Introduction

In Mexico, there are ca. 105 species of Mimosa L. (Leguminosae), 54% of which are endemic; thus, it is considered the most diverse genus of the mimosoids in the country (Grether et al. 2015). Mimosa luisana Brandegee is a species endemic to the semiarid Tehuacán–Cuicatlán Valley, which is located in the states of Puebla and Oaxaca, Mexico (Martínez-Bernal and Grether 2006). Mimosa luisana is considered a multipurpose species (Camargo-Ricalde et al. 2001) that forms resource islands (Camargo-Ricalde et al. 2002), which are reservoirs of arbuscular mycorrhizal fungal spores (Camargo-Ricalde and Dhillion 2003; Chimal-Sánchez 2015) and nitrogen-fixing bacteria (Camargo-Ricalde et al. 2010a) and also as a nurse plant for the columnar cactus Neobuxbaumia tetetzo (F.A.C.Weber ex K.Schum.) Backeb., a species endemic to this area (Valiente-Banuet and Ezcurra 1991).

Due to the fact that M. luisana provides ecosystem services, to its economic and cultural importance to local inhabitants (Camargo-Ricalde et al. 2001) and to its morphological, anatomical and physiological characteristics, this species has the potential to restore degraded environments within the Tehuacán–Cuicatlán Valley (Dhillion et al. 2004; Montaño-Arias et al. 2015, 2017). Through wood anatomical analysis, Montaño-Arias et al. (2017) determined that this species is resistant to drought events in adulthood, but its drought tolerance in other life cycle stages is unknown. There are studies that support the use of M. luisana in ecological restoration; however, the longevity of its seeds is unknown. These data would be particularly important to take in consideration, especially because the changes in precipitation regimens, predicted by the Intergovernmental Panel on Climate Change (IPCC).

The botanical characteristics of M. luisana are well known; however, the effect of seed longevity on germination is unknown. We consider that this knowledge is important because it could contribute to the understanding of the dynamic of plant establishment in an adverse environment, where Mimosa makes important association with micro- and macro-biota, as part of the ecosystem.

The seed is a very important stage of the life cycle of angiosperms since the establishment and growth of the species depend on it. Anatomical studies on the seed coat of M. luisana, described an external layer of macrosclereids and an internal layer of osteosclereids, whose make the seed coat hard and impermeable (Montaño-Arias 2016). According to some studies, seeds need scarification to germinate in a wide range of temperatures (Camargo-Ricalde et al. 2004; Montaño-Arias et al. 2015). Within the genus Mimosa, seed longevity has been poorly explored, although some studies on Leguminosae describe seeds with long life span, for 10 years (Moreno-Casasola 1973; Parra 1984), 50 years (Ewart 1908) and 81 years.

Research on germination of Mimosa seeds is increasing but it is still a research challenge. These studies investigate the optimal temperatures for germination (Camargo-Ricalde and Grether 1998; Camargo-Ricalde et al. 2004; Pavón et al. 2011; Montaño-Arias et al. 2015), and very few are focused on the effect of seed longevity on germination (Silveira et al. 2014).

In other mimosoid legumes, such as Leucaena leucocephala cv. Cunningham (González et al. 2012) and Albizia lebbeck (L.) Benth. (González et al. 2009), a negative correlation between seed age and germination percentage was found. According to González et al. (2009), seed age can affect the time of seedling emergence; for example, Gliricidia sepium (Jacq.) Kunth ex Walp. showed lower emergence percentage compared to Albizia lebbeck; however, at beginning of emergence, there were no differences between newly collected and older seeds. Seedling vigour is another variable associated with seed longevity; it was found that the seedlings size decreased as seeds get older González et al. (2009).

In the particular case of Mimosa, Gómez-Pompa et al. (1976) reported that naturally aged seeds of M. pudica L. showed a low germination percentage, while Silveira et al. (2014) found that the seeds of M. foliolosa Benth. subsp. pachycarpa (Benth.) Barneby, artificially stored for 12, 24 or 36 months, showed an accelerated germination process and an increased germination percentage.

Considering the biological and ecological relevance of M. luisana and the lack of studies referring the effect of seed longevity on germination, the present study explores the physiological implications of the seed longevity for plant establishment analysing: seed moisture content, seed imbibition rate, germination percentage and seedlings survival, under the assumption that seed age critically influences these parameters.

Methods

Mimosa luisana naturally occurs and is endemic to the scrublands of the semiarid area of Tehuacán-Cuicatlán Valley, in the Mexican states of Puebla and Oaxaca. A population located at the state of Puebla (18°15′ 23.7ʺ N, 97°09′03.3ʺ W, at 2,232 masl), was selected based on the predominance of Mimosa luisana. Mature fruits were collected in November of 2010, 2011, 2016 and 2017, the season of maximum productivity. Each year, samples were obtained from eight individuals (trees) with the same height and coverage (2.0 and 2.0 m, respectively). Voucher specimens were deposited at the official herbarium of the Universidad Autónoma Metropolitana, Iztapalapa campus (Herbario Metropolitano, UAMIZ) (Table 1).

Table 1 Voucher specimens of Mimosa luisana deposited at the “Herbario Metropolitano de la Universidad Autónoma Metropolitana, Iztapalapa” (UAMIZ)
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All seeds were extracted from mature fruits manually and those that showed no evidence of infestation by bruchids (healthy seeds) were selected under a stereomicroscope (Nikon, SMZ800, Japan) for the experiments. According to the year of collection, seeds were organised into four age groups: 84, 72, 12 and 0 months old (recently collected) as control.

Considering that seed size and weigh can influence the germination rate (Matilla 2004; Skogen et al. 2010), we measured the length, width and thickness of the seeds with a digital calliper (Absolute Digimatic, CD-6" CS) in all seeds of each age group. Individual seed were weighted (g) individually with an analytical balance (Denver Instrument, APX-100, US). Seeds in the range of 2.0–2.5 mm length, 2.0–2.4 mm width and 1.8–2.2 mm thickness, and 0.08–0.10 g weight were used for the experiments. Seeds were stored in airtight glass containers, in the dark at 20 °C, and 50–60% relative humidity (RH) until germination experiments.

Moisture content (MC)

Seed moisture content (%) was measured as follows: The “initial weight” was obtained with an analytical balance (Denver Instrument, APX-100), from three replicates of 30 seeds per age group (90 seeds per treatment); subsequently seeds were placed in an incubator (Rios Rocha, S.A., Model EC-33) at 65 °C until constant weight was achieved, to obtain the “Final weight”. The MC (%) was calculated according to ISTA (2013):

$$ {\text{MC }}\left( \% \right)\, = \,({\text{initial weight}}\, - \,{\text{final weight}})/{\text{initialweight}}\, \times \,100. $$

Treatments and experimental design

The experimental design was factorial 4 × 2: four seed age groups 84, 72, 12 and 0 months old (recently collected, control) and two scarification conditions: unscarified (UNS, control) and mechanically scarified (S) giving a total of eight treatments. Seeds were mechanically scarified, with a nail clipper, cutting the seed coat at the opposite side of the micropyle, to prevent the embryonic axis damage (Fig. 1a, b). All treatments were arranged completely randomised and replicates will be indicated for each experiment described below.

Fig. 1
figure 1

Seed and seedlings of Mimosa luisana. a unscarified seed; b scarified seed; c Mimosa seedling with cotyledons and protophyll, and d Mimosa plant with cotyledons persistent and metaphylls with two pairs of pinnae each with four pairs of leaflets. C cotyledon, P protophyll, M micropyle, ME metaphyll, LF leaflets, SS scarified seed. Scale: a, b = 0.9 mm; c, d = 1 cm

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Imbibition rate (IR)

There were three replicates with 30 seeds per treatment (90 per treatment). Each replicate was placed in 20 mL of water at the optimal germination temperature (25 °C) (Montaño-Arias et al. 2015). The imbibition process was monitored every 3, 6, 12, 24 and 48 h, until the seed stop imbibition. At each interval, the seeds were extracted, the excess water was removed using Whatman filter paper No. 2 and their initial weight was recorded with an analytical balance (Denver Instrument, APX-100). The seeds were then dried in an incubator (Rios Rocha, S.A., Model EC-33) at 65 °C until constant weight was achieved and this was considered as final weight.

The IR (%) was calculated according to Jacobo-Pereira et al. (2016) as follows:

$$ {\text{IR}}\, = \,\left( {{\text{final weight}}\, - \,{\text{initial weight}}/{\text{initial weight}}} \right)\, \times \,{1}00. $$

Germination experiment

A total of 100 seeds per age group were tested for germination. Twenty scarified seeds were sown in each of five sterile Petri dishes (9 cm diameter), with filter paper (Whatman no. 2), and seeds were moistened with deionised water and kept at 25 °C in a controlled environment chamber (Conviron T 38/Lb/AP); 25 °C, with 14 h light and 10 h darkness. Seeds were considered germinated when they showed emerged radicle of 1 mm (Bewley and Black 1994). All experiments lasted 8 days, when the scarified seeds reached 100% germination.

The following variables were evaluated according to Piedrahita (1997, 1998), Enríquez-Peña et al. (2004), Weng and Hsu (2006) and Montaño-Arias et al. (2015):

  1. I.

    Germination percentage (GP) was calculated as GP = Ng × 100/Ns,

    where Ng total germinated seeds and Ns Total sowed seeds.

  2. II.

    Half-maximal germination (G50) was the number of days after sowing needed to reach 50% of the total germination, estimated in this study through the interpolation of one day before and 1 day after 50% of total germination was achieved.

  3. III.

    Saturation rate (SR) was the germination time related to the total germination percentage: SR = Σ(niti)/N,

    where ni  number of seeds germinated in 1 day (i).

    ti number of days after sowing;

    N total number of seeds sown.

  4. IV.

    Germination rate (GR) was calculated as follows: GR = Σ (Ni)/t,

    where Ni number of seeds germinated in 1 day (i); t time from sowing until germination of the last seed.

Survival and growth

Seedlings (15 days after sowing, from the scarified seeds (treatments 84S, 72S, 12S, 0S)) were transplanted into 0.5 kg polypropylene pots (10 cm diameter × 50 cm hight) containing a mixture 3:1:1 of native soil:agrolita:volcanic rock (0.2 mm diameter). Native soil was obtained from Tehuacán–Cuicatlán Valley, the place of seed harvest. Pots were placed in the controlled environment chamber, at 25 °C, with 14 h light and 10 h darkness. The seedlings were watered every other day with 300 mL of deionised water. The seedlings were monitored daily, assessing (1) percentage of survival (green erect seedling and turgid), (2) seedling height (mm), measured from the base to the apex of its distal leaves, (3) appearance of the protophylls (Fig. 1c), and (4) appearance of the metaphylls (Fig. 1d). The terminology used is according to Duke and Polhill (1981).

Statistical analysis

Data were examined by a two-way ANOVA test (P < 0.05), followed by a comparison of means test (Tukey’s HSD, P < 0.05) (Sokal and Rohlf 1995). All analyses were performed with NCSS software (Hintze 2001).

Results

Moisture content (MC) and imbibition rate (IR)

The oldest seed groups (84 months and 7 years) exhibited the lowest MC, while there were no significant differences among the seeds of the other three ages (Table 2).

Table 2 Initial seed moisture content (MC) of Mimosa luisana seeds at four age groups
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Both, S and UNS seeds, imbibe up to 80% of their total absorption capacity after 3 h. It should be noted that the S seeds imbibed up to 0.5 mL (six times its size), while the UNS seeds (control) were only able to absorb 0.2 mL (three times its size). The calculation of the IR revealed a significant interaction between the treatments (F = 10.57, P < 0.001), which indicates that the IR depended on the age of the seed and the scarification treatment (Table 3).

Table 3 Imbibition rate (IR) and germination percentage (GP) of Mimosa luisana seeds at four seed age groups
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Germination experiment

Seed germination was epigeal and phanerocotylar. Our results showed that scarification promoted germination as expected (Table 3). The control-UNS treatment began its germination between the second and the seventh day, and its germination percentage (GP) was low (< 10%); while the germination of all scarified (S) seeds, regardless their age, began between the first and second day after sowing and their GP was significantly high (> 85%, F = 4.47, p < 0.05, Table 4).

Table 4 Result of the two-way ANOVA for analysis of the effect of Mimosa luisana seed age on germination percentage (%), half-maximal germination (G50), saturation rate (SR) and germination rate (GR)
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Factor analysis of the GP showed a significant interaction between treatments (Table 4); however, there was a difference between the S and UNS seeds, as scarification accelerated the germination process (Table 3).

In the S seed treatment, it was observed that the lowest GP was achieved by the 84- and 72-month-old seed groups, while the recently collected and the 12-month-old seeds achieved the highest GP (Table 3). It should be noted that the G50 showed no differences among treatments, indicating that 2 days are required to reach 50% germination (Table 4).

The same behaviour was observed in the SR; analysis also revealed a significant interaction in this variable (Table 4 and Fig. 2a), but there was a clear separation between the unscarified and scarified group. Intra-group analysis produced a similar pattern; the 0-UNS seeds required on average 0.5 days to reach only 5% of final germination; this group showed no significant differences compared to the other age’s categories. As for the S seeds, those that were recently collected required 3 days to reach a final GP of 99%, while seeds of 84, 72 and 12 months needed only 2 days to reach final GP of 86%, 86% and 100%, respectively.

Fig. 2
figure 2

Saturation rate (a) and germination rate (b) of Mimosa luisana seeds for four age groups

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Statistical analysis related to the GR revealed a significant interaction between the treatments (Table 4), regardless of seed age; it was observed that when the seeds were scarified, a higher number of seeds germinate every day. The 12-month-old seeds (12S) were the treatment that showed the highest germination rate (nine seeds per day) (Fig. 2b).

Survival and growth

The seedlings exhibited a low percentage of survival. Seedlings from the oldest seeds tended to live less; seedlings from the 84-month-old group showed a survival of 45% at 10 days but only 10% of seedlings survive 15 days. On the same number of days (10 days), seedlings from 72- and 12-month-old showed a 90% of survival, and both manage to reach 20 days (5% and 10%, respectively) (Table 5). Interestingly, the recently collected seeds (0 month old) showed a survival of 70% at 10 days, and they lived more (22 days) but with a very low percentage of survival (5%) (Table 5).

Table 5 Seedling survival percentage at four scarified seed age groups of Mimosa luisana
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The surviving seedlings, regardless of treatments grew between 0.5 and 1.0 mm per day. The cotyledons were foliar and they persisted after the appearance of the protophyll (Fig. 1c, d). The seedlings developed the protophyll, with four pairs of leaflets (Fig. 1c), 13 days after sowing. Eighteen days after sowing, the seedlings developed the first bipinnate leaf, with two pairs of pinnae, each with three or four pairs of leaflets. Between 20 and 22 days after sowing, the metaphylls emerged with two pairs of pinnae each with four pairs of leaflets (Fig. 1d).

Discussion

Moisture content (MC) and imbibition rate (IR)

The MC is an important parameter for the preservation of seeds viability, so it is the first aspect to consider in their storage (Giamminola et al. 2012). Seeds loose their MC with time. Our seeds from the 84-month-old group showed a low moisture content (4.65% ± 0.34%), while the recently harvest seeds (0 months) had a MC of 6.48% ± 0.06%, which suggests that they are orthodox seeds (Roberts 1973; Giamminola et al. 2012). The MC data together with the germination results indicate that M. luisana seeds remain viable 7 years after collection indicating a large life span as other mimosoids like M. glomerata Forssk.[= Dichrostachys cinerea (L.) Wight & Arn.] (Crocker 1938), M. pudica (Ewart 1908; Moreno-Casasola 1973) and M. foliolosa subsp. pachycarpa (Silveira et al. 2014) and others legumes like Astragalus massiliensis Lain., Cassia multljuga Rich., Cytisus austriacus Linn., Leucaena leucocephala Linn. and Melilotus lutea Gueld (Crocker 1938).

Seed longevity has been attributed to the presence of a hard and impermeable testa. According to Montaño-Arias (2016), M. luisana exhibits an impermeable testa with two layers of sclereid, characteristic that supports the notion of their long life span. Silveira et al. (2014) reported that the seeds of M. foliolosa subsp. pachycarpa have a long life span; however, they mentioned that older and unscarified seeds showed higher germination percentage than younger unscarified seeds. In contrast, in the present study, there were no significant differences in GP between seed age groups of unscarified seeds; which indicate that intact seeds of M. luisana do not deteriorate with time but for scarified seeds GP showed statistical difference between groups 72, 84 and 0, 12.

Nevertheless, MC is inversely correlated with IR since lower moisture gives the seed a greater ability to imbibition, the first step in the initiation of the germination process (Bewley and Black 1994). According to Moreno et al. (2006), imbibition is determined by the permeability of the testa. But in this study, IR was evaluated in S and UNS seeds of different ages groups and it was observed that the IR depended on seed age: the older the seed, the lower the MC and consequently the greater the imbibition. Furthermore, IR depends on the scarification; if the seed is scarified, water enters easily, while without scarification, the testa function as a barrier and it inhibits water entrance.

Germination experiment

Epigeal and phanerocotylar germination has been reported in other Mimosa species such as M. adenantheroides (M. Martens & Galeotti) Benth., M. calcicola B. L. Rob., M. lacerata Rose, M. polyantha Benth., M. purpusii Brandegee and M. texana (A. Gray) Small var. filipes (Britton & Rose) Barneby (Camargo-Ricalde et al. 2004) and also M. luisana (Montaño-Arias et al. 2015). Our study confirms that M. luisana seed germination required scarification, as was observed in other species of the genus by Camargo-Ricalde and Grether (1998), Leal and Biondi (2007), Biondi and Leal (2008), Chauhan and Johnson (2008) and Jayasuriya et al. (2013). When our seeds were scarified, germination began at the first days after sowing, and showed the highest GP, similar to the results reported in M. bimucronata (DC.) O. Kuntze (Ribas et al. 1996), M. aculeaticarpa Ortega var. biuncifera (Benth.) Barneby (Pavón et al. 2011), M. setosa Benth. (Sperandio et al. 2013), M. quitensis Benth. (Achipiz-Fajardo et al. 2014) and M. aculeaticarpa var. aculeaticarpa (Montaño-Arias et al. 2015). As a general conclusion, we found that there is a significant interaction between the age of the seed and the treatment received (scarification), so the GP depends on these two factors. Our results showed that the scarified seeds always germinated quicker than unscarified seeds. Recently collected and scarified seeds took more 3 days to reach their final GP, while the older (12, 72 and 84 months old) scarified seeds, took only 2 days. Similar results were reported for M. adenantheroides, M. calcicola, M. lacerata, M polyantha, M. purpusii and M. texana var. filipes (Camargo-Ricalde et al. 2004), M. luisana (Camargo-Ricalde et al. 2004; Montaño-Arias et al. 2015) and M. foliolosa subsp. pachycarpa (Silveira et al. 2014). In our study, seeds of 84 and 72 months showed decreased in the final-GP, which is consistent with the report for M. pudica (Gómez-Pompa et al. 1976).

Survival and growth

In Mimosa seedlings, these aspects are practically unexplored (Parra 1984; Camargo-Ricalde and Grether 1998; Santiago et al. 2001; Niroula et al. 2009; Silveira et al. 2014). When the seeds of M. luisana were scarified they exhibited a rapid GR; however, the growth of the seedling, under the same germination conditions (25 °C, 14 h light and 10 h darkness) was slow. The seedling survival is very important and it has several ecological implications. According to Sánchez et al. (2005), a rapid GR implies an early emergence of seedlings in the field that could ensure the establishment of the plants. But in the case of M. luisana, their seedlings and adults are consumed by goats, which are considered one of the dispersers of this species (Giordani et al. 2015). If M. luisana seedlings are slow-growing and are eaten by goats, this may explain why very few seedlings or young plants have been observed in the field (pers. obs.). Additionally, it is necessary to consider the low water availability in its natural ambient, characterised by a semiarid climate with low precipitation; which is another limiting factor for the emergence and establishment in natural conditions. It should be borne in mind that in this study, 25 °C was established as the optimal temperature for germination (Montaño-Arias et al. 2015) and plants were maintained at this temperature the whole experiment; however, the seedlings mortality rate was high, suggesting that this temperature may not be optimal for their growth. Soil is known to be an important factor for seedling establishment; according to Pavón et al. (2011), when native soil is used, there is a greater seedling survival. However, this was not the case for M. luisana in our study, although we use a mixture of native soil. Other important factor that could be affecting seedling survival is the failure to form mycorrhizae, this mutualistic association improves uptake of nutrients and water (Camargo-Ricalde et al. 2010b; Peña-Becerril et al. 2016). It has been found that arbuscular mycorrhizal fungi play a key role in the establishment, growth and survival of some Mimosa species in semi-arid environments, providing protection against drought, pathogenic fungi and nematode (de Souza et al. 2016). No information was found about the optimal growing temperature for mimosas, and in our knowledge, this is the first study to evaluate the effect of seed longevity on germination, and growth of M. luisana. The information generated in the present study is an important variable that needs to be taken into consideration, given the predictions of the IPCC, where fluctuations in climatic parameters are expected (Christensen et al. 2007).

Mimosa luisana seedling displayed the characteristics of an adult plant after 22 days. For future research, it is recommended to study the variables affecting seedling establishment (temperature, water, soil and light). An important conclusion is that M. luisana seeds have a long life span, desirable characteristic for restoration of semi-arid zones in Mexico in the context of Global warming.