The Lichenologist 50(3): 329–340 (2018) © British Lichen Society, 2018 doi:10.1017/S002428291800021X The draft genome of the lichen-forming fungus Lasallia hispanica (Frey) Sancho & A. Crespo Francesco DAL GRANDE, Anjuli MEISER, Bastian GRESHAKE TZOVARAS, Jürgen OTTE, Ingo EBERSBERGER and Imke SCHMITT Abstract: Lasallia hispanica (Frey) Sancho & A. Crespo is one of three Lasallia species occurring in central-western Europe. It is an orophytic, photophilous Mediterranean endemic which is sympatric with the closely related, widely distributed, highly clonal sister taxon L. pustulata in the supra- and oro-Mediterranean belts. We sequenced the genome of L. hispanica from a multispore isolate. The total genome length is 41·2 Mb, including 8488 gene models. We present the annotation of a variety of genes that are involved in protein secretion, mating processes and secondary metabolism, and we report transposable elements. Additionally, we compared the genome of L. hispanica to the closely related, yet ecologically distant, L. pustulata and found high synteny in gene content and order. The newly assembled and annotated L. hispanica genome represents a useful resource for future investigations into niche differentiation, speciation and microevolution in L. hispanica and other members of the genus. Key words: functional annotation, mating type, polyketide synthase, secretome, synteny, transposable elements Accepted for publication 7 January 2017 Introduction distribution, occurring in Mediterranean to Lasallia hispanica (Frey) Sancho & A. Crespo boreal-montane habitats from southern represents one of three species of the genus Europe to northern Scandinavia (Hestmark Lasallia which occur in central Western 1992; Rolshausen et al.2018).Theothertwo Europe (Sancho & Crespo 1989). The three congeners are endemic to the Mediterranean Lasallia species differ in distribution, region: L. hispanica prefers supra- and habitat preference, morphology and mode of oro-Mediterranean habitats in the Iberian reproduction. Lasallia pustulata has the widest Peninsula, southern Italy and northern Morocco, and L. brigantium is confined to coastal areas in west Corsica and north-west F. Dal Grande, A. Meiser, J. Otte, I. Ebersberger and I. Schmitt: Senckenberg Biodiversity and Climate Sardinia below 300 m a.s.l. (Sancho & Crespo Research Centre (SBiK-F), Senckenberganlage 25, 60325 1989). Lasallia hispanica is sympatric with Frankfurt am Main, Germany. L. pustulata in the supra- and oro- B. Greshake Tzovaras and I. Ebersberger: Institute of Mediterranean bioclimatic belts (Sancho & Cell Biology and Neuroscience, Goethe University Frankfurt, Max-von-Laue Str. 13, 60438 Frankfurt am Crespo 1989) where the two species often Main, Germany. share the same photobiont (Dal Grande et al. A. Meiser and I. Schmitt: Institute of Ecology, 2017). Lasallia hispanica and L. pustulata Evolution and Diversity, Goethe University Frankfurt differ in their water acquisition strategies: am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am L. pustulata relies on surface run-offs, whereas Main, Germany. B. Greshake Tzovaras: Environmental Genomics and L. hispanica takes up moisture directly from Systems Biology, Lawrence Berkeley National Labo- fog and low-lying clouds, therefore becoming ratory, Berkeley, CA 94720, USA. desiccated more rapidly and more frequently Current address for F. Dal Grande (corresponding (Vivas et al. 2017). A recent study comparing author): Departamento de Farmacología, Farm- acognosia y Botánica, Facultad de Farmacia, Uni- the photosynthetic performance of the two versidad Complutense de Madrid, 28040 Madrid, species in nature and under laboratory condi- Spain. Email: [email protected] tionssuggeststhatL. hispanica might be more Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.14, on 01 Oct 2021 at 17:18:30, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002428291800021X 330 THE LICHENOLOGIST Vol. 50 resistant to environmental stress than L. pustu- altitudinal gradient (Dal Grande et al. 2017). lata. This is probably due to the more efficient In this study we revealed the existence of two and rapid activation of stress-related repair locally adapted ecotypes using correlations mechanisms in L. hispanica (Vivas et al. 2017). between single-nucleotide polymorphisms The three Lasallia species have a mixed asexual (SNPs) and environmental parameters. and sexual reproductive strategy. However, Lichen metagenomics (i.e. the direct reproduction in L. pustulata is predominantly sequencing of mixed genomic material from vegetative, by means of isidia, while L. hispanica lichen thalli) represents a cultivation- and L. brigantium predominantly reproduce independent approach to explore the sexually (Sancho & Crespo 1989). While L. diversity and functional aspects of the lichen pustulata hasbeenusedasamodeltoexplore symbiosis. For instance, it is possible to climate adaptation in lichens (Dal Grande et al. reconstruct the genomes of the individual 2018) and symbiont-driven ecological expan- symbiotic partners using a single, short-read sion (Rolshausen et al. 2018), molecular studies sequencing library layout (i.e. metagenome on L. hispanica are lacking. The genetic differ- skimming; Greshake Tsovaras et al. 2016; entiation among the three species has yet to be Meiser et al. 2017). Metagenomic lichen explored. samples have also been used to apply restric- The genomics revolution is transforming tion site-associated DNA sequencing (RAD- the way we study evolution and ecology seq) for phylogenetic reconstructions of (Wolfe & Li 2003; Grube et al. 2014). lichenized fungi based on genomic sequence Evolutionary genomics and phylogenomics information (Grewe et al. 2017). Genome further our understanding of speciation, mining is increasingly employed to survey phylogenetic relationships and the evolu- lichens for genes associated with the bio- tionary origin of functional traits in synthesis of active metabolites, revealing in lichenized fungi. Phylogenomic datasets have some cases unexpected biosynthetic potential been used to resolve evolutionary relation- (e.g. Kampa et al. 2013). For example, ships in the Rhizoplaca melanophthalma Cladonia uncialis contained a gene cluster species complex (Chan & Ragan 2013; responsible for the biosynthesis of a haloge- Leavitt et al. 2016). Comparative genomics nated isocoumarin (Abdel-Hameed et al. has been used to reveal gene family size 2016). The advent of long-read sequencing changes and gene deletions associated with technologies from Pacific Biosciences (Pac- lichenization in Endocarpon pusillum (Wang Bio) and Oxford Nanopore Technologies will et al. 2014), to derive phylogenetic markers drastically improve the assembly process as useful for resolving relationships among well as the in-silico separation of organisms close relatives (Magain et al. 2017), and to from mixed DNA samples. study the properties and evolution of Here we present the de novo assembly and mitochondrial genomes (Xavier et al. 2012). annotation of the genome of L. hispanica.Using Ecological genomics is an emerging field Illumina next-generation sequencing technol- in lichenology. It allows questions to be ogy we obtained and annotated a high-quality addressed related to, for example, niche dif- draft genome. We identified gene clusters ferentiation, ecological specialization and associated with secondary metabolite bio- local adaptation. Transcriptomics has been synthesis, mating-type loci and transposable employed to infer the response of Peltigera elements, and compared them to the closely membranacea and its cyanobiont to thermal related L. pustulata (Davydov et al. 2010). stress (Steinhäuser et al. 2016), and of Finally, we established synteny and orthology Trebouxia to desiccation (Candotto Carniel between L. hispanica and L. pustulata.Inaddi- et al. 2016). Recently, we used a population tion to providing structured data for various genomics approach based on whole-genome phylogenetic studies, the work presented here resequencing of pools of DNA from lichen will provide a genomic resource for further populations to study the genomic signatures studies aiming to 1) understand the basis of of adaptation in L. pustulata along an polygenic adaptation in L. hispanica based on Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.14, on 01 Oct 2021 at 17:18:30, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002428291800021X 2018 Lasallia hispanica genome—Dal Grande et al. 331 population genomic resequencing of natural in Petri dishes (Denison 2003). Upon germination, the populations, 2) study the impact of different spores were transferred to malt yeast extract medium. reproductive strategies on the evolution of The mycobiont colonies were maintained at room temperature in darkness and were sub-cultured monthly genomes and populations in L. hispanica and L. onto fresh medium until sufficient biomass for genomic pustulata, and 3) infer the genomic footprints of analysis was obtained (c. 6 months; Fig. 1). niche differentiation of the two species. DNA isolation and sequencing Materials and Methods About 0·5 g of mycobiont mycelia was collected and ground in liquid nitrogen with a mortar and pestle. In vitro cultivation of the lichen-forming Genomic DNA was isolated using the CTAB Maxi-prep fungus Lasallia hispanica method (Cubero & Crespo 2002), resulting in a total yield of c.5µg