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Plecotus Macrobullaris Kuzjakin, 1965) in Lombardy (Northern Italy) Revealed by Dna Analysis

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Plecotus Macrobullaris Kuzjakin, 1965) in Lombardy (Northern Italy) Revealed by Dna Analysis 171-176.QXD4 31-08-2005 15:16 Pagina 171 «NATURA BRESCIANA» Ann. Mus. Civ. Sc. Nat. Brescia, 2005, 34: 171-175 FIRST RECORD OF THE ALPINE LONG-EARED BAT (PLECOTUS MACROBULLARIS KUZJAKIN, 1965) IN LOMBARDY (NORTHERN ITALY) REVEALED BY DNA ANALYSIS ILARIA TRIZIO, DAMIANO PREATONI, ROBERTA CHIRICHELLA, SABRINA MATTIROLI, MOSÈ NODARI, SIMONE CREMA, GUIDO TOSI, ADRIANO MARTINOLI1 Parole chiave – Chiroptera, Plecotus macrobullaris, distribu- Key words – Chiroptera, Plecotus macrobullaris, distribution, zione, conservazione, Lombardia. conservation, Lombardy. Riassunto – Prima segnalazione in Lombardia (Nord-Italia) di Abstract – Mitochondrial DNA sequencing of 63 samples of Orecchione alpino Plecotus macrobullaris, KUZJAKIN, 1965 de- bats from the genus Plecotus, from Lombardy and Trentino terminato mediante analisi genetica. Viene riportata la prima Alto Adige, allowed us to determine for the first time the pre- segnalazione per la Lombardia della specie Plecotus macro- sence of the new species Plecotus macrobullaris Kuzjakin, bullaris recentemente descritta, mediante l’impiego di tecni- 1965, in Lombardy. This taxon has been recently described, che genetiche, utilizzando anche campioni di animali prove- using genetic techniques, as a new species, also using tissue nienti dalla provincia di Trento, che rappresentano la prima se- samples from individuals captured in Trento Province, that re- gnalazione della specie per l’Italia. Il genere Plecotus include, presented the first record in Italy. Recent taxonomic studies on per l’Italia, quattro specie sorelle: oltre a P. macrobullaris so- the genus Plecotus have revealed the presence of four cryptic no contemplate anche P. austriacus, P. auritus, P. sardus, la so- species in Italy: in addition to P. macrobullaris there are P. au- la specie di chirottero endemica dell’Italia. Per l’esatta deter- striacus, P. auritus, P. sardus, the only endemic Italian bat spe- minazione delle specie è necessario quindi ricorrere a tecniche cies. Since no discriminant function based on morphometric genetiche in quanto non sono state ancora messe a punto fun- parameters is currently available, only genetic techniques al- zioni discriminanti basate su parametri biometrici, sebbene low to discriminate exactly the different species. Mitochon- siano state identificate alcune caratteristiche morfologiche uti- drial DNA sequencing allowed us to assign 18 out of 33 bats li per la distinzione. Nel presente lavoro sono riportate le bio- (55%) of the genus Plecotus caught in Lombardy to P. macro- metrie di base della specie. Per la determinazione della specie, bullaris, the others to P. auritus. Moreover, four reproductive nel presente lavoro è stato utilizzato il metodo del sequenzia- roosts of P. alpinus were identified, all in buildings. The spe- mento di una regione del DNA mitocondriale, analizzando 50 cies was never found in caves, in contrast with P. auritus. campioni di tessuto di animali provenienti da Lombardia e The presence of this new species in Lombardy should activate Trentino Alto Adige. Sul totale dei campioni analizzati il 55% large-scale monitoring to define species distribution and habi- è risultato appartenere alla specie P. macrobullaris. Sono stati tat preferences, finalised to define population status and con- individuati, inoltre, 4 rifugi riproduttivi della specie che, in re- servation strategies. lazione ai dati attualmente a disposizione, sembra preferire ri- fugi presso edifici mentre non ne è mai stata segnalata la pre- senza in grotta, a differenza di P. auritus. La presenza di questa nuova specie in Lombardia dovrebbe in- durre ad un monitoraggio a larga scala, per definire in detta- glio la distribuzione della specie e le preferenze di habitat, fi- nalizzate anche alla definizione dello status delle popolazioni presenti. INTRODUCTION al., 2000; RUEDI and ARLETTAZ, in press) and M. al- cathoe (HELVERSEN et al., 2001), and, particularly, in There has recently been growing interest in the taxo- the genus Plecotus, where two new species have been nomy of European bats, thanks to the development of described: P. macrobullaris and P. sardus (KIEFER and molecular genetic techniques, in particular mitochon- VEITH, 2001; MUCEDDA et al., 2002; SPITZENBERGER drial DNA sequencing. As a consequence, several et al., 2003). Hence, the geographic distribution of new species have been described or proposed: e.g. for the two “historic” sibling long-eared bat species in the genus Myotis, with Myotis punicus (CASTELLA et Europe, P. auritus and P. austriacus, needs to be revi- 1 Dipartimento Ambiente-Salute-Sicurezza, Università degli Studi dell’Insubria, via J. H. Dunant 3, I-21100 Varese, Italy, e-mail: [email protected] 171-176.QXD4 31-08-2005 15:16 Pagina 172 172 I. Trizio, D. Preatoni, R. Chirichella, S. Mattiroli, M. Nodari, S. Crema, G. Tosi, A. Martinoli sed. Moreover the distribution, habitat preferences can be subdivided into three major landscape clas- and potential niche segregation of four of these spe- ses: alpine (76.9% of total area), hilly (17.6%) and cies, which seem to occur sympatrically over large southern plains (5.5%), the latter coinciding with the areas in Europe, is still controversial. northern areas of the upper Po Plain. Bats were pre- The Plecotus species can be considered sibling dominantly monitored in the alpine and hilly parts of species, a phenomenon well known among other bat the study area. Plecotus spp. were monitored at 23 genera, particularly, among European Myotis species: sampling sites, 14 in Lombardy and 9 in Trentino Al- e.g. M. mystacinus and M. brandtii, M. myotis and M. to Adige. blythii (ARLETTAZ, 1996; ARLETTAZ et al., 1997). Si- bling species are in general difficult to discriminate based on traditional morphometric parameters or bioacoustic analysis, and this also holds for the genus Plecotus. In 2001 SPITZENBERGER et al. described P. kolombatovici Dulic (1980) as a third species of Ple- cotus, different from P. auritus (although originally described as a subspecies of P. austriacus). In the sa- me year, KIEFER and VEITH (2001) described another new species of Plecotus allied to P. auritus: P. alpinus (KIEFER and VEITH, 2001). Shortly afterwards, SPIT- ZENBERGER et al. (2002) described a new species of Fig. 1 – The five provinces included in the study area (thin line). Plecotus allied to P. auritus: P. microdontus SPITZEN- Thick line represents boundary between Italy and Switzerland. BERGER et al. 2002. When MAYER and VON HELVER- SEN (2001) demonstrated a sibling relationship between P. kolombatovici and P. austriacus, SPITZEN- MATERIAL AND METHODS BERGER et al., (2002) also revised their former opi- nion and agreed that the name kolombatovici should Trapping and handling bats be used for the species sibling to P. austriacus, and Mistnet censuses were carried out during the sum- described the sibling species of P. auritus as a new mers of 2000 to 2002, using nylon twisted-thread mi- species, P. microdontus, a synonym of P. alpinus, na- stnets (TUTTLE, 1976), with a 36 mm wide square med in 2003 (SPITZENBERGER et al.) P. macrobullaris. mesh. Each net was composed of five pockets, 60 cm In a wider context addressing ecological preferen- high each, for a total length of 3 to 10 metres. ces and distribution of the new species P. macrobulla- Mistnetting sites were chosen on the basis of a ris in Northern Italy, we here report the first record of homogeneous sampling scheme, covering different P. macrobullaris in Lombardy, verified using genetic habitat types and aimed at an exhaustive coverage analysis. Its relative abundance with respect to P. au- both of number of species and number of animals. ritus is estimated, and basic biometric parameters are Fixed mistnets were placed in sites where obligatory presented. or preferential fly lanes were identified, in particular near or across streams, or at the entrance of caves, by hooking each net to a pair of aluminium poles. Nets STUDY AREA were opened at dusk, just before bats left their roo- sting sites, and closed and dismounted after a clear The study area includes the alpine and prealpine decrease in bat activity, identified both by the absen- areas of the provinces of Como, Lecco, Sondrio, Va- ce of catches for at least an hour and by the absence rese (Lombardy) and Trento (Trentino Alto Adige) (or high decrease) of ultrasonic signals, monitored (Fig. 1). Due to the wide extension of the area, bat using an ultrasound detector in frequency division habitats varied from highly urbanised lowlands and mode. After opening the nets, they were checked con- lower hills, typical of the southern parts of most pro- tinuously. Trapped individuals were removed imme- vinces in Lombardy, to the sparsely inhabited moun- diately and held in cotton sacks until the nets were tains, in the provinces of Sondrio and Trento, and in closed. After that, animals were measured and tissue the northern and central parts of the provinces of Va- samples were taken, then releasing each animal at the rese, Como and Lecco. The study area as a whole point of capture. 171-176.QXD4 31-08-2005 15:17 Pagina 173 First record of the alpine long-eared bat in Lombardy revealed by DNA analysis. 173 Other catches were made during surveys in poten- from the tail membrane (uropatagium) of each skin- tial roost sites, using both mistnets and hand nets sampled bat using a sterile biopsy punch, and placed with 1 cm mesh, in the latter case when animals were in a 0.2 ml Eppendorf vial filled with 70% ethanol hiding in crevices or suspended from the ceiling. Sur- and stored at –20 °C until the moment of genetic veys were conducted in old buildings, where it is ea- analysis. The patagium has a high regenerating capa- siest to find bat roosts. Visits to roost sites were con- city and lesion were completely regenerated in less ducted according to LUTZ et al. (1986). Preliminary than a month. inspections were done by visiting as many buildings as possible, at all elevations.
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