Philippa Kaur

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Genome characteristics of ancient obligate symbiontsWe first tested the hypothesis that each of the ant lineages sequenced in our study (Cardiocondyla, Formica, and Plagiolepis) hosts its own ancient strictly vertically transmitted symbiont that have co-speciated with its host, which has been shown previously in the Camponotus- Blochmannia symbiosis [17]. To address this aim, we compared the genomes of symbionts from 13 species of ants, 8 from our study combined with 5 previously published genomes, representing four independently evolved symbioses. This includes symbionts from three Formica, two Plagiolepis, and an additional three Cardiocondyla species that we sequenced, in addition to four previously published genomes from Blochmannia, the obligate symbiont of Camponotus ants, and the one pre-existing Westeberhardia genome from Cardiocondyla obscurior [8, 18,19,20,21].We found the gene order of single copy orthologs in symbionts is highly conserved in ant species belonging to the same genus (Fig. 1). This type of structural stability of genomes is typically found in symbionts that have been strictly vertically transmitted within a matriline [22] and has been documented in the obligate symbionts of whiteflies, psyllids, cockroaches, and aphids [23,24,25,26]. In contrast, genome structure differed substantially between symbionts from different ant genera (Fig. 1, Fig. S1). We also find that the host and symbiont phylogenies are in general concordance in Cardiocondyla (TreeMap: p = 0.00100 CI95% = [0.00000, 0.00424]), and in Formica the topologies suggest co-segregation, although there were too few nodes to confirm this statistically (Fig. S2). Together, this strongly suggests the symbioses in all four ant lineages are independently acquired ancient associations that have co-speciated with their hosts.Fig. 1: Structural stability of ant symbiont genomes.A Ant lineages known to host bacteriocyte-associated symbionts (red font) and lineages not known to (black font), based on [91]. Outgroup (grey font) not examined in this study. B Visualisation of symbiont genomes showing conservation of gene order in the symbionts of ant species that belong to the same genus. Blocks show the locations of single copy orthologs in the symbiont genome, lines connect shared single copy orthologs between genomes. All genomes and annotations were generated in this study except the Blochmannia symbionts and the Westeberhardia strain from C. obscurior [8, 18,19,20,21]. *Evidence of symbionts were detected in embryos of Anoplolepis [91] but it is unclear if they are localised in bacteriocytes in larvae and adults.Full size imageIn addition, our phylogenetic analysis reveals that all four symbiont lineages originate from a single clade, the Sodalis-allied bacteria (Fig. 2). This demonstrates that ant lineages that host bacteriocytes-associated symbionts have convergently acquired related bacteria, which differs from previous findings based on limited taxa and genes [27]. All of the symbionts have evidence of advanced genome reduction, which is characterized by reduced genome size, GC content, and number of coding sequences, similar to other ancient obligate symbionts of insects [4]. The three strains of Westeberhardia we analysed have extremely small (0.45–0.53 Mb) GC depleted genomes (22–26%) that are similar to the figures reported for the strain in Cardiocondyla obscurior [8]; confirming that they have some of the smallest genomes of any known gammaproteobacterial endosymbiont (Fig. 2). By comparison, the symbionts in Formica and Plagiolepis have genomes around twice the size (1.37–1.38 Mb) and GC content (~41%) of Westeberhardia (Fig. 2) raising the possibility that they are in an earlier stage of genome reduction than both Westeberhardia and Blochmannia. The Formica and Plagiolepis symbionts have a similar size, GC range, and number of coding sequences as known obligate symbionts such as Candidatus Doolittlea endobia [28], and several Serratia symbiotica lineages that are co-obligate symbionts in aphids [29].Fig. 2: Phylogenetic origins of the bacteriocyte-associated symbionts of ants.A pruned phylogeny of gammaproteobacterial endosymbionts based on Fig. S8. The phylogeny is based on a dayhoff6 recoded amino acid alignment of 72 genes analysed using phylobayes. Bar plots represent the size (in Mbp) and GC content of symbiont genomes. Bars are colour coded to represent hypothesised relationships between symbionts and hosts. Species names highlighted in red in the phylogeny indicate the four bacteriocyte-associated symbionts of ants. Genomes sequenced and assembled for this paper are referenced as ‘novel symbiont’ lineages. Full phylogenies with node support and branch lengths are available as Fig. S8 and Fig. S9, respectively.Full size imageBacteriocyte-associated endosymbiontsUsing fluorescent in situ hybridisation, we determine whether the Sodalis-allied symbionts we sequenced are localised in bacteriocytes to confirm they are the associations first observed by Lillienstern and Jungen in the early 1900’s [10, 11].Consistent with Lilienstern’s findings [11], we found the Sodalis symbiont in Formica ants is distributed in bacteriocytes surrounding the midgut in adult queens (Fig. 3A). The symbionts are also found in eggs and ovaries of adult queens, indicating they are vertically transmitted from queens to offspring (Fig. 3B–C). Sectioning of F. cinerea larvae shows the bacteriocytes to be arranged in a single layer of cells surrounding the midgut, as well as in clusters of bacteriocytes closely situated to the midgut (Fig. 3D–D’). In adult Plagiolepis queens, the symbiont was not present in bacteriocytes around the midgut, suggesting the symbiont may play a more substantive role in larval development or pupation and then migrates to the ovaries prior to or during metamorphosis. Apart from that, the localisation of the symbiont in Plagiolepis was the same as in Formica – symbionts in larval midgut bacteriocytes, ovaries and eggs (Fig. S3) – supporting Jungen’s cytological findings [10]. Bacteriocytes are also found surrounding the midgut in Camponotus and Cardiocondyla ants [8, 30, 31] indicating the symbionts are localised in a similar manner in all four ant lineages.Fig. 3: Anatomical localisation of symbiont in Formica ants.Fluorescent in situ hybridisation (FISH) generated images showing the localisation of symbionts in Formica ants. A–C Whole mount FISH of Formica fusca: queen gut (A, crop and proventriculus on the right, midgut in the middle, hindgut and Malpighian tubules on the left), ovaries (B) and egg (C). DAPI staining of host tissue in blue, symbiont stained in red. D–D’. FISH on transverse cytological sections of Formica cinerea larva midgut. DAPI staining only, showing host nuclei of bacteriocytes in a single layer surrounding the midgut (D), and a magnified region highlighting symbionts in red localised within bacteriocytes and in a bacteriome (D’). A FISH image of the symbiont-free midgut of a Formica lemani queen is available as Fig. S11.Full size imageConservation of metabolic functions in ant endosymbiontsDespite on-going genome reduction, obligate symbionts of insects typically retain gene networks required for maintaining the symbiosis with their host, such as pathways for synthesising essential nutrients. This has resulted in the symbionts of sap- and blood-feeding insects converging on genomes that have retained the same sets of metabolic pathways – to synthesise essential nutrients missing in their hosts’ diets [32, 33]. Here we compare the metabolic pathways retained in the reduced genomes of the four bacteriocyte-associated symbionts of ants to test the hypothesis that have been acquired to perform similar functions. For this, we assess whether they have consistently retained metabolic pathways to synthesise the same key nutrients. Two major patterns stand out.The first major pattern we find is that the four ant symbionts have all retained the shikimate pathway, which produces chorismate, along with most of the steps necessary to produce tyrosine from this precursor (Tables 1 and S2). Both the symbiont of Formica and Westeberhardia each lack one of the genes required to produce tyrosine. However, in Westeberhardia it is believed the host encodes the missing gene, supplying the enzyme to fulfil the final step of the pathway [8]. Intriguingly, we find that this gene is also present in the Formica ant genomes (Fig. S4). In addition, all symbionts except Westeberhardia can produce phenylalanine which is a precursor that can be converted to tyrosine by their hosts [5, 34, 35]. Tyrosine is important for insect development as it is used to produce L-DOPA, which is a key component of insect cuticles [5]. Tyrosine is also a precursor for melanin synthesis, which is important in protection against pathogens, and plays a fundamental role in neurotransmitters and hormone production [36, 37]. In several species of ants, weevils, and other beetles, symbionts are believed to provision hosts with tyrosine, and it has been shown experimentally in several of these species that removal or inhibition of their symbionts causes cuticle development to suffer [38,39,40,41,42,43,44,45,46,47]. A thicker cuticle has been shown to help symbiont-carrying grain beetles resist desiccation [43], and defend against natural enemies [48]. However, female reproduction is delayed at higher humidity, suggesting a metabolic cost to carrying their Bacteroidetes symbiont. Tyrosine provisioning is also the likely function of Westeberhardia in Cardiocondyla ants, as this is one of the few nutrient pathways retained in this symbiont. Our analysis confirms the shikimate pathway, and the symbiont portions of the tyrosine pathway, have been retained in Westeberhardia from three phylogenetically diverse Cardiocondyla lineages, providing additional support for this hypothesis. In addition to tyrosine, most of the symbionts have retained the capacity to produce vitamin B9 (tetrahydrofolate) and all can perform the single step conversions necessary to produce alanine and glycine. However, our gene enrichment analysis indicates that tyrosine, and the associated chorismate biosynthetic process, are the only enriched vitamin or amino acid pathways that are shared by all of the symbiont genomes (Table S1). This suggests that provisioning of tyrosine by symbionts, or tyrosine precursors, is of general importance across all bacteriocyte-associated symbioses of ants.Table 1 Comparison of the retention and losses of metabolic pathways for key nutrients across ant symbionts.Full size tableThe second major pattern emerging from our comparative analysis is that there are clear differences in the pathways lost or retained across symbionts (Tables 1 and S2). This is most evident when comparing Blochmannia with Westeberhardia, the latter of which has lost the capacity to synthesise most essential nutrients. The symbionts of Formica or Plagiolepis, in contrast, have retained the capacity to synthesise many of the same amino acids and B vitamins as Blochmannia, suggesting they may perform similar functions for their hosts. However, Blochmannia has retained more biosynthetic pathways, particularly those involved in the synthesis of essential amino acids. Previous experimental studies have confirmed that Blochmannia provisions hosts with essential amino acids [1]. The absence of several core essential amino acids in the Formica and Plagiolepis symbionts may reflect differences in the dietary ecology of the different ant genera. The retention of the full complement of essential amino acids biosynthetic pathways in the highly reduced genome of Blochmannia does however indicate it plays a more substantive nutrient-provisioning role for its hosts than the other ant symbionts we investigated.Previous work on the extracellular gut symbionts of several arboreal ant lineages identified nitrogen recycling via the urease operon as a function that may be of key importance for ant symbioses [1, 2, 49, 50]. However, we do not find any evidence that the symbionts of Formica, Plagiolepis, or Cardiocondyla play a role in nitrogen recycling via the urease operon (Table 1). This suggests that nitrogen recycling may play an important role for more strictly herbivorous ants, such as Cephalotes. Our results indicate tyrosine supplementation by symbionts may be universally required for essential physiological process across a broader range of ant lineages.The origins and losses of symbioses in Formica and Cardiocondyla
We investigated the presence of the symbiont in phylogenetically diverse Formica and Cardiocondyla species to identify the evolutionary origins and losses of the symbiosis. Although the symbiont in Plagiolepis was present in P. pygmaea and two unknown Plagiolepis species we investigated, we did not have sufficient phylogenetic sampling to assess the origins of the symbiosis.In Formica, we find the symbiont is restricted to a single clade in the paraphyletic Serviformica group (Fig. 4A). The species in this clade are socially polymorphic, forming both multi-queen and single-queen colonies [51]. Based on a previously dated phylogeny of Formica ants, we estimate the symbiosis originated approximately 12–22 million years ago [52]. In Cardiocondyla, the symbiosis is widespread throughout the genus. The prevalence of the symbiont in Cardiocondyla, in combination with its highly reduced genome, suggests it is a very old association that likely dates back to the origins of the ant genus some 50–75 million years ago [53]. The symbiont was also absent in two clades, the argentea and palearctic groups (Fig. 4B). This may represent true evolutionary losses in these clades. It may be that these losses are linked to a notable change in social structure in these two Cardiocondyla clades, having gone from the ancestral state of maintaining multi-queen colonies to single-queen colonies [54], however it is not clear how this could impact the symbiosis.Fig. 4: Phylogenetic distribution of symbionts in queens of Formica and Cardiocondyla ants.Pie charts represent the proportion of Formica (A) and Cardiocondyla (B) queens sampled that carried the symbiont (red) and those that did not (grey). Numbers represent the number of queens positive for the symbiont over the total number of queens sampled (intracolony infection frequencies in Table S5). See the supplementary material for the statistical testing of differences in prevalence within Serviformica Clade 1. The Formica phylogeny is based on [81] and the Cardiocondyla phylogeny is based on [83], with major clades highlighted. Dashed lines indicate species added to the original source phylogeny based on additional published phylogenies (specified in the Taxonomic Analysis section of the methods). Starred names are provisional names of a recognised morphospecies to be described by B. Seifert.Full size imageEvidence of variation in colony-level dependence on symbiontsObservations from individual studies on F. cinerea and F. lemani [10, 11], as well as Cardiocondyla obscurior [8], reported rare cases of ant queens not harbouring their symbionts in nature. This called into question the degree to which these insects depend on symbionts for nutrients, and whether the symbiosis may be breaking down in certain host lineages. However, given the limited number of species and populations studied, it is unclear how often colonies are maintained with uninfected queens, and whether this varies across species, suggesting species may differ in their dependence on their symbiont. To answer this question, we assessed the presence of the symbionts in 838 samples from 147 colonies of phylogenetically diverse Formica and Cardiocondyla species collected across 8 countries.Our investigation reveals the natural occurrence of uninfected queens is a widespread phenomenon in many Formica and Cardiocondyla species (Fig. 4). We confirmed the absence of symbionts in queens, and that they have not been replaced with another bacterial or fungal symbiont, using multiple approaches including diagnostic PCR, metagenomic and deep-coverage amplicon sequencing (Tables S3,  S4, Figs. S5, S6). Wolbachia was high in relative abundance, especially in Formica ants, but was not sufficiently present across samples to be a feasible replacement. There was also clear evidence of variation across host species. In Formica, queens and workers of F. fusca always carried the symbiont, whereas queens and workers of F. lemani, F. cinerea, and F. selysi showed varying degrees of individuals not carrying the symbionts (Fig. 4A, Table S5). A similar pattern can be seen in Cardiocondyla, where queens of several species, such as C. obscurior, always carry the symbiont, compared to lower incidences in other species (Fig. 4B). Klein et al [8] identified a single C. obscurior colony with uninfected queens in Japan. However, queens of this species nearly always carry the symbiont in nature.The degradation and eventual loss of symbionts from bacteriocytes has been reported in males, and in sterile castes of aphids and ants [55], which do not transmit symbionts to offspring. In reproductive females, bacteriocytes may degrade as a female ages; however, symbionts are typically retained at high bacterial loads in the ovaries, as this is required to maintain the symbionts within the germline [31]. All of the symbiotic ant species we investigated maintain multi-queen colonies, and the vast majority had at least one queen, often more, within a colony that carried the symbiont (Table S5). We hypothesize that species that maintain colonies with uninfected queens may be able to retain sufficient colony-level fitness with only a fraction of queens harbouring the symbiont and receiving its nutritive benefits.Dependence on symbionts in a socioecological contextThe retention of symbionts in queens and workers of some species, but not others, suggests species either differ in their dependence on symbiont-derived nutrients, or that symbionts have lost the capacity to make nutrients in certain host lineages. Our analysis of symbiont genomes did not reveal any structural differences, such as the disruption of metabolic pathways, which could explain differences in symbiont retention between host species (Table S2). This suggests differences in the retention of symbionts may reflect differences in host ecologies.In ants, which occupy a wide range of feeding niches, reliance on symbiont-derived nutrients will largely depend on lineage-specific feeding ecologies. For example, several species of arboreal Camponotus ants have been shown to be predominantly herbivorous [56]. Blochmannia, in turn, has retained the capacity to synthesise key nutrients lacking in their plant-based diets, such as essential amino acids [1]. Blochmannia is also always present in queens and workers [31], which is a testament to the importance of these nutrients for the survival of its primarily herbivorous host [13]. In contrast, Formica and Cardiocondyla species are thought to have a more varied diet [14]. Diet flexibility and altered foraging efforts may therefore reduce their reliance on a limited number of symbiont-derived nutrients allowing colonies of some species to persist with uninfected queens in certain contexts. Silvanid beetles and grain weevils, for example, can survive in the absence of their tyrosine-provisioning symbionts [38, 57, 58] when provided nutritionally balanced diets, in the laboratory [57] or in cereal grain elevators [59, 60]. Similarly, studies on Cardiocondyla and Camponotus ants have shown they can maintain sufficient colony health in the absence of their symbionts, if provided a balanced diet [31, 61]. It would be interesting to know whether species of Formica and Cardiocondyla that always carry the symbiont in nature, such as F. fusca and C. obscurior, have more restricted diets with less access to nutrients such as tyrosine, as this may explain their dependence on their symbiont for nutrients and tendency to harbour them in queens.Although it is unusual for bacteriocyte-associated symbionts to be absent in reproductive females, the fact that it is simultaneously occurring in phylogenetically diverse species from many locations suggests the symbiosis may have persisted in this manner over evolutionary time. Perhaps through diet flexibility colonies can be maintained with uninfected queens in some contexts, however we expect them to be disadvantaged in other ecological scenarios. Fluctuating environmental conditions may therefore eventually purge asymbiotic queens from lineages, allowing the symbiosis to be retained over longer periods of evolutionary time. The multiple-queen colony lifestyle in all symbiotic Formica and Cardiocondyla species we investigated may also provide an additional social buffer that limits the costs to individual queens being asymbiotic. Workers will still nourish larvae and queens without symbionts and colony fitness may be maintained through the reproductive output of nestmate queens that carry the symbiont. There may also be an adaptive explanation for the losses if, for example, metabolic costs to maintain the symbiosis trade off in a context dependent manner [44, 62, 63]. Under this scenario, maintaining a mix of infected and uninfected queens may benefit a colony by allowing for optimal reproduction under a broader range of environmental scenarios.Our data suggest that symbiotic relationships can evolve to solve common problems but also rapidly break down if the symbiosis is no longer required, or potentially when costs are too high [44]. We have identified tyrosine provisioning as a possible unifying function across bacteriocyte-associated symbionts of ants. But we have also shown species can vary in how much they depend on symbionts for nutrients. Our results demonstrate that ants have a unique labile symbiotic system, allowing us to better understand the evolutionary forces that influence the persistence and breakdown of long-term endosymbiotic mutualisms.
Candidatus Liliensternia hugann and Candidatus Jungenella plagiolepisWe propose the names Candidatus Liliensternia hugann for the Sodalis-allied symbiont found in Formica. The genus name is in honour of Margarete Lilienstern who first identified the symbiont [11]. The species name is derived from the combined first names of the first authors parents. Similarly, we propose the name of Candidatus Jungenella plagiolepis for the Plagiolepis-bound symbiont. The genus name is in honour of Hans Jungen who originally discovered the symbiont [10], and the species name is derived from Plagiolepis, the genus in which the symbiont can be found. More

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Plant biomarker distributionsPlant lipid biomarkers are widely used to reconstruct vegetation and (hydro)climate in ancient environments, and previous studies at Olduvai15,17 have used biomarkers in spatiotemporal landscape reconstructions, although not at the resolution of this study (i.e.,  More

Truffles are socially and economically important in parts of Croatia. They can be worth up to €5,000 (US$5,300) per kilogram. The truffle industry and related tourism provides jobs, supplements incomes and boosts local economies. It’s not just about money, however; many people just love being out in the forest looking for them.My fascination with fungi began at the age of six, when my father and grandfather began taking me out to hunt for game and to collect mushrooms near our home in Istria. Today, I focus mainly on truffles and other hypogeous fungi, which produce their fruiting bodies underground. I spend 50–100 days a year in the field with my dogs, collecting samples and data on the life cycles, ecology and geographical spread of fungi across Croatia. Here, I’m with my dog Masha. I love the work.Thirty years ago, rainfall used to be more predictable across the year in Istria. Now, the climate is more extreme, and includes droughts. Truffles require a specific amount of water to grow. And warm winters have increased the population of wild boars, which damage the soil and eat the truffles. The truffles are becoming harder to find.Truffle plantations could take the pressure off natural habitats. There, the soil water content can be controlled, agricultural methods can be used to enhance production and boars can be kept out. We’re studying the viability of farming black truffles, in part by experimenting with different ways to inoculate tree seedlings with their spores.We’re using DNA barcoding to identify fungi in soil from their spores and root-like mycelium in protected areas. We’re finding that there are often many more species present than previously thought.Our comparisons of areas with and without truffles could help to reveal why they grow in some areas but not others. Our work is also helping to show the importance of biodiversity in places such as the Adriatic islands of Brijuni National Park. More

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This study reveals that various measures of bee diversity-including abundance, richness, and assemblage patterns are influenced by vane color and light reflectance patterns when passively sampling bees with vane traps. In particular, brightly colored vanes with higher light reflectance within 400–600 nm range attracted a greater diversity of bees in traps placed in a livestock pasture ecosystem. Effectiveness of blue and yellow vane traps had been compared previously in different ecosystems, for instance in apple orchards17, both woodland and open agriculture farmland13, and adjacent to Helianthus spp. (Asteraceae) field27. In all these studies, blue vane trap captured more bee species and 5–6 times more individuals compared to yellow vane trap.In the current study, we assessed a different design and size of vanes and a wider array of vane colors and reflectance patterns attached to sample collection jars. In particular, we used bright blue and yellow vanes that were made of plastic sheets covered with a micro-prismatic retro-reflective sheeting that provides better daytime and nighttime brightness as well as high visibility and durability. These vanes showed higher light reflectance and captured the most bees and bee species in this study (Table 2). Similar material was used on red vanes as well, but the light reflectance from those vanes was relatively lower, and as a result captured fewer bees. Traps with bright blue vanes performed especially well in terms of rates of bee capture (Fig. 2; 11.1 bees per trap per sampling date) and rates of species accumulation (Fig. 3). Bright yellow traps exhibited the second highest values for capture rates (Fig. 2; 6.6 bees per trap per sampling date) and species accumulation (Fig. 3), but these rates were not deemed significantly different from some other colors in which the reflective sheeting was not used, such as dark yellow, dark blue and purple.Bees use visual clues for detection, recognition, and memorization of floral resources in the foraging landscape7,28. The intensity of light reflected from different colors of vanes in traps affect number of bees attracted toward the trap10. Most bees can recognize colors that fall between 300 to 600 nm visual spectrums29. While the information related to the vision of many solitary and wild bees is not available, in the case of honey bees (Apis mellifera), color vision is trichromatic with highly sensitive photoreceptors at 344 nm (ultraviolet), 436 nm (blue) and 544 nm (green)30.In this study, colored vanes at a higher light reflectance between 400 to 600 nm attracted the highest number bee species in these passive traps. Capture rate differed among traps with different colored vanes in the current study, which can be explained by sensitivity of visual spectrum of bees and variation in the light reflectance of vanes of these traps. For example, bright blue vanes had two peaks of higher light reflectance, initially in 450–455 nm range and second peak with  > 800 nm. Such higher reflectance peak within the optimal range of bee vision may have played an important role in attracting abundant and diverse bee species to these passive traps. Similarly, bright yellow captured second largest number of bees, also had higher light reflectance peak within 600 nm but gradually decreased with increasing wavelength. Though bees have color spectrum from UV to orange31, they are sensitive to color spectrum between blue, green and ultraviolet32, which is a type of trichromatic vision system28. In one study33, red color vanes showed relatively lower light reflectance within 600 nm range, but had higher reflectance later in the spectrum, and this could be a reason why a low number of bees were collected in the traps. Past research showed contradictory views regarding the ability of bees to perceive red color. For instance, an early researcher in this field33, reported that bees recognize red color objects; however, other researchers had reported inability of bees to perceive34 or discriminate red from other colors35,36. It was argued that the bees see up to 650 nm in the visual spectrum and may not miss red colored flowers while foraging. However, other factors such as background (vegetation) color could also be contributing to bees’ ability to navigate different vane or flower colors in a livestock pasture landscape. Generally bees use color contrast to locate flower source, and hence neutral colors such as white are usually ignored29. Ultraviolet signal can make flowers more or less attractive to bees depending on whether it increases or decreases color contrast37. For example, UV color component in yellow38 and red39 flower increases chromatic contrast of these colored flowers with their background contributing attractiveness to the flowers. However, UV-reflecting white flowers decreases attractiveness for bees40.Different species of bees responded to different colors of vane traps. Out of the 49 bee species collected in this study, only nine bee species were found in all vane color types, whereas 14 species were found in only one trap color. For instance, out of five bumble bee species, two were found in all six vane colors, one was found in five colors, and two species (Bombus bimaculatus and B. fervidus) were only found in the traps with bright blue vanes. Many of the species that were only found in one trap color- Calliopsis andreniformis (1, bright yellow), Ceratina dupla (1, bright yellow), Diadasia afflicta (1, bright blue), Diadasia enavata (1, dark blue), Halictus rubicundus (1, dark yellow), Hylaeus mesillae (1, red), Lasioglossum tegulare (1, bright blue), Lasioglossum trigeminum (1, purple), Megachile montivaga (1, dark yellow), Melitoma taurea (1, bright blue), Svastra atripes (1, bright blue), and Triepeolus lunatus (1, dark yellow) were singletons and it was impossible to know if this represented a true preference or pattern. Our analysis of assemblage patterns after aggregating bees at the genus level, did show a gradient-like response in bee-color associations (Fig. 4), ranging from dark blue to yellows (with no strong associations found with red vanes). These patterns may be used to guide future (passive trap-based) sampling efforts to monitor bee diversity or to target specific bee species in livestock pastures or other ecosystems. While the bright blue and yellow traps with reflective sheeting were particularly attractive to bees, dark blue and purple traps also had relatively high levels of abundance and richness and collected higher number of Melissodes. Purple, as a color, is less commonly used than blue and yellow traps in bee monitoring. While this study shows that purple may be a viable option for bee collection, it’s similar assemblage pattern (Fig. 4) and low level of complementarity with dark blue traps (Table 2) suggests that it may be redundant with blue traps that are already commonly used. Differences in species- and sex-specific associations of bees with different colors of sampling traps had also been reported in previous studies41.Most of the bees collected in the current study were from Halictidae family (77.6%) followed by Apidae. However, few bee species in the families Andrenidae, Colletidae, and Megachilidae were collected. Consistent with our findings, others42 reported that bees of the Halictidae family were the most abundant bees in rangeland of Texas. The most common species found in this study were Au. aurata, L. disparile, L. imitatum, and Ag. texanus). In our previous studies we have found similar bee diversity in this study region18. Pollinator species richness and diversity as well as population distribution in livestock pasture vary during the season43. Mid-July to mid-August is the latter half of the summer season in the Southeastern USA, and the sampling period may have missed bee species that emerge earlier in the season and are reported in other studies42,43.Overall, the findings of this study showed that the wild bees responded differently to passive traps with colored vanes of different light wavelength and reflectivity when deployed in a livestock pasture ecosystem. Among six different colors of vanes (dark blue, bright blue, dark yellow, bright yellow, purple and red), the bright blue traps captured the highest number of individuals and species of bees. This could be due to an appropriate match between the visual spectrum of bees and the light reflectance spectrum of vanes, which were made of a micro-prismatic retro-reflective material. Bees responded similarly to traps with other colors of vanes, except for red vane traps, which captured the lowest number of bees. The findings of this study would be useful in understanding bee vision and responses to passive traps, and, such information would help in optimizing bee sampling methods for future monitoring efforts. More