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(American Journal of Botany. 2003;90:561-570.)
© 2003 Botanical Society of America, Inc.

Population Biology

Contrasting selection modes at the Adh1 locus in outcrossing Miscanthus sinensis vs. inbreeding Miscanthus condensatus (Poaceae)1

Yu-Chung Chiang2, Barbara A. Schaal2, Chang-Hung Chou3, Shong Huang4 and Tzen-Yuh Chiang5,6

2Department of Biology, Washington University, St. Louis, Missouri 63119 USA; 3National Pingtung University of Science and Technology, Pingtung, Taiwan; 4Department of Biology, Taiwan Normal University, Taipei, Taiwan 116; 5Department of Biology, Cheng-Kung University, Tainan, Taiwan 701

Received for publication June 20, 2002. Accepted for publication November 12, 2002.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We estimated DNA sequence variation of the Adh1 locus in the outcrossing Miscanthus sinensis (Poaceae) and its close selfing relative, M. condensatus. Tajima's test of selection is significantly negative for both overall exons and replacement sites in M. sinensis. Among its entire sample, nucleotide diversity of nonsynonymous sites is higher than that of synonymous sites. A McDonald and Kreitman test of neutrality indicates an excess of intraspecific replacement polymorphisms, suggesting possible directional selection toward advantageous mutants. However, frequent intragenic recombination suggests both purifying and positive selection is unlikely. Recent demographic expansions coupled with relaxation of purifying selection may have resulted in elevated genetic diversity at the Adh1 locus as well as the trnL-trnF intergenic spacer of cpDNA in this outcrossing species. In contrast, low levels of genetic diversity were detected at both the Adh1 locus and the cpDNA spacer in M. condensatus, consistent with bottlenecks associated with selfing in all populations. While Tajima's D and Fu and Li's F statistics did not reveal deviation from neutrality at the Adh1 locus in M. condensatus, 12 replacements vs. 10 synonymous changes were detected. Based on pairwise comparisons of the dN/dS ratio, lineages of closely related populations of the species distributed along saline habitats appeared to be under directional selection.

Key Words: alcohol dehydrogenase 1 • mating systems • Miscanthus condensatus • Miscanthus sinensis • modes of selection • Poaceae • population demography


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
One of the fundamental goals in the study of molecular evolution is to understand mechanisms that create and maintain genetic differences among evolutionary lineages. The levels and apportionment of genetic variation at a functional gene are governed not only by natural selection and recombination at the locus itself but are also inevitably affected by population demography, systems of mating, and migration, as the genes are carried by individuals that exist within populations. Homologous DNA sequences contain information about genealogical relationships and the imprints of the evolutionary forces that have shaped the genealogy. Gene genealogies have been widely used for the assessment of historical processes, such as hybridization (Small et al., 1998 ), domestication (Eyre-Walker et al., 1998 ; Small et al., 1999 ), and geographical assortment of lineages (Avise, 2000 ). But untangling the effects of processes such as selection and recombination, which operate directly on a sequence, from population processes such as genetic bottlenecks and demographic expansions has been difficult due to both a paucity of loci under documented selection and the limits of statistical resolution (Simonsen et al., 1995 ). In this study we examine selection at the alcohol dehydrogenase one (Adh1) locus in a pair of grass species with contrasting breeding systems and compare the effects of molecular vs. population level processes on the maintenance of polymorphisms.

The Adh gene family encodes a key enzyme, alcohol dehydrogenase, in the glycolytic pathway and provides an ideal model for examining effects of evolutionary forces operating directly on a gene vs. population-level history and demography. It is well known that gene duplication has frequently occurred in plants. Most angiosperms possess two Adh loci (Adh1 and Adh2), while Asteraceae, Fabaceae, and Arabidopsis thaliana have only a single locus (Clegg et al., 1997 ) and rice and Leavenworthia (Brassicaceae) contain a third locus, Adh3, a recent duplication of Adh2. To date, nucleotide sequences of Adh genes have been documented across a wide range of dicots (Leavenworthia: Charlesworth et al., 1998 ; peonies: Sang et al., 1997 ; yam: Terauchi et al., 1997 ; Gossypium: Small et al., 1998 ; Arabis and Arabidopsis: Miyashita et al., 1998 ) and monocots (maize: Eyre-Walker et al., 1998 ; barley: Cummings and Clegg, 1998 ; Lin et al., 2001 ; Pennisetum: Gaut and Clegg, 1993 ; Poaceae: Gaut et al., 1999 ; Arecaceae: Morton et al., 1996 ). Most studies have found low levels of genetic variation, with nucleotide diversity ({theta}) in the range of 0.0027 (Adh1 in barley) to 0.0063 (Adh1 in an inbreeding Arabidopsis), with the two exceptions of maize (0.0250 at Adh1) and barley (0.0137 at Adh3). Balancing selection and intralocus recombination were hypothesized to account for the maintenance of genetic polymorphism in these exceptional cases (Lin et al., 2001) . In other species with low levels of polymorphism, purifying selection could yield low diversity at the Adh locus if it were neutral and had little or no recombination (cf. Charlesworth et al., 1995 ), although hitchhiking and bottlenecks can also be factors (Savolainen et al., 2000 ).

In order to discern among these evolutionary forces, the sequences of unlinked loci can be compared to the candidate gene (Simonsen et al., 1995 ). Using multiple loci in recent studies has been exceptionally informative for molecular population genetics (Purugganan and Suddith, 1999 ; Kuittinen and Aguade, 2000 ). In this study, we analyze the sequences of a candidate gene, the Adh1 locus, as well as the intergenic spacer between the trnF and trnL genes of the chloroplast genome in Miscanthus sinensis and its close relative, M. condensatus, a grass genus closely related to sugarcane and maize (Chou et al., 1999 ). The organelle-DNA noncoding spacer was chosen because it is nearly neutral (cf. Chiang and Schaal, 2001) and is unlinked to the Adh locus. The pattern of cpDNA spacer evolution should follow neutral expectations (Tajima, 1989 ).

Ecological preferences and systems of mating distinguish these two closely related taxa. The outcrossing species M. sinensis is an assemblage of well-documented, morphologically distinct intraspecific taxa that are distributed along an altitudinal gradient. Besides the widespread var. sinensis of the Chinese mainland and Japan, taxa that are distributed in high mountains (var. transmorrisonensis), middle elevation grasslands (var. formosanus), and low elevation wastelands (var. glaber) in Taiwan have been recorded. Like many other angiosperms of Taiwan, the evolution of M. sinensis was shaped by historical glaciation events (cf. Lu et al., 2001 , 2002 ). Recent investigations revealed that var. transmorrisonensis was possibly the first taxon invading at high elevations of the island during the postglacial recolonization (Chou et al., 1999 ). Range expansion followed with the generation of two other taxa in habitats of middle and low elevations. In contrast, the selfing M. condensatus is morphologically homogeneous. Populations of this highly selfing species are distributed in saline habitats, along the islands of Japan, Ryukyu, and two offshore islands of Taiwan (Chou et al., 1999 ).

Although species-wide genetic variation may not necessarily be low in selfing species (cf. Savolainen et al., 2000) , inbreeding generally predicts a reduction in genetic diversity and effective recombination. Similar to many other inbreeding species (Liu et al., 1999 ), low diversity was suggested by random amplified polymorphic DNAs (RAPD) fingerprinting within and between populations of the self-fertilizing M. condensatus (Chou et al., 2000) . Given the contrasting mating systems among Miscanthus species, a strong association between the levels of genetic variation at both cpDNA and the Adh1 locus and outcrossing mating may be expected.

The function of the alcohol dehydrogenase 1, a protein encoded by Adh1, is associated with physiological responses to oxygen (Freeling and Bennett, 1985 ) and cold stress in plants (Dolferus et al., 1994 ). In the M. sinensis complex, which has apparently undergone adaptation to various elevations, purifying or positive selection (i.e., extrinsic forces) operating on the functional Adh1 gene should reduce the genetic variation within intraspecific taxa. However, demographic processes (i.e., intrinsic forces), such as possible population expansions in M. sinensis complex coupled with relaxation in purifying selection (cf. Ballard and Kreitman, 1994 ) and genetic recombination, may ameliorate the effects of natural selection on the locus and result in high levels of genetic diversity. Based on discrepancies between the cpDNA locus and the Adh1 gene, the relative contribution of the two evolutionary processes, i.e., natural selection and population demography, can be discerned.

The objectives of the current study are to investigate the following: (1) Does the outcrossing M. sinensis possess higher levels of genetic variation at the Adh1 locus than selfing M. condensatus? (2) Is the variation of Adh1 locus shaped by the same mode of selection in both species, showing a common pattern in the apportionment of genetic variation? (3) What is the relative effect of selection vs. population demography on molecular evolution of the Adh1 gene? (4) Does the Adh1 gene in M. condensatus also show low levels of variation, as was detected previously by RAPD analysis? (5) Can organelle DNA provide sufficient information for discerning intrinsic from extrinsic evolutionary forces?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant materials
A large collection of Miscanthus, representing a broad altitudinal range of M. sinensis (45 accessions) and a latitudinal range of M. condensatus (20 accessions), was obtained from Japan, mainland China, and Taiwan. Two individuals of the outgroup, M. floridulus, were also collected. Seven to 16 individuals were collected from 2–3 populations from each taxon of the M. sinensis complex, and 3–5 plants from each of five island populations of M. condensatus across Ryukyu and Green Islet of Taiwan. Sources of all plant materials and GenBank accession numbers for DNA sequences have been archived at the American Journal of Botany Supplementary Data website (http://ajbsupp.botany.org/v90/). Fresh leaf tissue was ground in liquid nitrogen. Total genomic DNA was extracted with a cetyltrimethylammonium bromide (CTAB) protocol of Murray and Thompson (1980) .

Polymerase chain reaction (PCR) amplification and DNA sequencing
The nuclear Adh1 locus and the intergenic spacer between trnL and trnF genes of cpDNA were amplified and sequenced. Ten nanograms of template DNA was used for each 100 µL amplification reaction. Primers specifically corresponding to the sequences in exon 4 (Adh1-F, 5'-ATAGAGAGTGTTGGAGAGG-3') and exon 9 (Adh1-R, 5'-GTTCTCCATGCGGATGATGC-3') (Dennis et al., 1984 , 1985 ) were used for amplification of the Adh1 locus. For the amplification of the intergenic spacer between the trnL and trnF genes of cpDNA, two universal primers were used (Taberlet et al., 1991 ). The reaction consisted of one cycle of denaturation at 95°C for 4 min, 30 cycles of 1 min denaturation at 92°C, 1 min 15 s annealing at 52°C, and 1 min 30 s extension at 72°C, followed by 10 min extension at 72°C. The PCR products were purified by electrophoresis in 1.0% agarose gel using 1x tris-acetate-EDTA (TAE) buffer. The gel was stained with ethidium bromide and the desired DNA band was cut and eluted using agarose gel purification (QIAGEN, Valencia, California, USA). Purified DNAs were sequenced in both directions by standard methods of the Taq dye deoxy terminator cycle sequencing kit (Perkin Elmer, Wellesley, Massachusetts, USA) on an Applied Biosystems Model 377A automated sequencer (Applied Biosystems, Foster, California, USA). Additional primers were used to complete the sequencing.

Data analysis
Nucleotide sequences were aligned with the program Genetics Computer Group (GCG) Wisconsin Package (version 10.0; Madison, Wisconsin, USA). Neighbor-joining (NJ) 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 a pairwise comparison was used to construct a minimum spanning network in a hierarchical manner (cf. Chiang and Schaal, 1999 ) with the aid of MINSPNET (Excoffier and Smouse, 1994 ). Nucleotide diversity was estimated by Tajima's (1989) {pi} and Watterson's (1975) {theta} statistics. Tests of neutrality and determination of the associated significance as well as the coalescent-based estimations of minimum recombination events were done by using the program DnaSP (Version 3.51, Rozas and Rozas, 1999 ). 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 + 4 Nm) where N is the effective population size and m is the migration rate (Slatkin, 1993 ).

The nonsynonymous/synonymous rate ratio (=dN/dS) is usually used to measure selection pressure at the protein level. If nonsynonymous mutations are beneficial and favored by natural selection, the ratio should be greater than one. The corrected proportions of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) were estimated from the sequences based on maximum likelihood methodology of Yang and Nielsen (1998) , in a pairwise manner for comparisons, using the software PAML (Yang, 1999 ). A McDonald and Kreitman (1991) test of neutrality was employed by comparing the distribution of synonymous and nonsynonymous (replacement) variation within and between species. Under neutrality, the ratio of replacement to synonymous fixed substitutions (differences) between species should be the same as the ratio of replacement to synonymous polymorphisms within species.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Nucleotide sequence polymorphism and tests for genetic recombination
DNA sequences were determined for all 67 samples for both loci. The Adh1 region is 1470 base pairs (bp) in length, including 912 bp of exon sequence (partial sequence of exons 4 and 10 and complete sequence of exons 5–9) and 558 bp of intron sequence. A high level of polymorphism was detected in the exons: 145 sites (84 replacements and 61 synonymous changes) in M. sinensis and 22 sites (12 replacements and 10 synonymous substitutions) in M. condensatus (Table 1). Intron polymorphism was also high; 121 sites in M. sinensis and 13 sites in M. condensatus were polymorphic (Table 1). In total, 39 haplotypes of 45 samples (86.7%) of M. sinensis and 10 haplotypes of 20 samples (50.0%) of M. condensatus were identified based on polymorphic sites of exon sequences, while 41 intron haplotypes (91.1%) of M. sinensis and 13 intron haplotypes (65%) of M. condensatus were identified.


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  		Table 1. Estimates of nucleotide diversity of Adh1 gene and trnL-trnF intergenic spacer of cpDNA and test statistics for selection at Adh1 locus in Miscanthus. Observed number (O) of singletons is compared to neutral expectation (E). bp = base pairs; *P < 0.05; **P < 0.02; ***P < 0.01

 
The distributions of polymorphic sites across haplotypes in the entire exon sequences and in partitions of replacement and synonymous sites as well as introns are summarized in Table 1. The distributions of polymorphic sites in exons are illustrated in Fig. 1. For the whole exon sample of 10 haplotypes of M. condensatus, 13 mutations of the 22 polymorphic sites occurred once, six occurred twice, two occurred three times, and one occurred four times (Fig. 1). Likewise, the number of singletons of the 13 Adh1 intron haplotypes was five in M. condensatus and was 85 of 39 exon haplotypes and 56 of 41 haplotypes in M. sinensis (Table 1; Fig. 1). The distribution of allelic frequencies at polymorphic sites was compared to the expectation under a neutral mutation model, based on the methods of Tajima (1989) . An excess over the expectation under the neutrality model, 35.2 in M. sinensis and 8.6 in M. condensatus, was estimated in the class of unique nucleotide polymorphisms in exons of each species. The allelic frequency distribution is significantly skewed toward rare alleles (Fig. 1). Excessive singletons over the neutral expectation were also observed in nonsynonymous sites (9 relative to the expectation of 4.7) of M. condensatus as well as in replacements (62 relative to the expectation of 20.9) and synonymous sites (23 relative to the expectation of 14.3) of M. sinensis (Table 1). Likewise, an excess of singletons (56 relative to the expectation of 28.8) was detected at Adh1 introns of M. sinensis (Table 1). Four haplotypes in M. condensatus and eight haplotypes in M. sinensis were identified based on sequences of the trnL-trnF intergenic spacer. There were three and six polymorphic sites for nucleotide substitutions in M. condensatus and M. sinensis, respectively (Table 1).



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  		Fig. 1. Expected number of polymorphic sites (exp) under a neutral model calculated by using equation 51 of Tajima (1989) and observed number of polymorphic sites (obs) at various times of occurrence (1–5) in the entire Adh1 gene in 10 haplotypes of Miscanthus condensatus (A) and at times of occurrence (1–20) in 39 haplotypes of M. sinensis (B). s, number of segregating sites; n, number of haplotypes. Figure abbreviations: FL = M. floridulus; c = M. condensatus; s = var. sinensis; t = var. transmorrisonensis; f = var. formosanus; g = var. glaber

 
Generally, recombination in inbreeding species should be more constrained compared to outcrossing plants. In the predominantly self-fertilizing M. condensatus, no recombination events were detected at either region studied (Table 1). In contrast, recombination may have occurred frequently at both introns (Rm = 11) and exons (Rm = 11) of the Adh1 gene of M. sinensis. A single intralocus recombination was detected at the cpDNA locus of M. sinensis.

Estimates of nucleotide diversity and statistical tests of neutrality
Nucleotide diversity of the Adh1 locus in M. sinensis ({theta} = 0.064 and {pi} = 0.040 for introns and {theta} = 0.037 and {pi} = 0.020 for exons) was much higher than that in M. condensatus ({theta} = 0.010 and {pi} = 0.009 for introns and {theta} = 0.008 and {pi} = 0.006 for exons) (Table 1). The variation at all silent sites (introns plus synonymous positions of exons) was higher in M. sinensis ({theta} = 0.0329 and {pi} = 0.0228) than in M. condensatus ({theta} = 0.0061 and {pi} = 0.0059). Nonsynonymous substitutions were also more variable in the outcrossing species. Within M. sinensis var. sinensis, higher levels of nucleotide diversity ({theta} = 0.020) were observed relative to other varieties (Table 2). Likewise, nucleotide diversity of the trnL-trnF noncoding spacer was higher in M. sinensis, {theta} = 0.030 and {pi} = 0.048, than in M. condensatus, {theta} = 0.0020 and {pi} = 0.0019.


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  		Table 2. Estimates of nucleotide diversity ({theta}) within and between taxa (above diagonal) and of FST/Nm between taxa of Miscanthus

 
A number of statistical tests were used to determine significant departures from neutrality at the Adh1 locus. Tajima's test has been found to be most powerful for testing alternative hypotheses involving selective sweeps, population bottlenecks, and population subdivision (Simonsen et al., 1995 ). For the exon sequence data of M. sinensis, all statistic test values, Fu and Li's D* = –3.06 and Tajima's D = –1.80, are significant (Table 1), indicating a deviation from neutrality due to an excess of singletons. In contrast, none of these values are significant for M. condensatus. When exon sequence data are partitioned into replacements and synonymous substitutions, a nonsignificant Tajima's D statistic (–0.876) is obtained for the synonymous sites. In addition, all values for both tests are nonsignificant for intron sequences of Adh1 and the cpDNA intergenic spacer.

A McDonald and Kreitman test for neutrality was conducted, based on a prediction that the relative levels of intraspecific replacement and synonymous polymorphisms are correlated with interspecific levels of replacement and synonymous substitutions. Sixty-three synonymous substitutions and 94 replacement sites of the 162 variable substitutions in the Adh1 locus occurred within species, suggesting an excess of intraspecific replacements. The comparison between M. sinensis and M. condensatus revealed one fixed replacement and four fixed synonymous differences. Nevertheless, the excess of polymorphic replacement sites within M. sinensis was not significant (G = 3.39, P = 0.066).

Phylogeny of Adh1 and trnL-trnF cpDNA spacer and population differentiation
Neighbor-joining trees of the Adh1 locus and the cpDNA spacer were congruent at the species level. The monophyly of each species was supported, indicating separate lineages (Figs. 2 and 3). Based on the minimum spanning network, seven clades of the 39 Adh1 haplotypes in M. sinensis were identified (Fig. 2). Most clades are widespread across intraspecific taxa, while clades B and C are restricted to M. sinensis var. sinensis and clade F is restricted to var. transmorrisonensis. In the Adh1 network, most interior nodes consisted of haplotypes of var. sinensis (e.g., a07, s09, s04, s03, and s01) and var. transmorrisonensis (e.g., t13, t08, and t16), indicating their likely ancestral status (cf. Donnelly and Tavaré, 1986 ; Golding, 1987 ; Crandall and Templeton, 1993 ). The cpDNA network revealed a similar pattern of allelic apportionment among taxa (Fig. 3). Haplotypes of var. sinensis were all nested in the gene network as interior nodes. Interior nodes of s01 and g01 were distributed in var. glaber and var. transmorrisonensis, while all exterior nodes were restricted to one taxon, such as t01 and t07 of var. transmorrisonensis and g09 of var. glaber. In var. condensatus, haplotypes c08 of Green and Ishigaki Islets and c10 of Amami and Miyako Islets were nested in the network as interior nodes.



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  		Fig. 2. Minimum spanning network of the Adh1 gene of M. condensatus and M. sinensis with a root of M. floridulus. Clade A of group I and clades B–H of group II are indicated. Bootstrap values (%) of each group are indicated. "0" represents a hypothetical common ancestor

 


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  		Fig. 3. Minimum spanning network of the trnL-trnF intergenic spacer of cpDNA of M. condensatus and M. sinensis with a root of M. floridulus. Bootstrap values (in percentages) of major groups are indicated. "0" represents a hypothetical common ancestor. In the group I, the geographical distribution of each haplotype is indicated in parentheses: G = Green Islet, I = Ishigaki Islet, A = Amami-O-Shima, M = Miyako Islet, T = Tanega Islet. In the group II, the distribution of haplotypes among taxa is indicated in parentheses

 
Based on nucleotide variation of the Adh1 locus, M. sinensis was significantly differentiated from M. condensatus. At this locus, intraspecific taxa of M. sinensis complex endemic to Taiwan were differentiated from var. sinensis, with FST ranging from 0.26 to 0.31, while they showed no significant differentiation among each other (Table 2). High levels of genetic differentiation were also detected among populations of M. condensatus, with FST values ranging from 0.53 to 0.96 (Table 3).


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  		Table 3. Estimates of genetic differentiation (FST, below diagonal) and migration (Nm, above diagonal) between island populations of M. condensatus based on Adh1 nucleotide variation

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Reduced variability at Adh1 locus and cpDNA intergenic spacer in inbreeding M. condensatus
The Adh1 region of the inbreeding species M. condensatus contains nucleotide variation at levels comparable to other plant species, such as Arabis and Arabidopsis (Table 4). Nevertheless, the variability at the Adh1 locus in M. condensatus is much lower than that of outcrossing M. sinensis. In M. condensatus, the levels of nucleotide diversity at exons and introns of Adh1 locus are reduced 4.7–7-fold relative to those in M. sinensis (Table 1). The difference between inbreeding and outcrossing Miscanthus is larger than has been empirically found in comparisons of other outcrossing and selfing species (Hamrick and Godt, 1996 ). These data raise the possibility that other evolutionary forces besides the breeding system have influenced the distribution of genetic variation.


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  		Table 4. Estimates of nucleotide diversity, {theta}, and test statistics of selection on Adh loci across angiosperms (*P < 0.05; **P < 0.02)

 
With limited recombination, reduction of genetic variability can be the result of purifying selection, hitchhiking, and genetic bottlenecks (Charlesworth et al., 1993 , 1995 ; Hudson and Kaplan, 1994 ). Background selection against deleterious mutations also reduces neutral polymorphism at closely linked sites. Charlesworth et al. (1995) point out that when a deleterious mutation is eliminated from a population, selectively neutral mutations linked to it are eliminated as well. Moreover, when an advantageous mutation is selected, the neutral variant sites linked to it will also be fixed by hitchhiking. Selective sweeps will eliminate variation from DNA segments that have lower rates of recombination (Wiehe and Stephan, 1993 ). Nevertheless, substantial differentiation among populations in M. condensatus shown by FST values for Adh1 (Table 3) is consistent with the effects of purifying selection (Stephan et al., 1998 ; Pamilo et al., 1999 ). Like Adh1 in barley (Cummings and Clegg, 1998 ) and Arabis (Kawabe et al., 1997 ), negative but nonsignificant values of Tajima's tests at the overall Adh1 sites as well as nonsynonymous sites (Table 4) indicate an excess of rare variants (cf. Charlesworth et al., 1995 ), suggesting purifying selection or population expansion.

Populations of selfing plants can be founded by single individuals, so that severe bottlenecks could contribute to the reduced genetic variability within populations (Charlesworth et al., 1993 ). Selfing plants also display elevated levels of genetic differentiation between populations as alternate genotypes are fixed in different populations by stochastic drift and natural selection (cf. Purugganan and Suddith, 1999 ). In contrast to the effects of purifying selection, which is restricted to a single locus and tightly linked DNA segments, bottlenecks usually cause reduced genetic variation at loci across the genome. In this study, levels of nucleotide diversity at the trnL-trnF spacer of the chloroplast genome in M. condensatus are reduced 15-fold relative to nucleotide diversity in M. sinensis. Low levels of cpDNA sequence variation in M. condensatus are consistent with the hypothesis of genetic bottlenecks. However, in the circumstances as observed here (low levels of genetic variation and significant population differentiation), statistical tests provide little power in distinguishing purifying selection from bottlenecks. Generally, purifying selection acts exclusively on a single gene and would have no effect on unlinked loci.

Our previous study of RAPD variation in M. condensatus found low levels of genetic polymorphisms across populations, which favors a bottleneck hypothesis (Chou et al., 2000 ). Repeated extinction and recolonization can yield complete bottlenecks (Charlesworth et al., 1993 ), which lead to a large decline in both within-population and overall nucleotide diversity (Pannell and Charlesworth, 1999 ; Savolainen et al., 2000 ).

Although strong selective sweeps cannot alone explain the reduction of genetic diversity both at Adh1 and cpDNA intergenic spacer loci in M. condensatus, the number of replacements vs. synonymous changes among lineages (12 : 10) suggests some level of selection favoring some specific mutants. The nonsynonymous/synonymous rate ratio (= dN/dS) is usually used to measure selection pressure at the protein level. If nonsynonymous mutations are beneficial and favored by natural selection, the ratio should be greater than one. In our investigation, pairwise comparisons of Adh1 sequences of M. condensatus reveal that dN/dS > 1 occurs exclusively between closely related haplotypes, including pairs of (1, 2), (3, 5), (6, 7), and (8, 9) (Table 5; Fig. 4). Variable but weak selective pressure can provide an explanation for the pattern of dN/dS distribution, in which a ratio >1 occurred between closely related haplotypes and a ratio <1 occurred between distantly related haplotypes, while all having low levels of radical nonsynonymous changes (Yang et al., 2000) . Geographically, all closely related haplotypes are sympatrically distributed (Fig. 4). Alcohol dehydrogenase may be under pressure to evolve in response to the different ecological conditions at saline habitats, in which M. condensatus is distributed (cf. Purugganan and Suddith, 1998 ).


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  		Table 5. Pairwise comparisons of dN (x1000):dS (x1000) among haplotypes of Adh1 gene in M. condensatus

 


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  		Fig. 4. Neighbor-joining tree of the Adh1 haplotypes of M. condensatus. Branches with dN/dS > 1 are in bold

 
We also calculated guanine and cytosine (G + C) content at third-position sites for each sequence of M. sinensis. Adh1 exons appear to have higher G + C content (48.0%) relative to introns (37.1%) and the cpDNA spacer (33.3%). High G + C content leads to biased codon usage, which is indicated with effective number of codons (ENC) = 48 and codon bias index (CBI) = 0.4. Both the excess of G and C codons and slight codon usage bias suggest that some levels of selection play a role in determining the rate of substitution at synonymous sites in Adh1 lineages of M. condensatus (cf. Comeron and Kreitman, 1998 ; Silva and Kidwell, 2000 ).

Relaxation of background selection at Adh1 locus in M. sinensis
This study found that Tajima's D was significantly negative for nucleotide variation at both overall and nonsynonymous sites (D = –1.803 [P < 0.05] and –2.380 [P < 0.01], respectively) in the Adh1 region of outcrossing M. sinensis (Table 1). The negative deviation was manifested as an excess of singleton nucleotide sites. Four possible evolutionary forces can result in the excess of low-frequency polymorphisms: hitchhiking (Kaplan et al., 1989 ), purifying selection (Tajima, 1989 ), recent bottlenecks (Charlesworth et al., 1993 ), and recent population expansion (Cummings and Clegg, 1998 ). While purifying selection usually eliminates deleterious mutations in a population, positive selection fixes alleles that are advantageous. Both effects will be striking in regions of low recombination and lead to reduced genetic variability. Elevated genetic polymorphism at the Adh1 locus (Tables 1, 4), associated with 11 minimum recombination events among lineages of M. sinensis, cannot be a result of negative selection or hitchhiking.

Although the restoration of neutral polymorphism by genetic drift and mutation following population bottlenecks can result in a transient excess of rare alleles (Charlesworth et al., 1993 ), both partial and complete bottlenecks usually reduce the nucleotide diversity of all loci. In M. sinensis, not only Adh1 exons, but also intron regions and the cpDNA noncoding spacer have comparatively high levels of variability. In addition, bottlenecks usually result in significant genetic differentiation between populations due to an extinction and recolonization process among metapopulations (Pannell and Charlesworth, 1999 ; Savolainen et al., 2000 ). In this study, within the M. sinensis complex, although var. sinensis is genetically distinct from other intraspecific taxa, low levels of population subdivision were detected among var. glaber, var. formosanus, and var. transmorrisonensis (Table 2). Altogether, the data are not consistent with bottleneck expectations.

Nevertheless, the evolution of Adh1 locus in M. sinensis is non-neutral. A McDonald and Kreitman test indicated an excess of intraspecific replacement polymorphisms, although nonsignificant. As both background and positive selection seem less likely, recent demographic expansion can account for both the excess of singletons and replacement polymorphisms within M. sinensis (cf. Cummings and Clegg, 1998 ). According to the phylogeographic evidence, most intraspecific taxa had experienced extinction-recolonization processes before they expanded (Chou et al., 1999 ). Such demographic expansion should lead to an excess of rare variants at all sites (both synonymous and nonsynonymous) and at all loci (Purugganan and Suddith, 1999 ), due to a relaxation of purifying selection (Ohta, 1992 ; Ballard and Kreitman, 1994 ). In this study, the negative Tajima's D value, which occurs at introns and exons of the nuclear Adh1 gene as well as the cpDNA locus in M. sinensis, supports the hypothesis of demographic expansion.

Diversification of Adh1 in Miscanthus
The scenario indicated by the consistent phylogenies of both Adh1 and the cpDNA trnL-trnF spacer loci and low estimated between-species Nm values (Table 2) suggests a long history of isolation between M. condensatus and M. sinensis (Figs. 1 and 2). Based on a mutation rate of 6.5–7.9 x 10–9 substitutions per site per year at synonymous sites of Adh1 in grasses (Gaut and Clegg, 1991 ; Gaut et al., 1996 ), the coalescence time of populations of M. condensatus with an average dS of 0.0169 (Table 5) can be estimated to be 1.1–1.3 million years ago. The rate of nonsynonymous changes of the species is found to be 1.70 x 10–9 substitutions · site–1 · yr–1, which appears to be higher than the synonymous rate of 2.50 x 10–10 substitutions · site–1 · yr–1 in grasses, due to replacement changes favored by positive selection.

Rates of synonymous changes at Adh1 in M. sinensis are about threefold of that in M. condensatus; this is mostly due to genetic recombination (Table 1). Based on the dS = 0.0457 among haplotypes, the coalescence time of populations in M. sinensis can be estimated at 1.17 x 106 yr before present. The rate of nonsynonymous changes of the species is 4.15 x 10–9 substitutions · site–1 · yr–1.

In contrast to the remarkable genetic differentiation among populations in M. condensatus associated with predominant selfing, most intraspecific taxa of M. sinensis have not attained coalescence at the Adh1 locus (Figs. 2 and 3) due to an apparently short time span since recent demographic expansions. Nevertheless, most endemic taxa, which occupy habitats of different altitudinal zones, were differentiated from the ancestral M. sinensis var. sinensis (Fig. 2 and Table 2). Geological evidence indicates that Taiwan had been linked for a long time to mainland Asia before the formation of the Taiwan Strait (Lin, 1966 ). Given such geological and geographical history, most species of the continental island were originally from the mainland. The minimum spanning network provides insights into migration history. Based on coalescence theory, ancestral haplotypes should occur at the interior nodes of the network and would have a greater probability of producing mutational derivatives (Golding, 1987 ; Crandall and Templeton, 1993 ). Accordingly, var. transmorrisonensis, which possesses haplotypes of interior nodes such as t13 of the clade E, t08 of the clade F, and t16 of the clade H in the network (Fig. 2), was probably the first taxon that migrated into high-elevation Taiwan during the postglacial recolonization. The connection of t13 to the ancestral s01 of var. sinensis suggests such a migratory route.

During glaciation, drastic temperature oscillation pushed a wide range of species toward extinction (cf. Petit et al., 1997 ; Lu et al., 2001 , 2002 ), thereby opening niches to the new invaders, including ancestral populations of var. transmorrisonensis. Following colonization, a demographic expansion possibly resulted in the predominance of the endemic taxon at the high mountain grasslands and elevated the genetic diversity. Based on the connection of most haplotypes to those of var. transmorrisonensis in the Adh1 gene network, an adaptive radiation might have occurred that led to the formation of var. formosanus and var. glaber at middle and low elevations, with subsequent morphological differentiation.

Conclusion
In this investigation, we examined the mode of selection in inbreeding M. condensatus and outcrossing M. sinensis. Systems of mating appear to play a major role in determining the levels of genetic variation. Frequent genetic recombination coupled with relaxation of selection may have contributed to the high polymorphisms in M. sinensis complex. Interpretation of the statistical tests that detect selection is not straightforward. Our study shows that the resolution of Tajima's test is limited for predominantly inbreeding species, even for sequences of cpDNA genome. The depleted genetic variation at Adh1 and cpDNA loci and significant population differentiation provide insight into the identification of bottlenecks as the most likely force in shaping the polymorphism apportionment in M. condensatus. In addition, based on pairwise comparisons of the dN/dS ratio, lineages of closely related populations of the species distributed along saline habitats appear to be under positive selection.


   FOOTNOTES
 
1 The authors thank James Beck, Ana Caicedo, Kuo-Fang Chung, and John Gaskin for critical comments on the manuscript. This research was supported by grants given to C. H. Chou (NSC 84-2311-B001-016, 85-2311-B001-014, and 86-2311-B001-013) and T. Y. Chiang (NSC-89-2311-B006-002-B27 and NSC-88-2311-B006-001-B27) from the National Science Council, Taiwan. Back

6 Author for reprint requests (tychiang{at}mail.ncku.edu.tw ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Avise J. C. 2000 Phylogeography: the history and formation of species. Harvard University Press, Cambridge, Massachusetts, USA

Ballard J. W. O. M. Kreitman 1994 Unraveling selection in the mitochondrial genome of Drosophila. Genetics 138: 757-772[Abstract]

Charlesworth D. B. Charlesworth M. T. Morgan 1995 The pattern of neutral molecular variation under the background selection model. Genetics 141: 1619-1632[Abstract]

Charlesworth D. F. L. Liu L. Zhang 1998 The evolution of the alcohol dehydrogenase gene family by loss of introns in plants of the genus Leavenworthia (Brassicaceae). Molecular Biology and Evolution 15: 552-559[Abstract]

Charlesworth B. M. T. Morgan D. Charlesworth 1993 The effect of deleterious mutations on neutral molecular variation. Genetics 134: 1289-1303[Abstract]

Chiang T. Y. B. A. Schaal 1999 Phylogeography of North American populations of the moss species Hylocomium splendens based on the nucleotide sequence of internal transcribed spacer 2 of nuclear ribosomal DNA. Molecular Ecology 8: 1037-1042[CrossRef]

Chou C. H. Y. C. Chiang T. Y. Chiang 2000 Genetic variability of Miscanthus sinensis var. condensatus based on the RAPD fingerprints. Canadian Journal of Botany 78: 1262-1268[CrossRef]

Chou C. H. S. Huang S. H. Chen C. S. Kuoh T. Y. Chiang Y. C. Chiang 1999 Ecology and evolution of Miscanthus of Taiwan. National Science Council Monthly 27: 1158-1169

Clegg M. T. M. P. Cummings M. L. Durbin 1997 The evolution of plant nuclear genes. Proceedings of the National Academy of Sciences, USA 94: 7791-7798[Abstract/Free Full Text]

Comeron J. N. M. Kreitman 1998 The correlation between synonymous and nonsynonymous substitutions in Drosophila: mutation, selection or relaxed constraints?. Genetics 150: 767-775[Abstract/Free Full Text]

Crandall K. A. A. R. Templeton 1993 Empirical tests of some predictions from coalescent theory with applications to intraspecific phylogeny reconstruction. Genetics 134: 959-969[Abstract]

Cummings M. P. M. T. Clegg 1998 Nucleotide sequence diversity at the alcohol dehydrogenase 1 locus in wild barley (Hordeum vulgare ssp. spontaneum): an evaluation of the background selection hypothesis. Proceedings of the National Academy of Sciences, USA 95: 5637-5642[Abstract/Free Full Text]

Dennis E. S. M. M. Sachs W. L. Gerlach E. J. Finnegan W. J. Peacock 1984 Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize. Nuclear Acids Research 12: 3983-4000[CrossRef]

Dennis E. S. M. M. Sachs W. L. Gerlach E. J. Finnegan W. J. Peacock 1985 Molecular analysis of the alcohol dehydrogenase2 (Adh2) gene of maize. Nuclear Acids Research 13: 727-743[CrossRef]

Dolferus R. M. Jacobs W. J. Peacock E. S. Dennis 1994 Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiology 105: 1075-1087[Abstract]

Donnelly P. S. Tavaré 1986 The ages of alleles and a coalescent. Advanced Applied Probability 18: 1-19

Excoffier L. P. E. Smouse 1994 Using allele frequencies and geographic subdivision to reconstruct gene trees within a species: molecular variance parsimony. Genetics 136: 343-359[Abstract]

Eyre-Walker A. R. L. Gaut H. Hilton D. L. Feldman B. S. Gaut 1998 Investigation of the bottleneck leading to the domestication of maize. Proceedings of the National Academy of Sciences, USA 95: 4441-4446[Abstract/Free Full Text]

Freeling M. D. C. Bennett 1985 Maize Adh1. Annual Review of Genetics 19: 297-323[ISI][Medline]

Gaut B. S. M. T. Clegg 1991 Molecular evolution of alcohol dehydrogenase 1 in members of the grass family. Proceedings of the National Academy of Sciences, USA 88: 2060-2064[Abstract/Free Full Text]

Gaut B. S. M. T. Clegg 1993 Molecular evolution of the Adh1 locus in the genus Zea. Proceedings of the National Academy of Sciences, USA 90: 5095-5099[Abstract/Free Full Text]

Gaut B. S. B. R. Morton B. C. MaCaig M. T. Clegg 1996 Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proceedings of the National Academy of Sciences, USA 93: 10274-10279[Abstract/Free Full Text]

Gaut B. S. A. S. Peek B. R. Morton M. T. Clegg 1999 Patterns of genetic diversification within the Adh gene family in the grasses (Poaceae). Molecular Biology and Evolution 16: 1086-1097[Abstract]

Golding G. B. 1987 Multiple substitutions create biased estimates of divergence times and small increases in the variance to mean ratio. Heredity 58: 331-339

Hamrick J. L. M. J. Godt 1996 Effects of life history traits on genetic diversity in plant species. Philosophical Transactions: Biological Sciences (the Royal Society) 351: 1291-1298[CrossRef]

Hudson R. R. N. L. Kaplan 1994 Gene trees with background selection. In B. Golding [ed.], Non-neutral evolution: theories and molecular data, 140–153. Chapman and Hall, New York, New York, USA

Kaplan N. L. R. R. Hudson C. H. Langley 1989 The ‘hitchhiking’ effect revisited. Genetics 123: 887-899[Abstract/Free Full Text]

Kawabe A. H. Innan R. Terauchi N. T. Miyashita 1997 Nucleotide polymorphism in the acidic chitinase locus (ChiA) region of the wild plant Arabidopsis thaliana. Molecular Biology and Evolution 14: 1303-1315[Abstract]

Kimura M. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 10: 111-120

Kuittinen H. M. Aguade 2000 Nucleotide variation at the CHALCONE ISOMERASE locus in Arabidopsis thaliana. Genetics 155: 863-872[Abstract/Free Full Text]

Kumar S. K. Tamura I. B. Jakobsen M. Nei 2001 MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17: 1244-1245[Abstract/Free Full Text]

Lin C. C. 1966 An outline of Taiwan's Quaternary geohistory with a special discussion of the relation between natural history and cultural history in Taiwan. Bulletin of the Department of Archaeology and Anthropology 23: 7-44

Lin J. A. H. D. Brown M. T. Clegg 2001 Heterogeneous geographic patterns of nucleotide sequence diversity between two alcohol dehydrogenase genes in wild barley (Hordeum vulgare subspecies spontaneum). Proceedings of the National Academy of Sciences, USA 98: 531-536[Abstract/Free Full Text]

Liu F. D. Charlesworth M. Kreitman 1999 The effects of mating system differences on nucleotide diversity at the phosphoglucose isomerase locus in the plant genus Leavenworthia. Genetics 151: 343-357[Abstract/Free Full Text]

Lu S. Y. K. H. Hong S. L. Liu W. P. Cheng W. L. Wu T. Y. Chiang 2002 Genetic variation and population differentiation of Michelia formosana (Magnoliaceae) based on cpDNA variation and RAPD fingerprints. Journal of Plant Research 115: 203-216[CrossRef][ISI][Medline]

Lu S. Y. C. I. Peng Y. P. Cheng K. H. Hong T. Y. Chiang 2001 Chloroplast DNA phylogeography of Cunninghamia konishii (Cupressaceae), an endemic conifer of Taiwan. Genome 44: 797-807[Medline]

McDonald J. H. M. Kreitman 1991 Adaptive protein evolution at the Adh locus in Drosophila. Nature 351: 652-654[CrossRef][Medline]

Miyashita N. A. Kawabe H. Innan R. Terauchi 1998 Intra- and interspecific DNA variation and codon bias of the alcohol dehydrogenase (Adh) locus in Arabis and Arabidopsis species. Molecular Biology and Evolution 15: 1420-1429[Free Full Text]

Morton B. R. B. S. Gaut M. T. Clegg 1996 Evolution of alcohol dehydrogenase genes in the palm and grass family. Proceedings of the National Academy of Sciences, USA 93: 11735-11739[Abstract/Free Full Text]

Murray M. G. W. F. Thompson 1980 Rapid isolation of high molecular weight plant DNA. Nuclear Acids Research 8: 4321-4325[CrossRef]

Ohta T. 1992 Theoretical study of near neutrality. II. Effect of subdivided population structure with local extinction and recolonization. Genetics 130: 917-923[Abstract]

Pamilo P. S. Pálsson O. Savolainen 1999 Deleterious mutations can reduce differentiation in small, subdivided populations. Hereditas 130: 257-264[CrossRef][ISI]

Pannell J. R. B. Charlesworth 1999 Neutral genetic diversity in a metapopulation with recurrent local extinction and recolonization. Evolution 53: 664-676[CrossRef][ISI]

Petit R. J. E. Pineau B. Demesure R. Bacilieri A. Ducousso A. Kremer 1997 Chloroplast DNA foot-prints of postglacial recolonization by oaks. Proceedings of the National Academy of Sciences, USA 94: 9996-10001[Abstract/Free Full Text]

Purugganan M. J. I. Suddith 1998 Molecular population genetics of the Arabidopsis CAULIFLOWER regulatory gene: nonneutral evolution and naturally occurring variation in floral homeotic function. Proceedings of the National Academy of Sciences, USA 95: 8130-8134[Abstract/Free Full Text]

Purugganan M. J. I. Suddith 1999 Molecular population genetics of floral homeotic loci: departure from the equilibrium-neutral model at the APETALA3 and PISTILLATA genes of Arabidopsis thaliana. Genetics 151: 839-848[Abstract/Free Full Text]

Rozas J. R. Rozas 1999 DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174-175[Abstract/Free Full Text]

Sang T. M. J. Donoghue D. Zhang 1997 Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species. Molecular Biology and Evolution 14: 994-1007[Abstract]

Savolainen O. C. H. Langley B. P. Lazzaro H. Freville 2000 Contrasting patterns of nucleotide polymorphism at the alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsis thaliana. Molecular Biology and Evolution 17: 645-655[Abstract/Free Full Text]

Silva J. C. M. G. Kidwell 2000 Horizontal transfer and selection in the evolution of P elements. Molecular Biology and Evolution 17: 1542-1557[Abstract/Free Full Text]

Simonsen K. L. G. A. Churchill C. F. Aquadro 1995 Properties of statistical tests of neutrality for DNA polymorphism data. Genetics 141: 413-429[Abstract]

Slatkin M. 1993 Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47: 264-279[CrossRef][ISI]

Small R. L. J. A. Ryburn J. F. Wendel 1999 Low levels of nucleotide diversity at homoeologous Adh loci in allotetraploid cotton (Gossypium L). Molecular Biology and Evolution 16: 491-501[Abstract]

Small R. L. J. A. Ryburn R. C. Cronn T. Seelanan J. F. Wendel 1998 The tortoise and the hare: choosing between noncoding plastome and nuclear adh sequences for phylogeny reconstruction in a recently diverged plant group. American Journal of Botany 85: 1301-1315[Abstract/Free Full Text]

Stephan W. L. Xing D. Kirby J. M. Braverman 1998 A test of the background selection hypothesis based on nucleotide data from Drosophila ananassae. Proceedings of the National Academy of Sciences, USA 95: 5649-5654[Abstract/Free Full Text]

Taberlet P. L. Gielly G. Pautou J. Bouvet 1991 Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109[CrossRef][ISI][Medline]

Tajima F. 1989 Statistical method for testing the neutral mutation of hypothesis by DNA polymorphism. Genetics 123: 585-595[Abstract/Free Full Text]

Terauchi R. T. Terachi N. T. Miyashita 1997 DNA polymorphism at the Pgi locus of a wild yam, Dioscora tokoro. Genetics 147: 1899-1914[Abstract]

Watterson G. A. 1975 On the number of segregating sites in genetical models without recombination. Theoretical Population Biology 7: 256-276[CrossRef][ISI][Medline]

Wiehe T. H. E. W. Stephan 1993 Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster. Molecular Biology and Evolution 10: 842-854[Abstract]

Yang Z. 1999 Phylogenetic analysis by maximum likelihood (PAML) (http://abacus.gene.ucl.ac.uk/software/paml.html). University College London, London, UK

Yang Z. R. Nielsen 1998 Synonymous and nonsynonymous substitution rate variation in nuclear genes of mammals. Journal of Molecular Evolution 17: 32-43

Yang Z. W. J. Swanson V. D. Vacquier 2000 Maximum-likelihood analysis of molecular adaptation in abalone sperm lysine reveals variable selective pressures among lineages and sites. Molecular Biology and Evolution 17: 1446-1455[Abstract/Free Full Text]




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