Mycological Progress (2019) 18:1411–1421 https://doi.org/10.1007/s11557-019-01523-0 ORIGINAL ARTICLE Two new species of Halophytophthora from Brazil Ana L. Jesus 1 & Agostina V. Marano 1 & Danilo R. Gonçalves 1 & Gustavo H. Jerônimo 1 & Carmen L. A. Pires-Zottarelli 1 Received: 4 October 2018 / Revised: 27 August 2019 / Accepted: 10 September 2019 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract During a survey of oomycetes in a mangrove area of São Paulo state, Brazil, a high number of isolates of Halophytophthora and related genera were recovered from leaves and water samples. In this study, we analysed the morphology and phylogenetic relationships of the isolates of Halophytophthora along with four ex-types provided by CBS-KNAW Culture Collection. Maximum likelihood and Bayesian analyses of the internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions confirmed previous evidence of the polyphyly of the genus and revealed the presence of two yet undescribed species. The two new species, Halophytophthora souzae and H. insularis, belong to the Halophytophthora sensu stricto clade and are described herein based on their morphology and/or phylogenetic position. Keywords ITS . LSU . Mangroves . Oomycota . Peronosporales Introduction The genus Halophytophthora was described by Ho and Jong (1990) to accommodate all Phytophthora species commonly found in marine habitats. Species of Halophytophthora play important ecological roles in the environment as first colonizers of fallen mangrove leaves (Newell et al. 1987; Nakagiri et al. 1989; Newell and Fell 1992; Tan and Pek 1997) and their abundance in saline habitats is generally attributed to the high tolerance to fluctuating salinity and pH conditions as well as to the production of large amounts of chemotactic zoospores that rapidly locate suitable substrates for colonization (Nakagiri et al. 1989; Leaño et al. 1998; Nakagiri 2000). Recently, some studies have demonstrated that Halophytophthora is polyphyletic (Hulvey et al. 2010; Robideau et al. 2011; Lara and Belbahri 2011; Nigrelli and Thines 2013; Yang and Hong 2014; Marano et al. 2014a; Jesus et al. 2016) and its distribution is not restricted to marine and estuarine habitats (Yang and Hong 2014). Currently, 14 species of Halophytophthora sensu lato are considered valid Section Editor: Marco Thines * Ana L. Jesus analuciajesus@hotmail.com 1 Núcleo de Pesquisa em Micologia, Instituto de Botânica, Av. Miguel Estéfano 3687, São Paulo, SP CEP 04301-012, Brazil (www.mycobank.org); however, only five (H. avicenniae, H. batemanensis, H. fluviatilis, H. polymorphica and H. vesicula) are grouped in a well-defined clade commonly referred to as Halophytophthora sensu stricto (Yang and Hong 2014; Marano et al. 2015; Jesus et al. 2016). Due to the poor phylogenetic resolution of this genus, some taxonomic changes have been proposed in the last few years. Halophytophthora kandeliae has been transferred to Phytopythium (Marano et al. 2014a, b; Thines 2014), and evidence supports that H. porrigovesica clusters within Phytophthora (Lara and Belbahri 2011; Marano et al. 2014a; de Cock et al. 2015) and that H. exoprolifera, H. elongata, H. bahamensis, H. epistomium, H. masteri and H. mycoparasitica might belong to yet undescribed genera (Marano et al. 2015). In addition, H. spinosa was transferred to the genus Salispina, which was proposed for grouping the two varieties of H. spinosa and the new species Salispina intermedia (Li et al. 2016). Most recently, H. operculata has been transferred to Calycofera, which is a sister genus to Phytopythium (Bennett et al. 2017). Despite all the taxonomic rearrangements performed hitherto, there is still much to resolve regarding the species assigned to Halophytophthora sensu lato, such as the position of some species that fall outside the Halophytophthora sensu stricto clade and are closely related to Pythium, Phytopythium and Phytophthora (Lara and Belbahri 2011; Marano et al. 2015; Li et al. 2016). During a survey of oomycetes at “Parque Estadual da Ilha do Cardoso”, Brazil, samples of mangrove leaf litter and water of different salinities were analysed and 101 specimens of Mycol Progress (2019) 18:1411–1421 1412 Halophytophthora were isolated, among them, two new species that are described herein based on a combination of morphological and/or molecular analyses of the ITS and LSU rDNA regions. Material and methods Sampling, isolation and morphological studies Samples of fallen mangrove leaves (approximately 400 g) of Rhizophora mangle L. and Laguncularia racemosa (L.) C. F. Gaertn. were collected between Aug 2012 and Jun 2013, at different salinity ranges (determined with Horiba®U-10 and U-51) along the Perequê river (S0, 0.5–1‰, S1, 0.7–0.8‰, S2, 13.5–16.2‰, S3, 21.7–29.1‰ and S4, 26.2–30‰). This river is located in a mangrove area at “Parque Estadual da Ilha do Cardoso” (PEIC), Cananéia, São Paulo state, Brazil. In the laboratory, leaves were cut with a cork borer into discs (6-mm diameter) and washed five times with a dilution of 50% seawater. After washing, leaves were processed as follows: (i) placed into Petri dishes with 30 mL of diluted seawater (the dilution was prepared according to the salinity recorded in the field) and baited with five Sorghum spp. seeds; (ii) placed onto Petri dishes with PYGs (meat peptone 1.25 g L−1, yeast extract 1.25 g L−1, glucose 3 g L−1, agar 6 g L−1, H2O 500 mL) prepared with 50% sterile seawater and 0.1 g L−1 of each penicillin G and streptomycin sulphate. After 3–4 days of incubation, the plates were examined under the microscope to check for the presence of Halophytophthora. The strains were purified by aseptically inoculating single hyphal tips onto PYGs. Once purified, fragments of the colonies were transferred to clarified V8s (V8 juice 50 mL, calcium carbonate 1.5 g L−1, agar 6 g L−1, H2O 450 mL) prepared with 50% sterile seawater and 0.1 g L−1 of each penicillin G and streptomycin sulphate. Plates were incubated in the dark for 4–7 days. Thereafter, fragments from the margins of purified colonies actively growing onto V8s were placed into new Petri dishes with 50% sterile seawater in order to stimulate zoospore release. Purified cultures were maintained on PYGs at room temperature in the dark for further use (morphological characterization and DNA extraction) and transferred to a new culture medium periodically. Identifications were made according to the original descriptions of Halophytophthora species (Anastasiou and Churchland 1969; Fell and Master 1975; Gerrettson-Cornell and Simpsom 1984; Ho et al. 1992; Ho et al. 2003; Nakagiri et al. 2001; Yang and Hong 2014), and the examination of ex-types (Halophytophthora batemanensis CBS 679.84, H. exoprolifera CBS 251.93 and CBS 252.93 and H. polymorphica CBS 680.84) and the voucher specimens H. vesicula CBS 152.96 and CBS 393.81 imported from the CBS-KNAW Culture Collection. DNA extraction, amplification and sequencing Biomass production was according to Marano et al. (2014a). DNA genomic extraction followed the protocol d es cr i be d in t he P ur eL i n k G en o m i c D N A K i t (Invitrogen™). Electrophoresis was performed using 1% (p/v) agarose gel. The partial LSU, complete ITS1-5.8S/ ITS2 (rDNA) and COI regions were amplified using the PCR SuperMix kit (Invitrogen™) and the primers SR1R/NS4, LR0R/LR6-O (Riethmüller et al. 2002), UNup 18S42/UN-up and OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011), respectively, in a C1000 Touch™ Thermal Cycler Bio-Rad. The PCR amplification was performed according to the conditions described in Marano et al. (2014a) and Robideau et al. (2011). Amplicons were purified with AxyPrep PCR Clean-up kit (Axygen®). PCR products were analysed by electrophoresis on a 1% agarose gel and stored frozen at − 20 °C. Sequencing was performed in an ABI 3730 DNA Analyser (Life Technologies™). Assembly of contigs and correction of ambiguous bases were performed manually using Sequencher 4.1.4. Phylogenetic analyses For phylogenetic reconstruction, two independent analyses were performed. First, we analysed LSU rDNA sequences of Halophytophthora (Table 1) and closely related genera (Phytophthora, Phytopythium and Pythium) deposited at GenBank, with Albugo candida as outgroup. Then, we analysed ITS rDNA sequences of Halophytophthora obtained in this study and other sequences of the clade Halophytophthora s. s. deposited at GenBank, using Phytophthora infestans and P. mirabilis as outgroup. Sequences were aligned using MAFFT 7 with default settings (Katoh and Standley 2013). The best-fitting model of evolution was selected using the Akaike Information Criterion in jModelTest 0.1.1 (Posada 2008). The maximum likelihood (ML) phylogenies were reconstructed with GARLI 2.0.1 (Bazinet and Cummings 2008) using the best model for nucleotide substitution (Tamura-Nei+G for ITS and General Time Reversible+G for LSU), heuristic search using TBR and support for modes obtained by 1000 bootstrap (bs) pseudo-replicates. Bayesian inference (BI) was performed with MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003) using the Markov Chain Monte Carlo (MCMC) methodology to calculate the posterior probabilities (pp) of the phylogenetic trees. The program was run for five million generations with the same model of evolution used for the maximum likelihood phylogeny. The first 10% of the iterations were discarded as burn-in and sampled every 1000th iterations from the remainder. Mycol Progress (2019) 18:1411–1421 Table 1 1413 Species considered in the present study, strain numbers, clades and GenBank accession numbers for each of the regions analysed Species Strain number Clade LSU ITS Albugo candida AC2V AC7A AJM 23 CCIBt 4113 Type AJM 125 AJM 45 CBS 188.85 IMB164 IMB160 CBS 679.84 Type MG 25–3 WPC7778A282 IMI327602 CBS 590.85 Type CBS 251.93 Type CBS 252.93 59B9 Albugo Albugo Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora Halophytophthora Halophytophthora Halophytophthora s.s. HQ665049 HQ665050 KY327268 KY327269 NA KU052240 HQ665146 NA NA KT455414 DQ361227 NA NA HQ665279 KT455415 KT455416 NA NA NA KY320199 KY320200 KY320201 KU052238 HQ643147 KM205206 KM205205 KT455400 NA GU258914 AF271223 NA NA NA KF734966 57A9 Type 57B9 59B8 AJM 74 CCIBt 4159 CCIBt 4114 Type CBS 241.83 Type CCIBt 4111 CCIBt 4112 CBS 680.84 Type EMTS10 Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. NA NA NA KY327270 KY327271 KY327272 JX115217 NA KT455404 KT455417 NA KF734963 KF734964 KF734965 KY320202 KY320203 KY320204 NA KT455390 KT455391 KT455403 JX910907 EMTS19 EMTS4 EMTS6 CBS393.81 CCIBt 4143 CCIBt 4148 CCIBt 4140 CCIBt 4141 Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. NA NA NA KT455418 KT455408 KT455413 KT455406 KT582099 JX910908 JX910918 JX910917 JF750389 KT455396 KT455399 KT455393 KT455394 CBS 152.96 CCIBt 4144 CCIBt 4142 CCIBt 4147 CCIBt 4110 CCIBt 4146 CCIBt 4145 CCIBt 4138 CBS 305.62 CBS 291.29 CBS 554.88 P10971 CBS 554.67 Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Halophytophthora s.s. Phytophthora Clade 4 Phytophthora Clade 10 Phytophthora Clade 2 Phytophthora Clade 8 Phytophthora Clade 6 HQ232463 KT455409 KT455407 KT455412 KY327273 KT455411 KT455410 KT455405 HQ665200 HQ665190 HQ665266 EU079704 HQ665265 GU258914 KT455397 KT455395 KT455398 NA NA NA NA NA NA NA NA NA Halophytophthora souzae Halophytophthora avicenniae Halophytophthora batemanensis Halophytophthora epistomium Halophytophthora exoprolifera Halophytophthora fluviatilis Halophytophthora insularis Calycofera operculata Halophytophthora polymorphica Halophytophthora sp. EMTS10 Halophytophthora sp. EMTS19 Halophytophthora sp. EMTS4 Halophytophthora sp. EMTS6 Halophytophthora vesicula Phytophthora arecae Phytophthora boehmeriae Phytophthora capsici Phytophthora foliorum Phytophthora gonapodyides Mycol Progress (2019) 18:1411–1421 1414 Table 1 (continued) Species Strain number Clade LSU ITS Phytophthora heveae Phytophthora humicola Phytophthora infestans CBS 296.29 CBS 200.81 CBS 366.51 Phytophthora Clade 5 Phytophthora Clade 6 Phytophthora Clade 1 HQ665195 HQ665148 HQ665217 NA NA HQ643247 Phytophthora katsurae Phytophthora kernoviae Phytophthora lateralis Phytophthora meadii Phytophthora megasperma Phytophthora mirabilis Phytophthora nemorosa Phytophthora niederhauserii Phytophthora palmivora Phytophthora phaseoli Phytophthora polonica Phytophthora pseudosyringae Phytophthora quercetorum Phytophthora quininea Phytophthora ramorum Phytophthora rubi Phytophthora sinensis Phytophthora tropicalis CBS 587.85 P10958 CBS 168.42 CBS 219.88 CBS 402.72 CBS 678.85 MG 42–7 PD 01121 CBS 298.29 CBS 556.88 P15004 PD 00159 PD 01105 CBS 407.48 CBS 101.553 CBS 967.95 CBS 557.88 CBS 434.91 Phytophthora Clade 5 Phytophthora Clade 10 Phytophthora Clade 8 Phytophthora Clade 2 Phytophthora Clade 6 Phytophthora Clade 1 Phytophthora Clade 3 Phytophthora Clade 7 Phytophthora Clade 4 Phytophthora Clade 1 Phytophthora Clade 9 Phytophthora Clade 3 Phytophthora Clade 4 Phytophthora Clade 9 Phytophthora Clade 8 Phytophthora Clade 7 Phytophthora Clade 7 Phytophthora Clade 2 HQ665278 EU080057 KJ128037 HQ665159 HQ665228 HQ665285 DQ361240 EU080247 HQ665195 HQ665267 EU080268 EU080026 EU080905 HQ665230 HQ665053 HQ665306 HQ665269 HQ665233 NA NA NA NA NA AF266777 NA NA NA NA NA NA NA NA NA NA NA NA Phytopythium boreale Phytopythium chamaehyphon Phytopythium cucurbitacearum Phytopythium fagopyri CBS 551.88 CBS 259.30 CBS 748.96 CBS 293.35 Phytopythium Clade I Phytopythium Clade II Phytopythium Clade III HQ665261 HQ665177 HQ665292 NA NA NA Phytopythium oedochilum Phytopythium sindhum Phytopythium vexans Pythium acrogynum Pythium aquatile Pythium attrantheridium Pythium buismaniae Pythium coloratum Pythium cryptoirregulare Pythium cystogenes FP1 CBS 286.31 GUCC5003 CCIBt 4023 CCIBt 4025 CBS 292.37 DAOM238986 CBS 261.30 CBS 549.88 CBS 215.80 DAOM230383 CBS 288.31 CBS 154.64 CBS 118.731 CBS 675.85 Phytopythium Clade II Phytopythium Clade II Phytopythium Clade II Phytopythium Clade II Phytopythium Clade I Phytopythium Clade I Phytopythium Clade I Phytopythium Clade I Phytopythium Clade III Pythium Clade E Pythium Clade B Pythium Clade F Pythium Clade J Pythium Clade B Pythium Clade F Pythium Clade J AB690590 AB690599 HQ665186 AB690589 KJ399965 KJ399966 HQ665191 HQ665309 HQ665178 HQ665258 HQ665153 HQ665308 HQ665188 HQ665128 HQ665083 HQ665284 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Pythium deliense Pythium dimorphum Pythium echinulatum Pythium glomeratum Pythium grandisporangium Pythium hydnosporum Pythium insidiosum Pythium irregulare Pythium iwayamai CBS 314.33 CBS 406.72 CBS 281.64 CBS 120.914 CBS 286.79 CBS 253.60 CBS 574.85 CBS 250.28 CBS 156.64 Pythium Clade A Pythium Clade H Pythium Clade E Pythium Clade I Pythium Clade C Pythium Clade D Pythium Clade C Pythium Clade F Pythium Clade G HQ665204 HQ665229 HQ665183 HQ665091 HQ665187 HQ665175 HQ665273 HQ665172 HQ665131 NA NA NA NA NA NA NA NA NA Phytopythium helicoides Phytopythium kandeliae Mycol Progress (2019) 18:1411–1421 1415 Table 1 (continued) Species Strain number Clade LSU ITS Pythium mastophorum Pythium monospermum Pythium nagaii CBS 375.72 CBS 158.73 CBS 779.96 Pythium Clade J Pythium Clade B Pythium Clade G HQ665220 HQ665137 HQ665299 NA NA NA Pythium okanoganense Pythium oligandrum Pythium ornamentatum Pythium periilum Pythium periplocum Pythium porphyrae Pythium prolatum Pythium senticosum Pythium splendens CBS 315.81 CBS 382.34 CBS 122.665 CBS 169.68 CBS 289.31 CBS 369.79 CBS 845.68 CBS 122.490 CBS 462.48 Pythium Clade G Pythium Clade D Pythium Clade D Pythium Clade B Pythium Clade D Pythium Clade A Pythium Clade H Pythium Clade H Pythium Clade I HQ665205 HQ665223 HQ665117 HQ665141 HQ665189 HQ665218 HQ665303 HQ665093 HQ665237 NA NA NA NA NA NA NA NA NA **Pythium and Phytophthora clades established by Lévesque and de Cock (2004) and Kroon et al. (2012), respectively Temperature tests Isolates of the new species proposed were subcultured onto PYGs plates with 15‰ salinity (prepared with diluted seawater) and incubated at 21 °C for 7 days. Agar discs (6-mm diameter) containing mycelium were cut with a sterile cork borer and inoculated onto 15‰ PYGs plates that were incubated at 0, 5, 10, 15, 20, 25, 30, 35 and 40 °C. Five replicates of plates were prepared and, after incubation in the dark for 120 h, the mycelial diameter of each colony was measured in four directions diametrically opposed, with the aid of a millimetre ruler. Salinity tests Isolates of the new species proposed were subcultured onto 15‰ PYGs plates at the optimum growth temperature, which was determined by the temperature tests. Agar discs (6-mm diameter) containing mycelium were cut with a sterile cork borer and inoculated onto PYG prepared either with distilled water or 10, 20, 30 and 40‰ seawater. Five replicates of plates were prepared and after 100 h of incubation in the dark, the mycelial diameter of each colony was measured in four directions diametrically opposed, with the aid of a millimetre ruler. Results Phylogenetic analyses The phylogenetic analysis of the LSU rDNA region (Fig. 1) shows that Halophytophthora species are distributed among four clades: Halophytophthora s. s., Phytophthora, Pythium and Phytopythium. The Halophytophthora s. s. clade is well supported (bs 100% and pp. 1.00) and is composed of six species: H. avicenniae, H. batemanensis, H. polymorphica, H. vesicula and the new species H. souzae and H. insularis herein described. Interestingly, Halophytophthora exoprolifera appears as sister to the Phytophthora clade (bs 56% and pp. 0.99). The single sequence of Halophytophthora operculata available at GenBank clustered within the Phytopythium clade (bs 99% and pp. 1.00), although this species has been recently transferred to the new genus Calycofera, which is sister to Phytopythium. The only sequence of Halophytophthora epistomium available at GenBank did not cluster together with any of the genera considered in this analysis (i.e. Halophytophthora, Pythium, Phytophthora or Phytopythium). The ITS phylogenetic tree (Fig. 2) shows a well-supported clade (bs 100% and pp. 1.00) composed of H. avicenniae, H. batemanensis, H. fluviatilis, H. polymorphica, H. vesicula, the two new species, H. souzae and H. insularis, and other two potentially new species yet undescribed (species 1: Halophytophthora sp. EMTS4 and EMTS6, and species 2: EMTS10 and EMTS19). The three specimens of each new species grouped together into two highly supported subclades (H. souzae: bs 99% and pp. 1.00; H. insularis: bs 100% and pp. 1.00). As shown in Figs. 1 and 2, the voucher specimen CBS 393.81 putatively assigned to H. vesicula and collected at the type locality by Anastasiou and Churchland (1969), appeared as sister to the clade of H. polymorphica (LSU: bs 99% and pp. 1.00; ITS: bs 89% and pp. 0.99) that contains the exholotype sequence of this species (CBS 680.84). Taxonomy Halophytophthora souzae A. V. Marano, A. L. Jesus & C. L. A. Pires-Zottarelli, sp. nov. Fig. 3 1416 Mycol Progress (2019) 18:1411–1421 Fig. 1 Phylogram generated by maximum likelihood based on the partial LSU rDNA region. Maximum likelihood bootstrap support values < 50% are indicated with (−). Bayesian posterior probability values > 0.70 are labelled numerically. The clades that were not recovered in the Bayesian tree are indicated with (0). The scale bar indicates the average number of substitutions per site. Sequences from this study are indicated in boldface MycoBank: MB 832391 Etymology: “souzae” is in memory of our colleague and mycologist of the “Instituto de Botânica, Dr. José Ivanildo de Souza, who contributed significantly to the studies of Halophytophthora in Brazil. Type: Brazil, São Paulo: Cananéia, “Parque Estadual da Ilha do Cardoso” (25° 03′ 05″–25° 18′ 18″ S; 47° 53′ 48″– 48° 05′ 42″W), Perequê river, from submerged leaves of Laguncularia racemosa L., August 30, 2012, A. L. Jesus, A. V. Marano & C. L. A. Pires-Zottarelli (holotype SP 466404, dried culture), ex-holotype culture CCIBt 4113. GenBank: LSU: KY327269, ITS: KY320200 and COI: KY327275. Mycelium hyaline, irregularly branched, with a few septa. Sporangiophores branched, undifferentiated from the vegetative hyphae, with terminal zoosporangia. Sporangium separated from the sporangiophore by a hyaline plug of variable thickness, 1.25–5.0 μm thick (av 3.6 μm thick). Zoosporangia formed abundantly, limoniform, ovoid, obpyriform 52.5–162.5 × 37.5–77.5 μm (av 93.5 × 56.5 μm) or irregular; sometimes with two apices (but only one discharge pore) and an operculum-like structure usually present. Internal proliferation absent. Zoospores differentiated inside the zoosporangium and discharged into vesicle. Vesicle always present, globose or elongate, 62.5–175 × 20–35 μm (av 119.9 × 28 μm), which disappears completely within 1 h after zoospore release; encysted zoospores, 8.7– 12.5-μm diameter (av 10.2-μm diameter). Oogonia terminal, smooth, globose, 25–45-μm diameter (av 38.6-μm diameter). Oospore single, plerotic, yellowish-brown with age, smooth, globose, 25–45-μm diameter (av 38.3-μm diameter); wall, 2.5–5.0 μm thick (av 3 μm thick). Antheridia strictly amphigynous; one per oogonium. Chlamydospores not observed. Cardinal temperatures (at 15‰ of salinity): optimum growth at 15 °C. No growth was observed at 0, 35 and 40 °C after 120 h of incubation in the dark; however, when cultures at these temperatures were brought back to 21 °C, the growth was reactivated, indicating that 0, 35 and 40 °C are not lethal temperatures for this species. Salinity tolerance (15 °C): optimum salinity for growth was 20‰ (65.5-mm colony diameter). All isolates grew well at all salinities tested (0 to 40‰). Mycol Progress (2019) 18:1411–1421 1417 Fig. 2 Phylogram generated by maximum likelihood based on the ITS rDNA region. Maximum likelihood bootstrap support values < 50% are indicated with (−). Bayesian posterior probability values > 0.60 are labelled numerically. The scale bar indicates the average number of substitutions per site. Sequences from this study are indicated in boldface Notes: this species is characterized by the presence of oogonia with a single oospore that is yellowish with age and strictly amphigynous antheridia. Zoosporangia are similar to most species of the genus (see Table 2). Other specimens examined: Brazil, São Paulo: Cananéia, “Parque Estadual da Ilha do Cardoso” (PEIC), 25° 03′ 05″– 25° 18′ 18″ S; 47° 53′ 48″–48° 05′ 42″ W, Perequê river, from submerged leaves of Laguncularia racemosa and Rhizophora mangle L., August 30, 2012, A. L. Jesus, A. V. Marano & C. L. A. Pires-Zottarelli (AJM 23) and February 27, 2013, A. L. Jesus, A. V. Marano & C. L. A. Pires-Zottarelli (AJM 125), lost cultures. Laguncularia racemosa L., November 08, 2012, A. V. Marano, A. L. Jesus & C. L. A. Pires-Zottarelli (holotype SP 466405, dried culture), ex-holotype culture CCIBt 4114; GenBank: LSU KY327272, ITS KY320204. Idem (paratype SP 466406, lyophilized and dried culture), ex-paratype culture CCIBt 4159; GenBank: LSU KY327271, ITS KY327278 and COI KY320203. Mycelium hyaline, irregularly branched, with a few septa. Sporangiophores branched, undifferentiated from the vegetative hyphae. Zoosporangia terminal, formed abundantly, limoniform, ovoid, obpyriform 38.9–105.3 × 28.2– 80.9 μm (av. 71.1 × 51.6 μm) or irregular; separated from the sporangiophore by a hyaline plug of variable thickness 2.5–10 μm thick (av 5.3 μm thick); sometimes with two apices (but only one discharge pore) and an operculumlike structure. Internal proliferation absent. Zoospores differentiated inside the zoosporangium and discharged into a globose or elongate vesicle, 55–110 × 20–42.5 μm (av 77.8 × 28.4 μm) that disappears completely within 1 h after zoospore release; encysted zoospores, 10–12.5-μm diameter (av 11.2-μm diameter). Chlamydospores and sexual structures not observed. Halophytophthora insularis A. L. Jesus, A.V. Marano & C. L. A. Pires-Zottarelli, sp. nov. Fig. 4. MycoBank: MB 832392. Etymology: from the Latin “insularis”, in reference to the island (“Ilha do Cardoso”) from where this species was isolated. Type: Brazil, São Paulo: Cananéia, “Parque Estadual da Ilha do Cardoso” (25° 03′ 05″–25° 18′ 18″ S; 47° 53′ 48″– 48° 05′ 42″ W), Perequê river, from submerged leaves of Mycol Progress (2019) 18:1411–1421 1418 Fig. 3 Halophytophthora souzae. a–c Terminal zoosporangia. d–e Zoosporangium with two apices but only one discharge pore. f–g Zoosporangium with operculum-like structure (arrow). h Zoosporangium with operculum-like structure and vesicle (arrow). i–j Oogonium with amphigynous antheridium. Bars = 10 μm Cardinal temperatures (at 15‰ of salinity): optimum growth at 25 °C; minimum at 10 °C and maximum at 35 °C. Little growth was observed at 5 °C and no growth was recorded at 0 and 40 °C after incubation in the dark for 120 h; however, when cultures from these temperatures were brought back to 21 °C, mycelial growth was reactivated, indicating that 0 and 40 °C are not lethal temperatures for this species. Salinity tolerance (25 °C): optimum salinity was 20‰ (73.5-mm colony diameter). Both isolates (CCIBt 4114 and 4159) grew well in all salinities tested (0 to 40‰). Notes: since the asexual structures of species in the Halophytophthora s. s. clade are very similar (Table 2) and H. insularis did not produce sexual structures, it was distinguished as a new species only based on its phylogenetic position. Other specimens examined: Brazil, São Paulo: Cananéia, “Parque Estadual da Ilha do Cardoso” (25° 03′ 05″–25° 18′ 18″ S; 47° 53′ 48″–48° 05′ 42″ W), Perequê river, from submerged leaves of Laguncularia racemosa L., November 08, 2012, A. V. Marano, A. L. Jesus & C. L. A. Pires-Zottarelli (AJM 74), lost culture. Discussion The specimens of Halophytophthora souzae and H. insularis were grouped together into two well-supported subclades within the Halophytophthora s. s. clade and showed morphological features common to all species of this clade, such as abundant sporangia production, zoospore discharge with vesicle formation—except in the case of H. fluviatilis, which releases zoospores directly from the zoosporangia without a vesicle (Yang and Hong 2014), absence of internal proliferation and presence of amphigynous antheridia in some of the isolates, as shown in Table 2. The identification of H. insularis as a new species was based only on its phylogenetic placement since none of the specimens produced sexual structures. The two new species grew well in all salinities tested (0–40‰), but with regard to temperature, only H. insularis was able to grow at 35 °C. In general, the two new species had the ability to survive under different salinity and temperature ranges, evidencing their capacity to adapt to the fluctuating conditions of mangrove habitats. Our analysis of the LSU rDNA region showed that Halophytophthora exoprolifera (CBS 251.93 and CBS Morphological features H. vesicula*, **, *** Growth pattern on Petaloid/rosette PYGs culture medium Zoosporangium Ovoid, obpyriform, shape obclavate, sometimes fusiform, irregular Zoosporangium 35–275 × 24–100 size (μm) Internal Absent proliferation Papilla Present Basal plug Present Vesicle Present H. polymorphica*, **, *** H. avicenniae*, **, *** H. batemanensis*, H. fluviatilis* *** H. souzae H. insularis Petaloid Petaloid Petaloid Petaloid Petaloid Globose, ovoid, obpyriform, ellipsoid, limoniform, irregular 44–178 × 33–85 Ovoid, obpyriform, obclavate, limoniform, botuliform, reniform, irregular 44–121 × 18–52.5 Globose, ellipsoid Globose, ovoid, Ovoid, obpyriform, Ovoid, obpyriform, and limoniform limoniform, limoniform and limoniform and obovoid, irregular irregular irregular 33–96 × 26–81 28.3–58.2 × 20.1–41 52.5–162.5 × 37.5–77.5 38.9–105.3 × 28.2–80.9 Absent Absent Absent Absent Absent Absent Absent Present Present Absent Present Present/sometimes absent1 Absent Present Present Present Present Absent Present Present Present Present Present Present Petaloid Operculum-like structure Antheridia Often present Often present Often present Often present Absent Often present Often present Paragynous Absent Absent Absent Amphigynous Not observed Oogonia Terminal, globose Absent Absent Absent Terminal, globose Not observed Oospores Plerotic Absent Absent Absent Plerotic Not observed Chlamydospores Rare Coralloid hyphae Absent Absent Absent Absent Present Absent Absent Paragynous and rarely amphigynous Lateral or terminal, globose Plerotic, sometimes abortive Absent Present Absent Absent Not observed Not observed 1 Mycol Progress (2019) 18:1411–1421 Table 2 Main morphological features of the species of Halophytophthora sensu stricto, according to the original description of the authors (*), and our observations of Brazilian isolates (**, see Jesus et al. 2016) and of voucher or type specimens (***) from the CBS-KNAW culture collection Not observed in our isolates or in the ex-type culture from the CBS-KNAW collection 1419 1420 Mycol Progress (2019) 18:1411–1421 Fig. 4 Halophytophthora insularis. a, b Terminal zoosporangia. c Zoosporangia, some of them with two apices. d Elongate vesicle (arrow). e Globose vesicle (arrow). f–g Zoosporangium with elongate vesicle and operculum-like structure (arrow). Bars = 10 μm 252.93) and Phytophthora are closely related. According to Ho et al. (1992), H. exoprolifera presents external proliferation of zoosporangia and an unusual mode of zoospore discharge in which zoospores are not retained inside the vesicle after being released. Nevertheless, the examined CBS extypes of H. exoprolifera presented a mode of zoospore discharge similar to most species in the Halophytophthora s. s. clade and no external proliferation of sporangia was observed. Therefore, further morphological and molecular studies are needed to comprehend the position of H. exoprolifera and reassess the taxonomic status of other species of Halophytophthora sensu lato. The ITS and LSU rDNA analyses presented herein and our previous study (Jesus et al. 2016) showed that the voucher CBS 393.81 falls outside the H. vesicula clade and is sister to the clade of H. polymorphica. After a careful analysis of the morphology of CBS 393.81 and of the ex-holotype H. polymorphica CBS 680.84, we found that both isolates are strikingly similar. In addition, the presence of a semi-persistent vesicle that forms an exit tube by the inversion of an internal plug, originally described for H. vesicula (Anastasiou and Churchland 1969), was observed in all Brazilian isolates that fall into the H. vesicula subclade, but not in the voucher CBS 393.81. It is possible that this feature in CBS 393.81 might have changed over time or being lost due to preservation and continuous subculturing. The possibility that CBS 393.81 and H. polymorphica belong to sister clades, with very similar morphological characteristics, should also be considered and further analysed. In any case, a more comprehensive study including new isolates from the two localities of the types of both species should be carried out in order to clarify their identity and phylogenetic position. In this study, we described two new species of Halophytophthora s. s. from a mangrove swamp in southeastern Brazil. Halophytophthora species are widespread in tropical and subtropical mangroves; however, studies are still scarce worldwide and particularly in South America. The Brazilian coast has the second largest mangrove area in the world (Pelage et al. 2019) and the diversity of members of Halophytophthora s. l. has not been surveyed yet in most of the mangrove areas. Therefore, it is highly likely that there are more undescribed species awaiting discovery. Since the loss of mangrove biodiversity is accelerating at a rapid pace due to habitat degradation by land use activities and climate change, inventories are required urgently. Mycol Progress (2019) 18:1411–1421 Acknowledgements We are indebted to “Instituto Florestal” for the permission given to collect samples at “Ilha do Cardoso”, Cananéia, São Paulo, SP; to Manoel Osorio Neves Junior for his valuable help during sampling and to Marcela Castilho Boro for preparing dried cultures and depositing the isolates in the CCIBt culture collection. Funding information We also wish to thank São Paulo Research Foundation – FAPESP for the fellowships given to A.L. Jesus (Process No. 2013/01409-0) and for the financial support given to C.L.A. PiresZottarelli (Process No. 2012/50222-7), and CAPES (“Coordenação de Aperfeiçoamento de Pessoal de Nível Superior”) for the fellowship and support given to A.V. 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