Four New Fungal Species Revealed as Genomes Expose Hidden Diversity in Leaf-Spot Genus Phyllosticta

Researchers surveying diseased and dead leaves in the tropical and subtropical forests of southern China have described four previously unknown species of Phyllosticta, a fungal genus whose members include some of the world's most economically damaging plant pathogens. Phyllosticta is a genus with a long and tangled taxonomic history. Established by Persoon in 1818, it has been shuffled between families and orders over the decades: Seaver placed it in Phyllostictaceae in 1922, later studies moved it into Botryosphaeriaceae within the Botryosphaeriales, and phylogenetic work eventually restored it to Phyllostictaceae as a sister clade to the botryosphaeriaceous fungi.

Today, Index Fungorum lists 3,228 names under the genus, a figure that reflects both genuine diversity and centuries of morphological ambiguity. The genus matters far beyond taxonomy: P. citricarpa causes citrus black spot, P. ampelicida drives grape black rot in North America, and P. capitalensis acts as a widespread endophyte with antagonistic activity against other pathogens, deploying melanized appressoria to penetrate host tissue. The challenge for mycologists is that Phyllosticta species look frustratingly alike.

Sexual morphs produce erumpent, uniloculate ascomata with pseudoparaphyses, clavate to ellipsoidal asci, and aseptate hyaline ascospores often capped with mucilage. Asexual morphs generate hyaline, aseptate conidia wrapped in a mucilaginous sheath and bearing variable apical appendages, while spermatia are cylindrical to dumbbell-shaped with terminal guttules. Because these traits overlap extensively across species, researchers have come to rely on multi-locus phylogenetic analysis for reliable species delimitation.

A 2020 study proposed six species complexes within the genus—centered on P. capitalensis, P. concentrica, P. cruenta, P. owaniana, P. rhodorae, and P. vaccinii—based on a five-locus dataset, and additional taxa have continued to emerge since. In the new work, the team collected 32 specimens between 2023 and 2024, excising 5 by 5 millimeter fragments from the margins of leaf lesions and surface-sterilizing them through sequential washes in 75% ethanol and 5% sodium hypochlorite before plating on potato dextrose agar at 25 degrees Celsius. Pure cultures were established from actively growing hyphal tips, and colony morphology was documented at 7 and 14 days.

Micromorphological features were measured with 30 replicate measurements per structural character, and holotype specimens were deposited in the Herbarium of Plant Pathology at Shandong Agricultural University, with living cultures preserved at the China General Microbiological Culture Collection Center and the Shandong Agricultural University Culture Collection. For the phylogenetic backbone, the researchers amplified and sequenced five genetic regions: the internal transcribed spacer including the 5.8S rRNA gene, the large subunit of the rRNA gene, and the protein-coding loci tef1, act, and gpdh. The final concatenated alignment of 159 isolates spanned 2,911 characters, of which 981 were parsimony-informative.

Maximum likelihood analysis, run with 1,000 rapid bootstrap replicates under the GTR substitution model selected via the Bayesian information criterion, and Bayesian inference produced topologically congruent trees. The analysis assigned the 159 isolates to 103 species and placed the four new taxa in well-supported clades: P. clematidea, P. decaspermi, P. pittosporicola, and P. morellae, each named for its host plant. The nucleotide differences separating the new species from their closest relatives are substantial.

P. decaspermi, isolated from diseased leaves of Decaspermum montanum in Hainan's Diaoluoshan National Forest Park, differs from P. guangdongensis by 68 nucleotides across the five loci, from P. mangiferae by 75, and from P. morellae by 114. P. morellae, from Morella rubra in Jianfengling National Forest Park, differs from P. guangdongensis by 36 nucleotides and from P. mangiferae by 40. P. clematidea, collected on Clematis vitalba in Yunnan, differs from P. concentrica by 55 nucleotides, while P. pittosporicola, from Pittosporum illicioides on Fengming Mountain at 1,926 meters elevation, differs from P. elliptica by 55.

Morphology corroborates the genetic splits: the new species show distinct conidial dimensions, with P. pittosporicola producing conidia of 7 to 9 by 4 to 6 micrometers, narrower and shorter than those of its relatives, and each occupies a different host. Notably, no sexual morph was observed for any of the four species; all were characterized from their asexual, pycnidial forms. The genomic component of the study addressed a striking resource gap: as of 6 October 2025, only seven Phyllosticta species had publicly available genomes on NCBI.

The team sequenced genomes of ten isolates, including the new species, and combined them with the seven published genomes, using Diplodia corticola as an outgroup. Genome assembly was performed with SPAdes, and annotation integrated repeat masking, non-coding RNA detection, and gene prediction combining RNA-seq-based, ab initio, and homology-based approaches. OrthoFinder analysis identified 6,364 clusters of orthologous proteins, and a maximum likelihood phylogenomic tree built from 5,399 of these clusters recovered distinct clusters corresponding to the P. concentrica and P. capitalensis species complexes, mirroring the five-gene topology while adding genome-scale resolution.

Gene family analysis revealed a mixture of conservation and divergence. Single-copy orthologous genes make up a large proportion of homologs, indicating a conserved core genetic framework, while each strain carries unique gene families that may underpin ecological adaptation—particularly in host interaction and secondary metabolite synthesis. UpSet analysis of six strains highlighted gene family modules shared across species, likely tied to core metabolism, alongside lineage-specific expansions potentially acquired during adaptation to particular host plants and environmental conditions.

Kyoto Encyclopedia of Genes and Genomes pathway annotation of ten species added a functional dimension. P. fujianensis showed relatively low activity in cysteine and methionine metabolism, suggesting conservation in sulfur-containing amino acid processing. P. saprophytica displayed elevated activity in protein processing in the endoplasmic reticulum, a pathway that mediates folding and modification of secreted hydrolytic enzymes such as cellulases and ligninases—consistent with its saprophytic lifestyle of decomposing dead plant tissue, a pattern the authors note parallels findings in Trichoderma reesei.

P. rhododendri stood out for heightened amino sugar and nucleotide sugar metabolism, which supplies precursors for chitin and glycoprotein synthesis in fungal cell walls, potentially aiding host interaction in a manner comparable to patterns documented in Colletotrichum gloeosporioides. These species-specific metabolic profiles, the researchers argue, reflect evolutionary and environmental pressures shaping distinct ecological strategies within a single genus. The study also refined the species-complex framework, expanding the P. capitalensis complex to 34 species and the P. concentrica complex to 34, while the P. cruenta complex holds 22, P. owaniana 6, P. rhodorae 3, and P. vaccinii 2—100 accepted species in total.

With the USDA Fungal Database documenting more than 7,500 records of Phyllosticta-host associations, and P. capitalensis alone exceeding 400 host records as a globally distributed generalist endophyte and weak pathogen, the genus's ecological versatility is clear. The authors caution that morphological overlap among species remains the central obstacle to accurate identification, reinforcing the necessity of multi-locus phylogeny and phylogenomics—a lesson echoed in studies of Diaporthe, Fusarium, and Bipolaris.

Subject of Research: Taxonomic description and genomic characterization of four new Phyllosticta fungal species from China

Article Title: Molecular and morphological characterization of four new Phyllosticta species ( Botryosphaeriales , Phyllostictaceae ): Genomic insights into evolutionary dynamics and metabolic adaptation

Article References: Zhang, M.-Y., Zhang, Z.-X., Wang, Y.-L., Zhang, X.-G., & Li, Z. (2025). Molecular and morphological characterization of four new Phyllosticta species (Botryosphaeriales, Phyllostictaceae): Genomic insights into evolutionary dynamics and metabolic adaptation. IMA Fungus, 16, Article e168055. https://doi.org/10.3897/imafungus.16.168055

Image Credits: AI Generated

DOI: 10.3897/imafungus.16.168055

Keywords: Phyllosticta, fungi, taxonomy, phylogenomics, new species, plant pathology, genomics, KEGG, endophytes, China, Botryosphaeriales, species complexes