Chromosome localization and gene synteny of the major histocompatibility complex in the owl monkey,Aotus

Rodent cells were hybridized with owl monkey (Aotus) cells of karyotypes II, III, V, and VI. Aotus-rodent somatic hybrid lines preferentially segregating Aotus chromosomes were selected to determine the chromosomal location of the major histocompatibility complex and other genes with which it is syntenic in man. Based on correlation between concordant segregation of the chromosome as visualized by G-banding and expression of the Aotus antigens or enzymes in independent Aotus-rodent hybrid clones, we have assigned Aotus gene loci for the MHC, GLO, ME₁, SOD₂, and PGM₃ to Aotus chromosome 9 of karyotype VI (2n=49/50), chromosome 10 of karyotype V (2n=46), and chromosome 7 of karyotypes II and III (2n = 54 and 53). On the basis of banding patterns we previously postulated that these chromosomes of the different karyotypes were homologous. The gene assignments reported here provide independent evidence for that hypothesis. Aotus chromosomes 9 (K-VI), 10 (K-V), and 7 (K-II, III) are homologous to human chromosome 6 in that they all code for the MHC, GLO, ME₁, SOD₂, and PGM₃. The structural differences between these homologous chromosomes probably resulted from a pericentric inversion.Abbreviations used in this paper MHC major histocompatibility complex - HLA human lymphocyte antigen - PGM3 phosphoglucomutase-3 - ME₁ cytoplasmic malic enzyme-1 - SOD₂ superoxide dismutase-B - GLO glyoxalase 1 - OMLA owl monkey leukocyte antigens - K karyotype - ₂-M ₂-microglobulin - DMEM Dulbecco's modification of Eagle's medium - PEG polyethylene glycol - HAT hypoxanthine, aminopterin, and thymidine - KC1 potassium chloride - G-band-trypsin Giemsa band     

Two new karyotypes in the Peruvian owl monkey (Aotus trivirgatus)

The observation of remarkable karyotypic variation in owl monkeys (Aotus trivirgatus) stimulated us to study the chromosomal evolution of this New World genus. As an extension of this project, we examined the chromosome complement of a “phenotype-B” Aotus population from Peru. In addition to karyotype V(2n = 46), two new karyotypes with diploid numbers of 47 and 48 were identified. A G-band comparison of these karyotypes indicated that the chromosome number polymorphism in these Peruvian owl monkeys resulted from a single fusion or fission event involving a single metacentric and two acrocentric chromosome pairs. This mechanism is also known to be responsible for the chromosome number polymorphism in at least two other populations of phenotype B Aotus, one from Colombia and the other from Panama.     

Aotus from the southwestern Amazon region is geographically and chromosomally intermediate betweenA. azarae boliviensis andA. infulatus

We described the karyotypes detected in 23Aotus specimens captured on Southwestern Amazon region (63°30'W, 9°00'S). This region should be occupied byA. nigriceps. However, the animals described here have a fur pattern and a karyotypes similar to those ofA. azarae boliviensis andA. infulatus. The females presented 50 chromosomes and the males 49 as a consequence of fusion between the chromosome Y and an autosome. This sample presented polymorphisms of G- and C-banding and NOR-staining. These animals are karyotypically intermediate betweenA. a. boliviensis andA. infulatus. The taxonomy and karyotypic evolution of these taxa are discussed.     

Molecular Phylogenetics of <Emphasis Type="Italic">Aotus</Emphasis> (Platyrrhini,Cebidae)

The accurate identification of taxa of Aotus is essential for 1) the development of precise biomedical assays, 2) the determination of potential illegal traffic of this genus, and 3) conservation. Although many studies have contributed to what we know about the phylogenetics of Aotus, none used a sufficiently large number of samples to clarify its complexity. To address this need, we sequenced 696 base pairs of the mitochondrial cytochrome-oxidase II gene (mtCOII) in 69 specimens of 7 taxa of Aotus. We also analyzed 8 microsatellite loci in 136 individuals of 6 taxa. In contrast to previous studies, we sampled only wild individuals and have a precise geographical origin for each one. The mtDNA results showed that: 1) the northern gray-necked group of Aotus is genetically more homogeneous than the polyphyletic red-necked group of Aotus; 2) the ancestors of Aotus vociferans seem to be the original species candidate for the current Aotus; 3) Aotus azarae azarae and A. a. boliviensis are the most differentiated taxa, likely a result of extreme genetic drift during stasipatric speciation; 4) the first genetic splits found among taxa of Aotus occurred during the Pliocene (or even Miocene) while the most recent ones happened during the Pleistocene, when forest refugia may have played an important role in speciation. The mean number of microsatellite alleles was 3–5.33 alleles per locus. We found some private alleles that could be useful in helping to identify illegal trade, although a larger sample size is needed to ensure that these alleles are really private to the relevant taxa. These new findings increase our understanding of the phylogeny of Aotus and the level of genetic diversity within different taxa of Aotus.     

Multi-directional chromosome painting maps homologies between species belonging to three genera of New World monkeys and humans

We mapped chromosomal homologies in two species of Chiropotes (Pitheciini, Saki Monkeys) and one species of Aotus (Aotinae, Owl Monkey) by multi-directional chromosome painting. Human chromosome probes were hybridized to Chiropotes utahicki, C. israelita and Aotus nancymae metaphases. Wooly Monkey chromosome paints were also hybridized to Owl Monkey metaphases. We established Owl Monkey chromosome paint probes by flow sorting and reciprocally hybridized them to human chromosomes. The karyotypes of the Bearded Saki Monkeys studied here are close to the hypothesized ancestral platyrrhine karytoype, while that of the Owl Monkey appears to be highly derived. The A. nancymae karyotype is highly shuffled and only three human syntenic groups were found conserved coexisting with 17 derived human homologous associations. A minimum of 14 fissions and 13 fusions would be required to derive the A. nancymae karyotype from that of the ancestral New World primate karyotype. An inversion between homologs to segments of human 10 and 16 suggests a link between Callicebus and Chiropotes, while the syntenic association of 10/11 found in Aotus and Callicebus suggests a link between these two genera. Future molecular cytogenetic work will be needed to determine whether these rearrangements represent synapomorphic chromosomal traits.     

Reference strand conformational analysis (RSCA) is a valuable tool in identifying MHC-DRB sequences in three species of Aotus monkeys

The Aotus monkey has been of great value in the pre-clinical study of malaria vaccine candidates. Several components of this primate’s immune system have been studied and they display great similarity to their human counterparts. Cloning and sequencing studies have revealed extensive sequence polymorphisms in Aotus MHC-DRB with very high similarities to several human allelic lineages, grouping at least nine distinct MHC-DRB lineages. As the efficacy of peptide vaccines in this animal model may be strongly influenced by exon 2 MHC-DRB polymorphism, the availability of a reliable and rapid MHC-DRB typing method for three species of Aotus (Aotus nancymaae, Aotus vociferans and Aotus nigriceps) is necessary. Reference strand conformational analysis (RSCA) was used here for differentiating the distinctive Aotus MHC-DRB sequences’ mobility using five fluorescently labelled references proved to be very useful for resolving closely related sequences, establishing the number of sequences transcribed in a particular monkey and their identity. The RSCA method’s reliability in terms of identifying Aotus MHC-DRB sequences will facilitate evaluating individual responsiveness to vaccines and prompt studies associating susceptibility/resistance to infectious agents or auto-immune disease, for which Aotus monkeys may be considered to be an appropriate animal model.     

Karyological study of the dolly vardenSalvelinus malma from Alaska

The karyotype of the dolly vardenSalvelinus malma from Alaska was analysed. A pair of huge acrocentrics, which could have resulted from either tandem fusion or centric fusion followed by pericentric inversion, was observed. Ag-NORs were observed at the terminal regions of the second largest chromosome pair, and individual size-differences in Ag-NORs and in chromosomes with Ag-bands were found. From a comparison of the karyotypes of the dolly varden from Alaska and Hokkaido (Japan), karyotype differentiation by pericentric inversion and translocation was discussed.     

Owl monkeys (Aotus) are highly divergent in mitochondrial Cytochromec Oxidase (COII) sequences

The evolutionary history and differentiation of the owl or night monkeys (Aotus),remain poorly resolved. Variation in pelage and skeletal morphology is relatively minor across their broad range, but cytogenetic studies have revealed that at least 12 karyotypically distinct forms exist, with 2Nchromosome numbers ranging from 46 to 58. We obtained DNA samples from three putative species- A. lemurinus, A. nancymae,and A. azarae-and five karyotypes (I, II, III, IV and VI), amplified the mitochondrial cytochrome coxidase subunit II gene (COII) via PCR and sequenced it. Comparisons of the sequences indicate relatively high levels of sequence divergence among the putative species (4.3 to 6.3%), and suggest that the taxa are genetically quite distinct and have likely experienced extended periods of isolated evolution. The levels of COII divergence represent approximately one- third of the levels found between divergent platyrrhine genera, such as Aotus, Saimiriand Callimico.Using estimates of substitution rates of COII evolution in hominoid primates,the estimated date of divergence for the AotusCOII sequences is 3.6 million years. The AotusCOII data support the existence of multiple species of Aotus,with origins predating late Pleistocene climatic events. Although A. nancymaeand A. azaraeboth live south of the Amazon River and have been considered members of the same species group, phylogenetic analysis of the COII sequences does not support a close relationship between them.     

Evolution of karyotypes and differentiation in 13 Rattus Species

Karyotypes of 13 Rattus species collected in Asia and Oceania were analysed with special interest to karyotype evolution and species differentiation. They were classified into three groups according to their karyotype similarity. Four species (R. annandalei, R. exulans, R. muelleri and R. norvegicus) with 2n=42 and a karyotype similar to some of the polymorphic karyotypes in the Asian black rats (R. rattus) are classified into the first group. Pericentric inversion of some acrocentrics seemed to have caused the differentiation of these species. The other four species (R. bowersii, R. fuscipes, R. leucopus and R. conatus) with similar karyotypes as the above group, but lower chromosome numbers than 2n=42 are classified into the second group. Robertsonian fusion in some acrocentrics observed in the first group are suggested to have caused the development of the species in this group. The remaining four species (R. sabanus, R. canus, R. huang and R. niviventer) with karyotypes markedly different from the above two in having a fewer number of small metacentrics are classified into the third group. They seemed to be more primitive karyotypes than the other Rattus species. By the comparison between the polymorphic karyotypes in the black rat, and karyotypes in its related species it was suggested that the former had occurred as primary events to the differentiation of the latter. Parallelism between the karyotype evolution and the species differentiation was discussed.Contribution No. 874 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (No. 92159 and 92332).     

MHC class I genes in the owl monkey: mosaic organisation, convergence and loci diversity

The MHC class I molecule plays an important role in immune response, pathogen recognition and response against vaccines and self- versus non-self-recognition. Studying MHC class I characteristics thus became a priority when dealing with Aotus to ensure its use as an animal model for biomedical research. Isolation, cloning and sequencing of exons 1–8 from 27 MHC class I alleles obtained from 13 individuals classified as belonging to three owl monkey species (A. nancymaae, A. nigriceps and A. vociferans) were carried out to establish similarities between Aotus MHC class I genes and those expressed by other New and Old World primates. Six Aotus MHC class I sequence groups (Ao-g1, Ao-g2, Ao-g3, Ao-g4, Ao-g5 and Ao-g6) weakly related to non-classical Catarrhini MHC were identified. An allelic lineage was also identified in one A. nancymaae and two A. vociferans monkeys, exhibiting a high degree of conservation, negative selection along the molecule and premature termination of the open reading frame at exon 5 (Ao-g5). These sequences high conservation suggests that they more likely correspond to a soluble form of Aotus MHC class I molecules than to a new group of processed pseudogenes. Another group, named Ao-g6, exhibited a strong relationship with Catarrhinis classical MHC-B-C loci. Sequence evolution and variability analysis indicated that Aotus MHC class I molecules experience inter-locus gene conversion phenomena, contributing towards their high variability.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

Karyotypic comparison among Cebuella pygmaea,Callithrix jacchus and C. emiliae (Callitrichidae,Primates) and its taxonomic implications

The karyotype of Cebuella pygmaea (2n=44) obtained by G-, C-banding, and NOR-staining is described. This species presents a heteromorphic C band in the intersticial region of the short arm of chromosome 2. The data obtained were compared with those previously described for the karyotypes of Callithrix jacchus and Callithrix emiliae. The three species differ in the amount and distribution of non-centromeric constitutive heterochromatin. The importance of the variation in constitutive heterochromatin for the phylogeny of the group is discussed. Comparison of the karyotypes in terms of G-banding patterns showed that C. pygmaea and C. emiliae differ from C. jacchus by a Robertsonian translocation and a paracentric inversion, whereas C. pygmaea and C. emiliae differ from each other by a reciprocal translocation between an acrocentric autosome and the short arm of the submetacentric chromosomes that distinguishes their karyotypes from that of C. jacchus. The possible evolutionary paths followed by the karyotypes of the three species are discussed.     

Karyotypic evolution in Aotus

Great karyotypic diversity exists within the platyrrhine genus Aotus. Primarily by comparing banded karyograms of different forms of Aotus, the pattern of karyotypic evolution can be assessed. Out-group comparisons are used to establish primitive and derived states of particular chromosomes, and a parsimonious cladogram is constructed. Other karyotypic changes are then positioned at appropriate nodes of the cladogram. The resulting phylogenetic hypothesis is entirely self-consistent, is in accord with out-group comparisons, does not invoke hybridization between ancestral forms, and, importantly, indicates a single origin for each rearrangement of euchromatic segments. Moreover, it is consistent with the hypothesis, derived from pelage studies, that the more southerly Aotus taxa constitute a holophyletic group. The reconstructed ancestral karyotype had a diploid number of 54. There has been little loss of euchromatic material during Aotus evolution.     

INVERSION POLYMORPHISM,LONGEVITY, AND BODY SIZE IN A NATURAL POPULATION OF DROSOPHILA BUZZATII

In this study we present the results of an analysis of differential longevity associated with Drosophila buzzatii second chromosome inversion karyotypes based on the assessment of more than 1000 individuals collected in a natural population. Comparisons of inversion frequencies between emerged and bait-collected flies showed not only that inversion arrangements were associated with differential longevity, but also that selection was sex specific. Because each individual fly was scored for thorax length and karyotype, we were able to show that longevity selection favoring larger flies coupled with the average effect of inversions on thorax length can account for the change of inversion frequencies due to longevity in females. The observed genotypic-by-sex interaction could be an important mechanism involved in the maintenance of the polymorphism. Arrangement 2Jz³, which was shown to impaired fecundity in two independent previous studies, exhibited a positive effect on longevity. This pattern of negative pleiotropy may be another plausible mechanism accounting for the maintenance of the polymorphism.     

An inversion supergene in Drosophila underpins latitudinal clines in survival traits

Chromosomal inversions often contribute to local adaptation across latitudinal clines, but the underlying selective mechanisms remain poorly understood. We and others have previously shown that a clinal inversion polymorphism in Drosophila melanogaster, In(3R)Payne, underpins body size clines along the North American and Australian east coasts. Here, we ask whether this polymorphism also contributes to clinal variation in other fitness‐related traits, namely survival traits (lifespan, survival upon starvation and survival upon cold shock). We generated homokaryon lines, either carrying the inverted or standard chromosomal arrangement, isolated from populations approximating the endpoints of the North American cline (Florida, Maine) and phenotyped the flies at two growth temperatures (18 °C, 25 °C). Across both temperatures, high‐latitude flies from Maine lived longer and were more stress resistant than low‐latitude flies from Florida, as previously observed. Interestingly, we find that this latitudinal pattern is partly explained by the clinal distribution of the In(3R)P polymorphism, which is at ~ 50% frequency in Florida but absent in Maine: inverted karyotypes tended to be shorter‐lived and less stress resistant than uninverted karyotypes. We also detected an interaction between karyotype and temperature on survival traits. As In(3R)P influences body size and multiple survival traits, it can be viewed as a ‘supergene’, a cluster of tightly linked loci affecting multiple complex phenotypes. We conjecture that the inversion cline is maintained by fitness trade‐offs and balancing selection across geography; elucidating the mechanisms whereby this inversion affects alternative, locally adapted phenotypes across the cline is an important task for future work.     

Similarity of giemsa banding patterns of chromosomes in several species of the Genus Rattus

Giemsa banding patterns of chromosomes in seven Rattus species were compared. Four species (R. rattus tanezumi, R. norvegicus, R. exulans and R. muelleri) had all 2n=42 and their karyotypes and banding patterns were similar, although slight differences were observed. Another subspecies (R. rattus rattus) and two other species (R. fuscipes and R. conatus) had fewer chromosomes than the above species by having large biarmed chromosomes developed probably by Robertsonian fusion. The origin of the arms of biarmed chromosomes was recognized by their characteristic banding patterns. The remaining species, R. sabanus, had a karyotype markedly different from the other species by having two small metacentrics although in the others their number was 7. Banding patterns of the chromosomes in this species, however, were also very similar to those of the other, and therefore the 7 small metacentrics seemed to have originated by pericentric inversion of small acrocentrics.Contribution No. 912 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan, Nos. 90183, 90375 and 744002.     

Robertsonian fusion and centric fission in karyotype evolution of higher plants

Robertsonian fusion and centric fission are uniquely detectable in comparative studies of karyotype patterns. They are the most important types of karyotype change in animals but seem to be relatively uncommon in higher plants. Both modify intra- and interchromosomal recombination and linkage relationships and consequently patterns of genetic variation. When differentiating populations or species they can produce postulating barriers to gene flow. The number of reported cases of fusion or fission in higher plants has increased over the years but remains low, and most of these are casual comparisons of karyotypes without any follow-up investigation. This review focuses on more adequate studies made in a few groups. Studies in the Tradescantieae produce the strongest evidence for fusion as a type of orthoselection in the subfamily. Some species ofLycoris are also considered to have evolved their karyotypes in that way. Some genera of slipper orchids and the cycad genusZamia have populations where atypical chromosome number increase can be attributed to fission probably as a result of stressful influences. It is suggested that fusion may have been involved in the evolution of many stable karyotypes and that fission is generally a secondary destabilizing mechanism which may lead to refusion in the long term. Their proven incidence remains making it unwise to suggest that they have been major influences in karyotype evolution in higher plants.     

Structural Alteration of the Membrane of Erythrocytes Infected with Plasmodium falciparum1

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30–40 nm in height and 90–100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

Identification,classification and evolution of Owl Monkeys (<Emphasis Type="Italic">Aotus</Emphasis>, Illiger 1811)

Background  

Owl monkeys, belonging to the genus Aotus, have been extensively used as animal models in biomedical research but few reports have focused on the taxonomy and phylogeography of this genus. Moreover, the morphological similarity of several Aotus species has led to frequent misidentifications, mainly at the boundaries of their distribution. In this study, sequence data from five mitochondrial regions and the nuclear, Y-linked, SRY gene were used for species identification and phylogenetic reconstructions using well characterized specimens of Aotus nancymaae, A. vociferans, A. lemurinus, A. griseimembra, A. trivirgatus, A. nigriceps, A. azarae boliviensis and A. infulatus.