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[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.
Genetics and Molecular Biology In Press, Accepted Manuscript
Copyright © 2019, Sociedade Brasileira de Genética.
This manuscript has been approved and it is published as a provisional version while it is
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Heterochromatin and numeric chromosome evolution in Bignoniaceae, with emphasis on the
Neotropical clade Tabebuia alliance
Joel M. P. Cordeiro1, Miriam Kaehler2, Luiz Gustavo Souza3 and Leonardo P. Felix1
1Universidade Federal da Paraíba, Campus II, Centro de Ciências Agrárias, Departamento de
Ciências Biológicas. Rodovia PB 079, Km 12, CEP: 58397-000, Areia, Paraíba, Brazil.
2Mulleriana: Sociedade Fritz Müller de Ciências Naturais. Rua Humberto Morona, 26, CEP:
80050-420, Curitiba, PR, Brazil.
3Universidade Federal de Pernambuco, Campus I, Centro de Ciências Biológicas, Departamento
de Botânica, Av. Prof. Moraes Rego, 1235, CEP: 50670-901, Recife, Pernambuco, Brazil.
Send correspondence to Leonardo P. Felix. Universidade Federal da Paraíba, Campus II, Centro de
Ciências Agrárias. Rodovia PB 079, Km 12, CEP: 58397-000, Areia, Paraíba, Brazil. e-mail:
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.
Keywords: Chromosome number, CMA/DAPI, Handroanthus, Polyploidy.
Received: June 12, 2018; Accepted: March 05, 2019.
In Press, Accepted Manuscript
Bignoniaceae is a Pantropical family composed mostly of trees and lianas, and includes 82
genera and 840 species (Fisher et al., 2004; Lohmann and Ulloa, 2016). The family is nested in eight
tribes: 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.
Bignoniaceae family, from a cytogenetic point of view, comprises two groups with distinct
karyotypes. The first group has a wide range of chromosome numbers (2n = 22, 28, 30, 36, 38, 40
and 42) and includes the tribes Jacarandeae, Tecomeae, Oroxyleae, and the two genera Argylia D.Don
and Delostoma D.Don (Moore, 1974; Goldblatt and Gentry, 1979; Piazzano, 1998; Piazzano et al.,
2015). The second group has the prevailing chromosome number 2n = 40, and includes Bignonieae,
Catalpeae, and the Crescentiina clade (Goldblatt and Gentry, 1979; Piazzano, 1998; Alcorcés de
Guerra, 2002; Ortolani et al., 2008; Firetti-Leggieri et al., 2011; Piazzano et al., 2015; Cordeiro et
al., 2016a, 2017). Ploidy variations (2n = 60, 80 and 120) were found for a few species of the tribe
Bignonieae and the clade Tabebuia alliance from the second group (Piazzano, 1998; Alves et al.,
2013; Piazzano et al., 2015; Cordeiro et al., 2017).
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 µm) (Guerra,
2000, 2012). Bignoniaceae have chromosome sizes of ~2 µm, 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., 2016b, 2017). Banding pattern characterizations can therefore often help
discriminate between cytotypes with stable chromosome numbers, sizes and morphologies. The
fluorochromes Chromomycin A3 (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
In Press, Accepted Manuscript
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 & 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
Materials and Methods
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, fifteen roots tips per
specimen were excised and analyzed.
Cytogenetic analyses
Mitosis was examined in root tips that had been pre-treated with 0.002 M 8-hydroxyquinoline
(8-HQ) for 24 hours at 4 ºC, fixed in 3:1 (v/v) absolute ethanol/glacial acetic acid for 30 minutes, 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 µL of CMA (0.1 mg/ mL) for one hour, and then with 10 µL of DAPI
(1 µg/ mL) for 30 minutes. 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 Image Tool v 3.0 software (Donald et al., 2008). 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).
In Press, Accepted Manuscript
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).
Chromosome numbers
The chromosome number of 12 species of the Tabebuia Alliance clade was analyzed, as well
as those of three species of Jacarandeae, two species of Tecomeae, and two species of Bignonieae
tribes. The karyotypes of the 19 species analyzed were predominantly symmetrical, principally with
metacentric or sub-metacentric chromosomes. Their sizes ranged from 1.02 µm ± 0.13 in Tabebuia
aurea (Silva Manso) Benth. & Hook.f. ex S. Moore to 2.19 µm ± 0.3 in J. praetermissa. The
chromosome number of most of the Tabebuia alliance was 2n = 40 (Crescentia L., Sparattosperma
Mart. ex Meisner, Tabebuia Gomez, and Zeyheria Mart.). However, Handroanthus Mattos showed
2n = 40 [H. impetiginosus (Mart. ex DC.) Mattos and H. umbellatus] as well as 2n = 80 [H.
chrysotrichus (Mart. ex DC.) Mattos and H. ochraceus (Cham.) Mattos], and 2n = 120 [H.
serratifolius (Vahl.) S.O.Grose]. The remaining species showed 2n = 36 [Jacaranda mimosifolia
D.Don., J. jasminoides (Thunb.) Sandwith., J. praetermissa, and Tecoma stans (L.) Juss. ex Kunth],
2n = 38 [Podranea ricasoliana (Tafani) Sprague], or 2n = 40 (A. citrinum and F. chica) (Table 1).
New chromosome records are described for Handroanthus umbellatus (Sond.) Mattos,
Sparattosperma leucanthum (Vell.) K.Schum, Tabebuia elliptica (DC.) Sandwith, T. roseoalba
(Ridl.) Sand., and Z. tuberculosa (Vell.) Bureau ex Verl. (2n = 40; Tabebuia alliance), as well as for
Fridericia chica (Bonpl.) L.G.Lohmann (2n = 40; Bignonieae tribe) and Jacaranda praetermissa
Sandwith (2n = 36; Jacarandeae tribe). Additionally, a new cytotype is described for Anemopaegma
citrinum Mart. ex DC. (2n = 40; Bignonieae tribe).
Base chromosome number and karyotype evolution
The chromosome numbers of 179 species of Bignoniaceae (belonging to all of its clades) were
compared (Table S1). Overall, most species showed 2n = 40 (67%) and 2n = 36 (19%). Chromosome
numbers were compiled in a phylogeny adapted from Olmstead et al. (2009) to infer chromosome
number evolution (Figure 2). The chromosome number 2n = 36 (n = 18) was principally distributed
within the tribe Jacarandeae, while 2n = 40 (n = 20) appeared especially in the tribes Bignonieae and
Catalpeae, in the clade Crescentiina, and in Tourrettieae. Other chromosome numbers occurred in
Argylia (2n = 30) and Delostoma (2n = 42), and in the tribes Oroxyleae (2n = 28 and 30) and
Tecomeae (2n = 22, 38 and 48).
In Press, Accepted Manuscript
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+/DAPI bands. 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+/DAPI telomeric 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 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).
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.
Molecular phylogeny, however, suggests a different story. Paulowniaceae and Schlegeliaceae
are not closely related to Bignoniaceae (Olmstead et al., 2009; Refulio-Rodriguez and Olmstead,
2014). According to Olmstead et al. (2009), the first diverging lineage within Bignoniaceae was
Jacarandeae (2n = 36), followed by a strongly supported clade (core Bignoniaceae) with Tourrettieae
(2n = 40), and then Argylia (2n = 30), Tecomeae (2n = 18, 22, 34, 36, 38, and 40), and a large clade
including Oroxyleae (2n = 28, 30), Crescentiina (mostly 2n = 40, but also 36, 38, 80 and 120), and
Bignonieae (mostly 2n = 40, but also 38, 60, and 80) (Figure 2). Among the most basal lineages
(Jacarandeae, Tourrettieae, Argylia, Tecomeae, and Delostoma) only 8.7% of the species (five
species) have 2n = 40, whereas 56.1% (32 species) show 2n = 36 (Table S1, Figure 2). Consequently,
In Press, Accepted Manuscript
the haploid base number for the family is x 20. Very likely, the haploid number is x = 18, which
was followed by an ascendant dysploidy (n = 18 n = 20) in the most derived and diversified clades
of the family.
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
Tecomae is placed between the basal (Jacarandeae and Tourrettieae) and most derived clades
of the Bignoniaceae (Crescentiina, Bignonieae, Catalpeae). The tribe is characterized by wide
variations in chromosome numbers (2n = 22, 36, 38, 40, and 48), unlike other tribes where 2n = 36
(Jacarandeae) or 2n = 40 (Bignonieae, Catalpeae, and Crescentiina clade) predominate (Table S1,
Figure 2). Variations in chromosome numbers in Tecomeae represent events of ascending and
descending disploidy resulting in different chromosome numbers. The presence of n = 20 in
Tourrettieae suggests that this number could have arisen at the Core Bignoniaceae by ascending
disploidy, while the other numbers could have arisen by ascending (n = 21, 24) and descending (n =
11, 14, 15, 19) disploidy.
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 40 [two species of Mansoa DC. in Bignonieae, two species of Oroxyleae, and Spathodea
campanulata P. Beauv. and Radermachera xylocarpa (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 (Fisher
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.
Reported chromosome numbers suggest that polyploidy is restricted to the clades Tabebuia
alliance and Bignonieae (Goldblatt and Gentry, 1979; Piazzano, 1998; Firetti-Leggieri et al., 2011,
2013; Cordeiro et al., 2017). Reproductive analyses of Handroanthus and Anemopaegma Mart. ex
Meisn. indicated self-pollination, sporophytic and pseudogamous apomixis, and polyembryonic seeds
(Piazzano, 1998; Bittencourt Jr and Moraes, 2010; Firetti-Leggierri et al., 2013) which are common
In Press, Accepted Manuscript
features in polyploid species (Piazzano, 1998; Firetti-Leggieri et al., 2013). Piazzano et al. (2015)
suggested that the polyploidy observed in those species probably originated by meiotic alteration,
leading to the production of non-reduced gametes. The absence of a morphological continuum
between sympatric species of the same genera (personal observations) suggests an autopolyploid
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, 2017; Figures 3, 4). In certain plant groups, such as
the Caesalpinia group (Van-Lume et al., 2017), and sect. Acanthophora of Solanum L. (Chiarini et
al., 2013) 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 to chromosome 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; Bittencourt Jr and Moraes
In Press, Accepted Manuscript
2010). However these species have a distinctive heterochromatin banding pattern 4A + 4B + 4C in
H. chrysotrichus and 4B in H. ochraceus. Similarly, T. roseoalba and T. elliptica have very similar
flowers and fruits, although they can be differentiated by their 3- or 5-foliolate leaves respectively
(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 Morawertz, 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 Morawertz, 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 it one of the major challenges in Bignoniaceae.
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
In Press, Accepted Manuscript
variation observed, heterochromatin banding patterns offer a promising tool for distinguishing
species, especially in the morphologically complex genus Handroanthus.
We thank the Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq) and
the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support,
and the Instituto Nacional do Semiárido (INSA) for their technical support.
Acosta MC, Moscone EA and Cocucci AA (2016) Using chromosomal data in the phylogenetic and
molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae)
influenced by historical changes in sea level. Plant Biol 18: 514-526.
Alcorcés de Guerra N (2002) Cariología de dos especies del gênero Tabebuia Gomes (Bignoniaceae).
UDO Agrícola 2: 4-21.
Almeida CCS, Carvalho PCL and Guerra M (2007) Karyotype differentiation among Spondias
species and the putative hybrid umbu-cajá (Anacardiaceae). Bot J Linn Soc 155: 541-547.
Almeida EM, Wanderley AM, Nollet F, Costa FR, Souza LGR, and Felix LP (2016) A new species
of Ameroglossum (Schrophulariaceae) growing on inselbergs in Northeastern Brazil. Syst Bot 41:
Alves MF, Duarte MO, Oliveira PE and Sampaio DS (2013) Self-sterility in the hexaploid
Handroanthus serratifolius (Bignoniaceae), the national flower of Brazil. Acta Bot Brasilica 27: 714-
Barros e Silva AE, Marques AS, Karla GB and Guerra M (2010) The evolution of CMA bands in
Citrus and related genera. Chromosome Res 18: 503-514.
Berjano R, Roa F, Talavera S and Guerra M (2009) Cytotaxonomy of diploid and polyploid
Aristolochia (Aristolochiaceae) species based on the distribution of CMA/DAPI bands and 5S and
45S rDNA sites. Plant Syst Evol 280: 219-227.
Bittencourt Jr NS and Moraes CIG (2010) Self-fertility and polyembryony in South American yellow
trumpet trees (Handroanthus chrysotrichus and H. ochraceus, Bignoniaceae): a histological study of
postpollination events. Plant Syst Evol 288: 59-76.
Carvalho R, Soares-Filho WS, Brasileiro-Vidal AC and Guerra M (2005) The relationships among
lemons, limes and citron: a chromosomal comparison. Cytogenet Genome Res 109: 276-282.
Castro JP, Medeiros-Neto E, Souza G, Alves LIF, Batista FRC and Felix LP (2016) CMA band
variability and physical mapping of 5S and 45S rDNA sites in Brazilian Cactaceae: Pereskioideae
and Opuntioideae. Braz J Bot. DOI: 10.1007/s40415-015-0248-5
Chen ST, Zhou ZK, Guan KY and Nakata M (2004) Karyomorphology of Incarvillea (Bignoniaceae)
and its implications in distribution and taxonomy. Bot J Linn Soc 144: 113-121.
In Press, Accepted Manuscript
Chiarini FE, Santiñaque FF, Urdampilleta JD and Las Peñas ML (2013) Genome size and karyotype
diversity in Solanum sect. Acanthophora (Solanaceae). Plant Syst Evol. DOI: 10.1007/s00606-013-
Cordeiro JMP, Almeida EM, Lima SAA, Assis FNM, Souza LGR and Felix LP (2016a)
Bignoniaceae, Bignonieae. In: Marhold K and Kučera J (eds) IAPT/IOPB chromosome data 23.
Taxon 65: 1455-1458.
Cordeiro JMP, Lima SAA, Paz SN, Santos MAS and Felix LP (2016b) Karyotype evolution in the
genus Jacaranda Juss. (Jacarandeae, Bignoniaceae): chromosome numbers and heterocromatin.
Genet Mol Res 15: gmr15048973.
Cordeiro JMP, Kaehler M, Souza G and Felix LP (2017) Karyotype analysis in Bignonieae
(Bignoniaceae): chromosome numbers and heterochromatin. An Acad Bras Cienc 89: 2697-2706.
Donald C, Brent DS, Mcdavid WD and Greer DB (2008) Uthscsa. Image Tool (IT) Version 3.0.
Available at: (accessed 10 April 2014).
Evtushenko EV, Levitsky VG, Elisafenko EA, Gunbin KV, Belousov AI, Šafář J, Doležel J and
Vershinin AV (2016) The expansion of heterochromatin blocks in rye reflects the co-amplification
of tandem repeats and adjacent transposable elements. BMC Genomics 17: 337.
Figueredo A, Oliveira ÁWL, Carvalho-Sobrinho JG and Souza G (2016) Karyotypic stability in the
paleopolyploid genus Ceiba Mill. (Bombacoideae, Malvaceae). Braz J Bot 39: 1087-1093.
Firetti-Leggieri F, Costa IR, Lohmann LG, Semir J and Martins ERF (2011) Chromosome studies in
Bignonieae (Bignoniaceae): the first record of polyploidy in Anemopaegma. Cytologia 76: 185-191.
Firetti-Leggieri F, Lohmann LG, Alcantara S, Costa IR and Semir J (2013) Polyploidy and
polyembryony in Anemopaegma (Bignonieae, Bignoniaceae). Plant Reproduction 26: 43-53.
Fischer E, Theisen I and Lohmann LG (2004) Bignoniaceae. In: Kadereit JW and Kubitzki K (eds)
The families and genera of vascular plants. Springer, Berlin 7: 9-98.
Gentry AH (1980) Bignoniaceae - Part I. Tribes Crescentieae and Tourrettieae. Flora Neotrop,
Monograph 25 (I): 1-131.
Gentry AH (1992) Bignoniaceae - Part II. Tribe Tecomae. Flora Neotrop, Monograph 25 (II): 1-370.
Gentry AH and Morawetz W (1992) Jacaranda. In: Bignoniaceae - Part II. Tribe Tecomae. Flora
Neotrop, Monograph 25 (II): 51-105.
Goldblatt P and Gentry AH (1979) Cytology of Bignoniaceae. Bot Not 132: 475-482.
Grabiele M, Debat HJ, Scaldaferro MA, Aguilera PM, Moscone EA, Seijo JG and Ducasse DA (2018)
Highly GC-rich heterochromatin in chili peppers (Capsicum-Solanaceae): a cytogenetic and
molecular characterization. Sci Hortic 238: 391-399
Grose SO and Olmstead RG (2007) Taxonomic revisions in the polyphyletic genus Tabebuia s. l.
(Bignoniaceae). Syst Bot 32: 660-670.
In Press, Accepted Manuscript
Guerra M (1986) Reviewing the chromosome nomenclature of Levan et al. Rev Brasil Genet 9: 741-
Guerra M (2000) Patterns of heterochromatin distribution in plant chromosomes. Genet Mol Biol 23:
Guerra M (2008) Chromosome numbers in plant cytotaxonomy: concepts and implications.
Cytogenet Genome Res 120: 339-350.
Guerra M (2012) Cytotaxonomy: the end of childhood. Plant Biosyst 146: 703-710.
Lohmann LG and Ulloa CU (2016) Bignoniaceae. In “Checklist of the World,” MOBOT/NYBG/Kew
Gardens. iPlants prototype Checklist. Available at: (accessed 10 April 2018).
Machado MC, Carvalho PCL and van den Berg C (2015) Domestication, hybridization, speciation,
and the origins of an economically important tree crop of Spondias (Anacardiaceae) from the
Brazilian caatinga dry forest. Neodiversity 8: 8-49.
Marinho ACTA, Vasconcelos S, Vasconcelos EV, Marques DA, Benko-Iseppon and Brasileiro-Vidal
AC (2018) Karyotype and genome size comparative analyses among six species of the oilseed-
bearing genus Jatropha (Euphorbiaceae). Genet Mol Biol 41: 442-449.
Mehrotra S and Goyal V (2014) Repetitive sequences in plant nuclear DNA: types, distribution,
evolution and function. Genomics Proteomics Bioinformatics 12: 164-171.
Moore RJ (org) (1974) Index to plant chromosome numbers for 1972. Regnum Veg 91: 1-108.
Morawetz W (1982) Morphologisch-ökologische differenzierung, biologie, systematic und evolution
der Neotropischen gattung Jacaranda (Bignoniaceae). Denkschriften der Österreichischen Akademie
der Wissenschaften. Springerverlag, Wien.
Oliveira IG, Moraes AP, Almeida EM, Assis FNM, Cabral JS, Barros F and Felix LP (2015)
Chromosomal evolution in Pleurothallidinae (Orchidaceae: Epidendroideae) with an emphasis on the
genus Acianthera: chromosome number and heterochromatin. Bot J Linn Soc 178: 102-120.
Olmstead RG (2013) Phylogeny and biogeography in Solanaceae, Verbenaceae and Bignoniaceae: a
comparison of continental and intercontinental diversification patterns. Bot J Linn Soc 171: 80-102.
Olmstead RG, Zjhra ML, Lohmann LG, Grose SO and Eckert AJ (2009) A molecular phylogeny and
classification of Bignoniaceae. Am J Bot 96: 1731-1743.
Olsen RT and Kirkbride Jr JH (2017) Taxonomic revision of the genus Catalpa (Bignoniaceae).
Brittonia 69: 387-421.
Ortolani FA, Mataqueiro MF, Moro JR, Moro FV and Damião Filho CF (2008) Morfo-anatomia de
plântulas e número cromossômico de Cybistax antisyphilitica (Mart.) Mart. (Bignoniaceae). Acta Bot
Brasilica 22: 345-353.
Pessoa E, Felix LP and Alves M (2014) A new Epidendrum (Laeliinae Orchidaceae) from the Atlantic
Forest of northeastern Brazil: evidence from morphology and cytogenetics. Brittonia 66: 347-352.
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Piazzano M (1998) Números cromosómicos em Bignoniaceae de Argentina. Kurtziana 26: 179-219.
Piazzano M, Las Peñas ML, Chiarini F and Bernardello G (2015) Karyotypes and DNA content in
Bignoniaceae. Caryologia 68: 175-183.
Raven PH (1975) The bases of the Angiosperm phylogeny: Cytology. Ann Missouri Bot Gard 62:
Refulio-Rodriguez NF and Olmstead RG (2014) Phylogeny of Lamiidae. Am J Bot 101: 287-299.
Roa F and Guerra M (2012) Distribution of 45S rDNA sites in chromosomes of plants: structural and
evolutionary implications. BMC Evol Biol 12: 225.
Saze H (2018) Epigenetic regulation of intragenic transposable elements: a two-edged sword. J
Biochem. DOI:10.1093/jb/mvy060
Scaldaferro MA, Grabiele M and Moscone EA (2012) Heterochromatin type, amount and distribution
in wild species of chili peppers (Capsicum, Solanaceae). Genet Resour Crop Evol. DOI:
Schweizer D (1976) Reverse fluorescent chromosome banding with Chromomycin and DAPI.
Chromosoma 58: 307-324
Stiefkens LM, Las Peñas ML, Bernardello G, Levin RA and Miller JS (2010) Karyotypes and
fluorescent chromosome banding patterns in southern African Lycium (Solanaceae). Caryologia 63:
Van-Lume B and Souza G (2018) Cytomolecular analysis of species in the Peltophorum clade
(Caesalpinioideae, Leguminosae). Braz J Bot. DOI: 10.1007/s40415-018-0449-9
Van-Lume B, Esposito T, Diniz-Filho JAF, Gagnon E, Lewis GP and Souza G (2017)
Heterochromatic and cytomolecular diversification in the Caesalpinia group (Leguminosae):
relationship between phylogenetic and cytogeographical data. Perspect Plant Ecol Evol Syst 29: 51-
Supplementary material
The supplementary material will be available in the final version of the article.
In Press, Accepted Manuscript
Figure 1 - Some of the species of the Neotropical lineage Tabebuia alliance clade sampled. A.
Crescentia cujete, B. Handroanthus chrysotrichus, C. H. impetiginosus, D. H. ochraceus, E. H.
serratifolius, F. H. umbellatus, G. Sparattosperma leucanthum, H. Tabebuia aurea, I. T. elliptica, J.
T. rosea, K. T. roseoalba, L. Zeyheria tuberculosa.
In Press, Accepted Manuscript
Figure 2 - Chromosome numbers of the Bignoniaceae clades. Values on the branches indicate
bootstrap parsimony analysis and the posterior probability of Bayesian inference; Asterisks indicate
100% posterior probabilities (topology and support values following Olmstead et al., 2009). Circle
sizes correspond to the numbers of species with chromosome records in each clade. Chromosomes
types A, B, C, D, E and F follow Cordeiro et al. (2017).
In Press, Accepted Manuscript
Figure 3 - Distribution of heterochromatic bands (CMA+, in yellow) of species of Jaracandeae,
Tecomeae, Bignonieae and the Tabebuia alliance. A. Jacaranda jasminoides (2n = 36), B. J.
mimosifolia (2n = 36), C. J. praetermissa (2n = 36), D. Podranea ricasoliana (2n = 38), E. Tecoma
stans (2n = 36), F. Anemopaegma citrinum (2n = 40), G. Fridericia chica (2n = 40), H. Handroanthus
chrysotrichus (2n = 80), I. H. impetiginosus (2n = 40), J. H. ochraceus (2n = 80). Scale bar in J
corresponds to 10 µm. Arrow heads indicate minor CMA bands; inserts in D, H and J highlight
chromosomes with inconspicuous CMA bands.
In Press, Accepted Manuscript
Figure 4 - Distribution of heterochromatic bands (CMA+, in yellow) of species of the Tabebuia
alliance (including Crescentieae). A. Handroanthus serratifolius (2n = 120), B. H. umbellatus (2n =
40), C. Sparattosperma leucanthum (2n = 40), D. Tabebuia aurea (2n = 40), E. T. elliptica (2n = 40),
F. T. rosea (2n = 40), G. T. roseoalba (2n = 40), H. Zeyheria tuberculosa (2n = 40), I. Crescentia
cujete (2n = 40). Scale bar in I corresponds to 10 µm. Arrow heads indicate minor CMA bands; inserts
in A highlight chromosomes with inconspicuous CMA bands.
In Press, Accepted Manuscript
Table 1. Species of Bignoniaceae analyzed and their main karyological parameters. Heterochromatin
patterns: A - large telomeric CMA+ bands, B - small telomeric CMA+ bands, C - proximal CMA+
bands, F - lack of heterochromatic bands. Abbreviations of the Voucher: JMPC - Joel Maciel Pereira
Cordeiro, LPF - Leonardo Pessoa Felix, EMA - Erton Mendonça de Almeida, SAAL - Saulo Antonio
Alves de Lima. Abbreviations in the Origin: PB - Paraíba State, BA - Bahia State, PI - Piauí State,
and MG - Minas Gerais State, Brazil.
Tribe/Alliance/species Voucher Origin 2n Median size
(µm) Heterochromatin
patterns Figure
Jacaranda jasminoides (Thunb.)
Sandwith. JMPC, 131 Sertãozinho-PB 36 2.09 6A + 4B + 26F 3A
J. mimosifolia D.Don LPF, 14457 Areia-PB 36 1.84 6A + 2B + 28F 3B
J. praetermissa Sandwith* LPF, 17606 Serra da Capivara-PI 36 2.19 2A + 8B + 26F 3C
Podranea ricasoliana (Tanfani)
Sprague JMPC, 135 Areia-PB 38 1.07 6B + 32F 3D
Tecoma stans (L.) Juss. ex Kunth LPF, 14412 Paulo Afonso-BA 36 1.16 2A + 4C + 30F 3E
Anemopaegma citrinum Mart. ex
DC.** JMPC, 1254 Pico do Jabre-PB 40 1.32 2A + 2B + 2D +
34F 3F
Fridericia chica (Bonpl.)
L.G.Lohmann* Manaus, AM 40 1.76 6A + 26B + 6E + 2F 3G
Tabebuia alliance
Handroanthus chrysotrichus
(Mart. ex DC.) EMA, 814 Campina Grande-PB 80 1.44 4A + 4B + 4C +
68F 1B, 3H
H. impetiginosus (Mart. ex DC.)
Mattos SAAL, 86 Areia-PB 40 1.39 2A + 2C + 36F 1C, 3I
H. ochraceus (Cham.) Mattos SAAL, 84 João Pessoa-PB 80 1.42 6B + 74F 1D, 3J
In Press, Accepted Manuscript
Tribe/Alliance/species Voucher Origin 2n Median size
(µm) Heterochromatin
patterns Figure
H. serratifolius (Vahl.) S. O.
Grose. Mattos JMPC, 251 Areia-PB 120 1.63 4A + 6B + 4C +
106F 1E, 4A
H. umbellatus (Sond.) Mattos* JMPC, 1043 Sertãozinho-PB 40 1.66 2A + 2B + 4C +
32F 1F, 4B
Sparattosperma leucanthum
(Vell.) K.Schum.* LPF, 15402 Alvorada de Minas-
MG 40 1.55 2A + 38F 1G, 4C
Tabebuia aurea (Silva Manso)
Benth. & Hook.f. ex S. Moore JMPC, 1078 Pirpirituba-PB 40 1.02 2A + 38F 1H, 4D
T. elliptica (DC.) Sandwith* SAAL, 81 Santa Rita-PB 40 1.86 2A + 38F 1I, 4E
T. rosea (Bertol.) Bertero ex A.
DC. JMPC, 154 Areia-PB 40 1.51 2A + 2C + 36F 1J, 4F
T. roseoalba (Ridl.) Sandwith* LPF, 14590 Campina Grande-PB 40 1.67 2A + 2C + 36F 1K, 4G
Zeyheria tuberculosa (Vell.)
Bureau ex Verl.* LPF, 14468 Maracás-BA 40 1.85 2A + 2C + 36F 1L, 4H
Crescentia cujete L. JMPC, 137 Serra da Raiz-PB 40 1.21 2A + 38F 1A, 4I
*First chromosome count for the species.
**New cytotype for the species.
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In Press, Accepted Manuscript
... Epidendrum nocturnum has been recorded as 2n = 40 and 80 (Tanaka and Kamemoto 1984;Felix and Guerra 2010;Assis et al. 2013), which highlights the occurrence of polyploidy, possibly due to ancestral genome combinations (Assis et al. 2013) and probably associated with self-pollination, apomixis, and polyembryony (Brieger and Bicalho 1977;Pavanelli 1985, 1986). Karyotype differentiation in morphologically complex groups of species can be better assessed with the use of specific markers such as heterochromatin bands (see, for example, Barros e Silva et al. 2010;Melo et al. 2014;Cordeiro et al. 2020). Cromomycin A3 (CMA) and 4 0 6-diaminidino-2-phenylindol (DAPI) are among the most used fluorochromes (Guerra 2000), providing evidence of genomic regions rich in guanine-cytosine (GC) and adeninethymine (AT) (Guerra 2000; Barros e Silva et al. 2010). ...
... The material was squashed using a coverslip in 60% acetic acid and frozen in liquid nitrogen to remove the coverslip. The slides were aged for three days at room temperature, stained for one hour with 10 lL of CMA (0.1 mg·ml -1 ), washed in distilled water, and then stained with 10 lL of DAPI (1 lg·ml -1 ) for half an hour, mounted in glycerol/ McIlvaine buffer (pH 7.0) (1/1, v/v), and stored for three days in the dark to stabilize the fluorochromes (Cordeiro et al. 2020). ...
Full-text available
The Epidendrum nocturnum Jacq. group comprises about 60 species. Nine species occur in Brazil, predominantly in the Atlantic and Amazon Forests. The group is taxonomically complex because its species are morphologically similar and not easily distinguished. The main objective of this work is to characterize their chromosome evolution and how this has affected species delimitation in Brazilian representatives of the E. nocturnum group. We used chromosome numbers, heterochromatin band patterns, and genome size variation to better understand the chromosome variation, species delimitation, and the relationship among seven representatives of this group. A new species from the Cerrado/Amazon Rainforest ecotone is described based on cytological and morphological characters. The new species, Epidendrum pareciense sp. nov., is a diploid (2n = 40), Epidendrum bahiense Rchb.f., Epidendrum carpophorum Barb.Rodr., Epidendrum micronocturnum Carnevali & G.A.Romero, Epidendrum purpureocaulis Sambin & Essers, and E. nocturnum are tetraploids (2n = 80), and Epidendrum tumuc-humaciense (Veyret) Carnevali & G.A.Romero is hexaploid (2n = 120), the last a new ploidy for the group. Heterochromatin in these species is characterized by guanine-cytosine-rich regions (cromomycin A3 (CMA ⁺ ) bands) on proximal and terminal regions associated with NORs. Genome size and numbers of CMA terminal bands are directly related to ploidy, suggesting that diploidization has not yet occurred and supporting the hypothesis of a recent polyploid origin for the E. nocturnum group.
... This is the first time that the karyotypes of the three species from the complex have been studied, because previous chromosome counts of Brazilian populations attributed to the T. cepula complex were misidentified as T. cebolleta (Felix & Guerra, 2000), which is not part of the complex but has the same chromosome number (Phang et al., 1979). Cytogenetic data, especially chromosome banding, has been important for taxonomy (reviewed in Guerra, 2008Guerra, , 2012, providing genetic evidence for the delimitation of closely related species (Acosta et al., 2012;Cordeiro et al., 2018Cordeiro et al., , 2020Pessoa et al., 2012Pessoa et al., , 2014Pessoa et al., , 2021Souza et al., 2012). However, our results show that even in groups in apparent initial stages of evolutionary divergence, the genome organization, represented by heterochromatin, can be extremely variable and not informative for taxon delimitation, as observed in the Pinus nigra J.F.Arnold complex (Boguni c et al., 2011). ...
The Trichocentrum cepula complex comprises three species, T. caatingaense, T. cepula and T. sprucei, endemic to tropical forests east of the Andes in South America. The delimitation of these species has been diversely interpreted due to the extensive morphological variation in the complex. We applied an integrative approach to achieve a better understanding of these biological units, using geometric morphometrics, cytogenetic analysis (chromosome counts and CMA/DAPI banding) and molecular phylogenetics (ITS and rpl32-trnL). An initial morphometric analysis using the pre-identified specimens into three taxa suggested that T. sprucei is distinct from the other two, which show some overlap. A subsequent analysis of the labellum, including only T. caatingaense and T. cepula organized in six pseudo-populations, suggested the existence of four morphological groups. All analysed specimens presented 2n = 36 chromosomes, CMA⁺/DAPI⁻ terminal bands and CMA⁻/DAPI⁺ pericentromeric bands, which varied in number across species, localities or even individuals from the same locality. The notable variation in DAPI⁺ pericentromeric bands may be related to transposable elements that could also be a factor influencing the wide morphological variation in the flowers. In the phylogenetic analysis, the specimens belonging to T. caatingaense formed a strongly supported clade sister to the rest, whereas the specimens belonging to T. cepula and T. sprucei emerged together, with their relationships tending to be determined by geographic proximity. The evidence we generated suggests that treating the Brazilian populations of this species complex under a single name, T. cepula, provides more taxonomic stability and utility, thus the necessary taxonomic changes are implemented.
... Chromosomal analysis has been extensively used for the karyotypic characterisation of different groups of plants, allowing the understanding of phylogenetic relationships and the detection of genetic diversity among taxa. Information such as chromosome number and size, morphology, and heterochromatin distribution, among other characteristics, is important for this type of study (Martins et al., 2018;Cordeiro et al., 2020;Querino et al., 2020). It is known that the genus Allium exhibits variation in regards to the chromosome number of its members and wide plasticity in base chromosome number, with reports of x = 7, 8, 10 and 11 (Xie-Kui et al., 2008; Khar et al., 2020). ...
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Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the “Branco Mineiro Piauí” accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.
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A new Epidendrum species of the Nocturnum group is proposed for the Brazilian central Amazon, near Manaus. It is described, illustrated, and compared with similar species. Epidendrum dayseae can be recognized by the pendent stem, the long, narrow leaves, a relatively long floral pedicel, and the lateral lobes of the lip smaller than the mid-lobe and deeply separated. The new species resembles E. longicolle, but is distinguished by the union between of the lateral and mid-lobes of the lip being under half the mid-lobe length. The new species is also compared with E. plurifolionocturnum. Its chromosome number is 2n = 4x = 80, with a band pattern similar to other species of the Nocturnum group.
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Jatropha is an important genus of Euphorbiaceae, with species largely used for various purposes, including the manufacturing of soaps and pharmaceutical products and applications in the bioenergetic industry. Although there have been several studies focusing J. curcas in various aspects, the karyotype features of Jatropha species are poorly known. Therefore, we analyzed six Jatropha species through fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with 5S and 45S rDNA probes and genome size estimation by flow cytometry. Our results revealed several chromosome markers by both CMA/DAPI and FISH for the analyzed species. Five Jatropha species (J. curcas, J. gossypiifolia, J. integerrima, J. multifida and J. podagrica) showed four CMA-positive (CMA+) bands associated with the 5S and 45S rDNA sites (one and two pairs, respectively). However, J. mollissima displayed six CMA+/DAPI- bands co-localized with both 5S and 45S rDNA, which showed a FISH superposition. A gradual variation in the genome sizes was observed (2C = 0.64 to 0.86 pg), although an association between evidenced heterochromatin and genome sizes was not found among species. Except for the unique banding pattern of J. mollissima and the pericentromeric heterochromatin of J. curcas and J. podagrica, our data evidenced relatively conserved karyotypes.
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The Peltophorum clade (Caesalpinioideae, Leguminosae) comprises 16 genera and approximately 105 species with overall pantropical distributions. The genera Peltophorum (Vogel) Benth. (9 spp.) and Delonix Raf. (11 spp.) are notable for having arboreal habits, ornamental uses, and karyotypes of 2n = 26 and 2n = 28, respectively. The present work sought to characterize the patterns of heterochromatin distribution and rDNA sites, as well as the genome sizes of three representatives of the Peltophorum clade. We analyzed P. pterocarpum (DC.) Backer ex K. Heyne, P. dubium (Spreng.) Taub., and D. regia (Bojer ex 121 Hook.) Raf. using double staining with the fluorochromes CMA and DAPI, fluorescent in situ hybridization (FISH) to detect rDNA sites, as well as flow cytometry to determine their DNA contents. The two species of Peltophorum have a chromosome number of 2n = 26, with meta/submetacentric chromosomes measuring on average 2.33 µm. On the other hand, D. regia has a number of 2n = 28, with larger chromosomes. Genome sizes varied from 1C = 0.90 pg in the two Peltophorum species to 1C = 1.55 pg in D. regia. CMA/DAPI staining showed CMA⁺/DAPI⁻ bands predominantly in the terminal regions of the chromosomes, with six, four, and three pairs being observed in P. pterocarpum, P. dubium, and D. regia, respectively. FISH revealed the presence of a chromosome pair with 5S rDNA sites in each of the two species of Peltophorum, and on two chromosome pairs in D. regia. The numbers of 35S sites varied from 6 to 24, with synteny being observed with 5S rDNA sites in a pair of chromosomes of P. pterocarpum and in two pairs in D. regia. Our data suggest that cytomolecular analysis could be useful tool for differentiating the species within the Peltophorum clade and that comparative analyses with other species could provide new insights into the karyotypic diversification of the subfamily Caesalpinioideae.
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Chromosome numbers and heterochromatin banding pattern variability have been shown to be useful for taxonomic and evolutionary studies of different plant taxa. Bignonieae is the largest tribe of Bignoniaceae, composed mostly by woody climber species whose taxonomies are quite complicated. We reviewed and added new data concerning chromosome numbers in Bignonieae and performed the first analyses of heterochromatin banding patterns in that tribe based on the fluorochromes chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI). We confirmed the predominant diploid number 2n = 40, as well as variations reported in the literature (dysploidy in Mansoa [2n = 38] and polyploidy in Dolichandra ungis-cati [2n = 80] and Pyrostegia venusta [2n = 80]). We also found a new cytotype for the genus Anemopaegma (Anemopaegma citrinum, 2n = 60) and provide the first chromosome counts for five species (Adenocalymma divaricatum, Amphilophium scabriusculum, Fridericia limae, F. subverticillata, and Xylophragma myrianthum). Heterochromatin analyses revealed only GC-rich regions, with six different arrangements of those bands. The A-type (one large and distal telomeric band) were the most common, although the presence and combinations of the other types appear to be the most promising for taxonomic studies.
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A taxonomic revision of Catalpa (Bignoiaceae), a genus of perennial trees frequently used in horticulture as garden and street trees, is provided. Eight natural species and two hybrid species are recognized, four in sect. Catalpa, four in sect. Macrocatalpa, and two hybrid species in sect. Catalpa. Although C. punctata has been used for one of the tropical species, C. macrocarpa is the correct scientific name. Catalpa tibetica is synonymous with C. bignonioides, C. fargesii with C. bungei, and C. obovata with C. macrocarpa. Lectotypes are designated for: Bignonia cassinoides, Bignonia longisiliqua, Bignonia longissima, Catalpa Walter, Catalpa subsect. Corymbosae, Catalpa bignonioides var. kaempferi, Catalpa bungei, Catalpa bungei var. heterophylla, Catalpa bungei var. intermedia, Catalpa domingensis, Catalpa fargesii, Catalpa henryi, Catalpa ×hybrida, Catalpa ovata var. flavescens, Catalpa punctata var. lepidota, Catalpa purpurea, Catalpa syringifolia var. pulverulenta, Catalpa sutchuensis, Catalpa ×teasii, and Cumbulu. Second-step lectotypes are designated for: Catalpa duclouxii, Catalpa ekmaniana, Catalpa oblongata, Catalpa obovata, and Catalpa ovata. Neotypes are designated for: Bignonia triloba, Catalpa aureovittata, Catalpa bignonioides var. variegata, Catalpa ×erubescens, Catalpa ×erubescens f. purpurea, Catalpa ×galleana, Catalpa ×hybrida var. atropurpurea, Catalpa japonica, Catalpa syringifolia var. aurea, Catalpa syringifolia var. koehnei, Catalpa syringifolia var. nana, Catalpa ×teasiana, and Catalpa umbraculifera.
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Most taxa in the Bignoniaceae have 2n = 40, but the basal clade Jacarandeae has 2n = 36, suggesting that x = 18 is the ancestral basic number for the family. Variations in heterochromatin band patterns in genera that are numerically stable, such as Jacaranda, could facilitate our understanding of the chromosomal and karyotypic evolution of the family. We characterized heterochromatin distributions in six Jacaranda species using chromomycin A3 (CMA) and 4’6-diamidino-2-phenylindole (DAPI). All of them had 2n = 36, including first counts for Jacaranda bracteata Bureau & K. Schum., Jacaranda irwinii A.H. Gentry, Jacaranda jasminoides (Thunb.) Sandwith, and Jacaranda rugosa A.H. Gentry. Their karyotypes had four to eight terminal CMA⁺/DAPI– bands per monoploid set. In the section Monolobos, Jacaranda brasiliana (Lam.) Pers. had eight terminal bands and Jacaranda mimosifolia D. Don had four; in the section Dilobos, J. bracteata had six bands per monoploid set, with the other species having five. While three species in the section Dilobos had the same number of terminal bands, J. irwinii had two additional pericentromeric bands and a proximal heterozygotic band, and J. bracteata had two distended CMA bands. The consistent records of 2n = 36 in Jacaranda may represent a plesiomorphic condition for the Bignoniaceae; therefore, the family originated from an ancestor with x = 18. However, 2n = 36 may represent a derived condition, and the family could have had an ancestral basic number of x = 20 that is still conserved in most representatives of the family.
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The genus Ameroglossum was known only from A. pernambucense, a species found on inselbergs in the states of Paraiba and Pernambuco, Brazil. Ameroglossum manoel-felixii, a new species of Ameroglossum, is described and illustrated here, and morphologically compared with A. pernambucense. Information about the geographic distribution of the new species and karyotypic comparisons of both species using CMA/DAPI banding and nuclear DNA content are also presented.
Genomes of animals and plants contain a large number of transposable elements (TEs). TEs often transpose into genic regions, affecting expression of surrounding genes. Intragenic TEs mostly reside in introns, and in much the same way as intergenic TEs, they are targeted by repressive epigenetic marks for transcriptional silencing. Silenced intragenic TEs generally co-repress expression of associated genes, while in some cases they significantly enhance splicing and transcript elongation. Genomes have evolved molecular mechanisms that allow the presence of silenced TEs within transcriptionally permissive chromatin environments. Epigenetic modulation of intragenic TEs often contributes to gene regulation, phenotypic expression, and genome evolution.
Capsicum comprises 35 species of chili peppers and five of them are cultivated worldwide as spices or vegetables. Diploid karyotypes based in x = 12 and x = 13 are common in Capsicum and the constitutive heterochromatin (cHet) is of particular interest in the genus since it is largely variable, particularly its highly GC-rich fraction. However, the repetitive DNA components of this heterochromatic regions are unknown. Given the co-locali-zation of rDNA loci with the CMA + DAPI-heterochromatic bands, we tested the hypothesis that the highly GC-rich cHet fraction is composed of the whole 18S-25S ribosomal DNA (rDNA) unit or some of its components. Here we report on a novel satellite for Capsicum and Solanaceae composed of the complete rDNA unit. We physically mapped six Capsicum derived specific 18S-25S rDNA probes that covered the entire span of the rDNA unit and analysed a DraI restriction product on eight chromosomally different taxa of Capsicum, representative of the major phylogenetic clades of chili peppers. The co-localization of every gene and spacer probes of the 18S-25S rDNA unit suggest their structural function as a major repetitive component of the highly GC-rich cHet in Capsicum species with x = 12. In addition, analyses of the clones derived from restriction assays in C. pubescens suggested that the differential functional status of 18S-25S rDNA loci (nucleolar organizer regions-NORs-or cHet) in this species is related to a divergence in a short sequence upstream the regulatory transcription initiation site (TIS) of the intergenic spacer (IGS). The results here provided evidence that an rDNA mega satellite played a significant role in the evolution of the karyotype features of x = 12 Capsicum species. The finding of a mega satellite family derived from the whole rDNA unit is a novelty for plant genomes.
The pantropical Caesalpinia group comprises 27 genera and approximately 205 species. The Neotropical species of the group are distributed in three main centers of diversity: the Andes, Mesoamerica (including Mexico, Central America, the southern USA and the Caribbean), and Northeastern Brazil. Our study investigates patterns of phylogenetic, environmental and geographical variation in an attempt to explain the karyotypic diversity which occurs within the Caesalpinia group. Cytogenetic analyses were based on chromosome number and morphology, CMA and DAPI heterochromatic bands, distribution of rDNA sites, and their molecular phylogenetic relationships based on the plastid markers matK, rps16, trnDT, trnL and ycf6 and the nuclear ribosomal marker ITS. Ecological differentiation among cytotypes was tested by comparing sets of climatic variables. In total, twenty species from ten genera of the Caesalpinia group were analysed, and all of them were found to have the same chromosome number (2n = 24). CMA⁺/DAPI⁻ bands were observed on the short arms of the acrocentric chromosomes in all of the species. Additionally, three different patterns were observed on the metacentric chromosomes: proximal CMA⁺/DAPI⁻ bands, proximal CMA⁰/DAPI⁻ bands and terminal CMA⁺/DAPI⁻ bands. The 45S rDNA sites varied from eight to 14, always co-localizing with CMA⁺/DAPI⁻ bands of acrocentrics, while 5S rDNA was localized in only a single chromosome pair in most species. A correlation between heterochromatic patterns and the geographic distributions/ecological niche of species was observed. Our analyses support some shared effects of the environment, when integrated with phylogeny and geography, on the CMA/DAPI variability. Thus, CMA⁺ heterochromatin is highly dynamic and its amplification/deamplification might be non-random, generating similar karyotypes in species which are not sister taxa in the molecular phylogeny but which co-occur in similar environments.