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

Julia Kruse; Volker Kummer; Roger G. Shivas; Marco Thines

doi:10.3897/mycokeys.41.28454

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).

Table 1.

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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
Thecaphora affinis	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
Th. molluginis	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

Table 2.

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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
Th. affinis	Astragalus glycyphyllos	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
Th. frezii	Arachis hypogaea	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
Th. italica	Silene italica	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
Th. molluginis	Mollugo cerviana	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
Th. oxytropis	Oxytropis pilosa	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
Th. schwarzmaniana	Rheum ribes	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

¹ Acronyms: AFTOL = Assembling the Fungal Tree Of Life, http://aftol.org; BASU: Herbarium of Bu-Ali Sina University, Iran; CUP = Plant Pathology Herbarium, Cornell University, New York, USA; GLM = Herbarium Senckenbergianum, Görlitz, Germany; HUV = Herbarium Ustilaginales Vánky, deposited in BRIP = Queensland Plant Pathology Herbarium, Brisbane, Australia; KRAM F = Mycological Collection of the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków, Poland; M = Botanische Staatssammlung, Munich, Germany; TUB = Herbarium Tubingense, Eberhard-Karls-Universität Tübingen, Germany; * not deposited in any public herbaria.

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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