Molecular Ecology (2011)
doi: 10.1111/j.1365-294X.2011.05245.x
FROM THE COVER
An ancient icon reveals new mysteries: mummy DNA
resurrects a cryptic species within the Nile crocodile
E V O N H E K K A L A , * †1 M A T T H E W H . S H I R L E Y , ‡1 G E O R G E A M A T O , † J A M E S D . A U S T I N , ‡
S U E L L E N C H A R T E R , § J O H N T H O R B J A R N A R S O N , ‡– K E N T A . V L I E T , * * M A R L Y S L . H O U C K , §
R O B D E S A L L E , † and M I C H A E L J . B L U M ††
*Department of Biological Sciences, Fordham University, New York, NY, USA, †Sackler Institute for Comparative Genomics,
American Museum of Natural History, New York, NY, USA, ‡Department of Wildlife Ecology & Conservation, University of
Florida, Gainesville, FL, USA, §Institute for Conservation Research, San Diego Zoo, San Diego, CA, USA, –Wildlife
Conservation Society, New York, NY, USA, **Department of Biological Sciences, University of Florida, Gainesville, FL, USA,
††Department of Ecology & Evolutionary Biology, Tulane University, New Orleans, LA, USA
Abstract
The Nile crocodile (Crocodylus niloticus) is an ancient icon of both cultural and scientific
interest. The species is emblematic of the great civilizations of the Nile River valley and
serves as a model for international wildlife conservation. Despite its familiarity, a
centuries-long dispute over the taxonomic status of the Nile crocodile remains
unresolved. This dispute not only confounds our understanding of the origins and
biogeography of the ‘true crocodiles’ of the crown genus Crocodylus, but also complicates
conservation and management of this commercially valuable species. We have taken a
total evidence approach involving phylogenetic analysis of mitochondrial and nuclear
markers, as well as karyotype analysis of chromosome number and structure, to assess
the monophyletic status of the Nile crocodile. Samples were collected from throughout
Africa, covering all major bioregions. We also utilized specimens from museum
collections, including mummified crocodiles from the ancient Egyptian temples at
Thebes and the Grottes de Samoun, to reconstruct the genetic profiles of extirpated
populations. Our analyses reveal a cryptic evolutionary lineage within the Nile crocodile
that elucidates the biogeographic history of the genus and clarifies long-standing
arguments over the species’ taxonomic identity and conservation status. An examination
of crocodile mummy haplotypes indicates that the cryptic lineage corresponds to an
earlier description of C. suchus and suggests that both African Crocodylus lineages
historically inhabited the Nile River. Recent survey efforts indicate that C. suchus is
declining or extirpated throughout much of its distribution. Without proper recognition
of this cryptic species, current sustainable use-based management policies for the Nile
crocodile may do more harm than good.
Keywords: ancient DNA, African biogeography, Crocodylus, C. niloticus, C. suchus, mummy
Received 30 January 2011; revision received 6 July 2011; accepted 7 July 2011
Introduction
Correspondence: Evon Hekkala,
E-mail: Ehekkala@Fordham.edu
We dedicate this work to our co-author, John Thorbjarnarson,
who passed during the final preparation of this manuscript
and whose unwavering commitment to crocodile conservation
has been an inspiration to all of us.
1
Contributed equally as joint first authors.
2011 Blackwell Publishing Ltd
The idea that taxonomy is destiny (May 1990) is particularly relevant to the conservation and management of
crocodilians (Hutton 2000). Current policies intended to
promote sustainable harvest of managed crocodile populations are based predominantly on morphological criteria that provide limited taxonomic and phylogenetic
2 E. HEKKALA ET AL.
resolution (Brazaitis 1973; Ross 1998). Assumptions of
genetic homogeneity and continuing taxonomic uncertainty within this group raise the concern that management plans may not adequately protect extant diversity
and evolutionary potential, especially in more widespread species. This situation is exemplified by the Nile
crocodile (Crocodylus niloticus), a widespread, commercially exploited species that has become a model of
international wildlife conservation (Ross 1998; Hutton
2000; Fergusson 2010) despite a history of taxonomic
discord that has persisted since the eighteenth century
(Table 1; Fuchs et al. 1974, King & Burke 1989).
The Nile crocodile is comprised of 11 synonymized,
historically described species and seven previously proposed subspecies (Table 1). As currently managed, the
species is recognized as a single entity, although recent
molecular studies provide evidence to the contrary.
Limited phylogenetic studies indicate that C. niloticus is
paraphyletic (Schmitz et al. 2003; Meredith et al. 2011),
and multilocus microsatellite comparisons have shown
that populations across Africa are geographically differentiated (Hekkala et al. 2009).
Although the Nile crocodile is considered widespread
with a largely sub-Saharan distribution, managing this
culturally and commercially valuable species as a single, widespread evolutionary lineage may be contributing to a globally significant loss of crocodilian diversity
(Hekkala et al. 2009; Shirley et al. 2009). This concern is
particularly important in western regions with popula-
tions that are increasingly susceptible to range contraction and local extirpation (Shirley et al. 2009). For
example, populations were found in the central Sahara
until the late nineteenth century (de Smet 1999) though
only small isolates may persist in some locales today
(Shine et al. 2001).
Here we test the hypothesis that the Nile crocodile is
a single, homogeneous evolutionary lineage through
total evidence molecular analysis of 5016 bp of mitochondrial and nuclear sequence data from samples collected from wild populations across Africa and
Madagascar (Fig. 1, Table 2). We provide a complementary
temporal
perspective
spanning
over
2 200 years through diagnostic haplotype analysis of
historical specimens from museum holdings, including
crocodile mummies from the ancient Egyptian sites of
Thebes and the Grottes de Samoun. Finally, we compare our sequence-based conclusions with karyotype
analysis.
Methods
Contemporary samples and markers
We collected 123 samples of Nile crocodiles from
throughout Africa (Fig. 1, Table 2). Collections were
made from wild or wild-caught, ranch-held individuals
and consisted of tail tissue or fresh blood (<0.5 mL)
either in lysis buffer or dried on Whatman filter paper.
Table 1 Taxonomic History of the Nile Crocodile. Locality refers to the type locality designation in the literature description, which
may not be the same as the origin of the type specimen for that taxon
Taxon
Author and Year
Locality
Crocodylus niloticus
Synonyms
Crocodylus vulgaris
Crocodylus suchus
Crocodilus multiscutatus
Crocodilus marginatus
Crocodilus lacunosus
Crocodilus complanatus
Crocodilus octophractus
Alligator cowieii
Crocodylus binuensis
Crocodilus madagascariensis
Crocodilus vulgaris var. madagascariensis
Crocodilus hexaphractos
Proposed subspecies
Crocodylus niloticus niloticus
Crocodylus niloticus africanus
Crocodylus niloticus chamses
Crocodylus niloticus cowiei
Crocodylus niloticus madagascariensis
Crocodylus niloticus pauciscutatus
Crocodylus niloticus suchus
Laurenti 1768
Egypt
Cüvier 1807
Geoffroy Saint-Hilaire 1807
RÜPPELL in Cretzschmar 1826
GEOFFROY 1827
GEOFFROY 1827
GEOFFROY 1827
RÜPPELL in GRAY in Griffith & Pidgeon 1831
SMITH in Hewitt 1937
Baikie 1857
Grandidier 1872
Boettger 1877
RÜPPELL in SCHMIDT 1886 (nomen nudum)
Egypt
Nile and Niger Rivers
Sudan
Egypt
Egypt
Egypt
Sudan
South Africa
Nigeria
Madagascar
Madagascar
Sudan
LAURENTI 1768
LAURENTI 1768
Bory de Saint-Vincent 1824
SMITH in Hewitt 1937
Grandidier 1872
Deraniyagala 1948
Geoffroy Saint-Hilaire 1807
Egypt
East Africa
Southern Congo
South Africa
Madagascar
Kenya
West Africa
2011 Blackwell Publishing Ltd
CRYPTIC AFRICAN CROCODYLUS SPECIES REVEALED 3
(a)
(b)
16
19
7
1
8
2
13 1 2
14
18
19
9
20
10
21
12
11
3
4
C. niloticus, ancestral
15
3
12
5
6
7
22
34
8
9
13
10 11
17
14
22
23
15
6
18
23
20
24
16
21
25 26
24
27
1 – 250
250 – 500
500 – 1000
1000 – 1500
1500 – 2500
2500 – 3500
3500 – 4500
4500 – 5825
1250
17
5
C. niloticus, derived
Rivers
Lakes
Country boundaries
Elevation (meters)
0
4
25
31
32
28
29
26
33
27
30
2500
Kilometers
Fig. 1 Map of sample localities showing the distribution of ancestral (white) and derived (red) haplotypes for historical pre-1975 (a)
and contemporary post-1975 (b) specimens.
To better understand the evolutionary history of C.
niloticus in relation to true crocodiles, our analyses
included data from samples of seven other Crocodylus
species representing both Asian and New World lineages. The remaining members of the Crocodylinae
(Osteolaemus tetraspis and Mecistops cataphractus) and
Alligator mississippiensis served as outgroups, reflecting
the most recent phylogenetic hypotheses for the crown
group of the Crocodylidae and the Order Crocodylia
(Gatesy & Amato 1992; Brochu 2003; McAliley et al.
2006; Meredith et al. 2011). These taxa were included
from samples taken from captive specimens (St. Augustine Alligator Farm, St. Augustine, FL, USA) or previously published sequences available on Genbank as
follows: C. rhombifer, C. acutus, C. moreletii, Mecistops
cataphractus and Osteolaemus tetraspis (all amplified and
sequenced as part of this study), C. intermedius
(12s—AY239132, 16s—AY239146, dloop—AF460207,
rag1—AY239173), C. porosus (12s—AY770534, 16s—
EU621805, dloop—AF460213, WANCY—DQ273698,
ND4—AJ810453), C. siamensis (mtDNA—EF581859,
rag1—AY136677) (Ray & Densmore 2002; Gatesy et al.
2003).
We examined sequence variation across a total of
5 016 bp from nine gene regions. Five regions (2761 bp)
were mitochondrial (mtDNA) and four were nuclear
(nDNA) (2254 bp), as follows: control region ⁄ dloop
(735 bp); 12s rRNA (421 bp); 16s rRNA (415 bp); WANCY tRNA cluster (Seutin et al. 1994) from the ND2flanking region including tRNA_Trp, tRNA_Ala,
tRNA_Asn, tRNA_Cys, and part of tRNA_Tyr (330 bp);
NADH dehydrogenase 4 (ND4, 860 bp); recombination 2011 Blackwell Publishing Ltd
activating gene 1 (rag1, 469 bp); ribosomal protein S6
(693 bp); and introns for tropomyosin (330 bp) and
ornithine decarboxylase (762 bp) (Friesen et al. 1999).
Contemporary sample data collection
DNA was extracted using Qiagen Easy-DNA extraction
kits or standard phenol–chloroform methods. Extraction
products were stored at 50 ng ⁄ lL. PCR cocktails and
cycling conditions were optimized for each marker
(Table S1, Supporting information) and amplifications
were performed on an ABI 9700 thermocycler in 20–
25 lL volumes. Sanger sequencing reactions were carried out using BigDye v3.1 sequencing kits in 6–8 lL
volumes. Gene regions were sequenced in both directions on either an ABI 3700 or 3730XL automated capillary sequencer. Base calling was performed with
Sequencher v4.1 (Genecodes Corp.). Consensus
sequences were produced with CLC v3.6.2. Marker
datasets were compiled and aligned individually in
MEGA4 (Tamura et al. 2007) utilizing Clustal W (Larkin
et al. 2007) (Gap penalties = 50, Gap Extension penalties = 25) and checked by eye prior to concatenation.
Contemporary sample analyses
Sequence data were first analyzed for fixed characters
using Population Aggregation Analysis (Davis & Nixon
1992) and terminal taxa with unique and fixed characters
were subsequently examined for phylogenetic structure
with data from all species combined by genome and concatenated for total evidence analysis (Maddison 1997;
Map
Number
Terminal Label
Year
Collected
2011 Blackwell Publishing Ltd
Museum
Specimen#
1825–1829
MNHN
1977_1606
1934
approx. 1824
1885
1886
FMNH
MNHN
MNHN
MNHN
20798
2175
1885407
1886_182
1882
1924
MNHN
AMNH
1886_186
28904
Cailloud
700–2200 YBP
MNHN
1986_1471
brought from Egypt 1820’s
Cailloud
700–2200 YBP
MNHN
1986_1473
brought from Egypt 1820’s
Cailloud
700–2200 YBP
MNHN
1986_1479
brought from Egypt 1820’s
V. Schoelcher
Gervais
700–2200 YBP
700–2200 YBP
MNHN
MNHN
1886_445
1986_1475
Gervais
700–2200 YBP
MNHN
1986_1478
Pariset
700–2200 YBP
MNHN
1986_1480
1927
1922
1922
1911
AMNH
AMNH
AMNH
AMNH
42962
23464
23465
10079
1803–1827
1749–1754
1966
approx. 1822
MNHN
MNHN
CAS
MNHN
7364
7524
133814
7546
Country
Locality
Collector
Figure 1A*
1
SENEGAL_1825
Senegal
UNK
2
3
4
5
SENEGAL_1934
SENEGAL_1824
IVORY COAST_1885
REP CONGO_1882
Senegal
Senegal
Cote-d’Ivoire
Republic of Congo
Kedougou(a)
UNK
Assinie
N’ganchou
G.S. Perrottet &
F.M.R. Leprieur
F.C. Wonder
Brongniart
Chaper
P.S. de Brazza
5
6
REP CONGO_1886
DEM REP CONGO_1924
N’gouchou
Kasai River
P.S. de Brazza
Father R. Callewaert
7
MUMMY_THEBES_A
Republic of Congo
Dem. Republic of
Congo
Egypt
7
MUMMY_THEBES_B
Egypt
7
MUMMY_THEBES_C
Egypt
7
8
MUMMY_HAUTE
MUMMY_SAMOUN_A
Egypt
Egypt
8
MUMMY_SAMOUN_B
Egypt
8
MUMMY_SAMOUN_C
Egypt
9
10
11
12
SUDAN_MELUT_1922
SUDAN_WNA_1922
SUDAN_WNB_1922
ZIMBABWE_1911
Sudan
Sudan
Sudan
Zimbabwe
Mummy - Grottes de
Thebes
Mummy - Grottes de
Thebes
Mummy - Grottes de
Thebes
Mummy, Haute Egypt
Mummy - Grottes de
Samoun
Mummy - Grottes de
Samoun
Mummy - Grottes de
Samoun
Melut
White Nile
White Nile
Faradje
13
14
15
16
SENEGAL_1803
SENEGAL_1768
CAMEROON_1966
EGYPT_1822
Senegal
Senegal
Cameroon
Egypt
UNK
UNK
Edea, Sanaga River
Nile
Anthony
Taylor
Taylor
Lang - Chapin
Expedition
C. Heudelot
Adanson
T.J. Papenfuss
T. Duvant
Notes
Not included in analysis,
partial sequence identical to
REP CONGO_1886
Crocodylus vert TYPE
Crocodylus vulgaris
PARATYPE
4 E. HEKKALA ET AL.
Table 2 Contemporary and Historical Samples Utilized in This Study. Locality and sampling data for each specimen utilized in this study. For archival material, both the original collection locality and the museum accession information are listed. Terminal Label refers to the specimen ID given in Fig. 2, Figs S1 and S2, Table 3, and Table S3. Museum
acronyms: AMNH—American Museum of Natural History (New York, NY, USA), CAS—California Academy of Sciences (San Francisco, CA, USA), FLMNH—Florida Museum
of Natural History (Gainesville, FL, USA), MNHN—Museum Nationale d’Histoire Naturelle (Paris, France), USNM—National Museum of Natural History, Smithsonian Institution, Washington, DC, ZFKM—Alexander Koenig Zoological Research Museum, Bonn, Germany). Genbank accession numbers are listed next to individuals from Schmitz et al.
2003. * Indicates only short 12s and ⁄ or dloop fragments were sequenced
2011 Blackwell Publishing Ltd
Table 2 (Continued)
Map
Number
Terminal Label
Country
Locality
Collector
17
ZIMBABWE_1912
Zimbabwe
Faradje
18
19
20
21
22
23
24
25
26
SUDAN_UN_1922
SUDAN_WNC_1922
SUDAN_WND_1922
SUDAN_WNE_1922
KENYA_1960
KENYA_1919
TANZANIA_1972
BOTSWANA_1967
MADAGASCAR_1885
Sudan
Sudan
Sudan
Sudan
Kenya
Kenya
Tanzania
Botswana
Madagascar
Zeraf, Upper Nile
White Nile
White Nile
White Nile
Garissa
Nairobi
UNK
Shakawe
Tulear
Lang - Chapin
Expedition
Taylor
Taylor
Taylor
Taylor
R.H. Pine
H.C. Raven
USFWS Confiscation
T. Liversedge
A. Grandidier
Year
Collected
Museum
Specimen#
1912
AMNH
10081
1922
1922
1922
1922
1960
1919–1920
1972
1967
1870
AMNH
AMNH
AMNH
AMNH
AMNH
USNM
AMNH
USNM
MNHN
23471
23466
23469
23470
88634
63592
108941
195448
6498
Madagascar
Madagascar
Madagascar
Amboasary
Amboasary
Amboasary
H. Bluntschli
H. Bluntschli
H. Bluntschli
1931
1931
1931
AMNH
AMNH
AMNH
71192
142496
71191
Mauritania
Matmata
S. Robin
1993
MNHN
1993_5805*
2
MAURITANIA_2
Mauritania
Aioun el-Atrouss
Bohme
UNK
ZFMK
Uncatalogued
3
SENEGAL
Senegal
Casamance River
M.H. Shirley
2008
FLMNH
Uncatalogued
4
GAMBIA_1
The Gambia
W. Bohme
UNK
N⁄A
4
4
5
6
GAMBIA_2
GAMBIA_3
BURKINA FASO
IVORY COAST_1
The Gambia
The Gambia
Burkina Faso
Cote-d’Ivoire
Kedougou, Gambia
River
River Gambia NP
River Gambia NP
UNK
Abi Lagoon
M.H. Shirley
M.H. Shirley
Bohme
M.H. Shirley
2008
2008
UNK
2006
FLMNH
FLMNH
N⁄A
FLMNH
Uncatalogued
Uncatalogued
7
8
9
IVORY COAST_2
GHANA_1
GHANA_2
Cote-d’Ivoire
Ghana
Ghana
Go River
Mole National Park
Legon Farms Dam,
Accra
M.H. Shirley
M.H. Shirley
M.H. Shirley
2006
2006
2006
FLMNH
FLMNH
FLMNH
Uncatalogued
Uncatalogued
Uncatalogued
10
11
BENIN*
NIGERIA
Benin
Nigeria
R. Bourgat
M.P.O. Dore
1978
2009
MNHN
FLMNH
1978_2051
Uncatalogued
12
CHAD
Chad
UNK
Escravos River,
Niger Delta
Ennedi
M. Klemens
1997
AMNH
145361*
Uncatalogued
Crocodylus madagascariensis
TYPE
Short 12S and dloop
sequences only
From 4 specimens utilized in
Schmitz et al. 2003
Djibelor Crocodile Farm wild stock
Specimen utilized in Schmitz
et al. 2003
Not included in phylogenetic
analysis, same as haplotype
found at Site 7
Not included in phylogenetic
analysis, same as haplotype
found at Site 8
Bushmeat sample collected in
Benin City
Short 12S and dloop
sequences only
CRYPTIC AFRICAN CROCODYLUS SPECIES REVEALED 5
27
MADAGASCAR_A_1931
27
MADAGASCAR_B_1931
27
MADAGASCAR_C_1931
Figure 1B
1
MAURITANIA_1
Notes
2011 Blackwell Publishing Ltd
Map
Number
Terminal Label
Country
Locality
Collector
Year
Collected
Museum
Specimen#
Notes
13
CENTRAL AFR REP
Berberati, near Bangui
L. Chirio
1995
MNHN
1997_3171*
MNHN 1997_3171, Short 12S
and dloop sequences only
14
14
14
15
16
REP CONGO_1*
REP CONGO_2*
REP CONGO_3*
REP CONGO_4
DEM REP CONGO
Dougou, Oubangi River
Likouala (Edzala?)
Lukouala, Congo
Likouala aux Herbes
Lac Mai Ndombe
V. de Buffrenil
V. de Buffrenil
V. de Buffrenil
M.J. Eaton
R. Fergusson
1986
1986
1986
2004
2002
MNHN
MNHN
MNHN
FLMNH
N⁄A
1987_1120
1987_1114
1986_1945
Uncatalogued
17
17
18
18
19
19
19
19
19
UGANDA_1
UGANDA_2
GABON_1
GABON_2
EGYPT_1
EGYPT_2
EGYPT_3
EGYPT_4
EGYPT_5
Central African
Republic
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Dem. Republic of
Congo
Uganda
Uganda
Gabon
Gabon
Egypt
Egypt
Egypt
Egypt
Egypt
Kidepo Valley NP
Kidepo Valley NP
Petit Loango, Loango NP
Petit Loango, Loango NP
Lake Nasser, near Aswan
Lake Nasser, near Aswan
Lake Nasser, near Aswan
Lake Nasser, near Aswan
Lake Nasser
M.H. Shirley
M.H. Shirley
M.J. Eaton
M.J. Eaton
M.H. Shirley
M.H. Shirley
M.H. Shirley
M.H. Shirley
UNK
2009
2009
2006
2006
2008
2008
2008
2008
UNK
FLMNH
FLMNH
FLMNH
FLMNH
FLMNH
FLMNH
FLMNH
FLMNH
ZFMK
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
Uncatalogued
20
21
21
22
KENYA_1
KENYA_2
KENYA_3
UGANDA_3
Kenya
Kenya
Kenya
Uganda
R. Fergusson
R. Fergusson
R. Fergusson
M.H. Shirley
2001
2001
2001
2010
N⁄A
N⁄A
N⁄A
FLMNH
Uncatalogued
22
23
UGANDA_4
UGANDA_5
Uganda
Uganda
M.H. Shirley
M.H. Shirley
2010
2010
FLMNH
FLMNH
Uncatalogued
Uncatalogued
24
UGANDA_6
Uganda
M.H. Shirley
2010
FLMNH
Uncatalogued
25
26
27
28
TANZANIA_2
TANZANIA_1
MALAWI
ZIMBABWE_1
Tanzania
Tanzania
Malawi
Zimbabwe
Tana River
Tana River
Tana River
Victoria Nile, Murchison
Falls NP
Semliki River, Semuliki NP
Lake Edward, Queen
Elizabeth NP
Lake Mburo, Ruizi
Drainage, Lake Mburo NP
Lake Rukwa
Rufiji River
Salima Bay
Lake Kariba
R. Fergusson
R. Fergusson
R. Fergusson
UNK
2001
2001
2001
UNK
N⁄A
N⁄A
N⁄A
N⁄A
28
ZIMBABWE_2
Zimbabwe
Lake Kariba
UNK
UNK
N⁄A
29
30
31
31
32
33
34
ZIMBABWE_3
SOUTH AFRICA
MADAGASCAR_1
MADAGASCAR_2
MADAGASCAR_3
MADAGASCAR_4
SUDAN
Zimbabwe
South Africa
Madagascar
Madagascar
Madagascar
Madagascar
Sudan
Lake Kariba
Lake St. Lucia
Ankarana Caves
Ankarana Caves
Betsiboka River
Estuary, Fort Dauphin
Chor Melk en-Nasir
R. Fergusson
A. Leslie
Garcia
Garcia
E. Hekkala
de Huelme
UNK
2002
UNK
2002
2002
2000
2002
UNK
N⁄A
N⁄A
N⁄A
N⁄A
N⁄A
N⁄A
ZFMK
Bushmeat sample collected in
Inongo
12s sequence from Schmitz
et al. 2003 (AY195943)
12s
et
12s
et
50489
sequence from Schmitz
al. 2003 (AY 195954 ⁄ 55)
sequence from Schmitz
al. 2003 (AY 195954 ⁄ 55)
12s sequence from Schmitz
et al. 2003 (AY195953)
6 E. HEKKALA ET AL.
Table 2 (Continued)
CRYPTIC AFRICAN CROCODYLUS SPECIES REVEALED 7
Kluge 1998). Prior to analysis, individual marker datasets
were tested for the maximum likelihood model of evolution with jModelTest 0.1.1 (Posada 2008) and MrModelTest2.3 (Nylander et al. 2004) for a C. niloticus-only
dataset and a dataset including Crocodylus outgroups.
Where the inferred model of evolution was not consistent
between datasets, we chose the model selected for the C.
niloticus-only data. Datasets were tested for congruence
and analyzed in PhyML (Guindon & Gascuel 2003) and
MrBayes (Ronquist & Huelsenbeck 2003) to generate
hypotheses of phylogenetic structure under maximum
likelihood and Bayesian algorithms as follows:
Maximum likelihood. A PhyML search was implemented
on the Montpellier Bioinformatics Platform (http://
www.atgc-montpellier.fr/phyml). The full, concatenated
dataset was analyzed under HKY85+I+G substitution
model as per the recommendation of jModelTest 0.1.1.
Trees were searched from a starting tree created by
BIONJ using the best of the SPR and NNI options with
topologies and branch lengths optimized. Branch support was determined with both the SH-Like and Chi2based options of the Approximate Likelihood Ratio Test
(aLRT) method (Anisimova & Gascuel 2006), as well as
nonparametric bootstrapping over 100 replicates. To test
the hypothesis of C. niloticus monophyly, we compared
the resulting topology to a constrained tree compiled
in MacClade4.01 (Maddison 1997) wherein C. niloticus
represented a monophyletic group. Additional ML
searches were conducted and the likelihood values for
the constrained and unconstrained topologies were
compared using the Shimoduro–Hasegawa option in
PAUP4.0b10 (Swofford 2002). Statistical measures for
rejection of the hypothesis of no difference were set at
95%.
Bayesian inference. The concatenated dataset was partitioned by gene region with the substitution model
implemented for each gene (12s—HKY+I, 16s—GTR+G,
dloop—HKY+I,
ND4—GTR+G,
WANCY—HKY,
rag1—JC, OD—F81, TROP—F81, S6—F81, mtDNA—HKY+I+G, nDNA—HKY+I) where all model parameters
were estimated by MrModelTest2.3 (Nylander et al.
2004). Gaps (indels) were coded as restriction site binary characters. Three simultaneous Markov Chain
Monte Carlo searches were run with five chains for
12 000 000 generations with trees sampled every 500
generations. A 50% majority rule consensus tree was
created after discarding the first 2000 ‘burn-in’ trees.
Trees were rooted by both outgroup and mid-point
rooting methods; both methods produced the same root
point (Hess et al. 2007).
We used BEAST v1.5beta2 (Drummond et al. 2006),
which implements a Bayesian MCMC method and a
2011 Blackwell Publishing Ltd
relaxed molecular clock approach (Drummond 2007),
to estimate divergence times. We assumed a relaxed
lognormal model of lineage variation and a Yule prior
for branching rates. We examined rates using the
combined dataset (nuDNA and mtDNA) partitioned
by gene region, as well as by coding versus non-coding regions. The coding regions were further partitioned according to 1 + 2 and 3 codon positions and
the substitution model, rate heterogeneity and base
frequencies were unlinked across codon positions
[(1 + 2), 3].
For calibration, we used fossil record-based estimates
of the divergence between Alligator and Crocodylus (ca.
79 mya), Crocodylus and Mecistops ⁄ Osteolaemus (at ca.
20–24 mya), as well as the earliest fossil appearances of
C. niloticus in Africa (ca. 3–7 mya) (Brochu 2004c; Brochu personal communication), and Crocodylus in the
Caribbean (conservatively estimated at 4–5 mya; Miller
1980). We used these dates as lognormal distribution
priors for each respective node setting the offset as the
minimum age (A. Drummond personal communication). We placed monophyly constraints on the New
World clade and on eastern C. niloticus, respectively,
thus attaining the same general topology as assessed by
the full phylogenetic analyses. Three replicates were
run for 100 000 000 generations each with tree and
parameter sampling occurring every 1000 generations.
The adequacy of a 10% burn in and convergence of all
parameters were assessed using the software TRACER
v1.4.1 (Rambaut & Drummond 2005). The sampling distributions of the three independent replicates were then
combined using the software LogCombiner v1.5 and the
resulting 360 000 000 samples summarized and visualized using the software Tree Annotator v1.5 and FigTree v1.2 (Rambaut 2006).
Mean intra- and inter-clade distances (i.e. number of
base substitutions per site from averaging over all
sequence pairs within and between groups) were calculated in MEGA4 for both the combined and the
mtDNA only datasets (Tamura et al. 2007). Sequences
for captive individuals were removed from all analyses, and divergence estimates for pairs not including
Alligator were estimated with the preceding datasets
minus Alligator. Analyses were conducted using Maximum Composite Likelihood (Tamura et al. 2004). The
rate variation among sites was modeled with a gamma
distribution (shape parameter = 1). The differences in
the composition bias among sequences were considered in evolutionary comparisons (Tamura & Kumar
2002).
Codon
positions
included
were
1st+2nd+3rd+Noncoding. All ambiguous positions
were removed for each sequence pair. Standard error
estimates were obtained by bootstrapping over 500 replicates.
8 E. HEKKALA ET AL.
Ancient DNA methods
Tissue was harvested from 57 dried or ethanol preserved museum specimens from eight institutions,
including both natural history and anthroplogical collections (Table 2). We sampled Egyptian crocodile
mummies from the Phoebe Hearst Museum (PHM) at
the University of California, Berkley; the University of
Pennsylvania Museum of Anthroplogy (UPenn); the
British Museum (BM); and the Musée National d’Histoire Naturelle (MNHN) (Table S3, Supporting information). During all archival tissue collections, surgical
utensils were sterilized and work areas were wiped
with DNAaway (Molecular Bioproducts) between samples. Specimen surfaces were wiped with 20% Clorox
bleach and air dried prior to sampling.
Mummified crocodile hatchlings from MNHN, PHM
and UPenn were very fragile and handled separately.
Individuals from MNHN were originally collected from
two sealed tombs (Grotte de Samoun and Grotte de
Thebes) in the early 1800s and are estimated to have
been interred between 200 BC and 200 AD (S. Ikram,
Cairo Museum, personal communication.). One hatchling from PHM was from collections noted as ‘predynastic’ Egypt (estimated ‡3100 BC), while one from
Upenn was undated. For each hatchling a cross section
of the tail, including bone and muscle tissue, was sampled, rinsed with 20% Clorox bleach and sterile water
prior to hydration in glycine buffer for 1 week to
3 months with regular fluid changes (Shedlock et al.
1997). Samples from adult mummies and more recent
specimens (nineteenth and twentieth centuries), were
soaked for 36–76 h in PBS with multiple fluid changes.
All museum samples were processed in clean room
facilities, separate from contemporary samples. Processing of each specimen was replicated in at least one
additional institution [either American Museum of Natural History aDNA Laboratory (AMNH), University of
Nevada Reno (UNR), U.S. EPA aDNA Laboratory, Cincinnati, OH (EPA), or Tulane University (TU)]. At each
institution DNA extraction, PCR setup and post-PCR
handling of archival samples took place in physically
separate locations with procedures following precautionary protocols recommended for use with degraded
or ancient DNA (Cooper & Poinar 2000; Paabo et al.
2004; Gilbert et al. 2005; Willerslev & Cooper 2005).
Facilities at AMNH and EPA were equipped with positive air pressure, wall mounted UV lamps, protective
disposable lab attire, and direct shipping of all equipment and reagents, while those at TU and UNR consisted of separate, dedicated lab space.
DNA extraction from archival museum specimens
consisted of a modified Qiagen DNeasy tissue protocol
after extended hydration in either PBS or Glycine buf-
fer. All samples were handled in batches of 6 with the
exception of mummies, which were processed as
batches ‘per institution’ of 4–8 samples. Negative controls were included throughout the process for each
batch of samples. During tissue digestion, 5 lL of 1 M
dithiothreitol (DTT) was added along with proteinase K
to enhance protein digestion. Care was taken to mix
reagents by hand at each step rather than risk shearing
the DNA by vortexing. Samples were eluted in two separate volumes of 75 lL with elution buffer warmed to
56 C after resting in the column for 15 min.
All pre- and post PCR handling was physically separated, and involved use of both positive and negative
controls. Positive PCR controls were added after archival tubes were sealed and placed on the thermocycler.
Primers were designed from modern crocodile
sequences to amplify ±187–200 bp each of mitochondrial 12s rRNA and d-loop gene regions covering previously identified hypervariable sites (12s183 5¢TTGCCCT
AAGCAGCCTGTAT3¢, 12s375 5¢CCGTCTTTGACAGTC
CTGGT3¢; and ncdlpFs 5¢GCCGACATTCTTATTAAACTAC3¢, ncdlpRs 5¢GCAGATAAATGAATGCCTTAT3¢,
Table S1). In addition, we attempted to amplify a
600 bp gene region using crocodile specific 12s primers
to confirm that no contemporary DNA was present in
aDNA extracts (Paabo et al. 2004).
Template DNA was amplified using GE Illustra puretaq PCR beads in 25 lL volumes and amplification
products were visualized on a 1% agarose gel with EtBr
staining. Successfully amplified PCR products were
cleaned using ExoSAP-IT (Affymetrix). Sanger
sequencing reactions were carried out using BigDye
v3.1 sequencing kits in 6–8 lL volumes. Gene regions
were sequenced in both directions on either an ABI
3100, 3700 or 3730XL automated capillary sequencer.
Base calling was performed with Sequencher v4.1
(Genecodes Corp.). In case of sequence ambiguity,
archival tissue samples were re-extracted, amplified and
sequenced up to three times for verification (Paabo
et al. 2004).
Historical specimen sequence analyses
Both 12s and d-loop sequences from archival specimens
were individually aligned with sequences from contemporary specimens. Assignment of each archival specimen to an evolutionary lineage was based on diagnostic
characters found in sequences from contemporary specimens. Nucleotide sites were considered diagnostic if
they were variable with fixed base differences between
clades. We utilized a PAA (Davis & Nixon 1992)
approach to assign historical specimens to clades with
the program CAOS (Character Attribute Organization
System; Sarkar et al. 2009).
2011 Blackwell Publishing Ltd
CRYPTIC AFRICAN CROCODYLUS SPECIES REVEALED 9
As an exploratory measure, we performed a phylogenetic analysis of the aligned short fragment sequence
data using a maximum likelihood approach as implemented in PhyML with the substitution model implemented HKY+I, as previously estimated by jModelTest
0.1.1 (Posada 2008).
Karyotyping
Samples for karyotype analysis were collected from
Nile crocodiles at the St. Augustine Alligator Farm Zoological Park and had the following accession numbers:
SAAF_1—93220, SAAF_edpool—A01026, and SAAF_
2—93044. Karyotyping was conducted on four cell lines.
Skin biopsies were taken from the toe webbing of captive individuals and primary fibroblast cell lines were
established and preserved in the San Diego Zoo’s Frozen Zoo cell repository. Harvests and chromosome
banding followed Kumamoto et al. (1996) with the
exception of a 33 C cell culture incubation temperature.
We also obtained DNA sequence data from these indi-
viduals, following the protocols for contemporary specimens presented above, for comparison to natural
populations and to address concerns about potential
hybridization in captivity.
Results
All phylogenetic methods used to examine our combined mtDNA and nDNA sequence dataset recovered a
paraphyletic C. niloticus, with a predominantly western
African clade sister to a monophyletic clade comprised
of a predominantly eastern African C. niloticus plus the
four New World Crocodylus species (Fig. 2). Tree topologies with significantly weaker support values were
recovered when C. niloticus monophyly was imposed.
Mean, corrected sequence divergence estimates showed
little intraclade divergence (<0.3%) for both the total,
concatenated dataset and the mtDNA dataset in both
C. niloticus clades (Table S2, Supporting information).
Mean intraclade divergence estimates between the
eastern and western clades did not overlap with mean
REP CONGO_4
UGANDA_1
GAMBIA_2
GAMBIA_3
SENEGAL
GAMBIA_1
MAURITANIA_2
BURKINA FASO
IVORY COAST_2
GHANA_1
DEM REP CONGO
SAAF_1
UGANDA_2
NIGERIA
C. rhombifer
C. moreletii
C. acutus
C. intermedius
EGYPT_1
EGYPT_2
EGYPT_3
EGYPT_4
SAAF_2
GABON_1
UGANDA_6
UGANDA_5
UGANDA_3
KENYA_1
KENYA_2
KENYA_3
SOUTH AFRICA
MALAWI
ZIMBABWE_3
TANZANIA_1
TANZANIA_2
MADAGASCAR_1
MADAGASCAR_2
MADAGASCAR_3
MADAGASCAR_4
C. siamensis
C. porosus
O.tetraspis
M. cataphractus
A. mississippiensis
Fig. 2 Phylogenetic tree illustrating results of the Bayesian analysis of the full dataset, with karyotype insets. As illustrated, both the
phylogenetic and karyotype analyses support a paraphyletic C. niloticus with the predominantly western clade (light grey) as sister
to a monophyletic New World and Eastern C. niloticus clade. Posterior Probabilities (PP) are indicated above branches. Significant
support is indicated by PP > 0.90. Individuals SAAF_1, SAAF_P (western) and SAAF_2 (eastern) exhibit the karyotypes displayed in
the insets. Both BY and ML analyses resulted in similar tree topologies.
2011 Blackwell Publishing Ltd
10 E . H E K K A L A E T A L .
interclade divergence values, which were more than
an order of magnitude higher (>4%), for both the
total concatenated dataset and the mtDNA dataset
(Table S2).
Karyotyping of representative captive individuals
from each clade affirmed sequence-based evidence of
evolutionary divergence between the two C. niloticus
lineages (Fig. 2, inset). Consistent with prior findings,
the derived eastern C. niloticus clade exhibits 32 chromosomes, comprised of 26 metacentric-submetacentric
and six acrocentric elements. The ancestral western
C. niloticus clade exhibits 34 chromosomes consisting
of 24 metacentric–submetacentric and 10 acrocentric
elements.
Divergence time estimates from the BEAST analyses
of the full dataset partitioned by gene and partitioned
by coding region and codon position were similar (i.e.
the mean estimated dates from one analysis fell within
the 95% confidence intervals of the other analysis),
though mean ages were generally older and the confidence intervals were larger when the data were partitioned by gene region. Hence, we report only the
outcome of the analysis based on coding region and
codon position. Divergence time estimates suggest that
the western C. niloticus lineage last shared a common
ancestor with the New World-Eastern C. niloticus clade
approximately 8.13 mya (5.24–12.64 mya, 95% CI tmrca)
(Fig. S1, Supporting information). The western clade
was estimated to have arisen ca. 2.455 mya (0.903–
4.722 mya, 95% CI tmrca) (Fig. S1). The eastern C. niloticus lineage was estimated to have last shared a common ancestor with the New World clade approximately
5.7 mya (3.69–8.44 mya, 95% CI tmrca) (Fig. S1).
We sequenced up to 197 bp of the 12s rRNA and up
to 219 bp of the dloop from mtDNA regions for 40 of
57 museum specimens (Table 2). We were able to
obtain sequence data for 8 of 22 crocodile mummies.
Only the mummified hatchlings from MNHN yielded
DNA (Table S3). Our attempts to amplify the larger 12s
fragment in the mummy and other museum specimens
failed, indicating that there was no contamination with
contemporary crocodile DNA. An alignment of the
short 12s and d-loop sequences from contemporary
specimens found 11 and 14 diagnostic sites, respectively, for the two C. niloticus clades (Table 3). Comparison of sequences obtained from the historical
specimens to these diagnostic sites enabled us to assign
24 individuals, including all 8 mummy sequences, to
the western clade and 16 individuals to the eastern
clade (Fig. 1, Table 3). Phylogenetic analysis of the
short aDNA dataset recovered a western clade including all mummies and placement of all other museum
specimens consistent with the haplotype based clade
assignment (Fig. S2, Supporting information).
Haplotype assignments of mummy specimens and
well documented collections from the Sudanese Nile
valley indicate that the two lineages of C. niloticus have
had overlapping distributions in the Nile drainage for
nearly two millennia (Fig. 1b, Table 3). In addition,
derived eastern haplotypes were recovered from two
historical specimens from coastal Senegal. Contemporary distributions suggest that little geographical
overlap now occurs (Fig. 1a). For example, all contemporary Egyptian specimens possess derived haplotypes,
whereas no derived eastern haplotypes have been
found in contemporary populations thus far sampled in
West Africa.
Discussion
Our total evidence based phylogenetic analysis revealed
a cryptic evolutionary lineage within the Nile crocodile.
This finding not only clarifies recent and historic disputes regarding both C. niloticus’ taxonomy and the biogeographic history of the genus, but also stands to
improve conservation and management of crocodilian
diversity across Africa and elsewhere.
Crocodylus diversity and taxonomy
Extant crocodiles are often portrayed as ‘living fossils,’
reflecting perceptions of morphological homogeneity
and evolutionary stasis, but evidence of greater crocodilian diversity and evolutionary dynamism is beginning to emerge. Eaton et al. (2009), for example, has
found cryptic diversity within the African dwarf crocodiles of the genus Osteolaemus. Our results also indicate
that greater diversity occurs within the crown genus
Crocodylus than is currently recognized.
Recognition of subspecies (e.g. Fuchs et al. 1974) does
not adequately reflect the degree or nature of divergence between the two recovered C. niloticus clades.
Our findings show that the two C. niloticus lineages are
distant relatives, and their paraphyletic relationship relative to New World congeners indicates that the two C.
niloticus clades are not sister taxa. Additionally, fixed
differences across sequence-based marker sets and chromosomes, as well as interclade distances, offer a basis
for diagnosing the two C. niloticus lineages as distinct
species (Moritz 1994; Goldstein & DeSalle 2000).
Although molecular divergence estimates between
members of the genus Crocodylus vary by clade and
marker, recognized Crocodylus species generally exhibit
<1% intraspecies divergence and 2.5–7.5% interspecies
divergence (White & Densmore 2001; McAliley et al.
2006). Similarly, newly diagnosed species within the
genus Osteolaemus exhibit within-clade divergence of
<0.4% and between-clade divergences of 4–16%,
2011 Blackwell Publishing Ltd
C R Y P T I C A F R I C A N C R O C O D Y L U S S P E C I E S R E V E A L E D 11
Table 3 Population Aggregation Analysis (PAA) Assigning Archival Specimens to Western or Eastern Clade. Diagnostic nucleotide
positions within the short 12s (11 sites) and d-loop (14 sites) sequences. Specimens in bold represent archival material. Eight mummy
specimens are highlighted in grey, all correspond to the western lineage. Sequences with question marks across one marker represent
failed amplification success for that specimen. D-loop site 206 is an indel event in the eastern clade. The miscoding error observed at
d-loop site 226 due to DNA degradation
Gene region
position
12s
dloop
187 193 204 206 209 221 225 229 258 274 303 121 122 128 147 156 201 203 206 209 223 226 227 234 240
Western Consensus
A
G
A
C
C
A
C
A
T
C
G
A
T
T
C
A
T
A
A
A
T
C
T
C
T
SAAFedpool
BURKINAFAS
DRCONGO
GHANA
GAMBIA
GAMBIAA
GAMBIAB
IVORYCOAST
MAURITANIA
NIGERIA
SENEGAL
RCONGO
KARAMOJAA
KARAMOJAB
MummyHaute
MummySamA
MummySamB
MummySamC
MummySamD
MummyThebA
mummyThebB
mummyThebC
Benin1990
SanghaCAR
Chad1993
DRCEdz1986
DRCLukuelu
DRCKas1924
CIAssi1885
RCNgou1886
Matmat1993
DRCNE1911
Oubang1986
Senega1824
Senega1825
SudMel1922
SudWNA1922
SudWNB1922
Senaga1934
Eastern Consensus
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SAAF2
GABONa
NASSERA
NASSERB
NASSERC
NASSERD
MADAGASCNW
MADAGASCSE
2011 Blackwell Publishing Ltd
12 E . H E K K A L A E T A L .
Table 3 (Continued)
Gene region
position
Eastern MADAGASCAA
MADAGASCAB
SAFRICA
KENYAA
KENYAB
KENYAC
QUEENNP02
MURCHISON2
LAKEMBURO2
ZIMBABWE
TANZANIAA
TANZANIAB
MALAWI
Sudan
Nasser
Kariba1
Kariba2
DRCNE1912
Botswa1967
SWCam1966
KenGar1960
KenNai1919
MadAmA1931
MadAmB1931
MadAMC1931
madTYP1885
vulTYP1822
VerTYP1768
Senega1803
SudWNC1922
SudWND1922
SunWNE1922
SudUN1922
Tanz1972
12s
dloop
187 193 204 206 209 221 225 229 258 274 303 121 122 128 147 156 201 203 206 209 223 226 227 234 240
T
T
T
T
T
T
?
?
?
T
T
T
T
T
T
T
T
T
?
T
T
?
T
T
?
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
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?
?
A
A
A
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A
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A
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A
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A
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?
A
A
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A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
?
?
?
G
G
G
G
G
G
G
G
G
?
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
?
?
?
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
T
T
T
T
T
T
?
?
?
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
?
?
?
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
?
?
?
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
?
?
?
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
?
?
?
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
depending on the marker (Eaton et al. 2009). In comparison, the two C. niloticus clades exhibited 0.3% withinclade and 4% between-clade divergence across 5 kbp
(Table S2). Preliminary morphometrics of C. niloticus
from museum collections representing sites from Kenya
and the Congo showing fixed, discrete and non-overlapping continuous character variation (R. Sadlier,
unpublished data) also support this conclusion.
That all mummy crocodiles from Thebes and Samoun
exhibit the western haplotype suggests both lineages
historically occurred in the lower Nile River (Fig. 1).
These findings are consistent with early arguments of
two Crocodylus species in Egypt, including historical
accounts that ancient Egyptian priests were cognizant
of two forms and selectively used the smaller, more
tractable form in temples and ceremonies (Herodotus in
Geoffroy Saint-Hilaire 1807). Analysis of museum speci-
T
T
T
T
T
T
?
?
?
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
?
?
?
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
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G
G
G
G
?
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G
?
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G
G
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C
C
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C
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C
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C
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C
C
C
C
C
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C
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C
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C
C
C
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G
G
G
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G
G
G
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?
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G
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A
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A
A
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A
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G
G
G
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G
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G
G
G
G
G
G
G
?
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G
?
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G
G
?
G
?
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C
C
C
C
C
C
C
C
C
C
C
C
C
?
?
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C
?
?
?
?
?
?
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C
C
?
C
?
?
?
?
?
.
.
.
G
.
G
G
G
G
.
.
.
.
?
?
?
?
G
?
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?
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?
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?
.
G
?
G
?
?
?
?
?
G
A
A
G
G
G
G
G
G
A
A
A
G
?
?
?
?
G
?
?
?
?
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G
G
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C
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C
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C
C
C
C
C
?
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C
?
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C
C
?
C
?
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T
T
T
T
T
T
T
T
T
T
T
T
T
?
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T
?
?
?
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T
T
?
T
?
?
?
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C
C
C
C
C
C
C
C
C
C
C
C
C
?
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C
?
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C
C
?
C
?
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?
mens from more recent collections (Fig. 1b, Table 2)
provides additional evidence that both lineages were
present in the upper Nile in Sudan until as recently as
the 1920s.
Molecular assignment of the eight crocodile mummies
to the western C. niloticus clade and Geoffroy Saint-Hilaire’s (1807) description of a mummified crocodile skull
from the same cache as a separate species, C. suchus, provides support for ascribing the western C. niloticus lineage to this taxon. The description of C. suchus included
the argument, disputed by Cuvier at the time (Cüvier
1807), that both C. niloticus and C. suchus were present in
the Nile and that the range of C. suchus likely extended
into the western Sahara (Geoffroy Saint-Hilaire 1807).
Geoffroy Saint-Hilaire (1807) went so far as to argue that
the distribution of both species likely overlapped in areas
of ancient Lake Chad during geologic times.
2011 Blackwell Publishing Ltd
C R Y P T I C A F R I C A N C R O C O D Y L U S S P E C I E S R E V E A L E D 13
Crocodylus biogeography and conservation
Evidence for cryptic diversity within C. niloticus provides key information on the evolution and distribution
of the genus Crocodylus. Fossils of Crocodylus checchiai in
Libya (ca. 5–6 mya) (Brochu 2001, 2003) and the Gargano Crocodylus sp. (ca. 5–6 mya) of southeastern Italy
(Delfino et al. 2007) provide evidence of dispersal and
diversification within the genus in north Africa and the
Mediterranean after the Miocene-Pliocene transition. In
light of the fossil record (e.g. Brochu 2003) and estimated divergence dates based on our molecular data,
our well-supported phylogenetic hypothesis of a paraphyletic C. niloticus bracketing New World congeners
provides further support for the hypothesis that the global distribution of Crocodylus reflects geologically recent
marine and transoceanic dispersal events (Brochu et al.
2007; Willis 2009; Meredith et al. 2011; Oaks 2011).
These findings are consistent with hypothesized transoceanic marine dispersal in other taxa including geckos
and parrots (e.g. de Queiroz 2005).
While our divergence estimates are preliminary and
partially based on uncertainties in the fossil record for
C. niloticus in Africa (C. Brochu personal communication), the pattern of divergence we recovered is consistent with many well recognized aspects of African
biogeography. The position of Congo Basin samples as
basal within the western lineage, and preliminary divergence estimates dating to 8.13 mya for the most recent
common ancestor of the western and eastern (including
New World species) clades, suggest that the newly identified African Crocodylus lineage evolved in the interior
of Central Africa during the late Miocene when the closing of the Tethys Sea brought about the climatic trend of
increasing aridity we see on the continent today (Axelrod & Raven 1978; Coetzee 1993; Plana 2004). Increasing
aridity resulted in the recession of forested areas and the
advancement of savannah and woodlands with associated sandy shores necessary for nesting. The contemporary and historical presence of the western lineage at the
northeastern margin of the Congo Basin, the Kidepo Valley in northeastern Uganda, and the Sangha River drainage in Central African Republic indicate that dispersal
may have begun in a northerly direction and then along
an east-west axis facilitated by drainage evolution (Goudie 2005; Drake et al. 2011). Further divergence in the
western clade occurred throughout the mid to late Pleistocene (0.035–1.43 mya) likely owing to the gradual drying of the ‘green Sahara’ and subsequent population
isolation (Drake et al. 2011). During this period, a series
of alluvial fans and paleolakes effectively connected the
Niger Delta (including the Senegal River) to the Nile
basin largely through what was Mega Lake Chad and
what is now the Sudd wetland in southern Sudan
2011 Blackwell Publishing Ltd
(Drake et al. 2011). Relict populations and rock paintings
indicate that crocodile populations were more abundant
across northern Africa during wetter climatic periods
(de Smet 1999; Shine et al. 2001; Drake et al. 2011).
Within-lineage genetic structure provides more
detailed understanding of connectivity across western
Africa. One of the two clades recovered within the western lineage consists largely of Sahelian localities structured by the drying of paleodrainages towards the end
of the Pleistocene (Drake et al. 2011) (Fig. S1). The
other clade is composed of localities in the Upper Guinea Forest Basin countries (e.g. Nigeria, Ghana, Cote
d’Ivoire), as well as coastal localities in Senegal and
Gambia (Fig. S1). River drainages in this region run
north to south draining into the Gulf of Guinea or
Atlantic Ocean, and therefore have had little connection
with paleodrainages of the Sahara. The observed phylogenetic structure also likely reflects drainage isolation
with infrequent marine dispersal, a pattern seen in
some coastal fishes (e.g. Falk et al. 2003; Agnese et al.
2006). Nile crocodiles are abundant in coastal lagoons
in this region and are regularly observed in marine
environments (Shirley et al. 2009; Fergusson 2010).
Similarly, the eastern clade of C. niloticus is broken
into two sister groups dating to around 3.274 mya with
likely origins in the Nile valley. Prior analyses of eastern populations based on nuclear markers revealed substantial sub-structuring corresponding to major barriers
to dispersal such as the Mozambique Channel (East
Africa and Madagascar), and to river drainages in
Kenya, Tanzania, Zimbabwe and South Africa (the Turkana, Ruaha, Zambezi and Limpopo river basins,
respectively) (Hekkala et al. 2009). It is possible that the
geographic structure exhibited by eastern C. niloticus
may be related to patterns of natal philopatry-associated
breeding and nesting behaviors (Hekkala et al. 2009).
Similar patterns of sub-structuring by drainage basin
have been observed in faunal assemblages found in
East African forest remnants (Azeria et al. 2007).
Our recovery of the eastern haplotype in two samples
from western Central Africa (i.e. the Ogooué
Basin—Gabon and Cameroon) likely reflects northward
dispersal from coastal Angola and the Kunene River.
The Cameroon Volcanic Line is a major biogeographic
feature separating this region from coastal West Africa
(Cantagrel et al. 1978; Lee et al. 1994; Meyers et al.
1998), and a similar pattern occurs in the Osteolaemus
dwarf crocodiles (Eaton et al. 2009).
On a continental scale, the cryptic east ⁄ west split
found in our study of African Crocodylus parallels patterns of differentiation observed between sister taxa in
several African faunal assemblages following the formation of the Rift Valley (de Menocal 2004; Moodley &
Bruford 2007). However, the geographic distributions of
14 E . H E K K A L A E T A L .
the ancestral and derived lineages (Fig. 1a, b) belie a
history of greater sympatry in Africa. The occurrence of
the derived lineage in historical specimens from Senegal
suggests the possibility of either greater sympatry in
western Africa in the past or a pattern of coastal dispersal by the Eastern lineage, though no contemporary
specimens from West Africa to date, coastal or otherwise, support either argument (Fig. 1). Individuals from
historical collections from the Sudanese Nile valley
(1822–1922) and northeastern DRC (1911–1912) also possess both lineages. While further sampling in Sudan
and NE DRC is needed to determine the extent of
sympatry today, the presence of the western clade in
the Kidepo Valley (Uganda) and anecdotal evidence of
similar crocodile populations in Ethiopia suggests that
the western clade is still distributed in this region
though it may be restricted to marginal habitats.
Previously, researchers using molecular data from
paleontological collections have shown evidence that
genetic diversity in wide ranging species has been lost
over historical and paleoecological time periods (Ramakrishnan & Hadley 2009 and references therein). This
growing field has been termed ‘phylochronology’ due
to the emphasis on reconstructing patterns of genetic
variation over time. Much of this work has focused on
Holocene patterns of faunal turnover and range contractions in northern latitudes (Ramakrishnan & Hadley
2009; examples therein, e.g. Shapiro et al. 2004; Hofreiter et al. 2004). While these studies are invaluable in
advancing understanding of the genetic consequences
of environmental change, our study reveals a much
more recent pattern of local extirpation with potentially
global consequences for loss of crocodilian biodiversity.
authenticating ancient DNA. The mummified crocodile
hatchings, with the exception of the ‘pre-dynastic’
hatchling from PHM, proved to be an exceptional
source for ancient DNA. The specimens came from
dry, sealed, relatively cool burial chambers and are
young (only 1 800–2 200 years old) in comparison to
source materials used in many other ancient DNA
studies (e.g. Hofreiter et al. 2004; Shapiro et al. 2004).
Importantly, our samples have two additional,
uniquely crocodilian advantages over samples comprised of mammalian bone and mummy tissue: nucleated red blood cells and a thick keratinized skin layer.
Both of these attributes likely serve as sources and
protective repositories for mtDNA.
Our combined analyses of museum and contemporary specimens indicate that, as formulated, major
national and international conservation agreements
intended to promote sustainable harvest of Nile crocodiles may instead accelerate extirpation because quotas
and translocation policies are based on erroneous taxonomy and assumptions of genetic homogeneity. This is
particularly relevant in countries that harbour populations of both lineages and have long running harvest
programs (e.g. Uganda) or are looking to initiate new
harvest programs (e.g. Ethiopia and Sudan). The newly
discovered evolutionary lineage of African Crocodylus is
particularly vulnerable to extinction because of its relative rarity and restricted occurrence in countries where
illegal harvest of skins, the bushmeat trade, and damage to wetlands are largely unregulated (Shirley et al.
2009). Taking precautionary measures, such as recognizing the ancestral lineage as C. suchus on the IUCN Red
List and reviewing its status, could reduce further loss
of at-risk populations.
Conclusion
This study emphasizes once again the utility of nontraditional archival specimens in contributing to our
understanding of evolutionary relationships and biogeographic history (Leonard 2008). As techniques for
accessing nucleic acids from archival materials become
more readily and reliably available, materials found in
ever more diverse repositories stand to provide greater
insight into changes over time related to natural and
anthropogenic processes. Our success in accessing
DNA from archival materials adds to the growing
body of work demonstrating the role of museum collections as banks of ‘ancient’ DNA that can be used to
establish baseline genetic profiles against which change
can be measured (Leonard 2008; Ramakrishnan &
Hadley 2009 and references therein). However, use of
archival materials is not without risk (Cooper & Poinar
2000). Many researchers examining genetic characteristics of paleomaterial have difficulty retrieving and
Acknowledgements
We thank the wildlife and CITES management authorities of
Ghana, Cote d’Ivoire, Senegal, Gambia, Nigeria, Gabon,
Republic of Congo, Egypt, Uganda, Kenya, Tanzania, Zimbabwe and Madagascar for permission to collect and export
samples. Funding was provided by the University of Florida,
Wildlife Conservation Society, The Sackler Institute for Conservation Genetics, Columbia University, Conservation, Food,
and Health Foundation, Columbus Zoo, Idea Wild, Conservation Leadership Programme, St. Augustine Alligator Farm
Zoological Park, Disney Wildlife Conservation Grant, US EPA
Star Fellowship, and the Zoological Society of San Diego. We
thank M.J. Eaton, R. Fergusson, T. Shine, W. Boehme, M.P.O.
Dore, M. Klemens, A. Leslie, G. Garcia and the St. Augustine
Alligator Farm Zoological Park for providing contemporary
samples, and the California Academy of Sciences (J. Vindum),
American Museum of Natural History, Field Museum, Royal
Museum for Central Africa, Musée National du Histoire
Naturelle (R. Bour), and Yale Peabody Museum (J. Gauthier)
for permission to collect tissue from museum specimens.
2011 Blackwell Publishing Ltd
C R Y P T I C A F R I C A N C R O C O D Y L U S S P E C I E S R E V E A L E D 15
Christopher Raxworthy and Salima Ikram provided insight
into historical biogeography and animal mummies, respectively. We thank E. Derryberry for her assistance with BEAST
analyses and three anonymous reviewers for suggested
improvements to the work.
Author contributions
EH and MHS, who contributed equally to this work and are
considered co-primary authors, designed the study, and conducted all lab work and phylogenetic analyses. EH collected
samples from Madagascar and conducted all museum sampling and aDNA work. MHS collected all samples from Ghana,
Cote-d’Ivoire, Senegal, Gambia, Uganda and Egypt. GA and
RD, and JDA are the dissertation supervisors of EH and MHS,
respectively, and contributed to the development of methods
and provided funding support. JT was an avid conservationist
and the MSc advisor for MHS. He contributed significantly to
our understanding of the taxonomic history of Nile crocodiles,
sampling strategy, design of fieldwork, and funding support.
SC and MH conducted all karyotype analyses. KV contributed
to the karyotype analysis of captive animals. MB contributed
analytical expertise and lab support. Sampling protocols were
reviewed by the University of Florida IACUC (#E-423).
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M.H.S.’s research utilizes multiple inferential tools to elucidate
population-level processes over different temporal and spatial
scales to facilitate the conservation of wildlife in Africa and
elsewhere. He is particularly interested in the interaction
between historic, landscape features and contemporary human
pressures in structuring wildlife populations.
Data accessibility
DNA sequences: DRYAD entry (datadryad.org; doi:10.5061/
dryad.s1m9h)
Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Gene regions and protocols used for amplification of
mtDNA and nuclear introns for Crocodylus niloticus and affiliated specimens used in the study
Table S2 Estimated Molecular Divergence. Mean distance estimates with S.E. for the full, concatenated dataset (below diagonal) and mtDNA-only dataset (above diagonal). Values in the
diagonal are intragroup mean distance estimates with S.E. for
the full, concatenated dataset (left) and mtDNA-only dataset
(right)
Table S3 All mummy specimens examined for this study.
Locality and date information is from museum accession notes
unless otherwise noted
Fig. S1 Estimated divergence dates for the two Crocodylus niloticus clades under a relaxed clock model as implemented in
BEAST v.4.3. The displayed estimates for mean divergence
date and 95% confidence intervals are based on the full dataset
partitioned by coding region with subsequent codon position
partitioning.
Fig. S2 Phylogenetic tree resulting from maximum likelihood
analysis of concatenated 12s and d-loop short fragments for
contemporary and archival specimens. Mummy specimens
have blue terminal labels.
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
REP CONGO_4
SAAF_1
DEM REP CONGO
2.45
UGANDA_1
GAMBIA_1
1.43
BURKINA FASO
MAURITANIA_2
NIGERIA
1.09
IVORY COAST_2
GHANA_1
0.78
8.13
UGANDA_2
GAMBIA_2
GAMBIA_3
SENEGAL
4.10
1.67
C. moreletii
C. rhombifer
C. acutus
2.99
C. intermedius
EGYPT_1
EGYPT_2
EGYPT_3
5.72
EGYPT_4
UGANDA_5
UGANDA_6
UGANDA_3
3.27
SAAF_2
GABON_1
11.50
SOUTH AFRICA
2.07
KENYA_1
KENYA_3
KENYA_2
TANZANIA_1
1.24
TANZANIA_2
ZIMBABWE_3
MALAWI
29.16
MADAGASCAR_1
MADAGASCAR_2
MADAGASCAR_3
MADAGASCAR_4
C. porosus
8.04
18.00
C. siamensis
O.tetraspis
M. cataphractus
A. mississippiensis
O.tetraspis
M. cataphractus
C. siamensis
C. porosus
SENEGAL_1934
SUDAN_MELUT_1922
SUDAN_WNA_1922
SUDAN_WNB_1922
MUMMY_HAUTE
MUMMY_THEBES_B
IVORY COAST_1885
DEM REP CONGO_1924
SENEGAL_1825
CHAD
GAMBIA_3
SENEGAL
GAMBIA_2
UGANDA_1
REP CONGO_3
MAURITANIA_1
REP CONGO_1
SENEGAL_1824
BENIN
NIGERIA
IVORY COAST
SAAF_1
GHANA_1
REP CONGO_4
UGANDA_2
DEM REP CONGO
MAURITANIA_2
BURKINA FASO
GAMBIA_1
MUMMY_THEBES_C
REP CONGO_2
ZIMBABWE_1911
REP CONGO_1886
CENTRAL AFR REP
MUMMY_SAMOUN_A
MUMMY_SAMOUN_B
MUMMY_SAMOUN_C
MUMMY_THEBES_A
C. intermedius
C. acutus
C. moreletii
C. rhombifer
EGYPT_1
EGYPT_2
EGYPT_3
EGYPT_4
SUDAN_WNC_1922
UGANDA_5
ZIMBABWE_2
ZIMBABWE_1
EGYPT_5
MADAGASCAR_A_1931
SUDAN
MADAGASCAR_B_1931
SENEGAL_1768
EGYPT_1822
KENYA_1960
SENEGAL_1803
SAAF_1
GABON_1
ZIMBABWE_1912
UGANDA_6
UGANDA_3
KENYA_1919
KENYA_1
KENYA_3
MADAGASCAR_C_1931
KENYA_2
BOTSWANA_1967
MALAWI
ZIMBABWE_3
SOUTH AFRICA
TANZANIA_1
TANZANIA_2
MADAGASCAR_2
SUDAN_WNE_1922
SUDAN_WND_1922
SUDAN_UN_1922
CAMEROON_1966
TANZANIA_1972
MADAGASCAR_1
MADAGASCAR_4
MADAGASCAR_1885
MADAGASCAR_3
Table S1
PCR Reaction Cocktail
Gene
12S
12s (short)
16S
Control Region/dloop
d-dloop short
ND4
Wancy
Rag-1
Trop
OD
S6
Vol.
Reaction
Buffer
µL
15
25
25
15
25
25
15
15
20
20
15
5X
mM
1
1.5
Illustra puretaq beads
Illustra puretaq beads
1
1.5
Illustra puretaq beads
Illustra puretaq beads
1
1.75
1
1.5
0.85
1.5
0.9
1.5
0.9
1.5
MgCl2
PCR Cycle Conditions
dNTP's Primer
Taq
mM
0.2
µM
0.5
U/µM
0.03
0.2
0.5
1
0.03
0.2
0.2
0.2
0.2
0.2
0.5
0.5
0.5
0.5
0.5
0.03
0.03
0.03
0.03
0.03
Extended
Denature
Minutes
4:00
5:00
5:00
4:00
4:00
5:00
4:00
4:00
4:00
4:00
4:00
Denature
Anneal
Extension
Extended
Extension
#
ͦ
C
Minutes ͦC Minutes ͦC Minutes ͦC Minutes ͦC #
94
1:00
94
1:00
52
1:30
74
4:00
72 35
94
1:00
94
1:00
52
1:30
72
4:00
72 35
94
1:00
94
1:00
52
1:30
72
4:00
72 33
94
1:00
94
1:00
54
1:30
72
4:00
72 35
94
1:00
94
1:00
54
1:30
72
4:00
72 35
94
1:00
94
1:00
52
1:30
72
4:00
72 33
94
1:00
94
1:00
55
1:30
72
4:00
72 35
94
1:00
94
1:00
56
1:30
72
4:00
72 35
94
1:00
94
1:15
56
1:30
76
4:00
74 35
94
1:00
94
1:00
54
1:30
74
4:00
72 35
94
1:00
94
1:00
60
1:30
74
4:00
72 35
Table S2
Eastern C. niloticus
Western C. niloticus
New World
Asia
Mecistops cataphractus
Osteolaemus tetraspis
Alligator mississippiensis
Eastern C. niloticus
Western C.
niloticus
New World
Asia
M. cataphractus
O. tetraspis
A. mississippiensis
0.003
0.007
0.045
0.032
0.066
0.125
0.166
0.484
±0.0008
±0.00169
±0.00921
±0.00647
±0.01247
±0.02228
±0.03081
±0.14319
0.044
0.004
0.007
0.057
0.065
0.123
0.174
0.506
±0.01136
±0.00094
0.002
±0.01158
±0.01239
±0.02240
±0.03332
±0.16007
0.071
0.135
0.178
0.514
±0.01329
±0.02426
±0.03282
±15967
0.138
0.188
0.492
±0.02463
±0.03480
±0.15171
0.155
0.513
±0.02876
±0.15830
0.039
0.056
±0.00892
±0.01325
±0.00463 ±0.00577
0.075
0.067
0.076
±0.01670
±0.01603
±0.01680
±0.0149 ±0.01374
0.144
0.144
0.140
0.153
±0.03427
±0.03492
±0.03296
±0.03589
0.175
0.162
0.158
0.197
0.159
±0.04625
0.042
±0.04152
±0.05273
±0.03966
0.293
0.332
0.332
0.396
0.338
0.316
0.086
±0.11862
±0.10341
±0.14893
±0.10481
±0.09330
0.023
0.031
0.076
0.077
N/A
N/A
0.471
±0.13390
N/A
Museum
MNHN
MNHN
MNHN
MNHN
MNHN
MNHN
MNHN
Specimen Number
1986_1475
1986_1478
1986_1480
1986_1471
1986_1473
1986_1479
1886_445
PHM
PHM
PHM
BM
BM
BM
BM
BM
BM
Upenn
Upenn
Upenn
Upenn
Upenn
620101
55121
514
35734
35726
35747
35751
6837
6847
E521
2965563
E2832
L12112
L12113
Terminal Label
MUMMY_SAMOUN_A
MUMMY_SAMOUN_B
MUMMY_SAMOUN_C
MUMMY_THEBES_A
MUMMY_THEBES_B
MUMMY_THEBES_C
MUMMY_HAUTE
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Specimen Name
mummySamA
mummySamB
mummySamC
mummyThebA
mummyThebB
mummyThebC
MummyHaute
PHM620101
PHM55121
PHM514
BM35734
BM35726
BM35747
BM35751
BM6837
BM6847
UpennE521
Upenn2965563
UpennE2832
UpennL12112
UpennL12113
Site Number
8
8
8
7
7
7
7
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
MNHN= Musee National d'Histoire Naturelle, PHM=Phoebe Heart Museum UCBerkeley, BM=British Museum, Upenn=Penn Museum
* estimated dates as per S. Ikram Cairo Museum
Locality
Mummy - Grottes de Samoun
Mummy - Grottes de Samoun
Mummy - Grottes de Samoun
Mummy - Grottes de Thebes
Mummy - Grottes de Thebes
Mummy - Grottes de Thebes
Mummy, Haute Egypt
Country
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Collector
Gervais
Gervais
Pariset
Cailloud - collected 1820s
Cailloud - collected 1820s
Cailloud - collected 1820s
V. Schoelcher
Mummy unknown
Mummy unknown
Mummy unknown
Mummy Manfalut
Mummy unknown
Mummy Manfalut
Mummy Manfalut
Mummy Manfalut
Mummy Manfalut
Mummy unknown
Mummy-Dindereh
Mummy-Tel El Yehudiyeh
Mummy-Maabdah (Samoun)
Mummy-Thebes?
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Unknown
Unknown
Unknown
E.J. Andrews
Unknown
Unknown
Unknown
E.J. Andrews
E.J. Andrews
Unknown
Cox Expedition 1918
Flinders Petrie
Unknown
G.R. Glidden 1848
Date Collected
200BC-200AD*
200BC-200AD*
200BC-200AD*
200BC-200AD*
200BC-200AD*
200BC-200AD*
200BC-200AD*
Haplotype
W
W
W
W
W
W
W
pre-dynastic
pre-dynastic
pre-dynastic
Roman
pre-dynastic
pre-dynastic
pre-dynastic
Roman
Roman
ND
ND
ND
Late period
Late period
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND