An expanded phylogeny for the genus Phytophthora

Abstract

A comprehensive phylogeny representing 142 described and 43 provisionally named Phytophthora species is reported here for this rapidly expanding genus. This phylogeny features signature sequences of 114 ex-types and numerous authentic isolates that were designated as representative isolates by the originators of the respective species. Multiple new subclades were assigned in clades 2, 6, 7, and 9. A single species P. lilii was placed basal to clades 1 to 5, and 7. Phytophthora stricta was placed basal to other clade 8 species, P. asparagi to clade 6 and P. intercalaris to clade 10. On the basis of this phylogeny and ancestral state reconstructions, new hypotheses were proposed for the evolutionary history of sporangial papillation of Phytophthora species. Non-papillate ancestral Phytophthora species were inferred to evolve through separate evolutionary paths to either papillate or semi-papillate species.

INTRODUCTION

The genus Phytophthora has had profound impacts on human history by causing agriculturally and ecologically important plant diseases (Erwin & Ribeiro 1996). Among the most notorious Phytophthora species is P. infestans, cause of the late blight disease, which was the primary cause of the Irish potato famine from 1845 to 1852 in which approximately one million people died and 1.5 million emigrated from Ireland (Turner 2005). Another example is the sudden oak death pathogen, P. ramorum, that has killed millions of coast live oak, tanoak and Japanese larch trees, and has permanently altered the forest ecosystems in California and Oregon, USA (Goheen et al. 2002, Rizzo et al. 2002, Rizzo et al. 2005). Other species, such as P. cinnamomi, P. nicotianae, and P. sojae, can also cause highly destructive plant diseases (Erwin & Ribeiro 1996). The impact caused by Phytophthora species has continued to increase with the emergence of new pathogens and diseases. The number of species known in the genus has doubled during the past decade due to extensive surveys in previously unexplored ecosystems such as natural forests (Jung et al. 2011, 2017, Rea et al. 2010, Reeser et al. 2013, Vettraino et al. 2011), streams (Bezuidenhout et al. 2010, Brazee et al. 2017, Reeser et al. 2007, Yang et al. 2016), riparian ecosystems (Brasier et al. 2003a, 2004, Hansen et al. 2012), and irrigation systems (Hong et al. 2010, 2012, Yang et al. 2014a, b). The total number of formally named species in the genus was about 58 in 1996 (Erwin & Ribeiro 1996), but now is more than 150. In addition, some provisionally or informally named species are also expected to be formally described in the near future.

A sound taxonomic system is foundational for correctly identifying Phytophthora species and safeguarding agriculture, forestry, and natural ecosystems. Traditionally, taxonomy of the genus was based on morphological characters. A fundamental morphology-based classification of Phytophthora species was established by Waterhouse (1963) who classified the species into six groups based on the morphology of sporangia, homothallism, and configuration of antheridia. However, plasticity in morphological characters amongst isolates of individual species is significant, so is homology or homoplasy among different species. For example, isolates of P. constricta (Rea et al. 2011), P. gibbosa (Jung et al. 2011), P. lateralis (Kroon et al. 2012), P. mississippiae (Yang et al. 2013), and P. multivesiculata (Ilieva et al. 1998) all produce a mixture of semi-papillate and non-papillate sporangia. Many non-papillate species recovered from irrigation water such as Phytophthora hydropathica (Hong et al. 2010) and P. irrigata (Hong et al. 2008) were morphologically inseparable from P. drechsleri, while sequence analyses demonstrated that they are distinct species. Also, production of many morphological structures and physiological features needs specific environmental conditions, while observation of these features requires substantial training and expertise. Difficulty in obtaining important morphological data can impair accurate species identification.

With the advent of DNA sequencing, the taxonomic concept for the genus has evolved from morphology to molecular phylogeny-based (Blair et al. 2008, Cooke et al. 2000, Kroon et al. 2004, Lara & Belbahri 2011, Martin et al. 2014, Martin & Tooley 2003, Robideau et al. 2011, Villa et al. 2006). In particular, the availability of whole genome sequences from P. sojae, P. ramorum (Tyler et al. 2006) and P. infestans (Haas et al. 2009) enabled the identification of genetic markers useful for multi-locus phylogenies (Blair et al. 2008).

Cooke et al. (2000) developed the first molecular phylogeny for the genus by analyzing sequences of the internal transcribed spacer region (ITS) of 51 species. Kroon et al. (2004) constructed a phylogeny based on sequences of four nuclear and mitochondrial genes of 48 species, and Blair et al. (2008) produced a sophisticated phylogeny based on sequences of seven nuclear genetic markers. That multi-locus phylogeny divided 82 Phytophthora species into 10 phylogenetically well-supported clades. Martin et al. (2014) analyzed sequences of seven nuclear and four mitochondrial genes of 90 formally named and 17 provisional species and provided phylogenies including 10 clades, almost identical to that of Blair et al. (2008), except that P. quercina and P. sp. ohioensis were excluded from clade 4 and grouped into a potentially new clade.

A comprehensive molecular phylogeny is required to understanding the evolution of Phytophthora species. Although discordance has been found between the molecular phylogeny and the morphology-based taxonomy (Cooke et al. 2000, Ersek & Ribeiro 2010), correlations have been observed between molecular phylogenies and individual morphological and physiological traits. Recent studies indicated that species in individual clades or subclades are mostly identical in sporangial papillation, and optimum and maximum growth temperatures (Cooke et al. 2000, Kroon et al. 2012, Martin et al. 2012, Yang 2014). However, there was limited to no correlation between phylogeny and the morphology of sexual organs, such as antheridial configuration (Cooke et al. 2000, Kroon et al. 2012, Martin et al. 2012, Yang 2014). These studies have implied that divergence in sporangial morphology and variation in environmental specialization may be the keys in the evolutionary history of Phytophthora species. Nevertheless, these hypotheses need to be further tested and the exact evolutionary history of the genus Phytophthora warranted more investigation.

In this study, an expanded phylogeny, including more than 180 Phytophthora taxa, many not included in any previous phylogeny, was constructed. Sequences of seven nuclear genetic markers were used for construction of the phylogeny. In light of this phylogeny, ancestral state reconstructions were conducted on the sporangial papillation of Phytophthora species. Important evolutionary divergence events and associated changes in the sporangial morphology of Phytophthora species are discussed.

MATERIALS AND METHODS

Isolate selection

A total of 376 Phytophthora isolates representing 142 described and 43 provisionally named species, plus one isolate of each Elongisporangium undulatum (basionym: Pythium undulatum), Halophytophthora fluviatilis, and Phytopythium vexans (basionym: Pythium vexans) as outgroup taxa were included (Table 1). These included 114 ex-types (Table 2). Also included were 164 authentic isolates that were designated as representative isolates by the originators of the respective species names (Table 1). The majority of these isolates were provided by the originators of the respective species, while the rest were purchased from the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, The Netherlands.

Table 1.

Information regarding isolates used in this study. GenBank accession numbers are listed in Table S1.

			Isolate identificationd							Isolate origins
(Sub)cladea	Speciesb	Papillac	CH	CBS	ATCC	IMI	WPC	MG	Typee	Host or Substrate	Location	Year	Reference
1a	P. cactorum	P	22E6				P10194	p25		Rhododendron sp.	Ohio, USA	n.a.f	(Schröter 1886)
			22E7		16693	21168	P0715	p6		n.a.	UK	n.a.
			22E8		16694, MYA-3653	50470	P10193	p7		Malus sp.	Zimbabwe	n.a.
	P. hedraiandra	P	33F3		MYA-4165			p225		Rhododendron sp.	Minnesota, USA	2002	(de Cock & Lévesque 2004)
			38C2							Irrigation water	Virginia, USA	2006
			62A5	111725			P19523		T	Viburnum sp.	The Netherlands	2001
	P. idaei	P	34D4	971.95	MYA-4065	313728	P6767	p220	T	Rubus idaeus	Scotland, UK	1987	(Kennedy & Duncan 1995)
			62A1	968.95					A	Rubus idaeus	Scotland, UK	1985
	P. pseudotsugae	P			52938	331662	P10339		T	Psendotsuga menziesii	Oregon, USA	n.a.	(Hamm & Hansen 1983)
	P. aff. hedraiandra	P	33F4					p226		Rhododendron sp.	Minnesota, USA	2003	n.a.
	P. aff. pseudotsugae	P	29B3					p185	A	Psendotsuga menziesii	Oregon, USA	1975	n.a.
1b	P. clandestina	P	32G1	347.86	58713, 60438	278933	P3943	p200	T	Trifolium subterraneum	Australia	1985	(Taylor et al. 1985)
			33D8		MYA-4064	287317		p215	A	Trifolium subterranea	Australia	1985
			38D4					p304		n.a.	Australia	n.a.
	P. iranica	P	61J4	374.72	60237	158964	P3882	p218	T	Solanum melongena	Iran	1969	(Ershad 1971)
	P. tentaculata	P	29F2	552.96			P8497		A	Chrysanthemum leucanthemum	Germany	n.a.	(Kröber & Marwitz 1993)
			30D5							Bacopa sp.	The Netherlands	2004
			30G8		MYA-3655					Argyranthemum frutescens	Germany	2004
1c	P. andina	SP	60A2					p460	A	Solanum betaceum	Ecuador	n.a.	(Oliva et al. 2010)
			60A3					p461	A	Solanum betaceum	Ecuador	n.a.
							P13365		T	Solanum brevifolium	Ecuador	2001
	P. infestans	SP	27A8							Solanum tuberosum	Mexico	1992	(De Bary 1876)
							P10650			Solanum tuberosum	Mexico	n.a.
	P. ipomoeae	SP	31B4				P10226		A	Ipomoea longipedunculata	Mexico	n.a.	(Flier et al. 2002)
			31B5	109229			P10225		T	Ipomoea longipedunculata	Mexico	1999
			31B6				P10227		A	Ipomoea longipedunculata	Mexico	n.a.
	P. mirabilis	SP	30C1		64069, MYA-4062		P3006	p145	A	Mirabilis jalapa	Mexico	n.a.	(Galindo-A & Hohl 1985)
			30C2		64070, MYA-4063		P3007	p153	A	Mirabilis jalapa	Mexico	n.a.
	P. phaseoli	SP	23B4					p106		Phaseolus lunatus	Delaware, USA	2000	(Thaxter 1889)
			35B6							Phaseolus sp.	Delaware, USA	2000
							P10145			Phaseolus lunatus	Delaware, USA	n.a.
							P10150			Phaseolus lunatus	Delaware, USA	n.a.
1	P. nicotianae	P	22F9		15410, MYA-4037			p23		Nicotiana tabacum	North Carolina, USA	n.a.	(Breda de Haan 1896)
			22G1		15409, MYA-4036			p22		Nicotiana tabacum	North Carolina, USA	n.a.
							P10116			Metrosideros excelsa	California, USA	2002
							P1452			Citrus sp.	California, USA	n.a.
2a	P. botryosa	P	22H8		MYA-4059			p44		Heavae sp.	Thailand	n.a.	(Chee 1969)
			46C2		26481			p384	A	Hevea brasiliensis	Thailand	n.a.
			62C6	581.69		136915	P3425		T	Hevea brasiliensis	Malaysia	1966
						130422	P6945			Hevea brasiliensis	Malaysia	1986
	P. citrophthora	P	03E5					p132		Irrigation water	Virginia, USA	2000	(Smith & Smith 1906)
			26H3					p31		n.a.	n.a.	n.a.
	P. colocasiae	SP	22F8		MYA-4159			p47		Colocasia esculenta	n.a.	1992	(Raciborski 1900)
			35D3					p276		Colocasia esculenta	Hawaii, USA	2005
	P. himalsilva	P	61G2	128767					T	Quercus leucotricophora	Nepal	2005	(Vettraino et al. 2011)
			61G3	128753					A	Quercus leucotricophora	Nepal	2005
	P. meadii	P	22G5		MYA-4043			p75		Citrus sp.	India	1992	(McRae 1918)
			61J9	219.88		129185				Hevea brasiliensis	India	1987
	P. occultans	SP	65B9	101557					T	Buxus sempervirens	The Netherlands	1998	(Man In’t Veld et al. 2015)
	P. terminalis	SP	65B8	133865					T	Pachysandra terminalis	The Netherlands	2010	(Man In’t Veld et al. 2015)
	P. aff. citrophthora	P	26H4					p32	A	n.a.	n.a.	n.a.	n.a.
						342898	P10341		A	Syringa sp.	England, UK	1990
	P. aff. himalsilva	P	61G4	128754					A	Castanopsis sp.	Nepal	2005	n.a.
	P. sp. 46C3	n.a.	46C3		66767		P6713	p385	A	Hevea brasiliensis	Malaysia	n.a.	n.a.
	P. sp. P6262	n.a.					P6262		A	Hevea brasiliensis	India	n.a.	n.a.
	P. sp. P6310	n.a.					P6310		A	Theobroma cacao	Indonesia	n.a.	n.a.
2b	P. capsici	P	22F4		15399, MYA-4034			p8	A	Capsicum annum	New Mexico, USA	1948	(Leonian 1922)
					46012		P0253			Theobroma cacao	Mexico	1964
				121656			P10386			Cucumis sativus	Michigan, USA	1997
	P. glovera	SP	31E5					p167	A	Nicotiana tabacum	Brazil	n.a.	(Abad et al. 2011)
			62B4	121969			P11685		T	Nicotiana tabacum	Brazil	1995
	P. mengei	SP	42B2		MYA-4554			p340	T	Persea americana	California, USA	n.a.	(Hong et al. 2009)
			42B3		MYA-4555			p341	A	Persea americana	California, USA	n.a.
	P. mexicana	P	45G4	554.88	46731	92550	P0646	p355		Solanum lycopersicum	Argentina	n.a.	(Hotson & Hartge 1923)
	P. siskiyouensis	SP	41B7	122779	MYA-4187		P15122		T	Stream water	Oregon, USA	2003	(Reeser et al. 2007)
			41B8						A	Soil	Oregon, USA	2003
	P. tropicalis	P	22H5					p27		Vanila sp.	Tahiti	n.a.	(Aragaki & Uchida 2001)
			35C8	434.91	76651, MYA-4218			p272	T	Macadamia integrifolia	Hawaii, USA	n.a.
	P. aff. capsici	P	22F5		15427, MYA-4035			p9		Nicotiana tabacum	North Carolina, USA	n.a.	n.a.
	P. sp. brasiliensis	n.a.			46705		P0630		A	Theobroma cacao	Brazil	1969	(Oudemans & Coffey 1991)
2c	P. acerina	SP	61H1	133931					T	Acer pseudoplatanus	Italy	2010	(Ginetti et al. 2014)
			61H2						A	Soil	Italy	2010
	P. capensis	SP	62C1	128319			P1819		T	Curtisia dentata	South Africa	n.a.	(Bezuidenhout et al. 2010)
			62C2	128320			P1822		A	Stream water	South Africa	n.a.
			62C3	128321			P1823		A	Olea campensis	South Africa	1986
	P. citricola	SP	33H8	221.88	60440	21173	P0716	p396	T	Citrus sinensis	Taiwan	1987	(Sawada 1927)
			33J2	295.29				p375	A	Citrus sp.	Japan	1929
	P. multivora	SP	55C5	124094					T	Soil	Western Australia, Australia	2007	(Scott et al. 2009)
	P. pachypleura	SP	61H6						A	Soil	UK	2006	(Henricot et al. 2014)
			61H7			502404			T	Acuba japonica	UK	2008
			61H8						A	Soil	UK	2009
	P. pini	SP	22F1		MYA-3656			p53	A	Rhododendron sp.	West Virginia, USA	1987	(Hong et al. 2011)
			45F1		64532			p343	T	Pinus resinosa	Minnesota, USA	1925
	P. plurivora	SP	22E9		MYA-3657			p101		Kalmia latifolia	Western Australia, Australia	1998	(Jung & Burgess 2009)
			22F2					p52		Rhododendron sp. cv. “Olga Mezitt”	New York, USA	n.a.
			33H9	379.61						Rhododendron sp.	Germany	1958
	P. sp. 22F3	SP	22F3					p33	A	n.a.	Ohio, USA	n.a.	n.a.
	P. sp. 28D1	SP	28D1					p119	A	Fagus sylvatica	New York, USA	n.a.	n.a.
			28D3					p121	A	Fagus sylvatica	New York, USA	n.a.
	P. sp. citricola VIII	SP	27D9						A	Unidentified leaf	Hainan, China	n.a.	n.a.
	P. sp. pini-like	SP	56G1						A	Taxus sp.	Pennsylvania, USA	2011	n.a.
	P. taxon emzansi	SP	61F2						A	Agathosma betulina	South Africa	2005	(Bezuidenhout et al. 2010)
			61F3						A	Agathosma betulina	South Africa	2005
2d	P. bisheria	SP	29D2							Rubus idaeus cv. Canby	Wisconsin, USA	1989	(Abad et al. 2008)
			31E6	122081			P10117		T	Fragaria ×ananassa	North Carolina, USA	1999
							P1620			Rhododendron sp.	North Carolina, USA	n.a.
	P. elongata	SP	33J3						A	n.a.	Australia	1995	(Rea et al. 2010)
			33J4						A	n.a.	Australia	1995
			55C4	125799					T	Soil	Western Australia, Australia	2004
	P. frigida	P	47G6						A	Eucalyptus smithi	South Africa	n.a.	(Maseko et al. 2007)
			47G7						A	Eucalyptus smithi	South Africa	n.a.
			47G8						T	Eucalyptus smithi	South Africa	2001
2e	P. multivesiculata	SP to NP	29E3	545.96			P10410		T	Cymbidium sp.	The Netherlands	n.a.	(Ilieva et al. 1998)
			30D4						A	Cymbidium sp.	The Netherlands	n.a.
	P. taxon aquatilis	SP	38J5		MYA-4577				A	Stream water	Virginia, USA	2006	(Hong et al. 2012)
3	P. ilicis	SP	23A7		56615, MYA-3897		P3939	p113		Ilex sp.	Canada	n.a.	(Buddenhagen & Young 1957)
			34D6							Quercus sp.	Germany	1999
			62A7	114348					T	Ilex aquifolium	The Netherlands	n.a.
	P. nemorosa	SP	28J3		MYA-4061			p141		Umbellularia californica	California, USA	n.a.	(Hansen et al. 2003)
			41C4		MYA-2948			p320	T	Lithocarpus densiflorus	California, USA	n.a.
	P. pluvialis	SP	60B3		MYA-4930				T	Rainwater	Oregon, USA	2008	(Reeser et al. 2013)
	P. pseudosyringae	SP	30A8	111772	MYA-4222			p284	T	Quercus robur	Germany	1997	(Jung et al. 2003)
			30B1					Pp285	A	Quercus robur	Germany	1997
	P. psychrophila	SP	29J5	803.95					T	Quercus robur	Germany	1995	(Jung et al. 2002)
			29J6		MYA-4083			p288	A	Quercus ilex	France	1996
4	P. alticola	P	47G5	121939			P16948		A	Eucalyptus dunnii	South Africa	n.a.	(Maseko et al. 2007)
	P. arenaria	P	55C2	127950					T	Soil	Western Australia, Australia	2009	(Rea et al. 2011)
			62B7	125800					A	Soil	Western Australia, Australia	2009
	P. megakarya	P	22H7		MYA-4040			p42		Theobroma cacao	Africa	n.a.	(Brasier & Griffin 1979)
			61J5	238.83	42100	202077			T	Theobroma cacao	Cameroon	n.a.
			61J6	239.83	42099	106327			A	Theobroma cacao	Nigeria	n.a.
	P. palmivora	P	22G8		MYA-4039		P10213	p65		Citrus sp.	Florida, USA	n.a.	(Butler 1910)
			22G9		MYA-4038			p26		Theobroma cacao	Costa Rica	n.a.
	P. quercetorum	P	15C7							Soil	South Carolina, USA	1997	(Balci et al. 2008)
			15C8							Soil	South Carolina, USA	1997
	P. quercina	P	30A4	783.95					A	Quercus robur	Germany	1995	(Jung et al. 1999)
			30A5	784.95	MYA-4084				T	Quercus robur	Germany	1995
			30A7							Quercus sp.	Serbia	2003
	P. sp. ohioensis	n.a.					P16050		A	Soil	Ohio, USA	2006	n.a.
5	P. agathidicida	P	67D5						T	Agathis australis	New Zealand	2006	(Weir et al. 2015)
	P. castaneae	P	22H6		MYA-4060			p45		Castanea sp.	Japan	n.a.	(Katsura 1976)
			30E7							Soil	Hainan, China	n.a.
			61J7	587.85	36818	325914			T	Soil	Taiwan	n.a.
	P. cocois	P	67D6						T	Cocos nucifera	Hawaii, USA	1990	(Weir et al. 2015)
	P. heveae	P	22J1			180616		p28	T	Heavae sp.	Malaysia	n.a.	(Thompson 1929)
			22J2		16701, MYA-3895			p17		soil	Tennessee, USA	1964
6a	P. gemini	NP	46H1	123382					A	Zostera marina	The Netherlands	1999	(Man in’t Veld et al. 2011)
			46H2	123383					A	Zostera marina	The Netherlands	1999
	P. humicola	NP	32F8	200.81	52179, MYA-4080		P3826	p198	T	Soil	Taiwan	1976	(Ko & Ann 1985)
			32F9				P6702	p199	A	Phaseolus vulgaris	Taiwan	n.a.
	P. inundata	NP	30J3			390121		p291	T	Olea sp.	Spain	1996	(Brasier et al. 2003b)
			30J4			389751		p298	T	Salix matsudana	UK	1972
							P8619			Pistacia vera	Iran	n.a.
	P. rosacearum	NP	22J9		MYA-3662			p82	A	Prunus sp.	California, USA	1987	(Hansen et al. 2009)
			41C1					p321	A	Prunus sp.	California, USA	n.a.
			47J1		MYA-4456				T	Malus domestica	California, USA	n.a.
	P. sp. 48H2	NP	48H2						A	Stream water	Virginia, USA	2008	n.a.
	P. sp. 62C9	NP	62C9						A	Stream water	Taiwan	2013	n.a.
	P. sp. personii	n.a.					P11555		A	Nicotiana tabacum	North Carolina, USA	n.a.	n.a.
	P. taxon walnut	NP	40A7						A	Irrigation water	Virginia, USA	2006	(Brasier et al. 2003a)
			43G1						A	Irrigation water	Virginia, USA	2007
6b	P. amnicola	NP	61G6	131652					T	Stream water	Western Australia, Australia	2009	(Crous et al. 2012)
			62C5	133867						Pachysandra sp.	The Netherlands	n.a.
	P. bilorbang	NP	61G8	131653					T	Soil	Western Australia, Australia	2010	(Aghighi et al. 2012)
	P. borealis	NP	60B2	132023	MYA-4881				T	Stream water	Alaska, USA	2008	(Hansen et al. 2012)
	P. crassamura	NP	66C9						A	Picea abies	Italy	2012	(Scanu et al. 2015)
			66D1	140357					T	Soil	Italy	2011
	P. fluvialis	NP	55B6	129424					T	Stream water	Western Australia, Australia	2009	(Crous et al. 2011)
	P. gibbosa	NP to SP	55B7						A	Soil	Western Australia, Australia	2009	(Jung et al. 2011)
			62B8	127951					T	Soil	Western Australia, Australia	2009
	P. gonapodyides	NP	21J5		46726			p117		Water	England, UK	n.a.	(Buisman 1927, Petersen 1910)
			34A8	554.67	60351		P6872			Reservoir water	n.a.	1967
	P. gregata	NP	55B8						A	Soil	Western Australia, Australia	2009	(Jung et al. 2011)
			62B9	127952					T	Soil	Western Australia, Australia	2009
	P. lacustris	NP	61D6						A	Soil	Germany	2003	(Nechwatal et al. 2013)
			61D8						A	Soil	Germany	2003
		NP	61E1						A	Soil	Germany	2006
						389725	P10337		T	Salix matsudana	England, UK	1972
	P. litoralis	NP	55B9	127953					T	Soil	Western Australia, Australia	2008	(Jung et al. 2011)
	P. megasperma	NP	62C7	402.72	58817	32035	P3599		T	Althaea rosea	Washington DC, USA	1931	(Drechsler 1931)
	P. mississippiae	NP to SP	57J1						A	Irrigation water	Mississippi, USA	2012	(Yang et al. 2013)
			57J2						A	Irrigation water	Mississippi, USA	2012
			57J3		MYA-4946				T	Irrigation water	Mississippi, USA	2012
			57J4						A	Irrigation water	Mississippi, USA	2012
	P. ornamentata	NP	66D2	140647					T	Soil	Italy	2012	(Scanu et al. 2015)
			66D3						A	Soil	Italy	2012
	P. pinifolia	NP	47H1	122924					T	Pinus radiata	Chile	2007	(Duran et al. 2008)
			47H2	122922					A	Pinus radiata	Chile	2007
	P. riparia	NP	60B1	132024	MYA-4882				T	Stream water	Oregon, USA	2006	(Hansen et al. 2012)
	P. thermophila	NP	55C1	127954					T	Soil	Western Australia, Australia	2004	(Jung et al. 2011)
	P. ×stagnum	NP	36H8						A	Irrigation water	Virginia, USA	2006	(Yang et al. 2014c)
			36J7						A	Irrigation water	Virginia, USA	2006
			43F3		MYA-4926				T	Irrigation water	Virginia, USA	2007
			44F9						A	Irrigation water	Virginia, USA	2007
	P. sp. 26E1	NP	26E1					p116	A	Malus domestica	New York, USA	n.a.	n.a.
	P. sp. canalensis	n.a.					P10456		A	Canal water	California, USA	2002	n.a.
	P. sp. delaware	NP	63H4						A	Pond water	Delaware, USA	2014	n.a.
			63H7						A	Pond water	Delaware, USA	2014
	P. sp. gregata-like	NP	22J5		16698			p16	A	n.a.	n.a.	n.a.	n.a.
	P. sp. megasperma-like	NP	23A1					p81	A	Prunus sp.	California, USA	n.a.	n.a.
			23A3		MYA-3660			p79	A	Actinidia chinensis	California, USA	1987
6	P. asparagi	NP	33D7			384046			A	Asparagus officinalis	New Zealand	1980	(Crous et al. 2012)
			62C4	132095	MYA-4826				T	Asparagus officinalis	Michigan, USA	2006
	P. sp. sulawesiensis	n.a.					P6306		A	Syzygium aromaticum	Indonesia	1989	n.a.
7a	P. attenuata	NP	67C5						T	Soil	Taiwan	2013	(Jung et al. 2017)
	P. europaea	NP	30A3							Quercus sp.	France	1998	(Jung et al. 2002)
			34C2							Quercus sp.	Germany	1999
			62A2	109049					T	Soil	France	1998
	P. flexuosa	NP	67C3						T	Soil	Taiwan	2013	(Jung et al. 2017)
	P. formosa	NP	67C4						T	Soil	Taiwan	2013	(Jung et al. 2017)
	P. fragariae	NP	22G6		11374		P3570	p114		Fragaria ×ananassa	Maryland, USA	n.a.	(Hickman 1940)
			30C5							Fragaria ×ananassa	Virginia, USA	n.a.
			61J3	209.46		181417	P6231		T	Fragaria ×ananassa	England, UK	n.a.
	P. intricata	NP	67B9						T	Soil	Taiwan	2013	(Jung et al. 2017)
	P. rubi	NP	30D7					p186	A	Rubus sp.	Australia	n.a.	(Man in ‘t Veld 2007)
			41D5							Rubus sp.	Norway	2005
			46C7		90442			p389	T	Rubus idaeus cv. "Glen Clova"	Scotland, UK	n.a.
	P. uliginosa	NP	62A3	109054			P10413		T	Soil	Poland	1998	(Jung et al. 2002)
			62A4	109055			P10328		A	Soil	Germany	1998
	P. ×alni	NP	32J6	392317	MYA-4081			p205	A	Alnus glutinosa	France	1996	(Brasier et al. 2004, Husson et al. 2015)
			32J7	392318				p206	A	Alnus sp.	Austria	1996
			47A7			392314			T	Alnus sp.	UK	1994
			47A8						A	Alnus sp.	The Netherlands	n.a.
	P. ×cambivora	NP	22F6		46719, MYA-4076			p64		Abies sp.	Oregon, USA	n.a.	(Buisman 1927, Jung et al. 2017)
			26F8		MYA-4075			p38		n.a.	New York, USA	n.a.
	P. ×heterohybrida	NP	67C1						T	Stream water	Taiwan	2013	(Jung et al. 2017)
	P. ×incrassata	NP	67C2						T	Stream water	Taiwan	2013	(Jung et al. 2017)
	P. sp. europaea SW	NP	33F7					p229	A	Soil	West Virginia, USA	2005	n.a.
7b	P. asiatica	NP	45G1		90455			p352	A	Robinia pseudoacacia	Jiangsu, China	n.a.	(Rahman et al. 2014a)
			46C6		56194			p388	A	Robinia pseudoacacia	Jiangsu, China	n.a.
			61H3	133347					T	Pueraria lobata	Japan	2005
	P. cajani	NP	33D9					p214		Cajanus cajani	India	n.a.	(Amin et al. 1978)
			45F6		44389			p348	A	Cajanus cajani	India	n.a.
			45F7		44388		P3105	p349	T	Cajanus cajani	India	n.a.
	P. melonis	NP	32F6		MYA-4079		P1371	p196	A	Cucumis sativus	China	n.a.	(Katsura 1976)
			41B4					p318	A	Cucumis sativus	Iran	n.a.
			45F3	582.69	52854				T	Cucumis sativus	Japan	n.a.
	P. niederhauserii	NP	01D5					p312	A	Irrigation water	Virginia, USA	2000	(Abad et al. 2014)
			23J6		MYA-4163			p57	A	Unknown ornamental	Israel	n.a.
			31E7				P10617	p169	A	Thuja occidentalis	North Carolina, USA	2001
	P. pisi	NP	60A4						T	Pea	Sweden	2009	(Heyman et al. 2013)
			60A5						A	Pea	Sweden	2009
	P. pistaciae	NP	33D6		MYA-4082	386658		p216	T	Pistacia vera	Iran	1986	(Mirabolfathy et al. 2001)
			41A9					p314	A	Pistacia vera	Iran	n.a.
	P. sojae	NP	22D8	312.62	16705, MYA-3899	131375		p19		Glycine max	Ontario, Canada	1959	(Kaufmann & Gerdemann 1958)
			28F9					p236		Glycine max	Mississippi, USA	1970
	P. vignae	NP	45G6		46735			p357	A	Glycine max	n.a.	n.a.	(Purss 1957)
			45G9		64832	316196	P3420	p379		Vigna unguiculata	Sri Lanka	n.a.
			46C1	112.76	64129			p380		Vigna sinensis	n.a.	n.a.
7c	P. cinnamomi	NP	23B1		15400, MYA-4057			p10		Camellia japonica	South Carolina, USA	n.a.	(Rands 1922)
			23B2		15401, MYA-4058			p11		Persea americana	Puerto Rico	1960
			61J1	144.22	46671	22938	P2110		T	Cinnamomum burmannii	Indonesia	1922
	P. parvispora	NP	30G9		MYA-4078			p178	A	Beaucarnea sp.	Germany	1991	(Scanu et al. 2014)
			46F6						A	Beaucarnea sp.	Germany	1992
			66C7	132771					A	Arbutus unedo	Italy	2008
			66C8	132772					T	Arbutus unedo	Italy	2011
	P. sp. ax	NP	46H5						A	Ilex glabra cv. “Shamrock”	Virginia, USA	2008	n.a.
7d	P. fragariaefolia	NP	61H4	135747					T	Fragaria ×ananassa	Japan	2005	(Rahman et al. 2014b)
	P. nagaii	NP	61H5	133248					T	Rosa sp.	Japan	1968	(Rahman et al. 2014b)
8a	P. cryptogea	NP	61H9	113.19		180615	P1738		T	Solanum lycopersicum	Ireland	n.a.	(Pethybridge & Lafferty 1919)
	P. drechsleri	NP	15E5							Soil	South Carolina, USA	1997	(Tucker 1931)
			15E6							Soil	South Carolina, USA	1998
			23J5	292.35	46724		P1087	p41	T	Beta vulgaris var. altissima	California, USA	n.a.
							P10331			Gerbera jamesonii	New Hampshire, USA	2003
	P. erythroseptica	NP	61J2	129.23		34684	P1693		T	Solanum tuberosum	Ireland	n.a.	(Pethybridge 1913)
	P. medicaginis	NP	23A4		MYA-3900			p37		Medicago sativa	Ohio, USA	n.a.	(Hansen & Maxwell 1991)
			28F1		44390		P1057	p124		Medicago sativa	California, USA	1975
	P. pseudocryptogea	NP			52402		P3103			Solanum marginatum	Ecuador	n.a.	(Safaiefarahani et al. 2015)
	P. richardiae	NP	31E8				P10355	p170		Zantedeschia sp.	Japan	1989	(Buisman 1927)
			45F5	240.30	60353, 46734	325930		p347	T	Zantedeschia aethiopica	USA	n.a.
							P10811			Zantedeschia aethiopica	Japan	1989
	P. sansomeana	NP	47H3		MYA-4455				T	Glycine sp.	Indiana, USA	n.a.	(Hansen et al. 2009)
			47H4						A	Glycine sp.	Indiana, USA	n.a.
			47H5						A	Glycine sp.	Indiana, USA	n.a.
	P. trifolii	NP	29B2		MYA-3901			p142	A	Trifolium vesiculosum	Mississippi, USA	1978	(Hansen & Maxwell 1991)
			62A9	117687					T	Trifolium sp.	Mississippi, USA	n.a.
	P. aff. cryptogea	NP	22G2	308.62	15402, MYA-4161	325907		p12		Aster sp.	California, USA	n.a.	n.a.
	P. aff. erythroseptica	NP	22J4		MYA-4041			p50		n.a.	Ohio, USA	n.a.	n.a.
			33A1					p207		Solanum tuberosum	Maine, USA	2004
	P. sp. kelmania	NP	24A7		MYA-4162			p102	A	Abies concolor	West Virginia, USA	1998	n.a.
			31E4				P10613	p166	A	Abes fraseri	North Carolina, USA	2002
8b	P. brassicae	SP	29D8	686.95					A	Brassica oleracea	The Netherlands	1995	(Man in’t Veld et al. 2002)
			61J8	179.87			P7517, P19521		T	Brassica oleracea	The Netherlands	1986
	P. cichorii	SP	62A8	115029					T	Cichorium intybus var. foliosum	The Netherlands	2004	(Bertier et al. 2013)
	P. dauci	SP	61E5	127102					T	Daucus carota	France	2009	(Bertier et al. 2013)
			32E5							Duscus carota	France	2004
			32E6				P10728			Duscus carota	France	2004
			32E7					p194		Duscus carota	France	2004
	P. lactucae	SP	61F4						T	Lactuca sativa	Greece	2001	(Bertier et al. 2013)
			61F7						A	Lactuca sativa	Greece	2002
			61F8						A	Lactuca sativa	Greece	2003
	P. primulae	SP	29E9	620.97				p286		Primula acaulis	Germany	1997	(Tomlinson 1952)
			29F1					p287		Primula sp.	The Netherlands	1998
	P. aff. brassicae-2	n.a.		112968			P6207		A	Allium cepa	Switzerland	n.a.	n.a.
	P. aff. cichorii	SP	61E3	133815					A	Cichorium intybus var. foliosum	UK	1999	n.a.
	P. sp. 29E7	SP	29E7						A	Allium porrum	The Netherlands	n.a.	n.a.
	P. taxon castitis	SP	61E7	131246					A	Fragaria ×ananassa	Sweden	1995	(Bertier et al. 2013)
	P. taxon parsley	SP	61G1						A	Petroselinum crispum	Greece	2006	(Bertier et al. 2013)
8c	P. foliorum	SP	49J8	121655	MYA-3638		P10974		T	Rhododendron sp.	Tennessee, USA	2004	(Donahoo et al. 2006)
	P. hibernalis	SP	22H1	270.31	60352	36906	P6871	p115		Citrus sinensis	Portugal	1931	(Carne 1925)
			32F7	114104	56353, MYA-3896	134760	P3822	p197		Citrus sinensis	Western Australia, Australia	1958
	P. lateralis	NP to SP	22H9		MYA-3898			p51	A	Chamaecyparis lawsoniana	Oregon, USA	n.a.	(Tucker & Milbrath 1942)
			29A9		201856			p128		Chamaecyparis lawsoniana	California, USA	1997
	P. ramorum	SP	32G2							Camellia japonica	South Carolina, USA	n.a.	(Werres et al. 2001)
			33F2							Quercus agrifolia	California, USA	n.a.
8d	P. austrocedrae	SP	41B5		MYA-4073				A	Austrocedrus chilensis	Argentina	n.a.	(Greslebin et al. 2007)
			41B6	122911	MYA-4074				T	Austrocedrus chilensis	Argentina	2005
	P. obscura	SP	60E9	129273					T	Soil	Germany	1994	(Grünwald et al. 2012)
			60F1						A	Pieris sp.	Oregon, USA	2009
			60F2						A	Kalmia latifolia	Oregon, USA	n.a.
	P. syringae	SP	21H9		34002		P0649	p187		Citrus sp.	California, USA	n.a.	(Klebahn 1905)
			23A6		MYA-3659			p35		n.a.	New York, USA	n.a.
8	P. stricta	NP	58A1		MYA-4944				T	Irrigation water	Mississippi, USA	2012	(Yang et al. 2014a)
			58A2						A	Irrigation water	Mississippi, USA	2012
			58A3						A	Irrigation water	Mississippi, USA	2012
			58A4						A	Irrigation water	Mississippi, USA	2012
9a (cluster 9a1)	P. aquimorbida	NP	40A6		MYA-4578				T	Irrigation water	Virginia, USA	2006	(Hong et al. 2012)
			40E3						A	Irrigation water	Virginia, USA	2006
			44G9						A	Irrigation water	Virginia, USA	2007
	P. chrysanthemi	NP	61E9						A	Chrysanthemum sp.	Japan	1998	(Naher et al. 2011)
			61F1	123163					T	Chrysanthemum ×morifolium	Japan	2000
	P. hydrogena	NP	44G8						A	Irrigation water	Virginia, USA	2007	(Yang et al. 2014b)
			46A3		MYA-4919				T	Irrigation water	Virginia, USA	2007
			46A4						A	Irrigation water	Virginia, USA	2007
	P. hydropathica	NP	05D1		MYA-4460			p366	T	Irrigation water	Virginia, USA	2000	(Hong et al. 2010)
			5C11		MYA-4459			p365	A	Irrigation water	Virginia, USA	2000
	P. irrigata	NP	04E4		MYA-4458			p335	A	Irrigation water	Virginia, USA	2000	(Hong et al. 2008)
			23J7		MYA-4457			p108	T	Irrigation water	Virginia, USA	2000
			44E4						A	Stream water	Virginia, USA	2007
	P. macilentosa	NP	58A5						A	Irrigation water	Mississippi, USA	2012	(Yang et al. 2014a)
			58A6						A	Irrigation water	Mississippi, USA	2012
			58A7		MYA-4945				T	Irrigation water	Mississippi, USA	2012
			58A8						A	Irrigation water	Mississippi, USA	2012
	P. parsiana	NP	47C3			395329			T	Ficus carica	Iran	1991	(Mostowfizadeh-Ghalamfarsa et al. 2008)
	P. virginiana	NP	40A9						A	Irrigation water	Virginia, USA	2006	(Yang & Hong 2013)
			44G6						A	Irrigation water	Virginia, USA	2007
			46A2		MYA-4927				T	Irrigation water	Virginia, USA	2007
	P. aff. parsiana G1	NP	47C7						A	Pistacia vera	Iran	n.a.	n.a.
			47C8						A	Pistacia vera	Iran	n.a.
						395328	P8618		A	Pistacia vera	Iran	1992
	P. aff. parsiana G2	NP	47C5			395330			A	Pistacia vera	Iran	1992	n.a.
			47C6			395331			A	Pistacia vera	Iran	1992
	P. aff. parsiana G3	NP	47D5						A	Pistacia vera	Iran	n.a.	n.a.
			47D8						A	Pistacia vera	Iran	n.a.
			47E1						A	Pistacia vera	Iran	n.a.
	P. sp. 35G4	NP	35G4						A	Irrigation water	Virginia, USA	2005	n.a.
	P. sp. 38D9	NP	38D9						A	Dianthus caryophyllus	Taiwan	n.a.	n.a.
	P. sp. 40J5	NP	40J5						A	Unknown leaf in seawater	Hainan, China	n.a.	n.a.
	P. sp. cuyabensis	n.a.					P8213		A	n.a.	Ecuador	1993	n.a.
	P. sp. lagoariana	NP	60B4				P8220		A	n.a.	Ecuador	n.a.	n.a.
			60B5				P8217		T	n.a.	Ecuador	n.a.
							P8223		A	n.a.	Ecuador	1993
9a (cluster 9a2)	P. macrochlamydospora-G1	SP	33E1				P10264			Glycine max	New South Wales, Australia	n.a.	(Irwin 1991)
							P10267			Glycine max	New South Wales, Australia	1994
	P. macrochlamydospora-G2	SP	31E9			351473	P8017	p171		Glycine max	Queensland, Australia	n.a.	(Irwin 1991)
			33D5	240.30	60353	340618				Zantedeschia aethiopica	The Netherlands	1927
	P. quininea	NP	45F2	406.48	56964			p344	A	Cinchona officinalis	Peru	n.a.	(Crandall 1947)
			46C4	407.48	46733			p386	T	Cinchona officinalis	Peru	n.a.
9a (cluster 9a3)	P. insolita	NP	327E1		MYA-4077			p123		Waterfall water	Hainan, China	n.a.	(Ann & Ko 1980)
			38E1	691.79	38789	288805			T	Soil	Taiwan	1980
							P6703		A	Soil	Taiwan	n.a.
	P. polonica	NP	40G9							Irrigation water	Virginia, USA	2006	(Belbahri et al. 2006)
			43F9							Irrigation water	Virginia, USA	2007
			49J9				P15005		A	Soil	Poland	2006
9b	P. captiosa	NP	46H6						A	Eucalyptus saligna	New Zealand	1999	(Dick et al. 2006)
			46H7				P10719		T	Eucalyptus saligna	New Zealand	1992
			46H8						A	Eucalyptus saligna	New Zealand	2000
							P10721		A	Eucalyptus saligna	New Zealand	1998
	P. constricta	NP to SP	55C3	125801					T	Soil	Western Australia, Australia	2006	(Rea et al. 2011)
	P. fallax	NP	46J2				P10722		T	Eucalyptus delegatensis	New Zealand	1997	(Dick et al. 2006)
			46J3						A	Eucalyptus nitens	New Zealand	2000
			46J5						A	Eucalyptus nitens	New Zealand	2000
							P10725		A	Eucalyptus fastigata	New Zealand	2004
10	P. boehmeriae	P	45F9	291.29		180614	P6950		T	Boehmeriae nivea	Taiwan	1927	(Sawada 1927)
	P. gallica	NP	50A1	111474			P16826		T	Quercus robur	France	1998	(Jung & Nechwatal 2008)
			61D5	111475			P16827		A	Phragmites australis	Germany	2004
	P. gondwanensis	P	22G7		MYA-3893					n.a.	Ohio, USA	n.a.	(Crous et al. 2015)
	P. intercalaris	NP	45B7	140632	TSD-7				T	Stream water	Virginia, USA	2007	(Yang et al. 2016)
			48A1						A	Stream water	Virginia, USA	2008
			49A7	140631					A	Stream water	Virginia, USA	2009
	P. kernoviae	P	46C8				P10956	p390		Rhododendron ponticum	England, UK	2004	(Brasier et al. 2005)
			46J6				P10681			Annona cherimola	New Zealand	2002
			46J8				P10671			Soil	New Zealand	2003
	P. morindae	P	62B5	121982					T	Morinda citrifolia var. citrifolia	Hawaii, USA	2005	(Nelson & Abad 2010)
	P. sp. boehmeriae-like	P	45F8	357.52	60173	32199	P1378	p350	A	Citrus sinensis	Argentina	1939	n.a.
n.a.	P. lilii	NP		135746					T	Lilium sp.	Japan	1987	(Rahman et al. 2015)
outgroup	Elongisporangium undulatum	P		101728		337230	P10342		T	Larix sp.	Scotland, UK	1989	(Uzuhashi et al. 2010)
	Phytopythium vexans	P		340.49	12194		P3980		T	n.a.	n.a.	n.a.	(de Cock et al. 2015)
	Halophytophthora fluviatilis	P	57A9		MYA-4961				T	Stream water	Virginia, USA	2011	(Yang & Hong 2014)

^a Molecular (sub)clade as designated in Fig. 1

^b Names of taxa informally designated for the first time in this study are underlined.

^c Sporangial papillation: NP = non-papillate, P = papillate, and SP = semi-papillate.

^d Isolate identification abbreviations: CH, Chuanxue Hong laboratory at Virginia Polytechnic Institute and State University, Virginia Beach, VA, USA; CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; ATCC, American Type Culture Collection, Manassas, VA, USA; IMI, CABI Biosciences, UK; WPC, the World Phytophthora Genetic Resource Collection at University of California, Riverside, USA; MG, Mannon E. Gallegly laboratory at West Virginia University, USA. Local identifications of respective isolates are provided in Table S1.

^e Ex-types (T) or authentic (A) isolates (designated as representative isolates by the originators of the respective species).

^f n.a.= not available.

Table 2.

Numbers of species and ex-types included in phylogenies for the genus Phytophthora in previous studies and this study.

	Number of species
Phylogeny in	Formal	Provisional	Number of ex-types
Cooke et al. (2000)	49	2	9
Kroon et al. (2004)	46	2	18
Blair et al. (2008)	72	10	16
Martin et al. (2014)	90	17	31
This study	142	43	114

DNA extraction

To extract genomic DNA (gDNA), an approximately 5 × 5 mm culture plug of each isolate was taken from the actively growing area of a fresh culture. This was then grown in 20 % clarified V8 broth (lima bean broth for growing a P. infestans isolate 27A8) at room temperature (ca. 23 °C) for 7–14 d to produce a mycelial mass. The mass was then blot-dried using sterile tissue paper and then lysed in liquid nitrogen or using a FastPrep^®-24 system (MP Biomedicals, Santa Ana, CA). gDNA was extracted using the DNeasy^® Plant Mini kit (Qiagen, Valencia, CA) or the Maxwell^® Plant DNA kit in combination with a Maxwell^® Rapid Sample Concentrator (Promega, Madison, WI).

DNA amplification and sequencing

A set of primers for seven genetic markers were used for DNA amplification including 60S Ribosomal protein L10 (60S), beta-tubulin (Btub), elongation factor 1 alpha (EF1α), enolase (Enl), heat shock protein 90 (HSP90), 28S ribosomal DNA (28S), and tigA gene fusion protein (TigA) as indicated in Blair et al. (2008). PCR reaction mixtures were prepared with the Takara Taq DNA polymerase (Takara Shuzo, Shiga, Japan) according to the manufacturer’s instructions. The PCR cycling protocol was the same as indicated by Blair et al. (2008), except that the Eppendorf^® Mastercycler^® Pro thermal cycler (Eppendorf, Hamburg) was used in this study. All PCR products were evaluated for successful amplification using agarose gel electrophoresis. Unsuccessful PCR amplifications were repeated using a modified protocol to attempt successful amplifications by optimizing annealing temperature using gradient PCR (typically with lower annealing temperatures) or using the GoTaq^® Flexi DNA Polymerase (Promega, Madison, WI) PCR mixture system.

Prior to sequencing, excess primer and dNTPs were removed from successful PCR products with shrimp alkaline phosphatase and exonuclease I (USB Catalog # 70092Y and 70073Z). One unit of each enzyme was added to 15 μL PCR product, incubated at 37 °C for 30 min, followed by heat inactivation at 65 °C for 15 min. Sequencing was performed with both amplifying primers as well as internal primers, if any, for individual genetic markers at the University of Kentucky Advanced Genetic Technologies Center (Lexington, KY). Derived sequencing files were visualized with FinchTV version 1.4.0 (Geospiza, Seattle, WA). Sequences of each isolate with all primers for individual genetic markers were aligned with Clustal W (Larkin et al. 2007) and edited manually to correct obvious sequencing errors and code ambiguous sites according to the International Union of Pure and Applied Chemistry (IUPAC) nucleotide ambiguity codes to produce a consensus sequence. All sequences produced in this study have been deposited in GenBank (Supplementary Table 1).

Among 379 isolates (including three isolates of the outgroup taxa) in the following phylogenetic analyses, all seven phylogenetic markers from 321 isolates were sequenced in this study. Sequences of all markers from 49 isolates by Blair et al. (2008) were also included in the analyses. Additionally, for seven isolates, sequences of one or two genes were newly produced in this study while the remaining gene sequences were from Blair et al. (2008). Sequences from P. lilii (CBS 135746) and P. sp. ohioensis (ST18-37) were obtained from Rahman et al. (2015) and from the Phytophthora Database (Park et al. 2013), respectively.

Phylogenetic analyses

Concatenated sequences of all isolates were aligned using Clustal X version 2.1 (Larkin et al. 2007). The alignment was edited in BioEdit version 7.2.5 (Hall 1999) to trim aligned concatenated sequences to an equal size and set missing data to question marks. The edited alignment was then analyzed in jModelTest version 2.1.7 (Posada 2008) to select the most appropriate model for the following phylogenetic analyses. Maximum likelihood (ML) analysis was performed using RAxML version 8.2.0 (Stamatakis 2014) with the selected model and 1000 bootstrap replicates. Maximum parsimony (MP) analysis was conducted using PAUP version 4.0a147 (Swofford 2002) with 1000 bootstrap replicates. Bayesian analysis (BA) was performed using MrBayes version 3.2.6 (Ronquist et al. 2012) for two million generations with the selected model. Phylogenetic trees were viewed and edited in FigTree version 1.4.2. Alignment and phylogenetic trees from all methods have been deposited in TreeBASE (S19303).

Ancestral character state reconstructions of sporangial papillation

Information on the sporangial papillation of individual species was compiled from the literature (Erwin & Ribeiro 1996, Gallegly & Hong 2008, Kroon et al. 2012, Martin et al. 2012) with emphasis given to their respective original descriptions (Table 1). Both likelihood and parsimony ancestral state reconstructions were performed on the ML tree from the phylogenetic analyses using Mesquite version 3.03 (Maddison & Maddison 2017).

RESULTS

Sequences, alignment, and phylogenetic model

PCR amplification and sequencing was successful for almost all isolates and seven genetic markers. Failure to obtain sequences only occurred occasionally for a few isolates, such as the EF1α gene of Phytophthora bilorbang (61G8), the Enl gene of P. macrochlamydospora (33E1, 31E9, and 33D5), and P. quininea (45F2), and TigA of P. megasperma (62C7) (Supplementary Table 1). These failures were set as missing data in the alignment. After trimming, each isolate was represented by an 8435-bp concatenated sequence in the alignment including gaps and missing data. This included 496 bp for 60S, 1136 bp for Btub, 965 bp for EF1α, 1169 bp for Enl, 1758 bp for HSP90, 1270 bp for 28S, and 1641 bp for TigA (TreeBASE S19303). The general time reversible nucleotide substitution model with gamma-distributed rate variation and a proportion of invariable sites (GTR+I+G) was identified by jModelTest as the most appropriate model for the phylogenetic analyses.

An expanded phylogeny including 10 clades and basal taxa

The three phylogenetic analysis methods, including ML, MP, and BA analyses (TreeBASE S19303), resulted in similar tree topologies. The topology and branch lengths of the ML inference are shown in Fig. 1. The monophyly of each of the previously recognized 10 clades was generally well supported with a few exceptions. Specifically, all clades except for clade 4 were highly supported by > 95 % bootstrap values in ML analysis and 100 % posterior probability (PP) in BA analysis (Fig. 1). Clades 1–3, 5, 7, and 10 were also highly supported by > 95 % bootstrap values in the MP analysis (Fig. 1). However, clades 6, 8, and 9, were only moderately supported with bootstrap numbers of 68, 61, and 52 in the MP analysis, respectively (Fig. 1).

Fig. 1.

A phylogeny for the genus Phytophthora based on concatenated sequences of seven nuclear genetic markers. Topology and branch lengths of maximum likelihood analysis are shown. Bootstrap values for maximum likelihood and maximum parsimony, and Bayesian posterior probabilities (percentages) are indicated on individual nodes and separated by a forward slash. An asterisk is used in place of nodes with unambiguous (100 %) support in all three analyses. A dash is used in place of a topology from an analysis ambiguous to the other two analyses and these sets of numbers with ambiguity in one analysis are also highlighted in red. Detailed structures of clades 2, 6, 7, and 9 are shown in Fig. 2, 3, 4, 5, respectively. Species represented by ex-types and authentic isolates are written in brown and blue, respectively. Branches indicating three hypothesized evolutionary paths with all species producing papillate or semi-papillate sporangia are drawn in red or orange, respectively. Scale bar indicates number of substitutions per site.

As nearly half of all taxa included in this phylogeny were recently described, all clades in this phylogeny are expanded here to various extents compared to previously published phylogenies. The general structure of clades 1, 3, 5, 8 and 10 remained as previously assigned by Blair et al. (2008) and Martin et al. (2014) with additions of new species. For example, clade 1 was divided into three well-supported subclades and P. nicotianae was placed basal to subclades 1b and 1c (Fig. 1). Clade 8 was divided into four generally well-supported subclades, except P. stricta, which was placed basal to all clade 8 species (Fig. 1). New subclades were assigned to clade 2 (Fig. 2), clade 6 (Fig. 3), clade 7 (Fig. 4) and clade 9 (Fig. 5).

Fig. 2.

Structure of Phytophthora clade 2 in a genus-wide phylogeny for the genus Phytophthora based on concatenated sequences of seven nuclear genetic markers. Topology and branch lengths of maximum likelihood analysis are shown. Bootstrap values for maximum likelihood and maximum parsimony, and Bayesian posterior probabilities (percentages) are indicated on individual nodes and separated by a forward slash. An asterisk is used in place of nodes with unambiguous (100 %) support in all three analyses. A dash is used in place of a topology from an analysis ambiguous to the other two analyses and these sets of numbers with ambiguity in one analysis are also highlighted in red. Species represented by ex-types and authentic isolates are written in brown and blue, respectively. Scale bar indicates number of substitutions per site.

Fig. 3.

Structure of Phytophthora clade 6 in a genus-wide phylogeny for the genus Phytophthora based on concatenated sequences of seven nuclear genetic markers. Topology and branch lengths of maximum likelihood analysis are shown. Bootstrap values for maximum likelihood and maximum parsimony, and Bayesian posterior probabilities (percentages) are indicated on individual nodes and separated by a forward slash. An asterisk is used in place of nodes with unambiguous (100 %) support in all three analyses. A dash is used in place of a topology from an analysis ambiguous to the other two analyses and these sets of numbers with ambiguity in one analysis are also highlighted in red. Species represented by ex-types and authentic isolates are written in brown and blue, respectively. Scale bar indicates number of substitutions per site.

Fig. 4.

Structure of Phytophthora clade 7 in a genus-wide phylogeny for the genus Phytophthora based on concatenated sequences of seven nuclear genetic markers. Topology and branch lengths of maximum likelihood analysis are shown. Bootstrap values for maximum likelihood and maximum parsimony, and Bayesian posterior probabilities (percentages) are indicated on individual nodes and separated by a forward slash. An asterisk is used in place of nodes with unambiguous (100 %) support in all three analyses. A dash is used in place of a topology from an analysis ambiguous to the other two analyses and these sets of numbers with ambiguity in one analysis are also highlighted in red. Species represented by ex-types and authentic isolates are written in brown and blue, respectively. Scale bar indicates number of substitutions per site.

Fig. 5.

Structure of Phytophthora clade 9 in a genus-wide phylogeny for the genus Phytophthora based on concatenated sequences of seven nuclear genetic markers. Topology and branch lengths of maximum likelihood analysis are shown. Bootstrap values for maximum likelihood and maximum parsimony, and Bayesian posterior probabilities (percentages) are indicated on individual nodes and separated by a forward slash. An asterisk is used in place of nodes with unambiguous (100 %) support in all three analyses. A dash is used in place of a topology from an analysis ambiguous to the other two analyses and these sets of numbers with ambiguity in one analysis are also highlighted in red. Species represented by ex-types and authentic isolates are written in brown and blue, respectively. Scale bar indicates number of substitutions per site.

Several species were placed basal to other species in their respective clades. First, the cluster of P. quercina and P. sp. ohioensis was placed basal to other species of clade 4 in all three analyses. The bootstrap supports of the ML and MP analyses, and PP (percentage) for the separation of this cluster from that of P. alticola, P. arenaria, P. megakarya, P. palmivora, and P. quercetorum in clade 4 were only 48, 78, and 84, respectively (Fig. 1). Second, P. lilii was excluded from all known clades; it was placed basal to clades 1–5 and 7 (Fig. 1). Third, in clade 6, bootstrap support for the ML and MP analyses, and PP for all species except P. asparagi and P. sp. sulawesiensis were 100/100/100 (Fig. 3). This set of support numbers decreased to 99/92/100 when P. sp. sulawesiensis was included, and to 100/68/100 when further including P. asparagi (Fig. 3). Fourth, the support numbers for clade 8 species excluding P. stricta was 100/100/100, but 96/61/100 when P. stricta was included (Fig. 1). Fifth, all papillate species in clade 10 (Table 1) formed a well-supported main cluster, while two more recently described non-papillate species, P. gallica and P. intercalaris, were placed basal to the main cluster (Fig. 1).

New subclades in clades 2, 6, 7, and 9

(a) Clade 2

In addition to the previously recognized subclades 2a and 2b, many species, such as P. acerina, P. capensis, P. citricola, P. multivora, P. pachypleura, P. plurivora, and P. pini in the commonly referred to “Phytophthora citricola-complex” defined a new subclade 2c (Fig. 2). Furthermore, P. bisheria, P. frigida, and P. elongata formed new subclade 2d and the cluster of P. multivesiculata and P. taxon aquatilis formed new subclade 2e, with maximum support values in each case (Fig. 2).

(b) Clade 6

Subclade 6a included P. gemini, P. humicola, P. inundata, P. rosacearum, P. sp. personii, P. sp. 48H2, P. sp. 62C9 and P. taxon walnut. The cluster of P. rosacearum and P. taxon walnut could not be separated from that represented by P. gemini with only moderate support values for separation (82/61/100) (Fig. 3). Isolates 62C9 and 48H2, belonging to two new species, had ambiguous placements within subclade 6a among the three analyses (Fig. 3). With approximately 20 species newly included in the present phylogeny, the previously recognized “P. megasperma-P. gonapodyides complex” (Brasier et al. 2003a), subclade II of clade 6 (Jung et al. 2011), or subclade 6b (Kroon et al. 2012) expanded and its separation from subclade 6a was well-supported by 100/100/100 values (Fig. 3). Within subclade 6b, separation of the cluster of P. bilorbang, P. lacustris, and P. riparia from the other subclade 6b species was highly supported by 97/94/100 (Fig. 3), indicating that these three species may define a new subclade, although this is not done in this study. Phytophthora sp. sulawesiensis was placed basal to other clade 6 species except for P. asparagi, while P. asparagi was basal to all other species in clade 6 (Fig. 3). Phytophthora asparagi was previously assigned as subclade 6c (Kroon et al. 2012) and subclade III of clade 6 (Jung et al. 2011); considering that the support value of MP analysis was only moderate (68 %) when this single taxon was included (Fig. 3), this previous assignation as a subclade was not adopted here. In addition, in order to be consistent with subclade names in other clades, subclades 6a and 6b were used here instead of subclades I and II by Jung et al. (2011).

(c) Clade 7

Four subclades were distinguished in clade 7. Separation of the previously assigned subclades 7a and 7b was only moderately supported by values 71/56/100 (Fig. 4). The general structure of subclade 7a remained the same even with the addition of seven new taxa. Six of these new species, including P. attenuata, P. flexuosa, P. formosa, P. intricata, P. ×heterohybrida, and P. ×incrassata were recently recovered from forest soils and streamwater in Taiwan (Jung et al. 2017). On the other hand, P. cinnamomi and P. parvispora were separated from subclade 7b. They, along with a provisional species, P. sp. ax from Virginia, USA (Table 1), formed a distinct new subclade 7c (Fig. 4). The new subclade 7d, including two recently described species from Japan (Rahman et al. 2014b), P. fragariaefolia and P. nagaii, was placed basal to other subclades in clade 7 (Fig. 4).

(d) Clade 9

The split of clade 9 into two subclades 9a and 9b was highly supported in ML (98 %) and BA (100 %) analyses and moderately supported in the MP (52 %) analysis (Fig. 5). However, monophyly was highly supported for subclade 9b (100/100/100) but not for subclade 9a (44/-/95) (Fig. 5). Within subclade 9a, three monophyletic clusters were formed: 9a1, 9a2, and 9a3. However, support for the separation of these three clusters was moderate or ambiguous. In particular, the MP results did not produce any consistent separation of the three clusters (Fig. 5). Cluster 9a1 included many recently described high-temperature tolerant species, such as P. aquimorbida, P. chrysanthemi, P. hydropathica, P. macilentosa, P. parsiana, and P. virginiana). The cluster of P. macrochlamydospora (two lineages with two isolates in each lineage, Table 1) and P. quininea constituted 9a2 (Fig. 5). The cluster of two other high-temperature tolerant species P. insolita and P. polonica constituted 9a3 (Fig. 5). The well-supported cluster of P. captiosa, P. constricta, and P. fallax was assigned as subclade 9b (Fig. 5).

Evolutionary history of sporangial papillation inferred from ancestral character state reconstructions

Sporangial papillation of individual species is indicated in Table 1 and Fig. 6. Due to the size of the cladograms, clusters including species with the same sporangial papillation within each (sub)clade were compressed in Mesquite. Both likelihood and parsimony methods suggested that non-papillate is the progenitor state of Phytophthora species, and that semi-papillate and papillate types were derived from the non-papillate. The analyses indicated three major clusters of semi-papillate and (or) papillate species diverged from the non-papillate ancestors. First, species in clades 1 to 5 (semi-papillate or papillate) diverged from non-papillate species in clade 7 and P. lilii (Fig. 6). Second, species in subclades 8b to 8d (semi-papillate) diverged from non-papillate subclade 8a species (Fig. 6). Third, papillate clade 10 species including P. boehmeriae, P. gondwanensis, P. kernoviae, and P. morindae diverged from the non-papillate P. gallica and P. intercalaris (Fig. 6). Several species such as P. macrochlamydospora, P. mississippiae, P. gibbosa, and P. constricta also evolved to produce partially semi-papillate sporangia (Fig. 6).

Fig. 6.

Ancestral state reconstructions of sporangial papillation for the genus Phytophthora based on likelihood (left cladogram) and parsimony (right cladogram). Trace character history analyses were performed on the maximum likelihood phylogeny in Mesquite. Clusters including species of uniform sporangial papillation within individual (sub)clades were compressed in Mesquite.

DISCUSSION

Here we presented an expanded phylogeny for the genus Phytophthora, encompassing 142 formally named and 43 provisionally recognized species (Table 2). In addition to this comprehensive coverage, this expanded phylogeny features over 1500 signature sequences generated from 278 ex-type and authentic isolates of 162 Phytophthora taxa (Supplementary Table 1). Furthermore, this study provided new insights into the evolutionary history of sporangial papillation in Phytophthora.

The expanded phylogeny provides a sound taxonomic framework for this agriculturally and ecologically important genus. One hundred and fourteen ex-types were included, representing 80 % of the 142 formally named species in this phylogeny. The majority of the 29 species not represented by ex-types, such as P. gonapodyides, P. infestans, P. meadii, P. mexicana, and P. nicotianae, were described long ago without designation of an ex-type culture. Likewise, almost all the 43 provisional species in this phylogeny were represented by authentic isolates from the originators of the respective species (Table 1 and Supplementary Table 1). This new framework will facilitate identification of new taxa in the future. As the genus continues to rapidly expand, some recently described species were not included in this study: P. mekongensis in subclade 2a (Puglisi et al. 2017), P. amaranthi in subclade 2b (Ann et al. 2016), P. boodjera in clade 4 (Simamora et al. 2015), P. chlamydospora in subclade 6b (Hansen et al. 2015), P. uniformis (basionym: P. alni subsp. uniformis) and P. ×multiformis (basionym: P. alni subsp. multiformis) in subclade 7a (Brasier et al. 2004, Husson et al. 2015), P. pseudolactucae in subclade 8b (Rahman et al. 2015), and P. prodigiosa (Puglisi et al. 2017) and P. pseudopolonica (Li et al. 2017) in subclade 9a. Likewise, some informally designated species also were not included: such as P. taxon humicola-like, P. taxon kwongan, and P. taxon rosacearum-like in subclade 6a (Jung et al. 2011). These and other emerging species are yet to be incorporated in the overall phylogeny of the genus.

The generation of over 1500 signature sequences from ex-types and authentic isolates in this study will aid researchers and first responders in correctly identifying Phytophthora cultures to the species level. DNA sequencing of selected genetic markers has become common practice in the identification of Phytophthora cultures (Kang et al. 2010). However, it is recognized that the accuracy of culture identity determined by this approach depends on the quality of the reference sequences used – and currently many sequence deposits are erroneously identified in public repositories, including GenBank (Kang et al. 2010). These errors originated in sequence deposits of cultures that were identified by morphological characters alone, and compounded by those identified through sequence matches to erroneous reference sequences or by single DNA markers (Kang et al. 2010). In this study, 29 isolates were found associated with an erroneous or modified identity (Supplementary Table 2). For instance, isolate 29B3 in clade 1 was identified as P. pseudotsugae and used as a key isolate for this species by Gallegly & Hong (2008). However, its sequences were distinct from those of the P. pseudotsugae ex-type (ATCC 52938). In the phylogenetic tree, it was basal to the cluster of P. cactorum and P. hedraiandra, thus its species identity was changed to P. aff. pseudotsugae (Fig. 1). In clade 2, isolate 26H4 was identified as P. citrophthora (Gallegly & Hong 2008) but sequences and phylogeny showed that it was close to but distinct from P. citrophthora isolates 03E5 and 26H3. It formed a cluster with isolate IMI 342898 (P10341), which was coded as P. sp. aff. colocasiae-1 by Martin et al. (2014). The identity of both isolates was then changed to P. aff. citrophthora (Fig. 2). Similarly, in clade 8, isolate 22G2 had been identified as P. cryptogea, although it was distinct from the P. cryptogea ex-type 61H9 (CBS 113.19). In the phylogenetic tree, it was basal to the cluster of P. cryptogea and P. erythroseptica, and the species identity was consequently changed to P. aff. cryptogea (Fig. 1). Changes in the identifications of these isolates, including the new and original names used, are indicated in Supplementary Table 2. The changes in the naming of these isolates highlights the importance of using signature sequences from ex-type or authentic isolates as references in future culture identification. In order to facilitate this practice, the signature sequences generated from ex-types or authentic isolates in the present study are marked as ‘(ex-type)’ or ‘(authentic)’, respectively, under the ‘isolate’ section in the ‘feature’ table of GenBank deposits. The research, diagnostic and regulatory communities are encouraged to use these sequences as references in future culture identification.

This study provided new insights into the evolutionary history of sporangial morphology in the genus Phytophthora, a subject that has fascinated generations of mycologists and plant pathologists. There have been three major hypotheses regarding the development of papillation, as illustrated in Fig. 7a, b, and c, respectively. First, papillate species were considered as descendants of Pythium-like, non-papillate ancestors and semi-papillation has been considered as intermediate between non-papillation and papillation (Blackwell 1949, Cooke et al. 2000, Erwin & Ribeiro 1996). Second, some semi-papillate species, exemplified by P. primulae in the group III of Waterhouse (1963) are primitive; they were suggested to have evolved to papillate and non-papillate species through two distinct evolutionary lines (Brasier 1983). Third, semi-papillate sporangia are morphological variants of papillate and non-papillate types (Cooke et al. 2000). Here we suggest that the non-papillate type is ancestral, and that non-papillate species could have evolved directly into either semi-papillate or papillate species (Fig. 7d). The evolution to semi-papillate species is exemplified by those in subclades 8b–d (Fig. 1), while evolution to papillate species is illustrated by P. boehmeriae and other papillate species in clade 10 (Fig. 1).The relationship between semi-papillate and papillate species appears to be more complicated (Fig. 7d). We also hypothesize that some semi-papillate species, such as those in subclade 1c, may have diverged from papillate ancestors, while some papillate species such as P. frigida may have evolved from semi-papillate ancestors of subclade 2d (Fig. 6).

Fig. 7.

Illustration of hypotheses on evolution of Phytophthora and associated changes in sporangial papillation: (a) species producing papillate sporangia evolved from non-papillate ancestors. Semi-papillation is considered as intermediate between non-papillation and papillation (Blackwell 1949, Cooke et al. 2000, Erwin & Ribeiro 1996); (b) some semi-papillate species, exemplified by P. primulae in the group III of Waterhouse (1963), are primitive and evolved to be non-papillate and papillate through two evolutionary paths, by Brasier (1983); (c) papillate species evolved from non-papillate ancestors. Semi-papillate species have been considered as morphological variants of papillate or non-papillate species, by Cooke et al. (2000); (d) a new hypothesis developed in this study that non-papillate ancestors evolved directly to either papillate or semi-papillate species. Some semi-papillate species further evolved to be papillate, or vice versa.

These new hypotheses are supported by the results from phylogeny and ancestral state reconstructions that suggest three major evolutionary paths in sporangial papillation of Phytophthora species (Fig. 1). First, the ancestor of modern species in clades 1–5 evolved to be papillate or semi-papillate (Figs 1, 6) while diverging from the common non-papillate ancestor of clade 7 species (Figs 1, 6). Second, the common ancestor of species in subclades 8b–d diverged from that of subclade 8a species while acquiring semi-papillation (Figs 1, 6). Third, the common ancestor of five clade 10 species in the main cluster including P. boehmeriae, P. gondwanensis, P. kernoviae, P. morindae, and P. sp. boehmeriae-like, acquired papillate sporangia while diverging from two non-papillate clade 10 species, P. gallica and P. intercalaris (Figs 1, 6). Besides these three major groups of papillate or semi-papillate species, a few species may have evolved to acquire semi-papillation independently, such as P. macrochlamydospora in clade 9 (Fig. 6). This evolutionary process may be underway for some other species including P. constricta, P. gibbosa, and P. mississippiae, which all produce both semi-papillate and non-papillate sporangia (Fig. 6). Furthermore, evolutionary reversion to partial production of non-papillate sporangia may have occurred in P. multivesiculata and P. lateralis in two semi-papillate subclades 2e and 8c, respectively (Fig. 6). However, that conclusion is uncertain due to limited and ambiguous data from species in these two subclades. Specifically, P. lateralis was ambiguously reported as non-papillate (Erwin & Ribeiro 1996, Gallegly & Hong 2008, Martin et al. 2012, Tucker & Milbrath 1942) or non- to semi-papillate (Kroon et al. 2012) in different studies. In subclade 2e, the only sister taxon of P. multivesiculata, P. taxon aquatilis, was provisionally described as semi-papillate, but only based on a single isolate (Hong et al. 2012). Evolutionary reversion in the sporangial papillation of these two species requires validation in the future. Also, more studies are warranted to analyze additional characters based on phylogenies with better clade-to-clade resolutions and provide a more comprehensive picture on the evolutionary history of Phytophthora species.

That a number of species were placed basal to other species in their respective clades in this expanded phylogeny presents a significant challenge to the monophyly of their respective clades and the current 10-clade system. First, P. stricta was initially placed close to other species in subclade 8a based on sequences of the cytochrome c oxidase 1 (cox1) gene, but was not grouped in any ITS clade (Yang et al. 2014a). This species was grouped in clade 8 in our expanded phylogeny by ML and BA analyses (Fig. 1); the monophyly of this clade was only moderately supported (61 %) in the MP analysis (Fig. 1). Second, the monophyly of clade 6 including P. asparagi was only moderately supported (68 %) in the MP analysis (Fig. 3). Third, although the inclusion of P. intercalaris in clade 10 was supported with maximum values, the exact positions of this species and P. gallica were still unresolved since the next node was only moderately supported (53 %) in the ML analysis and ambiguous in the MP analysis (Fig. 1). Fourth, similar to the finding of Blair et al. (2008), support for the monophyly of clade 4 including P. quercina and P. sp. ohioensis was only moderate (48/78/84). Also, similar ambiguity in the placement of the ‘P. quercina – P. sp. ohioensis’ cluster was observed among different phylogenetic approaches, and using different datasets including nuclear, mitochondrial, and combined nuclear and mitochondrial sequences (Martin et al. 2014). Fifth, this phylogeny confirmed the finding by Rahman et al. (2015) that P. lilii was not grouped in any clade of the current 10-clade system (Fig. 1). This species was not assigned as a distinct clade in our study, due to the relatively low clade-to-clade resolutions (Fig. 1). Further analyses are warranted to determine whether this unique species should be assigned as a new clade.

Although many branches in the expanded phylogeny have consistent maximum support in all three methods, some have only moderate to low or inconsistent support. These results highlight the challenges of correctly inferring the evolutionary separation of many closely related Phytophthora species, even when concatenated sequences from seven phylogenetic markers were used. It can be expected that as the cost of gene sequencing drops further, it will become possible to increase phylogenetic resolution among Phytophthora species by using concatenations of much larger numbers of genes. For example, Ye et al. (2016) used 293 concatenated housekeeping proteins to infer a robust phylogeny of seven fully sequenced Phytophthora species and confirmed that downy mildews (represented by three genome sequences) are nested within the genus Phytophthora, close to Phytophthora clade 4 (Ye et al. 2016). However, even with full genome sequences, ambiguity may not be completely resolved in cases where speciation has involved large populations of sexually reproducing individuals, for example, as a result of geographic separation. In these cases, there may be many sequence polymorphisms shared among separated species and these may confound the inference of a reliable phylogeny. Resolution of this level of ambiguity may require sequencing the whole genome of many isolates from the species of interest as well as using improved phylogenetic and coalescent methods.

With the number of described Phytophthora species increasing, recent studies have raised an important concern in the accurate detection of species boundaries using phylogenetic data (Jung & Burgess 2009, Pánek et al. 2016, Safaiefarahani et al. 2015). One example is the status of P. hedraiandra as a distinct species in subclade 1a (Pánek et al. 2016). As evidenced by the amplified fragment length polymorphism (AFLP) and phylogenetic analysis based on sequences of ITS, phenolic acid decarboxylase, and cox1 genes, a recent study concluded that P. hedraiandra was just one lineage of P. cactorum, while morphological data provided only limited information to delimitate these two species (Pánek et al. 2016). Also, phylogenetic analyses in this study indicated that P. cactorum and P. hedraiandra cluster with strong support (98/100/100), and P. aff. hedraiandra isolate 33F4 (previously identified as P. hedraiandra Supplementary Table 2), was clustered with P. cactorum (Fig. 1). Phylogenies based on nuclear sequences prior to this study also supported P. hedraiandra as closely related to P. cactorum (Blair et al. 2008, Martin et al. 2014). However, in the phylogenies based on concatenated sequences of four mitochondrial loci, and combined seven nuclear and four mitochondrial loci, P. hedraiandra was basal to the cluster of P. cactorum and P. pseudotsugae, and clustered with P. idaei, respectively (Martin et al. 2014). Phytophthora cactorum and P. hedraiandra also have very distinctive single-strand-conformation polymorphism patterns (Gallegly & Hong 2008). Apparently, more investigations are warranted to resolve the P. cactorum complex. Likewise, indistinct boundaries are present among species in other subclades, such as the ‘P. citricola complex’ or subclade 2c (Brazee et al. 2017, Jung & Burgess 2009), the ‘P. cryptogea complex’ in subclade 8a (Safaiefarahani et al. 2015, 2016) and cluster 9a1 in subclade 9a including P. hydropathica (Hong et al. 2010), P. parsiana (Mostowfizadeh-Ghalamfarsa et al. 2008), P. virginiana (Yang & Hong 2013) and other provisionally designated species. Accurately delimiting these closely related species within the genus remains an important task.

This expanded phylogeny has highlighted the importance and difficulty of accurately interpreting the position of hybrid Phytophthora species. As exemplified by P. ×alni (Brasier et al. 2004, Husson et al. 2015), many hybrid species have been identified among emerging plant pathogens (Jung et al. 2017, Man in’t Veld et al. 2012, Nirenberg et al. 2009). Due to the presence of multiple alleles originated from parent species in their nuclear genes, phylogenetic analysis of these hybrids based on nuclear sequences alone may not produce a robust placement. As illustrated in this phylogeny, the placement of hybrid species may be ambiguous. Specifically, in subclade 6b, support values for the placement of P. ×stagnum and its closely related species, P. mississippiae, P. borealis, and P. sp. delaware were moderate in the ML and BA analyses and ambiguous in the MP analysis (Fig. 3). Similarly, in subclade 7a, the placement of P. ×alni, P. ×cambivora, P. ×heterohybrida, and P. ×incrassata’ cluster was not well resolved due to ambiguous placement in the MP analysis and moderate support values in the other two analyses (Fig. 4). Adding mitochondrial sequences into the phylogenetic analyses may be a solution to this problem. However, due to the uniparental inheritance of mitochondria, the hybrids and their maternal parents are inseparable by mitochondrial sequences and their placements could conflict with nuclear analyses (Martin et al. 2014).