Introduction

Jahnulales (Dothideomycetes), an order of freshwater lignicolous ascomycetes, was established by Pang et al. (2002). This order is phylogenetically related to the order Dothideales, Patellariales, and Pleosporales (Campbell et al. 2007). Most species of the Jahnulales occur on rotting or soft submerged corticated or decorticated wood (Inderbitzin et al. 2001; Pang et al. 2002; Shenoy et al. 2006, 2010; Campbell et al. 2007; Suetrong et al. 2011; Tanaka et al. 2015). The order Jahnulales is characterized by ascomata with peridial walls multilayered, composed of large cells, stalked and (or) sessile bitunicate asci, and one-septate ascospores with gelatinous sheaths or appendages (Pang et al. 2002; Campbell et al. 2007; Suetrong et al. 2011; Jones et al. 2015). Most authors accept two families in this order, Aliquandostipitaceae and Manglicolaceae (Suetrong et al. 2011; Hyde et al. 2013; Jones et al. 2015). The family, Aliquandostipitaceae within this order was proposed based on the genus Aliquandostipite, which contains only tropical species and is characterized by broad septate hyphae, globose to subglobose ascomata, and one-septate ascospores (Inderbitzin et al. 2001). Six genera are accommodated in this family including Aliquandostipite, Jahnula and Megalohypha (Kirschstein 1936; Inderbitzin et al. 2001; Ferrer et al. 2007), known by its sexual morph, whereas Brachiosphaera, Speiropsis and Xylomyces are exclusively asexual morph (Tubaki 1958; Descals et al. 1976; Goos et al. 1977; Campbell et al. 2007; Suetrong et al. 2011). The other family of the order, Manglicolaceae, also present cylindrical asci and appendaged ascospores (Suetrong et al. 2011). The Manglicolaceae, represented by the marine fungus Manglicola guatemalensis (type) and the terrestrial fungus M. samuelsii, has been classified as an independent family based on LSU and SSU rRNA sequence analyses (Kohlmeyer and Kohlmeyer 1971; Huhndorf 1994; Jones et al. 2015). In addition, it differs from the members of the Aliquandostipitaceae by the production of obtusely clavate to fusiform, stipitate ascomata bearing a broad ostiole and unequally one-septate ascospores (Suetrong et al. 2011).

The largest genus within Jahnulales, Jahnula, was introduced by Kirschstein (1936) and is typified by Jahnula aquatica. All 16 species of Jahnula have been reported from wood or decorticated wood in fresh-water habitats (Hawksworth 1984; Hyde 1993; Hyde and Goh 1998; Hyde and Wong 1999; Ho et al. 2002; Pang et al. 2002; Pinruan et al. 2002; Raja and Shearer 2006; Campbell et al. 2007; Raja et al. 2008; Sivichai and Boonyuen 2010; Suetrong et al. 2011; Fournier et al. 2015). The genus is currently polyphyletic, and Jahnula sensu stricto accommodates Jahnula aquatica, J. granulosa, J. potamophila, and J. rostrata (Suetrong et al. 2011).

We are carrying out a survey of freshwater fungi along a north south latitudinal gradient in China and Thailand (Luo et al. 2017; Hyde et al. 2016a). Members of Jahnulales are major fungal species colonizing substrates in freshwater habitats, and recent taxonomic studies in freshwater habitats have revealed new asexual species (Luo et al. 2017). In this study, we report on two collections of Jahnula in China. The specimens were studied morphologically to enable identification, and regions of complete ITS and partial LSU and SSU rRNA gene were analyzed to determine their phylogenetic affinity to previously known Jahnula species.

Materials and methods

Sample collection, morphological studies, and isolation

Submerged dead wood pieces were collected from the Dianchi Lake, Yunnan Province in China in October 2016 and brought to the laboratory in zip lock plastic bags. Incubation of specimens was performed as outlined by Vrijmoed (2000). Fruiting bodies were found growing on decayed wood in a sterile plastic box after 2 weeks and were subsequently isolated based on the method of Chomnunti et al. (2014). Morphological characters were examined using an Olympus SZ61 stereoscope, and ascomata were sectioned by free-hand with a razor-blade. These sections were examined by a Nikon ECLIPSE Ni compound microscope, and images were taken with a Canon EOS 600D digital camera. Measurements were made with Tarosoft® Image Frame Work program v. 0.9.7. The specimens are deposited in the Kunming Institute of Botany, Academia Sinica and duplicated in Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Facesoffungi (FoF) and Index Fungorum (IF) numbers were registered as explained in Jayasiri et al. (2015) and Index Fungorum (2017) (http://www.indexfungorum.org/names/names.asp). New species are established based on the recommendations outlined by Jeewon and Hyde (2016).

DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted directly from mycelium using a Trelief™ Plant Genomic DNA Kit following the instructions of the manufacturer. The genomic DNA was amplified by using polymerase chain reaction (PCR) in a 25 μL reaction mixture. Regions of the internal transcribed spacers (ITS1–5.8S-ITS2), partial large subunit rRNA (LSU), and partial small subunit rRNA (SSU) were amplified using primer pair ITS5 and ITS4 (White et al. 1990), LROR and LR5 (Vilgalys and Hester 1990) and NS1 and NS4 (White et al. 1990) respectively. Each PCR reactions contained 12.5 μL of 2 × Power Taq PCR MasterMix, 10.5 μL ddH2O, and 0.5 μL of each primer (10 μM); and 1 μL genomic DNA extract and amplifications were carried out in an Applied Biosystems 2720 thermocycler (Foster City, CA, USA) with the following profile: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 50 s, annealing at 50 °C (SSU) or 53 °C (ITS and LSU) for 50 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 8 min. The PCR products were visualized by loading 5 μL on 1% agarose electrophoresis gels and bands viewed in Gel documentation system. The PCR products were sent to a commercial sequencing provider Tsingke Company, Beijing, P.R. China.

Phylogenetic analysis

The quality of our amplified nucleotide sequences were checked by Finch TV version 1.4.0 (www.geospiza.com/finchtv), and subjected to the BLAST search under the National Center for Biotechnology Information (NCBI). Then the closest matches for our strains were retrieved from the NCBI database (Pang et al. 2002; Campbell et al. 2007; Prihatini et al. 2008; Suetrong et al. 2011; Phookamsak et al. 2014; Wanasinghe et al. 2014; Ariyawansa et al. 2015; Hyde et al. 2016b). Sequences were aligned using the default setting of MAFFT v. 7.310 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley 2016), and manually corrected using Bioedit 7.0.9.0 (Hall 1999).

The phylogenetic analyses of combined gene regions were performed using maximum-likelihood (ML), Bayesian inference (BI), and maximum parsimony (MP) methods. The evolutionary model was obtained using MrModeltest v. 2.3 (Nylander et al. 2008) under the Akaike Information Criterion (AIC) performed in PAUP v. 4.0b10.

The maximum-likelihood analysis was run or performed using RAxML-HPC v.8 on XSEDE in CIPRES Science Gateway (Stamatakis 2014; Miller et al. 2010, 2015) with 1000 rapid bootstrap replicates using the General Time Reversible model incorporating invariant sites and a gamma distribution (GTR + I + G).

Bayesian inference was implemented by MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003) with the best-fit model (GTR + I + G for ITS and LSU; SYM + I + G for SSU) of sequence evolution estimated with MrModeltest 2.3 (Nylander et al. 2008). Posterior probabilities (PP) were estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck and Ronquist 2001; Zhaxybayeva and Gogarten 2002; Rannala and Yang 2008). Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run from random trees for 100,000,000 generations and sampled every 1000 generations. The temperature value was lowered to 0.15, burn-in was set to 0.25, and the run was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01.

Phylogenetic analyses were also performed with maximum parsimony in PAUP v. 4.0b10 (Swofford 2002). Details are outlined as in Jeewon et al. (2002, 2003) and Cai et al. (2006). In brief, ambiguously aligned regions were excluded by hand and gaps were treated as missing data. Trees were inferred with the heuristic search option with TBR branch swapping and 1000 random sequence additions. Maxtrees were unlimited; branches of zero length were collapsed, and all parsimonious trees were saved. Tree length [TL], consistency index [CI], homoplasy index [HI], retention index [RI], and rescaled consistency index [RC] were calculated for the MP tree. Clade stability was assessed using a bootstrap (BT) analyses with 1000 replicates, each with ten replicates of random stepwise addition of taxa.

Phylogenetic trees were viewed with FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: 20896). The nucleotide sequence data of new taxa have been deposited in GenBank (Table 1).

Table 1 Strains and GenBank accession numbers of the isolates used in this study. Isolates from this study are in bold underline and the ex-type strains are in bold
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Results

Phylogenetic analyses

The alignment comprised 2347 total characters including gaps. The tree was rooted with Farlowiella carmichaeliana. The Bayesian analysis resulted in a tree with the same topology and clades as the ML and MP trees. RAxML analysis yielded a best scoring tree with a final optimization likelihood value of − 16,010.965307 (Fig. 1). Parsimony analyses indicated that 1411 characters were constant; 264 variable characters were parsimony-uninformative, and 672 characters were parsimony-informative. The parsimony analysis of the data matrix resulted in a single most parsimonious tree (TL = 2691, CI = 0.544, RI = 0.751, RC = 0.409, HI = 0.456) is shown here. In the phylogenetic tree, the two isolates of Jahnula dianchia constitute a well-supported clade (100%, ML/1.0, PP/100%, MP) that is sister to J. sangamonensis (100%, ML/1.0, PP/100%, MP).

Fig. 1
figure 1

Maximum likelihood phylogenetic tree generated from analysis of a combined ITS, LSU, and SSU sequences dataset for 51 taxa of Dothideomycetes and Farlowiella carmichaeliana as the outgroup taxon. ML support values greater than 70% (BSML, left), Bayesian posterior probabilities greater than 0.95 (BYPP, middle) and MP bootstrap value higher than 70% (BSMP, right) are indicated above the nodes. The strain numbers are noted after the species names. Ex-type strains are indicated in bold. Isolates from this study are indicated in red bold

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Taxonomy

Jahnula dianchia S.K. Huang & K.D. Hyde, sp. nov .

Index Fungorum number: IF553200; Facesoffungi number: FoF 03149. Fig. 2

Fig. 2
figure 2

Jahnula dianchia (HKAS96327, holotype). a Material. b Ascoma on host. c Ascoma in vertical section. d Peridium. e Ostiole in vertical section with periphyses. f Asci with pseudoparaphyses. g–i Asci. j–l Ascospores. m Germinating ascospores. Note: g–h stained in Melzer’s reagent, arrow showing mammiform ascospores apex. Scale bars c, d = 100 μm, e–i = 50 μm, j–m = 20 μm

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Etymology: The name dianchia refers to the geographic location were the specimen was collected.

Holotype: HKAS96327

Saprobic on dead wood. Sexual morph: Ascomata 307–418 × 248–374 μm (\( \overline{x}=380\times 325\ \upmu \mathrm{m} \), n = 5), perithecial, solitary, superficial to sub-immersed, unilocular, obpyriform to subglobose, dark brown to black, papillate, ostiolate. Ostiole central, short, lined with hyaline periphyses. Peridium 35–75 μm thick, membranous, composed of brown to hyaline cells of textura angularis (Fig. 2d). Hamathecium comprising 2–3 μm wide, septate, branched, filiform pseudoparaphyses, embedded in a gelatinous matrix. Asci 198–238 × 16–20 μm (\( \overline{x}=217\times 19\ \upmu \mathrm{m} \), n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, pedicellate, rounded at apex, with a distinct ocular chamber. Ascospores 24–32 × 10–21 μm (\( \overline{x}=27.5\times 13\ \upmu \mathrm{m} \), n = 50), uniseriate, initially hyaline, becoming dark brown at maturity, oval to broadly ellipsoid, 1-septate, with a mammiform apex, slightly curved, smooth to verruculose, multiguttulate, apiculate, rounded at lower end. Asexual morph: undetermined.

Culture characteristics: Ascospores germinating on potato dextrose agar (PDA) within 2 weeks at 23 °C, colony 1.5 cm diam., hyphae 3–10 μm thick, cream to faint yellow from above and reverse, with concentric zonation, with filamentous mycelium, filiform at margin, with rough surface and raised elevation.

Material examined: CHINA, Yunnan, Kunming, Dianchi Lake (24° 51′ 29.18″ N, 102° 39′ 58.19″ E); on dead wood, 1 October 2016; S.K. Huang (KUN HKAS 96327, holotype), ibid. (MFLU 17-0693, isotype); Ex-type KUMCC 17-0034, MFLUCC 17-0887. ibid. (KUN HKAS 97459, MFLU 17-0698; KUMCC 17-0039, MFLUCC 17-0891).

Key to species of Jahnula based on the sexual morph

  • 1. Ascospores with appendages ......................................2

  • 1. Ascospores without appendages ...............................10

  • 2. Ascospores with long appendages arising from both poles ................................................................................3

  • 2. Ascospores surrounded by mucilaginous sheath .........4

  • 2. Ascospores with mucilaginous pads at both poles ......7

  • 3. Ascospores 47.5–55 × 23.5–26.5 μm4 .....................................................Jahnula appendiculata

  • 3. Ascospores 17.5–20 × 5–6.5 μm7  .......................................................................J. morakotii

  • 4. Two types of ascospores morphologies2 .........J. systyla

  • 4. One type of ascospores morphology ...........................5

  • 5. Ascospores light brown2 ......................J. potamophila

  • 5. Ascospores dark brown ...............................................6

  • 6. Ascospores 26–37.5 × 15–18 μm2  .........J. granulosa

  • 6. Ascospores 32–45 × 12–15 μm5  ................J. rostrata

  • 7. There are two types of ascospores2 .....J. seychellensis

  • 7. There are only one type of ascospores ........................8

  • 8. Ascospores dark brown5 .............................J. bipileata

  • 8. Ascospores light brown ...............................................9

  • 9. Asci cylindrical, ascospores uniseriate2 ......J. bipolaris

  • 9. Asci obclavate, ascospores biseriate3, 8 ..J. sunyatsenii

  • 10. Ascospores ellipsoid to fusiform..............................11

  • 10. Ascospores oval to oblong ......................................14

  • 11. Ascospores basal cell shorter than apical cell6 .......................................................................J. apiospora

  • 11. Ascospores symmetrical .........................................12

  • 12. Ascospores longer than 30 μm5  .........................................................................J. aquatica

  • 12. Ascospores shorter than 30 μm ...............................13

  • 13. Ascospores with tapering apices2 ...........J. poonythii

  • 13. Ascospores with rounded apices5 ..............................................................J. sangamonensis

  • 14. Asci cylindrical .......................................................15

  • 14. Asci obclavate9 ........................................J. purpurea

  • 15. Ascospores without mammiform apex, 19–30 × 6–8 μm1 .......................................................J. australiensis

  • 15. Ascospores with mammiform apex, 24–32 × 10.5–21 μm .............................................................J. dianchia

1 Hyde (1993); 2 Hyde and Wong (1999); 3 Inderbitzin et al. (2001); 4 Pinruan et al. (2002); 5 Raja and Shearer (2006); 6 Raja et al. (2008); 7 Sivichai and Boonyuen (2010); 8 Suetrong et al. (2011); 9 Fournier et al. (2015)

Discussion

As part of our studies on freshwater fungi in the Yunnan Province of China, we collected a fungal species that is morphologically similar to the genus Jahnula or Jahnula spp. Species of Jahnula are predominantly collected or isolated from freshwater habitats (Hyde 1993; Hyde and Wong 1999; Tsui et al. 2000; Ho et al. 2001; Pang et al. 2002; Pinruan et al. 2002; Raja and Shearer 2006; Raja et al. 2008; Sivichai and Boonyuen 2010; Suetrong et al. 2011; Fournier et al. 2015). Further, the two specimens in this study fit well with the diagnosis of Jahnula as previously circumscribed, but are morphologically distinct from other Jahnula species such as to justify the description and naming of a new species. In addition, DNA sequence data support that the new taxon, J. dianchia, is phylogenetically distinct from other known species. Cai et al. (2002) reported Jahnula poonythii K.D. Hyde & S.W. Wong from Dianchi Lake, which may be an earlier misidentified collection of our new species. However, fusiform ascospores are seen in J. poonythii whereas they are broadly ellipsoidal to oval in J. dianchia.

The phylogeny (Fig. 1) reveals a close relationship of the newly collected fungus to J. sangamonensis which has been reported from decorticated submerged woody debris in the USA (Raja and Shearer 2006). Jahnula dianchia is similar to J. sangamonensis but differs in the following aspects: (i) most of the ascomata of J. dianchia are superficial and J. sangamonensis is sub-immersed in the wood, (ii) the peridium of J. dianchia is 35–75 μm wide with the inner wall layer of ostiole lined with hyaline periphyses while those of J. sangamonensis are 40–44 μm wide with the inner wall layer of ostiole lined with reddish brown periphyses, (iii) the asci of two taxa are different in that endoascus of J. sangamonensis extends up to 500 μm long in water, and (iv) the ascospores of J. dianchia are similar to those of J. sangamonensis except that these are with mammiform apex. Seventeen species are included in the genus Jahnula (Suetrong et al. 2010, 2011; Fournier et al. 2015). Ten of them are different from J. dianchia by having ascospores with a gelatinous sheath, pads, or appendages (viz. Jahnula appendiculata, J. bipileata, J. bipolaris, J. granulosa, J. morakotii, J. potamophila, J. rostrata, J. seychellensis, J. sunyatsenii and J. systyla) (Fournier et al. 2015; Hyde and Wong 1999; Pang et al. 2002; Pinruan et al. 2002; Sivichai and Boonyuen 2010). The shape of ascospores in Jahnula apiospora, J. australiensis, J. poonythii, and J. purpurea is fusiform or oblong while in our new taxon, it is oval to broadly ellipsoid (Fournier et al. 2015; Hyde and Wong 1999; Raja et al. 2008). Jahnula dianchia is also different from J. aquatica, the type species of Jahnula, collected from Germany and South Africa, in terms of ascospore length (> 30 μm) (Hyde and Wong 1999).

To further support our new taxon, we follow the recommendations by Jeewon and Hyde (2016). Comparison of the 463 base pairs (bp) across the nuclear ribosomal DNA ITS region, including the spacers ITS1 and ITS2 together with the 5.8S rRNA gene reveals that there are 20 bp (4.3%) differences when compared to J. sangamonensis. In the same way, comparison of the 862 bp of the 28S ribosomal RNA gene region reveals 11 bp (1.3%) difference compared to J. sangamonensis that justifies our new species.

Despite having only two strains herein, J. dianchia constitutes a strong monophyletic subclade with high support and therefore establishment of a new species is justified. The phylogeny generated herein (Fig. 1) agrees with other published studies in recovering Jahnula as a non-monophyletic group within the Jahnulales. Five clades are recognized: Clade I comprises species that belongs to Jahnula sensu lato with high statistical support (Campbell et al. 2007; Suetrong et al. 2011) together with those anamorphic genus Speiropsis (typified by S. pedatospora) and Brachiosphaera taxa (typified by B. tropicalis) (Suetrong et al. 2011). Clade II includes species of Aliquandostipite, while clade III comprises species of Jahnula sensu stricto such as J. aquatica, J. granulosa, J. potamophila, and J. rostrata (Campbell et al. 2007, Suetrong et al. 2011). Clade IV represents the genus Manglicola (Suetrong et al. 2010, 2011), whereas Clade V accommodates the genus Megalohypha (Raja et al. 2011).