Nucleotide Sequence Diversity at the Methionine Synthase Locus in Endangered Dunnia sinensis (Rubiaceae): An Evaluation of the Positive Selection Hypothesis

Yu-Chung Chiang*, Xue-Jun Ge{dagger}, Chang-Hung Chou{ddagger}, Wen-Lung Wu§ and Tzen-Yuh Chiang§

*Department of Biology, Taiwan Normal University, Taipei, Taiwan, ROC;
{dagger}South China Institute of Botany, Chinese Academy of Sciences, Guangzhou, People's Republic of China;
{ddagger}Institute of Botany, Academia Sinica, Taipei, Taiwan, ROC;
§Department of Biology, Cheng-Kung University, Tainan, Taiwan, ROC


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Methionine synthase is a key enzyme for the synthesis of the aspartate-derived methionine, the immediate precursor of S-adenosyl-methionine, which has been illustrated to be associated with plant growth and pathogen interactions. In this study we tested the positive selection hypothesis of molecular evolution of the methionine synthase gene in Dunnia sinensis. In the entire sample of 87 sequences, 22 haplotypes of introns and 16 haplotypes of exons were identified. An excess of polymorphism over the neutral expectation for the class of unique nucleotide polymorphisms was observed in both exon and intron sequences. Ten replacement substitutions versus six synonymous substitutions among lineages, although nonsignificant, revealed that some advantageous mutants might have been favored. The distribution of dN/dS > 1 at nodes between closely related haplotypes in the gene network also indicated weak and variable positive selection. Nevertheless, low levels of genetic diversity in exons ({theta} = 0.0052) and introns ({theta} = 0.0070) of the methionine synthase gene of the outcrossing Dunnia were also attributed to the endangered status of the species. The atpB-rbcL intergenic spacer of cpDNA and the ribosomal internal transcribed spacer of mtDNA were used to discern the relative effectiveness of natural selection from intrinsic evolutionary forces. The low levels of nucleotide polymorphisms in both organelle spacers and the significant population differentiation reflected the effect of population-species history and demography. Two major lineages of the methionine synthase gene genealogy were recovered corresponding to two geographic regions, a result that was consistent with organelle phylogenies. Both past fragmentation and recent habitat disturbance causing complete bottlenecks may have resulted in population decline and geographic isolation and may have led to the depletion of genetic variation at loci in nuclear and organelle genomes.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Methionine, a de novo synthesized aspartate-derived amino acid in plants, plays a central role in cellular metabolism. In addition to serving as a building block for protein synthesis, methionine, the immediate precursor of S-adenosyl-methionine (AdoMet), is also involved in the biosynthesis of polyamines and phytohormone ethylene (Walden, Cordeiro, and Tiburcio 1997Citation ). For methionine metabolism the synthesis of AdoMet is the major route, with 80% of this amino acid engaged in this reaction (Giovanelli, Mudd, and Datko 1985Citation ). Methionine synthase not only catalyzes the last reaction in methionine biosynthesis but also serves to regenerate the methyl group of AdoMet. AdoMet metabolism has been found to be linked with plant growth (Eckermann, Eichel, and Schröder 2000Citation ) and pathogen interactions in plants (Ravanel et al. 1998Citation ). Host-pathogen interactions are assuredly a force that shapes organismal diversity and the evolution of genes responsible for resistance to pathogens. Bishop, Dean, and Mitchell-Olds (2000)Citation revealed that the variable effectiveness of alleles of many pathogen recognition genes and antipathogen loci, such as plant R-genes (McDowell et al. 1998Citation ) and chitinases, in response to variation in pathogen defenses was subject to positive selection and underwent adaptive evolution. Genetically, the methionine synthase is encoded by a single nuclear gene, which is composed of 11 exons (2,328 bp) and 10 introns (1,097 bp) in Arabidopsis (Kaneko et al. 1998Citation ). Despite the well-understood methionine biosynthesis and metabolism in plants (Ravanel et al. 1998Citation ; Eckermann, Eichel, and Schröder 2000Citation ; Hesse et al. 2001Citation ), to date this gene has been cloned from only a few species, such as Arabidopsis, Solanum, and coffee.

For detecting Darwinian selection at candidate loci, various methods based on the relative rate of nonsynonymous substitutions to synonymous substitutions have been developed (Hughes et al. 2000Citation ; Yang, Swanson, and Vacquier 2000Citation ). Nevertheless, many other evolutionary forces may mask or blur the effects of natural selection. The evolution of functional loci is not only shaped by the levels of functional constraints on genes and modes of natural selection but is also inevitably affected by population demography, systems of mating, and migratory history. Various statistical analyses have to be applied for distinguishing these forces. Technically, highly conserved DNA sequences provide no information on the evolutionary forces that shape the genealogy and fail to discern the effects of population history (such as bottlenecks and demographic expansion) from the modes and effects of natural selection at a single locus (Simonsen, Churchill, and Aquadro 1995Citation ). For the estimates of the relative effects of these evolutionary forces, some genetic variation is certainly required. Kaneko et al. (1998)Citation speculated that the evolution of the methionine synthase gene is fast. This gene thus provides an ideal model for investigating the apportionment of genetic variation associated with natural selection, population history, and demography.

Different evolutionary forces may result in a similar outcome of genetic variability and apportioning of patterns at a functional locus. For example, both background selection and bottlenecks can lead to the depletion of genetic variation within populations. Nonetheless, in gene genealogies the imprints recording organismal and gene histories are preserved. Sequencing loci unlinked to the candidate gene provide supplementary information for discerning the intrinsic from the extrinsic evolutionary forces (Simonsen, Churchill, and Aquadro 1995Citation ). Use of multiple loci has thus recently become informative in molecular population genetics (Purugganan and Suddith 1999Citation ). In this study we sequenced the methionine synthase gene, the atpB-rbcL intergenic spacer of the cpDNA, and the ribosomal internal transcribed spacer (ITS) region of the mtDNA in Dunnia sinensis. These organelle-DNA noncoding spacers were chosen because of their near neutrality and the lack of linkage to the methionine synthase gene, thus providing neutral expectations (Tajima 1989Citation ).

Dunnia sinensis Tutch. (Rubiaceae), an endangered species, is restricted to the southern part of Guangdong Province in the People's Republic of China (Ridsdale 1978Citation ). Distylous flowers of D. sinensis offer nectar and pollen to insects as pollination rewards. Spherical capsules bear winged and light seeds, which are dispersed by wind and gravity, resulting in short-range dispersal mostly within demes. During the past decades the health of the endemic species has been seriously threatened. Most of the habitats where D. sinensis resides, usually along streams or on hillsides at 100–600 m of altitude, have been recently destroyed for economic uses. After such human exploitation the patch-like distribution has kept the five remaining populations of the threatened species isolated from each other, especially between Zhuhai and Taishan along the coasts (fig. 1 ). The population and geographic structure of D. sinensis have been affected by habitat fragmentation. Given the limited between-population dispersal and the small population size, a great many genetic elements are expected to be lost via stochastic processes, leading to a low level of within-population variation and pronounced genetic differentiation between populations. Such demographic effects are expected to be reflected in the maternally inherited organelle DNAs (Harris and Ingram 1991Citation ) as well as the methionine synthase in D. sinensis.



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Fig. 1.—D. sinensis sample locations and distribution. Abbreviations of population names are given in table 1 .

 
In the present study we sequenced methionine synthase genes and noncoding spacers of organelle DNAs to evaluate the relative effectiveness of natural selection and demographic history on the functional locus. The objectives of this study were to investigate the following. (1) Does the genetic variation of organelle DNAs in D. sinensis tend to become depauperate because of their small effective population size, as in many endangered species? (2) When a low level of organelle variation is detected, does the methionine synthase gene vary at low levels as well? (3) Do the methionine synthase locus and the organelle DNAs yield consistent apportioning patterns? (4) Has Darwinian natural selection caused the methionine synthase gene to deviate from neutral expectation?


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Sample Collection, DNA Extraction, Primer Design, and PCR
Dunnia sinensis, a monotypic genus of the Rubiaceae, is restricted to the southern part of Guangdong Province in the People's Republic of China. A total of 87 individuals of D. sinensis, representing five extant populations, were sampled (table 1 ). Young and healthy shoots were collected in the field, rinsed with tap water, and dried in silica gel. All samples were stored at -70°C until they were processed. Leaf tissue was ground to powder in liquid nitrogen and stored in a -70°C freezer. Genomic DNA was extracted from the powdered tissue following a CTAB procedure (Murray and Thompson 1980Citation ).


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Table 1 Population Location, Number of Samples (N), and Site Coordination Used for a Phylogeographic Analysis of Dunnia sinensis

 
In this study the methionine synthase gene, the intergenic spacer between the atpB and rbcL genes of cpDNA, and the rDNA ITS of mtDNA were amplified and sequenced. One set of primers was designed for amplifying the methionine synthase gene by comparing sequences of Arabidopsis (Kaneko et al. 1998Citation ), Solenostemon scutellarioides (Lamiaceae, Z49150), Mesembryanthemum cystallinum (Aizoaceae, U84889), Catharanthus roseus (Apocynaceae, X83499), Solanum tuberosum (AF082893), and coffee (AF220054). Primers, met1-F (5'-CCATGGCTAGAGGAAATGCC-3') and met4-R (5'-GCCCAGATGTTCCTTCCATC-3'), correspond to the sequences in exon 1 (sites 36606–36625 of Arabidopsis [AB011480]) and exon 4 (sites 37682–37662), respectively. Complete sequence of exons 2 and 3 and partial sequence of exons 1 and 4 were obtained.

PCR amplification was carried out in a volume of 100 µl, using 10 ng of template DNA, 10 µl of 10x reaction buffer, 10 µl of MgCl2 (25 mM), 10 µl of dNTP mix (8 mM), 10 pmol of each primer, 10 µl of 10% NP-40, and 2 U of Taq polymerase (Promega, Madison, Wis). The reaction was optimized and programmed on an MJ thermal cycler (PTC 100) as one cycle of denaturation at 95°C for 4 min, 30 cycles of denaturation at 92°C for 45 s, annealing at 52°C for 1 min 15 s, and extension at 72°C for 1 min 30 s, followed by a 10-min extension at 72°C. Template DNA was denatured with reaction buffer, MgCl2, NP-40, and ddH2O for 4 min (first cycle) and cooled on ice immediately. Primers (for methionine synthase gene, cpDNA atpB-rbcL spacer [Chiang, Schaal, and Peng 1998Citation ], or mtDNA rITS [Chao, Sederoff, and Levings 1984Citation ]), dNTP, and Taq polymerase were added to this ice-cold mix. The reaction restarted at the first annealing at 52°C.

T-vector Cloning and Nucleotide Sequencing
PCR products were purified by electrophoresis in 1.0% agarose gel with 1x TAE buffer. The gel was stained with ethidium bromide, and the desired DNA band was cut out and eluted using agarose gel purification (QIAGEN). Purified DNAs were ligated to a pGEM-T easy vector (Promega). Plasmid DNAs were selected randomly with five clones and purified using a plasmid mini kit (QIAGEN). Purified plasmid DNAs were sequenced in both directions by standard methods of the Taq dye deoxy terminator cycle sequencing kit (Perkin Elmer) on an Applied Biosystems Model 377A automated sequencer (Applied Biosystems). Primers for sequence determination were T7-promoter and SP6-promoter, located on the p-GEM-T Easy Vector termination site.

Data Analysis
Nucleotide sequences were aligned with the Genetics Computer Group Wisconsin Package (Version 10.0; Madison, Wis.). Neighbor-Joining analysis was performed using MEGA2 (Kumar et al. 2001Citation ) by calculating Kimura's (1980)Citation two-parameter distance. The number of mutations between DNA genotypes in pairwise comparisons was used to construct a minimum spanning network using MINSPNET (Excoffier and Smouse 1994Citation ) in a hierarchical manner (cf. Chiang and Schaal 1999Citation ).

Levels of genetic diversity within populations were quantified with estimates of nucleotide divergence ({theta}) from S (Watterson 1975Citation ) using DnaSP (Version 3.14; Rozas and Rozas 1999Citation ), where {theta} = 4Nm for an autosome gene of a diploid organism (N and m are the effective population size and the mutation rate per nucleotide site per generation, respectively), and S is the number of segregating sites. Patterns of geographical subdivision and gene flow were estimated hierarchically using DnaSP. Gene flow within and among regions (populations) was approximated as Nm, the number of migrants per generation between populations, and was estimated using the expression FST = 1/(1 + 4Nm), where N is the effective population size, and m is the migration rate (Slatkin 1993Citation ). Tests of neutrality and determination of the associated significance as well as the coalescent-based estimations of minimum recombination events were done using DnaSP.

The corrected proportions of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) were estimated from the sequences on the basis of the maximum likelihood methodology of Yang and Nielsen (1998)Citation , in a pairwise manner for comparisons, using PAML (Yang 1999Citation ). Hughes, Ota, and Nei (1990)Citation developed a method for examining the properties of nonsynonymous substitutions. The method involves computing the proportion of radical nonsynonymous difference per radical nonsynonymous site (pNR) and the proportion of conservative nonsynonymous difference per conservative nonsynonymous site (pNC) (cf. Hughes et al. 2000Citation ). When pNR > pNC, a directional change in amino acid sequence is indicated, thus suggesting positive selection.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Nucleotide Polymorphism in D. sinensis and Tests for Genetic Recombination
Dunnia sinensis has a single methionine synthase gene that is homologous to the methionine synthase gene in Arabidopsis thaliana. DNA sequence was determined for all 87 samples of D. sinensis. No within-individual variation was detected. The methionine synthase gene region sequenced was 1,289 bp in length, including 611 bp of exon sequence (partial sequence of exons 1 and 4 and complete sequence of exons 2 and 3) and 678 bp of intron sequence. No indels occurred in the exons, whereas three indels (two 2 bp and one 48 bp) were detected in the introns. In the exons, 16 sites were polymorphic for nucleotide substitutions, including 10 replacement and 6 synonymous substitutions, in D. sinensis. In the introns, 25 sites were polymorphic (table 2 ). In total, 16 haplotypes of exons and 22 haplotypes of introns were identified on the basis of methionine synthase gene sequences. All DNA sequences were registered with the EMBL accession numbers AJ439217–AJ439248.


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Table 2 Genetic Diversity ({theta}), Tests of Neutrality, Number of Replacement (Rep) Versus Synonymous (Syn) Sites, and Minimum Recombination Events (Rm) in the Methionine Synthase Gene in Dunnia and Arabidopsis and the AdoMet Synthase Gene of Actinidia

 
The distribution of allelic frequencies of polymorphic sites was compared with the expectation (Gn(i)) under a neutral mutation model, which was calculated by using equation (51) of Tajima (1989)Citation :


where S is the number of segregating sites, n is the number of haplotypes, and i = 1 to n - 1. For the whole sample, mutations at 9 of the 16 polymorphic sites in the entire exon region and at 19 of the 25 polymorphic sites in the intron region occurred once in the sample. An excess over the expectations, 5.14 in exons and 6.89 in introns, under the neutral mutation model was observed in the class of unique nucleotide polymorphisms in both the exons ({chi}2 = 4.27, P = 0.029) and the introns ({chi}2 = 33.80, P < 0.005) of D. sinensis.

Seven haplotypes (h = 0.690 ± 0.0022) were detected at the atpB-rbcL intergenic spacer of cpDNA in D. sinensis. The region sequenced was 456 bp in length. Four point mutations (0.88%) and two indels ([137, 153] and [303, 311]) contributed to the polymorphism. Six haplotypes of mtDNA were identified, with haplotype diversity (h) of 0.476 ± 0.0011, from the 263-bp sequence of rITS. Differences between mitochondrial sequences were ascribed to four point mutations (0.15%). An excess of singles over neutral expectations was detected in the cpDNA spacer (three out of six polymorphic sites) and the mtDNA rITS (two out of four polymorphic sites). No recombination was detected in either organelle spacer, whereas single recombination events occurred in the exons and introns of the methionine synthase gene, respectively.

Estimates of Nucleotide Diversity and Statistical Tests of Neutrality
Nucleotide diversity for the whole sequenced methionine synthase gene region was estimated by Watterson's {theta} statistic. As expected, there was a higher diversity in the intron region ({theta} = 0.0070) than in the exon region ({theta} = 0.0052) (table 2 ). In the exons, nucleotide diversity was higher at synonymous sites ({theta} = 0.00815) than at nonsynonymous sites ({theta} = 0.00429). For nonsynonymous sites the conservative nonsynonymous difference (pNC = 0.0045) was also greater than the radical nonsynonymous difference (pNR = 0.0015). Nevertheless, when the sequence was partitioned into exon segments, the genetic diversity ({theta}) was higher at nonsynonymous sites than at synonymous sites in all exons except exon 1. Radical nonsynonymous changes were also greater than conservative nonsynonymous changes in exon 2 (pNC = 0.0066 vs. pNR = 0.0170) and in exon 4 (pNC = 0.0062 vs. pNR = 0.0081) (table 3 ). For the organelle noncoding spacers sequenced, the genetic diversity ({theta}) was estimated to be 0.0022 at the atpB-rbcL intergenic spacer of cpDNA and 0.0019 at the mtDNA rITS.


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Table 3 Genetic Diversity ({theta}), Number of Polymorphic (SS) and Singleton Sites of Neutrality, Number of Synonymous Versus Replacement Sites, and Rates of Radical Changes (pNR) and Conservative Differences (pNC) in Each Exon of the Methionine Synthase Gene of Dunnia sinensis

 
A number of statistical tests were used to determine significant departures from the neutrality hypothesis at the methionine synthase locus. Among them, Tajima's test was found to be the most powerful against the alternative hypotheses of selective sweep, population bottleneck, and population subdivision (Simonsen, Churchill, and Aquadro 1995Citation ). In this study, for the exon sequence data of D. sinensis, Fu & Li's D* statistic (-2.4108) was significant (P < 0.05), whereas a nonsignificant negative Tajima's D value (-1.30894) was obtained (table 2 ). In the entire sample of the methionine synthase gene, more replacement substitutions than synonymous substitutions were detected among the overall exons sequenced (10 vs. 6) and also for each exon (table 3 ). For example, no synonymous substitutions occurred in exons 2 and 3 because four and three replacement substitutions were detected, respectively.

Phylogeny of Methionine Synthase Gene and Organelle DNAs
To illustrate the genealogical relationships of haplotypes of the methionine synthase gene in D. sinensis, both conventional and nested clade analyses were conducted. A reconstructed neighbor-joining tree of this gene (fig. 2 ) identified two major clades that corresponded to two geographical regions, the eastern region (LM, TS, XH, ZH) versus the western region (YC), a result in agreement with phylogenies of the organelle DNAs (fig. 3 ). A minimum spanning network (fig. 4 ) was reconstructed on the basis of the mutational changes between haplotypes of the methionine synthase gene. Four dominant haplotypes of the methionine synthase gene, 1-1 (14 samples), 1-8 (32 samples), 1-9 (14 samples), and 1-12 (12 samples), were nested in the gene network as interior nodes. According to the coalescence theory, they tended to represent some ancestral haplotypes that had a greater probability of producing mutational derivatives (cf. Donnelly and Tavaré 1986Citation ). Spatially, the clade 3-1 was exclusively distributed in the YC population, whereas the clade 4-1 was widespread across populations of the eastern region (fig. 4 ). All haplotypes were restricted to a single population, except for 1-8 (LM, XH, and ZH) and 1-12 (LM and XH) (table 4 ).



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Fig. 2.—Neighbor-Joining tree, based on Kimura's (1980)Citation two-parameter distance, of haplotypes of the methionine synthase gene in D. sinensis. Numbers at the nodes indicate bootstrap values (expressed as a percentage) with 400 replicates

 


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Fig. 3.—A, Minimum spanning network generated using the method of Excoffier and Smouse (1994)Citation for haplotypes of the atpB-rbcL spacer of cpDNA of populations (in parentheses) of D. sinensis. Number of mutational change is indicated at the nodes. B, Minimum spanning network of haplotypes of the mtDNA ITS of populations (in parentheses) of D. sinensis

 


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Fig. 4.—Minimum spanning network generated using the method of Excoffier and Smouse (1994)Citation for haplotypes of the methionine synthase gene of populations of D. sinensis. Mutational changes are indicated at the nodes. Nodes with dN/dS > 1 at the exons (E1, E2, E3, or E4) are indicated. The replicate number of haplotypes is also indicated in parentheses when more than one

 

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Table 4 Distribution of Clades of the Methionine Synthase Gene in Populations of Dunnia sinensis

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Outcrossing Versus Selfing Alone Does Not Account for Differences in Polymorphism Between Species
System of mating is known to be one of the evolutionary forces that determine the levels and distribution of genetic variation in plant populations. Generally, predominant self-fertilization is likely to cause the depletion of genetic variability, because of the lack of crossing-over and the reduction of the effective population size. In this study, DNA polymorphism in the methionine synthase gene of D. sinensis, an insect-pollinated outcrosser, was measured and compared with the only available taxon, A. thaliana. Only 16 haplotypes of the methionine synthase gene were identified on the basis of exon sequences of 87 Dunnia samples, whereas two types of three individuals existed in Arabidopsis (Peleman et al. 1989Citation ). Earlier allozyme and microsatellite investigations showed that inbreeding Arabidopsis possessed lower within-population variation than did the average selfing species (cf. Savolainen et al. 2000Citation ). Nevertheless, the outcrossing D. sinensis, in spite of having a relatively higher level of genetic recombination (Rm = 1 in exons and introns, respectively), contained remarkably lower variation at the methionine synthase gene, with {theta} = 0.0052 in exons and {theta} = 0.0070 in introns, than did exons of A. thaliana ({theta} = 0.0676) (table 2 ). Apparently, outcrossing versus selfing alone does not determine the levels of genetic variability. Other evolutionary forces besides mating system must have governed the level of DNA polymorphism in D. sinensis.

Modes of natural selection perhaps contributed to such bewildering differences at the levels of genetic variation between Dunnia and Arabidopsis. However, comparisons between the two taxa exhibited common patterns of apportionment and maintenance of genetic variability, based on an excess of rare polymorphisms, which was indicated by significant negative Fu & Li's D* values and negative Tajima's D values, and more replacement substitutions than synonymous substitutions among sequences (table 2 ), thus reflecting the common extrinsic evolutionary forces acting on the methionine synthase gene of Arabidopsis and Dunnia. Under such circumstances, given the contrast systems of mating, Dunnia was expected to possess higher genetic divergence than Arabidopsis does. The unexpectedly low levels of polymorphism in the methionine synthase gene, therefore, implied the effects of intrinsic forces, particularly population demography and history.

Low Levels of Organelle DNAs Polymorphism and Consistent Nuclear and Organelle Phylogenies in D. sinensis, Indicating Common Species-Population History
To further evaluate the effects of internal evolutionary forces, genes unlinked to the candidate locus, such as the ribosomal ITS of mtDNA and the intergenic spacer between the atpB and rbcL genes of the cpDNA sequenced in this study, provide essential information. In D. sinensis, low levels of nucleotide diversity were detected at the rITS of mtDNA ({theta} = 0.0019) and the atpB-rbcL intergenic spacer of cpDNA ({theta} = 0.0022). Compared with the other flowering plants, such as Daucus carota (25 variants from 80 plants), Thymus vulgaris (50 mitotypes from about 400 plants), and Hevea brasiliensis (212 mtDNA variants in 395 accessions screened) (cf. Chiang et al. 2001Citation ), only six mitotypes were detected from 87 samples in D. sinensis. The genetic divergence of the rITS mtDNA was much lower than that of Cycas taitungensis ({theta} = 0.0264) (Huang et al. 2001Citation ), whereas it was close to that of Kandelia candel ({theta} = 0.0021), a species evolving through a possible bottleneck in East Asia (Chiang et al. 2001Citation ).

cpDNA haplotype variation (7 out of 87 individuals) in D. sinensis was also lower than in other angiosperms, e.g., 13 cpDNA haplotypes in Beta vulgaris ssp. maritima, 23 in white oaks, 11 in Argania, and 13 in Alnus (cf. Huang et al. 2001Citation ). Genetic divergence of cpDNA was much lower than that in C. taitungensis ({theta} = 0.0127) at the atpB-rbcL noncoding spacer (Huang et al. 2001Citation ).

Consistent results showing low levels of genetic variation at the two unlinked organelle genes and the candidate locus, methionine synthase gene, reflected the effects of the common history and demography that these genes evolved through. Consistent phylogenies of the three genes (figs. 3 and 4 ) at the level of geographical regions supported the hypothesis of a long isolation between populations of the western region (YC) and the eastern region (LM, TS, XH, and ZH). Nested clade analyses on organelle DNAs using the program GeoDis (Posada, Crandall, and Templeton 2000Citation ) all indicated past fragmentation between the two geographical regions and restricted gene flow within regions (data not shown). The monophyly of clade 3-1 and that of clade 4-1 of the gene tree of the methionine synthase gene (figs. 2 and 4 ), both of which were significantly supported by bootstrapping, agreed with the phylogeographic pattern described. Significant genetic differentiation between the western and eastern regions, with FST = 0.702 and Nm = 0.11 (table 5 ), also supported the isolation hypothesis.


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Table 5 Pairwise Comparisons of Genetic Differentiation (FST) and Deduced Gene Flow (Nm) Between Populations and Geographic Regions of Dunnia sinensis

 
The endangered status of D. sinensis may have contributed to the low levels of genetic variation at the three unlinked loci. Five extant populations were severely disturbed during the past century and remained small sized. On the basis of the observation of Tajima's D < 0, complete bottlenecks across all populations may have occurred after destruction of the habitat (cf. Charlesworth B., Morgan, and Charlesworth D. 1993Citation ). Extinction and recolonization processes are invoked to explain the effect of complete bottlenecks (Charlesworth B., Morgan, and Charlesworth D. 1993Citation ), which led to a large decline in both within-population and overall nucleotide diversity (Savolainen et al. 2000Citation ); that is, many genetic elements within populations may have been thereby lost via genetic drift. Elevated levels of genetic differentiation between populations would be encountered because alternate genotypes are fixed in different populations by stochastic drift and natural selection (cf. Purugganan and Suddith 1999Citation ). The estimated FST and deduced Nm values, based on the variation of the methionine synthase gene sequence, revealed the effects of such random evolutionary processes (table 5 ). Gene flow among most populations was limited to about 0.04–0.77 migrants per generation, except for that between LM and XH (Nm = 25.39), which indicated coancestry of the constituting alleles because of a possible recent division.

Replacement Versus Synonymous Substitutions and Positive Selection
In addition to the effects of bottlenecks, given the limited recombination (in this study Rm = 1 in exon sequences), reduction of genetic variability can also be caused by background selection and hitchhiking (Charlesworth B., Morgan, and Charlesworth D. 1993Citation ; Charlesworth D., Charlesworth B., and Morgan 1995Citation ). Background selection against deleterious mutations reduces neutral polymorphism at closely linked sites. Charlesworth D., Charlesworth B., and Morgan (1995)Citation pointed out that when a deleterious mutation is eliminated from a population, selectively neutral mutations linked to it are eliminated as well. In contrast, when an advantageous mutation is selected, the neutral variant sites linked to it will also be fixed by hitchhiking (Wiehe and Stephan 1993Citation ). Both effects are striking in regions of low recombination and lead to the depletion of genetic variability within populations.

Negative values of Tajima's tests, although nonsignificant, at the methionine synthase gene in both Dunnia and Arabidopsis (table 2 ) suggested an excess of rare variants (cf. Charlesworth D., Charlesworth B., and Morgan 1995Citation ), a pattern that fits with the background selection hypothesis. Nevertheless, recovery from a population bottleneck or recent demographic expansion can also lead to a transient excess of rare variants. Generally, background selection acts exclusively on a single gene, and would have no effect on unlinked loci, whereas the demongraphic processes affect all genomes profoundly. In D. sinensis the fact that all three unlinked loci possessed low levels of genetic variation and excessive singletons among sequences contradicted the background selection model. In contrast to the effects of background selection restricted to a single locus and tightly linked DNA segments, the bottleneck hypothesis, with reduced genetic variation at loci from all genomes, seems more plausible in this study.

Methionine synthase is a key enzyme for the synthesis of the aspartate-derived methionine, which is the immediate precursor of AdoMet. AdoMet metabolism has been illustrated to be associated with plant growth and pathogen interactions. Thus, the evolution of the methionine synthase gene in D. sinensis allowed a test of the positive selection hypothesis. That is, on the basis of all available statistical tests, one of the central interests of the study is to investigate if the Dunnia data reject the null hypothesis of a pure drift mutation process. Although nucleotide diversity at synonymous sites ({theta} = 0.00815) was higher than that at nonsynonymous sites ({theta} = 0.00429), indicating that strong positive selection might not be the major force causing the reduction of genetic diversity at the methionine synthase gene in D. sinensis, 10 replacement substitutions versus 6 synonymous substitutions among lineages reveals that advantageous mutations might be favored. In addition, excessive singletons in intron sequences, which usually evolve in accordance with neutral expectations, are also indicative of the effects of such an extrinsic force. Likewise, 57 replacement changes versus 10 synonymous changes in the same DNA region of the locus of Arabidopsis and 337 replacements versus 68 synonymous changes in the AdoMet synthase gene of Actinidia were detected, indicating a similar mode of natural selection in the aforementioned functional loci.

The relative rates of nonsynonymous (dN) and synonymous (dS) substitutions can yield important clues as to the nature of selection acting to shape nucleotide variation. A higher rate of dN than of dS (i.e., dN/dS > 1) is generally considered as unequivocal evidence of positive selection (Kimura 1983Citation ). At the methionine synthase locus in Arabidopsis, positive selection was ascertained on the basis of dN/dS (=0.4284/0.3697) > 1 among ecotypes (Peleman et al. 1989Citation ). Likewise, dN/dS (ranging from 1.76 to 1.94) occurred in the AdoMet synthase gene of Actidinia chinensis (Whittaker, Smith, and Gardner 1995Citation ). Dominant Darwinian selection may have made different haplotypes favored in various habitats, leading to high levels of species-wide polymorphism in inbreeding Arabidopsis ({theta} = 0.0676) and outcrossing Actidinia ({theta} = 0.2289). In contrast, most comparisons between sequences of D. sinensis demonstrated higher synonymous changes than replacements.

However, when the coding region was partitioned into exon segments, higher nucleotide variation was measured at nonsynonymous sites than at synonymous sites in most exons except exon 1 (table 3 ). Four and three replacement substitutions were detected in exons 2 and 3, respectively, whereas no synonymous changes occurred. In exons 2 and 4, among the nonsynonymous changes, the rate of radical changes (pNR) was greater than that of conservative differences (pNC), according to analyses based on the methodology of Hughes, Ota, and Nei (1990)Citation . Exons were seemingly under different selective pressures.

In our investigation, pairwise comparisons of methionine synthase gene sequences of D. sinensis revealed that dN/dS at 13 out of 15 nodes between closely related haplotypes was greater than 1 for one exon or two (fig. 4 ). Variable but weak selective pressure on each exon provides an explanation for such a dN/dS distribution pattern (Yang, Swanson, and Vacquier 2000Citation ). Geographically, the most closely related haplotypes are sympatrically distributed (fig. 4 ). One explanation is that the methionine synthase gene may be under pressure to evolve in order to colonize in response to the different habitats (cf. Purugganan and Suddith 1998Citation ). Within clade 3-1 of YC (western region), exons 2 and 3 were involved in most of the evolution of Darwinian selection, whereas exons 2 and 4 contributed to the positive selection in the eastern region. The haplotype network revealed patterns of adaptive radiation from geographically widespread ancestral haplotypes, i.e., 1-8 and 1-12 (fig. 4 ). For example, haplotype 1-8 of populations LM, XH, and ZH diverged to haplotype 1-11 (XH) and clade 2-3 (TS); haplotype 1-12 of populations LM and XH diverged to 1-13 (LM), 1-14 (XH), and clade 2-4 (XH). Divergence from the ancestral haplotype 1-1 may also suggest adaptive radiation to microhabitats in the YC population (western geographic region).


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
In this investigation we examined the selection modes at the methionine synthase gene. To date, sequences of multiple samples are only available in Dunnia (of this study) and Arabidopsis. In both species significant negative Fu & Li's D* statistic and nonsignificant negative Tajima's D statistic were obtained, indicating a similar apportioning pattern of DNA polymorphisms caused by common selective pressure. Unambiguous positive selection was detected in Arabidopsis on the basis of the ratio of nonsynonymous and synonymous substitutions. In contrast, weak and variable positive selection was manifested in D. sinensis on the basis of replacement sites rather than synonymous sites in each exon, pNR/pNC > 1 in exons 2 and 4, and the distribution of dN/dS > 1 at one exon or two between closely allied haplotypes. The effects of positive selection may have been blurred to some extent by species demography, as indicated by the lower level of nucleotide diversity than in the inbreeding Arabidopsis and the low variation of organelle DNAs. Significant genetic differentiation among populations of D. sinensis and consistent gene phylogenies among genes at geographical levels suggested that the evolution of the methionine synthase gene has been shaped by past fragmentation and recent bottlenecks, which led to the loss of DNA polymorphism and to elevated population differentiation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
This work was supported by grant NSC 90-2311-B-006-001 from the National Science Council, Taiwan, and grant 30170070 from the Chinese National Science Foundation.


    Footnotes
 
Richard Thomas, Reviewing Editor

Keywords: Dunnia sinensis gene genealogy methionine synthase gene organelle DNAs population demography positive selection Back

Address for correspondence and reprints: T. Y. Chiang, Department of Biology, Cheng-Kung University, Tainan, Taiwan 701, ROC. tychiang{at}mail.ncku.edu.tw Back


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 Introduction
 Materials and Methods
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 Acknowledgements
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Accepted for publication April 17, 2002.





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