全 文 ：Journal of Systematics and Evolution 46 (3): 391–395 (2008) doi: 10.3724/SP.J.1002.2008.08023
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com

Molecular evidence for natural hybridization between
Sonneratia alba and S. griffithii
1Suo QIU 1Ren-Chao ZHOU 2Yun-Qin LI 3Sonjai HAVANOND
3Chanop JAENGJAI 1Su-Hua SHI*
1(State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-Sen University,
Guangzhou 510275, China)
2(Experimental Center of Fundamental Teaching, Zhuhai Campus, Sun Yat-Sen University, Zhuhai 519802, China)
3(Department of Marine and Coastal Resources, Phayathai, Bangkok 10400, Thailand)
Abstract Interspecific hybridization has been frequently observed in the mangrove genus Sonneratia. However,
no natural hybridization has been reported between Sonneratia alba and S. griffithii to date, despite their overlap-
ping distribution in the coast of Andaman Sea. In this study, cysteine proteinase inhibitor gene (cpi) from the
nuclear genome, and two intergenic spacers (trnL-trnF and trnV-trnM) from the chloroplast genome, were se-
quenced to determine whether natural hybridization took place between the two species. Our results revealed two
distinct types of cpi sequences from the putative hybrid matching those acquired from S. griffithii and S. alba,
respectively. Sequencing of the chloroplast trnL-trnF and trnV-trnM regions showed that S. alba differed from S.
griffithii by one nucleotide in each region, and the putative hybrid had the identical sequences with S. griffithii.
Molecular data demonstrated clearly that there indeed existed natural hybridization between S. alba and S. grif-
fithii, and that S. griffithii was the maternal parent in this hybridization event.
Key words chloroplast DNA, mangroves, natural hybridization, nuclear gene, Sonneratia.
Natural hybridization is ubiquitous in flowering
plants and plays a significant role in plant evolution
and diversification (Arnold, 1997). Among man-
groves, interspecific hybrids have been reported in
four genera, namely, Rhizophora L. (Duke & Bunt,
1979; Parani et al., 1997; Lo, 2003), Bruguiera La-
marck (Ge, 2001), Lumnitzera Willd. (Tomlinson et
al., 1978; Tomlinson, 1986), and Sonneratia L. f.
(Duke, 1984, 1994; Tomlinson, 1986; Duke & Jackes,
1987; Wang et al., 1999; Zhou et al., 2005). Sonnera-
tia (Lythraceae sensu lato), a typical mangrove genus
comprising about six species (Duke & Jackes, 1987;
Tomlinson, 1986), is widely distributed from eastern
Africa through Indo-Malaya to northeastern Australia
and some islands in the west Pacific Ocean. There are
frequent reports of natural hybridization between
species of Sonneratia across the Indo-West Pacific
region. Natural hybridization in Sonneratia was first
reported by Muller and Hou-Liu (1966) in northwest-
ern Borneo, where two hybrids, S. alba×S. ovata and
S. alba×S. caseolaris, were postulated based on a
study of morphology and cytology. However, they
were not formally named. Later, the taxon S. alba×S.
caseolaris was observed in northeastern Australia as
having widespread distribution and exhibiting consis-
tent morphological characteristics, and it was named
S. ×gulngai N. C. Duke (Duke, 1984). S. ×gulngai has
also been found in China and Sri Lanka (Ko, 1993;
Jayatissa et al., 2001). The other hybrid taxon, S.
alba×S. ovata, was also found in Hainan, China, and
named as S. ×hainanensis W. C. Ko, E. Y. Chen & W.
Y. Chen (Ko, 1985; Wang et al., 1999). A third hy-
brid, S. ×urama N. C. Duke, was described in north-
eastern Australia and southern New Guinea (Duke,
1994). It was proposed as a distinct hybrid entity
originating from S. alba and S. lanceolata. Hybrid
origins of S. ×gulngai and S. ×hainanensis have been
documented by molecular data (Zhou et al., 2005).
During our field survey in the western coast of
Thailand, we found that S. alba J. Smith and S. grif-
fithii Kurz coexisted in Ranong Mangrove Forest
Center, Ranong. In Sonneratia, S. alba is the most
widespread species, occurring in almost the whole
range of Sonneratia, whereas S. griffithii is restricted
to the coast of Andaman Sea, from the upper Malay
Peninsula to Bengal (Tomlinson, 1986). Both species
grow in low intertidal zones of downstream estuaries
(Duke et al., 1998). However, we found that S. grif-
fithii was slightly upward in the habitat relative to S.
alba in our field survey. Habitat differentiation be-
tween the two species may reflect their different levels

———————————
Received: 20 February 2008 Accepted: 17 March 2008
* Author for correspondence. E-mail: lssssh@mail.sysu.edu.cn;
Tel.: 86-20-84113677; Fax: 86-20-34022356
Journal of Systematics and Evolution Vol. 46 No. 3 2008

392
of salt tolerance, as observed in other species of
Sonneratia (Duke et al., 1998; Zhou et al., 2007).
Sonneratia alba has white petals while S. griffithii
lacks any petal. The leaves of S. alba are oblong to
obovate whereas those of S. griffithii are ovate to
almost orbicular. A few morphologically intermediate
individuals occur in the overlapping areas of S. alba
and S. griffithii. These individuals usually have de-
generated white petals and broadly ovate leaves. We
consider that these individuals are probably hybrids
between the two species. Although many hybrids have
intermediate morphological features between their
parents (Rieseberg & Ellstrand, 1993), morphological
intermediacy is not invariably associated with hybrids
(Morrell & Rieseberg, 1998; Wolfe et al., 1998a, b;
Park et al., 2003). Therefore, the status of the putative
hybrid requires further investigation.
Single or low-copy nuclear genes have been suc-
cessfully used in identifying hybrids in plants (Sang &
Zhang, 1999; Ferguson & Sang, 2001; Gaskin &
Schaal, 2002; Pan et al., 2007). In addition, chloro-
plast DNA, usually maternally transmitted in angio-
sperms (Morgensen, 1996), can be used to determine
the maternal parent of hybrids (e.g., Ferris et al., 1997;
Moody & Les, 2002). In the present study, a nuclear
cysteine proteinase inhibitor (cpi) gene from S. alba,
S. griffithii and the putative hybrid was sequenced to
determine the hybrid status of the morphologically
intermediate taxon. Once its hybrid status was con-
firmed, we sequenced two intergenic spacers of
chloroplast DNA (trnL-trnF and trnV-trnM) from the
three taxa to determine the direction of hybridization.
1 Material and Methods
1.1 Plant materials
We collected Sonneratia alba, S. griffithii and the
putative hybrid in Ranong Mangrove Forest Center,
Ranong, Thailand (N 09°52′33″, E 98°35′87″). Leaves
from two individuals of each taxon were collected in
plastic bags with silica gels for DNA extraction. Table
1 listed the details of the samples collected. Voucher
specimens were deposited in the Herbaria of Sun
Yat-Sen University (SYS).
1.2 DNA extraction
Total cellular DNAs were extracted from dried
leaf tissues using the CTAB method according to
Doyle and Doyle (1987).
1.3 Sequencing of cysteine proteinase inhibitor
gene (cpi)
Cysteine proteinase inhibitor genes (cpi) consti-
tute a small multi-gene family in plants and are in-
volved in plant defense against insects (Lim et al.,
1996). There are three one-intron members within this
family in both rice and arabidopsis (Martinez et al.,
2005). Cysteine proteinase inhibitor gene (cpi) was
amplified using primers cpi-F (5′ AACAGCCTCG-
AGATCGAAG 3′) and cpi-R (5′ GAACTCCTGCA-
ACTCCTTG 3′), which were designed according to
the sequence of cpi from a cDNA library of Sonnera-
tia caseolaris (Zhou et al., 2007). PCR was conducted
with the following conditions: 94 ℃ (4 min); 30
cycles of 94 ℃ (1 min), 55 ℃ (1 min), 72 ℃ (1.5
min); and a final extension of 8 min at 72 ℃. PCR
products were purified by electrophoresis through a
1.2% agarose gel followed by use of the Pearl Gel
Extraction Kit (Pearl Bio-tech). While direct sequenc-
ing is feasible for both S. alba and S. griffithii, it
produced chimeric or unreadable peaks in the chro-
matogram for the putative hybrid. Hence, cloning
sequencing was performed subsequently for the
putative hybrid. Purified PCR products were cloned
into plasmids using the pMD18-T Vector System
(Takara). Twenty positive clones were randomly
selected for each amplification product and cultured
for isolating plasmids. Positive clones with the inserts
of correct size were confirmed by colony PCR. The
plasmids with correct inserts were sequenced using
universal M13-47 and RV-M primers. Sequencing
was conducted in ABI 3730 DNA automated se-
quencer with Bigdye Terminator Cycle Sequencing
Ready Reaction Kit (Applied Biosystems). Clones that
clearly resulted from PCR-mediated recombination
were excluded.
1.4 Chloroplast trnL-trnF and trnV-trnM se-
quencing
All samples from Sonneratia alba, S. griffithii,
and the putative hybrid were used in the chloroplast
trnL-trnF and trnV-trnM sequencing. Chloroplast
trnL-trnF and trnV-trnM regions were amplified
using universal primers trn-c and f (Taberlet et al.,
1991), and trnV and trnM (Cheng et al., 2005), re-
spectively. PCR products were purified and then
directly sequenced using the methods mentioned
above.
2 Results
2.1 Sequences of the cpi gene in S. alba, S. grif-
fithii, and the putative hybrid
For both S. alba and S. griffithii, the cpi gene
could be directly sequenced and clear sequences were
obtained. Neither species showed sequence variation
between accessions. The length of cpi gene of S. alba
QIU et al.: Natural hybridization between Sonneratia alba and S. griffithii


393
Table 1 Taxa of Sonneratia used in this study
Accession number
Taxon Voucher
cpi trnL-trnF trnV-trnM
S. alba 1 S. Shi 200709-11 (SYS) EU418796 EU418841 EU418831
S. alba 2 S. Shi 200709-15 (SYS) EU418797 EU418842 EU418832
S. griffithii 1 S. Shi 200709-01 (SYS) EU418798 EU418845 EU418833
S. griffithii 2 S. Shi 200709-16 (SYS) EU418799 EU418846 EU418834
The putative hybrid 1 S. Shi 200709-04 (SYS) EU418800-EU418816 EU418843 EU418835
The putative hybrid 2 S. Shi 200709-09 (SYS) EU418817-EU418830 EU418844 EU418836
SYS = Sun Yat-Sen University.


Table 2 cpi haplotypes of the putative Sonneratia hybrid revealed by cloning sequencing
Taxon Clone number Sequence length cpi haplotype
1, 3, 5, 7, 10, 12, 13, 14, 17, 19 645 bp SA The putative hybrid 1
2, 6, 9, 11, 15, 16, 20 641 bp SG
1, 4, 7, 10, 11, 15, 18, 20 645 bp SA The putative hybrid 2
2, 3, 6, 9, 16, 17 641 bp SG
SA, haplotype that is identical or highly similar to S. alba; SG, haplotype that is identical or highly similar to S. griffithii.


Table 3 Variable sites of the nucleotide sequences of chloroplast trnL-trnF and trnV-trnM regions in the three taxa of Sonneratia
trnL-trnF trnV-trnM
Taxon
Length (bp) Variable sites Length (bp) Variable sites
S. alba 1000 T237 840 G150
S. griffithii 1000 G237 840 A150
The putative hybrid 1000 G237 840 A150
The number subscript the variable sites are the positions of variable sites.


was 645 bp, but 641 bp for S. griffithii. There were 18
nucleotide substitutions and a 4-bp indel between S.
alba and S. griffithii. By contrast, direct sequencing of
the cpi gene generated chimeric or unreadable se-
quences for both accessions of the putative hybrid. In
the subsequent cloning sequencing, we obtained two
distinct types (designated as Type SA and Type SG,
respectively) of cpi sequences from both accessions of
the putative hybrid after excluding nine clones clearly
resulting from PCR-mediated recombination. The two
distinct types of sequences corresponded to that of S.
alba and S. griffithii, respectively. There were ten
clones of Type SA and seven clones of Type SG for
the first accession. For the second accession of the
putative hybrid, there were eight clones of Type SA
and six clones of Type SG (Table 2). Most clones of
Type SA shared identical sequence with S. alba, and
most clones of Type SG shared the same sequence as
S. griffithii. In the other few clones, one or two nu-
cleotide point mutations were observed. These minor
variations may be due to PCR error caused by Taq
DNA polymerase or unsampled intraspecific poly-
morphism.
2.2 Sequences of the chloroplast trnL-trnF and
trnV-trnM in S. alba, S. griffithii, and the putative
hybrid
The chloroplast trnL-trnF and trnV-trnM regions
in Sonneratia alba, S. griffithii, and the putative
hybrid exhibited limited variation. Sequence lengths
are consistently 1000 bp in the chloroplast trnL-trnF
region, and 840 bp in the trnV-trnM region for the
three taxa. Sequences of both accessions from each
taxon were identical. In total, there were two nucleo-
tide substitutions between S. alba and S. griffithii, one
in the trnL-trnF region and the other in the trnV-trnM
region (Table 3). Both accessions of the putative
hybrid had the same trnL-trnF and trnV-trnM se-
quences as S. griffithii.
3 Discussion
So far there has been no report describing natural
hybridization between S. alba and S. griffithii. The
putative hybrid studied here possesses two types of
cpi sequence, each corresponding to that of S. alba
Journal of Systematics and Evolution Vol. 46 No. 3 2008

394
and S. griffithii, and chloroplast haplotypes of S.
griffithii. Our results provide compelling evidence for
natural hybridization between S. alba and S. griffithii.
Thus there are four interspecific hybrids that have
been reported in Sonneratia and three have been
documented by molecular data. With the exception of
S. apetala, all other species in Sonneratia are involved
in interspecific hybridization. Interestingly, we find
that S. alba is involved in all four cases of interspeci-
fic hybridization. There are at least two factors that
contribute to frequent occurrence of interspecific
hybridization in Sonneratia.
One factor is that Sonneratia species often have
partially overlapping geographic distribution and
slightly overlapping habitats. For example, S. alba and
S. caseolaris are sympatric across almost the whole
range of S. caseolaris, from India to China and Aus-
tralia. Sonneratia griffithii and S. alba coexist in the
coast of Andaman Sea. All species in Sonneratia grow
within estuaries (Duke et al., 1998). The physiological
tolerance of each species to salinity determines its
habitat. Sonneratia alba occurs on the more salty
seaward side of mangrove forests, whereas S. caseo-
laris grows on the less salty inland side (Duke et al.,
1998). Sonneratia alba and S. griffithii occupy similar
habitats, but S. griffithii was slightly upward along
streams in comparison with S. alba. There is consid-
erable overlap in geographic distribution and habitats
between species of Sonneratia, providing spatial
chances for hybridization.
The other factor that may contribute to natural
hybridization is the long and partially overlapping
flowering periods, and shared pollinators between
species of Sonneratia. Most species of Sonneratia
taxa flower more than six months each year and have
a long overlap of flowering season. For example, S.
alba flowers almost throughout the year. All the
species of Sonneratia are mainly pollinated by bats
(Tomlinson, 1986), and this also provides great
chances for interspecific hybridization in this genus.
Sonneratia alba has the widest range and the longest
flowering period among species of Sonneratia, which
is probably the reason why it is involved in all cases
of hybridization.
Despite frequent hybridization in Sonneratia, it
seems that all hybrids are simple F1s. All the indi-
viduals of S.×gulngai and S.×hainanensis have been
identified as F1s by AFLP markers (Zhou et al., 2005).
With respect to the hybrid S. alba×S. griffithii, direct
sequencing of five other nuclear genes also produced
chimeric or unreadable sequences (data not shown). It
is reasonable to speculate that S. alba×S. griffithii are
heterozygous at all the six nuclear genes examined.
Maintenance of biparental sequences at all six ran-
domly selected nuclear loci in the hybrid individuals
implies that they are likely to be simple F1s, because
the chance of randomly sampling six heterozygous
loci from a non-F1 hybrid is very low [P≤(1/2)6].
The restriction of hybrids to F1s appears to be a
general phenomenon in Sonneratia and can be ex-
plained by strong postzygotic isolation between
hybridizing species or hybrid breakdown. For exam-
ple, the proportion of sterile pollen in either S.
×gulngai (95.6%) or S. ×hainanensis (54.4%) is much
higher than in the parental species, S. alba (8.8%), S.
caseolaris (5.7%), and S. ovata (3.3%) (Wang et al.,
1999). Since all interspecific hybrids of Sonneratia
appear to be F1s, these species seem to be able to
maintain genetic integrity in spite of interspecific
hybridization. Therefore, these non-hybrid species in
Sonneratia should be considered as well defined
biological species. As species in Sonneratia are very
likely subject to parapatric speciation (Zhou et al.,
2007), the hybrid zones of Sonneratia are probably
primary hybrid zones (not secondary ones by allo-
patric divergence and subsequently secondary con-
tact). Thus, the occurrence of hybridization and strong
postmating isolation between parental species suggest
that these species may be at the last stage of speciation
and can achieve complete reproductive isolation via
reinforcement.
Maternal inheritance of the chloroplast DNA in
Sonneratia has been identified using DAPI (4′,
6-diamidino-2-phenylindole) staining technology (R.
Zhou, unpublished data). Based on sequence data of
chloroplast trnL-trnF and trnV-trnM, S. griffithii was
identified as the maternal parent of the hybrid, at least
in the two individuals of the hybrid. Sonneratia alba
is thus the paternal parent. If the trend continues, the
direction of hybridization should be unidirectional.
Acknowledgements We thank Shong HUANG for
his help during the field survey. This study was sup-
ported by grants from the National Basic Research
Program of China (2007CB815701), the National
Natural Science Foundation of China (30730008,
30470119), the Chang Hung-Ta Science Foundation of
Sun Yat-sen University and the Young Teacher Foun-
dation of Sun Yat-Sen University (2006-33000-
1131357).
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