A new species of Stamnaria (Leotiomycetes, Helotiales) from Western Siberia

Abstract A new species of Stamnaria is described based on morphology and molecular data from a collection made in West Siberia. Stamnaria yugrana is differentiated by lanceolate, strongly protruding paraphyses and comparatively narrow, fusoid-clavate ascospores. The apothecia are urn-shaped due to a prominent and even collar as in S. persoonii. The species grows on fallen side branches of Equisetum sylvaticum, a rarely recorded host for Stamnaria. The authors formally describe the new species and provide colour illustrations. In addition, the literature is reviewed on previously described species of Stamnaria. Phylogenetic reconstruction of the Stamnaria lineage, based on the ITS ribosomal DNA, strongly supports the three currently recognised species: S. americana, S. persoonii and S. yugrana.


Introduction
During ongoing studies of the Helotiales (see , material of a species of Stamnaria Fuckel, which had been collected and morphologically documented by one of the authors (NVF), was investigated by molecular methods. Collections were made during surveys in the vicinities of Shapsha field station of Yugra State University in Western Siberia, Russia. After the first collection of the species in June 2008, the same locality was visited in June 2012, February 2014 and May 2015. During all these visits, abundant apothecia were observed. The species was always found growing on Equisetum sylvaticum. This plant species had rarely been mentioned before as a host for the genus Stamnaria (Sydow 1898: 432, S. equiseti;Jaap 1922: 15, S. persoonii). Moreover, the microscopic features of the collections differed markedly from all earlier taxa described in the genus Stamnaria. Therefore, it was decided to formally describe this fungus as a new species based on morphological and molecular characteristics.
Members of the genus Stamnaria (Ascomycota, Leotiomycetes, Helotiales) share the following characteristics: 1) the presence of a thick hyaline gelatinised layer of textura oblita outside the ectal excipulum of textura prismatica, 2) cells of ectal excipulum and paraphyses containing yellow-orange carotenoids, 3) apothecia erumpent through epidermis, 4) all species growing on members of Equisetum and 5) with asexual state in the genus Titaeospora Bubák (von Höhnel 1928, Gruber 2006, Baral in Jaklitsch et al. 2016). According to Johnston et al. (2014), the older name Stamnaria is recommended to be used for the holomorph, instead of Titaeospora. Based on the currently available sequence data, the genus forms its own clade within the Helotiales . Its isolated position was emphasised by Baral in Jaklitsch et al. (2016) (Gruber 2006). Stamnaria hyalopus occurs on decaying leaves of Carex vesicaria and S. thujae on Thuja occidentalis. Stamnaria pusio grows on rotting debris in the soil and was placed in tentative synonymy with Sarcoscypha occidentalis (Schwein.) Sacc. by Harrington (1990). Stamnaria herjedalensis appears to be a synonym of Roseodiscus equisetinus (Velen.) Baral (O. Eriksson pers. comm.). Stamnaria equiseti is considered a synonym of S. persoonii by accepting the older name equiseti (Saccardo 1889) but S. equiseti is of uncertain identity whereas that of S. persoonii could be clarified based on the lectotype (Gruber 2006). Taken together, only two species are currently accepted in the genus Stamnaria: S. americana and S. persoonii.

Study site ecology
The study site is located in the middle taiga sub-zone of Western Siberia, in Russia. The area is characterised by a subarctic climate with average yearly temperatures of -1.1 °C, ranging from averages of -20 °C in January to 18 °C in July. The total annual precipitation is 553 mm. The period without snow cover usually lasts from May until October (Bulatov et al. 2007).
The collections were made in a mixed coniferous-deciduous forest close to a stream and a forest path 1.2 km SSE of Shapsha village, 61.07929"N, 69.46925"E. The tree canopy was dominated by Pinus sibirica, Picea obovata and Abies sibirica with admixture of Betula pubescens, Populus tremula, Sorbus sibirica and Salix spp. The plant layer was made up of Equisetum sylvaticum, Rubus arcticus, Milium effusum and Luzula pilosa.
Several years ago, a fire took place in this forest, resulting in the dominance of E. sylvaticum. Ground fires generally result in completely new successional trajectories and one dominant component of early post-fire vegetation communities is E. sylvaticum. Its rhizomes are buried deep in the soil and thus are resistant to fire (Viereck 1983). Interestingly, the new Stamnaria species was only collected from these post-fire mass populations of E. sylvaticum. Elsewhere in coniferous forests in Khanty-Mansi Autonomous Okrug -Yugra, E. sylvaticum is common but occurs in sparse densities and no growth of Stamnaria has been observed.

Morphological studies
The species was discovered after examining the litter of the host plant in situ by the naked eye. Time of collection was always at the beginning of summer (about three weeks after snow melt). The substrate (Equisetum sylvaticum side branches) extracted from under the snow in February 2014 also gave abundant fruiting after three weeks of incubation in a moist chamber.
The litter was collected and brought to the laboratory where it was studied and documented the same day. Hereafter, the material (side branches with attached fruiting bodies) was dried at room temperature and stored as dry collection at Yugra State University Biological Collection (YSU). Voucher specimens are also preserved at Farlow Herbarium of Harvard University, Cambridge, Massachusetts (FH) and at V.L. Komarov Botanical Institute, Saint-Petersburg (LE).
The morphological features of the species were studied using a Zeiss Stemi 2000-C stereomicroscope, with magnification from 6 to 50× and a Zeiss Axiostar transmitted light microscope (with Achromat 10/0.25, 40/0.65 dry and 100/1.25 oil immersion objectives). Microstructures were studied and measured from living material in tap water and later compared to dead material from dried specimens. The iodine reaction was tested with Lugol's solution and Congo Red in water was used to stain the sections and the structure of the excipulum.
Macro-and micro-photographs were obtained using a Canon EOS 50D digital camera and Axiocam ERc 5s digital camera. Abbreviations used: * = living state, † = dead state, CR = Congo Red, VB = vacuolar body.

DNA extraction, PCR and sequencing
DNA was extracted from dry apothecia with the help of the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich, St Louis, MO), DNeasy Plant Mini Kit (Qiagen, Valencia, CA) and the QIAamp DNA Micro Kit (Qiagen). Per extraction, 1 to 4 apothecia were used. Apothecia were crushed in 1.5 mL tubes using a 1.5 mL pellet pestle (Kimble, Rockwood, TN, #749521-1500) or cut in half using a sterile no. 10 surgical blade on a disposable Bard-Parker handle (Aspen Surgical, Caledonia, MI). Undiluted DNA was used for PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The ITS was amplified using the forward primer ITS1f (5'-CTTGGTCATTTAGAGGAAGTAA-3') in combination with either ITS4 (5'-TCCTCCGCTTATTGATATGC-3') or the Ascomycota-specific primer ITS4A (5'-CGCCGTTACTGGGGCAATCCCTG-3') (White et al. 1990, Gardes and Bruns 1993, Larena et al. 1999). All PCR reactions were done in a Mastercycler ep gradient thermocycler (Eppendorf, Hauppauge, NY) using Sigma-Aldrich's REDTaq DNA Polymerase enzyme. PCR conditions were as follows: an initial denaturation step at 94 °C for 3 min, then 35 cycles of 94 °C for 1 min, 50 °C for 45 s and 72 °C for 90 s and a final extension step at 72 °C for 10 min (ITS).
Products with clear bands on agarose gel were cleaned with the Qiaquick PCR Purification Kit (Qiagen) and subsequently sequenced with the same primers (3 μl of purified PCR product per 10 μl sequencing reaction). Sequencing reactions were performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA). Sequences were trimmed, edited and assembled in Sequencher v. 4.10.1. All generated sequences have been deposited in GenBank (Table 1). BLAST searches for similar sequences were undertaken at: http://ncbi.nlm.nih.gov/blast/Blast.cgi.

Sequence alignment, nucleotide divergence and phylogenetic analyses
An ITS dataset was constructed to investigate the phylogenetic structure within the genus Stamnaria. Sequences were aligned using Muscle v3.7 (Edgar 2004), available on the Cipres Science Gateway version 3.3 (Miller et al. 2010). Pairwise evolutionary distances, the amounts of genetic variation between two species, were calculated using Paup on XSEDE, also on the Cipres Science Gateway. The p-distance is calculated as the proportion of sites in a pairwise alignment at which the compared sequences are different. This is the number of nucleotide differences divided by the number of nucleotides compared. In addition, Jukes-Cantor (JC69) and Kimura 2-parameter (K2P) distance metrics (Jukes andCantor 1969, Kimura 1980) were calculated. Neighbour joining (NJ) analyses (Saitou and Nei 1987) were performed to cluster taxa based on the respective distance matrices. The three resulting NJ trees were statistically compared with the SH test (Shimodaira and Hasegawa 1999).
Distribution. Known only from the type locality.
(2) and Sarcoleotia S. Ito & S. Imai (1) served as outgroup taxa (Geoglossomycetes, Geoglossales, sensu Schoch et al. 2006). This dataset includes the three currently recognised species in the genus Stamnaria. Intra-specific divergence in the ITS region ranges from 0.0 to 0.9 % while interspecific divergences range between 8.0 and 13.7 % (p-distances) or between 8.4-8.5 and 15.1 % (JC69, K2P). The p-distances are 8.0 % between S. americana and S. persoonii, 10.6 % between S. persoonii and S. yugrana and 13.7 % between S. americana and S. yugrana. JC69 and K2P distances are almost identical, higher but equivalent to the p-distances ( Table 2). The NJ phenogram constructed from the Jukes-Cantor distance matrix has a higher likelihood score (-lnL = 791.63111) compared to the analyses based on p-distances and Kimura 2-parameter distances, but the differences are insignificant. The genus Stamnaria is retrieved as a monophyletic clade in all three phylogenetic reconstructions (MP BS = 74, ML BS = 98, pp = 1.0). All morphologically delineated species of Stamnaria have maximum support. The position of the new species within the genus is unresolved. Under MP and ML inference, S. yugrana is sister to S. persoonii but this sister relationship is only supported by ML BS = 81. In the Bayesian analysis, on the other hand, S. yugrana is sister to (S. americana, S. persoonii), with moderate support for the sister relationship between the two latter species (pp = 0.8).

Discussion
The morphological and ecological features of S. yugrana (Table 3) are considerably different compared to the two previously recognised species, the most pronounced features being paraphyses of two types, including strongly protruding, broadly lanceolate ones and heteropolar, comparatively narrow ascospores. No previous reports of a Stamnaria species with ascertained identity are known from E. sylvaticum, when applying the revised species concept as reviewed in the introduction. Jaap (1922) presented a report of S. persoonii on E. sylvaticum but did not provide a morphological description. It is impossible to assess whether this report represents S. persoonii or S. yugrana because the collection does not seem to have been preserved (no voucher number cited).
Stamnaria yugrana differs from S. americana by solitary, never fasciculate, distinctly stalked apothecia, presence of a pronounced raised collar with free-ending hyphae at its inner excipulum, presence of two types of paraphyses (cylindrical and lanceolate) and shorter and especially narrower, fusoid-clavate ascospores ( Table 3). The ecology of these two species is also quite different: the apothecia of S. americana occur on living stems of Equisetum hyemale, while those of S. yugrana are found on dead fallen side branches of E. sylvaticum. Both S. yugrana and S. persoonii have stipitate apothecia with a distinct collar. However, S. yugrana differs by the presence of paraphyses of two types and much narrower, heteropolar spores with higher length/width ratio (Table 3). Apothecia of S. persoonii also grow on dead stems, but on another host plant, E. fluviatile (Gruber 2006).
Many Stamnaria collections have been misidentified and/or reported under misidentified host plants. Currently, the Stamnaria lineage has no taxonomic assignment at the family level. In their ITS+LSU rDNA phylogeny,  retrieved it as a sister clade to Erysiphales, but without support for this sister relationship.  found a sister relationship of Stamnaria and Roseodiscus, although only moderately supported by Bayesian analysis (pp = 0.83). Hosoya et al. (2013) suggested a transfer of the genus Stamnaria from Helotiaceae to the small family Leotiaceae. This was based on the strongly supported sister relationship with Leotia lubrica (Scop.) Pers. obtained in the maximum parsimony multigene analysis using rDNA, EF1 and RPB genes, with bootstrap values of 100 from neighbour-joining and 99 from maximum parsimony. Morphologically, Hosoya et al. (2013) mentioned "a gelatinized layer in the external part of apothecia" as a synapomorphic character and suggested to include Stamnaria in Leotiaceae in a restricted sense, which traces back to a concept introduced by Korf & Lizoň (2001). However, recent molecular research indicated that the family Leotiaceae includes solely soil-inhabiting species with large ascomata with convex, pileate or clavate fertile part. Amongst the genera in this family, Leotia Pers. is the only one showing a gelatinised external layer outside the ectal excipulum (Baral in Jaklitsch et al. 2016). In addition, the ascospores of Leotiaceae consistently contain large oil drops (Baral in Jaklitsch et al. 2016), unlike Stamnaria. Detailed morphological studies in the genus Stamnaria by Gruber (2006) revealed several other species, including collections that had been misidentified as S. americana or S. persoonii (sensu Magnes and Hafellner 1991, Brunelli 1992, Künkele et al. 2005).
The author proposed several new names for species that he considered undescribed, but they have not been validly published. Formal descriptions of these new species along with molecular phylogenetic analyses are planned in the near future. It is clear that the genus Stamnaria is in need of thorough revision. The discovery and detailed description of S. yugrana will help in the further delimitation of the genus.