PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USA10-PONE-RA-1963910.1371/journal.pone.0018071Research ArticleBiologyEcologyPlant ecologyEvolutionary biologyEvolutionary processesCoevolutionEvolutionary ecologyEcologyEvolutionary BiologyInherited Biotic Protection in a Neotropical Pioneer
PlantInherited Protection in PlantsDejeanAlain12*CorbaraBruno34LeroyCéline1DelabieJacques H. C.5RossiVivien6CéréghinoRégis78CNRS, Écologie des Forêts de Guyane (UMR-CNRS 8172), Campus
Agronomique, Kourou, FranceUniversité de Toulouse, UPS, Toulouse, FranceCNRS, UMR 6023, Laboratoire Microorganismes Génome et
Environnement (LMGE), Aubière, FranceClermont Université, Université Blaise Pascal, LMGE,
Clermont-Ferrand, FranceLaboratório de Mirmecología, Convênio UESC-CEPEC,
Centro de Pesquisas do Cacau, CEPLAC, Itabuna-BA, BrazilCIRAD, Écologie des Forêts de Guyane (UMR-CNRS 8172), Campus
Agronomique, Kourou, FranceCNRS, UMR 5245, EcoLab (Laboratoire d'Ecologie Fonctionnelle),
Toulouse, FranceUniversité de Toulouse, UPS, INPT, EcoLab, Toulouse,
FranceRaineNigelEditorRoyal Holloway University of London, United Kingdom* E-mail: alain.dejean@wanadoo.fr
Conceived and designed the experiments: AD. Performed the experiments: AD CL.
Analyzed the data: JHCD AD RC VR. Contributed reagents/materials/analysis
tools: BC JHCD RC CL. Wrote the paper: AD BC CL RC.
The authors have declared that no competing interests exist.
2011313201163e180713011201024220112011Dejean et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.
Chelonanthus alatus is a bat-pollinated, pioneer Gentianaceae
that clusters in patches where still-standing, dried-out stems are interspersed
among live individuals. Flowers bear circum-floral nectaries (CFNs) that are
attractive to ants, and seed dispersal is both barochorous and anemochorous.
Although, in this study, live individuals never sheltered ant colonies,
dried-out hollow stems - that can remain standing for 2 years - did. Workers
from species nesting in dried-out stems as well as from ground-nesting species
exploited the CFNs of live C. alatus individuals in the same
patches during the daytime, but were absent at night (when bat pollination
occurs) on 60.5% of the plants. By visiting the CFNs, the ants indirectly
protect the flowers - but not the plant foliage - from herbivorous insects. We
show that this protection is provided mostly by species nesting in dried-out
stems, predominantly Pseudomyrmex gracilis. That dried-out
stems remain standing for years and are regularly replaced results in an
opportunistic, but stable association where colonies are sheltered by one
generation of dead C. alatus while the live individuals nearby,
belonging to the next generation, provide them with nectar; in turn, the ants
protect their flowers from herbivores. We suggest that the investment in wood by
C. alatus individuals permitting still-standing, dried-out
stems to shelter ant colonies constitutes an extended phenotype because foraging
workers protect the flowers of live individuals in the same patch. Also, through
this process these dried-out stems indirectly favor the reproduction (and so the
fitness) of the next generation including both their own offspring and that of
their siblings, all adding up to a potential case of inclusive fitness in
plants.
Financial support for this study was provided by the Programme Amazonie II of the
French Centre National de la Recherche Scientifique (project 2ID) and the
Programme Convergence 2007-2013, Region Guyane from the European Community
(project DEGA). JHCD acknowledges the Conselho Nacional de Desenvolvimento
Cientifico e Technologico(CNPq) for his research grant. The funders had no role
in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.Introduction
It is thought that ants were initially ground-dwelling predators or scavengers and
that they adopted an arboreal way of life with the rise of angiosperms by the
mid-Eocene ≈50 million years ago [1], [2]. By preying on insects that they discovered on plant
foliage while they were foraging, the workers of ground-nesting species probably
constituted the first cases of biotic plant protection. Later, tight evolutionary
bonds developed between ants and plants. In what is known as a facultative
mutualism, plants induce ants to patrol their foliage by producing energy-rich food
rewards such as extra-floral nectar (EFN) and food bodies (FBs), reserving proteins
for their own metabolism. By providing the ants the fuel with which to patrol, the
plants' foliage is protected through the ants' predatory and territorial
defense activities [3]. Myrmecophytes, however, live in an obligatory association
with only a small number of plant-ants for which they provide a nesting place in
pre-existing cavities (domatia) in live plant organs, such as leaf pouches and
hollow stems or thorns, and frequently also food (i.e., EFN and/or FBs). In return,
plant-ants protect myrmecophytes from several kinds of enemies, particularly
defoliating insects [3].
As the basis of most food webs, plants have had to evolve defensive strategies
against herbivorous insects. These defenses can be “constitutive”
through physical barriers and the continuous production of toxic compounds, or
“induced” following attacks by herbivorous insects that trigger the
production of defensive chemicals or the emission of volatiles that attract the
natural enemies of the attacking insects [4]. Among plant defensive
strategies, the biotic, indirect defense provided by ants is of particular interest
because herbivorous insects have rarely developed counter-adaptations against ants
[5], [6]. Indeed, the
positive effects of biotic defense by ants on their host plant's fitness have
been unambiguously shown through a meta-analysis [7]–[9].
By concentrating ants on their crowns through the presence there of domatia,
myrmecophytes benefit from greater protection if compared with plants bearing EFNs
alone. This protection is even better when myrmecophytes also bear only EFN and/or
FBs [7],
[10]. EFN
production can be induced through herbivore damage [6] and, in myrmecophytes, the induced
recruitment of nestmates by ants discovering a leaf wound suggests the presence of
an induced defense (induced response) [6], [11].
The optimal defense theory predicts that, due to their costs, defenses are deployed
in direct proportion to the value and/or risk of the plant parts being attacked.
These costs correspond to the production of secondary compounds and/or the formation
of mechanical structures which would otherwise be allocated to plant growth and/or
reproduction [12]–[14]. In other words, plants invest in constitutive defenses
for organs of high value (e.g., reproductive organs, stems) and likelihood of attack
(e.g., young parts), while parts of lower value or likelihood of attack (e.g.,
leaves) are typically protected through induced defenses [15]–[18]. Because of their partnership
with ants, many plants bear EFNs not only on their vegetative parts, but also on
organs related to reproduction such as inflorescences, sepals, petals, and fruits
[18]–[22]. Yet, due to their predatory ability and/or their
territorial aggressiveness, ants can attack pollinators, limiting their access to
flowers. These ant-pollinator conflicts can disrupt plant reproduction, something
particularly true when the EFNs are situated close to flowers. Several processes can
attenuate these conflicts such as (1) EFNs distracting ants from floral nectar, (2)
flowers attracting pollinators when ants are less active, (3) EFNs active on young
plant parts while inflorescences develop on old shoots, and (4) flowers producing
pollen repellent to ants [22]–[26].
The focal species of this study, Chelonanthus alatus (Gentianaceae),
is a Neotropical bi-annual to perennial pioneer geophyte that colonizes both human-
and naturally-disturbed sites, as well as inselbergs (i.e., a mountain or rocky mass
that has resisted erosion and stands isolated in an essentially level area; also
called ‘monadnock’) [22], [27], [28]. The terminal inflorescences bloom year-round and are
pollinated by bats [29]. Like for some other bat-pollinated plants, the flowers
have petals that do not open completely at anthesis, forming a pseudo-tubular
corolla at the base, while the distal part flares into a wide opening (Fig. 1). The sepals of the calyx
dorsally bear blunt, thickened, longitudinal keels where ‘circum-floral
nectaries’ (CFNs) are located. Like EFNs, CFNs do not play a role in
pollination [20];
instead, they attract and retain ants in locations where they can best protect
flowers from herbivorous insects. Chelonanthus alatus is
self-compatible, with seed dispersal by gravity (barochory) or wind (anemochory)
[20], [29].
10.1371/journal.pone.0018071.g001
Chelonanthus alatus inflorescences showing the different
steps from bud formation to withering.
A. An inflorescence with an open flower. B. Extremity of an inflorescence
with a bud just before blooming. One Pseudomyrmex gracilis
worker is exploiting the surface of the nectaries situated at the tips of
each of the five petals. C. Illustration of the different circum-floral
nectaries: (1) on the sepals, and (2) externally on the petals where their
tips merge together. An examination of the surface of the nectaries with a
stereomicroscope showed numerous stomatal pores where droplets of nectar had
been excreted. D. Flower that had begun to wither with a P.
gracilis worker licking nectar from a sepal. The stomatal pores
situated at the tip of the petals are no longer active at this stage.
In this study, we hypothesized that C. alatus has evolved a
relationship with ants such that they protect the plant from herbivorous insects in
return for nectar rewards. To test this hypothesis, we first verified the
distribution of C. alatus individuals that seem dispersed in
patches where still-standing, dried-out hollow stems (hereafter “dried-out
stems”) are interspersed among live individuals at different stages of
development. Second, we verified the lifespan of live C. alatus
individuals, measured the stem diameter and height of ones that had recently died
and verified the longevity of dried-out stems. Third, we compared the ant species
visiting the CFNs with the ant community in the areas where C.
alatus grows. Fourth, we also examined the nest site selection of the
ants in the area to verify whether some of them nest in live, hollow C.
alatus stems and/or in dried-out C. alatus stems.
Finally, we sought to determine if ants nesting in the dried-out stems protect the
surrounding live C. alatus plants from herbivores.
ResultsFormation of Chelonanthus alatus patches
We monitored the changes occurring over 6 years in 15 patches for which we had
witnessed the development of the first C. alatus individuals in
areas that had been recently-cleared (Fig. 2). Seven patches were founded by only
one individual plant, the eight others by two to seven individual plants growing
in a 3 m radius. Despite this variation in the number of founding plants, in 13
patches the numbers of young seedlings and individuals producing flowers were
very similar from the third year (a 4-year-old patch is presented in Fig. 3). Yet, the fate of new
seedlings in two other patches was different as only dried-out stems remained
after the second year. In that case, a second generation of numerous young
seedlings appeared, but only during the fourth year; individuals bearing flowers
and fruits appeared during the fifth year (Fig. 2). Given the large numbers of
seedlings, they probably originated from the generation of C.
alatus that had died in the patches rather than from dead
individuals from other patches through anemochory.
10.1371/journal.pone.0018071.g002
Formation of the Chelonanthus alatus patches during
a 6-year-long survey.
The numbers between parentheses correspond to the numbers of individuals
from each case described in the corresponding line of the second
column.
10.1371/journal.pone.0018071.g003
A 4-year-old patch of Chelonanthus alatus showing
individuals at different stages of development.
Three individuals (a, b, c) are dried out, seven others (1, 2 …, 7)
are in bloom, three more (α, β, γ) have begun to grow, while
the 31 remaining are seedlings with 4-to-6 leaves.
The formation of patches in recently-cleared areas can be summarized as follows.
When one windborne C. alatus seed germinates in a favorable
area, a ‘founding’ individual develops. Through barochorous seed
dispersal, its offspring cluster all around it forming a patch of related
C. alatus individuals. Then, several generations succeed
one another. In all cases, some of the seeds produced can be carried by the wind
to a new pioneer area, permitting a new cycle to begin. Concerning pollination,
because C. alatus is self-compatible and mostly pollinated by
bats [20],
[29]
known to visit open flowers in succession, the opportunities for intra-patch
pollination are numerous and help to maintain relatedness between individuals.
Yet, inter-patch pollination also occurs as nectarivorous bats can travel over
relatively long distances [29].
Lifespan of Chelonanthus alatus and longevity of dried-out
stems
Among the 15 live C. alatus individuals tagged in January 2001,
11 lived for 18 months and then died during the dry season, and the remaining
four were still alive 2 years later (i.e., 3.5 years from the beginning of the
survey). Among the dried-out stems tagged at the beginning of the experiment,
six decayed after 12–18 months during their second rainy season. The same
fate was noted for the 11 dried-out stems corresponding to the individuals from
the previous survey that died after 18 months. The other nine remained upright
during the 2-year survey; the diameter of their stems was significantly larger
than those that decayed faster (means±SE; 0.8±0.04 cm
vs. 0.45±0.03 cm, respectively;
df = 23; t = 6.07; P<0.0001).
By measuring the stem diameter and height of 150 C. alatus
individuals that had recently died, we were able to establish a relationship
curve between these two variables (Fig. 4A). If compared to the measurements of the C.
alatus that had died earlier, those that produced persistent,
dried-out stems (i.e., 0.8±0.04 cm in diameter) were, based on this
curve, among the tallest individuals (see also Fig. 4B). Also, the stem diameters of 150
C. alatus individuals that had recently died were
significantly smaller than those of 90 dried-out stems selected at random and
for which we do not know how much time separated the measurement from the death
of the plant (means±SE; 0.55±0.01 cm vs.
0.61±0.02 cm; t = 2.44;
df = 238; P<0.05). We can therefore distinguish small
individuals with a short lifespan from taller individuals with a longer lifespan
and larger diameter at their base.
10.1371/journal.pone.0018071.g004
Stem diameter and height of Chelonanthus
alatus.
A. Blue diamonds correspond to the relationship between the stem diameter
and height of 150 C. alatus individuals that had
recently died (note that these individuals had lost their leaves and
were turning brown; the diameter of their stems, taken 5 cm from the
ground, will vary only very slightly as they will dry and will remain
still standing). Red circles correspond to 53 isolated, founding
C. alatus individuals. Note that both their stem
diameter and height are higher than those from the patches (see text for
statistics). B. Distribution of the number of individuals based on the
diameter of their stems.
We also monitored 53 founding C. alatus individuals at the stage
when they were turning brown. They had a wider stem diameter and were taller
than the 150 individuals from the previous lot (Fig. 4A) (means±SE; stem diameter:
1.053±0.037 cm vs. 0.55±0.01 cm;
t = 15.68; df = 201; P<0.0001;
height: 159.8±3.2 cm vs. 76.0±1.9 cm;
t = 22.39; df = 201; P<0.0001).
Ant diversity and activity
We found no ants inside any of the 98 live stems recorded in the 6-year-old
patches surveyed. In contrast, 114 of the 214 (i.e., 53%) dried-out stems
contained ants. Occupied stems had significantly larger diameters than
uninhabited, dried-out stems (0.52±0.02 versus 0.30±0.01;
df = 208; t = 9.24; P<0.0001).
Compared to the diameters of plants that had recently died (Fig. 4A), dried-out, ant-inhabited stems
correspond to medium to large-sized C. alatus individuals.
Of the 44 ant species inventoried during this study, as expected, most are
species characteristic of perturbed habitats. Among these ants, none nested in
live C. alatus individuals; six nested in the hollow,
above-ground parts of dried-out stems (predominantly Pseudomyrmex
gracilis found in 83 of the 114 inhabited, dried-out stems;
73%; Table 1) and
12 in the base (e.g., less than 5 cm high) of these dried-out stems (mostly
ground-nesting species with tiny workers expanding their colony to the root
area); 20 were recorded in the surrounding plant foliage; and 26 were captured
using pit-fall traps. We noted low Sørensen similarity indices between
ant species related to C. alatus and those recorded on adjacent
trees or captured using pit-fall traps (Table 1).
10.1371/journal.pone.0018071.t001
Ants inhabiting dried-out, hollow Chelonanthus
alatus stems among those noted in the area studied.
Nested in the dry C.
alatus stems (% of 114 cases)
Exploited C. alatus
EFNs (% of 80 cases)
Noted on adjacent trees
Captured in pit-fall traps (% of 40
cases)
No. species
SF
Nest.
Ant species
Base
aerial parts
diurnally
nocturnally
1
M
G
Atta sexdens
2.5
2
D
A
Azteca bequaerti
+
3
D
A
Azteca chartifex
+
4
F
M
Brachymyrmex sp. 1
2.6
18.75
11.25
+
5
F
Camponotus sp. 1
1.25
10.0
6
F
G
Camponotus blandus
100.00
+
10.0
7
F
Camponotus crassus
+
8
F
A
Camponotus femoratus
+
9
F
A
Camponotus latangulus
0.9
1.25
+
2.5
10
F
G
Camponotus melanoticus
32.50
+
5.0
11
F
Camponotus novogranadensis
+
12
M
G
Cardiocondyla obscurior
1.75
13
M
A
Crematogaster sp. 1
4.4
15.00
7.50
+
7.5
14
M
Crematogaster sp. 2
2.5
15
M
Crematogaster sp. 5
+
16
M
Crematogaster sp. 9
2.5
17
D
G
Dorymyrmex pyramicus guy.
7.5
18
E
G
Ectatomma brunneum
3.75
+
15.0
19
E
G
Ectatomma tuberculatum
15.00
11.25
+
20
F
G
Gigantiops destructor
1.25
+
2.5
21
P
G
Hypoponera opaciceps
1.75
22
M
G
Nesomyrmex tristani
5.3
23
D
Linepithema sp.
+
24
P
G
Odontomachus caelatus
2.5
25
P
G
Pachycondyla mesonotalis
0.9
26
F
Nylanderia sp. 1
3.5
1.25
2.5
27
F
Nylanderia sp. 2
0.9
15.0
28
F
Nylanderia sp. 3
0.9
29
M
G
Pheidole fallax
15.00
21.25
7.5
30
M
Pheidole sp. 4
2.5
31
M
Pheidole sp. 9
17.5
32
M
Pheidole sp. 29
10.0
33
M
G
Pheidole sp. 30
0.9
2.50
34
M
Pheidole sp. 31
+
7.5
35
M
Pheidole sp. 37
7.5
36
Ps
A
Pseudomyrmex ethicus
0.9
1.25
37
Ps
A
Pseudomyrmex gracilis
73.0
100.00
+
7.5
38
Ps
A
Pseudomyrmex sp. gr.
pallidus
5.3
7.50
+
2.5
39
Ps
G
Pseudomyrmex termitarius
2.6*
7.50
+
12.5
40
M
G
Solenopsis saevissima
1.25
12.5
41
M
G
Solenopsis sp. 1
0.9
42
M
G
Strumigenys louisianae
2.5
43
M
G
Wasmannia auropunctata
0.9
3.75
+
10.0
44
M
G
Wasmannia sp.
0.9
Number of ant species
12
6
17
5
20
26
No. ant species (EFNs pooled)
12
6
18
20
26
Sørensen sim. Ind. Pit-fall traps
0.21
0.25
0.64
0.43
-
on adjacent trees
0.18
0.31
0.63
-
-
on C. alatus EFNs
0.33
0.42
-
-
-
Base versus hollow stems
0.00
-
-
-
Note: List of ant species living inside of 144 dried-out, hollow
Chelonanthus alatus stems; foraging on
C. alatus, or on adjacent trees; and recorded
inside of 18 pit-fall traps. A total of 44 ant species recorded.
Subfamilies (SF) = D: Dolichoderinae; E:
Ectatomminae; F: Formicinae; M: Myrmicinae; P: Ponerinae; Ps:
Pseudomyrmecinae. Nesting habit (Nest.) = G:
ground-nesting species; A: arboreal species; M: generalist able to
nest in different situations. * only incipient colonies. For
EFNs, the percentages were obtained from the presence of workers of
corresponding species on at least one plant in the patch (eight
patches; 10 series of observations).
Ants visited the CFNs situated on the sepals from the beginning of the formation
of the buds until the formation of the fruits. They also visited the surface of
the nectaries situated externally at the tip of each petal that are active only
prior to the opening of the flowers (Fig. 1B–C).
Five of the six ant species nesting in the above-ground parts of the dried-out
stems exploited the CFNs of live C. alatus in the same patch,
and therefore situated in the vicinity (namely, Camponotus
latangulus, Crematogaster sp.1,
Pseudomyrmex ethicus, P.
gracilis, and Pseudomyrmex sp.,gr
pallidus). This was also the case for five of the 12
species nesting in the base of the dried-out stems (namely,
Brachymyrmex sp., Nylanderia sp. 1,
Pheidole sp. 30, Pseudomyrmex termitarius,
and Wasmannia auropunctata) and for seven ground-nesting
species (namely, Camponotus blandus, Camponotus
melanoticus, Ectatomma brunneum, Ectatomma
tuberculatum, Gigantiops destructor,
Pheidole fallax, and Solenopsis
saevissima; see also the Sørensen similarity index; Table 1). Camponotus
blandus (Formicinae) and P. gracilis
(Pseudomyrmicinae) workers were the most numerous diurnally, exploiting the CFNs
of several C. alatus in all of the patches, while the other 15
species recorded were much less numerous. Nocturnally, the CFNs were exploited
by only five species of which Camponotus melanoticus
pre-dominated (Table 1).
By scoring the number of times the ants visited the CFNs per C.
alatus individual, we noted that during the daytime P.
gracilis workers were the most frequent, followed by C.
blandus. At night, C. melanoticus pre-dominated as
previously noted, but 60.5% of the C. alatus individuals
were not visited by ants, which was unusual during the daytime (Fig. 5).
10.1371/journal.pone.0018071.g005
Ant visitation of Chelonanthus alatus.
Mean (±SD) number of C. alatus individuals
visited by ants (or individuals not visited) for their CFNs (98 plants
surveyed; 10 non-consecutive days of observation). A: during the daytime
(3 hours of observation per day during two periods: 10:00–11:00
and 17:00–18:00). B: At night (one 2-hour period of observation
per night: 23:00–01:00). Statistical comparisons. One-way analyses
of variance during the daytime: F = 580.4;
P<0.0001 and at night: F = 414.5; P<0.0001;
Newman-Keuls' post-hoc tests: different letters
indicate significant differences at P<0.05 between the daytime and at
night.
We recorded between five and 30 dried-out C. alatus sheltering
P. gracilis individuals per patch (10.38±8.4 on
average), corresponding to 82 and 411 workers, respectively (143±111
workers per patch on average; 8 patches). In total, of the 83 dried-out stems
sheltering P. gracilis, four contained an incipient colony
(i.e., a queen, up to 4 ‘small’ workers and brood), while the 79
others sheltered 14.3±3.7 workers plus abundant brood; the queens were
present in only one to four dried-out stems per patch.
Plant protection
Observations conducted during 10 non-consecutive days on 98 live C.
alatus individuals from eight patches revealed that the
inflorescences were attacked by cockroaches (diurnally in 60 cases; nocturnally
in 179 cases), and by curculionid and chrysomelid beetles (diurnally in 25
cases; nocturnally in 23 cases). Adjusted to the 12 h/12 h distribution of the
nycthemeron, the number of observations per day and the 10 days of observation,
this corresponds to a total of ca. 0.35 daily visits by defoliating insects per
inflorescence during the daytime and ca. 2.47 at night.
Of the more than 500 live C. alatus observed in total during
preliminary experiments and during this study, the foliage of only one
individual had been attacked by caterpillars. Concerning hemipterans,
colonization by Coccidae attended by Crematogaster sp.2 workers
was noted once, while other cases corresponded to isolated individuals
(Cicadellidae: four times; Fulgoridae: once; Membracidae: twice).
We also conducted an experiment comparing C. alatus individuals
bearing flowers from unaltered patches (control) with those from two
experimental treatments. The first experimental treatment corresponded to
patches where we had torn out all of the dried-out stems to eliminate their ant
inhabitants, and so their anti-defoliator activity. In the second experimental
treatment, we spread a ring of Tanglefoot® at the base of the plants to
prevent any ants from climbing up (including species nesting in the ground and
in nearby dried-out stems plus those coming from neighboring areas).
We modeled the rate at which the petals of flowers were attacked by defoliating
insects with a generalized linear model (GLM) using an ordinal probit link
function of the treatment. The experimental treatments had a significant effect
on the rate at which the petals were attacked by defoliating insects (Likelihood
ratio test: P = 0.006); the difference between the two
experimental treatments was not significant (Likelihood ratio test:
P = 0.4) (Fig.
6). In other words, the rate at which the petals were attacked by
defoliating insects was significantly lower for the C. alatus
in the unaltered patches than for those from either experimental treatment.
Thus, it is likely that much of the flower protection was provided by ants,
mostly P. gracilis, nesting in dried-out stems.
10.1371/journal.pone.0018071.g006
Ant protection of Chelonanthus alatus.
Percentages of petal (Flowers) and leaf (Leaves) surface destroyed by
defoliating insects in three situations. Control: unaltered patches;
first experimental treatment: individuals from patches whose standing,
dried-out stems were all torn out (eliminating their ant inhabitants);
second experimental treatment: individuals for which a ring of
Tanglefoot® was spread around their base to prevent any ant from
climbing up. Statistical comparisons (Likelihood ratio test for nested
models): different letters indicate significant differences at
P<0.05. Because we surveyed the two last, opposite leaves, the
statistics were calculated from the 30 pairs.
Yet, the ants appear to provide only weak biotic protection to the foliage as we
saw no significant difference in the percentage of foliar surface eaten by
insects between plants from unaltered patches and those from either experimental
treatment (Likelihood ratio test: P = 0.25; Fig. 6).
Discussion
This study indicates the existence of a facultative mutualism between live C.
alatus individuals and the workers of various opportunistic ant species
that visit their CFNs and/or forage for prey on their foliage. Among them,
P. gracilis, C. blandus (both diurnal) and
C. melanoticus (nocturnal) pre-dominated (Fig. 1; Table 1). These species were also recorded in the
same habitat exploiting the EFNs of Passiflora spp. [30].
The circum-floral position of these nectaries encourages ants to actively defend the
reproductive - but apparently not the vegetative - C. alatus
tissues (see [19] for a similar case for a Mexican orchid). This is
consistent with the optimal defense theory predicting that plants invest in the
defense of parts with a high fitness value, such as reproductive organs [12]–[18]. Yet,
concerning the foliage, the plants' anti-insect compounds seem sufficient (see
[31] for
secondary compounds in Gentianaceae), so that biotic protection was not demonstrated
and the rate of defoliation was low in all cases. This can explain why we noted only
a few cases of hemipterans attacking C. alatus.
Also, ant-pollinator conflicts, which can disrupt plant reproduction [22]–[26], seem to be
resolved in this case as the flowers attract pollinators when the ants are less
active (see also [24]). Indeed, it is unlikely that C.
melanoticus workers perturbed bat pollination because 60.5% of
the plants' inflorescences were not visited by ants at night (Fig. 1), whereas nuptial nectar
production is mostly nocturnal [29]. Furthermore, the pollinating bat, Glossophaga
soricina, very common in French Guiana, is also insectivorous, and its
hovering visits are extremely short [29], [32].
Although C. blandus and C. melanoticus also visited
the CFNs, the case of P. gracilis merits particular attention as
its workers were by far the most frequently noted. Known as the
“twig-ant”, this species nests in dried-out, hollow twigs into which the
workers are able to gnaw entrance holes [33], [34]. In this study, P.
gracilis colonies, known to be polydomous (multiple nests) and
polygynous (multiple queens) [35]–[37], nested in several dried-out C. alatus
stems, and each patch probably contained only one colony (confrontation tests;
unpublished results). The workers are territorial and are known to be efficient
predators [36]–[38], attacking other ants experimentally placed on their host
plant [34]; they
can even prey on other ant species (see Fig 1C).
Concerning the plant, by spreading a ring of Tanglefoot® at the base of
C. alatus individuals, we showed that, in general, the ants
protect the flowers, but not the vegetative tissues. Because very similar results
were obtained by eliminating only those ant species nesting in dried-out stems (that
were torn out), we can deduce that the latter, mostly P. gracilis,
account for most of the protection of the C. alatus flowers. We
also noted that dried-out stems sheltering ant colonies can persist for several
years thanks to their structure (they are typically tall, long-lived C.
alatus individuals), the quality of the wood that contains fungicides
[39], and the
anti-termite action of the ants nesting in their base.
Consequently, although it corresponds to a facultative mutualism, this situation is
similar to that involving myrmecophytes and plant-ants. Indeed, C.
alatus likely derives a benefit in terms of fitness because its flowers
are protected, while furnishing food (CFNs) and a favorable nesting site to a
limited number of ant species, mostly P. gracilis. Yet, the hollow,
dried-out internodes lodging ants, related to the phenology of C.
alatus, are not real ‘ant-domatia’ based on the following
definition which applies to the live parts of plants: “plant structures that
appear to be specific adaptations for ant occupation, often formed by the
hypertrophy of internal tissue at particular locations on the plant, creating
internal cavities attractive to ants” [40]. Although they may be located
in hollow thorns or leaf petioles, in leaf pouches and on fruits, most ant domatia
are caulinary; that is to say, live hollow stems and shoots [6], [10]. The facultative but persistent
associations noted in the present study suggest an evolving mutualism, and can shed
light on how such interactions might develop over evolutionary timescales. Indeed,
another case of a non-myrmecophytic plant sheltering ants in persistent, hollow
structures related to the plant's phenology - here inflorescence production -
has been reported in the Araceae [41].
Therefore, we suggest that the character “still standing, dried-out hollow
stems” favors – through facultatively mutualistic ants - the
reproduction (and so the fitness) of both their own offspring and those of their
siblings, all of which corresponds to a case of inclusive fitness in plants.
Inclusive fitness can be defined as the adaptive value (fitness) of an individual,
taking into account not only that individual's own reproductive success, but
also the success of its entire kin (i.e., those bearing some portion of the same
genotype [42]).
The development of the founding C. alatus individuals improves the
ecological niche for future generations through the persistence of their dried-out
stems that provide a nesting site for colonies of a facultative, mutualistic ant.
Then, some individuals from each generation bequeath this improvement to the next
generation. Because the C. alatus genes are expressed beyond their
immediate boundaries through these persistent dried-out stems, one can consider that
this example corresponds to an “extended phenotype” [43] rather than
“niche construction” which implies that genes are not involved in the
legacy [44]
(see also the controversy on this subject in [45]).
Materials and MethodsStudy site
This study was conducted between 2001 and 2010 in French Guiana near the Petit
Saut dam (5°03′39″N, 53°02′36″W). Surveys on the
formation of the C. alatus patches and on the relationship
between stem diameter and plant height were conducted along the last 15 km of
the road leading to the dam, plus the areas of Keren Roch and
Base vie situated 0.4 km and 1 km from the dam,
respectively. The other surveys were conducted on individuals forming patches
along the dirt road leading to Crique Plomb constituting a
narrow, cleared area situated in the middle of the rainforest.
The research undertaken meets all applicable standards for the ethics of
experimentation and research integrity.
Formation of Chelonanthus alatus patches
Between 2001 and 2002, we registered the formation of 15 new C.
alatus patches in different, recently-cleared areas. In each case,
we noted the number of individuals and mapped them. Then, we noted the fate of
the formation of these patches over 6 years, recording three times a year the
numbers of young seedlings, individuals bearing flowers and still-standing,
dried-out stems.
Lifespan of C. alatus individuals and longevity of dried-out
stems
In January 2001, we tagged 15 young individuals bearing four leaves and 15 stems
on dead plants starting to turn brown in order to know the lifespan of
C. alatus individuals and why the dried-out stems do not
decay, but remain standing in the patches. We verified if they were still
standing every 6 months during 2 years. Using calipers, we measured the diameter
5 cm from the ground of all of the stems at the beginning of the study; using
the unpaired t-test, we compared the diameter of the stems that had dried-out
and decayed 12–18 months later with those that remained standing.
We also measured the stem diameter and height of (i) 150
C. alatus individuals that had recently died to establish a
relationship curve between these two variables as well as (ii)
90 “old” dried-out stems selected at random, and
(iii) 53 founding C. alatus individuals
that had also recently died. Using the unpaired t-test, we compared the first
two lots to know if still-standing, dried-out stems are among the tallest and/or
the widest; then the first and the third lots to know if founding individuals
are taller and have a wider diameter than individuals chosen at random. Because
the first lot was compared twice, probabilities were adjusted using the
sequential Bonferroni procedure.
Ant diversity and activity
In order to know which ants species are present in eight C.
alatus patches (2.5-to-5 m×2 m) as well as in the surrounding
vegetation over a 2-m-wide area bordering each side of each patch, we did the
following. First, we placed five pit-fall traps in each patch for 24 h (a total
of 40 pit-fall traps) as it has been demonstrated that the data gathered through
the use of 20 pit-fall traps is robust enough to characterize a habitat in
French Guiana. This method also permits the comparison of sites whose habitat is
disturbed to different degrees (see [46]). We then conducted two
periods of observation during the daytime (10:00–11:00 and
17:00–18:00), and another at night (23:00–01:00) for 10
non-consecutive days (five observers). These periods of observation were chosen
because they correspond to the major periods of activity of diurnal and
nocturnal ant species, respectively, in this area (see [47], [48]).
We also noted which ant species visited the CFNs of each C.
alatus plant. Finally, we collected the ants sheltering in the
hollow stems of all of the C. alatus from the eight patches,
including dried-out individuals, by cutting them at their base and putting each
plant into a separate plastic bag; we then transported everything to the
laboratory.
We used the Sørensen similarity index to compare the ant species visiting
different plants or patches because it gives low weights to outlier values (see
Table 1). In the
Sørensen similarity index (QS = 2C/A+B), A and
B are the number of species recorded in samples A and B, respectively, and C is
the number of species shared by the two samples.
We compared the number of C. alatus individuals visited
diurnally and nocturnally for their CFNs by the different ant species using a
one-way ANOVA followed by a Newman-Keuls post-hoc test for
multiple comparisons.
In order to know if ants nest randomly in the dried-out stems or if they rather
select wide individuals, using the unpaired t-test, we also compared the
diameter of 127 uninhabited stems with 83 others sheltering ants.
Voucher specimens of the ants were deposited in the Laboratório de
Mirmecologia (CPDC collection, CEPEC-CEPLAC, Itabuna, Bahia,
Brazil).
Plant protection
We verified the impact of the ants on C. alatus flowers and
leaves by comparing the percentage of surface eaten by defoliating insects for
three groups of 30 C. alatus plants (55–70 cm tall)
bearing flowers. The objective of the experiment was to eliminate the
possibility for ants to protect live C. alatus from defoliators
through their predatory and/or their territorial behavior. Our experimental
design included three treatments: unaltered patches (control) and two
experimental treatments. Each of these three treatments were included in each of
three different patches (i.e., 3×3 = 9 patches in
total) along 700 m of the Crique Plomb dirt road. In the first
experimental treatment, we tore out all of the dried-out stems, thus eliminating
their ant inhabitants (mostly Pseudomyrmex gracilis). So,
ground-nesting species plus those from the neighboring areas were free to
exploit the C. alatus CFNs. In the second experimental
treatment, we spread a ring of Tanglefoot® at the base of the stems of live
individuals to prevent any ants from climbing up. Because Tanglefoot® is
toxic for plants, we first rolled a 5 cm wide band of aluminum foil around the
base of the stem, and then deposited the Tanglefoot® on the aluminum.
We used the two youngest leaves on each C. alatus (total of 30
pairs of leaves in each of the three replicates: control and the two
experimental treatments; i.e., 30×3 = 90 pairs of
leaves assessed) and verified the percentages of leaf surface destroyed after 20
days following the start of the experiment (at which time, both the flowers and
the leaves were intact). Due to their short lifespan, we obtained only 26
flowers (each from a separate plant individual) from each of the three
replicates. The experiment lasted 5 days, starting before the buds were ready to
open (see Fig 1B) until the
flowers began to wither.
We defined three rates of attack: (1) not at all attacked; (2) up to 25%
of the petals or leaf surface destroyed; and (3) more than 25% of the
petals or leaf surface destroyed.
The results were analyzed using an ordinal regression since the rate of attack
was an ordinal response. The relationship between the rate of attack of a flower
(or a leaf) and the treatment was modeled with a GLM using a probit link [49]. The
link function was selected, among the usual adapted link functions for ordinal
data, according to the Akaike Information Criterion. To avoid confusion due to
an eventual micro-site effect, we alternatively attributed treatments to the
nine patches: control treatment, first treatment, second treatment, control
treatment, etc. We verified the homogeneity of this experimental design and did
not detect a ‘patch effect’ (Likelihood ratio tests;
P = 0.9 for the flowers and P = 0.74
for the leaves).
Statistical analyses were conducted using GraphPad Prism 4.03, Inc. software and
R 2.10.1 software [50].
We are grateful to Andrea Yockey-Dejean for proofreading the manuscript and to five
graduate students (Sarah Groc, David Oudjani, Jean-Michel Martin, Michael Negrini,
and Pierre Uzac) for their participation in the field surveys during which the
preliminary experiments were conducted (2001-2004). We would also like to thank the
Laboratoire Environnement de Petit Saut for furnishing
logistical help.
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