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Received: 7 September 2023
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Accepted: 29 January 2024
DOI: 10.1002/ajb2.16303
RESEARCH ARTICLE
Vertically stratified interactions of nectarivores and
nectar‐inhabiting bacteria in a liana flowering across
forest strata*
Sarina Thiel
1
|Malika Gottstein
2
|Eckhard W. Heymann
3
|Jana Kroszewski
1
|
Narges Lieker
1
|Ney Shahuano Tello
4
|Marco Tschapka
5,6
|Robert R. Junker
7
|
Katrin Heer
2
1
Department of Biology, Conservation Ecology,
Philipps‐Universität Marburg, Karl‐von‐Frisch‐
Str, 8, Marburg, Germany
2
Eva Mayr‐Stihl Professorship for Forest Genetics,
Albert‐Ludwigs‐Universität Freiburg, Bertoldstr.
17, Freiburg, Germany
3
Verhaltensökologie & Soziobiologie, Deutsches
Primatenzentrum –Leibniz‐Institut für
Primatenforschung, Kellnerweg 4, Göttingen,
Germany
4
Estación Biológica Quebrada Blanco, Loreto,
Río Tahuayo, Peru
5
Institute of Evolutionary Ecology and
Conservation Genomics, University of Ulm,
Albert Einstein Allee 11, Ulm, Germany
6
Smithsonian Tropical Research Institute,
Apartado, 0843‐03092, Balboa, Ancon, Republic
of Panama
7
Evolutionary Ecology of Plants, Department of
Biology, Philipps‐Universität Marburg, Karl‐von‐
Frisch‐Str. 8, Marburg, Germany
Correspondence
Sarina Thiel, Department of Biology,
Conservation Ecology, Philipps‐Universität
Marburg, Karl‐von‐Frisch‐Str. 8, Marburg,
Germany.
Email: sarina.thiel@googlemail.com
Katrin Heer, Eva Mayr‐Stihl Professorship for
Forest Genetics, Albert‐Ludwigs‐Universität
Freiburg, Bertoldstr. 17, Freiburg, Germany.
Email: katrin.heer@forgen.uni-freiburg.de
Abstract
Premise: Vertical stratification is a key feature of tropical forests and plant–frugivore
interactions. However, it is unclear whether equally strong patterns of vertical stratification
exist for plant–nectarivore interactions and, if so, which factors drive these patterns.
Further, nectar‐inhabiting bacteria, acting as “hidden players”in plant–nectarivore
interactions, might be vertically stratified, either in response to differences among strata
in microenvironmental conditions or to the nectarivore community serving as vectors.
Methods: We observed visitations by a diverse nectarivore community to the liana
Marcgravia longifolia in a Peruvian rainforest and characterized diversity and community
composition of nectar‐inhabiting bacteria. Unlike most other plants, M. longifolia produces
inflorescences across forest strata, enabling us to study effects of vertical stratification on
plant–nectarivore interactions without confounding effects of plant species and stratum.
Results: Asignificantly higher number of visits were by nectarivorous bats and humming-
birds in the midstory than in the understory and canopy, and the visits were strongly
correlated to flower availability and nectar quantity and quality. Trochiline hummingbirds
foraged across all strata, whereas hermits remained in the lower strata. The Shannon
diversity index for nectar‐inhabiting bacterial communities was highest in the midstory.
Conclusions: Our findings suggest that vertical niche differentiation in
plant–nectarivore interactions seems to be partly driven by resource abundance,
but other factors such as species‐specific preferences of hummingbirds, likely caused
by competition, play an important role. We conclude that vertical stratification is an
important driver of a species’interaction niche highlighting its role for promoting
biodiversity and ecosystem functioning.
KEYWORDS
Bats, hummingbirds, Marcgraviaceae, nectar traits, nectarivores, nectar‐inhabiting bacteria, plant–animal
interactions, rain forest
Vertical stratification is a wide‐spread phenomenon in plant
and animal communities and a key factor for structuring
biodiversity, particularly in tropical forests (Basset et al., 2003;
Chmel et al., 2016;Thieletal.,2021). Plants and animals
occupy a variety of niches along the vertical forest gradient
(Allee et al., 1949; Richards, 1952;Smith,1973;Bongers,2001).
Patterns of vertical stratification for nectarivores and frugi-
vores have been observed in terms of species abundance,
Am J Bot. 2024;111:e16303. wileyonlinelibrary.com/journal/AJB
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https://doi.org/10.1002/ajb2.16303
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited and is not used for commercial purposes.
© 2024 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.
*We dedicate this paper to the memory of our colleague and friend Dr. Stefan Dressler, an international leading expert for the Marcgraviaceae, who died on 14 September 2023.
richness, and community composition (Buchanan‐Smith
et al., 2000; Kalko and Handley, 2001;Chmeletal.,2016;
Thiel et al., 2021). Thus, this vertical structure also affects
associated ecological processes such as pollination and seed
dispersal (Howe and Smallwood, 1982;Fleming,1993;
Jordano, 2000; Fleming and Kress, 2013).
In tropical rainforests, vertebrates play crucial roles as
pollinators and seed dispersers (Fleming and Kress, 2013). For
instance, nectarivorous bats and hummingbirds are among the
key pollinators whereas frugivorous bats, birds, and primates are
key seed dispersers (Fleming and Muchhala, 2008; Lobova
et al., 2009). However, studies focusing on the actual
interactions among taxa across the vertical gradient are scarce.
For plant–frugivore interactions, for instance, it was shown that
networks differ profoundly among strata in terms of mutual
specialization and interaction frequency and community
composition of frugivores (Shanahan and Compton, 2001;
Schleuning et al., 2011; Thiel et al., 2023). Few studies have
examined the vertical stratification of interactions among plants
and nectarivores although there are known differences in strata
use among taxa. In tropical lowland forests, hermit humming-
birds (subfamily Phaethornithinae) are primarily understory
foragers, whereas trochiline hummingbirds (subfamily Trochi-
linae) forage in the canopy and in the understory (Fleming and
Kress, 2013). This separation in vertical space (Feinsinger and
Colwell, 1978;Stiles,1981;Flemingetal.,2005) might have been
driven by the high level of interspecific competition among
hummingbirds in tropical lowland forests and the subsequent
specialization on differing floral resources (Maglianesi
et al., 2015). For nectarivorous phyllostomid bats, such a clear
distinction between understory‐and canopy‐feeding bats does
not seem to exist. Some studies indicate that bats prefer to feed
on flowers higher up due to better accessibility and conspicuity
(Diniz et al., 2019; Kobayashi et al., 2020), whereas others report
bats feeding on plants flowering in the understory (Czenze and
Thurley, 2021;Amorimetal.,2023). Generally, it seems that
most neotropical, nectarivorous bats are rather flexible and use
all levels of the forest for foraging (Kalko and Handley, 2001;
Fleming et al., 2005).
Plant–nectarivore interactions are determined by a series of
ecological factors and evolutionary processes (Carnicer
et al., 2009). One crucial ecological factor is resource availability
(González‐Castro et al., 2012). Hummingbirds and nectarivor-
ous bats are highly dependent on nectar resources with a high
sugar content (Stiles, 1981; Temeles et al., 2005;Suarezand
Welch, 2017), and competition for suitable nectar resources
plays an important role in structuring their community
organization (Feinsinger and Colwell, 1978; Kalko and
Handley, 2001). Nectar components such as sugars, amino
acids, lipids, and other nutrients (Carter et al., 2006), influence
the attractiveness of nectar for nectarivores. However, the
relationship between nectar and pollinators is far more complex
than originally assumed. For instance, by adjusting nectar
attributes and nectar secretion of already pollinated flowers,
plants manipulate pollinators to rather visit unpollinated flowers
with high pollen availability (Pyke et al., 2020;Domingo‐Mellos
et al., 2023). Moreover, nectar harbors an abundant and diverse
microbial community. These microorganisms might be impor-
tanthiddenplayersinplant–nectarivore interactions as their
metabolism can profoundly alter nectar chemistry and thus,
influence nectar consumption by nectarivores (Vannette
et al., 2013). So far, most studies on the nectar microbiome
concentrated on nectar‐inhabiting fungi and yeast (Lachance
et al., 2001a;Brysch‐Herzberg, 2004; Herrera et al., 2008;de
Vega et al., 2009;Pozoetal.,2009,2011), even though another
group of microbes, bacteria, are also frequently found in nectar
with a high abundance and diversity (Álvarez‐Pérez et al., 2012;
Fridman et al., 2012; Junker and Keller, 2015;Lievensetal.,2015;
Vannette, 2020; Gaube et al., 2021). Bacterial metabolism results
in a decline in total sugar concentration (Herrera et al., 2008;de
Vega et al., 2009), changes the sugar and amino acid
composition (Herrera et al., 2008; Canto and Herrera, 2012),
and increases nectar temperature (Herrera and Pozo, 2010).
Further, they emit volatiles that can modify flower and nectar
scent (Golonka et al., 2014;Pozoetal.,2014;Helletsgruber
et al., 2017;Reringetal.,2018). As these nectar traits are key
mediators of interactions between plants and nectarivores, such
changes in physiochemical properties of the floral nectar can
alter the attractiveness of a given flower to pollinators (Herrera
et al., 2008,2009; Herrera and Pozo, 2010; Vannette et al., 2013;
Junker et al., 2014;Lievensetal.,2015;Stevensonetal.,2017).
These bacterial effectsmayevenbestrongerthanthosebyyeast
(Vannette et al., 2013). At the same time, nectarivores
themselves are important vectors for the dispersal of nectar‐
inhabiting bacteria among plants (Sandhu and Waraich, 1985;
de Vega et al., 2009; Vannette et al., 2013). Thus, nectarivores
and microenvironmental conditions such as temperature,
rainfall, and vegetation density influence the structure and
diversity of nectar‐inhabiting bacteria communities, which in
turn affect nectar characteristics (Samuni‐Blank et al., 2014;
Sharaby et al., 2020;Bogoetal.,2021). If nectarivores and
microenvironmental conditions are vertically stratified
(Shaw, 2004; Fleming and Kress, 2013), it is plausible that
there is vertical stratification of nectar‐inhabiting bacterial
communities, which in turn might enhance vertical differences
of plant–nectarivore interactions.
Although there is extensive information about plant–
nectarivore interactions, to our knowledge, all studies investi-
gating the foraging behavior of nectarivores have focused on
their specialization on plant species, which presented flowers
within a single stratum, implying that species and stratum are
partially confounding variables. Yet, some plant species, most of
them cauliflorous, produce flowers and fruits over more than
one stratum. The neotropical liana Marcgravia longifolia
(Marcgraviaceae) is an ideal study organism to investigate the
vertical stratification in plant–animal interactions because it
provides inflorescences and infructescences from ground level
to the canopy. We already showed that frugivorous birds
feeding on M. longifolia fruits stayed within their preferred
vertical niche, even though the same resource was available
across strata (Thiel et al., 2023). Based on these results, we
concluded that vertical stratification is driven by inherent
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preferences of avian frugivores for specific strata rather than by
differences in the composition of plant communities. In the
frugivore community, species dependence on fruit in their diet
and species‐specifictraitssuchasbodysizeandwingshapein
combination with microenvironmental characteristics such as
vegetation density and canopy cover played an important role
(Thiel et al., 2023).Hereweusedthesamesystemtotest
whether nectarivores also prefer a certain vertical niche for
foraging or whether an attractive resource that is available across
strata drives nectarivorous hummingbirds and bats toward
foraging across the whole vertical gradient. We further
investigated whether the nectar‐inhabiting bacterial communi-
ties of M. longifolia have any vertical stratification.
So far, the few available studies did not unequivocally test
whether the use of vertical space by nectarivores is driven by
differential resource availability or is due to species‐specific
preferences for a certain vertical niche. Our unique study system
will allow us to test these possibilities in the presence of the
same resources along the entire vertical gradient. Specifically, for
the diurnal and nocturnal nectarivores, we consider two
contrasting hypotheses (here: H1 and H2) as relevant.
According to H1 nectarivores preferentially forage in a distinct
vertical niche based on species‐specificpreferences.Ifsucha
preference exists, the number of visits, species diversity, and
community composition are expected to differ among strata in
our study system. In contrast, according to H2, resource
quantity and quality are decisive for determining the vertical
foraging niche. In that case, we would expect that nectarivores
forage across strata with an increasing number of visits with
increasing inflorescence abundance, nectar quantity, and sugar
concentration. Because hummingbirds have been shown to use
differing foraging strategies to reduce interspecificcompetition
and because we also found vertical patterns for frugivorous
birds, we assumed that for hummingbirds both H1 and H2
provide realistic scenarios. For nectarivorous bats, on the other
hand,weexpectH2tobemorelikelyasbatshavebeenshown
to be more flexible and to use all vertical strata for foraging.
Finally, vertically stratified foraging behavior of nectarivores,
microclimatic conditions, and nectar properties influence
communities of nectar‐inhabiting bacteria. Thus, we hypothe-
sized that their diversity and composition is also vertically
stratified (H3). In our study system, we expected that
microenvironmental differences among strata are a strong
determinant of the vertical stratification of bacteria communi-
ties, which might either be fortified or homogenized depending
on how frequently nectarivores forage across strata. Since we
assumed that nectar properties only differ negligibly among
strata, they should not have an influence in our specificcase.All
hypotheses are tested against H0 of no differences among strata.
To test our hypotheses and expectations, we investigated
flower quality and nectarivore‐foraging behavior across the
vertical gradient by collecting microenvironmental data,
sampling nectar from M. longifolia individuals, and analysing
nectar quantity and sugar concentration across strata and
during anthesis and the 24 h‐cycle. We further recorded visits
of hummingbirds and nectarivorous bats and diversity and
community composition of the nectar‐inhabiting bacteria in
M. longifolia individuals across forest strata (understory,
midstory, canopy).
MATERIALS AND METHODS
Study site
The study was conducted at the Estación Biológica Quebrada
Blanco in northeastern Peruvian Amazonia (4°21′S 73°09′W;
EBQB, Loreto, Peru) on high ground terra firme rainforest
(“bosque de altura”; following Encarnación [1985]), inter-
spersed with swampy areas. Annual precipitation is ca.
3000 mm, with December–May the wettest months and
July–August the driest (Lüffeetal.,2018). Mean monthly
temperatures in the area range between 25° and 27°C
(Klingbeil and Willig, 2008). Further details of the study site
have been described by Heymann et al. (2021) and Heymann
and Tirado Herrera (2021).
Plant species
Marcgravia longifolia (Marcgraviaceae) is a woody liana
species only known from western Amazonia (Tropicos, 2020).
It produces long pedunculate and flagelliflorous inflores-
cences arising from the unbranched stem of the liana all the
way from ground level to the canopy, which is an extremely
rare phenomenon within the Marcgraviaceae and for plants
in general (Appendices S1,S2).
Data collection
Selection of Marcgravia longifolia individuals
We selected 29 of the 100 known M. longifolia individuals at the
study site for further observations of nectarivores and for nectar
sampling based on resource availability (sufficient number of
inflorescences) and visibility, and accessibility of the host tree
with climbing equipment. Data on flowering M. longifolia
individuals were collected in October 2017, from August to
September 2018, and from July to September 2019.
Classification of height and microenvironmental
variables
We determined host tree height for each selected individual and
additionally characterized canopy cover along the vertical
gradient by collecting diagonal hemispherical color photographs
to assess canopy cover. Additionally, we installed data loggers in
11 M. longifolia individuals in different heights to collect data on
temperature and light intensity (Appendix S1). We classified
three forest strata (understory, midstory, and canopy) in relation
to the vertical distribution of foliage and density of the
surrounding vegetation. We found that vegetation density was
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clearly distinct among these strata, and we assume that
nectarivores rather orient themselves along the vegetation
structure than based on absolute height (McCaig et al., 2020).
The understory was classified according to the height of the
dense surrounding shrub and palm tree layer (0 m until
between 3 m and 10 m). For the classification of the canopy
stratum, the height of the first canopy branch of the host tree
and the height of the surrounding canopies was decisive (from
between9mand22mtobetween12mand32m).The
midstory was then defined as the space between the understory
and the canopy, where vegetation density was lower (from
between 3 m and 10 m to between 9 m and 22 m; Appendix S3).
Nectar sampling across strata
To characterize diversity and community composition
of bacteria communities, we collected nectar from one
inflorescence per stratum from five plants of M. long-
ifolia in 2018. For each individual, samples were
collected on the same day. We selected inflorescences
in which some flowers had already shed their calyptra to
ensure that the inflorescences were anthetic and had
already produced nectar (Appendix S1).
To assess differences in nectar production, sugar concen-
tration, and pH values among strata, we collected nectar from
one inflorescence per stratum from seven M. longifolia
individuals in 2019. We selected inflorescences within anthesis.
In each M. longifolia individual, samples were collected on the
samedayandalwaysinthelatemorningorearlyafternoon,
respectively, when differences among strata in terms of
temperature and light intensity were greatest (see Results,
Microenvironmental variables). We measured nectar volume,
sugar concentrations, and pH. We then calculated mean nectar
volume as the sum of nectar from all nectaries of one
inflorescence divided by the number of nectaries. Mean pH and
mean sugar concentration were calculated as the mean of the
values for all sampled nectaries from one inflorescence. Lastly,
mean sugar quantity was calculated by multiplying mean nectar
quantity and mean sugar concentration (Appendix S1).
Nectar sampling during anthesis and over 24 h
In 2019, we selected a total of 13 inflorescences from 10 M.
longifolia individuals, which were accessible from the
ground, to examine nectar volume, sugar concentration,
and pH during anthesis. Nectar measurements started with
the onset of flowering until nectaries were shed (due to time
constraints, some measurements had to be stopped earlier).
Measurements were always done at the same time of day.
To determine whether nectar quantity and sugar concen-
tration show a variation over the 24‐h cycle, we selected a total
of nine inflorescences from six M. longifolia individuals at
ground level, which were approximately at the same stage of
anthesis. Measurements were made at 2‐hintervalsfor24h.For
each inflorescence, we determined the sampling height, the
number of nectaries, the total number of flowers, and the
number of flowers that had already shed their calyptra
(Appendix S1).
Identity and visitation rate of diurnal nectar
consumers
To record nectarivorous species feeding on M. longifolia
during the day, four researchers equipped with binoculars
(10 × 42 mm) recorded animal visits to 26 flowering M.
longifolia individuals in the morning (06:00 to 11:00 hours)
and in the afternoon (12:00 to 17:00 hours; Appendix S4).
The number of visits by each hummingbird species was
calculated per stratum fireachM. longifolia individual and
summed across years.
Furthermore, by selecting one to two nectar‐producing
inflorescences per stratum (indicated by hummingbird
visits), for all 26 M. longifolia individuals, and counting
the number of visits at those inflorescences, we determined
the visitation rate of diurnal nectarivores in a way that is
comparable to the nocturnal visitation rate derived from the
wildlife cameras (see below; we did not use the wildlife
cameras to monitor diurnal visitation because humming-
birds did not trigger them reliably, probably due to the
insulating characteristic of the hummingbirds’plumage and
the small difference between hummingbird body and
ambient temperature). For all 26 M. longifolia individuals,
the number of all inflorescences irrespective of their
phenology state was estimated for each height class of
4 m, on each observation day, and then assigned to strata.
We calculated mean number of inflorescences per stratum
of each M. longifolia individual across observation days. To
estimate the availability of alternative flower resources, close
to the observed M. longifolia individuals, we searched for
other plant species bearing flowers in a radius of 15 m
around the M. longifolia individuals (Appendix S1).
Identity and visitation of nocturnal nectarivores
To determine the visitation rate of nocturnal nectarivores,
we fixed automatic wildlife cameras (HyperFire 2TM HF2X;
Reconyx, Holmen, WI, USA; with additional IR‐LED) on
the liana stem close to inflorescences during anthesis at
heights between 0.5 and 23 m of 14 flowering M. longifolia
individuals (Appendix S1). We further monitored the
inflorescences sampled during anthesis with the wildlife
cameras, enabling us to directly observe nocturnal visita-
tions by nectarivores to these inflorescences during anthesis
and to directly correlate visitation to nectar volume and
sugar concentration. We counted each individual animal
that contacted the monitored inflorescences and calculated
the number of visits per M. longifolia individuals and
summed the visits to the individual across years.
To identify the nectarivorous bat species visiting flower-
ing M. longifolia individuals, we set up nocturnal mist nets in
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heights from 0.2 to 7.6 m as close as possible to 10 flowering
M. longifolia individuals and at control sites. We collected
pollen samples from nectarivorous bats using a small block of
fuchsin‐stained gelatine (Appendix S1).
Metabarcoding of bacteria communities in the
nectar
In the lab, the sample tubes containing the nectar were stored at
–20°C until extraction. We extracted microbial DNA from
nectar samples following the protocol of the ZymoBIOMICS
DNA Miniprep Kit (D4300; Zymo Research, Irvine, CA, USA).
ThemicrobiomeforeachsamplewasanalyzedbyEurofins
Genomics (Ebersberg, Germany) using the company's standard
procedure. Sequencing was done using Illumina MiSeq, and the
sequenced regions were V3‐V4 region of the 16 S rRNA gene to
identify bacterial operational taxonomic units (OTUs) using the
standard procedure by the service INVIEW Microbiome
Profiling 3.0 with MiSeq (Eurofins Genomics, Luxembourg)
(for detailed methods, see Junker et al., 2020). Abundances of
bacterial OTUs were normalized using lineage‐specificcopy
numbers of the relevant marker genes to improve estimates
(Angly et al., 2014). The Shannon diversity index (Shannon,
1948) was calculated based on the OTU composition (without
CSS normalization) after rarefying the data to the minimum
number of reads (N= 17,502) available in the samples
(repeats = 999) (Appendix S1).
Network analysis
We built an interaction matrix between hummingbird
species and the three forest strata (understory, midstory,
and canopy), summed across M. longifolia individuals. This
way, we calculated interaction frequencies of each hum-
mingbird species for each stratum (number of visits of
feeding animals within that stratum). Following traditional
network analyses, the interaction matrix was analyzed using
the R package bipartite (Dormann et al., 2009) in R version
4.2.2 (R Core Team, 2019). To build the matrix, we used a
quantitative matrix of interactions between hummingbirds
and strata, in which the nodes represent hummingbird
species or plant strata, respectively, and the links represent
the interaction frequency between them. The total fre-
quency of a hummingbird species was defined as the
number of visits to all strata, whereas the total frequency
from the perspective of a particular stratum was given as the
number of all hummingbird visits to this stratum (Blüthgen
et al., 2007).
To characterize hummingbird‐foraging behavior across
strata, we first assessed the degree of specialization for each
species by calculating Pielou's evenness index for visited strata
per species. Values of 0 indicate specialist species, values of 1
indicate generalist species. Second, we calculated the Shannon
diversity index for hummingbird interactions in each stratum
(Dormann, 2011).
Statistical analyses
Microenvironmental variables, resource quantity
and quality
To examine the factors that might influence the foraging
behavior of nectarivores, we analyzed whether micro-
environmental conditions and resource quantity and quality
differed among strata. First, we compared canopy closure
and number of inflorescences among strata by calculating
linear mixed effect models with canopy closure or number
of inflorescences as response variables, stratum as explana-
tory variable, and IDs of individual lianas as random factors
(Table 1). Residuals of canopy closure and number of
inflorescences were normally distributed. Then, for those
individuals that were sampled for nectar, we compared
nectar attributes among strata by calculating linear mixed
effect models with mean nectar volume, mean sugar
concentration, mean sugar volume, or mean pH value as
response variables; stratum, total number of flowers per
inflorescence, and percentage of open flowers as explanatory
variables; and IDs of individual lianas as random factors
(Table 1). Further, to identify whether nectar production
changed during anthesis, we calculated linear mixed effect
models with mean nectar volume, mean sugar concentra-
tion, or mean pH value as response variables; days of
anthesis, total number of flowers per inflorescence, and
percentage of open flowers as explanatory variables; and IDs
of individual lianas and of inflorescences nested in IDs of
individual lianas as random factors (Table 1). To test for
differences over the 24‐h cycle, we ran a Watson–Williams
test for circular distributions to analyze whether there is a
non‐uniformity in mean nectar quantity and mean sugar
concentration over the 24‐h cycle, pooled across all sampled
M. longifolia individuals.
Foraging behavior of nocturnal and diurnal
nectarivores
To test whether hummingbirds prefer a certain vertical niche
for foraging (Hypothesis 1 (H1)), we first analyzed whether the
degree of specialization differs among hermits and trochilines.
For this purpose, we calculated a linear mixed effect model
with Pilou's evenness index as response variable, hummingbird
subfamily as explanatory variable, and species identity as
random factor (Table 1). Then, we compared the zero‐inflated
count data of hummingbird visitation among strata by
running a generalized linear mixed effect model with number
of hummingbird visits as response variable; the interaction of
stratum, subfamily and number of inflorescences as explana-
tory variables; and IDs of individual lianas and species identity
as random factors. We also added a zero‐inflation term for
stratum, an offset for observation hours (to account for
differing number of hours of observation among M. longifolia
individuals) and fitted the model with a negative binomial
distribution and a logit link (Table 1). For the analyses,
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TABLE 1 Results of the linear and generalized linear mixed effect models examining differences in microenvironmental variables and resource quantity and
quality among strata, days of anthesis, and inflorescence characteristics. Further models tested the effect of stratum, hummingbird subfamily or the interaction of strata
and hummingbird subfamilies/nocturnal and diurnal taxa on hummingbird visitation, Pilou's evenness index, and nocturnal and diurnal visitation rate. The
Marcgravia longifolia individual, hummingbird species identity, or the individual inflorescences, respectively, were included as random factors. Pwas derived using the
glmmTMB() function.
Model Variable χ
2
df P
Microenvironmental variables
Canopy closure ~ Stratum + (1|Marcgravia ID) Stratum 41.82 2 <0.0001
Resource quantity and quality
Number of inflorescences ~ Stratum + (1|
Marcgravia ID)
Stratum 200.39 2 <0.0001
Mean nectar volume ~ Stratum + Percentage open
flowers + Total number of flowers + (1|
Marcgravia ID)
Stratum 0.39 2 0.821
Percentage open flowers 6.098 1 0.014
Total number of flowers 1.376 1 0.241
Mean sugar concentration ~ Stratum + Percentage
open flowers + Total number of flowers + (1|
Marcgravia ID)
Stratum 2.126 2 0.345
Percentage open flowers 29.631 1 <0.0001
Total number of flowers 8.163 1 0.004
Mean sugar volume ~ Stratum + Percentage open
flowers + Total number of flowers (1|
Marcgravia ID)
Stratum 0.444 2 0.801
Percentage open flowers 14.749 1 0.0001
Total number of flowers 1.479 1 0.224
Mean pH value ~ Stratum + Percentage open
flowers + Total number of flowers + (1|
Marcgravia ID)
Stratum 3.787 2 0.151
Percentage open flowers 0.186 1 0.667
Total number of flowers 0.13 1 0.719
Mean nectar volume ~ Day of anthesis + Percentage
open flowers + Total number of flowers + (1|
Marcgravia ID/Inflorescence ID)
Day of anthesis 16.84 2 0.0002
Percentage open flowers 8.19 1 0.004
Total number of flowers 4.485 1 0.027
Mean sugar concentration ~ Day of
anthesis + Percentage open flowers + Total
number of flowers + (1|Marcgravia ID/
Inflorescence ID)
Day of anthesis 58.136 2 <0.0001
Percentage open flowers 15.168 1 <0.0001
Total number of flowers 0.859 1 0.354
Mean pH ~ Day of anthesis + Percentage open
flowers + Total number of flowers + (1|Marcgravia
ID/Inflorescence ID)
Day of anthesis 2.785 2 0.248
Percentage open flowers 0.127 1 0.721
Total number of flowers 1.013 1 0.314
Hummingbird visitation
Number of hummingbird
visits ~ Stratum × Subfamily + Number of
inflorescences + (1| Species) + (1|Marcgravia
ID) + offset = (Hours), zi = ~ Stratum,
family = nbinom2
Stratum × Subfamily 8.102 2 0.017
Stratum 6.201 2 0.045
Subfamily 0.134 1 0.714
Number of inflorescences 0.378 1 0.539
Pilou's evenness index
Pilou's evenness index ~ Subfamily + (1| Species) Subfamily 5.97 1 0.015
Nocturnal and diurnal visitation rate
Number of visits ~ Stratum × Taxon + Number of
inflorescences + (1|Marcgravia ID) + offset
(Hours), zi = Stratum, family = Poisson
Stratum * Taxon 120.06 6 <0.0001
Stratum 918.46 2 <0.0001
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number of inflorescences and observation hours were log‐
transformed to approximate normal distribution of residuals.
In all models, we used a contrast to compare the estimated
marginal means of the response variables among strata or
among strata and differing hummingbird subfamilies
(Table 2). To quantify the differences in the hummingbird
community among forest strata, we computed pairwise
Bray–Curtis distances among strata and analyzed differences
in community composition among strata using a MANOVA
approach on our interaction matrix. We tested significance by
permuting the raw data (1000 permutations) using the
function adonis() in R package vegan 2.5‐6(Oksanen
et al., 2019).
Totestwhetherresourcequantityandqualityaredecisive
for determining the vertical foraging niche (H2), we compared
the visitation rate of nocturnal and diurnal nectarivores among
strata by calculating a generalized linear mixed effect model
with the number of visits per taxon as response variable, the
interaction of stratum and taxon (bat, bird, marsupial, or
moth) and the number of inflorescences as explanatory
variables, and IDs of individual lianas as random factors. We
also added a zero‐inflation term for stratum, an offset for
observation hours, and fitted the model with a Poisson
distribution and a logit link (Table 1). For the analyses,
number of inflorescences was log‐transformed to approximate
normal distribution of residuals. Further, the visitation rate of
nocturnal nectarivores during the anthesis was compared by
calculating a generalized linear mixed effect model with
number of visits as response variable; day of anthesis, mean
nectar quantity, and mean sugar concentration as explanatory
variables; and IDs of individual lianas and of inflorescences
nested in IDs of individual lianas as random factors. We also
added an offset for observation hours and fitted the model
with a Poisson distribution and a logit link (Table 1). In all
models, a contrast was used to compare among strata or days
of anthesis (Table 2; Appendix S5).
Diversity and community composition of nectar‐
inhabiting bacteria
To test whether the diversity and community composition of
nectar‐inhabiting bacteria differ among strata (H3), we first
performed a nonmetric multidimensional scaling (NMDS)
based on the Bray–Curtis distances between nectar samples
using the function vegdist() in R package vegan 2.5‐7
(Oksanen et al., 2019) to compare the community composition
among strata. Before the NMDS, we performed a cumulative
sum scaling (CSS) normalization in R package metagenome-
Seq. 1.28.2 (Paulson et al., 2013) on the read count data to
account for differences in sequencing depth among samples.
On top of ordination, we fitted microenvironmental vectors
and factors (plant individual, height, nectar volume, number of
flowers during anthesis, total number of flowers, and
proportion of flowering plants) to test for their effects on
bacterial composition using the function envfit() in R package
vegan 2.5‐7(Oksanenetal.,2019). Next, we compared the
diversity of nectar‐inhabiting bacteria among strata. To
account for different numbers of sequencing reads per samples
we first rarefied (999 repeats) the unscaled data set (raw
number of reads) to the minimum number of reads available
in the samples (N = 17,502) and then calculated the Shannon
diversity index for each sample based on the OTU assignment
of each read using the R package rtk 0.2.6.1 (Saary et al., 2017).
Diversity was correlated to height using a Spearman correla-
tion analysis. Visual inspection of the relationship between
height and bacterial diversity suggested a nonmonotonic
relationship with highest diversity values in the midstory.
Accordingly, we used the function qad() in R package qad
1.0.0 (Junker et al., 2021;Griessenbergeretal.,2022)totestfor
a nonmonotonic relationship. The package qad estimates
scale‐invariant‐directed and asymmetric dependence of
bivariate distributions with no underlying assumptions on
the distribution of the data and the function type.
Linear and generalized linear mixed models were calculated
using the function glmmTMB() in R package glmmTMB 1.0.2.1
(Brooks et al., 2020). We used the function Anova() in R
package car3.0‐10 (Fox and Weisberg, 2019)forWaldχ
2
tests
and determined contrast comparisons with the function
emmeans() in R package emmeans 1.4.7 (Lenth et al., 2020).
RESULTS
Microenvironmental variables
Microenvironmental conditions differed among strata. Canopy
closure significantly decreased from the understory to the
canopy (Tables 1and 2; Appendix S5). Even though we could
not compare temperature and light intensity measurements
TABLE 1 (Continued)
Model Variable χ
2
df P
Taxon 4302.97 3 <0.0001
Number of inflorescences 15.789 1 <0.0001
Number of visits ~ Day of anthesis + mean nectar
quantity + mean sugar concentration + (1|
Marcgravia ID/Inflorescence ID) + offset (Hours),
family = Poisson
Day of anthesis 1318.58 2 <0.0001
Mean nectar quantity 28.81 1 0.784
Mean sugar concentration 0.08 1 <0.0001
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TABLE 2 Standardized estimates, standard errors (SE), and Pvalues for all models derived from the emmeans() function. These contrast comparisons
were conducted for the glmmTMB() models in Table 1examining differences among strata, among strata and hummingbird subfamilies/taxa, or among strata
and inflorescence characteristics, respectively, on microenvironmental variables, day of anthesis, hummingbird visitation, or nocturnal and diurnal visitation.
The Marcgravia longifolia individual, the individual inflorescences, or hummingbird species identity, respectively, were included as random factors.
Response variable Contrast Standardized estimates SE P
Microenvironmental variables
Canopy closure High –Low –5.01 0.81506 <0.0001
High –Middle –2.15 0.808 0.009
Low –Middle 2.87 0.679 0.0001
Number of inflorescences High –Low –56.2 4.04 <0.0001
High –Middle –29.1 4.04 <0.0001
Low –Middle 27.1 3.33 <0.0001
Day of anthesis
Nectar quantity Before –After –0.005 0.017 0.774
During –After 0.029 0.011 0.016
Before –During –0.033 0.012 0.016
Sugar concentration Before –After –3.525 0.85 0.0002
During –After 0.873 0.536 0.109
Before –During –4.399 0.607 <0.0001
Hummingbird visitation
Hermits Low –High 7.175 4.211 0.012
Middle –High 3.36 1.945 0.146
Low –Middle 2.135 0.624 0.071
Trochilines Low –High 1.355 0.615 0.755
Middle –High 2.274 0.555 <0.0001
Low ‐Middle 1.064 0.221 0.834
Nocturnal and diurnal visitation
Stratum Low –High 1.896 0.177 <0.0001
Middle –High 2.299 0.212 <0.0001
Low –Middle 0.825 0.056 0.004
Bird Low –High 2.688 0.399 <0.0001
Middle –High 2.274 0.339 <0.0001
Low –Middle 1.182 0.07 0.007
Bat Low –High 2.638 0.146 <0.0001
Middle –High 4.051 0.203 <0.0001
Low –Middle 0.65 0.017 <0.0001
Moth Low –High 1.799 0.438 0.018
Middle –High 1.812 0.45 0.019
Low –Middle 0.993 0.178 0.968
Marsupial Low –High 1.014 0.213 0.964
Middle –High 1.673 0.355 0.018
Low –Middle 0.606 0.113 0.009
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statisticallyduetoinsufficient data, for many individuals, the
temperature and light intensity were, as expected, greater in
the canopy than in the lower strata (Appendix S6). Flowering
individuals from five plant species grew in the vicinity of the
observed M. longifolia individuals (Appendix S7). However, in
comparison to M. longifolia,theyproducedfewflowers and
were thus not further considered in our analysis (N= seven
individuals with <50 flowers, N=1 with >50 flowers). All
individuals produced their flowers high in the canopy.
Resource quantity and quality
Resource quantity differed among strata with more inflor-
escences in the under‐, and midstory than in the canopy
(Tables 1and 2,Figure1A;AppendixS5). Generally, nectar
production by M. longifolia ranged from 0.15 to 0.65 mL
(mean 0.475 ± 0.028 mL) of nectar per inflorescence with a pH
between 4 and 9. Mean nectar volume, mean sugar
concentration, and mean pH did not significantly differ
among strata (Table 1,Figure1; Appendix S5). For mean sugar
volume, we found a nonsignificant trend with a slightly higher
mean sugar volume in the canopy than in the under‐and
midstory (Table 1,Figure1; Appendix S5). Nectar production
varied temporally with higher mean volume and sugar
concentration during anthesis compared to before or after
anthesis (Tables 1and 2,Figure2A, B; Appendix S5). The pH
remained relatively constant over time (Table 1;AppendixS5).
We also found a circadian variation; mean nectar volume
(Watson–Williams test: F = 13.35, df = 8, P < 0.0001) and
mean sugar concentration (Watson–Williams test: F = 6.02,
df = 8, P < 0.0001) was highest during the night (nectar mean:
0.043 ± 0.004; sugar mean: 8.26 ± 0.48) and early morning
(nectar mean: 0.042 ± 0.004; sugar mean: 8.42 ± 0.37;
Figure 2C, D). In all models, mean nectar volume and mean
sugar concentration significantly increased with the percentage
of open flowers (Table 1; Appendix S5).
Identity of nocturnal and diurnal nectarivores
In total, during focal observations when all inflorescences of
M. longifolia visited by hummingbirds were considered, we
observed 1146 interactions between 26 flowering individuals
and 12 hummingbird species (three hermit and nine
trochiline species, Figure 3; Appendix S8). The most frequent
hummingbird species on flowers were the needle‐billed
hermit (Phaethornis philippii, N = 344 observations), Gould's
jewelfront (Heliodoxa aurescens, N = 284), and the fork‐tailed
woodnymph (Thalurania furcata, N = 276). During the
observations of visitations to the focal inflorescences per
stratum, we observed 1233 visits by hummingbirds.
The wildlife cameras documented 7946 visits by
nectarivorous bats (96.3% of the recorded visits; 2.7 visits
per observation hour), 155 visits by marsupials (1.9%; 0.05
visits per observation hour), and 152 visits by moths (1.8%;
0.05 visits per observation hour) to flowering M. longifolia
individuals. All marsupials that could be identified from
wildlife camera photos belonged to the genera Marmosa and
Micoureus (Didelphidae), and the only identifiable moth
species (40% of observed moth individuals) was Feigeria
scops (Erebidae).
In front of 10 flowering M. longifolia individuals, we
captured 89 bat individuals belonging to 16 species all from
the family Phyllostomidae. Of these, 66 individuals were
nectarivores (74.2%), 20 frugivores (22.5%), and three
animalivores (3.4%; Appendix S7). Among the nectarivor-
ous species, Thomas's nectar bat was the most abundant
species (Hsunycteris thomasi, N = 53 captures), followed by
Choeroniscus minor (N = 8), and the tailed tailless bat
(Anoura caudifer, N = 5). Of 37 pollen samples collected
from H. thomasi, 27 samples contained pollen from
M. longifolia. All three of the pollen samples from C. minor
and two of three from A. caudifer contained M. longifolia
pollen. In contrast, the percentage of nectarivores was much
lower at control sites, where we caught 38 bat individuals, of
TABLE 2 (Continued)
Response variable Contrast Standardized estimates SE P
Taxa Bat –Bird 4.44 0.259 <0.0001
Bat –Marsupial 19.17 1.604 <0.0001
Bat –Moth 28.35 2.683 <0.0001
Bird –Marsupial 4.31 0.431 <0.0001
Bird –Moth 6.38 0.687 <0.0001
Marsupial –Moth 1.48 0.182 0.002
Day of anthesis After –Before –21.97 0.311 <0.0001
After –During –22.98 0.196 <0.0001
Before –During –1.01 0.244 <0.0001
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which 23 were frugivores (60.5%), nine were animalivores
(23.7%), and six were nectarivores (15.8%; Appendix S9).
Foraging behavior of nocturnal and diurnal
nectarivores
In line with H1 (nectarivores prefer a certain vertical niche
for foraging), hummingbird visitation differed among strata
(Table 1, Figure 4; Appendix S5). While hermits foraged with
significantly fewer visits to the canopy, trochilines foraged
with equal frequency across strata (Tables 1and 2, Figure 4A;
Appendix S5). The differing visitation of hermits and
trochilines among strata was not correlated with number of
inflorescences (Table 1). Also, the Pilou's evenness index
differed among hermits and trochilines and indicated greater
specialization for a specific stratum for hermits than for
trochilines (Table 1,Figure4B;AppendixS5). In contradic-
tion to H1, neither the Shannon diversity index (understory:
1.5, midstory: 1.63, canopy: 1.43), nor the hummingbird
community composition differed among strata (adonis:
r
2
=0.033, F= 0.95, P=0.53; Figure 3).
In line with H2 (resource volume and quality are decisive
for determining the vertical foraging niche of nectarivores), the
visitation rate of nocturnal and diurnal nectarivores among
strata was positively correlated with inflorescence abundance
(Tables 1and 2,Figure5A;AppendixS5), which were both
higher in the understory than in the canopy. Similarly,
visitation rate was higher during anthesis than before or after
anthesis. Visitation further increased with sugar concentration
(Tables 1and 2,Figure5C, D; Appendix S5). Bats had the
highest visitation rates among all taxa in all strata but foraged
more often in the midstory than in the understory and canopy.
Birds, on the other hand, foraged less frequently in the canopy
(Tables 1and 2,Figure5B;AppendixS5).
Diversity and community composition of nectar‐
inhabiting bacteria
In contradiction to H3, the bacterial community composition
neither differed among strata nor was it influenced by any
other of the tested explanatory variables (Figure 6A). The plant
individual,itsheight,oranyflower or inflorescence character-
istics (nectar volume, number of flowers during anthesis, total
number of flowers, and proportion of flowering plants) did not
affect the composition of bacteria in flower nectar (fitting of
vectors or factor onto an ordination: r
2
≤0.218, P≥0.899). We
did not find a monotonic relationship between height and
bacterial Shannon diversity index (Spearman's correlation
coefficient: ρ=–0.248, P= 0.254), but, in line with H3, a
unimodal distribution of bacterial Shannon diversity along the
height gradient with highest diversity in mid‐elevation
(q(height, diversity) = 0.420, P= 0.011, Figure 6B).
FIGURE 1 Resource abundance and nectar attributes for flowers of Marcgravia longifolia across strata. (A) Number of inflorescences, (B) mean nectar
volume, (C) mean sugar concentration (% Brix), (D) mean sugar volume, and (E) mean pH of nectar for flowers in understory (Low), midstory (Middle),
and canopy (High). Different letters indicate significant differences among strata. Shown are the predicted means with their 95% confidence intervals derived
from the linear mixed effect models. Dots are individual raw data for (A) M. longifolia individuals or (B–E) inflorescences.
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DISCUSSION
By offering flowers across a vertical gradient, M. longifolia
attracted a broad spectrum of different nectarivorous species.
Higher visitation of nectarivores in the under‐and midstory
were highly correlated with inflorescence abundance. How-
ever, the differing inflorescence abundance among strata was
not the only factor influencing the differences in the
interaction frequency of hummingbirds among strata. Even
though the hummingbird community composition did not
differ among strata, the differences between subfamilies were
significant, with hermits mainly foraging in the lower strata,
whereas trochilines foraged across the whole vertical gradient.
Further, the diversity of nectar‐inhabiting bacteria community
significantly differed among strata with the highest Shannon
diversity index in the midstory. Our results illustrate the
importance of differentiating among forest strata when
analyzing plant–nectarivore interactions and highlight that
more than one determinant drives their vertical stratification.
Our findings indicate that, unlike frugivores that have
strong preferences for a certain vertical foraging niche
(Thiel et al., 2023), nectarivores are more flexible in their
use of vertical strata. Resource quantity and quality indeed
were decisive for influencing the vertical foraging niche.
They have a stronger influence on the foraging behavior of
nectarivores than on frugivores, and the presence of an
attractive resource across the whole vertical gradient drives
them to forage across strata. Resource availability is
considered as one of the most important drivers of network
structure, especially in communities of nectarivorous bats
and hummingbirds (Vázquez, et al., 2009a,2009b). These
hummingbirds derive around 90% of their dietary require-
ments from floral nectar (Gass and Montgomerie, 1981)
and, due to their extremely high metabolic rates imposed by
small size and hovering flight (Suarez and Welch, 2017),
need to visit hundreds of flowers per day (Hurly, 1996).
Thus, for the small nectarivores, optimal foraging plays a
crucial role; for instance, they prefer to forage where many
rewarding flowers are spatially close, and they tend to
proceed to the nearest, largest flower (Pyke, 1981). The
presence of a nearby attractive resource likely drives
nectarivorous species to leave their preferred vertical
foraging niche and to forage across all vertical strata. Most
observed hummingbird species visited M. longifolia flowers
FIGURE 2 Nectar attributes for flowers of Marcgravia longifolia over time. (A) Mean nectar volume. (B) Mean sugar concentration during anthesis. (C)
Relative nectar volume and (D) relative sugar concentration over 24 h. In A and B, different letters indicate significant differences among strata. Shown are
the predicted means with 95% confidence intervals derived from the linear mixed effect models. Dots are individual raw data points for inflorescences. In C
and D, black arrows represent the mean angle; bars represent the relative intensity of mean nectar production or mean sugar concentration for each time.
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FIGURE 3 Bipartite diagram depicting the interaction matrix of hummingbirds with Marcgravia longifolia in a tropical forest in northeastern Peruvian
Amazonia.Intotal,weobservedninetrochilineandthreehermithummingbirdspeciesvisitinginflorescences in the three strata of M. longifolia.Thethickness
of the grey lines connecting hummingbird species and strata corresponds to the interaction frequency with which hummingbirds fed in the respective stratum.
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across strata. Even species previously described as typical
understory foragers, such as H. aurescens, P. philippii, and
P. superciliosus (Schulenberg et al., 2010), occasionally
moved up to forage in the canopy. Nevertheless, humming-
bird visitation decreased toward the canopy, most likely due
to the lower resource abundance. Although nectar volume
and sugar concentration did not differ among strata,
the abundance of inflorescences was significantly lower in
the canopy. Further, we assume that flying up and down the
vertical gradient comes with a high energy cost for small
FIGURE 4 Foraging behavior of hermit and trochiline hummingbirds across strata. (A) Hummingbird visitation, (B) Pilou's evenness index of hermits
and trochilines among strata. Different letters indicate significant differences among strata. Shown are the predicted means with their 95% confidence
intervals derived from the linear, or generalized linear mixed effect models, respectively. Dots are individual raw data points for (A) M. longifolia individuals
or (B) hummingbird species, respectively.
FIGURE 5 Visitation to flowers of Marcgravia longifolia by nectarivores. (A) Total number of visitations, (B) number of visitations by different
nocturnal and diurnal species (“focal visitation”) shown for the understory (Low), midstory (Middle), and canopy (High), (C) nocturnal visitation rate
shown for before, during, and after anthesis, and (D) nocturnal visitation rate in relation to sugar concentration. Different letters (a–g) indicate significant
differences among strata. Shown are the predicted means with 95% confidence intervals derived from the linear or generalized linear mixed effect models.
Dots are raw data points for M. longifolia individuals in A and B and for inflorescences in C and D. The scatterplot shows fitted data (circles), effect size
(solid line) and 95% confidence intervals (dashed lines) of the linear mixed effects model.
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hummingbirds. Moreover, the microenvironmental condi-
tions in the canopy (Fetcher et al., 1985; Shaw, 2004) are
considered less favorable for their energy budget (Shankar
et al., 2019). In our system, the maximum temperature and
light intensity during the day were higher in the canopy
than at lower strata, whereas canopy cover was significantly
lower. The more extreme microenvironmental conditions in
the canopy did not seem to influence nectar attributes
across strata because nectar quantity and quality did not
significantly differ among strata, even though they were
collected when conditions were more extreme. Further,
because hummingbirds have very good spatial memory
capabilities (Hurly, 1996), they likely remember the location
of rewarding flowers and that the low abundance of
inflorescences in the canopy renders it a non‐or less‐
rewarding foraging location. The visitation of nectarivorous
bats was higher than that of hummingbirds across all strata.
This higher bat visitation across strata is crucial for
M. longifolia; nocturnal nectarivores are their main
pollinators, whereas hummingbirds act only as nectar
thieves. According to previous studies (Kalko and
Handley, 2001; Fleming et al., 2005), we assume that the
three identified nectarivorous bat species H. thomasi, C.
minor, and A. caudifer also foraged across all strata of M.
longifolia. Generally, bats are rather flexible and frequently
use all forest strata (Kalko and Handley, 2001). In our
system, their visitation was highest in the midstory and thus
not as concurring with inflorescence abundance as the
visitation of hummingbirds. Nectar production of M.
longifolia has a circadian rhythm, and the higher nectar
volume and sugar concentration during the night rendered
bats less dependent on the mere number of inflorescences.
Even though temporal data on nectar could only be
collected for inflorescences in the understory, we assume
this data to be representative because nectar attributes did
not even differ during the day when microenvironmental
differences among strata were highest. Furthermore, it
might be easier for bats to approach inflorescences in the
midstory where the surrounding vegetation is less dense
(Kalko and Handley, 2001; Diniz et al., 2019). Even in dry
forests where the vegetation is primarily low shrubs, bats
prefer the higher layers of the vegetation, whereas
hummingbirds are also found in the ground layer (Martins
and Batalha, 2007; Gottsberger and Silberbauer‐
Gottsberger, 2018; Domingos‐Melo et al., 2023). Consider-
ing the importance of bats as pollinators for M. longifolia,
collecting higher‐resolution data such as identifying indi-
vidual species and analyzing their community composition
across strata would be highly interesting.
Even though hummingbirds foraged across strata on an
attractive resource that was available along the entire vertical
gradient, the previously observed structure of neotropical
hummingbird communities was not dissolved, and different
species preferred a certain vertical niche for foraging.
Marcgravia longifolia attracted both hermit and trochiline
species and, in line with previous studies (Fleming et al., 2005;
Fleming and Kress, 2013), we still found the basic dichotomy
between hermit and non‐hermit species. The three observed
hermit species mainly foraged in the under‐and midstory, only
rarely moving into the canopy. In contrast, the trochilines
frequently foraged across the whole vertical gradient. Competi-
tion for food resources has been described as one of the primary
drivers determining community organization in hummingbirds
(Feinsinger and Colwell, 1978;Wolf,1978; Ornelas et al., 2007).
Thus, especially in tropical lowland forests, high degrees of
specialization on specificfloral resources are crucial for the
coexistence of the highly diverse hummingbird species
(Maglianesi et al., 2015). Our results suggest that niche division
among tropical hummingbirds might not only be defined by
specialization for certain flowering plant species, but that
hummingbirds also divide the vertical gradient of a plant that is
attractive to both hermit and non‐hermit species into distinct
vertical foraging niches. These observations are in line with the
studybyNaikatinietal.(2022) reporting that the vertical
foraging height of honeyeaters was formed by interspecific
competition. A further driver of niche division among hermit
FIGURE 6 Characteristics of bacterial communities in the nectar of M. longifolia across strata. (A) Composition across different heights, derived from
the NMDS model. Blue: low heights, red: high heights. (B) Shannon diversity index for bacterial communities in nectar from flowers at different heights.
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15372197, 2024, 3, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.1002/ajb2.16303, Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
and trochiline species is their differing feeding behavior.
Whereas territorial trochilines establish and vigorously defend
territories (Feinsinger and Colwell, 1978), trap‐lining hermits
travel among clumps of flowers, probably following a regular
route and visiting these clumps of flowers in a particular
sequence (Stiles, 1975). We also observed that trochiline species
established and defended territories around M. longifolia
individuals. They frequently chased away other hummingbirds,
their own and other species, which gave them exclusive access
to inflorescences across all vertical strata. Hermits, on the other
hand, visited M. longifolia individuals quickly, always feeding on
the same under‐,andmidstoryinflorescences in a regular,
sequential fashion. This behavior highlights how important
niche differentiation, not only among plant species but also
among strata, is for reducing competition in tropical humming-
bird communities. Thus, the vertical niche can be a strong
structuring factor in plant–animal interactions and needs to be
further studied.
Because nectar‐inhabiting bacteria can be important
influencers of plant–animal interactions (Vannette et al., 2013),
studying their communities could reveal important processes
driving the structure of plant–animal networks. Crucially, we
found that the diversity but not the composition of nectar‐
inhabiting bacteria communities of M. longifolia differed along
the vertical gradient with the highest Shannon diversity index
in the midstory. While this somewhat unclear pattern might
partially be attributed to the rather small number of samples,
we here discuss potential mechanisms that could lead to a
pattern of higher diversity in the midstory. We assume that the
many different nectarivorous species that frequently forage for
nectar of M. longifolia and move throughout the vertical
gradient had transferred bacteria to the nectar across strata,
with the highest likelihood of transfer to the midstory, which
they visited most frequently. Nectarivores are known to act as
vectors and transport microorganisms between flowers
(Lachance et al., 2001b;Belisleetal.,2012;Sharabyetal.,2020).
Due to differences in foraging patterns and morphology,
different nectarivores can transfer different microorganisms to
different spaces (Sharaby et al., 2020). For instance, Belisle et al.
(2012) attributed the nonrandom distribution of microfungi
inhabiting the nectar of Mimulus aurantiacus to spatially
nonrandom foraging by pollinators. In our study, the hermit
species supposedly followed a regular route from one
M. longifolia individual to the next, likely transferring bacteria
among individuals and thereby contributing to the high
bacterial diversity in the midstory. The trochiline species, on
the other hand, frequently foraged across strata and likely
transferred bacteria within M. longifolia individuals. Further,
the higher temperatures and light intensities in the less‐dense
canopy may also have contributed to the lower bacterial
diversity in this stratum. In keeping with the potential of
nectar‐inhabiting bacteria as “hidden players”in
plant–nectarivore interactions (Vannette et al., 2013), the
vertical stratification of the nectar‐inhabiting bacterial commu-
nities thus likely influences differences among strata in the
patterning of plant–nectarivore interactions.
CONCLUSIONS
We found differences among strata in the patterning of
plant–nectarivore interactions within a single plant species.
Our results suggest that vertical stratification in
plant–nectarivore interactions is driven by resource abun-
dance, but that other factors such as species‐specific
preferences of nectarivores for certain strata due to competi-
tion might play an important role. Further, the diversity of
nectar‐inhabiting bacteria communities was vertically strati-
fied. Considering the understudied influence of bacteria on
plant–nectarivore interactions (Vannette et al., 2013), their
vertically stratified structure might further drive differences
in plant–nectarivore interactions among strata. It is a long‐
held tenet in ecology that tropical species are very specialized
and have very fine‐grained niche partitioning, which may
facilitate the coexistence of this high number of species in
tropics (Schemske, 2002). Niche differentiation of species
along a vertical gradient may thus be a key factor promoting
diversity in tropical forests.
AUTHOR CONTRIBUTIONS
S.T., M.G., E.W.H., M.T., R.J., and K.H. conceived the ideas
and designed the methodology. S.T., M.G., J.K., N.L., and
N.S. collected the data. S.T., M.G., J.K., N.L., R.J., and K.H.
analyzed and interpreted the data. S.T., E.W.H., R.J., and
K.H. led the writing of the manuscript. All authors
contributed critically to the drafts and gave final approval
for publication.
ACKNOWLEDGMENTS
We thank the German Research Foundation (Deutsche
Forschungsgemeinschaft, DFG) for funding the project
“Vertical stratification of plant–animal interactions and their
impact on pollination and seed dispersal within a single
Neotropical plant species”to K.H. (HE 7345/5‐1), E.W.H. (HE
1870/27‐1), and M.T. (TS 81/14‐1). We further thank the Eva‐
Mayr‐Stihl foundation for their support. We are especially
grateful to Madita Jappe and Luca Hahn for their support
during the field work and to Milagros Rimachi for identifying
the recorded plant species, to Robert S. Voss (American
Museum of Natural History) for identifying marsupials, Axel
Hausmann and Hubert Thony (Zoologische Staatssammlung
München) for identifying moths from camera‐trap photos,
Alessandro Mainardi for advice with the circular statistics, and
to the reviewers for their constructive comments. We are
grateful to the Servicio Forestal y de Fauna Silvestre (SERFOR)
of the Peruvian Ministry of Agriculture for issuing research
permits (nos. 304‐2018‐MINAGRI‐SERFOR‐DGGSPFFS and
528‐2019‐MINAGRI‐SERFOR‐DGGSPFFS). Open Access
funding enabled and organized by Projekt DEAL.
DATA AVAILABILITY STATEMENT
Data of this paper are available from https://zenodo.org/
records/10527420 and https://qiita.ucsd.edu/study/description/
15418 (accession PRJEB72152 ERP156935).
VERTICAL STRATIFICATION IN A LIANA FLOWERING ACROSS STRATA
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ORCID
Sarina Thiel http://orcid.org/0000-0002-3652-9083
Malika Gottstein http://orcid.org/0000-0003-0359-7730
Eckhard W. Heymann http://orcid.org/0000-0002-
4259-8018
Marco Tschapka http://orcid.org/0000-0001-9511-6775
Robert R. Junker http://orcid.org/0000-0002-7919-9678
Katrin Heer http://orcid.org/0000-0002-1036-599X
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