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Chromosome painting in the manatee supports Afrotheria and Paenungulata

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Background Sirenia (manatees, dugongs and Stellar's sea cow) have no evolutionary relationship with other marine mammals, despite similarities in adaptations and body shape. Recent phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida manatee. Results The human autosomal and X chromosome paints delimited a total of 44 homologous segments in the manatee genome. The synteny of nine of the 22 human autosomal chromosomes (4, 5, 6, 9, 11, 14, 17, 18 and 20) and the X chromosome were found intact in the manatee. The syntenies of other human chromosomes were disrupted in the manatee genome into two to five segments. The hybridization pattern revealed that 20 (15 unique) associations of human chromosome segments are found in the manatee genome: 1/15, 1/19, 2/3 (twice), 3/7 (twice), 3/13, 3/21, 5/21, 7/16, 8/22, 10/12 (twice), 11/20, 12/22 (three times), 14/15, 16/19 and 18/19. Conclusion There are five derived chromosome traits that strongly link elephants with manatees in Tethytheria and give implicit support to Paenungulata: the associations 2/3, 3/13, 8/22, 18/19 and the loss of the ancestral eutherian 4/8 association. It would be useful to test these conclusions with chromosome painting in hyraxes. The manatee chromosome painting data confirm that the associations 1/19 and 5/21 phylogenetically link afrotherian species and show that Afrotheria is a natural clade. The association 10/12/22 is also ubiquitous in Afrotheria (clade I), present in Laurasiatheria (clade IV), only partially present in Xenarthra (10/12, clade II) and absent in Euarchontoglires (clade III). If Afrotheria is basal to eutherians, this association could be part of the ancestral eutherian karyotype. If afrotherians are not at the root of the eutherian tree, then the 10/12/22 association could be one of a suite of derived associations linking afrotherian taxa.
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BMC Evolutionary Biology
Open Access
Research article
Chromosome painting in the manatee supports Afrotheria and
Paenungulata
Margaret E Kellogg1, Sandra Burkett2, Thomas R Dennis3, Gary Stone2,
Brian A Gray3, Peter M McGuire4, Roberto T Zori3 and Roscoe Stanyon*5
Address: 1College of Veterinary Medicine, University of Florida, PO BOX 100245, Gainesville, FL 32610-0245, USA, 2Comparative Molecular
Cytogenetics Core, National Cancer Institute, Frederick, MD 21702, USA, 3Department of Pediatrics, Division of Genetics, University of Florida,
PO Box 100296, UFHSC, Gainesville, FL 32610, USA, 4Department of Biochemistry and Molecular Biology, University of Florida, PO Box 100245,
College of Medicine, Gainesville, FL 32610, USA and 5Department of Animal Biology and Genetics, University of Florence, Florence, Italy, Via del
Proconsolo 12, 50122 Florence, Italy (formerly at NCI, Frederick)
Email: Margaret E Kellogg - kelloggm@gmail.com; Sandra Burkett - sburkett@ncifcrf.gov; Thomas R Dennis - tdennis@tgen.org;
Gary Stone - gstone@ncifcrf.gov; Brian A Gray - grayb@pathology.ufl.edu; Peter M McGuire - pmcguire@biochem.med.ufl.edu;
Roberto T Zori - zorirt@peds.ufl.edu; Roscoe Stanyon* - roscoe.stanyon@unifi.it
* Corresponding author
Abstract
Background: Sirenia (manatees, dugongs and Stellar's sea cow) have no evolutionary relationship
with other marine mammals, despite similarities in adaptations and body shape. Recent
phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in
Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by
comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida
manatee.
Results: The human autosomal and X chromosome paints delimited a total of 44 homologous
segments in the manatee genome. The synteny of nine of the 22 human autosomal chromosomes
(4, 5, 6, 9, 11, 14, 17, 18 and 20) and the X chromosome were found intact in the manatee. The
syntenies of other human chromosomes were disrupted in the manatee genome into two to five
segments. The hybridization pattern revealed that 20 (15 unique) associations of human
chromosome segments are found in the manatee genome: 1/15, 1/19, 2/3 (twice), 3/7 (twice), 3/13,
3/21, 5/21, 7/16, 8/22, 10/12 (twice), 11/20, 12/22 (three times), 14/15, 16/19 and 18/19.
Conclusion: There are five derived chromosome traits that strongly link elephants with manatees
in Tethytheria and give implicit support to Paenungulata: the associations 2/3, 3/13, 8/22, 18/19 and
the loss of the ancestral eutherian 4/8 association. It would be useful to test these conclusions with
chromosome painting in hyraxes. The manatee chromosome painting data confirm that the
associations 1/19 and 5/21 phylogenetically link afrotherian species and show that Afrotheria is a
natural clade. The association 10/12/22 is also ubiquitous in Afrotheria (clade I), present in
Laurasiatheria (clade IV), only partially present in Xenarthra (10/12, clade II) and absent in
Euarchontoglires (clade III). If Afrotheria is basal to eutherians, this association could be part of the
ancestral eutherian karyotype. If afrotherians are not at the root of the eutherian tree, then the 10/
12/22 association could be one of a suite of derived associations linking afrotherian taxa.
Published: 23 January 2007
BMC Evolutionary Biology 2007, 7:6 doi:10.1186/1471-2148-7-6
Received: 18 October 2006
Accepted: 23 January 2007
This article is available from: http://www.biomedcentral.com/1471-2148/7/6
© 2007 Kellogg et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Background
Recently the molecular based approaches of super-ordinal
grouping of extant eutherians (Afrotheria, Euarchontog-
lires, Laurasiatheria and Xenarthra) has gained popularity
[1-3]. However, one of the four proposed super-orders,
Afrotheria, is controversial because it unites morphologi-
cally distinct species of African placentals (golden moles,
tenrecs, otter shrews, elephant shrews, aardvarks, hyraxes,
elephants and sirenians). Within Afrotheria, sirenians,
elephants and hyraxes form a clade called Paenungulata.
There is little morphological or paleontological evidence
that provides support for Afrotheria [4]. A movable snout
was hypothesized as a synapomorphic trait, but this fea-
ture is apparently not homologous across different afroth-
erian lineages[5]. More recently, it was proposed that
aspects of placentation could provide a synapomorphy for
this assemblage [6,7]. Some outstanding issues in higher
eutherian phylogenomics include the exact root of the
placental tree, the relationships within the super-ordinal
clade Laurasiatheria (moles, hedgehogs, shrews, bats,
cetaceans, ungulates, pangolins and carnivores), and
resolving the trichotomy of sirenians, elephants and
hyraxes [8].
Sirenia and Hyracoidea are the two afrotherian orders
remaining to be investigated with molecular cytogenetic
techniques. In this paper, the chromosome painting of the
Florida manatee (Trichechus manatus latirostris) is
reported. These data should be a valuable addition to our
understanding of afrotherian relationships and the euthe-
rian ancestral karyotype.
The Florida manatee
The endangered Florida manatee is a subspecies of the
West Indian manatee (Trichechus manatus) in the order
Sirenia. Sirenians are often considered phylogenetic out-
liers. Despite similarities in adaptations, habitat, and
body shape, they have no evolutionary relationship with
the other orders of marine mammals. Extant sirenians are
the only herbivorous marine mammals and live in fresh,
brackish or marine habitats dispersed along tropical and
subtropical environments.
Previous cytogenetic reports on manatees
Solid stained chromosome studies were completed on a
limited number of individual manatees, establishing the
chromosome number as 2N = 48 for the Florida manatee
[9,10] and 2N = 56 for the Amazonian manatee (Tri-
chechus inunguis) [11]. Following solid staining, chromo-
some-banding procedures allowed for the identification
of individual chromosome regions. Giemsa and trypsin
staining, or GTG-banding, was used to create karyotypes
and ideograms for the Florida manatee [12] and the Ama-
zonian manatee [13].
Comparisons of chromosome painting data provide an
independent test of the contrasting hypotheses on mam-
malian evolution and phylogeny. The research presented
here clarifies the phylogenetic position of the manatee
and tests the validity of the radical taxonomic assemblage
known as Afrotheria. The results are then compared to
other chromosome painting data in Afrotheria. In light of
the findings, the relationships within Afrotheria and the
alternative organizations of the ancestral eutherian karyo-
type are assessed.
Results
Examples of human chromosome paints (HSA) hybrid-
ized to manatee (TMA) metaphase chromosomes are
shown in Figure 1. Synteny was found intact in nine (4, 5,
6, 9, 11, 14, 17, 18 and 20) of the 22 human autosomal
and X chromosomes (Figure 2). Two hybridization signals
were evident on separate manatee chromosomes for ten
human chromosomes (1, 7, 8, 10, 12, 13, 15, 16, 21 and
22). The human 19 paint hybridized to three TMA chro-
mosomes (2, 12 and 14). Human chromosomes 2 and 3
were highly fragmented in the manatee genome and
painted four and five chromosomes, respectively (Table
1). Due to the small signals involved and the quality of
the metaphases, it was more difficult to assign the hybrid-
ization pattern for these two chromosomes. Human chro-
mosome paint 12 provided three signals on TMA 7, most
likely due to an inversion. Chromosome paints with peri-
centromeric signals on both arms of the same chromo-
some were considered as one signal. Centromere areas on
the manatee karyotype were not hybridized. The Y chro-
mosome was the only human probe that failed to provide
a signal in the manatee. Altogether, the human autosomal
chromosome paints and the X chromosome paint delim-
ited a total of 44 homologous segments in the manatee
genome. Human chromosome paints hybridized to 20
(15 unique) segments in the manatee genome: 1/15, 1/
19, 2/3 (twice), 3/7 (thrice), 3/13, 3/21, 5/21, 7/16, 8/22,
10/12 (twice), 11/20, 12/22 (thrice), 14/15, 16/19 and
18/19.
Discussion
The painting map of the manatee genome was compared
with results published on other Afrotheria taxa: aardvark,
elephant, elephant shrew and golden mole [14-17]. An
assessment of the associations found in each taxa are
shown in Table 1. All species have eight associations in
common (1/19, 3/21, 5/21, 7/16, 10/12, 12/22, 14/15
and 16/19). Five of these associations are considered
ancestral to all eutherians by most proposals (3/21, 7/16,
12/22 twice, 14/15 and 16/19). It appears that the associ-
ations 1/19 and 5/21 can be used to link afrotherian spe-
cies [14-16,18]. These associations provide cytogenetic
support, in agreement with molecular studies, that Afroth-
eria is a natural clade.
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New chromosome painting data in Xenarthra (anteaters,
sloths and armadillos) are also informative towards the
ancestral eutherian karyotype. Of the four species studied,
Tamandua tetradactyla, Choloepus didactylus, C. hoffmanii
and Dasypus novemcinctus [18,19], only the anteater has a
1/19 association. It is not likely that this association is
homologous to Afrotheria, because the anteater has the
most highly rearranged karyotype known in Xenarthra
[18].
The manatee data indicate that the association 10/12/22
is most likely ubiquitous throughout Afrotheria. A combi-
nation HSA10p/12p/22q and a single HSA10q were
found in the aardvark and elephant karyotypes [14,17].
An apparently identical association was later found in the
elephant shrew and golden mole [15]. The question is,
whether this association is a third cytogenetic landmark
for the Afrotheria clade, or instead should be considered
part of the ancestral eutherian karyotype.
The entire 10/12/22 association appears to be present in
clades I, Afrotheria, and IV, Laurasiatheria, only partially
present in clade II, Xenarthra (10/12), and absent in clade
III, Euarchontoglires (primates, rabbits, rodents, tree
shrews and flying lemurs). Carnivores have a homologous
10/12/22 association to Afrotheria, as demonstrated by
reciprocal chromosome painting [20,21]. Eulipotyphla
(shrews, solenodons, moles, hedgehogs, and Nesophontes)
also have the 10/12/22 association [19,22]. Chromosome
painting data in Xenarthra show that a 10/12 association
is present in the armadillo (D. novemcinctus) [18]. To date,
the 10/12 association has been found in three of the four
eutherian mammal clades. Yet, there is no reciprocal
painting in Xenarthra to prove that the 10/12 association
is truly homologous to that found in Afrotheria. Several
hypotheses can be developed with different implications
if Afrotheria or Xenarthra is considered basal. If Afrotheria
is basal, the occurrence of 10/12/22 in clades I and IV
would suggest that this association is part of the ancestral
eutherian karyotype with a subsequent, independent loss
in clades II and III. The occurrence of the 10/12/22 asso-
ciation clades I and IV, could be considered a phyloge-
netic link. Alternatively, the association could have been
independently acquired in the two clades. If Xenarthra is
basal, this association could have originated in Afrotheria
and was then lost in clade III.
Association 3/13 was found in the manatee, elephant and
elephant shrew. However, there are no reciprocal painting
data between human and manatee or human and ele-
phant shrew. Therefore, it is not possible to confirm that
the 3/13 association is homologous (involves the same
segments of both chromosomes 3 and 13). In view of the
afrotherian molecular data, this association was inde-
pendently derived in the Macroscelidae (elephant shrews)
and Paenungulata phylogenetic lineages [8].
Support for the Tethytheria and Paenungulata assemblage
Before the advent of molecular studies, some morpholo-
gists placed sirenians, elephants and hyraxes under Ungu-
lata. Elephants and sirenians were grouped together in
Tethytheria, while hyraxes were placed in Phenacodonta
along with perissodactyls [23]. Results in molecular stud-
ies are inconsistent and fail to resolve the Paenungulate
trifurcation [8] and some data do not support Tethytheria
[2,24-26]. Mitochondrial genome analyses do support
Tethytheria, but exclude Hyracoidea [1]. SINE insertion
data produced incongruent phylogenetic relationships
within Paenungulata, most likely due to a rapid diver-
gence from a highly polymorphic last common ancestor
[27].
The chromosome mapping data strongly support Tethyth-
eria (sirenians and elephants) and implies support for the
clade Paenungulata (Sirenia, Proboscidea, and Hyracoi-
dea). There appear to be four derived associations linking
elephants with manatees: 2/3, 3/13, 8/22 and 18/19. HSA
4/8p was not present in the manatee and may represent a
derived trait of Paenungulata. Both publications on the
elephant indicate that this association is also lacking
[14,17]. It is possible that the 4/8 association went unde-
tected in our study, as well as in elephants. Although, the
widespread occurrence of the 4/8 association in all mam-
mals, outside of elephants and most primates, lends cre-
dence to its inclusion in the ancestral eutherian karyotype.
Examples of hybridizations in the manatee a) human 12, b) human 13, c) human 14 in green and 15 in red d) human 17 in green and 18 in redFigure 1
Examples of hybridizations in the manatee a) human 12, b)
human 13, c) human 14 in green and 15 in red d) human 17 in
green and 18 in red.
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The karyotype of the manatee is shown to the left and the color coded idiogram to the right (modified from Gray et alFigure 2
The karyotype of the manatee is shown to the left and the color coded idiogram to the right (modified from Gray et al. 2002).
Manatee chromosomes are numbered below and human chromosome homology is shown laterally.
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It would be useful to test these hypotheses with rock hyrax
chromosome painting data.
Branching order in Afrotheria
The branching order within Afrotheria has not reached a
consensus. Some authors have viewed Macroscelidae, the
elephant shrews, as the most basal and early divergent
order within Afrotheria [2,28]. However, Murphy et al.
(2001) placed the triumvirate of sirenians, elephants and
hyraxes (Paenungulata) as basal, verified by additional
molecular data [1,3,29]. It is difficult to determine which
order is most basal because sirenians and elephants, like
other afrotherian species, have fairly derived karyotypes.
According to Robinson et al. (2003), associations 2/8, 3/
20 and 10/17 link elephant shrews, golden moles/tenrecs
and aardvarks. Only the association 2/8 is present in all
three. Recently, the association 2/8 was also found in ant-
eater (T. tetradactyla), sloth (Choloepus didactylus) and pan-
golin (Manis javanica) [18,19]. Associations 3/20 and 10/
17 are lacking in golden moles/tenrecs. Murphy et al.
(2004) proposed that the associations 3/20 and 10/17
were probably lost in golden moles/tenrecs. No reciprocal
painting was done in elephant shrews or golden moles/
tenrecs and it is therefore unknown if these associations
are actually homologous. There is weak cytogenetic evi-
dence linking elephant shrews and golden moles/tenrecs.
An alternate hypothesis might be a sister relationship
between elephant shrews and aardvarks. Perhaps a rapid
divergence in elephant shrews, golden moles/tenrecs and
aardvarks also resulted in limited phylogenetic signals for
these chromosome associations.
The root of the Eutherian tree
Although the super-order assemblies appear well estab-
lished, the most basal position on the eutherian tree has
not been determined with certainty [2,25,30]. Afrotheria
and Xenarthra are the two oldest eutherian clades and
probably emerged from the Southern Hemisphere in
excess of 100 million years ago [31,32]. Molecular dating
and biogeography have provided evidence that crown-
group Eutheria may have their most recent common
ancestry in the Southern Hemisphere (Gondwana) [32].
The other two clades (Laurasiatheria and Euarchontog-
lires) can be grouped as Boreoeutheria [33].
There are currently three hypotheses for the root of the
eutherian tree. Most discussions from molecular studies
place emphasis on either Afrotheria or Xenarthra as the
most basal clade [25,34]. A third hypothesis states that the
ancestral eutherian karyotype is a combination of both
clades. This hypothesis cannot be completely ruled out
and is preferred in some studies [35,36]. However, the
suite of derived chromosomal associations found in all
studied Afrotheria argues against the hypothesis that a
combination of the two clades is basal to the eutherians.
Recently, a report on retroelements gives support for the
hypothesis that Xenarthra is the sister group to all other
placentals [37]. Indeed, new cytogenetic comparisons
show that the proposed ancestral eutherian karyotype is
essentially conserved in Xenarthra, specifically in the two-
toed sloth (Choloepus hoffmanii) [16]. These two studies
should be given attention because both take into consid-
eration rare genomic events in which convergence is par-
ticularly limited. The conserved xenarthran karyotype
may well be indicative of their phylogenomic position
among eutherians. However, an essential point is that all
reconstructions of the ancestral eutherian karyotype are
preliminary until a relevant outgroup is studied with chro-
mosome painting. A taxonomically rich array of species
supported by appropriate out-groups is vital to the recon-
struction of mammalian genome evolution. The defi-
ciency of comparative chromosome painting data
between eutherians and marsupials is a severe limitation
on attempts to delineate the mammalian ancestral
genome. The analyses of other afrotherians, xenarthrans
and marsupials may clarify these unresolved questions.
Conclusion
The chromosome painting data presented here leave little
doubt that Tethytheria is a clade within Afrotheria and
implies support for the Paenungulata assemblage. Recent
retroposon data also confirmed Paenungulata, but could
Table 1:
2n12345678910111213141516171819202122
AEK 48121111221 2 1 2 1 1 1 2 1 1 2 1 1 2
manatee 48245111221 2 1 2(4) 2 1 2 2 1 1 3 1 2 2
golden mole 30121111221 2 1 2 1 1 1 2 1 1 2 1 1 2
elephant shrew 2612411122121 2 1112(3)112(3)112
aardvark 20121111221 2 1 2 1 1 1 2 1 1 2 1 1 2
elephant 56445311221 2 3 2 2 1 2 2 1 1 3 1 2 2
Number of segments homologous to human chromosome found in Afrotheria species. The taxa in the first, left column: AEK = ancestral eutherian
karyotype [14, 16, 40]. The second column list the 2n, diploid numbers for each species and the remaining columns refer to signals found for each
human chromosome. The number in brackets refers to higher number of hybridization signals due to pericentric inversions.
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not resolve the phylogenetic relationships among ele-
phants, sirenians and hyraxes [27]. It is generally appreci-
ated that characters with high evolutionary rates provide
good phylogenetic resolution. Afrotherian karyotypes
demonstrate high rates of chromosome evolution and
numerous derived inter-chromosomal rearrangements
link elephants and manatees. It is therefore likely that
additional chromosome painting in rock hyraxes could
shed light on the divergence sequence and resolve the Pae-
nungulata trichotomy.
Methods
Chromosome preparations of a male Florida manatee
(Trichechus manatus latirostris, TMA) were obtained from
peripheral blood mononuclear cells (PBMCs) and pri-
mary fibroblast cartilage cell culture. Cells were cultured
in RPMI 1640 (Hyclone) supplemented with 20% fetal
bovine serum (FBS), L-glutamine (0.01%) and gen-
tamicin (25 µg/ml). PBMCs were incubated in-vivo using
phytohemagglutinin (PHA, 0.25 mg/mL) as a mitotic
stimulant for 72 to 96 hr at 36°C in 5% carbon dioxide,
95% air and 100% relative humidity. Routine procedures
were used for chromosome preparations. We followed the
chromosome nomenclature as previously published [12]
pairing and grouping chromosomes by banding patterns,
relative lengths and morphology.
Human chromosome paints were obtained as previously
described by chromosome flow sorting followed by
degenerate oligonucleotide primed PCR amplification
[38,39]. Paints were labeled with either biotin-dUTP, dig-
oxigen-dUTP (both from Roche Applied Science) or Spec-
trum Orange-dUTP (Vysis).
Interspecific in-situ hybridizations of Florida manatee
chromosomes with human probes were performed with
300 to 500 ng of each biotin-labeled probe, 10 µg of
human Cot-1 DNA and 5 µg of ssDNA. The mixture was
precipitated and dissolved in 13–15 µl of hybridization
mixture (formamide 50%, dextran sulfate 10%, 2 × SSC).
Direct labeling with Spectrum Orange followed a Nick
Translation protocol (Vysis) using 1 µg of each amplified
human DNA probe, 0.2 mM Spectrum Orange and 25 µg
each of human and manatee Cot-1 DNA (Applied Genet-
ics Laboratories, Inc.). The mixture was precipitated and
dissolved in 10 µl distilled water. Approximately 300 ng
of probe from this mixture were dissolved in 10.5 µl
Hybrizol VII (Q-BIOgene) and 0.75 µg each of human
and manatee Cot-1 DNA.
The labeled probe mixture was denatured at 80°C for 10
min and reannealed at 37°C for 90 min before hybridiza-
tion. Slides were aged at 37°C for 30 min followed by
dehydration in a room temperature 70, 80, 90, and 100%
ethanol series. The DNA was denatured in 70% forma-
mide/2 × SSC, at 65°C for 90–120 s, and quenched in an
ice-cold ethanol series. Hybridization was carried out in a
humidity chamber at 37°C for five days. Post-hybridiza-
tion washes followed standard procedures at 40°C. Biotin
detection was performed with avidin-conjugated FITC
(Vector) for 45 min at 37°C. Counterstaining was per-
formed with DAPI (0.8 ng/µl) for 10 min and the slides
were mounted with antifade (100 mg p-phenylenedi-
amine in 80 ml glycerine, 20 ml PBS, pH 8).
Analyses were performed under a Zeiss Axiophot 2 or Axi-
oskop fluorescence microscope coupled with a CCD cam-
era (Photometrics), and images were captured with the
Smart Capture software (Digital Scientific Inc.).
Authors' contributions
RS and RZ conceived and designed the experiments. MK,
SB, TD and RS performed the experiments. BG, MK, RZ,
GS and PM prepared and contributed reagents/cell cul-
tures/analysis tools. RS, MK and SB analysed the data. RS,
MK and PM wrote the paper. All author read and
approved the final manuscript.
Acknowledgements
RS was supported by a grant "Mobility of Italian and foreign researchers
residing abroad" from MIUR (Ministero Italiano della Universita' e della
Ricerca). The U.S. Geological Survey and the University of Florida College
of Veterinary Medicine provided support to MK. Samples of manatee tissue
were collected under the U.S. Fish and Wildlife Service federal research
Table 2:
1/19 2/3 2/8 3/13 3/20 3/21 4/8 5/21 7/16 8/22 10/12 10/17 12/22 14/15 16/19 18/19 total
AEK XX X ? XXX 67
elephant shrew X X X X X X X X X X X X X 21
golden mole X X X X X X X X X X 14
Aardvark X X X XXX X X X X X X 20
Elephant XX X X XXXX XXXX17
Manatee XX X X XXXX XXXX15
Chromosomal associations found in two or more Afrotheria taxa. AEK = ancestral eutherian karyotype [14, 16, 40]. Associations were counted
only once to derive the total.
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permit MA791721 issued to the USGS, Sirenia Project. Many thanks for the
expertise and support of the UF Cytogenetics Laboratory staff.
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... However, branching within the Paenungulata is still debated (a phylogenetic time tree is provided in Figure 1). Rapid radiation, a deep divergence, and an extensive morphological diversification has led to limited phylogenetic signal confounding resolution at morphological and nucleotide levels (Amrine- Madsen et al., 2003;Kellogg et al., 2007;Nishihara et al., 2005;Pardini et al., 2007;Seiffert, 2007). Based on nine molecular loci, Sirenia is sister to Hyracoidea plus Proboscidea , whereas morphological evidence supports a Sirenian-Proboscidean clade (Tethytheria) within Paenungulata (Tassy & Shoshani, 1988). ...
... Songs are used as advertisement signals and possess geographical dialects with regard to syntax and syllable order (Kershenbaum et al., 2012). Research suggests that dispersing males introduce (Kellogg et al., 2007). Very few studies have been conducted on Paenungulata vocal learning. ...
... Because of the social life of elephants, the social cohesion function of VPL (Sewall et al., 2016) appears to be more reasonable (at least for the extant species). Yet an alternative explanation for the origin of VPL should not be ruled out, as one could argue that ancient three-dimensionality played a role in elephant evolution (Kellogg et al., 2007). Theoretically, this could be consistent with the mate choice and spatial-dimensional environment origin of complex VPL. ...
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Vocal production learning is the ability to modify a vocal output in response to auditory experience. It is essential for human speech production and language acquisition. Vocal learning evolved independently several times in vertebrates, indicating evolutionary pressure in favor of this trait. This enables cross-species comparative analysis to be used to test evolutionary hypotheses. Humans share this ability with a versatile but limited group of species: songbirds, parrots and hummingbirds, bats, cetaceans, seals, and elephants. Although case studies demonstrate that African savanna and Asian elephants are capable of heterospecific imitation, including imitation of human words, our understanding of both the underlying mechanisms and the adaptive relevance within the elephant’s natural communication system is limited. Even though comparing phylogenetically distant species is intriguing, it is also worthwhile to investigate whether and to what extent learned vocal behavior is apparent in species phylogenetically close to an established vocal learner. For elephants, this entails determining whether their living relatives share their special ability for (complex) vocal learning. In this review, we address vocal learning in Elephantidea and Sirenia, sister groups within the Paenungulata. So far, no research has been done on vocal learning in Sirenians. Because of their aquatic lifestyle, vocalization structure, and evolutionary relationship to elephants, we believe Sirenians are a particularly interesting group to study. This review covers the most important acoustic aspects related to vocal learning in elephants, manatees, and dugongs, as well as knowledge gaps that must be filled for one to fully comprehend why vocal learning evolved (or did not) in these distinctive but phylogenetically related taxa.
... To date, only T. manatus latirostris (2n = 48), T. manatus manatus (2n = 48), and T. inunguis (2n = 56) have briefly described karyotypes, with pair 20 carrying the nucleolus organizer region (NOR) in the terminal region and exclusively pericentromeric constitutive heterochromatin [3][4][5]. Despite the existence of G-banded karyotypes and total chromosome probes for genomic mapping of the Trichechus species, no comparative analysis has been performed to infer evolutionary relationships within the genus [6]. ...
... The analyzed TIN specimens presented a karyotype with 2n = 56 and an autosomal fundamental number (NFa) equal to 92, composed of 19 pairs of metacentric/submetacentric chromosomes (pairs [1][2][3][4][5][6][7][8][9][10][11][12][13][14][17][18][20][21]23), and eight acrocentric pairs (pairs [15][16]19,22,[24][25][26][27] ( Figure 1A); the sex-determination system is of the XY type, with submetacentric X and acrocentric Y ( Figure 1A). In turn, TMM specimens presented 2n = 48 and Nfa = 84, with 19 pairs of metacentric/submetacentric morphology (pairs 1 to 19), and four pairs of acrocentric chromosomes (pairs 20 to 23) ( Figure 1C); the sex pair is of the XY type, with submetacentric X and acrocentric Y ( Figure 1C). ...
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Great efforts have been made to preserve manatees. Recently, a hybrid zone was described between Trichechus inunguis (TIN) and the Trichechus manatus manatus (TMM) in the Amazon estuary. Cytogenetic data on these sirenians are limited, despite being fundamental to understanding the hybridization/introgression dynamics and genomic organization in Trichechus. We analyzed the karyotype of TMM, TIN, and two hybrid specimens (“Poque” and “Vitor”) by classical and molecular cytogenetics. G-band analysis revealed that TMM (2n = 48) and TIN (2n = 56) diverge by at least six Robertsonian translocations and a pericentric inversion. Hybrids had 2n = 50, however, with Autosomal Fundamental Number (FNA) = 88 in “Poque” and FNA = 74 in “Vitor”, and chromosomal distinct pairs in heterozygous; additionally, “Vitor” exhibited heteromorphisms and chromosomes whose pairs could not be determined. The U2 snDNA and Histone H3 multi genes are distributed in small clusters along TIN and TMM chromosomes and have transposable Keno and Helitron elements (TEs) in their sequences. The different karyotypes observed among manatee hybrids may indicate that they represent different generations formed by crossing between fertile hybrids and TIN. On the other hand, it is also possible that all hybrids recorded represent F1 and the observed karyotype differences must result from mechanisms of elimination.
... Considering the number of chromosomal arms, the different karyotypes observed between these two species could only be explained by the occurrence of both intrachromosomal and interchromosomal rearrangements, such as inversions, translocations and fusions, although the real process will only be elucidated after applying comparative chromosome painting to confirm homeology among chromosome pairs [13,14]. Unfortunately, up to now, ZOO-FISH is restricted to T. manatus [17,18], and the results strongly supported the close relationship between sirenians and elephants (Tethytheria) supporting the clade Paenungulata, which includes Sirenia, Proboscidea, and Hyracoidea [18]. Interestingly, although these two species show clear chromosomal differences [11][12][13][14][15][16][17][18], there are no karyological studies in individuals from the sympatric area, except for one male found in illegal captivity, which was confirmed as being a hybrid between T. manatus and T. inunguis by mitochondrial and nuclear DNA data, as well as cytogenetic analysis [1,8]. ...
... Unfortunately, up to now, ZOO-FISH is restricted to T. manatus [17,18], and the results strongly supported the close relationship between sirenians and elephants (Tethytheria) supporting the clade Paenungulata, which includes Sirenia, Proboscidea, and Hyracoidea [18]. Interestingly, although these two species show clear chromosomal differences [11][12][13][14][15][16][17][18], there are no karyological studies in individuals from the sympatric area, except for one male found in illegal captivity, which was confirmed as being a hybrid between T. manatus and T. inunguis by mitochondrial and nuclear DNA data, as well as cytogenetic analysis [1,8]. ...
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Two species of manatees are found in Northern Brazil—the Antillean manatee (Trichechus manatus), which is found along the coast from Florida to Northeastern Brazil, and the Amazonian manatee (Trichechus inunguis), endemic to the Amazon drainage basin. These species show a sympatric distribution in the region of the Marajó Archipelago, an estuarine area surrounding the Amazon River mouth. There is evidence of the occurrence of interspecific hybrids in this area, based on mitochondrial DNA analyses, although the use of nuclear markers has not corroborated this proposal. Considering that these species show very distinct karyotypes, despite being closely related (2n = 48 in T. manatus and 2n = 56 in T. inunguis), hybrids would present distinct chromosome numbers. Based on this, we conducted cytogenetic analyses using classic and molecular techniques in three calves found stranded in the Marajó Island and Amapá coast. The results showed that one of them, morphologically classified as T. inunguis, presented the correspondent karyotype, with 2n = 56. However, the other two, which were phenotypically similar to T. manatus, showed 2n = 49. Despite the same diploid number, their G-banding patterns revealed some differences. The results of the distribution of some microsatellite sequences have also confirmed the heterozygosity of some chromosomal pairs in these two individuals. These results are the first indubitable confirmation of the occurrence of natural hybrids between T. manatus and T. inunguis, and also brings about some issues concerning the viability of hybrids, considering that these two individuals do not correspond to an F1 hybrid, but instead, both presented a possible F2 karyotype.
... Regarding phylogenetics, the closest extant relatives of elephants are the aquatic sea-cows (dugongs and manatees; order Sirenia) and the terrestrial hyraxes (order Hyracoidea) (Kellogg et al. 2007). Very few studies have been conducted on seacow and hyrax vocal learning behavior and abilities. ...
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Complex vocal learning, the capacity to imitate new sounds, underpins the evolution of animal vocal cultures and song dialects and is a key prerequisite for human speech and song. Due to its relevance for the understanding of cultural evolution and the biology and evolution of language and music, the trait has gained much scholarly attention. However, while we have seen tremendous progress with respect to our understanding of its morphological, neurological and genetic aspects, its peculiar phylogenetic distribution has remained elusive. Intriguingly, animals as distinct as hummingbirds and humpback whales share well-developed vocal learning capacity in common with humans, while this ability is quite limited in nonhuman primates. Yet, solving this ‘vocal learning conundrum’ may shed light on the constraints ancestral humans overcame to unleash their vocal capacities. To this end I consider major constraints and functions that have been proposed. I highlight an especially promising ecological constraint, namely the spatial dimensionality of the environment. Based on an informal comparative review, I suggest that complex vocal learning is associated with three-dimensional habitats such as air and water. I argue that this is consistent with recent theoretical advances—i.e., the coercion-avoidance and dimensionality hypotheses—and with the long-standing hypothesis that mate choice is a major driver of the evolution and origin of complex vocal learning. However, I stress that multiple functions may apply and that quantitative phylogenetic comparative methods should be employed to finally resolve the issue.
... Regarding phylogenetics, the closest extant relatives of elephants are the aquatic sea-cows (dugongs and manatees; order Sirenia) and the terrestrial hyraxes (order Hyracoidea) (Kellogg et al., 2007). Very few studies have been conducted on sea-cow and hyrax vocal learning behavior and abilities. ...
Preprint
Full-text available
Complex vocal learning, the capacity to imitate new sounds, underpins the evolution of animal vocal cultures and song dialects and is a key prerequisite for human speech and song. Due to its relevance for the understanding of cultural evolution and the biology and evolution of language and music, the trait has gained much scholarly attention. However, while we have seen tremendous progress with respect to our understanding of its morphological, neurological and genetic aspects, its peculiar phylogenetic distribution has remained elusive. Intriguingly, animals as distinct as hummingbirds and humpback whales share well-developed vocal learning capacity in common with humans, while this ability is quite limited in nonhuman primates. Yet, solving this ‘vocal learning conundrum’ may shed light on the constraints ancestral humans overcame to unleash their vocal capacities. To this end I consider major constraints and functions that have been proposed. I highlight an especially promising ecological constraint, namely the spatial dimensionality of the environment. Based on an informal comparative review, I suggest that complex vocal learning is associated with three-dimensional habitats such as air and water. I argue that this is consistent with recent theoretical advances – i.e., the coercion-avoidance and dimensionality hypotheses – and with the long-standing hypothesis that mate choice is a major driver of the origin and evolution of complex vocal learning. However, I stress that multiple functions may apply and that quantitative phylogenetic comparative methods should be employed to finally resolve the issue.
... Morphological, developmental, paleontological, and, more recently, molecular evidence for an aquatic 167 ancestry of elephants has been steadily accumulating and now constitutes a solid case (Gaeth, Short, & 168 Renfree, 1999;Liu, Seiffert, & Simons, 2008;Mirceta et al., 2013). Of relevance here, the closest extant 169 relatives of elephants are the aquatic dugongs and manatees (order Sirenia) and the terrestrial and vocally 170 apt hyraxes (order Hyracoidea) ( Kellogg et al., 2007). Tellingly, reconstruction based on, among other 171 things, net surface charge of myoglobin indicates that the last common ancestor of sea cows, elephants, 172 and hyraxes had diving capacities of the magnitude otherwise only observed in lineages of expert divers, 173 such as cetaceans and pinnipeds (Mirceta et al., 2013). ...
Preprint
Full-text available
Vocal learning, the capacity to add new vocalizations to one’s repertoire, has gained much research attention because it is a key prerequisite for spoken language and vocal music. As a result, major progress has been made regarding its developmental, genetic and morphological underpinnings. However, why it evolved and, more specifically, why it is so peculiarly distributed across species, remain long standing questions. For instance, animals as distinct as hummingbirds and humpback whales share well-developed vocal learning capacity in common with humans, while this ability appears quite limited in nonhuman primates. In order to tackle these questions, this article brings together existing hypotheses, such as about the evolutionary functions of vocal learning and the impact of the spatial environment on sexual selection processes. Consistent with these hypotheses, I suggest, based on a systematic review of the latest evidence, that the cross-species distribution of vocal learning is associated with the spatial dimensionality of ancestral environments, and that this association is mediated by mate choice. This new perspective on the evolution of vocal learning is expected to open up novel avenues for further research on this pivotal cognitive ability. One implication is that human vocal learning may have evolved in (semi-)arboreal hominins at the base of the human clade, such as Ardipithecus ramidus, and thus much earlier than commonly assumed.
... Based on genomic resources analyzed here, the manatees share with other afrotherians a unique DR translocation separating it from the core class II region. Despite manatee genes being distributed over four main scaffolds, all class II MHC sequences, including translocated DR loci, presumably lie on the same chromosome, based on other afrotherian class II regions and data from chromosome painting in manatee (44). The similarity between the manatee and elephant class II organization suggests that elephant may serve as a model for understanding the manatee MHC function. ...
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Sirenians share with cetaceans and pinnipeds several convergent traits selected for the aquatic lifestyle. Living in water poses new challenges not only for locomotion and feeding but also for combating new pathogens, which may render the immune system one of the best tools aquatic mammals have for dealing with aquatic microbial threats. So far, only cetaceans have had their class II Major Histocompatibility Complex (MHC) organization characterized, despite the importance of MHC genes for adaptive immune responses. This study aims to characterize the organization of the marine mammal class II MHC using publicly available genomes. We located class II sequences in the genomes of one sirenian, four pinnipeds and eight cetaceans using NCBI-BLAST and reannotated the sequences using local BLAST search with exon and intron libraries. Scaffolds containing class II sequences were compared using dotplot analysis and introns were used for phylogenetic analysis. The manatee class II region shares overall synteny with other mammals, however most DR loci were translocated from the canonical location, past the extended class II region. Detailed analysis of the genomes of closely related taxa revealed that this presumed translocation is shared with all other living afrotherians. Other presumptive chromosome rearrangements in Afrotheria are the deletion of DQ loci in Afrosoricida and deletion of DP in E. telfairi. Pinnipeds share the main features of dog MHC: lack of a functional pair of DPA/DPB genes and inverted DRB locus between DQ and DO subregions. All cetaceans share the Cetartiodactyla inversion separating class II genes into two subregions: class IIa, with DR and DQ genes, and class IIb, with non-classic genes and a DRB pseudogene. These results point to three distinct and unheralded class II MHC structures in marine mammals: one canonical organization but lacking DP genes in pinnipeds; one bearing an inversion separating IIa and IIb subregions lacking DP genes found in cetaceans; and one with a translocation separating the most diverse class II gene from the MHC found in afrotherians and presumptive functional DR, DQ, and DP genes. Future functional research will reveal how these aquatic mammals cope with pathogen pressures with these divergent MHC organizations.
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The history of each human chromosome can be studied through comparative cytogenetic approaches in mammals which permit the identification of human chromosomal homologies and rearrangements between species. Comparative banding, chromosome painting, Bacterial Artificial Chromosome (BAC) mapping and genome data permit researchers to formulate hypotheses about ancestral chromosome forms. Human chromosome 13 has been previously shown to be conserved as a single syntenic element in the Ancestral Primate Karyotype; in this context, in order to study and verify the conservation of primate chromosomes homologous to human chromosome 13, we mapped a selected set of BAC probes in three platyrrhine species, characterised by a high level of rearrangements, using fluorescence in situ hybridisation (FISH). Our mapping data on Saguinus oedipus, Callithrix argentata and Alouatta belzebul provide insight into synteny of human chromosome 13 evolution in a comparative perspective among primate species, showing rearrangements across taxa. Furthermore, in a wider perspective, we have revised previous cytogenomic literature data on chromosome 13 evolution in eutherian mammals, showing a complex origin of the eutherian mammal ancestral karyotype which has still not been completely clarified. Moreover, we analysed biomedical aspects (the OMIM and Mitelman databases) regarding human chromosome 13, showing that this autosome is characterised by a certain level of plasticity that has been implicated in many human cancers and diseases.
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The precise hierarchy of ancient divergence events that led to the present assemblage of modern placental mammals has been an area of controversy among morphologists, palaeontologists and molecular evolutionists. Here we address the potential weaknesses of limited character and taxon sampling in a comprehensive molecular phylogenetic analysis of 64 species sampled across all extant orders of placental mammals. We examined sequence variation in 18 homologous gene segments (including nearly 10,000 base pairs) that were selected for maximal phylogenetic informativeness in resolving the hierarchy of early mammalian divergence. Phylogenetic analyses identify four primary superordinal clades: (I) Afrotheria (elephants, manatees, hyraxes, tenrecs, aardvark and elephant shrews); (II) Xenarthra (sloths, anteaters and armadillos); (III) Glires (rodents and lagomorphs), as a sister taxon to primates, flying lemurs and tree shrews; and (IV) the remaining orders of placental mammals (cetaceans, artiodactyls, perissodactyls, carnivores, pangolins, bats and core insectivores). Our results provide new insight into the pattern of the early placental mammal radiation.
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Correction for ‘Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant ( Loxodonta africana ) generated by chromosome painting’ by L. Fronicke, J. Wienberg, G. Stone, L. Adams and R. Stanyon (Proc. R. Soc. Lond. B 270 , 1331–1340. (doi: [10.1098/rspb.2003.2388][1])). On page 1336, errors were contained in figure 3a and the (a) and (b) labels in the figure 3 caption were incorrectly transposed. [1]: /lookup/doi/10.1098/rspb.2003.2388
Article
We concatenated sequences for four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes [aquaporin, alpha 2B adrenergic receptor (A2AB), interphotoreceptor retinoid-binding protein (IRBP), von Willebrand factor (vWF)] into a multigene data set representing 11 eutherian orders (Artiodactyla, Hyracoidea, Insectivora, Lagomorpha, Macroscelidea, Perissodactyla, Primates, Proboscidea, Rodentia, Sirenia, Tubulidentata). Within this data set, we recognized nine mitochondrial partitions (both stems and loops, for each of 12S rRNA, tRNA valine, and 16S rRNA; and first, second, and third codon positions of cytochrome b) and 12 nuclear partitions (first, second, and third codon positions, respectively, of each of the four nuclear genes). Four of the 21 partitions (third positions of cytochrome b, A2AB, IRBP, and vWF) showed significant heterogeneity in base composition across taxa. Phylogenetic analyses (parsimony, minimum evolution, maximum likelihood) based on sequences for all 21 partitions provide 99–100% bootstrap support for Afrotheria and Paenungulata. With the elimination of the four partitions exhibiting heterogeneity in base composition, there is also high bootstrap support (89–100%) for cow + horse. Statistical tests reject Altungulata, Anagalida, and Ungulata. Data set heterogeneity between mitochondrial and nuclear genes is most evident when all partitions are included in the phylogenetic analyses. Mitochondrial-gene trees associate cow with horse, whereas nuclear-gene trees associate cow with hedgehog and these two with horse. However, after eliminating third positions of A2AB, IRBP, and vWF, nuclear data agree with mitochondrial data in supporting cow + horse. Nuclear genes provide stronger support for both Afrotheria and Paenungulata. Removal of third positions of cytochrome b results in improved performance for the mitochondrial genes in recovering these clades.
Chapter
Periodically it is worthwhile to assess our knowledge and understanding of mammalian phylogeny and one of its expressions, classification. This short paper is yet another attempt to do so, taking into account the results of recently published paleontological research and drawing heavily on work in progress by many researchers in many fields and in various parts of the world. Concepts of mammalian phylogeny and classification have changed markedly during the last few years. A good many of the ideas expressed here are frankly speculative, but they are presented anyway in order to determine how well they will stand scrutiny, especially by nonpaleontologists. A few years ago I prepared a paper with a similar aim (McKenna, 1969), but that paper is now outdated. In the present offering I attempt to update certain aspects of my previous review by taking into account research published since 1969, as well as work being incorporated into a new classification of the Mammalia now being prepared which wall deal with all taxonomic levels down to the subgeneric level in essentially the same style as Simpson’s (1945) classification.
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Detailed chromosome studies were conducted for the Florida manatee (Trichechus manatus latirostris) utilizing primary chromosome banding techniques (G- and Q-banding). Digital microscopic imaging methods were employed and a standard G-banded karyotype was constructed for both sexes. Based on chromosome banding patterns and measurements obtained in these studies, a standard karyotype and ideogram are proposed. Characterization of additional cytogenetic features of this species by supplemental chromosome banding techniques, C-banding (constitutive heterochromatin), Ag-NOR staining (nucleolar organizer regions), and DA/DAPI staining, was also performed. These studies provide detailed cytogenetic data for T. manatus latirostris, which could enhance future genetic mapping projects and interspecific and intraspecific genomic comparisons by techniques such as zoo–FISH.
Article
As currently recognized, the mammalian order Lipotyphla contains six extant families: Chrysochloridae, Erinaceidae, Solenodontidae, Soricidae, Talpidae, and Tenrecidae. Although most mammalogists have accepted this taxon, the morphological support for Lipotyphla is relatively weak, and recent phylogenetic studies using molecular data have concluded that it is not monophyletic. Instead, these molecular studies place chrysochlorids and tenrecids in the proposed clade Afrotheria, together with aardvarks, elephants, elephant shrews, hyraxes, and sirenians. Despite strong molecular support, Afrotheria has received little or no morphological support. It was recently suggested that a mobile snout might be a morphological feature uniting afrotherians. To test this proposal, I dissected the extrinsic snout musculature in an assortment of lipotyphlan and afrotherian mammals. These muscles provide support for Lipotyphla but not for Afrotheria. The snout is moved by different muscles in different afrotherian taxa, suggesting that the mobile snout is not homologous across different afrotherian lineages. In contrast, lipotyphlans have a distinctive set of five snout muscles moving the snout tip that appears to be unique to these six families. In addition, in soricids and talpids, four of the five snout muscles originate posterior to the zygomatic arch, supporting sister-taxon status for these two lineages. Although the extrinsic snout musculature does not support Afrotheria as presently proposed, it is consistent with an Afrotheria that does not include chrysochlorids and tenrecids.