Phylogeny of the genus Apodemus with a special emphasis on the subgenus Sylvaemus using the nuclear IRBP gene and two mitochondrial markers: cytochrome b and 12S rRNA

Abstract

Phylogenetic relationships among 17 extant species of Murinae, with special reference to the genus Apodemus, were investigated using sequence data from the nuclear protein-coding gene IRBP (15 species) and the two mitochondrial genes cytochrome b and 12S rRNA (17 species). The analysis of the three genes does not resolve the relationships between Mus, Apodemus, and Rattus but separates Micromys from these three genera. The analysis of the two mitochondrial regions supported an association between Apodemus and Tokudaia and indicated that these two genera are more closely related to Mus than to Rattus or Micromys. Within Apodemus, the mitochondrial data sets indicated that 8 of the 9 species analyzed can be sorted into two main groups: an Apodemus group, with A. agrarius, semotus, and peninsulae, and a Sylvaemus group, with uralensis, flavicollis, alpicola, sylvaticus, and hermonensis. The position of Apodemus mystacinus is ambiguous and might be either included in Sylvaemus or considered a distinct subgenus, Karstomys, more closely related to Sylvaemus than to Apodemus. Estimation of the divergence time for these taxa suggests a separation between 7 and 8 My ago for the three groups (mystacinus and the two subgenera Apodemus and Sylvaemus). Within each subgenus, divergence times are between 5.4 and 6 My for Apodemus and between 2.2 and 3.5 My for Sylvaemus and mystacinus.

Introduction

The speciose genus Apodemus is widespread throughout the Palearctic region. Morphologically, Apodemus seems distinct from both Mus and Rattus, perhaps indicative of an ancient isolation (early Vallesian) of its ancestor (Martin-Suarez and Mein, 1998). Nevertheless, DNA/DNA hybridization data (Catzeflis, 1987; Catzeflis and Denys, 1992; Catzeflis et al., 1993; P. Chevret, unpublished data) and sequence analyses (Martin et al., 2000; Suzuki et al., 2000) tend to associate Apodemus with Mus.

Since the review of Musser et al. (1996) all but one of the Apodemus species have been divided into two subgenera: Sylvaemus (including most of the western Palearctic species) and Apodemus (in which A. agrarius and the ancient eastern Palearctic Alsomys subgenus, excluding A. argenteus, are included). The remaining A. argenteus seems to be distinct from the others. This hypothesis has been confirmed by Serizawa et al. (2000) based on sequences from the mitochondrial cytochrome b and the nuclear IRBP genes. Moreover, these authors proposed a fourth monotypic group, A. gurkha, the Himalayan field mouse.

At present, many questions concerning the phylogenetic relationships both of the genus Apodemus within the Murinae and between the different species within each of the Apodemus subgenera still remain unanswered.

According to several authors (Filippucci et al., 1989, Filippucci et al., 1996; Musser and Carleton, 1993; Musser et al., 1996; Orlov et al., 1996; Vorontsov et al., 1992), 13 different species are presently recognized within the subgenus Sylvaemus in the western Palearctic region: A. sylvaticus, Linnaeus (1758); A. flavicollis, Melchior (1834); A. alpicola, Heinrich (1952); A. uralensis, Pallas (1881); A. mystacinus, Danford and Alston (1877); A. hermonensis, Filippucci et al. (1989); A. fulvipectus, Ognev (1924); A. mosquensis, Orlov et al. (1996); A. ciscaucasicus, Orlov et al. (1996); A. ponticus, Sviridenko (1936); A. hyrcanicus, Vorontsov et al., 1992; A. arianus, Blanford (1881); and A. wardi, Wroughton (1908). Species within the subgenus Sylvaemus are phenotypically very similar, and traditional morphometrics are often at a loss to distinguish between them (Michaux et al., 2002; Zagorodnyuk, 1996). For this reason, several authors have employed protein electrophoresis (Britton-Davidian et al., 1991; Csaikl et al., 1980; Engel et al., 1973; Filippucci, 1992; Filippucci et al., 1996, Filippucci et al., 2002; Gemmeke, 1980; Hartl et al., 1992; Mezhzherin and Zykov, 1991) and traditional cytogenetics (Bulatova et al., 1991; Nadjafova et al., 1993; Zima, 1984) in an attempt to unravel relationships. Unfortunately, similar to morphometrics, cytogenetics were not successful in delimiting species, given the uniformity in karyotypic characteristics of this subgenus. More recently, new molecular methods such as restriction fragment length polymorphism (Chelomina, 1996; Michaux et al., 1996, Michaux et al., 1998a, Michaux et al., 1998b), random amplified polymorphic DNA (RAPD) (Bellinvia et al., 1999), and sequencing of mitochondrial and/or nuclear genes (Chelomina, 1998; Chelomina et al., 1998; Martin et al., 2000; Serizawa et al., 2000) were used with greater success. However, most of these studies included only a limited number of Sylvaemus species and although they gave very interesting results concerning the intraspecific and interspecific phylogenetic relationships within this subgenus, many inconsistencies among the different studies remain.

The aims of the present study were fourfold. First, we wanted to determine the sister group to the genus Apodemus, adding novel 12S rRNA mitochondrial DNA sequences to the already existing data of Serizawa et al. (2000) and Suzuki et al. (2000). In addition, we followed a combined data approach (Kluge, 1989) using three genes (12S rRNA, cytochrome b, and IRBP, available for 15 taxa) in concert. Second, we wanted to clarify the phylogenetic relationships among six European and Near Eastern Sylvaemus species, A. sylvaticus, A. flavicollis, A. uralensis, A. alpicola, A. mystacinus, and A. hermonensis, using new sequences from two mitochondrial regions (cytochrome b and 12S rRNA). Third, we wanted to propose more robust estimations of divergence dates between the different murine genera, the Apodemus subgenera, and the Sylvaemus species. Finally, we wanted to discuss the evolutionary history of this speciose genus.