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Nectar inhabiting yeasts in Virginian populations of Silene latifolia (Caryophyllaceae) and coflowering species.

INTRODUCTION

Nectar is one of the most common floral rewards a plant produces to attract pollinators (Simpson and Neff, 1983) and is used by pollinators as an energy source (Carpenter, 1983; Heinrich, 1983). It is high in sugars, amino acids, proteins, lipids, essential oils, polysaccharides, antioxidants, alkaloids, and vitamins (Baker and Baker, 1983; Dafni, 1992). Nectar is not only an attractant and food resource for pollinators, it is also a potential habitat for microorganisms (Gruess, 1917; Hautmann, 1924; Nadson and Krassilnikov, 1927; Lund, 1954; Phaff, 1978; Eisikowitch et al, 1990; Brysch-Herzberg, 2004 and references within; Herrera et al., 2008, 2009; de Vega et al., 2009; Belisle et al., 2011). Although these microorganisms have been found in nectar by microbial ecologists and taxonomists, only recently have pollination biologists and ecologists included these microorganisms in their study on plants and pollinators (Kevan et al., 1988, 1989; Eisikowitch et al., 1989, 1990; Brysch-Herzberg, 2004; Herrera et al., 2008, 2009, 2010; de Vega et al., 2009; Pozo et al, 2011, 2012).

Communities of nectar inhabiting microorganisms (NIMs) include bacteria (Alverez-Perez et al., 2012; Fridman et al., 2012), filamentous fungi (including molds), and yeasts (Gruess, 1917; Hautmann, 1924; Nadson and Krassilnikov, 1927; Lund, 1954; Eisikowitch et al., 1989; Kurtzman and Fell, 1998; LaChance et al., 2001c; Brysch-Herzberg, 2004; Belisle et al., 2011; Pozo et al., 2011, 2012). The focus of this study is the on yeasts which may be referred to as NIMs in the remainder of this study. Interest in NIMs began in the 1880s and early 1900s with descriptive studies that described the presence or absence of yeast in nectar and changes in the abundance of yeast throughout the year (Boutroux, 1884; Gruess, 1917; Hautmann, 1924; Nadson and Krassilnikov, 1927). These early studies demonstrated that yeast cell counts per flower may be relatively high, on the order of 106 cells per flower. More recent studies confirm the high prevalence of yeast in floral nectar (Brysch-Herzberg, 2004; de Vega et al., 2009; Herrera et al., 2008, 2009; Pozo et al., 2011).

Nectar inhabiting microorganisms have been reported in several different plant families, including the milkweed Asclepias (Kevan et al., 1989), mint Teucrium (Phaff et al., 1978), vetch Vicia and clover Trifolium (Lund, 1954), as well as several other plant families (Rosa et al., 1995; Brysch-Herzberg, 2004 and references within; Herrera et al., 2008, 2009; de Vega et al., 2009; Belisle et al., 2011; Pozo et al., 2011, 2012). NIMs have also been found on or in pollinators (Gilliam et al., 1977; Sandhu and Waraich, 1985; LaChance et al., 1990, 1998b, 2011a, 2001b, 2001c, 2003; Brysch-Herzberg, 2004; Pozo et al., 2012), in the nests of insects (Rosa et al., 2003; Pimentel et al., 2005; Herrera et al., 2010), and even on the eggs (Gibson and Hunter, 2005). Nectar inhabiting yeasts have been isolated mostly from hermaphroditic plant species (Gruess, 1917; Hautmann, 1924; Nadson and Krassilnikov, 1927; Lund, 1954; Phaff et al., 1978; LaChance et al., 1990, 1998a, b, 1999, 2003; Hong et al., 2003; Brysch-Herzberg, 2004; Herrera et al., 2008, 2009; Pozo et al., 2011), and a few ecological studies have been performed on hermaphroditic plants as well (Starmer et al., 1980; Eisikowitch et al., 1989; Kevan et al., 1989; Brysch-Herzberg, 2004; Herrera et al., 2008, 2010; de Vega et al., 2009). However, no study has separately examined nectar inhabiting microbial communities of male and female flowers in dioecious plants.

Sexual dimorphism is a common feature of dioecious plants, including sex based traits (Lloyd and Webb, 1977; Bell, 1985; Delph and Meagher, 1995; Delph et al., 1996; Geber et al., 1999) and differences in nectar (Kay et al., 1984; Jolls et al, 1994; Delph et al., 1996; Shykoff, 1997; Geber et al., 1999; Witt et al., 1999). Silene latifolia (white campion) is dioecious, with separate male and female individuals. It is insect pollinated and the flowers open at approximately 20:00 h and remain open until 12:00 h the next day, depending on humidity and temperature, so both nocturnal and diurnal pollinators have the potential to affect the fitness of S. latifolia and to disperse microorganisms. Nocturnal floral visitors include many moth species (sphingid, noctuid, and geometrid moths), while diurnal pollinators include honeybees, bumblebees, butterflies, and some fly species (Altizer et al., 1998; Golonka, pers. obs.). Male flowers of S. latifolia have a shorter life span (1-2 d) than female flowers (4-5 d) (Kaltz and Shykoff, 2001); therefore, yeast species may have longer to grow in female flowers than in male flowers. Male and female plants also produce flowers with different qualities and quantities of nectar. Male and female S. latifolia nectars are hexose dominated with little sucrose present (Witt et al., 1999). The sugar concentration of nectar extracted from bagged male and female flowers ranges from 20% to 54% and 10% to 33%, respectively (w:w; Shykoff and Bucheli, 1995; Shykoff 1997; Witt et al., 1999; Golonka, unpub, data). The nectar of this plant contains volatile compounds contributing to flower scent (Jurgens et al., 2002). Although male flowers produce more sugar per [micro]L of nectar, male flowers produce less nectar than female flowers (Shykoff and Bucheli, 1995; Shykoff, 1997; Witt et al., 1999; Golonka, unpub, data), and male flowers are visited more often than female flowers (Shykoff and Bucheli, 1995).

Given the different structures and floral longevities of male and female flowers, these differences could have a significant influence on the floral biology of the NIMs, which may in turn influence male and female flowers differently. Different yeast species may do better in the higher sugar environment of male flowers whereas other yeast species may do better in the lower sugar environment of female flowers (Shykoff, 1997; Witt et al., 1999). This study examines whether there is sexual dimorphism in the microbial communities of Silene latifolia floral nectar. In addition, this study examines the microbial flora in nectar of coflowering plant species, and as this plant species is known to be infected by the anther smut Microbotryum violaceum, this study also examines the effects of this disease on yeast community composition and if M. violaceum is able to establish populations of its haploid yeastlike sporidial stage in the nectar of healthy plants.

METHODS

THE PLANT SPECIES

Silene latifolia Poiret [= Silene alba (Miller) Krause] is a dioecious, perennial plant found in disturbed areas along roadsides and fields. It was introduced from Europe to the U.S. and Canada early in the 19th century and has since become common in the central and southern Appalachians as well as other parts of the eastern U.S. (McNeill, 1977).

POPULATIONS

Eight populations of Silene latifolia near Mountain Lake Biological Station, Giles County, VA were sampled for NIMs during the months of Jun., Jul., and Aug., the main flowering period for this plant across all sampling years of 1999, 2000, and 2001. Three of these populations contained plants diseased with the anther smut, Microbotryum violaceum. Populations were chosen on the basis of access to plants, health status (healthy populations had been healthy for at least two summer seasons, i.e., no anther smut), and location (near or far from other populations). Diseased sites were also chosen for the presence of healthy plants among diseased plants (within 2 m, and approximately 10 m away) to examine effects of disease on yeast community composition and to determine if M. violaceum is able to establish populations of its haploid yeastlike sporidial stage in the nectar of healthy plants. The months were chosen based on the flowering time of S. latifolia and sampling methodology in 1997 and 1998. Based on this previous research, the sampling scheme and dilution procedures used in 1999-2001 were standardized with minor changes (see below). 1999 and 2000 were drought years as indicated by lower volumes of nectar in male and female flowers than in previous years sampled or in 2001. Because of this drought, sample number and nectar volume were lower in 1999 and 2000 (.see Table 4), as indicated by average standing crop nectar volumes which were 1.11 [micro]L in 1998, 0.54 in 1999, 0.85 in 2000, and 1.16 in 2001 for male flowers and 2.23 [micro]L in 1998, 1.59 in 1999, 1.35 in 2000, and 1.70 in 2001 for female flowers.

NECTAR COLLECTION

Nectar collection was carried out for one week periods each month. When possible, a male nectar sample consisted of up to three flowers from the same male plant and a separate female nectar sample consisted of up to three flowers from the same female plant. A total of four male nectar samples and four female nectar samples were collected at each site. A total of 247 female samples (611 flowers total, 2.5 [+ or -] 0.8 flowers/sample) and 222 male samples (615 flowers total, 2.8 [+ or -] 0.5 flowers/sample) were collected during the months of Jun.-Aug. of 1999-2001. Nectar from coflowering plant species was collected in the same way; however, sometimes multiple plants were not available. A total of 120 coflowering plant samples were collected in the months of Jun.-Aug. of 1999-2001. The total number of yeast samples from coflowering plants (Tables 2 and 3) reflects the relative abundance of these plant species in populations of Silene latifolia. For S. latifolia, flowers were removed from plants and sepals were peeled away from the rest of the flower to better see and collect nectar at the base of the petals; this also prevented contamination of nectar sample. All S. latifolia flowers collected were 24-48 h old; with male flowers collected with stamens dehisced and fully reflexed and female flowers collected with fully elongated stigmas which occurs approximately 24-48 h after the flower initially opens (Golonka, unpub, data). For coflowering species, flowers were removed from plants and petals or sepals were removed from the rest of the flower, if needed, then nectar was collected. Sterile 1.0 or 5.0 [micro]L microcapillary tubes were used to collect nectar from flowers. After nectar was collected, the length of the nectar filled microcapillary, robe was measured and recorded, then the contents of the microcapillary robes were blown into sterile Eppendorf tubes using a bulb.

NECTAR PROCESSING

Due to possible changes in or breakdown of nectar sugars or other constituents, samples were plated out within 8-14 h of collection and kept at ambient temperature before plating. Nectar samples less than or equal to 3 [micro]L were diluted with 250 [micro]L of sterile water whereas nectar samples greater than 3 [micro]L were diluted with 500 [micro]L of sterile water in 1999 and 2000. In 2001, nectar samples less than or equal to 1.0 [micro]L were diluted by 150 [micro]L of sterile water; the rest of the nectar samples were processed the same as in 1999 and 2000. Post hoc t tests indicated this modification of the sampling procedure did not produce significant differences in the number of colony forming units on media for samples less than or equal to 1.0 [micro]L across the years.

After dilution, the nectar-water samples were vortexed for 1 min, serially diluted (1, [10.sup.-1], [10.sup.-2], and [10.sup.-3]), then plated onto plates of potato dextrose agar with 0.1% yeast extract (w:v; PDAY) and 0.5% (w:v) each penicillin and streptomycin. After diluting, for each dilution, two aliquots of 50 [micro]L was spread onto two plates, and plates were incubated at ambient temperature (15-24 [degrees] C) for up to 2 wk or until individual colonies were visible. The number of colony forming units (CFUs) was determined for each plate and an average CFU per sample was calculated based on all dilution plates. This method is commonly used in NIM studies and although there is some indication this methodology underestimates cell density, this method does accurately reflect species composition and relative cell densities (Brysch-Herzberg, 2004; Belisle et al., 2011; Peay et al., 2011; Pozo el al., 2011, 2012).

MORPHOSPECIES AND MOLECULAR IDENTIFICATION

Once yeast colonies were visible on serial dilution plates, yeast colonies were separated into morphospecies by the following characteristics: colony color (e.g., pigmentation, lack of pigmentation), colony shape (e.g., amorphous, circular), colony margin (e.g., entire, undulating, lobed, filamentous), colony surface (e.g., shiny, dull, wrinkled, smooth), colony texture (e.g., mucoid, viscous), colony elevation (e.g., flat, convex, raised), cell shape [e.g., ovoidal (oval), ellipsoidal, cylindrical (rod), elongate (long and narrow), triangular, globose (spherical)], cell size (tiny cells <1.0 lain, small cells 1.0-2.0 [micro]m, medium cells 2.0-3.5 [micro]m, and large cells >3.5 [micro]m), filamentation (e.g., pseudohyphae or hyphae), and vegetative reproduction method (budding, fission, conidia formation). This terminology and categorization was taken from Kurtzman and Fell (1998). Because chromosomal DNA was extracted from the majority of isolates, cultures were not maintained.

Molecular systematic techniques were used to identify and verify the morphospecies. This method is used extensively in identification of both culturable and unculturable strains of fungi (Head et al., 1998; Kurtzman and Blanz, 1998; Kurtzman and Fell, 1998; Sugita et al., 1999; Arnold et al., 2000; Herzberg et al., 2002; LaChance et al., 2003; Hong et al., 2003; Brysch-Herzberg, 2004; Pozo et al., 2011). Several representative isolates were streaked on PDAY multiple times to ensure a pure culture before nuclear DNA was extracted using the method of Xu et al. (2000). Yeast species were identified by PCR amplification and sequencing approximately a 1.2 kb section of the internal transcribed spacers (ITS1 and ITS2) and the D1/D2 region of the large subunit nrDNA following the methods of Kurtzman and Robnett (1997) and Fell et al. (2000). The primers used were ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and NL4 (5'-GGT CCG TGT TTC AAG ACG G-3'). PCR was performed for 35 cycles with denaturation at 94 C for 1 min, annealing at 52 C for 30 sec, and extension at 72 C for 1 min. Cycle sequencing was performed with the forward primers ITS1 and 5.8SR (5'-TCG ATG AAG AAC GCA. GCG-3') and the reverse primer NL4.

The consensus sequences for D1/D2 and ITS1/5.8S/ITS2 were initially aligned in BioEdit (Hall, 1997-2011) and grouped into operational taxonomic units (OTUs). OTUs were defined as groups of sequences sharing at least 98.5% pairwise similarity (modified from Kurtzman and Robnett, 1998). A representative sequence of each OTU was used to perform GenBank BLAST searches for the D1/D2 region of the large subunit and the ITS1/ 5.8S/ITS2 region. The most probable taxonomic match for each sequence was recorded (Table 1; BLAST searches last performed in Jan. 2011; following Kurtzman and Robnett, 1997, 1998; Fell et al., 2000; Scorzetti et al., 2002). If nucleotide substitutions occurred in less than 1.5% of the D1/D2 region when compared to BLAST search results, isolates were identified to the closest species (modified from Peterson and Kurtzman, 1991; Kurtzman and Robnett, 1997, 1998). When base substitutions occurred in more than 1.5% but less than 5% of the D1/D2 nrDNA region, an isolate was identified to the species of closest affinity, indicated by "aff" in the species name. When base substitutions occurred in more than 5% of the D1/D2 nrDNA region, isolates were identified to the closest genus only. Because there are so few ITS sequences in the database at present, results for the ITS BLAST searches generally only supported the genus level identification or species level identification if the organism is well studied (e.g., Cryptococcus spp.).

DATA ANALYSIS

Species accumulation curves were used to determine if there were differences in the numbers of species present in male and female samples across the years in Jun.-Aug. only. Chi-square tests of independence or Fisher exact chi-square tests of independence (N < 5; Stokes et al., 2000) were used to determine if there were differences among the sexes, within year, across the season (Jun.-Aug. only), across years, and across sites for presence (of any yeast species) and absence of NIMs. Chi-square tests of independence were also used to determine whether there were differences among the sexes, across sites, within season, and between years for the presence and absence of the anther smut, Microbotryum violaceum. Sites with this disease were analyzed for differences in the presence and absence of other NIMs among the sexes and the diseased sites.

Due to the inability to normalize counts of species, non-parametric Wilcoxon and Kruskal-Wallis tests (NPARIWAY, SAS Institute Inc., 2001) were used to compare species richness within and across years, among sexes, across sites, and across dates.

Due to the large number of samples with no yeast species present (44% of Silene latifolia samples; see Table 4), only samples with yeast species present (indicating pollinator visitation) were used in the following analyses. Within each year, a three way analysis of variance was used to determine if there were differences in the number of CFUs per [micro]L of nectar and per flower between the sexes, among sampling dates, and among the sites. Data were log-transformed before analysis, [log.sub.10] (number of CFUs). In this model, the main effects were sex, site, date, and all possible interaction terms. Among years, a four way analysis of variance was used to determine if there were differences in the number of CFUs per [micro]L of nectar and per flower among the sexes, between sampling dates, and among the sites. The main effects were sex, site, date, year, and all possible interactions except sex*site*date*year.

RESULTS

SPECIES ISOLATED

Thirty-seven morphospecies were isolated from populations of Silene latifolia (Table 1). Based on nrDNA sequences, the 37 morphospecies belong to 26 ascomycete and basidiomycete species (Table 1). Isolates were identified to the closest species when possible; however, in a few cases, base substitutions occurred in more than 5% of the D1/D2 nrDNA region and these isolates were identified to closest genus (see Table 1). There was no overlap among the genetic identifications, except for Cryptococcus flavescens, strains AA and J, which original genetic data indicated were separate species (Table 1). Several morphospecies showed distinct polymorphisms which may represent unique ecotypes (Golonka, 2002). Nearly all the yeast species isolated were present in S. latifolia nectar (Tables 2 and 3). Of the yeast samples extracted from Silene latifolia plants, 17% were classified as Metschnikowia species (112 out of 666 isolates; Tables 2 and 3).

SEXUAL DIMORPHISM

Species numbers.--An examination of the relationship between number of samples and cumulative number of yeast species found indicated that the numbers of species were beginning to plateau and that the sampling was relatively complete (Fig. 1). In 1999, the asymptote for males was less than for females (Fig. 1A). In 2000, both sexes had similar cumulative species curves (Fig. 1B); however, in 2001, male samples had a higher asymptote than female samples (Fig. 1C).

Presence and absence of yeast.--On average yeasts were present in 56% of all Silene latifolia samples across all three years. The presence and absence of yeasts was not dependent on the sex of the sample but was dependent on the year, with 1999 having yeasts present in fewer samples than 2000 and 2001 ([chi square] = 47.3, df = 2, P < 0.0001; Fig. 2, Table 4). The differences in presence and absence of yeast across years may be due to differences in rainfall (1999: 197 ram; 2000:372 mm; 2001:166 mm; Nagy, 2010). Presence and absence of yeasts was dependent on date only in 1999 with Aug. and Jul. having a similar and higher frequency of yeasts than Jun. (Table 4), and again this difference may be due to differences in rainfall (for 1999, Jun.: 11 mm, Jul. 117 mm, Aug.: 69 ram). Analysis also indicated that the occurrence of yeasts in nectar was dependent on site within year with all sites having a significant or marginal P value greater than 0.06, with the exception of site H5 (P = 0.13; overall: [chi square] = 66.0, df = 2, P < 0.0001).

Presence and absence of yeasts were not dependent on the sex (male or female) of the nectar (Fig. 2; 1999: [chi square] = 0.80, df = 1; 2000: [chi square] = 0.73, df = 1; 2001: [chi square] = 1.21, df = 1; P values in Fig. 2; Table 4 contains data). Presence and absence of yeasts were also not dependent on site within each sex; however, there was some indication that presence and absence of yeasts were dependent on date within females for all three years (1999:[chi square] = 8.79, df = 2, P = 0.012; 2000: [chi square] = 6.13, df = 2, P = 0.047; 2001: [chi square] = 10.8, df = 2, P = 0.004; Table 4), and only marginally significant within males for 1999 [chi square] = 5.36, df = 2, P = 0.069; Table 4). However, the peak month for yeast abundance was different in every year (1999: Aug.; 2000: Jun.; 2001: Jul.; Table 4). For 2000 and 2001, the peak yeast abundance month also had the highest amount of rainfall (2000, Jun.: 127 mm compared to Jul.: 120 mm and Aug.: 124 mm; 2001, Jul.: 169 mm, Jun.: 56 mm, Aug.: 40 mm).

Species richness.--There was a significant difference between the sexes for species richness within 2000, with males having a higher species richness than females (males: 1.48 [+ or -] 0.15; females: 1.01 [+ or -] 0.11; Fig. 3), but not within 1999 or 2001 (Fig. 3). Species richness differed significantly across the months for 1999 ([chi square] = 21.3, df = 2, P < 0.0001) and 2000 ([chi square] = 11.85, df = 2, P = 0.003) and approached significance for 2001 ([chi square] = 4.93, df = 2, P = 0.085). Species richness was significantly different across sites for 2001 ([chi square] = 19.6, df = 7, P = 0.0065). There was also some indication that species richness differed across sites for 2000 ([chi square] = 13.4, df = 7, P = 0.065) but not for 1999 ([chi square] = 6.15, df = 7, P = 0.52). Species richness differed across years ([chi square] = 68.5, df = 2, P < 0.0001), with 1999 having the lowest species richness (0.40 [+ or -] 0.07 species) compared to 2000 (1.23 [+ or -] 0.09 species) and 2001 (1.21 [+ or -] 0.10 species).

Cell density.--When yeasts were present in samples, the number of CFUs (summed over all yeast species) per [micro]L and per flower varied significantly among sites and across years (Table 5), with 2000 having the highest number of CFUs per [micro]L followed by 2001 and 1999 (Fig. 4). This difference again may be due to rainfall differences across the years (Fig. 4). There was also a significant interaction of date and year (Table 5). For brevity, Table 5 only contains the statistical results for the number of CFUs per [micro]L as similar results were obtained for the number of cells per flower. The effect of sex was not significant, although the interaction of sex*site (Table 5), ill terms of number of CFUs per [micro]L, approached significance. Results for date and sex*site*year also approached significance levels (Table 5). Overall, there is no clear evidence that the overall abundance of NIMs was substantially different between males and females (Table 5).

ANTHER SMUT: EFFECT OF AND DISPERSAL

Effect of disease on yeast communities.--Diseased and healthy sites were equally likely to have NIMs present in the nectar of Silene latifolia flowers in 1999, 2000, and 2001 (P > 0.20 for all years; Fig. 5). An analysis of diseased sites only for comparison of all yeast species present in these sites indicated that the presence of Microbotryum violaceum in these populations did not affect whether NIMs were present or absent in male or female nectar, across sites. However, in 1999, the presence and absence of NIMs were dependent on date within these three diseased sites ([chi square] = 9.27, df = 2, P = 0.0097). This dependence held for all sites, so this may not be a factor of whether M. violaceum was present in nectar at these sites but an effect of sampling year (see above data regarding rainfall in the various sampling months).

Spatial and temporal differences.--The yeast or sporidial stage of this pathogen itself was found in all of the diseased sites, and in four of the five healthy sites with consistent presence in H2, a healthy population near to a diseased population, D2 (Fig. 5). Chi square tests of independence across all sites and within each year indicated that the presence and absence of Microbotryum violaceum was not dependent on the sex of the nectar sample (P > 0.20 for all years) but was dependent on sampling date for 2000 ([chi square] = 11.4, df = 2, P = 0.003) with Jun. having a higher presence of M. violaceum in nectar than either Jul. or Aug. There was also a significant effect of site within 2001 (Fisher's exact test: P = 0.0003) but not within 1999 or 2000. When all years were analyzed together, presence and absence of M. violaceum was significantly dependent on site ([chi square] = 26.5, df = 7, P = 0.0004) and year ([chi square] = 13.1, df = 2, P = 0.0015) with 2000 having a higher presence of M. violaceum than either 1999 or 2001 (see Fig. 5). Presence and absence of M. violaceum, across all years, approached significance for date ([chi square] = 5.35, df = 2, P = 0.068) with Jun. having a higher presence of M. violaceumin nectar than either Jul. or Aug. but was not significantly dependent on the sex of the nectar sample.

Analyses within the three diseased sites and within each year indicated that the presence and absence of Microbotryum violaceum was not dependent on site or sampling date (P > 0.28 for all years and both variables). However, the presence of M. violaceum in nectar was +dependent on the sex of the nectar sample in 2000 ([chi square] = 3.85, df = 1, P = 0.049) with M. violaceum present more often in male nectar than female nectar. When all years were analyzed together, presence and absence was significantly dependent on sex of the nectar sample ([chi square] = 4.04, df = 2, P = 0.044), with M. violaceum present more often in male nectar than female nectar. The presence of this basidiomycete in nectar samples was also significantly dependent on year ([chi square] = 9.6, df = 2, P = 0.008), with 2000 and 2001 having higher and similar frequencies of this species compared to 1999 (Fig. 5). There was no significant effect of site or date on presence and absence of this species within diseased sites.

YEAST COMMUNITY ASSOCIATED WITH COFLOWERING PLANTS

Presence and absence of yeast.--Most of the 27 plant species sampled (59%) were capable of hosting both ascomycete and basidiomycete yeast species, with more plant species hosting ascomycetes (74%) than basidiomycetes (59%; Tables 2 and 3). In a few plant species (26%; Tables 2 and 3), no yeasts were present in nectar, but in all of these cases only one or two samples had been obtained.

Yeast distribution and specialization.--To assess if there was a difference in the distribution of yeast species among the plant species, an analysis was carried out on a subset of the data, excluding all plants or yeast species with fewer than 10 records. The eight plant species used in this analysis were Coronilla varia, Impatiens capensis, Lathyrus latifolius, Leonurus cardiaca, Lonicera japonica, Nepeta cataria, Saponaria officinalis, and Trifolium pratense, and the six fungi used were Aureobasidium pullulans, Metschnikowia aff. reukaufii, Metschnikowia koreensis, Metschnikowia aff. vanudenii, Cryptococcus flavescens, and Microbotryum violaceum. The results showed a significant overall yeast*plant species heterogeneity ([chi square] = 75.6, df = 35 P < 0.0001). Tests of heterogeneity between Silene latifolia and each of the other plant species indicated that yeast distribution was significantly different in two out of eight cases (Leonurus: Fisher's Exact Test, P = 0.016; Nepeta: Fisher's Exact Test, P < 0.0001) and approached significance in two other cases (Lonicera: Fisher's Exact Test, P = 0.058; Saponaria: Fisher's Exact Test, P = 0.075). There is therefore evidence of a degree of specialization in yeast community for the six fungal species examined.

Cell densities.--Table 6 shows the percentages of and abundance range and mean for each yeast species across all plant samples. The most frequently found yeast species was Aureobasidium pullulans which also had a maximum abundance of 33,000 CFUs per [micro]L of nectar. The most abundant floral associated microorganism was Metschnikowia aff. reukaufii with a maximum number of 128,000 CFUs per [micro]L of nectar.

Anther smut in coflowering plant species.--Microbotryum violaceum was present in 37% of the 27 plant species sampled (Table 3), including plants that were not present in diseased sites and plants that do not become infected by this plant pathogen. The number of M. violaceum CFUs per [micro]L of nectar ranged from 1.0 to 10,700 CFUs (mean: 419 CFUs/[micro]L, Table 6). Statistical analysis of the reduced table (see previous paragraphs) showed no significant heterogeneity in the distribution of M. violaceum. This lack of heterogeneity indicated that hosts other than Silene latifolia have equal frequencies of this pathogen.

Other interesting yeast species.--Although the number of samples from Asclepias syriaca was small, it is interesting that the yeast species included Metschnikowia aff. reukaufii, a yeast that was found previously to inhibit pollen germination of this plant (Eisikowitch et al., 1990).

DISCUSSION

SPECIES ISOLATED

Using traditional morphological criteria, morphotyping was found to discriminate well among species, as indicated by genetic (DNA) evidence. In several cases, morphotyping revealed more distinct species than was indicated by analysis of DNA. The use of morphological traits to separate isolates into morphospecies is a common technique and relatively accurate (Lund, 1954; Phaff et al., 1978; Oliver and Beattie, 1996; Arnold et al., 2000; Brysch-Herzberg, 2004). Prior studies utilizing morphotyping have also found a hyperdiversity of morphotypes present in samples (Arnold et al., 2000). The differences in morphology were consistent across strains and probably reflect true genetic variation. Further evidence comes from a nectar growth experiment (Golonka, 2002) where strains of Metschnikowia (strains M and N1; Metschnikowia koreensis) varied in their ability to grow in nectar. Nevertheless, it was not possible from these data to determine whether strains M and N1 were distinct molecular species or morphologically and ecologically variable strains of the same genetic species. The use of ITS and 26S nrDNA sequences are generally not considered sensitive enough to detect morphologically variable strains of the same genetic species (Fell et al., 2000; Scorzetti et al., 2002).

IS THERE SEXUAL DIMORPHISM IN YEAST COMMUNITIES IN S. LATIFOLIA?

There were no consistent year to year and site to site differences between male and female flowers of Silene latifolia in their yeast communities or in their abundance as estimated from colony number. A difference in presence and absence was seen in 1999 within the sexes for date, and a difference in species richness occurred among the sexes in 2000. In 1999, males had fewer species than females; but in 2000 males had higher species richness, and in 2001 males contained more species per sample than females, based on species accumulation curves. It therefore appears that the trend is for there to be more NIMs in male flowers, based on the 2000 and 2001 data; however, under severe conditions, such as the drought conditions of 1999, female flowers may have more NIMs present than male flowers (perhaps due to an offer of more nectar to pollinators than male flowers).

There is some indication that male flowers are visited more often than female flowers (Shykoff and Bucheli, 1995). With this increase in pollinator visitation to male flowers, more NIMs may be deposited within the nectar of male flowers than female flowers. Nectar was collected early in the morning, after both nocturnal and diurnal visitors, so more species may be present in male flowers due to a higher visitation of male flowers by pollinators. Male flowers are only open 1-2 nights (Kaltz and Shykoff, 2001; Golonka, unpub, data), so all male flowers had been open approximately the same time when nectar was collected. However, female flowers are open around 4-8 nights, producing nectar approximately 24-48 h after opening (Kaltz and Shykott, 2001; Golonka, unpub, data), and it was difficult to determine the age of a flower when nectar was collected, so flowers could have been open 2-8 nights. If a female flower was collected late in its life, then the microorganisms isolated from that flower may reflect yeast species truly capable of growing in nectar or may reflect a priority effect as seen in Peay et al. (2011), whereas the yeast isolated from male flowers may reflect yeast species deposited in nectar but not necessarily true nectar inhabiting species. This would also mean that male flowers would have higher species richness than female flowers. In a study by Golonka (2002), some of the isolated yeast species found in Silene latifolia nectar are capable of growing in nectar as was also demonstrated in similar species by Pozo et al. (2011, 2012) and Peay et al. (2011). However, further research needs to be done to determine if and how well other floral yeast species grow in S. latifolia nectar.

KNOWN NECTAR INHABITING YEASTS

The results of this study are consistent with previous studies in Germany (BryschHerzberg, 2004), South Africa (de Vega et al., 2009), and Spain (Herrera et al., 2009; Pozo et al, 2011) where species diversity and richness are low but cell density of yeasts is often relatively high in nectar. At least one species isolated, Microbotryum violaceum, and several species of the genus Metschnikowia (Gruess, 1917; Hautmann, 1924; Nadson and Krassilnikov, 1927; Lund, 1954; Phaff, 1978; Eisikowitch et al, 1990; Brysch-Herzberg, 2004 and references within; Herrera et al., 2010; Pozo et al, 2011), are well known as nectar inhabiting organisms, although the precise role of M. violaceum sporidia found in nectar is unclear in terms of its life cycle (Hood and Antonovics, 2000). In the present study, it was not possible to distinguish M. violaceum teliospore stages after germination on media from yeast stages actually growing in nectar. But, regardless, the results confirm that the spores of M. violaceum are widely dispersed, well away from diseased plants and populations as indicated by the prevalence of this microbe in coflowering plant species and in healthy populations distant from known diseased populations. Nectar of other plant species may well be a suitable medium for growth of sporidia and, therefore, act as a source of inoculum for healthy Silene latifolia. Other than this study, the role of sporidia in nonhost plants has never been investigated. The wide dispersal of spores or sporidia as indicated in the present study, is also evidence for widespread dispersal of NIMs by pollinators.

Compared to the Germany (Brysch-Herzberg, 2004) and Spain (Herrera et al., 2010; Pozo et al., 2011) studies, Metschnikowia species were not as prevalent in Virginia, USA, with Metschnikowia aff. reukaufii comprising only 10% of all isolates from all plant species compared to 47% in Germany and 65% in Spain. However, the goal of this study was not to survey all plant species in Giles County, Virginia for the presence of yeasts, so the plant samples are slightly skewed towards Silene latifolia. Metschnikowia aff. reukaufii is present in only 8% of all isolates extracted from S. latifolia nectar and 16% of all isolates from all other plant species sampled (see Tables 2 and 3). All three Metschnikowia species isolated in Virginia account for 21% of all isolates among all plants sampled, but only 17% of all S. latifolia samples and account for a higher percentage, 34%, of all other plant samples. The two mentioned Metschnikowia species in the Germany and Spain study, Met. reukaufii and Met. gruessii, comprise 64% of samples in Germany and 88% of samples in Spain. At the current time it is not known why there is such a difference in the percentage of samples containing this particular genus of yeast in Europe versus southeastern U.S. Future studies in southeastern U.S. should sample a wider range of plants and have a higher sampling of these other plant species.

OTHER YEAST SPECIES

Within Silene latifolia the most common yeast species was Aureobasidium pullulans, comprising 31% of yeast isolates, but only 22% of yeasts from other plant species, a lower percentage compared to Metschnikowia species (34%) in plants other than S. latifolia. Aureobasidium pullulans has been shown to be found in several other habitats in plants and in the environment (Lund, 1954; Phaff, 1978; Kurtzman and Fell, 1998; Andrews et al., 2002) and may be prevalent in nectar samples due to having alternative habitats available in southeastern Virginia, and may even be a transient species in nectar. Cryptococcus species were also more prevalent than Metschnikowia species, being present in 23% of isolates from all plant species, 24% of S. latifolia yeasts, and 18% of isolates from other plant species. This genus is more prevalent in S. latifolia nectar but is comparable in other plant species to isolates from Germany (18%; Brysch-Herzberg, 2004), but is much more prevalent in nectar from other plant species in Virginia than in isolates from Spain (2%; Herrera et al., 2010; Pozo et al., 2011). However, again, due to the focus of this study, the sampling was skewed towards S. latifolia plants and more sampling would be needed to verify the prevalence of this yeast in other plant species in Virginia.

ASCOMYCETES VS. BASIDIOMYCETES

The majority of isolates in this study were ascomycetous yeasts (510, Table 2) compared to basidiomycetous yeasts (389, Table 3). This is comparable to other studies (Pozo et al., 2011) that suggest that ascomycetous species are more prevalent because the nectar habitat favors microorganisms capable of fermenting sugars and being tolerant of an osmotically difficult habitat, the "nectar-filtering" idea proposed by Herrera et al. (2010). Studies by Herrera et al. (2010) and Pozo et al. (2011, 2012) indicate yeasts that are osmotolerant are normal inhabitants of nectar. Although we did not examine the osmotolerance of the yeast species isolated in this study, several of the ascomycete yeasts isolated are known to be osmotolerant (see Pozo et al., 2011). Research by Golonka (2002) supports the idea that the ascomycete species are more tolerant of the high sugar concentration in nectar than the basidiomycetes isolated from Silene latifolia. Additional work on common yeast species isolated from S. latifolia needs to be done to help corroborate the work started by Herrera et al. (2011), Peay et al. (2011), and Pozo et al. (2011, 2012) on osmotolerance of NIMs.

Even though more ascomycete isolates were found, the floral yeasts include representative species in all the major groups of fungi (Tables 2 and 3). Several yeastlike species (Table 1) were found in the nectar of this plant whereas only six species with a true yeast stage were found (23% Saccharomycotina, Table 1). Several of these species produced pseudomycelium or mycelium during growth, even a few of the true yeast species (Golonka, unpub, data; Kurtzman and Fell, 1998). This study shows not only the diversity of nectar inhabiting microorganisms but also confirms the commonality and abundance of yeasts in nectar, as prior studies have shown (Lund, 1954; Eisikowitch, et al., 1990; LaChance, et al., 2001a, b, c; Brysch-Herzberg, 2004; Pozo et al., 2011).

CONCLUDING THOUGHTS

The results indicate that microbial communities may be spatially and temporally variable in the nectar of Silene latifolia, as indicated by differences across the years and sites in species richness and cell densities. An examination of rainfall conditions across the three years sampled in this particular study suggests that rainfall may impact the presence and quantity of these microorganisms (see Fig. 4); however, it is unclear as to how drought conditions impact the microorganisms. The impact does not appear to be associated only with changes in nectar volume as 2001 had more standing crop nectar volume in both males and females than in 2000, but 2000 had the highest cell densities (see Results and Fig. 4). Perhaps the differences across years may be associated more with the number of flowers a plant is able to produce and keep open during a resource limited period as well as the quality of nectar, all factors that were not measured in this particular study. A recent study on a hummingbird pollinated plant indicated that a host plant's location and flower density may be associated with spatial distribution of yeast species (Belisle et al., 2011). It is obvious from these results and recent studies that more research needs to be conducted on how these microorganisms are affected by a number of different parameters, including flower density, rainfall, humidity, spatial distance from other plants, and several other factors.

A number of yeast species were associated with flower nectar and some occurred in relatively high frequencies and abundances (see Table 6). Some of these NIMs, such as Metschnikowia spp., are often only associated with flowers and insects (LaChance et al, 1990, 1998b, 2001b, 2003; Kurtzman and Fell, 1998). These frequently occurring yeast species appear to form a complex yeast community and may have an important role in floral biology. The roles of these microorganisms in floral nectar have been shown to be important (e.g., the yeast Metschnikowia reukaufii having a negative effect on the germination of pollen in the plant Asclepias syriaca; Eisikowitch et al., 1989; Kevan et al., 1989), and it is likely nectar inhabiting yeasts play important roles in other plant species. Several recent studies suggest that nectar inhabiting yeasts may alter the chemistry of the nectar (Canto et al., 2008; Herrera et al., 2008, 2009; de Vega et al., 2009; Herrera and Pozo, 2010), but it is difficult to ascertain the roles these microbes play in the ecology of these plants and the interaction with the pollinators without a detailed study of each species. Moreover, the identification of these organisms to "species" level necessarily encompasses uniform units, and that morphotypes seen in this culturing experiment may represent further biological subdivisions, each with a separate biology and perhaps a separate role in the nectar microbial community.

Several species of yeast are capable of existing on the same plant, potentially in the same flower; however, this study did not examine individual flower samples. Plants represent metapopulations of these yeast species and theoretically, it is possible for single flower samples to contain multiple yeast species, as seen in recent studies (Herzberg et al., 2002; Brysch-Herzberg, 2004; Herrera et al., 2010; Belisle et al., 2011; Peay et al., 2011; Pozo et al., 2011). There has only been a single study on the competition and coexistence of these microorganisms for resources within nectar or sugar solutions (Peay et al., 2011), and an examination of how these species interact at the community level may lead to a better understanding of the species diversity within nectar as well as their interactions with the pollinators and the plants. The growth of some species may be dependent on prior colonization of nectar by other species (Starmer, 1981; Peay et al., 2011), or they may exploit different subhabitats within the flower or the nectary itself. Further studies on the nectar microbial communities may provide insight into the co- evolution of these microorganisms with host plants, other yeast species, and potentially with pollinators.

Acknowledgments.--The authors thank Janis Antonovics for his feedback and constructive criticism and Mountain Lake Biological Station (MLBS), run by the University of Virginia, for facilitating the research at the station. This work was supported by funds from an NSF Graduate Research Fellowship for A. M. Golonka, a SigmaXi Grant-in-Aid awarded in 1997, a Mountain Lake Biological Station Gift Grant awarded in 2000, and a grant from the Catherine Keever Botany Fund awarded in 2000 through Duke University.

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SUBMITTED 10 JANUARY 2012 ACCEPTED 31 MAY 2012

ANNETTE M. GOLONKA (1)

Math, Science, Nursing, and Public Health, University of South Carolina Lancaster, Lancaster 29720

AND

RYTAS VILGALYS

Duke University, Department of Biology, P.O. Box 90338, Durham, North Carolina 27708

(l) Corresponding author: Telephone: (803) 313-7019; e-mail: golonkam@mailbox.sc.edu


Table 1.--Species isolated in 1999-2001. Results are GenBank BLAST
searches with base pair  matches for D1/D2 of 26S and ITSMTS2. If
multiple morphospecies were isolated, multiple strains are  listed in
the first column (OTUs), with the first morphospecies' BLAST results
listed. Ascomycetes are in  the top half of the table, Basidiomycetes
in the lower half below the line. An "*" indicates D1/D2 and  ITS1-2
are from different species or genera (see Results). P-values were all
less than [4e.sup.-37] and most were  0.0. N.S. indicates this species
or genus was not sequenced prior for this section of mDNA. <93%
through <96% means that base pair matches were less than or equal to
these percentages

                                                          GenBank
Morphotypes    Closest species identification          accession #

A, B, F, G,
  I, CC        Aureobasidium pultulans                 AY1815811
GG             Candida bombi                           AS788358
LL             Hyalodendriella sp.                     AY188359
BB             * Sphaeriothyriunzsp. (DI/2) OR         AY188366
                 Epicoccum sp. (ITS)
C              * Dothidea (D1/2) or Homonema aff.      AY188367
                 prunorum (ITS)
M, N1          Metsrhnikowia koreensis                 AY188370
P, HH          Metschnikowia aff. reukaufii            AY188383
N, N2          Metsch-nikou4a aff. vanudenii           AY188369
II             Phialemonium aff. cumatum               AY188371
Q              Pichia guilliermondii                   AY188372
JJ             Starmerella aff. bombicola              AY188377
L              Lalaria aff. inositophila               AY188378
KK             Cryptoroccus dimennae                   AY188365
J              Cryptococcus flavescens                 AY188361
AA             Cryptococcus flavescens                 AY188363
D, W           Cryptorocrus aff. laureraii             AY188360
R              Cryptococcus aff. rnaceruns             AY188385
T              Cryptoroccus magnus                     AY188384
X              Cryptoroccus oeirensis                  AN188362
K              Cryptococcus victoriae                  AY188380
Z              Microbotqurn violacmrni                 AYI88368
Y              Pseudozyma sp. 1                        AY188381
O              Psmrdozynra gmminicola                  AY188374
U, V           Rhodotorula glutinis                    AY188373
EE             Sporobolomyres aff. phaffi              AY188375
E              Sporoboloniyces ruberrirnus             AY188376
DD             Ustilago rnvydis OR Pseudozyma          MI 88382
                 prolifica (617/625)

Morphotypes    DI/D2        ITS1-2

A, B, F, G,
  I, CC        587/588      533/534
GG             476/482      351/374
LL             <94%         N.S.
BB             568/594      261/268

C              571/584      510/514

M, N1          517/524      N.S.
P, HH          497/514      334/346
N, N2          483/504      N.S.
II             564/576      504/516
Q              585/589      559/560
JJ             475/490      N.S.
L              562/569      548/563
KK             601/607      437/454
J              616/617      488/489
AA             618/620      480/481
D, W           592/610      463/491
R              572/603      150/179
T              609/612      580/580
X              614/616      558/558
K              614/615      472/474
Z              593/595      611/614
Y              <93%         <94%
O              599/606      678/702
U, V           591/593      558/558
EE             579/585      N.S.
E              589/594      552/556
DD             608/616      <94%

TABLE 2.--Number of times a given ascomycete yeast species was
recovered from male (M) and female (F) Silene latifolia samples and 26
coflowering  plant species. Data are from Jun.-Aug. of 1999-2001.
Total sample N is the number of samples of each plant species taken
during this period of time. Total  number of samples and isolates
listed at bottom of table. Blanks indicate 0 yeast samples. Yeast
species marked by an "[dagger]" are known to be potential plant
pathogens. Plant species that contained no yeast were removed from
list: Hemerocallis fulva, Melilotus alba, Silene noctiflora, Silene
vulgaris, Trifolium repens,  Vitia cracca, Viola striata

                         Total
                         sample       Aureobasidium       Candida
Plant species              N       pullulans [dagger]      bombi

Silene latifolia (F)        366                   107      5
Silene latifolia (M)        340                   101      6
Asclepias syriaca             3                     1
Barbarea vulgaris             3                     2
Campsis radicans              1
Coronilla varia              16                     8      1
Dianthus armeria              5
Impatiens eapensis            6                     3
Iris versicolor               1                     1
Lathyrus latifolius           7                     3
Leonurus cardiaca             9                     2
Lonicera japonica            19                     9      1
Lonicera tatarica             1                     1
Medicago sativa               2
Melilotus officinalis         2                     1
Nepeta cataria               29                     9      1
Prunella vulgaris             2                            1
Saponana officinalis         17                     5      2
Satureja vulgaris             9
Trifolium pratense           18                     5
Vicia villosa                 2                     1
Tota1 sample                858                   259      17

                                           Sphaeriothyrium
                                               sp. OR
                         Hyalodendriella   Epicoccum sp.
Plant species                  sp.            [dagger]

Silene latifolia (F)            3                 2
Silene latifolia (M)            1                 3
Asclepias syriaca
Barbarea vulgaris
Campsis radicans
Coronilla varia
Dianthus armeria
Impatiens eapensis
Iris versicolor
Lathyrus latifolius
Leonurus cardiaca
Lonicera japonica
Lonicera tatarica
Medicago sativa
Melilotus officinalis
Nepeta cataria                                   11
Prunella vulgaris
Saponana officinalis            1                 1
Satureja vulgaris                                 1
Trifolium pratense
Vicia villosa
Tota1 sample                    5                19

                                           Met.         Met.
                         Metschnikoma      aff.         aff.
Plant species             kareensis     reukaufii    vanudenii

Silene latifolia (F)          25            25           7
Silene latifolia (M)          19            27           9
Asclepias syriaca                           1            1
Barbarea vulgaris             1
Campsis radicans                            1
Coronilla varia               3             1
Dianthus armeria                            1
Impatiens eapensis            1             3
Iris versicolor
Lathyrus latifolius           2             2            2
Leonurus cardiaca                           3            2
Lonicera japonica             6             4            2
Lonicera tatarica                           1
Medicago sativa                             1
Melilotus officinalis
Nepeta cataria                7             12           8
Prunella vulgaris
Saponana officinalis          1             4
Satureja vulgaris             1                          1
Trifolium pratense            3             2            1
Vicia villosa                 1
Tota1 sample                  70            88           33

                         Phialemonium                      Starmerella
                             aff.            Pichia            aff.
Plant species              curvatum      guilliermondii     bombicola

Silene latifolia (F)           4                5               3
Silene latifolia (M)           1                1               1
Asclepias syriaca
Barbarea vulgaris
Campsis radicans
Coronilla varia                1
Dianthus armeria
Impatiens eapensis
Iris versicolor
Lathyrus latifolius                                             1
Leonurus cardiaca
Lonicera japonica                                               1
Lonicera tatarica
Medicago sativa                                 1
Melilotus officinalis
Nepeta cataria                                                  1
Prunella vulgaris
Saponana officinalis
Satureja vulgaris              1
Trifolium pratense
Vicia villosa
Tota1 sample                   7                7               7

                                Lalaria
                                 aff.
Plant species            inositophila [dagger]

Silene latifolia (F)               1
Silene latifolia (M)               5
Asclepias syriaca
Barbarea vulgaris
Campsis radicans
Coronilla varia
Dianthus armeria
Impatiens eapensis
Iris versicolor
Lathyrus latifolius
Leonurus cardiaca
Lonicera japonica                  1
Lonicera tatarica
Medicago sativa
Melilotus officinalis
Nepeta cataria
Prunella vulgaris
Saponana officinalis
Satureja vulgaris
Trifolium pratense
Vicia villosa
Tota1 sample                       7

TABLE 3.--Number of times a given basidiomycete yeast species was
recovered from male (M) and female (F) Silene latifolia samples and 26
coflowering  plant species. Data are from Jun.-Aug. of 1999-2001.
Total sample N is the number of samples of each plant species taken
during this period of time. Total  number of samples and isolates
listed at bottom of table. Blanks indicate 0 yeast samples. Yeast
species marked by an "[dagger]" are known to be plant pathogens.
Plant species that contained no yeast were removed from list:
Asclepias syriaca, Campsis radicans, Dianthus armeria, Hemerocallis
fulva, Melilotus alba, Prunella  vulgaris, Silene noctiflora, Silene
vulgaris, Trifolium repens, Vicia cracca, Viola striata.

                        Total                     Crypto.
Plant species           sample    Cryptococcus   flavescens
                           N        dimennae        (J)

Silene latifolia (F)        366        1             4
Silene latifolia (M)        340                      4
Barbarea vulgaris             3                      1
Coronilla varia              16                      1
Impatiens capensis            6
Iris versicolor               1
Lathyrus latifolius           7
Leonurus cardiaca             9
Lonicera japonica            19                      1
Lonicera tatarica             1
Medicago sativa               2
Melilotus officinalis         2
Nepeta cataria               29
Saponaria                    17
  officinalis
Satureja vulgaris             9
Trifolium pratense           18
Vicia villosa                 2                      1
Total samples               847        1             12

                         Crypto.      Crypto.    Crypto.
Plant species           flavesrens     aff.        aff.     Crypto.
                           (AA)      laurentii   macerans   magnus

Silene latifolia (F)        20           7          3         6
Silene latifolia (M)        22          12          2         6
Barbarea vulgaris           1
Coronilla varia             1                       1         2
Impatiens capensis          1            1
Iris versicolor
Lathyrus latifolius
Leonurus cardiaca
Lonicera japonica
Lonicera tatarica
Medicago sativa
Melilotus officinalis       1
Nepeta cataria                           2
Saponaria                   5
  officinalis
Satureja vulgaris
Trifolium pratense          4            2                    1
Vicia villosa
Total samples               55          24          6         15

                                                Micro-
                                                bntryum
Plant species            Crypto.    Crypto.     violareum   Pseudozyma
                        oeirensis   victoriae   [dagger]      sp. 1

Silene latifolia (F)        1       31          24              3
Silene latifolia (M)        4       38          43              3
Barbarea vulgaris                   1
Coronilla varia                     3           2
Impatiens capensis
Iris versicolor                                                 1
Lathyrus latifolius                             3
Leonurus cardiaca                   1
Lonicera japonica                   3           2
Lonicera tatarica                               1
Medicago sativa             1
Melilotus officinalis               1           1
Nepeta cataria                      1           4
Saponaria                                       5
  officinalis
Satureja vulgaris                               1
Trifolium pratense          1       3           2
Vicia villosa
Total samples               7       82          88              7

                                                      Sporobo-
                        Pseudozyma    Rhodotorula   lomyces aff.
Plant species           graminicola    glutinis        phaffii

Silene latifolia (F)         2            13              9
Silene latifolia (M)         2            12             12
Barbarea vulgaris
Coronilla varia
Impatiens capensis                         1              1
Iris versicolor
Lathyrus latifolius
Leonurus cardiaca                                         1
Lonicera japonica                          1
Lonicera tatarica
Medicago sativa
Melilotus officinalis
Nepeta cataria                             2              1
Saponaria                    1                            1
  officinalis
Satureja vulgaris
Trifolium pratense                         2              1
Vicia villosa
Total samples                5            35             26

                                     Ustilago maydis
                          Sporo.      OR Pseudozyma
                        ruberrimus      prolifica
Plant species            [dagger]        [dagger]

Silene latifolia (F)        9               2
Silene latifolia (M)        8               2
Barbarea vulgaris
Coronilla varia
Impatiens capensis          1
Iris versicolor
Lathyrus latifolius
Leonurus cardiaca           1
Lonicera japonica
Lonicera tatarica
Medicago sativa
Melilotus officinalis
Nepeta cataria
Saponaria                   1
  officinalis
Satureja vulgaris
Trifolium pratense          2
Vicia villosa
Total samples               22              4

TABLE 4.--Tables showing yeast presence and absence in Silene
latifolia samples sorted by year, month,  and sex of nectar sample.
(A) For 1999: [chi square] = 6.81, df = 2, P =0.033. (B) For 2000:
[chi square] = 3.71, df = 2, P =  0.16. (C) For 2001: [chi square] =
3.01, df = 2, P = 0.22. Chi-square results are for comparison of month
within  each year

Month                       Sex       Absent   Present   Total

(A) Contingency table for 1999

  Jun.                      Female      19        5       24
                            Male        20        4       24
  Jul.                      Female      7        12       19
                            Male        12        3       15
  Aug.                      Female      22       10       32
                            Male        17       13       30
                            Total       97       47       144

(B) Contingency table for 2000

  Jun.                      Female      5        23       28
                            Male        7        18       25
  Jul.                      Female      12       18       30
                            Male        8        17       25
  Aug.                      Female      14       15       29
                            Male        7        18       25
                            Total       53       109      162

(C) Contingency table for 2001

  Jun.                      Female      15       11       26
                            Male        7        20       27
  Jul.                      Female      12       19       31
                            Male        7        22       29
  Aug.                      Female      5        23       28
                            Male        9        13       22
                            Total       55       108      163

TABLE 5.--Analysis of variance table for log 10 (CFUs/[micro]L) for
Silene latifotia samples. Similar  significant results were obtained
for log 10(CFUs/flower) which is not shown here. The main effects were
sex, site,  date, year, and interaction terms. The dependent was log
transformed CFUs/[micro]L. Significant results are in bold

Source                  df         SS    F value         P

Log10(CFUs /[micro]L)

  Sex                     1     0.009       0.01     0.91
  Site                    7    13.1         2.58     0.015
  Date                    2     3.73        2.58     0.08
  Year                    2    13.8         9.51     0.0001
  Sex*Date                2     0.40        0.27     0.76
  Sex*Site                7     9.92        1.96     0.063
  Sex*Year                2     0.098       0.07     0.93
  Date*Year               4    25.2         8.69    <0.001
  Site*Year              13     9.41        1.00     0.46
  Site*Date              14     7.74        0.76     0.71
  Sex*Site*Year          12    12.9         1.49     0.13
  Error                 195   141.1
Total                   261   242.6

TABLE 6--Table showing the percentage of each yeast species in all
plant samples listed in Tables 1 and 2 (=# of samples with that yeast
species present /total number of yeast samples present in all plant
samples * 100). Range of abundance for each yeast species across all
plant samples listed in Tables 2 and  3/. Ascomycetes are in the top
half of the table, Basidiomycetes are in the lower half

Yeast species                    % of Each yeast species

Aureobasidium pullulans                   28.8
Candida bombi                             1.89
Hyalodendriella sp.                       0.57
Sphaeriothyrium/Epicoccum                 0.11
Dothidea/Hormonema                        1.00
Metschnikouria koreensis                  7.79
Metschnikozvia aff. reukaufi              9.79
Metschnikouria aff. vanudenii             3.67
Phialemonium aff. curvatum                0.78
Pichia guilliermondii                     0.78
Starmerella aff. bombicola                0.78
Lalariaaff. inositiophila                 0.78
Cryptococcus dimennae                     0.11
Cryptococcus favescens (J)                1.35
Cryptococcus favescens (AA)               6.12
Cryptococcus aff. laurentii               2.67
Cryptococcus aff. macerans                0.67
Cryptococcus magnus                       1.67
Cryptococcus oeirensis                    0.78
Cryptococcus victoriae                    9.12
Microbotryum violaceum                    9.79
Pseudozyma sp. 1                          0.79
Pseudozyma graminicola                    0.56
Rhodotorula glutinis                      3.89
Sporobolomyces aff. phafii                2.89
Sporobolomyces ruberrimus                 2.45
Ustilago/Pseudozyma                       0.44

                                 Abundance range (mean [+ or -] SE)
Yeast species                    (CFUs / [micro]L nectar)

Aureobasidium pullulans          0.58-33,000  (292 [+ or -] 152)
Candida bombi                     1.4-24,000  (1400 [+ or -] 1330)
Hyalodendriella sp.                  1.8-106  (30 [+ or -] 20)
Sphaeriothyrium/Epicoccum                8.4  (N.A.)
Dothidea/Hormonema                   1.6-350  (93 [+ or -] 43)
Metschnikouria koreensis           0.98-5170  (236 [+ or -] 68)
Metschnikozvia aff. reukaufi    0.46-128,000  (3250 [+ or -] 1880)
Metschnikouria aff. vanudenii      0.42-3580  (208 [+ or -] 93)
Phialemonium aff. curvatum          1/1/1914  (6.5 [+ or -] 1.6)
Pichia guilliermondii                10-1450  (80 [+ or -] 47)
Starmerella aff. bombicola          2/1/5910  (1070 [+ or -] 832)
Lalariaaff. inositiophila            0.88-46  (1.3 [+ or -] 6.8)
Cryptococcus dimennae                    0.8  (N.A.)
Cryptococcus favescens (J)           2.2-522  (74 [+ or -] 46)
Cryptococcus favescens (AA)         1/5/2420  (79 [+ or -] 45)
Cryptococcus aff. laurentii         0.58-458  (55 [+ or -] 25)
Cryptococcus aff. macerans           2.6-246  (52 [+ or -] 39)
Cryptococcus magnus                  1.5-581  (93 [+ or -] 40)
Cryptococcus oeirensis               3.1-166  (40 [+ or -] 26)
Cryptococcus victoriae              2/3/6500  (208 [+ or -] 87)
Microbotryum violaceum            1.0-10,700  (419 [+ or -] 182)
Pseudozyma sp. 1                     1.3-178  (51 [+ or -] 31)
Pseudozyma graminicola               2.4-418  (89 [+ or -] 82)
Rhodotorula glutinis                0.67-370  (44 [+ or -] 12)
Sporobolomyces aff. phafii          0.91-190  (20 [+ or -] 87)
Sporobolomyces ruberrimus           0.56-141  (27 [+ or -] 8.0)
Ustilago/Pseudozyma                   21-379  (153 [+ or -] 83)
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Author:Golonka, Annette M.; Vilgalys, Rytas
Publication:The American Midland Naturalist
Article Type:Report
Geographic Code:1U5VA
Date:Apr 1, 2013
Words:11663
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