Cytogenet Genome Res 2010;128:177–187
DOI: 10.1159/000298819
Elucidating the Evolution of the Red
Brocket Deer Mazama americana
Complex (Artiodactyla; Cervidae)
V.V. Abril a E.A.G. Carnelossi a S. González b J.M.B. Duarte a
a
Faculdade de Ciências Agrárias e Veterinárias, UNESP – Universidade Estadual Paulista, Departamento de
Zootecnia, Núcleo de Pesquisa e Conservação de Cervídeos (NUPECCE), Jaboticabal, Brasil; b Genética de la
Conservación, Departamento de Genética, IIBCE-Facultad de Ciencias-UdelaR, Montevideo, Uruguay
Key Words
Cervidae ⴢ Cytogenetics ⴢ Evolutionarily significant unit ⴢ
Mazama americana ⴢ Mitochondrial DNA ⴢ Phylogeny ⴢ
Phylogeography ⴢ Red brocket deer
Abstract
The red brocket deer Mazama americana is a neotropical
species that exhibits extensive karyotype variation under an
unvarying morphotype. In order to deduce red brocket deer
genetic units for conservation, gene flow between popula-
tions, and genetic variation, we initiated a cytogenetic and
molecular genetic study based on representative samples
from throughout their Brazilian geographic range. These
data represent the first cytotaxonomical and molecular sys-
tematics, and although sample sizes are limited, our results
clearly suggest that red brocket deer populations are sig-
nificantly differentiated with respect to karyotypes and the
mitochondrial sequences analyzed. We clearly recognized 2
independent species, and we will be focusing further re-
search in analyzing the meiotic dynamic to determine the
existence of other evolutionarily significant units under the
red brocket complex. Copyright © 2010 S. Karger AG, Basel
© 2010 S. Karger AG, Basel
1424–8581/10/1283–0177$26.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/cgr
Published online: April 20, 2010
The red brocket deer (Mazama americana) is the larg-
est deer species of the genus Mazama, with 30 to 40 kg
weight and 65 cm of height [Duarte, 1996], with a wide
geographic range from México to the north of Argentina
[Eisenberg, 1989; Emmons, 1990]. The species inhabits
dense forest and is less commonly found in altered areas
[Duarte, 1996; Bodmer, 1997; Varela et al., 2010].
Many aspects of the species taxonomy are uncertain,
such as the number of subspecies, and whether there are
one or more cryptic species under the red brocket deer
name. Allen [1915], described 8 species under the red
brocket deer complex, but Cabrera [1960] and Czernay
[1987], only considered one species composed of 9–15
subspecies, respectively. A recent morphological review
of M. americana by Rossi [2000] determined the exis-
tence of only one species. Another cryptic species, the
small red brocket deer M. bororo was erronously classi-
fied by morphological analysis as M. americana. M. boro-
ro was recently described based on chromosomal and
morphological differences [Duarte, 1996; Duarte and
Merino, 1997; Duarte and Jorge, 2003]. Cytotaxonomy
and DNA sequencing have provided excellent diagnostic
markers for species identification, thus improving the as-
sessment of taxonomic diversity within the genus [Du-
arte et al., 2008].
Prof. Dr. José Maurício Barbanti Duarte
Departamento de Zootecnia, Faculdade de Ciências Agrárias e Veterinárias
Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane s/n
Jaboticabal, SP 14884-900 (Brasil)
Tel. +55 16 3209 2678, Fax +55 16 3209 2682, ext. 220, E-Mail barbanti @ fcav.unesp.br
 The cytogenetic analysis of M. americana showed
wide variation in this species. The first description was
initially performed by Taylor et al. [1969], who reported
2n = 68 and NF = 74. Jorge and Bernirschke [1977] ana-
lyzed 3 individuals of M. americana temama and de-
scribed the basic karyotype as 2n = 50 and FN = 70. The
X chromosome was submetacentric and approximately
the same size as chromosome pair 7. The metacentric Y
was the smallest chromosome. The vast differences
among the animals analyzed by Jorge and Benirschke
[1977] and Taylor [1969] raised questions concerning
these species’ classification. Neitzel [1987] analyzed a
Paraguayan female and described yet another pattern for
the species. It had 2n = 52, plus 4–5 B chromosomes and
NF = 56. The submetacentric X was the largest chromo-
some. More recently, Duarte [1992] analyzed 4 Brazilian
animals, and obtained diploid numbers varying from 48,
50, 52 and 54, and NF = 54, 54, 56 and 56, respectively.
These results showed an amazing and wide karyotype
variation. According to Duarte and Merino [1997], this
wide variation appears to suggest the existence of several
cryptic species under the red brocket deer morphological
pattern. Furthermore, Duarte [1998], analyzed 33 indi-
viduals belonging to M. americana from several Brazilian
locations, finding again a wide range of variation, with a
diploid number ranging from 42–53 chromosomes and a
fundamental number of 48–57, not including many su-
pernumerary chromosomes (Bs).
Furthermore, the red brocket exhibited a multiple sex
chromosome system XX/XY1Y2 described in a G-band-
ing study by Sarria-Perea [2004]. This multiple sex chro-
mosome system possibly occurred when an X-autosome
translocation took place in an ancestral form of the spe-
cies. This finding was also corroborated by analyzing
the synaptonemal complex, in which the sex chromo-
somes appeared as a trivalent configuration (Aquino CI,
unpublished). A multiple XY1Y2 sex chromosome sys-
tem has also been previously described in other deer spe-
cies, as in the case of the Muntiacus muntjak [Pathak and
Lin, 1981].
The high karyotypic differences among these individ-
uals followed by a decreasing trend in chromosome num-
ber are suggestive of a synapomorphy that appeared in
the red brocket species complex, which is not correlated
with the rate of morphological and molecular differen-
tiation [Duarte et al., 2008]. However, the chromosomal
variation showed a geographic correlation suggesting a
possible regional karyotypic partition.
In-parallel molecular studies using mitochondrial
DNA (mtDNA) showed that the red brocket has a sharp
178 Cytogenet Genome Res 2010;128:177–187
trend to subdivide into several clusters in the phyloge-
netic tree, suggesting the existence of more than one
species or evolutionarily significant units in the group
[Duarte et al., 2008]. mtDNA has been the most widely
used tool for reconstructing population and species his-
tories, presumably because it is relatively easy to amplify,
not duplicated, typically non-recombining, supposedly
nearly neutral, and highly variable between and within
species [Taberlet, 1996; Avise,1998]. Furthermore this
molecular marker of maternal inheritance is useful for
population genetic analysis in neotropical deer living in
fragmented habitat [Avise, 1992; 1995]. Most deer spe-
cies usually have: (1) asymmetric genetic flow and dis-
persal rate, females being frequently phylopatric; (2) spa-
tial association of female and fawn, and (3) a strong ma-
ternal lineage structure consisting of demographic
autonomy among populations in an ecological scale
[González et al., 1998; Márquez et al., 2006; González et
al., 2010].
In deriving strategies for saving diminishing flora
and fauna, conservation biologists continue to search for
methods that can distinguish unambiguous units for
conservation, and this has resulted in the reevaluation
of the taxonomy of poorly studied groups. ‘Evolution-
arily significant units’ (ESUs) have been proposed and
with the increasing sophistication of molecular tech-
niques and genetic data analysis in the scientific conser-
vation literature, have evolved over the past few decades
[Ryder, 1986; Waples, 1991; Moritz, 1994; 1995; Vogler
and DeSalle, 1994; Crandall et al., 2000]. ESUs now rely
on measures that reflect genetic isolation rather than
adaptive diversity, a more holistic concept of species,
consisting of populations with varying levels of gene
flow evolving through drift and natural selection [Cran-
dall et al., 2000].
In order to deduce red brocket deer genetic units for
conservation [Moritz, 1994, 1995] and to better under-
stand the gene flow and genetic variation between popu-
lations, we initiated a cytogenetic and molecular genetic
study based on representative samples from throughout
the Brazilian geographic range. To determine levels of ge-
netic differentiation among isolated populations, we ex-
amined karyotypes and DNA sequences from the mito-
chondrial control region and cytochrome b partial gene
of 18 individuals from 6 localities (fig.1). These data rep-
resent the first cytotaxonomy and molecular systematics,
and although sample sizes are limited, our results suggest
that red brocket deer populations are significantly differ-
entiated with respect to karyotypes and the mitochon-
drial sequences analyzed.
Abril /Carnelossi /González /Duarte
 Material and Methods

Samples
The sampling areas were chosen taking into account the previ-
ous cytogenetic and molecular findings in Duarte et al. [2008].
Blood and skin samples of 18 animals of the M. americana species
from different regions of Brazil (fig. 1) were collected and ana-
lyzed. The studied individuals represent around 80% of the cap-
tive breeding stock of red broket deer from Brazil (see details in
table 1).

Cytogenetic Analysis
Metaphase chromosome slides were prepared from fibroblast
tissue cultures that were established from skin biopsies according
to Verma and Babu [1995] and the chromosome preparations were
submitted to G-banding using trypsin digestion [Seabright, 1971].
The chromosomes were classified as metacentric, submeta-
centric or acrocentric according to their arm ratios and were or-
ganized into groups according to their relative lengths (RL): A
(biarmed chromosomes with RL 12.5%), C (biarmed chromo-
somes with RL ! 2.5%), D (acrocentric chromosomes with RL
!3.0%), E (acrocentric chromosomes with RL 13.0%) and B (mi-
crochromosomes or extranumerary chromosomes with RL
11.0%). B chromosomes were not considered in calculating the
diploid and fundamental numbers because there was intraindi-
vidual variation [Abril and Duarte, 2008].
Molecular Genetics
Tissue or blood samples (50 mg or 100 ␮l) were used to isolate
genomic DNA according to Zadworny and Kuhnlein [1990]. A
partial fragment of the cytochrome b gene (480 bp) was amplified
using the primer L14724 5ⴕCGAAGCTTGATATGAAAAAC-
CATCGTTG3ⴕ and H15149 5ⴕAAACTGCAGCCCCTCAGAA-
TGATATTTGTCCTCA3ⴕ [Irwin et al., 1991]. For amplifying the
partial cytochrome b, we followed Duarte et al. [2008].
Universal primers Thr-L15910 (5ⴕ-GAATTCCCCGGTCTT-
GTAAACC-3ⴕ) and DL-H16498 (5ⴕCCTGAACTAGGAACCA-
GATG-3ⴕ) [based on Kocher et al., 1989] were used to amplify a
690 bp fragment of the control region, following the protocol de-
scribed by González et al. [1998]. Extraction controls and no-tem-
plate polymerase chain reaction (PCR) controls were used in each
amplification.
The PCR purification was performed using Shrimp Alkaline
Phosphatase (SAP) and an Exonuclease I (EXO), for a total volume
of 10 ␮l, 5 ␮l for PCR plus 0.5 ␮l for Exonuclease I (10 U/␮l), 1 ␮l
SAP (1 U/␮l) enzyme and 0.5 ␮l of SAP dilution buffer and 3 ␮l
of deionized water. Both DNA strands were sequenced forward
and reverse (3ⴕ–5ⴕ and 5ⴕ–3ⴕ) using an ABI automated sequencer
ABI 377 and 3100 (Applied Biosystems).
Molecular Data Analyses
Chromatograms open at MEGA 4 [Tamura et al., 2007], were
first aligned manually and then using Clustal X [Thompson et al.,
1997]. The alignments for all haplotypes were unambiguous. All
unique haplotypes were defined and subsequently submitted to
GenBank (accession numbers: GU305915–GU305939). Odocoi-
leus virginianus (DQ379370.1/EF644645-1) and Rangifer taran-
dus (AJ000029.1/AY970667) were used as outgroups.
The matrix of both regions were concatenated and submitted
to MODELTEST 3.7 [Pousada and Crandall, 1998] for the Akaike
Fig. 1. Map of Brazil showing the sampling locations. References:
The diamonds indicate individuals with the identification collec-
tion number (table 2).
Information Criterion (AIC). Nucleotide sequence data were ana-
lyzed using ‘neighbor joining’ (NJ) with PAUP*4.0b10 [Swofford,
2001], 10,000 bootstrap pseudoreplicates and with the model
TrN+I+G.
Additionally the sequences were organized by geographic lo-
cation to analyze the genetic distance between them and with the
closer species M. bororo and M. nana. The parameters used were
average distance between groups, ‘gaps/missing’ = ‘pairwise dele-
tion’ model and the ‘Kimura-2-parameter’. The pairwise genetic
distance was estimated between M. americana clades and M.
bororo and M. nana from GenBank data (accession numbers: M.
bororo: DQ789231.2 and DQ789187.2; M. nana: DQ789214.2,
DQ789227.2 and DQ789210.2) using ARLEQUIM 3.1 [Excoffier
et al. 2005].
The number of mutations between DNA genotypes in pair-
wise comparisons was used to construct a Minimum Spanning
Network in which sequences are the nodes of a network rather
than the terminal tips of a tree. Networks may more effectively
portray the relationships among sequences for populations, in
which many sequences may be derived from the same ancestral
genotype, using the number of substitutions Median Joining Net-
work [Bandelt et al., 1999] applying Network 4.5.0.0.
Results
Cytogenetic Analysis
From the 18 individuals analyzed we found 6 cyto-
types (Rondônia, Juína, Jarí, Carajás, Santarém and
Paraná) with a diploid number ranging from 42–53 and
fundamental number ranging from 48–56 (table 1).
Additionally we described the basic karyotype of each
cytotype (fig. 2), and the variants inside the different cy-

Chromosomal and Molecular Evolution Cytogenet Genome Res 2010;128:177–187 179

of the Red Brocket Deer
Table 1. Cytogenetic results of individuals from M. americana, grouped by cytotypes

No Sex Cytotype 2n/NF B Captivity origin Wildlife origin

235 F PRv 2n = 51/NF = 56 4 Pres. Prudente/SP Unknown

255 F PRb 2n = 52/NF = 56 1–4 Criadouro Teuto/GO Unknown
256 F PRb 2n = 52/NF = 56 0, 2–4 Criadouro Itaipu /PR Born in captivity
257 F PRb 2n = 52/NF = 56 2–5 Zôo de Cascavel/PR Pq. Nac. Iguaçu/PR
205 M PRb 2n = 53/NF = 56 1–5 Criadouro Itaipu/PR Born in captivity
259 M SAb 2n = 51/NF = 56 3, 4 or 6 Criadouro Santarém/PA Unknown
260 M SAb 2n = 51/NF = 56 3–4 IBAMA Santarém/PA Itaituba/PA
258 M JAb 2n = 49/NF = 56 4–5 Criadouro Santarém/PA Unknown
254 F CAb 2n = 50/NF = 54 3–6 IBAMA Imperatriz/MA Açailândia-MA
274 M CAv 2n = 50/NF = 54 3 IBAMA Imperatriz-MA Born in captivity
252 F JUv 2n = 43/NF = 48 3 IBAMA Juína/MT Juína-MT
253 F JUv 2n = 43/NF = 48 3 or 5 IBAMA Juína/MT Juína-MT
247 M JUv 2n = 44/NF = 46 3–5 IBAMA Juína /MT Juína-MT
248 F JUb 2n = 44/NF = 48 2–6 IBAMA Juína/MT Juína-MT
251 M JUb 2n = 45/NF = 48 2–3 IBAMA Juína/MT Juína-MT
211 F ROv 2n = 42/NF = 49 0, 2–5 Zôo de Ariquemes/RO Unknown
269 M ROv 2n = 42/NF = 46 3–5 Buritis-RO Buritis/RO
021 M ROb 2n = 43/NF = 46 4–6 Zôo de Ariquemes/RO Unknown

2n = diploid number; NF = fundamental number; B = B chromosomes; RO = Rondônia; JU = Juína; JA = Jarí; CA = Carajás; PR =

Paraná; SA = Santarém; v variant karyotype; b basic karyotype.

totypes, consisting of individuals carrying centric fu-
sions, inversions and aneuploidies (table 1).
There was also another important source of inter- and
intraindividual variation if we also included the B chro-
mosome variation (ranging, from 0–6) with the chromo-
some polymorphisms described in the basic complement.
The ‘Paraná cytotype’ was recognized as the more an-
cestral karyotype for having the higher diploid and fun-
damental number (2n = 52/53; FN = 56) and was used to
perform comparisons among the other cytotypes. The
banding pattern analysis let us identify the rearrange-
ments (fig. 3) and infer an ancestral karyotype in the an-
alyzed sample (2n = 52/53; FN = 54) and distinguish be-
tween the chromosome lineages of the red brocket deer
from Brazil (fig. 4).
The ‘Paraná cytotype’ originated from the inferred
ancestral cytotype with a pericentric inversion in one ac-
rocentric chromosome. The basic karyotype from Cara-
jás evolved from fixation of a tandem fusion between 2
acrocentric chromosomes of the Paraná cytotype. The
Santarém cytotype also originated from the Paraná cyto-
type by fixation of a first centric fusion, and a second
centric fusion evolved into the Jarí cytotype. Another lin-
eage with a low diploid and fundamental number may be
due to the accumulation of a great number of tandem fu-
sions that evolved into Juína and Rondônia cytotypes
which are very close chromosomally.
Molecular Phylogeny Inferred from Cytochrome b and
D-Loop Sequences
The individuals employed to describe the cytotypes
were analyzed with mitochondrial molecular markers.
From the 18 individuals analyzed in the D loop fragment
(488 bp), each had a unique haplotype (accession num-
bers: GU305922–GU305939). In the cytochrome b frag-
ment (386 bp), we identified 7 new haplotypes (accession
numbers: GU305915–GU305921).
A concatenated matrix was constructed with 386 bp of
the cytochrome b and the D-loop 488 bp sequences. We
found a high number of polymorphic sites S = 108, the
total number of mutations was Eta = 114, the nucleotide
diversity index was ␲ = 0.04633 with a standard deviation
of 0.00244, and the average of nucleotide differences was
k = 40.12418 (table 2).

180 Cytogenet Genome Res 2010;128:177–187 Abril /Carnelossi /González /Duarte

       
 a b

c d

e f

Fig. 2. Basic karyotypes of the 6 cytotypes described in M. americana. a Paraná cytotype (2n = 52/53 and
FN = 56); b Santarém cytotype (2n = 51/FN = 56); c Jarí cytotype (2n = 49/FN = 56); d Carajás cytotype (2n =
50/FN = 54); e Juína cytotype (2n = 45/FN = 48); f Rondônia cytotype (2n = 43/FN = 46).

Chromosomal and Molecular Evolution Cytogenet Genome Res 2010;128:177–187 181

of the Red Brocket Deer
 Centric fusion
Tandem fusion JU RO
2n = 44/45; 2n = 42/43;
a b Pericentric inversion FN = 48 FN = 46

c JA
2n = 48/49;
Ancestral FN = 56
M. americana
SA
2n = 52/53;
2n = 50/51;
FN = 54
FN = 56

PR
2n = 52/53;
d FN = 56

CA
2n = 50/51;
FN = 54

Fig. 3. G-banding chromosome analysis of the relationships be-
tween the chromosomes from the Paraná cytotype (ancestral cy-
totype) with chromosomes of other analyzed cytotypes. a Paraná
(PR) and Santarém (SA); b Paraná (PR) and Jarí (JA); c Paraná
(PR) and Carajás (CA); d Paraná (PR) and Juína (JU); e Paraná
(PR) and Rondônia (RO).
Fig. 4. Chromosomal evolution network showing the relation-
ships of the 6 analyzed cytotypes of M. americana (PR = Paraná;
CA = Carajás; SA = Santarém; JA = Jarí; JU = Juína; RO = Rondô-
nia; 2n = diploid number; FN = fundamental number).

Table 2. Number of sequences of each

analyzed cytotype for the concatenated N h S G+C ␲ K
sequences cytochrome b and D-loop (N);
haplotype numbers (h); number of Santarém 2 2 61 0.344 0.0702880.001211 61.0000
polymorphic sites (S); content of guanine Jari 1 1 – – – –
and cytosine (G + C); nucleotide diversity Rondônia 3 3 32 0.342 0.0245580.0001111 21.3333
and standard deviation (␲) and number Juína 5 5 33 0.343 0.0190880.0000139 16.6000
of average nucleotide differences (K) Paraná 5 5 6 0.349 0.0027680.0000007 2.4
Carajás 2 2 9 0.352 0.0103480.0000268 9.0

The phylogenetic analysis using the cytochrome b and
D loop concatenated fragment sequences showed 2 inde-
pendent clusters (A and B) with high bootstrap value sup-
port (fig. 5). Clade A was composed of Rondônia, Juína
and one haplotype from Santarém cytotypes and clade B
was composed of Paraná, Carajás and another haplotype
from the Santarém cytotypes. Considering phylogeogra-
phy assignments clade A is composed exclusively of indi-
viduals from the North of Brazil. However, in clade
B there are 3 individuals from the north of Brazil (Santa-
rém – 259, Carajás – 254 and 274) clustering with the
Paraná cytotype.
Furthermore, the pairwise computations of Fst using
AMOVA indicate that both clades are significantly dif-

182 Cytogenet Genome Res 2010;128:177–187 Abril /Carnelossi /González /Duarte

       
 100 021
Rondônia
211

89 251
Juína
247

100 248
Juína
100 252

100 269 Rondônia

253 Juína
258 Jari
260 Santarém
99

235
54 256
257 Paraná
98
205
82
255
100
259 Santarém

98 254
Carajás
274
Odocoileus virginianus
Rangifer tarandus

0.7

Fig. 5. Phylogenetic relationships of the red brocket deer M. americana inferred from concatenated partial cy-
tochrome b and D-loop sequences and the relationships with the cytotypes (Rondônia, Juína, Jarí, Santarém,
Carajás and Paraná), applying ‘Neighbor-Joining’ with the model TrN+I+G, and the bootstrap posterior prob-
ability values. References: individual number with the geographic location and the cytotype.

ferentiated relative to a random collection of genotypes
(table 3). Additionally, we performed a comparative ge-
netic analysis including the closest phylogenetic species,
the small red brocket deer M. bororo and the Brazilian
dwarf brocket M. nana (table 3). We also observed that
the highest differentiation values and genetic distances
were obtained among both clades (A and B), and clade A
was genetically closer to the small red brocket deer and
the Brazilian dwarf brocket deer.
Discussion
Cytotypes and Molecular Genetic Differentiation
The chromosome variation and differentiation found
in M. americana was clustered in the cytotypes and cor-
related with geographic location. This chromosome vari-
ation had geographic coherence, and we found more dif-
ferences between locations than inside them. However,
the cytogenetic finding was not possible to correlate with
external morphological differentiation [Duarte et al.,
2008].
The chromosome evolution in M. americana was
based on tandem and centric fusions, and clearly shows
the deer pattern for chromosome evolution as described
for the genus Muntiacus [Yang et al., 1997a, b, c; Huang
et al., 2006; Duarte et al., 2008]. To confirm the chromo-
some evolution by tandem and centric fusions we are in
the process of chromosome painting in the described cy-
totypes.
Among the neotropical deer, in addition to the M.
americana species, there are reports of B chromosomes
in M. nana (1 to 6 chromosomes) [Abril and Duarte,
2008], M. bororo (4 to 5 chromosomes) [Duarte and Jorge,
2003], M. gouazoubira (0 to 3 chromosomes) and M.
nemorivaga (2 to 8 chromosomes) [Duarte, 1998]. The be-

Chromosomal and Molecular Evolution Cytogenet Genome Res 2010;128:177–187 183

of the Red Brocket Deer
Table 3. Sequence statistics for red brocket deer M. americana (cluster A and B) among the species M. bororo and M. nana (below the
diagonal the p value and above the diagonal the pairwise distance FST)

Clade A (north) Clade B (north and south) M. bororo M. nana

Clade A (north) – 0.0000080.00000* 0.0781080.00240* 0.0097780.0038*

Clade B (north and south) 0.77937* – 0.0312580.005301* 0.0068480.0023*
M. bororo 0.71106* 0.96375* – 0.0976680.0096
M. nana 0.55484 0.93258 0.82857 –

* p < 0.05.

havior of these chromosomes was variable under differ-
ent banding techniques [Abril and Duarte 2008] reflect-
ing their instability and the different origin possibilities
[Camacho et al., 2000; Palestis et al., 2004]. Camacho
[2005] explained that small Bs exhibit a tendency to be
mitotically unstable, and thus may vary in number from
cell to cell within the same individual. We are now in the
process of implementing in situ hybridization in an at-
tempt to get new information on the role of B chromo-
some evolution in the Mazama genus.
The molecular phylogenetic relationships among indi-
viduals of red brocket deer revealed 2 clearly separated
lineages that are well correlated with the cytogenetic
findings. One lineage (clade A) included cytotypes
Rondônia and Juína with the low diploid number ranging
from 42–45 chromosomes and a fundamental number of
46–48. The other lineage (clade B) included Paraná, Cara-
jás and Santarém cytotypes with a higher diploid number
ranging from 48–53 chromosomes and a fundamental
number of 54–56.
Furthermore the cluster with high bootstrap values
between individual pairs 021/211, 251/247; 248/252 may
be related to the source of these animals that also shared
similar karyotypes (table 2).
However, clade A also included 2 animals that accord-
ing to cytogenetic data should be in clade B (individual
number 260, Santarém cytotype 2n = 51, FN = 56 and in-
dividual number 258 Jarí cytotype with 2n = 49, FN = 56).
The other animal from Santarém (259) had an identical
karyotype (2n = 51, FN = 56) and had a differentiated po-
sition in all the phylogenetic analysis.
The karyotypes of these animals (258 and 260) are
closer to the chromosome constitution of the animals of
clade B. Meanwhile, it is difficult to explain the phyloge-
netic position of these animals with unpaired chromo-
some constitution. With historic introgression by females
from West to East it would be possible to introduce this
cytochrome b haplotype in the Jarí and Santarém cyto-
types. Another possibility is the retention or preservation
of the ancestral haplotype during evolution [Dowling and
Secor, 1997; Donnelly et al., 2004].
Furthermore, in spite of the geographic distance the
Carajás cytotype is closely related to the Paraná. The G
banding pattern showed that karyotypically they are very
similar. The only difference we detected was a tandem
fusion that had reduced one chromosome pair.
We found closely related cytotypes that are now lo-
cated in far distant geographic areas suggesting that in
the recent past a sympatric area existed joining the Ama-
zonian East to the Southern populations. Currently the
cytotype geographic isolation array may have occurred or
be due to historic population fragmentation that may also
have promoted substantial differences in the mtDNA
[Avise et al., 1987; Duarte et al., 2008].
Chromosome rearrangements and evolution in the
red brocket deer complex seem to occur at a faster rate
than the molecular changes [Sarria-Perea, 2004]. Fur-
thermore the results do not show an independent mono-
phyly from the cytotypes Juína, Rondônia and San-
tarém.
Cytogenetics and mtDNA markers proved to be effi-
cient tools to analyze, understand, and elucidate the evo-
lution of the red brocket deer complex. The comparison
between cytogenetic and molecular data showed that the
Paraná cytotype seems to have more genetic stability
than other cytotypes, showing lower nucleotide diversity
and suggesting equilibrium (table 2). It is noteworthy that
the individuals belonging to this cytotype are genetically
closer in the network haplotype (fig. 3) and the chromo-
somes were shown to be more stable than the other cyto-
types. However, only one animal (female 235, table 1) ex-
hibited variation from the basic diploid number (2n =
52/53; NF = 56).

184 Cytogenet Genome Res 2010;128:177–187 Abril /Carnelossi /González /Duarte

       
 021 211 252
248
258
253
251
260
247
269
Paraná Carajás
Substitution
Fig. 6. Median Joining Network performed including the concatenated cytochrome b and D-loop partial se-
quences. The size of the branch represents the genetic distance among haplotypes and the small white circles
represent the probable ancestral haplotype. References: individual number with the geographic location and the
cytotype.
Patterns of Molecular Evolution and Differentiation
Another interesting result is that the average genetic
distance comparisons between clades A and B is higher
than the genetic distance between the species M. bororo
and M. nana. Furthermore the individuals from clade A
are more closely related to the species M. bororo and
M.  nana while the individuals from clade B showed a
greater genetic distance from them, corroborating at least
the existence in the red brocket complex of 2 species.
These values suggest a separate evolution of both clades,
which was previously dated by Duarte et al. [2008] for
clade A at the beginning of the Pleistocene (1 MYA), and
for clade B M. americana variants began separation prob-
ably in the late Pliocene (2 MYA).
In the Muntiacus genus species, a similar example of
extraordinary chromosomal variation occurs [Amato et
al., 2000; Huang et al., 2006]. The phylogenetic analysis
of Muntiacus species also suggested a recent radiation.
Furthermore, the species also showed a high karyotype
divergence (M. muntjak 2n = 6/7, M. crinifrons and
M. gongshanensis 2n = 8/9, M. feae 2n = 13/14, M. reeve-
si with 2n = 46), as a result of multiple events of centric
and tandem fusions, derived from an ancestral karyo-
type similar to M. reevesi [Yang et al., 1997a, b, c; Amato
et al., 2000; Groves and Schaller, 2000; Huang et al.,
2006].
Chromosomal and Molecular Evolution
of the Red Brocket Deer
256 235
255 205
257
259
274
254
Santarém Rondônia Juína Jari
The red brockets also had a recent radiation when they
migrated from North to South America and the karyo-
type and DNA constitution have not yet reached a stabi-
lization state and coalescence. However, our analyzed
sample will not let us elucidate a cytogenetic cline. It
would be necessary to do an extensive and detailed sam-
pling along the Brazilian territory to corroborate this hy-
pothesis.
Conservation and Management Implications
Although the sample sizes are limited, our results sug-
gest that red brocket deer populations are significantly
differentiated with respect to karyotypes and the mito-
chondrial sequences analyzed are crucial for identifying
units for management and conservation. For conserva-
tion management planning strategies, Moritz [1994] de-
fined 2 categories that are useful to identify conservation
units: the ESU is used to define a group of populations in
which significant divergence is recognized, and a man-
agement unit is used to define those populations with sig-
nificant differentiation in allelic frequencies. Using these
criteria, we evaluate the presence of different conserva-
tion units of red brocket deer in Brazil.
Our hypothesis and phylogenetic inferences are based
on the mutation rate of mtDNA, revealing a common his-
tory of the maternal lineage. As in some neotropical deer
Cytogenet Genome Res 2010;128:177–187 185
species, the sex dispersal rate is unbiased, since females
are frequently phylopatric and have a low dispersal rate
[Cathey et al., 1998; González et al., 1998; Randi et al.,
1998; Purdue et al., 2000; Oliveira et al., 2005; Márquez
et al., 2006]. The cytogenetic analysis, representing nucle-
ar inheritance, can complement the mutation rate to ana-
lyze, understand and elucidate the ‘red brocket deer com-
plex’ evolution. Both approaches showed a concordant
evolution history for discriminating species and possible
ESUs in red brockets.
This study reveals at least 2 species for M. americana
red brocket deer: (a) haplotypes belonging to populations
from the west Amazonian biome with low chromosome
numbers clustering in clade B; (b) haplotypes belonging
to populations from the eastern Amazonian biome
through the south of Brazil at the Parana River basin,
with high chromosome numbers clustering in clade A.
However, the existence of other ESUs or management
units in the red brocket deer complex are pending to be
elucidated, and should be confirmed based on an exten-
sive South American sampling and analyzing the mei-
otic dynamics of these cytotypes.
Would the current cytotypes described in the red
brocket deer complex be distinctive species, ESUs or
management units? We are conducting crossing experi-
ments with these individuals, to evaluate the existence of
post zygotic reproductive barriers between cytotypes. If
this barrier is present, the chromosome constitution
would be the limiting factor to population admixture
(hybridization), characterizing valuable biologic species.
Finally, our results highlight the concordance of the
cytogenetic and molecular findings that clearly validate
at least 2 independent species, which need to be formally
described. Moreover we will be focusing further research
in analyzing the meiotic dynamic to determine the exis-
tence of other ESUs or species under the red brocket deer
complex.
Acknowledgements
Our study was funded by: FAPESP, CNPq, PROBIO/MMA,
from Brazil, CSIC-UdelaR; PEDECIBA, from Uruguay, and the
Wildlife Trust Alliance. The authors are grateful to Paulo Anto-
nio Tosta and João Airton Boer, for laboratory assistance and an
anonymous reviewer for helpful suggestions. We also want to
thank to Drs. Ricardo Benavente and Gustavo Folle who kindly
invited us to be part of this special issue celebrating Dr. Maximo
Drets 80th birthday.

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