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Research Article
The insights into the evolutionary history of Translucidithyrium: based on a newly-discovered species
expand article infoXinhao Li, Hai-Xia Wu, Jinchen Li, Hang Chen, Wei Wang
‡ The Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming, China
Open Access

Abstract

During the field studies, a Translucidithyrium-like taxon was collected in Xishuangbanna of Yunnan Province, during an investigation into the diversity of microfungi in the southwest of China. Morphological observations and phylogenetic analysis of combined LSU and ITS sequences revealed that the new taxon is a member of the genus Translucidithyrium and it is distinct from other species. Therefore, Translucidithyrium chinense sp. nov. is introduced here. The Maximum Clade Credibility (MCC) tree from LSU rDNA of Translucidithyrium and related species indicated the divergence time of existing and new species of Translucidithyrium was crown age at 16 (4–33) Mya. Combining the estimated divergence time, paleoecology and plate tectonic movements with the corresponding geological time scale, we proposed a hypothesis that the speciation (estimated divergence time) of T. chinense was earlier than T. thailandicum. Our findings provided new insights into the species of Translucidithyrium about ecological adaptation and speciation in two separate areas.

Keywords

Divergence time, morphological characteristics, new species, Phaeothecoidiellaceae, phylogeny, speciation, taxonomy

Introduction

The sooty blotch and flyspeck fungi are widespread species and commonly occur on the surface of leaves, stems and fruits in tropical and subtropical zones (Yang et al. 2010; Gleason et al. 2011; Hongsanan et al. 2017; Zeng et al. 2018). Although these fungi do not directly harm host plants, they may affect the economic value of fruit sales ability and reduce photosynthesis in plants (Gleason et al. 2011). Sooty blotch fungi can form dark mycelial mats, whereas flyspeck fungi lack mycelial mats, form shiny and small, black spots (Batzer et al. 2005; Yang et al. 2010; Gleason et al. 2011; Zhang et al. 2015; Singtripop et al. 2016; Hongsanan et al. 2017). However, these fungi are poorly known, because of the difficulty in obtaining the strain which grows slowly (Yang et al. 2010; Hongsanan et al. 2017; Zeng et al. 2018).

Phaeothecoidiellaceae K.D. Hyde & Hongsanan was introduced by Hongsanan et al. (2017) and accommodated three genera Chaetothyrina, Houjia and Phaeothecoidiella in the order Capnodiales. Currently, it includes eight genera: Chaetothyrina, Exopassalora, Houjia, Nowamyces, Phaeothecoidiella, Rivilata, Sporidesmajora and Translucidithyrium (Hongsanan et al. 2020). Members of Phaeothecoidiellaceae are related to sooty blotch and flyspeck fungi and characterised by thyriothecia with setae, bitunicate asci and 1-septate ascospores (Singtripop et al. 2016; Hongsanan et al. 2017; Zeng et al. 2019; Hongsanan et al. 2020). Chaetothyrina is morphologically similar to the family Micropeltidaceae (Reynolds and Gilbert 2005), but is distinguishable by its brown upper wall of ascomata (Wu et al. 2019; Zeng et al. 2019). The genus Rivilata is placed in this family on the basis of morphological characters by Doilom et al. (2018). The Nowamyces was introduced as a new genus in the new family Nowamycetaceae by Crous et al. (2019) and Hongsanan et al. (2020) placed this genus into Phaeothecoidiellaceae by phylogenetic analysis. Hongsanan et al. (2020) listed Houjia, Exopassalora, Sporidesmajora and Phaeothecoidiella as asexual genera in Phaeothecoidiellaceae.

Translucidithyrium X.Y. Zeng & K.D. Hyde (2018) was introduced as a monotypic genus in Phaeothecoidiellaceae, which is represented by T. thailandicum X.Y. Zeng & K.D. Hyde (2018). It was characterised by epiphytes on the reverse of living leaves, semi-transparent ascomata, globose to subglobose asci and fusiform ascospores with verrucose and appendages. Ascospores germinated on MEA (Malt Extract Agar Medium) within 24 h. The colonies slowly grow on media, white to grey, circular and villiform (Zeng et al. 2018).

Liu et al. (2017) used the molecular clock approach to estimate the divergence time of the order Capnodiales crown age at 151–283 Mya (million years ago). Zeng et al. (2019) estimated the divergence time of the family Phaeothecoidiellaceae crown age at 40–60 Mya. The molecular clock approach for estimating divergence time might be used to predict speciation, historical climate change or other environmental events (Hélène and Arne 2014; Louca and Pennell 2020).

In this study, we collected an extraordinary new species of Translucidithyrium in Xishuangbanna, Yunnan Province, China. We described the morphological characteristics and built a phylogenetic tree to determine the classification of the new taxon. We compared and analysed the estimated divergence time of Translucidithyrium with the environmental changes around the corresponding time range to propose the evolutional history hypothesis of Translucidithyrium distributed in two different regions (China and Thailand).

Methods

Morphological

Fresh living leaves with olivaceous dots were collected at Xishuangbanna, China 21°55'51"N, 101°15'08"E, 540 m alt.) and delivered to the laboratory for observation. According to Wu et al. (2014), the collected samples were processed and examined by microscopes: the photos of ascomata were taken by using a compound stereomicroscope (KEYENCE CORPORATION V.1.10 with camera VH-Z20R). Hand sections were made under a stereomicroscope (OLYMPUS SZ61) and mounted in water and blue cotton and photomicrographs of fungal structures were taken with a compound microscope (Nikon ECLIPSE 80i). The single spore isolation was implemented by the methods of Choi et al. (1999) and Chomnunti et al. (2014). Germinated spores were individually transferred to PDA (Potato Dextrose Agar Medium) and incubated at 26 °C for 48 h. Colony characteristics were observed and measured after 4 weeks at 26 °C. Images used for figures were processed with Adobe Photoshop CC v. 2015.5.0 software (Adobe Systems, USA). The holotype was deposited at the herbarium of IFRD (International Fungal Research & Development Centre; Research Institute of Resource Insects, Kunming), reference number IFRD 9208. The ex-type strain was deposited at IFRDCC, reference number IFRDCC 3000.

DNA isolation, amplification and sequencing

According to the manufacturer’s instructions, genomic DNA was extracted from mycelium growing on PDA at room temperature by using the Forensic DNA Kit (OMEGA, USA). The primer pair LR0R and LR5 was used to amplify the large subunit (LSU) rDNA (Vilgalys and Hester 1990). The primer pair ITS5 and ITS4 was used to amplify the internal transcribed spacer (ITS) rDNA (White et al. 1990). The primer pair NS1 and NS4 was used to amplify the partial small subunit (SSU) rDNA (White et al. 1990). The PCR reactions were in accordance with instructions from Golden Mix, Beijing TsingKe Biotech Co. Ltd, Beijing, China: initial denaturation at 98 °C for 2 min, then 30 cycles of 98 °C denaturation for 10 s, 56 °C annealing for 10 s and 72 °C extension for 10 s (ITS and SSU) or 20 s (LSU) and a final extension at 72 °C for 1 min. All PCR products were sequenced by Biomed (Beijing, China).

Sequences alignments and phylogenetic analysis

BioEdit version 7.0.5.3 (Hall 1999) was used to re-assemble sequences generated from forward and reverse primers for obtaining the integrated sequences. Sequences were downloaded from GenBank using data from the publications of Zeng et al. (2018), Crous et al. (2019), Hongsanan et al. (2020) and Renard et al. (2020) and aligned using BioEdit version 7.0.5.3 (Hall 1999): in addition, sequences were adjusted manually.

Maximum Likelihood (ML) analysis was conducted by using RAxMLGUI v.1.0 (Silvestro and Michalak 2012). Aligned sequences were input into the software and Dothidea sambuci was selected as the outgroup taxon. One thousand non-parametric bootstrap iterations were employed with the “ML + rapid bootstrap” tools and “GTRGAMMA” arithmetic.

For Bayesian analysis, MrModeltest 2.3 (Nylander 2004) was used to estimate the best-fitting model for the combined LSU and ITS genes. Posterior probabilities (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo (MCMC) sampling in MrBayes v.3.2 (Ronquist and Huelsenbeck 2003). Six simultaneous Markov chains were run for 2,000,000 generations; trees were printed every 1,000 generations; trees were sampled every 100 generations. The first 5,000 trees submitted to the burn-in phase and were discarded; the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (Cai et al. 2006, 2008; Liu et al. 2012).

Fossil calibrations and divergence time estimations

The fossil Protographum luttrellii (Renard et al. 2020) was used to calibrate the divergence time of Asterotexiales and Aulographaceae (normal distribution, mean = 119.0, SD = 3.7). The secondary calibration from the family Phaeothecoidiellaceae with a crown age of 58 Mya (normal distribution, mean = 50.0, SD = 6.1) was used (Zeng et al. 2019). The additional secondary calibration of Capnodiales was used, based on the result from Liu et al. (2017) (normal distribution, mean = 217.0, SD = 40.0).

Divergence time analysis was carried out using BEAST v1.8.4 (Drummond et al. 2012). Aligned LSU sequence data were loaded into the BEAUti v1.10.4 for generating an XML file. An uncorrelated relaxed clock model (Drummond et al. 2006) with a lognormal distribution of rates was used for the analysis. We used a Yule Process tree prior (Yule 1925; Gernhard 2008), which assumes a constant speciation rate per lineage and a randomly-generated starting tree. The length of chain was set as 50 million generations and sampling parameters were set at every 5,000 generations in MCMC. Subsequent divergence time analysis was carried out using BEAST v.1.10.4 (Drummond et al. 2012). Tracer v.1.7.1 was used to check the effective sample sizes (ESS) and acceptable values were higher than 200. The .log files and .tree files generated by BEAST were combined in LogCombiner v1.10.4 after removing a proportion of states as burn-in. The Maximum Clade Credibility (MCC) tree was given by obtained data and was estimated in TreeAnnotator v.1.10.4 (Liu et al. 2017; Zeng et al. 2019, 2020; Renard et al. 2020).

The phylogenetic tree and MCC tree were visualized in FigTree v.1.4.3 (Rambaut 2012) and Adobe Illustrator CS6 v. 16.0.0 (Adobe Systems, USA).

Results

Phylogenetic study

The dataset of combined LSU and ITS sequences comprised 1350 characters after alignment. Bayesian Inference, in total, generated 20,001 trees and the average standard deviation of split frequencies reached 0.0096. A total of 15,001 trees were finally used to calculate posterior probabilities. Phylogenetic analysis showed that the new collection clusters with T. thailandicum with 100% Maximum Likelihood bootstrap support and 1.00 posterior probabilities (Fig. 1).

Figure 1. 

The topology shows family relationships of Capnodiales, based on combined LSU and ITS dataset analysis. Bootstrap values of Maximum Likelihood higher than 60% are shown on the left, while values of Bayesian posterior probabilities above 80% are shown on the right. New species is given in bold. Clades of the key species or family are given in bold. The tree is rooted with Dothidea sambuci (Dothideaceae, Dothideales).

Table 1.

Selected taxa in this study with their corresponding GenBank accession numbers. The newly-generated sequences are shown in bold.

No. Species Vouncher /strain no. LSU ITS
1 Acidomyces acidophilus MH1085 JQ172741 JQ172741
2 Asterina phenacis TH 589 GU586217
3 Asterotexiaceae sp. VUL.535 MG844162
4 Aulographum sp. VUL.457 MG844158
5 Batcheloromyces proteae CBS 110696 JF746163 JF746163
6 Baudoinia compniacensis CBS 123031 GQ852580
7 Brunneosphaerella protearum CPC 16338 GU214397 GU214626
8 Buelliella minimula Lendemer 42237(NY) KX244961
9 Camarosporula persooniae CBS 116258 JF770461 JF770449
10 Capnobotryella renispora CBS 214.90 GU214398 AY220612
11 Capnodium coffeae CBS 147.52 GU214400 DQ491515
12 Catenulostroma protearum CPC 15368 GU214402 GU214628
13 Chaetothyrina guttulata MFLUCC15–1080 KU358917 KX372277
14 Chaetothyrina guttulata MFLUCC15–1081 KU358914 KX372276
15 Chaetothyrina musarum MFLUCC 15–0383 KU710171
16 Cladosporium herbarum CBS 121621 KJ564331 EF679363
17 Cladosporium hillianum CBS 125988 KJ564334 HM148097
18 Cladosporium ramotenellum CBS 170.54 DQ678057 AY213640
19 Colletogloeum sp. NY1_3.2F1c FJ031986 FJ425193
20 Conidiocarpus (Phragmocapnias) betle MFLUCC 10–0050 JN832605
21 Devriesia staurophora ATCC 200934 KF901963 AF393723
22 Dissoconium aciculare CBS 204.89 GU214419 AY725520
23 Dothidea sambuci AFTOL-ID 274 AY544681 DQ491505
24 Dothistroma pini CBS 121011 JX901821 JX901734
25 Elasticomyces elasticus CCFEE 5547 KF309991
26 Exopassalora zambiae YHJN13 GQ433631 GQ433628
27 Extremus adstrictus TRN96 KF310022
28 Friedmanniomyces endolithicus CCFEE 5199 KF310007 JN885547
29 Hispidoconidioma alpinum L2–1/2 FJ997286 FJ997285
30 Hortaea werneckii CBS 100496 GU301817 AY128703
31 Houjia yanglingensis YHJN13 GQ433631 GQ433628
32 Lecanosticta pini CBS 871.95 GQ852598
33 Lembosia albersii MFLUCC 13–0377 KM386982
34 Lembosina sp. VUL.644 MG844165
35 Leptoxyphium cacuminum MFLUCC 10–0049 JN832602
36 Melanodothis caricis CBS 860.72 GU214431 GU214638
37 Microcyclosporella mali CPC 16171 GU570545 GU570528
38 Microxyphium citri CBS 451.66 KF902094
39 Morenoina calamicola MFLUCC 14–1162 NG059779 NR154210
40 Mycosphaerella pneumatophorae AFTOL-ID 762 KJ176856
41 Neodevriesia coryneliae CPC 23534 KJ869211 KJ869154
42 Neodevriesia hilliana CPC 15382 GU214414 GU214633
43 Neodevriesia xanthorrhoeae CBS 128219 HQ599606 HQ599605
44 Neopseudocercosporella capsellae CBS 127.29 KF251830 KF251326
45 Nowamyces globulus CBS 144598 MN162196 MN161935
46 Nowamyces piperitae CBS 143490 MN162200 MN161944
47 Parapenidiella tasmaniensis CBS 124991 KF901844 KF901522
48 Passalora eucalypti CBS 111318 KF901938 KF901613
49 Penidiella columbiana CBS 486.80 EU019274 KF901630
50 Periconiella velutina CBS 101950 EU041840 EU041783
51 Petrophila incerta TRN 77 GU323963
52 Phaeophleospora eugeniae CPC 15159 KF902095 KF901742
53 Phaeothecoidea eucalypti CBS 120831 KF901848 KF901526
54 Phaeothecoidiella illinoisensis CBS 125223 GU117901 GU117897
55 Phaeothecoidiella missouriensis CBS 125222 AY598917 AY598878
56 Phloeospora maculans CBS 115123 GU214670 GU214670
57 Piedraia hortae CBS 480.64 GU214466 GU214647
58 Piedraia quintanilhae CBS 327.63 GU214468
59 Pseudocercospora vitis CPC 11595 GU214483 GU269829
60 Pseudoramichloridium henryi CBS 124775 KF442561 KF442521
61 Pseudotaeniolina globosa CCFEE 5734 KF310010 KF309976
62 Pseudoveronaea obclavata CBS 132086 JQ622102
63 Racodium rupestre L346 EU048583 GU067666
64 Racodium rupestre L424 EU048582 GU067669
65 Ramichloridium apiculatum CBS 156.59 EU041848 EU041791
66 Ramularia endophylla CBS 113265 AY490776 AY490763
67 Ramularia pusilla CBS 124973 KP894141 KP894248
68 Ramulispora sorghi CBS 110578 GQ852653 -
69 Readeriella mirabilis CBS 125000 KF251836 KF251332
70 Recurvomyces mirabilis CBS 119434 GU250372 FJ415477
71 Repetophragma zygopetali VIC42946 KT732418
72 Schizothyrium pomi CBS 486.50 EF134948 EF134948
73 Scolecostigmina mangiferae CBS 125467 GU253877 GU269870
74 Scorias spongiosa CBS 325.33 GU214696 GU214696
75 Septoria cytisi USO 378994 JF700954 JF700932
76 Septoria lysimachiae CBS 123794 KF251972 KF251468
77 Sonderhenia eucalyptorum CBS 120220 KF901822 KF901505
78 Sphaerulina myriadea CBS 124646 JF770468 JF770455
79 Sporidesmajira pennsylvaniensis CBS 125229 MH874965 MF951287
80 Stenella araguata CBS 105.75 EU019250 EU019250
81 Teratoramularia kirschneriana CBS 113093 GU214669 GU214669
82 Teratosphaeria fibrillosa CBS 1217.07 GU323213 KF901728
83 Toxicocladosporium irritans CBS 185.58 EU040243 EU040243
84 Toxicocladosporium rubrigenum CBS 124158 FJ790305 FJ790287
85 Translucidithyrium chinense IFRDCC 3000 MT659404 MT659671
86 Translucidithyrium thailandicum MFLUCC 16–0362 MG993048 MG993045
87 Tripospermum myrti CBS 437.68 GU323216
88 Trochophora simplex CBS 124744 GU253880 GU269872
89 Uwebraunia communis CBS 114238 EU019267 AY725541
90 Vermiconia foris CCFEE 5459 GU250390 KF309981
91 Xenoconiothyrium catenatum CMW 22113 JN712570 JN712502
92 Zasmidium cellare CBS 146.36 EU041878 EU041821
93 Zygophiala cryptogama OH4_1A1a FJ147157 FJ425208
94 Zygophiala tardicrescens MWA1a EF164901 AY598856
95 Zygophiala wisconsinensis OH4_9A1c FJ147158 FJ425209

Taxonomy

Translucidithyrium chinense H. X. Wu & X. H. Li, sp. nov.

Figures 2, 3

Etymology

Refer to the location of species, China.

Holotype

IFRD9208

Description

Epiphytic on living leaves, ascomata with papillate. Superficial hyphae absent. Sexual morph: Ascomata solitary or scattered, 480–870 μm diam. (x̄ = 741 μm, n = 6), 65–82 µm high (x̄ = 72 μm, n = 8), olivaceous to brown, slightly semi-transparent under highlighted background, circular to suborbicular, with slightly prominent papilla, membranous, without ostiole (Fig. 2A–C). Peridium 8.3–10 μm thick, (x̄ = 9 μm, n = 11), composed of irregular, meandering, interwoven arranged cells, two layers: from brown to hyaline, outer layer composed of closely-arranged cells, brown; inner layer composed of hyaline, oblong, subdense arranged cells, poorly developed at the base (Fig. 2D–F). Asci evenly distributed and parallel arranged in hamathecium (Fig. 2D–F), 65–90 × 51–81 μm (x̄ = 77 × 60 μm, n = 10), 8-spored, bitunicate, hyaline, with an ocular chamber, ovoid at immature state, globose to subglobose at mature state, lacking pedicel, paraphyses absent (Fig. 2G–K). Ascospores 41–65 × 10–13 μm (x̄ = 50 × 11 μm, n = 20), irregularly overlapping, hyaline, ovoid at young state, fusiform with both ends tapered at mature state, 1-septate, constricted at the septum, upper cell a little larger than lower, with guttules at both ends, verrucose (Fig. 2L–P). Asexual morph: Undetermined.

Figure 2. 

Translucidithyrium chinense (IFRD 9208, holotype) A plant leaves B acscoma on leaves surface C squash of ascoma at 20 times amplification D cross section of ascoma in blue cotton at 20 times amplification E, F cross section of ascoma in blue cotton at 40 times amplification G asci at 100 times amplification H–K asci in blue cotton at 100 times amplification L ascospore at 100 times amplification M–P ascospore in blue cotton at 100 times amplification. Scale bars: 200 µm (B); 100 µm (C, D); 50 µm (E, F); 20 µm (G–K); 10 µm (L–P). We slightly adjusted the contrast, saturation and hue of images and removed the contaminants around main object in images in PS software without obscuration, erasure or distortion of any information existing in the original document.

Culture characteristics

Ascospores germinating on MEA at 36 h after spore-isolation, germinating on PDA at 48 h after spore-isolation. Colonies slow growing on MEA and PDA, irregular, villiform, convex, white on surface, yellow to brown at base. After a long period of growth, the pigments produced by culture discolour the medium, roots generate at the bottom (Fig. 3A–D). Culture hyphae hyaline, branched, constricted at the septum, 3 μm wide (Fig. 3E, F).

Figure 3. 

Culture of Translucidithyrium chinense (IFRDCC3000) A, B culture growing on the medium C, D the bottom of the medium with culture growing E, F the mycelium of culture at 100 times amplification. Scale bars: 10 µm (E, F).

Material examined

China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Xishuangbanna Botanical Garden; 21°55'51"N, 101°15'08"E, 540 m alt.; 21 Apr 2019; Haixia Wu and Xinhao Li leg; collected on living leaves of Alpinia blepharocalyx (IFRD 9208, holotype), ex-type living culture (IFRDCC 3000).

Notes

This new species is morphologically similar to Translucidithyrium thailandicum in having semi-transparent and largish ascomata, globose asci and hyaline ascospores with 1-septate. However, Translucidithyrium chinense has a slightly papilla thyriothecium with weaker transmittance and ascospores with guttules at both ends, while T. thailandicum has a flattened thyriothecium with higher transmittance and ascospores with appendages at both ends; besides, the size of ascomata and asci of T. chinense are slightly larger than those of T. thailandicum (795 μm vs. 621 μm; 77 μm vs. 64 μm). The culture characteristics of both species are different: the culture of T. chinense grows more slowly, has roots inserting into medium and turn the bottom brown. Phylogenetically, T. chinense clusters with T. thailandicum as a distinct clade with high support (100% ML / 1.00 PP, Fig. 1).

Divergence times estimates

The Maximum Clade Credibility (MCC) tree was similar to the major lineages in the Bayesian and ML trees. The crown age of Translucidithyrium showed 16 Mya (4–33), which was earlier than the divergence time of most genera in Phaeothecoidiellaceae. The estimated divergence time of Phaeothecoidiellaceae from Zeng et al. (2019) is 58 Mya, which corresponds to our results.

Discussion

Translucidithyrium thailandicum was found in the north of Thailand (Zeng et al. 2018). Translucidithyrium chinense was found in the Xishuangbanna Region, southwest of China, which lies on the northern border of a rainforest with rich microfungal resources. The new species is characterised by brown to olivaceous ascomata and slightly semi-transparent, subglobose asci without pedicel and fusiform ascospores with verrucose and guttules (Fig. 2). T. chinense is introduced as a new species in Translucidithyrium by morphological and phylogenetic studies (Figs 13).

The ascomata of Translucidithyrium are different from related genera of Phaeothecoidiellaceae: Nowamyces has immersed ascomata, Chaetothyrina has ascomata with setae and Rivilata has subcuticular ascomata (Singtripop et al. 2016; Doilom et al. 2018; Zeng et al. 2018; Crous et al. 2019; Hongsanan et al. 2020). Translucidithyrium is similar to the family Schizothyriaceae in having semi-transparent ascomata, globose to subglobose asci and hyaline ascospores with guttules. Schizothyriaceae includes Schizothyrium, Plochmopeltis, Hexagonella, Lecideopsella, Mycerema, Kerniomyces, Metathyriella, Myriangiella, Amazonotheca and Vonarxella (Phookamsak et al. 2016; Wijayawardene et al. 2020). The morphology of T. chinense is most similar to Lecideopsella by having globose asci and 1-septate ascospores, but Lecideopsella has a short pedicel at the bottom of the asci (Phookamsak et al. 2016; Zeng et al. 2018). Phylogenetically, Translucidithyrium formed a long clade and clustered within the family Phaeothecoidiellaceae. It indicated the existing certain genetic distance amongst Translucidithyrium, Phaeothecoidiellaceae and Schizothyriaceae. Phaeothecoidiellaceae and Schizothyriaceae are poorly studied families (Batzer et al. 2008; Phookamsak et al. 2016; Singtripop et al. 2016; Hongsanan et al. 2017; Zeng et al. 2018). Therefore, more fresh specimens with molecular data are needed to confirm the classification of Translucidithyrium, Phaeothecoidiellaceae and Schizothyriaceae.

Zuckerkandl and Pauling (1962) suggested that the number of differences amongst amino acids was proportional to species divergence time. We estimated the divergence time using BEAST analysis. The divergence time of Translucidithyrium crown age was estimated at 16 Mya (4–33), which was earlier than the crown ages of Chaetothyrina at 2 Mya (0–5), the crown ages of Repetophragma at 9 Mya (2–20), the crown ages of Nowamyces at 7 Mya (1–20) and the crown ages of Phaeothecoidiella at 4 Mya (0–14) within Phaeothecoidiellaceae (Fig. 4). The divergence time of Translucidithyrium is earlier than other genera in Phaeothecoidiellaceae. We estimate that the long divergence time should affect the genetic variation (Pauling 1964; Hall and Hallgrímsson 2008). Additionally, the evolutionary molecular clock approach confirmed the long clades of Translucidithyrium in the phylogenetic tree (Fig. 1).

Figure 4. 

The MCC tree with divergence times estimates of Phaeothecoidiellaceae obtained from a Bayesian approach (BEAST). Numbers at nodes indicate posterior probabilities (pp) for node support; bars correspond to the 95% highest posterior density (HPD) intervals. The key species are given in blue.

Historical events amongst different biological groups could then be compared with the dates of plate tectonic movements and paleoecology, according to the corresponding geological time scale (Lomolino et al. 2006; Berbee and Taylor 2010). Through relevant studies on the Qinghai-Tibet Plateau, it was found that the time of intense tectonic uplift and denudation is concentrated in 60–35 Mya, 25–17 Mya, 12–8 Mya and 5 Mya. Global cooling might have an impact on climate change in East Asia, especially at 15 Mya and 8 Mya (Lu et al. 2010). Rising plateaus and global cooling were drying up Asia (Liu 2000; Garzione et al. 2015). The time of the Qinghai-Tibet Plateau uplift and global cooling corresponded to the interval of the species in Translucidithyrium divergence time. We predict that the speciation of T. chinense was earlier than the speciation of T. thailandicum, as the divergence of Translucidithyrium was related to the Qinghai-Tibet Plateau uplift and global cooling. According to the evolution history of Translucidithyrium, it could be speculated that the speciation of T. chinense was earlier than T. thailandicum. With the climate becoming colder and with increased drought, T. chinense migrated from China to Thailand gradually to find a suitable area, then T. thailandicum formed. Due to the end of global cooling, the distribution pattern of Translucidithyrium in two different countries formed. Increasing fresh collections and application of new methodologies may result in modified conclusions.

Acknowledgements

Funds for research were provided by the Grant for Essential Scientific Research of National Nonprofit Institute (no. CAFYBB2019QB005), the Yunnan Province Ten Thousand Plan of Youth Top Talent Project (no. YNWR-QNBJ-2018-267) and the Yunnan Fundamental Research Projects (grant NO. 202001AT070014). The authors are deeply grateful to Prof. K.D. Hyde (Mae Fah Luang University, Thailand, MFU) for editing the English language of the manuscript, to Dr. Xiang-Yu Zeng and Dr. Nawaz Haider for revising this manuscript and to Dr. Rungtiwa Phookamsak for guiding experiment operation.

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