Fricke et al. BMC Ecol (2019) 19:29 https://doi.org/10.1186/s12898-019-0245-9 BMC Ecology

RESEARCH ARTICLE Open Access No evidence of fowering synchronization upon foral volatiles for a short lived annual species: revisiting an appealing hypothesis Ute Fricke1,2,3, Dani Lucas‑Barbosa2,4 and Jacob C. Douma1,2*

Abstract Background: Self-incompatible require simultaneous fowering mates for crosspollination and reproduc‑ tion. Though the presence of fowering conspecifcs and agents are important for reproductive success, so far no cues that signal the fowering state of potential mates have been identifed. Here, we empirically tested the hypothesis that plant foral volatiles induce fowering synchrony among self-incompatible conspecifcs by accelera‑ tion of fowering and fower opening rate of non-fowering conspecifcs. We exposed Brassica rapa Maarssen, a self- incompatible, in rather dense patches growing annual, to (1) fowering or non-fowering conspecifcs or to (2) foral volatiles of conspecifcs by isolating plants in separate containers with a directional airfow. In the latter, odors emitted by non-fowering conspecifcs were used as control. Results: Date of frst bud, duration of frst fower bud, date of frst fower, maximum number of open fowers and fower opening rate were not afected by the presence of conspecifc fowering neighbors nor by foral volatiles directly. Conclusions: This study presents a compelling approach to empirically test the role of fower synchronization by fo‑ ral volatiles and challenges the premises that are underlying this hypothesis. We argue that the life history of the plant as well as its interaction with pollinators and , as well as the distance over which volatiles may serve as synchronization cue, set constraints on the ftness benefts of synchronized fowering which needs to be taken into account when testing the role of foral volatiles in synchronized fowering. Keywords: Flowering synchronization, Flowering onset, Phenology, Plant–plant communication

Background [3]. Te most important exogenous signals are tempera- Te transition from the vegetative to fowering stage is ture, light conditions (such as photoperiod) and water irreversible and its timing is crucial [1, 2]: environmental availability [4]. For example, prolonged drought can be conditions should be favorable, and for obligate outcross- associated with accelerated fowering [5]. Shade and light ing species, fowering conspecifcs and pollinating agents quality can both play a role in fowering onset accelerat- to transfer need to be present in the environment. ing and delaying fowering, depending on the plant spe- Tus, fowering synchronization is critical for outcross- cies [3]. Furthermore, fungal, viral and bacterial pathogen ing species. infection as well as attack can alter fowering Te fowering phenology of a plant depends on the phenology [6]. Endogenous factors that play a role in interplay of endogenous signals and exogenous signals fowering phenology are plant hormones, sugar levels and the genetic makeup of the plant [3]. Diferences in the genetic makeup may lead to variation in the indi- *Correspondence: [email protected] vidual timing of fowering within a population [7], while 1 Centre for Crop Systems Analysis, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands exogenous biotic and abiotic signals and their interac- Full list of author information is available at the end of the article tions may constrain fowering time of populations [6].

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fricke et al. BMC Ecol (2019) 19:29 Page 2 of 9

Te intraspecifc variation in fowering onset can be P = 0.210]. Te frst fower opened at about large [8] and constrains pollen transfer between individu- 47.4 ± 4.7 days from sowing [ANOVA, F(1,38) = 1.385, als. Given the need of pollen from conspecifcs, cues that P = 0.247]. As in the neighboring experiment, the traits signal the presence of fowering mates might have estab- related to fowering phenology measured in the two- lished to tune fowering among mates [9, 10]. cylinder experiment were similar irrespectively of the Floral volatiles are outstanding candidate cues to signal foral volatiles exposure [ANOVA, E: F(1,25) = 0.008, the presence of fowering mates, for a number of reasons. P = 0.931; FB: F(1,24) = 1.704, P = 0.204; duration of First, foral volatiles are known to afect the physiology of frst fower bud (FD): F(1,24) = 2.544, P = 0.124; FF: neighboring plants, e.g. by inhibiting root growth [11]. F(1,24) = 0.415, P = 0.526]. Importantly, no interactions Second, plant volatiles can promote defenses and growth were found between the trial number and the tested in neighboring plants [12–15]. Tird, foral volatiles are traits, but the traits related to fowering phenology dif- often produced in higher quantity than leaf volatiles fered between trials in the neighboring experiment on which makes them likely detectable by nearby conspecif- average up to 8.5 days. In the two-cylinder experiment ics [16]. Finally, foral volatiles are a reliable cue exploited only FD and FF difered between trials by an average of by pollinators to locate fowers [17–19]. Floral volatile up to 2.5 days. Most of the traits related to fowering emission is often highest when a fower is ready for pol- phenology had slightly lower values in the two-cylin- lination, which makes unfertilized fowers particularly der experiment when compared with the neighboring attractive to pollinators [20, 21]. Unfertilized fowers are, experiment presented above. therefore, more attractive to pollinating agents than ferti- When modeling the number of open fowers over time, lized fowers and indeed foral scent changes or levels of the exposure to fowering or non-fowering (1) neighbor- after fertilization [22, 23]. ing plants or (2) their odors only did not add explanatory Whether cues of conspecifcs directly afect fower- value to the model [lr-test: (1) P = 0.627, (2) P = 0.818]. ing time has never been investigated, although there is Both groups of B. rapa plants, exposed to fowering some indirect evidence that supports this idea [10]. For and non-fowering neighboring conspecifcs, reached instance, butterfy egg deposition on Brassica nigra sped an equal maximum number of open fowers [Asym, (1) up fowering [24] and seed production of herbivore- P = 0.687, (2) P = 0.468], started fowering at a similar infested plants and its non-infested neighbors, whereas time [xmid, (1) P = 0.226, (2) P = 0.639] and opened their foral volatile composition was changed upon exposure fowers similarly quickly [scal, (1) P = 0.558, (2) P = 0.706] to butterfy eggs and caterpillars [16, 25]. Tus, additional (Fig. 1; Additional fle 1). However, the onset of fowering foral volatile-based mechanisms may underlie fowering difered between the two subsequent trials in both exper- phenology. iments [xmid, (1) P < 0.001, (2) P = 0.007]. A model on the In this study, we explored for the frst time whether fo- neighboring experiment data that included trial number ral volatiles can change fowering phenology of younger in scal and the other parameters led to ftting problems, conspecifcs, i.e. whether foral volatiles can afect the wherefore the covariate trial number was excluded from onset of fowering and the rate of fower opening. We the explanation of scal in the neighboring experiment revisited the hypothesis by [10] of fower synchronization model. In case of the (2) two-cylinder experiment, these and challenged its premise that it is benefcal for veg- observations were made under consideration of a fac- etative plants to advance the onset of fowering and the tor describing the number of plants elongated prior to fower opening rate in response to foral volatiles. exposure (preE). Tis factor was added as plants, which just had started to elongate, were used in the experiment Results due to a shortage of not yet elongated plants. A model Te traits related to fowering phenology days from with preE as explanatory factor was preferred (lr-test: sowing until stem elongation (E), days from sow- P < 0.001). Plants showing elongated stems at the initia- ing until the frst fower bud (FB) and days from sow- tion of the experiment tended to reach a higher NOF­ max ing until the frst fower opening (FF, fowering time) than plants initiating stem elongation later (P = 0.074). were not afected by the exposure to fowering or Maximum fower opening rate (MFR) was simi- non-fowering neighboring conspecifcs (neighbor- lar between plants exposed to (odors of) fowering or ing experiment). Stem elongation manifested on non-fowering emitters in both experiments (Fig. 2). In average 38.3 5.1 (mean SD) days from sowing the neighboring experiment MFR reached on average ± ± about 6.4 1.4 (mean SD) fowers per day [ANOVA, [ANOVA, F(1,41) = 0.214, P = 0.646]. Te frst fower ± ± bud appeared at about 42.1 5.3 days from sow- F(1,37) = 0.014, P = 0.905], while in the two-cylinder ± experiment a maximal rate of average about 11.5 5.7 ing [ANOVA, F(1,41) = 0.002, P = 0.967] and lasted ± for about 6.1 ± 1.1 days [ANOVA, F(1,38) = 1.629, Fricke et al. BMC Ecol (2019) 19:29 Page 3 of 9 r F a NF P = 0.247 Emitte 35 40 45 50 55 60 65 Days till the first flower

b c er s

ow P = 0.744 60 60

40 40

20 20 Number of open flowers 0 0 Maximum number of open fl 35 40 45 50 55 60 65 NF F Days since sowing Emitter

F d NF P = 0.093 Emitter FNF Emitte r 35 40 45 50 55 60 65 Days till the maximum number of open flowers Fig. 1 Relation between the number of open fowers and time when Brassica rapa plants were exposed to fowering (F; yellow) or non-fowering (NF; green) conspecifc emitters in the neighboring experiment. a Maximum number of days until the frst fower of B. rapa; b relationship between the number of open fowers and time until the maximum number of open fowers was reached for individual plants exposed to F or NF emitters modeled by a nonlinear mixed efects model based on the logistic function. Likelihood-ratio test showed no diferences between Emitters; c

maximum number of open fowers (NOFmax); d number of days until the maximum number of open fowers was reached; Presented P-values for the type of emitter are based on a two-way ANOVA including the trial number at α 0.05 =

fowers per day was reached [ANOVA, F(1,24) = 2.221, 25 a b P = 0.149]. P = 0.158 20 Discussion

15 Tis study is, to the best of our knowledge, the frst to

er opening rate evaluate the potential role of foral volatiles as cues to

ow 10 P = 0.905 synchronize fowering among conspecifcs. We designed two diferent types of experiments, one mimicking a feld 5 situation with fowering and non-fowering plants next to each other, and one in which plants were exposed to 0 Maximum fl volatiles only, using a system of connected vessels, to test NF F NF F the efect of foral volatiles on conspecifc neighboring Emitter Emitter plants of B. rapa. Flowering phenology of B. rapa was not Fig. 2 Maximum fower opening rates (mean SD) of B. rapa ± afected by foral volatiles under the conditions tested. upon exposure to (odors of) fowering (F) and non-fowering (NF) conspecifc emitters; a neighboring experiment; b two-cylinder Flowering synchronization may be achieved in difer- experiment; P-values presented are based on the type of emitter in a ent ways. Plants may shorten the time between the veg- two-way ANOVA with α 0.05 including the trial number etative stage and bud formation; they may shorten the = Fricke et al. BMC Ecol (2019) 19:29 Page 4 of 9

time between bud and fowering onset or increase the Second, the payof of synchronous fowering may rate in which new fowers open. To date, it is not known as well depend on the life history of the plant. A study if and when during the transition from the vegetative to demonstrated that plants that produce a large number of the fowering stage a plant is responsive to volatile cues. fowers in a short time (mass fowering) showed highest Terefore, we measured a number of indices that could synchrony [34]. Indeed, an ofset of a few days in fow- point to synchronization from as early as 8 days prior ering in mass fowering plants will have a larger efect to the formation of the frst bud till 12 days after fow- on pollination success compared to constant fowering ering started. Brassica rapa needs a short period of time plants. In addition, synchrony was observed in species between bud formation and fower formation (4 days) that fowered in response to an unambiguous fowering which leaves little room for adjusting the fowering onset cue (e.g. heavy rain), or where buds remained dormant upon a volatile cue when the buds are already formed. until a specifc cue became available [34]. For species Tus, any efect on synchronization would have likely that attain synchrony through responding to a specifc been observed in the date of the frst bud or the open- environmental cue, it is not clear what information value ing rate of the fowers. A power analysis showed that can be added by foral volatiles, and whether there will sample sizes of > 80 would have been needed to be able be selection for fower synchronization by foral vola- to show diferences in day of frst bud with 90% certainty tiles. Furthermore, one could argue that a minimal at 0.05 signifcance level. Terefore, if any diference was fower duration of the emitter plant is needed to be able detected, the ftness beneft of the earlier onset of fower- to induce fowering of conspecifcs by its volatiles and ing could be questioned. exchange pollen. B. rapa [35] fowers for ~ 30–40 days Floral volatiles may be perceived by any organism pre- with the majority of the fowers produced in ~ 20 days sent in the environment. Conspecifc plants may use this and substantial variation across individuals [8], which cues and beneft of the fact that foral volatiles play a pri- should give a window of opportunity for synchroniza- mary role in mediating interactions with pollinators. Vol- tion. Alternatively, synchronization by volatiles may be atile production is often highest prior to pollination, and not needed as the chance that the fowering schedules of the blend may change upon pollination [20, 23] and in two individuals will overlap is large. Tus, for this spe- this way plants maximize reproduction by guiding polli- cies, crosspollination may be assured by fowering over a nators to fowers that have not been yet pollinated. In our relatively long period [25] and responding to foral vola- experiment, exclusion of pollinators from the experiment tiles may only become critical for this species to ensure prevented post-pollination efects and we assume we had reproduction in the presence of herbivores. maximized the availability of foral volatiles cues. Tird, adjusting the fowering time to an earlier fow- Our study questions the hypothesis of fower synchro- ering plant may imply that a plant should fower before nization by volatile cues. Tis hypothesis is based on the it is optimal in terms of accumulated leaf biomass to premises that it is benefcial for conspecifcs to fower maximize seed production [36]. Hence, earlier fowering simultaneously, and that foral volatiles provide reliable may be benefcial in terms of increased pollination suc- cues of the presence of fowering conspecifcs plants. cess, but may be suboptimal in terms of the amount of First, the beneft of synchronous fowering depends very biomass accumulated and the maximum seed produc- much on other trophic levels [26, 27]. Synchronous fow- tion attainable, unless plants are subjected to stress with ering may result in more efective transfer of pollen by the risk of missing out on an opportunity to reproduce. pollinators compared to asynchronous fowering plants Te cost of fowering earlier probably limits the extent to [28], but too many fowering plants at the same time may which plants synchronize or invest in synchronization. lead to competition for pollinators such that asynchro- Finally, even though there is substantial evidence that nous fowering is an advantage [26, 29, 30]. Te same foral volatiles provide reliable cues of fowering and that holds for forivores; synchronous fowering may decrease pollinators can use olfactory cues to locate host plants the probability of being eaten [28], but the opposite has [37, 38], evidences, however, showing that the distance also been recorded: asynchronous fowering plants are over which foral volatiles disperse in meaningful con- more likely to escape fower herbivores [31]. B. rapa is centrations for conspecifc plants is limited, and may eaten by specialist herbivores, such as Pieris rapae and depend on the environmental conditions, such as ozone, Pieris brassicae, who feed both on leaves and fowers. wind speed and other canopy conditions [39, 40]. Plant However, later instars prefer fowers over leaves despite volatiles as induced upon herbivory have been shown the fact that fowers contain up to fve times more glu- to induce a response in neighboring plants at distances cosinolates than leaves [32, 33]. However, whether syn- up to 60 cm in bushy vegetation [41]. Even though foral chronous fowering is benefcial for B. rapa has not been volatiles are often emitted in higher amounts compared studied. to volatiles emitted from leaves [16], its ability to serve Fricke et al. BMC Ecol (2019) 19:29 Page 5 of 9

as synchronization cue does probably not extend to the in diameter were used. Seeds were stratifed at 5 °C for patch level. However, from a gene fow perspective, syn- 4 days. One-week old seedlings were transplanted indi- chronization between patches is probably more impor- vidually to 3-L pots (φ 17 cm) flled with potting soil. tant than within patches [25, 42]. Te greenhouse compartment was conditioned to 21 °C Tus, the life history of the plant as well as its interac- during the day and to 15 °C at night. Te plants were tion with pollinators and insect herbivores, and the dis- watered when needed. Plantings were staggered over a tance over which volatiles may serve as synchronization period of 5 weeks with a weekly sowing to achieve con- cue may set constraints on the ftness benefts of fower- tinuously available vegetative and fowering plants for the ing in synchrony. Tis raises the question which species experiment. likely synchronize upon foral volatiles. We suggest to use Te frst experiment was carried out in a separate representatives of the bromeliads, as they fower once greenhouse compartment conditioned to 20 °C during in a lifespan and die after sexual reproduction [43] and, the day and to 16 °C at night. Te second experiment exceptionally, fowering can be induced through ethylene was run in the same compartment as used for growing exposure [44, 45]. Alternatively, a stress-induced change the plants prior to the experiment. To avoid foral volatile in foral volatile composition, such as, upon plant expo- exposure of the receiver plants prior to the experiment, sure to herbivore attack may induce fowering both in planned receiver plants were kept up-wind of the fower- the attacked plant as well as its surrounding conspecif- ing plants with respect to the greenhouse ventilation and ics, such that reproduction is guaranteed before forivory at least at 2.5 m distance. Te smell of fowering B. rapa takes place [25]. could not be perceived by the human nose at the posi- tions, where receiver plants were grown, and we assume that receiver plants were not exposed to foral volatiles Conclusions prior to the experiment or, if so, at lower dose than under Results of this study showed that foral volatiles did not natural conditions where B. rapa plants generally occur accelerate foral transition or fowering and did not afect in dense patches. the fower opening rate of neighboring 2-week younger conspecifc B. rapa plants. Tis study revisits the hypoth- Set ups of the experiments esis of fower synchronization by foral volatiles by chal- Two experiments were carried out to test whether (ini- lenging the premises that are underlying this hypothesis. tially) vegetative B. rapa plants respond to fowering We argue that the role of foral volatiles in attaining syn- conspecifc neighboring plants and foral volatiles of con- chrony must be seen in the context of the benefts and specifcs. In the frst experiment, plants were placed in constraints that afect fowering synchrony, in particu- proximity of fowering conspecifc plants, hereafter called lar the life history of the plant and its interaction with neighboring experiment (Fig. 3a). In the consecutive sec- mutualists and antagonists. Our study ofers an approach ond experiment, plants were placed in separate contain- to empirically test the role of fower synchronization by ers with directional airfow (two-cylinder setup, Fig. 3b). foral volatiles and we discuss when we expect synchro- Both experiments involved emitter and receiver plants. nizing fowering to be advantageous, opening further Emitter plants had at least two fower heads each, and in research avenues. total at least 30 open fowers at the start of the exposure. Te fowering emitter plants were on average 14 days Methods older than conspecifc receiver plants. Non-fowering Plant cultivation conspecifc neighboring plants, or their odors, served as Brassica rapa Maarssen is an insect-pollinated, obligate the control. Tese non-fowering emitter plants included outcrossing annual (biannual) plant, which in nature plants ranging from no stem elongation until branch- grows as an early successional plant in high density ing and were replaced before they started fowering by patches [Lucas-Barbosa D, personal observation]. Fur- another non-fowering plant. Receiver plants were in thermore, the fowering phenology and foral volatiles of their vegetative stage and did not show stem elongation B. rapa are relatively well studied as well as its fowering at the day plants were randomly selected for the experi- traits [8, 33, 46, 47]. B. rapa seeds were obtained from ment. Te initial vegetative receiver plants and the non- a self-incompatible wild accession (following national fowering emitter plants were randomly selected from the guidelines for seed collection and storage). Tese B. available plants fulflling the criteria 32 to 39 days from rapa seeds were sown in germination boxes with a mix- sowing. ture of potting soil and sand (1:1 v/v). To narrow down the genetic variance and to fatten diferences in start- ing conditions of the plants, only seeds of 1.1 to 1.3 mm Fricke et al. BMC Ecol (2019) 19:29 Page 6 of 9

Fig. 3 Layout of the two experiments used to test the impact of a fowering emitter plants (E) and b foral volatiles on a conspecifc neighboring receiver plant (R). Non-fowering emitter plants and their odor were used as control

(1) Neighboring setup system (Fig. 3b). Te cylinders were closed at the top with To investigate the impact of fowering plants on traits transparent foil forming a dome. Te two-cylinder setup related to fowering phenology and the fower opening encompassed a total volume of about 1.5 m3. A ventila- rate of neighboring conspecifcs, two fowering or two tor blew air from the greenhouse compartment through non-fowering emitter plants were placed next to a veg- the emitter container into the receiver container where etative receiver plant at a distance of 7 cm for a period the air was then sucked out. Te ventilator was fxed in of 21 days (Fig. 3a). Te exposure of vegetative plants to the inlet to provide overpressure in the connected ves- other vegetative or non-fowering plants was used as con- sels and make sure that air moved from the emitter to the trol. Two fowering or two non-fowering (control) emit- receiver. Te suction was set to about 400 mL min−1. Te ter plants were chosen to increase scent intensity and exposure was maintained for 19 days. Data was collected to mimic the patchy conditions in which they naturally in two trials with seven replicates per type of emitter occur. Each replication was enclosed by transparent foil and trial. Te setup was validated through volatile col- to block horizontal air movement between diferent types lection and subsequent GC–MS (Additional fles 2, 3) of of emitter and replicates. During the experiment, we an empty receiver container, when two fowering plants made sure that the receiver and emitter plants were not were placed in the emitter container and when the whole touching each other and that the emitter plants were not system was empty. As positive control a dynamic head- overtopping the transparent enclosure. Te experiment space collection was done on two single fowering B. rapa was repeated twice in time with 10 and 12 replicates per plants. type of emitter, respectively. Measurements (2) Two‑cylinder setup Troughout the experiments, traits related to fowering Te aim of this experiment was to test the efect of phenology were measured on (focal) receiver plants, or foral volatiles on fower phenology of neighboring were derived from these measurements, to get a complete conspecifcs. Equivalent to the neighboring set up experi- picture of the hypothesized efect of foral volatiles on mental plants were exposed to odors of either two fower- fowering phenology. Te following traits were measured: ing plants or to two non-fowering plants. In the previous Te number of days (i) from sowing until stem elongation neighboring experiment, however, factors besides the (E), (ii) until the frst fower bud (FB) and (iii) until the foral volatiles, such as shading in the fowering emitter frst fower opening (FF, fowering time). From this, the treatment might have confounded the fowering emitter duration of the frst fower bud (FD) was calculated as the efect. To mitigate side efects along the emitter efect, time diference between the frst fower bud and the frst this two-cylinder, more controlled set up was used as fower opening. In addition, the number of open fow- follow up. In this case, the receiver plant and the two ers (NOF) was counted on a daily basis to investigate the emitter plants were placed in two separate polyethylene impact of foral volatiles on the fower opening rate. For cylinders (φ 42 cm, height: 1 m) connected through a this, a fower was considered open until the style became tube (φ 10 cm, length ~ 3 cm) and a ventilation-sucking clearly longer than the anthers, and the petals started to Fricke et al. BMC Ecol (2019) 19:29 Page 7 of 9

wilt or bend from their orthogonal position. From NOF distributions (through so-called generalized nonlinear the maximum number of open fowers ­(NOFmax) was mixed efects models), are currently not implemented in derived per plant individually. In some cases, ­NOFmax commonly available statistics packages, the relationship was clearly reached, but in other cases only a relative between NOF and time was assumed to have a normal maximum might have been reached as the NOF­ max was error distribution with a power variance structure (var- limited by the observation period. Power) [48]. Te logistic relationship between NOF and time (x) Data analysis was modeled through three parameters: Te impact of fowering conspecifc neighboring plants Asym or foral volatiles on non-fowering conspecifcs was f (x) = 1 + e(xmid−x)/scal (1) tested by testing diferences in a number of traits (E, FB, FF and FD). For these traits a two-way ANOVA was used Upper asymptote (Asym), x-intercept of the infection with type of emitter and trial as factors. point (xmid) and steepness of the logistic curve (scal) Furthermore, a nonlinear mixed efects (nlme) model (Eq. 1, Fig. 4). A higher value for the parameter Asym was used to ft a logistic relationship between the num- leads to a higher right-side asymptote. An increase of ber of open fowers and time when exposed to fowering xmid is linked to a right shift of the entire curve on the and non-fowering plants. Te nonlinear mixed efect x-axis. A lower scal causes a steeper curve, which is all model allows to analyze the non-linear relationship in the else equal. data more precisely based on the multiple parameters, For both experiments, the covariates “type of emit- which makes it advantageous over a repeated measures ter” and trial number were used to explain Asym, xmid ANOVA. A mixed efects model was taken as multiple and scal. In the two-cylinder experiment, an additional observations were made on individual plants requiring covariate was introduced correcting for the use of already the individual plant as random efect. Te trial num- elongated plants prior to the exposure (preE). Te signif- ber was included as fxed efect. Observations made on cance of the covariates was compared with a Likelihood NOF after plant individuals reached their maximum ratio test (lr-test) at α = 0.05. number of fowers were discarded to allow comparison To assess the impact of foral volatiles on the maximum between individuals. Because models allowing for count fower opening rate (MFR), the frst derivative (Eq. 2) of the sigmoidal model (Eq. 1) was taken and evaluated under the assumption that all fowers had equal lifespan. 0 Asym maximum flower opening rate : f ′(x) = Asym 4 · scal (2) 05 MFR was calculated for each plant by summing the fxed efects and random efects of the nlme models, which included the covariates type of emitter, trial num- 04 xmid ber and additionally preE in the two-cylinder experiment as well as the individual plant as random efect. MFRs y scal*2 were tested on an emitter efect by a one-way ANOVA at 03 α = 0.05. For statistical analysis, the software R was used [49] with the packages nlme [50] and lmtest [51]. 02

Additional fles 01

Additional fle 1. Relation between the number of open fowers and 40 42 44 46 48 50 time when Brassica rapa plants were exposed to odors of fowering or x non-fowering conspecifc emitters in the two-cylinder setup (Pendant to Fig. 4 Describes the relationship between the number of open Fig. 1 for the two-cylinder setup). Relation between the number of open fowers and time that results in a sigmoid curve. The three parameters fowers and time when Brassica rapa plants were exposed to odors of describe the upper asymptote (Asym, blue), the x-intercept of the fowering (F; yellow) or non-fowering (NF; green) conspecifc emitters in the two-cylinder setup. a) Maximum number of days until the frst fower infection point (xmid, red) and the steepness (scal, grey) of the of B. rapa; b) Relationship between the number of open fowers and time sigmoid curve Fricke et al. BMC Ecol (2019) 19:29 Page 8 of 9

Consent for publication until the maximum number of fowers was reached for individual plants Not applicable. exposed to F or NF emitters modeled by a nonlinear mixed efects model based on the logistic function. Likelihood-ratio test showed no diferences Competing interests between Emitters; c) Maximum number of open fowers; d) Number of The authors declare that they have no competing interests. days until the maximum number of open fowers was reached; Presented P-values for the type of emitter are based on a two-way ANOVA including Author details the trial number at α 0.05. 1 = Centre for Crop Systems Analysis, Wageningen University, Droevendaalses‑ Additional fle 2. Volatile organic compounds collected in the headspace teeg 1, 6708 PB Wageningen, The Netherlands. 2 Laboratory of Entomology, of Brassica rapa. Volatile organic compounds (VOCs) collected from the Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The headspace of Brassica rapa (BR) (for details on the method see Bruinsma Netherlands. 3 Present Address: Department of Animal Ecology and Tropical et al. [16]). The ratio of the peak areas in the gas chromatogram was calcu‑ Biology, Biocentre, University of Würzburg, Würzburg, Germany. 4 Present lated of VOCs collected from an empty receiver cylinder exposed to two Address: Laboratory of Bio‑Communication & Ecology, ETH Zurich, Zurich, fowering plants in the emitter cylinder (BR) and from an empty receiver Switzerland. cylinder when the emitter cylinder was left empty (BG). VOCs with a ratio of 0.8 (not grey) in a sample were determined by total ion composition Received: 8 November 2018 Accepted: 19 July 2019 (TIC)≤ and retention index (RI). The sample number is indicated when the identity of a VOC was confrmed (conf.). VOCs with a BG/BR ratio 0.8, which were confrmed at least in one sample, are marked yellow.≤ Con‑ frmed VOCs by TIC in the B. rapa samples were additionally confrmed by squaring RI with literature and their peak areas in the gas chromatogram References are presented by BR and BG in Additional fle 3. 1. Huijser P, Schmid M. The control of developmental phase transitions in plants. Development. 2011;138:19. Additional fle 3. Testing of the two-cylinder experiment - Peak areas of 2. Friedman J, Rubin MJ. All in good time: understanding annual and peren‑ volatile organic compounds collected from an empty receiver cylinder nial strategies in plants. Am J Bot. 2015;102:4. exposed to odors of two fowering Brassica rapa and from an empty (emit‑ 3. Srikanth A, Schmid M. Regulation of fowering time: all roads lead to ter) cylinder. Peak areas of volatile organic compounds collected from an Rome. 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