The first smut fungus, Thecaphoraanthemidis sp. nov. (Glomosporiaceae), described from Anthemis (Asteraceae)

Abstract There are 63 known species of Thecaphora (Glomosporiaceae, Ustilaginomycotina), a third of which occur on Asteraceae. These smut fungi produce yellowish-brown to reddish-brown masses of spore balls in specific, mostly regenerative, plant organs. A species of Thecaphora was collected in the flower heads of Anthemischia (Anthemideae, Asteraceae) on Rhodes Island, Greece, in 2015 and 2017, which represents the first smut record of a smut fungus on a host plant species in this tribe. Based on its distinctive morphology, host species and genetic divergence, this species is described as Thecaphoraanthemidissp. nov. Molecular barcodes of the ITS region are provided for this and several other species of Thecaphora. A phylogenetic and morphological comparison to closely related species showed that Th.anthemidis differed from other species of Thecaphora. Thecaphoraanthemidis produced loose spore balls in the flower heads and peduncles of Anthemischia unlike other flower-infecting species.


Introduction
Thecaphora species belong to the Glomosporiaceae (Urocystidales, Ustilaginomycotina). The type species is Th. seminis-convolvuli described from Convolvulus arvensis (Convolvulaceae) collected in France (Desmazièrs 1827). Until now, 63 species of Thecaphora have been recognised (Vánky 2012), infecting host plant species in 16 different eudicot families (Vánky and Lutz 2007, Roets et al. 2008, Vánky et al. 2008, Vánky 2012. Species of Thecaphora produce sori in flowers, fruits, seeds, stems, leaves or roots, often in galls or pustules. The granular to powdery spore balls are yellowishbrown to reddish-brown, but never black. The majority of Thecaphora species produce loose or permanent spore balls without sterile cells. An exception to this is Th. smallanthi, which was reported to have large spore balls with outer spores and an internal layer of hyaline (sterile) cells (Piepenbring 2001). Three species have single spores (not united in spore balls), namely, Th. thlaspeos, Th. oxalidis (Vánky et al. 2008) and Th. capensis (Roets et al. 2008).
The Asteraceae is the largest family of eudicots with an estimated number of 30,000 species (Funk et al. 2009). The Asteraceae is divided into 13 subfamilies, including four (Asteroideae, Cichorioideae, Carduoideae and Mutisioideae) that contain about 99% of all taxa. Anthemis is a large genus in the tribe Anthemideae (subfamily Asteroideae), along with Cota, Gonospermum (including Lugoa), Nananthea, Tanacetum and Tripleurospermum (Bremer and Humphries 1993, Oberprieler et al. 2009, Presti et al. 2010. Species of Anthemis are distributed in western Eurasia, including the Mediterranean region, northern Africa and a small part of eastern Africa (Oberprieler 1998, Oberprieler et al. 2009, Presti et al. 2010). There are 62 species of Anthemis in Europe. Anthemis chia belongs to the section Chiae and is a Mediterranean species common on Rhodes Island, Greece.
About 20 species of Thecaphora infect host plant species in six tribes of the Asteraceae. Taxa of the tribes Astereae and Heliantheae in the subfamily Asteroideae are often hosts of several Thecaphora species. Some less species-rich tribes, e.g. Coreopsideae, Millerieae, Polymnieae and Cynareae (subfamily Carduoideae) are also hosts of Thecaphora species. The species of Thecaphora on Asteraceae have not been studied by molecular phylogenetic methods, in contrast to species of Thecaphora on Caryophyllaceae (Vánky and Lutz 2007), Polygonaceae (Vasighzadeh et al. 2014) and Oxalidaceae (Roets et al. 2008(Roets et al. , 2012. Plants of Anthemis chia with distorted flower heads containing mostly ligulate (ray) florets and swollen peduncles were collected near Tsambika, Rhodes Island, Greece, in 2015 and 2017. The swollen flower heads contained reddish-brown granular to powdery spore ball masses, typical of species of Thecaphora. The aim of this study was to identify the fungus and to determine its taxonomic assignment based on morphological and phylogenetic analyses of the internal transcribed spacer (ITS, barcoding locus) sequence data.

Specimens
Herbarium specimens (23) of Thecaphora on a range of host plant species from across Europe and North America were examined (Tables 1, 2). The ITS sequences of specimens available on GenBank (19) and published in previous studies (Table 2) were included in the phylogenetic analysis. The nomenclature of the host plant species follows Euro+Med PlantBase (http://www.emplantbase.org/home.html) and the nomenclature of the fungi is according to Vánky (2012).
The morphology of the spore balls and spores of one specimen (GLM-F112531) of Thecaphora on Anthemis chia was microscopically examined at 1000× in 80% lactic acid heated to the boiling point on a glass slide. Measurements of 30 spore balls and 100 spores were made with the Zeiss AxioVision software and micrographs were taken with an Olympus FE-120 camera on a Seben SBX-5 compound microscope (Seben GmbH, Berlin). The measurements are reported as maxima and minima in parentheses and the means are placed in italics.

DNA extraction, amplification and sequencing
Genomic DNA was extracted from 23 herbarium specimens of Thecaphora (Table 1) using the methods reported by Kruse et al. (2017). The ITS nrDNA was amplified by PCR as reported in Kruse et al. (2018), using M-ITS1 (Stoll et al. 2003) as forward primer and either smITS-R1 or smITS-R2 (Kruse et al. 2017) as reverse primer. The ITS of host plants was amplified using primer pair ITS1P/ITS4 (Ridgway et al. 2003) with an annealing temperature of 53 °C. The resulting amplicons were sequenced at the Senckenberg Biodiversity and Climate Research Centre (BiK-F, Senckenberg) using the ITS4 primer (White et al. 1990). Sequences were deposited in GenBank (Table 2).

Phylogenetic analysis
In total, 42 ITS sequences from 21 Thecaphora species were used in the phylogenetic analyses. Sequences were aligned with MAFFT v.7 (Katoh and Standley 2013) employing the G-INS-I algorithm and leading and trailing gaps were trimmed. The resulting alignment length was 534 bp. The methods of phylogenetic analysis were according to Kruse et al. (2018) using Minimum Evolution (ME), Maximum Likelihood (ML) and Bayesian Inference (BA). Thecaphora italica and allied species were selected as an outgroup, on the basis of the phylogeny presented by Vánky and Lutz (2007). Host plant species determination was verified by comparison with published sequences from Asteraceae deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using BLASTN (Altschul et al. 1997).

Discussion
The present study is the first to identify a species of Thecaphora on a host plant species in the tribe Anthemideae (Asteraceae) (see Vánky 2012). Thecaphora anthemidis was recovered in a monophyletic group of Thecaphora species on Asteraceae, sister to Thecaphora solani on Solanum lycopersicum (Solanaceae). Our phylogenetic hypothesis, based on the ITS region, was similar to the analyses of the LSU locus of these taxa in Vánky and Lutz (2007) and Roets et al. (2008). In the latter study, Thecaphora polymniae, which is known only from the type collection on Polymnia riparia (Polymnieae, Asteroideae, Asteraceae) from South America (Vánky 2012), clustered within a clade of taxa that infect Fabaceae, Caryophyllaceae and Amaranthaceae (Roets et al. 2008). Thecaphora polymniae has spores with a reticulate ornamentation and this may be evidence of a host jump from one of these plant families to Asteraceae. Host jumps have been reported before in the Ustilaginomycotina (e.g. Begerow et al. 2002, Piątek et al. 2017 and are thought to be a driver of plant pathogen diversification (Choi and Thines 2015).
Previously, only two ITS sequences of Thecaphora species infecting Asteraceae (Th. spilanthis and Th. hennenea) were available on GenBank, which together with the new sequences reported in this study, represents only 20% of all Thecaphora species known to occur on Asteraceae. In addition to the sequence of Th. anthemidis, we have provided barcode sequences of the ITS region for eight other taxa not previously available on GenBank (Table I). Future studies should address whether species of Thecaphora that infect the flower heads of Asteraceae form a monophyletic group.