Chromosomal variability in Brazilian species of Anthurium Schott (Araceae): Heterochromatin, polyploidy, and B chromosomes

Abstract The genus Anthurium has a Neotropical distribution, with karyotype predominance of x = 15, although some species show disploidy or polyploid variations. The karyotypes of seven species and different populations of Anthurium were analyzed using fluorochrome CMA and DAPI staining. The karyotypes were composed of meta- and submetacentric chromosomes, with numbers varying from 2n = 30 to 2n = 60. Supernumerary euchromatic chromosomes were observed in A. affine, and supernumerary heterochromatic chromosomes were observed in A. gladiifolium and A. petrophilum. Polyploidy was recurrent in the Anthurium species analyzed, with records of 2n = 30 and 60 in different A. pentaphyllum populations. Fluorochrome staining revealed different CMA+ banding distributions between diploid and polyploid cytotypes of A. pentaphyllum, suggesting structural alteration events. Anthurium petrophilum, on the other hand, showed a more consistent banding profile, with 10 to 12 proximal CMA bands in the three populations analyzed. DAPI+/CMA0 regions occurred exclusively in populations of A. gracile and A. pentaphyllum. The heterochromatic fraction in Anthurium was found to be quantitatively variable among species and populations, which may be related with adaptive aspects, different environmental conditions, or phylogenetic position.


Introduction
The genus Anthurium is a strictly Neotropical monophyletic group that occurs from Mexico to Argentina, and is included within the subfamily Pothoideae, tribe Potheae (Croat, 1986;Coelho et al., 2009;Cusimano et al., 2011;Carlsen and Croat, 2013;Govaerts et al., 2016). This group comprises approximately 950 species (Boyce and Croat, 2011 onwards), 134 of which are known in Brazil (Coelho et al., 2018). The genus is taxonomically complex and subdivided into 18 sections  showing wide intra-and interspecific morphological diversity (Coelho and Mayo, 2007). The plants can have a terrestrial habit in the case of forest species, or be rupiculous, epiphytic, or hemiepiphytic vines, but they are rarely found in aquatic environments (Coelho et al., 2009;Gonçalves and Jardim, 2009); there are numerous helophytic species that can be found growing on exposed rock surfaces (Gonçalves, 2005;Haigh et al., 2011). The genus is monophyletic, with 18 clades that are easily distinguishable morphologically or geographically, and show low divergence in their trnG intron, trnH-psbA and trnC-ycf6 sequences, and in the CHS intron regions of their DNA, suggesting a rapid radiation of the group (Carlsen and Croat, 2013).
The most complete study of B chromosomes in Anthurium was undertaken by Marutani and Kamemoto (1983), and included examining both somatic and meiotic cells in A. warocqueanum Moore. These authors observed that the numbers of B chromosomes in somatic cells in the species was constant (2n = 30 + 3B), although there were different associations during metaphase I of meiosis (one trivalent, one bivalent and one univalent, or three univalents), resulting in variable numbers of B chromosomes in selfed offspring (ranging from 0 to 6) and indicating their transmission from both male and female gametes. The diversity of B chromosomes in Anthurium was noted by Marutani et al. (1993), who reported them in A. ochranthum K.Koch, A. cerrocampanense Croat, and A. garagaranum Standl., as  B chromosomes are commonly heterochromatic, although they appear euchromatic in some species (Camacho et al., 2000;Banaei-Moghaddam et al., 2014). No differential staining of Anthurium chromosomes has yet been undertaken, and the chromatin compositions of B chromo-somes among its different species have not been examined. We therefore analyzed chromosome number variability and CMA/DAPI banding distributions in seven Brazilian species of Anthurium to identify interspecific variations and supernumerary chromosomes in different populations and cytotypes. The main objective of this work was to identify karyotype variability in Brazilian species of Anthurium to determine the importance of that variability to chromosome evolution in the genus.

Collections and botanical documentation
Seven species of Anthurium harvested in various regions of Brazil were investigated, including individuals from three different populations. Intraspecific variations were investigated in four of the seven species. Information concerning all of the samples and their respective collection localities, populations, and collectors are summarized in Table 1. Specimens were maintained alive in the experimental gardens of the Plant Cytogenetic Laboratory of the Department of Biological Sciences of the Agrarian Sciences Center at the Federal University of Paraíba (UFPB), Brazil. Exsiccates were deposited in the Prof. Jayme Coelho de Moraes Herbarium (EAN).

Chromosomal analyses
Root tips were pretreated with 0.2% colchicine for 24 h at 10 C, fixed in 3:1 ethanol -acetic acid (v:v) for 2 h at room temperature, and subsequently stored at -20 C until 636 Nascimento et al. analyzed. The material was then washed in distilled water and digested in an enzymatic solution containing 2% cellulase (Onozuka) and 20% pectinase (Sigma) (w/v) for 1 h at 37°C. Slides were prepared using the squashing method in a drop of 45% acetic acid. Coverslips were subsequently removed in liquid nitrogen and samples were then air dried and kept for three days at room temperature (Guerra and Souza, 2002). Fluorochrome staining followed the protocol described by Carvalho et al. (2005). Samples were stained with 10 mL chromomycin A3 (CMA) (0.1 mg/mL) and stored for 1 h in the dark, before staining with 10 mL de DAPI (2 mg/mL), were again stored in the dark for 30 min before mounting with glycerol/Mcllvaine. The slides were aged for three days in the dark to stabilize the fluorochromes. Metaphases were photographed using a AxioCam MRm epifluorescence microscope (Zeiss) equipped with a video camera, utilizing Axiovision 4.8 software (Zeiss). Images were processed using Adobe Photoshop CS3 Software (Adobe Systems). Chromosome measurements were made using Image Tool 3.0 software (Brent et al., 2008). Chromosome morphology was determined using the centromeric index, following Guerra (1986a).

Results
Chromosome numbers and heterochromatin characteristics are summarized in Table 1. All species exhibited symmetrical karyotypes, with chromosomes varying from submetacentric to metacentric (Figures 1 and 2). Chromosome numbers varied from 2n = 30 to 2n = 60, with most species showing 2n = 30; 2n = 40 was observed in two populations of A. gracile ( Figure 1H, I) and 2n = 60 in two populations of A. pentaphyllum ( Figures 2C-D). Euchromatic B chromosomes were observed in a population of A. affine Schott from Queimadas, Paraíba State ( Figure 1A), and in populations from Águas Belas, Pernambuco State ( Figure  1C) and Andaraí, Bahia State ( Figure 1E). Anthurium gladiifolium Schott, on the other hand, showed three heterochromatic B chromosomes ( Figure 1F), while the population of A. petrophilum K.Krause from São João do Tigre, Paraíba, showed a single heterochromatic B chromosome ( Figure 2G). The species of Anthurium with B chromosomes analyzed here, their respective populations, and the frequency of B chromosomes in mitotic cells are presented in Table 2. None of the other species exhibited supernumerary chromosomes.
Staining with fluorochromes revealed from one to two proximal CMA + /DAPIbands on the short arm of A. affine ( Figure 1A-E), in two populations of A. gracile Lindl. with 2n = 40 ( Figure 1H-I), in A. jilekii Schott (Figure 2A), and in Anthurium sp. (Figure 2H). The population of A. gracile from Senhor do Bonfim, Bahia (2n = 30) showed up to 10 conspicuous proximal CMA bands ( Figure 1G), while A. gladiifolium showed up to 12 proximal bands ( Figure  1F), and up to 13 CMA bands were seen in a diploid population of A. pentaphyllum ( Figure 2B). The tetraploid popu-lations of A. pentaphyllum examined, however, exhibited five proximal CMA bands in a population from Mamanguape, Paraíba ( Figure 2C) and three bands in a population from Itabaiana, Sergipe ( Figure 2D). Anthurium petrophilum, on the other hand, demonstrated a more consistent banding profile, with 10 to 12 proximal CMA bands in the three populations analyzed. DAPI + /CMAbands were not clearly observed, except in the A. gracile population from Peruíbe ( Figure 1I) and in A. pentaphyllum from Meruoca ( Figure 2B), where the terminal regions of some chromosomes appeared stained with DAPI rather than with CMA, which were interpreted as DAPI + /CMA 0 regions.
The chromosome number 2n = 30 is the most frequent in the genus Anthurium, although other chromosome numbers, such as 2n = 26, 28, 32, 36, and 40, also occur (Sheffer and Kamemoto, 1976;Sheffer and Croat, 1983;Viégas et al., 2006). Those variations may represent cases of ascending or descending disploidy or different euploidy series of n = 15. Similarly, reports of polyploidy generally follow two distinct models (2n = 30-60-90 and 2n = 28-56) Viégas et al., 2006). Among the polyploid species analyzed, A. pentaphyllum follows the 30-60-90 model, the most common in the genus . In the 2n = 30, 40 and 60 series reported for A. gracile Guerra, 1986b;present work), however, 2n = 40 may have resulted from ascending or descending disploidy, although there are no intermediate chromosome numbers in the literature in support of those events.

Cytogenetics of Anthurium (Araceae) 637
Nascimento et al. The basic number x = 15 appears as the most probable for Anthurium based on the wide occurrence of 2n = 30 in the genus (Marchant, 1973). Sheffer and Kamemoto (1976) and Sheffer and Croat (1983)  ancestral number due to records of 2n = 24 and 48 in species of the section Tetraspermium Schott. Molecular phylogenetic data nonetheless suggest that the section Tetraspermium occupies a derived position in the genus (Carlsen and Croat, 2013). Anthurium flexile Schott, with 2n = 60 (Sheffer and Kamemoto, 1976), and A. clidemioides Standl. with 2n = 30 (Petersen, 1989) have been considered the most basal species (Carlsen and Croat, 2013), in support of x = 15 as the basic number of Anthurium. However, the hypothesis of x = 12 cannot be discarded offhand, as species of the genus Pothos L. (a sister group to Anthurium) show 2n = 24 and 26 (Rice et al., 2015), suggesting a relationship of those numbers to the karyotypic evolution of Anthurium.

B chromosomes
Of the 153 species of the genus Anthurium with known chromosome numbers, B chromosomes have been identified in 20 (approximately 13%). Among the species found to have B chromosomes, there are records for A. affine (Cotias-de- Oliveira et al., 1999) and the new occurrences in A. gladiifolium (30+3Bs) and A. petrophilum (30+1B). However, the occurrence of B chromosomes in the genus may be underestimated, whereas the numbers of B chromosomes may have been interpreted as intraspecific disploidy variation in chromosome numbers. For example, A. obtusum (Engl.) Grayum with 2n = 24, 30, A. durandii Engl. with 2n = 28, 30 , and A. conspicuum Sodiro with 2n = 28, 32 (Rice et al., 2015) may reflect B chromosomes interpreted as A chromosomes.
The B chromosomes of Anthurium, besides varying in number, can also vary in their origin and chromatin composition. Anthurium affine is distinct from other species because its B chromosomes were euchromatic, while A. gladiifolium and A. petrophilum show B chromosomes composed principally of GC-rich heterochromatin. Anthurium affine is characterized by having only small quantities of GC-rich heterochromatin, which are observed only in the NORs of one or two chromosomes. Anthurium gladiifolium and A. petrophilum, on the other hand, show large CMA bands in the pericentromeric regions of five to six chromosome pairs. Although the origins of B chromosomes are not yet certain, one well-accepted hypotheses is their derivation from A chromosomes (Jones and Houben, 2003;Houben et al., 2013). In that sense, it is reasonable to suppose that GC-rich heterochromatin regions of the A chromosomes of A. gladiifolium and A. petrophilum were incorporated into (and amplified in) their B chromosomes.
The occurrence of B chromosomes in Anthurium, as well as other groups of plants, seems to be independent phenomena (Camacho et al., 2000;Levin et al., 2005), without any clear effects above the species level. Phylogenetic analyses corroborate that hypothesis, as one can see in Anthurium species that have B chromosomes, but are placed in different clades (see the phylogenetic hypothesis proposed by Carlsen and Croat, 2013). As in Anthurium, the occurrences of B chromosomes in Picea A.Dietr. (Pinaceae) do not show clear phylogenetic relationships (Lockwood et al., 2013). All of the species of Calochortus Pursh (Liliaceae) that have B chromosomes (D'Ambrosio et al., 2017), on the other hand, are in the same clade (Subsection Venusti, Patterson and Givnish, 2003), suggesting that the occurrence of B chromosomes in different plants reflects different causes.
The presence of B chromosomes can produce phenotypic effects at the level of individuals, especially related to vigor, fertility and fecundity, increased germination vigor or speed, or the appearance of morphological traits (leaf striping in maize, for example) (Camacho et al., 2000;Banaei-Moghaddam et al., 2014;Houben et al., 2014). Studies involving correlations of B chromosomes and ecological/adaptive aspects will be extremely important to the understanding of their evolutionary relationships in plants, making Anthurium an excellent genus for testing hypotheses.

Heterochromatin in Anthurium
Heterochromatin distribution appears to be relatively variable among different species and populations of Anthurium. Heterochromatin is most frequently located in the subtelomeric and pericentromeric regions of plant chromosomes and in NORs (Lamb et al., 2007). Heterochromatin associated with NORs in plants frequently appears as CMA + /DAPIbands (Guerra, 2000). Those sequences can be differentially amplified, forming characteristic patterns useful in differentiating between the karyotypes of closely related taxa, such as in Citrus L. (Carvalho et al., 2005), Acianthera Scheidw. (Oliveira et al., 2015), the Bignonieae tribe (Cordeiro et al., 2017), Spondias L. (Almeida et al.,640 Nascimento et al.  (Almeida et al., 2016), and Vigna Savi (Shamurailatpam et al., 2014). Differential amplification of heterochromatin was observed in all of the species analyzed in the present work, especially in A. gracile, which exhibited from 2 to 10 CMA + bands in different populations. The phenomena responsible for variation in the heterochromatic portions of different plant species are not well known. The diverse CMA banding patterns observed in genera such as Caesalpinia L. sensu latu (Fabaceae) appear to be related to geographic distribution, ecological niches, and the phylogenetic relationships between the species ( Van-Lume et al., 2017). The heterochromatic fraction in Anthurium is quantitatively variable among species and populations, and may be related to adaptive aspects, reflecting environmental or phylogenetic factors in those taxa. Corroborating this hypothesis, the population of A. gracile from Senhor do Bonfim in the semiarid region of Bahia showed large numbers of CMA bands (10) when compared to populations from the humid coastal areas of Paraíba and São Paulo (each with only one pair of bands). Chromosome studies involving larger numbers of species and populations, in conjunction with evolutionary phylogenetic methodologies, could aid in understanding the karyotypic diversity observed in Anthurium, one of the most diversified groups of Neotropical monocotyledons.