Culturable endophytes from carnivorous plant traps in the UK: commonality of endophyte species across host species and sites.

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Brandon James Paul Shaw, David B Ryves, Helen Glanville, Erica B Young, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4021835/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Fungal endophytes are ubiquitous plant symbionts existing asymptomatically inside plant tissues but playing a crucial role in plant health and function. Endophytes have been extensively studied in many plants, such as agricultural systems, but in carnivorous plants which may engage microbes to aid in digestion, they are poorly understood. To address this deficiency, this study aims to assess the presence and host specificity of fungal endophytes in carnivorous plant traps. Five carnivorous plant species were sampled from two in-situ sites and greenhouse grown plants. Fungal endophytes were isolated from the traps of five carnivorous plant species: Drosera rotundifolia , Drosera anglica , Pinguicula vulgaris , Dionaea muscipula , and Sarracenia purpurea . These represent a range of trapping mechanisms, native and introduced species. We included different carnivorous plant species growing at the same site, and the same species at different sites to test for host and site specificity. Endophytes were cultured on malt extract agar and identified by sequencing the internal transcribed spacer (ITS) region. The isolated fungal endophytes were composed of species from common Ascomycota genera ( Alternaria , Cladosporium , Colletotrichum , and Didymocyrtis ). Fungal endophytes were present in all plant species, this expands our understanding of endophyte distribution in carnivorous plants, representing the first endophytes isolated from D. anglica and D. muscipula and the first endophytes from the traps of D. rotundifolia and P. vulgaris , along with five new fungal endophyte species in carnivorous plants. There was some host specificity; S. purpurea was the only host plant with Colletotrichum spp., whereas Alternaria spp. were found across multiple hosts and sites. The trap endophytes cultured here differed to root endophytes of Drosera and Pinguicula found in previous studies of plants at the same site, suggesting that above and below ground endophytes may differ, perhaps offering different functions in separate plant tissues. Endophytes Carnivorous plants Symbiosis Specificity Culture dependent Figures Figure 1 Introduction 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 8.4 o C) 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 8.7 o C). 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 ✔ Materials and Methods Sampling and Culturing Five individuals of each plant species from each site were collected for fungal endophyte isolation, these were distributed across each site and were within 100 m of each other. Plant tissue was immediately placed in a cool box in the field, and stored in a 4 o C fridge once returned to the laboratory. Within 24 hours of collection, plants were surface sterilised to remove any fungi contaminating the plant surface. This process was carried out underneath a biological safety cabinet to reduce contamination. For Drosera spp, P. vulgaris and D. muscipula any prey or obvious debris was removed from the plant leaves. Sarracenia purpurea pitchers were cut in half length ways and rinsed under running water with dish soap (clover chemicals LTD) for at least 30 minutes, the pitcher was then cut into smaller sections approximately 2.5 cm 2 to facilitate submerging during surface sterilisation. For Drosera spp, P. vulgaris and D. muscipula we used a three-step sterilisation approach modified from Hill et al., 2021 : plants were first submerged in 90% ethanol for 30 seconds, they were then immediately submerged in a 10% bleach solution for 180 seconds, finally they were immediately submerged in 70% ethanol for 60 seconds. For Sarracenia purpurea we followed the approach used by Glenn and Bodri ( 2012 ), because of the need to remove the waxy inner surface: pitcher segments were submerged in 90% ethanol for 60 seconds, then submerged in 10% bleach for 600 seconds, then in 70% ethanol for 30 seconds. Immediately after surface sterilisation, leaves were rinsed in ultrapure water (18.2MΩ-cm) and left at room temperature, under a biological safety cabinet, until the leaf surface was dry. Once dried, small sections of leaf (approx. 5 mm 2 ) were cut out, and both sides were briefly pressed onto malt extract agar (MEA) in a petri dish (‘control plate’) before being placed in a new petri dish with 10% MEA (‘culture plate’). The leaf press control was used to test the success of surface sterilisation. If colonies formed on the control plates, then it was assumed that the surface sterilisation had failed, and it could not be determined whether any fungi growing on culture plates were endophytic. Plates were stored in the dark at room temperature and monitored for fungal growth, checking on plates approximately every three days for two to three weeks. Fungal growth emanating from leaf sections on culture plates were replated onto clean MEA plates so that each plate contained a single fungal isolate. After colonies had grown to a sufficient size for description, they were assigned a morphotype based on observations of macro characteristics of the colony growth, such as colour(s), size, texture. DNA extraction and sequencing Mycelium (0.05 g) was scraped from fungal isolates using a sterile scalpel and placed in 2 ml tubes. A stainless-steel bead was then placed in each tube before freezing in liquid nitrogen and homogenising samples using a tissue lyser. DNA was extracted using the NucleoSpin Plant II kit, following the PL1 pipeline, the only alterations being 1 hour of lysis time and 50 µl of elution buffer used total. PCR amplifications were carried out with ITS primers, the forward primer ITS1(5′-TCCGTAGGTGAACCTGCG) (White et al., 1990 ), and the reverse ITS4 (5′-TCCTCCGCTTATTGATATGC) (White et al., 1990 ) in 20 µl reactions with the following reagents: 10 µl Qmix, 2 µl forward primer (at 5 µM), 2 µl reverse primer (at 5 µM), 4 µl ddH 2 O, and 2 µl DNA. The thermocycler program followed Hill et al., 2021 : 94 o C for 3 minutes followed by 35 cycles of 94 o C for 30 s, 53 o C for 35 s, and 72 o C for 60 s (with 5 seconds added to the 72 o C extension phase per cycle), finally 72 o C for 4 minutes. 4 µl of PCR product was then run on a 1% agarose gel to confirm a successful reaction. DNA extractions were sequenced using Sanger sequencing at NEOF (Sheffield University), after PCR product clean up using BigDye™. Poor quality reads were trimmed from the ends of the forward and reverse sequences by visual inspection on BioEdit (v7.2.5). Forward and reverse sequences were then aligned by ClustalW on MEGA11 (v11.0.13) and merged on AliView (v1.28). Sequences were searched using the nucleotide BLAST algorithm, and the UNITE database (Abarenkov et al. 2024), and in every case, the top hit was recorded. In cases where BLAST and UNITE differed in their identification of the fungal isolate, the database with the greatest % similarity was chosen. Results Culturing Out of 30 leaves, 11 contained culturable fungal endophytes, a total of 24 fungal isolates were obtained, 18 filamentous fungi and 6 yeasts (Table 2 ). Agar plates used as leaf press controls remained free from fungal growth, which confirms leaf surface sterilisation was successful and that fungal isolates were endophytic. Fungal endophytes were predominantly found in greenhouse container-grown S. purpurea (11 out of 24 endophytes), followed by container-grown D. musciupla (4 out of 24 endophytes ), in-situ S. purpurea and P. vulgaris each contained 3 endophytes, D. rotundifolia contained 2 endophytes, and D. anglica contained a single endophyte. Eight morphotypes were distinguished, of 11 plants containing endophytes five contained more than one morphotype in their leaves two of these morphotypes were yeast species, six were filamentous fungi. DNA sequencing PCR products ranging from 500–1000 bp were obtained from all isolates and DNA sequence analysis identified seven species of filamentous fungi from four genera ( Alternaria , Cladosporium, Colletotrichum , an d Didymocyrtis ), all of the filamentous fungi identified are ascomycetes. Sequencing of the yeast morphotypes was unsuccessful, genomic DNA present in the samples prevented successful sequencing of the ITS region, even with serial dilutions of extraction (Table 3 ). Table 2 Fungal cultures isolated from traps of carnivorous plants growing in-situ in the UK and greenhouse grown plants: Fungal isolate Site Host plant Morphotype BP_Da3_E1 Brathay Drosera anglica II BP_P60_E2 Brathay Sarracenia purpurea II BP_P60_E3 Brathay Sarracenia purpurea II BP_P62_E1 Brathay Sarracenia purpurea VI ERY_Pi7_E1 Eryri Pinguicula vulgaris IV ERY_Pi1_E1a Eryri Pinguicula vulgaris I ERY_Pi1_E1b Eryri Pinguicula vulgaris I ERY_Dr9_E1 Eryri Drosera rotundifolia V ERY_Dr9_E2 Eryri Drosera rotundifolia V LO_P3_E3 Greenhouse Sarracenia purpurea II LO_P0_E5 Greenhouse Sarracenia purpurea III LO_P0_E12 Greenhouse Sarracenia purpurea IV LO_P0_E1 Greenhouse Sarracenia purpurea I LO_P0_E8 Greenhouse Sarracenia purpurea I LO_P0_E3 Greenhouse Sarracenia purpurea VII LO_P0_E7 Greenhouse Sarracenia purpurea III LO_P0_E6 Greenhouse Sarracenia purpurea VIII LO_P0_E4 Greenhouse Sarracenia purpurea VII LO_P0_E9 Greenhouse Sarracenia purpurea IV LO_P2_E1 Greenhouse Sarracenia purpurea V LO_Vft2_E1 Greenhouse Dionaea muscipula I LO_Vft2_E2 Greenhouse Dionaea muscipula I LO_Vft2_E3 Greenhouse Dionaea muscipula III LO_Vft2_E4 Greenhouse Dionaea muscipula VIII Table 3 Sequence analysis of ITS region of fungal isolates from carnivorous plants growing in-situ in the UK and greenhouse grown plants. Isolate Host Site Top match Top match accession Max identity (%) Sequence NCBI Accession BP_Da3_E1 D. anglica Brathay Cladosporium allicinum MT573471.1 100 PP437089 BP_P60_E2 S. purpurea Brathay Colletotrichum spaethianum KP127987.1 100 PP437088 BP_P60_E3 S. purpurea Brathay Colletotrichum spaethianum KP127987.1 99.41 PP437090 BP_P62_E1 S. purpurea Brathay Colletotrichum spaethianum KP127987.1 99.8 PP437100 ERY_Pi7_E1 P. vulgaris Eryri N/A ERY_Pi1_E1a P. vulgaris Eryri Alternaria infectoria MT548683.1 100 PP437092 ERY_Pi1_E1b P. vulgaris Eryri Alternaria infectoria MT548683.1 100 PP437098 ERY_Dr9_E1 D. rotundifolia Eryri N/A ERY_Dr9_E2 D. rotundifolia Eryri N/A LO_P3_E3 S. purpurea Greenhouse Didymocyrtis cladoniicola Unite: UDB0801806 91.94 PP437086 LO_P0_E1 S. purpurea Greenhouse Alternaria infectoria MT635276.1 99.78 PP437091 LO_P0_E3 S. purpurea Greenhouse Alternaria infectoria MN534845.1 99.81 PP437094 LO_P0_E4 S. purpurea Greenhouse Colletotrichum acutatum MT364496.1 97.58 PP437099 LO_P0_E5 S. purpurea Greenhouse Alternaria infectoria MT573465.1 99.79 PP437087 LO_P0_E6 S. purpurea Greenhouse Cladosporium cladosporioides Unite: UDB0799158 99.01 PP437097 LO_P0_E7 S. purpurea Greenhouse Alternaria infectoria MT561399.1 99.81 PP437095 LO_P0_E8 S. purpurea Greenhouse Alternaria conjuncta MH861940.1 100 PP437093 LO_P0_E9 S, purpurea Greenhouse N/A LO_P0_E12 S. purpurea Greenhouse N/A LO_P2_E1 S. purpurea Greenhouse N/A LO_Vft2_E1 D. muscipula Greenhouse Alternaria infectoria MN534845.1 99.64 PP437102 LO_Vft2_E2 D. muscipula Greenhouse Alternaria infectoria MN534845.1 99.81 PP437085 LO_Vft2_E3 D. muscipula Greenhouse Alternaria infectoria MK911688.1 100 PP437096 LO_Vft2_E4 D. muscipula Greenhouse Cladosporium allicinum OW982756.1 99.6 PP437101 Discussion We found culturable fungal endophytes in the traps of all the species of carnivorous plant we investigated. This is the first study to explore these associations in the trapping mechanisms of European carnivorous plants. We found some evidence of host specificity of fungal endophytes between co-occurring carnivorous plant species: Sarracenia purpurea was the only plant to contain Colletotrichum spp., suggesting an affinity for pitcher plants from these endophytes. Common species such as Alternaria spp. were present in multiple hosts and across sites. These data extend existing knowledge of carnivorous plant associated fungal endophytes with the first identification of fungal endophytes in Drosera anglica and Dionaea muscipula and trap endophytes in Drosera rotundifolia and Pinguicula vulgaris . We confirm previous studies demonstrating that carnivorous plants contain fungal endophytes (Quilliam and Jones 2012 ; Lee, Ting, and Tan 2014; Glenn and Bodri 2012 ; Rueda-Almazán et al. 2021; Cheon et al. 2016 ) and add to a growing understanding that fungal endophytes are ubiquitous in carnivorous plant traps. We also report five new endophyte species known to infect carnivorous plants, contributing to a growing knowledge of the diversity of plant-fungal interactions in this unique group of plants. The endophytes identified are primarily common species, three of the four genera have previously been reported from carnivorous plants growing in French Guiana, India, Malaysia, Mexico, South Korea, the USA, and Wales: Alternaria (Naseem et al., 2021 ), Cladosporium (Leroy et al., 2021 ; Naseem et al., 2021 ; Quilliam and Jones 2012 ; Rueda-Almazán et al. , 2021; Cheon et al., 2016 )), and Colletotrichum (Leroy et al., 2021 ; Naseem et al., 2021 ; Lee, et al., 2014 ; Rueda-Almazán et al. , 2021; Glenn and Bodri 2012 ; Cheon et al., 2016 ). All of the species identified in this study have previously been identified as endophytes of non-carnivorous plant species including, tree, grass, and crop species (Supplementary table 1 ). Five species are also known pathogens of plants, these same five species are also species complexes (species delimitation that likely contains multiple species but identical morphology and insuficcient molecular techniques prevents resolution) (Supplementary table 1 ). Our study adds Alternaria infectoria , A. conjuncta , Cladosporium allicinum, C. cladosporioides , and Didymocyrtis cladoniicola to the list of known endophytes of carnivorous plants, providing more evidence to suggest that carnivorous plants host a wide variety of endophyte species. Colletotrichum acutatum and Colletotrichum spaethianum have both previously been reported from carnivorous plants: C. acutatum has been reported from the leaves of S. purpurea in the USA (Leroy et al., 2021 ; Glenn and Bodri 2012 ), and now S. purpurea in the UK. Colletotrichum spaethianum has been reported from the roots of Utricularia racemosa in South Korea (Cheon et al., 2016 ) and was found in S. purpurea in this study. A key question for fungal endophyte research is whether endophytes are host specific, the variety of functional traits (trapping mechanisms) in carnivorous plants are a useful study to test this. Using similar culturing methods to ours, Quilliam and Jones ( 2012 ) found evidence for host specificity in the root endophytes of D. rotundifolia and P. vulgaris growing at the same site. Specifically, only Trichoderma was isolated from the roots of both plant species, all other endophytes (4 species from Pinguicula and 7 from Drosera ) were specific to their host plant. We did not find any of the same endophytes in the traps of these species as Quilliam and Jones ( 2012 ) found in the roots. This suggests that above ground and below ground endophyte communities differ in these plants, previous studies have found similar differences (Guevara-Araya et al., 2020 ; Martins et al., 2016 ), perhaps caused by differing functional needs of the above- and below-ground plant tissues, in this case above ground aiding carnivorous function and below ground survival in waterlogged, low pH substrate. While Alternaria infectoria was found in P. vulgaris at this site and not D. rotundifolia , it is hard to see this as evidence for host specificity considering A. infectoria was found in other plant species at Brathay and in container grown plants, with A. infectoria being the most common isolate found in this study (9 of 24 isolates). This study found Colletotrichum spp. present only in Sarracenia purpurea at Brathay plantation and in greenhouse grown plants, in particular, C. acutatum has now been observed in S. purpurea ’s native range in the USA and in plants in the UK, suggesting C. acutatum may be considered a true endophyte of this species. Colletotrichum spp. have been found in other pitcher plants, Catopsis berteroniana , and Nepenthes spp (Lee, et al., 2014 ; Leroy et al., 2021 ; Naseem et al., 2021 ), perhaps Colletotrichum spp. are better adapted as pitcher plant endophytes over other carnivorous plant trap types. Fungal endophytes are known to provide a variety of functions to their host plants through the production of secondary metabolites. Alternaria infectoria and Cladosporium cladosporioides are both known to produce antimicrobial secondary metabolites (Casella et al., 2013 ; Scott et al., 1971 ), broadly these can be used by plants for pathogen resistance (Collinge, et al., 2022 ). It has previously been shown that carnivorous plants synthesise metabolites with antimicrobial properties, which may serve a prey preservation function (Hatcher, et al., 2020 ). The intriguing possibility is that these antimicrobial metabolites are synthesised by fungal endophytes, rather than the plant itself and may also represent a source of novel compounds. Additionally, fungi inhabiting the mucilage secretions of Drosera enhance prey digestion (Sun et al., 2023 ), it seems possible that fungi inhabiting these plants endophytically would also be able to contribute to prey digestion via enzyme production. Other fungi are known to inhabit the water contained within carnivorous pitcher plants forming part of a food web that aids in prey digestion, this is seen in Sarracenia purpurea and Nepenthes (Grothjan and Young 2019 ; Leonora S. Bittleston et al., 2018 ). Inquiline fungi are able to contribute to plant carnivory via prey digestion, it seems plausible that endophytes may also help with digestion, or perhaps inquiline fungi are capable of existing as endophytes within plant tissue. The culture-dependent approach revealed a low species diversity of endophytes in carnivorous plants. This is not consistent with expectations of plants in extreme environments hosting unique endophytes to mitigate extreme stresses. In carnivorous plants we may expect to see endophytes aiding in survival in a low nutrient environment. A culture dependent approach for carnivorous plants presents extra challenges owing to the nature of their leaves: Drosera spp. and P. vulgaris have mucosal leaves which may degrade quickly or be permeable during surface sterilisation, resulting in endophyte death; S. purpurea has a waxy layer over the inside of its pitcher leaves which, if not thoroughly removed, can block endophyte emergence during culturing. Additionally, many fungi may not be culturable at all (Siddique, et al., 2017 ; Dissanayake et al., 2018 ) or require more specialised media to grow. Growth media containing benomyl, for example, facilitates growth of basidiomycetes (Thorn et al., 1996 ) which are frequently underrepresented in culture dependent methods. High throughput next generation sequencing methods are increasingly being used in endophyte studies, and reveal more diverse communities than previously thought(Donald et al. , 2020; Siddique, et al., 2017 ; Dissanayake et al., 2018 ). These methods are not limited by culturing success and may prove useful to test hypotheses of endophyte diversity and host/ site specificity in the future. To understand the ecological role of these endophytes in plant carnivory multi-omics approaches and EpicPCR can show what fungal produced metabolites are used in plant carnivory. Conclusion In this study, for the first time, we cultured trap fungal endophytes from in-situ grown carnivorous plant species in the UK. We add to growing understanding of the ubiquity and diversity of carnivorous plant endophytes by reporting the presence of five new species of carnivorous plant endophytes. In addition, endophytes are reported for the first time from Drosera anglica and Dionaea muscipula . We found some evidence of host specificity between trap types from Colletotrichum spp. only infecting Sarracenia purpurea . Our understanding of the mechanisms of plant carnivory is incomplete. It seems likely that fungal endophytes play a key role in the various processes of carnivory: prey attraction, capture, and digestion. We have a limited knowledge of the functional role and diversity of endophytes in plant carnivory, this must be a priority for future research for Darwin’s “most wonderful plants in the World”. Statements & Declarations For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. This work was supported by Central England NERC Training Alliance (CENTA) PhD studentship (NE/S007350/1) and the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). All sequence data is deposited in NCBI, accession numbers detailed in Table 3 . The Authors have no relevant financial or non-financial interests to disclose. Author contributions as follows, Brandon Shaw : Conceptualization, Funding acquisition, Methodology, Investigation, Writing - Original Draft Jonathan Millett : Conceptualization, Funding acquisition, Supervision, Writing - Review & Editing David Ryves : Conceptualization, Supervision, Writing - Review & Editing Helen Glanville : Supervision, Writing - Review & Editing Erica Young : Supervision, Writing - Review & Editing. Declarations For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. This work was supported by Central England NERC Training Alliance (CENTA) PhD studentship (NE/S007350/1) and the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). All sequence data is deposited in NCBI, accession numbers detailed in Table 3. The Authors have no relevant financial or non-financial interests to disclose. Author contributions as follows, Brandon Shaw: Conceptualization, Funding acquisition, Methodology, Investigation, Writing - Original Draft Jonathan Millett: Conceptualization, Funding acquisition, Supervision, Writing - Review & Editing David Ryves: Conceptualization, Supervision, Writing - Review & Editing Helen Glanville: Supervision, Writing - Review & Editing Erica Young: Supervision, Writing - Review & Editing. Acknowledgements: Laboratory work was supported by the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). The authors would like to thank the technical staff of the facility: Tom Holden; Lucy Knowles; Gavin Horsburgh for their excellent support during the DNA extraction, amplification, and sequencing process. Thanks to the National Trust and private landowners for permission to access and sample the plants. 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PCR Protocols Supplementary Files SupplementaryTable1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 Aug, 2024 Reviewers invited by journal 10 May, 2024 Editor invited by journal 05 Apr, 2024 Editor assigned by journal 27 Mar, 2024 First submitted to journal 26 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4021835","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301094711,"identity":"d8e6478a-023c-423d-8b01-2cc5d812bc12","order_by":0,"name":"Brandon James Paul 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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 \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Molina-Montenegro et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lugtenberg et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bittleston et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 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., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The presence of endophytes in the fossil record of early land plants suggests co-evolution (Krings \u003cem\u003eet al.\u003c/em\u003e, 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.\u003c/p\u003e \u003cp\u003eCarnivorous 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 \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fleischmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Plant carnivory has evolved independently at least 11 times across the angiosperm phylogeny (Fleischmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with striking examples of convergent and divergent evolution of trapping mechanisms (Ellison and Gotelli, 2009; Thorogood \u003cem\u003eet al.\u003c/em\u003e, 2018). Carnivorous plants are typically found in extreme conditions, living in generally nutrient poor wetland environments, often with low pH (Pavlovič and Saganov\u0026aacute; \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the 150 years since Charles Darwin published his book \u0026lsquo;Insectivorous Plants\u0026rsquo; (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 \u0026ndash; fungal endophyte associations exist. For example, new research has demonstrated that \u003cem\u003eAcrodontium crateriforme, a\u003c/em\u003e fungus living in secretions of traps of \u003cem\u003eDrosera spatulata\u003c/em\u003e 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., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is not known, however, whether \u003cem\u003eA. crateriforme\u003c/em\u003e exists endophytically inside \u003cem\u003eDrosera\u003c/em\u003e, 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., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), contain a unique assembly of endophytes which aid survival in extreme conditions (Chadha et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Knowledge of carnivorous plant fungal endophytes is, however, remarkably limited, considering our understanding of endophyte importance in other plants (Collinge et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Eid et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), perhaps owing to sampling difficulties in remote wetland environments, or difficulties with culturing efforts.\u003c/p\u003e \u003cp\u003eOver 800 carnivorous plant species have been described (Fleischmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), but remarkably few have been studied for their fungal endophytes: four species of \u003cem\u003eSarracenia\u003c/em\u003e (Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), three species of \u003cem\u003eNepenthes\u003c/em\u003e (Lee, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), two species of \u003cem\u003ePinguicula\u003c/em\u003e (Quilliam and Jones \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rueda-Almaz\u0026aacute;n \u003cem\u003eet al.\u003c/em\u003e, 2021), and one species each of \u003cem\u003eCatopsis\u003c/em\u003e (Leroy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eDrosera\u003c/em\u003e (Quilliam and Jones 2010), \u003cem\u003eand Utricularia\u003c/em\u003e (Cheon et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 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 \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eColletotrichum, Penicillium\u003c/em\u003e, and \u003cem\u003eCladosporium\u003c/em\u003e (Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rueda-Almaz\u0026aacute;n \u003cem\u003eet al.\u003c/em\u003e, 2021). Some fungal endophytes have been found across different plant genera, for example \u003cem\u003eColletotrichum gloeosporoides\u003c/em\u003e has been found in both \u003cem\u003eSarracenia\u003c/em\u003e and \u003cem\u003eNepenthes\u003c/em\u003e (Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lee, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), both of which are independently evolved pitfall trap pitcher plants (Thorogood, \u003cem\u003eet al.\u003c/em\u003e,2018). It has been suggested that \u003cem\u003eC. gloeosporoides\u003c/em\u003e may be an endophyte adapted to life as a pitcher plant symbiont (Lee, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom these studies some insight into the ecology of plant-endophyte associations has been gained. Quilliam and Jones (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) were able to demonstrate host specificity in the root endophytes of \u003cem\u003eDrosera\u003c/em\u003e and \u003cem\u003ePinguicula\u003c/em\u003e at a single site in North Wales. Lee \u003cem\u003eet al.\u003c/em\u003e,, (2014) studied the endophytes of two different \u003cem\u003eNepenthes\u003c/em\u003e species growing at the same site in Malaysia and found low endophyte diversity in one of their study species. Rueda-Almaz\u0026aacute;n \u003cem\u003eet al.\u003c/em\u003e, 2021 studied the endophytes of the traps and roots of \u003cem\u003ePinguicula moranensis\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the culturable endophyte community from traps of five species of carnivorous plant growing in the United Kingdom, at \u003cem\u003etwo in-situ\u003c/em\u003e sites and greenhouse grown: \u003cem\u003eDrosera rotundifolia\u003c/em\u003e, \u003cem\u003eD. anglica\u003c/em\u003e, \u003cem\u003ePinguicula vulgaris\u003c/em\u003e, \u003cem\u003eDionaea muscipula\u003c/em\u003e, and \u003cem\u003eSarracenia purpurea\u003c/em\u003e. These species represent different trapping mechanisms (flypaper traps: \u003cem\u003eD. rotundifolia\u003c/em\u003e, \u003cem\u003eD. anglica\u003c/em\u003e, and \u003cem\u003eP. vulgaris\u003c/em\u003e; Pitfall: \u003cem\u003eS. purpurea\u003c/em\u003e; Snap trap: \u003cem\u003eD. muscipula\u003c/em\u003e), native (\u003cem\u003eD. rotundifolia\u003c/em\u003e, \u003cem\u003eD. anglica\u003c/em\u003e, and \u003cem\u003eP. vulgaris\u003c/em\u003e) and introduced (\u003cem\u003eS. purpurea\u003c/em\u003e, Walker, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) 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.\u003c/p\u003e \u003cp\u003eStudy species and sites\u003c/p\u003e \u003cp\u003eDetails of study species are as follows (summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cem\u003eSarracenia purpurea\u003c/em\u003e 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., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) attracted to nectar produced in extrafloral nectaries (Bennett and Ellison \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), prey fall into the pitcher and cannot escape due to downward pointing hairs and waxy surfaces (Heard \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Prey digestion is primarily undertaken by a microbial-dominated inquiline community living in the rainwater which collects in the pitcher (Heard \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). \u003cem\u003eSarracenia purpurea\u003c/em\u003e 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 \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), 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 \u003cem\u003eSarracenia\u003c/em\u003e in North America (Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), using culturing methods. They found that of four \u003cem\u003eSarracenia\u003c/em\u003e species \u003cem\u003eS. purpurea\u003c/em\u003e contained the highest diversity of endophytes, containing six culturable endophyte species, the three other \u003cem\u003eSarracenia\u003c/em\u003e species containing four or fewer species. No studies exist of the endophytes of \u003cem\u003eSarracenia\u003c/em\u003e in its introduced range in the UK.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDrosera rotundifolia\u003c/em\u003e L. and \u003cem\u003eDrosera anglica\u003c/em\u003e Huds. (Caryophyllales) are short-lived carnivorous plants (Crowder et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) 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 \u003cem\u003eet al.\u003c/em\u003e,, 2014). Once trapped their leaves close around the prey to form an external stomach, into which digestive enzymes (such as chitinolytic enzymes; Libantov\u0026aacute; et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) are excreted. Both \u003cem\u003eDrosera rotundifolia\u003c/em\u003e and \u003cem\u003eD. anglica\u003c/em\u003e have cosmopolitan circumboreal distributions are often co-occurring and have similar habitat affinities, growing in low nutrient, low pH conditions on \u003cem\u003eSphagnum\u003c/em\u003e dominated raised bogs (Crowder et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). \u003cem\u003eDrosera anglica\u003c/em\u003e is found in wetter microhabitats than \u003cem\u003eD. rotundifolia\u003c/em\u003e (Nordbakken \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). No studies of the trap endophytes of these species have been previously conducted. Root endophytes of \u003cem\u003eD. rotundifolia\u003c/em\u003e have previously been studied from one site in North Wales (Quilliam and Jones 2010).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePinguicula vulgaris\u003c/em\u003e 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 \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Prey are digested through the secretion of digestive enzymes, the main prey captured are Collembola and Nematocera (Karlsson, \u003cem\u003eet al.\u003c/em\u003e,1994). \u003cem\u003eP. vulgaris\u003c/em\u003e 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 \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). No studies of the trap endophytes of \u003cem\u003eP. vulgaris\u003c/em\u003e have been previously conducted. Root endophytes of \u003cem\u003eP. vulgaris\u003c/em\u003e have been studied from one site in North Wales (Quilliam and Jones \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and found that despite using the same trapping mechanism as \u003cem\u003eD. rotundifolia\u003c/em\u003e, each plant\u0026rsquo;s roots hosted unique endophytes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDionaea muscipula\u003c/em\u003e J. Ellis (Caryophyllales) uses a snap-trap mechanism to trap predominantly spiders and larger insects, \u003cem\u003eD. muscipula\u003c/em\u003e shares a common \u003cem\u003eDrosera\u003c/em\u003e-like carnivorous ancestor with \u003cem\u003eDrosera\u003c/em\u003e 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., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), creating an external stomach into which digestive enzymes are secreted. \u003cem\u003eDionaea muscipula\u003c/em\u003e has a very narrow habitat affinity, restricting the plant to habitats in the coastal plains and sandhills of North and South Carolina (Luken \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). 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 \u003cem\u003eDionaea\u003c/em\u003e endophytes.\u003c/p\u003e \u003cp\u003ePlants were sampled from two \u003cem\u003ein-situ\u003c/em\u003e locations, and one \u003cem\u003eex-situ\u003c/em\u003e: Brathay Plantation (Lat: 54.403\u0026deg;N, Long: -2.982\u0026deg;E, mean annual rainfall 1,390 mm, mean annual temperature 8.4\u003csup\u003eo\u003c/sup\u003eC) is a small minerotrophic mire, from which we collected co-occurring \u003cem\u003eS. purpurea\u003c/em\u003e and \u003cem\u003eD. anglica\u003c/em\u003e and \u003cem\u003eD. rotundifolia\u003c/em\u003e in July 2022. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At Eryri National Park (Snowdonia National Park) we sampled plants from a seepage mire on a steep hillside (Long: -4.011\u0026deg;E, Lat: 53.121\u0026deg;N, mean annual rainfall 1,423 mm, mean annual temperature 8.7\u003csup\u003eo\u003c/sup\u003eC). Here we collected co-occurring \u003cem\u003eD. rotundifolia\u003c/em\u003e and \u003cem\u003eP. vulgaris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in June 2022 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This same population has previously been studied, to investigate root endophytes (Quilliam and Jones 2010, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These locations were chosen because of the presence of different species of carnivorous plant in close proximity, and because \u003cem\u003eD. rotundifolia\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eGreenhouse plants: A number of \u003cem\u003eS. purpurea\u003c/em\u003e have been kept at Loughborough University for research purposes. These have been collected from \u003cem\u003enumerous in-situ\u003c/em\u003e 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 \u003cem\u003efrom in-situ\u003c/em\u003e plants in 2022. Additionally, a number of \u003cem\u003eDionaea muscipula\u003c/em\u003e were briefly kept for scientific purposes, plants were used to confirm the presence of endophytes in \u003cem\u003eD. muscipula\u003c/em\u003e, plants were sourced from a nursery.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy species information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHost species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrder\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTrapping mechanism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEricales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePitfall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDrosera rotundifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaryophyllales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlypaper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDrosera anglica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaryophyllales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlypaper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePinguicula vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLamiales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlypaper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDionaea muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaryophyllales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSnap trap\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e✔\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSampling and Culturing\u003c/p\u003e \u003cp\u003eFive individuals of each plant species from each site were collected for fungal endophyte isolation, these were distributed across each site and were within 100 m of each other. Plant tissue was immediately placed in a cool box in the field, and stored in a 4\u003csup\u003eo\u003c/sup\u003eC fridge once returned to the laboratory. Within 24 hours of collection, plants were surface sterilised to remove any fungi contaminating the plant surface. This process was carried out underneath a biological safety cabinet to reduce contamination. For \u003cem\u003eDrosera\u003c/em\u003e spp, \u003cem\u003eP. vulgaris\u003c/em\u003e and \u003cem\u003eD. muscipula\u003c/em\u003e any prey or obvious debris was removed from the plant leaves. \u003cem\u003eSarracenia purpurea\u003c/em\u003e pitchers were cut in half length ways and rinsed under running water with dish soap (clover chemicals LTD) for at least 30 minutes, the pitcher was then cut into smaller sections approximately 2.5 cm\u003csup\u003e2\u003c/sup\u003e to facilitate submerging during surface sterilisation. For \u003cem\u003eDrosera\u003c/em\u003e spp, \u003cem\u003eP. vulgaris\u003c/em\u003e and \u003cem\u003eD. muscipula\u003c/em\u003e we used a three-step sterilisation approach modified from Hill et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e: plants were first submerged in 90% ethanol for 30 seconds, they were then immediately submerged in a 10% bleach solution for 180 seconds, finally they were immediately submerged in 70% ethanol for 60 seconds. For \u003cem\u003eSarracenia purpurea\u003c/em\u003e we followed the approach used by Glenn and Bodri (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), because of the need to remove the waxy inner surface: pitcher segments were submerged in 90% ethanol for 60 seconds, then submerged in 10% bleach for 600 seconds, then in 70% ethanol for 30 seconds.\u003c/p\u003e \u003cp\u003eImmediately after surface sterilisation, leaves were rinsed in ultrapure water (18.2MΩ-cm) and left at room temperature, under a biological safety cabinet, until the leaf surface was dry. Once dried, small sections of leaf (approx. 5 mm\u003csup\u003e2\u003c/sup\u003e) were cut out, and both sides were briefly pressed onto malt extract agar (MEA) in a petri dish (\u0026lsquo;control plate\u0026rsquo;) before being placed in a new petri dish with 10% MEA (\u0026lsquo;culture plate\u0026rsquo;). The leaf press control was used to test the success of surface sterilisation. If colonies formed on the control plates, then it was assumed that the surface sterilisation had failed, and it could not be determined whether any fungi growing on culture plates were endophytic. Plates were stored in the dark at room temperature and monitored for fungal growth, checking on plates approximately every three days for two to three weeks. Fungal growth emanating from leaf sections on culture plates were replated onto clean MEA plates so that each plate contained a single fungal isolate. After colonies had grown to a sufficient size for description, they were assigned a morphotype based on observations of macro characteristics of the colony growth, such as colour(s), size, texture.\u003c/p\u003e \u003cp\u003eDNA extraction and sequencing\u003c/p\u003e \u003cp\u003eMycelium (0.05 g) was scraped from fungal isolates using a sterile scalpel and placed in 2 ml tubes. A stainless-steel bead was then placed in each tube before freezing in liquid nitrogen and homogenising samples using a tissue lyser. DNA was extracted using the NucleoSpin Plant II kit, following the PL1 pipeline, the only alterations being 1 hour of lysis time and 50 \u0026micro;l of elution buffer used total. PCR amplifications were carried out with ITS primers, the forward primer ITS1(5\u0026prime;-TCCGTAGGTGAACCTGCG) (White et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), and the reverse ITS4 (5\u0026prime;-TCCTCCGCTTATTGATATGC) (White et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) in 20 \u0026micro;l reactions with the following reagents: 10 \u0026micro;l Qmix, 2 \u0026micro;l forward primer (at 5 \u0026micro;M), 2 \u0026micro;l reverse primer (at 5 \u0026micro;M), 4 \u0026micro;l ddH\u003csub\u003e2\u003c/sub\u003eO, and 2 \u0026micro;l DNA. The thermocycler program followed Hill et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e: 94\u003csup\u003eo\u003c/sup\u003eC for 3 minutes followed by 35 cycles of 94\u003csup\u003eo\u003c/sup\u003eC for 30 s, 53\u003csup\u003eo\u003c/sup\u003eC for 35 s, and 72\u003csup\u003eo\u003c/sup\u003eC for 60 s (with 5 seconds added to the 72\u003csup\u003eo\u003c/sup\u003eC extension phase per cycle), finally 72\u003csup\u003eo\u003c/sup\u003eC for 4 minutes. 4 \u0026micro;l of PCR product was then run on a 1% agarose gel to confirm a successful reaction.\u003c/p\u003e \u003cp\u003eDNA extractions were sequenced using Sanger sequencing at NEOF (Sheffield University), after PCR product clean up using BigDye\u0026trade;. Poor quality reads were trimmed from the ends of the forward and reverse sequences by visual inspection on BioEdit (v7.2.5). Forward and reverse sequences were then aligned by ClustalW on MEGA11 (v11.0.13) and merged on AliView (v1.28). Sequences were searched using the nucleotide BLAST algorithm, and the UNITE database (Abarenkov et al. 2024), and in every case, the top hit was recorded. In cases where BLAST and UNITE differed in their identification of the fungal isolate, the database with the greatest % similarity was chosen.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCulturing\u003c/p\u003e \u003cp\u003eOut of 30 leaves, 11 contained culturable fungal endophytes, a total of 24 fungal isolates were obtained, 18 filamentous fungi and 6 yeasts (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Agar plates used as leaf press controls remained free from fungal growth, which confirms leaf surface sterilisation was successful and that fungal isolates were endophytic. Fungal endophytes were predominantly found in greenhouse container-grown \u003cem\u003eS. purpurea\u003c/em\u003e (11 out of 24 endophytes), followed by container-grown \u003cem\u003eD. musciupla\u003c/em\u003e (4 out of 24 endophytes\u003cem\u003e), in-situ S. purpurea\u003c/em\u003e and \u003cem\u003eP. vulgaris\u003c/em\u003e each contained 3 endophytes, \u003cem\u003eD. rotundifolia\u003c/em\u003e contained 2 endophytes, and \u003cem\u003eD. anglica\u003c/em\u003e contained a single endophyte. Eight morphotypes were distinguished, of 11 plants containing endophytes five contained more than one morphotype in their leaves two of these morphotypes were yeast species, six were filamentous fungi.\u003c/p\u003e \u003cp\u003eDNA sequencing\u003c/p\u003e \u003cp\u003ePCR products ranging from 500\u0026ndash;1000 bp were obtained from all isolates and DNA sequence analysis identified seven species of filamentous fungi from four genera (\u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eCladosporium, Colletotrichum\u003c/em\u003e, an\u003cem\u003ed Didymocyrtis\u003c/em\u003e), all of the filamentous fungi identified are ascomycetes. Sequencing of the yeast morphotypes was unsuccessful, genomic DNA present in the samples prevented successful sequencing of the ITS region, even with serial dilutions of extraction (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFungal cultures isolated from traps of carnivorous plants growing \u003cem\u003ein-situ\u003c/em\u003e in the UK and greenhouse grown plants:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungal isolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHost plant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMorphotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_Da3_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDrosera anglica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P60_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P60_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P62_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi7_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinguicula vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi1_E1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinguicula vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi1_E1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinguicula vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Dr9_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDrosera rotundifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Dr9_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDrosera rotundifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P3_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVIII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P2_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSarracenia purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDionaea muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDionaea muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDionaea muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDionaea muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVIII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence analysis of ITS region of fungal isolates from carnivorous plants growing \u003cem\u003ein-situ\u003c/em\u003e in the UK and greenhouse grown plants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTop match\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTop match accession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMax identity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSequence NCBI Accession\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_Da3_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. anglica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCladosporium allicinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT573471.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437089\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P60_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum spaethianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKP127987.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P60_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum spaethianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKP127987.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP_P62_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrathay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum spaethianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKP127987.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi7_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi1_E1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT548683.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Pi1_E1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT548683.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437098\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Dr9_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. rotundifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERY_Dr9_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. rotundifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEryri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P3_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDidymocyrtis cladoniicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnite: UDB0801806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e91.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT635276.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMN534845.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437094\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum acutatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT364496.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT573465.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437087\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCladosporium cladosporioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnite: UDB0799158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437097\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT561399.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437095\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria conjuncta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMH861940.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437093\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS, purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P0_E12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_P2_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMN534845.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMN534845.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAlternaria infectoria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMK911688.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO_Vft2_E4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD. muscipula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCladosporium allicinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOW982756.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePP437101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found culturable fungal endophytes in the traps of all the species of carnivorous plant we investigated. This is the first study to explore these associations in the trapping mechanisms of European carnivorous plants. We found some evidence of host specificity of fungal endophytes between co-occurring carnivorous plant species: \u003cem\u003eSarracenia purpurea\u003c/em\u003e was the only plant to contain \u003cem\u003eColletotrichum\u003c/em\u003e spp., suggesting an affinity for pitcher plants from these endophytes. Common species such as \u003cem\u003eAlternaria\u003c/em\u003e spp. were present in multiple hosts and across sites. These data extend existing knowledge of carnivorous plant associated fungal endophytes with the first identification of fungal endophytes in \u003cem\u003eDrosera anglica\u003c/em\u003e and \u003cem\u003eDionaea muscipula\u003c/em\u003e and trap endophytes in \u003cem\u003eDrosera rotundifolia\u003c/em\u003e and \u003cem\u003ePinguicula vulgaris\u003c/em\u003e. We confirm previous studies demonstrating that carnivorous plants contain fungal endophytes (Quilliam and Jones \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lee, Ting, and Tan 2014; Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rueda-Almaz\u0026aacute;n et al. 2021; Cheon et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and add to a growing understanding that fungal endophytes are ubiquitous in carnivorous plant traps. We also report five new endophyte species known to infect carnivorous plants, contributing to a growing knowledge of the diversity of plant-fungal interactions in this unique group of plants.\u003c/p\u003e \u003cp\u003eThe endophytes identified are primarily common species, three of the four genera have previously been reported from carnivorous plants growing in French Guiana, India, Malaysia, Mexico, South Korea, the USA, and Wales: \u003cem\u003eAlternaria\u003c/em\u003e (Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eCladosporium\u003c/em\u003e (Leroy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Quilliam and Jones \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rueda-Almaz\u0026aacute;n \u003cem\u003eet al.\u003c/em\u003e, 2021; Cheon et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)), and \u003cem\u003eColletotrichum\u003c/em\u003e (Leroy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rueda-Almaz\u0026aacute;n \u003cem\u003eet al.\u003c/em\u003e, 2021; Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cheon et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All of the species identified in this study have previously been identified as endophytes of non-carnivorous plant species including, tree, grass, and crop species (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Five species are also known pathogens of plants, these same five species are also species complexes (species delimitation that likely contains multiple species but identical morphology and insuficcient molecular techniques prevents resolution) (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our study adds \u003cem\u003eAlternaria infectoria\u003c/em\u003e, \u003cem\u003eA. conjuncta\u003c/em\u003e, \u003cem\u003eCladosporium allicinum, C. cladosporioides\u003c/em\u003e, and \u003cem\u003eDidymocyrtis cladoniicola\u003c/em\u003e to the list of known endophytes of carnivorous plants, providing more evidence to suggest that carnivorous plants host a wide variety of endophyte species. \u003cem\u003eColletotrichum acutatum\u003c/em\u003e and \u003cem\u003eColletotrichum spaethianum\u003c/em\u003e have both previously been reported from carnivorous plants: \u003cem\u003eC. acutatum\u003c/em\u003e has been reported from the leaves of \u003cem\u003eS. purpurea\u003c/em\u003e in the USA (Leroy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Glenn and Bodri \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and now \u003cem\u003eS. purpurea\u003c/em\u003e in the UK. \u003cem\u003eColletotrichum spaethianum\u003c/em\u003e has been reported from the roots of \u003cem\u003eUtricularia racemosa\u003c/em\u003e in South Korea (Cheon et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and was found in \u003cem\u003eS. purpurea\u003c/em\u003e in this study.\u003c/p\u003e \u003cp\u003eA key question for fungal endophyte research is whether endophytes are host specific, the variety of functional traits (trapping mechanisms) in carnivorous plants are a useful study to test this. Using similar culturing methods to ours, Quilliam and Jones (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found evidence for host specificity in the root endophytes of \u003cem\u003eD. rotundifolia\u003c/em\u003e and \u003cem\u003eP. vulgaris\u003c/em\u003e growing at the same site. Specifically, only \u003cem\u003eTrichoderma\u003c/em\u003e was isolated from the roots of both plant species, all other endophytes (4 species from \u003cem\u003ePinguicula\u003c/em\u003e and 7 from \u003cem\u003eDrosera\u003c/em\u003e) were specific to their host plant. We did not find any of the same endophytes in the traps of these species as Quilliam and Jones (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found in the roots. This suggests that above ground and below ground endophyte communities differ in these plants, previous studies have found similar differences (Guevara-Araya et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Martins et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), perhaps caused by differing functional needs of the above- and below-ground plant tissues, in this case above ground aiding carnivorous function and below ground survival in waterlogged, low pH substrate. While \u003cem\u003eAlternaria infectoria\u003c/em\u003e was found in \u003cem\u003eP. vulgaris\u003c/em\u003e at this site and not \u003cem\u003eD. rotundifolia\u003c/em\u003e, it is hard to see this as evidence for host specificity considering \u003cem\u003eA. infectoria\u003c/em\u003e was found in other plant species at Brathay and in container grown plants, with \u003cem\u003eA. infectoria\u003c/em\u003e being the most common isolate found in this study (9 of 24 isolates). This study found \u003cem\u003eColletotrichum\u003c/em\u003e spp. present only in \u003cem\u003eSarracenia purpurea\u003c/em\u003e at Brathay plantation and in greenhouse grown plants, in particular, \u003cem\u003eC. acutatum\u003c/em\u003e has now been observed in \u003cem\u003eS. purpurea\u003c/em\u003e\u0026rsquo;s native range in the USA and in plants in the UK, suggesting \u003cem\u003eC. acutatum\u003c/em\u003e may be considered a true endophyte of this species. \u003cem\u003eColletotrichum\u003c/em\u003e spp. have been found in other pitcher plants, \u003cem\u003eCatopsis berteroniana\u003c/em\u003e, and \u003cem\u003eNepenthes\u003c/em\u003e spp (Lee, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Leroy et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Naseem et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), perhaps \u003cem\u003eColletotrichum\u003c/em\u003e spp. are better adapted as pitcher plant endophytes over other carnivorous plant trap types.\u003c/p\u003e \u003cp\u003eFungal endophytes are known to provide a variety of functions to their host plants through the production of secondary metabolites. \u003cem\u003eAlternaria infectoria\u003c/em\u003e and \u003cem\u003eCladosporium cladosporioides\u003c/em\u003e are both known to produce antimicrobial secondary metabolites (Casella et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Scott et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), broadly these can be used by plants for pathogen resistance (Collinge, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has previously been shown that carnivorous plants synthesise metabolites with antimicrobial properties, which may serve a prey preservation function (Hatcher, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The intriguing possibility is that these antimicrobial metabolites are synthesised by fungal endophytes, rather than the plant itself and may also represent a source of novel compounds. Additionally, fungi inhabiting the mucilage secretions of \u003cem\u003eDrosera\u003c/em\u003e enhance prey digestion (Sun et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), it seems possible that fungi inhabiting these plants endophytically would also be able to contribute to prey digestion via enzyme production. Other fungi are known to inhabit the water contained within carnivorous pitcher plants forming part of a food web that aids in prey digestion, this is seen in \u003cem\u003eSarracenia purpurea\u003c/em\u003e and \u003cem\u003eNepenthes\u003c/em\u003e (Grothjan and Young \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Leonora S. Bittleston et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Inquiline fungi are able to contribute to plant carnivory via prey digestion, it seems plausible that endophytes may also help with digestion, or perhaps inquiline fungi are capable of existing as endophytes within plant tissue.\u003c/p\u003e \u003cp\u003eThe culture-dependent approach revealed a low species diversity of endophytes in carnivorous plants. This is not consistent with expectations of plants in extreme environments hosting unique endophytes to mitigate extreme stresses. In carnivorous plants we may expect to see endophytes aiding in survival in a low nutrient environment. A culture dependent approach for carnivorous plants presents extra challenges owing to the nature of their leaves: \u003cem\u003eDrosera\u003c/em\u003e spp. and \u003cem\u003eP. vulgaris\u003c/em\u003e have mucosal leaves which may degrade quickly or be permeable during surface sterilisation, resulting in endophyte death; \u003cem\u003eS. purpurea\u003c/em\u003e has a waxy layer over the inside of its pitcher leaves which, if not thoroughly removed, can block endophyte emergence during culturing. Additionally, many fungi may not be culturable at all (Siddique, et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dissanayake et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) or require more specialised media to grow. Growth media containing benomyl, for example, facilitates growth of basidiomycetes (Thorn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) which are frequently underrepresented in culture dependent methods. High throughput next generation sequencing methods are increasingly being used in endophyte studies, and reveal more diverse communities than previously thought(Donald \u003cem\u003eet al.\u003c/em\u003e, 2020; Siddique, et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dissanayake et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These methods are not limited by culturing success and may prove useful to test hypotheses of endophyte diversity and host/ site specificity in the future. To understand the ecological role of these endophytes in plant carnivory multi-omics approaches and EpicPCR can show what fungal produced metabolites are used in plant carnivory.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, for the first time, we cultured trap fungal endophytes \u003cem\u003efrom in-situ\u003c/em\u003e grown carnivorous plant species in the UK. We add to growing understanding of the ubiquity and diversity of carnivorous plant endophytes by reporting the presence of five new species of carnivorous plant endophytes. In addition, endophytes are reported for the first time from \u003cem\u003eDrosera anglica\u003c/em\u003e and \u003cem\u003eDionaea muscipula\u003c/em\u003e. We found some evidence of host specificity between trap types from \u003cem\u003eColletotrichum\u003c/em\u003e spp. only infecting \u003cem\u003eSarracenia purpurea\u003c/em\u003e. Our understanding of the mechanisms of plant carnivory is incomplete. It seems likely that fungal endophytes play a key role in the various processes of carnivory: prey attraction, capture, and digestion. We have a limited knowledge of the functional role and diversity of endophytes in plant carnivory, this must be a priority for future research for Darwin\u0026rsquo;s \u0026ldquo;most wonderful plants in the World\u0026rdquo;.\u003c/p\u003e \u003cp\u003eStatements \u0026amp; Declarations\u003c/p\u003e \u003cp\u003eFor the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e \u003cp\u003eThis work was supported by Central England NERC Training Alliance (CENTA) PhD studentship (NE/S007350/1) and the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). All sequence data is deposited in NCBI, accession numbers detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe Authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003cp\u003eAuthor contributions as follows, \u003cb\u003eBrandon Shaw\u003c/b\u003e: Conceptualization, Funding acquisition, Methodology, Investigation, Writing - Original Draft \u003cb\u003eJonathan Millett\u003c/b\u003e: Conceptualization, Funding acquisition, Supervision, Writing - Review \u0026amp; Editing \u003cb\u003eDavid Ryves\u003c/b\u003e: Conceptualization, Supervision, Writing - Review \u0026amp; Editing \u003cb\u003eHelen Glanville\u003c/b\u003e: Supervision, Writing - Review \u0026amp; Editing \u003cb\u003eErica Young\u003c/b\u003e: Supervision, Writing - Review \u0026amp; Editing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFor the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e\n\u003cp\u003eThis work was supported by Central England NERC Training Alliance (CENTA) PhD studentship (NE/S007350/1) and the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). All sequence data is deposited in NCBI, accession numbers detailed in Table 3.\u003c/p\u003e\n\u003cp\u003eThe Authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthor contributions as follows, \u003cstrong\u003eBrandon Shaw:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Methodology, Investigation, Writing - Original Draft \u003cstrong\u003eJonathan Millett:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Supervision, Writing - Review \u0026amp; Editing \u003cstrong\u003eDavid Ryves:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, Writing - Review \u0026amp; Editing \u003cstrong\u003eHelen Glanville:\u0026nbsp;\u003c/strong\u003eSupervision, Writing - Review \u0026amp; Editing \u003cstrong\u003eErica Young:\u0026nbsp;\u003c/strong\u003eSupervision, Writing - Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eLaboratory work was supported by the UK Natural Environment Research Council (NERC) Environmental Omics Facility (NEOF, grant number: NEOF1465). The authors would like to thank the technical staff of the facility: Tom Holden; Lucy Knowles; Gavin Horsburgh for their excellent support during the DNA extraction, amplification, and sequencing process. Thanks to the National Trust and private landowners for permission to access and sample the plants. The research was carried out as part of a Central England NERC Training Alliance (CENTA) PhD studentship (NE/S007350/1).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbarenkov, Kessy RH, Nilsson KH, Larsson, Andy FS, Taylor TW, May et al (2024) Tobias Guldberg Fr\u0026oslash;slev, Julia Pawlowska,. 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PCR Protocols\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycological-progress","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mypr","sideBox":"Learn more about [Mycological Progress](https://www.springer.com/journal/11557)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mypr/default.aspx","title":"Mycological Progress","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Endophytes, Carnivorous plants, Symbiosis, Specificity, Culture dependent","lastPublishedDoi":"10.21203/rs.3.rs-4021835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4021835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFungal endophytes are ubiquitous plant symbionts existing asymptomatically inside plant tissues but playing a crucial role in plant health and function. Endophytes have been extensively studied in many plants, such as agricultural systems, but in carnivorous plants which may engage microbes to aid in digestion, they are poorly understood. To address this deficiency, this study aims to assess the presence and host specificity of fungal endophytes in carnivorous plant traps. Five carnivorous plant species were sampled from two \u003cem\u003ein-situ\u003c/em\u003e sites and greenhouse grown plants.\u003c/p\u003e \u003cp\u003eFungal endophytes were isolated from the traps of five carnivorous plant species: \u003cem\u003eDrosera rotundifolia\u003c/em\u003e, \u003cem\u003eDrosera anglica\u003c/em\u003e, \u003cem\u003ePinguicula vulgaris\u003c/em\u003e, \u003cem\u003eDionaea muscipula\u003c/em\u003e, and \u003cem\u003eSarracenia purpurea\u003c/em\u003e. These represent a range of trapping mechanisms, native and introduced species. We included different carnivorous plant species growing at the same site, and the same species at different sites to test for host and site specificity. Endophytes were cultured on malt extract agar and identified by sequencing the internal transcribed spacer (ITS) region. The isolated fungal endophytes were composed of species from common Ascomycota genera (\u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eCladosporium\u003c/em\u003e, \u003cem\u003eColletotrichum\u003c/em\u003e, and \u003cem\u003eDidymocyrtis\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFungal endophytes were present in all plant species, this expands our understanding of endophyte distribution in carnivorous plants, representing the first endophytes isolated from \u003cem\u003eD. anglica\u003c/em\u003e and \u003cem\u003eD. muscipula\u003c/em\u003e and the first endophytes from the traps of \u003cem\u003eD. rotundifolia\u003c/em\u003e and \u003cem\u003eP. vulgaris\u003c/em\u003e, along with five new fungal endophyte species in carnivorous plants. There was some host specificity; \u003cem\u003eS. purpurea\u003c/em\u003e was the only host plant with \u003cem\u003eColletotrichum\u003c/em\u003e spp., whereas \u003cem\u003eAlternaria spp.\u003c/em\u003e were found across multiple hosts and sites. The trap endophytes cultured here differed to root endophytes of \u003cem\u003eDrosera\u003c/em\u003e and \u003cem\u003ePinguicula\u003c/em\u003e found in previous studies of plants at the same site, suggesting that above and below ground endophytes may differ, perhaps offering different functions in separate plant tissues.\u003c/p\u003e","manuscriptTitle":"Culturable endophytes from carnivorous plant traps in the UK: commonality of endophyte species across host species and sites.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-21 12:13:04","doi":"10.21203/rs.3.rs-4021835/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-08-06T08:40:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-10T14:48:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycological Progress","date":"2024-04-05T09:47:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-27T07:46:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycological Progress","date":"2024-03-26T11:51:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycological-progress","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mypr","sideBox":"Learn more about [Mycological Progress](https://www.springer.com/journal/11557)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mypr/default.aspx","title":"Mycological Progress","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ee3c81f8-b221-42ab-b3f2-0a569fc6c5b8","owner":[],"postedDate":"May 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-21T12:13:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-21 12:13:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4021835","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4021835","identity":"rs-4021835","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}