The Evolution of Genome Size and Distinct Distribution Patterns of Rdna in Phalaenopsis (Orchidaceae)

Botanical Journal of the Linnean Society, 2017, 185, 65–80. With 4 figures.

The evolution of genome size and distinct distribution patterns of rDNA in Phalaenopsis (Orchidaceae)

YUNG-I. LEE1,2*, MEI-CHU CHUNG3, HAO-CHIH KUO4, CHUN-NENG WANG4, YI-CHING LEE1, CHIEN-YU LIN1, HONG JIANG5 and CHIH-HSIN YEH6

1Department of Biology, National Museum of Natural Science, 40453 Taichung, Taiwan, ROC 2Department of Life Sciences, National Chung Hsing University, 40227 Taichung, Taiwan, ROC 3Institute of Plant and Microbial Biology, Academia Sinica, 115 Taipei, Taiwan, ROC 4Department of Life Science, National Taiwan University, Taipei, Taiwan, ROC 5Yunnan Academy of Forestry, Kunming, People’s Republic of China 6Taoyuan District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Taoyuan 327, Taiwan, ROC

Received 20 September 2016; revised 13 May 2017; accepted for publication 23 June 2017

Phalaenopsis is a horticulturally important genus of c. 63 spp. native to Southeast Asia. Aiming to understand the genome differentiation and phylogenetic relationships in diploid Phalaenopsis spp., we investigated the nuclear DNA content by flow cytometry karyomorphology and mapped rDNA loci by fluorescence in situ hybridization to address genome differentiation. Most species have a chromosome complement of 2n = 38 (but have different karyotype com- positions). Chromosome reduction is only observed in Phalaenopsis section Aphyllae (2n = 34 and 36). The genome sizes differ 6.3-fold, from 1C = 1.39 to 8.74 pg. Reconstruction of genome size indicates the genome size for the most recent common ancestor of Phalaenopsis subgenus Parishianae to be 3.92 pg/1C and for subgenus Phalaenopsis to be 2.42 pg/1C. The 45S rDNA sites are polymorphic and correlate positively with genome size, but 5S rDNA sites do not. Our results suggest independent evolutionary processes of genome size increase or decrease in each Phalaenopsis lineage. The polymorphism in the number of rDNA loci reveals that complex chromosomal rearrangements, e.g. gain or loss of repetitive DNA or chromosomes, inversion and/or transposition events, may have contributed to create the various karyotypes observed in Phalaenopsis spp.

ADDITIONAL KEYWORDS: chromosome – fluorescence in situ hybridization – karyotype – molecular phylogenetic analysis – rRNA genes.

INTRODUCTION plastid DNA sequences (Yukawa et al., 2005) have identified two distinct clades with different numbers Phalaenopsis Blume consists of c. 63 spp. distributed of pollinia and biogeographical distributions. Species from the Himalayas, through southern China and in the first clade [Phalaenopsis subgenus Parishianae Southeast Asia, to northern Australia (Sweet, 1980; (H.R.Sweet) Christenson] have four pollinia and the Christenson, 2001). Phalaenopsis spp. are extensively species are distributed throughout the Himalayas and sold in the floricultural trade as potted plants or cut Indochina; species in the second clade (Phalaenopsis flowers because of their colourful and long-lasting flow- subgenus Phalaenopsis) have two pollinia and are ers (US Department of Agriculture, 2015). Phylogenetic mainly distributed across the islands of tropical Asia analyses of Phalaenopsis spp. using nuclear ribosomal and Australia. Although many Phalaenopsis spp. are DNA internal transcribed spacer (ITS) sequences (Tsai, extensively used in breeding programmes, available Huang & Chou, 2006) or ITS sequences combined with cytogenetic information is incomplete. Previous cytological studies of Phalaenopsis indicated that although all species that had been studied had a chromosome *Corresponding author. E-mail: [email protected] complement of 2n = 2x = 38, there was considerable

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variation in chromosome sizes (Sagawa, 1962; Shindo de Sá, 1995; Garcia et al., 2017). The DNA sequence & Kamemoto, 1963; Sagawa & Shoji, 1968). Most transcribed for the 5S rRNA is c. 120 base pairs long, Phalaenopsis spp. have a symmetrical karyotype, but and is highly conserved among organisms. A non-tran- a few species, such as P. amboinensis J.J.Sm., P. venosa scribed spacer region (5S-NTS) separates adjacent 5S Shim & Fowlie and P. violacea Teijsm. & Binn., have rDNA repeats (Appels & Baum, 1992). Variation in the bimodal karyotypes with heterochromatin blocks that number and distribution of 5S and 45S rRNA genes as have accumulated at the ends of the large metacentric/ revealed by fluorescence in situ hybridization (FISH) in submetacentric chromosomes. Such karyotype vari- closely related species has provided valuable informa- ation in Phalaenopsis spp. has been considered to be tion about chromosomal evolution and genome organi- most probably due to differential accumulation of con- zation (e.g. Adams et al., 2000; Raskina, Belyayev & stitutive heterochromatin (Kao et al., 2001). Nevo, 2004; Lamb et al., 2007; Iovene et al., 2008; for a Knowledge of genome size data is an important con- review, see Gill, Hans & Jackson, 2008). Furthermore, sideration when applying various molecular tools to the distribution of rDNA loci also provides useful land- study genetic diversity (Fay, Cowan & Leitch, 2005; marks for chromosome identification and for tracing Fay et al., 2009). For example, AFLP methods to esti- karyotype evolution among related species (Jiang & mate genetic diversity usually fail for species with Gill, 1994; Linares et al., 1996; Moscone et al., 1999; large genome sizes as they do not produce a sufficient Taketa, Harrison & Heslop-Harrison, 1999; Hasterok number of bands to enable genetic diversity to be esti- et al., 2001; Hizume et al., 2002; Bogunić et al., 2011). mated (Fay et al., 2009). Genome size has also been To date, cytological data of Phalaenopsis have been shown to have considerable biological implications. largely focused on species in Phalaenopsis subgenus For example, several studies have identified correla- Phalaenopsis (Kao et al., 2001; Lin et al., 2001; Lee, tions between genome size and cellular volume or 2002, 2007; Chen, 2009; Tseng, 2009) and only P. pul- organ size (Van’t Hof & Sparrow, 1963; Price, Sparrow cherrima (Lindl.) J.J.Sm. has been studied cytoge- & Nauman, 1973), such as seed size and leaf size. netically in subgenus Parishianae (Peng, 2007). In Genome size is also correlated with the rate of growth this study, we examined the nuclear DNA contents and development, which are useful parameters for of seven Phalaenopsis spp. with the aim of achiev- breeding (Doležel, Doleželova & Novak, 1994; Cerbah ing a more comprehensive sampling of taxa in sub- et al., 2001). According to previous reports by Lin genus Parishianae than previously undertaken. We et al. (2001) and Chen et al. (2013), genome sizes of 50 also characterized the chromosomal distribution of Phalaenopsis spp. showed a 6.3-fold range in nuclear 5S rDNA and 45S rDNA loci in 45 Phalaenopsis spp. DNA amount (2C = 2.77–17.47 pg). However, the using FISH. These comprehensive cytogenetic studies genome size data for species belonging to Phalaenopsis and new genome size data reported here, analysed in a subgenus Parishianae are incomplete. molecular phylogenetic framework, provide important Repetitive DNA sequences (e.g. transposable ele- insights into the molecular aspects of chromosome ments and tandem repeats) are a major component of evolution in Phalaenopsis and valuable information plant nuclear genomes and may make up to 50–75% of for breeding programmes. the genome (Kubis, Schmidt & Heslop-Harrison, 1998; Heslop-Harrison & Schwarzacher, 2011). Repetitive DNA sequences show variation in sequence and copy MATERIAL AND METHODS number during evolution (Flavell, 1982) and thus knowledge of repetitive sequences can provide insights Plant material into the evolution of plant genomes and add support to We selected 45 representative taxa from both sub- taxonomic and phylogenetic studies (Heslop-Harrison genera and all sections of Phalaenopsis for this study. & Schmidt, 2012). In higher eukaryotes, ribosomal The plants were maintained in the greenhouses of genes (rDNAs) are organized into two distinct mul- the National Museum of Natural Science and voucher tigene families, coding for 45S rRNA and 5S rRNA, specimens were deposited in the herbarium of the respectively. Both types of rDNAs occur as tandemly National Museum of Nature and Science, Taiwan. repetitive sequences that encode non-translated rRNA Information on chromosome numbers and the vouch- involved in ribosome formation (Suzuki, Sakurai & ers are given in Table 1. Matsuda, 1996). Typically, repetitive 45S rDNAs with a non-coding intergenic spacer (IGS) between adjacent 45S rDNA units are tandemly arrayed at one or Genome size measurement several loci on chromosomes (Roa & Guerra, 2012). Young leaves of each sample were chopped using a The 5S rDNA locus also contains a tandem array of sharp razor blade in a Petri dish containing 3 mL of hundreds or even thousands of repeats, usually at a Galbraith’s buffer (Galbraith et al., 1983). Then, the different location from the 45S rDNA locus (Drouin & suspension of nuclei was filtered through a 30-μm

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Table 1. The voucher, diploid chromosome number, genome size, and the signals of rDNA FISH experiments in Phalaenopsis species. The infrageneric treatment follows Cribb and Schuiteman (2012).

Taxon Voucher 2n 1C-value (pg) Number of rDNA Positions of rDNA sites sites

5S rDNA 45S rDNA 5S rDNA 45S rDNA

Subgenus Parishianae Section Aphyllae P. honghenensis F.Y.Liu Yung-I Lee201381 36 5.92 ± 0.16a 2 4 proximal distal P. lowii Rchb.f. Yung-I Lee201382 36 2.46 ± 0.08a 2 2 distal distal P. stobartiana Rchb.f. Yung-I Lee201383 36 4.25 ± 0.11a 2 2 proximal distal P. taenialis (Lindl.) Yung-I Lee201384 36 3.56 ± 0.12a 2 2 proximal distal Christenson & Pradhan P. wilsonii Rolfe Yung-I Lee201385 36 4.38 ± 0.14b 2 2 proximal distal Section Esmeraldae P. chibae T.Yukawa Yung-I Lee201377 38 5.47 ± 0.19b 2 4 distal distal P. deliciosa Rchb.f. Yung-I Lee201378 38 4.37 ± 0.14b 2 2 distal distal P. finleyi Christenson Yung-I Lee201379 34 3.03 ± 0.11a 2 2 distal distal P. pulcherrima (Lindl.) Yung-I Lee201380 38 6.16 ± 0.18b 2 8 proximal distal J.J.Sm. Section Parishianae P. appendiculata Carr Yung-I Lee201386 38 3.43 ± 0.07a 4 4 distal distal P. gibbosa H.R.Sweet Yung-I Lee201387 38 5.16 ± 0.16a 2 6 proximal distal P. lobbii (Rchb.f.) Aver. Yung-I Lee201388 38 8.74 ± 0.16b 2 8 proximal distal P. parishii Rchb.f. Yung-I Lee201389 38 7.36 ± 0.27b 2 8 distal distal Subgenus Phalaenopsis Section Polychilos P. amboinensis J.J.Sm. Yung-I Lee201351 38 4.88 ± 0.21b 2 14 proximal distal P. bastianii O.Gruss & Yung-I Lee201352 38 2.89 ± 0.14b 2 2 distal distal Roellke P. bellina (Rchb.f.) Yung-I Lee201353 38 6.52 ± 0.24b 4 8 distal distal Christenson P. cochlearis Holttum Yung-I Lee201354 38 2.08 ± 0.07b 2 2 distal distal P. corningiana Rchb.f. Yung-I Lee201355 38 3.99 ± 0.19b 2 8 distal distal P. cornu-cervi Blume & Yung-I Lee201356 38 2.71 ± 0.13b 2 4 distal distal Rchb.f. P. fasciata Rchb.f. Yung-I Lee201357 38 2.89 ± 0.18b 2 2 distal distal P. fuscata Rchb.f. Yung-I Lee201359 38 2.02 ± 0.10b 2 4 distal distal P. gigantea J.J.Sm. Yung-I Lee201360 38 2.27 ± 0.12b 2 2 distal distal P. hieroglyphica (Rchb.f.) Yung-I Lee201361 38 2.67 ± 0.15b 2 2 distal distal H.R.Sweet P. lueddemanniana Rchb.f. Yung-I Lee201364 38 2.85 ± 0.15b 2 2 distal distal P. mannii Rchb.f. Yung-I Lee201365 38 5.72 ± 0.14b 2 2 distal distal P. mariae Burb. Yung-I Lee201366 38 2.87 ± 0.08b 2 2 distal distal P. micholitzii Rolfe Yung-I Lee201367 38 2.78 ± 0.08b 2 2 distal distal P. modesta J.J.Sm. Yung-I Lee201368 38 2.27 ± 0.11b 2 2 distal distal P. pallens Rchb.f. Yung-I Lee201369 38 2.99 ± 0.11b 2 2 distal distal P. pantherina Rchb.f. Yung-I Lee201370 38 2.87 ± 0.13b 2 2 distal distal P. pulchra (Rchb.f.) Yung-I Lee201371 38 2.32 ± 0.17b 2 2 distal distal H.R.Sweet P. sumatrana Korth. & Rchb.f. Yung-I Lee201372 38 4.37 ± 0.15b 2 8 distal distal P. tetraspis Rchb.f. Yung-I Lee201373 38 4.29 ± 0.14a 2 10 distal distal P. venosa Shim & Fowlie Yung-I Lee201374 38 4.29 ± 0.12b 2 10 proximal distal P. violacea Teijsm. & Binn. Yung-I Lee201375 38 6.45 ± 0.20b 2 10 distal distal P. viridis J.J.Sm. Yung-I Lee201376 38 1.97 ± 0.10b 2 2 distal distal

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Table 1. Continued

Taxon Voucher 2n 1C-value (pg) Number of rDNA Positions of rDNA sites sites

5S rDNA 45S rDNA 5S rDNA 45S rDNA

Section Phalaenopsis P. amabilis Blume Yung-I Lee201342 38 1.40 ± 0.06b 4 2 distal distal P. aphrodite Rchb.f. Yung-I Lee201343 38 1.40 ± 0.06b 4 2 distal, proximal distal P. celebensis H.R.Sweet Yung-I Lee201344 38 1.77 ± 0.06b 2 2 distal distal P. equestris (Schauer) Rchb.f. Yung-I Lee201345 38 1.48 ± 0.06b 2 2 distal distal P. lindenii Loker Yung-I Lee201346 38 1.46 ± 0.05b 2 2 distal distal P. philippinensis Golamco ex Yung-I Lee201347 38 1.38 ± 0.04b 6 2 distal, proximal distal Fowlie & C.Z.Tang P. sanderiana Rchb.f. Yung-I Lee201348 38 1.41 ± 0.06b 4 2 distal, proximal distal P. schilleriana Rchb.f. Yung-I Lee201349 38 1.39 ± 0.05b 6 2 distal, proximal distal P. stuartiana Rchb.f. Yung-I Lee201350 38 1.43 ± 0.06b 6 2 distal, proximal distal

a Data measured in the present study. In this study, three individual samples were measured separately for each species, and three replicates of each sample were processed. Chicken erythrocyte nuclei (2C = 3 pg) and Pisum sativum ‘Minerva Maple’ (2C = 9.56 pg) were used as the internal standard. b Data measured by Chen et al. (2013).

nylon mesh, treated with RNase A (Sigma Chemical MrModeltest 2.2 (Nylander, 2004). Two separate runs Co.), and stained with propidium iodide (PI) (Sigma). of four Markov chains Monte Carlo (MCMC; Yang Both PI and RNase A were adjusted to a final con- & Rannala, 1997) were performed for 3 000 000 gen- centration of 50 μg ml−1. Samples were kept on ice erations until the mean deviation of split frequency for 30 min and 10 000 nuclei were measured using a dropped below 0.01; a tree was sampled every 100th flow cytometer (Coulter EPICS Elite ESP, Beckman generation. Trees from the first 25% of generations Coulter) equipped with an air-cooled argon-ion laser were discarded using the ‘burn-in’ command and the tuned at 15 mW and operating at 488 nm. PI fluo- remaining trees were used to calculate an all-com- rescence was collected through a 645-nm dichroic patible consensus topology and posterior probability long-pass filter and a 620-nm band-pass filter. Flow (PP) values for individual branches. The convergence histograms were analysed with the FlowMax software of each run was estimated using the program Tracer (v. 2.4, Partec GmbH). Three individual samples were v1.4 (http://beast.bio.ed.ac.uk/Tracer) and the effective measured separately for each species and three repli- sample size values of > 300 were confirmed to have cates of each sample were processed. Chicken eryth- a sufficient level of sampling. The trees obtained in rocyte nuclei (2C = 3 pg; Biosure) and Pisum sativum these analyses were drawn with the TreeGraph 2 soft- L. ‘Minerva Maple’ (2C = 9.56 pg) were used as inter- ware (Stöver & Müller, 2010). nal standards.

Ancestral genome size reconstruction Phylogenetic analysis The genome size data of this study in combination with The nuclear ribosomal spacer regions, ITS1 and ITS2, the previous investigations (Lin et al., 2001; Chen et al., and the 5.8S ribosomal gene (ITS) of Phalaenopsis 2013) were used to reconstruct the ancestral state for spp. were obtained from the NCBI sequence data- genome size in Phalaenopsis. The Bayesian method base (National Center for Biotechnology Information, using the software BayesTraits v2 (Pagel & Meade, GenBank) for phylogenetic analysis by referring to 2014) was applied to investigate evolution of genome Carlsward et al. (2006) and Tsai et al. (2006), and size of Phalaenopsis. In brief, we modelled the genome the sequence of Amesiella philippinensis (Ames) size evolution of Phalaenopsis spp. across the phyloge- Garay was used as an outgroup taxon (Supporting netic tree inferred from MrBayes 3.2.1 and we recon- Information, Table S1). DNA sequences were aligned structed the ancestral states for genome size at internal using CLUSTALW (Thompson et al., 1997) and phy- nodes of the tree using the MCMC method. Given the logenetic relationships were analysed using a model- uncertainty of tree topology and the associated branch based Bayesian approach using MrBayes 3.2.1 lengths, we randomly sampled 3000 trees from the post (Ronquist & Huelsenbeck, 2003). The ‘best-fit’ model burn-in phase of MrBayes analysis that were used to HKY + I +Γ was selected under the Akaike informa- guide the BayesTraits analyses (Pagel, Meade & Barker, tion criterion test (Akaike, 1974) as implemented in 2004). We performed this tree-sampling procedure

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twice to obtain consistent results from the BayesTraits models where the relevant parameter was free to be analyses. The root-to-tip branch lengths were central in estimated in turn with the null model. BayesTraits analysis to inform tempo and mode of trait evolution (Pagel, 1999). This was undertaken with the drop.tip function of the ape package (Paradis, Claude & Chromosome preparation and FISH Strimmer, 2004) developed in R (R Core Team, 2015). Chromosome samples were prepared according to

In this study, we applied a log10 transformation to the the method of Chung et al. (2008) with minor modifi- genome size data of Phalaenopsis spp. to model the cations. Young and healthy root tips were harvested evolution of this trait in the manner of proportional and pretreated in 2 mM 8-hydroxyquinoline at 20 °C changes (see Lysák et al., 2009). We first explored two for 5 h, rinsed with distilled water, then fixed in BayesTraits models, corresponding to constant-vari- freshly prepared Carnoy’s solution (three parts eth- ance random-walk processes of genome size evolution anol to one part glacial acetic acid). Root tips were without (neutral-drift model) and with (directional macerated with 6% cellulose (Onozuka R-10, Yakult drift model) a directional trend across the phylogenetic Honsha) and 6% pectinase (Sigma) in 75 mM KCl, tree, respectively. The former model was specified with pH 4.0 at 37 °C for 90 min and then squashed on

a parameter α for the log10-transformed 1C value at slides in the same fixative. Slides were air-dried and the root of Phalaenopsis spp. and the latter model was stored at –80 °C until required. The FISH procedure introduced with an additional parameter β for a puta- was essentially the same as that described by Lee, tively non-zero mean of the random-walk process. We Chang & Chung (2011). The rDNA probes used in set uniform priors to both α and β, with boundaries at this study were: (1) pTA71 containing a repetitive (0, 2) and (−2, 2), respectively. Under each model, two unit of 45S rDNA (c. 9 kb) from Triticum aestivum independent MCMC runs were conducted, each run for L. (Gerlach & Bedbrook, 1979) and (2) pTA794 con- 10 million iterations (1000 iterations per sample) with taining a 5S rDNA repeat unit (410 bp) from T. aes- the first 10% discarded as burn-in. Traces of independ- tivum (Gerlach & Dyer, 1980). Both probes were ent runs for MCMC convergence were examined using labelled by nick translation with either digoxigenin- Tracer 1.6 (Rambaut et al., 2013). For model selection, 11-dUTP or biotin-16-dUTP (Roche Applied Science). we continued MCMC analyses described above with an The digoxigenin-labelled probes were detected additional 10 000 iterations of steppingstone sampling by anti digoxigenin-rhodamine (Roche Applied procedures (Xie et al., 2011) containing 200 stones to Science). The biotin-labelled probes were detected acquire marginal likelihood estimates (MLEs) for the by fluorescein isothiocyanate (FITC)-conjugated two models. The MLE values estimated were used to avidin (Vector Laboratories). Chromosomes were compute a Bayes factor (BF) (Kass & Raftery, 1995) for counterstained with 4′, 6-diamidino-2-phenylindole interpreting the strength of support for one model over (DAPI) in an antifade solution (Vector Laboratories).

the other. The statistic 2×logeBF was computed and a All images were captured digitally by a CCD camera value of > 2 was interpreted as positive evidence, sup- attached to an epifluorescence microscope (Axioskop porting an alternative model over a null one. 2, Carl Zeiss AG). The final image adjustments were In this study, the neutral-drift model was chosen made with Adobe Photoshop CS2 (version 9.0.2, from the model selection procedure. Using a null Adobe Systems Incorporated). For each species, we model, we explored three alternative models (each of examined three individual samples, and more than which specified a specific manner of tree branch trans- three root tips of each sample were processed. In formation). The first two alternative models allowed the FISH experiments, we counted nearly 100 cells the investigation of how the tempo of genome size evo- and repeated the experiment at least three times for lution varied across the tree, whereas the third tested each species. the extent to which genome size evolution reflected the phylogenetic tree. Specifically, the first alternative model was introduced with a parameter κ; values RESULTS of κ > 1 and < 1 inflated and shrank the difference between long and short branches of a tree. The second Chromosome numbers alternative model was introduced with a parameter δ; The results of the genome size and chromosome num- values of δ > 1 and < 1 stretched individual branches ber analyses are presented in Table 1 and Figure 1. near the root and tips, respectively. The third alterna- Most Phalaenopsis spp. showed a consistent chro- tive model was introduced with a parameter λ which mosome number of 2n = 2x = 38, except for spe- had a range between zero and one, and a λ value closer cies in Phalaenopsis section Aphyllae (H.R.Sweet) to zero transformed the tree further towards a star Christenson and one species in section Esmeraldae phylogeny. We replicated model selection procedures Rchb.f. In section Aphyllae, P. honghenensis F.Y.Liu as described above and compared the three alternative (Fig. 2A), P. lowii Rchb.f. (Fig. 2B), P. stobartiana

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Figure 1. Ancestral genome size reconstruction of diploid Phalaenopsis spp. and evidence for genome expansion and con- traction across the phylogenetic tree. The phylogenetic tree topology was generated from Bayesian inference using the nuclear internal transcribed spacer. The tree is rooted with Amesiella philippinensis and the values above branches indicate node support (posterior probabilities ≥ 0.95). Ancestral genome sizes for the most recent common ancestor (MRCA) of each major Phalaenopsis clade are shown. Closed circles indicate the haploid genome size (1C values in pg) for extant species; dashed lines indicate the ancestral genome sizes for the MRCA of subgenus Parishianae (red) and subgenus Phalaenopsis (blue) clades. For each Phalaenopsis sp., the increase/decrease in genome size relative to the MRCA of its clade is shown.

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Figure 2. Location of 5S and 45S rDNA sites on somatic chromosomes of Phalaenopsis spp. determined by fluorescence in situ hybridization. (A) P. honghenesis, (B) P. lowii, (C) P. stobartiana, (D) P. taenialis, (E) P. wilsonii, (F) P. chiba, (G) P. deliciosa, (H) P. finleyi, (I) P. pulcherrima, (J) P. appendiculata, (K) P. gibbosa, (L) P. lobbii, (M) P. parishii, (N) P. amboinensis and (O) P. bastianii. Arrows indicate the 45S rDNA signals (green) and arrowheads indicate the 5S rDNA signals (red). Scale bars = 10 μm.

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Rchb.f. (Fig. 2C), P. taenialis (Lindl.) Christenson Section Aphyllae & Pradhan (Fig. 2D) and P. wilsonii Rolfe (Fig. 2E) In P. honghenensis, four distal 45S rDNA sites were had 2n = 2x = 36 and P. finleyi Christenson (section detected (Fig. 2A), whereas in the other species of sec- Esmeraldae) had 2n = 2x = 34 (Fig. 2H). The kar- tion Aphyllae analysed, i.e. P. lowii (Fig. 2B), P. stobar- yotypes of most Phalaenopsis spp. were symmetri- tiana (Fig. 2C), P. taenialis (Fig. 2D) and P. wilsonii cal and uniform in size. The exception to this was (Fig. 2E), only two distal 45S rDNA sites were observed. encounted in Phalaenopsis section Polychilos (Breda) All these species had two proximal 5S rDNA sites, Rchb.f., in which a lineage containing P. amboinen- except for P. lowii which had two distal 5S rDNA sites. sis J.J.Sm. (Fig. 2N), P. bellina (Rchb.f.) Christenson (Fig. 3A), P. corningiana Rchb.f. (Fig. 3C), P. sumatrana Korth. & Rchb.f. (Fig. 4B), P. tetraspis Rchb.f. Section Esmeraldae (Fig. 4C), P. venosa Shim & Fowlie (Fig. 4D) and P. deliciosa Rchb.f. (Fig. 2G) and P. finleyi (Fig. 2H) had P. violacea Teijsm. & Binn. (Fig. 4E) possessed asym- two distal 45S rDNA sites and two distal 5S rDNA metrical and bimodal karyotypes. In these species, sites, P. chiba T.Yukawa (Fig. 2F) had four distal 45S larger chromosomes were usually metacentric or rDNA sites and two distal 5S rDNA sites, and P. pul- submetacentric, whereas smaller chromosomes were cherrima (Fig. 2I) had eight distal 45S rDNA sites and subtelocentric or acrocentric. two proximal 5S rDNA sites.

Genome size Section Parishianae Combining the genome size data generated in this P. appendiculata Carr (Fig. 2J) had four distal 45S study with previous investigations (Lin et al., 2001; rDNA sites and four distal 5S rDNA sites, P. gibbosa Chen et al., 2013) means that data are now availa- H.R.Sweet (Fig. 2K) had six distal 45S rDNA sites and ble for 56 out of 63 species (c. 89 %) in Phalaenopsis. two proximal 5S rDNA sites, while P. lobbii (Fig. 2L) This improved sampling relative to previous studies and P. parishii Rchb.f. (Fig. 2M) had eight distal 45S shows that genome size in Phalaenopsis ranges from rDNA sites. One pair of chromosomes in P. lobbii and 1C = 1.38 pg in P. philippinensis Golamco ex Fowlie P. parishii had four strong, distal 45S rDNA signals & C.Z.Tang to 1C = 8.74 pg in P. lobbii (Rchb.f.) Aver. at both ends. Phalaeopsis lobbii had two proximal (Table 1). The genome sizes in subgenus Parishianae 5S rDNA sites, whereas P. parishii had two distal 5S were relatively high (mean 1C value = 4.95 pg), rDNA sites. whereas those for species belonging to subgenus Phalaenopsis were lower (mean 1C value = 2.88 pg). To verify whether the genome sizes increased or Subgenus Phalaenopsis decreased independently in Phalaenopsis spp., we The species of subgenus Phalaenopsis were grouped reconstructed the genome size for the most recent into two sections and varied in the number of rDNA common ancestor (MRCA) of each clade. The results sites from two to 14 45S rDNA sites and two to six 5S of our analysis indicated that the evolution of rDNA sites per chromosome complement. genome size in Phalaenopsis showed no directional trend and no heterogeneous tempos in different parts of the phylogenetic tree. The ancestral genome Section Polychilos size for the MRCA of subgenus Parishianae was Most species of section Polychilos showed constancy reconstructed as 3.92 pg/1C, whereas that for the in 45S rDNA and 5S rDNA distributions, with two MRCA of subgenus Phalaenopsis was reconstructed distal 45S rDNA and two distal 5S rDNA sites (Figs as 2.42 pg/1C (Fig. 1). 2O, 3B, E, G–O, 4A, F). A few species, i.e. P. cornu- cervi Blume & Rchb.f. (Fig. 3D) and P. fuscata Rchb.f. (Fig. 3F), showed duplication of 45S rDNA loci, hav- Mapping 5S and 45S rDNA loci ing four distal 45S rDNA sites. In section Polychilos, The 5S and 45S rDNA sites are summarized in Table 1 massive duplication of 45S rDNA loci could only be and described in detail below. detected in the phylogenetic lineage containing P. amboinensis (Fig. 2N), P. bellina (Fig. 3A), P. corningiana (Fig. 3C), P. sumatrana (Fig. 4B), P. tet- Subgenus Parishianae raspis (Fig. 4C), P. venosa (Fig. 4D) and P. violacea The species of subgenus Parishianae were character- (Fig. 4E). These species had between eight and 14 ized by possessing two to eight 45S rDNA sites and two distal 45S rDNA sites. The highest number of sites to four 5S rDNA sites per chromosome complement. was observed in P. amboinensis, in which among the

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Figure 3. Location of 5S and 45S rDNA sites on somatic chromosomes of Phalaenopsis spp. determined by fluorescence in situ hybridization. (A) P. bellina, (B) P. cochlearis, (C) P. corningiana, (D) P. cornu-cervi, (E) P. fasciata, (F) P. fuscata, (G) P. gigantea, (H) P. hieroglyphica, (I) P. lueddemanniana, (J) P. mannii, (K) P. mariae, (L) P. micholitzii, (M) P. modesta, (N) P. pallens and (O) P. pantherina. Arrows indicate the 45S rDNA signals (green), and arrowheads indicate the 5S rDNA signals (red). Scale bars = 10 μm.

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Figure 4. Location of 5S and 45S rDNA sites on somatic chromosomes of Phalaenopsis spp. determined by fluorescence in situ hybridization. (A) P. pulchra, (B) P. sumatrana, (C) P. tetraspis, (D) P. venosa, (E) P. violacea, (F) P. viridis, (G) P. amabilis, (H) P. aphrodite, (I) P. celebensis, (J) P. equestris, (K) P. lindenii, (L) P. philippinensis, (M) P. sanderiana, (N) P. schilleriana and (O) P. stuartiana. Arrows indicate the 45S rDNA signals (green), and arrowheads indicate the 5S rDNA signals (red). Scale bars = 10 μm.

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14 sites, four were detected at the two distal ends of Several terminal and intercalary DAPI-positive one pair of chromosomes (Fig. 2N). In this section, bands are evident on their large and symmetrical most species showed two distal 5S rDNA sites, except chromosomes. The increase in genome size by add- for P. bellina with four distal sites (Fig. 3A), and P. ing repetitive DNA more or less equally to all chro- amboinensis and P. venosa with two proximal sites mosomes of the complement has also been observed (Figs 2N, 4D). in Cypripedium L. (Leitch et al., 2009), Lathryus L. (Narayan & Durrant, 1983), Nicotiana L. (Narayan, 1987) and Vicia L. (Raina, 1990). In contrast, in sec- Section Phalaenopsis tion Polychilos of subgenus Phalaenopsis, the lineage Most species of section Phalaenopsis showed constancy comprising species that have all undergone genome in 45S rDNA distribution, with two distal 45S rDNA size increases, i.e. P. amboinensis (Fig. 2N), P. bell- sites (Fig. 4G–O). In contrast, two to six 5S rDNA sites ina (Fig. 3A), P. corningiana (Fig. 3C), P. sumatrana were detected, in which two distal sites were detected (Fig. 4B), P. tetraspis (Fig. 4C), P. venosa (Fig. 4D) and in the lineage containing P. celebensis H.R.Sweet P. violacea (Fig. 4E), except for P. modesta, all pos- (Fig. 4I), P. equestris (Schauer) Rchb.f. (Fig. 4J) and sesses bimodal and asymmetrical karyotypes. In these P. lindenii Loker (Fig. 4K); four sites were detected in species, large DAPI-positive bands are mainly located the lineage containing P. amabilis Blume (four distal in the terminal regions of two large metacentric chro- sites; Fig. 4G), P. aphrodite Loker (two distal sites and mosomes and/or the long arms of large subtelocentric two proximal sites; Fig. 4H) and P. sanderiana Rchb.f. chromosomes. Only a few or no bright DAPI-positive (four distal sites; Fig. 4M); four distal sites and two bands were observed on small chromosomes of these proximal sites were detected in the lineage contain- species. In Oxalis L. (de Azkue & Martinez, 1988), ing P. philippinensis (Fig. 4L), P. schilleriana Rchb.f. Cypella Klatt and Hesperoxiphion Baker (Kenton et al., (Fig. 4N) and P. stuartiana Rchb.f. (Fig. 4O). 1990), Paphiopedilum Pfitzer (Cox et al., 1998) and Cephalanthera Rich. (Moscone et al., 2007), complex chromosomal rearrangements, such as Robertsonian fusions and fissions, and/or unequal gains or losses DISCUSSION of repetitive DNA to specific chromosomes and chromosome regions have been shown to contribute to the Karyotypes changes of chromosome size and karyotype symmetry. A consistent somatic chromosome number of 2n = 38 Our study comprising a comprehensive sampling in Phalaenopsis and other groups of the Aeridinae of taxa demonstrates that the amplification of repeti- has been reported in previous studies (Woodard, 1951; tive DNA (e.g. constitutive heterochromatin) could be Storey, 1952; Sagawa, 1962; Shindo & Kamemoto, specific to chromosomes and/or chromosome regions 1963; Storey, Kamemoto & Shindo, 1963; Kamemoto, in the different phylogenetic groups which have inde- 1965; Jones, 1967; Tara & Kamemoto, 1970; Arends & pendently undergone genome size expansion, result- Van der Laan, 1986; Aoyama, 1993). Here we report a ing in changes in chromosome size and symmetry, as reduction of chromosome number (to 2n = 34 or 36) in also reported by Kao et al. (2001) in their karyological section Aphyllae (P. honghenensis, P. lowii, P. sto- study of Phalaenopsis. bartiana, P. taenialis and P. wilsonii) and in section Esmeraldae (P. finleyi). From the phylogenetic analysis (Fig. 1), it is clear that P. finleyi is closely related to sec- Genome size evolution in Phalaenopsis tion Aphyllae, and they form a monophyletic lineage The present study has revealed that Phalaenopsis pos- sharing the character of chromosome number reduc- sesses a relative large range of genome sizes compared tion. This agrees with the classification of Christenson with many other genera in Orchidaceae (Cypripedium (2001), who considered that P. finleyi should belong being an exception; Leitch et al., 2009), with maxi- to section Aphyllae based on morphological features. mum and minimum C-values differing 6.3-fold. The Although Cribb & Schuiteman (2012) placed P. finleyi reconstruction of ancestral genome sizes inferred the in section Esmeraldae, our cytological data suggest genome size for the MRCA of subgenus Parishianae that section Aphyllae should include P. finleyi. to be 3.92 pg/1C and for subgenus Phalaenopsis to be The karyomorphology in Phalaenopsis reflects the 2.42 pg/1C (Fig. 1) with decreases and/or increases in close phylogenetic relationships of those species that genome size inferred in the different lineages in each have undergone increases in genome size (Fig. 1). In subgenus during diversification and speciation. subgenus Parishianae, the species with larger genome In subgenus Parishianae, the genome sizes of most sizes, e.g. P. honghenensis (Fig. 2A), P. pulcherrima species are higher than the reconstructed ancestral (Fig. 2I), P. gibbosa (Fig. 2K), P. lobbii (Fig. 2L) and state, suggesting a general trend towards genome size P. parishii (Fig. 2M), possess symmetrical karyotypes. expansion (Fig. 1). A feature of the growth habit of

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many species in subgenus Parishianae is that they are Beaulieu, 2008). However, species with smaller genome deciduous during the harsh dry season. Nuclear DNA sizes also occur in these Malesian islands (Christenson, content has been shown to affect cellular and organis- 2001). It is not clear whether the species with larger mal characters (e.g. nuclear volume, cell size, cell cycle, genome sizes occupy a specific ecological niche in these generation time and growth rate) and thus can influ- islands or whether other factors such as genetic drift are ence life form, development, phenology and the ecologi- involved in driving genome expansion (Lynch & Conery, cal performance of plant species, particularly in species 2003; Whitney et al., 2010). with larger genomes (reviewed by Leitch & Bennett, Apart from the genome increases noted above, the 2007; Leitch & Leitch, 2012; Greilhuber & Leitch, 2013). evolution of other Phalaenopsis spp. is predicted to In subgenus Parishianae, speciation is predicted to have been accompanied by genome decreases (Fig. 1). have occurred mostly in the Himalayas and Indochina, This is particularly notable in section Phalaenopsis whereas in subgenus Phalaenopsis it is more likely to (subgenus Phalaenopsis), species of which have the have occurred in Malesia (Yukawa et al., 2005). These smallest genome sizes so far reported and with little two regions differ significantly in their climate with variation (1.38–1.77 pg/1C). In this lineage, the genome Indochina being characterized by distinct dry and rainy sizes of all species are lower than the reconstructed seasons and Malesia having a more constant climate, ancestral state (2.42 pg/1C), suggesting genome down- with high precipitation throughout the year. Since sizing. Another example of this section is seen in sub- genome size has been shown to be broadly correlated genus Parishianae. Here, although most species have with guard cell size (Beaulieu et al., 2008), the species larger genomes (mean 1C value = 4.95 pg) than that with larger genome sizes in subgenus Parishianae are of the MRCA (3.92 pg/1C), the genome sizes of a few predicted to have larger guard cells that may show a species in this subgenus, i.e. P. appendiculata (sec- slower response to water stress compared with those tion Parishianae), P. lowii (section Aphyllae), P. finleyi species with smaller genome size and this may contrib- (section Aphyllae) and P. taenialis (section Aphyllae), ute to the reason why they shed leaves during the dry are lower, indicating genome downsizing. In section season. In relation to this, it is perhaps worth noting Aphyllae, the reduction of genome size may be due in that P. pulcherrima (section Esmeraldae) is the only part to the loss of one or two pairs of chromosomes (e.g. terrestrial Phalaenopsis spp. with an evergreen habit P. finleyi 2n = 34, P. lowii 2n = 36). among the species with larger genome sizes in subge- In angiosperms, genome size evolution has been nus Parishianae. Compared to terrestrial species, the shown to include both increases and decreases (Soltis epiphytes are likely to be under considerable water et al., 2003), which are predominantly driven by stress and it has been suggested that orchid species changes in the abundance of repetitive DNA sequences, with larger genome sizes may be restricted to a terres- e.g. tandem repeats and transposable elements which trial rather than epiphytic habit (Leitch et al., 2009). In are ubiquitous in plant genomes (Heslop-Harrison & P. pulcherrima, a shift in life-style to a terrestrial habit Schmidt, 2012). The amplification of repeat families, could represent an adaptive strategy to enable continu- typically retroelements, and the lack of DNA removal ous growth year-round in Indochina with its distinct mechanisms may result in the genome size expansion dry/rainy seasons. Possessing evergreen leaves ensures in plants (Kelly et al., 2015; Leitch & Leitch, 2012), the supply of carbohydrates year-round that would be whereas DNA removal via recombination-based pro- advantageous for enabling species to grow quickly in cesses such as unequal homologous recombination the tropics. In savanna tree species, evergreen species and/or illegitimate recombination are considered to showed greater leaf mass fraction and relative growth be important for genome downsizing (Bennetzen, Ma rate than deciduous species (Tomlinson et al., 2014). & Devos, 2005; Bennetzen & Wang, 2014). Since all In subgenus Phalaenopsis, the species with larger Phalaenopsis spp. are considered to be diploid, varia- genome size were found in the P. amboinensis– tion in genome size is caused by changes in the amount P. bellina–P. venosa–P. violacea–P. corningiana– of DNA per chromosome (chromosome size), without P. sumatrana–P. tetraspis lineage (except for significantly altering the number of chromosomes per P. modesta J.J.Sm.) and P. mannii Rchb.f. in section genome (polyploidization). Further studies are needed Polychilos compared with the ancestral genome size to compare the composition of repetitive DNA families reconstructed for the MRCA for the subgenus, indicating in different lineages of Phalaenopsis. genome size expansion. Species of the P. amboinensis– P. bellina–P. venosa–P. violacea–P. corningiana–P. sumatrana–P. tetraspis lineage are restricted to individual Variation in numbers and chromosomal Malesian islands (Andaman and Nicobar Islands, Borneo, locations of rDNA Sumatra and Sulawesi). It has been suggested that spe- In Phalaenopsis, the number of 5S and 45S rDNA sites cies with larger genome sizes may have a more restricted can vary from two to six and two to 14, respectively ecological distribution (Knight & Ackerly, 2002; Knight & (Table 1). In subgenus Parishianae, polymorphisms in

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the number and location of 45S rDNA sites are most rDNA sites (Fig. 4G, H, M) and the P. philippinensis– extensive in the species with larger genome sizes (e.g. P. schilleriana–P. stuartiana lineage has six 5S rDNA P. pulcherrima, P. gibbosa, P. lobbii and P. parishii). In P. sites (Fig. 4L, N, O). Nevertheless, overall the number lobbii and P. parishii, it is particularly notable that one and distribution of 5S rDNA loci detected by FISH in pair of chromosomes has four strong 45S rDNA sites at Phalaenopsis are generally less polymorphic than the both distal ends (Fig. 2L, M). The strong FISH signals 45S rDNA loci, a pattern also observed across many indicate high copy numbers of the 45S rDNA repeats, plant species studied with the exception of a few mono- and their presence at both chromosome ends could cots, i.e. Alstroemeria L. (Baeza, Schrader & Budahn, serve as excellent markers for chromosome identifica- 2007), Iris L. (Martinez et al., 2010), Paphiopedilum tion in these species. Furthermore, in section Polychilos, (Lan & Albert, 2011) and Tulipa L. (Mizuochi, Marasek duplication of 45S rDNA sites also occurs in the line- & Okazaki, 2007). In Paphiopedilum, 5S rDNA sites age with large genome sizes and bimodal karyotypes, i.e. were shown to be hemizygous, suggesting that gene P. amboinensis (Fig. 2N), P. bellina (Fig. 3A), P. venosa conversion via double-strand break repair events may (Fig. 4D), P. violacea (Fig. 4E), P. corningiana (Fig. 3C), P. be involved in the diversification of 5S rDNA distribu- sumatrana (Fig. 4B) and P. tetraspis (Fig. 4C). In this lin- tion in that genus (Lan & Albert, 2011). Nevertheless, eage, the multiple 45S rDNA sites are only detected at in section Phalaenopsis, polymorphic 5S rDNA sites the distal end of small chromosomes that have little or did not display hemizygosity, suggesting that the no constitutive heterochromatin. The increased genome increased number of 5S rDNA sites in some species size associated with a larger cell volume may require is more likely to be caused by transpositional events. higher rDNA transcriptional activity to maintain opti- In conclusion, our results demonstrate variations in mal cellular metabolisms through the amplification of genome size, karyotype symmetry and rDNA sites in rDNA repeats or by up-regulating transcription of indi- Phalaenopsis lineages, suggesting that evolutionary vidual rDNA copies (Prokopowich, Gregory & Crease, events leading to genome expansion and downsizing 2003). However, the transcriptional activity of rDNA have occurred independently in the different lineages. genes may be regulated epigenetically by dosage effect, Increases in genome size have been accompanied either such that only a subset of rDNA genes are functional. It by equal or by unequal additions of repetitive DNA to is also possible that some rDNA loci are non-functional chromosomes and this has resulted in changes in chromo- because of the insertion of transposable elements (Weber some size and karyotype symmetry. The diversification et al., 2013). In contrast to the above pattern, P. mannii, of rDNA loci, in terms of their number and distribution also in section Polychilos, has a larger genome size but patterns, suggests that the occurrence of chromosomal without a bimodal karyotype, and with only two distal rearrangements, the amplification of DNA sequences 45S rDNA sites (Fig. 3J). Thus, duplication of 45S rDNA and their transposition have contributed to the mobility sites may not necessarily accompany genome expansion of rDNA loci in different Phalaenopsis lineages. or perhaps elimination of 45S rDNA sites occurred during species divergence. Together, these examples illus- trate how the evolutionary events associated with rDNA ACKNOWLEDGEMENTS array mobility have occurred independently in the different lineages of section Polychilos. This work was supported by Institute of Plant and Variations in the number, position and size of rDNA Microbial Biology Academia Sinica, Taiwan, ROC, to sites between related species have been attributed to M.-C.C. and by Ministry of Science and Technology, chromosome rearrangements, such as translocations, Taiwan, ROC (NSC 102-2313-B-178-001-MY3) and inversions, duplications, deletions and transpositional National Museum of Natural Science, Taiwan, ROC, to events (Schubert & Wobus, 1985; Raskina et al., 2004, Y.-I.L. 2008; Altinkut et al., 2006). 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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. GenBank accession numbers of nrITS sequences obtained from the NCBI sequence database (National Center for Biotechnology Information, GenBank) for phylogenetic analysis in this study.

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