Introduction

Mangroves are distinctive coastal ecosystems comprising a diverse group of predominantly tropical trees and shrubs that are adapted to life in coastal intertidal marine locations (Tomlinson 1986). Breathing roots, salt-excreting leaves, and viviparous water-dispersed propagules are the three most important morphological and physiological traits (Duke 1992; Shi et al. 2005). Most studies on fungi colonizing mangroves are regarding to taxonomy (Cribb and Cribb 1955; Kohlmeyer and Kohlmeyer 1971, 1977; Kohlmeyer 1981, 1985; Kohlmeyer and Schatz 1985; Kohlmeyer and Volkmann-Kohlmeyer 1991), ecology and inventory (Kohlmeyer 1966, 1969; Hyde 1988; Hyde and Jones 1988; Hyde 1989a, b, c; Jones and Kuthubutheen 1989; Hyde et al. 1992; Hyde and Lee 1995; Alias et al. 2010; Pang et al. 2011), as well as a series of reviews (Jones 2000; Sarma and Hyde 2001; Jones 2011a, b; Jones et al. 2015; Sivakumar 2016), while other studies have considered the molecular phylogeny of mangrove fungi (Jones et al. 2009; Suetrong et al. 2009; Jones and Pang 2012). There are 74 mangrove species in 53 genera and 35 families that occurred along the protected shorelines of Thailand (Plathong and Plathong 2011), and 184 fungal species have been documented from Thai mangroves, of which approximately 85% are Ascomycota (Suetrong et al. 2017).

Pleosporales Luttr. ex M.E. Barr is the largest and most diverse group in Dothideomycetes (Ascomycota), including 75 families, 400 genera, and 52 genera incertae sedis (Schoch et al. 2006b, 2009; Hyde et al. 2013; Ariyawansa et al. 2015; Liu et al. 2017; Wijayawardene et al. 2018), and four new families were recently proposed in this order using multi-locus phylogenetic evidence (Valenzuela-Lopez et al. 2018). Since molecular phylogeny has been used to rank marine fungi, most of the species found from intertidal mangrove wood, twigs, and leaves were identified as members of Pleosporales and are distributed in 12 accepted families: Aigialaceae, Biatriosporaceae, Caryosporaceae, Halojulellaceae, Halotthiaceae, Lophiostomataceae, Morosphaeriaceae, Pseudoastrosphaeriellaceae, Salsugineaceae, Testudinaceae, Trematosphaeriaceae, and Zopfiaceae (Suetrong et al. 2009; Zhang et al. 2012b; Hyde et al. 2013; Jones et al. 2015; Devadatha et al. 2017; Hyde et al. 2017; Suetrong et al. 2017; Hashimoto et al. 2018; Wijayawardene et al. 2018). Four families: Didymellaceae, Leptosphaeriaceae, Lindgomycetaceae, and Melanommataceae also include manglicolous taxa but some genera lack molecular data to confirm their phylogenetic positions (Suetrong et al. 2009; Jones and Pang 2012; Jones et al. 2015).

During examination of collections of intertidal fungi from Nypa fruticans (brackish water palm) and Phoenix paludosa (mangrove date palm) in Thailand, a novel ascomycete species, Acuminatispora palmarum, was discovered with total four isolates obtained from both hosts. Morphological comparison and multi-gene phylogenetic analysis were carried out to reveal their taxonomical classification and delineate the phylogenetic relationships with related groups. A monotypic genus Acuminatispora gen. nov. (Pleosporales, incertae sedis) is introduced to accommodate the new taxon Acuminatispora palmarum. In addition, the phylogenetic relationships of the mangrove species Acrocordiopsis patilii and the genus Caryospora are discussed.

Materials and methods

Specimen collection, examination, and single spore isolation

Decayed petioles and rachides of palms were collected from mangrove habitats in Ranong, Trat, and Chanthaburi provinces, Thailand. The specimens were packaged in plastic bags in the field and washed under running water, and then examined via laboratory procedures following those of Jones and Hyde (1988). Morphological characters were observed using a Carl Zeiss stereo microscope fitted with an AxioCam ERC 5S camera and photographed by a Nikon ECLIPSE 80i compound microscope fitted with a Canon EOS 600D digital camera. Free hand sections of fruiting bodies were made into slides within water mounts and observed under Motic SMZ 168 stereo microscope. Measurements were taken by Tarosoft Image Frame Work program v. 0.9.7 and images used for figures were processed with Adobe Photoshop CS6 Extended v. 13.0 software. Isolations were obtained from single spores as described in Choi et al. (1999). The strains isolated in this study were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and Guizhou Culture Collection (GZCC). Herbarium specimens were deposited at the herbaria of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. MycoBank numbers and Facesoffungi numbers (Jayasiri et al. 2015) of the new taxa were provided, and the new taxa were established following recommendations outlined by Jeewon and Hyde (2016).

DNA extraction, PCR amplification, and sequencing

Fungal genomic DNA was extracted from fresh mycelia scraped from the margin of a colony on PDA that was incubated at 25–28 °C for 30 days, followed by the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech (Shanghai) Co., Ltd., China) manufacturer instructions. Two partial gene portions and two protein coding genes were used in this study: the large subunits of the nuclear ribosomal RNA genes (LSU), the small subunits of the nuclear ribosomal RNA (SSU), the translation elongation factor-1 alpha (TEF1α), and the second largest subunit of RNA polymerase II (RPB2). The primers used were LROR and LR5 for LSU (Vilgalys and Hester 1990), NS1/NS4 for SSU (White et al. 1990), EF1-983F/EF1-2218R for TEF1α (Rehner and Buckley 2005), and fRPB2-5F/fRPB2-7cR for RPB2 (Liu et al. 1999). The amplification reactions were performed in 25 μL of PCR mixtures containing 9.5 μL ddH2O, 12.5 μL 2× PCR MasterMix (TIANGEN Co., China), 1 μL DNA temple, and 1 μL of each primer. The PCR thermal cycle programs for LSU, SSU, and TEF1α amplification were as follows: initially denaturing step of 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 50 s, elongation at 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR thermal cycle program for the partial RNA polymerase second largest subunit (RPB2) was followed as initially 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 2 min, elongation at 72 °C for 90 s, and final extension at 72 °C for 10 min. PCR products were observed on 1% Agarose gel electrophoresis strained with ethidium bromide. Purification and sequencing of PCR products were carried out at Sangon Biotech (Shanghai) Co., Ltd., China.

Sequence alignment and phylogeny analyses

The closely related strains of the new taxa were retrieved using nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and other strains included in this study are mainly referred to Tanaka et al. (2009), Ariyawansa et al. (2015), Hashimoto et al. (2017), and Liu et al. (2017). The final dataset of LSU, SSU, TEF1α, and RPB2 sequence data used for the phylogenetic analyses along with original references and GenBank accession numbers is listed in Table 1.

Table 1 Taxa used in this study and their GenBank accession numbers. The type species of each genus are marked with superscript T and ex-type strains are in bold
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Sequences were aligned using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and then checked visually and manually optimized using BioEdit v.7.0.9 (Hall 1999). A maximum likelihood (ML) analysis was performed at the CIPRES web portal (Miller et al. 2010) using RAxML v.7.2.8 as part of the “RAxML-HPC Blackbox (8.2.10)” tool (Stamatakis 2006; Stamatakis et al. 2008). A general time-reversible (GTR) model was applied with a discrete gamma distribution and four rate classes. Fifty thorough ML tree searches were carried out in RAxML v.7.2.7 under the same model. One thousand non-parametric bootstrap iterations were run with the GTR model and a discrete gamma distribution. The resulting replicates were plotted onto the best scoring tree obtained previously.

Maximum parsimony (MP) analyses were performed using the heuristic search option with 1000 random taxa additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equally weight; gaps were treated as missing data. Maxtrees setting was 1000, and zero-length branches were collapsed, and all parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa (Hillis and Bull 1993). Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated.

The Bayesian analysis was performed using PAUP v.4.0b10 (Swofford 2002) and MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003). The best model for different gene partition in the concatenated dataset was determined by MrModeltest 2.3 (Nylander 2004). Posterior probabilities (Rannala and Yang 1996) were determined by Markov Chain Monte Carlo (MCMC) sampling (Larget and Simon 1999) in MrBayes v.3.1.2. Four simultaneous Markov chains were run for 10 million generations and trees were sampled every 1000th generation; thus, 10,000 trees were obtained. The suitable burn-in phases were determined by inspecting likelihoods and parameters in Tracer version 1.6 (Rambaut et al. 2013). Based on the tracer analysis, the first 1000 trees representing 10% were discarded as the burn-in phase in the analysis. The remaining trees were used to calculate posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic set to 0.01). Phylogenetic tree was visualized by FigTree v.1.4.0 (Rambaut 2012), and the alignment was deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S22887).

Results

Phylogeny

Four fungal isolates from palms were obtained from mangrove-inhabiting decayed substrates and identified as members of the order Pleosporales. LSU, SSU, TEF1α, and RPB2 sequence data and morphological characters were used to determine their placement and to describe the novel taxon. The combined dataset comprised 126 taxa with Mytilinidion andinense (CBS 123562) and Mytilinidion mytilinellum (CBS 303.34) as the outgroup taxa. The dataset comprises 3980 characters (LSU 1–902; SSU 903–2129; TEF1α 2130–3032; RPB2 3033–3980) after alignment, including gaps. The maximum parsimonious dataset consists of 3980 characters, of which 2462 characters were constant and 279 variable characters parsimony uninformative. Maximum parsimony analysis of the remaining 1239 parsimony-informative characters resulted in 1000 trees with TL = 9053, CI = 0.277, RI = 0.614, RC = 0.170, and HI = 0.723. RAxML, maximum-parsimony (MP), and Bayesian analysis of the combined dataset resulted in phylogenetic reconstructions with similar topologies, and the best sorting RAxML tree is shown in Fig. 1.

Fig. 1
figure 1figure 1

RAxML tree of Pleosporales based on analysis of combined LSU, SSU, TEF1α, and RPB2 sequence data. Bootstrap values for ML and MP equal to or greater than 75 are placed above and below the branches respectively. Branches with Bayesian posterior probabilities (PP) from MCMC analysis equal or greater than 0.95 are in bold. Newly generated sequences are indicated in blue. The tree is rooted with Mytilinidion mytilinellum (CBS 303.34) and Mytilinidion andinense (CBS 123562)

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Representatives of the sequenced families (with molecular data) of Pleosporales were included in our phylogenetic analysis (Fig. 1). The phylogenetic analysis based on the combined LSU, SSU, TEF1α, and RPB2 sequence data showed that the two suborders Massarineae and Pleosporineae are well-supported, and the familial assignment of Pleosporales is similar to previous studies (Schoch et al. 2009; Hyde et al. 2013; Ariyawansa et al. 2015; Liu et al. 2017). The four newly obtained fungal isolates formed a well-supported monotypic clade and can be identified as a new genus (namely Acuminatispora) in Pleosporales, which clustered together with Acrocordiopsis Borse & K.D. Hyde, Astrosphaeriella Syd. & P. Syd., Astrosphaeriellopsis Phook., Jian K. Liu & K.D. Hyde, and Caryospora De Not., and placed as basal lineages in the order. Additionally, Acuminatispora palmarum formed a sister clade to Caryospora, while Acrocordiopsis patilii clustered together with Astrosphaeriella and Astrosphaeriellopsis in an unsupported clade. However, the phylogenetic relationships of Acrocordiopsis, Astrosphaeriella, Astrosphaeriellopsis, Acuminatispora, and Caryospora do not form a stable clade and further taxon sampling is required to resolve their phylogenetic relationships.

Taxonomy

Acuminatispora S.N. Zhang., K.D. Hyde & J.K. Liu, gen. nov.

MycoBank: MB 825525; Facesoffungi number: FoF 04671

Etymology: Name refers to the ascospores with acute or narrowly pointed ending cells.

Saprobic in mangrove habitats. Sexual morph: Ascomata black, subglobose, solitary, scattered, immersed, with an erumpent short neck. Ostiole central, periphysate, cylinder-like opening. Peridium composed several layers with cells of textura angularis. Hamathecium comprising numerous, filamentous, hyaline, branched, trabeculate pseudoparaphyses, embedded in a gelatinous matrix. Asci 8-spored, bitunicate, cylindrical, slightly curved, long pedicellate, apically rounded with an ocular chamber. Ascospores overlapping biseriate to triseriate, hyaline to brown, fusiform with acute or narrowly pointed ending cells, 1-(rarely 3) septate, constricted at the central septa, guttulate, smooth-walled. Asexual morph: Undetermined.

Type species: Acuminatispora palmarum S.N. Zhang, K.D. Hyde & J.K. Liu

Acuminatispora palmarum S.N. Zhang, K.D. Hyde & J.K. Liu, sp. nov. Fig. 2.

Fig. 2
figure 2

Acuminatispora palmarum (MFLU 18-1068, holotype; MFLU 18-1071, paratype). a–c Appearance of ascomata on host surface. d Vertical section of ascoma. e Ostiole with periphyses. f Structure of peridium. g Trabeculate pseudoparaphyses. h–k Asci. l–q Ascospores. q, 3-septate ascospore in lactophenol cotton blue reagent with clearly acute ends. s–r Germinating ascospores. t Colony on PDA. Scale bars: a = 500 μm, b, c = 100 μm, d = 200 μm, f, h–k = 20 μm, e, g, l–s = 10 μm

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MycoBank: MB 825526; Facesoffungi number: FoF 04672

Etymology: The epithet referring to the host on which the fungus was collected.

Holotype: MFLU 18-1068

Saprobic on petioles and rachides of palms in mangrove habitats. Sexual morph: Ascomata 380–610 μm high (including neck), 150–395 μm diam. (\( \overline{x} \) = 531.2 × 275.8 μm, n = 10), black, subglobose, solitary, scattered, immersed, with an erumpent short neck. Ostiole 72–85 μm diam., central, periphysate, cylinder-like opening. Peridium 10–20 μm wide, composed several brown outside layers and inter layers with hyaline cells of textura angularis. Hamathecium up to 2.5 μm wide, comprising numerous, filamentous, hyaline, branched, trabeculate pseudoparaphyses, embedded in a gelatinous matrix. Asci 93–125 × 13–22 μm (\( \overline{x} \) = 109.5 × 16 μm, n = 15), 8-spored, bitunicate, cylindrical, slightly curved, long pedicellate, apically rounded with an ocular chamber. Ascospores 24–30 × 7–10 μm (\( \overline{x} \) = 27.2 × 8.1 μm, n = 30), overlapping biseriate to triseriate, hyaline to brown, fusiform with small, hyaline, acute, or narrowly pointed ending cells, 1-(rarely 3) septate, strongly constricted at the central septa, the upper cell broader, each cell with one large guttule and sometimes several small ones, smooth-walled, lacking a sheath or appendages. Asexual morph: Undetermined.

Culture characteristics: Ascospores germinating on PDA within 24 h at 25–28 °C under natural light. Germ tubes produced from each end. Colonies growing well on both PDA and MEA media and attaining a diameter about 1.5 cm on PDA after 21 days at 25 °C, obverse olive to gray-green or light gray-green, tufted colony center elevated, reverse dark green. Mycelium 2.5–3.5 μm wide, hyaline to pale brown, aerial, septate, branched, and anastomosing, producing chlamydospores.

Material examined: Thailand, Ranong, Ngao Mangrove Forest Research Center, intermittently submerged on decayed rachis of Phoenix paludosa (Roxb. 1832), 6 December 2016, S.N. Zhang, SNT53, holotype (MFLU 18-1068), isotype (HKAS 97478), ex-type living culture MFLUCC 18-0264 = GZCC 18-0001; Thailand, Trat, Ko Chang District, 12° 1′ 14″ N, 102° 23′ 19″ E, intermittently submerged on decayed rachis of Phoenix paludosa, 27 April 2017, S.N. Zhang, SNT107, paratype (MFLU 18-1069), living culture MFLUCC 18-0460 = GZCC 18-0002; Thailand, Trat, Ko Chang District, 12° 1′ 14″ N, 102° 23′ 19″ E, submerged on decayed petiole of Nypa fruticans (Wurmb 1779), 27 April 2017, S.N. Zhang, SNT111, paratype (MFLU 18-1070), living culture MFLUCC 18-0461 = GZCC 18-0003; Thailand, Chanthaburi, 12° 26′ 43″ N, 102° 15′ 47″ E, intermittently submerged on decayed petiole of Phoenix paludosa, 25 April 2017, S.N. Zhang, SNT133, paratype (MFLU 18-1071), living culture MFLUCC 18-0462 = GZCC 18-0004.

Habitat and distribution: Inhabiting mangrove forests. The Gulf of Thailand (east) and Andaman sea (west) coastline, Thailand.

Notes: Acuminatispora palmarum is phylogenetically distinct from other members in Pleosporales (Fig. 1) and its unique morphological features of deeply immersed ascomata with an erumpent short neck, 8-spored, long pedicellate asci, and biseriate to triseriate, brown, fusiform ascospores, 1-(rarely 3) septate, with a broader upper cell and small hyaline acute or conical ending cells, also distinguish it from its phylogenetically closely related genera Acrocordiopsis, Astrosphaeriella, Astrosphaeriellopsis, and Caryospora (Barr 1979; Borse and Hyde 1989; Alias et al. 1999; Phookamsak et al. 2015), as well as two other genera Caryosporella Kohlm. and Zopfia Rabenh (Arnaud 1913; Kohlmeyer 1985), which are distinct from A. palmarum in phylogeny but having similarity in ascospore morphology. Acuminatispora palmarum shares similar immersed ascomatal morphology and 3-septate brown ascospores with tapering ends to Coronopapilla mangrovei (K.D. Hyde) Kohlm. & Volkm.-Kohlm. (≡ Caryospora mangrovei K.D. Hyde), and another mangrove species Passeriniella savoryellopsis Hyde & Mouzouras (Hyde and Mouzouras 1988; Hyde 1989c; Kohlmeyer and Volkmann-Kohlmeyer 1990, 1991). However, Acuminatispora palmarum differs from Co. mangrovei in having distinctly smaller (24–30 × 7–10 μm) biseriate to triseriate, acute or narrowly pointed ending cells of fusiform ascospores, while the latter has large (36–60 × 16–24 μm) uniseriate, ellipsoidal, thick-walled ascospores with tapering rounded ends. In addition, the permutation and the number of ascospores in asci, and the position of secondary septa are also reliable to distinguish Ac. palmarum and P. savoryellopsis (Table 2), especially the obviously different size of ascospores (24–30 × 7–10 μm vs. 64–88 × 24–28 μm). Phylogenetically, Ac. palmarum is distinct from P. savoryellopsis based on multi-gene phylogeny (data not shown), and the phylogenetic relationship of Ac. palmarum and Co. mangrovei is unresolved as the molecular data of Coronopapilla spp. is not available. We hereby introduce Acuminatispora palmarum as a new species and establish the monotypic genus Acuminatispora to accommodate it.

Table 2 Morphological comparison of Acuminatispora palmarum, Coronopapilla mangrovei and Passeriniella savoryellopsis
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Discussion

The phylogenetic placement of Acuminatispora palmarum is problematic and even equivocal in this study because of (i) the grouping pattern of the Acuminatispora, Acrocordiopsis, Caryospora, and Astrosphaeriellaceae, which is inconsistent with the phylogenetic analysis of single- and multi-gene studies (Suetrong et al. 2009; Ariyawansa et al. 2015; Phookamsak et al. 2015); (ii) the family Caryosporaceae was established mainly based on the phylogeny with one species representing each genus (Acrocordiopsis and Caryospora) (Ariyawansa et al. 2015), which was not justified enough to define the family boundary; (iii) the relatively low corresponding bootstrap support values of Acuminatispora, Acrocordiopsis, and Caryospora might due to the few discovered species and little available molecular data of these groups. Therefore, it is difficult to justify the phylogenetic placement of these groups, and further taxon sampling and re-isolation of species is required before the taxonomic position of the new genus can be resolved.

The phylogeny based on multi-gene analysis also indicated that Acuminatispora palmarum clustered together with Caryospora, and formed a monospecific clade distinct from Acrocordiopsis and Caryospora. It is reasonable that with more taxa included, the phylogenetic positions of some groups become unstable and their phylogenetic relationships with others even change in a phylogenetic tree. Furthermore, the corresponding taxonomical classification may need to be modified.

Acrocordiopsis

The initial phylogenetic study of Acrocordiopsis patilii was conducted by Suetrong et al. (2009) who noted it grouped as a residual paraphyletic assemblage and was not assigned to any family. Subsequently, the genus was assigned to Salsugineaceae K.D. Hyde & S. Tibpromma (Hyde et al. 2013) based on the same sequence data as Suetrong et al. (2009), while Ariyawansa et al. (2015) placed Acrocordiopsis in the new family, Caryosporaceae with Caryospora and both of the above two treatments were based on phylogenetic analyses with few taxa representing the genera and families.

Wijayawardene et al. (2018) referred Ac. patilii in Salsugineaceae following Hyde et al. (2013). Jones and Pang (2012) also consider the phylogenetic placement of Ac. patilii unresolved. In our study, the phylogenetic position of the new taxon is not stable, as well as the taxa Acrocordiopsis, Astrosphaeriella, Astrosphaeriellopsis, and Caryospora. Currently, Ac. patilii and Ac. sphaerica are referred to the Salsugineaceae, although morphologically they have little in common with Salsuginea ramicola (website: marinefungi.org). Similarly, Acrocordiopsis is distinct from Astrosphaeriellopsis bakeriana both morphologically and phylogenetically. Further collections of Ac. patilii, Ac. sphaerica, and Salsuginea ramicola and new sequence data are required for all these species before their taxonomic positions are resolved.

Caryospora

The genus Caryospora and Acuminatispora formed an unsupported sister clade (ML/31, MP/17, and BYPP/0.55). Caryospora is an old genus, which currently includes approximately 12 species (Caryospora aquatic Huang Zhang, K.D. Hyde & Ariyaw., C. australiensis Abdel-Wahab & E.B.G. Jones, C. callicarpa (Curr.) Nitschke ex Fuckel, C. coffeae Pat. & Gaillard, C. daweiensis G.C. Zhao & R.L. Zhao, C. langloisii Ellis & Everh., C. masonii D. Hawksw., C. minima Jeffers, C. obclavata Raja & Shearer, C. olearum (Castagne) Sacc., C. phyllostachydis (Hara) I. Hino & Katum., C. putaminum (Schwein.) De Not.), and all have superficial or erumpent to nearly superficial ascomata, a carbonaceous peridium, and trabeculate pseudoparaphyses (Jeffers 1940; Barr 1979; Hawksworth 1982; Abdel-Wahab and Jones 2000; Raja and Shearer 2008; Hawksworth et al. 2010; Hu 2010; Ariyawansa et al. 2015). Only two species have been sequenced and further studies are required.