First cytomolecular characterization of three Neotropical woody bamboos (Bambusoideae, Poaceae) suggests ancient diploidized karyotypes

ABSTRACT

Although karyotype features are useful data for evolutionary studies, cytogenetic data in Bambusoideae are mainly based only on chromosome counts. The first comparative cytogenetic analysis of three Neotropical woody bamboo species - Guadua chacoensis, G. angustifolia and Chusquea tenella - was undertaken based on new and reviewed chromosome counts, CMA/DAPI double staining, fluorescent in situ hybridization (FISH) with 35S and 5S rDNA probes, and genome size estimation. The two species of Guadua were found to have 2n = 46 chromosomes, while the first record for C. tenella was 2n = 44. Only one pair of CMA⁺/DAPI^- was detected on the terminal region of metacentric chromosomes in all three species. Likewise, one pair of 5S and 35S rDNA sites was detected in all three species, with the 35S rDNA sites always collocated with the CMA⁺ bands. Genome sizes ranged from 2C ≈ 3.99 pg for the species of Guadua, to 2C = 4.77 pg for C. tenella. Considering the Miocene origin of Neotropical woody bamboos, the observed karyotype stability suggests that the analyzed species are diploidized paleopolyploids. The results reveal the conservative cytomolecular organization of Neotropical woody bamboo karyotypes, which helps to improve our understanding of the evolution of this group.

Keywords:
Chusquea; cytogenetics; diploidization; evolution; Guadua; polyploidy

Introduction

Representing the major clade of grasses with diversification in forest habitats, the bamboos (subfamily Bambusoideae) are perennial and multipurpose plants and one of the major non-timber forest products worldwide (Clark et al. 2015). The monophyletic Bambusoideae (127 genera/1,680+ species) is currently classified in three tribes, encompassing the herbaceous (Olyreae) and woody bamboos (Arundinarieae and Bambuseae) (Kelchner & BPG 2013; Soreng et al. 2017; Clark & Oliveira 2018). Within Bambuseae tribe, two major lineages are recognized [Paleotropical and Neotropical], with Brazil as one of the main centers of diversity and endemism of Neotropical bamboos (Greco et al. 2015; Soreng et al. 2017).

Both belonging to the Bambuseae tribe, the genera Guadua and Chusquea occur from Mexico to Argentina and represent relevant ecological component with economic potential within the Neotropical lineage (Clark et al. 2015). The genus Guadua (33 species) comprises species of greater height (up to 30 m) within the group, such as G. chacoensis and G. angustifolia, which has great suitability for construction (Clark et al. 2015; Vorontsova et al. 2016). The most diverse genus among woody bamboos (more than 180 species), Chusquea occurs primarily in montane forests and high-elevation grasslands, where they tend to occupy specialized habitats (Fisher et al. 2014; Clark & Mason 2019; Ruiz-Sanchez et al. 2020). Chusquea tenella is a common species of Brazilian Atlantic forest’s understory, frequently associated with altered and endangered environments (Schmidt & Longhi-Wagner 2009).

Woody bamboos are complex plants, with a tree-like habit with highly lignified culms and peculiar flowering behavior, displaying a long vegetative phase and typically gregarious and monocarpic cycles (Clark et al. 2015). Their evolutionary history involves independent rounds of polyploidy preceding the diversification of the main current lineages (Triplet et al. 2014; Guo et al. 2019). Assuming the basic chromosome number x = 12 proposed for Bambusoideae (Hilu 2004; Zhou et al. 2017), tetraploids are observed in Arundinarieae and Neotropical Bambuseae, while Paleotropical Bambuseae are considered hexaploids (Hilu 2004; Clark et al. 2015).

Polyploidy has been recognized as one of the major evolutionary forces in Angiosperms diversification and evolution, which is frequently associated with the process of cytological and genetic diploidization (Wendel 2015; Dodsworth et al. 2016). In Poaceae, it is estimated that all the members share at least two events of polyploidization followed by diploidization evolution (paleopolyploids) in some species, such as Sorghum bicolor and Oryza sativa (Jiao et al. 2011; Paterson et al. 2012). Thus, considering the ancient polyploid origin of Bambuseae tribe (ca. 33 Mya; Ruiz-Sanchez 2011; Guo et al. 2019), Neotropical lineages represent excellent models for investigating the cytogenetics consequences of diploidization processes in paleopolyploids (see Figueredo et al. 2016).

Karyotype analyses are proven to be useful tools for detailed genome characterization, providing relevant information such as chromosomal rearrangements, allopolyploid origin, and phylogenetic and evolutionary relationships of many plant groups (Berjano et al. 2009; Guerra 2012; Kolano et al. 2013; Souza et al. 2015; Carvalho et al. 2017). However, especially due to their long-life cycle and unpredictable flowering in woody bamboos, karyotype evolution is a complex and neglected subject, relying basically on chromosome counts (Nirmala et al. 2014).

The present work analyzed the karyotypic diversity of the three Neotropical woody bamboo species based on chromosome counts, morphology, double staining with the fluorochromes chromomycin A₃ (CMA) and 4’,6-diamidino-2- phenylindole (DAPI), and fluorescent in situ hybridization (FISH) with 5S and 45S rDNA. Besides that, we estimated the genome size of these species by flow cytometry. By the new data obtained here, we aimed to address three questions in the woody bamboos: (1) Does cytomolecular data corroborate a paleopolyploid origin of this lineage? (2) What is the degree of diploidization of the analyzed karyotypes? (3) Did the species of Guadua and Chusquea show similar trends in the evolution of the numbers of rDNA sites?

Materials and methods

Plant materials

Specimens of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and G. angustifolia Kunth were obtained from the collection of bamboo species from “Ressacada” Experimental Farm of the Federal University of Santa Catarina (UFSC) (27º41.1’ S; 48º32.63’ O), while Chusquea tenella Nees samples were collected from a natural population at “Ponta do Goulart” locality (27º33.68’ S; 48º31.33’ O), both in Florianópolis, Santa Catarina state (SC), Brazil. Voucher specimens were deposited in the Herbarium FLOR (UFSC) with the following numbers: G. chacoensis (FLOR 58620; Rossarolla MD & Venturi M., 16); G. angustifolia (FLOR 58626, Rossarolla MD, Venturi M., 26) and C. tenella (FLOR 58638; Rossarolla et al. 5).

Root tips were pretreated with 2 mM 8-hydroxyquinoleine for 24 h at 10 ºC, fixed in a solution of ethanol:acetic acid (3:1, v/v) for 2 to 24 h at room temperature, and stored at (20 ºC.

Cytogenetic analyzes

Chromosome banding

After washing with distilled water, root tips were digested in a solution of 2 % (w/v) cellulase (Onozuka)/20 % (v/v) pectinase (Sigma) for 1 h at 37 ºC. For slide preparation of chromosomes spread, the root was squashed in a drop of 45 % acetic acid (v/v) and the coverslip was later removed in liquid nitrogen.

For the CMA/DAPI double staining technique, the slides were aged for three days, stained with 10 µL CMA (0.1 mg mL^-1) for 1 h, and restained with 10 µL DAPI (2 µg mL^-1) for 30 min (Barros-e-Silva & Guerra 2010). The slides were mounted in glycerol:McIlvaine buffer pH 7.0 (1:1, v/v), and aged for three more days before analysis in an epifluorescence Leica DMLB microscope. Images were captured with Cohu CCD video camera using Leica QFISH software and then edited using Adobe Photoshop CS3 version 10.0 for better brightness and contrast.

Fluorescent in situ hybridization (FISH)

For the subsequent FISH, selected slides were destained in a solution of ethanol:acetic acid (3:1, v/v) (for 30 min) and absolute ethanol (for 1 h), and pretreated as described by Pedrosa et al. (2001). In order to localize rDNA sites, a 500 bp 5S rDNA clone (D2) of Lotus japonicus (Regel) K. Larsen labeled with Cy3-dUTP (Amersham) and a 6.5 kb 18S-5,8S-25S rDNA clone (R2) from Arabidopsis thaliana (L.) Heynh. labeled with digoxigenin-11-dUTP (Roche) were used as probes (Pedrosa et al. 2002). Both labeling techniques were performed by nick translation. The 35S rDNA probe was detected with sheep anti-digoxigenin FITC conjugate (Roche), and amplified with donkey anti-sheep FITC conjugate (Vector).

FISH analysis was performed according to Pedrosa et al. (2002), with minor modifications. The hybridization mix contained 50 % (v/v) formamide, 10 % (w/v) dextran sulfate, 2 × SSC and 5 ng µL^-1 of each probe. The slides were denatured at 75 ºC for 5 min and hybridized for up to 48 h at 37 ºC. Post-hybridization washes were performed in 0.1 × SSC at 42 ºC, reaching a final stringency of approximately 76 %. Afterward, the slides were counterstained and mounted in DAPI (2 µg mL^-1):Vectashield (Vector) (1:1, v/v) solution, and images of the cells were acquired as described above.

Chromosome morphometry and idiograms

For each species, at least 10 well-spread metaphases were analyzed using Adobe Photoshop CS3 version 10.0. The chromosome arm ratio (AR = length of the long arm / length of the short arm) was used to classify the chromosomes as metacentric (AR = 1-1.4), submetacentric (AR = 1.5-2.9), or acrocentric (AR> 3.0), following Guerra (1986). Karyotype symmetry was evaluated by the intrachromosomal (A₁) and interchromosomal (A₂) asymmetry indices (Zarco 1986). Mean lengths of total chromosome complement (T) of each chromosome pair, of short (SA) and long arms (LA), as well as number and position of heterochromatic bands and of the 35S and 5S rDNA sites were used to construct idiograms representing the haploid complement of each analyzed species, using CorelDRAW version X6 software.

Flow Cytometry

DNA content of three independent specimens was estimated by flow cytometry (FCM) using Glycine max var. Polanka (L.) Merr. as internal standard (2C = 2.5 pg; Doležel et al. 1994). Nuclei suspensions were obtained by co-chopping leaf fragments (30 mg) of each bamboo species and the internal standard in a Petri dish containing 1.5 mL of Woody Plant Buffer (Galbraith et al. 1983; Loureiro et al. 2007). Afterward, the nuclei suspension was stained with 20 µL propidium iodide (1 mg mL^-1) and analyzed on a flow cytometer PARTEC CyFlow. Histograms of relative fluorescence intensity of the G₀/G₁ peak were analyzed using FlowMax software version 2.4 (Partec). The genome size, or nuclear DNA content (pg), of each species was calculated according to the formula:

$$\begin{array}{l}\text{Nuclear DNA content }\left(\frac{\text{pg}}{\text{2C}}\right)\\ \text{=}\left[\frac{\text{sample G1 peak mean}}{\text{standard G1 peak mean}}\right]\text{x standard nuclear DNA content }\left(\frac{\text{pg}}{\text{2C}}\right)\end{array}$$

Results

The cytogenetic data of Guadua chacoensis, G. angustifolia, and C. tenella revealed similar and symmetrical karyotypes (see A₁ and A₂ index), regarding length of total chromosome complement (T) and predominance of metacentric (M) and submetacentric (SM) chromosomes. Guadua chacoensis and G. angustifolia equally showed 2n = 46 chromosomes (Figs. 1A-D, 2A-B), with the karyotype formula 14SM + 9M and 12SM + 11M, and T = 72.86 µm and T = 73.97 µm, respectively. In turn, C. tenella showed 2n = 44 (Figs. 1E-F, 2C) with 9SM + 13M, and similar mean length of total chromosome complement (T = 73.68 µm). Concerning the karyotype symmetry, C. tenella was slightly more symmetric (A₁ = 0.249; A₂ = 0.203) compared to G. chacoensis (A₁ = 0.323; A₂ = 0.342) and G. angustifolia (A₁ = 0.308; A₂ = 0.319).

Figure 1
Distribution of heterochromatin and rDNA sites in Guadua chacoensis (A, B), G. angustifolia (C, D), and Chusquea tenella (E, F). Arrows in A, C, and E indicate CMA⁺/DAPI^- bands (yellow). In B, D, and F, arrows show 35S rDNA sites (green) and arrowheads show 5S rDNA sites (red). Bar = 5 µm.

Figure 2
Idiograms of Guadua chacoensis (A), G. angustifolia (B), and C. tenella (C). Chromosomes are arranged by the position of centromere and by decreasing order of length. The 35S and 5S rDNA sites are represented by the green and red marks, respectively. CO: chromosome order; L: chromosome length; AR: arm ratio.

Figure 3
Histograms of relative fluorescence intensities obtained by FCM of nuclei suspension of Glycine max var. Polanka (internal standard; 2C = 2.5 pg) and (A) G. chacoensis (2C = 3.98 pg), (B) G. angustifolia (2C = 3.99 pg), and (C) C. tenella (2C = 4.77 pg).

Chromosome banding with CMA/DAPI fluorochromes showed only one pair of CMA⁺/DAPI^- bands for each species, revealing a remarkably stable heterochromatic pattern (Fig. 1A, C, E). The single pair of CMA⁺/DAPI^- bands was located on the terminal region of the long arm in G. chacoensis (Fig. 2A) and G. angustifolia (Fig. 2B), and short arm in C. tenella (Fig. 2C). No CMA^-/DAPI⁺ bands were detected in all three species. The 5S and 35S rDNA sites, detected by FISH, were located in different chromosomes: the 35S rDNA sites were collocated with CMA⁺/DAPI^- bands (Fig. 2A-C), whilst the 5S were on the interstitial region of the long arm in both Guadua species (Fig. 2A, B) and on the short arm of C. tenella (Fig. 2C).

The nuclei suspensions of all bamboo species and G. max var. Polanka (internal standard) resulted in histograms of G₀/G₁ peak showing coefficients of variation below 5 %, providing reliable and high quality of flow cytometric data (Fig. 3). The mean nuclear DNA content was estimated as 2C = 3.98 pg for G. chacoensis, 3.99 pg for G. angustifolia, and 4.77 pg for C. tenella. This is the first genome size estimation for G. chacoensis, as well as for the genus Chusquea.

Discussion

In woody bamboos, the prevailing basic chromosome number is x = 12, although x = 10, 11 numbers were also reported, especially for species of the genus Chusquea (Hilu 2004). The chromosome number observed for G. chacoensis and G. angustifolia (2n = 46) confirms previous counts for both species (Quarín 1977; Chen et al. 2003), while 2n = 44 is the first report for C. tenella. The chromosome numbers reported herein support the tetraploid karyotype in Neotropical woody bamboo species (Triplett et al. 2014; Guo et al. 2019).

In Guadua, 2n = 46 was also observed for G. paraguayana and G. capitata (Davidse & Pohl 1992; Gould & Soderstrom 1967), with the only deviation reported in G. macclurei (2n = 48; Davidse & Pohl 1992). For Chusquea species, the most common chromosome number reported is 2n = 40, based on x = 10. Variation from the expected number was also reported to C. oxylepsis (2n = 44; Hunziker et al. 1989). Those variations from the expected chromosome numbers must be then related to dysploid events from an ancient tetraploid karyotype, since the basic chromosome number for the Bambuseae tribe ranges from x = 10 to 12 (Schubert & Lysak 2011; Clark et al. 2015).

Polyploidy is recognized as a major evolutionary force in plants and it seems to have played an important role in radiation and habitat exploitation of woody bamboos (Nirmala et al. 2014; Soltis et al. 2014). Genomic data suggest that polyploidy plays an important role regarding the origin of woody traits and regulation of flowering behavior within the group (Guo et al. 2019). In fact, woody bamboo diversity is a consequence of three independent allopolyploid events involving four monophyletic and ancestral subgenomes, revealing a complex pattern of reticulate evolution (Triplett et al. 2014; Guo et al. 2019).

Fundamentally, the cytogenetic assumption of a recently formed polyploid is the additive pattern of rDNA sites/ heterochromatic blocks (Clarkson et al. 2005; Hasterok et al. 2006; Zhang et al. 2016). For instance, a hexaploid origin proposed for the Paleotropical woody bamboo lineage was corroborated by the presence of three pairs of 35S rDNA sites in Bambusa oldhamii, B. gibboides, and Melocanna baccifera (Zhou et al. 2017). Following this additive pattern in polyploids, two pairs of rDNA sites in tetraploids temperate and Neotropical woody bamboos would be expected (Triplett et al. 2014; Clark et al. 2015). However, our data revealed only one pair of rDNA sites and heterochromatic bands in three Neotropical woody bamboo species, suggesting a karyotype evolution shaped by diploidization events. The diploidization hypothesis is corroborated by analysis of temperate tetrapolyploids (Arundinaria tribe; 2n = 48) Phyllostachys heterocycla, P. vivax, Indosasa gigantea, and Pleioblastus gramineus, that indicate also only one pair of 35S rDNA sites detected by FISH (Peng et al. 2013; Zhou et al. 2017).

Since phylogenetic relationships and polyploid origin within woody bamboo lineages are currently well established, the group could represent a model to understand the evolution of paleopolyploid karyotypes (Triplett et al. 2014; Guo et al. 2019). Therefore, given the Miocene origin (20 - 25 Mya) of the Neotropical woody bamboo lineage (Ruiz-Sanchez 2011; Guo et al. 2019), as well as the karyotype features and reduced number of heterochromatic bands and rDNA sites, it is reasonable to consider the three analyzed species as diploidized paleopolyploids (Clarkson et al. 2005; Berjano et al. 2009; Figueredo et al. 2016).

Both processes of genetic and cytological diploidization are frequently associated with the evolution of polyploid genomes, which has been widely recognized as an important evolutionary force across plant species (Ma & Gustafson 2005; Jiao et al. 2011). Genetic diploidization involves gene loss or rearrangement, while cytological diploidization leads to reduced ploidy level and/or chromosome number, restoring the diploid-like meiotic behavior of the polyploid genome (Wolfe 2001; Hollister 2015; Dodsworth et al. 2016). Further karyotype and meiotic behavior analysis including a wide range of Neotropical woody bamboo species would be required to confirm the diploidized state of the group, which would be of great interest to resolve the complex genome evolution within the group.

Chromosome number and genome size variations have been widely suggested as an important character in grass evolution, mainly due to the effects of independent rounds of polyploidization (Bennetzen 2007; Devos 2010). Genome size estimations of woody bamboo species relied mainly on Paleotropical and temperate lineages, ranging from 2C = 1.67 pg (Bambusa affinis) to 2C = 5.61 pg (Pseudosasa japonica) (Gielis et al. 1997; Gui et al. 2007; Kumar et al. 2011; Jia et al. 2016; Zhou et al. 2017). The genome size of Neotropical woody bamboo had only been reported for G. angustifolia (2C = 3.03-3.98 pg; Gielis et al. 1997; Guo et al. 2019). Thus, the genome size data presented here are the first measurements for G. chacoensis and the genus Chusquea.

Even though polyploidization events can directly affect karyotype features (e.g. chromosome number, genome size, etc.), these traits are shaped by independent evolutionary processes (Weiss-Schneeweiss & Schneeweiss 2013). Chusquea tenella (2n = 44) showed a larger genome size (2C = 4.77 pg) when compared to G. chacoensis and G. angustifolia (both with 2n = 46 and 2C ≈ 3.99 pg), corroborating the independent evolution of chromosome number/genome size (Zhou et al. 2017; Guo et al. 2019). On the other hand, both intrachromosomal and interchromosomal asymmetry indices revealed a symmetric karyotype in C. tenella and Guadua species (see Peruzzi & Eroglu 2013), suggesting that this is not a good cytogenetic parameter to differentiate the karyotype of these species.

Genome size and the total length of chromosomes (T) are often evolutionarily correlated (Schubert & Lysak 2011; Weiss-Schneeweiss & Schneeweiss 2013). The three bamboo species analyzed showed a very similar total chromosome length (T) despite differences in genome size. Differential chromatin condensation patterns can explain this apparent incongruity (see Feitoza et al. 2017). In general, early-condensed proximal and late-condensed terminal chromatin are related to smaller genomes (as observed here in Guadua species), and a uniformly condensed chromatin is usually found in species with larger genomes (as C. tenella).

The results herein presented the first chromosome count for C. tenella, as well as the first genome size estimation for G. chacoensis and the genus Chusquea, revealing the first insights into karyotype evolution within the Neotropical woody bamboo lineage. This finding highlights the complexity of genetic analysis within the group, regarding chromosome number (polyploidy), genome size, and karyotype features. Furthermore, considering the diploidization hypothesis must be crucial for cytogenetic/genomic studies.

Acknowledgments

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Proc. 457726/2013-0, 302798/2018-8, 407974/2018-0, and 302105/2017-4, as well as the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and fellowships. We also thank the Laboratório de Citogenética e Evolução Vegetal of Universidade Federal de Pernambuco (UFPE), Recife - PE, Brazil.

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Publication Dates

• Publication in this collection
  22 Mar 2021
• Date of issue
  Oct-Dec 2020

History

• Received
  25 July 2019
• Accepted
  03 Aug 2020