Contents lists available at SciVerse ScienceDirect
Zoologischer Anzeiger
journa l h omepa g e: www.elsev ier .de / jcz
dentification of the sibling species Apodemus sylvaticus and Apodemus flavicollisRodentia, Muridae)—Comparison of molecular methods
anja Bugarski-Stanojevic ∗, Jelena Blagojevic, Tanja Adnadevic, Vladimir Jovanovic, Mladen Vujosevicepartment of Genetic Research, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
r t i c l e i n f o
rticle history:eceived 29 February 2012eceived in revised form4 September 2012ccepted 15 November 2012vailable online xxx
eywords:P-PCR
SSR-PCRpecies-specific primersytochrome bitochondrial DNA
-banding
a b s t r a c t
The yellow-necked field mouse, Apodemus flavicollis (Melchior, 1934), and the long-tailed field mouse,Apodemus sylvaticus (Linnaeus, 1758) are morphologically similar species, with largely overlapping geo-graphical areas and almost equal ecological requirements. This makes reliable species diagnosis basedon external characters a real challenge. When advanced multivariate methods of skull and/or dentalmorphometrics are employed, specimens can be successfully distinguished in most cases, but not all, mak-ing single specimen identification impossible. Application of C-band karyotyping clearly distinguishesbetween these two species. However, it can be applied only to live animals. Several molecular methodshave also allowed successful species diagnosis, but their universality has not been tested. Here we com-pare the diagnostic power of three molecular approaches and their applicability to populations from aspecies wide distributional area, as well as their simplicity of use, both for live animals and for samplesstored in alcohol for a considerable period, without DNA sequencing. A total of 200 tissue samples fromMorocco, France, Belgium, Serbia, Romania, Greece, Russia, Turkey and Kazakhstan, were analyzed byAP-PCR, ISSR-PCR and species-specific primers from the mitochondrial cytochrome b gene. The first two
methods gave clear species-specific DNA profiles in complete agreement with previous C-band results.However, only partial agreement was observed between the third, species-specific primer method andthe other two approaches and C-banding, as there were 4.5–10.5% false positive results. Hence we pro-pose AP-PCR and ISSR-PCR techniques with particular chosen primers for quick and reliable diagnosis ofthese two species. A large number of samples can be assayed in a short period, with minimal cost andeffort. Moreover, these techniques can be applied to a single specimen.
. Introduction
Morphological variability of the yellow-necked field mousepodemus flavicollis (Melchior, 1934) and the long-tailed fieldouse Apodemus sylvaticus (Linnaeus, 1758) and methods for their
istinction are the subject of long lasting discussions in the mam-alogical literature. In the absence of established and widely
ccepted discrimination criteria for these two species, zoologistsnd taxonomists still debate about their distribution and pres-nce in some areas. A. sylvaticus and A. flavicollis are wide-rangingpecies, with largely overlapping geographical areas and similarcological requirements (Filippucci et al., 1989; Musser et al., 1996;rlov et al., 1996). Together with other members of the westernalearctic subgenus Sylvaemus (Ognev, 1929), they are character-
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
zed by a low level of interspecies morphological differentiationhat can be interpreted to result from a bush-like radiation eventSerizawa et al., 2000) leading to the simultaneous emergence of
many species (Orlov et al., 1996; Chelomina et al., 1998; Michauxet al., 2002; Matsubara et al., 2004). In the western part of theirdistributional areas both species inhabit a large range of latitude,from North Africa to Scandinavia (Wilson and Reeder, 2005). Addi-tionally, A. flavicollis and A. sylvaticus express a clinal variationin body size (Niethammer, 1969; Renaud and Michaux, 2003).On average individuals of A. flavicollis are larger than those of A.sylvaticus, although in the southern parts of their distributionalareas, an opposite clinal variation in body size and pelage color isexpressed (Filippucci et al., 1984). These characteristics are deter-mined mainly by landscape and microclimatic conditions of theparticular population environment.
As the most widespread Western Palearctic species of smallmammals, they may be expected to be rather heterogeneous (Orlovet al., 1996). Indeed, they are characterized by fairly complexgenetic differentiation (Michaux et al., 2003; Hoofer et al., 2007;Bugarski-Stanojevic et al., 2011). Both interspecies similarity and
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
high intraspecies variability in morphological characters have oftenled to controversial discussions about their taxonomic status, pos-sible introgression and natural hybridization (Engel et al., 1973).Reliable species diagnosis on the basis of external characters is a
eal challenge (Zagorodnyuk, 1996; Michaux et al., 2001; Michauxt al., 2002). A morphological approach (Ðulic and Tvrtkovic, 1974;odorovic et al., 1971; Krystufek and Janzekovic, 2005; Fryntat al., 2006; C olak et al., 2007; Barciová and Macholán, 2009) issually insufficient for species characterization, as morphological
ntraspecies variability may obscure the real patterns of varia-ion (Orlov et al., 1996). However, when advanced multivariate
ethods of skull and/or dental morphometrics are employed, spec-mens can be successfully distinguished in most cases, but not allJanzekovic and Krystufek, 2004; Frynta et al., 2006; Jojic et al.,012). These methods require the reference group – a sufficientumber of individuals previosly determinated by both, molecularnd morphometric methods. Without a reference group, a singlenknown individual cannot be identified with these methods. Fur-hermore, specific ranges of the particular measurements showreater or smaller overlap and no single character per se can serves a diagnostic criterion. There are always individuals considered asntermediates between the two forms. Moreover, the identificationey and the discrimination power can be guaranteed only for theopulations under study, so one should be careful in extrapolatinghe results to other parts of the A. sylvaticus and A. flavicollis rangesFrynta et al., 2006; Barciová and Macholán, 2009). Consequently,
isinterpretations persist in the biological literature to this day.Besides their high morphological and ecological similarities,
hese species also have similar karyotypes (Zima and Král,984). They have 48 acrocentric chromosomes, with similar G-anding patterns (Vujosevic et al., 1984). However, a majorifference was found in the amount and distribution of constitu-ive heterochromatin (C-bands). While in A. flavicollis constitutiveeterochromatin is located exclusively in the centromeric area, in. sylvaticus, besides variable centromeric bands, some chro-osomes feature additional telomeric and/or interstitial bands.etaphases sequentially stained with the Q/C banding technique
Engel et al., 1973; Hirning et al., 1989; Reutter et al., 2001)ere proposed as a possible way to improve species identification.lthough application of the C-banding technique, as well as severalolecular methods, like protein electrophoresis (Britton-Davidian
t al., 1991; Orlov et al., 1996; Filippucci et al., 2002) and DNA anal-sis (Michaux et al., 1996, 1998, 2001, 2002, 2005; Chelomina et al.,998, 2007; Serizawa et al., 2000; Hoofer et al., 2007; Suzuki et al.,008; Dubey et al., 2009), clearly distinguished between these twopecies, they all have certain disadvantages. There is still a necessityor a simple approach that can be completed quickly and reliably,oth on live animals and on samples stored in alcohol for a longeriod, without DNA sequencing. The method should be univer-ally applicable to all populations from the species’ distributionalrea. Here we explore and compare three such approaches.
The first two molecular techniques are quick and effectiveulti-locus methods for producing species-specific fingerprints.
hey both have many advantages: no previous knowledge of theenome under study is necessary, many independent loci dis-ersed throughout the genome can be examined, the effort andost involved are modest, so many individuals can be assayed. Therst method, arbitrarily primed-polymerase chain reaction (AP-CR), has been used successfully in studying genetic variability,axonomy and phylogeny, especially in closely related species andopulations (Welsh and McClelland, 1990; Borowsky et al., 1995;uramoto et al., 1995; Vidigal et al., 1998; Atienzar and Jha, 2006;ing et al., 2006; Prieto et al., 2007; Bugarski-Stanojevic et al., 2008).he second one, inter simple sequence repeat-PCR (ISSR-PCR), haslso been effectively employed to study polymorphism and evolu-ionary relationships of lineages among higher plants and animals
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
Zietkiewicz et al., 1994; Bornet et al., 2002; Archibald et al., 2006;oy and Chakraborty, 2009; Wang et al., 2009; Bugarski-Stanojevict al., 2011). Besides their hypervariable nature, these types ofarkers are especially attractive due to the vast numbers of loci that
PRESSher Anzeiger xxx (2012) xxx– xxx
can be examined and the small amount of fresh or dried materialneeded per sample. The use of longer primers and a higher anneal-ing temperature makes these markers much more reproducible,compared with other fingerprinting techniques (e.g. RAPD).
The third discrimination method examined here is moleculartyping with species-specific primers designed from the mitochon-drial cytochrome b gene (Michaux et al., 2001). The technique isfast and simple, with clear results. Namely, the product of each PCRreaction, a species specific part of cytochrome b, is either presentor absent. The authors suggested that any specimen of the threewest European Apodemus species (A. flavicollis, A. sylvaticus andApodemus alpicola) can be confidently identified using this method.However, a control test on Eastern European samples was doneonly on five specimens of Apodemus uralensis, but has never beenperformed on A. flavicollis or A. sylvaticus from that area.
All three approaches require low quantities of total genomicDNA. They can be performed on a small piece of ear, a tail fragmentor a finger that can be taken from live animals, making it avail-able for identification in field investigations. This study intends tosurvey the diagnostic power of these three molecular methods fordiscrimination of Apodemus samples from a wide geographic areaof the Palearctic.
2. Materials and methods
2.1. Samples and locations
We performed DNA analysis of 200 specimens (Table 1) sup-posed to belong to three Apodemus species, 103 A. flavicollis, 79A. sylvaticus and 18 A. uralensis (Pallas, 1811). A total of 134individuals were collected in Serbia from 18 different localitiesin the period 1995–2011, using Longworth traps. Tissue sam-ples of 66 specimens, preserved in alcohol, were provided fromMorocco, France, Belgium, Romania, Greece, Russia, Turkey andKazakhstan (Fig. 1). A part of the results (Table 1) was previouslypublished and includes 59 samples analyzed by AP-PCR methodfrom Bugarski-Stanojevic et al. (2008), and 109 samples by ISSR-PCR from Bugarski-Stanojevic et al. (2011), with 46 individuals fromseven sample localities being identical for both methods. 753 sam-ples from nine localities were screened by C-banding techniqueonly (Table 1).
A. uralensis was tested as a control group, since, being a mem-ber of the subgenus Sylvaemus, is morphologically similar to theother two species. Their distribution overlaps in central and east-ern Europe and its occurrence was also credited to Serbia (Wilsonand Reeder, 2005). Moreover, Michaux et al. (2001) also tested fivespecimens of A. uralensis as a control group.
2.2. Chromosome analyses
Chromosome preparations were obtained from bone marrowusing the standard technique for all specimens collected in Serbia.C-banding was performed according to the method of Sumner(1972), slightly modified (treatment with barium hydroxide wasshortened to average 45 s). Specimens with only centromeric bandson all chromosomes were defined as A. flavicollis, while those withdifferent C-band distribution were ascribed to A. sylvaticus.
2.3. DNA extraction
Total DNA was extracted from frozen livers as described byManiatis et al. (1982) using a DNA extraction kit (DNeasy Blood
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
and Tissue Kit, Qiagen). Samples provided from other countriesincluded mouse liver, tails, ears and legs preserved in absoluteethanol. Since precise determination of the quality and purity ofDNA is essential for reproducible and reliable patterns (McClelland
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identification of the sibling species Apodemus sylvaticus and Apodemus flavicollis(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012), http://dx.doi.org/10.1016/j.jcz.2012.11.004
ARTICLE IN PRESSG Model
JCZ-25233; No. of Pages 9
V. Bugarski-Stanojevic et al. / Zoologischer Anzeiger xxx (2012) xxx– xxx 3
Table 1All mice specimens analyzed by four diagnostic methods. Sampling localities are specified by numbers (1–56) as in Fig. 1. Samples marked with * were previously analyzedand published in Bugarski-Stanojevic et al. (2008, 2011).
Species Geographic region Sampling localities Number of samples/diagnostic method
4 V. Bugarski-Stanojevic et al. / Zoologischer Anzeiger xxx (2012) xxx– xxx
F rs frod
asdm
2
cbnfitpiwttNc
(n(bd
PwT(aofi(
ig. 1. Sampling localities of all analyzed Apodemus individuals, specified by numbeiagnosed by C-banding only (�); A. sylvaticus (�); and A. uralensis (�).
nd Welsh, 1994; Tyler et al., 1997), DNA solutions were first mea-ured by spectrophotometry and the degree of degradation wasetermined using 0.8% agarose gels with 0.5 �g/ml ethidium bro-ide.
.4. AP-PCR analysis
The isolated DNA was amplified by AP-PCR. This method is oftenriticized for reproducibility, requiring subjective judgment of aand’s presence and being difficult to analyze because of the largeumber of products. Therefore, the chemical and temperature pro-les of each reaction, for each primer, were optimized accordingo Cobb and Clarkson (1994) in order to generate reproduciblerofiles of moderate complexity. We used repeated reactions and
ncreased sample size for confirmation of the reproducibility. DNAas tested at three template concentrations (50, 100 and 250 ng)
o exclude the possibility that template quality and impurities inhe DNA preparations affect the interpretation of the DNA profiles.egative controls were set up for each reaction to check for DNAontamination.
In this study we used one of the 22 previously optimized primersfor details see Bugarski-Stanojevic et al., 2008). The chosen 20-ucleotide primer E8S (exone 8) 5′-TAAATGGGACAGGTAGGACC-3′
Metabion) demonstrated the ability to generate the highest num-er of reproducible species-specific DNA fragments and thus toifferentiate precisely between the analyzed Apodemus species.
The reactions took place in 200 �l microtubes in a Gene AmpCR System 2700 (Applied Biosystems). Arbitrarily primed PCRith primer E8S was performed with: PCR reaction buffer [750 mM
ris–HCl (pH 8.8 at 25 ◦C), 200 mM (NH4)2SO4, 0.1% Tween 20]Fermentas), 0.4 mM of each of the dNTPs, 2.5 mM MgCl2, 5 �M
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
mplimer, 1 U of DreamTaq DNA polymerase (Fermentas) and 50 ngf genomic DNA, in a final volume of 25 �l. The temperature pro-le was: (94 ◦C for 4 min), four cycles at low stringency conditions94 ◦C for 1 min; 47 ◦C for 2 min; and 72 ◦C for 2 min), 35 cycles at
m Table 1. Symbols: A. flavicollis diagnosed by molecular methods (�); A. fllavicollis
high stringency conditions (94 ◦C for 30 s; 60 ◦C for 30 s; and 72 ◦Cfor 1.5 min) and a final extension (72 ◦C for 7 min).
2.5. ISSR-PCR analysis
ISSR markers are DNA sequences delimited by two invertedsimple sequence repeats (SSRs) that are amplified by a single PCRprimer, composed of a few complementary SSR units, with or with-out an anchored end. With an anchored sequence, ISSR primers aremore selective and amplification patterns are more reproducible(Kochieva et al., 2002). The use of longer primers (16–25 mers) com-pared to RAPD primers and a higher annealing temperature, makesthese markers very reproducible. In the previous study (Bugarski-Stanojevic et al., 2011) we have optimized 12 primers according toCobb and Clarkson (1994) for the ability to generate informativeand reproducible multilocus profiles and chosen six that producedthe DNA profiles of moderate complexity with a clear band pat-tern and the presence of species-specific fragments. Here we usedone primer (CAA)5GC (Metabion) that showed a clear band patternand the presence of the highest proportion of species-specific frag-ments for reliable discrimination of the studied Apodemus species.Optimized conditions for this selected primer were: PCR reactionbuffer [750 mM Tris–HCl (pH 8.8 at 25 ◦C), 200 mM (NH4)2SO4, 0.1%Tween 20] (Fermentas) 1.0 mM of each of the dNTPs, 2.5 mM MgCl2,0.5 �M amplimer, 1 U of DreamTaq DNA Polymerase (Fermentas)and 20 ng of genomic DNA, in a final volume of 20 �l. The tempera-ture profile was: initial denaturation at 94 ◦C for 5 min, followed by45 cycles (94 ◦C for 30 s; 50 ◦C for 30 s; and 72 ◦C for 1.5 min) and afinal extension at 72 ◦C for 7 min in 200 �l microtubes in a ThermalCycler 2720 (Applied Biosystems).
2.6. Species-specific primers from the mitochondrial cytochrome
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
b gene
The third method of species molecular discrimination wasemployed according to Michaux et al. (2001). From the
itochondrial cytochrome b gene these authors designed a pairf specific primers, which contain the nucleotidic characteristicsf each targeted species: A. flavicollis, A. sylvaticus and A. alpicola.n our study, each individual was tested with two pairs of specificrimers:
for A. sylvaticusSylUP: 5′-GAGGAGGATTCTCAGTAGAC-3′;SylDN: 5′-TTAATATGGGGTGGGGTGTTA-3′;for A. flavicollisFlaUP: 5′-AGCTACACTAACACGTTTC-3′;FlaDN: 5′-GCGTATGCAAATAGGAAGTAC-3′.The product of each PCR reaction represents a species specific
art of cytochrome b. Amplification reactions were similar as in theentioned study, carried out in 15 �l volumes including: PCR reac-
ion buffer [750 mM Tris–HCl (pH 8.8 at 25 ◦C), 200 mM (NH4)2SO4,.1% Tween 20] (Fermentas) 0.2 mM of each of the dNTPs, 2.5 mMgCl2, 0.5 �M amplimers, 1 U of DreamTaq DNA Polymerase (Fer-entas) and 50 ng of genomic DNA, in a final volume of 15 �l. All
eactions were performed at: 94 ◦C for 4 min, followed by 30 cycles94 ◦C for 20 s; 55 ◦C for 30 s; and 68 ◦C for 1.5 min) and a finalxtension at 68 ◦C for 10 min.
Additionally, an extensive search through all GenBankucleotide sequences of cytochrome b genes and pseudogenes of A.ylvaticus was done. The search was performed with BLASTN 2.2.27Altschul et al., 1997), having primer sequences (for FlaUP) ornverse and complementary sequence of primer FlaDN as queries.he GenBank database was accessed on 11 September 2012.
.7. Data collection and analysis
At the end of the reactions, the total volume of each PCR productas separated by horizontal gel electrophoresis (1.2% agarose in 1×
BE, with 0.5 �g/ml ethidium bromide), using the standard molecu-ar weight marker, GeneRuler 100 bp DNA Ladder Plus (Fermentas)n each electrophoretic run. The bands were visualized with UVight in the Bio-Rad Gel Doc XR + System and the results photo-ocumented using Quantity One 1-D Analysis Software (Bio-Radaboratories). Only distinct and reproducible, well-marked elec-rophoretic bands were included in the analysis. Faintly stainedands that were not clearly resolved and their relative intensityere not considered in the data collection. The bands were detected
nd calibrated using Total Lab v.1.10 software (Phoretix).
. Results
.1. Chromosome analyses
All captured animals from Serbia were karyotyped and appli-ation of C-banding confirmed the existence of two differentypes of C-band distribution. A total of 82 animals had C-bandsocated exclusively in the centromeric region, which is character-stic for A. flavicollis. The other 52 animals had additional distal andntercalar heterochromatin, present on some chromosomes, whileentromeric bands were totally absent, or present only on somehromosomes, which is specific for A. sylvaticus.
.2. AP-PCR analysis
DNA isolated from three Apodemus species was analyzed byhe previously selected AP-PCR primer, E8S. The profiles that wereested at three template concentrations, yielded minimal differ-nces, presented as unequal band intensities. This excluded the
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
ossibility that template quality and discrepancy between exper-ments could affect interpretation of the DNA profiles. The lengthf amplified bands ranged from 188 to 968 bp. DNA profiles weref moderate complexity, with distinct and reproducible bands.
PRESSher Anzeiger xxx (2012) xxx– xxx 5
The band number per individual varied from 9 to 14. Certainamplified bands were found to be specific to a particular species,i.e. present in one but absent from the other two species andthey were used for species identification purposes (indicated witharrows in Fig. 2A). DNA fragments specific for species A. flavicol-lis were: 968 bp, 495 bp, 435 bp, 387 bp and 272 bp. The species A.sylvaticus could be recognized by: 682 bp, 618 bp, 413 bp, 358 bp,240 bp and 218 bp. DNA bands characteristic for A. uralensis were:661 bp, 636 bp, 537 bp and 438 bp. The most obvious and distinc-tive species-specific DNA markers for A. flavicollis and A. sylvaticusare indicated with arrows in Fig. 2A.
3.3. ISSR-PCR analysis
The primer (CAA)5GC also gave a reproducible DNA pattern,specific for each species. The possibility that template quality andquantity, or differences between experiments affected interpre-tation of the DNA profiles was excluded, as any discrepancy inprofiles, appearing as unequal band intensities, was negligible.The range of DNA markers was 927–2049 bp. These profiles weresimple, with 6–8 bands per individual line. Species specific DNAfragments were: for A. flavicollis 1970 bp, 1696 bp, 1574 bp, 1368 bp,1275 bp, 1093 bp and 933 bp; for A. sylvaticus 2049 bp, 1652 bp,1560 bp, 1103 bp, 1060 bp and 954 bp; for A. uralensis DNA pro-files were more complex, with characteristic bands of: 2143 bp,1591 bp, 1494 bp, 1437 bp, 1324 bp, 1248 bp, 1076 bp and 927 bp.The most apparent species-specific DNA bands for A. flavicollis andA. sylvaticus are indicated with arrows in Fig. 2B.
3.4. Species-specific primers from the mitochondrial cytochromeb gene
Each individual was tested with both pairs of specific primers,FlaUP/DN and SylUP/DN. The product of each PCR reaction shouldrepresent the species specific part of cytochrome b. When amplifi-cation was positive, the specific primer amplified 351–386 bp DNAfragments. Electrophoretic scans were clear, with only one specificband present or absent in each line. Out of 200 analyzed individ-uals, 21 (10.5%) showed a false positive result, when amplifiedwith FlaUP/DN primers, and 9 (4.5%) gained false positive bandswhen amplified with the second primer pair, SylUP/DN. Thirteenspecimens of A. sylvaticus from Serbia and Greece were incorrectlydiagnosed as A. flavicollis. Three individuals of A. flavicollis fromSerbia were mistakenly recognized as A. sylvaticus (Table 2). Sev-eral false amplifications can be observed on electropherograms inFig. 2: (C) false A. sylvaticus, lines: 3, 4, 32, 33, 34, 35, 36, 37 and 38;and (D) false A. flavicollis, lines: 22, 23, 33, 34, 36, 37, 38 and 39.
Six specimens of the A. uralensis control group from Russia(locality numbers: 48, 50, 51, 52, and 53) gained both A. sylvaticusand A. flavicollis markers, while two more specimens from Russia(54) and Kazakhstan (55) amplified specific markers of A. flavicol-lis. However, 10 samples of A. uralensis from Ural Mt. (56) showednegative results with both primer pairs.
In BLASTn analysis, species-specific primers were obtained innonspecific species. For instance, some individuals of A. sylvati-cus captured in Russia (GenBank accession numbers AF429819 andAF429820) had a sequence 100% matched for the A. flavicollis-specific primer (FlaUP) and 90% for FlaDN. Some other individuals ofA. sylvaticus had sequences with a difference in a single nucleotide
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
(the last one) in the cytochrome b gene (AF159395) or evena cytochrome b pseudogene (the most numerous group, withindividuals collected across Europe, GenBank accession numbersFJ389582–FJ389619).
6 V. Bugarski-Stanojevic et al. / Zoologischer Anzeiger xxx (2012) xxx– xxx
Fig. 2. Several pooled electrophoretic scans of four PCR reactions. Besides the line numbers, locality numbers from Table 1 and Fig. 1 are given in brackets. Arrows indicatesome representative species-specific bands. Nt – negative control; M – standard molecular weight marker. (A) AP-PCR primer E8S: A. flavicollis: 1-(29), 2-(34), 3-(35), 4,7-(36), 9, 10-(14); A. sylvaticus: 5-(31), 6-(32), 8-(33), 11, 12-(3); A. flavicollis-specific bands: line 1: 495 bp and 968 bp; A. sylvaticus-specific bands: line 8: 240 bp and 413 bp;(B) ISSR-PCR primer (CAA)5GC: A. flavicollis: 1-(25), 2, 3-(19), 4-(34), 7-(33), 14, 15-(39); A. sylvaticus: 5-(31), 6-(32), 8-(33), 9, 10-(22), 11, 12-(23), 13-(9). A. flavicollis-specificbands: line 4: 1275 bp and 1696 bp; A. sylvaticus-specific bands: line 9: 1103 bp, 2049 bp and 1560 bp. (C) SylUP/DN primers: A. sylvaticus: 1, 2-(26); 3, 4, 5, 6-(5); 7-(28);8, 10-(30); 9-(29), 15-(31); 16-(32); 18-(33); 23, 24-(9); 26, 27, 28, 29-(49); 30-(44); 31-(28); A. flavicollis: 11, 20-(29); 12-(34); 13-(35); 14, 17-(36); 19-(10); 21, 22-(19);25-(9); A. uralensis: 32-(55); 33-(50); 34-(51); 35-(52); 36-(53); 37, 38-(48). Arrows indicate: the absence of species-specific band (A. sylvaticus lines 5, 6); the presence offalse positive band (A. flavicollis: lines 14, 20, 25 and A. uralensis: lines 33, 34, 35, 36, 37). (D) FlaUP/DN primers: A. sylvaticus: 5, 8-(26); 10-(28); 11, 13-(30); 12-(29); 18-(31);19-(32); 21-(33); 22, 23-(9); 25, 26, 27, 28-(3); 29, 30, 31, 32-(49); 33-(44); 34-(14); 36-(40); 37-(43); A. flavicollis: 1, 2, 7-(26); 3, 4, 6, 9-(6); 14-(29); 15-(34); 16-(35); 17,2 the p3
4
sssr
0-(36); 24-(9); 35-(14); A. uralensis: 38-(50); 39-(51); 40, 41-(56). Arrows indicate6, 37 and A. uralensis: lines 38, 39).
. Discussion
The molecular AP-PCR and ISSR-PCR techniques, with one cho-
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
en primer each, gave clear multilocus DNA profiles with severalpecies specific DNA markers. The molecular profiles of 134 mouseamples from Serbia were in complete agreement with C-bandesults. Three types of DNA profiles were amplified, characteristic
resence of false positive band (A. sylvaticus: lines 5, 8, 22, 23, 25, 26, 27, 28, 33, 34,
for A. flavicollis, A. sylvaticus and A. uralensis. Furthermore, tissuesamples of 66 specimens from Morocco, France, Belgium, Romania,Greece, Russia, Turkey and Kazakhstan (Fig. 1 and Table 1), earlier
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
identified as A. flavicollis, A. sylvaticus or A. uralensis by other authorsand methods, were absolutely confirmed by two of the appliedmolecular methods. All three species were clearly identified byspecies-specific DNA profiles.
V. Bugarski-Stanojevic et al. / Zoologischer Anzeiger xxx (2012) xxx– xxx 7
Table 2Individuals of all three species with presence (+) or absence (−) of specific DNA markers, amplified with both primer pairs, FlaUp/DN and SylUP/DN. Locality numbers fromFig. 1 and Table 1 are given in brackets; n – number of individuals.
Species Specimen locality n Fla UP/DN marker Syl UP/DN marker
A. flavicollis Serbia Cer Mt. (5) 2 + +Kozjak Mt. (14) 1 + +
A. uralensis Russia Sochi city (50) 1 + +Krasnaya pol. (51) 1 + +Nalchik city (52) 1 + +Orenburg region (53) 1 + +Belgorod region (48) 2 + +
MIsflcwsgpaRsmboncsga
ewsGtas
wfitrhCaEoeflpbmrt
Gorno-Altaisk c. (54)
Kazakhstan Almaty reg. (55)
Nevertheless, the third molecular technique, designed byichaux et al. (2001) showed partial inconsistency with AP-PCR,
SSR-PCR and C-banding. Our search through GenBank databasehowed existence of sequences identical or almost identical to A.avicollis-specific primer sequences in another species, A. sylvati-us. These sequences possibly give a high yield of PCR productsith FlaUP/DN. Besides total or partial sequence match with non-
pecific primers, the presence of nuclear copies of mitochondrialenes (pseudogenes) is another explanation for emerging falseositive results. They have been described in a variety of animalsnd plants, and revealed to be common in mammals, particularlyodentia (Dubey et al., 2009). Incorrect species identification inome instances is a consequence of inclusion of nuclear DNA intDNA data sets, among others. A solution to this problem could
e to use extraction methods enabling purification of mtDNA with-ut nuclear DNA. However, such procedures are often complex andot suitable for samples stored in ethanol, thus reducing their appli-ability. Another solution to this problem could be improving thepecies-specific primers in order to avoid amplification of pseudo-enes and to test them on the samples from the whole distributionalreas of the studied species.
In our analysis, 16 individuals of A. flavicollis and A. sylvaticusxpressed species-specific DNA markers – part of cytochrome b,hen amplified with each primer pair. They originated from the
outh eastern part of their distributional area, from Serbia andreece (Table 2). Thus, 10 specimens of A. sylvaticus from Serbia and
hree from Greece incorrectly showed A. flavicollis-specific bandsnd three samples of A. flavicollis from Serbia wrongly gained A.ylvaticus-specific bands.
Although the study of Michaux et al. (2001) was focused onest European Apodemus, they conducted a small control test onve specimens of A. uralensis and PCR results were always nega-ive. However, our analysis of this species showed miscellaneousesults. According to karyological and mtDNA analysis, this speciesas a noticeable phylogeographic structure (Balakirev et al., 2007;helomina et al., 2007; Bogdanov et al., 2009). Three forms, namedccording to their geographical distribution, can be distinguished:ast European, South European and Asian. Our control samplef A. uralensis included all three subgroups (Bugarski-Stanojevict al., 2011). Eight samples belonging to all three forms showed A.avicollis-specific DNA marker. The SylUP/DN primer pair gave falseositive results for the European form, but negative for samples
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
elonging to the Asian form (localities 54 and 55). Only speci-ens from the south Ural region (locality 56) showed negative PCR
esults when amplified with both primer pairs. These results showhat applying species-specific primers from the mitochondrial
1 + −1 + −
cytochrome b gene method in its present form to these three oftensympatric, or sometimes even syntopic species from eastern andsoutheastern European parts of their ranges could lead to confusionregarding their presence and distribution.
A. flavicollis and A. sylvaticus are common species in many west-ern and central European countries, although A. flavicollis is notfound in the southern Iberian Peninsula, western France, north-ern Belgium and the Netherlands (Mitchell-Jones et al., 1999). Thegeographic range of A. sylvaticus in Europe extends north to Scan-dinavia, east to central Belarus, eastern Ukraine and west Russia,which is its easternmost limit, and south to northwestern Turkey(Wilson and Reeder, 2005). Information on the geographic distribu-tion and taxonomic status of these species on the Balkan Peninsulais still not clear, because published data are based on morphologicalcharacterization only and molecular methods were performed at alimited number of localities. Distribution of A. sylvaticus in centralSerbia, south of the rivers Sava and Danube, was never validatedby molecular methods, but only by traditional morphometrics. Insurrounding countries it was confirmed in Greece by C-band kary-otyping (Rovatsos et al., 2008), and later established by molecularanalysis (Bugarski-Stanojevic et al., 2011). We have never ques-tioned the presence of A. sylvaticus in the Balkans, as erroneouslyquoted by Bego et al. (2008). Michaux et al. (2003) detected threeindividuals of A. sylvaticus by molecular assessment only at onelocality in Serbia, Susara, Deliblato sands, which is north of the riverDanube, in Vojvodina. The same three specimens from the Susaralocality were previously analyzed by allozyme studies (Filippucciet al., 2002). This species was confirmed at five localities in Vojvod-ina in our studies (Bugarski-Stanojevic et al., 2008, 2011). AlthoughKrystufek et al. (2012) argue that absence of A. sylvaticus from cen-tral Serbia is surprising for the scientific community, absence frompart of a generally established distributional area is not exclusivelyrelated to central Serbia. For instance, while A. sylvaticus is widelydistributed throughout Ukraine, including the southern mainland,in spite of extensive sampling, absence from the entire CrimeanPeninsula has been documented by many authors (Mezhzherin andLashkova, 1992; Orlov et al., 1996; Zagorodnyuk, 1996; Mezhzherinet al., 2002; Hoofer et al., 2007). According to Macholán et al. (2001),A. sylvaticus is also absent from the entire area of the Middle East. Insouth-eastern Europe and the Middle East, several taxa, previouslyidentified as A. sylvaticus, have been recognized as distinct species.In western Anatolia, four Sylvaemus species were diagnosed by elec-
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
trophoretic and morphological analyses (Filippucci et al., 1996).It was shown that A. sylvaticus, previously considered widely dis-tributed in Asian Turkey, is a very rare species there, limited to asmall area near the coast of the Black Sea. In the same way, it was
reviously commonly believed that A. sylvaticus is the most fre-uent species in Israel. This was altered after allozyme studies ofhe distribution of Apodemus species by Filippucci et al. (1989), whooncluded that, instead of A. sylvaticus, A. flavicollis is the most com-on species in Israel (apart from A. mystacinus). They also identified
new species, A. hermonensis.According to an extended sample of 1383 individuals from 27
ocalities in Serbia, identified by C-banding, and in some cases usingwo precise diagnostic methods (Table 1), we can conclude that A.ylvaticus is certainly not a widespread species in central Serbias is generally considered. It is evidently extensively distributedn Vojvodina. Recently we captured one specimen in sympatry
ith A. flavicollis on Kozjak Mt. on the southern border of Serbia.ts presence was already proven in Greece (Rovatsos et al., 2008;ugarski-Stanojevic et al., 2011). This kind of distribution has aomparable pattern with the geographic range of the species, Musortulanus, (Petrov, 1992), with dense distribution north of theivers Sava and Danube and only a slender link along the riverselika Morava and Juzna Morava in central Serbia, spreading to
he south toward Macedonia and to the east toward Bulgaria andomania. In our future field work it should be demonstrated if thisypothesis is true for A. sylvaticus.
Our results show that the AP-PCR and ISSR-PCR methods areuick and reliable diagnostic techniques for distinguishing amongll three species examined here. They are universally applicableo all populations in the whole distributional area. Large samplesan be assayed in a short period, with a minimal cost and effort,ithout DNA sequencing and killing the animals. At the same time,
hese techniques can always be applied to a single specimen, whichvercomes shortcomings of the morphological approach. Never-heless, considering the length of the species-specific bands (forhe AP-PCR primer from 188 to 968 bp and for the ISSR-PCR primeretween 927 and 2049 bp) these methods may have their limita-ions too. The amplification of such long DNA fragments may beifficult using degraded material, like old museum specimens oread animals collected in the fields. Consequently, these methodsppear more applicable for fresh tissues or live animals. Thus, itould be interesting to improve species-specific primer method,hich amplifies approximately 350 bp fragments, in order to avoid
mplification of pseudogenes and to become relevant on samplesrom the whole distributional areas of the studied species.
Distinctions between phenotypically similar and at the sameime morphologically and genetically highly variable species byraditional morphometrics and “expert knowledge” have causedonfusion and misinterpretations concerning their taxonomy, dis-ribution and presence in some areas. Today with a variety ofechniques available, it is necessary to use widely accepted andniversally applicable discrimination criteria
cknowledgements
We are most grateful to A.S. Bogdanov, E.B. Giagia-thanasopoulou, E. C olak, R.M. Libois, C. Nieberding, N. Kolchevand J.R. Michaux, who kindly provided us with tissue samples. Thisork was supported by the Ministry of Education and Science of
he Republic of Serbia, Grant No. 173003.
eferences
ltschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman,D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
search programs. Nucleic Acids Research 25 (17), 3389–3402.rchibald, J.K., Crawford, D.J., Santos-Guerra, A., Mort, M.E., 2006. The utility of
automated analysis of inter-simple sequence repeat (ISSR) loci for resolving rela-tionships in the Canary island species of Tolpis (Asteraceae). American Journalof Botany 93, 1154–1162.
PRESSher Anzeiger xxx (2012) xxx– xxx
Atienzar, A.F., Jha, N.A., 2006. The random amplified polymorphic DNA (RAPD) assayand related techniques applied to genotoxicity and carcinogenesis studies: acritical review. Mutation Research 613, 76–102.
Balakirev, A.E., Baskevich, M.L., Gmyl, A.P., Okulova, N.M., Andreeva, T.A., Sokolenko,O.V., Malygin, V.M., Khliap, L.A., Opatin, M.L., Orlov, V.N., 2007. On the taxonomicrank of ciscaucasicus and its relationships with the pygmy wood mouse Sylvae-mus uralensis inferred from the mtDNA cytochrome b gene sequence. Genetika43 (12), 1651–1666.
Barciová, L., Macholán, M., 2009. Morphometric key for the discrimination oftwo wood mice species, Apodemus sylvaticus and A. flavicollis. Acta ZoologicaAcademiae Scientiarum Hungaricae 55 (1), 31–38.
Bego, F., Krystufek, B., Paspali, G., Rogozi, E., 2008. Small terrestrial mammals ofAlbania: annotated list and distribution. Italian Journal of Mammalogy 19 (2),83–101.
Bogdanov, A.S., Atopkin, D.M., Chelomina, G.N., 2009. Analysis of genetic variationand differentiation in the pygmy wood mouse Sylvaemus uralensis (RodentiaMuridae) aided by the RAPD-PCR. Biological Bulletin 36, 227–324.
Bornet, B., Goraguer, F., Branchard, M., 2002. Genetic diversity in European andArgentinean cultivated potatoes (Solanum tuberosum subsp tuberosum) detectedby inter-simple sequence repeats (ISSR). Genome 45, 481–484.
Borowsky, R.L., McClelland, M., Cheng, R., Welsh, J., 1995. Arbitrarily primed DNA fin-gerprinting for phylogenetic reconstruction in vertebrates: Xiphophorus model.Molecular Biology and Evolution 12, 1022–1032.
Britton-Davidian, J., Vahdati, M., Benmehdi, F., Gros, P., Nancé, V., Croset, H.,Guerassimov, S., Triantaphyllidis, C., 1991. Genetic differentiation in fourspecies of Apodemus from southern Europe: A. sylvaticus, A. flavicollis, A. agrar-ius and A. mystacinus (Muridae Rodentia). Zeitschrift für Säugetierkunde 56,25–33.
Bugarski-Stanojevic, V., Blagojevic, J., Adna –devic, T., Jojic, V., Vujosevic, M., 2008.Molecular phylogeny and distribution of three Apodemus species (MuridaeRodentia) in Serbia. Journal of Zoological Systematics and Evolutionary Research46, 278–286.
Bugarski-Stanojevic, V., Blagojevic, J., Stamenkovic, G., Adna –devic, T., Gia-Gia-Athanasopoulou, E.B., Vujosevic, M., 2011. Comparative study ofthe phylogenetic structure in six Apodemus species (Mammalia Roden-tia) inferred from ISSR-PCR data. Systematics and Biodiversity 9 (1),95–106.
Chelomina, G.N., Suzuki, H., Tsuchiya, K., Moriwaki, K., Lyapunova, E.A., Vorontsov,N.N., 1998. Sequencing of the mtDNA cytochrome b gene and reconstructionof the maternal relationships of wood and field mice of the genus Apodemus(Muridae Rodentia). Russian Journal of Genetics 34, 529–539.
Chelomina, G.N., Atopkin, D.M., Bogdanov, A.S., 2007. Phylogenetic relationshipsbetween species and intraspecific forms of forest mice from the genus Sylvaemusas determined by partial sequencing of the cytochrome b gene of mitochondrialDNA. Doklady Biological Sciences 416, 356–359.
Cobb, B.D., Clarkson, J.M., 1994. A simple procedure for optimizing the polymerasechain reaction (PCR) using modified Taguchi methods. Nucleic Acids Research22, 3801–3805.
C olak, R., C olak, E., Yigit, N., Kandemir, I., Sözen, M., 2007. Morphometric and bio-chemical variation and the distribution of the genus Apodemus (Mammalia:Rodentia) in Turkey. Acta Zoologica Academiae Scientiarum Hungaricae 53 (3),239–256.
Dubey, S., Michaux, J.R., Brünner, H., Hutterer, R., Vogel, P., 2009. False phylogenies onwood mice due to cryptic cytochrome-b pseudogene. Molecular Phylogeneticsand Evolution 50, 633–641.
Ðulic, B., Tvrtkovic, N., 1974. Some problems of taxonomy of Apodemus sylvati-cus Linnaeus, 1758 and Apodemus flavicollis Melchior, 1834 in Yugoslavia. In:Kratochvíl, J., Obrtel, R. (Eds.), Symposium Theriologicum II. Proceedings of theInternational Symposium on Species and Zoogeography of European Mammals.Academia, Praha, Brno, pp. 183–189.
Engel, W., Vogel, W., Voiculescu, I., Ropers, H.H., Zenzes, M.T., Bender, K., 1973. Cyto-genetic and biochemical differences between Apodemus sylvaticus and Apodemusflavicollis, possibly responsible for the failure to interbreed. Comparative Bio-chemistry and Physiology 4, 1163–1173.
Filippucci, M.G., Cristaldi, M., Tizi, L., Conotli, L., 1984. Dati morfologici e morfo-metrici in popolazioni di Apodemus (Sylvaemus) dell’Italia centro-meridionaledeterminati elettroforeticamente. In: Contoli, L., Cristaldi, M., Filippucci, M.G.,Tizi, L., Vigna-Taglianti, A. (Eds.), Recenti acquisizionisul genere Apodemus inItalia. Supplemento alle Ricerche di Biologia della Selvaggina IX. Bologna, pp.85–126.
Filippucci, M.G., Simson, S., Nevo, E., 1989. Evolutionary biology of the genus Apode-mus Kaup, 1829 in Israel Allozymic and biometric analyses with description ofa new species: Apodemus hermonensis (Rodentia, Muridae). Italian Journal ofZoology 56, 361–376.
Filippucci, M.G., Storch, G., Macholán, M., 1996. Taxonomy of the genus Sylvaemusin western Anatolia – morphological and electrophoretic evidence. Senckenber-giana Biologica 75, 1–14.
Filippucci, M.G., Macholán, M., Michaux, J.R., 2002. Genetic variation and evolutionin the genus Apodemus (Muridae: Rodentia). Biological Journal of the LinneanSociety 75, 395–419.
Frynta, D., Mikulová, P., Vohralík, V., 2006. Skull shape in the genus Apodemus: phy-
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
logenetic conservatism and/or adaptation to local conditions. Acta Theriologica51, 139–153.
Hirning, U., Schulz, W.A., Just, W., Adolph, S., Vogel, W., 1989. A comparative studyof the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis. Chro-mosoma 8, 450–455.
oofer, S.R., Gaschak, S., Dunina-Barskovskaya, Y., Makluk, J., Meeks, H.N., Wickliffe,J.K., Baker, R.J., 2007. New information for systematics, taxonomy, and phylo-geography of the rodent genus Apodemus (Sylvaemus) in Ukraine. Journal ofMammalogy 88, 330–342.
anzekovic, F., Krystufek, B., 2004. Geometric morphometry of the upper molars inEuropean wood mice Apodemus. Folia Zoologica 53 (1), 47–55.
ojic, V., Nenadovic, J., Blagojevic, J., Paunovic, M., Cvetkovic, D., Vujosevic, M.,2012. Phenetic relationships among four Apodemus species (Rodentia Muridae)inferred from skull variation. Zoologische Anzeiger 251, 26–37.
ochieva, E.Z., Ryzhova, N.N., Khrapalpva, I.A., Pukhalskyi, V.A., 2002. Genetic diver-sity and phylogenetic relationships in the genus Lycopersicon (Tourn) Mill asrevealed by intersimple sequence repeat (ISSR) analysis. Russian Journal ofGenetics 38, 958–966.
rystufek, B., Janzekovic, F., 2005. Relative warp analysis of cranial and upper molarshape in rock mice Apodemus mystacinus sensu lato. Acta Theriologica 50 (4),493–504.
rystufek, B., Luznik, M., Buzan, E.V., 2012. Mitochondrial cytochrome b sequencesresolve taxonomy of field mice (Apodemus) in the western Balkan refugium. ActaTheriologica 57 (1), 1–7.
ing, Y., Lu, W.-F., Lu, F., Wang, Y.-G., Chen, J.-K., Zhang, W.-J., 2006. PCR-RFLP andAP-PCR of rbcL and ITS of rDNA show that Taxodiomeria peizhongii (Taxodium,Cryptomeria) is not an intergeneric hybrid. Journal of Integrative Plant Biology48, 468–472.
acholán, M., Filippucci, M.G., Benda, P., Frynta, D., Sádlová, J., 2001. Allozyme vari-ation and systematics of the genus Apodemus (Muridae, Rodentia) in Asia Minorand Iran. Journal of Mammalogy 82 (3), 799–813.
aniatis, T., Fritsch, E., Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
atsubara, K., Nishida-Umehara, C., Tsuchiya, K., Nukaya, D., Matsuda, Y., 2004.Karyotypic evolution of Apodemus (Muridae Rodentia) inferred from compar-ative FISH analyses. Chromosome Research 12, 383–395.
cClelland, M., Welsh, J., 1994. DNA fingerprinting by arbitrarily primed PCR. PCRMethods and Applications 4, 59–65.
ezhzherin, S.V., Lashkova, E.I., 1992. Two closely related mice species – Sylvaemussylvaticus and S. flavicollis (Rodentia, Muridae) in an area of their overlappingoccurrence. Vestnik Zoologii 26 (3), 33–41.
ezhzherin, S.V., Lashkova, E.I., Tovpinets, N.N., 2002. Geographic distribution, pop-ulation densities and habitat preference of the wood mice genus Sylvaemus(Rodentia, Muridae) on the Territory of Ukraine. Vestnik Zoologii 36 (6), 39–49.
ichaux, J.R., Filippucci, M.G., Libois, R.M., Fons, R., Matagne, R.F., 1996. Biogeogra-phy and taxonomy of Apodemus sylvaticus (the woodmouse) in the Tyrrhenianregion: enzymatic variations and mitochondrial DNA restriction pattern analy-sis. Heredity 76, 267–277.
ichaux, J.R., Libois, R., Ramalhinho, M.G., Maurois, C., 1998. On the mtDNArestriction patterns variation of the Iberian wood mouse (Apodemus sylvati-cus) Comparison with other west Mediterranean populations. Hereditas 129,187–194.
ichaux, J.R., Kinet, S., Filippucci, M.G., Libois, R., Besnard, A., Catzeflis, F., 2001.Molecular identification of three sympatric species of wood mice (Apodemus syl-vaticus, A flavicollis, A alpicola) in western Europe (Muridae, Rodentia). MolecularEcology Notes 1, 260–263.
ichaux, J.R., Chevret, P., Filippucci, M.G., Macholán, M., 2002. Phylogeny of thegenus Apodemus with a special emphasis on the subgenus Sylvaemus using thenuclear IRBP gene and two mitochondrial markers: cytochrome b and 12S rRNA.Molecular Phylogenetics and Evolution 23, 123–136.
ichaux, J.R., Magnanou, E., Paradis, E., Nieberding, C., Libois, R.M., 2003. Mitochon-drial phylogeography of the woodmouse (Apodemus sylvaticus) in the Western
Please cite this article in press as: Bugarski-Stanojevic, V., et al., Identificati(Rodentia, Muridae)—Comparison of molecular methods. Zool. Anz. (2012)
Palearctic region. Molecular Ecology 12, 685–697.ichaux, J.R., Libois, R., Filippucci, M.G., 2005. So close and so different: comparative
phylogeography of two small mammal species, the yellow-necked fieldmouse(Apodemus flavicollis) and the woodmouse (Apodemus sylvaticus) in the WesternPalearctic region. Heredity 94, 52–63.
PRESSher Anzeiger xxx (2012) xxx– xxx 9
Mitchell-Jones, A.J., Amori, G., Bogdanowitz, W., Krystufek, B., Reijnders, P.J.H.,Spitzenberger, F., Stubbe, M., Thissen, J.B.M., Vohralík, V., Zima, J., 1999. TheAtlas of European Mammals. Poyser Ltd., London.
Musser, G.G., Brothers, E.M., Carleton, M.D., Hutterer, R., 1996. Taxonomy and dis-tributional records of Oriental and European Apodemus, with a review of theApodemus–Sylvaemus problem. Bonner Zoologische Beiträge 46, 143–190.
Niethammer, J., 1969. Zur Frage der Introgression bei den Waldmäusen Apodemussylvaticus und A. flavicollis (Mammalia, Rodentia). Zeitschrift fuer ZoologischeSystematik und Evolutionsforschung 7, 77–127.
Orlov, V.N., Bulatova, N.Sh., Nadjafova, R.S., Kozlovsky, A.I., 1996. Evolutionary clas-sification of European wood mice of the subgenus Sylvaemus based on allozymeand chromosome data. Bonner Zoologische Beiträge 46, 191–202.
Petrov, B.M., 1992. Mammals of Yugoslavia – Insectivores and Rodents. NaturalHistory Museum, Supplementa 37, Belgrade.
Prieto, C., Jara, C., Mas, A., Romero, J., 2007. Application of molecular methods foranalysing the distribution and diversity of acetic acid bacteria in Chilean vine-yards. International Journal of Food Microbiology 115, 348–355.
Renaud, S., Michaux, J.R., 2003. Adaptive latitudinal trends in the mandible shape ofApodemus wood mice. Journal of Biogeography 30, 1617–1628.
Reutter, B.A., Nová, P., Vogel, P., Zima, J., 2001. Karyotypic variation between woodmouse species: banded chromosomes of Apodemus alpicola and A. microps. ActaTheriologica 46, 353–362.
Rovatsos, M.T., Mitsainas, G.P., Tryfonopoulos, G.A., Stamatopoulos, C., Gia-GiaAthanasopoulou, E.B., 2008. A chromosomal study on Greek populations of thegenus Apodemus (Rodentia, Muridae) reveals new data on B chromosome dis-tribution. Acta Theriologica 53 (2), 157–167.
Roy, S.C., Chakraborty, B.N., 2009. Genetic diversity and relationships among tea(Camellia sinensis) cultivars as revealed by RADP and ISSR based fingerprinting.Indian Journal of Biotechnology 8, 370–376.
Serizawa, K., Suzuki, H., Tsuchiya, K., 2000. A phylogenetic view on species radia-tion in Apodemus inferred from variation of nuclear and mitochondrial genes.Biochemical Genetics 38, 27–40.
Sumner, A.T., 1972. A simple technique for demonstrating centromeric heterochro-matin. Experimental Cell Research 75, 166–168.
Suzuki, H., Filippucci, M.G., Chelomina, G.N., Sato, J.J., Serizawa, K., Nevo, E., 2008. Abiogeographic view of Apodemus in Asia and Europe inferred from nuclear andmitochondrial gene sequences. Biochemical Genetics 46, 329–346.
Todorovic, M., Mikes, M., Savic, I., 1971. Biometric analysis of the genus Apode-mus in Vojvodina (Yugoslavia). In: Kratochvíl, J., Obrtel, R. (Eds.), SymposiumTheriologicum II. Proceedings of the International Symposium on Species andZoogeography of European Mammals. Academia, Praha, Brno, pp. 173–181.
Tyler, K.D., Wang, G., Tyler, S.D., Johnson, W.M., 1997. Factors affecting reliabilityand reproducibility of amplification-based DNA fingerprinting of representativebacterial pathogens. Journal of Clinical Microbiology 35, 339–346.
Vidigal, T.H.D.A., Dias Neto, E., Spatz, L., Nunes, D.N., Pires, E.R., Simpson, A.J.G., Car-valho, O.S., 1998. Genetic variability and identification of the intermediate snailhosts of Schisostoma mansoni. Memorias do Instituto Oswaldo Cruz 93, 103–110.
Vujosevic, M., Rimsa, D., Zivkovic, S., 1984. Patterns of G- and C-bands distributionon chromosomes of three Apodemus species. Zeitschrift für Säugetierkunde 49,234–238.
Wang, H.Z., Wu, Z.X., Lu, J.J., Shi, N.N., Zhao, Y., Zhang, Z.T., Liu, J.J., 2009. Moleculardiversity and relationships among Cymbidium goeringii cultivars based on inter-simple sequence repeat (ISSR) markers. Genetica 136, 391–399.
Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitraryprimers. Nucleic Acids Research 18, 7213–7218.
Wilson, D.E., Reeder, D.M., 2005. Mammal Species of the World: A Taxonomic andGeographic Reference, 3rd ed. John Hopkins University Press, Baltimore.
Zagorodnyuk, I.V., 1996. Sibling species of mice from Eastern Europe: taxonomy,diagnostics and distribution. Reports of the National Academy of Sciences ofUkraine 12, 166–173.
on of the sibling species Apodemus sylvaticus and Apodemus flavicollis, http://dx.doi.org/10.1016/j.jcz.2012.11.004
Zietkiewicz, E., Rafalski, A., Labuda, D., 1994. Genome fingerprinting by sim-ple sequence repeat (SSR)-anchored polymerase chain reaction amplification.Genomics 20, 176–183.
Zima, J., Král, B., 1984. Karyotypes of European mammals II. Acta Scientiarum Natu-ralium Academiae Scientiarum Bohemicae Brno 18 (8), 1–62.
![[B chromosome morphotypes of Apodemus peninsulae (Rodentia) from the Russian Far East]](https://static.documents.pub/doc/80x56/636204bf7cfdc0c82205cd6d/b-chromosome-morphotypes-of-apodemus-peninsulae-rodentia-from-the-russian-far.jpg)
