A novel approach for reliable qualitative and quantitative prey spectra identification of carnivorous plants combining DNA metabarcoding and macro photography
A combined DNA metabarcoding/in-situ macro photography approach to reliably analyse carnivorous plant prey spectraResults indicate that DNA metabarcoding allows for reliable analysis of prey spectra composition in carnivorous plants at a taxonomic resolution and level of completeness unachievable by traditional morphology-based approaches (as performed, for example, by4,5,6,7,9,10,11). Even in remote tropical northern Western Australia, where many (if not most) arthropod species have not yet been accessioned into the BOLD or GenBank barcode reference libraries, this method identified over 90% of obtained OTUs from our sample set; most of them at family-level, but 41% to genus-level, and 17% even down to species rank (Supplementary Data S1). Lekesyte et al.27 were able to identify 80% of the analysed prey items found on D. rotundifolia in England to species-level. However, their sampling was performed in western Europe, whose entomofauna is comparatively well studied taxonomically and has an excellent coverage in the BOLD reference library of DNA barcodes41. New insect barcodes are regularly added to the BOLD library through large-scale initiatives such as the international Barcode of Life Project (iBOL; https://ibol.org/) and its Australian node Australian Barcode of Life Network (ABOLN), hence accuracy of future metabarcoding research performed in Australia can be expected to increase to similar levels soon.In-situ macro photography was found to provide a valuable plausibility control tool for the prey taxa identified by metabarcoding. While many of the smaller prey taxa detected by metabarcoding were impossible to identify in the in-situ macro photographs due to their tendency to quickly degenerate after digestion into small, shapeless “crumbs”8, this control method considerably reduced the amount of prey taxa detected which were not actually present as prey in the Drosera samples. This flaw of metabarcoding is most commonly a consequence of procedural errors resulting in cross-contamination within the DNA extraction procedure27, usually resulting in low read numbers. However, in-situ macro photographs may also fail to detect species if prey captured by the sundew escaped from the trap33,42, or was stolen by larger animals. In both cases, a DNA imprint left on the Drosera leaves as excretions, detached scales, hairs or, frequently, as autotomised (shedded) body parts42 could have been detected by metabarcoding. Additionally, some barcoding-detected taxa may not constitute prey if they were associated with another captured prey taxon (either as part of its diet, or as a parasite). The latter may explain some barcode hits for taxa not immediately apparent from the in-situ macro photographs, as they are (endo)parasites of captured prey taxa. This was likely the case in the detected Strepsiptera (stylops) which are frequently contained as larvae and adult females in their hymenopteran and orthopteran hosts43. However, insect endoparasites and other non-obvious prey taxa were by default not excluded by the very conservative approach of pictorial plausibility control. Additionally, in the case of endoparasites, these organisms would also contribute to plant nutrition as “bycatch” after being digested together with their host, despite not having been actively attracted to the carnivorous traps. Finally, the control method tested in this study showed that even heavily digested prey items in the samples had sufficient amounts of intact (mitochondrial) DNA present to be detected by metabarcoding, as we found no instance of any prey item being clearly identifiable in the macro photographs but not present in the barcoding data.Prey spectra composition of the studied Drosera speciesThe analysed prey spectra of the three studied species from D. sect. Arachnopus most commonly contained flying insects (especially of the orders Diptera and Hemiptera, both present in 100% of the samples; Fig. 3), thus confirming earlier in-situ macro photography-based studies of closely-related D. sect. Arachnopus species by Krueger et al.8. All members of D. sect. Arachnopus are characterised by a large, erect growth habit and thread-like aerial leaves which usually do not contact the ground8,32, thereby excluding most ground-dwelling arthropods as prey. This result is also similar to other prey spectra studies of erect-leaved Drosera from different geographic areas, where flying insects (particularly Diptera) unanimously comprised almost the entire recorded prey5,11,44. Furthermore, this study confirmed the result of Krueger et al.8 that Hemiptera—and within this order especially the Cicadellidae—are exceptionally common in the prey spectra of D. sect. Arachnopus compared with all other, previously studied Drosera. A possible explanation for this may be the relatively high abundance of Cicadellidae in tropical habitats45 compared to subtropical or temperate habitats where the above-mentioned previous Drosera prey spectra studies were conducted.Of the five most commonly detected orders, Lepidoptera generally comprised the largest prey items in terms of body size or wingspan, respectively. This prey order was exceptionally common in D. finlaysoniana, being present in 100% of samples and also visually conspicuous in the in-situ photographs. Since this Drosera species had by far the largest trapping leaves among the three species studied with an average leaf length of 10.4 ± 0.6 cm (Suppl Appendix S7), and exhibits the largest leaves in D. section Arachnopus32, this may represent an example of large prey items being more easily captured by species with larger trapping leaves33. Additionally, the sampled population of D. finlaysoniana was huge and dense (see Supplementary Figure S1), probably attracting larger prey and enabling capture of larger prey items by “collective” trapping46. Alternatively, Fleischmann30 suggested that captured Lepidoptera themselves could attract further individuals of the same species by pheromone release, potentially explaining the very high numbers of this insect order observed in D. finlaysoniana.Differences among observed prey spectraComparison of prey spectra between the three studied Drosera species revealed significant differences at arthropod family-level but not at the higher level of arthropod orders, indicating that at a coarse taxonomic resolution, the same five arthropod orders (Diptera, Hemiptera, Hymenoptera, Lepidoptera and Thysanoptera) generally comprise most of the prey in D. sect. Arachnopus, regardless of given Drosera species or habitat. However, as strong differences were discovered in the ANOSIM comparison at family-level, it can be concluded that differences might likely increase with finer taxonomic resolution of prey taxa, a conclusion also reached by the carnivorous plant prey spectra meta-analysis of Ellison & Gotelli47. While these differences may be partially attributed to different morphological traits of the three species such as leaf scent8,30 or eglandular appendages31, the very high ANOSIM R-values returned and the large number of prey families contributing nearly equally to dissimilarity (Table 2) indicate that the most likely explanation is very different available prey spectra at the three study sites. Indeed, significant differences among different study sites, even within the same species, were previously reported for Drosera rotundifolia by Lekesyte et al.27 and for four species from D. sect. Arachnopus by Krueger et al.8. Notably, the three study sites feature different habitat types and climate regimes (Supplementary Fig. S1).Analyses indicate that there is likely little specialisation in prey capture by the three studied Drosera species. For example, the relatively high detection rate of Lepidoptera in the samples of D. finlaysoniana and D. hartmeyerorum compared to D. margaritacea may be explained by the lake margin habitats of the former two species, while the latter species was found in a completely dry drainage channel lacking any nearby waterbodies (Supplementary Fig. S1). Lepidoptera are likely to occur in much higher concentrations near water sources, especially during the dry season (May to November) when the surrounding areas are lacking other water sources (G. Bourke in Fleischmann30).Estimating prey quantityIn addition to providing a plausibility control for the compositional prey analysis by metabarcoding, the in-situ macro photography method facilitated an estimation of prey quantity per sample. Metabarcoding by itself is currently not a reliable tool for prey quantification due to the lack of a linear relationship between the number of sequence reads and organism biomass26,27.In contrast to Krueger et al.8, who generally found more prey items on larger trapping leaves in species of D. sect. Arachnopus (even when values were compared as per cm of trapping leaf length), the species with the largest leaves studied here (D. finlaysoniana) captured significantly less prey items than the smaller-leaved species D. margaritacea and D. hartmeyerorum (Fig. 4). However, while Krueger et al.8 was able to compare sympatric species (thus minimising any potential effects of the habitat or region on prey spectra), the three species in this study were studied at three different, geographically distant sites. While it is possible that overall prey abundance in the habitat was much lower at the D. finlaysoniana study site (Site 1), it can be hypothesised that the low total prey capture observed in this species may be due to the very large and extremely dense population resulting in strong intraspecific competition for prey (see Supplementary Fig. S1). This effect of population structure on prey capture has also been observed by Gibson48 and Tagawa and Watanabe46 who found a significant negative correlation between total prey capture and population density in different species of Drosera.Conclusions and outlookOur study is the first to employ a DNA metabarcoding approach supported by controls for species presence to analyse carnivorous plant prey spectra. When combined with in-situ macro photography, this method is clearly superior in terms of taxonomic resolution and completeness for analysis of environmental bulk samples (containing different organisms in highly variable states of preservation), as used here for the reconstruction of prey spectra of carnivorous plants. The capability of this method increases with new reference barcodes being regularly added to DNA barcode libraries (such as BOLD and NCBI GenBank) and it thus has the potential to become the standard methodology for future carnivorous plant prey spectra research.Additional studies are needed to test this method for other carnivorous plant species and genera, especially those possessing different trap types. Within Western Australia, three additional trap types occur: snap traps (Aldrovanda), suction traps (Utricularia) and pitfall traps (Cephalotus). In particular, it might be expected that in-situ macro photography will not work as well for the extremely small, typically submerged traps of Aldrovanda and Utricularia (which also completely enclose their captured, microscopic prey items49), potentially necessitating usage of alternative control methods for metabarcoding data. Furthermore, even within Drosera (adhesive traps) some species may require adjustments to the methodology presented here as they accumulate captured prey in a central point via tentacle movement (e.g., many climbing tuberous Drosera) or their leaves may be very difficult to place on paper sheets with the sticky side facing upwards (e.g., all pygmy Drosera). The latter problem may be solved by using reverse action forceps and photographing the leaves while held in place by the forceps.Extensive sampling of sites with co-occurring species from D. sect. Arachnopus is clearly required to better understand the ecological role of trap scent and eglandular appendages in this section. For example, manipulation experiments involving the removal of all yellow blackberry-shaped appendages of D. hartmeyerorum (which have been hypothesised to function as visual prey attractants31) and subsequent metabarcoding prey spectra comparisons of mutilated plants lacking emergences with control plants are proposed. Potential effects of population density on prey spectra (as hypothesised here for D. finlaysoniana) could be studied by comparing prey spectra of individual plants from within mass populations with more exposed-growing individuals of the same population. More
