Cytogenetic analysis shows extensive genomic rearrangements between red howler (Alouatta seniculus Linnaeus) subspecies.код для вставкиСкачать
American Journal of Primatology 35171-183 (1995) RESEARCH ARTICLES Cytogenetic Analysis Shows Extensive Genomic Rearrangements Between Red Howler (Alouatta seniculus, Linnaeus) Subspecies R. STANYON'.. S. - TOFANELLI'. M.A. MORESCALCHI'. G. AGORAMOORTHY3. O.A. RYDER4, AND J. WIENBERG' ~~ 'Institute of Physical Anthropology, university of Genoa, Genoa, Italy; 2Znstitute of Anthropology, University of Florence, Italy; 3Conseruation a n d Research Center, National ZooloPical Park, Smithsonian Institution, U.S.A.; *Center for Reproduction ofEndanpered Speci&, Zoological Society of Sun Diego, U.S.A.; 51nstitute. for Anthropology a n d Human Genetics, University of Munich, Germany A comparison of the G-banded karyotypes of two red howler subspecies, Alouatta seniculus arctoidea and A. s. sara, showed that they differed by at least 14 chromosomal rearrangements. Genomic reshuffling is so great that homologs between subspecies could not be found for some chromosome, while the assignment of homology for other chro'mosomes remains uncertain. The two red howlers, however, share a n unusual X,X,Y lY2/ XlXlX2X, sex-chromosome system that resulted from a Y-autosome translocation, probably in a common ancestor. The great chromosomal variability resulting from rapid chromosomal evolution in howlers indicates that cytogenetic data could make an important contribution to resolving phylogenetic and conservation problems in this group of highly conspicuous New World Monkeys. o 1995 Wiley-Liss, Inc. Key words: new world monkeys, platyrrhini, phylogeny, speciation, chromosomes, Y-autosome translocation INTRODUCTION Cytogenetic studies of New World primates (Playtrrhini) have demonstrated that karyological data can provide essential evolutionary information for primatologists and taxonomists. Chromosomal studies of the genus Alouatta (howler monkeys) have revealed an unexpected amount of variability both between and within traditionally recognized species. However, considering th a t howler monkeys are highly conspicuous with the widest geographic distributions of any neotropical monkey, cytogenetic reports are few and far from complete. There is no information for some recognized species and karyological description of subspecies are wholly inadequate. The genus is traditionally divided into six species: Alouatta belzebul, A. caraya, A. fusca, A , palliata, A . pigra, and A . seniculus. Their distribution runs from Received for publication January 24, 1994; revision accepted J u n e 14, 1994. Address reprint requests to R. Stanyon, Institute of Physical Anthropology, University of Genoa, Via Balbi 4, Genoa, Italy. 0 1995 Wiley-Liss, Inc. 172 I Stanyon et al. southern Mexico to northern Argentina, including parts of Peru and Bolivia [Hill, 1960; Wolfheim, 19831. Karyological studies even before the introduction of banding methods suggested that howler monkeys were karyologically variable [Chu & Bender, 1961; Bender & Chu, 1963; Egozcue & Egozcue, 19661. This karyological variability was confirmed when chromosomal banding methods were used to study the several species of the single subfamily genus, Alouatta [Koiffmann & Saldanha, 1974; Ma e t al., 1975; Yunis e t al., 1976; Armada et al., 1987; Lima & Seuanez, 1989, 19911. Briefly, diploid numbers in these monkeys range from 2n = 43 to 2n = 54, and fundamental numbers (FNs, number of chromosome arms) go from 56 to 76. The number of NOR (nucleolar organizing region) bearing chromosomes has been reported as 2 (A. seniculus stramineus, A. belzebul nigerrima), 6 (A. belzebul belzebul),or 8 ( A . belzebul) [Armada et al., 1987; Lima & Seuanez, 1989, 19911. Howler monkeys also have unusual karyological traits such as microchromosomes and various Y-autosome translocations. Alouatta seniculus (red howler) has previously been the subject of five research reports: two reports used classical staining [Chu & Bender, 1961; Bender & Chu, 19631 while the other three employed banding techniques [Yunis et al., 1976; Minezawa et al., 1985; Lima & Seuanez, 19911. If we accept the division of A. seniculus into 9 subspecies [Hill, 1960; see Fig. 11, then there have been reports on 4 subspecies: A. s. seniculus [Chu & Bender, 1961; Bender & Chu, 1963; Yunis et al., 19761,A. s. stramineus [Lima & Seuanez, 19911,A. s. macconnelZi [Lima et al., 19901, and A. s. Sara [Minezawa et al., 19851. In red howlers, the diploid numbers vary from 43 to 51 and FNs vary from 56 to 76. Various sex chromosome systems have been reported: the typical mammalian XY/XX (male/female) sex determination system, and a number of Y-autosome translocations. Here we present the first cytogenetic data for A. seniculus arctoidea and compare these chromosomes with those of A. s. Sara. Both subspecies present Y-autosome translocations and a n identical X,X,Y 1Y2/X,X,X,X2 (male/female) sex determination system. However, their karyotypes are surprisingly different. MATERIALS AND METHODS The Alouatta seniculus arctoidea (ASA) samples consisted of four heparinized blood draws that were taken immediately after capture at Mato Masaguaral (8'31" 67'35'W), Estado Guarico, Venezuela, from two males and two females. The samples were hand-carried to the Institute of Anthropology, Florence, Italy, where standard whole-blood cultures were established [Small et al., 19851. The tissue culture consisted of 0.3 ml of whole blood inoculated into 8 ml of tissue culture medium (RPMI 1640,15% FCS, 50 ug/ml Concavalin-A type I11 [SIGMA]). After 90 hours of culture a t 38.5"C, colcemid (0.05 ug/ml) was added for 60 minutes. The cultures were then harvested according to standard methods (hypotonic treatment with 0.075 M KC1 for 15 minutes and fixation in methanol: acetic acid). Fibroblasts from a single male Alouatta seniculus Sara (ASS) were established by traditional methods. This A . s. Sara was captured from the wild in Bolivia and purchased by the San Diego Zoo from World Wide Primates. The cells were cultured in DMEM supplemented with 10% fetal bovine serum. Cell harvesting was a s previously reported [Stanyon & Galleni, 19911. Chromosome spreads were banded as follows: G-banding with trypsin according to Small et al. , C-banding according to Sumner 119721, Ag-NOR banding according to Goodpasture and Bloom . The Ag-NOR preparations were sequentially G-banded with trypsin to identify the NOR bearing chromosomes. To aid comparisons between individuals and subspecies, photographic enlarge- .pa!pn?s s~eur!ue lie ~ 0 . 1 palood 3 s a d k + o h q uoy?nlosaJ uinrpaur uo pasvq SRM we~Zo!py ay? 30 u i q i e d Zuypueq ay? PUB say3ads y m a ~ 0 . 1 3sadL?od.Isy aag ~ 0 . 1 3sr+uauramsRaur 30 srseq a q uo papnqsuo3 aiaM surs.~Zo!p!. ayA .aurosowo.np 'x ay? 03 paz!p.~spue?s aiaM squaur wx OOZT 009 OOP 0 174 I Stanyon et al. Fig. 2. a: Shows the G-banded karyotype and b: Shows the idiogram of Alouatta seniculus arctoidea (ASA). The asterisks in the idiogram show the location of nucleolar organizing regions (NORs). RESULTS G-banded Karyotypes of Alouatta seniculus arctoidea Figure 2a shows the G-banded karyotype and 2b shows the idiogram of ASA. The two male ASAs had a diploid number of 2n = 45, while the two females had a diploid number of 44. The difference in diploid numbers was due to microchromosomes (four in the females and five in the two males). Therefore, the diploid number of both sexes, if microchromosomes are not considered, is 40. The fundamental number (FN) of both males is 58 (10 biarmed chromosomes, 26 acrocentric autosomes, 4 microchromosomes, and 4 sex chromosomes (the Xs are biarmed, while one Y is acrocentric and the other is submetacentric)).The fundamental number for females is also 58 due to the fact that our two females had one less microchromosome. If microchromosomes are not considered, then the FN is 53 in males and 54 in females. Sex chromosome system. The male sex chromosome complement can be described a s X,X,Y,Y, and the female complement a s X,X,X,X,. The X, is a typical primate X-chromosome, while X, was originally a submetacentric autosome. The Y, chromosome is a borderline submetacentric while Y, is a n acrocentric. The Y, derives from the q arm of the same autosome as X,, and Y, is the translocation product of the original Y chromosome with the p arm of this autosome. G-banded Karyotypes of Alouatta seniculus Sara Figure 3a shows a G-banded karyotype and 3b the idiogram from ASS. The single male ASS had a diploid number of 2n = 50 and a FN of 67 (14 biarmed and 28 acrocentric autosomes, 4 microchromosomes and 4 sex chromosomes (the Xs are biarmed, while 1 Y is acrocentric and the other is submetacentric)). The sex chromosome system was the same as in ASA. If microchromosomes are not considered then the diploid number is 46 and the FN = 63. Sequential G and AG-NOR Staining In ASA, three chromosome pairs show AG-NOR banding. One NOR is located on the short arm of chromosome 3 next to the centromere. Another NOR is located lRi w ......... /////j$j ..,:_:.:, ...... 3 1 I! .....:.:. ::/:j:;:; .:.:.:.:. ..... ......... ..... .... _:.:.:.:. :.:.: ..:... .. R H ::::::::: i:::::::: ! 1 2 l :::::_:.:. :z;:;:s; ........ U El 3 4 :::;::::: €j 5 I :.:.:...: i .:.:.:.:. ..... ;:::.:::. .:.:::.:. 6 ::/:.:::. 7 8 tl 9 €I ::::::::: H 10 U 11 12 13 14 15 16 17 18 Y1 s g H ::::..::. b 19 20 Fig. 2b y2 176 I Stanyon et al. Fig, 3. a: Shows the G-banded karyotype and b: Shows the idiogram of Alouatta seniculus Sara (ASS). The asterisks in the idiogram show the location of nucleolar organizing regions (NORs). a t the centromere of the medium acrocentric chromosome (n 9) and is polymorphic with Ag-NOR staining. Finally, a small acrocentric pair of chromosomes (n 17) shows a polymorphism for the NOR location. The NOR can be located either at the centromere or a t the terminal part of the long arm (see Fig. 4). In ASS, two pairs of biarmed chromosomes showed NORs: n 9 and n 20 (Figure 4). There is a polymorphism for the size of the NOR regions, but not for position as in ASA. Comparison of Karyotypes in A. s. arctoidea and A. s. Sara A comparison of diploid and fundamental numbers makes i t clear that these two howlers have quite distinct karyotypes: 2n = 44, 45, FN = 58 in A. s. arctoidea, and 2n = 50 and FN = 67 in A . s. Sara. If microchromosomes are not included in the diploid numbers would be 40 and 46 respectively. On the basis of G-banding, homologs between the chromosomes of the two red howlers can be proposed for a majority of the chromosomes with the exception of ASA18 and ASS13. However, it must be noted that the homologies proposed are working hypotheses that need to be verified by molecular methods. In some cases more than one hypothesis could be proposed, with one author or another favoring alternate solutions. Here we present only one set of proposals (Fig. 5). Whole chromosomal homologs. There are very close if not identical banding patterns between chromosomes ASAIASS: 4 5 , 9/8,11/14, 16/12, 17/20, and the sex chromosomes. A paracentric inversion apparently has occurred between ASA 8 and ASS 7. ASA 14 appears similar to ASS 17 except for the location of the centromere. Other proposed homologs are not as clear cut: ASA13 could derive from ASS11 by a pericentric inversion; ASAl2 may differ by a paracentric inversion from ASS18. Homologs involved in translocations. Two clear Robertsonian translocations have occurred between ASA and ASS: Chromosome ASAlp is homologous to ASS4 while ASAlq is homologous to ASS3; ASA l o p = ASS21 and ASAlOq = ASS19. Additionally, there appears to have been 7 tandem translocations: ASA2p and a small part of the long arm next to the centromere derive from ASSlp, the w ::::::::. w H ::::::::: ::::::::: :::::::::. Ii I .:.:.:... i :.::>::. ::::::::; c::::::: ::::::::: .;.:.:.:. ..... ::::::::: .:.:.:.:_ 1 10 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 178 I Stanyon et al. Fig. 4. A The sequential NOR and trypsin G-banding of NOR bearing chromosomes in Alouatta seniculus arctoidea (ASA); B: A partial metaphase from Alouatta seniculus Sara (ASS) after sequential NOR and G-banding. The arrows point to the NORs. remainder of the long arm is composed of ASS10 and ASS6; ASA3p and the 3q next to the centromere is composed of ASS9 while most of the long arm is homologous to ASSlq; ASA5 may derive from ASS16 and ASS23; ASA7 may derive from ASS15 and ASS22. Finally, ASS2 may derive from a fusion of ASA6 and ASA15. NOR-bearing chromosomes. We found six NOR-bearing chromosomes in ASA and four in ASS. If the between taxon homologs are correct then four NORs were derived from a common ancestor: NORs on ASA31ASS9 and ASA171ASS20. There are additional NORs on ASA9 not present in ASS. DISCUSSION Our results confirm that howler monkeys have high karyological variability. However, the extent of differences we revealed between supposed subspecies of red howlers was beyond that expected. The number of chromosomal mutations is impressive. A conservative estimate would include: 9 translocations (2 Robertsonian translocations, 7 tandem translocations); 4 intrachromosomal rearrangements (1 pericentric inversion and 3 paracentric inversions); a n additional NOR site in ASA; a number of unidentified changes to account for the chromosomes for which no homologs were found, ASA18 and ASS13. Sex Chromosome System in Howlers On the other hand, both subspecies have apparently conserved a n unusual X,X,Y ,Y,/X,X,X,X, sex-chromosome system. An identical system has been reported for some other red howler subspecies, A. seniculus stramineus and A . s. macconnelli, and the published chromosomes appear to have very similar banding patterns to the sex chromosomes of our two “subspecies.” In this previous report Red Howler Subspecies Genomic Rearrangements / 179 Fig. 5. A comparison of Alouatta seniculus arctoidea (ASA) and A. s. Sara (ASS) G-banded chromosomes. The homologies proposed here are one set of working hypotheses. The ASA chromosomes are numbered below in bold numbers and the ASS chromosomes are numbered to the side. the Y-autosome translocation was confirmed by meiotic studies [Lima & Seuanez, 19911. However, the only prior report on A. s. Sara proposed a Y-autosome translocation which resulted in a X,X,Y/X,X,X,X, sex-chromosome system. The Y was a large acrocentric that resulted from a n insertion of the Y with a n autosome; male and females differed in chromosome number 46 in females and 45 in males (if microchromosomes are not counted). Our single male had, not counting microchromosomes, 46 chromosomes. However, we were not able to directly compare banding patterns with the A. s. Sara previously published and it is unclear if any other difference exists between these samples. A. seniculus seniculus has been reported to have a normal mammalian XYIXX sex chromosome system [Yunis et al., 19761. However, Y-autosome translocations have been reported for other howler species: A. palliata [Ma et al., 19751;A. fusca clamitans [Lima & Seuanez, 19911; A. belzebul belzebul with a X,X,Y/X,X,X,X, system [Armanda et al., 19871. In primates, Y-autosome translocations are rare. In catarrhines there is only one report [Presbytis cristatus, Dutrillaux et al., 19841. In platyrrhines there have been a number of reports of Y-autosome translocations in several species (i.e., in Aotus triuirgatus [Ma et al., 19761; Cacajao caluus rubicundus [Dutrillaux et al., 19861, C . melanocephalus [Koiffmann & Saldanha, 19813), and Callimico goeldii [Seuanez et al., 19891. There are no reports on Y-autosome translocations in prosimians. Microchromosomes Red howlers, including our samples of A . seniculus arctoidea and A. s. Sara, are all distinguished by having microchromosomes (MCs). This cytogenetic trait is 180 I Stanyon et al. unknown in other monkeys, including other howler species, but i t is common in lemurs [Rumpler & Dutrillaux, 19861. The difference in the number of MCs between males (5) and females (4) in our sample of ASA is probably more apparent than real. We studied only two individuals from each sex and further studies with larger samples are needed to establish the actual variability in the number of MCs in ASA. Previous reports (cited above) show that the number of MCs in red howlers may vary from one to five. However, it is clear that the MCs are responsible for the major part of the variability in diploid numbers within red howler populations. If MCs are not considered the diploid numbers in A. seniculus range from 40 to 46. On this basis Columbian populations of A. s. seniculus (40) are closest to the Venezuelian populations of A . s. arctoidea (401, while the other populations of A . seniculus (A. s. Sara, A . s. stramineus, A. s. macconelli) studied so far all have a diploid number of 45 or 46. The similarity in diploid numbers, however, may hide profound karyological differences. Studies using banding methods should be used to compare various subspecies and local populations. The origin and significance of the MCs is not known. The within population variability in the number of MCs in red howlers, suggests that they contain little, if any, essential genetic information. Future research is needed to determine if they are composed of repetitive DNA sequences (i.e., late replication heterochromatin). Theoretically, MCs could be produced during Robertsonian translocations, but they are then usually lost [King, 19931. The presence of MCs in red howlers may indicate that their origin is recent. Significance of the Cytogenetic Variability Within AZouuttu seniculus The cytogenetic differences shown between A. s. arctoidea and A . s. Sara are more typical of differences seen between species belonging to different genera. For example, the differences in karyotypes are greater than those found between humans and any great ape [Stanyon, 19921. It is most likely that the cytogenetic differences that separate the two red howler “subspecies” are sufficient to insure reproductive isolation. Minezawa et al.  state that a t least 10 chromosomal rearrangements separate A . s. Sara (2n = 45,46 not counting MCs) from A. s. seniculus (2n = 401, and this is in line with the difference we found between A. s. Sara (2n = 46) and A. s. arctoidea (2n = 40). Armanda e t al.  in their original report on Alouatta belzebul belzebul (2n = 49, 50) and A . b. nigerrima (2n = 50) report that the karyotypes were “drastically different”: no homologs were found for four chromosome pairs in each subspecies. Although Lima et al.  describe the karyotype of A. s. macconnelli (2n = 46) and Lima and Seuanez [19911 present chromosomal data from A . s. stramineus (2n = 46), outside of the sex chromosomes, there is no direct comparison of these two red howlers. Lima and Sueanez  do state, however, that these two species are more similar to each other more than either is to A. s. seniculus or to A . s. Sara. Taxonomic implications. The most obvious implication of the marked karyological variability in red howlers is that i t would be worthwhile to investigate with a fuller range of criteria, the taxonomic status of A. seniculus “subspecies” [Minezawa et al., 1985; Lima & Seuanez, 19911. The conservation implications are clear: as for other organisms in tropical forest regions, i t is probable that the biological diversity and number of species have been underestimated. The use of cases of rapid karyological evolution for phylogenetic studies. A second significance of the chromosome data is that the variability between howler species and “subspecies” indicates that the karyotype in these primates has Red Howler Subspecies Genomic Rearrangements / 181 evolved rapidly. It is well appreciated that rapidly evolving systems permit high phylogenetic resolution. However, counting this report, only five of the nine seniculus “subspecies” have been examined, unfortunately in most cases by different workers using different methods with a bare minimum of comparisons. It is probable that cytogenetic data in howlers could provide significant evolutionary information and help clarify how chromosomes are related to population divergence and speciation. Clearly, i t would be worthwhile to study cytogenetically additional howler species, “subspecies”, and populations, preferably sampled from the wild. Possible general implications for platyrrhine evolution. In general, New World monkeys appear to be karyotypically variable. In squirrel monkeys (Saimiri) three different pericentric inversions have distinct geographic distribution among subspecies [Jones et al., 1973; Moore e t al., 19901. Within and between the various species and subspecies of Ateles four different pericentric inversions can be found [Kunkel et al., 1980; de Boer & de Bruijn, 19901. Subspecies of Ateles paniscus (A.p . paniscus, 2n = 32, and A . p . chamek, 2n = 34) differ by a probable tandem fusion and two pericentric inversions [de Boer & de Bruijn, 19901. Among primates, the howler cytogenetic situation most closely resembles that of the owl or night monkeys, genus Aotus [Brumback et al., 1971; Reumer & de Boer, 1980; Ma, 1981; Galbreath, 1983; Hershkovitz, 1983; Ma e t al., 19851. This genus is one of the most karyotypically diverse genera of mammals and there are 14 karyotypically definable populations [Ma et al., 19911. It is probable that when howler monkeys are better known they will prove to be a t least, if not more, karyotypically differentiated than the owl monkeys. Hypotheses can be developed to help explain why New World monkeys and especially Aotus and Alouatta are so chromosomally variable. One hypothesis is that chromosomal differentiation occurred as a result of isolation in forest refugia during the quaternary [Ma, 1981, 1976; Kunkel e t al., 19801. Chromosomal mutations would have been more easily fixed due to inbreeding among small groups of isolated monkeys [Ma, 19801. A . seniculus is a forest monkey and can be found in a range of forest habitats including small isolated patches. Even when other species of primates have disappeared because of deforestation, the red howlers are found occupying the last isolated stands of timber [Wolfheim, 19831. The fact that two different subfamilies have undergone extensive genomic rearrangements may suggest a common cause and similar adaptive capacities. Since chromosomal variability is still found within populations, it is an open question whether the karyological transformation process is punctuated or still taking place. On the other hand, the striking karyological diversity in both Aotus and A l ouatta may be more apparent simply because traditional taxonomic methods based on morphology, especially pelage, have not been sufficient to recognize the real biological divisions and diversity of South American primates. Clearly, taxonomic and phylogenetic decisions must attempt to explain and integrate all available data, including the chromosomes. CONCLUSIONS 1. We found that the karyotypes of two “subspecies” of red howlers were surprisingly different. Alouatta seniculus arctoidea had a diploid number of 2n = 44, 45 with a fundamental number (FN) of 58. A . seniculus Sara had a diploid number of 2n = 50 with a FN = 67. 2. A conservative estimate of chromosomal rearrangements between these two red howlers would include nine translocations and four inversions, but homologs could not be proposed for several chromosomes. Further, work using molecular 182 I Stanyon et al. methods, particularly in situ hybridization, will be needed to completely define the differences between karyotypes. 3. On the other hand, these two seniculus taxa are both characterized by unusual cytogenetic traits, such as microchromosomes and a XlX,Y,Y,/X,XlX2X, sex-chromosome system (resulting from a Y -autosome translocation), that were probably inherited from a common ancestor. 4.The variability between howler species and “subspecies” indicates that the karyotype in these primates has evolved rapidly and that cytogenetic data in howlers could help clarify how chromosomes are related to population divergence and speciation. It is most likely that the cytogenetic differences that separate the two red howler “subspecies” are sufficient to insure reproductive isolation. It would be worthwhile to have more complete cytogenetic data on Alouatta, in part because rapidly evolving systems permit high phylogenetic resolution. 5. Taxonomic and phylogenetic decisions must attempt to explain and integrate all available data, including the chromosomes. ACKNOWLEDGMENTS R.S. and M.A.M. thank the director and personnel of the Institute of Comparative Anatomy, University of Genoa, for access to the institute’s facilities. The authors also thank the three unnamed reviewers for valuable suggestions. This research was funded in part by the MURST 60% Grants, 1992. REFERENCES Armada, J.L.A.; Barroso, C.M.L.; Lima M.M.C.; Muniz, J.A.P.C.; Seuanez, H.N. Chromosome studies in Alouatta belzebul. AMERICAN JOURNAL OF PRIMATOLOGY 13:283-296, 1987. Bender, M.A.; Chu, E.H.Y. The chromosomes of primates. Pp. 261-310 in EVOLUTIONARY BIOLOGY AND GENETIC BIOLOGY OF THE PRIMATES. J . Buettner-Janusch, ed. New York, Academic Press, 1963. Brumback, R.A.; Staton R.D.; Benjamin, S.A.; Lang, C.M. The chromosomes of Aotus triuiergatus Humboldt 1812. FOLIA PRIMATOLOGICA 15:264-273, 1971. Chu, E.H.Y.; Bender, M.A. Chromosome cytology and evolution in primates. SCIENCE 133:1,399-1,405, 1961. de Boer, L.E.M.; de Bruijn, M. Chromosomal distinction between the red-faced and black-faced spider monkeys (Ateles paniscus paniscus and A . p . charnek). ZOO BIOLOGY 9:307-316, 1990. Dutrillaux, B.; Couturier, J.; Viegas-Pequignot, E. Evolution chromosomique de Platyrhiniens. MAMMALIA 5056-81., 1986. Dutrillaux, B.; Webb, G.; Muleris, M.; Couturier, J.; Butler, R. Chromosome study of Presbytis cristatus: Presence of a complex Y-autosome rearrangement in the male. ANNALES DE GENETIQUE 27:l34, 1984. Egozcue, J.; Egozcue, M.V. The chromosome complement of the howler monkey (Al- ouatta caraya Humboldt, 1812). CYTOGENETICS 520-294, 1966. Galbreath, G.L. Karyotypic evolution in Aotus. AMERICAN JOURNAL OF PRIMATOLOGY 4:245-251, 1983. Goodpasture, C.; Bloom, S.E. Visualization of nucleolar organizer regions in mammalian chromosome using silver staining. CHROMOSOMA 53:37-50, 1975. Hershkovitz, P. Two new species of night monkeys, genus Aotus (Cebidae, Platyrrhini): a preliminary report on Aotus taxonomy. AMERICAN JOURNAL OF PRIMATOLOGY 4:209-243, 1983. Hill, W.C.O. PRIMATE COMPARATIVE ANATOMY AND TAXONOMY, IV. CEBIDAE, PART A. Edinburgh, Edinburgh University Press, 1960. Jones, T.C.; Thorington, R.W.; Hu, M.M.; Adams, E.; Cooper, R.W. Karyotypes of Squirrel Monkeys (Saimiri sciureus) from different geographic regions. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 38:269-278, 1973. King, M. SPECIES EVOLUTION: THE ROLE OF CHROMOSOME CHANGE. Cambridge, Cambridge University Press, 1993. Koiffmann C.P.; Saldanha, P.H. Cytogenetics of Brazilian monkeys. JOURNAL OF HUMAN EVOLUTION 3:275-282, 1974. Koiffmann, C.P.; Saldanha, P.H. The karyotype of Cacajao metanocephatus (Platyrrhini, Primates). FOLIA PRIMATOLOGICA 36:150-155, 1981. Red Howler Subspecies Genomic Rearrangements / 183 Kunkel, L.M.; Heltne, P.G.; Borgaonkar, D.S. Chromosomal variation and Zoogeography i n Atleles. INTERNATIONAL JOURNAL OF PRIMATOLOGY 1:223232,1980. Lima, M.M.C.; Sampaio, M.1.C .; Schneider, M.P.C.: Scheffrahn. W.: Schneider,, H.:. Salzano, F.M. Chromosome and protein variation i n red howler monkeys. BRAZILIAN JOURNAL OF GENETICS 13(4): 789-802,1990. Lima, M.M.C.; Seuanez, H.N. Chromosome studies in t h e red howler monkey, Alouatta seniculus stramineus (Platyrrhini, Primates): description of a n X,X,Y,Y,/ X,X,X,X, sex-chromosome system and karyological comparison with other subspecies. CYTOGENETICS AND CELL GENETICS 57~151-156, 1991. Lima, M.M.C.; Seuanez, H.N. Cytogenetic characterization of Aluoatta belzebul with atypical pelage coloration. FOLIA PRIMATOLOGICA 52:97-101, 1989. Ma, N.S.F. Chromosome evolution in the owl monkey, Aotus. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 54: 293-303, 1981. Ma, N.S.F.; Elliot, M.W.; Morgan, L.; Miller, A.: Jones. T.C. Translocation of Y chromosome to an autosome in t h e Bolivian owl monkey, Aotus. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 45: 191-202, 1976. Ma, N.S.F.; Jones, T.C.; Thorington, R.W.; Miller. A,: Morean. L. Y-autosome translocation in the Howler Monkey, Alouatta palliata. JOURNAL O F MEDICAL PRIMATOLOGY 4:299-307, 1975. Ma, N.S.F.; Aquino, R.; Collins, W.E. Two new karyotypes in the Peruvian owl monkey (Aotus triuieraatus). AMERICAN JOURNAL O F PRIMATOLOGY 9:333341, 1985. Ma. N.S.F.: Gerhard. D.S.: Bock. S.C. Homologs of eleven loci on 'human chromosome 11 assigned to two owl monkey chromosomes. CYTOGENETICS AND CELL GENETICS 56:206-211, 1991. Minezawa, M.; Harada, M.; Jordan, O.C.; Borda, C.J.V. Cytogenetics of Bolivian endemic red howler monkeys (Alouatta seniculus sara): Accessory chromosome and , , I I Y-autosome translocation related numerical variations. KYOTO UNIVERSITY OVERSEAS RESEARCH REPORT O F NEW WORLD MONKEYS, 5:7-16, 1985. Moore, C.M.; Harris, C.P.; Abee, C.R. Distribution of chromosomal polymorphism in three subspecies of squirrel monkeys (genus Saimiri). CYTOGENETICS AND CELL GENETICS 53:118-122, 1990. Reumer, J.W.F.; de Boer, L.E.M. Standarization of Aotus chromosome nomenclature with description of the 2n = 49-50 karyotype and that of a new hybrid. JOURNAL OF HUMAN EVOLUTION 9:461-482. 1980. Rumpler, Y.; Dutrillaux, B. Evolution chromosomique des Prosimiens. MAMMALIA 50182-107, 1986. Seuanez, H.F.; Forman, L.; Matoyoshi, T.; Fanning, T.G. The Callimico goeldii (Primates, Platyrrnini) genome: Karyology and middle repetitive (Line-1) DNA seqeunces. CHROMOSOMA 98:389-395,1989. Small, M.F.; Stanyon, R.; Smith, D.; Sineo, L. High resolution chromosomes of rhesus macaques. AMERICAN JOURNAL O F PRIMATOLOGY 9:63-67,1985. Stanyon, R. How polymorphisms and homoplasy can be informative about the evolution and phylogeny of humans and apes. Pp. 423-439 in TOPICS IN PRIMATOLOGY 1. T. Nishida; W.C. McGrew; P. Marler; M. Pickford; F.B.M. de Waal, eds. Tokyo, University of Tokyo Press, 1992. Stanyon, R.; Galleni, L. A rapid fibroblast culture method for Mammalian chromosome. BOLLETION ZOOLOGIC0 582383, 1991. Sumner, A.T. A simple technique for demonstrating centromeric heterochromatin. EXPERIMENTAL CELL RESEARCH 75: 304-306, 1972. Wollfheim, J.H. PRIMATES OF THE WORLD: DISTRIBUTION. ABUNDANCE AND CONSERVATION. Seattle, University of Washihgton Press, 1983. Yunis, E.J.; Torres de Caballero, O.M.; Ramirez, C.; Ramirez, E. Chromosomal variations in the Primate Alouatta seniculus seniculus. FOLIA PRIMATOLOGICA 25:215-224, 1976.