A new species of Balansia (Clavicipitaceae) associated with a cyperaceous plant in Brazil

Fungal species belonging to the genus Balansia (Clavicipitaceae) are well known as endophytic and epibiotic species commonly found on grasses or sedges. Among the 36 species of Balansia described worldwide, ten have been reported in Brazil. While most species of balansoid fungi were described on graminaceous plants, only four were characterized on cyperaceous hosts. To correctly identify the species of balansoid fungi associated with Scleria bracteata (Cyperaceae), specimens were collected in the state of Alagoas, Brazil, in 2014 and 2016. Nucleotide partial sequences of the second-largest subunit of RNA polymerase II (RPB2), translation elongation factor 1-α (TEF1), 18S subunit ribosomal DNA (SSU), 28S subunit ribosomal DNA (LSU), and internal transcribed spacers (ITS) were obtained from each balansoid specimen. Based on morphology and molecular data, the specimens were identied as a putative new species of Balansia, herein referred to as Balansia scleriae sp. nov.


Introduction
Balansia Speg. (Clavicipitaceae) includes both endophytic and epibiotic species commonly found on grasses or sedges. Balansia species are, in general, characterized by capitate ascostromata that follow a well-de ned ephelidial-stage (=Ephelis) in some instances, and sometimes arising within it (Diehl 1950). The genus was introduced by Spegazzini, with Balansia claviceps as the type species, based on a fungus developing on in orescences of a graminaceous host showing a close a nity with Claviceps Tul., but distinguished from the latter for its conidial stage (Spegazzini 1885). Later Diehl (1950) monographed Balansia and related genera within the tribe Balansiae, and emended Spegazzini's concept of the genus, expanding the circumscription to include characteristics of the ephelidial-stage, an essential taxonomic trait to distinguish species.
Morphological characteristics such as stromata and ascomata morphology, symptomatology, features of asci, ascospores, together with conidial characteristics, geographic distribution, and the host a nities are traditionally used to differentiate Balansia species. Currently, species demarcation into this group is based on additional morphological characteristics combined with sequence and phylogenetic data (White et al. 2003). However, most nucleotide sequence information available in public databases such as GenBank-NCBI were obtained from species associated with plant hosts in the Poaceae botanical family (Kuldau et al. 1997, Reddy et al. 1998, 2000, Sung et al. 2007a, 2007b. Here, balansoid specimens infecting Scleria bracteata (Cyperaceae) in the state of Alagoas, Brazil, were morphologically and molecularly characterized as a novel Balansia species, herein referred to as Balansia scleriae sp. nov.

Sample collection and morphological characterization
Isolates of balansoid fungi associated with Scleria bracteata (Cyperaceae) were collected in the city of Maceio, Alagoas State, Brazil, in 2014 and 2016, and were deposited in the Mycological Collection of Herbarium Universidade de Brasília (UB), Brasília, Federal District, Brazil. To morphologically characterize these specimens, the fungal structures were initially observed using a Leica 205C model stereomicroscope. Representative materials were sectioned using a Leica CM 1850 freezing microtome, yielding 20-30 µm thick sections that were placed on slides containing colorless lactoglycerol and visualized on a Leica DM 2500 microscope coupled to a Leica DFC 490 digital camera using Nomarski interference microscopy. Size estimations for the structural components were based on at least 30 measurements when possible. Comparisons with type descriptions and illustrations were carried out using the available literature.
The PCR thermocycling conditions were: initial DNA denaturation at 94 o C for 1 min 30 s; 35 cycles of DNA denaturation at 94 o C for 30 s, primer annealing at 53 o C for 30 s, and extension at 72 o C for 45 s, and a nal extension step at 72 o C for 5 min. The PCR products were analyzed on 1% agarose gel and puri ed using ExoSAP-IT® PCR Product Cleanup (Affymetrix Inc.). Then, the amplicons were directly Sanger sequenced at Macrogen Inc. (Seoul, South Korea; http://www.macrogen.com). The electropherograms were manually/visually evaluated, and ambiguous positions were clari ed considering forward and reverse sequences. The contigs were individually assembled and annotated using GENEIOUS 9.0.5 (Kearse et al. 2012) and deposited in GenBank (http://www.ncbi.nlm.nih.gov).

Taxa sampling and alignment
The phylogenetic relationship of the specimens reported here and other species into the genus was assessed using the ITS sequence data (Table 1) because it was the genomic regions from which more sequences were available. The nucleotide sequences were aligned using the E-INS-i method (in MAFFT 7.305; Katoh and Standley 2013) and manually adjusted in AliView (Larsson 2014). The ITS matrix was partitioned as ITS1-5.8S-ITS2, and Claviceps purpurea was selected as the outgroup. The sequences described in the present study, and sequences retrieved from GenBank, are shown in Table 1. The alignment was deposited in TreeBASE (www.treebase.org).

Phylogenetic analyses
Maximum likelihood (ML) analysis was performed using RAxML 8.2.9 (Stamatakis 2014), starting with a randomized, stepwise addition parsimony tree under a GTR+G model. The branch support values were calculated using 1000 bootstrapping (BS) and replicated under the same model. Bayesian Inference (BI) was carried out using MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003), following 4x4 mode of the general time reversible (GTR) model for all partitions. Two independent chains were run, each one initiating from random trees and four simultaneous independent chains at 10 6 generations, with trees being sampled at every 10 3 generations. Four rate categories were used to approximate the gamma distribution. Average standard deviations of split frequencies (ASDSF) were used as a chain convergence criterion. Twenty-ve percent of all sampled trees were discarded as burn-in, and the remaining 75% employed to estimate the Bayesian posterior probabilities (BPPs) for branches. Both MrBayes and RAxML were ran through the CIPRES Science Gateway 3.1 web portal (Miller et al. 2010).

Results
The PCR ampli cation and sequencing of the regions SSU, LSU, ITS, TEF1-α, and RPB2 (GenBank Accession Nos. MK256221-MK256226, and MK249873-MK249875) yielded sequences of 990, 850, 400, 910, and 880 bp in length, respectively. The ITS nucleotide matrix contained 695 aligned sites, including gaps, with 268 variable sites of which 189 were parsimony informative (70% of variable sites). For each partition (ITS1, 5.8S, and ITS2) different evolutionary models were selected ( Table 2). The nucleotide matrix and phylogenetic trees reconstructed in this study are available in TreeBASE (study number S23703). Although the SSU, LSU, TEF1-α, and RPB2 sequences were not used in the phylogenetic analyses, they were deposited in GenBank for future studies and identi cation purposes.

Phylogenetic analyses
Balansia formed a monophyletic group in the BI reconstruction (0.95 Bayesian posterior probabilities -BPP). Both ML and BI analyses of the genus Balansia con rmed the genetic differences of the balansoid specimens on S. bracteata collected in Brazil from previously known Balansia species. Also, the new isolates were placed apart from B. cyperi, the only Balansia species that has been reported infecting cyperaceous hosts (Figure 1).

Taxonomy
Morphological comparisons together with phylogenetic analyses using sequence data from nuclear ITS rDNA con rmed that the isolates described here are different from previously reported Balansia species, and therefore are classi ed as belonging to a new species, herein referred to as Balansia scleriae sp. nov.
Balansia scleriae Guterres DC, Ramos-Sobrinho R, Pinho, Assunção IP, and Lima GSA, sp. nov. Fig. 2 Differs from Balansia spp. by having smaller asci and ascospores, and a thicker ascoma wall. Additionally, stroma of B. scleriae completely encloses the culms of the host at the internodes and is restricted to this tissue.

MycobanK: MB 829055
Etymology: referring to plant genus on which the fungus was found.

Discussion
Balansia spp. are usually described as host speci c or having a narrow host range. This feature still plays a major role in the taxonomy of the genus including species de nition. Four species of Balansia have been previously described from Cyperaceae hosts, Balansia carecis Hosag. on Carex licina in India (Hosagoudar 1994 (Diehl 1950;Al eri Jr. et al. 1984;Farr and Rossman 2021), and on Scleria chinensis (Zhuang 2001), and on an unidenti ed species of Carex (Teng 1996) both in China, B. cyperi Edgerton on Cyperus rotundus, on in orescences of Cy. virens and Cyperus sp. in the USA (Edgerton 1919;Diehl 1950;Clay 1986;Leuchtmann & Clay 1988;Farr and Rossman 2021), and B. borealis Tranzchel found on fruits of Carex sparsi ora in the former Soviet Union. Balansia carecis, B. borealis and B. cyperi are found only in in orescences and spikelet of the hosts (Table 3).
Phylogenetically related species, B. claviceps and B. granulosa are found on Panicum sp., Setaria palmicola, and on an indeterminate graminaceous host. Balansia claviceps has stipitate stroma, arising from the hypothallus, ascoma wall narrower than B. scleriae sp. nov, and a known conidial stage. Balansia granulosa, rst described as Dothichlöe granulosa, is not provided with a de nite ascoma wall, has narrower perithecia, and longer ascospores.
Balansia species infecting culms and leaves are not rare in hosts of the family Poaceae, but they usually form a scythe shape stroma linked to the host tissue only at the base, which sometimes partly surrounds the culm or petioles. On the other hand, on sedges, it occurs less often, been only recorded for B. cyperacearum, which type on an unidenti ed species of Cyperaceae from Surinam is unique in infecting abaxial leaf surfaces, whereas all the other specimens occur on culms and leaf bases (Berkeley and Curtis 1853). Based on these differences, Diehl (1950) suggested that B. cyperacearum could be an assembly of two completely different species, an idea reinforced by White et al. (1997). The stromata of B. scleriae, described here, slightly resemble a specimen collected by Chardón in Venezuela in 1939 and treated by Diehl (1950) as B. cyperacearum.
Perithecial stroma morphology was also traditionally reinforced as a stable characteristic of taxonomic value. Diehl (1950) accommodated Balansia species within the subgenera Eubalansia and Dothichloë, the former characterized by the possession of attened ascomatal stromata that develop on leaves or culms of grasses, while the latter included species with pulvinate or stipitate ascomatal stromata. Among the species infecting sedges, B. cyperi shows spherical to subspherical perithecial stromata, and B. cyperacearum has effuse and attened stromata. Balansia scleriae seems to be an intermediate between those two species, with perithecia immersed in coalescent cushion-like stromata with punctiform ostioles (Figure 2 B-C).
Balansia species on Cyperaceae usually cause dwarfness, witch's broom, or alter the bloom of the host. Balansia cyperi causes sterility, dwarfness and foliage deformation, characterized by terminal bracts swollen, while B. cyperacearum causes sterility. Plants infected by B. carecis show stunted growth reduced to half of their normal size and a characteristic incense candle-like in orescence due to the colonization by the fungus and abundant production of stroma (Hosagoudar 1994). Balansia borealis is known only for its type and its infection is restricted to fruits (Elenkin 1914). Although the stromatic development of B. scleriae sp. nov. along the internodes of S. bracteata, no apparent physiological symptoms were observed on this host.
The specimens of Balansia infecting S. bracteata were morphologically and genetically different from the four Balansia species known on Cyperaceae (Table 3). When compared to the two closest species, Balansia cyperi has larger ascospores and conidial stage, while B. cyperaceaerum is distinguished from the new species by smaller asci and ascoma wall. Further, Balansia scleriae can be morphologically differentiated from all currently recognized Balansia species by having very thick stroma, which completely encloses the culms of the host at the internodes and is restricted to this tissue.

Data availability
The datasets generated for this study can be found in Genbank: MK249873-MK249875 and MK256221-MK256226; Treebase: S23703.The results obtained in this study are included in the contents of this report.