Phylogenomic analyses of Alternaria section Alternaria: A high-resolution, genome-wide study of lineage sorting and gene tree discordance

Figures & data

Table 1. Summary of information and statistics for the analyzed section Alternaria genomes

Given species name at time of genome submission	Taxonomy notes^a	Isolate name	Sampling notes	Reference	Accession source	Data type	No. of contigs (>500 bp)	N50 (kb)	L50 (contigs)	Length (Mb)	GC%	Complete BUSCO genes (%)	Missing BUSCO genes (%)
A. alternata		ATCC 11680 (BMP 0238)	Allium, USA	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	253	450.6	25	33.52	51.13	99.00	0.88
A. alternata		ATCC 34957	Sorghum, USA	Nguyen et al. 2016	LMXP01000000	PacBio	27	2828.0	5	33.50	50.95	98.94	0.94
A. alternata	A. alternata ex-type	ATCC 66891 (EGS 34.016, BMP 0269, CBS 916.96)	Peanut, India	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	278	303.9	33	33.15	51.15	99.00	0.64
A. alternata		BMP 0270 (ATCC 66982, EGS 34.039)		Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	171	757.3	14	33.40	51.02	99.12	0.70
A. alternata		DET2001	Wheat, Canada	This study	JAAOQX010000000	Illumina	111	1206.5	11	33.47	51.03	99.36	0.59
A. alternata		DET2010	Wheat, Canada	This study	JAAOQV010000000	Illumina	216	1366.9	8	33.79	51.01	99.12	0.82
A. alternata		DET2019	Wheat, Canada	This study	JAAOQU010000000	Illumina	449	824.4	12	35.17	51.00	99.18	0.76
A. alternata	Apple pathotype	FERA 1177	Apple	Armitage et al. 2020	PDXD01000000	Illumina	735	536.6	17	35.63	51.11	99.18	0.82
A. alternata		JS-1623	Fir, Korea	Park et al. 2020	VZUP01000000	Illumina + PacBio	11	4961.7	3	33.67	50.97	92.85	6.21
A. alternata		KAS5516	Rye, Canada	This study	JAAOQT010000000	Illumina	174	1478.4	8	33.95	51.02	99.12	0.82
A. alternata		MOD1-FUNGI5	Grapes, USA	Gebru et al. 2020	SJDQ01000000	Illumina	1085	71.5	140	33.40	51.31	98.18	1.23
A. alternata	Peach pathotype	NAP07	Peach, Japan	Ushijima and Yamamoto 2019	BJEP01000000	Nanopore	19	3070.6	4	35.84	50.98	99.12	0.76
A. alternata		PPRI 21032	Onion, South Africa	Bihon et al. 2016	LSHC01000000	Illumina	530	244.4	41	33.01	51.30	98.01	1.76
A. alternata		SRC1lrK2f	Soil, USA	Zeiner et al. 2016	LXPP01000000	Illumina	91	1097.9	12	32.99	51.40	99.36	0.59
A. alternata	Tangerine pathotype	Z7	Tangerine	Wang et al. 2016	LPVP01000000	Illumina + PacBio	156	1182.7	9	34.36	50.99	99.36	0.59
A. arborescens		DET2008	Wheat, Canada	This study	JAAOQW010000000	Illumina	127	816.7	14	33.30	50.99	99.24	0.76
A. arborescens	Tomato pathotype, A. arborescens ex-type	EGS 39.128 (CBS 102605)	Tomato, USA	Hu et al. 2012	AIIC01000000	Illumina	493	314.3	30	33.79	50.93	99.30	0.64
A. arborescens		FERA 675	Asian Pear	Armitage et al. 2020	PDUP01000000	Illumina	325	862.6	14	33.94	51.06	99.24	0.70
A. arborescens		MOD1-FUNGI6	Apple, USA	Gebru et al. 2020	SJDP01000000	Illumina	459	302.5	30	33.75	51.10	99.12	0.70
A. arborescens		RGR 97.0013	Apple	Armitage et al. 2020	PDWY01000000	Illumina	287	684.7	17	33.80	50.89	99.36	0.64
A. arborescens		RGR 97.0016	Apple	Armitage et al. 2020	PEJP01000000	Illumina	339	528.3	23	33.77	51.08	99.30	0.70
A. citriarbusti	Moved to A. alternata	BMP 2343 (EGS 46.140)	Citrus, USA	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	1269	48.0	212	33.85	50.98	96.95	1.88
A. fragariae	A. alternata f. sp. fragariae	BMP 3062	Strawberry	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	779	78.2	126	33.13	50.99	97.95	1.29
A. gaisen	A. gaisen reference	EGS 90.391 (CBS 118488)	Asian Pear, Japan	Woudenberg et al. 2015	contact authors	Illumina	270	529.5	19	35.15	50.90	99.36	0.59
A. gaisen	Pear pathotype	FERA 650	Pear	Armitage et al. 2020	PDWZ02000000	Illumina + Nanopore	27	2110.0	6	34.35	51.14	99.36	0.59
A. limoniasperae	Rough lemon pathotype, moved to A. alternata	BMP 2335 (EGS 44.159)	Citrus	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	1596	50.4	213	34.76	50.98	96.95	2.17
A. longipes	A. longipes reference	BMP 0313 (EGS 30.033, CBS 540.94)	Tobacco, USA	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	1079	138.5	68	35.68	51.00	99.06	0.94
A. mali	Apple pathoype, moved to A. alternata	BMP 3063	Apple	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	2697	21.2	511	33.61	50.78	91.03	5.80
A. mali	Apple pathoype, moved to A. alternata	BMP 3064	Apple	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	1669	35.8	297	34.33	50.84	94.43	3.69
A. tangelonis	Tangerine pathotype, moved to A. gossypina	BMP 2327 (EGS 45.080)	Citrus	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	1639	37.4	270	33.75	50.87	94.72	3.46
A. tenuissima	A. tenuissima reference, moved to A. alternata	BMP 0304 (ATCC 96828, EGS 34.015, CBS 918.96)	Dianthus, UK	Dang et al. 2015	mycocosm.jgi.doe.gov	Illumina	216	662.1	18	33.36	51.03	99.12	0.76
A. tenuissima	Moved to A. alternata	FERA 1082	Apple	Armitage et al. 2020	PDXA01000000	Illumina	345	547.4	21	33.94	51.14	99.12	0.76
A. tenuissima	Moved to A. alternata	FERA 1164	Apple	Armitage et al. 2020	PDXB01000000	Illumina	250	709.1	15	34.72	50.90	99.24	0.70
A. tenuissima	Apple pathotype, moved to A. alternata	FERA 1166	Apple	Armitage et al. 2020	PDXC01000000	Illumina + Nanopore	22	2583.9	6	35.70	51.08	99.12	0.76
A. tenuissima	Moved to A. alternata	FERA 24350	Asian pear	Armitage et al. 2020	PDXE01000000	Illumina	167	608.1	17	33.07	51.32	99.12	0.76
A. tenuissima	Apple pathotype, moved to A. alternata	FERA 635	Apple	Armitage et al. 2020	PDXF01000000	Illumina	912	318.6	25	36.06	50.92	99.12	0.70
A. tenuissima	Moved to A. alternata	FERA 648	Pear	Armitage et al. 2020	PDXG01000000	Illumina	124	1341.0	10	33.51	50.99	99.36	0.59
A. tenuissima	Apple pathotype, moved to A. alternata	FERA 743	Apple	Armitage et al. 2020	PDXH01000000	Illumina	788	622.1	15	35.92	51.05	99.12	0.70
Average							531.18	903.46	61.03	34.11	51.04	98.36	1.27

Note. New genomes from Canadian isolates are shown in bold type.

^a Nomenclatural changes resulted from synonymization of original species names into A. alternata by Woudenberg et al. (2015).

Figure 1. Circular plots showing macrosynteny between section Alternaria genomes sequenced with long-read technology. Four genomes were compared with the NAP07 genome. NAP07 chromosomes are color-coded and shown on the right side of each plot. Scaffolds from other genomes are in black and shown on the left side of each plot. Tick marks along chromosomes/scaffolds indicate 1 Mb distances

Figure 2. Maximum likelihood tree constructed from the custom data set of 6209 genes concatenated. Shading of squares at nodes represents the level of support each node received from bootstrapping, but also from other data sets or analyses methods (see inset). For shading, black indicates that the branch was present and significantly supported, gray indicates that the branch was present but not significantly supported, and white indicates that the branch was absent. Significant support was considered >95% in ML ultrafast bootstrapping for the BUSCO and custom data sets, >95% in posterior probability for the custom MSC data set, and presence in consenses tree for OrthoFinder data set. Numbers on branches indicate the gene concordance factor (GCF) for the 6209 genes trees compared with the displayed species tree. Current species name is indicated in brackets in taxon labels, if different from original species name. ^T indicates ex-type strain

Figure 3. Plots of branch-specific concordance factor as a function of genomic location. Lines represent moving averages based on a sliding window of 20 genes. A concordance factor of 100 indicates that the branch was present in all included gene trees, whereas 0 indicates that the branch was absent in all included gene trees. The mean for the entire chromosome is indicated within each panel

Table 2. List of 20 candidate genes that are diagnostic for the four analyzed clades in section Alternaria

Locus	Related NCBI accession	Gene product	Chromosome	Gene midpoint (NAP07 coordinate)
ASA-01	XM_018527094	ClpP/crotonase (caseinolytic protease)	Chr01	804 003
ASA-02	XM_018532566	Glycoside hydrolase	Chr01	3 419 152
ASA-03	XM_018525396	Thermostable alpha-amylase	Chr01	4 422 548
ASA-04	XM_018526656	Hypothetical protein	Chr02	1 798 824
ASA-05	XM_018523574	Hypothetical protein	Chr02	2 280 898
ASA-06	XM_018525342	MFS general substrate transporter (major facilitator superfamily)	Chr02	5 311 887
ASA-07	XM_018527519	Hypothetical protein	Chr03	893 828
ASA-08	XM_018532806	RTA1-domain-containing protein	Chr03	1 251 119
ASA-09	XM_018524513	Hypothetical protein predicted sulfite efflux pump	Chr03	1 359 323
ASA-10	XM_018531969	Hypothetical protein	Chr04	819 652
ASA-11	XM_018533777	Class I glutamine amidotransferase-like protein	Chr04	2 294 759
ASA-12	XM_018527323	Homogentisate 1,2-dioxygenase	Chr05	650 876
ASA-13	XM_018534328	Hypothetical protein	Chr05	2 032 935
ASA-14	XM_018534844	Hypothetical protein	Chr06	773 854
ASA-15	XM_018524984	Non-ribosomal peptide synthetase C-terminal	Chr07	1 253 208
ASA-16	XM_018529476	Hypothetical protein	Chr07	2 178 554
ASA-17	XM_018527994	Acetyl-CoA synthetase-like protein	Chr09	1 007 905
ASA-18	XM_018524187	AhpC/TSA family protein-like protein (alkyl hydroperoxide reductase/thiol specific antioxidant)	Chr09	1 421 701
ASA-19	XM_018524743	Histone-fold-containing protein	Chr10	828 763
ASA-20	XM_018533177	zf-ZPR1-domain-containing protein (zinc-finger domain)	Chr10	1 038 833

Table 3. Existing markers and their ability to discriminate the four analyzed clades in section Alternaria.

				Gene tree supports clade?
Divergence level	Locus	Annotation	Chromosome	gaisen	alt/ten	arborescens	longipes
Deep	28S	28S nuclear rRNA (large subunit)	Chr07	N	N	N	N
Deep	ITS	Internal transcribed spacer (ITS1-5.8S-ITS2)	Chr07	N	N	N	N
Deep	18S	18S nuclear rRNA (small subunit)	Chr07	N	N	N	N
Medium	gapdh	Glyceraldehyde-3-phosphate dehydrogenase	Chr06	Y	N	N	Y
Medium	rpb2	RNA polymerase, second largest subunit	Chr05	Y	N	Y	Y
Medium	tef1	Translation elongation factor 1-alpha	Chr03	N	N	N	Y
Shallow	Alta1	Major allergen Alt a 1	Chr02	Y	N	Y	Y
Shallow	endoPG	Endopolygalacturonase	Chr01	Y	N	Y	Y
Shallow	KOG1058	Coatemer subunit beta	Chr10	Y	N	N	N
Shallow	KOG1077	AP-2 adaptor complex subunit alpha	Chr01	Y	N	Y	Y
Shallow	OPA10-2	Anonymous region (putative FAD-binding-like protein)	Chr01	Y	N	Y	Y
Shallow	OPA1-3	Anonymous region	Chr01	N	N	N	Y
Shallow	Pgs1	CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase	Chr07	Y	N	N	N
Shallow	Rev3	Catalytic subunit of DNA polymerase zeta	Chr04	Y	N	Y	Y
Shallow	Tma22	Translation machinery associated protein	Chr05	Y	Y	Y	Y

Figure 4. Numbers of orthogroups that were clade specific (present/absent). Numbers within circles indicate orthogroup patterns for single clades (e.g., present in all taxa in clade but absent from all other taxa/absent from all taxa in clade but present in all other taxa). Numbers between circles indicate orthogroup patterns shared between two clades

Dang HX, Pryor B, Peever T, Lawrence CB. 2015. The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species. BMC Genomics 16:239.

 PubMed Web of Science ®Google Scholar

Nguyen HDT, Lewis CT, Lévesque CA, Gräfenhan T. 2016. Draft genome sequence of Alternaria alternata ATCC 34957. Genome Announcements 4:e01554–15.

 PubMedGoogle Scholar

Armitage AD, Cockerton HM, Sreenivasaprasad S, Woodhall J, Lane CR, Harrison RJ, Clarkson JP. 2020. Genomics evolutionary history and diagnostics of the Alternaria alternata species group including apple and Asian pear pathotypes. Frontiers in Microbiology 10:3124.

 PubMed Web of Science ®Google Scholar

Park SY, Jeon J, Kim JA, Jeon MJ, Jeong MH, Kim Y, Lee Y, Chung H, Lee YH, Kim S. 2020. Draft genome sequence of Alternaria alternata JS-1623, a fungal endophyte of Abies koreana. Mycobiology, doi:https://doi.org/10.1080/12298093.2020.1756134

 Google Scholar

Gebru ST, Gangiredla J, Tournas VH, Tartera C, Mammel MK. 2020. Draft genome sequences of Alternaria strains isolated from grapes and apples. Microbiology Resource Announcements 9:e01491–19.

 Google Scholar

Ushijima K, Yamamoto M. 2019. A sequence resource of autosomes and additional chromosomes in the peach pathotype of Alternaria alternata. Molecular Plant-Microbe Interactions 32:1273–1276.

 PubMed Web of Science ®Google Scholar

Bihon W, Cloete M, Gerrano AS, Oelofse D, Adebola P. 2016. Draft genome sequence of Alternaria alternata isolated from onion leaves in South Africa. Genome Announcements 4:301022–16.

 Google Scholar

Zeiner CA, Purvine SO, Zink EM, Paša-Tolić L, Chaput DL, Haridas S, Wu S, LaButti K, Grigoriev IV, Henrissat B, Santelli CM, Hansel CM. 2016. Comparative analysis of secretome profiles of manganese(II)-oxidizing ascomycete fungi. PLoS ONE 1:e0157844.

 Google Scholar

Wang M, Sun X, Yu D, Xu J, Chung K, Li H. 2016. Genomic and transcriptomic analyses of the tangerine pathotype of Alternaria alternata in response to oxidative stress. Scientific Reports 6:32437.

 PubMed Web of Science ®Google Scholar

Hu J, Chen C, Peever T, Dang H, Lawrence C, Mitchell T. 2012. Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer. BMC Genomics 13:171.

 PubMed Web of Science ®Google Scholar

Woudenberg JHC, Seidl MF, Groenewald JZ, de Vries M, Stielow JB, Thomma BPHJ, Crous PW. 2015. Alternaria section Alternaria: species, formae speciales or pathotypes? Studies in Mycology 82:1–21.

 Google Scholar