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

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interpretable as the basal condition since this state is shared by the outgroup genera Mexipedium and Phragmipedium (unpublished data;[23]). With 2 signals being the inferred primitive condition, rearrangement by duplication 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. This phenomenon is also seen in P. malipoense, with its two chromosomes that show hemizygous 25S and 5S rDNA signals, respectively.

		Number of rDNA sites				Positions of rDNA sitesb
			5S		25S+5S
Taxon	2n	25S	major	visible sitesa	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

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 Parvisepalum, 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 suggested to descend via centric fission from 25 diploid metacentrics [2]. According to phylogenetic relationships known at present (Figure 1), and the centric fission hypothesis, sections Cochlopetalum and Barbata (with telocentrics descended from 26 diploid metacentrics) have evolved aneuploid increase independently. All four species studied here have two telomeric 25S rDNA signals, and 4 major 5S rDNA signals (Figure 4; Table 1). 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, pericentromeric 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 same chromosome in the 2n = 34 species P. moquettianum (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 pericentromeric 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 chromosomes 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 Coryopedilum/Pardalopetalum clade, all species having 2n = 26, is the most dynamic in Paphiopedilum regarding chromosomal 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. In P. supardii (Figure 6G), one hemizygous chromosome has telomeric 25S signals on each arm. P. adductum also shows 25S hemizygosity, and both this species and P. supardii show the maximum number of signals. Species of the Coryopedilum/Pardalopetalum group show at least 4 major 5S rDNA signals (up to 8 in P. parishii (Figure 7B)) and multiple 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 chromosome. 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 distribution, with 2 telomeric signals (Figure 8; Table 1). Major 5S signals number 2-4, and extremely few dispersed repeats 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 dispersed repeats. P. sukhakulii (Figure 8C), P. venustum (Figure 8F) and P. wardii (Figure 8A) show linked 5S signals. 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, 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 Parvisepalum 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) or 8 (all others) per species, were sequenced (Additional file 1). Only a few clones were identical to each other (2 sequences from P. acmodontum, 2 from P. henryanum, 2 from P. hirsutissimum, 2 from P. stonei, 4 from P. dayanum, 4 from P. malipoense, and one sequence each of P. 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 alignment was accomplished using MAFFT and default settings. Numbers of polymorphic loci within species, and phylogenetic relationships, were assessed in order to estimate 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 phylogenetic tree outgroup-rooted using Phragmipedium besseae showed 2 major groups of sequences: section Parvisepalum versus the remainder of the genus. The single tree of maximum likelihood is shown (as a phylogram, Additional file 2), as is the majority-rule consensus tree based on 100 bootstrap replicates (Additional file 3). Some large species-specific clades were observed, as well as some section-specific clades. Overall, however, the phylogenetic tree was poorly representative of phylogenetic relationships due to extensive duplication of 5S loci.

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, such that when multiple loci do appear, species are commonly polyploid relatives of diploids. There is no evidence at all, however, for polyploidy in Paphiopedilum, where the only chromosome number differences are aneuploid, in a series reflective of centric fission or fusion.

In general, FISH patterns of 25S rDNA loci are reported to be more polymorphic than those of the 5S rDNA [12–14, 26, 28–32]. Conversely, in all sections of Paphiopedilum, except for Parvisepalum and Concoloria, 5S rDNA sites showed much more variability both in number and physical location than did 25S rDNA sites.

The most parsimonious ancestral number of 25S rDNA sites in Paphiopedilum is two, based on outgroup comparison to the genera Mexipedium and Phragmipedium (unpublished results;[3, 22]). Duplication of 25S rDNA sites was observed only in three of the seven sections of Paphiopedilum: Parvisepalum (2n = 26), Coryopedilum (2n = 26) and Pardalopetalum (2n = 26) (Table 1). The physical positions of 25S rDNA loci are relatively conservative. In most Paphiopedilum species we analyzed, 25S rDNA signals are located in terminal chromosome positions. Variation was only observed in three species, Paphiopedilum adductum, P. randsii and P. lowii, which showed 1-4 subtelomeric 25S rDNA signals (Figures 6E, F and 7A, respectively). The ancestral number of 5S rDNA sites, again by outgroup comparison, is 2 (unpublished results from Mexipedium and Phragmipedium), and is only observed in sections Parvisepalum and Concoloria. Massive duplication and amplification of 5S rDNA loci, leading to large-scale polymorphism of numbers, sizes and physical positions of signals, was found prevalent in the remaining five sections. The numbers and distribution of rDNA loci vary widely among plants; however, usually less than one-third of chromosomes display either 45S rDNA or 5S rDNA [13]. It is therefore noteworthy that in some lineages of Paphiopedilum, up to 24 of the 26 chromosomes bear at least one rDNA locus, and a single chromosome can bear up to five major 5S rDNA loci.

Apparently, 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. A similar situation has also been described in many other diploid species, e.g. the diploid lineage of Brassicaceae [10], Cyperaceae [11, 12], Iris[13], and Rosaceae [14]. The massive duplication of rDNA loci in Paphiopedilum sections Cochlopetalum, Paphiopedilum, Coryopedilum and Pardalopetalum could partly contribute to the increase of genome size. Perhaps paradoxically, species with the smallest (P. exul; section Paphiopedilum) and largest (P. dianthum; section Pardalopetalum) haploid genome sizes are both members of groups that show considerable 25S and 5S locus duplication in our FISH experiments. These two species differ more than two-fold in genome size, 16.1 to 35.1 Mb, respectively [33]. If we assume that the number of distinct genes among Paphiopedilum species is roughly constant, this would suggest that genome size increase is primarily due to repetitive element amplification, but that since rDNA duplication is associated with both smaller and larger genomes in the genus, size differences may be more logically traceable to other repetitive DNAs, such as mobile elements. However, a possible tendency for elimination of rDNA loci was found in section Barbata, which has the greatest average genome sizes and chromosome numbers [4]. The number of 25S rDNA loci in Barbata remains two through all the species we studied, while the distribution pattern of 5S rDNA is less dispersed than its sister group, Coryopedilum plus Pardalopetalum. Due to the derived phylogenetic position of section Barbata (Figure 1), it is most parsimonious to conclude that unique chromosomal conditions seen in the group would be similarly derived (autapomorphic). As such, centric fission in Barbata appears to be associated with loss of rDNA loci, while in other systems, centric fission has led to rDNA gains [34]. 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, additional traceable chromosome markers are needed to provide further evidence that chromosomal rearrangements are related to rDNA loss.

A combination of different mechanisms causes high mobility of rDNA

Different mechanisms have been postulated to account for the mobility and polymorphism of numbers, sizes and positions of rDNA sites, such as transposon-mediated transpositional events [36–38], and chromosome rearrangements (translocation, inversion, duplication, deletion) caused by homologous or non-homologous unequal crossing-over and gene conversion [9, 28, 30, 36]. These processes could act alone or in combination, and they do not necessarily imply changes in overall chromosome morphology [31, 34].

The great degree of 5S repeat dispersion seen in sections Cochlopetalum, Paphiopedilum, Coryopedilum and Pardalopetalum has, to our knowledge, only been observed in the monocots Alstroemeria, Tulipa, and Iris[13, 39, 40]. 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. Indeed, some of the signals we observed may be pseudogenes transported within the genomes by retroelements, therefore leading to the false interpretation that we are visualizing entire and active rDNA arrays. Both subtelomeric and pericentromeric regions are well known as hot spots of breakpoints and are also enriched for TEs [5, 6]. Considering the abundant minor loci we observed in these regions, a contribution of transpositions to the dispersed 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 situ amplification cycles via rearrangement could lead to the origin of FISH-detectable loci. Furthermore, hemizygous 5S rDNA sites have been widely observed in many Paphiopedilum species. A double-strand break occurring in a hemizygous region would increase the probability of causing other rearrangements, owing to the absence of a homologous template for its repair [5]. 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. However, in the case of 5S rDNA, there is in fact strong evidence for NTS sequence diversity, which could either be accounted for by the presence or small loci below the FISH detection limit or perhaps by considerable within-array diversity. One future experimental approach to determine whether considerable intra-array diversity indeed exists would be to perform FISH using 5S-NTS-specific probes.

Diversification of 25S rDNA distribution patterns is also observed in Paphiopedilum, but the numbers of loci and degree of dispersion is much lower than for 5S rDNA. Therefore, 5S rDNA might be more frequently seeded by TEs via transpositional events, or, amplification or maintenance of 5S rDNA loci via rearrangement could be more effective and tolerated during the chromosome evolution process. 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].

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. Within-array 5S rDNA diversity appears very likely in Paphiopedilum as well, since many species (e.g., all Parvisepalum and Concoloria) have only one observable 5S locus. For example, 6 species of section Parvisepalum are represented in our phylogenetic analysis by 6-8 distinct sequence variants. These 5S-NTS variants can be concluded to occur within at least partial arrays, pseudogenized or not, since the amplified pieces include sections of 5S rDNA at their 5' and 3' ends. Recent within-species duplication events may be indicated by single-species clades of 5S-NTS sequences, such as P. dayanum, P. lowii, P. sangii, but these could just as well indicate within-array variation, as single-species clades of Parvisepalum (e.g., P. malipoense) and Concoloria (P. bellatulum) most likely do. In many cases, it can be readily seen that duplication of 5S loci has occurred prior to speciation, for example, within Coryopedilum (a large group of sequences representing P. sanderianum, P. stonei, and P. supardii; similarly also within a group of P. adductum and P. randsii sequences). In some cases, ancient duplications must be much older than the major phylogenetic groups of Paphiopedilum, since, for example, P. delenatii shares sequence variants similar to other Parvisepalum species yet has at least one other variant that is more similar to sequences from all other sections. We investigated the possibility of contamination regarding this finding, but discovered similar repeats across 8 distinct P. delenatii accessions (results not shown). Another explanation for multispecies clades, e.g., within well-defined groups such as Parvisepalum could be ancient hybridization.

We observed that increasing within-species 5S NTS sequence diversity correlates with increasing minimum numbers of visible 5S rDNA loci in Paphiopedilum (Figure 9); therefore we infer that interlocus concerted evolution is weak within the genus. Our conclusion concurs with previous findings in many plant genera, such as Gossypium[17], Triticum[18], Chenopodium[19], Nicotiana [20] and Pinus[27]. So far, to our knowledge, noticeable interlocus concerted evolution of 5S rDNA arrays has not been demonstrated in plants.

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, 20, 44–47]. Arrays located in subtelomeric regions are thought to undergo stronger interlocus homogenization forces than ones located in proximal regions. Potential evidence was observed in section Barbata, in which all of six species studied possess two 5S loci. These six species can be categorized into two groups according to the locations of 5S loci. One group harboring proximal 5S loci includes P. wardii, P. dayanum, P. venustum and P. acmodontum, while the other group harboring subtelomeric 5S loci includes P. purpuratum and P. sangii (Figure 8; Table 1). Considering that all six species are closely related and possess the same number of loci, 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. The fact that the average number of polymorphic sites in the proximal-loci group (151.5) is 18% more than that in the subtelomeric-loci group (128), indicates that proximally located loci seem less homogenized than the subtelomerically located loci.

Additionally, we found that not only interlocus but also intralocus concerted evolution is also influenced by chromosomal localization. In section Concoloria, two closely related species, P. bellatulum and P. niveum, both have one 5S locus, but with different localizations. The difference in sequence polymorphism between the two species may be caused by the different locations of the 5S loci. P. niveum, which has a pericentromeric locus, showed 1.68 - fold more polymorphic sites than P. bellatulum, which has a subtelomeric locus (Table 1).

It is well-known that meiotic homologous recombination has been largely suppressed in pericentromeric and centromeric regions. 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]. 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, this may result in the exchange of not only a fraction of the rDNA but also the centromeres themselves. Such an event is more likely to have significantly greater negative consequences to the organism than if the event occurred in the subtelomeric region, which then might result in exchange of telomeres [17, 47]; loss of centromeres would prohibit cell division, whereas loss of telomeres might not restrict mitosis or meiosis. As such, centromerically-located rDNA arrays are expected to show weaker homogenization forces, since fewer individuals with unequal crossovers in this region are expected to survive. In contrast, the subtelomeric region is characterized by a higher rate of interchromosomal exchange [5], thus stronger concerted evolutionary forces could be expected in this region.

All species of section Parvisepalum, as with P. bellatulum, have subtelomeric 5S loci, some of which are closely linked with 25S loci. If 5S localization correlates significantly with homogenization, as with 25S, which is always telomeric-subtelomerically located, we should expect subtelomeric 5S repeats to show decreased sequence diversity due to stronger concerted evolutionary forces. However, this is not the case, since variation in the number of polymorphic sites is not significantly different by section (with or without Pardalopetalum included in Coryopedilum; single factor ANOVA P = 0.06 and 0.1, respectively). We therefore infer that localization of the 5S rDNA arrays only partially contributes to the weak concerted evolution observed in Paphiopedilum.

There are several other hypothesized mechanisms that could lead to the weak concerted evolutionary force on 5S rDNA arrays. For example, ongoing chromosomal rearrangement such as insertion, deletion, or transposition could occur within arrays too frequently for interlocus concerted evolution to be effective. Another possibility is that concerted evolutionary processes homogenize 5S rDNA arrays at rates lower than the rate of speciation, thus novel mutations cannot be fixed or removed and high levels of intralocus polymorphism are expected within arrays [17]. Additionally, the base composition and secondary structure of rDNA sequences may also affect the rate of concerted evolution [53]. It is unknown whether weak concerted evolutionary forces are shared by other Paphiopedilum tandem repeats, or if this is characteristic of 5S rDNA arrays only. This issue can be elucidated by further studies on other tandem repeats, such as 25S rDNA arrays.

Plant materials

Thirty-seven species of the Paphiopedilum genus covering all seven sections were analyzed in this study. Information on the species and sections is provided in Table 1. Actively growing roots were used for chromosome preparation, while leaves were used for genomic DNA extraction.

Chromosome preparation

Root tips were pre-treated with 0.004 M 8-hydroxyquinoline for 4-6 h at 10°C, and fixed in freshly prepared fixative (3:1 ethanol: acetic acid) for 48 h at 10°C. The fixed root tips were then rinsed thoroughly with tap water and macerated in an enzyme mixture containing 2% cellulase (Onozuka R-10, Rpi) and 1% pectolyase (Aspergillus japonicus Y-23, MP) at 37°C for 30 min. After re-fixation in fixative for 15 min, the meristematic cells were squashed in a drop of 45% acetic acid under a coverslip (22 × 22 mm) on a microscope slide. Slides were then dipped into liquid nitrogen and air dried after the coverslips were carefully removed by a blade.

Probe labelling and Fluorescence in situ hybridization (FISH)

25S rDNA, a 2.3-kb ClaI subclones of the 25S rDNA coding region of Arabidopsis thaliana [54] and 5S rDNA (pTa794) [55]were used as probes. 25S rDNA was labelled with biotin-16-dUTP (Roche) and 5S rDNA was labelled with digoxigenin-11-dUTP (Roche), all by nick translation method using the kit from Roche. The hybridization buffer consisted of 50% deionized formamide, 2 × SSC, 50 mM sodium phosphate (pH 7.0), 10% dextran sulfate and sheared salmon sperm DNA (Invitrogen) in 100 × excess of labeled probes. 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. Slides with metaphase spreads were treated with 70% deionized formamide in 2 × SSC at 70°C for 2 min. Denatured probes in hybridization buffer were then applied to the slides, which were incubated at 37°C for 10 h in a humid chamber. Post-hybridization washes and immunodetection were carried out in an automated in situ hybridization instrument, the InsituPro VSi (Intavis Bionanalytical Instruments). The slides were washed in 2 × SSC at room temperature for 5 min and twice in 2 ×SSC at 50°C for 10 min. Fluorescence signal was detected using anti-Digoxigenin-Rhodamine conjugate (Roche) and streptavidin-fluorescein conjugate (Invitrogen). The preparations were mounted and counterstained in Vectashield containing 1.5 μg/ml DAPI (4', 6-diamidino-2-phenylindole) (Vector Laboratories). Images were taken by a Zeiss AxioCam MRm black-and-white CCD camera on a Zeiss Imager. Z1 fluorescence microscope and then processed uniformly using Zeiss AxioVision software. FISH signals were false-colored, and DAPI fluorescence was left in gray-tone.

PCR amplification, cloning and sequencing

Total genomic DNA was extracted from fresh leaves using Qiagen DNeasy Plant Mini kit. The 5S-NTS region was amplified by PCR using the universal degenerate primers: 5'-TGGGAAGTCCTYGTGTTGCA-3' and 5'-KTMGYGCTGGTATGATCGCA-3'[56]. Touchdown amplification was performed as follows: an initial step at 94°C for 5 min, followed by 10 cycles of 94°C for 1 min, annealing for 1 min (start at 60°C, and decreased by 1°C per cycle), and 72°C for 1 min, then 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, the final step at 72°C was extended to 10 min. After gel purification using QIAquick Gel Extraction Kit (Qiagen), PCR products were ligated into pDrive Cloning 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.

Data analysis

Sequences were aligned using the MAFFT (Multiple Alignment using Fast Fourier Transform) web server at the European Bioinformatics Institute [57]. Default parameters were used: gap opening penalty = 1.53, gap extension penalty = 0.123, tree rebuilding number = 1, maxiterate = 0, and perform FFTS = localpair. The sequence alignment is available as a supplementary FASTA file (Additional file 4).

Within-species 5S-NTS polymorphism was estimated, based on the aforementioned multiple alignment, using DnaSP version 5.10.01 [58]. The relationship between numbers of polymorphic sites and minimum numbers of visible 5S rDNA signals was investigated using linear regression analysis (in Microsoft Excel).

Phylogenetic reconstruction was performed using maximum likelihood optimization available through the RaxML BlackBox web server [59] running RaxML version 7.2.8 [60]. Default settings were used. The 8 Phragmipedium besseae sequences were indicated as the outgroup. RaxML was called using the following commands: raxml -# 100 -n pasted -o bess1, bess2, bess3, bess4, bess5, bess6, bess7, bess8 -f a -m GTRGAMMA -x 564547904 -p 564547904 -s 0VaDTW. All search information, as was output on the web site, is included in Additional file 5.