Density dependence in reproduction plays an important role in stabilising population dynamics via immediate negative feedback from population density to reproductive output. Although previous studies have shown that negative density-dependent reproduction is associated with strong spacing behaviour and social interaction between individuals, the proximal mechanism for generating negative density-dependent reproduction remains unclear. In this study, we investigated the effects of density-induced stress on reproduction in root voles. Enclosed Founder populations were established by introducing six (low density) and 30 (high density) adults per sex into per enclosure (four enclosures per density in total) during the breeding season from April to July 2012 and from May to August 2015. Faecal corticosterone metabolite (FCM) levels, reproductive traits (recruitment rate and the proportion of reproductively active indivuduals), and founder population numbers were measured following repeated live-trapping in both years. The number of founders was negatively associated with recruitment rates and the proportion of reproductively active indivuduals, displaying a negative density-dependent reproduction. FCM level was positively associated with the number of founders. The number of founder females directly affected the proportion of reproductive females , and directly and indirectly through their FCM levels affected the recruitment rate; the effect of the number of male founders on the proportion of reproductive males was mediated by their FCM level. Our results showed that density-induced stress negatively affected reproductive traits and that density-induced stress is one ecological factor generating negative density-dependent reproduction.

Root voles in the study area

Our study was conducted at Haibei Alpine Meadow Ecosystem Research Station, Menyuan County, approximately 155 km north of Xining, the capital city of Qinghai province, People’s Republic of China (37°370´N, 101°120´E). The area is a secondary vegetation type meadow with a dense leaf layer. The major plant species include Elymus nutans, Poa sp., Kobresia humilis, and Potentila fruticosa. The root vole is the most common rodent in the study area. Root-vole populations in this area fluctuate only seasonally, with the lowest levels occurring in early spring; multiyear cycles are weak or absent (Jiang et al., 1991). Root voles have a preference for dense vegetation (mainly E. nutans) (Liu et al., 1991; Bian et al., 1994). The average population size across the study sites ranged from 70 to 170 voles ha-¹ during the past 20 years, while in certain dense grassland sites, where grazing activities were limited and vegetation consisted mainly of E. nutans, the density reached to c. 400 voles ha^-1 in late autumn (high level season, Jiang et al.,1991; Bian et al., 1994; Sun et al., 2002). The breeding season typically lasts from April to late October. Females have exclusive territoriality during the breeding season; males, conversely, have large area ranges that extensively overlap with those of other males (Sun et al., 1982). The lifetime of free-ranging individuals is < 1 year. Spring-born individuals attain sexual maturity in the year they are born; fall-born voles remain reproductively inactive during winter (Bian et al., 2015).

Experimental facility

The experiment was undertaken in eight 0.15-ha (50 × 30 m) outdoor enclosures in 2012 and 2015. The enclosures were constructed using galvanised steel panels (1.5 m aboveground and 0.5 m belowground), which prevented mammalian predators from gaining entry. Avian predators were excluded by a 3 × 3 cm grid wire mesh held aloft by a central pillar (10 × 250 cm) in each enclosure. Each enclosure was equipped with 60 laboratory-made wooden traps (Bian et al., 2015), spaced in a 5 × 5 m grid. Each trap was covered with a wooden sheet to protect it from exposure to precipitation and temperature extremes.

Establishment of populations and live-trapping

A total of 288 voles of each sex, 6 months of age or older, were separately used to establish the enclosure populations in 2012 and 2015. They were either F2 generations born in the laboratory or captured as juveniles in the previous year. All individuals were tagged in the ear with identifying metal tags. The populations were introduced into the enclosures in April 2012 and May 2015 at two density conditions. According to the the low- and high- density levels observed in nature (Jiang et al.,1991; Bian et al., 1994; Sun et al., 2002), the low-density condition consisted of six adults per sex in each of the four enclosures, and the high-density condition consisted of 30 adults per sex in each of the other four enclosures in 2012 and 2015. The initial body weights did not differ among the voles in the different enclosures (F₇,₂₈₀ = 1.72, P = 0.103 in 2012, F₇,₂₈₀ = 0.192, and P = 0.987 in 2015). Live-trapping started after allowing the animals to acclimate to their new environments for two weeks and lasted until late July 2012 and August 2015, respectively. Standard capture–mark–recapture methods were used throughout the study. Six trapping sessions were conducted in 2012 and seven in 2015; each consisted of three trapping days. The time interval between any two trapping sessions was one week. The traps were set between 7 AM and 7 PM, baited with a bit of carrot, checked every 2 h, and locked closed when trapping did not occur. Following each capture, we recorded animal identification, sex, and body mass. Females were considered reproductive if they had enlarged nipples and teats barren of hair. Males were considered in breeding conditions if their testes were scrotal rather than abdominal. The animal was, then, released at the point of capture after handling. The F1 offspring born in the enclosures were captured at 20–30 days of age and permanently moved to the laboratory for use in subsequent experiments (Bian et al., 2015; Yang et al., 2018).

Fecal corticosterone metabolite (FCM) measurement

FCM levels reflect the level of circulating corticosterone that occurred 10–12 h previously in root voles (He et al., 2013), and FCM is derived primarily from plasma-free corticosterone in rodents (Sheriff et al., 2010). Faecal samples for the FCM analysis were collected during the first 2 h of trapping (09:00–11:00 AM), and each captured animal was sampled once within a 3-day trapping session; thus, all animals provided only a single sample in each trapping session. Meanwhile, each trap was cleaned with water before collecting the faecal sample, ensuring that the samples were not influenced by the previous trapping or time of day. Traps used to sample faeces only had a few carrots. Faecal samples from pregnant females were not collected to avoid confounding effects of reproduction states on FCM levels (McDonald, 1998; Edwards & Boonstra, 2018). The total number of faecal samples was 546 and 832 in 2012 and 2015, respectively, throughout each experiment, and they accounted for 59% and 67% of the sum of minimum number known alive in each trapping session throughout the duration of experiments in both years (excluding reproductive females). The collected samples were, then, frozen in ice, transported to the laboratory, and stored in a −20°C freezer until analysis. FCM was measured following the methods outlined by Yang et al. (2018), previously validated for root voles. First, the collected faecal samples were lyophilised (Labconco, Kansas City, MO, USA) for 14–18 h, ground into particles, and homogenised in 0.5 mL NaOH solution (0.04 M). The extraction of FCM was performed by adding 5 mL of CH₂Cl₂ to the sample (0.1 g), followed by sonication for 15 min (Pihl & Hau, 2003) and centrifugation for 15 min at 3,000 g. After centrifugation, 1 mL of the solution was taken from the organic layer, diluted with 3 mL CH₂Cl₂, and then mixed with 4 mL of a mixed solution of sulfuric acid and ethanol (7:3, v:v). The samples were, then, shaken for 2 min and rested for 30 min before separation of the sulfuric acid layer for fluorescence detection. The fluorescence density in each sample was measured using an RF-540 IPC Fluorometer (Shimadzu, Japan) at excitation and emission wavelengths of 470 and 520 nm, respectively, and the FCM concentration in each sample was calculated based on the fluorescence densities produced by varying concentrations of the standard (Chen et al., 2012).

Statistical analysis

We used the minimum number known alive (MNKA) method to estimate the founder numbers. The recruitment rate was calculated as the recruits captured in a trapping session divided by the adult females captured in the second preceding session in each enclosure.. The proportion of the reproductively active individuals was evaluated using the numbers of reproductively active voles divided by the total numbers of adults captured for each sex in a trapping session.  Recapture rate was calculated as the numbers of captured individual divided by MNKA in a trapping session. 

We used generalized linear mixed models (GLMMs) in SPSS v.19 (IBM, Armonk, NY, USA) to test the effects of population density on founder number, reproduction and recapture rate. We combined the both years data to increase the statistical power. Founder number, proportion of reproductively active individual, recruitment rate and recapture rate as response variables, the treatment, sex and time as predictor variables for founder numbers analyses and treatment, time as predictor variables for other data analyses, predictor variables were entered in all the models to test separately the main and interactive effects. In all data analyses, fence and year were both specified as random effects, which allowed for correlated responses within years and fences. Factor fence was nested within factor year. Because founder number is subject to poisson distribution, response variables were analysed using poisson distribution and log-link function. Because the number, recruitment rate, proportion of reproductively active individuals and recapture rate were repeatedly sampled during the experiment, these data were analysed using GLMM repeated measures to take account of the change in variables over time. For this method, because response variables may be correlated to observational units (enclosures) at different time points (trapping session), we first conducted a comparison of candidate models with various covariance structures using the corrected Akaike information criterion (AICc). The model with the smallest AICc value was then selected. Post hoc comparisons for significant treatment effects were followed the Bonferroni test. Comparisons of the means were considered significant at P < 0.05. All data are expressed as mean ± standard error.

Recursive model in SEM is a structural equation model that described the complex relationship between variables by simultaneous equations, all the paths flow one way with no feedback or reciprocal loops and the errors are uncorrelated (Schreiber, 2008). The variables in recursive model include endogenous variables (outcome variables) and exogenous variables (predictor variables). Different from regression analysis, the recursive model can explain the direct and indirect effects between variables, and the path graph can more intuitively represent the complex relationship between variables. To tease apart which factors of density and FCM levels induced reproductive suppression, we used the model to explore the pathways of how density, through FCM level of founder voles, affected reproductive traits (recruitment rate and proportion of reproductively active indivuduals). We first considered a full model that included all possible pathways, and, then, sequentially eliminated non-significant pathways until we attained the final model. We reported path coefficients as standardised effect sizes. This analysis was performed with a longitudinal data set, which included cumulative time of trapping session (CT), recapture rate (RR), founder number (N), recruitment rate (R), proportion of reproductively active individuals (P) and mean FCM level per trapping session in 2012 and 2015, respectively. In analyses, RR, N, FCM, R and P were entered in the model as endogenous variables, and CT as exogenous variables. Because founder number, recapture rate and proportion of reproductively active individuals are not subject to normal distribution, so the data were sqrt-transformed and arcsine transformed prior to the analyses, respectively. We used the χ2 test (if p > 0.05, then no paths were missing, and the model was a good fit) and root mean square error of approximation (RMSEA) (if p < 0.05, then no paths were missing, and the model was a very good fit) to evaluate the fit of the model.

GENERAL INFORMATION:

1. Title of Dataset: Data from: Is negative density-dependent reproduction regulated by density-induced stress in root voles? Two field experiments.

2. Author Information:

Author 1

Name: Dr Guozhen Shang

Institution: Northwest Institute of Plateau Biology Chinese Academy of Sciences, Xining 810001, China

Email: qhsgz@qq.com

Author 2

Name: Dr Shouyang Du

Institution: College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China

Author 3

Name: Dr Yanbin Yang

Institution: College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Henan 45000, China

Author 4

Name: Yifan Cao

Institution: Northwest Institute of Plateau Biology Chinese Academy of Sciences, Xining 810001, China

Corresponding author 1

Name: Dr Janghui Bian

Institution: Northwest Institute of Plateau Biology Chinese Academy of Sciences, Xining 810001, China

Email: bjh@nwipb.cas.cn

Corresponding author 2

Name: Dr Yan Wu

Institution: School of Life and Environment Sciences, Hangzhou Normal University, Hangzhou 310012, China.

Email: wuyanqh@163.com

3. Date of data collection: 2012-2015

4. Geographic location of data collection: Haibei Alpine Meadow Ecosystem Research Station, Menyuan County, approximately 155 km north of Xining, the capital city of Qinghai province, People’s Republic of China (37°370´N, 101°120´E)

5. Funding sources that supported the collection of the data:  the National Natural Science Foundation of China (Grant Numbers 31870397, 31800339 and 31570421), the Natural Science Foundation of Qinghai Province (Grant Number 2018-ZJ-906 and 2021-ZJ-945Q), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant/Award Number XDA2005010406).

DATA & FILE OVERVIEW

1. Description of dataset

These data were generated to investigate the effects of density-induced stress on reproduction in root voles (Microtus oeconomus). Enclosed Founder populations were established by introducing six (low density) and 30 (high density) adults per sex into per enclosure (four enclosures per density in total) during the breeding season from April to July 2012 and from May to August 2015. Faecal corticosterone metabolite (FCM) levels, reproductive traits (recruitment rate and the proportion of reproductively active indivuduals), and founder population numbers were measured following repeated live-trapping in both years. We separately tested the difference in reproductive traits between high-and low-density enclosure populations, and used the recursive model in the structural equation model (SEM) to analysed how the number of founders and FCM levels affected reproduction.

2. File List:

File 1 Name: MSA-female.csv

File 1 Description: the trapping session, FCM, proportion of reproductively active individuals, number, recruitment rate and rate for females in 2012 and 2015.

File 2 Name: MSA-male.csv

File 2 Description: the trapping session, FCM, proportion of reproductively active individuals, number, recruitment rate and rate for males in 2012 and 2015.

File 3 Name:  population_number.csv

File 3 Description: the number of founder populations in low- and high-density enclosures in 2012 and 2015.

File 4 Name: recapture_rate.csv

File 4 Description: Recapture rate of enclosures founder populations in 2012 and 2015, Recapture rate was calculated as the numbers of captured individual divided by MNKA in a trapping session.

File 5 Name: recruitment.csv

File 5 Description: recruitment rate in 2012 and 2015, the recruitment rate was calculated as the recruits captured in a trapping session divided by the adult females captured in the second preceding session in each enclosure.

File 6 Name: reproductive_condition_of_male.csv

File 6: Description: The proportion of the reproductively male in low- and high-density enclosures in 2012 and 2015. it was evaluated using the numbers of reproductively active male voles divided by the total numbers of adults captured for male in a trapping session.

File 7 Name: reproductive_condition_of_female.csv

File 7 Description: The proportion of the reproductively female in low- and high-density enclosures in 2012 and 2015. it was evaluated using the numbers of reproductively active female voles divided by the total numbers of adults captured for female in a trapping session.