In situ or ex situ seed conservation: which is the

Plant Species Biology (2016)

doi: 10.1111/1442-1984.12131

In situ or ex situ seed conservation: which is the more effective way to maintain seed longevity of an endangered cactus? JOANA PAULA BISPO NASCIMENTO* and MARCOS VINICIUS MEIADO† *Postgraduate Program in Ecology and Conservation, Campus Prof. José Aloísio de Campos, Federal University of Sergipe, Av. Marechal Rondon, s/n, Jardim Rosa Elze, São Cristóvão, 49100-000; and †Department of Bioscience, Campus Prof. Alberto Carvalho, Federal University of Sergipe, Av. Vereador Olímpio Grande, s/n, Centro, 49500-000, Itabaiana, Sergipe, Brazil

Abstract With restricted populations and a small number of individuals, Discocactus bahiensis Britton & Rose (Cactaceae) is an endangered species in Brazil and its capacity for the formation of seed banks in the soil and the maintenance of seed viability remains unknown. Thus, the aim of the present study was to determine the most efficient way to maintain viability during storage of seeds of D. bahiensis. Seeds were stored in paper bags and either kept in a cold chamber (7 2 C) in the dark (ex situ conservation) or buried in the soil to a depth of 5 cm in an area of natural occurrence of the species (in situ conservation). Germinability of the seed banks was evaluated monthly for 20 months. During the first 10 months of storage, germinability of the seeds conserved in situ and ex situ was similar to that of recently collected seeds. After this period, a 70% reduction in germinability was found for the seeds maintained in situ and there was nearly complete loss of viability after 12 months of storage in the field (germinability < 10% in the last 8 months of the experiment), indicating the ability to form persistent soil seed banks. In contrast, the seeds stored in the cold chamber maintained greater than 70% germinability throughout the entire analysis period, demonstrating that ex situ conservation is the most efficient way to maintain the viability of the seeds of this endangered species. Keywords: Cactaceae, Discocactus bahiensis, seed germination, soil seed bank, storage. Received 8 December 2014; revision received 7 March 2015; accepted 28 February 2016

Introduction As ecosystems face climate change, strong anthropogenic pressures, genetic erosion and loss of diversity, the conservation of genetic resources should be seen as a worldwide priority and a very important future research issue (Draper et al. 2004). The conservation of both plant and animal organisms is traditionally grouped into two categories, namely in situ and ex situ conservation, in which the conservation of populations, species and/or germplasms occurs, respectively, within or outside its site of origin (Draper et al. 2004). There are different ways to preserve all or part of a vegetal organism, such as in vivo Correspondence: Marcos V. Meiado Email: [email protected]. © 2016 The Society for the Study of Species Biology

conservation, in vitro conservation, cryopreservation and seed banks (Assis et al. 2011). The presence of viable seed banks in the soil (in situ) is one of the main indicators of the potential for regeneration of an ecosystem. Species that form seed banks in the soil exhibit different longevity periods and germination behaviors. Simpson et al. (1989) classified seed banks as either transient (germination within 1 year after dispersal) or persistent (viability for more than 1 year). The formation of seed banks is an important reproductive strategy for plants, especially those that undergo long periods of severe climate conditions, such as in semi-arid ecosystems, which can be unfavorable to seedling development and survival (Moles et al. 2003). However, the physiological ecology of seeds of native species under natural conditions is under-investigated and it is

J. P. B. NASCIMENTO AND M. V. MEIADO necessary to determine the factors that contribute to the formation of seed banks in the soil as well as the period for which seeds remain viable in order to ensure the natural regeneration of ecosystems. On the other hand, the aim of an artificial seed bank or germplasm bank (ex situ) is to preserve the physical and physiological quality of seeds (Assis et al. 2011). When stored correctly, seeds can remain viable for a long period of time and can be used for different purposes, such as genetic diversity studies, reforestation and the recovery of degraded areas. This conservation method provides information on the period for which seeds remain viable in order to ensure the natural regeneration of environments (Assis et al. 2011). Seeds stored either in situ or ex situ must maintain their germination potential (germinability), which is determined by the percentage of seeds germinated in a given time period and under given conditions (Draper et al. 2004). In Brazil, some species of the family Cactaceae have been studied to determine seed germination behavior and the potential for the formation of seed banks in the soil. These studies have demonstrated considerable variation in germination behavior and longevity (Cheib & Garcia 2012; Meiado 2012, 2014; Meiado et al. 2012b, 2015). However, knowledge on the ecophysiology of seeds from species of Cactaceae, especially regarding germination and longevity under natural and artificial conditions, has received little attention in recent decades, despite the fundamental importance of these aspects for restoring natural ecosystems (Goodman et al. 2012; Meiado et al. 2012b). There is a lack of knowledge on the potential for the formation of seed banks and longevity of long-stored seeds from Discocactus bahiensis Britton & Rose (Cactaceae), which is an endemic species of cactus currently listed as endangered because of the restriction of its populations and the small number of individuals. Thus, the aim of the present study was to evaluate the germination of seeds conserved in the soil seed banks (in situ conservation) and stored in a cold chamber (ex situ conservation) to determine the most efficient way to maintain the seed viability of this endangered cactus. Because of the decreased metabolism of seeds at low temperatures, we believe that ex situ seed conservation maintains seed viability for a longer period of time. If our predictions are correct, this strategy for seed conservation may also be used to preserve seeds from other cacti that are also endangered and have similar germination behavior.

Materials and methods Species and study site In Brazil, the family Cactaceae is represented by 260 species distributed throughout all ecosystems in the country © 2016 The Society for the Study of Species Biology

(Taylor et al. 2015). The Caatinga ecosystem is a tropical dry forest located in northeastern Brazil and is home to approximately 90 species of this family, including some cacti of the genus Discocactus Pfeiff. (Meiado et al. 2012a; Taylor et al. 2015). This genus has 11 species distributed in the Caatinga, Cerrado (savannah) and Pantanal (wetland) ecosystems (Machado et al. 2005; Taylor et al. 2015). However, most species are found in the state of Bahia (northeastern Brazil), where a high degree of floristic endemism of the family Cactaceae is found (Taylor & Zappi 2004). Four species of the genus Discocactus are found in this state: Discocactus bahiensis Britton & Rose, Discocactus catingicola Buining & Brederoo, Discocactus petr-halfari Zachar and Discocactus zehntneri Britton & Rose, the latter of which has two subspecies, Discocactus zehntneri subsp. boomianus (Buining & Brederoo) N.P. Taylor & Zappi and Discocactus zehntneri Britton & Rose subsp. zehntneri. The present study was conducted with D. bahiensis, which occurs in areas of the Caatinga ecosystem in the states of Bahia, Ceará, Pernambuco and Piauí (northeastern Brazil) (Taylor et al. 2015). Caatinga consists of patches of seasonally dry forest and sclerophyll vegetation (sensu Mooney et al. 1995; Pennington et al. 2000) covering a 850,000 km2 semi-arid region (Sampaio 1995). Variation in the vegetation structure is conditioned by topography, human disturbance and, most importantly, by a combination of the average annual rainfall and soil attributes (Sampaio 1995; Prado 2003). Rainfall ranges from 240 to 900 mm per year throughout the Caatinga, and soils range from moderately fertile, saline and shallow to impoverished deep sandy soils, at both landscape and regional levels (Sampaio 1995). October and May are the months that have the lowest and highest rainfalls, respectively. However, the small number of known populations, the small number of individuals per population and habitat specificity limit the occurrence of this species to a restricted area and contribute to its classification as endangered (Meiado et al. 2012a). In Brazil, the species is commonly known as ‘frade-de-cavalo’ and the plants have a discoid habit, with a cephalium at the apex of the stem from which nocturnal white flowers emerge, which are pollinated by moths. The fruits exhibit a lateral dehiscence and white coloration and are dispersed primarily by ants (Taylor & Zappi 2004; Machado et al. 2005). Seeds were collected from an area of natural occurrence of the species in the municipality of Juazeiro in the state of Bahia (09 280 35.400 S, 040 340 44.400 W; 390 m asl) in 2012. The area has typical sclerophyllous vegetation of the Caatinga ecosystem and eutrophic, haplic, planosol soils (Brasil 2006a,b). About 150 reproductive plants of this species are found in the study area. Each fruit contains about 80 seeds. The seeds have a moisture content Plant Species Biology

SEED CONSERVATION OF AN ENDANGERED CACTUS of 12% and germination is more than 95% when the seeds are subjected to light and a temperature of 30 C (Meiado et al. 2015). During the period in which the seeds remained in the soil seed bank, total precipitation at the site where the seeds were buried was 206.2 mm and the mean temperature ranged from 19.8 C to 35.4 C.

Conservation of seeds To determine whether seeds maintain their viability and germination capacity when stored in the soil and when stored in ex situ cold conditions, seeds were removed from the 50 fruits (one fruit per plant), washed with running water for 10 min to remove the funicular pulp and divided into two groups. In the first group, seeds were stored in paper bags in a cold chamber (5 2 C) and kept in the dark at the Seed Laboratory of the Reference Center for the Recovery of Degraded Areas of the Caatinga of the Federal University of the São Francisco Valley, Brazil (ex situ conservation). In the second group, seeds were placed in nylon bags (10cm × 10 cm) containing soil from the collection site and buried in the soil at a depth of 5 cm in an area of natural occurrence of the species from where the seeds had originally been collected (in situ conservation) (Cheib & Garcia 2012). Seeds were buried after the period of seed dispersal that occurred in the dry season.

Germination parameters and statistical analyses Germination of the seeds buried in the soil was evaluated on a monthly basis for 20 months, from March 2012 to November 2013. At each evaluation, four bags containing 25 seeds were exhumed (100 seeds for each evaluation period, totaling 2000 exhumed seeds). In the laboratory, the seeds stored in the field and those stored in the cold chamber were washed in running water, distributed in four repetitions of 25 seeds and set to germinate in Petri dishes measuring 5 cm in diameter containing filter paper moistened with 3 mL of distilled water. The Petri dishes were kept under the optimum germination conditions for the species, under white light with a 12-h light/dark photoperiod at a temperature of 30 C (Meiado et al. 2015), for 30 days. The emergence of the radicle was the criterion for the determination of germination. At the end of the experiment, germinability (%), mean germination time X X t= ni:ti= ni, in which ti is the time since the onset of the experiment to the nth observation [days] and ni is the number of seeds germinated in time i), the emergence rate index adapted from Maguire (1962) (ERI = (G1/ N1) + (G2/N2) + … + (Gn/Nn), in which G1, G2 and Gn correspond to the number of seeds germinated at Plant Species Biology

the first, second and last count, respectively, and N1, N2 and Nn represent the number of days elapsed to the first, second and last count, respectively and the synchroniP zation index (E = − fi.log2fi, in which fi is the relative germination [i.e. the proportion of seeds germinated in a time interval]) were calculated based on Ranal and Santana (2006). High values of ERI and low values of E indicate fast and more synchronized germination, respectively (Ranal & Santana 2006). Germinability was transformed into arc-sine √%. Data on mean germination time, the emergence rate index and the synchronization index of the treatments with germinability less than 5% were excluded from the analysis (Meiado et al. 2010). The results were submitted to twofactor (type of seed conservation and storage time) analysis of variance (two-way ANOVA) and means were compared using Tukey’s test (Ranal & Santana 2006). The Shapiro–Wilk and Levene tests were used to determine the normality of the data and equal variance, respectively (Zar 2010). All statistical analyses were performed with the aid of the STATISTICA 10.0 program (StatSoft 2012), with the level of significance set to 5% (p < 0.05).

Results All the exhumed D. bahiensis seeds were intact, with no signs of physical deterioration after the in situ conservation time. The two factors assessed (type of conservation and storage time) exerted a significant influence on germinability (F = 498.887, d.f. = 1, P < 0.0001 and F = 23.398, d.f. = 20, P < 0.0001, respectively). In the first 10 months of storage, germinability of the seeds conserved both in situ and ex situ was similar to that of recently collected seeds. After this period, a 70% reduction in germinability was found for the seeds maintained in situ (1st month, 82.0 9.5%; 20th month, 7.0 8.2%) until the nearly complete loss of viability after 12 months of storage in the field (germinability < 10% in the last 8 months of analysis). In contrast, the seeds stored in the cold chamber (ex situ conservation) maintained germinability greater than 70% throughout the entire analysis period (1st month, 83.0 6.8%; 20th month, 71.0 8.2%), indicating a significant interaction between the factors analyzed (F = 16.833; d.f. = 20, P < 0.0001) (Fig. 1). The mean germination time ranged from 5 to 9 days, with no significant difference in relation to storage time for either the seeds stored in situ or those stored ex situ (F = 3.536, d.f. = 1, P = 0.0632) (Table 1). However, the type of seed conservation and storage time influenced the emergence rate index, which was faster for the seeds stored in the cold chamber in comparison to those that © 2016 The Society for the Study of Species Biology

J. P. B. NASCIMENTO AND M. V. MEIADO more synchronized germination was influenced by the decrease in the number of germinated seeds during the storage time. In the last months of analysis, we observed a decrease in germination of seeds stored in the soil (in situ); however, these seeds germinated almost on the same day, providing a more synchronized germination.

Discussion The findings of the present study demonstrate a significant difference between the types of conservation of seeds from D. bahiensis. Independently of storage time, ex situ conservation is the most effective way to maintain the viability of the seeds of this endangered species of cactus. No signs of deterioration were found among the seeds with either conservation method and no type of dormancy was acquired during the storage time. Thus, seeds of D. bahiensis remain viable in the soil up to 20 months. The present findings are in agreement with Cheib and Garcia (2012), who evaluated the seeds of four cacti species of Arthrocereus A. Berger and also found that the seeds conserved in both soil and a cold chamber remained intact with no signs of decomposition or physiological death, thereby demonstrating the capacity to form seed banks. However, not all species have this ability. In a study conducted with the arboreal species Senegalia polyphylla (DC.) Britton & Rose (Fabaceae), Araújo Neto et al. (2005) found that none of the seeds remained

Fig. 1 Germinability (mean confidence interval, %) of seeds from Discocactus bahiensis Britton & Rose (Cactaceae) conserved in situ (white circles) in areas of the Caatinga ecosystem in the municipality of Juazeiro (state of Bahia, Brazil) and ex situ (black circles) in a cold chamber with a temperature of 7 2 C for 20 months.

remained in the soil of the Caatinga ecosystem (F = 53.727, d.f. = 1, P < 0.0001 and F = 16.497, d.f. = 14, P < 0.0001, respectively) (Table 1). The synchronization index was also significantly influenced by the storage time and type of conservation, with more synchronized germination with the increase in time (F = 5.84, df = 14, p < 0.0001) and greater synchronized germination among seeds stored in situ in comparison with those stored ex situ (F = 36.21, d.f. = 1, P < 0.0001) (Table 1). However,

Table 1 Mean germination time (MGT, days), emergence rate index (ERI) and germination synchronization index (E) of seeds from Discocactus bahiensis Britton & Rose (Cactaceae) conserved in situ in areas of the Caatinga ecosystem in the municipality of Juazeiro (state of Bahia, Brazil) and ex situ in a cold chamber with a temperature of 7 2 C for 20 months MGT Time (month) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

In situ 7.6 5.9 6.4 6.7 5.8 6.7 6.3 5.9 6.1 8.4 6.2 5.8 6.5 11.1 5.6

1.5a 0.8a 1.0a 0.9a 0.2a 0.6a 0.6a 0.8a 0.5a 1.4a 0.7a 0.9a 1.1a 1.8a 0.5a

ERI Ex situ 7.6 9.2 6.2 6.0 5.5 5.7 5.9 5.8 6.4 7.3 9.0 8.3 8.4 8.0 6.9

1.5a 1.4b 0.9a 0.4a 0.2a 0.7a 0.5a 0.5a 1.0a 1.2a 1.2b 0.3a 1.4a 0.3b 0.6a

In situ 3.3 4.1 3.0 3.4 3.8 3.4 3.9 4.1 3.3 2.7 3.2 2.2 0.6 0.4 0.3


E Ex situ 3.3 2.7 4.1 4.0 4.4 4.3 4.7 4.5 3.6 3.5 2.9 2.6 2.7 2.8 3.8

0.4a 0.6a 0.5a 0.4a 0.4a 0.7a 0.6a 0.8a 0.6a 0.5a 0.5a 0.5a 0.7b 0.6b 0.3b

In situ 3.0 2.3 2.3 2.7 2.6 2.6 2.6 2.6 2.5 2.6 1.9 2.0 1.7 1.7 1.5


Ex situ 3.0 2.8 2.3 2.4 2.2 2.3 2.7 2.6 2.6 2.7 3.1 2.3 2.6 2.7 2.8

0.3a 0.2a 0.3a 0.3a 0.2a 0.1a 0.1a 0.1a 0.2a 0.1a 0.5b 0.2a 0.3b 0.1b 0.2b

Data are represented by mean standard deviation. Different letters indicate significant differences between in situ and ex situ seed conservation at P < 0.05 (Tukey’s honestly significant difference test) © 2016 The Society for the Study of Species Biology

Plant Species Biology

SEED CONSERVATION OF AN ENDANGERED CACTUS intact after storage in soil (in situ conservation), demonstrating an inability to form seed banks. According to Rojas-Aréchiga and Batis (2001), for cactus seeds to form a seed bank and remain viable in the soil, it is necessary to exhibit the proper physiological, morphological and ecological characteristics, such as the requirement of light for germination, a small size (about 2 mm or less in length), a post-maturation period for germination or ecological longevity. Studies have associated seed size with the requirement of light for germination and the formation of seed banks in the soil (i.e. Baskin & Baskin 2014). According to Thompson et al. (1993), small seeds, such as those from species of Discocactus (