Seed viability, germination and seedling emergence ...

South African Journal of Botany 109 (2017) 237–245

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Seed viability, germination and seedling emergence of the critically endangered stem succulent, Adenium swazicum, in South Africa K. Van der Walt a,b,⁎, E.T.F. Witkowski b a b

South African National Biodiversity Institute (SANBI), Private Bag X101, Pretoria 0001, South Africa Restoration and Conservation Biology Research Group, School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Private Bag 3, Wits, Johannesburg 2050, South Africa

a r t i c l e

i n f o

Article history: Received 8 August 2016 Received in revised form 14 December 2016 Accepted 8 January 2017 Available online 2 February 2017 Edited by C Seal Keywords: Apocynaceae Ex situ conservation Germination rate Regeneration ecology Seedling establishment Shading Summer impala lily Temperature Tetrazolium staining

a b s t r a c t The regeneration ecology of many southern African threatened plant species is poorly understood. Temperature is considered the most important environmental factor governing seed germination. The germination temperature requirements of the critically endangered stem succulent, Adenium swazicum, were determined under controlled conditions, together with tetrazolium staining protocol tests to assess seed viability. The effects of soil medium, watering levels, depth of sowing and shading on germination/seedling emergence were also determined at the Lowveld National Botanical Garden (LNBG). Germination/seedling emergence was compared under nursery conditions within (Skukuza Nursery in Kruger National Park) and outside (LNBG) the natural distribution range of A. swazicum. The germination of viable seeds ranged 96.5–100% for a broad range of temperatures from 20 to 35 °C as well as for 30/20 °C (light/dark) alternating temperature. No germination was recorded at 5 °C and 10 °C, and relatively limited germination at 15 °C (42.9%) and 40 °C (15.4%). These results were modelled using a Generalized non-linear model, with a binomial error and a logit link function, best-fit models were second-order polynomials (i.e., quadratic). Mean Germination Time ranged from 2 to 6 days among temperature treatments. Tetrazolium together with germination results for the 20−35 °C temperatures showed moderate to high seed viability across populations and between years within the same population. Seedlings readily emerged irrespective of soil media (3 types) or planting depth (surface, 5 and 10 mm) but higher emergence occurred under more frequent watering. Seedlings emerged equally well under sun or shade conditions, but only subsequently survived in the shade. Despite lower temperatures recorded during October/November 2010 at LNBG (outside range: 27.2 ± 0.9 °C) compared to Skukuza (within range: 32.6 ± 0.9 °C), final germination/seedling emergence was high at both locations (LNBG: 82%; Skukuza: 94%). Together with field observations, this study shows that (a) seed germination is rapid and with high percentages after being cued by warm to hot summer temperatures, a period when rainfall is also at its highest, and (b) that seedling recruitment depends on the availability of suitably shaded microsites. This is the first study on the regeneration ecology of an Adenium sp. and will aid both in situ and ex situ conservation of A. swazicum. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction South Africa has the world's richest temperate flora (Germishuizen et al., 2006), with more than 20,450 indigenous vascular plant species, but of these, 4809 (24%) are of conservation concern. Despite various conservation efforts, loss of biodiversity continues on regional and global scales due to increasing intensity of disturbances such as overexploitation of species, habitat destruction, climate change, invasion by alien species and plant diseases (Botha et al., 2004; Knowles and Witkowski, 2000; Mouillot et al., 2013; Royal Botanic Gardens (RBG) Kew, 2016). Plant conservation biology has largely been based on decades of research into plant population dynamics and distributions and the factors ⁎ Corresponding author at: Wellington Gardens, 101 Glenmore Street, Wellington, 6012, New Zealand.

http://dx.doi.org/10.1016/j.sajb.2017.01.011 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

that affect them (Van Dyke, 2003; Heard and Ancheta, 2011). However, a species long-term persistence often depends on reproduction, seed dispersal within the same community, expansion into new habitats and survival through times unfavourable for growth (Vasquez-Yanes and Orozco-Segovia, 1993; Weiersbye and Witkowski, 2002; Cousins et al., 2013). Variation in population size relates to underlying vital rates such as germination, seedling growth, reproduction and death, which in turn are influenced by multiple environmental factors including fire, herbivory and weather (Buckley et al., 2010). Two basic alternatives that limit plant recruitment include the availability of viable seed and the availability of suitable microsites at which seedling establishment is possible (Eriksson and Ehrlѐn, 1992; Lamont et al., 1993). Areas under tree canopies in savannas have reduced soil temperature and higher nutrient levels compared to adjacent open spaces, which improves the survival and growth of seedlings (Kos and Poschlod, 2007). In the succulent thicket biome, nearly all the endemic succulent species

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K. Van der Walt, E.T.F. Witkowski / South African Journal of Botany 109 (2017) 237–245

were recorded under Euclea shrubs (Moolman and Cowling, 1994). It has been argued that seedling establishment under or close to the mother plant could be indicative of localized seed dispersal and seed trapping (seed trapped in branches of mother plants or surrounding vegetation) rather than nurse effects per se on the seedlings (Haussmann et al., 2010; Soliveres et al., 2012). Temperature is considered the most important environmental factor governing the maximum germination percentage and germination rate, and germination is usually only possible within well-defined temperature limits (Garcia-Huidobro et al., 1982; Hardegree, 2006; Mattana et al., 2010). The optimum germination temperature for a species is characterised by maximum germination in the shortest time, while no germination will occur beyond maximum and minimum temperatures (Probert, 2000; Shen et al., 2010). Once the temperature requirement for germination is reached, some species have a fast germination response to rain (Fenner and Thompson, 2005), with species such as Colophospermum mopane, Acacia tortilis, A. karroo and Combretum apiculatum having 90–100% seed germination within 4–5 days at 30 °C (Stevens et al., 2014). However, the availability of water is another major factor limiting germination, early seedling establishment and growth (Wilson and Witkowski, 1998; Carvajal et al., 2014). A germinated seed is highly vulnerable to lack of moisture for growth, fire, herbivores, burial under litter, being washed away by rain, and heat on bare soil, and hence up to 90% of released seed will not make it past the seedling stage (Leck, 2008). Sodic or brackish areas add another stress to seedlings due to high salt content of the soil (Medinski et al., 2010), as well as being hard when dry and lack structure (hence poor aeration) when wet. Such extreme conditions cause some species to become specialized by forming corms, bulbs or tubers under the soil surface to avoid drought or heat (Holmgren et al., 2006). Adenium swazicum (Apocynaceae) is a critically endangered southern African savanna stem succulent species largely found on sodic soils. Although Adenium originally comprised six species, Plaizer (1980) in his revision of the genus reduced it to five, namely A. obesum Roem & Schult, A. multiflorum Klotzsch, A. boehmianum Schinz, A. oleifolium Stapf and A. swazicum Stapf. With the exception of A. obesum, all species are restricted to small areas in southern Africa. All Adenium species occur in savanna or open forests on sandy or rocky soils, which are often brackish (Plaizer, 1980). Adenium swazicum is approximately 0.2–0.7 m tall with a carrot-like tuber, which can be up to 1 m in diameter. The leaves are narrowly oblong, rounded and mucronate at the apex. The inflorescence is approximately 1.5–3.5 × 1–2.5 cm in size and tinged with red or pink, flowering occurs between October and April (Plaizer, 1980). The fruit are follicles 15–20 cm in length, with two follicles produced per flower. Follicles mature from late September. Seeds have a woody outer layer which protects the soft embryo; individual seeds weigh approximately 0.002 g and are 9–13 mm in length with hairy tufts on both ends (KvdW, unpublished data). No research has been conducted on its seed dormancy and germination, or requirements for seedling establishment, and this is the case for all Adenium spp. In order to better manage both in situ and ex situ conservation of A. swazicum, the aim was to study its seed germination, and seedling establishment requirements. The objectives were to determine and compare (a) seed viability between A. swazicum populations, (b) germination temperature requirements, (c) the importance of soil medium, depth of planting, moisture stress and shading on seedling emergence and establishment, and (d) to compare germination response and seedling establishment under nursery conditions within and outside the natural distribution range of A. swazicum. 2. Methods 2.1. Study area and species Adenium swazicum occurs from the Kruger National Park (KNP) and Timbavati Private Nature Reserve in the north, extending to

Komatipoort/Malelane and Swaziland in the south and Mozambique in the east. The area has strong summer (N 80%) rainfall (October to April), receiving an average of 620 mm per annum (Mucina and Rutherford, 2006). The mean maximum temperature recorded in the Lowveld National Botanical Garden (LNBG) in Nelspruit, Mpumalanga (approximately 60 km west of the closest known population of A. swazicum) and KNP (Skukuza indigenous nursery) during the experimental period (12th of October 2010 until 10th of November 2010) was 27.2 ± 0.9 °C and 32.6 ± 0.9 °C, while the mean minimum temperature was 15.0 °C ± 0.4 °C and 17.7 ± 0.6 °C, respectively (KNP Scientific Services; LNBG weather records; TuTiempo weather; World Weather Online). 2.2. Seed collection and storage Seeds were collected from three representative populations (Populations A–C; Table 1) in the lowveld of Mpumalanga (Komatipoort/ Malelane), based on land use (protected areas and private land) and size of population (minimum of 50 plants/population). Given that A. swazicum is a critically endangered species, large numbers of seeds could not be collected. Details for all seed collections during the experimental period (October 2009 to October 2010 are provided in Table 2. All seeds were collected at the point of natural dispersal by securing nylon stockings over developing follicles, securing both ends lightly with a cable tie to avoid seed dispersal by wind. All seeds were stored under ambient conditions in a cool, dry storeroom in brown paper bags at the LNBG prior to the trials. 2.3. Seed viability Standard tetrazolium staining protocol was used to assess seed viability (AOSA/SCST, 2010). The initial tetrazolium (TZ) tests were conducted using 25 seeds from each of the three seed batches in order to test the method for A. swazicum seeds (Table 2). Seeds were prepared for the TZ test by placing them in 50 mm diameter Petri dishes, which were suspended over distilled water inside an airtight container for 24 h at 20 °C. After 24 h, the seeds were transferred to 90 mm diameter Petri dishes that contained a 1% water-agar solution and stored at 20 °C for an additional three days. The seeds were then placed in plastic vials containing a 1% 2, 3, 5-triphenyltetrazolium chloride solution and each vial was wrapped in heavy-duty aluminium foil to prevent reaction to light. The vials were incubated at 30 °C in an 8/16 h light/dark cycle for two days. The seeds were then washed with distilled water and evaluated immediately. Seed viability was based on tissue characteristics as described in Patil and Dadlani (2009); red and uniform staining of the embryo was taken as viable. Seed batches were kept separate throughout the experiment. 2.4. Seed germination A permit was obtained from Mpumalanga Tourism and Parks Agency (MTPA) to export the seeds to the Millennium Seed Bank of the Royal Botanic Gardens, Kew, at Wakehurst Place, England for the germination trial, in June 2010. Seeds were not treated or rinsed before germination experiments. Seeds were placed in 90 mm Petri dishes (5–10 seeds/ dish) containing a 1% water-agar solution within nine different environmental control incubators set at a 8/16 h light/dark cycle at 5 °C constant temperature intervals between 5 °C and 40 °C as well as one alternating temperature of 30/20 °C, also at 8/16 h light/dark cycle (the 8 h light period coincided with 30 °C) The light/dark cycle was applied by means of fluorescent lamps providing a photosynthetic photon flux density of 80 μmol m−2 s−1. Due to the limited number of seeds available, especially from population A, the more extreme constant temperatures of 5 °C, 10 °C, 15 °C and 40 °C only used seeds from population C (2009) (Table 3). Furthermore, the seed batches were run in sequence on the incubator (population C before A), and hence it was decided to prioritise


239

Table 1 Characteristics of the three Adenium swazicum populations from which seed was collected from 2009 to 2010. Population characteristic

Population A

Population B

Population C

Quarter degree grid cell Vegetation type (Mucina and Rutherford, 2006) Altitude (meters above sea level) Land use Associated vegetation

2531 BC Malelane Mountain Bushveld 278 Protected area (natural) Combretum hereroense Diospyros mespiliformis Euclea divinorum Grewia bicolor Grewia flava Schotia capitata 0.95 121

2531 BC Granite Lowveld 276 Protected area (natural) Acacia borleae Combretum hereroense Euclea divinorum Grewia bicolor Schotia brachypetala Xanthocercis zambesiaca 2.10 137

2531 DB Tshokwane-Hlane Basalt Bushveld 183 Private sugarcane farm (cultivated) Aloe marlothii Elaeodendron transvaalense Euclea divinorum Gymnosporia senegalensis Pappea capensis

Surface area of population (ha) Number of plants

high germination temperatures only for population A due to low seed availability (H. Pritchard, pers. comm.). Temperatures selected for the trial were based on temperatures found across the natural distribution of A. swazicum. Germination scoring was done three times a day for three weeks or until 100% germination was achieved. Germination was defined as emergence of the radicle by 2 mm. The seeds that did not germinate were then tested for viability using tetrazolium, as already described. Final germination scores were based on the percentage of viable seed that germinated to enable comparisons to be made between seed batches. Seed batches were kept separate throughout the experiments. 2.5. Seedling emergence Seedling emergence experiments at the LNBG were conducted on population C to test (a) germination medium, (b) depth of planting, (c) available moisture and (d) shading on seedling emergence and initial establishment. Emergence was defined as appearance of the stem above the ground, while cotyledon and stem colour change (from green to brown) indicated initial establishment up to 12 weeks. Trays were prepared on the 18th of January 2010 and seedling emergence was scored on a daily basis at 14:00 from 29th January to 5th March 2010, Monday to Friday. Total seedling emergence was expressed as a percentage of the total number of seeds sown per treatment. Three germination media were compared: (a) 100% seedling mix (Braaks, South Africa), (b) mixture of 50% seedling mix (Braaks, South Africa) and 50% river sand, and (c) 50% river sand mixed with 50% red, loamy soil from the grounds of the LNBG. Each tray had 30 evenly distributed seeds sown at 5 mm depth, kept under 40% shade net and watered daily at 14:00. To assess planting depth, seeds were planted into one of three trays, either (a) on the surface, (b) 5 mm or (c) 10 mm below the surface. Each tray had 30 evenly distributed seeds sown into 100% seedling mix (Braaks, South Africa), under 40% shade net and watered daily at 14:00. The effect of available moisture/watering regime was determined in four trays containing 100% seedling mix, each with 30 seeds planted 5 mm below the surface and placed under 40% shade net. The trays were either watered once in (a) 14, (b) seven (c) or three days or (d) kept moist by a mist spray for 20 s every 2 h, 24 h a day. The effect of shading was determined by sowing 50 A. swazicum seeds in full sun in the LNBG and another 50 into a 25 cm × 25 cm

0.37 141

tray covered with 40% shade netting and same soil medium (100% seedling mix — Braaks, South Africa) placed next to the full sun treatment. These trays only received natural rainfall (no artificial watering), total rainfall during the experimental period was 36 mm (LNBG weather records) 2.6. Comparison within and outside the natural distribution range To determine differences in germination/seedling emergence within (Skukuza) and outside the A. swazicum distribution range (LNBG), three seedling trays were prepared on 12th October 2010. Two trays, one containing seed from population B and the other from population C, were kept in the nursery at LNBG. The third tray, containing seed from population B, was maintained at Skukuza. Each tray had 50 evenly distributed seeds sown at 5 mm depth into 100% seedling mix, kept under 40% shade net and watered daily at 14:00. For all trials, seedling emergence was scored daily at 14:00, from 12th October until 10th November 2010, Monday to Friday. Seedlings were scored as emerged once the cotyledons were visible above the ground. Total seedling emergence was expressed as a percentage of the total number of seeds sown per tray. 2.7. Data and statistical analyses Mean germination time (MGT) was calculated as: MGT = ∑(G′t′)/ Gm, where G′ is the number of seed which germinate at hour t′, and Gm is the maximum number of seed that germinated at the temperature interval. The minimum time (hours) to onset of germination (Timemin) and time (hours) to maximum germination (Timemax) per temperature interval were also recorded. To determine the effects of temperature and population/seed batch (covariates) on seed germination, we modelled the proportion of viable seeds (following tetrazolium testing) that germinated using a Generalized non-linear model (GzNLM), with a binomial error and a logit link function. The resultant best-fit models were second-order polynomials (i.e., quadratic). Model suitability was assessed graphically and also statistically, using an F-test, comparing the best-fit model to a NULL model. Model parameters were then interpreted under the logit function and by using odds ratios which significantly improve model interpretation. All models were implemented using the Microsoft R Open (MRAN), enhanced R distribution (V 3.3.1). One-way ANOVAs followed by Fisher's LSD were used to compare the

Table 2 The date, population and number of seed collected, as well as description and date of trial/experiment, on germination and seedling emergence / establishment of Adenium swazicum. Date collected

Number of Seed

Population

Experiment details

Date of experiment(s)

October 2009

175 235 115 490 100 50

A C C C B C

Germination temperature requirements and tetrazolium tests at Kew Millennium Seed Bank

June 2010

Seedling emergence and establishment experiments at Lowveld National Botanical Garden Seedling emergence and establishment inside (Skukuza) and outside (Lowveld National Botanical Garden) the natural distribution of Adenium swazicum

January to March 2010 October to November 2010

May 2010 October 2009 October 2010

240


Table 3 The number of seeds within replicate Petri-dishes and in total for each temperature interval for the three seed batches (year collected in parentheses), used to test the germination temperature requirements of Adenium swazicum. Experiments were conducted at the Kew Millennium Seed Bank in June 2010. Temperature

Population C (2009) number of seeds (petri-dishes × seeds/dish)

Population C (2010) number of seeds (Petri-dishes × seeds/dish)

Population A (2009) number of seeds (petri-dishes × seeds/dish)

Total seeds

40 °C 35 °C 30 °C 25 °C 20 °C 15 °C 10 °C 5 °C 30/20 °C Total seeds

15 (3 30 (3 30 (3 30 (3 30 (3 15 (3 15 (3 15 (3 30 (3 210

10 (2 10 (2 10 (2 10 (2 10 (2 10 (2 10 (2 10 (2 10 (2 90

0 30 (3 30 (3 30 (3 30 (3 0 0 0 30 (3 150

25 70 70 70 70 25 25 25 70 450

× × × × × × × × ×

5) 10) 10) 10) 10) 5) 5) 5) 10)

× × × × × × × × ×

5) 5) 5) 5) 5) 5) 5) 5) 5)

MGT between temperature treatments. A series of χ2 tests compared germination/seedling emergence between (a) different soil media, (b) depth of planting, (c) watering regime, (d) shading and, (e) emergence within and outside the natural distribution range, under nursery conditions. Results were generally represented as mean ± S.E. Apart from the GzNLM, all statistical analyses were conducted using Analyse-it for Microsoft Excel (version 2.30). 3. Results 3.1. Seed viability Population A in 2009 had the highest percentage of viable seed (93%) compared to population C in 2009 (78%) and 2010 (41%; χ22 = 85.53; p b 0.0001). Seed viability within population C also varied significantly between 2009 and 2010 (χ21 = 38.51; p b 0.0001). 3.2. Seed germination Temperature was a highly significant predictor of the proportion of viable A. swazicum seeds that germinated (GzNLM, F = 116.73, deviance(resid) = 3.7, d.f. = 17, P b 0.001) (Fig. 1). Specifically, there is a high probability (0.967 to 0.998) that temperatures of between 20 and 35 °C resulted in germination of viable seeds, whereas the probability declines steeply at temperatures in excess of 35 °C and below 20 °C. Population/seed batch was not a significant predictor of germination and thus this covariate was removed from the resultant best-fit model in a step-wise fashion. A small residual deviance (3.7) together with a low Akaike Information Criteria (AIC) value (23.7) and no pattern evident in a plot of fitted versus residual values, indicates an excellent model fit. The germination for temperature treatments between 20 and 35 °C ranged 96–100% across the three seed batches, with 98.1% (model prediction, 98.6%), 100% (99.8%), 100% (99.8%) and 96.5% (96.7%) overall for 20, 25, 30 and 35 °C respectively (on a mean seed basis). The 30/20 °C alternating temperature treatment yielded very similar/the same results as the 20, 25 and 30 °C treatments for each of the three seed batches. No seeds germinated at 5 °C (model prediction, 0.000006%) or 10 °C (0.07%), while % germination was 42.9% (42.1%) at 15 °C and 15.4% (15.1%) at 40 °C. The model also predicted 0.01% germination at 45 °C and 0% at 50 °C, although these two temperatures were not tested in the laboratory. Hence A. swazicum has a relatively broad highly effective germination temperature range of 20–35 °C, which may reflect its broad distribution range from Mpumalanga, Swaziland and Mozambique where summers are warm to hot. Minimum time (hours) to the onset of germination (Timemin) was significantly different among temperature treatments (F6,11 = 17.80; P b 0.0001) with the shortest and longesttime to germination recorded at 35 °C (30.0 ± 3.5 h) and 15 °C (199.0 ± 7.0 h), respectively. Timemax was also significantly different among temperature treatments (F6,11 = 3.39; P = 0.034), with shortest and longest time to maximum germination recorded at 35 °C

× × × ×

10) 10) 10) 10)

× 10)

(80.0 ± 24.6 h) and 15 °C (236.0 ± 30.1 h), respectively (Fig. 2). Mean Germination Time (MGT) was significantly different among temperature treatments (F6,11 = 4.76; P b 0.0104), being fastest at 35 °C (50.1 ± 2.6 h) and slowest at 15 °C (149.0 ± 56.8 h). 3.3. Seedling emergence and seedling establishment It is acknowledged that seedling emergence will be less than or equal to actual germination percentages, as some germinated seeds might not emerge above the soil surface. In addition, because germination is expressed on the basis of viable seeds, whereas seedling emergence is based on the number of seeds sown, emergence will always be lower than germination using this method of analysis. Seedling emergence percentages were similar for the three soil media, 100% seedlings mix (39%), 50% seedlings mix: 50% sand (39%) and 50% sand: 50% local soil (30.6%) (χ22 = 1.83; P = 0.4005; Fig. 3a) as well as depth of planting, surface (26.5%), 5 mm below surface (35%) and 10 mm below surface (26.5%) (χ22 = 2.32; P = 0.3135; Fig. 3b). However, seed planted at 10 mm below the soil surface emerged two weeks after seed that were planted on the surface or 5 mm below the surface.

Fig. 1. Proportion of viable seeds that germinated (observed = bars) and the Generalized non-linear model (GzNLM) fit (predicted = line) of 8 temperature treatments from 5 to 40 °C at 5 °C intervals for Adenium swazicum. Seed population/batch did not differ significantly and hence was removed from the final model. Viable seed replication was 19, 18, 14, 55, 52, 59, 57 and 13 seeds for the temperatures 5 °C through to 40 °C respectively. Seed germination for the alternating 20/30 °C treatment was exactly the same as the 20 °C treatment.


241

Germination (%)

(a) 100 90 80 70 60 50 40 30 20 10 0

5°C 10°C 15°C 20°C 25°C

0

20 40 60 80 100 120 140 160 180 200 220 240

Time (hours)

(b)

30°C

Germination (%)

35°C

100 90 80 70 60 50 40 30 20 10 0

40°C Alternate

0

20 40 60 80 100 120 140 160 180 200 220 240

Time (hours)

Fig. 2. The course of cumulative germination of viable seeds for Adenium swazicum at (a) 5 °C, 10 °C, 15 °C, 20 °C and 25 °C, as well as (b) 30 °C, 35 °C, 40 °C and alternating temperatures of 30/20 °C (light/dark). Total seeds employed is provided in Table 3, whereas viable seed replication was 19, 18, 14, 55, 52, 59, 57 and 13 seeds for temperatures 5 °C through to 40 °C respectively, and 55 seeds for 30/20 °C.

Watering regime had a significant effect, with the highest seedling emergence for the mist spray (46%), followed by watering once every three (24%) and seven days (16.4%), with no emergence for watering once every 14 days (χ23 = 64.75; p b 0.0001; Fig. 3c). In most treatments, initial seedling establishment (over 12 weeks) was similar to seedling emergence, except that 100% of emerged seedlings (Fig. 3d) died by week 2 when in full sun (χ21 = 36.1; p b 0.0001; Fig. 4). 3.4. Comparison within and outside the natural distribution range Despite lower temperatures recorded at LNBG (27.2 ± 0.9 °C) compared to Skukuza (32.6 ± 0.9 °C), MGT was shortest for seed from population B (11.5 days) and population C (9.9 days) at LNBG, and longest at Skukuza (17.4 days). Seedling emergence was high for seed from population B (LNBG-B = 82%; SKUK-B = 94%), compared to population C (LNBG-C = 42%; χ22 = 37.2; p b 0.0001; Fig. 5). 4. Discussion The germination together with the Tetrazolium results showed that seed viability was moderate to high across populations/seed batches. The nursery trials showed (a) high germination percentages/seedling

emergence at both sites, (b) rapid germination rates and (c) that seedling establishment is highly dependent on the presence of shade. Seedlings readily emerged irrespective of soil media (3 types) or planting depth (surface, 5 and 10 mm depth) but were intolerant of very low water availability. According to Crawley and Ross (1990) there are four potential limiting factors in plant recruitment: limited viable seed production, competition, microsites and herbivory. Low seed viability may be related to inbreeding depression and reduced fitness resulting from homozygosity which can be severe enough to affect the viability of small and isolated wild populations (Keller and Waller, 2002; Frankham, 2005; Ronce, 2007; Le Cadre et al., 2008; Mattana et al., 2010). Seed viability was moderate to low for population C in 2009 and 2010, germination and tetrazolium tests revealed 78 and 93% viability for seeds collected in October 2009 from population C and A respectively. No seed was available from population B in 2009 for the germination/tetrazolium trial, but nursery trials conducted in October/November 2010 recorded more than 80% seedling emergence compared to 42% for seed collected from population C. Population C consisted of approximately 140 individuals, the largest of the four studied populations, but also had the smallest aerial extent of 0.37 ha. In contrast to populations A and B which were located in a

242


Seedling Mix

1

(c)

Sand:Soil

2

3

4

Constant

5 6 7 8 Number of Weeks 3 Days

9

10

11

2

3

4

5

7

8

10mm

1

2

3

4

5

6

7

8

9

10

11

12

9

10

11

12

Number of Weeks

7 Days

6

5mm

50 45 40 35 30 25 20 15 10 5 0

12

(d)

14 Days

50 45 40 35 30 25 20 15 10 5 0 1

On surface

(b)

Percentage seedling emergence


Sand:Seedling Mix

50 45 40 35 30 25 20 15 10 5 0



(a)

9

10

11

Shade

Open (Full Sun)

50 45 40 35 30 25 20 15 10 5 0

12

1

2

3

4

Number of Weeks

5

6

7

8

Number of Weeks

Fig. 3. Percentage seedling emergence over time (12 weeks) for (a) soil media (seedling mix, equal parts sand and seedling mix, equal parts sand and soil), (b) depth of planting (surface, 5 mm below, and 10 mm below soil surface, (c) watering regime (constant, every 3, 7, or 14 days), and (d) shading (under 40% shade cloth or open/full sun). Seed replication was 90 (30/ treatment), 90 (30), 120 (30) and 100 (50/treatment) for soil media, depth of planting, watering regime and shading respectively.

formally protected area, population C was isolated through habitat transformation (sugarcane fields) and located more than 25 km away from the nearest known neighbouring population. Anthropogenic pressures such as deforestation, urbanisation agriculture and habitat

Seedling emergence

Seedling Mix

(c)

1 Sand: 1 Seedling Mix Soil Media

Seedling emergence

3 Days

Surface

(d)

Seedling establishment

7 Days

Watering Regime


5mm Below

10 Below

Depth of Planting

50 45 40 35 30 25 20 15 10 5 0 Constant

Seedling emergence 50 45 40 35 30 25 20 15 10 5 0

1 Sand: 1 Soil

Percentage seedling emergence/establishment


(b)


50 45 40 35 30 25 20 15 10 5 0



(a)

fragmentation can affect pollination and seed dispersal, thereby altering gene flow patterns in plant populations (Bennett et al., 2006; Baldauf et al., 2014). Although genetic connectivity and seed dispersal were not part of this study, recorded distances of wind dispersed seeds

14 Days

Seedling emergence


50 45 40 35 30 25 20 15 10 5 0 Shade

Open (Full Sun) Shading Aspect

Fig. 4. The percentage seedling emergence and initial ‘seedling establishment’ during the 12 week trial testing differences between (a) soil media, (b) depth of planting, (c) watering regime and (d) shading for Adenium swazicum under ambient nursery conditions at the Lowveld National Botanical Garden, Nelspruit, South Africa. Seed replication was 90 (30/treatment), 90 (30), 120 (30) and 100 (50/treatment) for soil media, watering regime, depth of planting and shading respectively.


(a)

LNBG - B

LNBG - C

SKUK - B

Seedling emergence (%)

100 90 80 70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

Days

Fig. 5. The course of cumulative seedling emergence over time (days) for Adenium swazicum seeds from populations B and C at Lowveld National Botanical Garden (LNBG) and for population B at Skukuza Nursery. Fifty seeds were used for each seed batch.

range from close (Herrera, 1991) to over 100 m (Soons and Bullock, 2008; Baldauf et al., 2014). The height from which seeds are released as well as the height and structure of surrounding vegetation play a crucial role in determining seed dispersal distances (Soons et al., 2004), with many seeds trapped in the parent plant or neighbouring vegetation (Herrera, 1991). Adenium swazicum is a low growing shrub with an average height of only 34.6 ± 2.3 cm for the monitored plants in population C (KvdW, unpublished data). The low plant height, distance to closest known population (25 km), high level of habitat transformation surrounding population C, and results from this study indicate the likelihood of inbreeding depression impacting population C. Temperature is considered the most common primary cue for seed germination as this increases the possibility of seedling emergence coinciding with the time of the year when conditions are most favourable (Fenner and Thompson, 2005). Studies confirm that seed germination of savanna species seldom occurs below 10 °C, while the optimal temperatures are between 20 °C and 35 °C (Hoffman et al., 1989; Qu et al., 2008; Stevens et al., 2014; Mackenzie et al., 2016). Similarly field seedling emergence (germination) trials showed that baobab seeds also germinate in mid-summer when temperatures are high and at the peak of the summer rainfall season (Venter and Witkowski, 2013). This was confirmed to be the case for A. swazicum where high germination success was achieved at temperatures between 20 °C and 35 °C, with no germination recorded at 10 °C or 5 °C and low germination recorded at 15 °C and 40 °C. High germination percentages across a wide temperature range are an indication that it is unlikely that seeds exhibit dormancy (Baskin and Baskin, 2004) but also indicates that a species is likely to have a seasonal preference for germination, rather than a seasonal requirement (Mackenzie et al., 2016). Once suitable germination temperatures were reached, seed germination of A. swazicum was initiated within 48 h. Rapid germination is a common occurrence for arid-adapted plants, which allows the seed to take advantage of short window periods when water is available (Wilson and Witkowski, 1998; Daws et al., 2002; Kos and Poschlod, 2007). In the presence of moisture, A. swazicum reached maximum germination in less than 90 h at 20, 25, 30, and 35 °C and took slightly longer to reach maximum germination (120 h) at the alternating temperature of 20/30 °C, indicating that germination is rapid, allowing germination during short window periods of suitable conditions. Seedlings of many species overcome the lethal effect of high soil temperatures by growing below the canopy of adult shrubs or trees, although this is more common in desert species, it has been recorded in various vegetation types (i.e. Callaway et al., 2002; Flores and Enrique, 2003; López et al., 2007). Shading had an important effect on seeding survival. Although 40% seedling emergence was recorded in full sun as well as those under 40% shade netting, the seedlings in full sun all

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died by the second week, while all the seedlings under 40% shade survived and persisted for the whole 12 week observation period. These seedlings were not artificially watered and received rain only (36 mm), and so lack of moisture may have been the critical limit in the sun. Seedling emergence within and outside the natural distribution range matched the germination temperature results well, with the low emergence of seed from population C (LNBG-C; 42%) likely due to its low seed viability. In summary, once minimum temperatures for germination (≥20 °C) have been reached, (a) moisture is essential to initiate germination, (b) emerged seedlings do not require constant moisture to survive, and (c) shading is not essential for emergence, but it is essential for seedlings to persist. Seedlings observed in the wild were located either under the mother plant canopy or other vegetation, which provided a suitable microclimate for seedling establishment. This has also been confirmed in desert species where the early survival of dominant shrub species is dependent on nurse plants (López et al., 2016). Since adult A. swazicum plants were often recorded in full sun, it is possible that the seedlings which germinate under nurse plants eventually outcompete these plants over time. It is however also possible that the sodic soils with higher clay content could retain moisture for a longer period compared to the Braaks seedlings mix which was used in the ex situ experiments. Although not studied in this paper, vegetative propagation appeared to be important for A. swazicum (KvdW, pers.obs.), and this persistence niche in which species survive through resprouting is often associated with unproductive/desert sites where recruitment is restricted to sporadic, high precipitation years (Holmgren et al., 2006; López et al., 2016). In situ seedling observations for Nerium oleander, another member of the Apocynaceae revealed that soil media did not affect germination, although seedlings in open areas died soon after germination (Herrera, 1991). The cultivation of A. swazicum from seed was highly successful, with the different soil media tested having no apparent influence on seedling emergence and establishment. The depth, at which seeds were planted, did not influence final seedling emergence, although seeds that were planted 10 mm below the soil surface took longer to emerge. This indicates that A. swazicum seeds have the ability to germinate even if covered with soil and/or litter. Bradstock and Auld (1995) found that soil temperatures on the blackened post-fire surface, with little or no vegetation cover, could be as high as 70 °C. Although soil temperature was not measured during this study, it is likely that the barren areas associated with sodic sites will also reach very high temperatures. This was confirmed by in situ observations, in which all seedlings were located underneath nurse plants with no seedlings observed in full sun during the survey period. Seed germination and seedling emergence experiments for A. swazicum revealed that the factors which are likely to limit recruitment for this species are the availability of microsites which provide suitable germination temperatures and protection of the seedlings from direct sunlight. Although germination in A. swazicum was triggered by moisture and suitable temperatures, ex situ seedling emergence and seedling establishment experiments revealed that seedlings would only survive at suitable microsites that provide sufficient shade. It should be noted that microsite availability is episodic in most habitats, and in many cases, the carrying capacity of an area might fluctuate from generation to generation with seedling establishment in woody plants likely to be episodic (Crawley and Ross, 1990; E.T.F. Witkowski, unpublished). There were a limited number of seedlings present in the studied A. swazicum populations from 2009 to 2011, but all were located under nurse plants, including mother plants. 5. Conclusion This study revealed that an integrated approach to ex situ and in situ conservation can greatly reduce the extinction risk of A. swazicum. Moderate to high seed viability, lack of seed dormancy and broad germination/seedling emergence abilities of A. swazicum make this species a good candidate for ex situ conservation. Ex situ conservation methods

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should include a combination of seed banking and living collections, prioritizing sustainable seed collection from populations which are not currently represented as viable ex situ conservation populations. Longterm survival of these ex situ populations should be ensured by addressing ecological, evolutionary, anthropogenic and genetic issues through the prevention of hybridization in living collections, genetic drift and inbreeding. Seeds which are stored in seed banks should be regenerated regularly to determine the extent by which seed viability decreases over time. In situ conservation should aim to investigate and address low seed viability of populations which are isolated and not under formal protection. Anthropogenic impacts affecting A. swazicum in the wild have not been neutralized and are likely to continue increasing (habitat transformation, overgrazing, medicinal plant harvesting), restoration efforts within these areas are unlikely to be affective. Management of populations in protected areas should focus on long term regeneration monitoring. Acknowledgements Funding for fieldwork was provided by the NORAD/SANBI threatened species fund. Willem Froneman (LNBG), and Michele Hofmeyer (KNP) are thanked for their assistance during fieldwork and germination trails. Prof Neil Crouch (SANBI) and Guin Zambatis (KNP) are thanked for literature assistance. Dr. Mervyn Lotter and Blackie Swart (MTPA) provided the permits for seed collection and export. Prof Hugh Pritchard (Kew Millennium Seed Bank Project (MSBP)) provided guidance with seed viability testing and germination trails, while Dr. Tiziana Ulian provided flight tickets and accommodation which made my (KvdW) stay at MSBP possible. Wesley Hattingh provided expert assistance with the Generalized non-linear seed germination model. References Association of Official Seed Analysis (AOSA)/Society of Commercial Seed Technologies (SCST), 2010m. Tetrazolium Testing Handbook. Baldauf, C., Guillardi, C., Aguirra, T.J., Correa, C.E., Dos Santos, A.M., Souza, A.P., Sebbenn, A.M., 2014. Genetic diversity, spatial genetic structure and realised seed and pollen dispersal of Himatanthus drasticus (Apocynaceae) in the Brazilian savanna. Conservation Genetics 15, 2364–2379. Baskin, J.M., Baskin, C.C., 2004. A classification system for seed dormancy. Seed Science Research 14, 1–16. Bennett, A.F., Radford, J.Q., Haslem, A., 2006. Properties of land mosaics: Implications for nature conservation in agricultural environments. Biological Conservation 133, 250–264. Botha, J., Witkowski, E.T.F., Shackleton, C.M., 2004. Market profiles and trade in medicinal plants in the Lowveld, South Africa. Environmental Conservation 31, 38–46. Bradstock, R.A., Auld, T.A., 1995. Soil temperatures during experimental bushfires in relation to fire intensity: consequences for legume germination and fire management in South-eastern Australia. Journal of Applied Ecology 32, 76–84. Buckley, Y.M., Ramula, S., Blomberg, S.P., Burns, J.H., Crone, E.E., Ehrlѐn, J., Knight, T.M., Pichancourt, J.B., Quested, H., Wardle, G.M., 2010. Causes and consequences of variation in plant population growth rate: a synthesis of matrix population models in a phylogenetic context. Ecology Letters 13, 1182–1197. Callaway, R., Brooker, R.W., Choler, P., Kikvidze, Z., Lortie, C.J., Michalet, R., Paolini, L., Pugnaire, F.I., Newingham, B., Aschehouh, E.T., Armas, C., Kikodze, D., Cook, B.J., 2002. Positive interactions among Alpine plants increase with stress. Nature 417, 844–848. Carvajal, D.E., Loayza, A.P., López, R.P., Toro, P.J., Squeo, F.A., 2014. Growth and early seedling survival of four Atacama Desert shrub species under experimental light and water availability regimes. Revista Chilena de Historia Natural 87, 28. Cousins, S.R., Witkowski, E.T.F., Pfab, M.F., Riddles, R.E., Mycock, D.J., 2013. Reproductive ecology of Aloe plicatilis, a fynbos tree aloe endemic to the Cape Winelands, South Africa. South African Journal of Botany 87, 52–65. Crawley, M.J., Ross, G.J.S., 1990. The population dynamics of plants (and discussion). Philosophical Transactions of the Royal Society, B: Biological Sciences 330, 125–140. Daws, M.I., Burslem, D.F.R.P., Crabtree, L.M., Kirkman, P., Mullins, C.E., Dalling, J.W., 2002. Differences in seed germination responses may promote coexistence of four sympatric Piper species. Functional Ecology 16, 258–267. Eriksson, O., Ehrlѐn, J., 1992. Seed and microsite limitation of recruitment in plant populations. Oecologia 91, 360–364. Fenner, M., Thompson, K., 2005. The Ecology of Seeds. Cambridge University Press, Cambridge. Flores, J., Enrique, J., 2003. Are nurse-protégé interactions more common among plants from arid environments? Journal of Vegetation Science 14, 911–916.

Frankham, R., 2005. Genetics and extinction. Biological Conservation 126, 131–140. Garcia-Huidobro, J., Monteith, J.L., Squire, G.R., 1982. Time, temperature and germination of pearl millet (Pennisetum typhoides S.H.). Journal of Experimental Botany 33, 288–296. Germishuizen, G., Meyer, N.L., Steenkamp, Y., Keith, M. (Eds.), 2006. A Checklist of South African PlantsSouthern African Botanical Diversity Network Report no Vol. 41. SABONET, Pretoria. Hardegree, S.P., 2006. Predicting germination response to temperature. I. Cardinaltemperature models and subpopulation-specific regression. Annals of Botany 97, 1115–1125. Haussmann, N.S., McGeoch, M.A., Boelhouwers, J.C., 2010. Contrasting nurse plants and nurse rocks: the spatial distribution of seedlings of two sub-Antartic species. Acta Oecologica 36, 229–305. Heard, S.B., Ancheta, J., 2011. Effects of salinity and temperature on ex situ germination of the threatened Gulf of St.Lawrence Aster, Symphyotrichum laurentianum Fernald (Nesom). Plant Species Biology 26, 158–162. Herrera, J., 1991. The reproductive biology of a Mediterranean riparian shrub, Nerium oleander L. (Apocynaceae). Botanical Journal of the Linnean Society 106, 147–172. Hoffman, M.T., Cowling, R.M., Douie, C., Pierce, S.M., 1989. Seed predation and germination of Acacia erioloba in the Kuiseb River Valley, Namib Desert. South African Journal of Botany 55, 103–106. Holmgren, M., Strapp, P., Dickman, C.R., Gracia, C., Graham, S., Gutierrez, J.R., Hice, C., Jaksic, F., Kelt, D.A., Letnic, M., Lima, M., Lòpez, B.C., Meserve, P.L., Milstead, W.B., Polis, G.A., Previtali, M.A., Richter, M., Sabate, S., Squeo, F.A., 2006. Extreme climatic events shape arid and semiarid ecosystems. Frontiers in Ecology and the Environment 4, 87–95. Keller, L.F., Waller, D., 2002. Inbreeding effects of wild populations. Trends in Ecology & Evolution 17, 2230–2241. Knowles, L., Witkowski, E.T.F., 2000. Conservation biology of the succulent shrub, Euphorbia barnardii, a serpentine endemic of the Northern Province, South Africa. Austral Ecology 25, 241–252. Kos, M., Poschlod, P., 2007. Seeds use temperature cues to ensure germination under nurse-plant shade in Xeric Kalahari Savannah. Annals of Botany 99, 667–675. Lamont, B.B., Witkowski, E.T.F., Enright, N.J., 1993. Post-fire litter microsites: safe for seeds, unsafe for seedlings. Ecology 74, 501–512. Le Cadre, S., Tully, T., Mazer, S.J., Ferdy, J.B., Moret, J., Machon, N., 2008. Allee effects within small populations of Aconitum napellus ssp. lusitanicum, a protected subspecies in northern France. New Phytologist 179, 1171–1182. Leck, M.A., 2008. Seedling Ecology and Evolution. University Press, Cambridge. López, R.P., Valdivia, S., Sanjines, N., de la Quintana, D., 2007. The role of nurse plants in the establishment of shrub seedlings in the semi-arid subtropical Andes. Oecologia 152, 779–790. López, R.P., Squeo, F.A., Gutiérrez, J.R., 2016. Differential effect of shade, water and soil type on emergence and early survival of three dominant species of the Atacama Desert. Austral Ecology 41, 428–436. Mackenzie, B.D.E., Auld, T.D., Keith, D.A., Hui, F.K.C., Ooi, M.K.J., 2016. The effect of seasonal ambient temperatures on fire-stimulated germination of species with physiological dormancy: a case study using Boronia (Rutaceae). PloS One 11, e0156142. http:// dx.doi.org/10.1371/journal.pone.0156142. Mattana, E., Daws, M.I., Bacchetta, G., 2010. Comparative germination ecology of the endemic Centranthus amazonum (Valerianaceae) and its widespread congener Centranthus ruber. Plant Species Biology 25, 165–172. Medinski, T.V., Mills, A.J., Esler, K.J., Schmiedel, U., Jürgens, N., 2010. Do soil properties constrain species richness? Insights from boundary line analysis across several biomes in south western Africa. Journal of Arid Environments 74, 1052–1060. Moolman, H.J., Cowling, R.M., 1994. The impact of goat grazing on the endemic flora of South African succulent thicket. Biological Conservation 68, 53–61. Mouillot, D., Graham, N.J., Villѐger, S., Mason, N.W.H., Bellwod, D.R., 2013. A functional approach reveals community responses to disturbances. Trends in Ecology & Evolution 28, 167–177. Mucina, L., Rutherford, M.C. (Eds.), 2006. The vegetation of South Africa, Lesotho and Swaziland. Strelitzia 19. South African National Biodiversity Institute, Pretoria. Patil, V.N., Dadlani, M.V., 2009. Tetrazolium test for seed viability and vigour. In: McDonald, M.B., Kwon, F.Y. (Eds.), Flower Seeds: Biology and Technology Handbook. Oxfordshire, UK. Plaizer, A.C., 1980. A Revision of Adenium Roem & Schults and of Diplorhynchus Welw. Ex Fic & Hiern (Apocynaceae). Mededlingen Landbouwhogesch. Wageningen Ned. pp. 80–120. Probert, R.J., 2000. The role of temperature in the regulation of seed dormancy and germination. In: Fenner, M. (Ed.), Seeds: the Ecology and Regeneration in Plant Communities. CAB International, Wallingford. Qu, X.X., Huang, Z.Y., Baskin, J.M., Baskin, C.C., 2008. Effect of temperature, light and salinity on seed germination and radicle growth of the geographically widespread halophyte shrub Halocnemum strobilaceum. Annals of Botany 101, 293–299. RBG Kew, 2016. The State of the World's Plants Report. Royal Botanic Gardens, Kew. Ronce, O., 2007. How does it feel to be like a rolling stone? Ten questions about dispersal evolution. Annual Review of Ecology, Evolution, and Systematics 38, 231–253. Shen, S.K., Wang, Y.H., Ma, H.Y., 2010. Seed germination requirements and responses to desiccation and storage of Apterosperma oblate (Theaceae), an endangered tree from south-eastern China: implications for restoration. Plant Species Biology 25, 158–163. Soliveres, S., Eldridge, D.J., Hemmings, F., Maestre, F.T., 2012. Nurse plant effects on plant species richness in drylands: the role of grazing, rainfall and species specificity. Perspectives in Plant Ecology, Evolution and Systematics 14, 402–410. Soons, M.B., Bullock, J.M., 2008. Non-random seed abscission, long-distance wind dispersal and plant migration rates. Journal of Ecology 96, 581–590.

K. Van der Walt, E.T.F. Witkowski / South African Journal of Botany 109 (2017) 237–245 Soons, M.B., Heil, G.W., Nathan, R., Katul, G.G., 2004. Determinants of long-distance seed dispersal by wind in grasslands. Ecology 85, 3056–3068. Stevens, N., Seal, C.E., Archibald, S., Bond, W., 2014. Increasing temperatures can improve seedling establishment in arid-adapted savanna trees. Oecologia 175, 1029–1040. Van Dyke, F., 2003. Conservation Biology: Foundations, Concepts, Applications. McGraw Hill Companies, Boston. Vasquez-Yanes, C., Orozco-Segovia, A., 1993. Patterns of seed longevity and germination in the tropical rainforest. Annual Review of Ecology, Evolution, and Systematics 24, 69–87. Venter, S.M., Witkowski, E.T.F., 2013. Where are the young baobabs? Factors affecting regeneration of Adansonia digitata L. in a communally managed region of southern Africa. Journal of Arid Environments 92, 1–13.

245

Weiersbye, I.M., Witkowski, E.T.F., 2002. Seed fate and practical germination methods for 46 perennial species that colonize gold mine tailings and acid mine drainage-polluted soils in the grassland biome. In: Seydack, A.H.W., Vorster, T., Vermeulen, W.J., van der Merwe, I.J. (Eds.), Multiple Use Management of Natural Forests and Woodlands: Policy Refinements and Scientific ProgressProceedings of the Natural Forests and Savanna Woodlands Symposium III, KNP (May 2002). Department of Water Affairs and Forestry Indigenous Forest Management, Pretoria, pp. 221–255. Wilson, T.B., Witkowski, E.T.F., 1998. Water requirements for germination and early seedling establishment in four African savanna woody plant species. Journal of Arid Environments 38, 541–550.