Effects of Great Bustard (Otis Tarda) Gut Passage on Black Nightshade (Solanum Nigrum) Seed Germination

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Seed Science Research (2014) 24, 265–271 doi:10.1017/S0960258514000178 q Cambridge University Press 2014

Effects of great bustard (Otis tarda) gut passage on black nightshade (Solanum nigrum) seed germination

C. Bravo1*, S. Velilla2, L.M. Bautista1 and B. Peco2 1Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales-CSIC, C/Jose´ Gutie´rrez Abascal, 2. 28006 Madrid, Spain; 2Department of Ecology, Universidad Auto´noma de Madrid, C/Darwin, Ciudad Universitaria Cantoblanco, 28049 Madrid, Spain

(Received 1 February 2014; accepted after revision 13 April 2014; ﬁrst published online 22 May 2014)

Abstract Introduction

Birds are important seed dispersers for fleshy fruits Seed dispersal and plant disperser interactions play through their transportation of ingested seeds. The an important role in plant community structure, seeds of many species germinate faster and in greater population dynamics and plant species diversity. proportions after passing through a digestive tract, Understanding how frugivores affect germination of although the effects of this passage vary amongst bird endozoochorous plant species is of great relevance and plant species. Many factors determine the from ecological and evolutionary perspectives (Howe germination success of ingested seeds, such as and Smallwood, 1982; Traveset et al., 2007). The seed scarification during the digestion process, the consumption of fleshy fruits by vertebrates may be fertilizing effect of droppings and the removal of pulp regarded as a mutualistic relationship in which a plant surrounding the seeds. In central Spain, the great aims to disperse its seeds surrounded by edible and bustard (Otis tarda) may act as a disperser of nutritious fruit (Estrada and Fleming, 1986). This European black nightshade (Solanum nigrum). We mutualistic relationship is generally quantified in analysed the germination success of ingested and terms of seed dispersal effectiveness (SDE), which is non-ingested S. nigrum seeds. The fertilizing effect of determined by quantity (i.e. number of dispersed bustard droppings and the disinhibition effect of the seed) and quality (i.e. condition of the dispersed seeds removal of Solanum pulp on final germination or probability of seeds reaching an adult stage) percentage, germination speed and viability were components (Schupp, 1993; Schupp et al., 2010). The also assessed. Although ingested seeds germinated quality component of dispersal is influenced by the faster than non-ingested seeds, the former showed a quality of the treatment, such as seed scarification, lower germination percentage than the latter: 80–87% during the digestion process, and by the quality of seed versus 99%. Droppings and fruit pulp showed no effect deposition, such as the fertilizing effect of manure and on germination enhancement, except in one aspect: chemical disinhibition by frugivory, i.e. removal of the germination speed of non-ingested seeds pulp surrounding the seeds (Traveset et al., 2007). decreased when they were sprayed with a fruit extract. Many characteristics of animal dispersers, such as We confirm that seeds ingested by great bustards had feeding behaviour (diet type) and physiology (body lower germination success than non-ingested seeds. mass, retention time), may influence the scarification of Although seed ingestion by great bustards reduced the seed coat when passing through the gut (Jackson, seedling emergence, the number of emerged seed- 1992; Afik and Karasov, 1995; Traveset et al., 2007). lings was still quite large. Thus, great bustards may Retention time varies with diet type (shorter in play a role as a S. nigrum seed dispersal vector. frugivores) and body size (longer in large animals) (Jackson, 1992; Afik and Karasov, 1995). There is a Keywords: agricultural ecosystems, endozoochorous greater risk of digestive damage to seeds in animals relationship, germination success, Mediterranean, seed with long retention times (Jordano and Schupp, 2000). dispersal, weed Scarification can be disadvantageous because the digestive fluid to which seeds are exposed may damage the embryo and thus decrease seed viability, *Correspondence but it can also help germination due to the scarification Email: [email protected] effect on hard-coated seeds (Traveset et al., 2007). 266 C. Bravo et al.

Ingested seeds may germinate better than non- of great bustards and S. nigrum are widely distributed. ingested seeds due to the presence of manure The habitat is mainly cultivated for cereal production (Dinerstein and Wemmer, 1988; Traveset et al., 2001). (Triticum aestivum and Hordeum vulgare), with a few The fertilizing function of manure has been acknow- fields used for other agricultural crops (legumes, ledged by seed dispersal experts (Willson and Traveset, olives, sunflowers and grape vines). S. nigrum is 2000) and supported by experimental evidence distributed on the edges of fields and in stubble. The (Traveset et al., 2007; Carmona et al., 2013). However, climate is continental Mediterranean, with an annual the germination success of seeds may be reduced if precipitation (mean ^ SD) of 442.5 ^ 125.5 mm, and a manure facilitates the growth of microorganisms mean annual temperature of 14.48C (maximum and (fungi and bacteria) (Meyer and Witmer, 1998). minimum temperatures, 42.28C and 214.88C). Maxi- The compounds in fruit pulp can reduce germina- mum temperatures and minimum precipitation are tion success by blocking biochemical pathways of reached during July and August (24.58C, 50 mm3), germination. By the disinhibition effect (Samuels and coinciding with the fruiting period of S. nigrum. All Levey, 2005; Traveset et al., 2007), ingested seeds may climatic data were taken from AEMET (2014). germinate better than non-ingested ones when the Great bustard droppings and mature fruits of pulp surrounding seed is removed during gut passage. S. nigrum were collected in August 2012. Three flocks The excreted seeds are released from the blockage, and of females (totalling 84 individuals) were located and also from the osmotic and light regimes within the fruit tracked from afternoon to sunset to detect their pulp, which can reduce and even suppress germina- roosting sites. Fresh droppings (n ¼ 62) were collected tion (Traveset et al., 2007). The disinhibition effect from these sites at the following sunrise (Bautista et al., derived from the elimination of the fruit pulp may 2013). We did not use a fixed transect because great differ between disperser species, since the potentially bustards are gregarious and have no fixed roosting significant effects of fruit pulp elimination are sites at night, nor can the midday resting sites be inherently part of the ‘gut passage’ process. predicted, forcing the collection of droppings at a new Among disperser birds, seed dispersal effectiveness site each time. We collected fewer droppings than birds is particularly well-known in frugivorous passerine to limit the chances of pseudo-replication. Seeds were species (Herrera, 2002; Traveset et al., 2007). It has been separated from each dropping with the aid of binocular reported that the European black nightshade (Solanum microscopes (20£), immediately washed with de- nigrum) is commonly dispersed in the western Palaeartic ionized water, gently dried with laboratory blotting by medium-sized (,0.2 kg) frugivorous birds, such as paper and stored in paper bags in a dark room at Eurasian blackbirds (Turdus merula) and European constant temperature (48C). Darkroom humidity was starlings (Sturnus vulgaris)(Barneaet al., 1990; Clergeau, minimized by bags of silica gel. Non-ingested seeds 1992; Rodrı´guez-Pe´rez et al., 2005). In contrast, dispersal were collected in the same crop fields or nearby (within of S. nigrum seeds ingested by omnivorous non- 500 m) of the dropping collection point. We harvested passerine bird species is poorly documented. On the 4–5 intact mature fruits per Solanum individual, Iberian Peninsula, the great bustard (Otis tarda), one of totalling 260 mature fruits from 61 individuals. In the the world’s heaviest flying birds (5–10 kg) (Alonso et al., lab, the seeds were removed from the fruits and the 2009), is an occasional feeder on S. nigrum (roughly same cleaning and storage protocol was applied. 5–28% presence of S. nigrum in its summer diet) (Lucio, To assess the fertilizing and disinhibition effects, an 1985; Lane et al., 1999; Bravo et al.,2012).Thegreat extract of bustard droppings and S. nigrum fruits was bustard may thus play a major, but still unknown, role as obtained. A random sample of 349 mature fruits and a seed dispersal vector of S. nigrum. 5 droppings were oven-dried (808C; 72 h). Dry In this context, our goals were to verify: (1) the droppings (6.72 g) and dry fruits (12.01 g) were effect of the great bustard digestive tract on seed mashed, weighed and diluted with deionized water germination rate and ratio, as well as on seed viability at 1:10. The effects of gut passage on the seed coat were and seed-coat damage; (2) the fertilizing effect of also evaluated visually by scanning electron bustard droppings; and (3) the disinhibition effect of microscopy (SEM) (Samuels and Levey, 2005). Seeds fruit pulp removal on the final germination percentage were defined as damaged or undamaged when more and germination speed of S. nigrum seeds. or less than 50% of the seed coat was harmed. Coats of 20 ingested and 20 uningested S. nigrum seeds were chosen at random for examination by SEM. Materials and methods

Study site and sampling procedure Germination experiment

The ﬁeldwork was conducted on agricultural farmland S. nigrum is a summer annual species with spring- and in central Spain (298400N, 38280W), where the populations summer-germinating seeds (Baskin and Baskin, 2001). Seed dispersal by great bustard 267

In our study area there are no S. nigrum seedlings The significant effect of treatment on the number of in autumn or winter (unpublished results), leading to germinated and viable seeds at the end of the the assumption of winter dormancy for the collected experiment and the germination speed was calculated seeds. Seeds were therefore subjected to a cold with generalized linear models. We assumed binomial stratification treatment before the start of the germina- error distribution and logit link function for the tion experiment, consisting of groups of 50 seeds number of germinated and viable seeds at the end of spread on two sheets of filter paper soaked with the experiment. The significant effect of treatment on deionized water in Petri dishes, and kept in cold germination speed was analysed by quasi-Poisson storage at 48C for 7 weeks. Generalized Linear Model to deal with overdispersion, At the end of the dormancy-breaking treatment, assuming Poisson error distribution and log-link 50 full and firm seeds were set to germinate in each Petri function (Zuur et al., 2009). Post-hoc comparisons dish, laid on two sheets of soaked filter paper. Seeds between treatments were calculated with the Tukey extracted from different droppings were mixed before test for all models. The effect of passage through the starting the germination experiment. Four treatments digestive tract on ingested and non-ingested seed were carried out: Treatment 1 (T1, n ¼ 9), excreted seeds coat damage was analysed with a chi-square test. soaked with deionized water (3 ml); Treatment 2 All the above-mentioned analyses were calculated (T2, n ¼ 10), excreted seeds soaked with bustard manure with R, version 2.15.0 (http://cran.r-project.org/bin/ extract (3 ml); Treatment 3 (T3, n ¼ 10), seeds from windows/base/old/2.15.0/). Results are reported as mature fruits soaked with deionized water (3 ml); and mean ^ SD in the text and mean ^ SE in the figures. Treatment 4 (T4, n ¼ 10), seeds from mature fruits soaked with pulp extract (3 ml). Petri dishes were randomly distributed in temperature-controlled chambers pro- Results grammed for a 16 h light/8 h dark photoperiod at 258C/158C, respectively (Baskin and Baskin, 2001). Over Most droppings (83.9%) contained S. nigrum seeds. We a 4-week period, germinated seeds were counted and counted 59.8 ^ 44.3 seeds per dropping and 53 ^ 12 removed every 3 d. Radicle emergence was the criterion seeds per fruit (mean ^ SD). Final germination used to record a seed as germinated (Taab and percentages differed significantly between treatments Andersson, 2009). After each count, the Petri dishes (x2 ¼ 64.32, df ¼ 35, P , 0.001; Fig. 1A). Germination were randomly moved between chambers to avoid a was significantly lower in ingested seeds than non- chamber position effect. The viability of non-germinated ingested seeds (Fig. 1A; T1 versus T3, Z ¼ 5.887, seeds was checked by crushing them with tweezers at P , 0.001). There was no significant effect of manure the end of the experiment. Empty or rotten seeds were extract on germination percentage of ingested seeds considered not viable. Final germination percentage was (T1 versus T2, Z ¼ 22.074, P ¼ 0.122). There was defined as germinated seeds divided by total viable also no significant effect of Solanum pulp extract seeds and multiplied by 100. Seed viability percentage on germination percentage of non-ingested seeds was defined as viable seeds divided by total seeds and (T3 versus T4, Z ¼ 0.008, P ¼ 1.000). multiplied by 100. The germination speed differed significantly between treatments (x2 ¼ 1.48, df ¼ 35, P , 0.001; Fig. 1B). The ingested seeds germinated faster than non-ingested seeds (Fig. 1B; T1 versus T4: Z ¼ 4.933, Data analysis P , 0.001; T1 versus T3: Z ¼ 1.892, P ¼ 0.231; T2 versus T3: Z ¼ 2.802, P ¼ 0.026; T2 versus T4: At the end of the experiment, we calculated the Z ¼ 5.928, P , 0.001). There was no significant effect germination speed as the time in days until 50% of of manure extract on germination speed of ingested germinations occurred in each Petri dish (T50). For seeds (T1 versus T2: Z ¼ 20.837, P ¼ 0.836). There was this purpose, a logistic regression model y ¼ A[(1 þ exp a significant effect of pulp extract on germination (b–k·days)21] was fitted to the time–germination speed, the germination of non-ingested seeds with profiles, where y stands for the cumulative percentage pulp extract was slower than that of non-ingested of germinated seeds and A, b and k are the estimated seeds without pulp (T3 versus T4: Z ¼ 3.162, parameters of maximum germination percentage (A), P ¼ 0.008). Viability percentage differed significantly the rate parameter (k) and the position of the curve in between treatments (x2 ¼ 32.806, df ¼ 35, P , 0.001; relation to the time axis (b) (Torres and Frutos, 1990). Fig. 1C). Ingested seeds had a lower viability Non-linear regression analyses were calculated for each percentage than non-ingested seeds (T1 versus T3: Petri dish with JMP statistical analysis software (2009; Z ¼ 4.866, P , 0.001). There was no significant effect SAS Institute Inc., Cary, North Carolina, USA). The of manure extract on the viability of ingested seeds median germination time was calculated for each Petri (T1 versus T2: Z ¼ 1.826, P ¼ 0.222). There was also dish as T50 ¼ b/k (Torres and Frutos, 1989, 1990). no significant effect of pulp extract on viability of 268 C. Bravo et al.

ABC100 3.6 100 T3 T4 T3 T4 T4 95 95 3.2

90 90 2.9 T1 T1 85 T50 (d) T3 85 Viability (%) T2 2.6 T2

Final germination (%) 80 80 T1 T2 75 2.3 75 No Yes No Yes No Yes Liquid extract Liquid extract Liquid extract

Figure 1. (A) Final germination percentage, (B) germination speed (T50) and (C) viability percentage of S. nigrum seeds. There were four treatments: ingested seeds (filled symbols) with deionized water (T1) and with bustard manure extract (T2); and non-ingested seeds (empty symbols) with deionized water (T3) and with pulp fruit extract (T4). Means ^ SE are shown. non-ingested seeds (T3 versus T4: Z ¼ 0.000, Interestingly, in the case of blackbirds and starlings, P ¼ 1.000). There were significant differences in the germination percentage increased when the seed coat presence/absence of damaged seed coats between was eroded (Clergeau, 1992) but did not increase when ingested and non-ingested seeds (x2 ¼ 19.25, df ¼ 38, this was not the case (Barnea et al., 1990; Mas and P , 0.001) (see supplementary Fig. S1, available online). Traveset, 1999). The positive effect of scarification on germination in these species could be due to their much smaller digestive systems than that of the great Discussion bustard, and thus the physical and chemical scarification of the seeds should also be less intense. This study confirms that a high proportion (84%) of Furthermore, small pebbles, grit, cereal seeds and great bustard droppings contain S. nigrum seeds in even olive drupes (Olea europaea) have been found in large quantities (up to . 200 seeds/dropping). the bustard gut and diet (Bailey et al., 1997; Lane et al., Although great bustard ingestion of S. nigrum seeds 1999; Delibes et al., 2012). These items, along with the decreased the final germination and viability percen- vigorous gizzard of the great bustard, can physically tages, most seeds survived passage through the great erode the seed coat. In fact, SEM inspection revealed bustard gut and were able to germinate in the that the ingested seeds used in our experiment laboratory. Great bustards may thus play an important bore evidence of physical scarification of the seed role as S. nigrum seed-disperser vectors on the Iberian coat (see supplementary Fig. S1). Peninsula. Seed retention time in the digestive tract also The great bustard is 30 times bigger than the main influences the germination of ingested seeds and S. nigrum avian dispersers (blackbirds and starlings). depends on the disperser’s diet and body size (Jackson, Body size is generally positively correlated with home 1992; Afik and Karasov, 1995). Longer gut retention range area, seed retention time in the gut and quantity may reduce seed viability and germination percen- of dispersed seeds (Spiegel and Nathan, 2007). Great tages over shorter periods (Charalambidou et al., 2003). bustards move across several hectares daily (unpub- To our knowledge, seed retention time is unknown in lished results). In late summer, between 15 and 39% of any bustard species, but is probably longer than in bustard individuals travel roughly 50–80 km (Palacıń blackbirds and starlings due to their different body et al., 2009), which provides an opportunity for size and diet type. It has been shown that herbivorous Solanum seeds to disperse over longer distances than bird species with caecal fermentation, such as the via blackbirds and starlings, whose home range areas bustard family (Otididae), have a longer retention time (Tobler and Smith, 2004; Minderman et al., 2010) are than frugivorous or omnivorous species (Karasov, usually smaller than those of great bustards. 1990; DeGolier et al., 1999). In fact, some studies of seed The lower viability and final germination percen- dispersal effectiveness in geese (herbivorous diet and tages of ingested seeds in comparison with non- caecal fermentation) have described a decrease in seed ingested ones contrast with previous studies of major germination percentage after gut passage (Chang et al., S. nigrum disperser species such as blackbirds and 2005; Figuerola et al., 2010). starlings. The germination percentage of seeds Germination speed of ingested seeds was higher ingested by these two bird species either increased than that of non-ingested seeds. Roughly 80% of the (Clergeau, 1992; Rodrı´guez-Pe´rez et al., 2005) or was ingested seeds germinated in 3 d, in contrast to 6 d in unaffected (Barnea et al., 1990; Mas and Traveset, 1999). the case of 80% of the non-ingested seeds (Fig. 2). Seed dispersal by great bustard 269

fungi and/or phytotoxicity, facilitated by some com- 100 ponents of the excrement (Hoekstra et al., 2002). The 80 same preparation and spraying of a manure extract was successfully applied in previous studies (Carmona et al., 60 T1 2013). We also discard the lack of effect due to a low manure concentration in our extract, given that the 40 T2 T3 presence of the manure extract (regardless of concen- Germination (%) 20 T4 tration) did affect germination in previous studies (Carmona et al., 2013). 0 The releasing effect of the elimination of the fruit 0 36912151821 24 27 pulp was not detected, since the germination Time (d) percentages in clean seeds and seeds sprayed with pulp extract were not significantly different. The pulp Figure 2. Cumulative mean proportions of germinated Solanum nigrum seeds for each treatment. There were four extract probably lost key volatile secondary meta- treatments: ingested seeds (filled symbols) with deionized bolites responsible for inhibition (Cipollini and Levey, water (T1) and with bustard manure extract (T2); and non- 1997) when the fruits were dried at 808C for 72 h. ingested seeds (empty symbols) with deionized water (T3) Nonetheless, the germination speed was reduced by and with pulp fruit extract (T4). Dashed lines show seeds the pulp extract (Fig. 1B), which indicates that the pulp with manure or pulp extract. Solid lines show seeds without extract worked as hypothesized. The lack of an extract. inhibitory effect of the pulp on final germination is contrary to the results of previous studies, where final Likewise, more than 80% of S. nigrum seeds germination of seeds with pulp is usually lower than germinated within 3 d after ingestion by blackbirds that of clean seeds (Cipollini and Levey, 1997; Meyer and Sardinian warblers (Sylvia melanocephala) (Mas and Witmer, 1998; Robertson et al., 2006). The method and Traveset, 1999). Although these bird species did used to assess the pulp effect was probably not the not accelerate germination in this experiment, they did most suitable, as previous studies exploring this aspect so in a second one (Rodrı´guez-Pe´rez et al., 2005). The used intact fruits (Barnea et al., 1991; Cipollini and increase in germination speed due to endozoochory Levey, 1997; Meyer and Witmer, 1998; Robertson et al., is a recurrent result in most studies of germination 2006). The pulp extract concentration (1:10) was (Traveset and Verdu´ , 2002), and great bustards follow probably not strong enough to inhibit germination or this pattern. Increased germination speed might not be break the high osmotic pressure of the seed, and thus related to fitness because early germinated seeds could did not prevent its water absorption (Mayer and be more likely to die due to other factors, such as Poljakoff-Mayber, 1989). Removal of S. nigrum pulp desiccation, pathogens or predators (Traveset, 1998; slowed germination, but no further conclusions can be Traveset et al., 2007). It is unclear whether short time drawn from the effects of pulp extract on the periods, such as the difference in germination speed germination percentage or viability. between ingested and non-ingested seeds induced by In order to understand the role of the great bustard great bustard (3 d) or even by blackbirds (Rodrı´guez- in plant recruitment, we need to dissect the com- Pe´rez et al., 2005), are meaningful in an ecological ponents of dispersal effectiveness. In our study, we context. In central Spain, S. nigrum germinates in late estimated some of the quality components of seed spring, a period of high desiccation risk, and unless an dispersal effectiveness. Great bustards have a negative increase in plant fitness can be shown, it is difficult to effect on the germination of S. nigrum seeds, although support the claim that an increase in germination the seeds still germinate in large numbers and double speed is advantageous (Traveset, 1998). On the other their germination speed. Further research combining hand, the deposition site of the ingested seed may the effect of ingestion on seed germination with the determine the ultimate success of the seedling and microhabitat effect of the seed deposition site should future plant (Schupp et al., 2010; Rey and Alcańtara, be carried out to understand the disperser quality of 2014). the seed consumer species. In addition, it must be The fertilizing effect of the great bustard’s droppings stressed that a fuller understanding of seed dispersal on final germination, germination speed and viability of mechanisms is necessary in order to define the factors the ingested seeds was not significant, contrary to our involved in agricultural ecosystems (Benvenuti, 2007). prediction and previous studies (Traveset et al., 2007). The null effect was unusual because manure surrounding deposited seeds is considered to enhance germina- Supplementary material tion by providing nutrients, such as nitrogen, which stimulate germination (Luna and Moreno, 2009), or to To view supplementary material for this article, please diminish germination due to the growth of bacteria and visit http://dx.doi.org/10.1017/S0960258514000178 270 C. Bravo et al.

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