Reconstruction of ancestral genome size in Pitcairnioideae (Bromeliaceae): what can genome size tell us about the evolutionary history of its five genera?

Reconstruction of ancestral genome size in Pitcairnioideae (Bromeliaceae): what can genome size... Abstract We expand the genome size (GS) database for Bromeliaceae, specifically for subfamily Pitcairnioideae, and verify whether GS can provide information on the diversification of the five genera in this subfamily. We also provide a phylogenetic perspective on GS evolution in the subfamily and reconstruct the ancestral state for this character. We show that the evolutionary path of GS from the origin of angiosperms to the origin of Pitcairnioideae agrees with the proportional model of GS evolution. Furthermore, we propose that the high phenotypic diversity that is found across Bromeliaceae and that is well represented in Pitcairnioideae is both correlated with high rates of GS evolution of the species and associated with a short period of diversification. The paper also highlights the value of flow cytometry as a rapid and reliable technique for generating GS data which can be analysed in conjunction with other molecular and morphological data to help elucidate patterns of evolution and phylogenetic relationships within this family. INTRODUCTION Taxon delimitation is a fundamental step for conservation, studying evolution and undertaking systematic studies of an organism (Sites & Marshall, 2003, 2004). There is a consensus that species should correspond to independent evolutionary lineages (Wilson, 1978; Duminil & Michele, 2009; Padial et al., 2010). The use of different types of characters (e.g. morphological, physiological, molecular or karyotypic) in a given taxon can help to provide an accurate classification (Dayrat, 2005; Schlick-Steiner et al., 2010; Cristiano, Cardoso & Fernandes-Salomão, 2013). Alternatively, using the same character in a large number of taxa provides opportunities for studying the evolution of that character. The evolution of a particular character may even help in understanding the evolutionary patterns and processes underpinning phylogenetic relationships among existing organisms (Le Quesne, 1974; Maddison & Maddison, 1989). Bromeliaceae, a predominantly Neotropical family (Benzing, 2000), comprising 73 genera and nearly 3600 species (Givnish et al., 2011; Butcher & Gouda, cont. updated), are considered to be a natural group. The monophyly of Bromeliaceae has been consistently confirmed in several studies (Stevenson & Loconte, 1995; Chase et al., 1995, 2000; Givnish et al., 2011). Givnish et al. (2007) suggested that a recent diversification of modern lineages of Bromeliaceae occurred c. 19 Mya. These authors inferred that such lineages first appeared in the Guayana Shield, northern South America, and then spread centripetally throughout the New World. In terms of chromosome number, most species present the basic number x = 25, with few exceptions (Gitaí, Horres & Benko-Iseppon, 2005; Ceita et al., 2008; Gitaí et al., 2014; Nunes & Clarindo, 2014). Available data also suggest that species of Bromeliaceae have small genome sizes (GSs), as their 2C-values range from 0.6 pg to 2.52 pg ( x¯ = 1.18 ± 0.37) (Angiosperm DNA C-values Database; Bennett & Leitch, 2012; 1 pg = 978 Mbp). The subfamilies and genera of Bromeliaceae have frequently been questioned and changed over the last few decades (Grant, 1992, 1993, 1996; Gouda, 1994; Givnish et al., 2004, 2007), as most genera have poor morphological delimitation and low molecular differentiation (Escobedo-Sarti et al., 2013). Pitcairnioideae, one of these subfamilies, comprise the genera Pitcairnia L’Hér. (405 species), Fosterella L.B.Sm. (31 species), Dyckia Schult. & Schult.f. (168 species), Encholirium Mart. ex Schult. & Schult.f. (29 species) and Deuterocohnia Mez (18 species) (Butcher and Gouda, cont. updated; BFG, 2015; Forzza, 2017). The first two genera exhibit mesic morphological and physiological characteristics, whereas the other three exhibit xeric morphological and physiological characteristics and belong to a monophyletic group informally known as ‘the xeric clade’ of Pitcairnioideae (Givnish et al., 2011; Santos-Silva et al., 2013; Schütz et al., 2016). The relationships between the genera of Pitcairnioideae have been questioned and studied in recent years. Rex et al. (2009) presented a multilocus plastid DNA phylogenetic analysis of Fosterella, showing it to be monophyletic, but infrageneric relationships in other genera of the subfamily remained unclear. Similar results were reported by Schütz (2011) in a taxonomic revision based on plastid and nuclear DNA data for Deuterocohnia. The results showed low genetic variability in the genus, resulting in low-resolution trees and networks. A phylogenetic analysis by Saraiva, Mantovani & Forzza (2015), based on morphological characters alone, supported the monophyly of Pitcairnia s.l. and agreed with previous findings (Terry, Brown & Olmstead, 1997; Givnish et al., 2004, 2007, 2011; Horres et al., 2007), but their analysis did not resolve all species relationships within the genus. These relationships also remained unresolved in the molecular phylogenetic analysis of Schütz et al. (2016), as did the delimitation of Dyckia and Encholirium. M. N. Moura et al. (unpubl. data) conducted a more comprehensive molecular and morphological phylogenetic analysis of Encholirium, which supports the idea of the genus being paraphyletic and corroborates the hypothesis regarding the recent divergence of Pitcairnioideae (the crown age of Pitcairnioideae was 11.8 Mya according to Givnish et al., 2011). To study relationships between species that have recently diversified, DNA sequences, commonly used in phylogenetic studies, may not have had enough time to accumulate detectable differences to resolve phylogenetic relationships (Givnish et al., 2011). On the other hand, abrupt changes in nuclear DNA content can occur over short periods of time (from one generation to another) as a result of numerical (euploidy and/or aneuploidy) and/or structural (deletion, inversion, duplication and/or translocation) alterations. These changes are common in many lineages of plants and are considered to be key factors in the evolution of the genomes of several species (e.g. Bennett & Smith, 1976; Kunkel, 1990; Soltis & Soltis, 2009; Campos et al., 2011; Lee, Chang & Chung, 2011; Lepers-Andrzejewski et al., 2011; Szadkowski et al., 2011; Chester et al., 2012). According to Doležel, Greilhuber & Suda (2007), flow cytometry (FCM) is a fast and reliable method to estimate GS in plants. Information on nuclear GS in Bromeliaceae has toadied in studies regarding their taxonomy, evolution, genetic diversity and reproductive biology (Ebert & Till, 1997; Ramírez-Morillo & Brown, 2001; Sgorbati et al., 2004; Favoreto et al., 2012). Nevetheless, given the diversity of the family and ongoing taxonomic problems, as outlined above, there is still insufficient GS data available (< 3% for species of Bromeliaceae and < 7% for species of Pitcairnioideae), and hence our understanding of related evolutionary processes is poorly understood (Gitaí et al., 2014). In the present study, we aim to expand the GS database for Bromeliaceae, specifically Pitcairnioideae, and examine whether GS data can help to provide insights into the diversification of the five genera in the subfamily. We also provide a phylogenetic perspective on GS evolution in the subfamily and reconstruct the ancestral state of this character. MATERIAL AND METHODS Genome size estimations Taxon sampling and flow cytometry FCM analyses were carried out at the Laboratório de Genética da Universidade Federal de Juiz de Fora (UFJF). Leaf samples of 53 species of the five genera of Pitcairnioideae were collected from the living collection at the Jardim Botânico do Rio de Janeiro: one Deuterocohnia sp., 17 Dyckia spp., 16 Encholirium spp., one Fosterella sp. and 18 Pitcairnia spp. Vouchers have been deposited in herbarium RB (Jardim Botânico do Rio de Janeiro) and herbarium SPF (Universidade de São Paulo) under the numbers presented in Table 1. The nuclear DNA content of three samples of each studied species was measured using the DNA 2C-value of Pisum sativum L. as an internal standard (9.09 pg; Doležel et al., 1998). To obtain a suspension of nuclei, 1.0 cm2 leaf tissue of the standard and each sample were simultaneously chopped in 1.0 mL cold LB01 buffer (Doležel, Binarová & Lucretti, 1989) with the aid of a cutting blade. The suspension of nuclei was subsequently filtered through 30-µm nylon mesh. The solution was supplemented with 5.0 µL of RNAse at a concentration of 100 µg mL−1 and then stained with 50 µL propidium iodide (PI) solution at a concentration of 1 mg mL−1. The samples were stored in the dark and analysed within 1 h of preparation. Table 1. Genome sizes estimated for all studied specimens, the mean calculated for each species, standard deviation and herbarium voucher Species  2C (pg)  Reference  Voucher  Deuterocohnia longipetala (Baker) Mez  0.74  Bennett and Leitch (2011)  –  Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm.  1.73 ± 0.021  Gitaí et al. (2014)  Leg. Nr. 130007†  Deuterocohnia meziana Kuntze ex Mez  1.20 ± 0.02  This study  RB 382284*  Deuterocohnia schreiteri A.Cast.  0.8  Bennett and Leitch (2011)  –  Dyckia brevifolia Baker  2.01 ± 0.08  This study  RB 571588*  Dyckia choristaminea Mez  1.78 ± 0.02  This study  RB 434135*  Dyckia consimilis Mez  2.17 ± 0.01  This study  RB 673612*  Dyckia distachya Hassler  1.86 ± 0.06  This study  RB 593320*  Dyckia estevesii Rauh  1.6  Bennett and Leitch (2011)  –  Dyckia floribunda Griseb.  1.58  Bennett and Leitch (2011  –  Dyckia granmogulensis Rauh  2.18 ± 0.01  This study  RB 458314*  Dyckia ibiramensis Reitz  2.13 ± 0.08  This study  RB 571595*  Dyckia maritima Baker  2.07 ± 0.06  This study  RB 1230313*  Dyckia marnier-lapostollei L.B.Sm.  1.95 ± 0.02  This study  RB 547282*  Dyckia minarum Mez  1.88 ± 0.05  This study  RB 377972*  Dyckia monticola L.B.Sm. & Reitz  2.08 ± 0.00  This study  RB 329151*  Dyckia pseudococcinea L.B.Sm.  2.10 ± 0.04  This study  RB 723296*  Dyckia pulquinensis Wittm.  2.38 ± 0.45  This study  RB 588007*  Dyckia reitzii L.B.Sm.  1.66 ± 0.00  This study  RB 583483*  Dyckia saxatilis Mez  1.88 ± 0.12  This study  RB 462377*  Dyckia sthreliana H.Büneker & R.Pontes  1.80 ± 0.03  This study  RB 595146*  Dyckia tenebrosa Leme & H.Luther  2.13 ± 0.01  This study  RB 484461*  Dyckia tuberosa (Vell.) Beer  1.82 ± 0.08  This study  RB 594245*  Encholirium agavoides Forzza & Zappi  2.11 ± 0.01  This study  RB 504154*  Encholirium biflorum (Mez) Forzza  1.40 ± 0.04  This study  RB 583280*  Encholirium ctenophyllum Forzza & Zappi  2.90 ± 0.06  This study  RB 504157*  Encholirium diamantinum Forzza  1.92 ± 0.04  This study  RB 458335*  Encholirium cf. diamantinum Forzza  2.05 ± 0.06  This study  RB 637204*  Encholirium gracile L.B.Sm.  1.56 ± 0.02  This study  RB 570910*  Encholirium heloisae (L.B.Sm.) Forzza & Wand.  1.94 ± 0.11  This study  RB 563567*  Encholirium horridum L.B.Sm.  1.57 ± 0.02  This study  RB 462618*  Encholirium irwinii L.B.Sm.  1.74  Bennett & Leitch (2011)  –  Encholirium irwinii L.B.Sm.  1.94 ± 0.03  This study  SPF 131942§  Encholirium luxor L.B.Sm. & R.W.Read  2.02 ± 0.03  This study  RB 391473*  Encholirium magalhaesii L.B.Sm.  1.94 ± 0.01  This study  RB 670779*  Encholirium pedicellatum (Mez) Rauh  1.69 ± 0.02  This study  RB 670694*  Encholirium pulchrum Forzza, Leme & O.B.C.Ribeiro  1.83 ± 0.01  This study  RB 504202*  Encholirium scrutor (L.B.Sm.) Rauh  1.96 ± 0.04  This study  RB 563568*  Encholirium spectabile Mart. ex Schult. & Schult.f.  2.16 ± 0.01  This study  RB 331722*  Encholirium subsecundum (Baker) Mez  2.02 ± 0.13  This study  RB 747925*  Fosterella penduliflora (C.H.Wright) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella villosula (Harms) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella windischii L.B.Sm. & R.W.Read  0.91 ± 0.01  This study  RB 452098*  Pitcairnia albiflos Herb.  1.39 ± 0.05  This study  RB 498556*  Pitcairnia andreana Linden  1.3  Bennett & Leitch (2011)  –  Pitcairnia angustifolia Aiton  1.06  Bennett & Leitch (2011)  –  Pitcairnia aphelandriflora Lem.  1.24  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.2  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.29 ± 0.013  Gitaí et al. (2014_  Leg. Nr. 16095†  Pitcairnia aureobrunnea Rauh  1.12  Bennett & Leitch (2011)  –  Pitcairnia azouryi Martinelli & Forzza  1.37 ± 0.10  This study  RB 484459*  Pitcairnia barbatostigma Leme & A.P.Fontana  1.26 ± 0.02  This study  RB 462404*  Pitcairnia bradei Markgr.  1.62 ± 0.09  This study  RB 586285*  Pitcairnia burchellii Mez  1.41 ± 0.02  This study  RB 526713*  Pitcairnia burle-marxii Braga & Sucre  1.43 ± 0.13  This study  RB 509698*  Pitcairnia cardenasii L.B.Sm.  1.02  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.22  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.31 ± 0.005  Gitaí et al. (2014)  Leg. Nr. 19495†  Pitcairnia decidua L.B.Sm.  1.78 ± 0.32  This study  RB 534853*  Pitcairnia feliciana (A.Chev.) Harms & Mildbr.  0.6  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.28  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.44  Nunes et al. (2013)  CESJ 5569‡  Pitcairnia flammea Lindl.  1.69 ± 0.14  This study  RB 597781*  Pitcairnia glauca Leme & A.P.Fontana  1.31 ± 0.06  This study  RB 565871*  Pitcairnia glaziovii Baker  1.56 ± 0.07  This study  RB 488168*  Pitcairnia grafii Rauh  1.34  Bennett & Leitch (2011)  –  Pitcairnia halophila L.B.Sm  1.08  Bennett & Leitch (2011)  –  Pitcairnia heerdeae E.Gross & Rauh  1.18  Bennett & Leitch (2011)  –  Pitcairnia heterophylla (Lindl.) Beer  0.88  Bennett & Leitch (2011)  –  Pitcairnia hitchcockiana L.B.Sm.  1.28  Bennett & Leitch (2011)  –  Pitcairnia irwiniana L.B.Sm.  1.51 ± 0.07  This study  RB 547217*  Pitcairnia macrochlamys Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia macrochlamys Mez  1.25 ± 0.014  Gitaí et al. (2014  Leg. Nr. 12596†  Pitcairnia micotrinensis Read  1.1  Bennett & Leitch (2011)  –  Pitcairnia nortefluminensis Leme  1.50 ± 0.05  This study  RB 511495*  Pitcairnia palmoides Mez & Sodiro  1.18  Bennett & Leitch (2011)  –  Pitcairnia paraguayensis L.B.Sm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia patentiflora L.B.Sm.  1.09 ± 0.01  This study  RB 549440*  Pitcairnia piepenbringii Rauh & E.Gross  1.2  Bennett & Leitch (2011)  –  Pitcairnia poeppigiana Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia pomacochae Rauh  1.24  Bennett & Leitch (2011)  –  Pitcairnia prolifera Rauh  0.84  Bennett & Leitch (2011)  –  Pitcairnia rectiflora Rauh  1.2  Bennett & Leitch (2011)  –  Pitcairnia riparia Mez  1.14  Bennett & Leitch (2011)  –  Pitcairnia rubiginosa Baker  1.29 ± 0.01  This study  RB 547129*  Pitcairnia sceptrigera Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia sceptrigera Mez  1.2  Gitaí et al. (2014  Leg. Nr. F009†  Pitcairnia schultzei Harms  1.32  Bennett & Leitch (2011)  –  Pitcairnia spicata (Lam.) Mez  1.22  Bennett & Leitch (2011)  –  Pitcairnia staminea Lodd.  1.64 ± 0.05  This study  RB 427244*  Pitcairnia suaveolens Lindl.  1.20 ± 0.01  This study  RB 554600*  Pitcairnia tabuliformis Linden  1.1  Bennett & Leitch (2011)  –  Pitcairnia uaupensis Baker  1.57 ± 0.01  This study  RB 547093*  Pitcairnia ulei L.B.Sm.  1.54 ± 0.13  This study  RB 547266*  Pitcairnia venezuelana L.B.Sm. & Steyerm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia villetaensis Rauh  1.26  Bennett & Leitch (2011)  –  Pitcairnia yaupibajaensis Rauh  1.12  Bennett & Leitch (2011)  –  Tillandsia usneoides (L.) L.  2.52  Zonneveld et al. (2005)  –  Species  2C (pg)  Reference  Voucher  Deuterocohnia longipetala (Baker) Mez  0.74  Bennett and Leitch (2011)  –  Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm.  1.73 ± 0.021  Gitaí et al. (2014)  Leg. Nr. 130007†  Deuterocohnia meziana Kuntze ex Mez  1.20 ± 0.02  This study  RB 382284*  Deuterocohnia schreiteri A.Cast.  0.8  Bennett and Leitch (2011)  –  Dyckia brevifolia Baker  2.01 ± 0.08  This study  RB 571588*  Dyckia choristaminea Mez  1.78 ± 0.02  This study  RB 434135*  Dyckia consimilis Mez  2.17 ± 0.01  This study  RB 673612*  Dyckia distachya Hassler  1.86 ± 0.06  This study  RB 593320*  Dyckia estevesii Rauh  1.6  Bennett and Leitch (2011)  –  Dyckia floribunda Griseb.  1.58  Bennett and Leitch (2011  –  Dyckia granmogulensis Rauh  2.18 ± 0.01  This study  RB 458314*  Dyckia ibiramensis Reitz  2.13 ± 0.08  This study  RB 571595*  Dyckia maritima Baker  2.07 ± 0.06  This study  RB 1230313*  Dyckia marnier-lapostollei L.B.Sm.  1.95 ± 0.02  This study  RB 547282*  Dyckia minarum Mez  1.88 ± 0.05  This study  RB 377972*  Dyckia monticola L.B.Sm. & Reitz  2.08 ± 0.00  This study  RB 329151*  Dyckia pseudococcinea L.B.Sm.  2.10 ± 0.04  This study  RB 723296*  Dyckia pulquinensis Wittm.  2.38 ± 0.45  This study  RB 588007*  Dyckia reitzii L.B.Sm.  1.66 ± 0.00  This study  RB 583483*  Dyckia saxatilis Mez  1.88 ± 0.12  This study  RB 462377*  Dyckia sthreliana H.Büneker & R.Pontes  1.80 ± 0.03  This study  RB 595146*  Dyckia tenebrosa Leme & H.Luther  2.13 ± 0.01  This study  RB 484461*  Dyckia tuberosa (Vell.) Beer  1.82 ± 0.08  This study  RB 594245*  Encholirium agavoides Forzza & Zappi  2.11 ± 0.01  This study  RB 504154*  Encholirium biflorum (Mez) Forzza  1.40 ± 0.04  This study  RB 583280*  Encholirium ctenophyllum Forzza & Zappi  2.90 ± 0.06  This study  RB 504157*  Encholirium diamantinum Forzza  1.92 ± 0.04  This study  RB 458335*  Encholirium cf. diamantinum Forzza  2.05 ± 0.06  This study  RB 637204*  Encholirium gracile L.B.Sm.  1.56 ± 0.02  This study  RB 570910*  Encholirium heloisae (L.B.Sm.) Forzza & Wand.  1.94 ± 0.11  This study  RB 563567*  Encholirium horridum L.B.Sm.  1.57 ± 0.02  This study  RB 462618*  Encholirium irwinii L.B.Sm.  1.74  Bennett & Leitch (2011)  –  Encholirium irwinii L.B.Sm.  1.94 ± 0.03  This study  SPF 131942§  Encholirium luxor L.B.Sm. & R.W.Read  2.02 ± 0.03  This study  RB 391473*  Encholirium magalhaesii L.B.Sm.  1.94 ± 0.01  This study  RB 670779*  Encholirium pedicellatum (Mez) Rauh  1.69 ± 0.02  This study  RB 670694*  Encholirium pulchrum Forzza, Leme & O.B.C.Ribeiro  1.83 ± 0.01  This study  RB 504202*  Encholirium scrutor (L.B.Sm.) Rauh  1.96 ± 0.04  This study  RB 563568*  Encholirium spectabile Mart. ex Schult. & Schult.f.  2.16 ± 0.01  This study  RB 331722*  Encholirium subsecundum (Baker) Mez  2.02 ± 0.13  This study  RB 747925*  Fosterella penduliflora (C.H.Wright) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella villosula (Harms) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella windischii L.B.Sm. & R.W.Read  0.91 ± 0.01  This study  RB 452098*  Pitcairnia albiflos Herb.  1.39 ± 0.05  This study  RB 498556*  Pitcairnia andreana Linden  1.3  Bennett & Leitch (2011)  –  Pitcairnia angustifolia Aiton  1.06  Bennett & Leitch (2011)  –  Pitcairnia aphelandriflora Lem.  1.24  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.2  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.29 ± 0.013  Gitaí et al. (2014_  Leg. Nr. 16095†  Pitcairnia aureobrunnea Rauh  1.12  Bennett & Leitch (2011)  –  Pitcairnia azouryi Martinelli & Forzza  1.37 ± 0.10  This study  RB 484459*  Pitcairnia barbatostigma Leme & A.P.Fontana  1.26 ± 0.02  This study  RB 462404*  Pitcairnia bradei Markgr.  1.62 ± 0.09  This study  RB 586285*  Pitcairnia burchellii Mez  1.41 ± 0.02  This study  RB 526713*  Pitcairnia burle-marxii Braga & Sucre  1.43 ± 0.13  This study  RB 509698*  Pitcairnia cardenasii L.B.Sm.  1.02  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.22  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.31 ± 0.005  Gitaí et al. (2014)  Leg. Nr. 19495†  Pitcairnia decidua L.B.Sm.  1.78 ± 0.32  This study  RB 534853*  Pitcairnia feliciana (A.Chev.) Harms & Mildbr.  0.6  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.28  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.44  Nunes et al. (2013)  CESJ 5569‡  Pitcairnia flammea Lindl.  1.69 ± 0.14  This study  RB 597781*  Pitcairnia glauca Leme & A.P.Fontana  1.31 ± 0.06  This study  RB 565871*  Pitcairnia glaziovii Baker  1.56 ± 0.07  This study  RB 488168*  Pitcairnia grafii Rauh  1.34  Bennett & Leitch (2011)  –  Pitcairnia halophila L.B.Sm  1.08  Bennett & Leitch (2011)  –  Pitcairnia heerdeae E.Gross & Rauh  1.18  Bennett & Leitch (2011)  –  Pitcairnia heterophylla (Lindl.) Beer  0.88  Bennett & Leitch (2011)  –  Pitcairnia hitchcockiana L.B.Sm.  1.28  Bennett & Leitch (2011)  –  Pitcairnia irwiniana L.B.Sm.  1.51 ± 0.07  This study  RB 547217*  Pitcairnia macrochlamys Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia macrochlamys Mez  1.25 ± 0.014  Gitaí et al. (2014  Leg. Nr. 12596†  Pitcairnia micotrinensis Read  1.1  Bennett & Leitch (2011)  –  Pitcairnia nortefluminensis Leme  1.50 ± 0.05  This study  RB 511495*  Pitcairnia palmoides Mez & Sodiro  1.18  Bennett & Leitch (2011)  –  Pitcairnia paraguayensis L.B.Sm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia patentiflora L.B.Sm.  1.09 ± 0.01  This study  RB 549440*  Pitcairnia piepenbringii Rauh & E.Gross  1.2  Bennett & Leitch (2011)  –  Pitcairnia poeppigiana Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia pomacochae Rauh  1.24  Bennett & Leitch (2011)  –  Pitcairnia prolifera Rauh  0.84  Bennett & Leitch (2011)  –  Pitcairnia rectiflora Rauh  1.2  Bennett & Leitch (2011)  –  Pitcairnia riparia Mez  1.14  Bennett & Leitch (2011)  –  Pitcairnia rubiginosa Baker  1.29 ± 0.01  This study  RB 547129*  Pitcairnia sceptrigera Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia sceptrigera Mez  1.2  Gitaí et al. (2014  Leg. Nr. F009†  Pitcairnia schultzei Harms  1.32  Bennett & Leitch (2011)  –  Pitcairnia spicata (Lam.) Mez  1.22  Bennett & Leitch (2011)  –  Pitcairnia staminea Lodd.  1.64 ± 0.05  This study  RB 427244*  Pitcairnia suaveolens Lindl.  1.20 ± 0.01  This study  RB 554600*  Pitcairnia tabuliformis Linden  1.1  Bennett & Leitch (2011)  –  Pitcairnia uaupensis Baker  1.57 ± 0.01  This study  RB 547093*  Pitcairnia ulei L.B.Sm.  1.54 ± 0.13  This study  RB 547266*  Pitcairnia venezuelana L.B.Sm. & Steyerm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia villetaensis Rauh  1.26  Bennett & Leitch (2011)  –  Pitcairnia yaupibajaensis Rauh  1.12  Bennett & Leitch (2011)  –  Tillandsia usneoides (L.) L.  2.52  Zonneveld et al. (2005)  –  *Voucher of species of the present study, deposited in the herbarium of the Jardim Botânico do Rio de Janeiro - Herbarium RB. †Cultivated, Palmengarten Frankfurt. ‡Herbarium CESJ of Universidade Federal de Juiz de Fora, Minas Gerais, Brazil. §Herbarium SPF of Universidade de São Paulo. View Large For GS estimates, the suspension was analysed using a FacsCalibur (Becton Dickinson) flow cytometer equipped with a laser source (488 nm). From each sample, 10000 PI-stained nuclei were analysed for their relative fluorescence intensity. Three independent replications were performed, and histograms with a coefficient of variation > 5% were rejected. The nuclear GS average (pg) of each sample was measured in accordance with the formula given by Doležel & Bartos (2005). Histograms were analysed using Flowing 2.5.1 software (http://www.flowingsoftware.com). Additional GS data for 40 species were extracted from the Plant DNA C-values Database (http://data.kew.org/cvalues/) and previously published data (Gitaí et al., 2005, 2014; Zonneveld, Leitch & Bennett, 2005; Bennett & Leitch, 2011; Nunes et al., 2013): three Deuterocohnia spp., two Dyckia spp., one Encholirium sp., two Fosterella spp. and 31 Pitcairnia spp., with five duplicate values, and one value for the outgroup species Tillandsia usneoides (L.) L. in the phylogenetic tree. Statistical analyses To check for differences between the average GS measurements of the sampled genera, statistical calculations were performed using R 2.15.1 (R Core Team, 2013). Because the variables did not fulfil the normality assumption (Shapiro–Wilk test), differences in 2C-values between pairs of genera were assessed using the non-parametric Wilcoxon rank sum test. Sequence alignment and phylogeny Taxon sampling Ninety-seven samples were analysed, including 14 taxa of Encholirium, 24 of Dyckia, 11 of Deuterocohnia, 22 of Pitcairnia and 23 of Fosterella. Two species were included as outgroups: Catopsis nitida (Hook.) Griseb. and Tillandsia usneoides, both belonging to Tillandsioideae. Some of the molecular operational taxonomic units were obtained from M. N. Moura et al. (unpubl. data), and 84 sequences were obtained from GenBank (Supporting Information Appendix S1). Phylogenetic analyses The matK sequences were aligned using the Muscle algorithm (Edgar, 2004) provided in MEGA 5.0 (Tamura et al., 2011). We performed the analysis using Bayesian inference (BI). To infer the best nucleotide substitution model, we used the program MrModelTest 2.3 (Nylander, 2004) with the Akaike information criterion, and the substitution model GTR + I + G was selected. The trees were queried using the software MrBayes 3.2.2 (Ronquist & Huelsenbeck, 2003) with two independent runs and four Markov chains each, one cold and three heated. Each chain was run for 50 million generations and sampled every 5000 generations. The convergence of the cold chains was checked using the program Tracer 1.6 (Drummond & Rambaut, 2007), and a burn-in on the first 25% of the trees was performed before using the remaining topologies to build a consensus topology with its respective branch lengths; this topology was then visualized using the FigTree 1.3 software program (Raumbaut, 2008). Ancestral genome size We plotted all 2C-values estimated in this study plus the 2C-values extracted from the Plant DNA C-values Database (http://data.kew.org/cvalues/) on the plastid phylogenetic tree. We used three different methods to estimate ancestral GS across the phylogenetic tree, aiming to ensure the consistency of the generated data: maximum parsimony (MP) analysis using Mesquite 3.04 (Maddison & Maddison, 2011); maximum likelihood (ML) reconstruction implemented in Stable Traits (Elliot, 2014); and BI using Markov chain Monte Carlo (MCMC) in BayesTraits 2.0 (Pagel, Meade & Barker, 2004) using the ‘continuous random walk’ model. We also verified whether the variables evolved according to a Brownian model of evolution across the phylogenetic tree using BayesTraits 2.0 (Pagel et al., 2004). RESULTS Genome size estimations We present new GS estimates for 53 species belonging to the five genera of Pitcairnioideae (Table 1), 51 of which correspond to taxa that have not been previously studied, plus 45 species from the literature. From 405 Pitcairnia spp., we now have 2C-value estimates for 48 species (c. 12% of the total); for Fosterella, only three out of the 31 species have GS estimates (9.7%); for Deuterocohnia 22% (four species out of 18); for Dyckia 11% (19 species out of 168) and for Encholirium we have estimates of GS for 16 species (55% of 29) (Gouda et al., 2015, cont. updated; BFG, 2015; Forzza, 2017). We have therefore expanded the genome database for Pitcairnioideae by 15% and this now enables us investigate whether GS data can be used as a diagnostic character to distinguish between the five genera in this subfamily. The lowest 2C-value was found in Pitcairnia feliciana (A.Chev.) Harms & Mildbr. (0.6 pg) and the highest value was found in Encholirium ctenophyllum Forzza & Zappi (2.90 pg). Overall, the estimated 2C-values varied between genera, ranging from 0.74 to 1.73 pg in Deuterocohnia (average 1.11 pg), 1.58 to 2.38 pg in Dyckia (average 1.95 pg), 1.4 to 2.9 pg in Encholirium (average 1.93 pg), 0.91 to 1.86 pg in Fosterella (average 1.54 pg) and 0.6 to 1.78 pg in Pitcairnia (average 1.27 pg). From the 98 values estimated, 77.5% range from 1.0 to 2.0 pg (Table 1). FCM histograms used to infer the GS of each specimen showed peaks corresponding to the G0/G1 nuclei of the targeted species and the G0/G1 and G2 nuclei of the internal standard used here (Fig. 1). The G0/G1 peaks of all specimens included in this study can be clearly discriminated, and their coefficients of variation were always < 5%, which is considered suitable for GS determination using FCM (Cardoso, Martinelli & Latado, 2012). Figure 1. View largeDownload slide Fluorescence intensity histograms obtained in a) Deuterocohnia meziana Kuntze ex Mez, b) Dyckia pulquinensis Wittm., c) Encholirium luxor L. B. Sm. & R. W. Read, d) Fosterella windischii L. B. Sm. & R. W. Read, and e) Pitcairnia flammea Lindl. The x axis corresponds to an arbitrary scale of fluorescence intensity (proportional to the size of the genome), and the y axis represents the number of nuclei with that fluorescence intensity. Figure 1. View largeDownload slide Fluorescence intensity histograms obtained in a) Deuterocohnia meziana Kuntze ex Mez, b) Dyckia pulquinensis Wittm., c) Encholirium luxor L. B. Sm. & R. W. Read, d) Fosterella windischii L. B. Sm. & R. W. Read, and e) Pitcairnia flammea Lindl. The x axis corresponds to an arbitrary scale of fluorescence intensity (proportional to the size of the genome), and the y axis represents the number of nuclei with that fluorescence intensity. We used the Wilcoxon rank sum test to compare the average GS values of the five genera of Pitcairnioideae (Fig. 2). Significant differences in GS were observed between Deuterocohnia and Dyckia (W = 3, P < 0.01), Deuterocohnia and Encholirium (W = 5, P < 0.01), Dyckia and Pitcairnia (W = 1150, P < 0.01), and Encholirium and Pitcairnia (W = 1035.5, P < 0.01). All the other relationships tested presented P-values > 5%. Figure 2. View largeDownload slide Box plots showing the range of genome sizes (GSs) encountered in Deuterocohnia, Dyckia, Encholirium, Fosterella and Pitcairnia. The x-axis shows the genera and the y-axis shows GS (2C-values). Numbers in boxes represent the number of species sampled within each genus. Letters A and B above the boxplots indicate the different averages. Figure 2. View largeDownload slide Box plots showing the range of genome sizes (GSs) encountered in Deuterocohnia, Dyckia, Encholirium, Fosterella and Pitcairnia. The x-axis shows the genera and the y-axis shows GS (2C-values). Numbers in boxes represent the number of species sampled within each genus. Letters A and B above the boxplots indicate the different averages. Phylogenetic analysis The alignment length of 824 bp was obtained for the matK plastid region using 94 sequences from species belonging to Pitcairnioideae and sequences from three accessions of two species as outgroups, which included 152 variable sites (18.45%). Figure 3 shows the Bayesian consensus phylogenetic tree based on the matK gene. The majority of the species of the xeric clade (Deuterocohnia, Dyckia and Encholirium) were grouped into one clade with a high posterior probability (PP) (n2, PP = 1). In this clade, another containing all species of Dyckia and Encholirium was recovered (n3, PP = 0.95) in a polytomy. Figure 3. View large Download slide Bayesian consensus tree resulting from the matK alignment (824 bp length). Coloured dots on the branches indicate the posterior probability (PP) values: green dots represent values between 1 and 0.95; yellow dots represent values between 0.94 and 0.90; and red dots represent values smaller or equal to 0.89. The nodes are indicated with numbers. Values above and below the branches represent the ancestral genome size (GS; 2C-values in pg) at particular nodes: in blue is the value generated by the maximum likelihood (ML) method using StableTraits (asterisks are related to confidence interval values shown in Supporting Information, Appendix S2); orange is the value generated by the maximum parsimony (MP) method using Mesquite; black, given below the branches, is the value generated by Bayesian inference (BI) using BayesTraits. Different genera are indicated by coloured boxes. Light pink and Dark pink: Deuterocohnia; Purple: Dyckia; Yellow: Encholirium; Blue: Fosterella; Dark green and Light green: Pitcairnia. Genome size data (2C-values) obtained in this study () or taken from Bennett & Leitch (2011) (), Gitaí et al. (2014) () and Zonneveld et al. (2005) () are presented on the right of the phylogenetic tree. Figure 3. View large Download slide Bayesian consensus tree resulting from the matK alignment (824 bp length). Coloured dots on the branches indicate the posterior probability (PP) values: green dots represent values between 1 and 0.95; yellow dots represent values between 0.94 and 0.90; and red dots represent values smaller or equal to 0.89. The nodes are indicated with numbers. Values above and below the branches represent the ancestral genome size (GS; 2C-values in pg) at particular nodes: in blue is the value generated by the maximum likelihood (ML) method using StableTraits (asterisks are related to confidence interval values shown in Supporting Information, Appendix S2); orange is the value generated by the maximum parsimony (MP) method using Mesquite; black, given below the branches, is the value generated by Bayesian inference (BI) using BayesTraits. Different genera are indicated by coloured boxes. Light pink and Dark pink: Deuterocohnia; Purple: Dyckia; Yellow: Encholirium; Blue: Fosterella; Dark green and Light green: Pitcairnia. Genome size data (2C-values) obtained in this study () or taken from Bennett & Leitch (2011) (), Gitaí et al. (2014) () and Zonneveld et al. (2005) () are presented on the right of the phylogenetic tree. The species of the mesic genera, Fosterella and Pitcairnia, were grouped into one clade (n6, PP = 0.79), but in Pitcairnia the species grouped into two clades: n9, which had a PP = 1 and contained the most species (N = 13); and n15, which had a PP = 1 and contained nine species. All Fosterella spp. formed a single clade with a high probability value (n7, PP = 1). Deuterocohnia spp. were divided in the phylogenetic tree: some species [Deuterocohnia brevispicata Rauh & L. Hrom., Deuterocohnia meziana Kuntze ex Mez and Deuterocohnia scapigera (Rauh & L.Hrom.) M.A.Spencer & L.B.Sm.] appear in a polytomy with the other species of the xeric clade. The other species [Deuterocohnia brevifolia (Griseb.) M.A.Spencer & L.B.Sm., Deuterocohnia longipetala Mez, Deuterocohnia schreiteri A.Cast. and Deuterocohnia glandulosa E.Gross] form a sister group of the remaining Pitcairnioideae (n18, PP = 1). Ancestral genome size Many of the methods commonly used to reconstruct the ancestral state of a character assume a Brownian model of character evolution across the phylogenetic tree (Webster & Purvis, 2002). However, when certain evolutionary processes are involved in the evolution of the characters analysed, the Brownian model may not be adequate (Pagel, 1998; Freckleton & Harvey, 2006). In the present study, we verified whether the variables evolved according to a Brownian model of evolution, and the test revealed that a Brownian model is indeed sufficient according to the available data (P > 0.05). To further check this we chose to analyse the data using three methods, but we found no significant difference between them. The ancestral GS for Pitcairnioideae (n1, Fig. 3) reconstructed using MP was 1.29 pg, using ML was 1.24 pg (0.75–1.71, 95% highest posterior density) and based on BI was 1.21 ± 0.15 pg. Across the phylogenetic tree, both increases and decreases in GS were reconstructed relative to the ancestral value. The values obtained with the three methods varied little among themselves, as observed, for example, for node 2 (MP = 1.50, ML = 1.49 and MCMC = 1.49 ± 0.16), node 3 (MP = 1.84, ML = 1.81 and MCMC = 1.84 ± 0.04) and node 7 (MP = 1.50, ML = 1.50 and MCMC = 1.52 ± 0.23). DISCUSSION The GS values reported here (Table 1) for species that have previously been estimated by other researchers (and available in the Plant DNA C-values database) were found to be close. For example, the published values of Dyckia estevesii Rauh and Dyckia floribunda Griseb. are 1.60 and 1.58 pg, respectively, and that of Encholirium irwinii L.B.Sm. is 1.74 pg (Ebert & Till, 1997). No significant differences were observed between the GS of species classified as (1) Dyckia and Encholirium, (2) Dyckia, Encholirium and Fosterella, (3) Deuterocohnia and Pitcairnia, (4) Deuterocohnia and Fosterella or (5) Pitcairnia and Fosterella. This may be due to the low number of Fosterella samples analysed, and because the variation was higher in Deuterocohnia than in the other three genera. Considering the problems of species delimitation in the genera belonging to the xeric clade (Deuterocohnia and Dyckia plus Encholirium) (Schütz, 2011; Santos-Silva et al., 2013; Krapp et al., 2014; Schütz et al., 2016; M. N. Moura et al., unpubl. data), differences in GS might be expected if the morphological characteristics that differentiate the genera had evolved rapidly from a sudden change in GS, possibly as a result of numerical and structural chromosomal alterations. In fact, despite the small sample available for Deuterocohnia, it appears that changes in GS may have occurred during its divergence (average = 1.00 pg) from Dyckia and Encholirium (average = 1.95 and 1.93 pg/2C, respectively), given the significant differences in GS observed between these lineages (Fig. 2). It is possible that changes in GS may have been abrupt and were accompanied by morphological and anatomical changes in these two groups of the xeric clades (Deuterocohnia and Dyckia plus Encholirium) shortly after their separation. Alternatively, the changes in GS could have accumulated slowly in both groups (Santos-Silva et al., 2013). In the mesic genera (Pitcairnia and Fosterella), a similar scenario may perhaps be envisaged, because despite the broadly similar average 2C-values in Pitcairnia and Fosterella (1.27 and 1.54 pg, respectively), apomorphies have been identified in each of these genera (Santos-Silva et al., 2013; Saraiva et al., 2015). Our molecular phylogenetic results highlighted a low level of nucleotide divergence between the species of Pitcairnioideae and support the hypothesis that the genera of the xeric clade have diverged recently. Nevertheless, the results did not resolve Deuterocohnia as monophyletic, as recovered by Givnish et al. (2011) and Santos-Silva et al. (2013). Instead, these results corroborate those of Schütz (2011), Schütz et al. (2016) and M. N. Moura et al. (unpubl. data), reinforcing the possible division of Deuterocohnia; some species appear in a polytomy with the other taxa of the xeric clade, whereas the other species form a group that is distinct from all the remaining species of the subfamily (Fig. 3). This separation is also apparent in the GS data for the Deuterocohnia spp. as the average GS for the species that appear in the polytomy with Dyckia and Encholirium is 1.2 pg/2C, whereas the average GS for the species that form a distinct clade is about half of this value, 0.77 pg/2C. In the study by Schütz (2011), Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm. was recovered in the clade containing Deuterocohnia longipetala and Deuterocohnia glandulosa, and in Gitaí et al. (2014) this species was reported with a GS of 1.73 pg/2C (Table 1), notably higher than the 2C-values reported for the other two species of the same clade (i.e. 0.74 and 0.80 pg/2C, respectively). However, polyploidy has been observed for this species in Gitaí et al. (2005) since the chromosome number reported was 2n = 50 and 2n = 100, and this may explain the large GS variation observed in the species in this clade. For Dyckia and Encholirium the chromosome number is usually 2n = 50 (Gitaí et al., 2005, 2014). Pitcairnia spp. formed two clades, as reported by Schütz et al. (2016) and Rex et al. (2009). In some studies using molecular (Givnish et al., 2004, 2007, 2011) and morphological (Saraiva et al., 2015) datasets, this genus has been shown to be monophyletic. In the study by Schütz et al. (2016), which comprises the most recent and complete phylogenetic analysis of Pitcairnioideae, monophyly of Pitcairnia was recovered only using nuclear DNA sequences. When plastid data were used, the results were the same as those found in the present study. Therefore, the delimitation of the genus remains controversial, despite Pitcairnia being recovered as monophyletic using a morphological dataset (Saraiva et al., 2015). The occurrence of cytotype diversity (i.e. different ploidies within a species) is apparent for two species of Pitcairnia for which GS is presented in this study (Table 1; Gitaí et al., 2014): Pitcairnia flammea Lindl. (2C = 1.69 pg, this study; 2n = 50/2n ≈ 100, Gitaí et al., 2014) and Pitcairnia sceptrigera Mez (2C = 1.2 pg; 2n = 50/2n ≈ 100, Gitaí et al., 2014). Without chromosome counts for the plants used to estimate GS it is not possible to determine whether the 2C-values reported are for the diploid or tetraploid cytotype of the species. For the other species presented in Table 1 that have karyotype data available, the diploid chromosome number is 2n = 50 (Gitaí et al., 2014). All species of Fosterella, the other mesic genus, grouped into one clade and GS was estimated for only three species (Table 1): Fosterella penduliflora (C.H.Wright) L.B.Sm. (2C = 1.86 pg, Bennett & Leitch, 2011), Fosterella villosula (Harms) L.B.Sm. (2C = 1.86 pg, Bennett & Leitch, 2011) and Fosterella windischii L.B.Sm. & R.W.Read (2C = 0.91 pg, this study). Polyploidy has also been reported for the first two species (2n = 100 and 2n = 150, respectively, Gitaí et al., 2014) and no karyotype data are available for the third. The reconstructed ancestral GS for Pitcairnioideae was 1.21 pg/2C according to the MCMC method, 1.24 pg according to the ML method and 1.29 pg according to the parsimony method. These values are smaller than the reconstructed ancestral GS of 1.45 pg/2C for all angiosperms (Puttick, Clark & Donoghue, 2015) and also smaller than 1.99 pg/2C estimated for the ancestral spermatophyte (Puttick et al., 2015). Overall, our analysis of GS evolution in Pitcairnioideae reported here fits the model of proportional GS evolution proposed by Oliver et al. (2007) which predicts that ‘the rate of GS evolution is proportional to the GS’, i.e. if a given taxon has an ancestor with a large GS, the descendants of the ancestor can have a small or large GS, giving rise to a large variation in GS. By contrast, if the GS of the ancestor is small, the descendants of the ancestor are likely to have small genomes, which results in only limited variance in GS. In this sense, we do not expect drastic changes in GS in a group that has arisen from ancestors with small GS, as shown in Figure 3. GS values have also been reported for other subfamilies of Bromeliaceae (Brocchinioideae, Bromelioideae, Puyoideae and Tillandsioideae) (Ebert & Till, 1997; Ramírez-Morillo & Brown, 2001; Zonneveld et al., 2005; Favoreto et al., 2012; Gitaí et al., 2014) and the 2C-values range from 0.64 pg [Orthophytum saxicola (Ule) L.B.Sm., Bromelioideae; Ramírez-Morillo & Brown, 2001] to 2.52 pg [Tillandsia usneoides (L.) L., Tillandsioideae; Zonneveld et al., 2005] (Gitaí et al., 2014). A notably higher 2C-value was reported by Favoreto et al. (2012) for Tillandsia loliacea Mart. ex Schult. & Schult.f. (3.34 pg), but most 2C-values fall between 1.0 and 2.0 pg (c. 70% of estimates) as observed in the present study for Pitcairnioideae. This indicates that the ancestral Bromeliaceae may also have had a small GS with the variation of 2C-values in the family tending to be skewed towards smaller values (Oliver et al., 2007). Bromeliaceae are the most species-rich angiosperm family, almost exclusively native to the New World (Givnish et al., 2011) and, although most 2C-values reported for the family show little variation, we found in this study with Pitcairnioideae some examples of larger variation in genera (Deuterocohnia and Pitcairnia; Table 1). This variation could be related to chromosomal numerical or structural changes, as reported by Gitaí et al. (2005, 2014). Kraaijeveld (2010) suggested that allopatric populations of organisms with small GS may have a higher rate of divergence than those with larger GS. He attributed this phenomenon to the assumption that in a small genome, the probability of phenotypic changes caused by mutations is larger than in a large genome, which has accumulated more non-coding DNA and repetitive DNA. In addition, Puttick et al. (2015) reported that the ability to alter GS (i.e. the rate of change of GS over time) exhibits the strongest correlation with species diversification rather than the size of the genome itself. In relation to the data presented here, we can perhaps assume that the high phenotypic diversity observed across Bromeliaceae and that is well represented in Pitcairnioideae may indeed be correlated with high rates of GS evolution of the species, given the short period of species diversification that is estimated for the family. In summary, the results obtained in this study improve our knowledge concerning GS in Pitcairnioideae and will contribute to a better understanding of the natural history of this subfamily and Bromeliaceae as a whole. In addition, the FCM data reported here contribute to our knowledge of GS diversity in Bromeliaceae, which is still poorly known given the large numbers of species in this family. The paper also highlights the value of FCM as a rapid and reliable technique for generating GS data, which can be analysed in conjunction with other molecular and morphological data to help elucidate patterns of evolution and phylogenetic relationships in this family. ACKNOWLEDGMENTS This study was carried out as part of a Masters and PhD degree in Ecology by M.N.M. at the Universidade Federal de Viçosa, who is grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Edital MCT/CNPq/MEC/CAPES No. 52/2010 – PROTAX) and the Coordenação de Pessoal de Nıível Superior (CAPES) for the scholarship. We thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the postdoctoral fellowship to M.P.C. (Process number: BPD-00037-13). This work was supported by grants from FAPEMIG (APQ-00366-12 - Edital 01/2012), CAPES and CNPq. R.C.F. received a Research Productivity Fellowship from CNPq (Proc. 303420/2016-2). We thank Prof. Lyderson Facio Viccini (Laboratório de Genética da Universidade Federal de Juiz de Fora) for enabling FCM measurements. We also thank Karla S. C. Yotoko (Laboratório de Bioinformática e Evolução da Universidade Federal de Viçosa) for her support in the laboratory and Janaína Gomes-da-Silva, Danon C. Cardoso, Guilherme M. A. Carvalho, Leandro Licursi and Natállia M. F. Vicente for critical reading and helpful suggestions. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Appendix S1. Sequences used in the phylogenetic analysis with their specimen voucher and GenBank accession number Appendix S2. Confidence interval of the values generated by the maximum likelihood method in StableTraits. 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Annals of Botany  96: 229– 244. Google Scholar CrossRef Search ADS PubMed  © 2018 The Linnean Society of London, Botanical Journal of the Linnean Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Botanical Journal of the Linnean Society Oxford University Press

Reconstruction of ancestral genome size in Pitcairnioideae (Bromeliaceae): what can genome size tell us about the evolutionary history of its five genera?

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The Linnean Society of London
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© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society
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0024-4074
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Abstract

Abstract We expand the genome size (GS) database for Bromeliaceae, specifically for subfamily Pitcairnioideae, and verify whether GS can provide information on the diversification of the five genera in this subfamily. We also provide a phylogenetic perspective on GS evolution in the subfamily and reconstruct the ancestral state for this character. We show that the evolutionary path of GS from the origin of angiosperms to the origin of Pitcairnioideae agrees with the proportional model of GS evolution. Furthermore, we propose that the high phenotypic diversity that is found across Bromeliaceae and that is well represented in Pitcairnioideae is both correlated with high rates of GS evolution of the species and associated with a short period of diversification. The paper also highlights the value of flow cytometry as a rapid and reliable technique for generating GS data which can be analysed in conjunction with other molecular and morphological data to help elucidate patterns of evolution and phylogenetic relationships within this family. INTRODUCTION Taxon delimitation is a fundamental step for conservation, studying evolution and undertaking systematic studies of an organism (Sites & Marshall, 2003, 2004). There is a consensus that species should correspond to independent evolutionary lineages (Wilson, 1978; Duminil & Michele, 2009; Padial et al., 2010). The use of different types of characters (e.g. morphological, physiological, molecular or karyotypic) in a given taxon can help to provide an accurate classification (Dayrat, 2005; Schlick-Steiner et al., 2010; Cristiano, Cardoso & Fernandes-Salomão, 2013). Alternatively, using the same character in a large number of taxa provides opportunities for studying the evolution of that character. The evolution of a particular character may even help in understanding the evolutionary patterns and processes underpinning phylogenetic relationships among existing organisms (Le Quesne, 1974; Maddison & Maddison, 1989). Bromeliaceae, a predominantly Neotropical family (Benzing, 2000), comprising 73 genera and nearly 3600 species (Givnish et al., 2011; Butcher & Gouda, cont. updated), are considered to be a natural group. The monophyly of Bromeliaceae has been consistently confirmed in several studies (Stevenson & Loconte, 1995; Chase et al., 1995, 2000; Givnish et al., 2011). Givnish et al. (2007) suggested that a recent diversification of modern lineages of Bromeliaceae occurred c. 19 Mya. These authors inferred that such lineages first appeared in the Guayana Shield, northern South America, and then spread centripetally throughout the New World. In terms of chromosome number, most species present the basic number x = 25, with few exceptions (Gitaí, Horres & Benko-Iseppon, 2005; Ceita et al., 2008; Gitaí et al., 2014; Nunes & Clarindo, 2014). Available data also suggest that species of Bromeliaceae have small genome sizes (GSs), as their 2C-values range from 0.6 pg to 2.52 pg ( x¯ = 1.18 ± 0.37) (Angiosperm DNA C-values Database; Bennett & Leitch, 2012; 1 pg = 978 Mbp). The subfamilies and genera of Bromeliaceae have frequently been questioned and changed over the last few decades (Grant, 1992, 1993, 1996; Gouda, 1994; Givnish et al., 2004, 2007), as most genera have poor morphological delimitation and low molecular differentiation (Escobedo-Sarti et al., 2013). Pitcairnioideae, one of these subfamilies, comprise the genera Pitcairnia L’Hér. (405 species), Fosterella L.B.Sm. (31 species), Dyckia Schult. & Schult.f. (168 species), Encholirium Mart. ex Schult. & Schult.f. (29 species) and Deuterocohnia Mez (18 species) (Butcher and Gouda, cont. updated; BFG, 2015; Forzza, 2017). The first two genera exhibit mesic morphological and physiological characteristics, whereas the other three exhibit xeric morphological and physiological characteristics and belong to a monophyletic group informally known as ‘the xeric clade’ of Pitcairnioideae (Givnish et al., 2011; Santos-Silva et al., 2013; Schütz et al., 2016). The relationships between the genera of Pitcairnioideae have been questioned and studied in recent years. Rex et al. (2009) presented a multilocus plastid DNA phylogenetic analysis of Fosterella, showing it to be monophyletic, but infrageneric relationships in other genera of the subfamily remained unclear. Similar results were reported by Schütz (2011) in a taxonomic revision based on plastid and nuclear DNA data for Deuterocohnia. The results showed low genetic variability in the genus, resulting in low-resolution trees and networks. A phylogenetic analysis by Saraiva, Mantovani & Forzza (2015), based on morphological characters alone, supported the monophyly of Pitcairnia s.l. and agreed with previous findings (Terry, Brown & Olmstead, 1997; Givnish et al., 2004, 2007, 2011; Horres et al., 2007), but their analysis did not resolve all species relationships within the genus. These relationships also remained unresolved in the molecular phylogenetic analysis of Schütz et al. (2016), as did the delimitation of Dyckia and Encholirium. M. N. Moura et al. (unpubl. data) conducted a more comprehensive molecular and morphological phylogenetic analysis of Encholirium, which supports the idea of the genus being paraphyletic and corroborates the hypothesis regarding the recent divergence of Pitcairnioideae (the crown age of Pitcairnioideae was 11.8 Mya according to Givnish et al., 2011). To study relationships between species that have recently diversified, DNA sequences, commonly used in phylogenetic studies, may not have had enough time to accumulate detectable differences to resolve phylogenetic relationships (Givnish et al., 2011). On the other hand, abrupt changes in nuclear DNA content can occur over short periods of time (from one generation to another) as a result of numerical (euploidy and/or aneuploidy) and/or structural (deletion, inversion, duplication and/or translocation) alterations. These changes are common in many lineages of plants and are considered to be key factors in the evolution of the genomes of several species (e.g. Bennett & Smith, 1976; Kunkel, 1990; Soltis & Soltis, 2009; Campos et al., 2011; Lee, Chang & Chung, 2011; Lepers-Andrzejewski et al., 2011; Szadkowski et al., 2011; Chester et al., 2012). According to Doležel, Greilhuber & Suda (2007), flow cytometry (FCM) is a fast and reliable method to estimate GS in plants. Information on nuclear GS in Bromeliaceae has toadied in studies regarding their taxonomy, evolution, genetic diversity and reproductive biology (Ebert & Till, 1997; Ramírez-Morillo & Brown, 2001; Sgorbati et al., 2004; Favoreto et al., 2012). Nevetheless, given the diversity of the family and ongoing taxonomic problems, as outlined above, there is still insufficient GS data available (< 3% for species of Bromeliaceae and < 7% for species of Pitcairnioideae), and hence our understanding of related evolutionary processes is poorly understood (Gitaí et al., 2014). In the present study, we aim to expand the GS database for Bromeliaceae, specifically Pitcairnioideae, and examine whether GS data can help to provide insights into the diversification of the five genera in the subfamily. We also provide a phylogenetic perspective on GS evolution in the subfamily and reconstruct the ancestral state of this character. MATERIAL AND METHODS Genome size estimations Taxon sampling and flow cytometry FCM analyses were carried out at the Laboratório de Genética da Universidade Federal de Juiz de Fora (UFJF). Leaf samples of 53 species of the five genera of Pitcairnioideae were collected from the living collection at the Jardim Botânico do Rio de Janeiro: one Deuterocohnia sp., 17 Dyckia spp., 16 Encholirium spp., one Fosterella sp. and 18 Pitcairnia spp. Vouchers have been deposited in herbarium RB (Jardim Botânico do Rio de Janeiro) and herbarium SPF (Universidade de São Paulo) under the numbers presented in Table 1. The nuclear DNA content of three samples of each studied species was measured using the DNA 2C-value of Pisum sativum L. as an internal standard (9.09 pg; Doležel et al., 1998). To obtain a suspension of nuclei, 1.0 cm2 leaf tissue of the standard and each sample were simultaneously chopped in 1.0 mL cold LB01 buffer (Doležel, Binarová & Lucretti, 1989) with the aid of a cutting blade. The suspension of nuclei was subsequently filtered through 30-µm nylon mesh. The solution was supplemented with 5.0 µL of RNAse at a concentration of 100 µg mL−1 and then stained with 50 µL propidium iodide (PI) solution at a concentration of 1 mg mL−1. The samples were stored in the dark and analysed within 1 h of preparation. Table 1. Genome sizes estimated for all studied specimens, the mean calculated for each species, standard deviation and herbarium voucher Species  2C (pg)  Reference  Voucher  Deuterocohnia longipetala (Baker) Mez  0.74  Bennett and Leitch (2011)  –  Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm.  1.73 ± 0.021  Gitaí et al. (2014)  Leg. Nr. 130007†  Deuterocohnia meziana Kuntze ex Mez  1.20 ± 0.02  This study  RB 382284*  Deuterocohnia schreiteri A.Cast.  0.8  Bennett and Leitch (2011)  –  Dyckia brevifolia Baker  2.01 ± 0.08  This study  RB 571588*  Dyckia choristaminea Mez  1.78 ± 0.02  This study  RB 434135*  Dyckia consimilis Mez  2.17 ± 0.01  This study  RB 673612*  Dyckia distachya Hassler  1.86 ± 0.06  This study  RB 593320*  Dyckia estevesii Rauh  1.6  Bennett and Leitch (2011)  –  Dyckia floribunda Griseb.  1.58  Bennett and Leitch (2011  –  Dyckia granmogulensis Rauh  2.18 ± 0.01  This study  RB 458314*  Dyckia ibiramensis Reitz  2.13 ± 0.08  This study  RB 571595*  Dyckia maritima Baker  2.07 ± 0.06  This study  RB 1230313*  Dyckia marnier-lapostollei L.B.Sm.  1.95 ± 0.02  This study  RB 547282*  Dyckia minarum Mez  1.88 ± 0.05  This study  RB 377972*  Dyckia monticola L.B.Sm. & Reitz  2.08 ± 0.00  This study  RB 329151*  Dyckia pseudococcinea L.B.Sm.  2.10 ± 0.04  This study  RB 723296*  Dyckia pulquinensis Wittm.  2.38 ± 0.45  This study  RB 588007*  Dyckia reitzii L.B.Sm.  1.66 ± 0.00  This study  RB 583483*  Dyckia saxatilis Mez  1.88 ± 0.12  This study  RB 462377*  Dyckia sthreliana H.Büneker & R.Pontes  1.80 ± 0.03  This study  RB 595146*  Dyckia tenebrosa Leme & H.Luther  2.13 ± 0.01  This study  RB 484461*  Dyckia tuberosa (Vell.) Beer  1.82 ± 0.08  This study  RB 594245*  Encholirium agavoides Forzza & Zappi  2.11 ± 0.01  This study  RB 504154*  Encholirium biflorum (Mez) Forzza  1.40 ± 0.04  This study  RB 583280*  Encholirium ctenophyllum Forzza & Zappi  2.90 ± 0.06  This study  RB 504157*  Encholirium diamantinum Forzza  1.92 ± 0.04  This study  RB 458335*  Encholirium cf. diamantinum Forzza  2.05 ± 0.06  This study  RB 637204*  Encholirium gracile L.B.Sm.  1.56 ± 0.02  This study  RB 570910*  Encholirium heloisae (L.B.Sm.) Forzza & Wand.  1.94 ± 0.11  This study  RB 563567*  Encholirium horridum L.B.Sm.  1.57 ± 0.02  This study  RB 462618*  Encholirium irwinii L.B.Sm.  1.74  Bennett & Leitch (2011)  –  Encholirium irwinii L.B.Sm.  1.94 ± 0.03  This study  SPF 131942§  Encholirium luxor L.B.Sm. & R.W.Read  2.02 ± 0.03  This study  RB 391473*  Encholirium magalhaesii L.B.Sm.  1.94 ± 0.01  This study  RB 670779*  Encholirium pedicellatum (Mez) Rauh  1.69 ± 0.02  This study  RB 670694*  Encholirium pulchrum Forzza, Leme & O.B.C.Ribeiro  1.83 ± 0.01  This study  RB 504202*  Encholirium scrutor (L.B.Sm.) Rauh  1.96 ± 0.04  This study  RB 563568*  Encholirium spectabile Mart. ex Schult. & Schult.f.  2.16 ± 0.01  This study  RB 331722*  Encholirium subsecundum (Baker) Mez  2.02 ± 0.13  This study  RB 747925*  Fosterella penduliflora (C.H.Wright) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella villosula (Harms) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella windischii L.B.Sm. & R.W.Read  0.91 ± 0.01  This study  RB 452098*  Pitcairnia albiflos Herb.  1.39 ± 0.05  This study  RB 498556*  Pitcairnia andreana Linden  1.3  Bennett & Leitch (2011)  –  Pitcairnia angustifolia Aiton  1.06  Bennett & Leitch (2011)  –  Pitcairnia aphelandriflora Lem.  1.24  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.2  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.29 ± 0.013  Gitaí et al. (2014_  Leg. Nr. 16095†  Pitcairnia aureobrunnea Rauh  1.12  Bennett & Leitch (2011)  –  Pitcairnia azouryi Martinelli & Forzza  1.37 ± 0.10  This study  RB 484459*  Pitcairnia barbatostigma Leme & A.P.Fontana  1.26 ± 0.02  This study  RB 462404*  Pitcairnia bradei Markgr.  1.62 ± 0.09  This study  RB 586285*  Pitcairnia burchellii Mez  1.41 ± 0.02  This study  RB 526713*  Pitcairnia burle-marxii Braga & Sucre  1.43 ± 0.13  This study  RB 509698*  Pitcairnia cardenasii L.B.Sm.  1.02  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.22  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.31 ± 0.005  Gitaí et al. (2014)  Leg. Nr. 19495†  Pitcairnia decidua L.B.Sm.  1.78 ± 0.32  This study  RB 534853*  Pitcairnia feliciana (A.Chev.) Harms & Mildbr.  0.6  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.28  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.44  Nunes et al. (2013)  CESJ 5569‡  Pitcairnia flammea Lindl.  1.69 ± 0.14  This study  RB 597781*  Pitcairnia glauca Leme & A.P.Fontana  1.31 ± 0.06  This study  RB 565871*  Pitcairnia glaziovii Baker  1.56 ± 0.07  This study  RB 488168*  Pitcairnia grafii Rauh  1.34  Bennett & Leitch (2011)  –  Pitcairnia halophila L.B.Sm  1.08  Bennett & Leitch (2011)  –  Pitcairnia heerdeae E.Gross & Rauh  1.18  Bennett & Leitch (2011)  –  Pitcairnia heterophylla (Lindl.) Beer  0.88  Bennett & Leitch (2011)  –  Pitcairnia hitchcockiana L.B.Sm.  1.28  Bennett & Leitch (2011)  –  Pitcairnia irwiniana L.B.Sm.  1.51 ± 0.07  This study  RB 547217*  Pitcairnia macrochlamys Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia macrochlamys Mez  1.25 ± 0.014  Gitaí et al. (2014  Leg. Nr. 12596†  Pitcairnia micotrinensis Read  1.1  Bennett & Leitch (2011)  –  Pitcairnia nortefluminensis Leme  1.50 ± 0.05  This study  RB 511495*  Pitcairnia palmoides Mez & Sodiro  1.18  Bennett & Leitch (2011)  –  Pitcairnia paraguayensis L.B.Sm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia patentiflora L.B.Sm.  1.09 ± 0.01  This study  RB 549440*  Pitcairnia piepenbringii Rauh & E.Gross  1.2  Bennett & Leitch (2011)  –  Pitcairnia poeppigiana Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia pomacochae Rauh  1.24  Bennett & Leitch (2011)  –  Pitcairnia prolifera Rauh  0.84  Bennett & Leitch (2011)  –  Pitcairnia rectiflora Rauh  1.2  Bennett & Leitch (2011)  –  Pitcairnia riparia Mez  1.14  Bennett & Leitch (2011)  –  Pitcairnia rubiginosa Baker  1.29 ± 0.01  This study  RB 547129*  Pitcairnia sceptrigera Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia sceptrigera Mez  1.2  Gitaí et al. (2014  Leg. Nr. F009†  Pitcairnia schultzei Harms  1.32  Bennett & Leitch (2011)  –  Pitcairnia spicata (Lam.) Mez  1.22  Bennett & Leitch (2011)  –  Pitcairnia staminea Lodd.  1.64 ± 0.05  This study  RB 427244*  Pitcairnia suaveolens Lindl.  1.20 ± 0.01  This study  RB 554600*  Pitcairnia tabuliformis Linden  1.1  Bennett & Leitch (2011)  –  Pitcairnia uaupensis Baker  1.57 ± 0.01  This study  RB 547093*  Pitcairnia ulei L.B.Sm.  1.54 ± 0.13  This study  RB 547266*  Pitcairnia venezuelana L.B.Sm. & Steyerm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia villetaensis Rauh  1.26  Bennett & Leitch (2011)  –  Pitcairnia yaupibajaensis Rauh  1.12  Bennett & Leitch (2011)  –  Tillandsia usneoides (L.) L.  2.52  Zonneveld et al. (2005)  –  Species  2C (pg)  Reference  Voucher  Deuterocohnia longipetala (Baker) Mez  0.74  Bennett and Leitch (2011)  –  Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm.  1.73 ± 0.021  Gitaí et al. (2014)  Leg. Nr. 130007†  Deuterocohnia meziana Kuntze ex Mez  1.20 ± 0.02  This study  RB 382284*  Deuterocohnia schreiteri A.Cast.  0.8  Bennett and Leitch (2011)  –  Dyckia brevifolia Baker  2.01 ± 0.08  This study  RB 571588*  Dyckia choristaminea Mez  1.78 ± 0.02  This study  RB 434135*  Dyckia consimilis Mez  2.17 ± 0.01  This study  RB 673612*  Dyckia distachya Hassler  1.86 ± 0.06  This study  RB 593320*  Dyckia estevesii Rauh  1.6  Bennett and Leitch (2011)  –  Dyckia floribunda Griseb.  1.58  Bennett and Leitch (2011  –  Dyckia granmogulensis Rauh  2.18 ± 0.01  This study  RB 458314*  Dyckia ibiramensis Reitz  2.13 ± 0.08  This study  RB 571595*  Dyckia maritima Baker  2.07 ± 0.06  This study  RB 1230313*  Dyckia marnier-lapostollei L.B.Sm.  1.95 ± 0.02  This study  RB 547282*  Dyckia minarum Mez  1.88 ± 0.05  This study  RB 377972*  Dyckia monticola L.B.Sm. & Reitz  2.08 ± 0.00  This study  RB 329151*  Dyckia pseudococcinea L.B.Sm.  2.10 ± 0.04  This study  RB 723296*  Dyckia pulquinensis Wittm.  2.38 ± 0.45  This study  RB 588007*  Dyckia reitzii L.B.Sm.  1.66 ± 0.00  This study  RB 583483*  Dyckia saxatilis Mez  1.88 ± 0.12  This study  RB 462377*  Dyckia sthreliana H.Büneker & R.Pontes  1.80 ± 0.03  This study  RB 595146*  Dyckia tenebrosa Leme & H.Luther  2.13 ± 0.01  This study  RB 484461*  Dyckia tuberosa (Vell.) Beer  1.82 ± 0.08  This study  RB 594245*  Encholirium agavoides Forzza & Zappi  2.11 ± 0.01  This study  RB 504154*  Encholirium biflorum (Mez) Forzza  1.40 ± 0.04  This study  RB 583280*  Encholirium ctenophyllum Forzza & Zappi  2.90 ± 0.06  This study  RB 504157*  Encholirium diamantinum Forzza  1.92 ± 0.04  This study  RB 458335*  Encholirium cf. diamantinum Forzza  2.05 ± 0.06  This study  RB 637204*  Encholirium gracile L.B.Sm.  1.56 ± 0.02  This study  RB 570910*  Encholirium heloisae (L.B.Sm.) Forzza & Wand.  1.94 ± 0.11  This study  RB 563567*  Encholirium horridum L.B.Sm.  1.57 ± 0.02  This study  RB 462618*  Encholirium irwinii L.B.Sm.  1.74  Bennett & Leitch (2011)  –  Encholirium irwinii L.B.Sm.  1.94 ± 0.03  This study  SPF 131942§  Encholirium luxor L.B.Sm. & R.W.Read  2.02 ± 0.03  This study  RB 391473*  Encholirium magalhaesii L.B.Sm.  1.94 ± 0.01  This study  RB 670779*  Encholirium pedicellatum (Mez) Rauh  1.69 ± 0.02  This study  RB 670694*  Encholirium pulchrum Forzza, Leme & O.B.C.Ribeiro  1.83 ± 0.01  This study  RB 504202*  Encholirium scrutor (L.B.Sm.) Rauh  1.96 ± 0.04  This study  RB 563568*  Encholirium spectabile Mart. ex Schult. & Schult.f.  2.16 ± 0.01  This study  RB 331722*  Encholirium subsecundum (Baker) Mez  2.02 ± 0.13  This study  RB 747925*  Fosterella penduliflora (C.H.Wright) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella villosula (Harms) L.B.Sm.  1.86  Bennett & Leitch (2011)  –  Fosterella windischii L.B.Sm. & R.W.Read  0.91 ± 0.01  This study  RB 452098*  Pitcairnia albiflos Herb.  1.39 ± 0.05  This study  RB 498556*  Pitcairnia andreana Linden  1.3  Bennett & Leitch (2011)  –  Pitcairnia angustifolia Aiton  1.06  Bennett & Leitch (2011)  –  Pitcairnia aphelandriflora Lem.  1.24  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.2  Bennett & Leitch (2011)  –  Pitcairnia atrorubens (Beer) Baker  1.29 ± 0.013  Gitaí et al. (2014_  Leg. Nr. 16095†  Pitcairnia aureobrunnea Rauh  1.12  Bennett & Leitch (2011)  –  Pitcairnia azouryi Martinelli & Forzza  1.37 ± 0.10  This study  RB 484459*  Pitcairnia barbatostigma Leme & A.P.Fontana  1.26 ± 0.02  This study  RB 462404*  Pitcairnia bradei Markgr.  1.62 ± 0.09  This study  RB 586285*  Pitcairnia burchellii Mez  1.41 ± 0.02  This study  RB 526713*  Pitcairnia burle-marxii Braga & Sucre  1.43 ± 0.13  This study  RB 509698*  Pitcairnia cardenasii L.B.Sm.  1.02  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.22  Bennett & Leitch (2011)  –  Pitcairnia chiapensis Miranda  1.31 ± 0.005  Gitaí et al. (2014)  Leg. Nr. 19495†  Pitcairnia decidua L.B.Sm.  1.78 ± 0.32  This study  RB 534853*  Pitcairnia feliciana (A.Chev.) Harms & Mildbr.  0.6  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.28  Bennett & Leitch (2011)  –  Pitcairnia flammea Lindl.  1.44  Nunes et al. (2013)  CESJ 5569‡  Pitcairnia flammea Lindl.  1.69 ± 0.14  This study  RB 597781*  Pitcairnia glauca Leme & A.P.Fontana  1.31 ± 0.06  This study  RB 565871*  Pitcairnia glaziovii Baker  1.56 ± 0.07  This study  RB 488168*  Pitcairnia grafii Rauh  1.34  Bennett & Leitch (2011)  –  Pitcairnia halophila L.B.Sm  1.08  Bennett & Leitch (2011)  –  Pitcairnia heerdeae E.Gross & Rauh  1.18  Bennett & Leitch (2011)  –  Pitcairnia heterophylla (Lindl.) Beer  0.88  Bennett & Leitch (2011)  –  Pitcairnia hitchcockiana L.B.Sm.  1.28  Bennett & Leitch (2011)  –  Pitcairnia irwiniana L.B.Sm.  1.51 ± 0.07  This study  RB 547217*  Pitcairnia macrochlamys Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia macrochlamys Mez  1.25 ± 0.014  Gitaí et al. (2014  Leg. Nr. 12596†  Pitcairnia micotrinensis Read  1.1  Bennett & Leitch (2011)  –  Pitcairnia nortefluminensis Leme  1.50 ± 0.05  This study  RB 511495*  Pitcairnia palmoides Mez & Sodiro  1.18  Bennett & Leitch (2011)  –  Pitcairnia paraguayensis L.B.Sm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia patentiflora L.B.Sm.  1.09 ± 0.01  This study  RB 549440*  Pitcairnia piepenbringii Rauh & E.Gross  1.2  Bennett & Leitch (2011)  –  Pitcairnia poeppigiana Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia pomacochae Rauh  1.24  Bennett & Leitch (2011)  –  Pitcairnia prolifera Rauh  0.84  Bennett & Leitch (2011)  –  Pitcairnia rectiflora Rauh  1.2  Bennett & Leitch (2011)  –  Pitcairnia riparia Mez  1.14  Bennett & Leitch (2011)  –  Pitcairnia rubiginosa Baker  1.29 ± 0.01  This study  RB 547129*  Pitcairnia sceptrigera Mez  1.2  Bennett & Leitch (2011)  –  Pitcairnia sceptrigera Mez  1.2  Gitaí et al. (2014  Leg. Nr. F009†  Pitcairnia schultzei Harms  1.32  Bennett & Leitch (2011)  –  Pitcairnia spicata (Lam.) Mez  1.22  Bennett & Leitch (2011)  –  Pitcairnia staminea Lodd.  1.64 ± 0.05  This study  RB 427244*  Pitcairnia suaveolens Lindl.  1.20 ± 0.01  This study  RB 554600*  Pitcairnia tabuliformis Linden  1.1  Bennett & Leitch (2011)  –  Pitcairnia uaupensis Baker  1.57 ± 0.01  This study  RB 547093*  Pitcairnia ulei L.B.Sm.  1.54 ± 0.13  This study  RB 547266*  Pitcairnia venezuelana L.B.Sm. & Steyerm.  1.36  Bennett & Leitch (2011)  –  Pitcairnia villetaensis Rauh  1.26  Bennett & Leitch (2011)  –  Pitcairnia yaupibajaensis Rauh  1.12  Bennett & Leitch (2011)  –  Tillandsia usneoides (L.) L.  2.52  Zonneveld et al. (2005)  –  *Voucher of species of the present study, deposited in the herbarium of the Jardim Botânico do Rio de Janeiro - Herbarium RB. †Cultivated, Palmengarten Frankfurt. ‡Herbarium CESJ of Universidade Federal de Juiz de Fora, Minas Gerais, Brazil. §Herbarium SPF of Universidade de São Paulo. View Large For GS estimates, the suspension was analysed using a FacsCalibur (Becton Dickinson) flow cytometer equipped with a laser source (488 nm). From each sample, 10000 PI-stained nuclei were analysed for their relative fluorescence intensity. Three independent replications were performed, and histograms with a coefficient of variation > 5% were rejected. The nuclear GS average (pg) of each sample was measured in accordance with the formula given by Doležel & Bartos (2005). Histograms were analysed using Flowing 2.5.1 software (http://www.flowingsoftware.com). Additional GS data for 40 species were extracted from the Plant DNA C-values Database (http://data.kew.org/cvalues/) and previously published data (Gitaí et al., 2005, 2014; Zonneveld, Leitch & Bennett, 2005; Bennett & Leitch, 2011; Nunes et al., 2013): three Deuterocohnia spp., two Dyckia spp., one Encholirium sp., two Fosterella spp. and 31 Pitcairnia spp., with five duplicate values, and one value for the outgroup species Tillandsia usneoides (L.) L. in the phylogenetic tree. Statistical analyses To check for differences between the average GS measurements of the sampled genera, statistical calculations were performed using R 2.15.1 (R Core Team, 2013). Because the variables did not fulfil the normality assumption (Shapiro–Wilk test), differences in 2C-values between pairs of genera were assessed using the non-parametric Wilcoxon rank sum test. Sequence alignment and phylogeny Taxon sampling Ninety-seven samples were analysed, including 14 taxa of Encholirium, 24 of Dyckia, 11 of Deuterocohnia, 22 of Pitcairnia and 23 of Fosterella. Two species were included as outgroups: Catopsis nitida (Hook.) Griseb. and Tillandsia usneoides, both belonging to Tillandsioideae. Some of the molecular operational taxonomic units were obtained from M. N. Moura et al. (unpubl. data), and 84 sequences were obtained from GenBank (Supporting Information Appendix S1). Phylogenetic analyses The matK sequences were aligned using the Muscle algorithm (Edgar, 2004) provided in MEGA 5.0 (Tamura et al., 2011). We performed the analysis using Bayesian inference (BI). To infer the best nucleotide substitution model, we used the program MrModelTest 2.3 (Nylander, 2004) with the Akaike information criterion, and the substitution model GTR + I + G was selected. The trees were queried using the software MrBayes 3.2.2 (Ronquist & Huelsenbeck, 2003) with two independent runs and four Markov chains each, one cold and three heated. Each chain was run for 50 million generations and sampled every 5000 generations. The convergence of the cold chains was checked using the program Tracer 1.6 (Drummond & Rambaut, 2007), and a burn-in on the first 25% of the trees was performed before using the remaining topologies to build a consensus topology with its respective branch lengths; this topology was then visualized using the FigTree 1.3 software program (Raumbaut, 2008). Ancestral genome size We plotted all 2C-values estimated in this study plus the 2C-values extracted from the Plant DNA C-values Database (http://data.kew.org/cvalues/) on the plastid phylogenetic tree. We used three different methods to estimate ancestral GS across the phylogenetic tree, aiming to ensure the consistency of the generated data: maximum parsimony (MP) analysis using Mesquite 3.04 (Maddison & Maddison, 2011); maximum likelihood (ML) reconstruction implemented in Stable Traits (Elliot, 2014); and BI using Markov chain Monte Carlo (MCMC) in BayesTraits 2.0 (Pagel, Meade & Barker, 2004) using the ‘continuous random walk’ model. We also verified whether the variables evolved according to a Brownian model of evolution across the phylogenetic tree using BayesTraits 2.0 (Pagel et al., 2004). RESULTS Genome size estimations We present new GS estimates for 53 species belonging to the five genera of Pitcairnioideae (Table 1), 51 of which correspond to taxa that have not been previously studied, plus 45 species from the literature. From 405 Pitcairnia spp., we now have 2C-value estimates for 48 species (c. 12% of the total); for Fosterella, only three out of the 31 species have GS estimates (9.7%); for Deuterocohnia 22% (four species out of 18); for Dyckia 11% (19 species out of 168) and for Encholirium we have estimates of GS for 16 species (55% of 29) (Gouda et al., 2015, cont. updated; BFG, 2015; Forzza, 2017). We have therefore expanded the genome database for Pitcairnioideae by 15% and this now enables us investigate whether GS data can be used as a diagnostic character to distinguish between the five genera in this subfamily. The lowest 2C-value was found in Pitcairnia feliciana (A.Chev.) Harms & Mildbr. (0.6 pg) and the highest value was found in Encholirium ctenophyllum Forzza & Zappi (2.90 pg). Overall, the estimated 2C-values varied between genera, ranging from 0.74 to 1.73 pg in Deuterocohnia (average 1.11 pg), 1.58 to 2.38 pg in Dyckia (average 1.95 pg), 1.4 to 2.9 pg in Encholirium (average 1.93 pg), 0.91 to 1.86 pg in Fosterella (average 1.54 pg) and 0.6 to 1.78 pg in Pitcairnia (average 1.27 pg). From the 98 values estimated, 77.5% range from 1.0 to 2.0 pg (Table 1). FCM histograms used to infer the GS of each specimen showed peaks corresponding to the G0/G1 nuclei of the targeted species and the G0/G1 and G2 nuclei of the internal standard used here (Fig. 1). The G0/G1 peaks of all specimens included in this study can be clearly discriminated, and their coefficients of variation were always < 5%, which is considered suitable for GS determination using FCM (Cardoso, Martinelli & Latado, 2012). Figure 1. View largeDownload slide Fluorescence intensity histograms obtained in a) Deuterocohnia meziana Kuntze ex Mez, b) Dyckia pulquinensis Wittm., c) Encholirium luxor L. B. Sm. & R. W. Read, d) Fosterella windischii L. B. Sm. & R. W. Read, and e) Pitcairnia flammea Lindl. The x axis corresponds to an arbitrary scale of fluorescence intensity (proportional to the size of the genome), and the y axis represents the number of nuclei with that fluorescence intensity. Figure 1. View largeDownload slide Fluorescence intensity histograms obtained in a) Deuterocohnia meziana Kuntze ex Mez, b) Dyckia pulquinensis Wittm., c) Encholirium luxor L. B. Sm. & R. W. Read, d) Fosterella windischii L. B. Sm. & R. W. Read, and e) Pitcairnia flammea Lindl. The x axis corresponds to an arbitrary scale of fluorescence intensity (proportional to the size of the genome), and the y axis represents the number of nuclei with that fluorescence intensity. We used the Wilcoxon rank sum test to compare the average GS values of the five genera of Pitcairnioideae (Fig. 2). Significant differences in GS were observed between Deuterocohnia and Dyckia (W = 3, P < 0.01), Deuterocohnia and Encholirium (W = 5, P < 0.01), Dyckia and Pitcairnia (W = 1150, P < 0.01), and Encholirium and Pitcairnia (W = 1035.5, P < 0.01). All the other relationships tested presented P-values > 5%. Figure 2. View largeDownload slide Box plots showing the range of genome sizes (GSs) encountered in Deuterocohnia, Dyckia, Encholirium, Fosterella and Pitcairnia. The x-axis shows the genera and the y-axis shows GS (2C-values). Numbers in boxes represent the number of species sampled within each genus. Letters A and B above the boxplots indicate the different averages. Figure 2. View largeDownload slide Box plots showing the range of genome sizes (GSs) encountered in Deuterocohnia, Dyckia, Encholirium, Fosterella and Pitcairnia. The x-axis shows the genera and the y-axis shows GS (2C-values). Numbers in boxes represent the number of species sampled within each genus. Letters A and B above the boxplots indicate the different averages. Phylogenetic analysis The alignment length of 824 bp was obtained for the matK plastid region using 94 sequences from species belonging to Pitcairnioideae and sequences from three accessions of two species as outgroups, which included 152 variable sites (18.45%). Figure 3 shows the Bayesian consensus phylogenetic tree based on the matK gene. The majority of the species of the xeric clade (Deuterocohnia, Dyckia and Encholirium) were grouped into one clade with a high posterior probability (PP) (n2, PP = 1). In this clade, another containing all species of Dyckia and Encholirium was recovered (n3, PP = 0.95) in a polytomy. Figure 3. View large Download slide Bayesian consensus tree resulting from the matK alignment (824 bp length). Coloured dots on the branches indicate the posterior probability (PP) values: green dots represent values between 1 and 0.95; yellow dots represent values between 0.94 and 0.90; and red dots represent values smaller or equal to 0.89. The nodes are indicated with numbers. Values above and below the branches represent the ancestral genome size (GS; 2C-values in pg) at particular nodes: in blue is the value generated by the maximum likelihood (ML) method using StableTraits (asterisks are related to confidence interval values shown in Supporting Information, Appendix S2); orange is the value generated by the maximum parsimony (MP) method using Mesquite; black, given below the branches, is the value generated by Bayesian inference (BI) using BayesTraits. Different genera are indicated by coloured boxes. Light pink and Dark pink: Deuterocohnia; Purple: Dyckia; Yellow: Encholirium; Blue: Fosterella; Dark green and Light green: Pitcairnia. Genome size data (2C-values) obtained in this study () or taken from Bennett & Leitch (2011) (), Gitaí et al. (2014) () and Zonneveld et al. (2005) () are presented on the right of the phylogenetic tree. Figure 3. View large Download slide Bayesian consensus tree resulting from the matK alignment (824 bp length). Coloured dots on the branches indicate the posterior probability (PP) values: green dots represent values between 1 and 0.95; yellow dots represent values between 0.94 and 0.90; and red dots represent values smaller or equal to 0.89. The nodes are indicated with numbers. Values above and below the branches represent the ancestral genome size (GS; 2C-values in pg) at particular nodes: in blue is the value generated by the maximum likelihood (ML) method using StableTraits (asterisks are related to confidence interval values shown in Supporting Information, Appendix S2); orange is the value generated by the maximum parsimony (MP) method using Mesquite; black, given below the branches, is the value generated by Bayesian inference (BI) using BayesTraits. Different genera are indicated by coloured boxes. Light pink and Dark pink: Deuterocohnia; Purple: Dyckia; Yellow: Encholirium; Blue: Fosterella; Dark green and Light green: Pitcairnia. Genome size data (2C-values) obtained in this study () or taken from Bennett & Leitch (2011) (), Gitaí et al. (2014) () and Zonneveld et al. (2005) () are presented on the right of the phylogenetic tree. The species of the mesic genera, Fosterella and Pitcairnia, were grouped into one clade (n6, PP = 0.79), but in Pitcairnia the species grouped into two clades: n9, which had a PP = 1 and contained the most species (N = 13); and n15, which had a PP = 1 and contained nine species. All Fosterella spp. formed a single clade with a high probability value (n7, PP = 1). Deuterocohnia spp. were divided in the phylogenetic tree: some species [Deuterocohnia brevispicata Rauh & L. Hrom., Deuterocohnia meziana Kuntze ex Mez and Deuterocohnia scapigera (Rauh & L.Hrom.) M.A.Spencer & L.B.Sm.] appear in a polytomy with the other species of the xeric clade. The other species [Deuterocohnia brevifolia (Griseb.) M.A.Spencer & L.B.Sm., Deuterocohnia longipetala Mez, Deuterocohnia schreiteri A.Cast. and Deuterocohnia glandulosa E.Gross] form a sister group of the remaining Pitcairnioideae (n18, PP = 1). Ancestral genome size Many of the methods commonly used to reconstruct the ancestral state of a character assume a Brownian model of character evolution across the phylogenetic tree (Webster & Purvis, 2002). However, when certain evolutionary processes are involved in the evolution of the characters analysed, the Brownian model may not be adequate (Pagel, 1998; Freckleton & Harvey, 2006). In the present study, we verified whether the variables evolved according to a Brownian model of evolution, and the test revealed that a Brownian model is indeed sufficient according to the available data (P > 0.05). To further check this we chose to analyse the data using three methods, but we found no significant difference between them. The ancestral GS for Pitcairnioideae (n1, Fig. 3) reconstructed using MP was 1.29 pg, using ML was 1.24 pg (0.75–1.71, 95% highest posterior density) and based on BI was 1.21 ± 0.15 pg. Across the phylogenetic tree, both increases and decreases in GS were reconstructed relative to the ancestral value. The values obtained with the three methods varied little among themselves, as observed, for example, for node 2 (MP = 1.50, ML = 1.49 and MCMC = 1.49 ± 0.16), node 3 (MP = 1.84, ML = 1.81 and MCMC = 1.84 ± 0.04) and node 7 (MP = 1.50, ML = 1.50 and MCMC = 1.52 ± 0.23). DISCUSSION The GS values reported here (Table 1) for species that have previously been estimated by other researchers (and available in the Plant DNA C-values database) were found to be close. For example, the published values of Dyckia estevesii Rauh and Dyckia floribunda Griseb. are 1.60 and 1.58 pg, respectively, and that of Encholirium irwinii L.B.Sm. is 1.74 pg (Ebert & Till, 1997). No significant differences were observed between the GS of species classified as (1) Dyckia and Encholirium, (2) Dyckia, Encholirium and Fosterella, (3) Deuterocohnia and Pitcairnia, (4) Deuterocohnia and Fosterella or (5) Pitcairnia and Fosterella. This may be due to the low number of Fosterella samples analysed, and because the variation was higher in Deuterocohnia than in the other three genera. Considering the problems of species delimitation in the genera belonging to the xeric clade (Deuterocohnia and Dyckia plus Encholirium) (Schütz, 2011; Santos-Silva et al., 2013; Krapp et al., 2014; Schütz et al., 2016; M. N. Moura et al., unpubl. data), differences in GS might be expected if the morphological characteristics that differentiate the genera had evolved rapidly from a sudden change in GS, possibly as a result of numerical and structural chromosomal alterations. In fact, despite the small sample available for Deuterocohnia, it appears that changes in GS may have occurred during its divergence (average = 1.00 pg) from Dyckia and Encholirium (average = 1.95 and 1.93 pg/2C, respectively), given the significant differences in GS observed between these lineages (Fig. 2). It is possible that changes in GS may have been abrupt and were accompanied by morphological and anatomical changes in these two groups of the xeric clades (Deuterocohnia and Dyckia plus Encholirium) shortly after their separation. Alternatively, the changes in GS could have accumulated slowly in both groups (Santos-Silva et al., 2013). In the mesic genera (Pitcairnia and Fosterella), a similar scenario may perhaps be envisaged, because despite the broadly similar average 2C-values in Pitcairnia and Fosterella (1.27 and 1.54 pg, respectively), apomorphies have been identified in each of these genera (Santos-Silva et al., 2013; Saraiva et al., 2015). Our molecular phylogenetic results highlighted a low level of nucleotide divergence between the species of Pitcairnioideae and support the hypothesis that the genera of the xeric clade have diverged recently. Nevertheless, the results did not resolve Deuterocohnia as monophyletic, as recovered by Givnish et al. (2011) and Santos-Silva et al. (2013). Instead, these results corroborate those of Schütz (2011), Schütz et al. (2016) and M. N. Moura et al. (unpubl. data), reinforcing the possible division of Deuterocohnia; some species appear in a polytomy with the other taxa of the xeric clade, whereas the other species form a group that is distinct from all the remaining species of the subfamily (Fig. 3). This separation is also apparent in the GS data for the Deuterocohnia spp. as the average GS for the species that appear in the polytomy with Dyckia and Encholirium is 1.2 pg/2C, whereas the average GS for the species that form a distinct clade is about half of this value, 0.77 pg/2C. In the study by Schütz (2011), Deuterocohnia lorentziana (Mez) M.A.Spencer & L.B.Sm. was recovered in the clade containing Deuterocohnia longipetala and Deuterocohnia glandulosa, and in Gitaí et al. (2014) this species was reported with a GS of 1.73 pg/2C (Table 1), notably higher than the 2C-values reported for the other two species of the same clade (i.e. 0.74 and 0.80 pg/2C, respectively). However, polyploidy has been observed for this species in Gitaí et al. (2005) since the chromosome number reported was 2n = 50 and 2n = 100, and this may explain the large GS variation observed in the species in this clade. For Dyckia and Encholirium the chromosome number is usually 2n = 50 (Gitaí et al., 2005, 2014). Pitcairnia spp. formed two clades, as reported by Schütz et al. (2016) and Rex et al. (2009). In some studies using molecular (Givnish et al., 2004, 2007, 2011) and morphological (Saraiva et al., 2015) datasets, this genus has been shown to be monophyletic. In the study by Schütz et al. (2016), which comprises the most recent and complete phylogenetic analysis of Pitcairnioideae, monophyly of Pitcairnia was recovered only using nuclear DNA sequences. When plastid data were used, the results were the same as those found in the present study. Therefore, the delimitation of the genus remains controversial, despite Pitcairnia being recovered as monophyletic using a morphological dataset (Saraiva et al., 2015). The occurrence of cytotype diversity (i.e. different ploidies within a species) is apparent for two species of Pitcairnia for which GS is presented in this study (Table 1; Gitaí et al., 2014): Pitcairnia flammea Lindl. (2C = 1.69 pg, this study; 2n = 50/2n ≈ 100, Gitaí et al., 2014) and Pitcairnia sceptrigera Mez (2C = 1.2 pg; 2n = 50/2n ≈ 100, Gitaí et al., 2014). Without chromosome counts for the plants used to estimate GS it is not possible to determine whether the 2C-values reported are for the diploid or tetraploid cytotype of the species. For the other species presented in Table 1 that have karyotype data available, the diploid chromosome number is 2n = 50 (Gitaí et al., 2014). All species of Fosterella, the other mesic genus, grouped into one clade and GS was estimated for only three species (Table 1): Fosterella penduliflora (C.H.Wright) L.B.Sm. (2C = 1.86 pg, Bennett & Leitch, 2011), Fosterella villosula (Harms) L.B.Sm. (2C = 1.86 pg, Bennett & Leitch, 2011) and Fosterella windischii L.B.Sm. & R.W.Read (2C = 0.91 pg, this study). Polyploidy has also been reported for the first two species (2n = 100 and 2n = 150, respectively, Gitaí et al., 2014) and no karyotype data are available for the third. The reconstructed ancestral GS for Pitcairnioideae was 1.21 pg/2C according to the MCMC method, 1.24 pg according to the ML method and 1.29 pg according to the parsimony method. These values are smaller than the reconstructed ancestral GS of 1.45 pg/2C for all angiosperms (Puttick, Clark & Donoghue, 2015) and also smaller than 1.99 pg/2C estimated for the ancestral spermatophyte (Puttick et al., 2015). Overall, our analysis of GS evolution in Pitcairnioideae reported here fits the model of proportional GS evolution proposed by Oliver et al. (2007) which predicts that ‘the rate of GS evolution is proportional to the GS’, i.e. if a given taxon has an ancestor with a large GS, the descendants of the ancestor can have a small or large GS, giving rise to a large variation in GS. By contrast, if the GS of the ancestor is small, the descendants of the ancestor are likely to have small genomes, which results in only limited variance in GS. In this sense, we do not expect drastic changes in GS in a group that has arisen from ancestors with small GS, as shown in Figure 3. GS values have also been reported for other subfamilies of Bromeliaceae (Brocchinioideae, Bromelioideae, Puyoideae and Tillandsioideae) (Ebert & Till, 1997; Ramírez-Morillo & Brown, 2001; Zonneveld et al., 2005; Favoreto et al., 2012; Gitaí et al., 2014) and the 2C-values range from 0.64 pg [Orthophytum saxicola (Ule) L.B.Sm., Bromelioideae; Ramírez-Morillo & Brown, 2001] to 2.52 pg [Tillandsia usneoides (L.) L., Tillandsioideae; Zonneveld et al., 2005] (Gitaí et al., 2014). A notably higher 2C-value was reported by Favoreto et al. (2012) for Tillandsia loliacea Mart. ex Schult. & Schult.f. (3.34 pg), but most 2C-values fall between 1.0 and 2.0 pg (c. 70% of estimates) as observed in the present study for Pitcairnioideae. This indicates that the ancestral Bromeliaceae may also have had a small GS with the variation of 2C-values in the family tending to be skewed towards smaller values (Oliver et al., 2007). Bromeliaceae are the most species-rich angiosperm family, almost exclusively native to the New World (Givnish et al., 2011) and, although most 2C-values reported for the family show little variation, we found in this study with Pitcairnioideae some examples of larger variation in genera (Deuterocohnia and Pitcairnia; Table 1). This variation could be related to chromosomal numerical or structural changes, as reported by Gitaí et al. (2005, 2014). Kraaijeveld (2010) suggested that allopatric populations of organisms with small GS may have a higher rate of divergence than those with larger GS. He attributed this phenomenon to the assumption that in a small genome, the probability of phenotypic changes caused by mutations is larger than in a large genome, which has accumulated more non-coding DNA and repetitive DNA. In addition, Puttick et al. (2015) reported that the ability to alter GS (i.e. the rate of change of GS over time) exhibits the strongest correlation with species diversification rather than the size of the genome itself. In relation to the data presented here, we can perhaps assume that the high phenotypic diversity observed across Bromeliaceae and that is well represented in Pitcairnioideae may indeed be correlated with high rates of GS evolution of the species, given the short period of species diversification that is estimated for the family. In summary, the results obtained in this study improve our knowledge concerning GS in Pitcairnioideae and will contribute to a better understanding of the natural history of this subfamily and Bromeliaceae as a whole. In addition, the FCM data reported here contribute to our knowledge of GS diversity in Bromeliaceae, which is still poorly known given the large numbers of species in this family. The paper also highlights the value of FCM as a rapid and reliable technique for generating GS data, which can be analysed in conjunction with other molecular and morphological data to help elucidate patterns of evolution and phylogenetic relationships in this family. ACKNOWLEDGMENTS This study was carried out as part of a Masters and PhD degree in Ecology by M.N.M. at the Universidade Federal de Viçosa, who is grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Edital MCT/CNPq/MEC/CAPES No. 52/2010 – PROTAX) and the Coordenação de Pessoal de Nıível Superior (CAPES) for the scholarship. We thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the postdoctoral fellowship to M.P.C. (Process number: BPD-00037-13). This work was supported by grants from FAPEMIG (APQ-00366-12 - Edital 01/2012), CAPES and CNPq. R.C.F. received a Research Productivity Fellowship from CNPq (Proc. 303420/2016-2). We thank Prof. Lyderson Facio Viccini (Laboratório de Genética da Universidade Federal de Juiz de Fora) for enabling FCM measurements. We also thank Karla S. C. Yotoko (Laboratório de Bioinformática e Evolução da Universidade Federal de Viçosa) for her support in the laboratory and Janaína Gomes-da-Silva, Danon C. Cardoso, Guilherme M. A. Carvalho, Leandro Licursi and Natállia M. F. Vicente for critical reading and helpful suggestions. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Appendix S1. Sequences used in the phylogenetic analysis with their specimen voucher and GenBank accession number Appendix S2. Confidence interval of the values generated by the maximum likelihood method in StableTraits. 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Botanical Journal of the Linnean SocietyOxford University Press

Published: Mar 1, 2018

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