Fax +41 61 306 12 34E-Mail karger@karger.chwww.karger.com

Cytogenet Genome Res 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

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].

Key Words

Cervidae � Cytogenetics � Evolutionarily significant unit  � Mazama americana � Mitochondrial DNA � Phylogeny � Phylogeography � 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

Published online: April 20, 2010

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/nJaboticabal, SP 14884-900 (Brasil) Tel. 5516 3209 2678, Fax 5516 3209 2682, ext. 220, E-Mail barbanti   @   fcav.unesp.br

© 2010 S. Karger AG, Basel1424–8581/10/0000–0000$26.00/0

Accessible online at:www.karger.com/cgr

Abril   /Carnelossi   /González   /Duarte   Cytogenet Genome Res 2

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/XY 1 Y 2 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 XY 1 Y 2 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

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.

Chromosomal and Molecular Evolution of the Red Brocket Deer

Cytogenet Genome Res 3

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 1 2.5%), C (biarmed chromo-somes with RL ! 2.5%), D (acrocentric chromosomes with RL ! 3.0%), E (acrocentric chromosomes with RL 1 3.0%) and B (mi-crochromosomes or extranumerary chromosomes with RL 1 1.0%). 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 5 � CGAAGCTTGATATGAAAAAC-CATCGTTG3 � and H15149 5 � AAACTGCAGCCCCTCAGAA-TGATATTTGTCCTCA3 � [Irwin et al., 1991]. For amplifying the partial cytochrome b, we followed Duarte et al. [2008].

Universal primers Thr-L15910 (5 � -GAATTCCCCGGTCTT-GTAAACC-3 � ) and DL-H16498 (5 � CCTGAACTAGGAACCA-GATG-3 � ) [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 (3 � –5 � and 5 � –3 � ) 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

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-

Fig. 1. Map of Brazil showing the sampling locations. References: The diamonds indicate individuals with the identification collec-tion number (table 2).

Abril   /Carnelossi   /González   /Duarte   Cytogenet Genome Res 4

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 ).

Table 1. C ytogenetic 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 Unknown255 F PRb 2n = 52/NF = 56 1–4 Criadouro Teuto/GO Unknown256 F PRb 2n = 52/NF = 56 0, 2–4 Criadouro Itaipu /PR Born in captivity257 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 Unknown260 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-MT247 M JUv 2n = 44/NF = 46 3–5 IBAMA Juína /MT Juína-MT248 F JUb 2n = 44/NF = 48 2–6 IBAMA Juína/MT Juína-MT251 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 Unknown269 M ROv 2n = 42/NF = 46 3–5 Buritis-RO Buritis/RO021 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.

Chromosomal and Molecular Evolution of the Red Brocket Deer

Cytogenet Genome Res 5

a b

c d

e f

Fig. 2. Basic karyotypes of the 6 cytotypes described in M. americana. a Paraná cytotype (2n = 52/53 andFN = 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).

Abril   /Carnelossi   /González   /Duarte   Cytogenet Genome Res 6

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 cladeB 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-

N h S G + C � K

Santarém 2 2 61 0.344 0.0702880.001211 61.0000Jari 1 1 – – – –Rondônia 3 3 32 0.342 0.0245580.0001111 21.3333Juína 5 5 33 0.343 0.0190880.0000139 16.6000Paraná 5 5 6 0.349 0.0027680.0000007 2.4Carajás 2 2 9 0.352 0.0103480.0000268 9.0

a b

c

d

e

JU2n = 44/45;

FN = 48

RO2n = 42/43;

FN = 46

JA2n = 48/49;

FN = 56

SA2n = 50/51;

FN = 56

PR2n = 52/53;

FN = 56

CA2n = 50/51;

FN = 54

AncestralM. americana2n = 52/53;

FN = 54

Centric fusion

Tandem fusion

Pericentric inversion

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. N umber of sequences of each analyzed cytotype for the concatenated sequences cytochrome b and D-loop (N); haplotype numbers (h); number of polymorphic sites (S); content of guanine and cytosine (G + C); nucleotide diversity and standard deviation (�) and number of average nucleotide differences (K)

Chromosomal and Molecular Evolution of the Red Brocket Deer

Cytogenet Genome Res 7

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-

100

99

0.7

100

82

98

98

100

89

100

100

54

021

211

251

247

248

252

269253

258260

235

256

257205

255

259

254274

Rondônia

Rondônia

Juína

Juína

Juína

Jari

Paraná

Santarém

Santarém

Carajás

Odocoileus virginianus

Rangifer tarandus

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.

Abril   /Carnelossi   /González   /Duarte   Cytogenet Genome Res 8

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).

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* – 0.0976680.0096M. nana 0.55484 0.93258 0.82857 –

* p < 0.05.

Chromosomal and Molecular Evolution of the Red Brocket Deer

Cytogenet Genome Res 9

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].

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

021 211 252

248

258253

260 251

247

269

255256 235

205

257

259

274

254

Paraná Carajás Santarém Rondônia Juína Jari

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.

Abril   /Carnelossi   /González   /Duarte   Cytogenet Genome Res 10

References

Abril VV, Duarte JMB: Chromosome polymor-phism in the Brazilian dwarf brocket deer, Mazama nana (Mammalia, Cervidae). Gen-et Mol Biol 31: 53–57 (2008).

Allen JA: Notes on american deer of the genus Mazama . Bull Am Mus Nat Hist 34: 521–553 (1915).

Amato G, Egan MG, Shaller BG: Mitochondrial DNA variation in Muntjac: evidence for dis-covery, and phylogenetic relationships, in Vrba ES, Schaller GB (eds): Antelopes, Deer, and Relatives: Fossil Record, Behavioral Ecology, Systematics, and Conservation, pp 285–295 (Yale University Press, London 2000).

Avise JC: Molecular population structure and the biogeographic history of a regional fau-na: a case history with lessons for conserva-tion biology. Oikos 63: 62–76 (1992).

Avise JC: Mitochondrial DNA polymorphism and a connection between genetics and de-mography of relevance to conservation. Conserv Biol 9: 686–690 (1995).

Avise JC: The history and purview of phylogeog-raphy: a personal reflection. Mol Ecol 7: 371–379 (1998).

Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, et al: Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Ann Rev Ecolog Syst 18: 489–522 (1987).

Bandelt HJ, Forster P, Röhl A: Median-joining networks for inferring intraspecific phylog-enies. Mol Phylogenet Evol 16: 37–48 (1999).

Bodmer R: Ecologia e conservação dos veados mateiro e catingueiro na Amazônia, in Du-arte JMB (ed): Biologia e Conservação de Cervídeos Sul-Americanos: Blastocerus, Ozotoceros e Mazama , pp 69–77 (FUNEP, Ja-boticabal 1997).

Cabrera A: Catálogo de los mamíferos de Améri-ca del Sur. Revista Museo Argentino Ber-nardino Rivadavia 4: 309–732 (1960).

Camacho JPM: B chromosomes, in Gregory TR (ed): The Evolution of Genome, pp 223–286 (Elsevier, San Diego 2005).

Camacho JPM, Sharbel TF, Beukeboom LW: B chromosome evolution. Phil Trans R Soc Lond B 355: 163–178 (2000).

Cathey JC, Bickham JW, Patton JC: Introgressive hybridization and nonconcordant evolu-tionary history of maternal and paternal lin-eages in North American deer. Evolution 52: 1224–1229 (1998).

Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK: Considering evolutionary pro-cesses in conservation biology. Trends Ecol Evol 15: 290–295 (2000).

Czernay S: Spiesshirsche und Pudus. Die Neue Brehm Bucherei 581: 1–84 (1987).

Donnelly MJ, Pinto J, Girod R, Besansky NJ, Lehmann T: Revisiting the role of introgres-sion vs shared ancestral polymorphisms as key processes shaping genetic diversity in the recently separated sibling species of the Anopheles gambiae complex. Heredity 92: 61–68 (2004).

Dowling TE, Secor CL: The role of hybridization and introgression in the diversification of animals. Ann Rev Ecol Evol Syst 28: 593–619 (1997).

Duarte JMB: Aspectos Taxonômicos e Citogené-ticos de Algumas Espécies de Cervídeos Brasileiros. (Master Dissertation, Faculdade de Ciências Agrárias e Veterinárias, Univer-sidade Estadual Paulista, Jaboticabal 1992).

Duarte JMB: Guia de Identificação de Cervíde-os Brasileiros. 14 pp (FUNEP, Jaboticabal 1996).

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 80 th birthday.

Chromosomal and Molecular Evolution of the Red Brocket Deer

Cytogenet Genome Res 11

Duarte JMB: Análise Citogenética e Taxonômica do Gênero Mazama (Cervidae; Artiodacty-la) no Brasil. (PhD Thesis, Instituto de Bio-ciências, Universidade Estadual Paulista, Botucatu 1998).

Duarte JMB, Jorge W: Morphologic and cytoge-netic description of the small red brocket ( Mazama bororo Duarte, 1996) in Brazil. Mammalia 67: 403–410 (2003).

Duarte JMB, Merino ML: Taxonomia e evolução, in Duarte JMB (ed): Biologia e Conservação de Cervídeos Sul-Americanos: Blastocerus, Ozotoceros e Mazama , pp 1–21 (FUNEP, Ja-boticabal 1997).

Duarte JMB, González S, Maldonado JE: The surprising evolutionary history of South American deer. Mol Phylogenet Evol 49: 17–22 (2008).

Eisenberg JF: Mammals of the Neotropics. V 1, 449 pp (The University of Chicago Press, Chicago 1989).

Emmons LH: Neotropical Rainforest Mammals, a Field Guide, 281 pp (University of Chicago Press, Chicago 1990).

Excoffier L, Laval G, Schneider S: Arlequin ver. 3.0: an integrated software package for pop-ulation genetics data analysis. Evol Bioin-form Online 1: 47–50 (2005).

González S, Maldonado JE, Leonard JA, Vilà C, Barbanti Duarte JM, et al: Conservation ge-netics of the endangered Pampas deer ( Ozotoceros bezoarticus ). Mol Ecol 7: 47–56 (1998).

González S, Garcia JE: Fecal DNA, in Duarte JMB, Gonzalez S (eds): Neotropical Cervi-dology, pp 306–312 (IUCN/FUNEP, Gland 2010).

Groves CP, Schaller GB: The phylogeny and bio-geography of the newly discovered Anna-mite Artiodactyls, in Vrba ES, Schaller GB (eds): Antelopes, Deer, and Relatives: Fossil Record, Behavioral Ecology, Systematics, and Conservation, pp 261–285 (Yale Univer-sity Press, London 2000).

Huang L, Wang J, Nie W, Su W, Yang F: Tandem chromosome fusions in karyotypic evolu-tion of Muntiacus : evidence from M. feae and M. gongshanensis. Chromosome Res 14: 637–647 (2006).

Irwin DM, Kocher TD, Wilson AC: Evolution of the cytochrome b gene of mammals. J Mol Evol 32: 128–144 (1991).

Jorge W, Benirschke K: Centromeric hetero-chromatin and G-banding of the red brocket deer Mazama americana temama ( Cervoid-ea, Artiodactyla ) with probable non-Robert-sonian translocation. Cytologia 42: 711–721 (1977).

Kocher TD, Thomas WK, Edwards A, Pääbo SV, Villablanca FX, Wilson AC: Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with con-served primers. Proc Natl Acad Sci USA 86: 6196–6200 (1989).

Márquez A, Maldonado JE, González S, Bec-caceci MD, Garcia JE, Duarte JMB: Phylo-geography and Pleistocene demographic his-tory of the endangered marsh deer ( Blasto-cerus dichotomus ) from the Río de la Plata Basin. Conserv Genetic 7: 563–575 (2006).

Moritz C: Defining ‘evolutionarily significant units’ for conservation. Trends Ecol Evol 9: 373–375 (1994).

Moritz C: Uses of molecular phylogenies for con-servation. Proc R Soc Lond B Biol Sci 349: 113–118 (1995).

Neitzel H: Chromosome evolution of Cervidae: karyotypic and molecular aspects, in Obe G, Basler A (eds): Cytogenetics, Basic and Ap-plied Aspects, pp 90–112 (Springer Verlag, 1987).

Oliveira EJF, Garcia JE, Contel EPB, Duarte JMB: Genetic structure of Blastocerus dicho-tomus populations in the Paraná River basin (Brazil) based on protein variability. Bio-chem Genet 43: 5–6 (2005).

Palestis BG, Burt A, Jones RN, Trivers R: The distribution of B chromosomes across spe-cies. Cytogenet Genome Res 106: 147–148 (2004).

Pathak S, Lin CC: Synaptonemal complex of the sex-autosome trivalent in a male Indian muntjac. Chromosoma 82: 367–376 (1981).

Pousada D, Crandall KA: Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818 (1998).

Purdue JR, Smith MH, Patton JC: Female phy-lopatry and extreme spatial genetic hetero-geneity in white-tailed deer. J Mammal 81: 179–185 (2000).

Randi E, Mucci N, Pierpaoli M, Douzery E: New phylogenetic perspectives on the Cervidae (Artiodactyla) are provided by the mito-chondrial cytochrome b gene. Proc R Soc Lond B Biol Sci 265: 793–801 (1998).

Rossi RV: Taxonomia de Mazama Ranfinesque, 1817 do Brasil (Artyodactyla, Cervidae). (Master Dissertation, Instituto de Biociên-cias, Universidade de São Paulo, Sao Paulo 2000).

Ryder OA: Species conservation and systematics: the dilemma of subspecies. Trends Ecol Evol 1: 9–10 (1986).

Sarria-Perea JA: Comparação Entre Alguns Citótipos de Mazama americana (Artiodac-tyla; Cervidae): Quão Grande é a Diferença Entre Eles? (Master Dissertation, Faculdade de Ciências Agrárias e Veterinárias, Univer-sidade Estadual Paulista, Jaboticabal 2004).

Seabright M: A rapid banding technique for hu-man chromosomes. Lancet:1: 971–972 (1971).

Swofford DL: PAUP Phylogenetic Analysis Us-ing Parsimony. Version 4.0b10 (Sinauer As-sociates, Sunderland 2001).

Taberlet P: The use of mitochondrial DNA con-trol region sequencing in conservation ge-netics, in Smith TK, Wayne RK (eds): Mo-lecular Approaches in Conservation, pp 125–142 (Oxford University Press, New York 1996).

Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599 (2007).

Taylor KM, Hungerford DA, Snyder RL: Artio-dactyl mammals: their chromosome cytolo-gy in relation to patterns of evolution, in Be-nirschke K (ed): Comparative Mammalian Evolution, pp 346–356 (Springer, Berlin 1969).

Thompson JD, Gibson TJ, Plewniak F, Jeanmou-gin F, Higgins DG: The ClustalX windows interface: f lexible strategies for multiple se-quence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882 (1997).

Varela DM, Trovati RG, Guzman KR, Rossi RV, Duarte JMB: Red brocket deer Mazama americana (Erxleben 1777), in: Duarte JMB, González S (eds): Neotropical Cervidology, pp 151–159 (IUCN/FUNEP, Gland 2010).

Verma RS, Babu A: Human Chromosomes: Principles and Techniques, 2nd ed, 419 pp (McGraw-Hill, New York 1995).

Vogler AP, DeSalle R: Diagnosing units of con-servation management. Conserv Biol 8: 354–363 (1994).

Waples RS: Pacific salmon, Oncorhynchus spp ., and the definition of ‘species’ under the En-dangered Species Act. Mar Fish Rev 53: 11–22 (1991).

Yang F, Müller S, Just R, Ferguson-Smith MA, Wienberg J: Comparative chromosome painting in mammals: human and the Indi-an muntjac ( Muntiacus muntjak vaginalis ). Genomics 39: 396–401 (1997a).

Yang F, O’Brien PCM, Wienberg J, Ferguson-Smith MA: A reappraisal of the tandem fu-sion theory of karyotype evolution in the In-dian muntjac using chromosome painting. Chromosome Res 5: 109–117 (1997b).

Yang F, O’Brien PCM, Wienberg J, Ferguson-Smith MA: Evolution of the black muntjac ( Muntiacus crinifrons ) karyotype revealed by comparative chromosome painting. Cy-togenet Cell Genet 76: 159–163 (1997c).

Zadworny D; Kuhnlein U: The identification of the kappa-casein genotype in Holstein dairy cattle using the polymerase chain reaction. Theor Appl Genet 80: 631–634 (1990).