Molecular Confirmation of Natural Hybridization Between Lumnitzera

Aquatic Botany 95 (2011) 59–64

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Aquatic Botany

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Short communication Molecular conﬁrmation of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea

Miaomiao Guo, Renchao Zhou ∗, Yelin Huang, Jianhua Ouyang, Suhua Shi ∗

State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-Sen University, Guangzhou 510275, China article info abstract

Article history: The hypothesis of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea, two man- Received 19 November 2010 grove species distributed in the Indo-West Pacific region, was proposed in 1970s based on morphological Received in revised form 28 February 2011 traits; however, no molecular evidence has been reported to support it. In this study, we sequenced Accepted 1 March 2011 two low-copy nuclear genes and one chloroplast intergenic spacer (trnS-trnG) in the two Lumnitzera Available online 9 March 2011 species and their putative hybrid to test this hypothesis. Our results revealed that there were 9 and 27 nucleotide substitutions at the two nuclear loci, respectively, between one haplotype of L. racemosa and L. Keywords: littorea, and that the putative hybrid showed additivity in chromatograms at these sites. Sequencing the Mangrove Natural hybridization chloroplast intergenic region trnS-trnG showed that the two Lumnitzera species differed by seven fixed Nuclear gene nucleotide substitutions and four fixed insertions/deletions in this region, while the putative hybrid had Chloroplast DNA identical sequences to L. racemosa. Molecular data clearly demonstrated that there indeed existed natural Lumnitzera hybridization between L. racemosa and L. littorea and that L. racemosa was the maternal parent in this

1. Introduction Lumnitzera (Combretaceae) is a typical mangrove genus in the Indo-West Pacific region (Tomlinson, 1986). This genus comprises Natural hybridization in flowering plants is ubiquitous and two species, Lumnitzera racemosa and Lumnitzera littorea. The two plays important roles in plant evolution and diversification (Arnold, species differ strikingly in petal color, with white petals in L. race- 1997). About 11% of the described plant species originate from mosa and red petals in L. littorea. L. racemosa is widely distributed interspecific or intergenic hybridization (Rieseberg, 1997), but from East Africa to the West Pacific, including Fiji, Tonga, and north- the distribution is uneven in different plant lineages (Ellstrand ern Australia, while L. littorea largely overlaps with L. racemosa, et al., 1996). Consisting of approximately 70 species from about except in East Africa, where L. littorea is not found (Tomlinson, 20 families, mangroves are a unique group of woody plants that 1986; Duke, 2006). In habitat, L. littorea is better suited to well- inhabit the intertidal zones of tropical and subtropical coasts (Duke, drained sites with less salinity, while L. racemosa is more resistant 1992). Interspecific hybridization has also been frequently reported to saline conditions and occurs at the margin of bare salt pans among mangroves, especially in the four genera Sonneratia (Duke, (Tomlinson et al., 1978). Despite habitat differentiation, sympatry 1984, 1994; Tomlinson, 1986; Zhou et al., 2005; Qiu et al., 2008), of these two species is frequently observed in mangrove stands Rhizophora (Duke and Bunt, 1979; Parani et al., 1997; Duke, 2010; from Southeast Asia. In addition to the two species, there is a dis- Lo, 2010), Bruguiera (Ge, 2001), and Lumnitzera (Tomlinson et al., tinct form with pink flowers in this genus found in the Philippines, 1978; Tomlinson, 1986; Duke, 2006). Molecular means have been New Guinea, Vietnam, and Australia, where the two species over- used to confirm the occurrence of hybridization in some cases lap (Duke, 2006; Nguyen et al., 2010). The form was first reported (Parani et al., 1997; Ge, 2001; Zhou et al., 2005; Qiu et al., 2008; as a new species L. rosea [(Gaud.) Presl., 1834 in Tomlinson et al., Wu et al., 2009; Lo, 2010). 1978] and was later interpreted by Tomlinson et al. (1978) as a hybrid, L. × rosea (we use L. × rosea for the distinct form there- after), between L. racemosa and L. littorea, based on morphological intermediacy in flower color and other traits. Lumnitzera × rosea possesses different traits from its putative parents, including termi- ∗ nal racemes of flowers with long and reflexed petals nearly as long Corresponding authors at: School of Life Science, Sun Yat-Sen University, as the stamens and slightly eccentric style placement (Duke, 2006). Guangzhou 510275, China. Tel.: +86 20 84113677; fax: +86 20 34022356. × E-mail addresses: [email protected] (R. Zhou), [email protected] Lumnitzera rosea commonly borders mid- to high-intermediate (S. Shi). estuarine locations of notably moderate to wet climatic regions

(Duke, 2006). Furthermore, L. × rosea is a diploid, with the same chromosome number (2n = 22) as its putative parental species (Le and Hoang, 2009). Confirmation of hybrid status of taxa is a prerequisite for taxonomical and evolutionary studies and also for scientific conservation and management. Because morphological criteria alone are sometimes misleading in the determination of hybrid status (Morrell and Rieseberg, 1998), molecular evidence is usually nec- essary to obtain convincing conclusions. Single or low-copy nuclear genes hold great promise in identifying hybrids and have been suc- cessfully used in plants (Sang and Zhang, 1999; Qiu et al., 2008; Wu et al., 2010). As an adjunct, chloroplast DNA, which is mater- nally transmitted in the majority of angiosperms (Mogensen, 1996), can be used to determine the maternal parent of hybrids. In this study, we sequenced two low-copy nuclear genes (nab and ppr) and one chloroplast intergenic spacer (trnS-trnG) in Lumnitzera to test the hybrid status of L. × rosea. The nab gene is a member of the nuclear acid binding gene family, which includes four members in Arabidopsis thaliana. It is considered to be of crucial importance in the regulation of gene expression, for example, response to cold Fig. 1. Haplotype networks of the two nuclear genes and chloroplast trnS-trnG stress and cytokinin signal transduction (Hwang and Sheen, 2001; region for L. racemosa (A: nab;B:ppr;C:trnS-trnG). Circles represent haplotypes, and numbers in the circles show the number of each haplotype. Numbers close to Bae et al., 2003). The ppr gene family, which encodes proteins the solid lines represent mutation steps between haplotypes. Gray shading circles with pentatricopeptide repeat motifs, is involved in RNA process- represent the haplotypes of L. racemosa that involved in hybridization. ing and organelle biogenesis (Lurin et al., 2004). Only 7% of the members of this family in Arabidopsis contain more than one intron (Lurin et al., 2004), and the ppr gene in Lumnitzera comprises three two nuclear genes (nab F: 5 AGCAGCACAGTGAGGTTG 3; nab R: introns within the examined regions. Samples were collected from 5GCCTTCTGAACGGGTCT 3; ppr F: 5 CCTTAGGCTACTGTGGGA 3; Khao-Than, Tha Chang, Surat Thani, Thailand, where L. racemosa ppr R: 5 GAGGGATGTTAGGCTGAT 3) were designed with Primer and L. littorea are sympatric, and two individuals of L. × rosea were Premier 5.0. Chloroplast trnS-trnG regions were amplified using found during our field survey. Once we confirmed its hybrid status, universal primers trnS and trnG(Hamilton, 1999). PCR amplifi- chloroplast markers were used to infer the direction of hybridiza- cations were conducted for the samples mentioned above using tion. TaKaRa HS PrimeStar DNA polymerase with the following conditions: 32 cycles of 98 ◦C for 10 s, 58 ◦C for 10 s, 72 ◦C for 2 min, and 2. Material and methods a final extension at 72 ◦C for 8 min. PCR products were purified by electrophoresis through a 1.2% agarose gel followed by use of 2.1. Plant materials the Pearl Gel Extraction Kit (Pearl Bio-tech) and they were then sequenced on an ABI 3730 DNA automated sequencer with the During a field survey of mangroves on the eastern coasts BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied of the Malay Peninsula in 2009, we found three taxa of Lum- Biosystems). All of the sequences were deposited in GenBank with nitzera in sympatry in Khao-Than, Tha Chang, Surat Thani, Thailand accession numbers HQ456231–HQ456247. (9◦1731.42N, 99◦1157.23E). L. racemosa was dominant in this location, while both L. littorea and the putative hybrid L. × rosea were rare; only two individuals of each taxon were found. These 3. Results taxa are easily recognized by their distinct petal colors, with white petals in L. racemosa, red petals in L. littorea, and pink 3.1. Sequences of the two nuclear genes in L. racemosa, L. littorea, petals in L. × rosea. We sampled 25 individuals of L. racemosa and L. × rosea and two individuals each of L. littorea and L. × rosea. To pre- vent underestimation of the intraspecific polymorphism of L. The length of the nab gene of L. littorea was 615 bp, and no littorea, we also collected five, two and one individuals, respec- sequence variation was observed across the four populations of tively, from other three Thailand populations, namely, Bo, Khlung, this species. In contrast, L. racemosa exhibited a higher level of Chanthaburi (12◦2355.92N, 102◦1603.57E), Khanom, Nakhon Si polymorphism, with two divergent haplotypes detected (Fig. 1A). Thammarat (9◦1259.10N, 99◦4910.02E) and Chaiya, Surat Thani Haplotypes A1 and A2 differed by eight nucleotides and one inser- (9◦2221.72N, 99◦1551.24E). Leaves from each individual were tion/deletion (indel) (Table 1). The nab gene sequences of both collected in plastic bags with silica gel for DNA extraction. individuals of L. × rosea showed chromatogram additivity between haplotype A2 of L. racemosa and L. littorea (Fig. 2), which differed 2.2. DNA extraction by nine nucleotide substitutions (Table 1). The ppr gene was 811 bp long in L. racemosa and 810 bp long Total genomic DNAs were extracted from dried leaf tissues using in L. littorea. Again, no sequence variation was observed across the the CTAB method (Doyle and Doyle, 1987). four populations of L. littorea, while L. racemosa exhibited a higher level of polymorphism. There were four haplotypes in L. racemosa 2.3. Sequencing of the two nuclear genes and the chloroplast for this gene, and haplotypes B1 and B2 were dominant, while B3 trnS-trnG region and B4 were rare (Fig. 1B). B1 and B3 differed by 1 nucleotide sub- stitution, as did B2 and B4. For the two individuals of L. × rosea, Two randomly selected low-copy nuclear genes (nab and double peaks on the chromatograms were observed at all 27 sites ppr) from a cDNA library of L. racemosa (Guo et al., unpub- where haplotype B2 of L. racemosa and L. littorea differed (Fig. 2 and lished data) were used for the experiments. PCR primers for the Table 2). M. Guo et al. / Aquatic Botany 95 (2011) 59–64 61

Table 1 Variable sites of the nab gene in L. littorea, L. racemosa, and L. × rosea. Numbers represent the positions of variable sites. There are two haplotypes (A1 and A2) for L. racemosa, and the individuals with haplotype A2 are involved in the hybridization event.

Taxon Variable site

69 90 160 200 342 355 369 377 385 394 482 506 598 609

L. littorea CTGGAGCGATAACT L. × rosea CKGKRRCGRWAWYW L. racemosa (A1) T G T T A A – T A T T A T A L. racemosa (A2) C G G T G A C G G A A T T A

Thus, L. × rosea showed a perfect additivity between L. racemosa used to determine the direction of the hybridization event. While and L. littorea at two randomly selected nuclear genes, providing there was no variation in this region across the four populations compelling evidence for the hypothesis of hybridization between of L. littorea, three haplotypes were observed for L. racemosa. The L. racemosa and L. littorea. Interestingly, for both nuclear genes, only network of the three haplotypes was shown in Fig. 1C. Haplotypes one of the two common haplotypes of L. racemosa was involved in C2 and C3 differed by five nucleotide substitutions and two indels, the hybridization event. while C1 and C3 differed by three nucleotide substitutions. L. littorea differed from L. racemosa at seven fixed nucleotide substitutions 3.2. Sequences of the chloroplast trnS-trnG region in L. racemosa, and four fixed indels (Table 3). Both individuals of L. × rosea had the L. littorea, and L. × rosea same trnS-trnG sequences as haplotype C2 of the common species L. racemosa, indicating that L. racemosa was the maternal parent Since the hybrid status of L. × rosea was confirmed based on the and that L. littorea was the pollen donor for the two hybrid individ- nuclear genes, the chloroplast intergenic region trnS-trnG could be uals.

Fig. 2. Chromatogram additivity at two nuclear loci (nab and ppr)inL. × rosea. Black triangles represent the variable sites between L. racemosa and L. littorea, and the numbers represent the positions of these sites. Only ﬁve sites for each gene are shown. 62 M. Guo et al. / Aquatic Botany 95 (2011) 59–64 TT TT TT TT ...... GGGA GGGA AA C – ... , and individuals with haplotype C2 are involved , and individuals with haplotype B2 are involved in the L. racemosa AG–ATT GAAGA– ATT GA ATT GAAGA– CGAAGA– T C – A TT L. racemosa ...... TC AA TC AA ...... AAAGTTAATTCTA AAAGTTAATTCTA GGGGAAT T GAT T T CAACGC GGGGAAT T GAT T CCAACGC TTTATT TTTATT . Numbers represent the positions of variable sites. There are three haplotypes (C1–C3) for rosea × . Numbers represent the positions of variable sites. There are four haplotypes (B1–B4) for L. rosea , and × L. , and L. racemosa , L. racemosa , L. littorea Gin trn L. littorea S- trn gene in A T C C T T C – C T T A TCGTT – – A G A AA A C A T C G A TTTT G T G T – TA 65 101 113 146 153 221 222 223–226 276 293 296 312 351–355 371–416 480–505 597 604 605 709–723 739 821–840 ppr 33 39 40 73 119 155 163 170 197 268 326 328 335 352 404 455 509 523 538 544 555 569 606 632 641 649 658 682 703 710 714 724 731 748 753 787 T-A T CT GT GCGGAAT AGGAGCAGGCCT TATT-AAT AGAA RWKTMRTWYYGYKYRGWRKARRRKYARKYCTWRGRA (C1)(C2) G(C3) A C G C C A A A C T C T C T G G G A A A – TTTT – G G G T G G G T T T T T – – – TA – – AA AA (B1) G(B2) G A A G G T T C C G G C T T A C T C C A G C T T A G T G G (B3) G(B4) G A A G G T T C C G G C C T A C T C C A G C T T A G T G G rosea rosea × × L. racemosa L. racemosa L. racemosa L. L. racemosa TaxonL. littorea Variable site L. racemosa TaxonL. littorea Variable site L. L. racemosa L. racemosa hybridization event. Table 2 Variable sites of the Table 3 Variable sites of chloroplast in the hybridization event. M. Guo et al. / Aquatic Botany 95 (2011) 59–64 63

4. Discussion been reported among mangroves, and almost all of these hybrid individuals are described as pure F1s(Zhou et al., 2005; Qiu et al., Our molecular data provide compelling evidence for the 2008; Duke, 2010; Lo, 2010). The prevalence of F1 hybrids in man- hybrid status of Lumnitzera × rosea, which was proposed based groves can be explained by strong postzygotic isolation between on morphological traits. Other lines of evidence also support this hybridizing species or hybrid breakdown. The formation of only F1s hypothesis. For example, the mean pollen fertility of L. × rosea is means that hybrid speciation is impossible for these hybrids with- 19.5 ± 1.3%, while it is 71.7 ± 0.8% for the parental species L. race- out asexual reproduction, however, the existence of hybridization mosa (Tran and Ngugen, 2009; Tran et al., 2009). In addition, the can strengthen reproductive isolation between hybridizing species fruits of L. × rosea are mostly of immature development and are by reinforcement (Dobzhansky, 1937). apparently sterile (Duke, 2006). These observations are consis- tent with the prediction that hybrids usually have reduced fertility (Gottlieb, 1972). Acknowledgements Strikingly, the common species L. racemosa has two divergent common haplotypes in the three loci examined here. This may This work was supported by National Natural Science Foun- be caused by recent admixture of two divergent populations. The dation of China (30730008, 30800060, 31070290, 40876075, decline in sea level in the Pleistocene glaciation period may have 40976081), the Doctoral Foundation of Ministry of Education caused population shrinkage and isolation of populations of the of China (200805581042), SRF for ROCS, SEM and the Funda- mangroves. These isolated and thus differentiated populations may mental Research Funds for the Central Universities (09lgpy35, have come across later when the sea level rose. 10lgpy20), the Natural Science Foundation of Guangdong Province There are at least three factors that contribute to the interspe- (8151027501000089, 8451027501001492). cific hybridization in Lumnitzera. First, L. racemosa and L. littorea have partially overlapping geographic distribution and habitats. L. References littorea largely overlaps with L. racemosa from India through South- east Asia, north to China, south to Australia (Tomlinson, 1986; Duke, Arnold, M.L., 1997. Natural Hybridization and Evolution. Oxford University Press, 2006). The two species differ slightly in habitat, with L. littorea New York/Oxford. occurring at sites with lower salinity and L. racemosa growing in Arnold, M.L., Hamrick, J.L., Bennett, B.D., 1993. Interspecific pollen competition and reproductive isolation in Iris. J. 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