NOTA BOTNICA

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

ating patterns in plant populations have a crucial role in establishing 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 influence on individual reproductive ability, effective
population size, and degree of population subdivision; affecting the levels of
inbreeding, genetic variation, genetic
structure, and speciation (Barrett, 2003,
Charlesworth, 2003). Mating system is
a complex trait that reflects the interaction between floral 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 diversity among its natural populations
and for successful long term conservation efforts (Charlesworth, 2003).
Mangrove swamps are highly
productive ecosystems that provide
numerous environmental services,
including protection from flood and
coastal erosion, habitat for reproduction, and nurseries for a variety of organisms many of which are critical
for fisheries maintenance and biodiversity conservation (Polidoro et al.,
2010). Because of their high productivity, mangrove swamps are a nutrient 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 species is distributed extensively along
tropical and subtropical coasts of the
eastern Pacific and the Atlantic coasts
of the American Continent and West
Africa (Tomlinson, 1986). Avicennia
germinans is a cryptoviviparous species with buoyant propagules, permitting effective long distance dispersal
(Rabinowitz, 1976; Nettel and Dodd,
2007). The perfect flowers of A. germi-

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-GALLEGOS1
1

Universidad de Ciencias y Artes de Chiapas (UNICACH),

Campus del Mar, Chiapas, Mexico
2
University of California, Berkeley, Department of Environmental Science,
Policy and Management, California, United States of America
3
Universidad de Ciencias y Artes de Chiapas (UNICACH),
Facultad de Ciencias Biolgicas, Chiapas, Mexico.
4
El Colegio de la Frontera Sur (ECOSUR), Unidad Tapachula, Chiapas, Mexico
4
Corresponding author: a.nettel.h@gmail.com

nans are bee pollinated; self-fertilization is probably avoided in individual

flowers by the early development of
male organs and late development
of female organs (Tomlinson, 1986).
Studies on floral biology and pollination system in Avicennia marina
(Forssk.) Vierh. the main representative of the genus in East Africa,
Australia, and Asia found indications of partial self-incompatibility
(Clarke and Myerscough, 1991). Furthermore, genetic studies on A. germinans and A. marina suggest that
tropical populations tend to be outcrossed (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 mating 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 Chiapas, 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 constructions, and agricultural activity;

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 coefficient. Standard error in parenthesis.
Location

Code

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

32

0.44 (0.04)

0.43 (0.04)

4.42 (0.33)

-0.04 (0.01)

PAR

115

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 northwest of Chiapas, this location has a drier
regime and mangroves are less developed, reaching just five to eight meters
tall. At each locality, we collected leaves
from around 30 potential parental trees
and leaves and fruiting bodies from five
to nine mother trees; selected trees were
located at least 30 m apart. The number 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 material 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 Thangjam et al. (2003). We PCR amplified
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 cocktail included 1X PCR buffer, 2.0 mm
MgCl2, 0.2 mm of each dNTP, 25 mg/
mL BSA, 250 nm del each forward
and reverse primer (forward primers
were fluorescently labeled), 1U of Taq
Gold Polymerase (Applied Biosystems), and 5 ng of template DNA for
a final 20 L volume. Amplification
and electrophoresis conditions fol-

lowed Nettel et al. (2005), performing 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 estimating expected heterozygosity (HE),
observed heterozygosity (HO), and
allelic richness (Rt) using the FSTAT
ver 2.9.3.2 (Goudet et al., 2002)
software. We also estimated the inbreeding coefficient and tested for deviations from Hardy-Weinberg expectations 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 selfing, of biparental inbreeding, and the effects of
intrapopulation genetic substructure
on population mating patterns. We
estimated the following parameters:
multilocus population outcrossing
rate (tm); inbreeding coefficient of
mother trees (F); minimum estimate
of the apparent selfing due to biparental inbreeding (tm ts); the proportion
of siblings that share the same father
(rp); and the effect of population substructure on outcrossing events [rp (s)rp (m)]. Default values were used for
initial conditions (tm = 0.9, F = 0.1,
and rp = 0.1). Standard errors were
calculated by 100 bootstrap repetitions, re-sampling individuals within
families.
Estimated levels of genetic diversity were intermediate in Avicennia
germinans sampled populations compared to those detected with microsat-

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 examined per population; F, inbreeding coefficient 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.
Code

Families/
propagules n

tm

tm-ts

rp(m)

[rp (s) rp (m)]

CIG

8/159

0.049 (0.09)

0.583 (0.09)

0.043 (0.04)

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)

116

0.180 (0.05) -0.032 (0.09)

ellite markers for A. marina (ArnaudHaond et al. 2006); allelic richness

ranged from 3.17 (CAT) to 4.42 (PAR)
and observed and expected heterozygocity ranged from 0.39 (CAT) to 0.44
(PAR) and 0.41 (CIG) to 0.43 (CAT
and PAR), respectively. Estimates of
inbreeding coefficients were close to
zero, ranging from -0.04 (PAR) to 0.07
(CAT); none of the studied populations presented significant deviations
from Hardy-Weinberg equilibrium.
The estimated outcrossing rate (tm)
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). Estimates for the effect of population substructure on outcrossing events were
negative for all localities, indicating
that outcrossing events were not influenced significantly by genetic substructuring among pollen donors. Our
results confirm 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 population CAT. Our results are consistent
with previous inferences regarding
the mating system of Avicennia; a
mixture of outbreeding and selfing in
tropical populations, where outcrossing events predominate but are mostly at random with very little effect of
population substructure and biparental 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
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 Secretara 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.
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Received: September 10, 2012

Accepted: October 30, 2012

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

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