ISSN 1179-3155 (print edition) Phytotaxa 307 (3): 191–198 http://www.mapress.com/j/pt/ Copyright © 2017 Magnolia Press Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.307.3.3 New insights into Plectospira genus (Oomycetes, Straminipila): morphological and molecular analyses GUSTAVO HENRIQUE JERÔNIMO1,2, ANA L. JESUS1, SARAH C.O. ROCHA1, DANILO R. GONÇALVES & CARMEN L.A. PIRES-ZOTTARELLI1 1 2 Instituto de Botânica, Núcleo de Pesquisa em Micologia, Av. Miguel Stéfano 3687, CEP 04301-912, São Paulo, SP, Brazil E-mail: gejeronimo@hotmail.com Abstract We analyzed the morphology and phylogenetic placement of three isolates of Plectospira belonging to Plectospira gemmifera and P. myriandra that were isolated from soil, water bodies and reservoir sediment. The molecular data are from the partial large subunit and the complete internal transcribed regions of the ribosomal DNA. The genus is characterized by the presence of lobulated zoosporangia and mode of zoospore discharge forming an evanescent cluster in the exit orifice. Our isolates clustered together with the single isolate of the genus available in GenBank, however, our phylogenetic analysis and morphological characterization showed that the isolate identified as Plectospira myriandra CBS 523.87 is possibly another taxon. In addition, P. gemmifera is recorded for the first time in Brazil and its sequences were inserted for the first time in a phylogenetic reconstruction. Keywords: morphology, phylogeny, Plectospira Introduction The genus Plectospira was proposed by Drechsler (1927) to include Plectospira myriandra, a phytopathogenic species originally isolated from sugarcane roots in USA. Initially placed in Saprolegniaceae due its morphological characteristics, the genus was transferred to Leptolegniaceae by Dick et al. (1999) and more recently to Verrucalvaceae by Beakes et al. (2014) considering its phylogenetic position together with the genera Aphanomyces, Pachymetra and Verrucalvus. The genus present mycelial growth and a type of zoosporangium composed typically of a complex of inflated elements together with well-developed evacuation hyphae (Drechsler 1927). Currently, according to MycoBank, four phytopathogenic species of Plectospira are described and considered legitimate (Plectospira myriandra Drechsler, P. gemmifera Drechsler, P. agama Drechsler and P. plagiocaula Drechsler) since Plectospira dubia Atkins was transferred to Atkinsiella by Vishiniac (1958) considering the morphological characteristics. The species of Plectospira are responsible for causing diseases in crops of economic importance such as tomato (Solanum lycopersicum L.) (Drechsler 1927) and sugarcane (Saccharum officinarum L.), and also in higher-plants such as Prunella vulgaris L. and Rumex acetosella L. (Watanabe 1987), although its infectious mechanism remains poorly investigated. Considering the importance of Plectospira species as parasite of different crops, is surprising as mentioned by Johnson et al. (2002), that only few studies have reported the occurrence of the genus worldwide. After their original descriptions, only P. myriandra were reported again in literature, firstly in Japan (Watanabe 1987) and then in Brazil (Gomes et al. 2003, Rocha 2006, Miranda & Pires-Zottarelli 2012, Jesus et al. 2013), but always as saprotrophs in vegetal debris. On the other hand, there is no record of P. gemmifera and the other species in literature after their publication. According to Johnson et al. (2002), the few reports can be related to a frequent misidentification of the species as belonging to Aphanomyces, another genus from Verrucalvaceae that presents morphological similarities and is composed by species considered parasites of plants (Scott 1961, Gaulin et al. 2007). The aim of this study was to characterize morphologically the isolates of P. myriandra and P. gemmifera and include both species in a phylogenetic reconstruction. Accepted by Kevin Hyde: 9 May 2017; published: 26 May 2017 191 Material & Methods Origin of isolates We used three Brazilian isolates of Plectospira in our analysis, which were deposited in CCIBt culture collection (“Coleção de Culturas de Algas, Cianobactérias e Fungos do Instituto de Botânica”, São Paulo, SP, Brazil) and MMBF culture collection (“Micoteca Mário Barreto Figueiredo”) belonging to WFCC (World Federation for Culture Collections). The isolates CCIBt 3992 and CCIBt 3372 of Plectospira myriandra was isolated from soil (23°03’05’’–25°18’18’’S and 47°53’48’’–48°05’42’’W) and freshwater samples (23º38’08”–23º40’18”S and 46º36’48”–46º38’00”W) respectively, of the Atlantic Rainforest areas, São Paulo State, Brazil. The isolate of Plectospira gemmifera (CCIBt 4250, MMBF 04/16) was isolated from sediment sample of Itupararanga reservoir (23°37’11.58”S and 47°13’59.34”W). Morphological studies Sexual and asexual structures of the isolates were characterized and measured once the pure cultures were obtained. Identification was made according to the main literatures of the genus including Drechsler (1927, 1929) and Johnson et al. (2002). Due to the similarity of the species of the genus, a comparative table has been prepared to help in the morphological identification of these two species. The morphological description of P. myriandra was made considering the isolates CCIBt 3992 and CCIBt 3372, the latter described in Jesus et al. (2013). DNA extraction, PCR amplification and sequencing For DNA extraction, the isolates were cultivated initially onto solid medium with 0.20 gL-1 of each streptomycin sulphate and penicillin G, as antibiotics. A small piece of agar with mycelium from the pure culture was transferred to Erlenmeyer’s containing 50 mL of MP5 liquid medium (maltose-peptone) prepared with autoclaved reverse-osmosis water. After incubation for 5–10 days at 21°C, the mycelium was transferred to 2.0 mL microfuge tubes in order to obtain mycelial pellets with enough biomass for DNA extraction. DNA genomic extraction followed the protocol described in the “PureLink Genomic DNA Kit” (InvitrogenTM). Electrophoresis was performed using 1% (p/v) agarose gel. The partial LSU and complete ITS regions were amplified using the primers LR0R/LR6-O (Riethmüller et al. 2002) and UN-up 18S42/UN-up 28S22 (Robideau et al. 2011) respectively. DNA was amplified with the PCR SuperMix kit (Invitrogen®) for a final volume of 25 μl in a C1000 Touch™ Thermal Cycler Bio-Rad. The PCR amplification technique was performed following the conditions described by Marano et al. (2014). Amplicons were purified with AxyPrep PCR Clean-up kit (Axygen®). Phylogenetic analysis For phylogenetic reconstruction were selected 23 isolates of Verrucalvaceae, which represents the genera with sequences available in GenBank (Table 1). The isolates Achlya primoachlya (Coker & Couch) T.W. Johnson & R.L. Seymour (CCIBt 3982) and Achlya caroliniana Coker (CCIBt 3870) were used as the outgroup taxa. Initially, the contiguous sequences were assembled using Sequencher™ version 4.1.4 (Gene Codes®) and the ambiguous bases were manually edited. The alignment was performed in MAFFT version 7 (Kazutaka & Daron 2013) following standart default. JModelTest 0.1.1 (Posada 2008), with Akaike Information Criterion, was used to determine the best model of base substitution. For sequences of ITS and LSU of rDNA regions the Transversion model (TVM+G) plus gamma distribution (G) was indicated as the best substitution model. The complete ITS (763 bp) and partial LSU regions (1261 bp) were concatenated in SequenceMatrix 1.8 (Vaidya et al. 2010) resulting in a fragment of 2024 bp in length. The Maximum likelihood (ML) analysis were performed in Garli 2.01 (Bazinet & Cummings 2008) using partition models and branch swapping by best TBR, while the support of nodes, was determined with bootstrap analysis with 1000 replications. Tree was rooted with members of the Achlya Nees. TABLE 1. Taxa, voucher and GenBank accession number of isolates used in phylogenetic reconstruction. NA: not available. *Isolates from this study. Taxa Voucher Aphanomyces repetans Aphanomyces repetans Aphanomyces laevis Aphanomyces laevis CBS 1268.86 CBS 1268.87 CBS 478.71 CCIBt 4070 192 • Phytotaxa 307 (3) © 2017 Magnolia Press GenBank accession number ITS LSU NA HQ395662 NA HQ395671 HQ643122 HQ665242 KP006463 KP006453 ...continued on the next page JERÔNIMO ET AL. TABLE 1. (Continued) Taxa Voucher Aphanomyces sinensis Aphanomyces stellatus Aphanomyces stellatus Aphanomyces salsuginosus Aphanomyces astaci Aphanomyces iridis Aphanomyces iridis Aphanomyces euteiches Aphanomyces euteiches Aphanomyces euteiches Aphanomyces euteiches Aphanomyces cochlioides Pachymetra chaunorhiza Plectospira myriandra* Plectospira myriandra* Plectospira myriandra Plectospira gemmifera* Achlya primoachlya Achlya caroliniana ATCC 4825 CCIBt 3994 AR 51 ATCC 4775 CBS 1215.37 SAP 356 CBS 524.87 CBS 154.73 315 1309 CBS 156.73 CBS 477.71 CBS 960.87 CCIBt 3992 CCIBt 3372 CBS 523.87 CCIBt 4244 CCIBt 3982 CCIBt 3870 GenBank accession number ITS LSU JQ070116 JQ070142 KP006462 KP006454 NA AF119587 JQ070107 JQ070132 NA JX115216 FM999227 NA HQ643121 HQ665248 NA HQ665129 KM486065 NA KM486066 NA NA HQ665132 HQ643115 HQ665241 NA AF119598 KR063220 KR063219 KT935288 KX353796 HQ643402 HQ665247 KX084705 KX084707 KM058757 KM058754 KP006458 KP006451 Results Plectospira gemmifera and P. myriandra had common characteristics of the genus such as the presence of lobulate zoosporangia and mode of zoospore discharge forming an evanescent cluster in exit orifice (Fig. 1–2). Although these species are morphologically similar, some structures can be used to help in distinguishing them (Table 2). Plectospira myriandra presents an evident pellucid spot and scattered pits in the internal wall of the oospores (Fig. 2) while P. gemmifera produces abundant gemmae in the whole life cycle (Fig. 1). Besides that, our isolates presented proportion of antheridia production different that was described by Drechsler (1927, 1929). We observed that our isolates of P. myriandra frequently present abundant antheridia while in P. gemmifera they are rare or not frequent. Therefore, we advise that antheridia proportion should be carefully observed in future taxonomic studies. This is the second record of P. gemmifera in the world and the first record in Brazil. Morphological analysis Plectospira gemmifera Drechsler (1929: 358) Fig 1. A–M Mycelium dense; hyphae slender, sparingly branched. Inflated elements of sporangia abundant, sometimes very extensive, which produces a long tube to release the zoospores. Zoospores forming an evanescent cluster in exit orifice and encysting with usually 10–12 μm diam. Gemmae abundant, spherical, 20–35 μm diam. and frequently pyriform or obpyriform, 40–35 × 25–28 μm diam. Oogonia mostly terminal on short branches or on longer hyphae, smooth, subsphaerical, 18–22 × 19–24 μm diam., spherical, 20–30 μm diam. provided with wall 2.5–3.0 μm in thickness. Antheridia absent or when present, diclinous. Oospore single, colorless, subcentric, spherical, 15–20 μm diam. and subsphaerical, 18–20 × 20–22 μm diam. Specimen examined—BRAZIL. São Paulo: Votorantim, Itupararanga reservoir, from sample of surface sediment located 2 meters under the water column, 29 august 2012, CCIBt 4250 and MMBF 04/16. Notes: The isolate CCIBt 4250 produces abundant gemmae of different formats when cultivated in culture medium (CMA + glucose) and in water cultures growing in Sorghum sp. seeds. In contrast with the original description of Drechsler (1929), that observed antheridia always present, in the isolate studied the antheridia production were rare or not frequent and most oogonia are abortive and/or parthenogenetic. Despite this, the abundant production of gemmae and the characteristics of oogonium, oospores and zoosporangium are in agreement with Drechsler (1929) and confirm our identification as Plectospira gemmifera. NEW INSIGHTS INTO PLECTOSPIRA GENUS Phytotaxa 307 (3) © 2017 Magnolia Press • 193 TABLE 2. Comparison of morphological characteristics between isolates of Plectospira myriandra and P. gemmifera collected in this study. Taxa (number of the culture collection) Plectospira myriandra (CCIBt 3772/CCIBt 3992) Plectospira gemmifera (CCIBt 4250) Antheridia diclinous, monoclinous, frequent diclinous, rare Oogonium scattered pits in the wall unpitted wall Oospores presence of pellucid spot absence of pellucid spot Gemmae absent present Notes: Johnson et al. (2002) mentioned that P. gemmifera presents zoosporangia larger and with more lobules than P. myriandra, however, we did not include this characteristic in the table above once it varies depending on the bait used and thus it is difficult to observe and compare. Structures FIGURE 1. A–M. Plectospira gemmifera CCIBt 4250. A–D. Different formats of gemmae. E. Lobulated zoosporangia. F–G. Zoosporangium with a long tube and zoospores cluster. H. Catenulate gemmae. I. Spherical gemmae with germinative tube. J. Oogonium and antheridia. K. Subcentric oospores. L. Empty gemmae. M. Piriform gemmae germinating. Bars: 10 μm. 194 • Phytotaxa 307 (3) © 2017 Magnolia Press JERÔNIMO ET AL. FIGURE 2. A–J. Plectospira myriandra. A–E. Plectospira myriandra CCIBt 3992. A. Lobulated zoosporangium. B. Oogonium and subcentric oospore. C. Oogonium showing pellucid spot (ps). D. Oogonium showing scattered pits in the wall (pw). E. Oogonium and abundant antheridia. F–J. Plectospira myriandra CCIBt 3372. F. Lobulated zoosporagium. G. Oogonium and antheridia. H. Oogonium and pellucid spot (ps) showed in indirect focus (arrow). I. Oogonium and subcentric oospore. J. Oogonia with pellucid spot indicated by the arrow. ps: pellucid spot. pw: scattered pits in the wall. Bars: 10 μm. Plectospira myriandra Drechsler (1927: 294) Fig 2. A–J Mycelium dense; hyphae slender, sparingly branched. Inflated elements of sporangia abundant, which produces a long tube to release the zoospores. Zoospores forming an evanescent cluster in exit orifice and encysting with usually 8–11 μm diam. Gemmae absent. Oogonia mostly laterally, rarely terminal on short branches, smooth, spherical, 20–30 μm diam., provided with wall of 1.0–2.0 μm in thickness. Antheridia always present, diclinous, rarely monoclinous, ranging from 2 to 15 or more. Oospore single, yellow, subcentric, spherical, 13.7–22 μm in diam. Specimen examined:—BRAZIL. São Paulo: Cananéia, “Parque Estadual da Ilha do Cardoso (PEIC)”, from soil sample, 20 August 2012, CCIBt 3992. NEW INSIGHTS INTO PLECTOSPIRA GENUS Phytotaxa 307 (3) © 2017 Magnolia Press • 195 Notes:—The isolates CCIBt 3992 and CCIBt 3372 (Jesus et al. 2013) of Plectospira myriandra presented all characteristics mentioned in its original descriptions (Drechsler 1927), and are in agreement with other three studies previously published in Brazil (Rocha 2006, Gomes et al. 2003, Miranda & Pires-Zottarelli 2012). In addition, the features mentioned above, which are shown in Table 2 and Figure 2, were crucial for a correct identification of this species. Phylogenetic placement Our concatenate analysis showed two major clades (Fig. 3) which presented the same configuration of the functional groups mentioned by Diéguez-Uribeondo et al. (2009). The clade I is composed by saprophytic and/or opportunistic taxa (subclade I-a) and by species considered parasites of animals (subclade I-b). The clade II is composed by parasites of plants, and is divided in two subclades (II-a and II-b) strongly supported (100% and 100%, respectively). The subclade II-b is composed by four isolates of Plectospira genus (P. myriandra CCIBt 3372, CCIBt 3992, CBS 523.87 and P. gemmifera CCIBt 4250) and can be considered as sister group of the Aphanomyces and Pachymetra species parasites of plants (clade II-a). Besides that, our phylogenetic reconstruction indicated with high bootstrap value (100%) that the isolate of P. myriandra CBS 523.87 possibly do not belong to this taxon. FIGURE 3. Concatenate analysis of complete ITS and partial LSU rDNA regions of Verrucalvaceae family inferred by Maximum Likelihood method. The numbers next the branches indicate the bootstrap values (%) and the scale bar the numbers of substitution per site. In bold the three Brazilian isolates of Plectospira and in grey the whole clade. Discussion Our morphological and phylogenetic analyses added important contributions to a poorly investigated genus inside the Verrucalvaceae sensu Beakes et al. (2014). Both species (P. gemmifera and P. myriandra) analyzed in this study showed morphological features that contributed to their identification. In addition, their position in the phylogenetic analysis of two DNA combined regions (complete ITS and partial LSU of rDNA) corroborated our morphological characterization. The Plectospira species formed a well-supported monophyletic subclade (100%) positioned as a sister group of subclade II-a, composed by parasites of plants (Riethmüller et al. 2002, Diéguez-Uribeondo et al. 2009). Although the phylogenetic analysis showed a closely relation between the isolates from this study with plant pathogenic species (subclade II-a), it is interesting to mention that our isolates were isolated as saprophytes from soil, freshwater and surface sediment of reservoirs, which shows the great adaptability of these species in different environments. Inside the genus Plectospira, the three isolates from this study clustered with the single isolate of the genus identified as P. myriandra (CBS 523.87) available in GenBank, although our two isolates of P. myriandra are not very closely related with the specimen from GenBank. Considering the position of P. myriandra from GenBank together 196 • Phytotaxa 307 (3) © 2017 Magnolia Press JERÔNIMO ET AL. with P. gemmifera with high support value (100%), as well as the presence in our P. myriandra isolates of singular characteristics mentioned in Table 2, we suggest that P. myriandra (CBS 523.87) described by Watanabe (1987) is possibly another species of Plectospira. This study brings important information about the occurrence and provides new molecular data of a poorly studied genus. Although both species herein related have been previously described as parasites of plants, we found them acting as saprophytes in soil, freshwater habitats and sediment reservoir. The ability of both species as facultative parasites shows the great flexibility of these taxa to different environmental pressures. Acknowledgments We would like to thank FAPESP (“Fundação de Amparo à Pesquisa do Estado de São Paulo), for the fellowships given to G.H. 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