ZOOLOGICAL RESEARCH

Species identification of crested (Nomascus) in captivity in China using karyotyping- and PCR-based approaches

Wen-Hui Nie1,*, Jin-Huan Wang1, Wei-Ting Su1, Yu Hu1, Shui-Wang He1, Xue-Long Jiang1,*, Kai He1,2,*

1 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 2 Kyoto University Museum, Kyoto University, Kyoto 606-8417, Japan

ABSTRACT Keywords: F2 hybrid ; Fluorescence in situ hybridization; Nomascus; Pericentric inversion; Gibbons and (Hylobatidae) are well-known for Species identification; welfare their rapid chromosomal evolution, which has resulted in high speciation rate within the family. On the other INTRODUCTION hand, distinct karyotypes do not prevent speciation, The rates of chromosomal evolution are an order of magnitude allowing interbreeding between individuals in captivity, higher in Mammalia than in most other classes of vertebrates and the unwanted hybrids are ethically problematic as all and are strongly correlated with high speciation rates (Bush gibbon species are endangered or critically endangered. et al., 1977). High karyotypic diversity and rapid speciation are often observed in taxa characterized by small effective Thus, accurate species identification is crucial for captive population sizes, which includes the gibbons and siamangs of breeding, particularly in China where studbooks are the small family Hylobatidae (). These are unavailable. Identification based on external morphology endemic to southern China, South Asia, and Southeast Asia, is difficult, especially for hybrids, because species and have been assigned into four genera (Hoolock, , are usually similar in appearance. In this study, we Nomascus, and Symphalangus; Brandon-Jones et al., 2004; Roos & Geissmann, 2001). While siamangs (Symphalangus) employed G-banding karyotyping and fluorescence in possess a unique and large gular sac (throat pouch), gibbons situ hybridization (FISH) as well as a PCR-based share very similar external morphology, and were long approach to examine karyotypic characteristics and acknowledged as a single genus (Hylobates). However, identify crested gibbons of the genus Nomascus from Hoolock and Nomascus were subsequently recognized as full zoos and nature reserves in China. We characterized genera based on karyotypic studies, which revealed a unique and identified five karyotypes from 21 individuals of diploid number (2n) of chromosomes among each genus: Hoolock (2n=38), Hylobates (2n=44), Nomascus (2n=52), Nomascus. Using karyotypes and mitochondrial and and Symphalangus (2n=50) (Chiarelli, 1972; Prouty et al., nuclear genes, we identified three purebred species 1983; Van Tuinen & Ledbetter, 1983). This subdivision, as and three hybrids, including one F2 hybrid between N. supported by molecular phylogenetic studies, occurred in the gabriellae and N. siki. Our results also supported that N. leucogenys and N. siki shared the same inversion on chromosome 7, which resolves arguments from previous Received: 21 November 2017; Accepted: 19 March 2018; Online: 03 studies. Our results demonstrated that both karyotyping April 2018 and DNA-based approaches were suitable for identifying Foundation items: This study was supported by the Wildlife Conservation purebred species, though neither was ideal for hybrid Program of Yunnan Province, China. K.H. was supported by a JSPS identification. The advantages and disadvantages of Postdoctoral Fellowship for Overseas Researchers (P16092). both approaches are discussed. Our results further *Corresponding authors, E-mail: [email protected]; jiangxl@mail. highlight the importance of animal ethics and welfare, kiz.ac.cn; [email protected] which are critical for endangered species in captivity. DOI: 10.24272/j.issn.2095-8137.2018.036

356 Science Press Zoological Research 39(5): 356–363, 2018 Late Miocene approximately 7 Ma (Fan et al., 2017; Springer their original habitat is a goal for conservation but requires et al., 2012). Today, 20 species of gibbons and siamangs are accurate identification of purebred animals. Extreme caution recognized, though the number continues to increase due to must be undertaken in captive breeding as interspecific and newly discovered species (Fan et al., 2017). intergeneric hybridization can occur between gibbons and Speciation in this family is considerably faster than that siamangs (Hirai et al., 2007; Myers & Shafer, 1979), which observed in many other groups of and is considered do not resulting in reduced fertility among offspring in certain to be the result of high frequency of chromosomal rearrangement cases (Van Tuinen et al., 1999). Hybrid offspring are difficult to (Misceo et al., 2008), including compound inversion/translocation visually identify due to their similar external morphology, with (Jauch et al., 1992; Koehler et al., 1995a, 1995b; Marks, identification using the karyotypic approach considered more 1982; Nie et al., 2001; Stanyon & Chiarelli, 1982, 1983; reliable. Co-housing of different species has been avoided in Van Tuinen & Ledbetter, 1983; Yu et al., 1997). Previous many countries and animals are karyotyped for identification, comparison between the gibbon (N. leucogenys) and human with studbooks maintained as a database for appropriate genomes revealed 100 synteny breakpoints, which can facilitate management. However, this system is not yet well established chromosome breakage and rearrangement (Carbone et al., 2006). in China and different species are usually housed together Gibbons exhibit highly structured society in their populations, resulting in hybridization and unknown interbreeding history. which may promote chromosome rearrangement fixation, thereby In the current study, we examined 21 gibbons using a allowing rapid speciation (Wilson et al., 1975). combination of G-banding karyotyping and fluorescence in situ The crested gibbon genus Nomascus comprises seven hybridization (FISH). We also sequenced mitochondrial and recognized species, including N. concolor (western nuclear fragments across a known inversion breakpoint on black-crested gibbon), N. gabriellae (yellow-cheeked chromosome 7 identified in a previous study (Carbone et al., gibbon), N. hainanus (Hainan gibbon), N. leucogenys 2009). We examined whether the combination of G-banding (northern white-cheeked gibbon), N. nasutus (eastern and FISH is a reliable approach for species identification. black-crested gibbon), N. siki (southern white-cheeked We evaluated the efficiency and accuracy of karyotyping- gibbon) (Brandon-Jones et al., 2004; Roos & Geissmann, and PCR-based approaches for the identification of purebred 2001), and the recently discovered N. annamensis (northern animals and hybrids. We also re-examined whether the buffed-cheeked gibbon) (Thinh et al., 2010). These species pericentric inversion on chromosome 7 is species-specific in are characterized by a variety of morphological, anatomical, N. leucogenys. karyological, and vocal features (Couturier & Lernould, 1991; MATERIALS AND METHODS Garza & Woodruff, 1994; Schilling, 1984; Thinh et al., 2011). Although all studied Nomascus species have a unique Samples diploid number of 52 (Couturier et al., 1982; Dutrillaux We collected peripheral blood from 20 gibbon individuals raised et al., 1975; Van Tuinen & Ledbetter, 1983), they also in Nanning Zoo, Ningbo Zoo, Kunming Zoo, and Huanglianshan exhibit considerable interspecific karyotypic variation. Using National Natural Reserve, and obtained one sample (No.14 in the R-banding technique, Couturier & Lernould(1991) Table1) of cultured gibbon cells maintained at the Kunming distinguished the karyotypes for six individuals of N. Institute of Zoology, Chinese Academy of Sciences. Fourteen leucogenys, two N. siki, three N. gabriellae, and several hybrids individuals were female and seven were male. All experimental using a combination of an inversion on chromosome 7 and a procedures and animal care were performed according to the translocation between chromosomes 1 and 22. The inversion protocols approved by the Ethics Committee of the Kunming on chromosome 7 was also confirmed based on the number of Institute of Zoology, Chinese Academy of Sciences. hybridization signals provided by human chromosome probes Cell culture, metaphase preparation, and G-banding 4 and 22. On unrearranged chromosome 7, one hybridization Chromosome suspensions were obtained from lymphocyte signal of probe 4 on 7q and one signal of probe 22 on 7p were cultures. Cell culture and metaphase preparations were detected. On rearranged chromosome 7, two signals of each performed following conventional procedures (Hungerford, probe were detected, one on each arm of the chromosomes 1965). Briefly, whole blood was cultivated in the presence of (Koehler et al., 1995b). Heterozygous translocation t (1; 22) phytohaemagglutinin in RPMI 1640 medium supplemented with has also been identified in hybrids of N. leucogenys and N. 10% fetal bovine serum. After 68–70 h of growth, colchicine gabriellae (Couturier et al., 1982; Turleau et al., 1983). Using was added to the cell cultures to a final concentration of a PCR-based approach, Carbone et al.(2009) detected 0.4–0.8 µg/mL. The cell cultures were incubated for another the inversion on chromosome 7 in N. leucogenys but not in 2–4 h before harvest. We treated the cells with hypotonic N. siki. These results disagreed with those of Couturier & solution (0.075 mol/L KCl) for 20 min, with thrice fixation in 3:1 Lernould(1991), and thus need to be revisited and clarified methanol/glacial acetic acid. Slides were prepared by applying using additional samples. 10 µL of metaphase suspension onto dry and clean slides, Currently, most gibbon species are endangered or critically which were then allowed to air dry. G-banding was performed endangered. Several species, including Hylobates lar and following Seabright(1971). All karyotypes were analyzed after N. leucogenys, may have been extirpated from China largely G-banding. due to poaching. Reintroducing animals from zoos to

Zoological Research 39(5): 356–363, 2018 357 FISH, image capture, and processing were mounted in Vectashield mounting medium with DAPI We used a biotin-labeled probe of the human 22 chromosome. (406-diamidino-2-phenylindole, Vector Laboratories, USA). Fluorescence in situ hybridization, detection, image capture, Digital images were acquired using a CytoVision system and processing were carried out following Yang et al. (Applied Imaging Co., USA) with a CCD camera mounted (2000) and Nie et al.(2002). We detected fluorescence on a Zeiss microscope (Germany). We associated the signals using a layer of Cy3-avidin (1:1 000 dilution; hybridization signals with specific chromosome regions based Amersham Pharmacia Biotech, USA). After detection, slides on DAPI-banding patterns.

Table 1 Samples used in this study and a summary of karyotypic characteristics including the FISH signals on the chromosome 7, the lengths of chromosomes 1 and 22, the results of PCR using primer sets BP and HSA, as well as the identification results based on each approach FISH Nick Name House (signal on chr. 7) Chr. 1 Chr. 22 Karyotype BP HSA Cyt b genes Final identification (pairs)

Long Short Su-Su Nanning Zoo 2 N. leucogenys N/A N/A N/A N/A (Normal) (Normal) Long Short Actuators 2018, 7, x FOR PEER REVIEW 8 of 22 Bei-Li Nanning Zoo 2 N. leucogenys + − N. leucogenys N. leucogenys (Normal) (Normal) 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different Long Short Gou-Dan Nanning Zoo 2 N. leucogenys179 lengths+ of chromosomes− N. leucogenys 1 and 22, and FISH N.revealed leucogenys two, three, or four fluorescence (Normal) (Normal) Long Short 180 signals on chromosome 7 (Figure 1). Considering the G-banded karyotyping and FISH San-Mei Nanning Zoo 2 N. leucogenys N/A N/A N/A N/A (Normal) (Normal) 181 results, we recognized five karyotypes representing three karyotypic species and two Long Short No14 Missing 2 N. leucogenys182 hybrids,+ with− the latterN. cf. twoleucogenys distinguished by N nonhomologous. cf. leucogenys pairing. (Normal) (Normal) 183 Nomascus leucogenys (Figure 2) Long Short No name Kunming Zoo 2 N. leucogenys N/A N/A N/A N/A (Normal) (Normal) 184 The G-banded karyotypes of 10 individuals (7♀, 3♂) were similar to the G-banded Long Short 185 (Van Tuinen & Ledbetter, 1983) and R-banded karyotypes for N. leucogenys (Couturier & NB1 Ningbo Zoo 2 N. leucogenys N/A N/A N/A N/A (Normal) (Normal) 186 Lernould, 1991). Chromosomes 1 and 22 were normal. Two pairs of FISH signals were Long Short NB2 Ningbo Zoo 2 N. leucogenys187 foundN/A on chromosome N/A 7, supporting N/A a pericentric inversion N/A event (Figure 1A). (Normal) (Normal) Huanglianshan 188 Nomascus siki (Figure 3) Long Short HLS3 Nature 2 N. leucogenys189 N/AThe karyotypes N/A of five N/A samples (3♀, 2♂) were N/A identical to those of N. siki reported by (Normal) (Normal) Reserve 190 Couturier & Lernould (1991). Compared with N. leucogenys, a balanced translocation Huanglianshan Actuators 2018, 7, x FOR PEER REVIEW 8 of 22 Long Short 191 between chromosome pairs 1 and 22 was detected, resulting in one pair of shortened HLS4 Nature 2 N. leucogenys N/A N/A N/A N/A (Normal) (Normal) 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different Reserve 192 chromosome 1 and one pair of derived chromosome 22 [t (1;22)]. Similar to that observed 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence Fang-Fang Nanning Zoo 2 Short Long N. siki 193 in N.N/A leucogenys N/A, FISH demonstrated N/A a pericentric N/Ainversion event on chromosome 7 E’gui Nanning Zoo 2 Short Long N. siki 180+ signals− on chromosomeN. siki 7 (Figure 1). Considering N. siki the G-banded karyotyping and FISH 194 (Figure 1B). The sample “Da-shan” had a typical siki-karyotype. Qingguangyan Nanning Zoo 2 Short Long N. siki 181N/A results, N/A we recognized N/A five karyotypes representing N/A three karyotypic species and two 195 Nomascus gabriellae (Figure 4) Laoer Nanning Zoo 2 Short Long N. siki N/A N/A N/A N/A 182 hybrids, with the latter two distinguished by nonhomologous pairing. 196 The G-banded karyotypes of three animalsN. (1♀, siki 2♂)× were the same as that of N. 183 Nomascus leucogenys (Figure 2) Da-Shan Nanning Zoo 2 Short Long N. siki 197 gabriellae+ (Couturier− N. & gabriellaeLernould, 1991). This karyotype(N. siki was similar to N. siki in the 184 The G-banded karyotypes of× 10N. individuals gabriellae (7♀,) 3♂) were similar to the G-banded 198 presence of t (1;22). Only one pair of FISH signals were observed on chromosome 7, thus 316 Nanning Zoo 1 Short Long N. gabriellae 185− (Van+ TuinenN. & gabriellaeLedbetter, 1983) and R-banded N. gabriellae karyotypes for N. leucogenys (Couturier & 199 differing− from N. siki and N. leucogenys (Figure 1C). Bai-Shou Nanning Zoo 1 Short Long N. gabriellae 186 Lernould,+ 1991).N. gabriellae Chromosomes 1 and N. 22gabriellae were normal. Two pairs of FISH signals were Jing-Jing Nanning Zoo 1 Short Long N. gabriellae200 Nomascus− leucogenys+ N.×Nomascus gabriellae siki hybrid N. (Figure gabriellae 5) 187 found on chromosome 7, supporting a pericentric inversion event (Figure 1A). 1 short, 1 short, N. leucogenys× Mei-Mei Nanning Zoo 2 N. leucogenys×N.201 siki +The G-banded− karyotypeN. leucogenys of one specimen was unique, characterized by a 1 long 1 Long 188 Nomascus siki (Figure 3) N. siki 202 heterozygous translocation. One chromosome 1 and 22 were normal, similar to that of N. 1 and 189 The karyotypes of five samplesN. (3♀, gabriellae 2♂) were× identical to those of N. siki reported by Xiao-Xiao Nanning Zoo Short Long N. gabriellae×N.203 siki leucogenys+, +and the N.other leucogenys chromosome 1 and 22 were similar to that of N. siki, indicating a half 190 Couturier & Lernould (1991). Compared N.with siki N. leucogenys, a balanced translocation 1 and 204 one translocation t (1; 22). Two pairs of FISH signals were observed on chromosome 7, A-Huang Nanning Zoo Short Long N. gabriellae×N. siki 191N/A between N/A chromosome N/A pairs 1 and 22 was detected, N/A resulting in one pair of shortened a half 205 indicating a pericentric inversion event (Figure 1D). This specimen was identified as a 192 chromosome 1 and one pair of derived chromosome 22 [t (1;22)]. Similar to that observed

The karyotype and mitochondrial of Da-Shan suggest different specific affinities. +: The206 positive hybrid193 result of inN. N. leucogenysof leucogenys PCR× usingN., FISH siki.primer demonstrated set BPa pericentric or HAS. inversion−: event on chromosome 7

The negative result of PCR using primer set BP or HAS. N/A: Not available. 194 (Figure 1B). The sample “Da-shan” had a typical siki-karyotype.

195 Nomascus gabriellae (Figure 4)

196 The G-banded karyotypes of three animals (1♀, 2♂) were the same as that of N.

358 www.zoores.ac.cn 197 gabriellae (Couturier & Lernould, 1991). This karyotype was similar to N. siki in the

198 presence of t (1;22). Only one pair of FISH signals were observed on chromosome 7, thus

199 differing from N. siki and N. leucogenys (Figure 1C).

200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5)

201 The G-banded karyotype of one specimen was unique, characterized by a

202 heterozygous translocation. One chromosome 1 and 22 were normal, similar to that of N.

203 leucogenys, and the other chromosome 1 and 22 were similar to that of N. siki, indicating

204 one translocation t (1; 22). Two pairs of FISH signals were observed on chromosome 7,

205 indicating a pericentric inversion event (Figure 1D). This specimen was identified as a

206 hybrid of N. leucogenys×N. siki.

Amplification, sequencing, and species identification A

After karyotyping, we extracted total DNA from the cultured cells Actuators 2018, 7, x FOR PEER REVIEW 8 of 22 using a commercial DNA extraction kit (Blood & Cell Culture DNA Mini Kit, Qiagen, Germany). The DNA extracted from 10 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different samples was considered acceptable for subsequent analyses as 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence B other samples were not well preserved after karyotyping. We 180 signals on chromosome 7 (Figure 1). Considering the G-banded karyotyping and FISH amplified and sequenced cyt b genes using the primer pair L14724_hk3 (50-GGACTTATGACATGAAAAATCATCGTTG-30) 181 results, we recognized five karyotypes representing three karyotypic species and two 0 0 and H15915_hk3 (5 -GATTCCCCATTTCTGGTTTACAAGAC-3 ). 182 hybrids,C with the latter two distinguished by nonhomologous pairing. We also conducted PCR using the primers provided in Carbone 183 Nomascus leucogenys (Figure 2) et al.(2009) (263C9_BP_L with 263C9_BP_R (BP hereafter) and 263C9_BP_L with 263C9_HSA22_R (HSA hereafter)), targeting 184 The G-banded karyotypes of 10 individuals (7♀, 3♂) were similar to the G-banded D a fragment across a breakpoint on chromosome 7. According 185 (Van Tuinen & Ledbetter, 1983) and R-banded karyotypes for N. leucogenys (Couturier & to Carbone et al.(2009), HSA primers should amplify a 186 Lernould, 1991). Chromosomes 1 and 22 were normal. Two pairs of FISH signals were fragment across a breakpoint in species without an inversion 187 found on chromosome 7, supporting a pericentric inversion event (Figure 1A). (e.g., Hylobates spp., N. gabriellae, and N. siki), and BP primers E should result in positive amplification only for N. leucogenys. The 188 Nomascus siki (Figure 3) mitochondrial and nuclear amplicons were sequenced using a ABI 3730 sequencer (Applied Biosystems, USA). 189 The karyotypes of five samples (3♀, 2♂) were identical to those of N. siki reported by Actuators 2018, 7, x FOR PEER REVIEW 8 of 22 We identified species based on the karyotyping and 190 Couturier & Lernould (1991). Compared with N. leucogenys, a balanced translocation Figure 1 Comparison of G-banded chromosomes 1, 7, and 22 sequencing results using the following strategy: (1) we 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different 191and FISHbetween results chromosome with pairs human 1 and 22 22 chromosome-specificwas detected, resulting in one probe pair of shortened identified the species based on its karyotype; (2) we verified 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence 192in differentchromosome Nomascus 1 and one species pair of derived and theirchromosome hybrids 22 [t (1;22)]. Similar to that observed the karyotyping results using PCR with primer sets BP and 180 signals on chromosome 7 (Figure 1). Considering the G-banded karyotyping and FISH HSA; (3) we BLAST each cyt b gene against the GenBank 193A: Nomascusin N. leucogenys leucogenys, FISH; B: demonstratedNomascus sikia pericentric; C: Nomascus inversion gabriellae event on; chromosome D: 7 181 results, we recognized five karyotypes representing three karyotypic species and two nucleotide database; (4) we estimated a gene tree to verify 194hybrid (Figure of N. leucogenys 1B). The sample×N. siki “Da-shan”; E: hybrid had of N.a typical gabriellae siki-karyotype.×N. siki. the BLAST results using our cyt b genes accompanied by 182 hybrids, with the latter two distinguished by nonhomologous pairing. a set of sequences representing the recognized Nomascus 195Nomascus 183Nomascus Nomascus leucogenys gabriellae leucogenys (Figure ((FigureFigure 4) 2) 2) species downloaded from GenBank; and (5) we repeated 196The 184 G-banded The TheG-banded karyotypesG-banded karyotypes karyotypes of 10 of of individualsthree 10 individuals animals (7(1♀,(7♀,, 3♂) 32♂) )were were similar the similar same to the as G-banded that of N. the karyotyping and amplification/sequencing in triplicate in 197to the185gabriellae G-banded(Van Tuinen (Couturier ( Van& Ledbetter, Tuinen& Lernould, 1983) & Ledbetterand 1991). R-banded This, karyotypekaryotypes1983) and forwas N. R-banded similar leucogenys to N. (Couturier siki in the & cases where the mitochondrial, nuclear sequencing, and karyotypes186 Lernould, for 1991).N. leucogenys Chromosomes 1( andCouturier 22 were normal. & Lernould Two pairs ,of 1991FISH signals). were karyotyping results conflicted (e.g., sample Da-Shan, see 198Chromosomes presence of t 1(1;22). and 22Only were one pair normal. of FISH Two signals pairs were of observ FISHed signals on chromosome 7, thus 187 found on chromosome 7, supporting a pericentric inversion event (Figure 1A). Results and Discussion). A final purebred or hybrid and 199were differing found on from chromosome N. siki and N. 7,leucogenys supporting (Figure apericentric 1C). inversion species identification was determined. 188 Nomascus siki (Figure 3) 200event Nomascus (Figure1 leucogenysA). ×Nomascus siki hybrid (Figure 5) We constructed the maximum likelihood gene tree Actuators189 2018, 7, x FORThe PEER karyotypes REVIEW of five samples (3♀, 2♂) were identical to those of N. siki17 reported of 22 by using RAxML v8.2.10 and the CIPRES Science Gateway 201 The G-banded karyotype of one specimen was unique, characterized by a 190 Couturier & Lernould (1991). Compared with N. leucogenys, a balanced translocation (Stamatakis, 2014). In addition to the obtained cyt b 202 heterozygous translocation. One chromosome 1 and 22 were normal, similar to that of N. sequences, 17 cyt b sequences representing seven recognized 191 between chromosome pairs 1 and 22 was detected, resulting in one pair of shortened species were downloaded from GenBank (Supplementary 203 192leucogenys chromosome, and 1the and other one pairchromosome of derived chromosome1 and 22 were 22 [tsimilar (1;22)]. to Similar that of to N. that siki observed, indicating Table S1) and aligned with our sequences using MAFFT v7.3 204 193one translocationin N. leucogenys t (1;, FISH 22). demonstrated Two pairs of a pericentricFISH signals inversion were eventobserved on chromosome on chromosome 7 7, (Katoh & Standley, 2014). We partitioned the alignments 205 194indicating (Figure a 1B). pericentric The sample inversi “Da-shan”on event had (Figure a typical 1D). siki-karyotype. This specimen was identified as a by codon positions (three partitions). We used GTR+G as 195 Nomascus gabriellae (Figure 4) the evolutionary model for each partition as RAxML does 206 hybrid of N. leucogenys×N. siki. 196 The G-banded karyotypes of three animals (1♀, 2♂) were the same as that of N. not accept models other than GTR or GTR+G. We ran each analysis using the rapid bootstrapping algorithm and let RAxML 197 gabriellae (Couturier & Lernould, 1991). This karyotype was similar to N. siki in the halt bootstrapping automatically. 198 presence of t (1;22). Only one pair of FISH signals were observed on chromosome 7, thus

199 differing from N. siki and N. leucogenys (Figure 1C). RESULTS 200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5) Identified karyotypic species 201 The G-banded karyotype of one specimen was unique, characterized by a The diploid number (2n) of all analyzed gibbons was 52. 453 202 heterozygous translocation. One chromosome 1 and 22 were normal, similar to that of N. 454 FigureFigure 2 2G-banded G-banded karyotype karyotype of a northern of awhite-cheeked northern white-cheekedgibbon (N. leucogenys) G-banding revealed different lengths of chromosomes 1 and 203 leucogenys, and the other chromosome 1 and 22 were similar to that of N. siki, indicating gibbon (N. leucogenys) 22, and FISH revealed two, three, or four fluorescence signals 204 one translocation t (1; 22). Two pairs of FISH signals were observed on chromosome 7, on chromosome 7 (Figure1). Considering the G-banded 205 indicating a pericentric inversion event (Figure 1D). This specimen was identified as a karyotyping and FISH results, we recognized five karyotypes Nomascus siki (Figure3) representing three karyotypic species and two hybrids, with the The206 karyotypes hybrid of N. ofleucogenys five samples×N. siki. (3 , 2 ) were identical to latter two distinguished by nonhomologous pairing. those of N. siki reported by Couturier & Lernould(1991).

Zoological Research 39(5): 356–363, 2018 359

455 456 Figure 3 G-banded karyotype of a southern white-cheeked gibbon (N. siki) 457

Actuators 2018, 7, x FOR PEER REVIEW 18 of 22

Actuators 2018, 7, x FOR PEER REVIEW 17 of 22

Compared with N. leucogenys, a balanced translocation a pericentric inversion event (Figure1D). This specimen was between chromosome pairs 1 and 22 was detected, resulting 458 identified as a hybrid of N. leucogenys×N. siki. in one pair of shortened chromosome 1 and one pair of derived 459 Figure 4 G-banded karyotype of a yellow-cheeked gibbon (N. gabriellae) chromosome 22 (t (1; 22)). Similar to that observed in N.

leucogenys, FISHActuators demonstrated2018, 7, x FOR PEER REVIEW a pericentric inversion event 8 of 22 on chromosome 7 (Figure1B). The sample “Da-Shan” had a 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different typical siki-karyotype. 453 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence 454 Figure 2 G-banded karyotype of a northern white-cheeked gibbon (N. leucogenys) 180 signals on chromosome 7 (Figure 1). Considering the G-banded karyotyping and FISH

181 results, we recognized five karyotypes representing three karyotypic species and two

182 hybrids, with the latter two distinguished by nonhomologous pairing.

183 Nomascus leucogenys (Figure 2)

184 The G-banded karyotypes of 10 individuals (7♀, 3♂) were similar to the G-banded

185 (Van Tuinen & Ledbetter, 1983) and R-banded karyotypes for N. leucogenys (Couturier &

186 Lernould, 1991). Chromosomes 1 and 22 were normal. Two pairs of FISH signals were

187 found on chromosome 7, supporting a pericentric inversion event (Figure 1A).460 Actuators 2018, 7, x FOR PEER REVIEW 8 of 22 461 FigureFigure 5 G-banded G-banded karyotype karyotype of a N. leucogenys×N. of a N. leucogenys siki hybrid ×N. siki 188 Nomascus siki (Figure 3) 462 hybrid 178 189The diploidThe number karyotypes (2 nof) fiveof all samples analyzed (3♀, 2♂)gibbons were identicalwas 52. toG-banding those of N. revealedsiki reported different by 179 lengths190 of Couturierchromosomes & Lernould 1 and (1991). 22, and Compared FISH withrevealed N. leucogenys, two, three, a balanced or four translocation fluorescenceNomascus gabriellae×Nomascus siki hybrid (Figure6) 180 signals191 onbetween chromosome chromosome 7 (Figure pairs 11). and Considering 22 was detected, the resultingG-banded in on kae ryotypingpair of shortened andThe FISH distinct G-banded karyotype of two individuals was similar 455 192 chromosome 1 and one pair of derived chromosome 22 [t (1;22)]. Similar to that observedto that of N. siki and N. gabriellae in the presence of Figure181 results, 3 G-banded we recognized karyotype five karyotypes of a southern representing white-cheeked three karyotypic species and two 456gibbon Figure 3 ( N.G-banded193 siki in) karyotype N. leucogenys of a ,s outhernFISH demonstrated white-cheeked a pericentric gibbon (N. inversion siki) event on chromosomet 7 (1; 22). FISH detected three fluorescence signals on 457 182 hybrids, with the latter two distinguished by nonhomologous pairing. 194 (Figure 1B). The sample “Da-shan” had a typical siki-karyotype. chromosome 7, indicating heterozygous pericentric inversion 183 Nomascus leucogenys (Figure 2) (Figure1E). These two individuals were identified as hybrids of Nomascus195 gabriellaeNomascus gabriellae(Figure (Figure4) 4) N.Actuators gabriellae 2018, 7, x FOR× PEERN. REVIEW siki. 19 of 22 The184 G-banded196The G-banded karyotypesThe G-banded karyotypes ofkaryotypes three of 10 of i animalsndividuals three animals (1(7♀, (1♀,, 3♂) 22♂)) were were thesimilar same the asto thatthe ofG-banded N.

same185 (Van as197 that Tuinen gabriellae of &N. Ledbetter, gabriellae(Couturier 1983) & Lernould, (andCouturier R-banded 1991). This& karyotypes karyotype Lernould wasfor, similarN.1991 leucogenys to). N. siki in(Couturier the & This karyotype was similar to N. siki in the presence of t (1; 22). 186 Lernould,198 presence1991). Chromosomesof t (1;22). Only one1 and pair 22 of wereFISH signalsnormal. were Two observ pairsed ofon chromosomeFISH signals 7, thuswere Only one pair of FISH signals were observed on chromosome 199 differing from N. siki and N. leucogenys (Figure 1C). 7,187 thus found differing on chromosome from N. siki7, supportingand N. leucogenysa pericentric inversion(Figure event1C). (Figure 1A). 200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5) 188Actuators 2018Nomascus, 7, x FOR PEER siki REVIEW (Figure 3) 18 of 22 201 The G-banded karyotype of one specimen was unique, characterized by a

189 202The karyotypesheterozygous of translocation. five samples One (3♀, chromosome 2♂) were 1 and identical 22 were tonormal those, similar of N. tosiki that reported of N. by 190 Couturier203 leucogenys& Lernould, and (1991). the other Compared chromosome with 1 and N. 22 leucogenys, were similar ato balanced that of N. sikitranslocation, indicating

191 between204 chromosomeone translocation pairs t (1; 1 22). and Two 22 waspairs detected,of FISH signals resulting were obsin onervede pair on chromosome of shortened 7, 205 indicating a pericentric inversion event (Figure 1D). This specimen was identified as a 192 chromosome 1 and one pair of derived chromosome 22 [t (1;22)]. Similar to that observed 206 hybrid of N. leucogenys×N. siki. 193 in N. leucogenys, FISH demonstrated a pericentric inversion event on chromosome 7

194 (Figure 1B). The sample “Da-shan” had a typical siki-karyotype. 463 195 Nomascus gabriellae (Figure 4) 464 FigureFigure 6 6G-banded G-banded karyotype karyotype of a N. gabriellae×N. of a N. siki leucogenys hybrid ×N. siki hybrid 196 The G-banded karyotypes of three animals (1♀, 2♂) were the same as that of N.

197 gabriellae (Couturier & Lernould, 1991). This karyotype was similar to N. siki in the Mitochondrial and nuclear sequences 198 presence of t (1;22). Only one pair of FISH signals were observed on chromosomeWe 7, determined thus the complete cyt b sequences for 10 individuals 458 Figure 4 G-banded karyotype of a yellow-cheeked gibbon (N. 459 199Figure 4differing G-banded from karyotype N. siki of aand yellow-cheeked N. leucogenys gibbon (Figure (N. gabriellae 1C). ) representing the three purebred species (n=7) and two gabriellae) 200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5) hybrids (n=3) identified in the karyotypic analyses. All new sequences are available in GenBank under accession Nos. Nomascus201 The leucogenys G-banded karyotype×Nomascus of one specimensiki hybrid was unique, (Figure characterize5) d by a MH188408–MH188428 (Supplementary Table S1). Five out The202 G-bandedheterozygous karyotypetranslocation. One of chromosome one specimen 1 and 22 waswere normal unique,, similar to ofthat the of N. seven purebred animals were supported by BLAST and characterized by a heterozygous translocation. One phylogenetic analyses using the cyt b gene and the other two 203 leucogenys, and the other chromosome 1 and 22 were similar to that of N. siki, indicating chromosome 1 and 22 were normal, similar to that of N. were not (Figure7). One sample (Da-Shan) was identified leucogenys204 one translocation, and the t other(1; 22). chromosome Two pairs of FISH 1 andsignals 22 were were obs similarerved on chromosomeas N. 7, siki based on its karyotype, but its mitochondrial gene to205 that indicating of N. siki a, pericentric indicating inversi oneon translocation event (Figure 1D). t (1; This 22). spec Twoimen pairs was identifiedwas as a typical of N. gabriellae, which also supported by our of FISH signals were observed on chromosome 7, indicating phylogenetic analyses (bootstrap value (BS)=98). Sample 206 hybrid of N. leucogenys×N. siki.

360 www.zoores.ac.cn

460

461 Figure 5 G-banded karyotype of a N. leucogenys×N. siki hybrid 462

14 was identified as N. leucogenys based on its karyotype its mitochondrial gene results. but could not be identified unambiguously as it was equally Because the karyotype and PCR results were congruent, we similar to N. leucogenys and N. siki. It was closely related assigned seven samples as purebred species and identified to (BS=84) and formed a sister lineage of these two species another two as hybrids. One sample (14) was identified on the phylogenetic tree (BS=52). The cyt b genes of the as N. cf. leucogenys as it did not cluster with the other two hybrids identified in the karyotypic analyses (Xiao-Xiao N. leucogenys sequences downloaded from GenBank. We and Mei-Mei) corresponded to one of their parental species considered Da-Shan as an F2 hybrid of N. gabriellae and N. (BS≥88). On our cyt b genetic tree (Figure7), sample 14 was siki due to the inconsistent patterns revealed by karyotyping a close relative to (BS=84) and formed a sister lineage with N. and PCR using the HSA and BP primers (siki), and the siki+N. leucogenys (BS=52). mitochondrial phylogeny (gabriellae) (Table1).

DISCUSSION

We used karyotypic- and PCR-based approaches to examine the affinities of Nomascus species characterized by high karyotypic diversity. We determined the specific karyotypes of each species as well as the existence of an inversion on chromosome 7 in N. siki and N. leucogenys. We also revealed the definitive existence of hybrids in zoos in China, which calls attention to the animal ethics and welfare issues related to endangered species breeding in captivity. Using the Nomascus species as our focal taxa, we evaluated the accuracy and efficiency of both approaches in identifying purebred and hybrid Xiao-xiao-hybrid species. Our results supported that N. gabriellae, N. leucogenys, and N. siki are distinguishable based on translocation t (1; 22) and inversion on chromosome 7 (Figures1–5), even though N. leucogenys and N. siki diverged from each other only recently. Couturier & Lernould(1991) revealed that N. hainanus does not have an inversion on chromosome 7 or translocation t (1; 22), and therefore has a distinct karyotype. It would be interesting to examine the G-banded karyotypes and FISH results of N. concolor as well as N. annamensis, which is sister to N. gabriellae and recognized only recently (Thinh et al., 2010). Due to interspecific karyotypic variation, hybrids can be easily distinguished based on heterozygous R-, G-banded karyotypes and FISH results (Couturier & Lernould, 1991; this study). In wild populations, hybridization of Hylobates species such as H. Figure 7 Maximum-likelihood gene tree estimated using the lar×H. pileatus, H. lar×H. agilis, and H. muelleri×H. agilis/H. cyt b genes of Nomascus species albibarbis has been observed (Brockelman & Gittins, 1984). Hybridization between Nomascus species in the wild has not Branch lengths correspond to substitution rates. Numbers on branches are been reported, which is likely due to their allopatric distribution. bootstrap values. Sequences downloaded from GenBank are in gray and However, we observed heterozygous karyotypes in captive GenBank accession Nos. are shown. Nomascus animals, congruent with previous study (Couturier & Lernould, 1991). Our results support the possibility of The PCR results using the HSA and BP primer sets were nonhomologous pairing during meiophase (Figures5,6), which concordant with the karyotypic results. The PCR results using the is an interesting characteristic in Hylobatidae. Because BP primers (confirming the inversion) were positive for purebred gibbons have very strong social structures and usually live N. leucogenys, N. siki, and the N. leucogenys×N. siki hybrid in small family groups, such nonhomologous pairing may be (Mei-Mei) and were negative when using the HSA primers for easily fixed during evolutionary histories and further promote these samples (Table1). The PCR results using the BP primers diversification and speciation, resulting in high species diversity were negative for purebred N. gabriellae but positive when using within the family. the HSA primers. For the hybrid identified as N. gabriellae×N. Our FISH and DNA analyses supported the existence of siki (Xiao-Xiao), PCR and sequencing were successful using an inversion on chromosome 7 in both N. leucogenys and N. both BP and HSA primers. For Da-Shan, the PCR results were siki (Figures1–3), consistent with the findings of Couturier positive using BP primers and negative using HSA primers, which & Lernould(1991), though not supported by Carbone et al. were congruent with its karyotype (siki type) but disagreed with (2009). The negative results obtained in the latter study may

Zoological Research 39(5): 356–363, 2018 361 be due to the hair sample used to represent N. siki, which is but could not easily distinguish N. leucogenys/N. siki from the known for low DNA quantity as well as the existence of PCR hybrid of N. leucogenys×N. siki. inhibitors. The phylogenetic position of sample 14 was an interesting COMPETING INTERESTS finding. Its karyotype was identical to the other N. leucogenys The authors declare that they have no competing interests. samples (Table1), which was not supported by the mitochondrial gene tree (Figure7). We repeated amplification AUTHORS’ CONTRIBUTIONS and sequencing three times for this sample. The sequences were identical, indicating no cross-sample contamination, W.H.N., K.H. and X.L.J. conceived of the experiments. J.H.W., W.T.S. and no premature codon was observed, indicating it was and Y.H. cultured the cells, prepared the chromosomal suspensions and not a pseudogene. There are two hypothetical scenarios performed G-banding and chromosome-painting experiments. S.W.H. that may explain these results. One is incomplete lineage completed all molecular experiments. W.H.N., K.H. and X.L.J. analyzed the sorting between N. leucogenys and N. siki, resulting in data, wrote and revised the paper. All of the authors read and approved the non-monophyletic relationships. This situation has never been final manuscript. observed because the effective population sizes of gibbon species are usually small. Alternatively, this sample might ACKNOWLEDGEMENTS represent a distinct and unknown taxon, which is a close We are grateful to the Directors of Nanning Zoo, Ningbo Zoo, Kunming Zoo, relative but not identical to N. leucogenys or N. siki. The and Huanglianshan National Nature Reserve for allowing us to sample blood sample was obtained from the Kunming Institute of Zoology, from the gibbons for this study. 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