MycoKeys 6 | : 27-37 (20 | 9) A peer-reviewed open-access journal doi: 10.3897/mycokeys.6 1.36444 RESEARCH ARTICLE oO Mycokeys http://mycokeys.pensoft.net Launched to accelerate biodiversity research A novel sequestrate species from Mexico: Aroramyces guanajuatensis sp. nov. (Hysterangiaceae, Hysterangiales) Rafael Pefia-Ramirez'”, Zai-Wei Ge’, Rigoberto Gaitan-Hernandez’*, César Ramiro Martinez-Gonzalez*, Gonzalo Guevara-Guerrero! | Instituto Tecnolégico de Cd. Victoria, Av. Portes Gil 1301 Pte. C.P 87010, Cd. Victoria Tam, Mexico 2 Kunming Institute of Botany, Chinese Academy of Sciences, 132, Lanhei Road, Kunming 650201, China 3 Instituto de Ecologia, A.C., Carretera antigua a Coatepec # 351, El Haya, C.P 91070, Xalapa, Veracruz, Mexico 4 Universidad Nacional Auténoma de México, Profesor de Asignatura B, Departamento de Biologia, Facultad de Ciencias Ciudad Universitaria, Delegacién Coyoacdn, 04510, Ciudad de México, Mexico Corresponding author: Gonzalo Guevara-Guerrero (guevaragg@hotmail.com) Academic editor: Marc Stadler | Received 23 May 2019 | Accepted 16 October 2019 | Published 11 December 2019 Citation: Peha-Ramirez R, Ge Z-W, Gaitan-Hernandez R, Martinez-Gonzalez CR, Guevara-Guerrero G (2019) A novel sequestrate species from Mexico: Aroramyces guanajuatensis sp. nov. (Hysterangiaceae, Hysterangiales). MycoKeys 61: 27-37. https://doi.org/10.3897/mycokeys.61.36444 Abstract Knowledge of sequestrate Hysterangiaceae fungi in Mexico is very limited. In the present study, a new member of the family, Aroramyces guanajuatensis sp. nov., is described. This speciesis closely related to A. balanosporus, but differs from the latter in possessing a tomentose peridium 165-240 um thick, with occasional large terminal hyphae up to 170 um, a variable mesocutis (isodiametric to angular), and distinct bright yellowish subcutis. In contrast, A. balanosporus possesses a fibrillose peridial surface with shorter hyphae, a peridium 200-450 pm thick, and a mainly hyaline isodiametric mesocutis with a slightly wider subcutis. The phylogenetic analysis of the LSU gene separated A. guanajuatensis from A, balanosporus with a Bayesian posterior probability (PP) = 1. This is the third Aroramyces species described for the American continent. Keywords Truffle, truffle-like, sequestrate fungi, hypogeous fungi Copyright Rafael Pefia-Ramirez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 28 Rafael Pena-Ramirez et al. / MycoKeys 61: 27-37 (2019) Introduction Aroramyces Castellano and Verbeken was coined to settle Hymenogaster radiatus (Lloyd, 1925) and Hysterangium gelatinosporum (Cribb, 1957) from two different genera (Cas- tellano et al. 2000). Phylogenetic analysis places Avoramyces near, but different to Hyster- angium (Hosaka et al. 2006, 2008). Aroramyces is characterized by its unique combina- tion of a brown gleba, spiny spores with distinctly inflated utricles, gelatinized gleba, and basidiome with a tomentose surface with numerous soil particles adhering to all sides. At present, there are four species in this group (Kirk 2018): Aroramyces radiatus (Lloyd) Castellano, Verbeken & Walleyn, A. gelatinosporus (J.W. Cribb) Castellano (Castellano et al. 2000), A. balanosporus G. Guevara & Castellano, and A. herrerae G. Guevara, Gomez-Reyes & Castellano (Guevara-Guerrero et al. 2016). Aroramyces guanajuatensis was discovered during a survey aiming to document the fugal diversity in Guanajuato, Mexico. It is therefore determined that the number of Aroramyces species described in the American continent is now three. Materials and methods Sampling and morphological characterization The collections were discovered with a cultivator, digging around trees up to a depth of 15 cm. All encountered fruiting bodies were photographed fresh and then dried at 50 °C. The chosen material was cut by hand and rehydrated with 5% KOH for morphological studies. Thirty spores were measured. Peridial slices were made and observed under optical microscopy (Castellano et al. 1989). For scanning electron microscopy pictures (JSM5600LYV, JOEL, Tokyo, Japan), the spores were coated with gold and palladium. Voucher collections are deposited at José Castillo Tovar (ITCV) Herbarium, Instituto Tecndlogico de Ciudad Victoria, Mexico. DNA extraction, amplification, sequencing and phylogenetic analyses Genomic DNA was obtained with CTAB (Martinez-Gonzalez et al. 2017) ) or using Fungal DNA extraction Kit (Bio Teke Corporation, China) from 2—3 mg of dry tis- sue. DNA quantification was performed with Nanodrop (Thermo, USA). Each sample was diluted to 20 ng/uL for PCR amplification. LROR and LR5 primers were used to amplify the LSU gene (Vilgalys and Hester 1990). The PCR reaction contained the following: enzyme buffer 1x, Zag DNA polymerase, 0.8 mM deoxynucleoside triphos- phates (0.2 mM each), 100 ng DNA, 20 pmol of each primer, and 2 units of Go 7aq DNA (Promega, USA), with a final volume of 15 pL. The amplification program was run as follows: denaturalization at 96 °C for 2 min, 35 cycles of denaturalization at 94 °C for 1 min, annealing at 57 °C for 1 min, polymerization at 72 °C for 1 min, and final elongation at 72 °C for 5 min. All PCR reactions were carried out in a Peltier Aroramyces guanajuatensis sp. nov From Mexico 29 Thermal Cycler PT'C-200 (BIORAD, Mexico). The PCR products were verified by agarose gel electrophoresis run for 1 h at 95 V cm? in 1.5% agarose and 1x TAE buff- er (Tris Acetate-EDTA). The products were then dyed with GelRed (Biotium, USA) and viewed in a transilluminator (Infinity 3000 Vilber, Loumat, Germany). Finally, the products were purified using the ExoSap Kit (Affymetrix, USA) according to the manufacturer's instructions and were prepared for the sequencing reaction using the BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied BioSystems). Sequencing was carried out in a genetic analyzer (Model 3130XL, Applied Bio- Systems, USA) at the Biology Institute of the National Autonomous University of Mexico (UNAM). The sequences of both strains of each sample were analyzed, edited, and assembled using BioEdit v. 1.0.5 (Hall 1999) to create consensus sequences. The consensus sequences were compared with those in the GenBank database of the Na- tional Center for Biotechnology Information (NCBI) using the BLASTN 2.2.19 tool (Zhang et al. 2000). The LSU region was aligned using the online version of MAFFT v. 7 (Katoh et al. 2002, 2017; Katoh and Standley 2013). The alignment was revised in PhyDE (Miller et al. 2005), and small manual adjustments were then made to maximize the similarity between characters. The matrix was composed of 30 taxa (640 characters) (Table 1). Ramaria gelatinosa (access number AF213091) was used as the outgroup. Ihe phylogeny was performed using Bayesian inference in MrBayes v. 3.2.6 64x (Huelsenbeck and Ronquist 2001). The information block matrix included two independent runs of the MC3 chains for ten million generations (standard deviation < 0.01); the reversible-jump strategy was used (Huelsenbeck et al. 2004). An evolu- tionary model was used, so a proportion of invariable sites were designated, and the other proportion came from a gamma distribution (invgamma). The convergence of chains was visualized in Tracer v. 1 (Rambaut et al. 2014). The phylogram of maximum credibility for the clades was recovered in TreeAnotator v. 1.8 (Bouckaert et al. 2014) based on the burning of 2.5 million trees. Table |. List of NCBI accession numbers for LSU and ITS sequences of Aroramyces guanajuatensis. Herbarium number LSU NCBI number ITS NCBI number ITCV 1689 MK761021 MN392935 ITCV 1691 MK761022 MN392936 ITCV 1694 MK761023 MN392937 ITCV 1711 MK761024 MN392938 ITCV 1610 MK761025 MN392939 ITCV 1610 MK811035 - ITCV 1613 MK761026 MN392940 ITCV 1613 MK811036 - LEG 129: MK761027 MN392941 ITCV 1731 MK761028 MN392942 ITCV 1734 MK761029 MN392943 ITCVel738 MK761030 MN392944 PEG WAEFS9: MK761031 MN392945 ITCV 1741 MK761032 MN392946 30 Rafael Pena-Ramirez et al. / MycoKeys 61: 27-37 (2019) Results Molecular analyses ITS and LSU sequences of 12 samples of A. guanajuatensis were obtained (Table 1). ITS and LSU sequences are respectively identical. Based on this, only four sequences were selected for phylogenetic analysis. Then after, alignment was performed with 6 sequences of Aroramyces and 22 sequences of Hysterangium (Table 2). Phylogenetic results were as follows: According to the Bayesian analysis, after 10 million gen- erations, 25% trees were discarder as the burn-in. The standard deviation between the chains stabilized at 0.002, indicating that MC3 reached a stationary phase. To confirm that the sample size was enough, the “parameter” file was analyzed using Tracer v. 1.6 (Rambaut et al. 2014), verifying that all parameters had an estimated sample size above 1,500. The subsequent probabilities (SP) were estimated based on the strict consensus rule produced by MrBayes and indicated on the maximum credibility clade tree. The Bayesian inference analysis recovered A. guanajuatensis as a monophyletic group, with a posterior probability of 1. (Fig. 1). Ramaria gelatinosa was used to root the tree. Avoramyces balanosporus and A. guanajuatensis showed the closest relationship but were branched with a probability of 1 and a dissimilarity of 2.19, supporting the existence of a new taxon. The Hysterangium species segregated and formed two different branches. Taxonomy Aroramyces guanajuatensis Peha-Ramirez, Guevara-Guerrero, Z. W. Ge & Mar- tinez-Gonzalez, sp. nov. MycoBank No: 830329 Figures 2—4 Type. MEXICO. State of Guanajuato, municipality of Guanajuato, Cuenca de la Esperanza Protected Natural Area, 7 November. 2016Pefa-Ramirez 108 (Holotype: ILGY 1613). Diagnosis. Aroramyces guanajuatensis is characterized by a peridium 167—240 um thick, of cotton-like hyphae, up to 170 um, long, variably structured mesocutis, yel- lowish subcutis, spores with irregular and inflated utricle. Etymology. "guanajuatensis" in reference to the site (Guanajuato state) where the new taxon was discovered. Description. Basidiome 4.4—17x3.9—13.5x3.2-11.2 mm, globose or subglobose to irregular, sometimes compressed when growing together. Peridial surface white, pale brown, fibrillose or tomentose, often with cotton-like patches of white hyphae en- compassing some debris soil, stones, leaves, and roots. Peridium Separable and fragile, exposing portions of the gleba. < 0.5 mm thick, mostly hyaline, outer portion pale Aroramyces guanajuatensis sp. nov From Mexico 31 Table 2. List of Aroramyces and Hysterangium species, GenBank accession numbers, and references for LSU sequences used in the phylogenetic analysis. Sequences of new taxon are in bold. Species Aroramyces balanosporus G. Guevara & Castellano A. guanajuatensis A. gelatinosporus (J.W. Cribb) Castellano A. radiatus (Lloyd) Castellano, Verbeken & Walleyn Aroramyces sp. Hysterangium affine Mase & Rodway H. album Zeller & C.W. Dodge H. aureum Zeller H. caleareum R. Hesse H. clathroides Vittad H. coriaceum R. Hesse H. crassum (Tul & C. Tul) E, Fisch FH. epiroticum Pacioni H. fragile Vittad H. hallingi Castellano & J.J. Muchovej H. inflatun Rodway H.. membranaceum Vittad H. neotunicatun Castellano & Beever H.. pompholyx Tul. & C. Tul. H. rugisporum Castellano & Beever H. salmonaceum G.W. Beaton, Pegler & T.W.K. Young H. separabile Zeller H. spegazzinii Castellano & J.J. Muchovej H. stoloniferum Tul. & C. Tul. H. strobilus Zeller & C.W. Dodge GenBank MK811031 MK761024 MK811036 MK811035 MK761031 DQ218524 DQ218525 KY686203 DQ218527 DQ218530 DQ218546 DQ218490 DQ218491 DQ218492 AF213121 AF213122 AY574686 AY574687 DQ218495 DQ218496 DQ218497 DQ218549 DQ218498 DQ218550 DQ218499 DQ218500 DQ218501 DQ974810 DQ218502 DQ218503 AF336259 DQ218504 Reference This paper This paper This paper This paper This paper Hosaka et al. 2008 Hosaka et al. 2008 Nuske & Abell unpublished Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Humpert et al. unpublished Humpert et al. unpublished Giachini et al. 2010 Giachini et al. 2010 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Hosaka et al. 2008 Smith et al. 2007 Hosaka et al. 2008 Hosaka et al. 2006 Binder and Bresinsky 2002 Hosaka et al. 2006 brown, with a dark ring next to the gleba. Gleba brown, trama gelatinized, locules irregularly shaped, columella dendroid, translucent gray. Odor fungoid; taste not re- corded. Basidiomata hard wen dried. Peridium three layered, 165-240 pm thick. Epicutis 7.5—22.5 um thick of hyaline to reddish brown, thin-walled, interwoven to repent or erect hyphae, 2-9 um wide, forming scattered caespitose groups of erect, branched, setal hyphae up to 170 um long, with abundant crystalline structures adherent on hyphal walls, clamp connec- tions present. Mesocutis 55-105 um thick, abundant hyaline, isodiametric, globose to subglobose, angular pseudoparenchyma like cells. 4—35x3—24 um, also with some irregularly shaped, interwoven hyphae, 3-11 wm wide. walls 1-2 um pm wide, clamp 32 Rafael Pena-Ramtrez et al. / MycoKeys 61: 27-37 (2019) ned 0.89 DQ218492_Hysterangium_calcareum__Europe 0.34 - DQ218549_Hysterangium_inflatum__Tasmania - DQ218546_Hysterangium_affine__Australia 0.72 DQ218500_Hysterangium_rugisporum__New Zealand 1 DQ218497_Hysterangium_hallingii__Argentina DQ218503_Hysterangium_spegazzinii__Argentina DQ218499_Hysterangium_pompholyx__West and North Europe 1 DQ218490_Hysterangium_album__USA 0.18 : DQ218498_Hysterangium_membranaceum__Holartic and India DQ218504_Hysterangium_strobilus__USA 1 DQ218501_Hysterangium_salmonaceum__ Australia 0.34 DQ218550_Hysterangium_neotunicatum__New Zealand DQ974810_Hysterangium_separabile__USA ny MK76102_Aroramyces_guanajuatensis__ Mexico ail 0.26 MK811036 Aroramyces guanajuatensis Mexico MK811035_Aroramyces_guanajuatensis__ Mexico MK761031_Aroramyces_guanajuatensis_ Mexico MK811034_Aroramyces_balanosporus__ Mexico 0.55 DQ218524_Aroramyces_gelatinosporus__Australia 0.51 0.99 0.99 KY686203_Aroramyces_sp__Australia DQ218527_Aroramyces_sp__India DQ218530_Aroramyces_sp___Guyana DQ218525_Aroramyces_radiafus__ Zimbabwe 0.32 AF213122_Hysterangium_coriaceum__Holartic AY574686_Hysterangium_coriaceum__Holartic 0.3 DQ218491_Hysterangium_aureum__USA 0.81 AF336259_Hysterangium_stoloniferum__Holartic DQ218496_Hysterangium_fragile__Holartic 0.29 DQ218495_Hysterangium_epiroticum__South Europe DQ218502_Hysterangium_separabile__USA 1 AF213121_Hysterangium_cilathroides__Holartic AY574687_Hysterangium_crassum__Central Europe AF213090_Ramaria_gelatinosa 0.002 Figure 1. Maximum probability phylogram of clades obtained with Bayesian inference. The posterior probabilities for each clade are shown on the branches. The accession numbers in the sequence labels indicate the GenBank accession numbers. connection absent. Subcutis 22.5—95 um thick, of interwoven prostrate hyphae, 3—4 um broad, with scattered large pseudoparenchyma like cells up to 37.5x30 um, clamp connection absent. Trama of hyaline, interwoven hyphae 4 um wide, embedded in a gelatinized ma- trix, clamp connections present. Basidia fusoid to clavate, hyaline, 14-48 x 9-12 um, mean = 32.5 x 10.3 um, wall 1 um thick. Basidiospores ellipsoid to broadly ellipsoid, symmetrical, hyaline to pale brown, slightly reddish in KOH, pale brown in mass, excluding utricle 9-13 x 6-7 um long, mean = 11 x 6.1 um, Q range = 1.5—2.17, Q mean = 1.8; with utricle 12-17 x 7-10 um long, mean = 14.47 x 8.27 um, Q = 1.5—2.13, mean = 1.76. Ornamentation of ir- regular crest contained within an inflated utricle, hilar appendage in cross-section appears rectangular, 1-3 x 4—6 um, mean = 1.97 x 5.2 um. Apex obtuse. Utricle inflated up to 3 um from spore wall, mean = 1.43 um, occasionally the utricle is asymmetrically inflated. Distribution, habit and ecology. MEXICO, state of Guanajuato. Cuenca de la Es- peranza Protected Natural Area. Hypogeous, under Quercus spp. at 2530 m. 21°03.87'N, 101°13.193'W. October to December. The March collection was dried in situ. Aroramyces guanajuatensis sp. nov From Mexico oP Figure 2. a-f Avoramyces guanajuatensis (holotype [TCV 1613) a, b basidiome showing the peridial sur- face C basidiome in cross-section showing glebal surface d cross-section of peridium showing three-layered peridium (1 epicutis, 2 mesocutis, 3 subcutis) at 400x e=f basidiospore 1000x f irregular crest contained within an inflated utricle. Scale bars: 0.5 cm (a=c, f), 20 um (d), 5 um (e). Additional material examined. Mexico, state of Guanajuato, Cuenca de la Esper- anza Protected Natural Area: 21°04.075'N, 101°13.193'W 2500 m, 26 October 2016, Pefta-Ramirez 102, paratype (ITCV 1610). 21°03891'N, 101°13.531'W 2457 m, 5 October 2016, Pefa-Ramirez 70 (ITCV 1689). 21°03.891'N, 101°13.533'W, 2464 m, 5 October 2016, Peha-Ramirez 72 (ITCV 1691). 21°03.88'N, 101°13.561'W, 2471 m, 5 October 2016, Pefa-Ramirez 75 (ITCV 1694). 21°03.85'N, 101°13.278'W. 2533 m, 19 October 2016 Pefia-Ramirez 92 (ITCV 1711). 21°03.741'N, 101°13.461'W. 2500 m, 14 November 2016, Peha-Ramirez 110 (ITCV 1729). 21°03.901'N, 101°13.551'W. 22 December 2016, Pefa-Ramirez 119 (ITCV 1738). 21°03.905'N, 101°13.018'W. 2458 m. 22 December 2016, Pefa-Ramirez 120 (ITCV 1739). 21°04.045'N, 101°13.018'W. 2508 m, 6 March 2017, Pefia-Ramirez 122 (ITCV 1741). 34 Rafael Pena-Ramirez et al. / MycoKeys 61: 27-37 (2019) Jim BREE @SM-368 Figure 3. Electronic photomicrograph of basidiospores a Aroramyces balanosporus (ITCV 848) and b Aroramyces guanajuatensis (ITTCV 1739). Scale bars: 5 um. Figure 4. a-f Aroramyces guanajuatensis (holotype ITCV 1613) a=c Cross-section of peridium a epicutis of hyphae postrate cells and angular pseudoparenchyma like mesocutis b arrow head showing postrate cell subcutis € arrow head showing large cell scattered in subcutis d arrow head showing epicutis hyphae with attached crystals e basidia and basidioles f epicutis clamp connection. Scale bar: 10 um (a=, e), 25 um (d), 5 um (f). Aroramyces guanajuatensis sp. nov From Mexico 35 Discussion In the Bayesian inference analysis, Various Aroramyces nest together along with unde- scribed species mentioned in Nuske & Abell (unpublished) and Hosaka et al. (2008). The clade of the genus Aroramyces segregated between two clades that group species of Hysterangium. The close relationship of Aroramyces to Hysterangium in our study agrees with Hosaka et al. (2006, 2008). Aroramyces balanosporus and A. guanajuatensis are closely related but are morphologically and molecularly distinct (Figure 1). Although, the objective of the current assignment is not inferring the phylogenetic relationships in Hysterangiaceae, the result clearly supports Aroramyces guanajuatensis to be an inde- pendent species within the genus Avoramyces with a posterior probability of 1. The hilar appendage is larger in Aroramyces guanajuatensis compared to A. balanospo- rus. Ihe isodiametric mesocutis cells of A. guanajuatensis differ from the variously shaped cells in the mesocutis of A. balanosporus. The asymmetric utricle of Aroramyces herrerae up to 6 um wide whereas the asymmetric utricle of A. guanajuatensis rarely reaches 3 um wide. Mexican Aroramyces are associated with Quercus spp. Interestingly A. radiatus from Africa has smaller spores, 10—12(—13.5) x 6—7(—8) pm, and is associated with Brachyste- gia spiciformis (Caesalpinioideae) and Upaca sp. (Euphorbiaceae). Aroramyces gelatinospo- rus from Australia has similar sized spores but a single-layered peridium and is associated with Eucalyptus spp. (Myrtaceae) (Castellano et al. 2000; Lloyd 1925). The collections were discovered in the Cuenca de la Esperanza Protected Natural Area in Guanajuato, Mexico, located north of Michoacan and east of Jalisco. The pres- ence of unidentified species in this region highlights the importance of this protected natural area and as an area to search for additional new fungal taxa. Acknowledgements Pefa-Ramirez acknowledges ‘TecNM (Tecnolégico Nacional de México) for fellowship PRODEP 1264-2015-2017. Guevara-Guerrero thanks CONACyT and TecNM for re- search support. Martinez-Gonzalez acknowledges Laura Marquez y Nelly Lopez, LaNa- Bio, of Instituto de Biologia, Universidad Nacional Auto6noma de México and to Maria Eugenia Muniz Diaz de Ledn, for giving us access to the Laboratory of Molecular Biol- ogy. 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