生命科学学院-范黎.pdf
生命科学学院-范黎.pdf
Phytotaxa 427 (4): 270–278 https://www.mapress.com/j/pt/ Copyright © 2019 Magnolia Press Article ISSN 1179-3155 (print edition) PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.427.4.5 Geopora sinensis, a new truffle species from North China based on morphological and molecular data LI-JIE GUO, YU-YAN XU & LI FAN* College of Life Science, Capital Normal University, Xisanhuanbeilu 105, Haidian, Beijing 100048, China * Email: fanli@mail.cnu.edu.cn Abstract Geopora sinensis sp. nov. was described and illustrated from the soil under Picea wilsonii trees in North China based on molecular and morphological analyses in this paper. Morphologically G. sinensis is similar to G. cooperi, G. gilkeyae and G. tolucana, but distinguished from them by the combination of broadly ellipsoid ascospores and white hymenium. ITS-based phylogenetic analysis supports the establishment of the new species. Keywords: ITS, Pezizales, taxonomy, hypogeous fungi Introduction When we investigated hypogeous fungi from northern China in 2017, some medium to large-sized, brown truffle-like specimens were collected in soil under Picea wilsonii Mast. in autumn from Shanxi Province. The ascomatal gross morphology of these truffle samples look like the hypogeous species Geopora cooperi Harkn. and its allies, including G. gilkeyae (Burds.) G. Guevara, Göker & Stielow and G. tolucana Guevara, Göker & Stielow (Harkness 1885, Burdsall 1968, Fogel 1992, Guevara et al. 2012), however, the results of microscopic examinations and molecular phylogenetic analyses supported that they belong to an undescribed species. Material and Methods Morphological studies Fresh specimens were collected in soil under P. wilsonii from Shanxi Province. They were dried and deposited in BJTC (Herbarium of Biology Department, Capital Normal University, Beijing, China). Macroscopic characters were recorded from fresh samples. Microscopic characters were examined from dried materials by mounting free-hand sections of ascomata in 5% KOH and Melzer’s reagent (Dring 1971). One hundred and fifty mature ascospores were measured in KOH from 5 ascomata of 5 collections and the symbol Q is used to indicate mean length/width ratios of ascospores in side view. DNA extraction, PCR amplification and sequencing Dried ascomata were crushed by shaking for 45 s at 30 Hz 2−4 times (Mixer Mill MM301, Retsch, Haan, Germany) in a 1.5 ml tube together with 3 mm-diam tungsten carbide balls, and total genomic DNA was extracted using the modified CTAB method (Gardes and Bruns 1993). The ITS region was amplified using the primers ITS1-F/ITS4 (White et al. 1990; Gardes and Bruns 1993). Polymerase Chain Reactions (PCR) were performed in 25 µl reaction containing 2 μl DNA template, 1 µl primer (10 µM) each, 12.5 µl of 2 × Master Mix [Tiangen Biotech (Beijing) Co.], 8.5 μl ddH2O. PCR reactions were implemented as follows: an initial denaturation at 94 ℃ for 3 min; followed by 35 cycles at 94 ℃ for 45 s, 55 ℃ for 45 s, 72 ℃ for 1 min; and a final extension at 72 ℃ for 10 min. The PCR products were sent to Beijing Zhongkexilin Biotechnology Co. Ltd. for purification, sequencing, and editing. Validated sequences were 270 Accepted by Wen-Ying Zhuang: 3 Dec. 2019; published: 30 Dec. 2019 deposited in GenBank database (http://www.ncbi.nlm.nih.gov/). Other sequences of Geopora and the related fungi were mainly from those used by Guevara et al. (2012) and Flores-Renteria et al. (2014), or retrieved from GenBank using the BLAST tool with a threshold of 90% sequence identity. Wilcoxina mikolae (GQ267499) and Pyronema domesticum (HM016895) were selected as out-group taxa (TABLE 1). FIGURE 1. Phylogeny derived from most parsimonious analysis of the ITS rDNA sequences from Geopora and related species. Wilcoxina mikolae and Pyronema domesticum as outgroup taxa. Bootstrap support values≥70% and posterior probability values≥0.95 are showing at notes. Phylogenetic analysis Sequences were aligned and edited in Clustal X (Thompson et al. 1997) and BioEdit7.1.3 (Hall 1999), and manually adjusted for maximum sequence similarity in Se-Al version.2.03a. (Rambaut 2000). Ambiguously aligned regions identified by Gblocks 0.91b on line (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) in the alignment were excluded from the analyses. The following relaxed parameters were used in Gblocks: minimum number of sequences for a conserved position = 40, minimum number of sequences for a flanking position = 40, maximum Geopora sinensis, a new truffle species Phytotaxa 427 (4) © 2019 Magnolia Press • 271 number of contiguous nonconserved positions = 8, minimum length of a block = 5, and allowed gap positions = ‘‘with half’’. A most parsimonious (MP) tree was constructed with PAUP* 4.0b10 (Swofford 2002). The bootstrap values were determined using the following settings: 1000 replicate searches on all parsimoniously informative characters using 100 random sequence addition replications and TBR (tree-bisection reconnection) branch swapping algorithms in PAUP*. Consistency index (CI), retention index (RI) and homoplasy index (HI) were also calculated. Tree was viewed with TreeView32 (Page 2001). The bootstrap values (BS) ≥ 70% were considered to be significant (Hillis and Bull 1993). TABLE 1. Sources of specimens and GenBank accession numbers for sequences used in this study. Newly generated sequences are in bold. Species Voucher Origin ITS Reference Geopora arenicola TAA:179692 Estonia FM206461 Tamm et al. 2010 G. arenicola TAA:116793 Estonia FM206459 Tamm et al. 2010 G. arenicola TAA:114852 Estonia FM206458 Tamm et al. 2010 G. cercocarpi Kropp2 USA HQ283097 Southworth et al. 2011 G. cervina H:JP3106 Finland FM206414 Tamm et al. 2010 G. cervina TAA:188517 Estonia FM206418 Tamm et al. 2010 G. cervina TAA:117472 Estonia FM206415 Tamm et al. 2010 G. cooperi SOC1051 USA FJ789595 Gladdish et al. 2010 G. clausa 17002 Italy JF908766 Osmundson et al. 2013 G. cooperi src515 USA DQ974731 Smith et al. 2007 G. cooperi MO-205834 USA KT968604 Unpublished G. cooperi 16977 Italy JF908023 Osmundson et al. 2013 G. cooperi VK1783 Greece JN812045 Unpublished G. cooperi (S) F23212 Spain FR694203 Guevara-Guerrero et al. 2012 G. cooperi AH9065 Spain JN812044 Unpublished G. cooperi AH9846 Spain JN812041 Unpublished G. cooperi AH39106 Morocco JN812043 Unpublished G. cooperi AH39089 Morocco JN812042 Unpublished G. cooperi BJTC FAN727 China MN137239 This study G. cooperi f. cooperi (S) F48895 Sweden FR694202 Guevara-Guerrero et al. 2012 G. cooperi f. cooperi TA50 Pakistan JN558642 Ashraf et al. 2012 G. nicaeensis 14596 Italy JF908022 Osmundson et al. 2013 G. pinyonensis DGB 27586 USA KF768652 Flores-Renteria et al. 2014 G. pinyonensis DGB 27586 USA KF768653 Flores-Renteria et al. 2014 G. sepulta - - KU991187 Unpublished G. sinensis BJTC FAN1011 China MN148104 This study G. sinensis BJTC FAN1029 China MN148110 This study G. sinensis BJTC FAN1013 China MN148105 This study G. sinensis BJTC FAN1028 China MN148109 This study G. sinensis BJTC FAN1018 China MN148108 This study G. sinensis BJTC FAN765 China MN148113 This study G. sinensis BJTC FAN759 China MN148112 This study G. sinensis BJTC FAN769, holotype China MN148101 This study G. sinensis BJTC FAN778 China MN148103 This study G. sinensis BJTC FAN775 China MN148102 This study G. sinensis BJTC FAN1016 China MN148107 This study ......continued on the next page 272 • Phytotaxa 427 (4) © 2019 Magnolia Press guo et al. TABLE 1. (Continued) Species Voucher Origin ITS Reference G. sinensis BJTC FAN1015 China MN148106 This study Geopora sp. TAA:192232 Estonia FM206420 Tamm et al. 2010 Geopora sp. 3-MAS-2016/ECM - KU991189 Unpublished G. sumneriana 16978 Italy JF908024 Osmundson et al. 2013 G. tenuis TAA:117404 Russia FM206403 Tamm et al. 2010 G. tenuis H:RS09584 Finland FM206402 Tamm et al. 2010 G. tenuis TAA:192293 Estonia FM206401 Tamm et al. 2010 G. tolucana 1082IICV_frb2 Mexico HQ184961 Guevara-Guerrero et al. 2012 G. tolucana 1082IICV_frb1 Mexico HQ184960 Guevara-Guerrero et al. 2012 Pezizales sp. d334 USA AF266709 Bidartondo et al. 2001 Phaeangium lefebvrei 17051 Botswana JF908768 Osmundson et al. 2013 P. lefebvrei BMBO18 Algeria KR073966 Zitouni-Haouar et al. 2015 P. lefebvrei STBL1 - GQ228095 Unpublished P. lefebvrei 102PJ - AF387652 Unpublished P. lefebvrei 84PJ - AF387654 Unpublished P. lefebvrei BMBC13 Algeria KR073973 Zitouni-Haouar et al. 2015 P. lefebvrei BMBC11 Spain KR073965 Zitouni-Haouar et al. 2015 P. lefebvrei 84PJ - AF387653 Unpublished Picoa juniperi AH39247 Spain JN392154 Zitouni-Haouar et al. 2015 P. juniperi gv-pa-16 Saudi Arabia KX262703 Unpublished P. juniperi IRA-MBA sb7 Tunisia GU391565 Sbissi et al. 2010 P. juniperi IRA-MBA sb1 Tunisia GU391559 Sbissi et al. 2010 P. juniperi IRA-MBA sb8 Tunisia GU391566 Sbissi et al. 2010 P. juniperi AH38893 Spain JN392175 Zitouni-Haouar et al. 2015 Picoa sp. IRA-MBASba Medenine KT350943 Zitouni-Haouar et al. 2015 Pyronema domesticum A10 China HM016895 Unpublished Pyronemataceae sp. JW96a China GQ281481 Wei et al. 2010 uncultured Ascomycota RT238 USA DQ822805 Peay et al. 2007 uncultured fungus TP21_OTU58 USA EF434150 Taylor et al. 2007 uncultured fungus B2_OTU58 USA EF433967 Taylor et al. 2007 uncultured fungus TK1774A USA AY702785 Izzo et al. 2005 uncultured Geopora 7704 Germany EU668289 Bidartondo et al. 2008 uncultured Geopora se1A-01 USA EU334890 Unpublished uncultured Geopora RFLP type Z USA HQ630380 Gordon et al. 2011 uncultured Geopora RFLP type E USA HQ630377 Gordon et al. 2011 uncultured Geopora RFLP type K USA HQ630379 Gordon et al. 2011 uncultured Geopora RFLP type C USA HQ630376 Gordon et al. 2011 uncultured Pezizales OTU7 USA GQ221623 Wolfe et al. 2009 uncultured Tricharina LO31 B3 - EU726332 Hynes et al. 2010 uncultured Tricharina PP7 F10 - EU726331 Hynes 2009 uncultured Tricharina Pine6_a1 - EU649085 Unpublished uncultured Tricharina PP10 D5 - EU726333 Hynes 2009 Wilcoxina mikolae K04C38T193 New Zealand GQ267499 Walbert et al. 2010 Geopora sinensis, a new truffle species Phytotaxa 427 (4) © 2019 Magnolia Press • 273 Bayesian inference (BI) was conducted using MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) as an additional method of evaluating branch support. In the BI analysis, after selecting the best substitution model (GTR+GAMMA+I) determined by MrModeltest v2.3 (Nylander 2004), two independent runs of four chains were conducted for 1000000 Markov Chain Monte Carlo (MCMC) generations using the default settings and sampled every 100 generations. The temperature value was lowered to 0.20, burn-in was set to 0.25, and the run was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01. A 50% majority-rule consensus tree was constructed and visualized with TreeView32 (Page 2001). Bayesian posterior probability (PP) ≥ 95% were considered as significantly supported (Alfaro et al. 2003). Sequences derived in this study were deposited in GenBank (Table 1), the alignment in TreeBASE (www.treebase.org; study S24738). Results Molecular phylogenetics The nuc-ITS dataset contained 79 taxa and comprised 571 characters including the alignment gaps, of which 271 were parsimony-informative, 358 variable but parsimony-uninformative, and 213 were conserved. The 100 equally most parsimonious trees obtained in the maximum parsimony analysis (MP) with limited number of saved trees were 1212 steps long with a consistency index (CI) of 0.525, a retention index (RI) of 0.838 and a homoplasy index (HI) of 0.475. One of the most parsimonious trees is presented in (Fig. 1). MP and BI analyses yielded identical tree topologies and only the tree inferred from the MP analysis is shown (Fig. 1). The phylogenetic analysis revealed that the sequences of Geopora sinensis were clustered, forming an independent branch with strong statistic supports, and further clustered to G. tolucana with low support. The G. cooperi sequence of the Xinjiang (China) sample (BJTC FAN727) was grouped together with those from Europe, Pakistan and U.S.A with strong statistic support (Fig. 1). FIGURE 2. Geopora sinensis (BJTC FAN769, holotype). a. Ascomata. b-c. Asci and ascospores. d. Excipular hairs. e. Ectal excipulum. f. Medullary excipulum. 274 • Phytotaxa 427 (4) © 2019 Magnolia Press guo et al. Taxonomy Geopora sinensis L. Fan & L.J. Guo, sp. nov. (Fig. 2) Fungal Names: FN570661 Etymology:—sinensis, China, referring to the country of the type locality. Holotype: China. Shanxi Province, Ningwu County, Guancen Mountain, 25 August 2017, in soil under P. wilsonii, K. B. Huang HKB064 (BJTC FAN769). Ascomata hypogeous, irregularly subglobose to ellipsoid, 15–48 × 28–43 mm, yellow brown, brown to earth brown, fragile, surface covered by dense tomentum, encrusted with adhering soil particles, convolute to slightly brainlike, furrows filled with mycelia and debris. Gleba compact, with some labyrinthoid chambers, infolded strongly, lambry thin formed by brown peridium and whitish to cream hymenium. Peridium 280–347 μm thick, not detachable, two-layered. Outer layer (ectal excipulum) 84–144 μm thick, textura angularis to angularis-prismatica, composed of oval, ovate or more or less globose cells of 13.5–26.0 × 12.5–28.0 μm, walls light brown to reddish brown towards outer side surface. Inner layer (medullary excipulum) 178–200 μm thick, textura intricata, hyphae interwoven, hyaline, thin-walled, 6.0–7.0 μm in diam. Subhymenium not well differentiated. Excipular hairs 203–352 × 6.5–11.0 μm, hyphae-like, straight or curved, occasionally twisted, slightly thick-walled, septate, with obtuse tips. Asci cylindrical, 213–280 × 20–32 μm, thin-walled, hyaline, 8-spored, no Melzer’s reaction. Ascospore uniseriate, broadly ellipsoid to subglobose, 18.0–25.0 × 17.0–19.5 μm ( x = 21.4 ± 1.7 × 18.1 ± 0.8), Q = 1.07–1.30 ( x = 1.18 ± 0.06), hyaline, thin-walled, smooth, with a large-guttule. Habit, habitat and distribution:—hypogeous, in soil under Picea wilsonii, Shanxi Province, China. Additional specimens examined:—China, Shanxi Province, Ningwu County, Guancen Mountain, elev. 2099 m, 25 August 2017, in soil under P. wilsonii, M. Chen CM025 (BJTC FAN765); ibid. T. Li LT170835 (BJTC FAN759); ibid. K. B. Huang HKB070 (BJTC FAN775); ibid. K. B. Huang HKB073 (BJTC FAN778); ibid. elev. 2026m, 12 October 2017, in soil under P. wilsonii, L. J. Guo GLJ002 (BJTC FAN1011); ibid. L. J. Guo GLJ004 (BJTC FAN1013); ibid. Y. Y. Xu XYY053 (BJTC FAN1015); ibid. Y. Y. Xu XYY054 (BJTC FAN1016); ibid. Y. Y. Xu XYY056 (BJTC FAN1018). ibid. B. D. He HBD019 (BJTC FAN1028); ibid. B. D. He HBD020 (BJTC FAN1029). Discussion Geopora sinensis is similar to G. cooperi, G. gilkeyae and G. tolucana in ascomatal appearance (Burdsall 1968, Fogel 1992, Guevara et al. 2012) as they all have hypogeous truffle-like fruitbodies. Geopora cooperi is differentiated from G. sinensis by its ellipsoid to long-ellipsoid ascospores (Q = 1.49–1.77) (Fogel 1992), while G. tolucana can be distinguished by its light-brown hymenium and large-sized ascospores (20–30 × 16–23 μm) (Guevara et al. 2012). The North American G. gilkeyae shared the same types of ascospores and hymenium (Fogel 1992). However, G. gilkeyae was distantly related to G. sinensis in the ITS tree (Fig. 1), and shared less than 95% ITS sequence similarity. Our phylogenetic analysis showed that the sequences of G. sinensis were grouped together as an independent branch with a strong supporting value (Fig.1). Although the sequences further clustered in three subclades (Fig. 1), we treated them as intraspecific variations, as all these sequences have more than 99% similarities, and their voucher specimens are collected from the same locality. Zhuang (2014) listed G. cooperi f. cooperi Harkn. (= G. cooperi), and G. cooperi f. gilkeyae Burds. (= G. gilkeyae) in the Flora Fungorum Sinicorum (Pyronemataceae). According to Zhuang, G. cooperi was originally recorded from Xinjiang (Liu and Tao 1988) and G. cooperi f. gilkeyae was from Inner Mongolia (Song 1993). We are not able to locate the specimen MHSU438 (G. cooperi from Xinjiang), currently we found a collection (BJTC FAN727) from Xinjiang which belongs to the G. cooperi complex. Our phylogenetic analysis revealed that the sequences of G. cooperi from Europe, Asia and North America were not identical (Fig.1), and agree with Guevara et al. (2012) that they probably represent different species. We are not able to determine which cluster represents the authentic G. cooperi before examination of the type material. Thus, we treat the Chinese material as G. cooperi-like species (BJTC FAN727). We also studied the specimen (HKAS 23369) of G. cooperi f. gilkeyae from Inner Mongolia, China (Song 1993). However, its ascospores are not completely mature, and its DNA sequence was not successfully sequenced in this study, we are therefore not sure if this specimen was identified correctly. Geopora sinensis, a new truffle species Phytotaxa 427 (4) © 2019 Magnolia Press • 275 Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 31750001) and Beijing Natural Science Foundation (No. 5172003). References Alfaro, M.E., Zoller, S. & Lutzoni, F. (2003) Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. 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