115

NOTA BOTNICA

MATING SYSTEM ANALYSES OF TROPICAL POPULATIONS OF THE BLACK MANGROVE,

AVICENNIA GERMINANS (L.) L. (AVICENNIACEAE)

ALEJANDRO NETTEL-HERNANZ1, 3, 4, RICHARD S.DODD2, MARIED OCHOA-ZAVALA3, CRISTIAN TOVILLA-HERNNDEZ 4

AND JOS REYES DAS-GALLEGOS11Universidad de Ciencias y Artes de Chiapas (UNICACH),

Campus del Mar, Chiapas, Mexico2University of California, Berkeley, Department of Environmental Science,

Policy and Management, California, United States of America3Universidad de Ciencias y Artes de Chiapas (UNICACH),

Facultad de Ciencias Biolgicas, Chiapas, Mexico.4El Colegio de la Frontera Sur (ECOSUR), Unidad Tapachula, Chiapas, Mexico

4Corresponding author: a.nettel.h@gmail.com

Location Code n Ho He Rt FIS

La Cigea CIG 32 0.40 (0.04) 0.41 (0.04) 3.46 (0.38) 0.04 (0.01)

Santa Catalina CAT 30 0.39 (0.02) 0.43 (0.03) 3.17 (0.17) 0.07 (0.02)

El Paraso PAR 32 0.44 (0.04) 0.43 (0.04) 4.42 (0.33) -0.04 (0.01)

Table 1. Genetic diversity and inbreeding estimates of three Avicennia germinans populations from Chiapas, Mexico. n, number of adult trees; Ho, observed heterozygocity; He, expected heterozygocity; Rt, allelic richness; FIS, inbreeding coeffi cient. Standard error in parenthesis.

Botanical Sciences 91 (1): 115-117, 2013

Mating patterns in plant popula-tions have a crucial role in es-tablishing spatio-temporal patterns of genetic diversity within populations as well as in their evolutionary dynamics (Barrett, 2003, Glmin et al., 2006). Mating system and pollen dispersal patterns have a direct infl uence on in-dividual reproductive ability, effective population size, and degree of popula-tion subdivision; affecting the levels of inbreeding, genetic variation, genetic structure, and speciation (Barrett, 2003, Charlesworth, 2003). Mating system is a complex trait that refl ects the interac-tion between fl oral traits, demography, genetics, population structure, and a number of environmental factors that affect pollination (Barrett and Eckert, 1990, Ritland 2002). Understanding a species mating system is crucial for a meaningful analysis of the genetic di-versity among its natural populations and for successful long term conserva-tion efforts (Charlesworth, 2003). Mangrove swamps are highly productive ecosystems that provide numerous environmental services, including protection from fl ood and coastal erosion, habitat for reproduc-tion, and nurseries for a variety of or-ganisms many of which are critical for fi sheries maintenance and biodi-versity conservation (Polidoro et al., 2010). Because of their high produc-tivity, mangrove swamps are a nutri-ent source for other coastal and marine ecosystems such as marine prairies and coral reefs (Duke et al., 2007). The black mangrove, Avicennia germinans (L.) L. (Avicenniaceae), is one of the most important mangrove tree species in the western hemisphere; this spe-cies is distributed extensively along tropical and subtropical coasts of the eastern Pacifi c and the Atlantic coasts of the American Continent and West Africa (Tomlinson, 1986). Avicennia germinans is a cryptoviviparous spe-cies with buoyant propagules, permit-ting effective long distance dispersal (Rabinowitz, 1976; Nettel and Dodd, 2007). The perfect fl owers of A. germi-

nans are bee pollinated; self-fertiliza-tion is probably avoided in individual fl owers by the early development of male organs and late development of female organs (Tomlinson, 1986). Studies on fl oral biology and pol-lination system in Avicennia marina (Forssk.) Vierh. the main represen-tative of the genus in East Africa, Australia, and Asia found indica-tions of partial self-incompatibility (Clarke and Myerscough, 1991). Fur-thermore, genetic studies on A. ger-minans and A. marina suggest that tropical populations tend to be out-crossed (Arnaud-Haond et al., 2006; Giang et al., 2003; Cern-Souza et al., 2005). However, direct evidence of the proportion of outcrossing and

self-pollination are lacking for the genus. Moreover, little is known about the effect of population substructure and inbreeding caused by mating with relatives (biparental inbreeding). The pre-sent study aims to analyze the mat-ing system of A. germinans populations using progeny arrays and microsatellite genetic markers. During Fall 2005, we collected fruiting bodies (one propagule per fruiting body) and leaves from mother trees at three localities along the Chi-apas, Mexico tropical coastline: (1) La Cigea [CIG] (14 37 00 N, 92 19 00 W), close to the border with Guatemala, in an area highly impacted by population expansion, road con-structions, and agricultural activity;

116

Code Families/ F tm tm-ts rp(m) [rp (s) rp (m)] propagules n

CIG 8/159 0.049 (0.09) 0.583 (0.09) 0.043 (0.04) 0.180 (0.05) -0.032 (0.09)

CAT 9/157 -0.200 (0.07) 0.774 (0.09) 0.062 (0.027) 0.231 (0.08) -1.230 (0.09)

PAR 5/74 -0.200 (0.09) 0.770 (0.12) 0.013 (0.038) 0.056 (0.14) -0.037 (0.17)

Table 2. Mating system parameters of three populations of Avicennia germinans from Chiapas, Mexico. Families/propagules n, total number of parental trees and total number of seedlings ex-amined per population; F, inbreeding coeffi cient of mother trees; tm, multilocus outcrossing rate; tm-ts, biparental inbreeding; rp(m), proportion of siblings that share the same father ; [rp (s) rp (m)], effect of population substructure on outcrossing events. Standard errors in parentheses.

Botanical Sciences 91 (1): 115-117, 2013

ALEJANDRO NETTEL-HERNANZ ET AL.

(2) Santa Catalina [CAT] (15 18 00 N, 92 52 00 W), found within the Biosphere Reserve of La Encrucijada, this area presents the best developed mangrove forests in Mexico with trees reaching 30 meters in height; and (3) Paraso [PAR] (15 57 00 N, 93 49 00 W), located in the extreme north-west of Chiapas, this location has a drier regime and mangroves are less devel-oped, reaching just fi ve to eight meters tall. At each locality, we collected leaves from around 30 potential parental trees and leaves and fruiting bodies from fi ve to nine mother trees; selected trees were located at least 30 m apart. The num-ber of fruiting bodies per mother tree analyzed ranged from 14 to 20 at CIG, from 7 to 20 at CAT, and from 13 to 14 at PAR; average number of fruiting bodies analyzed per mother tree was 18, 16, and 14, respectively. Plant ma-terial was dried using silica gel and a herbarium oven. Genomic DNA from leaves was extracted using the CTAB method (Cullings, 1992); genomic DNA from cotyledons was extracted using the method described by Thang-jam et al. (2003). We PCR amplifi ed six microsatellites designed for the black mangrove (Nettel et al., 2005) in three groups: (a) Te1, (b) Di13, Te8, Te9, and (c) Di6t, Te4t. PCR cock-tail included 1X PCR buffer, 2.0 mm MgCl

2, 0.2 mm of each dNTP, 25 mg/

mL BSA, 250 nm del each forward and reverse primer (forward primers were fl uorescently labeled), 1U of Taq Gold Polymerase (Applied Biosys-tems), and 5 ng of template DNA for a fi nal 20 L volume. Amplifi cation and electrophoresis conditions fol-

lowed Nettel et al. (2005), perform-ing an annealing temperature of 58 C for group (a) and 50 C for groups (b) and (c). Results were analyzed with GENESCAN 3.7 and GENOTYPER 3.7 software (Applied Biosystems). Genetic diversity of parental trees was evaluated at each location by esti-mating expected heterozygosity (H

E),

observed heterozygosity (HO), and

allelic richness (Rt) using the FSTAT

ver 2.9.3.2 (Goudet et al., 2002) software. We also estimated the in-breeding coeffi cient and tested for de-viations from Hardy-Weinberg expec-tations with FSTAT. Mating system analyses were performed with MLTR ver 3.2 (Ritland, 2002). MLTR takes advantage of multilocus information to infer levels of true selfi ng, of bipa-rental inbreeding, and the effects of intrapopulation genetic substructure on population mating patterns. We estimated the following parameters: multilocus population outcrossing rate (tm); inbreeding coeffi cient of mother trees (F); minimum estimate of the apparent selfi ng due to biparen-tal inbreeding (t

m t

s); the proportion

of siblings that share the same father (r

p); and the effect of population sub-

structure on outcrossing events [rp (s)-

rp (m)]. Default values were used for

initial conditions (tm = 0.9, F = 0.1, and r

p = 0.1). Standard errors were

calculated by 100 bootstrap repeti-tions, re-sampling individuals within families. Estimated levels of genetic diver-sity were intermediate in Avicennia germinans sampled populations com-pared to those detected with microsat-

ellite markers for A. marina (Arnaud-Haond et al. 2006); allelic richness ranged from 3.17 (CAT) to 4.42 (PAR) and observed and expected heterozy-gocity ranged from 0.39 (CAT) to 0.44 (PAR) and 0.41 (CIG) to 0.43 (CAT and PAR), respectively. Estimates of inbreeding coeffi cients were close to zero, ranging from -0.04 (PAR) to 0.07 (CAT); none of the studied popula-tions presented signifi cant deviations from Hardy-Weinberg equilibrium. The estimated outcrossing rate (t

m)

ranged from 0.583 (Standard Error, SE = 0.09) in CIG to 0.774 (SE = 0.09) in CAT (Table 2). The detected level of biparental inbreeding was very low, ranging from 0.062 (SE = 0.03) in CAT to 0.013 (SE = 0.04) in PAR. The proportion of siblings that share the same father was low but present in CIG and CAT (0.18 [SE = 0.05] and 0.23 [SE = 0.08], respectively). Esti-mates for the effect of population sub-structure on outcrossing events were negative for all localities, indicating that outcrossing events were not in-fl uenced signifi cantly by genetic sub-structuring among pollen donors. Our results confi rm with direct evidence that Avicennia germinans populations are predominantly outcrossing but that they can support moderate levels of self-fertilization; up to almost 50% in the impacted population CIG and about 30% in the preserved popula-tion CAT. Our results are consistent with previous inferences regarding the mating system of Avicennia; a mixture of outbreeding and selfi ng in tropical populations, where outcross-ing events predominate but are most-ly at random with very little effect of population substructure and biparen-tal inbreeding.

Acknowledgments

This work was funded by a grant from the University of California Institute for Mexico and the United States (UC MEXUS). It was also sponsored by the U.S. Department of State under the Fulbright program; the Consejo

117

Received: September 10, 2012Accepted: October 30, 2012

Botanical Sciences 91 (1): 115-117, 2013

MATING SYSTEM ANALYSES OF TROPICAL POPULATIONS OF THE BLACK MANGROVE, AVICENNIA GERMINANS (L.) L. (AVICENNIACEAE)

Nacional de la Ciencia y Tecnologa (CONACYT) and UC MEXUS; and the Programa de Mejoramiento del Profesorado (PROMEP) of the Sec-retara de Educacin Pblica (SEP), Mxico. We thank Wasima Mayer, Luis Roberto Prez Marceln, Juan Carlos de la Presa Prez for technical help. A collection and export permit issued by the Direccin General de Vida Silvestre, Mexico (permit no. SGPA/DGVS/10862) was used for this project.

Literature cited

Arnaud-Haond S., Teixeira S., Massa S.I., Billot C., Saenger P., Coupland G., Du-arte C.M. and Serro E.A. 2006. Genetic structure at range edge: low diversity and high inbreeding in Southeast Asian man-grove (Avicennia marina) populations. Molecular Ecology 15:3515-3525.

Barrett S.C.H. 2003. Mating strategies in fl owering plants: the outcrossing-self-ing paradigm and beyond. Philosophi-cal Transactions of the Royal Society of London, Series B 358:991-1004.

Barrett S.C.H. and Eckert C.G. 1990. Vari-ation and evolution of mating systems in seed plants. In: Kawano S. Ed. Bio-logical Approaches and Evolutionary Trends in Plants, pp. 229-254, Academ-ic Press, London.

Cern-Souza I., Toro-Perea N. and Crde-nas-Henao H. 2005. Population genetic structure of neotropical mangrove spe-cies on the colombian Pacifi c Coast:

Avicennia germinans (Avicenniaceae). Biotropica 37:258-265.

Charlesworth D. 2003. Effects of inbreed-ing on the genetic diversity of popula-tions. Philosophical Transactions of the Royal Society of London, Series B 358:1051-1070.

Clarke P.J. and Myerscough P.J. 1991. Floral biology and reproductive phe-nology of Avicennia marina in South Eastern Australia. Australian Journal of Botany 39:238-239.

Cullings K.W. 1992. Design and testing of a plant-specifi c PCR primer for eco-logical and evolutionary studies. Mo-lecular Ecology 1:233-240.

Duke N.C., Meynecke J.O., Dittmann S., Ellison A.M., Anger K., Berger U., Cannicci S., Diele K., Ewel K.C., Field C.D., Koedam N., Lee S. Y., Marchand C., Nordhaus I. and Dahdouh-Guebas F. 2007. A world without mangroves? Science 317:41-42.

Giang L.H., Nguyen H.P., Sy T.M. and Ko H. Genetic variation of Avicennia marina (Forsk.) Viert. (Avicenniaceae) in Vietnam revealed by microsatellite and AFLP markers. 2003. Genes and Genetic Systems 78:399-407.

Glmin S., Bazin E. and Charlesworth D. 2006. Impact of mating systems on patterns of sequence polymorphism in fl owering plants. Proceedings of the Royal Society B 273:3011-3019.

Goudet J., Perrin N. and Waser P. 2002. Tests for sex-biased dispersal using bi-parentally inherited genetic markers. Molecular Ecology 11:1103-1114.

Nettel A. and Dodd R.S. 2007. Drifting propagules and receding swamps: Ge-netic footprints of mangrove recolo-nization and dispersal along tropical coasts. Evolution 61:958-971.

Nettel A., Rafi i F. and Dodd R.S. 2005. Characterization of microsatellite markers for the mangrove tree Avicen-nia germinans L. (Avicenniaceae). Mo-lecular Ecology Notes 5:103105.

Polidoro A.B., Carpenter K.E., Collins L., Duke N.C., Ellison A.M., Ellison J.C., Farnsworth E.J., Fernando E.S., Kathiresan K., Koedam N.E., Living-stone S.R., Miyagi T., Moore G.E., Ngoc Nam V., Eong Ong J., Primavera J.H., Salmo III S.G., Sanciangco J.C., Sukardjo S., Wang Y. and Hong-Yong J.W. 2010. The loss of species: Man-grove extinction risk and geographic areas of global concern. Plos One 5:e10095.

Ritland K. 2002. Extensions of models for the estimation of mating systems using n independent loci. Heredity 88:221-228.

Tomlinson P.B. 1986. The Botany of Man-groves. Cambridge University Press, Cambridge.

Rabinowitz, D. 1978. Dispersal properties of mangrove propagules. Biotropica 10:4757.

Thangjam, R., Maibam, D. and Sharma, J. 2003. A simple and rapid method for isolation of DNA from Imbibed Em-bryos of Parkia timoriana (DC.) Merr. for PCR analysis. Food, Agriculture and Environment 1: 36-38.

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages false /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Average /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure true /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles true /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice