2.1 Study area
Our study was conducted in Xinjiang Uygur Autonomous Region, center of Asia-Europe continent (Fig. 1). This region extends from the south slope of the Altai Mountains to the hinderland of the Tianshan Mountains. The Junggar basin between the two mountains is covered by the Gurbantunggut desert, the second largest desert in China. This region belongs to semi-arid and arid climatic zone, with the elevation ranging from 189 to 7435 m, and encompasses complex landforms and microclimate between mountains and basins. Due to the characteristics of geography, environment and climate, Xinjiang region provides biological community with a variety of habitats and has been listed as one of the priority areas for biodiversity conservation in China. The Irtysh River, Emin River, Ili River and Bortala River originate from Xinjiang Uygur Autonomous Region, northwest China (Fig. 1) and ultimately flow towards the Republic of Kazakhstan. In particular, the Ili River is the largest river in Xinjiang region. The Irtysh River is the only Chinese river that disgorges into the Arctic Ocean. Ulungur Lake covers an area of 1035 square kilometers. These rivers and their affiliated water bodies are the most important water resources in northwest China and play an important role in maintaining the biodiversity of aquatic ecosystems (Wang et al., 2010).
2.2 Sampling and experimental material
Aquatic insects are absolutely predominant faunas in the transboundary rivers of northwest China (Wang et al., 2014). Despite extraordinarily abundant resources for aquatic insects, few studies have been carried out in these transboundary rivers (Wang et al., 2014). Furthermore, transboundary rivers such as the Ili River and Irtysh River are suffering from the intervening of anthropogenic activities, and hence accurate classification of macroinvertebrates is essential for the environmental and biodiversity assessment. As for the organisms used for water quality assessment, macroinvertebrates are one of the most optimal categories due to a large size, weak dispersal and the sensitiveness to the change of water quality (Resh et al., 1995).
We collected macroinvertebrate samples in four transboundary rivers (the Irtysh River, Emin River, Ili River and Bortala River) between China and Kazakhstan, Mongolia and Russia (Fig. 1). Study sites were set in the main stems, tributaries and affiliated water bodies (lakes and reservoirs). Due to a long frozen season (November to next April), samples of Mayfly (order Ephemeroptera), stonefly (order Plecoptera), caddisfly (order Trichoptera), robber fly (order Diptera), true bugs (order Hemiptera), Coleoptera, Odonata, Mollusca and Annelida were collected in May, July, August and October of 2013-2017. Macroinvertebrates were collected by Surber net, Kick-net, Peterson grab and D-framed dip net according to the habitat type of study areas. Standardized sampling protocols were executed at different habitats (i.e., lentic and lotic water bodies) from upstream to downstream. Approximately 30,000 specimens were preserved in 95% ethanol to allow for the morphological identification and molecular analyses. In particular, when the color of ethanol solution for macroinvertebrate soaking became dark yellow during the field survey, ethanol needed to be replaced for two or three times in order to warrant a good preservation of the specimens for further molecular analysis. Macroinvertebrate samples were sorted and identified under a dissecting microscope in the laboratory, and stored at -20 °C at the College of Fisheries, Huazhong Agricultural University (China). In the lab, specimens were identified based on morphological characteristics, and then intact individuals were selected to conduct the DNA barcoding analysis for nine taxonomic groups (Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, Odonata, Mollusca and Annelida).
2.3 DNA extractions, amplification and sequencing
Total genomic DNA was extracted from legs, cerci, half or whole body according to a specimen’s size, using the phenol–chloroform protocol (Barnett and Larson, 2012). and quantified in a NanoDrop 2000c (Thermo Scientific, USA). The primer pair LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al., 1994) was used to amplify the fragment DNA of COI (mitochondrial cytochrome C oxidase I) with the length of about 658 bp. Polymerase Chain Reactions (PCR) were performed in 50 µL volumes using the following reagents and concentrations: a volume of 50 μL containing 10×PCR buffer 5 μL, Mg2+ (2.5 mmol/L) 5 μL, dNTP (2.5 mmol/L) 3.5 μL, l.5 μL (10 nmol/L) each primer, 0.5 U Taq polymerase (Takara, Dalian, China), 2 μL (50 ng/μL) DNA template and complete ddH2O to 50 μL. The PCRs were run as follows: 94 °C for 3 min; then 94 °C for 1 min, 45 °C for 2 min, and 72 °C for 3 min, for 40 cycles; and 72 °C for another 5 min (Folmer et al. 1994). PCR products were checked by electrophoresis at 1% agarose with an ethidium bromide stain, and if successful, the PCR products were subsequently purified and directly sequenced by Invitrogen Corporation in China.