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) RESEARCH ARTIC LE Open Access Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid Tianying Lan and Victor A Albert * Abstract Background: Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady’s slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that conce rted evolutionary forces may homogenize diversity. Results: 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions: Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants. Background Paphiopedilum, a genus of approximately 80 species indi- genous to tropical and s ubtropical Southeast Asia, is among the most widely grown and hybridized of all orch- ids. Species of Paphiopedilum are also ecologically impor- tant narrow endemics in various mainland and island habitats, which range from montane rainforest to seaside cliffs [1]. Karyological studies of Paph iopedilum have revealed considerable chromosomal variation, which ranges from 2n = 26 to 2n = 42, in aneuploid increments sugges- tive of centric fission [2]. Ba sic molecular phylogenetic information on the genus is available [3]. Subgenus Parvisepalum, which is sister to the rest of the genus, has 2n = 26 metacentric chromosomes, whereas the type sub- genus Paphiopedilum includes both clades of 2n = 26 spe- cies and two distinct lin eages of species that bear greater than 26 chromosomes, with the number of telocentrics equal to twice the number of metacentrics that ostensibly split [3]. Hap loid genome size is extremely larg e in these orchids, ranging from 16.1 to 35.1 megabases (Mb) [4]. Chromosome number has been shown to be positively cor- related with genome size [4], so rearrangement processes must include either insertion or deletion of DNA segments. General issues in plant chromosomal evolution include the contribution of rearrangements to genome * Correspondence: vaalbert@buffalo.edu Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260, USA Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 © 2011 Lan and Albert; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reprodu ction in any medium, provided the original work is properly cited. structure and size. Rearrangement proc esses involve double-strand break repair, which occurs frequently at hotspots in pericentromeric and telomeric regions [5,6]. Gene duplications may be caused by unequal c rossing over, retrotransposition, or genome duplication [7]. Tan- dem repeats duplication or segmental duplication is one of the possible outcomes of unequal crossing over [7,8]. These phenomena may be investigated empirically through use of Fluorescence In Situ Hybridization (FISH) on highly repetitive DNA loci subject to con- certed evolution, such as the 18S-5.8S-25S (45S) and 5S ribosomal DNA (rDNA) arrays, w hich may sho w dupli- cation or evidence for rearrangement-producing hetero- logous recombination [9]. Infrageneric comparative rDNA FISH analyses, in which mobility and patterning have been systematically investigated as species-specific karyotype markers, are co mmon in the literature [10-14]. We use such analyses here to document chro- mosomal dynamics in Paphiopedilum. FISH has been applied previously to Paphiopedilum, bu t in a limited manner only, and especially in hybrids [15,16]. Both 45S and 5S rDNAs in plants are characterize d by intergenic spacers. 5S rDNA non-tra nscribed spacer (5S- NTS) sequences have seen some use as phylogenetic mar- kers [17-21]. However, most studies of 5S-NTS to-date have employed direc t sequencing of PCR products, and thereisevidencethattheNTSbothwithinandamong arrays can show polymorphism. We have cloned 5S-NTS segments in Paphiopedilum in order to study pas t and ongoing gene duplication events and the possibili ty of gene conversion both within arrays and among duplicated loci. We briefly report distribution patterns of rDNA signals from a phylogenetic systematic perspective [22] according to accepted section -level classification. We do not aim to provide complete karyotypic comparisons, nor a full cyto- taxonomic treatment; rather, we concern ourselves with demonstrable evidence for dynamic rearrangements dur- ing the evolution of Paphiopedilum.5S-NTSsequence data are also compared with a phylogenetic hypothesis in order to ascertain duplication history of paralogs. Results Distribution patterns of ribosomal DNA by Fluorescence In Situ Hybridization, according to phylogeny and section-level classification Section Parvisepalum Section Parvisepalum is the sister group of all other Paphiopedilum species (Figure 1). Two to four 25S rDNA signals are apparent (Figure 2) among 2n = 26 chromosomes, with two signals most parsimoniously interpretable as the basal condition since this state is shared by the outgroup genera Mexipedium and Phra g- mipedium (unpublished data; [23]). With 2 signals being the inferred primi tive condition, rearrangement by dupli- cation is observed in Paphiopedilum armeniacum, P. emersonii and P. hangianum, which have more loci. 5S rDNA patterns are stable, showing 2 subtelomeric signals that are usually closely linked with one pair of 25S signals (Table 1). In P. delenatii, translocation of either the 5S or 25S rDNA locus has occurred.Thisphenomenonisalso seen in P. malipoense, with its two chromosomes that show hemizygous 25S and 5S rDNA signals, respectively. Section Concoloria Species of section Concoloria show two 25S and 5S signals (Table 1), each on separate chromosomes (2n = 26 total), similarly to Paphiopedilum delenatii of section Parvisepa- lum, except in that the 5S signals are interstitially instead of subtelomerically placed (Figure 3). Section Cochlopetalum Section Cochlopetalum displays an aneuploid number of chromosomes, the telocentrics of which have been sug- gested to descend via centric fission from 25 diploid metacentrics [2]. According to phylogenetic relati onships known at present (Figure 1), and the centric f ission hypothesis, sections Cochlopetalum and Barbata (with telocentrics descended from 26 diploid metacentrics) have evolved aneuploid increa se independently. All four species studied here have two telomeric 25S rDNA sig- nals, and 4 major 5S rDNA signals (Figure 4; Table 1). P arv i sepa l um Concoloria Cochlopetalum Paphiopedilum Coryopedilum Coryopedilum Pardalopetalum B a r bata Figure 1 Section-level phylogenetic tree of genus Paphiopedilum. Section-level phylogenetic tree based on rDNA ITS sequences published b y Cox [3]. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 2 of 15 All 4 species have multiple dispersed 5S signals, rather unlike species of sections Parviflora and Concoloria,and these, like the major loci, are mostly subtelomeric, peri- centromeric and centromeric in position. The 2 species with 2n = 32 chromosomes, Paphiopedilum liemianum (Figure 4C) and P. primulinum (Figure 4A), both have two 5S bands localized on the same chromosomes as the 25S signals, whereas only a single 5S band is seen on the Figure 2 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Parvisepa lum.(A)Paphiopedilum emersonii,(B) P. delenatii, (C) P. malipoense, (D) P. hangianum, (E) P. armeniacum, (F) P. micranthum. 25S rDNA (green) and 5S rDNA (red) probes were simultaneously detected in all Paphiopedilum species. Chromosomes were counterstained with DAPI. All scale bars = 10 μm. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 3 of 15 Table 1 Paphiopedilum species studied, diploid chromosome numbers, rDNA FISH patterns, and 5S-NTS sequence polymorphic sites Number of rDNA sites Positions of rDNA sites b 5S 25S+5S Taxon 2n 25S major visible sites a Co-localization 5S 25S 5S-NTS Polymorphic sites Paphiopedilum Subg. Parvisepalum Sect. Parvisepalum armeniacum 26 4 2 2 2 st t 104 delenatii 26 2 2 2 0 st t 178 emersonii 26 4 2 2 2 st t 124 hangianum 26 4 2 2 2 st t 120 malipoense 26 2 2 2 1 st t 94 micranthum 26 4 2 2 2 st t 59 Subg. Paphiopedilum Sect. Concoloria bellatulum 26 2 2 2 0 i t 118 niveum 26 2 2 2 0 i t 198 Sect. Cochlopetalum liemianum 32 2 4 22 2 st, i, p, c t 162 moquettianum 34 2 4 20 2 st, i, p, c t 225 primulinum 32 2 4 25 2 st, i, p, c t 71 victoria-regina 34 2 4 24 2 st, i, p, c t 137 Sect. Paphiopedilum druryi 30 2 4 16 0 st, i, p, c t 184 fairrieanum 26 2 2 14 2 st, i, p, c t 146 henryanum 26 2 2 17 2 st, i, p, c t 180 hirsutissimum 26 2 6 21 2 st, i, p, c t 182 tigrinum 26 2 6 17 2 st, i, p, c t 141 Sect. Coryopedilum adductum 26 9 4 28 6 st, i, p, c t, st 180 gigantifolium 26 6 6 32 6 st, i, p, c t 210 glanduliferum 26 4 4 26 4 st, i, p, c t 202 randsii 26 4 4 30 4 st, i, p, c t, st 187 sanderianum 26 2 4 16 0 st, i, p, c t 143 stonei 26 2 4 25 2 st, i, p, c t 114 supardii 26 9 4 26 7 st, i, p, c t 226 Sect. Pardalopetalum dianthum 26 2 4 28 2 st, i, p, c t 251 haynaldianum 26 4 4 8 2 st, i, p, c t 110 lowii 26 6 4 28 4 st, i, p, c t, st 161 parishii 26 4 4 34 4 st, i, p, c t 189 Sect. Barbata acmodontum 38 2 4 4 0 i t 169 curtisii 36 2 2 2 0 i t 164 dayanum 36 2 4 6 0 i, p t 169 hennisianum 34 2 2 6 0 i t 153 purpuratum 40 2 4 8 0 st, i t 138 sangii 38 2 4 18 0 st, i t 118 sukhakulii 40 2 2 13 0 st, i t 151 venustum 40 2 4 8 2 i t 109 wardii 42 2 4 4 0 i t 159 a Minimum numbers of visible 5S rDNA FISH signals, including numbers of both major and visible dispersed sites. b st, subtelomeric; t, telomeric; i, interstitial; p, pericentromeric; c, centromeric Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 4 of 15 Figure 3 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Concoloria. (A) Paphiopedilum be llatulum,(B)P. niveum. Figure 4 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Cochlopetalum.(A)Paphiopedilum primulinum, (B) P. moquettianum,(C)P. liemianum,(D)P. victoria-regina. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 5 of 15 same chromosome in the 2n = 34 species P. moquettia- num (Figure 4B) and P. victoria-regina (Figure 4D). Section Paphiopedilum All 5 species of section Paphiopedilum studied show two 25S signals in the telomeric region (Figure 5; Table 1). All species, which are 2n = 26 except for P. druryi (Figure 5E) at 2n = 30, show at least 2 specific 5S rDNA bands, as many as 6, and numerous dispersed signals in the pericen- tromeric and centromeric regions. In all but P. druryi the major signals are closely linked with the 25S arrays. In P. druryi, 4 of the major signals appear to be located on different arms and on morphologically different chromo- somes that may only be partly homologous (this condition was observed in at least 4 cells). Sections Coryopedilum and Pardalopetalum In current phylogenetic results, section Pardalopetalum is derived within section Coryopedilum (Figure 1); as such, they will be discussed together here. Together, the Coryo- pedilum/Pardalopetalum clade, all species having 2n = 26, is the most dynamic in Paphiopedilum regarding chromo- somal rearrangements (Figure 6, 7; Table 1). 25S signals vary from 2 to 9, the latter showing hemizygosity. Signals in all species except Paphiopedilum lowii (Figure 7A), P. adductum (Figure 6E) and P. randsii (Figure 6F) are telomeric. 1-4 subtelomeric 25S signals were observed in P. lowii, P. adductum and P. randsii.InP. supardii (Figure 6G), one hemizygous chromosome has telomeric 25S signals on each arm. P. addu ctum also shows 25S hemizygosity, and both this spec ies and P. supardii show the maximum number of signal s. Species of the Coryope- dilum/Pardalopetalum groupshowatleast4major5S rDNA signals (up to 8 in P. parishii (Figure 7B)) and mul- tiple dispersed repeats in pericentromeric and centromeric regions. In the Pardalopetalum group, all species show at least 2 strong (up to 5) 5S bands located on one chromo- some. Close linkage with 25S occurs throughout the group, other than in P. sanderianum (Figure 6A), either with major or minor 5S bands, and appearing in different placements along chromosome arms. Section Barbata Species of section Barbata, which have 2n = 28-42 and the largest genome sizes, show constancy in 25S rDNA distri- bution, with 2 telomeric signals (Figure 8; Table 1). Major 5S signals number 2-4, and extremely few dispersed Figure 5 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Paphiopedilum. (A) Paphiopedilum fairrieanum, (B) P. hirsutissimum, (C) P. tigrinum, (D) P. henryanum, (E) P. druryi. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 6 of 15 repeat s were observed. Most 5S loci are not centromeric , whereas telomeric, subtelomeric, pericentromeric, and interstitial placements are observed. Only Paphiopedilum curtisii (Figure 8G) and P. hennisianum(Figure 8B) have two major 5S signals, and the first species shows no dis- persed repeat s. P. sukhakulii (Figure 8C), P. venustum (Figure 8F) and P. wardii (Figure 8A) show linked 5S sig- nals. Only in P. venustum is close linkage of 25S and 5S observed, and then only involving a minor 5S band. Because Barbata is the most derived section in the genus (Figure 1), either its species have lost 25S and 5S rDNA loci, since Cochlopetalum, Paphiopedilum, Coryopedilum, Figure 6 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Coryopedilum. (A) Paphiopedilum sanderianum, (B) P. gigantifolium, (C) P. stonei, (D) P. glanduliferum, (E) P. adductum, (F) P. randsii, (G) P. supardii. Arrows indicate subtelomeric 25S rDNA signals. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 7 of 15 and Pardalopetalum usually have more, or the species of the latter sections have increased the number of rDNA loci independently given the low number in sections Par- visepalum and Concoloria. Diversity of 5S ribosomal DNA non-transcribed spacer sequences We investigated duplication history correlated with the dynamic rearrangements observed in 5S rDNA loci. In order to survey sequence variation in 5S-NTS, random clones, 7 (Paphiopedilum niveum)or8(allothers)per species, were sequenced (Additional file 1). Only a few clones were identical to each other (2 sequences from P. acmodontum,2fromP. henryanum,2fromP. hirsutissi- mum,2fromP. stonei,4fromP. dayanum,4from P. malipoense,andonesequenceeachofP. stonei and P. supardii). Sequences of 5S-NTS ranged from 283 bp (P. micranthum 1) to 455 bp (P. bellatulum 5). Given extensive sequence divergence of 5S-NTS and our desire not to manually adjust alignment [24], an objective align- ment was accomplished using MAFFT and default settings. Numbers of polymorphic loci within species, and Figure 7 FISH of 25S and 5S rDNA to metaphase chromosomes of Paphiopedilum section Pardalopetalum. (A) Paphio pedilum lowii, (B) P. parishii, (C) P. dianthum, (D) P. haynaldianum. Arrows indicate subtelomeric 25S rDNA signals. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 8 of 15 phylogen etic relationships, were assessed in order t o esti- mate the strength of gene conversion and the extent of paralogy, respectively. Numbers of polymorphic sites within species positively correlated with minimum numbers of visible 5S signals (P < 0.01, R^2 = 0.21; Figure 9), suggesting that interlocus gene conversion is relatively weak. A phylo- genetic tree outgroup-rooted using Phragmipedium besseae showed 2 major groups of sequences: section Parvisepalum Figure 8 FISH distribution pattern of 25S and 5S rDNA on metaphase chromosomes of Paphiopedilum section Barbata. (A) Paphiopedilum wardii, (B) P. hennisianum, (C) P. sukhakulii, (D) P. purpuratum, (E) P. dayanum, (F) P. venustum, (G) P. curtisii, (H) P. acmodontum, (I) P. sangii. Lan and Albert BMC Plant Biology 2011, 11:126 http://www.biomedcentral.com/1471-2229/11/126 Page 9 of 15 [...]... duplication of 5S loci 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, including Brassicaceae [10], Cyperaceae [11], Asteraceae [25,26], Leguminosae [27], Pinus [28], and Rosaceae [14] Plants typically show some degree of conservatism of rDNA repeat duplication,... Elimination of rDNA loci during chromosomal evolution has been documented in, e.g., Brassicaceae and Rosaceae [10,14] The mechanism that accounts for such loss of rDNA loci, however, remains unclear 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] In the case of section Barbata,... molecular data Lindleyana 1994, 9:115-132 23 Anisimova M, Cannarozzi GM, Liberles DA: Finding the balance between the mathematical and biological optima in multiple sequence alignment Trends in Evolutionary Biology 2010, 2(1):e7 24 Garcia S, Panero JL, Siroky J, Kovarik A: Repeated reunions and splits freature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family... 10 Hasterok R, Wolny E, Hosiawa M, Kowalczyk M, Kulak-Ksiazczyk S, Ksiazczyk T, Heneen WK, Maluszynska J: Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae Annals of Botany 2006, 97(2):205-216 11 Da Silva C, Quintas CC, Vanzela AL: Distribution of 45S and 5S rDNA sites in 23 species of Eleocharis (Cyperaceae) Genetica 2010, 138(910):951-957 12 Sousa A, Barros... structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species Heredity 2002, 88(1):19-25 21 Liu Z, Zhang DM, Wang XQ, Ma XF, Wang XR: Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines American Journal of Botany 2003, 90:17-24 22 Albert V: Cladistic relationships of the slipper orchids (Cypripedioideae: Orchidaceae) from congruent morphological and. .. peripheral population of Aegilops speltoides Tausch Chromosome Research 2004, 12(2):153 39 Baeza C, Schrader O, Budahn H: Characterization of geographically isolated accessions in five Alstroemeria L species (Chile) using FISH of tandemly repeated DNA sequences and RAPD analysis Plant Syst Evol 2007, 269(1-2):1-14 40 Mizuochi H, Marasek A, Okazaki K: Molecular cloning of Tulipa fosteriana rDNA and subsequent... FISH analysis yields cytogenetic organization of 5S rDNA and 45S rDNA in T gesneriana and T fosteriana Euphytica 2007, 155(1-2):235-248 41 Kalendar R, Tanskanen J, Chang W, Antonius K, Sela H, Peleg O, Schulman AH: Cassandra retrotransposons carry independently transcribed 5S RNA Proc Natl Acad Sci USA 2008, 105(15):5833-5838 42 Kapitonov V, Jurka J: A novel class of SINE elements derived from 5S rRNA... distribution pattern is tenable, and TEs containing 5S rDNA-derived sequences have in fact been observed in many plants [41] and animals [42] It is nonetheless possible that due to the similarity of rDNA arrays, chromosomal rearrangement could be induced via heterologous recombination, and in turn, rearrangement could generate repeated sequences through unequal crossovers After generation of a novel locus, in. .. BMC Plant Biology 2010, 10:176 25 Malinska H, Tate JA, Matyasek R, Leitch AR, Soltis DE, Soltis PS, Kovarik A: Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae) allotetraploids BMC Evolutionary Biology 2010, 10:291 26 Moscone E, Klein F, Lambrow M, Fuchs J, Schweizer D: Quantitative karyotyping and dual-color FISH mapping of 5S and 18S-25S... vector and transformed into QIAGEN EZ competent cells (Qiagen PCR Cloning kit) Recombinant clones were screened by colony direct PCR method and were sequenced 7-8 clones per each species using T7 (5’-TAATACGACTCACTATAGGG-3’) primer Probe labelling and Fluorescence in situ hybridization (FISH) Data analysis 25S rDNA, a 2.3-kb ClaI subclones of the 25S rDNA coding region of Arabidopsis thaliana [54] and . 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. Tulipa fosteriana rDNA and subsequent FISH analysis yields cytogenetic organization of 5S rDNA and 45S rDNA in T. gesneriana and T. fosteriana. Euphytica 2007, 155(1-2):235-248. 41. Kalendar R, Tanskanen. 57(5):758-771. doi:10.1186/1471-2229-11-126 Cite this article as: Lan and Albert: Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid. BMC Plant Biology 2011 11:126. Submit