Ribosomal and Protein Gene Phylogeny Reveals Novel Saprobic Fungal Species From Juglans regia and Urtica dioica.

During an ongoing investigation of Ascomycetes from plant substrates, three saprobic species were found from plant substrates. Two new species, Leptosphaeria regiae and Neomicrosphaeropsis juglandis were isolated from dead branches of Juglans regia from Turkey. Another species is introduced herein as Subplenodomus urticae sp. nov within the family Leptosphaeriaceae found on Urtica dioica in Italy. Multigene phylogenies based on combined LSU, ITS, SSU, and β-tubulin DNA sequence data generated from maximum likelihood and MrBayes analyses indicate that Leptosphaeria regiae is related to L. slovacica and forms an independent lineage within the genus Leptosphaeria. Subplenodomus urticae is basal to S. iridicola and its establishment as a new species is strongly supported. Neomicrosphaeropsis juglandis forms a moderately supported lineage in between N. italica and N. elaeagni in the Didymellaceae. Full morphological details are provided herein and phylogenetic relationships of the three new species are also discussed.

Introduction

The bitunicate fungi commonly known as Dothideomycetes is one of the largest group of fungi with a high level of diversity (Zhang et al., 2012; Hyde et al., 2013, 2018, 2019; Tibpromma et al., 2017; Jayasiri et al., 2019). Most of them exist as decomposers, endophytes, epiphytes, fungicolous, lichenized, or lichenicolous fungi in diverse environments (Jeewon et al., 2013, 2017; Phukhamsakda et al., 2016; Doilom et al., 2017; Wanasinghe et al., 2018; Pem et al., 2019a; Phookamsak et al., 2019). They can reproduce either sexually or asexually (Doilom et al., 2014, 2018; Wijayawardene et al., 2014, 2018; Pem et al., 2019c, d). The role of fungi as decomposers is crucial as it helps in recycling of nutrients and releasing enzymes hence maintaining the nutrient compositions of the ecosystem (Hyde and Jeewon, 2003; Hyde et al., 2005; Tang et al., 2005). Decomposers also play vital role in the eco-system such as breakdown of rock to form soils, protection against pathogens and as a food source and alteration of pollutants (Dighton, 2016; Singh et al., 2016). Leptosphaeriaceae is a family in the order Pleosporales (Dothideomycetes, Ascomycota) introduced by Barr (1987) and typified by the genus Leptosphaeria. There are 1,800 epithets of Leptosphaeriaceae recorded in Index Fungorum (2020) with the largest number of species (1,669) occurring in the genus Leptosphaeria and 130 epithets in Mycobank (2020) but most of them lack molecular data. The family Leptosphaeriaceae is characterized by immersed to superficial ascomata, cylindrical to oblong pedicellate asci and reddish brown or yellowish brown, septate ascospores (Hyde et al., 2013; Ariyawansa et al., 2015; Dayarathne et al., 2015; Wanasinghe et al., 2016). Asexual morph are coelomycetous producing phialidic or annellidic conidiogenous cells (Wijayawardene et al., 2018). Leptosphaeriaceae species differ from other closely related families in the Dothideomycetes by the presence of a scleroplectenchymatous peridium. During our ongoing survey, another species resembling those of the asexual genus Neomicrosphaeropsis was observed. The genus Neomicrosphaeropsis was introduced by Thambugala et al. (2016) to accommodate four species namely; N. italica, N. novorossica, N. rossica, and N. tamaricicola. The type species is N. italica and was isolated from dead branches of Tamarix (Tamaricaceae) in Italy. Species of Neomicrosphaeropsis are pathogens or endophytes (Wijayawardene et al., 2017) and are morphologically characterized by hyaline to light brown, aseptate, obovoid to ellipsoidal conidia (Wijayawardene et al., 2018). The genus Neomicrosphaeropsis has been reported to comprise complex species which are morphologically similar but phylogenetically different (Thambugala et al., 2016). Neomicrosphaeropsis resembles species of Microsphaeropsis which is also accommodated in Didymellaceae as well as species of Coniothyrium in having hyaline to light brown, aseptate, obovoid to ellipsoidal, smooth-walled conidia (Verkley et al., 2014; Crous et al., 2019). There are currently 10 epithets in the genus Neomicrosphaeropsis (Index Fungorum, 2020). In this study, we introduce a new asexual species in the genus Neomicrosphaeropsis isolated from stems of Juglans regia (Juglandaceae) in Turkey using multi-gene (LSU, ITS, SSU, and β-tubulin) phylogenetic data. To the best of our knowledge, there have been no fungal species of Leptosphaeriaceae and Didymellaceae associated with Juglans regia in Turkey. We also report on a new species of Leptosphaeriaceae specifically in the genus Subplenodomus found on Urtica dioica from Italy. The aim of this study is to characterize these three fungal isolates in terms of morphology and phylogeny based on multi-gene sequence data.

Materials and Methods

Samples Collection, Morphological Examination, and Isolation

Specimens were collected from dead stems and branches of Juglans regia in the Corum and Kirikkale province of Turkey and on Urtica dioica in the province of Forlì-Cesena (FC) Italy (Figure 1). Samples were stored in Zip-lock bags and returned to the laboratories for examination and description of morphological characters. The specimens were observed under a Motic SMZ 168 series dissecting stereo-microscope. Free hand sections of fungal structures were taken and mounted in water for microscopic study. Photomicrography was carried out using a Canon 750D digital camera fitted to the microscope. Measurements were made with the Tarosoft (R) Image Frame Work software. The images were processed with Adobe Photoshop CS5 v. 12.0 software (Adobe Systems, United States) to illustrate fungal characters using a photoplate. Pure cultures were established from single ascospores/conidia on 2% malt extract agar (MEA; 62 g/L Criterion in distilled water) as described in Vijaykrishna et al. (2004) and Pem et al. (2019d). Cultures were incubated at 25°C for up to 5 weeks and cultural characters were observed and measured after a week and again after 4 weeks following Liu et al. (2014). Holotype specimens are deposited in the herbarium located at Mae Fah Luang University (MFLU) and isotype specimens are deposited at the Kunming Institute of Botany, Academia Sinica Herbarium (HKAS), China. Ex-type living cultures are deposited at the Mae Fah Luang Culture Collection (MFLUCC) and duplicates at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell cultures (DSMZ). Faces of fungi numbers (Jayasiri et al., 2015) and Index Fungorum number[1] are provided.

FIGURE 1

Geographical coordinates and collecting site of specimen (a,b: Turkey, c: Juglans regia; d,e: Italy, f: Urtica dioica).

DNA Extraction, Amplification, and Sequencing

Isolates were grown on MEA at 16 ± 2°C for 8 weeks. DNA was extracted from fresh fungal mycelium using the DNA extraction kit (E.Z.N.A Fungal DNA Mini Kit, D3390-02, Omega Bio-Tek) following the manufacturer’s protocol. Polymerase Chain Reaction (PCR) was performed to amplify specific gene regions. Primers LR0R and LR5 were used to amplify part of the nuclear ribosomal large subunit 28S rRNA gene (LSU) (Vilgalys and Hester, 1990; Rehner and Samuels, 1994). The Internal transcribed spacer rDNA region (ITS1, 5.8S rDNA, and ITS2) was determined using the primer ITS5/ITS4 and the 18S small subunit ribosomal RNA (SSU) was amplified using NS1/NS4 (White et al., 1990). For the beta-tubulin (TUB) gene, partial cds region, the primers Bt2a/Bt2b was used (Woudenberg et al., 2009). Primer sequences are available at the WASABI (Web Accessible Sequence Analysis for Biological Inference) database at the AFTOL website (aftol.org). Amplification reactions profiles for LSU, ITS, SSU, and β-tubulin gene followed Tang et al. (2007), Wang et al. (2007), and Luo et al. (2017). The analysis of PCR amplification products (amplimers) were performed by the commercial sequencing provider (BGI, Ltd Shenzhen, PR China). Same primer pairs used for amplification process were used for sequencing. The nucleotide sequence data acquired is deposited in GenBank (Table 1). The final alignment and tree are deposited in the TreeBASE repository site[2]. The recommendations outlined by Jeewon and Hyde (2016) were used to establish the new taxa.

TABLE 1

Isolates used in this study. Newly generated sequences are indicated in bold.

Taxon	Strain/Culture No.	Status	GenBank accession numbers
			LSU	ITS	SSU	BTUB
Alloleptosphaeria italica	MFLUCC 14-0934		KT454714	KT454722
Allophoma labilis	CBS 124.93		GU238092	GU237868		GU237620
Alternariaster bidentis	CBS 134021	T	KC609341	KC609333
Alternariaster centaureae diffusae	MFLUCC 14-0992	T	KT454715	KT454723	KT454730
Alternariaster helianthi	CBS 327.69	T	KC584369	KC609335	KC584627
Alternariaster trigonosporus	MFLU 15-2237	T	KY674858	KY674857
Ascochyta pisi	CBS 126.54		MH868800	MH857263	EU754038	GU237531
Ascochyta pisi	CBS 122751		KP330444	KP330432		KP330388
Ascochyta pisi	CBS 122785	T	GU237969	GU237763	EU754039	GU237532
Boeremia exigua var exigua	CBS 431.74		JX681074	FJ427001	EU754084	FJ427112
Boeremia exigua var pseudolilacis	CBS 101207		GU237941	GU237721		GU237503
Briansuttonomyces eucalypti	CBS 114887		KU728520	KU728480		KU728596
Briansuttonomyces eucalypti	CBS 114879	T	KU728519	KU728479		KU728595
Calophoma aquilegiicola	CBS 108.96		GU238042	GU237736		GU237582
Calophoma aquilegiicola	CBS 107.96		GU238041	GU237735		GU237581
Calophoma clematidina	CBS 108.79		MH872951	FJ426989
Calophoma clematidis-rectae	CBS 507.63		MH869956	FJ515606		FJ515624
Calophoma glaucii	CBS 112.96		GU238077	GU237750		GU237610
Calophoma vodakii	CBS 173.53	T	MH868686	MH857149		KT389791
Cumuliphoma indica	CBS 654.77		GU238122	FJ427043		FJ427153
Cumuliphoma indica	CBS 991.95		GU238121	FJ427044		FJ427154
Cumuliphoma omnivirens	CBS 341.86	T	LT623214	MH861962		FJ427152
Cumuliphoma pneumoniae	CBS 142454		LN907392	LT592925		T592994
Didymella aliena	CBS 379.93		GU238037	GU237851		GU237578
Didymella anserina	CBS 360.84		GU237993	GU237839		GU237551
Didymella chenopodii	CBS 128.93		GU238055	GU237775		GU237591
Didymella exigua	CBS 183.55	T	MH868977	MH857436	GU296147	GU237525
Didymella glomerata	DAOM 214575		JN938876	JN942893	JN939031
Didymella glomerata	CBS 464.97		GU238009	FJ427012	EU754086	FJ427123
Didymella negriana	CBS 358.71		MH871931	GU237838		GU237635
Didymella sp.	CPC 21698		KF777232	KF777180
Didymella sp.	CPC 22587		KJ869191	KJ869134		KJ869246
Didymellocamarosporium tamaricis	MFLUCC 15-0067	T	KY496733	KY496753	KY501116
Didymellocamarosporium tamaricis		T	KU848183		KU848182
Didysimulans italica	MFLUCC 15-0059		KY496730	KY496750	KY501118
Didysimulans mezzanensis	MFLUCC 15-0078		KY496733	KY496753	KY501116
Dothidotthia aspera	CPC 12933		EU673276	MK442601	EU673228
Dothidotthia symphoricarpi	CBS 119687		EU673273	MH863064	EU673224
Ectophoma multirostrata	CBS 110.79		GU238110	FJ427030		FJ427140
Ectophoma multirostrata	CBS 274.60	T	MH869536	MH857982		FJ427141
Ectophoma multirostrata	CBS 368.65		MH870257	MH858615		FJ427143
Ectophoma pomi	CBS 267.92	T	GU238128	GU237814		GU237643
Epicoccum nigrum	CBS 173.73	T	MH872357	MH860655	GU238206	FJ427107
Heterophoma novae-verbasicola	CBS 127.93	T	GU238120	GU237774		GU237639
Heterophoma sylvatica	CBS 874.97		GU238147	GU237907		GU237662
Heterospora chenopodii	CBS 115.96		EU754188	JF740227	EU754089
Heterospora chenopodii	CBS 448.68	T	EU754188	JF740227	EU754089
Heterospora dimorphospora	CBS 165.78		JF740281	JF740204	JF740098
Heterospora dimorphospora	CBS 345.78		GU238069	JF740203	GU238213
Juxtiphoma eupyrena	CBS 374.91		GU238072	FJ426999		FJ427110
Juxtiphoma eupyrena	CBS 527.66		MH870524	FJ427000		FJ427111
Leptosphaeria cichorium	MFLUCC 14-1063		KT454712	KT454720	KT454728
Leptosphaeria conoidea	CBS 616.75		MH872726	MH860957	JF740099	KT389804
Leptosphaeria doliolum	CBS 505.75	T	GU301827	MH860947	GU296159	JF740144
Leptosphaeria doliolum	MFLUCC 15-1875		MH870536	MH858879	GU296159
Leptosphaeria ebuli	MFLUCC 14-0828	T	KP744488	KP744446	KP753954
Leptosphaeria errabunda	CBS 617.75		JF740289	JF740216		JF740150
Leptosphaeria italica	MFLU 15-0174		KT783670
Leptosphaeria macrocapsa	CBS 640.93		JF740304	JF740237		JF740156
Leptosphaeria pedicularis	CBS 390.80		JF740294	JF740224		JF740155
Leptosphaeria regiae	MFLUCC 18-1137	T	MN244171	MN244201	MN244177
Leptosphaeria sclerotioides	CBS 148.84		JF740270	JF740193
Leptosphaeria slovacica	CBS 125975		JF740316	JF740248
Leptosphaeria sydowii	CBS 385.80		JF740313	JF740244		JF740157
Leptosphaeria urticae	MFLU 18-0591	T	MK123332	MK123333	MK123329
Leptosphaeria veronicae	CBS 126583		MH875625	MH864163		JF740161
Leptosphaerulina arachidicola	CBS 275.59		MH869401	MH857863		GU237543
Leptosphaerulina australis	CBS 317.83		MH873322	MH861604	GU296160	GU237540
Leptosphaerulina trifolii	CBS 235.58		MH869300	MH857767		GU237542
Loratospora aestuarii	JK 5535B		GU301838		GU296168
Macroventuria anomochaeta	CBS 502.72		MH872250	GU237873		GU237545
Macroventuria anomochaeta	CBS 525.71		MH872013	GU237881	GU238208	GU237544
Macroventuria wentii	CBS 526.71	T	MH872014	MH860250		GU237546
Microsphaeropsis olivacea	CBS 432.71		GU237987	GU237863		GU237548
Microsphaeropsis olivacea	CBS 442.83		EU754171	GU237865	EU754072	GU237547
Microsphaeropsis olivacea	CBS 233.77		GU237988	GU237803		GU237549
Microsphaeropsis ononidicola	MFLUCC 15-0459, ICMP 21575	T	MG967668	MG967670		MG973087
Microsphaeropsis proteae	CBS 111303		JN712561	JN712495
Microsphaeropsis proteae	CBS 111320		JN712562	JN712496		JN712649
Microsphaeropsis proteae	CBS 111319	T	JN712563	JN712497		JN712650
Microsphaeropsis spartii-juncei	MFLU 16-0100	T	MH069668	NR160346	MH069674	MH069687
Microsphaeropsis spartii-juncei	MFLU 16-0097		MH069669	MH069663	MH069675	MH069688
Neoas. europaea	CBS 820.84	T	KT389729	NR136131		KT389809
Neoas. exitalis	CBS 389.86		KT389733	MH861971		KT389813
Neodid. cannabis	CBS 121.75	T	GU237972	GU237761		GU237535
Neodid. polemonii	CBS 109181	T	GU238133	GU237746		GU237648
Neodid. xanthina	CBS 383.68		GU238157	GU237855		GU237669
Neoleptosphaeria jonesii	MFLUCC 16-1442	T	KY211870	KY211869	KY211871
Neoleptosphaeria rubefaciens	CBS 223.77	T	JF740312	JF740243
Neoleptosphaeria rubefaciens	CBS 387.80		JF740311	JF740242
Neomicrosphaeropsis alhagi-pseudalhagi	MFLUCC 17-0825	T	MH069670	MH069664	MH069676	MH069689
Neomicrosphaeropsis cystisi	MFLUCC 13-0396	T	KX572342	KX572337	KX572347
Neomicrosphaeropsis cystisicola	MFLUCC 18-0355	T	MH069671	MH069665		MH069690
Neomicrosphaeropsis cystisinus	MFLUCC 16-0790	T	KX611241	KX611243	KX611242
Neomicrosphaeropsis elaeagni	MFLUCC 17-0740	T	MH069672	MH069666	MH069678	MH069691
Neomicrosphaeropsis italica	MFLUCC 15-0485	T	KU729854	KU900318	KU900309
Neomicrosphaeropsis italica	MFLUCC 16-0284		KU900296	KU900321	KU900311	KX453299
Neomicrosphaeropsis italica	MFLUCC 15-0484		KU729853	KU900319		KX453298
Neomicrosphaeropsis italica	MFLUCC 15-0487		KU729852	KU900320	KU900310
Neomicrosphaeropsis juglandis	MFLUCC 18-0795	T	MN244206	MN244223	MN244183	MN871954
Neomicrosphaeropsis minima	MFLUCC 13-0394		KX572341	KX572336	KX572346
Neomicrosphaeropsis novorossica	MFLUCC 14-0578	T	KX198710	KX198709	KX198711
Neomicrosphaeropsis rossica	MFLUCC 14-0586	T	KU729855	KU752192	KU870914
Neomicrosphaeropsis tamaricicola	MFLUCC 14-0443		KU729851	KU900322	KU900312
Neomicrosphaeropsis tamaricicola	MFLUCC 14-0439		KU729858	KU900323	KU900313
Neomicrosphaeropsis tamaricicola	MFLUCC 14-0602	T	KM408754	KM408753	KM408755	MH069692
Nothophoma anigozanthi	CBS 381.91	T	GU238039	GU237852		GU237580
Nothophoma arachidia-hypogaeae	CBS 125.93		MH874048	MH862388		GU237583
Nothophoma infossa	CBS 123395	T	GU238089	MH863295		FJ427135
Nothophoma infossa	CBS 123394		GU238088	FJ427024		FJ427134
Ophiosphaerella herpotricha	CBS 620.86		DQ678062	KF498728	DQ678010
Paraboeremia adianticola	CBS 187.83		GU238035	GU237796		GU237576
Paraboeremia putaminum	CBS 130.69		MH871005	MH859273		GU237652
Paraboeremia selaginellae	CBS 122.93	T	GU238142	NR 135980		GU237656
Paraleptosphaeria dryadis	CBS 643.86		MH873696	MH862007	KC584632
Paraleptosphaeria macrospora	CBS 114198		MH874520	MH862957
Paraleptosphaeria nitschkei	CBS 306 51	T	MH868393	MH856873		KT389833
Paraleptosphaeria padi	MFLU 15-2756	T	KY554198	KY554203	KY554201
Paraleptosphaeria rubi	MFLUCC 14-0211	T	KT454718	KT454726	KT454733
Paraphoma radicina	CBS 111.79		MH872952	MH861183	EU754092	KF252667
Phaeosphaeria elongata	CBS 120250		GU456327	MH863080	GU456306
Phaeosphaeria oryzae	CBS 110110	T	MH874442	MH862850	GQ387530	KF252680
Phaeosphaeriopsis glaucopunctata	MFLUCC 13-0265		KJ522477	KJ522473	KJ522481
Phoma herbarum	CBS 615.75		KF251715	KF251212	EU754087	KF252703
Phoma herbarum	CBS 274.37		KT389754	KT389537		KT389835
Phoma herbarum	CBS 502.91		GU238082	GU237874		GU237613
Phoma neerlandica	CBS 134.96		KT389753	KT389535		KT389834
Phomatodes aubrietiae	CBS 627.97	T	MH874272	GU237895		GU237585
Phomatodes aubrietiae	CBS 383.67		GU238044	GU237854		GU237584
Phomatodes nebulosa	CBS 127776		MH876211	MH864771		GU237634
Plenodomus agnitus	CBS 121. 89		MH875626	KP744459		KY064053
Plenodomus artemisiae	KUMCC 18-0151	T	MK387958	MK387920	MK387928
Plenodomus chrysanthemi	CBS 539.63	T	MH869970	MH858349	GU238230	KY064055
Plenodomus collinsoniae	CBS 120227		JF740276	JF740200		KY064056
Plenodomus collinsoniae	VT2	T		MN653010
Plenodomus congestus	CBS 244.64	T	JF740278	AF439460		KY064058
Plenodomus deqinensis	CGMCC 3.18221	T	KY064031	KY064027		KY064052
Plenodomus enteroleucus	CBS 142.84	T	JF740287	JF740214		KT266266
Plenodomus fallaciosus	CBS 414.62		MH869793	JF740222
Plenodomus guttulatus	MFLUCC 15-1876	T	KT454713	KT454721	KT454729
Plenodomus hendersoniae	CBS 113702		MH874506	MH862939		KT266271
Plenodomus influorescens	CBS 143.84		JF740297	JF740228		KT266267
Plenodomus libanotidis	CBS 113795		MH874508	MH862943		KY064059
Plenodomus lijiangensis	KUMCC 18-0186	T	MK387959	MK387921	MK387929
Plenodomus lindquistii	CBS 381.67		MH870699	MH858999
Plenodomus lingam	CBS 260.94	T	JX681096	MH862462		KY064060
Plenodomus lupini	CBS 248.92		JF740303	JF740236		KY064061
Plenodomus pimpinellae	CBS 101637		MH874352	JF740240		KY064062
Plenodomus salviae	MFLUCC 13-0219	T	KT454717	KT454725	KT454732
Plenodomus sinensis	KUMCC 18-0152		MK387961	MK387923	MK387931
Plenodomus sinensis	KUMCC 18-0153		MK387960	MK387922	MK387930
Plenodomus sinensis	KUN HKAS 102227		MK387962	MK387924	MK387932
Plenodomus sinensis	MFLUCC 17-0757		MF072718	MF072722	MF072720
Plenodomus tracheiphilus	CBS 127250		JF740318	JF740250
Plenodomus visci	CBS 122783	T	EU754195	JF740256	EU754096	KY064063
Plenodomus wasabiae	CBS 120119			JF740257		KT266272
Pseudoascochyta novae-zelandiae	CBS 141689		LT592893	LT592892		LT592894
Pseudoleptosphaeria etheridgei	CBS 125980		MH875320	JF740221
Remotididymella anthropophila	CBS 142462			LT592936		LT593005
Remotididymella desctructiva	CBS 378.73	T	MH872414	MH860707		GU237601
Remotididymella desctructiva	CBS 133.93		GU238064	GU237779		GU237602
Remotididymella desctructiva	CBS 162.78		GU238062	GU237788		GU237600
Setomelanomma holmii	CBS 110217		GU301871	KT389542	GU296196
Similiphoma crystallifera	CBS 193.82	T	GU238060	GU237797		GU237598
Sphaerellopsis filum	CBS 234.51		KP170723	KP170655		KP170704
Sphaerellopsis hakeae	CPC 29566	T	KY173555	KY173466
Sphaerellopsis isthmospora	HKAS 102225A	T	MK387963	MK387925	MK387933
Sphaerellopsis isthmospora	HKAS 102225B	T	MK387964	MK387926	MK387934
Sphaerellopsis macroconidiale	CBS 233.51		KP170726	KP170658		KP170707
Sphaerellopsis macroconidiale	CBS 658.78		MH868352	KP170659		KP170708
Sphaerellopsis paraphysata	CPC 21841	T	KP170729	KP170662		KP170710
Stagono. cucurbitacearum	CBS 133.96		GU238181	GU237780	GU238234	GU237686
Stagono. hortensis	CBS 104.42		GU238198	MH856097		GU237703
Subplenodomus apiicola	CBS 285.72		GU238040	MH860477	GU238211
Subplenodomus drobnjacensis	CBS 269.92		JF740285	JF740211	JF740100
Subplenodomus galicola	MFLU 15-1368	T	KY554199	MF467894
Subplenodomus iridicola	CBS 143395	T	MH107965	MH107919
Subplenodomus urticae	MFLUCC 17-2311	T	MN597995	MN597998	MN597997
Subplenodomus valerianae	CBS 630.68		MH870914	JF740251	GU238229
Subplenodomus violicola	CBS 306.68		MH870849	MH859138	GU238231	KT389849
Vacuiphoma bulgarica	CBS 357.84	T	GU238050	GU237837		GU237589
Vacuiphoma oculihominis	URTHSC D116-308	T	LN907451	LT592954		LT593023
Xenodidymella applanata	CBS 195.36	T	KT389764	MH855770		KT389852

Phylogenetic Analysis

SeqMan v. 7.0.0 (DNASTAR, Madison, WI, United States) was used to assemble consensus sequences. Sequences of closely related strains were recovered from BLAST searches of GenBank[3] together with sequences of representative species used by Chen et al. (2015), Thambugala et al. (2016), and Phookamsak et al. (2019) and these are listed in Table 1. Sequences were aligned with online MAFFT v. 7 (Kuraku and Katoh, 2013; Katoh et al., 2019)[4]. The alignments were checked visually and improved manually where necessary using BioEdit v. 7.0.5.2 (Hall, 1999). Ambiguous regions were excluded from the analyses and gaps were treated as missing data. All novel sequences were deposited in GenBank and the final alignment and tree deposited in TreeBASE2. Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) methods. Maximum likelihood analyses (ML), for single and combined gene alignments included 1,000 bootstrap replicates and was performed using RAxML-HPC2 run on XSEDE (8.2.8) (Stamatakis, 2014) in the CIPRES Science Gateway platform (Miller et al., 2011) using GTR+I+G model of evolution. The final tree was selected among suboptimal trees from each run by comparing likelihood scores with the GTRGAMMA nucleotide substitution model. The best fitting substitution model for each single gene partition and the concatenated data set was determined in MrModeltest 2.3 (Nylander, 2004) for Bayesian inference posterior probabilities (PP). GTR+I+G model was used for each partition or each gene separately, and incorporated into the analysis. The Bayesian inference posterior probabilities (PP) distribution (Zhaxybayeva and Gogarten, 2002) was estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.2 (Ronquist et al., 2012). The MCMC analyses, with six chains were run, started from random tree topology and lasted 1,000,000 generations and sampled every 100 generations (Nylander et al., 2008). The Tracer v. 1.5.0 software program was used to calculate the distribution of log-likelihood scores in order to determine the stationary phase for each search, to check whether extra runs were required to achieve convergence, the stable likelihood plateaus and burn–in value (Drummond et al., 2012). The first 2,000 generations were excluded as burn-in and 10,000 trees were obtained. Maximum likelihood bootstrap values equal or greater than 50% and Bayesian inference posterior probabilities (PP) equal or greater than 0.90 are given in black below or above each node (Figure 1). The phylograms were viewed in FigTree v1.4[5] and edited using Microsoft PowerPoint 2016.

Results

Taxonomy

D. Pem, Selcuk, Jeewon & K.D. Hyde, sp. nov. Figure 2

FIGURE 2

Leptosphaeria regiae (MFLU 17-0523, holotype). (a) Specimen. (b,c) Appearance of ascomata on host surface. (d) Vertical section through the ascoma. (e) Peridium. (f) Hamathecium. (g–k) Asci. (l–o) Ascospores. (p) Germinating ascospore. (q,r) Culture characteristics on MEA (q: above view; r: reverse view). Scale bars: (b–d) = 200 μm, (e,g,h,k) = 50 μm, (f) = 5 μm, (i,j) = 20 μm, (l–o) = 10 μm.

[urn:lsid:indexfungorum.org:names: 557056]

Facesoffungi Number: FoF 06229

Etymology – Name reflects the host from which the fungus was isolated.

Holotype – MFLU 17-0523

Saprobic on dead stem of Juglans regia. Sexual morph: Ascomata 315–377 μm high, 364–410 μm diam., solitary or gregarious, superficial or semi-immersed on host tissue, visible as black spots on host surface, brown to dark brown. Ostiole apex dark brown to black, ostiolar canal filled with periphyses, papilla not conspicuous. Peridium 41–50 μm wide, comprising two cell types, outer layer composed of large, heavily pigmented, thick-walled cells of textura angularis, inner layer composed of scleroplectenchymatous cells of textura angularis. Hamathecium comprising numerous, 1.4–2.6 μm diam., slime coated, branched, cellular pseudoparaphyses. Asci 99–130 × 9–10 μm ( = 104.8 × 9.9 μm, n = 30), 8-spored, bitunicate, numerous, cylindrical to cylindric-clavate, short pedicellate, apically rounded, with indistinct ocular chamber. Ascospores 15–18 × 6–7 μm ( = 17.1 × 7.1 μm, n = 30), uni to bi-seriate, hyaline brown when immature, becoming yellowish brown to brown at maturity, ellipsoid to broadly fusiform, with rounded to acute ends, slightly clavate, narrow toward the base, 3-septate, constricted at septum, widest above the central septum, smooth-walled. Asexual morph: Undetermined.

Culture characteristics – Colonies on MEA, 17–20 mm diam. after 7 days at 16°C, margin irregular, aerial mycelia thinly hairy, sparse, white and flat; reverse dark brown, white at the margin.

Material examined – TURKEY, The Middle Kizilirmak river basin, Kirşehir province, Kaman district, Savcili small town, 911 m a.s.l., 39° 13′ 684″N, 33° 41′ 034″E, on dead stem of Juglans regia (Juglandaceae), 8 June 2012, Faruk Selcuk (MFLU 17-0523 holotype); ibid. (isotype in HKAS), ex-type living culture MFLUCC 18-1137.

GenBank accession numbers: LSU: MN244171, SSU: MN244177, ITS: MN244201

Notes – The new isolate Leptosphaeria regiae was obtained from dead stem of Juglans regia. In the NCBI BLASTn search of ITS sequence L. regiae has a closest match with L. sclerotioides (Preuss ex Sacc.) de Gruyter et al. (2013) (LP7-MRL) with identities 477/492 (97%) and 1% gaps. In our multigene phylogenetic analysis, L. regiae clusters close to L. slovacica (CBS 125975) with strong bootstrap support (100% ML, 1.00 PP). A comparison of 528 base pairs across the ITS (+5.8S) regions shows 71 (11.9%) base pair differences between L. regiae and L. slovacica. Morphologically, L. regiae differs from L. slovacica in its smaller ascospores (15–18 μm vs. 18–22 μm). We therefore, introduce Leptosphaeria regiae as a new species in the genus Leptosphaeria based on differences in morphology and DNA sequence data.

D. Pem, Selcuk F, Jeewon & K.D. Hyde, sp. nov. Figure 3

FIGURE 3

Neomicrosphaeropsis juglandis (MFLU 17-0517, holotype). (a,b) Appearance of conidiomata on host surface. (c) Vertical section through conidioma. (d–g) Conidiogenous cells and developing conidia. (h–m) Conidia. (n) Germinating conidia. (o,p) Culture characters on MEA (o: Above view; p: Reverse view). Scale bars: (a,b) = 1000 μm, (c) = 50 μm, (d) = 5 μm, (e–g) = 10 μm, (h–m) = 5 μm, (n) = 15 μm.

[urn:lsid:indexfungorum.org:names: 556688]

Facesoffungi number– FoF 06211

Etymology – Name reflects the host from which the fungus was isolated

Holotype – MFLU 17-0517

Saprobic on stems of Juglans regia. Sexual morph: Undetermined. Asexual morph: Conidiomata 158–189 μm high × 172–228 μm diam. ( = 170.6 × 202.9 μm, n = 10), pycnidial, scattered, solitary, aggregated or gregarious, immersed, slightly erumpent, black, globose to subglobose, uni- to bi-loculate, non-ostiolate. Conidiomatal wall 10–20 μm wide comprising light to dark brown, thick-walled cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells enteroblastic, phialidic, light brown, integrated, smooth. Conidia 8–11 × 6–7 μm ( = 9.8 × 6.9 μm, n = 50), yellowish or greenish brown, aseptate, obovoid to ellipsoidal, smooth-walled, sometimes guttulate.

Culture characteristics – Colonies growing on MEA, reaching a diameter of 25 mm after 7 days at 25°C, circular to irregular, flat to slightly raised, mycelium medium sparse, surface initially white, becoming pale saffron to pale white, reverse dark-gray with whitish edge, smooth at surface with entire to slightly filamentous edge, thinly hairy.

Material examined – TURKEY, The Middle Kizilirmak river-basin, Kirikkale province, Delice district, Çerikli small town, 682 ma.s.l., 39° 53′ 689″N, 33° 59′ 769″E, on dead aerial stems of Juglans regia L. (Juglandaceae), 8 August 2012, Faruk Selcuk (MFLU 17–0517, holotype); ibid. (isotype in HKAS), ex-type living culture MFLUCC 18-0795, DSM 109836.

GenBank accession numbers – LSU: MN244206, SSU: MN244183, ITS: MN244223, BTUB: MN871955.

Notes – Our new taxon Neomicrosphaeropsis juglandis is characterized by large, aseptate conidia with a unique yellowish or greenish brown color and measures 8–11 × 6–7 μm, compared to N. italica (3.6–6.2 μm × 2.9–4.6 μm), the type species of the genus Neomicrosphaeropsis. The ITS sequence comparison of N. juglandis with N. tamaricicola, N. italica and N. rossica reveals a difference of 0.8% (5 base pairs difference). However, strong evidence to support N. juglandis as a new species comes from the comparison of the RPB2 gene of our new species, N. juglandis to that of N. italica which shows a pairwise difference of 1.6% as well as that of β-tubulin with N. italica showing a difference of 2.1%. We therefore, introduce N. juglandis as a new species in the genus Neomicrosphaeropsis (Didymellaceae) based on morphological and phylogenetic evidence derived especially from protein coding genes.

D. Pem, Camporesi, Jeewon & K.D. Hyde, sp. nov. Figure 4

FIGURE 4

Subplenodomus urticae (MFLU 17-1694, holotype). (a,b) Appearance of ascomata on host surface. (c) Vertical section through the ascoma. (d) Peridium. (e) Hamathecium. (f–i) Asci. (j–p) Ascospores. (q) Germinating ascospore. (r,s) Culture characteristics on MEA (r: above view; s: reverse view). Scale bars: (b–d) = 200 μm, (e,g,h,k) = 50 μm, (f) = 5 μm, (i,j) = 20 μm, (l–o) = 10 μm.

[urn:lsid:indexfungorum.org:names: 557057]

Facesoffungi Number: FoF 06855

Etymology – Name reflects the host from which the fungus was isolated

Holotype – MFLU 17-1694

Saprobic on dead stem of Urtica dioica. Sexual morph: Ascomata 98–162 μm high, 111–200 μm diam., solitary or gregarious, superficial or semi-immersed on host tissue, visible as black spots on host surface, dark brown to black, papillate. Ostiole 29–32 × 48–66 μm, smooth, ostiolar canal filled with periphyses. Peridium 10–28 μm wide, comprising two cell types, outer layer composed of large, heavily pigmented, thick-walled cells of textura angularis, inner layer composed of hyaline cells of textura prismatica. Hamathecium comprising numerous, long, 1.7–2.4 μm ( = 2.2 μm, n = 50) wide, broad, transversely septate, branched, cellular pseudoparaphyses. Asci 44–65 × 7.3–10.7 μm ( = 58.4 × 8.9 μm, n = 30), 8-spored, bitunicate, numerous, cylindrical to cylindric-clavate, short pedicellate, apically rounded, with indistinct ocular chamber. Ascospores 19–24 × 4.4–5.6 μm ( = 21.9 × 5.2 μm, n = 30), overlapping biseriate, hyaline when immature, becoming yellowish brown to brown at maturity, ellipsoidal to broadly fusiform, tapering at the ends, 3–septate, constricted at septum, widest at second septum, smooth-walled. Asexual morph: Undetermined.

Culture characteristics – Circular, surface rough, entire edge, in the middle powdery, on the edge thinly hairy, margin well-defined and slightly radiating, white and slightly raised in the middle, greenish gray at the edge; reverse white in the middle, strongly radiating, cracking the media, greenish-gray at the edges.

Material examined – ITALY, near Balze – Verghereto [province of Forlì-Cesena (FC)], on stem of Urtica dioica (Urticaceae), 19 September 2017, Erio Camporesi (MFLU 17-1694, holotype), ibid. (isotype in HKAS), ex-type living culture MFLUCC 17-2311.

GenBank accession numbers – LSU: MN597995, SSU: MN597997, ITS: MN597998.

Notes – Subplenodomus urticae was collected from dead stem of Urtica dioica. Morphologically, the present collection matches the description of S. galiicola and S. iridicola in having broad cylindrical, with club-shaped pedicel. However, S. urticae is distinct from S. galiicola in having smaller ascomata (98–162 × 111–200 μm vs. 254–285 × 311–314 μm), smaller ostiole (29–32 × 48–66 μm vs. 70–98 × 98–117 μm), shorter asci (44–65 × 7.3–10.7 μm vs. 66–120 × 12–17 μm) and smaller ascospores (19–24 × 4.4–5.6 μm vs. 30–40 × 6–9 μm. Phylogenetically, S. urticae forms an independent lineage distinct from S. iridicola and other Subplenodomus species. Subplenodomus urticae differs from S. iridicola in having shorter asci (44–65 × 7.3–10.7 μm vs. 80–100 × 10–15 μm) and shorter ascospores (19–24 × 4.4–5.6 μm vs. 21–25 × 6–7 μm). Other species of Subplenodomus are in their asexual morph and thus cannot be compared. A comparison of 528 ITS (+5.8S) nucleotides between S. urticae and S. galiicola shows 57 (9.5%) base pair difference while that of S. urticae and S. iridicola shows 69 (13.1%) base pair difference. Thus, a new taxon is introduced as S. urticae based on the recommendations provided by Jeewon and Hyde (2016).

Phylogenetic Analyses

In the multi-locus phylogeny inferred from the combined dataset of LSU, ITS, SSU, and β-tubulin, several well-supported clades can be recognized which are used for the delimitation of the 10 genera namely, Plenodomus, Alternariaster, Sphaerellopsis, Leptosphaeria, Alloleptosphaeria, Pseudoleptosphaeria, Subplenodomus, Paraleptosphaeria, and Heterospora (Figure 5). The genus Plenodomus forms a well-supported clade within the family Leptosphaeriaceae and comprised 26 strains as well as the type species Plenodomus lingam (Tode: Fr.) Höhn. The genus Alternariaster also forms a well-supported clade sister to the Plenodomus clade and included four strains namely, A. trigonosporus, A. centaureae-diffusae, A. bidentis along with A. helianthi, the type species of the genus Alternariaster. Sphaerellopsis strains together with the type strain, Sphaerellopsis filum (Biv.) B. Sutton clustered in a distinct clade, fully supported in all analyses (89% ML, 1.00 PP). Leptosphaeria sensu stricto forms a well-supported clade in the family Leptosphaeriaceae comprising L. doliolum strains, the type species, strains of 12 other species along with the new species Leptosphaeria regiae (MFLUCC 18-1137). Our new taxon, L. regiae is close to L. slovacica (CBS 125975). The monotypic genera Alloleptosphaeria and Pseudoleptosphaeria form a distinct clade sister to each other and close to the genus Neoleptosphaeria which comprise three strains with N. rubefaciens as type species. Our new taxon, Subplenodomus urticae constitutes an independent lineage and fits within the genus Subplenodomus. The genus Paraleptosphaeria forms a well-supported monophyletic clade (89% ML, 1.00 PP) with four strains together with the type species Paraleptosphaeria nitschkei. The new species Neomicrosphaeropsis juglandis clusters in the family Didymellaceae with moderate support (85% ML, 1.00 PP). The multigene analyses show that N. juglandis (MFLUCC 18-0795) is phylogenetically related to species in the genus Neomicrosphaeropsis in particular to N. italica (Figure 5). We therefore, describe the three taxa as new based on the recommendations outlined by Jeewon and Hyde (2016).

FIGURE 5

Phylogram generated from maximum likelihood and Bayesian analysis based on combined LSU, ITS, SSU, and β-tubulin sequence data retrieved from GenBank. Related sequences were referred to Chen et al. (2015), Thambugala et al. (2016), and Phookamsak et al. (2019). One hundred and sixty-nine different taxa are included in the combined genes sequence analyses which comprised 2528 characters (LSU: 1–899, ITS: 900–1510, SSU: 1511–2515, β-tubulin: 2516–2878) including gaps. Dothidotthia symphoricarpi (CPC 12929) and Neodothidotthia negundinicola (CPC 12933) are used as the out-group taxa. Maximum likelihood (ML) analysis was conducted in the CIPRES Science Gateway V.3.3. The best sorting RAxML tree with a final likelihood value of -25154.560152 is presented. Estimated base frequencies were as follows: A = 0.242800, C = 0.228484, G = 0.270168, T = 0.258548; substitution rates AC = 1.463454, AG = 3.304206, AT = 1.844561, CG = 0.804481, CT = 7.244176, GT = 1.000000; gamma distribution shape parameter α = 0.629245; proportion of invariant 0.688986. ML bootstrap values ≥50% are given as the first set of numbers and approximate likelihood-ratio test (aLRT) ≥0.90 values as the second set of numbers above the nodes. Voucher/strain numbers are given after the taxon names, the one from type material are indicated in bold face. The new taxa are given in bold and blue. The bar length indicates the number of nucleotide substitutions per site.

Discussion

Several recent papers have described saprobic fungi from the class Dothideomycetes from different hosts across the world (Pem et al., 2018, 2019a,b; Hyde et al., 2019, 2020; Phookamsak et al., 2018, 2019). This study reports on three species that are new to science collected from Turkey and Italy. They are Leptosphaeria regiae, Subplenodomus urticae from the family Leptopshaeriaceae and Neomicrosphaeropsis juglandis from the family Didymellaceae. Both Leptosphaeriaceae and Didymellaceae are highly diverse family with more than hundred species discovered during the last 10 years.

Leptosphaeria regiae forms a distinct lineage basal to L. slovacica (CBS 125975). Phylogeny recovered herein depict a close association of Leptosphaeria regiae to L. slovacica but the affinities of the latter with other species is obscure. Despite a close phylogenetic link, these two species are morphologically different. Leptosphaeria regiae differs from L. slovacica in having relatively shorter ascospores (15–18 μm vs. 18–22 μm) (de Gruyter et al., 2013). Leptosphaeria pedicularis is in its asexual state characterized by black globose perithecia, short ostiole and hyaline cylindrical conidia and cannot be morphologically compared to L. regiae. Comparison of the ITS sequences of these species shows 6.7% (L. regiae vs. L. slovacica) and 7.3% (L. regiae vs. L. pedicularis) nucleotides differences, respectively. Leptosphaeria regiae seems to be most closely allied to L. cichorium by resemblance of general morphological features such as superficial or semi-immersed globose to subglobose ascomata, peridium of schleroplectenchymatous cells and yellowish brown, fusoid, 3-septate ascospores (Ariyawansa et al., 2015). However, L. regiae has longer asci (99–130 × 9–10 μm vs. 71–115 × 5–8 μm) compared to L. cichorium. A pairwise comparison of 523 ITS (+5.8S) sequence data reveals 34 (6.8%) base pair differences between L. regiae and L. cichorium which confirms the two species as distinct. Leptosphaeria regiae also resembles L. italica in sharing superficial or semi-immersed globose ascomata and fusiform ascospores while the former differs in having longer asci (99–130 × 9–10 μm vs. 60–112 μm × 7–12 μm) (Dayarathne et al., 2015). Furthermore, L. italica occurs on Rhamnus alpinus in the Province of Forlì-Cesena in Italy while L. regiae was found on Juglans regia in the Çorum province of Turkey and they are phylogenetically apart (Figure 5). It is also worth to compare the new species L. regiae to that of L. doliolum, the type species of the genus Leptosphaeria. The former has shorter asci (99–130 × 9–10 μm vs. 105–150 × 7–10 μm) and smaller ascospores (15–18 × 6–7 μm vs. 25–30 × 4–6 μm) compared to L. doliolum which was recorded on dead stem in England (Shearer et al., 1990; Liu et al., 2015). Comparison of ITS sequence data between L. regiae and L. doliolum shows 4.4% base pair differences and these two species are far apart in the phylogenetic tree. Likewise, our new species L. regiae also bears morphological resemblance to L. urticae in having cylindric-clavate, short pedicellate asci but markedly differs from L. urticae in having shorter ascospores (15–18 × 6–7 μm vs. 35–40 × 4–6 μm) and in the number of ascospore septa (3 vs. 8–9) (Phookamsak et al., 2019). Moreover, the multi-locus phylogenetic study demonstrates that both species could be clearly differentiated with 25 (5.1%) ITS nucleotides differences between them. Leptosphaeria regiae also shares similarities to L. ebuli in having cylindrical to cylindric-clavate, short pedicellate asci. However, L. regiae has larger ascomata (315–377 × 364–410 μm vs. 226–396 × 241–251 μm) and shorter ascospores (15–18 × 6–7 vs. 23–28 × 4–5 μm) compared to L. ebuli (Liu et al., 2015). Leptosphaeria regiae and L. ebuli are phylogenetically distant and ITS DNA sequence comparison reveals 28 (5.9%) base pair differences. There are 605 estimated species in the genus Leptosphaeria but only 15 species have DNA sequence data (Species Fungorum, 2020). A synopsis of all recognized species having molecular data are provided in Table 2.

TABLE 2

Synopsis of Leptosphaeria species having DNA sequence data in GenBank.

Leptosphaeria species	Country	Sexual morph (μm)						Asexual morph (μm)			References
		Host	Ascomata	Peridium	Asci	Ascospores	Septate	Conidiomata	Conidiogeneous cells	Conidia
Leptosphaeria cichorium	Italy	Cichorium intybus	206–240 × 251–363	2.5–7.5	71–115 × 5–8	11–20 × 3– 6	3-septate	189–200 × 196–220	2–5 × 2–4	3–6 × 1–3	Ariyawansa et al., 2015
Leptosphaeria conoidea	Italy	stems of angelica (Apiaceae)			90 × 5–5.5	15–20 × 4					Saccardo, 1875
Leptosphaeria doliolum	England	dead stem	340–460 × 360–500	85–110	105–150 × 7–10	25–30 × 4–6	3-septate	Undetermined.			Ariyawansa et al., 2015
Leptosphaeria ebuli	Italy	Sambucus ebulus (Adoxaceae)	226–396 × 241–251	24–26	80–109 × 8–9	23–28 × 4–5	3-septate	Undetermined.			Liu et al., 2015
Leptosphaeria errabunda	Netherlands	Delphinium sp. (Ranunculaceae)	Description not available								de Gruyter et al., 2013
Leptosphaeria italica	Italy	Rhamnus alpinus L. ssp. Fallax (Boiss.) Marie and Petitmangin (Rhamnaceae)	285–294 × 248–260	38–40	60–112 × 7–12	12–18 × 4–6	3-septate	Undetermined.			Dayarathne et al., 2015
Leptosphaeria macrocapsa	Netherlands	Mercurialis perennis (Euphorbiaceae)									de Gruyter et al., 2013
Leptosphaeria pedicularis	Switzerland	Pedicularis sp. (Scrophulariaceae)	Description not available								de Gruyter et al., 2013
Leptosphaeria regiae	Turkey	Juglans regia (Juglandaceae)	315–377 × 364–410	41–50	99–130 × 9–10	15–18 × 6–7	3-septate				This study
Leptosphaeria sclerotioides	Canada	Medicago sativa (Fabaceae)								5–6 × 2	de Gruyter et al., 2013
Leptosphaeria slovacica	Czech Republic	Ballota nigra (Lamiaceae)	Description not available								de Gruyter et al., 2013
Leptosphaeria sydowii	Switzerland, Netherlands	Papaver rhoeas (Papaveraceae), Senecio Jacobaea (Asteraceae)	Description not available								de Gruyter et al., 2013
Leptosphaeria urticae	England	Urtica dioica (Urticaceae)	100–130 × 70–110	25–50	60–140 × 9.9–11	35–40 × 4–6	(8–)9-septate				Phookamsak et al., 2019
Leptosphaeria veronicae	Netherlands	stem of Veronica “Shirley Blue” (Scrophulariaceae)	Description not available								de Gruyter et al., 2013

There are six morphological species in the genus Subplenodomus (Species Fungorum, 2020) and all six species are described based on DNA sequence data (Tibpromma et al., 2017; Crous et al., 2018). The new species Subplenodomus urticae is morphologically similar to S. iridicola in sharing superficial or semi-immersed black ascomata and cylindrical asci with club-shaped pedicel but distinct in that the latter was described from Iris sp. (Iridaceae) from UK and has larger ascospores (21–25 × 5–7 μm vs. 19–24 × 4.4–5.6 μm) (Crous et al., 2018). A synopsis of Subplenodomus species is provided in Table 3. Phylogenetically, S. urticae clusters in the genus Subplenodomus basal to S. iridicola. Subplenodomus violicola is the type species of Subplenodomus and was established by de Gruyter et al. (2013). Since then, five additional species have been described in the genus. Subplenodomus urticae differs from S. iridicola by 13.1% nucleotide differences in the ITS regions. In our multi-gene analysis, the affinities of Subplenodomus corroborates those reported by previous studies (Schoch et al., 2009; Zhang et al., 2012; Crous et al., 2018; Phookamsak et al., 2019). Subplenodomus apiicola, S. drobnjacensis, S. valerianae, and S. violicola all produce pycnidia with an elongated neck. The pycnidial wall is pseudoparenchymatous. The new species S. urticae is unique and well-distinct among all the species reported in the genus Subplenodomus. Subplenodomus urticae is the first Subplenodomus species reported from Urtica dioica (Urticaceae) and is unique in having shorter cylindrical asci as well as ascospores compared to other species of Subplenodomus.

TABLE 3

Synopsis of Subplenodomus species having DNA sequence data in GenBank.

Subplenodomus species	Host	Country	Ascomata	Ostioles	Peridium	Asci	Ascospores	References
Subplenodomus apiicola	Apium graveolens var. rapaceum (Apiaceae)	Germany	Description not available					de Gruyter et al., 2013
Subplenodomus drobnjacensis	Gentiana makinoi “Royal Blue” (Gentianaceae)	Netherlands	Description not available					de Gruyter et al., 2013
Subplenodomus galiicola	Galium sp. (Rubiaceae)	Italy	254–285 × 311–314	70–98 × 98–117	32–60	66–120 × 12– 17	30–40 × 6–9	Tibpromma et al., 2017
Subplenodomus iridicola	Iris sp. (Iridaceae)	UK	150–250 diam	20–30 μm diam		80–100 × 10–15	(19–) 21–25 (–27) × (5–) 6 (–7) μm	Crous et al., 2018
Subplenodomus valerianae	Valeriana phu (Valerianaceae)	Netherlands	Description not available					de Gruyter et al., 2013
Subplenodomus violicola	Viola tricolor (Violaceae)	Netherlands	Description not available					de Gruyter et al., 2013
Subplenodomus urticae	Urtica dioica (Urticaceae)	Italy	98–162 × 111–200	29–32 × 48–66	10–28	44–65 × 7.3–10.7	19–24 × 4.4–5.6	This study

Our new species Neomicrosphaeropsis juglandis is an independent lineage close to N. italica. Among the several genes regions analyzed, it was noted that β-tubulin DNA sequence data generated relatively well-resolved topologies to support intergeneric relationships within the Didymellaceae and particularly in connection with Neomicrosphaeropsis (data not shown). Our new taxon is an addition to Neomicrosphaeropsis and is also the first record of the genus on Juglans regia in Turkey. A synopsis of the asexual morph of existing species of Neomicrosphaeropsis is provided in Table 4. Our new taxon is unique in that it produces larger aseptate conidia compared to other Neomicrosphaeropsis species and has been reported from a different host. Among the phenotypically diverse species, the genus Neomicrosphaeropsis as well as Didymellocamarosporium in Didymellaceae produce pigmented, muriform spores (Hyde et al., 2016; Thambugala et al., 2016). One interesting finding is also that Neomicrosphaeropsis cytisi, N. cytisicola and N. cytisina are morphologically similar with conidial measurement ranging between 4–7.9 × 2.5–3.5 μm and all of them were isolated from Cytisus sp. (Fabaceae). The authors differentiate the two species based on size and form of conidiomata. However, it is highly probable that N. cytisi, N. Cytisicola, and N. cytisina are all same species as the ITS sequences of the three species are same (no base pair difference). RPB2 comparison of N. cytisi and N. cytisicola shows only three base pair difference across 1089 nucleotides examined. No TEF and BTUB sequences are available for the three species for comparison. Likewise, N. tamaricicola, N. rossica, N. Novorossica, and N. italica have been isolated from Tamarix species and are morphologically similar with conidial measurements ranging from 3.5–6.6 × 2.5–4.6 μm. Comparison of RPB2 and BTUB gene sequences between N. italica and N. tamaricola shows no base pair difference. However, comparison of TEF gene sequences between N. italica to N. rossica and N. tamaricicola shows 7 or 1.0% and 9 or 1.3% base pair differences, respectively, while there was no base pair differences between N. rossica and N. tamaricicola. It can be possible that some of these species are the same and need to be synonymised in future studies. In our phylogenetic analyses, N. minima is closely related to N. cytisi with strong bootstrap support. Comparison of available ITS and TEF sequences of N. minima and N. cytisi reveals zero base pair difference, however, there are some slight differences in conidial sizes (N. minima: 2.8–5.4 × 9.2–3.6 μm vs. N. cytisi: 4.5–7.9 × 3–5 μm). Neomicrosphaeropsis minima also differs from N. cytisi in conidiomatal size (N. minima: 60–80 μm diam., 60–95 μm high vs. N. cytisi: 75–155 μm diam., 75–130 μm high) and were isolated from different hosts. Whether these species are distinct and merit a specific taxonomic rank warrant further investigations given that minor conidial and conidiomatal size differences could vary under cultural and host conditions. Recollecting and sequencing of more coniothyrium-like and more fungi similar to Neomicrosphaeropsis from different geographical regions are also essential to clarify the placement of species and to infer relationships in Neomicrosphaeropsis and Neomicrosphaeropsis-like genera within the Didymellaceae (Pleosporales).

TABLE 4

Synopsis of the asexual morph of existing species of Neomicrosphaeropsis.

Neomicrosphaeropsis species	Asexual morph		Hosts	Country	References
	Conidiogenous cells (μ m)	Conidial size (μ m)
Neomicrosphaeropsis alhagi-pseudalhagi	7–12 × 8–10	30–45 × 18–22	Alhagi pseudalhagi (M. Bieb.)Desv (Fabaceae)	Uzbekistan	Wanasinghe et al., 2018
Neomicrosphaeropsis cytisi	2– 4 × 9 3.5–6	4.5–7.9 × 3–5	Cytisus sp. (Fabaceae)	Italy	Hyde et al., 2016
Neomicrosphaeropsis cytisicola	–	4–7 × 2.5–3.5	Cytisus sp. (Fabaceae)	Italy	Wanasinghe et al., 2018
Neomicrosphaeropsis cytisina	1–2 × 1–2	5–7.9 × 3–5	Cytisus scoparius L. (Fabaceae)	Italy	Wanasinghe et al., 2018
Neomicrosphaeropsis elaeagni	–	16-20 × 7-9	Elaeagnus angustifolia L. (Elaeagnaceae)	Russia	Wanasinghe et al., 2018
Neomicrosphaeropsis italica	2.5–5 × 1.8–3.1	3.6–6.2 × 2.9–4.6	Tamarix sp. (Tamaricaceae)	Italy	Thambugala et al., 2016
Neomicrosphaeropsis minima	2.6–5.5 × 9.2–3.5	2.8–5.4 × 9 2–3.6	Verbascum sp. (Scrophulariaceae)	Italy	Hyde et al., 2016
Neomicrosphaeropsis novorossica	2.6–4.2 × 2–3.4	4.3–7.5 × 3.6–5.1	Tamarix ramosissima Ledeb. (Tamaricaceae)	Russia	Thambugala et al., 2016
Neomicrosphaeropsis juglandis	–	8–11 × 6–7	Juglans regiae (Juglandaceae)	Turkey	This study.
Neomicrosphaeropsis rossica	3–4.6 × 2–4	4.4–5.7 × 2.9–3.9	Tamarix ramosissima Ledeb. (Tamaricaceae)	Russia	Thambugala et al., 2016
Neomicrosphaeropsis tamaricicola	2–4 × 1.6–3.2	3.5–6.6 × 2.5–3.4	Tamarix gallica L. (Tamaricaceae)	Italy	Thambugala et al., 2016

Data Availability Statement

The datasets generated for this study can be found in the sequences retrieved from the 28S rRNA, Internal transcribed spacer rDNA region (ITS1, 5.8S rDNA, and ITS2), 18S rRNA and beta-tubulin (TUB) gene sequencing and were deposited at GenBank-NCBI under the nucleotide accession number as follows: MN244171, MN244201, MN244177 (Leptosphaeria regiae), MN244206, MN244223, MN244183, MN871954 (Neomicrosphaeropsis juglandis), MN597995, MN597998, MN597997 (Subplenodomus urticae).

Author Contributions

DP, RJ, and KH designed the study. FS did the sample collections. DP and RJ were involved in the phylogenetic analyses. SL contributed to the research funds. All authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.