MORPHOLOGY, BIOMETRICS AND GERMINATIVE POTENTIAL OF Mollinedia clavigera Tul

This study aimed to evaluate the morphology and biometry of fruits and seeds, as well as germination potential of Mollinedia clavigera Tul. seeds, of the family Monimiaceae, an endemic species from Brazil. The internal and external structures have been identified for the evaluation of morphology. For biometric characteristics of fruits and seeds, we considered the diameter and length, plus the weight and number of seeds per kilogram. Germination was evaluated under the temperature of 20, 25 and 30oC in two substrates (paper and vermiculite). As a morphological result, we identified common features to the Monimiaceae family, as well as the presence of Curculionidae larvae in the seeds. Biometric data showed mean values of 7.42 mm in length and 6.13 mm in diameter of fruits, 6.91 mm in length and 5.07 mm in diameter of seeds. Results have identified the temperatures of 20 and 25o C are the are the most suitable for germinaon, wich happend 23 days after experiment beginning.


INTRODUCTION
The historical paths that resulted in the development of the science of natural resources and, at the same time, the current resourcefulness of legislative policies to encourage the preservation of species, especially vegetables, with the restoration and preservation of the Legal Reserve and Riparian Forests, according to the requirements of the New Forest Code Federal Law No. 12,651/2012(BRAZIL, 2012, showed that 12 million hectares of forests need to be restored, and the Brazilian government has committed to achieve this goal by 2030 (BRAZIL, 2016). Thus, it is expected an increase in the production of native tree species seedlings, however the productive reality of the tree seed sector in the country is not prepared to meet this demand, with limitations on the production scale, legal regularity of companies, financial incentives (FREIRE et al., 2017), production methodologies and determination of potential species.
These mishaps refer to the need for new alternatives in the production of seedlings of native species in Brazil, ensuring the genetic variability of seedlings inserted in these environments, in addition to the knowledge of the characteristics of the seeds of existing species in order to contribute, according to Haliski et al. (2013), both for its conservation and for its use. Among the attributes necessary for characterization, the morphology and biometrics of the fruit and seed, in addition to the germination potential that can be under different temperature or substrate conditions, are composed of information necessary for the production of seedlings.
In the morphological description of seeds and seedlings, the structures are differentiated, making it possible to obtain information on germination, in addition to an understanding of the functionality of these structures in ecosystems, evidencing the potential for ecological use in conservation, environmental restoration (COSMO et al., 2017;GOMES et al., 2015) and identification of the successional stage, (GOGOSZ et al., 2015) dispersion and regeneration.
Seed and fruit biometrics, in turn, is an information tool that allows the aggregation of these characteristics mentioned above. However, it occurs more intrinsically, with regard to the dynamics of dispersion and establishment, the genetic variability of the species within the population, the relationships between this variability and environmental factors (RICKLI-HORST et al., 2017), and the differentiation of species of the same genus (ZUFFO et al., 2016). FLORESTA, Curitiba, PR, v. 50, n. 2, p. 1363-1372 In the characterization of fruit morphology, the following aspects were evaluated: type, color, dimensions and consistency of the pericarp. The seeds, in turn, were evaluated for external and internal aspects. In the external ones, we considered the color, appearance of the endocarp surface, seed shape, hilum location, micropyle and seed raphe, length and diameter. From the internal structures, the cotyledons and embryonic axis (embryo components) and endosperm were identified.
Some germinated seeds were separated and observed during the development of the radicle until the emission of cotyledons. These were gathered for a photographic record of the seed and from the emission of the radicle until the exposure of the cotyledons.

Biometrics, weight and humidity
To obtain the biometrics of fruits and seeds, the measurements consisted of the length corresponding to the longest measurement, starting from the hilum to the opposite end, and the diameter corresponding to the thickest measure surrounding the seed or fruit.
Fruit biometrics were performed considering the seed and pericarp. In the case of seeds, biometrics were performed considering embryo, endosperm, tegument and endocarp. For this purpose, 50 units were measured for each measurement performed using a digital caliper considering two decimal places.
To estimate the weight of 1,000 seeds and quantity of seeds per kilogram, 16 replicates of 50 seeds and 8 replicates for the fruits were weighed on a precision scale (0.0001 g) (BRAZIL, 2009).
In obtaining the moisture content of seeds and fruits, three replicates of 50 seeds and two replications of 50 fruits were considered. Using the oven-drying method at 105 ±3 °C for 24 hours, the results were expressed as a percentage based on the wet weight of the seeds.

Germination
Before the installation of the germination test, the soaking curve was developed in order to evaluate the water absorption and the possibility of seed dormancy to later define the treatments used in germination. For the soaking curve, four replicates of 25 seeds each were used, arranged in Petri dishes, moistened with distilled water. The Petri dishes were packed in a BOD incubator with a constant temperature of 25 °C and their weights measured until stabilization in the following time intervals: 0, 3, 6, 9, 12, 24, 36, 48, 60 and 72 hours. For the assembly of the germination test, the seeds were disinfected with 5% sodium hypochlorite solution for 5 minutes and then washed with distilled water, according to RAS methodology (BRAZIL, 2009). The seeds were placed on the substrates fine vermiculite (20 g) and blotter paper (two leaves) previously sterilized in an oven. Vermiculite was moistened with 60 ml of distilled water, and the paper until saturation was then distributed in properly identified transparent plastic boxes (gerbox®). In each repetition -gerbox® -40 seeds were inserted.
The tests were conducted in seed germinators regulated for constant temperature regimes of 20 °C, 25 °C and 30 °C, under illumination of fluorescent lamps connected 24 hours a day, inserted in an air-conditioned room at a temperature of 18 ºC, and the number of germinated seeds was evaluated daily, adopting as germination criterion the emission of radicle equal to or greater than 2 mm.

Analysis of the data
For seed and fruit biometrics, the average, the standard deviation, coefficient of variation and amplitude were calculated, in addition to the weight of 1,000 seeds and number of seeds per kilogram.
In obtaining the moisture content (MC), the average percentage for fruits and seeds was calculated by the ratio of wet weight to dry weight. The germination speed index (GSI) was determined according to Maguire (1962); average germination time (AGT) according to Silva and Nakagawa (1995), with the result expressed in days after sowing.
where: P = wet weight of the seed with total weight of the container, w = dry weight of the seed with total weight of the container, t = total weight of the container, nº S = number of germinated seeds, nº G i = number of non-germinated seeds, t i = time in days.
The experiment was conducted in a completely randomized design, with the treatments distributed in a factorial arrangement 3 x 2 (three temperatures and two substrates), with five replications of forty seeds each.
After verifying its normality by Bartlett's test, the percentage and germination speed index data were submitted to variance analysis, and when there was significance, the averages were compared by the Tukey test at 5% probability (α=0.05). There was no need for data transformations, according to the tests of normality and homogeneity of variances.

Morphology
The fruits ( Figure 1) were classified as free multiple indeiscent head, black-nigrescent in the mature phase, with dimensions between 5-7 mm in diameter and 6-9 mm in length. In the seed processing, it was noticed that the endocarp has rigid consistency, being difficult to separate them from the seed episperm, which for this reason was considered part of the external structures of the seed.  Figure 2A) of the seeds, the endocarp presents an opaque surface, of spotted color with a mixture of black and brown colors, dimensions of 4-6 mm in diameter and 5-9 mm length, ovalelliptical shape, with hilum and micropyle in the same arrangement, in addition to the absence of the seed raphe.
Among the internal structures of the seeds ( Figure 2B), it was observed episperm of light brown color, with extremely thin thickness (0.5 mm), endosperm of transparent-white coloration and semicarnosal consistency occupying most of the seed (100%), finding to be reserve endosperm.
The embryonic axis and the cotyledons (to a lesser extent) have yellowish-white coloration, located with axial position.
During seed processing, it was observed the presence of an insect larvae only confirmed in the seed germination process; in this study attributed as a pathogen of the species. The presence of this larva was confirmed in all seeds whose endocarp is marked by a scar that at tact presents roughness (Figure 3).   After analysis, it was noticed that the larvae settle between the episperm and the endocarp and later invade the endosperm. The larvae were identified at the family level and belong to Curculionidae of the order Coleoptera. The larvae present yellowish-white coloration soon after hatching, which occurred mainly when still stored.
Another characteristic observed, both before the installation of the experiments and later, during the course of the tests, was the ease of proliferation of fungi that covered the seeds randomly in all the replications evaluated in the period, making them impossible to germinate. Even the hypochlorite solution did not prevent the development of fungi, which suggests a longer immersion time in the solution.
The seeds started the germination process with the emission of the radicle 23 days after the beginning of the experiment. It was observed that, close to this date, the seed began tobloat, indicating the absorption of water, staying in this state for a period of 3 days until pointing out the radicle. After 4 days, the radicle reached 2 mm, size necessary to consider the effective germination of the seed. The growth of the radicle until the opening of the cotyledons occurred in the period of 8 weeks (Figure 4).

Biometrics
Fruit biometrics showed an average length of 7.42 ±0.50 mm and amplitude of 2.14 mm, the average diameter was 6.13 ±0.32 mm with amplitude of 1.23 mm. The average weight found for repetitions was 17.45 ±0.50 g with an amplitude of 1.54 g. In each kilogram of fruit there are on average 2,865.12 units and the average moisture content was 34.15% (Table 1).
Regarding seed biometrics, an average length of 6.91 ±0.66 mm with amplitude of 3.05 mm was found, and the average diameter was 5.07 ±0.36 mm with an amplitude of 1.77 mm. The average weight found was 9.97 ±0.32 g, with an amplitude of 1.13 g. The weight of 1,000 seeds is 199.49 and the number of seeds per kilogram is approximately 5,013. The seeds presented a moisture content of 51.62% (Table 1).

Germination
Because it is a species lacking bibliographic records regarding its structures, germination potential and dormancy, the soaking test was performed to verify the permeability or impermeability of the endocarp and episperm. The results obtained can be seen in Figure 5, in which the seeds presented higher water absorption in the first nine hours, tending to stabilize later.
The stabilization of the curve started 36 hours after the beginning of the test, which indicates the absence of episperm impermeability of the seed, thus, the germination test was performed without any dormancy breaking methodology. About the average germination percentage results (G%) ( Table 2), there was no significant interaction (P>0.05) between substrate and temperature for any of the variables evaluated, thus, the effects of the factors studied for germination percentage proved to be independent. It is observed that the vermiculite substrate is superior and differs statistically from the paper at all temperatures. The temperatures of 20 and 25 °C show the best results, not differing from each other and being statistically higher than the temperature of 30 °C.
Similarly, among the treatments evaluated, the percentage of non-germinated seeds for the total seeds tested reached 66.83% on paper and 52.17% for vermiculite substrate, considering all temperatures. Possibly the presence of fungi caused the high rate of non-germinated seeds. The percentage of non-germinated seeds at each temperature for each substrate, presented in Table 2, shows lower germination potential for the temperature of 30 ºC in both substrates.
Regarding the germination speed index (GSI) ( Table 2), vermiculite at temperatures of 20 or 25 °C was higher than the paper, with no statistical difference between temperatures that, however, differed from 30 °C, in which no significant differences were also observed between the substrates tested. However, the average germination time (AGT) did not present statistically significant differences in the temperatures evaluated, nor in the substrates tested. Germination started on the 23rd day after the installation of the experiment and it lasted a total of 57 days, being interrupted when there was no germination for 4 consecutive days ( Figure 6).

Morphology
In general, the identified characteristics are similar to the observed ones in other studies on the Monimiaceae family, or the genus Mollinedia (PEIXOTO, 2002;PEIXOTO, 2001;BARROSO et al., 1999). On the external structures observed for fruits and seeds of the species M. clavigera, the hilum and micropyle are arranged at the base of the seed at one end, the hilum being considered ventral, followed by the micropyle represented by a small hole imperceptible to the naked eye (BARROSO et al., 1999). The presence of rigid endocarp added to the episperm (GOGOSZ et al., 2015) possibly favors the germination delay of the seeds of Mollinedia, since the endocarp assumes additional characteristics of the episperm, of seed protection. According to Barroso et al. (1999), seeds do not always present a very distinct testa and episperm, and sometimes at maturity the episperms are confused into one and the structure and consistency of the episperms vary according to the type of fruit that encloses the seed.
Cotyledons are well differentiated, oval in shape, as described by Barroso et al. (1999), and possibly exert haustorial function within the seed, that is, at the beginning of the germination process, they carry out the transport of food reserves, a characteristic of most late secondary species, in which cotyledons remain for a long period trapped inside the seed (GOGOSZ et al., 2015).  As for the occurrence of the larvae, it was observed that it was higher over time, but in a short period, due to the hatching of many larvae in only 24 hours. To hatch, the larvae require moisture, a condition offered by the high moisture of the seed when compared to the fruit in which it favored the hatching of the larvae after the removal of the existing rigid pericarp.
The larvae of the order of the Coleoptera are of the Curculioniform type, of the family Curculionidae, the most numerous of the animal kingdom. Classified as phytophagous, that is, it feeds on plant materials, the characterization of the family demonstrates that most species have endophytic posture (GALLO et al., 2002). This description evidences the possible posture in reproductive tissues when the fruit was still in formation, since no holes or perforations were observed in the ripe fruits both with the naked eye and in a stereoscopic magnifying glass.
By considering these characteristics in the field, we can subsidize the beneficial idea of insertion of this species in forest restoration projects. The fruits can serve as food to the fauna, while, in contact with the soil, will easily degrade or germinate. With the hatching of larvae, in addition to fruits, small insects and larvae serve as food, essentially for the avifauna. Thus, it is inferred about the dispersal syndrome of the species, which is characterized as zoochoric (GOGOSZ et al., 2015;SOUTO, SOUTO;BOEGER, 2011).

Biometrics and moisture content
According to the averages obtained, the observed form of fruits and seeds is similar to the dimensions described by Peixoto (2001) in which the drupes are between 0.9 to 1.2 cm long and 0.6 to 1.0 cm wide. Thus, surely the shape of the fruits and seeds are characterized from oval to elliptical, also confirmed by the average values of diameter and the highest coefficient of variation identified for the length. The greater variation of the values for the length of fruits and seeds evidences the greater distribution of the values with less frequency of the data.
It is evident that the length varies more from one seed to another and that both the fruit and the seeds do not seem to be influenced by the conditions of the environment with regard to weight and diameter, but by the characteristics of the species. The length, however, is possibly influenced by these conditions, which does not prevent or hinder the identification of fruits in the field. Similarly, when comparing the biometrics of fruits and seeds, it is noted that the coefficient of variation is higher for the seeds than for the fruits, due to the fact of morphological protection, from fruit to seed, which evidences the lower influence that the fruits suffer from the environment in relation to the seeds.
As mentioned earlier, the processing showed resistance of fruit removal. This resistance, in turn, protects the seed from the environmental conditions that, by the results, indicate influencing their dimensions. However, the fruit of this species did not exercise protection of the seeds against larvae, since many seeds presented larvae that hatched and derailed germination.
The weight of fruits and seeds did not vary much, ensuring the number of seeds or fruits per kilogram, which expresses approximately twice as many seeds in the absence of fruits.
The moisture content of the seeds obtained was higher than for the fruits. This fact can be explained by the thickness that the pericarp presents, that is, the thickness of the pericarp because it is much lower than the volume of the seed that occupies most of the fruit, resulting in this difference in the percentage of moisture content.
In addition, the higher degree of moisture in the seeds justifies the ease of fungal proliferation that occurred in the germination process, in which further studies should address the best way to conduct germination in addition to paying attention to the storage of seeds of M. clavigera.

Germination
The soaking curve that demonstrated water absorption, evident in the first nine hours, reflects in a episperm characteristic of permeability. Despite these characteristics, the seed does not present rapid germination speed, possibly justified by the presence of the endocarp, in addition to susceptibility to fungi and the presence of larva of the family Curculionidae. The germination speed index (GSI), higher at 20 ºC, and the highest number of seeds germinated in the vermiculite substrate, for 20 and 25 ºC, express the characteristics of the species when inserted in the forest environment. When considering these germination results, concomitantly the characteristics of the parent trees in which the seeds were collected, an ecological behavior of a species belonging to initial and late succession stages is evidenced, a characteristic also described by Gogosz et al. (2015).
The location of the parent trees near the trails, evidences the conditions of temperature, substrate and luminosity necessary for germination and development of the species. According to Peixoto (2001); Avila et al. (2013), the species of the Monimiaceae family adapt to environments with diffuse light and do not present pronounced requirements regarding edaphic conditions, which favors their wide distribution and abundance in the understory. In this sense, M. clavigera can occur quite densely in the regenerative stratum of secondary succession areas.
This fact is still confirmed by observing the germination test of this study, at a temperature of 30 ºC, which presented the lowest number of seeds germinated for both vermiculite and paper, being an unsatisfactory result. In another study to verify the biodiversity of the herbaceous-shrub stratum in capons in the Vila Velha State Park, it was found that the species M. clavigera is associated with the shrubby stratum of more biodiverse fragments, assuming that the plant has great ecological importance due to the significant occurrence in the understory of the Mixed Ombrophilous Forest. Thus, it can be an interesting species in the process of natural regeneration among secondary species, since it develops in gradients with lower temperature variations, preferring small openings (natural gaps) inside the forest (MORO; MILAN; MORO, 2012).