Aquatic Botany 95 (2011) 59–64 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot 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 hybridization event. The uncommon direction of hybridization and F1 nature of hybrids in this case, and in mangroves in general, is discussed. © 2011 Elsevier B.V. All rights reserved. 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 0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.03.001 60 M. Guo et al. / Aquatic Botany 95 (2011) 59–64 (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 con- servation 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 condi- tions: 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).