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RESEARCH ARTICLE
Diversity and distribution of microbial
communities in floral nectar of two night-
blooming plants of the Sonoran Desert
Martin von Arx
1
, Autumn Moore
1
, Goggy Davidowitz
1
, A. Elizabeth ArnoldID
2
*
1Department of Entomology, The University of Arizona, Tucson, AZ, United States of America, 2School of
Plant Sciences and Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson, AZ,
United States of America
*arnold@ag.arizona.edu
Abstract
Nectar-inhabiting microbes are increasingly appreciated as important components of plant-
pollinator interactions. We quantified the incidence, abundance, diversity, and composition
of bacterial and fungal communities in floral nectar of two night-blooming plants of the Sono-
ran Desert over the course of a flowering season: Datura wrightii (Solanaceae), which is pol-
linated by hawkmoths, and Agave palmeri (Agavaceae), which is pollinated by bats but
visited by hawkmoths that forage for nectar. We examined the relevance of growing environ-
ment (greenhouse vs. field), time (before and after anthesis), season (from early to late in
the flowering season), and flower visitors (excluded via mesh sleeves or allowed to visit flow-
ers naturally) in shaping microbial assemblages in nectar. We isolated and identified bacte-
ria and fungi from >300 nectar samples to estimate richness and taxonomic composition.
Our results show that microbes were common in D.wrightii and A.palmeri nectar in the
greenhouse but more so in field environments, both before and especially after anthesis.
Bacteria were isolated more frequently than fungi. The abundance of microbes in nectar of
D.wrightii peaked near the middle of the flowering season. Microbes generally were more
abundant as time for floral visitation increased. The composition of bacterial and especially
fungal communities differed significantly between nectars of D.wrightii and A.palmeri,
opening the door to future studies examining their functional roles in shaping nectar chemis-
try, attractiveness, and pollinator specialization.
Introduction
Nectar-inhabiting microbes are increasingly appreciated as important components in many
plant-pollinator systems [1,2]. Bacteria and fungi, including both yeasts and filamentous fungi,
have been identified in nectar of diverse plant species [3–4]. They can influence nectar chemis-
try, volatiles, pollen germination, floral attractiveness, and nutritional quality for pollinators
[2,5–7]. Diverse studies have identified microbial communities in nectar in various temperate
and tropical plants [1–8], but relatively little is known about nectar-inhabiting microbes in
desert flowers.
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OPEN ACCESS
Citation: von Arx M, Moore A, Davidowitz G,
Arnold AE (2019) Diversity and distribution of
microbial communities in floral nectar of two night-
blooming plants of the Sonoran Desert. PLoS ONE
14(12): e0225309. https://doi.org/10.1371/journal.
pone.0225309
Editor: Renee M. Borges, Indian Institute of
Science, INDIA
Received: February 2, 2019
Accepted: November 1, 2019
Published: December 12, 2019
Copyright: ©2019 von Arx et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Sequence data are
available at GenBank (accessions KJ543743 -
KJ544084) and in S1 Table, which also contains
metadata for each microbial isolate considered
here.
Funding: This research was supported by the
College of Agriculture and Life Sciences at the
University of Arizona; the National Science
Foundation (USA), grant IOS-1053318 to GD; and
the National Institute of Food and Agriculture
(USA), award ARZT-1361340-H25-242 to AEA and
In areas such as the Sonoran Desert, known for richness both of flowering plants and polli-
nators, a rich history of studies has characterized plant-pollinator associations [9–11]. How-
ever, to our knowledge only two studies have examined the diversity of nectar-inhabiting
microbes in the Sonoran Desert bioregion: [12] examined nectar of saguaro cactus (Carnegiea
gigantea) and cultivars of Citrus, and [13] examined nectar from cultivars of cotton in field-
and greenhouse settings, as well as saguaro, prickly pear cactus (Opuntia), and Citrus. These
studies showed that bacteria (Staphylococcus, other Gram-positive bacteria, and some Gram-
negative strains) were present but not highly diverse or abundant in nectar of saguaro [12,13].
In Citrus they recorded bacteria, but only rarely, with the sum of their work highlighting infre-
quent infections by Gram-negative bacteria, one fungus, and one actinomycete [12,13]. These
studies did not identify nectar-inhabiting microbes to species nor compare and contrast them
among host plants, evaluate their seasonality, or explore their abundance before and after
anthesis. Thus little is known regarding the microbial diversity in nectar of Sonoran Desert
plants.
Understanding the diversity and distributions of nectar-inhabiting microbes is a key step in
understanding the interactions that shape the diverse biotic communities of the arid south-
west, which are threatened increasingly by human activity at local scales [9,10] as well as cli-
mate change more broadly [14]. The aims of this study were to characterize the diversity and
distributions of microbes associated with nectar of two iconic plants of the Sonoran Desert
region, where previous studies of nectar microbiomes have been limited in scope. Specifically,
we quantified the frequency, abundance, diversity, and composition of bacterial and fungal
communities in floral nectar of two species of night-blooming plants: Datura wrightii (Solana-
ceae), which is pollinated by hawkmoths [15], and Agave palmeri (Agavaceae), which is polli-
nated by bats but visited by hawkmoths that forage for nectar [15–17]. These two species differ
in their nectar composition, concentration, floral display, flower longevity, and flowering phe-
nology [15], yet are both a major source of nectar for the hawkmoth community of the south-
west [16,17]. Here we describe the relevance of growing environment (greenhouse vs. field),
time (before and after anthesis), season (from early to late in the flowering season), and flower
visitors (excluded via mesh sleeves or allowed to visit flowers naturally) in shaping microbial
communities in nectar. Our work complements the growing literature regarding nectar-inhab-
iting microbes in other biotic communities [1–8,18–24] while also addressing key gaps in
existing knowledge of the biodiversity and biotic interactions between plants and other organ-
isms in the Sonoran Desert region [9,10].
Materials and methods
Nectar was collected from D.wrightii and A.palmeri flowers on plants in field and greenhouse
environments (S1 Table). Collections were conducted either on University of Arizona property
or in public lands for which nectar is not listed as requiring a collection permit (for details see
S1 Table). Neither plant species is endangered or protected. We evaluated D.wrightii through-
out a summer flowering season (31 May to 16 October, 2013) and A.palmeri during peak flow-
ering in that season (14–24 July, 2013). Flowers were collected 1 h before or 16 h after anthesis
(D.wrightii) or 24 h before anthesis, at anthesis, or at 24 h intervals up to 72 h after anthesis
(A.palmeri). Floral visitors were allowed or were excluded from flowers with a fine mesh sleeve
that excluded bats and large insects.
Flowers of D.wrightii collected in the field were processed for nectar collection within 1 h
of harvesting. Nectar collection from A.palmeri flowers was performed directly on site. To col-
lect nectar from D.wrightii we sliced each flower longitudinally and used a sterile syringe to
Nectar microbes of Sonoran Desert plants
PLOS ONE | https://doi.org/10.1371/journal.pone.0225309 December 12, 2019 2 / 14
colleagues. The funders played no role in the study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
remove all available nectar (ca. 20 uL/flower). Nectar from A.palmeri flowers was collected
directly with a sterile syringe (ca. 50 uL/flower).
Each nectar sample was diluted twice (1:10 each time) and then partitioned for plating on
four media (Saboraud’s agar; lysogeny broth agar, LBA; yeast potato dextrose agar, YPDA;
malt extract agar, MEA). All media were prepared from standard products according to the
manufacturer’s instructions (Fisher Scientific). Plates were incubated for 70 h under laboratory
conditions. We then counted the number of colony forming units (CFU) per plate. Values
were log-transformed for analysis and were compared by ANOVA (analyses of environment,
anthesis, and floral visitors) or a generalized linear model (seasonality for D.wrightii). We
measured sucrose content for flowers that had a large enough nectar volume to support micro-
bial sampling as well as sucrose measurement (65 flowers representing six individuals of A.
palmeri, and 116 flowers representing 12 individuals of D.wrightii, ecnompassing representa-
tive flowers in all study sites and throughout the duration of the study) (S1 Table).
Characterization of microbial isolates
Representatives of each morphotype observed in each nectar sample were vouchered at the
Robert L. Gilbertson Mycological Herbarium at the University of Arizona (accession numbers
are listed in S1 Table). DNA was extracted from a fresh culture of each isolate following [25],
with sampling of morphotypes proportional to their occurrence. To characterize bacteria we
used primers 27F and 1492R to amplify a ca. 1400 basepair fragment of the 16S ribosomal
RNA (16S rRNA) as described in [26]. To characterize fungi we used primers ITS1F and LR3
to amplify a ca. 1200 bp fragment consisting of the nuclear ribosomal internal transcribed
spacers, 5.8S region, and the first ca. 600 bp of the nuclear ribosomal large subunit (ITS-partial
LSUrDNA). PCR conditions are described in [26]. Products were evaluated by electrophoresis
on a 1% agarose gel. Positive products were cleaned with Exo-SAP-IT and diluted 1:1 with
molecular grade water prior to bidirectional sequencing with the above primers (5 uM) on an
Applied Biosystems AB3730XL (Foster City, CA) at the University of Arizona Genetics Core.
Sequences were edited and assembled as described in [27]. Consensus sequences for each iso-
late were submitted to GenBank (accessions KJ543743—KJ544084). The final data set con-
sisted of 270 isolates from D.wrightii nectar (of which 210 were bacteria) and 76 isolates from
A.palmeri nectar (of which 60 were bacteria).
Operational taxonomic units and taxonomic placement
Operational taxonomic units (OTU) were designated at four levels of sequence similarity
(95%, 97%, 99%, and 100%; S1 Table) in Sequencher 5.0 as described by [28]. Each sequence
was compared by BLAST against the NCBI GenBank database to estimate taxonomic place-
ment at the genus level and above. Each match was scrutinized to avoid spurious identification.
We used OTU records at 97% similarity (bacteria) and 95% similarity (fungi) to estimate rich-
ness, diversity, and taxonomic composition [25–29]. We evaluated the completeness of sam-
pling by constructing species accumulation curves in EstimateS v. 8.0 (http://viceroy.eeb.
uconn.edu/estimates/). Diversity was calculated as Fisher’s alpha, which is robust to variation
in sample size [29]. We compared communities of microbes in nectar via analyses of similarity
(ANOSIM) in PAST (https://folk.uio.no/ohammer/past) with 999 permutations and similarity
defined by the Jaccard index. Only non-singleton OTU were included. Stress values
were �0.20 in each analysis. Results were visualized by non-metric multidimensional scaling
(NMDS).
Nectar microbes of Sonoran Desert plants
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Results
Microbes were found frequently in nectar of D.wrightii and A.palmeri flowers. Microbes were
isolated on all four media. Bacteria were isolated most frequently on LBA and least frequently
on MEA and Sabouraud’s agar (S1 Table). Fungi were isolated most frequently on Sabouraud’s
agar and least frequently on LBA (S1 Table). These patterns were consistent for samples from
both plant species (S1 Table).
Nectar-inhabiting microbes of D.wrightii
Microbes were isolated from D.wrightii flowers under field- and greenhouse conditions,
before and after anthesis, and in the presence and absence of floral visitors (Fig 1). Bacteria
were isolated from D.wrightii nectar more frequently than fungi (77.8% of isolates were bacte-
ria). The frequency of nectar samples containing microbes was greater in field conditions than
in greenhouse conditions and generally was higher after anthesis than before anthesis (Fig 1).
Overall, the abundance of microbes per nectar sample (i.e., CFU/uL) was greatest in flowers
after anthesis that were open to floral visitors (Fig 2). Exclusion of visitors resulted in abun-
dances of microbes after anthesis that were similar to those in flowers before anthesis (Fig 2).
Overall, the percent of nectar samples and agar plates showing evidence of microbial growth
was greatest in the middle of the flowering season (mid-July to mid-August) relative to earlier
or later in the season (Fig 3).
Composition of bacterial communities in nectar differed marginally as a function of grow-
ing environment and floral visitation after anthesis, but did not differ between flowers before
or after anthesis when pollinators were excluded, or as a function of pollinator visitation before
flowers opened (Table 1). In contrast, composition of fungal communities in nectar samples
differed in flowers before vs. after anthesis and, after anthesis, as a function of flower visitation
Fig 1. Proportion of D.wrightii and A.palmeri flowers containing nectar microbes (bacteria and fungi; one nectar
sample per flower). Nectar was collected from plants in field or greenhouse environments, at different time points
relative to anthesis, and with or without exclusion of flower visitors ("sleeve", "no sleeve"). Different letters assigned
within a time category indicate statistically significant differences (Tukey HSD, p <0.05).
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Nectar microbes of Sonoran Desert plants
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(Table 1). Taxonomic affinities of representative bacteria and all fungi from D.wrightii nectar
are shown in Table 2. The full list of isolates is presented in S1 Table. Before anthesis, Pseudo-
zyma,Rosenbergiella, and Micrococcus were particularly common in D.wrightii nectar (S1
Table). Candida was isolated frequently from nectar after anthesis, but the bacterial commu-
nity became diverse after anthesis (S1 Table).
Nectar-inhabiting microbes of A.palmeri
Microbes were isolated from A.palmeri flowers at and after anthesis, but they were very rare
before anthesis (Fig 1). Bacteria were isolated more frequently from A.palmeri nectar than
fungi (76.7% of isolates were bacteria). Nectar-inhabiting microbes were more common in
flowers of field-grown vs. greenhouse-grown plants (Fig 1). Flowers that were exposed to floral
visitors tended to harbor more microbes than flowers from which visitors were excluded (Fig
4). The abundance of microbes per sample increased in abundance over time for flowers from
which visitors were excluded (Fig 5). This increase was not as pronounced in flowers that were
accessible to visitors (Fig 5). Overall, we observed no shift in bacterial communities as a func-
tion of floral visitation (Table 1). The taxonomic affinities of bacteria and fungi from A.pal-
meri are shown in Table 2. The full list of isolates is presented in S1 Table. We did not observe
strong dominance by particular fungi or bacteria before vs. after anthesis (S1 Table).
Comparison of nectar-inhabiting microbes of D.wrightii and A.palmeri
The species richness of microbes isolated from nectar of D.wrightii generally was similar to
that observed in A.palmeri (Fig 5). Although the most common bacterial OTU were found in
Fig 2. Concentration of colony forming units (CFU) in D.wrightii nectar collected before and after anthesis from
plants with and without flower visitor exclusion (N = 14, 47, 12, 24 for -1 h/sleeve, -1 h/no sleeve, +16 h/sleeve and
+16 h/no sleeve, respectively). Different letters indicate significant differences (Tukey HSD, p <0.05).
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Nectar microbes of Sonoran Desert plants
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Fig 3. Change in abundance of nectar microbes in D.wrightii flowers during one flowering season (31 May (5/
31)– 16 October (10/16), 2013). Nectar was collected from plants in the field 1 h before anthesis and without
exclusion of flower visitors. (A) Proportion of D.wrightii nectar samples with microbes (N = 9 each, except for 25 June,
19 August and 16 October, with N = 8, 5, and 8, respectively). (B) Proportion of agar plates showing microbe growth
(N = 72 each, except for 25 June, 19 August and 16 October, with N = 64, 40, and 64, respectively). Dashed lines
describe second-order polynomial regressions. Different letters indicate significant differences between collection
dates (GLM, p <0.05).
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both species (Table 2), the overall composition of bacterial communities (Fig 6A) and fungal
communities (Fig 6B) differed between the two plant species. The bacteria that were found in
both plants species included ubiquitous genera such as Pantoea/Erwinia and the nectar-inhab-
iting genus Rosenbergiella [30]. Two yeasts made up >70% of the fungal isolates from D.
wrightii nectar, whereas two filamentous fungi (one with a yeast form, Aureobasidium) com-
prised >50% of the fungal isolates from A.palmeri (Table 2).
Discussion
In their review of biotic interactions between plants and other organisms in the Sonoran Des-
ert, [9] described plant-microbe interactions as one of the largest gaps in knowledge of the
regional flora and its dynamics. This study contributes to filling that gap by documenting nec-
tar-inhabiting microbes in two night-blooming plant species for which pollination biology has
been characterized previously [15–17,31–33]. This is the first systematic survey of nectar
microbiomes in the region and it provides a perspective on the microbial assemblages in
ephemeral flowers of perennial plants. The culture collection and temporal perspectives gener-
ated by this study provide a basis for future work regarding how such microbes may influence
nectar quality, pollinator nutrition, and pollinator specialization [1,2,15,20].
In addition to carbohydrates, floral nectars also contain amino acids and fatty acids [34–
37]. Hawkmoths, which forage on both plants studied here [15–17,32], are able to use these
amino acids and fatty acids for metabolic fuel or incorporate them to their somatic or repro-
ductive tissues [38–40] with differential allocation strategies between the sexes [38]. It is possi-
ble that these amino acids and fatty acids in nectar are derived from routine senescence cycles
of the nectar microbiome. If so, it is possible that pollinator foraging decisions may be related,
if indirectly, to nectar microbial communities for reasons beyond carbohydrates alone.
Relative to two previous studies in the region [12,13], the present study shows that nectar-
inhabiting microbes are common and diverse at a regional level in flowers of two night-
Table 1. Effects of growing environment, age and flower visitors on nectar microbe community composition in Datura wrightii and Agave palmeri in southeastern
Arizona.
Factor group
1
N
1
group
2
N
2
R
a
p
a
Bacteria, D.wrightii
growing environment greenhouse 7 field 67 0.088 0.068
age (no sleeve only)
b
before anthesis
d
31 after anthesis
e
21 -0.004 0.510
age (sleeve only)
c
before anthesis 6 after anthesis 10 0.052 0.190
flower visitors (before anthesis) no sleeve 31 sleeve 6 -0.006 0.510
flower visitors (after anthesis) no sleeve 21 sleeve 10 0.07 0.093
Fungi, D.wrightii
age (no sleeve only) before anthesis 13 after anthesis 12 0.15 0.008
flower visitors (before anthesis) no sleeve 13 sleeve 8 0.062 0.160
flower visitors (after anthesis) no sleeve 12 sleeve 6 0.56 0.001
Bacteria, A.palmeri
f
flower visitors no sleeve 26 sleeve 5 0.056 0.220
a
Calculated with one-way ANOSIM, with Jaccard’s index.
b
"no sleeve only" = included only nectar samples from flowers with no flower visitor exclusion.
c
"sleeve only" = included only nectar samples from flowers with flower visitor exclusion.
d
"before anthesis" = nectar samples collected 1 h before anthesis.
e
"after anthesis" = nectar samples collected 16 h after anthesis.
f
fungi from A.palmeri were not included because only 16 isolates were obtained, most of which represented OTU that occurred only one time.
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blooming species. We found that fungi and bacteria were more common in nectar of D.
wrightii and A.palmeri than in the species evaluated previously [12,13], and here highlight
their abundance throughout the growing season (D.wrightii), their dynamics with respect to
floral visitors (both species) and environment (greenhouse vs. field, both species), and their
taxonomic composition. Our analyses reveal significant differences in the microbial communi-
ties in nectar of D.wrightii and A.palmeri, even though the most common bacterial genera
occurred in both species (Table 2). Such differences may reflect many factors, including nectar
chemistry and the microbiomes of pollinators. We noted that yeasts were common in nectar
of D.wrightii flowers, whereas they were considered rare to absent in other plants of the region
in previous work [13]. The fungal genus Aureobasidium, observed here in nectar of A.palmeri,
Table 2. Top BLAST matches (genera) for the most common bacteria and all fungi isolated from nectar of D.
wrightii and A.palmeri (see S1 Table for full list and details). Taxa are presented in decreasing order of abundance
as isolated from D.wrightii. (%) = isolates relative to total isolates of bacteria (above) or fungi (below).
Top BLAST match D.wrightii: isolates (%) A.palmeri: isolates (%)
Bacteria
Rosenbergiella sp. 21 (10.0) 13 (21.7)
Pseudomonas sp. 18 (8.6) 1 (1.7)
Pantoea and Erwinia sp. 15 (7.1) 4 (6.7)
Enterobacter sp. 15 (7.1) 6 (10.0)
Micrococcus sp. 14 (6.7) 1 (1.7)
Staphylococcus sp. 14 (6.7) 1 (1.7)
Kocuria sp. 11 (5.2) 3 (5.0)
Pluralibacter sp. 8 (3.8) 0 (0)
Paenibacillus sp. 7 (3.3) 0 (0)
Bacillus sp. 7 (3.3) 2 (3.3)
Cronobacter sp. 6 (2.9) 0 (0)
Klebsiella sp. 6 (2.9) 0 (0)
Serratia sp. 6 (2.9) 0 (0)
Lactobacillus sp. 5 (2.4) 1 (1.7)
Acinetobacter sp. 4 (1.9) 6 (10.0)
Enterococcus sp. 1 (0.5) 5 (8.3)
Fungi
Pseudozyma sp. 31 (51.7) 0 (0)
Candida sp. 12 (20.0) 0 (0)
Cryptococcus sp. 4 (6.7) 0 (0)
Wickerhamiella sp. 4 (6.7) 0 (0)
Alternaria sp. 2 (3.3) 2 (12.5)
Cladosporium sp. 2 (3.3) 1 (6.3)
Kodamaea sp. 2 (3.3) 0 (0)
Leptosphaeria sp. 1 (1.7) 0 (0)
Metschnikowia sp. 1 (1.7) 0 (0)
Naganishia sp. 1 (1.7) 0 (0)
Aspergillus sp. 0 (0) 1 (6.3)
Aureobasidium sp. 0 (0) 4 (25.0)
Clavispora sp. 0 (0) 1 (6.3)
Diatrypella sp. 0 (0) 1 (6.3)
Fusarium sp. 0 (0) 1 (6.3)
Thyronectria sp. 0 (0) 5 (31.3)
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appears to occur frequently in nectar of diverse plant species in other biomes [7]. In future
work we anticipate examining nectar communities with culture-free methods, as these
Fig 4. Concentration of colony forming units in A.palmeri nectar. (A) CFU concentration in the nectar of 0, 1, 2,
and 3 d old A.palmeri flowers with flower visitor exclusion (N = 2, 3, 3, and 7, respectively). (B) CFU concentration in
the nectar of 0, 1, 2, and 3 d old A.palmeri flowers without flower visitor exclusion (N = 5, 12, 8, and 8, respectively).
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typically reveal diverse communities with members that may be recalcitrant to culturing under
the methods used here [41,42]. We also suggest that future culture-based surveys should use
cultivation media that have been used in previous studies of nectar microbiomes in other
plants and geographic locations [1–8,18–24,41–44]: such approaches will clarify the novelty
and distinctiveness of microbiomes in nectar of the night-blooming plants of the Sonoran Des-
ert as studied here. Different media fostered growth by distinctive portions of the microbiome
in the present study, consistent with differences in pH and nutrient content and underscoring
the importance of considering media carefully for culture-based studies of nectar communi-
ties. Commonly used media in nectar microbiome studies (e.g., trypticase soy agar [4], or R2A
and yeast medium [45]) could be especially useful in future work.
Nectar microbes have been studied most extensively in plants with flowers that are receptive
over multiple days, and in those cases microbes appear to colonize nectar mostly after anthesis
via pollinators and airborne deposition [3,22,43]. Here we documented the occurrence of
microbial communities in nectar before anthesis in two species, but especially in D.wrightii,
which has a flower longevity of only one night. Hence, our results suggest that microbes could
Fig 5. Richness of nectar microbes in D.wrightii and A.palmeri flowers. (A) Species accumulation curve for fungi
in D.wrightii nectar samples (N = 59 isolates). (B) Species accumulation curve for fungi in A.palmeri nectar samples
(N = 16 isolates). (C) Species accumulation curve for bacteria in D.wrightii nectar samples (N = 210 isolates). (D)
Species accumulation curve for bacteria in A.palmeri nectar samples (N = 60 isolates). Figures show the number of
fungi and bacteria species observed (here estimated as OTU) (Mao Tau; black lines), lower and upper 95% confidence
intervals (light gray lines), and bootstrap estimate of richness (dashed lines).
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alter nectar composition and chemistry in the absence of pollinators. We observed that thrips
were common in and on these flowers before anthesis and suspect that they may serve as vec-
tors for movement of microbes among flowers before pollinators can access them [44]. In
future work we suggest quantifying thrips on flowers after anthesis, on bagged flowers, and
under different settings to understand their roles. Measuring visitation rates by pollinators and
other insects to flowers also could inform our results and will be important in future work.
After anthesis, microbes generally increased in abundance in nectar, a pattern observed in
flowers with and without pollinator visitation. In D.wrightii such visitation was associated
with significant changes in fungal community composition, as was the transition from pre- to
post-anthesis (Table 1). Our results are broadly consistent with microbial communities devel-
oping with input from pollinators but also with population growth of microbial populations in
nectar over time. In general bacterial community composition was less sensitive to such fac-
tors, a topic to be explored in further work.
Although we were not able to sample nectar from A.palmeri and D.wrightii in the same
location, samples for these species came primarily from the Tucson area, where both species
occur as ornamentals and in small patches of native vegetation throughout the city. These spe-
cies have overlapping distributions and flower concurrently in the Tucson region [15].
Removal of data from Box Canyon (A.palmeri only; ca. 30 km southeast of Tucson; S1 Table)
did not change our main conclusions. The bats that pollinate A.palmeri occur in the Tucson
basin and cover large distances nightly. For example it has been shown that the foraging radius
of Leptonycteris colonies can be 30–50 km, and these bats frequently move pollen over long-
distances [46]. Therefore, we do not have reason to expect a strong geographic pattern to the
distribution of nectar microbes; however, further sampling to evaluate this pattern is needed.
Similar studies concerning hawkmoth foraging behavior are lacking and knowledge about
flight range in a natural setting is scarce, but M.sexta that forage on A.palmeri and pollinate
D.wrightii are numerous in Tucson and were observed commonly at all of our study sites. In a
laboratory setting it has been shown that M.sexta can cover distances of several kilometers
(5.8 ±2.7 km) within 3 h [47], which indicates that they can act as long-distance pollen
Fig 6. Community analysis of nectar microbe communities for D.wrightii and A.palmeri.Figure shows the results
of non-metric multidimensional scaling based on Jaccard’s index computed with non-singleton OTU only, and
ANOSIM results for (A) bacterial (N = 31 and 47 for A.palmeri and D.wrightii, respectively) and (B) for fungal
communities (N = 8 and 39 for A.palmeri and D.wrightii, respectively).
https://doi.org/10.1371/journal.pone.0225309.g006
Nectar microbes of Sonoran Desert plants
PLOS ONE | https://doi.org/10.1371/journal.pone.0225309 December 12, 2019 11 / 14
dispersers as well and encounter different D.wrightii and A.palmeri populations during forag-
ing bouts.
The Sonoran Desert is known for its iconic mutualisms between plants and pollinators [9,
10]. In this biodiverse region, both flowering plants and their native pollinators are diverse,
frequently endemic, and often threatened by human activity and climate shifts. Our long-term
aim is to integrate a perspective based on microbes to help understand the dynamics of such
mutualisms, their ecological traits, and their evolution, and the cryptic ways in which they are
sensitive to anthropogenic activities at local and regional scales.
Supporting information
S1 Table. Data regarding microbial isolates.
(XLSX)
Acknowledgments
We thank N. Massimo and M. Gunatilaka for laboratory assistance.
Author Contributions
Conceptualization: Martin von Arx, Goggy Davidowitz, A. Elizabeth Arnold.
Data curation: Martin von Arx, Autumn Moore, A. Elizabeth Arnold.
Formal analysis: Autumn Moore, A. Elizabeth Arnold.
Funding acquisition: Goggy Davidowitz, A. Elizabeth Arnold.
Investigation: Martin von Arx, Autumn Moore, Goggy Davidowitz, A. Elizabeth Arnold.
Methodology: Martin von Arx, Autumn Moore, Goggy Davidowitz, A. Elizabeth Arnold.
Project administration: Goggy Davidowitz, A. Elizabeth Arnold.
Supervision: Martin von Arx, Goggy Davidowitz.
Writing – original draft: Martin von Arx, A. Elizabeth Arnold.
Writing – review & editing: Martin von Arx, Autumn Moore, Goggy Davidowitz, A. Eliza-
beth Arnold.
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