Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
375
Vol.53, n. 2: pp. 375-387, March-April 2010
ISSN 1516-8913 Printed in Brazil
BRAZILIAN ARCHIVES OF
BIOLOGY AND TECHNOLOGY

A N I N T E R N A T I O N A L J O U R N A L



Genetic Analysis of Species in the Genus Catasetum
(ORCHIDACEAE) using RAPD Markers

Luciana do Valle Rego Oliveira1, Ricardo Tadeu de Faria1*, Claudete de Fátima Ruas2,
Paulo Maurício Ruas2, Melissa de Oliveira Santos2 and Valdemar P. Carvalho2
1Departamento de Agronomia; Universidade Estadual de Londrina; Celso Garcia Cid Km 379; 86051-990;
Londrina - PR - Brasil. 2Departamento de Biologia Geral; Universidade Estadual de Londrina; Celso Garcia Cid
Km 379; 86051-990; Londrina - PR - Brasil


ABSTRACT

In this work, RAPD molecular markers were used to access the genetic variability and to study the inter and
intraespecifc relationship in a group of 37 species, including 56 individuals. A total of 15 RAPD primers were
selected for DNA amplification. From a total of 221 bands analyzed, 209 (95%) were polymorphics. The level of
interespecifc genetic similarity ranged from 37% between Catasetum complanatum and Catasetum laminatum to
83% between Catasetum triodon and Catasetum uncatum. The intraspecifc genetic similarity varied 88% for the
individuals of Catasetum triodon to 93% between the individuals of Catasetum atratum and Catasetum
macrocarpum. These results would contribute to understand the genetic relationship in Catasetum, to define the
strategies to establish a germplasm core collection for the genus and to provide support for breeding programs.

Key words: Catasetum, molecular marker, orchid, RAPD, genetic similarity



*Author for correspondence: faria@uel.br
INTRODUCTION

The family Orchidaceae has unique characteristics
that do not resemble with any other group of plants
(Rittershausen, 1998). It is considered the largest
family within the group of flowering plants, and
comprises approximately 35,000 species
distributed across six tribes, 80 subtribes, and
about 750 genera (Heywood, 1993). It constitutes
a very successful group, with more than 100,000
hybrids developed in the last 150 years, both by
natural and artificial crosses (Suttleworth et al.,
1993; Rittershausen, 1998; Faria et al., 2001;
Moreira and Isaias, 2008).
The classification of the family Orchidaceae is not
yet well defined. The classification system has
been developed for centuries and was established
mainly by taxonomists. It includes a number of
divisions and subdivisions that group the plants
based on their similarities in structure and flower
appearance. The most accepted classification at
present takes into account other plant
characteristics, such as cytological and molecular
data (Dressler, 1993).
The genus Catasetum has more than 100 species
(Raposo, 1992), of which the major part is
terrestrial or epiphytic (Scaglia, 1998). The habitat
of this genus extends from Mexico down to
Argentina, with a center of radiation in Brazil
(Raposo, 1992). It includes some of the most
beautiful and rare species (ABRACC, 1998a). The
plant considered as typical in the genus Catasetum

Oliveira, L. do V. R. et al
Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
376
is Catasetum macrocarpum. Other synonyms of
the genus that were often used in the past are:
Catachaeteum Hoffmansegg. 1842; Cuculina Raf.
1836; Monacanthus G. Don 1839; Monachanthus
Lindley 1832; Myanthus Lindley 1832; and
Warczewitzia Skinner 1850 (Orchidaceae
brasiliensis, 2004).
The genus Catasetum, as well as the majority of
the family Orchidaceae, presents chromosomal
variation, such as: n= 27, 28, 54, and 81 (Tanaka
and Kamemoto, 1984). However, for some genera,
the basic number of chromosomes is usually
constant: 20 in Cattleya, 15 in Vanda, 19 in
Dendrobium and Phalaenopsis (Carnier, 1996).
Occasionally, changes in the number of
chromosomes do occur, resulting in an increase in
the degree of ploidy. The increase in ploidy in
orchids is often accompanied by an increase in the
size of plant parts; plants are more robust and
flowers generally have a better shape, and could
become gigantic (Carnier, 1996). According to
Tanaka and Kamemoto (1984), chromosome
number determination is an important tool to
conduct the breeding studies in this family.
Molecular markers have been widely used in
genetic diversity studies, and also as an aid in the
classification of orchid species and genera (Tsai et
al., 2002; Obara-Okeyo and Kako, 1998; Williams
et al, 2001).
Many studies have tried to establish a molecular
phylogeny in the family Orchidaceae.
Freudenstein and Doyle (1994) used chloroplast
DNA as part of a phylogenetic study in
Corallorhiza, while Squirrel et al. (2001) used
chloroplast genes and the variability obtained by
isozymes in different Epipactis helleborine
populations. Ribosomal ITS sequences have been
employed in other studies, either individually or
together with sequences to make inferences about
interspecific relationships in different groups of
orchids (Gravendeel et al., 2001; Pridgeon et al.,
2001; Williams et al., 2001).
The RAPD technique (Random Amplified
Polymorphic DNA) has been used in certain
orchid groups in order to study their structure and
genetic diversity. Tsai et al. (2002) studied several
genotypes in the Tribe Oncidiinae using RAPD
markers and revealed a genetic similarity that
ranged between 25 and 71%. Obara - Okeyo and
Kako (1998) used 132 RAPD markers to analyze
the efficacy of this technique in the identification
of Cymbidium cultivars. Their results showed that
it was possible to discriminate all the cultivars
when an adequate quantity of primers was used. In
other studies, RAPD markers were used in
conjunction with isozymes, in order to analyze the
genetic structure and conservation of orchid
populations. A correlation between data obtained
with the use of both types of markers was
observed (Case et al., 1998; Sun and Wong, 2001).
The objective of this work was to determine the
genetic similarity between 37 species in the genus
Catasetum, including 56 individuals, and to clarify
the intra and interspecific relationships in this
genus using RAPD molecular markers.


MATERIAL AND METHODS

Plant material
The plant material was obtained from the orchid
collection of the Agronomy Department at Centro de
Ciências Agrárias of Universidade Estadual de
Londrina, in Brazil. The species and subspecies
studied, as well as their corresponding names in the
collection, chromosome numbers (2n), general
geographical distribution and specific origin (Table
1).

DNA extraction
Genomic DNA was extracted following the
methodology described by Doyle and Doyle
(1987), with some modifications. DNA was
extracted from young leaves of individual plants
macerated in liquid nitrogen, and mixed with 2.5
mL of 3% CTAB extraction buffer (3% CTAB;
150 mM Tris – HCL; 30 mM EDTA, ph 7.5; 2.2
mM NaCl, 1.5% PVP) and 4 µL β –
mercaptoethanol. The samples were then
incubated in a double boiler at 65ºC for 30 minutes
and centrifuged for five minutes at 14,000 rpm.
After centrifugation, the supernatant was isolated
and 1.5 volume of isopropanol was added,
followed by incubation at –20ºC for 30 minutes.
At the end of incubation, the samples were
submitted to centrifugation for five minutes at
14,000 rpm and then the supernatant was
discarded. The resulting precipitate was washed
twice in 70% alcohol, resuspended in 0.1 mL TE
buffer (Tris-HCl 1 mM; EDTA 0.1 mM, pH 8.0)
and added of 1 µl proteinase K (10 mg/ µL). After
incubation for one hour at 37ºC, one volume of
3M ammonia acetate was added and the samples
were incubated on ice for 20 minutes. After this

Genetic Analysis of Species in the Genus Catasetum (ORCHIDACEAE)
Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
377
period, the same volume of isopropanol wasadded,
followed by incubation for at least 20 minutes at –
20ºC. The DNA was recovered by centrifugation
at 14,000 rpm for five minutes, washed in 70%
alcohol and dissolved in TE buffer. The DNA was
quantified with a fluorometer (Dyna-Quant,
Hoefer B Pharmacia) and diluted to a final
concentration of 10 ng/µl.

Table 1 - Species in the genus Catasetum, subspecies, collection, number of chromosomes (2n), geographical
distribution, and origin of species studied by means of the RAPD technique.






no Genus species subspecies Collection Geographical Distribution Origin of the Species under Study
1 Ctsm. arietinum Hideto Ctsm. 2 PE PE
2 Ctsm. aripuanense UEL Ctsm. 37 MS and MT MT
3 Ctsm. ariquimense Igor Ctsm. 6 RO RO
4 Ctsm. atratum UEL Ctsm. 11 e 24 SP, PR, SC, RS and MG PR
5 Ctsm. atratum UEL Ctsm. 66 SP, PR, SC, RS and MG Unknown origin
6 Ctsm. atratum UEL Ctsm. 58 SP, PR, SC, RS and MG Unknown origin
7 Ctsm. barbatum semicuculatum UEL Ctsm. 3 TO, PA, AM, AC, MT, MA, MS, CE and PI Unknown origin
8 Ctsm. barbatum spinosum UEL Ctsm. 10 e 12 TO, PA, AM, AC, MT, MA, MS, CE and PI Unknown origin
9 Ctsm. barbatum UEL Ctsm. 26 Hideto Ctsm. 3 TO, PA, AM, AC, MT, MA, MS, CE and PI Unknown origin
10 Ctsm. cernuum UEL Ctsm. 47 SP, RJ, PR, ES and MG RJ
11 Ctsm. ciliatum UEL Ctsm. 63 PA and AM PA
12 Ctsm. complanatum UEL Ctsm. 6 e 40 RO RO
13 Ctsm. confusum Igor Ctsm. 2 GO GO
14 Ctsm. cristatum UEL. Ctsm. 34 RR, AP and PA Unknown origin
15 Ctsm. denticulatum Igor Ctsm. 1 RO RO
16 Ctsm. discolor

UEL. Ctsm. 32 e 35 AM, PA, MA, CE, MT, PE, BA, ES, EJ,
Suriname and Guianas
BA
17 Ctsm. discolor

UEL. Ctsm. 27 e 22 AM, PA, MA, CE, MT, PE, BA, ES, EJ,
Suriname and Guianas
PA
18 Ctsm. fimbriatum UEL. Ctsm. 30 e 42 MT, MS, SP, PR, MG and GO MG
19 Ctsm. fimbriatum ornithorhynchum UEL Ctsm. 49 MT, MS, SP, PR, MG and GO Unknown origin
20 Ctsm. fimbriatum UEL Ctsm. 56 e 64 MT, MS, SP, PR, MG and GO Unknown origin
21 Ctsm. fimbriatum fissum UEL Ctsm. 23 MT, MS, SP, PR, MG and GO PR
22 Ctsm. fimbriatum (flor vermelha) UEL Ctsm. 67 MT, MS, SP, PR, MG and GO Unknown origin
23 Ctsm. galeritum Hideto Ctsm.4 AM, PA, GO and TO Unknown origin
24 Ctsm. gardneri UEL Ctsm. 5 e 19 PE to RJ BA
25 Ctsm. gladiatorium UEL Ctsm. 9 GO and MT(Brasil's central plateau) GO
26 Ctsm. gladiatorium UEL Ctsm. 62 GO and MT(Brasil's central plateau) Unknown origin
27 Ctsm. gnomus UEL Ctsm. 45 e 31 AM AM
28 Ctsm. hookeri UEL Ctsm. 39 SP and RJ Unknown origin
39 Ctsm. hookeri UEL Ctsm. 36 e 53 SP and RJ Unknown origin
30 Ctsm. juruenense UEL Ctsm. 38 MT MT
31 Ctsm. juruenense UEL Ctsm. 28 MT MT
32 Ctsm. laminatum UEL Ctsm. 51 México Unknown origin
33 Ctsm. lemosii Igor Ctsm. 5 PAand MT Unknown origin
34 Ctsm. macrocarpum

UEL Ctsm. 2 RJ, BA, PB, PE, RN, PI, PA, AM, TO, MA,
RR and ES
PA
35 Ctsm. macrocarpum

UEL Ctsm. 25 e 29 RJ, BA, PB, PE, RN, PI, PA, AM, TO, MA,
RR and ES
BA
36 Ctsm. macrocarpum

UEL Ctsm. 41 RJ, BA, PB, PE, RN, PI, PA, AM, TO, MA,
RR and ES
Unknown origin
37 Ctsm. matogrossense Igor Ctsm. 11 MT MT
38 Ctsm. moreanum Igor Ctsm. 9 Brazil BA
39 Ctsm. osculatum UEL Ctsm. 13 MS, MT, RO and GO MT
40 Ctsm. osculatum UEL Ctsm. 55 MS, MT, RO and GO MS
41 Ctsm. osculatum UEL Ctsm. 20 MS, MT, RO and GO RO
42 Ctsm. parguazense Igor Ctsm. 12 RO RO
43 Ctsm. pileatum UEL Ctsm.68 AM Unknown origin
44 Ctsm. planiceps

Igor Ctsm. 8 AM (norte do Brasil), Venezuela, Guianas,
Suriname e Perú PA
45 Ctsm. pulchorom Hideto Ctsm. 5 AM, PA and MT MT
46 Ctsm. purum UEL Ctsm. 16 e 43 BA, ES and MG BA
47 Ctsm. saccatum UEL Ctsm. 65 AC and AM Unknown origin
48 Ctsm. sanguineum UEL Ctsm. 4 Colombia, Guiana, Venezuela, Brazil, Costa Rica and Panama
AC
49 Ctsm. schmidtianum Igor Ctsm. 4 MT MT
50 Ctsm. spitzii UEL Ctsm. 18, 54 e 59 GO Unknown origin
51 Ctsm. spitzii UEL Ctsm. 14 GO Unknown origin
52 Ctsm. triodom UEL Ctsm. 8 e 57 SP, PR and SC PR
53 Ctsm. triodom UEL Ctsm. 33 SP, PR and SC PR
54 Ctsm. uncatum UEL Ctsm. 21, 61 e 50 PE Unknown origin
55 Ctsm. vinaceum

UEL Ctsm. 15, 60 e 48
Hideto Ctsm. 6
MT, MS and GO MT
56 Ctsm. vinaceum UEL Ctsm. 1 MT, MS and GO Unknown origin

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378
DNA amplification via PCR (Polymerase Chain
Reaction)
The RAPD reactions were conducted in a final
volume of 15 µl containing 7.92 µl sterile water;
1.5 µl amplification buffer (50 mM KCl, 15 mM
MgCl2, 100mM Tris-Cl, pH 9.0); 1.8 µl dNTPs
(0.1 mM of each dGTP, dATP, dCTP, dTTP); 1.8
µl primer –Operon Technologies (4 mM); 1U Taq
DNA polymerase (Biotools); and 20 ng DNA
(polymerization), followed by a final extension for
7 min at 72o C. The amplification products were
separated by electrophoresis in 1% agarose gel,
stained with ethidium bromide (0.15 µl/ml), and
visualized under UV light. Gel images were
captured using a photographic documentation and
thermoprinting system.

Data analysis
The amplification products were analyzed by
marking their presence (1) or absence (0) for each
DNA fragment generated. The data obtained were
analyzed with the NTSYS-PC software (Rohlf,
2000) and utilized to obtain the genetic similarity
matrix using DICE coefficient. The UPGMA
clustering method (Unweighted Pair Group
Method using Arithmetic Averages) was used to
construct a dendrogram. The reliability of the
associations shown on the dendrogram was
evaluated by bootstrap probability with 1,000
permutations, using the bood v. 3.0 software
(Coelho 2001). The cophenetic coefficient
between the similarity matrix and the dendrogram
was computed using the NTSYS program.


RESULTS AND DISCUSSION

A total of 15 RAPD primers (Table 2) were
selected and used for DNA amplification. Two
hundred and twenty-one markers were generated,
of which 209 were polymorphic (95%). The
electrophoretic pattern obtained with primer
OPAE – 16 is shown in Figure 1.

Table 2 – Primers used sequence of primers (Operon Technologies), number of bands, and percentage of
polymorphic bands for species in the genus Catasetum under study.
Primer Primer Sequence Number of bands Clustered polymorphic bands
OPAW-01 5’ ACCTAGGGGA 3’ 6 83%
OPAW-03 5’ CCATGCGGAG 3’ 20 90%
OPAW-05 5’ CTGCTTCGAG 3’ 21 95%
OPAW-10 5’ GGTGTTTGCC 3’ 14 86%
OPAW-15 5’ CCAGTCCCAA 3’ 14 93%
OPAE-16 5’ TCCGTGCTGA 3’ 19 100%
OPAI-01 5’ GGCATCGGCT 3’ 8 88%
OPAI-02 5’ AGCCGTTCAG 3’ 15 92%
OPAD-07 5’ CCCTACTGGT 3’ 16 93%
OPAD-15 5’ TTTGCCCCGT 3’ 23 96%
OPAD-13 5’ GGTTCCTCTG 3’ 17 100%
OPAD-19 5’ CTTGGCACGA 3’ 12 92%
OPAS-18 5’ GTTGCGCAGT 3’ 20 100%
OPAX-04 5’ TCCCCAGGAG 3’ 8 100%
OPAX-07 5’ ACGCGACAGA 3’ 9 100%
Total 251 95%


The RAPD data were used to generate a similarity
matrix (Table 3) and the dendrogram (Fig. 2). In
the dendrogram, most associations between
groups obtained bootstrap values (BO) higher
than 50%, thus attesting to the reliability of this
experiment.
The polymorphism generated with RAPD markers
allowed the species under analysis to be divided
into several distinct groups. The high value
obtained in the cophenetic analysis (r=0.922)
ensured the reliability of the clusters observed in
the dendrogram. The significance of the
correlation, tested according to Mantel’s test,
showed that the number of markers used was
sufficient to assess the genetic diversity of the
species analyzed.

Genetic Analysis of Species in the Genus Catasetum (ORCHIDACEAE)
Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
379










Figure 1 - Electrophoretic pattern obtained with primer OPAE – 16. Samples are identified by the
numbers presented in Table 2. M – Molecular weight marker (DNA ladder Invitrogen
100pb), C – Control sample.



































Figure 2 - Genetic similarity dendrogram of 37 species studied in the Genus Catasetum, including
56 individuals, calculated by DICE index, based on RAPD markers. The number
between parentheses corresponds to the sequence presented in table 1. Bootstrap values
below 50% are not shown. Londrina (PR, Brazil), UEL, 2004.

-Ctsm. arietinum (1)
- Ctsm. atratum (4,5, 6)
-Ctsm. barbatum semi. (7)
-Ctsm. aripuanense (2)
-Ctsm. cernuum (10)
-Ctsm. confusum (13)
-Ctsm. hookeri (28, 29)
-Ctsm. barbatum
(9) -Ctsm. barbatum spin. (8)
-Ctsm. ariquimense (3)
-Ctsm. uncatum (54)
-Ctsm. triodon (52, 53)
-Ctsm. pulchorom (45)
-Ctsm. fimbriatum
(18, 19, 20, 21)
-Ctsm. spitzii (50, 51)
-Ctsm. galeritum (23)
-Ctsm. fimbriatum verm.(22)
-Ctsm. lemosii (33)
-Ctsm. matogrossense (37)
-Ctsm. cristatum (14)
-Ctsm. gnomus (27)
-Ctsm. vinaceum (55, 56)
-Ctsm. denticulatum (15)
-Ctsm. discolor (16, 17)
-Ctsm. shimidtianum (49)
-Ctsm. saccatum (47)
-Ctsm. Macrocarpum
(34, 35, 36)
-Ctsm. moreanum (38)
-Ctsm. osculatum
(39, 40, 41)
-Ctsm. planiceps (46)
0.51
-Ctsm. gladiatorum (25, 26)
0.76 0.63 0.88 1.00
-Ctsm. purum (44)
-Ctsm. pileatum (43)
-Ctsm. parguazense (42)
-Ctsm. juruenense (30, 31)
-Ctsm. gardineri (24)
-Ctsm. ciliatum (11)
-Ctsm. complanatum (12)
-Ctsm. sanguineum (48)
-Ctsm. laminatum (32)
100
100
100
100
100
82 83
98
70
92
72
66
77 59
58
77
84
95
60
79
90

98

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380
Ctsm. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1 1,00
2 0,74 1,00
3 0,64 0,60 1,00
4 0,67 0,68 0,56 1,00
5 0,63 0,65 0,54 0,94 1,00
6 0,64 0,63 0,56 0,93 0,91 1,00
7 0,71 0,75 0,52 0,62 0,61 0,57 1,00
8 0,68 0,66 0,59 0,57 0,57 0,59 0,73 1,00
9 0,70 0,75 0,51 0,64 0,61 0,60 0,94 0,76 1,00
10 0,62 0,62 0,54 0,76 0,74 0,71 0,62 0,54 0,61 1,00
11 0,45 0,53 0,49 0,57 0,56 0,55 0,60 0,56 0,61 0,55 1,00
12 0,58 0,54 0,52 0,54 0,54 0,52 0,62 0,60 0,60 0,51 0,50 1,00
13 0,67 0,66 0,55 0,77 0,74 0,73 0,70 0,60 0,69 0,78 0,52 0,58 1,00
14 0,60 0,61 0,58 0,55 0,54 0,52 0,62 0,68 0,60 0,57 0,53 0,58 0,60 1,00
15 0,51 0,51 0,58 0,47 0,47 0,51 0,51 0,55 0,50 0,46 0,45 0,51 0,51 0,59 1,00
16 0,54 0,52 0,59 0,52 0,52 0,50 0,55 0,62 0,55 0,51 0,64 0,57 0,50 0,64 0,53 1,00
17 0,54 0,50 0,59 0,54 0,53 0,50 0,53 0,60 0,53 0,51 0,66 0,54 0,50 0,60 0,53 0,92 1,00
18 0,61 0,54 0,57 0,59 0,55 0,54 0,57 0,60 0,58 0,57 0,54 0,53 0,52 0,63 0,57 0,51 0,58 1,00
19 0,59 0,57 0,57 0,54 0,50 0,49 0,57 0,61 0,58 0,50 0,50 0,52 0,47 0,60 0,59 0,52 0,56 0,88 1,00
20 0,65 0,60 0,57 0,60 0,56 0,55 0,61 0,66 0,62 0,53 0,53 0,53 0,51 0,63 0,58 0,53 0,58 0,87 0,90
21 0,65 0,59 0,59 0,58 0,56 0,55 0,57 0,64 0,59 0,52 0,54 0,57 0,51 0,65 0,57 0,55 0,58 0,82 0,85
22 0,60 0,55 0,58 0,55 0,54 0,51 0,58 0,55 0,57 0,50 0,55 0,63 0,51 0,65 0,60 0,59 0,59 0,73 0,81
23 0,60 0,66 0,60 0,55 0,57 0,56 0,59 0,61 0,59 0,55 0,57 0,58 0,60 0,58 0,56 0,61 0,62 0,58 0,65
24 0,56 0,54 0,53 0,57 0,57 0,56 0,55 0,53 0,55 0,59 0,59 0,47 0,56 0,50 0,48 0,67 0,65 0,53 0,51
25 0,60 0,59 0,61 0,55 0,53 0,53 0,61 0,66 0,62 0,57 0,55 0,55 0,57 0,64 0,54 0,57 0,57 0,63 0,65
26 0,58 0,61 0,57 0,51 0,51 0,48 0,59 0,62 0,61 0,52 0,51 0,51 0,53 0,61 0,52 0,55 0,57 0,64 0,67
27 0,59 0,58 0,60 0,56 0,54 0,53 0,56 0,59 0,57 0,56 0,56 0,57 0,48 0,61 0,47 0,69 0,67 0,61 0,67
28 0,57 0,59 0,55 0,71 0,73 0,69 0,60 0,54 0,58 0,68 0,55 0,50 0,67 0,50 0,51 0,52 0,54 0,58 0,58
29 0,58 0,61 0,54 0,72 0,71 0,70 0,61 0,51 0,59 0,69 0,58 0,53 0,70 0,53 0,50 0,53 0,55 0,57 0,57
30 0,55 0,52 0,56 0,54 0,54 0,53 0,52 0,49 0,51 0,56 0,45 0,54 0,55 0,56 0,54 0,53 0,53 0,55 0,55
31 0,58 0,56 0,60 0,54 0,54 0,52 0,55 0,51 0,55 0,54 0,44 0,56 0,56 0,63 0,60 0,58 0,58 0,56 0,54
32 0,53 0,53 0,48 0,50 0,50 0,48 0,47 0,52 0,49 0,55 0,48 0,48 0,53 0,51 0,37 0,51 0,53 0,50 0,48
33 0,66 0,64 0,58 0,66 0,65 0,64 0,64 0,61 0,65 0,57 0,53 0,61 0,61 0,57 0,57 0,54 0,57 0,59 0,61
34 0,61 0,57 0,56 0,50 0,50 0,50 0,55 0,64 0,57 0,52 0,57 0,57 0,50 0,62 0,46 0,66 0,69 0,59 0,58
35 0,61 0,52 0,55 0,46 0,47 0,47 0,53 0,59 0,55 0,51 0,55 0,59 0,48 0,57 0,45 0,62 0,65 0,58 0,58
36 0,61 0,53 0,54 0,46 0,46 0,46 0,55 0,60 0,56 0,51 0,57 0,58 0,48 0,58 0,46 0,59 0,63 0,57 0,57
37 0,59 0,61 0,65 0,60 0,59 0,57 0,54 0,58 0,55 0,60 0,51 0,56 0,57 0,53 0,50 0,53 0,53 0,55 0,59
38 0,57 0,53 0,53 0,58 0,56 0,54 0,55 0,58 0,53 0,56 0,57 0,52 0,54 0,59 0,46 0,67 0,65 0,48 0,51
39 0,62 0,56 0,57 0,58 0,58 0,58 0,50 0,59 0,51 0,58 0,56 0,51 0,53 0,55 0,49 0,59 0,63 0,57 0,57
40 0,62 0,57 0,58 0,57 0,55 0,55 0,51 0,59 0,52 0,58 0,60 0,51 0,51 0,53 0,50 0,59 0,63 0,55 0,57
41 0,58 0,56 0,57 0,56 0,57 0,58 0,50 0,56 0,51 0,59 0,57 0,54 0,54 0,54 0,49 0,56 0,58 0,54 0,54
42 0,63 0,52 0,57 0,58 0,54 0,54 0,60 0,57 0,59 0,58 0,54 0,63 0,59 0,54 0,49 0,57 0,57 0,57 0,57
43 0,62 0,53 0,55 0,49 0,47 0,47 0,54 0,56 0,52 0,51 0,53 0,56 0,53 0,63 0,51 0,61 0,59 0,54 0,53
44 0,59 0,58 0,56 0,56 0,54 0,54 0,56 0,58 0,54 0,60 0,53 0,53 0,58 0,64 0,52 0,55 0,53 0,56 0,57
45 0,50 0,57 0,53 0,54 0,52 0,52 0,53 0,56 0,58 0,59 0,52 0,55 0,50 0,51 0,47 0,48 0,50 0,52 0,56
46 0,59 0,56 0,56 0,57 0,59 0,58 0,56 0,61 0,56 0,60 0,47 0,48 0,63 0,57 0,55 0,52 0,51 0,48 0,50
47 0,53 0,55 0,50 0,51 0,53 0,53 0,53 0,56 0,54 0,57 0,57 0,55 0,51 0,55 0,45 0,59 0,59 0,54 0,55
48 0,53 0,54 0,55 0,49 0,47 0,48 0,52 0,55 0,52 0,50 0,53 0,51 0,51 0,53 0,52 0,55 0,57 0,53 0,51
49 0,60 0,55 0,66 0,51 0,51 0,51 0,59 0,60 0,56 0,55 0,59 0,53 0,58 0,70 0,50 0,64 0,69 0,60 0,56
50 0,57 0,56 0,52 0,50 0,55 0,50 0,56 0,52 0,57 0,51 0,56 0,55 0,48 0,51 0,49 0,53 0,53 0,59 0,57
51 0,53 0,54 0,55 0,56 0,59 0,56 0,52 0,53 0,53 0,52 0,51 0,54 0,53 0,50 0,52 0,46 0,47 0,60 0,56
52 0,59 0,59 0,47 0,62 0,65 0,60 0,60 0,56 0,61 0,60 0,50 0,47 0,61 0,52 0,48 0,47 0,50 0,56 0,54
53 0,58 0,58 0,52 0,65 0,66 0,63 0,58 0,55 0,57 0,64 0,46 0,50 0,64 0,54 0,49 0,46 0,50 0,53 0,50
54 0,61 0,55 0,55 0,64 0,66 0,63 0,59 0,54 0,59 0,61 0,48 0,56 0,60 0,51 0,51 0,47 0,52 0,59 0,56
55 0,63 0,61 0,60 0,55 0,57 0,54 0,65 0,63 0,63 0,59 0,55 0,55 0,59 0,55 0,54 0,51 0,53 0,62 0,61
56 0,62 0,59 0,56 0,55 0,54 0,52 0,59 0,57 0,58 0,53 0,49 0,60 0,52 0,58 0,52 0,53 0,51 0,58 0,60
Ctsm. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
20 1,00
21 0,87 1,00
22 0,76 0,81 1,00
23 0,60 0,61 0,64 1,00
24 0,55 0,51 0,50 0,58 1,00
25 0,64 0,64 0,70 0,67 0,58 1,00
26 0,68 0,63 0,63 0,66 0,55 0,85 1,00
27 0,65 0,67 0,69 0,65 0,63 0,68 0,64 1,00
28 0,57 0,58 0,57 0,65 0,60 0,65 0,60 0,59 1,00
29 0,56 0,59 0,54 0,66 0,59 0,65 0,61 0,58 0,88 1,00
30 0,52 0,53 0,56 0,60 0,55 0,68 0,64 0,60 0,65 0,68 1,00
31 0,51 0,57 0,60 0,60 0,57 0,67 0,61 0,60 0,60 0,63 0,87 1,00
32 0,47 0,52 0,49 0,53 0,48 0,51 0,49 0,56 0,50 0,49 0,52 0,51 1,00
33 0,65 0,63 0,62 0,61 0,56 0,59 0,58 0,59 0,61 0,60 0,63 0,62 0,60 1,00
34 0,57 0,61 0,61 0,63 0,54 0,64 0,59 0,73 0,50 0,51 0,59 0,58 0,58 0,56 1,00
35 0,58 0,62 0,61 0,62 0,52 0,60 0,57 0,69 0,50 0,49 0,57 0,56 0,61 0,57 0,92 1,00
36 0,58 0,62 0,63 0,59 0,50 0,62 0,58 0,67 0,48 0,49 0,57 0,56 0,59 0,57 0,91 0,95 1,00
37 0,57 0,55 0,59 0,61 0,61 0,62 0,57 0,62 0,64 0,56 0,54 0,51 0,49 0,67 0,55 0,55 0,54 1,00
38 0,52 0,50 0,56 0,59 0,58 0,62 0,59 0,73 0,53 0,54 0,56 0,56 0,52 0,55 0,64 0,60 0,58 0,66 1,00
39 0,59 0,59 0,60 0,61 0,59 0,58 0,54 0,69 0,55 0,55 0,56 0,55 0,56 0,64 0,73 0,73 0,71 0,61 0,70
40 0,60 0,59 0,62 0,61 0,57 0,59 0,56 0,69 0,54 0,54 0,56 0,56 0,62 0,62 0,72 0,76 0,73 0,57 0,65
41 0,53 0,57 0,59 0,60 0,53 0,58 0,55 0,65 0,56 0,53 0,61 0,59 0,63 0,65 0,72 0,77 0,71 0,56 0,62
42 0,55 0,57 0,62 0,60 0,61 0,63 0,57 0,63 0,61 0,60 0,65 0,62 0,49 0,63 0,57 0,54 0,54 0,66 0,65
43 0,49 0,56 0,64 0,62 0,51 0,59 0,52 0,64 0,55 0,56 0,59 0,61 0,49 0,53 0,72 0,67 0,67 0,58 0,69
44 0,54 0,57 0,67 0,58 0,52 0,72 0,63 0,63 0,61 0,57 0,66 0,68 0,56 0,64 0,64 0,63 0,64 0,66 0,69
45 0,53 0,55 0,53 0,65 0,49 0,64 0,6 0,57 0,59 0,60 0,61 0,54 0,55 0,61 0,54 0,58 0,57 0,61 0,55
46 0,47 0,53 0,54 0,55 0,45 0,66 0,59 0,56 0,62 0,61 0,63 0,64 0,46 0,53 0,58 0,53 0,52 0,57 0,65
47 0,54 0,59 0,60 0,55 0,50 0,59 0,54 0,63 0,50 0,52 0,60 0,58 0,58 0,61 0,70 0,72 0,71 0,54 0,61
48 0,54 0,48 0,50 0,60 0,57 0,58 0,56 0,56 0,53 0,56 0,52 0,53 0,47 0,53 0,54 0,53 0,50 0,59 0,58
49 0,57 0,60 0,61 0,62 0,54 0,68 0,61 0,67 0,54 0,57 0,62 0,63 0,55 0,62 0,75 0,69 0,71 0,57 0,64
50 0,58 0,61 0,58 0,69 0,52 0,55 0,59 0,56 0,60 0,58 0,56 0,56 0,51 0,60 0,50 0,53 0,53 0,53 0,49
51 0,57 0,57 0,57 0,70 0,44 0,54 0,55 0,51 0,61 0,57 0,55 0,51 0,52 0,58 0,47 0,49 0,47 0,54 0,48
52 0,56 0,55 0,51 0,60 0,54 0,59 0,60 0,51 0,72 0,72 0,58 0,54 0,52 0,61 0,48 0,50 0,50 0,53 0,49
53 0,51 0,50 0,47 0,61 0,52 0,55 0,52 0,52 0,74 0,72 0,58 0,53 0,54 0,57 0,49 0,51 0,47 0,56 0,53
54 0,55 0,56 0,53 0,60 0,47 0,61 0,56 0,53 0,79 0,77 0,62 0,59 0,57 0,61 0,49 0,51 0,48 0,55 0,53
55 0,64 0,57 0,64 0,70 0,54 0,70 0,64 0,58 0,70 0,66 0,58 0,53 0,55 0,60 0,56 0,58 0,57 0,64 0,52
56 0,64 0,59 0,65 0,65 0,52 0,61 0,57 0,61 0,62 0,61 0,61 0,58 0,51 0,61 0,55 0,52 0,53 0,64 0,52
Table 3 - Genetic similarity matrix obtained for 37 species of Catasetum with 56 individuals obtained from RAPD
markers. Londrina (PR, Brazil), UEL, 2004.























































(cont…)

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381
Ctsm. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
39 1,00
40 0,86 1,00
41 0,78 0,87 1,00
42 0,62 0,62 0,59 1,00
43 0,73 0,68 0,65 0,67 1,00
44 0,68 0,66 0,69 0,70 0,70 1,00
45 0,57 0,59 0,62 0,61 0,53 0,62 1,00
46 0,60 0,62 0,63 0,66 0,69 0,72 0,59 1,00
47 0,66 0,68 0,72 0,58 0,60 0,68 0,63 0,65 1,00
48 0,57 0,58 0,57 0,60 0,52 0,61 0,60 0,57 0,60 1,00
49 0,66 0,64 0,65 0,62 0,68 0,65 0,56 0,62 0,66 0,61 1,00
50 0,58 0,60 0,59 0,60 0,55 0,52 0,60 0,56 0,56 0,54 0,53 1,00
51 0,55 0,59 0,56 0,59 0,51 0,52 0,61 0,57 0,52 0,53 0,50 0,81 1,00
52 0,56 0,55 0,56 0,54 0,51 0,53 0,63 0,59 0,53 0,53 0,50 0,66 0,61 1,00
53 0,56 0,55 0,57 0,56 0,54 0,52 0,64 0,60 0,51 0,54 0,51 0,61 0,66 0,88 1,00
54 0,57 0,56 0,57 0,59 0,53 0,55 0,64 0,65 0,54 0,56 0,54 0,67 0,69 0,80 0,85 1,00
55 0,60 0,63 0,60 0,62 0,56 0,60 0,65 0,57 0,53 0,58 0,59 0,73 0,71 0,70 0,67 0,75 1,00
56 0,59 0,59 0,52 0,63 0,54 0,52 0,59 0,53 0,53 0,57 0,55 0,70 0,67 0,60 0,57 0,65 0,80 1,00
(Cont. Table 3)








































Figure 3 - Principal coordinates dispersion graph obtained from DICE genetic similarity matrix,
estimated by RAPD markers, between 37 species in the genus Catasetum including 56
individuals, according to the sequence presented in table 3. Londrina (PR, Brazil),
UEL, 2004.


It was possible to visualize that cluster data based
on DICE’s genetic similarity matrix (Table 3)
showed interspecific genetic similarity index
values ranging from 37% between Ctsm.
laminatum and Ctsm. denticulatum to 83% (81%
Bootstrap) between Ctsm. triodon and Ctsm.
uncatum. Species represented by more than one
individual (Table 1) showed an intraspecific
genetic similarity index ranging from 88% (82%
Bootstrap) for the individuals of the species Ctsm.
triodon to 93% (100% Bootstrap) between
individuals in the Ctsm. atratum group and Ctsm.
macrocarpum.
In the principal coordinates dispersion graph
obtained from DICE genetic similarity matrix, it
was observed that the orchid species under study
obeyed almost the same association pattern
obtained in the dendrogram, they were distributed

C
o
o
r
di
n
a
te

2

Coordinate 1

Oliveira, L. do V. R. et al
Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
382
according to the clusters formed (Fig. 2 and 3).
The associations observed in the dendrogram
allowed the identification of 11 distinct groups;
some groups were formed by species that shared
morphological characteristics or occupied the
same geographical area.
In the first group, the species Ctsm. aripuanense,
Ctsm. Arietinum, and Ctsm. Barbatum were
associated with a similarity index of 71% (58%
bootstrap), and within this group, two Ctsm.
barbatum plants (Ctsm. barbatum semicuculatum
and Ctsm. barbatum sp.) were associated with
94% (100% bootstrap) similarity. The two plants
were collected from distinct geographical regions
(Minas Gerais and Amazonas). The ecological
conditions in these two Brazilian states are
distinct: the State of Minas Gerais (MG) presents a
somewhat subtropical hot climate, while the State
of Amazonas (AM) presents a more tropical
climate with little temperature variation
throughout the year and a short dry season with
high humidity during the night (Holst, 1999). The
climatic differences between both the collection
sites and their high genetic similarity value (94%)
suggested that Ctsm. barbatum semicuculatum and
Ctsm. barbatum could have split not long ago in
evolutionary terms, or that these differences were
due to variation in the habitats only.
The species Ctsm. arietinum is very common in its
region of origin living under palm trees.
Characteristically, its pseudobulbs are arched
downward and leaves are longer than usual for the
genus. These characteristics are kept constant
during cultivation (Holst, 1999). Based on the
quantity of cilia on the labellum, Ctsm. arietunum
immediately evokes the species Catasetum
barbatum, justifying the association herein
observed. Differently from this group, however,
the flowers are arranged strongly clustered at the
distal section in relation to the base. The shape of
the labellum is also completely different from
other species in the group cited above, as well as
the color of its flowers. Another characteristic is
that the dispersion area of Ctsm. arietinum is
specifically located in the Brazilian Northeastern
region (PE), where other species of this
previously-mentioned complex cannot be found.
In this genus, the only other species confirmed in
the region was Ctsm. discolor Lindl. (ABRACC,
1995), but it was not associated with this group,
which showed a CS of 54% with Ctsm. arietinum.
The species Ctsm. arietinum is described in the
literature as belonging to the Ctsm. cristatum
complex (Holst, 1999). However, in this study,
Ctsm. cristatum appeared associated with another
group, presenting a genetic similarity of only 60%
with Ctsm. arietinum. In spite of the fact that they
have ciliated labella, which was a distinctive trait,
these species were not linked in the Dendrogram.
This could have happened due to the fact that both
species have distinct geographical distributions,
with Ctsm. arietinum distributed mainly in the
State of Pernambuco, while Ctsm. cristatum is
distributed across the States of Roraima, Amapá,
and Pará. However, although these species have
different concentration areas, they share the same
ecological climate distribution, consisting of a hot,
humid, and tropical climate which remains
unchanged almost the entire year, with high
moisture during the night, even during dry periods,
which are quite short (Holst, 1999).
In the first group, there was also Ctsm.
aripuanense, which was associated with species of
Ctsm. barbatum and Ctsm. arietinum, with a
genetic coefficient of similarity of 72%. These
three species present some similarities, and are
found in the States of MT and MS (with a wider
distribution for Ctsm. barbatum). They show
medium flowering in summer, and their main trait
is the presence of numerous cilia on the labellum
(Raposo, 1992; Holst, 1999). The second group
was associated with a mean similarity of 69%
(44% Bootstrap) to the species: Ctsm. atratum,
Ctsm. cernnum, Ctsm. confusum, Ctsm. hookeri,
Ctsm. triodon, and Ctsm. uncatum. In this group,
the intraspecific genetic similarity was high (93%),
with 100% bootstrap between Ctsm. atratum
plants. The species Ctsm. atratum can be found in
the states of SP, PR, SC, RS, and MG (Raposo,
1992); it occurs in mountainous regions under
common subtropical climate ecological conditions
(Holst, 1999), which may justify their high genetic
similarity value.
Ctsm. cernnum and Ctsm. confusum were
associated with 78% similarity (bootstrap=70).
Although these two species are not
morphologically alike, clustering may have
occurred because both Ctsm. cernuum and
confusum present concave petals and sepals with
dark red spots, medium flowering in summer, and
an epiphytic growth habit (Holst, 1999). The
species Ctsm. triodon and Ctsm. uncatum were
associated with 82% similarity (bootstrap=82).
However, these species present morphological
differences and cannot be found in the same
geographical region. Ctsm. triodon is found in the

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383
States of SC, PR, and RS, while Ctsm. uncatum
occurs in the States of PE, AL, CE, and BA.
A 3rd group to become associated involved Ctsm.
ariquimense, Ctsm. lemosii, and Ctsm.
matogrossense, with a mean similarity of 63%. In
general, the species in this group have few
morphological similarities, in agreement with the
RAPD data. In addition, these species are
generally found in distinct geographical regions,
except Ctsm matogrossense and lemosii, which
can be found in MT, submitted in this case to the
same ecological conditions, thus justifying the
higher similarity between these species (CS=
67%). Catasetum ariquimense could be linked to
Ctsm. barbatum due to the presence of fimbriae;
representatives of the barbatum complex, however,
do not occur in the same geographical region as
Ctsm. ariquimense (ABRACC, 1996). These two
species obtained a coefficient of similarity of 54%,
reinforcing the responses obtained in this
experiment.
The fourth group to become linked, with a mean
similarity of 93%, was the group containing Ctsm.
fimbriatum species (bootstrap=92). Ctsm.
fimbriatum with dark red flowers was a little more
isolated than the others. Because this color is not
very common in this species, it is suggested that it
could be a mutation; therefore, it could be a new
variety.
The species Ctsm. galeritum, Ctsm. spitzii, and
Ctsm. vinaceum were associated in the fifth group,
with a mean similarity of 70%. These three species
can be found in the State of GO; however, Ctsm.
galeritum and Ctsm. vinaceum have a wider
geographical distribution. Catasetum pulchrum
shows few morphological similarities with the
other two species in the group, having
differentiated flowers, particularly due to brown
stripes disposed across petals and sepals on a light
background (Holst, 1999). Catasetum pulchrum
was associated with species in this group with a
mean similarity of 61%.
Although associated with the species Ctsm. spitzii
and Ctsm vinaceum, Ctsm. galeritum did not
present common morphological characteristics
either. However, this species can bloom in the
same season as Ctsm. spitzii and can also be found
under the same climatic and ecological conditions
of occurrence of the species Ctsm. spitzii and
Ctsm. vinaceum. These climatic and ecological
conditions are represented by an essentially
continental climate, where drought periods are
long and moisture is very low through almost the
whole year, and days are hot and dry, while nights
are reasonably cold (Holst, 1999).
The association demonstrated in the dendrogram
(mean similarity of 70%) between the species
Ctsm. spitzii and Ctsm. vinaceum confirmed what
had been previously described in the literature.
According to Holst (1999) and ABRACC (1996),
greater affinity was found between the species
Ctsm. spitzii and the species Ctsm. vinaceum and
Ctsm. trulla Lindl. (not used in this experiment).
However, Ctsm. spitzii has a more convex than
sack-shaped labellum on its male flowers, having
only a light depression below the middle, and long
fleshy bristles on the margins of the lower half.
According to Holst (1999), both species are
morphologically similar at the vegetative stage,
and discrimination can only be made after
flowering.
The 6th group clustered Ctsm cristatum, Ctsm.
shimidtianum, Ctsm. discolor, Ctsm. gnomus, and
Ctsm. moreanum species with a mean similarity of
67%. These data are in agreement with the small
morphological similarity among these species.
According to Miranda and Lacerda (1992), while
the genus Catasetum presented taxonomic
difficulties, some of its representative species,
including Ctsm. cristatum Lindl., were even more
complicated due to the apparent similarity of their
flowers, both male and female. A knowledge about
their geographical distribution has been a
facilitating factor in understanding the group, and
at present, many species can be clearly defined.
Catasetum cristatum is the northernmost of these
species in Brazil, occurring from Venezuela to the
States of Roraima, Amapá, and Pará. Ctsm
barbatum (Lindl.) Lindl. presents the largest
dispersion area, along the Solimões, Negro, and
Amazonas Rivers and their tributaries in a region
with elevations lower than 300 m, until the coastal
region with higher-moisture microclimates of the
Brazilian Northeast. Species related to this genus
are found in the States of Minas Gerais and São
Paulo; however, these are still under study, since
descriptions already made for plants in this region
present many controversies. Some species and
varieties described in the 19th century had their
types destroyed and were described in little detail,
with not very enlightening drawings, and vague
references to the site of origin, which prevented
them from being validated and making it difficult
to correlate what had been published with the
present knowledge (ABRACC, 1998). From this
work, it could be, therefore, concluded that the

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384
species in the Ctsm. cristatum complex were
distinct species, since they clustered separately.
The 7th group clustered the species Ctsm.
macrocarpum, Ctsm. osculatum, and Ctsm.
saccatum. The intraspecific level of variation for
Ctsm. macrocarpum was 93% (100% bootstrap),
and 84% for Ctsm. osculatum. The mean genetic
similarity among the three species was 71%.
However, the species Ctsm macrocarpum was
morphologically different from the species Ctsm.
saccatum and Ctsm. osculatum, which were
morphologically more similar. Two of the
analyzed Ctsm. macrocarpum plants were
collected in the States of PA and BA; the
geographical distribution of this species is very
wide, comprising the States of RJ, BA, PB, PE,
RN, PI, PA, AM, TO, MT, MA, RR, and ES. The
ecological conditions in these states are more or
less similar, with the climate practically identical
throughout the year, with temperature ranging
from 30oC to 15oC (Holst, 1999). Because they
were found in nearby regions and under the same
ecological condition, the high CS value found
(93%) was justified.
Within the same group, the species Ctsm.
saccatum and Ctsm. osculatum were clustered with
69% genetic similarity. Although this similarity
was not very high, these species had a lot in
common, but were also different in many points,
justifying the CS value found in this experiment.
According to ABRACC (1996), the morphological
differences between Ctsm. osculatum and Ctsm.
saccatum are as follows:
-Ctsm. osculatum presents a flower stalk that
begins erect but becomes arched under the weight
of the flowers, while Ctsm. saccatum presents a
pendant flower stalk;
-The male and female flower pedicels in Ctsm.
osculatum are up to 3.5 cm in length, while in
Ctsm. saccatum they have a length of up to 6.2
cm;
-Male flowers in Ctsm. osculatum are smaller and
usually yellowish-green or greenish-brown in
color, with small spots when present, while Ctsm.
saccatum has larger, dark, brown or reddish-brown
flowers, always presenting evident, larger spots;
-The floral elements proportions in both species
are significantly different: Ctsm. saccatum has
long and narrow sepals and petals, while in Ctsm.
osculatum they are short and broad;
-The labellum in Ctsm. osculatum is subcordate
(heart-shaped) while in Ctsm. saccatum it is
trilobate. The labellum in Ctsm. osculatum is
broader than long;
-Female flowers in Ctsm. osculatum are much
smaller than in Ctsm. saccatum;
The 8th group associated the species Ctsm.
gladiatorum and Ctsm. juruenense with 65%
similarity; these apparently did not have much in
common beyond the absence of female flowers
and the presence of cilia on the labellum, which
were, however, much smaller in size in Ctsm.
juruenense (Raposo, 1992; ABRACC, 1996;
Lacerda, 1998 a, b; Holst, 1999).
Catasetum gladiatorium is distributed across a
wide area in the Brazilian Central Plateau, in the
States of Mato Grosso, Goiás, and Tocantins, and
is epiphytic in palm trees that occur in regions
under artificial pastures and cerrados, at elevations
between 230 and 790m. The species emits two to
three consecutive flower stalks per year and
blooms in the summer and fall. The species Ctsm.
spitzii, Ctsm. fimbriatum, Ctsm. rooseveltianum,
Ctsm. galeritum, Ctsm. fuchsii, Ctsm. ornithoides,
Ctsm. osculatum, Ctsm. schmitdianum, and Ctsm.
vinaceum also occur in the region (Lacerda, 1998
a, b). Catasetum gladiatorum showed a 63%
coefficient of similarity with Ctsm. cristatum, and
has been considered a member of the cristatum
complex in the literature, but has also been
previously classified as Ctsm barbatum, with
which it obtained 60% similarity (Table 4).
The 9th group included the species Ctsm.
parguazense, Ctsm. pileatum, Ctsm. purum and
Ctsm. planiceps. The greatest genetic proximity
was verified between the species Ctsm. purum and
Ctsm planiceps, with a 72% similarity association.
These species presented marked morphological
differences and a very distinct geographical
distribution. While Ctsm. planiceps can be found
in the State of AM (north of Brazil), Venezuela,
the Guianas, Suriname, and Peru, Ctsm. purum is
found in the States of BA, ES, and MG.
Although Ctsm. purum and Ctsm uncatum were
not clustered in the same group in the dendrogram
(Genetic coefficient of similarity=55%), they have
traits in common that could differ in some points.
According to Lacerda (1997) these differences are
as follows:
-both have roots, rhizomes, pseudobulbs, and
leaves typical of the genus, but Ctsm. purum
attains a larger size;
-male flowers are green in both, but a little smaller
in Ctsm. uncatum;

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385
-both species occur in Bahia, with Ctsm Purum
extending until Minas Gerais and Espírito Santo,
and Ctsm. uncatum extending until Pernambuco.
The species Ctsm. pileatum and Ctsm parguazense
are considered unique, and do not show many
similarities, especially with regard to their flowers
in relation to other species in the genus. The
species Ctsm. complanatum was displayed
individually between groups 9 and 10. This
isolation could be due to a not very significant
morphological similarity with the other species.
Ctsm. complanatum was a species related to Ctsm.
cirrhaeoides, which was not used in this
experiment. The only differences between them
are: the presence of an arched, non-pendulous
flower stalk, flowers concentrated at the distal end
of the inflorescence (whereas in the other orchid
the flowers are located very close to the
pseudobulb), a more sharply-pointed and flat
conical labellum, and a quite distinct geographical
distribution in both species (ABRACC, 1999).
Ctsm. denticulatum is a species that occupies the
same geographical and climatic region as Ctsm.
complanatum; these species obtained a similarity
matrix value of 51% between themselves, but were
not associated in the dendrogram.
The 10th group showing associations contained the
species Ctsm. ciliatum, Ctsm. gardineri, and Ctsm.
sanguineum. Ctsm. ciliatum and Ctsm. gardineri
were associated with 59% similarity. Ctsm.
sanguineum was a little more distant from the
group, with 56% similarity (Table 4).
Ctsm. ciliatum is considered a synonym of Ctsm.
roseo-album, which is treated as a variety of Ctsm.
discolor. Ctsm. gardineri is also considered a
synonym of Ctsm. discolor, with only a few
differences, such as smaller and more yellow
flowers in the species Ctsm. gardineri than in
Ctsm. discolor; the Ctsm. gardineri plant as a
whole is more robust (Holst, 1999).
The association between Ctsm. discolor and Ctsm.
gardineri and Ctsm. ciliatum was not shown in the
dendrogram; however, the three species were
associated in the matrix with 69% similarity; also,
Ctsm. discolor and Ctsm. gardineri obtained 66%
similarity, while Ctsm. discolor and Ctsm. ciliatum
obtained 65% similarity. This association showed
that the species Ctsm. gardineri and Ctsm. ciliatum
were more similar to Ctsm. discolor than between
themselves.
In the dendrogram, the species Ctsm. laminatum
and Ctsm denticulatum were isolated from other
species in the same genus. The isolation observed
for Ctsm. laminatum could be due to its
geographical distribution; it occurs in Mexico, in
the States of Michoacan, Guerrero and Oaxaca,
and cannot be found in Brazil as the others (Holst,
1999).
Ctsm denticulatum, together with Ctsm. pulchrum
N.E. Brown and Ctsm. cirrhaeoides Hoehne., form
a group with pendant and somewhat dense
inflorescences. The denticulate margins of the
labellum are the most important aspect in
discriminating Ctsm. denticulatum from the other
two, which have smooth margins (Lacerda, 2001).
The species Ctsm. pulchrum and Ctsm
denticulatum were not in the same group and
obtained a low similarity index of 47%. This low
index could be explained because these species
came from distinct geographical regions (States of
AM, PA, and MT for Ctsm. pulchrum, and RO for
Ctsm. denticulatum), and also because they were
under different ecological and climatic conditions.


CONCLUSIONS

These results demonstrated the great genetic
diversity of the species analyzed. The high
polymorphism index obtained showed that RAPD
markers could also aid to determine the relations
in the genus Catasetum, in addition to being
effective in identifying the polymorphism itself.
The information generated based on these data
could contribute to define the strategies for the
establishment of germplasm collections, as well as
to elucidate the problems related to the
classification of species. It is still important to
emphasize that additional studies with a bigger
number of plants and other species would be
important and necessary for a better definition of
strategies.


ACKNOWLEDGEMENTS

We are grateful to Conselho Nacional de
Desenvolvimento Científico e Tecnológico
(CNPq) and to Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES) for
financial support.

Oliveira, L. do V. R. et al
Braz. Arch. Biol. Technol. v.53 n.2: pp. 375-387, Mar/Apr 2010
386
RESUMO

Neste trabalho, marcadores moleculares de RAPD
foram utilizados para acessar a variabilidade
genética e estudar as relações interespecíficas e
intraespecífica em um grupo de 37 espécies,
compreendendo 56 plantas individuais. Um total
de 15 primers foram selecionados para
amplificação do DNA. De um total de 221 bandas
analisadas, 209 (95%) foram polimórficas. O nível
de similaridade genética interespecífica variou de
37% entre Catasetum complanatum e Catasetum
laminatums a 83% entre Catasetum triodon e
Catasetum uncatum. A similaridade genética
intraespecífica variou de 88% entre os indivíduos
de Catasetum triodon a 93% entre os indivíduos
de Catasetum atratum e Catasetum macrocarpum.
Os resultados deste trabalho contribuem para o
entendimento das relações interespecíficas no
gênero Catasetum, para definir estratégias para o
estabelecimento de um banco de germoplasma e
para dar suporte a programas de melhoramento.


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Received: May 17, 2007;
Revised: February 26, 2008;
Accepted: May 05, 2009.