J Mol Evol (2003) 57:623–635 DOI: 10.1007/s00239-003-2512-8

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in Larix potaninii ()

Xiao-Xin Wei, Xiao-Quan Wang, De-Yuan Hong

Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China

Received: 9 December 2002 / Accepted: 16 June 2003

Abstract. Nuclear ribosomal DNA (nrDNA) of Key words: nrDNA ITS — Pseudogene — Re- gymnosperms, especially Pinaceae, is characterized combination — Concerted evolution — Geo- by slow concerted evolution and exhibits substantial graphical differentiation — Larix ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angio- sperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed Introduction spacer (ITS) region in four varieties of Larix potani- nii, a species from the mountainous areas of western Nuclear ribosomal DNA (nrDNA) occurs as tan- China. Two clones with more than a 100-bp deletion demly repeated units at one or several loci, with copy in ITS1 were detected in L. potaninii var. chinensis numbers varyingfrom several hundreds to thousands and L. potaninii var. australis, respectively. The de- per haploid genome (Long and Dawid 1980). Each letion resulted in the loss of most part, includingthe unit, separated by intergenic spacers (IGS), consists motif sequence, of subrepeat 1 (SR1). Sequence di- of the 18S, 5.8S, and 26S codingregionsand two vergence and phylogenetic analyses showed that some internal transcribed spacers (ITS1 and ITS2) in clones would be pseudogenes given their low GC . Repeats within one array are more closely content, high substitution rates, unique positions in related to one another than to repeats in arrays on the phylogenetic trees, or significant length variation. other chromosomes (Schlo¨ tterer and Tautz 1994; These clones might represent orphons or paralogues Copenhaver et al. 1995; Muir et al. 2001) or to re- at minor loci resulting from large-scale gene or peats in other taxa (Arnhein 1983) due to concerted chromosome reorganization. Some recombinants evolution by unequal crossing-over (Smith 1976) and characterized by chimeric structure and discordant gene conversion (Nagylaki and Petes 1982; Nagylaki phylogenetic positions in partitioned sequence anal- 1984; Hillis et al. 1991). For nrDNA of angiosperms, yses indicate that unequal crossing-over plays an intragenomic diversity is generally low and the length important role in the process of nrDNA concerted of the ITS region remains in the relatively narrow evolution. In addition, some varieties of L. potaninii range of 565 to 700 bp (Baldwin et al. 1995; Wendel might have experienced an nrDNA founder effect et al. 1995). Though some polymorphism was ob- parallel to their geographical isolation. served within or amongindividuals, species, or even genera, the ITS repeats do not differ significantly in length, but solely in nucleotide substitutions. The Correspondence to: Dr. Xiao-Quan Wang; email: xiaoq_wang@ only exception, a variant carryinga 100-bp insertion ns.ibcas.ac.cn in ITS1, was reported within and amongindividuals 624 from five populations of the Lisianthius skinneri the seed scales, and Sect. Multiserialis includes species species complex of the family Gentianaceae (Sytsma with bracts extendingfar beyond the seed scales and Schaal 1990). (Patschke 1913; Farjon 1990; LePage and Basinger The concerted evolution of nrDNA in gymno- 1991, 1995; Schorn 1994). Larix potaninii, a species of sperms, however, is much slower than that in angio- Sect. Multiserialis, is endemic to the Himalayas and sperms. Substantial length variation is commonly adjacent regions. Due to morphological radiation observed. For example, the family Pinaceae exhibits and geographical isolation, it has differentiated into the greatest variation in ITS length, ranging from four varieties, namely, var. potaninii (Pot), var. aus- 1550 bp in Pseudotsuga to between 3150 and 3660 bp tralis (Aus), var. chinensis (Chi), and var. himalaica in Picea (Liston et al. 1996; Marrocco et al. 1996; (Him) (Farjon 1990, 2001; Fu et al. 1999). The former Germano and Klein 1999; Maggini et al. 1998, 2000; two varieties are distributed mainly in the Hengduan Gernandt et al. 2001). Besides, inter- or intraspecific Mountains, the eastern Himalayan diversity ‘‘hot length difference in the ITS region as large as several spot’’ (Wilson 1992), and they are sympatric in SW hundred base pairs was reported in Abies (Xianget al. Sichuan and NW Yunnan. Var. himalaica is confined 2000), Picea (Maggini et al. 1998, 2000), and Pinus to the Himalayas (SW Tibet and Nepal), and var. (Quijada et al. 1998; Gernandt et al. 2001). Espe- chinensis has a very limited distribution in the Taibai cially, extensive intragenomic length variation (>100 Mountains, S Shaanxi. However, no clear molecular bp) has been found in Cedrus deodara (Liston et al. differentiation was found amongthe four varieties 1996) and Pinus sylvestris (Karvonen and Savolainen (Wei and Wang2003). In the present study, we 1993) through RFLP or PCR-RFLP analysis. It is cloned the nrDNA ITS region of all the varieties of L. interesting that the large and extensive length varia- potaninii to investigate its intra- and intergenomic tion always results from ITS1, while 5.8S and ITS2 heterogeneity and to explore the distribution pattern have very conserved lengths, i.e., 162 and 232–245 bp, and phylogenetic history of ITS paralogues, which respectively (Gernandt and Liston 1999; Gernandt would shed a new light on the evolutionary dynamics et al. 2001). Sequence structure analysis showed that of nrDNA family in gymnosperms. a conserved motif embedded in two to six subrepeats occurs in all Pinaceae ITS1 and is synapomorphic for the family. In particular, Pinaceae ITS1 length vari- Materials and Methods ation is highly positively correlated with subrepeat number (Vining 1999). The high heterogeneity of Materials nrDNA in Pinaceae provides us with an ideal mate- rial to retrieve the evolutionary process of the The four varieties of Larix potaninii were all sampled in this study. multigene family and to explore the mechanism of L. laricina, a North American species of Sect. Larix, was also concerted evolution. However, previous studies fo- analyzed. The origins of materials are shown in Table 1. cused mainly on two old genera, i.e., Pinus and Picea, with an early Cretaceous origin (Florin 1963; Millar DNA Extraction,ITS Region Amplification, 1993; Wang et al. 2000), and no large intragenomic Cloning,and Sequencing length variation of the ITS region was found at the sequence level (Bobola et al. 1992; Karvonen and Total DNA was extracted from silica gel dried needles using the CTAB method followingthe protocol of Rogersand Bendich Savolainen 1993; Quijada et al. 1998; Gernandt et al. (1988) and used as template in the polymerase chain reaction. 2001). In addition, Pinus and Picea have multiple The ITS region was amplified with primers ITS1N (50- rDNA loci, ranging between five and eight pairs in GTCGTAACAAGGTTTCCGTAGG) located on the 18S rDNA the diploid genomes (Brown and Carlson 1997; and ITS4 of White et al. (1990). The PCR reaction was carried out Gernandt and Liston 1999). Some authors suggested in a volume of 25 ll containing5–50 ngof DNA template, 6.25 pmol of each primer, a 0.2 mM concentration of each dNTP, 2 mM that the ITS heterogeneity may be related to the high MgCl2, and 0.75 U of Taq DNA polymerase. Amplification was number of rDNA loci (Liston et al. 1996; Gernandt conducted in a Peltier Thermal Cycler (PTC-200, MJ Research). and Liston 1999; Gernandt et al. 2001). Further PCR cycles were as follows: 1 cycle of 4 min at 70°C, 4 cycles of 40 studies on the relationship between concerted evolu- sat94°C, 20 s at 52°C, and 2 min 30 s at 72°C, followed by 36 tion rate and rDNA loci number are needed. cycles of 20 s at 94°C, 20 s at 52°C, and 2 min 30 s at 72°C, with a final extension step for 10 min at 72°C. PCR products were sepa- Contrary to Pinus and Picea, Larix is a young rated by 1.5% agarose gel electrophoresis. The band with the right genus with an Eocene origin (LePage and Basinger size was cut out and purified usingthe GFX PCR DNA and Gel 1995; Wanget al. 2000) and has fewer rDNA loci Band Purification Kit (Pharmacia) and then cloned with the (three pairs) (Lubaretz et al. 1996). It consists of pGEM-T Easy Vector System II (Promega). about 10 species, which fall into two morphologically For each taxon, at least 30 clones with correct insertion (de- termined by digestion with EcoRI) were screened by comparing distinct groups based on the female cone. Sect. Larix restriction fragments of MspI or both HaeIII and HinfI. All distinct (or Pauciserialis) comprises species characterized by clones were sequenced with the two PCR primers and several in- bracts on the cone that did not extend well beyond ternal primers (Fig. 1), i.e., LITS3 (50-CTTCTTGCCTCGA- 625

Table 1. Sources of materials used in the present study

Taxon Source Latitude/longitude Clone Nos. GenBank accession Nos.

L. potaninii Var. australis (Aus) Lijiang, Yunnan, China 26°080/100°020 1, 2, 18, 20, 7, 17 AY188526–AY188529, AY188550–AY188551 Var. chinensis (Chi) Changan, Shaanxi, China 34°010/108°090 1, 5, 9, 12, 26, 27 AY188530–AY188534, AY188552 Var. himalaica (Him) Jilong, Xizang, China 28°090/85°020 5, 7, 9, 13, 18, 22, 14 AY188535–AY188540, AY188553 Var. potaninii (Pot) Kangding, Sichuan, China 30°000/101°090 1, 2, 3, 5, 8, 10, 16 AY188541–AY188547 L. laricina Arboretum of Shenyang, 1, 2 AY188548–AY188549 Liaoning, China/cultivated

Fig. 1. Schematic representation and primer position for ITS region in Larix. Shaded boxes represent the two subre- peats. Horizontal arrows indicate the locations and directions of primers used in the present study. The bases in bold- face show the conserved motif sequence in the two subrepeats. The underlined sequences represent an 11-bp indel. Numbers below the vertical arrows in- dicate base position in the aligned ITS data set. The arrowhead represents the core position.

GATTTCC), ITS1R1 (50-CATAACAAGCACACCCATCAC), option, and a maximum of 1000 trees saved per round. To evaluate ITS1R2 (50-CCTCGTGCAAGACAAAGCAC), and P2N (50- relative robustness of the clades found in the most parsimonious GAGAGCCGAGATATCCGTTG), usingthe ABI Prism Bigdye trees, the bootstrap analysis (Felsenstein 1985) employed 100 rep- Terminator Cycle SequencingReady Reaction Kit. After purifi- licates, with a maximum of 100 trees saved per round usingthe cation through precipitation with 95% EtOH and 3 M NaAc (pH heuristic search with random sequence addition, TBR branch 5.2), the sequencingreaction products were applied to an ABI 377 swapping, and the MULTREES option. The neighbor-joining (NJ) automatic sequencer (PE Applied Biosystems, Inc.). tree was also constructed with MEGA version 2.1 (Kumar et al. 2001) based on the Kimura (1980) two-parameter genetic distance. Data Analyses Results Boundaries of the ITS, ITS1, 5.8S, and ITS2 regions were identified in comparison with other available sequences (Gernandt and Liston 1999). Sequence alignments were made with CLUSTAL X Length Heterogeneity and Structure of ITS in (Thompson et al. 1997) and refined manually. MEGA v.2.1 (Ku- Larix potaninii mar et al. 2001) was applied to estimate GC content and nucleotide substitutions (d) for the ITS1, 5.8S, and ITS2 regions separately Twenty-eight distinct clones were recognized by re- accordingto Kimura’s (1980) two-parameter model. The standard errors of the estimates were obtained applying500 bootstrap striction analysis and completely sequenced. The en- replicates. tire ITS region length of the clones ranges from 1653 Three data sets—the whole ITS region, 50 ITS1 partition, and 30 to 1772 bp. Except Him-22, a clone with a 1-bp de- ITS1+5.8S+ITS2 partition—were analyzed with the maximum letion in the 5.8S codingregion,the others have a parsimony method usingPAUP (version 4.0) (Swofford 1998). L. conserved length of 5.8S region (162 bp) and ITS2 laricina was used as the outgroup considering the female cone morphology (Patschke 1913; Farjon 1990, 2001; Fu et al. 1999; (232 bp), respectively. Extensive length variation in LePage and Basinger 1991, 1995; Schorn 1994) and the congruent these clones results from the ITS1 region (Table 2). result of previous molecular and allozyme locus analyses that Larix For example, Aus-17 and Chi-26 have much shorter was divided into a North American and Eurasian clade (Gernandt ITS1, i.e., 1259 and 1260 bp, respectively, than the and Liston 1999; Semerikov and Lascoux 1999; Wei and Wang others (1364–1378 bp). The two clones also have an 2003). All character states were specified as unordered and equally weighted with indels as missing data. Heuristic search was imple- 11-bp deletion together with Him-5 and Him-22. mented with 100 random addition sequence replicates, tree However, the intragenomic ITS1 length variation was bisection–reconnection (TBR) branch swapping, the MULTREES no more than 2 bp for the rest of the clones except 626

Table 2. GC content of ITS1, 5.8S, and ITS2 in different taxa (average) and some divergent clones

ITS1 5.8S gene region ITS2

Taxa/clones Length (bp) GC% Length (bp) GC% Length (bp) GC%

L. potaninii Var. australis 1368 57.0 162 51.3 232 59.5 Var. chinensis 1372 or 1374 57.5 162 51.6 232 59.9 Var. himalaica 1366, 1367, or 1378a 57.0 162 51.3 232 59.5 Var. potaninii 1372 or 1374 57.1 162 51.6 232 59.6 Divergent clones Aus-7 1365 56.1 162 51.3 232 59.5 Aus-17 1259 53.1 162 51.3 232 59.9 Chi-12 1364 56.0 162 51.9 232 60.4 Chi-26 1260 55.4 162 48.1 232 59.9 Him-22 1370 51.3 161 42.2 232 53.9 L. laricina 1365 58.2 162 51.3 232 60.2 a Length of Him-5.

Chi-12, with a length of 1364 bp. Generally, except Sequence divergence of ITS1, 5.8S, and ITS2 was for some unique sequences, the ITS1 region length is calculated, respectively, and is shown in Table 3. The relatively conserved in each variety. five unique clones were not included in the intervari- Structure analysis showed that the two subrepeats etal divergence calculation in which all clones from the (SR1 and SR2), with a highly conserved center core same variety were treated as a group. As a result, great of GGCCACCCTAGTC embedded, respectively divergence of the whole or partial ITS region was (Gernandt and Liston 1999), also exist in Larix po- observed amongthe five clones (not shown) and be- taninii ITS1 except the two clones Aus-l7 and Chi-26 tween the five clones and the four varieties of L. po- (Fig. 1). In the alignment matrix of ITS data, SR1 taninii. Especially, the genetic distance between the and SR2 covered nucleotide positions 815–878 and three clones, i.e., Chi-26, Aus-17, and Him-22, and the 1108–1185 separately, and sequences between posi- four varieties was two to six times higher than the tion 745 and position 861, includingthe core of SR1, intervarietal divergence. Surprisingly, Chi-26 and were deleted for Aus-17 and Chi-26. An 11-bp indel Him-22 showed great divergence of the 5.8S region between SR1 and SR2 (positions 1023–1033) was besides the two spacers. In contrast, Aus-7 and Aus-17 observed in four clones of L. potaninii, namely, Aus- had a low divergence of both the 5.8S region and ITS2. 17, Chi-26, Him-5, and Him-22, as well as two clones The highest sequence divergence was found in Him-22. of L. laricina (Fig. 1). In addition, three GC-rich boxes were detected between nucleotide positions Phylogenetic Analyses 831–884, 1357–1378, and 1673–1702, with 78, 91, and 77 GC contents, respectively. % The aligned full-length sequences of the 28 clones from L. potaninii and L. laricina had 1793 characters. GC Content and Sequence Divergence Parsimony analysis usingheuristic search generated 340 most parsimonious trees with tree length = 379 From Fig. 2 we can see that five clones, Aus-7, Aus-l7, steps, consistency index = 0.8522, and retention in- Chi-12, Chi-26, and Him-22, have much more varia- dex = 0.7838. One tree that was almost congruent ble nucleotide sites. Therefore, we calculated their with the strict consensus tree in topology is shown in GC content and sequence divergence independently. Fig. 3. A clade comprising Chi-26, Aus-17, and Him- Excludingthe five clones, averageGC contents of 22, three clones with extraordinarily longbranch ITS1, 5.8S, and ITS2 in different varieties were 57.0– lengths, diverged out first. Aus-7 separated out next. 57.5, 51.3–51.6, and 59.5–59.9%, respectively (Table Then the others formed a clade, in which the clones of 2). In general, the GC content in ITS2 was slightly var. potaninii and var. australis mixed together and higher than that in ITS1, while that in the 5.8S region formed a monophyletic group (bootstrap value = was significantly lower than in the two spacers. For 70%) as sister to var. chinensis clade. The clones from the five clones, the GC content in ITS1 (51.3–56.1%) var. himalaica grouped together as sister clade of the was a little or much lower than the mean value of other three varieties. Consideringthat long-branch each taxon. Additionally, Chi-26 had a relatively low attraction is likely to cause phylogenetic errors, we GC content (48.1%) in the 5.8S region. In particular, deleted the three clones Chi-26, Him-22, and Aus-17 a markedly low GC content was found in each part and reanalyzed the data. The resultingphylogenywas (ITS1, 5.8S, and ITS2) of the Him-22 ITS region. topologically congruent with Fig. 3. 627

For Aus-7, Aus-17, Chi-12, and Chi-26, substitu- end was retained (Fig. 1). Liston et al. (1996) detected tions were under a great bias toward one of the two that the ITS1 of Pinaceae is much longer than that of ITS fragments separated between SR1 and SR2, and other seed plants and ranges from 1170–1177 bp in some chimeric structures were clearly observed (Fig. Pseudotsuga (Gernandt and Liston 1999) to 2784– 2). Therefore, the aligned ITS data set was parti- 3263 bp in Picea (Maggini et al. 2000), which is due in tioned, between the two subrepeats, into the 50ITS1 part to the presence of multiple, dispersed subrepeats (878 bp) and 30ITS1+5.8S+ITS2 (915 bp) for fur- (Vining1999). Marrocco et al. (1996) first observed ther phylogenetic analyses in order to retrieve some five subrepeats with sequence homology in Pinus pi- recombination events. As expected, the partitioned nea, and the sixth subrepeat in this species was re- analyses yielded most parsimonious trees conflicting ported by Gernandt and Liston (1999). In addition, in topologies, in particular, the positions of the three five to six subrepeats were found in the other species clones Chi-12, Chi-26, and Aus-7. In the strict con- of Pinus (Gernandt et al. 2001), two in Larix (Ger- sensus tree of 81 most parsimonious trees found in nandt and Liston 1999) and three in both Pseudot- the 50ITS1 analysis (Fig. 4A), a strongly supported suga and Tsuga (Viningand Campbell 1997; clade, comprising Aus-7, Aus-17, and Him-22, sepa- Gernandt and Liston 1999). All these subrepeats rated out at the base. The other clones from L. po- range from 68 to 265 bp in size with a conserved taninii formed a clade, in which three subclades, i.e., motif sequence 50-GGCCACCCTAGTC-30 (Ger- var. chinensis, var. himalaica, and var. potaninii + nandt and Liston 1999; Vining1999). var. australis, were found. Chi-12 and Chi-26 were Gernandt and Liston (1999) suggested that sub- nested in the var. chinensis subclade. However, in the repeats might play a role in directing rRNA 30ITS1 + 5.8S + ITS2 phylogeny (the strict con- processingconsideringits highlevel of sequence sensus tree of 1000 most parsimonious trees) (Fig. conservation and robust behavior in secondary fold- 4B), Chi-26, rather than Aus-7, formed a solidly ingmodels in Larix and Pseudotsuga. If that is true, supported basal clade (87% bootstrap value) together the loss of most of SR1 in Aus-17 and Chi-26 may with Aus-17 and Him-22. The clone Chi-12 came out imply that the two divergent copies have been re- next rather than beingnested in var. chinensis subc- leased from selective constraint and, therefore, may lade. The NJ tree of the complete ITS region was represent pesudogenes. ITS pseudogenes have been almost same as the MP tree (Fig. 3) in topology. Chi- reported in Zea mays (Buckler and Holtsford 1996a, 26, Aus-17, and Him-22 formed a solid clade basal to b), Aconitum (Kita and Ito 2000), and Pinus (Ger- the other clones of L. potaninii. nandt et al. 2001). In particular, two of three diver- gent rDNA clusters exist as pseudogenes and predate Discussion the species divergence of two Quercus species (Muir et al. 2001). These pseudogenes are characterized by an Marked Intragenomic Heterogeneity of ITS and increased rate of substitution or transitional bias, a nrDNA Pseudogenes decreased GC content, a reduction in the spontaneity of secondary structure formation, a decreased num- Attributed to rapid concerted evolution of nrDNA, ber of methylation sites, and a basal or unique posi- the ITS paralogues in angiosperms do not differ sig- tion in the phylogenetic trees with long terminal nificantly in length except an intra- and intergenomic branch lengths. The three clones Aus-17, Chi-26, and length variation of 100 bp in ITS1 documented from Him-22 found in the present study show a clear ten- the tetraploid Lisianthius skinneri species complex dency toward pseudogenes in these characters, espe- (Gentianaceae) (Sytsma and Schaal 1990). Although cially for Him-22, a unique clone with a markedly low interspecific ITS length variation of several hundred GC content, the greatest sequence divergence, and an base pairs in size has been observed in some taxa of extraordinarily long branch length (Table 3, Fig. 3). gymnosperms, especially in Pinaceae (Maggini et al. 1998; Xianget al. 2000; Gernandt et al. 2001), ex- ITS Recombinants and Their Implications for tensive intragenomic length variation of ITS (>100 Concerted Evolution bp) was reported only in Cedrus deodara (Liston et al. 1996) and 1 of 97 individuals of Pinus sylvestris nrDNA recombination could generate chimeric (Karvonen and Savolainen 1993) through RFLP or molecules, which will most likely be homogenized to PCR-RFLP analysis. In the present study, we found a single rDNA molecule, with a sequence intermedi- that two divergent clones, Chi-26 and Aus-17, were ate between those of the two ancestral sequences more than 100 bp shorter than the other clones from (Muir et al. 2001). The three clones Aus-7, Chi-12, the same individual due to a major deletion in the and Chi-26 show great nucleotide substitution bias ITS1 region. The deletion resulted in the loss of most toward 50ITS1 or 30ITS1+5.8S+ITS2 (Fig. 2), and of subrepeat 1 (SR1), includingthe conserved motif; they have discordant positions in phylogenies of the only a very small fragment of SR1 (16 bp) at the 30 two ITS partitions separated between SR1 and SR2 628 Fig. 2. Variable nucleotide sites in the aligned ITS sequences of the four varieties of Larix potaninii. Dots indicate identity to the first taxon sequence and indels are indicated as dashes. Shaded sequences denote the unique clones. 629 630

Table 3. Sequence divergence (lower-left matrices) of ITS1, 5.8S, and ITS2 among L. potaninii varieties and some divergent clones; upper- right matrices are standard errors

ITS1 5.8S region ITS2

Taxon Pot Aus Chi Him Pot Aus Chi Him Pot Aus Chi Him

Pot — 0.002 0.002 0.002 — 0.002 0.005 0.002 — 0.001 0.005 0.001 Aus 0.007 — 0.002 0.002 0.003 — 0.005 0.000 0.003 — 0.005 0.001 Chi 0.012 0.009 — 0.002 0.012 0.009 — 0.005 0.008 0.008 — 0.005 Him 0.013 0.011 0.010 — 0.003 0.000 0.009 — 0.002 0.001 0.007 — Aus-7 0.024 0.021 0.024 0.024 0.003 0.000 0.009 0.000 0.002 0.001 0.007 0.000 Chi-12 0.022 0.019 0.015 0.018 0.009 0.006 0.006 0.006 0.011 0.010 0.007 0.009 Chi-26 0.033 0.032 0.028 0.030 0.047 0.045 0.055 0.045 0.015 0.014 0.011 0.013 Aus-17 0.066 0.065 0.066 0.064 0.003 0.000 0.009 0.000 0.006 0.005 0.011 0.004 Him-22 0.083 0.083 0.081 0.080 0.097 0.097 0.105 0.093 0.075 0.074 0.080 0.073

Fig. 3. One of the 340 most parsimonious trees constructed from sequence analysis of the ITS region with L. laricina as the outgroup (length = 379, CI = 0.8522, RI = 0.7838). The numbers above and below the branches denote branch lengths and bootstrap percentages greater than 50%, respectively. The numbers followingtaxa indicate different clones.

(Figs. 4A and B), which gives strong evidence for PCR reaction, such as Aus-7 and Aus-17, had differ- their origin from recombination between divergent ent recombination break points and carried specific nrDNA paralogues. Another clone, Aus-17, shares a substitutions. Therefore, they were considered to be major deletion (>100 bp) with Chi-26 besides many true recombinants rather than PCR recombinants synapomorphies between the two clones around the (Bradley and Hillis 1997). ITS recombinants have deletion (Figs. 1 and 2), and therefore, it could also been reported from fungi (Hughes and Petersen 2001) be a recombinant sequence. Visual inspection indi- and several angiosperm groups (Buckler et al. 1997; cated that the unique clones obtained from the same Muir et al. 2001) besides Pinus (Gernandt et al. 2001). 631

Fig. 4. Comparison of the strict con- sensus trees obtained from analyses of partitioned ITS data sets. The clones with gray shading indicate putative re- combinants. The numbers above the branches represent bootstrap values. A 50ITS1 partition (tree length = 181, CI = 0.8895, RI = 0.8519). B 30ITS1 + 5.8S + ITS2 partition (tree length = 182, CI = 0.8901,RI = 0.8387).

Interestingly, an 11-bp indel (Fig. 1) was shared by the highly conserved core sequence and that unequal Chi-26, Aus-17, Him-5, and Him-22 from L. potaninii crossing-over resulted in the number variation of and all clones of the outgroup L. laricina, a short- tandem subrepeats in Pinaceae. This hypothesis was bract species endemic to North America, and thus, strongly supported by our finding that most of SR1, the indel is very likely characteristic of some ancestral includingthe core, was deleted in the two clones Aus- ITS copies of Larix. Besides the synplesiomorphy of 17 and Chi-26. It is interestingthat our data present the 11-bp indel, Aus-17 and Chi-26 diverged first in an apparent paradox: concerted evolution plays an the ITS region phylogeny (Fig. 3), implying that the important role in the homogenization of diverse two clones could be evolutionary relicts resulted from nrDNA repeats, whereas it results in a high level of recombination amongancient ITS paraloguesat repeat heterogeneity at the same time. The paradox nonhomologous loci. In contrast, Aus-7 and Chi-12 could be explained by slow concerted evolution in the are likely to be products of recent recombination pine family, which may be attributed to the charac- events in that they have much shorter branch lengths teristic genome size, chromosomal organization, and than Aus-17 and Chi-26 (Figs. 4A and B). structure of nrDNA repeats and the relatively Two mechanisms of recombination commonly in- longgeneration times (Karvonen et al. 1993; Ger- voked as beingresponsible for concerted evolution nandt et al. 2001). are unequal crossing-over (Smith 1976) and gene The number of major 18S–25S rDNA loci in dip- conversion (Nagylaki and Petes 1982; Nagylaki 1984; loid gymnosperm genomes can vary between 3 and 10 Hillis et al. 1991). Gernandt et al. (2001) suggested (Doudrick et al. 1995; Lubaretz et al. 1996; Brown that the most likely site of recombination, particu- and Carlson 1997; Tagashira and Kondo 2001; Liu larly between nonhomologous subrepeats, might be et al. 2003), whereas nonpolyploid angiosperms gen- 632

Fig. 4. Continued.

erally have only 1 or 2 NOR loci (Longand Dawid some rearrangements. The recombinant clones found 1980; Rogers and Bendich 1987), rarely 3 or 4 (Zhang in the present study, especially the three putative and Sang1999). The largenumber of rDNA loci and pseudogenes Aus-17, Chi-26, and Him-22, are likely more subrepeats could slow the rate of repeat ho- from the minor loci given their chimeric structure and mogenization. Moreover, the family Pinaceae is re- high sequence divergence. Furthermore, these clones markable for its large nuclear genome (Murray 1998) have atypical restriction digestion profiles compared and frequent interspecific hybridization (Carlson and to the other 35 screened clones of respective taxa, a Theroux 1993; Gernandt et al. 2001), whereas it has a greater AT content and high rate of deamination- highly conserved karyotype (2n = 24, except Pseu- type substitutions (Fig. 2, Table 2), indicating that dolarix), with longmetacentric chromosomes of they, particularly Him-22, may exist in only one similar size (Brown and Carlson 1997). In order to rDNA locus or represent dispersed solitary genetic maintain chromosome stability, divergence and spe- elements, that is, orphons (Childs et al. 1981). Kar- ciation must be accompanied by more complicated vonen and Savolainen (1993) found that an rDNA chromosome rearrangements in Pinaceae than in variant carryinga 400-bp deletion in ITS1 was pre- angiosperms, which would give rise to some minor sent in only one of eight rDNA loci of Pinus sylves- loci (Buckler et al. 1997). Liu et al. (2003) observed tris. Ribosomal DNA (rDNA) orphons have been some weak signals of 18S–25S rDNA loci in some reported in some angiosperms such as Tripsacum and pines using the FISH method and suggested that the Winteraceae (Buckler et al. 1997). If the orphon genes weak signals may indicate remnants of primary sites or paralogues at minor loci are selectively neutral, of 18S–25S rDNA that once existed at the centrom- they would fasten the divergence and become pseu- eres but later moved out to distal sites by chromo- dogenes and, thus, result in the rDNA heterogeneity. 633

Nevertheless, the concerted evolution rate of the distributed in the Hengduan Mountains and geo- nrDNA family is faster in Larix than in Pinus. All the graphically located between the former two varieties ITS clones, except some unique ones, show a con- (Fu et al. 1999; Farjon 2001). The complicated to- served length and more synapomorphies within each pography of the Himalayan region caused by the taxon of Larix (Table 2, Fig. 3). This may be at- uplift of the Tibetan Plateau provided ecological tributed to the less complex nrDNA organization, conditions for a nrDNA founder effect in the differ- i.e., three NOR loci (Lubaretz et al. 1996), and the entiation process of L. potaninii varieties. Although short history of Larix (LePage and Basinger 1995; maximum dispersal distances of the Pinaceae pollen Wanget al. 2000). In contrast, Pinus has an early can be 300–1300 km in strongair currents (Potter and Cretaceous origin and as many as six to eight NOR Rowley 1960), the nonsaccate pollen (Owens et al. loci, which could result in the very slow concerted 1998), wet mountainous habitat, and small popula- evolution and extensive intragenomic heterogeneity tion size of L. potaninii make long-distance pollen of nrDNA (Karvonen et al. 1993; Listen et al. 1996; dispersal impossible, which may further result in the Marrocco et al. 1996; Gernandt et al. 2001). For ex- fixation and extensive geographical isolation of ample, the maximum number of ITS types detected in founder nrDNA copies due to the absence of gene a single individual was 11, based on 13 clones from P. flow amongthe varieties. A founder effect of multiple culminicola (Gernandt et al. 2001). gene families has been reported in 5S rDNA of Triticum (Allaby and Brown 2001) and Ophiopogon Founder Effect and Geographical Differentiation of (Niu and Zhang2002). As for var. australis and var. nrDNA in Larix potaninii potaninii, they are sympatric in SW Sichuan and NW Yunnan, and show only minor differences in the size As discussed above, extraordinary intra- or interge- of the female cone. Therefore, it is not surprisingthat nomic nrDNA heterogeneity exists in gymnosperms, ITS clones of the two varieties mixed together in the especially Pinaceae, and has not been completely phylogeny. homogenized by concerted evolution. The nrDNA polymorphisms can disperse amongdifferent popu- lations or species through gene flow followed by Acknowledgments. We thank Drs. Zhou Shi-Liangand DingKai- frequent recombination amongnrDNA loci. That is, Yu for help in field collection and Miss Sun Ying-Xue for help in rDNA paralogues could exist among different species sequencing. This work was supported by grants from the State Key Basic Research and Development Plan (Grant G2000046804) and without reproductive isolation. For instance, different Chinese Academy of Sciences (Grant kscxz-sw-101A and the spe- ITS clones from the same (or different) individuals of cial fund to WangXiao-Quan). a species were polyphyletic in Pinus subsection Cembroides, a group characterized by frequent interspecific hybridization and very slow concerted References evolution (Gernandt et al. 2001). The similar phe- nomenon was also reported in two closely related Allaby RG, Brown TA (2001) Network analysis provides insights Quercus species that frequently hybridize (Muir et al. into evolution of 5S rDNA arrays in Triticum and Aegilops. Genetics 157:1331–1341 2001). 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