Phylogeny and taxonomic revision of Kernia and Acaulium

The genera Kernia and Acaulium comprise species commonly isolated from dung, soil, decaying meat and skin of animal. The taxonomy of these fungi has been controversial and relies mainly on morphological criteria. With the aim to clarify the taxonomy and phylogeny of these fungi, we studied all the available ex-type strains of a large set of species by means of morphological and molecular phylogenetic analyses. Phylogenetic analysis of the partial internal transcribed spacer region (ITS) and the partial 28S rDNA (LSU) showed that the genera Kernia and Acaulium were found to be separated in two distinct lineages in Microascaceae. Based on morphological characters and multilocus phylogenetic analysis of the ITS, LSU, translation elongation factor 1α and β-tubulin genes, the species in Kernia and Acaulium were well separated and two new combinations are introduced, i.e. Acaulium peruvianum and Acaulium retardatum, a new species of Kernia is described, namely Kernia anthracina. Descriptions of the phenotypic features and molecular phylogeny for identification are discussed for accepted species in two genera in this study.

. Multilocus phylogenetic analysis have considerably improved our understanding of species concepts in many fungal groups [18][19][20][21][22][23][24][25] , but the studies for revising the genera of Kernia and Acaulium are relatively limited. Besides, during our investigation of intestinal fungi in animals in China, three particular Kernia isolates from the dung of marmot were isolated. The present work also aims to clarify the taxonomic position of these strains as putative new species using the genealogical concordance analysis 24 . We provide a multigene (ITS, LSU, TEF, TUB) phylogeny of Kernia and Acaulium and related fungi based on a large set of strains, which includes all available ex-type cultures and well-identified reference strains from international culture collections.

Results
Generic circumscription. DNA sequences determined in this study are deposited in GenBank, and accession numbers are listed in Table 1. To delineate generic boundaries, we conducted a phylogenetic analysis using the combined LSU and ITS datasets including 29 currently accepted species belonging to nine genera of Microascaceae and one species of the family Graphiaceae. Graphium penicillioides were selected as outgroup (Fig. 1).
The final alignment consisted of 31 strains and contained 1,385 characters (LSU 796, ITS 589). Figure 1 shows the ML tree including ML bootstrap values (bs) and posterior probabilities (pp) values. The trees obtained from ML and Bayesian analyses of the individual loci and the combined analysis showed congruent topologies. The phylogenetic inferences (Fig. 1) showed that Kernia and Acaulium were monophyletic, the species of Kernia and Acaulium clustered into a single, well-supported lineage (bs = 100%/pp = 100%), respectively. Figure 2 is demonstrated that the colonies of K. peruviana  www.nature.com/scientificreports/  www.nature.com/scientificreports/ to dark brown colonies apparently different with the Acaulium species. Among with the K. geniculotricha and K. nitida were located in the same clade with the value of (bs = 94%/pp = 91%) and combined with morphological characters, they could be identified as the same species in this research. Three Kernia strains which were isolated from the dung of marmot, were clustered with the species of K. hippocrepida and have a well-supported value (bs = 100%/pp = 100%).
Colonies on PDA reaching 5 mm diameter after 10 days at 20 °C, slow growing, raised centrally, with flat and irregular margin, white. On SDA reaching 9 mm diameter, moderately growing, raised centrally, aerial mycelium absent or sparse, white to cream. On CMA reaching 9 mm diameter, moderately growing, planar, white, margin discrete. On OA reaching 15 mm diameter, planar, white.
Identification keys. According to the morphological features, identification keys were constructed for the different genera including all the phylogenetic species recognised in this study (Supplementary information 1).

Discussion
The family Microascaceae was established by Luttrell 26 , comprising saprobic and plant pathogenic species. Some species of Microascaceae are opportunistic pathogens and show intrinsic resistance to antifungal agents 11,20,27 . Recent molecular studies have demonstrated that the Microascaceae contains several closely related genera that are difficult to separate morphologically 11 including Microascus, Scedosporium and Scopulariopsis. Recently, three of the most debated genera of the family, Microascus, Scopulariopsis and Pithoascus were revised by morphology and multigene phylogeny 11,16 . As a result, several taxa were excluded from these genera and appeared as a new lineage within the Microascaceae as Acaulium 11 .
In this study we have reviewed the taxonomic circumscription of species in the genera of Kernia and Acaulium, traditionally referred to as sexual and asexual morphs, respectively, and two genera using a polyphasic approach based on the genealogical concordance analysis, phylogeny and morphological data. These results show that Kernia and Acaulium constitute two phylogenetically distant lineages, combining the results of phenotypic data, delineate the accepted species of the two new combination species, proposing a new species, which are clarifying the identity of species as Kernia nitida and K. geniculotricha, reclassifying the white synnematous species as Acaulium peruvianum and A. retardatum, and describing a new species K. anthracina isolated from the dung of marmot. The species of Kernia are mainly isolated from dung of various animals, while the species of Acaulium have a worldwide distribution and are mainly isolated from dung, litter, soil, skin of a horse and decaying meat [4][5][6][7][8][9][10]16 .
Sandoval-Denis et al. 11 was firstly attempts to clarify phylogenetically the relationships among the different genera of the Microascaceae by the use of partial LSU and ITS sequences. Subsequently, Microascaceae was revised by Sandoval-Denis et al. 16 based on morphological, physiological and molecular phylogenetic analyses using DNA sequence data of four loci (ITS, LSU, TEF and TUB). These studies demonstrated that several genera of Microascaceae raised questions concerning correct positions of several members of the family and their generic circumscriptions, suggesting a possible subdivision of Microascus and Scopulariopsis into several smaller genera as Kernia and Acaulium. Our results based on the phylogenetic reconstructions of two loci (LSU, ITS) indicated that Kernia and Acaulium fall into two groups (Fig. 1). Besides, it also shows that Kernia and Acaulium species can be well separate by phylogentic analysis of four loci (LSU, ITS, TEF and TUB). As known that Acaulium is characterised by the formation of pale colonies with dense hyphal fascicles and the presence of abundant oil drops in the mycelium, conidia and ascospores, showing a guttulate appearance 16 . The new combination species A. peruvianum and A. retardatum clustered in Acaulium group, in which the species produce white and pale grey colonies, and have a wide isolation source. A new species was clustered in Kernia group, which form compact dark brown or black colonies, and mainly isolated from dung (Figs. 2, 3). In addition, species delineation was also assessed in the genus of Kernia as the closed species of K. anthracina and K. hippocrepida under the genealogical concordance analysis using DNA sequence data of four loci (Fig. 4).
The absence of clear diagnostic morphological characters can be used to identify species which belonging to the Kernia and Acaulium species 2,11,16 . The species of 'K. geniculotricha' and K. nitida have been identified two different species according to morphologically characters, but molecular data can easily identified them as the same species, using any of the four genes studied here. Some species as A. peruvianum and A. retardatum isolates were initially identified as K. peruviana, K. retardate at CBS based on their morphology. Combined the molecular data, the group of species that would previously have been included in Kernia and Acaulium are easily recognized. Our phylogeny demonstrates that, although Kernia and Acaulium share similar morphological and ecological traits, they are in fact genetically distant. The phylogenetic data is supported by relevant morphological differences, such as the color of colonies, the shape of ascospores or conspicuously hairy ascomata 2,[8][9][10] .
The new species, K. anthracina and K. hippocrepida, are very similar in ITS but easily distinguished by TUB and TEF sequences. All Kernia species can be well separated with TUB and TEF partial gene sequences. Based on ITS alone, K. anthracina and K. hippocrepida cannot be distinguished (Fig. 4), but morphology and TUB and TEF sequences clearly differentiate them. The lack of the isotype herbarium specimens examined here prevented us from conclusively characterizing five of the other described species K. bifurcotricha, K. setadispersa, K. cauquensis, K. irregularis, K. ovata, leaving them as nomena dubia. From our study, we found that it is easy to identify the species of Kernia and Acaulium by polyphasic approach.
The delimitation of the two genera in this study contributes to an integrated phylogeny of the family Microascaceae. The two monophyletic genera currently accepted are statistically supported in the four-locus phylogeny (Fig. 3). There are seven species included in Acaulium by our revision, while ten species in Kernia. It is regret that some species of Kernia absent holotype material and unavailable for these species. Therefore, further studies are needed to establish a comprehensive modern classification of the Kernia and to give better insight into the evolutionary relationships among the species in the genus.

Materials and methods
Eight Kernia and Acaulium ex-type strains were obtained from the CBS culture collection (CBS) housed at the Westerdijk Fungal Biodiversity Institute (WI), Utrecht, the Netherlands. More isolates potentially related to the obtained Acaulium strains were selected based on a preliminary phylogenetic analysis of LSU + ITS sequences from GenBank, as well as several cultures of the Kernia, which isolated from the feces of Marmota monax 25  DNA extraction, PCR amplification and sequencing. Total genomic DNA was extracted from mycelia grown on PDA or OA plates using the protocol of Guo et al. 28 . Primers ITS1 and ITS4 were used to amplify the ITS region of the nuclear rRNA gene 29 , primers LROR/LR5 primers were used for the partial 28S rDNA (LSU) 30 , primers 983F and 2218R 31 for the elongation factor 1-α gene (TEF), and primers Bt2a and Bt2b 32 for the partial β-tubulin gene (TUB phylogenetic analysis. Sequence data of the four loci were aligned with Clustal X 33 . Reference sequences were retrieved from GenBank and the accession numbers indicated in Table 1. Manual editing of sequences was performed in MEGA6 34 . The concatenated sequences (LSU + ITS) or (LSU + TUB + TEF + ITS) were assembled using SeaView 35 and alignments were deposited in TreeBASE (www. treebase.org, submission no.: S25764). The combined dataset of two or four loci was analyzed phylogenetically using Bayesian MCMC 36 and Maximum Likelihood 37 , respectively. For the Bayesian analyses, the models of evolution were estimated by using MrModeltest 2.3 38 . Posterior probabilities (PP) 39,40 were determined by Markov Chain Monte Carlo sampling (MCMC), Six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation (resulting in 20,000 total trees). The first 4,000 trees represented the burn-in phase of the analyses and were discarded and the remaining 16,000 trees were used for calculating PP in the majority rule consensus tree. For the ML analysis in RAxML 37 , the GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites. Analyses were performed using the CIPRES web portal 41 . Trees were visualised in TreeView 1.6.6 42 .