Lawrence Berkeley National Laboratory Recent Work Title Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics. Permalink https://escholarship.org/uc/item/25k8j699 Journal Fungal diversity, 104(1) ISSN 1560-2745 Authors Vandepol, Natalie Liber, Julian Desirò, Alessandro et al. Publication Date 2020-09-16 DOI 10.1007/s13225-020-00455-5 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Fungal Diversity https://doi.org/10.1007/s13225-020-00455-5 Resolving the Mortierellaceae phylogeny through synthesis of multi‑gene phylogenetics and phylogenomics Natalie Vandepol1 · Julian Liber2 · Alessandro Desirò3 · Hyunsoo Na4 · Megan Kennedy4 · Kerrie Barry4 · Igor V. Grigoriev4 · Andrew N. Miller5 · Kerry O’Donnell6 · Jason E. Stajich7 · Gregory Bonito1,3 Received: 17 February 2020 / Accepted: 25 July 2020 © MUSHROOM RESEARCH FOUNDATION 2020 Abstract Early eforts to classify Mortierellaceae were based on macro- and micromorphology, but sequencing and phylogenetic studies with ribosomal DNA (rDNA) markers have demonstrated conficting taxonomic groupings and polyphyletic genera. Although some taxonomic confusion in the family has been clarifed, rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae. In this study, we applied two parallel approaches to resolve the Mortierel- laceae phylogeny: low coverage genome (LCG) sequencing and high-throughput, multiplexed targeted amplicon sequenc- ing to generate sequence data for multi-gene phylogenetics. We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset. Resolving the Mortierellaceae phylogeny into monophyletic genera resulted in 13 genera, 7 of which are newly proposed. Low-coverage genome sequencing proved to be a relatively cost-efective means of generating a high-confdence phylogeny. The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach, but has limitations too. We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity and evolution. Keywords Mortierellaceae · Phylogenomics · Molecular systematics · Taxonomy · Multi-locus sequence typing Introduction Electronic supplementary material The online version of this Early diverging fungi belonging to Mortierellomycotina article (https ://doi.org/10.1007/s1322 5-020-00455 -5) contains are diverse in ecology and species richness, and are classi- supplementary material, which is available to authorized users. fed within a single order (Mortierellales) and as belonging * Gregory Bonito to a single family (Mortierellaceae). Phylogenetically, this [email protected] lineage is closely related to arbuscular mycorrhizal fungi (Glomeromycotina) and Mucoromycotina, and are among 1 Department of Microbiology and Molecular Genetics, the earliest diverging lineages of fungi to have independently Michigan State University, East Lansing, MI 48824, USA evolved diferentiated macroscopic fruiting body structures, 2 Department of Plant Biology, Michigan State University, in the form of ~ 1 cm sporocarps (Smith et al. 2013; Spata- East Lansing, MI 48824, USA fora et al. 2016; Chang et al. 2019). Several species of Mor- 3 Department of Plant Soil and Microbial Sciences, Michigan tierella are prolifc producers of polyunsaturated fatty acids State University, East Lansing, MI 48824, USA and have relevance to nutraceutical industries and bioenergy 4 Joint Genome Institute, Berkeley, CA 94720, USA research (Goyzueta et al. 2019). Mortierellaceae are com- 5 Illinois Natural History Survey, University of Illinois monly detected and isolated from soils, plant debris, insect Urbana-Champaign, Champaign, IL 61820, USA guts, mosses and living plant roots (Dixon-Stewart 1932; 6 United States Department of Agriculture, Agricultural Gams 1977; Domsch et al. 1980), and have been found on Research Service, Peoria, IL 61604, USA every continent, including Antarctica (Gams 1977; GBIF.org 7 Department of Microbiology and Plant Pathology & 2019). Yet, the inability to resolve phylogenetic relationships Institute for Integrative Genome Biology, University within Mortierellaceae has limited inferences pertaining to of California-Riverside, Riverside, CA 92521, USA Vol.:(0123456789)1 3 Fungal Diversity species distributions, diversity and functional ecology of commonly observed. Macromorphology, micromorphology, Mortierellaceae (Gams 1977; Petkovits et al. 2011; Wagner and the production of all three spore types may vary con- et al. 2013). siderably between growth media and conditions, which can Early eforts to classify Mortierellaceae were based on complicate morphological species identifcations (Fig. 3). macro- and micromorphology, including colony growth Species and their groupings were repeatedly redefned in patterns, hyphal branching, and spore production (Gams the mid 1900s, which was concluded by Gams in 1977, who 1977). Most species within Mortierellaceae have distinct divided the lineage into two subgenera: Micromucor and macromorphological growth patterns on agar media, with Mortierella. Within Mortierella subgenus Mortierella, Gams colonies forming rounded to slightly pointed rosette “pet- recognized 9 sections: Alpina, Actinomortierella, Haplospo- als”, although some species grow in simple rings and others rangium, Hygrophila, Schmuckeri, Simplex, Spinosa, Sty- are completely devoid of visible growth rings (Fig. 1). Mor- lospora, and Mortierella, the last of which contained the tierellaceae fungi can produce three types of spores: asexual type genus and species for the Mortierellaceae, Mortierella sporangiospores born in sporangia, asexual chlamydospores polycephala (Gams 1977). Micromucor was later reclassi- that can be terminal or intercalary, and sexual zygospores fed to belong within Mucoromycota in the genus Umbe- (Fig. 2). One or more spore types may be absent in some lopsis (Meyer and Gams 2003). Additional genera, Gam- species, such as M. chlamydospora which lacks sporan- siella, Dissophora, Modicella, and Lobosporangium were giospores and M. parvispora which lacks chlamydospores subsequently described and accepted as Mortierellaceae, but (Gams 1977). In some species, chlamydospores may be dec- remained polyphyletic with respect to Mortierella (Thaxter orated with spines or other ornamentations and are referred 1914; Benjamin 1978; Benny and Blackwell 2004; Petkovits to as stylospores (Chien et al. 1974). Both heterothallism et al. 2011; Smith et al. 2013). The genus Haplosporangium and homothallism have been observed in Mortierellaceae was retired by Gams (1977) and all included species, includ- and sexuality varies by species, although mating is not ing the type species H. bisporale, have been transferred to Fig. 1 Macromorphology spe- cies panel. a Mortierella sp. JL58 on MEA + YE, 11 days; b–d Mortierella sp. JL29, AP5, and JL1 on MEA + YE, 11 days; e Mortierella elongata NVP64-on PDA/2 + YE, 6 days; f–h M. alpina NVP153, JL109, and KOD1002 on PDA/2 + YE, 6 days; i M. humilis PMI1414- on PDA/2 + YE, 6 days 1 3 Fungal Diversity Fig. 2 Micromorphology panel. Mortierella elongata NVP64 a spo- giophore bearing sporangiospores; and d sexual zygospore from het- rangiospores and bent sporangiophores on agar surface; b intercalary erothallic mating with M. elongata NVP5. Scale bars: a 100 µm; b chlamydospore and septate, evacuated hyphae; c branched sporan- 10 µm; c 200 µm; d 20 µm Fig. 3 Media-dependent morphology panel. Mortierella af. ambigua JL86, 10 days, on three media a PDA/2 + YE, b MEA + YE, c CZA Mortierella, in various clades. The frst modern sequencing- efort expanded the diversity of sequenced species and reor- based revision of the Mortierellaceae established that the ganized and combined several of the Petkovits et al. (2011) morphological classifcation system was largely unsupported clades into a total of seven clades: selenospora, verticillata- and defned 12 new clades (Petkovits et al. 2011). Only sect. humilis, lignicola, dissophora, capitata, alpina, and gamsii Schmuckeri, sect. Actinomortierella, and sect. Mortierella (Wagner et al. 2013). were retained in the new clades. A second rDNA sequencing 1 3 Fungal Diversity Currently, there are close to 125 accepted Mortierellaceae Materials and methods species, and new species continue to be formally described each year (see Supplementary Table 1). Estimates based on Sampling, isolation, and culture conditions environmental sequencing predict there to be more than 170 Mortierellaceae species worldwide, indicating at least one Diverse isolates were obtained from established culture quarter of the species in this family remain to be described collections including the Agricultural Research Service (Gams 1977; Benny 2009; Nagy et al. 2011; Smith et al. 2013; Culture Collection (NRRL) and Westerdijk Fungal Bio- Wagner et al. 2013; Degawa et al. 2014; Takashima et al. diversity Institute (CBS-Centraalbureau voor Schimmel- 2018a, b). The current Mortierellaceae classifcation divides cultures) and from collaborators to broaden geographic species into six genera: Aquamortierella, Dissophora, Gam- and biodiversity sampling. Fresh isolates for this study siella, Lobosporangium, Mortierella, and Modicella (Benny were also obtained from roots, soils and plant substrates 2009). However, most of these genera are monotypic or ditypic collected across Australia,