Ana Paula Moraes

Tatiana T Souza-Chies

Eudes M Stiehl-Alves

Paula Burchardt

Lilian Eggers

Sonja Siljak-Yakovlev

Spencer C Brown

Olivier Chauveau

Sophie Nadot

Mickaël Bourge

Lyderson Facio Viccini

Eliane Kaltchuk-Santos

Calydorea Herb C Alba

J A Castillo

C Approximata

R C Foster

H Crosae Roitman

H Darwinii Roitman

Gelasine G Herb

Coerulea

Evolutionary trends in Iridaceae: new cytogenetic findings from the New World

Keywords:

With the present work, we aim to provide a better understanding of chromosome evolutionary trends among southern Brazilian species of Iridoideae. Chromosome numbers and genome sizes were determined for 21 and 22 species belonging to eight genera of Tigridieae and two genera of Trimezieae, respectively. The chromosome numbers of nine species belonging to five genera are reported here for the first time. Analyses of meiotic behaviour, tetrad normality and pollen viability in 14 species revealed regular meiosis and high meiotic indexes and pollen viability (> 90%). The chromosome data obtained here and compiled from the literature were plotted onto a phylogenetic framework to identify major events of chromosome rearrangements across the phylogenetic tree of Iridoideae. Following this approach, we propose that the ancestral base chromosome number for Iridoideae is x =8 and that polyploidy and dysploidy events have occurred throughout evolution. Despite the variation in chromosome numbers observed in Tigridieae and Trimezieae, for these two tribes our data provide support for an ancestral base number of x = 7, largely conserved in Tigridieae, but a polyploidy event may have occurred prior to the diversification of Trimezieae, giving rise to a base number of x2 = 14 (detected by maximum-parsimony using haploid number and maximum likelihood). In Tigridieae, polyploid cytotypes were commonly observed (2x,4x,6x and 8x), whereas in Trimezieae, dysploidy seems to have been the most important event. This feature is reflected in the genome size, which varied greatly among species of Iridoideae, 4.2-fold in Tigridieae and 1.5-fold in Trimezieae. Although no clear difference was observed among the genome sizes of Tigridieae and Trimezieae, an important distinction was observed between these two tribes and Sisyrinchieae, with the latter possessing the smallest genome sizes in Iridoideae.

INTRODUCTION

Iridaceae (Asparagales; APG III, 2009) are a monocot family composed of 65-75 genera and > 2030 species divided into seven monophyletic subfamilies (Goldblatt et al., 2008). Among these subfamilies, Iridoideae is the only one with representatives in the Neotropics, the second most important centre of diversity for Iridaceae, with 900 known species in 30 genera (Goldblatt & Manning, 2008). In South America, three of the five tribes of Iridoideae (Sisyrinchieae, Tigridieae and Trimezieae) are represented (Goldblatt & Manning, 2008;Goldblatt et al., 2008). The 165 native species of Iridoideae in Brazil, including those endemic to the region, belong to 18 genera, 12 occurring in southern Brazil [START_REF] Eggers | Lista do Brasil -Iridaceae. Lista de Espécies da Flora do Brasil[END_REF]: Calydorea Herb., Catila Ravenna, Cypella Herb., Eleutherine Herb., Gelasine Herb., Herbertia Sweet, Kelissa Ravenna, Onira Ravenna, Phalocallis Herb. (Tigridieae); Neomarica Sprague and Trimezia Salisb. ex Herb. (Trimezieae); and Sisyrinchium L. (Sisyrinchieae) (Fig. 1).

Iridoideae, as in other subfamilies of Iridaceae, have diverse karyotypes, with polyploidy and dysploidy playing an important role in karyotype diversification [START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]. Iridaceae are cytologically well known, with chromosome numbers determined for > 1330 species (almost 65% of the species in the family), although most records are from the Northern Hemisphere and Africa [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF][START_REF] Heywood | Cytological studies in South American Iridaceae[END_REF][START_REF] Rudall | An anatomical and chromosomal investigation of Sisyrinchium and allied genera[END_REF][START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]. Chromosome data from South American species are scarce [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF][START_REF] Kenton | Genome size variation in Sisyrinchium L. (Iridaceae) and its relationship to phenotype and habitat[END_REF], particularly for natural populations [START_REF] Alves | Chromosome characterization and variability in some Iridaceae from northeastern Brazil[END_REF][START_REF] Souza-Chies | Studies on diversity and evolution of Iridaceae species in southern Brazil[END_REF][START_REF] Tacuatiá | Cytogenetic and molecular characterization of morphologically variable Sisyrinchium micranthum (Iridaceae) in southern Brazil[END_REF], 2012), and this has hampered any comprehensive analysis of chromosome evolution in subfamily Iridoideae. Furthermore, although the analysis of chromosome data in a phylogenetic context is essential for understanding chromosome evolution and its importance in species diversification, such studies have not been carried out in Iridaceae to date.

Despite the numerous species that have been analysed in this family, the base chromosome number is still uncertain, probably due to frequent chromosomal rearrangements throughout the family. [START_REF] Goldblatt | Phylogeny and classification of Iridaceae[END_REF] suggested x = 10, with polyploidy associated with dysploidy giving rise to derived numbers such as x 2 = 14, 12, 11, 9, 8, 7 and 5 [START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]. In addition to mitotic chromosome number, meiotic stability, tetrad normality (meiotic index) and pollen grain viability from natural populations are important characteristics that provide insight into the reproductive success of natural populations. Through an analysis of meiosis, chromosome number and chromosome behaviour are observed, and the morphology and viability of pollen grains can also be evaluated. Such analyses have been largely neglected in species of Iridaceae.

The nuclear DNA content, or genome size (GS or 2C), plays a role during species evolution (Bennett & Leitch, 2005) and has become a valuable trait in biosystematics [START_REF] Šmarda | Random distribution pattern and non-adaptivity of genome size in a highly variable population of Festuca pallens[END_REF][START_REF] Bennett | Nuclear DNA amounts in angiosperms: targets, trends and tomorrow[END_REF][START_REF] Pellicer | Insights into the dynamics of genome size and chromosome evolution in the early diverging angiosperm lineage Nymphaeales (water lilies)[END_REF][START_REF] Pellicer | A universe of dwarfs and giants: genome size and chromosome evolution in the monocot family Melanthiaceae[END_REF]. However, despite its importance, genome sizes have been estimated for only c. 1.8% of all angiosperm species [START_REF] Bennett | Plant DNA C-values database[END_REF]. Specifically in Iridaceae, DNA content has been reported for c. 150 out of 2030 species (7.4%), with 2C values ranging from 0.96 to 62.76 pg [START_REF] Siljak-Yakovlev | Towards a genome size and chromosome number database of Balkan Flora: C-values in 343 taxa with novel values for 242[END_REF][START_REF] Bennett | Plant DNA C-values database[END_REF][START_REF] Dagher-Kharrat | Nuclear DNA C-values for biodiversity screening: case of the Lebanese flora[END_REF][START_REF] Pustahija | Small genomes dominate in plants growing on serpentine soils in west Balkans, an exhaustive study of 8 habitats covering 308 taxa[END_REF]. In Iridoideae, no data are available for 42 species of Trimezieae (Goldblatt et al., 2008), and the GS values of just 12 of 1725 species (Goldblatt et al., 2008) of Tigridieae (0.69%) have been reported: two Cypella spp. and three species of Hesperoxiphion Baker (2C values ranging from 2.48 to 8.76 pg; [START_REF] Kenton | Nuclear DNA amount and karyotype symmetry in Cypella and Hesperoxiphion (Tigridieae: Iridaceae)[END_REF], Kelissa brasiliensis (Baker) Ravenna (2C = 17.50 pg), Gelasine elongata (Graham) Ravenna (= G. azurea Herb.) (2C = 3.50 pg), Cipura paludosa Aubl. (2C = 16.9 pg) and four species of Tigridia Juss. (from 7.75 to 16.20 pg) [START_REF] Zonneveld | First nuclear DNA amounts in more than 300 angiosperms[END_REF][START_REF] Bennett | Plant DNA C-values database[END_REF]. Concerning Sisyrinchieae, most reports are for Sisyrinchium L., with 33 estimates of 2C ranging between 1.0 and 8.4 pg [START_REF] Bennett | Plant DNA C-values database[END_REF].

Given the paucity of karyotype information about species of Iridoideae from southern Brazil, the present work was undertaken to increase our knowledge concerning (1) chromosome number and ploidy, (2) genome size and (3) meiotic behaviour, meiotic index and pollen viability of species of Iridoideae. Our data, combined with information taken from the literature, have been analysed in a phylogenetic framework [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF] to understand chromosome evolution better and reconstruct the ancestral base chromosome number in Iridoideae, with special attention paid to Tigridieae and Trimezieae.

MATERIAL AND METHODS

Most samples were collected in southern Brazil between 2006 and 2009 (Table 1). Part of the studied material is held in the Botanical Garden of Porto Alegre, Fundação Zoobotânica do Rio Grande do Sul, Brazil. Flower buds, seeds and bulbs were collected for chromosome studies and fresh leaves for flow cytometry analyses. Vouchers were deposited in the herbarium ICN (acronym according to [START_REF] Thiers | Index Herbariorum: a global directory of public herbaria and associated staff[END_REF]. Scientific names follow [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF].

MITOTIC ANALYSIS

Mitotic analyses were performed using root meristems and ovary walls. Materials were pre-treated with 8-hydroxyquinoline (2 mM) for 24 h at 10 °C, fixed in absolute ethanol/glacial acetic acid (3:1, v/v) for 24 h at room temperature and stored at -20 °C. For squash chromosome preparation, roots were hydrolysed in 5 M HCl for 20 min at room temperature and macerated in a drop of 45% acetic acid. Slides were frozen in liquid nitrogen and air dried. The best slides were stained in 2% Giemsa and mounted in Entellan (Merck). Alternatively, some slides were prepared following the standard Feulgen method. All observations were conducted using a Zeiss Axioplan Universal photomicroscope.

MEIOTIC ANALYSIS

Collected flower buds were fixed in ethanol/glacial acetic acid (3:1, v/v) for 24 h at room temperature and kept thereafter at -20 °C. Anthers were dissected, and pollen mother cells were mounted on a slide in a drop of 60% acetic acid. Slides were frozen in liquid nitrogen and air dried. The best slides were stained with 2% Giemsa and mounted in Entellan. Meiotic behaviour was evaluated by analysing the first 500 pollen mother cells observed. All available phases of meiosis I and II were analysed. Abnormalities, such as non-orientated bivalents and multivalents in metaphase I, bridges and laggards in anaphase and telophases I and II, were evaluated. Meiotic indexes were calculated using the formula: MI = (number of normal tetrads observed/total number of tetrads observed) × 100. Microcytes and micronuclei, bridges,

POLLEN STAINABILITY

Pollen stainability and pollen morphology were used to assess pollen viability. Flowers at anthesis were collected, fixed and stored as described above. Slides were prepared following Alexander's method [START_REF] Alexander | A versatile stain for pollen, fungi, yeast and bacteria[END_REF], in which empty, non-viable pollen grains stain green, and full, viable pollen grains stain purple. Samples of 500 pollen grains per flower were analysed from at least five individuals per species.

GENOME SIZE ESTIMATION

Total DNA content was assessed by flow cytometry according to [START_REF] Marie | A cytometric exercise in plant DNA histograms, with 2C values for 70 species[END_REF] and [START_REF] Doležel | Plant genome size estimation by flow cytometry: inter-laboratory comparison[END_REF]. Petunia hybrida Vilm. 'PxPc6' (2C = 2.85 pg; [START_REF] Marie | A cytometric exercise in plant DNA histograms, with 2C values for 70 species[END_REF] and Solanum lycopersicum 'Stupicke' (1.96 pg; [START_REF] Doležel | Plant genome size estimation by flow cytometry: inter-laboratory comparison[END_REF] were used as internal standards. Details about which standard species was used for each species of Iridaceae analysed are available upon request. Leaves of the studied species and the internal standards were chopped using a razor blade in a Petri dish with 600 μL [START_REF] Galbraith | Rapid flow cytometric analysis of the cell cycle in intact plant tissues[END_REF] nuclear-isolation buffer, supplemented with 10 mM sodium metabisulphite and 1% polyvinylpyrrolidone 10 000. The suspension was passed through a 50-μm mesh nylon filter (Cell-Trics, Partec) and nuclei were stained with 50-70 μg mL -1 propidium iodide with RNAse (2.5 U mL -1 ) at 4 °C. The DNA content of 5000-10 000 stained nuclei was determined for each sample using an Elite ESP flow cytometer (Beckman-Coulter) with an argon 488-nm laser, a Partec CyFlow 532-nm laser cytometer or a FACSCalibur 488-nm laser (Becton, Dickinson). The total 2C DNA value was calculated using the linear relationship between the fluorescent signals from the stained nuclei of the unknown specimen and the internal standard. Generally, three or five samples were assessed for each taxon and the standard deviation (SD) was calculated. However, for seven species [Calydorea campestris Baker, C. crocoides Ravenna, Catila amabilis Ravenna, Herbertia zebrina Deble, Phalocallis coelestis (Lehm.) Ravenna, Neomarica coerulea (Ker Gawl.) Sprague and Neomarica candida (Hassl.) Sprague] only one individual was available, and no SD could be calculated. The term 'monoploid genome size' (1Cx) was used to represent the DNA content in a basic chromosome set (x) of a somatic cell [START_REF] Greilhuber | Intraspecific variation in genome size in angiosperms: identifying its existence[END_REF], whereas 2C refers to the whole GS of a somatic cell.

CHROMOSOME EVOLUTION

The updated chromosome numbers described for Iridoideae were compiled from the literature and the IPCN (Index to Plant Chromosome Numbers -Missouri Botanical Garden; http://www.tropicos.org/ Project/IPCN) (Table 2). For each taxon, the haploid (n) and base chromosome numbers (x) were recorded. Following standard conventions, throughout this paper, we refer to 'x' as the ancestral base chromosome number of a lineage, x 2,3 . . . as the derived secondary, tertiary (. . .) base chromosome number of a [START_REF] Doležel | Nuclear DNA content and genome size of trout and human[END_REF]. †Due to the dynamic chromosome number variation with recurrent dysploidy, we could not be sure about the ploidy level for the majority of species. When just one species is uncertain in the genus, the ploidy level was indicated by '*'.

A. drummondii (Graham) R.C.Foster 14 (2x) +1B - - - A11 28 (2x) - - - A11, GT97 42 (4x) - - - A11 56 (6x) - - - A11 A. silvestris (Loes.) Goldblatt 28 (2x) - - - G82 A. veracruzana Goldblatt & T.M.Howard 28 (2x) - - - GH82 Cobana Ravenna: (1 / 2n = 28 / x = 14) C. guatemalensis (Standl.) Ravenna 28 (2x) - - - GT97 Eleutherine Herb.: (4 / 2n = 12 / x = 6) E. bulbosa (Mill.) Urb. 12 (2x) - - - BG90, G82, G88, G91, GS91 E. latifolia (Standl. & L.O.Williams) Ravenna 12 (2x) - - - GS91 Ennealophus N.E.Br.: (5 / 2n = 14 / x = 7) E. euryandrus (Griseb.) Ravenna 14 (2x) - - - GT97 E. foliosus (Kunth) Ravenna 14 (2x) - - - GT97 Gelasine
(2x) - - - G82 T. hallbergii Molseed 28 (2x) - - - G82 T. flammea (Lindl.) Ravenna 28 (2x) - - - G82 T. meleagris (Lindl.) G. Nicholson 28 (2x) - - - G82 T. mexicana Molseed 28 (2x) - - - G82 T. multiflora (Baker) Ravenna 28 (2x) - - - G82 T. orthantha (Lem.) Ravenna 28 (2x) - - - G82
. castaneomaculata A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. decora (Ravenna) A.Gil 18 (2x) - - - GIL N. decumbes (Ravenna) A.Gil 18 (2x) - - - GIL N. eburnea A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. floscella A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. glauca (Seub. ex Klatt) Sprague 18 (2x) - - - A11, GIL, G82, GT97, KH84 N. gracilis (Herb.) Sprague 16 (2x)* - - - TRB08 18 (2x) - - - GIL, GH92 N. humilis (Klatt) Capell. 18 (2x) - - - A11, GIL N. involuta A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. imbricata (Hand. Mazz.) Sprague 16 (2x)* - - - GIL N. itatiaica (Ravenna) A.Gil 18 (2x) - - - GIL N. longifolia (Link et Otto) Sprague 18 (2x) - - - GIL N. mauroi A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. northiana (Schneev.) Sprague 18 (2x) - - - A07, GIL, GT97, TRB08 N. portosecurensis (Ravenna) Chukr 18 (2x) - - - GIL N. cf. paradoxa (Ravenna) Chukr 28 (2x)* - - - A11 N. rupestres (Ravenna) Chukr 28 (2x) - - - CG08 N. sabinei (Lindley) Churk 18 (2x) - - - GIL N. sancti-vicentei A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. sergipensis A.Gil & M.C.E.Amaral 18 (2x) - - - GIL N. silvestres (Vellozo) Churk 18 (2x) - - - GIL N. unca (Ravenna) A.Gil 18 (2x) - - - GIL Neomarica sp. 1 28 (2x)* - - - PW Neomarica sp. 2 18 (2x) - - - A11 Neomarica sp. 3 28 (2x
‡The list of species from Sisyrinchieae is not an exhaustive collection, especially for Sisyrinchium. Species were chosen according to phylogeny (used in [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF] [START_REF] Chukr | Revisão de Trimezia Salisb. ex. Herb. (Iridaceae) para o Brasil[END_REF]; G75, [START_REF] Goldblatt | Revision of the bulbous plants of North America[END_REF]; G82, [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF]; GWZ84, [START_REF] Goldblatt | Index to plant chromosome numbers[END_REF]; GS91, [START_REF] Goldblatt | Systematics and chromosome cytology of Eleutherine Herbert (Iridaceae)[END_REF]; GH92, [START_REF] Goldblatt | Notes on Alophia (Iridaceae) and a new species, A. veracruzana, from Veracruz, Mexico[END_REF]; GT97, [START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]; G88, [START_REF] Guerra | Mitotic and meiotic analysis of a pericentric inversion associated with a tandem duplication in Eleutherine bulbosa[END_REF]; G91, [START_REF] Guerra | Cis-acting regulation of the NOR cistrons in Eleutherine bulbosa (Iridaceae)[END_REF]; GIL, A. Gil (unpubl. data); K02, [START_REF] Kalaiselvi | Karyomorphological studies in varieties of Gladiolus primulinus and Tigridia pavoniana[END_REF]; KH84, [START_REF] Heywood | Cytological studies in South American Iridaceae[END_REF]; KRJ86, [START_REF] Kenton | Genome size variation in Sisyrinchium L. (Iridaceae) and its relationship to phenotype and habitat[END_REF]; K87, [START_REF] Kenton | An unusual case of complexheterozygosity in Gelasine azurea (Iridaceae), and ITS implications for reproductive biology[END_REF]; K90, [START_REF] Kenton | Nuclear DNA amount and karyotype symmetry in Cypella and Hesperoxiphion (Tigridieae: Iridaceae)[END_REF] lineage, 2n as the chromosome number in somatic tissues and n as the haploid chromosome number observed in gametes [START_REF] Stebbins | Chromosomal variation and evolution[END_REF][START_REF] Guerra | Chromosome numbers in plant cytotaxonomy: concepts and implications[END_REF][START_REF] Peruzzi | x' is not a bias, but a number with real biological significance[END_REF]. Based on the recent phylogenetic hypothesis of [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF], the ancestral haploid and base chromosome numbers were reconstructed as categorical data with Mesquite v.2.73 [START_REF] Maddison | Mesquite: a modular system for evolutionary analysis[END_REF] under unordered maximum-parsimony (MP), applying ancestral character reconstruction analysis. For species without published chromosome data, but with consistent chromosome numbers across the genus, those chromosome numbers were assumed for the species without data (for codification details, see supporting information, Appendix S1).

In addition, the reconstruction of ancestral chromosome numbers was analysed with CHROM-EVOL v.2 (http://www.zoology.ubc.ca/prog/chromEvol .html) [START_REF] Mayrose | Probabilistic models of chromosome number evolution and the inference of polyploidy[END_REF][START_REF] Glick | ChromEvol: assessing the pattern of chromosome number evolution and the inference of polyploidy along a phylogeny[END_REF], which uses a maximum likelihood estimation (MLE) approach. This likelihood-based method tests a number of different models to evaluate chromosome number changes along the phylogenetic branches and infers which model best explains the chromosome number variation in extant species. We ran all four constant rate models because the best model has usually been found to be one of these four models [START_REF] Escudero | Karyotypic changes through dysploidy persist longer over evolutionary time than polyploid changes[END_REF]. For each species, the haploid chromosome number was defined and plotted into a counts file (Appendix S2). These models were fitted to our data, each with 10 000 simulations, and the one that best fitted the dataset was selected under the Akaike information criterion (AIC). The maximum number of chromosomes was set to 500 higher than the highest number found in the empirical data, and the minimum was set to 1.

RESULTS

Fifty-four accessions of 25 species of Iridaceae were collected in southern Brazil (Table 1). Chromosome numbers for 21 species belonging to ten genera, together with genome size for 22 species (Table 2) and meiotic behaviour/pollen viability for 14 species were determined (Table 3). Chromosome numbers were determined for nine species for the first time.

CHROMOSOME NUMBERS, KARYOTYPES AND C-VALUE DATA

Tigridieae species

Clade A (from [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF] All species of Tigridieae analysed by mitotic and/or meiotic chromosome counts had n = 7. The GS values obtained here varied four-fold, ranging from 2C = 3.90 to 15.43 pg, and the monoploid genome size (1Cx) varied 2.2-fold, ranging from 1.93 to 4.17 pg (Table 2). The chromosome numbers of five Calydorea spp. are reported here for the first time, with all having 2n = 14/n = 7: C. alba Roitman & J.A.Castillo, C. approximata R.C.Foster (Fig. 2A), C. basaltica Ravenna (Fig. 3A), C. campestris (Fig. 2B) and C. crocoides (Fig. 3B). Calydorea approximata had a bimodal karyotype with two pairs of large metacentric/submetacentric chromosomes and five pairs of small submetacentrics, one of them with a satellite (Fig. 2A). In agreement with observations of mitotic chromosomes, the bivalent configurations observed in C. campestris suggest that the karyotype is composed of submetacentric and metacentric chromosome pairs (Fig. 2B). By contrast, C. crocoides had six ring bivalents and one rod bivalent, indicating a pair of telocentric chromosomes in addition to the metacentrics and submetacentrics (Fig. 3B). Although all Calydorea spp. had the same diploid chromosome number and similar karyotypes, their GS varied 1.82fold. Calydorea approximata had a 2C value of 4.58 pg, whereas C. alba and C. basaltica had approximately twice this DNA content (8.34 and 7.72 pg, respectively); C. campestris and C. crocoides presented intermediate GS values, 6.96 and 6.90 pg, respectively (Table 2). Chromosome number was not determined for Catila amabilis in the present work, but its GS was 2C = 4.08 pg.

The chromosome number was 2n = 14 for all Cypella spp.: C. hauthalii (Kuntze) R.C.Foster subsp. opalina Ravenna (Fig. 2C), C. fucata Ravenna (Fig. 2D), C. aff. fucata and C. herbertii Herb. (Fig. 2E). Cypella spp., much like Calydorea, usually had two pairs of large chromosomes and five smaller ones, with one satellited pair. Cypella hauthalii subsp. opalina and C. herbertii had 2C values of c. 4 pg (Table 2), whereas C. laxa Ravenna had a slightly higher DNA content (4.83 pg).

In the present work, six Herbertia spp. were investigated, and four ploidies (2x, 4x, 6x and 8x) were found. Herbertia crosae Roitman & J.A.Castillo and H. darwinii Roitman & J.A.Castillo are diploids (2n = 14; Fig. 2I), whereas H. quareimana Ravenna (Fig. 2J) and H. pulchella Sweet (Fig. 3C,D) are tetraploids (2n = 28 and n = 14, respectively). Two cytotypes were found in H. lahue (Molina) Goldblatt; two populations were hexaploid (2n = 42) and another six were octoploid (2n = 56; Fig. 2K). Herbertia zebrina had its chromosome number determined here for the first time (2n = 14; Fig. 2L). Herbertia chromosomes were medium to large in size, with diploids presenting both large and small chromosome pairs, whereas polyploids presented a more gradual size decrease among chromosome pairs (compare Fig. 2I-L). Herbertia darwinii had two pairs of large chromosomes, one medium and other small with a satellited pair (Fig. 2I). The tetraploid H. quareimana had satellites on two pairs (Fig. 2J). Concerning DNA content, the 2C values in Herbertia increased with increasing ploidy: approximately 4.0 pg in diploids, 8.6 pg in tetraploids, 11.6 pg in the hexaploid and 15.4 pg in the octoploid. However, 1Cx decreased with ploidy, ranging from 1.93 to 2.34 pg, with the lowest content observed in the hexaploid and octoploid cytotypes of H. lahue. The Brazilian specimens of Kelissa brasiliensis and Onira unguiculata (Baker) Ravenna analysed here were diploids with 2n = 14 (Fig. 2G,H) and 2C = 4.73 and 4.57 pg, respectively.

Clade B

The first count for Gelasine coerulea (Vell.) Ravenna is presented here (2n = 14; Fig. 2F). The karyotype showed two large meta-and submetacentric chromosome pairs and up to three satellited chromosomes; hence, two chromosome pairs seem to bear satellites.

The DNA content was estimated at 2C = 5.05 pg but with a high coefficient of variation. Chromosome counting was not performed for Phalocallis coelestis, and the GS was determined to be 2C = 7.21 pg.

Trimezieae species

Three species were analysed in Trimezieae, and two haploid chromosome numbers (n = 9 and n = 14) were found. The 2C DNA values ranged from 7.6 to 8.7 pg, and 1Cx from 3.80 to 4.35 pg (Table 2).

The chromosome numbers of two Neomarica spp. were determined: Neomarica candida had 2n = 18/ n = 9 (Figs 2M,3E), whereas the other species (Neomarica sp.) had n = 14 (Fig. 3F). Genome size was estimated for N. caerulea and N. candida at 2C = 7.92 pg and 2C = 7.60 pg, respectively. The last species of Trimezieae investigated was Trimezia spathata Baker, with 2n = 28/n = 14 (Figs 2N,3G,H). This species had a symmetrical karyotype with metacentric and submetacentric chromosomes and a DNA content of 2C = 8.7 pg.

MEIOTIC BEHAVIOUR AND POLLEN VIABILITY

Species from eight genera were analysed for meiotic chromosome pairing and segregation, tetrad normality (meiotic index) and pollen grain stainability as an indicator of pollen grain viability (Table 3; Fig. 3). The chromosome numbers of three species (Calydorea crocoides, Herbertia pulchella and Neomarica sp.) could be determined through just meiotic analysis (Fig. 3B,C,F); for the remaining species, meiotic analysis confirmed the somatic chromosome number. All of the species presented highly regular meiosis (91.7-99.6%; Table 3), usually with bivalent (II) formation at diakinesis and metaphase I and regular disjunction (anaphase and telophase I and II). The most common abnormalities were non-orientated bivalents (Fig. 3H,I) and anaphase bridges (Fig. 3K), particularly in Calydorea basaltica, in either anaphase I or anaphase II, sometimes with fragments or laggards (Fig. 3L). In some cases, it was possible to observe multivalents in Neomarica candida (Fig. 3M).

Nine species showed meiotic indexes > 98% (Fig. 3N-P), and 12 had at least 90% viable pollen grains (Table 3); only Calydorea basaltica and Neomarica candida had < 90% fully stained pollen grains (Fig. 3P). Although Calydorea alba, Gelasine coerulea, Herbertia lahue and Onira unguiculata underwent no meiotic analysis, their high pollen stainability (> 98.8%) indicates regular meiosis for all.

RECONSTRUCTION OF ANCESTRAL CHROMOSOME

NUMBERS IN IRIDOIDEAE Chromosome numbers were recovered for 142 species of Iridoideae (151 species including the present work), and haploid and base chromosome numbers were determined for each genus and plotted against the phylogenetic tree of [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF] (Figs 4 and 5). Chromosome counts for Dietes Salisbury ex Klatt (Irideae) and Diplarrena Labill. (Diplarreneae), used as outgroups, were also recovered from the literature.

Haploid and base chromosome numbers reconstructed under MP (Mesquite; Fig. 4) and base chromosome numbers reconstructed under MLE (ChromEvol; Fig. 5) were broadly similar across simulations. For haploid number, an unordered MP analysis recovered 680 equally parsimonious trees, all with 14 steps, whereas for base chromosome number, 264 equally parsimonious trees with 11 steps were recovered. ChromEvol performed 10 000 simulations, and the best model, according to the AIC, was constant rate demi_est (Table 4; Fig. 5). Based on these results, polyploidy and descending dysploidy seem to have played important roles during the diversification of Iridoideae (Table 4). The base chromosome number reconstructed for Iridoideae was x = 8. Some derived base numbers, such as x = 5, 6, 7 and 9, probably arose through dysploidy, whereas x = 14, 18 and 19 were probably generated via polyploidy (Figs 4 and5).

A base number of x = 7 for Tigridieae was supported by both the MP and the MLE analyses. From the MLE analysis (Fig. 5), the number x 2 = 14 appears to have originated twice by polyploidy in clade B: first in the clade formed by Alophia Herb., which is a sister to the rest of clade B, and second, in the large derived clade comprising Cardiostigma Baker/Tigridia Juss./ Sessilanthera Molseed & Cruden/Cobana Ravenna (Fig. 5). MP also predicted these two polyploid events, but only when haploid chromosome numbers were reconstructed (i.e. in a shift from n = 7 to n = 14; Fig. 4).

Trimezieae showed considerable variation in chromosome number (Table 2), but due to the limited chromosome data available, contrasting results were obtained from the different analyses. The MP analysis using haploid number suggested an ancestral state of n = 10, but x = 7 was suggested when using the base chromosome number (Fig. 4). The MLE analysis indicated x = 14 for the tribe, double the ancestral haploid chromosome number suggested by MP (Fig. 5).

Sisyrinchieae also presented variability in haploid/ base chromosome number, as reconstructed by MP For species with polyploidy series, the lower haploid chromosome number was applied. Reconstructions of character ancestral states were conducted under MP following the phylogeny hypothesis proposed by [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF]. For tree 1 (n) arrows indicate events of polyploidization, while for tree 2 (x), arrows indicate the probably ancestral state in the origin of Tigridieae/Trimezieae tribes. Arrowhead/plus signal ascendant disploidy, while arrowhead/minus signal descendant disploidy events. Morphological synapomorphies for Tigridieae are represented (plicate leaves, bulb and monosulcate pollen grain). For data sources and codification details, see Appendix S1. and MLE, with an ancestral haploid number of n = 10 using MP and ancestral base numbers of x = 8 (MP, Fig. 4) or x = 9 (MLE, Fig. 5). A range of secondary base numbers, i.e. x 2 = 5, 9 and 19 (MP) and 18 and 19 (MLE), was also reconstructed for some derived lineages. Even in Sisyrinchieae, polyploidy was important, underlying x 2 = 19 in Libertia Raf. and x2 = 18 in Orthrosanthus Sweet (MLE, Fig. 5), although both were probably also associated with dysploidy. However, it is important to note the discrepancy between models relating to Orthrosanthus. An important difference was detected when using haploid number (n = 27, MP in Fig. 4) from MLE (x 2 = 18; Fig. 5) and the base chromosome number from MP (x = 5; Fig. 4). However, these differences are probably the consequence of a paucity of data.

DISCUSSION

The data obtained herein demonstrate the large variability in chromosome numbers, karyotype features and DNA content among species of Iridoideae. The ancestral base chromosome number suggested for the subfamily, x = 8, was concordant among analyses. Chromosome rearrangements gave rise by ascending dysploidy to x = 9 (MLE) or x = 8 (MP) in Sisyrinchieae or by descending dysploidy to x = 7 in Tigridieae (MP and MLE), which in turn, via polyploidy, gave rise to x = 14 in Trimezieae (MLE only). The MP haploid chromosome number analysis further suggested that polyploidy has been important in the evolution of Tigridieae, as both n = 7 and n = 14 were common. This trend was also reflected in the MLE statistical analysis. However, dysploidy is also clearly an important evolutionary mechanism, and reconstruction models suggest that it is operating in all three tribes.

TIGRIDIEAE SPECIES

The conserved base chromosome number for Tigridieae, x = 7, was supported by all analyses, reinforcing the Tigridieae monophyly hypothesis, as did the presence of a bimodal karyotype composed of two long and five short chromosome pairs [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF][START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]. Based on morphological and cytogenetic criteria, [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF] divided Tigridieae into two subtribes, Cipurinae and Tigridiinae, both exclusively from the New World. Such a division is partly supported by [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF]: Clade A corresponds to Cipurinae and Clade B to Tigridiinae. Some discrepancies were observed between these two proposals. Ainea conzattii (R.C.Foster) Ravenna, previously associated with Tigridia (Tigridiinae; Goldblatt et al., 2008), was grouped with Calydorea undulata Ravenna, C. pallens Griseb. and Catila amabilis/Nemastylis Nutt. by [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF], consistent with the present analysis. Secondly, Phalocallis coelestis, commonly associated with Cypella (Cipurinae, Clade A; Goldblatt et al., 2008), was grouped with Gelasine elongata (Graham) Ravenna and Eleutherine Herb. (Tigridiinae, Clade B in [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF].

Considering the clades within Tigridieae, the base chromosome number suggested for Cipurinae/Clade A was x = 7, in accordance with previous authors, presenting a ploidy series without dysploidy events [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF][START_REF] Kenton | Nuclear DNA amount and karyotype symmetry in Cypella and Hesperoxiphion (Tigridieae: Iridaceae)[END_REF][START_REF] Alves | Chromosome characterization and variability in some Iridaceae from northeastern Brazil[END_REF]. On the other hand, Tigridiinae/Clade B presents more variable haploid numbers, which probably arose from both polyploidy and descending dysploidy events, two important events suggested by both the MP and the MLE analyses. [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF] suggested a palaeopolyploidy event at the base of Tigridiinae, giving rise to n = x 2 = 14. However, our data suggest two independent polyploidy events across the clade (see circles 16 and 17 in Fig. 5), in addition to two descending dysploidy events [x 2 = 6 in Eleutherine (circle 18 in Fig. 5) and x 2 = 5 in Phalocallis]. These results suggest that a secondary base number of x 2 = 14 is a derived rather than an ancestral state in Tigridiinae/Clade B.

Ploidy series were commonly observed among genera of Tigridieae, e.g. Calydorea [C. azurea Klatt and C. pallens,2n = 4x = 28,and C. xiphioides (Poepp.) Espinosa, 2n = 6x = 42 [START_REF] Goldblatt | Chromosome cytology in relation to suprageneric systematics of Neotropical Iridaceae[END_REF][START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF][START_REF] De Tullio | Tamia (Iridaceae), a synonym of Calydorea: cytological and morphological evidence[END_REF]] and Herbertia in particular. In accordance with the pat- terns found here, four ploidies (2x, 4x, 6x and 8x) have been reported for Herbertia: 2x, 4x and 6x individuals in H. pulchella (4x was found here; also see [START_REF] Winge | Studies on cytotaxonomy and polymorphism of the genus Alophia (Iridaceae)[END_REF][START_REF] Heywood | Cytological studies in South American Iridaceae[END_REF][START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]) and 2x, 4x, 6x and 8x individuals in H. lahue (6x and 8x were found here; also see [START_REF] Winge | Studies on cytotaxonomy and polymorphism of the genus Alophia (Iridaceae)[END_REF][START_REF] Heywood | Cytological studies in South American Iridaceae[END_REF][START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF][START_REF] Moreno | Cytogenetic studies in Herbertia Sw. (Iridaceae)[END_REF]. The other three Herbetia spp. studied here (H. crosae, H. darwinii and H. quareimana) showed just one ploidy. As expected, in Herbertia, the GS ranged according to ploidy, with diploids and octoploids presenting lower and higher contents, respectively (Fig. 6). However, considering the 1Cx values, Herbertia seems to have undergone the wellknown genome downsizing phenomenon following polyploidy [START_REF] Leitch | Genome downsizing in polyploid plants[END_REF][START_REF] Leitch | Genomic plasticity and the diversity of polyploid plants[END_REF][START_REF] Bennett | Nuclear DNA amounts in angiosperms: targets, trends and tomorrow[END_REF], with hexaploids and octoploids having lower 1Cx values than their diploid relatives. Similar results were found in Kelissa brasiliensis (Fig. 6): diploid, 2n = 2x = 14, had 2C = 4.73 pg/ 1Cx = 2.36 pg, whereas the octoploid reported in the literature, 2n = 8x = 56, had 2C = 17.36 pg/ 1Cx = 2.16 pg [START_REF] Bennett | Plant DNA C-values database[END_REF]. Despite the recent improvements in understanding the phylogenetic relationships in Iridoideae, some taxonomic uncertainties still persist in Tigridieae. The alliance formed among Calydorea, Cypella, Herbertia, Kelissa and Onira needs more attention in view of the phylogenetic principles of monophyly. The reconstruction performed by [START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF] revealed that Cypella and Calydorea were not monophyletic but rather split into unrelated lineages and grouped with species of different genera. No morphological apomorphy has been described for Cypella, and, further, Cypella, Herbertia, Kelissa and Onira do not have contrasting karyotypes. These observations highlight the need to identify and employ other markers to elucidate such relationships and thus increase our understanding of the diversification of Iridaceae in South America. On the other hand, chromosome and GS data did support the taxonomic separation of Phalocallis from Cypella (Figs 4-6), contra Goldblatt & Manning (2008). Cypella coelestis and C. plumbea were previously considered synonyms of Phalocallis coelestis (2n = 10; 2C = 7.21 pg) by [START_REF] Ravenna | Notas sobre Iridaceae V[END_REF] and the divergence of chromosome data between this taxon and other Brazilian Cypella spp. (2n = 14; 2C = about 4 pg) reinforces the position of this species in the genus Phalocallis, as also accepted by [START_REF] Barker | World checklist of Iridaceae[END_REF] and [START_REF] Eggers | Lista do Brasil -Iridaceae. Lista de Espécies da Flora do Brasil[END_REF].

TRIMEZIEAE SPECIES

Species of Trimezieae show a large range of base numbers (x = 8, 9, 10, 13, 14; see Table 2). Such dynamic chromosome evolution is probably a result of cycles of polyploidy followed by increasing or decreasing dysploidy events [START_REF] Goldblatt | Chromosome cytology of Iridaceae -patterns of variation, determination of base numbers, and modes of karyotype change[END_REF]Goldblatt & Manning, 2008). The molecular evidence strongly supports Trimezieae as monophyletic and being composed of four major clades [START_REF] Lovo | New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?[END_REF], but, morphologically, the tribe lacks robust synapomorphies (Goldblatt et al., 2008;[START_REF] Chauveau | Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera[END_REF].

Concerning Neomarica, chromosome numbers and karyotype features have been obtained for ten species, seven of which have been taxonomically well determined (Table 2). Despite the chromosome number variation, based on counts for 22 Neomarica spp., 2n = 2x = 18 is suggested for the genus (A. Gil, unpubl. data). If so, then some species with alternative chromosome numbers may need to be transferred to new genera. Neomarica sp., 2n = 2x = 28, despite lacking taxonomic definition, showed the same chromosome number as Neomarica cf. paradoxa, n = x 2 = 14, and it is possibly the same species as that analysed by [START_REF] Alves | Chromosome characterization and variability in some Iridaceae from northeastern Brazil[END_REF]. However, taking into account such discordant base numbers and some morphological differences in N. cf. paradoxa, the revision of the generic taxonomy of this genus is important. With respect to Trimezia spathata, [START_REF] Chukr | Revisão de Trimezia Salisb. ex. Herb. (Iridaceae) para o Brasil[END_REF] reported two chromosome numbers: 2n = 28 and 60 (x = 7 and 5; Table 2). Nevertheless, [START_REF] Heywood | Cytological studies in South American Iridaceae[END_REF] considered T. spathata (cited as T. martii) as a diploid (x 2 = 14) because 14 bivalents are formed with high chiasma frequency, in agreement with our observations (Table 3). In this case, T. spathata, with 2n = 28 and 60, is most probably a diploidized palaeopolyploid. The polyphyletic Trimezia varies greatly in chromosome numbers (2n = 26, 28, 40, 52, 54, 60, 80, 82; Table 2), making it difficult to establish a relationship between chromosome number and species and to define the ancestral base number.

The MP and MLE analyses performed here suggested x = 7/14 for Trimezieae, with recurrent dys-ploidy within the tribe. The x = 14 suggested by MLE is probably a result of a polyploidy event prior to the diversification of the tribe. However, considering the haploid number of n = 10 reconstructed by MP, this incongruence is most probably a result of the diverse chromosome numbers reported for the tribe. Morphologically, Trimezieae are diverse, and no morphological character has so far been defined, even for the four molecular clades defined in the tribe [START_REF] Lovo | New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?[END_REF].

The large chromosome number variation detected in Trimezieae could not be fully represented in our MP and MLE analyses, particularly given the underrepresentation of species in the phylogenetic tree. Additionally, no chromosome data were available for Pseudotrimezia, resulting in a gap of data for the Trimezieae. In addition, molecular studies by [START_REF] Lovo | New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?[END_REF] revealed a complex organization of Trimezieae, with four clades that do not correspond to specific genera. Furthermore, Neomarica and Trimezia were both split into three different clades, suggesting that they are not monophyletic (see also [START_REF] Gil | Seven new combinations in the genus Neomarica (Trimezieae-Iridaceae)[END_REF]. Without a clear taxonomic description with defined autopomorphies for each genus and a well-supported and sampled phylogenetic tree, such diverse chromosome data created by successive cycles of polyploidy and dysploidy are difficult to analyse, even using statistical models, and the mechanisms underlying the chromosome number variations in Trimezieae remain unknown.

CONCLUDING REMARKS

Species of Iridaceae show broad variation in morphological features, which has resulted in taxonomic controversies. Different approaches are therefore needed to provide additional clues about species relationships. Despite the large variability in chromosome number and size in this family, cytological research and GS estimations have been useful tools in understanding the evolution of taxa at different taxonomic levels and groups with different geographical origins or ecological specializations [START_REF] Šmarda | Random distribution pattern and non-adaptivity of genome size in a highly variable population of Festuca pallens[END_REF]Goldblatt & Manning, 2008;[START_REF] Sonnleitner | Distribution and habitat segregation on different spatial scales among diploid, tetraploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the eastern Alps[END_REF][START_REF] Pellicer | Insights into the dynamics of genome size and chromosome evolution in the early diverging angiosperm lineage Nymphaeales (water lilies)[END_REF][START_REF] Pellicer | A universe of dwarfs and giants: genome size and chromosome evolution in the monocot family Melanthiaceae[END_REF].

In light of the paucity of chromosome and genome size data for South American Iridaceae, this study provides new data and corroborates some published data. Above all, the present study has brought together, in a phylogenetic context, all of the new and previous chromosome data related to Iridoideae. The diversity of ploidy and haploid and base chromosome numbers, analysed using two independent statistical modelling approaches, demonstrates the complexity of cytogenetic character evolution in this family, underlining the importance of polyploidy and descending dysploidy as primary mechanisms of chromosome evolution, but with different events occurring in different tribes. Polyploidy events are frequent in Iridoideae, and the occurrence of intrageneric/ intraspecific polyploidy suggests that polyploid cytotypes are common, particularly in Tigridieae/Clade A. However, the dysploidy events usually associated with Iridaceae appear to be especially important in Tigridieae/Clade B and in Trimezieae.

Figure 1 .

 1 Figure 1. Iridaceae flowers. A, Calydorea campestris; B, C. crocoides; C, Cypella fucata; D, C. hauthalii subsp. opalina; E, Gelasine coerulea; F, Herbertia pulchella; G, H. quareimana; H, H. zebrina; I, Kelissa brasiliensis; J, Onira unguiculata; K, Phalocallis coelestis; L, Trimezia spathata. All photographs were taken by A. P. Moraes, except D, H and K taken by L. Eggers. Bar in K and L represents 1 cm.

Figure 2 .

 2 Figure 2. Mitotic metaphases of Iridaceae species. A, Calydorea approximata,2n = 14; B, C. campestris,2n = 14; C, Cypella hauthalii subsp. opalina,2n = 14; D, C. fucata,2n = 14; E, C. herbertii,2n = 14; F, Gelasine coerulea,2n = 14; G, Kelissa brasiliensis,2n = 14; H, Onira unguiculata,2n = 14; I, Herbertia darwinii,2n = 14; J, H. quareimana,2n = 28; K, H. lahue,2n = 56; L, H. zebrina,2n = 14; M, Neomarica candida,2n = 18; N, Trimezia spathata,2n = 28. Arrows in A, F, I and J indicate secondary constrictions. Bar in N represents 10 μm.

Figure 3 .

 3 Figure 3. See caption on next page.

Figure 3 .Figure 4 .

 34 Figure 3. Meiotic analysis of Iridaceae species -haploid chromosome numbers and observed meiotic abnormalities. A, Calydorea basaltica, n = 7 (metaphase I); B, C. crocoides, n = 7 (metaphase I); C, Herbertia pulchella, n = 14 (metaphase I); D, H. pulchella n = 14 (prophase II); E, Neomarica candida, n = 9 (metaphase II); F, Neomarica sp., n = 14 (prophase I -diakinesis I); G, Trimezia spathata, n = 14 (prophase II); H, T. spathata in metaphase I with non-orientated bivalents (arrows); I, C. campestris in metaphase I with univalents (arrows); J, T. spathata in anaphase I with bridge (arrow); K, N. candida in anaphase I with bridge (arrow) and two laggard chromosomes (arrowheads); L, Calydorea basaltica in anaphase II with bridge (arrow); M, N. candida in metaphase I with tetravalent ring (arrow); N, C. crocoides showing 'tetrad' with seven microspores; O, N. candida showing tetrad with microcyte (arrow); P, N. candida showing viable pollen grain (V) and an unviable micro pollen grain (Unv). Bar in P represents 10 μm.◀

Figure 5 .

 5 Figure 5. Base chromosome number estimate and evolution in Iridoideae inferred under Bayesian optimization. For species with polyploidy series, different haploid numbers were plotted considering the frequency of each number. The phylogeny hypothesis used was proposed by Chauveau et al. (2012) and simulation of base chromosome number followed the constant rate demi_est model. Base number and posterior probabilities are mainly indicated at nodes. For data sources and codification details, see Appendix S2.

Figure 6 .

 6 Figure 6. Genome size (2C) and base chromosome number (x) variation in Iridoideae. Filled circles represent the 2C value described for species and the number below species indicates the base chromosome number. Genera are organized according to the phylogeny hypothesis proposed by Chauveau et al. (2012). For detailed information see Appendix S1.

Table 1 .

 1 Species investigated in Tigridieae and Trimezieae tribes of Iridaceae, voucher specimen and geographical origin

	Taxa	Collector number	Origin [State, City]	Latitude	Longitude

and unequally sized cells were considered abnormal. Chromosome numbers were determined in diakinesis (prophase I).

Table 1 .

 1 Continued

	Taxa	Collector number	Origin [State, City]	Latitude	Longitude
	Trimezieae
	Neomarica Sprague
	N. caerulea (Ker Gawl.) Sprague	IRI 02 †
	N. candida (Hassl.) Sprague	IRI 04 †
		IRI 43	RS, Dom Pedro de Alcântara	-29.363*	-49.853*
	Neomarica sp.	ESC 338	PR, Sengés	-24.061	-49.521
	Trimezia Salisb. ex Herb.
	T. spathata Baker	ESC 368	PR, Guarapuava	-25.367	-51.334
		IRI 09	RS, Derrubadas	-27.264*	-53.865*
		IRI 12	RS, Julio de Castilhos	-29.230*	-53.677*
		IRI 61	RS, Lajeado	-29.451*	-52.010*
		TS (no number) ‡	RS, Caçapava do Sul	-30.589*	-53.478*
	ESC, Eggers, L. & Souza-Chies, T.T.; IRI, Porto Alegre Botanical Garden collection; RS, Rio Grande do Sul; PR, Paraná;
	SC, Santa Catarina.
	*Municipality geographical coordinates obtained from GoogleEarth.
	†Without data of origin and in cultivation at Botanical Garden.
	‡No number available, live specimen in cultivation, awaiting voucher.

Table 2 .

 2 Continued

	Taxa	2n (ploidy level) 2C (pg) 1Cx (pg) 2C (Mbp) Reference(s)
	N. tenuis subsp. pringlei (S.Watson)	14 (2x)	-	-	-	G75
	Goldblatt
	N. tenuis var. nana (S.Watson)	14 (2x)	-	-	-	G82
	R.C.Foster
	Onira unguiculata (Baker) Ravenna:	14 (2x)	4.57	2.28	4470	PW
	(1 / 2n = 14 / x = 7 / 2.28)
	Clade B
	Alophia Herb.: (5 / 2n = 28 / x = 14)

Table 2 .

 2 Continued

	Taxa	2n (ploidy level) 2C (pg) 1Cx (pg) 2C (Mbp) Reference(s)
	T. pavonia (L.f.) DC.	26 (2x)*	8.70	4.35	8508	BL12, K02
		28 (2x)	-	-	-	S70
	T. vanhouttei Roezl ex Van Houtte	28 (2x)	-	-	-	G82
	Trimezieae (3 / 42)
	Neomarica Sprague: (21 / 2n = 16, 18, 22,
	28, 32 / x = 8, 9, 14 / 3.80-3.96)
	N. altivallis (Ravenna) A.Gil	18 (2x)	-	-	-	GIL
	N. brachypus (Baker) Sprague	18 (2x)	-	-	-	GIL
	N. caerulea (Ker Gawl.) Sprague	18 (2x)	7.92	-	7750	A11
		32 (4x)	-	-	-	GIL, G82, GT97,
						KH84, ZBS85
	N. candida (Hassl.) Sprague	18 (2x)	7.60	3.80	7430	A07, A11, PW
	N

Table 2 .

 2 Continued

	Taxa	2n (ploidy level) 2C (pg) 1Cx (pg) 2C (Mbp) Reference(s)
	T. spathata Baker	28 (2x)	8.70	4.35	8510	PW
		60 (6x)				CG08
	As Trimezia martii and T. sincorana	60 (6x)	-	-	-	KH84
	T. steyermarkii R.C.Foster	52 (5x)	-	-	-	GT97, TRB08
	T. truncata Ravenna	26 (2x)	-	-	-	CG08
		28 (2x)	-	-	-	CG08
	Sisyrinchieae ‡ (6 / c. 177)
	Sisyrinchium L. (194 / 2n = 18, 36, 34, 48,
	96 / x 2 = 8; 9; 17 / 0.35-2.10)
	S. angustifolium Miller	96 (12x)				MM76
	S. californicum (Ker Gawl.) Dryand.	32 (4x)	1.40	0.35	1369	KRJ86
	S. chilense Hook.	16 (2x)	1.46	0.73	1427	G82, KH84, KRJ86
	S. convolutum Nooca	36 (4x)	1.90	0.48	1858	G82, KH84, KRJ86
		72 (9x)	4.20	0.53	4107	KRJ86
	S. longipes (E.P.Bicknell) Kearney &	34 (4x)	1.40	-	1369	KRJ86
	Peebles
	S. micranthum Cav.	32 (4x)	-	-	-	G82
		48 (6x)	2.90	0.48	2836	KRJ86
	S. minus Engelmann & A.Gray	10 (?)				OL62
	S. pachyrhizum Baker	96 (12x)	7.00	0.58	6846	KRJ86
	S. platense I.M.Johnst.	48 (6x)	3.40	0.57	3325	KRJ86
	S. striatum Sm.	18 (2x)	2.50	1.25	2445	KRJ86
	S. tenuifolium Humb. & Bonpl. ex Willd.	36 (4x)	2.40	0.60	2347	KRJ86
	S. tinctorium Kunth	36 (4x)	1.00	0.25	978	KRJ86
	S. vaginatum Spreng. (cited as	36 (4x)	8.40	2.10	8215	KRJ86
	Sisyrinchium alatum Hook.)
	Olsynium Raf.: (14 / 2n = 18 / x = 8, 9 / 3.55)
	O. biflorum Goldblatt (cited as	18 (2x)	7.10	3.55	6943	G82, KRJ86
	Phaiophleps biflora (Thunb.) R.C.Foster)
	O. douglasii (A.Dietr.) E.P.Bicknell	64 (8x)	-	-	-	CH02
	O. douglasii (A.Dietr.) E.P.Bicknell (cited	18 (2x)	-	-	-	KH84
	as Sisyrinchium grandiflorum Dougl. ex
	Lindl.)
	O. douglasii (A.Dietr.) E.P.Bicknell (cited	96 (12x)	-	-	-	KRJ86, RKL86
	as Sisyrinchium douglasii A. Ditr.)
	O. filifolium (Gaudich.) Goldblatt (cited as	18 (2x)	-	-	-	KH84, KRJ86
	Sisyrinchium filifolium Gaudich.)
	O. junceum (E.Mey. ex C.Presl)	20 (2x)	-	-	-	KH84, R86, RKL86
	Goldblatt (cited as Sisyrinchium
	junceum E.Mey.)
	O. scirpoideum subsp. scirpeum (Phil.)
	R.A.Rodr. & Martic. [cited as
	Sisyrinchium scirpoideum subsp.
	scirpeum (Phil.) Rodr.Rios.]
	O. scirpoideum subsp. scirpoideum	18 (2x)				R86
	(Poepp.) Goldblatt (cited as
	Sisyrinchium scirpoideum subsp.
	scirpoideum Poepp.)
	Libertia Spreng.: (14 / 2n = 18, 38, 76, 228 /
	x = 9 / x 2 = 19 / 3.55)
	L. biflorum (Thunb.) Goldblatt [cited as	18 (2x)	7.10	3.55	6943	G82, KRJ86
	Phaiophleps biflora (Thunb.) R.C.Foster]

L. cranwelliae Blanchon, B.G.Murray & Braggins

Table 2 .

 2 Continued

	Taxa	2n (ploidy level) 2C (pg) 1Cx (pg) 2C (Mbp) Reference(s)
	L. chilensis (Molina) Gunckel [cited as	76 (4x)				B00
	L. formosa Graham]
	L. ixioides (G.Forst.) Spreng.	228 (12x)				B00
	L. edgariae B lanchon, B.G.Murray &
	Braggins
	L. grandiflora (R.Br.) Sweet
	L. micrantha A.Cunn.
	L. mooreae Blanchon, B.G.Murray &
	Braggins
	L. peregrinans Ckn. & Allan
	L. paniculata (R.Br.) Spreng.
	L. pulchella Spreng.
	L. sessiliflora Skottsb. [cited as	38 (2x)				B00
	L. coerulescens Kunth & Bouché]
	Orthrosanthus Sweet: (9 / 2n = 40, 50, 54
	and 84/ x = 10) †
	O. acorifolius (Kunth) Ravenna	54 (5x)	-	-	-	GT97
	O. chimboracensis (Kunth) Baker	54 (5x)	-	-	-	GT97
	O. var. exsertus R.C.Foster	50 (5x)	-	-	-	KH84
	O. polystachyus Benth.	40 (4x)	-	-	-	KH84
	Solenomelus Miers:
	S. segethi Kuntze [cited as Solenomelus	48 (2x)				RKL86
	sisyrinchium (Griseb. in Lechler) Pax]
	Outgroup
	Dietes Salisbury ex Klatt (Irideae): (6 /
	2n = 60 / x = 10)
	D. robinsoniana (F.Meull.) Klatt	60 (2x)	-	-	-	GT97
	Diplarrena Labill. (Diplarreneae): (2 /
	2n = 32 / x = 16)
	D. moraea Labill.	32 (2x)	-	-	-	KH84
	D. latifolia Benth.	32 (2x)	-	-	-	KH84

Unpublished counts are in bold type. Parentheses after tribes indicate (total number of genera/total number of species), while parentheses after genera indicate (total number of species/chromosome number recorded for the genera/base chromosome number suggested/1Cx range). *1 pg = 978 Mbp

  and those that have both chromosome number and genome size published.No Iris species are listed here as the genus presents many diploid chromosome numbers, and both polyploidy and dysploidy are frequent, hampering analysis of chromosome number evolution. Underlined number in Neomarica and Trimezia indicates the most common data observed.

	References: A07, Alves & Felix (2007); A11, Alves et al. (2011); AOC02, Aaron & Ortiz-Catedral (2002); BG90, Beltrão &
	Guerra (1990); B00, Blanchon et al. (2000); BL12, Bennett & Leitch (2012); CH02, Cholewa & Henderson (2002); CG08,

Table 3 .

 3 Haploid chromosome number, meiotic normality, meiotic indexes and pollen fertility in analysed species of Iridaceae

			Meiosis I and II	Meiotic index		Pollen viability
	Taxa	N	N*	% †	N*	% †	N*	% †
	Tigridieae
	Clade A
	Calydorea
	C. alba	7					5 (2500)	98.96
	C. approximata	7	2 (160)	96.25			5 (2500)	98.52
	C. basaltica	7	1 (500)	98.90			5 (2500)	87.48
	C. campestris	7	4 (2000)	98.75	12 (2400)	99.80	20 (10.000)	91.06
	C. crocoides	7	2 (1000)	99.00	4 (800)	98.70	10 (5000)	93.58
	Cypella
	C. herbertii	7	4 (144)	91.70	2 (200)	99.00	5 (2500)	98.70
	Herbertia
	H. lahue	28					3 (1500)	98.07
	H. pulchella	14	3 (300)	99.30	8 (1600)	100	10 (5000)	99.02
	Kelissa
	K. brasiliensis	7	1 (500)	99.20	5 (1000)	100	5 (2500)	97.90
	Onira
	O. unguiculata	7			5 (1000)	100	5 (2500)	98.40
	Clade B
	Gelasine
	G. coerulea	7					10 (5000)	98.88
	Trimezieae
	Neomarica
	N. candida	9	6 (3000)	99.55	6 (1200)	99.75	10 (5000)	72.30
	Neomarica sp	14	4 (2000)	98.00	3 (600)	99.8	5 (2500)	94.36
	Trimezia
	T. spathata	14	7 (4500)	99.60	17 (3400)	99.77	20 (10000)	90.26
	*N, number of individuals analysed (number of cells).
	†Percentage of normal cells.

Table 4 .

 4 Log-likelihood and AIC estimates for the data set analysed for the constant rate model implemented by ChromEvol software

		Log-				Demi-
	Model	likelihood AIC	Gain Loss	Duplication	duplication
	Constant gain, loss and duplication	-176	358.1
	Constant gain, loss and demi-duplication	-154.9	315.7
	Constant gain, loss, duplication and demi-duplication* -151.8	311.7 8.21	18.01 26.91	7.85
	Constant gain and loss	-294	592.1
	*Best-fit model.

A. P. MORAES ET AL.

ACKNOWLEDGEMENTS

We are grateful to CNPq (150307/2008CNPq (150307/ -3, 304197/ 2012-2)-2), FAPERGS, CAPES/COFECUB, Propesq/ UFRGS and FAPESP (2011/22215-3 to A.P.M.) for financial support. We thank Lauís B. Corrêa, Alessandra B. Marasini and Victória Muccillo for assistance with meiotic analyses, Dr. José Marcelo Campos (UFJF) for his help with the cytometric analyses and Dra. Magdalena Vaio (UDELAR) and Dr. José Luiz Rybarczyk Filho (UNESP, CNPq 473789/2013-2) for their help with bioinformatic analyses. We are also indebted to Dr Andrea Carneiro and Ari D. Nilson (Jardim Botânico de Porto Alegre, Fundação Zoobotânica do Rio Grande do Sul, Brazil) for generously providing seeds, inflorescences and leaf material from living collections for our analysis. Finally, we are grateful to the anonymous referees who helped to improve the manuscript.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Appendix S1. Haploid (n) and base chromosome number (x), both codified as categorical data, mapped along the phylogeny. Appendix S2. Haploid chromosome number (n) mapped along the phylogeny.