An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo

Although important advances have been made in recent years in the taxonomy of different families and subfamilies of Malagasy bats, those belonging to the Vespertilioninae remain partially unresolved. Herein using a mitochondrial marker (cytochrome b) as the point of departure for 76 specimens of Malagasy vespers and appropriate African taxa, we diagnose the six taxa of this subfamily on the island by overlaying different morphological and bioacoustic characters on the clade structure of sequenced animals. The species include: endemic Neoromicia matroka, which is sister to African N. capensis; endemics N. malagasyensis and N. robertsi, which form sister species; a member of the genus Hypsugo, which is sister to African H. anchietae and described herein as new to science; Pipistrellus hesperidus for which Madagascar animals are genetically close but distinct from African populations of the same species; and endemic P. raceyi, which shows segregation of eastern (mesic) and western (dry) populations and its sister species relationships are unresolved. While the external and craniodental measurements, as well as bioacoustics variables, allow only partial differentiation of these six species of Vespertilioninae, molecular characters provide definitive separation of the taxa, as do male bacular morphology.Irene D. Pritzker Foundation associated with the Field Museum of Natural History African Training Fund; John D and Catherine T.MacArthur Foundation; Volkswagen Foundation; and the Centre de Recherche et de Veille sur les maladies émergentes dans l’Océan Indien (CRVOI) through the Fonds Européen de Développement Régional Programme Opérationnel de Coopération Territoriale Réunion, pathogènes associés à la faune sauvage ocean Indien #31189. SYNTHESYS project http://synthesys.info/, which is financed by European Community Research Infrastructure Action under the FP7 Integrating Activities Program.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1096-36422016-04-30hb2015Zoology and EntomologyMammal Research Institut

Pipistrellus hesperidus

<i>PIPISTRELLUS HESPERIDUS</i> (TEMMINCK, 1840) <p> <i>Molecular genetics</i></p> <p> K2P distance within this lineage for animals obtained in dry forest habitats of western Madagascar was 0.001 (<i>N</i> = 14, Table 1); hence, these populations show little genetic variation at the nucleotide level. No haplotype was shared in common between sequenced samples of <i>P</i>. <i>hesperidus</i> from South Africa (TM 47666) and Swaziland (TM 47738), as compared to those from Madagascar (<i>N</i> = 28, Table 9). These lineages exhibited a divergence of 1.89% (K2P), reflecting 22 fixed mutations in the trimmed sequence. Haplotype diversity was reasonably high in the Malagasy population (Hd 0.744), in spite of a lower nucleotide diversity (Pi 0.00181), but these indices were not calculated for the African population due to limited sample size.</p> <p> <i>Morphometrics</i></p> <p>Measurements presented in Table 2.</p> <p> <i>Craniodental morphology</i></p> <p> Members of this genus are easily differentiated from known Malagasy members of the genus <i>Neoromicia</i> by the presence of five post-canine teeth as compared with four in the latter genus, which lacks a first upper premolar (P 3). Bates <i>et al</i>. (2006) noted that the upper canine (C 1) was not in contact with the second upper premolar (P 4). On the basis of specimens sequenced herein, this character holds, although in some cases these two teeth are almost in direct contact.</p> <p> <i>Bioacoustics</i></p> <p>Measurements presented in Table 6.</p> <p> <i>Bacular morphology</i></p> <p> As described by Bates <i>et al</i>. (2006) for Malagasy specimens of <i>P</i>. <i>hesperidus</i>, the baculum is relatively short (1.7–1.8 mm); here we add an additional four specimens that range in total length 1.60–2.10 mm (Table 8). The distal tip and proximal base show a bifid indentation, and the shaft is distinctly recurved (Fig. 8C). This is the same configuration found in African <i>P</i>. <i>hesperidus</i> (Fig. 8D; Kearney <i>et al</i>., 2002), which ranges in total length from 1.7 to 2.5 mm (T. Kearney, unpublished data, <i>N</i> = 23).</p> <p> <i>Known geographical range</i></p> <p> In Figure 1, localities are presented of sequenced specimens of <i>P</i>. <i>hesperidus</i>, which also include the sites mentioned by Bates <i>et al</i>. (2006) for verified records of this species on Madagascar.</p> <p> <i>Taxonomic comments</i></p> <p> Different lines of evidence, including karyological distinctions (e.g. Volleth <i>et al</i>., 2001), have been published indicating that <i>P</i>. <i>hesperidus</i> is most likely paraphyletic and best considered a species complex. However, because of a lack of resolution in character differences of holotypes representing different proposed names currently considered synonyms of <i>P</i>. <i>hesperidus</i>, Simmons (2005) recognised several subspecies, which include <i>P</i>. <i>h</i>. <i>fuscatus</i> Thomas, 1901 from much of the Afrotropics and <i>P</i>. <i>h</i>. <i>subtilis</i> Sundevall, 1846 from southern Africa and Madagascar. Greater genetic sampling is needed from southern Africa to resolve the differences found in the current study between this portion of the continent and Madagascar, but given the subtle genetic differences and seemingly continuous character states associated with bacular morphology, we maintain Malagasy populations as <i>P</i>. <i>hesperidus</i>.</p> <p> Milne-Edwards (1881) described a small species of vespertilionid from Madagascar under the name <i>Vespertilio humbloti</i>. Peterson <i>et al</i>. (1995) mentioned that the type series was apparently lost and the status of this taxon could not be assessed. In the MNHN, the syntype series associated with Milne- Edwards’ description of <i>V</i>. <i>humbloti</i> was located (MNHN 1986.1074 to 1986.1082); most of the nine specimens were immature. Through the courtesy of Dr. Cécile Callou of the MNHN, the skulls from two adult specimens were extracted, cleaned, and allocated the numbers MNHN 1986.1074 and 1986.1075. Both of these specimens have a prominent first upper premolar (P 3), typical of the genera <i>Pipistrellus</i> and <i>Hypsugo</i>. Further, 1986.1074 has a seemingly bicuspid second upper incisor (I 3) and 1986.1075 an unicuspid I 3 that, in both cases, the longest portion of the I 3 reaches the length of the first upper incisor (I 2) cingulum; a diastema between C and second upper premolar (P 4), a prominent first upper premolar (P 3) in lateral view (similar to the illustration in Bates <i>et al</i>., 2006, Fig. 5B); and craniodental measurements falling within the range of <i>P</i>. <i>hesperidus</i> (Tables 3 and 4). On the basis of these different features, we identify the Milne-Edwards series of <i>V</i>. <i>humbloti</i> as <i>P</i>. <i>hesperidus</i>; this awaits confirmation based on bacular structure or molecular genetics. Hence, <i>V</i>. <i>humbloti</i> is considered a junior synonym of <i>P</i>. <i>hesperidus</i>.</p> <p> Two unsexed specimens held in the BMNH (28.1.24.1, 28.1.24.2) were collected by Capt. K. Parcon in June 1922 on Europa Island (22.3683°S, 40.3633°E) positioned halfway across the Mozambique Channel between Mozambique and Madagascar. Using the craniodental characters outlined in Bates <i>et al</i>. (2006) and Monadjem <i>et al</i>. (2010), the two animals are referable to <i>Pipistrellus</i>, and one of the specimens in relatively good shape (BMNH 28.1.24.2), based on the form of the first upper premolar (P 3) and the second upper incisor (I 3), as well as cranial shape, is identified as <i>P</i>. <i>hesperidus</i> and not <i>Neoromicia nana</i>, as written on the specimen label. This identification needs to be verified based on molecular data or potentially bacular morphology. In the same accession of the <i>Pipistrellus</i> material is a specimen of <i>Coleura seychellensis</i> Peters, 1868 (BMNH 28.1.24.3) obtained by the same captain on 4 October 1924 near Amirante Island, Seychelles (6.000°S, 53.1667°E), indicating that Capt. Parcon navigated boats in the western Indian Ocean. The context that the <i>Pipistrellus</i> specimens were obtained is ambiguous and they could have been stowaways on the vessel after docking in a coastal area and then transported to Europa Island; the direction of travel from Africa towards Madagascar or vice versa is unknown. However, if they represent a natural occurrence on Europa Island, this would provide evidence of movements for this species spanning at least half the distance between southern Africa and Madagascar.</p>Published as part of <i>Goodman, Steven M., Rakotondramanana, Claude Fabienne, Ramasindrazana, Beza, Kearney, Teresa, Monadjem, Ara, Schoeman, M. Corrie, Taylor, Peter J., Naughton, Kate & Appleton, Belinda, 2015, An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo, pp. 988-1018 in Zoological Journal of the Linnean Society 173 (4)</i> on pages 1004-1005, DOI: 10.1111/zoj.12223, <a href="http://zenodo.org/record/5335115">http://zenodo.org/record/5335115</a&gt

Neoromicia matroka

<i>NEOROMICIA MATROKA</i> (THOMAS & SCHWANN, 1905) <p> <i>Molecular genetics</i></p> <p> Specimens assigned to <i>N</i>. <i>matroka</i> and collected across the geographical range of this species (Goodman & Ramasindrazana, 2013), form a monophyletic lineage (Fig. 2) and display little in the way of genetic variation. K2P distance within this lineage was 0.008 (<i>N</i> = 11, Table 1). This species forms the sister group to sub-Saharan <i>N</i>. <i>capensis</i> and these two clades are separated by 4.5% sequence divergence. <i>Neoromicia matroka</i> was formerly considered a subspecies of <i>N</i>. <i>capensis</i> (e.g. Koopman, 1994), but based on a variety of characters outlined herein, it warrants specific recognition and placement in the genus <i>Neoromicia</i>. In turn, two specimens from Botswana identified as <i>N</i>. cf. <i>melckorum</i> (TM 48485 and TM 48487) (see Monadjem <i>et al</i>., 2010 for a definition of <i>N</i>. cf. <i>melckorum</i>), are sister to the <i>matroka - capensis</i> clade and separated by about 12% sequence divergence.</p> <p>CHARACTERIZATION OF MALAGASY VESPER BATS 1001</p> <p> <i>Morphometrics</i></p> <p>Measurements presented in Table 2.</p> <p> <i>Craniodental morphology</i></p> <p> <i>Neoromicia malagasyensis</i> and <i>N</i>. <i>matroka</i> show little overlap in craniodental measurements and these two species are readily distinguished from <i>N</i>. <i>robertsi</i> (see Tables 3 and 4).</p> <p> <i>Bioacoustics</i></p> <p>Measurements presented in Table 6.</p> <p> <i>Bacular morphology</i></p> <p> As described by Bates <i>et al</i>. (2006), the baculum in <i>N</i>. <i>matroka</i> is moderate in length and averages 2.26 mm (2.12–2.49 mm, <i>N =</i> 6, see Table 8), the distal end is flattened and deflected ventrally, the dorsal surface has a vertical projection, and there is some intraspecific variation in the development of lateral flanges (Fig. 7A, B). The general shape of the baculum in <i>N</i>. <i>matroka</i> is similar to <i>N</i>. <i>capensis</i> (Fig. 7E; Kearney <i>et al</i>., 2002); the two taxa form sister species. The total length in <i>N</i>. <i>capensis</i> is slightly shorter, averaging 2.04 mm (1.59– 2.50 mm, <i>N</i> = 37) (T. Kearney, unpublished data).</p> <p> <i>Known geographical range</i></p> <p> In Figure 1, localities are presented of sequenced specimens of <i>N</i>. <i>matroka</i>. Additional specimens presented in Bates <i>et al</i>. (2006) and Goodman <i>et al</i>. (2012a) using other molecular results or bacula morphology include: Manambolo, Ambavafatra, 47°1′25″S, 22°8′58″E (FMNH 167660); Parc National de Mantadia, 18°49.94′S, 48°25.63′E (UADBA 43674); and Ambohipo, Antananarivo, 18°55.60′S, 47°33.80′E (UADBA 43675). On the basis of an unpublished study by Malalatiana Michèle Ratsimbazafy and Alexandre Hassanin of the Muséum National d’Histoire Naturelle de Paris, there is molecular evidence of this species in the Kianjavato area, 21.3818°S, 47.8928°E, 90 m asl (Goodman <i>et al</i>., 2014) and females placed in OTU 3 of ‘ <i>N</i>. <i>matroka</i> ’ in Goodman <i>et al</i>. (2014, Fig. 7) are actually <i>P</i>. <i>raceyi</i>.</p>Published as part of <i>Goodman, Steven M., Rakotondramanana, Claude Fabienne, Ramasindrazana, Beza, Kearney, Teresa, Monadjem, Ara, Schoeman, M. Corrie, Taylor, Peter J., Naughton, Kate & Appleton, Belinda, 2015, An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo, pp. 988-1018 in Zoological Journal of the Linnean Society 173 (4)</i> on pages 1000-1002, DOI: 10.1111/zoj.12223, <a href="http://zenodo.org/record/5335115">http://zenodo.org/record/5335115</a&gt

Pipistrellus raceyi Bates, Ratrimomanarivo, Harrison & Goodman 2006

<i>PIPISTRELLUS RACEYI</i> BATES <i>ET AL</i>., 2006 <p> <i>Molecular genetics</i></p> <p> This endemic species is known from two distinct portions of Madagascar. The samples used in the intraspecific genetic analysis include ten animals from dry deciduous western forests and nine specimens from humid eastern area. The K2P distance within <i>P</i>. <i>raceyi</i> when these lineages were pooled was 0.004 (<i>N</i> = 7, Table 1). No haplotypes were shared between the eastern and western populations (<i>N</i> = 17, Table 9). These populations exhibited a divergence of 0.74% (K2P), reflecting five fixed mutations in the trimmed cytochrome <i>b</i> sequences (680 bp). When the full sequence set of <i>P</i>. <i>raceyi</i> was examined (<i>N</i> = 17, Table 9), diversity was reasonably high in the western population (Hd 0.722, Pi 0.00287), but was noticeably low in the eastern population (Hd 0.429, Pi 0.00057), which was only represented by two different haplotypes and distinguished by a single base change.</p> <p> This species is notably divergent from the other Vespertilioninae samples included in the analysis, specifically members of the genus <i>Pipistrellus</i>, and without more extensive taxonomic and geographic sampling, little can be advanced concerning possible sister taxa or the geographical region the ancestral form that colonized Madagascar was from. Bates <i>et al</i>. (2006) suggested, based on baculum morphology, that <i>P</i>. <i>raceyi</i> might be closely related to Asiatic <i>P</i>. <i>endoi</i> Imaizumi, 1959. This interesting hypothesis warrants further molecular investigation.</p> <p> <i>Morphometrics</i></p> <p>Measurements presented in Table 2. Animals from eastern populations are in general larger than those from western populations.</p> <p> * <i>Hypsugo bemainty</i> was only sampled as a single population.</p> <p> †Only two samples were available from the African population of <i>Pipistrellus hesperidus</i> and intraspecific diversity could not be calculated.</p> <p> <i>Craniodental morphology</i></p> <p> As mentioned under this header for <i>P</i>. <i>hesperidus</i>, members of this genus occurring on Madagascar are easily differentiated from <i>Neoromicia</i> based on upper toothrow counts. Further, Bates <i>et al</i>. (2006) have provided a few characters to differentiate <i>P</i>. <i>hesperidus</i> from <i>P</i>. <i>raceyi</i>, the other member of the genus occurring on Madagascar: (1) the second upper incisor (I 3) ≤½ the length of the first upper incisor (I 2) (in <i>hesperidus</i>) vs. I 3 nearly the length of I 2 (in <i>raceyi</i>); (2) I 2 is unicuspid (in <i>hesperidus</i>) vs. bicuspid (in <i>raceyi</i>); and (3) that the first upper premolar (P 3) is notably small (in <i>hesperidus</i>) as compared to large and prominent (in <i>raceyi</i>). A reevaluation of these characters based on the specimens of <i>P</i>. <i>hesperidus</i> sequenced in this study (<i>N =</i> 13) found: characters 1 – consistent in all cases, character 2 – almost uniformly consistent, with the exception of MNHN 1986.1074 with a seemingly bicuspid I 2, character 3 – consistent in all cases, with some variation in size of the P 3, which was always smaller than in <i>P</i>. <i>raceyi</i>. Bates <i>et al</i>. (2006) also noted that the relative length of I 3 to I 2 between <i>P</i>. <i>hesperidus</i> and <i>Hypsugo anchietae</i> [= <i>H</i>. <i>bemainty</i> sp. nov., see below] were similar and the best means to differentiate these two taxa based on craniodental characters was the former has a distinctly thin zygomata and without a jugal eminence (fig. 11 in Bates <i>et al</i>., 2006).</p> <p> <i>Bioacoustics</i></p> <p> Bioacoustics (average values, sexes combined, <i>N</i> = 4, which include from the west 2 ♂♂ and 1 ♀ and from the east 1 ♂): PF: 56.6 (range 53.4–58.2) kHz, Fmax: 114.8 (range 91.7–128.3) kHz, Fmin: 46.0 (range 41.7– 51.4) kHz, Dur: 2.6 (range 2.3–3.1) ms, IPI: 67.3 (range 14.8–125.0) ms (Table 6). The single sequenced female with associated bioacoustic recordings had several parameters that fell outside the range for three males (Fig. 6). Further work is needed to verify this pattern. <i>Bacular morphology</i></p> <p> In their description of <i>P</i>. <i>raceyi</i>, Bates <i>et al</i>. (2006) provided details on its bacular morphology. The length of this structure in <i>P</i>. <i>raceyi</i> is distinctly longer than any other Malagasy vesper and measures 8.90 and 10.00 mm (Table 8). It has a long and narrow shaft, with a slight curvature, and variable deflection at the distal end that seems to indicate some intraspecific variation (Fig. 8A, B). A specimen obtained outside of Andasibe (FMNH 222722) had a distinctly shorter baculum (6.10 mm), which may be related to age and lack of ossification of the structure (Table 8, Fig. 8B).</p> <p> <i>Known geographical range</i></p> <p> Localities from which sequenced specimens of this species are known are presented in Figure 1. One specimen from another site referred to this species includes Forêt de Mikea, 22°16.00′S, 43°28.70′E (FMNH 176165) (Bates <i>et al</i>., 2006).</p> <p> <i>Taxonomic comments</i></p> <p> Notable genetic differences were found between eastern and western populations of <i>P</i>. <i>raceyi</i>, with the type specimen being from the eastern locality of Kianjavato (Fig. 1). Further, statistically significant differences were identified between specimens from these two different portions of the island in several external measurements (Table 2) and in all cranial and dental measurements (Tables 3 and 4). Further work is needed to assess the level of genetic divergence and possible reproductive isolation between eastern and western populations; herein we maintain them as a single taxon, with divergent populations.</p>Published as part of <i>Goodman, Steven M., Rakotondramanana, Claude Fabienne, Ramasindrazana, Beza, Kearney, Teresa, Monadjem, Ara, Schoeman, M. Corrie, Taylor, Peter J., Naughton, Kate & Appleton, Belinda, 2015, An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo, pp. 988-1018 in Zoological Journal of the Linnean Society 173 (4)</i> on pages 1007-1008, DOI: 10.1111/zoj.12223, <a href="http://zenodo.org/record/5335115">http://zenodo.org/record/5335115</a&gt