Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid

Lan and Albert
Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 (12 September 2011)

so rearrangement processes must include either insertion or deletion of DNA segments. These plants have long been of interest regarding their chromosomal evolution. Basic molecular phylogenetic * Correspondence: vaalbert@buffalo. The dispersed signals mainly occur at centromeric and subtelomeric positions. NY 14260. Karyological studies of Paphiopedilum have revealed considerable chromosomal variation. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements. provided the original work is properly cited. duplications. whereas the type subgenus Paphiopedilum includes both clades of 2n = 26 species and two distinct lineages of species that bear greater than 26 chromosomes. and reproduction in any medium. the latter representing hemizygosity. has 2n = 26 metacentric chromosomes. and show from 2 specific signals to many. USA information on the genus is available [3]. is among the most widely grown and hybridized of all orchids. in aneuploid increments suggestive of centric fission [2]. Chromosome number is positively correlated with genome size. including the possibility that concerted evolutionary forces may homogenize diversity. Buffalo. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history. so rearrangement processes must include either insertion or deletion of DNA segments. Results: 5S and 25S rDNA loci among Paphiopedilum species.com/1471-2229/11/126 RESEARCH ARTICLE Open Access Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum. Chromosome number has been shown to be positively correlated with genome size [4]. representing all key phylogenetic lineages. duplications. both major and minor and highly dispersed. licensee BioMed Central Ltd. exhibit a considerable diversity that correlates well with recognized evolutionary groups. . and phylogenetically-correlated variation within Paphiopedilum. Haploid genome size is extremely large in these orchids. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events. These results make the genus a model system for the study of complex chromosomal evolution in plants. and inversions . which is sister to the rest of the genus. Species of Paphiopedilum are also ecologically important narrow endemics in various mainland and island habitats.edu Department of Biological Sciences. which range from montane rainforest to seaside cliffs [1].0). Background Paphiopedilum. Conclusions: Paphiopedilum species display many chromosomal rearrangements . distribution.org/licenses/by/2. ranging from 16. a genus of approximately 80 species indigenous to tropical and subtropical Southeast Asia. as well as interlocus and intralocus diversity. translocations.1 megabases (Mb) [4]. with the number of telocentrics equal to twice the number of metacentrics that ostensibly split [3].Lan and Albert BMC Plant Biology 2011.biomedcentral. 11:126 http://www.but only weak concerted evolutionary forces among highly duplicated 5S arrays. 80 species of lady’s slipper orchids native to Southeast Asia. a lady’s slipper orchid Tianying Lan and Victor A Albert* Abstract Background: Paphiopedilum is a horticulturally and ecologically important genus of ca. Subgenus Parvisepalum. which are hotspots for chromosomal breakpoints.for example. University at Buffalo.1 to 35. General issues in plant chromosomal evolution include the contribution of rearrangements to genome © 2011 Lan and Albert. which suggests that double-strand break repair processes are dynamic and ongoing. which ranges from 2n = 26 to 2n = 42. which permits unrestricted use. which involves a progressive aneuploid series based on either fission or fusion of centromeres. 25S rDNA signals range from 2 (representing 1 locus) to 9. 5S loci display extensive structural variation.

and especially in hybrids [15. Table 1). or genome duplication [7]. 11:126 http://www. we concern ourselves with demonstrable evidence for dynamic rearrangements during the evolution of Paphiopedilum. We use such analyses here to document chromosomal dynamics in Paphiopedilum. and there is evidence that the NTS both within and among arrays can show polymorphism. according to phylogeny and section-level classification Section Parvisepalum Species of section Concoloria show two 25S and 5S signals (Table 1). and the centric fission hypothesis. which have more loci. FISH has been applied previously to Paphiopedilum. each on separate chromosomes (2n = 26 total). However. 5S rDNA patterns are stable. Rearrangement processes involve double-strand break repair. Section-level phylogenetic tree based on rDNA ITS sequences published by Cox [3]. Tandem repeats duplication or segmental duplication is one of the possible outcomes of unequal crossing over [7.com/1471-2229/11/126 Page 2 of 15 structure and size. 5S rDNA non-transcribed spacer (5SNTS) sequences have seen some use as phylogenetic markers [17-21].16]. in which mobility and patterning have been systematically investigated as species-specific karyotype markers. Two to four 25S rDNA signals are apparent (Figure 2) among 2n = 26 chromosomes. which occurs frequently at hotspots in pericentromeric and telomeric regions [5. Parvisepalum Concoloria Cochlopetalum Paphiopedilum Coryopedilum Coryopedilum Pardalopetalum Barbata Figure 1 Section-level phylogenetic tree of genus Paphiopedilum. rearrangement by duplication is observed in Paphiopedilum armeniacum.biomedcentral. These phenomena may be investigated empirically through use of Fluorescence In Situ Hybridization (FISH) on highly repetitive DNA loci subject to concerted evolution. with two signals most parsimoniously interpretable as the basal condition since this state is shared by the outgroup genera Mexipedium and Phragmipedium (unpublished data. delenatii. We have cloned 5S-NTS segments in Paphiopedilum in order to study past and ongoing gene duplication events and the possibility of gene conversion both within arrays and among duplicated loci.8]. Section Cochlopetalum Section Parvisepalum is the sister group of all other Paphiopedilum species (Figure 1). are common in the literature [10-14]. Both 45S and 5S rDNAs in plants are characterized by intergenic spacers.8S-25S (45S) and 5S ribosomal DNA (rDNA) arrays. We do not aim to provide complete karyotypic comparisons. most studies of 5S-NTS to-date have employed direct sequencing of PCR products. All four species studied here have two telomeric 25S rDNA signals. retrotransposition. showing 2 subtelomeric signals that are usually closely linked with one pair of 25S signals (Table 1). the telocentrics of which have been suggested to descend via centric fission from 25 diploid metacentrics [2]. According to phylogenetic relationships known at present (Figure 1). With 2 signals being Section Cochlopetalum displays an aneuploid number of chromosomes.Lan and Albert BMC Plant Biology 2011.6]. 5S-NTS sequence data are also compared with a phylogenetic hypothesis in order to ascertain duplication history of paralogs. nor a full cytotaxonomic treatment. rather. and 4 major 5S rDNA signals (Figure 4. Gene duplications may be caused by unequal crossing over. Infrageneric comparative rDNA FISH analyses. such as the 18S-5. P. the inferred primitive condition. [23]). This phenomenon is also seen in P. except in that the 5S signals are interstitially instead of subtelomerically placed (Figure 3). with its two chromosomes that show hemizygous 25S and 5S rDNA signals. emersonii and P. malipoense. respectively. We briefly report distribution patterns of rDNA signals from a phylogenetic systematic perspective [22] according to accepted section-level classification. sections Cochlopetalum and Barbata (with telocentrics descended from 26 diploid metacentrics) have evolved aneuploid increase independently. . In P. translocation of either the 5S or 25S rDNA locus has occurred. but in a limited manner only. similarly to Paphiopedilum delenatii of section Parvisepalum. hangianum. which may show duplication or evidence for rearrangement-producing heterologous recombination [9]. Section Concoloria Results Distribution patterns of ribosomal DNA by Fluorescence In Situ Hybridization.

com/1471-2229/11/126 Page 3 of 15 Figure 2 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Parvisepalum. and these. Chromosomes were counterstained with DAPI. (E) P.Lan and Albert BMC Plant Biology 2011. The 2 species with 2n = 32 chromosomes. (D) P. (C) P. both have two 5S bands localized on the same chromosomes as the 25S signals. armeniacum. All 4 species have multiple dispersed 5S signals. (A) Paphiopedilum emersonii. (F) P. 25S rDNA (green) and 5S rDNA (red) probes were simultaneously detected in all Paphiopedilum species. 11:126 http://www.biomedcentral. All scale bars = 10 μm. Paphiopedilum liemianum (Figure 4C) and P. malipoense. like the major loci. (B) P. micranthum. whereas only a single 5S band is seen on the . pericentromeric and centromeric in position. are mostly subtelomeric. delenatii. primulinum (Figure 4A). rather unlike species of sections Parviflora and Concoloria. hangianum.

p. p. i. including numbers of both major and visible dispersed sites. Cochlopetalum liemianum moquettianum primulinum victoria-regina Sect. centromeric . 11:126 http://www. c st. c st. i i i t t t t t t t t t t t t. i. c st. i st. p. i. p. rDNA FISH patterns. st t t t. c st. Parvisepalum Sect.Lan and Albert BMC Plant Biology 2011. p. p. c st. telomeric. pericentromeric. c st. p. b st. Pardalopetalum dianthum haynaldianum lowii parishii Sect. p. c st. i. c st. p. c st. i. i. c st. i. i st. c. subtelomeric. i. i. i. c st. i. Paphiopedilum Sect. i. p. Paphiopedilum druryi fairrieanum henryanum hirsutissimum tigrinum Sect. p. p. i. i.biomedcentral.com/1471-2229/11/126 Page 4 of 15 Table 1 Paphiopedilum species studied. Barbata acmodontum curtisii dayanum hennisianum purpuratum sangii sukhakulii venustum wardii a Positions of rDNA sitesb 25S+5S 5S 25S 5S-NTS Polymorphic sites 2n 25S major visible sitesa Co-localization 26 26 26 26 26 26 4 2 4 4 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 1 2 st st st st st st t t t t t t 104 178 124 120 94 59 26 26 32 34 32 34 30 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 38 36 36 34 40 38 40 40 42 2 2 2 2 2 2 2 2 2 2 2 9 6 4 4 2 2 9 2 4 6 4 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 4 2 2 6 6 4 6 4 4 4 4 4 4 4 4 4 4 2 4 2 4 4 2 4 4 2 2 22 20 25 24 16 14 17 21 17 28 32 26 30 16 25 26 28 8 28 34 4 2 6 6 8 18 13 8 4 0 0 2 2 2 2 0 2 2 2 2 6 6 4 4 0 2 7 2 2 4 4 0 0 0 0 0 0 0 2 0 i i st. i. c st. c st. i. p. t. and 5S-NTS sequence polymorphic sites Number of rDNA sites 5S Taxon Paphiopedilum Subg. st t t t t t t. diploid chromosome numbers. c i i i. i. interstitial. Coryopedilum adductum gigantifolium glanduliferum randsii sanderianum stonei supardii Sect. p. c st. i. c st. p. p. p. c st. st t t t t t t t t t t 118 198 162 225 71 137 184 146 180 182 141 180 210 202 187 143 114 226 251 110 161 189 169 164 169 153 138 118 151 109 159 Minimum numbers of visible 5S rDNA FISH signals. c st. p. i. p i st. p. i. Parvisepalum armeniacum delenatii emersonii hangianum malipoense micranthum Subg. i. p. p. Concoloria bellatulum niveum Sect. c st. c st.

(D) P. 11:126 http://www. Figure 4 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Cochlopetalum.Lan and Albert BMC Plant Biology 2011. (C) P.com/1471-2229/11/126 Page 5 of 15 Figure 3 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Concoloria. .biomedcentral. (A) Paphiopedilum primulinum. (B) P. liemianum. niveum. (B) P. moquettianum. victoria-regina. (A) Paphiopedilum bellatulum.

Species of the Coryopedilum/Pardalopetalum group show at least 4 major 5S rDNA signals (up to 8 in P. 11:126 http://www. All species. Table 1).com/1471-2229/11/126 Page 6 of 15 same chromosome in the 2n = 34 species P. victoria-regina (Figure 4D). Section Paphiopedilum All 5 species of section Paphiopedilum studied show two 25S signals in the telomeric region (Figure 5. (E) P. all species show at least 2 strong (up to 5) 5S bands located on one chromosome. druryi. all species having 2n = 26. and appearing in different placements along chromosome arms. 25S signals vary from 2 to 9. section Pardalopetalum is derived within section Coryopedilum (Figure 1). and numerous dispersed signals in the pericentromeric and centromeric regions. (D) P. as such. with 2 telomeric signals (Figure 8.biomedcentral. lowii. other than in P. P. randsii (Figure 6F) are telomeric. (A) Paphiopedilum fairrieanum. Sections Coryopedilum and Pardalopetalum In current phylogenetic results. In P. and both this species and P. which have 2n = 28-42 and the largest genome sizes. (B) P. adductum (Figure 6E) and P. In P. 4 of the major signals appear to be located on different arms and on morphologically different chromosomes that may only be partly homologous (this condition was observed in at least 4 cells). In all but P. randsii. parishii (Figure 7B)) and multiple dispersed repeats in pericentromeric and centromeric regions. 1-4 subtelomeric 25S signals were observed in P. Table 1). 7. druryi the major signals are closely linked with the 25S arrays. In the Pardalopetalum group. supardii (Figure 6G). which are 2n = 26 except for P. (C) P. show at least 2 specific 5S rDNA bands. druryi. P. Major 5S signals number 2-4. the latter showing hemizygosity. henryanum. moquettianum (Figure 4B) and P. as many as 6. Section Barbata Species of section Barbata. druryi (Figure 5E) at 2n = 30. show constancy in 25S rDNA distribution. either with major or minor 5S bands. hirsutissimum. tigrinum. they will be discussed together here. . sanderianum (Figure 6A). Together. is the most dynamic in Paphiopedilum regarding chromosomal rearrangements (Figure 6. P.Lan and Albert BMC Plant Biology 2011. one hemizygous chromosome has telomeric 25S signals on each arm. Table 1). and extremely few dispersed Figure 5 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Paphiopedilum. Signals in all species except Paphiopedilum lowii (Figure 7A). the Coryopedilum/Pardalopetalum clade. supardii show the maximum number of signals. Close linkage with 25S occurs throughout the group. adductum also shows 25S hemizygosity. adductum and P.

Only Paphiopedilum curtisii (Figure 8G) and P. P.Lan and Albert BMC Plant Biology 2011. randsii. (E) P. . 11:126 http://www. (G) P. and interstitial placements are observed.biomedcentral. (A) Paphiopedilum sanderianum. since Cochlopetalum. pericentromeric. Paphiopedilum. repeats were observed. glanduliferum. (D) P. sukhakulii (Figure 8C). gigantifolium. and the first species shows no dispersed repeats. Only in P. (B) P. venustum (Figure 8F) and P.com/1471-2229/11/126 Page 7 of 15 Figure 6 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Coryopedilum. Because Barbata is the most derived section in the genus (Figure 1). venustum is close linkage of 25S and 5S observed. stonei. adductum. and then only involving a minor 5S band. Coryopedilum. supardii. whereas telomeric. (C) P. Arrows indicate subtelomeric 25S rDNA signals. (F) P. subtelomeric. wardii (Figure 8A) show linked 5S signals. hennisianum(Figure 8B) have two major 5S signals. either its species have lost 25S and 5S rDNA loci. Most 5S loci are not centromeric. P.

random clones. (D) P. 7 (Paphiopedilum niveum) or 8 (all others) per species.com/1471-2229/11/126 Page 8 of 15 Figure 7 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Pardalopetalum. 4 from P. micranthum 1) to 455 bp (P. acmodontum. Arrows indicate subtelomeric 25S rDNA signals. 4 from P. Diversity of 5S ribosomal DNA non-transcribed spacer sequences We investigated duplication history correlated with the dynamic rearrangements observed in 5S rDNA loci. 2 from P. haynaldianum.Lan and Albert BMC Plant Biology 2011. were sequenced (Additional file 1). 2 from P. stonei. supardii). In order to survey sequence variation in 5S-NTS.biomedcentral. and Pardalopetalum usually have more. and . or the species of the latter sections have increased the number of rDNA loci independently given the low number in sections Parvisepalum and Concoloria. parishii. (B) P. hirsutissimum. (A) Paphiopedilum lowii. 11:126 http://www. Sequences of 5S-NTS ranged from 283 bp (P. (C) P. an objective alignment was accomplished using MAFFT and default settings. bellatulum 5). 2 from P. stonei and P. henryanum. and one sequence each of P. dianthum. Numbers of polymorphic loci within species. malipoense. Given extensive sequence divergence of 5S-NTS and our desire not to manually adjust alignment [24]. Only a few clones were identical to each other (2 sequences from P. dayanum.

(I) P. acmodontum. (B) P. venustum. (G) P. sangii. Figure 9). respectively. (C) P.01. phylogenetic relationships. (D) P.biomedcentral.Lan and Albert BMC Plant Biology 2011. (A) Paphiopedilum wardii. suggesting that interlocus gene conversion is relatively weak. (E) P. A phylogenetic tree outgroup-rooted using Phragmipedium besseae showed 2 major groups of sequences: section Parvisepalum .com/1471-2229/11/126 Page 9 of 15 Figure 8 FISH distribution pattern of 25S and 5S rDNA on metaphase chromosomes of Paphiopedilum section Barbata. Numbers of polymorphic sites within species positively correlated with minimum numbers of visible 5S signals (P < 0. hennisianum. sukhakulii. (F) P. were assessed in order to estimate the strength of gene conversion and the extent of paralogy. (H) P. curtisii.21. R^2 = 0. dayanum. purpuratum. 11:126 http://www.

Paphiopedilum. was found prevalent in the remaining five sections. F and 7A. Coryopedilum (2n = 26) and Pardalopetalum (2n = 26) (Table 1). respectively). In most Paphiopedilum species we analyzed. usually less than one-third of chromosomes display either 45S rDNA or 5S rDNA [13]. respectively [33]. Iris [13]. is 2 (unpublished results from Mexipedium and Phragmipedium). It is therefore noteworthy that in some lineages of Paphiopedilum. Discussion Variation in numbers and chromosomal locations of rDNA Variation in numbers and distribution patterns of rDNA loci among related species is commonly observed in many different plant genera. In general. There is no evidence at all. Paphiopedilum adductum.26.biomedcentral. Coryopedilum plus Pardalopetalum. up to 24 of the 26 chromosomes bear at least one rDNA locus. Leguminosae [27]. Overall. Due to the derived phylogenetic position of section Barbata (Figure 1). however. which has the greatest average genome sizes and chromosome numbers [4].28-32]. These two species differ more than two-fold in genome size. the phylogenetic tree was poorly representative of phylogenetic relationships due to extensive duplication of 5S loci. Cyperaceae [11. FISH patterns of 25S rDNA loci are reported to be more polymorphic than those of the 5S rDNA [12-14. 5S rDNA sites showed much more variability both in number and physical location than did 25S rDNA sites. Pinus [28]. The number of 25S rDNA loci in Barbata remains two through all the species we studied. and Rosaceae [14]. Variation was only observed in three species. randsii and P. Apparently. for polyploidy in Paphiopedilum.g. and a single chromosome can bear up to five major 5S rDNA loci. species with the smallest (P. Some large species-specific clades were observed. in all sections of Paphiopedilum. The numbers and distribution of rDNA loci vary widely among plants. Additional file 2). Coryopedilum and Pardalopetalum could partly contribute to the increase of genome size.26]. Asteraceae [25. lowii. P < 0. A similar situation has also been described in many other diploid species. sizes and physical positions of signals. but that since rDNA duplication is associated with both smaller and larger genomes in the genus. versus the remainder of the genus. Cyperaceae [11]. and Rosaceae [14]. Line indicates trend derived from linear regression analysis based on 5SNTS within-species polymorphic sites and minimum numbers of visible 5S signals (data from Table 1. again by outgroup comparison. it is most parsimonious to conclude that unique chromosomal .1 to 35.21. as is the majority-rule consensus tree based on 100 bootstrap replicates (Additional file 3). exul. such that when multiple loci do appear. this would suggest that genome size increase is primarily due to repetitive element amplification.). such as mobile elements. and is only observed in sections Parvisepalum and Concoloria.22]). 16. The massive duplication of rDNA loci in Paphiopedilum sections Cochlopetalum.1 Mb. Plants typically show some degree of conservatism of rDNA repeat duplication. however. which showed 1-4 subtelomeric 25S rDNA signals (Figures 6E. in a series reflective of centric fission or fusion. 25S rDNA signals are located in terminal chromosome positions. The ancestral number of 5S rDNA sites.12]. P. including Brassicaceae [10]. while the distribution pattern of 5S rDNA is less dispersed than its sister group. Duplication of 25S rDNA sites was observed only in three of the seven sections of Paphiopedilum: Parvisepalum (2n = 26). The physical positions of 25S rDNA loci are relatively conservative. section Paphiopedilum) and largest (P. as well as some sectionspecific clades. size differences may be more logically traceable to other repetitive DNAs. e.com/1471-2229/11/126 Page 10 of 15 Figure 9 The relationship between polymorphism of 5S-NTS sequences and numbers of observed 5S rDNA signals. However. 11:126 http://www. based on outgroup comparison to the genera Mexipedium and Phragmipedium (unpublished results. leading to large-scale polymorphism of numbers. R^2 = 0. where the only chromosome number differences are aneuploid.Lan and Albert BMC Plant Biology 2011. If we assume that the number of distinct genes among Paphiopedilum species is roughly constant. the diploid lineage of Brassicaceae [10].01. however. Conversely. [3. dianthum. there is no strong correlation between the increase in the number of rDNA sites and the increase in the number of chromosomes or genome size. The most parsimonious ancestral number of 25S rDNA sites in Paphiopedilum is two. The single tree of maximum likelihood is shown (as a phylogram. Perhaps paradoxically. section Pardalopetalum) haploid genome sizes are both members of groups that show considerable 25S and 5S locus duplication in our FISH experiments. Massive duplication and amplification of 5S rDNA loci. except for Parvisepalum and Concoloria. species are commonly polyploid relatives of diploids. a possible tendency for elimination of rDNA loci was found in section Barbata.

duplication. The lack of dispersed repeats in the basalmost section Parvisepalum may reflect either a lack of seeding events or slow amplification processes that do not yield hybridization-visible arrays. In some cases. In many cases. additional traceable chromosome markers are needed to provide further evidence that chromosomal rearrangements are related to rDNA loss. in the case of 5S rDNA.. since many species (e. 11:126 http://www. and chromosome rearrangements (translocation. there is in fact strong evidence for NTS sequence diversity. it can be readily seen that duplication of 5S loci has occurred prior to speciation.14]. Diversification of 25S rDNA distribution patterns is also observed in Paphiopedilum. and Iris [13. dayanum. while in other systems. A double-strand break occurring in a hemizygous region would increase the probability of causing other rearrangements. a contribution of transpositions to the dispersed distribution pattern is tenable. as single-species clades of Parvisepalum (e. A combination of different mechanisms causes high mobility of rDNA Different mechanisms have been postulated to account for the mobility and polymorphism of numbers. 5S-NTS sequences highlight interlocus and intralocus diversity and weak concerted evolutionary forces Previous studies of other angiosperm species have suggested that intralocus 5S rDNA diversity occurs. P. owing to the absence of a homologous template for its repair [5]. The mechanism that accounts for such loss of rDNA loci. and in turn. therefore leading to the false interpretation that we are visualizing entire and active rDNA arrays. malipoense) and Concoloria (P. lowii. adductum and P. centric fission has led to rDNA gains [34]. Recent within-species duplication events may be indicated by single-species clades of 5S-NTS sequences. Elimination of rDNA loci during chromosomal evolution has been documented in..biomedcentral. The original seeding of rDNA repeats to ectopic locations in the genome could be the result of transposable element activity or perhaps incorporation of array segments into breakpoints as part of non-homologous end joining during DNA repair. centric fission in Barbata appears to be associated with loss of rDNA loci. Considering the abundant minor loci we observed in these regions. One future experimental approach to determine whether considerable intra-array diversity indeed exists would be to perform FISH using 5S-NTS-specific probes. remains unclear. Indeed. in situ amplification cycles via rearrangement could lead to the origin of FISH-detectable loci. such as P.6]. to our knowledge.34]. bellatulum) most likely do. Coryopedilum and Pardalopetalum has. which could either be accounted for by the presence or small loci below the FISH detection limit or perhaps by considerable within-array diversity. but the numbers of loci and degree of dispersion is much lower than for 5S rDNA. stonei. only been observed in the monocots Alstroemeria. Therefore. delenatii shares sequence variants similar to other Parvisepalum species yet has at least one other variant . These processes could act alone or in combination. deletion) caused by homologous or non-homologous unequal crossing-over and gene conversion [9.g. for example. The different evolutionary tendencies between 25S and 5S rDNA might be caused by their function and sequence divergence or localization in distinct nuclear compartments [43]. For example. P. Both subtelomeric and pericentromeric regions are well known as hot spots of breakpoints and are also enriched for TEs [5.30. sangii. In the case of section Barbata. since the amplified pieces include sections of 5S rDNA at their 5’ and 3’ ends. however. ancient duplications must be much older than the major phylogenetic groups of Paphiopedilum. Brassicaceae and Rosaceae [10. but these could just as well indicate within-array variation. since.40]. such as transposon-mediated transpositional events [36-38]. Tulipa. inversion. The great degree of 5S repeat dispersion seen in sections Cochlopetalum. After generation of a novel locus.. 6 species of section Parvisepalum are represented in our phylogenetic analysis by 6-8 distinct sequence variants. within Coryopedilum (a large group of sequences representing P. chromosomal rearrangement could be induced via heterologous recombination. and TEs containing 5S rDNA-derived sequences have in fact been observed in many plants [41] and animals [42]. and they do not necessarily imply changes in overall chromosome morphology [31. Withinarray 5S rDNA diversity appears very likely in Paphiopedilum as well. amplification or maintenance of 5S rDNA loci via rearrangement could be more effective and tolerated during the chromosome evolution process. sanderianum.g. all Parvisepalum and Concoloria) have only one observable 5S locus. As such.com/1471-2229/11/126 Page 11 of 15 conditions seen in the group would be similarly derived (autapomorphic). e. pseudogenized or not. It is nonetheless possible that due to the similarity of rDNA arrays. randsii sequences).39. Furthermore. rearrangement could generate repeated sequences through unequal crossovers. some of the signals we observed may be pseudogenes transported within the genomes by retroelements. sizes and positions of rDNA sites. for example.g. similarly also within a group of P. Paphiopedilum.28. A presumed evolutionary loss of abundant terminal nucleolar organizing regions (NOR) in Arabidopsis has been hypothesized to be the consequence of an ancient fusion event [35]. and P. These 5S-NTS variants can be concluded to occur within at least partial arrays. P.36]. P. hemizygous 5S rDNA sites have been widely observed in many Paphiopedilum species. or. P. supardii. 5S rDNA might be more frequently seeded by TEs via transpositional events.Lan and Albert BMC Plant Biology 2011. However.

within well-defined groups such as Parvisepalum could be ancient hybridization. dayanum. respectively).06 and 0. therefore we infer that interlocus concerted evolution is weak within the genus. In contrast. centromerically-located rDNA arrays are expected to show weaker homogenization forces. As such. acmodontum.com/1471-2229/11/126 Page 12 of 15 that is more similar to sequences from all other sections. since fewer individuals with unequal crossovers in this region are expected to survive. noticeable interlocus concerted evolution of 5S rDNA arrays has not been demonstrated in plants. to our knowledge. We investigated the possibility of contamination regarding this finding. as with P. both have one 5S locus. Chenopodium [19]. this may result in the exchange of not only a fraction of the rDNA but also the centromeres themselves. thus stronger concerted evolutionary forces could be expected in this region. but discovered similar repeats across 8 distinct P. indicates that proximally located loci seem less homogenized than the subtelomerically located loci. purpuratum and P. niveum. P. The difference in sequence polymorphism between the two species may be caused by the different locations of the 5S loci.1. which then might result in exchange of telomeres [17. . Nicotiana [20] and Pinus [27].5) is 18% more than that in the subtelomeric-loci group (128). but with different localizations. as with 25S. bellatulum and P. we should expect subtelomeric 5S repeats to show decreased sequence diversity due to stronger concerted evolutionary forces. such as Gossypium [17]. this is not the case. loss of centromeres would prohibit cell division.biomedcentral.g. The best supported hypothesis to explain weak homogenization forces on 5S rDNA arrays is that the chromosomal location of rDNA arrays has a substantial impact on interlocus concerted evolution [17. while the other group harboring subtelomeric 5S loci includes P. the base composition and secondary structure of rDNA sequences may also affect the rate of concerted evolution [53]. 11:126 http://www. P. In section Concoloria.20. It is unknown whether weak concerted evolutionary forces are shared by other Paphiopedilum tandem repeats. deletion. We therefore infer that localization of the 5S rDNA arrays only partially contributes to the weak concerted evolution observed in Paphiopedilum. Triticum [18]. venustum and P. which has a subtelomeric locus (Table 1). showed 1. One group harboring proximal 5S loci includes P. Additionally. some of which are closely linked with 25S loci. However. niveum. have subtelomeric 5S loci. Our conclusion concurs with previous findings in many plant genera. So far. For example. This issue can be elucidated by further studies on other tandem repeats. bellatulum. e. Table 1). There are several other hypothesized mechanisms that could lead to the weak concerted evolutionary force on 5S rDNA arrays. ongoing chromosomal rearrangement such as insertion. we found that not only interlocus but also intralocus concerted evolution is also influenced by chromosomal localization. or transposition could occur within arrays too frequently for interlocus concerted evolution to be effective. sangii (Figure 8. If 5S localization correlates significantly with homogenization. since variation in the number of polymorphic sites is not significantly different by section (with or without Pardalopetalum included in Coryopedilum. These six species can be categorized into two groups according to the locations of 5S loci.. bellatulum. which is always telomeric-subtelomerically located. which has a pericentromeric locus. whereas loss of telomeres might not restrict mitosis or meiosis. The fact that the average number of polymorphic sites in the proximal-loci group (151. two closely related species. Another explanation for multispecies clades. in which all of six species studied possess two 5S loci.47]. Considering that all six species are closely related and possess the same number of loci. Such an event is more likely to have significantly greater negative consequences to the organism than if the event occurred in the subtelomeric region. Potential evidence was observed in section Barbata. It is well-known that meiotic homologous recombination has been largely suppressed in pericentromeric and centromeric regions. delenatii accessions (results not shown).Lan and Albert BMC Plant Biology 2011. such as 25S rDNA arrays.68 . the subtelomeric region is characterized by a higher rate of interchromosomal exchange [5]. Unequal crossovers between sister chromatids and gene conversion documented in the centromeres of many organisms have been postulated as the major homogenization force for tandem repeats located in these areas [48-52].fold more polymorphic sites than P. Additionally. A plausible explanation for this has been proposed previously: if unequal crossover events between rDNAs of two chromosomes occurred in the proximal region to centromeres. Arrays located in subtelomeric regions are thought to undergo stronger interlocus homogenization forces than ones located in proximal regions. Another possibility is that concerted evolutionary processes homogenize 5S rDNA arrays at rates lower than the rate of speciation. We observed that increasing within-species 5S NTS sequence diversity correlates with increasing minimum numbers of visible 5S rDNA loci in Paphiopedilum (Figure 9). All species of section Parvisepalum. single factor ANOVA P = 0. P. thus novel mutations cannot be fixed or removed and high levels of intralocus polymorphism are expected within arrays [17]. P.44-47]. or if this is characteristic of 5S rDNA arrays only. it can be logically assumed that the difference in sequence polymorphism between the two groups is caused by the different locations of the 5S loci. wardii.

Recombinant clones were screened by colony direct PCR method and were sequenced 7-8 clones per each species using T7 (5’-TAATACGACTCACTATAGGG-3’) primer.Lan and Albert BMC Plant Biology 2011. bess2. 50°C for 1 min. translocations. The slides were washed in 2 × SSC at room temperature for 5 min and twice in 2 ×SSC at 50°C for Sequences were aligned using the MAFFT (Multiple Alignment using Fast Fourier Transform) web server at the European Bioinformatics Institute [57]. and 72°C for 1 min. which suggests that double-strand break repair processes are dynamic and ongoing. a 2. and fixed in freshly prepared fixative (3:1 ethanol: acetic acid) for 48 h at 10°C. Information on the species and sections is provided in Table 1. RaxML was called using the following commands: raxml -# 100 -n pasted -o bess1. 50 mM sodium phosphate (pH 7. the final step at 72°C was extended to 10 min. Rpi) and 1% pectolyase (Aspergillus japonicus Y-23. and perform FFTS = localpair. the InsituPro VSi (Intavis Bionanalytical Instruments). all by nick translation method using the kit from Roche. cloning and sequencing Thirty-seven species of the Paphiopedilum genus covering all seven sections were analyzed in this study. maxiterate = 0. Slides with metaphase spreads were treated with 70% deionized formamide in 2 × SSC at 70°C for 2 min. The hybridization buffer consisted of 50% deionized formamide. Images were taken by a Zeiss AxioCam MRm black-and-white CCD camera on a Zeiss Imager.0).5 μg/ml DAPI (4’.but only weak concerted evolutionary forces among highly duplicated 5S arrays. and DAPI fluorescence was left in gray-tone. The preparations were mounted and counterstained in Vectashield containing 1. MP) at 37°C for 30 min. Default parameters were used: gap opening penalty = 1.004 M 8-hydroxyquinoline for 4-6 h at 10°C. Actively growing roots were used for chromosome preparation.8 [60]. Methods Plant materials 10 min. 6-diamidino-2-phenylindole) (Vector Laboratories). and inversions . The fixed root tips were then rinsed thoroughly with tap water and macerated in an enzyme mixture containing 2% cellulase (Onozuka R-10. Fluorescence signal was detected using antiDigoxigenin-Rhodamine conjugate (Roche) and streptavidin-fluorescein conjugate (Invitrogen). Default settings were used. The relationship between numbers of polymorphic sites and minimum numbers of visible 5S rDNA signals was investigated using linear regression analysis (in Microsoft Excel). and decreased by 1°C per cycle). Probe labelling and Fluorescence in situ hybridization (FISH) Total genomic DNA was extracted from fresh leaves using Qiagen DNeasy Plant Mini kit. Phylogenetic reconstruction was performed using maximum likelihood optimization available through the RaxML BlackBox web server [59] running RaxML version 7. gap extension penalty = 0. The 8 Phragmipedium besseae sequences were indicated as the outgroup. FISH signals were false-colored. PCR products were ligated into pDrive Cloning vector and transformed into QIAGEN EZ competent cells (Qiagen PCR Cloning kit). duplications.com/1471-2229/11/126 Page 13 of 15 Conclusions Paphiopedilum species display many chromosomal rearrangements . Within-species 5S-NTS polymorphism was estimated. Chromosome preparation Root tips were pre-treated with 0. then 35 cycles of 94°C for 1 min. 25S rDNA was labelled with biotin-16-dUTP (Roche) and 5S rDNA was labelled with digoxigenin-11-dUTP (Roche). Post-hybridization washes and immunodetection were carried out in an automated in situ hybridization instrument.10. the meristematic cells were squashed in a drop of 45% acetic acid under a coverslip (22 × 22 mm) on a microscope slide.biomedcentral.2. Data analysis 25S rDNA. followed by 10 cycles of 94°C for 1 min.for example. Z1 fluorescence microscope and then processed uniformly using Zeiss AxioVision software. tree rebuilding number = 1.3-kb ClaI subclones of the 25S rDNA coding region of Arabidopsis thaliana [54] and 5S rDNA (pTa794) [55]were used as probes. 11:126 http://www. 2 × SSC. using DnaSP version 5. which were incubated at 37°C for 10 h in a humid chamber. . PCR amplification.01 [58]. The 25S and 5S rDNA probes were mixed to a final concentration of about 2 ng/μl and then denatured at 94°C for 10 min before being used. 10% dextran sulfate and sheared salmon sperm DNA (Invitrogen) in 100 × excess of labeled probes. and 72°C for 1 min. Slides were then dipped into liquid nitrogen and air dried after the coverslips were carefully removed by a blade. The sequence alignment is available as a supplementary FASTA file (Additional file 4). Touchdown amplification was performed as follows: an initial step at 94°C for 5 min. After re-fixation in fixative for 15 min.53. annealing for 1 min (start at 60°C. Denatured probes in hybridization buffer were then applied to the slides. based on the aforementioned multiple alignment.123. After gel purification using QIAquick Gel Extraction Kit (Qiagen). The 5S-NTS region was amplified by PCR using the universal degenerate primers: 5’-TGGGAAGTCCTYGTGTTGCA-3’ and 5’-KTMGYGC TGGTATGATCGCA-3’ [56]. These results make the genus a model system for the study of complex chromosomal evolution in plants. while leaves were used for genomic DNA extraction.

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