From White to Black, from Darkness to Light: Species Delimitation and UNITE Species Hypothesis Testing in the Russula Albonigra Species Complex. Ruben De Lange ( [email protected] ) Ghent University https://orcid.org/0000-0001-5328-2791 Slavomír Adamčík Slovak Academy of Sciences Institute of Botany: Botanicky ustav Slovenskej akademie vied Katarína Adamčíkova Slovak Academy of Sciences: Slovenska akademia vied Pieter Asselman Ghent University: Universiteit Gent Jan Borovička Czech Academy of Sciences: Akademie ved Ceske republiky Lynn Delgat Ghent University: Universiteit Gent Felix Hampe Ghent University: Universiteit Gent Annemieke Verbeken Ghent University: Universiteit Gent Research Keywords: Basidiomycota, Ectomycorrhizal fungi, New species, Phylogeny, Russulaceae, Russulales, subg. Compactae, Integrative taxonomy, Typication Posted Date: December 8th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-118250/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at IMA Fungus on August 2nd, 2021. See the published version at https://doi.org/10.1186/s43008-021-00064-0. Page 1/64 Abstract Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata that is blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological amplitude and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R. albonigra s.l. represents a species complex consisting of at least ve European, three North-American and one Chinese species. Morphological study shows traditional characters used to delimit R. albonigra are not always reliable. Therefore, a new delimitation of the R. albonigra lineage is proposed and a key to the described European species of R. subg. Compactae is presented. A lectotype and an epitype are designated for R. albonigra and three new European species are described: R. ambusta, R. nigrifacta and R. ustulata. UNITE species hypotheses at different thresholds were tested against the taxonomic data. The species hypotheses at the similarity threshold 0.5% give a perfect match to the phylogenetically dened species within the R. albonigra lineage. Publicly available sequence data can contribute to species delimitation and expand knowledge on ecology and distribution, but the pitfalls are short and low quality sequences. The importance of updating public taxonomic data and using correct sequence similarity thresholds is emphasised. Introduction The UNITE database The concept of a species has been troubling the minds of biologists throughout history (Hey et al. 2003, Hey 2006). A multitude of different denitions have been proposed to dene what a species is exactly, starting with the use of mainly morphological characteristics and evolving toward genetics (Mayden 1997, De Queiroz 2007). Regardless of the diculties in providing a true satisfactory denition, the concept of a species remains the basis of many biological studies as many researchers and conservationists need this taxonomic information (Hofstetter et al. 2019). Molecular identication of species gained importance over the last decades. As new techniques became available and more easily accessible, the number of publications increased immensely (Hibbett et al. 2011, Kõljalg et al. 2013, Nilsson et al. 2018). The main problems with molecular identication are poor taxon coverage, misidentications and incorrect use of names in many public sequence databases (Kõljalg et al. 2005, Hofstetter et al. 2019). This is why the UNITE database was created. The UNITE database was originally dedicated to the reliable identication of ectomycorrhizal (ECM) fungi. Over the years it has expanded greatly, covering all fungal lineages in all geographical areas represented by ITS sequences (Kõljalg et al. 2005, Nilsson et al. 2018). UNITE targets the still most widely used, universal fungal barcode: the nuclear internal transcribed spacer (ITS) region to provide high-quality reference records (Nilsson et al. 2018). Taxonomic reliability is one of the founding principles of the database, therefore the term “species hypotheses” (SHs) was introduced. A species hypothesis is an approximate species-level OTU assigned with a unique digital object identier (DOI) which allows unambiguous reference across studies. These species hypotheses are available at several sequence similarity thresholds (i.e. from 97–99.5%) and are either assigned automatically with a representative sequence or manually by a taxonomic expert with a reference sequence (Kõljalg et al. 2013, Nilsson et al. 2018). Furthermore UNITE supports third-party annotation of the sequence data to match new nomenclatural and taxonomic insights or to correct suboptimal taxonomic annotation or other metadata (Nilsson et al. 2018). Page 2/64 The Russula albonigra lineage Russula Pers. is a large genus of ectomycorrhizal fungi with a worldwide distribution (Buyck et al. 2018, Looney et al. 2018). Species within Russula subg. Compactae (Fr.) Bon sensu stricto (Buyck et al. 2018) are characterised macroscopically by thick-eshed basidiomata with a dull-coloured (i.e. white, brown, grey to black) pileus and a context that is typically blackening with intermediate reddening, rarely browning or without colour change. In contrast to most other Russula species, numerous lamellulae are present in between the lamellae (also present within R. subg. Malodorae Buyck & V. Hofst., R. subg. Brevipedum Buyck & V. Hofst., R. subg. Archaeae Buyck & V. Hofst. and R. subg. Glutinosae Buyck & X.H. Wang), and they are arranged in a regular pattern, with shorter ones in between the longer ones. Microscopically, the representatives of this subgenus have spores with an inamyloid suprahilar spot. When present, hymenial cystidia are mostly minutely capitate with one central knob, while pileocystidia often have two eccentrical knobs (Buyck et al. 2018). Leonhardt et al. (2019) have shown that basidiomata of R. subg. Compactae accumulate remarkably lower Zn concentrations than most other European species of the genus. In Europe, eleven species (and some varieties) are described within this subgenus (Romagnesi 1967, 1985, Sarnari 1998, Pidlich-Aigner 2015), but phylogenetic data suggest a much higher diversity due to the presence of multiple species complexes (unpublished data). All European species within this subgenus are placed within R. sect. Nigricantinae Bataille (as in Buyck et al. (2018)). This study focuses on Russula albonigra (Krombh.) Fr., a species within R. subg. Compactae that is recognised by its moderately distant, relatively narrow lamellae, the surface of the basidiomata and especially of the lamellae that is strongly and rapidly blackening and resulting in black-and-white contrast of wounded and untouched parts, and the menthol- cooling taste of the lamellae. Most publications report blackening without intermediate reddening of the context and the surface, although Romagnesi described a strongly reddening form: Russula albonigra forma pseudonigricans Romagn. Microscopically, the species was dened by a low reticulate spore ornamentation and pileocystidia without apical knobs (Romagnesi 1967). Our observations on morphological variability and broad ecological amplitude of this species, together with the large distribution area, being reported also from the USA (Singer 1958, Hesler 1961, Shaffer 1962), suggest that the traditional concept may correspond to more than one species. In this study, we use sequence data of 4 DNA markers and a detailed morphological revision to test the taxonomic status of R. albonigra. Multilocus sequence data and morphological differences are used to test UNITE species hypotheses at different thresholds. Material And Methods Sampling This study is based on collections from sampling trips in Belgium (2016, 2017 and 2018), Italy (1997, 2000 and 2016), Norway (2016), Slovakia (2003, 2006, 2008, 2009, 2011, 2015 and 2017) and Sweden (2016 and 2018). All collections are deposited in the Herbarium Universitatis Gandavensis (GENT) or the herbarium of the Slovak Academy of Sciences (SAV). Supplementary collections were requested from the herbarium of the Mycological Department of the National Museum in Prague, Czech Republic (PRM), and from the personal herbaria of Felix Hampe, Jesko Kleine and Helga Marxmüller. Samples that could belong in the Russula albonigra complex were selected based on morphology: (1) collections that were identied as R. albonigra in the eld based on macro-morphology; (2) collections from herbaria that were labelled as R. albonigra based on both macro- and micro-morphological observations. The selected samples were Page 3/64 molecularly tested using the ITS marker as a guideline and not included for further study when not placed within the R. albonigra complex. Morphological analysis The macroscopic description is based on observations from fresh material, with colour codes referring to Kornerup and Wanscher (1978) and guaiac reactions referring to Chalange (2014). The microscopic description and terminology follow Adamčík et al. (2019). Microscopic characters were studied from dried material, spores were observed in Melzer’s reagent, elements of the hymenium and pileipellis were observed in Congo red in L4 after ca. 10 seconds in KOH 10%. Basidiospores were measured using a crosshair