Karyotype comparison of five African Vespertilionini species with comments on phylogenetic relationships and proposal of a new subtribe Author Volleth, Marianne Department of Human Genetics, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany & Present address: Triesdorf Bahnhof 8, 91732 Merkendorf, Germany & Corresponding author: E-mail: mvolleth 19 @ gmail. com mvolleth19@gmail.com Author Mayer, Frieder Leibnitz Institute for Evolution and Biodiversity Science, Museum für Naturkunde, Invalidenstr. 43, 10115 Berlin, Germany Author Heller, Klaus-Gerhard Triesdorf Bahnhof 8, 91732 Merkendorf, Germany Author Müller, Stefan Institute of Human Genetics, Munich University Hospital, Ludwig-Maximilian University, Goethestr. 29, 80336 Munich, Germany Author Fahr, Jakob Department of Experimental Ecology and Conservation Genomics, University of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany & Present address: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, Göttinger Chaussee 76 a, 30453 Hannover, Germany text Acta Chiropterologica 2023 2023-08-03 25 1 35 52 journal article 280654 10.3161/15081109ACC2023.25.1.002 78082210-95c4-4565-8b3f-734a661e613d 1733-5329 10265140 Nycticeinops happoldorum ( Hutterer et al ., 2019 ) The examined male of N. happoldorum (NHA) showed a karyotype with 2n = 24 chromosomes (FNa = 44) with eight large- to medium-sized meta- to submetacentric autosomal pairs and three medium-sized subtelocentric pairs. The X chromosome was a medium-sized submetacentric and the Y chromosome was a small metacentric chromosome ( Fig. 8 ). The composition of meta- and submetacentric autosomal pairs was analysed by G-band pattern comparison with the basic vespertilionid karyotype and with karyotypes of other Vespertilionini species. None of the four metacentric pairs of Myotis was conserved in N. happoldorum . Instead, the following arm combinations were found in pairs NHA1 to NHA8: 1/13, 2/7, 3/6, 4/9, 5/12, 8/10, 14/19, and 15/18. The composition of the three subtelocentric pairs was verified using MMY painting probes. NHA9 showed homology to three small Myotis chromosomes, MMY 23 in the short arm, MMY20 and MMY 22 in the long arm, separated by a small heterochromatic segment. NHA10 was homologous to the combined MMY24 and MMY25 painting probe in the short and MMY 11 in the long arm. FISH with tree shrew TBE30 probe confirmed homologous sequences to MMY 24 in the distal part of the NHA10 short arm. The smallest subtelocentric pair, NHA11, was shown to bear MMY21 homologous sequences in the short arm and the proximal part of the long arm, and MMY16/17 (TBE5) homologous sequences in the distal part of the long arm. The G-banding pattern of the X chromosome differed clearly from that of state II, which characterizes Vespertilionini and Pipistrellini species ( Fig. 7 ). FIG . 5. Rearranged bi-armed pair 3/4 of P. brunnea depicted from left to right after G-banding, C-banding, FISH with E. macaco painting probe EMA8 and T. belangeri probe TBE6. The extant centromere is indicated by a dash, the ancestral centromere position by an asterisk. In the basic karyotype, EMA8 homologous sequences were located in the proximal region of the MMY3 homologous chromosomal arms, whereas here they are found in the long arm of P. brunnea chromosome 3/4 (TBE6, homologous to parts of MMY4, was used to delimit the proximal and distal regions of the MMY4 homologous segment in P. brunnea chromosome 3/4) C-banding revealed a nearly completely heterochromatic Y chromosome and few small non-centromeric heterochromatic segments in addition to weak centromere staining. Only one homologue of NHA1 showed a terminally located heterochromatic band on the long arm. In NHA3 and NHA5, pericentromeric heterochromatin was present, but the homologs differed concerning the position, either in the short or the long arm. In NHA9, an interstitial C-positive band was present in the middle of the long arm, whereas a C-band positive segment was present at the long arm telomere in NHA11 on both homologs ( Fig. 6B ). FIG . 6. C-banded metaphase spreads of (A) P. brunnea , (B) N. happoldorum , and (C) N. schlieffenii . X and Y chromosomes and heterochromatic segments of interest are indicated, numbers refer to MMY homologies. In (B), arrows point to those heterochromatic segments which showed differences between the homologs and arrowheads point to C-positive segments on pairs NHA11 and NHA13. For further explanation see main text FIG . 7. Examples of G-banded X chromosomes: The banding patterns of the X chromosomes from L. kirinyaga (LKI) and N. schlieffeni (NSC) were similar to that of state II of the basic vespertilionid karyotype. The N. guineensis (NGU) X was the product of an X-autosome translocation and the X chromosomes from P. brunnea (PBR) and N. happoldorum (NHA) showed unique derived G-banding patterns The NORs are positioned at the SC of the NHA8 long arm. Interestingly, NHA8, the fusion product of MMY18 and MMY15 homologs, showed a heteromorphism concerning the position of the centromere ( Fig. 9 ). One homologue conserved the ancestral condition concerning the position of NOR and centromere. In the second homologue, however, the centromere is located within the MMY18 homologous arm, resulting in a subtelocentric shape. As the G-banding pattern was preserved, a centromere repositioning event is assumed as mode of rearrangement. The chromosomes 11, 12, and 15 were present in vespertilionid state II. The baculum of this specimen is depicted in Fig. 10 .