Revisiting Salisapiliaceae.

Of the diverse lineages of the Phylum Oomycota, saprotrophic oomycetes from the salt marsh and mangrove habitats are still understudied, despite their ecological importance. Salisapiliaceae, a monophyletic and monogeneric taxon of the marine and estuarine oomycetes, was introduced to accommodate species with a protruding hyaline apical plug, small hyphal diameter and lack of vesicle formation during zoospore release. At the time of description of Salisapilia, only few species of Halophytophthora, an ecologically similar, phylogenetically heterogeneous genus from which Salisapilia was segregated, were included. In this study, a revision of the genus Salisapilia is presented, and five new combinations (S. bahamensis, S. elongata, S. epistomia, S. masteri, and S. mycoparasitica) and one new species (S. coffeyi) are proposed. Further, the species description of S. nakagirii is emended for some exceptional morphological and developmental characteristics. A key to the genus Salisapilia is provided and its generic circumscription and character evolution in cultivable Peronosporales are discussed.

INTRODUCTION

The Phylum Oomycota is a monophyletic group of fungal-like eukaryotes of the Kingdom Straminipila (Beakes & Thines 2017). Members of this group are saprotrophs, pathogens, or parasites of various plant and animal species in both aquatic and terrestrial environments. Habitats in which oomycetes seem to play a major role are the mangrove and salt marshes (Marano ). Fallen senescent leaves of mangrove and salt marsh plants have proven to be rich in oomycete decomposers, which were originally subsumed as estuarine or marine Phytophthora (Fell & Master 1975, Pegg & Alcorn 1982, Nakagiri ). Based on their environmental preference, they were later assigned to a morphologically diverse genus of their own, Halophytophthora (Ho & Jong 1990).

Halophytophthora was found to be polyphyletic on the basis of phylogenetic studies (Hulvey , Lara & Belbahri 2011). Based on recent phylogenetic analyses, there are only five known species of the Halophytophthora s. str., namely H. vesicula (the type species of the genus), H. avicenniae, H. batemanensis, H. polymorphica (Hulvey , Lara & Belbahri 2011, Nigrelli & Thines 2013, Marano , Thines 2014), and the freshwater isolate, H. fluviatilis (Yang & Hong 2014) – the only known congener to date which was isolated from a freshwater biome. A few species of Halophytophthora were transferred to Phytopythium (Phytopythium kandeliae, basionym: H. kandeliae) (Thines 2014), and Salispina (Salispina lobata, basionym: Phytophthora spinosa var. lobata, and Salispina spinosa, basionym: Phytophthora spinosa var. spinosa) (Li ); whereas some species were either associated with Phytophthora or Salisapilia, or are forming separate lineages (Li , Marano , Jung ).

The genus Salisapilia, which type species, Salisapilia sapeloensis, was isolated from Spartina alterniflora, was described based on the following features contrasting to Halophytophthora: a small hyphal diameter, the formation of an apical or protruding hyaline plug, the absence of an evanescent or persistent vesicle during zoospore release, and homothallism. However, Salisapilia nakagirii, a homothallic species described by Hulvey , did not develop sporangia under the cultivation conditions applied, so the description of this species was based only on the morphology of gametangia and its phylogenetic placement within Salisapiliaceae. However, Hulvey included only a small fraction of the species described in Halophytophthora in their dataset. Thus, it cannot be ruled out that several lineages not strongly supported as nested within Halophytophthora (Lara & Belbahri 2011) represent members of the genus Salisapilia. It was the aim of this study to close this knowledge gap by detailed phylogenetic and morphological analyses.

MATERIALS AND METHODS

Acquisition of strains and sporulation

Ex-type strains of Halophytophthora and Salisapilia were either acquired from NBRC in Japan or the Westerdijk Fungal Biodiversity Institute (formerly CBS-KNAW) in the Netherlands. Strains were cultivated and maintained on clarified-vegetable juice agar (VJA) (Medium No. 15 NBRC, using Alnatura Gemüsesaft or Campbell V8 Juice) (http://www.nite.go.jp/en/nbrc/cultures/media/culture-list-e.html) with or without antibiotics: Nystatin (500 mg/mL), as well as Rifampicin (30 mg/mL) or Streptomycin (0.5 mg/mL).

All strains used in this study were tested for sporulation in saline solution at 0, 10, 20 and 30 promille (w/v) from 3–7-d-old cultures in 60 mm Petri plates. Plates were incubated in the dark at room temperature for 18–24 h or until sporangia were formed. Morphological characteristics were observed using a Motic AE31 trinocular inverted microscope (Motic, Wetzlar, Germany) and photos were taken using a Canon Digital Camera EOS 500D (Canon, Tokyo, Japan). Isolates were also grown on agarised media: Potato Carrot Agar (PCA), Peptone Yeast Glucose Agar (PYGA) and Potato Dextrose Agar (PDA) at room temperature (~20–25 °C) (Crous ).

DNA extraction, PCR, and phylogenetic reconstruction

Cultures were grown on VJA plates at room temperature in a dark compartment. After 7–10 d, mycelia were harvested and subjected to DNA extraction following the method outlined in Bennett ). Extracted genomic DNA for all samples was amplified by PCR for the internal transcribed spacers (ITS), and the large nuclear ribosomal subunit (LSU). The primers ITS1-O (Bachofer 2004) and LR0 (Moncalvo ) were used for the ITS region, while LR0R (Moncalvo ) and LR6-O (Riethmüller ) were used for the LSU region.

The 25 µL PCR reaction mixes contained 1× PCR Buffer, 0.2 mM dNTPs, 2.0 mM MgCl2, 0.8 µg bovine serum albumin, 0.4 µM of each primer, 0.5 U Taq polymerase and 10–50 ng of DNA. Cycling conditions for the ITS included an initial denaturation at 94 °C for 4 min, followed by 36 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 20 s, and elongation at 72 °C for 60 s; and a final elongation at 72 °C for 4 min. For the LSU region, initial denaturation was set at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 53 °C for 20 s, and elongation at 72 °C for 2 min; and a final elongation at 72 °C for 7 min. All amplification reactions were carried out in an Eppendorf Mastercycler Pro equipped with a vapoprotect lid (Eppendorf AG, Hamburg, Germany).

PCR amplicons were sequenced by the laboratory centre of the Senckenberg Biodiversity and Climate Research Centre (SBiK-F, Frankfurt am Main, Germany) using the primer used in PCR. Sequences were analysed, assembled into contigs, and edited using Geneious v. 5.0.4 (Biomatters Ltd., USA). Edited contigs in FASTA format and ex-type sequences from the NCBI (https://www.ncbi.nlm.nih.gov/nuccore) and the Phytophthora database (http://www.phytophthoradb.org/) (Table S1) were uploaded to the TrEase webserver (http://www.thines-lab.senckenberg.de/trease/) for multiple sequence alignment using MAFFT, version 7 (Katoh ). A primary phylogenetic tree computation using Minimum Evolution (ME) was generated using FastTree, version 1 (Price ) as implemented on the TrEase webserver following the Generalized Time-Reversible (GTR) algorithm and 1 000 bootstrap replicates. Maximum Likelihood (ML) inference was done as the secondary tree using the FastTree, version 2 (Price ) with the GTR algorithm model and 1 000 bootstrap replicates. A third phylogenetic reconstruction was done using Bayesian Inference (BI) as implemented in the TrEase webserver using MrBayes, version 3.2 (Ronquist ). For Bayesian analysis the 6-GTR substitution model was used and 1 M generations were run, with trees sampled at every 10 000th generation, discarding the first 30 % of the sampled trees to ensure sampling always reached the stationary phase. After checking that there were no supported conflicts between the datasets, alignments of each locus were concatenated into a single alignment file using SequenceMatrix (Vaidya ) and phylogenetic trees of concatenated alignments were generated following the above-mentioned protocols. Phylogenetic trees were viewed using MEGA v. 6 or 7 (Tamura ).

Ancestral state reconstruction for papilla and hyaline apical plug

The ancestral state reconstruction of the papilla and the hyaline apical plug was done using observed or recorded characteristics for Halophytophthora (Anastasiou & Churchland 1969, Gerrettson-Cornel & Simpson 1984), Salisapilia (Table 1), and other members of Peronosporaceae (e.g. Phytophthora, Phytopythium, and Pythium) (van der Plaats-Niterink 1981, de Cock , Paul 1987, Erwin & Ribeiro 1996, Paul , Paul 2000, Nechwatal & Oßwald 2003, Uzuhashi , Kroon , de Cock ). The traits were mapped on the Bayesian phylogeny of the concatenated dataset using Mesquite v. 3.2, and the likelihood ancestral reconstruction algorithm (Maddison & Maddison 2018) was run using the following the character data: (0) Papilla (P) forming an apical plug (AP); (1) P not forming an AP; (2) Semi-papilla (SP) forming an AP; (3) SP not forming an AP; (4) Non-papillate; and, “?” when sporangial germination was neither reported nor observed.

Table S1.

GenBank numbers of sequences used in this study.

Species	Strain information	Other strain no.	ITS	LSU
Halophytophthora
H. bahamensis	NBRC 32557	IFO 32557 ATCC28297 P3931*	MF979510	MF979503
H. bahamensis T	NBRC 32556	IFO 32556 ATCC 28296 CBS 586.85 IMI 330182 P3930*	MF979511	MF979504
H. batemanensis T	NBRC 32616	CBS 679.84 NBRC 32616 MG 25-3 MG 33-5 DAR 41559 IMI 327602 ATCC 56965	AF271223	DQ361227
H. elongata T	NBRC 100786	BCRC 33983	MF979512	MF979505
H. epistomia	CBS 590.85	NBRC 32617	HQ643220	HQ665279
H. epistomia T	NBRC 32617	IFO 32617 ATCC 28293 IMI 330183 CBS 590.85	MF979513	MF979506
H. masteri T	NBRC 32604	IFO 32604 ATCC 96906 CBS 207.95	MF979514	MF979507
H. mycoparasitica	NBRC 32967	IFO 32967	MF979515	MF979508
H. mycoparasitica	NBRC 32966	IFO 32966	MF979516	MF979509
H. polymorphica T	CBS 680.84	DAR 41562 IFO 32619 ATCC 56966 NBRC 32619	HQ643313	HQ665288
H. vesicula T	NBRC 32216	IFO 32216 CBS 393.81	JF750389	KT455418
H. vesicula	CBS 152.96		HQ232472	HQ232463
Phytopythium
P. helicoides	CBS 286.31		HQ643383	HQ665186
P. kandeliae	AJM26		KJ399962	KJ399965
P. kandeliae	CBS 113.91		KJ399961	HQ665079
P. megacarpum	CBS 112351		AB725881	HQ665067
P. montanum	CBS 111349		AB725883	HQ665064
P. ostracodes	CBS 768.73		HQ643395	HQ665295
P. palingenes	CCIBt 3981		KR092139	KR092143
P. vexans	CBS 119.80		AY598713	HQ665090
Salisapilia
S. nakagirii T	LT6456	CBS 127947 NBRC 108757	HQ232467	HQ232458
S. sapeloensis T	LT6440	CBS 127946 NBRC 108756	HQ232466	HQ232457
S. tartarea T	CBS 208.95	IFO 32606 NBRC 32606 ATCC 96905	HQ232473	HQ232464
Saprolegnia
S. parasitica	CBS 540.67		AY310504	HQ665256
S. parasitica	CBS 127041		HQ111458	HQ395663
Phytophthora
P. boehmeriae	CBS 291.29	PD 00181 P6950	NR147884	HQ665190
P. capsici	CBS 128.23		DQ464056	HQ665120
P. cinnamomi	CBS 144.22		KC478663	HQ665126
P. clandestina	CBS 349.86	P3942	PD_00134	PD_00134
P. colocasiae	P6317		PD_00139	PD_00139
P. idaei	CBS 971.95	P6767 IMI 313728	PD_00177	PD_00177
P. ilicis	P3939		PD_00133	PD_00133
P. infestans	CBS 366.51		HQ643247	HQ665217
P. insolita	IMI 288805	PD 00175 P6195	NR147858	EU080180
P. ipomoeae	P10225		PD_00078	PD_00078
P. iranica	CBS 374.72	P3882	PD_00173	PD_00173
P. kernoviae	P10958		PD_00105	PD_00105
P. Mexicana	CBS 554.88	P0646	PD_00061	PD_00061
P. phaseoli	P10145		PD_00067	PD_00067
P. polonica	P15005		PD_01107	PD_01107
P. quininea	CBS 406.48	P3247	PD_00126	PD_00126
P. ramorum	CBS 101553	PD 00065 P10103	NR147877	HQ665053
Pythium
P. acanthicum	CBS 377.34		HQ643409	HQ665222
P. aphanidermatum	CBS 118.80		AY598622	HQ665084
P. apleroticum	CBS 772.81		AY598631	HQ665296
P. aquatile	CBS 215.80		AY598632	HQ665153
P. capillosum	CBS 222.94		AY598635	HQ665164
P. catenulatum	CBS 842.68		AY598675	HQ665302
P. dissotocum	CBS 166.68		AY598634	HQ665139
P. graminicola	CBS 327.62		HQ643545	HQ665211
P. inflatum	CBS 168.68		AY598626	HQ665140
P. insidiosum	CBS 574.85		AY598637	HQ665273
P. monospermum	CBS 158.73		AY598621	HQ665137
P. oligandrum	CBS 382.34		AY598618	HQ665223
P. torulosum	CBS 316.33		AY598624	HQ665206
P. vanterpoolii	CBS 295.37		AY598685	HQ665193
P. volutum	CBS 699.83		AY598686	HQ665291
Globisporangium
G. echinulatum	CBS 281.64		AY598639	HQ665183
G. heterothallicum	CBS 450.67		AY598654	AY598654
G. irregulare	CBS 250.28		AY598702	HQ665172
G. macrosporum	CBS 574.80		AY598646	HQ665272
G. multisporum	CBS 470.50		AY598641	HQ665239
G. paddicum	CBS 698.83		AY598707	HQ665290
G. perplexum	CBS 674.85		AY598658	HQ665283
G. pleroticum	CBS 776.81		AY598642	HQ665298
G. polymastum	CBS 811.70		AY598660	HQ665301
G. spinosum	CBS 275.67		AY598701	HQ665181
G. sylvaticum	CBS 453.67		AY598645	HQ665236
G. ultimum	CBS 122650		HQ643864	HQ665103
G. ultimum	CBS 398.51		AY598657	HQ665227
Elongisporangium
E. anandrum	CBS 285.31		AY598650	HQ665185
E. dimorphum	CBS 406.72		AY598651	HQ665229
E. helicandrum	CBS 393.54		AY598653	HQ665225
E. prolatum	CBS 845.68		AY598652	HQ665303
E. undulatum	CBS 157.69		AY598708	HQ665134

Strain information and abbreviation

T – ex-Type specimen

ATCC – American Type Culture Collection, USA

BCRC – Bioresource Collection and Research Center, Taiwan

CBS – Westerdijk Fungal Biodiversity (formerly Centraalbureau voor Schimmelcultures), The Netherlands

NBRC – NITE Biological Resource Centre, Japan

IMI – CABI Bioscience, part of the United Kingdom National Culture Collection

IFO – Institute for Fermentation Osaka, Japan

PD – sequence strains obtained from the Phytophthora database (http://www.phytophthoradb.org/)

*information obtained from the Phytophthora WOC Database, World Phytophthora Genetic Resource Collection (http://phytophthora.ucr.edu/)

RESULTS

Phylogenetic reconstructions

According to the phylogeny based on concatenated sequences of ITS and LSU in this study (Fig. 1), strains Halophytophthora bahamensis NBRC 32556 (Fig. 2), the strain NBRC 32557 (Fig. 4), which was named as H. bahamensis, but is not conspecific with the ex-type strain, H. elongata NBRC 100786 (Fig. 3), H. epistomia NBRC 32617 (Fig. 5), H. masteri NBRC 32604 (Fig. 6), and H. mycoparasitica NBRC 32966 (= NBRC 32967) (Fig. 7) clustered with other members of the Salisapiliaceae with strong to maximum support (Fig. 1). Further, these strains were distinct from S. nakagirii LT6456 (= CBS 127947) (Fig. 8), S. sapeloensis LT6440 (= CBS 127946) (Fig. 9), and S. tartarea CBS 208.95 (Fig. 10).

Fig. 1.

Phylogenetic tree based on concatenated ITS and LSU alignments based on Minimum Evolution (ME) inference, with bootstrap support values from ME and Maximum Likelihood, as well as posterior probabilities from Bayesian Inference, in the respective order. (-) indicates support below 50 % (bootstrap) or 0.8 (posterior probability), or alternating but not strongly supported topology (support below 70 % bootstrap or 0.9 posterior probability). The scale bar indicates the number of nucleotide substitutions per site.

Fig. 2.

Salisapilia bahamensis NBRC 32256. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E, F. Mature, vacuolated sporangia, (inset figure, sporangium showing hyaline apical plug). Scale bars: A–D. = 30 mm, E, F. = 20 µm.

Fig. 4.

Salisapilia elongata NBRC 100786. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Mature sporangium; hyaline apical plug (inset). D. Mature sporangium releasing zoospores through a tubular vesicle. Scale bars: A, B = 30 mm, C, D. = 20 µm.

Fig. 3.

Salisapilia coffeyi NBRC 32557. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E. Immature sporangium. F, G. Mature sporangia, (inset figure) sporangium showing hyaline apical plug. H. Empty sporangium; inset, elevated or umbonate basal plug. Scale bars: A–D = 30 mm, E–H = 20 µm.

Fig. 5.

Salisapilia epistomia NBRC 32617. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E–F. Mature sporangia; hyaline apical plug (inset, Fig. 4E). Scale bars: A–D. = 30 mm, E, F. = 20 µm.

Fig. 6.

Salisapilia masteri NBRC 32604. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C, D. Mature sporangia. Scale bars: A, B = 30 mm, C, D = 20 µm.

Fig. 7.

Salisapilia mycoparasitica NBRC 32966. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E, F. Mature sporangia. Scale bars: A–D = 30 mm, E–F = 20 µm.

Fig. 8.

Salisapilia nakagirii CBS 127947. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E. Immature sporangium. F–I. Mature sporangia, (inset figure) modified shape of a sporangium. H. Empty sporangium. I. Mature sporangium with two discharge tubes. Scale bars: A–D = 30 mm, E–I = 20 µm.

Fig. 9.

Salisapilia sapeloensis CBS 127946. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E. Mature sporangium. F, G. Oogonia. Scale bars: A–D = 30 mm, E–G = 20 µm.

Fig. 10.

Salisapilia tartarea CBS 208.95. Colony patterns on A. Vegetable juice agar. B. Potato carrot agar. C. Peptone yeast glucose agar. D. Potato dextrose agar. E, F. Mature sporangia. Scale bars: A–D = 30 mm, E, F = 20 µm.

Morphology

Halophytophthora bahamensis NBRC 32556 (Fig. 2E–F), H. elongata NBRC 100786 (Fig. 4C–D), H. epistomia NBRC 32617 (Fig. 5E–F), H. masteri NBRC 32604 (Fig. 6C–D), and H. mycoparasitica NBRC 32966 (= NBRC 32967) (Fig. 7E–F), were all forming a distinct hyaline apical plug at the apex of the discharge tube similar to S. sapeloensis CBS 127946 (Fig. 9E) and S. tartarea CBS 208.95 (Fig. 10E–F). The apical hyaline plug was indistinct in S. nakagirii CBS 127947 (Fig. 8F–G). The shape of sporangia varied among species. The mode of zoospore release was either directly through a discharge tube or by the formation of an evanescent vesicle. A summary of the morphology of Salisapilia spp. is presented in Table 1.

The shape of sporangia of NBRC 32557 (Fig. 3E–H) was different from the ex-type culture of H. bahamensis (NBRC 32556) to which it had been assigned. The sporangia of the ex-type culture were bursiform, obclavate, obpyriform to highly variable and multi-lobed; whereas strain NBRC 32557 has narrowly bursiform, obpyriform to narrowly-elongated and obclavate sporangia. Variation of the shape of the sporangium was pronounced for H. bahamensis NBRC 32556, and some sporangia bore two discharge tubes. In contrast, NBRC 32557 always formed a single discharge tube and its sporangial shape was more stable. The strain NBRC 32557 releases its zoospore after extrusion of the small hyaline apical plug from the discharge tube. Zoospores are released directly out from the discharge pore and a vesicle was absent. After the sporangia had released zoospores, an umbonate or elevated basal plug was observed. Gametangia and chlamydospores were not observed for the strain NBRC 32557.

The ancestral trait reconstruction (Fig. 11) of the papilla and hyaline apical plug suggested that papillate and semi-papillate sporangia were putatively derived from non-papillate sporangia. Further, a sporangium with papilla forming a discharge tube and a hyaline apical plug appeared to be a synapomorphic trait for Salisapilia.

Fig. 11.

Ancestral trait reconstruction of the papilla and hyaline apical plug for Elongisporangium, Globisporangium, Halophytophthora, Phytophthora, Phytopythium, Pythium, and Salisapilia. White-coloured branches represent lineages with papillate sporangia bearing a hyaline apical plug; blue – papillate sporangia with no hyaline apical plug; yellow – semi-papillate sporangia with no hyaline apical plug; black – non-papillate sporangia. The scale corresponds to species divergence relative to nucleotide substitution rates based on the Bayesian phylogenetic inference.

Taxonomy

Based on the presented phylogenetic and morphological analyses of the different taxa included in this study, the genus Salisapilia contains several additional species previously treated as members of Halophytophthora. As a consequence, five new combinations (i.e. S. bahamensis, S. elongata, S. epistomia, S. masteri, and S. mycoparasitica) and a new species (S. coffeyi) for the genus Salisapilia are introduced here. Measurements for sporangia are given as (min.–)average_minus_SD–SD–average_plus_SD(–max.).

Hulvey et al., Persoonia 25: 112 (2010), MycoBank MB517465.

Colonies on VJ agar stellate, indistinct, petalloid; aerial hyphae limited; vegetative hyphae with regular branching, septae occur at maturity; hyphal swellings present, shape variable; sporangia produced in saline water, shape obpyriform, ovate, obovate, elongate to irregular; proliferation often external; dehiscence or discharge tube present, usually with a hyaline plug at the apex; zoospore release occurs after dehiscence of the hyaline plug at the apex of the discharge tube, or zoospores exit directly from the discharge pore or through an evanescent tubular to vase-like vesicle; gametangia observed for some species; antheridial attachment paragynous, diclinous; oogonia smooth-walled; oospores spherical to ovoid, terminal or intercalary.

Type species: Salisapilia sapeloenesis Hulvey et al.

Synopsis of species included in Salisapilia

(Fell & Master) R. Bennett & Thines, MycoBank MB823448. Fig. 2.

Basionym: Phytophthora bahamensis Fell & Master, Canad. J. Bot. 53: 2913. 1975. MB320472.

Synonym: Halophytophthora bahamensis (Fell & Master) Ho & Jong, Mycotaxon 36: 381. 1990. MB126014.

Typus: Holotype ATCC 28296, cultures ex-type = CBS 586.85 = IMI 330182 = NBRC 32556 , voucher ex ex-type strain NBRC3256 = USTH 014147, University of Santo Tomas Herbarium, Manila, Philippines.

Distribution: Bahamas, Philippines.

R. Bennett & Thines, MycoBank MB823342. Fig. 3.

Etymology: Dedicated to Michael Coffey for his contributions to the study of cultivable oomycetes.

Colony pattern on vegetable juice agar and potato carrot agar petaloid to rosette-like; vegetative hyphae highly branched, with septae at maturity; sporangiogenic hyphae undifferentiated from vegetative hyphae, bearing a single sporangium; sporangia smooth and thin-walled, with vacuoles, non-deciduous, (25.5–) 44–74–107(–126) × (4–)6.5–10.5–17(–19.5) µm, bursiform, narrowly bursiform, obpyriform to narrowly-elongate and obclavate, mostly with a tapering apex; dehiscence tube present; 5–13 × 2.5–4 µm; dehiscence plug present, hyaline, 1–3 µm in diameter; basal plug present, hyaline, raised to umbonate; proliferation external; zoospore release directly through the dehiscence tube after ejection of the dehiscence plug; vesicle not observed; chlamydospores not observed; gametangia not observed.

Typus: Bahamas, Conception Island, isolated from decaying leaf of Rhizophora mangle, Oct. 1972, J.W. Fell & I.M. Master (holotype USTH 014149, ex-type culture NBRC 32557, GenBank: ITS, MF979510; LSU, MF979503).

(Ho & Chang) R. Bennett & Thines, MycoBank MB823450. Fig. 4.

Basionym: Halophytophthora elongata Ho & Chang, Mycotaxon 85: 417. 2003. MB372647.

Typus: Holotype 17II2001, Y.M. Ju, Institute of Botany, Academica Sinica, Taipei, Taiwan, cultures ex-type BCRC 33983 = NBRC 100786.

Distribution: Taiwan, Philippines.

(Fell & Master) R. Bennett & Thines, MycoBank MB823449. Fig. 5.

Basionym: Phytophthora epistomium Fell & Master, Canad. J. Bot. 53: 2913. 1975. MB320475.

Synonym: Halophytophthora epistomia (Fell & Master) Ho & Jong, Abstracts IMC-4, Regensburg, 1990. MB126016.

Typus: Holotype ATCC 28293, cultures ex-type IMI 330183 = CBS 590.85 = NBRC 32617, voucher ex ex-type strain NBRC32617 = USTH 014147, University of Santo Tomas Herbarium, Manila, Philippines.

Distribution: USA.

(Nakagiri & Newell) R. Bennett & Thines, MycoBank MB823447. Fig. 6.

Basionym: Halophytophthora masteri Nakagiri & Newell, Mycoscience 35: 227. 1994. MB363473.

Typus: Holotype NBRC H-12169, NITE Biological Resource Center, Japan, cultures ex-type IFO 32604 = ATCC 96906 = CBS 207.95 = NBRC 32604.

Distribution: Bahamas.

(Fell & Master) R. Bennett & Thines, MycoBank MB824539. Fig. 7.

Basionym: Phytophthora mycoparasitica Fell & Master, Canad. J. Bot. 53: 2916. 1975. MB320485.

Synonym: Halophytophthora mycoparasitica (Fell & Master) Ho & Jong, Mycotaxon 36: 381. 1990. MB126017.

Typus: Holotype ATCC 28292 (discarded), (lectotype designated here fig. 16, Canad. J. Bot. 53: 2918 (1975), MBT386266; epitype designated here NBRC H-12221, MBT386249, ex-epitype culture NBRC 32966, NITE Bioresource Centre, Japan).

Other materials examined: NBRC 32967, NITE Bioresource Centre, Tokyo Japan.

Distribution: Malaysia, Japan.

Notes: The designated type, ATCC 28292, is no longer available, and no additional specimen was deposited in any recognised fungarium at the time Phytophthora mycoparasitica was proposed. Since neither inactive nor living material appears to remain from the collection of Fell & Master (1975), fig. 16 from that publication is designated as the lectotype, the specimen NBRC H-12221 is designated as the epitype and NBRC 32996 (NBRC, Japan) as the ex-epitype culture.

Hulvey et al., Persoonia 25: 113. 2010, MycoBank MB517466. Fig. 8.

Typus: Holotype CBS H-20478, Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands, ex-type cultures CBS 127947 = NBRC 108757 = LT6456.

Distribution: USA.

Colony pattern on Vegetable juice agar and potato carrot agar indistinct; stellate to rosette-like on peptone yeast agar; hyphae branched with septae at maturity; sporangia ovoid, globose to obpyriform, (26–)81.5–137–205(–231) × (11.5–)32–66.5–113(–133.5) µm; dehiscence tube present, filled with non-sporogenous protoplasmic mass, size 6–18.0 × 4.5–8.5 µm; hyaline apical plug indistinct; sporangial wall wrinkled in some sporangia; basal plug present in few sporangia; proliferation external; zoospore release through an evanescent vesicle; vesicle vase-shaped; gametangia present; antheridia diclinous, paragynous, club-shaped or lobed, 3–10 µm in length; oogonia hyaline, spherical, 33–48 µm; oospores 28–44 µm, hyaline, with a uniformly refractile ooplast vacuole; wall 1–7 µm.

Hulvey et al., Persoonia 25: 113. 2010. MycoBank MB517467. Fig. 9.

Typus: Holotype CBS H-20477, Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands, ex-type cultures NBRC 108756 = LT6440 = CBS 127946.

Distribution: USA.

(Nakagiri & Newell) Hulvey, Nigrelli, Telle, Lamour & Thines, MycoBank MB517468. Fig. 10.

Basionym: Halophytophthora tartarea Nakagiri & Newell, Mycoscience 35: 224. 1994. MB363474.

Synonym: Salisapilia tartarea (Nakagiri & S.Y. Newell)

Hulvey et al., Persoonia 25: 114. 2010. Nom. inval., Art. 41.5 (Melbourne).

Typus: Holotype NBRC H-12168, NITE Biological Resource Center, Japan, ex-type cultures NBRC 32606 = ATCC 96905 = CBS 208.95.

Distribution: USA.

Note: Invalidly proposed in Persoonia 25: 114 (2010), as the date of publication of the basionym was omitted.

DISCUSSION

Estuarine and saltmarsh oomycetes are a diverse group of heterokonts that recently received much attention. Members of this ecological group are in the genera Halophytophthora (Ho & Jong 1990), Phytopythium (Bala ), Salisapilia (Hulvey ), Salispina (Li ), and Calycofera (Bennett ). Of these taxa, Halophytophthora and Salisapilia were regarded to be in need of taxonomic revision (Nigrelli & Thines 2013, Marano , Beakes & Thines 2017), and the latter genus was resolved in this study.

Members of the monogeneric Salisapiliaceae are characterised by a small hyphal diameter, a protruding hyaline apical plug, and the absence of a vesicle during zoospore release (Hulvey ). However, the sporangia of S. nakagirii CBS 127947 were reported to release zoospores into a semi-persistent vesicle and that the typical hyaline apical plug was absent (Marano ). These observations are largely confirmed in this study, demonstrating that S. nakagirii has an exceptional mode of sporulation, even though we classify the vesicle as evanescent, as the structure is not readily observable sometime after zoospore release. Marano reached the conclusion that S. nakagirii is papillate; however, it appears that the discharge tube is rather filled with some non-sporogenous mass, which is protoplasmic of origin, and its distalmost part is probably homologous to the apical plug observed in other species of Salisapilia, giving the impression of a papilla (Gerrettson-Cornell & Simpson 1984).

Hulvey suggested that the intricacies of zoospore release might be of phylogenetic relevance and, thus, useful for resolving some systematic complexities of saprotrophic oomycetes. However, the example of S. nakagirii, in line with observations on other species of saprotrophic or hemibiotrophic Peronosporales, demonstrates the necessity to combine morphological and ontogenetic data with molecular phylogenetics, as the process of zoospore release might be variable within genera (Gisi , Gerrettson-Cornell & Simpson 1984, Bala , de Cock ). Difficulty in finding clade specific-synapomorphies is common in saprotrophic and hemibiotrophic oomycetes. A good example of this is the paraphyletic genus Phytophthora, where the classification by Waterhouse (1963) or Stamps does not reflect natural groupings resolved by multigene-phylogenies (Cooke , Kroon , Blair , Runge ). Halophytophthora elongata (Ho ) and H. masteri (Nakagiri ) formed elongated to tubular-shaped, discharge-tube-like vesicles, similar to the vase-like vesicle of S. nakagirii prior to zoospore release.

While the absence of a vesicle does not seem to be a characteristic useful for delineating Salisapilia, the hyaline apical plug is a feature that seems to be of more diagnostic value. It is a usually readily observable cone-like structure nested at and eventually protruding from the apex of the discharge tube. Prior to zoospore release, the hyaline plug is ejected or detached from the discharge tube (Nakagiri , Ho ) giving way for the release of zoospores. The only species in which this feature does not manifest is S. nakagirii. Haowever, variation in size of the hyaline apical plug is present among members of Salisapilia (Table 1). Based on the ancestral trait reconstruction analysis, it was observed that non-papillate sporangia appear as an ancestral trait to papillate and semi-papillate sporangia (Yang ). In the present ancestral state reconstruction (Fig. 11), the absence of the hyaline apical plug seems to be a derived feature in S. nakagirii, and otherwise appears to be an exclusive synapomorphy for the genus Salisapilia.

Phylogenetically, Salisapiliaceae is a well-supported clade that appears to be a sister group to Peronosporaceae and Pythiaceae (Hulvey , this study). Hulvey suggested that H. bahamensis, H. epistomia, H. exoprolfiera, and H. operculata might belong to the genus Salisapilia, but as no sequence data were available at that time to support this, Hulvey refrained from proposing new combinations for any of these taxa. Of these species, Halophytophthora operculata was recently transferred to the genus Calycofera (Bennett b), which was inferred to be the sister taxon to Phytopythium. Jung suggested that H. epistomia might need to be accommodated in a genus of its own, but in the present study, it could be demonstrated that the morphology of H. epistomia fits well to the emended diagnosis of Salisapilia. Thus, it was combined into that genus instead of erecting a new one.

Key to the species of Salisapilia

1. Sporangia non-papillate; hyaline plug absent .........................................

1. Sporangia papillate; hyaline plug present ......................................... 2

2. Zoospore release through an evanescent vesicle ......................................... 3

2. Zoospore release directly through the discharge tube ......................................... 4

3. Dehiscence tube ragged appearance, with collar-like folds; sporangium shape ovoid, obpyriform, spherical .........................................

3. Dehiscence tube smooth with cone-like plug; sporangium shape, mainly elongated, bursiform, cylindrical-elongated .........................................

4. Sporangia vacuolated ......................................... 5

4. Sporangia non-vacuolated ......................................... 6

5. Sporangium shape bursiform; multi-lobed with aseptate or septate setiform appendages .........................................

5. Sporangium shape narrowly bursiform, obpyriform, elongate to obclavate; single-lobed, setiform appendages absent .........................................

6. Sexual structures absent; sporangium surface denticulate with few spines .........................................

6. Sexual structures present, homothallic; sporangium surface smooth, spines absent ......................................... 7

7. Oospores aplerotic .........................................

7. Oospores plerotic ......................................... 8

8. Hyaline apical plug protruding through the discharge tube, 3–8 µm long; sporangium shape ovoid to obpyriform .........................................

8. Hyaline apical plug nested at the discharge tube, 14–90 µm long; sporangium shape langeniform to obpyriform .........................................

Table 1.

Morphological comparison of Salisapilia spp. Measurements for sporangia are given as (min.–)average_minus_SD–SD–average_plus_SD(–max.).

Structure	S. sapeloensis (Hulvey et al. 2010)	S. coffeyi (This study)	S. bahamensis (Fell & Master 1975)	S. elongata (Ho et al. 2003)	S. epistomia (Fell & Master 1975, Ho et al. 1990a)	S. nakagirii (Hulvey et all. 2010, This studyb)	S. masteri (Nakagiri et al. 1994)	S. mycoparasitica (Fell & Master 1975)	S. tartarea (Nakagiri et al. 1994)
Hyphal diam (μm)	1–2	1–3	1–3	3–9	2–4	1–2	2–10	2–9	1–3(–9)
Septa	Occurs at maturity	Occurs at maturity	Occurs at maturity	Occurs at maturity	Occurs at maturity	Occurs at maturity	Occurs at maturity	Develop numerous septa with age	Non-septate, or septate with age
Branching pattern	Branched or unbranched	Branched or unbranched	Branching, rare	Unbranched	Branching, rare	Branched or unbranched	Branched or unbranched	Branched or unbranched	Unbranched or branched
Sporangiogenic hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae	Undifferentiated to vegetative hyphae
Sporangia Size (μm)	34–97 (av. 59)	44.05–107.33 × 6.51–16.92 (av. 74.01 × 10.32)	26–119 × 19–43 (av. 61 × 28) 39–97 × 14–31 (av. 68 × 23)	115–530 × 32–64	43–184 × 56–107 (av. 127.6 × 63.3)	81.5–205.25 × 32.25–113 (av. 136.88 × 66.43)b	26–92 × 18–91 (av. 64 × 62.6)	26–131 × 14–111 (av. 82 × 61)	20–104 × 18–96 (av. 55.6 × 47.6)
Discharge tube size (μm)	6–18	4.81–13 × 2.58–3.94 (av. 9.07 × 3.12)	3–7	-	10–51 × 9–10	6.18–18.02 × 4.3–8.7 (av. 11.95 × 6.63) b	5–28 × 6–10	Av. 22, tapering	10–22 × 4–8
Apical plug size (μm)	3–8, protruding	~1–3	1–2 (width)	10 × 5.6	14–90 × 9–10	Indistinct b	5–24 × 5–14	5–15 × 3–10	11–29 × 5–8
Surface	Smooth, partly rough	Smooth	Smooth	Smooth	Smooth	Non-smooth b	Smooth	Denticulate, few spines	Smooth
Vacuole	Absent	Present	Present	Absent	Absent	Absent b	Absent	Absent	Absent
Basal plug	Present in some, hyaline	Present, hyaline	Present, hyaline	Present, hyaline	Present, hyaline	Present, hyaline b	Present, hyaline	Present, hyaline	Present, hyaline
Detachment	Non-caducous	Non-caducous	Non-caducous	Non-caducous	Non-caducous	Non-caducous b	Non-caducous	Non-caducous	Non-caducous
Shape	Ovoid, obpyriform	Bursiform to often narrowly bursiform, obpyriform to narrowlyelongate and obclavate; Setiform appendages, absent	Highly variable, bursiform, multi-lobed, obclavate, obpyriform; Setiform appendages, present, aseptate to septate	Obovoid, obclavate, bursiform, cylindrical, elongated	Lageniform, obpyriform	Ovoid, globose, obpyriform b	Spherical, ovoid, obpyriform	Obnapiform	Spherical, ovoid to obpyriform
Zoospore release	Zoospores exit through the discharge tube after ejection of the apical plug	Zoospores exit through the discharge tube after ejection of the apical plug	Zoospores exit through the discharge tube after ejection of the apical plug	Zoospores exit through the discharge tube after ejection of the apical plug	Zoospores exit through the discharge tube after ejection of the apical plug	Zoospores are released in a vase-like discharge vesicle b	The apical plug is extruded, and a tubular vesicle is ejected. Zoospores exit through the opening	Zoospores exit through the discharge tube after ejection of the apical plug. Plug evanesces rapidly.	Zoospores exit through the discharge tube after ejection of the apical plug
Vesicle	Absent	Absent	Absent	Present, tubular	Absent	Present, vaselikeb	Present, tubular	Absent	Absent
Oogonia
Size (μm)	35–60, 49	Not observed	Not observed	Not observed	34–40, 37 a	33–48, 39	Not observed	Not observed	33–66
Surface	Smooth				Smooth a	Smooth			Smooth
Shape	Spherical, ovoid				Spherical a	Spherical			Spherical, tapered base
Oospore	Plerotic	Not observed	Not observed	Not observed	Plerotic a	Plerotic	Not observed	Not observed	Aplerotic
Size (μm)	28–56, 48				- a	28–44			24–62
Wall (μm)	2–9				4–5 a	1–7			3–10
Antheridia	Paragynous	Not observed	Not observed	Not observed	Paragynous a	Paragynous	Not observed	Not observed	Diclinous, paragynous
Size (μm)	2–9				6–24 × 2–8, 12–6 a	3–10			4–10
Shape	Simple, lobed or branched				- a	Club-shaped, lobed			Partly enwraps oogonia

- no data provided.

Data from Ho et al. (1990).

Characteristics of S. nakagirii observed in this study.