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The first smut fungus, Thecaphora anthemidis sp. nov. (Glomosporiaceae), described from Anthemis (Asteraceae)
expand article infoJulia Kruse, Volker Kummer§, Roger G. Shivas|, Marco Thines#¤
‡ University of Southern Queensland, Toowoomba, Australia
§ University Potsdam, Potsdam, Germany
| University of Southern Queensland, Queensland, Australia
¶ Goethe University Frankfurt am Main, Frankfurt, Germany
# Senckenberg Gesellschaft für Naturforschung, Frankfurt, Germany
¤ Integrative Fungal Research Cluster, Frankfurt, Germany
Open Access

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 Anthemis chia (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 Thecaphora anthemidis sp. 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. Thecaphora anthemidis produced loose spore balls in the flower heads and peduncles of Anthemis chia unlike other flower-infecting species.

Keywords

Glomosporiaceae, host specificity, internal transcribed spacer, molecular phylogenetics, smut fungi

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 yellowish-brown 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, 2001, 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, 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.

Materials and methods

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).

Collection records for specimens of Thecaphora examined in this study.

Species Host Country Location Date Collector Herbarium accession no.*
Thecaphora affinis Astragalus glycyphyllos Slovenia Lower Styria, region Savinjska, N of Ljubno ob Savinjii, trail to Mt. Greben Smrekovec-Komen from Primož pri Ljubnem, wayside, 46°24'21"N, 14°49'54"E, 1150 m asl 14 July 2015 J. Kruse GLM F112522
A. glycyphyllos Germany Saxony-Anhalt, SW of Zschornewitz, forestry trail nearby SW-shore of „Gürke“ (Zschornewitzer Lake) 26 June 2007 H. Jage GLM F094059
Th. anthemidis Anthemis chia Greece Island Rhodes, 3.5 km NE Archangelos, Tsambika, way up to monastery, northeastslope, 36°14'03"N, 28°09'19"E, 90 m asl 26 April 2017 V. Kummer GLM F112531
Th. haumanii Iresine diffusa Costa Rica Prov. Guanacaste, 6 km NW de la barrada de la Laguna de Arenal 1 April 1992 R. Berndt, M. Piepenbring M 0236177
Th. leptideum Chenopodium album France Lotharingia, Forbach, Kreuzberg Mt. Aug.-Oct. 1912/1913 A. Ludwig M 0230099
Th. molluginis Mollugo cerviana Romania Bratovesti, Oltenia 15 July 1963 K. Lug. Eliart M 0236178
M. cerviana Romania Oltenia, Timburesti 19 Sept. 1958 L. Pop M 0236180
Th. oxalidis Oxalis stricta Austria Upper Austria, Braunau at Inn, Hagenau Inncounty, Hagenauer Street, wayside, 48°16'24"N, 13°06'03"E, 340 m asl 18 Aug. 2014 J. Kruse GLM F112523
O. stricta Germany Bavaria, Upper Franconia, Fichtelmountains, Fichtelberg, Sandgrubenway, cemetery, 605 m asl 17 Sept. 2012 J. Kruse GLM F112524
O. stricta Germany Saxony-Anhalt, county Anhalt-Bitterfeld, Bitterfeld-Wolfen, Mühlstreet, allotment garden area „Kühler Grund“, 51°37'23"N, 12°20'08"E 13 July 2014 J. Kruse & H. Jage GLM F112525
Th. pustulata Bidens pilosa Puerto Rico, USA Mayagüez 13 Mar. 1920 H. H. Whetzel, E. W. Olive CUP PR000458
Th. seminis-convolvuli Convolvulus arvensis Germany Saxony, Middlesaxony, Freiberg, Halsbrücker Street, roadside, 50°55'31"N, 13°20'56"E, 400 m asl 11 Aug. 2017 J. Kruse GLM F112527
C. arvensis Germany Hesse, c. 8.5 km SE Eschwege, Weißenborn, Sandhöfe, path, 51°07'35"N, 10°07'25"E, 250 m asl 22 July 2017 J. Kruse GLM F112528
C. arvensis Germany Saxony-Anhalt, SSE Seeben, at Franzosenstein, wayside 26 Aug. 2002 H. Jage GLM F065278
Calystegia sepium Germany Mecklenburg-Western Pomerania, county Vorpommern-Rügen, 1,5 km NE of Barth, Glöwitz, rest area, 54°22'15"N, 12°45'38"E, 0 m asl 24 Aug. 2014 J. Kruse GLM F112526
C. sepium Germany North Rhine-Westphalia, county Steinfurt, Rheine, castle grounds Bentlage, between parking area and Gradierwerk, 52°17'49"N, 07°25'11"E, 35 m asl 14 July 2017 J. Kruse GLM F112529
Th. seminis-convolvuli C. sepium Germany Schleswig-Holstein, county Schleswig-Flensburg, Schaalby, W of Winningmay, parking area at „Reesholm“, wayside, 54°31'44"N, 09°37'53"E, 2 m asl 30 Aug. 2014 J. Kruse GLM F112530
Th. thlaspeos Arabis ciliata Austria Tyrol, district Kufstein, county Walchsee, Kaiserwinkel, track from hickinghut towards Niederkaseralm, over Hintere Abendpoit, eastslope Mt. Hochköpfl, 47°41'25"N, 12°19'37"E, 1300 m asl 21 July 2014 J. Kruse GLM F112533
A. ciliata Germany Bavaria, Chiemgauer Alps, county Rosenheim, Priener Hut, track 8,20, way up towards Kampenwand, alpine meadow, 47°42'29"N, 12°19'27"E, 1570 m asl 18 July 2014 J. Kruse GLM F112536
A. ciliata Germany Bavaria, Chiemgauer Alps, county Traunstein, Priener Hut, track 8,20 towards Priener Hut, alpine meadow, 47°42'07"N, 12°20'36"E, 1310 m asl 19 July 2014 J. Kruse GLM F112537
A. hirsuta Germany Hesse, Meißnerfoothills, Werra-Meißner-county, Großalmerode, S of Weißenbach, “Bühlchen”, calcareous grassland, 51°14'55"N, 09°51'08"E, 500 m asl 13 June 2015 J. Kruse GLM F112532
A. hirsuta Germany Bavaria, county Donau-Ries, Harburg, N of Ronheim, dry grassland, 435 m asl 20 June 2013 J. Kruse GLM F112534
A. hirsuta Germany Bavaria, Upper Bavaria, county Weilheim, N of Pähl, E at Hartschimmelhof, N „Goaslweide“, wayside, 720 m asl 20 July 2013 J. Kruse GLM F112535

Specimens and GenBank sequences used for phylogenetic analyses. Sequences generated in this study are shown in bold.

Thecaphora species Host Herbarium accession no. 1 ITS GenBank accession no. Reference
Th. affinis Astragalus glycyphyllos GLM F112522 MH399748 this paper
GLM F094059 MH399749 this paper
Th. alsinearum Stellaria holostea HUV 10535 EF200032 Vánky and Lutz 2007
Th. amaranthi Amaranthus hybridus HUV 20727 EF200013 Vánky and Lutz 2007
Th. anthemidis Anthemis chia GLM F112531 MH399758 this paper
Th. frezii Arachis hypogaea Sa-EM1* KP994420 Cazón et al. 2016
Cba-GD2* KP994419 Cazón et al. 2016
Th. haumanii Iresine diffusa M 0236177 MH399764 this paper
Th. hennenea Melampodium divaricatum HUV 14434 EF200014 Vánky and Lutz 2007
Th. italica Silene italica HUV 20345 EF200026 Vánky and Lutz 2007
HUV 20344 EF200025 Vánky and Lutz 2007
Th. leptideum Chenopodium album M 0230099 MH399756 this paper
Th. melandrii Silene alba HUV 12677 EF200024 Vánky and Lutz 2007
Th. molluginis Mollugo cerviana M 0236178 MH399762 this paper
M 0236180 MH399763 this paper
Th. oxalidis Oxalis stricta GLM F112524 MH399759 this paper
GLM F112523 MH399760 this paper
GLM F112525 MH399761 this paper
Th. oxytropis Oxytropis pilosa Kummer P 1146/3* KF640685 Kummer et al. 2014
Kummer P 1146/2* KF640684 Kummer et al. 2014
Th. pustulata Bidens pilosa CUP PR000458 MH399757 this paper
Th. saponariae Saponaria officinalis TUB 012796 EF200022 Vánky and Lutz 2007
Th. schwarzmaniana Rheum ribes BASU 4242 JX006079 Vasighzadeh et al. 2014
KRAM F-49788 KF297811 Vasighzadeh et al. 2014
Th. seminis-convolvuli Calystegia sepium GLM F112529 MH399742 this paper
GLM F112526 MH399743 this paper
GLM F112530 MH399744 this paper
Convolvulus arvensis GLM F112527 MH399745 this paper
GLM F112528 MH399746 this paper
GLM F065278 MH399747 this paper
Th. solani Solanum lycopersicum HUV 11180 EF200037 Vánky and Lutz 2007
Th. sp. Rheum palmatum S. Wang 1991* KJ579177 Piątek et al. unpublished
Y. Wang 2013* KJ579176 Piątek et al. unpublished
HUV 21117 KF297812 Vasighzadeh et al. 2014
Th. spilanthis Acmella sp. AFTOL 1913 DQ832243 Matheny et al. 2006
Th. thlaspeos Arabis hirsuta GLM F112532 MH399752 this paper
TUB 015857 KJ579178 Vasighzadeh et al. 2014
GLM F112534 MH399750 this paper
GLM F112535 MH399751 this paper
Arabis ciliata GLM F112537 MH399753 this paper
GLM F112533 MH399754 this paper
GLM F112536 MH399755 this paper

Results

Molecular phylogenetic reconstruction

The ML and BA trees yielded consistent topologies with the ME tree (Fig. 1). The Thecaphora sp. on Anthemis chia, together with three Asteracious species (Th. pustulata, Th. hennenea and Th. spilanthis) and Th. solani from Solanum lycopersicum (Solanaceae), formed a sister clade to the species on other host plant families with strong to intermediate bootstrap support (83% in ME, 93% in ML). The Thecaphora sp. on Anthemis chia was sister to the other Asteracious species with low bootstrap support (59% in ME, 59% in ML), but high Bayesian posterior probability (96%). The Thecaphora species on Fabaceae were polyphyletic, with Th. frezii on Arachis hypogaea sister to Th. oxalidis on Oxalis stricta (Oxalidaceae). Thecaphora frezii was distant to a monophyletic lineage on Oxytropis pilosa and Astragalus glycyphyllos, which was sister to Th. seminis-convolvuli, the type of the genus. All specimens of Th. seminis-convolvuli collected on Calystegia sepium and Convolvulus arvensis (Convolvulaceae) had identical ITS sequences, as was the case with Thecaphora thlaspeos on Arabis hirsuta and A. ciliata (Brassicaceae). Within the clade of mostly Caryophyllaceae-infecting species, two species of Thecaphora infected other families of the Caryophyllales, namely Th. molluginis on Mollugo cerviana (Molluginaceae) and Th. haumanii on Iresine diffusa (Amaranthaceae).

Figure 1. 

Phylogenetic tree of Thecaphora species based on ME analysis of the ITS locus. Numbers on branches denote support in ME, ML and BA, respectively. Values below 50% are denoted by ‘–‘. The bar indicates the number of substitutions per site. Ex-type sequences are highlighted with an asterisk.

Taxonomy

Thecaphora anthemidis J. Kruse, V. Kumm. & Thines, sp. nov.

MycoBank No: 827067
Figure 2A–H

Type

Greece, Rhodes Island, 3.5 km NE Archangelos, Tsambika, on path to monastery, northeast slope, 36°14'03"N, 28°09'19"E, 90 m a.s.l, on Anthemis chia, 26 Apr. 2017, V. Kummer. Holotype GLM-F112531, isotype Herbarium V. Kummer P 1971/chia4; ITS sequence GenBank MH399758.

Etymology

From the host plant genus Anthemis.

Description

Sori in swollen and distorted flower heads and peduncles; spore ball mass initially white, later reddish-brown, granular to powdery; spore balls subglobose to ellipsoidal, rarely ovoid, mostly regular in shape, (31–) 36–41–47 (–52) × (28–) 31–38–44 (–50) µm, length/width ratio 0.9–1.1–1.2 (n=30), under light microscopy yellowish-brown to pale yellowish-brown, composed of 2–10 (–12) loosely united spores that separate easily; spores ellipsoidal, subglobose, ovoid or cuneiform, (18–) 20–21–23 (–25) × (14–) 17–18–20 (–23) µm, length/width ratio of 1.1–1.2–1.4 (n=100), with flattened contact surfaces and rounded exposed surfaces; wall at contact surface up to 0.5 µm thick, wall at free surface up to 3 µm thick, densely verrucose with warts 0.5–1 µm high, often confluent and sometimes irregular.

Host range

Anthemis chia.

Distribution

Greece.

Notes

Thecaphora anthemidis has sori in the flower heads and the peduncles, which differentiates it from the following species that produce pustules, galls or swellings on the stems of Asteraceae: Th. ambrosiae, Th. denticulata, Th. heliopsidis, Th. hennenea, Th. melampodii, Th. mexicana, Th. neomexicana, Th. piluliformis, Th. polymniae, Th. pulcherrima, Th. pustulata, Th. smallanthi and Th. spilanthis. Four of the seven previously known species of Thecaphora that infect the flower heads of Asteraceae, namely Th. arnicae, Th. burkartii, Th. californica and Th. cuneata have firmly united spores that only separate after considerable pressure, which differentiate them from Th. anthemidis that has loose spore balls. Further, Th. arnicae (spore balls comprised of up to 25 spores), Th. californica (6–20 spores) and Th. solidaginis (8 to 50 or more spores) have larger spore balls with larger numbers of spores than Th. anthemidis. The spores of Th. cuneata are radially arranged within the spore balls and Th. burkartii has spores with an outer wall 5–9 µm thick, which is more than three times thicker than in Th. anthemidis. Thecaphora lagenophorae and Th. trailii are morphologically most similar to Th. anthemidis. Thecaphora lagenophorae is only known to infect Solenogyne gunnii (tribe Astereae) in Australia (Vánky 2012). Thecaphora trailii infects species of Carduus, Cirsium and Saussurea (Asteraceae, tribe Cynareae, Carduoideae) (Vánky 2012) and further differs from Th. anthemidis by having smaller spore balls (12–30 µm) and fewer spores (2–8) per spore ball.

Figure 2. 

Sori, spore balls and spores of Thecaphora anthemidis on Anthemis chia (GLM-F112531) (A–H), A habit B–C swollen flower heads and peduncles D dissected flower head with reddish granular powdery spore ball mass E young spore balls F mature spore balls G–H single spores. Scale bars: 10 µm.

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.

Acknowledgements

The authors are grateful to Ulrike Damm and Michaela Schwager of the Herbarium Senckenbergianum Görlitz (GLM) for providing us with herbarium numbers for private collections and to the curators Dagmar Triebel of the Herbarium Munich (M), Ulrike Damm of the Herbarium Senckenbergianum Görlitz (GLM) and Scott LaGreca of the Cornell University New York (CUP) for loaning specimens from their keeping. We also thank all private collectors of the specimens investigated in this study for giving material to public herbaria. Furthermore, we want to thank the Ministry for Environment and Energy, Directorate for Forest Management for the collection permission in Rhodes (Greece). Alistair McTaggart is thanked for proofreading the paper and helpful advice.

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