<i>Tuber rugosum</i>, a new species from northeastern North America: Slug mycophagy aides in electron microscopy of ascospores

Species in the genus <i>Tuber</i> are ascomycetous fungi that produce hypogeous fruiting bodies commonly called truffles. These fungi are ecologically relevant owing to the ectomycorrhizal symbiosis they establish with plants. One of the most speciose lineages within <i>Tuber</i> is the Rufum clade, which is widely distributed throughout Asia, Europe, and North America and is estimated to include more than 43 species. Most species in this clade have spiny spores, and many still have not been formally described. Here, we describe <i>T. rugosum</i> based on multigene phylogenetic analysis and its unique morphological characters. <i>Tuber rugosum</i> (previously designated in literature as <i>Tuber</i> sp. 69) has been collected throughout the Midwest, USA, and Quebec, Canada, and is an ectomycorrhizal symbiont of <i>Quercus</i> trees, as confirmed through morphological and molecular analyses of root tips presented here. We also present a novel method for preparing <i>Tuber</i> ascospores for scanning electron microscope imaging that includes feeding, digestion, and spore excretion by the slug <i>Arion subfuscus</i>. Following this method, spores become free from ascus and other mycelial debris that could obscure morphological traits during their passage through the snail gut while maintaining ornamentation. Finally, we report the fatty acid analysis, a fungicolous species association, and we provide an updated taxonomic key of the Rufum clade.块菌属（Tuber）的物种均为子囊菌，其产生的地下子实体通常被称为松露。这类真菌因与植物形成外生菌根共生关系而具有重要的生态意义。块菌属内物种多样性最高的支系之一为鲁弗姆支系（Rufum clade），该支系广泛分布于亚洲、欧洲及北美，据估计包含超过43个物种。该支系的多数物种具有带刺孢子，且其中许多尚未被正式定名与发表。本研究基于多基因系统发育分析以及独特的形态特征，对皱块菌（T. rugosum）进行了正式描述。皱块菌（Tuber rugosum，此前文献中记为块菌属未定种69（Tuber sp. 69））的采集区域覆盖美国中西部与加拿大魁北克省；本研究通过对根尖的形态学与分子生物学分析证实，该物种是栎属（Quercus）植物的外生菌根共生伙伴。本研究同时报道了一种用于扫描电子显微镜（scanning electron microscope, SEM）成像的块菌属子囊孢子制样新方法，该方法利用蛞蝓（Arion subfuscus）取食、消化并排泄孢子。经该方法处理后，孢子可脱离子囊及其他可能掩盖其形态特征的菌丝碎屑，且在通过蛞蝓消化道的过程中能够保留表面纹饰。最后，本研究报道了皱块菌的脂肪酸分析结果、一种伴生真菌物种关联，并提供了更新后的鲁弗姆支系分类检索表。