REGULAR ARTICLE

Biogenic ethylene promotes seedling emergence from the sedimentseed bank in an ephemeral tropical rock pool habitat

Adam T. Cross & Gregory R. Cawthray & David J. Merritt &Shane R. Turner & Michael Renton & Kingsley W. Dixon

Received: 12 December 2013 /Accepted: 28 February 2014# Springer International Publishing Switzerland 2014

AbstractBackground and aims Ethylene has been increasinglyimplicated as a regulatory mechanism in plant germina-tion, growth, and development, and is produced fromthe sediments of freshwater habitats. In this paper, weanalyse the production and origin of ethylene fromephemeral freshwater rock pool sediments, and explorethe role of ethylene in regulating seedling emergencefrom the seed bank.Methods The production of ethylene from rock poolsediments subjected to variable moisture content andantibiotic treatments was assessed through gas chroma-tography, and the role of ethylene in regulating seedlingemergence was determined by seedling emergence as-says and seed germination experiments.Results Biogenic ethylene production from rock poolsediments occurred rapidly (3–6 h) following inunda-tion, with the majority of seedling emergence occurringbetween 36 and 72 h. Inoculation of sediments with

streptomycin and amphotericin B resulted in significant-ly reduced ethylene production (up to 60 % and 84 %respectively), and completely inhibited seedling emer-gence. Additionally, the exposure of dormant seeds toethylene resulted in significantly increased seed germi-nation percentage in five out of six rock pool species.Conclusions Biogenic ethylene production may play animportant role in regulating seed dormancy and thetiming of seedling emergence from the sediment seedbank following inundation events in rock pools andother freshwater aquatic communities.

Keywords Sandstone . Ephemeral freshwater wetland .

Seed germination . Kimberley . Vernal pool

Introduction

The role of ethylene (C2H4) as an important regulator ofmany plant physiological responses has become increas-ingly well demonstrated throughout the scientific litera-ture in recent years (Bleecker and Kende 2000;Ladygina et al. 2006; Lin et al. 2009). Ethylene in soilsand sediments originates primarily from the decompo-sition of organic matter by soil microbes, primarilybacteria and fungi (Smith and Restall 1971; Arshadand Frankenberger 1990; Jackel et al. 2004; Ladyginaet al. 2006). Ethylene is likely to play a significantecological role even at low levels (Smith 1976),possessing near-full biological activity at extremelylow concentrations (6.5×10−6 nM at 25 °C; Arshadand Frankenberger 2002). The production of ethylene

Plant SoilDOI 10.1007/s11104-014-2083-z

Responsible Editor: Jeff R. Powell.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-014-2083-z) contains supplementarymaterial, which is available to authorized users.

A. T. Cross (*) :G. R. Cawthray :D. J. Merritt :S. R. Turner :M. Renton :K. W. DixonSchool of Plant Biology, The University ofWestern Australia,35 Stirling Highway, CrawleyWA 6009 Perth, Australiae-mail: Adam.cross@bgpa.wa.gov.au

A. T. Cross :D. J. Merritt : S. R. Turner :K. W. DixonKings Park and Botanic Garden,Fraser Avenue, West PerthWA 6005 Perth, Australia

is strongly influenced by soil environment (Smith andRestall 1971; Arshad and Frankenberger 1990, 2002),with the highest rates of ethylene production havingbeen observed at shallow depth (Jackel et al. 2004), insubstrates rich in organic matter (Zeikus and Winfrey1976; Goodlass and Smith 1978; VanCleemput et al.1983), and under anaerobic conditions (Lynch andHarper 1974; Smith 1976; VanCleemput et al. 1983;Zechme i s t e r -Bo l t ens t e rn and Smi th 1998;Zechmeister-Boltenstern and Nikodim 1999; Jackelet al. 2004). Freshwater wetlands generally possessshallow, chemically reduced, organic-rich sediments(Keddy 2010), harbouring an active microbial commu-nity (Smith and Restall 1971; Pedersen and Sayler 1981;Boon and Mitchell 1995; Devai and Delaune 1996; Qiuet al. 2005). Redox potential is an indicator of oxygen-ation in flooded sediments (Zeikus 1977; Chen andAvnimelech 1986), and influences the rate and compo-sition of hydrocarbon production (Devai and Delaune1996). Sediment aeration is dependent upon depth ofinundation, duration of flooding, and rate of water flow(Keddy 2010). Therefore, it is likely that ethylene pro-duction occurs readily from the anaerobic sediments ofephemeral freshwater wetlands in response to floodingevents.

Although ethylene has been shown to be producedfrom the mud, sediment, and flooded soil of freshwaterecosystems (Smith and Restall 1971; Zeikus andWinfrey 1976; Zeikus 1977; Devai and Delaune1996), it is unknown whether similar processes areactive in the shallow sediments of highly ephemeralhabitats such as rock pools. These habitats, particularlyin semiarid tropical environments, can experience infre-quent, episodic, or seasonal flooding, often of shortduration (days to weeks; Deil 2005; A.T. Cross,unpubl. data), in strong contrast to the more regularhydro-periods experienced by previously studied eco-systems (Smith and Restall 1971; Lynch and Harper1974; Smith 1976; VanCleemput et al. 1983;Zechme i s t e r -Bo l t ens t e rn and Smi th 1998;Zechmeister-Boltenstern and Nikodim 1999; Arshadand Frankenberger 1990, 2002; Jackel et al. 2004).Sediment moisture content is regarded as a major factoraffecting ethylene production and stability, potentiallydue to reduced microbial degradation and decreasedoxygen availability under waterlogged conditions(Arshad and Frankenberger 1990). While desiccationtolerance is well demonstrated in prokaryotes (Hollandand Coleman 1987; Potts 1994; Sterflinger and

Krumbeinn 1995; Johnson et al. 1996; Sterflinger1998), the effect of seasonal desiccation cycles on mi-crobial activity and the responsiveness of microbes tofluctuating soil moisture remains largely unexplored.Ethylene is involved in the regulation of seed dormancyand germination in many plant species (Kepczynski andKepczynska 1997; Baskin and Baskin 1998), and mayplay a particularly significant role in aquatic ecosystemswhere inhabitants display specific adaptations toflooding (Blom 1999). Ephemeral wetland ecosystemsare commonly characterised by sediment seed banks(Bonis et al. 1995; Deil 2005; James et al. 2007;Brock 2011), and the germination of many species fromaquatic sediments appears to be cued by flooding orinundation (Baskin and Baskin 1998; Brock andRogers 1998; Leck and Brock 2000; Deil 2005;Aponte et al. 2010; Brock 2011; Carta et al. 2013; Geet al. 2013).

Recent studies have suggested that freshwater rockpools are model ecosystems, offering unique opportuni-ties to investigate specific research questions regardingcommunity level ecological and successional mecha-nisms. Consequently, such ecosystems have becomemicrocosms of increasingly high interest for ecologistsand evolutionary biologists (Deil 2005; Brendonck et al.2010). Rock pool ecosystems harbour a high diversityof specialist and endemic species, and contribute signif-icantly to regional biodiversity (Pinder et al. 2000;Aponte et al. 2010; Jocque et al. 2010; Tuckett et al.2010). Rock pools are characterised by highly variableabiotic and biotic conditions that continuously test thetolerance limits of inhabiting organisms (Brendoncket al. 2000; Jocque et al. 2010), with the availability ofwater being perhaps the most ecologically limiting fac-tor (Deil 2005). Hydro-regime is reliant almost exclu-sively upon the balance between precipitation and evap-oration (Deil 2005; Vanschoenwinkel et al. 2009;Brendonck et al. 2010), and the small volume of poolsresults in episodic wetting of short duration (Brendoncket al. 2010). Irregular and prolonged dry phase eventsare a recurring disturbance phase (Brock et al. 2003),and resident organisms are often taxa with short lifehistories, which reproduce rapidly over short genera-tional times to exploit transient periods of favourableconditions (Brock et al. 2003; Deil 2005). Rock poolstherefore offer the unique opportunity to study the ecol-ogy and disturbance response of an ephemeral aquaticecosystem at the community level within definedboundaries, allowing inferences to be made about

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ecosystem functioning of other temporary or seasonalaquatic habitats occurring at larger scales.

Desiccation tolerance and dormancy has been dem-onstrated in the microbial communities of various hab-itats (Potts 1994; Gottlieb et al. 2005; Thomas et al.2006; Knowles and Castenholz 2008; Jones and Lennon2010; McKew et al. 2011), and the activity of microor-ganisms has been examined in large freshwater ecosys-tems (Groffman et al. 1996; Gutknecht et al. 2006).However, the composition, resilience, and ecologicalrole of microbes in ephemeral wetland sediments remainunknown, particularly in relation to interactions with thesediment seed bank. The effect of microbial activity onthe longevity of seed banks has been previously inves-tigated (Chee-Sanford et al. 2006; Wagner andMitschunas 2008; Dalling et al. 2011), however theimportance of microbial products such as ethylene ininducing germination and emergence from the sedimentseed bank has not received much attention and is notwell understood. To address this knowledge gap, thisstudy investigated the capacity for ethylene productionin ephemeral rock pool sediments and explored the roleof ethylene in regulating seedling emergence from theseed bank by examining (i) the suitability of the sedi-ment environment to microbial activity, in terms ofchemical composition, organic matter content, and ox-ygenation at different flooding depths; (ii) ethylene pro-duction in sediments of different texture in response towetting; (iii) the contribution of different microbialgroups to ethylene production following exposure toantibacterial and antifungal treatments; (iv) emergencefrom the sediment seed bank after inundation and insediments treated with antibacterial and antifungal treat-ments; and (v) the ability of ethylene gas to stimulategermination in the freshly collected seeds of six rockpool species.

Materials and methods

Study site

The study site was located in the catchment of theMorganRiver, on the northwest tip of the Gardner Plateau in thearid tropical North Kimberley region of northern WesternAustralia (14° 47′ 46″ S, 126° 31′ 27″E). Over 3,000 rockpools are situated within the study site on approximately10 km2 of discontinuous sandstone pavement, includingca. 200 which possess vegetation. Rock pools consisted

of geologically weathered depressions of varying size anddepth in the sandstone bedrock (Appendix 1), and weredistributed almost exclusively on flat or gently slopingsandstone pavement. Rainfall in the North Kimberley isunpredictable and highly seasonal, with around 95 %falling during the November to April summer wet season(ca. 1,200 mm annually; McKenzie et al. 2009), and isconsistently exceeded by evapotranspiration rates(>2,000 mm annually; Luke et al. 1997). For each rockpool, depth and sediment depth were measured usingdigital callipers and a small metal rod, and total and basalsurface area were estimated from length and widthmeasurements.

Sediment sampling and chemical analyses

Desiccated sediments from 41 vegetated rock poolswere collected at the end of the dry season, in earlyOctober 2011, shortly before the onset of the summerwet growing season. Sites were selected in order tocreate a representative sample of the size, depth, andvegetation association of rock pool habitats. Ten drysediment core subsamples were collected from the top1 cm of the sediment within each rock pool (each 10 cm3

in volume), and combined to provide one sample perpool. These were stored dry in paper bags for severaldays, at ambient temperature (~30 °C), before transportback to the laboratory (Kings Park and Botanic Garden,Perth, Western Australia). Prior to use in experimentaltrials, all sediment samples were sieved (2.86 mmgauge) to remove large debris and stony material. Toexamine the chemical composition of sediments, 400 gsamples from all rock pools were sent to CSBP Plantand Soil Laboratories (Bibra Lake, Western Australia)for analytical determination of sediment properties andchemical factors. Due to the small size and shallowsediment depth of most rock pools, only six poolsyielded enough sediment to conduct field capacity, re-dox potential, and ethylene production experiments.

Seed collection and quality

Mature seeds (brown and dehiscing) of six annual rockpool species, including Eriocaulon sp. Morgan River(A.T.Cross 62) (Eriocaulaceae), Microcarpaea minima(Retz . ) Merr. (Phrymaceae) , Myriophyl lumcallitrichoides subsp. striatum Orchard (referred tohereafter as M. callitrichoides) and M. sp. HardingRange (M.D.Barrett and R.L.Barrett MDB 1825)

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(Haloragaceae), Portulaca bicolor F.Muell., and P. sp.rock pools (K.A.Menkhorst 310) (Portulacaceae), werecollected from field sites in the North Kimberley,Western Australia, in March–April 2011 and March–May 2012. Species were selected based on their emer-gence from incubated sediment samples and comprisedthe dominant flora of rock pools in the study site. Allseed collections were cleaned manually by gentlyrubbing material over steel sieves (2 mm to 250 μmgauge) with a handheld rubber stopper, before separat-ing seeds from chaff using a vacuum aspirator(SELECTA BV Gravity Seed Separator, Netherlands).Seeds were stored in a controlled environment (CE)room at 15 °C and 15 % relative humidity (RH) priorto experimental use. Seed quality was determined non-destructively via X-ray analysis (MX-20 digital X-raycabinet, Faxitron, USA), with three replicates of 100seeds examined for each species. Seeds were scored asfilled if the endosperm was complete, not shrunken orretracted from the testa, and if no significant internaldamage was evident.

Determination of sediment moisture content and fieldcapacity

The moisture content of field-collected sediments,and sediments at field capacity, was determinedfollowing the methods of Somasegaran and Hoben(1985). Three 50 g replicates of field-collected sed-iment from six sites, representing three with a sub-strate of loamy sand, and three with a substrate ofloam, were placed into aluminium weigh boats andweighed. Samples were then dried at 105 °C in adrying oven until reaching a stable weight, withfinal dry weight recorded. A further three 50 greplicates from each site were poured into 100 mlclear plastic cylinders, and incrementally irrigatedwith small volumes of de-ionised (DI) water untiltwo-thirds of the soil volume had been wetted.Samples were allowed to equilibrate for 48 h, be-fore the top 1 cm of wetted sediment was removedand discarded, and the middle 5 cm of wettedsediment (at field capacity) was removed andweighed. These sub-samples were then dried tostable weight at 105 °C, with final dry weight recorded.Field-collected and field capacity moisture levels (on adry weight basis) for each site were averaged, informingthe adjustment of dry sediments to field capacity insubsequent experiments.

Redox potential following inundation

The redox potential of rock pool sediments afterflooding was examined as an indicator of oxygenation(Zeikus 1977; Chen and Avnimelech 1986). Three rep-licate samples of 10 g sediment were maintained atvarying depths of inundation and incubated at 30 °Cfor 7 days. Sediment samples were placed into testtubes, and irrigated with DI water to form a head of 2,3, or 5 cm of water above the sediment surface.Additionally, air and nitrogen gas were continuouslybubbled through separate 2 L flasks containing DI waterfor the duration of the experiment, acting as aerated andoxygen-deprived control solutions, respectively.Measurements of redox potential were taken at thesediment surface (or at 3 cm depth for control solutions)after 0, 2, 6, 12, and 24 h, and after 2, 3, 4, 5, 6, and7 day using a water quality meter with ORP electrode(D-54 Handheld Meter, HORIBA, Japan).

Ethylene production and quantification

The production of ethylene from rock pool sedimentsbefore and after flooding was assessed using methodsadapted from Smith and Restall (1971), Zeikus (1977),Arshad and Frankenberger (1990), and Devai andDelaune (1996). Samples of dry sediment from the samesix sites used for moisture content determination (6 geach, three of loam and three of loamy sand) wereplaced into 10 ml flat-bottomed headspace vials(Agilent Technologies, Australia). Replicates of sedi-ment from each site were irrigated with DI water toeither field capacity (as determined previously) or wa-terlogged (twice field capacity). Replicates of dry sedi-ment acted as a control treatment for each site. Vialswere sealed with rubber butyl septa and aluminiumcrimp caps (Supelco, USA) to prevent gas loss, andincubated at 30 °C (sensu Zeikus and Winfrey 1976;Zeikus 1977). After 1, 3, 6, 12, and 24 h, and 2, 3, 4, 5,6, and 7 day, 1.0 ml of headspace was removed fromvials with a gas-tight syringe (BDUltrafine 1 mlNeedle;Sigma, Australia), and ethylene content analysed by gaschromatography using a Shimadzu GC-8A gas chro-matograph (Kyoto, Japan) equipped with a flame ioni-zation detector. Separation of hydrocarbons wasachieved with a Porapak N (100–200 mesh) column(100 cm in length, with an internal diameter of2.0 mm). Carrier gas (high purity nitrogen) flow-ratewas 20 ml min−1. The temperature of both injector and

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detector were 150 °C, and oven temperature was heldisothermally at 40 °C. Five replicates were analysed ateach time period for all treatments. Working standardsconsisted of ethylene gas of high purity (99.8 %; BOCGases, Perth, WA), and a high purity hydrocarbon gasmix (methane, ethane, propane, butane; BOC Gases,Perth, WA), diluted in air. Preliminary trials using eth-ylene standards injected into sealed vials determined therate of ethylene loss over 7 days to be ca. 0.008 nmolC2H4 h

−1 (data not presented).To assess the role that soil microbial diversity plays

in the production of hydrocarbons, additional 6 g sedi-ment samples from four sites (two each of loam andloamy sand, a subset of the previous six sites) wereplaced into vials and irrigated to field capacity with DIwater, as previously described. Sediments were theninoculated with either amphotericin B (an antifungalagent; Sigma, Australia) or streptomycin (an antibacte-rial agent), both at a final concentration of 1 mM (sensuArshad and Frankenberger 1990). Additional replicateswere irrigated to field capacity with PPM solution (PlantPreservative Mixture, a biocide targeting both fungi andbacteria; Plant Cell Technology, USA), at a concentra-tion of 1 ml PPM L−1 DI water. To determine thecontribution of non-biological processes to hydrocarbonproduction, additional samples of dry sediment weresterilised by autoclaving at 121 °C before being irrigatedto field capacity with sterile DI water. All vials weresealed and capped, and incubated for 7 days at 30 °C.The production of ethylene in sterilised and antibiotic-treated sediments was determined by gas chromatogra-phy as previously described after 3, 5, 6, and 7 day, andcompared to untreated controls.

To determine the capacity for endogenous ethyleneproduction from seeds, three replicates of 0.1 g of fresh,dry seeds from each of the six species was placed into10 ml flat-bottomed headspace vials. Seeds were hy-drated with 1 ml of DI water, before vials were sealedand incubated as previously described. Control vialscontaining DI water but no seeds were also prepared.After 7 days, 1.0 ml of headspace was removed fromvials with a gas-tight syringe, and ethylene analysed bygas chromatography.

Emergence from the sediment seed bank

To assess the timing and abundance of seedling emer-gence from rock pool sediments, as well as the role ofbiogenic ethylene in stimulating germination, dry

sediment from 12 sites of known vegetation assemblage(three E. sp. Morgan River, two M. callitrichoides, twoM. sp. Harding Range, two P. bicolor, two P. sp. rockpools, and one M. minima) was irrigated with DI waterto either field capacity or waterlogged conditions aspreviously described, and incubated at constant 30 °Con a 12/12 h light/dark cycle (light source 30 W coolwhite fluorescent tubes [400–700 nm] with a photonflux density at seed level of ca. 50 μmol m−2 s−1).Four replicates were used for each site, with 50 g ofsediment spread across the bottom of 100 mm roundplastic containers sealed with airtight plastic lids afterirrigation. Additional replicates for each site were irri-gated to field capacity and inoculated with eitheramphotericin B or streptomycin, both at a final concen-tration of 1 mM. Emergence in all treatments was scoredafter 6, 12, 24 and 48 h, and then daily for 4 weeks.

Seed germination experiments

To assess the effect of ethylene exposure on the germi-nation of freshly collected seeds, seeds of each specieswere incubated in 90-mm Petri dishes on water agar(0.7 % w/v) sealed with Glad Wrap ®. Methods ofethylene exposure followed Kepczynski et al. (2003),with 100 seeds of each species sealed in fine nylon meshbags, moistened with DI water, and placed in 100 cm3

glass vials. Vials were closed with rubber septa (Suba-Seal, Sigma Aldrich Chemicals, Australia), with ethyl-ene gas injected into each (final concentration ca.50 nmol) and the concentration of ethylene verifiedusing gas chromatography as previously described.Seeds were incubated in the ethylene atmosphere for24 h, before being removed for germination experi-ments. Petri dishes were maintained in temperature-and light-controlled incubators at 30 °C, exposed to a12 h daily photoperiod. Four replicates of 25 seeds wereused for each species, with dishes scored daily for4 weeks. Germination was defined as the emergenceof the radicle from the testa to a length of at least1 mm. All non-germinated seeds were cut tested todetermine viability, with firm, white seeds deemed via-ble. Data presented is cut-test adjusted, and is thereforebased only on the numbers of viable seeds.

Statistical analysis

One-way ANOVA (SPSS Statistics 21, IBM, USA),with Tukey HSD post-hoc tests where possible, were

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employed to test the significance of sedimentchemistry factors, moisture content, and field ca-pacity values between sediment texture groups,and of depth and control treatments on redoxpotential (after 7 day). Two-way ANOVA wereemployed to test the significance of sediment tex-ture type and either inundation treatment orsterilisation treatment on the headspace concentra-tion of ethylene. Poisson regression was employedto test the significance of sterilisation treatmentand flooding depth on counts of seedling emer-gence from the seed bank. Binary logistic regres-sion (backward stepwise [Wald]) was employed toassess the effect of ethylene exposure on seedgermination percentage. All statistical tests wereconducted using 95 % CI, with significance deter-mined by P<0.05. Data are presented as mean ±standard error where possible.

Results

Sediment characters, moisture content, and fieldcapacity

The mean depth of rock pools within the study sitewas 28±2 mm, with a mean sediment depth of 12±1 mm. Average pool size was 1.7±0.1 m2, with abasal area of 1.3±0.1 m2. Two distinct sedimenttypes were distinguished by sediment texture andcomposition analyses, each constituting roughlyhalf of the rock pools sampled (Appendix 1).Pools possessed a substrate either of loam, varyingin colour from black to grey or light brown (22sites), or predominantly grey loamy sand (19 sites).No spatial relationship was discernable in the dis-tribution of the two sediment types. Loam sedi-ments possessed significantly higher organic mattercontent and considerably elevated levels of nitro-gen, phosphorus, sulphur and calcium as well as ahigher electrical conductivity and pH (Table 1).Moisture content in dry field-collected sedimentsvaried from 0.6 to 1.6 % (mean 1.1±0.2 %), andwas not significantly influenced by the main effectof sediment texture (P=0.658). Moisture content insediments at field capacity ranged from 11 to 25 %(mean 18±3 %), and also displayed no significantrelationship to sediment texture type (P=0.432).

Redox potential of flooded sediments

Redox potential (Eh) decreased markedly over 7 days ofincubation after flooding in all sediment samples(Fig. 1), and was not significantly influenced either bythe main effects of inundation depth (P=0.324) or sed-iment texture (P=0.219), or the interaction of thesefactors (P=0.301). Initial redox values at the surface offlooded sediments were lower than in the aerated controlsolution (P=0.01, mean Eh=246±14 mV), and begandecreasing after 12 h until not significantly differentfrom the oxygen-deprived control solution after 7 days(P=0.198, mean Eh=−172±5 mV).

Ethylene production from sediment and seeds

Ethylene was produced rapidly from field capacity andwaterlogged sediments following wetting, and head-space ethylene concentration increased in all sites testedover 7 days of incubation (Fig. 2). The baseline rate ofethylene production from dry sediments was extremelylow, and was significantly lower (P=0.047) in sites witha loam substrate (37±6 % lower than loamy sand).Ethylene evolved at a significantly higher rate underfield capacity and waterlogged conditions comparedwith dry sediments (P<0.001), and although mean eth-ylene production was also higher in loamy sand sedi-ments for both field capacity and waterlogged

Table 1 Significant physical and chemical factors distinguishingephemeral sandstone rock pools by sediment texture type

Texture type Loam Loamy sand Sig.

NH4-N (mg kg−1) 243.7±52.8 39.6±9.3 ***

NO3-N (mg kg−1) 79.6±18.3 11.1±3.5 ***

Total phosphorus (mg kg−1) 81.2±10.9 24.6±3.7 ***

Sulphur (mg kg−1) 36.8±7.6 10.8±1.6 ***

Calcium (mg kg−1) 20.3±3.3 6.6±0.8 ***

Conductivity (μS m−1) 330±60 90±10 ***

Potassium (mg kg−1) 150.3±25.6 64.6±9.3 **

Magnesium (mg kg−1) 6.8±1.1 2.8±0.4 **

Manganese (mg kg−1) 15.1±3.3 5.4±0.8 **

Zinc (mg kg−1) 1.5±0.3 0.6±0.1 **

Iron (mg kg−1) 177.8±17.7 126.4±44.1 NS

Organic matter (%) 3.4±0.5 2.1±0.3 *

pH (CaCl2) 5.42±0.05 5.28±0.04 *

NS not significant

Significance: ***P<0.001; **P<0.01; *P<0.05

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treatments (168 % and 136 % respectively), markedvariation was noted within sediment types and thisrelationship was not significant (P=0.233).

Ethylene production from antibiotic-treated sedi-ments was significantly lower than in untreated fieldcapacity controls (Fig. 3), and the effectiveness of eachtreatment was not significantly affected by the maineffects of sediment type (P>0.05). Streptomycin treat-ment resulted in a 32 % and 60 % decrease in ethyleneproduction, compared with untreated field capacityloam and loamy sand sediments, respectively.However, streptomycin was significantly less effectivein depressing ethylene production than bothamphotericin B (P=0.009) and PPM (P=0.008).Ethylene production was similar from sediments treatedwith amphotericin B and PPM (P=0.545), ca. 72 % and84 % lower than in untreated loam and sandy loamsediments respectively, and no significant interactioneffect was present between sediment texture type andantibiotic treatments on ethylene production. Ethyleneproduction from autoclaved sediments was below de-tection limits (Fig. 3).

In addition to ethylene, other hydrocarbons detectedat low concentrations included methane (highest con-centrations 10.8, 13.3, and 13.2 nmol CH4 kg−1 dryweight sediment in untreated dry, field capacity, andwaterlogged sediments respectively), ethane (9.9 and7.6 nmol C2H6 kg

−1 dry weight sediment in untreatedfield capacity and waterlogged sediments respectively),and propane (15.0 and 17.1 nmol C3H8 kg

−1 dry weightsediment in untreated field capacity and waterloggedsediments respectively). These hydrocarbons were notdetected in sediments treated with antibacterial and an-tifungal treatments with the exception of methane,which was occasionally present at very low levels(<5 nmol CH4 kg

−1 dry weight sediment). No hydrocar-bon production was detected from autoclavedsediments.

Endogenous ethylene was produced at low concen-trations from the seeds of rock pool flora, and constitutesless than 0.06 % of total ethylene production in fieldcapacity or waterlogged sediments based on approxima-tion of sediment seed bank size. Ethylene was detected

Fig. 2 Production of ethylene gas from dry (circle), field capacity(square), and waterlogged (triangle) loam (closed symbols) andloamy sand (open symbols) rock pool sediments (nmol C2H4 kg

−1

dry sediment) over a 7 day incubation period at 30 °C. Datarepresents mean ethylene production from five 6 g sedimentreplicates for each sediment type and for each inundation treat-ment. Error bars indicate one standard error of the mean

Fig. 1 Redox potential (Eh, mV) in ephemeral rock pool sedimentsfollowing inundation to 2 cm (open circle), 3 cm (open triangle) and5 cm (open square) depths and incubation for 7 day at 30 °C, withoxygenated (closed circle) and de-oxygenated (closed triangle)controls. Data represents mean redox potential from three 10 gsediment replicates for each inundation treatment. As no signifi-cance was determined between sediments of differing texture type,pooled values of both texture types are presented for each depthtreatment. Error bars indicate one standard error of the mean

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from seeds of E. sp. Morgan River (3.2±1.6 nmol C2H4

g−1 dry seed d−1),M. sp. Harding Range (2.0±1.3 nmolC2H4 g−1 dry seed d−1), and M. callitrichoides(2.0±2.0 nmol C2H4 g

−1 dry seed d−1), but was belowdetection limits for all other species tested.

Emergence from the sediment seed bank

Seedling emergence from the sediment seed bank oc-curred rapidly following wetting in all sediment samplestested (Fig. 4). High rates of seedling emergence wererecorded within the first 72 h in all species except E. sp.Morgan River, and the majority of all emergence oc-curred within 7 days. The main effects of inundation onemergence were highly significant (P<0.001), with onaverage 444±95 and 41±9 seedlings recorded in fieldcapacity and inundated treatments respectively for E. sp.Morgan River after 4 weeks, 273±52 and 1.3±0.9 re-spectively forM. minima, 78±19 and 17±1 respectivelyfor M. sp. Harding Range, 65±8 and 12±4 forM. callitrichoides, 48.3±13 and 3.2±1 respectively forP. bicolor, and 14±3 and 0.3±0.3 respectively for P. sp.rock pools. No emergence was recorded for any speciesin sediments treated with either streptomycin oramphotericin B over a 4-week period.

Effect of ethylene on seed germination

Five of the six species tested displayed no germinationin water only, with germination only recorded for E. sp.Morgan River (1.8±1.2 %). For all species except E. sp.

Morgan River, germination increased to varyingdegrees following exposure to ethylene gas, andthe main effect of ethylene exposure on germina-tion percentage was highly significant (P<0.001).The highest germination percentages observed afterethylene exposure were in P. bicolor (30±4 %) and P. sp.rock pools (13±4%), followed byM. minima (10±4%),M. callitrichoides (9±5 %), and M. sp. Harding Range(4±2 %). High seed viability was recorded for all spe-cies, including 100 % for E. sp. Morgan River andM. minima, 97.7±0.3 % for P. bicolor, 97.0±0.6 % forM. callitrichoides, 88.7±4.1 % for P. sp. rock pools, and87.3±0.3 for M. sp. Harding Range.

Discussion

This study demonstrates that biogenic ethylene is pro-duced rapidly from sandstone rock pool sediments fol-lowing inundation, as a result of both bacterial andfungal activity under reducing conditions. The produc-tion of ethylene is followed within hours by widespreademergence of seedlings from a dormant sediment seedbank. Rapid and synchronous germination afterflooding is a common feature of ephemeral wetlandtaxa, with individuals maximising fitness by growingquickly to maturity and seed production (Deil 2005;Brock 2011). Seed dormancy is an adaptation to preventgermination when environmental conditions areunfavourable for seedling establishment and survival,and conditional dormancy (which allows seeds to

Fig. 3 Production of ethylene gas(nmol C2H4 kg

−1 dry sediment)from control and treated rock poolloam (open bars) and loamy sand(solid bars) sediments, after 7 dayincubation at 30 °C. Datarepresents mean ethyleneproduction from five 6 g sedimentreplicates for each sediment typeand for each antibiotic treatment.Annotation indicates significancebetween treatments for eachsediment type. Error bars indicateone standard error of the mean

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germinate over a narrow range of conditions) is a com-mon characteristic of seeds from climatically unpredict-able habitats (Baskin and Baskin 1998). Seasonal andunpredictable periods of desiccation are a regular dis-turbance phase from which ephemeral wetland commu-nities must recover (Brock et al. 2003). Ethylene pulsesresult from stimulation of the microbial communityfollowing wetting, and may therefore cue germinationin rock pools and other ephemeral wetland habitats byproviding indication to seeds that sediments are suitablyflooded, and therefore conducive to seedlingestablishment.

Flooding commonly controls emergence from theseed bank in temporary wetlands (Bonis et al. 1995;Brock et al. 2003; Deil 2005; Brock 2011; Carta et al.2013), and wetlands in seasonally arid tropical environ-ments are often tuned to seasonal pulses of inundation(Mitsch et al. 2010). Bet hedging is common in the

sediment seed bank of these habitats, with only a smallproportion of seeds emerging after each wetting event(Deil 2005). It is therefore likely that the alleviation ofseed dormancy in ephemeral habitats is reliant on acombination of factors, possibly including after-ripening (Carta et al. 2013), seasonal dormancy cycling(Merritt et al. 2007), hydro-regime (Casanova andBrock 2000; James et al. 2007; Boudell and Stromberg2008), or exposure to other germination stimulants(Baskin and Baskin 1998; Merritt et al. 2007).Individual species respond differently to different com-binations of cues, as diversity and community compo-sition in ephemeral habitats is strongly influenced by theduration, extent, and timing of inundation events(Casanova and Brock 2000; Deil 2005; James et al.2007). Results from this study suggest that the seedsof rock pool flora are tuned to seasonal and unpredict-able flooding cycles, with ethylene exposure stimulating

Fig. 4 Seedling emergence of sixspecies from rock pool sedimentsirrigated to either field capacity(circle) and waterlogged(triangle), over a 7 day incubationperiod at 30 °C. Data representsmean seedling emergence fromfour 50 g sediment replicates,with seedling emergence frommultiple sediment samples pooledfor each species. Error barsindicate one standard error of themean

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seed germination, and field capacity treatments resultingin significantly higher seedling emergence and the emer-gence of more species compared to waterlogged sedi-ments. The duration and periodicity of flooding havebeen identified as crucial mechanisms regulating emer-gence in freshwater wetlands, particularly in ephemeralhabitats from a number of different regions (Willis andMitsch 1995; Casanova and Brock 2000; Peterson andBaldwin 2004; Liu et al. 2005; Capon 2007; Aponteet al. 2010; Brock 2011). For many aquatic plants,particularly submerged species of permanent water bod-ies, germination and seedling emergence appear to becued by flooding and anaerobic conditions (Baskin andBaskin 1998; Leck and Brock 2000). However, in thisstudy, seedling emergence from rock pool sedimentswas significantly reduced in flooded treatments com-pared with sediments maintained at field capacity.Reduced emergence under flooded conditions has alsobeen reported from the sediments of intermittently wettidal freshwater wetlands in Maryland and Delaware,North America (Peterson and Baldwin 2004), and anephemeral marsh in northern Belize (Johnson 2004).Additionally, damp rather than flooded conditions insediments of intermittently flooded and seasonal wet-lands in North America, eastern Australia, and SouthChina have been observed to yield higher species diver-sity and overall emergent seedling biomass (Willis andMitsch 1995; Casanova and Brock 2000; Liu et al.2005). This emergence strategy may be common inhabitats experiencing an unpredictable hydroregime orextreme ephemerality, and may favour the rapid estab-lishment of dwarf annual species with short life histo-ries, a characteristic component of ephemeral wetlandflora from several disjunct environments (Deil 2005;Alvarez et al. 2012).

Rate of emergence and number of seedlings differedsignificantly between species, with very rapid emer-gence (ca. 80 % in 24 h) and low seedling densityobserved for P. sp. rock pools, a transitional speciesoften found growing in shallow pool margins and onskeletal sands at the edge of sandstone pavements;slower emergence (ca. 80 % in 36–72 h) and higherseedling density in M. callitrichoides, M. sp. HardingRange, and M. minima (full aquatics inhabiting deeperrock pools); and much slower emergence (ca. 80 % in 6to 8 days) but extremely high seedling density in E. sp.Morgan River, a ubiquitous species throughout thestudy area occurring in rock pools of all sizes anddepths. The proportion of freshly collected seeds that

germinated upon exposure to ethylene in the laboratorywas low (between 4 % and 30 % depending uponspecies), suggesting that seed dormancy may not simplybe broken by exposure to ethylene, but that additionalabiotic factors may influence the sensitivity of seeds toethylene (Ribeiro and Barross 2006), and the timing ofgermination in rock pool flora from the sediment seedbank. The comparatively high numbers of emergingseedlings from incubated sediments may indicate thatthe sediment seed bank comprises a seed population ofmixed age and with varying states of dormancy, with aproportion possessing high sensitivity to ethylene.Future studies examining the diversity, resilience, andpersistence of the sediment seed bank through numerouswetting events in rock pool communities are required.

Redox potentials below Eh −150 to −170 mV indi-cate anaerobic conditions in freshwater sediments(Zeikus and Winfrey 1976; Devai and Delaune 1996),with facultative anaerobic respiration occurring betweenEh −100 to −300 mV (Chen and Avnimelech 1986).Conditions facilitating facultative anaerobic respirationwere reached in rock pool sediments after 48 h post-flooding (Fig. 1); however ethylene production wasdetected as early as three hours post-flooding (Fig. 2).Ethylene is metabolised by soil microorganisms underwell aerated conditions, but not under anoxia (deBont1976), and it has been suggested that aerobic microor-ganisms synthesise ethylene during the early stages ofwaterlogging, while O2 is abundant, and that over timeas water enters a subsequent anoxic phase, ethylenebecomes trapped and is preserved by slow rates ofdegradation under reducing conditions (Jackson 1985;Arshad and Frankenberger 1990; Zechmeister-Boltenstern and Nikodim 1999). The observed rate ofethylene production from waterlogged loam (6.7 nmolC2H4 kg

−1 d−1 dry sediment) and loamy sand (11.3 nmolC2H4 kg

−1 d−1 dry sediment) sediments fromKimberleyrock pools is markedly lower than recorded previouslyfor other freshwater systems. Ethylene was evolved insignificantly higher quantities from sandy loam fresh-water marsh sediments in the Mississippi River(570.4 nmol C2H4 kg−1 d−1 dry sediment; Devai andDelaune 1996), and from fen peat in the UnitedKingdom (855.6 nmol C2H4kg

−1 d−1 dry sediment;Smith and Restall 1971), though it must be noted thatboth these habitats are characterised by predictablehydro-regime and extended periods of inundation.Organic matter content was markedly higher in both ofthe latter sites (ca. 40–50 % and 38 % respectively) than

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in rock pool sediments (3.4 % in loam and 2.1 % inloamy sand).

Our results indicate that ethylene production fromrock pool sediments originates primarily from biologicalsources, with no hydrocarbon production observed fromautoclaved sediments. Fungi appear to play a moredominant role than bacteria in ethylene production, asindicated by the greater suppressive action ofamphotericin B compared with streptomycin (Fig. 3).As ethylene production by organisms of both groupsappears to be similar (Qiu et al. 2005), the fungal tobacterial ratio in Kimberley rock pool sediments couldbe estimated from this study to be approximately 2:1.Qiu et al. (2005) suggest that fungi are significantlymore dominant in the surface litter of seasonal wetlands,while bacteria become more dominant in deeper soilsand exposed sediments, and provide similar fungal tobacterial ratios (2.9–3.2) for the litter of a seasonalwetland in southwestern Western Australia (Qiu et al.2005). Fungi are regarded as more tolerant of dry soilconditions (Holland and Coleman 1987; Johnson et al.1996), although bacteria also possess desiccation toler-ance (Potts 1994; Gottlieb et al. 2005; Thomas et al.2006). It might be expected that organisms possessing agreater capacity for desiccation tolerance will be morefunctionally dominant in rock pool habitats, given thatsediments are generally very shallow (<2 cm), experi-ence extremes in temperature (>50 °C; A.T. Crossunpubl. data), often possess thin organic crusts (Deil2005), and remain in a continuously desiccated statefor up to 8 months each year (A.T. Cross, unpubl. data).

Extensive black microbial crusts are common on thebare-rock peripheries of sandstone rock pools in thestudy sites in the northern Kimberley region ofAustralia (Fig. 5), although their composition has notbeen determined. The diversity and functional role ofalgae, bacteria, and fungi in ephemeral wetland habitatsis poorly understood (Deil 2005), however lichens,cyanobacteria, chemoorganotrophic bacteria, and fungiare common inhabitants of rock surfaces (Krumbein andJens 1981; Nienow and Fr iedmann 1993) .Dematiaceous fungi, particularly those with yeast-likegrowth patterns, are among the most successful organ-isms on rock surfaces in arid and semi-arid environ-ments, and are characterised by the formation of black,clump-like communities capable of tolerating high tem-peratures and low water activity (Sterflinger andKrumbeinn 1995; Sterflinger 1998). Black yeasts maybe a component of rock and sediment microflora in

Kimberley rock pool habitats, however it must be notedthat cyanobacterial crusts of similar appearance(Scytonema spp. and Stigonema spp.) have been report-ed on the rock surfaces of granite inselbergs in FrenchGuiana (Sarthou and Grimaldi 1992 in Deil 2005) andVenezuela (Budel et al. 1994), and sandstone pavementsin Utah (Jocque et al. 2007). Cyanobacterial crusts onrock surfaces change water chemistry though N- and C-fixation (Sarthou and Grimaldi 1992), and links havebeen drawn between cyanobacterial activity and nitro-gen concentration in sandstone habitats at similar levelsto those recorded in this study (Chan et al. 2005; Jocqueet al. 2007).

The confirmation of ethylene occurring at notableconcentrations in the immediate vicinity of the sedimentseed bank may implicate it as an important factor in therecruitment of rock pool flora, given the apparent per-manence of rock pool ecosystems over long geologicalperiods (Deil 2005; Jocque et al. 2010), and the positivegermination responses of several rock pool specieswhen exposed to ethylene under ex situ conditions. Inthe closed system of a small, ephemeral, rainwater-fedrock pool, microbial activity may therefore contribute tocommunity ecology through influencing seedling emer-gence. In this regard, the composition and functionalityof microbial communities in rock pools deserves futurestudy, particularly in relation to the effect of microbial-derived ethylene on the germination, early growth, anddevelopment of rock pool flora. Ethylene is a key reg-ulator of plant growth (Frankenberger and Arshad 1995)and bacterial activity (Smith 1976), and is also a germi-nation cue for many plant species, particularly wetlandtaxa (Kepczynski and Kepczynska 1997; Baskin and

Fig. 5 Typical sandstone rock pool habitat in the North Kimber-ley, displaying extensive black microbial crusts between andaround pools on bare rock

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Baskin 1998; Blom 1999; Ladygina et al. 2006).Abundant and active microbial communities are presentin the sediment of freshwater wetlands (Smith and Restall1971; Pedersen and Sayler 1981; Boon and Mitchell1995; Devai and Delaune 1996; Qiu et al. 2005), andgiven the apparent significance of flooding events inregulating emergence from the sediment seed bank inephemeral habitats (Brock et al. 2003; Deil 2005; Brock2011; Carta et al. 2013), the response of the microbialcommunity in these habitats to wetting, and the role ofthe microbial community in regulating other ecologicalmechanisms such as germination, must be examined ingreater detail. Rock pools display high levels of floralendemism (Pinder et al. 2000; Aponte et al. 2010; Jocqueet al. 2010; Tuckett et al. 2010), suggesting that speciespresent possess specific adaptations to the biotic andabiotic factors of the rock pool environment.

A number of ephemeral wetland habitats have beenidentified globally that possess ecological and hydro-logical similarities to Kimberley sandstone rock pools,most notably the seasonal pool and ephemeral flushhabitats defined by Deil (2005). These include the sea-sonal wetlands, rock pools, and vernal pools of tropicalSouth America (Deil et al. 2011; Tabosa et al. 2012),eastern tropical Africa and South Africa (Deil 2005;Muller 2007; Vanschoenwinkel et al . 2008;Vanschoenwinkel et al. 2009; Lumbreras et al. 2012),southwest Western Australia (Pignatti and Pignatti1994, 2005), west and northeast United States (Deil2005; Lathrop et al. 2005), and the Mediterranean(Deil 2005; Bagella et al. 2009; Aponte et al. 2010;Zacharias and Zamparas 2010), and the ephemeral flushhabitats of French Guiana and South Africa (Sarthouand Grimaldi 1992; Sarthou and Villiers 1998). Thesehabitats often support unique vegetation assemblageswell adapted to ephemeral conditions, and experiencethe ecological challenges associated with highly season-al and often unpredictable rainfall, short inundationperiods (days to months), and inter-annual variabilityin hydroregime (Bliss and Zedler 1998; Pyke 2004;Bauder 2005; Deil 2005; Hulsmans et al. 2008;Vanschoenwinkel et al. 2009). Recurring seasonaldrought events are recognised as perhaps the most overtselective mechanism in ephemeral habitats (Brock et al.2003), and it has been suggested that these conditionsmay have driven the widespread nanism andephemerism observed in the flora of vernal pools androck pools globally (Deil 2005; Bornette and Puijalon2011; Alvarez et al. 2012). As annual therophytes are

reported to constitute a high proportion of the inhabitantflora of ephemeral wetlands from many regions, includ-ing Brazil (Tabosa et al. 2012), California (Bliss andZedler 1998), Chile (Alvarez et al. 2012), theMediterranean (Aponte et al. 2010), and southwestWestern Australia (Tuckett et al. 2010), reliance upona persistent and resilient seed bank capable ofresponding rapidly to environmental cues is likely tobe a convergent character of ephemeral wetland com-munities. Seasonal timing appears to be an importantcue in the emergence of seedlings from Californianvernal pools (Bliss and Zedler 1998), and for manyannual species rapid and widespread seedling emer-gence from desiccated temporary wetland sedimentsoccurs after wetting (Bonis et al. 1995; Casanova andBrock 2000; Warwick and Brock 2003; James et al.2007; Aponte et al. 2010; Brock 2011). However, thespecific germination cues associated with wetting havepreviously remained largely unexplored. Results fromthis study suggest that emergence from the seed bank inephemeral wetlands is regulated by the response of thesediment microbial community to the depth and dura-tion of flooding, signalling favourable conditions forseedling establishment.

Acknowledgments This work was supported by an AustralianPostgraduate Award to ATC from the Commonwealth of Austra-lia, a grant from the Friends of Kings Park, Perth, Western Aus-tralia, and a personal donation from John Crone. Assistance withfieldwork from Celia Mitchell, Mark Warrington, and KatherineChuk is gratefully acknowledged. The Myers family and staff atTheda Station are particularly thanked for their hospitality andsupport. The manuscript was improved by valuable commentsfrom Jeff Powel and two anonymous reviewers.

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