Mixed-species forests are often more productive than monocultures because of a lower niche overlap and higher taxonomic and functional diversity of soil microbial communities. Males and females of dioecious plants have sex-specific adaptations to diverse habitats. The potential of using sexual differences in establishing more diverse poplar plantations has not been explored in degraded areas. We conducted a series of greenhouse and field experiments to investigate how belowground competition, soil microbial communities and seasonal variation nitrogen content differ among female, male and mixed-sex Populus cathayana plantations. In the greenhouse experiment, female neighbors suppressed the growth of males under optimal nitrogen conditions. However, male neighbors enhanced δ¹⁵N of females under inter-sexual competition. In the field, the root length density, root area density and biomass of fine roots were lower in female plantations than in male or mixed-sex plantations. Bacterial networks of female, male and mixed-sex plantations were characterized by different composition of hub nodes, including connectors, module and network hubs. The sex composition of plantations altered bacterial and fungal community structures according to Bray-Curtis distances, with 44% and 65% of variance explained by the root biomass, respectively. The total soil nitrogen content of mixed-sex plantation was higher than that in female plantation in spring and summer. The mixed-sex plantation also had a higher β-1,4-N-acetyl-glucosaminidase activity in summer and a higher nitrification rate in autumn than the other two plantations. The seasonal soil N content, nitrification rate and root distribution traits demonstrated spatiotemporal niche separation in the mixed-sex plantation. We argue that a strong female-female competition and limited nitrogen content could strongly impede plant growth and reduce the resistance of monosex plantations to climate change and the mixed-sex plantations constitutes a promising way to restore degraded land.

The four experiments as following:

Experiment 1. Plant-plant competition under different N levels

To investigate the effects of the sex identity on the growth of neighboring plants, one-year-old cuttings were sampled from male and female plantations and then cultivated in pots in a naturally-lit greenhouse at the Hangzhou Normal University (30°17′N, 120°0′E). Thereafter, cultivated plants of a similar size were randomly chosen and transplanted in plastic pots (diameter 36 cm, height 27 cm). Each pot was filled with sand, vermiculite, and perlite (1:1:1). Each pot contained two rooted cuttings, either male-male, female-female, or male-female. Half of the plants were subject to an optimal N treatment (control) and half of the plants to a limited N treatment (N-). Each treatment had five replicates. The N levels were controlled with the Long Ashton solution (supplementary material for detailed information of the Long Ashton solution). After growing for 14 weeks, the plants were harvested, separated between biomass fractions and dried at 75 °C for 72 h, after which the dry mass of each fraction was measured. The root N content was measured by semimicro Kjeldahl method (Wu et al. 2021). The dried leaf samples (n = 3) were used to determine natural δ13C and δ15N with an isotope ratio mass spectrometer (DELTA V Advantage, Thermo Fisher Scientific, Inc., USA). The Pee Dee Belemnite and N2 were used as standards to calculate δ13C and δ15N, respectively.

Experiment 2. Variation in root features along soil vertical profile in different plantations

The root architecture and distribution along the soil profile was explored among the three plantation types. Twenty 10 m × 8 m plots were set up in the male and female plantations, and 17 plots in the mixed-sex plantation. A soil core sampler (diameter 70 mm) was used to sample soil and roots at soil depths of 0-10 cm, 10-20 cm, 20-30 cm, 30-40 cm, 40-50 cm and 50-60 cm. The soil cores were sampled at a distance of 1 m from each plant. All soil samples were immediately put in plastic bags and taken to the lab. Roots in each sample were carefully separated from soil and washed by deionized water. Coarse roots (diameter ≥ 2 mm) of each sample were separated and dried at 75 °C for 72 h to measure their biomass. Fine roots (diameter < 2 mm) were immediately scanned, and the Win-Rhizo system (Régent instrument Inc., Québec, Canada) was used to measure the total fine root length. After scanning, fine roots of each sample were separated into two fractions based on the diameter 1-2 mm or <1 mm. Both root fractions were dried at 75 °C for 72 h and their dry mass was estimated. Nitrogen contents of coarse and fine roots were measured by semimicro Kjeldahl method (Wu et al. 2021).

Experiment 3. Seasonal dynamics in phospholipid fatty acids (PLFAs) and N cycle

This experiment aimed to explore seasonal dynamics in the microbial biomass, extracellular enzyme activities and net nitrification rate among the three plantations. Sampling was conducted in April, July and October, 2021 from the female, male and mixed plantations. As the Experiment 2 demonstrated that the bulk of the root biomass was located in 0-10 and 10-20 cm soil layers (Fig. 1), the samples were taken from these soil layers. From each stand, ten replicate samples were collection.

The soil microbial biomass was assessed by PLFA analyses according to Schindlbacher et al. (2011). All measurements included a blank sample with the internal standard (peak 19:0, nonadecanoic fatty acid). The PLFA biomarkers including iso-, anteiso-(i13:0, i14:0, i15:0, a15:0, i16:0, i17:0 and a17:0) and 10Me-(10Me17:0 and 10Me18:0) branched PLFAs were attributed to Gram-positive bacteria (G+ bacteria) (Luo et al. 2022); monounsaturated and cyclopropyl PLFAs including 17:1ω8c, 18:1ω5c, 18:1ω7c, cy17:0 and cy19:0 were attributed to Gram-negative bacteria (G– bacteria) (Luo et al. 2022); and 16:1ω5c, 18:1ω9c and 18:3ω6c were used for fungi (Joergensen 2022; Willers et al. 2015). The PLFAs including 14:0, 16:0, 17:0, 18:0 and 20:0 were used as non-specific markers present in all microorganisms (Joergensen 2022). The sum of all selected PLFAs was used to characterize the total microbial biomass. The sum of G+ and G− bacterial PLFAs was used to characterize the bacterial biomass. The G+ : G– ratio and fungal : bacterial (F:B) ratio were separately calculated as the ratio of G+ PLFAs to G– PLFAs and the ratio of fungal PLFAs to bacterial PLFAs. The activities of three hydrolytic enzymes, including β-1,4-glucosidase (BG), β-1,4-N-acetyl-glucosaminidase (NAG), and acid phosphatase,  were measured following the methods in Jing et al. (2020). The net nitrification rate was assessed by quantifying changes in NO3- concentration before and after a 10-day aerobic incubation (Durán et al. 2014). Soil-water suspension (1:2.5 w/v) was used for pH determination.

Experiment 4. Microbiota assembly in different sex plantations

In the beginning of August, 2020, soil samples were taken from the depths 0-20 cm, 20-40 cm and 40-60 cm to explore their chemical and microbial traits of different soil layers. Five plots (25 m × 15 m) were established in each plantation. In each plot, three soil samples were collected at each soil depth and pooled to form a composite sample. Five soil samples at each depth were also collected in the degraded area without any artificial management practices. A total of 45 samples were collected. After sampling, the soil samples were kept on ice before transportation to the laboratory, where a part was kept at -80 °C until DNA extraction and a part was air-dried for chemical analyses, including contents of soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP) and available phosphorus (AP) contents. The remaining part of soil was kept at 4 °C to measure the contents of NH4+, NO3-, dissolved organic carbon (DOC), dissolved organic nitrogen (DON), microbial carbon biomass (MBC) and microbial nitrogen biomass (MBN). SOM was determined by the potassium dichromate external heating method. Soil AP was determined by the molybdenum-antimony colorimetric method after extraction with sodium bicarbonate (Guo et al. 2021). TN was measured by the Kjeldahl method and TP by Ultraviolet spectrophotometer. Fresh soil (10 g) was extracted with 2 M KCl, and the extractions were used to determine NH4+ and NO3- contents. The contents of DOC and DON were estimated according to Luo et al. (2022). MBC and MBN were determined by the chloroform fumigation extraction method (Vance et al. 1987).