Host specialization and molecular evidence support a distinct species of smut fungus, Anthracoidea hallerianae (Anthracoideaceae), on Carex halleriana (Cyperaceae)

Abstract: The species of Anthracoidea on Carex are host-specific smut fungi restricted to sedges belonging to the same or closely related sections. They are characterized by sori that form black, hard bodies around aborted nuts of their hosts. In Carex sect. Hallerianae, only one species, C. halleriana, is known as a host of Anthracoidea. The taxonomic status of this smut fungus was problematic due to a lack of molecular data. It has been reported under different names, mainly as “A. caricis” or “A. irregularis”. A comparative morphological study and molecular phylogenetic analysis, using LSU (large subunit) nuclear rDNA sequences, supported the recognition of a distinct species, A. hallerianae. The new species is described and illustrated based on material from Central Europe, the Iberian Peninsula, the Balkan Peninsula, the Mediterranean area and Transcaucasia. Citation: Denchev T. T., Denchev C. M., Koopman J., Begerow D. & Kemler M. 2021: Host specialization and molecular evidence support a distinct species of smut fungus, Anthracoidea hallerianae (Anthracoideaceae), on Carex halleriana (Cyperaceae). – Willdenowia 51: 57–67. Version of record first published online on 9 March 2021 ahead of inclusion in April 2021 issue.


Introduction
The smut fungi in the genus Anthracoidea Bref. are characterized by sori that form globose to broadly ellipsoidal or ovoid, black, hard bodies around aborted nuts of cyperaceous plants. In Carex L. (Cyperaceae), the sori are scattered in female spikes or in female flowers of mixed spikes, depending on the Carex species. The sori are covered initially by a thin peridium, which later ruptures to expose the spore mass. This spore mass is firmly agglutinated at first, at maturity becoming powdery on the surface of the sorus. A few species of Anthracoi-dea have mature sori with an agglutinated spore mass, which cracks into small, irregular pieces. The spores are formed singly and are usually flattened. Mature spores are liberated and dispersed by the wind after the peridium ruptures. At an early stage of host flowering, spores germinate to produce basidiospores that may infect flowers. The infection is local and confined to individual flowers (Kukkonen 1963;Vánky 1979Vánky , 2013Denchev & al. 2013;Denchev & Denchev 2016).

DNA extraction, PCR amplification, and sequencing
-For DNA extraction, one sorus per infected Carex halleriana was removed. The samples were milled in the Fastprep-24 TM Sample Preparation Instrument (MP Biomedicals), using two steel beads. Genomic DNA was isolated using the my-Budget Plant DNA Kit TM (Bio-Budget Technologies GmbH, Germany), according to the manufacturer's protocol (protocol 1: "Isolation of DNA from plant material using lysis buffer SLS"). PCR using GoTaq TM Master Mix (Promega, U.S.A.) with the primer combination LR0R/LR6 (Vilgalys & Hester 1990;Moncalvo & al. 1995) was performed to amplify the LSU (large subunit) of nuclear rDNA, which is the standard molecular marker for Anthracoidea (e.g. Hendrichs & al. 2005;Piątek & al. 2015). Standard thermal cycling conditions with an annealing temperature of 52° C were used for amplification. Five μl of PCR products were purified using ExoSAP (1 : 5 diluted in ddH2O; New England Biolabs, Sequences were deposited in the NCBI nucleotide database (see Table 1 for accession numbers).
Phylogenetic analysis -The newly generated Anthracoidea sequences and representative sequences downloaded from GenBank (Table 1) were aligned using the e-ins-i option in MAFFT v7.450 (Katoh & Standley 2013). Ambiguous alignment regions were removed using GBLOCKs (Castresana 2000) implemented in Sea-View (Gouy & al. 2010 Morphological examination -Dried specimens from B, BUCM, GZU, K, L, MA, P, SOMF and W (herbarium codes according to Thiers 2020+) were examined under light microscope (LM) and scanning electron microscope (SEM). For LM observations and measurements, spores were mounted in lactoglycerol solution (w : la : gl = 1 : 1 : 2) on glass slides, gently heated to boiling point to rehydrate the spores and then cooled. The measurements of spores are given as min -max (extreme values) (mean ± 1 standard deviation). The total number of spores (n) from all collections (x) measured are given in the form "(n/x)". The spore length range is assigned to one of the groups distinguished by Denchev & al. (2020: 11): very small-sized, small-sized, medium-sized and large-sized. For SEM, spores were attached to specimen holders by double-sided adhesive tape and coated with gold in an ion sputter. The surface structure of spores was observed and photographed at 10 kV accelerating voltage using a ZEISS SIGMA VP scanning electron microscope. The description of spore ornamentation is in accordance with Denchev & al. (2013). The description below is based on the specimens examined. The shapes of spores are arranged in descending order of frequency.
Morphology -The Anthracoidea species have very few diagnostic morphological characteristics. The morphology of the sori bears no diagnostic value, with the exception of very few species (e.g. A. intercedens Nannf., A. pseudofoetidae L. Guo and A. subinclusa (Körn.) Bref.; Denchev & Minter 2011;Vánky 2011;Denchev & al. 2020). The most important characteristics are spore-based: sizes, shape (in plane view, since most species have flattened spores), wall thickness and wall ornamentation (pattern and height). Characteristics of less taxonomic significance include internal swellings, lightrefractive areas, and protuberances. Their presence and frequency may vary between different collections of one species, but due to the scarcity of morphological characteristics, their careful examination and use in combination with the diagnostic morphological features is still very important. The morphological description of the studied smut fungus on Carex halleriana was based on the examination of 21 specimens from Central Europe, the Iberian Peninsula, the Balkan Peninsula, the Mediterranean area and Transcaucasia. The specimens were characterized by irregularly rounded to angular or elongated to irregularly elongated spores (as seen in plane view), with an unevenly thickened, 1 -3.3(-3.7) μm thick spore wall, that was minutely to moderately verruculose (warts up to 0.4(-0.5) μm high). The mean values of the spore length and width of the examined specimens fell into a range of 20.8 -22.5 μm and 15.5 -18.5 μm, respectively (Table 2). Spores longer than 26 μm were usually with elongated or irregularly elongated shape. As an exception, single spores with a length up to 31 μm were observed. The spores often had light-refractive areas and 1(-3) protuberances and sometimes had 1 or 2(-4) internal swellings.

Taxonomy
Based on the host specialization and molecular data, we propose a new species of Anthracoidea on Carex halleriana.
Diagnosis -Differs from other Anthracoidea species by specialization on sedges in Carex sect. Hallerianae.
Etymology -The epithet is derived from the host plant, Carex halleriana.
Remarks -The examined specimens share the same morphology, with only small variations. The Austrian specimen (GZU 222890) has spores with more regular shape and lower ornamentation, but the other characteristics match well with the morphology of the new species. Some specimens (like SOMF 30199; R. Böcker s.n., B; P00283665) possess spore walls with common and wellvisible internal swellings, while most of the studied specimens have spore walls with uncommon and inconspicuous internal swellings. When numerous specimens of a particular species are examined, it may turn out that the presence and conspicuousness of internal swellings can vary considerably (cf. the case of Anthracoidea eburneae Denchev & Denchev 2016: 77). Internal swellings are more visible in the lightly coloured immature spores.
It was found that spores longer than 26 μm usually had an elongated or irregularly elongated shape (Fig. 3F). As an exception, single spores with length up to 31 μm were also observed.
Carex halleriana is distributed from C and S Europe, the Mediterranean area and Crimea to the Caucasus and W Asia (to Afghanistan) (Kukkonen 1987(Kukkonen , 1998Egorova 1999;Luceño 2008). It is a lowland to montane species, usually occurring on dry mountain slopes or in dry broad-leaved mountain forests, usually on limestone (Egorova 1999;Luceño 2008). Based on the available distribution data, it can be assumed that Anthracoidea hallerianae is coextensive with its host.