Integrating Different Lines of Evidence to Establish a Novel Ascomycete Genus and Family (Anastomitrabeculia, Anastomitrabeculiaceae) in Pleosporales

Abstract

A novel genus, Anastomitrabeculia, is introduced herein for a distinct species, Anastomitrabeculia didymospora, collected as a saprobe on dead bamboo culms from a freshwater stream in Thailand. Anastomitrabeculia is distinct in its trabeculate pseudoparaphyses and ascospores with longitudinally striate wall ornamentation. A new family, Anastomitrabeculiaceae, is introduced to accommodate Anastomitrabeculia. Anastomitrabeculiaceae forms an independent lineage basal to Halojulellaceae in Pleosporales and it is closely related to Neohendersoniaceae based on phylogenetic analyses of a combined LSU, SSU and TEF1α dataset. In addition, divergence time estimates provide further support for the establishment of Anastomitrabeculiaceae. The family diverged around 84 million years ago (MYA) during the Cretaceous period, which supports the establishment of the new family. The crown and stem age of Anastomitrabeculiaceae was also compared to morphologically similar pleosporalean families.

1. Introduction

Pleosporales is the largest order within Dothideomycetes (Ascomycota) [1]. The taxonomic and phylogenetic relationships of families and genera within this order are well documented [1,2,3,4,5,6,7]. Pleosporales comprises two suborders, Massarineae and Pleosporineae [1]. Pleosporineae includes economically important plant pathogens and Massarineae includes mainly saprobes from terrestrial or aquatic environments [1,3]. Zhang et al. [1] revised 174 genera and accepted 26 families in Pleosporales. The suborder Massarineae was resurrected to accommodate five families, the Lentitheciaceae, Massarinaceae, Montagnulaceae (Didymosphaeriaceae), Morosphaeriaceae and Trematosphaeriaceae [1]. Hyde et al. [2] correlated morphology with phylogenetic evidence and accepted 41 families in this order. Tanaka et al. [3] introduced two new families, Parabambusicolaceae and Sulcatisporaceae, accepting 12 families in Massarineae. The family Longipedicellataceae was introduced, and the divergence time in Pleosporales was estimated with emphasis on Massarineae [4]. The crown age of Pleosporales was dated to 211 MYA and Massarineae was dated to 130 MYA [4]. Species boundaries in Cucurbitariaceae were revised [5] and the family, Lentimurisporaceae, was introduced in Pleosporales [6].

Species in this order are abundant and occur in terrestrial, marine and freshwater habitats [7,8,9]. The species can be epiphytes, endophytes or parasites of living leaves or stems, hyperparasites on fungi or insects, lichenized, or saprobes of dead plant stems, leaves or bark [7,8,9]. Currently, about 400 genera in 64 families are known in Pleosporales [1,2,7,10,11,12,13], with numerous coelomycetous and hyphomycetous taxa as their asexual morphs [1,13,14,15].

Several pleosporalean taxa are pathogens associated with a broad range of hosts including bamboo. Bamboo (Poaceae) comprises over 115 genera with around 1500 species [16,17,18], can be found in diverse climates [17], and are widely distributed in various forest types in Thailand [18,19]. It has been estimated that around 1100 fungal species belonging to over 200 genera have been described or recorded worldwide on bamboo and most of these bamboo-associated fungi are ascomycetes [20,21].

Divergence time estimates using molecular clock methodologies have been widely used in fungal taxonomy [4,11,22,23,24,25,26,27]. Several studies have applied molecular dating to provide additional evidence for higher taxa ranking in Pleosporales [4,6,7,11]. In this study, we introduce a novel bambusicolous species, Anastomitrabeculia didymospora within Anastomitrabeculia, which is accommodated in a new family, Anastomitrabeculiaceae, based on morphology, multi-loci phylogeny and divergence times estimates.

2. Materials and Methods

2.1. Sample Collection, Isolation and Identification

Dead bamboo culms were collected from a freshwater stream from Krabi province, Thailand, in 2015. The samples were incubated in plastic boxes with sterile and moist tissue at 25–30 °C for 3 days. Pure fungal colonies were obtained using single-spore isolation [28]. Germinating spores were transferred aseptically to potato dextrose agar (PDA) and malt extract agar (MEA) (Difco™). The cultures were incubated at 25 °C with frequent observations. Fungal characters were observed using a stereo microscope (Zeiss SteREO Discovery v. 8) fitted with an Axio Cam ERc5S and a Leica DM2500 compound microscope attached with a Leica MC190 HD camera. All microscopic measurements were carried out using Tarosoft (R) Image Frame Work program and the images were processed with Adobe Photoshop CS6 version 13.0 software (Adobe Systems, San Jose, CA, USA). The type specimens were deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand, and pure cultures were deposited at the Mae Fah Luang University Culture Collection (MFLUCC). The new taxon was linked with Facesoffungi numbers (FoF) [29] and Index Fungorum (Index Fungorum 2020, http://www.indexfungorum.org/, accessed on 2 December 2020) and established based on guidelines recommended by Jeewon and Hyde [30].

2.2. DNA Extraction, PCR Amplification and DNA Sequencing

DNA extraction, PCR amplification, DNA sequencing and phylogenetic analysis were carried out as detailed in Dissanayake et al. [31]. Total genomic DNA was extracted from fresh mycelium with a Biospin Fungus Genomic DNA Extraction Kit (BioFlux^®) (Hangzhou, P.R. China) following the manufacturer’s protocol. The nuclear ribosomal large subunit 28S rRNA gene (LSU) [32], the nuclear ribosomal small subunit 18S rRNA gene (SSU) [33] and the translation elongation factor 1-alpha gene (TEF1α) [34] were amplified using primers (LSU: LROR/LR5, SSU: NS1/NS4 and TEF1α: 983F/2218R). Polymerase chain reaction (PCR) was performed using PCR mixtures containing 5–10 ng DNA, 1X PCR buffer, 0.8 units Taq polymerase, 0.3 μM of each primer, 0.2 mM dNTP and 1.5 mM MgCl₂. PCR conditions were set at an initial denaturation for 3 min at 94 °C, followed by 40 cycles of 45 s of denaturation at 94 °C, annealing for 50 s at 56 °C for LSU, SSU and 52 °C for TEF1α and extension for 1 min at 72 °C, with a final extension of 10 min at 72 °C. All the PCR products were visualised on 1% Agarose gels with added 6 μL of 4S green dyes, per each 100 mL. Successful PCR products were purified and sequencing was performed by Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai, P.R. China). All sequences generated in this study were submitted to GenBank (Table 1) and the ITS region of Anastomitrabeculia didymospora was deposited with the accession number MW413900 (MFLUCC 16-0412) and MW413897 (MFLUCC 16-0417).

Table 1.

DNA sequences and GenBank numbers used for the phylogenetic analyses in this study. The ex-type strains are in bold and the new taxon introduced in this study is indicated in blue.

Taxon	Strain Number	GenBank Accession Numbers
		LSU	SSU	TEF1α
Acrocalymma aquatica	MFLUCC 11-0208	JX276952	JX276953	-
Acrocalymma fici	CBS 317.76	KP170712	-	-
Acrocalymma medicaginis	CPC 24340	KP170713	-	-
Acrocalymma medicaginis	CPC 24341	KP170714	-	-
Acrocalymma medicaginis	CPC 24345	KP170718	-	-
Acrocalymma pterocarpi	MFLUCC 17-0926	MK347949	MK347840	-
Aigialus grandis	BCC 20000	GU479775	GU479739	GU479839
Aigialus mangrovis	BCC 33563	GU479776	GU479741	GU479840
Aigialus parvus	BCC 18403	GU479778	GU479743	GU479842
Aigialus rhizophorae	BCC 33572	GU479780	GU479745	GU479844
Aliquandostipite khaoyaiensis	CBS 118232	GU301796	-	GU349048
Amniculicola immersa	CBS 123083	FJ795498	GU456295	GU456273
Amniculicola lignicola	CBS 123094	EF493861	EF493863	-
Amniculicola parva	CBS 123092	GU301797	GU296134	GU349065
Amorosia littoralis	NN 6654	AM292055	AM292056	-
Anastomitrabeculia didymospora	MFLUCC 16-0412	MW412978	MW412977	MW411338
Anastomitrabeculia didymospora	MFLUCC 16-0417	MW413899	MW413898	MW411339
Angustimassarina populi	MFLUCC 13-0034	KP888642	KP899128	KR075164
Angustimassarina quercicola	MFLUCC 14-0506	KP888638	KP899124	KR075169
Anteaglonium abbreviatum	ANM 925a	GQ221877	-	-
Anteaglonium globosum	SMH 5283	GQ221911	-	GQ221919
Anteaglonium parvulum	MFLUCC 14-0821	KU922915	KU922916	-
Antealophiotrema brunneosporum	CBS 123095	LC194340	LC194298	LC194382
Aquasubmersa japonica	HHUF 30468	LC061586	LC061581	-
Aquasubmersa japonica	HHUF 30469	LC061587	LC061582	-
Aquasubmersa mircensis	MFLUCC 11-0401	JX276955	JX276956	-
Arthonia dispersa	UPSC 2583	AY571381	AY571379	-
Ascocratera manglicola	BCC 09270	GU479782	GU479747	GU479846
Ascocylindrica marina	MD6011	KT252905	KT252907	-
Ascocylindrica marina	MD6012	KT252906	-	-
Ascocylindrica marina	MF416	MK007123	MK007124	-
Bahusandhika indica	GUFCC 18001	KF460274	-	-
Bambusicola massarinia	MFLUCC 11-0389	JX442037	JX442041	-
Berkleasmium micronesicum	BCC 8141	DQ280272	DQ280268	-
Berkleasmium nigroapicale	BCC 8220	DQ280273	DQ280269	-
Bimuria novae-zelandiae	CBS 107.79	AY016356	AY016338	DQ471087
Botryosphaeria dothidea	CBS 115476	AY928047	EU673173	AY236898
Brevicollum hyalosporum	MAFF 243400	LC271239	LC271236	LC271245
Brevicollum hyalosporum	MFLUCC 17-0071	MG602200	MG602202	MG739516
Brevicollum hyalosporum	PUFNI 17628	MH918671	-	-
Brevicollum versicolor	HHUF 30591	LC271240	LC271237	LC271246
Capnodium salicinum	CBS 131.34	DQ678050	DQ677997	-
Cladosporium cladosporioides	CBS 170.54	DQ678057	DQ678004	-
Clematidis italica	MFLUCC 15-0084	KU842381	KU842382	-
Corynespora cassiicola	CBS 100822	GU301808	GU296144	GU349052
Corynespora smithii	CABI 5649b	GU323201	-	GU349018
Crassiparies quadrisporus	HHUF 30590	LC271241	LC271238	LC271248
Crassiparies quadrisporus	HHUF 30409	LC100025	LC100017	-
Crassiperidium octosporum	KT 2144	LC373108	LC373084	LC373120
Crassiperidium octosporum	KT 2894	LC373109	LC373085	LC373121
Crassiperidium octosporum	KT 3008	LC373110	LC373086	LC373122
Crassiperidium octosporum	KT 3029	LC373111	LC373087	LC373123
Crassiperidium octosporum	KT 3046	LC373112	LC373088	LC373124
Crassiperidium octosporum	KT 3188	LC373113	LC373089	LC373125
Crassiperidium octosporum	KT 3468	LC373114	LC373090	LC373126
Crassiperidium octosporum	KT 3604	LC373115	LC373091	LC373127
Crassiperidium octosporum	KT 3605	LC373116	LC373092	LC373128
Crassiperidium octosporum	MM 9	LC373117	LC373093	LC373129
Crassiperidium quadrisporum	KT 27981	LC373118	LC373094	LC373130
Crassiperidium quadrisporum	KT 27982	LC373119	LC373095	LC373131
Cryptoclypeus oxysporus	HHUF 30507	LC194345	LC194303	LC194390
Cryptocoryneum akitaense	MAFF 245365	LC194348	LC194306	LC096136
Cryptocoryneum japonicum	MAFF 245370	LC194356	LC194314	LC096144
Cryptocoryneum longicondensatum	MAFF 245374	LC194360	LC194318	LC096148
Cyclothyriella rubronotata	CBS 141486	KX650544	KX650507	KX650519
Cyclothyriella rubronotata	CBS 121892	KX650541	-	KX650516
Cyclothyriella rubronotata	CBS 385.39	MH867543	-	-
Cyclothyriella rubronotata	CBS 419 85	GU301875	-	GU349002
Delitschia didyma	UME 31411	DQ384090	AF242264	-
Delitschia winteri	CBS 225.62	DQ678077	DQ678026	DQ677922
Dendrographa decolorans	Ertz 5003	AY548815	AY548809	-
Dendrographa leucophaea f. minor		AF279382	AF279381	-
Dendryphion europaeum	CPC 22943	KJ869203	-	-
Dendryphion europaeum	CPC 23231	NG_059120	-	-
Dendryphion nanum	MFLUCC 16-0975	MG208132	-	MG207983
Didymosphaeria rubi-ulmifolii	MFLUCC 14-0023	KJ436586	KJ436588	-
Dissoconium aciculare	CBS 204.89	GU214419	GU214523	-
Ernakulamia cochinensis	PRC 3992	LT964670	-	-
Flavomyces fulophazii	CBS 135761	KP184040	KP184082	-
Fuscostagonospora cytisi	MFLUCC 16-0622	KY770978	KY770977	KY770979
Fuscostagonospora sasae	CBS 139687	AB807548	AB797258	-
Fusculina eucalyptorum	CBS 145083	MK047499	-	-
Gordonomyces mucovaginatus	CBS 127273	JN712552
Halojulella avicenniae	JK 5326A	GU479790	GU479756	-
Halojulella avicenniae	BCC 20173	GU371822	GU371830	GU371815
Halojulella avicenniae	PUFD542	MK026757	MK026754	-
Halojulella avicenniae	BCC 18422	GU371823	GU371831	GU371816
Halojulella avicenniae	BCC28357	KC555567	KC555565	-
Halojulella avicenniae	GR00584	KC555568	KC555566	-
Halotthia posidoniae	BBH 22481	GU479786	GU479752	-
Helminthosporium aquaticum	MFLUCC 15-0357	KU697306	KU697310	-
Helminthosporium velutinum	MAFF 243854	AB807530	AB797240	-
Helminthosporium velutinum	MFLUCC 13-0243	KU697305	-	-
Helminthosporium velutinum	MFLUCC 15-0423	KU697304	-	-
Hermatomyces iriomotensis	HHUF 30518	LC194367	LC194325	LC194394
Hermatomyces tectonae	MFLUCC 14-1140	KU764695	KU712465	KU872757
Hermatomyces thailandica	MFLUCC 14-1143	KU764692	KU712468	KU872754
Hobus wogradensis	TI	KX650546	KX650508	KX650521
Hysterium angustatum	CBS 236.34	FJ161180	GU397359	FJ161096
Hysterium angustatum	MFLUCC 16-0623	MH535893	MH535885	MH535878
Jahnula seychellensis	SS2113	EF175665	EF175643	-
Latorua caligans	CBS 576.65	KR873266	-	-
Latorua grootfonteinensis	CBS 369.72	KR873267	-	-
Lentimurispora urniformis	MFLUCC 18-0497	MH179144	MH179160	MH188055
Leptosphaeria doliolum	CBS 505.75	GQ387576	GQ387515	GU349069
Leptoxyphium cacuminum	MFLUCC 10-0049	JN832602	JN832587	-
Leucaenicola phraeana	MFLUCC 18-0472	MK348003	MK347892	-
Lignosphaeria fusispora	MFLUCC 11-0377	KP888646	-	-
Lignosphaeria thailandica	MFLUCC 11-0376	KP888645	-	-
Lindgomyces ingoldianus	ATCC 200398	AB521736	AB521719	-
Longiostiolum tectonae	MFLUCC 12 0562	KU764700	KU712459	-
Lophiotrema eburnoides	HHUF 30079	LC001707	LC001706	-
Lophiotrema nucula	CBS 627.86	GU301837	GU296167	GU349073
Macrodiplodiopsis desmazieri	CBS 140062	KR873272	-	-
Magnicamarosporium diospyricola	MFLUCC 16-0419	KY554212	KY554211	KY554209
Massarina eburnea	CBS 473.64	GU301840	GU296170	-
Massariosphaeria phaeospora	CBS 611.86	GU301843	GU296173	-
Mauritiana rhizophorae	BCC 28866	GU371824	GU371832	GU371817
Medicopsis romeroi	CBS 122784	EU754208	EU754109	KF015679
Medicopsis romeroi	CBS 252.60	EU754207	EU754108	KF015678
Medicopsis romeroi	CBS 132878	KF015622	KF015648	KF015682
Murispora rubicunda	IFRD 2017	FJ795507	GU456308	GU456289
Neoastrosphaeriella krabiensis	MFLUCC 11-0025	JN846729	JN846739	-
Neohendersonia kickxii	CBS 112403	KX820266	-	-
Neohendersonia kickxii	CBS 122938	KX820268	-	-
Neohendersonia kickxii	CBS 114276	KX820267	-	-
Neohendersonia kickxii	CPC 24865	KX820270	-	-
Neohendersonia kickxii	CBS 122941	KX820269	-	-
Neomassaria fabacearum	MFLUCC 16-1875	KX524145	KX524147	KX524149
Neomassaria formosana	NTUCC 17-007	MH714756	MH714759	MH714762
Neomassarina chromolaenae	MFLUCC 17-1480	MT214466	MT214419	MT235785
Neomassarina pandanicola	MFLUCC 16-0270	MG298945	-	MG298947
Neomassarina thailandica	MFLUCC 10-0552	KX672157	KX672160	KX672163
Neomassarina thailandica	MFLUCC 17-1432	MT214467	MT214420	MT235786
Neotorula aquatica	MFLUCC 150342	KU500576	KU500583	-
Neotorula submersa	KUMCC 15-0280	KX789217	-	-
Occultibambusa bambusae	MFLUCC 13-0855	KU863112	KU872116	-
Occultibambusa pustula	MFLUCC 11-0502	KU863115	KU872118	-
Ohleria modesta	MGC	KX650562	-	KX650533
Ohleria modesta	CBS 141480	KX650563	KX650513	KX650534
Paradictyoarthrinium diffractum	MFLUCC 13-0466	KP744498	KP753960	-
Paradictyoarthrinium diffractum	MFLUCC 12-0557	KP744497	-	-
Paradictyoarthrinium hydei	MFLUCC 13-0465	MG747497	-	-
Paradictyoarthrinium tectonicola	MFLUCC 13-0465	KP744500	KP753961	-
Paradictyoarthrinium tectonicola	MFLUCC 12-0556	KP744499	-	-
Periconia thailandica	MFLUCC 17-0065	KY753888	KY753889	-
Phaeoseptum aquaticum	CBS 123113	JN644072	-	-
Phaeoseptum terricola	MFLUCC 10-0102	MH105779	MH105780	MH105781
Phyllosticta capitalensis	CBS 226.77	KF206289	KF766300	-
Piedraia hortae	CBS 480.64	GU214466	-	-
Polyplosphaeria fusca	CBS 125425	AB524607	AB524466	AB524822
Preussia lignicola	CBS 363.69	DQ384098	DQ384087	-
Preussia lignicola	CBS 264.69	GU301872	GU296197	GU349027
Pseudoastrosphaeriella bambusae	MFLUCC 11-0205	KT955475	KT955455	KT955437
Pseudoastrosphaeriella longicolla	MFLUCC 11-0171	KT955476	KT955456	KT955438
Pseudoastrosphaeriella thailandensis	MFLUCC 10-0553	KT955477	KT955456	KT955439
Pseudolophiotrema elymicola	HHUF 28984	LC194381	LC194339	LC194418
Pseudomassariosphaeria bromicola	MFLUCC 15-0031	KT305994	KT305996	KT305999
Pseudotetraploa curviappendiculata	CBS 125426	AB524610	AB524469	AB524825
Quadricrura septentrionalis	CBS 125428	AB524617	AB524476	AB524832
Racodium rupestre	L346	EU048583	EU048575	-
Racodium rupestre	L424	EU048582	EU048577	-
Ramusculicola thailandica	MFLUCC 13-0284	KP888647	KP899131	KR075167
Rimora mangrovei	JK 5246A	GU301868	GU296193
Roccella fuciformis	Tehler 8171	FJ638979	-	-
Rostriconidium aquaticum	KUMCC 15-0297	MG208144	-	MG207995
Rostriconidium aquaticum	MFLUCC 16-1113	MG208143	-	MG207994
Salsuginea ramicola	KT 2597.1	GU479800	GU479767	GU479861
Salsuginea ramicola	CBS 125781	MH877872	-	-
Scorias spongiosa	CBS 325.33	MH866910	GU214696	-
Seriascoma didymospora	MFLUCC 11-0179	KU863116	KU872119	-
Sigarispora arundinis	JCM 13550	AB618998	AB618679	LC001737
Sigarispora ravennica	MFLUCC 14-0005	KP698414	KP698415	-
Splanchnonema platani	CBS 222.37	KR909316	KR909318	KR909319
Sporidesmioides thailandica	KUMCC 16-0012	KX437758	KX437760	KX437767
Sporidesmioides thailandica	MFLUCC 13-0840	NG_059703	NG_061242	KX437766
Sporormia fimetaria	UPS:Dissing Gr.81.194	GQ203729	-	-
Sporormiella minima	CBS 52450	DQ468046	-	DQ468003
Stagonospora pseudocaricis	CBS 135132	KF251762	KF251259	KF252741
Stemphylium vesicarium	CBS 191.86	DQ247804	DQ247812	DQ471090
Stemphylium vesicarium	CBS 714.68	DQ678049	DQ767648	DQ677888
Sulcatispora acerina	KT2982	LC014610	LC014605	LC014615
Sulcosporium thailandicum	MFLUCC 12-0004	KT426563	KT426564	-
Teichospora quercus	CBS 143396	MH107966	-	MH108030
Tetraplosphaeria sasicola	KT 563	AB524631	AB524490	AB524838
Torula gaodangensis	MFLUCC 17-0234	NG_059827	NG_063641	-
Torula herbarum	CBS 111855	KF443386	KF443391	KF443403
Triplosphaeria maxima	MAFF 239682	AB524637	AB524496	-
Tubeufia chiangmaiensis	MFLUCC 11-0514	KF301538	KF301543	KF301557
Tubeufia javanica	MFLUCC 12-0545	KJ880036	KJ880035	KJ880037
Vargamyces aquaticus	CBS 639.63	KY853539	-	-
Vargamyces aquaticus	HKUCC 10830	DQ408575	-	-
Versicolorisporium triseptatum	HHUF 28815	AB330081	AB524501	-
Westerdykella dispersa	CBS 297.56	MH869191	-	-
Westerdykella ornata	CBS 379.55	GU301880	GU296208	GU349021
Xenomassariosphaeria rosae	MFLUCC 15-0179	MG829092	MG829192	-

2.3. Phylogenetic Analysis

The sequence data were assembled using BioEdit v. 7.2.5 [35] and subjected to a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find the closest matches with taxa in Pleosporales. Reference sequence data of this order and some representatives of other orders of Dothideomycetes were downloaded from previously published studies [1,6,36,37,38,39]. The sequences were automatically aligned using default settings in MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/) [40]. A combined dataset of three gene regions (LSU, SSU and TEF1α) was prepared and manually adjusted using BioEdit and AliView [41]. Phylogenetic analyses of the combined dataset were performed using maximum likelihood, maximum parsimony and Bayesian inference method. Maximum likelihood analyses (ML), including 1000 bootstrap pseudoreplicates, were performed at the CIPRES web portal [42] using RAxML v. 8.2.12 [43]. Maximum parsimony analysis was conducted using PAUP v.4.0b 10 [44] with the heuristic search option and number of replications 1000 each. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were documented.

The best model for different genes partition was determined in JModelTest version 2.1.10 [45] for posterior probability (PP). The general time reversible (GTR) model with a discrete gamma distribution plus invariant site (GTR+I+G) substitution model was used for the combined dataset. Posterior probabilities [46] were estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.6 [47]. Four simultaneous Markov chains were run for 10 million generations and trees were sampled every 1000th generation, thus resulting in 10,000 trees. The suitable burn-in phase was determined by inspecting traces in Tracer version 1.7 [48]. The first 10% of generated trees representing the burn-in phase of the analyses were discarded, while the remaining trees were used to calculate posterior probabilities (PP) in the majority rule consensus tree. The phylograms were visualized with FigTree v1.4.0 program [49] and edited using Adobe Illustrator CS6 v15.0 (Adobe Systems, USA).

2.4. Fossil Calibration and Divergence Time Estimates

Divergence times were estimated with BEAST 2.6.2 [50] based on the methodology described in Phukhamsakda et al. [4]. The aligned sequence dataset (LSU, SSU and TEF1α) used for the phylogenetic analyses were loaded into BEAUTI 2.6.2 to prepare the XML file. Nucleotide substitution models were determined using JModelTest version 2.1.10. The GTR+I+G nucleotide substitution model was applied to LSU and TEF1α partitions. The symmetrical (SYM) model with a discrete gamma distribution plus invariant site (SYM+I+G) substitution model was applied to the SSU partition. The data partitions were set with unlinked substitution, linked clock model and linked tree. An uncorrelated relaxed clock model with lognormal distribution was used. The Yule speciation process, which assumes a constant rate of speciation divergence, was used as the tree prior [51]. The analysis was performed in BEAST 2.6.2 for 100 million generations, sampling every 1000 generations. The effective sample size (ESS) was analysed with Tracer version 1.7 to check that the values were greater than 200, as recommended by Drummond et al. [52]. The first 20% trees were discarded as the burn-in phase and the remaining trees were combined in LogCombiner 2.6.2. The maximum clade credibility was calculated in TreeAnnotator v 2.6.2. The phylograms were visualized with FigTree v.1.4.0 program.

To estimate the divergence time for Anastomitrabeculiaceae, the fossil Metacapnodium succinum (Metacapnodiaceae) was used to set the crown age of Capnodiales using a normal distribution, mean of 100 MYA, SD of 150 MYA, giving 95% credibility interval of 346 MYA [4,23,53,54]. The fossil Margaretbarromyces dictyosporus was used to calibrate the crown age of Aigialus (Aigialaceae) using a gamma distribution, with an offset of 35 MYA, a shape of 1.0, scale of 25, providing 95% credibility interval of 110 MYA [4,55,56,57]. The split between Arthoniomycetes (outgroup) and Dothideomycetes was used as the secondary calibration using a normal distribution, mean of 300 MYA, SD of 50 MYA, giving 95% credibility interval of 382 MYA [22,36,53,54].

3. Results

3.1. Phylogenetic Analyses

The combined gene alignment comprised 196 strains and 2800 characters (LSU: 860 characters, SSU: 1039 characters and TEF1α: 901 characters). Among the 2800 characters, there were 1492 conserved sites (53%), 364 variable sites (13%) and 944 parsimony informative sites (34%). The parsimony analysis of the data matrix yielded one most parsimonious tree out of 1000 (CI = 0.265, RI = 0.659, RC = 0.175, HI = 0.735, Tree Length = 7606). Based on BLAST search in the NCBI GenBank of the LSU gene, the newly generated taxon MFLUCC 16-0412 and MFLUCC 16-0417 show 95% similarity to Crassiperidium quadrisporum (KT 27981 and KT 27982). The topology of the phylogenetic tree based on the LSU gene was generally congruent with the overall topology of the tree based on the combined dataset. Phylogenetic trees generated from maximum likelihood, maximum parsimony and Bayesian analysis of the combined dataset resulted in similar topologies with some exception. The position of Cyclothyriellaceae and Longiostiolaceae differed between the three methods. The best scoring RAxML tree had a final likelihood value of −40,523.297855 (Figure 1). The new taxon formed an independent lineage basal to the Halojulellaceae with strong Bayesian inference support and moderate support from maximum likelihood (0.99 PP/65% MLBT). A new genus Anastomitrabeculia is therefore introduced within Anastomitrabeculiaceae to accommodate the new species.

Figure 1.

The best scoring RAxML tree based on a combined LSU, SSU and TEF1α dataset. RAxML bootstrap support and maximum parsimony values ≥60% (BT), as well as Bayesian posterior probabilities ≥0.90 (BYPP) are shown, respectively, near the nodes. The ex-type strains are in bold and the scale bar indicates 0.06 changes per site. The tree is rooted with species of Arthoniomycetes and the new taxon is indicated in blue.

3.2. Fossil Calibration and Divergence Time Estimates

The topology of the maximum clade credibility (MCC) tree (Figure 2) was congruent with the tree obtained from the Bayesian inference analysis and the maximum likelihood analysis. The divergence times of the dating analysis are listed in Table 2. The crown age of Dothideomycetes is estimated at 263 MYA during the Permian period based on the MCC tree. The split of Arthoniomycetes and Dothideomycetes occurred around 323 MYA during the Carboniferous period. The crown age of Pleosporales is estimated at 206 MYA, and Hysteriales diverged from Pleosporales approximately 236 MYA during the Triassic period. The crown age of Anastomitrabeculiaceae is estimated at around 2.6 MYA, and it diverged from Halojulellaceae at around 84 (52–116) MYA. Anastomitrabeculiaceae formed an independent lineage with close relationship to Halojulellaceae with strong posterior probability in the MCC tree (0.99 BYPP). The divergence time of Anastomitrabeculiaceae was compared to Pleosporalean families with trabeculate pseudoparaphyses, cylindrical asci and ascospores with a sheath (Table 3). The divergence time of Anastomitrabeculiaceae was also compared to Didymosphaeriaceae as they are morphologically similar by having trabeculate pseudoparaphyses and cylindrical asci.

Figure 2.

Maximum clade credibility (MCC) tree of families in Dothideomycetes using BEAST. Numbers at nodes indicate posterior probabilities (PP) for node support. Bars correspond to the 95% highest posterior density (HPD) intervals. Posterior probabilities greater than 0.95 are given near the nodes. The new taxon is indicated in blue. Geological time scales are given at the base together with scale in million years ago (MYA) [58].

Table 2.

Divergence time estimates obtained from BEAST analysis. The median and the 95% Highest Posterior Density are provided in million years ago (MYA). The geological time scales are given based on the median node age.

Nodes	Node Age	Geological Time Period
Arthoniomycetes–Dothideomycetes	323 (310–349)	Carboniferous
Dothideomycetes crown group	263 (216–313)	Permian
Hysteriales–Pleosporales	236 (188–300)	Triassic
Pleosporales crown group	206 (171–254)	Triassic
Capnodiales crown group	147 (99–200)	Jurassic
Anastomitrabeculiaceae stem group	84 (52–116)	Cretaceous
Aigialaceae–Aigialus sp.	37 (18–56)	Eocene
Anastomitrabeculiaceae crown group	2.6 (0.19–6.61)	Neogene

Table 3.

Divergence time estimates obtained from BEAST analysis for families with similar morphology to Anastomitrabeculiaceae. The crown age and the stem age are provided in million years ago (MYA).

Families	Crown Age	Stem Age
Aigialaceae	102	141
Amniculicolaceae	90	177
Anastomitrabeculiaceae	2.6	84
Anteagloniaceae	52	98
Bambusicolaceae	29	57
Cyclothyriellaceae	66	95
Delitschiaceae	78	131
Didymosphaeriaceae	47	81
Fuscostagonosporaceae	26	63
Lindgomycetaceae	31	92
Neomassariaceae	82	131
Pseudoastrosphaeriellaceae	56	147
Tetraplosphaeriaceae	91	189

3.3. Taxonomy

Anastomitrabeculiaceae Bhunjun, Phukhams and K.D. Hyde, fam. nov.

Index Fungorum number: IF556817, Facesoffungi number: FoF 09521.

Etymology: Referring to the name of the type genus.

Saprobic on dead bamboo culms submerged in freshwater. Sexual morph: Ascomata immersed under a clypeus to semi-immersed, gregarious, uniloculate, globose to subglobose, carbonaceous, black. Ostiole central, apex well developed. Peridium multi-layered, sub-carbonaceous or coriaceous, with dark brown to hyaline cells arranged in a textura angularis. Hamathecium composed of numerous, filamentous, trabeculate pseudoparaphyses, septate, anastomosing between the asci and at the apex. Asci bitunicate, fissitunicate, broad cylindrical to cylindrical-clavate, bulbous pedicel, with an ocular chamber. Ascospores biseriate, broadly fusiform, septate, smooth-walled, with wall ornamentation, surrounded by mucilaginous sheath.

Note: Anastomitrabeculiaceae is introduced to include Anastomitrabeculia, which is reported as a saprobe on bamboo culms. Anastomitrabeculiaceae is characterised by semi-immersed, coriaceous or carbonaceous ascomata with septate, trabeculate pseudoparaphyses and hyaline ascospores with longitudinally striate wall ornamentation, surrounded by mucilaginous sheath. Anastomitrabeculiaceae formed a well-supported independent lineage closely related to Halojulellaceae, but Halojulellaceae differs by its cellular pseudoparaphyses and golden-brown ascospores.

Type genus: Anastomitrabeculia Bhunjun, Phukhams and K.D. Hyde.

• Anastomitrabeculia Bhunjun, Phukhams. and K.D. Hyde, gen. nov.

• Index Fungorum number: IF556560, Facesoffungi number: FoF 09522.

• Etymology: Referring to the trabeculate pseudoparaphyses anastomosing between the asci and at the apex.

Colonies on natural substrate umbonate at the centre, circular, black shiny dots are visible on the host surface. Ascomata on surface of the host, immersed under a clypeus, gregarious, uniloculate, subglobose, carbonaceous. Ostiole orange pigment near ostiole. Peridium comprising multilayers of brown to hyaline cells of textura angularis, inner layers composed of thin, hyaline cells. Asci 8–spored, bitunicate, fissitunicate, broad cylindrical to cylindrical-clavate, with a bulbous pedicellate, rounded at the apex, with an ocular chamber. Ascospores biseriate, broadly fusiform, tapering towards the ends, hyaline, with guttules in each cell, constricted at the septa, with longitudinally striate wall ornamentation, surrounded by mucilaginous sheath.

Note: Anastomitrabeculia is established as a monotypic genus. It is characterised by the presence of carbonaceous ascomata, with orange pigment near ostiole and ascospores with longitudinally striate wall ornamentation. Anastomitrabeculia is morphologically similar to members of Pleosporales in having perithecioid ascomata, bitunicate asci and hyaline ascospores.

Type species: Anastomitrabeculia didymospora Bhunjun, Phukhams and K.D. Hyde.

• Anastomitrabeculia didymospora Bhunjun, Phukhams and K.D. Hyde, sp. nov.

• Index Fungorum number: IF556559; Facesoffungi number: FoF 09523 Figure 3.

• Etymology: Referring to the didymosporous ascospores.

• Holotype–MFLU 20-0694.

Figure 3.

Anastomitrabeculia didymospora (MFLU 20-0694, holotype). (a) Ascomata on bamboo. (b) Close-up of ascomata. (c) Vertical section of ascoma. (d) Ostiolar canal. (e) Peridium layer. (f) Trabeculate pseudoparaphyses. (g–i) Asci. (j) Pedicel. (k–o) Ascospores showing mucilaginous sheath. (p) Culture characteristics on PDA from above and below (9 cm diameter petri dish). Scale bar: (b) = 500 µm, (c) = 200 µm, (d–i) = 50 µm, (j–o) = 10 µm.

Saprobic on dead bamboo culms submerged in freshwater. Sexual morph: Ascomata 430–460 μm high, 435–575 μm diam., immersed under a clypeus to semi-immersed, gregarious, uniloculate, globose to subglobose, carbonaceous, rough, black, ostiolate. Ostiole 160 μm high, 270 μm diam., central, apex well developed, papillate, with pore-like opening, with periphyses filling the ostiolar canal, dark brown to black, orange pigment near ostiole. Peridium 6–18 μm wide, comprising 3–5 layers of brown to hyaline cells of textura angularis, inner layers composed of thin, hyaline cells. Hamathecium of dense, long, 0.8–1.25 µm wide ($\overline{x}$ = 1 μm, n = 50), filiform, filamentous, trabeculate pseudoparaphyses, septate, branched, embedded in a gelatinous matrix, anastomosing between the asci and at the apex. Asci 125–160 × 15–20 μm ($\overline{x}$ = 145 × 17 μm, n = 20), 8–spored, bitunicate, fissitunicate, broad cylindrical to cylindrical-clavate, with bulbous pedicellate, rounded at the apex, with an ocular chamber. Ascospores 18–28 × 7–10 μm ($\overline{x}$ = 22.5× 9 μm, n = 20), biseriate, broadly fusiform, tapering towards the ends, hyaline, 1-septate at the centre, constricted at the septum, cell above septate enlarged, straight, smooth-walled, with longitudinally striate wall ornamentation, surrounded by mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Ascospores germinating on MEA and PDA within 24 h with germ tubes developing from basal cells. Colonies on MEA and PDA umbonate at the centre, circular, friable, reaching 20 mm diameter after four weeks of incubation at 25 °C. Culture on MEA with white aerial mycelium, dark brown at the centre and paler towards the edge from above and below. Culture on PDA dark brown from above and below.

Material examined: THAILAND, Krabi province (8.1° N, 98.9° E), on dead bamboo culms, 15 December 2015, C. Phukhamsakda, KR001 (MFLU 20-0694, holotype), ibid, 18 December 2015 (MFLU 20-0695, paratype); ex-type living culture MFLUCC 16-0412; ex-paratype living culture, MFLUCC 16-0417.

4. Discussion

In this study, we introduce a new species, genus and family for a collection of Pleosporales found on bamboo. The introduction of new taxa, even at the family level, is not surprising, considering that about 93% of fungi remain unknown to science despite ca. 2000 species described every year [59,60]. Pleosporalean species can occur in terrestrial, marine and freshwater habitats [7,8,9]. Several studies have reported new pleosporalean taxa from freshwater or marine habitats or from bambusicolous hosts [1,3]. Pleosporales have unique characters such as perithecioid ascomata typically with a papilla and bitunicate, generally fissitunicate asci, bearing mostly septate ascospores of different colours and shapes, with or without a gelatinous sheath [7]. The morphology of Anastomitrabeculiaceae is similar to members of the Pleosporales based on the presence of pseudoparaphyses, perithecioid ascomata, bitunicate asci and hyaline ascospores. Anastomitrabeculiaceae is characterised by semi-immersed to superficial ascomata, trabeculate pseudoparaphyses, cylindrical asci and ascospores with longitudinally striate wall ornamentation, surrounded by mucilaginous sheath. The newly discovered species formed a well-supported independent lineage basal to the Halojulellaceae based on phylogenetic analyses of the combined dataset (0.99 PP/65% MLBT). Halojulellaceae differs by its cellular pseudoparaphyses and golden brown ascospores [2]. The new taxon is also phylogenetically closely related to Neohendersoniaceae, which differs by its cellular pseudoparaphyses and smooth-walled ascospore [61]. A novel genus Anastomitrabeculia is therefore introduced to accommodate one new species, Anastomitrabeculia didymospora. A new family, Anastomitrabeculiaceae, is also introduced to accommodate this independent lineage.

Several pleosporalean families such as Aigialaceae, Amniculicolaceae, Anteagloniaceae, Astrosphaeriellaceae, Bambusicolaceae, Biatriosporaceae, Caryosporaceae, Cyclothyriellaceae, Delitschiaceae, Didymosphaeriaceae, Fuscostagonosporaceae, Lindgomycetaceae, Melanommataceae, Neomassariaceae, Pseudoastrosphaeriellaceae, Striatiguttulaceae and Tetraplosphaeriaceae share similar characters to Anastomitrabeculiaceae in having trabeculate pseudoparaphyses, cylindrical asci and ascospores with a sheath [7]. The nature of pseudoparaphyses is often overlooked, but they have taxonomic relevance at the genus and possibly family levels [7], but not at the ordinal level [62]. These families differ from Anastomitrabeculiaceae mainly by their ascospores, for example, Aigialaceae and Amniculicolaceae have brown and muriform ascospores [7]. Anteagloniaceae differs by having a peridium composed of dark brown cells of textura epidermoidea, cellular or trabeculate pseudoparaphyses and small, uniseriate ascospores [2]. Astrosphaeriellaceae differs by its brown, sub-fusiform to fusiform, obclavate to ellipsoidal, or limoniform ascospores [63] and Biatriosporaceae differs by its immersed ascomata and fusiform, dark brown ascospores [2]. Caryosporaceae differs by its broad-fusiform, ovoid or ellipsoid, brown ascospores [64]. Bambusicolaceae species have also been isolated from dead bamboo culms, but they differ from Anastomitrabeculiaceae by their cellular pseudoparaphyses and multi-seriate, smooth-walled ascospores [2]. Cyclothyriellaceae differs by its uniseriate, ellipsoid to fusiform, brown ascospores with several eusepta [65]. Fuscostagonosporaceae differs in having globose to subglobose ascomata, fissitunicate asci with long stipes and narrowly fusiform ascospores [66]. Anastomitrabeculiaceae shares several characters with Didymosphaeriaceae in having immersed ascomata formed under a clypeus, trabeculate pseudoparaphyses and cylindrical asci. Didymosphaeriaceae and Melanommataceae differ in having cellular or trabeculate pseudoparaphyses and brown, multi-septate, muriform ascospores [7]. Lindgomycetaceae differs by the presence of cellular or trabeculate pseudoparaphyses and brown, multi-septate ascospores with bipolar mucilaginous appendages [7]. Neomassariaceae differs by its immersed ascomata and ellipsoid ascospores. Pseudoastrosphaeriellaceae differs by its brown to reddish-brown ascospores with longitudinal ridges towards the ends and Striatiguttulaceae differs in having brown, ellipsoid ascospores with paler end cells. Tetraplosphaeriaceae differs by its immersed ascomata and slightly curved, pale brown ascospores [7].

Divergence time estimate has been widely used as supporting evidence to clarify taxonomic status of extant or novel families in fungal taxonomy [4,6,23,24,26,27,67]. In this study, the MCC tree was congruent with the topology of the trees generated from Bayesian inference analysis and maximum likelihood analyses. The divergence time estimates for the crown age of Dothideomycetes (263 MYA), the split of Dothideomycetes and Arthoniomycetes (323 MYA), the crown age of Pleosporales (206 MYA) and the divergence of Hysteriales from Pleosporales (236 MYA) are similar to previous studies [4,7,11]. Hyde et al. [27] recommended that the divergence times of families should be between 50 and 150 MYA. The stem age is usually preferred to the crown age in taxa ranking as it is not affected by the sample size of the clade [27]. Based on the MCC tree, Anastomitrabeculiaceae and Halojulellaceae share the stem age of 84 MYA which supports the establishment of Anastomitrabeculiaceae.

The divergence time of Anastomitrabeculiaceae was also compared to Pleosporalean families with trabeculate pseudoparaphyses, cylindrical asci and ascospores with a sheath (Table 3). Cyclothyriellaceae has an estimated crown age of 66 MYA and it diverged at 95 MYA. Fuscostagonosporaceae has a crown age of approximately 26 MYA and it diverged around 63 MYA. Bambusicolaceae, which was also isolated from dead bamboo culms, has a crown age of 29 MYA and a stem age of about 57 MYA. The stem age of Anastomitrabeculiaceae lies within the range of divergence times of those with similar morphology, but the crown age of Anastomitrabeculiaceae (2.6 MYA) is much earlier compared to these families. Bambusicolaceae was introduced by Hyde at al. [2] to include three bambusicolous taxa, and it currently has 15 species [7]. Fuscostagonosporaceae was introduced by Hyde at al. [66] to accommodate one bambusicolous taxon and it currently has four species [7]. Ariyawansa et al. [64] introduced the pleosporalean family, Caryosporaceae, which is morphologically similar to Astrosphaeriellaceae and Trematosphaeriaceae [7]. Based on Liu et al. [11], the stem age of Caryosporaceae (85 MYA) is similar to Trematosphaeriaceae (88 MYA) compared to Astrosphaeriellaceae (113 MYA), but the crown age of Caryosporaceae (2 MYA) is much earlier compared to Astrosphaeriellaceae (55 MYA) and Trematosphaeriaceae (65 MYA). Astrosphaeriellaceae currently has 111 species, and Trematosphaeriaceae has 103 species, whereas Caryosporaceae has ten species [7]. Compared to their morphologically similar families, the early crown of Anastomitrabeculiaceae and Caryosporaceae could be due to their smaller sample size. Therefore, further collections are needed for an accurate estimation of the crown age as it is affected by the sample size of the clade [27]. This could also be due to rapid speciation of pleosporalean fungal species given their high adaptation capabilities.

The estimated crown age of Pleosporales (206 MYA) lies within the early Triassic period. The origin of monocotyledons is estimated within the late Cretaceous period (around 145 MYA) [68]. This period is associated with the diversification of pleosporalean families, which continued during the early Cretaceous period when there was a major diversification and radiation of angiosperms, which favoured further diversification of Pleosporalean families to adapt to various hosts [69].

Hosts and their symbionts can speciate in parallel, which relates to a high level of congruence between the phylogeny of the hosts and their symbionts [70,71]. Therefore, studies focusing on divergence time is important for a better understanding of host–pathogen interaction as well as co-evolutionary interactions [72]. This study uses a polyphasic approach based on morphology, multi-locus phylogenetic analyses and divergence time estimates. By implementing a polyphasic approach, we provide strong evidence for introducing the new family based on congruent results supporting the establishment of a new family.

Author Contributions

Conceptualization, C.S.B., C.P.; methodology, C.S.B., C.P.; resources, K.D.H.; writing—original draft preparation, C.S.B.; writing—review and editing, C.S.B., C.P., R.J., I.P. and K.D.H.; supervision, K.D.H.; funding acquisition, K.D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Thailand Research Fund, grant RDG6130001 entitled “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences generated in this study were submitted to GenBank.

Conflicts of Interest

The authors declare no conflict of interest.