Three New Species of Fusicolla (Hypocreales) from China

To explore the species diversity of the genus Fusicolla, specimens from Henan, Hubei and Jiangsu Provinces in China are examined, and three undescribed taxa are encountered. The morphological characteristics and DNA sequence analyses of the combined acl1, ITS, LSU, rpb2 and tub2 regions support their placement in Fusicolla and their recognition as new species. Fusicolla aeria sp. nov. is distinguished by the formation of abundant aerial mycelia on PDA, falcate, (1–)3-septate macroconidia 16–35 × 1.5–2.8 μm and subcylindrical, aseptate microconidia 7.5–13 × 0.8–1.1 μm. Fusicolla coralloidea sp. nov. has a coralloid colony on PDA, falcate, 2–5-septate macroconidia 38–70 × 2–4.5 μm and rod-shaped to ellipisoidal, aseptate microconidia 2–7 × 1–1.9 μm. Fusicolla filiformis sp. nov. is characterized by filiform, 2–6-septate macroconidia 28–58 × 1.5–2.3 μm and lacking microconidia. Morphological differences between these novel species and their close relatives are compared in detail. The previously recorded species of the genus in China are listed and a key to these taxa is provided.

Within the scope of our current study on the Chinese Fungus Flora, fresh hypocrealean specimens are examined. Based on the morphology and phylogenetic analyses of the combined sequences of the larger subunit of the ATP citrate lyase (acl1), nuclear ribosomal DNA ITS1-5.8S-ITS2 (ITS), the large subunit of nuclear ribosomal DNA (LSU), the second largest subunit of RNA polymerase II (rpb2) and β-tubulin (tub2), three novel species of Fusicolla are introduced. Comparisons between these taxa and their close relatives are performed. The previously recorded Fusicolla species in China are also listed.

Sampling and Morphological Studies
Specimens on wood substrates were collected from Henan, Hubei and Jiangsu Provinces in China and deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). Lactophenol cotton blue solution was used as a mounting medium for the examination of features and measurements of conidiophores, macroconidia and microconidia. Photographs were taken with a Zeiss AxioCam MRc 5 digital camera (Jena, Germany) attached to a Zeiss Axio Imager A2 microscope (Göttingen, Germany). Cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC). For colony features and growth rates, strains were grown on potato dextrose agar (PDA, 20% (w/v) potato + 2% (w/v) dextrose + 2% (w/v) agar) and synthetic nutrient-poor agar (SNA) [14] in 90 mm plastic Petri dishes at 25 • C for 14 d with alternating periods of light and darkness (12 h/12 h).

DNA Extraction, PCR Amplification, Sequencing and Phylogenetic Analyses
Genomic DNA was extracted from fresh mycelium following the method of Wang and Zhuang [15].
Newly acquired sequences and those retrieved from GenBank are listed in Table 1. The sequences were assembled and aligned, and the primer sequences were trimmed by BioEdit 7.0.5 [22] and converted to NEXUS files by ClustalX 1.83 [23]. The sequences of acl1, ITS, LSU, rpb2 and tub2 were combined and analyzed by Bayesian inference (BI) and maximum likelihood (ML) methods to determine the phylogenetic positions of these strains. The BI analysis was conducted by MrBayes 3.1.2 [24] using a Markov chain Monte Carlo (MCMC) algorithm. Nucleotide substitution models were determined by MrModeltest 2.3 [25]. The ML analysis was performed via IQ-Tree 1.6.12 [26] using the best model for each locus chosen by ModelFinder [27]. Trees were examined by TreeView 1.6.6 [28]. The Bayesian inference posterior probability (BIPP) values greater than 0.9 and maximum likelihood bootstrap (MLBP) values greater than 70% were shown at the nodes.

Phylogeny
The acl1, ITS, LSU, rpb2 and tub2 sequences of 24 Fusicolla species were analyzed. The resulting BI tree is shown in Figure 1. The topology of the ML tree was similar to that of the BI tree.

Discussion
Since the establishment of Fusarium Link in 1809, many fusarioid species have been assigned to the genus and the generic boundary has become obscure. The accumulated morphological and phylogenetic data suggested that the genus was heterogeneous [30]. Efforts were made toward the construction of a monophyletic Fusarium as well as its allies [31,32]. The previously recognized members classified in Fusarium sensu lato are now treated as separate genera, i.e., Albonectria Rossman & Samuels, Atractium Link, Bisifusarium L. Lombard, Crous [2,33,34].
The phylogenetic overview of Fusicolla based on multilocus sequence analyses showed that the genus is monophyletic [2]. The present phylogeny, including the newly added taxa, inferred from sequences of the acl1, ITS, LSU, rpb2 and tub2 regions, resulted in a similar tree topology to that demonstrated in the previous studies [8,9,46,47]. The result indicated that the four Chinese strains (CGMCC 3.24907, 3.24908, 3.24909 and 3.24910) grouped with the known species of Fusicolla (BIBP/MLBP = 1.0/96%), which confirmed their taxonomic placements. Fusicolla filiformis is associated with, but clearly separated from, F. gigas (BIBP/MLBP = 1.0/100%) and is characterized by filiform macroconidia. Fusicolla aeria is grouped with F. acetilerea and F. elongata, all three species forming abundant aerial mycelia on PDA. Fusicolla coralloidea, representing an independent linage, can be easily distinguished by its coralloid synnemata in culture and rod-shaped to ellipsoidal microconidia.
Among the known species of Fusicolla, F. aquaeductuum, F. betae, F. bharatavarshae  [2,5,8,10,34,46,47,49], as well as the newly described species. Large-scale surveys covering different ecosystems and substrates in unexplored regions will further improve our knowledge of the species diversity of the genus and establish connections between the sexual and asexual stages of Fusicolla species, which will permit a better understanding of the whole fungus.