Maximum Parsimony Methods Research Articles

Morphological and molecular identification for four new wood-inhabiting species of Lyomyces (Basidiomycota) from China.

Fungi are one of the most diverse groups of organisms on Earth, in which the wood-inhabiting fungi play an important role in forest ecosystem processes and functions. Four new wood-inhabiting fungi, Lyomyceshengduanensis, L.niveomarginatus, L.wumengshanensis and L.zhaotongensis, are proposed, based on morphological features and molecular evidence. Lyomyceshengduanensis differs in the brittle basidiomata with pruinose hymenial surface, a monomitic hyphal system and ellipsoid basidiospores (3.5-6 × 3-4.5 µm). Lyomycesniveomarginatus is distinguished by the subceraceous basidiomata with crackled hymenial surface, a monomitic hyphal system and ellipsoid basidiospores (4.5-7 × 3-4 µm). Lyomyceswumengshanensis is distinguished by the grandinioid hymenial surface, a monomitic hyphal system and ellipsoid to broad ellipsoid basidiospores (4-6 × 3-5 µm). Lyomyceszhaotongensis is unique in the grandinioid hymenial surface, a monomitic hyphal system and broadly ellipsoid basidiospores measuring as 2.6-3.5 × 2.5-3 µm. Sequences of ITS and nLSU rRNA markers of the studied samples were generated and phylogenetic analyses were performed using the Maximum Likelihood, Maximum Parsimony and Bayesian Inference methods. The phylogram, based on the ITS+nLSU rDNA gene regions, included three genera within the Schizoporaceae viz. Fasciodontia, Lyomyces and Xylodon, in which the four new species were grouped into Lyomyces. The phylogenetic tree inferred from the ITS sequences highlighted that L.hengduanensis group with L.zhaotongensis and then closely grouped with L.crustosus, L.ochraceoalbus, and L.vietnamensis. The new taxon L.niveomarginatus was retrieved as a sister to L.juniperi. The new species L.wumengshanensis was sister to L.macrosporus. The new taxon L.zhaotongensis grouped with L.hengduanensis and then closely grouped with L.crustosus, L.ochraceoalbus and L.vietnamensis.

Never ending diversity: two new species of the genus Allocreadium (Digenea: Allocreadiidae) including new keys to the genus.

Two new species of the genus Allocreadium were isolated from the intestines of the Lake minnow Rhynchocypris percnura caught in the backwater of the Komissarovka River in the South of the Russian Far East. The morphology of A. anastasii n. sp. corresponds to that of Allocreadium sp. from Lake Khar (Mongolia) and Allocreadium sp. Belous, 1952 from the Primorsky region of Russia except for the preacetabular anterior border of the vitelline follicles in A. anastasii n. sp. from the Komissarovka River vs. at anterior half of ventral sucker in Allocreadium sp. Genetic analysis revealed the identity of A. anastasii n. sp. to Allocreadium sp. 1 from the Nezhinka River and Lake Khar. Allocreadium macrolecithum n. sp. was differentiated from Palaearctic Allocreadium spp. by having the following features: respectively large vitelline follicles extending from posterior extremity to anterior margin of the ventral sucker; relatively short caeca reaching the border of middle and posterior thirds of hindbody; and small testes in the middle of hindbody. Interspecific genetic p-distances between Allocreadium spp. were 0.16-7.23% in 28S gene and 18.62-31.54% in Cox1 mtDNA gene. In the phylogenetic tree reconstructed with Maximum parsimony and Bayesian Inference methods, A. anastasii n. sp. and A. macrolecithum n. sp. were nested into different species groups of the genus Allocreadium - sister to A. khankaiense and A. bursense, respectively. Modified dichotomous keys were prepared for 31 Palaearctic species of Allocreadium including A. crassum, A. dogieli, A. papilligerum, A. bursense, A. anastasii n. sp., and A. macrolecithum n. sp.

Morphological characteristics and phylogenetic analyses revealed Geastrum yunnanense sp. nov. (Geastrales, Basidiomycota) from Southwest China

A new species (Geastrum yunnanense) from Yunnan Province in Southwest China is described and illustrated based on morphological and molecular evidence. This species is characterized by the globular or ovate endoperidial body, shallowly saccate exoperidium, and spherical basidiospores (2.0–2.5 × 1.9–2.5 µm in diameter). Sequences of the internal transcribed spacers (ITS) and large subunit (nrLSU) of the nuclear ribosomal DNA (rDNA) markers of the studied samples were generated, and the phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic analyses inferred from ITS+nrLSU dataset indicated that G. yunnanense nested within the genus Geastrum, forming a monophyletic lineage sister to G. velutinum, and G. javanicum. A full description, illustrations, and phylogenetic analysis results for the new species are provided.

Morphological characteristics and phylogenetic analyses reveal Scopuloides yunnanensis (Polyporales, Basidiomycota), a new wood-decaying fungus from China

Wood-decaying fungi, as crucial decomposers in forest ecosystems, have been the subject of meticulous research. In this study, a new wood-decaying fungal species, Scopuloides yunnanensis, found in Yunnan, China, is proposed. Scopuloides yunnanensis was identified based on a comprehensive analysis of its membranaceous basidiomata, generative hyphae, lamprocystidia, projecting cystidia, and allantoid basidiospores. In addition to the morphology, phylogenetic analyses of ITS and nLSU rRNA markers were performed with Maximum Likelihood, Maximum Parsimony, and Bayesian inference methods, ensuring the robustness of our findings. The phylogenetic analysis showed that S. yunnanensis is sister to S. hydnoides. The new species’ description, illustrations, and results of the phylogenetic analysis are provided. In addition, an identification key to all Scopuloides species worldwide is provided.

The diversity and taxonomy of Tomentella (Thelephoraceae, Thelephorales) with descriptions of four new species from Southwestern China.

Taxonomy plays a central role in understanding the diversity of life, translating the products of biological exploration and discovery specimens and observations into systems of names that settle a "classification home" to taxa. Up to this point, studies on the taxonomy and phylogeny of the basidiomycetous genus Tomentella stemmed mainly from the temperate to boreal zones of the North Hemisphere, but were scarce in tropical Asia. In this study, four new species, viz. Tomentellaolivaceobasidiosa, T.velutina, T.wumenshanensis and T.yunnanensis from China, are described and illustrated based on the morphological characteristics and molecular phylogenetic analyses, in which the sequences of ITS+nLSU+mtSSU+RPB2 genes were used for the phylogenetic analyses using Maximum Likelihood, Maximum Parsimony and Bayesian Inference methods. All the new species can be well recognised by their macroscopical and anatomical characteristics. The four new species, closely related taxa in the phylogenetic tree and morphologically similar species are discussed.

REDESCRIPTION OF HASSALSTRONGYLUS ADUNCUS CHANDLER, 1932 (NEMATODA: HELIGMONELLIDAE), WITH THE DESCRIPTION OF A NEW SPECIES OF THE GENUS AND THEIR PHYLOGENETIC POSITION WITH OTHER HELIGMOSOMOIDEA TRAVASSOS, 1914.

Hassalstrongylus Durette-Desset, 1971 (Nematoda: Heligmonellidae), includes 19 species that are distributed from the southwestern United States to central-western Argentina. Hassalstrongylus aduncus is a parasitic nematode of rodents from the subfamilies Arvicolinae, Murinae, and Sigmodontinae, and has been recorded from southern Virginia and Oklahoma to Costa Rica. This species was described by Chandler in 1932; the morphology of the synlophe was not included. Subsequently, in 1972, Durette-Desset described only the synlophe of the middle region of the body in both sexes. Despite its wide geographical distribution, to date, there has been no redescription that includes information complementary to the morphology of the synlophe, such as a study of the body surface or a molecular phylogenetic analysis. We reevaluated the morphology of some specimens that were presumably similar to H. aduncus parasite of Sigmodon sp. from Jalisco, Mexico, and it was determined that these corresponded to an undescribed species of the genus. Herein, we present a redescription of H. aduncus parasite of Sigmodon toltecus from Hidalgo, Mexico, with morphological traits such as the excretory pore, deirids, and ovijector, and provide a description of the synlophe in the anterior and posterior regions of both sexes and include scanning electron microscopy images. Hassalstrongylus geolayarum n. sp. is differentiated from H. aduncus by the number of ridges in the middle region of the body (23 vs. 21), as well as proportions between some traits of males and females such as total length/spicule length, total length/gubernaculum length, total length/length of the esophagus and total length/distance of the vulva and the size of the eggs (42 vs. 58 μm). Phylogenetic analysis is based on partial sequences of the nuclear ribosomal internal transcribed spacer region (ITS1 + 5.8S + ITS2) of the rDNA, using the maximum-parsimony, maximum-likelihood, and Bayesian inference methods revealed the close relationship of H. aduncus + H. geolayarum n. sp. within the Heligmosomoidea and confirmed the placement of the Hassalstrongylus monophyletic clade well-supported within Heligmonellidae. The new species presented a genetic divergence of 3.4-3.8% relative to H. aduncus. This is the first species of the genus described in Mexico. Presumably, there are more species not yet described throughout the geographic range of H. aduncus. A taxonomic review and molecular phylogenetic analysis are required in which more species and genes are analyzed in Heligmosomoidea to confirm the status of the nonmonophyletic groups recovered here.

First Report of Colletotrichum aenigma Causing Anthracnose on Dragon Fruit in Korea.

Dragon fruit (Selenicereus undatus) is a valuable fruit crop in tropical and subtropical regions. It is renowned for its nutritional benefits, such as high sodium, potassium, and vitamin levels, and as a source of prebiotics and antioxidants (Balendres et al. 2019). In July 2023, anthracnose symptoms on stems were detected on dragon fruit plants in Jeju, South Korea. The typical anthracnose symptoms, such as sunken necrotic lesions (5-20 mm in diameter), were seen on the mature stems. The disease incidence ranged from 10% to 12% among the three surveyed greenhouses. To isolate the causative organism, infected stem samples were surface sterilized, cut into small pieces, and placed on potato dextrose agar (PDA). After two days of incubation at 24ºC, white hyphae appeared on the PDA around the plant tissues. Isolates CNU H23009 and CNU H23010 were purified from a single hypha under a stereoscope (e-Xtra Figure 1). Conidial morphology was examined from two-day-old fungal cultures grown on V8 juice agar. The conidia were transparent, aseptate, cylindrical to clavate, with a rounded apex and base, and measured 11.9 - 16.85 × 5.17 - 6.91 μm (mean = 15.28 × 5.93 μm, n = 30). No appressoria was observed. Morphological characteristics indicated the isolates were Colletotrichum sp. matching the description of the C. gloeosporioides species complex (Weir et al. 2012). To further identify the isolates, genomic DNA was extracted and the ribosomal internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified using ITS1/ITS4, GDF/GDR, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Based on phylogenetic analysis, the isolates clustered with C. aenigma (strains ICMP18608, ICMP18686, CSH2, and QSG1), with 71% bootstrap support, as determined using the maximum parsimony method in PAUP 4.0 (e-Xtra Figure 2). Based on morphological and molecular characteristics, isolates were identified as C. aenigma. Sequences of CNU H23009 and CNU H23010 were deposited in GenBank with accession numbers OR535144 and OR535145 for ITS, OR540725 and OR540726 for GAPDH, and OR540723 and OR540724 for ACT. The pathogenicity was tested on healthy dragon fruit stems using wound inoculation with mycelial plugs of the CNU H23009 isolate. Controls were inoculated with PDA plugs. The plants were covered with plastic bags to maintain humidity and incubated in a greenhouse at 25ºC. After two days, necrotic spots had developed on the inoculated tissues; after four days, black, irregular, and sunken necrotic lesions similar to those seen in the field were observed. No symptoms occurred in the controls. C. aenigma was re-isolated from the artificially inoculated plants and re-identified based on conidial morphology. The pathogenicity test was repeated three times with three replications for each treatment. Previous studies have reported that C. aenigma, C. gloeosoporioides, C. siamense, C. truncatum, and C. karsti cause anthracnose in dragon fruit. However, C. aenigma has been reported only in Thailand (Balendres et al. 2019; Meetum et al. 2015). To our knowledge, this is the first report of C. aenigma causing anthracnose in dragon fruit in Korea.

Morphology and multigene phylogeny revealed four new species of Xylodon (Schizoporaceae, Basidiomycota) from southern China.

Fungi are one of the most diverse groups of organisms on Earth, amongst which wood-inhabiting fungi play a crucial role in ecosystem processes and functions. Four new wood-inhabiting fungi, Xylodoncremeoparinaceus, X.luteodontioides, X.poroides and X.wumengshanensis are proposed, based on morphological features and molecular evidence. Xylodoncremeoparinaceus is distinguished by a cream hymenial surface with a pruinose hymenophore, a monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Xylodonluteodontioides is characterised by flavescens hymenophore surface with odontioid hymenophore, monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Xylodonporoides bears coriaceous basidiomata with a poroid hymenophore surface, monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Xylodonwumengshanensis is a distinct taxon by its grandinoid hymenophore surface, monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Sequences of ITS and nLSU rRNA markers of the studied samples were generated and phylogenetic analyses were performed using the Maximum Likelihood, Maximum Parsimony, and Bayesian Inference methods. The phylogram, based on the ITS+nLSU rDNA gene regions, included three genera within the Schizoporaceae as Fasciodontia, Lyomyces and Xylodon. The four new species were grouped into the genus Xylodon. The topology, based on the ITS sequences, revealed that Xylodoncremeoparinaceus was grouped closely with X.pruinosus, X.detriticus and X.ussuriensis. The taxon X.luteodontioides was sister to X.nesporii. The species X.poroides separated from X.pseudotropicus, while X.wumengshanensis was grouped with four taxa: X.patagonicus, X.radula, X.subtropicus and X.taiwanianus.

Detection and analysis of parvovirus B19 among blood donors in a regional blood center in Eastern China

Background and objectiveThe B19 virus is mainly transmitted through the respiratory tract; however, studies have shown that it can also be transmitted through blood transfusions or plasma products. This study investigated B19V antibodies, DNA, and gene typing in blood donors at a central blood station in China to evaluate the status of B19V infection. Materials and methodsA total of 7728 samples from Suzhou Blood Center were collected from July 2022 to April 2023. Samples were detected for the B19V DNA using real-time polymerase chain reaction. Furthermore, 893 selected samples were screened for the seroprevalence of B19V antibodies using enzyme-linked immunosorbent assay. The NS1-VP1u fragment of the B19V DNA-positive samples was amplified using nested PCR, and the sequences were determined. A B19V phylogenetic tree was constructed using neighborhood joint and maximum parsimony methods to discriminate genotypes using the NS1-VP1u sequences. ResultsThe percentages of IgG, IgM, and DNA were 19.4 %, 1.9 %, and 0.09 %, respectively. IgG positivity increased with age, and there was a significant difference among the blood groups. The IgG levels of repeat donors were greater than those of first-time donors. There were no apparent differences in the IgM levels in all the participants. Genotyping revealed that the B19 genotype was 1. ConclusionsThe prevalence of B19V antibodies and DNA was lower in these areas than in rest of China, indicating that the risk of B19V transmission via transfusion may be relatively low. However, during transfusion, particular attention should be paid to the B19V-susceptible populations, especially those in high-risk groups.

The wood-decaying fungal diversity unveiled by morphology and phylogeny in Ailaoshan Mountain, Yunnan, China

Seven field surveys were carried out in the Ailaoshan area of Yunnan Province, China, from 2016 to 2021, in which 2,814 specimens of wood-decaying fungal species were collected and identified based on morphology and phylogenetic analyses. One hundred and three wood-decaying species were identified, belonging to 64 genera, 29 families, and nine orders. Polyporales is the most abundant order in this area, accounting for 55.34% of all species, while Polyporaceae is the most abundant family, accounting for 18.45% of all species discovered in this area. The wood-decaying fungal checklist is provided according to the latest fungi classification system. Of these species, 11 are edible and therapeutic, four are forest pathogens, eight are threatened, and nine have just been reported from Ailaoshan Mountain. These species comprise 10.68%, 3.88%, 7.77%, and 8.74% of all species, respectively, while 71 species, or 68.93%, have unclear functional relationships. The sequences of the Internal Transcribed Spacer (ITS) gene region of the studied specimens were generated, and the phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. This study enriches the diversity of wood-decaying fungi in the Ailaoshan area. Also, it provides a theoretical basis for the future protection and utilization of wood-decaying fungal resources in this area.

Lycium barbarum Is a New Host of Botryosphaeria dothidea Associated with Soft Rot Disease in China.

Goji berry (Lycium barbarum) is a plant of the Solanaceae family that is cultivated in the Chinese provinces of Xinjiang, Ningxia, Gansu, and Qinghai, and its fruit is used as a traditional Chinese medicine (Yossa Nzeuwa et al. 2019). In July 2019, fruit rot was observed at an incidence of 20 to 25% on the Goji berry at a fruit market in Yinchuan, Ningxia, China. The fruit symptoms began as slightly shriveled areas on fruit peel, with noticeable softening of the infested portion of the tissue, followed by rotting and a sour odor. To isolate the pathogen, ten symptomatic tissues were randomly collected from different boxes, surface-sterilized for 30 s with 75% ethanol, followed by 0.1% mercuric chloride, then rinsed in sterile distilled water three times and plated onto PDA. The plates were incubated at 25°C in the dark for 7 days. Five purified fungal isolates from different fruit were obtained and single-spores. Emergent fungal colonies were white with 1 to 3 mm white margins and abundant aerial hyphae, 1 to 6 mm high, that became dark gray after 4 to 5 days. Conidia were hyaline, unicellular, fusiform, and measured 19.3 to 28.2 μm× 3.8 to 6.4 μm (n=50). All the morphological characteristics were consistent with Botryosphaeria spp. (Slippers et al. 2004). Five representative isolates, BJN1-BJN5, were selected for molecular identification. Total genomic DNA of the isolates was extracted with a Plant/Fungi DNA Isolation Kit. Translation elongation factor 1-alpha (EF1) gene and internal transcribed spacer (ITS) regions were amplified with primers EF1-728F/986R (Carbone and Kohn 1999) and ITS1/ITS4, respectively. The sequencing results of the five isolates were consistent, and those of the isolate BJN1 we deposited in the NCBI GeneBank database for EF1 (MK733274) and ITS (MK359291). A BLAST search of the GenBank database indicated that the EF1 and ITS sequences had 100% and 99% similarity, respectively, to B. dothidea ex-type strain (AY236898 and KF766151). A phylogenetic tree was constructed using maximum parsimony methods in MEGA11 and BJN1 isolate clustered with the reference sequence of B. dothidea. Pathogenicity tests were performed, inoculating healthy fruit with both mycelial plugs (7 days old) and conidial suspension (1 × 106 conidia/ml), repeated three times. Mycelial plugs of five isolates (BJN1-BJN5) growing on PDA with a colony diameter of 4 mm were placed on the sterilized surface of 20 Goji berry fruit. Sterile PDA plugs were placed on 12 healthy fruit as a control. In a second test, conidial suspensions of five isolates were sprayed on the surface of 20 healthy fruit and sterilized distilled water was used as a control. The inoculated fruits were maintained in an artificial climate chamber at 25°C and 80% to 85% relative humidity with a 12-h photoperiod for 7 days. The development of soft rot, similar to that observed on the original samples, was observed on inoculated fruit while control fruits remained asymptomatic. The pathogen was reisolated from infected fruit and confirmed as B. dothidea based on morphological characteristics and molecular sequences. To our knowledge, this is the first report of B. dothidea causing postharvest fruit rot of Goji berry, and this pathogen has been reported to cause fruit rot in Kiwifruit (Li et al. 2016) and Yellowhorn (Liu et al. 2018). This study provides information on a new postharvest fruit rot of Goji berry in China that has the potential to cause economic losses.

Morphological and molecular identification for four new wood-inhabiting species of Trechispora (Basidiomycota) from China.

Four new wood-inhabiting fungi, Trechisporaalbofarinosa, T.bisterigmata, T.pileata and T.wenshanensisspp. nov., are proposed based on a combination of morphological features and molecular evidence. Trechisporaalbofarinosa is characterized by the farinose basidiomata with flocculence hymenial surface, a monomitic hyphal system with clamped generative hyphae, and ellipsoid, warted basidiospores. Trechisporabisterigmata is characterized by the membranous basidiomata with odontioid hymenial surface, rhizomorphic sterile margin, barrelled basidia and subglobose to broad ellipsoid, smooth basidiospores. Trechisporapileata is characterized by the laterally contracted base, solitary or imbricate basidiomata, fan shaped pileus, radially striate-covered surface with appressed scales, odontioid hymenophore surface, and subglobose to broad ellipsoid, thin-walled, smooth basidiospores. Trechisporawenshanensis is characterized by a cottony basidiomata with a smooth hymenial surface, and ellipsoid, thin-walled, warted basidiospores. Sequences of ITS and LSU marker of the studied samples were generated, and phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic tree inferred from the ITS+nLSU sequences highlighted that four new species were grouped into the genus Trechispora.

Common Methods for Phylogenetic Tree Construction and Their Implementation in R.

A phylogenetic tree can reflect the evolutionary relationships between species or gene families, and they play a critical role in modern biological research. In this review, we summarize common methods for constructing phylogenetic trees, including distance methods, maximum parsimony, maximum likelihood, Bayesian inference, and tree-integration methods (supermatrix and supertree). Here we discuss the advantages, shortcomings, and applications of each method and offer relevant codes to construct phylogenetic trees from molecular data using packages and algorithms in R. This review aims to provide comprehensive guidance and reference for researchers seeking to construct phylogenetic trees while also promoting further development and innovation in this field. By offering a clear and concise overview of the different methods available, we hope to enable researchers to select the most appropriate approach for their specific research questions and datasets.

Morphological and molecular identification of Dissingia confusa based on the first record of the species in Türkiye

In the present study, specimens kept in Fungarium of Van Yüzüncü Yıl University - Türkiye (VANF) were evaluated based on morphological and molecular characters, and identified as Dissingia confusa as a new record from Eastern Türkiye. Ascocarp, hymenium, stipe, asco-spores, ascus and paraphyses were considered as diagnostic morphological characters and the nucleotide sequences of the large subunit (LSU) ribosomal DNA and the translation elonga-tion factor 1-alpha (TEF1-α) were referred as molecular data. A phylogenetic analysis was performed with Maximum parsimony and Bayesian inference methods. The DNA sequences of 15 (7 for TEF1-α) Dissingia and 27 (14 for TEF1-α) Helvella specimens downloaded from the GenBank database were included in the analysis to estimate phylogeny between the two close genera. Morphological evaluations and genetic evidences confirmed that Dissingia is phylogenetically separated from Helvella at genus level and helped to identify the studied specimens as D. confusa as a new record in Türkiye. Detailed description, colour images of fresh and dried ascomata, along with photographs of microscopic characters and the obtained phylogenetic trees are given.

Plastid phylogenomics and fossil evidence provide new insights into the evolutionary complexity of the ‘woody clade’ in Saxifragales

BackgroundThe “woody clade” in Saxifragales (WCS), encompassing four woody families (Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae), is a phylogenetically recalcitrant node in the angiosperm tree of life, as the interfamilial relationships of the WCS remain contentious. Based on a comprehensive sampling of WCS genera, this study aims to recover a robust maternal backbone phylogeny of the WCS by analyzing plastid genome (plastome) sequence data using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods, and to explore the possible causes of the phylogenetic recalcitrance with respect to deep relationships within the WCS, in combination with molecular and fossil evidence.ResultsAlthough the four WCS families were identically resolved as monophyletic, the MP analysis recovered different tree topologies for the relationships among Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae from the ML and BI phylogenies. The fossil-calibrated plastome phylogeny showed that the WCS underwent a rapid divergence of crown groups in the early Cretaceous (between 104.79 and 100.23 Ma), leading to the origin of the stem lineage ancestors of Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae within a very short time span (∼4.56 Ma). Compared with the tree topology recovered in a previous study based on nuclear genome data, cytonuclear discordance regarding the interfamilial relationships of the WCS was detected.ConclusionsMolecular and fossil evidence imply that the early divergence of the WCS might have experienced radiative diversification of crown groups, extensive extinctions at the genus and species levels around the Cretaceous/Paleocene boundary, and ancient hybridization. Such evolutionarily complex events may introduce biases in topological estimations within the WCS due to incomplete lineage sorting, cytonuclear discordance, and long-branch attraction, potentially impacting the accurate reconstruction of deep relationships.

First report of Dactylonectria pauciseptata causing root rot on Eucalyptus cinerea in China.

Eucalyptus cinerea is an evergreen tree in the Myrtaceae. It is native to southern and eastern New South Wales and northern and eastern Victoria, Australia. It was introduced into China in the 1980s (Silva et al. 2011). Because of its unique shape, flexible stems, and rapid growth characteristics, it is widely used in the pulp industry and in decorative materials such as flower bouquets. In July 2022, 5- to 10-year-old E. cinerea showing symptoms of dehydration, withering and yellowing leaves, were found in forests and nurseries in Kunming and Songming, China. More than 37% of the trees showed these symptoms at each location, and disease severity was about 30%. Sixty symptomatic plants were collected from five tree nurseries. Diseased roots with 2-cm-long lesions were soaked in 75% ethanol for 15 s, 0.1% mercuric chloride for 2 min, rinsed with sterilized water, and placed on potato dextrose agar (PDA) at 25℃ for 3 days. Thirty samples were plated, and 21 isolates (YJLGF01 to YJLGF21) obtained, 11 strains with similar colony morphology (including representative strains YJLGF03 to YJLGF05). Three isolates (YJLGF03 to YJLGF05) were obtained by single-spore purification. On PDA, the colonies were circular with fluffy white to light yellow mycelium; the underside was yellowish brown. Conidiophores were bifurcated, with macroconidia borne terminally. The macroconidia were cylindrical with rounded, blunt ends, yellow to transparent, 1 to 3 septate (22.5 to 47.6 × 4.5 to 7.1 µm); microconidia were 0 to 1 septate (12.5 to 19.6 × 4.7 to 6.4 µm). Chlamydospores were spherical, rosary-like, and light yellow. Morphological characteristics were consistent with published descriptions of Dactylonectria pauciseptata (Piperkova et al. 2017). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1- alpha (ef1-α) gene, and the beta-tubulin 2 (β-tub2) gene were amplified and sequenced (ITS accessions OR735053, OR735054, OR735055; β-tub2 accessios OR757447, OR757448, OR757449; ef1-α accessions OR757450, OR757451, OR757451) using published primers (White et al. 1990; Carbone et al. 1999). A phylogenetic tree was developed by Maximum Parsimony (MP) and Maximum Likelihood (ML) methods. These three isolates fell into the D. pauciseptata clade and were distinguished clearly from other species. Pathogenicity tests were performed using the same three isolates. Each isolate was cultured on PDA, and then subcultured in V8 juice broth on an orbital shaker at 180 RPM for 5 days. Conidia were collected by centrifugation at 6,000 RPM for 5 min, and then resuspended in sterilized distilled water (1×106 conidia/ml). Injured roots of one-year-old E. cinerea were soaked in the spore suspension for 1 h before being transplanted in sterile vermiculite. The plants were incubated at 25℃ with a 12 h photoperiod and 90% humidity. Five plants were inoculated as a group for each treatment and the entire experiment was completed three times. Among the inoculated plants, the incidence of disease development was 100%. A small sot appeared after 4 days, with a water-soaked lesion appearing and gradually expanding during days 5 to 7. After 10 days symptoms of root necrosis were similar to the those observed in the nursery, and aboveground plant parts had yellow, withering leaves and defoliation after 10 to 15 days. Control plants treated with sterile water showed no disease symptoms. The three strains were successfully reisolated from inoculated seedlings and confirmed them using DNA sequencing. No isolates were obtained from the control plants, thus fulfilling Koch's postulates. Dactylonectria pauciseptata was first reported from necrotic tissue of infected grape roots (Schroers et al. 2008). So far, it has been reported in Turkey, Canada, Brazil, Italy, and other countries (Erper et al. 2013; Úrbez-Torres et al. 2014; Santos et al. 2014). Based on our results, E. cinerea is a new host plant of D. pauciseptata in China. This disease is a threat to the nursery production of E. cinerea, potentially leading to a reduction in yields and economic losses.

First Report of Golovinomyces bolayi Causing Powdery Mildew on Lettuce in Mexico.

In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish powdery masses on leaves. Disease incidence was about 100% and signs covered up to 40% of leaf surface. Mycelium was amphigenous forming white patches. Hyphal appressoria were indistinct or nipple-shaped and solitary. Conidiophores (n= 30) were hyaline, erect, arising from the upper surface of hyphal mother cells or lateral, and of 90 to 201 μm long. Foot cells were cylindrical, of 49 to 92 × 10-15 μm, followed by 1-3 shorter cells, and forming conidia in chains. Conidia (n= 100) were hyaline, ellipsoid-ovoid, doliiform-subcylindrical, 27 to 40 × 14 to 20 μm. Conidial germination belonging to the Euoidium type. Chasmothecia were not observed. The morphological characters were consistent with those of Golovinomyces bolayi (Braun et al. 2019). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH451. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 506 bp sequence had 100% identity to those of G. bolayi (LC417109 and LC417106). Phylogenetic analyses using the Maximum Likelihood and Maximum Parsimony methods were performed and confirmed the results obtained in the morphological analysis. The isolate UACH451 grouped in a clade with isolates of G. bolayi. The ITS sequence was deposited in GenBank under accession number OR467546. Pathogenicity was confirmed by gently dusting conidia onto ten leaves of healthy lettuce plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 70%. All inoculated leaves developed similar symptoms to the original observation after 10 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. Based on morphological data and phylogenetic analysis, the fungus was identified as G. bolayi. This pathogen has been previously reported causing powdery mildew on lettuce in Argentina, Canada, Chile, Ecuador, Peru, USA and Venezuela (Braun et al. 2019; Mieslerová et al. 2020). To our knowledge, this is the first report of G. bolayi causing powdery mildew on lettuce in Mexico.

Species diversity and taxonomy of Vararia (Russulales, Basidiomycota) with descriptions of six species from Southwestern China.

Vararia is a species-rich genus in the family Peniophoraceae and has been shown to be polyphyletic. In this study, sequences of ITS and LSU rRNA markers of the studied samples were generated and phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. Seventeen lineages including six new species from China, i.e., V.fissurata, V.lincangensis, V.punctata, V.isabellina, V.sinensis, and V.yaoshanensis were recognized, in which V.fissurata is characterized by the brittle basidiomata with pruinose and cracking hymenophore having white to olivaceous buff hymenial surface, the clamped generative hyphae, presence of the two types gloeocystidia; V.lincangensis is characterized by the simple-septa generative hyphae, and thick-walled skeletal hyphae, and ellipsoid basidiospores; V.punctata is delimited by its thin to slightly thick-walled generative hyphae, and thick-walled skeletal hyphae, present thick-walled, clavate to cylindrical gloeocystidia; V.isabellina is characterized by having the cream to isabelline to slightly brown hymenial surface, thin to slightly thick-walled generative hyphae, and sub-fusiform to navicular basidiospores; V.sinensis is distinguishable by its white to slightly pink hymenial surface, thick-walled skeletal hyphae, and sub-fusiform to navicular basidiospores; V.yaoshanensis is characterized by cream to pinkish buff to cinnamon-buff hymenial surface, slightly thick-walled generative hyphae, the presence of two types gloeocystidia, and slightly thick-walled, ellipsoid basidiospores. Phylogram based on the ITS+nLSU rDNA gene regions included nine genera within the family Peniophoraceae as Amylostereum, Asterostroma, Baltazaria, Dichostereum, Michenera, Peniophora, Scytinostroma and Vararia, in which the six new wood-inhabiting fungi species were grouped into genus Vararia. The phylogenetic tree inferred from the combined ITS and LSU tree sequences highlighted that V.fissurata was found to be the sister to V.ellipsospora with strong supports. Additionally, V.lincangensis was clustered with V.fragilis. Furthermore, V.punctata was retrieved as a sister to V.ambigua. Moreover, V.sinensis was grouped with five taxa as V.breviphysa, V.pirispora, V.fusispora, V.abortiphysa and V.insolita. The new species V.isabellina formed a monophyletic lineage, in which it was then grouped closely with V.daweishanensis, and V.gracilispora. In addition, V.yaoshanensis was found to be the sister to V.gallica with strong supports. The present results increased the knowledge of Vararia species diversity and taxonomy of corticioid fungi in China. An identification key to 17 species of Vararia in China is provided.

Phylogenetic and Taxonomic Analyses of Five New Wood-Inhabiting Fungi of Botryobasidium, Coltricia and Coltriciella (Basidiomycota) from China.

In this present study, five new wood-inhabiting fungal taxa, Botryobasidium gossypirubiginosum, Botryobasidium incanum, Botryobasidium yunnanense, Coltricia zixishanensis, and Coltriciella yunnanensis are proposed. Botryobasidium gossypirubiginosum is distinguished by its slightly rubiginous hymenial surface, monomitic hyphal system, which branches at right angles, and subglobose, smooth basidiospores (14-17.5 × 13-15.5 µm); B. incanum is characterized by its white to incanus basidiomata having a hypochnoid hymenial surface, and ellipsoid, smooth basidiospores (6.5-8.5 × 3.5-5 µm); B. yunnanense is characterized by its buff to slightly yellowish hymenial surface, monomitic hyphal system, and broadly ellipsoid to globose, smooth, thick-walled basidiospores (11.5-14.5 × 9.5-10.5 µm); Coltricia zixishanensis differs in its rust brown pileal surface, and ellipsoid, thick-walled basidiospores (5-6.5 × 4-4.5 µm). Coltriciella yunnanensis is distinguished by its tiny pilei, short stipe, and navicular, verrucose basidiospores (10.5-12.5 × 6-7 µm). Sequences of ITS and nLSU genes were used for phylogenetic analyses using the maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic results inferred from ITS sequences revealed that B. gossypirubiginosum was closely related to B. robustius; the species B. incanum was grouped with B. vagum; B. yunnanense was related to B. indicum. The species C. zixishanensis was grouped with C. confluens and C. perennis. ITS sequences revealed that C. zixishanensis was grouped into the genus Coltriciella, in which it was grouped with Co. globosa and Co. pseudodependens.

DNA Barcoding of Invasive Terrestrial Plant Species in India.

Invasive plants are known to cause biodiversity loss and pose a major risk to human health and environment. Identification of invasive plants and distinguishing them from native species has been relied on morphological examination. Stringent requirement of floral characters and decreasing number of expert taxonomists are making conventional morphology-based identification system tedious and resource-intensive. DNA barcoding may help in quick identification of invasive species if distinct sequence divergence pattern at various taxonomic levels is observed. The present work evaluates the utility of four molecular markers; rbcL, matK, their combination (rbcL + matK), and psbA-trnH for identification of 37 invasive plant species from India and also in distinguishing them from 97 native species. A psbA-trnH locus was found to be of restricted utility in this work as it was represented by the members of a single family. A hierarchical increase in K2P mean divergence across different taxonomic levels was found to be the maximum for matK alone followed by rbcL + matK and rbcL alone, respectively. NJ clustering analysis, however, confirmed the suitability of combined locus (rbcL + matK) over individual rbcL and matK as the DNA barcode. RbcL showed the lowest resolution power among the three markers studied. MatK exhibited much better performance compared to rbcL alone in identifying most of the species accurately although it failed to show monophyly of genus Dinebra. Two families; Asteraceae and Poaceae, remained polyphyletic in the trees constructed by all three markers. Combined locus (rbcL + matK) was found to be the most suitable marker as it raised the resolution power of both the markers and could identify more than 90% of genera correctly. Phylogenetic tree constructed by Maximum-Parsimony method using combined locus as a molecular marker exhibited the best resolution, thus, supporting the significance of two-locus combination of rbcL + matK for barcoding invasive plant species from India. Present study contributes to the global barcode data of invasive plant species by adding fifty-one new sequences to it. Effective barcoding of additional number of native as well as invasive plant species from India is possible using this dual locus if it is combined with one or more new molecular plastid markers. Expansion of barcode database with a focus on barcode performance optimisation to improve discrimination ability at species level can be undertaken in future.