Saturday, October 31, 2015

[Mammalogy • 2015] Rhinolophus luctoides • Surprising Genetic Diversity in Rhinolophus luctus (Chiroptera: Rhinolophidae) from Peninsular Malaysia: Description of a New Species Based on Genetic and Morphological Characters

Rhinolophus luctoides
Volleth, Loidl, Mayer, Yong, Müller & Heller, 2015
 DOI: 10.3161/15081109ACC2015.17.1.001

In the family Rhinolophidae, the members of the trifoliatus clade are easily recognisable by a unique noseleaf structure and a fluffy fur. Within this group, Rhinolophus luctus is the largest species with currently six recognized subspecies, distributed from India to Bali. We investigated genetic (karyotype, mitochondrial DNA sequence) and morphological characters from a Peninsular Malaysian sample. Although the diploid number was 2n = 32 in all specimens, karyotype analysis revealed two largely different chromosomal sets, with a Y-autosome translocation present only in one of the taxa. Morphological examination revealed differences concerning size of the baculum and length of the lower toothrow. Based on these results, a new species is described [Rhinolophus luctoides] and the former subspecies distributed on the Malayan Peninsula, Rhinolophus luctus morio, is elevated to species rank, Rhinolophus morio.

 Keywords: Rhinolophus luctoidesRhinolophus morioRhinolophus trifoliatus, Y-autosome translocation, synaptonemal complex, baculum, echolocation frequency, FISH, mtDNA


In 2005, the number of recognized species of the monotypic family Rhinolophidae (horseshoe bats) was 77 (Simmons, 2005). Since then, at least 19 new species have been described on the basis of morphological differences, corroborated by molecular (12 species) and karyological (one species) data, at present resulting in a total of about 96 species (Yoshiyuki and Lim, 2005; Soisook et al., 2008; Wu et al., 2008, 2009, 2011; Zhou et al., 2009; Benda and Vallo, 2012; Taylor et al., 2012; Kerbis Peterhans et al., 2013; Patrick et al., 2013, Soisook et al., 2015). This rapid increase in species number reflects the notion that the general morphological uniformity in the genus Rhinolophus masks subtle species specific differences, which can be recognized only through detailed studies. Representative examples, where species initially have been proposed on the basis of DNA sequence divergences that were subsequently confirmed by morphological data, can be found in South Africa (Taylor et al., 2012; Jacobs et al., 2013) and South-East Asia (Patrick et al., 2013). Based on morphological features, the genus Rhinolophus is divided into 15 groups (Csorba et al., 2003). Among these, most easily recognized are the members of the R. trifoliatus group by their long, fluffy fur and a unique noseleaf structure with lateral lappets at the base of the sella. This clade, which corresponds to the subgenus Aquias Gray 1847 (Guillén-Servent et al., 2003), is distributed from the Indian subcontinent to Southeast Asia. The mem bers of the trifoliatus group are clearly distinguished by their body size. In addition to the smallsized species R. sedulus and the medium-sized R. trifoliatus, large-sized members are found throughout the whole distributional range, from Sri Lanka to Nepal on the Indian subcontinent in the west, to the southern parts of China in the east and north, and to the Indonesian islands Java, Sumatra and Bali in the south. The first large-sized specimen from Java was described as R. luctus by Temminck in 1835.

However, quite a large number of subspecies or closely related species has been described subsequently, which were all subsumed as subspecies of R. luctus by Tate in 1943. A short summary of the complicated history of this taxon can be found in Topál and Csorba (1992). The Indian R. beddomei, formerly a subspecies of R. luctus, was elevated to species rank for the reason of a different shape of the upper canine and general size differences (Topál and Csorba, 1992). A deviating diploid chromosome number (see below) and smaller body size led Yoshiyuki and Harada (1995) to re-establish the specific rank of R. formosae Sanborn, 1939. However, there are still six different names, which have originally been designated as names for species, subspecies or races but are now all subsumed under the species name Rhinolophus luctus. Simmons (2005) accepted perniger, lanosus, spurcus as inhabitants of the northern parts of the distributional range, as well as luctus, morio and foetidus as subspecies of R. luctus, whereas geminus was considered as synonymous with luctus. The assignment of a specimen to a certain R. luctus subspecies can presently be done only by the sampling locality as distinct morphological differences have not been described.

The members of the trifoliatus clade are not only clearly separated by morphological features from their congeners, but also by a cytogenetic feature, i.e., a low diploid chromosome number (2n). Typically, the genus Rhinolophus is karyologically characterized by a high 2n with the majority of species showing a diploid number higher than 56. Apart from the exceptional case of R. hipposideros with its three karyotypic variants 2n = 54, 56 and 58 (reviewed in Volleth et al., 2013), only a small number of species with a diploid chromosome number smaller than 56 has been reported so far. According to Csorba et al. (2003) they belong to four species groups: (1) the rouxi group (R. rouxi 2n = 56, R. sinicus 2n = 36, R. thomasi 2n = 36), (2) the pearsoni group (R. pearsoni 2n = 42 and 44, R. yunanensis 2n = 46) and (3) the euryotis group (R. rufus 2n = 40) (Zhang, 1985; Zima et al., 1992; Rickart et al., 1999; Gu et al., 2003; Ao et al., 2007; Mao et al., 2007; Wu et al., 2009). The fourth group with 2n lower than 56 is the R. trifoliatus clade.

Up to now, only conventionally stained chromosomes of two R. luctus subspecies have been described. A non-differentially stained karyotype with 2n = 32, a submetacentric X and an acrocentric Y from a single male specimen assigned to R. l. perniger was reported by Harada et al. (1985) from northern Thailand. From a central Thailand province, a female specimen designated as R. l. morio with 2n = 32 was described having a karyotype similar to R. l. perniger, however, without presenting a karyotype image (Hood et al., 1988). A karyotype comprising 2n = 32 chromosomes has also been reported for R. beddomei from India (Naidu and Gururaj, 1984; Koubínová et al., 2010). Further, according to the differing diploid number of 52 (Ando et al., 1980, 1983), the former R. luctus subspecies formosae is now treated as a separate species (Yoshiyuki and Harada, 1995). The only species from the trifoliatus group for which a differentially stained karyotype has been published is the smallest species of the clade, R. sedulus, with a diploid number of 2n = 28 (Volleth et al., 2014).

During our chromosomal study of members of the trifoliatus group from Peninsular Malaysia, we were intrigued to find two distinctly different chromosomal sets among our ‘R. luctus’ sample. Initially, the discovery of an unusual sex chromosome system in the first specimen called for the investigation of additional specimens. The second individual, however, unexpectedly carried a different karyotype. In the present paper we report on morphological, karyological and mitochondrial DNA sequence differences found between these two cryptic rhinolophid species from Peninsular Malaysia. The results show that two forms exist in close geographic proximity, which according to genetic features represent distinct species.


Rhinolophus luctoides sp. nov.
 Volleth, Loidl, Mayer, Yong, Müller & Heller, 2015

Etymology: The name luctoides was chosen because this species, regarding external appearance, is very similar to subspecies of R. luctus.

Habitat: Rhinolophus luctoides was found in selectively logged Dip tero carp Rain Forest at elevations higher than 600 m, 5 km NE of the Field Studies Centre (FSC) of Ulu Gombak, and in Montane Rain Forest of Gent ing Highlands and Cameron Highlands. The hab itat of the Gombak valley, where the FSC is situated, has been described in detail by Medway (1966). The surroundings of the FSC have been reported as one of the locations with the highest species richness of bats in the Old World (Sing et al., 2013).

Rhinolophus morio Gray, 1842 status revivisco

The skull dimensions of the holotype of R. morio Gray, 1842 from Singapore, deposited in the Natural History Museum London, are similar to those of the two lowland specimens collected by us in the vicinity of Kuala Lumpur (Templer Park, Rawang). Concerning the ratio of lower toothrow to mandible length, the taxon morio comes close to subspecies of R. luctus (perniger, foetidus, lanosus). However, morio differs clearly in the ratio zygomatic width to mandible length from the above mentioned subspecies. In this respect, morio resembles other genera in the trifoliatus clade, i.e. R. trifoliatus, R. sedulus and R. beddomei (Table 3). By reason of these cranial proportions and the characteristic karyotype with the unique Y-autosomal translocation (see below), we elevate the taxon morio to species rank (Rhinolophus morio stat. rev.).

Marianne Volleth, Josef Loidl, Frieder Mayer, Hoi-Sen Yong, Stefan Müller and Klaus-Gerhard Heller. 2015. Surprising Genetic Diversity in Rhinolophus luctus (Chiroptera: Rhinolophidae) from Peninsular Malaysia: Description of a New Species Based on Genetic and Morphological Characters.  Acta Chiropterologica. 17(1):1-20. DOI: 10.3161/15081109ACC2015.17.1.001