While high efficiency and cost‐effectiveness are two merits of environmental DNA (eDNA) techniques for detecting aquatic organisms, the difficulty of designing species‐specific primers can result in significant expenditure of time and money. During the in silico stage of primer development, primer specificity is predicted with alignment techniques such as BLAST that is based on the number and position of the primer/nontarget template mismatches. However, we speculate that nonspecific amplification is influenced by additional parameters, which lead to inaccuracies of in silico prediction. We performed in vitro specificity tests for 38 species‐specific primers selected for seven fishes and six turtles, using single‐plex conventional PCR (cPCR). A subset of 12 primer pairs were further tested with SYBR Green‐based or TaqMan‐based single‐plex quantitative PCR (qPCR). We disentangle the relative importance of mismatch properties (types and positions), primer properties (length, GC content, and 3′ end stability), PCR conditions (template concentrations and annealing temperatures), and PCR technique (cPCR, TaqMan‐based, or SYBR Green‐based qPCR) in determining the occurrence of amplifications. We then compared the PCR outcomes with the specificity check under two stringency scenarios based on alignment (i.e., BLAST search). We conducted a total of 679 cPCR and 226 qPCR analyses, with 90% of the reactions tested with nontarget templates. Primer pairs predicted by Primer‐BLAST to be specific rarely showed such specificity during the in vitro testing. BLAST searches correctly predicted the outcomes of around 67% of cPCR and qPCR, but had low sensitivity in detection of nontarget amplification (29–57%). Primer specificity increased significantly with total number of mismatches and annealing temperature, but decreased with higher GC content in the primer sequence. Mismatches that consisted of A‐A, G‐A, and C‐C pairings exerted 56% stronger reduction in nonspecific amplification effects than other mismatches. To conclude, we show that the prediction of primer specificity based only on the number and position of mismatches can be misleading. Our findings can be applied to increase the efficiency of the in silico primer selection process to maintain the relatively high efficiency and cost‐effectiveness of eDNA techniques.
The vegetation grown on grasscretes along channelized rivers have been regularly mown in Hong Kong. However, no baseline information on the relationship between different mowing regimes and the biodiversity of such riverbed vegetation is available. We therefore carried out a manipulative experiment along a channelized river to test the effect of mowing frequency and intensity on the abundance and species richness of terrestrial biodiversity. We conducted point counts and transect counts to survey birds, butterflies and Odonates, night surveys for amphibians and reptiles, quadrat surveys for vegetation and sweep net and pan traps for other macroinvertebrates. The results from Generalized Linear Mixed Models (GLMMs) showed a taxon-specific effect of mowing regime. Bird species richness was significantly suppressed in plots mown with high frequency. Both butterfly abundance and species richness were greatly enhanced by low intensity and low frequency mowing. Odonate abundance, and the abundance and species richness of other macroinvertebrates remained high whenever a portion of vegetation was retained as refuge. Amphibians and reptiles did not prefer to utilize the vegetation grown on grasscretes, and thus showed no impact from different mowing regimes. The overall species richness of vegetation was not affected by mowing regimes, but the domination of tall invasive Brachiaria mutica was suppressed by any mowing activity. To cater for the need of most taxa, we propose a mosaic mowing regime, in which most parts along the channelized river could be mown infrequently to 600 mm tall while some of the patches remain unmown.
1. Active and abandoned paddy fields are valuable habitats for aquatic fauna in monsoonal Asia. Changes in land use and farming practices have caused substantial losses of paddy-derived marshes in recent decades. Few of those remaining have been designated as protected areas for biodiversity or are managed for conservation. 2. Between 2014 and 2017, 35 paddy-derived marshes (13 protected and 22 unprotected) in Hong Kong were visited with the aim of sampling aquatic macroinvertebrates and assessing the vulnerability of sites to different threats. Twenty of them had been sampled in 1996, allowing the investigation of biodiversity change over time in relation to protection status. The representativeness of protected marshes was evaluated based on their α-, βand γ-diversity.3. In total, 272 macroinvertebrate taxa were recorded (mean, 57 morphospecies per site). Out of the 20 resurveyed sites, five (three protected) were terrestrialized and four (all unprotected) had been filled in between 1996 and 2014-2017. The relative changes in αand β-diversity of the remaining 11 resurveyed marshes were unaffected by their protection status.4. The 10 remaining protected marshes had similar αand β-diversity to those of unprotected sites and, in total, hosted fewer species than the mean value obtained in 9,999 random selections of the same number of sites, indicating that macroinvertebrate diversity was not very well represented within the protected marshes.5. Protection alone was not successful as a conservation measure because it failed to prevent the degradation and loss of paddy-derived marshlands in Hong Kong. Furthermore, a failure to consider biodiversity when the sites were designated as protected reduced the representativeness and effectiveness of the protected area network for achieving the conservation objectives.6. Conservation planners need to take account of freshwater biodiversity (especially aquatic insects) during the selection of marshland sites for protection and ensure that protected sites are appropriately managed.
1. Semi-natural marshland is becoming increasingly prevalent in Asia as a result of the continuing abandonment of rice cultivation. Although these marshes are important habitats for aquatic animals, they are susceptible to terrestrialization. Large mammalian herbivores that can retard terrestrialization are in decline globally, but domesticated bovids may serve as their surrogates, and could be used for managing semi-natural marshes. Relevant research in Asia is lacking, however. 2. Aquatic macroinvertebrates were sampled in both the wet and dry seasons from 26 freshwater marshes (abandoned paddy fields) across Hong Kong, encompassing 15 sites grazed by feral bovids (yellow cattle and water buffalo) and 11 ungrazed sites. The aim was to investigate seasonal variation in the effects of bovids on macroinvertebrate communities in monsoonal marshes. 3. Four decades after paddy cultivation had been abandoned, semi-natural marshes with low-intensity bovid grazing (0.06-0.14 cattle ha −1) had significantly higher (16%) site-scale γ-diversity. Macroinvertebrate communities at grazed sites had more Coleoptera and larval Odonata, and differed markedly from those at ungrazed sites. The effects of grazing on diversity and composition were unaffected by season, but season itself was a significant predictor of αand β-diversity and species composition. 4. This study is the first to record the responses of aquatic macroinvertebrate diversity and composition to large-mammal grazing in Asian marshlands. Given that bovid grazing at low intensity can control plant growth, with concomitant benefits for wetland diversity, it is suggested that targeted grazing of short duration could be used for conservation management of abandoned paddy fields.
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