Karyological Analysis and NOR Polymorphism of Phayre's Langur

Nucleus DOI 10.1007/s13237-017-0220-9

ORIGINAL ARTICLE

Karyological analysis and NOR polymorphism of phayre’s langur, Trachypithecus phayrei crepuscula (Primate, Colobinae) in Thailand

1 2,3 2 4,5 Krit Pinthong • A. Tanomtong • S. Khunsook • I. Patawang • 6 7 W. Wonkaonoi • P. Supanuam

Received: 13 August 2016 / Accepted: 19 September 2017 Ó Archana Sharma Foundation of Calcutta 2017

Abstract Karyological analysis of Phayre’s langur T. phayrei crepuscula are 224 and 259, respectively, and (Trachypithecus phayrei crepuscula) at Songkhla Zoo, each chromosome pair could be clearly differentiated. In Thailand has been reported in this present article. Con- addition, the long arm of subcentromeric chromosome pair ventional staining, GTG-banding, and high-resolution 17 showed clearly observable nucleolar organizer regions/ technical analysis were carried out on standard whole NORs. This is the ﬁrst report on polymorphism of NORs in blood T-lymphocyte culture from 4 specimens of T. phayrei crepuscula. Thus the results indicated that a T. phayrei crepuscula. The results showed that the diploid heteromorphic chromosome pair with different sizes of number is 2n = 44 and the fundamental number is 88 in NORs is present for chromosome pair 17 (17a 17b) in both both male and female. The autosomes consisted of 6 large male and female animal. The results provide useful infor- metacentric, 10 large submetacentric, 2 large acrocentric, 6 mation for further cytogenetic study of this species. The m medium metacentric, 14 medium submetacentric, 2 small karyotype formula could be deduced as: 2nðÞ¼44 L6 þ sm a m sm sm a submetacentric, and 2 small acrocentric chromosomes. The L10 þ L2 þ M6 þ M14 þ S2 þ S2 þ 2 Sex-chromosomes. X chromosome is the large submetacentric chromosome, while the Y chromosome is medium metacentric chromo- Keywords Phayre’s langur Á Trachypithecus phayrei some. From GTG-banding and using high-resolution crepuscula Á Karyotype Á Chromosome techniques, the numbers of bands and locations in

& A. Tanomtong 3 Toxic Substances in Livestock and Aquatic Animals [email protected] Research Group, Khon Kaen University, Muang, Khon Kaen 40002, Thailand Krit Pinthong [email protected] 4 Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai 50200, Thailand S. Khunsook [email protected] 5 Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Muang, I. Patawang Chiang Mai 50200, Thailand [email protected] 6 Major of Biology, Faculty of Science and Technology, W. Wonkaonoi Mahasarakham Rajabhat University, Muang, [email protected] Mahasarakham 44000, Thailand P. Supanuam 7 Program of Biology, Faculty of Science, Ubon Ratchathani [email protected] Rajabhat University, Ubon Ratchathani, Muang 34000, Thailand 1 Program of Biology, Faculty of Science and Technology, Surindra Rajabhat University, Surin, Muang 32000, Thailand 2 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 123 Nucleus

Introduction Procolobus, Presbytis, Semnopithecus, Pygathrix, Rhinop- ithecus, Nasalis, and Simias. The genus Trachypithecus was Phayre’s langur (Trachypithecus phayrei), as known as suggested the following 17 species: T. auratus, T. barbei, T. Phayre’s leaf monkey or Phayre’s lutung, is old world cristatus, T. delacouri, T. ebenus, T. francoisi, T. geei, T. monkey in subfamily Colobinae. In addition, this subfamily germaini, T. hatinhensis, T. johnii, T. laotum, T. obscurus, T. includes the genera Trachypithecus, Colobus, Piliocolobus, phayrei, T. pileatus, T. poliocephalus, T. shortridgei, and T. vetulus [13, 14]. The Phayre’s langur is a long tailed herbivorous primate animal. The colour of its body is rusty-brown to gray with paler anterior part; hind legs and tail are usually the same darker colour as its back. A whitish ring around each of the eyes and white patch on both lips are distinctive characters to identify the animal (Fig. 1). Three subspecies of Trachyp- ithecus phayrei have been found in the world. These are (1) T. p. phayrei from India, Bangladesh and western Myanmar, (2) T. p. crepuscula from central, southern to southeastern Yunnan, Thailand, Laos and Viet Nam and (3) T. p. shanicus from western to southwestern Yunnan and northern to east- ern Myanmar [11, 12]. This species is listed under CITES appendix II and IUCN endengered species [22]. To date, few cytogenetic reports of the Trachypithecus phayrei [7, 20] have been recorded for its karyotypes with G-band, C-band and chromosome painting. In the present research, we also presented a comparative study with our findings and previous reports. Moreover, this is the first report on standard karyotype and idiogram by high-resolution, G-banding as well as presence of polymorphic NORs on the chromosome pair 17 in this species. This finding is an Fig. 1 General characteristics of the Phayre’s langur, Trachypithecus phayrei crepuscula (Primate, Colobinae) from Thailand important basic knowledge that can be applied in the studies on animal diversity and taxonomic classifications.

Fig. 2 Metaphase chromosome plates and karyotypes from female (a) and male (b) specimens of Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 by conventional staining technique. Arrows indicate sex- chromosomes and satellite chromosomes/NORs (scale bars = 10 lm)

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Fig. 3 Metaphase chromosome plates and karyotypes from female (a) and male (b) specimens of Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 by GTG- banding technique. Arrows indicate sex-chromosomes and satellite chromosomes/NORs (scale bars = 10 lm)

Materials and methods culture of whole blood samples. The culture cells were treated with colchicine-hypotonic-ﬁxation-air-drying tech- Blood samples of the T. phayrei crepuscula (two males and nique followed by conventional staining, GTG and high- two females) were collected from Songkhla Zoo, Thailand resolution techniques [5, 24]. We used a total of best 20 and then subjected to cytogenetic studies by lymphocyte metaphases selected from 200 metaphases screened for

Fig. 4 Metaphase chromosome plates and karyotypes from female (a) and male (b) specimens of Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 by high- resolution technique. Arrows indicate sex-chromosomes and satellite chromosomes/NORs (scale bars = 10 lm)

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Fig. 5 Idiogram showing lengths and shapes of chromosomes of Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 by conventional staining technique. The arrow indicates NOR-bearing Fig. 7 Idiogram of Phayre’s langur (Trachypithecus phayrei crepus- chromosome pair 17a cula), 2n = 44 by high-resolution technique. Chromosome pair 17a show nucleolar organizer region (satellite chromosome)

cytogenetic study using a light microscope as described earlier [6]. The individual chromosome length measure- ment, karyotype and idiogram analyses were accomplished from these 20 best metaphases selected for analysis.

Results and discussions

The cytogenetic study of T. phayrei crepuscula using lymphocyte culture demonstrated that the chromosome number is 2n (diploid) = 44 and the fundamental number (NF, number of chromosome arms) for both sexes is 88 (Fig. 2). These results agree with those reported by Chen et al. [7] and Nie et al. [20]. Regarding the diploid chromosome number and NF reported by others for the subfamily Colobinae in Thailand, including species like T. cristatus (2n = 44, NF = 88) [1, 15, 23] and T. obscurus (2n = 44, NF = 88) [8, 16, 25, 32], all of them found to have the same 2n = 44 and most have NF = 88. Conventional, GTG-banding and high-resolution GTG- banding have been demonstrated in karyotypes of Thai Phayre’s langur in Figs. 2, 3 and 4, respectively along with idiograms of those in Figs. 5, 6 and 7, respectively. The Fig. 6 Idiogram of Phayre’s langur (Trachypithecus phayrei), autosomes consisted of 6 large metacentric, 10 large sub- 2n = 44 by GTG-banding technique. Chromosome pair 17a show nucleolar organizer region (satellite chromosome) metacentric, 2 large acrocentric, 6 medium metacentric, 14 123 Nucleus

Table 1 Mean length of short arm chromosome (Ls), long arm calculated from metaphase chromosomes in 20 different cells of chromosome (Ll), total arm chromosome (LT), relative length (RL), Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 centromeric index (CI) and standard deviation (SD) of RL, CI Chromosome pair Ls Ll LT RL ± SD CI ± SD Chromosome size Chromosome type

1 0.495 0.613 1.108 0.032 ± 0.001 0.553 ± 0.011 Large Metacentric 2 0.473 0.565 1.038 0.031 ± 0.001 0.541 ± 0.026 Large Metacentric 3 0.400 0.625 1.025 0.030 ± 0.003 0.605 ± 0.026 Large Submetacentric 4 0.245 0.748 0.993 0.029 ± 0.002 0.749 ± 0.024 Large Acrocentric 5 0.373 0.568 0.941 0.028 ± 0.003 0.604 ± 0.031 Large Submetacentric 6 0.315 0.605 0.920 0.027 ± 0.004 0.657 ± 0.020 Large Submetacentric 7 0.305 0.540 0.845 0.025 ± 0.002 0.641 ± 0.014 Large Submetacentric 8 0.318 0.505 0.823 0.024 ± 0.003 0.616 ± 0.037 Large Submetacentric 9 0.375 0.433 0.808 0.024 ± 0.002 0.537 ± 0.036 Large Metacentric 10 0.290 0.488 0.778 0.023 ± 0.002 0.629 ± 0.015 Medium Submetacentric 11 0.265 0.485 0.750 0.022 ± 0.003 0.635 ± 0.087 Medium Submetacentric 12 0.368 0.375 0.743 0.021 ± 0.002 0.505 ± 0.018 Medium Metacentric 13 0.280 0.440 0.720 0.021 ± 0.002 0.611 ± 0.031 Medium Submetacentric 14 0.200 0.505 0.705 0.020 ± 0.002 0.685 ± 0.035 Medium Submetacentric 15 0.240 0.418 0.658 0.019 ± 0.002 0.647 ± 0.058 Medium Submetacentric 16 0.303 0.338 0.640 0.019 ± 0.001 0.522 ± 0.064 Medium Metacentric 17a 0.273 0.323 0.595 0.018 ± 0.002 0.606 ± 0.027 Medium Submetacentric 18 0.233 0.358 0.591 0.017 ± 0.002 0.548 ± 0.029 Medium Metacentric 19 0.215 0.353 0.568 0.016 ± 0.002 0.619 ± 0.055 Medium Submetacentric 20 0.195 0.355 0.550 0.014 ± 0.001 0.645 ± 0.021 Small Submetacentric 21 0.128 0.323 0.450 0.013 ± 0.001 0.716 ± 0.015 Small Acrocentric X 0.432 0.680 1.112 0.031 ± 0.003 0.612 ± 0.025 Large Submetacentric Y 0.240 0.450 0.690 0.020 ± 0.002 0.695 ± 0.010 Medium Submetacentric aSatellite chromosome (nucleolar organizer region, NOR) medium submetacentric, 2 small submetacentric, and 2 NORs in dusky langur (T. obscurus) on pair 19 and 17 small acrocentric chromosomes (Table 1). This differs respectively. The NOR polymorphism was found in few from the reports of Chen et al. [7] which showed that the species of Asian non-human primates for instance, white- autosomes were consisted of 22 metacentric, 16 submeta- handed gibbon (Hylobates lar), dark-handed gibbon (H. centric, and 4 acrocentric chromosomes. The X was agilis), moloch gibbon (H. moloch)[19, 27–31, 33, 35]. metacentric chromosome and morphology of Y-chromo- The NORs are consisted of numerous repetitive 28S and some was not reported. The previous results disagree with 18S ribosomal gene. Multiplication of ribosomal DNA is those from Nie et al. [20] which reported that the auto- able to increase or decrease size of NORs in pair 17 of somes were comprised of 22 metacentric, 18 submetacen- T. phayrei [5]. tric, and 2 acrocentric chromosomes. Sex chromosome Use of GTG-banding technique in chromosomes of showed in only metacentric X-chromosome. Review Phayre’s langur shows a pattern of transverse light and reports on karyotype of langur are shown in Table 2. heavy bands. The banding pattern on a set of chromosomes Nucleolar organizer regions (NORs) of Thai T. phayrei with haploid number (n) including autosomes and sex- are clearly located on pericentromeric position of chro- chromosomes showed 224 bands in metaphase and 259 mosome pair 17 which were found to be heteromorphic. bands in prometaphase stages (Figs. 6, 7). This result can This result showed presence of unequal sizes of NORs on be compared with the report by Yunis and Prakash [36] pair 17 with longer NOR in 17a than that in 17b. The which showed that the number of bands in GTG-banding heteromorphic NORs in 17a and 17b were found in both pattern is over 1000 per haploid set of chromosomes of male and female animal (Fig. 8). This is the ﬁrst discovery prometaphase stage in human and apes. The reason of of heteromorphism of NORs in this species. Our present reporting lower number of GTG bands in this study is report is consistent with those in Sangpakdee et al. [25] and because only clear and detectable bands were taken into Tanomtong et al. [32] which reported heteromorphism of account during analysis of results.

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Table 2 Review reports of Species 2n NF m sm a t X Y References cytogenetic study of the langur (leaf monkey), subfamily African langurs Colobinae (Primate, Cercopithecidae) Colobus polycomos 44 88 28 14 0 0 m t Buettner-Janusch [4] 44 88 24 18 0 0 m – Chiarelli [9] C. guereza 44 88 42 0 0 m – Bigoni et al. [2] True langurs Trachypithecus obscurus 44 88 26 14 2 0 sm a Chiarelli [8] 44 88 40 2 0 sm sm Hsu and Benirschke [16] 44 88 28 12 2 0 sm sm Ponsa et al. [23] 44 88 22 18 2 0 m sm Sangpakdee et al. [25] 44 88 12 26 4 0 sm sm Tanomtong et al. [32] T. cristatus 44 88 40 2 0 m m/ Bigoni et al. [1] a 44 88 40 2 0 sm sm Hsu and Benirschke [15] 44 88 28 12 2 0 sm sm Ponsa et al. [23] T. francoisi 44 88 22 16 4 0 m m Chen et al. [7] 44 88 22 18 2 0 m – Nie et al. [20] T. phayrei 44 88 22 16 4 0 m – Chen et al. [7] 44 88 22 18 2 0 m – Nie et al. [20] 44 88 12 26 4 0 sm sm This study Semnopithecus entrellus 44 88 28 12 2 0 m a Buettner-Janusch [4] 50 – – – – – m m Chu and Bender [10] 44 88 28 12 2 0 sm sm Ushijima et al. [34] Odd-nose langurs Nasalis larvatus 48 96 46 0 0 sm sm Bigoni et al. [3] 48 96 46 0 0 sm a Hsu and Benirschke [17] 48 96 46 0 0 sm a Stanyon et al. [26] Rhinopithecus 44 88 18 22 2 0 sm a Chen et al. [7] roxellanae Pygatrix nemaeus 44 88 40 2 0 m a Hsu and Benirschke [18] 44 88 42 0 0 sm – O’Brien et al. [21] 2n, diploid chromosome number; NF, fundamental number; m, metacentric; sm, submetacentric; a, acrocentric; t, telocentric chromosomes; X, X chromosome; Y, chromosome; –not available

acrocentric. Thus the karyotype formula of Thai Phayre’s langur is as follow: m sm a m sm sm a 2nðÞ¼44 L6 þ L10 þ L2 þ M6 þ M14 þ S2 þ S2 þ 2 Sex - chromosomes:

Acknowledgements The ﬁnancial support from The Zoological Park Organization under the Royal Patronage of His Majesty the King is Fig. 8 Examples cited from three different cells: the new discovery gratefully acknowledged. We are thankful to the Director of Songkhla polymorphism found in chromosome pair 17 (17a17b) of male and Zoo for valuable help. female Phayre’s langur (Trachypithecus phayrei crepuscula), 2n = 44 by GTG-banding technique References Chromosome markers of T. phayrei crepuscula are autosome pairs 1 and 21 which are largest metacentric 1. Bigoni F, Koehler U, Stanyon R, Ishida T, Wienberg J. Fluo- chromosome and smallest acrocentric chromosome rescence in situ hybridization establishes homology between human and silvered leaf monkey chromosomes, reveals recipro- respectively. An asymmetrical karyotype is an important cal translocations between chromosomes homologous to human appearance in Phayre’s langur, comprised of three types of Y/5, 1/9 and 6/16 and delineates an X1X2Y1Y2/X1X1X2X2 sex- chromosomes, metacentric, submetacentric and chromosome system. Am J Phys Anthropol. 1997;23:315–27. 123 Nucleus

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