[PROVISIONAL] Heterochromatin and numeric chromosome evolution in Bignoniaceae, with emphasis on the Neotropical clade Tabebuia alliance.

Bignoniaceae is a diverse family composed of 840 species with Pantropical distribution. The chromosome number 2n = 40 is predominant in most species of the family, with n = 20 formerly being considered the haploid base number. We discuss here the haploid base number of Bignoniaceae and examine heterochromatin distributions revealed by CMA/DAPI fluorochromes in the Tabebuia alliance as well as in some species of the Bignonieae, Tecomeae, and Jacarandeae tribes. When comparing the chromosome records and the phylogenies of Bignoniaceae it can be deduced that the base number of Bignoniaceae is probably n = 18, followed by an ascendant dysploidy (n = 18 → n = 20) in the most derived and diverse clades. The predominant heterochromatin banding patterns in the Tabebuia alliance were found to be two terminal CMA+ bands or two terminal and two proximal CMA+ bands. The banding pattern in the Tabebuia alliance clade was more variable than seen in Jacarandeae, but less variable than Bignonieae. Despite the intermediate level of variation observed, heterochromatin banding patterns offer a promising tool for distinguishing species, especially in the morphologically complex genus Handroanthus.


Introduction
Bignoniaceae is a Pantropical family composed mostly of trees and lianas, and includes 82 genera and 840 species (Fischer et al., 2004;Lohmann and Ulloa, 2016). Eight tribes are nested within the family: Bignonieae, Catalpeae, Coleeae, Crescentieae, Jacarandeae, Oroxyleae, Tecomeae, and Tourrettieae, plus the informal Crescentiina clade, that comprises the Neotropical and Palaeotropical subclades (Olmstead et al., 2009). While the morphological features of most tribes of Bignoniaceae are well-characterized, the Crescentiina clade and its subclades are well-sustained lineages, although without clear morphological synapomorphies (Grose and Olmstead, 2007;Olmstead et al., 2009). The Crescentiina clade comprises two informal lineages: the exclusively Neotropical Tabebuia alliance and the Paleotropical clade with Asian and African genera (Olmstead et al., 2009). The Tabebuia alliance has 14 genera and 147 species of trees and shrubs that have composite and palmate leaves (Grose and Olmstead, 2007). Most species within that clade belong to Tabebuia Gomes ex DC. and Handroanthus Mattos, while the remaining genera are smaller but widely-distributed in the Americas (Gentry, 1992;Grose and Olmstead, 2007). There is great morphological variability within the Tabebuia alliance, so that the delimitation of its species is often difficult.
Most species of Bignoniaceae show n = 20, and it has been proposed that x = 20 is the haploid base number for the family (Goldblatt and Gentry, 1979;Piazzano, 1998;Piazzano et al., 2015). However, when confronting the known chromosome numbers of Bignoniaceae and the phylogenetic analyses of Olmstead et al. (2009), it became evident that the most primitive clades (such as Jacarandeae) are x = 18, suggesting that a different number from 20 could be the haploid base number of the family.
Chromosome numbers and morphologies are the features most used in karyotype analyses and ground cytotaxonomy studies (Guerra, 2008), although those characters can be uninformative in groups where chromosome numbers are stable and the chromosomes are small (< 3 mm) (Guerra, 2000(Guerra, , 2012. Bignoniaceae have chromosome sizes of~2 mm, meta-submetacentric morphology, and 2n = 36 or 40 is predominant in the majority of species (Goldblatt and Gentry, 1979;Piazzano et al., 2015;Cordeiro et al., 2016bCordeiro et al., , 2017. Banding pattern characterizations can therefore often help discriminate between cytotypes with stable chromosome numbers, sizes and morphologies. The fluorochromes Chromomycin A 3 (CMA) and 4'6-diamidino-2-phenylindole (DAPI) are specific for GC-rich (CMA) or AT-rich (DAPI) regions respectively, and usually stain regions with tandem repeats of non-coding DNA (Schweizer, 1976;Guerra, 2000). They have been used mainly to characterize karyotypes with chromosomes that have the same size and morphology, and to differentiate the karyotypes of species with identical chromosome numbers (see Almeida et al., 2007;Barros e Silva et al., 2010;Cordeiro et al., 2016b;Almeida et al., 2016). The different patterns found can help determine taxonomic distinctions and clarify relationships among species (Carvalho et al., 2005;Almeida et al., 2007;Oliveira et al., 2015), as well as contribute to the description of new taxa, such as Epidendrum sanchezii E.Pessoa & L.P.Felix (Pessoa et al., 2014), Ameroglossum manoel-felixii L.P.Felix & E.M.Almeida (Almeida et al., 2016), and Spondias bahiensis P.Carvalho, Van den Berg and M.Machado (Almeida et al., 2007;Machado et al., 2015). Preliminary studies in the tribe Jacarandeae (Cordeiro et al., 2016b) indicated that heterochromatin distribution appeared to follow a specific pattern (8-16 CMA + terminal bands), while in the tribe Bignonieae (Cordeiro et al., 2017) heterochromatin distribution is quite variable among the species. That result demonstrates that regions rich in GC base pairs (CMA + ) can be variable even among closely related species of Bignoniaceae, and that a specific pattern for each group or tribe may not exist.
The main objective of this work was to describe the cytotaxonomic differences between related species of Bignoniaceae (mainly in the Neotropical lineage of the Tabebuia alliance clade) by examining their heterochromatin distributions, and discuss the haploid base number of the Bignoniaceae based on compilations of the chromosome numbers known for all lineages of the family.

Taxon sampling
Heterochromatin banding patterns of 12 species of the Tabebuia alliance clade were analyzed (Figure 1), as well as those of three species of Jacarandeae, two species of Tecomeae, and two species of Bignonieae tribes. The species, vouchers, and primarily karyological information are presented in Table 1. The vouchers were deposited in the EAN herbarium. An average of three specimens of each species were grown in plastic pots in the experimental garden of the Centro de Ciências Agrárias of the Universidade Federal da Paraíba. When the roots reached 2 cm in length, 15 root tips per specimen were excised and analyzed.

Cytogenetic analyses
Mitosis was examined in root tips that had been pretreated with 0.002 M 8-hydroxyquinoline (8-HQ) for 24 h at 4 ºC, fixed in 3:1 (v/v) absolute ethanol/glacial acetic acid for 30 min, and then stored in a freezer at -20 ºC. The roots were digested with an enzymatic solution (2% cellulase and 20% pectinase) for one hour at 37 ºC. Root tips were squashed in 45% acetic acid and coverslips were removed by freezing in liquid nitrogen. The samples were aged for three days at room temperature and stained with 10 mL of CMA (0.1 mg/mL) for 1 h, and then with 10 mL of DAPI (1 mg/mL) for 30 min. The samples were mounted in glycerol/McIlvaine's buffer at pH 7.0 (1:1, v/v) and kept in the dark for three days (Cordeiro et al., 2017).
The best metaphases were photographed using an AxioCam MRC5 digital camera and AxioVision 4.8 software (Carl Zeiss Microscopy GmbH, Jena Germany). Measurements were made using Uthscsa Image Tool (IT) v 3.0 software. The final images were prepared using Adobe Photoshop CS3 v 10.0 (Adobe Systems Incorporated, San Jose, USA). Chromosome morphology was determined using the centromeric index, following Guerra (1986).

Base chromosome number and karyotype evolution
The base chromosome number analysis is based on 179 species of Bignoniaceae, distributed in all of the clades retrieved by Olmstead et al. (2009) for the family. The list of samples, and their chromosome numbers and respective references are presented in Table S1 (Supplementary Material). Karyotype and molecular phylogenetic data were compiled for representatives of the Bignoniaceae. The numbers of species analyzed in each Bignoniaceae clade and their chromosome numbers and frequencies are presented in a phylogeny adapted from Olmstead et al. (2009) to demonstrate their putative chromosome number evolution. Information concerning heterochromatin patterns is presented for Bignonieae, Tabebuia alliance, Tecomeae, and Jacarandeae. The chromosomes types (A, B, C, D, E and F) follow Cordeiro et al. (2017).

Heterochromatin patterns
The heterochromatin banding patterns of the 19 species analyzed showed GC-rich (CMA + /DAPI -) bands located on the telomeric or proximal regions of the chromosomes (Figures 3 and 4). The species belonging to Jacarandeae, Tecomeae, and Bignonieae tribes had distinct patterns of CMA + /DAPIbands. Jacarandeae had five pairs of telomeric bands in J. jasminoides ( Figure 3A) and J. praetermissa ( Figure 3C), and four telomeric pairs in J. mimosifolia ( Figure 3B). Tecomeae had three pairs of inconspicuous telomeric bands in P. ricasoliana ( Figure 3D), and one telomeric pair plus two proximal pairs in T. stans ( Figure 3E). Bignonieae displayed two telomeric pairs as well as two telomeric and proximal pairs in A. citrinum ( Figure 3F), and 16 telomeric pairs and three telomeric pairs with bands on the short and long arm in F. chica (Figure 3G). Most species in the Tabebuia alliance had karyotypes with a pair of chromosomes with large CMA + /DAPItelomeric bands, as seen in Crescentia cujete L. (Figure 4I), S. leucanthum ( Figure 4C), T. elliptica ( Figure 4E), and T. aurea ( Figure 4D). Karyotypes with two telomeric and two proximal bands were observed in H. impetiginosus ( Figure  3I), Tabebuia rosea (Bertol.) Bertero ex A.DC. (Figure 4F), T. roseoalba ( Figure 4G), and Z. tuberculosa ( Figure 4H). The remaining species of Handroanthus showed distinct heterochromatin patterns: four telomeric bands (two large 6 Cordeiro et al. and two small) and four proximal bands in H. umbellatus ( Figure 4B), four small telomeric bands in H. ochraceus ( Figure 3J), eight telomeric bands (four large and four small) and four proximal bands in H. chrysotrichus ( Figure  3H), and ten telomeric bands (four large and six small) and four proximal bands in H. serratifolius ( Figure 4A).

Discussion
Chromosome number evolution in Bignoniaceae Raven (1975) suggested x = 7 as the ancestral base number for Bignoniaceae, with the most common n = 20 being generated by a six-fold polyploidization followed by the loss of one pair of chromosomes; that base number was suggested because he considered Oroxyleae (n = 14 and 15) to be the most primitive tribe in Bignoniaceae. Several cytological studies in Bignoniaceae (Goldblatt and Gentry, 1979;Piazzano, 1998;Chen et al., 2004) agreed with the hypothesis of Raven (1975). More recent works, such as Piazzano et al. (2015), however, suggested x = 20 as the basic number of Bignoniaceae. The principal justification for that would be the large number of species with 2n = 40, and groups considered correlated with Bignoniaceae, such as Paulowniaceae and Schlegeliaceae, which also share the haploid number n = 20.
Jacarandeae and Tourretieae are the most primitive group for Bignoniaceae. Jacarandeae include two genera (Jacaranda Juss. and Digomphia Benth.) and approximately 55 species that are widely distributed throughout the Neotropics (Gentry, 1980;Olmstead et al., 2009). The chromosome number in the Jacaranda is very well characterized by the 2n = 36 (Cordeiro et al., 2016b). Tourrettieae include two small genera subwoody to herbaceous vines (Eccremocarpus Ruiz & Pav. and Tourrettia DC.) and six species distributed in the Andes and north in the Central American Cordilleras to Mexico (Gentry, 1980;Olmstead et al., 2009). There are chromosomal records in this tribe only for Tourrettia lappacea (L'Hér.) Willd. (2n = 40) (Goldblatt and Gentry, 1979). Although the chromosomal record for Tourrettieae and Jacarandeae are different, these two basal tribes share some traits, as the doubly compound leaves and pollen that is psilate and tricolpate (Olmstead et al., 2009). Further sampling in Tourrettieae can confirm whether 2n = 40 is a typical chromosomal number for the tribe species or if there may be other chromosome numbers, as also observed in Tecomeae.
Most species of the derived clade comprising Catalpeae, Oroxyleae, Crescentiina, and Bignonieae (Olmstead et al., 2009) have the karyotype 2n = 40. Among the 122 species with known chromosome numbers within this clade, 92.6% (113 species) show 2n = 40. Only six species show 2n ¹ 40 [two species of Mansoa DC. in Bignonieae, two species of Oroxyleae, and Spathodea campanulata P. Beauv. and Radermacheraxylocarpa (Roxb.) Roxb. ex K. Schum.; Table S1]. The remaining species are polyploids of the haplotype n = 20 (2n = 60, 80, 120). This large clade comprises around 80% of the species of Bignoniaceae (Olmstead et al., 2009), which makes 2n = 40 the most common karyotype in the family. The four tribes and informal groups in this derived clade show marked geographical patterns. The most species-rich tribe (Bignonieae) is Neotropical (Fischer et al., 2004) as is one lineage of the informal Crescetiina (which also has one Paleotropical clade) (Grose and Olmstead, 2007). Catalpeae is from temperate North America and China and the tropical Greater Antilles (Gentry, 1980;Olsen and Kirkbride Jr, 2017), while the smallest tribe, Oroxyleae, is from tropical southern and southeastern Asia and Malaysia (Olmstead, 2013). Their wide distribution around the world and the high numbers of species in those tribes make n = 20 the most common haploid number in Bignoniaceae. The haploid number n = 20 could be related to actual diversity and the occupation of a wide variety of habitats.

Heterochromatin patterns
Heterochromatin in the basal lineage of Jacarandeae (Bignoniaceae) is composed exclusively by 8-16 terminal CMA + bands, while the following lineages (Tecomeae, Bignonieae, Tabebuia alliance) also demonstrate pericentromeric CMA + bands, but with reductions in the numbers of terminal CMA + blocks (Cordeiro et al., 2016b(Cordeiro et al., , 2017; Figures 3 and 4). In certain plant groups, such as the Caesalpinia group (Van-Lume et al., 2017), and sect. Acan-thophora of Solanum L. (Chiarini et al., 2014) and Nierembergia Ruiz & Pav. (Acosta et al., 2016), the heterochromatin patterns appear to follow a specific evolutionary pattern for the species in the different clades. In genera such as Lycium L. (Stiefkens et al., 2010), Pereskia Mill. (Castro et al., 2016), and Ceiba Mill. (Figueredo et al., 2016), however, the heterochromatin pattern appears to be quite conserved and demonstrate only small variability among the different species. In most plant groups, however, heterochromatin patterns tend to be random and quite distinct, even among closely related species (Berjano et al., 2009;Scaldaferro et al., 2012;Grabiele et al., 2018;Van-Lume and Souza, 2018). For Bignoniaceae as a whole, three heterochromatin patterns can be seen, with the occurrence of a specific pattern for Jacarandeae (terminal CMA + blocks), conserved patterns for the diploid species of the Tabebuia alliance (two terminal CMA + blocks and 0-2 pericentromeric CMA + blocks), and a pattern of random attributions in relation to the numbers and positions of CMA + blocks in the tribe Binonieae (Cordeiro et al., 2017).
The heterochromatin banding patterns of the Bignonieae tribe in Bignoniaceae (Cordeiro et al., 2017) are characterized by strong differences in the sizes and locations of the CMA + blocks, and six chromosome types are recognized based on heterochromatic regions. The two species of Bignonieae analyzed here confirm the patterns described before for the tribe, with the occurrence of type A (large telomeric CMA + bands), type B (small telomeric CMA + bands), type D (telomeric and proximal CMA + bands), and type F chromosomes (showing a lack of heterochromatic bands) in A. citrinum, and type A, B, E (two telomeric CMA + bands) and F chromosomes in F. chica (Table 1, Figure 2). The species sampled in Tabebuia alliance, Jacarandeae, and Tecomeae have four chromosome types (according to Cordeiro et al., 2017): type A, type B, type C (proximal CMA + bands), and type F.
The pattern of two CMA + telomeric bands (chromosome type A) seen in most species of the Tabebuia alliance is very common among Angiosperms, and usually corresponds to a nucleolar organizer region (Guerra, 2000;Roa and Guerra, 2012). Telomeric CMA bands are most likely related to rDNA sites as seen in most plant species (Barros e Silva et al., 2010;Castro et al., 2016;Marinho et al., 2018). Differences among species could be related tochromosome rearrangements and the amplification and reduction of rDNA sites caused by satellites or transposable sequences (Mehrotra and Goyal, 2014;Evtushenko et al., 2016;Saze, 2018).
The vegetative morphologies of Handroanthus species having yellow corollas are very similar (Gentry, 1992), with H. chrysotrichus and H. ochraceus being very close, even when comparing their flowers, leaves, and fruits. Those two species show a continuum of morphological variations, and hybridization or introgression has therefore been suggested (Gentry, 1992 (Gentry, 1992). The heterochromatin banding patterns of those two species are distinct, with the former having two proximal plus two telomeric bands (2A + 2C), while the latter has only two telomeric bands (2A). While banding patterns are still seldom-used in taxonomic studies, the results reported here support their utility in such analyses.
The chromosome numbers and heterochromatin banding patterns of J. jasminoides, J. praetermissa and J. mimosifolia support published data for the genus (Cordeiro et al., 2016b). Jacaranda is one of the largest genera of Bignoniaceae, with more than 50 species widely distributed in the Neotropics (Gentry and Morawetz, 1992). The genus is very well characterized by the chromosome number 2n = 36 (Morawetz, 1982;Cordeiro et al., 2016b) and by having 8 to 16 small and terminal CMA + bands (Cordeiro et al., 2016b). In addition to its stable chromosome features, Jacaranda has a very consistent morphology, with all of its species having pinnate or bipinnate leaves, calyx lobes that are deeply divided, staminodes longer than the stamens, and oblong and strongly flattened capsules opening through a rupture perpendicular to the septum (Morawetz, 1982;Gentry and Morawetz, 1992;Olmstead et al., 2009).
The heterochromatin banding patterns of Tecomeae have been poorly studied. The karyotypes of the two species of the tribe analyzed here, however, were relatively distinct from the species belonging to Jacarandeae, the Tabebuia alliance clades, and Bignonieae species (Cordeiro et al., 2017). Although T. stans shows 2n = 36, its heterochromatin banding pattern (2A + 4C) is quite distinct from species with similar chromosome numbers, such as Jacaranda (8-16 A + B; Cordeiro et al., 2016b). Regarding P. ricasoliana, this species has an uncommon chromosome number for the Bignoniaceae (2n = 38) and six small terminal CMA + bands -a unique pattern in the family (which usually has at least two large terminal CMA + bands) (Cordeiro et al., 2016b(Cordeiro et al., , 2017. Although there is still little data available concerning banding patterns in Tecomeae, their lack of synapomorphies (Olmstead et al., 2009), wide distributions (Olmstead, 2013), high variability of life forms (including herbs, shrubs, trees, and lianas), environments occupied (from tropical to temperate forests, to both Andean and Himalayan mountains), and variations in chromosome numbers of the species within this clade, make this tribe one of the major challenges in Bignoniaceae.

Conclusion
The revision of the chromosome numbers previously reported for Bignoniaceae, allied to previous phylogenetic studies for the family, support a basic haploid chromosome number different from 20 for the family. The most likely primary base number for the family is x = 18, which is the most common haploid number among its basal lineages. Ascending disploidy leading to x = 20 is consistent with the chromosome numbers found in the most derived and diversified lineages, where that number predominates. A broad study involving reconstructions of chromosome counts in families related to Bignoniaceae, as well as in all of its clades, would help clarify the evolution of the karyotype of the family.
The chromosomes of the Tabebuia alliance showed only GC-rich bands (CMA + /DAPI -) located in telomeric or proximal regions. The banding pattern within that clade was more variable than seen in Jacaranda, but less variable than in Bignonieae. Despite the intermediate level of variation observed, heterochromatin banding patterns offer a promising tool for distinguishing species, especially in the morphologically complex genus Handroanthus.

Supplementary material
The following online material is available for this article: Table S1 -Chromosome numbers recorded for the Bignoniaceae family and their respective bibliographic references.