New and Interesting Pine-Associated Hyphomycetes from China

Pine trees play a crucial role in the forests of Sichuan Province, boasting rich species diversity and a lengthy evolutionary history. However, research and investigation on fungi associated with pine trees are insufficient. This study investigated the diversity of hyphomycetes fungi associated with pine trees in Sichuan Province, China. During the survey, we collected five specimens of hyphomycetes from branches and bark of species of Pinus. Five barcodes were selected for study and sequenced, including ITS, SSU, LSU, TEF1, and RPB2. Morphological examination and multi-locus phylogenetic analyses revealed three new species, viz. Catenulostroma pini sp. nov. within Teratosphaeriaceae, Kirschsteiniothelia longisporum sp. nov. within Kirschsteiniotheliaceae, Sporidesmiella sichuanensis sp. nov. within Junewangiaceae, and two known species, Paradictyoarthrinium diffractum and P. hydei within Paradictyoarthriniaceae, which are the new host records from Pinus species. Catenulostroma pini, distinguished from other species in the genus by its unique morphology, has three conidial morphologies: small terminal helicoconidia, scolecoconidia with many septa, and phragmoconidia conidia. Kirschsteiniothelia longisporum has longer spores when compared to the other species in the genus. According to phylogenetic analysis, Sporidesmiella sichuanensis formed an independent clade sister to S. aquatica and S. juncicola, distinguished by differences in conidial size.


Introduction
Hyphomycetes are a polyphyletic group of fungi that lack fruiting bodies (conidiomata), have hyphae that may be immersed in the substrate or not, and where sporulation mainly occurs on differentiated septate hyphae [1,2].Hyphomycetes represent the asexual forms of many fungal species and are highly diverse, with more than 2265 genera and 13,800 species reported worldwide [2,3].Hyphomycetes are widely distributed and can be saprophytic in freshwater, marine, and terrestrial ecosystems, or they can parasitize animals and plants as pathogens [4][5][6][7].Many of the fungi in this group are aquatic and characterized by the production of conidia that are passively discharged from the hyphae, facilitating dispersal [8][9][10].Since most conidia are characteristically shaped, species can often be identified by morphology, a common practice in the study of dematiaceous hyphomycetes [11][12][13][14].However, since species may have undergone convergent evolution in morphology, phylogenetic analysis should also be incorporated into classification.
Catenulostroma (Teratosphaeriaceae, Dothideomycetes) was introduced by Crous et al., with the type C. protearum, which was previously placed in the genus Trimmatostroma [15].Trimmatostroma and Catenulostroma are morphologically similar; however, phylogenetically, they appear as distinct genera, with the type species of Trimmatostroma belonging to the was made following the method described by Senanayake et al. [42].Germinated conidia were individually transferred to potato dextrose agar (PDA) media plates and incubated in the dark at 25 • C. Culture characteristics were examined and recorded regularly after 1-3 weeks.
The holotype specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and all specimens were deposited in the Herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China.The living ex-type cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and all living cultures were deposited in the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China.The taxonomic descriptions of the new taxa have been deposited in MycoBank.

DNA Extraction, PCR Amplification and Sequencing
Fungal genomic DNA was extracted from mycelia using the Trelief TM Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, China) according to the manufacturer's protocol.For Sporidesmiella sichuanensis specimens (HKAS 136267), obtaining a culture was not feasible, necessitating the direct extraction of DNA from fruiting structures using the method used by Wanasinghe et al. [43].Five loci, the nuclear ribosomal internal transcribed spacer (ITS: ITS1-5.8S-ITS2), the nuclear ribosomal small subunit rRNA (SSU), the nuclear ribosomal large subunit rRNA (LSU), the partial translation elongation factor 1-alpha (TEF1), the partial second largest subunit of RNA polymerase II (RPB2), were selected for study and amplified by polymerase chain reaction (PCR).The corresponding primer pairs and PCR conditions are listed in Table 1.The final reaction volume of the PCR reagent was 25 µL, containing 2 µL of DNA template, 1 µL each of the forward and reverse primer, 8.5 µL of double-distilled water (ddH 2 O), and 12.5 µL of 2×Flash PCR MasterMix (mixture of DNA Polymerase, dNTPs, Mg 2+ and optimized buffer; CoWin Biosciences, Taizhou, China).The PCR products were visualized by 1% agarose gel electrophoresis.Sanger sequencing was conducted by Tsingke Biological Technology (Beijing, China).Newly generated sequences were deposited in GenBank, and the accession numbers are listed in Tables 2-5.

Phylogenetic Analyses
According to the corresponding Sanger sequencing chromatograms, misleading data from the ends of raw sequencing fragments were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0(DNASTAR, Inc., Madison, WI, USA).Barcode sequences of all species (Tables 2-5) were downloaded from the NCBI nucleotide database using the R package Analysis of Phylogenetics and Evolution 5.0 (APE, http://ape-package.ird.fr,7 July 2024) [52].
The multiple sequence alignments were conducted using MAFFT version 7.310 [53] with options "--adjustdirection --auto", and the alignment files were further trimmed using trimAl version 1.4 [54] with the option "-gapthreshold 0.5", which only allows 50% of taxa with a gap in each site.The best-fit nucleotide substitution models for each locus were selected using ModelFinder version 2.1.1 [55] under the Corrected Akaike Information Criterion (AICC).All sequence alignments were combined using an in-house Python script.
Maximum Likelihood (ML) and Bayesian analysis (BI) were conducted based on individual and combined datasets.Four phylogenetic trees were constructed by multi-locus phylogenetic analyses.The first tree represents the phylogenetic analysis of Catenulostroma, the second tree represents the phylogenetic analysis of Kirschsteiniothelia, the third tree represents the phylogenetic analysis of Paradictyoarthrinium, and the fourth tree represents the phylogenetic analysis of Sporidesmiella within the Junewangiaceae.ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [56], and the topology was evaluated using 1000 ultrafast bootstrap replicates.The BI was conducted using parallel MrBayes version 3.2.7a[57].The ML trees were visualized using ggtree version 2.4.1 [58] and further edited in Adobe Illustrator version 16.0.0.

Phylogenetic Analyses
Sequences of three loci were successfully obtained for the Catenulostroma pini (UESTCC 24.0185).Nine taxa were included in the combined ITS, LSU and SSU sequence data, with Teratosphaeria fibrillosa (CPC 1876) as the outgroup (Figure 1).The combined dataset (ITS: 1-644, LSU: 645-1871, SSU: 1872-3619) was composed of 273 distinct patterns, 136 parsimony-informative sites, 324 singleton sites and 3159 constant sites.The bestfit evolution models were K2P + G4 for the ITS partitions, K2P + I for the LSU partition, K2P for the SSU partition.The best-scoring ML tree (lnL = −7762.005)with support values from ML and Bayesian analysis at the node is shown in Figure 1.
from the ends of raw sequencing fragments were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0(DNASTAR, Inc., Madison, WI, USA).Barcode sequences of all species (Tables 2-5) were downloaded from the NCBI nucleotide database using the R package Analysis of Phylogenetics and Evolution 5.0 (APE, http://ape-package.ird.fr,7 July 2024) [52].
The multiple sequence alignments were conducted using MAFFT version 7.310 [53] with options "--adjustdirection --auto", and the alignment files were further trimmed using trimAl version 1.4 [54] with the option "-gapthreshold 0.5", which only allows 50% of taxa with a gap in each site.The best-fit nucleotide substitution models for each locus were selected using ModelFinder version 2.1.1 [55] under the Corrected Akaike Information Criterion (AICC).All sequence alignments were combined using an in-house Python script.
Maximum Likelihood (ML) and Bayesian analysis (BI) were conducted based on individual and combined datasets.Four phylogenetic trees were constructed by multi-locus phylogenetic analyses.The first tree represents the phylogenetic analysis of Catenulostroma, the second tree represents the phylogenetic analysis of Kirschsteiniothelia, the third tree represents the phylogenetic analysis of Paradictyoarthrinium, and the fourth tree represents the phylogenetic analysis of Sporidesmiella within the Junewangiaceae.ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [56], and the topology was evaluated using 1000 ultrafast bootstrap replicates.The BI was conducted using parallel MrBayes version 3.2.7a[57].The ML trees were visualized using ggtree version 2.4.1 [58] and further edited in Adobe Illustrator version 16.0.0.

Phylogenetic Analyses
Sequences of three loci were successfully obtained for the Catenulostroma pini (UESTCC 24.0185).Nine taxa were included in the combined ITS, LSU and SSU sequence data, with Teratosphaeria fibrillosa (CPC 1876) as the outgroup (Figure 1).The combined dataset (ITS: 1-644, LSU: 645-1871, SSU: 1872-3619) was composed of 273 distinct patterns, 136 parsimony-informative sites, 324 singleton sites and 3159 constant sites.The best-fit evolution models were K2P + G4 for the ITS partitions, K2P + I for the LSU partition, K2P for the SSU partition.The best-scoring ML tree (lnL = −7762.005)with support values from ML and Bayesian analysis at the node is shown in Figure 1.
According to the multi-locus phylogeny (Figure 1), our collection (UESTCC 24.0185) formed an independent clade sister to Catenulostroma hermanusense (CBS 128768) and C. protearum (CBS 125421) with 99% ML, 1.00 PP statistical support.Combining the morphological evidence with phylogeny, we propose a new species, C. pini, isolated from Pinus massoniana.Sequences of three loci were successfully obtained for the Kirschsteiniothelia longisporum (UESTCC 24.0190).A phylogenetic tree of species in Kirschsteiniothelia was constructed (Figure 2), including 48 taxa, with Tenuitholiascus porinoides (HMAS-L0139638) as the out-group.The combined dataset (ITS: 1-507, LSU: 508-1368, SSU: 1369-2392) was composed of 988 distinct patterns, 585 parsimony-informative sites, 252 singleton sites and 1555 constant sites.The best-fit evolution models were GTR + F + G4 for the ITS partitions, GTR + F + G4 for the LSU partition, and K2P + I + G4 for the SSU partition.The best-scoring ML tree (lnL = −14,653.774)with support values from ML and Bayesian analysis at the node is shown in Figure 2.  According to the multi-locus phylogeny (Figure 2), our collection (UESTCC 24.0190) formed a branch sister to Kirschsteiniothelia aquatica (MFLUCC 16-1685).Based on the morphological evidence and phylogeny, we propose a new species, K. longisporum, isolated from Pinus taeda.
Sequences of three loci were successfully obtained for the Paradictyoarthrinium diffractum (UESTCC 24.0187) and Paradictyoarthrinium hydei (UESTCC 24.0188).A phylogenetic tree of species in Paradictyoarthrinium was constructed (Figure 3), including 13 taxa, with Nigrograna obliqua (CBS 141477) as the outgroup.The combined dataset (ITS: 1-518, LSU: 519-1364, SSU: 1365-2418) was composed of 314 distinct patterns, 73 parsimonyinformative sites, 347 singleton sites and 1998 constant sites.The best-fit evolution models were K2P + I for the ITS partitions, K2P + I for the LSU partition, and K2P + I for the RPB2 partition.The best-scoring ML tree (lnL = −5397.698)with support values from ML and Bayesian analysis at the node is shown in Figure 3.
According to the multi-locus phylogeny (Figure 3), our collection (UESTCC 24.0188) nest with P. hydei strains with 99% ML, 1.00 PP statistical support and our collection (UESTCC 24.0187) nest with P. diffractum strains.Based on the morphological evidence and phylogeny, we report our collections (UESTCC 24.0188 and UESTCC 24.0187) as new host records of P. hydei and P. diffractum from Pinus spp.According to the multi-locus phylogeny (Figure 3), our collection (UESTCC 24.0188) nest with P. hydei strains with 99% ML, 1.00 PP statistical support and our collection (UESTCC 24.0187) nest with P. diffractum strains.Based on the morphological evidence and phylogeny, we report our collections (UESTCC 24.0188 and UESTCC 24.0187) as new host records of P. hydei and P. diffractum from Pinus spp.Sequences of four loci were successfully obtained for the Sporidesmiella sichuanensis (HKAS 136267).A phylogenetic tree of species in Sporidesmiella within the Junewangiaceae was constructed (Figure 4), including 15 taxa, with Junewangia thailandica (MFLU 15-2682) as the outgroup.The combined dataset (ITS: 1-520, LSU: 521-1326, RPB2: 1327-2323, TEF1: 2324-3181) was composed of 576 distinct patterns, 375 parsimony-informative sites, 398 singleton sites and 2408 constant sites.The best-fit evolution models were GTR + F + G4 for the ITS partitions, K2P + I for the LSU partition, HKY + F + I for the RPB2 partition, GTR + F + G4 for the TEF1 partition.The best-scoring ML tree (lnL = −9362.397)with support values from ML and Bayesian analysis at the node is shown in Figure 4.
According to the multi-locus phylogeny (Figure 4), our collection (HKAS 136267) formed an independent clade sister to Sporidesmiella aquatica (DLUCC 0777) and Sporidesmiella juncicola strains.Based on the morphological evidence and phylogeny, we identified S. sichuanensis as a novel species from Pinus taeda.Sequences of four loci were successfully obtained for the Sporidesmiella sichuanensis (HKAS 136267).A phylogenetic tree of species in Sporidesmiella within the Junewangiaceae was constructed (Figure 4), including 15 taxa, with Junewangia thailandica (MFLU 15-2682) as the outgroup.The combined dataset (ITS: 1-520, LSU: 521-1326, RPB2: 1327-2323, TEF1: 2324-3181) was composed of 576 distinct patterns, 375 parsimony-informative sites, 398 singleton sites and 2408 constant sites.The best-fit evolution models were GTR + F + G4 for the ITS partitions, K2P + I for the LSU partition, HKY + F + I for the RPB2 partition, GTR + F + G4 for the TEF1 partition.The best-scoring ML tree (lnL = −9362.397)with support values from ML and Bayesian analysis at the node is shown in Figure 4.

Discussion
In this study, two rarely seen species from Pinus spp.were reported.Catenulostroma pini sp.nov., isolated from the dead bark of Pinus massoniana, is the seventh species of the genus and the first time that the genus Catenulostroma has been recorded in China [15,16,59].Sporidesmiella sichuanensis sp.nov., isolated from a dead branch of Pinus taeda, is the eleventh species in the genus to have sequence data.Sporidesmiella is an old genus with origins dating back to the 18th century (previously as Sporidesmium); hence, molecular data are unavailable for most species [32].Sporidesmiella pini, was isolated from needles of Pinus sylvestris in the Netherlands [63].This suggests that many unique and rare taxa on Pinus spp.may still await discovery and exploration.
Catenulostroma pini sp.nov. is an interesting species in which three conidial morphologies were observed.The first type is a helicoconidia small terminal conidia, characterized

Discussion
In this study, two rarely seen species from Pinus spp.were reported.Catenulostroma pini sp.nov., isolated from the dead bark of Pinus massoniana, is the seventh species of the genus and the first time that the genus Catenulostroma has been recorded in China [15,16,59].Sporidesmiella sichuanensis sp.nov., isolated from a dead branch of Pinus taeda, is the eleventh species in the genus to have sequence data.Sporidesmiella is an old genus with origins dating back to the 18th century (previously as Sporidesmium); hence, molecular data are unavailable for most species [32].Sporidesmiella pini, was isolated from needles of Pinus sylvestris in the Netherlands [63].This suggests that many unique and rare taxa on Pinus spp.may still await discovery and exploration.
Catenulostroma pini sp.nov. is an interesting species in which three conidial morphologies were observed.The first type is a helicoconidia small terminal conidia, characterized by helicospores are solitary, acrogenous, euseptate, constricted at the septa, circinate, and spherical.This morphology is slightly similar to that of C. protearum obtained from OA, as reported by Crous et al. [60].However, the conidia in our collection are circinate and spherical, whereas the conidia of C. protearum are variably muriform to transversely septate [60].The transversely septate conidia of C. protearum are very similar to the second conidial morphology in our collection [60].They all occur in branched chains, forming chain-like, septate, and scolecospores, which are also similar to the conidia of C. chromoblastomycosum [15,60].After germination and sporulation of helicoconidia on PDA, we observed a third morphology of conidia characterized by simple or branched chains, cylindric-obclavate, straight to flexuous, hyaline to pale brown, septate, constricted at the septate.Similar to C. hermanusense, conidia are in simple or branched chains but with different morphologies: cylindric-obclavate and constricted at the septa, compared to subcylindrical to ellipsoid [59].Besides the unique and diverse morphology of the genus Catenulostroma, it is noteworthy that C. chromoblastomycosum was described as a case of human chromoblastomycosis [15].Currently, no other species of this genus have been found capable of infecting humans and causing disease.
In this study, four hyphomycetes fungi collected from Pinus spp.were isolated and identified: Catenulostroma pini sp.nov.(Teratosphaeriaceae), Kirschsteiniothelia longisporum sp.nov.(Kirschsteiniotheliaceae), Paradictyoarthrinium diffractum and P. hydei of Paradictyoarthriniaceae, all within Dothideomycetes.Dothideomycetes represent the largest and most diverse class of ascomycete fungi [6,25,64,65].This class includes over 25 orders, 110 families and more than 19,000 species [66].Their representatives have an incredibly diverse lifestyle and can be associated with various hosts and substrates [6].Another collection was identified as Sporidesmiella sichuanensis sp.nov., a saprophyte in the Junewangiaceae family within Sordariomycetes.Sporidesmiella is a polyphyletic genus.Based on phylogenetic analysis of the LSU and RPB2 datasets, Shenoy et al. [67] classified S. fusiformis into the family Melanommataceae within Dothideomycetes.Subsequently, using combined ITS, LSU, RPB2, and TEF1 sequence data, many species in the genus were reassigned to Junewangiaceae within Sordariomycetes [10,[33][34][35][36].The diversity of fungal groups in Sordariomycetes is high, and most species are saprophytic fungi that can degrade organic matter in nature and promote the material cycle of the ecosystem [68][69][70].

Figure 1 .
Figure 1.The phylogram of the genus Catenulostroma (Teratosphaeriaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-SSU.The tree is rooted with Teratosphaeria fibrillosa (CPC 1876).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.90 were displayed at the nodes as ML/PP.Support values below 95 and 0.90 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 2 .
Figure 2. The phylogram of the genus Kirschsteiniothelia (Kirschsteiniotheliaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-SSU.The tree is rooted with Tenuitholiascus porinoides (HMAS-L0139638).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.Sequences of three loci were successfully obtained for the Paradictyoarthrinium diffractum (UESTCC 24.0187) and Paradictyoarthrinium hydei (UESTCC 24.0188).A phylogenetic

Figure 2 .
Figure 2. The phylogram of the genus Kirschsteiniothelia (Kirschsteiniotheliaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-SSU.The tree is rooted with Tenuitholiascus porinoides (HMAS-L0139638).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.
J. Fungi 2024, 10, 546 9 of 23 tree of species in Paradictyoarthrinium was constructed (Figure 3), including 13 taxa, with Nigrograna obliqua (CBS 141477) as the outgroup.The combined dataset (ITS: 1-518, LSU: 519-1364, SSU: 1365-2418) was composed of 314 distinct patterns, 73 parsimony-informative sites, 347 singleton sites and 1998 constant sites.The best-fit evolution models were K2P + I for the ITS partitions, K2P + I for the LSU partition, and K2P + I for the RPB2 partition.The best-scoring ML tree (lnL = −5397.698)with support values from ML and Bayesian analysis at the node is shown in Figure 3.

Figure 3 .
Figure 3.The phylogram of the genus Paradictyoarthrinium (Paradictyoarthriniaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2.The tree is rooted with Nigrograna obliqua (CBS 141477).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 3 .
Figure 3.The phylogram of the genus Paradictyoarthrinium (Paradictyoarthriniaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2.The tree is rooted with Nigrograna obliqua (CBS 141477).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 4 .
Figure 4.The phylogram of the genus Sporidesmiella (Junewangiaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2-TEF1.The tree is rooted with Junewangia thailandica (MFLU 15-2682).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 4 .
Figure 4.The phylogram of the genus Sporidesmiella (Junewangiaceae) from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2-TEF1.The tree is rooted with Junewangia thailandica (MFLU 15-2682).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Table 1 .
Loci used in this study with the corresponding PCR primers and conditions.

Table 2 .
Species details and their GenBank accession numbers used in phylogenetic analyses of Catenulostroma.Type strains are in bold, and newly generated sequences are in red.

Table 5 .
Species details and their GenBank accession numbers used in phylogenetic analyses of Sporidesmiella.Type strains are in bold, and newly generated sequences are in red.