Introduction

The Yucatán Peninsula (YP), is a region in south-eastern Mexico, covering 450 000 km2 bordered by the Gulf of Mexico and the Caribbean Sea, including the states of Campeche, Quintana Roo and Yucatan. It is considered a biotic province in the Neotropical region showing a high diversity (^{Morrone, 2005}). In the YP, 2300 plant species have been documented across 956 genera and 161 families, of which the family Fabaceae is the best represented, with 78 genera and 225 species (^{Fernández Carnevali et al., 2012}).

Haematoxylum campechianum L. (Fabaceae), commonly known as Palo de Tinte, is a species of tree native to the tropical region of the American continent and distributed across Guatemala, Belize, and the YP in Mexico (^{Niembro, 2002}). In the YP, this species grows on deep clay substrata with poor drainage and a high moisture content, generally known as lowlands (^{Niembro, 2002}). As a significant biocultural resource, H. campechianum is raw source for the extraction of dyes from pre-Columbian times, exponentially increasing during the Spanish colonization at the beginning of the 16th century (^{Dampier, 2004}; ^{Villegas & Torras, 2014}). It is used for bioprospecting anti-inflammatory, antioxidant, and antiseptic active compounds (^{Norton, 1996}; ^{Duke, 2008}), histochemical properties (reviewed by ^{Avwioro, 2011}) as well as anticarcinogens (^{Peng et al., 2014}). Moreover, its trunks are used to produce posts for perimeter fences on rural land, coal and as firewood, while its leaves and shoots are used for fodder and ornamental purposes (^{Plasencia-Vázquez, Villegas, Ferrer-Sánchez, & Zamora-Crescencio, 2017}). The latter due to its colorful flowers, nectar-polleniferous and visited by bees of the species Apis mellifera L. (^{Quezada-Euán, May-Itzá, & González-Acereto, 2001}); thus, H. campechianum is a potential resource in the Mexican meliponiculture to production of honey and wax.

Studies undertaken on H. campechianum have mainly focused on aspects of its natural history (^{Niembro, 2002}; ^{Villegas & Torras, 2014}), ecological restoration (^{Zamora-Cornelio, 2010}), resilience in degraded environments (^{Pérez, 2014}), as well as its distribution (^{Plasencia-Vázquez et al., 2017}; ^{Chablé-Vega et al., 2019}), tree morphometric structure (^{Chablé-Vega et al., 2019}) and ethnobotanical characteristics (^{Contreras, 2010}). However, research seeking knowledge on the germination of the H. campechianum seeds has received little attention (^{Zamora-Cornelio, Ochoa-Gaona, Vargas Simón, Castellanos Albores, & de Jong, 2010}).

The germination of seeds is one of the most crucial stages in plant development (^{Baskin & Baskin, 2014}). Environmental factors, such as temperature, water, light, and pH determine the success of the germination process (^{Baskin & Baskin, 2014}). However, as seeds do not indefinitely conserve their germination capacity, a high viability and germination rate depends, in large part, on storage conditions (^{Baskin & Baskin, 2014}). For example, a high viability rate in dry-stored seeds (45 °C - 50 °C) depends, in large part, on the balance between the storage container moisture and oxygen (^{Ellis & Hong, 2007}). However, this balance varies from species to species and depends on the seeds’ chemical composition and seed type.

Various adaptation strategies are observed in seed morphometry (e.g., size, biomass and/or fresh weight) and play an important role in the germination process and the establishment of seedlings (^{Rojas-Aréchiga, Mandujano, & Golubov, 2013}). Large seeds and/or those with greater fresh weight present a higher level of nutrient reserves in the cotyledons, which tends to increase their viability, germination, survival, and emergence speed, making them more tolerant to adverse conditions than small seeds (^{Khurana & Singh, 2001}; ^{Loza-Cornejo, López-Mata, & Terrazas, 2008}; ^{Wang, Baskin, Cui, & Du, 2009}).

Since many plant species develop under adverse environmental conditions (e.g., water scarcity as well as high temperatures and light intensity; Nobel, 2010), an important aspect to consider in the natural dynamics of the germination process is the differential response observed in seeds’ desiccation tolerance over time (^{Farrant, Pammenter, & Berjak, 1993}; ^{De Oliveira-Gentil, 2001}). Therefore, desiccation tolerance is a crucial functional characteristic of the ecology of plant regeneration (^{Tweddle, Dickie, Baskin, & Baskin, 2003}).

Typically, seeds can be classified as orthodox (^{Roberts, 1973}; ^{Tweddle et al., 2003}; ^{Wyse & Dickie, 2017}), meaning that they are able to tolerate desiccation under low humidity conditions (3% - 7%), thus drastically reducing metabolic activity and, by means of prior rehydration, conserving a high viability rate (^{Roberts, 1973}; ^{Rangel Fajardo et al., 2011}). These seeds can be easily conserved either in situ, via the formation of persistent seed banks in the soil (^{Thompson, 2000}), or ex situ, where they are stored in germplasm banks (^{Rangel Fajardo et al., 2011}).

Seeds can also, in contrast, be classified as recalcitrant when they remain metabolically active and do not tolerate desiccation, rapidly losing their viability below humidity levels of 12% - 31% (^{Roberts, 1973}; ^{Tweddle et al., 2003}; ^{Wyse & Dickie, 2017}). Consequently, these seeds do not form persistent seed banks in the soil, they are not able to be stored in germplasm banks (Tweddle et al., 2003; ^{Rangel Fajardo et al., 2011}). A third seed type can be classified as intermediate, with these seeds capable of tolerating desiccation within a range of 7% - 20% humidity; however, their viability decreases more rapidly when stored at low (0 °C) rather than warm (12 °C - 21 °C) temperatures, over the long term (^{Roberts, 1973}; ^{Pritchard, 2004}).

To date, few studies have focused on evaluating the germination of H. campechianum seeds. ^{Zamora-Cornelio et al. (2010)}, found a high and speed germination (86%) in freshly harvested seeds of this species. These authors suggest a recalcitrant effect in the seeds of this species. Nevertheless, to test this hypothesis, long-term studies focused on assess variations in fresh weight, viability, and germination of the H. campechianum stored seeds are necessary.

Objectives

The present study aimed to analyze the effect of storage time on the fresh weight, viability, and germination of H. campechianum seeds.

Materials and methods

Seed collection

The H. campechianum seeds were harvested in the municipality of Palizada, Campeche, Mexico (Fig. 1), in 2016, 2017, 2018, & 2019, between the months of March to July, the species’ fructification period (^{Centro de Investigación Científica de Yucatán [CICY], 2010}). The climate is tropical monsoon (Am), the average annual temperature at the site is 26.2 °C, with temperatures of over 46 °C in spring (March-May) and below 18 °C in winter (December-February), and a mean precipitation of 1634.5 mm is observed during the rainy season, occurring between June and October (^{Comisión Nacional del Agua [Conagua] 2010}); Servicio Meteorológico Nacional [SMN], 2020).

Figure 1 Location of the natural populations of Haematoxylum campechianum in the study area, in the municipality of Palizada, Campeche, Mexico. 

In the YP, this species forms dense monospecific groups in clay soils subject to periodic flooding that have poor drainage knowns as “tíntales” (^{Niembro, 2002}; ^{Plasencia-Vázquez et al., 2017}). For the four collection cycles, the seeds were obtained from pods of 20 mature individuals (at least 20 pods of three branches per mature individual were collected) pertaining to two adjacent tíntales in the study area (Fig. 1). A polyethylene mesh (1.83 m × 6.10 m) at 50% was placed under the tree canopy to retain the collected pods; subsequently, they were cleaned, and the seeds were extracted manually from pods, stored in muslin bags under 60% - 80% relative humidity conditions and a standard temperature of 25 °C, until the experiments inherent to this research began after the 2019 harvest.

Results

Fresh weight

The statistical results indicate significant fresh weight differences between the treatments (collection year), considering an X2 = 27.20 and p < 0.05. The highest fresh seed weight (4.94 g ± 0.03 g, SE) was obtained in 2016. The 2017 seeds showed low mean fresh weight values than 2016, but also statistically differed between collection years (3.84 g ± 0.01 g). For the 2018 and 2019 seeds, lower in fresh weight values were found (3.63 g ± 0.02 g, and 3.67 g ± 0.06 g, respectively), no statistical differences between both treatments (Fig. 2).

Figure 2 Mean fresh weight of Haematoxylum campechianum seed batches (± standard error) harvested over four years of collection (X2 = 27.20 and p < 0.05). 

Germination: 2018 vs 2019

Significant differences in germination were found for both years. The 2019 seeds germinated in a shorter time and showed a higher germination percentage than the 2018 seeds (66.5% ± 0.76% vs 5% ± 0.21%, respectively) in the first two days of the experiment (Fig. 3). The maximum germination percentage also differed significantly between both years (F(19,360) = 41. 919; p ≤ 0.05), with the 2019 sample reaching 98.5% ± 3.43% in eight days, while the 2018 sample reached maximum germination, 54% ± 2.84%, in 20 days. The residual difference (SCRR 2018= 16.435, SCRR 2019= 0.868, SCRNR= 17.303; F(2,102) = 204; p < 0.01) indicate significant differences in accumulated germination between the seeds collected in 2018 and 2019, with the latter presenting rapid germination, in contrast to the former (Fig. 4).

Figure 3 Comparison of the daily percentage of Haematoxylum campechianum seed germination from 2018 to 2019. 

The difference between models concurs with the comparison undertaken via the Chow test.

Figure 4 Adjusted models for the germination of Haematoxylum campechianum seeds for 2018 and 2019. 

Discussion

The seed viability and germination of H. campechianum were affected by storage time, with our results showing that the seeds collected in 2019 displayed higher viability and germination percentages than those collected in 2018 while viability and germination were null for the 2016 and 2017 samples.

The morphometric attributes of seeds, such as size, fresh weight, and biomass are important to the germination process, as they influence seedling development (^{Rojas-Aréchiga et al., 2013}). In this sense, in some species of the family Fabaceae, seeds with greater biomass present differential responses in germination (^{Huyghe, 1993}; ^{Lobos, Miranda, & Mera,2008}), although they also have reater commercial value (^{Lobos et al., 2008}); however, few seed morphometry studies have been conducted on this plant family.

^{Pérez (2014)}, found differences in several attributes, including mass and germination, in the seeds of wild species of the genus Lupinus (L. campestris, L. exaltatus, L. hintonii, and L. montanus) collected in 2011 and 2012. He documented that these four species presented intraspecific variations in seed weight (L. hintonii =35.51 g ± 0.087 g, L. montanus = 1.8 g ± 0.002 g, L. campestris = 1.6 g ± 0.002 g, and L. exaltatus = 1.33 g ± 0.014 g) and germination (with and without mechanical scarification), which was higher in treated seeds (90%).

Ecologically, all seeds differing in their desiccation tolerance (^{Farrant et al., 1993}; ^{De Oliveria-Gentil, 2001}); thus, seeds can be classified into orthodox, recalcitrant, and intermediate (^{Roberts, 1973}; ^{Pritchard, 2004}). Desiccation tolerance in seeds of species in the Fabaceae family has also been little researched. ^{Giamminola, Morandini, and de Viana, (2012)}, did not find significant differences in the germination of Prosopis nigra Hieron., with a storage time of three months applied prior to scarification. The seeds of this species were germinated 90% and presented a shorter germination time (4.8 days) and a higher moisture content (CH = 10% - 12%) than desiccated seeds (CH = 3% - 5%), which germinated at a level of 80%, results which indicate that the seeds of P. nigra are orthodox. ^{Galíndez et al. (2015)}, identified the presence of physical dormancy and the effect of storage on two subtropical species of Fabaceae, Amburana cearensis (Allemao) A.C. Sm and Myroxylon peruiferum Lf. seeds, using different scarification methods. They found that the A. cearensis seeds stored at -18 °C ± 2 °C for three and 12 months were orthodox, while the M. peruiferum seeds were sensitive to desiccation and storage at -18ºC.

Furthermore, ^{Morandini, Giamminola, and de Viana (2013)}, determined both desiccation tolerance and germination in Prosopis ferox Griseb and Pterogyne nitens Tul., subjecting the seeds to mechanical scarification and storing them for six months at -20 °C and at CH = 10% - 12% and 3% - 5%, respectively. Their results show that máximum germination occurred in seeds with a lower 3% - 5%, and that the effect of storage for six months at -20 °C did not influence germination; therefore, seeds of both species are classified as orthodox, given that the germination level was over 80% when CH was reduced to 3% - 5% and after storage for six months at -20 °C.

The findings found here are contrary to most results documented above. This is due to the fact that the studies described indicates that the majority of the Fabaceae species studied showed different strategies for increasing their perpetuation potential over time, despite being subject to adverse environmental conditions, as is the case with the induction of dormancy. Although the moisture content of the H. campechianum seeds was not evaluated due to poor seed availability over the years of collection, it is assumed that this was high in the collection years, given that H. campechianum trees in the YP are normally distributed in lowland areas (tíntales) characterized by a high-water content in the soil (^{Niembro, 2002}; ^{Plasencia-Vázquez et al., 2017}), and that moisture content reduced as storage time increased; however, this hypothesis should be tested in future research.

The results obtained in the present study indicate that the H. campechianum seeds could be considered as recalcitrant due to the high germination percentage in recently harvested seeds in 2019 and an accelerated ageing process, which was characterized by a low viability and germination rate in seeds stored in previous years. Based only in germination data during one collected year, ^{Zamora-Cornelio et al. (2010}), suggested that the H. campechianum seeds were recalcitrant because both a high and speed germination (86%). Our research is novel and incorporates variations in the fresh weight, biochemical viability tests as well as germination essays under a multi-year stored approach of the H. campechianum seeds and support the assumption proposed by ^{Zamora-Cornelio et al. (2010)}.

In the case of this study, it was found that independent of the year of collection, the seeds presented an inverse relationship between seed fresh weight and both viability and germination. The fresh weight results are contrary to those previously reported for some Fabaceae species (^{Huyghe, 1993}; ^{Lobos et al., 2008}) and show the great variability of morphometric and physiological attributes in these species. Moreover, seeds of this family show a great range of ecophysiological, genetic, and micro-environmental adaptations which enable them to establish themselves in most of the ecosystems worldwide (^{Langer & Hill, 1991}).

A possible explanation for the greater percentage fresh weight found in H. campechianum seeds subject to a longer storage time may be related to the increased number of toxic radicals at a cellular level, such as reactive oxygen species (ROS). They consist in free radicals (O2●-, superoxide anion radical; OH●, hydroxyl radical; HO2●, perhydroxy radical and RO●, alkoxy radicals) and non-radical (molecular) forms (H2O2, hydrogen peroxide and 1O2, singlet oxygen (^{Halliwell, 1987}; ^{Huang, Ullah, Zhou, Yi, & Zhao, 2019}). ROS are produced naturally during cellular metabolism in different parts of the plant cell (^{Huang et al., 2019}). In seeds, during the first stages of germination, the principal sources for the generation of ROS are mitochondria, peroxisomes, and cell membranes. Considering that, in most cases, the first stages of germination occur in darkness, chloroplasts do not contribute to ROS generation (^{Huang et al., 2019}); however, once the seedling is produced, it will begin to form chloroplasts, which are also a source of ROS.

According to the oxidative window model proposed by ^{Bailly, El-Maarouf-Bouteau, and Corbineau (2008)}, an adequate and optimal ROS level is required for germination to occur. Therefore, very high ROS levels, for example in seeds that have aged due to prolonged storage periods or those subject to high temperatures, would cause damage at an embryo level and prevent said seeds from germinating (^{Bailly et al., 2008}). It is possible that the higher percentage of fresh weight in older H. campechianum seeds documented in the present study is related to a potentially higher ROS level, and that this caused structural damage to the embryos and consequently, negatively affected the viability and germination in stored seeds of this species. However, more research is required to support this hypothesis.

Given the current high deforestation rate from anthropogenic activities documented in the YP (^{Rueda, 2010}), the present study seeks to promote the use and sustainable exploitation of H. campechianum within its natural area of distribution and contributing to the creation of effective conservation and restoration strategies as well as biotechnological purposes. Moreover, it could be the key to a paradigm shift about the exploitation of natural resources, not only of this species but also any other timber and non-timber resources in the state of Campeche and the YP. Future research on H. campechianum may be focused on evaluating different seed storage mechanisms considering variations in germination between a same year of collection in order to maximize the propagation both in vitro and ex situ of its natural populations; in addition, include other variables such as length, thickness, colour, number of seeds per pod and/or tree among others. It may also seek to analyze the effect of various environmental factors, such as drought, thermal tolerance thresholds, magnetism, photoblastism, salinity and dynamic of the ROS in different organelles of plant, on germination and seedlings establishment under both laboratory and field conditions. Future studies may also explore the capacity of H. campechianum as a soil restorer, evaluating its potential as a nitrogen fixer in the soil, which is typical of many Fabaceae species. This would require long-term research in the field, evaluating the regeneration cycle and reproductive characteristics of the natural populations of this species, the production and dispersal of seeds, dynamic of the seed bank in the soil as well as the establishment, survival, and performance of seedlings and young individuals.

Conclusions

The viability and germination of H. campechianum seeds were observed to be affected by storage time. The recently harvested seeds presented a higher viability and germination percentage than older seeds. As the findings of the present study indicate an accelerated ageing process in this species, it could be considered recalcitrant. The present research is the first long-term seed ecology study of H. campechianum, presenting an evaluation of the seed germination dynamics and delineating preliminarily the thresholds desiccation tolerance in H. campechianum, a charismatic and key forest species with high sociocultural value to the state of Campeche and the YP, Mexico.