On the biochemical systematics of three apodemus species

Abstract

-1. Electrophoretic patterns of various proteins were studied in samples of Apodemus silvaticus, Apodemus flavicollis and Apodemus microps populations. 2. According to electrophoretic results Apodemus microps is closer related to Apodemusflavicollis than to Apodemus sylvaticus. 3. From the data of 26 loci, Nei's coefficient of the overall genetic distance between Apodemus flavicollis and Apodemus sylvaticus was calculated and resulted in a divergence time of some 423,000 yr between the two species. I N T R O D U C T I O N Protein electrophoresis has proved to be a powerful tool for resolving systematic problems at the species and sub-species level. The advantages and disadvantages of this method have been discussed (Avise, 1974, 1976; Mayr 1975; Selander 1976). It is the aim of this study, using biochemical markers, to elucidate the systematic relationship between three species of the sub-genus Sylvaemus, a group of mice that are very difficult to classify morphologically, especially at younger age. Zimmermann (1962) has grouped into this sub-genus the three species Apodemus sylvaticus (L.), Apodemus flavicollis (Melchior), and Apodemus microps (Kratochvil et Rosicky). Karyotype analyses (Matthey, 1936, 1962; Kral 1970; Engel et al., 1973) show the close relationship among these species. As yet there is no single morphological feature known to the authors by which these species could be separated. The members of this species triad often can be distinguished only by correlation of some morphological features (Steiner 1968; Csaiki, manuscript in preparation). Together with the systematic results a new catching locality for Apodemus microps is described which seems to represent one of the most westerly points of the distribution of this species. MATERIALS AND METHODS Collection of material All the animals examined were caught live by means of wooden traps in the Steinfeld, a part of the Wiener Becken in Lower Austria. Apodemus sylvaticus (n = 23) and Apodemus microps (n = 2) were caught in the southwest of the village Tattendoff along hedges and windbreaks in the autumn of 1977. Apodemusflavicollis (n = 21) was caught at the same time in the woodlands of the river Leitha near the town Ebenfurt. Ecological studies on the fauna of these biotopes carried out by one of the authors (F.C.) revealed that Apo~ demus microps occurs in low densities and has a very large migration rate. The earlier work of Steiner (1968) also showed for other biotopes the low densities and the unsteady character of this species. 411 This may be the reason that among the normally very successful capture rates of Apodemus sylvaticus only two Apodemus microps individuals were caught. Electrophoretic methods Blood was taken by puncture of the sinus retro-orbitalis following anaesthesia of the animals. An equal volume of a 1.47~o glucose-0.44~ o citric acid-l.32~o sodium citrate solution was added, and after centrifugation at 2000 g for 2 min at 4°C, the supernatant serum was removed with a pipette. and frozen at -20°C. This provided the material for the investigation of serum proteins. To the erythrocytes was added an equal volume of 0.01 M phosphate buffer (pH = 7.4) containing 0.1 mM MgCI2. After freezing and thawing the hemolysates were centrifuged at 20,000 g for 30 min at 4°C. The clear supernatant was absorbed to filter pads and inserted into gel slots. Organs were homogenized in a glass/glass PotterElvenhjem homogenizer using the same phosphate buffer as described above. The samples were treated further as described for the lysate. For electrophoresis four different buffer systems were used: (1) gel: 13Yo starch in 0.006M potassium phosphate, pH = 7,4; bridge: 0.1 M potassium phosphate, pH = 7.4; for lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6-PGD), glucose-6-phosphate dehydrogenase (G-6-PD), adenosine deaminase (ADA), superoxide dismutase (SOD), aspartate aminotransferase (AAT), glyoxalase I (GLO I), NAD-dependent malate dehydrogenase (NAD-MDH), NADP-dependent malate dehydrogenase (NADP-MDH), esterase D (Est D), phosphoglucose isomerase (PGI), haemoglobin (Hb) (2) gel: 12Yo starch in 0.008M Tris-0.003 Citrate. pH = 8.0; bridge: 0.223 M Tris-0.068 M Citrate, pH = 8.0, for NADPH-dependent isocitrate dehydrogenase (NADPIDH). (3) gel: 12~o starch in 0.01 M Tris-0.01 M Malate0.001 M MgCI2, pH = 7.7; bridge: 0.1 M Tris-0.1 Malate0.01 M MgCI2, pH = 7.7, for creatine phosphokinase (CPK), adenylate kinase (AK), aldolase (Ald). (4) gel: 13~o starch in 0.043 M Tris-0.012 Citrate-0.05 M LiOH-0.0015 M Borate, pH = 7.4; bridge: 0.2 M Borate, pH = 8.0, for transferrine (TO and postalbumine (PA). After electrophoresis the gels were sliced and stained for Est D according to Hopkinson et al. (1973), for ADA according to Spencer et al. (1968), for GLO I according to KiSmpf et al. (1975) and according to Shaw & Prasad (1970) for all other enzymes.