Flora 267 (2020) 151601

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Flora
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Pollination modes and divergent flower traits in three species of Plantago T

subgenus Plantago (Plantaginaceae)
Stefan Abrahamczyk*, Lea Sophie Dannenberg, Maximilian Weigend
Nees-Institute for Biodiversity of Plants, University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany

A R T I C LE I N FO A B S T R A C T

Edited by Fei-Hai Yu Different pollination modes have been reported from the genus Plantago, including cleistogamy, autogamy,
Keywords: ambophily and anemophily. However, these categories are no fixed units, but broad overlap exists between
Ambophily them. For several widely distributed P. species rigorous tests of the pollination mode are missing. Therefore, we
Chasmogamous analysed six floral traits and conducted pollination experiments of three common P. species to quantify the
Cleistogamous different pollination modes and their impact on seed production. Plantago australis turned out to be obligately
Insect cleistogamous with extremely low pollen grain production. Instead, P. major is an autogamous species with
Ovules chasmogamous flowers and the ability to increase seed production by wind-mediated outcrossing. However, its
Pollen
short style-stigma complexes underline that seed production is mainly induced by active selfing. Finally, P. media
Wind pollination
is pollinated by wind and insects. Its long, conspicuously collourd filaments and high pollen grain production
attract bees but also allow wind pollination. Seed production of P. media increased by 42%, when insects in
addition to wind had access to the flowers. Cleistogamy and ambophily evolved from wind-pollinated ancestors
in P., probably in parallel to the colonization of new habitat types.

1. Introduction
Wind is the most common abiotic pollen vector: about 10% of the
angiosperm species are adapted to wind pollination (Linder, 1998;
Ackerman, 2000). However, wind pollination is phylogenetically con-
centrated to a few clades (min. 65 evolutionary events) with the ma-
jority of species in the Poales, but at least some representatives in most
larger angiosperm orders (Linder, 1998). Wind pollination is also con-
centrated in specific habitat types, especially in open vegetation, such
as grasslands but also in ruderal vegetation and on islands (Regal, 1982;
Lososová et al., 2006). Generally, the proportion of wind-pollinated
plants increases with increasing latitude and elevation (Regal, 1982).
Wind-pollinated plant species are characterized by typical floral traits,
such as absent or strongly reduced perianths and nectarines, long fila-
ments, exposed, feathery stigmas and the production of numerous, but
small and smooth pollen grains with little or no pollen kit (Culley et al.,
2002; Friedman and Barrett, 2008, 2009). Further, wind-pollinated
species often have unisexual flowers and dioecy is common (Culley
et al., 2002; Friedman and Barrett, 2008). Also, most wind-pollinated
species have one-seeded fruits and are either selfing or obligately out-
crossing (Goodwillie et al., 2005; Friedman and Barrett, 2009).
Wind pollination is a derived character in angiosperms because the
ancestors of wind-pollinated species are all insect-pollinated (Culley
et al., 2002; Friedman and Barrett, 2008). However, many plant species
are not purely pollinated by wind, but also by insects, to a larger or
smaller extend (e.g. Wragg and Johnson, 2011; Mangla and Gupta,
2015; Custodio et al., 2017). This mixed pollination system, called
ambophily, combines floral traits typical for wind and insect pollina-
tion, such as a reduced perianth and exposed stigmas with con-
spicuously coloured bracts or filaments. Ambophily is often interpreted
as a transition from insect to wind pollination (Culley et al., 2002;
Friedman and Barrett, 2008, 2009). But ambophily can also represent a
transition away from wind pollination in ancestrally wind-pollinated
clades. Reversals from wind to animal pollination are surprisingly rare:
the evolution of ambophily from wind-pollinated ancestors is only
known from seven angiosperm families, such as Cyperaceae, Poaceae or
Salicaceae (Culley et al., 2002; Wragg and Johnson, 2011; Dórea et al.,
2018). Pure insect pollination evolved only once from wind-pollinated
ancestors in the genus Ficus (Moraceae; Culley et al., 2002). Such
transitions away from pure wind pollination are often correlated to the
colonization of new, less wind-exposed and more humid habitats, such
as the undergrowth of tropical rainforests, where pollination also by
insects is more efficient than only by wind (Culley et al., 2002; Costa
and Machado, 2012; Dórea et al., 2018). Only rarely (in the case of
Cyperus, Cyperaceae) the evolution of ambophily from wind pollination
is not correlated to a habitat switch, but may be related to the

⁎
Corresponding author.
E-mail address: stefan.abrahamczyk@uni-bonn.de (S. Abrahamczyk).

https://doi.org/10.1016/j.flora.2020.151601
Received 13 December 2019; Received in revised form 15 April 2020; Accepted 20 April 2020
Available online 29 April 2020
0367-2530/ © 2020 Elsevier GmbH. All rights reserved.
S. Abrahamczyk, et al.
Fig. 1. Ultrastructures of: stigma-style complexes with stigma papillae of Plantago media (A, B, D), P. major (E and F) and P. australis (H and I); and a pollen grains of
P. media (C), P. major (G) and P. australis (J).
occurrence in lower densities of the ambophilous species, which may
render insect pollination more effective (Wragg and Johnson, 2011).
Other transitions from wind pollination – beside ambophily – involve
mostly a combination of wind pollination and active selfing (Culley
et al., 2002; Friedman and Barrett, 2009; Mangla and Gupta, 2015) or
the evolution of cleistogamy (self-pollination in closed flowers; Connor,
2
Flora 267 (2020) 151601
1981; Culley and Klooster, 2007). The evolution of autogamy or cleis-
togamy from wind-pollinated ancestors often occurred in colonizing
species, or species with low population densities. Here, the evolution of
selfing or cleistogamy ensures high seed set in the case of mating
partner and pollinator limitation.
Plantago (Plantaginaceae) is well known to be largely wind-
S. Abrahamczyk, et al. Flora 267 (2020) 151601

Fig. 2. Comparison of floral traits (A: filament length; B: style-stigma complex length; C: diameter of pollen grains; D: length of stigma papillae; E: pollen grain
number; F: ovule number) between Plantago media, P. major and P. australis by ANOVAs in combination with Tukey tests and Kruskal–Wallis tests in combination with
Dunn tests. Degrees of freedom always = 2. Different capital letters above/below boxes indicate significant differences between groups.

pollinated. The cosmopolitan genus consists of annual to perennial
herbs, occurring in different kinds of open habitats. It has hermaphro-
ditic, protogynous flowers with stigmas staying receptive for a while
after the anthers open (Hammer, 1978). The flowers show several traits
typical for wind-pollinated plants, such as reduced corollas, more or
less long anther filaments and feathery stigmas (e.g. Hammer, 1978;
Primack, 1978). The fruits, however, are multi-seeded capsules
(Hammer, 1978), which is untypical for wind-pollinated plants having
commonly one-seeded fruits (Friedman and Barrett, 2009). Some
species, such as P. lanceolata and P. media are completely self-in-
compatible, while P. lagopus and P. ovata are at least partly and P. major
and P. coronopus - among many other species - are fully self-compatible
(e.g. van Dijk and van Delden, 1981; Meeuse, 1984; Wolff et al., 1988;
Sharma et al., 1992, 1993).
For individual P. species different pollination modes have been re-
ported: Plantago lagopus, P. lanceolata, P. media and P. ovata have been
shown to be ambophilous (Meeuse, 1984; Stelleman, 1984; Sharma
et al., 1992). These species have long, conspicuously cream or pinkish

3
S. Abrahamczyk, et al. Flora 267 (2020) 151601

Table 1
Floral traits of three P. species and results of the different pollination experiments for fruit and seed set.
Plantago media Plantago major Plantago australis

Herbarium number 2718 – 2698

Collection number – LD-187-19-1 –

mean sd mean sd mean sd

Filament length [mm] 10.2 0.82 4.38 0.46 2.0 0.38

Style-stigma complex length [mm] 7.32 1.24 2.76 0.5 4.04 0.35
Length of stigma papillae [μm] 68.4 7.32 144.6 28.92 146.6 17.98
Pollen grain diameter [μm] 24.8 1.6 22.4 1.85 27.1 3.44
Pollen grain number per flower 51.784 11.664 19.545 9450 56 56.49
Ovule number per flower 6.12 1.05 12.16 2.61 3.16 0.37
P/O-ratio 8608 2134 2164 833 18.36 18.76
Fruit set open pollination [%] 100.0 0.0 100.0 0.0 – –
Fruit set insect exclusion wide mesh [%] 74.6 18.7 100.0 0.0 – –
Fruit set insect exclusion narrow mesh [%] 43.8 33.1 100.0 0.0 – –
Fruit set insect & wind exclusion [%] 0.0 0.0 100.0 0.0 100.0 0.0
Seed set open pollination 3.76 1.33 11.04 2.61 – –
Seed set insect exclusion wide mesh 2.92 1.63 10.84 2.39 – –
Seed set insect exclusion narrow mesh 1.4 0.58 11.72 2.45 – –
Seed set insect & wind exclusion 0.0 0.0 9.44 2.29 2.68 0.56

coloured filaments and anthers and produce large amounts of pollen
that attract insects, especially bees and syrphid flies (Leereveld et al.,
1976; Primack, 1978; Sharma et al., 1993) but are also pollinated by
wind. The degree of insect/wind pollination of these species can vary
between sites and seasons: wind pollination is most important in wind-
exposed sites and during times of low insect abundance (Stelleman,
1984; Sharma et al., 1992, 1993). However, the proposed high im-
portance of insect pollination for seed set of P. media (⁓85%) by
Meeuse (1984) seems questionable due to the missing statistical ana-
lysis and some inaccuracy in the interpretation of the results.
In addition to the possibility of being pollinated by wind and/or
insects some P. species, such as P. lagopus, P. major or P. ovata are also
known to be autogamous (Wolff, 1991; Sharma et al., 1992, 1993;
Tormo Molina et al., 2001). But the specific importance of these di-
vergent pollination modes has rarely been disentangled for individual
species. Several other species from different sections of the subgenera
Plantago and Psyllium have been mentioned to be obligately cleistoga-
mous, but these claims are mostly based on morphological observations
and have rarely been tested by pollination experiments (Primack, 1978;
Sharma et al., 1992). Further, the few studies analysing flower traits of
P. species with different pollination modes revealed highly divergent
results for the same species and trait, especially in pollen grain number
per flower and pollen grain size (e.g. Hammer, 1978; Primack, 1978;
Sharma et al., 1992, 1993). Therefore, it is necessary to critically re-
assess flower traits and pollination modes of some of the P. species.
In this study we compare reproductive traits and analyse the fruit
and seed sets under different pollination treatments of three widely
distributed P. species with putatively different pollination modes.
Specifically we ask:
was cultivated from seeds collected at ruderal sites in Bolivia and
purchased via the Millennium Seed Bank. We deposited vouchers of all
three species in the University Herbarium Bonn (BONN).
2.2. Floral traits
For the collation of floral traits we always used five different genetic
individuals per species and analysed all traits from five flowers of each
individual. We measured the lengths of filaments and of the style-
stigma complex to connect these morphological traits to the individual
pollination modes. For counting the number of pollen grains per flower
we collected the anthers of pre-anthetic, closed flowers in Eppendorf
tubes and let them dry for at least 48 h. After dehiscence we added 200
μL glycerol to each sample of P. major and P. media and 50 μL glycerol
to each sample of P. australis to account for the low pollen grain number
of this species. Then, we mixed the suspensions for 5 min with a la-
boratory mixer mill (Retsch MM 200; Retsch, Haan, Germany) at 30 Hz
and put the tubes into an ultrasound bath (Bandelin, Sonorex) for 10
min. Before counting the number of pollen grains per flower under a
microscope we vortexed all samples and transferred 20 μL of the sus-
pension into a haemocytometer containing a Fuchs-Rosenthal counting
chamber with 16 squares. We randomly chose five squares and counted
the pollen grains within these squares. In a last step we calculated the
total number of pollen grains per flower. Additionally, we counted the
number of ovules of the same flowers used for pollen grain counting.
We opened the ovary under a stereo microscope, took out the ovules
and counted them.
2.3. Scanning electron microscopy

1 How do the reproductive traits differ between P. species with dif- To study some ultrastructures of pollen grains and style-stigma
ferent pollination modes? complexes of all three P. species we collected fresh flowers, dissected
2 Is P. australis cleistogamous? the desired structures, froze them in liquid nitrogen and investigated
3 How much does wind contribute to the pollination of P. major? the samples with cryo-electron microscopy. This technique has the
4 How much do insects contribute to the pollination of P. media? advantage that less artefacts occur than with the traditional approach
and that soluble structures, such as pollen kit remains. From the ob-
2. Material and methods tained photos we measured the lengths of five stigma papillae and the
diameter of five pollen grains per species. Further, we searched for the
2.1. Plant material presence of pollen kit.

We used the living collection of Bonn University Botanical Gardens 2.4. Pollinator observations
as source for our study. Plantago major and P. media are growing wild in
ruderal areas respectively on meadows in the gardens. Plantago australis To verify the existing observations of pollination modes of the three

4
S. Abrahamczyk, et al. Flora 267 (2020) 151601

Fig. 3. Comparisons between fruit (A) and seed sets (B) of the different polli-
nation treatments for Plantago media and between seed sets (C) of the different
pollination treatments for P. major by an ANOVA in combination with Tukey
tests and Kruskal–Wallis tests in combination with Dunn tests. Degrees of
freedom always = 3. Different capital letters above/below boxes indicate sig-
nificant differences between groups. Fruit set of P. major was 100% for all
pollination treatments.

Table 2
Pollinators of P. media at two meadow sites in Bonn, Germany and their visi-
tation frequency at inflorescences during eight hours of observation.
Pollinator Insect order Number of visits

Bombus terrestris Hymenoptera 58

Apis mellifera Hymenoptera 46
Bombus hypnorum Hymenoptera 44
Lasioglossum spec. Hymenoptera 41
Bombus hortorum Hymenoptera 7
Bombus pascuorum Hymenoptera 5
Oedemera spec. Coleoptera 2
Scaeva spec. Diptera 1

P. species (Knuth, 1899/1904; Leereveld et al., 1976; Primack, 1978)

we conducted pollinator observations at P. major and P. media and
searched for chasmogamous flowers at P. australis. We observed polli-
nators of P. major at a ruderal site at Bonn University Botanical Gardens
between 10th and 18th of July 2019 for five hours in total. Further, we
observed pollinators of P. media at two natural populations in dry
meadows, one in Bonn University Botanical Gardens and the other three
kilometres away (Kiefernweg, Bonn) between 13th of May and 05th of
June 2019 for eight hours in total. We conducted all observations at
sunny days with low wind speed. When an insect visited the in-
florescence of one P. species we identified the species using Gokcezade
et al. (2017); Westrich (2018) and the AnimalBase Project Group
website (www.animalbase.uni-goettingen.de, May 2019), observed its
behaviour, noted between how many inflorescences it moved.

2.5. Pollination experiments

For P. major and P. media we conducted four different pollination

treatments: 1. Open pollination; 2. Insect exclusion by a net with wide
meshes (0.5 × 0.5 mm); 3. Insect exclusion by a net with narrow me-
shes (0.1 × 0.2 mm); 4. Insect and wind exclusion in a pollinator-free
greenhouse. Per species and treatment we always used five plant in-
dividuals each. The plants for treatment 1 were situated outside without
any kind of protection. The plants for treatment 2 and 3, which were
completely covered with a net, were situated outside but under a
transparent plastic roof to protect them against rain. We used nets with
two kinds of meth sizes for both species to get an idea on the effects of
wind speed reduction and thus potentially reduced pollen availability
induced by the nets. Both kinds of nets completely excluded insect
pollinators. For P. australis we only conducted treatment 4 because this
species exclusively produced cleistogamous flowers.
When the seeds started ripening, we counted fruit set from one in-
florescence per individual each (in total five inflorescences per species
and treatment). We opened all fruits and checked for normally-looking
seeds. We assessed only fruits containing at least one normally looking
seed as well-developed. Finally, we counted the seed set at all opened
fruits.

2.6. Statistical analyses

We compared floral traits between the three P. species and analysed

fruit, respectively seed set between pollination treatments for P. major
and P. media separately. Before comparisons we tested for homogeneity
of variance by Fligner tests. If this requirement was fulfilled we

5
S. Abrahamczyk, et al.
conducted ANOVAs with Tukey posthoc tests. If this requirement was
not fulfilled – even after log transforming the data – we conducted
Kruskal–Wallis tests with Dunn posthoc tests. To evaluate the effect that
pollination by insects only has on the seed production of P. media we
developed a formula, in which we divided the product of fruit and seed
set under insect exclusion (= pure wind pollination; treatment 2) by
the product of fruit and seed set without any exclusion (= open pol-
lination; treatment 1):
(fruit set treatment 2(%)* seed set treatment 2)
x=
(fruit set treatment 1(%)* seed set treatment 1)
All analyses were conducted in R version 3.4.3 (R Development
Core Team, 2017).
3. Results
The three Plantago species differed strongly in their floral traits: P.
australis had the significantly shortest filaments and significantly longer
style-stigma complexes than P. major but significantly shorter style-
stigma complexes than P. media (Figs. 1 and 2). The length of the stigma
papillae of P. australis was significantly longer than those of P. media
but not different from P. major. Further, P. australis produced the sig-
nificantly lowest amounts of ovules and pollen grains per flower (Fig. 2;
Table 1). The pollen grains of P. australis were significantly larger in
diameter than those of P. major, but not than those of P. media. Plantago
major differed from P. media by having significantly shorter filaments
and style-stigma complexes, but significantly longer stigma papillae
(Figs. 1 and 2). Plantago major also produced significantly more ovules
per flower than P. media, but significantly less pollen grains. The dia-
meter of pollen grains was not significantly different between both
species. We did not find pollen kit on the pollen grains of any of the
three P. species.
We exclusively observed cleistogamous flowers at P. australis. In the
insect and wind exclusion experiment P. australis had a fruit set of 100%
and 84.8% of the ovules developed to seeds (Table 1). In contrast to P.
australis, P. major and P. media only produced chasmogamous flowers.
The flowers of P. major were never visited by insects and had a fruit set
of 100% in all pollination treatments. Wind slightly increased seed
production in P. major (by 13.0–19.5% depending on pollination
treatment) compared to insect and wind exclusion but this increase was
not significant in most pollination treatments (Fig. 3).
In total we observed 204 visits of mostly bees (Bombus, Apis,
Lasioglossum) at the inflorescences of P. media within eight hours of
observation (Table 2). During each visit these insects frequented several
flowers, but we did not count the number of flowers visited. The insects
were attracted by the pollen grains and moved from one flower with
open anthers to the next within the same inflorescence, but they also
moved between inflorescences and plant individuals. By this activity
the pollen-carrying insects came in contact with the still receptive
stigmas of the flowers with freshly opened anthers and pollinated these
flowers. The very young, purely female stage flowers above the flowers
with open anthers were rarely visited.
The fruit set of P. media was highly variable between pollination
treatments (Fig. 3): In the insect and wind exclusion experiment no
fruits were produced at all. In contrast, open pollination led to a fruit
set of 100%. If insects were excluded by a net with large meshes fruit set
was slightly, but significantly reduced (mean 74.6%; Table 1). The
exclusion of insects by a net with fine mesh led to a strong, significant
reduction of fruit set (mean 43.8%). Seed set developed in parallel to
fruit set in the different pollination treatments in P. media: open polli-
nation and plant covering with wide-meshed nets led to statistically
indistinguishably, high seed set (mean 3.76 respectively 2.92 seed per
fruit; Table 1). Covering plants with fine-meshed nets led to a sig-
nificantly reduced fruit set (mean 1.4 seeds per fruit) and no seeds
developed if insects and wind were excluded. Combining the results of
fruit and seed set, pollination success of P. media increased by 42.07% if
6
Flora 267 (2020) 151601
insects and wind had access to the flowers compared to pure wind
pollination.
4. Discussion
We observed conspicuous variation in floral traits between Plantago
species in relation to their pollination mode: Plantago australis re-
presents a typical, obligately cleistogamous species with closed flowers,
short filaments and the production of extremely low amounts of com-
paratively large pollen grains, as already hypothesized by Primack
(1978). Surprisingly, the style-stigma complexes resigned from the
closed flowers of P. australis were similar in lengths and in the lengths of
their papillae to the style-stigma complexes of P. major, which only
produced chasmogamous flowers. These conserved traits as well as the
phylogenetic position of P. australis in the terminal section of P. sub-
genus Plantago (Hassemer et al., 2019) indicate that it evolved from
chasmogamous, probably wind-pollinated ancestors, possibly as an
adaptation to its early successional habitats (Primack, 1978).
Genetic analyses already indicated that P. major produced seeds
mostly by selfing (Wolff, 1991) even though its chasmogamous flowers
permit pollination by wind-mediated outcrossing. Indeed, we found
slightly, but mostly not significantly (13.0–19.5%) higher seed sets in P.
major if pollination by wind was allowed, which indicates that P. major
conducts some outcrossing if mating partners are available. This
strategy is common in plant species occurring in ephemeral habitats,
where the availability of sufficient mating partners is not guaranteed
(Barrett, 2002). Further, our results are in line with the floral traits of P.
major because their exerted filaments and style-stigma complexes are
surprisingly short and the pollen grain production per flower
(19,545 ± 9450) is comparatively low for a wind-pollinated species
(Friedman and Barrett, 2009).
In contrast to the other two P. species, P. media is not able to con-
duct active self-pollination and is mostly dependent on outcrossing for
seed production (van Dijk and van Delden, 1981). Instead, it is heavily
visited by insects, especially by bees (Bombus, Apis, Lasioglossum)
(Leereveld et al., 1976), which are attracted by the showy, violet fila-
ments and anthers, producing large amounts of pollen grains. In addi-
tion to the strong optical flower signal, also the comparatively short
stigma papillae indicate an adaptation to insect pollination. However,
the long filaments and stigma-stamen complexes represent typical
flower traits of wind-pollinated species. Our pollination experiment
revealed that wind is an important pollen vector for P. media but pol-
lination success increases by 42.07% if also insects have free access to
the flowers. These results refute the conclusion of Meeuse (1984) who
claimed that insect pollination increased seed set by 85%.
Based on these results, P. media is clearly an ambophilous species,
which is probably also true for the other species of the section
Lamprosantha and possibly also for the species of the closely related
section Eremopsyllium (Hassemer et al., 2019) because all of these
species have similar, showy inflorescences. Considering flower struc-
ture and colouration in combination with the reported pollination
modes (e.g. Primack, 1978; Tormo Molina et al., 2001; Nilsson, 2005)
of the species in the early-branching P. clades (Hassemer et al., 2019),
ambophily probably evolved from wind-pollinated ancestors in sub-
genus Plantago, representing only the eighth family (Plantaginaceae), in
which this happened (Wragg and Johnson, 2011; Dórea et al., 2018).
Commonly, the evolution of ambophily from wind-pollinated ancestors
is linked to the colonization of less wind-exposed habitats, such as the
floor of dense forests (Culley et al., 2002; Costa and Machado, 2012;
Dórea et al., 2018). However, all P. species, including the (likely) am-
bophilous ones occur in open vegetation types, leading to the question,
which other factor might have triggered the evolution of ambophily in
P.? One plausible hypothesis is that ambophily evolved in parallel to
the colonization of less ephemeral habitats, such as different kinds of
meadows, the habitats of the ambophilous P. species (Tutin et al.,
1964). In these more stable habitats mating partner and insect
S. Abrahamczyk, et al.
pollinator availability may be more reliable than in ephemeral habitats
and insect pollinators are much less wasteful pollen vectors than wind,
making ambophily a very effective pollination mode in these habitats.
Declaration of Competing Interest
None.
Acknowledgements
We thank the Millennium Seed Bank for providing seeds of P. aus-
tralis, Josef Manner and his colleagues from Bonn University Botanical
Gardens for cultivating our plants and the anonymous reviewers and
the editor Fei-Hai Yu for helpful comments.
References
Ackerman, J.D., 2000. Abiotic pollen and pollination: ecological, functional, and evolu-
tionary perspectives. Plant Syst. Evol. 222, 167–185.
Barrett, S.C., 2002. Evolution of sex: the evolution of plant sexual diversity. Nat. Rev.
Gen. 3, 274–284.
Connor, H.E., 1981. Evolution of reproductive systems in the Gramineae. Ann. Mo. Bot.
Gard. 68, 48–74.
Costa, A.C.G., Machado, I.C., 2012. Flowering dynamics and pollination system of the
sedge Rhynchospora ciliata (Vahl) Kükenth. (Cyperaceae): does ambophily enhance its
reproductive success? Plant Biol. 14, 881–887.
Culley, T.M., Klooster, M.R., 2007. The cleistogamous breeding system: a review of its
frequency, evolution, and ecology in angiosperms. Bot. Rev. 73, 1–29.
Culley, T.M., Weller, S.G., Sakai, A.K., 2002. The evolution of wind pollination in an-
giosperms. Trends Ecol. Evol. 17, 361–369.
Custodio, T., Comtois, P., Araujo, A.C., 2017. Reproductive biology and pollination
ecology of Triplaris gardneriana (Polygonaceae): a case of ambophily in the Brazilian
Chaco. Plant Biol. 19, 504–514.
Dórea, M.C., Santos, D.W.J., Oliveira, R.P., Funch, L.S., Santos, F.A.R., 2018.
Reproductive traits related to anemophily and insect visitors in two species of
Poaceae from the Brazilian Atlantic rainforest. Braz. J. Bot. 41, 425–434.
Friedman, J., Barrett, S.C., 2008. A phylogenetic analysis of the evolution of wind pol-
lination in the angiosperms. Int. J. Plant Sci. 169, 49–58.
Friedman, J., Barrett, S.C., 2009. Wind of change: new insights on the ecology and
evolution of pollination and mating in wind-pollinated plants. Ann. Bot. 103,
1515–1527.
Gokcezade, J.F., Gereben-Krenn, B.-A., Neumayer, J., 2017. Feldbestimmungsschlüssel
für die Hummeln Deutschlands, Österreichs und der Schweiz. Quelle & Meyer Verlag,
Wiebelsheim, pp. 19–45.
Goodwillie, C., Kalisz, S., Eckert, C.C., 2005. The evolutionary enigma of mixed mating
systems in plants: occurrence, theoretical explanations, and empirical evidence.
Flora 267 (2020) 151601
Annu. Rev. Ecol. Evol. Syst. 36, 47–79.
Hammer, K., 1978. Entwicklungstendenzen blütenökologischer Merkmale bei Plantago.
Flora 167, 41–56.
Hassemer, G., Bruun-Lund, S., Shipunov, A.B., Briggs, B.G., Meudt, H.M., Rønsted, N.,
2019. The application of high-throughput sequencing for taxonomy: the case of
Plantagosubg.Plantago (Plantaginaceae). Mol. Phyl. Evol. 138, 156–173.
Knuth, P., 1899. 1904. Handbuch der Blütenbiologie, Bd. II + III, 1 and 2. Teil.
Engelmann, Leipzig.
Leereveld, H., Meeuse, A.D.J., Stelleman, P., 1976. Anthecological relations between
reputedly anemophilous flowers and syrphid flies. II. Plantago media L. Acta Bot.
Neerl. 25, 205–211.
Linder, H.P., 1998. Morphology and the evolution of wind pollination. In: Owens, S.J.,
Rudall, P.J. (Eds.), Reproductive Biology in Systematics, Conservation and Economic
Botany. Royal Botanic Gardens, Kew, pp. 123–135.
Lososová, Z., Chytrý, M., Kühn, I., Hájek, O., Horáková, V., Pyšek, P., Tichý, L., 2006.
Patterns of plant traits in annual vegetation of man-made habitats in central Europe.
Pers. Plant Ecol. Evol. Syst. 8, 69–81.
Mangla, Y., Gupta, K., 2015. Love in the air: wind pollination ecological and evolutionary
considerations. In: Kapoor, R., Kaur, I., Koul, M. (Eds.), Plant Reproductive Biology
and Conservation. International Publishing House, New Delhi, pp. 234–245.
Meeuse, A.D.J., 1984. Rate of dependence of Plantago media L. On entomophilous re-
production – preliminary report. Acta Bot. Neerl. 33, 129–130.
Nilsson, E., 2005. Breeding System Evolution and Pollination Success in the Wind-polli-
nated Herb Plantago maritima (Doctoral Dissertation). University of Upsala.
Primack, R.B., 1978. Evolutionary aspects of wind pollination in the genus Plantago
(Plantaginaceae). New Phytol. 81, 449–458.
R Development Core Team, 2017. R: a Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna.
Regal, P.J., 1982. Pollination by wind and animals: ecology of geographic patterns. Annu.
Rev. Ecol. Syst. 13, 497–524.
Sharma, N., Koul, P., Koul, A.K., 1992. Reproductive biology of Plantago: shift from cross-
to self-pollination. Ann. Bot. 69, 7–11.
Sharma, N., Koul, P., Koul, A.K., 1993. Pollination biology of some species of genus
Plantago L. Bot. J. Linn. Soc. 111, 129–138.
Stelleman, P., 1984. Significance of biotic pollination in a nominally anemophilous plant:
Plantago lanceolata. Proc. Ned. Ak. Wet. Ser. C: Biol. Med. Sci. 87, 95–119.
Tormo Molina, R., Silva Palacios, I., Muñoz RodrÍguez, A.F., Tavira Muñoz, J., Moreno
Corchero, A., 2001. Environmental factors affecting airborne pollen concentration in
anemophilous species of Plantago. Ann. Bot. 87, 1–8.
Tutin, T.G., Heywood, V.H., Burges, N.A., Valentine, D.H., 1964. Flora Europaea:
Plantaginaceae to Compositae (and Rubiaceae) Vol. 4 Cambridge University Press,
Cambridge.
van Dijk, H., van Delden, W., 1981. Genetic variability in Plantago species in relation to
their ecology. Theor. Appl. Genet. 60, 285–290.
Westrich, P., 2018. Die Wildbienen Deutschlands. Ulmer Verlag, Stuttgart.
Wolff, K., 1991. Analysis of allozyme variability in three Plantago species and a com-
parison to morphological variability. Theor. Appl. Genet. 81, 119–126.
Wolff, K., Friso, B., van Damme, J.M.M., 1988. Outcrossing rates and male sterility in
natural populations of Plantago coronopus. Theor. Appl. Genet. 76, 190–196.
Wragg, P.D., Johnson, S.D., 2011. Transition from wind pollination to insect pollination
in sedges: experimental evidence and functional traits. New Phytol. 191, 1128–1140.