Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands
© Mittelbach et al.; licensee BioMed Central. 2015
Received: 3 September 2014
Accepted: 15 January 2015
Published: 1 February 2015
Studies on the diversity of yeasts in floral nectar were first carried out in the late 19th century. A narrow group of fermenting, osmophilous ascomycetes were regarded as exclusive specialists able to populate this unique and species poor environment. More recently, it became apparent that microorganisms might play an important role in the process of plant pollination. Despite the importance of these nectar dwelling yeasts, knowledge of the factors that drive their diversity and species composition is scarce.
In this study, we linked the frequencies of yeast species in floral nectars from various host plants on the Canary Islands to nectar traits and flower visitors. We estimated the structuring impact of pollination syndromes (nectar volume, sugar concentration and sugar composition) on yeast diversity.
The observed total yeast diversity was consistent with former studies, however, the present survey yielded additional basidiomycetous yeasts in unexpectedly high numbers. Our results show these basidiomycetes are significantly associated with ornithophilous flowers. Specialized ascomycetes inhabit sucrose-dominant nectars, but are surprisingly rare in nectar dominated by monosaccharides.
There are two conclusions from this study: (i) a shift of floral visitors towards ornithophily alters the likelihood of yeast inoculation in flowers, and (ii) low concentrated hexose-dominant nectar promotes colonization of flowers by basidiomycetes. In the studied floral system, basidiomycete yeasts are acknowledged as regular members of nectar. This challenges the current understanding that nectar is an ecological niche solely occupied by ascomycetous yeasts.
Several recent studies have invoked a resurgent interest in the importance of pollination to plant reproductive success and fertility [1,2]. Herrera et al.  and Vannette et al.  presented the first evidence for microbially-mediated impacts on plant pollination and fecundity by nectar dwelling yeast and bacteria, respectively. Nectar-dwelling unicellular fungi (yeasts) have fascinated researchers for over a hundred years. For example, ascomycetous yeasts, namely Metschnikowia reukaufii Pitt & M.W. Mill. and Metschnikowia gruessii Gim.-Jurado, were known since the late nineteenth century as common inhabitants of floral nectar in various host flowers . Subsequent studies addressed the distribution , ecology [7-9], and physiological properties [10-12] of these species. Research in the past years added knowledge on functionality , population structure  and epigenetic variability of Metschnikowia reukaufii . Studies on flowers all over the world strengthened the impression of a narrow and highly specific nectarivorous yeast community [16-18], which may consist up to 85% of fast growing ascomycetous specialists , adapted to sugar rich, temporally and spatially fragmented nectar environments.
A broader diversity of yeast species has been regularly reported from nectar [6,20,21], including both unicellular non-fermenting yeasts and yeast-like fungi [22,23]. Basidiomycetous yeasts are supposedly unable to persist in specific nectar environments based on their in vitro properties, such as growth preferences determined in culture media [24,25]. Thus, they are mostly regarded as autochthonous to non-flower habitats, such as plant surfaces or soils [23,26,27] and their presence in floral nectar is usually believed to be a contamination from other neighboring substrates .
The nectar of individual flowers represent species-poor habitats [18,19] characterized by both harsh physiological conditions  and strong species competition , which favors fast-growing microorganisms. The co-evolution of pollination syndromes between plants and flower visitors shaped a tremendous variety of floral habitats, which differ amongst other traits in nectar volume, and sugar concentration and composition [30,31]. Reukauf  and later Sandhu and Waraich  interpreted interspecific variation of nectar-traits as influential to nectar inhabiting fungi, however, Brysch-Herzberg  found no evidence for this in his exhaustive study. Since then, a few studies showed a correlation of main pollinator groups to yeast quantities [17,32], but not to the incidence of yeast nor their diversity .
The floral traits adapted to different pollination strategies should impose both direct and indirect effects on nectar dwellers. For example, Herrera et al.  showed the highly concentrated, sucrose-dominant nectar of Helleborus foetidus L. may act as a selective environmental filter for arriving yeast inoculum, which explains the dominance of osmophilic ascomycetes in nectar [16,34]. Responses of yeast community structure to nectar properties have received little attention so far, although different taxonomic groups of yeasts often possess distinct physiological characteristics  and assimilation tests of yeast strains demonstrated wide inter-specific differences in the ability to utilize various mono-, di, trisaccharides, as well as polyoles, alcohols etc. as a sole carbon source [36,37]. As a consequence, changes in the yeast environment, such as nectar chemistry, are likely to favor the growth of different yeast species, thereby directly affecting yeast community composition through the alteration of osmotic pressure, pH or availability of a particular nutrient.
The adaptation to environmental (nectar) habitats is only one factor responsible for a successful establishment of yeast populations. Equally important should be the propagation, the dispersal, and the inoculation of yeasts into nectar habitats, which are in turn indirectly driven by floral traits. Since nectar-dwelling yeasts are predominantly vectored by flower visitors , the composition and visitation frequencies of pollinator communities might conceivably govern the composition and abundances of the yeast-inoculum, respectively. Visitor anatomy and behavior should additionally impact yeast transfer and inoculation. Thus, a lower degree of visitor specialization signifies foraging on a wider variety of flowers or even food sources other than nectar. This might lead to a more heterogeneous pool of microorganisms, including ones normally not found in nectar, via the constant transfer of microorganisms between substrates .
To study the influence of pollination syndromes on nectar-dwelling yeasts, we analyzed floral nectars on the island of Tenerife, Spain. The Canary Islands provide a unique bird-pollination element , comprising opportunistic nectar feeding passerine birds . Different evolutionary scenarios for the origin of ornithophily in Macaronesia have been proposed. Most likely, bird-related floral traits are relictual in some plant groups and de-novo in others (see  for Discussion). Flowers adapted to bird pollination are generally characterized by large red to orange corollas, diurnal anthesis, the absence of scent and the provision of suitable landing platforms . Nectars are expected to be abundant, highly dilute with a dominance of monosaccharides . However, on the Canary Islands, morphological adaptations of ornithophilous flowers are inconsistent with entomophilous relatives, albeit, Canarian bird species tend to prefer hexose (monosaccharide-dominated) to sucrose (disaccharide-dominated) nectars . The de novo adaptation of ornithophily to passerine birds after island colonization is expected for the plant genus Echium L. (Boraginaceae) , which developed rather generalistic pollination syndromes and a variety of mixed bird/insect pollination systems .
In the present study we aim to link nectarivorous yeast diversity to different pollination-syndromes, addressing the impacts of nectar traits (volume, sugar concentration and composition) and floral visitors (frequency and composition). We hypothesize that yeast communities should be determined by two different sets of parameters: (i) alterations in the floral habitat itself, such as nectar concentration, abundance, and sugar composition, and (ii) the yeast transfer conditions as a result of different flower visitor assemblages.
Sampled host plants, nectar traits, flower visitors, & diversity index
E. plantagineum 1
E. plantagineum 2
Analysis of main nectar-sugars (sucrose, glucose, and fructose) revealed two major groups of host plants with either sucrose-dominant or hexose-dominant nectars (Table 1). Nectar-volumes ranged from 0.9 μl (±0.2 SD) in E. strictum to 22.7 μl (±17) in C. canariensis with sugar concentrations from 14.5% (±2.6) in E. leucophaeum to 42.7% (±10.4) in T. heterophyllum.
Observations of floral visitors
We observed a total of 7503 flower visits on the 4 focal Echium host species. Individual visitation rates differed between the observed species up to one magnitude (Table 1). Echium strictum received the highest visitation frequency in 2012 (0.00378 visits per flower per minute (v/f/min) ±0.052 SD) and Echium simplex the lowest in 2013 (0.00037 ± 0.004). Most abundant pollinator groups were bumblebees (0.0056 ± 0.001), consisting almost exclusively of visits by Bombus canariensis Pérez. Visitors of the functional group of bees (0.00485 ± 0.001) were classified as members of the genera Megachile and Osmia, while honeybees were only present in Echium strictum in 2012 and account for only 21% of all bee visits. Flower visiting birds were identified either as Common Chiffchaff (Phylloscopus collybita Vieillot), Atlantic Canary (Serinus canaria L.), or Blue Tit (Parus caeruleus L.; only on Echium leucophaeum) and have been observed on Echium leucophaeum in 2012 (0.0001 ± 0.003) and on Echium simplex in 2013 (0.00011 ± 0.003).
Yeast communities and pollination syndromes
The overall diversity of yeast species determined in this study is consistent with results of previous studies [19,48]. However, our study yielded numerous basidiomycetous yeasts regularly isolated from flowers and identified as Cryptococcus carnescens, Cr. heimaeyensis, and Cystofilobasidium capitatum (Additional file 1). Our results show that yeast communities are significantly mediated by the type of flower visitor and by nectar sugar concentration (Figure 4). This confirmed former hypotheses of flower-trait mediated yeast communities [7,22] and expanded the known effects of pollinator composition  and pollination syndromes  on the diversity and composition of yeast communities. Although floral traits and pollinator composition are naturally correlated, they may steer two different mechanisms of the yeast-colonization process, namely (i) the ability to grow in nectar and (ii) the probability of flower inoculation. Below, we discuss the two mechanisms in more details.
(ii) Selective effects of nectar can only partially explain the reduced occurrence of ascomycetes in hexose-dominant nectars. Specialized nectar yeasts are believed to grow equally well in different nectar-sugar compositions (, Figure 2), although data on host-genotype interaction of M. reukaufii provides some support to the diversifying selection hypothesis  and suggests growth characteristics to be rather strain-specific.
The dominance of basidiomycetes in hexose nectars might be a result of an altered flower visitor community (Figure 4) since the visitation frequency of insects, commonly vectoring and inoculating ascomycetes [24,28] is reduced. In addition, the inoculation probability of allochthonous species should be increased in ornithophilous flowers. This might be caused by the generalistic foraging behavior of nectar-feeding birds on the Canary Islands, which feed on a broad variety of resources of plant origin, such as fruits or plant tissues  in addition to hexose-rich nectar . Since these plant-related habitats harbor large numbers of basidiomycetous yeasts [26,27], the probability of yeast inoculation in nectar is increased by bird visitors. Indeed, South African plants, visited by passerine birds were found to harbor more yeasts (incidence and abundance) than sympatric plants visited by insects, only . Yeast diversity in our study is either high in flowers visited by birds or in flowers visited by bumblebees and other bees (Figure 4). Our observations mirror one common ecological law that selective pressure in the environment constrains species diversity, including microorganisms. Less strict conditions (sugar type and concentration) attract different flower visitors and allow a broader range of microbes to colonize flowers from a larger number of sources.
The shift from insect to passerine bird pollination on the Canary Islands resulted in various degrees of dependence to bird-visits, ranging from strict ornithophily in Isoplexis canariensis  to occasional visits by birds in Echium wildpretii . As a consequence, floral adaptations to ornithophilous pollinators might be imperfect in the sense of classic pollination ecology: for example Teucrium heterophyllum is believed to be pollinated by passerine birds, despite highly concentrated sucrose-dominant nectar. Taken together, these diverse and overlapping floral habitats provide a broad spectrum of available vectors and niches for microbial nectar-colonizers on a small regional scale. Our study reveals that the filter effect of nectar  might depend on nectar properties and on the diversity of the microbial inoculate. The pollination syndromes of the sampled host plants could in turn facilitate the inoculation and ease establishment of allochthonous microorganisms in nectars due to their species richness and overlapping diversity. These suggestions are supported by increased functional diversities of yeast communities in niches other than high-concentrated nectars (Figure 4).
Despite being combined in one ornithophilous pollination-syndrome, passerine bird pollination in the old world and hummingbird pollination in the new world evoked different floral adaptations by plants, impeding comparisons of the diversity of nectar-dwelling microbes. Sugar-concentrations of nectars in hummingbird-flowers have been found to be elevated (25%) in contrasts to sunbird pollinated flowers (21%) . In addition, hummingbirds commonly prefer sucrose-dominated nectars [44,55] and forage on flowers with long and narrow corollas, which impede the visitation of other floral visitor-groups .
Indeed, nectars of hummingbird-pollinated Mimulus aurantiacus are dominated by specialized ascomycetes, such as M. reukaufii and Candida rancensis , species prevailing in sucrose-dominated flowers in our study (Figure 2). Nonetheless, Belisle et al.  showed that hummingbirds transport a large diversity of microfungi, including yeasts species isolated in our survey, namely C. rancensis, S. bombicola, Cr. flavescens, Cr. carnescens, and A. pullulans (Additional file 1).
Basidiomycetes in nectar
According to our results, Cys. capitatum and Cr. carnescens are common inhabitants of sampled flowers and may directly profit from the sampled hexose-dominant nectar-environments (Figure 2). Brysch-Herzberg  also isolated Cys. capitatum from floral nectar regularly, without recognizing its potential nectar-related habit. Previously there was no evidence that Cys. capitatum might be competitive with fast-growing, fermenting ascomycetes in sugar-rich environments, due to characteristics of its former isolation habitats, such as soil [60,61] and marine surface water . However, recently, researchers have documented an affinity to sugar-enriched habitats by Cys. capitatum and report this species from fruits of Sorbus aucuparia L. and Rosa canina L. [63,64], tree exudates ( and references therein), and fruiting bodies of the tree parasite Cyttaria .
Little is known about the ecology of the second frequent basidiomycete, Cr. carnescens, which was isolated from flowers twice [57,67], and has been reported as frequent inhabitant of grapes  and phyllosphere in Mediterranean ecosystems . Despite these plant related sources, Cr. carnescens has been understood as a pervasive species isolated from seawater, soil, and glacial ice . Based on its phenotype, this yeast has been long considered a synonym of Cr. laurentii until Takashima et al.  demonstrated that this complex comprises several distinct and distantly related species. Although proper interpretation of both Cr. laurentii and Cr. carnescens from older studies is therefore precluded, several members of the two phylogenetic clades (Cr. laurentii and Cr. victoriae, respectively) inhabit substrates of plant origins, such as fruits and leaves  and were also isolated from nectar in the present study: Cr. laurentii, Cr. tephrensis, Cr. foliicola, and Cr. heimaeyensis. The latter species was also reported from flowers before  but mislabeled as Cryptococcus aff. victoriae (Additional file 2).
In this study, we present significant trends in the community structure of nectar dwelling yeast. Nectar sugar concentration, composition, and flower visitor assemblages were identified as main selective forces. Furthermore, we reveal the previously underestimated importance of basidiomycetous yeasts as inhabitants of ornithophilous flowers with hexose dominant nectar on Tenerife. Bird-pollination on the Canary Islands clearly represents an exotic case study in the evolution of floral traits, but the provision of hexose-rich or even dominant nectar is a common phenomenon and can be found in various plants , e.g. related to dipteran pollination syndromes  or due to phylogenetic history . More comprehensive data on yeast distribution across different pollination syndromes and nectar types would be clearly desirable to better comprehend the distribution, ecology, diversity and functions of basidiomycetous yeasts in floral nectar.
It is widely known that basidiomycetous and ascomycetous yeasts differ substantially in their lifestyles and physiological properties, suggesting different ecological strategies. While basidiomycetes have been hardly associated with nectar foraging insects, ascomycetous specialists have been almost exclusively isolated from flowers, honey pots and insects [24,73]. This leads to the conclusion that ascomycetes spend their whole life cycle inside the insect-flower system, whereas basidiomycetes might possess a broader variety of alternative substrates or even switch from saprobic to parasitic or fungicolous lifestyles [74,75]. Nonetheless, both groups highly depend on durable structures to overcome phases of transportation and rest in ephemeral nectar habitats. While the formation of ascospores in ascomycetous yeasts has been well studied, similar resistant structures of basidiomycetes in sugar-rich habitats have not been identified so far. Whether or not these ecological prerequisites together with the corresponding assimilation profiles provide basidiomycetes an advantage in colonizing nectars of ornithophilous plants requires detailed studies.
Inconsistencies in yeast incidences among years, the unbalanced experimental design, and the reliance on data from literature in this study clearly formulate the need for a more detailed and comprehensive sampling. Nonetheless, diversity patterns of nectar-borne yeasts remain stable during both years, validating our conclusions although impeding broader generalization.
Study sites & plant species
Fieldwork was conducted on the island of Tenerife in the eastern Anagar mountain region. In April 2012 a sympatric population of Echium strictum L.f., E. leucophaeum Webb ex Sprague & Hutch., and E. plantagineum L. was studied close to Chinamada (approx. 28°33.80, − 016°17.41'). In May 2013, we sampled a sympatric population (approx. 28°34.70', −016°08.75') of E. strictum, E. leucophaeum, E. simplex DC., and E. plantagineum (1 population in scrubland and 1 population close to forest). All other studied taxa were sampled within 500 m of the focal population. Plant species grow in natural sclerophyllous coastal scrubland, except for Isoplexis canariensis (L.) Lindl. and Canarina canariensis (L.) Vatke, which are part of the vegetation of lower laurel forests . A complete list of sampled plants can be found in Additional file 1.
Individual flowers (flowering branches or inflorescences) in fertile female stage were carefully removed (to avoid mechanical damages) from 3 plant individuals (except for C. canariensis (n = 6) and E. plantagineum (n = 4)) in the late afternoon and immediately covered in sterile plastic-bags until further processing in the lab. To account for biases due to different flower numbers of host plants, we randomly picked 40 flowers from collected plant material for nectar sampling. In adition, we covered 5 flowers of each host plant in bud stage and processed the nectar as controls. Nectar was removed from the flowers using sterile micro capillaries (Hirschmann Laborgeräte, Eberstadt, Germany) within a maximum of 4 hours after flower harvest. Total nectar volume of each flower was mixed with in 100 μl of sterile tap water and streaked out on modified solid YM medium (0.3% w/v Yeast extract, 0.5% w/v Peptone, 0.3% w/v Malt extract, 1% w/v Glucose, 2% w/v Agar) supplemented additionally with nectar-related sugars (1% w/v Fructose and 1% w/v Sucrose) and acidified with 1% v/v 80% Lactic acid (final pH = 4.5) to prevent bacterial growth. Plates were stored at room temperature for 4 days and then kept at lower temperature (4°C) to slow down the development of molds. Plates were examined after 7 days of incubation: colonies were differentiated into macro-morphological types using dissection microscopy and the respective counts were recorded as colony forming units (CFU). One representative per plate was transferred into pure culture. All isolated strains were stored at −80°C in glycerol/glucose (1:1, w:w). Nectar samples from covered buds (controls) did not yield any fungal or yeast cultures.
Pure cultures were transferred to liquid YM-medium and incubated for 48 hours at room temperature. DNA was extracted using a phenol-chloroform extraction method and the LSU ribosomal gene region (D1/D2 domains) was amplified (for detailed methods see ) and sequenced using the primers ITS1f or NL1 and NL4 [78,79] .
Sequences were edited manually and trimmed using Sequencher 5.0 following the criteria: (i) trimming no more than 25% of the sequence length until the first 25 nucleotides would contain less than 5 ambiguities, and (ii) trimming no more than 25% of the sequence length until the first 25 nucleotides would contain less than 5 nucleotides with confidences below 25. Two separate alignments for Basidiomycota and Ascomycota were created using MAFFT 7.110 , manually edited and curated with GBlocks allowing smaller final blocks and gap positions within it . For convenience, formal classification into operational taxonomic units (OTU) was conducted using MOTHUR 1.32.1  applying a 98% cut-off value and considering also different similarity values traditionally used to delimit yeast species in ascomycetes and basidiomycetes [18,83,84]. Results of the OTU analysis were confirmed by morphological inspections and interpretation of phylogenetic maximum likelihood trees obtained with raxmlGUI  using the GTRGAMMA model and 1000 bootstrap replications (Figure 6).
Three strains with reduced sequence qualities (MOM_217, MOM_232, MOM_859) were manually inspected again with the Sequencher 5.0 software and included into an alignment of closely related OTUs taking into account both their morphological characterization and phylogenetic placement. Representative sequences for each OTU were identified to the species level using NCBI GenBank and MycoBank databases . Sequences are stored at the EMBL nucleotide sequence database  and representative strains are deposited in the DSMZ, German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) (Table 1).
Standing crop nectar was removed from 25 randomly selected flowers per species and population, harvested at the same time and from the same individual plants as before. Volumes were measured with glass-capillary tubes. Sugar concentrations were estimated with a handheld refractometer (10-80% brix, neoLab Universal, Germany). To analyze sugar compositions, 5 flowers were covered with nylon meshes in bud stage to avoid nectar contamination. Nectar samples were harvested from open flowers and stored in sterile tubes, filled with 70% Ethanol until further analysis by HPLC (see  for detailed methods). Nectar samples for sugar analysis from Echium leucophaeum, E. simplex, E. strictum were collected at the Botanical Garden in Berlin-Dahlem, Germany and samples of E. plantagineum at the Botanical Garden of Bonn-University, Germany, following the same procedure. Nectar sugar compositions of Canarina canariensis, Teucrium heterophyllum L’Hér., Lavatea acerifolia Cav., and Isoplexis canariensis are taken from literature [45,47].
Observations of flower visitors
Observations of floral visitors were conducted prior to nectar yeast samplings on the same individual plants. Each flower visit was counted as a new and independent event without any regard to individual visitors probing on more than one flower per plant in a row. Flower observations were undertaken in 10 min intervals with 3 researchers simultaneously, each one observing a different plant species. Focal species and individuals were changed every 30 minutes to ensure an objective threshold and to provide coverage of all plant species during all times of one day. Pollinators were pooled to functional groups as proposed by Fenster et al. , since we believe this classification is suitable for the objectives of this study. To increase accuracy, large plants were divided into intercepts to reduce the number of flowers observed simultaneously. Observations on Canarina canariensis and Isolplexis canariensis did not yield visitor observations, data regarding visitation rates of these species was therefore taken from Ollerton et al.  and Rodríguez-Rodríguez & Valido , respectively. Teucrium heterophyllum is reported as generally bird-pollinated [88,89], and we rely on this information since no observational records were available for this plant species. Similarly, Lavetera acerifolia is considered insect-pollinated .
Growth tests were conducted in closed 96-well microplates  in 150 μl of artificial nectar medium, consisting of yeast nitrogen base (YNB, Difco BD) and 40% sugars mixture (Glucose, Sucrose, Fructose, 1:1:1 w/w) using Infinite 200 Pro microplate reader (Tecan Austria GmbH, Austria). Cells from 5-day cultures were harvested from solid YM media dissolved in 1% PBS buffer, filtered through 30 μm filter (Partec GmbH, Germany) and inoculated in the artificial nectar medium (20 cells per μL = 3000 cells per well) using BD FACSAria III cell sorter (BD Biosciences, USA) as starting cultures. Each strain was inoculated in 16 wells and a total of 32 wells were blank containing the medium only. Cultures were incubated for 10 days at 25°C and measured automatically every hour. Between measurements plates were incubated as static culture for 45 minutes followed by 15 minutes shaking at 1000 rpm with 4 mm amplitude prior to the next absorbance measurement. Absorbance was measured at 600 nm every hour using the following options: multiple per well (12 reads in circle (filled) pattern) and 5 flashes in a read. Values from the reads were averaged.
The incidence of species was determined in all 480 flowers and organized in a presence/absence ‘site*species’ matrix to analyze yeast diversity. A total of 291 flowers did not yield any culturable yeast and were excluded from the analysis. To avoid biased results in the final analysis due to inflated zero counts and unequal sample sizes, yeast incidences of single flowers of each host plant and year were summarized and handled as yeast frequencies per host plant and year. Relative incidences were determined as a proportion of a particular yeast species frequency in each host plant and year. We calculated Shannon’s index of diversity  to characterize the diversity and structure of host-specific yeast communities.
Mean nectar volume and sugar concentration for each host plant and year were standardized to account for differences in measured units. Except for nonparametric multidimensional scaling (nmds) analysis, the sugar composition was classified based on the percentages of sucrose and hexose into a factor variable (nectar type), providing 4 categories reaching from sucrose dominant to hexose-dominant nectar (for details, see ).
Pollinator observations of individual plants were pooled to achieve ‘visitation frequencies’ of each functional visitor group as visits/flower/minute (v/f/min) for each host plant and year. To assess diversity of flower visitors for each host plant, we calculated the generalization level according to  as Simpson diversity index.
Mantel tests of dissimilarity matrices of yeast species frequencies (Bray-Curtis), as implemented in the R package ‘vegan’  were used to evaluate the correlation between floral traits and flower-visitor frequencies. To create an ordination plot of yeast diversity, we applied the ‘metaMDS’ function for nonparametric multidimensional scaling (nmds) to the dissimilarity matrix. Floral traits and visitor frequencies were subsequently added to the nmds graphic using the implemented ‘envfit’ function. The function ‘adonis’ was used to partition the variation of yeast frequencies among factorized floral traits and visitation frequencies. To account for differences between samplings in 2012 and 2013, we constrained the subsequent permutation tests (10,000 replicates) to the respective sampling years. Both functions are also implemented in R package ‘vegan’.
We used recursive binary partitioning to regress yeast diversity according to environmental covariates incorporating nectar traits (volume + sugar concentration + nectar type), pollinator compositions (visitation frequencies of each functional group), and sampling year. The procedure constructs unified tests for independence by means of conditional distribution of linear statistics in the permutation test framework. Stopping criteria were set to the nominal level of the conditional independence tests as α = 0.01, using a simple Bonferroni correction. The analysis was conducted with the ‘ctree’ function implemented in the ‘party’ package . Subsequently, we extracted assembled yeast communities from the recursive partitioning analysis output and used the function ‘ses.mpd’, as implemented in the R package ‘picante’  to test whether these assemblages represent phylogenetically clustered subsamples. The ‘ses.mpd’ calculates the mean pairwise distance between all species in the subsamples, based on a provided phylogenetic maximum likelihood tree, and compares phylogenetic relatedness to patterns expected under a null model, allowing for randomized community data matrix abundances within samples (maintaining species occurrence frequencies). Additionally, we calculated the functional dispersion indices for all node assemblages (‘FDis’ in the ‘FD’ package ) of assimilation traits for each acknowledged species.
Differences in relative incidence between ascomycetes and basidiomycetes were calculated with the Kruskal Wallis Test, using nectar type as grouping factor. Significances for single factor combinations were identified by Wilcoxon pairwise tests with subsequent Holm correction for multiple tests .
We calculated the point-biserial correlation coefficient to analyze the species ecological preferences . The index measures the association of standardized species distributions for site groups as implemented in the r-package ‘indicspecies’ . All calculations were accomplished using the R framework 3.0.2. .
All research was conducted within an appropriate ethical framework. Field work on Tenerife was conducted with permission by the Área de Medio Ambiente, Sostenibilidad Territorial y Aguas (Expte: AFF 33/13, No Sigma: 2013–00172).
Availability of supporting data
The data set supporting the results of this article is available in the Data Dryad repository, doi:10.5061/dryad.0qp3q .
The authors like to thank O. Röhl, S. Lotze-Engelhardt, P. Testroet, and S. Mitulla for exhaustive help in the fieldwork and Dominik Schmidt for support in the lab. Sampling in Tenerife would have been impossible without Arnoldo Santos Guerra. Victoria Michael and Petra Henke (DSMZ) assisted with laboratory experiments, and Álvaro Fonseca and Marco Guerreiro (FCT/UNL) provided their experience in identification of basidiomycetous yeasts. We thank Carlos Herrera & Clara de Vega for critical discussions on this project and 2 anonymous reviewer for their helpful comments. The Ruhr-University Bochum partly funded the field and lab work, and enabled the open access publication.
- Garibaldi LA, Steffan-Dewenter I, Winfree R, Aizen MA, Bommarco R, Cunningham SA, et al. Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science. 2013;339:1608–11.PubMedView ArticleGoogle Scholar
- Bartomeus I, Ascher JS, Gibbs J, Danforth BN, Wagner DL, Hedtke SM, et al. Historical changes in northeastern US bee pollinators related to shared ecological traits. Proc Natl Acad Sci U S A. 2013;110:4656–60.PubMed CentralPubMedView ArticleGoogle Scholar
- Herrera CM, Pozo MJ, Medrano M. Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity. Ecology. 2013;94:273–9.PubMedView ArticleGoogle Scholar
- Vannette RL, Gauthier M-PL, Fukami T. Nectar bacteria, but not yeast, weaken a plant-pollinator mutualism. Proc Biol Sci. 2013;280:20122601.PubMed CentralPubMedView ArticleGoogle Scholar
- Boutroux L. Conservation des ferments alcooliques dans la nature. Ann des Sci Nat Série IV, Bot. 1884;17:145–209.Google Scholar
- Jimbo T. Yeasts isolated from flower nectar. Sci Reports Tohoku Imp Univ. 1926;2:161–87.Google Scholar
- Reukauf E. Die Nektarhefen. Die Kleinwelt. 1911;3:25–7.Google Scholar
- Hilkenbach R. Nektarhefen - Neue Beiträge zur Kenntnis der wilden Hefen in der Natur. Kiel: Christian-Albrechts-Universitaet; 1911.Google Scholar
- Nadson G, Krasilnikow N. La levure du nectar des fleurs: Anthomyces reukaufii Gruess. Bull la Société Mycol Fr. 1927;43:232–44.Google Scholar
- Hautmann F. Über die Nektarhefe Anthomyces Reukaufii. Arch für Protistenkd. 1921;48:213–44.Google Scholar
- Martin HH. Beitrag zur Kenntnis der Morphologie und Physiologie der Nektarhefe Candida Reukaufii (Grüss) Diddens et Lodder. Arch Mikrobiol. 1954;20:141–62.PubMedView ArticleGoogle Scholar
- Babjeva IP, Gorin S. About the sporulation and the life cycle of Metschnikowia pulcherrima and M. reukaufii in the nature. Moscow Univ Her. 1973;5:82–5.Google Scholar
- Herrera CM, Pozo MJ. Nectar yeasts warm the flowers of a winter-blooming plant. Proc Biol Sci. 2010;277:1827–34.PubMed CentralPubMedView ArticleGoogle Scholar
- Herrera CM, Pozo MJ, Bazaga P. Nonrandom genotype distribution among floral hosts contributes to local and regional genetic diversity in the nectar-living yeast Metschnikowia reukaufii. FEMS Microbiol Ecol. 2013;87:1–8.Google Scholar
- Herrera CM, Pozo MJ, Bazaga P. Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower- living yeast. Mol Ecol. 2012;21:2602–16.PubMedView ArticleGoogle Scholar
- Lachance M-A, Starmer WT, Rosa CA, Bowles JM, Barker JS, Janzen DH. Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Res. 2001;1:1–8.PubMedView ArticleGoogle Scholar
- Herrera CM, de Vega C, Canto A, Pozo MJ. Yeasts in floral nectar: a quantitative survey. Ann Bot. 2009;103:1415–23.PubMed CentralPubMedView ArticleGoogle Scholar
- Belisle M, Peay K, Fukami T. Flowers as islands: spatial distribution of nectar-inhabiting microfungi among plants of mimulus aurantiacus, a hummingbird-pollinated shrub. Microb Ecol. 2012;63:711–8.PubMed CentralPubMedView ArticleGoogle Scholar
- Pozo MJ, Herrera CM, Bazaga P. Species richness of yeast communities in floral nectar of southern Spanish plants. Microb Ecol. 2011;61:82–91.PubMedView ArticleGoogle Scholar
- Zinkernagel H. Untersuchungen über Nektarhefen. Berlin: Friedrich-Wilhelms-Universitaet; 1929.Google Scholar
- Wiens F, Zitzmann A, Lachance M-A, Yegles M, Pragst F, Wurst FM, et al. Chronic intake of fermented floral nectar by wild treeshrews. Proc Natl Acad Sci U S A. 2008;105:10426–31.PubMed CentralPubMedView ArticleGoogle Scholar
- Sandhu DK, Waraich MK. Yeasts associated with pollinating bees and flower nectar. Microb Ecol. 1985;11:51–8.PubMedView ArticleGoogle Scholar
- Pozo MJ, Lachance M-A, Herrera CM. Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly. FEMS Microbiol Ecol. 2012;80:281–93.PubMedView ArticleGoogle Scholar
- Brysch-Herzberg M. Ecology and taxonomy of yeasts associated with the plant-bumblebee mutualism in central Europe. FEMS Microbiol Ecol. 2004;50:87–100.PubMedView ArticleGoogle Scholar
- Lachance M-A. Yeast biodiversity: how many and how much? In: Rosa CA, Péter G, editors. Biodivers Ecophysiol Yeasts, Ser Yeast Handb. Heidelberg: Springer Berlin; 2006. p. 1–9.View ArticleGoogle Scholar
- Last FT, Price D. Yeasts associated with living plants and their environs. In: Rose A, Harrison J, editors. Yeasts, Vol 1. London: Academic; 1969. p. 183–218.Google Scholar
- Fonseca A, Inácio JJS. Phylloplane yeasts. In: Rosa CA, Peter G, editors. Biodivers Ecophysiol Yeasts, Ser Yeast Handb. Heidelberg: Springer Berlin; 2006. p. 263–301.View ArticleGoogle Scholar
- Herrera CM, Canto A, Pozo MJ, Bazaga P. Inhospitable sweetness: nectar filtering of pollinator-borne inocula leads to impoverished, phylogenetically clustered yeast communities. Proc Biol Sci. 2010;277:747–54.PubMed CentralPubMedView ArticleGoogle Scholar
- Peay KG, Belisle M, Fukami T. Phylogenetic relatedness predicts priority effects in nectar yeast communities. Proc Biol Sci B. 2012;279:749–58.View ArticleGoogle Scholar
- Baker HG, Baker I. Chemical constituents of nectar in relation to pollination mechanisms and phylogeny. In: Nitecki HM, editor. Biochem Asp Evol Biol. Chicago, IL: Chicago University Press; 1982. p. 131–71.Google Scholar
- Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD. Pollination syndromes and floral specialization. Annu Rev Ecol Evol Syst. 2004;35:375–403.View ArticleGoogle Scholar
- De Vega C, Herrera CM, Johnson SD. Yeasts in floral nectar of some South African plants: quantification and associations with pollinator type and sugar concentration. South African J Bot. 2009;75:798–806.View ArticleGoogle Scholar
- Pozo MJ, de Vega C, Canto A, Herrera CM. Presence of yeasts in floral nectar is consistent with the hypothesis of microbial-mediated signaling in plant-pollinator interactions. Plant Signal Behav psb. 2009;4:1102–4.View ArticleGoogle Scholar
- Alvarez-Pérez S, Herrera CM. Composition, richness and nonrandom assembly of culturable bacterial-microfungal communities in floral nectar of Mediterranean plants. FEMS Microbiol Ecol. 2013;83:685–99.PubMedView ArticleGoogle Scholar
- Bolotin-Fukuhara M. Genomics and biodiversity in yeasts. In: Rosa C, Peter G, editors. Biodivers Ecophysiol Yeasts, Ser Yeast Handb. Heidelberg: Springer Berlin; 2006. p. 45–66 [The Yeast Handbook].View ArticleGoogle Scholar
- Sampaio JP. Utilization of low molecular weight aromatic compounds by heterobasidiomycetous yeasts: taxonomic implications. Can J Microbiol. 1999;45:491–512.PubMedView ArticleGoogle Scholar
- Kurtzman CP, Fell J, Boekhout T. The Yeasts, a Taxonomic Study. 5th ed. Amsterdam: Elsevier; 2011. p. 1332–2080.Google Scholar
- Phaff HJ, Miller MW, Mrak EM. The Life of Yeasts. 2nd ed. Cambridge, Massachusetts: Harvard University Press; 1978. p. 320.View ArticleGoogle Scholar
- Ehlers BK, Olesen JM. The fruit-wasp route to toxic nectar in Epipactis orchids? Flora. 1997;192:223–9.Google Scholar
- Vogel S. Blütenbiologische Typen als Elemente der Sippengliederung. Bot Stud. 1954;1:1–338.Google Scholar
- Ollerton J, Cranmer L, Stelzer RJ, Sullivan S, Chittka L. Bird pollination of Canary Island endemic plants. Naturwissenschaften. 2009;96:221–32.PubMedView ArticleGoogle Scholar
- Rodríguez-Rodríguez MC, Valido A. Opportunistic nectar-feeding birds are effective pollinators of bird-flowers from Canary Islands: experimental evidence from Isoplexis canariensis (Scrophulariaceae). Am J Bot. 2008;95:1408–15.PubMedView ArticleGoogle Scholar
- Proctor M, Yeo P, Lack A. The Natural History of Pollination. Portland: Timber Press; 1996. p. 487.Google Scholar
- Baker HG, Baker I. Floral nectar sugar constituents in relation to pollinator type. In: Jones C, Little R, editors. Handb Exp Pollinat Biol. New York: Van Nostrand Reinholdt; 1983. p. 117–41.Google Scholar
- Dupont YL, Hansen DM, Rasmussen JT, Olesen JM. Evolutionary changes in nectar sugar composition associated with switches between bird and insect pollination: the Canarian bird-flower element revisited. Funct Ecol. 2004;18:670–6.View ArticleGoogle Scholar
- Böhle UR, Hilger HH, Martin WF. Island colonization and evolution of the insular woody habit in Echium L (Boraginaceae). P Natl Acad Sci USA. 1996;93:11740–5.View ArticleGoogle Scholar
- Valido A, Dupont YL, Olesen JM. Bird-flower interactions in the Macaronesian islands. J Biogeogr. 2004;31:1945–53.View ArticleGoogle Scholar
- Jacquemyn H, Lenaerts M, Brys R, Willems K, Honnay O, Lievens B. Among-population variation in microbial community structure in the floral nectar of the bee-pollinated forest herb pulmonaria officinalis L. PLoS One. 2013;8:e56917.PubMed CentralPubMedView ArticleGoogle Scholar
- Yurkov AM, Kemler M, Begerow D. Assessment of yeast diversity in soils under different management regimes. Fungal Ecol. 2012;5:24–35.View ArticleGoogle Scholar
- Heil M. Nectar: generation, regulation and ecological functions. Trends Plant Sci. 2011;16:191–200.PubMedView ArticleGoogle Scholar
- Herrera CM. Population growth of the floricolous yeast Metschnikowia reukaufii : effects of nectar host, yeast genotype and host x genotype interaction. FEMS Microbiol Ecol. 2014;88:250–57.PubMedView ArticleGoogle Scholar
- Martín A, Lorenzo JA. Aves Del Archipiélago Canario. La Laguna: Francisco Lemus; 2001.Google Scholar
- Olesen JM. Floral biology of the Canarian Echium wildpretii: bird-flower or a water resource to desert bees? Acta Bot Neerl. 1988;37:509–13.Google Scholar
- Nicolson SW, Fleming PA. Nectar as food for birds: the physiological consequences of drinking dilute sugar solutions. Plant Syst Evol. 2003;238:139–53.Google Scholar
- Nicolson SW, Thornburg RW. Nectar chemistry. In: Nicolson SW, Nepi M, Pacini E, editors. Nectaries and Nectar. Dordrecht: Springer; 2007. p. 215–63.View ArticleGoogle Scholar
- Leins P, Erbar C. Flower and Fruit. Stuttgart: Schweizerbart’sche Verlagsbuchhandlung; 2010. p. 439.Google Scholar
- Belisle M, Mendenhall CD, Oviedo Brenes F, Fukami T. Temporal variation in fungal communities associated with tropical hummingbirds and nectarivorous bats. Fungal Ecol. 2014;12:1–7.View ArticleGoogle Scholar
- Golonka AM, Vilgalys R. Nectar inhabiting yeasts in Virginian populations of silene latifolia (Caryophyllaceae) and coflowering species. Am Midl Nat. 2013;169:235–58.View ArticleGoogle Scholar
- Glushakova AM, Kachalkin AV, Chernov IY. Yeasts in the Flowers of Entomophilic Plants of the Moscow Region. Microbiology. 2014;83:125–34.View ArticleGoogle Scholar
- Vishniac H. A multivariate analysis of soil yeasts isolated from a latitudinal gradient. Microb Ecol. 2006;52:90–103.PubMedView ArticleGoogle Scholar
- Yurkov AM, Wehde T, Kahl T, Begerow D. Aboveground deadwood deposition supports development of soil yeasts. Diversity. 2012;4:453–74.View ArticleGoogle Scholar
- Fell JW, Hunter IL, Tallman AS. Marine basidiomycetous yeasts (Rhodosporidium spp. n.) with tetrapolar and multiple allelic bipolar mating systems. Can J Microbiol. 1973;19:643–57.PubMedView ArticleGoogle Scholar
- Maksimova IA, Yurkov AM, Chernov IY. Spatial structure of epiphytic yeast communities on fruits of Sorbus aucuparia L. Biol Bull. 2009;36:613–8.View ArticleGoogle Scholar
- Glushakova AM, Chernov I. Seasonal dynamics of the structure of epiphytic yeast communities. Microbiology. 2009;79:830–9.View ArticleGoogle Scholar
- Weber RWS. On the ecology of fungal consortia of spring sap-flows. Mycologist. 2006;20:140–3.View ArticleGoogle Scholar
- Libkind D, Tognetti C, Ruffini A, Sampaio JP, van Broock M. Xanthophyllomyces dendrorhous (Phaffia rhodozyma) on stromata of Cyttaria hariotii in northwestern Patagonian Nothofagus forests. Rev Argent Microbiol. 2011;43:226–32.PubMedGoogle Scholar
- De Vega C, Herrera CM. Relationships among nectar-dwelling yeasts, flowers and ants: patterns and incidence on nectar traits. Oikos. 2012;121:1878–88.View ArticleGoogle Scholar
- Verona O, Luchetti G. Ricerche microbiologiche su di alcuni vini ed alcune uve delle marche. Boll Reg Inst Super Agrar Pisa. 1936;12:256–311.Google Scholar
- Fonseca A, Boekhout T, Fell JW. Cryptococcus Vuillemin. In: Kurtzman CP, Fell JW, Boekhout T, editors. Yeasts, a Taxon study. 5th ed. Amsterdam: Elsevier Amsterdam; 2011. p. 1661–737.View ArticleGoogle Scholar
- Takashima M. Three new combinations from the Cryptococcus laurentii complex: Cryptococcus aureus, Cryptococcus carnescens and Cryptococcus peneaus. Int J Syst Evol Microbiol. 2003;53:1187–94.PubMedView ArticleGoogle Scholar
- Nocentini D, Pacini E, Guarnieri M, Martelli D, Nepi M. Intrapopulation heterogeneity in floral nectar attributes and foraging insects of an ecotonal Mediterranean species. Plant Ecol. 2013;214:799–809.View ArticleGoogle Scholar
- Nicolson SW, Van Wyk B-E. Nectar sugars in proteaceae: patterns and processes. Aust J Bot. 1998;46:489–504.View ArticleGoogle Scholar
- Magyar D, Gönczöl J, Révay A, Grillenzoni F, Seijo-Coello MDC. Stauro- and scolecoconidia in floral and honeydew honeys. Fungal Divers. 2005;20:103–20.Google Scholar
- Bandoni RJ. Dimorphic heterobasidiomycetes: taxonomy and parasitism. Stud Mycol. 1995;38:13–27.Google Scholar
- Meirinho C, Estevinho M, Choupina A. Phylogeny and character evolution in the jelly fungi (Tremellomycetes, Basidiomycota, Fungi). Mol Phylogenet Evol. 2011;61:12–28.View ArticleGoogle Scholar
- Bramwell D, Bramwell Z. Wild Flowers of the Canary Islands. 2nd ed. Alcorcón (Madrid): Editorial Rueda SL; 2001.Google Scholar
- Yurkov AM, Kemler M, Begerow D. Species accumulation curves and incidence-based species richness estimators to appraise the diversity of cultivable yeasts from beech forest soils. PLoS One. 2011;6:e23671.PubMed CentralPubMedView ArticleGoogle Scholar
- Gardes M, Bruns T. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol. 1993;2:113–8.PubMedView ArticleGoogle Scholar
- O’Donnell K. Fusarium and its near relatives. In: Fungal Holomorph Mitotic, Meiotic Pleomorphic Speciat Fungal Syst. Wallingford: CAB International Publishing; 1993. p. 225–33.Google Scholar
- Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.PubMed CentralPubMedView ArticleGoogle Scholar
- Castresana J. Selection of conserved blocks from multiple alignments for their Use in phylogenetic analysis. Mol Biol Evol. 2000;17:540–52.PubMedView ArticleGoogle Scholar
- Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75:7537–41.PubMed CentralPubMedView ArticleGoogle Scholar
- Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek. 1998;73:331–71.PubMedView ArticleGoogle Scholar
- Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res. 2002;2:495–517.PubMedView ArticleGoogle Scholar
- Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML. Org Divers Evol. 2011;12:335–7.View ArticleGoogle Scholar
- Schoch CL, Robbertse B, Robert V, Vu D, Cardinali G, Irinyi L, et al. Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database (Oxford). 2014;2014:1–21.View ArticleGoogle Scholar
- Kanz C, Aldebert P, Althorpe N, Baker W, Baldwin A, Bates K, et al. The EMBL nucleotide sequence database. Nucleic Acids Res. 2005;33(Database issue):D29–33.PubMed CentralPubMedView ArticleGoogle Scholar
- Mühlbauer I, Vogel S, Weber A. Blütenökologie makaronesischer Endemiten mit ornithophilen Merkmalen. Linzer biol Beitr. 2000;32:681–2.Google Scholar
- Valido A, Olesen JM. Pollination on islands : examples from the Macaronesian archipelagos. In: Serrano A, Borges P, Boieiro M, Oromí P, editors. Terr arthropods Macaronesia Biodiversity, Ecol Evol. Portugal: Sociedade Portuguesa de Entomologia; 2009. p. 249–83.Google Scholar
- Fuertes-Aguilar J, Ray MF, Francisco-Ortega J, Santos-Guerra A, Jansen RK. Molecular evidence from chloroplast and nuclear markers for multiple colonizations of Lavatera (Malvaceae) in the Canary Islands. Syst Bot. 2002;27:74–83.Google Scholar
- Kurtzman CP, Fell JW, Boekhout T, Robert V. Methods for Isolation, Phenotypic Characterization and Maintenance of Yeasts. In: Kurtzman CP, Fell J, Boekhout T, editors. Yeasts, a Taxon study. 5th ed. Amsterdam: Elsevier; 2011. p. 87–110.View ArticleGoogle Scholar
- Shannon CE. A Mathematical theory of communication. Bell Syst Tech J. 1948;27:379–423.View ArticleGoogle Scholar
- Sahli HF, Conner JK. Characterizing ecological generalization in plant-pollination systems. Oecologia. 2006;148:365–72.PubMedView ArticleGoogle Scholar
- Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O'Hara RB, et al. vegan: Community Ecology Package. R package version 2.0-10. http://CRAN.R-project.org/package=vegan
- Hothorn T, Hornik K, Zeileis A. Unbiased recursive partitioning: a conditional inference framework. J Comput Graph Stat. 2006;15:651–74.View ArticleGoogle Scholar
- Kembel SW, Cowan PD, Helmus MR, Cornwell WK, Morlon H, Ackerly DD, et al. Picante: R tools for integrating phylogenies and ecology. Bioinformatics. 2010;26:1463–4.PubMedView ArticleGoogle Scholar
- Laliberté E, Legendre P. A distance-based framework for measuring functional diversity from multiple traits. Ecology. 2010;91:299–305.PubMedView ArticleGoogle Scholar
- Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;6:65–70.Google Scholar
- De Cáceres M, Legendre P, Moretti M. Improving indicator species analysis by combining groups of sites. Oikos. 2010;119:1674–84.View ArticleGoogle Scholar
- De Cáceres M, Legendre P. Associations between species and groups of sites: indices and statistical inference. Ecology. 2009;90:3566–74.PubMedView ArticleGoogle Scholar
- R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. 2014. [http://www.R-project.org/]
- Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D. Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. Gigascience 2014. http://datadryad.org/review?doi=doi:10.5061/dryad.0qp3q
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.PubMed CentralPubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.