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Systematics and Phytogeography |
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 China; 3Graduate School, Chinese Academy of Sciences, Beijing 100039 China; and 4Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 USA
Received for publication December 11, 2005. Accepted for publication January 16, 2007.
ABSTRACT
There is growing evidence that hybridization not only by means of allopolyploidy but also at the homoploidy level was a major driving force of plant diversification. While allopolyploidy is known to be a common mode of speciation in Paeonia (Paeoniaceae), hybrid speciation at the diploid level needs further evaluation. Paeonia anomala was previously considered to be an interspecific hybrid but with an unknown ploidy level. In this study P. anomala is identified as a diploid (2n = 10). With increased sampling of populations and molecular markers, we showed that P. anomala is a homoploid hybrid that originated from a cross between P. veitchii and P. lactiflora. Five populations of P. anomala were sequenced for the following molecular markers: the matK gene and two intergenic spacers, psbA-trnH and rps16-trnQ, of the chloroplast genome; the internal transcribed spacers (ITS) of nuclear ribosomal DNA; and three low-copy nuclear genes, Adh1, Adh2, and Gpat. The populations of P. anomala were grouped together with P. veitchii on the ITS and Gpat phylogenies but with P. lactiflora on the chloroplast phylogeny. Sequence polymorphism was found at the Adh1 and Adh2 loci within individuals of P. anomala. These polymorphic sequences were grouped with P. veitchii and P. lactiflora, respectively. Phenetic analysis indicated that P. anomala is morphologically similar to P. veitchii. Phenotypic evolution resulting from the combination of two diverged genomes might have occurred primarily at the physiological level and allowed P. anomala to adapt to geographic regions different from those of its parents.
Key Words: fluorescent in situ hybridization • homoploid hybrid • meiosis • Paeonia anomala • phenetic analysis • phylogeny • speciation
Recent genetic studies of homoploid hybridization in sunflowers have indicated that the combination of diverged genomes allowed hybrid species to establish in novel environments (Rieseberg et al., 2003
). The breakthrough in the understanding of the genetic mechanisms of homoploid hybrid speciation encourages accelerated progress toward phylogenetic documentation of diploid hybrids. The theoretical and experimental challenges in reconstructing evolutionary histories of homoploid hybridization deserve greater attention from plant systematists.
Discerning between allopolyploidization and homoploid hybridization requires different strategies of phylogenetic analyses. Reconstruction of evolutionary origins of allopolyploids is relatively straightforward because diverged nuclear alleles from both parents are usually maintained in the hybrid genome as different loci. Molecular cloning and phylogenetic analyses of the parental alleles or homoeologous loci in the allopolyploids together with the genes from the extant diploid relatives have led to the reconstruction of allopolyploidy (Small et al., 1998
; Cronn et al., 1999
; Sang and Zhang, 1999
).
Difficulties in reconstructing homoploid hybridization arise when one of the parental alleles becomes fixed at the majority of nuclear loci of the hybrid. The more ancient the hybrid species, the larger the number of loci at which the fixation could have occurred through genetic drift. Fixation can be accelerated by population bottlenecks and frequent inbreeding. As a result, a hybrid species forms a sister group with one of the parents on a nuclear gene phylogeny. The hope to reconstruct homoploid hybridization, especially an ancient one, often comes from the observation of incongruent positions of the hybrid between multiple gene phylogenies (Rieseberg and Soltis, 1991
; Wendel and Doyle, 1998
; Sang and Zhong, 2000
).
Several challenges remain in this approach to reconstruct homoploid hybridization. First, the incongruence may be caused by factors other than hybridization, such as lineage sorting, especially when the parental species of the hybrid shared a short history of common ancestry (Doyle, 1997
). Second, we still lack an adequate number of nuclear markers for most plant groups. The fewer the nuclear phylogenies, the weaker the statistical power to detect hybridization. Third, an asymmetric fixation of parental alleles, possibly as a result of a backcross with one of the parents, also reduces the chance of finding phylogenetic incongruence. These complicating factors continue to challenge our ability to reliably identify a homoploid hybrid and most likely lead to an underestimation of the frequency of this mode of speciation.
Here we report multiple lines of evidence supporting the origin of a diploid hybrid species of Paeonia L. Paeonia anomala L. (previously named P. sinjiangensis K. Y. Pan; Hong and Pan, 2004
) was previously hypothesized to be a hybrid because of incongruence between the chloroplast DNA (cpDNA) and nuclear ribosomal DNA (nrDNA) phylogenies of Paeonia (Sang et al., 1997
). This species, with an unknown ploidy level at the time, formed a sister group with diploid species P. veitchii Lynch on the nrDNA ITS phylogeny, but formed a sister group with P. lactiflora Pall. in the cpDNA matK phylogeny. The subsequent study using Adh gene sequences revealed that the individual of P. anomala had sequence polymorphism for both Adh1 and Adh2, providing further support for a hybrid origin (Sang and Zhang, 1999
). Because a similar pattern of Adh sequence polymorphism has been found for several allotetraploid species of Paeonia (Sang and Zhang, 1999
), we speculated that P. anomala was an allotetraploid.
To test the hypothesis, we conducted a field investigation of P. anomala in the Aletai area in northwestern China. To our surprise, chromosome counts indicated that this is a diploid species. We thus examined the molecular phylogenies of four gene markers studied previously, with a larger population sample. In addition, we sequenced two new cpDNA regions to improve the support of the cpDNA phylogeny and a new single-copy nuclear marker, the Gpat gene (Tank and Sang, 2001
), to provide an independent assessment of the nuclear phylogenies. We also studied the morphology and cytogenetics of the hybrid and the closely related diploid species. The data were brought together to bear on questions concerning the origin, evolution, and phylogenetic reconstruction of a homoploid hybrid.
MATERIALS AND METHODS
Plant materials
First, note the recent nomenclatural changes for some of the Paeonia species studied here. Paeonia sinjiangensis has been renamed P. anomala, and P. anomala var. intermedia (C. A. Meyer) O. & B. Fedtsch. is now called P. intermedia C. A. Meyer (Hong and Pan, 2004
). For morphological analysis, we focus on P. anomala, its putative parents, P. veitchii and P. lactiflora, and the closely related species P. intermedia found to be nearly sympatric with P. anomala. Characters representing morphological variations among these species were measured from specimens either collected by us or previously deposited in the Herbarium (PE), Institute of Botany, the Chinese Academy of Sciences, Beijing. These include 17 specimens of P. intermedia, 12 of P. anomala, 20 of P. lactiflora, and 22 of P. veitchii (Appendix).
For molecular phylogenetic analyses, we sampled 11 individuals from five populations of P. anomala (Fig. 1, Table 1). Additional samples for the closely related species included two populations of P. intermedia, one population of P. lactiflora, and one population of P. veitchii (Table 1). Three of the populations of P. anomala collected were used for cytogenetic study. Voucher specimens have been deposited in the Herbarium (PE), Institute of Botany, the Chinese Academy of Sciences, Beijing.
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) was used in cluster analysis. Unweighted pair-group method using arithmetic average (UPGMA) analysis and principal coordinate analysis (PCoA) were performed with the program MVSP (version 3.13b, Kovach Computing Services, Anglesy, Wales, UK).
Molecular experiments
Total DNA was isolated from silica-gel-dried leaves using the CTAB method (Doyle and Doyle, 1987
). Conditions for the polymerase chain reaction (PCR) were reported previously (Sang et al., 1995
, 1997
; Tank and Sang, 2001
). PCR products were purified using a GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Buckinghamshire, UK). PCR products of nuclear genes were cloned with the pGEM-T Easy System (Promega Corporation, Madison, Wisconsin, USA). At least 16 clones with correct insertion (determined by digestion with EcoRI) were screened through comparison of their sequences generated from one of the PCR primers. All distinct clones were sequenced in both directions. Sequencing was done in an ABI377 automated DNA sequencer using ABI Prism Bigdye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA) and on MegaBACE1000 automated DNA sequencer using DYEnamic ET Dye Terminator Sequencing Kit (Amersham Biosciences).
Phylogenetic analyses
DNA sequence alignments were done with CLUSTAL X (Thompson et al., 1997
), followed by manual adjustment. Parsimony, as implemented in PAUP* version 4.0b10 (Swofford, 2002
), was used to infer phylogenies based on nucleotide substitutions in aligned sequences. Section Mutan of Paeonia was chosen as the outgroup (Sang et al., 1997
). Additional diploid species P. japonica (Makino) Miyabe & Takeda, P. obovata Maxim., and P. tenuifolia L. were included in analyses to encompass the diversity within section Paeonia.
Heuristic searches were performed with 1000 (cpDNA and ITS) or 100 (Adh1, Adh2, Gpat) random addition sequence replicates and the tree-bisection-reconnection (TBR) branch swapping and MULTREES option. Bootstrap analysis was carried out with 1000 replicates of heuristic search with TBR branch swapping, ACCTRAN optimization, and random taxon addition MaxTree was set at 500 for the Adh1, Adh2, and Gpat data sets).
Bayesian analyses for topology estimation were carried out using MrBayes version 2.0 (Huelsenbeck and Ronquist, 2001
). Modeltest 3.06 (Posada and Crandall, 1998
) was used to determine appropriate models of sequence evolution for all data sets (Table 2). One cold and three incrementally heated Markov Chain Monte Carlo (mcmc) chains were run each for 1 100 000 generations and were sampled every 1000 generations. For all analyses, the first 300 samples from each run were discarded as burn-in to ensure the chains reached stationarity. Phylogenetic inferences were based on those trees sampled after generation 300 000.
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). The global permutation P value was estimated using BLAST-like global scores obtained from 10 000 replicates. Recombination was considered to be present if P < 0.05. The possibility of gene conversion was also tested.
Cytogenetics
For meiotic studies, young floral buds were fixed in Carnoy's solution (absolute ethanol : acetic acid = 3 : 1) and then stored in 70% alcohol at 20°C. The samples for microscopic observation were prepared using squashing methods and stained with modified Carbol fuchsin (Hong et al., 1988
). For each individual, more than two anthers were studied for a meiotic stage, and at least 100 cells were recorded. The micrographs were taken using a Leitz (Wetzlar, German) Orthoplan microscope with a 100x oil lens. All negatives were scanned into a computer with a Scan Wit 2720S (Acer, Taiwan, China) scanner at a resolution of 2700 dots per inch. The scanned images were processed by using Photoshop (version 6.0, Adobe, San Jose, California, USA). The procedures for fluorescent in situ hybridization (FISH) were described in Zhang and Sang (1999)
. The relative length of fragments (Lf, the absolute length of fragment divided by the absolute length of the long arm of chromosome 1) was calculated to estimate inverted segments in P. anomala.
RESULTS
Morphology
In the UPGMA and PCoA analyses, the three diploid species, P. intermedia, P. lactiflora, and P. veitchii, were morphologically distinct, and P. intermedia and P. veitchii were more similar to each other than to P. lactiflora (Fig. 2A–B). All but one specimen of P. anomala was intermixed with those of P. veitchii in the results of both analyses.
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In the Adh1 phylogeny, the cloned sequences of P. anomala formed two diverged clades, with one (Adh1A1) being a sister group of P. veitchii and the other (Adh1A2) closely related to P. lactiflora (Fig. 4A). Also, the clones on the Adh1A2 clade had the same three indels of P. lactiflora. Four individuals had both types of sequences: one individual of population 1 (1B), one individual of population 3 (3B), one individual of population 4 (4B), and the individual studied previously. Two individuals from population 1 and 5 (1A and 5A) had only one type of Adh1 sequence closely related to that of P. lactiflora. The remaining six individuals, 1C, 2A, 2B, 3A, 4A, and 5B, had only the P. veitchii type of sequence.
Two diverged types of Adh2 sequences were also identified for P. anomala, with one forming a monophyletic group with P. veitchii and the other being closely related to P. lactiflora (Fig. 4B). All sampled individuals of P. anomala had these two types of Adh2 sequences.
On the Gpat phylogeny, all individuals of P. anomala formed a strongly supported monophyletic group (Fig. 4C). It was then grouped with P. veitchii and P. intermedia in an unresolved trichotomy in the strict consensus, with bootstrap support of 90%.
The global permutation P values (simulated P values) of three low-copy nuclear gene sequences of P. anomala were shown in Table 3. No recombination was detected for Gpat or Adh1, whereas it was detected for Adh2. The Adh2 recombinants identified were only from P. anomala. These sequences were then excluded from the phylogenetic analysis.
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The hybrid origin of P. anomala was initially hypothesized on the basis of its incongruent position between nrDNA ITS and cpDNA matK phylogenies. Because only one individual of P. anomala was included in the study and the support for the sister relationship of P. anomala and P. lactiflora was relatively low (68% bootstrap) on the chloroplast matK phylogeny, the immediate requirement for testing the hypothesis was to increase population sampling and obtain additional cpDNA sequences.
A field study was conducted in the Aletai area, and five additional populations of P. anomala were sampled. In the chloroplast genome, sequencing two additional fast-evolving regions yielded useful phylogenetic information. The fact that all populations of P. anomala formed monophyletic groups on the ITS and cpDNA phylogenies and the bootstrap value for the sister relationship between P. anomala and P. lactiflora increased to 97% in the cpDNA tree further supports the hypothesis of hybrid speciation. It is clear that P. anomala has a chloroplast genome more similar to that of P. lactiflora than to P. veitchii. Additionally, in situ hybridization showed that the number of 18S rDNA sites (eight) was also more similar to P. lactiflora (seven major and one minor site) than to P. veitchii (10 major sites) (Fig. 5C; Zhang and Sang, 1999
).
The Adh2 phylogeny provides stronger support for the hybrid origin of P. anomala than does the Adh1 phylogeny. Each individual of P. anomala sampled in this study has two diverged Adh2 sequences. One type of sequence forms a monophyletic group with P. veitchii, and the other is closely related to P. lactiflora, although it did not resolve into a monophyletic group. These two types of Adh2 sequences likely represent two loci rather than two alleles of a locus because the chance of randomly sampling only heterozygous individuals from 13 individuals of five populations is very low (P = 0.0001, assuming half the individuals in the populations are heterozygous).
For Adh1, although two types of sequences have been cloned from P. anomala, they are not as highly diverged from each other as are the Adh2 types and are found in fewer than half the sampled individuals of P. anomala. We cannot determine whether the two types represent two loci or two alleles of a locus. In any case, however, the Adh1 data are consistent with the hypothesis of the hybrid origin of P. anomala from diploid species closely related to P. veitchii and P. lactiflora.
The results here demonstrate how a diploid hybrid could maintain the sequence polymorphism derived from parental species. The polymorphic sequences may result from duplicated loci or diverged alleles from the parents at the time of hybridization. It is also possible that parental alleles ended up in the different chromosomal locations in a hybrid genome as a result of chromosomal rearrangement after homoploid hybridization. Chromosomal inversion heterozygotes, seen as the formation of bridges and fragments in meiosis, of P. anomala could provide the mechanism for the maintenance of the parental alleles (Fig. 5A, B).
A genetic analysis of nuclear gene sequences in an F2 population between two individuals with known genotypes can determine whether the polymorphic sequences represent different loci or alleles at the same locus. We obtained a few F1 seeds but have not developed F2 populations because of the long generation time of peonies (3–4 yr from seed to flowering). In any event, our results indicate that while phylogenetic incongruence has provided the primary source of evidence for homoploid hybrid speciation, sequence polymorphism of single- or low-copy nuclear genes can potentially serve as another line of evidence.
Among four nuclear genes sampled from P. anomala, only the P. veitchii type of sequence was found for the nrDNA and Gpat genes, suggesting the P. veitchii alleles have been fixed at these loci. The fixation of the ITS sequence may have resulted from concerted evolution among nrDNA sites near telomeres of eight chromosomes consistent with the previous findings in allotetraploid species of Paeonia (Zhang and Sang, 1999
).
It has been recently demonstrated that new adaptation could arise from homoploid hybridization through transgressive segregation (Rieseberg et al., 2003
). The populations of P. anomala have so far been unambiguously identified in the Aletai area of northwestern China. The distribution of the species may extend farther north but certainly not south, given the careful documentation of Paeonia populations in those areas in China (Hong and Pan, 2004
). The distribution of this species thus does not overlap with its parents, P. veitchii and P. lactiflora, which are found in southern and eastern regions (Fig. 1). The phenology of these regions is distinct, suggesting that a novel adaptation has most likely resulted from the hybridization.
The high level of morphological similarity between P. anomala and P. veitchii may be explained by a backcross with P. veitchii or by the dominance of the P. veitchii alleles for the morphological traits. The novel adaptive traits of P. anomala in comparison to P. veitchii may be primarily physiological rather than morphological. From the morphological point of view, it is reasonable or even more appealing to treat P. veitchii and P. anomala as conspecific taxa (Hong and Pan, 2004
). The questions of how to classify hybrids and how to effectively reflect the corresponding phylogenetic, physiological, and ecological information in a practically useful classification still need to be addressed.
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1 The authors thank C. Wang and J.-F. Mao for assistance with field and laboratory work and D.-Y. Hong and K.-Y. Pan for valuable suggestions. The research was supported by the National Natural Science Foundation of China (grant no. 30121003 and 39928003). 
2 Author for correspondence (e-mail: zhangdm{at}ibcas.ac.cn
)
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