Botanical Journal of the Linnean Society, 2016, 181, 532–541. With 3 figures Chromosomal features of Fosterella species (Bromeliaceae, Pitcairnioideae) 1 Departamento de Gen etica, Centro de Ci^ encias Biol ogicas, Universidade Federal de Pernambuco, CEP 50670-420 Recife, PE, Brazil 2 Department of Sciences, Institute of Biology, University of Kassel, D-34132 Kassel, Germany Received 9 August 2015; revised 1 December 2015; accepted for publication 20 January 2016 Fosterella (Bromeliaceae) comprises 31 species with rosulate leaves and mostly small, whitish flowers. Previous karyological studies were restricted to chromosome counts. In the present study, chromosomal variation in Fosterella was analysed using CMA3/DAPI staining and/or fluorescence in situ hybridization (FISH) using 45S and 5S rDNA probes, generating data for nine taxa with either 2n = 50 or 100 chromosomes. A single chromosome pair containing one CMA+/DAPI band was identified in all diploid species. In the tetraploid Fosterella hatschbachii one pair had a CMA+/DAPI band, whereas the other tetraploid studied, Fosterella yuvinkae, had two pairs with proximal bands. The presence of two CMA+/DAPI pairs in F. yuvinkae may indicate a recent polyploidization event. This paper also reports the application of FISH in Bromeliaceae. FISH using 45S rDNA as the probe revealed one pair of terminal sites in most species and a co-localization with CMA+/ DAPI bands in all analysed species. The 5S rDNA sites were terminal in the tetraploid F. hatschbachii and proximal in all other species studied. Our data indicate that Fosterella species have little heterochromatin and it is largely restricted to the vicinity of the nucleolus organizer region. The data also indicate that hybridization (sometimes associated with polyploidy) has probably played an important role in the evolution of Fosterella. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 ADDITIONAL KEYWORDS: diploidization – fluorescence in situ hybridization – NOR-associated heterochromatin – polyploidy – rDNA sites. INTRODUCTION Bromeliaceae are among the major families of Neotropical plants, with c. 3300 species in 58 genera (Luther, 2012). The natural distribution area of the family ranges from the south-eastern United States south to northern Chile and Argentina. A single species is found in West Africa [Pitcairnia feliciana (A.Chev.) Harms & Mildbr.], where it probably arrived as a consequence of long-distance dispersal (Jacques-Felix, 2000; Givnish et al., 2004). Representatives of Bromeliaceae generally have showy inflorescences and leaves arranged in rosettes that often form a water and nutrient reservoir, a so-called tank (Benzing, 2000). Waterimpounding tanks not only confer tolerance to *Corresponding author. E-mail: brasileirovidal.ac@gmail.com 532 drought, but they also constitute a microenvironment inhabited by a variety of animals, such as ants, frogs, arachnids and snakes (Reitz, 1983; Benzing, 2000). According to the most recent classifications by Givnish et al. (2007, 2011), Bromeliaceae are divided into eight monophyletic subfamilies, Brocchinioideae, Lindmanioideae, Navioideae, Tillandsioideae, Hechtioideae, Puyoideae, Bromelioideae and Pitcairnioideae. Fosterella L.B.Sm. belongs to the last of these, in which it forms a well-supported monophyletic group (Rex et al., 2009; Givnish et al., 2011). The genus comprises 31 species distributed from southern Argentina to northern Peru, with a disjunction in Mexico and a centre of diversity in the Bolivian Andes (Rex et al., 2009; Wagner et al., 2013). Fosterella species are terrestrial herbs with small leaves arranged in rosettes © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 ~ VASCONCELOS1, ANA  HEVILA MENDES DE LIMA SILVA1, EMANUELLE VARAO 1 2 MARIA BENKO-ISEPPON , NATASCHA WAGNER , KURT WEISING1 and ANA CHRISTINA BRASILEIRO-VIDAL1* CHROMOSOMES OF FOSTERELLA MATERIAL AND METHODS PLANT MATERIAL Chromosome numbers were counted for 11 accessions from nine Fosterella species. Eight of these species were analysed by CMA3/DAPI staining and seven by FISH (for details see Table 1). Seeds or fixed roots were sampled from the living collection kept at the Institute of Biology, Department of Sciences, University of Kassel (Kassel, Germany), and were preserved in the germplasm collection of the Laboratory of Plant Genetics and Biotechnology, Department of Genetics, UFPE (Pernambuco, Brazil). Vouchers of the analysed accessions have been deposited in the herbaria listed in Table 1. PREPARATION AND STAINING OF METAPHASE CHROMOSOMES For mitotic chromosome preparations, root tips obtained from plants cultivated in pots or from germinated seeds were pretreated with 2 mM 8-hydroxyquinoline at 8 °C for 24 h, fixed in absolute ethanol/glacial acetic acid (3:1, v/v) for 6 h at room temperature, and stored at 20 °C until use. Fixed materials were washed three times in distilled water, followed by digestion in a 2% (w/v) cellulase (Onozuka R-10, Serva) and 20% (v/v) pectinase (Sigma-Aldrich) solution for 5 h at 37 °C. Slides were prepared according to Carvalho & Saraiva (1993), with minor modifications. After staining with DAPI solution (2 lg mL–1)/glycerol (1:1, v/v), slides were destained and fixed in ethanol/glacial acetic acid (3:1, v/v) for 30 min and transferred to absolute ethanol for 1 h, both at room temperature. After air drying, the best slides were stored at 20 °C until further experiments were performed. Chromosome staining with DAPI allows characterization of AT-rich (DAPI+) or AT-poor (DAPI–) heterochromatic regions, and CMA3 preferentially binds to GC-rich DNA (CMA3+) (Schweizer, 1976; Barros e Silva & Guerra, 2010). For CMA3/DAPI staining, air-dried slides were aged for 3 days at room temperature. Staining was then performed with CMA3 (0.5 mg mL 1, 1 h) and DAPI (2 mg mL 1, 30 min), mounted in McIlvaine’s buffer (pH 7.0)/glycerol (1:1, v/v) and stored for another 3 days (Schweizer & Ambros, 1994). After image capture, slides were destained as previously described and stored at 20 °C for use in FISH experiments. RDNA PROBES The probes for FISH were R2, a 6.5-kb fragment of the 18S–5.8S–25S rDNA repeat unit from Arabidopsis thaliana (L.) Heynh., and D2, a 400-bp fragment containing two 5S rDNA repeats from Lotus japonicus (Regel) K.Larsen (Pedrosa et al., 2002). Plasmids were isolated using the Plasmid Mini Kit (Qiagen) and © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 and mostly whitish flowers. The fruit is a dehiscent capsule that releases winged, wind-dispersed seeds upon dehiscence (Ibisch et al., 2002; Peters, 2009). Fosterella differs from closely related genera by having nude petals and anthers that join the filament at their base (Ibisch et al., 2002). Molecular phylogenetic analyses based on plastid DNA have shown that Fosterella is subdivided into six wellsupported evolutionary lineages (Rex et al., 2009; Wagner et al., 2013). To date, chromosome counts are available for only c. 10% (c. 390 species) of species of Bromeliaceae (Gitaı et al., 2014) and triple staining with the fluorochromes chromomycin A3 (CMA3), actinomycin and 40 ,6-diamidino-2-phenylindole (DAPI) has only been carried out in three species (Gitaı, Horres & Benko-Iseppon, 2005). This scarcity of data reflects the difficulty of analysing extremely small chromosomes, which in the family range from 0.21 to 2.72 lm (Zanella et al., 2012). The basic chromosome number in the vast majority of species of Bromeliaceae is x = 25, with the majority of species analysed being diploids (68% of analysed species). Nevertheless, polyploids have been reported for several genera from three of the eight subfamilies of Bromeliaceae. Tetraploids (12 species) have been observed in Tillandsioideae, and tetra- and hexaploids have been found in Bromelioideae (nine tetraploids and three hexaploids) and Pitcairnioideae (six tetraploids and two hexaploids). Further polyploids will probably be detected if additional species are analysed (Gitaı et al., 2014). In Pitcairnioideae, chromosome counts indicative of polyploidy have so far been reported for some species of Fosterella (Delay, 1947a, b; Brown & Gilmartin, 1986; Brown, Palacı & Luther, 1997) and Deuterocohnia Mez and Pitcairnia L’Her. (Gitaı et al., 2005, 2014). In Fosterella, chromosome numbers have so far been determined in just six accessions from four species, with three polyploid accessions: (1) F. penduliflora (C.H.Wright) L.B.Sm. (one diploid accession, 2n = 2x = 50; one tetraploid, 2n = 4x = 100; and one hexaploid, 2n = 6x = 150); (2) F. rusbyi (Mez) L.B.Sm. (2n = 2x = 50); (3) F. villosula (Harms) L.B.Sm. (2n = 6x = 150); and (4) F. weberbaueri (Mez) L.B.Sm. (2n = 2x = 50) (Delay, 1947a, b; Brown & Gilmartin, 1984, 1986, 1989; Brown et al., 1997). In the present work, we applied CMA3/DAPI staining of metaphase chromosome preparations from nine Fosterella species and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes to reveal additional features of infrageneric chromosomal diversity in Fosterella. In addition, we report new chromosome counts for eight species. 533 534 SILVA ET AL. Table 1. Analysed species of Fosterella with specimen numbers of permanent herbarium vouchers, phylogenetic groups (according to Rex et al., 2009; Wagner et al., 2013), ploidy (PL), chromosome numbers (2n), quantity and localization of CMA+ bands, and 45S and 5S rDNA sites PL 2n CMA+ (pair) B, WU, HEID, albicans CUZ, LPB 2x 50 1 terminal 1 terminal 1 proximal 1A/2A KAS, LPB micrantha 2x 50 1 terminal 1 terminal 1 proximal 1B/2B P. Ibisch & LPB, FR, SEL, micrantha C. Nowicki USZ, WU 98.0173 NiSch CICY, KAS micrantha 11-12 2x 50 1 terminal 1 terminal 1 proximal – 2x 50 1 terminal n.a. Specimen Herbarium F. robertreadii Ibisch & J. Peters F. christophii Ibisch, R.V asquez & J. Peters F. christophii Ibisch, R.V asquez & J. Peters F. micrantha (Lindley) L.B. Smith F. gracilis (Rusby) L.B. Smith F. floridensis Ibisch & E.Gross F. hatschbachii L.B. Smith & R.W. Read F. rusbyi (Mez) L.B.Smith F. spectabilis H. Luther F. spectabilis H.Luther F. yuvinkae Ibisch, E. Gross & Reichle Rauh 20866 NW 09.030 Group 45S rDNA 5S rDNA (pair) (pair) n.a. Figures CMA/FISH 1C/– NW 09.022 LPB, KAS penduliflora 2x 50 1 proximal 1 proximal 1 proximal 1D/2C NW 09.003 LPB, KAS rusbyi 2x 50 n.a. Leme 7100 KAS rusbyi 4x 100 1 proximal 1 proximal 1 terminal 1E/2E NW 09.009 Peters 06.0048 Peters 06.0046 Reichle P-SR1 LPB, KAS rusbyi 2x 50* 1 proximal 1 proximal 1 proximal 1F/2F FR rusbyi 2x 50 1 terminal n.a. LPB rusbyi 2x 50 1 terminal 1 terminal 1 proximal 1G/2G LPB rusbyi 4x 100 2 proximal n.a. 1 terminal n.a. n.a. n.a. –/2D – 1H/– B, Herbarium Berolinense, Botanical Garden Berlin, Germany; CICY, Herbarium of the Centro de Investigaci on Cientıfica de Yucat an, Mexico; CUZ, Vargas Herbarium of the University of Cuzco, Peru; FR, Herbarium Senckenbergianum Frankfurt/Main, Germany; HEID, Herbarium of the Botanical Garden Heidelberg, Ruprecht-Karls-University, Germany; KAS, Herbarium of the University of Kassel, Germany; LPB, Herbario Nacional de Bolivia, Universidad Mayor de San Andr es, La Paz; SEL, Marie Selby Botanical Gardens, Sarasota, Florida; USZ, Herbario del Oriente Boliviano, Museo de Historia Natural Noel Kempff Mercado, Universidad de Santa Cruz (Aut onoma Gabriel Ren e Moreno); VASQ, Herbarium Vasquezianum, Santa Cruz, Bolivia (private collection Roberto Vasqu ez); WU, Herbarium of the University of Vienna, Institute of Botany, Austria; n.a., not analysed. *Chromosome number reported by Brown & Gilmartin (1984, 1989). labelled by nick translation with digoxigenin-11-dUTP and biotin-11-dUTP (both from Roche Diagnostics) for 45S and 5S rDNA, respectively. The manufacturers’ instructions for the kits were followed throughout. FLUORESCENT IN SITU HYBRIDIZATION FISH pretreatment washes were based on PedrosaHarand et al. (2009), in which the 77% stringency wash was performed with 0.19 saline sodium citrate (SSC) at 42 °C. Chromosome and probe denaturation and detection were performed according to HeslopHarrison et al. (1991). The hybridization mixture, containing 50% (v/v) formamide, 29 SSC, 10% (w/v) dextran sulphate and 5–10 ng lL 1 of that probe, was denatured at 75 °C for 10 min. Each slide received 10 lL of the hybridization mixture and was hybridized for at least 48 h at 37 °C. Digoxigenin- or © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 Species CHROMOSOMES OF FOSTERELLA biotin-labelled probes were detected using conjugated anti-digoxigenin rhodamine (Roche Diagnostics) and Alexa Fluor-conjugated streptavidin (Invitrogen), respectively, in a solution containing 1% (w/v) bovine serum albumin. Preparations were counterstained and mounted with 2 lg mL 1 DAPI in Vector’s Vectashield (1:1, v/v). ANALYSIS Images of the best preparations were acquired using a Leica DMLB epifluorescence microscope and a Leica DFC 340FX camera with the Leica CW4000 software. Images were pseudo-coloured and optimized for contrast and brightness with the Adobe Photoshop CS4 software (Adobe Systems). RESULTS A summary of the results obtained in the present work is provided in Table 1. Chromosome counts of the eight new analysed Fosterella species revealed six diploid species (2n = 2x = 50) and two tetraploid species (F. hatschbachii L.B.Sm. & R.W.Read and F. yuvinkae Ibisch, E.Gross & Reichle, 2n = 4x = 100). Additionally, 2n = 2x = 50 chromosomes was found for F. rusbyi, as previously described by Brown & Gilmartin (1984, 1989) (Supporting Information, Fig. S1). Representative results obtained by CMA3/DAPI staining (eight species) and FISH (seven species) are shown in Figures 1 and 2, respectively. CMA3/DAPI staining allowed the identification of one pair of CMA+/DAPI (i.e. GC-rich heterochromatin) bands in all diploid species (Fig. 1A–D, F, G), mostly in the terminal region. The exceptions are F. gracilis (Rusby) L.B.Sm. and F. rusbyi, which had a pair of proximal bands (Fig. 1D, Table 1). With regard to the tetraploid species, in F. hatschbachii one pair of CMA+/DAPI bands was evident in the proximal region (Fig. 1E), whereas in F. yuvinkae two proximal pairs were observed (Fig. 1H). For the latter species, different chromosome sizes and band positions could be discerned (considering their distance from the telomere). The FISH procedure revealed a single chromosome pair carrying 45S and 5S rDNA in all analysed species, including the tetraploid F. hatschbachii (Fig. 2E). Furthermore, 45S rDNA sites were co-localized with CMA+/DAPI bands (compare Figs 1B and 2B, 1E and 2E, and 1G and 2G). These results indicate that in Fosterella CMA+/DAPI heterochromatin is exclusively associated with the nucleolus organizer region (NOR). The 45S rDNA signals were generally located in terminal regions, except for F. gracilis (2n = 50; Fig. 2C), F. hatschbachii (2n = 100; Fig. 2E) and F. rusbyi (2n = 50; Fig. 2F), which displayed hybridization signals at proximal sites. In contrast, the 5S rDNA sites were generally located in the proximal chromosome regions (Fig. 2A–D, F, G), with the exception of F. hatschbachii (Fig. 2E) which displayed 5S rDNA sites in terminal regions. DISCUSSION Until now, there have been only a few cytogenetic studies of the genus Fosterella, with previous reports restricted to counts of meiotic bivalents or mitotic chromosomes of four species (six accessions) (Delay, 1947a, b; Brown & Gilmartin, 1984, 1986, 1989; Brown et al., 1997). Chromosome counts for eight additional species are provided in the present work (six diploids and two tetraploids), supporting previous studies concerning the predominance of diploid species and confirming x = 25 as the basic chromosome number for the group (Brown & Gilmartin, 1984, 1986, 1989). Fosterella species have high numbers of small chromosomes (2n = 50, 100, 150), and this severely complicates counting them and analysis by classical and molecular cytogenetic techniques, such as FISH. The latter technique was applied for the first time to chromosomes of Bromeliaceae in this work. Studies with base-specific fluorochromes have been performed only in three species of Bromelioideae so far. For two of them [Greigia sphacelata (Ruiz & Pav on) Regel and Ochagavia litoralis (Philippi) Zizka, Trumpler & Zoellner, both with 2n = 50], the presence of one pair of CMA+/DAPI bands was reported (Gitaı et al., 2005), which is the same as the CMA3/DAPI data obtained for the diploid species in this work. In contrast, two pairs of CMA+/DAPI bands were observed in metaphases of the diploid Aechmea bromeliifolia (Rudge) Baker (Gitaı et al., 2005), whereas in the present study two pairs of CMA+/DAPI bands were observed only in the tetraploid F. yuvinkae. Two pairs are actually expected in tetraploid species (2n = 4x = 100), but this feature did not hold for the other tetraploid studied, F. hatschbachii, which only had a single pair of chromosomes carrying CMA+/DAPI bands. The FISH technique revealed that the number of rDNA sites in Fosterella is well conserved, as all analysed species had one pair of chromosomes carrying 45S rDNA and one pair carrying 5S rDNA for diploid cells. The only exception was F. hatschbachii, which had one pair of sites for each marker for tetraploid cells. A similar conservation of rDNA site number was observed when diploid and polyploid subspecies of Paspalum quadrifarium Lam. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 DATA 535 536 SILVA ET AL. B C D E F G H Figure 1. Metaphase chromosomes of diploid (A–D, F–G) and tetraploid (E, H) Fosterella species stained with CMA (yellow) and counterstained with DAPI (pseudocoloured in grey). A, F. robertreadii; B, F. christophii; C, F. micrantha; D, F. gracilis; E, F. hatschbachii; F, F. rusbyi; G, F. spectabilis; H, F. yuvinkae. Arrows point to CMA3+ bands. Dotted lines indicate distended secondary constrictions in H. The bar in H represents 5 lm. (Poaceae; Vaio et al., 2005) and species of Daucus L. (Apiaceae; Iovene et al., 2008) and Iris L. (Iridaceae; Lim et al., 2007) were compared with each other. The results obtained here for the tetraploid F. hatschbachii could perhaps be explained by an ongoing diploidization process, possibly following silencing of some of the duplicated genes (Soltis & Soltis, 1999; Adams, Percifield & Wendel, 2004; see below). If this explanation is correct, then the observation that F. yuvinkae has two pairs of CMA+ bands suggests it might be a more recent polyploid than F. hatschbachii. This suggestion is reinforced by the observation that in F. yuvinkae both proximal CMA+ bands were observed on chromosome pairs that differ © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 A CHROMOSOMES OF FOSTERELLA A B 537 C E F G Figure 2. Fluorescence in situ hybridization of 45S (green) and 5S (red) rDNA probes in metaphase chromosomes of Fosterella species counterstained with DAPI (pseudocoloured in grey). A, F. robertreadii; B, F. christophii; C, F. gracilis; D, F. floridensis; E, F. hatschbachii; F, F. rusbyi; G, F. spectabilis. Arrows and arrowheads indicate 45S and 5S rDNA sites, respectively. The bar in G represents 5 lm. in size. This could in turn indicate that F. yuvinkae arose from a hybridization step followed by allopolyploidy. If so, then this represents the first clear docu- mented example of allopolyploidy in Bromeliaceae that is supported by karyological evidence. Mirzaghaderi, Houben & Badaeva (2014) observed a © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 D 538 SILVA ET AL. similar size polymorphism for 45S rDNA sites in the allotetraploid Aegilops triuncialis Ledb. ex Trautv. (Poaceae) and suggested that the signal size reduction could be the result of a suppression of NORs after hybridization. It is well known that polyploid plants have a tendency towards returning to their original monoploid DNA content during evolution. This process, named diploidization (Wolfe, 2001; Leitch & Bennett, 2004; Leitch et al., 2008), has been investigated in some detail in soybean [Fabaceae, Glycine max (L.) Merr; Schmutz et al., 2010]. Apparently, soybean has a partially diploidized tetraploid genome, which is the product of a diploid ancestor (n = 11) that later underwent aneuploidy (n = 10), polyploidization (n = 20) and diploidization (n = 20) (Shultz et al., 2006; Schlueter et al., 2007; Schmutz et al., 2010). Diploidization has been observed most often in polyploids that formed some time ago and hence have had a sufficiently long time for evolutionary divergence [e.g. see evidence from FISH with a 45S rDNA probe in Avena L. (Linares et al., 1996), Nicotiana L. (Kovarik et al., 2008) and Aristolochia L. (Berjano et al., 2009)]. In allopolyploids of Nicotiana, silencing of 45S rDNA from one parent was confirmed to have taken place during evolution, pointing to gene silencing as the major agent in the diploidization process in the group (Kovarik et al., 2008). Additionally, if transposable elements are found to be associated with the rDNA sites, it is possible that they may contribute to the elimination of rDNA sequences. Such a mechanism has previously been suggested as a possible explanation of how rDNA sites were lost in an allotetraploid species of Arabidopsis Heynh. in Holl & Heynh. (Pontes et al., 2004) following polyploidization. The chromosomal localization of CMA+/DAPI bands and 45S rDNA sites shows a certain preference for terminal regions, with some exceptions. In angiosperms, a tendency has been observed towards the localization of 45S rDNA sites in the terminal region of the short arm, generally occupying the whole short arm in acrocentric chromosomes, and of 5S rDNA sites in proximal regions (Roa & Guerra, 2012). A hypothesis for the preferentially terminal position of the 45S rDNA was suggested in an © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 Figure 3. Chromosomal positions of CMA+ bands and 45S and 5S rDNA sites for the studied Fosterella species, mapped onto the proposed phylogenetic tree for the genus (modified from Wagner et al., 2013). Blocks in yellow indicate CMA+/ DAPI bands, blocks in green indicate 45S rDNA sites co-localized with CMA+/DAPI bands, and blocks in red indicate 5S rDNA sites. CHROMOSOMES OF FOSTERELLA CONCLUSIONS In view of the data presented from both the current work and the literature, the karyological features in Fosterella can be summarized as: (1) a prevalence for speciation at the diploid level, although polyploids can also arise (e.g. F. yuvinkae which may have arisen through allopolyploidy and hybridization); (2) 2n = 50, 100, 150 corroborating x = 25 as the basic chromosome number for Fosterella; (3) low amounts of GC-rich heterochromatin (CMA+/DAPI ) associated with the NORs; (4) presence of a single pair of chromosomes bearing 45S rDNA sequences in diploid species, whereas polyploid species may contain either one or two 45S rDNA sites, depending on the state of ongoing re-diploidization; and (5) variation in the chromosomal position of rDNA marker sequences, with a prevalence for terminal positions for the 45S rDNA repeats (except in F. hatschbachii, F. gracilis and F. rusbyi) and proximal positions for 5S rDNA repeats (except for F. hatschbachii). Taken together, CMA/DAPI staining and FISH with 45S and 5S rDNA markers appear to be useful approaches for tracking karyoevolution in Fosterella, facilitating, for example, the identification of putative hybrids. ACKNOWLEDGEMENTS e and We thank colleagues Diego S. B. Pinang Rodrigo C. G. Oliveira for help during the collection of seeds and root tips. We also thank CNPq (Con- selho Nacional de Desenvolvimento Cientıfico e Tecogico, Brazil), DAAD (German Academic nol ~o de AperExchange Service), CAPES (Coordenacßa feicßoamento de Pessoal de Nıvel Superior, Brazil – ~o de PROBRAL Program) and FACEPE (Fundacßa  Pesquisa do Estado de Pernambuco, BraAmparo a zil) for financial support and fellowships. REFERENCES Adams K.L., Percifield R., Wendel J.F. 2004. Organ-specific silencing of duplicated genes in a newly synthesized allotetraploid. Genetics 168: 2217–2226. Barros e Silva A.E., Guerra M. 2010. The meaning of DAPI bands observed after C-banding and FISH procedures. Biotechnic and Histochemistry 85: 115–125. Benzing D.H. 2000. Bromeliaceae: profile of an adaptive radiation. Cambridge: Cambridge University Press. Berjano R., Roa F., Talavera S., Guerra M. 2009. Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species based on the distribution of CMA/DAPI bands and 5S and 45S rDNA sites. Plant Systematics and Evolution 280: 219–227. Brown G.K., Gilmartin A.J. 1984. Chromosome number reports LXXXV. Taxon 33: 756–760. Brown G.K., Gilmartin A.J. 1986. Chromosomes of the Bromeliaceae. Selbyana 9: 88–93. Brown G.K., Gilmartin A.J. 1989. Chromosome numbers in Bromeliaceae. American Journal of Botany 76: 657–665. Brown G.K., Palacı C.A., Luther H.E. 1997. Chromosome numbers in Bromeliaceae. Selbyana 18: 85–88. Carvalho C.R., Saraiva L.S. 1993. An air drying technique for maize chromosomes without enzymatic maceration. Biotechnic and Histochemistry 68: 142–145. Delay C. 1947a. Recherches sur la structure des noyaux quiescentes chez les phanerogames. Revue de Cytologie et de Cytophysiologie V eg etales 9: 169–222. Delay C. 1947b. Recherches sur la structure des noyaux quiescences chez les phanerogames. Revue de Cytologie et de Cytophysiologie V eg etales 10: 103–229. Gitaı J., Horres R., Benko-Iseppon A.M. 2005. Chromosomal features and evolution of Bromeliaceae. Plant Systematics and Evolution 253: 65–80. Gitaı J., Paule J., Zizka G., Schulte K., Benko-Iseppon A.M. 2014. Chromosome numbers and DNA content in Bromeliaceae: additional data and critical review. Botanical Journal of the Linnean Society 176: 349–368. Givnish T.J., Barfuss M.H.J., Benjamin E.E.V., Riina R., Schulte K., Horres R., Gonsiska P.A., Jabaily R.S., Crayn D.M., Smith J.A.C., Winter K., Brown G.K., Evans T.M., Holst B.K., Luther H., Till W., Zizka G., Berry P.E., Sytsma K.J. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight locus plastid phylogeny. American Journal of Botany 98: 872–895. Givnish T.J., Millam K.C., Berry P.E., Sytsma K.J. 2007. Phylogeny, adaptive radiation, and historical biogeography © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 analysis with Arabidopsis (Vaillant et al., 2007; see also McStay & Grummt, 2008). According to such work, the organization of the chromosome territories indicates that the telomeres are preferably situated around the nucleolus. Therefore, the localization of the 45S rDNA might be related to chromosome organization during interphase. In Fig. 3, we have mapped the CMA3/DAPI staining and FISH data generated in the present study onto the phylogenetic tree for Fosterella reported by Wagner et al. (2013). The 45S rDNA sites are terminal in all species except F. hatschbachii and F. rusbyi (which belong to the rusbyi group in clade IV) and F. gracilis (which belongs to the distantly related penduliflora group in clade I). Although we cannot draw any firm conclusions on the basis of the present dataset, we predict that the 45S rDNA sites were located in a terminal position in the ancestral karyotype, whereas their localization in proximal regions is a derived character that has emerged at least twice independently. 539 540 SILVA ET AL. Peters J. 2009. Revision of the genus Fosterella L.B.Sm. (Bromeliaceae). PhD Thesis, University of Kassel, Germany. Pontes O., Neves N., Silva M., Lewis M.S., Madlung A., Comai L., Viegas W., Pikaard C.S. 2004. Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proceedings of the National Academy of Sciences of the United States of America 101: 18240–18245. aria – bromelia endaceas e a mal Reitz R. 1983. Bromeli ^emica. In: Reitz R, ed. Flora Ilustrada Catarinense. Itajaı: Herb ario Barbosa Rodrigues. Rex M., Schulte K., Zizka G., Peters J., V asquez R., Ibisch P., Weising K. 2009. Phylogenetic analysis of Fosterella L.B.Sm. (Pitcairnioideae, Bromeliaceae) based on four chloroplast DNA regions. Molecular Phylogenetics and Evolution 51: 472–485. Roa F., Guerra M. 2012. Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications. BMC Evolutionary Biology 12: 225. Schlueter J.A., Lin J.Y., Schlueter S.D., Vasylenko-Sanders I.F., Deshpande S., Yi J., O’Bleness M., Roe B.A., Nelson R.T., Scheffler B.E., Jackson S.A., Shoemaker R.C. 2007. Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing. BMC Genomics 8: 330. Schmutz J., Cannon S.B., Schlueter J., Ma J., Mitros T., Nelson W., Hyten D.L., Song Q., Thelen J.J., Cheng J., Xu D., Hellsten U., May G.D., Yu Y., Sakurai T., Umezawa T., Bhattacharyya M.K., Sandhu D., Valliyodan B., Lindquist E., Peto M., Grant D., Shu S., Goodstein D., Barry K., Futrell-Griggs M., Abernathy B., Du J., Tian Z., Zhu L., Gill N., Joshi T., Libault M., Sethuraman A., Zhang X.-C., Shinozaki K., Nguyen H.T., Wing R.A., Cregan P., Specht J., Grimwood J., Rokhsar D., Stacey G., Shoemaker R.C., Jackson S.A. 2010. Genome sequence of the paleopolyploid soybean. Nature 463: 178–183. Schweizer D. 1976. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58: 307–324. Schweizer D., Ambros P.F. 1994. Chromosome banding. Stain combinations for specific regions. Methods in Molecular Biology 29: 97–112. Shultz J.L., Kurunam D., Shopinski K., Iqbal M.J., Kazi S., Zobrist K., Bashir R., Yaegashi S., Lavu N., Afzal A.J., Yesudas C.R., Kassem M.A., Wu C., Zhang H.B., Town C.D., Meksem K., Lightfoot D.A. 2006. The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max. Nucleic Acids Research 34: D758–D765T. Soltis D.E., Soltis P.S. 1999. Polyploidy: recurrent formation and genome evolution. Trends in Ecology & Evolution 14: 348–352. Vaillant I., Tutois S., Cuvillier C., Schubert I., Tourmente S. 2007. Regulation of Arabidopsis thaliana 5S rRNA genes. Plant and Cell Physiology 48: 745–752. Vaio M., Speranza P., Valls J.F., Guerra M., Mazzella C. 2005. Localization of the 5S and 45S rDNA sites and © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 of Bromeliaceae inferred from ndhF sequence data. Aliso 23: 3–26. Givnish T.J., Milliam T.M., Evans T.M., Hall J.C., Berry P.E., Terry R.G. 2004. Ancient vicariance or long-distance dispersal? Inferences about phylogeny and South American–African disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. International Journal of Plant Sciences 165: 35–54. Heslop-Harrison J.S., Schwarzacher T., AnamthawatJ onsson K., Leitch A.R., Shi M. 1991. In situ hybridization with automated chromosome denaturation. Technique 3: 109–115. Ibisch P.L., V asquez R., Gross E., Kr€ omer T., Rex M.. 2002 Novelties in Bolivian Fosterella (Bromeliaceae). Selbyana 23: 204–219. Iovene M., Grzebelus E., Carputo D., Jiang J., Simon P.W. 2008. Major cytogenetic landmarks and karyotype analysis in Daucus carota and other Apiaceae. American Journal of Botany 95: 793–804. Jacques-F elix H. 2000. The discovery of a bromeliad in Africa: Pitcairnia feliciana. Selbyana 21: 118–124. Kovarik A., Dadejova M., Lim Y.K., Chase M.W., Clarkson J.J., Knapp S., Leitch A.R. 2008. Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics. Annals of Botany 101: 815–823. Leitch I.J., Bennett M.D. 2004. Genome downsizing in polyploid plants. Biological Journal of the Linnean Society 82: 651–663. Leitch I.J., Hanson L., Lim K.Y., Kovarik A., Chase M.W., Clarkson J.J., Leitch A.R. 2008. The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Annals of Botany 101: 805–814. Lim K.Y., Matyasek R., Kovarik A., Leitch A. 2007. Parental origin and genome evolution in the allopolyploid Iris versicolor. Annals of Botany 100: 219–224. Linares C., Gonz alez J., Ferrer E., Fominaya A. 1996. The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S–5.8S–26S rDNA and a C genome specific DNA sequence in the genus Avena. Genome 39: 535–542. Luther H.E. 2012. An alphabetic list of bromeliad binomials, 13th edn. Sarasota, FL: The Sarasota Bromeliad Society & Marie Selby Botanical Gardens. McStay B., Grummt I. 2008. The epigenetics of rRNA genes: from molecular to chromosome biology. Annual Review of Cell and Developmental Biology 24: 131–157. Mirzaghaderi G., Houben A., Badaeva E.D.. 2014 Molecular–cytogenetic analysis of Aegilops triuncialis and identification of its chromosomes in the background of wheat. Molecular Cytogenetics 7: 91. Pedrosa A., Sandal N., Stougaard J., Schweizer D., Bachmair A. 2002. Chromosomal map of the model legume Lotus japonicus. Genetics 161: 1661–1672. Pedrosa-Harand A., Kami J., Geffroy V., Gepts P., Schweizer D. 2009. Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species. Chromosome Research 17: 405–417. CHROMOSOMES OF FOSTERELLA cpDNA sequence analysis in species of the quadrifaria group of Paspalum (Poaceae, Paniceae). Annals of Botany 96: 191–200. Wagner N., Silvestro D., Brie D., Ibisch P.L., Zizka G., Weising K., Schulte K. 2013. Spatio-temporal evolution of Fosterella (Bromeliaceae) in the Central Andean biodiversity hotspot. Journal of Biogeography 40: 869–880. 541 Wolfe K.H. 2001. Yesterday’s polyploids and the mystery of diploidization. Nature Reviews Genetics 2: 333–341. Zanella C.M., Janke A., Palma-Silva C., Kaltchuk-Santos E., Pinheiro F.G., Paggi G.M., Soares L.E.S., Goetze M., B€ uttow M.V., Bered F. 2012. Genetics, evolution and conservation of Bromeliaceae. Genetics and Molecular Biology 35: 1020–1026. Additional Supporting Information may be found in the online version of this article: Figure S1. Metaphase chromosomes of nine Fosterella species. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 181, 532–541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/181/3/532/2707801 by guest on 07 June 2020 SUPPORTING INFORMATION