moleCular CytogenetiCS revealS an unCommon

StruCtural and numeriCal ChromoSomal

heteromorPhiSm in ZePhyrantheS braChyandra

(amaryllidaCeae)

la CitogenétiCa moleCular revela un heteromorfiSmo CromoSómiCo

numériCo y eStruCtural PoCo Común en ZePhyrantheS braChyandra

(amaryllidaCeae)

1

Thiago Nascimento * , Raquel S. Gonçalves , Mariana Báez

,

2

1

and Marcelo Guerra

Guillermo Seijo

1

. Laboratório de Citogenética e

Summary

Evolução Vegetal, Departamento

de Botânica, Universidade Federal

de Pernambuco-UFPE, R. Prof.

Moraes Rego, s/n, Recife, PE 50670-

Background and aims: Zephyranthes brachyandra belongs to a tribe of ornamental

Amaryllidaceae native of South America, whose genera circumscription and

phylogenetic relationships are still unclear. Cytologically, Z. brachyandra is a

tetraploid whose chromosomes are of similar size and morphology, hindering the

identiﬁcation of its 2n = 24 chromosomes. The aim of this study was to investigate the

stability of the many CMA and DAPI bands and the occurrence of B chromosomes

by a cytomolecular approach.

M&M: For this investigation we conducted a cytomolecular analysis with CMA/DAPI

staining and ﬂuorescence in situ hybridization with 5S and 35S rDNA probes, and

the TTTAGGG telomeric probe.

4

2

20, Brazil.

.InstitutodeBotánicadelNordeste

+

(

UNNE - CONICET) and Facultad

de Ciencias Exactas y Naturales y

Agrimensura, Universidad Nacional

del Nordeste, Sargento Cabral

2

131, 3400 Corrientes, Argentina.

thiagoagtc@gmail.com

Citar este artículo

Results: In the present work, a cytomolecular analysis of Z. brachyandra, revealed a

+

large and variable number of CMA and DAPI heterochromatic bands and 5S and

5S rDNA sites, and a regular distribution of the TTTAGGG telomeric sequences.

*

3

In addition, one individual was monotrisomic with 2n = 24, and another one had a B

chromosome. Both numerical and structural chromosome alterations were clearly

characterized by CMA/DAPI bands and rDNA sites.

Conclusions: Comparing the present data with the cytological data for other species

of Zephyranthes, it becomes clear that a cytomolecular approach is fundamental

to the understanding of the chromosome variation and cytotaxonomy of the group.

NASCIMENTO, T., R. S. GONÇALVES,

M. BÁEZ, G. SEIJO AND M. GUERRA.

2

022. Molecular cytogenetics

reveals an uncommon structural

and numerical chromosomal

heteromorphism in Zephyranthes

brachyandra (Amaryllidaceae). Bol.

Soc. Argent. Bot. 57: 39-49.

Key WordS

CMA/DAPI banding, cytotaxonomy, FISH, monotrisomy, rDNA, telomeric DNA.

reSumen

DOI: https://doi.

org/10.31055/1851.2372.v57.

n1.34304

Introducción y objetivos: Zephyranthes brachyandra pertenece a una tribu de

Amaryllidaceae ornamentales nativa de América del Sur, cuya circunscripción

de géneros y relaciones ﬁlogenéticas aún no están claras. Citológicamente, Z.

brachyandra es un tetraploide cuyos cromosomas son de tamaño y morfología

similar, lo que diﬁculta la identiﬁcación de sus 2n = 24 cromosomas. El objetivo de

este estudio fue investigar la estabilidad de las numerosas bandas CMA+ y DAPI+

y la aparición de cromosomas B mediante un enfoque citomolecular.

M&M: Para esta investigación realizamos un análisis citomolecular con tinción CMA/

DAPI e hibridación ﬂuorescente in situ con sondas de ADNr 5S y 35S, y la sonda

telomérica TTTAGGG.

Resultados: En el presente trabajo se realizaron varios análisis citomoleculares de Z.

brachyandra, que revelaron un número alto y variable de bandas heterocromáticas

CMA+ y DAPI+ y de sitios de ADNr 5S y 35S, además de una distribución típica de

las secuencias teloméricas TTTAGGG. Además, un individuo era monotrisómico

con 2n = 24 y otro tenía un cromosoma B. Las alteraciones cromosómicas tanto

numéricas como estructurales se caracterizaron claramente por bandas CMA /

DAPI y sitios de ADNr.

Conclusión: Al comparar los datos actuales con la literatura citológica de otras

especies del género Zephyranthes, queda claro que un enfoque citomolecular es

fundamental para la comprensión de la variación cromosómica y la citotaxonomía

del grupo.

Recibido: 3 Ago 2021

Aceptado: 18 Feb 2022

Publicado impreso: 31 Mar 2022

Editora: Paola Gaiero

PalabraS Clave

Bandeamiento CMA/DAPI, citotaxonomía, FISH, monotrisomía, ADNr, ADN

telomérico

ISSN versión impresa 0373-580X

ISSN versión on-line 1851-2372

39

Bol. Soc. Argent. Bot. 57 (1) 2022

introduCtion

near the nucleolus organizer region (Guerra, 2000;

Roa & Guerra, 2015; Samoluk et al., 2017). Within

One of the taxonomic approaches most certain limits, the amount of heterochromatin

intensively explored in the last century was is not critical to genome function but in a few

cytotaxonomy [summarized by Stebbins (1971)]. species it is enormously expanded, forming

The rationale behind this approach is that the large heterochromatic blocks, as in Trithrinax

karyotype allows a “macroscopic” view of the campestris and Capsicum species (Gaiero et al.,

genetic material of a species, with some particular 2012; Grabiele et al., 2018), or numerous small

details of individuals or populations and reveals bands, as in Cuscuta monogyna (Ibiapino et al.,

many characters common to its genus or tribe. 2020). The tandemly repeated nature of rDNA

In contrast with various other morphological and telomeric DNA sites allows a considerable

descriptors, karyotype changes, such as polyploidy variation in number and size of these sites,

and chromosome inversions or translocations, may contributing to a better karyotype characterization

work as reproductive barriers among populations of species or populations (Pedrosa-Harand et

or species, playing therefore an important role in al., 2006; Robledo & Seijo, 2010; Rosato et al.,

the speciation process (Levin, 2002). Moreover, 2017, 2018; Silvestri et al., 2020). Currently,

the karyotype is the only morphological trait that chromosome staining with the fluorochromes

is not aﬀected by gene expression, environmental chromomycin A (CMA) and 4’,6-diamidino-2-

3

conditions, age, developmental phase, etc (Guerra, phenylindole (DAPI) is the most used method to

2

012). In general, using appropriate techniques, diﬀerentiate GC-rich and AT-rich heterochromatic

karyotype similarity among species of a genus is bands, respectively (Guerra, 2000). Other more

an indicative of their phylogenetic proximity. specific chromosome regions are revealed by

Karyotype description was initially restricted fluorescence in situ hybridization (FISH), a

to chromosome number, size and morphology well stablished method to localize any type of

(

Stebbins, 1971), but recent advances on DNA sequence along the chromosomes (Jiang,

molecular cytogenetics expanded the number of 2019). The analysis of CMA/DAPI bands and

chromosomal marks and allowed a more detailed rDNA sites, or other DNA sequences revealed by

karyotype description (Jiang, 2019). For example, FISH, into a phylogenetic context has ensured an

the chromosomes of all species of Citrus and enormous progress in cytotaxonomical analyses

related genera are very similar in number, size (e.g., Chalup et al., 2015; Silvestri et al., 2020;

and morphology but they diﬀer widely in the Ribeiro et al., 2020). However, cytomolecular

number and distribution of heterochromatic bands methods are more time-consuming and have been

and 5S and 35S rDNA sites (reviewed by Guerra, used in a still limited number of cytotaxonomical

2

009). Likewise, the assumption that the families works.

Juncaceae and Cyperaceae had holocentric Zephyranthes brachyandra (Baker) Backer

chromosomes as one of their synapomorphies (Amaryllidaceae) is an ornamental bulbous plant

Judd et al., 2016) was overturned by careful native of the Neotropics, from southern South

(

analyses of the chromosome morphology and America to Mexico and southwest USA, including

centromere immunostaining of some Juncus southern Brazil and West Indies, sometimes

species, revealing that in Juncaceae, at least, referred to as Habranthus brachyandrus (Baker)

holocentricity is rather a particularity of a few Sealy (Daviña, 2001; Daviña & Honﬁ, 2018;

genera (Guerra et al., 2019).

García et al. 2019). Phylogenetically, the former

Structural chromosome differentiation is genera Zephyranthes and Habranthus are now

most commonly revealed by identification of recognized as subgenera of Zephyranthes s.l.

heterochromaticbandsandsitesofhighlyconserved (García et al. 2019). The two subgenera share

DNA sequences, as the telomeric DNA and 5S and similar chromosome size and morphology, large

3

5S rRNA genes. These marks are composed by variation in chromosome numbers, mainly with x

repetitive DNA sequences densely concentrated = 6, several ploidy levels, and some species with

into a few blocks and located preferentially at the B chromosomes (Naranjo, 1974; Felix et al., 2008,

centromeric and terminal chromosome regions or 2011a; Daviña, 2001; Daviña & Honﬁ, 2018).

40

T. Nascimento et al. - Karyotype variability of Zephyranthes brachyandra

Cytologically, Z. brachyandra is a tetraploid with twice in destilled water, digested in a 2% cellulase

+

2

n = 24, characterized by many DAPI and CMA

(Onozuka)/ 20% pectinase (Sigma) solution at 37

heterochromatic bands located in the interstitial- °C for one hour, and macerated in a drop of 45%

terminal region of most chromosome arms (Felix acetic acid. The coverslips were removed in liquid

et al., 2011b). The individual analyzed by Felix nitrogen and the slides were dried in the air. The

et al. (2011b) presented 2n = 24 plus one B preparations were mounted and stained with a 1

chromosome, whereas the ﬁve bulbs examined μg/mL DAPI/glycerol (1:1) solution, in order to

by Daviña (2001) exhibited always 2n = 24. B select the best slides. Subsequently, they were

chromosomes are extra chromosome usually destained in ethanol/acetic acid (3:1), air dried,

smaller than the regular chromosomes of the and aged for three days at room temperature.

species (A chromosomes), mostly depleted of

genes, and present only in some individuals of the Chromosome staining with CMA/DAPI and FISH

species (see, e.g., Marques et al., 2013; Vanzela et

Chromosome double staining with the

al., 2017). They may occasionally be accumulated ﬂuorochromes CMA (Sigma) and DAPI (Sigma)

in some individuals aﬀecting their DNA content was performed as described by Vaio et al. (2018).

and some other phenotype characters (Huang et Aged slides were stained with CMA (0.1 mg/mL)

al., 2016).

for 1 h, counterstained with DAPI (2 µg/mL),

In the present work, we conducted a detailed mounted with glycerol-McIlvaine buﬀer pH 7.0

karyotype analysis of three individuals of Z. (1:1) and aged again for three days. After image

brachyandra collected in the ﬁeld, aiming to capture of the best metaphases the chromosomes

+

investigate: a, the stability of the many CMA

were destained and in situ hybridized according

+

and DAPI bands reported previously for a single to Pedrosa et al. (2002). The probes used for 5S

individual (Felix et al. 2011b); b, the distribution and 35S rDNA sites were, respectively, D2 from

of 5S rDNA, 35S rDNA and telomeric DNA sites; Lotus japonicus (Regel) K. Larsen and a clone

c, the occurrence of B chromosomes. We also from Triticum aestivum L. (plasmid Pta71). The

evaluated karyotype similarity in chromosome probes were direct and indirect labeled with

number, size and morphology, banding patterns Cy3 dUTP (GE Healthcare) (5S rDNA probe)

and rDNA sites of Z. brachiandra with other and digoxigenin-11-dUTP (Roche) (35S rDNA

species of the genus, in order to contribute to the probe) respectively, both by nick translation

cytotaxonomy of Zephyranthes.

(Invitrogen). The hybridization mixture contained

0% formamide (v/v), 10% dextran sulfate

w/v), 2x SSC, and 5–10 ng/μL of rDNA probe,

accomplishing a ﬁnal stringency of ~76%. The

5S rDNA probe was detected with sheep anti-

5

(

material and methodS

3

Plants material

digoxigenin–FITC (Roche).

Three bulbs of Z. brachyandra were collected in

For detection of telomeric sites, a synthetic

San Javier (27º52´17.9´´- 55º07´41.7´´), Misiones, TTTAGGG oligoprobe labeled with Cy3 by

Argentina, and cultivated in the Experimental Macrogen Inc. was in situ hybridized according

Garden of the Department of Botany, Federal to the protocol of Cuadrado et al. (2010), slightly

University of Pernambuco, Recife, Brazil. A modiﬁed. Brieﬂy, 10 µL of the probe solution (8

voucher was deposited in the herbarium Prof. ng/µL of the oligoprobe diluted in 2x SSC) were

Jayme Coelho de Moraes (Federal University of applied to each slide for 2 h at room temperature.

Paraiba, Brazil, voucher EAN 29460).

Afterwards, the slides were washed in 4x

SSC/0.2% Tween20 for 10 min. All preparations

were counterstained and mounted with 2 μg/

Slides preparation

For mitotic analyses, young root tips were mL DAPI in Vectashield (Vector). Images of the

pretreated with colchicine 0.2% at 10 °C for 24 best cells were captured with a Leica DM5500B

h, ﬁxed in ethanol-acetic acid (3:1, v/v) for 2 h at fluorescence microscope and later processed

room temperature, then stored at –20 °C until the with Adobe Photoshop CC 2020 for brightness,

moment of use. The ﬁxed meristems were washed contrast, and sharpness.

41

Bol. Soc. Argent. Bot. 57 (1) 2022

Chromosome measurements

and heterochromatic bands and rDNA sites

In order to characterize chromosome size common to all of them (Fig. 2). The individuals

and morphology, chromosomes of the three best without B chromosomes were heteromorphic

metaphases for each karyotype were measured for a chromosome pair formed by a meta-

using the software DRAWID version 0.26 (Kirov and a submetacentric chromosome and were

et al., 2017). The chromosome arm ratio [r = karyotypically identical regarding all other

length of long arm (l)/short arm (s)] was used chromosome characteristics, suggesting that they

to deﬁne chromosomes as metacentric (r = 1.0- were clones from a single individual. Therefore,

1

.49), submetacentric (r = 1.50-2.99), acrocentric they will be referred to herein as a single karyotype.

(

AR ≥ 3.0) or telocentric, according to Guerra Excluding the B chromosome, chromosome size

(

1986). Chromosome pairs were ordered from I to varied from 14.59 to 9.28 µm and the average

XII by decreasing size of the short arm.

haploid complement size was 146.75 µm (Fig.

and Table 1). The chromosome morphology in

2

the two karyotypes was very similar, except for

the arm ratio of chromosome pair X, which was

reSultS

1

.24 for the karyotype without B and 1.80 for

The plants collected in Argentina grew very the karyotype with B. This diﬀerence changed its

well and ﬂowered during the summer in Recife chromosome morphology from metacentric (AR

(

Fig. 1), but only produced sterile seeds. They = 1.00-1.49) to submetacentric (AR = 1.50-2.99)

revealed a high rate of asexual reproduction (Guerra, 1986) and the karyotype formula from

by lateral bulb production, providing plenty of 8M + 4SM for the individual with 2n = 24 to 9M

material for investigation. About 15 metaphases + 3SM for the individual with 2n = 24 + 1B (the

of each individual were analyzed for chromosome chromosome B was not included in the karyotype

number. Two individuals analyzed presented formula). The average arm ratio for pair X

2

n = 24 and a third presented 2n = 24 plus a B considering both karyotypes was 1.52 (Fig.2).

chromosome. The three best metaphases of each The remaining chromosomes were metacentrics

plant were selected for a careful comparison (pairs I to IX) or submetacentrics (pairs XI and

of CMA/DAPI bands and rDNA sites. Two XII). The most outstanding karyotype feature of

+

karyograms were mounted for each individual and Z. brachyandra was the large amount of DAPI

+

a single ideogram was constructed summarizing heterochromatin. There were large DAPI bands

the average sizes for each chromosome pair on the short arm of 10 chromosomes and one to

four small bands in the interstitial/terminal region

of the remaining chromosome arms (Fig. 3A). The

submetacentric pairs XI and XII presented similar

size and morphology, diﬀering mainly in the

+

number of DAPI bands on the long arm: two on

+

pair XI and three on pair XII. CMA bands were

identiﬁed only in the short arms of the smaller

submetacentrics (Fig. 3A’) and in the short arm

of pair VIII (not always visible). Besides these

general features, the two karyotypes observed

here had several small structural diﬀerences.

The number and position of the 5S and 35S

rDNA loci complemented the identiﬁcation of

each chromosome pair and were different in

the two karyotypes. The karyotype with the

heteromorphic pair had 19 sites of 5S and six

sites of 35S rDNA. The 5S rDNA sites were

Fig. 1. Flowers of Zephyranthes brachyandra

cultivated as ornamental plants in Recife, Brazil.

located on the interstitial (12 sites) or terminal

(7 sites) regions of the large metacentric and

42

T. Nascimento et al. - Karyotype variability of Zephyranthes brachyandra

Fig. 2. Idiogram of Z. brachyandra, including all characters observed in both karyotypes plus the B

chromosome. CO, chromosome order; S, chromosome size in µm; AR, arm ratio; CM, chromosome

+

morphology. The scale on the left side indicates the size of the chromosome arms. Blue = DAPI band;

+

yellow = CMA band; yellow with green bars = 35S rDNA sites co-localized with CMA+ bands; red dots = 5S

rDNA sites; very small purple dots = TTTAGGG sites.

Table 1. Chromosome data of Zephyranthes brachyandra karyotype, including chromosome order

(CO, larger to smaller short arm), short and long arm sizes, chromosome size, arm ratio, chromosome

morphology (CM), number and position of DAPI+ bands and CMA+ bands, and number and position of 5S

and 35S rDNA sites. DAPI+ bands were classiﬁed as large (L) or small (S), CMA+ bands as terminal (T),

and rDNA sites as terminal (T) or interstitial (i). Chromosomal marks on the short arms are superscript and

those on the long arms are subscript.

Short arm

µm)

Long arm

(µm)

Chromosome

Size (µm)

DAPI

bands

CMA 5S rDNA 35S rDNA

CO

Arm ratio CM

(

bands

sites

S

I

6.91 ± 0.98 7.68 ± 0.92

6.20 ± 0.78 7.28 ± 1.27

6.06 ± 1.13 7.42 ± 0.98

14.59 ± 1.82 1.12 ± 0.09

13.48 ± 1.97 1.70 ± 0.12

13.49 ± 1.88 1.25 ± 0.23

12.28 ± 1.83 1.09 ± 0.06

M

2 + 2_S

--

S

T

II

2 + 2_S

1

S

III

1 + 2_S

--

S

IV* 5.89 ± 0.92 6.39 ± 0.92

IV* 2.58 ± 0.23 7.33 ± 0.31

1 + 1_S

S

T

9.91 ± 0.42

2.87 ± 0.34 SM

2 + 2_S

1

L

T

V

5.65 ± 0.63 7.02 ± 0.65

5.49 ± 0.56 6.65 ± 0.85

12.67 ± 0.98 1.26 ± 0.19

12.15 ± 1.28 1.21 ± 0.13

12.29 ± 1.45 1.26 ± 0.09

12.94 ± 1.93 1.43 ± 0.32

12.06 ± 1.21 1.38 ± 0.24

M

1 + 2_S

--

1

--

L

i

VI

1 + 2_S

1

S

VII 5.44 ± 0.68 6.84 ± 0.85

VIII 5.40 ± 0.95 7.54 ± 1.21

3 + 2_S

--

L

i

1 + 2_S

1 + 1

1

i

L

i

IX

X

5.10 ± 0.60 6.95 ± 0.95

4.93 ± 0.95 7.29 ± 1.23

2.52 ± 0.38 6.78 ± 0.75

1 + 3_S

1 + 1

--

T

L

i

12.22 ± 1.77 1.52 ± 0.34 SM

1 + 3_S

1 + 1

1

i

T

XI

9.30 ± 0.93

9.28 ± 0.96

6.75 ± 0.45

2.73 ± 0.41 SM

2.94 ± 0.87 SM

2_S

3_S

--

1

--

1

T

XII 2.09 ± 0.28 7.19 ± 0.94

-- 6.75 ± 0.45

1

T

B

∞

A

1 + 1

1_T

T

43

Bol. Soc. Argent. Bot. 57 (1) 2022

Fig. 3. Metaphase of Z. brachyandra with a heteromorphic chromosome pair. Chromosomes were stained

with DAPI (A) and CMA (A’) and in situ hybridized with 5S (red) and 35S (green) rDNA probes (A’’). Arrows

+

in A’ point to CMA bands of submetacentrics. Inset in A’’ shows magniﬁed images of small rDNA sites.

Karyograms based on this metaphase allow the identiﬁcation of each chromosome pair by comparison of the

diﬀerent marks (B). Asterisks in B indicate heteromorphic pairs. Bar in A corresponds to 10 µm.

submetacentric chromosomes and a single site including an extra signal on the short arm of both

on one homologue of pair XI (Fig 3A’’). The ﬁve homologues of pair V and a heteromorphic site on

small submetacentrics had a 35S rDNA site on pair X, and 12 sites of 35S rDNA plus one in the

+

the short arm co-localized with the CMA band. B chromosome. Noteworthy, the additional 35S

One chromosome of pair VIII also displayed a rDNA sites, located on pairs VIII and X, were not

+

small 35S rDNA near the large DAPI band, but clearly detected as CMA+ bands. The 35S rDNA

+

it was not co-localized with a CMA band. The site on pair X was very close to the large DAPI

karyograms based on Fig. 3A-A’’ placed the extra band but they were not colocalized (see insets in

small submetacentric provisionally as pair IV Fig. 4A and respective chromosomes in Fig. 4B).

(

Fig. 3B), since one homologue of pair IV was

The TTTAGGG probe labelled the telomeric

absent. However, it was almost identical to pair regions of all chromosomes of both karyotypes and

XI in size and morphology as well as in banding no ectopic site was found (Fig. 5). No short arm

+

patterns, with two weakly diﬀerentiated DAPI

was visible on the B chromosome, suggesting that

+

bands on the long arm and a CMA band on the it was a telocentric. It was shorter (6.75 µm) than

short one. At least six other chromosome pairs the submetacentrics (Fig. 2), had no DAPI bands,

were heteromorphic for one or more bands or sites and presented two CMA bands: one interstitial and

+

(

asterisks in ﬁgure 3B).

one very small on its narrower end (inset in Fig.

The karyotype with a B chromosome exhibited 4A’). The latter could be the centromere itself or a

a similar pattern of CMA/DAPI bands and rDNA very small short arm, whereas the interstitial band

sites (Fig. 4A-A’). It had 19 sites of 5S rDNA, coincided with a 35S rDNA site.

44

T. Nascimento et al. - Karyotype variability of Zephyranthes brachyandra

+

Fig. 4. Metaphase of Z. brachyandra with 2n = 24 + 1B displaying DAPI bands (blue) plus 35S rDNA sites

+

(

green) (A) and CMA bands (white arrows) plus 5S rDNA sites (red dots) (A’). Insets show magniﬁed

chromosome regions with small rDNA sites (white frames) and B chromosome (yellow arrows and frames).

Karyograms (B) were based on the same metaphase. Bar in A corresponds to 10 µm.

diSCuSSion

The two karyotypes of Z. brachyandra analysed

here differed mainly in the occurrence of a

heteromorphic chromosome pair in one of them

and a B chromosome in the other. Regarding

chromosome sizes and haploid karyotype formula,

our data (14.59 to 9.28 µm and 8M + 4SM or

9

M + 3SM) differ in part from that reported

by Daviña (2001) (10.89 to 6.14 µm and 8M +

SM). The diﬀerence in chromosome size may be

4

due to the distinct anti-mitotic pre-treatment and

the condensation status of selected metaphases

(Guerra, 2012), whereas the karyotype formula 8M

+

4SM was the same found here for the karyotype

without B. Therefore, in spite of the small sample

size, our data revealed karyotype instability, which

was not detected in previous studies.

The two karyotypes displayed a similar number

of CMA/DAPI bands and diﬀered slightly in the

Fig. 5. In situ hybridization of the TTTAGGG probe

in a metaphase of Z. brachyandra with 2n = 24 +

number of the 5S and 35S rDNA sites. The complex

1B (arrow) revealed terminal sites only. Inset shows

+

magniﬁed B chromosome. Bar corresponds to 10 µm. DAPI banding pattern of Z. brachyandra was

45

Bol. Soc. Argent. Bot. 57 (1) 2022

similar to that reported by Felix et al. (2011b), while 1976) and Alloe rabaiensis (Bradham, 1983).

+

the number and position of CMA bands reported by However, the similarity of the extra submetacentric

these authors were quite distinct from the present with chromosome pair XI in size, morphology,

+

ones. They found eight or ten bright CMA bands and CMA/DAPI bands, as well as the absence

(excepting the B chromosome), only two of them of one homologue of pair IV, suggest that this

on small submetacentric chromosomes, whereas karyotype had a trisomy for pair XI combined

+

in our sample there were only four or six CMA

and a monosomy for pair IV. Such monotrisomic

bands (excepting the B and the extra submetacentric aneuploids are more commonly found among

+

chromosome). It is possible that some CMA bands chromosomically engineered crop plants, as wheat

in our sample had not been detected because they and barley, and most of them are compensating

were too small. Actually, the 35S rDNA sites of aneuploids involving homeologous chromosomes

+

pairs VIII and X were expected to be CMA , since (Singh, 2003). Differently, in Z. brachyandra

+

they are usually co-localized with CMA bands (e.g., the monotrisomy involved two quite different

Moraes et al., 2007; Gaiero et al., 2012; Silva et chromosomes, although the plant phenotype was

al., 2019). However, small rDNA sites, like those, identical to the normal plants. In this case, the normal

+

are sometimes not detected as CMA bands (e.g., appearance of the individual is most probably due to

Vaio et al., 2018). Variation in size and number of the buﬀer eﬀect of polyploidy, in which a deletion

heterochromatic bands and rDNA sites depends or a duplication of an entire chromosome of a

on the number of repetitive sequences per locus, tetraploid genome could not be suﬃcient to alter

which may be caused by recombination events with the phenotype (Deng et al., 2018). The occurrence

unequal exchanges (Lower et al., 2018). However, of aneuploid plants is more common in meiotically

+

the position of some interstitial CMA bands instable polyploids (Mandáková & Lysak, 2018),

reported by Felix et al. (2011b) did not coincide with while Z. brachyandra has a regular meiosis (Daviña,

position of the 35S rDNA sites found in our sample, 2001; Daviña & Honﬁ, 2018). Monotrisomy in wild

suggesting that if they correspond to 35S rDNA plants has rarely been reported, but it is probably

sites, they are not the same reported here.

underestimated because its detection demands a

B chromosomes probably arise from regular clear distinction of the chromosomes involved, as

A chromosomes and can alter their original DNA demonstrated in Tragopogon miscellus (Chester et

composition by rapidly accumulating or losing al., 2012) and Nothoschordum bonariense (Souza

several DNA sequences (Marques et al., 2013). In et al., 2019). Trisomy as well as B chromosomes

Z. brachyandra the only B chromosome observed have also been reported in some other species of

was a telocentric chromosome similar in size Zephyranthes (see e.g., Felix et al., 2008, 2011a,b).

and morphology to that reported by Felix et al.

(

Despite the polymorphisms observed here, Z.

brachyandra is karyotypically very well deﬁned,

+

2011a,b), but without the two interstitial DAPI

bands observed by those authors. Both Bs had a due to its many chromosome marks. The only other

+

small terminal CMA band, although only one Zephyranthes species where rDNA sites have been

+

reported here had an extra interstitial CMA band investigated is Z. robusta, a diploid with 2n = 12

co-localized with a 35S rDNA site. Polymorphisms (10M + 2SM) and similar chromosome sizes. The

on B chromosomes are more common than in molecular karyotype of Z. robusta is also similar

the regular chromosomes, probably due to the to that expected for a hypothetical diploid ancestor

dispensable nature of Bs (Marques et al., 2013; of Z. brachyanda, with two 35S rDNA sites co-

+

Vanzela et al., 2017).

localized with CMA bands on submetacentric

At first sight, the extra submetacentric short arms, ten 5S rDNA sites, some interstitial

+

chromosome found in a karyotype with the normal and subterminal DAPI bands and a few other

chromosome number seemed to be a partial deletion heterochromatic blocks detected by C-banding

of the short arm of a metacentric chromosome. (Barros e Silva & Guerra, 2010; Felix et al.,

Heteromorphism for large chromosome segments 2011b; A.E. Barros e Silva, unpublished results).

are rare but have already been clearly documented This similarity points to a possible autopolyploid

for several plant species, especially those with large origin for Z. brachiandra, in spite of its regular

chromosomes, as Crocus cancellatus (Brighton, meiotic pairing (Daviña and Honﬁ, 2018), which

46

T. Nascimento et al. - Karyotype variability of Zephyranthes brachyandra

is usually associated with allopolyploidy. Regular CARVALHO A., M. DELGADO, A. BARÃO, M.

bivalent formation has also been reported for other

autopolyploids, as Arabidopsis arenosa (Carvalho

et al., 2010), and autopolyploidy may be not as

rare as supposed earlier (Soltis et al., 2007). The

assumption of an autopolyploid origin for Z.

brachiandra could also explain the many duplicated

FRESCATADA, E. RIBEIRO, C. S. PIKAARD,

W. VIEGAS & N. NEVES. 2010. Chromosome

and DNA methylation dynamics during meiosis in

the autotetraploid Arabidopsis arenosa. Sex. Plant.

Reprod. 23:29-37.

https://doi.org/10.1007/s00497-009-0115-2

patterns of chromosome banding and rDNA site CHALUP, L., S. S. SAMOLUK,V. S. NEFFA& G. SEIJO.

distribution observed here, some of them blurred

by the intense heteromorphism characteristic of

heterochromatic bands and rDNA sites (Chalup

et al., 2015; Silvestri et al., 2020; Ribeiro et

al., 2021). Five other species of Zephyranthes

2015. Karyotype characterization and evolution in

South American species of Lathyrus (Notolathyrus,

Leguminosae) evidenced by heterochromatin and

rDNA mapping. J. Plant Res. 128: 893–908.

https://doi.org/10.1007/s10265-015-0756-1

analysed by Felix et al. (2011b) revealed a much CHESTER, M., J. P. GALLAGHER, V. V. SYMONDS,

+

smaller and variable number of CMA bands and

A. V. C. SILVA, E. V. MAVRODIEV, A. R. LEITGH,

P. S. SOLTIS & D. E. SOLTIS. 2012. Extensive

chromosomal variation in a recently formed natural

allopolyploid species, Tragopogon miscellus

(Asteraceae). Proc. Natl. Acad. Sci. 109: 1176–1181.

https://doi.org/10.1073/pnas.1112041109

+

no DAPI bands, suggesting that a cytomolecular

investigation of other Zephyranthes species would

be very helpful to understand the cytotaxonomy and

chromosomal evolution of this group.

CUADRADO, A. & N. JOUVE. 2010. Chromosomal

detection of Simple Sequence Repeats (SSRs) using

nondenaturing FISH (ND-FISH). Chromosoma 119:

495–503. https://doi.org/10.1007/s00412-010-0273-x

authorS Contribution

THN conducted the FISH experiments, analyzed

the data, constructed the ﬁgures and tables and DAVIÑA, J. R. 2001. Estudios citogenéticos en algunos

wrote the paper. RSG conducted FISH experiments

and helped to write the paper. MB collected the

material, conducted FISH experiments and helped

géneros argentinos de Amaryllidaceae. Doctoral

thesis, Universidade Nacional de Córdoba, Córdoba,

Argentina.

to write the paper. GS collected the material and DAVIÑA, J. R. & A. I. HONFI. 2018. IAPT chromosome

helped to write the paper. MG discussed the results,

provided resources for the FISH experiments,

designed this research and wrote the paper. All

data 28 [extended online version]. In: MARHOLD

K. AND KUČERA J. (Eds.). Taxon. 67: E1–E46.

https://doi.org/10.12705/676.39

authors read and approved the ﬁnal version of the DENG, X., Y. SHA, Z. LV, Y. WU, A. ZHANG, F.

paper.

WANG & B. LIU. 2018. The capacity to buﬀer and

sustain imbalanced D-subgenome chromosomes

by the BBAA component of hexaploid wheat is an

evolved dominant trait. Front. Plant Sci. 9: 1–12.

https://doi.org/10.3389/fpls.2018.01149

bibliograPhy

BARROS E SILVA, A. E. & M. GUERRA. 2010. The

meaning of DAPI bands observed after C-banding

and FISH procedures. Biotech. Histochem . 85: 115–

FELIX, W. J. P., J. H. A. DUTILH, N. F. M., ANDREA

A. F. & LEONARDO P. F. 2008. Intrapopulational

chromosome number variation in Zephyranthes

sylvatica Baker (Amaryllidaceae: Hippeastreae)

from Northeast Brazil. Rev. Bras. Bot. 31: 371–75.

https://doi.org/10.1590/S0100-84042008000200020

FELIX, W. J. P., L. P. FELIX, N. F. MELO, M. B. M.

OLIVEIRA, J. H. A. DUTILH & R. CARVALHO.

2011a. Karyotype variability in species of the

genus Zephyranthes Herb. (Amaryllidaceae–

Hippeastreae). Plant Syst. Evol. 294: 263.

1

25. https://doi.org/10.3109/10520290903149596

BRADHAM, P. 1983. Evolution in a stable chromosome

system. In: P.E. BRADHAM & M.D. BENNET

(

Eds). George Allen & Unwin (Publishers), Kew

Chromosome Conference II, pp. 251-260. London.

BRIGHTON, C. A. 1977. Cytological problems in the

genus Crocus (Iridaceae): II. Crocus Cancellatus

Aggregate. Kew Bulletin 32: 33-45.

https://doi.org/10.2307/4117257

https://doi.org/10.1007/s00606-011-0467-6

47

Bol. Soc. Argent. Bot. 57 (1) 2022

FELIX, W. J. P., L. P. FELIX, N. F. MELO, J. H. A.

DUTILH & R. CARVALHO. 2011b. Cytogenetics

of Amaryllidaceae species: heterochromatin

evolution in diﬀerent ploidy levels. Plant Syst.

Evol. 292: 215–221.

https://doi.org/10.1007/s00606-011-0418-2

GAIERO, P., C. MAZZELLA, M. VAIO, A. E. BARROS

E SILVA, F. F. SANTIÑAQUE, B. LÓPEZ-CARRO,

G. A. FOLLE & M. GUERRA. 2012. An unusually

high heterochromatin content and large genome size

in the palm tree Trithrinax campestris (Arecaceae).

Aust. J. Bot. 60: 378.

IBIAPINO, A., M. A. GARCÍA, M. COSTEA, S.

STEFANOVIĆ & M. GUERRA. 2020. Intense

proliferation of rDNA sites and heterochromatic

bands in two distantly related Cuscuta species

(Convolvulaceae) with very large genomes and

symmetric karyotypes. Genet. Mol. Biol. 43: 1–9.

https://doi.org/10.1590/1678-4685-GMB-2019-0068

JIANG, J. 2019. Fluorescence in situ hybridization in

plants: recent developments and future applications.

Chromosome Res. 27: 153–165.

https://doi.org/10.1007/s10577-019-09607-z

JUDD, W. S., CAMPBELL C. S., KELLOGG E. A.,

STEVENS P. F. & DONOGHUE M. J. 2016. Plant

systematics: a phylogenetic approach. 4th ed.

Sunderland, Sinauer Associates.

http://dx.doi.org/10.1071/BT12029

GARCÍA N., A. W. MEEROW, S. ARROYO-

LEUENBERGER, R. S. OLIVEIRA, J. H. DUTILH,

P. S. SOLTIS & W. S. JUDD. 2019. Generic

classiﬁcation of Amaryllidaceae tribe Hippeastreae.

Taxon 68: 481-498.

KIROV, I., L. KHRUSTALEVA, K. V. LAERE, A.

SOLOVIEV, S. MEEUS, D. ROMANOV & I.

FESENKO. 2017. DRAWID: user-friendly java

software for chromosome measurements and

idiogram drawing. Comp. Cytogenet. 11: 747–757.

https://doi.org/10.3897/compcytogen.v11i4.20830

LEVIN, D. A. 2002. The role of chromosomal change in

plant evolution. New York, Oxford University Press.

LOWER, S. S., M. P. MCGURK, A. G. CLARK & D.

A. BARBASH. 2018. Satellite DNA evolution: old

ideas, new approaches. Curr. Opin. Genet. Dev. 49:

70–78. https://doi.org/10.1016/j.gde.2018.03.003

MANDÁKOVÁ, T. & M. A. LYSAK. 2018. Post-

polyploid diploidization and diversiﬁcation through

dysploid changes. Curr. Opin. Plant Biol. 42: 55–65.

https://doi.org/10.1016/j.pbi.2018.03.001

https://doi.org/10.1002/tax.12062

GRABIELE, M.⁠, H. J. DEBAT⁠, M.A. SCALDAFERRO,

P. M. AGUILERA, E. A. MOSCONE, J. G.

SEIJO & D. A. DUCASSE. 2018. Highly GC-

rich heterochromatin in chili peppers (Capsicum-

Solanaceae): A cytogenetic and molecular

characterization. Sci. Hortic. 238: 391–399.

https://doi.org/10.1016/j.scienta.2018.04.060

GUERRA, M. S. 1986. Reviewing the chromosome

nomenclature of Levan et al. Braz. J. Genet. 9: 741-

7

43.

GUERRA, M. 2000. Patterns of heterochromatin

distribution in plant chromosomes. Genet. Mol. Biol.

2

3: 1029–1041.

MARQUES, A., A. M. BANAEI-MOGHADDAM,

S. KLEMME, F. R. BLATTNER, K. NIWA, M.

GUERRA & A. HOUBEN. 2013. B chromosomes

of rye are highly conserved and accompanied the

development of early agriculture. Ann. Bot. 112:

527–534. https://doi.org/10.1093/aob/mct121

https://doi.org/10.1590/S1415-47572000000400049

GUERRA, M. 2009. Chromosomal variability and

the origin of Citrus species. In: Mahoney C. L. &

Springer D. A. (Eds) Genetic diversity, pp. 51-68.,

Nova Science Publishers New York..

GUERRA, M. 2012. Cytotaxonomy: the end of childhood.

Plant Biosyst. 146: 703–710.

MORAES, A. P., R. R. LEMOS, A. C. BRASILEIRO-

VIDAL, W. S. S FILHO & M. GUERRA. 2007.

Chromosomal markers distinguish hybrids and non-

hybrid accessions of mandarin. Cytogenet. Genome

Res. 119: 275–281.

https://doi.org/10.1080/11263504.2012.717973

GUERRA, M., T. RIBEIRO & L. P. FELIX. 2019.

Monocentric chromosomes in Juncus (Juncaceae)

and implications for the chromosome evolution of

the family. Bot. J. Linn. Soc. 191: 475-483.

https://doi.org/10.1159/000112074

NARANJO, C. A. 1974. Karyotypes of four argentine

species of Habranthus and Zephyranthes

(Amaryllidaceae). Phyton. 32: 61–71.

PEDROSA, A., N. SANDAL, J. STOUGAARD,

D. SCHWEIZER & A. BACHMAIR. 2002.

Chromosomal map of the model legume Lotus

japonicus. Genetics 161:1661-1672.

https://doi.org/10.1093/botlinnean/boz065

HUANG, W., Y. DU, X. ZHAO & W. JIN. 2016. B

chromosome contains active genes and impacts the

transcription of A chromosomes in maize (Zea mays

L.). BMC Plant Biol. 16: 88.

https://doi.org/10.1186/s12870-016-0775-7

48

T. Nascimento et al. - Karyotype variability of Zephyranthes brachyandra

PEDROSA-HARAND, A., C. C. S. ALMEIDA, M.

MOSIOLEK, M. W. BLAIR, D. SCHWEIZER

M. GUERRA. 2006. Extensive ribosomal

DNA ampliﬁcation during Andean common bean

Phaseolus vulgaris L.) evolution. Theor. Appl.

SILVA, S. C., S. MENDES, T. RÉGIS, O. S. PASSOS,

W. S. S. FILHO & A. PEDROSA-HARAND. 2019.

Cytogenetic map of pummelo and chromosome

evolution of true Citrus species and the hybrid sweet

orange. J. Agric. Sci. 11: 148.

&

(

Genet. 112: 924–933.

https://doi.org/10.5539/jas.v11n14p148

https://doi.org/10.1007/s00122-005-0196-8

SILVESTRI, M .C., A. M. ORTIZ, G. A. R. DOBLADEZ

& G. I. LAVIA. 2020. Chromosome diversity in

species of the genus Arachis, revealed by FISH and

CMA/DAPI banding, and inferences about their

karyotype diﬀerentiation. An. Acad. Bras. Cienc.

92: 1–29.

https://doi.org/10.1590/0001-3765202020191364

SINGH, R. J. 2003. Plant cytogenetics. 3rd ed. Boca

Raton, Florida, USA .

SOLTIS, D. E., P. S. SOLTIS, D. W. SCHEMSKE, J. F.

HANCOCK, J. N. THOMPSON, B. C. HUSBAND

& W. S. JUDD. 2007. Autopolyploidy in angiosperms:

have we grossly underestimated the number of species?

Taxon. 56: 13-30. https://doi.org/10.2307/25065732.

SOUZA, G., A. MARQUES, T. RIBEIRO, L. G.

DANTAS, P. ESPERANZA, M GUERRA & O.

CROSA. 2019. Allopolyploidy and extensive

rDNA site variation underlie rapid karyotype

evolution in Nothoscordum section Nothoscordum

(Amaryllidaceae). Bot. J. Linn. Soc. 190: 215–228.

https://doi.org/10.1093/botlinnean/boz008

RIBEIRO, T., E. VASCONCELOS, K. G. B. SANTOS,

M. VAIO, A.C. BRASILEIRO-VIDAL & A.

PEDROSA-HARAND. 2020. Diversity of repetitive

sequences within compact genomes of Phaseolus L.

beans and allied genera Cajanus L. and Vigna Savi.

Chromosome Res. 28: 139–153.

https://doi.org/10.1007/s10577-019-09618-w

RIBEIRO, T., J. NASCIMENTO, A. SANTOS, L. P.

FÉLIX, & M. GUERRA. 2021. Origin and evolution

of highly polymorphic rDNA sites in Alstroemeria

longistaminea (Alstroemeriaceae) and selated

species. Genome 64: 833–45.

https://doi.org/10.1139/gen-2020-0159

ROA, F. & M. GUERRA. 2015. Non-random distribution

of 5S rDNA sites and its association with 45S rDNA

in plant chromosomes. Cytogenet. Genome Res. 146:

2

43–249. https://doi.org/10.1159/000440930

ROBLEDO, G. & G. SEIJO. 2010. Species relationships

among the wild non-B genome of Arachis species

(section Arachis) based on FISH mapping of rDNA

loci and heterochromatin detection: a new proposal for

genome arrangement. Theor. Appl. Genet. 121: 1033–

STEBBINS, G. L. 1971. Chromosomal evolution in

higher plants. Edward Arnold Ltd, London.

1

046. https://doi.org/10.1007/s00122-010-1369-7

VAIO, M., J. NASCIMENTO, S. MENDES, A.

IBIAPINO, L. P. FÉLIX, A. GARDNER, A.

GARDNER, E. EMSHWILLER, P. FIASCHI,

M. GUERRA. 2018. Multiple karyotype changes

distinguish two closely related species of

Oxalis (O. psoraleoides and O. rhombeo-ovata)

and suggest an artificial grouping of section

Polymorphae (Oxalidaceae). Bot. J. Linn. Soc.

188: 269–280.

https://doi.org/10.1093/botlinnean/boy054

VANZELA, A. L. L., A. A. PAULA, C. C. QUINTAS,

T. FERNANDES, J. N. C. BALDISSERA & T.

B. SOUZA. 2017. Cestrum strigilatum (Ruiz &

Pavón, 1799) B chromosome shares repetitive DNA

sequences with A chromosomes of diﬀerent Cestrum

(Linnaeus, 1753) species. Comp. Cytogenet. 11:

511–524.

ROSATO, M., I. ÁLVAREZ, G. N. FELINER & J. A.

ROSSELLÓ. 2017. High and uneven levels of 45S

rDNA site-number variation across wild populations

of a diploid plant genus (Anacyclus, Asteraceae).

PLoS One 12: e0187131.

https://doi.org/10.1371/journal.pone.0187131

ROSATO, M., I. ÁLVAREZ, G. N. FELINER & J.

A. ROSSELLÓ. 2018. Inter- and intraspecific

hypervariability in interstitial telomeric-like repeats

(TTTAGGG)n in Anacyclus (Asteraceae). Ann. Bot.

1

22: 387–395. https://doi.org/10.1093/aob/mcy079

SAMOLUK, S. S., L. M. I. CHALUP, C. CHAVARRO,

G. ROBLEDO, D. J. BERTIOLI, S. A. JACKSON

&

J. G. SEIJO. 2019. Heterochromatin evolution in

Arachis investigated through genome-wide analysis

of repetitive DNA. Planta 49: 1405-1415.

https://doi.org/10.1007/s00425-019-03096-4

https://doi.org/10.3897/CompCytogen.v11i3.13418

49