Melaleuca Deanei) and Three Common (Melaleuca Styphelioides, Melaleuca Thymifolia and Melaleuca Nodosa) Melaleuca (Myrtaceae) Species of the Sydney Region

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Austral Ecology (2015) 40, 661–671

Seed size and the regeneration niches of one rare (Melaleuca deanei) and three common (Melaleuca styphelioides, Melaleuca thymifolia and Melaleuca nodosa) Melaleuca (Myrtaceae) species of the Sydney region

ALISON HEWITT,1* PAUL HOLFORD,1 ADRIAN RENSHAW,1 GLENN STONE2 AND E. CHARLES MORRIS1 1School of Science and Health and 2School of Computing, Engineering and Mathematics, University of Western Sydney, Locked Bag 1797, Penrith, New South Wales 2751, Australia

Abstract Melaleuca species occupy varied habitats across Australia with 16 members of the genus occurring in the Sydney district including the rare species Melaleuca deanei F. Muell. Little is known of their germination and recruitment requirements.This paper reports on experiments assessing the effects of temperature, water potential, fire cues, light and shade levels on germination in four species of Melaleuca native to the Sydney region. For the shade experiment, seedling survival at 12 months is also reported. The experiments tested the hypothesis that M. deanei, which exhibits few seedlings in the field, has limited seedling recruitment because of its particular requirements for germination and establishment. Further, that it differs in these requirements from three common congenerics: M. nodosa (Sol ex Gaertn.) Sm., M. thymifolia Sm. and M. styphelioides Sm. Results indicate that M. deanei has a substantially similar temperature and water potential range for germination to the common congeners from 15 to 35°C, and 0 to −0.65 ψ. Melaleuca nodosa displayed the broadest regeneration niche on all factors assessed. Germination of M. styphelioides was significantly reduced in the dark and M. styphelioides and M. deanei were most sensitive to shade in the seedling establishment/post-germination phase. Melaleuca deanei had significantly larger seed that was slower to germinate in all experiments. Germination of the four species was unaffected by the application of heat and smoke.The substantially similar germination parameters exhibited by the four species, despite differences in habitat, may reflect their close phylogenetic affinities. The effects of fire on stimulating seed release from the canopy, the high-light environment post-fire together with adequate follow-up rainfall may all be critical in seedling establishment for M. deanei.

Key words: ﬁre cue, germination, light, phylogenetic niche, shade, temperature, water potential.

INTRODUCTION Melaleuca deanei F. Muell. is a rare, serotinous shrub of restricted distribution centred on the Sydney region Maintaining population numbers through time is of New SouthWales (DECCW 2010) where it occurs in important for any species and is particularly so for small, disjunct populations that are reproductively iso- those that are rare (Schemske et al. 1994; Yates lated from each other. Population sizes range from & Broadhurst 2002; Gaston & Fuller 2008; relatively small (<200 plants) to large (>700 plants), Commonwealth of Australia 2014). Failure to main- allowing comparison of reproductive and recruitment tain population numbers via replacement of senescent success between populations of different sizes. Earlier individuals can occur as a result of poor reproductive work (Hewitt et al. 2014) found poor reproductive success (flowering, pollination and seed set) or poor success within relatively smaller populations of recruitment success (dispersal, germination and estab- M. deanei; these populations had a lower incidence of lishment) (Cropper 1993; Hobbs & Yates 2003). Small flowering, lower proportions of fruiting plants and plant populations are particularly at risk of such fail- lower viable seed loads per unit area, compared with ures and further demographic decline, even without relatively larger populations or to populations of obvious external threats to their persistence (Gilpin & three common congeners (M. styphelioides Sm., Soule 1986; Kunin & Gaston 1997). M. thymifolia Sm. and M. nodosa (Sol ex Gaertn.) Sm.). However, poor recruitment was recorded in both large and small populations of M. deanei.When recruit- *Corresponding author. School of Science and Health, Uni- versity of Western Sydney, Locked Bag 1797, Penrith, NSW ment was observed, it appeared to be tightly coupled to 2751, Australia (Email: [email protected]) disturbance by fire and follow-up rainfall (Hewitt et al. Accepted for publication January 2015. 2014). An absence of seedlings and a predominance of

© 2015 The Authors doi:10.1111/aec.12233 Austral Ecology © 2015 Ecological Society of Australia 662 A. HEWITT ET AL. older individuals were also recorded within urban, METHODS streamside populations of M. styphelioides. We hypothesized that given adequate quantities of The study species viable seed, seedling recruitment of M. deanei and M. styphelioides may only occur under specific sets of The four Melaleuca species are serotinous resprouters that conditions. If these conditions rarely occur, recruit- produce fruit in summer (Hewitt et al. 2014). Melaleuca ment events will be few. For example, Robinson et al. styphelioides is weakly serotinous, retaining capsules for 1–2 (2006) reported relatively narrow conditions of seasons, whereas the other three species are strongly favourable salinity, temperature and light for success- serotinous, retaining fruit within the canopy for many years. ful germination of Melaleuca ericifolia Sm. (swamp Melaleuca nodosa is a shrub to 4 m that is found over clay and paperbark), corresponding to field conditions that shale soils; Melaleuca thymifolia a shrub to 2 m that inhabits occur infrequently. Conversely, germination could be poorly drained soils of shale or clay influence; Melaleuca occurring over a relatively wider range of conditions, styphelioides is a shrub or small tree to 15 m that is often found over alluvial soils along stream banks; and M. deanei is a shrub but at suboptimal times for subsequent seedling to 3 m that is restricted to disjunct, sandstone ridgetops found establishment. For example, Ramirez-Padilla and north and south of Sydney from Brisbane Waters to Nowra. Valverde (2005) reported less specific germination requirements in a rare species of cactus, compared with two common species, suggesting that germination Seed collection and preparation occurred at times of suboptimal seedling survival, a factor contributing to seedling death and declining Seed was collected from eight sites (two sites per species: abundance. Table 1) in February–March of 2009 (see Hewitt et al. 2014 If the four congeners have substantially similar ger- for comprehensive site details). At each site, five mature mination responses, it may be that differences in their infructescences were removed from 10 randomly chosen environments are influencing recruitment. Melaleuca fruiting plants from 2 patches per site (20 plants per site in deanei is found on Hawkesbury sandstone ridgetops, total). Infructescences were placed into brown paper bags in whereas the common congeners occur on low-lying the field. The bags were stored upright and open on a bench streambanks, or swampy or seasonally inundated posi- at ambient laboratory temperature and low humidity for 4 weeks. Each bag was then individually emptied onto a metal tions that are more typical of the genus. In a large study of ecological and phylogenetic correlates of germination in 633 species of alpine meadows, HaiYan et al. Table 1. Provenance of seeds used in experiments on water (2009) showed that taxonomic membership accounted potential (Ψ), temperature and fire cues (T), effect of light for the majority of variation in germination indices. (L) and effect of shade (S) Several other studies have reported congeners to share similarities in ecological traits despite heterogeneous Species Site Experiment habitats, including seed germination strategies Melaleuca Porto Ridge, Brooklyn T, Ψ (Thompson & Grime 1979), seed dispersal patterns deanei 033° 33′ 12′S (Smith-Ramirez et al. 1998), germination time 151° 12′ 33″E (Figueroa & Armesto 2001) and seed mass (Moles et al. Nepean Dam T, Ψ,S,L 2005). The idea that a shared ancestral history would 034° 15′ 13″ S have bearing on the current recruitment difficulties 150° 39′ 58″ E Ψ evident in M. deanei is intriguing and suggests that the Melaleuca Australian Defence T, nodosa Industry (ADI) rare species could be struggling at the edge of the 033° 43′ 27″S evolutionary range or margins of a phylogenetic niche 150° 38′ 42″E (sensu Wiens et al. 2010) for members of this genus. Rookwood Cemetery T, Ψ,S,L A series of experiments tested the effects of moisture, 033° 52′ 28″S temperature, and light and shade on germination and 151° 03′ 50″E recruitment of one rare (M. deanei) and three common Melaleuca Campbell Hill Reserve T, Ψ,L ′ ″ (M. styphelioides, M. nodosa and M. thymifolia) species styphelioides 033° 52 16 S 151° 00′ 09″E of Melaleuca in the Sydney region; these factors have Nurragingy Reserve T, Ψ,S long been considered the most important influences on 033° 45′ 42″S these processes (Harper 1977; Bewley et al. 2013). In 150° 51′ 22″E addition, the effects of heat and smoke were investi- Melaleuca Castlereagh T, Ψ,L gated (Morris 2000; Kenny 2003). The question thymifolia 033° 39′ 35″S ′ ″ common to all experiments was whether the regenera- 150° 40 48 E Ψ tion niche of M. deanei was comparable with that of the Pheasants Nest T, ,S 034° 16′ 23″S congeners, thus contributing to the low recruitment 150° 38′ 42″E success reported from the field. doi:10.1111/aec.12233 © 2015 The Authors Austral Ecology © 2015 Ecological Society of Australia SEED SIZE, REGENERATION NICHE OF MELALEUCA 663

tray and placed in an oven for 6 h at 40°C. After drying, of autoclaved germination medium (7 g L−1 agar (Sigma- coarse materials were removed and discarded.The remaining Aldrich) in dH2O) and the dishes sealed with Parafilm. Four seed and frass were returned to the bags and air dried for a replicate dishes were used per combination of site, species, further week before being sieved through metal sieves of temperature and treatment (fire cues or not).The dishes were progressively smaller pore aperture. Each seed lot was then then placed into six temperature-controlled growth cabinets sequentially passed through a Selecta Machinefabriek BV with a 12:12 h light : dark cycle; the position of the Petri Aspirator at a feed speed of 4/12 and air fan speeds of 60, 80, dishes was re-randomized every 24–48 h. Germination 100 and 110 Hz (common species) or 80, 100, 120 and counts were made every 5 days.The experiment was repeated 140 Hz for the larger and heavier seed of M. deanei. Cleaned 6 months later with temperature re-randomized among the seed was then placed into envelopes that were stored over cabinets. silica gel.Two weeks later, the seed was surface sterilized in a 0.5% sodium hypochlorite solution for 20 s, rinsed in dis- tilled water and air dried on paper towels for 24 h then transferred to labelled glass jars and kept in the dark. For the Light experiments, all seeds were individually selected under a stereomicroscope to exclude visibly damaged seed. The Seeds of each species were germinated either in the light or in viability of seed ranged from approximately 80% the dark in two replicate experiments. In each, there were (M. thymifolia) to approximately 95% (M. styphelioides) four Petri dishes for each species and treatment and 25 seeds (Hewitt et al. 2014). per Petri dish. Seeds were placed on filter papers moistened Average seed size per site and per species was determined with 40 mL of water (topped up after 5 days), and the dishes by weighing three lots of 500 seed per combination of site were maintained in constant light and temperature (22°C). and species. Dark treatments were triple wrapped in aluminium foil, set up and inspected under green safelight. Seeds were checked for germination after 7, 14, 21, 28 and 35 days. After 35 days, Water potential the aluminium foil was removed and germination observed for a further 10 days. The experiment factorially combined 11 treatments of water potential from 0 to −1.2 MPa, with the four species each from two sites. The water potentials were generated with Shade solutions of polyethylene glycol (PEG) (Sigma-Aldrich, MW 6000) per Michel (1983). Each Petri dish contained a sterile Shade cages were constructed by sewing layers of nylon filter paper, 40 mL of PEG solution and 20 seeds, with four shadecloth onto cylindrical wire frames (900 mm in height dishes per combination of species, site and water potential. and 680 mm diameter). Layers of shadecloth were added to The dishes were sealed with Parafilm to prevent evaporation achieve four shade levels (Table 2), with three cages per and placed in a temperature-controlled cabinet at 20°C and shade level. The cages were placed in a glasshouse without a 12:12 h light : dark photoperiod cycle. Germination, as environmental control. Pots (120-mm diameter, 100-mm assessed by radicle emergence, was recorded every 5 days. high) were filled to 80 mm with Debco RG–2 seedling The position of the Petri dishes in the cabinet was potting mix and 60 mL of ‘Osmocote Exact’ 8–9 month re-randomized every 24–48 h to minimize any effects of posi- release fertilizer. Vortex sprinkler spray heads were anchored tion in the cabinet on germination. The experiment was in large 200 mm pots of pebbles placed centrally in each repeated the following year using a second growth cabinet. shade cage. Eight pots (two pots per species) were placed equidistant around the sprinklers under each shade cage and watered, after which 25 seeds were placed on the surface of Temperature and fire cues each pot. Tap water was used for watering under an auto- mated system for 5 m at 8 am and 6 pm from October to The experimental design had four factors: species, fire cues April, and at 9 am and 4 pm from May to September. At the (with both heat and smoke or without either), temperature commencement of the experiment, it was ensured that the (10, 15, 20, 25, 30 and 35°C) and site, using two sites per spray heads gave equal amounts of water to each surrounding species.The temperatures were selected to extend above and pot.This was checked again at days 3, 7 and thereafter every below the average monthly maxima for summer (25°C) and winter (15°C) for the Sydney region. Half of the seed from each species received an application −2 of heat and then smoke (fire cues). Heat was applied to each Table 2. Mean light levels under the shade cages (μmol m ) μ −2 replicate by placing seeds into an open, glass Petri dish in a and as a percentage of full sunlight (1800 mol m ) fan-forced oven for 5 min at 80°C (Morrison & Morris Average light Comparison 2000). Smoke was generated by burning eucalyptus leaves in Treatment level (μmol m−2) to full sun a beekeeper’s burner in a fume hood, the smoke then being passed through a condensing tube to cool and dry it before Control (open) 1260 30% shade delivery to a partially sealed wooden box in which each Petri Low 1008 44% shade dish was placed. Medium 315 82.5% shade Treated and untreated seeds (25 per dish) were then indi- High 12.6 99.3% shade vidually placed into Petri dishes (90 mm) containing 40 mL

4 weeks. The number of seeds germinating and the number RESULTS and proportion of seedlings surviving were assessed every 6 weeks for 12 months. Light measurements were made under the shade cages Seed size inside the glasshouse (Table 2) using a handheld luxmetre (Li-Cor Environmental). These light measurements were Signiﬁcant (F3,24 = 69.3, P ≤ 0.001) differences were similar to those taken at the sites from which the seeds were found among the mean seed masses. The seed mass collected with a eucalypt canopy attenuating light at ground varied between 21 and 65 μg per seed with M. deanei level by 6–25% (equivalent to no shade/open-cage treatment having the largest seed and M. thymifolia the smallest in the glasshouse). Melaleuca thickets were found to attenuate (Table 3). light levels by 75–96%, these being equivalent to light levels in the medium and high-shade treatments.

Water potential Data analysis = Data with respect to the final percent germination in the There were significant effects of species (F3,659 851.8; < = < water potential experiment were subjected to linear mixed P 0.001) and water potential (F10,659 762.3; P modelling using the package, lme4 (Bates et al. 2013). 0.001) on germination, and a significant (F30,659 = Models used fixed effects of species and water potential. 53.19; P < 0.001) interaction between these factors. Random effects were site and repetition of the experiment. However, there was no significant effect of either site

Assessment of whether species had different responses to (F1,659 = 1.15; P = 0.285) or experimental repetition water potential was equivalent to testing for an interaction. (F1,659 = 2.43; P = 0.120; Appendix S1). The data for anova χ2 was used for the fixed effects, and approximate both repetitions were combined, and the mean values statistics for the random effects (this latter approximation is for each combination of species, water potential and justified due to the large number of residual degrees of site are presented in Figure 1. The effects of water freedom). The data regarding final percent germination in the tem- potential were similar for M. deanei, M. nodosa and perature experiment were subjected to arcsine transforma- M. styphelioides. At experimental water potentials from tions, and the data for each species were analyzed separately; 0.0 to −0.8 MPa, between 75% and 100% of seeds this analysis showed that there was no significant effect of germinated (Appendix S2). However, when the water repetition, fire cues or site. Therefore, the data relating to potential dropped below −0.8 MPa, germination these effects were combined, and the data for all four species quickly reduced to 0%. For M. thymifolia, fewer seeds were analyzed together using two-way anova with means germinated, and those that did were more greatly being separated using Tukey’s Honestly Significant Differ- affected by lowered water potential than for the other = ence (HSD) tests at P 0.05 using Statistica (Version 9.1; species. From 0.0 to −0.4 MPa, germination was only Stat Soft Inc., Tulsa, OK, USA). The analyses described 55–65%.When the water potential dropped below this below also used this package. The ‘times to 50% of final germination achieved’ (T ) value, germination quickly fell to below 20%; and after 50 − (Grime et al. 1981; Thompson & Grime 1983; Jurado & 0.95 MPa, no seeds germinated. Westoby 1992) for both the water potential and temperature Due to poor germination at −0.85 MPa and below, experiments were obtained from a curve of best fit plotted only the data estimating theT50 collected at −0.75 MPa through the germinant counts using TableCurve 2D (Version and above were analyzed. Initially, the data for the T50 5; AISN Software, Mapleton, OR, USA).The data were then for each species were examined separately and, for each subjected to the Box-Cox transformation procedure (Box & species, there were no significant effects of repetition Cox 1964) to reduce heteroscedasticity and then subjected to (P = 0.55–0.95) or site (P = 0.24–0.91).Therefore, the anova . Means were separated using Tukey’s HSD tests at data associated with these factors were combined, and = P 0.05. a second analysis using the data from all species Mean final germination data from the light experiment was conducted. All species showed a significantly were analyzed by three-way factorial anova, with species and light treated as fixed factors and repetition of the experiment as a random factor. The factor, repetition, was found to be non-significant, and the data re-analyzed with this factor Table 3. Mean (n = 6) seed masses for the four Melaleuca removed. species in the study. Means followed by the same letter are Data for mean germination and for seedling survival to 12 not significantly different from each other according to a = months from the shade experiment were analyzed by a Student–Newman–Keuls test at P 0.05 mixed-model anova, with species and shade level as fixed, Species Seed mass (μg) orthogonal factors, and application of the shade treatment (cage) as a third factor nested within shade level. M. deanei 64.6 ± 4.95a Heteroscedasticity was assessed using Levene’s test, and data M. nodosa 34.9 ± 2.51b were transformed as required. Post-hoc comparisons of M. styphelioides 33.9 ± 0.75b means were made using Student–Newman–Keuls tests at M. thymifolia 21.4 ± 2.8c P = 0.05. doi:10.1111/aec.12233 © 2015 The Authors Austral Ecology © 2015 Ecological Society of Australia SEED SIZE, REGENERATION NICHE OF MELALEUCA 665

Fig. 1. Mean (n = 8) percentage germination (solid line) and the time for 50% of the seed to germinate (T50; dashed line) of seeds from two sites for each of the four Melaleuca species used in the study at 11 different water potentials. The means are derived from the combined data from two repetitions.

(F7,472 = 346.2; P < 0.001) reduced speed of germina- potential range over which germination was achieved tion as water potential was reduced (Fig. 1). However, for M. deanei, compared with its congeners (Appendix the species showed differential responses in the reduc- S2). tion in the speed of germination (F3,472 = 519.7; P < 0.001), and there was a signiﬁcant species × water potential interaction (F21,472 = 6.95; P < 0.001). For Temperature and ﬁre cues

M. styphelioides and M. nodosa, the T50 was between 8 and 9 days at water potentials between 0 and There was a significant effect (F5,744 = 550.5; P < −0.35 MPa and then became longer as the water poten- 0.001) of temperature on final germination, and the tial reduced further. At 0 MPa, seed of M. thymifolia percentage final germination was different for each took about 11 days to reachT50.The seed of this species species (F3,744 = 392.79; P < 0.001); there was also a was more sensitive to reducing water potential, as the significant interaction (F15,744 = 23.045; P < 0.001).

T50 increased at all lower water potentials. Melaleuca Final germination was lowest at 10°C for all species. deanei was the slowest to germinate of the four species, A rise in temperature to 15oC increased germination in with a T50 of 12–15 days; however, the T50 did not all species; a rise to 20°C resulted in another increase increase until the water potential became less than in germination in M. thymifolia only, with ﬁnal germi- −0.45 kPa. nation of the remaining three species remaining Melaleuca thymifolia germinated at over 75% of its approximately constant at the level achieved at 15°C. maximum over the narrowest water potential range, Final percent germination varied little with further M. nodosa the widest, with M. deanei and increases in temperature for M. deanei and M. nodosa, M. styphelioides within the ranges of these two (Appen- but declined at 35°C for M. thymifolia and dix S2).Thus, there was no evidence of narrower water M. styphelioides (Fig. 2).

Fig. 2. Mean (n = 24) percent germination (solid line) and the time for 50% of the seed to germinate (T50; dashed line) of seeds either subjected or not to ﬁre cues (heat and smoke) from each of the four Melaleuca species germinated at six different temperatures. The means are derived from the combined data from two sites and two experimental repetitions.

Species showed considerable overlap in the tempera- Light ture range over which 75% of maximum germination was achieved, with no strong evidence of a narrower or There was a signiﬁcant effect of species (F3,56 = 64.9; broader temperature range for germination of P < 0.001) and light treatment (F1,56 = 107.3; P ≤ 0.001)

M. deanei compared with congeners (Appendix S3). on germination and a significant interaction (F3,56 = Melaleuca styphelioides and M. nodosa had a slightly 117.7; P < 0.001). Melaleuca deanei, M. thymifolia and broader temperature range (<15 to 33–35°C), M. nodosa germinated equally well under both light and M. thymifolia and M. deanei a slightly narrower (17–18 dark conditions, although the final percentage germina- to 33–34°C) over which 75% of maximum germination was higher for M. nodosa than for M. deanei and tion was achieved. M. thymifolia. However, light significantly affected the

T50 differed signiﬁcantly among species (F3,744 = germination of M. styphelioides with about 95% ﬁnal ger-

5.195; P < 0.001) and temperatures (F5,744 = 216.3; mination in the light, but only about 10% for the seed P < 0.001), and there was a significant species × tem- germinated in the dark (Fig. 3). When aluminium wrap perature interaction (F15,744 = 26.06; P < 0.001). The was removed from the Petri dishes at day 35, over the effects of site, season and fire cues were not significant following 10 days, the seed of M. styphelioides germinated for any of the four species. The ranking of the four to about 95% within 3 days, whereas there was no further species by speed of germination in this experiment was germination for the other three species. the same as in the experiment examining water potential with M. deanei having the slowest speed of germination. For all species, the fastest germination Shade occurred at 25–30°C; above or below this range, the speed of germination decreased especially at tempera- Germination and seedling emergence differed among tures at or below 15°C (Fig. 2). the four Melaleuca species (F3,24 = 30.48; P ≤ 0.001) doi:10.1111/aec.12233 © 2015 The Authors Austral Ecology © 2015 Ecological Society of Australia SEED SIZE, REGENERATION NICHE OF MELALEUCA 667

(a) a a a a a a 100 a a b b b b b b 80 b b

40 Seed germination (%) 20

(b) Control Low shade 100 a Medium shade High shade ab 80 ab Fig. 3. Mean (n = 8) percentage of seeds from the four ab b Melaleuca species germinating under continuous light or dark. The same letters above columns indicate that means 60 c are not signiﬁcantly different from each other at P = 0.05.

40 Seedling survival (%) but was unaffected by shade level (F3,24 = 0.638; P = 20 d d d d 0.612); there was no interaction between these factors de = = (F9,24 0.734; P 0.674) nor was there any effect of e e eee cage (F8,24 = 0.485; P = 0.855). Final germination ranged 92–96% for M. nodosa and M. styphelioides and 69–80% for M. deanei and M. thymifolia (Fig. 4A). M. deanei M. nodosa M. thymifolia However, the survival of seedlings significantly differed M. styphelioides among the species (F = 71.14; P ≤ 0.001) and 3,24 Fig. 4. Mean (n = 6) percentage of seeds germinating (a) = ≤ among the shade treatments (F3,24 1925.25; P and percentage of seedlings surviving 12 months (b) from the 0.001), and there was a significant interaction between four Melaleuca species subjected to four shade treatments. these factors (F9,24 = 41.779; P ≤ 0.001); again, there The same letters above columns indicate that means are not = was no significant effect of cage (F8,24 = 0.91; P = significantly different from each other at P 0.05. 0.85). Survival of seedlings in the control (unshaded) was high for all species (Fig. 4B) but, as shade and speed of germination, which may affect recruit- increased, the proportion of seedlings surviving ment of M. deanei in the field. A level of phylogenetic reduced, with the reduction being different for each of constraint on germination response to water potential the four species.The seedlings of M. deanei were most could also be limiting seedling recruitment in affected by shade, with no seedlings surviving under M. deanei and accounting for its rarity in these rela- the medium and high treatments and a substantial tively drier environments. reduction in survival in the low-shade treatment. The results were similar for M. styphelioides with only a few Seed size seedlings surviving in the low and medium treatments, and none in the high-shade treatment. Seedlings of The speed of germination is thought to be particularly M. nodosa and M. thymifolia were least affected by important in small seeded species (like Melaleuca) shade with many seedlings surviving in the low-shade because small seeds lack large food reserves and must, treatment; however, few seedlings survived under the therefore, quickly become autotrophic (Leishman medium-shade treatment, and none under the high- et al. 2000; Daws et al. 2008). In addition, Melaleuca shade treatment cages. species are known to have little or no endosperm to draw upon during seedling establishment (Beardsell et al. 1993; Zomlefer 1994).The speed of germination DISCUSSION is also critical in habitats where water availability is restricted, such as the sandstone ridgetops of In general, the germination and establishment of M. deanei, where growing roots must keep pace with M. deanei was similar to that of its congeners. water levels that can drop very quickly after rain (Hillel However, differences were found in its shade tolerance 1972).

Daws et al. (2008) correlated larger seed size with the proposed common ancestral origins of Melaleuca faster speed of germination, whereas others have in the swampy margins of rainforests (Barlow & reported the converse, with small seed germinating Cowley 1988), a retention of ecological characteristics faster to gain an advantage in capturing resources from these environments may be negatively impacting (Norden et al. 2009). In the present study, the larger seedling recruitment in M. deanei. Several authors seed of M. deanei germinated more slowly than the have provided evidence for closely related species dis- congeners. Some have reported larger seeds evident in playing ecological similarity or a so-called characteris- drought-prone regions (Westoby et al. 1992) and seed- tic ‘phylogenetic niche’ (Harvey & Pagel 1991; lings from larger seeds surviving longer until rain Smith-Ramirez et al. 1998; Losos 2008; Wiens et al. (Morrison et al. 1992). 2010). The predominance of clonal propagation by root suckering over sexual recruitment evident in M. deanei (Hewitt et al. 2014) may also be a conse- Water potential quence of failed seedling recruitment/environmental pressures in a relatively drier environment. The results for the Melaleuca species were consistent with the general pattern from a large number of Temperature studies, in that reductions in water potential reduce both the final percentage and speed of seed germina- There was considerable overlap in the temperature tion (Bradford 1995). However, there is usually a range over which the four species germinated, and no linear relationship between final germination and evidence of a broader or narrower temperature range water potential for most species (Bradford 1995). In for germination of M. deanei. Most species of Austral- contrast, the four Melaleuca species showed a pattern ian Melaleuca and Eucalyptus germinate within a range similar to those reported for Casuarina cristata Miq., of 20–35°C, germination taking 5–40 days (Turnbull Eucalyptus cambadgeana Maiden (Arnold et al. 2014a), & Doran 1987). Our results appear to fall within these Acacia harpophylla F. Muell. ex Benth. (Arnold et al. general parameters. Our results also accord with those 2014b) and species of Eucalyptus from Western Aus- of Gillespi et al. (1996) who reported germination of tralia (Schutz et al. 2002) in that final germination 40–70% at 20°C for seedlots of three species in our remained relatively constant or decreased only slightly study (M. nodosa, M. styphelioides and M. thymifolia). over the initial range of decreasing water potentials Our germination percentages at 20°C were around until species-specific values were reached. Beyond 60% in M. thymifolia and over 75% in M. nodosa and these values, steep declines in germination occurred as M. styphelioides and probably reflect reliable selection water potential reduced further. of highly viable seed. Although the water potential range for germination With maximum daily soil temperatures during of M. deanei fell within the range of the congeners, our summer above 30°C in the upper 10 mm of soil in the results suggest that M. deanei requires these conditions Sydney region and above 16°C in winter (Auld & be present for longer as itsT at 20°C was greater than 50 Bradstock 1996), our results suggest that some germi- its congeners. Soil derived from Hawkesbury sand- nation could occur for all study species in any season stone ridgetops where M. deanei grows is typically with adequate rainfall. Melaleuca thymifolia showed coarse textured, has high sand content, and dries and reduced germination below 20°C and would perhaps drains rapidly (Salter et al. 1966). Although rainfall is germinate less well in winter. spread fairly uniformly throughout the year in the Sydney region and a single storm is likely to produce a moist soil surface for 7–15 days, follow-up rainfall Fire cues within 20 days is highly unpredictable (Zammit & Westoby 1987). Water availability in the soils over Seeds of the four Melaleuca species did not respond Hawkesbury sandstone ridgetops is, therefore, either negatively or positively to fire cues with respect expected to be limiting unless rainfall events are sus- to final percentage germination or speed of germi- tained or seed is dispersed into slow-draining nation. Similarly, Sweedman and Merritt (2006) list microsites. Water potential models from these areas 83 species of Melaleuca requiring no pre-treatment of (Atlas of Living Australia website: http://spatial.ala. seed to germinate, and Lamont and Enright (2000) org.au, verified 23 December 2014) also suggest that reported that germination of many serotinous species water potential levels high enough to sustain germina- is unaffected by heating pre-treatments of 60–100°C. tion of M. deanei may occur infrequently. Dormancy in Myrtaceae is considered unusual The similarities evident in the germination (Lamont et al. 1991; Long et al. 2014), and seed responses to water potential/moisture levels of the four released from serotinous fruits are known to germinate Melaleuca species is most interesting given the differ- readily once dispersed (Lamont et al. 1991; Long et al. ences in water availability of the species habitats. Given 2014). doi:10.1111/aec.12233 © 2015 The Authors Austral Ecology © 2015 Ecological Society of Australia SEED SIZE, REGENERATION NICHE OF MELALEUCA 669

Light emergence occurring under all shade levels including the deepest shade. This concurs with a germination In laboratory experiments using coloured cellophane, and light quantity experiment conducted on M. deanei Virtue (1991) reported red light (as occurs in direct and M. armillaris by Virtue (1991), which reported no sunlight with no overtopping canopy) being necessary detectable effect of increased shading on total germi- for germination of M. deanei and M. armillaris (Sol. ex nation or on time to 50% germination. It is possible Gaertn.) Sm. These results appear to have been that the few hours of light exposure that the seeds strongly influenced by lower germination of M. deanei received after surface sowing in the glasshouse during seed from one provenance under all light conditions experimental set up or the exposure to light when and no germination from this provenance in the dark. cages were removed to inspect the irrigation system at In our experiments, M. deanei, M. thymifolia and days 3 and 7 were sufficient to end the dark-induced M. nodosa germinated equally under both light and dormancy of M. styphelioides. Sufficient light exposure dark conditions, whereas M. styphelioides showed a of imbibed seed to break dormancy can occur in a few requirement for light exposure. Differences in seed seconds in some species (Bewley et al. 2013). selection methods (Virtue 1991 used replicates of Shade at the highest levels caused seedling etiola- equal weights of mixed seed and chaff, whereas we tion and eventual death in all four species. Seedlings individually selected apparently viable seed under a of M. deanei were the most shade intolerant, and it is microscope) may also have led to the greater variability likely to be a poor competitor during seedling estab- in the previous results. lishment if strongly shaded by other plants. Fire would Melaleuca styphelioides is weakly serotinous and does benefit seedling establishment via removal of shading not retain seed capsules for more than two seasons; a vegetation. Even without fire, ridgetops with a level or light requirement for germination would cue germina- northerly aspect in south-east NSW are typically high- tion to the detection of a canopy gap. Melaleuca light environments, with high-incident radiation and quinquenervia (Cav.) S. T. Blake is also weakly high-light levels at the ground surface (Bale et al. serotinous and has a light requirement for germination 1998). The canopy on these ridgetops comprises (Baumann 2008). Thus, there could be a linkage Eucalypt species that are generally widely spaced in the between degree of serotiny and light-induced field (our percentage canopy cover at M. deanei sites germination. Strongly serotinous species like varied from 5% to 15%). In addition, Eucalypts M. deanei retain seed until fire triggers its release (see possess leaves of high reflectance and a pendant habit Hewitt et al. 2014), with germination following in the that allows both direct and scattered light into the post-fire high-light environment, whereas those that subcanopy space (Barrett & Ash 1992). The shade release seed more constantly during inter-fire intervals level at which M. styphelioides and M. deanei failed to (like M. styphelioides, M. quinquenervia) may await a survive is equivalent to field levels we recorded under light stimulus signalling a canopy gap. a Melaleuca thicket, but not under a eucalypt canopy. The subsequent, rapid germination we observed Melaleuca thymifolia and M. nodosa survived only in when M. styphelioides seed were re-exposed to light low numbers in the experiment under medium shade, was also reported by Baumann (2008) for which was consistent with shade levels measured M. quinquenervia. Melaleua styphelioides seed swelled under Melaleuca thickets. This is consistent with our while under light or dark treatments; however, radicle field observations, where seedlings of these two protrusion through the seed coat did not occur for species were recorded only in canopy gaps or stand seed in the dark, but rapidly occurred with removal to edges. light. This suggests an embryo-imposed block in the absence of light rather than seed coat dormancy. It is apparent that when seed of the four study species is ACKNOWLEDGEMENTS released, it is non-dormant, with M. styphelioides showing a level of dark-induced dormancy until We thank the staff of the NSW Seedbank at Mt Annan exposed to light. This may be a problem for recruit- for the use of seed cleaning equipment, advice on ment at urban waterway sites such as those we studied, germination media and helpful discussions. where weeds such as Tradescantia fluminensis Vell. and many exotic vines form dense mats casting heavy shade. REFERENCES

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