Hystrix It. J. Mamm. (n.s.) 21(2) (2010): 115-126 NEW RECORD OF PIGMY FIELD MICE (GENUS APODEMUS, MURIDAE, RODENTIA) FROM NORTHEASTERN IRAN 1 1 JAMSHID DARVISH *, SAFIE AKBARY RAD , ROOHOLLAH SIAHSAR- 2 3 VIE , MOHAMAD ALI HOSEIN POUR FEIZI , 1 FATEMEH GHORBANI 1Rodentology Research Department, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran, and Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran; *Corresponding author, E-mail: [email protected] 2Rodentology Research Department, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran, and Institut des Sciences de l’Evolution, Cc 064, Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France 3Department of Biology, Faculty of Sciences, Tabriz University, Tabriz, Iran Received 17 October 2009; accepted 20 June 2010 ABSTRACT - Field mice from eastern Elborz, Golestan province, were examined using mo- lecular, morphologic and morphometric characters. RFLP analysis showed the presence of three different haplotypes, corresponding to as many species: Apodemus cf. uralensis, A. cf. hyrcanicus and A. witherbeyi. While the two latter species have been previously reported from the studied area, the discovery of A. cf. uralensis in this region extends the eastern border of this species’ distribution. Key words: RFLP, new records, step field mouse, hyrcanian field mouse, pigmy field mouse, northeastern Iran RIASSUNTO - Nuova segnalazione di topo selvatico pigmeo (genere Apodemus) in Iran nord orientale. Topi selvatici dell’Elborz orientale, provincia di Golestan (Iran nord orien- tale) erano esaminati usando caratteri molecolari, morfologici e morfometrici. L’analisi RFLP ha rilevato la presenza di tre aplotipi, corrispondenti a altrettante specie: Apodemus cf. uralensis, A. cf. hyrcanicus e A. witherbeyi. Mentre le due ultime specie sono già state segnalate per l’area di studio, la scoperta di Apodemus cf. uralensis in questa regione am- plia i confini orientali dell’area di distribuzione. Parole chiave: RFLP, topi selvatici, genere Apodemus, nuove segnalazioni, Iran nord orien- tale DOI: 10.4404/Hystrix-21.2-4452 INTRODUCTION the genus Apodemus Kaup 1829, which is widespread in the western Palaearc- So far 79 species of rodents have been tic, are remnants of post glacial refu- reported from Iran (Karami et al., gees. Available information concerning 2008). Among them, the field mice of the taxonomy and range of Apodemus 115 Darvish et al. species in Iran is scarce (Filippucci, Musser and Carleton, 2005; Javidkar et 1992; Mezhzherin, 1997; Frynta et al., al., 2005 and 2007; Darvish et al., 2001; Filippucci et al., 2002; Michaux 2006; Siahsarvie and Darvish, 2008); et al., 2002; Darvish et al., 2006). Cur- A. avicennicus (Darvish et al., 2006), rently, five species have been reported recently described from Yazd province, from Iran (Musser and Carleton, 2005; central Iran (Darvish et al., 2006). Karami et al., 2008): A. flavicollis With the aim of improving the know- (Melchior, 1834) from the Zagros ledge about the geographic distribution Mountains (W Iran); A. hyrcanicus of field mice in northeastern Iran, we i) (Vorontsov et al., 1992), from the Hyr- applied a PCR-based restriction frag- canian forests along the southern bor- ment length polymorphism (RLFP) der of the Caspian Sea and, eastward, method for distinguishing the species up to Dasht (Bulatova et al., 1991; of the genus Apodemus by the analysis Musser and Carleton, 2005; Javidkar et of cytochrome b (mtDNA), and ii) ex- al., 2007); A. uralensis Palas1811, from amined 39 morphological and mor- Makidi in Arasbaran, NW Iran phometric diagnostic characters from (Kryštufek and Hutterer, 2006); A. wi- 13 specimens trapped in Golestan prov- therbyi (Thomas, 1902), from plains, ince. mountain and plateau steppes and high- land semi-deserts of north-eastern Iraq STUDY AREA AND METHODS and most of the central and northern Ira- nian Plateau, including Azerbaijan, Kur- Sampling was performed in three different distan, Lorestaan, Isfahan, Fars, Semnan, localities of Golestan, north-eastern Iran: Tehran, central and eastern Mazandaa- Touskahestan (36º 42' 35N, 54º 35' 19 E), ran, northern and eastern Khorasan and Garmabdasht (36º 42' 12 N, 54º 35' 06 E) Kopet Dagh Mountain (Zagros and El- and Gachian (36 º 43' 36N, 54 º 34' 60 E) burz provinces; Macholan et al., 2001; (Fig. 1). Figure 1 - Study area: 1- Tuskahestan, 2- Garmabdasht, 3- Gachian, 4- Azarbaijan. 116 Pigmy field mouse from Iran Table 1 - Museum code (ZMFUM), haplotype and sampling locality of the three studied species of the genus Apodemus. Species Museum code Haplotype Locality A. witherbyi 1816, 1820, 1822, 1823, 1824, 1825, C Azarbaijan 1826, 1830, 1832, 1839, 1840, 1842, 1855, 1857, 1896, 1897, 1898, 1899, 1900, 1914 1935 Gachian A. hyrcanicus 1797 B Garmabdasht 1860, 1865, 1910 Tuskahestan 1926, 1931 Gachian A. uralensis 1880, 1885, 1906, 1911, 1929, 1932 A Tuskahestan The first two areas are woodland, with oaks PCR products were incubated at 37˚ C for (Quercus spp.), beech (Fagus sylvatica) 3-4 hours to be completely digested by two and maples (Acer spp.), at an altitude of, restriction enzymes: AluI and HinfI. As respectively, 895 and 1045 m a.s.l. Gachian different species produce as many patterns, is a steppic region at 1888 m a.s.l. with to distinguish among species results were junipers (Juniperus sp.) and Sedum sp. analysed by electrophoresis on 1% agarose Molecular, morphological and morphome- gels. tric analyses were carried out on 13 speci- Overall 39 morphological traits and mor- mens. A further 20 individuals from Azer- phometric measurements were considered baijan, NW Iran, were included as a refer- (after Filippucci et al., 1996 and Frynta et al., ence for A. witherbyi (Hossein Pour Feizi et 2001), including in the analyses only adult al., 2009) (Tab. 1). specimens so as to rule out growth and evo- Genomic DNA was extracted from liver or lutionary effects (Frynta and Zizkova, muscle tissues preserved in 98% ethanol, 1992). using Genetbio DNA extraction kit. Stan- The following dental (N = 12), cranial (N = dard voucher specimens (skin and skull) 9) and external (N = 4) measures were were deposited at the Zoology Museum of filed: M1L: first upper molar length, M1W: Ferdowsi University of Mashhad first upper molar width, M2L: second upper (ZMFUM), Iran. Complete cytochrome b molar length, M2W: second upper molar gene was amplified using modified univer- width, M3L: third upper molar length, sal primers L7 (5΄-ACTAATGA- M3W: third upper molar width, LM1L: first CATGAAAAATCATCGTT-3΄) and H6 lower molar length, LM1W: first lower mo- (5΄-TCTTCATTTTTGGTTTACAAGAC- lar width, LM2L: second lower molar 3΄) (Montgelard et al., 2002). Amplifica- length, LM2W: second lower molar width, tions were carried out in a Primus 96 ther- LM3L: third lower molar length, LM3W: mal cycler with an initial denaturation step third lower molar width, RH: rostrum at 94°C for 2 minutes followed by 35 height, ZYGW: zygomatic width, RW: ro- cycles (45s at 94°C, 45s at 50°C and 90s at strum width, IOW: interorbital length, 68°C) and a final extension time of 10 mi- BCW: braincase width, CBL: condylobasal nutes at 68°C (Chevret et al., 2005). The length, BULL: bulla length, UML: upper 117 Darvish et al. molar tooth row length, LML: lower molar Moreover, the variation in size, shape or tooth row length, BL: body length, TL: tail position of 14 further parameters was also length, FL: foot length, EL: ear length. considered (Tab. 2). Table 2 - Variation in size, shape or position of the 14 parameters analysed. 1. Bulla a: massive and well developed b: medium size 2. Angular Process of the a: well developed and wide b: tender and blade shaped Mandible (Fig. 2A) 3. Fronto-Parietal Suture a: V shaped and angled b: U shaped and curved 4. Posterior edge of the Pala- a: curved b: rather straight tine (Fig. 2B) 5. Position of the Incisors a: ortho- b: semiorthodont c: opisthodont (Fig. 2C) dont 6. Connective plan of the c: t3 or t1 with b: t3 or t1 with a d: t3 or t1 are labial Anterocone (t3) and a: no con- a long enamel short enamel horn connected to Anterostyle (t1) to the proto- nection horn towards towards t5 the side of t5 cone (t5) (Fig. 3A). t5 7. Relative position of the a: both in a Enterostyle (t4) and paracone b: t4 is upper c: t4 is lower row (t6) in upper M1 (Fig. 3B) 8. Size of the Anteroconule c: well developed and similar to (t1bis-t3bis) in the upper M1 a: absent b present a real cusp (Fig. 3C). b: relatively smaller than t6; 9. Position of the Metacone a: massive, large and similar to straightly connected to the hypo- (t9) in the upper M1. the paracone (t6) cone (t8) 10. Position of the median a: well developed and similar to Anteroconid (tma) in the low- b: medium size c: Tiny lower cusps er M1 (Fig. 3D). 11. Relative position of tma a: tma is connected to paired labi- and paired Anteroconid cusps b: no connection al and lingual Anteroconid (Fig. 3E). 12. Number of Cingula in the a: 1 b: 2 c: 3 d: 4 lower M1 (Fig. 3F). 13. Number of Cingula in the a: 0 b: 1 c: 2 lower M2 (Fig. 3G). 14. Size of C1 in the lower a: large/me- b: small/tiny c: absent M2 (Fig. 3H). dium 118 Pigmy field mouse from Iran Figure 2 - Morphological states of cranial characters (see the text for explanations). Dental characters were measured by a ste- riance-covariance. The original data were reomicroscope accurate to the nearest 0.01 projected onto the functions defined by the mm. Cranial measurements were taken us- standardized linear discriminant coeffi- ing a caliper accurate to the nearest 0.05 cients to obtain individual scores (Claude, mm.