Karyotype comparison and phylogenetic relationships of Pipistrellus-like bats (Vespertilionidae; Chiroptera; Mammalia)

Karyotype comparison and phylogenetic relationships of Pipistrellus-like bats(Vespertilionidae; Chiroptera; Mammalia)

M. Volleth1;6, G. Bronner2, M. C. GÎpfert3, K.-G. Heller4, O. von Helversen4 & H.-S. Yong51 Institut fÏr Humangenetik, UniversitÌt Erlangen-NÏrnberg, Erlangen, Germany; 2 School ofEnvironmental Sciences & Development, Potchefstroom University, South Africa; 3 Institut fÏr Zoologie I,UniversitÌt Erlangen-NÏrnberg, Erlangen, Germany; 4 Institut fÏr Zoologie II, UniversitÌtErlangen-NÏrnberg, Erlangen, Germany; 5 Dept. of Zoology, Universiti Malaya, Kuala Lumpur,Malaysia;6 Present address: Institut fÏr Humangenetik, UniversitÌtsklinikum, Leipzigerstr. 44, D-39120Magdeburg,Germany; Tel: xx49 391 6715342; Fax: xx49 391 6715066;E-mail: [email protected]

Received 15 May 2000; received in revised form and accepted for publication by B. Dutrillaux 11 October 2000

Key words: Chiroptera, chromosomal evolution, Eptesicus, Pipistrellus, phylogenetic relationships

Abstract

Detailed karyotype descriptions of 20 Pipistrellus-like bat species belonging to the family Vespertilionidaeare presented. For the ¢rst time, chromosomal complements of four species, i.e. Pipistrellus stenopterus(2n� 32), P. javanicus (2n� 34), Hypsugo eisentrauti (2n� 42) and H. crassulus (2n� 30) are reported.A Pipistrellus kuhlii-like species from Madagascar represents a separate species distinguished from theEuropean Pipistrellus kuhlii (2n� 44) by a diploid chromosome number of 42. Banded karyotypes arepresented for the ¢rst time for Scotozous dormeri, Hypsugo capensis, Hesperoptenus blanfordi,Tylonycteris pachypus and robustula. Chromosomal evolution in the family Vespertilionidae is charac-terized by the conservation of entire chromosomal arms and reductions in diploid chromosome numbervia Robertsonian fusions. Less frequently, centric ¢ssions, para- and pericentric inversions and centromereshifts were found to have occurred. In several cases a certain type of chromosomal change predominates ina karyotype. Examples of this are the acquisition of interstitial heterochromatic bands in Tylonycterisrobustula, and centric shifts in P. javanicus, H. eisentrauti and Hesp. blanfordi. The species examinedhere belong to three tribes, i.e. Pipistrellini, Vespertilionini and Eptesicini, which are distinguished bychromosomal characteristics. According to our results, the species Pipistrellus (Neoromicia) capensisbelongs to the Vespertilionini and not to the Pipistrellini. We therefore propose to elevate the subgenusNeoromicia to generic rank.

Introduction

Vespertilionid bats of the cosmopolitan generaPipistrellus and Eptesicus are extremely dif¢cultto classify because of similar morphological

characteristics. Traditionally, generic distinctionbetween Pipistrellus and Eptesicus was based onpresence or absence of the second upperpremolar (see e.g. Ellerman & Morrison-Scott1951). This procedure has yielded increasingly

Chromosome Research 9: 25^46, 2001. 25# 2001 Kluwer Academic Publishers. Printed in the Netherlands

unsatisfactory and incongruent results, and it hasbecome clear that tooth reduction has taken placeindependently in several lineages of the familyVespertilionidae.

Recently, the shape of the baculum and certainchromosomal characteristics were found to besuitable for distinguishing between Pipistrellusand Eptesicus (Heller & Volleth 1984, Hill &Harrison 1987, Volleth & Heller 1994). Basedon baculum morphology, Hill & Harrison (1987)divided the genus Pipistrellus into sevensubgenera: Pipistrellus, Hypsugo, Falsistrellus,Perimyotis, Arielulus, and Vespadelus andNeoromicia, both of which were formerlyclassi¢ed under Eptesicus. In the meantime,some of these subgenera have been given fullgeneric rank after detailed morphological orbiochemical analyses (Hypsugo: Horacek &Hanak 1986, Ruedi & Arlettaz 1991;Perimyotis: Menu 1984; Arielulus: Csorba &Lee 1999).

To elucidate the unsolved phylogenetic relation-ships within this group, a comparative analysis ofthe karyotypes of about 50 vespertilionid specieswas made (Volleth & Heller 1994). This revealedthat members of the genus Pipistrellus (sensu Hill& Harrison 1987) differ in two chromosomalcharacters and belong either to the tribePipistrellini (subgenus Pipistrellus) or to the tribeVespertilionini (subgenera Hypsugo, Falsistrellus,Vespadelus). To avoid a polyphyletic condition,it was suggested to elevate the latter threesubgenera to generic rank. Together with thegenus Hesperoptenus, Eptesicus forms a thirdtribe, Eptesicini. The phylogenetic tree anddiagnostic characters are described in detailin Volleth & Heller (1994). Brie£y, thephylogenetic relationships for the three tribesmentioned read: (Eptesicini (VespertilioniniPipistrellini)).

In the present paper we give the completekaryotype descriptions of 16 Pipistrellus-likespecies included in the phylogenetic analysis inVolleth & Heller (1994), plus karyotypes and aninterpretation of phylogenetic relationships foran additional three African and one Asian species.Based on these karyological analyses, we concludethat the subgenus Neoromicia (sensu Hill &Harrison 1987) should be treated as a separategenus.

Materials and methods

Chromosome preparations

Metaphase spreads were obtained from ¢broblastcultures by conventional procedures (Volleth1987). GTG- and RBG-banding procedures werecarried out according to Seabright (1971) andCamargo & Cervenka (1980), respectively.Nucleolus organizer regions (NORs) were deter-mined by Ag-staining (Volleth 1987) andheterochromatic material was detected byCBG-banding (Sumner 1972). For identi¢cationof chromosomal arms in C-banded andsilver-stained metaphases, sequential stainingwas performed (QFQ-AgNOR, QFQ-CBG orAgNOR-GTG, GTG-CBG). The £uorescencedouble and triple staining with CMA, DAPIand distamycin A was performed according toSchweizer (1980). Chromosomal arms werenumbered using Bickham's scheme given in thepaper on the American Myotis species (Bickham1979a). Abbreviations used in the text: FN, fun-damental number (number of autosomal chromo-some arms); SC, secondary constriction; CS,centromere shift. Two chromosomal arm numbersseparated by a solidus (/) indicate the result of aRobertsonian translocation. A dash (^), however,indicates two connected arms without anycentromere between them. In some cases, £uor-escence in situ hybridization (FISH) with humanwhole chromosome probes (WCPs) was applied(for a description of the method, see Volleth etal. 1999).

Specimens examined

Data for the European species are given in Volleth(1987), those of the non-European species aregiven in Volleth & Heller (1994). Additional speci-mens included here are as follows(SMF� accession numbers of the SenckenbergMuseum, Frankfurt/Main): Hypsugo crassulus(HCR): Congo (Zaire), Prov. Kivu, Irangi, Centrede Recherches en Sciences Naturelles (01�540S,28�270E), 1M, 2F, SMF 79441-3 (Heller et al.1994); Pipistrellus cf. kuhlii (P-K): Madagascar,Morondava, Kirindy forest, 1M; Neoromiciacapensis (NCA): Republic of South Africa,

26 M. Volleth et al.

Mpumalanga, Badplaas, Farm Groenvaly(25�280S, 30�450E), 1M, 2F; and Wakkerstroom(27�210S, 30�090E), 1M, SMF 87493-6; Eptesicusbottae (EBO): Greece, Rhodes, Salakos(Helversen 1998), 1M.

Additionally, the numbers and sex of the speci-mens examined, together with the country oforigin, are given in Table 1.

Chromosomal features for cladistic analysis

Besides large-scale homology of chromosomalarms, karyotype comparisons of members of thefamily Vespertilionidae have revealed minor dif-ferences in 8 arms owing to small peri- orparacentric inversions. On the basis of differentG-banding patterns, two `states' (I and II) of thesechromosome arms can be discriminated, which aredescribed in detail in Volleth & Heller (1994). Forthe species treated here, four features are import-

ant (Table 1): the state of the chromosomal arms11 and 23, discriminating Pipistrellini from theVespertilionini, and, as synapomorphic featuresof both tribes, the state of chromosomal arm 15and of the X. Furthermore, a so-called `basickaryotype' with 2n� 44 and four metacentricchromosomes (i.e. 1/2, 3/4, 5/6 and 16/17) wascreated for comparative analysis (for details, seeVolleth & Heller 1994).

Results

Karyotypes of the Pipistrellini

Two features characterize the members of thistribe: chromosome 11 is present in state I (Figure1), whereas chromosome 23 is present in stateII (Table 1). The X is, unless otherwise indicated,present in state II.

Table 1. Species list with chromosomal characteristics, systematic classi¢cation and data of specimens. The classi¢cation of Pipistrellus-likebats into three tribes (P, V and E) is based upon the distribution of four chromosomal characteristics, i.e. chromosomal arms 11, 15, 23 andX, occurring in two states (I, II).

Chromosome no.

Species 11 15 23 X 2n/FN Tribe Number and sex Origin

Pipistrellus pipistrellus I II II II 44/50 P 7M, 5F EuropePipistrellus nathusii I II II II 44/50 P 1M GreecePipistrellus kuhlii I II II II 44/50 P 1F GreecePipistrellus cf. kuhlii I II II II 42/50 P 1M MadagascarPipistrellus mimus I II II II* 38/48 P 1F Sri LankaPipistrellus stenopterus I II II II 32/50 P 1M MalaysiaPipistrellus javanicus I II II II* 34/46 P 1F MalaysiaGlischropus tylopus I II II II* 30, 31/50 P 1M, 1F MalaysiaScotozous dormeri I II II II 30/50 P 1M, 1F (India)Hypsugo savii II II I II 44/50 V 3M Greece, TurkeyHypsugo eisentrauti II II ? II 42/58 V 1M RwandaHypsugo crassulus II II I II 30/56 V 1M, 2F CongoNeoromicia capensis II II I II 32/50 V 2M, 2F South AfricaTylonycteris pachypus II II I II* 46/50 V 2M, 2F MalaysiaTylonycteris robustula II ? I II* 32/50 V 2M, 2F MalaysiaEptesicus serotinus I I I I 50/48 E 1M, 1F Germany, GreeceEptesicus nilssonii I I I I 50/48 E 1F GermanyEptesicus bottae I I I I 50/48 E 1M GreeceArielulus circumdatus I I I I 50/48 E 1F MalaysiaHesperoptenus blanfordi I I I I* 32/54 E 5M, 2F Malaysia

For all four chromosomes mentioned here, state I was found to be the ancestral and state II the derived condition in the familyVespertilionidae (see Volleth & Heller 1994). ?, state unknown; *, rearranged form (see text); tribe: E, Eptesicini; P, Pipistrellini; V,Vespertilionini; Origin: country of origin; Pipistrellus pipistrellus: specimens fromGermany, Spain, Greece and Turkey; Scotozous dormeri:ancestors of the captive-born specimens came from India.

Karyotype comparison of Pipistrellus-like bats 27

Pipistrellus pipistrellus, kuhlii and nathusiiThe European Pipistrellus species all possess akaryotype with 2n� 44 and FN� 50. The locationof the NORs was described in detail in Volleth(1987), where a GTG-banded karyotype ofPipistrellus pipistrellus can also be found. AfterC-banding the centromeric heterochromatin isclearly stained. The Y chromosome of Pipistrelluspipistrellus and P. nathusii is only half the sizeof the smallest autosome and is not (P. pipistrellus)or only slightly (P. nathusii) stained afterC-banding. No male of P. kuhlii was availablefor study.

Pipistrellus cf. kuhliiIn contrast to the European Pipistrellus kuhlii with2n� 44, this species from Madagascar shows adiploid chromosomal complement with 2n� 42(FN� 50). This difference is the result of aRobertsonian fusion between arms numbers 11and 12 (Figure 2). The Y chromosome is very tiny,even smaller than chromosome 25, withheterochromatin present in the centromere only.The centromeric heterochromatin of all chromo-somes is clearly visible after C-banding. TheNORs are situated in the secondary constriction(SC) of chromosome 15.

Pipistrellus mimusThe karyotype of this species with 2n� 38 andFN� 48 consists of six meta- to submetacentricand 12 acrocentric autosomal pairs (Figure 3).

In addition to the three large metacentrics ofthe basic karyotype, three Robertsonian fusionproducts were found: 8/11, 9/13 and 10/12.Chromosome 3/4 shows an altered appearancedue to a pericentric inversion which has trans-ferred a small proximal segment of arm 3 toarm 4. The small metacentric chromosome 16/17is missing, but an acrocentric element is presentinstead. In the X chromosome, a pericentricinversion has moved a small proximal segmentof Xq to Xp, which thus becomes the longer arm.The NORs are located in the SC of chromosome15. With the exception of the two smallestchromosomes, 24 and 25, C-banding revealedcentromeric heterochromatin only in theacrocentric chromosomes. These chromosomesshowed bright CMA £uorescence in thejuxtacentromeric regions, where non-euchromaticmaterial seems to have accumulated.

Pipistrellus stenopterusThis species shows a karyotype with 2n� 32 andFN� 50 (Figure 3). The karyotype consists of ninemeta- to submetacentric, one subtelocentric and¢ve acrocentric autosomal pairs. P. stenopterusshares three fusion products with P. mimus andjavanicus, i.e. 8/11, 9/13 and 10/12. In addition,the following Robertsonian fusions were found:14/21, 15/18 and 19/20. Another similarity withP. mimus is the rearranged chromosome 3/4.Chromosome 16/17 shows an altered appearance;it is subtelocentric, probably as a result of a small

Figure 1. Comparison of the states of arm 11; state I left, state II right. The arrows point to the position of the distinctiveGTG-negative band. Species shown from left to right (with arm combinations of Robertsonian fusion chromosomes in brackets):ESE, Eptesicus serotinus; GTY, Glischropus tylopus; PPI, Pipistrellus pipistrellus; P-K, Pipistrellus cf. kuhlii (12/11); HSA, Hypsugosavii; HEI, Hypsugo eisentrauti (23/11); HCR, Hypsugo crassulus (3/11); NCA, Neoromicia capensis (7/11); and TRO, Tylonycterisrobustula (10/11).


pericentric inversion. The acrocentric Y chromo-some is of the size of chromosome 23 and nearlyentirely heterochromatic. The NORs are situatedin the SC of arm 15. C-banding revealed thatthe proximal segment of chromosome 24 isheterochromatic.

Pipistrellus javanicusThis species shows a karyotype with 2n� 34 andFN� 46. The autosomes are composed of sixmeta- to submetacentric, one subtelocentric andnine acrocentric elements (Figure 3). The compo-sition of the biarmed chromosomes is 1/2, 3/4,7/18, 8/11, 9/13, 10/12 and 14/15. Some chromo-somes have been altered by centromere shift (CS).In the previously metacentric chromosome 5/6,the centromere is now situated at the former

telomere of arm 5. In 9/13 the centromere has beenshifted into arm 9, and in 14/15 into arm 14.Furthermore, the X chromosome is an acrocentricelement with the centromere at the previoustelomere of the long arm (Figure 4). Chromosome16/17 of the basic karyotype is present in thisspecies as an acrocentric element. Chromosomes19 to 25 have remained acrocentrics. The NORsare situated in the SC of chromosome 21.C-banding revealed very weakly stainedcentromeric regions and heterochromatic materialin the proximal third of chromosome 24.

Glischropus tylopusThis species possesses a karyotype with 30chromosomes in the female and 31 chromosomesin the male. The difference between the sexes is

Figure 2. GTG-banded karyotype of a male Pipistrellus cf kuhlii. Please note that, according to Bickham's (1979a) scheme,chromosomal arms instead of chromosomes are numbered to facilitate comparison between different karyotypes. Scale bar� 5 mm.



due to the translocation of autosomal arm 23 tothe X chromosome. The fundamental number(excluding the X-autosome translocationchromosome) is 48, or 50 if arm 23 is taken intoaccount. The karyotype has been described pre-viously (Volleth & Yong 1987) but without givingthe arm combinations of the fusion chromosomes.These are: 1/7, 2/24, 3/15, 4/20, 5/8, 6/10, 9/14,12/13 and 21/25. Chromosome 16/17 shows analtered appearance compared with that of thebasic karyotype, probably due to a small peri-centric inversion. Arms 11, 18, 19 and 22 werefound in an acrocentric condition. A pericentricinversion of the ancestral X chromosome hasresulted in an acrocentric element with the pre-viously long arm now being the proximal partand the formerly short arm becoming the distalpart. A second step, X-autosome-translocation,

has resulted in a chromosome consisting ofX-chromosomal material in the long arm andchromosome 23 material in the short arm. Theautosomal part (i.e. arm 23) of the translocationchromosome is separated by a small interstitialheterochromatic segment from the centromereand the X-chromosomal material. The Ychromosome has nearly the size of chromosome22 and is composed of heterochromatinexcept for a small distal segment. The NORsare situated in the SC of chromosome 15. AfterC-banding, the centromeric heterochromatinwas clearly visible. Further, small non-euchromatic bands were found close to thecentromere of arm 1, 2, 3, 4, 11, 12, 18, 19 andthe X. These regions were GTG- andCBG-negative and CMA-positive and, exceptfor arm 4, also DistA/DAPI-positive.

Figure 3 (opposite). Comparison of GTG-banded chromosomes of the three Pipistrellus species, P. mimus (PMI, left chromosomes),P. stenopterus (PST, central chromosome) and P. javanicus (PJA, right chromosome of each triplet). No Y chromosomes can be shownfor PMI and PJA, because only females have been studied.

Figure 4. Centromere shift in Pipistrellus javanicus chromosomes (left or central chromosome) in comparison to P. mimus (rightchromosome of each pair, left and right chromosomes in the triplet). In the X, Vespadelus darlingtoni (GTG; called Pipistrellussagittula in Volleth & Tidemann 1989) and Pipistrellus pipistrellus (RBG) were chosen for comparison. The arrowheads pointto the centromeres.


Scotozous dormeriThe karyotype of this species shows 2n� 30 andFN� 50 (Figure 5). Except for the Y chromosome,all chromosomes are biarmed. The followingRobertsonian fusion chromosomes were found:7/13, 8/11, 9/12, 10/14, 15/20 and 24/25. Inchromosome 1/2, the centromere was found tobe translocated to arm 1, owing to a centric shiftor a pericentric inversion with the breakpoint inarm 2 close to the centromere. In the fusionproduct 18/19, a pericentric inversion hastransferred the proximal part of arm 18 toarm 19, which is now the long arm of therearranged element. The acrocentric Ychromosome is about the size of chromosome22 and consists partly of heterochromatin. The

NORs were found in narrow SCs located inchromosome 15 and in the tiny short arms ofchromosome 22 and 23. After C-banding thecentromere heterochromatin was only weaklystained. The tiny short arms of chromosome 21to 23 and the pericentromeric regions ofchromosome 24/25 stained clearly. Furthermore,both captive-born specimens showed cell-to-cellvariation in the centromere position of sixautosomal pairs. In 3/4, 5/6, 10/14, 7/13 and15/20, the centromere was shifted a smalldistance towards the short arm, and in 8/11towards the long arm. The mean number of alteredautosomal arms was 1.5 per cell. None of the cellsanalysed showed all autosomes in an unalteredcondition.

Figure 5. GTG-banded karyotype of a male Scotozous dormeri. Open triangles indicate the ancestral, and closed triangles the actual,position of the centromere in the chromosomes with pericentric inversions.


Karyotype descriptions of the Vespertilionini

The distinctive characteristics for this tribe arestate II of chromosome 11 (synapomorphicfeature, see Figure 1) and state I of chromosome23. The X chromosome and chromosome 15 are,as with the Pipistrellini, present as state II.

Hypsugo saviiThis species possesses a karyotype with 2n� 44and FN� 50. H. savii differs from `true'Pipistrellus species with 2n� 44 in the states ofchromosome 11 and 23 (see Table 1). The NORsare situated in the SC of chromosome 15. The very

tiny submetacentric Y chromosome is half the sizeof the smallest autosome and lacks CBG positiveheterochromatin.

Hypsugo eisentrautiThe karyotype consists of three large, sixmedium-to-small meta- to submetacentric and11 acrocentric autosomal pairs (Figure 6). The dip-loid chromosome number is 42 with a FN of 58.Six autosomal pairs differ from those of the basickaryotype: 7, 8, 11, 15, 16/17 and 20. For threepairs, a centromere shift (CS) is assumed. Inchromosome 7, the centromere has shifted intothe GTG-dark block in the middle of thechromosome. In chromosome 15, the centromerehas shifted to the distal part of the formerly

Figure 6. GTG-banded karyotype of a male Hypsugo eisentrauti. In the chromosomes with centromere shifts (cs), the open trianglesindicate the ancestral and the closed triangles the actual position of the centromere. Scale bar� 5 mm.


acrocentric chromosome, resulting in a sub-metacentric chromosome. The short arm of thischromosome represents the distal part of theancestral chromosome and the secondary con-striction formerly located near the centromere isnow located near the telomere of the long arm.In chromosome 20 the centromere has shifted evenmore towards the telomere, resulting in a sub-telocentric chromosome. In chromosome 8, whichshows a small euchromatic short arm, a small peri-centric inversion is assumed. A Robertsonianfusion has taken place between chromosome 11and chromosome 23. It was not possible to ascer-tain whether chromosome 23 represents state IIor state I. The smallest submetacentric chromo-some could have been derived from the ancestralchromosome 16/17 by a pericentric inversion.The NORs are located in the SC of chromosome

15. The Y chromosome is a very tiny, mostlyheterochromatic chromosome of the same sizeas chromosome 25. Centromeric heterochromatinwas only weakly stained after C-banding.

Hypsugo crassulusThe karyotype consists of 30 chromosomes with anFN of 56 (Figure 7). All autosomes and the X andY are biarmed. The composed chromosomes 1/13,3/11, 7/22, 8/20, 14/18, 15/25 and 19/21 are theresult of Robertsonian fusions. In the case of 2^24and 10^23, where the centromere lies withinarm 2 and 10 respectively, pericentric inversionsare assumed for chromosomes 2 and 10, inaddition to fusions with chromosomes 24 and23, respectively. Chromosomes 4, 5, and 6 are sub-metacentric chromosomes: in chromosomes 4 and5, a pericentric inversion, and, in chromosome

Figure 7. GTG-banded karyotype of a female Hypsugo crassulus. The inset shows the gonosomes of a male. In the fusion chromo-somes with an inversion (inv) in one arm, the dash indicates the border between the fused arms. The open triangles point to theformer, the ¢lled triangle to the actual, position of the centromere in chromosome 6 with a centromere shift (cs).


6, a centromere shift, are assumed to haveoccurred. As none of the metacentric chromo-somes 1/2, 3/4 and 5/6 of the basic karyotypeis present, Robertsonian ¢ssions must also haveoccurred in these cases. Active NORs were foundon three chromosomes, i.e. close to the centromereof arm 3 and arm 19 and in the short arm of thefusion product 15/25, separated from thecentromere by a small heterochromatic, poly-morphic segment. The pattern of NOR activityvaried between the specimens examined (see Table2). In addition to the centromeric dots, a smallheterochromatic band was found near thetelomere of arm 1 and between the centromereand the NOR in the short arm of 15/25. The smallY chromosome is heterochromatic except for thedistal part of the long arm.

Neoromicia capensisThe diploid chromosome number of this species is32, and the fundamental number is 50. Thereare nine large to medium meta- to submetacentric,one small metacentric and ¢ve small acrocentricautosomal pairs. The following composition of

chromosomal arms was found: 1/2, 3/4, 5/6,7/11, 8/9, 10/12, 13/18, 14/20, 15/21 and 16/17.The arms 19 and 22 to 25 are acrocentric (Figure8a). Chromosome 1/2 exists in two states in theVespertilionidae (Volleth & Heller 1994). Gen-erally, the members of the tribes treated here showstate II. In Neoromicia capensis, however,chromosome 1/2 belongs neither to state I norto state II. The GTG-positive band of arm 2,located close to the centromere in state II, islocated pericentromerically owing to a centromereshift (Figure 8b). The NORs are located in the SCof arm 15. In GTG-banded chromosomes, theSC is visible as a small weakly stained region closeto the centromere of arm 15 (see Figure 8a).AgNOR-staining revealed the presence of thenucleolus organizer region (NOR) at this location(see Figure 8c). The centromeric heterochromatinwas only weakly stained after C-banding. Thetiny Y chromosome is mostly heterochromatic.Of the four specimens studied, a polymorphic fea-ture was found in one female: one homologueof the pairs 24 and 25, which are not dis-tinguishable, was of the same size as pair 23,

Table 2. Distribution of nucleolus organizer regions (NORs): mean value of active NORs per chromosomal arm and cell.

Chromosomal arm no.

Species N 3 15 19 21 22 23 24� 25a

Pipistrellus cf. kuhlii 20 2.0Pipistrellus mimus 17 2.0Pipistrellus stenopterus 15 1.7Pipistrellus javanicus 31 1.7Glischropus tylopus 1 14 2.0Glischropus tylopus 2 13 2.0Scotozous dormeri 1 26 1.8 1.9 1.9Scotozous dormeri 2 31 1.6 1.7 1.1Hypsugo eisentrauti 26 2.0Hypsugo crassulus 1 53 0.7 1.2 1.9Hypsugo crassulus 2 35 0.4 1.2Hypsugo crassulus 3 27 1.0 1.9Neoromicia capensis 1 15 1.9Neoromicia capensis 2 13 1.9Tylonycteris pachypus 1 6 1.8Tylonycteris pachypus 2 5 2.0Tylonycteris robustula 14 2.0Eptesicus nilssoni 16 1.9Eptesicus bottae 25 2.0Hesperoptenus blanfordi 1 10 2.0Hesperoptenus blanfordi 2 5 2.0

N, number of cells analysed; a chromosomal arms 24 and 25 are indistinguishable; numbers following species refer to specimen number.



and subtelocentric in shape with the short arm andthe proximal portion of the long arm beingheterochromatic.

Tylonycteris pachypus and robustulaThe karyotypes of these species, which aremorphologically very similar, seem at ¢rst glanceto be very different. The diploid chromosomenumber is 46 for T. pachypus and 32 for T.

robustula. However, both species share threerearranged chromosomes: fusion chromosomes9/23 and 18/19 and an acrocentric Xchromosome.

The karyotype of T. pachypus (2n� 46,FN� 50) is dominated by acrocentric elements(Figure 9). Chromosomal arms 1 to 6 areacrocentric. The small metacentric 16/17, andthe two fusion chromosomes (9/23, 18/19), are

Figure 8 (opposite). GTG-banded karyotype of a maleNeoromicia capensis (a) and (b) comparison of chromosome 1/2 ofN. capensis(left chromosome of each pair) and Hypsugo eisentrauti (right chromosome of each pair). Note that the pericentromeric region isG-positive in N. capensis, but G-negative in H. eisentrauti. For explanation see text. (c) AgNOR-stained cell showing NORs atthe location of the SC in arm 15 (see arrows). The SC is also visible in the GTG-banded karyotype in (a).

Figure 9. Comparison of GTG-banded karyotypes of Tylonycteris robustula (left chromosome) and T. pachypus (right chromosomeof each pair). The bi-armed chromosomes in the triplets belong to T. pachypus.


the only biarmed elements. The NORs are situatedin the SC of chromosome 15. A pericentricinversion comprising the entire long arm hasresulted in an acrocentric X chromosome withthe former long arm becoming the proximal andthe former short arm becoming the distal part.The Y chromosome is similar in size to chromo-some 24 and only weakly stained after C-banding.Except the centromeric dots, tiny heterochromaticshort arms were also present on chromosome 6, 8and the X.

The karyotype of T. robustula consists of 32chromosomes and the FN is 50. The autosomesare composed of 11 meta- to submetacentric,one subtelocentric and three acrocentric pairs.In addition to the metacentric elements 1/2, 3/4,5/6 and 16/17, the following fusion products werefound: 9/23 and 18/19 (in common with T.pachypus), 8/14, 10/11, 12/20 and 13/15.

Chromosomes 24 and 25 show tinyheterochromatic short arms and chromosomes7, 21, and 22 are acrocentric (Figures 9 & 10)The NORs are situated in the SC of chromosome15. The metacentric Y chromosome is of the sizeof chromosome 25 with one arm beingheterochromatic (concerning X, see T. pachypus).In addition to clearly stained centromeric dots,C-banding revealed interstitial heterochromaticbands at 16 different locations, some being presentin a polymorphic condition (Figure 10). Theheterochromatic bands are QFQ- andDAPI-negative but a few are CMA-positive.The location of the heterochromatic bands isobvious, even in G-banded chromosomes, fromthe presence of GTG-negative segments. At mostlocations, both homologous chromosomes of allfour specimens examined showed theseheterochromatic bands. At a few sites, aninterstitial band was found in only one of thehomologues of a specimen (e.g. arm 3), or was evencompletely absent (e.g arm 1). One faintpolymorphic non-euchromatic band, located inthe central part of arm 9, distal to the clearlyCGB-positive band, was present only in onehomologue of two out of four specimens and,although being DAPI- and GTG-negative andCMA-positive, it was surprisingly CBG-negative(see Figure 10: left homologue showing twoGTG-negative bands in arm 9; the distal one isCBG-negative). In terms of chromosomal evol-

ution, these intercalary bands must be of fairlyrecent origin, having formed after separation fromthe T. pachypus lineage. It was not possible toestablish the state of chromosome 15 in T.robustula due to the presence of heterochromaticmaterial (see Table 1).

Karyotype descriptions of the Eptesicini

The members of this tribe differ from thePipistrellini and Vespertilionini by twochromosomal features, i.e. the states of chromo-some 15 and the X (see Table 1).

Eptesicus serotinus and nilssoniiThese species show a karyotype with 2n� 50 andFN� 48. Only the X chromosomes are biarmed.The large metacentrics of the basic karyotype, 1/2,3/4 and 5/6, are here present as six acrocentricelements owing to Robertsonian ¢ssions. Thefourth autosomal metacentric, 16/17, is presentas a single acrocentric element (17^16) with onlyone GTG-positive band in the proximal half. Somemetaphases of E. nilssoni showed a very weakGTG-positive band in the distal part of this smallacrocentric chromosome. In both species, theNORs are situated in the SC of chromosome15. The Y chromosome of E. serotinus is of thesame size as arm 24 and submetacentric with aheterochromatic short arm.

Eptesicus bottaeThe karyotype of E. bottae (Figure 11, 2n� 50,FN� 48) is similar to that of E. serotinus exceptthat chromosome 17^16 clearly shows twoGTG-positive bands. The Y chromosome is simi-lar in size to chromosome 25, metacentric, andcomposed of weakly staining heterochromatinwith a small euchromatic segment in one arm. Flu-orescence in-situ hybridization with human wholechromosome probes (WCPs) shows that theacrocentric element which has been derived fromthe metacentric chromosome 16/17, consists ofmaterial homologous to arm 17 in the proximalpart and homologous sequences to arm 16 inthe distal part.


Figure 10. GTG- and CBG-banded karyotype of Tylonycteris robustula. The GTG-banded cell and CBG-stained chromosomes ofrows 2, 5 and 8 are from male 266, CBG-banded chromosomes of rows 3, 6 and 9 from male 245. The interstitial heterochromaticblocks are represented in the GTG-banded chromosomes by GTG-negative bands. Note the differences in non-centromericheterochromatin between male 266 and male 245 in arms 1 and 3. For further explanations, see text.


Arielulus circumdatusThis Southeast-Asian species was described as amember of the genus Pipistrellus on the basis ofthe dental formula. According to morphologicaland karyological features (2n� 50, FN� 48),however, we considered it as belonging to thegenus Eptesicus or at least to the Eptesicini (Heller& Volleth 1984). In contrast, this species has stillbeen listed as a member of Pipistrellus, con-stituting a distinct subgenus, Arielulus by Hill &Harrison (1987). Due to morphologicalcharacteristics, the taxon Arielulus has recentlybeen given full generic rank by Csorba & Lee(1999). We follow the suggestion of the latterauthors, who also consider Arielulus to be closelyrelated to Eptesicus, not to Pipistrellus.

The G-banded karyotype of Arieluluscircumdatus is identical to that of E. bottae.

Hesperoptenus blanfordiThe diploid chromosomal number of this species is32 and the fundamental number 54. The auto-somes consist of nine meta- to submetacentric,two subtelocentric and three acrocentric pairs plusone polymorphic pair with a meta- or sub-telocentric form (Figure 12). This chromosomalcomplement differs considerably from the basickaryotype, with the result that some of therearrangements could not be fully clari¢ed byGTG-banding. None of the metacentric chromo-somes of the basic karyotype was present. Arm1 is fused with arm 13, which shows one or two

Figure 11. GTG-banded karyotype of Eptesicus bottae.


CBG-positive bands inserted in the proximalGTG-positive segment (polymorphism). Ad-ditional Robertsonian fusion products are 8/11,9/12, 7/14, 10/20 and 15/18. Three chromo-some arms, formerly acrocentric, occur here assubmetacentric elements, i.e. 2, 3 and 6. In each

case, a centromere shift has probably led to theseforms, but for arm 2 and 6 pericentric inversionscannot be completely excluded. Arms 4, 5 and19 are unchanged with an acrocentric condition.The smallest metacentric element is composedof arms 23 and 24, with a small heterochromatic

Figure 12. (a) Composed karyotype of Hesperoptenus blanfordi (GTG-banded left, RBG-banded right chromosome of each pair);(b) chromosome A of two specimens: both homologues are present in the subtelocentric form (left) or in the metacentric form (right).


segment separating them. The remainingchromosomes, referred to as À' and `B' in Figure12, must contain arms 16, 17, 21, 22 and 25. Exceptfor arm 22, which is clearly present in the shortarm of `B', banding analyses did not clarify theaf¢nities of the rest. Chromosome À' exists intwo forms, a metacentric and a subtelocentricone (polymorphism), which differ only in the pos-ition of the centromere (Figure 12b). The proximalpart of the long arm of the subtelocentric (ST)form, and nearly the whole short arm of themetacentric (M) form, consist of heterochromaticmaterial (see late-replicating segments in theRBG-banded chromosomes). Of the seven speci-mens studied, three males had two subtelocentrics,two males and one female showed one sub-telocentric and one metacentric each, and onefemale had two metacentrics.

ZOO-FISH experiments with human wholechromosome probes (WCPs) con¢rmed the resultof the banding analyses in showing that the shortarm of chromosome B contains arm 22. Further,the long arm contains chromosomal arms 16and 17 in the same order as described for E. bottae.The long arm of the metacentric form, and the dis-tal part of the long arm of the subtelocentric formof chromosome A are homologous to arm 21.The tiny euchromatic segment at the distal endof the short arm of the M form, making up theentire short arm of the ST form, is probably hom-ologous to arm 25 (no FISH data).

The X chromosome is metacentric and wasprobably derived from the state I X by aparacentric inversion of the short arm, followedby an extended pericentric inversion withbreakpoints in the distal parts of the long armand the short arm. The Y chromosome issubmetacentric, of the size of arm 18 andheterochromatic except a small segment in theshort arm. The NORs are situated in the SC ofarm 15. After CBG-banding the centromeres wereonly weakly stained.

Discussion

In general, the bat family Vespertilionidae ischromosomally relatively conservative since com-plete chromosomal arms are conserved and canbe identi¢ed in the individual species. Different

kinds of rearrangements have occurred duringkaryotype evolution of the family, namelyRobertsonian translocations, inversions,centromere shifts and heterochromatin additions.For phylogenetic analyses, Robertsonian fusionproducts should be used with caution becauseof the high possibility of homoplastic events. Incontrast, inversions are very useful for cladisticanalysis. In the Vespertilionidae, 8 chromosomalarms which have undergone peri- or paracentricinversions during karyotype evolution were found(Volleth & Heller 1994). Most of the otherrearrangements, however, are found in onekaryotype only, or in two closely related species,and are thus synapomorphies which shed littlelight on the phylogenetic af¢nities of these taxa.Only the cytogenetically most interesting examplesare discussed further below.

Centromere shift was ¢rst described as a modeof chromosomal rearrangement by Coleman(1948). One possible mechanism leading to CSis a series of two inversions, one peri- and oneparacentric, with break-points at similar positions.A second mechanism explaining an unchangedbanding pattern despite different centromerelocation is the formation of a neocentromere withsubsequent inactivation of the ancestralcentromere (Choo 1997). In recent years, severalexamples of neocentromere formation in humanshave been published (see e.g. Rivera et al. 1999,Voullaire et al. 1999).

Until now, centromere shifts have been foundonly very rarely in bat karyotypes (two presumedcases in the evolution of the phyllostomid sub-family Glossophaginae; Baker & Bass 1979). Inthe Vespertilionidae, centric transpositions haveoccurred in several species. In some, a centromereshift has occurred only in one chromosome ofthe respective karyotype, i.e. Hypsugo crassulus,Neoromicia capensis and Scotozous dormeri. Inthe last species, however, the rearrangement couldalso be explained by a pericentric inversion. Inthree unrelated species, more than one centric shifthas occurred during karyotype evolution: inPipistrellus javanicus (4), in Hypsugo eisentrauti(3) and in Hesperoptenus blanfordi (3).

In some cases, the distribution and location ofthe rearranged chromosomal segments can aidan understanding of the mode of karyotypeevolution. The karyotype of Hypsugo crassulus


exhibits many rearranged chromosomes. Inter-estingly, the break-points may have been locatedin close proximity to each other as a consequenceof non-random chromosomal arrangement at cer-tain stages of the cell cycle. Both in meioticprophase (when the telomeres are clustered dueto the formation of a bouquetösee, e.g., Dernburget al. 1995) and in interphase (when centromeresare clustered due to the Rabl orientationöseeCremer et al. 1982, Haaf & Ward 1995), chromo-some arms are arranged roughly in parallel. Thus,one could speculate that breakage at similarchromosomal regions (in chromosomes 4, 5, 6and 10 of H. crassulus; see Figure 7) is governedby nuclear architecture. Likewise, the C-bandingpattern in Pipistrellus mimus could be explainedby a speci¢c chromosomal arrangement withinthe nucleus. In this species, enlarged paracentricheterochromatic segments occur in the acrocentricmembers but not in the metacentric members ofthe chromosome complement. According to themodel by Schweizer & Loidl (1987), the spreadof repetitive sequences may be facilitated by thespatial proximity between the donor and therecipient chromosomal regions. Paracentric locion acrocentric chromosomes are assembled withthe telomeres near the nuclear periphery in themeiotic bouquet. The centromeres of metacentricchromosomes, on the other hand, are outsidethe telomeric cluster. Therefore, this modelsuggests that, in P. mimus, the acquisition ofheterochromatic material has occurred by a mech-anism acting during meiotic prophase.

An interesting case of divergent karyotype evol-ution is found in the two species of £at-headedbats. Both Tylonycteris species show extremelyspecialized morphological adaptations forroosting in bamboo stalks, i.e. £attened skulland disc-like pads on feet and hands (Nowak1999). Their unbanded karyotypes are quite differ-ent (2n� 32 and 2n� 46, Yong et al. 1971), butbanding analyses have revealed three synapo-morphic features: fusion chromosomes 9/23 and18/19 and an acrocentric X chromosome. Afteracquisition of these characteristics, however, thekaryotypic evolution of the two species has beenextremely different. In T. robustula, severalRobertsonian fusions have occurred in additionto acquisition of interstitial heterochromaticsegments at 16 different locations, some of which

are polymorphic. In T. pachypus, however,Robertsonian ¢ssions have occurred in thechromosomes 1/2, 3/4 and 5/6.

Comparison with published data

Out of the 20 pipistrelloid species presented here,karyotypes are shown for the ¢rst time for 4species (i.e. Pipistrellus stenopterus and javanicus,Hypsugo eisentrauti and crassulus), and bandingresults are given for another ¢ve species for whichonly conventionally stained karyotypes were pre-viously published. References can be found inZima & Horacek (1985) and in McBee et al.(1986); more recent papers are Harada et al. (1985,Tylonycteris robustula), Yoo & Yoon (1992,Hypsugo savii coreensis), Rautenbach et al. (1993,Neoromicia capensis). Except for the Europeanspecies, banded karyotypes have only been pub-lished for Glischropus tylopus (Volleth & Yong1987) and Pipistrellus mimus (Sreepada et al.1996).

Pipistrellus mimus was reported to have a dip-loid chromosome number of 34 (Manna &Talukdar 1965, McBee et al. 1986) or 38 (Pathak& Sharma 1969, Hsu & Benirschke 1973,Bhatnagar & Srivastava 1974, Naidu 1985,Sreepada et al. 1996, this paper). This may indicatethe existence of two cryptic species, as proposed byPathak & Sharma (1969).

A different karyotype from that presented herewas reported for Scotozous dormeri by Sreepadaet al. (1996; as Pipistrellus dormeri; 2n� 36,FN� 50). Again, this could suggest the presenceof cryptic species.

The European Pipistrellus and Eptesicusspecies, and also H. savii, have been well studied,mainly by conventional staining but also withbanding analyses in some cases (Zima 1982, Fedyk& Ruprecht 1983, Kasahara & Dutrillaux 1983,Ilioupoulou-Georgudaki & Giagia 1986, Zimaet al. 1989). These results concur with our dataexcept for the banding pattern on the acrocentricchromosome in Eptesicus, which originates fromthe small metacentric 16/17. The publishedEptesicus serotinus and nilssonii chromosomesare described as identical to that of E. fuscus(Bickham 1979b), which shows two bands on thischromosome. In contrast, the specimens of E.serotinus and nilssonii studied in this paper show


only one proximally situated GTG-positive band.A re-evaluation of the E. nilssonii specimenstudied by Zima (1982) resulted in a strong proxi-mal and a very weak distal band (Zima, personalcommunication). Thus, E. nilssoni differs fromthose species, such as E. circumdatus and E.bottae, which have two equally intensely stainingbands. The Japanese E. nilssonii parvus, however,shows a subtelocentric 16/17 (Ono & Yoshida1995). The reason for the differences betweenthe E. serotinus specimens studied by us and byFedyk and Ruprecht (1983) remains unclear.

Phylogenetic relationships

The formerly unresolved phylogenetic relation-ships of Pipistrellus-like bats can be elucidatedby chromosomal analysis. The features used forcladistic analysis and the resulting phylogenetictree of the Vespertilionidae have been describedin detail in Volleth & Heller (1994). ThePipistrellus-like bats, characterized morpho-logically by a shortened rostrum and reductionof tooth number, can be divided into three tribesaccording to karyological data: Pipistrellini,Vespertilionini and Eptesicini. The ¢rst and thesecond share two synapomorphic characters (stateII of chromosome 15, state II of the Xchromosome) and are distinguished from eachother by one autapomorphic feature each (stateII of chromosome 11 for Vespertilionini, stateII of chromosome 23 for Pipistrellini).

Surprisingly, not all members of the genusPipistrellus (sensu Hill & Harrison 1987) werefound to belong to the same tribe. Members ofthe subgenus Pipistrellus belong, together withspecies of the genera Glischropus, Scotozousand Nyctalus (data for the last genus in Volleth1992) to the Pipistrellini. Members of the formersubgenus Hypsugo make up the second group,Vespertilionini, together with Vespertilio,Tylonycteris, Philetor and ¢ve Australian genera(see Volleth & Tidemann 1989, 1991). Accordingto our results, Neoromicia also belongs to theVespertilionini. Therefore, to avoid a polyphyleticclassi¢cation for the genus Pipistrellus, we suggestthat Neoromicia should be afforded generic rank.

The species N. capensis studied in this papershows a rearrangement of chromosome 1/2.Whether this characteristic is an autapomorphic

feature for N. capensis or, more likely, asynapomorphic characteristic for the taxonNeoromicia, remains unresolved. However, thereis another characteristic which may be able toserve as a synapomorphic feature for this group:N. capensis and three additional South AfricanNeoromicia species (i.e. rendalli, nanus andzuluensis) have three Robertsonian fusionchromosomes in common (7/11, 8/9 and 10/12)(Kearney, personal communication).

The Pipistrellus species with 2n� 42 is the ¢rstPipistrellus kuhlii-like bat described forMadagascar (GÎpfert et al. 1995). A mor-phologically similar form from South Africa,which also possesses a diploid chromosomenumber of 42 (Rautenbach et al. 1993) and thefusion product 11/12 (Kearney, personal com-munication), and the Madagassic specimen couldbelong to the same species. Pipistrellus kuhliispecimens from Europe and North Africa,however, possess a karyotype with 2n� 44(references can be found in Zima &Horacek 1985).In the family Vespertilionidae, with only oneexception, i.e. the genus Rhogeessa (Baker et al.1985), a certain species possesses one distinctkaryotype. In several cases, one and the samekaryotype is even found in all members of a genus(e.g. Myotis, Eptesicus). Therefore, we stronglysuggest that that the Madagassic and SouthAfrican Pipistrellus kuhlii-like bats (2n� 42)should be separated from Pipistrellus kuhlii(2n� 44) owing to different karyotypes.

The present paper shows the great value of adetailed comparative cytogenetic analysis forsolving systematic relationships even when the rateof karyotype evolution of the taxon in question isbelieved to be rather low.

Acknowledgements

The material for this paper has been accumulatedover many years. During this time we receivedsupport from many people: We thank our friendsand colleagues for help with bat collecting: C.and A. Liegl, Dr. W. Metzner, Prof. G. Neuweilerand J. Sachteleben. We thank Dr. Steinhauer-Burkhart (GTZ) and our African guide BoniNdumbo for manyfold support during ourexpedition to the Kivu region.


GB undertook this research whilst serving asCurator of Mammals at the Transvaal Museum(South Africa), and thanks the Director (Dr. I.Rautenbach) and Council of Trustees for theirsupport of this project. D. Bellars and J. Konekindly provided ¢eld assistance.

Warm thanks for critical comments on themanuscript go to Dr. J. Loidl, Vienna. We alsothank Dr. K. Fredga for his review of the manu-script and many constructive suggestions.

Last but not least, my cordial thanks go to Prof.R.A. Pfeiffer and all members of the cytogeneticlaboratories of the Dept. of Human Genetics inErlangen who shared their facilities with meand my bat cultures for over a decade.

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Karyotype comparison and phylogenetic relationships of Pipistrellus-like bats (Vespertilionidae; Chiroptera; Mammalia)