Biochemical Systematics and Ecology 53 (2014) 816

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

Cross transferability of SSR markers to endangered Cedrela

species that grow in Argentinean subtropical forests, as a
valuable tool for population genetic studies
M. Cristina Soldati a, *, M. Virginia Inza a, Luis Fornes b, Noga Zelener a
a

Instituto de Recursos Biolgicos, INTA Castelar-CIRN-CNIA, De los Reseros y N. Repetto (ex Las Cabaas) s.n, Hurlingham 1686,
Buenos Aires, Argentina
b
INTA-EEA Famaill, Ruta 301 km 32 (4132), Famaill, Tucumn, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 May 2013
Accepted 14 December 2013
Available online 9 January 2014

Species of Cedrela with a high economic value from Northwest and Northeastern Argentina
are severely exploited. This work evaluates whether 51 nuclear SSRs, developed to study
phylogenetically close species in the Meliaceae family (Cedrela odorata, Cedrela ssilis,
Swietenia humilis and Swietenia macrophylla), can be used to study C. ssilis, Cedrela balansae, Cedrela saltensis and Cedrela angustifolia. A 62.8% of the total of 194 SSRs/species
combinations showed a successful, homologous and cross-species amplication. As expected, a great success in SSRs transferability among Cedrela species was observed.
Twenty-one screened SSRs showed a successful amplication pattern in all target species
and many of them were polymorphic (9, 13, 13 and 7 SSRs for C. ssilis, C. balansae, C.
saltensis and C. angustifolia, respectively). The high number of evaluated SSRs from the
Cedrela genus and Meliaceae family, allowed us to obtain a suitable set of validated
markers that are highly variable and easily scored, and also identify those which were less
sturdy. We were able to retain a useful set of markers for three of the target species, but
not for C. angustifolia. This could be due to its greater phylogenetic and morphological
distances to the other three species. The lack of SSRs developed for our target species,
transforms the transferred SSRs reported here in a valuable tool to monitor the genetic
consequences of forest overexploitation on Cedrela species.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Cedrela
Meliaceae
Microsatellite (SSR)
Transferability
Cross-species

1. Introduction
Cedrela genus (Meliaceae) is distributed throughout Latin America, from Mexico (24
N) to Argentina (27
S) (Styles, 1981)
and includes tropical and subtropical highly valuable trees that are currently threatened by overexploitation, habitat loss and
genetic erosion (Cavers et al., 2004; Kageyama et al., 2004; Muellner et al., 2009; Inza et al., 2012; Soldati et al., 2013).
Pennington and Muellner (2010) have recognized 17 species of Cedrela, ve of which were reported by Zapater et al. (2004) in
Argentina. Cedrela balansae C. DC., Cedrela saltensis Zapater & del Castillo and Cedrela angustifolia C. DC. (Cedrela lilloi,
Pennington and Muellner, 2010) are montane species of the Northwestern Yungas Rainforest which grow at different altitudinal levels; Cedrela ssilis Vell. and Cedrela odorata L. are lowland species of the Northeastern Paranaense Rainforest, the
latter species with a small and very restricted distribution (Brown et al., 2002; Zapater et al., 2004; Grau et al., 2006; Malizia

* Corresponding author. Tel.: 54 11 4621 1819/0840; fax: 54 11 4621 6903.

E-mail addresses: mcsoldati@yahoo.com.ar (M.C. Soldati), vinza@cnia.inta.gov.ar (M.V. Inza), lfornes@correo.inta.gov.ar (L. Fornes), nzelener@cnia.inta.
gov.ar (N. Zelener).
0305-1978/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bse.2013.12.003

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

et al., 2006). Population reductions of cedar species have been aggravated by low density, aggregated distribution and poor
regeneration properties of the genus (Patio Varela, 1997; Mostacedo and Fredericksen, 1999; Grau et al., 2003; Zamora Petri,
2006) as well as a regional context of unsustainable management and illegal trade (Grau and Brown, 2000; Brown and
Pacheco, 2006). C. ssilis and C. angustifolia are categorized as endangered while C. odorata is categorized as vulnerable
by the IUCN Red List (International Union for Conservation of Nature, 2012). Additionally, these three species are included in
appendix III of CITES (Convention of International Trade in Endangered Species of Wild Fauna and Flora, 2013).
Molecular characterization of genetic variability is a key tool in breeding and conservation strategies for forest resources
(Newton et al., 1999; Cavers et al., 2004; Gallo et al., 2009). Microsatellites or Simple Sequence Repeats (SSRs, Tautz, 1989) are
increasingly becoming the marker of choice in many plant species (Morgante and Olivieri, 1993) due to their abundance,
simplicity of detection, high degree of polymorphism and codominant inheritance (Caixeta et al., 2006; Oliveira et al., 2006).
They have been widely used in population genetics studies of endangered native forest species to interpret reproductive
systems (White et al., 2002; Kageyama et al., 2003; Hernndez, 2008) and to estimate gene ow (Lowe et al., 2003), genetic
diversity and population genetic structure (Novick et al., 2003; Peakall et al., 2003). SSRs have been developed for Swietenia
species Meliaceae (White and Powell, 1997a, 1997b; Lemes et al., 2002) and for C. odorata and C. ssilis (Hernndez, 2008;
Hernndez et al., 2008; Gandara, 2009), which are the most important and widely distributed species of the genus in Latin
America (Muellner et al., 2009; Pennington and Muellner, 2010). Lastly, despite the forest importance and threat condition of
C. angustifolia, C. balansae and C. saltensis, no valuable microsatellites are yet available.
Generally, the use of microsatellites has been limited due to the cost and time required to species-specic primers
development (Caixeta et al., 2006; Oliveira et al., 2006; Muchugi et al., 2008). However, it is known that the homology of
microsatellite anking regions among related species allows cross-species amplication (transferability) in the same taxa
(Peakall et al., 1998; Oliveira et al., 2006). For tropical trees, transferability has been observed in several genus (Lemes et al.,
2007; Corbo Guidugli et al., 2009; Nazareno et al., 2009) and in several families (White and Powell, 1997b; Rao et al., 2007;
Lemes et al., 2011). Consequently, SSR markers transferability emerges as a valuable tool for comparative studies among
related taxa species and as a helpful instrument in breeding and conservation programs.
The aim of this work was to examine the transferability of microsatellites developed in Cedrela species (C. odorata and C.
ssilis) and Meliaceae family (Swietenia humilis and Swietenia macrophylla) to C. ssilis, C. balansae, C. saltensis and C.
angustifolia. Our ndings provide information on the rates of cross species/genera transferability of SSRs and the relative
degrees of SSR polymorphism. A set of polymorphic microsatellites showing a successful transfer across species of cedar
would provide useful tools for genetic studies focused on preserving populations in their natural ranges of distribution in
Latin America.
2. Materials and methods
2.1. Plant material and DNA extraction
Young leaves from trees randomly chosen were collected from C. ssilis, C. balansae, C. saltensis and C. angustifolia
throughout the Paranaense Rainforest and the Yungas Rainforest (Jujuy, Salta and Misiones provinces), thus representing two
natural populations of each species (Fig. 1). The population sizes, ranging from 9 to 17 individuals, were dened according to
the relative size of each stand, taking into account a minimum distance of 80100 m between trees to minimize the probability of sampling related individuals (Gillies et al., 1999). Once collected, the leaves were dried in silica gel (Chase and Hills,

Fig. 1. Distribution of plant material: eight natural populations assessed. A1 (C. angustifolia, Barit National Park, Salta), A2 (C. angustifolia, La Ramada, Jujuy), B1
(C. balansae, Ro Seco Forestal Santa Brbara, Salta), B2 (C. balansae, Apolinario Saravia, Salta), S1 (C. saltensis, San Andrs, Salta), S2 (C. saltensis, Calilegua National
Park, Jujuy), F1 (C. ssilis, San Antonio, Misiones), F2 (C. ssilis, Campo Guaran, Misiones).

10

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

1991) and stored at 20
C in individual net bags until processing. Total genomic DNA extraction and DNA quantication were
carried out following the procedures described by Soldati et al. (2013). DNA samples were diluted with ultrapure water to
achieve the required concentrations (1.25 ng/ml or 2.5 ng/ml).
2.2. Primer pairs, PCR amplications and banding patterns
We assessed the transferability of 51 SSRs developed for different Meliaceae species belonging to Cedreloideae subfamily to C. ssilis, C. balansae, C. saltensis and C. angustifolia. Twenty-three from C. odorata (Hernndez et al., 2008;
Hernndez, 2008; Navarro, unpublished data), ten from S. macrophylla (Lemes et al., 2002) and eight from S. humilis
(White and Powell, 1997a, b) were tested in all target species. Additionally, nine primer pairs developed for C. ssilis (Gandara,
2009; Ciampi, unpublished data) were tested in C. balansae, C. saltensis and C. angustifolia, meaning that each SSR primer pair
was used to amplify the DNA for all genotypes except those of its own species.
The success of transferability as well as the clearness of resolution patterns and polymorphism levels was assessed on a
sample of six to eight individuals randomly chosen per target species. Different polymerase chain reaction (PCR) conditions were
tested on each species/primer pair to optimize the transferability. PCR was carried out in a nal volume of 12 ml, containing 2.5
10 ng of DNA, 1 X reaction Buffer (Inbio-Highway), 1.53 mM of MgCl2, 0.2 mM of each dNTP, 0.82 mM of each primer and 0.5
units of TPlus DNA polymerase (Inbio-Highway). PCR thermal proles for SSR markers from C. odorata, C. ssilis and S. macrophylla
were adjusted from the original protocols as follows: 94
C for 2 min followed by 30 cycles at 94
C for 1 min, 5560
C for 1 min,
72
C for 1 min and a nal extension at 72
C for 5 min. PCR amplications for S. humilis SSRs were performed using the
touchdown methodology under the following conditions: 94
C for 3 min followed by a ten-step touchdown decreasing by 1
C
each step (94
C for 30 s, 65
C to 56
C for 30 s and 72
C for 1 min) followed by 25 cycles of 94
C for 30 s, 56
C for 30 s and 72
C for
1 min, with a nal extension of 7 min at 72
C. PCR products were visualized at 6% (w/v) denaturing polyacrylamide gels stained
with silver nitrate (Silver Sequence Promega Biotech, Madison, WI) and sized by comparison to a 10 bp DNA Ladder (Invitrogen).
Results were classied as successful amplication (SA) and no amplication (NA) according to presence or absence of amplied
bands, respectively. Banding patterns were classied into three categories: polymorphic (P), when a number of sharp bands,
compatible with the species ploidy level in the expected size range were obtained; monomorphic (M), when the presence of a
single band of equal molecular weight was observed; and non-specic (NS), when there was an unclear banding pattern.
To assess the potential discrimination power of each polymorphic SSR and to accomplish a nal selection according to
Hardy-Weinberg equilibrium (HWE) validation, two detection systems were used according to the target species. For C.
angustifolia populations, polymorphism analysis was conducted at 6% (w/v) denaturing polyacrylamide gels stained with
silver nitrate (Silver Sequence Promega Biotech, Madison, WI). The molecular weight of the bands was estimated in base pairs
(bp) by comparison with 10 bp DNA Ladder (Invitrogen), using the GEL software (Dubcovsky, unpublished data) which is
based on the reciprocals method (Elder and Southern, 1987). For C. ssilis, C. balansae and C. saltensis populations, amplication products were resolved by capillary electrophoresis and uorescence detection, according to Soldati et al. (2013).
2.3. Data analysis
A total of 194 SSRs/species combinations were assessed estimating the percentages of SA, P, M and NS, both globally and
for each target species. In addition, we estimated the percentage of cross species amplication with an index that summarizes
information for each pair of source-target species (Moreno et al., 2011) considering SA (CSA: Cross Species Amplication) and
P (CSP: Cross Species Polymorphic Amplication) patterns.
Pre-selection of SSRs was performed according to patterns of polymorphism detected (at least two alleles at any frequency). Once the SSR markers were pre-selected, these were evaluated through two populations from each species (32, 26,
23 and 28 individuals for C. ssilis, C. balansae, C. saltensis and C. angustifolia, respectively) to assess their potential
discrimination power and to validate them correctly according to HWE. For each polymorphic locus in each species the
following parameters were estimated: (i) allele size range, (ii) number of alleles per locus or allelic multiplicity (Na), (iii) allelic
richness per population (Ar), (iv) expected heterozygosity (He), (v) observed heterozygosity (Ho), and (vi) polymorphism
information content (PIC). Parameters (iv) and (v) were estimated using GDA 1.1 software (Lewis and Zaykin, 2001) whereas
parameter (vi) was estimated using Cervus 3.0.3 software (Kalinowski et al., 2007). Finally, for a denitive selection of SSR
markers, possible deviations from HWE (being the assumptions an innitely large populations, random mating and absence
of evolutionary forces affecting populations) were assessed. The frequency of null alleles (NA), the inbreeding coefcient (Fis)
and the linkage disequilibrium (LD) were estimated using GENEPOP 4.0 (Rousset, 2008) and GDA 1.1 software. SSRs were
selected according to Soldati et al., (2013) criteria for these parameters.
3. Results
3.1. Amplication success
3.1.1. Global amplication success
One hundred and twenty-two SSRs/species combinations (62.8%) out of the total of 194 presented an SA. Of these combinations, 42 (34.4%), 42 (34.4%) and 38 (31.2%) showed P, M and NS patterns, respectively (Table 1). Furthermore, all

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

11

Table 1
Cross species comparison for 51 SSR loci tested in four Cedrela species. For each species two columns are shown: the left-hand column (N) indicates the
number of individuals tested for transferability analysis and polymorphism levels; the right-hand column (Q) contains the overall form of PCR amplications
(polymorphic (P); monomorphic (M); non-specic (NS); no amplication (NA)).
Source species

C. odorata

C. ssilis

S. macrophylla

S. humilis

Locus

Ced 2
Ced 4a
Ced18
Ced 22
Ced 26b
Ced 27
Ced 34a
Ced 34b
Ced 36
Ced 41
Ced 44
Ced 54
Ced 61a
Ced 61b
Ced 64
Ced 65
Ced 72
Ced 83
Ced83b
Ced 95
Ced 96
Ced 131
Ced 131a
CF 26
CF 34
CF 37
CF 63
CF 64
CF 66 A
CF 66 B
CF 75
CF 78
CF 83
Sm 01
Sm 22
Sm 31
Sm 32
Sm 34
Sm 40
Sm 45
Sm 46
Sm 47
Sm 51
MAC 38
MAC 39
MAC 44
MAC 45
MAC 52
MAC 59
MAC 63
MAC 69

C. ssilis

C. balansae

C. saltensis

C. angustifolia

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8

P
P
P
M
NS
P

NS
NS
P
P
NS

NS
P
P
NS
NS
M

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8

M
M

M
M

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8

P
P
P
M
NS
P

NS
NS
P
P
NS
P

M
NS

NS
P
P
NS
NS
M
P
NS
M
NS

M
P
NS

M
M

M
M

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8

P
P
P
M
NS
P

NS
NS
P
P
NS
P

M
NS

NS
P
P
NS
NS
M
P
NS
M

P
P

P
NS

M
M

M
M

8
8
8
8
8
8
8
6
8
8
8
8
8
8
8
8
8
8
8
8
8
7
6
8
8
8
8
8
8
8
8
8
8
6
8
8
8
6
8
8
8
8
8
6
8
8
8
8
8
8
8

P
P
M
M

M
NS
P
M
P
M
M
M
NS

NS
NS
M
NS
NS
NS

NS
M

M
P
M
M
M

NS

M
P

NS

M
M

polymorphic combinations produced fragments within the expected size range (<100 bp larger or smaller than the original
sequences, according to Arnold et al., 2002 criteria). Additionally, 21 (41.2%) out of 51 Meliaceae SSRs screened showed an SA
pattern in the four target species and four SSRs (Ced2, Ced4a, Ced27 and Ced41) showed a P pattern in all tested Cedrela
species (Table 1).
3.1.2. Amplication success according to target species
As a whole, the four target species showed similar amplication success, although a clear increase in number of markers
showing an M pattern was observed for C. angustifolia. For C. ssilis, 23 out of 41 SSR markers (56.1%) showed an SA pattern: 9
of these displayed a P pattern, 7 an M pattern and 7 an NS pattern. In the case of C. balansae, 33 out of 51 SSR markers (64.7%)
showed an SA pattern: 13 of these markers exhibited a P pattern, whereas 9 exhibited an M pattern and 11 an NS pattern.
Additionally, an SA pattern was observed in 32 out of 51 markers tested (62.7%) for C. Saltensis, 13 of these showing a P pattern,

12

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

9 an M pattern and 10 an NS pattern. Finally for C. angustifolia, 34 out of 51 SSR markers (66.6%) showed an SA pattern: 7 of
these markers exhibited a P pattern, 17 an M pattern and 10 an NS pattern.
3.2. Cross species amplication
The CSA for C. odorata markers in the four target species were high, ranging between 78.3% and 86.9%. This index was
slightly inferior for C. ssilis markers showing values ranging between 70% and 80%. For Swietenia SSRs, the CSA in the four
target species decreased notably, ranging between 30% 40% and 25%37.5% for S. macrophylla and S. humilis, respectively
(Table 2).
All target species were polymorphic at several loci. C. odorata SSRs once again were clearly differentiated, with a high
polymorphism degree. For these markers, the CSP ranged from 21.7% to 39.1%. For C. ssilis markers, lower CSP values were
observed in all target species except for C. saltensis which exhibited a higher value of this index (1040%). For the SSRs
assessed from Swietenia genus, a clear decrease in CSP was observed in all target species. For S. macrophylla SSRs, the CSP in
the target species ranged between 0% and 10%, whereas for S. humilis SSRs no polymorphic amplication was observed (Table
2). Additionally, the CSP indexes across source species SSRs showed in general lower values for C. angustifolia, ranging between 0% and 21.7% (Table 2).
3.3. Discrimination power, validation and selection of SSR markers
For C. ssilis, the nine polymorphic loci allowed us to detect a total of 106 allelic variants. The He and PIC values ranged
from 0.535 to 0.933 and from 0.418 to 0.912 for loci Ced83b and Ced95, respectively, being the highest values among the four
species analyzed (Table 3). Null alleles were found in all nine polymorphic SSRs transferred; however, they only reached
values greater than 0.05 at one locus (Ced4a). None of the loci revealed signicant LD (P > 0.05). Moreover, the Fis did not
reveal signicant deviations from HWE genotypic proportions, except for loci Ced4a and Ced83b (Table 3). Based on these
results, a set of seven SSRs (i.e. Ced2, Ced18, Ced27, Ced41, Ced44, Ced95 and Sm22) is proposed as a tool for genetic studies in
C. ssilis. In the case of C. balansae, the 13 polymorphic loci found revealed a total of 69 allelic variants. The He and PIC values
ranged from 0.117 to 0.836 and from 0.111 to 0.798 for loci Ced27 and Ced44, respectively (Table 3). A higher degree of NA was
found, reaching values greater than 0.05 in ve of thirteen loci. Additionally, one locus showed LD (Ced18) and Fis values
pointing out a deviation of Hardy-Weinberg proportions in three of the thirteen polymorphic loci (Table 3). Despite these
results, we were able to retain a set of eight polymorphic SSRs (i.e. Ced2, Ced27, Ced41, Ced44, Ced61a, Ced95, CF66A and
Sm22) that could be used for genetic studies. For C. saltensis a total of 56 allelic variants were identied through the 13
polymorphic loci. A very wide range was found for the He and PIC values (0.043 to 0.747 and 0.042 to 0.702 for loci CF26 and
Ced95, respectively) as shown in Table 3. Null alleles were found in 12 of 13 loci, reaching values that point out a signicant
deviation from HWE in eight of them. Only one locus showed LD (Ced61a), even though the Fis values revealed signicant
deviations from HWE genotypic proportions in four out of thirteen loci (Table 3). For this species, our results allowed the
selection of ve SSR markers (i.e. Ced2, Ced44, Ced83b, CF26 and CF66A) as a possible tool for genetic analyses. For C.
angustifolia, only 23 allelic variants were revealed by the seven transferred polymorphic loci. The He and PIC values ranged
from 0.259 to 0.637 and from 0.238 to 0.553 for loci Ced4a and Ced27, respectively (Table 3). There were important deviations
from HWE in all loci, either by NA presence or by higher Fis values; however, no LD was found (Table 3). Due to these results, it
was not possible to select a set of suitable markers for genetic studies in C. angustifolia. Finally, it is important to note that only
two SSR markers (Ced2 and Ced44) might be used to analyze three of the studied species (C. ssilis, C. balansae and C. saltensis)
as a whole.
4. Discussion
Successful cross-species transferability of SSRs across taxa is associated to the evolutionary distance existent between
source and target species. The transferability success decreases when the evolutionary distance between species increases
(Steinkellner et al., 1997; Zucchi et al., 2002; Barbar et al., 2007). Our results strongly support that SSRs transferability is

Table 2
Percentage of SSRs cross species transferability between source and target species.
Source species

Target species
C. ssilis
CSA

C. odorata
C. ssilis
S. macrophylla
S. humilis
a
b

78.3%

30%
25%

C. balansae
CSP

34.7%

10%
0%

C. saltensis

C. angustifolia

SA

CSP

SA

CSP

SA

CSP

86.9%
80%
30%
25%

39.1%
30%
10%
0%

86.9%
70%
30%
25%

39.1%
40%
0%
0%

86.9%
70%
40%
37.5%

21.7%
10%
10%
0%

Percentage of cross species amplication. Includes polymorphic, monomorphic and non-specic banding patterns.
Percentage of cross species polymorphic amplication. Includes only polymorphic banding patterns.

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

13

Table 3
Descriptive statistics for polymorphic loci successfully transferred to Cedrela species. In bold letters are highlighted the values for which the markers had to
be discarded.
SSR

C. ssilis

C. balansae

bpoa

Nb Nac Ar (F1F2)d He/Hoe

PICf

Ang

DLh

Fisi

Ced2
Ced4a
Ced18
Ced27
Ced41
Ced44
Ced54
Ced61a
Ced83b
Ced95
CF26
CF66A
CF66B
CF78
Sm22
Sm51

134164
195222
118156
147159
114146
162215

196200
180230

32
32
28
26
32
32

31
30

0.744
0.823
0.835
0.716
0.877
0.911

0.418
0.912

0.047
0.092
0.001
0.041
0.038
0.019

0.008
0.027

0.380
0.160
0.579
0.585
0.374
0.441

0.647
0.316

0.097
0.230
0.053
0.117
0.025
0.035

L0.249
0.072

SSR

C. saltensis

Ced2
Ced4a
Ced18
Ced27
Ced41
Ced44
Ced54
Ced61a
Ced83b
Ced95
CF26
CF66A
CF66B
CF78
Sm22
Sm51
a
b
c
d
e
f
g
h
i

10
10
16
6
12
22

3
21

119133 32 6

710
79
913
46
109
1518

32
1416

56

0.786/0.719
0.856/0.656
0.867/0.917
0.773/0.864
0.902/0.875
0.930/0.903

0.535/0.613
0.933/0.867

bpo

138153
200212
118140
153-157
116128
176188

243261
192200
192204
139154
130142

126138
0.711/0.719 0.651 0.019 0.434 0.002 122124

Na Ar
He/Ho
(B1B2)

PIC

AN

DL

Fis

26
25
25
25
26
26

26
22
26
26
26

26
25

5
4
8
3
5
10

4
4
9
5
5

5
2

0.438
0.178
0.773
0.111
0.702
0.798

0.418
0.392
0.795
0.600
0.360

0.734
0.136

0.008
0.893
0.304
0.001
0.002
0.003

0.000
0.156
0.003
0.249
0.000

0.351
0.000

0.569
0.473
0.025
0.493
0.476
0.324

0.548
0.570
0.383
0.073
0.555

0.343
0.498

0.094
0.048
0.639
0.037
0.119
0.046

-0.023
0.012
0.083
0.384
0.070

0.221
0.112

44
42
74
31
53
87

42
43
86
52
35

53
22

0.488/0.538
0.189/0.200
0.816/0.280
0.117/0.120
0.759/0.808
0.836/0.846

0.465/0.462
0.443/0.455
0.833/0.846
0.648/0.385
0.394/0.423

0.787/0.538
0.150/0.160

C. angustifolia

bpo

Na Ar (S1S2) He/Ho

PIC

AN

DL

Fis

bpo

Na Ar
He/Ho
(A1-A2)

PIC

AN

DL

Fis

139151
202216
118145
153157
116124
176219

243260
192202
195234
145158
130142
201216
126138

23
22
21
21
23
23

23
21
23
23
23
21
23

2
6
5
3
2
8

3
5
7
2
5
4
4

0.175
0.647
0.483
0.272
0.146
0.688

0.392
0.175
0.702
0.042
0.590
0.172
0.195

0.001
0.348
0.173
0.140
0.116
0.000

0.421
0.001
0.116
0.001
0.033
0.896
0.107

0.272
0.208
0.129
0.269
0.302
0.321

0.024
0.304
0.380
0.520
0.139
0.534
0.154

0.077
0.123
0.398
0.515
0.059
0.024

0.713
0.029
0.036
0.008
0.029
0.032
0.377

143149
211219

239259
94116

191197

184-208

122126

28
28

26
28

28

28

27

4
3

4
3

0.448
0.238

0.553
0.436

0.386

0.491

0.535

0.078
0.055

0.213
0.485

0.063

0.000

0.411

0.590
0.391

0.435
0.463

0.556

0.576

0.492

0.048
0.008

0.162
L0.521

-0.025

L0.460

-0.523

22
64
52
22
22
75

32
33
75
21
35
33
32

0.198/0.217
0.714/0.636
0.544/0.333
0.298/0.143
0.162/0.174
0.731/0.696

0.449/0.130
0.184/0.190
0.747/0.696
0.043/0.043
0.669/0.696
0.182/0.190
0.206/0.130

43
32

43
32

33

33

33

0.490/0.500
0.259/0.250

0.637/0.538
0.551/0.821

0.447/0.464

0.538/0.778

0.614/0.926

Observed size range in pair bases.

Number of individuals tested.
Number of alleles or Allelic multiplicity.
Allelic richness (for each population tested).
Expected heterozygosity/Observed heterozygosity.
Polymorphism information content.
Frequency of null alleles.
Linkage disequilibrium.
Inbreeding coefcient.

differentially successful across taxa. Transferability of primers developed in species of Meliaceae family was in general
moderate to high (62.8%), following Gasic et al. (2009) criteria. However, cross-species amplication showed a wide range for
both successful amplication and polymorphic amplication due to different source/target species combinations, with a clear
drop in transfer success from cross-species within genus to cross-genera. Transferability from Swietenia SSRs to Cedrela
species was low for successful amplication, and even lower for polymorphic amplication failing in most of the examined
SSRs. These results are consistent with proposed transferability percentages of amplied (35%) and polymorphic (10%)
markers between genera (Krishna and Puppala, 2004; Barbar et al., 2007). In contrast, Cedrela SSRs were successfully
transferred within the genus with higher rates for both successful amplication and polymorphic amplication patterns. Our
ndings are in complete agreement with studies that have reported that cross-genera transferability within family may be
limited or failed (Echt et al., 1999; Zucchi et al., 2002) and that transfer success improve when this occurs across species
within genus (Steinkellner et al., 1997; Terui et al., 2006; Lemes et al., 2007).
Swietenia and Cedrela genus are classied into the Meliaceae family and the Cedreloideae subfamily; however, they belong
to tribes Swietenieae and Cedreleae, respectively (Muellner et al., 2009; Pennington and Muellner, 2010). Lower conservation
degree of SSR anking regions due to higher phylogenetic and morphological distances (Powell et al., 1996; Collevatti Garcia

14

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

et al., 1999; Peakall et al., 1998) may explain lower transferability of primers between these genera. Previously, White and
Powell (1997b) have examined 11 S. humilis SSRs in several Meliaceae family members and reported that 7 SSRs amplied
in Swietenia species, 5 in C. odorata (same subfamily, different tribe) and only 4 in Melia sp. and Azadirachta sp. (different
subfamily). As expected, a greater success in SSR transferability within Cedrela genus may be explained by higher homology in
anking regions, leading to amplify putatively homologous microsatellite loci across Cedrela species. Additionally, the slightly
greater success in the transferability of primers developed for C. odorata might be due to inherent characteristics of those
primers.
Transferability variation between taxa is even more evident if this is assessed by focusing only on polymorphic amplication (a key factor to decide the nal use of SSR markers). Usually, when SSR markers are transferred the polymorphism may
decrease signicantly (Chagn et al., 2004; Barbar et al., 2007; Mnejja et al., 2010). The amount of transferred markers
which were polymorphic within genus was greater and similar between C. ssilis, C. balansae and C. saltensis, with moderate
polymorphic amplication rates ranging from 30 to 40%. Although polymorphism generally depends on genetic distance,
other factors such as mutation rate could make this relationship more confusing (Kuleung et al., 2004) and might explain
these similar results. A clear decrease in the percentage of polymorphic loci was particularly observed for Cedrela SSRs in C.
angustifolia, which had only 10% of the C. ssilis SSRs and 21.7% of the C. odorata SSRs showing a polymorphic pattern. These
values are in accordance with the lowest number of total alleles and the greatest number of monomorphic loci also detected
in this species. It is known that the level of microsatellite polymorphism is not directly transferable (Oliveira et al., 2006).
Thus, our results conrm that polymorphic SSRs from one species might display monomorphism in other related taxa. Lower
polymorphism in C. angustifolia strongly support the recent phylogenetic and morphological studies of Cedrela genus, that
proposed that C. angustifolia have the most distant relationship to the other Cedrela species assessed in this work (Muellner
et al., 2009; Pennington and Muellner, 2010). Furthermore, lower variability in C. angustifolia may be also attributable to an
overall lower level of genetic diversity of C. angustifolia populations in Argentina (Inza et al., 2012). A loss of variability in
transferred microsatellites may be also associated to a higher frequency of null alleles occurrence in more distantly related
species (Mnejja et al., 2010; Roa et al., 2000; Nazareno et al., 2009). Then, lower heterozygosity levels and higher frequencies
of null alleles in C. angustifolia and C. saltensis might be due to the fact that these species are more distant from the group
composed of C. ssilis, C. balansae and C. odorata as it has been described by Pennington and Muellner (2010).
Transferability-approach is an important pathway to improve the cost-effectiveness of SSRs, using them in species for
which have not yet been developed (Pandian et al., 2000; Arnold et al., 2002), as in several species of Cedrela genus. The
usefulness of the Cedrela primers to generate microsatellite markers across the genus was conrmed. Success of transferability was different according to the species, with a polymorphic SSRs set of potential use of 8, 7 and 5 for C. balansae, C.
ssilis and C. saltensis, respectively, and none for C. angustifolia. Particularly for C. balansae, most of these primers have been
successfully used in studies of population genetic variability (Soldati et al., 2013). Further studies are necessary to increase the
number of SSRs available to compare these related species and also to develop new SSRs for C. angustifolia. The number of
transferred SSRs might likely improve by developing markers from expressed sequences since these would be located in more
conserved genomic regions (Kuleung et al., 2004; Liewlaksaneeyanawin et al., 2004). To conclude, the potentially useful
transferred microsatellites reported here are now valuable tools for providing data on population genetic variability of these
endanger Cedrela species, to understand genetic erosion processes by overexploitation and to guide appropriate management
and conservation plans.
Acknowledgments
This research was nanced by INTA, under the framework of PNFOR 44331 project, and by Bioversity International/INTA/
CIAT, under the framework of the letter of agreement N
20049/2009-2011.
We would like to thank the technical teams of the EEA INTA Famaill, EEA Yuto, AER Tartagal and EEA Montecarlo, who
participated in plant material collection. We would also like to thank Alicia Cammarasana for her helpful comments regarding
language usage.
References
Arnold, C., Rossetto, M., McNally, J., Henry, R.J., 2002. The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae. Am. J.
Bot. 83 (1), 2228.
Barbar, T., Palma-Silva, C., Paggi, G.M., Bered, F., Fay, M.F., Lexer, C., 2007. Cross-species transfer of nuclear microsatellite markers: potential and limitations.
Mol. Ecol. 16, 37593767.
Brown, A.D., Grau, S.A., Lomscolo, T., Gasparri, N.I., 2002. Una estrategia de conservacin para las selvas subtropicales de montaa (Yungas) de Argentina.
Ecotrpicos 15, 147159.
Brown, A.D., Pacheco, S., 2006. Importancia del gnero Cedrela en la conservacin y desarrollo sustentable de las Yungas australes. In: Pacheco, S., Brown, A.
(Eds.), Ecologa y Produccin de cedro (gnero Cedrela) en las Yungas australes. LIEY-ProYungas, Tucumn, Argentina, pp. 918.
Caixeta, E.T., de Olivera, A.C.B., de Brito, G.G., Sakiyama, N.S., 2006. Tipos de marcadores moleculares. In: Borm, A., Caixeta, E.T. (Eds.), Marcadores
Moleculares. UFV, Brasil, Vicosa, pp. 978.
Cavers, S., Navarro, C., Lowe, A.J., 2004. Targeting genetic resource conservation in widespread species: a case of Cedrela odorata L. For. Ecol. Manage. 197,
285294.
Chagn, D., Chaumeil, P., Ramboer, A., Collada, C., Guevara, A., Cervera, M.T., Vendramin, G.G., Garcia, V., Frigerio, J.M., Echt, C., Richardson, T., Plomion, C.,
2004. Cross-species transferability and mapping of genomic and cDNA SSRs in pines. Theor. Appl. Genet. 109, 12041214.
Chase, M., Hills, H., 1991. Silica gel: an ideal material for eld preservation of leaf samples for DNA studies. Taxn 40 (2), 215220.

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

15

CITES, 2013. Convention of International Trade in Endangered Species of Wild Fauna and Flora. http://www.cites.org/esp/resources/species.html. Accessed
April 29 2013UNEP-WCMC. UNEP-WCMC Species Database: CITES-Listed Species (May 03, 2013).
Collevatti Garcia, R., Brondani, R.V., Grattapaglia, D., 1999. Development and characterization of microsatellite markers for genetic analysis of a Brazilian
endangered tree species Caryocar brasiliense. Heredity 83, 748756.
Corbo Guidugli, M., de Campos, T., Barbosa de Sousa, A.C., Feres, J.M., Sebbenn, A.M., Mestriner, M.A., Betioli Contel, E.P., Alzate-Marin, A.L., 2009. Development and characterization of 15 microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical trees
species. Conserv. Genet. 10, 10011004.
Echt, C.S., Vendramin, G.G., Nelson, C.D., Marquardt, P., 1999. Microsatellite DNA as shared genetic markers among conifer species. Can. J. For. Res. 29, 365371.
Elder, J.K., Southern, E.M., 1987. Computer-aided Analysis of One Dimensional Restriction Fragment Gels. Restriction Fragment Gels, pp. 165172. Chapt 7.
Gallo, L.A., Marchelli, P., Chauchard, L., Gonzalez Pealba, M.G., 2009. Knowing and doing: research leading to action in the conservation of forest genetic
diversity of Patagonian temperate forests. Conserv Biol. 23 (4), 895898.
Gandara, F.B., 2009. Diversidade gentica de populaes de Cedro (Cedrela ssilis Vell. Meliaceae) no Centro-Sul do Brasil. PhD. thesis. Escuela Superior de
Agricultura, Universidad de San Pablo, Brasil.
Gasic, K., Han, Y., Kertbundit, S., Shulaev, V., Iezzoni, A.F., Stover, E.W., Bell, R.L., Wisniewski, M.E., Korban, S.S., 2009. Characteristics and transferability of
new apple EST-derived SSRs to other Rosaceae species. Mol. Breed 23, 397411.
Gillies, A., Navarro, C., Lowe, A., Newton, A., Hernndez, M., Wilson, J., Cornelius, J.P., 1999. Genetic diversity in Mesoamerican populations of mahogany
(Swietenia macrophylla), assessed using RAPDs. Heredity 83, 722732.
Grau, A., Brown, A.D., 2000. Development threats to biodiversity and opportunities for conservation in the mountain ranges of the upper Bermejo River
Basin, NW Argentina and SW Bolivia. Ambio 29 (7), 445450.
Grau, H.R., Easdale, T.A., Paolini, L., 2003. Subtropical dendroecology dates disturbances and forest dynamics in northwestern Argentina montane ecosystems. For. Ecol. Manage. 177, 131143.
Grau, A., Zapater, M.A., Neumann, R., 2006. Botnica y distribucin del gnero Cedrela en el noroeste de Argentina. In: Pacheco, S., Brown, A. (Eds.), Ecologa
y Produccin de cedro (gnero Cedrela) en las Yungas australes. LIEY-ProYungas, Tucumn, Argentina, pp. 1930.
Hernndez, L.G., 2008. Genetic Diversity and Mating System Analysis of Cedrela Odorata L. (Meliaceae) Populations under Different Human Dominated
Landscapes and Primary Forests. Magister Scientiae thesis. Manejo y Conservacin de Bosques Naturales y Biodiversidad, Escuela de Posgrado, Programa de Educacin para el Desarrollo y la Conservacin del Centro Agronmico Tropical de Investigacin y Enseanza, Costa Rica.
Hernndez, L.G., Buonamici, A., Walker, K., Vendramin, G.G., Navarro, C., Cavers, S., 2008. Isolation and characterization of microsatellite markers for Cedrela
odorata L. (Meliaceae), a high value neotropical tree. Conserv. Genet. 9 (2), 457459.
Inza, M.V., Zelener, N., Forns, L., Gallo, L., 2012. Effect of latitudinal gradient and impact of logging on genetic diversity of Cedrela lilloi along the Argentine
Yungas rainforest. Ecol. Evol. 2 (11), 27222736.
IUCN, 2012. IUCN Red List of Threatened Species. Version 2012.2. http://www.iucnredlist.org (May 03, 2013).
Kageyama, P.Y., Sebbenn, A.M., Ribas, L.A., Gandara, F.B., Castellen, M., Perecim, M.B., Vencovsky, R., 2003. Diversidade gentica em espcies arbreas
tropicais de diferentes estgios sucessionais por marcadores genticos. Sci. Forum 64, 93107.
Kageyama, P., Caron, D., Gandara, F., Dagoberto do Santos, J., 2004. Conservation of Mata Atlntica forest fragments in the State of So Paulo, Brazil. In:
Vinceti, B., Amaral, W., Meilleur, B. (Eds.), Challenges in Managing Forest Genetic Resources for Livelihoods: Examples from Argentina and Brazil. International Plant Genetic Resources Institute, Rome, Italy, pp. 167185.
Kalinowski, S.T., Taper, M.L., Marshall, T.C., 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol. Ecol. 16, 10991106. http://dx.doi.org/10.1111/j.1365-294x.2007.03089.x.
Krishna, G., Puppala, N., 2004. Cross-taxa transferability of sequence tagged microsatellite site (STMS) primers from Pulses to Peanut (Arachis hypogaea L.).
In: Fourth International Crop Science Congress. URL. http://www.cropscience.org.au/icsc2004.
Kuleung, C., Baenziger, P.S., Dweikat, I., 2004. Transferability of SSR markers among wheat, rye, and triticale. Theor. Appl. Genet. 108, 11471150.
Lemes, M.R., Brondani, R.P., Grattapaglia, D., 2002. Multiplexed systems of microsatellite markers for genetic analysis of mahogany, Swietenia macrophylla
king (Meliaceae), a threatened neotropical timber species. J. Hered. 93 (4), 287291.
Lemes, M., Martiniano, T., Reis, V., Faria, C., Gribel, R., 2007. Crossamplication and characterization of microsatellite loci for three species of Theobroma
(Sterculiaceae) from the Brazilian Amazon. Genet. Resour. Crop. Evol. 54, 16531657.
Lemes, M.R., Esashika, T., Gaoue, O.G., 2011. Microsatellites for Mahoganies: twelve new loci for Swietenia macrophylla and its high transferability to Khaya
senegalensis. Am. J. Bot. 98 (8), e207e209.
Lewis, P.O., Zaykin, D., 2001. GDA 1.1, Genetic Data Analysis: Computer Program for the Analysis of Allelic Data. Available at: http://hydrodictyon.eeb.uconn.
edu/people/plewis/software (May 03, 2013).
Liewlaksaneeyanawin, C., Ritland, C.E., El-Kassaby, Y.A., Ritland, K., 2004. Single-copy, species-transferable microsatellite markers developed from loblolly
pine ESTs. Theor. Appl. Genet. 109, 361369.
Lowe, A.J., Jourde, B., Breyne, P., Colpaert, N., Navarro, C., Wilson, J., Cavers, S., 2003. Fine-scale genetic structure and gene ow within Costa Rican populations of mahogany (Swietenia macrophylla). Heredity 90, 268275.
Malizia, L., Blundo, C., Pacheco, S., 2006. Diversidad, estructura y distribucin de bosques con cedro en el noroeste de Argentina y sur de Bolivia. In:
Pacheco, S., Brown, A. (Eds.), Ecologa y Produccin de cedro (gnero Cedrela) en las Yungas australes. LIEY-ProYungas, Tucumn, Argentina, pp. 83104.
Mnejja, M., Garcia-Mas, J., Audergon, J., Ars, P., 2010. Prunus microsatellite marker transferability across rosaceous crops. Tree Genet. Genom. 6, 689700.
Moreno, A.C., Marchelli, P., Vendramin, G.G., Gallo, L.A., 2011. Cross transferability of SSRs to ve species of Araucariaceae: a useful tool for population
genetic studies in Araucaria araucana. Forest Syst. 20 (2), 303314.
Morgante, M., Olivieri, A.M., 1993. PCR-amplied microsatellites as markers in plant genetics. Plant J. 3 (1), 175182.
Mostacedo, B.C., Fredericksen, T.S., 1999. Regeneration status of important tropical forest tree species in Bolivia: assessment and recommendations. For.
Ecol. Manage. 124, 263273.
Muchugi, A., Kadu, C., Kindt, R., Kipruto, H., Lemurt, S., Olale, K., Nyadoi, P., Dawson, I., Jamnadass, R., 2008. Molecular markers for tropical trees, a practical
guide to principles and procedures. In: Dawson, I., Jamnadass, R. (Eds.), ICRAF Technical Manual no. 9. World Agroforestry Centre, Nairobi.
Muellner, A.N., Pennington, T.D., Chase, M.W., 2009. Molecular phylogenetics of Neotropical Cedreleae (mahogany family, Meliaceae) based on nuclear and
plastid DNA sequences reveal multiple origins of Cedrela odorata. Mol. Phylogenet. Evol. 52, 461469.
Nazareno, A.G., Pereira, R.A.S., Feres, J.M., Mestriner, M.A., Alzate-Marin, A.L., 2009. Transferability and characterization of microsatellite markers in two
Neotropical Ficus species. Genet. Mol. Biol. 32 (3), 568571.
Newton, A.C., Allnutt, T.R., Gillies, A.C.M., Lowe, A.J., Ennos, R.A., 1999. Molecular phylogeography, intraspecic variation and the conservation of tree
species. Tree 14 (4), 140145.
Novick, R.R., Dick, C.W., Lemes, M.R., Navarro, C., Caccone, A., Bermingham, E., 2003. Genetic structure of Mesoamerican populations of big leaf mahogany
(Swietenia macrophylla) inferred from microsatellite analysis. Mol. Ecol. 12, 28852893.
Oliveira, E.J., Gomes Pdua, J., Zucchi, M.I., Vencovsky, R., Carneiro Vieira, M.L., 2006. Origin, evolution and genome distribution of microsatellites. Genet.
Mol. Biol. 29 (2), 294307.
Pandian, A., Ford, R., Taylor, P.W.J., 2000. Transferability of sequence tagged microsatellite site (STMS) primers across four Major Pulses. Plant. Mol. Biol. Rep.
18, 395a395h.
Patio Varela, F., 1997. Recursos Genticos de Swietenia y Cedrela en los Neotrpicos: Propuestas para Acciones Coordinadas. Direccin de Recursos
Forestales, Departamento de Montes. FAO, Roma, Italia. Available at: http://www.fao.org/DOCREP/006/AD111S/AD111S00.HTM (May 03, 2013).
Peakall, R., Gilmore, S., Keys, W., Morgante, M., Rafalski, A., 1998. Crossspecies amplication of soybean (Glycine max) simple-sequence repeats (SSRs) within
the genus and other legume genera: implications for the transferability of SSRs in plants. Mol. Biol. Evol. 15, 12751287.

16

M.C. Soldati et al. / Biochemical Systematics and Ecology 53 (2014) 816

Peakall, R., Elbert, D., Scott, L.J., Meagher, P.F., Offord, C.A., 2003. Comparative genetic study conrms exceptionally low genetic variation in the ancient and
endangered relictual conifer, Wollemia nobilis (Araucariaceae). Mol. Ecol. 12, 23312343.
Pennington, T.D., Muellner, A.N., 2010. A Monograph of Cedrela (Meliaceae). dh books, The Manse, Chapel Lane, Milborne Port, England.
Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S., Rafalski, A., 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers
for germplasm analysis. Mol. Breed 2, 225238.
Rao, M.N., Ramesha, B.T., Ravikanth, G., Ganeshaiah, K.N., Uma Shaanker, R., 2007. Cross-species amplication of coconut microsatellite markers in rattans.
Silvae Genetica 56 (6), 282286.
Roa, A., Chavarriaga-Aguirre, P., Duque, M., Maya, M., Bonierbale, M., Iglesias, C., Tohme, J., 2000. Cross-species amplication of Cassava (Manihot esculenta)
(Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationships. Am. J. Bot. 87 (11), 16471655.
Rousset, F., 2008. Genepop Version 4.0: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103106.
Soldati, M.C., Fornes, L., van Zonneveld, M., Thomas, E., Zelener, N., 2013. An assessment of the genetic diversity of Cedrela balansae C. DC. (Meliaceae) in
Northwestern Argentina by means of combined use of SSR and AFLP molecular markers. Biochem. Syst. Ecol. 47, 4555.
Steinkellner, H., Lexer, C., Turetschek, E., Glssl, J., 1997. Conservation of (GA)n 607 microsatellite loci between Quercus species. Mol. Ecol. 6, 11891194.
Styles, B.T., 1981. Swietenioideae. In: Pennington, T.D., Styles, B.T., Taylor, D.A.H. (Eds.), Meliaceae. Flora Neotropica Monograph. Botanical Garden, New York,
USA, pp. 359418.
Tautz, D., 1989. Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 17, 64636471.
Terui, C., Lian, L., Saito, Y., Ide, Y., 2006. Development of microsatellite markers in Acer capillipes. Mol. Ecol. Notes 6, 7779.
White, G., Powell, W., 1997a. Isolation and characterization of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species.
Mol. Ecol. 6, 851860.
White, G., Powell, W., 1997b. Cross-species amplication of SSR loci in the Meliaceae family. Mol. Ecol. 6, 11951197.
White, G., Boshier, D., Powell, W., 2002. Increased pollen ow counteracts fragmentation in a tropical dry forest: an example from Swietenia humilis
Zuccarini. Proc. Natl. Acad. Sci. USA 99, 20382042.
Zamora Petri, M., 2006. Inuencia de la ganadera trashumante y la apertura de claros en la supervivencia y crecimiento de Cedrela lilloi en Tariqua, Bolivia. In:
Pacheco, S., Brown, A. (Eds.), Ecologa y Produccin de cedro (gnero Cedrela) en las Yungas australes. LIEY-ProYungas, Tucumn, Argentina, pp. 131142.
Zapater, M.A., Del Castillo, E.M., Pennington, T.D., 2004. El Gnero Cedrela (Meliaceae) en la Argentina. Darwiniana 42 (14), 347356.
Zucchi, M.I., Brondani, R.P.V., Pinheiro, J.B., Brondani, C., Vencovsky, R., 2002. Transferability of microsatellite markers from Eucalyptus spp. to Eugenia
dysenterica (Myrtaceae family). Mol. Ecol. Notes 2, 512513.