*Department of Biology, Taiwan Normal University, Taipei, Taiwan, ROC;
South China Institute of Botany, Chinese Academy of Sciences, Guangzhou, People's Republic of China;
Institute of Botany, Academia Sinica, Taipei, Taiwan, ROC;
Department of Biology, Cheng-Kung University, Tainan, Taiwan, ROC
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Abstract |
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Introduction |
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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. 2000
; Yang, Swanson, and Vacquier 2000
). 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 1995
). For the estimates of the relative effects of these evolutionary forces, some genetic variation is certainly required. Kaneko et al. (1998)
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 1995
). Use of multiple loci has thus recently become informative in molecular population genetics (Purugganan and Suddith 1999
). 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 1989
).
Dunnia sinensis Tutch. (Rubiaceae), an endangered species, is restricted to the southern part of Guangdong Province in the People's Republic of China (Ridsdale 1978
). 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 100600 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 1991
) as well as the methionine synthase in D. sinensis.
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Materials and Methods |
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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 1998
], or mtDNA rITS [Chao, Sederoff, and Levings 1984
]), 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. 2001
) by calculating Kimura's (1980)
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 1994
) in a hierarchical manner (cf. Chiang and Schaal 1999
).
Levels of genetic diversity within populations were quantified with estimates of nucleotide divergence () from S (Watterson 1975
) using DnaSP (Version 3.14; Rozas and Rozas 1999
), where
= 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 1993
). 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)
, in a pairwise manner for comparisons, using PAML (Yang 1999
). Hughes, Ota, and Nei (1990)
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. 2000
). When pNR > pNC, a directional change in amino acid sequence is indicated, thus suggesting positive selection.
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Results |
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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 statistic. As expected, there was a higher diversity in the intron region (
= 0.0070) than in the exon region (
= 0.0052) (table 2
). In the exons, nucleotide diversity was higher at synonymous sites (
= 0.00815) than at nonsynonymous sites (
= 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 (
) 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 (
) 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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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 1986
). 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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Discussion |
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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 ( = 0.0019) and the atpB-rbcL intergenic spacer of cpDNA (
= 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. 2001
), 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 (
= 0.0264) (Huang et al. 2001
), whereas it was close to that of Kandelia candel (
= 0.0021), a species evolving through a possible bottleneck in East Asia (Chiang et al. 2001
).
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. 2001
). Genetic divergence of cpDNA was much lower than that in C. taitungensis (
= 0.0127) at the atpB-rbcL noncoding spacer (Huang et al. 2001
).
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 2000
) 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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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. 1993
; Charlesworth D., Charlesworth B., and Morgan 1995
). Background selection against deleterious mutations reduces neutral polymorphism at closely linked sites. Charlesworth D., Charlesworth B., and Morgan (1995)
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 1993
). 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 1995
), 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 ( = 0.00815) was higher than that at nonsynonymous sites (
= 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 1983
). 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. 1989
). Likewise, dN/dS (ranging from 1.76 to 1.94) occurred in the AdoMet synthase gene of Actidinia chinensis (Whittaker, Smith, and Gardner 1995
). Dominant Darwinian selection may have made different haplotypes favored in various habitats, leading to high levels of species-wide polymorphism in inbreeding Arabidopsis (
= 0.0676) and outcrossing Actidinia (
= 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)
. 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 2000
). 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 1998
). 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).
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Conclusions |
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Acknowledgements |
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Footnotes |
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Keywords: Dunnia sinensis
gene genealogy
methionine synthase gene
organelle DNAs
population demography
positive selection
Address for correspondence and reprints: T. Y. Chiang, Department of Biology, Cheng-Kung University, Tainan, Taiwan 701, ROC. tychiang{at}mail.ncku.edu.tw
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