Seed germination of Microlicia fasciculata, an apomictic and aluminium accumulator species: Unexpected intraspecific variability in a restricted Neotropical savanna area

Flora 220 (2016) 8–16

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Seed germination of Microlicia fasciculata, an apomictic and aluminium accumulator species: Unexpected intraspecific variability in a restricted Neotropical savanna area Marli A. Ranal a,∗ , Clesnan Mendes-Rodrigues b , Walquíria Fernanda Teixeira c , Ana Paula de Oliveira d , Rosana Romero a a

Instituto de Biologia, Universidade Federal de Uberlândia, Avenida João Naves de Ávila 2121, Uberlândia, MG 38400-902, Brazil Instituto de Biologia, Faculdade de Matemática—Estatística, Hospital de Clínicas de Uberlândia, Universidade Federal de Uberlândia, Avenida João Naves de Ávila 2121, Uberlândia, MG 38400-902, Brazil c Programa de Pós-Graduac¸ão em Biologia Vegetal, Instituto de Biologia, Universidade Federal de Uberlândia, Avenida João Naves de Ávila 2121, Uberlândia, MG 38400-902, Brazil d Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Vic¸osa, Campus Rio Paranaíba, Rodovia MG-230, Km 8, Rio Paranaíba, MG 38810-000, Caixa Postal 22, Brazil b

a r t i c l e

i n f o

Article history: Received 1 May 2015 Received in revised form 30 December 2015 Accepted 1 February 2016 Edited by Hermann Heilmeier Available online 4 February 2016 Keywords: Apomixis Cerrado Melastomataceae Palm swamp Vereda

a b s t r a c t In the Ecological Station of Panga, Microlicia fasciculata (Melastomataceae) is restricted to areas with palm swamps, environments subjected to desiccation due to anthropogenic actions including global climate change. This can put the species at risk of local extinction. Studies related to seed germination of M. fasciculata are rare and could help in understanding its ability to survive in these environments. The present paper aims to (1) assess intraspecific variability of seed germination and (2) study the effect of aluminium on seed germination. Two experiments were conducted in completely randomized design. The first evaluated intraspecific variability based on the germination measurements. The second evaluated the effect of aluminium on seed germination (5, 20, 40, 80 and 160 mg L−1 Al(OH)3 at pH 4.0 and one aluminiumfree treatment at pH 7.0). The results showed high intraspecific variability for germinability (6–30%), coefficient of variation of germination time (5.2–29.6%) and uncertainty (0.92–2.59 bits). Seed germination was slow (0.08 ≤ v ≤ 0.11 day−1 ; v for quick species is 1 day−1 or some hours−1 ) and asynchronous (0.11 ≤ Z ≤ 0.38; perfect synchrony is equal to 1). These features guarantee the survival of the species in these unstable environments, especially in relation to the volume of water that accumulates in the rainy season and its decrease in the dry season or by anthropogenic action. Under the action of aluminium and pH 4.0 the germinability of seeds ranged from 5.5 to 9.5% for doses between 5 and 160 mg L−1 of Al(OH)3 , whereas in the aluminium-free treatment at pH 7.0 germinability reached 19%. Our hypothesis is that the presence of aluminium in the seeds, since the species is an accumulator (4000–6000 mg Al kg−1 in leaves), is sufficient to trigger the germination process and the external addition was a limiting factor for the seeds. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction

Abbreviations: CVt , coefficient of variation of the germination time; ESP, Ecological Station of Panga; G, germinability; t, mean germination time; tf , time to first germination; tl , time to last germination; U, uncertainty; v, mean germination rate; Z, synchrony of the germination process. ∗ Corresponding author. Fax: +55 3432325016. E-mail addresses: [email protected], [email protected] (M.A. Ranal), [email protected] (C. Mendes-Rodrigues), walquiria [email protected] (W.F. Teixeira), [email protected] (A.P. de Oliveira), [email protected] (R. Romero). http://dx.doi.org/10.1016/j.flora.2016.02.001 0367-2530/© 2016 Elsevier GmbH. All rights reserved.

Apomictic species have an expected low genetic variability as embryos are formed only with information of the maternal genitor (Koltunow, 1993). In environments of small extension and with specific climatic and soil conditions, as is the case of the palm swamps in the Cerrado biome, these species can form clonal populations as pointed out by Richards (1997) and Tweney and Mogie (1999) for other types of environment. The palm swamps occupy an estimated area of 1.61% of the Cerrado biome (Reatto et al., 2008) and comprise 4.63% of the Eco-

M.A. Ranal et al. / Flora 220 (2016) 8–16

logical Station of Panga (ESP) according to Cardoso et al. (2009). They are found mainly in the banks of the valleys of small drainages that are not areas of springs, despite having wet soil most of the year (Cardoso et al., 2009). These areas are becoming reduced due to the dryness caused by recent changes in land use in the vicinity of the ESP (Cardoso et al., 2009). The removal of natural vegetation and the intensive use for agriculture and livestock have entailed profound changes in areas where the water table emerges, such as palm swamps and the original wetlands in the interior of the ESP (Lopes and Schiavini, 2007; Cardoso et al., 2009). The soil of these environments is hydromorphic, with a thick dark layer of partially decomposed organic matter over a layer with gleization resulting from oxireduction processes (Reatto et al., 2008); medium acidity, high content of aluminium and organic matter (Ramos et al., 2006). The Melastomataceae family has several species tolerant to aluminium, including accumulators with foliar concentration above 1000 mg kg−1 (Haridasan, 1982, 2008; Haridasan and Araújo, 1988; Jansen et al., 2002). In Brazil Melastomataceae is the sixth most diverse family of angiosperms, with 66 genera and about 1370 species distributed around the country (Baumgratz et al., 2015) in different vegetation types with a variable number of species (Romero and Martins, 2002). Among the numerous tribes of this family is Microlicieae with 275–300 species, being ca. 90% endemic of the Brazilian Cerrado (Clausing and Renner, 2001; Romero, 2003). Microlicia fasciculata Mart. ex Naudin is a shrub (0.4–0.6 m high) that produces capsules in March, April, October and November (Silva and Romero, 2008), months of the wet season, except April that is the first of the dry season and, consequently, conserves residual water in the soil. The species occurs in different vegetation types of the Cerrado biome as palm swamps, rocky fields, grasslands, and wet grasslands close to gallery forests (Romero, 1996; Silva and Romero, 2008). M. fasciculata is apomictic, with pollen viability of only 1.9% (Santos et al., 2012), which may represent a good model for studies of environmental effects in function of the low expected genetic variability (Richards, 1997; Tweney and Mogie, 1999). According to these authors, the occurrence of apomixis leads to the production of clonal individuals in the population and the low pollen viability reduces the cross pollinization rate. Together, these characters induce low genetic variability, making the environmental effects to be more easily tested. In the ESP M. fasciculata occurs mainly in palm swamps (Araújo et al., 2002), which have suffered alterations in recent years due to global climate changes (Cardoso et al., 2009). Although there are no studies showing which category M. fasciculata belongs with respect to aluminium, its occurrence in soils rich in this element may be an indication that it is at least tolerant to it. We aim that with our results we will be able to infer about the vulnerability of M. fasciculata to local extinction in a restricted area of the Neotropical savanna included in an important hotspot (Myers et al., 2000). In this context, our objectives were to evaluate the intraspecific variability and the effect of aluminium on seed germination of M. fasciculata, associating the physiological quality of the seeds and the germination pattern with soil chemical composition and nutritional status of the adult plants. 2. Materials and methods 2.1. Studied area and collection period Seeds of M. fasciculata were collected in December 2011, during the rainy season, in the Ecological Station of Panga (ESP), Uberlândia, Minas Gerais, Brazil (19◦ 09 20 S and 48◦ 24 35 W). The ESP has an area of 409.5 ha and belongs to the Federal University of Uberlândia since 1986. It is a protected area included in the category of Private Natural Heritage Reserve (Schiavini and Araújo, 1989). The palm swamp studied has an area of 0.84 ha.

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The regional climate is classified as tropical savanna (Aw Megathermic), according to the updated classification of Köppen–Geiger (Kottek et al., 2006), with two distinct seasons; cool and dry winter from April to September and hot and rainy summer from October to March. In the period of 1981–1998, the average annual temperature was 22.2 ◦ C, with minimum and maximum absolute temperatures of 1 and 37.4 ◦ C and average annual rainfall of 1599.6 mm (climate diagram presented by Ranal (2003)). In the period of 2010–2013, the average annual temperature recorded was 24.5 ◦ C and the average annual rainfall was of 1413.4 mm (data provided by the Meteorological Station of the Federal University of Uberlândia), with minimum and maximum absolute temperatures of 10.8 and 35.4 ◦ C. The average rainfall recorded by the same Meteorological Station during this period was 38 mm for the winter (April–September) and 197.6 mm for the summer (October–March). The average temperature for the winter was 23.7 ◦ C and for the summer 25.3 ◦ C.

2.2. Intraspecific variability and effect of aluminium on the seed germination process The intraspecific variability of one M. fasciculata population established in one of the palm swamps of the ESP was evaluated in a completely randomized design with three replicates of 50 seeds for each one of the 25 mother plants studied. The collection was randomized in the palm swamp and all mother plants presented abundant and mature fruits. When the mother plants were too near, forming clumps, only one of them was chosen for the fruit collection. The distance between them varied from 0.5 to 2 m. The aluminium effect on seed germination was studied based on a previous analysis of the aluminium content in the original soil where the species occurs in the ESP. The soil (n = 10) presents 47.7 mg L−1 of Al3+ (the same as 5.3 mmolc L−1 ; see Section 2.4 for extraction and determination methods and results for more details). The quantities of aluminium for the germination test were determined from this result and from Haridasan (2000) pointing to 10 mg L−1 for Miconia fallax DC. as a good aluminium concentration for the growing of seedlings, flowering and fruiting. Based on the results of the first experiment, seeds from mother plants number 13, 14, 17, 18 and 21, with mean germinability of 26.27%, were mixed in disproportional quantities before being sown due to the limited number of seeds produced by some mother plants. Five aluminium concentrations were tested (5, 20, 40, 80 and 160 mg L−1 Al(OH)3 , equivalent to 1.7, 6.9, 13.8, 27.7 and 55.4 mg L−1 Al3+ ). One relative control treatment was included in the experiment, using distilled water. All treatments had four replicates of 50 seeds each. All seeds were washed with distilled water and sown over filter paper in Emanueli Chambers (Araújo and Ranal, 2005) with 30 mL of distilled water or the aluminium solutions. The aluminium solutions were monitored weekly to keep the pH equal to 4.0 ± 0.2, using NaOH or HCl, both of them at 1 mM to provide aluminium for absorption by the seeds (about relationships between aluminium and pH see Marschner (1995)). The distilled water of the relative control treatment was renewed weekly and its pH stayed at 7.0. The relative control treatment was included in the experiment only to demonstrate the physiological quality of the seeds, but not for comparisons related to the aluminium effect due to the difference in the pH values. Both experiments were maintained in a B.O.D. incubator (Biochemical Oxygen Demand), at 25 ◦ C and photoperiod of 12 h. The evaluations were made daily, at the same time of sowing, adopting the embryo protrusion as germination criterion. The experiments were concluded after a period equal to the highest interval between two germinations. As many replicates had zero germination dur-

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M.A. Ranal et al. / Flora 220 (2016) 8–16

Table 1 Relevant germination measurements (mean ± standard error; n = 3 replicates for mother plant) for seeds of Microlicia fasciculata from 25 mother plants collected in one palm swamp of the Ecological Station of Panga, Uberlândia, Minas Gerais, Brazil. Means followed by different letters in each column are significantly different based on the Scott–Knott test (P < 0.05), F: ANOVA statistics; P: probability. Mother plant

Germinability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

8.67 16.67 7.33 7.33 18.00 17.33 12.67 6.67 20.00 10.67 14.67 7.33 26.67 24.67 6.00 12.67 30.00 25.33 12.00 6.00 24.67 12.00 8.67 15.33 9.33

Mean F (P)

14.43 ± 0.91 10.72 (<0.0001)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.76 d 5.81 b 1.33 d 1.33 d 0.00 b 0.67 b 1.76 c 0.67 d 0.00 b 0.67 c 0.67 b 0.67 d 7.06 a 0.67 a 0.00 d 1.76 c 1.76 a 3.46 a 1.15 c 0.00 d 0.67 a 3.46 c 0.67 d 0.67 b 1.76 d

ing 20 days, observations were concluded after 40 days from sowing. 2.3. Characters evaluated Germinability (G); time to first germination (tf ), time to last germination (tl ) and mean germination time (t, Labouriau, 1983); mean germination rate (v, Labouriau, 1970; for quick species v is 1 day−1 or some hours−1 ; see table of time in Parsons (2012)); coefficient of variation of the germination time (CVt , Ranal and Santana, 2006); uncertainty, the entropy associated to the distribution of the relative frequency of germination through time, where numbers near to zero indicate that the germination is concentrated in time (U, Labouriau and Valadares, 1976) and synchrony of the germination process (Z, Ranal and Santana, 2006) were evaluated. The meaning, use and limitations of these measurements and spreadsheets for their calculations were presented by Ranal and Santana (2006) and Ranal et al. (2009). The germination measurements cited above were calculated for seeds collected from each mother plant and also for groups formed artificially, based on the results of the statistical analysis of the coefficient of variation of the germination time and uncertainty. Length and width of five seeds from five individuals were measured and the ranges between the smallest and largest measurements were provided. 2.4. Chemical and physical analysis of soil and chemical analysis of leaf samples Soil samples were collected at 0–20 cm of depth, near the mother plants studied, in November 2010 (n = 10). All soil samples were air dried and sieved through a 2 mm mesh. Qualitative attributes to the nutrients available and exchangeable were used according to the tables presented by Alvarez-V. et al. (1999)

Coefficient of variation of the germination time (%) 11.48 13.79 10.22 15.40 11.64 14.68 19.47 5.20 14.98 17.07 17.54 15.77 22.65 22.02 12.27 20.42 23.36 15.11 14.78 18.00 14.87 12.84 29.57 12.03 12.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.65 a 3.58 a 2.35 a 5.19 a 3.52 a 5.60 a 2.29 b 0.22 a 1.88 a 2.10 a 3.65 a 1.24 a 3.93 b 2.52 b 5.05 a 0.90 b 0.90 b 1.53 a 3.18 a 3.71 a 1.79 a 2.51 a 2.00 b 2.51 a 3.27 a

15.92 ± 0.77 2.67 (0.0017)

Uncertainty (bit) 1.29 1.67 1.25 1.07 1.50 1.65 1.96 0.95 1.61 1.70 1.72 1.08 2.55 2.59 0.92 1.56 2.33 1.75 1.61 0.92 2.02 1.40 1.46 1.76 1.40

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.19 a 0.38 a 0.33 a 0.15 a 0.44 a 0.58 a 0.30 b 0.03 a 0.35 a 0.22 a 0.30 a 0.21 a 0.28 b 0.17 b 0.00 a 0.20 a 0.20 b 0.09 a 0.32 a 0.00 a 0.13 b 0.27 a 0.04 a 0.20 a 0.29 a

1.59 ± 0.07 2.93 (0.0007)

for cultivated plants, due to lack of descriptions for native species. Leaf samples were collected in November 2010 (n = 6) and in September 2012 (n = 7) and stored in plastic bags in field conditions. In the laboratory they were washed in distilled water and dried in paper bags at 60 ◦ C with forced air circulation until constant mass. After that, they were ground in a Wiley type mill and sieved with a 1 mm mesh for chemical analysis. The nitrogen extraction of the soil and leaf samples was made by sulphuric acid digestion; the extraction of the other nutrients was made by nitroperchloric digestion (EMBRAPA, 2009). The extracts obtained were used to measure the levels of total P by colorimetry; Ca, Mg, Cu, Fe, Mn, Zn and Al (in the leaves) content by atomic absorption spectrophotometry; K by flame photometry and Al in the soil by titration according to EMBRAPA (2009). Boron was determined by the Azomethine H colorimetric method (EMBRAPA, 2009). All samples of soil and leaves were analysed in the Laboratory of Soil and Limestone Analysis of the Federal University of Uberlândia, according to EMBRAPA (2009). Also the most classic agronomic references to the analysis and interpretations were consulted, including Prado (1995), EMBRAPA (1997) and Raij et al. (2001), since they are the most common and comparable references in Brazil.

2.5. Statistical analysis The means were compared by the Scott–Knott test for robustness for the normality of the residuals of the ANOVA (Borges and Ferreira, 2003) and for capacity to form exclusive groups. The last character made comparisons easier for the 25 evaluated means. For the experiment with aluminium, excluding the relative control treatment, the data were tested for fitness to the regression model. The relative frequencies of germination were calculated according to Labouriau (1983).

M.A. Ranal et al. / Flora 220 (2016) 8–16

3. Results Seeds of M. fasciculata are very small, with 0.4–0.6 mm × 0.3–0.4 mm, and showed intraspecific variability in relation to germinability (G), coefficient of variation of the germination time (CVt ) and uncertainty (Table 1). The highest value of germinability was 30% and the lowest 6%. Germinability formed four groups (6–9.33%; 10.67–12.67%; 14.67–20% and 24.67–30%); two groups for spreading of germination through time (values of CVt from 5.20 to 18% and from 19.47 to 29.57%) and two groups for the uncertainty of the germination process (0.92–1.76 bits and 1.96–2.59 bits) according to the Scott–Knott test. Seeds with a low coefficient of variation of germination time had also low uncertainty and vice versa, except for seeds of the mother plants numbers 16, 21 and 23 (Table 1). In relation to the other characters without intraspecific variability, the germination process occurred with mean initial time of 8.53 ± 0.19 days and range of 6.67 ≤ tf ≤ 11 days (F24,50 = 1.73; P = 0.051); mean final time of 12.71 ± 0.25 days and range of 10.67 ≤ tl ≤ 14.33 days (F24,50 = 0.74; P = 0.787); mean of the mean germination time of 10.61 ± 0.16 and range of 9.47 ≤ t ≤ 12.44 days (F24,50 = 0.80; P = 0.724); mean of the mean germination rate of 0.10 ± 0.001 day−1 and range of 0.08 ≤ v ≤ 0.11 day−1 (F24,50 = 0.68; P = 0.845) and with mean synchrony of 0.26 ± 0.02, range of 0.11 ≤ Z ≤ 0.38 (F24,50 = 0.98; P = 0.503). Nearly 45% of the seeds that germinated completed the process between the eighth and tenth days after sowing, independently of the grouping (Fig. 1A–D), but this result is associated to the seeds of the few mother plants with germinabilities between 20 and 30% (Table 1). The seeds that germinated without aluminium (relative control) presented higher germinability (19.0 ± 0.87%; mean ± standard error), uncertainty (2.45 ± 0.02 bits) and coefficient of variation of the germination time (18.45 ± 0.98%) in relation to the mean of the 25 individuals evaluated in the first experiment (Table 1). The first germination time was 9.25 ± 0.24 days; the last germination time 16.25 ± 0.13 days; the mean germination time 13.01 ± 0.11 days; the mean germination rate 0.08 ± 0.00 day−1 and the synchrony 0.10 ± 0.01 (Z value). The high germinability of these seeds was a consequence of the choice of the better seeds to install the experiment (seeds of individuals 13, 14, 17, 18 and 21), but this choice did not prevent asynchrony (Fig. 2). Observing only the results of the treatments with different aluminium concentrations, this element did not affect the germination process (5.5 ≤ G ≤ 9.5%, F4,19 = 0.879, P = 0.500; 5.75 ≤ tf ≤ 13.0 days, F4,19 = 1.87, P = 0.167; 11.5 ≤ tl ≤ 15.0 days, F4,19 = 0.51, P = 0.725; 12.22 ≤ t ≤ 14.15 days, F4,19 = 1.08, P = 0.401; 7.30 ≤ CVt ≤ 20.80, F4,19 = 1.29, P = 0.316; 0.07 ≤ v ≤ 0.08 day−1 , F4,19 = 1.50, P = 0.252; 0.01 ≤ Z ≤ 0.29, F4,19 = 0.87, P = 0.504; 1.23 ≤ U ≤ 1.88, F4,19 = 0.92, P = 0.476). The germination process was restricted in all treatments (low germinability). In addition, none of the germination measurements adjusted to the regression models. The soil where the species is established is of medium texture class, presenting the sum of clay and silt greater than 15% and less than 35%. It has medium active acidity (pH in water), potential acidity (H+ + Al3+ ) and exchangeable acidity (Al3+ ); a very low sum of bases and effective cationic exchange capacity; medium cationic exchange capacity at pH 7.0; a very low saturation by bases; good aluminium saturation and medium organic matter content (Table 2). These characters are a consequence of the very low content of potassium, calcium and magnesium that are considered the most important bases of the soil. The soil also has a very low content of phosphorus, boron and manganese, a low content of zinc, medium of copper and high only for iron. This means that this soil is poor in nutrients as a consequence of the high saturation by aluminium (greater than 50%), low saturation by bases (less than 50%) and is therefore considered dystrophic (Table 2). The leaves pre-

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sented a high content of aluminium, manganese and iron, and a low content of calcium (Table 2).

4. Discussion Even though being apomictic and with low pollen viability (Santos et al., 2012), M. fasciculata has intraspecific variability for some germination measurements, in the same way as was observed for Miconia ferruginata DC. (Mendes-Rodrigues et al., 2010), which is also apomictic and with low pollen viability. Apparently, apomictic species with low pollen viability can produce progeny with variability, probably as a reflection of maternal effect, associated with environmental effects besides genetic influence. More studies are still needed to compare the intraspecific variability of species with absence of sexual reproduction and those strictly sexual to elucidate the source of variability within the Melastomataceae. Other species, as Miconia albicans (Sw.) Triana, are also apomictic with sterile pollen (Goldenberg and Shepherd, 1998) showing variability in the seeds collected in different years or in different vegetation types (Sales et al., 2013). Perhaps this feature is the key for survival of species and diversity of the family. Another important factor is the high frequency of seeds without embryos in species of Melastomataceae, ranging from 13 to 86.5% (Silveira et al., 2013). The low germinability of M. fasciculata seeds could be a consequence of the low frequency of seeds with embryos and not due to poor seed quality. This aspect was not evaluated in this study due to the small size of the seeds. Future studies should consider the presence of embryos, despite the difficulties generated by the very small seed size and the hardness of the tegument. The use of X-rays can be a good solution for small seeds as was recommended by ISTA (2015) to determine the physical quality of the seeds. From the practical point of view, the presence of intraspecific variability is a complicating factor when a preliminary evaluation of variability in the target population is not made and the focus of the research is to evaluate the influence of environmental effects on the population, as pointed out by Mendes-Rodrigues et al. (2010). The principal difficulty is related to the formation of homogeneous replicates, essential in experiments with environmental and experimental effect focus. However, from the biological point of view, this feature is a very important population aspect, since it is relevant for the survival of the species, especially those that are established in unstable environments such as palm swamps, due to local and global climate changes, as pointed out by Cardoso et al. (2009). Although producing numerous and tiny seeds, the mean germinability of M. fasciculata seeds was 14.4% (mean germinability of all mother plants analysed). This feature, together with its occurrence in wet grasslands, subject to dryness (see Cardoso et al. (2009)), can support the inclusion of M. fasciculata in the list of species with potential local risk, due to climate changes. Thus, M. fasciculata will be the second species of Melastomataceae at risk in the ESP. The other species at risk is Miconia theaezans (Bonpl.) Cogn. that disappeared from the gallery forest of the Panga Stream as adult individuals (Lopes and Schiavini, 2007), but was present in the soil seed bank (Pereira-Diniz and Ranal, 2006). According to Pereira-Diniz and Ranal (2006), the Cerradão limits are expanding towards the gallery forest, most likely due to macroclimatic changes of the region; and the live register of M. theaezans in the soil as viable seed is the chance for its survival if favourable conditions permit seed germination and seedling establishment. According to Simon et al. (2009), Microlicieae clade possibly had diversified from moist environments, about 10 million years ago. This hypothesis is supported by the occurrence of the species in wetlands, unfortunately subject to seasonal dryness intensified by anthropogenic activities (Cardoso et al., 2009).

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M.A. Ranal et al. / Flora 220 (2016) 8–16

Fig. 1. Relative frequency of seed germination of Microlicia fasciculata as a function of time. The seeds were collected in one palm swamp in the Ecological Station of Panga, Uberlândia, Minas Gerais, Brazil. (A) 25 mother plants pooled together; (B) for each mother plant sampled; (C) mother plants with CVt ≤ 18.00 or CVt ≥ 19.47; (D) mother plants with U ≤ 1.76 or U ≥ 1.96. G: germinability; t: mean germination time; CVt : coefficient of variation of the germination time; U: uncertainty Z: synchronization index.

Fig. 2. Relative frequency of seed germination of Microlicia fasciculata. Seeds collected in one palm swamp in the Ecological Station of Panga, Uberlândia, Minas Gerais, Brazil were exposed to different Al(OH)3 concentrations.

Among the drastic consequences of the anthropogenic actions are the climatic changes which are affecting the Cerrado biome. In the Uberlândia, for example, the mean temperature from 2010 to 2013 increased by 2.3 ◦ C, compared with 1981–1998 (Ranal, 2003) and rainfall decreased by 186.2 mm. It should be taken into

account that during the 18 years evaluated previously there were also drier and hotter years and this warming is part of natural cycles. Therefore, these observations should continue to give support to the management of these wetlands, which are directly affected by climate fluctuations as pointed out by Cardoso et al. (2009). The

M.A. Ranal et al. / Flora 220 (2016) 8–16

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Table 2 Chemical and physical analysis of soil and chemical analysis of leaf samples of Microlicia fasciculata established in a palm swamp of the Ecological Station of Panga, Uberlândia, Minas Gerais, Brazil. Soil chemical analysis 1

N

P

K −3

2010 n = 10

November

Mean sd

Mean sd

Ca

S-SO4 2−

Mg

−3

November

Cu

mmolc dm

3.43 1.98

0.74 0.40

1.10 0.30

0.40 0.50

0.70 1.06

0.14 0.05

0.87 0.36

pH water

H + Al

SB

t

T

V

m

O.M.

1:2.5

mmolc dm−3

5.32 0.43

46.1 14.0

– –

CS

Mn

Zn

mg dm

7.54 –

48.34 –

Al3+

130.20 120.39

2.05 2.51

0.51 0.34

5.30 1.50

g kg−1

% 2.24 –

Fe

mmolc dm−3

mg dm

4.63 –

Physical analysis of soil 2010

B

−3

70.29 –

37.90 21.20

Texture class

FS

S

C

346.75 106.21

52.13 63.89

199.25 131.24

−1

g kg n=8

Year

Mean sd Month

401.63 145.50

Medium

Leaf chemical analysis N

P

K

Ca

Mg

S

g kg−1 2010 n=6 2012 n=7

November September

Mean sd Mean sd

12.37 6.08 8.33 3.85

B

Cu

Fe

Mn

Zn

Al

6.58 2.30 6.03 0.93

378.98 71.68 272.93 56.54

517.18 88.91 385.26 61.54

25.57 7.08 15.51 2.79

6352.17 819.03 4359.90 430.45

mg kg−1 0.33 0.05 0.56 0.05

5.00 0.32 5.50 1.44

1.33 0.60 3.03 1.19

0.92 0.26 1.03 0.16

0.73 0.05 0.49 0.04

9.72 7.82 23.65 3.49

Soil analysis: 1 P available, Mehlich1 extractor; n: sample size; sd: standard deviation; SB: sum of bases (Ca + Mg + K); t: effective cationic exchange capacity (SB + Al); T: cationic exchange capacity at pH 7.0 [SB + (H + Al)]; V: saturation by bases [(SB/T) 100]; m: exchangeable aluminium saturation [(Al/t) 100]; O.M.: organic matter; CS: coarse sand; FS: fine sand; S: silt; C: clay; extraction and determination of P and K: HCl 0.05 mol L−1 + H2 SO4 0.0125 mol L−1 ; S-SO4 2− : monobasic calcium phosphate 0.01 mol L−1 ; Ca, Mg and Al: KCl 1 mol L−1 ; H + Al: buffer solution SMP at pH 7.5; O.M.: colorimetric method; B: BaCl2 ·2H2 O 0.0125% hot; Cu, Fe, Mn and Zn: DTPA (Diethylene Triamine Pentaacetic Acid) 0.005 mol L−1 + TEA (Triethanolamine) at 0.1 mol L−1 + CaCl2 0.01 mol L−1 at pH 7.3.

wetland management according to climate fluctuations can protect and perhaps save the species that occurs exclusively in the palm swamps of the ESP. Probably the intraspecific variability in the germination of the seeds of M. fasciculata has been important to spread out from wetlands to dryer areas such as rocky fields and grasslands where the species is frequently found (Matsumoto and Martins, 2005; Santos and Silva, 2005; Silva and Romero, 2008). The spread of germination through time, detected by relatively low synchrony (0.11 ≤ Z ≤ 0.38, remembering that perfect synchrony is equal to 1.0), even in a short period (6.67 ≤ tf ≤ 11.0 days and 10.67 ≤ tl ≤ 14.33 days), also indicates that M. fasciculata seeds have the potential to form a soil seed bank. Higher uncertainty values for seeds with higher germinabilities also reinforce this spreading of germination through time and express the chance of the seed to germinate in an appropriate moment. This feature offsets the low germination percentage being a strong indicator of the presence of relative dormancy sensu Labouriau (Labouriau, 1983), perhaps due to the thickness of the testa (mechanical dormancy according to the simplified version of Nikolaeva’s classification presented by Baskin and Baskin (1998)). As observed for other Melastomataceae, the presence of a mechanical sclerotic layer gives the testa this thickness (Groenendijk et al., 1996), resulting in staggering of germination over time, as observed for M. ferruginata seeds (Mendes-Rodrigues et al., 2010). Dormancy in other species of Melastomataceae, including Chaetostoma armatum (Spreng.) Cogn., was also recorded by Silveira et al. (2012) and Ribeiro et al. (2015). Seeds of Trembleya laniflora (D. Don) Cogn. have also the potential to form soil seed banks, maintaining their viability after 42 months of storage, although they have been considered as a case of non-dormant seeds (Rodrigues and Silveira, 2013). Some individuals of Microlicia inquinans Naudin from Serra da Canastra National Park disappear after a fire (R. Romero, pers. obs.), but it

is known that several species of Microlicia show fire adaptations such as xylopodium (Simon et al., 2009). So, species less resistant to seasonal drought and fire need to have resistant subterranean structures with regrowth possibilities or viable seeds in the soil to ensure the survival of the population. M. fasciculata seeds also had greater homogeneity (lower values of CVt ) and greater synchronization (Z values reached 0.38), compared with M. ferruginata seeds from dry environments, and this may be another advantage for the species. Species that occur in wet environments have greater synchrony in the germination process, as has been found by Ludewig et al. (2014) and Mendes-Rodrigues (unpublished data), allowing them to take advantage of specific environmental windows favourable to germination. As the fruiting of M. fasciculata occurs in March and April, October and November (Silva and Romero, 2008) and the collection of seeds used in the experiments of this study was made in December, it seems that the relative highest synchrony of germination is associated with the establishment of a greater number of seedlings still early in the rainy season, when the soil of the edge of the palm swamps is not saturated with water. It would be interesting to investigate whether the germination pattern described here is repeated for seeds released in March and April, the end of the rainy season. Probably these seeds will have greater asynchrony, slower and heterogeneous germination, which could keep some of them dormant during the dry season. The production of staggered crops with physiologically different seeds could be the key to the reproductive success of the species. However, it is still unclear whether what really leads to the low germinability in M. fasciculata is the poor quality of seeds, the high percentages of embryo absence or the presence/type of seed dormancy.

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Even in an area as restricted as the palm swamps, intraspecific variability in the germination of the seeds of M. fasciculata was observed, which can facilitate its survival. However, further evaluations related to the survival of seedlings and young plants of this species in these types of environment should be made. Some phytosociological studies have demonstrated the occurrence of zonation in palm swamps which shows that even in restricted environments, soil and microclimatic variations may occur by inducing the differential distribution of species (Oliveira et al., 2009). Seeds of M. fasciculata showed similar germinabilities when exposed to the different concentrations of aluminium and lower than the relative control treatment, but the confounding between pH and aluminium hinders comparisons and requires future experiments to test a second control with distilled water at pH 4.0. However, considering the mean and standard deviation of the potential acidity (H+ + Al3+ ) of the soil of the palm swamp (4.61 ± 1.40) where this species is established, germination could occur without problems at the pH tested (4.0). Besides, at pH 7.0 germinability reached 19%, showing seeds with better conditions to complete the germination process than that maintained under aluminium and low pH action. Thus, at least 19% of the seeds were in good conditions to germinate. These results suggest that the low germinability of the seeds under the action of aluminium could indicate that this element is present in considerable quantities in the seeds, sufficient to trigger the germination process, and exogenous application may have caused some toxicity in the embryo, affecting the cell division and/or elongation processes making the protrusion difficult. The rhizotoxicity of aluminium was demonstrated by several authors during the last 100 years (Sun et al., 2010; Bian et al., 2013; Kopittke et al., 2015; Shu et al., 2015; Roselló et al., 2015). Again, more experiments are necessary to support the hypothesis of toxicity of the exogenous aluminium. It is known that for accumulators the presence of aluminium is essential to the growing of seedlings and fruiting (Haridasan, 2000, 2008), including M. albicans (Haridasan, 1988), but in the case of seeds, our hypothesis is that the endogenous aluminium is sufficient to trigger the germination process. Probably the first green leaves produced by seedlings of M. albicans cultivated without aluminium received this element accumulated in the seeds and after the exhaustion of the Al reserve, chlorosis and necrosis appeared (see images presented by Haridasan (2008)). This indicates the importance of aluminium in the seeds for Al-accumulators. Many Cerrado species of Melastomataceae, Rubiaceae and Vochysiaceae accumulate aluminium in leaves and seeds (Haridasan, 2008). Specifically for seeds, the range found by Haridasan (2008) varied from 4800 to 40500 mg kg−1 of Al, as is the case of M. albicans with 6900 mg kg−1 , M. pohliana Cogn. with 5300 mg kg−1 and Vochysia tucanorum Mart. reaching up to 40500 mg kg−1 . This is not surprising because the family Vochysiaceae has Al-hyperaccumulators as Callisthene major Mart., Qualea grandiflora Mart. and Vochysia pyramidalis Mart. with Al content in leaves ranging from 3900 to 6500 mg kg−1 (Andrade et al., 2011). M. fasciculata is not mentioned in the literature as an accumulator, but the presence of more than 1000 mg kg−1 of aluminium in its leaves is sufficient to include this species in this group, according to the criterion proposed by Weeb (1954). Besides, many accumulator species are found in the Microlicieae tribe according to the colorimetric method adopted by Jansen et al. (2002). These authors concluded that Al accumulation represents a plesiomorphic character, a primitive status inherited from a common ancestor of Memecylaceae and Melastomataceae. M. ferruginata, occurring in a dystrophic soil of one cerradão in Brasilia, presented 3750 mg kg−1 of aluminium in its leaves (Haridasan and Araújo, 1988) and 610–790 mg kg−1 in seeds collected in the Serra de Caldas Novas—Goiás State (MendesRodrigues et al., 2010). If M. ferruginata has high aluminium content

in the seeds, although having about 600 mg kg−1 less aluminium in the leaves than M. fasciculata, it is expected that the seeds of the latter would also have the featured element in its composition. Two species of mistletoes, Phthirusa ovata (DC.) Eichler and Psittacanthus robustus (Mart.) Mart. infecting M. albicans, an Alaccumulator, and P. ovata infecting Byrsonima verbascifolia (L.) DC., a non-Al-accumulator, were used by Scalon et al. (2013) as models to study interactions among Al and mineral nutrition and Al re-translocation. When growing over B. verbascifolia, the non-Alaccumulator host species, the leaves, branches and seeds of P. ovata had 260, 132 and 122 mg Al kg−1 . High concentrations of Al in the leaves of P. ovata were observed only when parasitizing the Al-accumulating host (10869 mg kg−1 ), but without accumulation in branches (156 mg kg−1 ) or seeds (127 mg kg−1 ). In P. robustus, large concentrations of Al were found in leaves (8679 mg kg−1 ), branches (2173 mg kg−1 ) and seeds (2878 mg kg−1 ). Apparently, Alaccumulator species show high content of Al in their seeds, but the restricted number of species studied hinders generalizations regarding accumulation of Al in the seeds. In the same cerradão of Brasilia, Haridasan and Araújo (1988) found 6250 mg kg−1 in the leaves of M. albicans and 1560 mg kg−1 in the leaves of M. pohliana. The soil had 27 mmolc kg−1 of aluminium at 0–15 cm depth; 6 mmolc kg−1 at 15–25 cm depth and 2 mmolc kg−1 at 25–60 cm depth. In the red latosol (oxisol) of the Água Limpa Farm of the University of Brasília, with a pH between 3.7 and 4.9 from zero to 60 cm deep, low base saturation and aluminium from 6 to 9 mmolc kg−1 at the soil surface and around 3 mmolc kg−1 between 45 and 60 cm deep, Haridasan (1982) found M. ferruginata with 4310 mg kg−1 and M. pohliana with 6630 mg kg−1 of leaf aluminium content. Comparing the aluminium content of these two environments with those obtained in the palm swamp studied, the latter presented a lower quantity and still M. fasciculata accumulated aluminium in its leaf tissues as much as M. albicans in the cerradão of Brasilia and M. pohliana in Água Limpa Farm. The different results on the aluminium content in the leaves of the same species occurring in different places, associated with the small size of M. fasciculata leaves, the results of the aluminium content being presented for the first time, and the possibility of small portions of the branches being involuntarily included in the analyses indicate that more samples from different regions should be analysed to find the range of aluminium that this species is able to accumulate. M. fasciculata is also a manganese accumulator according to the criterion of Gauch (1972) because it has over 300 mg kg−1 of manganese in its tissues. This species may be considered calcifugous according to the criterion of Hou and Merkle (1950) and confirmed by Haridasan and Araújo (2005), because it presents 1.33 g kg−1 of calcium in its tissues (the limits given by the authors for this type of plants are between 4.8 and 10.8 g kg−1 ), and also has high iron levels compared to those found by Haridasan and Araújo (1988) for species of cerradão dystrophic and mesotrophic (30–180 mg kg−1 ), justified by the high content of the element in the soil. Accumulators appear to be common in Melastomataceae, as T. laniflora in ironstone soils from the Espinhac¸o Range in Minas Gerais (Teixeira and Lemos-Filho, 2002) and Cambessedesia hilariana (Kunth) DC., Miconia cf. sellowiana Naud. and Tibouchina multiflora Cogn. in rocky fields over hemathitic litholic canga at the Brucutu Mine, Barão de Cocais, Minas Gerais (Mourão and Stehmann, 2007), being probably an iron accumulator species. Concluding, seeds of M. fasciculata have intraspecific variability. Seed germination was slow and asynchronous. This species has a high content of iron, is calcifugous, and an accumulator of manganese and aluminium. This set of characteristics allows the species to survive in dystrophic and hydromorphic soils as palm swamps,

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