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Systematics and Phytogeography |
Plant Germ-plasm Institute, Graduate School of Agriculture, Kyoto University, Nakajo, Mozume, Muko 617-0001, Japan
Received for publication November 5, 2004. Accepted for publication July 24, 2005.
ABSTRACT
This study analyzes intra- and interspecific variation in chloroplast DNA (cpDNA) in diploid Triticum-Aegilops species. This analysis focused on DNA sequence variation in noncoding regions of cpDNA, which included base-pair substitutions, insertion/deletions (indels, 50 loci pooled), microsatellites (7 loci pooled), and inversions. Nine of 13 Triticum-Aegilops species were successfully identified and genotyped using these data. Sixty-two haplotypes were detected in 115 accessions of 13 diploid species. Because of the large number of characters examined, novel deep relationships within and among Triticum-Aegilops species could be identified and evaluated. Phylogenetic trees for the genus Triticum-Aegilops were constructed with Hordeum vulgare and Dasypyrum villosum as outgroups, and the results were compared to previous studies. These data support the following inferences: (1) Aegilops species should be included in Triticum; (2) groups D, T, M, N, U, and section Sitopsis (except Ae. speltoides) underwent speciation concurrently, but most diploid species evolved independently; (3) Ae. mutica does not occupy a basal position in Triticum-Aegilops; (4) Ae. speltoides is in a basal position and differs significantly from other Sitopsis species; (5) Ae. caudata is polyphyletic in all trees; (6) the genus Aegilops is paraphyletic with Secale.
Key Words: chloroplast microsatellite • indels • phylogeny • Triticum-Aegilops species
Plants belonging to the genera Triticum L. and Aegilops L. are important genetic and economic resources, because they are evolutionarily related to the major agricultural crop T. aestivum L. van Slageren (1994)
described 22 Aegilops and five Triticum species, including some diploid (2n = 2x = 14) and some polyploid (2n = 4x = 28 and 2n = 6x = 42) cytotypes. These species are distributed primarily in the Mediterranean-western Asiatic region. Based on morphology and genome analysis, Aegilops has been divided into the following six sections: Sitopsis (Jaub. & Spach) Zhuk., Amblyopyrum (Jaub. & Spach) Eig, Polyeides Zhuk., Cylindropyum (Jaub. & Spach) Zhuk., Comopyrum (Jaub. & Spach) Zhuk., and Vertebrata Zhuk. Emend Kihara, (Zhukovsky, 1928
; Eig, 1929
; Kihara, 1954
). van Slageren (1994)
separated Ae. mutica Boiss. from Aegilops and placed it into a monospecific genus called Amblyopyrum (Jaub. & Spach) Eig. van Slageren and others (Kellogg et al., 1996
) argue that Aegilops and Triticum are two distinct genera, while earlier researchers (e.g., Stebbins, 1956
) argued that these species should be considered members of the same genus. Here, data are presented to support the latter taxonomy, and in this study, Triticum-Aegilops is considered a single genus.
For the genus Triticum-Aegilops, initial analyses of meiotic chromosomes pairing in interspecific hybrids were done to identify species genome. Kihara (1954)
reviewed these numerous cytogenetic studies and established genomic formulae for various Triticum-Aegilops species. Later, many cytoplasmic and cytological studies (e.g., Tsunewaki et al., 1976
; Teoh and Hutchinson, 1983
) and an isozyme analysis (e.g., Benito et al., 1987
) were done to reveal the genome relationships of Triticum-Aegilops species. More recent analyses have been focused on molecular markers in nuclei (Dvo
ák and Zhang, 1992
; Sasanuma et al., 1996
; Wang et al., 2000
; Huang et al., 2002a
, b
; Sallares and Brown, 2004
) or organelles (Tsunewaki and Ogihara, 1983
; Terachi and Tsunewaki, 1986
; Ogihara and Tsunewaki, 1988
; Terachi and Tsunewaki, 1992
). However, questions remain about the taxonomic relationships between Triticum-Aegilops sections and species. For example, the phylogenetic position of Ae. mutica is unclear, as are the relationships between species in sections Sitopsis and Comopyrum.
It is clear that more data are needed to clarify the phylogenetic relationships within Triticum-Aegilops. This study is designed to address this need by systematically analyzing interspecific relationships for the 13 diploid species in Triticum-Aegilops as well as intraspecific variation using several accessions of each species. The importance of intraspecific variation in phylogenetic analysis has been described previously (Takahata and Nei, 1985
), but few studies of intraspecific variations in Triticum-Aegilops have been conducted (excepting mtDNA RFLP, Breiman, 1987
; cpRFLP, Miyashita et al., 1994
; PCR-SSCP, Ohsako et al., 1996
; chloroplast microsatellites, Provan et al., 2004
).
The chloroplast genome is uniparentally inherited and generally lacks heteroplasmy, making it useful for phylogenetic studies. However, because the rate of base-pair substitution (BPS) mutations in the chloroplast genome of Triticum-Aegilops species is low (Miyashita et al., 1994
), it is difficult to characterize intraspecific relationships or develop a robust phylogeny using BPS alone. This problem can be solved by characterizing other variable sequence features in the chloroplast intergenic sequences, such as insertion/deletions (indels) and inversions (Kelchner and Clark, 1997
; Kelchner, 2000
; Ingvarsson et al., 2003
). Furthermore, Ishii et al. (2001)
identified 24 chloroplast microsatellite loci (designated as WCt1– 24) with more than 10 mononucleotide repeats in T. aestivum cv. Chinese Spring, raising the possibility of including these variable sequences in phylogenetic analyses, as shown by Provan et al. (2004)
. The work of Provan et al. (2004)
suggests that microsatellites are an emergent tool for high-resolution analysis of inter- and intraspecific variation in Triticum-Aegilops. However, because they highlighted the assignation of a cytoplasm donor for cultivated species, their study included fewer informative characters than were available here. This is because diversification at the diploid level is considered the key event for evolution at the genus level, and, therefore, diploid species of Triticum-Aegilops are focused in this study.
The goal of this study is to clarify the phylogenetic relationships within Triticum-Aegilops. To this end, inter- and intra-specific genetic variation was analyzed for 13 diploid species and 115 accessions of Triticum-Aegilops. To obtain a sufficient number of sequence features for this analysis, BPS, indels, inversions, and microsatellites were characterized in the four cpDNA intergenic regions. The secondary goal of this study is to genotype multiple accessions of all diploid Triticum-Aegilops species. Using the same set of cpDNA sequence polymorphisms described above, 62 unique haplotypes were identified. The implications of these results for the evolutionary history of Triticum-Aegilops are discussed.
MATERIALS AND METHODS
Plant materials and DNA extraction
Thirteen diploid species from six sections of Aegilops and two of Triticum were examined. Five to 18 accessions of each Triticum-Aegilops species were selected from the collection maintained at the Laboratory of Crop Evolution, Plant Germ-plasm Institute, Graduate School of Agriculture, Kyoto University (Kawahara, 2005
), so that the accessions cover almost the whole area of its geographical distribution. Detailed passport data of each accession are available from the following web site: http://www.shigen.lab.nig.ac.jp/wheat/komugi/top/top.jsp. We used three genera, Hordeum (2n = 2x = 14), Dasypyrum (2n = 2x = 14), and Secale (2n = 2x = 14) as potential outgroups. These genera belong to tribe Triticeae, as does Triticum-Aegilops. In total, 115 accessions of Triticum-Aegilops species, one Hordeum L., one Dasypyrum (Cross. & Dwz.) Dur., and two Secale L. accessions were used (Table 1). Seed samples, except for Hordeum, were planted in the field at Kyoto University. A DNA sample of Hordeum vulgare cv. Betzes was provided by Dr. S. Nasuda, Kyoto University. We extracted genomic DNA from young leaves of a single plant for each accession using Plant DNAzol (Life Technologies Inc., Grand Island, NY, USA.), following the procedure recommended by the manufacturer. Vouchers of strains are deposited at the herbarium of the Plant Germ-plasm Institute.
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PCR conditions were as follows: 25 cycles of 45 s at 96°C for denaturation, 1 min at 56°C for annealing, and 1 min at 72°C for polymerization; final extension of 3 min at 72°C. PCR fragments were washed at 70% ethanol. PCR-amplified DNA fragments were analyzed by direct DNA sequencing using an Applied Biosystems 3730xl DNA analyzer (Hitachi Instruments Service, Tokyo, Japan).
Data analyses
Sequences from all four chloroplast regions were combined and aligned manually using DNASIS version 3.0 (Hitachi Software Engineering, Tokyo, Japan). Alignments were adjusted manually to minimize the number of gaps. For phylogenetic analyses, all gaps and inversions were removed from sequences. Insertions and deletions (indels) were scored as independent phylogenetic characters appended to the matrix of sequences; presence/absence (0 vs. 1) binary characters for large indels, and the number of repeat units (specified symbols = "pqrs...") for mononucleotide (microsatellite) and dinucleotide (TA) repeats (Graham et al., 2000
; Swofford, 2002
; Ingvarsson et al., 2003
). Three different analyses based on three different data sets were carried out: base-pair substitution (BPS) only; BPS + indels; or BPS + indels + microsatellites. Because the mutational process of chloroplast microsatellites is not clear (Provan et al., 2001
), microsatellite sequences were given "unordered" characters using the PAUP option. For unordered characters, change from any state to any other state is one step (Fitch parsimony; Fitch, 1971
).
Phylogenetic relationships were determined by maximum parsimony (MP) analysis using PAUP* version 4.0b10 (Swofford, 2002
). MP analyses were run with all sites weighted equally and unordered character states (i.e., Fitch parsimony). The heuristic search option was used with a stepwise addition sequence set at random; branch-swapping method tree bisection-reconnection (TBR) and MAXTREES were set to autoincrease. Because saved trees exceeded 20 000 when microsatellite data were added, MAXTREES were set to 20 000 in this data set. Strict consensus trees were constructed from multiple equally parsimonious trees. Phylogenetic relationships were also analyzed by the neighbor-joining (NJ) method (Saitou and Nei, 1987
) using PAUP*. The number of nucleotide substitutions per site was estimated by Kimura's (1980)
two-parameter method and used to estimate the genetic distance. The reliability of the clades on the shortest tree(s) was assessed using bootstrapping with PAUP (Felsenstein, 1985
) performed with 1000 replicates. The consistency index (CI) (Kluge and Farris, 1969
) and the retention index (RI) (Farris, 1989
) were calculated.
The incongruence length difference (ILD) test with PAUP* was used to determine the congruency of different phylogenetic trees (Farris et al., 1994
). The ILD test has been criticized by several authors (e.g., Yoder et al., 2001
; Darlu and Lecointre, 2002
), however, the test has its merit as a first assessment for the congruence of data sets (Hipp et al., 2004
). We performed the ILD test using removed invariable characters in this study (Cunningham, 1997a
, b
). The analysis was conducted with 100 replicates using a heuristic search strategy. The test indicates significant conflict when the score is 
1% of P value.
RESULTS
Sequence variation
Four regions of the chloroplast genome were sequenced in 115 accessions representing 13 diploid species of Triticum-Aegilops. The length of these regions varied (Table 2). There were 50 indels ranging from 1 to 45 bp in length. Six mononucleotide and one dinucleotide (TA) microsatellite repeats were detected, two of which were not detected in a previous study (Table 2; Ishii et al., 2001
). When indels, microsatellites, and inversions were excluded, 2740 bp of cpDNA were available for phylogenetic analyses. When indels, microsatellites, and inversions were included, 2806 bp of cpDNA were available. Sequence polymorphism including BPS, indels, inversions, and microsatellites were used to identify 62 chloroplast haplotypes in 111 accessions of Triticum-Aegilops species (Table 1). The number of haplotypes per species varied from two haplotypes in 11 accessions of Ae. umbellulata Zhuk., to eight haplotypes in 10 accessions of Ae. tauschii Coss. Triticum-Aegilops species were successfully identified and genotyped using these data, with minor exceptions (i.e., Ae. sharonensis Eig vs. Ae. longissima Schweinf. & Muschl.; Ae. mutica Boiss. vs. Ae. caudata L.). These data are important resources for understanding genome differentiation in diploid Aegilops species. Analysis of autapomorphic sequence variation in outgroup species showed the following: 52 substitutions and 14 indels for Hordeum, 16 substitutions and 4 indels for Dasypyrum, and 11 substitutions and 3 indels in two species of Secale. Thus, 66 haplotypes were available for the phylogenetic analyses described below.
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Outgroup choice
Previous phylogenetic analyses based on molecular data suggested that Secale is the closest relative of the genus Triticum-Aegilops (see Kellogg et al., 1996
). Moreover, Triticum-Aegilops was reported to be paraphyletic with Secale cereale L. in some of the molecular phylogenetic trees (Huang et al., 2002b
; Mason-Gamer et al., 2002
), indicating the relatively close relationship between the two genera. Therefore, two Secale species were used as ingroup accessions. As a result, the phylogenetic trees (Figs. 1, 2) showed that the genus Triticum-Aegilops constitutes a polyphyletic group with Secale. When all three genera, Hordeum, Dasypyrum, and Secale, were used as outgroups, the topology of the phylogenetic tree was completely disordered, i.e., it was incongruent with the widely accepted belief that Ae. speltoides Tausch is more closely related to other Aegilops species than to Hordeum (data not shown). This suggested that Secale should not be used as an outgroup for Triticum-Aegilops. Accordingly, Hordeum and Dasypyrum were used as outgroups in this study.
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; Vogl et al., 2003
). In this study, a phylogenetic tree was constructed for four chloroplast regions based on three data sets: BPS; BPS + indels; or BPS + indels + microsatellites. ILD tests showed no significant incongruence between the four chloroplast regions. Based on this result, and the fact that the combined data set had higher resolvability and reliability than individual data sets from each region (data not shown), the combined data sets were used for additional analysis, as described next.
Selective analysis with and without indels and microsatellites
Table 3 summarizes the characteristics of strict consensus trees that were constructed using the different data sets. When only BPS were used, there were 61 parsimony informative substitution characters in 2740 bp of aligned chloroplast sequence data. The strict consensus tree based on these data had lower resolution than the neighbor-joining tree, but the two trees were largely congruent with each other (Figs. 1a, b). The CI, after excluding uninformative characters and RI values (0.8442 and 0.9615, respectively), exhibited low homoplasy, indicating high reliability of the tree. A strict consensus tree was also constructed using BPS + indels. This tree was based on the 108 shortest trees, and CI = 0.7679 after excluding uninformative characters, and RI = 0.9353, both of which were slightly lower than for the tree based on BPS only (Fig. 2, left). The pairwise ILD test for these two data sets show no significant heterogeneity, meaning that little homoplasy arises when the data are combined. The topology of this tree recovers all nodes and provides considerably better resolution within ingroups. For similar analysis using BPS + indels + microsatellites, the number of equally parsimonious trees was very high (>20 000) (Table 3). Although lower CI after excluding uninformative characters and RI values (0.6154 and 0.8828, respectively) are obtained for this data set, phylogenetic relationships were almost congruent for this (Fig. 2, right) and other (Figs. 1b, 2, left) data sets. In addition, the topology of this tree (Fig. 2, right) recovers all nodes and provides considerably better resolution within ingroups than the other trees (Figs. 1b, 2, left). The only incongruency was in the position of Ae. caudata and Ae. tauschii. However, no significant incongruence was detected by ILD test for analyses with BPS only or BPS + indels + microsatellites.
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insisted in his review that inversions show high homoplasy and that they may not be particularly useful for phylogenetic studies. In this study, the inversion in the ndhF-rpl32 intergenic region showed high homoplasy in all trees from the rooted analysis (Figs. 1–3) and was not used for phylogenetic analysis for this reason. Thus, this study provides support for the views expressed in Kelchner (2000)
.
Phylogenetic relationships
Neighbor-joining trees were constructed using all data sets, but no significant differences from strict consensus trees were detected (Fig. 1a and data not shown). Heuristic searches of the 66 haplotype matrix based on BPS only produced strict consensus trees (Fig. 1b). Strict consensus trees were also constructed from BPS + indels (Fig. 2, left) and from BPS + indels + microsatellites (Fig. 2, right). All strict consensus trees showed that Ae. speltoides was basal to other Triticum-Aegilops species. Unexpectedly, all strict consensus trees showed that the two Secale species were completely included in the Triticum-Aegilops clade with high bootstrap values (>99%) for all three data sets (Figs. 1b, 2, left and right). This result was also consistent with a synapomorphic four-base deletion in the rpoB-trnC intergenic region.
The most terminal group is comprised of the A, C, D, M, N, T, and U genome accessions. Analyses based on BPS + indels (Fig. 2, left), or BPS + indels + microsatellites (Fig. 2, right) suggest that two Triticum species first branched out from Aegilops species in the terminal group. The section Sitopsis species, excluding Ae. speltoides (corresponding to subsection Emarginata by Eig [1929]
), clustered with high bootstrap values (83–92%) in all trees (Figs. 1b, 2, left and right). As for patterns of phylogenetic differentiation of each species, the potential monophyly of T. boeoticum Boiss., Ae. umbellulata, Ae. uniaristata Vis., Ae. speltoides, and Ae. tauschii was supported by all phylogenetic trees. In contrast, T. urartu Tum., and Ae. mutica were always paraphyletic, and Ae. comosa Sm. in Sibth & Sm., Ae. searsii Feldman & Kislev ex Hammer, Ae. bicornis (Forssk.) Jaub. & Spach, and Ae. longissima were paraphyletic in a subset of trees. Ae. caudata was polyphyletic in all trees (Figs. 1b, 2, left and right).
DISCUSSION
Analysis of DNA sequence variation in Triticum-Aegilops chloroplast DNA
The high CI values after excluding uninformative characters and RI values (0.8442 and 0.9615, respectively) indicated low homoplasy in the strict consensus tree based on BPS only (Fig. 1b, Table 3); this indicates high reliability of the tree. For the strict consensus tree based on BPS + indels (50 loci pooled), these indices were slightly lower (CI after excluding uninformative characters = 0.7679 and RI = 0.9353). However, there were few differences in large clustering patterns for these two trees (Figs. 1b, 2, right). Within the terminal group comprised of genome accessions A, C, D, M, N, T, and U, the tree based on BPS + indels (Fig. 2, left) has higher resolution than the tree based on BPS (Fig. 1b). This indicates that indels are useful for phylogenetic analyses of these species and for lower taxonomic levels in this genus. When all BPS + indels + microsatellites were considered in the analysis, the phylogenetic relationships remained largely congruent (Fig. 2, right vs. Figs. 1b, 2, left). The phylogenetic tree based on BPS + indels + microsatellites (Fig. 2, right) had higher resolution than other trees in the terminal group, especially within T. boeoticum, Ae. comosa, Ae. tauschii, and Ae. speltoides. The unrooted strict consensus tree in the terminal group comprised of C, D, M, N, T, and U also had higher resolution (Fig. 3). The CI values after excluding uninformative characters and RI values were also slightly lower in the strict consensus tree based on BPS + indels + microsatellites (Fig. 2, right, Table 3), indicating a higher level of homoplasy than for other trees. This result was expected, because size homoplasy of chloroplast microsatellite alleles has been reported previously (reviewed by Provan et al., 2001
).
The process by which mutations accumulate in chloroplast microsatellites is not well understood; however, these sequences appear to exhibit size homoplasy, based on the distribution of length variants within the phylogeny. Provan et al. (1999)
suggested that size homoplasy might not be a major problem for chloroplast microsatellites because the mutation rate is lower than in nuclear microsatellites. In this study, the use of microsatellite data strengthened the argument for several strong groupings in the phylogenetic trees and increased the amount of diversity within species (e.g., T. boeoticum, Ae.tauschii, and Ae. speltoides). These results suggest that microsatellite fragment size may be useful as a phylogenetic marker in Triticum-Aegilops.
Phylogenetic inferences in the genus Triticum-Aegilops
Phylogenetic inferences among diploid Triticum-Aegilops species
In previous studies, RFLPs in cpDNA (Ogihara and Tsunewaki, 1988
) and mtDNA (Terachi and Tsunewaki, 1992
) correlated with phylogenetic differences between Ae. speltoides, Triticum species, and the rest of Aegilops species. However, no outgroup taxa were included in their studies, so the precise phylogenetic relationships remained unclear. Because the phylogenic analysis presented here has higher resolution than previous analyses, it is clear in this study that Ae. speltoides branched out first, before other Triticum and Aegilops species (i.e., terminal groups comprising A, D, T, M, N, and U, and the subsection Emarginata) could be differentiated. Consequently, all phylogenetic trees presented here indicate that diploid Triticum species are completely included within Aegilops. In other words, there is no discrimination between Triticum and Aegilops. Stebbins (1956)
has argued for merging Triticum and Aegilops into a single genus, but Bowden (1959)
claimed that further studies are needed. Recently, van Slageren (1994)
and Kellogg et al. (1996)
expressed their opinion that Aegilops and Triticum should be retained as two distinct genera. Based on the present result, if Triticum and Aegilops are retained as separate genera, Ae. speltoides must be treated as a new genus, suggesting that consensus for Stebbins' view is growing and the idea of the merging Triticum and Aegilops into a single genus may eventually be generally accepted.
Zohary and Feldman (1962)
noticed that each diploid Aegilops species is genetically distinct from the remaining diploid Aegilops species. If each species has evolved more or less independently, unique mutations must exist in the genome of each species differentiating it from the other species. However, the phylogenetic relationships among groups D, T, M, N, and U and the subsection Emarginata have been unclear, possibly because the number of characters has been insufficient to reveal phylogenetic relationships. In the present study, a sufficient number of diagnostic markers was available to substantiate the idea that most of the diploid species evolved independently from one another.
The large number of characters used in this study provides new opportunities to estimate phylogenetic relationships within and among Triticum-Aegilops species. The following discussion reviews the major phylogenetic relationships in Triticum-Aegilops and compares the present results with those of earlier studies.
Phylogenetic position of Ae. mutica
Aegilops mutica is native to Turkey and Armenia. Because several morphological characters (e.g., awnless linear spikes) differentiate Ae. mutica from other Aegilops species, van Slageren (1994)
clamed that Ae. mutica should belong to genus Amblyopyrum instead of Aegilops. Ae.mutica and Ae. speltoides are the only predominantly cross-pollinating diploid species in the genus. Since most perennial relatives in the tribe Triticeae are allogamous, allogamy is generally accepted as an ancestral character. In addition, Ae. mutica bears a close resemblance to the perennial relatives, especially Agropyron, so it has been considered an ancestral species based on morphological and cytogenetical analyses (reviewed by Ohta, 1991
). However, accessions of Ae. mutica were included in the most terminal clade in the present study. Similar results were obtained by Dvoák and Zhang (1992)
and Ohsako et al. (1996)
. Moreover, the level of intraspecific variation in Ae. mutica was not only much lower than in Ae. speltoides, based on PCR-SSCP analyses (Ohsako et al., 1996
), but also much lower than other allogamous species based on allozyme analyses (Hegde, et al., 2002
). Those two results suggest that Ae. mutica may have originated relatively recently. Some molecular analyses suggest that Ae. mutica is close to Ae. umbellulata (Terachi et al., 1984
; Murai et al., 1989
, cpDNA RFLP) as well as to Ae. caudata, Ae.comosa, Ae.umbellulata, Ae.uniaristata (Dvoák and Zhang, repeated nucleotide sequences, 1992
), and Ae. tauschii (Terachi and Tsunewaki, mitochondrial DNA RFLP, 1992
). However, recent EST data (Sallares and Brown, 2004
) showed that Ae. mutica has a basal position and that it is close to Ae. speltoides. One explanation for these contradictory results was suggested by Sallares and Brown (2004)
as follows: Ae. mutica may have a complicated genome structure that is a result of ancestral hybridization and introgression events. Although the precise position of Ae. mutica in the genus Aegilops is not yet certain, we conclude that Ae. mutica should be included in Aegilops, based on all available molecular data.
Phylogenetic relationships in section Sitopsis
The section Sitopsis consists of five species, Ae. speltoides, Ae. searsii, Ae. bicornis, Ae. sharonensis, and Ae. longissima. It has been the focus of intensive studies because these species have been considered potential B genome donors to polyploid wheat. Eig (1929)
recognizes two subsections: subsection Truncata, with the top of the empty glume cut straight across, and subsection Emarginata, with the top of the empty glume shallowly notched. Subsection Truncata contains Ae. speltoides, and the rest of the species belong to subsection Emarginata. Namely, Eig claimed that Ae. speltoides had separated from other species of section Sitopsis. This claim is supported by various molecular data (cpDNA RFLP, Ogihara and Tsunewaki, 1988
; Murai et al., 1989
; Ogihara et al., 1994
; in situ hybridization and C-banding, Badaeva et al., 1996a
, b
; PCR-single-strand conformational polymorphism (PCR-SSCP), Ohsako et al., 1996
; nuclear RFLP, Sasanuma et al., 1996
; PCR-SSCP, Wang et al., 1997
; Provan et al., 2004
, chloroplast microsatellites; the entire 5' external transcribed spacer (ETS) region of the 18S rRNA gene, Sallares and Brown, 2004
). In most of these studies, differences between Ae. speltoides and the remaining Sitopsis species have been detected. Nevertheless, some studies suggest that the five Sitopsis species form a single group without clear differentiation (repeated nucleotide sequences, Dvoák and Zhang, 1992
; PCR-SSCP analyses of organellar DNAs, Wang et al., 1997
). Nor did van Slageren (1994)
recognize subsections within section Sitopsis. In this study, all phylogenetic trees show that Ae. speltoides does not form a monophyletic clade with other Sitopsis species and that Ae. speltoides is distant from the other four Sitopsis species. In our opinion, therefore, Ae. speltoides should be treated as a distinct group.
Phylogenetic relationships of section Comopyrum
Section Comopyrum was first described by Jaubert and Spach (1850– 1853)
and, at that time, it contained only Ae. comosa (Ae. comosa var. subventricosa). Later, Zhukovsky (1928)
classified Ae. caudata, Ae.comosa, Ae.heldreichii Holzm. (syn. of Ae. comosa), and Ae. uniaristata into section Comopyrum based on morphological characters. Kihara (1937
, 1954
) pointed out the close relationship between Ae. uniaristata and Ae. comosa based on genome analysis, but excluded Ae. caudata from sect. Comopyrum and designated the MM genome to Ae. comosa and MuMu to Ae. uniaristata. Chennaveeraiah (1960)
, however, proposed assigning a distinct NN genome to Ae. uniaristata based on karyotype analysis. Kimber et al. (1983)
also found no preferential pairing between the two genomes. Wang et al. (2000)
claimed that the classification of section Comopyrum should be reconsidered based on their study of ITS sequences. Badaeva et al. (1996a
, b
) also postulated that Ae. uniaristata should be removed from section Comopyrum based on in situ hybridization and chromosome morphology data. In this study, the phylogenetic trees show paraphyly between Ae. uniaristata and Ae. comosa (Figs. 1, 2). However, the unrooted strict consensus tree based on BPS + indels + microsatellites showed that Ae. uniaristata and Ae. comosa cluster together (Fig. 3). In addition, previous molecular studies based on chloroplast DNA RFLP analysis showed monophyly of sect. Comopyrum species, Ae. comosa, and Ae. uniaristata (Terachi et al., 1984
; Ogihara and Tsunewaki, 1988
), similar to our data. This suggests that a sufficient number of characters and an adequate sample size are needed for molecular phylogenetic analyses. Incongruence between different data sets is largely caused by too few characters. For example, Cronn et al. (2002)
reported unreliable phylogenetic performance when using a limited number of characters to analyze chloroplast DNA. In this study, the classification of section Comopyrum is supported by the maternal marker, but further study may be necessary to confirm this classification.
Polyphyly of Ae. caudata
In this study, it is noticeable that the polyphyly of Ae. caudata is supported by all trees. Ae. caudata occurs mainly in the Aegaeis and western Turkey, is less common elsewhere in Turkey and is sporadic along the Fertile Crescent (van Slageren, 1994
). Tanaka et al. (1967)
first discovered intraspecific sterility in Ae. caudata, and then Ohta (1992)
confirmed this by crossing experiments. In addition, he found that the intraspecific sterility developed in geographically separated populations. Recently, Ohta (2000)
confirmed this result, identifying intraspecific sterility in A. caudata from west and east of the mountains between west and central Anatolia.
In order to identify genetic differentiation among Ae. caudata accessions from west and central Anatolia, 12 accessions covering the entire distribution area were analyzed. The results show that Ae. caudata is divided into two distinct groups in all trees. The group in a more basal position (CAU-I, II, III, and IV) includes accessions from the entire distribution area, indicating that geographical location is not congruent with phylogenetic position (Table 1, Figs. 2, 3). However, there is intraspecific sterility in Ae. caudata and there are also two highly diverged chloroplast DNA sequences, suggesting that Ae. caudata comprises a distinct species. If this is correct, these accessions may represent convergent evolution of morphological features. Alternatively, introgression from Ae. umbellulata or Ae. mutica into Ae. caudata is still a possibility. This study is the first intraspecific study of Ae. caudata with molecular markers. Therefore, additional crossing experiments, as well as molecular studies with a larger sample size and more markers, are needed to confirm this result.
Phylogenetic position of Secale
An unexpected result from this study was that all analyses show the two Secale species lying within the Triticum-Aegilops clade. This occurs with high bootstrap values (>99%). A similar result was obtained by Sallares and Brown (2004)
using the nuclear genome, but with very weak node support. Similarly, several maternal (Murai et al., 1989
) and biparental (Huang et al., 2002a
, b
) markers also demonstrate a close relationship between Triticum-Aegilops and Secale. On the other hand, when most of the genera in the tribe were sampled, the data support separation of Secale from Triticum-Aegilops (Hsiao et al., 1995
; Kellogg and Appels, 1995
; Mason-Gamer, 2004
). Therefore, although our data suggest that the genus Secale be included within the Triticum-Aegilops clade, this result may reflect early introgression of organelle DNA between Triticum-Aegilops and Secale. If this were the case, cpDNA data could not resolve the deep phylogeny of Secale and Triticum-Aegilops. Because Secale is still part of an unresolved basal polytomy, more extensive studies are needed to clarify the phylogeny of genera in the Tribe Triticeae including Secale and Triticum-Aegilops.
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The authors thank Dr. S. Nasuda, Kyoto University, for his kindness in providing total DNA from H. vulgare; Dr. Y. Matsuoka, Fukui Prefectural University, for technical advice; Prof. O. Ohnishi, Kyoto University, for helpful advice; Dr. Y. Yasui, Kyoto University, for useful comments on an early version of this manuscript; Mr. J. Fawcett, Kyoto University for comments on the revision of the manuscript and the reviewers for comments that helped us improve the manuscript. This is Contribution No. 122 from the Plant Germ-plasm Institute, Graduate School of Agriculture, Kyoto University. This research was partly supported by a grant from the Ministry of Education, Culture and Technology, the National BioResource Project KOMUGI. 
2 Author for correspondence (e-mail: kawatai{at}mbox.kudpc.kyoto-u.ac.jp
)
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