Sampling area
Two river wetlands – Zhangcun River and Wenquan River of Qingdao City, Shandong Province, China were selected as the sampling area for the present study (the climatic factors see Table 1; Fig. 1; Fig. 2). Zhangcun River is located in the core residential region of Laoshao District, while Wenquan River is located in the sparsely populated suburb of Jimo District. The data of population density of Qingdao city at 10 a.m. on weekdays was obtained from Gaode Map (AutoNavi Software Co., Ltd., https://www.amap.com/) by API (Application Programming Interface). Gaode is one of the most famous navigation and location-based services provider in China. The population heatmap was then generated through Kernel Density method using ArcGIS 10.3 software(Environmental Systems Research Institute, California). The population heatmap shows that the population density was higher around the sampling section of Zhangcun River than that of Wenquan River (Fig. 1). Google imagery with 1 m spatial resolution of the two river sampling areas was downloaded from the Google Earth service using the BIGEMAP software (http://www.bigemap.com). 1 km buffer zone was created for each area based on the river bank and the land-use/cover was classified into four types of road, river, construction, and green space based on the visual interpretation method using ArcGIS 10.3 software (Environmental Systems Research Institute, California). The ratio of impervious surface area to pervious surface area is 6.22:1 for the 1 km range along the sampling section of Zhangcun River, while this ratio is 0.89:1 for the 1 km range along the sampling section of Wenquan River (Fig. 2). Based on the evaluation of population density and impervious surface area, the urbanization intensity is greater in the sampling section of Zhangcun River than that of Wenquan River (McKinney 2002; McDonnell et al. 2008). Therefore, Zhangcun River and Wenquan River are separately classified into urban and periurban river wetland types.
Table 1
Climatic factors of the two sampling sites
|
Latitude (°N)
|
Longitude
(°E)
|
MAP (mm)
|
MAT (℃)
|
HT (℃)
|
LT (℃)
|
Zhangcun River
|
36.1267
|
120.4597
|
73.9
|
12.5
|
24.0
|
0.2
|
Wenquan River
|
36.4404
|
120.6561
|
73.8
|
12.3
|
23.9
|
-0.5
|
MAP – mean annual precipitation, MAT – mean annual temperature, HT – highest temperature, LT – lowest temperature |
Sampling Approach
A total of 31 quadrats of macrophyte populations were sampled in these two rivers – 8 quadrats of Alternanthera philoxeroides and 4 quadrats of Typha angustifolia were investigated in the sampling section of Zhangcun River, and 11 quadrats of A. philoxeroides and 8 quadrats of T. angustifolia were monitored in the sampling section of Wenquan River. T. angustifolia is the native species co-occuring with the exotic invasive A. philoxeroides in these two rivers. The quadrat size is 1 m × 1 m within the center of respective pure mats of A. philoxeroides and mono-stands of T. angustifolia (Müllerová et al. 2020). The distance between any two quadrats of the same species is at least 20 m in order to avoid sampling of the same genet (Oduor et al. 2022).
The sediment underneath each plant of each quadrat was carefully collected beneath the center of quadrat surface (approximately 20 cm-deep from the surface). The sediment was immediately placed into labelled plastic sampling bags and 2-ml centrifuge tubes. Then, the sampling bags and centrifuge tubes were put into an ice box. The sediment in the sampling bags were naturally withered and then the dry materials were used for the elemental analyses of carbon, nitrogen and phosphorus. The centrifuge tubes were placed into a box with dry ice and then sent to Novogene Co. Ltd for the 16S rDNA amplicon sequencing analysis of soil microbial diversity and function.
Soil Elemental Analysis
The soil carbon and nitrogen contents (TC and TN) were measured using a EuroEA3000 CHNS-O analyser (EuroVector, Italy). The soil phosphorus content (TP) was determined according to National Standards of the People's Republic of China: method for determination of soil total phosphorus (GB 9837–88). Then, soil C/N, soil C/P and soil N/P was calculated. A one-way ANOVA with Tukey’s test was performed to detect the probable difference in soil TC, soil TN, soil TP, soil C/N, soil C/P and soil N/P between different species in respective urban and periurban river using SPSS 22.0 (SPSS, Chicago, IL, USA).
Sequencing
Extraction of genome DNA
Total genome DNA from samples was extracted using CTAB method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1ng/µL using sterile water.
Amplicon Generation
16S rRNA/18SrRNA/ITS genes of distinct regions (16S V4/16S V3/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified used specific primer (e.g. 16S V4: 515F806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, et. al) with the barcode. All PCR reactions were carried out with 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs); 2 µM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98℃ for 1 min, followed by 30 cycles of denaturation at 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 30 s. Finally 72℃ for 5 min.
Pcr Products Quantification And Qualification
Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with Qiagen Gel Extraction Kit (Qiagen, Germany).
Library preparation and sequencingSequencing libraries were generated usingTruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.
Data analysis
Paired-end reads assembly and quality control
Data split
Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence.
Sequence Assembly
Paired-end reads were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/), a very fast and accurate analysis tool, which was designed to merge paired-end reads when at least some of the reads overlap the read generated from the opposite end of the same DNA fragment, and the splicing sequences were called raw tags.
Data Filtration
Quality filtering on the raw tags were performed under specific filtering conditions to obtain the high-quality clean tags according to the QIIME(V1.9.1, http://qiime.org/scripts/split_libraries_fastq.html) quality controlled process.
Chimera Removal
Chimera removal
The tags were compared with the reference database (Silva database, using UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimera sequences, and then the chimera sequences were removed. Then the Effective Tags finally obtained.
Otu Cluster And Species Annotation
OTU Production
Sequences analysis were performed by Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/). Sequences with ≥ 97% similarity were assigned to the same OTUs. Representative sequence for each OTU was screened for further annotation.
Species Annotation
For each representative sequence, the Silva Database (http://www.arb-silva.de/) was used based on Mothur algorithm to annotate taxonomic information.
Data Normalization
OTUs abundance information were normalized using a standard of sequence number corresponding to the sample with the least sequences. Subsequent analysis of alpha diversity was performed based on this output normalized data.
Alpha Diversity
Alpha diversity is applied in analyzing complexity of species diversity for a sample through four indices including observed species, chao1, Shannon’s diversity index and Simpson’s diversity index. The four indices in our samples were calculated with QIIME (Version 1.7.0) and displayed with R Software (Version 2.15.3).