Structural variations of subterminal satellite blocks and their source mechanisms as inferred from the meiotic configurations of chimpanzee chromosome termini.

African great apes have large constitutive heterochromatin (C-band) blocks in subtelomeric regions of the majority of their chromosomes, but humans lack these. Additionally, the chimpanzee meiotic cell division process demonstrates unique partial terminal associations in the first meiotic prophase (pachytene). These are likely formed as a result of interaction among subtelomeric C-band blocks. We thus conducted an extensive study to define the features in the subtelomeric heterochromatic regions of chimpanzee chromosomes undergoing mitotic metaphase and meiotic cell division. Molecular cytogenetic analyses with probes of both subterminal satellite DNA (a main component of C-band) and rDNA demonstrated principles of interaction among DNA arrays. The results suggest that homologous and ectopic recombination through persistent subtelomeric associations (post-bouquet association observed in 32% of spermatocytes in the pachytene stage) appears to create variability in heterochromatin patterns and simultaneously restrain subtelomeric genome polymorphisms. That is, the meeting of non-homologous chromosome termini sets the stage for ectopic pairing which, in turn, is the mechanism for generating variability and genomic dispersion of subtelomeric C-band blocks through a system of concerted evolution. Comparison between the present study and previous reports indicated that the chromosomal distribution rate of sutelomeric regions seems to have antagonistic correlation with arm numbers holding subterminal satellite blocks in humans, chimpanzees, and gorillas. That is, the increase of subterminal satellite blocks probably reduces genomic diversity in the subtelomeric regions. The acquisition vs. loss of the subtelomeric C-band blocks is postulated as the underlying engine of this chromosomal differentiation yielded by meiotic chromosomal interaction.

Considerable Synteny and Sequence Similarity of Primate Chromosomal Region VIIq31.

Human chromosome 7 has been the focus of many behavioral, genetic, and medical studies because it carries genes related to cancer and neurodevelopment. We examined the evolution of the chromosome 7 homologs, and the 7q31 region in particular, using chromosome painting analyses and 3 paint probes derived from (i) the whole of chimpanzee chromosome VII (wcVII), (ii) human 7q31 (h7q31), and (iii) the chimpanzee homolog VIIq31 (cVIIq31). The wcVII probe was used instead of the whole human chromosome 7 because the chimpanzee contains additional C-bands and revealed large areas of synteny conservation as well as fragmentation across 20 primate species. Analyses focusing specifically on the 7q31 homolog and vicinity revealed considerable conservation across lineages with 2 exceptions. First, the probes verified an insertion of repetitive sequence at VIIq22 in chimpanzees and bonobos and also detected the sequence in most subtelomeres of the African apes. Second, a paracentric inversion with a breakpoint in the cVIIq31 block was found in the common marmoset, confirming earlier studies. Subsequent in silico comparative genome analysis of 17 primate species revealed that VIIq31.1 is more significantly conserved at the sequence level than other regions of chromosome VII, which indicates that its components are likely responsible for critical shared traits across the order, including conditions necessary for proper human development and wellbeing.

Hypervariability of Nucleolus Organizer Regions in Bengal Slow Lorises, Nycticebus bengalensis (Primates, Lorisidae).

Slow lorises are a cryptic species complex, and thus genetic markers are needed to identify distinct evolutionary lineages or species. We examined the nucleolus organizer regions (NORs) of Bengal slow lorises (Nycticebus bengalensis) using FISH with 18S rDNA (rDNA-FISH) and silver nitrate staining (Ag-NOR stain). Ten individuals of the putatively single species N. bengalensis showed higher variability in localization than 3 other congeners, though their overall karyotypes were similar. The rDNA-FISH analysis detected a total of 18 loci, in contrast to previous studies of other slow loris species that revealed far fewer (6-10) loci. Eight of the 18 loci detected in the present analysis were found to be semi-stable localizations at 4 different chromosomes, while 10 were found to be unstable localizations at 5 other chromosomes. The semi-stable locations showed occasional presence/absence of variations for rDNA-FISH, and unstable locations were polymorphic among individuals, contributing to the higher variability of NORs in this taxon. We hypothesize that the larger numbers of rDNA loci found in N. bengalensis were introduced by genomic dispersion through ectopic recombination in association with terminal regions including rDNA. Such differences are potentially very powerful chromosomal markers to be used in species identification and conservation.

The genome of the blood fluke Schistosoma mansoni.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes.

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

Higher-order repeat structure in alpha satellite DNA is an attribute of hominoids rather than hominids.

Alpha satellite DNA (AS), a major DNA component of primate centromeres, is composed of a tandem array of repeat units of approximately 170 bp. The AS of hominids (family Hominidae; humans and great apes) includes sequences organized into higher-order repeat (HOR) structures, with a periodic appearance of multiple copies of the basic repeat units. Here, we identified an HOR in AS of the siamang, a small ape phylogenetically distinct from hominids but included in hominoids (superfamily Hominoidea). We sequenced long stretches of genomic DNA, and found a repetition of blocks consisting of six and four basic repeat units. Thus, AS organization into HOR is an attribute of hominoids, rather than, as currently postulated, hominids. In addition to centromeres, siamangs carry AS in terminal heterochromatin blocks, and it cannot be determined at present whether these HOR-containing AS sequences originate from the centromere or from the terminal heterochromatin. Even if the latter is the case, these sequences might affect the composition of centromeric AS by being transferred to the centromere.

Tandem repeat sequences evolutionarily related to SVA-type retrotransposons are expanded in the centromere region of the western hoolock gibbon, a small ape.

Hoolock hoolock (the western hoolock gibbon) is a species of the family Hylobatidae (small apes), which constitutes the superfamily Hominoidea (hominoids) together with Hominidae (great apes and human). Here, we report that centromeres or their vicinities in this gibbon species contain tandem repeat sequences that consist of 35-50-bp repeat units, and exhibit a sequence similarity with the variable number of tandem repeat (VNTR) region of the SVA, LAVA and PVA transposons. SVA is a composite retrotransposon thought to have been formed by fusion of three solo elements in the common ancestor of hominoids. LAVA and PVA are recently identified retrotransposons that have the same basic structure as SVA. Thus, the large-scale tandem repeats in the centromere region may have been derived from one or more of SVA-type transposons, including the three mentioned above and other yet unknown elements, or the repeat sequences could have served as a source for such elements. Amplification of VNTR-related sequences in another gibbon species, Hoolock leuconedys (eastern hoolock gibbon), has recently been reported, but it is yet to be examined whether the large-scale tandem repeats observed in the two species originated from a single event that occurred in their common ancestor. The repeat sequences in the western hoolock gibbon are mostly 40 kb or more in length, are present in 28 of the 38 chromosomes of the somatic cells, and are homozygous for chromosomal presence/absence.

In situ hybridization analysis of gibbon chromosomes suggests that amplification of alpha satellite DNA in the telomere region is confined to two of the four genera.

The siamang (Symphalangus syndactylus), a species of the family Hylobatidae (gibbons), carries large blocks of constitutive heterochromatin in the telomere region of chromosomes. We recently found that alpha satellite DNA constitutes these heterochromatin blocks as a main component. Alpha satellite DNA, tandem repeat sequences of 171-bp repeat units, is a major component of centromeres in primates. In addition to the siamang, the white-cheeked gibbon (Nomascus leucogenys) was previously found to carry the alpha satellite DNA in the telomere region, although not as large a scale as the siamang. Gibbons comprise four genera: Hoolock, Hylobates, Nomascus, and Symphalangus. Here, we report that the amplification of alpha satellite DNA in the telomere region is probably confined to two genera: Nomascus and Symphalangus. We examined one species of Hoolock and four species of Hylobates and obtained evidence against such an amplification event in these species. The phylogenetic relationship of the four gibbon genera remains unclear. One simple explanation for the current distribution of the telomere region alpha satellite DNA would be that Nomascus and Symphalangus are relatively closely related and the amplification occurred in their common ancestor.

Frequent gene conversion events between the X and Y homologous chromosomal regions in primates.

BACKGROUND: Mammalian sex-chromosomes originated from a pair of autosomes. A step-wise cessation of recombination is necessary for the proper maintenance of sex-determination and, consequently, generates a four strata structure on the X chromosome. Each stratum shows a specific per-site nucleotide sequence difference (p-distance) between the X and Y chromosomes, depending on the time of recombination arrest. Stratum 4 covers the distal half of the human X chromosome short arm and the p-distance of the stratum is approximately 10%, on average. However, a 100-kb region, which includes KALX and VCX, in the middle of stratum 4 shows a significantly lower p-distance (1-5%), suggesting frequent sequence exchanges or gene conversions between the X and Y chromosomes in humans. To examine the evolutionary mechanism for this low p-distance region, sequences of a corresponding region including KALX/Y from seven species of non-human primates were analyzed. RESULTS: Phylogenetic analysis of this low p-distance region in humans and non-human primate species revealed that gene conversion like events have taken place at least ten times after the divergence of New World monkeys and Catarrhini (i.e., Old World monkeys and hominoids). A KALY-converted KALX allele in white-handed gibbons also suggests a possible recent gene conversion between the X and Y chromosomes. In these primate sequences, the proximal boundary of this low p-distance region is located in a LINE element shared between the X and Y chromosomes, suggesting the involvement of this element in frequent gene conversions. Together with a palindrome on the Y chromosome, a segmental palindrome structure on the X chromosome at the distal boundary near VCX, in humans and chimpanzees, may mediate frequent sequence exchanges between X and Y chromosomes. CONCLUSION: Gene conversion events between the X and Y homologous regions have been suggested, mainly in humans. Here, we found frequent gene conversions in the evolutionary course of primates. An insertion of a LINE element at the proximal end of the region may be a cause for these frequent conversions. This gene conversion in humans may also be one of the genetic causes of Kallmann syndrome.

Behavioral sleep in captive owl monkey (Aotus azarae) and squirrel monkey (Saimiri boliviensis).

The objective of this study was to test the hypothesis that activity-behavioral sleep parameters differ between nocturnallyactive owl monkeys and diurnally-active squirrel monkeys which are sympatric and of Bolivian origin. The total sleep time (TST) and sleep episode length (SEL) of 7 adult owl monkey siblings and 4 adult squirrel monkeys were quantitated by actigraphy for 7 days under captive conditions. The higher TST/24 h values and longer SEL/12 h quiescent phase quantitated for owl monkeys in comparison to that of squirrel monkeys clearly indicate that the behavioral sleep is markedly different between these two groups, though they are sympatric in wild. Significant differences noted in the sleep architecture between squirrel monkeys and owl monkeys can be attributed to the influences in the selected sleep niche, threat perception from predators, and disturbances from natural elements (especially rain) in the natural habitat.

Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases.

Egg production is responsible for life cycle progression and host immunopathology during schistosomiasis, with the associated parasite molecules being investigated as potential novel chemotherapeutic targets. Here, we characterize two Schistosoma mansoni products, tyrosinase 1 and tyrosinase 2 (SmTYR1/SmTYR2) and show that their diphenol oxidase enzyme activities are critical for eggshell formation and production. The genes encoding these bifunctional enzymes (monophenol and diphenol oxidases) result from a duplication event that likely occurred before speciation and exist in the parasite's genome as multiple copies, which are linked and localized to chromosomes 4 and W. SmTYR1/SmTYR2 transcription and diphenol oxidase action are developmentally regulated with most enzyme activity localized to the eggshell-producing cells contained within the vitellaria of adult female worms. Importantly, kojic-acid mediated inhibition (IC50=0.5 microM) of SmTYR1/SmTYR2's diphenol oxidase activity during in vitro culture of sexually mature adult worms resulted in a significant decrease in the production of phenotypically normal eggs. Therefore our data suggest that SmTYR1/2 inhibition represents a novel and potentially effective strategy for combating schistosomiasis and furthermore, it may point to new methods for combinatorial control of immunopathology and egg transmission during platyhelminth infection.

A most distant intergeneric hybrid offspring (Larcon) of lesser apes, Nomascus leucogenys and Hylobates lar.

Unlike humans, which are the sole remaining representatives of a once larger group of bipedal apes (hominins), the "lesser apes" (hylobatids) are a diverse radiation with numerous extant species. Consequently, the lesser apes can provide a valuable evolutionary window onto the possible interactions (e.g., interbreeding) of hominin lineages coexisting in the same time and place. In the present work, we employ chromosomal analyses to verify the hybrid ancestry of an individual (Larcon) produced by two of the most distant genera of lesser apes, Hylobates (lar-group gibbons) and Nomascus (concolor-group gibbons). In addition to a mixed pelage pattern, the hybrid animal carries a 48-chromosome karyotype that consists of the haploid complements of each parental species: Hylobates lar (n = 22) and Nomascus leucogenys leucogenys (n = 26). Studies of this animal's karyotype shed light onto the processes of speciation and genus-level divergence in the lesser apes and, by extension, across the Hominoidea.

Night Monkey Hybrids Exhibit De Novo Genomic and Karyotypic Alterations: The First Such Case in Primates.

Using molecular chromosomal analyses, we discovered night monkey hybrids produced in captivity from matings between a female Aotus azarae boliviensis (2n = 50) and a male Aotus lemurinus griseimembra (2n = 53). The parents produced seven offspring in total, including one male and six females-a pattern consistent with Haldane's rule. Chromosomal studies were conducted on four of the hybrid offspring. Two of them showed relatively "simple" mixture karyotypes, including different chromosome numbers (2n = 51, 52), which were formed because of a heteromorphic autosome pair in the father (n = 26, 27). The other two hybrid monkeys exhibited de novo genomic and karyotypic alterations. Detailed analysis of the alterations revealed that one individual carried a mixture karyotype of the two parental species and an X chromosome trisomy (53,XXX). The second individual displayed trisomy of chromosome 18 (52,XX,+18) and a reciprocal translocation between autosomes 21 and 23 (52,XX,+18,t(21;23)). Interestingly, the second monkey exhibited mosaicism among blood cells (mos52,XX,+18[87]/52,XX,+18,t(21;23)[85]), but only a single karyotype (52,XX,+18) in skin fibroblast cells. The X- and 18-trisomies were derived from a doubling of the mother's chromosomes in early embryonic cell division, and the reciprocal translocation likely developed in the bone marrow of the offspring, considering that it was observed only in blood cells. Such occurrence of trisomies in hybrid individuals is a unique finding in placental mammals.

Co-Opted Megasatellite DNA Drives Evolution of Secondary Night Vision in Azara's Owl Monkey.

Owl monkeys (genus Aotus) are the only taxon in simian primates that consists of nocturnal or otherwise cathemeral species. Their night vision is superior to that of other monkeys, apes, and humans but not as good as that of typical nocturnal mammals. This incomplete night vision has been used to conclude that these monkeys only secondarily adapted to a nocturnal lifestyle, or to their cathemeral lifestyle that involves high night-time activity. It is known that the rod cells of many nocturnal mammals possess a unique nuclear architecture in which heterochromatin is centrally located. This "inverted nuclear architecture", in contrast with "conventional nuclear architecture", provides elevated night vision by passing light efficiently to the outer segments of photoreceptors. Owl monkey rod cells exhibit an intermediate chromatin distribution, which may provide them with less efficient night vision than other nocturnal mammals. Recently, we identified three megasatellite DNAs in the genome of Azara's owl monkey (Aotus azarae). In the present study, we show that one of the three megasatellite DNAs, OwlRep, serves as the primary component of the heterochromatin block located in the central space of the rod nucleus in A. azarae. This satellite DNA is likely to have emerged in the Aotus lineage after its divergence from those of other platyrrhini taxa and underwent a rapid expansion in the genome. Our results indicate that the heterochromatin core in the A. azarae rod nucleus was newly formed in A. azarae or its recent ancestor, and supports the hypothesis that A. azarae, and with all probability other Aotus species, secondarily acquired night vision.

Higher-order repeat structure in alpha satellite DNA occurs in New World monkeys and is not confined to hominoids.

Centromeres usually contain large amounts of tandem repeat DNA. Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units. HOR-carrying AS also occurs in other hominoids, but results reported to date for phylogenetically more remote taxa have been negative. Here we show direct evidence for clear HOR structures in AS of the owl monkey and common marmoset. These monkeys are New World monkey species that are located phylogenetically outside of hominoids. It is currently postulated that the presence of HOR structures in AS is unique to hominoids. Our results suggest that this view must be modified. A plausible explanation is that generation of HOR structures is a general event that occurs occasionally or frequently in primate centromeres, and that, in humans, HOR-carrying AS became predominant in the central region of the centromere. It is often difficult to assemble sequence reads of tandem repeat DNAs into accurate contig sequences; our careful sequencing strategy allowed us to overcome this problem.

Adenylosuccinate lyase of Schistosoma mansoni: gene structure, mRNA expression, and analysis of the predicted peptide structure of a potential chemotherapeutic target.

Adenylosuccinate lyase is an enzyme used in parasite nucleotide salvage pathways that cleaves adenylosuccinate into adenosine 5'-monophosphate and fumarate. A cDNA encoding adenylosuccinate lyase from the trematode parasite Schistosoma mansoni has been cloned for analysis. Sequencing of the cDNA revealed an open reading frame of 1454 nucleotides that codes for a protein with a predicted mass of about 54.5 kDa. Comparative analysis of the predicted protein sequence shows that S. mansoni adenylosuccinate lyase has a lot of similarity with human adenylosuccinate lyase. Genomic analysis using S. mansoni adenylosuccinate lyase-containing bacterial artificial chromosome (BAC) clones revealed a gene of approximately 19.4 kb consisting of eight exons and seven introns. Intron 6 was found to contain a novel 2.9 kb long terminal repeat retrotransposon with direct terminal repeats of 500 nucleotides. Fluorescence in situ hybridisation mapping localised S. mansoni adenylosuccinate lyase to the Z and W chromosomes. Analysis of S. mansoni adenylosuccinate lyase mRNA expression levels using real time reverse transcriptase (RT)-PCR showed that S. mansoni adenylosuccinate lyase is expressed at higher levels in the female worms than in the male worms and is expressed at different levels than other purine nucleotide salvage enzymes. Male homogenate showed a specific activity of 10.3 units/mg protein while the female showed a specific activity of 24.2 units/mg protein. These data indicate that S. mansoni adenylosuccinate lyase is an important parasite enzyme and should be examined as a potential chemotherapeutic target.

FISH mapping for helminth genome.

Basic techniques for fluorescence in situ hybridization (FISH) mapping that have been used in genome projects on schistosomes and filariae are introduced. The chapter shows techniques specific for bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones and includes experiences of chromosome preparation, DNA labeling, hybridization, microscopy, and localization of BAC clones.

Evolutionary origin of higher-order repeat structure in alpha-satellite DNA of primate centromeres.

Alpha-satellite DNA (AS) is a main DNA component of primate centromeres, consisting of tandemly repeated units of ~170 bp. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, in which a block of multiple repeat units forms a larger repeat unit and the larger units are repeated tandemly. The presence of HOR in AS is widely thought to be unique to hominids (family Hominidae; humans and great apes). Recently, we have identified an HOR-containing AS in the siamang, which is a small ape species belonging to the genus Symphalangus in the family Hylobatidae. This result supports the view that HOR in AS is an attribute of hominoids (superfamily Hominoidea) rather than hominids. A single example is, however, not sufficient for discussion of the evolutionary origin of HOR-containing AS. In the present study, we developed an efficient method for detecting signs of large-scale HOR and demonstrated HOR of AS in all the three other genera. Thus, AS organized into HOR occurs widely in hominoids. Our results indicate that (i) HOR-containing AS was present in the last common ancestor of hominoids or (ii) HOR-containing AS emerged independently in most or all basal branches of hominoids. We have also confirmed HOR occurrence in centromeric AS in the Hylobatidae family, which remained unclear in our previous study because of the existence of AS in subtelomeric regions, in addition to centromeres, of siamang chromosomes.

Locational diversity of alpha satellite DNA and intergeneric hybridization aspects in the Nomascus and Hylobates genera of small apes.

Recently, we discovered that alpha satellite DNA has unique and genus-specific localizations on the chromosomes of small apes. This study describes the details of alpha satellite localization in the genera Nomascus and Hylobates and explores their usefulness in distinguishing parental genome sets in hybrids between these genera. Fluorescence in situ hybridization was used to establish diagnostic criteria of alpha satellite DNA markers in discriminating small ape genomes. In particular we established the genus specificity of alpha satellite distribution in three species of light-cheeked gibbons (Nomascus leucogenys, N. siki, and N. gabriellae) in comparison to that of Hylobates lar. Then we determined the localization of alpha satellite DNA in a hybrid individual which resulted from a cross between these two genera. In Nomascus the alpha satellite DNA blocks were located at the centromere, telomere, and four interstitial regions. In Hylobates detectable amounts of alpha satellite DNA were seen only at centromeric regions. The differences in alpha satellite DNA locations between Nomascus and Hylobates allowed us to easily distinguish the parental chromosomal sets in the genome of intergeneric hybrid individuals found in Thai and Japanese zoos. Our study illustrates how molecular cytogenetic markers can serve as diagnostic tools to identify the origin of individuals. These molecular tools can aid zoos, captive breeding programs and conservation efforts in managing small apes species. Discovering more information on alpha satellite distribution is also an opportunity to examine phylogenetic and evolutionary questions that are still controversial in small apes.

The first finding of chromosome variations in wild-born western hoolock gibbons.

Hoolock gibbons (genus Hoolock) are a group of very endangered primate species that belong to the small ape family (family Hylobatidae). The entire population that is distributed in the northeast and southeast of Bangladesh is estimated to include only around 350 individuals. A conservation program is thus necessary as soon as possible. Genetic markers are significant tools for planning such programs. In this study, we examined chromosomal characteristics of two western hoolock gibbons that were captured in a Bangladesh forest. During chromosome analysis, we encountered two chromosome variations that were observed for the first time in the wild-born western hoolock gibbons (Hoolock hoolock). The first one was a nonhomologous centromere position in chromosome 8 that was observed in the two examined individuals. The alteration was identical in the two individuals, which were examined by G-band and DAPI-band analyses. Chromosome paint analyses revealed that the difference in the centromere position was due to a single small pericentric inversion. The second variation was a heterozygous elongation in chromosome 9. Analysis by sequential techniques of fluorescence in situ hybridization with 18S rDNA and silver nitrate staining revealed a single and an inverted tandem duplication, respectively, of the nucleolus organizer region in two individuals. These chromosome variations provide useful information for the next steps to consider the evolution and conservation of the hoolock gibbon.