From Sardinia (Italy)
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Acta Chiropterologica, 4(2): 121135, 2002 PL ISSN 1508-1109 © Museum and Institute of Zoology PAS A new species of long-eared bat (Chiroptera, Vespertilionidae) from Sardinia (Italy) MAURO MUCEDDA1, ANDREAS KIEFER2, ERMANNO PIDINCHEDDA1, and MICHAEL VEITH2 1Centro per lo Studio e la Protezione dei Pipistrelli in Sardegna Gruppo Speleologico Sassarese, Via dei Navigatori 7, 07100 Sassari, Italy; E-mail of MM: [email protected] 2Institute of Zoology, Department of Ecology, Mainz University, Saarstraße 21, D-55099 Mainz, Germany We describe a new species of long-eared bat, genus Plecotus, from the island of Sardinia (Italy). The new species is clearly distinguishable from other European Plecotus species by its mitochondrial 16S rRNA gene (4.19.6% sequence divergence) as well as by a unique combination of morphological characters such as brownish colour of dorsal pelage, a relatively large thumb and thumb claw, an almost cylindrical form of the penis and the characteristic shape of the baculum. The most important morphological diagnostic character is a relatively long (≥ 18 mm) and wide (≥ 6 mm) tragus. The new species is currently known from three localities on Sardinia. In addition to the new species we discovered a lineage of P. auritus, which is substantially differentiated from continental P. auritus at subspecific level (1.22.7% of sequence divergence of the 16S rRNA gene). The existence of these two endemic bat taxa on Sardinia highlights the islands importance in the conservation of the European bat community. Key words: Plecotus sp. nov., Plecotus auritus, Plecotus austriacus, long-eared bat, Sardinia, DNA sequence analysis, 16S rRNA, morphology, conservation INTRODUCTION when applying molecular techniques to bats from Greece. Molecular techniques have greatly en- Recently, two new species of long-eared hanced our understanding of the evolution- bats (genus Plecotus) were discovered in ary relationships of organisms and applica- Europe. Species rank was assigned to P. ko- tion to European bats has recently enabled lombatovici Dulic, 1980, formerly consid- the detection of cryptic species. Two dis- ered a subspecies of P. austriacus Fischer, tinct phonic types (Weid and von Helversen, 1829 (Mayer and von Helversen, 2001), and 1987) of the widespread pipistrelle Pipi- a formerly unknown cryptic species P. alpi- strellus pipistrellus (Schreber, 1774), corre- nus Kiefer and Veith, 2002 (= P. micro- sponded to genetically well-defined lineag- dontus Spitzenberger, 2002 see Kock, es that are differentiated at the species level 2002) was discovered in the Alps and adja- (Jones and Parijs, 1993; Barratt et al., 1997; cent mountains of Southern Europe. Mayer and von Helversen, 2001). Within Knowledge of the Sardinian bat fauna European Myotis mystacinus group, the is currently expanding. The Centre for the morphologically cryptic Myotis alcathoe Study and Protection of Sardinian Bats (von Helversen et al., 2001) was discovered (Centro per lo Studio e la Protezione dei 122 M. Mucedda, A. Kiefer, E. Pidinchedda, and M. Veith Pipistrelli in Sardegna) recently mentioned length (including carpals); F2 = length of the 2nd fin- 18 species: four rhinolophids, 13 vespertil- ger (incl. carpals); F3 = length of 3rd finger (incl. ionids and one molossid (Mucedda et al., carpals); F4 = length of 4th finger (incl. carpals); F5 = length of 5th finger (incl. carpals); HF = hind foot 1999). All of these species also occur on the length (excl. claws); CaL = calcar length; Ear = ear European mainland. length; TL = tragus length (TLBlatt in Häussler and Until 1959, the only species of long- Braun 1991); TW = tragus width; TH = thumb length eared bats (genus Plecotus) known to occur excl. claw; CL = claw length; SL = skull length; CBL on Sardinia was the brown long-eared bat, = condylobasal length; SH = skull height (incl. bul- lae); IOW = interorbital constriction width; M3M3 = P. auritus (Linnaeus, 1758) (Lanza, 1959). width across upper molars; CM3 = length of maxil- Mucedda et al. (2002) reconfirmed its pres- lary toothrow; CM3 = length of mandibular tooth- ence and also recorded the grey long-eared row; M3M3 = width across lower molars; ML = bat, P. austriacus. To better define priorities mandible length; MW = mastoid width; CsupL = for conservation it was desirable to geneti- length of upper canines; MBD = maximal bulla diam- eter; ZW = zygomatic width; MDB = minimal dis- cally compare Sardinian Plecotus to con- tance between bullae; BL = length of baculum; BW = specific populations from the European basal width of baculum. mainland (Mitchell-Jones et al., 1999). The baculum of the holotype was extracted fol- More specifically, we were interested in de- lowing the procedure of Anderson (1960). It was pho- termining if Sardinian Plecotus are geneti- tographed with a Leitz photomicroscope DMRB to cally similar to mainland populations be- obtain the drawing and then measured with the same device to the nearest of 0.01 mm. cause they only recently colonised the is- land, or do they constitute derived, geneti- DNA Extraction and Sequencing cally distinct lineages that deserve special attention for national and international DNA was extracted using QiAmp tissue extrac- wildlife conservation? To clarify the genet- tion kits (Qiagen). Double-stranded PCR was used to ic identity of Sardinian brown and grey amplify mitochondrial DNA fragments. Primers and cycling procedures were as follows: 16SA (light long-eared bats we compared partial mito- chain; 5 - CGC CTG TTT ATC AAA AAC AT - 3) chondrial DNA sequences of Sardinian and 16SB (heavy chain; 5 - CCG GTC TGA ACT specimens to published sequences of all CAG ATC ACG T - 3) of Palumbi et al. (1991) am- currently known European species of Pleco- plified to a ca. 555 bp section of the mitochondrial tus (Kiefer et al., 2002). Here we report the 16S ribosomal RNA gene. PCR cycling procedure was as follows: initial denaturation step: 90 s at 94°C, discovery of two genetically distinct lineag- 33 cycles: denaturation for 45 s at 94°C, primer an- es of Sardinian Plecotus, one of which de- nealing for 45 s at 55°C, extension for 90 s at 72°C. serves recognition at the species level. PCR products were purified using the Qiaquick pu- rification kit (Qiagen). We sequenced single-stranded fragments on an ABI 377 automatic sequencer using MATERIALS AND METHODS standard protocols. We sequenced 555 bp of the 16S rRNA gene that are homologous to the base pair po- Specimens Studied sitions 22152490 of the Pipistrellus abramus com- plete mitochondrial genome (Nikaido et al., 2001). We sampled, under license, 22 specimens of These sequences were aligned to previously pub- long-eared bats from three localities in central Sar- lished sequences of all European Plecotus species dinia (Appendix). Samples for DNA extraction were (GenBank Accession Nos. AY134012134026, obtained from wing tissue, using sterile biopsy punch- AF529229529230 Kiefer et al., 2002) using the es (Worthington Wilmer and Barrett, 1996). We Clustal X software (Thompson et al., 1997). Only dif- recorded the following morphometric (hand-held cal- ferent haplotypes were included in the analysis. For liper measurements to the nearest 0.05 mm) and oth- hierarchical outgroup comparison we included Bar- er morphological characters, according to Stebbings bastella barbastellus (Schreber, 1774) and Myotis (1967) and Häussler and Braun (1991): HB = head- bechsteinii (Kuhl, 1817) (GenBank AF529231 and and-body length; Tail = tail length; FA = forearm AY134027, respectively Kiefer et al., 2002). A new species of long-eared bat from Sardinia 123 Molecular Data Analysis analyses reveal two major clades. The first major clade contains P. kolombatovici and We determined the number and distribution of P. austriacus (incl. the Sardinian samples 3, base substitutions. The amount of phylogenetic signal 6, 9, 10, 11 and 12); the second one com- was assessed by generating 106 random trees and cal- prises P. auritus [incl. the Sardinian sam- culating the skewness (g ) and kurtosis (g ) of the re- 1 2 ples 4, 5, 14, 16 and 17 (haplotypes 4, 5, 14, sulting tree length distribution (with PAUP*, version 4.08b Swofford, 2001). Prior to model assessment 17 see Appendix)], P. alpinus, and a Sar- we performed a χ2-test for base distribution across se- dinian clade consisting of samples 1, 2, 13, quences to rule out non-homogeneous base composi- 15, 20, 21, and 22 (haplotypes 1, 2, and 13). tions that require the use of the paralinear LogDet dis- All clades are supported by bootstrap values tance instead of specific substitution models (Lock- > 90%. Mean substitution rates and TrN hart et al., 1994). Using a hierarchical likelihood ratio distances among lineages of each of the test (LRT), we tested the goodness-of-fit of nested substitution models for homogeneous data partitions two major clades are in the same range (for ingroup taxa only). We used MODELTEST ver- (0.430.54 and 0.0570.067, respectively sion 3.06 (Posada and Crandall, 1998) to determine Table 1). Substitution rates for the 16S a specific substitution model to be used for fur- rRNA gene of ca. 5% correspond to substi- ther analyses. For our 16S rRNA gene a Tamura- tution rates of 1112% for protein coding Nei (TrN) substitution model (Tamura and Nei, mitochondrial genes like ND1, ND2 or cy- 1993) with no invariable sites (I = 0), and among site substitution rate variation with a γ-shape parameter tochrome b (authors own data). The latter = 0.4882 was selected. indicate differentiation at the species level We used the neighbor-joining algorithm (NJ (see Smith and Patton, 1993, and Bradley Saitou and Nei, 1987), applying the selected substitu- and Baker, 2001 for mammals in general, tion model, for phylogenetic tree reconstruction. We and Cooper et al., 2001, and Mayer and von calculated maximum parsimony tree (MP), treating gaps as missing characters and giving equal weight Helversen, 2001 for bats). Consequently, to transitions and transversions (heuristic search with and in accordance with morphological data the TBR branch swapping algorithm).