Botanical Studies (2010) 51: 337-342.
CYTOGENETICS
Cytogenetic analysis in the terrestrial orchid Sacoila
argentina (Griseb.) Garay from Paraguay
Mauro GRABIELE1,2, Ana I. HONFI1, Juan C. CERUTTI1, Virginia FERNANDEZ3, Deidamia
FRANCO3, and Julio R. DAVINA1*
1Programa de Estudios Floristicos y Genetica Vegetal (PEFyGV), Facultad de Ciencias Exactas, Quimicas y Naturales,
Universidad Nacional de Misiones, 3300 Posadas, Argentina
2Instituto Multidisciplinario de Biologia Vegetal (IMBIV-CONICET), Facultad de Ciencias Exactas, Fisicas y Naturales,
Universidad Nacional de Cordoba, 5000 Cordoba, Argentina
3Departamento de Biologia, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Asuncion, 2160 San
Lorenzo, Paraguay
(Received August 13, 2008; Accepted December 11, 2009)
ABSTRACT. Sacoila argentina (Griseb.) Garay is a terrestrial orchid with ornamental value found in northern Argentina, Bolivia, and Paraguay and is considered an object of conservation within the Paraguayan Chaco region, only found at Boqueron Department and chromosomally unknown until this study. Here, we present another natural population of Sacoila argentina for Paraguay (Paraguari Department, Villa Florida, 57°07'48.71" W - 26°23'32.16" S) so extending its area of distribution. Conventional cytogenetic techniques were applied in order to analyze its genetic system. Sacoila argentina is a diploid species with 2n = 2x = 46 small size chromosomes (1.21 - 4.45 μm) and 40.63 μm per haploid genome. Its karyotype, composed of 38m + 8sm, is bimodal (A2=0.36/R=3.69) due to a pair (# 20, sm) that is more than two times larger than the mean length of the chromosome (1.77 μm), and symmetric (Ai=0.18/i=44.58/r>2=0.13) belonging to Stebbins category 2B. The chromosome pair # 20 also carries a terminal macrosatellite on its short arm with an active NOR. Sporogenesis is normal and pollen grain viability is high (80%). Meiotic behaviour is regular and chromosomes pair up as 23 bivalents in the male (pollen mother cells, PMC) as they do in the female meiosis (megaspore mother cells, MMCs), confirming the basic number x = 23 for Sacoila. In PMCs and MMCs at diakinesis/metaphase I, ring bivalents (80%) with distal chiasmata (85%) are common, and the mean of chiasmata per bivalent is high (1.80) in both germinal lines. The recombination index, affected by the meiotic number (n = 23) and the mean of chiasmata/cell (41.5) is high (RI=64.5). Natural populations of Sacoila argentina possess low density, are scarce, interspersed and just found at flowering time. For these reasons, it is a rarely collected species.
Keywords: Chromosomes; Genetic system; Orchidaceae; Sacoila argentina.
INTRODUCTION
Sacoila Raf. is a small genus of orchids with ca. ten species native to the tropics and subtropics of America (Garay, 1980) and three of them are found in Paraguay: S. argentina Griseb. (Garay), S. lanceolata (Aubl.) Garay, and S. pedicellata (Cogn.) Garay. Sacoila argentina is a terrestrial and annual orchid with yellow-orange flowers and basal leaves, distributed from Bolivia to Paraguay and northern Argentina (Garay, 1980; Correa, 1996). Its habitat comprises open and low forests, the borders of high forests where the light is greater, and dunes and loose sandy grounds. In Paraguay, the orchid is found only in the
*Corresponding author: E-mail: julio@invs.unam.edu.ar; Tel: +54-3752-427776.
Boqueron Department and is considered a conservation object within the Paraguayan Chaco Region. Natural populations of S. argentina possess a low density, are scarce and interspersed, and are found only at flowering time. For these reasons, the species is rarely collected and chromosomally unknown.
This work reports the first cytogenetic analysis of S. argentina and extends its area of distribution in Paraguay.
MATERIALS AND METHODS
Plant material was collected from Paraguari Department, Villa Florida, at 57°07'48.71" W-26°23' 32.16" S, Paraguay. A voucher specimen, Davina & Honfi 612, was deposited at the Herbarium of the Universidad Nacional de Misiones, Argentina (MNES).
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Meiotic studies were carried out in young floral buds and ovules both fresh and fixed in absolute ethanol: glacial acetic acid (3:1) and stained with 2% aceto-carmine according to Davina (2001). Bivalent and chiasmata frequencies were estimated by analysis of 30 cells at diakinesis or metaphase I. Pollen stainability was estimated in 1000 grains stained with carmine-glycerine.
Mitotic studies were performed in young ovules pretreated with saturated 1-bromonaphthalene for 3 h at room temperature and hydrolyzed in HCl 1N for 2 min at 60°C according to Davina (2001). Ovaries were macerated in a drop of 2% aceto-orcein and then squashed.
For karyotype description the chromosomes were arranged in groups according to the position of the centromere (median, m; submedian, sm) and in order of decreasing size in each class. Chromosome nomenclature followed Levan et al. (1964). Satellites were classified according to Battaglia (1955). At least ten best metaphases were selected for making idiograms. The length of chromosome arms and satellites were measured on drawings made with a camera lucida (x2600). Karyotype asymmetry was estimated using the intrachromosome (A1) and interchromosome (A2) asymmetry indices of Romero Zarco (1986), as well as the categories of Stebbins (1971).
RESULTS
Sacoila argentina has been chromosomally studied for the first time showing 2n = 46 (Figure 1A). Its karyotype is composed of 38 metacentric and 8 submetacentric chromosomes (38 m + 8 sm), most of which can not be identified, but they were organized in pairs in order to present individual measurements (Tables 1, 2; Figure 2). A terminal macrosatellite in the short arm of the largest pair (# 20, sm) carrying the active NOR was observed (Figures 1A, 2). Total chromosome length is 81.25 [im, and mean chromosome length is 1.77 μm, ranging from 1.21 μm) to 4.45 μm (sm) (Table 2), a small size. Despite the uniformity in chromosome size, the karyotype of S. argentina is bimodal due to a pair (# 20, sm) more than two times larger than the mean chromosome length (Table 2). This is also evidenced by the interchromosome asymmetry index A2 value (0.36) and by the largest/ smallest chromosome ratio R value (3.69). Most S. argentina chromosomes are metacentrics, and the rest
Figure 1. A-C, Somatic and meiotic chromosomes of Sacoila argentina. A, Mitotic metaphase with 2n = 46; B, MMC at metaphase I with 23 II; C, PMC at metaphase I with 23 II. Arrowheads in A show satellites on pair # 20 sm, and in B, C show the largest ring bivalent formed by pair # 20. Scale bars = 5 μm.
Figure 2. Idiogram of Sacoila argentina (38 m + 8 sm). Scale bar = 1 μm.
GRABIELE et al. ― Cytogenetics of Sacoila argentina
339
Table 1. Quantitative parameters of chromosomes of Sacoila argentina.
Pair
s (im) ±SEa
l (im) ±SEa
c ((im) ±SEa
Ia
RL±SEa
Type
1
1.00±0.01
1.17±0.05
2.17±0.01
46.13
5.35±0.03
m
2
1.01±0.02
1.10±0.01
2.11±0.02
47.96
5.20±0.02
m
3
0.99±0.01
1.08±0.02
2.07±0.01
47.92
5.09±0.02
m
4
0.93±0.05
1.12±0.01
2.05±0.01
45.26
5.04±0.03
m
5
0.95±0.03
1.08±0.06
2.03±0.03
46.81
4.99±0.04
m
6
0.91±0.03
0.97±0.03
1.88±0.03
48.28
4.61±0.03
m
7
0.85±0.01
0.92±0.02
1.77±0.01
48.17
4.35±0.02
m
8
0.82±0.01
0.91±0.02
1.72±0.02
47.50
4.24±0.02
m
9
0.78±0.01
0.86±0.01
1.64±0.01
47.37
4.03±0.01
m
10
0.78±0.01
0.82±0.01
1.60±0.01
48.65
3.93±0.01
m
11
0.75±0.02
0.82±0.01
1.57±0.02
47.95
3.87±0.02
m
12
0.71±0.02
0.84±0.01
1.55±0.03
45.83
3.82±0.02
m
13
0.71±0.02
0.78±0.02
1.49±0.01
47.83
3.66±0.02
m
14
0.69±0.01
0.78±0.01
1.47±0.01
47.06
3.61±0.01
m
15
0.66±0.01
0.72±0.01
1.38±0.01
47.82
3.39±0.01
m
16
0.66±0.01
0.72±0.01
1.38±0.03
47.82
3.39±0.02
m
17
0.66±0.01
0.72±0.01
1.38±0.03
47.82
3.39±0.02
m
18
0.58±0.02
0.67±0.01
1.25±0.01
46.55
3.08±0.02
m
19
0.56±0.01
0.65±0.02
1.21±0.01
46.43
2.97±0.02
m
20
1.65±0.01
2.80±0.01
4.45±0.01
37.05
10.95±0.01
sm
21
0.52±0.01
1.16±0.01
1.68±0.02
30.77
4.14±0.02
sm
22
0.43±0.06
1.03±0.03
1.46±0.03
29.41
3.61±0.04
sm
23
0.39±0.03
0.95±0.06
1.34±0.04
29.03
3.29±0.04
sm
aI=mean centromeric index; c=mean chromosome length; l=mean long arm length; s=mean short arm length; RL=relative chromosome length expressed as percentages of total complement length; SE=standard error.
Table 2. Karyotype parameters of Sacoila argentina.
are submetacentrics, so its karyotype is symmetrical in respect to the centromere position on chromosomes as evidenced by the mean centromeric index (i) value (44.58), intrachromosome asymmetry index A1 value (0.18) and proportion of chromosome pairs with arm ratios higher than 2 (0.13) (Table 2). Thus, the karyotype of S. argentina, although bimodal, is rather symmetrical and belongs to category 2B of Stebbins (1971) (Table 2).
The sporogenesis of S. argentina has been normal in all its stages, not only in the female but also in the male germline. Since meiotic behaviour is identical in megaspore mother cells (MMCs) and pollen mother cells (PMCs) the meiotic chromosome associations at diakinesis and metaphase I for both lines are jointly presented (Table 3). Sacoila argentina's gametic number is n = 23 and its chromosomes are arranged as 23 bivalents (Table 3; Figures 1B, C). Most bivalents are rings (80%) with distal chiasmata (85%), and the mean of chiasmata per bivalent is high (1.80) (Table 3). Also, the recombination index, affected by the meiotic number (n = 23) and the mean of chiasmata/cell (41.5) is high (RI = 23 + 41.5 = 64.5) (Table3). Pollen grain viability is elevated (80%) (Table 3).
2n
46
x
23
Karyotype formula
38 m + 8 sm
TCL
81.25 (m
c
1.77 (m
c max
4.45 (m (sm)
c min
1.21 (m (m)
i
44.58
A
1
0.18
A
2
0.36
R
3.69
r>2
0.13
Stebbins category
2B
A1,A2=intrachromosomal and interchromosomal asymmetry indices; c=mean chromosome length; c max, c min=maximum and minimum chromosome length; i=mean centromeric index; r>2=proportion of chromosome pairs with arm ratio>2; R=largest/smallest chromosome ratio; TCL=total complement length.
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Table 3. Meiotic chromosome associations at diakinesis and metaphase I of Sacoila argentina.
of S. lanceolata from Catling (1987). This author describes sexual reproduction for S. lanceolata var. lanceolata and var. paludicola from Guyana and Florida respectively, in which a normal sequence of pollination, pollen tube growth, fertilization, and embryo seed development occurs; however, the variety lanceolata from Florida reproduces by agamospermy, in which the female gametophytes developed from reduced megaspores (n) degenerate, and integumental cells became embryos (Catling, 1987). Reduced megaspores (n) are pre conditions to expecting normal and reduced gametophythes, but for reasons described above, our findings of female meiotic behaviour do not definitively suggest a sexual mode of reproduction for S. argentina, which should be confirmed by cytoembryological studies. Sacoila lanceolata and S. argentina also possess differences in their range of distribution and population size. The former species is widespread in the Americas from Florida to northern and central Argentina, forming large populations while the latter species is restricted to Bolivia, Paraguay, and northern Argentina (Garay, 1980; Catling, 1987) with small and interspersed populations.
The high recombination index of S. argentina suggests that, if the species reproduces by sexual means, the genetic variability can be distributed throughout the population. However in S. argentina, there is an elevated chance that the consequences will occur, mainly because the species has small populations. Efforts to increase the number of individuals by biotechnological means and to look for new natural populations could be a good strategy for overcoming the severe breeding system barriers to natural conservation of the species.
Acknowledgements. We are grateful to the FACEN- UNA authorities who facilitated the collection trips within the Convenio de Cooperacion Cientifica between FACEN-
UNA (Paraguay) and FCEQyN-UNaM (Argentina). This
study was partially supported by Grant PICT-O 36907 (SECyT-Argentina). The authors also give thanks to Ms. Bib. Irma Stella Insaurralde for her taxonomical assistance.
LITERATURE CITED
Battaglia, E. 1955. Chromosome morphology and terminology. Caryologia 8: 179-187.
Catling, P.M. 1987. Notes on the breeding system of Sacoila lanceolata (Aublet) Garay (Orchidaceae). Ann. Missouri Bot. Gard. 74: 58-68.
Cocucci, A. 1956. Cromosomas gameticos de Stenorrhynchos australis Lindl. y Tradescantia radiata Clarke. Trabajos
Mus. Bot. Univ. Nac. Cordoba 2: 781-783.
Correa, M.N. 1 996. Orchidaceae. In F.O. Zuloaga and O. Morrone (eds.), Catalogo de las Plantas Vasculares de la Republica Argentina I. Pteridophyta, Gymnospermae y Angiospermae Monocotyledoneae. Monogr. Missouri Bot. Gard. 60: 242-271.
n
Bivalents per cell ±SE
23
Rings
18.40±0.22
Rods
4.60±0.23
Total
23
Chiasmata per cell ±SE
Distals
35.30±0.21
Interstitials
6.20±0.45
Total
41.50±0.22
Chiasmata per bivalent ±SE
1.80±0.02
Pollen stainability SE = standard error.
80 %
DISCUSSION
Sacoila argentina presented 2n = 46 and the same number was found in S. lanceolata by Cocucci (1956) (sub Stenorrynchos australis Lindl., n = 23), Grabiele et al. (2005) from natural populations of Argentina and Felix & Guerra (2005) from Brazil. At present, these are the only two species of Sacoila that have been cytogenetically analyzed. The chromosome number 2n = 46 was also found in species of the tribe Spirantheae Endl. other than Sacoila (Eltroplectris Raf., Mesadenella Pabst & Garay, Pelexia Poit. ex Rich., Sarcoglottis C. Presl, and Skeptrostachys Garay) by Martinez (1985), Dematteis and Davina (1999), Felix and Guerra (2005) and Grabiele et al.
(2005).
The karyotype formula and morphometric parameters of chromosomes of S. argentina (Tables 1, 2) are similar to those found in S. lanceolata by Grabiele et al. (2005) despite the fact that ovules were used in the former and root meristems in the latter species analysis. The general features of karyotype described for S. argentina are also shared by the other analyzed genera of subtribe Stenorrynchidinae Szlach. (Spirantheae), like Eltroplectris, Mesadenella and Skeptrostachys (Martinez, 1985; Dematteis and Davina, 1999; Grabiele et al., 2005), and this is despite the uncommon 2n = 26 from E. schlechteriana (Porto et Brade) Pabst (Martinez, 1985; Dematteis and Davina, 1999).
The presence of 23 bivalents confirms the basic chromosome number x = 23 for Sacoila proposed by Felix and Guerra (2005) and Grabiele et al. (2005), despite the fact that it may be a derived number.
The female meiosis behaviour found in S. argentina indicates reduced megaspore (n) production. However, it does not specify a particular mode of reproduction, if taking into account the results on the breeding system
GRABIELE et al. ― Cytogenetics of Sacoila argentina
341
Davina, J.R. 2001. Estudios citogeneticos en algunos generos argentinos de Amaryllidaceae. Tesis Doctoral UNC, pp.
184.
Dematteis, M. and J.R. Davina. 1999. Chromosome studies on some Orchids from South America. Selbyana 20: 235-238.
Felix, L.P. and M. Guerra. 2005. Basic chromosome numbers in terrestrial orchids. Pl. Sys. Evol. 254: 131-148.
Garay, L.A. 1980. A generic revision of the Spiranthinae. Bot. Mus. Leafl. 28: 278-425.
Grabiele, M., J.C. Cerutti, D.H. Hojsgaard, A.I. Honfi, and J.R. Davina. 2005. Caracterizacion cariotipica de especies
de Spirantheae Endl. (Spiranthoideae-Orchidaceae) de Argentina. J. Basic Appl. Genet. 17: 119.
Levan, A., K.L. Fredga, and A.A. Sandberg. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220.
Martinez, A. J. 1985. The chromosomes of Orchids VIII. Spiranthinae and Cranichidinae. Kew Bull. 40: 139-147.
Romero Zarco, C. 1986. A new method for estimating karyotype asymmetry. Taxon 3: 531-536.
Stebbins, G.L. 1971. Chromosomal Evolution in Higher Plants. New York: Addison-Wesley Co.
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產自巴拉圭之陸生蘭花Sacoila argentina (Griseb.) Garay
之細細胞遺傳學分析
Mauro GRABIELE1,2, Ana I. HONFI1, Juan C. CERUTTI1, Virginia FERNANDEZ3,
Deidamia FRANCO3, and Julio R. DAVINA1
1Programa de Estudios Floristicos y Genetica Vegetal (PEFyGV)
Facultad de Ciencias Exactas, Quimicas y Naturales
Universidad Nacional de Misiones, 3300 Posadas, Argentina
2Instituto Multidisciplinario de Biologia Vegetal (IMBIV-CONICET)
Facultad de Ciencias Exactas, Fisicas y Naturales
Universidad Nacional de Cordoba, 5000 Cordoba, Argentina
3Departamento de Biologia, Facultad de Ciencias Exactas y Naturales
Universidad Nacional de Asuncion, 2160 San Lorenzo, Paraguay
Sacoila argentina (Griseb.) Garay是一種具觀賞價值之陸生蘭花，分佈於北阿根廷，玻利維亞，及
巴拉圭。在巴拉圭之Chaco區域被定為保育對象，只見於Boqueron省，其染色體之資料直到本文才揭
曉。在此，我們介紹另一巴拉圭之自然族群(取自Paraguari 省，Florida 村'西經 57°07'48.71"- 南緯
26°23'32.16")因此擴展它的分佈地區。傳統之細胞遺傳技術被用來分析其基因系統。Sacoila argentina
及二倍體2n = 2x = 46小染色體（1.21 - 4.45 (im)'單倍體基因組長40.63 (im '它核型含38m + 8sm '乃
二型體（A2=0.36/R=3.69)'此係因爲有一對染色體(# 20, sm)比染色體之長度平均（1.77 (m)超過2倍
大。核型乃對稱的（A1=0.18/i=44.58/r>2=0.13)屬於Stebbins分類2B 。染色體對# 20也在具活躍之NOR
的短臂上帶一尾端大衛星DNA ，孢子形成正常，花粉粒之存活率高（80%)。減數分裂行爲正常，而染
色體對在雄(花粉細胞）爲23 二價體，如同雌（大孢子母細胞）性減數分裂時之數目，証實基本數目
x = 23 ，在花粉母細胞及大孢子母細胞之diakinesis/metaphase I期，環形二價體（80%)與遠方chiasmata
(85%)乃常見者，而每一個二價體之平均chiasmata數是高的（1.80)，無論是雄的或雌的部份。重組指
標[受減數分裂數n = 23 '及每一細胞之chiasmata平均數（41.5)所影響]是高的（RI=64.5) 。 Sacoila
argentina之自然族群稀少，密度低，分散，且只在開花期才看得到；基於上述理由，此乃很少被收集到
之物種。
關鍵詞：染色體；基因系統；Orchidaceae ； Sacoila argentina 。