ISSN 1067-4136, Russian Journal of Ecology, 2008, Vol. 39, No. 1, pp. 70–72. © Pleiades Publishing, Ltd., 2008.Original Russian Text © V.V. Rozhnov, A.L. Morgulis, M.V. Kholodova, I.G. Meshcherskii, 2008, published in Ekologiya, 2008, No. 1, pp. 73–76.

70

Advanced molecular genetic methods have substan-tially extended the possibilities of both faunistic analy-sis and ecological behavioral studies. For example,DNA isolated from feces of an animal can be used foridentifying its species and sex, genetic relationships,and the population to which it belongs, as well as, insome cases, for individual identification (Reed et al.,1997; Kohn and Wayne, 1997; Frantzen et al., 1998;Dalen et al., 2004; Bellemain et al., 2005; etc.). This, inturn, is a basis for studying many aspects of behavioralecology, which is especially important for nocturnaland elusive species (Wilson et al., 2003; Gomez-Moliner et al., 2004).

The use of feces as a source of DNA entails somedifficulties related to the small amount of host DNA,considerable admixture of heterogenic DNA (fromfood remains, parasites, etc.), high DNA degradationrate, and substances inhibiting the polymerase chainreaction (PCR). Therefore, many current studies areaimed at finding optimal methods for preserving feces(Frantzen et al., 1998; Piggot and Taylor, 2003) and iso-lating the amounts of fecal DNA that would be suffi-cient for the corresponding analyses. Much attention ispaid to the search for species-specific DNA fragmentsand selection of primers for their amplification.

Successful use of mtDNA for species identificationof mustelid feces (Gomez-Moliner et al., 2004) made itpossible to perform studies aimed at improvement andpractical use of the corresponding methodology.

The purpose of this study was to estimate the possi-bility of identifying the species of riverine mustelidsliving on the banks of the Mezha River in the CentralForest Biosphere Reserve (Tver oblast, Russia) by ana-lyzing DNA isolated from their feces, including thedevelopment of methods for collecting and preservingfeces and optimization of the conditions of DNA isola-tion and PCR.

Collection and preservation of feces.

DNA can beisolated from fresh, dried, or chemically preservedspecimens (Frantzen et al., 1998). We preserved fecesin 96% ethanol preventing DNA degradation, whichallowed us to store them for a long time at room tem-perature. Before isolation of DNA, the specimens weredried to remove ethanol.

DNA isolation

was performed by means of a Dia-tom® DNA Prep 100/200 reagent kit (Izogen, Moscow,Russia) according to the protocol recommended by themanufacturer, with some modifications related to thespecificity of the analyzed material: the initial incuba-tion in the lysing buffer solution was prolonged to 1 h;after the incubation, we centrifuged the test tubes twotimes for 10 min each, transferring the supernatant fluidinto a fresh test tube every time in order to completelyremove insoluble particles. We also tried the methodwith the Chelex-100 reagent (BioRad, United States),which is widely used for extracting DNA from differenttissues. However, it proved unsuitable for the samplesof feces.

We used the GenePak® PCR Core (Izogen, Mos-cow, Russia) or the MasterMIX 5

×

Mag

DD

Mix2025(Dialat, Moscow, Russia) reagent kit to perform PCR.According to the method employed in this study(Gomez-Moliner et al., 2004), the first reaction wasperformed using primers L15774 (gtaaaacgacggccagta-catgaattggaggacaaccagt) and H16498 (cctgaactaggaac-cagatg) amplifying an approximately 600-bp mtDNAfragment that included the 3'-terminal region of thecytochrome

b

gene, the genes of threonine and prolinetransport RNAs, and more than 300 bp of the controlregion. We used the following PCR profile: (1)

94°ë

for5 min; (2) 32 cycles of incubation at

94°ë

for 1 min,

53°C

for 1 min, and

72°C

for 1 min; and (3)

72°ë

for5 min. The resultant PCR product was used as a DNAsolution for the second reaction with primers L16007(cccaaagctaaaattctaa) and H16290 (ctcgaggcatggt-

SHORTCOMMUNICATIONS

The Use of Molecular Genetic Methods for Identificationof Mustelid Species by Analyzing Feces

V. V. Rozhnov, A. L. Morgulis, M. V. Kholodova, and I. G. Meshcherskii

Severtsov Institute of Ecology and Evolution, Russian Academy of SciencesLeninskii pr. 33, Moscow, 119071 Russia

e-mail: rozhnov@sevin.ru

Received March 20, 2007

DOI:

10.1134/S1067413608010128

Key words

: mitochondrial DNA, identification of species, feces, mustelids.

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THE USE OF MOLECULAR GENETIC METHODS FOR IDENTIFICATION 71

gataaagc) amplifying a shorter fragment (about 240 bp)within the first fragment, which comprised 35 bp of the3'-terminal region of the proline transport DNA geneand part of the hypervariable site of the mtDNA controlregion. The PCR profile was the same, except that theannealing temperature was increased from 53 to

56°C

.

The results of PCR were visualized by means of1.5% agarose gel electrophoresis with ethidium bro-mide staining. The stained gels were examined using aUV transilluminator.

In addition to the above primers, we preliminarilytested several primers amplifying other regions ofmammalian mtDNAs, including fragments of the genesof cytochrome

b

and the first unit of cytochrome

c

oxi-dase.

The solution of fecal DNA was also tested for sub-stances inhibiting PCR (the

Taq

polymerase activity).The presence of an inhibitor was evidenced by a stop ofamplification of a priori pure control DNA when thetest solution was added to the reaction mixture.

Purification of PCR products.

To purify the PCRproduct of primers and other components, we precipi-tated it with a 0.125 M ammonium acetate solution in96% ethanol for 30 min, with subsequent centrifugationand removal of the supernatant fluid. The DNA pelletwas washed with 70% ethanol cooled to –20

°

C, centri-

fuged, and, after removing the supernatant fluid, driedat 65

°

C and dissolved in deionized water.The amplified fragments were sequenced using the

L16007 primer by means of an ABI 310 automatedsequencer (Applied Biosystems) with the use of theBigDye Terminator v.3.1 Cycle Sequencing kit. Thespecies identity of the specimens was determined byautomatically estimating the similarity of the sequencesobtained (about 210 bp) to mtDNA sequences of differ-ent mustelid species stored in the NCBI GenBank inter-national database, using the NCBI/BLAST program.

Analysis of the results allowed us to draw somemethodological conclusions. Ethanol is the most suit-able preservative for mustelid feces collected in thefield: in contrast to some other preservatives, ethanol iseasy to remove and does not degrade DNA or interferewith its subsequent isolation from biological tissues.The Diatom® DNA Prep 100/200 reagent kits manu-factured by Izogen (Moscow, Russia) ensure a highpurity of the isolated DNA and may be used for itsextraction from feces.

Testing of the samples of the obtained DNA solutionshowed that they contained no inhibitors of

Taq

DNApolymerase. However, our attempts to amplify longerfragments (the genes of cytochrome

b

and the first sub-unit of cytochrome

c

oxidase) failed. Visual estimationalso showed that the amount of DNA left in the reactionmixture after the first reaction (using the primers ampli-fying the 600-bp fragment) was obviously insufficientfor analysis. One possible cause of this loss of DNA isits fragmentation by bacterial restriction nucleases dur-ing long exposure to air.

The duration of storage of the preserved specimensalso affected the efficiency of analysis. Analysis ofspecimens that had been stored for no more thanone year yielded a substantially greater number of pos-itive results than analysis of specimens stored for alonger time (table).

One more problem with fecal DNA is that the spec-imens contain much heterogenic DNA (compared tothe amount of host DNA), including the DNA of ani-mals eaten by mustelids. When the aforementionedprimers are used, the corresponding regions of thisDNA may also be amplified, with the result that furtheranalysis of the products of PCR that seemed successfulupon visualization shows the presence of severalsequences that cannot be separated.

As a result, the overall efficiency of our analysis wasabout 30%: species identity was assigned to 57 out of188 samples of mustelid feces. Since it is usually easyto collect samples, we think that this efficiency is highenough for most studies. For example, we found thatmost samples of feces collected in the floodplain of theMezha River belonged to American minks (

Neovisonvison

) and otters (

Lutra lutra

); in addition, this biotopewas visited by polecats (

Mustella putorius

) and mar-tens (

Martes martes

) (figure). We find no signs of thepresence of European minks (

Mustela lutreola

) in the

Efficiency of analysis of specimens stored for different periodsof time

Storage time,years

Numberof specimens

analyzed

Percentage of PCRs with positive results,

%

2 49 18

1.5 93 28

1 or less 46 48

Lutra lutra

Neovison vison

Martes martes

Mustela putorius

40%

9%

4%

47%

Proportions of mustelid species in the study area.

72

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ROZHNOV et al.

studied area, although this species was common in thisregion and, in particular, in the Central Forest Bio-sphere Reserve and on the banks of the Mezha(Tumanov and Zverev, 1986; Kachanovskii, 1999).This indicates that it is actively displaced by an ecolog-ically similar species, the American mink, whose feceswere most frequent among the test specimens. Thus,molecular genetic analysis of mustelid feces may beused for studying coadaptive faunistic groups of smallcarnivores, detecting the presence of individual speciesin the habitats studied, and estimating their relativeabundance. This method can also be used in ecologicalbehavioral studies, e.g., for analyzing some aspects ofindirect communication between species inhabiting thesame biotopes. A special study on the spatial distribu-tion of scent marks of riverine mustelids (their choiceof places and objects where they leave excretions)showed that this distribution was nonrandom (Rozhnovand Avilov, 2005): the marks were found at noticeableelements of macrorelief (mouths of streams, bound-aries of biotopes, and beaver dams) and were left onobjects standing out against the nearest environment(tree trunks fallen across streams, stones jutting out ofthe water, and sandbanks). Species identification offeces by molecular methods showed that representa-tives of different species left their scent marks on thesame places. The ability of small carnivores to deter-mine the species identity of scent marks, includingfecal marks (Sokolov and Rozhnov, 1983, 1989; Rozh-nov, 2004) provides a basis for interspecific communi-cation and reflects superposition of the biological sig-nal fields of different species. This is evidence for thenecessity of special studies on the structure and spatialarrangement of biological signal fields of mammalianspecies and their use by animals for communication.The method described above will be useful for thesestudies.

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