Eukaryotic algae, as the primary group of photosynthetic autotrophs, exert a significant influence on the development and functions of biological crusts in dryland ecosystems. Despite their importance, there are substantial knowledge gaps on the composition of eukaryotic algal communities and their effects on the distributions of bacteria and fungi in dryland soils.

This study examined the eukaryotic algal community along a successional sequence of biocrusts in the Gurbantunggut desert, while also investigating their patterns of co-occurrence with bacteria and fungi through high-throughput sequencing and bioinformatic analyses.

The results showed that nitrogen and phosphorus levels played a crucial role in the regulation of changes in the abundance and composition of the algal community. In particular, changes in the structure of the algal community arise primarily from fluctuations in the main species, rather than from loss and appearance of species during the biocrust succession. The accumulation of nitrogen and phosphorus in the biocrust led to increases in the relative abundance of algal species in the Chlorophyta. The results also indicated that eukaryotic algae played an important role in affecting bacterial and fungal communities and significantly improved the stability of the microbial community, reflected by the robustness of co-occurrence networks. The network analysis further indicated that eukaryotic algae affected the stability of microbial co-occurrence networks either by acting as keystone taxa or associating with the keystone bacterial and fungal taxa.

These findings reveal a clear mechanism by which soil nitrogen and phosphorus levels affected the composition of eukaryotic algae communities and further regulated bacterial and fungal communities during biocrust development, providing valuable information on the development and functional execution of biocrusts in dryland ecosystems.

The soil physicochemical factors were determined according to standard methods (Tan 1995). Briefly, nitrate and ammonium were extracted with 1 M KCl and determined using an Automated Discrete Analyzer (AQ2+, SEAL Analytical Inc., England). Soil organic carbon (SOC) was determined using the K₂Cr₂O₇ method. Total nitrogen (TN) was determined using the kjeldah method, total phosphorus (TP) using the NaOH Melting-Mo Te Sc Colorimetry method, and available phosphorus (AP) using the 0.5 mol/L NaHCO₃ Leaching-Mo Te Sc Colorimetry method. The soil pH was determined using a soil-to-water ratio of 1:2.5 using a pH meter (Sartorius PB-10, Germany).

Genomic DNA was extracted from 0.25 g of soil using the SPINeasy Soil DNA Kit (MP Biomedicals, USA) according to the manufacturer’s instructions. The quantity and purity of the extracted DNA were evaluated using the NanoDrop ND2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). To characterize the abundance of bacteria, fungi and algae in bare sand and biocrust soils, the 18S rRNA gene for microalgae, the internal transcribed spacer (ITS) region of the fungal and the 16S bacterial gene were amplified with the primer set V8f/1510R (Bradley, Pinto & Guest 2016), gITS7/ITS4 (Ihrmark et al. 2012) and 515F/806R (Bates et al. 2011) respectively on a QuantStudio3 Real-Time PCR system (ThermoFisher Scientific, MA, USA). PCR reactions were carried out in a 20 μl volume system containing 1 μl DNA (~10 ng), 1 μl of 10 μM each primer, and 10 μl of SYBR Green Premix (Takara, Japan). The PCR cycle conditions were 95 ° C for 5 min and followed by 35 cycles of 30 s at 95°C, 20 s at 58 ° C (for 16S rRNA), 30 s at 56 ° C (for ITS), and 30 s at 58 ° C (for 18S rRNA), 30 s at 72 ° C. Fluorescence data were collected at the end of the annealing stage. A standard curve was generated using a serial dilution of plasmids containing target gene fragments to calculate the unknown samples.