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Journal of Chemical Neuroanatomy 40 (2010) 210–222
Contents lists available at ScienceDirect
Journal of Chemical Neuroanatomy
journal homepage: www.elsevier.com/locate/jchemneu
Nuclear organization of cholinergic, putative catecholaminergic and
serotonergic systems in the brains of five microchiropteran species
Jean-Leigh Kruger a, Leigh-Anne Dell a, Adhil Bhagwandin a, Ngalla E. Jillani a,
John D. Pettigrew b, Paul R. Manger a,*
a
b
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa
Queensland Brain Institute, University of Queensland 4072, Australia
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 26 March 2010
Received in revised form 28 May 2010
Accepted 28 May 2010
Available online 4 June 2010
The current study describes, using immunohistochemical methods, the nuclear organization of the
cholinergic, catecholaminergic and serotonergic systems within the brains of five microchiropteran
species. For the vast majority of nuclei observed, direct homologies are evident in other mammalian
species; however, there were several distinctions in the presence or absence of specific nuclei that
provide important clues regarding the use of the brain in the analysis of chiropteran phylogenetic
affinities. Within the five species studied, three specific differences (presence of a parabigeminal nucleus,
dorsal caudal nucleus of the ventral tegmental area and the absence of the substantia nigra ventral)
found in two species from two different families (Cardioderma cor; Megadermatidae, and Coleura afra;
Emballonuridae), illustrates the diversity of microchiropteran phylogeny and the usefulness of brain
characters in phylogenetic reconstruction. A number of distinct differences separate the microchiropterans from the megachiropterans, supporting the diphyletic hypothesis of chiropteran phylogenetic
origins. These differences phylogenetically align the microchiropterans with the heterogenous grouping
of insectivores, in contrast to the alignment of megachiropterans with primates. The consistency of the
changes and stasis of neural characters with mammalian phylogeny indicate that the investigation of the
microchiropterans as a sister group to one of the five orders of insectivores to be a potentially fruitful
area of future research.
ß 2010 Elsevier B.V. All rights reserved.
Keywords:
Microbat
Chiroptera
Neuromodulatory systems
Diphyly
Evolution
Mammalia
1. Introduction
The proposed monophyletic order Chiroptera has been divided
into two suborders: megachiroptera and microchiroptera; however,
Linnaeus originally grouped the megachiropterans with primates.
This classification was largely ignored until the finding that primates
and megachiropterans share several advanced visual pathway
characteristics, in particular the retinotectal pathway, that are not
shared by other mammals (Pettigrew, 1986; Pettigrew et al., 1989,
2008). The ‘‘flying primate’’ hypothesis proposes that the megachiropterans, with the dermopterans, form a sister group to the
primates, and is based on several derived neural features that are
absent in microchiropterans and other mammals (Pettigrew et al.,
1989, 2008; Manger et al., 2001; Maseko and Manger, 2007; Maseko
Abbreviations: III, oculomotor nucleus; Vmot, motor division of trigeminal nucleus; VI, abducens nucleus; VIId, facial nerve nucleus, dorsal division; VIIv, facial nerve nucleus,
ventral division; A1, caudal ventrolateral medullary tegmental nucleus; A2, caudal dorsomedial medullary nucleus; A4, dorsal medial division of locus coeruleus; A5, fifth
arcuate nucleus; A6c, compact portion of locus coeruleus; A6d, diffuse portion of locus coeruleus; A7d, nucleus subcoeruleus, diffuse portion; A7sc, nucleus subcoeruleus,
compact portion; A8, retrorubral nucleus; A9l, substantia nigra, lateral; A9m, substantia nigra, medial; A9pc, substantia nigra, pars compacta; A9v, substantia nigra, ventral or
pars reticulata; A10, ventral tegmental area; A10c, ventral tegmental area, central; A10d, ventral tegmental area, dorsal; A10dc, ventral tegmental area, dorsal caudal; A11,
caudal diencephalic group; A12, tuberal cell group; A13, zona incerta; A14, rostral periventricular nucleus; A15d, anterior hypothalamic group, dorsal division; A15v, anterior
hypothalamic group, ventral division; A16, catecholaminergic neurons of the olfactory bulb; AP, area postrema; B9, supralemniscal serotonergic nucleus; C1, rostral
ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary nucleus; ca, cerebral aqueduct; CLi, caudal linear nucleus; CVL, caudal ventrolateral serotonergic
group; DRc, dorsal raphe nucleus, caudal division; DRd, dorsal raphe nucleus, dorsal division; DRif, dorsal raphe nucleus, interfascicular division; DRl, dorsal raphe nucleus,
lateral division; DRp, dorsal raphe nucleus, peripheral division; DRv, dorsal raphe nucleus, ventral division; EW, Edinger–Westphal nucleus; Fr, fasciculus retroflexus; GC,
periaqueductal grey matter; IC, inferior colliculus; IP, interpeduncular nucleus; LDT, laterodorsal tegmental nucleus; MnR, median raphe nucleus; PC, cerebral peduncle; pVII,
preganglionic motor neurons of the superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the inferior salivatory nucleus; PBg, parabigeminal
nucleus; PPT, pedunculopontine nucleus; Rmc, red nucleus, magnocellular division; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus;
RVL, rostral ventrolateral serotonergic group; SC, superior colliculus; scp, superior cerebellar peduncle.
* Corresponding author. Tel.: +27 11 717 2497; fax: +27 11 717 2422.
E-mail address: [email protected] (P.R. Manger).
0891-0618/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchemneu.2010.05.007
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
et al., 2007). The logical conclusion of this hypothesis is that the
Chiropteran order is actually diphyletic and flight evolved twice in
mammals. A summary of the evidence on each side of the debate
about whether bats are monophyletic or diphyletic is provided in the
accompanying paper (Dell et al., 2010).
Most studies that support the diphyletic origin of bats have
concentrated on specific neuroanatomical structures in megachiropterans, with little work to date having been done in
microchiropterans. Maseko and Manger (2007) and Maseko
et al. (2007) undertook investigations into the nuclear organization
of the cholinergic, catecholaminergic and serotonergic systems in
the microchiropteran Miniopterus schreibersii, and the megachiropteran Rousettus aegyptiacus, which defined several further
differences between the mega- and microchiropterans lending
further support for the diphyletic origin of the Chiroptera. In
contrast, studies favouring Chiropteran monophyly are usually
based on DNA and molecular findings (e.g. Murphy et al., 2001;
Teeling et al., 2005; Van Den Bussche and Hoofer, 2004).
Manger (2005) proposed, based on studies of the nuclear
organization of the cholinergic, catecholaminergic and serotonergic systems of a range of mammalian species (see also Maseko
et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008; Gravett
et al., 2009; Pieters et al., 2010; Bux et al., 2010), that all species
within an order will exhibit the same complement of homologous
nuclei of these systems. This proposal infers that if mega- and
microchiropterans belonged to the same mammalian order, they
should have the same nuclear organization of these systems;
however this is not the case as shown by Maseko and Manger
(2007) and Maseko et al. (2007).
While these previous studies lend support to the diphyletic origin
of the Chiropterans, it should be noted that the microchiropterans in
particular are one of the most species-rich suborders of mammals,
consisting of over 800 species (Nowack, 1999). Thus, before firm
conclusions regarding differences between the two suborders can be
made, further species should be investigated. The current study
describes the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems in the brains of five previously
unstudied microchiropteran species from a range of phylogenetically distant families (distant within the microchiropteran suborder). A speculative hypothesis proposed that the shared midbrain
binocular circuitry of primates and megachiropterans represented
two independent evolutionary events that were each driven by the
selective forces of the ‘‘small branch niche’’ (Martin, 1986). It is
important to note that the vast majority of the nuclei of the systems
under investigation here do not play any direct role in the neural
processes related to flight, vision or echolocation and as such the
findings cannot be ignored on the basis of sensory or motor
specialisations of the Chiroptera, an argument that has been
previously levelled at the studies of Chiropteran neuroanatomy
that support the diphyletic hypothesis (Martin, 1986; Allman, 1999).
2. Materials and methods
Three brains of each of the following microchiropteran species were used in this
study: Cardioderma cor (average body mass = 26 g; average brain mass = 670 mg),
Chaerophon pumilus (average body mass = 5.4 g; average brain mass = 122 mg),
Coleura afra (average body mass = 11.5 g; average brain mass = 257 mg), Hipposideros
commersoni (average body mass = 101.9 g; average brain mass = 750 mg) and
Triaenops persicus (average body mass = 13.7 g; average brain mass = 271 mg). All
animals were captured from wild populations in Kenya and were treated and used
according to the guidelines of the University of the Witwatersrand Animal Ethics
Committee, the Kenya National Museums and the Kenyan Wildlife Services. The
animals were euthanazed (Euthanaze, 1 ml/kg, i.p.) and upon cessation of respiration,
perfused intracardially with an initial rinse of 0.9% saline solution at 4 8C (1 ml/g body
mass) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4 8C (1 ml/g
body mass). After removal from the skull, each brain was post-fixed overnight in the
paraformaldehyde solution and subsequently stored in an anti-freeze solution at
20 8C. Before sectioning, the brains were allowed to equilibrate in 30% sucrose in
0.1 M PB at 4 8C. Each brain was then frozen in crushed dry ice and sectioned into
211
50 mm thick serial coronal sections on a freezing microtome. A one in four series of
stains was made for Nissl substance, choline-acyltransferase (ChAT), tyrosine
hydroxylase (TH) and serotonin (5-HT). Sections for Nissl staining were first mounted
on 0.5% gelatine coated slides, cleared in a solution of 1:1 absolute alcohol and
chloroform and then stained with 1% cresyl violet.
The sections used for immunohistochemical staining were treated for 30 min in
an endogenous peroxidase inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30%
hydrogen peroxide) followed by three 10 min rinses in 0.1 M PB. Sections were then
pre-incubated for 2 h, at room temperature, in blocking buffer (3% normal rabbit
serum, NRS, for ChAT sections or 3% normal goat serum, NGS, for TH and 5-HT
sections, 2% bovine serum albumin, BSA, and 0.25% Triton X-100 in 0.1 M PB).
Thereafter sections were incubated in the primary antibody solution in blocking
buffer for 48 h at 4 8C under gentle agitation. Anti-cholineacetyltransferase
(AB144P, Millipore, raised in goat) at a dilution of 1:3000 was used to reveal
cholinergic neurons. Anti-tyrosine hydroxylase (AB151, Millipore, raised in rabbit)
at a dilution of 1:7500 revealed the catecholaminergic neurons. Serotonergic
neurons were revealed using anti-serotonin (AB938, Millipore, raised in rabbit) at a
dilution of 1:10,000. This incubation was followed by three 10 min rinses in 0.1 M
PB and the sections were then incubated in a secondary antibody solution (1:750
dilution of biotinylated anti-goat IgG, BA 5000, Vector Labs, for ChAT sections or a
1:750 dilution of biotinylated anti-rabbit IgG, BA 1000, Vector Labs, for TH and 5-HT
sections, in a blocking buffer containing 3% NGS/NRS and 2% BSA in 0.1 M PB) for 2 h
at room temperature. This was followed by three 10 min rinses in 0.1 M PB, after
which sections were incubated for 1 h in AB solution (Vector Labs), followed by
three 10 min rinses in 0.1 M PB. Sections were then placed in a solution containing
0.05% diaminobenzidine (DAB) in 0.1 M PB for 5 min, followed by the addition of
3 ml of 30% hydrogen peroxide per 0.5 ml of solution. Chromatic precipitation was
visually monitored and verified under a low power stereomicroscope. Staining
continued until such time as the background stain was at a level that would allow
for accurate architectonic reconstruction without obscuring the immunopositive
neurons. Development was arrested by placing sections in 0.1 M PB, followed by
two more rinses in this solution.
Sections were then mounted on 0.5% gelatine coated glass slides, dried overnight,
dehydrated in a graded series of alcohols, cleared in xylene and coverslipped with
Depex. The controls employed in this experiment included the omission of the
primary antibody and the omission of the secondary antibody in selected sections.
As a further control for the cholinergic immunohistochemistry, we used choline
acetyltransferase (AG220, Millipore) at a dilution of 5 mg/ml in the primary
antibody solution (see above) as an inhibition assay. This solution was incubated for
3 h at 4 8C prior to being used on the sections. We also reacted adjacent sections that
were not inhibited. In the sections where the primary antibody had been inhibited,
no staining was evident. Sections were examined under a low power stereomicroscope and using a camera lucida the architectonic borders of the sections were
traced following the Nissl stained sections. Sections containing the immunopositive
neurons were then matched to the drawings and the neurons were marked. Select
drawings were then scanned and redrawn using the Canvas 8 drawing program.
Digital photomicrographs were captured using a Zeiss Axioskop and the Axiovision
software. No adjustments of pixels, or manipulation of the captured images were
undertaken, except for the adjustment of contrast, brightness, and levels using
Adobe Photoshop 7.
All architectonic nomenclature was taken from the atlas of a Microchiropteran
brain (Baron et al., 1996), while the nomenclature used to describe the
immunohistochemically revealed systems was based on Dahlström and Fuxe
(1964), Hökfelt et al. (1984), Törk (1990), Woolf (1991), Smeets and González
(2000), Manger et al. (2002a,b,c), Maseko and Manger (2007), Maseko et al. (2007),
Moon et al. (2007), Dwarika et al. (2008), Limacher et al. (2008), Bhagwandin et al.
(2008), Gravett et al. (2009) and Pieters et al. (2010).
3. Results
The nuclear organization of the cholinergic, catecholaminergic
and serotonergic neural systems generally followed that found by
Maseko and Manger (2007) in their study of M. schreibersii,
although there were some notable differences between species,
specifically in the cholinergic and catecholaminergic systems, and
these are explicitly described. The following description applies
generally to all the microchiropteran species studied, except where
specifically noted.
3.1. Cholinergic nuclei
The cholinergic system is generally subdivided into the cortical
cholinergic interneurons, the striatal region, basal forebrain,
diencephalon, pontomesencephalon and the cranial nerve nuclei
groups (Woolf, 1991). In the five species examined in the current
study, we identified cholineacetyltransferase immunoreactive
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(ChAT+) neurons in all these subdivisions, excluding the cortical
cholinergic interneurons, which have a variable occurrence across
mammalian species (e.g. Bhagwandin et al., 2006). Additionally,
there was no evidence of any ChAT+ neurons in the medullary
tegmental field.
3.1.1. Cholinergic striatal interneurons
ChAT+ neurons were found in the Islands of Calleja, the olfactory
tubercle, nucleus accumbens, the caudate/putamen complex and in
the globus pallidus. The distribution, density, topography and
nuclear organization of ChAT+ neurons in these areas was found to
be similar across all five microchiropteran species, as well as
following the distribution and morphology as described by Maseko
and Manger (2007) in their study of M. schreibersii.
tegmental (LDT) nuclei in all five species studied (Fig. 1). An
unusual dense aggregation of ChAT+ neurons in the PPT of C.
pumilus was observed in the postero-lateral portion of this nucleus
in a position lateral to the superior cerebellar peduncle (Fig. 2);
however, the remaining species evinced ‘‘typical’’ microchiropteran PPT and LDT morphology (Maseko and Manger, 2007). In
contrast to the findings of Maseko and Manger (2007), evidence of
the parabigeminal nucleus was found in C. cor and C. afra (Fig. 3).
ChAT+ neurons in the parabigeminal nucleus were most readily
observed in C. cor, with only weak staining occurring in this
nucleus in C. afra. In both species, the location of the parabigeminal
[(Fig._2)TD$IG]
3.1.2. Cholinergic basal forebrain nuclei
The basal forebrain nuclei are composed of the medial septal
nucleus, the Diagonal band of Broca and the nucleus basalis (Woolf,
1991; Maseko et al., 2007). ChAT+ neurons were observed in all of
these nuclei across all five microchiropteran species, as found by
Maseko and Manger (2007) in M. schreibersii. No notable
interspecies differences were observed.
3.1.3. Diencephalic cholinergic nuclei
ChAT+ neurons were found in the medial habenular nucleus, as
well as in the three hypothalamic nuclei (dorsal, lateral and
ventral) in all five microchiropteran species. In T. persicus there was
very strong ChAT immunoreactivity in the medial habenular
nucleus, as well as in the lateral hypothalamic cluster, while C. afra
and C. cor showed only a weak reactivity in the lateral
hypothalamic region. The ventral hypothalamic nucleus was most
well expressed in C. cor. In general, our findings were similar across
all five microchiropteran species and in terms of nuclear
organization are identical to that found in M. schreibersii (Maseko
and Manger, 2007). No evidence of ChAT+ neurons could be found
in the anterior nuclei of the dorsal thalamus as seen in the rock
hyrax (Gravett et al., 2009).
3.1.4. Midbrain/pontine cholinergic nuclei
As with the observations made in M. schreibersii, we observed
ChAT+ neurons in the pedunculopontine (PPT) and the laterodorsal
[(Fig._1)TD$IG]
Fig. 1. Photomicrograph of ChAT immunopositive neurons forming the laterodorsal
tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT) nuclei
in Cardioderma cor. The large inferior colliculus (IC) gives a slightly different
impression of the topological relations of LDT and PPT, although these nuclei still
maintain the same basic features as in all other mammals observed to date. Scale
bar = 500 mm. ca – cerebral aqueduct.
Fig. 2. Photomicrographs of three adjacent sections (250 mm apart, A the most
rostral, C the most caudal) through the caudal portion of the pedunculopontine
tegmental nucleus (PPT) in Chaerophon pumilus. Note the major density of ChAT
immunopositive neurons lateral to the superior cerebellar peduncle (scp). This is an
unusual feature of the PPT and was only seen in this species. Scale bar in C = 100 mm
and applies to all. Vmot – motor division of the trigeminal nucleus.
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[(Fig._3)TD$IG]
J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
213
Fig. 3. Five low power photomicrographs at the level of the ChAT immunoreactive oculomotor nucleus (III) and interpeduncular nucleus (IP) showing the strongly ChAT
immunoreactive parabigeminal nucleus (PBg) in Cardioderma cor (A), a weakly ChAT immunoreactive PBg in Coleura afra (B), and its complete absence in Chaerophon pumilus
(C), Hipposideros commersoni (D), and Triaenops persicus (E). (F) Higher power photomicrograph of the parabigeminal nucleus in Cardioderma cor. Scale bar in E = 500 mm and
applies to A–E. Scale bar in F = 250 mm. III – oculomotor nucleus; ca – cerebral aqueduct; EW – Edinger–Westphal nucleus.
nucleus was typical of that seen in other mammals. No evidence of
ChAT+ neurons within the parabigeminal nucleus could be found
in the other three species investigated, which is congruent with the
findings for M. schreibersii (Maseko and Manger, 2007). No
evidence of ChAT+ neurons could be found in the pedunculopontine parvocellular (PPTpc), the laterodorsal tegmental parvocellular (LDTpc) or the superior and inferior colliculus interneurons as
occasionally observed in other species (Gravett et al., 2009; Pieters
et al., 2010). The lack of ChAT+ neurons in these regions follows the
findings of Maseko and Manger (2007) for M. schreibersii.
observed in other mammalian species (e.g. Maseko et al., 2007;
Bhagwandin et al., 2008; Gravett et al., 2009). All other typically
ChAT immunoreactive cranial nerve nuclei were observed in all five
species, in line with earlier findings for M. schreibersii (Maseko and
Manger, 2007). ChAT+ expression was found to be particularly
strong in the oculomotor neurons in C. cor, while in T. persicus the
nucleus ambiguus was found to be particularly large. C. afra
possessed a large ventral division of the facial nerve nucleus, as well
as strong ChAT+ expression in pVII, pIX, the hypoglossal nucleus and
the ventral horn of the spinal cord.
3.1.5. Cranial nerve nuclei
As observed by Maseko and Manger (2007) in M. schreibersii, no
evidence of ChAT+ neurons were observed in the cochlear nucleus or
the medullary tegmental field in any of the five microchiropteran
species investigated; however, in the current study evidence of
ChAT+ neurons was found for the Edinger–Westphal nucleus (Fig. 4),
the preganglionic superior salivatory nucleus (pVII) and the
preganglionic inferior salivatory nucleus (pIX) (Fig. 5). These nuclei
were present in all five microchiropteran species investigated and
showed similar neuronal morphology, distributions and numbers as
3.2. Catecholaminergic nuclei
The catecholaminergic nuclei were found throughout the brains
of all five microchiropteran species investigated and were revealed
using tyrosine hydroxylase (TH) immunoreactivity. Generally our
findings mirrored those of Maseko and Manger (2007), although
differences in TH+ immunoreactivity were noted in the A15v
(anterior hypothalamic ventral group), A10d (dorsal ventral
tegmental area), A10dc (dorsal caudal ventral tegmental area)
and A9v (substantia nigra ventral) in specific species.
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[(Fig._4)TD$IG]
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Fig. 4. Photomicrographs of ChAT immunoreactive neurons within the Edinger–Westphal nucleus (EW) of three microchiropteran species: (A) Triaenops persicus; (B)
Cardioderma cor; and (C) Hipposideros commersoni. Note the ChAT immunoreactive fasciculus retroflexus (fr) on either side of the midline. Scale bar in B = 500 mm and applies
to all.
[(Fig._5)TD$IG]
Fig. 5. Photomicrographs of the ChAT immunoreactive neurons forming the preganglionic neurons of the superior salivatory (pVII) and inferior salivatory (pIX) nuclei in (A)
Cardioderma cor, (B) Triaenops persicus and (C) Chaerophon pumilus in relation to the ChAT immunoreactive neurons of the ventral (VIIv) and dorsal (VIId) subdivisions of the
facial nerve nucleus and the abducens nucleus (VI). Scale bar in C = 500 mm and applies to all.
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
3.2.1. A16 – olfactory bulb
These neurons, found in the stratum granulosum of the
olfactory bulb, showed TH+ immunoreactivity across all five
microchiropteran species investigated, which concurs with the
findings for M. schreibersii (Maseko and Manger, 2007).
3.2.2. Hypothalamic nuclei (A11–A15)
TH+ neurons were identified in the A11, A12, A13 and A14
nuclei (Fig. 6) in all species studied, in agreement with previous
findings in M. schreibersii (Maseko and Manger, 2007). The A14
(rostral periventricular cell group) and A11 (caudal diencephalic
group) nuclei were both strongly expressed in terms of neuronal
number in H. commersoni (Fig. 6D). No evidence was found for the
A15d (anterior hypothalamic group, dorsal division) nucleus in any
of the five species studied, as seen in M. schreibersii; although this
nucleus is present in many other mammals (e.g. Maseko et al.,
2007; Bhagwandin et al., 2008; Gravett et al., 2009). TH+ neurons
were found in the A15v (anterior hypothalamic group, ventral
[(Fig._6)TD$IG]
215
division) in C. pumilus, H. commersoni and T. persicus, but not in C.
cor or C. afra. The occurrence of A15v in three species is in contrast
to the findings of Maseko and Manger (2007) for M. schreibersii.
3.2.3. Midbrain catecholaminergic nuclei (A8–A10)
In the present study, there was evidence of TH+ immunoreactivity in many of the midbrain catecholaminergic nuclei typically
described for mammals (Fig. 7; Smeets and González, 2000), these
include the A8, A9m, A9pc, A9l, A10, A10c, and A10d nuclei, all of
which were found in the locations typically seen across all
mammals. In the prior study of M. schreibersii (Maseko and Manger,
2007) no evidence for the A10dc, A10d, or A9v nuclei was observed.
In the current study partial evidence of A10dc was found in C.
pumilus and C. cor in the form of occasional TH+ neurons in the
periaqueductal grey matter near the ventrolateral edge of the
cerebral aqueduct (Fig. 7); however, no evidence of this nucleus
could be found in the other microchiropteran species, in line with
the findings of Maseko and Manger (2007). Additionally, partial
Fig. 6. Photomicrographs of various TH immunoreactive neurons in the hypothalamus of different species of microchiropterans. (A) The A13 nucleus in Coleura afra, (B) A14
neurons in Cardioderma cor, (C) A13 neurons in Hipposideros commmersoni, (D) A11 neurons in Hipposideros commmersoni. Scale bar in A = 100 mm. Scale bar in B = 50 mm and
applies to B–D.
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[(Fig._7)TD$IG]
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
Fig. 7. Diagrammatic reconstructions of the midbrain catecholaminergic nuclei in four species of microchiropteran. Note the similarity between species, as well as the
possible A10dc in C. pumilus and C. cor. See list for abbreviations.
evidence for A9v could be found in all five microchiropteran
species under investigation, which differs from the findings for M.
schreibersii (Maseko and Manger, 2007). It should be noted though
that in all species studied herein, that the number of neurons that
could be assigned to the A9v nucleus was small, varied between
individuals of the same species and varied across species.
3.2.4. Rostral rhombencephalon (locus coeruleus complex, A4–A7)
TH+ neurons were observed in the A5 (fifth arcuate), A6d (locus
coeruleus diffuse), A7sc (subcoeruleus compact) and A7d (subcoeruleus diffuse) nuclei in all five microchiropteran species
investigated in this study, as well as in M. schreibersii (Maseko and
Manger, 2007). The location, densities, and morphology of these
neurons were similar across all species. No evidence for an A6c or
A4 nucleus was found (Fig. 8), as was also the case in M. schreibersii
(Maseko and Manger, 2007).
3.2.5. Caudal rhombencephalon (A1, A2, C1, C2, area postrema)
Evidence was found for all five caudal rhombencephalon nuclei,
across all five microchiropteran species studied, which concurs
with previous findings in M. schreibersii (Maseko and Manger,
2007) and across all mammals studied to date. The C1 nucleus
(rostral ventrolateral tegmental group) was weakly expressed in
terms of neuronal numbers in C. pumilus. For the remaining nuclei a
strong homogeneity in the expression of these nuclei across
species was observed. No evidence of the C3 nucleus, a nucleus that
has only been found in rodents (e.g. Bhagwandin et al., 2008), was
observed.
3.3. Serotonergic neurons
In mammals, the serotonergic neurons are typically subdivided
into rostral and caudal nuclear clusters, with both being located
entirely the brainstem (Törk, 1990; Jacobs and Azmitia, 1992). In
mammals in general, the rostral cluster consists of the CLi (caudal
linear), B9 (supralemniscal), MnR (median raphe) and the dorsal
raphe nuclei, while the caudal cluster is composed of the RMg
(raphe magnus), RPa (raphe pallidus), RVL (rostral ventrolateral
cell column), CVL (caudal ventrolateral cell column) and ROb
(raphe obscurus) nuclei.
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
217
Fig. 8. Photomicrographs of TH immunoreactive neurons in the locus coeruleus of different species of microchiropterans. (A) The A6d and A7d nuclei in Coleura afra, (B) A6d
and A7sc neurons in Triaenops persicus, (C) A6d neurons in Chaerophon pumilus, (D) A6d and A7d neurons in Cardioderma cor. Scale bar in D = 500 mm and applies to all.
[(Fig._9)TD$IG]
3.3.1. Rostral cluster
All serotonergic nuclei typically assigned to the rostral cluster
showed specific serotonergic immunoreactivity across all five
microchiropteran species investigated. This is consistent with the
previous findings for M. schreibersii (Maseko and Manger, 2007)
and for all Eutherian mammals (Maseko et al., 2007). The CLi
nucleus was strongly expressed in T. persicus, with the B9 nucleus
being strongly expressed in C. afra, and C. cor (Fig. 9) and C. pumilus.
The dorsal raphe cluster could be divided into dorsal (DRd), ventral
(DRv), interfascicular (DRif), lateral (DRl), caudal (DRc) and
peripheral (DRp) nuclei, all of which were observed in all five
species, all evincing a similar appearance (Fig. 10). As with all prior
observations, only a few neurons of the peripheral nucleus of the
dorsal raphe were located outside the periaqueductal grey matter.
3.3.2. Caudal cluster
All five microchiropteran species studied had 5-HT+ neurons in
the RMg, RPa, RVL, CVL and ROb nuclei, as was observed in M.
schreibersii (Maseko and Manger, 2007) and all other Eutherian
mammals studied to date. It was noted that RMg possessed few 5HT+ neurons in C. afra, and CVL was weakly expressed in terms of
Fig. 9. A photomicrograph showing the most rostral serotonergic nuclei in
Cardioderma cor, the caudal linear nucleus (CLi) and the supralemniscal
serotonergic neurons (B9) at the level of the interpeduncular nucleus (IP). Scale
bar = 100 mm.
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[(Fig._10)TD$IG]
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
Fig. 10. Photomicrographs of the nuclear subdivisions of dorsal raphe nuclear complex in four microchiropteran species: (A) Chaerophon pumilus; (B) Hipposideros
commersoni; (C) Coleura afra; and (D) Triaenops persicus. Scale bar in D = 500 mm and applies to all. See list for abbreviations.
neuronal numbers in T. persicus. In all other respects these nuclei
were similar to previous observations in other mammals.
4. Discussion
Within the cholinergic, catecholaminergic and serotonergic
systems many similarities in nuclear organization were found in
the five microchiropterans investigated in this study and that
reported for M. schreibersii (Maseko and Manger, 2007); however,
some notable differences in nuclear organization were found in the
cholinergic and catecholaminergic systems. Several differences
were observed when comparing the nuclear organization of these
systems with observations made on the megachiropterans
previously studied (Maseko et al., 2007; Dell et al., 2010), the
most notable of which occurred, again, in the cholinergic and
catecholaminergic systems. When making a broader comparison
across mammals, the general organization of these systems in the
microchiropterans were similar to that seen in other mammals, but
notable differences in the presence or absence of specific nuclei
within the cholinergic and catecholaminergic systems were found.
Previous studies of these systems in insectivores appear to show
the greatest number of similarities in terms of nuclear organization
of these systems with that observed in microchiropterans (see also
Dell et al., 2010), suggesting a close phylogenetic link between the
microchiropterans and the insectivores.
4.1. Comparisons amongst microchiropterans
4.1.1. Cholinergic nuclei
The nuclear organization of the cholinergic system was
generally found to be similar in all species; however there were
notable differences in the pontine and cranial nerve nuclei.
ChAT+ neurons were identified in the parabigeminal nucleus of C.
cor and C. afra, which were not seen in the other microchiropterans studied herein or in M. schreibersii (Maseko and Manger,
2007). While a strongly expressed parabigeminal nucleus was
observed in C. cor, in C. afra the immunoreactivity of these
neurons was weak. ChAT+ immunoreactivity also occurred in the
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
Edinger–Westphal, pVII and pIX cholinergic nuclei across all five
microchiropteran species studied, whereas no evidence for ChAT
immunoreactivity of the neurons within these nuclei was found
in M. schreibersii (Maseko and Manger, 2007). These latter
differences may be attributed to the antibody used in the current
study – Maseko and Manger (2007) utilised the ChAT antibody
AB143 (Chemicon), while the current study employed AB144P
(Chemicon). A study by Bhagwandin et al. (2006) looked at the
revelation of cortical cholinergic neurons in a range of rodent
species using three different antibodies (AB143, AB144P and
vChAT). It was shown that AB144P consistently revealed the
most cholinergic neurons, while the revelation was limited using
AB143 and vChAT. It is possible that these antibodies bind to
different regions of the cholineacetyltransferase molecule and
thus the molecule involved in producing acetylcholine may differ
in structure in different parts of the central nervous system and
differ within specific phylogenies (Bhagwandin et al., 2006). The
cholinergic neurons revealed in this study, but not in the study by
Maseko and Manger (2007), are mostly neurons that appear to be
involved with the autonomic nervous system (except for those
seen in the parabigeminal nucleus); thus, there may be, at least in
the microchiropterans and rodents, a differing morphology of the
cholineacetyltransferase molecule in the parts of the brain
associated with the ‘‘classical’’ cholinergic system and that part
of the cholinergic system associated with the autonomic nervous
system.
4.1.2. Catecholaminergic nuclei
The most notable difference in the catecholaminergic nuclei
was the presence of the A15v nucleus in all five microchiropteran
species in this study, as well as the possible existence of the A9v
nucleus; however A15v was only poorly expressed in C. cor and C.
afra, while it was strongly expressed in the other three species.
Neither of these nuclei were present in M. schreibersii (Maseko and
Manger, 2007). Some evidence was also found for A10dc but only
in C. cor and C. afra, with no other species expressing this nucleus,
yet all were observed to have the A10d nucleus which was
reported as absent in M. schreibersii. It is important to note that C.
cor and C. afra are also the only two microchiropteran species that
showed the presence of the parabigeminal nucleus, as well as a
very weak expression of A15v. These findings group these two
species, from the families Megadermatidae and Emballonuridae,
together, but slightly separate them from the other 17 or so
microchiropteran families, which is not in agreement with the
recently published molecular, paraphyletic phylogenies of the
microchiropterans, where megadermatids are aligned with
rhinolophoids and split from all other microbats (Van Den
Bussche and Hoofer, 2004; Jones et al., 2005; Teeling et al.,
2005; Gu et al., 2008). On the other hand, morphological studies
have consistently placed these two Old World microchiropteran
families together (e.g. Smith, 1976; Van Valen, 1979; Pierson,
1986). All other catecholaminergic nuclei were found to be similar
across all six microchiropteran species.
4.1.3. Serotonergic nuclei
The nuclear organization of the serotonergic system is
homogenous across all six microchiropteran species studied to
date, i.e. the five species in this study together with M. schreibersii
(Maseko and Manger, 2007). There were a few differences in
expression patterns of individual nuclei observed between the
species studied. C. afra had a notably small RMg nucleus, while CVL
was particularly small in T. persicus. As specific functions have not
been ascribed to these nuclei beyond the modulation of certain
portions of the neurons within the grey matter of the spinal cord
(Törk, 1990) it is difficult to speculate if these differences have any
particular functional implications.
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4.2. Comparisons with megachiropterans
4.2.1. Cholinergic nuclei
In the cholinergic system, the five microchiropterans examined
in this study and the three megachiropterans that have been
studied (Rousettus aegyptiacus, Eidolon helvum, Epomophorus
wahlbergi) have the expression of almost all nuclei in common,
including the Edinger–Westphal, pVII and pIX nuclei which were
reported as absent in M. schreibersii (Maseko et al., 2007; Dell et al.,
2010). The only difference in organization of the cholinergic nuclei
is found in the pontine region, this being the variable presence of
ChAT immunoreactivity in the neurons of the parabigeminal
nucleus. ChAT immunoreactivity of neurons within this nucleus
occurs in all megachiropterans studied (Maseko et al., 2007; Dell
et al., 2010), as well as in two of the microchiropterans C. cor and C.
afra; however, ChAT immunoreactivity the parabigeminal nucleus
was absent in the four other microchiropteran species that have
been examined, and in C. afra the ChAT immunoreactivity was
weak. The variability in the occurrence of ChAT immunoreactivity
of the neurons of the parabigeminal nucleus is difficult to explain
at present, and further examination of the presence or absence of
this immunoreactivity in other microchiropteran species may
elucidate this issue, determining whether this is a phylogenetic or
functional variation. It may also be possible that the parabigeminal
nucleus is actually absent in the species where we have not
detected any ChAT immunoreactivity. Connectional studies from
the superior colliculi of those species without ChAT immunoreactivity would determine the presence or absence of this nucleus.
4.2.2. Catecholaminergic nuclei
The major differences noted between megachiropterans
previously studied (Maseko et al., 2007; Dell et al., 2010) and
the five microchiropteran species examined in this study occur in
the presence or absence of nuclei assigned to the catecholaminergic system. The A15d, A6c and A4 nuclei were not present in the
microchiropterans, including M. schreibersii (Maseko and Manger,
2007); however these nuclei were found in the three megachiropteran species that have been studied (Maseko et al., 2007; Dell
et al., 2010). The A10dc nucleus is possibly present in C. cor and C.
afra, but not in the other three microchiropteran species
investigated, nor M. schreibersii (Maseko and Manger, 2007). This
nucleus is however found in the three megachiropteran species
that have been studied (Maseko et al., 2007; Dell et al., 2010). The
A9v nucleus was very weakly expressed in all five microchiropteran species, while it is strongly expressed in all megachiropteran species (Maseko et al., 2007; Dell et al., 2010). The A15v
nucleus is only weakly expressed in C. cor and C. afra, while it is
strongly expressed in the other three microchiropterans studied
and in the megachiropterans. All remaining catecholaminergic
nuclei were consistent across microchiropterans and megachiropterans.
4.2.3. Serotonergic nuclei
There were no differences between microchiropterans and
megachiropterans in terms of the nuclear organization of the
serotonergic system (Maseko and Manger, 2007; Maseko et al.,
2007; Dell et al., 2010).
4.3. Comparisons with other mammals
4.3.1. Cholinergic nuclei
The presence of all cholinergic striatal and basal forebrain nuclei
is homogenous across all mammalian species studied thus far,
including the microchiropterans (Ferreira et al., 2001; Manger et al.,
2002a; Maseko et al., 2007; Limacher et al., 2008; Bhagwandin et al.,
2008; Gravett et al., 2009; Pieters et al., 2010; Dell et al., 2010).
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Microchiropterans and all other Eutherian mammals have cholinergic neurons in the medial habenular nucleus and three nuclei
within the hypothalamus (Ferreira et al., 2001; Manger et al., 2002a;
Maseko et al., 2007; Limacher et al., 2008; Bhagwandin et al., 2008;
Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010; Dell et al.,
2010); however, the monotremes do not show this reactivity in the
dorsal, ventral and lateral hypothalamic nuclei (Manger et al.,
2002a). There are a number of differences and similarities within the
pontine cholinergic nuclei across mammalian species. The parabigeminal nucleus was found in two of the microchiropteran species
in this study: C. cor and C. afra. This nucleus has been found in the
giraffe, the rodents, the afrotherians (in this study represented by the
rock hyrax and elephant shrew), the carnivores, the megachiropterans and the primates (Jones and Cuello, 1989; Maseko et al., 2007;
Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009;
Pieters et al., 2010; Bux et al., 2010); however, it is absent in C.
pumilus, H. commersoni and T. persicus, as well as M. schreibersii
(Maseko and Manger, 2007), the laboratory shrew, the echidna and
the platypus (Manger et al., 2002a; Karasawa et al., 2003). The
superior collicular interneurons have, to date, only been seen in the
rodents, elephant shrew and tree shrew (Pieters et al., 2010), while
microchiropterans and other mammals do not express these
interneurons (Maseko and Manger, 2007; Gravett et al., 2009;
Limacher et al., 2008; Maseko et al., 2007). The inferior collicular
cholinergic interneurons do not occur in the microchiropterans or
other mammals (Maseko and Manger, 2007; Gravett et al., 2009;
Limacher et al., 2008; Maseko et al., 2007; Bhagwandin et al., 2008),
except for the elephant shrew (Pieters et al., 2010). The cranial nerve
nuclei generally follow a similar pattern throughout all mammals
with a few notable exceptions (Maseko and Manger, 2007; Gravett
et al., 2009; Limacher et al., 2008; Maseko et al., 2007; Bhagwandin
et al., 2008). In this study, the Edinger–Westphal, pVII and pIX nuclei
were present in all five microchiropteran species investigated.
These nuclei are not present in the echidna, the platypus and the
laboratory shrew (Manger et al., 2002a; Karasawa et al., 2003);
however all other mammals studied to date have these nuclei in
common (Maseko and Manger, 2007; Maseko et al., 2007; Limacher
et al., 2008; Bhagwandin et al., 2008; Gravett et al., 2009; Pieters
et al., 2010; Bux et al., 2010; Dell et al., 2010). The medullary
tegmental field is absent in microchiropterans, the elephant shrew,
the rock hyrax, the megachiropterans and primates (Maseko and
Manger, 2007; Maseko et al., 2007; Gravett et al., 2009; Pieters et al.,
2008), while it has been seen in the monotremes, rodents and
carnivores (Manger et al., 2002a; Maseko et al., 2007; Bhagwandin
et al., 2008).
4.3.2. Catecholaminergic nuclei
In general the hypothalamic catecholaminergic nuclei are
similar across mammals (Skagerberg et al., 1988; Tillet and
Thibault, 1989; Leshin et al., 1995; Tillet et al., 2000; Manger et al.,
2002b, 2004; Maseko et al., 2007; Bhagwandin et al., 2008;
Limacher et al., 2008; Gravett et al., 2009; Pieters et al., 2010; Bux
et al., 2010). The A15v nucleus shows a variable presence in the
microchiropterans studied and has also been reported to be
absent in the rabbit and the tree shrew (Maseko et al., 2007; Dell
et al., 2010). The A15d nucleus is absent in all microchiropterans,
as well as the insectivores, artiodactyls, the elephant shrew and
the tree shrew (Pieters et al., 2010; Bux et al., 2010; Dell et al.,
2010). The midbrain nuclei are also, for the most part, homogenous across mammalian species (Skagerberg et al., 1988; Tillet
and Thibault, 1989; Leshin et al., 1995; Tillet et al., 2000; Manger
et al., 2002b; Bhagwandin et al., 2008; Limacher et al., 2008;
Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010; Dell et al.,
2010); however differences occur in the microchiropterans, in
that the A10dc and A9v nuclei have a variable occurrence. The
A10dc nucleus was absent in C. pumilus, H. commersoni, T. persicus
and M. schreibersii, while C. cor and C. afra possibly possess this
nucleus but it is very weakly expressed in terms of neuronal
number. In the microchiropterans A9v may be present but again,
only weakly expressed in terms of neuronal number. The A9v
nucleus shows a similar morphology in the hedgehog, and is
absent in the rabbit and carnivores (Dell et al., 2010). The
microchiropterans lack the A6c and A4 within the locus coeruleus
complex. These two specific nuclei are also absent in the
monotremes, insectivores, artiodactyls, rodents, afrotherians
and carnivores, but are present in the rabbit, megachiropterans
and primates (Maseko et al., 2007; Dell et al., 2010). In the caudal
rhombencephalon nuclei, only C3 shows any variation across
mammalian species. This nucleus has only been seen in the
rodents (Skagerberg et al., 1988; Bhagwandin et al., 2008;
Limacher et al., 2008), while it is absent in all other mammalian
species.
4.3.3. Serotonergic nuclei
The serotonergic system is homogenous, in terms of nuclear
organization, across all Eutherian mammals that have been
previously studied, including microchiropterans. No Eutherian
mammals have been observed to possess serotonergic neurons
within the periventricular organ, which have only been found in
the monotremes (Manger et al., 2002c). In the rostral serotonergic
cluster, all 9 nuclei are present in microchiropterans and other
Eutherian mammals (Da Silva et al., 2006; Limacher et al., 2008;
Moon et al., 2007; Dwarika et al., 2008; Gravett et al., 2009; Pieters
et al., 2010; Bux et al., 2010; Dell et al., 2010). The monotremes
have a similar nuclear organization, although the DRc (dorsal
raphe, caudal, or B6) nucleus is not present in these mammals
(Manger et al., 2002c). A similar distribution is seen for the caudal
serotonergic cluster, with microchiropterans and other Eutherian
mammals having all 6 nuclei in common (Da Silva et al., 2006;
Badlangana et al., 2007; Moon et al., 2007; Dwarika et al., 2008;
Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009;
Pieters et al., 2010). The monotremes are again similar, however,
the CVL (caudal ventrolateral serotonergic group) nucleus is not
present (Manger et al., 2002c). CVL is also absent in the opossum
(Crutcher and Humbertson, 1978).
4.4. Bat phylogeny
Although there are many similarities in the cholinergic,
catecholaminergic and serotonergic systems between microchiropterans and megachiropterans, it is important to note that these
similarities are those that are found to be common to most
mammals studied (see the tables provided in Dell et al., 2010 for a
full summary). The differences between the microchiropterans and
megachiropterans are far more revealing, as, using an unbiased
phylogenetic analysis (see Dell et al., 2010), megachiropterans
align more closely with primates than any other group, while
microchiropterans align more readily with the insectivores. It is
also necessary to recall that the neural systems investigated in this
study are, for the most part, unrelated to flight, olfaction,
echolocation or vision and any differences can therefore be
considered related to the phylogenetic history of the two
chiropteran suborders and not related to adaptation associated
with chiropteran specialisations. The placement of the megachiropterans as a sister group to the primates has become standard in
the examination of the diphyletic origin of bats; however, our
proposal that the microchiropterans form a sister group to the
insectivores is a novel concept, with only Crosby and Woodburne
(1943) briefly touching on this possibility from a neural perspective. Although further study is required, as the insectivora is a large
and heterogeneous group (Symonds, 2005), it would seem that the
microchiropteran – insectivoran link is one of particular interest in
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J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222
the debate surrounding chiropteran phylogenetic history (Siemers
et al., 2009).
In addition to the order level phylogeny examined here, the
results of the current study are revealing in terms of internal
phylogeny of the microchiroptera. It is clear that two species of the
microchiropterans studied here share more in common that the
other three species, these two species being C. cor and C. afra. In
common, and to the exclusion of the other three microchiropterans studied, these two share the presence of the parabigeminal
nucleus (PBg), the dorsal caudal nucleus of the ventral tegmental
area (A10dc), and poor expression of the ventral division of the
anterior hypothalamic group (A15v). At first glance, this might
appear to support the ‘‘paraphyly of microbats’’, a new DNA-based
phylogeny in which three families of microchiropterans are
united with the megachiroptera in the Yinpterochiroptera, to the
exclusion of the remaining families of microchiropterans that are
lumped together into the Yangochiroptera (Teeling, 2009). Any
similarities, however, are superficial and slight. To begin with, the
brain data provide no support whatever for the most radical plank
of ‘‘microbat paraphyly’’, which is the inclusion of megachiropterans in Yinpterochiroptera. According to this DNA-based
hypothesis (from portions of 17 nuclear genes, Teeling et al.,
2005), the three rhinolophoids whose brains we have studied
should share neural features with the three megachiropteran
species we studied (Maseko et al., 2007; Dell et al., 2010). There
are no features of this kind, so there is no support from the brain
data for this aspect of ‘‘microbat paraphyly’’. Secondly, our data do
not conform to the composition of Yinpterochiroptera. While we
have two outlying microbats based on the brain nuclei, the
megadermatid Cardioderma and the emballonurid Coleura, only
the megadermatid Cardioderma belongs to the Yinpterochiroptera
as constituted, while the emballonurid Coleura belongs to
Yangochiroptera. Moreover, two rhinolophoids that we studied,
Hipposideros and Triaenops, should belong to Yinpterochiroptera
as constituted, but instead show no brain specialisations that
would set them apart from Yangochiropterans examined, such as
the molossid, Chaerophon, and the vespertilionid relative, Miniopterus (Maseko and Manger, 2007). Our brain data appears to
more in agreement with earlier morphological phylogenetic
studies of microchiropteran relationships (Smith, 1976; Van
Valen, 1979; Pierson, 1986); thus there appears to be a distinct
DNA vs morphology difference regarding the phylogeny of the
chiropterans.
Acknowledgments
This work was supported by funding from the South African
National Research Foundation (PRM and JDP, South African
Biosystematics Initiative, KFD2008052300005). The authors wish
to extend their gratitude to the members of the National Museums
of Kenya, especially Mr. Bernard ‘Risky’ Agwanda, without whom
this work would not have been possible.
Ethical statement: The microchiropterans used in the present
study were caught from wild populations in Kenya under
permission and supervision from the appropriate wildlife directorates. All animals were treated and used according to the
guidelines of the University of the Witwatersrand Animal Ethics
Committee, which parallel those of the NIH for the care and use of
animals in scientific experimentation.
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