Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and ¼ potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

中文翻译：

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Tulasnella 新种与澳大利亚陆生兰花 Cryptostylidinae 和 Drakaeinae 相关

许多兰花与 Tulasnella 菌根真菌在种子萌发和成年期支持方面具有必然关系。尽管 Tulasnella 作为菌根伙伴很重要，但许多物种仍未被描述。在这里，我们使用多序列基因座系统发育分析来界定和描述与来自亚部落 Cryptostylidinae 和 Drakaeinae 的澳大利亚陆生兰花相关的六个新的 Tulasnella 物种。五个新物种，Tulasnella australiensis、T. occidentalis、T. punctata、T. densa 和 T. concentrica，都与 Cryptostylis (Cryptostylidinae) 相关，而 T. rosea 与 Spiculaea ciliata (Drakaeinae) 相关。先前也报道了代表 T. australiensis 的分离株与 Arthrochilus (Drakaeinae) 的关联。所有新描述的 Tulasnella 物种都通过四个基因座（nuc rDNA 内部转录间隔区 ITS1-5.8S-ITS2 [ITS]、C14436 [ATP 合酶]、C4102 [谷氨酸合酶] 和 mt 16S rDNA [mtLSU]）的系统发育分析定界. ITS 区域物种之间的成对序列差异范围为 5.6% 至 25.2%，新描述物种内的最大序列差异范围为 1.64% 至 4.97%。种内和种间成对差异分布在 4-6% 的区域内存在差距，只有一个种内值为 4.97%（对于两个 T. australiensis 分离株）和一个种间值为 4.97% 5.6%（涉及 T. occidentalis 的分离株）属于该区域。根据荧光染色，所有六个新的 Tulasnella 物种都是双核的，并具有有隔膜的圆柱形菌丝。培养物形态有一些细微的变化，但在 3MN+维生素培养基上测量的菌落直径在生长 6 周后在物种之间没有差异。然而，T. australiensis 在 ½ FIM 和 ¼ 马铃薯葡萄糖琼脂 (PDA) 培养基上的生长显着（P < 0.02）比其他人慢。对这些 Tulasnella 物种的正式描述对记录 Tulasnella 的多样性做出了重大贡献，并提供了名称和界限，以支持进一步研究真菌及其与兰花的关系。