexicana at a low level of virulence, while the main pathogenic species cluster in a derived clade around S. schenckii s.str. In this paper, phylogeny and epidemiology of the members of this clade were investigated for 99 clinical and 36 environmental strains using four genetic loci, viz. rDNA ITS and partial CAL, TEF1, and TEF3; data are compared with amplified fragment length polymorphism (AFLP) genotyping. The four main species of the pathogenic clade were recognised. The species proved to show high degrees of endemicity, which enabled interpretation of literature data where live material or genetic information is lacking. The clade of four species comprised nine subclusters, which often had limited geographic distribution and were separate from each other in all partitions, suggesting low degrees of interbreeding between populations. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of dispersal. Sporothrix brasiliensis is known to be involved in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from putrid plant material, causing a sapronosis. Sporothrix schenckii s.str., the most variable species within the clade, also had a plant origin, with ecological similarities to that of S. globosa. A hypothesis was put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates repeated infection of mammals.

Fungal Planet description sheets: 281-319.

Novel species of fungi described in the present study include the following from South Africa: Alanphillipsia aloeicola from Aloe sp., Arxiella dolichandrae from Dolichandra unguiscati, Ganoderma austroafricanum from Jacaranda mimosifolia, Phacidiella podocarpi and Phaeosphaeria podocarpi from Podocarpus latifolius, Phyllosticta mimusopisicola from Mimusops zeyheri and Sphaerulina pelargonii from Pelargonium sp. Furthermore, Barssia maroccana is described from Cedrus atlantica (Morocco), Codinaea pini from Pinus patula (Uganda), Crucellisporiopsis marquesiae from Marquesia acuminata (Zambia), Dinemasporium ipomoeae from Ipomoea pes-caprae (Vietnam), Diaporthe phragmitis from Phragmites australis (China), Marasmius vladimirii from leaf litter (India), Melanconium hedericola from Hedera helix (Spain), Pluteus albotomentosus and Pluteus extremiorientalis from a mixed forest (Russia), Rachicladosporium eucalypti from Eucalyptus globulus (Ethiopia), Sistotrema epiphyllum from dead leaves of Fagus sylvatica in a forest (The Netherlands), Stagonospora chrysopyla from Scirpus microcarpus (USA) and Trichomerium dioscoreae from Dioscorea sp. (Japan). Novel species from Australia include: Corynespora endiandrae from Endiandra introrsa, Gonatophragmium triuniae from Triunia youngiana, Penicillium coccotrypicola from Archontophoenix cunninghamiana and Phytophthora moyootj from soil. Novelties from Iran include Neocamarosporium chichastianum from soil and Seimatosporium pistaciae from Pistacia vera. Xenosonderhenia eucalypti and Zasmidium eucalyptigenum are newly described from Eucalyptus urophylla in Indonesia. Diaporthe acaciarum and Roussoella acacia are newly described from Acacia tortilis in Tanzania. New species from Italy include Comoclathris spartii from Spartium junceum and Phoma tamaricicola from Tamarix gallica. Novel genera include (Ascomycetes): Acremoniopsis from forest soil and Collarina from water sediments (Spain), Phellinocrescentia from a Phellinus sp. (French Guiana), Neobambusicola from Strelitzia nicolai (South Africa), Neocladophialophora from Quercus robur (Germany), Neophysalospora from Corymbia henryi (Mozambique) and Xenophaeosphaeria from Grewia sp. (Tanzania). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA.

The basal fungal order Mortierellales constitutes one of the largest orders in the basal lineages. This group consists of one family and six genera. Most species are saprobic soil inhabiting fungi with the ability of diverse biotransformations or the accumulation of unsaturated fatty acids, making them attractive for biotechnological applications. Only few studies exist aiming at the revelation of the evolutionary relationships of this interesting fungal group. This study includes the largest dataset of LSU and ITS sequences for more than 400 specimens containing 63 type or reference strains. Based on a LSU phylogram, fungal groups were defined and evaluated using ITS sequences and morphological features. Traditional morphology-based classification schemes were rejected, because the morphology of the Mortierellales seems to depend on culture conditions, a fact, which makes the identification of synapomorphic characters tedious. This study belongs to the most comprehensive molecular phylogenetic analyses for the Mortierellales up to date and reveals unresolved species and species complexes.

Complete genome sequence of the first endornavirus from the ascocarp of the ectomycorrhizal fungus Tuber aestivum Vittad.

The genus Endornavirus was recently approved by the International Committee on Taxonomy of Viruses (ICTV) [1, 10] to be separated from the family Partitiviridae, and endornaviruses have only been described in plants and fungi [4, 15]. They share common properties such as symptom-free infection of their host, occurrence in lowcopy number in host cells, lack of true virions [7], and an efficient vertical transmission in their hosts [16]. Endornaviruses have unique plasmid-like properties that differ markedly from those of other conventionally encapsidated viruses [5, 12, 16]. The genomes of endornaviruses consist of linear double-stranded RNA (dsRNA) with a characteristic, single open reading frame (ORF) of up to 18 kb in length, often preceded by a site-specific nick at the 5’ end [5, 7, 8, 12, 18]. The predicted protein from this ORF harbours in its N-terminal part characteristic amino acid (aa) sequence motifs for viral RNA helicases (VHel) or viral methyltransferases (VMet), while aa sequence motifs for UDP-glycosyltransferases (UGT) and RNA-dependent RNA polymerases (RdRp) are located at close proximity to the 30 end [7]. The remaining polyprotein is unrelated to any known functional proteins in the database. Currently, the INSDC databases contain complete sequences of seven endornaviruses. Three are plant-associated viruses infecting cultivated and wild rice (Oryza sativa endornavirus [OsEV] and Oryza rufipogon endornavirus [OrEV]) [5, 11], respectively), as well as broad bean (Vicia faba endornavirus [VFV]) [13]. In addition, four fungal isolates are known. One isolate each was reported from the mycelia of the violet root rot fungus (Helicobasidium mompa endornavirus 1-670 [HmEV1-670]) [12] and from an informally designated plant-pathogenic oomycete Phytophtora spp. (Phytophtora endornavirus 1 [PEV1]) [6], while two isolates originate from the pathogenic ascomycete Gremmeniella abietina (Gremmeniella abietina type B RNA virus) [15]. Here, we report the complete nucleotide sequence of an isolate of a putatively novel species in the genus Endornavirus from an ectomycorrhizal fungus, isolated from the hypogeous ascomata of a black truffle (Tuber aestivum Vittad.). Accordingly, we propose the name Tuber aestivum endornavirus (TaEV). The TaEV genome structure is highly similar to that of other Ascomycetes-infecting endornaviruses, such as Gremmeniella abietina type B RNA virus [15], containing characteristic motifs for the DEAD-like helicase (DEXDc), VMet and RdRp.

Phylogenetic Findings Suggest Possible New Habitat and Routes of Infection of Human Eumyctoma

Eumycetoma is a traumatic fungal infection in tropical and subtropical areas that may lead to severe disability. Madurella mycetomatis is one of the prevalent etiologic agents in arid Northeastern Africa. The source of infection has not been clarified. Subcutaneous inoculation from plant thorns has been hypothesized, but attempts to detect the fungus in relevant material have remained unsuccessful. The present study aims to find clues to reveal the natural habitat of Madurella species using a phylogenetic approach, i.e. by comparison of neighboring taxa with known ecology. Four species of Madurella were included in a large data set of species of Chaetomium, Chaetomidium, Thielavia, and Papulaspora (n = 128) using sequences of the universal fungal barcode gene rDNA ITS and the partial LSU gene sequence. Our study demonstrates that Madurella species are nested within the Chaetomiaceae, a family of fungi that mainly inhabit animal dung, enriched soil, and indoor environments. We hypothesize that cattle dung, ubiquitously present in rural East Africa, plays a significant role in the ecology of Madurella. If cow dung is an essential factor in inoculation by Madurella, preventative measures may involve the use of appropriate footwear in addition to restructuring of villages to reduce the frequency of contact with etiologic agents of mycetoma. On the other hand, the Chaetomiaceae possess a hidden clinical potential which needs to be explored.

DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation

DNA barcoding is a global initiative for species identification through sequencing of short DNA sequence markers. Sequences of two loci, ITS and LSU, were generated as barcode data for all (ca. 9k) yeast strains included in the CBS collection, originally assigned to ca. 2 000 species. Taxonomic sequence validation turned out to be the most severe bottleneck due to the large volume of generated trace files and lack of reference sequences. We have analysed and validated CBS strains and barcode sequences automatically. Our analysis shows that there were 6 and 9.5 % of CBS yeast species that could not be distinguished by ITS and LSU, respectively. Among them, ∼3 % were indistinguishable by both loci. Except for those species, both loci were successfully resolving yeast species as the grouping of yeast DNA barcodes with the predicted taxonomic thresholds was more than 90 % similar to the grouping with respect to the expected taxon names. The taxonomic thresholds predicted to discriminate yeast species were 98.41 % for ITS and 99.51 % for LSU. To discriminate current yeast genera, thresholds were 96.31 % for ITS and 97.11 % for LSU. Using ITS and LSU barcodes, we were also able to show that the recent reclassifications of basidiomycetous yeasts in 2015 have made a significant improvement for the generic taxonomy of those organisms. The barcodes of 4 730 (51 %) CBS yeast strains of 1 351 (80 %) accepted yeast species that were manually validated have been released to GenBank and the CBS-KNAW website as reference sequences for yeast identification.

Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota)

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

A novel Tuber aestivum (Vittad.) mitovirus.

Double-stranded RNA (dsRNA) viruses have been reported from major phyla of the fungal kingdom [8, 11]. Mycovirus infections have been associated with obvious disease symptoms but often remain symptomless [3, 8, 11]. Fungal viruses are known from eleven families: Barnaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Hypoviridae, Metaviridae, Narnaviridae, Partitiviridae, Reoviridae, Totiviridae and recently also Endornaviridae [1, 8, 11, 14, 15]. The simplest mycoviruses are assigned to the genera Mitovirus and Narnavirus (family Narnaviridae). Members of both genera lack coat proteins, and their linear genomes are characterized by a single open reading frame (ORF) coding for a viral RNA-dependent RNA polymerase (RdRp). Mitoviruses, in contrast to cytoplasmatic narnaviruses, seem to replicate in strict association with mitochondria [2, 8, 11, 12]. Common characteristic features of all mitochondrial viruses are 50and 30-terminal untranslated regions (UTRs) of variable length. It has been suggested that these terminal residues act as stem-loop structures for RdRp recognition and initiation of replication [3, 4]. Mitoviruses have attracted significant attention, as they trigger fungal hypovirulence and phenotypic changes such as reduced growth, sporulation and pigmentation [3–6, 13, 20]. Most mitoviruses are known from phytopathogenic fungal genera only, such as Ophiostoma, Cryphonectria, and Botrytis, and a few from Gremmeniella, Sclerotinia, Thanatephorus, Thielaviopsis, Helicobasidium and the arbuscular mycorrhizae Glomus [4–6, 11, 19–21]. The development of biological control agents against fungal pathogens using mycoviruses has attracted significant scientific attention, whereas the exploration of virus diversity in non-pathogenic fungi has been widely neglected [15, 16]. In this paper, we report the third known virus infecting the ectomycorrhizal fungus Tuber aestivum Vittad. The genome described here is the largest one known in the genus Mitovirus, and based on its host, the name Tuber aestivum mitovirus (TaMV) is proposed. Genome characteristics are highly similar to those of other known mitoviruses, including a single ORF encoding a viral RdRp as well as typical 50 and 30 UTRs.

Species Delimitation in Taxonomically Difficult Fungi: The Case of Hymenogaster

Background False truffles are ecologically important as mycorrhizal partners of trees and evolutionarily highly interesting as the result of a shift from epigeous mushroom-like to underground fruiting bodies. Since its first description by Vittadini in 1831, inappropriate species concepts in the highly diverse false truffle genus Hymenogaster has led to continued confusion, caused by a large variety of prevailing taxonomical opinions. Methodology In this study, we reconsidered the species delimitations in Hymenogaster based on a comprehensive collection of Central European taxa comprising more than 140 fruiting bodies from 20 years of field work. The ITS rDNA sequence dataset was subjected to phylogenetic analysis as well as clustering optimization using OPTSIL software. Conclusions Among distinct species concepts from the literature used to create reference partitions for clustering optimization, the broadest concept resulted in the highest agreement with the ITS data. Our results indicate a highly variable morphology of H. citrinus and H. griseus, most likely linked to environmental influences on the phenology (maturity, habitat, soil type and growing season). In particular, taxa described in the 19th century frequently appear as conspecific. Conversely, H. niveus appears as species complex comprising seven cryptic species with almost identical macro- and micromorphology. H. intermedius and H. huthii are described as novel species, each of which with a distinct morphology intermediate between two species complexes. A revised taxonomy for one of the most taxonomically difficult genera of Basidiomycetes is proposed, including an updated identification key. The (semi-)automated selection among species concepts used here is of importance for the revision of taxonomically problematic organism groups in general.