The use of PCR-RFLP as an identification tool for three closely related species of rodents of the genus Akodon ( Sigmodontinae , Akodontini )

Three cryptic species of the rodent Akodon (A. cursor, A. montensis and Akodon sp.) were analyzed. The two former species are sympatric in the Brazilian states of São Paulo, Rio de Janeiro and Minas Gerais, where hybrids have already been found. The third species, Akodon sp., occurs in an isolated area in Central Brazil. The identification of these species is difficult by the need of living animals. At present, karyotyping is the only method used in the identification of specimens. We used PCR-RFLP of the mitochondrial cytochrome gene to test the distinctiveness of the three species, which was confirmed by the absence of shared species-specific haplotypes. We also detected a geographical pattern of haplotypes distribution with highly polymorphic populations of A. cursor from Espírito Santo and of A. montensis from Rio Grande do Sul.

Akodon Meyen, 1833 (Muridae, Sigmodontinae) is widely distributed throughout South America (Smith and Patton 1993) and is one of the richest genus of sigmodontines, with around 41 species (Musser and Carleton 2005).The taxonomy of the genus is complex because the limits of some species are not clear and morphology is not always useful in distinguishing related species.One specific case regards a group of three species of Akodon from Brazil, the so-called Akodon cursor species group, composed of A. cursor Winge, 1887;A. montensis Thomas, 1913; and an undescribed species Akodon sp.
For a long time, A. montensis was considered a junior synonym of A. cursor (Musser and Carleton, 2005) because of their morphological similarity in integumental, cranial and dental features (Ximenez and Langguth 1970).The re-current misidentification of A. montensis and A. cursor was an issue in the taxonomy of Atlantic species of Akodon, which were basically defined by their karyotypes: 2n = 14-16 in A. cursor and 2n = 24-26 in A. montensis.Currently, A. montensis is recognized as a species with 2n = 24-26, distributed from the Atlantic coast of Rio de Janeiro, Minas Gerais throughout southern Brazil, Uruguay, Misiones in Argentina and eastern Paraguay (Ximenez and Langguth 1970, Yonenaga-Yassuda 1979, Liaschovich and Reig 1989, Rieger et al. 1995, Geise et al. 2001, Pardiñas et al. 2003).
Both species are simpatric in the states of Rio de Janeiro, Minas Gerais, São Paulo and in northern Paraná.Natural hybrids have been found in São Paulo (Fagundes et al. 1997a, b) and sterile hybrids have been produced in the lab (Yonenaga et al. 1975).Christoff (2002) used external anatomy and 20 cranium-dental features to determine the morphological characteristics of A. cursor and A. montensis.This author concluded that no external or cranio-dental feature could be used to discriminate between both species, which could only be achieved through karyotyping.Geise et al. (2004) suggested that the absence of a gall bladder in A. montensis could be a good discriminating feature between this species and A. cursor but they noted the usefulness of karyotyping for species identification.There is no information about the presence of a gall bladder in Akodon sp.
A. cursor presents 2n = 14 to 16 with polymorphisms due to autosomes fusions and inversion (Fagundes et al. 1997a(Fagundes et al. , 1998)). A. montensis has 2n = 24, 25 and 26 and polymorphisms resulting from variable numbers of supernumerary chromosomes (Yonenaga-Yassuda 1979, Rieger et al. 1995, Fagundes et al. 1997b).Akodon sp., the third species of the A. cursor species group, presents 2n = 10 (Silva and Yonenaga-Yassuda 1998) and was collected in Mato Grosso State, Central Brazil, in a transitional area between the Amazonian forest and the Cerrado.This species is morphologically undistinguishable from the other two (A.U.Christoff, unpublished data).
The validity of the species status for these three taxons is questioned because they share a similar morphology, and two of them are sympatric in part of their distribution area.Karyotyping is currently the only way to discriminate each of these three species.
The major aim of this study was to develop molecular markers that would allow to distinguish these three Akodon species and thus avoid misidentifications.The PCR-RFLP is a rapid, easy and cheap technique used in species identification.Although it is a low cost technique, karyotyping requires the sacrifice of live animals or the establishment of cell cultures, which may be expensive and sometimes unfeasible due to ecological issues.DNA is easily obtained from blood, muscle, skin, ear or tail biopsies from living animals.The presence of distinct haplotypes in A. cursor, A. montensis and Akodon sp would allow their use for taxonomy and to genetically discriminate the three species, which would then be considered as full species under the biological species concept.
In this work, 78 samples were used: 50 samples of Akodon cursor from 12 localities, 23 of A. montensis from six localities, and five of Akodon sp from two localities.Specimens of A. cursor and A. montensis were from the Atlantic rainforest and specimens of Akodon sp. were from a transitional area between the Amazonian forest and the Cerrado (Table S1).The samples used are deposited at the Tissue Collection of "Laboratório de Genética Animal" at Universidade Federal do Espírito Santo (LGA-UFES) and "Laboratório de Citogenética de Vertebrados" at Universidade de São Paulo (CIT-USP).
Total genomic DNA was extracted from liver or muscle preserved in 70% ethanol following Bruford et al. (1992).Polymerase Chain Reaction (PCR) was used to amplify the complete sequence of the cytochrome b mitochondrial gene (1140 bp), with primers MVZ 05 (5'-CGAA GCTTGATATGAAAAACCATCGTTG-3') and MVZ 14 (5'-GGTCTTCATCTYHGGYTTACAAGAC-3') (Smith andPatton, 1991, 1993).PCR amplifications were carried out in a 50 μL reaction mix containing 1xTaq buffer, 3.0 mM MgCl 2, 0.4 mM of each dNTP, 0.4 mM of each primer, 0.6 U Platinum Taq DNA polymerase (Invitrogen) and 50 ng of DNA.PCR cycles were one cycle at 92 °C for 5 min, 37 cycles of 92 °C for 1 min, 47 °C for 1 min and 72 °C for 1 min.A final extension at 72 °C for 5 min was performed to completely extend the amplified product.After amplification, the PCR products were checked by electrophoresis in a 1% agarose gel and fragment sizes were estimated using a 100 bp or a 1 kb marker ladder (Invitrogen).
For PCR-RFLP analysis, the 1140 bp PCR products were digested with seven restriction endonucleases which recognize four to seven base pairs (AluI, BsaI, HaeIII, MboI HinfI, RsaI and TaqI, Invitrogen).Restriction fragments were separated by electrophoresis in a 5% acrylamide gel and their sizes were estimated using the molecular markers.The results were taken into account when the sum of all the restriction fragments for each enzyme were in the range of 1140bp ± 100.Bands generated for the same enzyme that migrated similarly in the same gel were considered homologous.
Each set of bands obtained after the digestion of the PCR product by each enzyme (the cleavage pattern) was at- tributed a letter.Some enzymes showed more than one cleavage pattern (polymorphisms).The mtDNA haplotype of each individual was defined as the cleavage patterns obtained with all of the enzymes used.
Four of the seven tested restriction endonucleases were considered informative (AluI, HaeIII HinfI and MboI), i.e., they generated species-specific band patterns (Table 1).The informative cleavage patterns were: AluI, four cleavage patterns for A. cursor Some of the cleavage patterns were shared by A. cursor and A. montensis, but the haplotypes (the cleavage patterns of all four enzymes) were distinct for each of the three species (Table 2).Haplotypes 1 to 13 belonged to A. cursor, 14 to 24 occurred in A. montensis and haplotype 25 was found in Akodon sp.Some intraspecific variability was detected in A. cursor and A. montensis.A phylogeographic pattern was observed with particular A. cursor haplotypes related to specific geographic areas: haplotypes 1 and 2 in the northern localities of Pernambuco and Bahia, haplotypes 3 to 13 in the southern localities of Espírito Santo and São Paulo.The haplotypes of A. montensis from the southern population of Rio Grande do Sul (haplotypes 17, 18, 19, 20, 21, 22, 23 and 24) were not shared by the northern populations of São Paulo (haplotypes 14, 15 and 16).This pattern of geographic variation may reflect hitherto unknown molecular differences between populations.This contrasts with results from karyologic and morphometric analyses that revealed heterogeneous population patterns across the distribution of A. cursor in Espírito Santo with ten haplotypes versus three in São Paulo and of A. montensis in Rio Grande do Sul with eight haplotypes versus three in São Paulo (Table 2).
The fact that all 25 haplotypes were species-specific (Table 2) and that only few cleavage patterns were shared by A. cursor and A. montensis (B for AluI, A, B and C for HinfI and A for MboI) indicates that the three species are genetically divergent, although the time since speciation was not sufficient to allow morphological differences to accumulate.
This methodology can be widely used to identify non-karyotyped animals (which represent over 80% of the non-identified specimens) from museums or private collections, if tissue samples are preserved in ethanol or liquid nitrogen.Identification of ancient specimens without tissue samples remains a difficult task, since only highly degraded DNA is obtainable from the skins kept in museums.
Our main conclusions are that molecular data support the hypothesis that A. cursor, A. montensis and Akodon sp.   are distinct and isolated species and allow to easily distinguishing each of the three Akodon species.
(A, B, C and D); four patterns for A. montensis (B, E, F and G) and one pattern for Akodon sp.(H); HaeIII, seven cleavage patterns for A. cursor (A, B, C, D, E, F and G); two for A. montensis (H and I) and one for Akodon sp.(J); HinfI, four cleavage patterns for A. cursor (A, B, C and D); four patterns for A. montensis (A, B, C and E) and one for Akodon sp (F).; and MboI, six cleavage patterns for A. cursor (A, B, C, D, E and F); seven for A. montensis (A, G, H, I, J and K) and one for Akodon sp (L).

Table 1 -
Cleavage patterns (identified by upper cases in the left) of four restriction endonucleases for the three species of Akodon.Band sizes are given in base pairs.

Table 2 -
Collection sites and haplotypes of Akodon cursor, A. montensis and Akodon sp.