Introduction

Mangroves are inhabited by saprotrophic oomycetes, fungal-like eukaryotes in the kingdom Straminipila (Fell & Master 1975, Leaño et al. 2000, Leaño 2001, Thines 2014, Marano et al. 2016, Bennett et al. 2017a). These organisms are the first colonisers of fallen senescent mangrove leaves and, thus, have an important role in the nutrient cycling in estuarine ecosystems (Newell et al. 1987, Nakagiri et al. 1989, Leaño et al. 2000). Of the diverse mangrove oomycetes, Salispina is a genus currently comprising three described species (Li et al. 2016): S. intermedia (type species), S. spinosa (syn. Phytophthora spinosa var. spinosa, Halophytophthora spinosa var. spinosa), and S. lobata (syn. Phytophthora spinosa var. lobata, Halophytophthora spinosa var. lobata). This genus was erected to accommodate saprotrophic mangrove oomycetes with aculeolate or spiny, variously shaped sporangia, and direct zoospore release through an apical discharge tube. However, the higher taxonomic affinity of Salispina remained uncertain, and the genus was not assigned to a family or order (Li et al. 2016).

In the Philippines, Leaño (2001) recognized S. lobata (as H. spinosa var. lobata) as the first record of Salispina for the Philippines, and we did not find any other report of these organisms in the Philippines. It was the aim of this study to investigate the presence of additional species of Salispina in Philippine mangroves, and to resolve the family and order classiication.

Materials and Methods

Isolation, morphological investigation, and sporulation

The isolation and purification of the isolate used in this study, which came from decaying leaves collected from mangroves at Davao del Sur, Philippines, followed the method of Bennett & Thines (2017). For morphological investigations, samples were processed as described in Bennett & Thines (2017), but values were rounded to the nearest half micron, except for mean values. For sporulation, the development of sporangia from agarised media plugs was observed in saline concentrations of 0–3 % incubated at room temperature (∼20–25 °C) in a dark compartment. Zoospore release was induced by placing mycelia with mature sporangia in a saline solution (≥ 3.5 %) and at 35 °C without light. Colony radial growth at 20, 25, 30, and 35 °C was tested in vegetable juice agar (VJA, commercial V8 Juice, Campbell or Alnatura Gemüsesaft, Alnatura; NBRC, medium no. 15), with or without seawater (http://www.nite.go.jp/en/nbrc/cultures/media/culture-list-e.html); and potato carrot agar (PCA; Crous et al. 2009), based on Alnatura Demeter Karotten mit Kartoffeln, Alnatura). Mean colony radial growth was measured for five days and expressed as mm/day following the method of Solis et al. (2010).

Salispina sp. USTCMS 1611, S. spinosa CBS 591.85, and S. lobata CBS 588.85 were tested for growth in VJA at room temperature (∼20–25 °C) for 5 d using a candle jar incubation method as described below and mean colony radial growth was measured according to Solis et al. (2010). For sporangium development under depleted oxygen conditions, mycelium in VJA from a 7 d-old culture plate (three per strain) was cut and the resulting pieces of ∼1–2 cm2 were placed in 60 mm Petri dishes containing 3 % saline solution. The Petri dishes were placed in a desiccator with a burning candle instead of silica gel. Then the desiccator was closed, allowing the candle to consume the oxygen until the flame could not be supported anymore. Subsequently, the desiccator was incubated at room temperature (∼20–25°C). Another set-up was incubated in ambient air on a work-bench at room temperature (∼20–25 °C). For zoospore release, the same settings were used, except for incubation at 35 °C and a saline solution of 3.5 %.

DNA Extraction and PCR amplification

For DNA extraction, a phenol-isoamyl-chloroform method was used (Bennett et al. 2017b). Subsequently, PCR amplification of cytochrome oxidase 1 (cox1), cytochrome oxidase 2 (cox2), and large nuclear ribosomal subunit (nrLSU) was done using the PCR primers listed in Table 1. The PCR reaction mix contained 1× PCR buffer, 0.2 mM dNTPs, 2.0 mM MgCl2, 0.8 µg BSA, 0.4 µM of each primer, 0.5 U Taq polymerase and 10–50 ng DNA. PCR amplification of the cox1 region was done with an initial denaturation at 95 °C for 4 min, followed by 36 cycles of denaturation at 95 °C for 40 s, annealing at 51 °C for 40 s, and elongation at 72 °C for 60 s. Afinal elongation was done at 72 °C for 5 min. The cycling conditions for the cox2 region included an initial denaturation at 94 °C for 4 min, followed by 36 cycles of denaturation at 94 °C for 40 s, annealing at 51 °C for 40 s, and elongation at 72 °C for 40 s. A final elongation was carried out at 72 °C for 4 min.

Table 1 PCR Primers used in this study.
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For the LSU region, the cycling conditions were as follows: — initial denaturation 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 120 s. Subsequently, a final extension was carried out at 72 °C for 7 min.

PCR amplicons were sent to the SBiK-F Central Laboratory for sequencing with the primers used for PCR amplification. Sequences were assembled, converted into contigs and edited using Geneious version 5.0.4 (Biomatters, New Zealand). The resulting contigs were exported in fasta file format along with reference sequences selected from NCBI (https://www.ncbi.nlm.nih.gov/nucleotide) (Table 2). The resulting sequences were uploaded to the TrEase phylogeny webserver (http://www.thines-lab.senckenberg.de/trease/) for sequence alignment and phylogenetic tree reconstruction. The program MAFFT (Katoh et al. 2002) was used for multiple sequence alignment of cox1, cox2, and nrLSU sequences. Specifically, the FFT-NS-1 (fast) model was the chosen algorithm for cox1 and cox2 due to the absence of gaps and because taxa used in the multiple sequence alignments were closely related species. The G-INS-i was the algorithm used for nrLSU sequences. The primary phylogenetic tree, Minimum Evolution (ME), was generated using FastTree (Price et al. 2009), with 1000 bootstrap replicates and following the Generalized Time-Reversible (GTR) algorithm. Maximum Likelihood (ML) was generated using RAxML (Stamatakis 2014) where GTR-GAMMA was the chosen algorithm supported by 1000 bootstrap replications. Bayesian Inference was done using MrBayes (Ronquist et al. 2012) with the GTR model of substitutions and running four incrementally heated chains for 1 000 000 generations, discarding the first 30 % of the resulting trees to ensure sampling of trees and posterior probability calculations from the stationary phase. After making sure no supported incongruences were present for the different loci, alignments of cox1, cox2, and nrLSU sequences were concatenated using SequenceMatrix (Vaidya et al. 2010) and phylogenetic trees were computed as outlined above. Phylogenetic trees were viewed and annotated using MEGA, version 6 and 7 (Tamura et al. 2013).

Table 2 GenBank* sequences (accession numbers) used in this study.
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Results

Morphology

Salispina sp. USTCMS 1611 was isolated from decaying leaves collected from mangroves at Davao del Sur. Colony morphology of the isolate was appressed on both VJA and PCA (Fig. 1A–B). The strain developed aculeolate sporangia similar to known taxa of Salispina (Fig. 1) (Table 3). Hyphae were 2–9 µm wide, with retraction septae forming in some hyphae in old cultures submerged in 2–3 % saline solution incubated at room temperature (∼20–25 °C). The branching pattern was irregular. Sporulation was achieved when plugs with mycelia were placed in 2–3 % saline solution and incubated at room temperature (∼20–25 °C) in the dark. Sporangiogenic hyphae are not differentiated from vegetative hyphae until the hyphal apex swells to form a protosporangium (Fig. 1C–D). The sporangia are ovoid, clavate, globose to obpyriform (Fig. 1E–J) but some were irregularly shaped (Fig. 1H); they measured (33.5−)43–57.5–77.5(−87) × (10.5−)20–36.5–66(−75.5) (n = 100). Spines were predominantly forming at the apex of the sporangia, resulting in a crown-like appearance (Fig. 1 D–E, J), while some sporangia were partially covered in spines, rarely entirely aculeolate (Fig. 1 F–H, J), or smooth-walled sporangia were observed (not depicted). The sporangia were non-caducous and non-papillate. The sporangial content was vacuolated. The inner base of the sporangia, where the basal plug is located, was concave (Fig. 1 I, K). The basal plug was observed to be hyaline, separating the sporangiogenic hypha from the sporangium. Zoospore release occurred only when mycelium with mature sporangia was placed in a saline solution with ≥ 3.5 % and incubated at 35 °C. The apex of the dehiscence tube (Fig. 1 I–J) deliquesces and zoospores swim directly out from the tube, i.e. no vesicle was observed. No chlamydospores and gametangia were observed. A summary of morphological features of known Salispina spp. is given in Table 3.

Fig. 1
figure 1

Morphology of Salispina hoi (USTCMS 1611). Colony pattern on: A. Potato carrot agar (PCA), and B. Vegetable juice agar (VJA). C. Protosporangium. D. Immature or young sporangium. E–H. Mature sporangia; spines are forming at the apex of sporangia, while others are either having scattered spines on the surface of the sporangia or a smooth surface. H. Irregularly-shaped aculeolate sporangium. I–J. Sporangia with dehiscence tube (arrow), zoospores differentiate inside the sporangia. K. Empty sporangium. Bars: A–B = 30 mm, C–K = 20 µm.

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Table 3 Morphology of Salispina species.
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The mean colony radial growth of Salispina sp. USTCMS 1611 in VJA and PCA at different temperatures is given in Fig. 2A. The growth and sporulation of the three Salispina spp. in VJA in candle jar incubation at room temperature (∼ 20–25 °C) are presented in Fig. 2B.

Fig. 2
figure 2

Mean colony radial growth. A. Mean colony radial growth of Salispina hoi (USTCMS 1611) on VJA and PCA at different temperatures. B. Mean colony radial growth of the three Salispina species on VJA at room temperature in a candle jar. (++) = sporulation both under candle jar and ambient air conditions; (+) = sporulation under ambient air condition.

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Phylogeny

The multigene phylogenetic analysis (Fig. 3) and the singlegene phylogenetic trees (Figs S1–S3) showed that USTCMS 1611 is a distinct member of the Salispina clade, with maximum support in all analyses. Salispina sp. USTCMS 1611 was not conspecific with any known species of Salispina (Figs S1, S3), and grouped as sister to S. lobata (Figs S1–S2). In addition, the genus Salispina was found to be sister to Sapromyces elongatus (Rhipidiaceae, Rhipidiales) with strong to maximum support in the phylogenetic reconstruction based on the concatenated dataset with nuclear and mitochondrial loci (Fig. 3).

Fig. 3
figure 3

Phylogenetic tree based on concatenated sequences of cox1, cox2, and LSU. Minimum Evolution (ME) was used as the primary tree with bootstrap support values from ME, and Maximum Likelihood (ML), and Bayesian posterior probability. (-) indicates bootstrap support values lower than 50 % or unsupported alternating topology from the corresponding primary tree. Scale bar indicates the number of substitutions per site.

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Discussion

The genus Salispina was proposed based on phylogenetics and sporangial characteristics with Salispina intermedia as the type species (Li et al. 2016). The two additional species, S. spinosa and S. lobata, were first considered to be members of Phytophthora (Fell & Master 1975; as Ph. spinosa var. spinosa, and Ph. spinosa var. lobata, respectively), and later transferred to Halophytophthora (Ho & Jong 1990; as H. spinosa var. spinosa, and H. spinosa var. lobata) based on their occurrence in estuarine environments. However, Nakagiri (2002) reported in a conference note that S. spinosa (referred to as H. spinosa) has close affinities to Sapromyces of Rhipidiales. Phylogenetic analyses in the present study revealed a strongly supported sister-group relationship between Sapromyces elongatus, which is the only species of Rhipidiales with sequences deposited at NCBI, and Salispina.

The family Rhipidiaceae includes Araiospora (Thaxter 1896), Aqualinderella (Emerson & Weston 1967), Mindeniella (Kanouse 1927), Nellymyces (Batko 1971), Rhipidium (Cornu 1871), and Sapromyces (Fritsch 1893). These taxa occur in freshwater habitats anchored to submerged twigs and fruits (Sparrow 1960, Beakes & Thines 2017). Most members of the family have arborescent thalli (except Mindeniella and Sapromyces) with a more or less distinct basal cell derived from a germinated zoospore (Minden 1916), a holdfast network, and all known members feature jointed or constricted hyphae, as well as stalked sporangia and gametangia (Sparrow 1960, Blackwell et al. 2015). Sporangia of members of the family are either aculeolate or smooth-walled. Examples with aculeolate sporangia include Araiospora spinosa (syn. Rhipidium spinosum) (Thaxter 1896), A. coronata (Linder 1926), A. pulchra (Kevorkian 1934), A. streptandra (Kevorkian 1934, Shanor & Olive 1942), M. spinospora (Kanouse 1927, Sparrow & Cutter 1941), M. asymmetria (Johnson 1951), and N. megaceros (Batko 1971). The formation of spines was believed to be influenced by the availability of nutrients in the substrate as outlined below. Mindeniella has the tendency to form aculeolate sporangia after colonies are well established in the substrate (Kanouse 1927, Sparrow & Cutter 1941). However, Sparrow (1960) mentioned that Ralph Emerson had informed him that there was a correlation between the formation of spines and the near absence of oxygen in axenic cultures. Zoospore release in the family is either directly through a discharge tube (e.g. Aqualinderella fermentans, M. asymmetria) or a vesicle (e.g. Araiospora coronata, M. spinospora, R. americanum). The discharge tube is generally formed at the sporangial apex, but its length varies in different species. Gametangia of Rhipidiaceae are often pedicellate, and some species apparently produce oospores parthenogenically (e.g. Aqualinderella fermentans, M. spinospora, N. megaceros, R. parthenosporum), similar to Phytophthora insolita (Ann & Ko 1980). Several members of Rhipidiaceae were reported to grow in low oxygen concentrations (e.g. Aqualinderella, Mindeniella, Rhipidium) (Emerson & Weston 1967, Gleason 1968, Dogma 1975, Natvig 1981) and, hence, can be considered as facultative anaerobes. Dick (2001) suggested in his diagnosis of the order Rhipidiales that members had either a facultative or an obligate fermentative metabolism.

In not displaying hyphal constrictions or stalked sporangia, Salispina is morphologically divergent from the accepted genera of Rhipidiaceae. Interestingly, Fell & Master (1975) inferred that nutrition plays an important role in the development of spines in S. spinosa (as Phytophthora spinosa var. spinosa), similar to the conclusions presented before by Kanouse (1927), Sparrow & Cutter (1941), and Sparrow (1960) for Rhipidiaceae. The three strains of Salispina (S. lobata CBS 588.85, S. spinosa CBS 591.85, and Salispina sp. USTCMS 1611) tested in this study were able to grow in a candle jar arrangement, where atmospheric oxygen is around 10–14 % and carbon dioxide about 2–5 % (Luechtefeld et al. 1982, El-Sherbeeny 1996). In a mangrove environment, abiotic factors (i.e. salinity, temperature, and oxygen concentration) constantly fluctuate (Leaño et al. 2000, Kathiresan 2004, Krauss et al. 2008). In particular, the oxygen concentration is often depleted during low tide, and gas production (e.g. CH4, NH3, H2S) by anaerobic bacteria can be observed (Kathiresan 2004). This provides suitable conditions for both obligate or facultative anaerobes and microaerophiles. In line with the fermentative or microaerophilic habit observed for various members of Rhipidiales, Salispina sp. USTCMS 1611 showed normal vegetative growth in candle jars, but sporulation of members of Salispina was triggered by normal oxygen levels, and increased salinity and temperature, conditions that probably correspond to the early rise of the sea level after a low tide. While the physiological properties of Salispina support placement in Rhipidiales, the high morphological and phylogenetic divergence between Salispina and members of the Rhipidiaceae does not support a placement of Salispina in that family. Such a taxonomic classification would render the morphologically well-delineated family highly heterogenous. We therefore introduce the new family name Salispinaceae to accommodate the genus Salispina.

Salispina sp. USTCMS 1611 is a sister taxon to S. lobata, which has sporangia with a peculiar shape. Initially obpyriform, the sporangia of S. lobata subsequently develop lateral lobes until the sporangium looks botryose (Fell & Master 1975). However, USTCMS 1611 has ovoid, clavate, globose, to obpyriform sporangia, with some sporangia showing variations in shape, but not becoming botryose. In addition, the formation of spines appears to be different between the two species, with most spines of USTCMS 1611 formed at the apex of the sporangium, while some sporangia have scattered spines or are even smooth-walled. In contrast, sporangia of S. lobata are either entirely or partially aculeolate (with no distinct pattern), or non-aculeolate (Table 3). Based on morphology and phylogenetic relationships, this strain cannot be assigned to any known taxon in Salispina, and so is described here as a new species.

This raises the number of known species in Salispina to four, but, given the still fragmentary knowledge regarding estuarine oomycetes in general and Salispina in particular, it seems likely that additional species of this genus will be discovered. In contrast to other orders of Oomycota, such as Albuginales (Choi et al. 2007, Thines et al. 2009, Ploch et al. 2010, Ploch & Thines 2011, Mirzaee et al. 2013), Peronosporales (Riethmüller et al. 2002, Voglmayr 2003, Voglmayr et al. 2004, Thines et al. 2006, 2007, Göker et al. 2007, Thines et al. 2008, 2015, Choi & Thines 2015), and Saprolegniales (Dick et al. 1999, Riethmüller et al. 1999, Leclerc et al. 2000, Spencer et al. 2002, Diéguez-Uribeondo et al. 2007, Hulvey et al. 2007, Steciow et al. 2013, Sandoval-Sierra et al. 2014, Steciow et al. 2014, Rocha et al. 2018), the Rhipidiales has received relatively little attention, probably owing to a lower degree of cultivation success from environmental samples due to their often microaerophilic to anaerobic nature. Thus, it seems promising to undertake targeted sampling in oxygen-depleted limnic environments in order to gain further insights into these understudied organisms which might play an important role in nutrient cycling.

Taxonomy

Rhipidiales M. W. Dick, Straminipilous Fungi: 305 (2001).

Salispinaceae R. Bennett & Thines, fam. nov. MycoBank MB824253

Diagnosis: Differs from Rhipidiaceae in the absence of conspicuous hyphal constrictions.

Type: Salispina Marano et al., Fungal Diversity 78: 198 (2016).

Salispina hoi R. Bennett & Thines, sp. nov.

MycoBank MB823076

Etymology: Dedicated to Hon Ho, for his pioneering studies into mangrove oomycetes.

Diagnosis: Differ from its sister taxon, S. lobata in sporangia that do not become botryose at maturity and from all species of the genus by a pronounced preference of spine formation at the apex and a quickly evanescing discharge tube.

Type: Philippines: Davao del Sur, 6.579667°N 125.453667°E, isolated from decaying mangrove leaf litter, 6 Sep. 2015, R.M. Bennett, M.K. Devanadera, & G.R. Dedeles (USTH 014145 — holotype; USTCMS 1611 – ex-type culture).

Description: Mycelium appressed on VJA and PCA. Hyphae 2–9 µm wide; septae forming at maturity, branching irregular; sporangiogenic hyphae not differentiated from vegetative hyphae, bearing a single terminal sporangium. Sporangia, shape ovoid, globose, obpyriform to variable; size (33.5−) 43–57.6–77.5(−87) × (10.5−)20–36.6–66(−75.5) µm; papilla absent, basal plug concave and hyaline; sporangial content vacuolate; surface aculeolate, with spines mostly forming at the apex of sporangia resulting in a crown-like appearance, some sporangia are smooth or with very few scattered spines. Zoospores discharge directly through a dehiscence tube; the apex of the tube deliquescent, allowing zoospores to escape from sporangia; vesicle absent. Chlamydospores not observed. Gametangia not observed.

Sequences: cox1 MG019399, cox2 MF991430, and LSU MG385863.