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

2018·9.1 MB·

by  Nie, Wen-Hui

ZOOLOGICAL RESEARCH
Species identification of crested gibbons (Nomascus) in
captivity in China using karyotyping- and PCR-based
approaches
Wen-HuiNie1,*,Jin-HuanWang1,Wei-TingSu1,YuHu1,Shui-WangHe1,Xue-LongJiang1,*,KaiHe1,2,*
1KunmingInstituteofZoology,ChineseAcademyofSciences,KunmingYunnan650223,China
2KyotoUniversityMuseum,KyotoUniversity,Kyoto606-8417,Japan
ABSTRACT Keywords: F2 hybrid gibbon; Fluorescence in
situ hybridization; Nomascus; Pericentric inversion;
Gibbonsandsiamangs(Hylobatidae)arewell-knownfor
Speciesidentification;Animalwelfare
theirrapidchromosomalevolution,whichhasresulted
INTRODUCTION
in high speciation rate within the family. On the other
hand, distinct karyotypes do not prevent speciation, Theratesofchromosomalevolutionareanorderofmagnitude
allowing interbreeding between individuals in captivity, higherinMammaliathaninmostotherclassesofvertebrates
andtheunwantedhybridsareethicallyproblematicasall and are strongly correlated with high speciation rates (Bush
et al., 1977). High karyotypic diversity and rapid speciation
gibbonspeciesareendangeredorcriticallyendangered.
are often observed in taxa characterized by small effective
Thus,accuratespeciesidentificationiscrucialforcaptive
populationsizes,whichincludesthegibbonsandsiamangsof
breeding, particularly in China where studbooks are
thesmallapefamilyHylobatidae(Primate).Theseanimalsare
unavailable. Identificationbasedonexternalmorphology endemic to southern China, South Asia, and Southeast Asia,
is difficult, especially for hybrids, because species andhavebeenassignedintofourgenera(Hoolock,Hylobates,
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
possessauniqueandlargegularsac(throatpouch),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 andNomascusweresubsequentlyrecognizedasfull
zoosandnaturereservesinChina. Wecharacterized generabasedonkaryotypicstudies, whichrevealedaunique
diploid number (2n) of chromosomes among each genus:
and identified five karyotypes from 21 individuals of
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
andthreehybrids,includingoneF2hybridbetweenN. supported by molecular phylogenetic studies, occurred in the
gabriellae andN.siki. Ourresultsalsosupportedthat
N.leucogenysandN.siki sharedthesameinversionon
chromosome7,whichresolvesargumentsfromprevious Received: 21November2017;Accepted: 19March2018;Online: 03
studies. Ourresultsdemonstratedthatbothkaryotyping April2018
andDNA-basedapproachesweresuitableforidentifying Foundationitems:ThisstudywassupportedbytheWildlifeConservation
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 PostdoctoralFellowshipforOverseasResearchers(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]
whicharecriticalforendangeredspeciesincaptivity. DOI:10.24272/j.issn.2095-8137.2018.036
356 SciencePress ZoologicalResearch39(5):356–363,2018
Late Miocene approximately 7 Ma (Fan et al., 2017; Springer their original habitat is a goal for conservation but requires
etal.,2012). Today,20speciesofgibbonsandsiamangsare 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
newlydiscoveredspecies(Fanetal.,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 mammals and is considered do not resulting in reduced fertility among offspring in certain
tobetheresultofhighfrequencyofchromosomalrearrangement cases(VanTuinenetal.,1999).Hybridoffspringaredifficultto
(Misceoetal.,2008),includingcompoundinversion/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
genomesrevealed100syntenybreakpoints, whichcanfacilitate management. However,thissystemisnotyetwellestablished
chromosomebreakageandrearrangement(Carboneetal.,2006). in China and different species are usually housed together
Gibbons exhibit highly structured society in their populations, resultinginhybridizationandunknowninterbreedinghistory.
whichmaypromotechromosomerearrangementfixation,thereby In the current study, we examined 21 gibbons using a
allowingrapidspeciation(Wilsonetal.,1975). combinationofG-bandingkaryotypingandfluorescenceinsitu
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;
MATERIALSANDMETHODS
Garza&Woodruff,1994;Schilling,1984;Thinhetal.,2011).
Although all studied Nomascus species have a unique Samples
diploid number of 52 (Couturier et al., 1982; Dutrillaux Wecollectedperipheralbloodfrom20gibbonindividualsraised
et al., 1975; Van Tuinen & Ledbetter, 1983), they also inNanningZoo,NingboZoo,KunmingZoo,andHuanglianshan
exhibit considerable interspecific karyotypic variation. Using NationalNaturalReserve,andobtainedonesample(No.14in
the R-banding technique, Couturier & Lernould (1991) Table 1) of cultured gibbon cells maintained at the Kunming
distinguished the karyotypes for six individuals of N. InstituteofZoology,ChineseAcademyofSciences. Fourteen
leucogenys,twoN.siki,threeN.gabriellae,andseveralhybrids individualswerefemaleandsevenweremale.Allexperimental
using a combination of an inversion on chromosome 7 and a proceduresandanimalcarewereperformedaccordingtothe
translocationbetweenchromosomes1and22. Theinversion protocols approved by the Ethics Committee of the Kunming
onchromosome7wasalsoconfirmedbasedonthenumberof InstituteofZoology,ChineseAcademyofSciences.
hybridization signals provided by human chromosome probes
Cellculture,metaphasepreparation,andG-banding
4and22. Onunrearrangedchromosome7,onehybridization
Chromosome suspensions were obtained from lymphocyte
signalofprobe4on7qandonesignalofprobe22on7pwere
cultures. Cell culture and metaphase preparations were
detected. Onrearrangedchromosome7, twosignalsofeach
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)
phytohaemagglutinininRPMI1640mediumsupplementedwith
has also been identified in hybrids of N. leucogenys and N.
10% fetal bovine serum. After 68–70 h of growth, colchicine
gabriellae (Couturieretal.,1982; Turleauetal.,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(0.075mol/LKCl)for20min,withthricefixationin3:1
Lernould (1991), and thus need to be revisited and clarified
methanol/glacialaceticacid.Slideswerepreparedbyapplying
usingadditionalsamples. 10 µL of metaphase suspension onto dry and clean slides,
Currently, mostgibbonspeciesareendangeredorcritically whichwerethenallowedtoairdry. G-bandingwasperformed
endangered. Several species, including Hylobates lar and followingSeabright(1971). Allkaryotypeswereanalyzedafter
N. leucogenys, may have been extirpated from China largely G-banding.
due to poaching. Reintroducing animals from zoos to
ZoologicalResearch39(5):356–363,2018 357
FISH,imagecapture,andprocessing were mounted in Vectashield mounting medium with DAPI
Weusedabiotin-labeledprobeofthehuman22chromosome. (4(cid:48)6-diamidino-2-phenylindole, Vector Laboratories, USA).
Fluorescence in situhybridization, 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; hybridizationsignalswithspecificchromosomeregionsbased
Amersham Pharmacia Biotech, USA). After detection, slides onDAPI-bandingpatterns.
Table1SamplesusedinthisstudyandasummaryofkaryotypiccharacteristicsincludingtheFISHsignalsonthechromosome7,
thelengthsofchromosomes1and22,theresultsofPCRusingprimersetsBPandHSA,aswellastheidentificationresults
basedoneachapproach
FISH
NickName House (signalonchr.7) Chr.1 Chr.22 Karyotype BP HSA Cytbgenes Finalidentification
(pairs)
Long Short
Su-Su NanningZoo 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 NanningZoo 2 N.leucogenys + − N.leucogenys N.leucogenys
(Normal) (Normal) Actuators 2018, 7, x FOR PEER REVIEW 8 of 22
178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different
Long Short
Gou-Dan NanningZoo 2 N.leucogeny1s7197 8 leng+thsT ohfe c dh−irpolmoiods noumNme.sble e1ru a(cn2odng )2e o2nf, yaaslnld a nFaISlyHze Nrde. vgleeibaulbecodon tgsw ewona,y sts h5r2e.e ,G o-rb faonudri nfglu roerveescaelendc ed ifferent
(Normal) (Normal)
Long Short 18107 9 siglennaglsth osn o cf hcrhormomosoosmome e7s ( 1F iagnudre 2 12), .a Cnodn FsIiSdeHr irnegv ethaele Gd -tbwaon,d tehdr ekea, royro ftyopuirn fglu aonrde sFcIeSnHce
San-Mei NanningZoo 2 N.leucogenys N/A N/A N/A N/A
(Normal) (Normal) 18118 0 ressiuglntsa,l ws oen r ecchorgonmizoesdo mfiev e7 k(aFriygoutryep 1e)s. rCeopnressidenertiinngg tthhree Ge -kbaarnydoetydp kica rsypoetcyipeisn agn adn tdw FoI SH
No14 Missing 2 Long Short N.leucogeny1s8128 1 hyrbesr+iudlsts, ,w wite−h r tehceo glanNtitz.eerc dtfw. filoev eud icksoatirgnygeountyiysphseesd r bepyNr ne.socenfn.htolienmguo ctlhoorggeoeeu nksya psryaiortiynpgi.c species and two
(Normal) (Normal)
18138 2 Nhoymbarsidcus,s w leituhc othgee nlaytste (rF tiwguor de i2st)i nguished by nonhomologous pairing.
Long Short
Noname KunmingZoo 2 (Normal) (Normal) N.leucogeny1s8148 3 NNo/mTAhaes cGuN-sb /lAaenudceodg eknaryyso (tFyNipg/eAusr eo f2 1) 0 individuals (7N♀/,A 3♂) were similar to the G-banded
Long Short 18158 4 (Van TTuhinee Gn -&ba Lneddebde kttaerry, o1t9y8p3e)s aonf d1 0R -inbdanivdieddu aklasr y(7o♀ty,p 3e♂s )f owr eNre. lseiumciolagre tnoy sth (eC Gou-btuarnidere d&
NB1 NingboZoo 2 N.leucogenys N/A N/A N/A N/A
(Normal) (Normal) 18168 5 Le(Vrnaonu lTdu, i1n9e9n1 &). CLhedrobmetotesro, m19es8 31) aanndd 2R2- bwaenrdee ndo krmaraylo. tTywpeos pfoairr Ns o. fl eFuIcSoHg esnigyns a(lCs owueturer ier &
Long Short
NB2 NingboZoo 2 N.leucogeny1s8178 6 foLuNenrd/nA oonu lcdhN, r1/oA9m9o1s).o Cmher o7m, soNusp/opAmoretsi n1g a an dp e2r2ic wenetrreic n ionNrvm/eArasli.o Tnw eov epnati r(sF oigfu FreIS 1HA s).i g nals were
(Normal) (Normal)
Huanglianshan 18188 7 Nfoomuansdc ouns scihkrio (mFiogsuorme e3 )7 , supporting a pericentric inversion event (Figure 1A).
Long Short
HLS3 Nature 2 N.leucogeny1s8198 8 NNo/mTAhaes ckuaNsr /ysAoiktyi p(Fesig oufr efi 3v)Ne s/Aamples (3♀, 2♂) wereN i/dAentical to those of N. siki reported by
(Normal) (Normal)
Reserve 19108 9 CouturTiehr e& k aLreyrontoyupleds (o1f9 f9iv1e). sCaommppleasr e(3d ♀w,i t2h♂ N) .w leeurec oidgeenntyicsa, la t ob atlhaonscee do ft rNan. ssliokci arteipoonr ted by
Huanglianshan Long Short 19119 0 beCtwoueetunr iceAhrcr tu&oaAmt ocLrtosu e2asrt0oon1rm8so, 27ue0, 1l xdp8 F, a 7O(i,1 Rrxs9 PF 9OE1E1R aR) Pn .RE dCEE VRo2I REm2EW Vpw Ia EarWse dd ewteitchte Nd,. rleeusuclotignegn iyns ,o an eb aplaainrc oefd s thraonrtselnoecdat ion 8 of 82 2o f 22
HLS4 Nature 2 N.leucogenys N/A N/A N/A N/A
Reserve (Normal) (Normal) 19129 1 chbr1eo7tm8w 1oe7se8on m ceh r1oT mahneodT sd ohoimpen ledeo i ipppdala oinirriu dsom f1n b duaeemnrr db(iv2 e2enr2 d)( 2owcnfha )ras ool ldmf a eaontlesal colaytmnezadeel,d y 2r zge2eis db[ut bl gt(oii1nbn;sbg2 o w2inn)as] s.o w Sn5ae2ims .p G5ial2ia-r.br Gaotnof- d bstiahhnnoagdtr tiroenebngvse eredaerl vveeedad dl eidff edrieffnetr ent
Fang-Fang NanningZoo 2 Short Long N.siki 19139 2 inc 1NNh7r./9o Al 1me7uo9csl oeoNgnmeg/lAneteh yn1ssg ,aot hFnf sdIc S ohoHfrn oc emdh eprooNmasmio/orAm onossefot srdm a1ete reasidnv 1 dea da 2pn 2cedh,r ia2rcon2emd,n aoFtnrsIidSoc mNHFinI /eSrvA e2Hevr2 esr ai[eoltve n(ed1 ae ;tlv2weed2on ),t]t w .to hSonr,ie mtcehh,i rloreaorer m, f toooour s trfoh ofmaluut eroo fr7bleu ssoecrrevenseccdee nce
E’gui NanningZoo 2 Short Long N.siki 19149 3 (Fii1ng8 u+N0r 1.e 8 l1e0uBs ic)go.− ngTsaiehlgnsen y oassnla,s mc FohIpnrSl oecHm hN“ rodDo.semaosm-mioskosheinoa s7mnt r”(eaF ht7ieag d(duF raaieg p tu1yer)rpe.i i Cc1ceoa)n.ln tCNssriiio.dkcne isis-rnikiikdnvaiegerryr itsonhitgeoy nGtph ee-e.bv Gaenn-dbt eaodnn d kceahdrr ykooamtryyoposiotnymgp iaenn 7gd aFnIdS HFI SH
Qingguangyan NanningZoo 2 Short Long N.siki 19159 4 N(o1FNm8i/g1aAu 1src8eu1 r s1e NBgsua/)rA.lbe tTsrs,iuh ewleltls eas, aerwem (ceFpo rilgegenc uN“iorzDg/eeAn ad4i- z)fs eihvdae nf ki”va ehr yakodat ryayp oteytsyp priceeapsl rNr esesi/pekArnie-tskineangrty itnohgrtye teph ekr.ea er ykoatryypoitcy sppice csipeesc aiensd atnwdo two
Laoer NanningZoo 2 Short Long N.siki N/A N/A N/A N/A
19169 5 N1o82mT 1ha8es2 cGh uy-sbb grhaiayndbbdsr,re iiwdde sliklt,a hawer ty ih(tohFet iyltgahputeetr eslera ot4ttwf)e troh trdweioset iadnnigsitumiinsaghlusNe id(s.1 hbs♀eyidk , n ib2oy♂n hn)× oowmnehoroelmo tghooleuo ssga opmuasier pianasgi r.ti hn agt. o f N.
Da-Shan NanningZoo 2 Short Long N.siki 19179 6 ga1b8r+i3e 1lTl8ah3eeN (GoC−m-Nobauoastmnucduraiessed crlN e uk&us.a c rlgLoeyagueoercbtnynoropygiuseee lsnl(d lFyoa,s if1ge (t9uFhr9ireg1e u2)e.r) ae Tn 2hi)ims ak(lasNr (y.1os♀tiyk, pi2e♂ w) aws esriem tihlaer s taom Ne. assi kthi aint othf eN .
19189 7 prge1as8eb4nr 1ice8el4 lo afe t( (C1To;h2ue2tT u)G.hr i-Oeeb rGan n&l-ydb eaLodnne dekren apdora yukiolradt ryo,y pf1o eF9tsy9I pSo1efH)× s.1 oTsN0if hgi .1nings0da a iklivsnbai rddwryiiueveoairltldleysau po(eae7bl ♀ssw e(,a) r73vs♀♂ es,di) m3 wo♂inel)a r crewh tseroiorm eNm isl.oi amssrio kitmloia rietn h t7 oet, h Gttheh- eub Gas n-dbeadn ded
316 NanningZoo 1 Short Long N.gabriellae 18−5 185( Va+n(V Tauni nTeuniN n&e.n gL &aedb Lbreieetdtleblare,et t1e9r8, 31)9 8a3n)d aRNn-d.b gaRna-dbbeardnie dklelaadr yekoatryypoetsy pfoers Nfo.r l eNu. cloeguecnoygse n(Cyso u(Ctuoruietur r&ie r &
19199 8 dipffreersienngc fer oomf t N(1.; s2i2k)i. aOnndl yN o. nleeu pcaoigre onfy Fs I(SFHig usirgen 1aCls) .w ere observed on chromosome 7, thus
Bai-Shou NanningZoo 1 Short Long N.gabriellae − + N.gabriellae N.gabriellae
186 186L ernLoeurlndo,u 1l9d9, 11)9.9 C1h).r oCmhroosmomoseosm 1e asn 1d a2n2d w 2e2r ew neroer mnoarl.m Tawl. oT pwaoir ps aoifr sF oISf HFI SsiHgn sailgsn walesr ew ere
Jing-Jing NanningZoo 1 Short Long N.gabriella2e0109 9 Ndomif−faesrcinugs flre+oumco Nge. nsyikNsi×. aNgnoadm bNrai.se lceluluasce soigkei nhyysb (rFiidNg u(.Frgeiga 1ubCrre)ie .5 l )la e
187 187f ounfdo uonnd c ohnr ocmhroosmomoseo 7m, es u7p, psourptpinogrt ian pge ar ipceenritcreicn tirnivc eirnsvioenrs ieovne netv (eFnitg (uFrieg u1rAe) 1. A ).
Mei-Mei NanningZoo 2 1short, 1short, N.leucogenys×N202.10s 0ik i No+mThaes cGu-s−b laenudceodg ekNnar.yysleo×tuNycpooem goaefs ncouynsse ssipkei chimyNber.ni ldwe u(aFsc iouggnuireqenu 5ye)s, c×haracterized by a
1long 1Long 20220 1 het1e8r8o 1zT8yh8geN o uGosm-N btaroaasmnncdusalessod csc uikakstai i rso(yiFnkoi.ig t Oy(uFprnieege u3ocr)fhe or 3on)me ospsoecmime 1en a nNwda. s2s 2uik nwiiqeuree ,n cohramraacl,t esriimzeilda rb tyo at hat of N.
1and 189 189 TheT khaer ykoatryypoetsy poef sf iovfe f sivaem spalmespN (l3e.♀sg (,a 32b♀♂r,i) e2 w♂llae)r eew× iedreen itdiceanlt itcoa lt htoo steh oosfe N o.f s Nik.i sriekpi orretpeodr bteyd by
Xiao-Xiao NanningZoo ahalf Short Long N.gabriellae×N2.02s30i k2i leuh1ec9t+o0egr 1oe9nz0yyC sgo,o +uauCtnsuod rtu irteathurne r&s iNoleo rtL.h c&eaelertr niL uocoecnhuro.rln odOgom u(en1loned9s y 9co(sh11m)r9.oe9 Cm 11o)o .am sCnopdoma m2ree2p d1 a wN rwaeen.idrtd shew i2sNkii2tim.h wl ieNluea.r crle oet ogun eoctnohrmygaseta ,no la,yf ssbN,ia ma.l a sibnliaakclreia ,dtn oi cnt retdahdinac tstar latooinfcn sagNlto i.oc nat ion
1and 20240 3 onleeu trcaongselnocyas,t iaonnd t t(h1e; o2t2h)e. rT cwhoro pmaoirsso omf eF 1IS aHnd s i2g2n awlse rwe esriem oilbasre trov ethda ot no fc hNr.o smikois, oinmdei c7a,t ing
A-Huang NanningZoo Short Long N.gabriellae×N.siki 1N9/1A 191b Netw/bAeeetwn ecehnr ocmhroosmNomo/Aseo pmaeir ps a1i rasn 1d a2n2d w 2a2s w dNaest/e Adcetetedc, treeds,u rletisnuglt iinng o inne opnaeir p oafi rs hoof rstheonretde ned
ahalf 20250 4 inodnicea ttrinangs alo pceartiicoenn tt r(i1c; i2n2v)e.r sTiwono epvaeinrst o(Ff iFgIuSreH 1 sDig)n. aTlsh iws esrpee coibmseernv weda so nid cehnrtoifmieods aosm ae 7,
192 192c hrocmhroosmomoseo 1m aen 1d aonnde opnaeir p oafi rd oefr idveerdi vcehdr ocmhroosmomoseo 2m2e [ 2t 2(1 [;t2 (21);]2. 2S)i]m. Siliamr itloa rt htoat tohbats eorbvseedr ved
ThekaryotypeandmitochondrialofDa-Shansuggestdifferentspecificaffinities.+:Th2e0260p 5o shityiinvbdreiidcr aoetfis nNug. l atl epouefcroiPcgeCenntRyrisc×u iNsnv.i nesirgksiipo. nr iemveenrt s(FeigtuBreP 1Dor). HThAisS s.p−eci:men was identified as a
193 193i n Ni.n l eNu. cloeguecnoygse,n FyIsS, HFI SdeHm doenmstorantsetdra ate pde ar ipceenritcreicn tirnivc eirnsvioenrs ieovne netv oennt cohnr ocmhroosmomoseo 7m e 7
ThenegativeresultofPCRusingprimersetBPorHAS.N/A:Notavailable. 206 h1y9b4r 1id9 4o( fF Nig(.u Flreiegu u1crBoe)g .1e TBnh)y.es T ×shaNem. spsailmke i“p. Dlea “-sDhaa-ns”h ahna”d haa tdy pa itcyapl isciakli -skikair-ykoatryypoet.y pe.

195 195N omNaosmcuass cguasb rgiaebllraiee l(laFeig (uFrieg u4r)e 4)
196 196 TheT Gh-eb Gan-dbeadn dkeadr ykoatryypoetsy poef st horfe teh areneim analism (a1l♀s (, 12♀♂,) 2 w♂e)r ew tehree sthaem sea amse t haast tohfa tN o.f N.
358 www.zoores.ac.cn
197 197g abrgiaebllraiee l(laCeo u(Ctuoruietur r&ie rL &er nLoeurlndo,u 1l9d9, 11)9.9 T1h).i sT khaisr ykoatryypoet ywpaes w siams isliamr itloa rN t.o s Nik.i siink it hine the
198 198p respernecsee nocfe t o(1f ;t2 (21);.2 O2)n.l yO nolnye opnaeir p oafi rF oISf HFI SsiHgn sailgsn walesr ew oerbes eorbvseedr voend c ohnr ocmhroosmomoseo 7m, et h7u, st hus
199 199d iffedriifnfegr ifnrogm fr oNm. s Nik.i sainkdi aNn.d l eNu. cloeguecnoygse n(Fysig (uFrieg u1rCe) .1 C ).
200 200N omNaosmcuass cleuus cloeguecnoygse×nNyso×mNaosmcuass csuiksi shikyib hriydb r(Fidig (uFrieg u5r)e 5)
201 201 TheT Gh-eb Gan-dbeadn dkeadr ykoatryypoet yopfe o onfe osnpee csipmeecnim weans w uansi quunei,q cuhea, rcahcaterariczteedri zbeyd ab y a
202 202h etehroetzeyrgoozyusg otruasn tsrlaoncsaltoiocnat.i Oonn.e O cnher ocmhroosmomoseo 1m aen 1d a2n2d w 2e2r ew neroer mnoarl,m saiml, isliamr itloa rt htoat tohfa tN o.f N.
203 203l eucloeguecnoygse,n aynsd, atnhde oththe eort hcherr ocmhroosmomoseo 1m aen 1d a2n2d w 2e2r ew seirme isliamr itloa rt htoat tohfa tN o.f s Nik.i ,s iiknid, iicnadtiincgat ing
204 204o ne otrnaen tsrlaoncsaltoiocnat ito (n1 ;t 2(12;) .2 T2w). oT pwaoir ps aoifr sF oISf HFI SsiHgn sailgsn walesr ew oerbes eorbvseedr voend c ohnr ocmhroosmomoseo 7m, e 7,
205 205i ndiicnadtiincgat ian pge ar ipceenritcreicn tirnivc eirnsvioenrs ieovne netv (eFnitg (uFrieg u1rDe) 1. DTh).i sT shpise csipmeecnim weans w idaesn itdifeinetdif aiesd a a s a
206 206h ybrhiydb orifd N o.f l eNu. cloeguecnoygse×nNys.× sNik.i .s iki.
Amplification,sequencing,andspeciesidentification
A
Afterkaryotyping,weextractedtotalDNAfromtheculturedcells 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
sampleswasconsideredacceptableforsubsequentanalysesas 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence
other samples were not well preserved after karyotyping. We B
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 (5(cid:48)-GGACTTATGACATGAAAAATCATCGTTG-3(cid:48)) 181 results, we recognized five karyotypes representing three karyotypic species and two
and H15915_hk3 (5(cid:48)-GATTCCCCATTTCTGGTTTACAAGAC-3(cid:48)). 182 hCybrids, with the latter two distinguished by nonhomologous pairing.
WealsoconductedPCRusingtheprimersprovidedinCarbone
183 Nomascus leucogenys (Figure 2)
etal.(2009)(263C9_BP_Lwith263C9_BP_R(BPhereafter)and
263C9_BP_Lwith263C9_HSA22_R(HSAhereafter)),targeting 184 The G-banded karyotypes of 10 individuals (7♀, 3♂) were similar to the G-banded
a fragment across a breakpoint on chromosome 7. According 185 (DVan 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
(e.g.,Hylobatesspp.,N.gabriellae,andN.siki),andBPprimers 187 found on chromosome 7, supporting a pericentric inversion event (Figure 1A).
E
shouldresultinpositiveamplificationonlyforN.leucogenys.The
188 Nomascus siki (Figure 3)
mitochondrial and nuclear amplicons were sequenced using a
189 The karyotypes of five samples (3♀, 2♂) were identical to those of N. siki reported by
ABI3730sequencer(AppliedBiosystems,USA).
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
Figure1ComparisonofG-bandedchromosomes1,7,and22
sequencing results using the following strategy: (1) we 178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different
1a91n dFbIeStwHeerne cshuroltmsowsoimthe phauirms 1a annd2 222 cwharso dmeteocsteodm, rees-usltpinegc iinfi ocnep rpoaibr eof 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
the karyotyping results using PCR with primer sets BP and 1i9n2 difcfehrroemnotsNomoem 1a asncdu osnes ppaeirc oife dseraivnedd tchhreoimrohsyobmrei d22s [t (1;22)]. Similar to that observed
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 1A93: Nomina Nsc. ulesucleougceongyes,n FyIsS;HB d:eNmoomnastsrcautesd sai kpie;riCce:nNtroicm iansvceurssiogna ebvreienltla oen; cDhr:omosome 7
181 results, we recognized five karyotypes representing three karyotypic species and two
nucleotide database; (4) we estimated a gene tree to verify 1h9y4b rid(oFfigNu.rele 1uBco).g Tehney ssa×mNp.lsei k“iD;Ea-:shhyabnr”i dhaodf Na .tygpaibcarile slliakei-×kNar.ysoitkyi.pe.
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 1N95o m18N3a somcNuaosscmualses cguuascb loreiugecleolagnee ny(yFssi g((FuFirgeiug r4eu) 2r)e 2)
species downloaded from GenBank; and (5) we repeated 1T96h e18G4 -baTnhdee TGdh-eb kGaan-brdayenodd tekydap kreyarosytoyotpyfeps1e so0 of fit nh10dre iievn diadinvuiimdaualasllss (((771♀♀,,, 332♂♂) ))w wwereeer ersei mthiselai msr atomil atehr ea sG t-hbaant doefd N .
the karyotyping and amplification/sequencing in triplicate in A1ctt9uoa7to trsh1 28e0g51 8aG, b7,r- (xibV eFalOalnanR edTP E(ueECiRdno eRun(E tVV&uIarE iLWneer d T&bue tLitneerer, n1no9u8&3ld) ,La 1ne9dd 9Rb1-)eb.a tTntedherids, kk1aa9rryy8oo3tty)yppeaesn fwodra sNR s. -ilbmeuaiclnoagrd etenody Ns (. Csoiku8it uiornfi e2tr2h &e
cases where the mitochondrial, nuclear sequencing, and karyotypes for N. leucogenys (Couturier & Lernould, 1991).
karyotyping results conflicted (e.g., sample Da-Shan, se1e78 1C98h Tr1ho8emp6 d roeipsselLooneicmrdne oneouusflm dt,1 b(11e9a;r92 n(122d)).n. C2)O ho2nrfol wyma looel snaroenem apenlaysoi z1rer oamdnf gdaF i2Ibl.S2b HTownw essrioe gw nnpaoasarlm s5i raw2sl..e GTorewf- booFab pInsSadeirriHsnv geosdf r i FegoIvnnSe Hacahl lsesridog nmdaiolfssf ewormeerneet 7, thus
Results and Discussion). A final purebred or hybrid an1d79 le1nw9g9et hr1se 8od7ffo i fcufhenrfrooidnumgnood f nsrooonmcm cheh Nrsroo 1.m m saoiknosoids m a2one2md 7, ,Nae sn.u d7pl ep,FuoIscrStuoiHngpg epr naeo yvprsee tar(iilnFceeigdgn tutarwriceop i1,ne Cvthre)irrc.es eie o,nn o terrv ifceonuitn r( Fvfileguurosrreei o1scAne)n. c e
speciesidentificationwasdetermined. 180 sigenvaels1n 8ot8n( FchigNrouommreoass1ocumAse )s. i7k i( (FFiigguurree 31)) . Considering the G-banded karyotyping and FISH
200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5)
We constructed the maximum likelihood gene tree
189 The karyotypes of five samples (3♀, 2♂) were identical to those of N. siki reported by
181 resuAlcttsu,a tworse 2 0r1e8,c 7o, xg FnOiRz ePdEE fRi RvEeV kIEaWr y otypes representing three karyotypic species and tw1o7 of 22
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 1b82 hybrids, with the latter two distinguished by nonhomologous pairing.
202 heterozygous translocation. One chromosome 1 and 22 were normal, similar to that of N.
191 between chromosome pairs 1 and 22 was detected, resulting in one pair of shortened
sequences,17cytbsequencesrepresentingsevenrecognized
183 Nomascus leucogenys (Figure 2)
species were downloaded from GenBank (Supplementary 203 19l2e ucochgreonmyoss, oamnde 1th aen do tohneer pcahirr oomf doesroivmede c1h raonmdo 2so2m wee 2r2e [sti m(1;i2la2r) ]t.o S tihmaitla or ft oN t.h asti koib, sienrdveicda ting
TableS1)andalignedwithoursequencesusingMAFFTv7.1384 204 T1h9eo3 G ne- btiranan Nnds.e lldoe cukacatoirgoyenon ttyy s(p,1 eF;s I2 So2Hf) .1d T0em winoodn ispvtariaidrteusd ao lafs pF(e7IrS♀icH,e n 3st♂rigic)n iwanlvese rwres iesorinme eoivlbaesrn ett roov nteh dceh orGonm- cbohasrnoodmmeedo 7 s ome 7,
(Katoh & Standley, 2014). We partitioned the alignment1s85 (V20a5n T1u9ii4nn ednic (&aFtii gLnuger eda b1 peBet)tr.e iTrc,eh 1ne 9tsr8aicm3 )ip nalevn e“dDr sRaio--sbnha aennvd”ee hndat dk( Faar itygyouptriyceap 1le Dssi k)f.io -Tkr ahNriys.o lsteypupecec.oi mgeenny ws (aCs oiduetunrtiiefire &d as a
by codon positions (three partitions). We used GTR+G as
the evolutionary model for each partition as RAxML doe1s86 L2e0r6n ou1l9dh5, y 1b9r9Nid1o )om.f aC Nshc.ur olse mguaocbosrogiemelnleayses 1×(F Naign.u dsr ie2k 42i.) were normal. Two pairs of FISH signals were
not accept models other than GTR or GTR+G. We ran eac1h87 found 1o9n 6 chromosTohme eG -7b,a snudpedp okratriynogt yap pese roifc tehnretrei ca niinmvaelrss (io1♀n ,e 2v♂en) tw (eFreig tuhree s a1mAe) .a s that of N.
analysisusingtherapidbootstrappingalgorithmandletRAxM1L88 Noma1sc9u7 s sigkai b(rFieiglluaree ( C3)o uturier & Lernould, 1991). This karyotype was similar to N. siki in the
haltbootstrappingautomatically. 189 Actuators 2T011h98e,8 7k , xa rFyOporRet PsyeEpnEecRse R ooEffV ItfE i(Wv1e; 2 s2a).m Opnlleys o(n3e♀ p,a 2ir♂ o)f FwIeSrHe siidgennatlisc wale rteo othbsoesrev eodf oNn. cshirkoim roepsoom8rt eeo fd7 2 , b2t hyu s
199 differing from N. siki and N. leucogenys (Figure 1C).
RESULTS 17189 0 CoTuthuer ideirp &lo iLde nrunomubledr ( (129n9)1 o).f Calol manpaalryezde wd igthib Nbo. nles uwcaosg e5n2y. sG, -ab baanldainncge rde tvreaanlseldo cdaitfifoenre nt
200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5)
17199 1 lenbgetthwse oefn cchhrroommoossoommees p1a airnsd 1 2 a2n, da n2d2 FwIaSsH d eretevcetaelde,d r tewsuol,t itnhgre ien, oonr efo puari rf loufo srehsocretennceed
Identifiedkaryotypicspecies 201 The G-banded karyotype of one specimen was unique, characterized by a
18109 2 sigcnharlosm oons cohmroe m1 oasnodm oen 7e (pFaiigr uorfe d 1e)r.i vCeodn csihdreorminogs othmee G 2-2b a[nt d(1e;d2 2k)a]r.y Soitmypilianrg t aon tdh aFt IoSbHs erved
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.
G-banding revealed different lengths of chromosomes 1 a1n81d19 3 4r5e4si unl FNtsFi,.g i wglu2eue0urr 3ecer eo2c2g GoelgGne-buny-acisbnzo, edgaFdeenIdn Sf dyikHvsae,e raddy kneodamkt yrtayohpoerne tysooytotfrph aateety rsen cpdorhe reartpho rpoemeersfonresi awcnoemthninniettog er1i- r ctcath hnhirendeeev e2kre 2eknr dsaw irwgoeyirnbohe b tesiyotvipmneei in-(clNca ts rh.op tlneeoe c uectihchekaorstego aoedmnfn doNys s.t)o ws mi koie , i7n dicating
22,andFISHrevealedtwo,three,orfourfluorescencesign1a8l12s9 4 hy(bFrgiigdiusb,r b2ew0 o1i4tBn h) t.(h NoTenh .elea lt etrstaaeunrmcs tlpoowclgeoa t e“idoDninsay tti- sn(s1gh);ua 2ni2s”h) .he Tdawd b oya p tnayoiprnsi hcoaofl mF sIoiSklHoi-g ksoiagurnysa olpstay wipreienr.e g o. b served on chromosome 7,
on chromosome 7 (Figure 1). Considering the G-banded
karyotyping and FISH results, we recognized five karyotyp1e813s9 5 NoNmoNamsocamu2sc0su 5al ses ugccaiuonbgdsrieicenasltyliiasnk eg(i F(aFi( pgFieguriurigecre eu2n 4)rtr )ei c 3in)version event (Figure 1D). This specimen was identified as a
representingthreekaryotypicspeciesandtwohybrids,with1th81e49 6 TThhTee2h G0e6k -G baa-rbnyhadyonebtddryei dkpd ao ekrfys aNory.t yoloeptfuyecpsfio eogvsef e no1yf0 sst ×hianNredm. eisv ipakidnil.eui maslasl (s(3 7(♀1♀,, 3, 2♂2♂) ))w wewreere esr itemheil iasdar emtone t thaicse atGhla-btt aoonf dNed.
lattertwodistinguishedbynonhomologouspairing. those of N. siki reported by Couturier & Lernould (1991).
18159 7 (Vgaanb Triueilnlaeen (&C oLuetdubrieetrte &r, 1L9e8rn3o) ualndd, 1R9-9b1a)n.d Tehdi sk akrayroytoytpyepse fwora sN s. ilmeuilcaor gtoen Nys. s(iCkoi uintu trhiee r &
18169 8 Leprnreosuelndc, e1 o9f9 1t )(.1 C;2h2r)o. mOonsloym oense 1p aainr do f2 2F IwSeHr es ingonramlsa lw. eTrwe oo bpsaeirrvs eodf oFnIS cHhr soimgnoaslosm wee 7re, thus
18179 9 foudnifdf eornin cgh frroommo Nso.m siek i7 ,a Znsuodp oNploo. rlgetiiunccgao lag Repneeyrsisce e(aFnirtgrciuchr ei3n 1v9Ce(r5)s.) io: n3 e5v6e–n3t (6F3ig,u2r0e 118A).3 59
18280 0 NoNmoamscauscsu ssi klie u(Fciogguerney 3s)× Nomascus siki hybrid (Figure 5)
18290 1 ThTeh kea Gry-obtaynpdeesd o kf afrivyeo tsyapme polfe so n(3e♀ sp, e2c♂im) wene rwe aisd eunntiiqcuael ,t oc htharoascet eorfi zNe.d s bikyi ar eported by
19200 2 Cohuettuerrioezr y&go Luesr tnroaunlsdlo (c1a9ti9o1n).. OConem cpharroemd wosiothm Ne .1 l eauncdo 2g2e nwyesr, ea nboarlmanacl,e dsi mtrailnasrl otoc atthiaotn o f N.
19210 3 beltewueceong cehnryosm, aonsdo mthee poathiresr 1c harnodm 2o2s owmase d1e atencdt e2d2, rweesurelt sinimg iilna ro tnoe tphaaitr o off N sh. osirktei,n iendd icating
19220 4 choronme otrsaonmsleo c1a atinodn ot n(e1 ;p 2a2ir) .o Tf wdeor ipvaeidrs c ohfr oFmISoHso smigen 2a2ls [wt (e1re;2 o2b)]s.e Srvimedi loanr tcoh trhoamt oosbosmerev e7d,
455
19230 5 in iNnd. ilceautcinogg ean pyesr, iFceISnHtri cd einmvoenrssitorant eedv ean pt e(rFicigeunrteri c1 Din)v. eTrhsiios ns peevceinmt eonn wcharso imdeonstoifmieed 7 a s a
456 Figure 3 G-banded karyotype of a southern white-cheeked gibbon (N. siki)
19240 6 4(5F7ih gyubr rei d1 Bof) .N T.h lee uscaomgpelney “sD×Na-.s hsiakni.” had a typical siki-karyotype.
195 No mascus gabriellae (Figure 4)
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
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.
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 (Figure 1D). This specimen was
between chromosome pairs 1 and 22 was detected, resulting 458 identifiedasahybridofN.leucogenys×N.siki.
inonepairofshortenedchromosome1andonepairofderived 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, FISH demonstrated a pericentric inversion event
Actuators 2018, 7, x FOR PEER REVIEW 8 of 22
on chromosome 7 (Figure 1B). The sample “Da-Shan” had a
178 The diploid number (2n) of all analyzed gibbons was 52. G-banding revealed different
typicalsiki-karyotype.
453 179 lengths of chromosomes 1 and 22, and FISH revealed two, three, or f our 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)4. 6 0
Actuators 2018, 7, x FOR PEER REVIEW Figure8 5of G22 -bandedkaryotypeofaN.leucogenys×N.siki
461 Figure 5 G-banded karyotype of a N. leucogenys×N. siki hybrid
188 Nomascus siki (Figure 3)
462 hy brid
178 18T9h e diploTidh en kuamryboteyrp (e2s nof) foivf ea slal manplaelsy (z3e♀d, 2g♂ib)b woenrse iwdeanst i5ca2l. tGo t-hboasne doifn Ng. rseikvie raelpeodrt eddi fbfye rent
179 leng1t9h0s of Ccohurtoumrieor s&o mLeersn o1u aldn (d1 92921,) .a Cnodm FpIaSreHd wreivthe Nal.e ldeu tcwogoe, ntyhsr,e ae b, aolra nfcoeudr t rfalnusolorecasNtcioeonnm cea scusgabriellae×Nomascussiki hybrid(Figure6)
180 sign1a9l1s onb cehtwroeemn ocshorommeo s7o m(Fei gpauirrse 11 a).n dC 2o2n wsiadse dreintegc ttehde, rGes-ubltainngd iend o knea rpyaoirt yofp sinhogr taennTeddh F eISdHi stinctG-bandedkaryotypeoftwoindividualswassimilar
455F 1i8g1u rer3esuG1l9t-s2b, awnec dhrreeocmdoogksnoaimzreey d1o fatinvydep o kneae ropyafoirta yofps deoesr uirvetephdr eechsrernonmtwionshgo mittheer 2e-2ce h[kt ae(1rey;2ko2te)y]d.p Sicim siplaerc tioe tsh aatn otdbo stew rvtohe da t of N. siki and N. gabriellae in the presence of
456g ibFbigourne 3( NG1-.9b3sa nidkeiidn) k Nar. yleoutycpoeg eonf yas ,s oFuItShHer dne wmhointes-tcrahteeedk ae dp egriibcbeonntr i(cN i.n svikeri)s ion event on chromosomte 7( 1; 22). FISH detected three fluorescence signals on
457 182 hybrids, with the latter two distinguished by nonhomologous pairing. chromosome 7, indicating heterozygous pericentric inversion
194 (Figure 1B). The sample “Da-shan” had a typical siki-karyotype.
(Figure1E).Thesetwoindividualswereidentifiedashybridsof
N18o3m asNcoum1s9a5s gcuasNb loermuieacsolclguaese ngya(bsF r(iFeiglilgaueu rr(Fee ig24u) r)e 4)
N.gabriellae×N.siki.
T1h8e4 G-ba1n9Td6h ee dG-kbaaTnrhydeeo Gdt y-kbpaanreydseodty okpaferysto hotyrfpe 1ees0 o ifan tdnhirivemeid aaunlaismlsa (l(s17 (♀1♀,, ,3 22♂♂))) wwweerreee r thseiem staihlmaeer atos tthhaet oGf -NAbc.ta unatodrse 2d01 8, 7, x FOR PEER REVIEW 19 of 22
s1a8m5 e (aVsa1nt9 hT7 auitngeoanbf r&iNe lLl.aeed g(bCaeobtuttreuirre,i e1lrl9 a&8e 3L)e (raCnnodou luRd,t -1ub9ar9ni1ed).re Tdh& kisa krLayeroyrtoyntypopeeus wlfdoasr, sNi1m.9 ille9aur1 tco)o .Nge. snikyis i(nC thoeu turier &
ThiskaryotypewassimilartoN.sikiinthepresenceoft(1;22).
198 presence of t (1;22). Only one pair of FISH signals were observed on chromosome 7, thus
186 Lernould, 1991). Chromosomes 1 and 22 were normal. Two pairs of FISH signals were
OnlyonepairofFISHsignalswereobservedonchromosome
199 differing from N. siki and N. leucogenys (Figure 1C).
71,8t7h usfoduifnfde roinn cghrforommosoNm.es 7i,k siuappnodrtiNng. lae puecriocegnetrnicy sinv(eFrisgiounr eev1enCt )(F.igure 1A).
200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5)
1A8c8tu ators 2N01o8, m7, xa FsOcRu PsE EsRi kREiV (IEFWig ure 3) 18 of 22
201 The G-banded karyotype of one specimen was unique, characterized by a
189 20T2h e khaerteyrootzyypgoeuss o tfra fnisvloec saatimonp. lOens e( 3ch♀ro, m2o♂so) mwee 1r ea nidd e2n2t wicearle tnoo rtmhoals,e s iomfi lNar. tsoi tkhia tr eopf oNr.t ed by
190 Cou2t0u3r ier l&eu cLoegrennoyus,l dan (d1 t9h9e 1o)th. eCr ochmropmaoresodm we i1th a nNd .2 l2e uwceoreg seinmyilsa,r ato b tahlaat nocf eNd. stirkain, sinlodiccaattiinogn
191 betw20e4e n cohnreo mtraonssolomcaeti opna itr (s1 ;1 2 a2n).d T 2w2o wpaairss doef tFeIcStHed s,i grneaslus lwtienrge oinbs oernvee dp oanir c ohfro smhoosrotmenee 7d,
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 FFigiguurree 6 6G-Gba-nbdeadn kdareyodtykpea orfy ao Nt.y gpaberioelflaae×NN.. slikeiu hcyborigd enys×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
Mitochondrialandnuclearsequences
198 presence of t (1;22). Only one pair of FISH signals were observed on chromosome 7, thus
Wedeterminedthecompletecytbsequencesfor10individuals
458 Figure4G-bandedkaryotypeofayellow-cheekedgibbon(N .
459 1F9i9g ure 4d iGff-ebarinndge df rkoamry oNty.p sei okfi aa nyedl lNow. -lcehueceokegde ngiybsb o(Fn i(gNu. rgea 1brCie)l.l a e) representing the three purebred species (n=7) and two
gabriellae) hybrids (n=3) identified in the karyotypic analyses. All new
200 Nomascus leucogenys×Nomascus siki hybrid (Figure 5)
sequences are available in GenBank under accession Nos.
N20o1m ascusThlee Gu-cboangdeend ykasr×yoNtyopem oaf sonceu sspesciimkeinh wyabsr uidniq(uFei,g cuharreac5te)rized by a MH188408–MH188428 (Supplementary Table S1). Five out
T2h0e2 Gh-ebtearonzdyegodus ktraanrsylooctyatpioen. Oonfe cohnroemossopmeec 1im anedn 22w waesre nuornmiqalu, esi,milar to othfatth oef Ns.e venpurebredanimalsweresupportedbyBLASTand
characterized by a heterozygous translocation. One phylogeneticanalysesusingthecytb geneandtheothertwo
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 (Figure 7). One sample (Da-Shan) was identified
le20u4c ogoenney tsra,naslnocdattihone t o(1th; 2e2r).c Thwroom paoirsso omf FeIS1H asingdnal2s2 wewree roebsserivmedil aorn chromoassomNe .7,s iki based on its karyotype, but its mitochondrial gene
to20t5h ationfdiNca.tisnigk ia, pinerdiciceanttriinc ginovenresiotrna envsenlot c(Faitgiuorne 1tD(1).; T2h2is) .spTewciomepna wirass identifwieda sas at ypical 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 itsmitochondrialgeneresults.
but could not be identified unambiguously as it was equally BecausethekaryotypeandPCRresultswerecongruent,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). Onourcytbgenetictree(Figure7),sample14was siki due to the inconsistent patterns revealed by karyotyping
acloserelativeto(BS=84)andformedasisterlineagewithN. and PCR using the HSA and BP primers (siki), and the
siki+N.leucogenys(BS=52). mitochondrialphylogeny(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
chromosome7inN.siki andN.leucogenys.Wealsorevealed
thedefinitiveexistenceofhybridsinzoosinChina,whichcalls
attention to the animal ethics and welfare issues related to
endangeredspeciesbreedingincaptivity.UsingtheNomascus
species as our focal taxa, we evaluated the accuracy and
efficiencyofbothapproachesinidentifyingpurebredandhybrid
Xiao-xiao-hybrid species.
OurresultssupportedthatN.gabriellae,N.leucogenys,and
N. siki are distinguishable based on translocation t (1; 22)
andinversiononchromosome7(Figures1–5),eventhoughN.
leucogenysandN.siki divergedfromeachotheronlyrecently.
Couturier&Lernould(1991)revealedthatN.hainanusdoesnot
haveaninversiononchromosome7ortranslocationt(1; 22),
andthereforehasadistinctkaryotype. Itwouldbeinteresting
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).
Duetointerspecifickaryotypicvariation,hybridscanbeeasily
distinguishedbasedonheterozygousR-,G-bandedkaryotypes
and FISH results (Couturier & Lernould, 1991; this study). In
wildpopulations,hybridizationofHylobatesspeciessuchasH.
Figure 7 Maximum-likelihood gene tree estimated using the lar×H.pileatus, H.lar×H.agilis, andH.muelleri×H.agilis/H.
cytbgenesofNomascusspecies albibarbis has been observed (Brockelman & Gittins, 1984).
Hybridization between Nomascus species in the wild has not
Branchlengthscorrespondtosubstitutionrates. Numbersonbranchesare
beenreported,whichislikelyduetotheirallopatricdistribution.
bootstrapvalues. SequencesdownloadedfromGenBankareingrayand
However, we observed heterozygous karyotypes in captive
GenBankaccessionNos.areshown. 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 nonhomologouspairingduringmeiophase(Figures5,6),which
concordantwiththekaryotypicresults.ThePCRresultsusingthe is an interesting characteristic in Hylobatidae. Because
BPprimers(confirmingtheinversion)werepositiveforpurebred 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
thesesamples(Table1). ThePCRresultsusingtheBPprimers diversificationandspeciation,resultinginhighspeciesdiversity
werenegativeforpurebredN.gabriellaebutpositivewhenusing withinthefamily.
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.
bothBPandHSAprimers. ForDa-Shan,thePCRresultswere siki (Figures 1–3), consistent with the findings of Couturier
positiveusingBPprimersandnegativeusingHSAprimers,which & Lernould (1991), though not supported by Carbone et al.
werecongruentwithitskaryotype(siki type)butdisagreedwith (2009). The negative results obtained in the latter study may
ZoologicalResearch39(5):356–363,2018 361
be due to the hair sample used to represent N. siki, which is butcouldnoteasilydistinguishN.leucogenys/N.siki fromthe
known for low DNA quantity as well as the existence of PCR hybridofN.leucogenys×N.siki.
inhibitors.
The phylogenetic position of sample 14 was an interesting COMPETINGINTERESTS
finding. ItskaryotypewasidenticaltotheotherN.leucogenys
Theauthorsdeclarethattheyhavenocompetinginterests.
samples (Table 1), which was not supported by the
mitochondrialgenetree(Figure7). Werepeatedamplification 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 completedallmolecularexperiments. W.H.N.,K.H.andX.L.J.analyzedthe
sorting between N. leucogenys and N. siki, resulting in data,wroteandrevisedthepaper.Alloftheauthorsreadandapprovedthe
non-monophyleticrelationships. Thissituationhasneverbeen finalmanuscript.
observed because the effective population sizes of gibbon
ACKNOWLEDGEMENTS
species are usually small. Alternatively, this sample might
represent a distinct and unknown taxon, which is a close
WearegratefultotheDirectorsofNanningZoo,NingboZoo,KunmingZoo,
relative but not identical to N. leucogenys or N. siki. The
andHuanglianshanNationalNatureReserveforallowingustosampleblood
sample was obtained from the Kunming Institute of Zoology,
fromthegibbonsforthisstudy.
ChineseAcademyofSciences,in1993andtheinformationon
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