CAS an essential Laboratory of Tropical woodland Ecology, Xishuangbanna Tropical botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan, 666303 China

Center for Plant Ecology, main point Botanical Gardens, Chinese Academy that Sciences, Mengla, 666303 China

Kunming inspection & style Institute that State Forestry and Grassland Administration, Kunming, Yunnan, 650216 China

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Spencer C. H. Barrett

Department of Ecology and also Evolutionary Biology, university of Toronto, 25 Willcocks Street, Toronto, ON, M5S 3B2 Canada

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Ji-Qin Yang

CAS an essential Laboratory the Tropical forest Ecology, Xishuangbanna Tropical factory Garden, Chinese Academy the Sciences, Mengla, Yunnan, 666303 China

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Jian-Li Zhao

Yunnan crucial Laboratory of tree Reproductive Adaption and also Evolutionary Ecology, Yunnan University, Kunming, Yunnan, 650091 China

Laboratory the Ecology and also Evolutionary Biology, State key Laboratory for Conservation and also Utilization that Bio-Resources in Yunnan, Yunnan University, Kunming, Yunnan, 650216 China

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Yong-Mei Xia

CAS crucial Laboratory that Tropical woodland Ecology, Xishuangbanna Tropical botanical Garden, Chinese Academy that Sciences, Mengla, Yunnan, 666303 China

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Corresponding Author

Qing-Jun Li

Yunnan key Laboratory of tree Reproductive Adaption and Evolutionary Ecology, Yunnan University, Kunming, Yunnan, 650091 China

Laboratory that Ecology and also Evolutionary Biology, State key Laboratory because that Conservation and Utilization the Bio-Resources in Yunnan, Yunnan University, Kunming, Yunnan, 650216 China

Author because that correspondence:

Qing-Jun Li

Tel: +86 871 65030660

Yong-Li Fan

CAS an essential Laboratory the Tropical forest Ecology, Xishuangbanna Tropical factory Garden, Chinese Academy that Sciences, Mengla, Yunnan, 666303 China

Center because that Plant Ecology, core Botanical Gardens, Chinese Academy the Sciences, Mengla, 666303 China

Kunming inspection & design Institute of State Forestry and Grassland Administration, Kunming, Yunnan, 650216 China

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Spencer C. H. Barrett

Department the Ecology and Evolutionary Biology, college of Toronto, 25 Willcocks Street, Toronto, ON, M5S 3B2 Canada

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Ji-Qin Yang

CAS key Laboratory the Tropical woodland Ecology, Xishuangbanna Tropical factory Garden, Chinese Academy of Sciences, Mengla, Yunnan, 666303 China

Search for an ext papers by this author

Jian-Li Zhao

Yunnan an essential Laboratory of tree Reproductive Adaption and Evolutionary Ecology, Yunnan University, Kunming, Yunnan, 650091 China

Laboratory that Ecology and also Evolutionary Biology, State vital Laboratory because that Conservation and also Utilization the Bio-Resources in Yunnan, Yunnan University, Kunming, Yunnan, 650216 China

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Yong-Mei Xia

CAS key Laboratory that Tropical forest Ecology, Xishuangbanna Tropical factory Garden, Chinese Academy that Sciences, Mengla, Yunnan, 666303 China

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Corresponding Author

Qing-Jun Li

Yunnan crucial Laboratory of plant Reproductive Adaption and also Evolutionary Ecology, Yunnan University, Kunming, Yunnan, 650091 China

Laboratory that Ecology and Evolutionary Biology, State an essential Laboratory because that Conservation and Utilization the Bio-Resources in Yunnan, Yunnan University, Kunming, Yunnan, 650216 China

Author because that correspondence:

Qing-Jun Li

Tel: +86 871 65030660

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Water-mediated fertilization is ubiquitous in early on land plants. This genealogical mode that fertilization has, however, typically been considered to have actually been lost during the evolutionary background of terrestrial flower plants. Us investigated reproductive mechanisms in the subtropical ginger Cautleya gracilis (Zingiberaceae), which has actually two pollen problems – granular and filiform masses – depending upon external conditions. We tested even if it is rain reinvented granular pollen right into filiform masses and also whether this then promoted pollen-tube growth and also fertilization that ovules. Using speculative manipulations in the field we investigated the donation of water-mediated fertilization come seed production. Rain resulted in granular pollen to kind filiform masses of germinating pollen tubes, i m sorry transported sperm come ovules, bring about fertilization and also seed set. Flowers exposed come rain developed significantly an ext seeds 보다 those safeguarded from the rain, which kept granular pollen. Insect pollination made just a limited contribution to seed set because merganser conditions limited pollinator service. Our outcomes reveal a formerly undescribed fertilization system in flowering plants entailing water-mediated fertilization created by rain. Water-mediated fertilization is likely to it is in adaptive in the subtropical monsoon settings in i m sorry C.gracilis occurs by ensuring reproductive assurance when persistent rain stays clear of insect-mediated pollination.


Introduction

Green algae space the far-off ancestors come terrestrial plants, and fertilization in this group occurs in aquatic atmospheres mediated by water (Bold & Wynne, 1985; Clifton & Clifton, 1999). In early on land plants, such as ferns, lycopods, horsetails and also bryophytes (mosses, liverworts and also hornworts), fertilization relies on a constant layer of moisture, making this taxa poorly adapted to terrestrial problems that experience expanded dry durations (Longton & Schuster, 1983; Glime, 2013). Thus, water-mediated fertilization has been taken into consideration a unique feature of early land plants in which motile sperm are the agents of male gamete dispersal (Niklas, 1997; raven etal., 2005). By contrast, in seed plants (gymnosperms and also angiosperms), this suspended on water is highly limited because pollen, the highly reduced male gametophyte generation, is the agent of male gamete dispersal and is more resistant to desiccation than sperm (Pacini, 2000). In most seed tree fertilization is accomplished following pollen dispersal by wind or animals (Faegri & valve der Pijl, 1971), although in the c.350 species of hydrophilous angiosperms (0.1% that the angiosperms), water is the primary agent the pollen dispersal (Sculthorpe, 1967; Cox, 1988; Ackerman, 2000).

Currently, the prevailing check out is that water-mediated fertilization has actually been lost throughout the evolutionary background of terrestrial flowering plants due to the fact that it is nonadaptive (Cronberg etal., 2006). Indeed, fertilization instrument independent that water represent a far-reaching reproductive invention that is intimately associated with the ecological and also evolutionary success of seeds plants. Nevertheless, flowers have actually numerous methods to encounter water, particularly in the kind of rain, increasing the opportunity that water-mediated fertilization may have been overlooked in some terrestrial angiosperms and could have actually adaptive value in settings that experience persistent rain.

Flowering plants release pollen to the external environment throughout anthesis. Pollen is dehydrated and also quiescent in ~ this stage, protected and encased in a fairly impermeable tough sporopollenin coat maintaining viability throughout dispersal. Pollen grains subsequently rehydrate and also germinate after ~ landing top top stigmatic surfaces, few of which room wet (reviewed in Taylor & Hepler, 1997; Franklin-Tong, 2010). It is typically fatal because that pollen to contact water prior to adhering come the stigma, and also diverse morphological and also biochemical instrument have advanced to safeguard pollen indigenous rain (Eisikowitch & Woodell, 1974; Mao & Huang, 2009). Pollen grains deserve to burst in much less than a few minutes in numerous types (Corbet & Plumridge, 1985; Mao & Huang, 2009), and indeed water is used as an emasculation device for the production of hybrid noodle (Gossypium hirsutum), because cotton pollen loser viability ~ a brief exposure to water (Burke, 2002). Water has actually been a significant abiotic selective force in the reproduction biological of flowering tree (Jones, 1967; sun etal., 2008; Mao & Huang, 2009; Wang etal., 2010), with most research focused on methods that ameliorate the harmful impacts of water on pollination and mating. By contrast, the possibly positive effects of water on plant reproduction in terrestrial angiosperms have been greatly ignored, as well as the noticeable requirements of water for plant growth.

Here, us report a formerly unknown water-mediated fertilization device in a terrestrial flower plant. Cautleya gracilis (Smith) Dandy (Fig.1a), a perennial herb in the ginger family members (Zingiberaceae), occurs in moist valleys over 1800m asl in subtropical southwestern China. Flowering occurs from June to August throughout the monsoon season. Individual flowers open in the at an early stage morning and wither during late evening. In spite of their brief duration of anthesis, many flowers endure rain owing to the season in which they flower. In ours preliminary monitorings of stamens in the field, we uncovered two pollen conditions: granular (Fig.1b) and also a filiform massive (Fig.1c) relying on external weather conditions. This exploration motivated united state to advice the theory that pollen transformation was induced by water (rain) and also played a vital role in reproduction.

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Figure 1
Open in figure viewerPowerPoint
Water-mediated fertilization in Cautleya gracilis. (a) Flower the C.gracilis; (b) anther in dry condition illustrating granular pollen on the anther and closely associated stigma; (c) anther and stigma from a flower subjected to rain mirroring mass filiform pollen tubes; (d) micrograph native a flower based on rain; the arrow identifies pollen pipe elongated indigenous a normally wetted anther in the direction of the stigma; (e) summary micrograph from a flower subjected to rain, showing countless pollen pipe that have grown native the anther v the stigma into the style. Because that (d) and (e), the tissue was stained v aniline blue and also observed under a fluorescence microscope.
We start by documenting the floral biological of C.gracilis, consisting of pollinator observations, compatibility status and the facility for autonomous self-pollination and also the timing of this process. Having established this an easy information, we then addressed 3 questions: under field conditions does rain stimulate pollen germination and pollen-tube growth; what is the contribution of water-mediated fertilization to open-pollinated seed manufacturing on rainy days; to what level does pollinator-mediated pollination add to seed production? Our outcomes revealed three distinctive reproductive mechanisms causing seed set in C.gracilis: pollinator-mediated pollination and also autonomous self-pollination throughout rainless days, and rain-mediated self-fertilization on merganser days. Rain brought about pollen come germinate and also develop pollen tubes transferring sperm come ovules and achieving fertilization. This novel fertilization strategy appears to it is in the primary cause of seed manufacturing in the species.

Materials and also Methods

study system and also sites

Our study on C.gracilis was conducted during five flowering seasons from 2009 to 2013. We selected populations at 3 sites come conduct ar studies in Yunnan province, southwestern China: Lincang (LC population, 24°10′N, 99°37′E, altitude 2260m), Yangbi (YB population, 25°40′N, 100°02′E, altitude 2340m) and also Ailao mountain (AM population, 24°32′N, 101°01′E, altitude 2480m). The distance in between each population is c.100km. The bulk of the fieldwork was carried out at LC. C. Gracilis inhabits rock wall surfaces in moist valleys at LC and YB, yet is epiphytic on trees at AM. People of C.gracilis can develop several inflorescences, with only one flower open up at a time on every inflorescence.

Floral biology, pollinator observations and also compatibility status

To collect simple information on the floral biology of C.gracilis, us randomly selected c.30 tree at LC and also recorded the variety of flowers every inflorescence, the timing of anthesis, floral longevity, pollen condition and also we likewise determined the autonomous self-pollination mechanism. We decided c.20 flowering people to record monitorings of insects visiting flowers and their foraging behavior from beforehand morning to so late evening for 6d in each population, in both rainy and rainless conditions during each July from 2009 come 2013.

To determine the compatibility status and also facility because that autonomous self-pollination, us randomly selected single flowers native c.90 plants scattered transparent the population at LC during July 2010, and also these to be bagged before anthesis and also evenly assigned to 3 treatments during rainless days: flowers hand-pollinated using pollen grains from the exact same flower and also bagged (self-pollination); flowers emasculated before anthesis, hand-pollinated throughout anthesis v pollen from other individuals and bagged (cross-pollination); and flowers unmanipulated and also bagged to stop insect tourists (autonomous self-pollination). We accumulated mature fruits 30d after ~ the therapies to recognize seed number. We used a generalized linear model to test for distinctions in seed number among the 3 treatments, adhered to by a least significant difference (LSD) test because that multiple comparisons. Finally, we also emasculated and also bagged c.30 flowers before anthesis come test because that apomixis, but we discovered no evidence for this phenomenon in the species.

when does autonomous self-pollination occur throughout rainless days?

To determine the time of autonomous self-pollination in C.gracilis, we selected single flower buds from c.210 tree at LC, and these to be bagged to protect against insect visitors throughout the entire 1d duration of anthesis. We randomly and evenly assigned flowers amongst seven groups: anthers removed 2.5h after anthesis; anthers eliminated 4h after anthesis; anthers gotten rid of 6h after anthesis; anthers eliminated 8h ~ anthesis; anthers gotten rid of 10h ~ anthesis; anthers eliminated 12h after anthesis; and also anthers left intact (NC). We accumulated mature fruits 30d after ~ the therapies to identify seed number. We supplied a generalised linear design to examine distinctions in seeds number adhered to by an LSD test because that multiple comparisons.

go rain-induced germination that pollen an outcome in ovule fertilization?

First, we examined the various pollen problems in C.gracilis to identify whether the filiform pollen consisted of germinated pollen through pollen tubes. To check this, we fixed samples the anthers v the granular and also the filiform pollen in FAA systems (formaldehyde, acetic acid, ethanol in ~ 1:1:18 ratio) in populaces at all three research sites. Every sample was later stained with aniline blue (0.1% in 0.1moll−1 CH3COOK) and observed under a fluorescence microscope (Zeiss LSM710; Carl Zeiss AG, Oberkochen, Germany).

We climate hypothesized the the insitu pollen germination in the ar was brought about by rain. To test this, us performed two separate experiments. In the very first experiment, solitary flowers native c.150 people were randomly selected on merganser days in LC during July 2011. Us bagged inflorescences with single flowers utilizing nylon mesh, i beg your pardon excluded visitors but permitted rain to permeate bags. Plants were evenly and also randomly allocated to two treatments: bagged flower were defended from rain making use of plastic covers; and also bagged flowers were left intact and wetted by rain. We then identified the problem of pollen seed at 2h ~ the therapies were collection up. In the second experiment, to additional investigate the result of water top top pollen germination, we built up granular pollen seed from single flowers native 20 tree on a clear morning in LC. We then performed two therapies under ar conditions: in the first, c. 50% of the pollen grains from every flower were put on a piece of dried filter record on a glass slide; and in the second, the remaining pollen grains were placed on moist filter record on a glass slide. We then examined the condition of pollen grains 2h after ~ the treatments under a stereo microscopic lense (Leica M50; Leica Camera AG, Wetzlar, Germany). The filter document with germinated pollen was resolved in FAA systems for more laboratory observation.

In addition, we figured out if rain-induced pollen germination results in ovule fertilization by tracking the expansion of pollen pipe in pistils. Solitary flowers native 30 people were randomly selected on days with early morning rain in LC throughout July 2011 and also 2012. We bagged inflorescences using nylon mesh before flowering, i m sorry excluded visitors but permitted rain to permeate bags. We collected the pistils and anthers v insitu germinated pollen at 08:00h when autonomous self-pollination had actually not occurred, therefore excluding pollen from autonomous self-pollination. We addressed the samples of anthers and also pistils in FAA solution. Every sample was later stained v aniline blue and also the growth of pollen tubes and ovule penetration to be observed under a fluorescence microscope.

What is the contribution of water-mediated fertilization come seed production?

We hypothesized that rain-mediated ovule fertilization would certainly promote seed production of C.gracilis throughout rainy conditions. To test this hypothesis, we compared the seed set of bagged flower either exposed to or sheltered from the rain in the LC population during the monsoon season. In addition, we evaluated even if it is delayed self-pollination might also promote seed manufacturing in C.gracilis. To identify this, we compared the seed set of bagged flower either with anthers gotten rid of at 16:00h or left intact, on both rainy and also rainless days. We selected 2 patches (A and also B) separated by 1.5km to conduct the experiment at LC in July 2011. In each patch, we randomly selected single flowers native c.320 plants and also visitors were excluded during the entire 1d period of anthesis. Us evenly divided flowers from every patch right into two groups: plants sheltered by plastic covers throughout the entire 1d period of anthesis to defend pollen from everyday rain and also thus pollen germination on anthers (group 1); and plants exposed to rain (group 2). We confirm each flower in ~ 16:00h top top the work of anthesis to confirm that pollen from group 1 was ungerminated and pollen from group 2 to be germinated on anthers. We accumulated mature fruits 30d ~ the treatments to recognize seed number. Note that a preliminary experiment demonstrated that sheltering on merganser days go not impact seed set. We supplied a generalised linear fixed results model to test for distinctions in particle number per fruit among treatments because of the nonnormality the data, with pollen condition, treatment, patch and also their interaction considered in the analysis.

We predicted that rain-mediated fertilization would certainly maximize ovule fertilization under rainy conditions. To check this, we contrasted seed manufacturing from rain-mediated fertilization with what occurs once flowers are hand self-pollinated. We randomly selected solitary flowers from c.100 tree at LC and also performed two therapies in July 2011: in the first, c.50 flowers were sheltered through plastic, bagged and also hand self-pollinated on rainy days; in the second, c.50 flowers were bagged and exposed to rain on merganser days. We collected fruits c.30d after therapies for particle counting. We compared seed number per fruit in between the two therapies using a t-test.

To additional evaluate the prestige of rain-mediated fertilization to seed production, we determined the donation of insect pollinators to seed production. This was assessed by quantifying the donation of seed manufacturing by pollinator-mediated pollination under both rainless and rainy conditions at LC and also YB in 2012. We evenly assigned single flowers from c.120 plants to two teams at each site: in one group, plants to be exposed come rainless days, and also in the other, plants were exposed to rainy days. For each group, flower were same allocated to 2 treatments: open-pollination (i.e. Intact and also unmanipulated) seed set, a treatment that provides information on open-pollinated seeds production; and emasculation of flowers prior to anther dehiscence and exposure come pollinators. The second treatment was designed to avoid intrafloral self-fertilization, but permitted pollination by pollinators. Therefore, the seed production for this treatment shows the potential for pet pollination and also the availability of pollinators under various weather conditions. We collected fruits c.30d after therapies for seed counting. We used a generalised linear fixed impacts model to check for distinctions in seed number per fruit among treatments since of the nonnormality that data, with anther removal, environment, populace and their interaction thought about in the analysis.

To evaluate whether rain had an effect on seed manufacturing apart from facilitating pollen germination, we randomly selected solitary flowers indigenous c.100 plants and performed two treatments during July 2011 in LC: in the first, c.50 flower were bagged and hand self-pollinated on sunny days; and in the second, c.50 flower were sheltered, bagged and also hand self-pollinated on rainy days. Us quantified seed collection per fruit and used a t-test to determine if there was a difference in particle number between the 2 treatments.

In ours study, every analyses to be performed utilizing the statistical software application R v.3.3.0 (R main point Team, 2016). Data for all t-tests were confirm for normality and homogeneity of variance and were changed as needed. All the generalized linear models were run making use of the car package v the quasi-Poisson distribution since of the overdispersed data (Fox etal., 2013).

Results

Floral biology, pollinator observations and compatibility status

Cautleya gracilis had 6.9±0.3 flowers (mean±SE) per inflorescence, v one flower open up at a time on every inflorescence. Flowers were scented and also opened synchronously in ~ c.04:00h in each population and withered in ~ c.21:00h, lasting C.gracilis pollen to be granular (Fig.1b; Supporting info Fig.S1a). In the LC population, two types of bees (Elaphropoda magrettii and also Amegilla yunnanensis) were the key pollinators of flowers contacting sex organs during foraging for nectar. Top top rainless days us observed a average of 2.8±0.31visitsh−1 (n=25h of observation). By contrast, on rainy days during the monsoon period, visitation through pollinators was lot reduced, 0.08±0.05 visitsh−1 over 33h of observation. In populaces YB and also AL, us observed no insect visitors, regardless of even if it is or no it rained.

We uncovered that stigmatic liquid was repeatedly secreted during the flowering of C.gracilis and also this developed a globule top top the stigma at c.10:00h (Fig.1b). Pollen grains close to the stigma adhered to the stigmatic fluid, thus achieving autonomous self-pollination on rainless days. This autonomous self-pollination mechanism has previously to be reported in another species of ginger (Roscoea debilis; pan & Li, 2012), i beg your pardon is the sister genus of Cautleya.

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There to be no significant difference in seeds number per fruit in between hand self- or cross-pollinated flowers (Fig.2), indicating that plants the C.gracilis are very self-compatible. Bagged flowers produced substantially lower seed set than hand-pollinated flowers throughout rainless job (Fig.2), describe that, back C.gracilis has some capacity for autonomous self-pollination throughout rainless days, it is not adequate to provide full reproductive assurance under pollen-limited conditions.