Population and genetic diversity of Tibetan red deer based on fecal DNA

Hejiao Hu·Bo Xing·Miao Yang·Hamenya Mpemba·Zhonghai Lv·Minghai Zhang

Introduction

Microsatellite DNA is also known as simple sequence repeats or short tandem repeats(Weber 1990).Large quantities of microsatellite markers are widely distributed and contain real genetic information,so they are widely used for individual identi fication and genetic diversity analyses;these markers are especially suitable for studying animal fecal DNA that is severely degraded(Yin et al.2007).

Tibetan red deer(Cervus elaphus wallichii)is one of eight subspecies of red deer in China.It is endemic to the Tibet Autonomous Region and is a national grade II key protected wild animal.The ‘China Red Data Book of Endangered Animals’lists red deer as an endangered species.C.elaphus is listed by the ‘International Union for Conservation of Nature’(IUCN)Red List of threatened species as a species of least concern(LC)(Lovari et al.2016).In 1992,C.elaphus was declared extinct in the wild by the World Wildlife Fund(Liu 2009).Since 1995,Chinese and foreign scholars rediscovered and con firmed its presence in Tibet.This rare and endangered subspecies currently remains in only a very small range of areas in Tibet,in Sangri County(Liu 2009).Thus the subspecies is a high conservation priority for international organizations and wildlife biologists.Currently,there is little existing information on Tibetan red deer abundance or genetic diversity.

In this study,we collected and extracted DNA from wild Tibetan red deer fecal pellets in the green-grass period during 2013–2014.We quanti fied genetic diversity of the Tibetan red deer population by individual microsatellite markers and we analyzed the population using capture–mark–recapture(CMR)methods to reveal its status and provide a valid scienti fic basis for protection of Tibetan red deer(Tian et al.2010).

Materials and methods

Sample collection

A line transect was sampled to cover the entire Tibet Shannan Red Deer Nature Reserve(hereafter,the reserve)in Tibet’sSangriCounty (92°04′–92°28′E,29°10′–29°20′N)in August 2013 and August 2014 during the green-grass period.We collected all fecal pellet groups encountered along the sample transect.From each group we loaded a few pellets into a centrifuge tube and preserved them by soaking in 95%alcohol(Goossens et al.1998;Whittier et al.1999;Zhang et al.2004).We collected 199 pellet groups during 2013 and 2014.

DNA extraction

The QIAamp DNA Stool Mini Kit(RT)was used to extract fecal DNA following manufacturer instructions.Also,the success of DNA extraction and purity of genomic DNA were examined by 1.0%agarose gel electrophoresis and the DNA was stored at-20°C.

Screening of microsatellite primers

Twelve pairs of microsatellite primers were screened based on their high polymorphism as determined in our previous work(C143,N,ETH225,T507,DM42,T530,DM45,T501,BM203,BM1225,T123,and T156)(Zhang et al.2010).Primers were synthesized by Sangon Biotech(Shanghai,China)and dissolved in deionized water,which was sterilized at a concentration of 100 μmol/L as a stock solution for standby(Table 1).

PCR

The total volume of the reaction system was 10 μL,and included 0.1 μL Taq;1 μL 10 × buffer;1 μL dNTP;1 μL BSA;0.2 μL each for upstream and downstream primers together with template DNA and sterilized deionized water to a volume of 10 μL.Ampli fication was conducted as follows:95 °C denaturation for 10 min;40 cycles of 95 °C denaturation for 30 s,54–57 °C annealing for 40 s,72 °C extension for 10 min;and a final extension at 72°C for 10 min.PCR products were stored at 4°C.All samples were ampli fied three times,and 3 μL of the ampli fication product was subjected to agarose gel electrophoresis.

Individual identi fication

Excel?Microsatellite Toolkit software was used to find the gene microsatellite genotyping data matches.The standard we used was described by Bellemain et al.(Eva et al.2005)and required that fecal samples could be ampli fied three times.When only one allele was found,the individual was considered homozygous for this allele;when two different alleles were found,then the individual was considered heterozygous.After a given sample was ampli fied three times,and if only one allele was ampli fied each time,the sample was then re-ampli fied.After ampli fication,the allele frequency was inferred.If the allele appeared two or more times,it was determined tobe heterozygous,and ifthe allele appeared once,it was determined to be homozygous.

Genetic diversity

Excel?software was used to evaluate genetic parameters for all sites and individuals(Sun et al.2011).We then calculated the number of alleles(Na),effective number of alleles(Ne),observed heterozygosity (Ho),expected heterozygosity(He),and polymorphism information content(PIC)(Zhan 2006).

Population analysis

The non-invasive mark–recapture(NMR)method was used to estimate population size.Individual deer identi fied from one pellet group and later identi fied as the same individual by another pellet group were ‘marked’by the first identi fication and ‘recaptured’by the second(Zhang et al.2010).Capwire in R was used to analyze such population samples.Capwire used two models because of different probabilities of capturing individual deer:the even capture probability model(ECM)and two innate rates models(TIRMs)(Miller et al.2005;Pennell et al.2013).One thousand bootstrap replicates were performed to generate con fidence intervals for population size in both models at the 95%level.To test the applicability of the distribution of collected samples for each model,a likelihood ratio test in Capwire was conducted.If a model test P value significantly deviated from 0,the number of samples distributed was consistent with model assumptions.Accordingly,these results were used to determine a suitable model for this population.

Table 1 Microsatellite primersequences

Results

DNA extraction

Because of the high elevation of the study area(c.4300 m a.s.l.)and its dry climate,DNA degradation confounded the processing of collected and preserved feces.We reextracted samples from which we were initially unable to obtain ampli fication products.This enabled the successful extraction of DNA from 87 feces samples that we used for subsequent analysis(Fig.1).

Fig.1 Ampli fication results.Lane 1 was a DNA ladder,2 was a positive control,3 was a negative control,and 4–25 were samples

Individual identi fication

The 12 sites had high joint distinguishability rates.Even in thecase oftwins,theprobabilityofdeterminingerrorP(sib)was only 0.000031(Fig.2).Final individual identi fication revealed that the 87 samples belonged to 50 individuals.

Population genetic diversity

The population average Na was 7.58±0.18 and Ne was 4.91±0.16;average Ne for each site was much lower than Na,and the difference was signi ficant(P＜0.01).Single microsatellite PICs ranged from 0.39 to 0.90,with an average of 0.67±0.013.Among the 12 sites,only T123 was a moderately polymorphic locus,and the other 11 sites were highly polymorphic.He ranged from 0.45 to 0.91,with an average of 0.72±0.01,and average performance of heterozygosity was 0.52±0.11(Table 2).

Population size

The 50 individuals were identi fied mainly from the 87 analyzed samples,which excluded ‘false recapture’samples.Pelletsfromthirty- fiveindividualswerecollectedonlyonce,while pellets from others were collected 2–7 times.Each individual was captured 1.74 times on average.The TIRMpopulationestimation module yielded anestimateof87deer(97.5%con fidence interval of 84–90),while the ECM module estimated 65 deer(97.5%con fidence interval,65–67).ThelikelihoodratiotestPvaluedidnotsigni ficantly deviatefrom0;thisshowedthatthecaptureratesofdifferent individuals differed across the survey area,so we accepted resultsproducedbytheTRIMmodule.Therefore,withinthe 200 km2reserve,we estimated that there were 87±3 red deer at 97.5%statistical con fidence.The population density was approximately 0.435–0.450 individuals/km2.

Fig.2 Decrease in probability of identity,P(ID),for Tibetan red deer genotypes as more microsatellite loci were added

Table 2 Genetic indices in the study population based on 12 microsatellite loci

Discussion

Non-invasively studying wildlife populations by sampling DNA in fecal samples is a common method in field ecology(Kohn et al.1999;Eggert et al.2003;Solberg et al.2006;Zhan et al.2006).NMR combines ideas regarding traditional mark–recapture methods with the advantages of non-invasive sampling.Our estimate of 84–90 red deer differed from that of the local forestry bureau,which estimated that the number of red deer was approximately 300 in 2008(Liu 2009).Our data were based on samples collected using systematic and repeatable field methods.Thus we conclude that the results presented here more accurately represent the actual red deer population size.

Analyzing conspeci fic but signi ficantly different populations and genetic variation between different individuals within the same population can elucidate the evolutionary potential of species(Tian et al.2010).In this study,the 12 microsatellite loci revealed that average He was 0.72±0.01 in the Tibetan red deer population.This is slightly lower than that reported by Tian et al.(2010)for red deer population diversity in the Wanda Mountains of northeast China(0.738±0.076,Tian et al.2010).High genetic diversity in other deer species was reported by Wu et al.(2008)in a comparative study:white-tailed deer Odocoileus virginianus(He=0.67–0.74)(Deyoung et al.2003),red deer C.elaphus(He=0.71)(Goodman et al.2001),and roe deer Capreolus capreolus(He=0.50–1.0)(Fickel and Reinsch 2000).Comparison of our results for red deer with those of Wu et al.(2008)con firms that the Tibetan red deer population,although small,has high genetic diversity.

Ne was informative for populations that randomly mate,and Na was fixed in offspring genes.The gap between Ne and Na can roughly estimate the risk of allelic loss(Tian et al.2010).Ne can also be re flected in population genetic differentiation.However,Na and Ne can be in fluenced by sample size(Maudetr et al.2002);these values increase as the number of samples increases(Yan and Zhang 2004).Zhang et al.concluded that for a given microsatellite locus to be suitable for assessment of genetic diversity,at least four alleles must be amplified(Zhang et al.2012).There were,at least four ampli fied alleles for each of the 12 microsatellite loci used in this study,and average Ne was 4.58±0.15.Thus,these 12 sites were suitable for use in assessing genetic diversity;they showed high genetic richness and variation,and the sample size used in this study ful filled the requirements for analysis of genetic diversity(Han et al.2013).

PIC is a measure of genetic diversity;the larger the value,the higher the gene abundance(Han et al.2013).Botstein et al.proposed that PIC could measure the degree of genetic variation:PIC values＞0.5 indicate highly polymorphic loci,values 0.25＜PIC＜0.5 re flect moderately polymorphic loci,and PIC values＜0.25 indicate lowpolymorphic loci(Botstein et al.1980).The average PIC value of the 12 microsatellite loci used in this study was approximately 0.67±0.013.Of the 12 sites,only T123 was moderately polymorphic,and the other 11 sites were all highly polymorphic.

Based on the above findings,we conclude that although the Tibetan red deer population was small,genetic diversity was high.High genetic diversity is expected to prove advantageous for population recovery.Therefore,the design and management of the reserve by the local forestry department should focus on protection of the Tibetan red deer and reduction of adverse anthropogenic impacts on red deer and their habitats.Effective protection and favorable conditions for the survival of the Tibetan red deer should be ensured to avoid the loss of genetic diversity thata prerequisiteforpopulation recovery.We also recommend that the local forestry bureau increase capital investment for further research on Tibetan red deer.

Botstein D,White RL,Skolnick M,Davis RW(1980)Construction of a genetic linkage map in man using restriction fragment length polymorphisms.Am J Hum Genet 32(3):314–331

Deyoung RW,Demarais S,Honeycutt RL,Rooney AP,Gonzales RA,Gee KL(2003)Genetic consequences of white-tailed deer(Odocoileus virginianus)restoration in Mississippi.Mol Ecol 12(12):3237–3252

Eggert LS,Eggert JA,Woodruff DS(2003)Estimating population sizes for elusive animals:the forest elephants of Kakum National Park,Ghana.Mol Ecol 12(6):1389–1402

Eva B,Swenson JE,David T,Sven B,Pierre T(2005)Estimating population size of elusive animals with DNA from huntercollected feces:four methods for brown bears.Conserv Biol 19(1):150–161

Fickel J,Reinsch A(2000)Microsatellite markers for the European Roe deer(Capreolus capreolus).Mol Ecol 9(7):994–995

Goodman SJ,Tamate HB,Wilson R,Nagata J,Tatsuzawa S,Swanson GM,Pemberton JM(2001)Bottlenecks,drift and differentiation:the population structure and demographic history of sika deer(Cervus nippon)in the Japanese archipelago.Mol Ecol 10(6):1357–1370

Goossens B,Waits LP,Taberlet P(1998)Plucked hair samples as a source of DNA:reliability of dinucleotide microsatellite genotyping.Mol Ecol 7(9):1237–1241

Han HB,Zhou XR,Pang BP,Zhang MZ,Li HP(2013)Microsatellite analysis of Asia Oedaleus genetic diversity in Inner Mongolia.Acta Entomol Sin 56(01):79–87

Kohn MH,York EC,Kamradt DA,Haught G,Sauvajot RM,Wayne RK(1999)Estimating population size by genotyping faeces.Proc R Soc B Biol Sci 266(1420):657–663

Liu WL(2009)Critically endangered rare animal species—Tibetan red deer.Tibet Sci Technol 6:65–68

Lovari S,Lorenzini R,Masseti M,Pereladova O,Carden RF,Brook SM(2016)Cervus elaphus.The IUCN red list of threatened species 2016:e.T55997072A22155320 http://dx.doi.org/10.2305/IUCN.UK.2016-2.RLTS.T55997072A22155320.en.Downloaded on 07 April 2017

Maudetr C,Miller C,Bassano B,Breitenmoserwürsten C,Gauthier D,Obexerruff G(2002)Microsatellite DNA and recent statistical methods in wildlife conservation management:applications in Alpine ibex[Capra ibex(ibex)].Mol Ecol 11(3):421–436

Miller CR,Joyce P,Walts LP(2005)A new method for estimating the size of small populations from genetic mark–recapture data.Mol Ecol 14(7):1991–2005

Pennell MW,Stansbury CR,Waits LP,Miller CR(2013)Capwire:a R package for estimating population census size from noninvasive genetic sampling.Mol Ecol Resour 13(1):154–157

Solberg KH,Bellemain E,Drageset OM,Taberlet P,Swenson JE(2006)An evaluation of field and non-invasive genetic methods to estimate brown bear(Ursus arctos)population size.Biol Conserv 128(2):158–168

Sun JR,Li Y,Yan S,Zhang QW,Xu HL(2011)Microsatellite marker analysis of genetic diversity of Cacopsylla chinensis(Yang et Li)(Hemiptera:Psyllidae)populations in China.Acta Entomol Sin 54(7):820–827

Tian XM,Zhang MH,Zhang H,Yang CW,Jin ZM(2010)Microsatellite analysis of red deer population genetic diversity in East Forest of Wanda Mountain in Heilongjiang Province.Chin J Ecol 29(3):543–548

Weber JL(1990)Informativeness of human(dC-dA)n.(dG-dT)n polymorphisms.Genomics 7(4):524–530

Whittier CA,Dhar AK,Stem C,Goodall J,Alcivar WA(1999)Comparison of DNA extraction methods for PCR ampli fication of mitochondrial cytochrome c oxidase subunit II(COII)DNA from primate fecal samples.Biotechnol Tech 13(11):771–779

Wu H,Hu J,Wan Q,Fang S,Liu W,Zhang S(2008)Microsatellite polymorphism and genetic structure of populations of sika deer.Acta Theriol Sin 28(2):109–116

Yan LN,Zhang DX(2004)Effects of sample size on various genetic diversity measures in population genetic study with microsatellite DNA markers.Curr Zool 50(2):279–290

Yin J,Zhang MH,Xie XC(2007)STR application in wild Siberian red deer individual identi fication.Chin J Wildl 28(3):42–44

Zhan XJ(2006)Using Noninvasive Genetic Sampling to Estimate the Population Size and Study Dispersal of Giant Pandas.Beijing,Institute of Zoology,Chinese Academy of Sciences

Zhan X,Li M,Zhang Z,Goossens B,Chen Y,Wang H,Bruford MW,Wei F(2006)Molecular censusing doubles giant panda population estimate in a key nature reserve.Curr Biol 16(12):451–452

Zhang BW,Wei FW,Li M,Lv XP(2004)Simple method of DNA extraction from giant Pandas and red pandas feces.Curr Zool 50(3):452–458

Zhang H,Zhang MH,Luo LY(2010)Screening of microsatellite loci of red deer in the eastern forest of Wanda Mountain in Heilongjiang Province.Chin J Wildl 31(2):59–62

Zhang NN,Wang YQ,Wang QY,Wang ZB,Zhang XH,Bai JY,Mao W(2012)Analysis on the genetic diversity of 9 microsatellite loci in Taihang native goat.Chin J Anim Sci 38(3):18–21