Research Article |
Corresponding author: Hong-Yan Su ( suhongyan16@163.com ) Academic editor: Rungtiwa Phookamsak
© 2020 Li Xu, Dan-Feng Bao, Zong-Long Luo, Xi-Jun Su, Hong-Wei Shen, Hong-Yan Su.
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Citation:
Xu L, Bao D-F, Luo Z-L, Su X-J, Shen H-W, Su H-Y (2020) Lignicolous freshwater ascomycota from Thailand: Phylogenetic and morphological characterisation of two new freshwater fungi: Tingoldiago hydei sp. nov. and T. clavata sp. nov. from Eastern Thailand. MycoKeys 65: 119-138. https://doi.org/10.3897/mycokeys.65.49769
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Lignicolous freshwater fungi represent one of the largest groups of Ascomycota. This taxonomically highly diverse group plays an important role in nutrient and carbon cycling, biological diversity and ecosystem functioning. The diversity of lignicolous freshwater fungi along a north-south latitudinal gradient is currently being studied in Asia. In this paper, we introduce two novel freshwater taxa viz. Tingoldiago hydei sp. nov. and T. clavata sp. nov. which were collected from freshwater substrates in Eastern Thailand. Morphological comparison based on the size of ascomata, asci and ascospores, as well as multi-gene phylogenetic analyses based on LSU, SSU, ITS and TEF1-α DNA sequences, supports their placement in Tingoldiago (Lentitheciaceae). Descriptions and illustrations of these two new species are provided.
2 new species, Lentitheciaceae, Freshwater fungi, phylogeny, taxonomy
Freshwater fungi are those which the whole or part of their life cycle is found in a freshwater habitat (
We are studying the diversity of lignicolous freshwater fungi in Thailand, in order to establish the phylogenetic relationships of lignicolous freshwater fungi, understanding the natural classification of this group and contributing to the biogeographical diversity of fungi (
Lentitheciaceae was introduced by
In this paper, we introduce two new freshwater species of Tingoldiago (Lentitheciaceae), based on morpho-molecular studies. Detailed descriptions and illustrations of these two new species are provided.
Submerged decaying wood samples were collected from That Phanom, Nakhon Phanom, Thailand and brought to the laboratory in plastic bags. The samples were incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Specimen observations and morphological studies were conducted, following the protocols provided by
Pure cultures were obtained by single spore isolation followed by Chomnunti et al. (2014). Germinating ascospores were transferred aseptically to potato dextrose agar (PDA) plates and grown at 16–25 °C in daylight. Colony colour and other characters were observed and measured after three weeks. The specimens were deposited in the herbarium of Mae Fah Luang University (
Fungal mycelium was scraped from the surface of colonies grown on a PDA plate or MEA plate at 25 °C for 4 weeks, transferred into a 1.5 ml centrifuge tube and ground using liquid nitrogen. The EZ geneTM fungal gDNA kit (GD2416) was used to extract DNA from the ground mycelium according to the manufacturer’s instructions. The gene regions of the large subunit of the nuclear ribosomal DNA (LSU), the internal transcribed spacers (ITS), the small subunit of the nuclear ribosomal DNA (SSU) and the translation elongation factor (TEF1-α) RNA were amplified using the primer pairs LR0R/LR7 (
The sequence was assembled by using BioEdit and sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Lentitheciaceae and from recently published data (
Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (
Phylogenetic tree based on RAxML analyses of combined LSU, SSU, ITS and TEF1-α sequence data. Bootstrap support values for maximum likelihood (ML, black) and maximum parsimony (MP, red) higher than 75% and Bayesian posterior probabilities (PP, black) greater than 0.95 are indicated above the nodes as MP / ML /PP. The ex-type strains are in bold and the newly obtained isolates are in red. The tree is rooted at Corynespora smithii (CABI5649b) and Corynespora cassiicola (CBS100822).
MP analyses were performed using the heuristic search option with 1000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap (BS) analysis with 1000 replicates, each with ten replicates of random stepwise addition of taxa (
The Bayesian analysis was performed with MrBayes v3.2 (
The phylograms were visualised in FigTree 1.4.2 (
Taxa used in this study and their GenBank accession numbers, the newly generated sequences are indicated wirh * and the type strains are indicated in bold.
Taxa | strain | GenBank accession number | |||
---|---|---|---|---|---|
LSU | SSU | ITS | TEF1 | ||
Bambusicola bambusae | MFLUCC 11–0614 | JX442035 | JX442039 | NR121546 | KP761722 |
B. irregulispora | MFLUCC 11–0437 | JX442036 | JX442040 | NR121547 | KP761723 |
B. massarinia | MFLUCC 11–0389 | JX442037 | JX442041 | NR121548 | – |
Bimuria novaezelandiae | AFTOL ID931 | – | – | – | DQ471087 |
Byssothecium circinans | CBS67592 | GU205217 | GU205235 | – | GU349061 |
Corynespora cassiicola | CBS100822 | GU301808 | GU296144 | – | GU349052 |
C. smithii | CABI5649b | GU323201 | – | – | GU349018 |
Dacampia engeliana | 72868 | KT383791 | – | – | – |
D. hookeri | 74269 | KT383793 | – | – | – |
D. hookeri | 81840 | KT383795 | – | – | – |
Darksidea alpha | CBS 135650 | KP184019 | KP184049 | NR137619 | KP184166 |
D. beta | CBS 135637 | KP184023 | KP184049 | NR137957 | KP184189 |
D. delta | CBS 135638 | – | – | NR137075 | – |
D. epsilon | CBS 135658 | KP184029 | KP184070 | NR137959 | KP184186 |
D. gamma | CBS 135634 | KP184031 | KP184073 | NR137587 | KP184188 |
D. zeta | CBS 135640 | KP184013 | KP184071 | NR137958 | KP184191 |
Falciformispora lignatilis | BCC 21117 | GU371826 | GU371834 | KF432942 | GU371819 |
F. lignatilis | BCC 21118 | GU371827 | GU371835 | KF432943 | GU371820 |
Halobyssothecium obiones | 27AV2385 | – | – | KX263864 | – |
H. obiones | MFLUCC 15–0381 | MH376744 | MH376745 | MH377060 | MH376746 |
Helicascus nypae | BCC36752 | GU479789 | GU479755 | – | GU479855 |
Kalmusia scabrispora | KT2202 | AB524594 | AB524453 | – | AB539107 |
Karstenula rhodostoma | CBS69094 | GU301821 | GU296154 | – | GU349067 |
Katumotoa bambusicola | KT1517a | AB524595 | AB524454 | LC014560 | AB539108 |
Keissleriella breviasca | KT649 | AB807588 | AB797298 | – | AB808567 |
K. culmifida | KT2642 | AB807592 | AB797302 | LC014562 | – |
K. gloeospora | KT829 | AB807589 | AB797299 | LC014563 | – |
K. poagena | CBS136767 | KJ869170 | – | KJ869112 | – |
K. quadriseptata | KT2292 | AB807593 | AB797303 | AB811456 | AB808572 |
K. taminensis | KT571 | AB807595 | AB797305 | LC014564 | AB808574 |
K. trichophoricola | CBS 136770 | KJ869171 | – | KJ869113 | – |
Lentithecium clionina | KT1149A | AB807540 | AB797250 | LC014566 | AB808515 |
L. fluviatile | CBS 123090 | FJ795450 | FJ795492 | – | – |
L. pseudoclioninum | KT1111 | AB807544 | AB797254 | AB809632 | AB808520 |
Massarina cisti | CBS 266 62 | FJ795447 | FJ795490 | LC014568 | AB808514 |
M. eburnea | CBS 473 64 | GU301840 | GU296170 | – | GU349040 |
Montagnula opulenta | AFTOLID1734 | DQ678086 | AF164370 | – | – |
Morosphaeria ramunculicola | JK5304B | GU479794 | GU479760 | – | – |
Murilentithecium clematidis | IT1078 | KM408758 | KM408760 | KM408756 | – |
M. clematidis | MFLUCC 14–0562 | KM408759 | KM408761 | KM408757 | KM454445 |
Neoophiosphaerella sasicola | KT1706 | AB524599 | AB524458 | LC014577 | AB539111 |
Palmiascoma gregariascomum | MFLUCC 11–0175 | KP744495 | KP753958 | KP744452 | – |
Paraconiothyrium brasiliense | CBS100299 | JX496124 | AY642523 | JX496011 | – |
Paraphaeosphaeria michotii | MFLUCC 13–0349 | KJ939282 | KJ939285 | KJ939279 | – |
P. minitans | CBS122788 | EU754173 | EU754074 | – | GU349083 |
Phaeodothis winteri | CBS18258 | – | GU296183 | – | – |
Phragmocamarosporium platani | MFLUCC 14–1191 | KP842915 | KP842918 | – | – |
Pleurophoma ossicola | CBS139905 | KR476769 | – | KR476736 | – |
P. ossicola | CPC24985 | KR476770 | – | NR137992 | – |
Pleurophoma pleurospora | CBS130329 | JF740327 | – | – | – |
Poaceascoma aquaticum | MFLUCC 14–0048 | KT324690 | KT324691 | – | – |
P. halophila | MFLUCC 15–0949 | MF615399 | MF615400 | – | – |
P. helicoides | MFLUCC 11–0136 | KP998462 | KP998463 | KP998459 | KP998461 |
Pseudomurilentithecium camporesii | MDLUCC 14-1118 | MN638846 | MN638850 | MN638861 | – |
Setoseptoria arundinacea | KT600 | AB807575 | AB797285 | LC014595 | AB808551 |
S. magniarundinacea | KT1174 | AB807576 | AB797286 | LC014596 | AB808552 |
S. phragmitis | CBS 114802 | KF251752 | – | KF251249 | – |
S. scirpi | MFUCC 14–0811 | KY770982 | KY770980 | MF939637 | KY770981 |
Stagonospora macropycnidia | CBS 114202 | GU301873 | GU296198 | – | GU349026 |
Tingoldiago graminicola | KH155 | AB521745 | AB521728 | LC014599 | AB808562 |
T. graminicola | KH68 | AB521743 | AB521726 | LC014598 | AB808561 |
T. graminicola | KT891 | AB521744 | AB521727 | – | AB808563 |
*T. hydei | MFLUCC 19-0499 | MN857177 | – | MN857181 | – |
*T. clavata | MFLUCC 19-0496 | MN857178 | MN857186 | MN857182 | – |
*T. clavata | MFLUCC 19-0498 | MN857179 | MN857187 | MN857183 | – |
*T. clavata | MFLUCC 19-0495 | MN857180 | MN857188 | MN857184 | – |
Towyspora aestuari | MFLUCC 15–1274 | KU248852 | KU248853 | NR148095 | – |
Trematosphaeria pertusa | CBS 122368 | FJ201990 | FJ201991 | NR132040 | KF015701 |
Trematosphaeria pertusa | CBS 122371 | GU301876 | GU348999 | KF015669 | KF015702 |
The aligned sequence matrix comprises LSU, SSU, ITS and TEF1-α sequence data for 69 taxa, with Corynespora smithii and Corynespora cassiicola as out-group taxa. The dataset comprises 3334 characters after alignment including gaps (LSU: 1–897; SSU: 898–1920; ITS: 1921–2522; TEF1-α: 2523–3479). The topologies of RAxML, MP and Bayesian are similar and the bootstrap support values for Maximum Likelihood (ML), Maximum Parsimony (MP) higher than 75% and Bayesian posterior probabilities (PP) greater than 0.95 are given above the nodes. Maximum parsimony analyses indicated that 2,442 characters were constant, 232 variable characters parsimony uninformative and 805 characters are parsimony-informative. The RAxML analysis of the combined dataset yielded the best scoring tree (Figure
The novel species Tingoldiago hydei and T. clavata, introduced in this paper, are supported by multi-phylogenetic analyses. Four newly generated strains clustered together within Tingoldiago with strong statistical support (100 ML/95 MP/1.00 PP, Figure. 1). Three strains of T. clavata clustered together and sister to T. hydei with strong bootstrap support (99 ML/97 MP/1 PP, Figure
Referring to Kevin D. Hyde for his contributions in fungal taxonomy.
Thailand, That Phanom, Nakhon Phanom, on submerged decaying wood, 13 November 2018, D.F. Bao, B-126 (
Saprobic on submerged decaying wood. Sexual morph: Ascomata 180–280 × 330–470 μm (x̄ = 400 × 420 μm, n = 10), immersed to semi-immersed, erumpentia, gregarious, scattered, depressed globose to conical with a flattened base, dark brown to black, as dark spots on host surface. Ostioles central, papillate, short, crest-like, dark brown. Peridium 33.5–50 μm wide, comprising 4–6 layers, brown to dark brown cells of textura anngularis. Hamathecium comprising 2–2.5 μm (n = 30) wide, numerous, branched, septate, hyaline, cellular pseudoparaphyses. Asci 95–164 × 18–22 μm (x̄ = 129 × 20 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, rounded at apex, with a short pedicellate. Ascospores 37.5–42 × 7.5–9 μm (x̄ = 40 × 8 μm, n = 30), overlapping, 2–3-seriate, clavate with round ends, straight, uniseptate, deeply constricted at septum, with broad and short upper cells 17.5–20 × 7–8.7 μm (x̄ = 18.7 × 7.9 μm, n = 30), narrow and long lower cells 20.6–23.3 × 5.9–7.4 μm (x̄ = 21.9 × 6.7 μm, n = 30), tapering towards the end, with short appendages at the septum, hyaline, guttulate, smooth, surrounded by a fusiform gelatinous sheath. Asexual morph: Undetermined.
Tingoldiago hydei (
Ascospores germinating on PDA within 24 hours. Colonies on MEA effuse, greyish-white to dark brown from above and below, reaching 3–4 cm diameter within 30 days at room temperature under natural light, composed of subhyaline to pale brown, septate, smooth hyphae.
Phylogenetic analysis showed that Tingoldiago hydei is related to T. clavata; however, they are in different lineages with significant support (99 ML/97 MP/1.00 PP, Figure
Tingoldiago clavata is similar to the type species, T. raminicola in having immersed to semi-immersed, depressed globose to conical ascomata with flattened base, bitunicate, fissitunicate, cylindrical-clavate asci and clavate, straight, uniseptate ascospores. However, T. clavata differs from T. raminicola in having longer asci (95–164 × 18–22 vs. 87.5–122 × 18.25–25 μm) and smaller ascospores (37.5–42 × 7.5–9 vs. 43.5–53 × 7.5–11 μm). Moreover, ascopores of T. clavata have short appendages at the septum while ascospores of T. raminicola lack appendages. In addition, we compared the base pairs of ITS regions between these two species and there were 25 base pairs without gaps (5.1%) differences. Therefore, we introduce our isolate as a new species based on both phylogeny and morphological characters.
Referring to the clavate ascospores of this fungus.
Thailand, That Phanom, Nakhon Phanom, on submerged decaying wood, 13 November 2018, D.F. Bao, B-161 (
Saprobic on submerged decaying wood. Sexual morph: Ascomata 145–210 × 145–195 μm (x̄ = 175 × 169 μm, n = 10), immersed to semi-immersed, gregarious, scattered, erumpentia, depressed globose to conical with a flattened base, dark brown to black, as dark spots on host surface. Ostiole central, round to papillate, short, crest-like, dark brown. Peridium 28–47 μm wide, comprising several layers, pale brown to brown cells of textura anngularis. Hamathecium comprising 1.5–2.0 μm (n = 30) wide, numerous, branched, septate, hyaline, cellular pseudoparaphyses. Asci 110–148 × 20–27 μm (x̄ = 129 × 23 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, rounded at apex, with a short pedicellate. Ascospores 48–51 × 7.5–9 μm (x̄ = 50.5 × 8.5 μm, n = 30), overlapping, 2–3-seriate, clavate, with round ends, straight, uniseptate, deeply constricted at septum, hyaline, with broad and short upper cells 16.6–18.9 × 7.8–9.0 μm (x̄ = 17.7 × 8.4 μm, n = 30), narrow and long lower cells 30–32.9 × 6.5–8.0 μm (x̄ = 31.5 × 7.3 μm, n = 30), tapering towards the end, guttulate, smooth, 2–4 equatorial appendages at the septum and surrounded by a fusiform gelatinous, sheath. Asexual morph:Undetermined.
Ascospores germinating on PDA within 24 hours. Colonies on MEA effuse, velvety, greyish-white to dark brown from above and below, reaching 2.5–3 cm diameter within 30 days at room temperature under natural light, composed of subhyaline to brown, septate, smooth hyphae.
Thailand, That Phanom, Nakhon Phanom, on submerged decaying wood, 13 November 2018, D.F. Bao, B160 (paratype:
Tingoldiago clavata resembles the type species, T. graminicola in having bitunicate, cylindrical-clavate asci with a short pedicellate and clavate, hyaline, 1-septate, ascospores with broad upper cells, narrow lower cells. However, we can distinguish them by the size of ascomata and asci and the colour, septate and appendages of ascospores. Tingoldiago clavata has smaller ascomata (110–148 ×145–195 vs. 150–250 × 250–450 μm) and larger asci (110–148 × 20–27 vs. 87.5–122 × 18.25–25 μm). Moreover, ascopsores of T. clavata are hyaline, uniseptate, with 2–4 equatorial appendages at the septum, while ascopspores of T. graminicola are brown and 3-septate at maturity and lacking appendages at the septum. In addition, a comparison of the 491 nucleotides across the ITS gene region of T. clavata and T. graminicola reveals 25 base-pair differences and therefore provides further evidence to introduce T. clavata as a new species as recommended by
During the last decade, freshwater fungi in Thailand have been mainly reported from north, south and northeast of Thailand (
The morphological comparisons of Tingoldiago species discussed in this study.
Taxa | Distribution | Ascomata (μm) | Pseudoparaphyses (μm) | Asci (μm) | Ascospores (μm) | References |
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Tingoldiago graminicola | Japan, UK | 150–250 × 250–450 | 1.5–4 | 87.5–122 × 18.25–25 | 43.5–53 × 7.5–11 |
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T. hydei | Thailand | 180–280 × 330–470 | 1.8–2.5 | 95–164 × 18–22 | 37.5–42 × 7.5–9 | This study |
T. clavata | Thailand | 145–210 × 145–195 | 1.4–2.0 | 110–148 × 20–27 | 48–51 × 7.5–8.5 | This study |
Tingoldiago is a well-resolved genus in this family with a stable clade within Lentitheciaceae. The genus can be distinguished from other genera in this family by having hyaline, uniseptate, upper cells are broad and basal cells are narrow ascospores with a large fusiform gelatinous sheath. The sheath is considered to be an adaptation by the genus that enables ascospores to attach to the substrates in moving water (Shearer 1993,
We would like to thank the National Natural Science Foundation of China (NSFC 31860006, 31660008) and the Fungal Diversity Conservation and Utilization Innovation team of Dali University (ZKLX2019213) for financial and laboratory support. Dan-Feng Bao thanks Shaun Pennycook from Landcare Research, Auckland, New Zealand, for advising on the taxon names. Wen-Li Li and Yan-Mei Zhang are acknowledged for their help on DNA extraction and PCR amplification.