And Ainpiificatior7 of Genes from Fecal Material Were Aimed at Devel

Home , Feces

n P984, Putman~ remarked in and ainpiificatior7 of genes from his review FM:& ikxn Faeres fecal material were aimed at devel- that ‘for any frustrated field opirng slew tools for clinicai diagno- mammalogist seeking observa- sisz7. Similar techniques allowed tional da’s on their elusive study parasitofogicai studies of domestic animals, dung may represent the and wild populations; for example, most readily-avaiiabie and easily- tbe amplification of DNA from tape- collected source of information worm (.Ecl~inococcm muMocul0ris) upon which they may fall back in in feces of red foxes (Vdpes udprs) despair’. Despair is frequent these showed this parasite had a high days because many mammals puevalence~x. are becoming increasingly elusive Feces contain cells shd from as their numbers decline. Rarity the intestinal lining; thus DNA fForn makes direct observation difficult the bust itself could potentially but the need for genetic, derno- be is&ted ad aanalyzea.l.The ap- graphic and life-history infor- p8iciltiOlI of this idea to an anilnal mation for species Sacimgextinc4ioul ~bopulatiore involved a saaalil popu- is more pressing than ever. Ad- lation of elusive, higbiy eoadan- ditionally, the great expense arPd gc~ctl brown bcnrs (Unua c~n’los)i~i extensive number of permits re- the Brenta mountains of northern quired to capture and transport 11alyx!‘-.“. The population size and samples makes studies of endan- Michael Kohn and Robert Wayne are at the sex composition were unknown, but gered species unpalatable to many Oept ot Glogy, University of California, anecdotal observations suggested 621 Circle Drive South. Los Angeles, biologists2-6. that only a few bears remained. A CR 90095-1606, tJSA. Even in abundant species, large- single mitochondrial DNA controi- scale survevs cf DorPufations are region sequence was found in bear logistically &ffic& ii they depend feces collected from the Brerlta on opportunistic sampting (e.g. Ref. 6). Moreover, “any study mountainassuggesting that ;he popu%ation was small and of a natural population has the unavoidable side effect of isolated. This sequence was present also at high frequencv altering the dynamics of the population in ways that may be in bear populations from Slovenia, Croatia and Bosnia, indk- difficult to predict” or that may change some characteris- eating that the Rrenta bears formerly were in contact with tics that a researcher is attempting to measure’.x. these populations. A 8(-cbromosomal nuclear marker SRY Must we lose hope if observation and biological sampling was used to show that at least one male and one female 1~11 are not possible? Perhaps blot: in this article we discuss a&- WWe [H?Wtl~ in r!::! alYil. TBWSe re’.4ts also implit ihal cations of recently introduced genetic techniques for the 0thcr WlCl?Z ~llilt%tTS SUCh AS InicroSatellil~s ElKQ tH> ClSOd analysis of feces (molecuPar scatology) when studying Iree-

----I._~_ - ranging mammal populations. These techniques allow for r the purification of DNA from feces, which is then followed by amplification of specific DNA sequences using the polymer- Mitochondrioi DNA’“(mtDNA) is a matemalty inhented. haplord.nun-recombmmg ase chain reaction (PCR). An array of genetic markers with and extra-nuclear genome. Each cell has multiple mtDNA copses crangc IC-250G different properties is available9-‘a and call be used to ad- copies) and thus mtDNA sequences are more eastly amplified than smgle copy. dress taxonomic questions, individual f(ler~tificat~on through nuclear genes. Because the mtDNA cytoclx~ne 0 (~~40) gene has only moderate mtraspeciflc variation. it is well suited for use In species identlficatlon. The mtDNA ‘genetic fingerprinting’, and sex determination with gender control Pegion IS commonly vanable on the rntraspecific bevel and is sultable for specific markers (Box I ), Moreover, specific sequences from studies oi genetic variability. phylogeography. ass@?ment to management units. other passengers through the gut such as food and patho- and forens,?,. Chloroplast DNA” (cpDNA) and mtDNA ran be used to amplify plant gens can be determined by using CR primers specific fcx and animal sequences from feces to study diet. Moreover, a vast number of their amplification. Consequently, molecular techniques ap- pathogen sequences (“(ruses. bactena. protists and macroparasItes) are avallable for ampltflcation from fecal material. plied to feces allow the identification of animal species, indi- Mlcrosatellites~.‘~ [also referred to as simple sequence length polymorphism viduals, their sex, pathogens and food habits. Furthermore, (SSLP). or slmpfe sequence repeats Jare nuclear. single-copy DNA conslstlng of tan- comprehensive sampling of an area for feces, or systematic demly repeated short sequence mohfs such as (CA),. They are scattered through fecal transects of populations. may allow estimates of home out :he genomo and are highly polymorphic. 3~ assaying several m!crosatellrte 100. 3 multllocus genotyDc:can he obtaIned: a .genetlc fingerprint’, which IS untque to Indk range, reproductive patterns, kinship structure and popu v~dual animals These markers are cudon~~na~tl~II?W:IIC.~ :alleks !rom both par l&ion size. Thk information may be augmented by tech- ents are traceable in the offsprl:d) and are useful for studying paternity and [tinship. niques for analysis of feces that do not use DNA1~‘5-25(Rox2). genetic varlatton. population genetrc structure and gene flow9.11. M~crosatellites Analysis of feces will potentially provide a means to study Identified in one species can usually be employed IO study a related species! ‘. PCR pnmers for the SHVgene on the Y chromosome can be used t0 dlstin genetics, life history and population dynamics of mammals. gu,ulsll male from female DNA”. Genetic maiicer sequences and WI? pnmers are published or accesslb:e throu$ GenBanlc or EMBL, which are electronic databases avallablr: on tile lW?rnet tha! PCR can produce large quantities of specific DNA se- can be searched for sequences by entering the taxon. genomlc lOCUS or SequenCC quences from small, degraded and impure samples”). Stimu- of Interest. lated by this property of RX, early attempts of isolation fecal studies showed that the source for reintroductlcn should be bears taken from the Slovenian p ation. Mitocbondrial DNA has been amplified from feces Mammal specres can often be tdentified based on their feces, allowtng stuu’ies of spec~cs iompos~t~on~. However, without addrtional information feces ara assigned of the Indian elephant (Elephas maximw), European bison correctiy :n only about 59-6606 cf the case2 ?j5, This uncertatnty IS owing to con- (Bison Bonasrrs), polar bear (Ursus nlak+itimus)3ci~3’and dugong sideraM? size-overlap of feces rn related tdxa In some cases, the pH value and (Dugong dugo.cJng)“z.Four studies Rave now confirmed that b~lc acids of feces have be‘sn use;~! to rdentl& specie\!‘. Relative abundance and individuals could be ~f~~ge~~~i~ted’ from their feces; these populatton SIZE have been estimated from fecal counts1 lE Hocever. the resdmultng studies successfully amplified microsatellites from feces of of Illdivrduals constrtutes a problem. Census data baser1 on teces can be supple menteci by techniques described elsewhere1,16,17. wild baboons (.Pnpio cynoce@~lus)~~~, bonobos (PM panis- Habitat use and range size may be investigated by examining spatial variation cus)34,brown bears (Ursus arclos)3” and pinnipeds?s. Although in feces abundance. Simrlarly, territory boundaries r- ‘y be marked by fecal aggre- some technical problems were encountered (Box 3), these gations:. These studies often require individuals to /I gest colored plastic markers studies F:esent convincing evidence that such problems or radioactive halt, which uniquely mark their feces. Analvsis of food habits and dietary quality have frequently used fece9.l;. In can be overcomexr-36 (see also Refs Z&37,38), and feces may carnIvore& undigested ploy Items can be recovered and Identified. Herbivore dung some day be as useful as blood or tissue samples for genetic often contains plant remams. In general, this approach lhas shortcomingsl,‘r,‘i,ls. analysis. In part, our intent in this article is to provide incen- tive for additional t&mica0 research. Previously, a variety of other potential sources for DIVA such as hair, bones, feathers, saliva, skin and nails have been collected for Pson-invasive genetic analy&P’. However, these SOtiCC(5 may be more difficult to obtain than fecal samples ant4 may provide less intormation. The g.enetic an fife history iaIformatiom that potentially ran be extracted from fecal samples is sunmiarizcd in Fig. 1,

on feces to identify individuai beam?. Finally, a glimpse of Two essential parameters that need to be assessed in the feeding habits of Brenta bears during late summer be~~~~v~o~a~studies are the number of offspring produced by (when feces were collected) was provided by e relationship of indivi plant chloroplast sequences of the &CL gene group to each other. This information allows behaviors feces. Fecal plant sequences were identified as belonging to lead to reproductive success to be identified and prov the hawthorn genusHzotini;13, thought to be important in the an assessment of the kinship, component of cooperative be- diet of brown bears. Although animals were never seen dur- haviors (e.g. Ref. 11). M ing the study, feces provided answers to establish paternity and usually require direct observation and the handling of ani- popu&ionsX,“.l l.:i’).~llland mals. The results also had conservation implications: the However, depending on polymorphism and levels of hetero- presence of a male and female bear left oprla the possibility Lygosily of anicr~sal4ite loci, about 20 microsatelli+e loci that thC small popUfatiOD might iDCrCiW?.Furthrmos-e, thf> may be needed to determine paternity and kinship and to dis- tingmisb relatives from non-relatives. ‘This estimate is based

on a study in wild-caught, outbred mice (MM n~usc~lus) where 20 uamlinked loci were sufficient to discriminate be- tweeal unrelated and full-sib dyads with about 97% accuracy, Fe:e:es contain cells shed (ram the gut and a complex mrxture of other con,- and to discriminate l~alf~si~~pairs from ularelated or full-sib pounds (microorganisms, undigested food. digestive enzymes, mucus, bile salts pairs with better than 80x accuracy’!’ (see also Ref. 40). and bilrrubr,V7. In addition. lhcrbrvore feces Contam plant polysaccharrdes”6. Imtral studies encountered it~hlbltiOnof the Taq polynierase durrng PCR by substances 111 Importantly, the number of loci FQ be scored increases the e:JracP” \lJJ, Results were improved when extraction methods relying on DNA as populdion ~ete~~~y~Qsi~y deer adsorbing beads were employed- ‘?,3L,h. Furthermore. addmon of bovine serum albu. ne kin& WI (BS& ~ell~l~~e or othe;substarices to DNA extractIon and PCR circumvented ver, in s III ibitron*‘J”Jfi. Hexadecyltnmethyl-ammomum bromide ;CIAB) rs commonfv empl,:ed to remove plant polysacchandes from DNA extracts. analyzed, which may compensate Incanslstency for the effort of scoring more loci. Because of the difficulty in Preliminary data show that DNA m feces IS present m low coav nunbers and lifying many single-copy loci IS degradedJO )1?.Amplification of short DNA segments resulted 111more consrstent of large social groups may not amplifrcatrofls than longcr fragmcn:s”“. Of specral concern m the amulificatlon of mrcrosatellites is the mrstypmgof mdrvrduals owing to PCR arttfacts and/or stoch. economically feasible ;f present through analysis of feces astic events when pipetting drlute and degraded DNA teniplatei,“.J” I’. Conse- alone. Even so, limited >audies of small social groups, es- quently, multrple extractions and PCR amplrfications from fecal samples should be pecially when augments :! by blood or hair samples, are fea- done’03 15 7’ sible. Maueovw. wbn c 9ducting paternity exclusion analy- ~~~t~o~~y It IS Important to select and test carefully PCR primers and to optrmrzc PCR sis, sometimes very Iev !oci suffic: to exclude most of the conditions!” )*. Specrfrclty IS mcrrased wlwn spmcs-s~~e~hc primers (desrgncd males of a troop of prim.*tes. especially *.rhen this troop has from publIshed sequences1 are c~sed~~. Nonetholcss, PCR products should be se- IXYU cxtollsiveay obsert ::tP.l”. quenced to ver& authentrcity of results‘ r” 3L. When amplifying m!crosatellites from feces. norbsbxliic products Oh+ii &curt results. Southern blotting36 or hot-start PCR condrtronsZs are useful to circumvent this problem. Can~aamlnation A common method to estimate population size isavoives During DNA extraction and PCR setup, contammation IS a potentcal source of extrapolation from anim;l counts on designated transects’“. er10r-6.Thus, wrth degraded or drlute DNA, a separate facihty dedrcated to DNA However, tkis terhniqut ;ener& assumes a uniform dcn- extractron and PCR setup should be used as well as ihiclusion of blank extractrons sity Of animals within their habitat and that individuals are and FCR blanks in all steps cf the experiment26 m. not double counted. In many species of large mammals, feces are deposited in concentrations along established routes

TREE uol. 12. no. 6 June 6.997 _.1. AGTTGATAATGG 2 : ACTTGATTAT!-& 3:ACCTGAACACAG

Fig. 1. A fecal sample collected in the field can be analyzed in many different wavs. Recently-mtroduced molecular techmoues i,mol~!cular scatoiogy) allow the lsolatlon and analysis of DNA from feces and thereby provide genetlc a?d life histoly Information on mdwiduals. (a) MtDNA sequencing leg cyrochrome b) allows the anlmal specw (In this case, Ursus arctos) to be identified; (b) mtDNA analysis reveals the phylogenetic relationshlp of the mdlvidual (underllned; to othei species ol’io~~@~f~cs: (c)the sex can be determined using primers specific for the mammdlian Y-chromosome; (d) pathogen sequences (viruses, bactena. nrohsts. macrooarasltcs) can be identified through amplification by stratn specific primers: (e) plant and animal food items revealed USIL~ species-specific plant chloro~last pnmers (e.g. rbcL) or anlmal mtDNA primers (e.g. from cytochrome b). Individuals can be fingerprinted through mlcrosatelllte typing, allrl population size, genetic vanatlon. lunshlp and patwnlly can be deduced through extensive microsatellite ger,otyp+ngof feces collected throughout an area. for warnpIe. scr :‘I ::KJ !.;’ :‘I ‘“Y ,.,.; (3(,~cl+.lnr.l- 41ul,l. I n. ,--_e“ll .,,‘.LII.““,*c t. finperprmt. In (g). male 1 (Ml) shares an allele with Imale 2 (MZ). but Ml can be excluded as father of the juvenile (I).The, frm;ilC ~p~wt11:S ~nd~;alK by f

such as roads or territorial boundaries8.‘C’. Consequently, a may mark the core area used by a61individual. Quantitative large proportion of the pcapulation may leave feces at spc- methods similar to those used to deduce core artaas and cific points that can be sampled repeatedly. The point w home range from radiote ry observations could be ap- al collection over a large area not reveal plied to these kinds of da m fecal studies (e.g. Pef. 41). tilocus microsateQlite genotyp y indicate that viduals have been sampled an census size is Effective ~~~~lat~~n size number of unique multilocus genotypes. ‘bee variables are critical to accurate assessments of Even in species that deposit feces in a more uniform or ran- effective population size: (1) the fraction of individuals that dom distribution across the landscape, population size may reproduce, (2) the sex ratio of those individuals, and (3) the beestimated using a transect method. Feces collected along variance in family sizelL,l:‘. in small populations, toa which transects would be typed for micr0sate~~~te loci and the re- feces can be obtained from all i ividuals, such variables can lationship of feces collected to new multilocus genotypes be estimated (Fig. 2). The num r of individuals can be tabu- should define a curve whose asymptote represents tbe cen- lated from the number of unique rn~~tiloc~s mic~osat~~f~te sus population size. Especialily for cryptic species, system- genotypes (see above), the sex ratio determined by use ol atic recovery of feces may allow more accurate estimates ol sex specific probes (Box I) and some information about population size than observatiora alone’. Moreover, NW family size deduced from paternity analysis (see Refs 8,39. analysis of feces may be ‘.he only way to prove the prescr~ce 40). In endangered specks. ofttw just any rvidencc of rcl~rn- of a cryptic, rare or pmta;ively extinct species in am area? duction is very ~m~~~ta~~ iniormatiola that microsatellite- based fecal analysis can provide. Hsrwe range and territory si7e BntewEtwia& species, the approximate dimensions of tea-- ritories and core-use area can potentially be uncovered by The extent of genetic variation dcperads on ofkctive popu- documenting the distribution and den Pation size and in dec.ining populations is an important vari- rosatellite genotypes found in feces. able t0 monitor because a goal ol many C0nserWtioll pW ighest density of feces from a given m grams is to maintain a certain fraction of genetic variation analysis of feces may be the only viable means to infer diet.

Relief from sampling despair has an uoexpeotc? source in feces. The combi- nation of data from conven- tional analysis of feces with

number of animals

Fjg~ 2. The actual number of mdlvlduals I” a population IS often much greater than the genetically effective pGpulatlOn Size. hensive picture of the hidden N,, AnaIrS&of fecal DNAI” conjunctto? with fecal steroid hormone analysis may allow the eStimatlOn Of tile number Of life oh elusive and rare mam- reproducing individuals, with IS related to N, Ths is Illustraied us~n:: d hypolhetlcal populdtlOn Of 10 bears: (a) indiv!duals .*,..I..lllOlJ. CorXenti :s,2l analysis are counted by cof~ect~on of feces followed by aniilysis t~slng mlcrosatelllte IOCI: (bl the Inumber of adults, and their :espect~\e sex (for\:ales. block: make, gn?y) 1s dntermlned by hormone analyst and w\th Y-chromosome markers: (c) ba;ed on tnelr can provide a portrait of re- estr&ol and proycslciiorle lkvels some of the adult dn~m;ils ale not reproclucbve; (d) patcrnlty analysis using mlcrosatellite productive cycles, pathogens lot find that ugly .! kc, b~:it~ .~rfu:~lli t!,/lrc).ill:.18 .inil !i31 IIIC ~lopllatlor~ one generatlon later. and diet. which can be aug- __--.._.__.._._ .-.- -I mented with data from DNA- based techniques. Ad ally, analysis of DNA sequences from feces may provide information on paternity, kinship. sex ratios. census and effective populalion size, gene flow and phylogeography. In conservation management, the Iack of such genetic, demogaphic ant! Pifehistory data has been a notorious pro invasive population assessment potentially The challenge of the future is to develop better methods for dealing with the problems of inhi ition, inconsistency, speci- ficity and co~~a~~~a~io~ such that molecular scatology can be routinely applied to large population samples. 14 O’Brien. S.J. et ul (1993) Anchore reference loci br ~~~~a~t~ve geeramnemapping in wammak, Eur. &net 3,103-l li 15 Halfpenny, J. and Biesot. E. (19%)A field Guide fo Mcmm~u/ Tracking in North America, Johnson Books 16 Sutherland. %‘..I.. ed. (1996) /%@rcul Cenws Teckhnrgrres. (I Handbook. Cambridge University Press

17 ;?lills, M.G 1. (1996) Methological advaaacc5 in capture. cenSw3, and

food-habits Sltdles of Iqge AWcm camivores. in Cflrnr~~orr~ Bekacror, Ecolo~. and El;okrtron (Vol. 2) (Gittleman. J.L.. cd.), pp. 223-242. Cornell University Press 18 Carss, D.N. and Parkinson, S.G. (1996) l%kws aswciated witb otter (Lurra futraj fecal analysis. 1. Assessing general diet from spraints. .I Zoo/ 238.301-317 19 Hobbs, N.T. (1987) Fecal indices to dietau quality: a criPique, J. W/d!. Mmage. 51.317-320 fQ Leslie, D.M. and Starkey, E.E. (1987) Fecal indices to dietary quality: a reply, J Wild!. Munuge, 51.321-325 21 Wasser, S.l<., Monfort. S.L. and Wildt. D.E. (1991) Rapid extraction aB faecal steroids for measuriiregreproductive cyclicity sod earUy pregnancy io f~ee~~ao~~~~ yellow baboons (Pqh cynocq9haQes cynocephofus), .I. Reprod. .C,r/. YZ. 4 15-423 22 Varley, J. (1991) ~o~~to~~~~ ovarian function in captive and free.rangiog wildlife by means iof urinary and fecal steroids, .I Zoo Wnll Med. 22, 23-31 23 Phillips. M.K. and Scbeck. .I. (1991) Parasitbim in cnptivc and reintroduced red wolves, J H’ikrl. flrsetrs~~ 27. 49X-501 24 Hirayama. K;. *!t ul. (1989) The kcal Wora of the gianUpanda. .I .\pp/ Burterrol 67.411-415 25 Mason. CF. and Ratford. J.R. (1994) PC5 congeners in tissues of European otter (h&o lulro), Bull. Erwrron. Conturn. To.uicol. 53. 548-554 26 Arnheim. N.. White, T. and Rainey. W.E. (1990) ~~~~~ca~o~ of PCB: orgaaisnnzd and population bioQy, Bio.Screwr 411, 174-182 27 Sidransky. 1). ef al. (19921 ~~e~t~~ca~o~ of ros mutations in ttae stool of patients with curable colorectable tumors, Suc~ncc~56, 1W-105 28 Bretagne. S. el ul. (1993) Detection of Ecckinocu~ccns rnu~tjco~~rj§ DNA irn ~QXfeces, fur~sih~~o~ 106. 193-199 29 Hess. M. <‘I rd. (1992) Excrement analysis by PCR, .Xulum 359. 199

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