Original article

Variability of fruits and seedlings of Coccoloba uvifera L. in coastal ecosystems of Cuba

Raúl C. López-Sánchez^{1  *} 

Mijail Bullain-Galardis¹ 

Juan Torres-Rodriguez¹ 

Ernesto Gómez-Padilla¹ 

Raúl Campos-Posada² 

Gloria Campos-Posada² 

Bettina Eichler-Löbermann³ 

Ludovic Pruneau⁴ 

Amadou Ba⁴ 

¹Universidad de Granma, carretera Manzanillo, km 17, Bayamo 85100, Granma, Cuba

²Universidad Autónoma de Coahuila, Barranquilla sin número, Colonia Guadalupe, Monclova, Coahuila, CP. 25730, México

³Departamento de Ciencias de las Plantas, Facultad de Agricultura, Universidad de Rostock. Justus-von Liebig-Weg 6 18059 Rostock, Alemania

⁴Université des Antilles, 97159, Pointe-à-Pitre, Guadalupe, Francia

ABSTRACT

Fruit of the seagrape (Coccoloba uvifera L.), in coastal areas north and south of Cuba, in three provenances were collected: Guardalavaca, Cayo Coco and las Coloradas with the objective of evaluating the morphological and physiological variability in fruits and seedlings of this species. The mass, diameter and length of the fruits and seeds were evaluated. The collected seeds were sown under nursery conditions, six months later morphological characteristics of the seedlings were evaluated: stem height, root length, stem and root dry mass, as well as physiological characteristics: water content in the leaves and the roots, fluorescence of chlorophyll, leaf area and specific leaf area. The correlations between all the evaluated indicators were calculated. The results showed significant variations in the characteristics of the fruits and the growth of the sea grape seedlings from the provenances studied. The fruits of the Guardalavaca provenance showed larger size and the seeds of the Cayo Coco locality were those of greater size and mass. In the case of the seedlings, those of Cayo Coco showed the highest values of biomass accumulation in the aerial part, in the root, and of water accumulation in their tissues. A direct relationship between the parameters of the seeds and morphological and physiological indicators were found. Molecular biology assays are recommended to study if there are genetic differences in the same species, as well as to identify the adaptive mechanisms of the seedlings to the stressful conditions where they develop.

Key words: diversity; growth; morphology; physiology; sea grape

INTRODUCTION

Coccoloba uvifera (L.), commonly known as sea grapes, is a small tree or shrub with ramifications at a low level, which grows up to 15 m in height. It has thick and smooth branches and a robust trunk that reaches up to 70 cm in diameter. It is common in coastal dunes and rocky coasts within its natural distribution in the Caribbean. It is recognized easily by its large, thick and almost circular leaves and bunches in the form of edible grapes. It can be found in Florida, Mexico, the Caribbean and in Central America even in South America ¹ and preferably throughout the coastal coast forming an important part of the coastal vegetation ². Its tolerance to saline soils and sea spray makes it an excellent native species on the coasts as barriers against the wind and for the recovery of coastal dunes ³. The sea grape has perspectives due to the use that can be given to it as wood, firewood, as well as the medicinal properties of its leaves and fruits ^2,4.

In the Cuban flora, 34 species of the Coccoloba genus and this has a high level of endemism (25 species) are reported. It is probably why there is a high presence of this in the islands bordering Cuba ⁵, on the other hand, for conservation purposes of the species and restoration programs; the best option is to reproduce it from seed to promote genetic variability ². Therefore, the domestication of the sea grape is important not only for the protection of coastal ecosystems, but also for use in animal feed, natural medicine and income generation. The domestication of tree species leads to the quantitative characterization of the fruits, their nutritional value, the morphology and the ecophysiology of the native populations ⁶. The use of native tree species is potentially useful in contributing to food security and nutrition ^6,7.

The distribution of variation between different geographical areas is of great interest for the conservation of biological diversity. This is especially important for species of commercial interest, where genetic diversity constitutes a source of genes that is incorporated into breeding programs both in plants and in animals ².

The diversity of morphological characters constitutes the backbone of studies on genetic diversity at any of its levels ³. In the case of cultivated species, this type of information has been widely used to discriminate between materials from different geographical regions and has allowed a more efficient management of germplasm banks in large international centers ⁴. These works have also allowed the establishment of genetic relationships between cultivars and local races of different origins and, in addition, allowed to relate the diversity observed with different geographical regions and other elements of the physical environment ⁸.

Taking into account what the previous reported, this research aims to evaluate the morphological and physiological variability in fruits and seedlings of sea grapes from three coastal ecosystems of Cuba.

MATERIALS AND METHODS

The collection of fresh fruits of C. uvifera were collected in three representative localities of coastal areas of the north and south of Cuba, on the basis of which populations of this species were established during the months of July to September of the year 2017. Playa Las Coloradas, Guardalavaca Beach and Cayo Coco (Table 1) were the localities where the collections were made. In each locality, 20 trees were selected, and in each all of them, the ripe fruits were collected without damage or malformations from different positions and placed in labeled plastic bags.

Table 1 Geographical location, annual precipitation, average annual temperature, pH and electrical conductivity of the localities studied 

Localities (Beaches)	Latitude (N)	Longitude (W)	Annual precipitation (mm)	Average annual temperature (ºC)	pH	Ce (dS m^-1)
Cayo Coco	22°32'10,06"	78°21'19,44"	855	25.6	8.75	7.84
Las Coloradas	19°56'00"	77°41'00"	942	27	8.91	7.62
Guardalavaca	21°07′30″	75°49′44″	974	26.4	8.54	7.31

The fresh fruits were determined the length (mm) and the diameter (mm) and were weighed with an electronic analytical balance (Denver Instrument) to determine its mass. Subsequently, the pulp from the seeds was separated which were washed and dried in the open air for five days in the shade for weighing, and in addition, the length (mm) and the diameter (mm) were determined.

Preparation for the exriments

After drying the fruits, we proceeded to scarify the seeds to eliminate the dormancy by immersing them in 95 % sulfuric acid for three hours and rinsing with distilled water. Seeds were placed to germinate in plastic containers (50x75 cm) with 1 kg of sterile river sand in a SNIJDER growth chamber (day/night temperature 35/25 °C, relative humidity 80 % and photoperiod of 12 light hours). The seeds were placed in containers for six weeks, following a completely randomized arrangement with 40 repetitions (one seed per repetition) per treatment (locality).

Each container once a week was removed and they were irrigated twice with the nutrient solution and once a week with normal water. The nutrient solution that was used was elaborated with the following composition in ppm: 4.28 K, 15.67 Na, 6.36 Ca, 4.99 Mg, 1.26 NH₄, 2.75 NO₃ - (H₂O-extractable), 0.12 Olsen-P. pH (H₂O) 8.41, pH (KCl) 7.2, total salts 0.11 g l^-1 and electrical conductivity 0.036 mScm^{-1 (8}.

At six weeks, chlorophyll fluorescence (Fv / Fm) was measured, measured with a fluorometer (Handy chlorophyll fluorometer). The measurements were taken in the morning (10:00 am). Two readings were made on each of the leaves fully expanded and exposed to light. Afterwards, all the leaves of the seedlings of each container were taken to determine the leaf area (cm²) using a foliar area meter (Area Meter AM 350). The specific leaf area (cm² g^-1) was calculated using the formula:

$Specific leaf area = Foliar area \left(c{m}^2\right)/dry mass \left(g\right)$

After these measurements, the seedlings were harvested and the stems were separated from the roots, to measure the height of the stem (cm) and the length of the roots (cm). The foliar and root samples were placed in the oven (Memmert) for 48 hours at 80 °C to later determine the dry mass of the stems (g) and the root (g) using an analytical balance with a resolution of ± 0.000 g.

The water content in the leaves (%) was determined by equation ⁹:

$Water content in the leaves = 100* (fresh mass \left(g\right)-dry mass \left(g\right)/fresh mass \left(g\right)$

Statistical analysis

A completely randomized design was used and the ANOVA test and the Tukey test were used to evaluate if there were significant differences between seeds and seedlings of the different localities studied. We calculated the correlations between all the indicators evaluated using the Spearman correlation range. All the data was processed with the Statistic for Windows package, version 10 ¹⁰.

RESULTS AND DISCUSSION

Variations in the morphology of fruit

Significant differences were found between the three localities studied in the morphological indicators of the fruits evaluated. The fruits from Guardalavaca showed the highest values of weight, diameter and length of the fruit and weight of the pulp. The seeds of the locality Las Coloradas were the ones that showed the lowest values in the indicators evaluated in the fruits (Table 2).

Tabla 2 Características de los frutos de C. uvifera provenientes de tres localidades costeras de Cuba 

Localities (Beaches)	Mass of the fruit (g)	Diameter of fruit (mm)	Length of the fruit (mm)	Diameter of the seed (mm)	Mass of the seed (g)	Length of the seed (mm)	Mass of the pulp (g)
Cayo Coco	2.47 b	15.40b	17.30 b	10.45 a	0.52 a	17.0 a	1.94 b
Guardalavaca	3.38 a	17.30 a	19.30 a	9.90 b	0.45 b	15.6 b	2.90 a
Las Coloradas	2.30 c	11.80 c	16.30 c	10.20 b	0.49 b	15.9 b	1.80 b
ESx	0.31	0.28	0.71	0.68	0.82	0.22	0.64

Means with equal letters are not statistically different when applying the Tukey Test (p≤0.05). ESx: Standard error of the mean

The fruits from the Cayo Coco locality showed the highest values of diameter, mass and length of the seed, there being no significant differences between the seeds from the localities Guardalavaca and Las Coloradas (Table 2). As you can see, the sea grape fruits from Guardalavaca are larger and contain more pulp, but they have small seeds compared to those of the Cayo Coco locality and do not differ statistically in terms of the characteristics of their seeds with the fruits of the locality Las Coloradas (Table 2).

It is necessary to point out that in the international literature consulted there are no reports about the comparison of this species in different localities, they are limited to describing the characteristics of the species. The values obtained are in the range of those reported for the same species in fruits harvested in Puerto Rico ²; nevertheless, it was observed that the seeds have less mass and less diameter of the fruit, as well as less seed mass. Seed length values of 10 mm are reported, in Hawaii for this species, lower than those collected in this investigation ¹¹.

The climatic data of the studied localities show that the locality of Guardalavaca presents high values of precipitations and lower temperatures, as well as a lower alkalinity of the soils, These results indicate that the phenotypic variability that shows the species could be influenced by the edaphoclimatic conditions that presents the geographical area where it develops.

Variations in the morphology of the seedlings

The stem height and root length indicators indicated significant differences between the three locations studied. Seedlings of fruits from Cayo Coco showed higher values; while those of Las Coloradas were the ones that showed the lowest values in the growth indicators evaluated. (Table 3).

Table 3 Morphological characteristics of C. uvifera seedlings from three locations in Cuba. 

Localities (Beaches )	Height of stem (cm)	Lenthg of the root (cm)	Dry mass of stem (g)	Dry mas of root (g)
Cayo Coco	15.620 a	10.200 a	0.526 a	0.120 a
Guardalavaca	14.920 b	9.800 b	0.510 b	0.080 b
Las Coloradas	13.380 c	8.860 c	0.490 b	0.070 b
ESx	0.150	0.19	0.004	0.002

Means with equal letters are not statistically different when applying the Tukey Test (p≤0.05). ESx: Standard error of the mean

The seedlings of C. uvifera from Cayo Coco showed the highest values of dry mass in the stem and the root. the seedlings of the locality of Guardalavaca did not show significant differences with the seedlings of the locality the Coloradas for the dry mass of the stem and the dry mass of the root (Table 3).

Although there are no reports for this species. other authors point out the genetic variability within the same arboreal species. finding differences in morphological indicators in the species Olea europaea L. from four regions ¹². and in seedlings of Adansonia digitata L. (baobab) with seeds from southern and southeastern Africa ¹³.

The values of the annual precipitation of of Cayo Coco town are lower than other localities; however. the best values in the morphological indicators evaluated are obtained in the seedlings from seeds of this locality. This could suggest the existence of genetic variability within the species. However. several authors hypothesize that several species of broadleaf trees can develop larger compounds in their leaves when they are in stressful conditions ^13-15. which would justify the criterion that the phenotypic variability shown by the species is influenced due to the edaphoclimatic conditions presented by the geographical area where it is developed.

Variations in ecophisiological indicators

The same trend of the morphological indicators where the results of the sea grape seedlings of Cayo Coco were statistically superior in comparison with the rest of the localities when evaluating the physiological indicators except in the indicator of leaf area were presented (Table 4).

Table 4 Physiological characteristics of C. uvifera seedlings from three locations in Cuba 

Localities (beaches)	Water content in leaves (%)	Water content in roots (%)	(Fv/Fm)	Specific foliar area (cm-^2.g^-1)	Leaf area (cm^2)
Cayo Coco	81.12 a	79.22 a	0.84 a	253.22 a	127.15 a
Guardalavaca	79.32 b	75.62 b	0.79 b	252.27 a	125.25 a
Las Coloradas	77.72 c	72.72 c	0.74 b	249.28 b	122.22 a
ESx	1.25	1.47	0.08	2.24	1.41

Means with equal letters are not statistically different when applying the Tukey Test (p≤0.05). ESx: Standard error of the mean

When analyzing the correlations established between the characteristics of the fruits and the rest of the indicators. the significant and negative correlations of the mass and diameter of the fruit stand out with the accumulation of biomass and the water content of the root and with the foliar area. This aspect is important when selecting ecotypes of this species through fruits for possible programs of reforestation of coastal dunes or for the production of phytopharmaceuticals or other natural products from this part of the plant. The mass of the seed correlated statistically significantly and positively with the accumulation of water in the leaves and the specific leaf area.

Regarding the length of the seed. it correlated in a positive and significant way with the height of the stem. this indicator that in turn correlates strongly and positively with the physiological indicators studied. This denotes the relationship between fruits with less mass. but longer seeds present in the drier areas. which in turn produce taller seedlings. and higher production of root biomass that allows them to survive in drought conditions (Table 5).

The information obtained in the correlations studied suggests that sea grape plants that achieve seeds of greater length and mass were able to accumulate more water in their tissues and higher biomass. If we relate this to what were the seedlings from the locality with lower levels of precipitation. it would be faced with an adaptive mechanism to tolerate these stressful conditions and that allow under normal conditions to achieve higher values than those of other localities with less drought problems. This demonstrates the hypothesis that plants from areas with lower rainfall (Table 1) have relatively higher root and water content in their roots compared to those from areas with higher rainfall (Table 4). This could help seedlings from dry environments survive periods of drought stress. which are prone to progressively increase.

Table 5 Correlation coefficients for the morphological and physiological indicators evaluated in the C. uvifera seedlings from three locations. 

	MF	DF	LF	DS	MS	LS	MP	LT	LR	MST	MSR	CAH	CAR	Fv/Fm	AFE	AF
MF	1.00
DF	0.80**	1.00
LF	0.45	0.49	1.00
DS	-0.71*	-0.86**	-0.43	1.00
MS	-0.42	-0.48	-0.26	0.57*	1.00
LS	0.39	0.45	0.15	-0.70*	-0.86**	1.00
MP	0.87**	0.93**	0.45	-0.92**	-0.42	0.53*	1.00
LT	-0.10	-0.29	-0.26	0.05	0.65*	0.59*	-0.14	1.00
LR	-0.22	-0.33	-0.10	0.49	0.67	0.01	-0.52*	0.29	1.00
MST	-0.13	-0.20	-0.16	0.48	0.58*	-0.02	-0.41	0.28	0.92**	1.00
MSR	-0.57*	-0.51*	-0.38	0.15	-0.21	0.45	-0.40	0.77*	0.16	0.04	1.00
CAH	-0.47	-0.37	-0.16	0.21	0.56*	0.38	-0.47	0.52*	0.69*	0.58*	0.66*	1.00
CAR	-0.54*	-0.35	-0.26	0.15	-0.48	0.49	-0.42	0.63*	0.51*	0.39	0.86**	0.90**	1.00
Fv/Fm	-0.42	-0.48	-0.34	0.23	-0.35	0.49	-0.42	0.91**	0.46	0.59*	0.90**	0.71**	0.87**	1.00
AFE	-0.25	-0.31	-0.15	0.29	0.57*	0.39	-0.42	0.64*	0.18	0.78*	0.53*	0.83**	0.79**	0.79**	1.00
AF	-0.59*	-0.36	-0.38	0.21	-0.40	0.36	-0.46	0.69*	0.47	0.69*	0.81**	0.93**	0.99**	0.80**	0.77*	1.00

**Significance to p < 0.01.

* Significance to p < 0.05.

MF: Mass of fruit. DF: Diameter of fruit. LF: Length of fruit. DS: Diameter of seed. MS: Mass of seed. LS: Length of seed. MP: Mass of pulp. LT: Height of stem. LR: Length of the root. MST: Aerial dry mass. MSR: Dry mass of root. CAH: Water content in leaves. CAR: water content of roots.

Fv/Fm: Fluorescence of chlorophyll. AFE: Specific aerial mass. AF: Foliar area

The results demonstrate the phenotypic variability present within the same species when differences are observed in the morphological and physiological indicators evaluated in sea grape seedlings from three locations. These give a measure of the influence of the environment on the morphology of the fruits and that is reflected subsequently in the growth of the seedlings in the same growing conditions.

Other similar investigations. but with other tree species. indicate that in curved Jatropha seedlings. under drought conditions. they produce new leaves using the water stored in the stems ¹⁶. While baobab seedlings can produce leaves. using stored water. in either the hypocotyl or the main root ¹⁷.

Everything seems to indicate that this adaptation also appears in the seedlings of sea grapes. which causes the differentiation in the behavior of the seedlings according to the characteristics of the ecosystem where they are developed. So for future investigations it would be evaluated through molecular biology techniques. if there were genetic differences that allow us to better adjust our hypotheses about these morphological and physiological responses.

CONCLUSIONS

• There is variability in the characteristics of the fruits and the growth of the sea grape seedlings from three different locations. There is a direct relationship between the parameters of the seeds and morphological and physiological indicators. apparently the seeds from the dry place produce more biomass and water accumulation in their tissues.

• The capacity of the seeds from the locality of lower cumulative rainfall values (Playa Cayo Coco) to show the highest values of length and mass and in the seedling phase of accumulating more water in their tissues and higher biomass is explained as an adaptive mechanism to tolerate stressful conditions.

RECOMMENDATIONS

Trials. to study with molecular biology tools if there are genetic differences between the same species. in addition to the adaptive mechanisms of the sea grape seedlings to the stressful conditions where they develop that can cause the phenotypic differences found are recommended.

BIBLIOGRAFÍA

1. Sene S. Analyse de la diversité des symbioses ectomycorhiziennes du Coccoloba uvifera (L.) en zones d'origine et en zones d'introduction [Internet] [These de Doctorat]. [France]: Universite Cheikh Anta Diop De Dakar; 2015. 159 p. Available from: http://isyeb.mnhn.fr/sites/isyeb/files/documents/manuscrit_the_se_de_doctorat-seynabou_sene.pdf

2. Nuñez MA, Dickie IA. Invasive belowground mutualists of woody plants. Biological Invasions. 2014;16(3):645-61. doi:10.1007/s10530-013-0612-y

3. Séne S, Avril R, Chaintreuil C, Geoffroy A, Ndiaye C, Diédhiou AG, et al. Ectomycorrhizal fungal communities of Coccoloba uvifera L. mature trees and seedlings in the neotropical coastal forests of Guadeloupe (Lesser Antilles). Mycorrhiza. 2015;25(7):547-59. doi:10.1007/s00572-015-0633-8

4. Dickie IA, Bufford JL, Cobb RC, Desprez-Loustau M-L, Grelet G, Hulme PE, et al. The emerging science of linked plant-fungal invasions. New Phytologist. 2017;215(4):1314-32. doi:10.1111/nph.14657

5. Moeller HV, Dickie IA, Peltzer DA, Fukami T. Mycorrhizal co-invasion and novel interactions depend on neighborhood context. Ecology. 2015;96(9):2336-47.

6. Noa IC. Coccoloba howardii (Polygonaceae), a new species from Cuba. Willdenowia. 2012;42(1):95-8. doi:10.3372/wi.42.42112

7. Põlme S, Bahram M, Kõljalg U, Tedersoo L. Biogeography and specificity of ectomycorrhizal fungi Coccoloba uvifera. In: Biogeography of Mycorrhizal Symbiosis [Internet]. Suiza: Springer, Cham; 2017 [cited 13/02/2019]. p. 345-59. (Ecological Studies; vol. 230). Available from: https://link.springer.com/chapter/10.1007/978-3-319-56363-3_16

8. Séne S, Selosse M-A, Forget M, Lambourdière J, Cissé K, Diédhiou AG, et al. A pantropically introduced tree is followed by specific ectomycorrhizal symbionts due to pseudo-vertical transmission. The ISME Journal. 2018;12(7):1806-16. doi:10.1038/s41396-018-0088-y

9. Bandou E, Lebailly F, Muller F, Dulormne M, Toribio A, Chabrol J, et al. The ectomycorrhizal fungus Scleroderma bermudense alleviates salt stress in seagrape (Coccoloba uvifera L.) seedlings. Mycorrhiza. 2006;16(8):559-65. doi:10.1007/s00572-006-0073-6

10. STATISTICA Software. StatSoft (data analysis software system) [Internet]. Version 12. Tulsa, USA; 2011 [cited 09/07/2018]. Available from: https://statisticasoftware.wordpress.com/2013/05/15/statsoft-releases-version-12-of-statistica-software/

11. Nageswara Rao RC, Talwar HS, Wright GC. Rapid assessment of specific leaf area and leaf nitrogen in peanut (Arachis hypogaea L.) using a chlorophyll meter. Journal of Agronomy and Crop Science. 2001;186(3):175-82. doi:10.1046/j.1439-037X.2001.00472.x

12. Dickie IA, Bennett BM, Burrows LE, Nuñez MA, Peltzer DA, Porté A, et al. Conflicting values: ecosystem services and invasive tree management. Biological Invasions. 2014;16(3):705-19. doi:10.1007/s10530-013-0609-6

13. Sorkheh K, Khaleghi E. Molecular characterization of genetic variability and structure of olive (Olea europaea L.) germplasm collection analyzed by agromorphological traits and microsatellite markers. Turkish Journal of Agriculture and Forestry. 2016;40(4):583-96.

14. Cuni Sanchez A, De Smedt S, Haq N, Samson R. Variation in baobab seedling morphology and its implications for selecting superior planting material. Scientia Horticulturae. 2011;130(1):109-17. doi:10.1016/j.scienta.2011.06.021

15. Bâ AM, McGuire KL, Diédhiou AG. Ectomycorrhizal symbioses in tropical and neotropical forests. Edición: 1. Boca Raton, USA: CRC Press; 2014. 300 p.

16. Vincenot L, Selosse M-A. Population biology and ecology of ectomycorrhizal fungi. In: Biogeography of Mycorrhizal Symbiosis [Internet]. Suiza: Springer, Cham; 2017 [cited 13/02/2019]. p. 39-59. (Ecological Studies; vol. 230). Available from: https://link.springer.com/chapter/10.1007/978-3-319-56363-3_2

17. Maes WH, Achten WMJ, Reubens B, Raes D, Samson R, Muys B. Plant-water relationships and growth strategies of Jatropha curcas L. seedlings under different levels of drought stress. Journal of Arid Environments. 2009;73(10):877-84. doi:10.1016/j.jaridenv.2009.04.013

Received: July 09, 2018; Accepted: January 30, 2019

^*Autor para correspondencia. [e-mail]