Reproductive biology of a threatened Australian saltmarsh plant – Wilsonia backhousei

Aquatic Botany 99 (2012) 1–10

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Reproductive biology of a threatened Australian saltmarsh plant – Wilsonia backhousei Karen Sommerville ∗ , Alex Pulkownik, Margaret Burchett School of Environment, Faculty of Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia

a r t i c l e

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Article history: Received 31 August 2011 Received in revised form 15 December 2011 Accepted 16 December 2011 Available online 26 December 2011 Keywords: Conservation Restoration Clonality Flowering Pollination Anemophily Seed yield Dispersal Dormancy Germination Salinity

a b s t r a c t The reproductive biology of a threatened saltmarsh plant, Wilsonia backhousei Hook.f., was investigated with a view to improving conservation and restoration outcomes for the species. Population phenology was studied every two weeks, over two consecutive flowering seasons, in three to six 0.25 m2 quadrats set in monocultures of the species at each of eight sites in New South Wales, Australia. Floral density (flowers m−2 ) ranged from 0 to 5800 ± 1400 m−2 and varied significantly among sites (P < 0.05). Peak flowering occurred in mid-late October, during a period of no tidal inundation; fruit maturation coincided with inundation by extreme high tides in December, January and February. Sediment samples collected from each quadrat post-flowering were analysed for water content, salinity, texture (clay content) and pH. Ordinal logistic regression (OLR) of floral density against foliage volume and sediment variables showed a significant positive response to increasing foliage volume and negative responses to increasing salinity and clay content (P < 0.05). Foliage volume, in turn, showed a significant positive response to sediment water content and again negative responses to increasing salinity and clay content (P < 0.05). Fruit development occurred at all sites; however, little to no seed was produced at five of the sites. When quadrats not producing seed were excluded (to limit the influence of self-incompatibility), OLR of seed yield against floral density and sediment variables indicated sediment water content was the only significant predictor of yield (P = 0.015). The morphology and phenology of individual flowers, potential pollination vectors, and general seed biology were also investigated. A combination of floral characteristics indicated that W. backhousei is wind-pollinated, likely to be outbreeding, and may be self-incompatible. The fruit is buoyant and, as maturation coincides with inundation by extreme high tides, may be dispersed within and among sites by tidal flow. The seed was found to be viable and long-lived (>70% germination in both fresh seed and seed aged for 6 years); however, seed germination is limited by physical dormancy and is significantly reduced by salinity levels ≥40 dS m−1 (6 ± 2.4% after 28 days, P < 0.001). Based on these outcomes, recommendations are made for maximising the reproductive potential of remnant and restored populations. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Saltmarshes are important ecological communities that contribute to the environmental health and economic function of estuaries around the globe (Valiela, 2006). Human activities over many centuries have unfortunately led to large-scale, worldwide, losses from these ecosystems in terms of both area and biodiversity (Jickells and Rae, 1997; Zedler et al., 2001; Adam, 2002; Valiela, 2006). In New South Wales (NSW), Australia, losses from individ-

∗ Corresponding author. Present address: The Australian Botanic Garden, Mount Annan Drive, Mount Annan, NSW 2567, Australia. Tel.: +61 2 4634 7942; fax: +61 2 4634 7950. E-mail addresses: [email protected] (K. Sommerville), [email protected] (A. Pulkownik), [email protected] (M. Burchett).

ual estuaries have been substantial with, for example, 80–100% of saltmarsh area lost from a number of bays in the vicinity of Sydney (Burchett and Pulkownik, 1996; Saintilan and Williams, 2000). Saltmarshes along the entire coast of NSW are now listed as ‘Endangered Ecological Communities’ under the NSW Threatened Species Conservation Act 1995. Growing awareness of the value of saltmarshes, and the extent of their decline, is leading to increasing interest in conserving remnants and restoring degraded areas (Zedler and Adam, 2002; Valiela, 2006). Successful conservation and restoration of saltmarsh, however, requires an understanding of the biology and ecology of the constituent species (Green et al., 2009). This is particularly so when those species are themselves threatened and do not readily recolonise restored areas, even when provided with conditions suitable for their establishment. In NSW, one such species is Wilsonia backhousei Hook.f. (Convolvulaceae), a plant listed as ‘Vulnerable’ under the NSW Threatened Species Conservation Act 1995.

0304-3770/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.12.010

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K. Sommerville et al. / Aquatic Botany 99 (2012) 1–10

This species is a perennial, rhizomatous sub-shrub, endemic to temperate coastal saltmarshes in Australia (Benson and McDougall, 1995). W. backhousei has a patchy distribution extending from the Central Coast of NSW south to Tasmania and west to Perth. The species was once relatively common in the Sydney region of NSW (Hamilton, 1919), but its abundance has now greatly declined, largely as a result of habitat destruction (Adam, 1996). A management plan developed for W. backhousei in 2004 identified a number of knowledge gaps hindering effective conservation and restoration for the species; among these was a lack of information on many aspects of its reproductive biology (Sydney Olympic Park Authority and Sainty and Associates, 2004). The capacity for natural recruitment and reproduction is essential for the longterm survival of any population and so effective management of a threatened species requires an adequate understanding of its reproductive biology (Vallee et al., 2004). Though W. backhousei is known to flower from October to December and produce one or two seeds per fruit (Benson and McDougall, 1995), the mechanisms for pollination and seed dispersal were unknown prior to this study. Flowering and fruiting have been observed to vary considerably among sites (pers. obs.), but factors influencing this variation were also previously unknown. Despite the species’ ability to produce seed, no seedlings have been observed in NSW over many years of monitoring (Clarke and Kerrigan, 1997). Whether this lack of recruitment is related to low seed viability, some form of seed dormancy, or edaphic factors inhibiting germination, has not been investigated previously. It has been suggested that the main mode of reproduction in W. backhousei is asexual (Benson and McDougall, 1995; Sydney Olympic Park Authority and Sainty and Associates, 2004). Given a stable environment, plants that reproduce asexually have a distinct advantage over sexual species in that a single clone may reproduce for many generations without the need for mates (Beebee and Rowe, 2004). Asexual reproduction also allows dispersal to varying degrees, both within and among sites (Adam, 1990; Sainty and Jacobs, 2003). To increase the chances of persistence in the long term, however, a population needs to possess sufficient genetic diversity to allow for evolution and adaptation to environmental change (Falk et al., 2006). For coastal saltmarsh plants such as W. backhousei, the threat of climate change and sea level rise means that the ability to adapt to changes in salinity and tidal inundation is likely to be essential to the survival of the species. As the distribution of genetic variation within and among populations is strongly linked to reproductive traits (Falk et al., 2006; Halkett et al., 2005), a first step in ensuring the maintenance of genetic diversity in restored populations of clonal species is to understand the factors that contribute to effective sexual reproduction, dispersal and recruitment. The aim of this study was thus to gain a better understanding of the reproductive biology of W. backhousei, both to assist

immediate efforts to conserve the species and to provide the foundation for future investigations. Specifically, we aimed to investigate: (i) population phenology, especially in relation to tidal inundation, (ii) edaphic factors influencing flower and seed production, (iii) morphology and phenology of individual flowers, (iv) potential pollination vectors, and (v) general seed biology, including dispersal and dormancy mechanisms, viability and the effect of saline substrates on germination. 2. Methods 2.1. Population phenology Phenology at the population level was examined in relation to tidal inundation in three estuaries in NSW, Australia (Table 1). Sampling was undertaken at eight saltmarsh stands within these localities, in belt transects established for a concurrent ecological study (manuscript in preparation). Investigations were conducted from early October to January at a total of six sampling sites in 2003, and eight in 2004 (Table 1). Each transect was set up to pass through the largest, continuous, undisturbed patch of W. backhousei at the site. Sampling area thus varied from 15 to 132 m2 (Table 1). Three to six 0.25 m2 quadrats were positioned randomly in each patch of W. backhousei; buds, flowers and fruits (developing and mature) were counted every two weeks in five (of 16) randomly selected subdivisions per quadrat. The timing of peak flowering and fruit maturation was then related to observations of tidal inundation at each site. Maximum floral density (flowers m−2 ) for the season was determined by summing the buds, flowers and fruits counted at each sampling event and using the highest of these values as an estimate of total flower production. Observations at the Newington Nature Reserve Nursery site (Table 1) did not commence till the onset of fruiting in 2003, therefore the proportion of developing fruits to flowers at this site in 2004 was used to estimate total flowering in 2003. This proportion was consistent between years (varying by only 2–11%) at other sites that flowered well in both 2003 and 2004. In 2003, a sub-sample of mature fruits from each site was checked for the presence of seed, and the proportion of fruit bearing seed was used to estimate seed production for each quadrat. In 2004, mature fruits were harvested from every quadrat and checked individually for the presence of seed. Differences in floral density and seed yield among sites, and between years within a site, were analysed by the non-parametric Kruskal–Wallis test in GenStat 10th ed. 2.2. Edaphic factors influencing flower production and seed yield At the end of each flowering season (late November–early December), projective foliage cover (PFC) was estimated for all

Table 1 Location and size of transects used to study flowering and fruiting in monocultures of Wilsonia backhousei. Locality

Site

Homebush Bay, Parramatta River ∼14 km NW of Sydney GPOa

Mason Park Waterbird Refuge Haslams Reach NNRb – Wharf Marsh NNR – Nursery Voyager Point

Voyager Point, Georges River ∼24 km SW of Sydney GPO Cararma Creek, Jervis Bay ∼150 km S of Sydney GPO a b c d

General Post Office. Newington Nature Reserve. Map datum = Australian Geodetic Datum 66. Sites studied in 2004 only.

Cabbage Tree Creekd Cararma Inletd

Transect size

Site coordinatesc

5m×3m 5m×3m 5m×3m 6m×5m 6m×3m 4 m × 15 m

S 33◦ 51 30.0 S 33◦ 50 25.0 S 33◦ 50 30.0 S 33◦ 49 33.7 S 33◦ 49 23.3 S 33◦ 57 20.7

E 151◦ 04 54.0 E 151◦ 05 03.0 E 151◦ 03 41.0 E 151◦ 03 55.8 E 151◦ 04 14.6 E 150◦ 58 17.7

12 m × 11 m 7 m × 13 m

S 34◦ 59 16.1 S 35◦ 00 13.0

E 150◦ 45 04.0 E 150◦ 46 42.8

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quadrats. Foliage height was measured in each of the 16 quadrat subdivisions and average height per quadrat was used in conjunction with PFC to calculate foliage volume (dm3 m−2 ). Four sediment cores (18 mm × 75 mm) were then taken randomly from within each quadrat and combined to produce a single composite sample for the quadrat. Sediment samples were air-dried at 40 ◦ C and analysed for water content, texture, salinity (measured as electrical conductivity), and pH. Water content was determined by loss of sample weight on initial drying at 40 ◦ C plus loss on further drying at 105 ◦ C; all other values were determined using sediment dried at 40 ◦ C. Sediment texture was determined following McDonald et al. (1990). Textural categories were converted to ordinal values of 1–6, representing increasing quantities of clay, following the Northcote interpretation (Hazelton and Murphy, 2007). Electrical conductivity (EC) and pH were measured in 1:5 sediment:water extracts. EC1:5 values were then used to estimate effective pore-water salinity (EPWS; the salinity surrounding the plant roots at the time of sampling) using the following equation: EPWS =

5 × EC1:5 WC40 ◦ C

EC1:5 is the electrical conductivity (dS m−1 ) of a 1:5 sediment:water extract and WC40 ◦ C is the water content of samples dried at 40 ◦ C, expressed as g per g sediment. EPWS provides a quantitative comparison of sediment salinity among samples, sites, and sampling periods. The equation assumes a linear relationship between the electrical conductivity of an undiluted sample and EC1:5 ; dilution and measurement of a series of artificial seawater solutions indicate that this assumption is valid for the range of EC1:5 values encountered in this study (≤13 dS m−1 ; data not shown). Differences in sediment characteristics among sites were tested by the non-parametric Kruskal–Wallis test in GenStat 10th ed. Raw data were normalised by log or arcsine transformations prior to further analysis; data that could not be normalised (e.g. floral density and seed yield) were converted to ordinal variables. Associations between response variables and sediment characteristics were then investigated using ordinal logistic regression in SPSS version 19. Ordinal predictor variables were analysed as co-variates rather than factors in order to preserve the ordering of the data. Deviance and Pearson Chi-square goodness-of-fit tests were used to check how well each model fitted the data. 2.3. Morphology and phenology of individual flowers Buds, flowers and fruit at various stages of development were examined using a dissecting microscope and an FEI XL30 environmental scanning electron microscope (ESEM; FEI, Oregon USA). The ESEM was operated at an accelerating voltage of 25 kV, and a pressure of 0.8–1.0 Torr, using a large field gaseous secondary electron detector. Particular attention was paid to the size and shape of pollen grains, the stigma surface, the presence of nectaries, and the relative arrangement of anthers and stigmas. An indication of breeding system was gained by calculating the pollen-ovule ratio (P/O) following Dafni (1992) for 10 buds collected randomly from the sampling sites at each of Newington Nature Reserve (NNR) and Mason Park (Table 1), and an additional site on a cliff top at Clovelly, NSW. The Kruskal–Wallis test was used to check for differences in P/O among sites. The sequence of floral development was examined in situ at NNR Wharf Marsh. Twenty flower buds close to opening were selected randomly, marked with flagging tape, and inspected daily for opening of the corolla, changes in colour and/or scent, relative position

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of stigma and anthers, pollen release, and the sequence of wilting. Observations commenced on 29 October 2004 and continued until each flower had wilted completely. Casual observations of insect visitation were made simultaneously. Fruit development was then observed every two weeks until fruit appeared mature. Internal seed structures were subsequently examined under ESEM as described above. 2.4. Pollination vectors Observations of floral morphology, phenology and insect visitation were used to give an indication of possible pollination vectors according to syndromes described by Fægri and van der Pijl (1979) and Ackerman (2000). As initial observations suggested W. backhousei was anemophilous, the potential for wind-pollination was tested in the field using glass microscope slides smeared with soft paraffin wax. Six slides were secured into each of two slide holders, uncovered slides alternating with slides enclosed by a double-layer poly-organza bag. The enclosing bags had a mesh size of 250 ␮m which has been shown to exclude most windborne pollen (Neal and Anderson, 2004), and should also have been small enough to exclude the range of insects observed at the site while allowing the passage of some windborne pollen. The slide holders were placed in a vertical position, at the approximate height of nearby W. backhousei flowers (50–100 mm), and positioned so that the paraffin-coated portion of the slides faced either due east or southeast, into the prevailing wind. After 24 h, the slides were collected and examined under a light microscope. The total number of pollen grains was counted in 10 randomly selected fields of view per slide. The presence of W. backhousei pollen on both covered and uncovered slides was considered an indication of wind transport of pollen. Differences resulting from the orientation of the slide holders were tested using the 2-sample T-test and compared to wind direction recorded at half-hourly intervals, for the duration of the test, at the Sydney Olympic Park weather station (∼3 km from the study site). 2.5. Seed biology 2.5.1. Dispersal The potential for tidal dispersal of propagules was first assayed by a flotation test. Sixty capsules were collected randomly from NNR Wharf Marsh and three replicates of ten capsules were placed in containers filled with tap water. The number of fruits floating in each container was recorded at intervals of approximately 0, 24, 48, 72 and 96 h. The ability of fruit to float was considered an indication of the capacity for tidal dispersal. To test for tidal dispersal in the field, small nets were placed in a semi circle about a patch of W. backhousei that had produced a good quantity of fruit, and was known to be covered by extreme high tides at the time of fruit maturity. Each net was 1 m in length, with an opening of 200 mm × 250 mm and a mesh size of 1 mm2 . The height of the opening was sufficient to ensure that the top of the net remained above water level during the high tide. Each net was secured to the saltmarsh surface with the opening facing into the ebb tide, approximately 1 m away from the nearest patch of W. backhousei. The nets were left in place for the duration of the king tides (three consecutive days) in both January and February, 2005. At the conclusion of each series of tides, the nets were collected and examined for the presence of W. backhousei fruit. The capture of seed-bearing fruit was considered to be an indication of dispersal by tides. 2.5.2. Dormancy and viability Approximately 900 seeds for dormancy and viability testing were collected randomly from NNR Wharf Marsh in January 2005.

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All seeds were removed from their capsules and stored dry at room temperature for six weeks prior to testing. As the seed coat appeared impermeable, an imbibition test (Baskin and Baskin, 2001) was conducted to check for physical dormancy. A random sub-sample of 30 seeds was placed on moist filter paper in a single Petri dish, then removed, blotted dry and weighed individually to the nearest 0.1 mg. The seeds were replaced randomly on the moist filter paper, held at room temperature in a laboratory for 72 h, then blotted dry and re-weighed. The 2-sample T-test was used to test for any significant change in weight between 0 and 72 h. Seed viability was investigated by germination testing. Two hundred seeds were sterilised for 5 min in 0.5% NaOCl (to control fungal growth observed in an earlier pilot experiment) and rinsed in sterile water; 100 of the sterilised seeds were scarified (nicked with a scalpel) to break any physical dormancy and the remainder were left intact. Intact and scarified seeds were sown onto filter paper moistened with sterile distilled water, in 5 replicates of 20 seeds per treatment, and incubated in a growth cabinet for 28 days with a light/dark cycle of 13/11 h and alternating temperatures of 27/17 ◦ C (based on average temperatures recorded for February/March – the months of highest mean rainfall – in the local area). Treatments were placed randomly within the growth chamber, inspected three times per week for germination, and re-randomised following each inspection. The filter paper was re-moistened with sterile distilled water as needed. Germinated seeds, or seeds infected with mould, were removed as observed. After 28 days, all non-germinating seeds were checked for viability by the cut-test (Baskin and Baskin, 2001). Seed was considered viable if the contents were still firm and creamy-white. 2.5.3. Germination on saline substrates The effect of salinity on germination was tested in 2010 on seed collected randomly from NNR Wharf Marsh and stored for 6 years at ambient temperatures in a non-air conditioned room. Seed was scarified (as in Section 2.5.2) then sown onto 0.7% (w/v) agar prepared with distilled water or one of three saline solutions. Saline solutions were prepared using NaCl and CaCl2 in the molar ratio 45:1 (the approximate ratio found in seawater) and diluted to produce media with electrical conductivities of 10, 20 and 40 dS m−1 (equivalent to approximately 0.2, 0.4 and 0.8 × the EC of natural seawater). The pH of the solutions was adjusted to 7.5–7.6, the average pH of sediment supporting monocultures of W. backhousei in the Wharf Marsh (Section 2.2). Seed was sown in five replicates of ten seeds, and incubated in a growth cabinet for 28 days at 27/12 ◦ C with a light/dark cycle of 12/12 h. These incubation conditions differed slightly from the original germination test (Section 2.5.2) as the experiment was conducted, of necessity, in shared incubators at the NSW SeedBank. Treatments were placed randomly within the growth chamber, inspected once per week for germination, and re-randomised following each inspection. Differences among treatment effects on total germination were analysed using Binary Logistic Regression in GenStat 10th ed. 3. Results 3.1. Population phenology Flower buds were visible from mid-September in both sampling years and anthesis commenced in early October. Peak flowering occurred in mid- to late-October; mature fruits appeared from December through January. Mean floral density (flowers m−2 ) varied significantly among sites (P < 0.001, Fig. 1), ranging from 0 at Mason Park (in 2004) to 5771 ± 1380 m−2 at NNR Wharf Marsh. Mean seed yield (number of seeds per flower) also varied

Fig. 1. Mean flower and seed densities in monocultures of Wilsonia backhousei at various sites in New South Wales, Australia. Data for 2004 presented. NNR: Newington Nature Reserve.

significantly among sites (P < 0.001, Fig. 1), and ranged from 0 at several sites to 3603 ± 1063 m−2 at NNR Wharf Marsh. Significant differences between years were only observed for floral density at Mason Park (P = 0.037) and for seed yield at NNR Wharf Marsh (P = 0.004). At three sites in Homebush Bay (Mason Park, Waterbird Refuge and Newington Nature Reserve Nursery) no seed was produced at all (despite the production of flowers in one or both years), while only a very few seeds were produced at Voyager Point. Flowers in the study transect at Cabbage Tree Creek also failed to produce seed though seed was observed elsewhere within the site. As the flowers in that transect were submerged under rainwater for the duration of flowering (approximately three weeks), it is likely that flooding prevented pollination. Tidal inundation of quadrats was observed at five of the eight sites. Inundation only occurred during extreme high tides and was therefore restricted to several consecutive days in each of November, December, January and February. Tidal inundation occurred after peak flowering and coincided with fruit maturation. 3.2. Edaphic influences on floral density and seed yield Mean values for sediment water content at each site ranged from 0.1 to 0.8 g g−1 , estimated pore water salinity (EPWS) ranged from 30 dS m−1 to 288 dS m−1 , and soil pH varied from 6.2 to 8.2. All three variables differed significantly among sites (P < 0.001). Differences were also observed between years at some sites. At Mason Park, water content was significantly lower (P < 0.001) and salinity significantly higher (P < 0.001) in 2004 than in 2003. Salinity was also significantly higher in 2004 at the Waterbird Refuge, Haslams Reach and Newington Nature Reserve Nursery (P < 0.05). Sediment texture varied both within and among sites and ranged from loamy sand (clay content 5–10%) at Cararma Inlet to heavy clay (clay content 45–55%+) at Mason Park, the Waterbird Refuge, Haslams Reach and Voyager Point. Floral density and seed yield were greatest in NNR Wharf Marsh (Table 1) where sediment water content ranged from 0.3 to 0.6 g g−1 dry weight, EPWS ranged from 43 to 127 dS m−1 , pH ranged from 7.5 to 8.5, and the texture varied from sandy loam to sandy clay loam (approx. 10–20% clay content). Regression of floral density against all measured variables identified a significant positive response to increasing foliage volume and negative responses to increasing sediment salinity and clay content (N = 58, P < 0.05). Foliage volume, in turn, showed a significant positive relationship to increasing sediment water content and, again, a negative response to increasing sediment salinity and

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Table 2 Edaphic and biotic variables influencing foliage volume, floral density and seed yield in monocultures of Wilsonia backhousei. Data analysed by ordinal logistic regression. Predictor

Foliage volumeb (N = 58) c

B Water contenta Salinitya Textureb pH Foliage volumea Floral densityb

13.178 −3.467 −0.453 −0.483 – –

d

Floral densityb (N = 58) e

P

Fit

0.001 0.000 0.046 0.261 – –

0.917

B −4.736 −2.262 −0.888 0.063 2.401 –

Seed yieldb (N = 20)

P

Fit

0.156 0.035 0.000 0.870 0.027 –

1.142

B 22.847 6.119 0.828 −0.431 – 1.123

P

Fit

0.015 0.088 0.225 0.861 – 0.092

0.901

Bold text has been used to highlight variables having a significant effect. a Log-transformed variables. b Ordinal variables. c B represents the regression coefficient such that for every one unit increase in the predictor we may expect a B increase (or decrease if the sign is negative) in the expected log-odds of a higher level of response. d P-values <0.05 indicate a significant effect. e P-value for the Pearson Chi-square goodness-of-fit test.

clay content (N = 58, P < 0.05). Seed yield appeared to have significant positive responses to increasing floral density and all sediment variables (N = 47, P ≤ 0.004); however, when quadrats that failed to produce seed were removed from the data set (to limit the potential influence of self-incompatibility), the only significant predictor of yield was sediment water content (N = 20, P = 0.015). The magnitude of the log-odds for each significant predictor variable (Table 2) suggested that foliage volume had the greatest influence over floral density, while sediment water content had a far greater influence over foliage volume and seed yield than any other variable. All goodness-of-fit tests yielded P-values >0.9 indicating the models fitted well with the data. 3.3. Morphology and phenology of individual flowers Individual flowers were white, apparently unscented, and did not appear to produce nectar though a ring of potentially nectarproducing tissue was present at the base of the ovary (Fig. 2a). Flower size was small in comparison with other Convolvulaceae species (corolla diameter ∼5 mm). Pollen grains were very small

(20–30 ␮m in length), had a smooth, dry surface (Fig. 2b and c), did not exhibit clumping and were easily dispersed by a puff of air. By comparison, the two stigmas were quite large and possessed a finely divided surface with numerous depressions into which a pollen grain might lodge (Fig. 2d). The mean P/O was found to be 23 000 ± 1500, a ratio that is consistent with that of an outcrossing, wind-pollinated, homoecious species (Cruden, 2000). There were no significant differences in P/O among sites (P = 0.883). Flowers remained open for the duration of the flowering period and exhibited herkogamy (spatial separation of anthers and stigma) and potential dichogamy (temporal separation of pollen release and stigma receptivity). On the first day of opening, pollen was released from anthers held well above the level of both the corolla and the stigmas (Fig. 3a). Pollen was fully released by the second or third day, after which the stamens began to wilt and the two styles lengthened, in most cases raising the stigmas above the level of the corolla and above the level of the spent anthers (Fig. 3b). If the stigmas do not become receptive until the styles have reached their full length, then anther dehiscence and stigma receptivity are

Fig. 2. Wilsonia backhousei: (a) immature bud showing developing anthers ‘A’, stigmas ‘S’, ovary ‘O’ and potential nectary ‘N’; (b and c) pollen grains in latitudinal and polar views, respectively; (d) stigmatic surface with adhering pollen grain ‘P’.

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Fig. 3. Wilsonia backhousei: (a) newly opened flower with dehiscing anthers ‘A’ and immature stigmas ‘S’; (b) flower with spent anthers ‘A’ and mature stigmas ‘S’; (c) cross-section of seed showing palisade cells in seed coat ‘P’ and folded embryo ‘E’; (d) cross-section of seed showing fully developed cotyledons ‘C’ and radicle ‘R’.

temporally separated. These floral features, combined with those described in the previous paragraph, are characteristic of windpollination (Dafni, 1992; Ackerman, 2000). The corolla began to wilt four to ten days after opening, with stigmas wilting simultaneously or soon after. Fruit development, indicated by a reddening and swelling of the calyx, was evident within a week after the corolla had wilted. Mature fruit, which began to appear in December, consisted of a small, papery, brown capsule that was easily detached from the parent plant and contained one or occasionally two seeds. 3.4. Pollination Observations made while studying floral phenology indicated that: (a) pollen could be easily dispersed by wind and (b) insect visitation was rare and the movements of occasional visiting insects did not appear sufficient to effect pollination. The few insects observed on the flowers over a period of 10 consecutive days – hover flies, honey bees, thrips and a single butterfly – either did not touch the anthers and stigmas, or simply collected pollen from dehiscing anthers. A single native hover fly was observed to touch both anthers and stigmas on one occasion only. W. backhousei pollen grains were found on all six of the open glass slides used in the field test; a smaller amount of pollen was also present on all covered slides. No pollen from any other species was observed. Slides facing south-east collected significantly more pollen grains (2.2 ± 0.3 mm−2 ) than slides facing due east (1.1 ± 0.2 mm−2 , P < 0.05). Wind direction over the 24 h test period ranged from SE-SSE in the late morning until early evening (11 h total, mean wind speed 23 km h−1 ), and S-WSW at other times, suggesting wind direction had an influence on pollen deposition. No evidence of insect visitation (e.g. tracks, scats or insect bodies) was observed on any of the slides. 3.5. Seed biology 3.5.1. Dispersal mechanisms Fruit placed in tap water floated for up to four days, indicating that dispersal of fruit by tides is possible. Nets placed in the field

to collect propagules travelling on outgoing tides did catch fruit containing seed; however, the number caught was very small – a total of five in January and two in February. 3.5.2. Dormancy and viability The seed possessed a hard seed coat consisting of two cell layers – the innermost composed of thickened palisade cells – enclosing a large, folded embryo with little surrounding endosperm (Fig. 3c and d); these are features associated with physical seed dormancy (Baskin and Baskin, 2001). The imbibition test confirmed the presence of physical dormancy with no significant change in weight of non-scarified seed after 72 h on moist filter paper (P = 0.504). Nonscarified seed incubated on moist filter paper for 28 days failed to germinate, while scarified seed germinated to 73 ± 6% within 2 weeks. Seed that did not germinate, but did not have to be removed because of fungal growth, appeared viable at the termination of the experiment. 3.5.3. Germination on saline substrates Scarified 6-year old seed was still able to germinate to 72 ± 10% in non-saline conditions (in this case, on water agar). Germination of the aged seed was not significantly reduced, compared with the control, by exposure to salinity levels of 10 dS m−1 (68 ± 7.3%, P = 0.663) or 20 dS m−1 (60 ± 4.5%, P = 0.207). Total germination at 40 dS m−1 was only 6 ± 2.4% after 28 days, significantly less than at the lower levels of salinity (P < 0.001). Seed exposed to 40 dS m−1 continued to germinate slowly over time, however, and after 71 days had reached 26% germination. Seeds that failed to germinate at this level of salinity were inspected by the cut-test after 100 days; the majority of non-germinating seeds were no longer viable due to fungal and/or bacterial infection. 4. Discussion As saltmarsh communities are important on a number of levels – from maintaining the environmental health of estuaries (Jickells and Rae, 1997; Valiela, 2006) to maintaining resources for commercial fisheries (Zimmerman et al., 2000; West, 2001a,b; Mazumder et al., 2006), and providing habitat and food-web support for a

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wide range of fauna (Laegdsgaard, 2006) – restoration of degraded saltmarsh communities can have multiple benefits, provided that the restoration is undertaken with the aim of establishing selfsustaining and functional communities. As this process may require supplementary planting for species that do not re-establish readily, successful restoration requires an understanding of the biology and ecology of the constituent species (Green et al., 2009). Here we present the results of an investigation into the reproductive biology of W. backhousei in order to determine the conditions that would need to be duplicated to ensure successful reproduction, dispersal and establishment in a restored community. This work formed part of a wider conservation-focused study of threatened saltmarsh communities on the mid-NSW coast of south-eastern Australia. 4.1. Population phenology Peak flowering in W. backhousei coincided with a period of relatively low high tides. In the populations studied, inundation only occurred during the extreme high (king) tides in both winter and summer. Flowering commenced two months after the last winter king tide, and was largely finished before the onset of the first of the summer king tides in November. As regular tidal submersion during the flowering period may interfere with both wind and insect pollination (Adam, 1990), the timing of these events ensures that flowers are kept dry during anthesis (notwithstanding rain events), allowing efficient dispersal of pollen. The importance of inundation during anthesis is reflected in the lack of seed set in the transect at Cabbage Tree Creek, where the flowers were submerged under rainwater for several weeks during peak flowering (Section 3.1). Fruit maturity, however, coincided with three king tide events in December, January and February, indicating that the dispersal of mature fruit may be facilitated by the ebb and flow of the tides. Restoration practitioners could thus improve the reproductive and dispersal potential of W. backhousei by ensuring the species is planted at such an elevation that it will only be submerged by extreme high tides in winter and summer. 4.2. Edaphic influences on flower and seed production Of the variables investigated, sediment water content was found to have the strongest influence on reproductive success via its influence on foliage volume (and hence floral density) and seed development (Section 3.2). The sediment water content of a saltmarsh is determined by a number of factors including rainfall, proximity to the water table, the frequency and duration of tidal inundation, the water holding capacity of the sediment and topography. Given that the frequency of tidal inundation of W. backhousei must be limited to ensure effective pollination (Section 4.1), the sediment water content at a restoration site may be improved by ensuring close proximity to the water table and/or or by maximising the infiltration and retention of rainwater. Infiltration is likely to be best on sites that are relatively flat (minimising surface runoff) and have sediments that are light in texture and fairly porous. Flat sites that are situated at the base of a slope would also receive the benefit of surface run-off. Water retention is influenced by the texture of the sediment, with greater retention in soils with a higher proportion of clay particles, though silts and fine sands have been reported to hold the most water in the available water range (Hazelton and Murphy, 2007). In this study, however, growth and flowering showed a negative response to increasing levels of clay (Section 3.2). This outcome was no doubt influenced by those sites situated on heavy clay soils (Mason Park, Waterbird Refuge and Voyager Point) which were also highly compacted. Such compaction limits water infiltration and

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also physically limits plant growth (Hazelton and Murphy, 2007). While the site at Haslams Reach (one of only three to produce good quantities of seed; Table 1) also had heavy clay soil, the high proportion of gravel in the soil (10–60% by weight, data not shown) would have reduced compaction and improved water infiltration. Heavy clay soils may also retain more salts (depending on the type of clay) than soils of lighter texture and increasing salinity was another factor observed to limit growth and flowering for this species (Section 3.2). The greatest flowering and seed yield was observed at Newington Nature Reserve Wharf Marsh, where the population was situated on flat land at the base of a slope, in close proximity to the water table, on sediment that varied from sandy loam to sandy clay loam (10–20% clay content). The sediment at this site was never dry and flowering was consequently profuse. These substrate characteristics would serve as a good model for restoration practitioners in the preparation of sites for planting W. backhousei.

4.3. Morphology and phenology of individual flowers W. backhousei flowers possess a number of structural and developmental features that are characteristic of the wind-pollination syndrome described by Ackerman (2000), including: spatial, and potentially temporal, separation of male and female reproductive structures; small, smooth and dry pollen; exposed anthers and stigmas; drab, reduced perianth; and an apparent lack of scent and nectar production (Section 3.3). This is atypical of Convolvulaceae species which often exhibit medium to large, showy flowers (Martin, 1970; Watson and Dallwitz, 2006), produce scent and/or nectar, and are pollinated by insects (Willmott and Búrquez, 1996; Ushimaru and Kikuzawa, 1999; Galetto and Bernardello, 2004). In addition, the flowers of Convolvulaceae species often last only a single day (Willmott and Búrquez, 1996; Ushimaru and Kikuzawa, 1999; Galetto and Bernardello, 2004; Fairley and Moore, 2000) whereas W. backhousei flowers remain open for up to seven days. The P/O of W. backhousei (23 000 ± 1500) was very close to the median P/O reported by Cruden (2000) for outcrossing, windpollinated, bisexual species. This ratio suggests that the species is chiefly outbreeding, a suggestion that is supported by the presence of herkogamy – a floral trait associated with minimising self-pollination (Dafni, 1992). The lack of seed set in some populations (e.g. Mason Park South, the Waterbird Refuge and Newington Nature Reserve Nursery), despite the production of flowers and the availability of a pollen vector, suggests that W. backhousei may also be self-incompatible. This condition has previously been found in a number of other perennial species belonging to the Convolvulaceae family (Devall and Thien, 1992; Stucky and Beckmann, 1982; Willmott and Búrquez, 1996; Ushimaru and Kikuzawa, 1999; Galetto and Bernardello, 2004). Self-incompatibility has important implications for a clonal species. In areas colonised by a single propagule, for example, or restored using cuttings from a single individual, the species may be able to spread vegetatively, but will be unable to produce seed due to the absence of compatible mates. In areas colonised by compatible clones, large distances between the clones may limit sexual reproduction by limiting pollen transfer (Wolf and Harrison, 2001). A self-incompatible mating system in W. backhousei would thus mean that the selection of a diverse array of clones, and their placement in such a way as to allow effective pollen transfer, would be very important to the long-term success of any restoration program. Conversely, the absence of seed set in a population could indicate to conservation managers that clonal diversity in that population was minimal. This issue was investigated further in a genetic study of the species (manuscript in preparation).

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4.4. Pollination vectors It has generally been assumed that many saltmarsh species are wind pollinated as this is the characteristic pollination mode in the major saltmarsh families Graminae, Cyperaceae, Juncaceae and Chenopodiaceae (Hamilton, 1919; Adam, 1990). Many Convolvulaceae species, however, are insect pollinated (see, for example, Willmott and Búrquez, 1996; Ushimaru and Kikuzawa, 1999; Galetto and Bernardello, 2004), and Convolvulaceae is not listed among the 60 angiosperm families in which anemophilous species have been recorded (Ackerman, 2000). Despite this, the pollenovule ratio of W. backhousei closely corresponds to the median P/O reported for outcrossing, wind-pollinated, homoecious species (Cruden, 2000). Though this P/O is not large compared to some anemophilous species, it is more than three times the P/O of the wind-pollinated tree Betula pendula (syn. B. verrucosa) (Fægri and van der Pijl, 1979). While some degree of biotic pollination cannot be ruled out without further study, the combination of anemophilous floral features, transport of pollen by wind, and limited insect visitation suggest the species is predominantly windpollinated. Airborne pollen may travel hundreds (van de Water et al., 2003) or even thousands (Bourgeois, 2000) of kilometres under the right conditions, yet the distance over which pollination actually occurs is much less. Successful pollination after transport is dependant on a number of factors, including the length of time the pollen remains viable. A recent review of genetic studies on tree pollination found that wind borne pollen commonly fertilised individuals only hundreds of metres away from the source (Ashley, 2010); however, a study on Populus trichocarpa found that one third of pollinations resulted from pollen that had travelled more than 16 km (Slavov et al., 2009). Even low growing species, such as grasses, have been found to effect pollination over distances of up to 21 km (Watrud et al., 2004). Once wind-borne pollen is released, transport and successful fertilisation are dependent on such factors as wind speed, turbulence and the frequency of updrafts (Tackenberg, 2003), local topography (van de Water et al., 2007), height of the vegetation (Fægri and van der Pijl, 1979) and the structure of surrounding vegetation (Dafni, 1992). For the low growing W. backhousei, pollen transport is likely to be affected by the presence of taller saltmarsh and terrestrial species that may act as filters to pollen movement. In addition, the fragmented saltmarsh communities in New South Wales are frequently surrounded by trees and/or buildings and open water (pers. obs.); therefore, the effective transport and deposition of W. backhousei pollen may be restricted to within populations, with minimal pollen flow among them. As wind appears to be an important pollination vector for this species, plants translocated for restoration work should be placed so as to allow effective air-borne pollen transport. This would involve placing compatible plants in close proximity, preferably without taller vegetation that might interfere with pollen movement. Plants should also be placed so as to be beyond the reach of normal high tides during the flowering season to prevent tidal inundation interfering with the pollination process. 4.5. Seed biology 4.5.1. Dispersal The ability of W. backhousei fruit to float for up to four days was unexpected, given that Hamilton (1919) noted the species lacked any equipment for lengthy flotation. The buoyancy of fruits of some species may be achieved by the presence of hairs that trap air bubbles (Huiskes et al., 1995) and this is certainly possible for W. backhousei fruits, which contain numerous hairs on the inner surface of each calyx lobe that persist in the mature fruit. A buoyancy period of four days may not be considered lengthy in comparison to

some saltmarsh species – the seed of eight species tested by ElseyQuirk et al. (2009), for example, had average flotation times ranging from 24 to 95 days in seawater – yet it is sufficient to ensure the potential for dispersal by tides, at least within the same estuary. The capture of fruit by nets placed in the field is an indication that W. backhousei fruit can be dispersed by tides; the small number of fruits captured may indicate rarity of dispersal or may be an artefact of the trap design. Huiskes et al. (1995) found that floating nets captured significantly more propagules than nets fixed to the saltmarsh surface (as used in the present study). The authors also found that, in the mid- to high-marsh, significantly more propagules were caught in nets facing into the flood tide than in nets facing the ebb tide, indicating that transport of propagules from this part of the marsh was predominantly landward. As the nets in this instance were placed so as to face into the ebb tide only, and were in place for several days, it is possible that more W. backhousei propagules were transported landward (away from the nets) than seaward, and/or that some of the propagules initially carried into the nets were washed back out on subsequent flood tides. Previous studies have found that the majority of saltmarsh seeds are dispersed only locally (Rand, 2000; Griffith and Forseth, 2002; Wolters et al., 2005, 2008; Morzaria-Luna and Zedler, 2007). While there is potential for seeds exported from a marsh to travel quite long distances (Koutstaal et al., 1987; Huiskes et al., 1995; Minchinton, 2006), the actual distance travelled is likely to be dependent on tidal amplitude (and, hence, velocity) and the presence of any vegetative barriers between the source population and the open water. In the sites used in this study, the tidal regime is microtidal (≤2 m) and tidal velocities are correspondingly slow. The distance travelled by any W. backhousei fruit exported from a marsh may therefore be relatively short. In addition, vegetative barriers created by taller species – such as Sarcocornia quinqueflora in Newington Nature Reserve Wharf Marsh and Juncus krausii at Voyager Point – have the potential to trap any fruit carried by the ebb tide and to limit the inward transport of propagules from neighbouring marshes. At sites where the W. backhousei population extends right to the water’s edge (e.g. Cararma Inlet and Cabbage Tree Creek), tidal export no doubt has a much greater chance of success.

4.5.2. Dormancy and viability W. backhousei seeds exhibit several features characteristic of physical dormancy (Baskin and Baskin, 2001; Fig. 3g) that are also found in other members of the Convolvulaceae that possess this barrier to germination (Sharma and Sen, 1974; Chandler et al., 1977; Hutchison and Ashton, 1979; Swan, 1980). In this study, the high rate of germination of nicked seed in non-saline conditions indicated that, though the seed is physically dormant, it is also highly viable (Section 3.5.2). In nature, seed coat damage sufficient to break dormancy may occur through the action of predators, through repeated cycles of wetting and drying, through tumbling over rocks or coarse sediment, through the activities of infauna such as crabs, or simply through gradual deterioration. In natural conditions, the germination of seed with an impermeable seed coat may spread over several years, potentially forming a long-lived soil seed bank. This has been found to be the case for other species in the Convolvulaceae such as Evolvulus nuttalianus (7 years) and Ipomoea purpurea (7+ years) (Baskin and Baskin, 2001). Given that six-year old W. backhousei seed germinated as well as fresh seed, after storage in uncontrolled conditions (Section 3.5.3), it is likely the seed may persist in the soil seed bank for many years. Certainly the seed appears to be orthodox in its storage behaviour and is thus suitable for ex situ conservation by seed banking.

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4.5.3. Germination on saline substrates The substantial reduction in germination of W. backhousei at salinity levels close to seawater (Section 3.5.3) is typical of many saltmarsh species (Woodell, 1985; Noe and Zedler, 2000; ElseyQuirk et al., 2009). Increasing levels of salinity increase external osmotic pressure, making it difficult for the embryo to take up the water required for germination. Germination therefore requires sufficient rainfall to dilute the salts present in the topsoil of the marsh (Adam, 1990; Noe and Zedler, 2001). The continued slow germination of W. backhousei at 40 dS m−1 over 60 days, however, suggests that this species has some capacity at least to germinate at high saltmarsh salinities, albeit at a much slower rate. This raises the interesting possibility that continued exposure of non-dormant seeds to salt may induce the production of osmoprotectants in the embryo, increasing its ability to take up sufficient water to germinate. This would make a useful future study. Though the seed of W. backhousei is obviously viable, germination appears to be a rare occurrence in the field (Clarke and Kerrigan, 1997). This is likely due to the combined effects of physical dormancy (Section 3.5.2) and very saline substrates (Section 3.2), i.e. germination may be occurring, but so infrequently as to escape detection. Germination in greater numbers would require damage or aging of the seed coat and sufficient rain to reduce the salinity of the sediment to half seawater or less. Other factors, such as temperature and light, are also likely to be important though this species has been observed to germinate equally well at alternating temperatures of 20/10, 27/12 and 27/17 ◦ C (unpubl. data). 4.6. Implications for conservation and restoration The results of this study have provided information on the reproductive biology of W. backhousei that will assist in the effective management of remnant populations and in the planning of future restoration projects. In summary, the reproductive potential of remnant or restored populations of the species may be maximised by ensuring the site has sufficient sediment water content to allow good flower and seed production, a diversity of clones to enable cross-pollination, compatible clones situated in close proximity to enable effective transfer of pollen by wind, and inundation by summer king tides to enable the dispersal of fruit. Acknowledgments We thank the Sydney Olympic Park Authority for providing financial support and access to Newington Nature Reserve. We thank Ray Kilduff, Jenny Wilkins, Gemma Armstrong, Janine Wech and Patti Kuhl for assistance in field sampling and sediment analysis. We thank the Microstructural Analysis Unit of The University of Technology, Sydney, for providing access to the FEI XL 30 Environmental Scanning Electron Microscope and training in its use. We also thank two anonymous reviewers for their valuable input. References Ackerman, J.D., 2000. Abiotic pollen and pollination: ecological, functional, and evolutionary perspectives. Plant Syst. Evol. 222, 167–185. Adam, P., 1990. Saltmarsh Ecology. Cambridge University Press, Cambridge. Adam, P., 1996. Saltmarsh vegetation study. In: Homebush Bay Ecological Studies, 1993–1995, vol. 2. CSIRO, Collingwood, Victoria. Adam, P., 2002. Saltmarshes in a time of change. Environ. Conserv. 29, 39–61. Ashley, M.V., 2010. Plant parentage, pollination and dispersal: how DNA microsatellites have altered the landscape. Crit. Rev. Plant Sci. 29, 148–161. Baskin, C.C., Baskin, J.M., 2001. Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego. Beebee, T.J.C., Rowe, G., 2004. An Introduction to Molecular Ecology. Oxford University Press, New York. Benson, D., McDougall, L., 1995. Ecology of Sydney plant species. Part 3. Dicotyledon families Cabombaceae to Eupomatiaceae. Cunninghamia 4, 217–297.

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