MBE Advance Access originally published online on January 30, 2006
Molecular Biology and Evolution 2006 23(4):807-816; doi:10.1093/molbev/msj100
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Published by Oxford University Press 2006.
Research Article |
Genetic Structure and Evolutionary History of a Diploid Hybrid Pine Pinus densata Inferred from the Nucleotide Variation at Seven Gene Loci
* State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China; and Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan
E-mail: xiao-ru.wang{at}niwl.se.
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Abstract |
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Although homoploid hybridization is increasingly recognized as an important phenomenon in plant evolution, its evolutionary genetic mechanisms are poorly documented and understood. Pinus densata, a pine native to the Tibetan Plateau, represents a good example of a homoploid hybrid speciation facilitated by adaptation to extreme environment and ecological isolation from the parents. Its ecologically and reproductively stabilized nature offers excellent opportunity for studying genetic processes associated with hybrid speciation. In this study, we investigated the levels and patterns of nucleotide variation in P. densata and its putative parents. Haplotype composition, gene genealogies, and the levels and patterns of nucleotide variation gave further support to the hybrid nature of P. densata. Allelic history, as revealed by our data, suggests the ancient nature of the hybrid preceding elevation of the Tibetan Plateau. We detected more deviations from neutrality in P. densata than in the parental species. Thus, at least some of the evolutionary forces that have shaped the genetic variation in P. densata are likely to be different from those acting upon parental species. We speculate that when populations of P. densata invaded new territories, they had elevated rates of response to selection in order to develop traits that help them to survive and adapt in the new environments.
Key Words: genealogy • hybrid speciation • nucleotide polymorphism • Pinus densata • population heterogeneity • selection
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Hybridization has long been considered as an important evolutionary force in plant evolution (Anderson 1948
; Stebbins 1950; Lewontin and Birch 1966; Grant 1981; Arnold 1997; Rieseberg 1997). It can quickly create evolutionary novelties that promote adaptive evolution and speciation (Arnold and Hodges 1995; Turelli, Barton, and Coyne 2001; Arnold, Bouck, and Cornman 2003; Rieseberg et al. 2003). Historically, polyploid speciation is thought to be the main form of hybrid speciation (Grant 1981; D. E. Soltis and P. S. Soltis 1995) while the role of homoploid hybridization has not been fully examined due to the difficulties in its detection that the most parsimonious conclusion was that it was not as prevalent (and therefore important) as polyploidy hybrid speciation. Only recently research employing molecular approaches has revealed several cases of homoploid hybrid speciation suggesting this form of speciation may be more common than previously thought (Wolfe, Xiang, and Kephart 1998; Gross and Rieseberg 2005; Howarth and Baum 2005). However, the genetic mechanisms underlying homoploid hybrid speciation in plants are poorly understood. A few investigations targeted traits that are distinct in hybrids and are thought to be important in species-specific adaptations (Rieseberg, Archer, and Wayne 1999; Rieseberg et al. 2003; Lexer, Lai, and Rieseberg 2004). These studies suggested that transgressive segregation appears to be sufficient to explain the origin of adaptations in hybrids. Another important mechanism underlying homoploid hybrid speciation is chromosomal rearrangement relative to parental species. Different models of chromosomal speciation have been proposed that facilitate reproductive isolation in the process of speciation (see review by Rieseberg 2001 and references therein). A different approach to understand the mechanisms of hybrid speciation is to study the patterns of nucleotide variation at randomly selected gene loci to infer the selective forces and demographic events involved in the history of speciation (Avise 1989; Kliman et al. 2000).
Pinus densata is a conifer tree with large distribution in the southeastern part of the Tibetan Plateau. It forms pure forest at high elevations (2700–4200 m asl) and regenerates well (Wu 1956; Guan 1981). The origin of P. densata has been investigated with allozyme, chloroplast (cp) DNA, and mitochondrial (mt) DNA markers (Wang and Szmidt 1994; Wang, Szmidt, and Savolainen 2001; Song et al. 2002, 2003). All results indicate that P. densata originated from hybridization between Pinus tabuliformis and Pinus yunnanensis without alteration in the ploidy level (Wang, Szmidt, and Savolainen 2001; Song et al. 2002, 2003; Liu et al. 2003). Pinus tabuliformis is widely distributed from northern to central China, and P. yunnanensis has a relatively limited range in southwestern China (Wu 1956). The geographic distribution of the three pines forms a succession, with P. tabuliformis in the north, P. densata in the middle, and P. yunnanensis in the south. The high elevation habitat occupied by P. densata is inaccessible to any other pine species growing in the region (Wu 1956; Guan 1981). Patterns of variation in allozymes, cp, and mt DNA show that individual populations of P. densata have very diverse genetic compositions, with varying degrees of genomic contribution from each parental species (Wang and Szmidt 1994; Wang, Szmidt, and Savolainen 2001; Song et al. 2002, 2003). In addition, populations of P. densata from different parts of the plateau show reciprocal parentage. These results suggest that populations of P. densata have unique evolutionary histories and most likely independent origins.
Habitat divergence plays a crucial role in plant speciation. It is even more important in homoploid hybrid speciation to facilitate reproductive isolation from the parents. Theory indicates that this process is most likely to be driven by ecological divergence (McCarthy, Asmussen, and Anserson 1995; Buerkle et al. 2000; Barton 2001). Indeed, all well-documented examples of homoploid hybrid plant species occur in habitats that are different from those of their parental species (Gross and Rieseberg 2005). Pinus densata represents a good example of a homoploid hybrid speciation facilitated by adaptation to extreme environment and ecological isolation from the parents. Its ecologically and reproductively stabilized nature offers excellent opportunities for studying genetic processes associated with the hybrid speciation and evolution.
To date, there are very few studies of polymorphism and divergence across several nuclear gene loci in conifers (Garcia-Gil, Mikkonen, and Savolainen 2003; Kado et al. 2003; Brown et al. 2004; Neale and Savolainen 2004; Bouillé and Bousquet 2005). Furthermore, there is particularly little information about the levels and patterns of nucleotide variation in plant species that evolved through interspecific hybridization. Studies on the levels and patterns of nucleotide variation in nuclear genes can help to better understand the evolutionary forces that have shaped genetic variation in P. densata and its parents and permit a genome-wide assessment of speciation, which cannot be revealed by uniparentally inherited cp and mt DNA markers.
In this study, we surveyed the nucleotide polymorphism and haplotype structure over seven loci in P. densata and its two putative parents. Our main objectives were (1) to determine the levels and patterns of DNA polymorphism in the investigated species complex, (2) to characterize the population heterogeneity and allelic coalescence history in P. densata and its parental species, and (3) to better understand the history of the hybrid speciation of P. densata.
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Materials and Methods |
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Population Sampling
Seeds from three, three, and five populations were sampled for P. tabuliformis, P. yunnanensis, and P. densata, respectively (fig. 1). For P. densata, seeds were collected from 14 to 17 individual trees in each population. One seed from each tree was germinated on a Petri dish and a haploid megagametophyte from each seed was used for genomic DNA isolation. For P. tabuliformis and P. yunnanensis, composite seed samples were collected from more than 100 trees per population. Megagametophytes from 13 to 16 seeds per population were used for DNA extraction. These megagametophytes were regarded as random gamete samples from each population. A total of 164 haploid DNA samples were analyzed. The geographic locations of the populations are shown in figure 1. Two populations of P. tabuliformis (PtDJ and PtSZ) were from the northern limit of its distribution, the other one (PtSX) was from the central part of its distribution. The three populations of P. yunnanensis (PyBS, PyDL, and PyYX) were from the Yunnan province. Of the five populations of P. densata, four (PdLX, PdMK, PdDB, and PdDC) were located along the eastern edge of the Tibetan Plateau, the other one (PdNX) was further into the center of the plateau. These populations were selected based on the previous investigations, which showed a diverse genetic composition of individual populations and verified their hybrid nature (Wang and Szmidt 1994
; Wang, Szmidt, and Savolainen 2001; Song et al. 2002, 2003).
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Pinus densata, 
Pinus yunnanensis.Sampled Loci and Sequencing
In a preliminary survey, about 80 gene loci were screened for amplification in the three species investigated in this study. Only the loci that were represented by a single polymerase chain reaction (PCR) band were selected. The PCR products were cloned into a pGEM T-easy vector (Promega Inc., Madison, Wisc.) and 7–8 clones were sequenced for each locus to examine whether they consisted of a single sequence. Finally, seven loci (ARA, DEH, PHO, POD, PtIFG2009, PtIFG8744, and PtIFG8887) were selected for sequence analysis. The putative function and structure of these loci and the PCR primers are described in Supplementary Table 1.
DNA of the haploid megagametophytes was used to amplify the seven loci. PCR was performed in a volume of 20 µl consisting of 0.2 µM of each primer, 2.0 mM of MgCl2, 200 µM of each dNTP, and 1.0 U of Ex Taq DNA polymerase (Takara Biotechnology, Dalian, China), using a PTC100 (MJ Research Inc., Waltham, Mass.) thermal cycler programmed for an initial denaturation at 94°C for 3 min followed by 34 cycles of 30 s at 94°C, annealing at a specific temperature (as listed in Supplementary Table 1) for 30 s and extension for 30 s at 72°C, and a final extension of 10 min at 72°C. Amplification products were first examined through electrophoresis in 1.0% agarose gel. The products were then cut from the gel and purified using a GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The purified products were sequenced directly using a BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Applied Biosystems, Foster City, Calif.). Unique haplotype sequences for each locus are deposited in the GenBank with the accession numbers DQ232904–DQ233201.
The genus Pinus is divided into two subgenera, Strobus and Pinus. Pinus tabuliformis, P. yunnanensis, and P. densata all belong to the subgenus Pinus. To estimate the divergence between the two subgenera, two haploid DNA samples from each of Pinus armandii and Pinus koraiensis, both from the subgenus Strobus, were sequenced for the seven loci included in this study. These sequences were used as outgroup.
Data Analysis
One hundred sixty-four haploid DNA samples were sequenced for each locus except for DEH and PtIFG8887 loci, for which due to the failure of PCR amplification in some samples only 163 and 140 samples were sequenced, respectively. Sequences were aligned with ClustalX (Thompson et al. 1997) and further manually adjusted using BioEdit program (Hall 1999). The total aligned sequence length over the seven loci was 3,040 bp, which included 1,463 bp of exons, 610 bp of introns, and 977 bp of the 3'UTR regions. Nucleotide polymorphism measured by 
w (Watterson 1975) and diversity measured by 
(Nei and Li 1979), intragenic minimum recombination events (Rm) (Hudson and Kaplan 1985) and haplotype diversity (Hd) (Nei and Li 1979), at each locus and in each population and species, were estimated using DnaSP v4.0 program (J. Rozas and R. Rozas 1999). The ratio of replacement (a) and synonymous (s) polymorphism (a/s) was calculated for each locus in each population. Population differentiation was estimated by Fst (Hudson, Slatkin, and Maddison 1992) as implemented in the ProSeq v2.9 program (Filatov 2002) with 1,000 permutations to obtain the significance estimates of Fst. Gaps were excluded in all analyses.
The measures of linkage disequilibrium (LD) D' and r2 among informative sites were calculated using DnaSP v4.0. The statistical significance of LD was determined by Fisher's exact test with and without Bonferroni correction.
Each locus was tested for departure from neutrality by Tajima's (1989) D and Fu and Li's (1993) D* and F* statistics using DnaSP v4.0. Except for DEH locus at which sequencing failed for the two pines of subgenus Strobus, the Fu and Li statistics were performed with a P. armandii sequence as outgroup. The use of the subgenus Strobus sequence as outgroup is to improve the power of the test as it is less related to the three pines investigated in this study. Under neutral model, the level of polymorphism within species correlates with the degree of divergence between species across loci. The HKA test (Hudson, Kreitman, and Aguade 1987) was developed to test this prediction. When applied to multilocus data, the HKA test assesses the overall fit of the data to the neutral model that assumes the same ratios of polymorphism and divergence at each locus. The HKA test was performed for P. densata versus P. tabuliformis, P. densata versus P. yunnanensis, and P. tabuliformis versus P. yunnanensis as implemented in Jody Heys' multilocus HKA program (http://lifesci.rutgers.edu/
heylab/HeylabSoftware.htm). The simulations were run 1,000 times.
Gene/allele genealogies of each locus were constructed by coalescent simulations using the Median-Joining model as implemented in the Network v4.0 program (Bandelt, Forster, and Röhl 1999). We treated the segregating sites as independent evolutionary events. All indels were excluded from the analysis. Sequences of the subgenus Strobus were used as outgroup in the network construction of each locus except for the DEH locus.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Nucleotide Polymorphism
Measures of nucleotide polymorphism at each locus and in each population and species are presented in Supplementary Table 2. The average estimates over the seven loci for each population and species (calculated using pooled population data for each species) are presented in table 1. At species level, the nucleotide polymorphism over all loci (
w) was similar in P. tabuliformis (0.0107) and P. densata (0.0101), which was nearly twofold higher than that in P. yunnanensis (0.0055). Silent polymorphism in the three species was four- to fivefold higher than the replacement polymorphism (table 1). Within each species, the levels of polymorphism ( and w) among loci differed by four- to sixfold. The POD locus was the most polymorphic in P. tabuliformis and P. densata, while the PtIFG8744 locus was the most polymorphic in P. yunnanensis. PHO was the least polymorphic locus in all the three pines (Supplementary Table 2).
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The ratios of replacement (
a) and synonymous (s) polymorphism at five of the seven loci are listed in Supplementary Table 3. Two loci (PtIFG8887 and PtIFG8744) were excluded from this analysis due to their very short coding regions. Most of the values for a/s at ARA, DEH, PHO, and PtIFG2009 (except for the PHO locus in population PyYX) were smaller than 1 in the investigated populations. At the POD locus, however, three populations (PtSX, PdLX, and PdMK) had a/s greater than 1. Minimum numbers of recombination events in the three pine species at the seven loci are listed in Supplementary Table 2. Among the seven loci, recombination was detected at five (ARA, DEH, POD, PtIFG2009, and PtIFG8744), four (ARA, DEH, PtIFH2009, and PtIFG8744) and three (ARA, PtIFG2009, and PtIFG8744) loci in P. tabuliformis, P. densata, and P. yunnanensis, respectively. No recombination was detected at the PHO and PtIFG8887 loci in any of the three pines. The lack of recombination at PHO and PtIFG8887 correlates with the relatively low haplotype diversity (Hd) at these loci.
Segregating sites at each locus were used to compute LD in each population and species. Among 1,367, 1,360, and 466 pairwise intragenic comparisons over the seven loci in P. tabuliformis, P. densata, and P. yunnanensis, 16%, 18%, and 33% comparisons were significant by Fisher's exact test and 4%, 8%, and 18% were still significant after Bonferroni correction (Weir 1996), respectively. Pinus yunnanensis showed the highest number of significantly associated sites (two- to fourfold higher than that in the other two pines). No intergenic LD was detected in any of the three pines.
There was significant population differentiation in P. tabuliformis and P. densata, with multilocus Fst values of 0.086 and 0.105, respectively (table 2). Population differentiation in P. yunnanensis was low (0.031) and not significant.
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Tests of Selective Neutrality
Tajima's D, Fu and Li's D* and F*, and HKA tests were used to detect the departure from the neutral model of molecular evolution at each locus. The Tajima's D and Fu and Li's F* values at the seven loci in each population are given in table 3. Fu and Li's D* test gave a similar trend as F* and thus is not shown. In P. tabuliformis, no significant D and F* values were detected at any locus in any population. In P. densata, however, significant departures from neutrality were detected by both Tajima's D and Fu and Li's F*. Populations PdMK and PdDB had positive significant D and F* values at the ARA and PtIFG8744 loci, respectively, and population PdNX had positive significant F* and positive but not significant D at the PtIFG2009 locus. In P. yunnanensis, population PyBS had positive significant D and F* at the PtIFG8744 locus while populations PyDL and PyYX had positive significant F* but not significant D at the ARA locus. In addition, in two populations of P. densata (PdNX and PdMK) both D and F* or only F* were significantly negative at the DEH and POD loci (table 3).
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We performed HKA tests for P. densata versus P. tabuliformis, P. densata versus P. yunnanensis, and P. tabuliformis versus P. yunnanensis over the seven loci. The results showed that none of the pairwise species comparisons gave significant chi-square statistic at any locus.
Effective Population Size
For neutral alleles in mutation-drift equilibrium, synonymous polymorphism ws in diploid genome is equal to 4Neµg, where Ne is the effective population size and µg the mutation rate per generation (Tajima 1989). We used P. armandii of subgenus Strobus to calculate the average divergence between the subgenera Pinus and Strobus at six of the seven loci, excluding DEH locus due to the failure in amplification in P. armandii. According to the fossil records, pines diversified into the two subgenera during the early Cretaceous ca. 130 MYA (Miller 1977). The estimated average divergence at silent sites (Ks) between P. armandii and P. tabuliformis and P. yunnanensis was 0.0507, which can be treated as the accumulation of silent mutations over 130 Myr. Thus, the mutation rate per year (µy) was estimated to be Ks/2T = 1.95 x 10–10 for P. tabuliformis, P. yunnanensis, and P. densata. Assuming pines take 25 years to reach full seed production, the mutation rate per generation (µg) can be estimated as 25µy = 4.875 x 10–9. Ne can then be estimated as ws/4µg. A summary of the estimated effective population sizes for individual populations of the three pines is listed in table 1. Pinus tabuliformis and P. densata had similar and large effective population sizes (7.85 x 105 and 7.32 x 105), which were approximately twofold larger than that of P. yunnanensis (3.95 x 105).
Genealogy of Each Locus
Genealogies of the haplotypes observed at each of the seven loci were constructed by coalescent simulations using the Median-Joining network (fig. 2). Based on the topology and the frequency of the haplotypes, the genealogies of the seven loci can be grouped into three classes. The first class includes simple and relatively small networks with short branches such as those obtained for the PHO and PtIFG8887 loci. The PHO locus harbored 12 haplotypes among the 164 samples of the three investigated pine species, with four haplotypes (H1, H2, H3, and H9) occurring at high frequencies. Excluding P. densata, the H1 haplotype was the main haplotype specific to P. tabuliformis and the H3 and H9 haplotypes were specific to P. yunnanensis. When P. densata was included, all these haplotypes become shared with P. densata. At the PtIFG8887 locus, 21 haplotypes were detected among which the H2 haplotype dominated in all the three pines. Thus, this haplotype could be regarded as the ancestral haplotype with many of the minor haplotypes derived from it. A smaller cluster surrounding the H10 haplotype was specific to P. yunnanensis and P. densata. The second class of genealogy included more complex networks composed of two to three main haplotypes with relatively deep coalescence shared by all the three species. This class included the ARA, PtIFG8744, DEH, and POD loci. The ARA locus harbored 22 haplotypes of which three (H4, H9, and H5) accounted for 75% of the total number of haplotypes. These three haplotypes were shared by all the three pines but the H9 haplotype was more common in P. yunnanensis and P. densata. Six mutation steps separated the haplotypes H9 and H4, and three mutation steps separated haplotypes H9 and H5. Similar to the PtIFG8887 locus, the topology of the PtIFG8744 locus had two clusters, one including the haplotypes H1 and H2, which were shared by all the three species, and another cluster surrounding the H11 haplotype, which was shared by only P. yunnanensis and P. densata. Six mutation steps separated the two clusters. At the DEH locus, 27 haplotypes were found among which 13 were unique to P. densata. The three common haplotypes (H1, H4, and H5) were shared by all the three pine species. A few intermediate frequency haplotypes were largely species-specific, like H2 and H3 to P. tabuliformis, H15 to P. densata, and H13, H11, and H14 to P. yunnanensis. At the POD locus, 25 haplotypes were detected of which two (H1 and H8) were the most frequent. The H1 haplotype was shared by all the three species, but H8 haplotype was most abundant in P. densata (65%). The cluster composed of H8, H21, H23, and H24 haplotypes was nearly specific to P. densata. The last class of genealogy, which included the PtIFG2009 locus, was particularly large and reticulated. A total of 48 haplotypes were found at this locus and the haplotypes were spread with no clear structure. The high reticulation of the haplotypes at this locus suggests more recombination events over the evolutionary history.
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In general, despite different types of genealogies among the seven loci, some common features can be noticed: (1) most of the P. densata haplotypes coalesced to P. tabuliformis and P. yunnanensis, (2) at five of the seven loci, P. densata harbored the largest number of haplotypes, (3) many haplotypes specific to either P. tabuliformis or P. yunnanensis were shared with P. densata, and (4) among the 175 haplotypes detected at the seven loci in the three investigated pines, 52 (30%) were unique to P. densata, 57 (33%) to P. tabuliformis, and 11 (6%) to P. yunnanensis.
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Discussion |
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Levels and Patterns of Nucleotide Polymorphism
Results from a few available studies on the patterns and levels of nucleotide polymorphism in conifers are summarized in table 4. Very low levels of polymorphism (
w or ) were observed in Pinus sylvestris (0.0004–0.0014) and Cryptomeria japonica (0.0002–0.0038), intermediate levels were observed in Pinus taeda (0.0041), P. yunnanensis (0.0055), Picea abies, Picea glauca, and Picea mariana (0.0066–0.0081). On the other hand, high levels were found in P. tabuliformis (0.0107), P. densata (0.0101), and Pseudotsuga menziesii (0.0085). The level of nucleotide polymorphism in P. tabuliformis and P. densata was about two- and sevenfold higher than that of P. taeda and P. sylvestris, respectively. Among the three pine species analyzed in this study, P. yunnanensis harbored about half of the polymorphism found in the other two pines. The level of polymorphism in P. tabuliformis and P. densata was comparable to that reported in Populus tremula (w = 0.0167, s = 0.0160, ns = 0.0059) (Ingvarsson 2005), a diecious, wind-pollinated leaf tree, and in outcrossing maize (w = 0.0096, ws = 0.0173, wns = 0.0039) (Tenaillon et al. 2001). Furthermore, our results show that within each species the levels of polymorphism among loci can differ by as much as four- to sixfold. Substantial locus-to-locus variation in the levels of polymorphism was also reported in other studies on conifers (Kado et al. 2003; Brown et al. 2004; Bouillé and Bousquet 2005). Thus, many more loci should be sampled to obtain a representative genome-wide estimate of the polymorphism in individual species of conifers.
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The level of polymorphism at neutral or near-neutral loci is determined by effective population size, mutation rate, recombination, and demography (Hedrick 1980
; Kimura 1983). The high polymorphism detected in P. tabuliformis is consistent with its larger Ne and suggests less drastic population size reduction in the Pleistocene glaciations as compared to P. sylvestris and P. taeda. Indeed, much of the region currently occupied by P. sylvestris has been covered by Pleistocene ice while the regions occupied by P. tabuliformis were ice-free (Prentice, Bartlein, and Webb 1991; Lindsey 2002). The high polymorphism observed in P. densata is rather unusual for the homoploid hybrid plant species. Among the case studies on homoploid hybrid speciation in plants, the hybrids often show lower (or roughly equivalent) genetic diversity than those found in parental species (Gallez and Gottlieb 1982; Maki and Murata 2001; Schwarzbach and Rieseberg 2002; Welch and Rieseberg 2002; Gross, Schwarzbach, and Rieseberg 2003). The low levels of genetic diversity in hybrids are explained by restricted distribution, small population sizes, and recent and single origins. Pinus densata, on the other hand, differs from the other hybrids in that it has an advanced evolutionary history with multiple origins, and its populations from different geographic regions show reciprocal parentage and varying degrees of genomic contribution from parental species (Wang and Szmidt 1994; Wang, Szmidt, and Savolainen 2001; Song et al. 2002, 2003). The complex evolutionary history and genetic composition of P. densata should have contributed to its present considerable polymorphism.
Very low levels of intragenic LD (after Bonferroni correction) were detected at the seven loci in P. tabuliformis (4%) and P. densata (8%). These results add further evidence for the high recombination rate expected for outcrossing species with large Ne. The higher LD (18%) in P. yunnanensis is consistent with its smaller Ne estimated in the present study. Substantial interspecific gene flow can also cause LD. However, our results did not show increased LD in P. densata, suggesting its populations are in drift-mutation equilibrium and there is no on-going hybridization with the parental species in the sampled populations. This conclusion is in accordance with the allozyme data which also did not detect increased LD in P. densata (Wang, Szmidt, and Savolainen 2001).
Signature of Selection in P. densata
Compared to P. tabuliformis and P. yunnanensis, we detected more departures from neutrality in P. densata. Significant positive Tajima's D and/or Fu and Li's F* were observed at the ARA, PtIFG8744, and PtIFG2009 loci in populations PdMK, PdDB, and PdNX, respectively. Significant positive D and F* would suggest either balancing selection, or a reduction in population size in the recent past. Whereas demographic processes are likely to affect all loci in a similar manner, the effects of selection are usually restricted to a specific locus. The significant positive D and F* at the ARA locus in PdMK contrast the negative D and F* observed at all the other six loci in this population, which suggests against the reduction in population size. In fact, PdMK had the largest Ne in P. densata (table 1). Thus, balancing selection seems the more likely reason for the observed significant departure from the neutral model. As for the PtIFG8744 and PtIFG2009 loci in populations PdDB and PdNX, without other supporting evidence it is difficult to distinguish between the possibilities of selection and demographic factors. Interestingly, significant positive F* at ARA locus was also observed in populations PyDL and PyYX and significant positive D and F* at PtIFG8744 locus in population PyBS of P. yunnanensis. This parallel pattern in departure from neutrality between P. yunnanensis and P. densata at these two loci may suggest certain functional significance. More information from the full gene sequences is needed to clarify the reasons for the non-neutral variation at these two loci.
Significant negative D and/or F* were observed at the DEH and POD loci in populations PdNX and PdMK, respectively. Significant negative D and F* may result from either some form of selective pressure or from population expansion. The ratio of a/s at the POD locus in the PdMK population was 1.626, suggesting selective advantage of the replacements. Thus, the observed significant negative F* in this population could be the result of positive selection. The POD gene encodes a peroxidase. Peroxidases are a family of enzymes that catalyze oxidation–reduction reactions and play important roles in detoxification, cell protection, and defense responses in plants. The functional significance of the observed departure from neutrality at this locus needs to be verified with full-length gene sequences. Together with the observation at the ARA locus in the PdMK population, we see an example of different evolutionary forces shaping the genetic variation at different loci in the same population. In contrast to the POD locus, a/s ratio at the DEH locus in the PdNX population was 0.141, which is consistent with the action of purifying selection. Taking into account the absence of negative D and F* values across other loci to signify expansion of the PdNX population, some form of negative selection could be the cause for the observed deviation from neutrality at this locus. More detailed studies are needed to better explain the observed locus-specific deviations from neutrality.
Based on the observed deviations, which may represent signatures of selection in P. densata, it is tempting to speculate that when populations of P. densata invaded new territories they had elevated rates of response to selection in order to develop traits that help them survive and adapt in the new environments. Populations with different genetic composition that have occupied different regions of the Tibetan Plateau might have experienced varying degrees and types of selective forces and demographic processes. The effects of diverse evolutionary forces on populations could be preserved by limited gene flow among populations. Indeed, P. densata showed the highest Fst (0.105) among the three pines. This result is in agreement with the Fst estimated from allozyme loci which also revealed high differentiation (0.086) in P. densata (Wang, Szmidt, and Savolainen 2001). The Fst estimates for P. tabuliformis, however, varied between allozyme (0.026) and DNA sequence (0.086) data. This difference was likely caused by the sampling of very distant populations used in the DNA study. The level of population differentiation in P. densata is much higher than those commonly found in outcrossing, wind-pollinated conifers (Fst < 0.05) (Hamrick and Godt 1996). This high differentiation could be related to two factors, the diverse genetic composition among populations and the complex geography of the southeastern Tibetan Plateau. The topology of the region occupied by P. densata is characterized by high mountains and deep valleys. Therefore, gene flow among populations from different regions is likely to have been limited in P. densata.
Hybrid Nature and Multiple Origins of P. densata
Haplotype distribution at individual loci showed that most of the major haplotypes are shared by the three pines. At some loci (e.g., PHO, PtIFG8887) species-specific haplotypes were found for P. tabuliformis and P. yunnanensis when P. densata was excluded. Inclusion of P. densata, however, resulted in sharing of all these formerly species-specific haplotypes. This pattern gives further support to the hybrid nature of P. densata as most of its haplotypes appeared to be inherited or derived from the two parental species. The fixation time of neutral polymorphisms in a population depends on the population size (Kimura and Ohta 1969; Tajima 1983; Hudson 1990). The average coalescence time of two randomly selected alleles at a gene locus is estimated by 2Ne generations (Tajima 1983). Assuming a generation time of 25 years in pines, the estimates of Ne in table 1 would translate into 19.7–39.3 MYA of allele coalescence time in the three pine species. Alleles in P. yunnanensis had shorter coalescence history (19.7 MYA) than those in P. tabuliformis (39.3 MYA). The coalescence history in P. densata varied among populations (21.3–38.5 MYA) but within the boundary set by the two parental species (table 1). This supports multiple origins of this species. The five populations of P. densata differ in the levels of gene admixture from each of the parental species (Wang, Szmidt, and Savolainen 2001; Song et al. 2003). Thus, it is not surprising to see much variation in allelic history among its populations. Our present estimates of the allele coalescence time suggest that the PdMK and PdLX populations have been much influenced by P. tabuliformis, while populations PdDB, PdDC, and PdNX by P. yunnanensis. This suggestion is in agreement with the previous allozyme and mt and cpDNA results, which showed ca. 50% of P. tabuliformis and 30% of P. yunnanensis cpDNA components in the PdMK population (Pd-1 and 2 in Wang, Szmidt, and Savolainen 2001), 65% of P. yunnanensis cpDNA component in PdDC (Pd-7 in Wang, Szmidt, and Savolainen 2001), and dominant P. yunnanensis mt and cpDNA in PdDB (Song et al. 2003). It should be mentioned, however, that due to the observed deviations from neutrality for some loci in P. densata and P. yunnanensis, the allelic coalescence estimates may be biased to some extent.
The estimated allele coalescence time in the three pines also indicates the ancient nature of the allelic polymorphism. When there is much-shared polymorphism among related species, the coalescence of many alleles would precede species divergence (Bouillé and Bousquet 2005). Hybridization between divergent species would make this phenomenon more pronounced. The speciation of P. densata is suggested to be related to its adaptation to a unique ecological niche at high elevations on the Tibetan Plateau, where neither of the two parental pines can normally grow (Wang and Szmidt 1994; Wang, Szmidt, and Savolainen 2001). The uplift of the Tibetan Plateau dates back at least 20 MYA (Ruddiman and Kutzbach 1991; Harrison et al. 1992; Ruddiman 1998). However, significant increases in altitude of the plateau are thought to have occurred only about 8–10 MYA followed by continued development of the plateau toward the north and east (Harrison et al. 1992; Zhisheng et al. 2001). Drastic geographic and climatic changes in that period could have either brought distant species together or separated sympatric species, and thus altered the flora (Florin 1963; Frenzel 1968). The hybrid nature of P. densata suggests gene exchange between P. tabuliformis and P. yunnanensis occurred before they became separated by the uplift of the plateau. Although we cannot date precisely the origin of each population in these geographic events, it is evident that the extant populations of P. densata from different parts of the plateau differ in their origin, and their allele coalescence history much exceeds the establishment of the hybrid in its final unique territory on the Tibetan Plateau. Further surveys of candidate genes associated with important adaptive traits in P. densata could provide better insights into the functional significance of its allelic diversity and evolution.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary Tables 1–3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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This study was supported by grants from the Natural Science Foundation of China (NSFC 30325006 and 30121003). A.E.S. was supported by grant 17405032 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Pekka Pamilo, Associate Editor
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