Are carnivorous plants mixotrophic?

Carnivorous plants, which capture animal prey to thrive in nutrient-poor environments, have independently originated 12 times in angiosperms (Fleischmann et al., 2018; Lin et al., 2021). These plants have evolved various types of traps, with sticky traps being the most common strategy (Freund et al., 2022). The sticky trap has independently evolved in at least six lineages, including the Caryophyllales and Lamiales (Freund et al., 2022), and all other trap types may have evolved from it (Albert et al., 1992; Fleischmann et al., 2018; Freund et al., 2022). Most carnivorous plants with sticky traps use modified leaves to capture insects and photosynthesize simultaneously, which may diminish their photosynthetic performance (Wicke et al., 2014; Ross et al., 2016; Fu et al., 2023). Additionally, previous studies demonstrate that terrestrial carnivorous plants have a lower photosynthetic rate than noncarnivorous plants (Méndez & Karlsson, 1999; Ellison, 2006). This is consistent with plastid gene loss, a trait observed in all tested carnivorous lineages, except for Brocchinia (Gruzdev et al., 2019; Nevill et al., 2019; Fu et al., 2023). Most plastid gene loss in carnivorous plants occurs within the NDH (=NAD(P)H dehydrogenase-like) complex, which is likely involved in photooxidative stress responses (Peltier et al., 2016; Yamori & Shikanai, 2016; Strand et al., 2019). Thus, it has been hypothesized that carnivorous plants assimilate organic carbon from prey to overcome environmental stress, which makes the NDH complex dispensable (Wicke et al., 2014; Silva et al., 2016, 2018; Fu et al., 2023). The degradation of photosynthetic functions and plastomes is also prevalent in heterotrophic plant lineages (Wicke et al., 2016; Graham et al., 2017), which include parasitic plants that obtain nutrients from plant hosts and mycoheterotrophic plants that rely on underground fungi (Merckx, 2012; Heide-Jørgensen, 2008). Therefore, the degradation of photosynthetic functions and plastomes may suggest that certain species of carnivorous plants are mixotrophs that combine autotrophic photosynthesis with heterotrophic nutrition (Tĕšitel et al., 2018). Carnivorous plants absorb essential nutrients like nitrogen (N) and phosphorus (P) from their animal prey, which suggests that they can also absorb organic carbon (C) like heterotrophic plants (Wicke et al., 2014; Ross et al., 2016; Fu et al., 2023). Former studies usually used a natural abundance of heavy isotopes to track the nutrient flow from animals to carnivorous plants (e.g. see Merckx, 2012; Klink et al., 2019), which may not be sensitive enough if the C transfer is low. By using a more sensitive labeled isotope method, direct uptake of carbon from prey has been demonstrated in several studies and fuels respiration in the Venus flytrap (Dixon et al., 1980; Greenway et al., 1990; Rischer et al., 2002; Kruse et al., 2014, 2017; Gao et al., 2015; Fasbender et al., 2017). However, further experiments comparing the ratio of C and N transfer from isotopically labeled prey are still needed to determine whether heterotrophy is widespread across multiple carnivorous plant lineages. Here, we used double-labeled (13C and 15N) insects to detect C transfer, which has been commonly used to confirm heterotrophy and trace the C flow in parasitic and mycoheterotrophic plants (e.g. see McKendrick et al., 2002; Merckx et al., 2010; Yang et al., 2012; Gebauer et al., 2016; Gomes et al., 2020). We also compared C transfer with N transfer across four carnivorous species belonging to two independent lineages. We used tissue-cultured plants grown under the same conditions within the lab to diminish any factor of variability in natural conditions. We tested four carnivorous species with sticky traps, two Drosera (Caryophyllales) and two Pinguicula (Lamiales), to demonstrate if heterotrophy is common among carnivorous plants and if there is any differentiation of heterotrophy among lineages. The δ15N for all control and carnivorous plants was within −5.19‰ to 2.22‰. One week after feeding the plants with labeled insects, the control plants showed no significant increase in δ15N levels; however, all four carnivorous species exhibited a large and significant increase in δ15N, with Drosera capensis experiencing an increase up to 62.16‰ (Fig. 1). The two Drosera species experienced a greater increase in δ15N levels than the two Pinguicula species. A simple mixing model suggests that up to 0.41% of N was transferred from insects to plants, and the two Drosera species got more than twice as much N as the two Pinguicula species (Fig. 2). The δ13C for all control and carnivorous plants was within −14.40‰ to −12.52‰. One week after feeding the plants with labeled insects, the control plants showed no significant increase in δ13C levels, but all four carnivorous species exhibited a significant increase in δ13C, with D. capensis experiencing an increase up to −10.09‰. A simple mixing model suggests that up to 0.29% of C was transferred from insects to plants. However, δ13C uptake is generally less than that of δ15N, except in the case of Pinguicula agnata, which has a more significant average δ13C uptake than δ15N (Fig. 2). Like animal stomachs, carnivorous plants use their traps to digest prey and enzymes similar to animal pepsin to break down proteins (Freund et al., 2022). The uptake of mineral nutrition like N and P has been well demonstrated in carnivorous plants (Adamec et al., 2018). It has also been shown that carnivorous plants take up organic substances like amino acids from prey or solutions (Juniper et al., 1989; Karagatzides et al., 2009). However, even though C uptake has been occasionally documented from absorption of amino acids (Dixon et al., 1980; Rischer et al., 2002), the magnitude of C uptake has yet to be quantified across different lineages; thus, whether carnivorous plants are heterotrophic is still undetermined (Givnish et al., 2018). Our results clearly show that four carnivorous species across two independent lineages assimilate C from prey, which strongly suggests that heterotrophy may be common in carnivorous plants (Fig. 1). It has been shown that Dionaea uses prey-derived amino acid C to fuel respiration and can be considered heterotrophic (Fasbender et al., 2017), but C is localized to the fed trap and petiole and does not move systemically through the plant (Kruse et al., 2017). However, here we clearly show that C is transferred from prey to nonfed traps in Drosera and Pinguicula. The utilization of C assimilation may vary among different carnivorous lineages, and the extent of which should be investigated in the future. Our results also show that the uptake of C is around half the amount of N in all carnivorous species tested, except for P. agnata (Fig. 2). Although carnivorous plants obtain more N than C from prey, C uptake is not trivial. In P. agnata, the uptake of C is even higher, about twice that of N, which indicates the magnitude of carbon assimilation, or the level of heterotrophy, may vary among carnivorous plants. Interestingly, alpha-amylase has been identified in the digestive fluid of P. × Tina (a hybrid of P. agnata and P. zecheri) (Kocáb et al., 2020). This enzyme has not been found to be present in the digestive fluid proteomes of other carnivorous genera and may point to heterotrophic tendencies within Pinguicula. It has been shown that aquatic Utricularia can grow in complete darkness with a carbohydrate-rich medium (Harder, 1970) and prey-derived organic carbon is important for aquatic carnivores when carbon dioxide (CO2) and light are limited (Adamec, 1997). This indicates that some carnivorous plants may acquire more C from prey than others or that they can become more heterotrophic under certain conditions, which also varies among hemiparasitic plants (Těšitel et al., 2010). In tuberous Drosera, for example, variants lacking stalked tentacles were found to be incapable of C uptake from prey, while normal variants with stalked tentacles absorbed and translocated C throughout the plant body and daughter rhizome (Dixon et al., 1980). In the case of Pinguicula, certain species are heterophyllous and form either a noncarnivorous winter rosette or hibernaculum (Legendre, 2000; Shimai et al., 2021), which may result in seasonal differences in C uptake. Furthermore, the production of carnivorous organs can be a phenotypically plastic trait influenced by nutrient availability (Dixon et al., 1980; Ellison & Gotelli, 2002; Englund & Harms, 2003; Thorén et al., 2003; Bott et al., 2008; Pavlovič et al., 2010; Gao et al., 2015). Research into the impact of environmental conditions on heterotrophy, and subsequently, on the evolution of related gene families in the carnivorous plants needs to be tested. Further insight could be taken from hemiparasitic Pedicularis (Orobanchaceae), a genus of relatively recent heterotrophs in which variation in plastome degradation exists (Li et al., 2021). It is unsurprising that carnivorous plants assimilate organic C from prey and have some level of heterotrophy, considering they can easily obtain nutrients following digestion. However, since all carnivorous plants investigated can photosynthesize (Pavlovič, 2022), they should be treated as partially heterotrophic or mixotrophic, similar to hemiparasitic and partially mycoheterotrophic plants. Therefore, we might expect that the magnitude of C uptake may differ across carnivorous lineages. We suggest repeating similar experiments to investigate levels of heterotrophy in additional species outside the Caryophyllales and Lamiales. Although we confirmed that C was transported from fed to unfed traps, further research is warranted to determine how carnivorous plants use prey-derived C and what proportion of total C comes from photosynthesis. We selected species circumscribed to two distantly related lineages of carnivorous plants, Caryophyllales: Droseraceae (Drosera capensis L. and Drosera regia Stephens) and Lamiales: Lentibulariaceae (Pinguicula agnata Casper and Pinguicula gigantea Luhrs). Carnivorous plants were sourced from The Institute of Ecology (Instituto de Ecología, A.C.). Plants were propagated by tissue culture on ½-strength Murashige & Skoog (½MS) media (50 ml per jar) with added activated charcoal (media recipe: ½MS, 20 g l−1 sucrose, 8 g l−1 agar, 1 g l−1 activated charcoal, 3 ml l−1 PPM, pH 5.5) to reduce genetic variability among individual plantlets. We chose Beta vulgaris L. (Caryophyllales; US National Plant Germplasm System; accession no PI531253) and Ocimum basilicum L. (Lamiales; US National Plant Germplasm System; accession no PI652061) as noncarnivorous plant controls. Control plants were grown from seeds on the same MS tissue culture media as carnivorous plants. We fed 200 fruit flies (Drosophila melanogaster Meigen) with 13C and 15N-labeled amino acids (100 mg algal amino acid mixture, Sigma-Aldrich, added to 100 g standard D. melanogaster medium). Another 100 fruit flies were fed a normal standard D. melanogaster medium as a control. Fruit flies were frozen before experimentation. We placed three labeled fruit flies on the most recent fully developed leaf of each plant, 10 plants for each carnivorous species, and five for each noncarnivorous species. A 0.05% Tween-20 solution was used to fix insects lightly to the leaf. Three unlabeled fruit flies were also placed on five plants for each species as controls. We kept plants with insects in the jar and collected whole leaves untouched by insects after 1 wk. For both Pinguicula species and D. regia, we collected three to five leaves per plant that were untouched by the insects and found lower in the whorl (older leaves). For D. capensis, we collected all leaf material that had not come in direct contact with the labeled insect. All samples were dried by silica gel, ground to powder, and sent to the University of California Davis Stable Isotope Facility (SIF), except for the unlabeled carnivorous plant and unlabeled D. melanogaster samples, which were sent to the Laboratory for Isotopes and Metals in the Environment (LIME) at The Pennsylvania State University for isotopic analysis. δ13N and δ15N were analyzed using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd, Cheshire, UK) at the SIF, and a Thermo Delta V Isotope Ratio Mass Spectrometer with Elemental Analyzer (Costech 4010, Costech Analytical Technologies Inc., Valencia, CA, USA) coupled to a Conflo IV interface at the LIME. δ13C was expressed in units of per mil (‰) where δ = [(R sample/R standard) − 1] × 1000, R = 13C : 12C, with carbon in international standards Vienna Pee Dee Belemnite as the standard. δ15N was expressed in units of per mil (‰) where δ = [(R sample/R standard) − 1] × 1000, R = 15N : 14N, with nitrogen in air as the standard. The precision was typically ±0.2‰ for δ13C and ±0.3‰ for δ14N (Supporting Information Dataset S1). In order to run a t-test, data were determined to be normal and the variances unequal (see isotope.ttest.R, Dataset S2). Then we utilized Welch's two sample t-test to determine statistical significance between average δ13C and δ15N measured in control and enriched treatments, for each species (Datasets S1, S2). Bar charts were created in R v.4.3.2 (R Core Team, 2023) using ggplot2 (Wickham, 2016) and figures were finalized in Adobe Illustrator (isotope.barcharts.R, Dataset S2). We thank Tomás Carlo for providing space and equipment for processing labeled plant samples. We also thank Stephen W. Schaeffer for providing the insect material. This material is based upon work supported by the National Science Foundation under grant no. DEB 2030871. Renner also acknowledges support from the Human Frontier Science Program Early Career Grant RGY0082/2021. This work was supported by the USDA National Institute of Food and Agriculture and Hatch Appropriations under project no. PEN04974 and accession no. 7006543. None declared. QL conceived of the project. QL and SSY conducted experiments, collected data, and analyzed results. SSY prepared the figures. QL, SSY and TR interpreted results and wrote the initial draft of the manuscript. QL, SSY, MM-R, EI-L and TR made comments on and final edits to the manuscript. TR contributed funding for the project. QL and SSY share joint first authorship of this work. Supporting Information is available for this paper. Dataset S1 Raw data for δ13C and δ15N (sheet 1), results of simple mixing model (sheet 2), and statistical results for Welch's t-test (sheet 3). Dataset S2 R scripts for Welch's t-test assumptions, statistical analysis, and bar chart generation. R software (R Core Team, 2023) is required to run scripts associated with Dataset S2. 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