Volume 1: Spring 2014: pp. 19-29.

R E V I E W

The invisible communities of nectar: How yeast and alter nectar characteristics Anthony Schmitt Abstract Plant - pollinator relationships are based off a binary reward system. Plants present pollinators with nectar as an energetic reward, while pollinators transfer genetic material to help plants achieve full reproductive success. The constituents of nectar play a crucial role in facilitating this mutual relationship. A new area of research is emerging that may change the way biologists view this binary system; it may no longer be a two-way interaction. Microorganisms - yeasts and bacteria - have been found to inhabit nectars across a wide geographic range and across a large range of plant species. These microorganisms change the characteristics of nectar in such a way to can alter pollinator behavior. For example, yeasts and bacteria can modify the sugar composition and concentration of nectar. Sugars in nectar are a chief reward for pollinator visitation. This review examines the new field of research presented by these microbes by breaking down changes in nectar as a result of microbial communities. The implications presented by these findings may significantly change the way biologists view plant-pollinator interactions as research continues to develop. *Biology Department, University of Minnesota Duluth *Writing Studies Department, University of Minnesota Duluth

Introduction facilitating co-evolution (Cronk and Ojeda Understanding the interactions of species 2008). It was long believed that the plant- within a community is important in pollinator system was a binary relationship. expanding our ecological and evolutionary However, new studies are finding a new knowledge, especially when two species factor in this relationship. rely on each other for reproductive The chemistry of nectar is understood to success. Many plants rely on the mutual have an important role in the plant-pollinator relationship between themselves and their binary system. Constituents attract and pollinators in order to produce a maximum deter specific organisms to help facilitate seed set. There are many ways that plant pollination in a way that will maximize species attract the pollinators that help fecundity (Adler 2000; Heil 2011). Although some constituents, such as H O , is thought plants achieve reproductive success. A 2 2 few examples include flower morphology, to sterilize nectar by killing off microbes (Adler 2000; Carter et al. 2007; Carter and Corresponding flower placement, flower color and scent. Thornburg 2004), but some microbes are Author: Perhaps the most important attractant is the still present in nectar. Anthony Schmitt production and enticement of nectar within It has long been known that [email protected] flowers. Nectar is a major energetic reward plants present to their pollinators in order to microorganisms, such as yeasts, inhabit help facilitate this desired interaction. The floral nectars (Sandhu and Waraich 1985), specificity of this system can be important in but their role was never closely examined. However, new research indicates that The Duluth Journal of Undergraduate Biology 119 R E V I E W Volume 1: Spring 2014 microorganisms are emerging as a source of pollinators. In his review, Heil (2011) of variation in the characteristics of nectar wrote that nectars rich in sucrose generally and therefore may have a significant role in attract hummingbirds, butterflies, moths, the plant-pollinator relationship. Microbial long-tongued , and ants whereas nectars community structure and ecology, in rich in hexose (glucose and fructose) attract general, is a field that is largely unexplored short – tongued bees and flies. Amino acids and research in this area may be beneficial have been reported to be more attractive to understanding the ecology of nature on a to insects rather than bird pollinators since wide spectrum (Furhman 2009). birds may be able to gain essential nutrients This review aims to present the current from other sources (Gonzalez-Teuber and knowledge of how yeasts and bacteria Heil 2009; Heil 2011). Some components shift nectar characteristics. Additionally, are thought to be specifically for defending the review will briefly discuss the potential against microbial infection and some are impact microorganisms may have on plant thought to be toxic to organisms that may – pollinator interactions. The purpose of this harm the plant (Adler 2000; Carter et al. paper is to bring awareness to this emerging 2007; Carter and Thornburg 2004). All the ecological niche within nectars that may different components within nectar provide hold larger implications in the important some type of function, which leads to the role pollinators have with plant fecundity. central purpose of this review. Microbes, such as yeasts, use nectar as a habitat and are Nectar chemical composition is interacting with the varying characteristics important for plant-pollinator that allow nectar to provide its ecological facilitation role in plant reproduction. The chemistry of nectar and its relation to Yeasts in Nectar pollinators has been extensively reviewed (for instance see reviews by Gonzalez- The approach to studying yeasts, and other Teuber and Heil 2009; Heil 2011). To fully microbiota, in nectar has resulted in two core understand all the components regarding fields of research. Recent studies support the function of nectar, some background the assertion that yeasts are present in flower information on nectar chemistry may be nectars across a wide range of plants species useful. It is well established that nectar is in separate plant communities. These mainly a solution of water and sugars (Baker studies provide evidence that nectar-living and Baker, 1983), although other constituents yeasts are geographically wide spread. including amino acids, proteins, lipids, Additionally, other recently published alkaloids and several other compounds studies support the notion that yeasts have have also been found in nectars (Baker and the ability to modify nectar chemistry. Baker 1983; Gonzalez-Teuber and Heil Nectar robber 2009). Some nectar compounds are thought Yeasts are common in nectar Organisms who to attract beneficial species (Brandenburg In order to establish the importance of consume a flowers et al. 2009), while some, such as alkaloids, yeasts in nectar, recent studies examined nectar generally by drilling a hole in the are thought to deter harmful species, such whether or not there was support for the corolla. Nectar robbers as nectar robbers. The concentration and postulate that yeasts are found throughout generally do not abundance of certain components that make a wide variety of plant nectars. Herrera et provide any benefit to up a flower’s nectar chemistry is believed to al. (2009) performed a quantitative survey the plant. be strongly correlated to plant – pollinator across two continents that helped establish mutualism. the wide-ranging occurrence of yeasts in the Sugars and free amino acids are nectar of insect-pollinated plants. understood to be important for the attraction Yeast communities were frequently

The Duluth Journal of Undergraduate Biology 20 Invisible communities in nectar R E V I E W Volume 1: Spring 2014 established throughout floral nectars in species (Herrera et al. 2010). This may the species and flowers surveyed reaching be due to the nature of nectar as a rather high densities within these nectars (Herrera hostile environment to yeasts (Adler 2000; et al. 2009). The study examined plant Carter and Thornburg 2004). The hostile communities from two separate continents environment may allow yeast species with and three different locations with different osmotolerance and resistance to secondary habitat variations (see study for further compounds found in nectar to have an explanation)– Donana (south-western advantage over other yeast species (Herrera Spain), Cazorla (south-eastern Spain) and et al. 2010). Yucatan (eastern Mexico). Overall, nectar Pozo et al. (2011) tested the osmophilic from 44 plant families and 130 plant species tolerance of nectar-living yeast species were examined. In Donana 31.8% of flowers by testing their growth response (positive contained yeasts, 42.3% of Cazorla flowers or negative response) in solutions of Metschnikowia contained yeasts, and 54.4% of flowers in 50% glucose in order to classify them as reukaufii Yucatan contained yeasts (Herrera et al. osmophilic species versus non – osmophilic A nectar living yeast 2009). Herrera et al. (2009) also found that species. There was a higher frequency of species that is found yeast cell densities of 104 cells mm-3 were osmophilic yeast species (35.5% of plant in the nectar of almost all plant species that quite common. It should be noted that other species surveyed) when compared to non – have been surveyed so studies have found that yeasts can reach osmophilic yeast species (4.8% of the plant far. It is a fungus part higher densities within nectar (de Vega et species surveyed). of the Ascomycota al. 2009), but generally densities were close As mentioned earlier, M. reukaufii has phylum and the to what was found in this survey (Brysch- been found in high densities across all the Metschnikowiaceae family. Herzberg, 2004; Herrera et al. 2009; de studies reviewed and may be a species Vega et al. 2009 and Belisle et al. 2011). with specialized traits to allow it to thrive DNA methylation Since yeasts are found in rather high in nectar. Once source of this nectar niche- The process of adding densities in nectar, researchers began filling characteristic ofM. reukaufiiseems to a methyl group to the to explore how community structures be due to DNA methylation polymorphisms cytosine or adenine in DNA nucleotides. developed in within nectar habitats. One that occur in nectar dwelling communities Methylation can alter key finding was that generally there was (Herrera et al. 2012). In short, M. reuakufii the expression of low species richness of yeasts. Pozo et al. undergoes epigenetic changes within its specific genes. (2011) explored the richness of yeast species genome as a result of variation in sugar within the nectars of different Spanish concentration and composition (sucrose, Epigenetics plants. The main assertion from the study glucose or fructose ratios in nectars). Both Changes in gene expression that are was that yeasts were present in all the nectar sugar concentration and composition vary caused by something droplets but there was low species richness both intra- and interspecifically among other than DNA among the nectar samples with 1.2 yeast plants. This genotypic plasticity, through sequence, i.e. DNA species per nectar sample on average. Pozo an alteration of DNA methylation, was methylation. et al. (2011) identified two species of yeasts, positively correlated with M. reukaufii’s Metschnikowia reukaufii (73.4% nectar ability to survive in these varying drops) and Metschnikowia gruessii (29.7% nectars. When the methylation inhibitor nectar drops) together having a frequency of 5-azacytidine was introduced to yeasts, 84.7% among the 216 yeast isolates – there their ability to cope with variations in nectar were 12 total yeast species isolated. The concentration and composition decreased yeast species M. reukaufii has been found as (Herrera et al. 2012). This plasticity in a chief inhabitant of nectar. M. reukaufii is most likely critical in their Low species richness could be an adaptability to inhabit and utilize nectars, indication that the habitat of nectar may especially since yeast species generally are play a selective role by filtering out yeast introduced to their nectar habitats through

The Duluth Journal of Undergraduate Biology 21 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 modes of transportation that are out of their The study found that sugar concentration control. was significantly altered among yeast- How yeasts arrive to the nectars they inhabited flowers (Herrera et al. 2008). inhabit is also an emerging field of research. Yeast cell density increased steadily as One hypothesis that has received support in flowers progressed through their floral several studies is that the pollinators are the stages, and as the density of cells changed, vectors that transfer the yeasts to the nectar. so did nectar chemistry. All combinations Belisle et al. (2011), Brysch-Herzberg of sucrose:fructose concentrations (glucose (2004), and de Vega and Herrera (2012) always had low concentrations) were found examined hummingbirds, bees and ants as among the plant species including pure- potential vectors and all found evidence sucrose nectars and pure-fructose nectars. that these pollinators may be the source for As cell density increased the level of sucrose nectar dwelling yeasts. Unsurprisingly, these decreased while, in contrast, fructose studies exemplify once again that the mutual increased. The average density found among relationship between plants and pollinators all late stage nectaries was about 104 – 105 is more complicated than a simple two-way cells/mm-3, which was common among most system. Pollinators are introducing yeasts studies. A pattern in sugar concentration into nectar. Once yeasts are established in also emerged between “clean” nectars with nectar they begin going to work in changing no yeasts and nectars that supported yeast their habitats characteristics, potentially communities. Clean nectars had higher altering the nectar in a way that may concentrations of sugar overall and were compete with the interests of the pollinators generally sucrose dominated, while nectars that acted as the yeasts vector. supporting dense yeast communities had lower overall concentrations of sugar and For further information about nectar yeast were either completely fructose (H. foetidus) community features and community or fructose dominated (Aquilegia). In distribution, the following studies may be summary, the researchers found a significant of interest: Belisle et al. (2011), Herrera et alteration of nectar sugar components that al. (2010), Peay et al. (2011), and Pozo and was correlated to the presence of increasing Herrera (2012). yeast cell density. The trend of sugar concentration varying Yeasts in nectar alter its inversely with yeast cell density was also characteristics found in a study done on 40 South African Yeasts alter the characteristics of nectar plant species (de Vega et al. 2009) providing in a variety of ways. Research is finding further support that there is a correlation that yeasts can alter the concentrations of between the presence of nectar-living yeasts sugars in nectar, as well as the temperature and alteration of chemical composition in of nectar. Both of these components have flower nectars. South African plants were important implications in facilitating lacking a quantitative study establishing the pollinator behavior. presence of yeasts in South African plant Herrera et al. (2008) performed a study species, so de Vega and colleagues surveyed involving nectar samples from three species 40 plant species (19 families) across several of -pollinated plants in southern Spain: locations that differed by geography and Helleborus foetidus, Aquilegia vulgaris and habitat type. They were able to confirm Aquilegia pyrenacia cazorlensis (family: that yeasts were prevalent in the flowers Ranunculaceae). Flowers of each plant they surveyed. Of the plants examined, were allowed to open before they were 51.3% individual plants had yeasts and collected to allow natural pollinator visits. 43.2% of the flowers had yeasts. In order to

The Duluth Journal of Undergraduate Biology 22 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 explore changes in nectar composition, the de Vega and Herrera 2012). researchers further investigated the nectar Nectar chemistry is not the only aspect of the bird-pollinated species Watsonia found to be altered by yeast inhabitants. pillansii (family: Iridaceae). Once again Temperature change in nectar may also there was a significant inverse correlation be a consequence of nectar-living yeasts. between yeast cell density and both sugar This was found in another examination of concentration (%) as well as sugar content the winter-blooming H. foetidus. Instead of (mg/ul); as yeast cell density increased looking at nectar chemistry, this time nectar sugar concentration and content decreased. temperature was investigated as a varying In a third study in southern Spain factor consequential of yeast inhabitance. researchers studied the plant species Cytinus Herrera and Pozo (2010) found that yeast hypocistis, a parasitic plant pollinated by presence partly contributed to internal floral ants, which emerged as another example temperature (other factors such as the plants of yeast-altered nectar chemistry. Nectar own metabolic functions were taken into samples were taken from flowers that were account). There was an observable change allowed to be visited by pollinators, and in interior floral temperature between yeast- these samples were evaluated for yeast inhabited nectar versus “clean” nectars frequency. As expected there was a high that did not have any yeasts. The change incidence of yeasts in both plants (94%) and in temperature was also correlated with flowers (77%) (de Vega and Herrera 2012). yeast cell density. As cell density increased Again there was a significant correlation so did temperature, proportionally. Yeast between increased yeast cell density cell densities of 105 cells mm-3 resulted and a drop in sugar concentration. The in an increase of 5-6º C (change in nectar

relationship between increased cell density temperature was measured as Tnect – Tair) and drop in nectar sugar concentration (Herrera and Pozo 2010). These few studies was only significant in the presence of M. clearly indicate that yeast cells, at high reukaufii (p = 0.01) perhaps again showing densities, are significantly altering the the specialization this species may have for characteristics of nectar and could have nectar microhabitats (de Vega and Herrera substantial effects on pollinator visitation 2012). and behavior, although to what extent these These studies together reveal a significant changes have on plant-pollinator ecology shift in sugar composition, content and still remains rather elusive. concentration in response to yeasts (Herrera The evidence of yeasts altering nectar et al. 2008; de Vega et al. 2009; de Vega temperature may have implications for and Herrera 2012). The energetic reward shifting pollinator behavior. Studies have provided by nectar sugars is fundamental found that bees tend to prefer warmer to the mutual relationship between a plant nectars (Dyer et al. 2006 and Norgate et and its pollinator. Both birds and bees prefer al. 2010). In addition to temperature, sugar nectars with higher sugar concentrations concentration may influence bee behavior (Cnaani et al. 2006 and Roberts 1996). independent of nectar temperature. Whitney Once yeasts are introduced into nectar et al. (2008) found that regardless of warmer they seemingly begin to degrade the sugar nectar, bees preferred nectars with higher reward provided by nectar that drives this concentrations of sucrose. Bumble bees, mutual relationship. Curiously though, Bombus terrstris, sense the temperature the pollinators themselves are the vectors and sugar concentration independently and for some yeast species and perhaps are would more often select for higher sugar introducing competitors into their precious concentration over a higher temperature source of energy (Brysch-Herzberg 2004; of nectar. Nectar concentration and

The Duluth Journal of Undergraduate Biology 23 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 temperature are believed to both provide in nectars across different plant species. metabolic rewards for the bumble bee. The study surveyed 27 South African The trio of interactions between yeasts, plant species (the same plant community plants and pollinators needs to be further that were examined for yeasts in de Vega explored in order to better understand et al. 2009) for bacterial establishment. all the ecological implications of these Twenty-one of the 27 plant species findings. The degradation of overall nectar harbored bacterial communities (77.8%). quality could potentially be significantly Although many plants contained bacterial influencing pollinator foraging behavior. communities, there was low species richness, similar to species richness patterns Bacteria in Nectar among nectar dwelling yeasts. Perhaps Perhaps an even less explored niche in this is a trend that will continue to emerge the microbiota of nectar is the effect of among microbial communities in nectar. bacteria on nectar. Five proteins, known as Only three different bacterial phyla were Nectarins I-V, have been found in nectar identified: , Actinobacteria, (Carter and Thornburg 2004). Nectarins are and Firmicutes (Alvarez-Perez et al. 2012). involved in the production of compounds, Within these phyla, seemed to be the most dominant. such as H2O2, that may play a pivotal role in nectar to prevent bacterial invasion These results were paralleled in Fridman within nectar (Carter et al. 2007; Carter and et al.’s (2012) study. Fridman and colleagues Thornburg 2004). The presence of these examined three plant species: Amygdalus constituents implies that nectar may be a communis, Citrus paradisi, and Nicotiana rather hostile environment for bacteria to glauca. Gammaproteobacteria were the inhabit. However, recent studies are finding dominant species in these nectars making contradicting evidence. up 59% of the isolates in A. communis, 82% of the isolates in C. paradisi, and 45% of Bacteria are evident in nectar much isolates in N. glauca. Another noteworthy like yeasts finding that emerged from work by Fridman et al. (2012) was the discovery of novel Two studies published in 2012 surveyed the bacteria species in each of the three plant prevalence of bacteria in nectar (Alvarez- species studied. The presence of novel Perez et al., 2012 and Fridman et al., bacteria species may be indicative of 2012). There were five key findings: (i) nectar playing a selective role in filtering bacteria were found among most plants out bacteria unsuited for the habitat within surveyed with low species richness in nectar nectar as well as leading to newly developed (Alvarez-Perez et al., 2012 and Fridman et species or genera that are better adapted for al., 2012), (ii) different plant species have the specific characteristics in nectar. unique bacterial communities (Fridman The bacteria isolated from nectar had et al., 2012), (iii) novel bacterial species key physiological characteristics. Species were identified in nectar (Alvarez-Perez et were able to grow in the presence of 3% al. 2013 and Fridman et al., 2012), (iv) key H O indicating catalase production in physiological adaptations may be evident 2 2 A common enzyme these bacterial isolates. Additionally the found in many species in nectar inhabiting bacteria (Alvarez- that breaks down H O isolates were able to grow at low levels 2 2 Perez et al. 2012) and (v) bacteria of the into water and oxygen. of oxygen (microbaerobiosis) and could Gammaproteobacteria class were the most Catalase activity may generally tolerate sucrose levels of 10%- prevalent in nectar. allow some microbes 30% (Alvarez-Perez et al. 2012). All of Alvarez-Perez et al. (2012) conducted to survive the presence these tested variables are characteristics of of H O in nectar. a study that tested and found support that 2 2 nectar that were previously thought to serve bacteria, much like yeasts, are widespread

The Duluth Journal of Undergraduate Biology 24 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 an anti-microbial function. In the study recognized as the most frequent pollinator done by Fridman et al. (2012), the plant for M. aurantiacus. The researchers explored species were specifically chosen for their what impact bacteria and yeasts may have secondary metabolites. N. glauca contains on Anna’s hummingbird foraging behavior nicotine and anabasine, A. communis and M. aurantiacus reproduction success. contains amygdalin and C. paradisi contains Gluconobacter reduced the proportion caffeine. These secondary metabolites were of closed stigma, which is a reproductive thought to potentially serve as a potential indicator for effective pollination in M. antimicrobial, but Fridman et al. (2012) aurantiacus on average by 23% on average found that there were no negative effects when compared to control nectar and yeast when these secondary metabolites were inoculated flowers. Gluconobacter reduced introduced to agar plates containing isolated the number of seeds produced by an average bacteria. More research is required to better of 18% (M. reukaufii had no significant understand the physiological characteristics reduction). Further, in Gluconobacter of bacterial nectar and their phylogenetic inoculated flowers on average 27% less relationships. nectar was removed, indicating less visitation by pollinators. Yeasts and bacteria both alter nectar The authors (Vannette et al. 2012) theorized and effect pollinator behavior that several factors may impact pollinator foraging behavior and pollination success Yeasts and bacteria have been found to as the result of Gluconobacter in nectar. coexist in the same nectar environment First, hummingbirds are fond of higher (Alvarez-Perez and Herrera 2012). Each sucrose concentrations and tend to avoid species alters nectar characteristics, but nectars with higher fructose concentrations. how these alterations affect plant fecundity Hummingbirds also tend to respond more and pollinator still requires further so to compounds within nectar rather than investigation. To address these relationships attractant signals produced flower nectars. a rather interesting study examined the Thus, the pollinating hummingbird may hummingbird pollinated plant species, need to taste the nectar before it is deterred. Mimulus aurantiacus (Phrymaceae), and This may be why less nectar was taken the difference in pollinator behavior and from Gluconobacter inoculated nectar and pollination success when nectars were flowers had decreased pollination success. inoculated with either Gluconobacter spp. Hummingbirds may have foraged on those (bacteria) or M. reukaufii (yeast) (Vannette flowers less, reducing pollen distribution. et al. 2012). Both species of microbes In addition to studying hummingbird altered nectar chemistry. Each species foraging, the behavior of bumble bee similarly reduced H O concentration by 2 2 foraging has also received recent attention to about 80% perhaps making the nectar a better understand nectar dwelling microbes more inhabitable environment though the and their implications for plant reproductive yeasts and bacteria differed in altering other ecology. Studies found differences in characteristics. Gluconobacter had a greater foraging behavior that were correlated with impact on both pH (reduced by 5 units) nectar microbial communities. Good et and sucrose (reduced by 35%) where as al. (2014) tested how bacteria and yeasts M. reukaufii had smaller reduction impacts modify feeding preference in Apis millifera, (2 pH units and 17%). Gluconobacter a honey bee species. Three bacterial species also increased fructose concentrations and were studied: Asaia astilbes, reduced glucose concentrations where M. tasmaniensis and Lactobaccillus kunkeei. reukaufii was not found to have any effect. Each of these bacterial species were selected Anna’s hummingbird (Calypte anna) is

The Duluth Journal of Undergraduate Biology 25 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 because they are known to be A. milifera of M. reukaufii in nectar or how bees sense symbiotic gut microflora and can survive the presence of yeasts, but they speculated in nectar as well as alter its characteristics. that it might be due to the way yeasts are The researchers examined the yeast species altering characteristics. Some sources of M. reukaufii since it is a central nectar attraction Herrera and colleagues suggested inhabiting yeast species, and this yeast include a shift in nectar temperature, yeast species is also found in the bee gut. It should metabolites, sugar or amino acid profiles, be noted that the researchers used artificial taste alterations and volatile emissions flowers and nectar, but the flowers and (Herrera et al. 2013). nectar were very similar to what is found Although plants with nectars containing in the field. Nectar removal was less (3– yeasts had more nectar removed and more 32%) in nectars inoculated with bacteria visits from foraging bees, they had lower versus the control (no microbes) and nectars reproductive success. H. feotidus plants inoculated with M. reukaufii. All species of with nectar inhabiting yeasts reduced the microbes studied were able to alter nectar number of pollen tubes, fruit set, seed set chemistry, although the researchers only and seed size, which are all indicators of examined A. astilbes (bacteria), M. reukaufii plant reproduction (Herrera et al. 2013). (yeast) and a different Erwinia (bacteria) Herrera et al. (2013) considered longer species than listed above. They no longer pollinator visitation as a potential source of had access to their bacterial species E. reduced plant fecundity when bees removed tasmaniensis and L. kunkeei. In summary, more nectar. The authors mentioned another

all microbes reduced pH, while H2O2 and study found that when H. feotidus is self- sucrose remained unchanged. A difference pollinated they produce fewer and smaller was observed in the glucose and fructose seed sets; longer visits by pollinators would levels of A. astilbes inoculated nectar. There facilitate more self-pollinations. were significantly higher concentrations of The new data emerging from the studies glucose and fructose than in the control, M. discussed in this section are just beginning reukaufiiinoculated flowers andErwinia sp. to break down the ecological role microbes inoculated nectars. The authors suggest that may play in plant reproduction. Researchers this alteration of sugar concentration may are continuing to find support that yeasts and be the driving force behind the preference bacteria play a significant part in facilitating of the bee pollinators to remove less nectar the foraging behavior of pollinators. More from bacteria inoculated nectars. It’s the follow up studies are needed to continue difference in nectar chemistry rather than to test the different roles nectar-living just the presence of bacteria that is altering microbes, pollinators, and plants have in bee behavior (Good et al. 2014). this intertwined system. M. reukaufii did not deter bees from removing nectar of the flowers they Conclusion inhabited. This is supported in another study The perception of plant-pollinator as well. Herrera et al. (2013) found that the relationships as being a binary system, bee species B. terrestris preferred nectars when a plant essentially attracts a specific inhabited by yeasts than nectars without pollinator, is changing with emerging yeasts when they examined the plant species evidence of microbial organisms altering H. feotidus. The statistical significance of nectar characteristics - a major energetic nectar consumed was in nectars inoculated reward for pollinators provided by flowers with M. reukaufii and not M. gruessi. The in exchange for reproductive services to authors were unsure as to the direct source of increase fecundity. This review analyzed attraction that was produced by the presence studies that present growing evidence of

The Duluth Journal of Undergraduate Biology 26 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 microbes inhabiting nectars across a wide geographic range as well as evidence that these microbial communities are altering the characteristics of nectar. In some cases, microbes can degrade the nectar reward produced by a plant by reducing sucrose concentrations. On the other hand, in bumble bee pollinated plants, microbe activity may be beneficial by warming the nectar. This field of research is still in its infancy and there is still much to be learned about the implications of microbial Author Biography communities in nectar. There is a need for Anthony Schmitt is a senior at the continued studies on a wider, global scale University of Minnesota Duluth. He will be that evaluate microbial communities and graduating with B.A. in both Biology and their ecological implications in flowers Writing Studies. He currently works as an throughout the world. Closer examination undergraduate research assistant for Dr. as to how microbial communities develop Clay Carter. Dr. Carter studies genes and and directly impact pollinator interactions other molecular processes that underly the is needed. Additionally, research should production of nectar in plants, specifically focus on how plant reproductive success is in the Brassicaceae family. After graduating influenced by microbial nectar alteration. he plans to attend graduate school and The complexity and lack of knowledge pursue a PhD in a field of biology, although involved with microbial ecology opens the he is currently undecided. Anthony studied door for further experimentation to better abroad in New Zealand his sophomore year understand this rather elusive biological and loves to travel. He also loves hunting, system. fishing and anything that gets him outdoors. Acknowledgements References I would like to thank Dr. David Beard, Dr. Adler LS. 2000. The ecological significance of Jennifer Liang, Dr. Shannon Stevenson, and toxic nectar. Oikos 91(3):409-20. Dr. Elizabethada Wright for their continued Alvarez-Perez S, Herrera CM, de Vega C. 2012. support and advisement throughout the Zooming-in on floral nectar: A first exploration process of preparing this review for final of nectar-associated bacteria in wild plant publication. I would also like to thank communities. FEMS Microbiol Ecol 80(3):591- Dr. Clay Carter for the inspiriation of this 602. review. His expertise and advice were Alvarez-Perez S, Lievens B, Jacquemyn H, crucial to the publication of this article. I Herrera CM. 2013. nectaris sp also have to achnowledge Dr. John Pastor nov and acinetobacter boissieri sp nov., isolated for helping me grow as a writer througout from floral nectar of wild mediterranean insect- my college career. Lastly, I would like to pollinated plants. Int J Syst Evol Microbiol thank the peer reviewers for their time and 63:1532-9. feedback to help me prepare a publishable Baker H and Baker I. 1983. A brief historical review. review of the chemistry of floral nectar. In: The biology of nectaries. Bentley B and Elias T, editors. 1st ed. New York: Columbia University Press. 126 p. Belisle M, Peay KG, Fukami T. 2012. Flowers

The Duluth Journal of Undergraduate Biology 27 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 as islands: Spatial distribution of nectar- Fridman S, Izhaki I, Gerchman Y, Halpern M. inhabiting microfungi among plants of mimulus 2012. Bacterial communities in floral nectar. aurantiacus, a hummingbird-pollinated shrub. Environmental Microbiology Reports 4(1):97- Microb Ecol 63(4):711-8. 104. Bentley B and Elias T, editors. 1983. The biology Fuhrman JA. 2009. Microbial community of nectaries. 1st ed. New York: Columbia structure and its functional implications. Nature University Press. 253 p. 459(7244):193-9. Brandenburg A, Dell’Olivo A, Bshary R, Gonzalez-Teuber M and Heil M. 2009. Nectar Kuhlemeier C. 2009. The sweetest thing chemistry is tailored for both attraction of advances in nectar research. Curr Opin Plant mutualists and protection from exploiters. Plant Biol 12(4):486-90. Signaling & Behavior 4(9):809-13. Brysch-Herzberg M. 2004. Ecology of yeasts in Good AP, Gauthier M-PL, Vannette RL, plant-bumblebee mutualism in central Europe. Fukami T (2014) Honey Bees Avoid Nectar FEMS Microbiol Ecol 50(2):87-100. Colonized by Three Bacterial Species, But Not Canto A and Herrera CM. 2012. Micro-organisms by a Yeast Species, Isolated from the Bee Gut. behind the pollination scenes: Microbial imprint PLoS ONE 9(1): e86494. doi:10.1371/journal. on floral nectar sugar variation in a tropical plant pone.0086494 community. Annals of Botany 110(6):1173-83. Heil M. 2011. Nectar: Generation, regulation, Carter C and Thornburg R. 2004. Is the nectar and ecological functions. Trends Plant Sci redox cycle a floral defense against microbial 16(4):191-200. attack? Trends Plant Sci 9(7):320-4. Herrera CM, Pozo MI, Bazaga P. 2012. Jack of Carter C, Shafir S, Yehonatan L, Palmer R, all nectars, master of most: DNA methylation Thornburg R. 2006. A novel role for proline and the epigenetic basis of niche width in a in plant floral nectars. Naturwissenschaften flower-living yeast. Mol Ecol 21(11):2602-16. 93(2):72-9. Herrera CM and Pozo MI. 2010. Nectar yeasts Carter C, Healy R, O’Tool NM, Naqvi SMS, warm the flowers of a winter-blooming plant. Ren G, Park S, Beattie GA, Horner HT, Proceedings of the Royal Society B-Biological Thornburg RW. 2007. Tobacco nectaries Sciences 277(1689):1827-34. express a novel NADPH oxidase implicated in Herrera CM, Pozo MI, Medrano M. 2013. Yeasts the defense of floral reproductive tissues against in nectar of an early-blooming herb: Sought by microorganisms. Plant Physiol 143(1):389-99. bumble bees, detrimental to plant fecundity. Cnaani J, Thomson JD, Papaj DR. 2006. Flower Ecology 94(2):273-9. choice and learning in foraging bumblebees: Herrera CM, Garcia IM, Perez R. 2008. Invisible Effects of variation in nectar volume and floral larcenies: Microbial communities degrade concentration. Ethology 112(3):278-85. floral nectar of bumble bee-pollinated plants. Cronk Q and Ojeda I. 2008. Bird-pollinated Ecology 89(9):2369-76. flowers in an evolutionary and molecular Herrera CM, Canto A, Pozo MI, Bazaga P. context. Journal of Experimental Botany 2010. Inhospitable sweetness: Nectar filtering of 59(4):715-27. pollinator-borne inocula leads to impoverished, de Vega C and Herrera CM. 2012. Relationships phylogenetically clustered yeast communities. among nectar-dwelling yeasts, flowers and ants: Proceedings of the Royal Society B: Biological Patterns and incidence on nectar traits. Oikos Sciences 277(1682):747-54. 121(11):1878-88. Herrera CM, de Vega C, Canto A, Pozo MI. de Vega C, Herrera CM, Johnson SD. 2009. 2009. Yeasts in floral nectar: A quantitative Yeasts in floral nectar of some South African survey. Annals of Botany 103(9):1415-23. plants: Quantification and associations with Peay KG, Belisle M, Fukami T. 2012. pollinator type and sugar concentration. S Afr J Phylogenetic relatedness predicts priority Bot 75(4):798-806. effects in nectar yeast communities. Proceedings

The Duluth Journal of Undergraduate Biology 28 Invisible communites in nectar R E V I E W Volume 1: Spring 2014 of the Royal Society B: Biological Sciences 279(1729):749-58. Pozo MI, Lachance M, Herrera CM. 2012. Nectar yeasts of two southern Spanish plants: The roles of immigration and physiological traits in community assembly. FEMS Microbiol Ecol 80(2):281-93. Pozo MI, Herrera CM, Bazaga P. 2011. Species richness of yeast communities in floral nectar of southern Spanish plants. Microb Ecol 61(1):82- 91. Roberts MW. 1996. Hummingbirds’ nectar concentration preferences at low volume: The importance of time scale. Anim Behav 52(2):361-70. Sandhu DK and Waraich MK. 1985. Yeasts associated with pollinating bees and flower nectar. Microb Ecol 11(1):51-8. Thornburg R, Carter C, Powell A, Mittler R, Rizhsky L, Horner H. 2003. A major function of the tobacco floral nectary is defense against microbial attack. Plant Syst Evol 238(1-4):211- 8. Vannette RL, Gauthier ML, Fukami T. 2013. Nectar bacteria, but not yeast, weaken a plant - pollinator mutualism. Proceedings of the Royal Society B-Biological Sciences 280(1752):20122601. Whitney HM, Dyer A, Chittka L, Rands SA, Glover BJ. 2008. The interaction of temperature and sucrose concentration on foraging preferences in bumblebees. Naturwissenschaften 95(9):845-50.

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