As a highly ecologically adaptable photosynthetic autotrophic organism, green algae have a wide range of habitats, not only distributed in conventional freshwater bodies, marine ecosystems, and terrestrial habitats, but also able to tenaciously colonize extremely harsh environments such as deserts, alpine snowfields, and polar regions (Sciuto et al. 2023). In recent years, with the widespread application and increasing depth of investigation of molecular phylogenetic techniques, many new evolutionary clades and taxonomic units in the Chlorophyta phylum have been continuously discovered and reported (Prochazkova et al. 2019; Andreoli et al. 1998; Sciuto et al. 2015; Gálvez et al. 2021; Trentin et al. 2022). However, this only reveals the tip of the iceberg of green algae biodiversity, and there is still enormous exploration space for its overall species richness and phylogenetic lineage. This study focuses on the unique ecologically fragile area of the Qinghai Tibet Plateau, where two green algae were successfully isolated from saltwater lakes and freshwater rivers. Given the limitations of traditional morphological identification in the classification of green algae, we adopted a multi gene joint analysis strategy to define the species of the isolates: we selected the nuclear 18S rRNA gene as a conserved molecular marker for higher-order taxa, and combined it with the nuclear core barcode gene – the nuclear transcription spacer (ITS) region, which has higher variation resolution, for sequencing; At the same time, in order to provide more detailed evidence from the perspective of functional and structural evolution, we also conducted in-depth annotation and comparison of the secondary structure of ITS2. Based on the comprehensive molecular evidence presented above, we have classified these two algal isolates as belonging to the genus Pleurastrum. Looking back at the classification history of this genus, Pleurastrum was first established by scholar Chodat as early as 1894 (Chodat 1894); Despite being over a hundred years old, the species diversity of this genus is still far from sufficient. According to the records of the Algaebase database (Guiry and Guiry 2023), currently this genus only covers 8 valid species worldwide. From the perspective of phylogenetic status, the genus Pleurastrum is the only member of the Pleurasraceae family in the Chlorophyta phylum, Chlorophyta class, and Chlorophyta order. The phenomenon of “single genus and single family” and the extremely low species description rate further highlight the research gap that urgently needs to be explored in terms of species diversity and evolutionary mechanisms of this genus.

For a long time, there has been a relative lack of research literature and reports on the genus Pleurastrum, which makes it extremely rare and easily overlooked in field sampling and taxonomic identification. However, as a unique group within the Chlorophyta phylum, the genus Pleurastrum exhibits strong ecological adaptability and a wide range of habitats. Its species not only inhabit conventional aquatic ecosystems, but are also widely distributed in diverse terrestrial habitats (Chodat 1894; Sciuto et al., 2023). At the morphological level, this genus exhibits extremely high phenotypic plasticity, with its body structure spanning a wide lineage from single-cell spheroids to complex branching filaments (Chodat 1894; Visher 1933; Sciuto et al., 2023). Looking back at the taxonomic history of this genus, since the first establishment of the Pleurastrum model species P. insigne, many scholars have mainly relied on phenotypic features to define and describe new species within the genus (Visher 1933; Deason and Bold 1960; Tupa 1974; Sluiman and Gärtner 1990). Although early cultivation observations and comparative morphological studies have to some extent expanded our understanding of the species diversity of Pleurastrum, relying solely on morphological standards has repeatedly caused confusion and controversy in taxonomy (Deason and Bold 1960; Tupa 1974; Sluiman and Gärtner 1990; Lukešová 1991). This limitation mainly stems from two aspects: on the one hand, some key morphological characteristics of green algae often only appear briefly at specific developmental stages of the cell growth cycle, and due to the extremely short observation window, it is easy to make misjudgments due to omissions; On the other hand, some of the morphological features used as identification criteria are actually convergent or conservative features widely present within the Chlorophyta phylum, lacking sufficient evolutionary specificity and difficult to use as reliable criteria for defining higher-order taxa or distinguishing closely related species. Compared to the limitations of traditional morphology, the phylogenetic analysis that integrates molecular markers with the secondary structure of the internal transcription spacer 2 (ITS2) can provide a more objective and comprehensive perspective on the dual dimensions of genome evolution and RNA structure conservation, thus revealing its hidden species diversity more realistically. In view of this, this study established a multidimensional identification strategy that integrates morphology and molecules: it retains the intuitive comparison of traditional morphological structures and deeply introduces molecular feature analysis based on ITS2 secondary structures; Meanwhile, a phylogenetic tree was constructed by combining multiple gene sequences such as 18SrDNA and ITS. The implementation of this comprehensive research framework aims to break through the bottleneck of traditional classification and provide a solid scientific basis for more accurate analysis and clarification of the species diversity and systematic evolutionary status of the genus Pleurastrum in China.

This study aims to combine morphological and molecular phylogenetic analysis of 18S rDNA and ITS sequences, and compare the ITS2 secondary structures of closely related species. Proposed a new species of Pleurastrum.

The genus Pleurastrum was established by Chodat in 1894. Although new species have been continuously reported in recent years (Sciuto et al. 2023; Kim et al. 2025), according to AlgaeBase records, this genus currently only contains 8 species. As a monotypic genus of the Pleurasraceae family, its species diversity urgently needs to be further explored; Throughout the entire Chlorophyta phylum, even with the emergence of new evolutionary clades and species (Andreoli et al. 1998; Sciuto et al. 2015; Prochazkova et al. 2019; Gálvez et al. 2021; Trentin et al. 2022), Its overall biodiversity is still far from fully revealed. Looking back at the taxonomic history of this genus, since the description of the Pleurastrum type species P. insigne, species definition has long relied on morphological features (Visher 1933), but a single phenotype criterion can easily lead to taxonomic confusion (Deason and Bold 1960). The introduction of molecular markers and phylogenetic analysis of ITS2 secondary structure can effectively compensate for this deficiency. Therefore, this study adopts a strategy that combines morphology and molecules, based on multi gene sequences such as nuclear SSU rDNA and ITS, supplemented by ITS2 secondary structural features, to more accurately analyze the species diversity of the Pleurastrum genus in China.

In the phylogenetic analysis, a Bayesian tree constructed based on 18S rDNA showed that multiple Pleurastrum species, including the isolated isolate in this study, clustered into a highly supported monophyletic branch, which was significantly distinguished from genera such as Protosiphon, Spongiochloris, and Spongioarcinopsis (Fig. 2). Therefore, these isolates were classified under the genus Pleurastrum. The phylogenetic tree constructed based on ITS sequences also confirmed this attribution (Fig. 3A), and showed high ML support and Bayesian posterior probability at the intra species, inter species, and closely related genus levels. At the intra genus level, P. gracilis has formed an independent branch: although it is most closely related to P. amblystomatis, P. aquaticum, and P. ellipsoideum, they have clear boundaries with each other. The ITS Bayesian tree also supports the phylogenetic independence of P. gracilis (Fig. 3A). In addition, ITS2 secondary structure analysis showed that the four main helix regions (Helix) of these two isolates had at least one compensatory base change (CBC) with other species within the genus, and had a unique branching structure on Helix-1. Given that the emergence of CBC is often regarded as an important indicator for defining species or genus level taxonomic units (Vanormelingen et al. 2007; Bock et al. 2011a, b), the differences in the above secondary structures further confirm the independent species status of P. gracilis.

In terms of morphology, P. gracilis exhibits significant differences: its cell wall is thinner than that of P. insigne and P. microstigmatum, which have different wall thicknesses; At the same time, this species exhibits the phenomenon of multiple ellipsoidal cells connected end-to-end, which has not been reported in other species within the genus. In terms of ecological habits, species of the genus Pleurastrum are widely distributed in aquatic and terrestrial ecosystems (Chodat 1894; Sciuto et al. 2023), while the isolates in this study were collected from both low altitude freshwater rivers and high-altitude saline lakes. This indicates that the species has a broad ecological range and is tolerant to extreme high-altitude environments. Previous studies have shown that green algae can thrive in high-altitude extreme habitats (Andersen 1992; Malcata et al. 2018), and this characteristic of P. gracilis endows it with potential ecological and economic research value.

Based on phylogenetic analysis, morphological features, and ITS2 secondary structure comparison, P. gracilis is proposed as a new Pleurastrum. As the only genus within its family, this genus covers a limited number of species, and there should be increasing research on the diversity of this genus and its family.