Journal of Heredity Advance Access originally published online on December 5, 2006
Journal of Heredity 2007 98(1):88-92; doi:10.1093/jhered/esl054
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Brief Communications |
Phylogeographic Analyses of Callicebus lugens (Platyrrhini, Primates)
From the Genetics Division, Instituto Nacional de Câncer, Rua André Cavalcanti, 37, 4th floor, 20231-050, Rio de Janeiro, RJ, Brazil (Casado, Bonvicino, and Seuánez); the Post-graduate programme in Zoology, Museu Nacional, Rio de Janeiro, Brazil (Casado); the Department of Tropical Medicine, IOC-FIOCRUZ, Rio de Janeiro, Brazil (Bonvicino); and the Department of Genetics, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (Seuánez)
Address correspondence to H. N. Seuánez at the address above, or e-mail: genetics{at}inca.gov.br.
A phylogeographic study of Callicebus lugens was carried out based on cytochrome b DNA sequence data. Here, we report, for the first time, the distribution of C. lugens south of the Rio Negro, in Barcelos municipality (Amazonas State, Brazil), indicating that this river is not the southern boundary of the distribution of this species as previously proposed. Specimens from the north and south banks showed the same diploid number (2n = 16), while phylogenetic reconstructions based on maximum parsimony, distance, and maximum likelihood analyses grouped all specimens in a strongly supported clade comprising 2 separate lineages, in coincidence with their geographic distribution along riverbanks. Median-joining analysis showed a similar separation, with 22 transitions between the 2 groups, whereas time of divergence estimates indicated that the splitting of the C. lugens lineages occurred some 2.2 million years before present. Conservation strategies should take into consideration that this species might be sympatric with Callicebus torquatus at the south bank of Rio Negro.
Titi monkeys (genus Callicebus Thomas, 1903) comprise the most complex and diverse group of neotropical primates, mainly distributed in the tropical forests of the Amazonas and Orinoco basins, part of the Atlantic forest region of Brazil, and the Chaco and dry forests of Bolivia and Paraguay (Herhskovitz 1990). Phylogenetic arrangements based on morphometric attributes showed 5 different species groups of titi monkeys: torquatus, personatus, moloch, cupreus, and donacophilus (Kobayashi 1995). The latest revision of this genus recognized 28 species within these 5 groups and further recognized 2 new species from central Brazilian Amazonia, Callicebus bernhardi and Callicebus stephennashi, and included Callicebus lugens in the torquatus group together with Callicebus torquatus, Callicebus lucifer, Callicebus purinus, Callicebus regulus, and Callicebus medemi (van Roosmalen et al. 2002). These authors postulated that the Rio Negro represents the southern limit of the distribution of C. lugens and the northern boundary of the distribution of C. torquatus.
Phylogenetic reconstructions, based on sequence data of the mitochondrial gene cytochrome b, demonstrated the monophyly of the torquatus group and its relationship with other Callicebus (Bonvicino et al. 2003). This report also showed that C. lugens exhibited the lowest diploid chromosome number in the primate order (2n = 16), whereas chromosome painting with human chromosome probes demonstrated that its karyotype was highly shuffled in respect to the human (Stanyon et al. 2003).
Although phylogeographic studies of Callicebus are not presently available, studies of other primate genera have shown that rivers might represent drastic geographic barriers, as was the case of the Saguinus niger populations across the Rio Tocantins (Pará State, Brazil). Analysis of the D-loop region of the mitochondrial genome showed a higher divergence between populations of different margins than between valid species like Saguinus mystax and Saguinus imperator (Vallinoto et al. 2006).
As our field studies demonstrated that C. lugens actually occurs along both banks of the Rio Negro (Amazonas State, Brazil), the molecular divergence of specimens across this river was analyzed based on DNA sequence data of the mitochondrial gene cytochrome b. Although the number of animals was small, genetic distance estimates between animals from different banks were several times higher than between animals of a same bank, indicating a time of divergence of some 2 million years before present (MYBP).
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Material and Methods |
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Eight C. lugens were captured along tributaries of Rio Negro, in Barcelos municipality, Amazonas State. Four animals were captured along the left (or north) bank and 4 other along the right (or south) bank of this river, some 190 km apart (Figure 1). Along the left bank of Rio Negro, 3 females (MN69142/CRB2205 [GenBank AF524888], MN69288/CRB2570 [DQ337711], and MN69365/CRB2667 [DQ337712]) were collected at Sítio da Mamãe, Igarapé Japomeri (left bank of Rio Padauari), some 80 km apart from the site where a fourth female specimen (MN69435/CRB2433 [AF524889]) was captured, at Três Barracas, Igarapé Jauari (left bank of Rio Aracá). Along the right bank of Rio Negro, 3 animals (CRB1696 [DQ337710], MN69388/CRB2697 [DQ337709], and MN69389/CRB2698 [DQ337708]) were collected at Igarapé do Bainaiá, and a fourth specimen (female CRB2837 [DQ337707]) some 20 km apart, at Road Barcelos-Caurés, both near the city of Barcelos.
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All specimens from the left bank of Rio Negro and one specimen (CRB2837) from the right bank were karyotyped. Chromosome preparations were obtained from bone marrow culture in RPMI 1640, 20% fetal calf serum, colchicine (10–6 M) and ethidium bromide (5 µg/ml) for 2 h (Ikeuchi 1984).
DNA was extracted from tissue samples preserved in ethanol following Sambrook et al. (1989) and Smith et al. (1987). The complete cytochrome b gene (1140 bp) was sequenced in 6 specimens, and partial sequence data of 2 other C. lugens (GenBank AF524888 and AF524889) and 1 C. torquatus (AF524890) were completed. Cytochrome b was amplified with primers Citb-1 (5'-CGAAGCTTGATATGAAAAACCATCGTTG-3') and MVZ-14 (5'-GGTCTTCATCTYHGGYTTACAAGAC-3'), under the following conditions: 94 °C (2 min); 35 cycles: 94 °C (1 min), 50 °C (1 min), and 72 °C (1 min/30 s). Purified amplified products were sequenced in MegaBace 1000 and ABI-Prism 377 automatic sequencers after labeling with the DYEnamic ET Dye Terminator Sequencing kit for MegaBace and ABI PrismTM 377 DNA Analysis Systems and primers Citb-1, Citb-Alo (5'-ATAGCCACAGCATTCATAGGC-3'), and Alo Aot R (5'-TGAATGCTGTGGCTATRGTT-3'). Sequences were manually aligned and edited using Sequencing Navigator 3.3 software (Applied Biosystems 1994).
Genetic distance was estimated with p-distance model and pairwise deletion using the Molecular Evolutionary Genetics Analyses software (MEGA 3.1; Kumar et al. 2004). Phylogenetic analyses were carried out with PAUP* 4.0 b10 (Swofford 2003) for maximum parsimony (MP) and maximum likelihood (ML) analyses. Construction of the MP and ML topologies was carried out by heuristic search with stepwise addition of branches with 10 random replicates implemented by the algorithm tree bisection reconnection. In MP analysis, we worked with the random branch addition, with transition (ts) and transversion (tv) ratios of 1:1 and 8:1. This latter ratio was provided by Mega 3.1; nucleotide pair frequencies and ts:tv ratio being estimated without out-group data. Bootstrap estimates were based on 1000 replicates. ML analysis was carried out with the best-fit model of evolution of base substitution chosen by MODELTEST 3.07 (Posada and Crandall 1998), general time reversible model of nucleotide substitution with estimation of invariable sites and gamma correction (Rodríguez et al. 1990). One specimen belonging to the torquatus group (C. torquatus—AF524890 [GenBank] whose sequence data were completed to 1140 bp) and one specimen of the personatus group (Callicebus nigrifrons—AF524884 [GenBank] ) were also included in this analysis, with one Aotus trivirgatus (AY250707 [GenBank] ) and one Alouatta belzebul (AY374343 [GenBank] ) as out-groups.
Median joining (MJ) was carried out with NETWORK 4.1.0.8 [EC] for analyzing intraspecific phylogenies and evaluating structure and geographic distribution patterns (Bandelt et al. 1999; Posada and Crandall 2001; http://www.fluxus-engineering.com).
To estimate the time of C. lugens divergence, we applied 2-cluster and branch-length tests implemented in LINTREE (Takezaki et al. 1995), to examine the constancy of evolutionary rates of nucleotide substitutions. Sequences that evolve very quickly or slowly at a high significance level (5%) were eliminated. Distance analysis measured by neighbor joining (NJ) was estimated assuming p-distance model using MEGA 3.1. The rate of divergence of cytochrome b was calibrated at 19.0 MYBP corresponding to the splitting of the subfamily Pitheciinae (Schneider et al. 2001). To improve the accuracy of distance estimates we included 4 other specimens, one Chiropotes israelita (AY226189 [GenBank] ), one Chiropotes utahicki (AY226185 [GenBank] ), one Pithecia irrorata (AY226183 [GenBank] ), and a Callicebus specimen representative of the moloch group, Callicebus hoffmannsi (AF524885 [GenBank] ).
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Results |
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All karyotyped specimens, from both banks of the Rio Negro, showed 2n = 16, XX; FNa = 22, as previously reported for this species (Bonvicino et al. 2003). Their chromosome complement showed 8 chromosome pairs (4 very large-sized, bi-armed pairs, 1 medium-sized metacentric pair, and 3 small acrocentric pairs). The medium-sized metacentric pair had been identified as the X chromosome pair by ZOO-FISH, using a human X chromosome probe (Bonvicino et al. 2003).
Analysis of the complete cytochrome b sequence (1140 bp) of 8 C. lugens showed 31 mutations in a region encompassed between nucleotides (nt) 21 and 1126. Genetic distance estimates between C. lugens of different river margins ranged from 1.9% to 2.2%, contrary to estimates between left-bank haplotypes (0.0–0.2%) and from right-bank haplotypes (0.1–0.7%). Genetic distance estimates between C. torquatus and C. lugens haplotypes varied from 4.0% to 4.4% (Table 1).
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MP and ML analyses (Figure 2A) showed the monophyly of Callicebus and, within this clade, of the torquatus group, both with bootstrap values of 95% to 100%. The torquatus clade was divided into 2 sister branches, one with C. torquatus and another leading to a clade grouping; the 8 C. lugens that was further divided in 2 well-supported groups, one with the 4 specimens from the left bank of Rio Negro and another, with the 4 specimens from the right bank of this river (with bootstrap values of 69% to 100% and 94%, respectively). MJ showed the same geographic pattern, indicating at least 22 transitions between C. lugens haplotypes of different margins (Figure 2B). A calibrated NJ tree indicated that these C. lugens lineages split approximately 2.2 MYBP, whereas the divergence of Pitheciini tribe occur some 15.0 MYBP, and the 2 species of Chiropotes at 2.6 MYBP (Figure 2C).
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Discussion |
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Callicebus lugens from both banks of Rio Negro were karyotypically identical, as previously reported in this species (Bonvicino et al. 2003). Moreover, MP and ML analyses grouped them in a strongly supported clade that split in 2 sister lineages, leading to a left-bank clade and a right-bank clade. Three specimens from the right bank showed different haplotypes despite that they were collected at the same time and locality, whereas another specimen (CRB2837) was captured 1 year later in a site 20 km apart. Among the 3 animals collected in Igarapé Japomeri (left bank of Rio Negro), 2 animals captured in different years shared the same haplotype. The forth, left-bank specimen was captured some 80 km apart, separated from Igarapé Japomeri by rivers and patches of "campinarana" (a kind of nonforest, nonsavanna vegetation growing on an extremely poor soil [oligotrophic], common to the Amazonas basin and representing a natural barrier to this species). Genetic distance estimates, however, showed a higher divergence estimates between right-bank haplotypes than between left-bank haplotypes. Distance estimates between left- and right-bank haplotypes indicated that these 2 groups represent different evolutionary lineages of a karyotypically homogeneous taxon, a fact that must be taken into consideration in the management of this species. MJ analysis showed that the C. lugens groups differed by a minimum of 22 transitions and 1 median vector between closest haplotypes, whereas another median vector (non-sampled haplotype or extinct haplotype) was postulated within the right-bank group (Figure 2B).
These findings point to the Rio Negro as a natural geographic barrier to gene flow between C. lugens, although gene flow at the high course of Rio Negro cannot be ruled out. This pattern was coincident with similar findings in other primates distributed along the Rio Negro, like Alouatta, with Alouatta senicula along the right bank and Alouatta straminea along the left bank of this river. Similarly, the Rio Tocantins split S. niger in 2 different groups, one of which, at the west bank of this river, being more similar to specimens of the east bank of the Rio Xingu than to specimens from the east bank of Rio Tocantins (Vallinoto et al. 2006).
Divergence time estimates indicated that C. lugens of different Rio Negro margins split approximately at 2.2 MYBP, a time that is compatible with the river dynamics of the Amazon region indicating that the Rio Amazonas originated approximately 16 MYBP and that its current dynamics was only defined some 6 MYBP later (Kaandorp et al. 2005). Divergence might thus be explained by the Riverine Barrier hypotheses. It postulates that the development of Amazonian river system due to the Cenozoic uplift of the Andes fragmented the ranges of once widespread species, resulting in a relevant causal process of speciation across riverbanks (Marroig and Cerqueira 1997). Finally, our findings also indicated that C. lugens has a larger geographic distribution than the one proposed by van Roosmalen et al. (2002), extending to the south, along the right bank of the Rio Negro, disproving the postulation that this river might represent the southern limit of C. lugens, separating this species from C. torquatus. As this finding suggests that the southern population of C. lugens might be sympatric with C. torquatus, this must be taken into consideration for eventual conservation strategies involving breeding and re-introduction of specimens in selected areas.
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Acknowledgments |
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This work was supported by CNPq, FUNASA-Barcelos, Depto de Medicina Tropical-IOC-FIOCRUZ, and FAPERJ (Brazil). We are grateful to A. Junqueira, A. Queiroga, P. Albajar, I. G. Garcia, and R. Neri for helping us in field work. Animals were wild caught according to Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis licences 005/2003-CGFAU/LIC, 171/2004/-CGFAU/LIC, and 147/2004//-CGFAU/LIC.
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Footnotes |
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Corresponding Editor: Stephen J. O'Brien
Received January 20, 2006
Accepted August 30, 2006
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