Molecular Data Reveals Rich Diversity of the Sequestrate Genus Melanogaster (Boletales, Paxillaceae) with Emphasis on the Species from China

Malanogaster are ectomycorrhizal fungi characterized by hypogeous fruitbodies. Many ITS rDNA sequences of Malanogaster are recovered from molecular surveys of fungal communities, and remain insuciently identied making it dicult to determine whether these sequences represent conspecic or novel taxa. In this study, the ITS sequences of Malanogaster were collected comprehensively and analyzed within ITS-based phylogenetic framework. Twenty-one distinct phylogenetic species can be distinguished based on the ITS phylogeny and a threshold of 98% ITS sequence identity, and most species of Melanogaster showed more than 98.1% intraspecic ITS identity and less than 97.9% interspecic identity. Ten species were recognized from China, but combined morphology, nine of which were described and illustrated in this manuscript, including 4 new species (M. minobovatus nov. sp., M. panzhihuaensis nov. sp., M. quercus nov. sp. and M. tomentellus nov. sp.), 1 new combination (M. obvatus comb. & stat. nov.), and 4 known species (M. broomeanus, M. shanxiensis, M. spinisporus, M. subglobisporus). Signicantly amendments were added to M. spinisporus and M. subglobisporus. M. fusisporus was added a new mature specimen from type location, and the description and exquisite illustrations were provided also. The diagnostic characteristics of the genus Melanogaster and a key to the Chinese species of the genus Melanogaster were provided. A 28 Sphylogeny was also conducted to conrm the phylogenetic locations of Chinese Melanogaster species and “Alpova trappei”, consistented with ITS analysis. gastroid Paxillineae (Moreau et al. 2011, 2013). In this study, the internal transcribed spacer region of ribosomal DNA (ITS) and nuclear ribosomal large subunit (nrLSU) were used to identify and analyze the taxonomy and systematics of Melnaogaster, and our aims are: (1) to clarify the phylogenetic diversity of Melanogaster species based on ITS and nrLSU sequences newly obtained from Chinese specimens in this study as well as downloaded from published databases (Genbank, https://www.ncbi.nlm.nih.gov/genbank/), and (2) to describe the Chinese species of Melnaogaster, including 4 new species and 1 new combination. in the present molecular analysis among the about 25 species accepted in Melanogaster before this study, including Melanogaster carolinensis (Bosc ex Spreng.) De Toni, Melanogaster cauvinianus (Corda) Mont. ex Berl., De Toni & E. Fisch., Melanogaster durissimus Cooke, Melanogaster eisenii Harkn., Melanogaster macrocarpus Zeller, Melanogaster minisporus Cázares, G. Guevara, J. García & Trappe, Melanogaster natsii, Melanogaster ovoidisporus, Melanogaster parksii Zeller & C.W. Dodge, Melanogaster trappei I.P.S. Thind & B.M. Sharma, Melanogaster tuberiformis Corda, Melanogaster umbriniglebus Trappe & Guzmán, Melanogaster utriculatus, Melanogaster variegates and Melanogaster vittadinii Soehner & Knapp. These putative novel species and old species without DNA data required more mycologists to pay attention to them in the future. Morphologically, the spore shape and size of a species is basically regular and stable, which is usually used in the species delimitation of Melanogaster, but all in all, the combination of spore characteristics, peridium structure, color of basidiomata and gleba, can perfectly help us distinguish species. For example, M. obovatus and M. quercus have similar spore shape and size, the gelba, however, is yellow green to yellow brown in M. obovatus, but black in M. quercus, by which the two species are completely differentiated from each other.


Introduction
The genus Melanogaster Corda. was erected by Corda (1831), belonging to Basidiomycota, Basidiomycetes, Boletales, Paxillaceae (Binder and Hibbett, 2006;Lacheva 2015). The species of this genus have partly or entirely hypogeous fruit bodies, commonly called false tru es, and found on almost all continents (Türkoğlu and Castellano, 2013). Their fruit bodies are tuber-like after dried especially, comprised of a central spore producing region (gleba) consisting of many global chambers surrounded by an outer skin (peridium), and spores are usually dark color. On ripening, their fruit bodies are gelatinous and elastic, the contents of the glebal chambers disintegrate and liquefy to produce a very dark and slimy spore mass, which is quite different with true tru e (Ainsworth 2005 Yuan and Sun (1996) reported M. broomeianus Berk. from Sichuan Province. Up to 1996, a total of 8 species and 2 varieties of the genus Melanogaster have been found in China. be sequenced were cloned into a pLB vector (Tiangen, Beijing) and then sequenced with primers: 23-mer (5' -CGACTCACTATAGGGAGAGCGGC-3') and M13R (5' -AAGAACATCGATTTTCCATGGCAG-3').
Sequence preparation Total 22 ITS and 14 nrLSU fragments newly generated sequences were assembled and edited using SeqMan (DNA STAR package; DNAStar Inc., Madison, WI, USA) with generic-level identities for sequences con rmed via Blast queries of GenBank. Additional ITS and nrLSU sequences of Melanogaster and Alpova species were downloaded from GenBank, combined with our sequence database to create ITS and nrLSU phylogeny to illustrate the phylogenetic diversity in Melanogaster. The genus search tool in emerencia (http://www.emerencia.org/) was used to retrieve insu ciently identi ed ITS sequences from GenBank, whose pairwise similarity was most similar to identi ed Melanogaster species (Nilsson et al. 2005; Ryberg et al. 2009). The data set of compiled ITS and nrLSU sequences were ltered by removing sequences of poor quality or short length as well as sequences not belonging to Melanogaster based on BLAST (Altschul et al. 1997). Details of the taxa, specimens, host, origin and GenBank sequence accessions of ITS and 28S sequences employed in the phylogeny analyses were provided in Table 1 and Table 2 Respectively. Table 1 Sources of specimens and GenBank accession numbers for sequences used in this study. Sequences newly generated for this study are in bold.   (Nylander 2004), which were GTR + I + G for the ITS data set and SYM + I + G for the nrLSU data set. Two independent runs of four chains were conducted for 1 400 000 (for ITS) and for 580 000 (for nrLSU) Metropolis-coupled Markov chain Monte Carlo (MCMCMC) generations with the default setting. The average standard deviations of split frequency (ASDSF) values were far lower than 0.01 at the end of the generations. Trees were sampled every 100 generations after burn-in (well after convergence), and 50% majority-rule consensus trees were constructed and visualized with TreeView (32 bit) (Page 2001

Phylogenetic analysis
The ITS dataset contained 128 sequences (22 generated in this study) and comprised 604 sites, of which 284 were variable sites, and 237 of these variable sites were informative. ML and BI analyses produced identical tree topologies, and only the tree derived from the ML analyses is shown (Fig. 1).
The sequences of Melanogaster were grouped into a clade together with 4 sequences labeled as "Alpova trappei" with support values of BS = 77 and PP = 1.00, then further clustered with the other sequences of Alpova with a strong support value (BS = 100). The sequences of Alpova employed in this analysis, except the samples of "Alpova trappei", formed two independent clades with strong support. In Melanogaster clade, a total 21 phylotypes (marked A-U) can be recognized based on a species delimitation of 98% ITS similarity and strong supported clades of the ITS-based ML tree (Fig. 1) Phylogeny based nrLSU sequences were analyzed because there were the samples of Alpova trappei grouped into Melanogaster clade rather than Alpova clade in ITS-based phylogeny although there were a small number of available sequences. The nrLSU dataset contained 35 sequences (14 generated in this study) and comprise772 sites, of which 103 variable sites, and 64 of these variable sites were informative. ML and BI analyses produced identical tree topologies, and only the tree derived from the ML analyses is shown (Fig. 2). Two clades were recognized in this tree, respectively representing Melanogaster and Alpova, and the sequences labeled as "Alpova trappei" (marked with pentagram ( ) symbol) were placed in Melanogaster clade rather than Alpova clade, consistent with that of the ITS-based phylogenetic analysis (Fig. 1). The other sequences of Alpova cluster an independent clade with a strong support clade (BS = 97, PP = 1.00). These further con rmed that the samples of Alpova trappei represented the species of the genus Melanogaster. Moreover, the Chinese Melanogaster sequences were grouped in Melanogaster clade as those observed on the ITS tree (Fig. 1).
Habit, habitat and distribution: August to September, in soil under Quercus moglica Fish., Q. acutissima Carr. Northeast and South China.
Notes: Melanogaster fusisporus is re-described in details based on the re-examinations for paratypes cited by Wang et al. (1995) and newly added specimen HMAS83274 from type location. In this study, the beautiful illustrations were provided, intuitively presented the shape of the fusiform spore to people. The newly added specimen, HMAS83274 ex MHSU2008 used to be cited as Melanogaster fusisporus var. obovatus (this variety has been revised to Melanogaster obovatus based on present study.) in Flora Fungorum Sinicorum (Vol.7) (Liu et al. 1998). Although we sequenced unsuccessfully, we noticed this specimen was mature and tted well with M. fusisporus in morphyology, besides it was collected from type location in Chinese Yunnan Provence. For the above reasons, we diagnosed this specimen as M. fusisporus. Melanogaster fusisporus is diagnosed by its fusiform basidiospores with a bluntly apiculate apex, orange brown or deep brown basidiospores at maturity, easy to be recognized. Melanogaster quercus differs from M. fusisporus by its brick red basidiomata and light yellowish basidiospores of 10. Basidioma 3.5 × 4.5 cm, hypogeous, subglobose, yellow-brown, without distinct basal zone; rhizomorphs invisible, Strong wine aroma or fruit aroma. Gleba black, liquefying to produce a very dark spore mass. Peridium two-layered, 20-50 µm thick, composed of interwoven hyphae, 4-7 µm in diam, smooth, clamp connections abundant; subcutis 300-450 µm thick, composed of interwoven hyphae, with a large number of in ated cells, ellipsoidal, irregular or puzzle-like, up to 6.5-17 µm wide, light yellowish to yellow toward outside surface. Trama plates of hyaline gelatinized hyphae. Basidia not observed. Basidiospores obovoid, 5.1-6.9 × 4.2-5.1 µm (avX = 6.1 ± 0.4 × 4.7 ± 0.2, n = 50), Q = 1.1-1.5 (avX = 1.3 ± 0.1, n = 50), deep brown at maturity, 0.5-1 µm thick, smooth, the base truncate-cupped, with very short remnants of sterigmata.
Habit, habitat and distribution: September, in soil under Pinus bungeana. Warm temperate zone of North China.

Habit, habitat and distribution
June to September, in soil under Quercus sp., Pinus tabuliformis Carr. Temperate zone of North and Northeast China.

Notes
Phylogenetic analysis based on the sequences of specimens newly collected from type locality and morphologically consistent with the type specimen of M. fusisporus var. obovatus, revealed that these sequences clustered the independent clade J with a strong support (BS = 96/PP = 1.00) (Fig. 1), supporting they represented a distinct species. Therefore, we raised M. fusisporus var. obvatus to species level as M. obvatus, and an epitype (BJTC FAN1091) was designated here. Phylogenetically, M. quercus and M. shanxiensis were related to M. obovatus closely. However, they morphologically differed from M. obovatus in black gleba and elongate-fusiform spores with apiculate apex for the former and white to pale yellow basidioma and elongate-obovatus basidiospores for the latter. Diagnosis: Melanogaster panzhihuaensis is diagnosed by its oval basidiospores with blunt and narrow apex and reddish brown basidiospores at maturity. Description: Basidioma1.5 × 1.7 cm, hypogeous, subglobose, brown to dark brown when dry; no rhizomorphs visible; odor no record. Gleba pale yellow-brown when fresh, rm, black after dried, with sterile white veins scattered throughout. Peridium single-layered, 150-220 µm thick, compactly interwoven hyphae,

Notes
In present study, the sequence of M. panzhihuaensis showed less than 89.89% ITS similarity to other species and formed an independent clade U (Fig. 1), indicating it was a distinct species. In addition, the sequences of M. minobvatus formed sister clade with M. panzhihuaensis. However, M. minobvatus is differentiated from M. panzhihuaensis by its yellow-brown basidiomata, obovoid and small basidiosporesof 5.1-6.9 × 4.2-5.1 µm. Morphyologically, Melanogaster natsii is similar to M. panzhihuaensis in the color of gleba, but M. natsii differs from the latter in its broadly citriform or obovatus and pale yellow brown basidiospores. Diagnosis: Melanogaster quercus is diagnosed by brick red appearance, black gleba and light yellow elongate-fusiform basidiospores with apiculate apex. M. obovatus is closely related to M. quercus. However, M. obovatus differs from M. quercus in its yellow green to yellow brown gleba and elongate-fusiform with rounded top to obovate spores.
Habit, habitat and distribution September, in soil under Quercus sp. Warm temperate zone of North China.

Notes
Phylogenetically, the sequences of Melanogaster quercus clustered together and branched as the sister group to the sequence of M. shanxiensisis with no support value, implying that M. quercus is a distinct species and related to M. shanxiensisis. However, M. shanxiensis differs from M. quercus in its white to pale yellow basidiomata and elongate-obovate basidiospores. Morphyologically and phylogenetically, M. obovatus is closely related to M. quercus. However, M. obovatus differs from M. quercus in its yellow green to yellow brown gleba and elongate-fusiform with rounded top to obovate spores. In addition, the Up to now, there are ten species and varieties to be described from China based on morphological data alone according to our data. In the present study the collections of Melanogaster from China, including the old specimens deposited in HMAS and KUN-HKAS, and the ones recently collected by us, were researched using morphological and molecular phylogenetic methods. Consequently, the occurrence of four known species in China was con rmed, including M. broomeanus, M. shanxiensis, M. spinisporus, M. subglobisporus. The variety M. ovoidisporus var. angustatisporus was treated as synonym of M. broomeanus, while M. fusisporus var. obvatus was raised to species level as M. obvatus. DNA sequences of the other four species, M. fusisporus, M. obovatisporus, M. ovoidisporus and M. natsii, were not successfully sequenced in this study because the type specimens and reliable specimens cited in Flora Fungorum Sinicorum (Vol. 7) (Liu et al., 1998) are old or poorly preserved, but they have distinct morphological features according to our reexaminations for their type specimens and reliable specimens. We therefore con rmed their occurrence in China. Moreover, four new species were proposed from China based on the newly collected specimens, including M. minobovatus, M. panzhihuaensis, M. quercus and M. tomentellus.
Totally, the occurrence of 13 Melanogaster species in China were con rmed in present study. In addition, there are still potential Melanogaster species that have not been discovered in China, such as the sequence of ECM (HM105535), which chould represent a potential new taxon (Phylotype R: labeled as M.sp.6, Fig. 1) from China.