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Seed ecology and germination treatments in Magnolia dealbata: An endangered species
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Flora 201 (2006) 227–232 www.elsevier.de/flora
Seed ecology and germination treatments in Magnolia dealbata: An endangered species Juan Corral-Aguirrea, La´zaro Rafael Sa´nchez-Vela´squezb, a
Laboratorio de Ecologı´a, Facultad de Biologı´a, Universidad Veracruzana. Circuito Gonzalo Aguirre B., Zona Universitaria. Xalapa, Ver., CP 91000, Mexico b Laboratorio de Biotecnologı´a y Ecologı´a Aplicada, Direccio´n General de Investigaciones, Universidad Veracruzana, Campus para la Cultura, las Artes y el Deporte, Circuito Av. Culturas Veracruzanas, Ap. Postal 250, Xalapa, Ver., CP 91000, Me´xico Received 30 June 2005; accepted 20 July 2005
Abstract Magnolia dealbata is a deciduous tree species in danger of extinction. Seed ecology data and successful germination treatments are important for conservation and species reintroduction programs. This study reports about seed ecology and germination treatments of M. dealbata Zucc. The results showed that the seeds, influence of vertebrates excluded, persisted viable after 1 year, suggesting the presence of a semi-persistent seed pool. There was no significant difference (p40:05) in survival of seeds with or without sarcotesta after a year. Significant differences (po0:05) were found in the number of seeds surviving after a year at different levels of burial. There was no significant interaction upon survival (p40:05) between sarcotesta presence or absence in the seed and burial depth. If vertebrates were not excluded the seeds had a removal rate of 100% 8 months after being exposed. Seed survival curve was of Type II, that is, with constant mortality throughout the time. The most successful germination treatment (po0:05) with 100% germination was soaking of seeds without sarcotesta for 24 h at room temperature (18–20 1C). r 2005 Elsevier GmbH. All rights reserved. Keywords: Endangered species; Magnolia dealbata; Germination treatment; Seed pool; Seed survival
Introduction The genus Magnolia is represented in Mexico by seven species and two subspecies (Va´zquez, 1994), all species being components of the cloud forest (Rzedowski, 1996). Magnolia dealbata Zucc. (Magnoliaceae) is a species endemic to Mexico, classified as endangered according to the International Union for Nature Conservation and the federal laws of Mexico. Habitat Corresponding author.
E-mail address: [email protected] (L.R. Sa´nchez-Vela´squez). 0367-2530/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2005.07.004
destruction and the limited natural distribution of M. dealbata are perhaps the principal causes that make it an endangered species. M. dealbata is used, among others, in traditional medicine by Mexican natives (flowers), for fences and firewood (CATIE, 1998; Gutierrez and Vovides, 1997; Pattison, 1985; Va´zquez, 1994). Several pre-germination treatments (stratified at 0 1C for 10 days, and stratified at 5 1C for 0, 6, 7, 8, 9 and 10 months) with M. dealbata seeds have been tested, with a result of approximately 40% germination (reported in both: Gutierrez and Vovides, 1997; Vovides and Iglesias, 1996). Mass production of M. dealbata seedlings is
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necessary for its reintroduction in impoverished areas of cloud forest (Ramirez-Bamonde et al., 2005). Seed dynamics in situ have not been studied to date. Some species of ants, mammals, birds and reptiles can play an important function removing the seeds (dispersion and predation) (Baskin and Baskin, 2001). Seed dynamics, dispersion, predation, and germination are ecological processes, which affect maintenance of populations and colonization in space and time (Alexander et al., 2001; Baskin and Baskin, 2001; Brown et al., 1975; Harper, 1977; Lorente-Adame and Sa´nchezVela´squez, 1996; Martinez, 2001; McEuen and Curran, 2004; Pizo and Oliveira, 1998). Seed pools may maintain the population growth rate in the long term and then prolong the time until extinction (McCue and Holstford, 1998). Stored seeds may act as buffers of genetic diversity loss in the populations. Therefore, understanding of the seed dynamic is one fundamental prerequisite in species conservation programs. Composition and color of seed sarcotesta are characteristics that can play an important part in seed dispersion and predation. The sarcotesta is a structure that can protect the seed from drying and predation (Camacho, 1994). The objective of this study was to know about basic aspects of ecology and dynamics of M. dealbata seeds and seed preservation. Specifically, the following questions were raised: (1) Can the seeds create a persistent seed pool in situ? (2) Is seed survival different with or without sarcotesta, when the seeds are buried at different soil depths? (3) Does the presence of the sarcotesta influence seed removal rate in situ? (4) What type of survival curve do the seeds present? (5) Is it possible that the reported germination percentage (o50%) could be increased? To answer these questions, three experiments were designed: (a) seed pool analysis, (b) quantification of removal and survival of seeds, and (c) pre-germination treatments.
Materials and methods
molecular phylogenies by ndhF sequence (Kim et al., 2001) and matK sequences (Azuma et al., 2001), and leaves, infrutescence and flower morphology (Va´zquez, 1994), suggest that both, M. dealbata and M. macrophylla, are separate species. M. dealbata is a deciduous tree that grows up to 25 m in height, with large white flowers and leaves; 20 and 60 cm long, respectively. Each adult tree can produce several new sprouts after having been cut, but only one or two main stems will survive. The altitudinal distribution range of M. dealbata is 1200–1500 m. M. dealbata is a species associated with the cloud forest and shares its habitat with Carpinus carolineana Walt, Clethra mexicana DC, Trema micrantha (L.) Blume, Ageratina ligustrina (DC) King & H. Rob., Litsea glaucescens HBK, Liquidambar macrophylla Oersted, Miconia glaberrima (Schlecht.) Naud., Quercus sartorii Liebm., Senecio arborescens Steetz, Ternstroemia sylvatica Schlecht. et Cham., Xylosma flexuosum (HBK) Hemsl., Psychotria pubescens Sw., Wigandia urens (Ruiz & Pav.) HBK and Duranta repens L. among other species (Sa´nchez-Vela´squez and Pineda-Lopez, in press). Seeds of M. dealbata are about 1.470.7 cm (mean7 standard deviation of the mean) long and have a red sarcotesta or exocarp, rich in oils. The seeds are inside compound fruits (polyfollicles), 9073 mm long and 5572 mm wide, which when ripening expose the seeds hanging from the follicle. Seed number per fruit is 4975.
Seed collection Fifty-six fresh ripe polyfollicles were picked from the canopies of the 10 healthy M. dealbata trees in Coyopolan, Veracruz, Mexico. The trees were spaced approximately 100 m from each other. The polyfollicles were kept for 5 days in a ventilated area of the laboratory; later, the seeds were extracted by hand. A viability test with tetrazolium chloride at 1% (Baskin and Baskin, 2001) was carried out in four groups of 25 seeds. Three experiments were designed with the lot of collected seeds.
Study area and species Seed pool This work was carried out in Coyopolan, Ixhuacan de los Reyes, Veracruz, Mexico (191210 N, 971040 W, 1560 m in elevation). The climate of the zone is humid and warm with an annual mean temperature of 18 1C and rainfall throughout the year. The type of the soil is an Andosol (Gobierno del Estado de Veracruz, 1998). M. dealbata is a member of the Rhytidospermum Section and it was described as Magnolia macrophylla Michaux var. dealbata (Johnson, 1989). However, evidence of chloroplast DNA (Qiu et al., 1995),
Six treatments of four replicates of 25 seeds (a total of 600 seeds) were applied; the treatments were: seeds with sarcotesta placed: (a) on the soil surface, (b) buried at 5 cm depth, and (c) buried at 10 cm depth from soil surface. Seeds without sarcotesta were placed the same way. Each replicate was kept in aluminum mesh bags (10 10 cm, with 2 mm light diameter) in order to avoid their getting lost or mixed up with other seeds of the same species. The treatments were placed at four sites randomly chosen
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within a cloud forest, where M. dealbata is dominant (Sa´nchez-Vela´squez and Pineda-Lopez, in press). After a year, the seeds were picked up and their viability was tested with tetrazolium chloride. The amount of surviving seeds was analyzed using log-linear models (Everitt, 1977) of the CATMOD procedure of SAS (1988).
Curves of seed survival and mortality Four treatments with four replicates of 25 seeds each (a total of 400 seeds) were applied; the treatments were: (1) seeds with sarcotesta remaining and vertebrate predators excluded, (2) seeds without sarcotesta and vertebrate predators excluded, (3) seeds with sarcotesta, not excluding vertebrates, and (4) seeds without sarcotesta and without excluding vertebrates. Exclusion of vertebrates was attained by deposition of the seeds in aluminum mesh boxes (25 25 25 cm, 2 mm light diameter). Seeds without exclusion were placed on screen trays (25 25 2 cm, 2 mm light diameter), fixed on the forest floor. Seed removal was registered every month over 1 year; after that year, the remaining seeds were picked up and viability was analyzed with tetrazolium chloride. For the analysis of survival curves of the seeds with and without sarcotesta, and without vertebrate exclusion (every removed seed was considered dead), mortality rates in specific times were estimated and their time relations analyzed (Sa´nchez-Vela´squez, 2001). By definition, the mortality rate is qN=qt ¼ kt N, where kt is the probability that an individual may die at time t. As 1=NqN ¼ q log N, then the first equation can be rewritten as q log N=qt ¼ kt . Now it may be ^ the estimated mortality rate, as the allowed to define k, differential equation k^ ¼ D log N=Dt. Then for any time t, the mortality rate, estimated for a specific time between time t and time t þ x can be calculated as k^ ¼ log N tþx log N t =x, where x is the time interval, for which the mortality rate was estimated. With this last equation, the mortality rates of both seed groups were calculated for each time interval. This method of analysis has the advantage over the standard survival curves of each point being really independent of ^ and t is the previous points. If the relation between k, linear, then k^ ¼ b0 þ b1 t, where b0 is the ordinate at origin and b1 is the steepness of the line that relates cohort mortality with time. If b1 does not significantly differ from zero, cohort mortality of seeds is independent of age (time). In demographic terms, this means that the survival rate is of Type II (constant mortality). If b1 is significantly greater than zero, mortality increases with cohort age (i.e. the survival curve is of Type I). If b1 is significantly less than zero, mortality decreases with the age of the cohort (i.e. the survival
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curve is of Type III). The linear relationship between mortality rate and time was analyzed by simple linear regression. All data were tested for homoscedasticity, independence, and normality of residual error (Neter et al., 1985). The procedure of generalized linear models (GLM) of the SAS statistic package (ver. 6.03; SAS, 1988) was employed, using Type III estimates of the square sum. For the seed groups with vertebrate exclusion, with or without sarcotesta, the mortality rate was calculated (after 1 year, that is, x is equal to 1 year), using the same above-mentioned equation (so, every removed seed was considered dead). A t-Student test was applied, by means of the TTEST with the Cochran and Cox approximation (SAS, 1988), in order to compare both treatments, that is, viability of seeds with sarcotesta vs. seeds without sarcotesta.
Germination treatments One thousand and two hundred seeds with sarcotesta were used, previously stratified in barren humid sand and exposed during 13 days between 4 and 10 1C (Saldan˜a-Acosta et al., 2001). Twelve treatments (with four replicates of 25 seeds each) were applied to these seeds without sarcotesta: at room temperature (18–20 1C), at 40, 60, and 80 1C, during 24, 48, and 72 h each, submerged seeds (using mesh bags, 10 10 cm, with 2 mm light diameter) in 1 l of water in all the cases (using glassware and a ceramic–aluminum hotplate). Once the treatments applied, the seeds were placed randomly in blocks in sterile substratum inside a glasshouse. The frequencies comparisons among treatments (absolute germination value), were made through Kruskal–Wallis test with multiple comparisons of Tukey-DVS (Zar, 1999).
Results The lot of collected seeds used for this study presented 100% viability.
Seed pool In the whole seed pool there was no significant difference between the number of live seeds (after 1 year) with and without sarcotesta (w2 ¼ 0:85, p40:05). Nevertheless, burial depth affected seed survival; the higher survival rate was found in the greater depth (w2 46:21; po0:05, for all cases, Fig. 1). There was no significant interaction between the number of viable seeds with or without sarcotesta and burial depth (w2 ¼ 0:36, p40:05).
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With sarcotesta
c b
25
20 + 12.6
20 10 5
14+ 7
a 4+4
0
0 0
5
100
100
32 + 10.8 29 + 9.9
30
Seed germination (%)
Seed viability (%)
35
15
a
Without sarcotesta
b 85 +5
80
b b 66 + 3.4
76+10.56
72h AMB
24h40 °C
60 40 20
10
Depth (cm)
Fig. 1. Seed pool. Mean percentage and standard deviation of viable seeds of Magnolia dealbata after 1 year placement on or in the soil in Coyopolan, Ver., Mexico. Different letters mean significant differences among burial depth (po0:05). However, between both treatments (with sarcotesta and without sarcotesta), no significant differences were found (p40:05).
0 24h AMB
48h AMB
Treatment
Fig. 2. Mean percentage of germination and standard deviation of Magnolia dealbata seeds under different germination treatments, in Coyopolan, Ver., Mexico. Different letters mean significant differences (po0:05). ‘‘AMB’’ means environment temperature (18–20 1C).
Survival and mortality curves of seeds
Discussion The linear equations between mortality rates of seeds without exclusion of vertebrates and time were MR with sarcotesta ¼ 0:00235 þ 0:00003ðtÞ; and MR without sarcotesta ¼ 0:0017 þ 0:000077ðtÞ, where MR is mortality rate and t is time in days. The slopes of the straight lines are not different from zero (p40:05), which suggests that the survival curves are of Type II, that is, the mortality rate is constant throughout the time. The seeds were found removed entirely after 8 months. The mortality rate between the group of seeds with sarcotesta and seeds without sarcotesta (excluding vertebrates) was not significantly different (t ¼ 0:84; p40:05); in other words, the presence or absence of sarcotesta in seeds did not influence their survival. In the treatment with exclusion of vertebrates, 0.5% of the total of seeds survived.
Germination treatments All the treatments at room temperature (18–20 1C, exposed at 24, 48 and 72 h), and the treatment of 24 h at 40 1C, i.e. these four treatments, were statistically analyzed; the remaining treatments showed 100% mortality. There were significant differences in germination among treatments (H ¼ 10:76; po0:05). The simplest treatment (po0:05), seeds soaked during 24 h at room temperature (18–20 1C), was the most successful (Fig. 2).
The percentage of survival of M. dealbata seeds after a year stratification in the soil increased with burial depth. Such a relationship has also been reported in other studies, for example, in an endangered wild relative of maize, Zea diploperennis (Lorente-Adame and Sa´nchezVela´squez, 1996; Sa´nchez-Vela´squez et al., 2002), and generally in a wide variety of species (sensu Baskin and Baskin, 2001). The Magnoliaceae family has species with embryos that are not very developed at the moment of fruit dehiscence. This suggests morphological or morpho-physiological latency (Saldan˜a-Acosta et al., 2001). However, with this study we prove that the M. dealbata seeds (previously stratified in barren humid sand) can germinate a few days after they were spread. Since the habitat of M. dealbata occupies zones having 10–20 days of frost per year (Zulueta and Soto, 1993), it is possible that the seeds remain subjected to low temperatures for several weeks, germinating later, when the time is propitious. Likewise, low temperatures release slowly the nutrient reserves of the endosperm for the growth of the embryo, allowing its survival during the winter (Camacho, 1994). The natural germination occurs in the rainy period (July–August), almost 9 months after seed dispersal. Seeds proved to be viable after 1 year of being buried (Fig. 1), suggesting that M. dealbata has a persistent seed pool. A seed bank may become established if the processes of seed removal and trampling of cattle in the cloud forest favor seed burial. Likewise, with artificial seed burial viable seeds can be obtained after a year, which – by contrast – has not been achieved under conditions of artificial storage (Vovides, pers. comm.).
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Presence or absence of the sarcotesta in M. dealbata seeds is a factor that does not influence removal rate. It was observed that ants had removed the sarcotesta after the first month of the experiment; such interactions with ants have been observed in other plant species (Brown et al., 1975; Pizo and Oliveira, 1998). This could explain why there are no differences in mortality rate between seeds with and without sarcotesta. In the cold season (January–February, 2002), sclerotestas were found showing signs of attack with a clear pattern of breaking. This suggests that they might have been removed by rodents, which were also reported as important in some other studies (Alexander et al., 2001; Brown et al., 1975; Pizo and Oliveira, 1998). Removal of those seeds, that did not suffer predation, may be considered an opportunity of secondary dispersal for germination in the hot season, allowing for the population to persist in time and space. Due to the high removal rates observed, the results indicate three possible ways for successfully acquiring seedlings: (1) not all the removed seeds are destroyed, (2) buried seeds can escape predators, and (3) mass production of seeds may satiate predators so that some seeds escape predation (Janzen, 1976). However, more detailed studies are necessary to understand the amount each of these processes contributes rendering seedlings. Type II and III survival curves are typical in various plant species (Harper, 1977; Sa´nchez-Vela´squez et al., 2002); however, type II is particularly frequent in clonal species (Harnett and Bazzaz, 1985). Within the framework of this study it was not possible to analyze in detail the fate of the seeds from a tree (dispersal or predation). This should be done by a specific study. Eight of the 12 germination treatments resulted in complete seed mortality. It was expected that the application of hot water at high temperatures and during long lapses of time would increase the permeability of the testa for gases and water, thus stimulating germination (Demel Teketay, 1996; Foroughbakch, 1989). The results of the treatments carried out here contrast with those of other authors, like those of Weaver (1987) who quotes unpublished studies, where no satisfactory germination was obtained for Magnolia splendens in Puerto Rico. Saldan˜a-Acosta et al. (2001) studied in a similar way M. iltisiana, and Vovides and Iglesias (1996) M. dealbata; the greatest success in germination of both studies was less than 50%. Perhaps one the most important aspects to successfully germinate magnolia seeds is that the seeds must be always maintained in a moist environment (Vovides and Iglesias, pers. comm.). This was the case in the present study where the seeds were always maintained in a moist environment. With the results obtained about different aspects of seed ecology of M. dealbata this work contributes to the advance in the search for the production of seedlings
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with the purpose of management and conservation of endangered taxa in Mexico.
Acknowledgements Thanks to the community of Coyopolan for their support during field work. This paper was part of the first author’s thesis of Master’s degree in Management of Forest Resources of the University Veracruzana, and is part of the project ‘‘Demography, Ecology, Management of Magnolia dealbata under Contrasting Conditions: An Endangered Species’’, financed by CONACYT 139240-V. Thanks for revision and valuable comments to Jorge Galindo-Gonza´lez, Maria del Rosario Pineda-Lo´pez and two anonymous reviewers.
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Johnson, D.L., 1989. Nomenclatural changes in Magnolia. Baileya 23, 55–56. Kim, S., Park, C.W., Kim, Y.D., Suh, Y., 2001. Phylogenetic relationships in family Magnoliaceae inferred from NDHF sequences. Am. J. Bot. 88, 717–728. Lorente-Adame, R.G., Sa´nchez-Vela´squez, L.R., 1996. Dina´mica estacional del banco de frutos del Teocintle Zea diploperennis (Gramineae). Biotropica 28, 267–272. Martı´ nez, M.R., 2001. Papel de las hormigas en la remocio´n de semillas de Ficus perforata excretadas por Alouatta palliata mexicana, en un fragmento de selva (Playa Escondida) en Los Tuxtlas, Veracruz. Bachelor Thesis, Universidad Veracruzana, Me´xico. McCue, K., Holstford, P., 1998. Seed bank influences on genetic diversity in the rare annual Clarkia springvillensis (Onagraceae). Am. J. Bot. 85, 30–36. McEuen, A.B., Curran, L.M., 2004. Seed dispersal and recruitment limitation across spatial scales in temperate forest fragments. Ecology 85, 507–518. Neter, J., Wasserman, W., Kutmer, M.H., 1985. Applied Linear Statistical Models, second ed. Richard D. Irwin, Inc., Homewood, IL. Pattison, G., 1985. Magnolia dealbata. Magnolia. J. Magnolia Soc. 21, 17–18. Pizo, M., Oliveira, P., 1998. Interaction between ants and seeds of a nonmyrmecochorous neotropical tree, Cabralea canjerana (Meliaceae), in the Atlantic forest of southeast Brazil. Am. J. Bot. 85, 669–674. Qiu, Y.L., Chase, M.W., Parks, C.R., 1995. A chloroplast DNA phylogenetic study of the eastern Asia–eastern North America disjunct section Rytiodospermum of Magnolia (Magnoliaceae). Am. J. Bot. 82, 1582–1588. Ramı´ rez-Bamonde, E., Sa´nchez-Vela´squez, L.R., AndradeTorres, A., 2005. Seedling survival and growth of three
species of mountain cloud forest in Mexico, under different canopy treatments. New Forests 30, 95–101. Rzedowski, J., 1996. Ana´lisis preliminar de la flora vascular de los bosques meso´filos de montan˜a de Me´xico. Acta Bot. Mex. 35, 25–44. Saldan˜a-Acosta, A., Zuloaga, M.S., Jardel, E., 2001. Germinacio´n de Acer skutchii Rehder y Magnolia iltisiana Va´zquez en la Reserva de la Biosfera Sierra de Manantla´n, Jalisco, Me´xico. Foresta Veracruzana 3, 1–8. Sa´nchez-Vela´squez, L.R., 2001. Efecto de las perturbaciones sobre la demografı´ a de Zea diploperennis. Ph.D. Thesis, Universidad Nacional Auto´noma de Me´xico, Me´xico. Sa´nchez-Vela´squez, L.R., Pineda-Lo´pez, M.R., in press. Associated species, structure and dynamics of two Magnolia dealbata populations: an endangered species. Rev. Biol. Trop. Sa´nchez-Vela´squez, L.R., Ezcurra, E., Martı´ nez-Ramos, M., A`lvarez-Buylla, E., Lorente, A.R., 2002. Population dynamics of Zea diploperennis, an endangered perennial herb: effect of slash and burn practice. J. Ecol. 90, 684–692. SAS, 1988. SAS Stat User’s Guide for Personal Computers, Version 6.08. SAS Institute, Cary, NC. Va´zquez, G.J.A., 1994. Magnolia (Magnoliaceae) in Mexico and Central America: a synopsis. Brittonia 46, 1–23. Vovides, A., Iglesias, C., 1996. Seed germination of Magnolia dealbata Zucc. (Magnoliaceae). An endangered species from Mexico. HortScience 31, 877. Weaver, P.L., 1987. Ecological observations on Magnolia splendens Urban in the Luquillo Mountains of Puerto Rico. Caribbean J. Sci. 23, 340–351. Zar, J.H., 1999. Biostatistical Analysis. Prentice-Hall, London. Zulueta, R., Soto, M., 1993. Bioclimatologı´ a de Flora de Veracruz. Magnoliaceae. Instituto de Ecologı´ a, A.C. University of California, Riverside, CA. No. 6, Me´xico.
Flora 201 (2006) 227–232 www.elsevier.de/flora
Seed ecology and germination treatments in Magnolia dealbata: An endangered species Juan Corral-Aguirrea, La´zaro Rafael Sa´nchez-Vela´squezb, a
Laboratorio de Ecologı´a, Facultad de Biologı´a, Universidad Veracruzana. Circuito Gonzalo Aguirre B., Zona Universitaria. Xalapa, Ver., CP 91000, Mexico b Laboratorio de Biotecnologı´a y Ecologı´a Aplicada, Direccio´n General de Investigaciones, Universidad Veracruzana, Campus para la Cultura, las Artes y el Deporte, Circuito Av. Culturas Veracruzanas, Ap. Postal 250, Xalapa, Ver., CP 91000, Me´xico Received 30 June 2005; accepted 20 July 2005
Abstract Magnolia dealbata is a deciduous tree species in danger of extinction. Seed ecology data and successful germination treatments are important for conservation and species reintroduction programs. This study reports about seed ecology and germination treatments of M. dealbata Zucc. The results showed that the seeds, influence of vertebrates excluded, persisted viable after 1 year, suggesting the presence of a semi-persistent seed pool. There was no significant difference (p40:05) in survival of seeds with or without sarcotesta after a year. Significant differences (po0:05) were found in the number of seeds surviving after a year at different levels of burial. There was no significant interaction upon survival (p40:05) between sarcotesta presence or absence in the seed and burial depth. If vertebrates were not excluded the seeds had a removal rate of 100% 8 months after being exposed. Seed survival curve was of Type II, that is, with constant mortality throughout the time. The most successful germination treatment (po0:05) with 100% germination was soaking of seeds without sarcotesta for 24 h at room temperature (18–20 1C). r 2005 Elsevier GmbH. All rights reserved. Keywords: Endangered species; Magnolia dealbata; Germination treatment; Seed pool; Seed survival
Introduction The genus Magnolia is represented in Mexico by seven species and two subspecies (Va´zquez, 1994), all species being components of the cloud forest (Rzedowski, 1996). Magnolia dealbata Zucc. (Magnoliaceae) is a species endemic to Mexico, classified as endangered according to the International Union for Nature Conservation and the federal laws of Mexico. Habitat Corresponding author.
E-mail address: [email protected] (L.R. Sa´nchez-Vela´squez). 0367-2530/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2005.07.004
destruction and the limited natural distribution of M. dealbata are perhaps the principal causes that make it an endangered species. M. dealbata is used, among others, in traditional medicine by Mexican natives (flowers), for fences and firewood (CATIE, 1998; Gutierrez and Vovides, 1997; Pattison, 1985; Va´zquez, 1994). Several pre-germination treatments (stratified at 0 1C for 10 days, and stratified at 5 1C for 0, 6, 7, 8, 9 and 10 months) with M. dealbata seeds have been tested, with a result of approximately 40% germination (reported in both: Gutierrez and Vovides, 1997; Vovides and Iglesias, 1996). Mass production of M. dealbata seedlings is
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necessary for its reintroduction in impoverished areas of cloud forest (Ramirez-Bamonde et al., 2005). Seed dynamics in situ have not been studied to date. Some species of ants, mammals, birds and reptiles can play an important function removing the seeds (dispersion and predation) (Baskin and Baskin, 2001). Seed dynamics, dispersion, predation, and germination are ecological processes, which affect maintenance of populations and colonization in space and time (Alexander et al., 2001; Baskin and Baskin, 2001; Brown et al., 1975; Harper, 1977; Lorente-Adame and Sa´nchezVela´squez, 1996; Martinez, 2001; McEuen and Curran, 2004; Pizo and Oliveira, 1998). Seed pools may maintain the population growth rate in the long term and then prolong the time until extinction (McCue and Holstford, 1998). Stored seeds may act as buffers of genetic diversity loss in the populations. Therefore, understanding of the seed dynamic is one fundamental prerequisite in species conservation programs. Composition and color of seed sarcotesta are characteristics that can play an important part in seed dispersion and predation. The sarcotesta is a structure that can protect the seed from drying and predation (Camacho, 1994). The objective of this study was to know about basic aspects of ecology and dynamics of M. dealbata seeds and seed preservation. Specifically, the following questions were raised: (1) Can the seeds create a persistent seed pool in situ? (2) Is seed survival different with or without sarcotesta, when the seeds are buried at different soil depths? (3) Does the presence of the sarcotesta influence seed removal rate in situ? (4) What type of survival curve do the seeds present? (5) Is it possible that the reported germination percentage (o50%) could be increased? To answer these questions, three experiments were designed: (a) seed pool analysis, (b) quantification of removal and survival of seeds, and (c) pre-germination treatments.
Materials and methods
molecular phylogenies by ndhF sequence (Kim et al., 2001) and matK sequences (Azuma et al., 2001), and leaves, infrutescence and flower morphology (Va´zquez, 1994), suggest that both, M. dealbata and M. macrophylla, are separate species. M. dealbata is a deciduous tree that grows up to 25 m in height, with large white flowers and leaves; 20 and 60 cm long, respectively. Each adult tree can produce several new sprouts after having been cut, but only one or two main stems will survive. The altitudinal distribution range of M. dealbata is 1200–1500 m. M. dealbata is a species associated with the cloud forest and shares its habitat with Carpinus carolineana Walt, Clethra mexicana DC, Trema micrantha (L.) Blume, Ageratina ligustrina (DC) King & H. Rob., Litsea glaucescens HBK, Liquidambar macrophylla Oersted, Miconia glaberrima (Schlecht.) Naud., Quercus sartorii Liebm., Senecio arborescens Steetz, Ternstroemia sylvatica Schlecht. et Cham., Xylosma flexuosum (HBK) Hemsl., Psychotria pubescens Sw., Wigandia urens (Ruiz & Pav.) HBK and Duranta repens L. among other species (Sa´nchez-Vela´squez and Pineda-Lopez, in press). Seeds of M. dealbata are about 1.470.7 cm (mean7 standard deviation of the mean) long and have a red sarcotesta or exocarp, rich in oils. The seeds are inside compound fruits (polyfollicles), 9073 mm long and 5572 mm wide, which when ripening expose the seeds hanging from the follicle. Seed number per fruit is 4975.
Seed collection Fifty-six fresh ripe polyfollicles were picked from the canopies of the 10 healthy M. dealbata trees in Coyopolan, Veracruz, Mexico. The trees were spaced approximately 100 m from each other. The polyfollicles were kept for 5 days in a ventilated area of the laboratory; later, the seeds were extracted by hand. A viability test with tetrazolium chloride at 1% (Baskin and Baskin, 2001) was carried out in four groups of 25 seeds. Three experiments were designed with the lot of collected seeds.
Study area and species Seed pool This work was carried out in Coyopolan, Ixhuacan de los Reyes, Veracruz, Mexico (191210 N, 971040 W, 1560 m in elevation). The climate of the zone is humid and warm with an annual mean temperature of 18 1C and rainfall throughout the year. The type of the soil is an Andosol (Gobierno del Estado de Veracruz, 1998). M. dealbata is a member of the Rhytidospermum Section and it was described as Magnolia macrophylla Michaux var. dealbata (Johnson, 1989). However, evidence of chloroplast DNA (Qiu et al., 1995),
Six treatments of four replicates of 25 seeds (a total of 600 seeds) were applied; the treatments were: seeds with sarcotesta placed: (a) on the soil surface, (b) buried at 5 cm depth, and (c) buried at 10 cm depth from soil surface. Seeds without sarcotesta were placed the same way. Each replicate was kept in aluminum mesh bags (10 10 cm, with 2 mm light diameter) in order to avoid their getting lost or mixed up with other seeds of the same species. The treatments were placed at four sites randomly chosen
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within a cloud forest, where M. dealbata is dominant (Sa´nchez-Vela´squez and Pineda-Lopez, in press). After a year, the seeds were picked up and their viability was tested with tetrazolium chloride. The amount of surviving seeds was analyzed using log-linear models (Everitt, 1977) of the CATMOD procedure of SAS (1988).
Curves of seed survival and mortality Four treatments with four replicates of 25 seeds each (a total of 400 seeds) were applied; the treatments were: (1) seeds with sarcotesta remaining and vertebrate predators excluded, (2) seeds without sarcotesta and vertebrate predators excluded, (3) seeds with sarcotesta, not excluding vertebrates, and (4) seeds without sarcotesta and without excluding vertebrates. Exclusion of vertebrates was attained by deposition of the seeds in aluminum mesh boxes (25 25 25 cm, 2 mm light diameter). Seeds without exclusion were placed on screen trays (25 25 2 cm, 2 mm light diameter), fixed on the forest floor. Seed removal was registered every month over 1 year; after that year, the remaining seeds were picked up and viability was analyzed with tetrazolium chloride. For the analysis of survival curves of the seeds with and without sarcotesta, and without vertebrate exclusion (every removed seed was considered dead), mortality rates in specific times were estimated and their time relations analyzed (Sa´nchez-Vela´squez, 2001). By definition, the mortality rate is qN=qt ¼ kt N, where kt is the probability that an individual may die at time t. As 1=NqN ¼ q log N, then the first equation can be rewritten as q log N=qt ¼ kt . Now it may be ^ the estimated mortality rate, as the allowed to define k, differential equation k^ ¼ D log N=Dt. Then for any time t, the mortality rate, estimated for a specific time between time t and time t þ x can be calculated as k^ ¼ log N tþx log N t =x, where x is the time interval, for which the mortality rate was estimated. With this last equation, the mortality rates of both seed groups were calculated for each time interval. This method of analysis has the advantage over the standard survival curves of each point being really independent of ^ and t is the previous points. If the relation between k, linear, then k^ ¼ b0 þ b1 t, where b0 is the ordinate at origin and b1 is the steepness of the line that relates cohort mortality with time. If b1 does not significantly differ from zero, cohort mortality of seeds is independent of age (time). In demographic terms, this means that the survival rate is of Type II (constant mortality). If b1 is significantly greater than zero, mortality increases with cohort age (i.e. the survival curve is of Type I). If b1 is significantly less than zero, mortality decreases with the age of the cohort (i.e. the survival
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curve is of Type III). The linear relationship between mortality rate and time was analyzed by simple linear regression. All data were tested for homoscedasticity, independence, and normality of residual error (Neter et al., 1985). The procedure of generalized linear models (GLM) of the SAS statistic package (ver. 6.03; SAS, 1988) was employed, using Type III estimates of the square sum. For the seed groups with vertebrate exclusion, with or without sarcotesta, the mortality rate was calculated (after 1 year, that is, x is equal to 1 year), using the same above-mentioned equation (so, every removed seed was considered dead). A t-Student test was applied, by means of the TTEST with the Cochran and Cox approximation (SAS, 1988), in order to compare both treatments, that is, viability of seeds with sarcotesta vs. seeds without sarcotesta.
Germination treatments One thousand and two hundred seeds with sarcotesta were used, previously stratified in barren humid sand and exposed during 13 days between 4 and 10 1C (Saldan˜a-Acosta et al., 2001). Twelve treatments (with four replicates of 25 seeds each) were applied to these seeds without sarcotesta: at room temperature (18–20 1C), at 40, 60, and 80 1C, during 24, 48, and 72 h each, submerged seeds (using mesh bags, 10 10 cm, with 2 mm light diameter) in 1 l of water in all the cases (using glassware and a ceramic–aluminum hotplate). Once the treatments applied, the seeds were placed randomly in blocks in sterile substratum inside a glasshouse. The frequencies comparisons among treatments (absolute germination value), were made through Kruskal–Wallis test with multiple comparisons of Tukey-DVS (Zar, 1999).
Results The lot of collected seeds used for this study presented 100% viability.
Seed pool In the whole seed pool there was no significant difference between the number of live seeds (after 1 year) with and without sarcotesta (w2 ¼ 0:85, p40:05). Nevertheless, burial depth affected seed survival; the higher survival rate was found in the greater depth (w2 46:21; po0:05, for all cases, Fig. 1). There was no significant interaction between the number of viable seeds with or without sarcotesta and burial depth (w2 ¼ 0:36, p40:05).
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With sarcotesta
c b
25
20 + 12.6
20 10 5
14+ 7
a 4+4
0
0 0
5
100
100
32 + 10.8 29 + 9.9
30
Seed germination (%)
Seed viability (%)
35
15
a
Without sarcotesta
b 85 +5
80
b b 66 + 3.4
76+10.56
72h AMB
24h40 °C
60 40 20
10
Depth (cm)
Fig. 1. Seed pool. Mean percentage and standard deviation of viable seeds of Magnolia dealbata after 1 year placement on or in the soil in Coyopolan, Ver., Mexico. Different letters mean significant differences among burial depth (po0:05). However, between both treatments (with sarcotesta and without sarcotesta), no significant differences were found (p40:05).
0 24h AMB
48h AMB
Treatment
Fig. 2. Mean percentage of germination and standard deviation of Magnolia dealbata seeds under different germination treatments, in Coyopolan, Ver., Mexico. Different letters mean significant differences (po0:05). ‘‘AMB’’ means environment temperature (18–20 1C).
Survival and mortality curves of seeds
Discussion The linear equations between mortality rates of seeds without exclusion of vertebrates and time were MR with sarcotesta ¼ 0:00235 þ 0:00003ðtÞ; and MR without sarcotesta ¼ 0:0017 þ 0:000077ðtÞ, where MR is mortality rate and t is time in days. The slopes of the straight lines are not different from zero (p40:05), which suggests that the survival curves are of Type II, that is, the mortality rate is constant throughout the time. The seeds were found removed entirely after 8 months. The mortality rate between the group of seeds with sarcotesta and seeds without sarcotesta (excluding vertebrates) was not significantly different (t ¼ 0:84; p40:05); in other words, the presence or absence of sarcotesta in seeds did not influence their survival. In the treatment with exclusion of vertebrates, 0.5% of the total of seeds survived.
Germination treatments All the treatments at room temperature (18–20 1C, exposed at 24, 48 and 72 h), and the treatment of 24 h at 40 1C, i.e. these four treatments, were statistically analyzed; the remaining treatments showed 100% mortality. There were significant differences in germination among treatments (H ¼ 10:76; po0:05). The simplest treatment (po0:05), seeds soaked during 24 h at room temperature (18–20 1C), was the most successful (Fig. 2).
The percentage of survival of M. dealbata seeds after a year stratification in the soil increased with burial depth. Such a relationship has also been reported in other studies, for example, in an endangered wild relative of maize, Zea diploperennis (Lorente-Adame and Sa´nchezVela´squez, 1996; Sa´nchez-Vela´squez et al., 2002), and generally in a wide variety of species (sensu Baskin and Baskin, 2001). The Magnoliaceae family has species with embryos that are not very developed at the moment of fruit dehiscence. This suggests morphological or morpho-physiological latency (Saldan˜a-Acosta et al., 2001). However, with this study we prove that the M. dealbata seeds (previously stratified in barren humid sand) can germinate a few days after they were spread. Since the habitat of M. dealbata occupies zones having 10–20 days of frost per year (Zulueta and Soto, 1993), it is possible that the seeds remain subjected to low temperatures for several weeks, germinating later, when the time is propitious. Likewise, low temperatures release slowly the nutrient reserves of the endosperm for the growth of the embryo, allowing its survival during the winter (Camacho, 1994). The natural germination occurs in the rainy period (July–August), almost 9 months after seed dispersal. Seeds proved to be viable after 1 year of being buried (Fig. 1), suggesting that M. dealbata has a persistent seed pool. A seed bank may become established if the processes of seed removal and trampling of cattle in the cloud forest favor seed burial. Likewise, with artificial seed burial viable seeds can be obtained after a year, which – by contrast – has not been achieved under conditions of artificial storage (Vovides, pers. comm.).
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Presence or absence of the sarcotesta in M. dealbata seeds is a factor that does not influence removal rate. It was observed that ants had removed the sarcotesta after the first month of the experiment; such interactions with ants have been observed in other plant species (Brown et al., 1975; Pizo and Oliveira, 1998). This could explain why there are no differences in mortality rate between seeds with and without sarcotesta. In the cold season (January–February, 2002), sclerotestas were found showing signs of attack with a clear pattern of breaking. This suggests that they might have been removed by rodents, which were also reported as important in some other studies (Alexander et al., 2001; Brown et al., 1975; Pizo and Oliveira, 1998). Removal of those seeds, that did not suffer predation, may be considered an opportunity of secondary dispersal for germination in the hot season, allowing for the population to persist in time and space. Due to the high removal rates observed, the results indicate three possible ways for successfully acquiring seedlings: (1) not all the removed seeds are destroyed, (2) buried seeds can escape predators, and (3) mass production of seeds may satiate predators so that some seeds escape predation (Janzen, 1976). However, more detailed studies are necessary to understand the amount each of these processes contributes rendering seedlings. Type II and III survival curves are typical in various plant species (Harper, 1977; Sa´nchez-Vela´squez et al., 2002); however, type II is particularly frequent in clonal species (Harnett and Bazzaz, 1985). Within the framework of this study it was not possible to analyze in detail the fate of the seeds from a tree (dispersal or predation). This should be done by a specific study. Eight of the 12 germination treatments resulted in complete seed mortality. It was expected that the application of hot water at high temperatures and during long lapses of time would increase the permeability of the testa for gases and water, thus stimulating germination (Demel Teketay, 1996; Foroughbakch, 1989). The results of the treatments carried out here contrast with those of other authors, like those of Weaver (1987) who quotes unpublished studies, where no satisfactory germination was obtained for Magnolia splendens in Puerto Rico. Saldan˜a-Acosta et al. (2001) studied in a similar way M. iltisiana, and Vovides and Iglesias (1996) M. dealbata; the greatest success in germination of both studies was less than 50%. Perhaps one the most important aspects to successfully germinate magnolia seeds is that the seeds must be always maintained in a moist environment (Vovides and Iglesias, pers. comm.). This was the case in the present study where the seeds were always maintained in a moist environment. With the results obtained about different aspects of seed ecology of M. dealbata this work contributes to the advance in the search for the production of seedlings
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with the purpose of management and conservation of endangered taxa in Mexico.
Acknowledgements Thanks to the community of Coyopolan for their support during field work. This paper was part of the first author’s thesis of Master’s degree in Management of Forest Resources of the University Veracruzana, and is part of the project ‘‘Demography, Ecology, Management of Magnolia dealbata under Contrasting Conditions: An Endangered Species’’, financed by CONACYT 139240-V. Thanks for revision and valuable comments to Jorge Galindo-Gonza´lez, Maria del Rosario Pineda-Lo´pez and two anonymous reviewers.
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