2.2.1 Study area and sites
Study sites were along four rivers in the Western Cape Province of South Africa (the Eerste (34°00'28.19’S; 19°00'6.46’E), Lourens (34°00'56.90’S; 18°58'42.54’E), Molenaars (33°42'00’S;
19°13'60’E) and Palmiet (34°03'24.95’S; 19°00'48.52’E) Rivers). All rivers had untransformed sections of natural fynbos vegetation, as well as sections of cultivated land, planted with vineyards and orchards. Untransformed sites of the Eerste, Lourens and Molenaars Rivers were in the upper river reaches, while agricultural sites were downstream (middle reaches). However, in the case of the Palmiet River, the untransformed sites were in the lower reaches, and agricultural sites in the upper reaches. The choice of a range of variously disturbed habitats was to ‘stretch’ the responses of the two life stages under conditions from transformed to fully historic.
Ninety sites were randomly selected per river using QGIS 2.14.3 (QGIS Development Team, 2014). These were divided between the natural and agricultural land uses. Each site comprised a
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10 x 3 m area of water, starting from the river’s edge. Sampling took place twice per site, once during spring/early summer and again in late summer/autumn, to account for both larvae and adult species seasonal differences. Sites were evenly distributed between three flow regimes: still deposition pools (< 0.01 m/sec), gently flowing glides (surface flow 0.01 - 0.4 m/sec), and fast flowing riffles (surface flow > 0.4 m/sec).
2.2.2 Sampling of adult, exuviae and larval dragonflies
On warm, cloudless days, two observers recorded the species and the abundance of mature male dragonflies present at each site. Observations were conducted for 30 min/site, using close-focus binoculars, between 10:00 and 15:00. Observers used a butterfly net to collect voucher specimens or individuals that were difficult to identify in the field. These were later identified according to Samways and Simaika (2016). The species Trithemis dorsalis and T. furva are indistinguishable in the field, and were recorded as one taxon (‘TD’). Exuviae were searched for along the same transects used for adults for 30min/site.
Aquatic larvae were sampled along the same transects used for adult observations. River substrate was disturbed by foot, and with a 30 x 30 cm, 1 000 μm mesh net, which simultaneously collected the suspended samples. To account for habitat differences between species, all microhabitats present at each site were sampled for flow rates, vegetation, and substrate. Each site was sampled for 3 minutes, after which, larvae were separated from debris on a tray, and preserved in 90%
ethanol. Individuals close to or in their final instar were identified to species according to Suhling et al. (2014). Specimens that were too small to accurately identify were excluded from the dataset.
2.2.3 Environmental variables
At each site, 24 environmental variables (EVs) were recorded. These included the categorical variables of river catchment (Eerste, Lourens, Palmiet, or Molenaars Rivers), land use (0 = natural, 1 = agricultural), flow type (pool, glide, or riffle). Percentage shade was visually estimated, and river width and depth at each site was determined with a tape measure. A handheld GPS recorded
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elevation and site positions. A multi-parameter water quality meter (Model: YSI 556 Multi Probe System; Make: YSI Fondriest Environmental) was used to measure water parameters (temperature, dissolved oxygen, conductivity, and pH) at each site. For both river edges and beds, two observers visually estimated percentages of rocks, sand, and detritus. For vegetation data, average vegetation height, as well as the percentage cover, of both alien and indigenous plant structures were estimated per site. Percentage cover of Prionium serratum (Palmiet Reed) and aquatic macrophytes were recorded as separate variables, as they have a strong influence on dragonflies (Samways & Sharratt, 2010). Water temperature and agricultural land use were strongly positively correlated, and so land use was excluded from analyses.
2.2.4 Data analyses
2.2.4.1 Analyses for the ‘father knows best’ hypothesis
To determine whether sampling effort was sufficient, the non-parametric species estimators ICE, Chao2, and second-order Jackknife were calculated in R 3.5.2 (R Core Team, 2018) using the fossil package (Vavrek, 2011). For insect assemblages where a large number of rare species occur, as in the CFR, non-parametric species estimators are recommended (Hortal et al., 2006). Rarefied species accumulation curves were constructed for the larvae, excuviae and adult dragonflies in R (R Core Team, 2018) using the vegan package (Oksanen et al., 2015). There were such poor returns from surveys of exuviae, that they were not included in any further analyses.
Three datasets were used for all analyses, and included larvae, adults, and physically adjusted adults. The adjusted adult dataset excluded six species (Zosteraeschna minuscula, Chlorolestes umbratus, Ceriagrion glabrum, Tramea limbata, Pantala flavescens, and Elatoneura glauca) that were only observed as adults, and not as larvae.
Whether the relationships between dragonfly larvae and adults are scale dependent is unknown, and therefore three different scales were used in analyses. The small scale comprised 360 sites of 10 x 3 m (90 per river), with each site at least 50 m from the nearest site. At the medium scale, there were 40 sites and each covered an area of 90 x 3 m. Here, for each site, data were pooled from nine adjacent small scale sites (10 sites per river). The large scale had eight sites and each
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covered an area of 450 x 3 m (2 per river). Here, for each site, data were pooled from five adjacent medium scale sites (2 sites per river).
Analyses were run using four assemblage measures: abundance, species richness, Shannon diversity index scores, and Dragonfly Biotic Index scores (DBI). The vegan package (Oksanen et al., 2015) in R (R Core team, 2018) was used to calculate Shannon diversity index scores per site.
For each observed species, DBI scores were obtained from Samways and Simaika (2016).
Thereafter, the average score per site was calculated by dividing the sum of all the species’ DBI scores at a site by the number of species observed at that site.
Spearman rank correlations in R (R Core Team, 2018) tested the degree of association between larvae vs. adults, and larvae vs. adjusted adults for abundance, species richness, Shannon diversity index scores, and DBI scores at the three spatial scales.
To determine whether larvae and adults of the same species occurred at the same sites, exact tests of goodness-of-fit were to be performed with the binom.test function in the native stats package in R (R Core Team, 2018). The hypothesis tested was that there would be significantly different proportions of matches and mismatches between the larvae and adults of a species, against the null hypothesis that there would be no differences, assuming an equal likelihood of both. The constituents of the function are the number of successes/matches (the number of matches between the same species of larvae and adult), the number of trials: number of matches + number of mismatches (sites where only the larvae or the adult of a species was found), and the hypothesized probability of success (here 0.5). Here, the exact test was selected over a chi-squared or G-test, as it is applicable to sample sizes of < 1 000 or when at least one of the expected values is small (five or less). Analyses were run at the three spatial scales, for all the species, and sites combined, and then for each species individually. Bar graphs of species proportions were plotted with ggplot2 (Wickham & Chang, 2008). Similarly, a bar graph of larvae and adult abundances per species was also plotted.
2.2.4.2 Analyses for the ‘shared environmental preferences’ hypothesis
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Further analyses aimed to determine whether larvae and adults were driven by the same environmental variables. For dragonfly abundance, species richness, Shannon index, and DBI scores, best fit models were determined for larvae, adults, and adjusted adults, with a stepwise forward selection method based on AIC values using the AICcmodavg package (Mazerolle &
Mazerolle, 2017) in R (R Core Team, 2018). Because all data were found to be non-normal in the lme4 package (Bates et al., 2014), generalized linear mixed-effect models (GLMMs) were run. As data were not over-dispersed, the GLMMs could accurately identify which environmental factors significantly influenced the four response variables of the three groups (larvae, adults, and adjusted adults). River identity, as well as elevation, were included as the random effects. Abundance and species richness data best fitted a Poisson distribution (Bolker et al., 2009), and Shannon and DBI score data were transformed using the formula: x = (x - minimum value)/(maximum value - minimum value), thereafter they best fitted a binomial distribution.
To select the groups of the most important environmental variables that influenced the larvae, adults, and adjusted adults assemblage structure, Canonical Correspondence Analyses (CCAs) were run in CANOCO 5 (ter Braak & Šmilauer, 2012). This was done separately for each assemblage using interactive forward selection analyses and 9999 permutations (ter Braak, 1990). In the mvabund package (Wang et al., 2012) in R (R Core Team, 2018), the function manyglm determined the best fit distribution for the data. Thereafter, GLMs with multivariate extensions were run with abundance data for larvae, adults and adjusted adults using the variables selected by the CCAs.
These were best-fitted with a negative binomial distribution, which assumes a quadratic mean-variance. This model-based approach, tests multiple effects at the assemblage level, by fitting a separate GLM to each species, using the given explanatory variables. This method accounts for between-species correlations, and uses resampling-based hypothesis testing to determine which environmental variables are associated with the abundances (Wang et al., 2012).