Fungal endophytes are fungi which spend at least part of their life history living asymptomatically inside the tissue of a host plant (Rodriguez et al., 2009), they are ubiquitous and highly diverse with estimates of one million polyphyletic species across the fungal phylogeny (Sun and Guo 2012). Endophytes provide tolerance to abiotic and biotic stresses via the production of secondary metabolites, which can confer drought and heat tolerance, pathogen resistance, and anti-herbivory mechanisms (Giauque and Hawkes 2013; Molina-Montenegro et al., 2016; Lugtenberg et al., 2016; Bittleston et al., 2010). As well as having mutualistic symbiotic relationships with host plants, endophytes exhibit a variety of life-history strategies; they may be latent saprobes and/or latent pathogens (Porras-Alfaro and Bayman 2011). Research into fungal endophytes has shown that they are essential for plant health and functionality (Rodriguez et al., 2009). The presence of endophytes in the fossil record of early land plants suggests co-evolution (Krings et al., 2012), resulting in unique, potentially obligate, relationships. As the study of fungal endophytes advances, more questions arise surrounding the evolution and role of this symbiosis for both plant and fungi, especially for plants with adaptations for life in extreme environments such as carnivorous plants.
Carnivorous plants attract, trap, and digest animal prey (typically arthropods) using modified leaves as traps, nutrients from digested prey supplements root uptake for growth and reproduction (Givnish 2015; Fleischmann et al., 2018). Plant carnivory has evolved independently at least 11 times across the angiosperm phylogeny (Fleischmann et al., 2018; Lin et al., 2021), with striking examples of convergent and divergent evolution of trapping mechanisms (Ellison and Gotelli, 2009; Thorogood et al., 2018). Carnivorous plants are typically found in extreme conditions, living in generally nutrient poor wetland environments, often with low pH (Pavlovič and Saganová 2015). In the 150 years since Charles Darwin published his book ‘Insectivorous Plants’ (Darwin 1874) many aspects of their biology have been well studied. The mechanisms of plant carnivory are, however, still not fully understood. Considering the ubiquity of fungal endophytes, it is likely that a range of carnivorous plant – fungal endophyte associations exist. For example, new research has demonstrated that Acrodontium crateriforme, a fungus living in secretions of traps of Drosera spatulata plays a key role in carnivory by aiding in prey digestion: the presence of this fungus reduces digestion time up to 25%, in part by the production of peptidases to aid in breakdown of large proteins (Sun et al., 2023). It is not known, however, whether A. crateriforme exists endophytically inside Drosera, it may only occur inside the external secretions of the plant. Fungal endophytes are conceivably similarly important to carnivorous plants, possibly producing enzymes or metabolites that could aid in prey digestion. Additionally, plants growing in other extreme environments, such as mangrove trees (Suryanarayanan et al., 2012), contain a unique assembly of endophytes which aid survival in extreme conditions (Chadha et al., 2015). Knowledge of carnivorous plant fungal endophytes is, however, remarkably limited, considering our understanding of endophyte importance in other plants (Collinge et al., 2022; Eid et al., 2019), perhaps owing to sampling difficulties in remote wetland environments, or difficulties with culturing efforts.
Over 800 carnivorous plant species have been described (Fleischmann et al., 2018), but remarkably few have been studied for their fungal endophytes: four species of Sarracenia (Glenn and Bodri 2012), three species of Nepenthes (Lee, et al., 2014; Naseem et al., 2021), two species of Pinguicula (Quilliam and Jones 2012; Rueda-Almazán et al., 2021), and one species each of Catopsis (Leroy et al., 2021), Drosera (Quilliam and Jones 2010), and Utricularia (Cheon et al., 2016). These studies typically focus on a single plant species growing at a single site. Successful culturing efforts of carnivorous plant endophytes typically reveal common species, including species from ubiquitous genera such as Alternaria, Colletotrichum, Penicillium, and Cladosporium (Glenn and Bodri 2012; Naseem et al., 2021; Rueda-Almazán et al., 2021). Some fungal endophytes have been found across different plant genera, for example Colletotrichum gloeosporoides has been found in both Sarracenia and Nepenthes (Glenn and Bodri 2012; Lee, et al., 2014), both of which are independently evolved pitfall trap pitcher plants (Thorogood, et al.,2018). It has been suggested that C. gloeosporoides may be an endophyte adapted to life as a pitcher plant symbiont (Lee, et al., 2014).
From these studies some insight into the ecology of plant-endophyte associations has been gained. Quilliam and Jones (2012) were able to demonstrate host specificity in the root endophytes of Drosera and Pinguicula at a single site in North Wales. Lee et al.,, (2014) studied the endophytes of two different Nepenthes species growing at the same site in Malaysia and found low endophyte diversity in one of their study species. Rueda-Almazán et al., 2021 studied the endophytes of the traps and roots of Pinguicula moranensis in Mexico at three different sites during the winter and summer and found that seasonality impacted endophyte assembly rather than differences between the three sites. To date, however, no studies have looked for host specificity between co-occurring carnivorous plant species at the same and different sites.
In this study, we investigated the culturable endophyte community from traps of five species of carnivorous plant growing in the United Kingdom, at two in-situ sites and greenhouse grown: Drosera rotundifolia, D. anglica, Pinguicula vulgaris, Dionaea muscipula, and Sarracenia purpurea. These species represent different trapping mechanisms (flypaper traps: D. rotundifolia, D. anglica, and P. vulgaris; Pitfall: S. purpurea; Snap trap: D. muscipula), native (D. rotundifolia, D. anglica, and P. vulgaris) and introduced (S. purpurea, Walker, 2014) species. We used culture-dependant methods to isolate fungi endophytically colonising trap tissues and sequencing of ITS region to identify isolated fungi. Owing to paucity of data of carnivorous plant endophytes in Europe, our aim was to confirm the presence and diversity of fungal endophytes in above ground (trap) tissue of carnivorous plants in the UK, and to establish the extent of host-specificity of these fungal endophytes.
Study species and sites
Details of study species are as follows (summarised in Table 1):
Sarracenia purpurea L. (Ericales) is a long-lived carnivorous pitcher plant, which uses pitfall traps developed from pitcher shaped leaves into which prey fall in and drown. Prey in introduced populations of the UK are primarily Formicidae, Diptera and Coleoptera (Whatmore et al., 2022) attracted to nectar produced in extrafloral nectaries (Bennett and Ellison 2009), prey fall into the pitcher and cannot escape due to downward pointing hairs and waxy surfaces (Heard 1998). Prey digestion is primarily undertaken by a microbial-dominated inquiline community living in the rainwater which collects in the pitcher (Heard 1998). Sarracenia purpurea is distributed across eastern North America where it grows primarily on raised bogs. The species was introduced to Europe from North America in the nineteenth century (Walker 2014), where it is naturalised and can be locally invasive but does not spread from site to site. One study has investigated the fungal endophytes of Sarracenia in North America (Glenn and Bodri 2012), using culturing methods. They found that of four Sarracenia species S. purpurea contained the highest diversity of endophytes, containing six culturable endophyte species, the three other Sarracenia species containing four or fewer species. No studies exist of the endophytes of Sarracenia in its introduced range in the UK.
Drosera rotundifolia L. and Drosera anglica Huds. (Caryophyllales) are short-lived carnivorous plants (Crowder et al., 1990) which grow in small rosettes, and use sticky flypaper traps formed by excreting a sticky polysaccharide from stalked glands (tentacles) on their leaves. Their primary prey are Diptera and Collembola (Foot et al.,, 2014). Once trapped their leaves close around the prey to form an external stomach, into which digestive enzymes (such as chitinolytic enzymes; Libantová et al., 2009) are excreted. Both Drosera rotundifolia and D. anglica have cosmopolitan circumboreal distributions are often co-occurring and have similar habitat affinities, growing in low nutrient, low pH conditions on Sphagnum dominated raised bogs (Crowder et al., 1990). Drosera anglica is found in wetter microhabitats than D. rotundifolia (Nordbakken 1996). No studies of the trap endophytes of these species have been previously conducted. Root endophytes of D. rotundifolia have previously been studied from one site in North Wales (Quilliam and Jones 2010).
Pinguicula vulgaris L. (Lamiales) is a small perennial rosette forming plant, which uses sticky flypaper traps with glandular hairs that secrete a sticky mucilage over the leaves and traps small insects, leaf margins roll inwards after prey capture (Heslop-Harrison 2004). Prey are digested through the secretion of digestive enzymes, the main prey captured are Collembola and Nematocera (Karlsson, et al.,1994). P. vulgaris has a circumboreal distribution where it grows in a variety of substrata where permanent water is present and has a wide pH tolerance (Heslop-Harrison 2004). No studies of the trap endophytes of P. vulgaris have been previously conducted. Root endophytes of P. vulgaris have been studied from one site in North Wales (Quilliam and Jones 2012) and found that despite using the same trapping mechanism as D. rotundifolia, each plant’s roots hosted unique endophytes.
Dionaea muscipula J. Ellis (Caryophyllales) uses a snap-trap mechanism to trap predominantly spiders and larger insects, D. muscipula shares a common Drosera-like carnivorous ancestor with Drosera spp (Gibson and Waller 2009) Prey trigger the snap trap by stimulating trigger hairs on the leaf surface, which causes the trap to close (Volkov et al., 2008), creating an external stomach into which digestive enzymes are secreted. Dionaea muscipula has a very narrow habitat affinity, restricting the plant to habitats in the coastal plains and sandhills of North and South Carolina (Luken 2005). This plant is kept commonly as a houseplant around the world where stock is often grown from sterile plant cultures. There have been no studies to date on Dionaea endophytes.
Plants were sampled from two in-situ locations, and one ex-situ: Brathay Plantation (Lat: 54.403°N, Long: -2.982°E, mean annual rainfall 1,390 mm, mean annual temperature 8.4oC) is a small minerotrophic mire, from which we collected co-occurring S. purpurea and D. anglica and D. rotundifolia in July 2022. (Fig. 1). At Eryri National Park (Snowdonia National Park) we sampled plants from a seepage mire on a steep hillside (Long: -4.011°E, Lat: 53.121°N, mean annual rainfall 1,423 mm, mean annual temperature 8.7oC). Here we collected co-occurring D. rotundifolia and P. vulgaris (Fig. 1) in June 2022 (Fig. 1). This same population has previously been studied, to investigate root endophytes (Quilliam and Jones 2010, 2012). These locations were chosen because of the presence of different species of carnivorous plant in close proximity, and because D. rotundifolia is present at both sites. This design enables us to address questions on host vs site specificity by comparing different species at the same site, and the same species at different sites.
Greenhouse plants: A number of S. purpurea have been kept at Loughborough University for research purposes. These have been collected from numerous in-situ populations across England, including Lower Hyde Bog in Dorset and Brathay Plantation in Cumbria. These plants were used in 2021 to refine the culturing process for the greatest success before collecting from in-situ plants in 2022. Additionally, a number of Dionaea muscipula were briefly kept for scientific purposes, plants were used to confirm the presence of endophytes in D. muscipula, plants were sourced from a nursery.
Table 1
Study species information
Host species | Order | Trapping mechanism | Site |
Brathay | Eryri | Greenhouse |
Sarracenia purpurea | Ericales | Pitfall | ✔ | | ✔ |
Drosera rotundifolia | Caryophyllales | Flypaper | ✔ | ✔ | |
Drosera anglica | Caryophyllales | Flypaper | ✔ | | |
Pinguicula vulgaris | Lamiales | Flypaper | | ✔ | |
Dionaea muscipula | Caryophyllales | Snap trap | | | ✔ |