Two new species of Geejayessia (Hypocreales) from Asia as evidenced by morphology and multi-gene analyses

Abstract Two new species of Geejayessia are introduced, based on materials collected from central China. Geejayessiaclavatasp. nov. is characterised by gregarious, red brownish to dark red, oval-subglobose to globose perithecia that are formed on a basal stroma; (4–7-)8-spored cylindrical asci; ellipsoidal or rarely broadly ellipsoidal, uniseptate, smooth or finely verruculose ascospores; clavate, aseptate microconidia and absence of macroconidia. Geejayessiasinicasp. nov. is characterised by red to bright red, pyriform, subglobose to globose, perithecia on a basal stroma, collapsing laterally when dry; subcylindrical to clavate asci with a rounded apex; ellipsoidal, uniseptate ascospores; and falcate, multiseptate macroconidia with an arcuate tip. Morphological distinctions of the new species from the related fungi are discussed. This is the first report of Geejayessia from Asia.

In our examinations of nectriaceous collections from central China, two cosmosporalike fungi were encountered. Judging by perithecial gross morphology, anatomic structures and culture characteristics, they represented two previously undescribed species of Geejayessia. Their taxonomic placements were confirmed by multigene phylogenetic analyses. Distinctions between the new species and their closely related fungi are discussed.

Sampling and morphological studies
Specimens were collected from Shennongjia National Nature Reserve and Longyuwan National Forest Park and were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). Methods used by Luo and Zhuang (2010) and Schroers et al. (2011) were generally followed for morphological observations. The test for colour changes of the perithecial wall was made with 3% KOH and 100% LA. To observe internal and microscopic characteristics of the perithecial wall, longitudinal sections through ascomata were made with a freezing microtome (YD-1508-III, Jinhua, China) at a thickness of 6-8 μm. Microscopic examinations and measurements were taken from longitudinal sections and squash mounts in lactophenol cotton blue solution using an Olympus BH-2 microscope (Tokyo, Japan). Photographs were taken with a Leica DFC450 digital camera (Wetzlar, Germany) attached to a Leica M125 stereomicroscope (Milton Keynes, UK) for gross morphology and a Zeiss AxioCam MRc 5 digital camera (Jena, Germany) attached to a Zeiss Axio Imager A2 microscope (Göttingen, Germany) for anatomical structures. Measurements of individual structures were based on 30 units, except when otherwise noted. Cultures were obtained by single ascospore isolation from fresh ascomata. To determine colony features, isolates were grown on cornmeal dextrose agar [CMD, 4% (w/v) cornmeal + 2% (w/v) dextrose + 2% (w/v) agar], potato dextrose agar [PDA, 20% (w/v) potato + 2% (w/v) dextrose + 2% (w/v) agar] and synthetic nutrient-poor agar (SNA; Nirenberg 1976) in 90 mm plastic dishes at 25 °C for 7 d. For the observation of conidiophores, macroconidia and microconidia, cultures were grown on SNA at 25 °C with alternating periods of light/darkness (12 h/12 h). Colony growth rates were measured after 7 d.

DNA extraction, PCR amplification and sequencing
The genomic DNA was extracted from fresh mycelium following the methods of Wang and Zhuang (2004). Three primer pairs, acl1-230up/acl1-1220low , ITS5/ITS4 (White et al. 1990) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999) were used to amplify the sequences or partial sequences of the larger subunit of the ATP citrate lyase (ACL1), the internal transcribed spacers with the 5.8S nuclear ribosomal DNA (ITS) and the second largest subunit of the RNA polymerase II (RPB2), respectively. PCR reactions were performed on an ABI 2720 Thermal Cycler (Applied Biosciences, Foster City, California, USA), based on the procedures detailed in Gräfenhan et al. (2011), White et al. (1990) and Liu et al. (1999). DNA sequencing was carried out in both directions on an ABI 3730XL DNA Sequencer (Applied Biosciences).

Sequence alignment and phylogenetic analyses
Newly generated sequences and those retrieved from GenBank are listed in Table 1 Voglmayr & Jaklitsch were used as outgroup taxa. Sequences were assembled, aligned and the primer sequences were trimmed with BioEdit 7.0.5 (Hall 1999) and converted to NEXUS files by ClustalX 1.8 (Thompson et al. 1997). The partition homogeneity test of ACL1, ITS and RPB2 regions was performed with PAUP 4.0b10 (Swofford 2002). To confirm the phylogenetic positions of the new species, sequences of ACL1, ITS and RPB2 were combined and analysed with Bayesian Inference (BI) and Maximum Parsimony (MP) analyses. The MP analysis was performed with PAUP 4.0b10 (Swofford 2002) using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR (tree bisection and reconnection) branch swapping. Topological confidence of resulted trees was tested by maximum parsimony bootstrap proportion (MPBP) with 1000 replications, each with 10 replicates of random addition of taxa. The BI analysis was conducted by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a Markov chain Monte Carlo algorithm. Nucleotide substitution models were determined by MrModeltest 2.3 (Nylander 2004). GTR+I+G was shown to be the best-fit model for the combined sequences in the BI analysis. Four Markov chains were run simultaneously for 1,000,000 generations with the trees sampled every 100 generations. A 50% majority rule consensus tree was computed after excluding the first 2500 trees as 'burn-in'. Bayesian inference posterior probability (BIPP) was determined from the remaining trees. Trees were examined in TreeView 1.6.6 (Page 1996). BIPP greater than 90% and MPBP greater than 50% are shown at the nodes.
The genus Geejayessia was previously known from Europe, North America and Oceania (Samuels and Rogerson 1984;Nirenberg and Samuels 2000;Schroers et al. 2011). The new species discovered from central China extends the distribution of the genus to Asia.