Multigene phylogenetic analyses to establish new Valsaria species and taxonomic significance of spore ornamentation

During our studies on fungal diversity from plant substrates, a new species of Valsaria was isolated from dead branches of Ostrya carpinifolia. The taxon is morphologically similar to other taxa in Valsariaceae and is characterized by pseudostromata, apically free pseudoparaphyses, bitunicate asci, and dark brown, 2-celled ascospores. However, it differs from extant species in number of guttules and ornamentation of spore. It is introduced herein as Valsaria ostryae sp. nov. within the family Valsariaceae. Multigene phylogenies based on combined LSU, ITS and RPB2 DNA sequence data generated from maximum likelihood, maximum parsimony and MrBayes analyses indicate that V. ostryae is basal to V. lopadostomoides and V. rudis and its establishment as a new species is strongly supported. No discordance was found between our morphological and phylogenetic species boundaries as postulated by other researchers and our molecular data strongly supports ornamentation of spore as useful for species delineation. Valsaria species do not appear to be host specific. Full morphological details are provided herein and phylogenetic relationships of Valsaria species are also discussed in light with host association.


Introduction
Valsariaceae was introduced by Jaklitsch et al. [1] and classified in the order Valsariales. Species of this family have a worldwide distribution occurring on sun-exposed, corticated logs, branches of coniferous and broadleaf trees and on bamboo [1] and exist as saprobes, plant pathogens or necrotrophs [2,3]. The family comprises three genera namely Bambusaria  [1,4]. The asexual morphs are known to be coelomycetous [4]. Valsaria was introduced by Cesati and De Notaris [5] with Valsaria insitiva (Tode) Ces. & De Not. as the type species. Ju et al. [2] examined numerous species to provide an update of its taxonomy. Multigene phylogenetic analyses have also been performed to clarify species taxonomy [1]. In this paper, we introduce one new species of Valsaria, which was found on dead terrestrial branches of Ostrya carpinifolia (Betulaceae). Combined analyses of LSU, ITS and RPB2 DNA sequence data using maximumlikelihood (ML), maximum-parsimony (MP) and Bayesian inference analyses clearly indicate that this species belongs to the Valsariaceae with strong statistical support. Phylogeny also provides important clues on the taxonomic usefulness of morphological features that can be used to differentiate among Valsaria species and its allies. In this paper, the new species is described, illustrated and compared with similar taxa.

Isolates and morphology
Specimens were collected from dead branches of Ostrya carpinifolia in the Province of Forlì-Cesena [FC], Santa Sofia, Camposonaldo, Italy and were pressed and dried individually between blotting papers and labelled. The geographic coordinates for the data collected is 43.952106, 11.873138 (Fig 1). Specimens were observed under a Motic SMZ 168 series dissecting stereo-microscope. Hand-cut sections of the fruiting structures were mounted in water for microscopic studies and photomicrography. Pure cultures were obtained from single spore isolation following the method designated in Chomnunti et al. [6]. Germinating ascospores were transferred aseptically to malt extract agar (MEA) plates and incubated at 16˚C. Colony characters on media were observed and growth rates were measured after one week and at weekly intervals. No specific permissions were required for the location because permissions are standardized only for macro-fungi such as only 3 fruiting-bodies for each collection, the indication of the name of the species that it needs to collect and other things valid only for macrofungi. Also, the field studies did not involve endangered or protected species. The holotype specimen was deposited in the Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand. Ex-type living cultures are deposited in the Culture Collection at Mae Fah Luang University (MFLUCC) and at the International Collection of Microorganisms from Plants (ICMP). Faces of Fungi and Index Fungorum numbers are provided as in Jayasiri et al. [7] and Index Fungorum [8]. New taxa were established based on recommendations outlined by Jeewon and Hyde [9].

DNA extraction, PCR amplification and sequencing
Fungal isolates were grown on MEA media at 16 ± 2˚C for 4 weeks. Fungal mycelium was extracted with Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China). Amplification reactions were performed using known primer pairs, LR0R/LR5 for the 28S large subunit rDNA (LSU) [10], ITS5/ITS4 for 5.8S nrRNA gene with the two flanking internal transcribed spacers (ITS) [11] and fRPB2-5F/ fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) [12]. The ultimate volume of the PCR reaction were 25 μl, containing 1 μl of DNA template (40 μg), 1 μl of each forward and reverse primer (0.2 μM) and 12.5 μl of 2xMaster Mix and 9.5 μl of ddH2O. The PCR condition was as follows: initial denaturation 95˚C for 3 min, 35 cycles of denaturation at 95˚C for 30 s, annealing at 54˚C for 48 s, elongation at 72˚C for 1 min, and final extension at 72˚C for 10 min for LSU, ITS and RPB2. PCR products were sequenced with the same primers mentioned above at Sangon Biotech, Shanghai, China.

Sequence alignment and phylogenetic analyses
Phylogenetic analyses were conducted on a combined dataset of LSU, ITS and RPB2 sequence data. Taxa used in the analyses were obtained through recent publications [1,13]. Separate LSU, ITS and RPB2 DNA sequences were also subjected to BLAST search engine tool of NCBI for verification and selection of taxa for subsequent phylogenetic analyses. Maximum likelihood analysis was performed by using raxmlGUIv.0.9b2 [14]. The search strategy was set to rapid bootstrapping and the analysis carried out using the GTRGAMMAI model of nucleotide substitution. The number of replicates was inferred using the stopping criterion [15]. Maximum Likelihood bootstrap values equal or greater than 90% are given as the first set of numbers above the nodes (Fig 2). PAUPv4.0b10 was used to conduct the parsimony analysis to obtain the phylogenetic trees. Trees were inferred using the heuristic search option with 1000 random sequence additions. Maxtrees were setup to 500 and branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony ( [16] were performed in order to determine whether trees were significantly different. Maximum-parsimony bootstrap values equal or greater than 90% are given as the second set of numbers above the nodes (Fig 2). Bayesian Inference (BI) analysis was conducted with MrBayes v. 3.1.2 [17] to evaluate Posterior probabilities (BYPP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). Two parallel runs were conducted, using the default settings, but with the following adjustments: Six simultaneous Markov chains were run for 5,000,000 generations and trees were sampled every 1000 generation. The distribution of log-likelihood scores was examined to determine stationary phase for each search and to decide if extra runs were required to achieve convergence, using the program Tracer 1.5 [18]. First 20% of generated trees were discarded and remaining 80% of trees were used to calculate posterior probabilities (PP) of the majority rule consensus tree. Phylograms were visualized with FigTree v1.4.0 program [19] and reorganized in Microsoft power point (2016) and Adobe Illustrator CS5 (Version 15.0.0, Adobe, San Jose, CA). The finalized alignment and tree is deposited in TreeBASE, submission ID: 22749 (http://purl.org/phylo/treebase/phylows/study/TB2:S22749).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to Index Fungorum from where they will be made available to the Global Names Index. The unique Index Fungorum number can be resolved and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix www.indexfungorum.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.

Compliance with ethical standards
There is no conflict of interest (financial or non-financial) and all authors have agreed to submission of paper. The authors also declare that they have no conflict of interest and confirm that the field studies did not involve endangered or protected species.

Discussion
Species of Valsaria occur primarily on woody parts of ornamental plants but is also distributed on a wide range of hosts. Our new taxon was isolated from Ostrya carpinifolia which belongs to Eudicots and Betulaceae family [20]. Substrates colonised by members of Valsaria recorded until now are all dicotyledons of the Fabaceae, Fagaceae and Vitaceae. Valsaria lopadostomoides can be found on evergreen oaks, such as Quercus ilex (Fagaceae) while V. rudis occur on deciduous oaks, such as Quercus cerris and Q. petrae (Fagaceae). Woody plant hosts of V. lopadostomoides and V. rudis are closely related to each other as both are accommodated in the Fagaceae which is a monophyletic family close to Quercoideae [21]. Valsaria neotropica has been isolated from unidentified hosts in the Neotropics and seems to be distributed in the  tropical terrestrial ecoregions of the Americas and the South American temperate zone. Valsaria insitiva was described mainly from non-fabaceous hosts and Vitis (Vitaceae) which is accommodated in its own order Vitales and closely related to Rhamnaceae in the order Rhamnales [22]. Valsaria robiniae have been reported from Amorpha, Caragana, Colutea, Hippocrepis and Robinia species in Central Europe, submediterranean regions and Eastern USA while V. spartii occur on various species of woody Fabaceae in the Sub-Mediterranean. Host distribution of V. robiniae and V. spartii seems to be related as most of the species are from Fabaceae which is the third largest family of angiosperms after Orchidaceae and Asteraceae [23]. Based on available literature, it is apparent that Valsaria has a wide host range and species are less likely to be host specific. As far as we are aware, no Valsaria species are known from Ostrya carpinifolia. We compare the new species with V. insitiva, the type species of Valsaria and they share similar characters. However, they differ in the number of guttules in each ascospore cell, as well as in the surface ornamentation of the ascospores (Fig 3). Our new taxon can also be compared to V. sparti and V. robiniae and differs in ascospores that are not constricted. Valsaria ostryae also shares similar morphological characters to V. rudis and has 2-3 guttules in each ascospore cell, while V. rudis has no guttule in its ascospore cells. Previous morphological studies and phylogenetic investigations based on SSU, ITS-LSU RPB2 and TEF genes data have provided insights into the taxonomy and classification of Valsaria and its allies [1]. The most recent taxonomic work dealt with more than 100 collections of Valsaria sensu lato, including two new species, which has helped to resolve generic delimitation, circumscription and classification of Valsaria [1]. In this study, a new species with a unique morphological character is illustrated and its relationships with other species are investigated based on multigene phylogenetic analyses. Our molecular data analyzed herein generated phylogenies that are congruent with those of Jaklitsch et al. [1] and helps to resolve intergeneric relationships between Bambusaria, Myrmaecium and Valsaria. Three well supported monophyletic groups are recovered which support Bambusaria, Myrmaecium and Valsaria as phylogenetically distinct genera (Fig 2). Nevertheless, we observe that several morphological characters such as ostiole, apical ring, and pseudoparaphyses are interspersed in several clades which could be examples of homoplasy in morphological characters within the Valsariaceae. Our molecular phylogeny provides an overview of the usefulness of specific morphs for species and generic delineation.
A similar phylogenetic scenario is reported herein. The three clades corresponding to different genera can be easily distinguished based on spore ornamentation. Valsaria is characterized by fine warted to reticulate or finely tuberculate ascospores surface, whereas Bambusaria and Myrmaecium species possess densely reticulate and longitudinally ribbed ornamentation respectively. Even at the species level, ornamentation can be used to a certain extent to segregate species. For example, V. insitiva the type species, can easily be delineated from other Valsaria species herein, except V.neotropica, based on finely tuberculate spore ornamentation. Valsaria rudis and Valsaria lopadostomoides are similar in having ascospores surface 'warted to reticulate' but additionally the ascospores of V. lopadostomoides is described as 'spotted' [1]. In addition, M. fulvopruinatum, characterized by densely reticulate spores can be distinguished from M. rubrum and M. rubricosum, whose spore surface has labyrinth ornamentation. We can therefore rely mostly on such morphology to circumscribe species at this taxonomic level. However, relationships based on other morphs, such as stromatal characteristics, presence or absence of ostioles/ pseudoparaphyses/apical ring, septum constriction and number of guttules show little congruence with findings from molecular data and are not phylogenetically significant for intergeneric segregation. Nevertheless some of these characters, especially the latter two, if used judiciously can be used to circumscribe species. For instance, V. lopadostomoides and M. fulvopruinatum can be distinguished from other Valsaria and Myrmaecium species on the basis of number of guttules. The number of guttules per spore has also been considered as a diagnostic feature to delineate taxa at the intraspecific level. For example, guttule character was found as important to separate species and varieties recognized in the genus Phoma [52] and helped to provide a key to differentiate among thirty taxa. Recently, Southworth et al. [53] demonstrated that number of guttules in each spore has been useful to delineate species of Balsamia when other characters overlap. Vander [54] reported that the occurrence of large guttules was useful to distinguish species of Phyllosticta, especially when used in combination with other characters. Presence of guttule is especially useful when confirming diagnosis of herbarium material from fresh specimen and this has helped to validate Lophiostoma diminuens (Pers.) Fuckel [55]. The diagnostic microscopical characters supporting ranks of different taxa of the family Sarcoscyphaceae were mainly based upon differences in the guttule (lipid) number and pattern of living ascospores [41]. To differentiate genera within Gnomoniaceae, Monod [56] used characters such as presence or absence of small guttules. The latter has also been considered as a diagnostic feature to separate Diaporthe species from grapevine [57] as well as freshwater fungal species [58,59]. Guttule characters can also be used to distinguish our new taxon, V. ostryae from others. Phylogeny herein indicates V. ostryae to be more closely related to V. lopadostomoides and V. rudis than any other species. Although these three species share several common morphological features (pseudostromatic stromata, apically free pseudoparaphyses, apical ring and dark brown ascospores), our new taxa is clearly distinct in number of guttules and surface ornamentation.
To further substantiate establishment of Valsaria ostryae as a new species, the complete ITS and RPB2 gene regions were compared. Nucleotides differences reveal clear cut differences that warrant new species status. For example, V. ostryae differs from V. lopadostomoides by 5.31% bp difference in the ITS gene regions. Considering both molecular and morphological data we regard V. ostryae as a new taxon that warrants species rank. The genus Valsaria is known to comprise taxa with minor morphological variations that result in taxonomic confusion. For example, the ascomata of Valsaria insitiva, V. lopadostomoides and V.neotropica are monostichously arranged in valsoid configuration measuring 250-450 μm high, 180-400 μm diam, peridium 14-25 μm thick, cylindrical asci with a short cylindrical to barrel-shaped ring measuring in between 96-112 μm × 11-18.5 μm and 2-celled, ellipsoid dark brown ascospores with the size ranging from 12-19 μm × 6.5-11.7 μm. However, when analyzing the gene regions, they are all genetically different. ITS base pair difference between Valsaria insitiva to V. lopadostomoides is 2.4%, V insitiva to V. neotropica is 11.8%, and V. lopadostomoides to V. neotropica is 14.3%. Likewise, other Valsaria species such as V. robiniae, V. rudis, V. spartii and our new taxon share similar morphological characters which are not really meaningful in species delineation.
This study provides delightful taxonomic insights across Valsaria species. We anticipate that taxonomists will pay particular attention to these characters that can potentially aid in species identification of other genera. Our study also reveals that Valsaria could be a speciose genus and there is a need for more collections targeting a wide variety of hosts which can help to answer pertinent questions with regards to fungal diversity issues.