DOI: 10.5281/zenodo.17397880
title: Dataset used in article Linking millipede trophic niche to litter–millipede elemental composition across an altitudinal gradient

description: 2.1 Sampling sites

The study area is located on the northern slope of the Changbai Mountain Natural Reserve in northeast China (41°43′-42°26′ N and 127°42′-128°17′ E). We established eight forest plots, ranging from 800 to 1850 m, were selected with an elevation difference of approximately 150 m between adjacent plots. Each plot was subdivided into four 10 m2 subplots, which were spaced more than 100 m apart to minimize spatial autocorrelation (Keitt et al., 2002). To ensure ecological representativeness, sampling plots were placed beneath dominant tree species characteristic for the respective elevation. This design ensured that sampling across sites and elevations was consistent and comparable.

2.2 Sampling, extraction and species determination

Millipedes were manually sampled from the litter layer in August/September 2021. At each plot, sampling was standardized by using eight collectors and a fixed sampling duration of approximately one hour, during which all visible millipede individuals were collected. A total of 32 samples were obtained. Millipedes were placed in 1.5 ml centrifuge tubes containing 70% ethanol, transferred to the laboratory and stored at -80 °C. In addition, five samples of litter material were collected from each subplot using a 5.5 cm diameter corer, thoroughly mixed to form one composite sample, and stored at -20 °C for subsequent analysis.

Millipedes were determined to species based on morphological characters using a stereomicroscope (Leica S8 APO, Wetzlar, Germany). The species were ascribed to five taxonomic groups based on their taxonomic affiliation and life form, i.e. Diplomaragnidae, Julidae, Paradoxosomatidae, Polydesmidae and Polyzoniidae (Mikhaljova, 2004).

2.3 Stable isotope and elemental concentration analysis

Millipedes were freeze-dried for 72 h, ground in a bead mill (Bead Ruptor 12, Omni International, Kennesaw, USA) and 1–2 mg weighed into tin capsules. Stable isotopes were measured using a coupled system of an elemental analyzer (NA 1500, Carlo Erba, Milan, Italy) and a mass spectrometer (MAT 251, Finnigan, Bremen, Germany) adopted for the analysis of small sample sizes (Langel and Dyckmans, 2014). Ratios of the heavy isotope to the light isotope (13C/12C,15N/14N, denoted as R) were expressed in parts per thousand relative to the standard using the delta notation with δ13C or δ15N = (Rsample/Rstandard − 1) × 1000 (‰). Vienna PD Belemnite and atmospheric nitrogen were used as standard for 13C and 15N, respectively. Acetanilide was used for internal calibration.

Total carbon (C) and nitrogen (N) concentrations of millipedes were analyzed using a vario EL cube CHNOS elemental analyzer (Elementar Analysensysteme, Langenselbold, Germany) from ca. 2 mg dry material. Litter samples were dried at 60°C for 48 h and ground. Approximately 8 mg of this material was used for the analysis of total C and total N concentrations using a Vario MACRO Cube Elemental Analyzer (Elementar Analysensysteme, Langenselbold, Germany). Furthermore, the concentration of Ca, Cu, K, Mg, Mn, Na, P and Zn of millipedes and litter material were measured after HNO3 digestion using an iCAP 6300 ICP-OES spectrometer (Thermo Fisher, Cambridge, England). All element concentrations were expressed as percentages (%) of dry weight.