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The identification of field mice Apodemus flavicollis, Apodemus sylvaticus, and Apodemus alpicola represents a challenge for field scientists due to their highly overlapping morphological traits and habitats. Here, we propose a new fast real‐time PCR method to discriminate the three species by species‐specific TaqMan assays. Primers and probes were designed based on the alignment of 54 cyt‐b partial sequences from 25 different European countries retrieved from GenBank. TaqMan assays were then tested on 133 samples from three different areas of Italy. Real‐time PCR analysis showed 92 samples classified as A. flavicollis, 13 as A. sylvaticus, and 28 as A. alpicola. We did not observe any double amplification and DNA sequencing confirmed species assignment obtained by the TaqMan assays. The method is implementable on different matrices (ear tissues, tail, and blood). It can be used on dead specimens or on alive animals with minimally invasive sampling, and given the high sensitivity, the assay may be also suitable for degraded or low‐DNA samples. The method proved to work well to discriminate between the species analyzed. Furthermore, it gives clear results (amplified or not) and it does not require any postamplification handling of PCR product, reducing the time needed for the analyses and the risk of carryover contamination. It therefore represents a valuable tool for field ecologists, conservationists, and epidemiologists.

Field identification of European wood mice Apodemus spp. is challenging due to their morphological resemblance and frequent sympatry. We developed discriminant functions based on body mass and acoustic variables of distress calls to identify three cryptic species of wood mice (Apodemus alpicola, Apodemus flavicollis and Apodemus sylvaticus) in Italy. We achieved an overall correct classification rate of 86–98%; the best results (100% correct classification) were obtained for Apodemus sylvaticus calls. This minimally invasive, effective and low-cost method highlights the potential role of bioacoustics as a powerful tool for field discrimination of cryptic species of terrestrial mammals.

Aim, Location Although the alpine mouse Apodemus alpicola has been given species status since 1989, no distribution map has ever been constructed for this endemic alpine rodent in Switzerland. Based on redetermined museum material and using the Ecological-Niche Factor Analysis (ENFA), habitat-suitability maps were computed for A. alpicola, and also for the co-occurring A. flavicollis and A. sylvaticus. Methods In the particular case of habitat suitability models, classical approaches (GLMs, GAMs, discriminant analysis, etc.) generally require presence and absence data. The presence records provided by museums can clearly give useful information about species distribution and ecology and have already been used for knowledge-based mapping. In this paper, we apply the ENFA which requires only presence data, to build a habitat-suitability map of three species of Apodemus on the basis of museum skull collections. Results Interspecific niche comparisons showed that A. alpicola is very specialized concerning habitat selection, meaning that its habitat differs unequivocally from the average conditions in Switzerland, while both A. flavicollis and A. sylvaticus could be considered as generalists in the study area.

Genetic variation was studied using protein electrophoresis of 28–38 gene loci in 1347 specimens of Apodemus agrar-ius, A. peninsulae, A. flavicollis, A. sylvaticus, A. alpicola, A. uralensis, A. cf. hyrcanicus, A. hermonensis, A. m. mystacinus and A. m. epimelas, representing 121 populations from Europe, the Middle East, and North Africa. Mean values of heterozygosity per locus for each species ranged from 0.02 to 0.04. Mean values of Nel's genetic distance (D) between the taxa ranged from 0.06 (between A. flavicollis and A. alpicola) to 1.34 (between A. uralensis and A. agrarius). The highest values of D were found between A. agrarius and other Apodemus species (0.62-1.34). These values correspond to those generally observed between genera in small mammals. Our data show that A. agrarius and A. peninsulae are sister species, well-differentiated from other taxa. High genetic distance between A. m. mystacinus and A. m. epimelas leads us to consider them distinct species and sister taxa to other Western Palaearctic species of the subgenus Sylvaemus. The data also suggest a recent separation of members of the latter group from a common ancestor, and subsequent rapid radiation, making it difficult to infer phylogenetic relationships. Some taxonomic implications of the results are discussed further.© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 395–419.

The ability of a PCR-based restriction fragment length polymorphism (RFLP) analysis of the cytochrome b (mtDNA) to distinguish Apodemus alpicola from two other Apodemus species was investigated. The partial sequencing of the cytochrome b allowed the identification of one enzyme as being potentially diagnostic. This was supported by an analysis of 131 specimens previously identified using morphometric and/or allozymic data, indicating that the PCR-based RFLP method provides a rapid and reliable tool for distinguishing A. alpicola from its two co-occurring congenerics. The method is applicable to samples taken in the field for ecological studies, and could easily be adapted to the identification of museum samples.

The banding pattern (G-, C-, AgNOR-staining) was described in karyotypes ofApodemus alpicola Heinrich, 1952 andA. microps Kratochvil et Rosicky, 1952 collected from the Alps and central Europe. Distinct differences between the two species were revealed in the distribution of C-heterochromatic regions in autosomes and the sex chromosomes, and the distribution of nucleolar organizer regions (NORs). Extensive variation in the distribution pattern of C-heterochromatin and NORs obviously exists among the wood mice of the subgenusSylvaemus, and individual species can be distinguished according to a specific variation pattern. However, it seems premature to designate individual karyotypic forms as separate species, because the extent of overall geographical interpopulation variation is still not sufficiently known.

A total of 108 Apodemus skulls from Switzerland, Austria, Italy, France and Germany was studied to determine morphological characteristics useful in identifying individuals as Apodemus sylvaticus (Linnaeus, 1758), A. flavicollis (Melchior, 1834) or A. alpicola Heinrich, 1952. The original assignment of the samples to the three species was based on molar cusp morphology, body proportions, pelage coloration, and allozyme analysis. The 24 measured cranial characters used together accurately discriminated between the three species and correctly classified 100% of the individuals to species. A stepwise discriminant function analysis showed that 6 cranial characters are sufficient to differentiate between the three species, with a correct classification above 97%. Fisher's linear discriminant function coefficients can be used directly for classification of unknown specimens.

Apodemus alpicola Heinrich, 1952. J. Mammal., 33:260. TYPE LOCALITY: Allgäu, Osterachtal, S Germany. DISTRIBUTION: NW parts of the Alps: S Germany, Austria, Liechtenstein, Switzerland, and N Italy. SYNONYMS: alpinus. COMMENTS: Subgenus Sylvaemus. Reviewed by Storch and Lütt (1989) who noted that S. alpicola occured syntopically with A. sylvaticus and A. flavicollis. The specific integrity of alpicola was biochemically confirmed by Vogel et al. (1991)., Published as part of Guy G. Musser & Michael D. Carleton, 1993, Order Rodentia - Family Muridae, pp. 501-755 in Mammal Species of the World (2 nd Edition), Washington and London :Smithsonian Institution Press on page 570, DOI: 10.5281/zenodo.7353098, {"references":["Storch, G., and O. Lutt. 1989. Artstatus der Alpenwaldmaus, Apodemus alpicola Heinrich, 1952. Zeitschrift fur Saugetierkunde, 54: 337 - 346.","Vogel, P., T. Maddalena, A. Mabille, and G. Paquet. 1991. Confirmation biochimique du statut specifique du mulot alpestre Apodemus alpicola Heinrich, 1952 (Mammalia, Rodentia). Bulletin Societie Vaudoise des Sciences Naturelles, 80: 471 - 481."]}