Estación de Biología "Los Tuxtlas" Instituto de Biología-UNAM



Genetic studies through fecal DNA



Population geneticists are relying increasingly on the study of DNA variation within and among populations (5). In the past, traditional genetics were dependent on visible differences between species. Usually only domesticated animals or cultivated plants could be used because controlled crosses had to be done to be able to do analyses by using Mendel´s principles of inheritance (8). With the coming of molecular techniques, genetic differences can now be obtained by direct analyses of DNA and proteins. The use of DNA in population genetics gives an enormous amount of possibilities to answer research questions. Genetic variation within and between populations, divergence between species and questions on phylogeny can be answered. Besides this, DNA fingerprinting can be done by which individual animals can be traced to answer questions on home range, territory and patterns of migration. Pedigree studies can be performed and histories of populations can be examined (8).

Non-invasive monitoring

New techniques have been developed to add to the possibilities to study primates. One of them is the use of non-invasive monitoring. With this technique DNA of mammals can be collected in the field without disturbing them. This can be done by collecting faeces of which DNA can be extracted. DNA is present in faeces because the intestinal lining is renewed every week, which causes body-cells to be excreted with the faeces. The extracted DNA can be analyzed using microsatellite-analysis. This technique enables researchers to individually type samples, although observations still have to be done to determine the origin of the sample. Besides the information DNA provides, other elements in faeces like hormones or parasites can be examined. The diagram below shows an overview of the possibilities.

Although the technique can be very useful, there are some disadvantages, especially when collecting fecal material. The most important is the small amount of DNA present in the feces. The DNA can be highly degraded due to passing through the intestinal track.The above indicates that with DNA, genetic studies can be done on any organism thinkable. However, when doing research on animal populations, blood or tissue samples for DNA can be hard to obtain. Capturing often causes a considerable amount of stress. Nocturnal animals, marine mammals or animals living in dense forest are particularly difficult to sample because they are hard to trace (4). Noninvasive sampling provides solutions for these problems. With this sampling method, DNA material can be obtained without capturing the animals. DNA is present in the roots of hairs, in feathers and in feces. Fecal material contains DNA because the epithelial cells of the intestinal lining are renewed regularly. The old epithelial cells are excreted with the feces. By collecting these materials, DNA can be extracted and analyzed. For herbivorous animals, the secondary compounds present in plants can have an inhibitory effect on the quality of the DNA. The table below gives an overview of the advantages and disadvantages of using noninvasive techniques

Table 1: an overview of the advantages and disadvantages of noninvasive sampling





Provides very useful genetic information

Hairs are difficult to get (2)


Capture not (always) necessary (6)



Genetic studies difficult due to arboreal life many animals (2)



Provides very useful genetic information

Limited amount of DNA (3)


Capture not (always) necessary (6)

Lack of microsatellite primers (2)


Genetic studies difficult due to arboreal life many animals (2)

Degradation of DNA (3)



Inhibiting secondary compounds

The use of non-invasive techniques to extract DNA has allowed primatologists to recently carry out studies of genetic variation, gene flow, and paternity, among other, in populations of primates in the wild. The collecting of fecal DNA from epithelial cells and the characterization of genetic markers (microsatellites) allow the identification of individuals, of their sex and of their contribution to the genetic pool in the population. Thus, these techniques have resulted in evidence about individual reproductive choices, about kinship structure and about population genetics (genetic variation and gene flow) in groups and populations of primates.


In undisturbed areas, migration can occur between populations of a specific species. Individuals can migrate to another population and reproduce. In this way, exchange of genes is possible. This maintenance of genetic variation is important and beneficial to a population. When environmental circumstances change, a population should answer to this by adjusting itself. Natural selection of the fittest individuals will occur. When genetic variation is high enough, the right combination of genes will be present in the population. Individuals with these genes will reproduce offspring, and the population will remain. The diagram to the left illustrates the "normal" process of gene flow in a population of primates living in extensive forest tracts. Individuals, males and females, leave their natal troop and join other troops where they reproduce. When the forest is fragmented, primate populations are also fragmented and the spatial separation among forest patches may disrupt the processess of emigration and immigration among remaining troops. Reduction of genetic variation, in animal generations, as a result of lack of "fresh" genes and inbreeding is the outcome.


Human destruction of forests causes fragmentation. Because of this, populations of animals or plants from the same species can become isolated from each other, thus reducing the possibilities for migration and genetic transfer. Organisms can become separated from resources or required habitats by fragmentation, thus reducing individual fitness. More important, habitat fragmentation is likely to increase extinction proneness due to inbreeding because small, isolated populations will become inbred overtime (Frankham, 1998). Because of inbreeding, populations will be less capable of adjusting to new situations. Inbreeding also increases the occurrence of diseases: dangerous genes are rare in populations with high genetic variation, but can take over when variation is low. Fragmented areas are also endangered by edge-effects. Because of the more open area, predation and habitat deterioration at the edges may be more intense.

The focus of our genetic research with with populations of wild primates in southern Mexico is to investigate, by using non-invasive techniques, the genetic variation and gene flow in populations of howler and spider monkeys existing in fragmented habitats and in extensive forest tracts. Forest fragmentation by human activity has resulted not only in significant reductions in the size of primate populations, but also in alterations in their demographic structure. As a corollary to these changes, physical and temporal isolation has diminished the flow, through processes of emigration and immigration, of individuals among remaining social units, a situation that may also contribute to a higher incidence of inbreeding. In the case of populations existing in extensive and protected forest tracts, the primate’s sociobiological events that lead to the emigration and immigration of males and females among troops in the population sustain gene flow and thus adequate genetic variation.

Our specific interest is to assess, through DNA extraction from primate fecal material, genetic variation and gene flow in primate populations existing in extensive forest tracts and in fragmented landscapes in particular regions of southern Mexico. Thus, our research is being carried out in Los Tuxtlas (Veracruz) and in the state of Tabasco with populations of mantled howling monkeys (Alouatta palliata) and in the states of Chiapas and Campeche with populations of the black howler monkey (Alouatta pigra). In these latter regions, spider monkeys (Ateles geoffroyi) are also under investigation. In the case of extensive and protected forest tracts we are focusing on the study of populations of the above primate species in the biosphere reserves of Los Tuxtlas (Veracruz), Calakmul (Campeche) and Montes Azules (Chiapas), as well as in the protected forest of Palenque National Park in Chiapas. The conceptual base of this program involves an esperimental design comparing primate populations in landscapes with different degrees of fragmentation and these with populations living in extensive forest tracts.

Evidence from well-designed studies supports the utility of corridors as a conservation tool (Beier & Noss, 1998). By connecting the isolated populations, migration can occur between them. Genetic transfer is again possible which increases fitness of the populations. Edge effects may be reduced by allowing vegetation to grow along the forest edges to increase cover (Estrada et al. 2002).

DNA extraction is done from the epithelial cells lining the intestine, cells which are carried by the fecal material as it is expelled from the digestive tract. About 5 g of fecal material are collected from identified individuals. Samples are stored in vials with about 35 ml of silica gel and are later processed in the laboratory for extraction of DNA.

Field techniques for collecting feces

At the Biological station at Los Tuxtlas and in other sites in southern Mexico we are collecting fecal material from wild howler and spider monkeys for DNA extraction. There are several aspects that should be taken into account when collecting fecal samples in the field. Table 2 shows these aspects and lists the advantages and disadvantages for each of the aspects listed.

Table 2: an overview of the advantages and disadvantages of field techniques for collecting fecal material




Use fresh samples

Higher success rate due to less degradation

More difficult to find

Process in field or in lab?

Higher success rate. Less degradation when processed in field

More difficult to bring gear to the field

Use outer part of feces (1)

More epithelial cells (1)

Not possible when feces is scattered

Work sterile (gloves, clean with alcohol)

Less contamination


Process as soon as possible (1)

Less degradation


Storage techniques for feces

Storing the samples for further analyses should be done very carefully. Degradation of DNA can occur easily. Degrading compounds in the feces have to be eliminated in order to conserve the DNA. There is a variety of techniques used to store fecal material. Below several techniques are listed (table 3).

Table 3: an overview of the advantages and disadvantages of storage techniques for fecal material




95% Ethanol

Most commonly used (5), cheap, good results (5)


Silica gel

High success rate



Cheaper than silica gel




Labor intensive

Buffers from special kits (eg. Qiagen)

Especially developed for fecal material, high success rate


20% dimethylsulfoxide, saturated with NaCl (5)

Storage at ambient temperature (5)



To be able to do analyses, the samples have to be processed. First, the DNA present in the fecal samples has to be isolated from the fecal material. Standard techniques are available for this (5). Because of the small amounts of DNA in the feces, amplification of the DNA is necessary to obtain larger amounts of DNA. With the Polymerase Chain Reaction (PCR) DNA can be amplified. Often amplification of microsatellites is done. These are small pieces of DNA. They are highly variable, so they can be used for individual typing. More explanation on microsatellites can be found below.

Microsatellite analysis. Microsatellite loci (also called Simple Sequence Repeats (SSRs)) are stretches of nuclear DNA, which are composed of tandemly repeated units of 2 to 6 base pairs (such as (CA)n or (CCTTAA)n). They are found in a wide variety of eukaryotes and also in the chloroplastic genome of plants (Jarne & Lagoda, 1996). Microsatellite loci are non-coding DNA-sequences. Their exact function is unknown. They are not subjected to selection and are highly polymorhpic. Pedigree analyses have shown that they are codominant and inherited in a Mendelian fashion. An individual inherits alleles from both parents. All together, microsatellites can be used to answer questions about genetic diversity within and between populations, individual identification, distribution and numbers as well as questions on population structure kinship structure, population dynamics and taxonomy (Parker et al. 1998). In order to be able to do microsatellite analyses, species specific primers are necessary. These primers attach to the DNA surrounding the microsatellites and form new strands of microsatellite DNA. Thus, enough DNA can be synthesized to be able to visualize and analyze it.

Once amplification is done, the parts of DNA with different molecular weights have to be separated from each other. This is done by electrophoresis. The DNA is inserted in a gel through which a current is passed. The DNA fragments with a low molecular weight will move faster through the gel than the DNA fragments with a high molecular weight. By adding a size standard, the molecular weights of the samples can be determined and analyses can be done.

Protocol for collecting fecal samples in the field used by the Primate Laboratory of the field station Los Tuxtlas of UNAM 

At Los Tuxtlas we developed, after many hours of field trials, a method for collecting and storing fecal samples for DNA. Below is a description of material and method used is given. We suggest that people rehearse collecting material before launching a field program.

Material: Sterile vials ( +/- 100 ml), Dehumidifier powder/cristals, Filter paper, Examination gloves, Sterile plastic spoons, Colored flags, Permanent marker, Alcohol

Preparation: Before going to the field, preparation of the vials has to be done. We use 100 ml sterile vials. Other vials can be used as well, but if they are not sterile they have to be cleaned with alcohol. To each vial, approximately one centimeter of a dehumidifier powder (e.g. silica gel or euqivalent) is added. The dehumidifier dries the sample very quickly, and stops degradation of the sample. The filter paper is cut in pieces of 10x10 cm, folded and placed in the vial. All this work is done with gloves to reduce contamination with human cells.

Fieldwork: Once we encounter a troop of howler monkeys in the field, we determine sex and age of the animals present in the troop. Since howlers tend to defecate at the same time, attention has to be paid at the locations of the individual monkeys. Once a monkey defecates, we locate the fecal material and place colored flags beside the feces. This way, we are able to distinguish between fecal samples of different individuals, even when two individuals defecated at the same spot. For each individual we use four vials to collect fecal material in (for backup and use in other studies). In order to work as sterile as possible, we use gloves to collect the samples. With a sterile plastic spoon a small piece of fecal material (appr. 1x0,5x0,5 cm) is placed on the filter paper. The paper is then folded two times, and the ends are folded into each other to make sure the dehumidifier stays separated from the sample and no contamination can occur. Date, site and individual are written on the lid of the vials and the four vials are taped together and placed in a bag.

Once back from the field, the data of the samples we collect are added in a database. Each vial is given a unique number and they stored at room temperature for further analyses.

Literature cited in tables above

  1. Marchant, L.F. et al, Highly successful non-invasive collection of DNA from wild Chimpanzees. Miami University, Dep. of Anthr and Zoology, Oxford, Ohio, USA
  2. Escobar, P. (2000) Microsatellite primers for the wild brown capuchin monkey Cebus apella. Molecular Ecology 9, 107
  3. Zhang, X. et al. Extraction of DNA and PCR analysis of DNA from free-ranging howling monkey (Alouatta  palliata) feces. University of Pittsburgh. Abstract
  4. Mowat, G. et al. Using genetic tagging to estimate animal population parameters (1999)
  5. Warner J.P.(1998) In Molecular Genetic Analysis of Populations, a practical approach (ed. Hoelzel A.R.), pp 33. IRL Press, Oxford

Other related literature DNA fecal samples

Constable J.J, Packer C., Collins D.A., Pusey A.E. (1995) Nuclear DNA from primate dung. Nature 373-393

Wasser S., Houston C., Koehler G., Cadd G., Fain S. (1997) Techniques for application of fecal DNA methods to field studies of ursids. Molecular Ecology 6: 1091-1097

Kohn M.H. and Wayne R.K. (1997) Facts from feces revisited. Tree 12: 223-227

Taberlet P., Waits L.P. and Lutkart G. (1999) Noninvasive genetic sampling: look before you leap. Tree 14: 323-327

Taberlet P., Griffin S., Goossens B., Questiau S., Manceau V., Escaravage N., Waits L.P., Bouvet J. (1996) Reliable geotyping of samples with very low DNA quantities using PCR. Nucl. Acids Res. 24: 3189-3194

Frantzen M.A., Silk J.B., Ferguson J.W., Wayne R.K., Kohn M.H. (1998) Empirical evaluation of preservation methods for fecal DNA. Molecular Ecology 7 (10): 1423-8. Abstract

Some other relevant references:

Beier P., Noss R.F. 1998. Do habitat corridors provide connectivity? Conservation Biology 12:6, 1241-1252

Bradley, BJ, Chambers, KE & Vigilant, L. 2001. Accurate DNA-based sex identification of apes using non-invasive samples. Conservation Genetics 2: 179.181.

Brandley, BJ, Boesch, C & Vigilant, L. 2001. Identification and redesign of human microsatellite markers for genotyping wild chimpanzee (Pan troglodytes verus) and gorilla (Gorilla gorilla gorilla) DNA from feces. Conservation Genetics 1: 289-292.

Estrada A.E., Rivera A., Coates-Estrada R. 2002. Predation of artificial nests in a fragmented landscape in the tropical region of Las Tuxtlas, Mexico. Biological Conservation 106, 199-209.

Frankham R. 1998. Inbreeding and extinction: island populations. Conservation Biology 12:3, 665-675

Morin PA, Chambers KE, Boesch, C, Vigilant, L. 2001. Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus). Molecular Ecology 10: 1835-1844.

Bradley, BJ & Viginlant, L. 2002. False alleles derived from microbial DNA pose a potential source of error in microsatellite genotyping of DNA from faeces. Molecular Ecology News 2: 602-605.

Vigilant, L. Hofreiter, M, Siedel, H, Boesch, C. 2001. Paternity and relatedness in wild chimpazee communities. PNAS 96: 12890-12895.

Recent references for Alouatta:

James RA; Leberg PL; Quattro JM; Vrijenhoek RC. Genetic diversity in black howler monkeys (Alouatta pigra) from Belize. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY. 1997. 102(3), Pgs: 329-336

James RA; Leberg PL; Quattro JM; Vrijenhoek RC.Genetic diversity in black howler monkeys (Alouatta pigra) from Belize. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY. 1997. 102(3), Pgs: 329-336

Ellsworth JA; Hoelzer GA. Characterization of microsatellite loci in a New World primate, the mantled howler monkey (Alouatta palliata). MOLECULAR ECOLOGY. 1998. 7(5), Pgs: 657-658

Torres OM; Leibovici M. [Characterization of the karyotype of the red howler monkey Alouatta seniculus that inhabits Colombia.]. CALDASIA. 2001. 23(2), Pgs: 537-548

Bonvicino CR; Lemos B; Seuanez HN. Molecular phylogenetics of howler monkeys (Alouatta, Platyrrhini): A comparison with karyotypic data.. CHROMOSOMA. 2001. 110(3), Pgs: 241-246

Torres OM; Leibovici M. [Characterization of the karyotype of the red howler monkey Alouatta seniculus that inhabits Colombia.] CALDASIA. 2001. 23(2), Pgs: 537-548

De Oliveira EHC; Suemitsu E; Da Silva AF; Sbalqueiro IJ; Ando S; Someya K; Suzaki Y; Honda M. Geographical variation of chromosomal number in Alouatta fusca clamitans (Primates, Atelidae). CARYOLOGIA. 2000. 53(2), Pgs: 163-168

Reproductive success increases with degree of kinship in cooperative coalitions of female red howler monkeys (Alouatta seniculus).. BEHAVIORAL ECOLOGY AND SOCIOBIOLOGY. 2000. 48(4), Pgs: 253-267

de Oliveira EHC; de Lima MMC; Sbalqueiro IJ; Pissinatti A. The karyotype of Alouatta fusca clamitans from Rio de Janeiro, Brazil: Evidence for a y-autosome translocation.GENETICS AND MOLECULAR BIOLOGY. 1998. 21(3), Pgs: 361-364

Figueiredo WB; Carvalho-Filho NM; Schneider H; Sampaio I.Mitochondrial DNA sequences and the taxonomic status of Alouatta seniculus populations in northeastern Amazonia.NEOTROPICAL PRIMATES. 1998. 6(3), Pgs: 73-77

Mudry MD; Rahn M; Gorostiaga M; Hick A; Merani MS; Solari AJRevised karyotype of Alouatta caraya (Primates: Platyrrhini) based on synaptonemal complex and banding analyses. HEREDITAS. 1998. 128(1), Pgs: 9-16

Pope TR Effects of demographic change on group kin structure and gene dynamics of populations of red howling monkeys. JOURNAL OF MAMMALOGY. 1998. 79(3), Pgs: 692-712

Consigliere S; Stanyon R; Koehler U; Agoramoorthy G; Wienberg J Chromosome painting defines genomic rearrangements between red howler monkey subspecies. CHROMOSOME RESEARCH. 1996. 4(4), Pgs: 264-270

de Oliveira E H C; de Lima M M C; Sbalqueiro IJ Chromosomal variation in Alouatta fusca. NEOTROPICAL PRIMATES. 1995. 3(4), Pgs: 181-183

Cortes-Ortiz L; Bermingham E; Rico C; Rodriguez-Luna E; Sampaio I; Ruiz-Garcia M. Molecular systematics and biogeography of the Neotropical monkey genus, Alouatta. MOLECULAR PHYLOGENETICS AND EVOLUTION. 2003. 26(1), Pgs: 64-81

de Oliveira EHC; de Lima MMC; Sbalqueiro IJ; da Silva ASF Analysis of polymorphic NORs in Alouatta species (Primates, Atelidae). CARYOLOGIA. 1999. 52(3-4), Pgs: 169-175

de Oliveria E H C Cytogenetic and phylogenetic studies of Alouatta from Brasil and Argentina. NEOTROPICAL PRIMATES. 1996. 4(4), Pgs: 156-157

Rahn MI; Mudry M; Merani MS; Solari AJ Meiotic behavior of the X1X2Y1Y2 quadrivalent of the primate Alouatta caraya.CHROMOSOME RESEARCH. 1996. 4(5), Pgs: 350-356

de Oliveira EHC Cytogenetic studies in the family Alouattinae. NEOTROPICAL PRIMATES. 1995. 3(2), Pgs: 52-53

Mudry MD; Rahn IM; Solari AJ Meiosis and chromosome painting of sex chromosome systems in Ceboidea. AMERICAN JOURNAL OF PRIMATOLOGY. 2001. 54(2), Pgs: 65-78

Schneider H; Canavez FC; Sampaio I; Moreira MAM; Tagliaro CH; Seuanez HN Can molecular data place each neotropical monkey in its own branch? CHROMOSOMA. 2001. 109(8), Pgs: 515-523

Stanyon R; Consigliere S; Bigoni F; Ferguson-Smith M; O'Brien PCM; Wienberg J Reciprocal chromosome painting between a New World primate, the woolly monkey, and humans. CHROMOSOME RESEARCH. 2001. 9(2), Pgs: 97-106

Escobar-Paramo P Microsatellite primers for the wild brown capuchin monkey Cebus apella. MOLECULAR ECOLOGY. 2000. 9(1), Pgs: 107-108

Groves C Why taxonomic stability is a bad idea, or why are there so few species of primates (or are there?). EVOLUTIONARY ANTHROPOLOGY. 2001. 10(6), Pgs: 192-198

Romagno D Primate tables chromosome. CARYOLOGIA. 2001. 54(4), Pgs: 285-297

Dietz JM Conservation of biodiversity in neotropical primates. BIODIVERSITY, II. UNDERSTANDING AND PROTECTING OUR BIOLOGICAL RESOURCES. M.L. Reaka-Kudla, D.E. Wilson, E.O. Wilson, Editors. Washington, DC: Joseph Henry Press. 1997, Pgs: 341-356

Pope TR Socioecology, population fragmentation, and patterns of genetic loss in endangered primates. CONSERVATION GENETICS: CASE HISTORIES FROM NATURE. J.C. Avise, J.L. Hamrick, Editors. New York: Chapman & Hall. 1996, Pgs: 119-159

Pope TR Socioecology, population fragmentation, and patterns of genetic loss in endangered primates. CONSERVATION GENETICS: CASE HISTORIES FROM NATURE. J.C. Avise, J.L. Hamrick, Editors. New York: Chapman & Hall. 1996, Pgs: 119-159

Gibbons E F Jr Conservation of primates in captivity. CONSERVATION OF ENDANGERED SPECIES IN CAPTIVITY. E.F. Gibbons, Jr., B.S. Durrant, A.J. Demarest, Editors. Albany: SUNY Press. 1995, Pgs: 485-501

Contribuye a la conservación de las selvas tropicales

Return to main page

Protege los primates Mesoamericanos


copyright@alejandro estrada