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Crocodilian phylogeny inferred from twelve mitochondrial protein-coding genes, with new complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae

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We report complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae, whose gene orders match those of other crocodilians. Phylogenetic analyses based on the sequences of 12 mitochondrial protein-coding genes support monophyly of two crocodilian taxonomic families, Alligatoridae (genera Alligator, Caiman, and Paleosuchus) and Crocodylidae (genera Crocodylus, Gavialis, Mecistops, Osteolaemus, and Tomistoma). Our results are consistent with monophyly of all crocodilian genera. Within Alligatoridae, genus Alligator is the sister taxon of a clade comprising Caiman and Paleosuchus. Within Crocodylidae, the basal phylogenetic split separates a clade comprising Gavialis and Tomistoma from a clade comprising Crocodylus, Mecistops, and Osteolaemus. Mecistops and Osteolaemus form the sister taxon to Crocodylus. Within Crocodylus, we sampled five Indopacific species, whose phylogenetic ordering is ((C. mindorensis, C. novaeguineae), (C. porosus, (C. siamensis, C. palustris))). The African species C. niloticus and New World species C. acutus form the sister taxon to the Indopacific species, although our sampling lacks three other New World species and an Australian species of Crocodylus.
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Crocodilian phylogeny inferred from twelve mitochondrial protein-coding genes,
with new complete mitochondrial genomic sequences for Crocodylus acutus and
Crocodylus novaeguineae
Zhang Man, Wang Yishu, Yan Peng, Wu Xiaobing
The College of Life Sciences, Anhui Normal University, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Wuhu 241000, China
article info
Article history:
Received 14 September 2010
Revised 29 March 2011
Accepted 29 March 2011
Available online 2 April 2011
Keywords:
Crocodylus acutus
Crocodylus novaeguineae
Mitochondrial genomes
Phylogeny
abstract
We report complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae,
whose gene orders match those of other crocodilians. Phylogenetic analyses based on the sequences of 12
mitochondrial protein-coding genes support monophyly of two crocodilian taxonomic families, Alligato-
ridae (genera Alligator,Caiman, and Paleosuchus) and Crocodylidae (genera Crocodylus,Gavialis,Mecistops,
Osteolaemus, and Tomistoma). Our results are consistent with monophyly of all crocodilian genera. Within
Alligatoridae, genus Alligator is the sister taxon of a clade comprising Caiman and Paleosuchus. Within
Crocodylidae, the basal phylogenetic split separates a clade comprising Gavialis and Tomistoma from a
clade comprising Crocodylus, Mecistops, and Osteolaemus.Mecistops and Osteolaemus form the sister taxon
to Crocodylus. Within Crocodylus, we sampled five Indopacific species, whose phylogenetic ordering is ((C.
mindorensis,C. novaeguineae), (C. porosus,(C. siamensis,C. palustris))). The African species C. niloticus and
New World species C. acutus form the sister taxon to the Indopacific species, although our sampling lacks
three other New World species and an Australian species of Crocodylus.
Crown Copyright Ó2011 Published by Elsevier Inc. All rights reserved.
1. Introduction
The American crocodile (Crocodylus acutus) is a crocodylian spe-
cies found primarily in Central America. It grows faster than the
American alligators and is much more tolerant of salt water. The
New Guinea crocodile (Crocodylus novaeguineae) is a small species
of crocodile found on the island of New Guinea.
Numerous molecular datasets have been compiled to examine
intergeneric phylogenetic relationships among crocodylians
(Brochu and Densmore, 2001; Gatesy and Amato, 1992; Gatesy
et al., 1993, 2003, 2004; Harshman et al., 2003; McAliley et al.,
2006; Ray and Densmore, 2002; White and Densmore, 2001; Willis
et al., 2007). The main controversies focus on the taxonomic status
of the African slender-snouted crocodile (Mecistops cataphractus),
the dwarf crocodile (Osteolaemus tetraspis), the false gharial
(Tomistoma schlegelii) and Gavialis gangeticus. The three primary
families commonly recognized within the Crocodylia were the Alli-
gatoridae (Alligator,Caiman,Melanosuchus and Paleosuchus), the
Crocodylidae (Crocodylus,Osteolaemus and Tomistoma) and the
Gavialidae (including only one species, G. gangeticus). Morphologi-
cal comparisons of extant and fossil samples consistently placed T.
schlegelii within the Crocodylidae (Brochu, 1997; Norell, 1989;
Vélez-Juarbe et al., 2007) and many previous studies placed G.
gangeticus in a separate family, Gavialidae (Brochu, 1997; Dessauer
and Densmore, 1983; Gatesy et al., 2004). However, every molecu-
lar analysis revealed the sister-group relationships between G.
gangeticus and T. schlegelii (Densmore, 1983; Densmore and Owen,
1989; Densmore and White, 1991; Feng et al., 2010; Gatesy and
Amato, 1992, 2008; Gatesy et al., 2003; Hass et al., 1992; Harsh-
man et al., 2003; Janke et al., 2005; Ji et al., 2006; Li et al., 2007;
Meganathan et al., 2010; Roos et al., 2007; White and Densmore,
2001; Willis et al., 2007). The African slender-snouted crocodile
was alternatively placed in Crocodylus (Norell, 1989) or isolated
as a new genus, Mecistops (Feng et al., 2010; Gatesy and Amato,
2008; Li et al., 2007; McAliley et al., 2006; Willis et al., 2007; Willis,
2009). Mecistops was alternatively grouped with Crocodylus
(Brochu, 2000; Densmore and White, 1991; Li et al., 2007) or with
the African dwarf crocodile (O. tetraspis)(Feng et al., 2010; Gatesy
et al., 2003; White and Densmore, 2001; Willis, 2009).
Phylogenetic relationships among the species of traditional
Crocodylus have received less attention, and many species lack
complete mitochondrial genomic sequence data. Molecular phylo-
genetic studies within Crocodylus often reveal contrasting relation-
ships (Feng et al., 2010; Gatesy and Amato, 2008; Ji et al., 2006; Li
et al., 2007; McAliley et al., 2006; Meganathan et al., 2010; Roos
et al., 2007; Willis, 2009). Here, we sequenced the complete mito-
chondrial genomes (mtDNA) of C. acutus and C. novaeguineae to
evaluate the phylogenetic relationships in Crocodylus. Our
1055-7903/$ - see front matter Crown Copyright Ó2011 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2011.03.029
Corresponding author. Fax: +86 553 3836873.
E-mail address: wuxb@mail.ahnu.edu.cn (W. Xiaobing).
Molecular Phylogenetics and Evolution 60 (2011) 62–67
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
phylogenetic analyses use 12 mitochondrial protein-coding genes
for five Indopacific species (C. mindorensis,C. novaeguineae,C. poro-
sus,C. siamensis and C. palustris), one New World species (C. acutus)
and C. niloticus. We focus our discussion of Crocodylus relationships
on the Indopacific group (Brochu, 2000; Meganathan et al., 2010).
2. Materials and methods
2.1. Samples and DNA extraction
The blood samples of C. acutus and C. novaeguineae were ac-
quired from the specimen storeroom of the laboratory in Anhui
Normal University (presented by Dr. George Amato from AMNH),
and were stored at 80 °C. The mtDNA was extracted following
the procedure described by Arnason et al. (1991). Extracted DNA
was diluted 20 times with doubly distilled water and stored at
20 °C.
2.2. PCR amplification and sequence assembling
The mtDNA sequences of Crocodylus niloticus,Crocodylus poro-
sus, Crocodylus siamensis and M. cataphractus (Genbank accession
NO. DQ273697, DQ273698, EF581859, EF551000, respectively)
were aligned using Clustal X 1.8 (Thompson et al., 1997) and con-
served primers were then designed using Oligo 6.0 (Rychlik and
Rychlik, 2000). Each pair of primers generated a product that over-
laps more than 100 bp.
PCR was performed in a 30
l
l volume containing 10mmpl/L
Tris–HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl
2
, 0.25 mmol/
L of each dNTP, 10
l
mol/L of each primer, 1 U Taq polymerase,
and 25 ng DNA templates, with the following conditions: an initial
denaturation step of 95 °C (5 min) followed by 30 cycles of 95 °C
(30 s), 52–60 °C (30 s), and 72 °C (1 min) followed by 72 °C for
10 min. Annealing temperature was changed from 52 to 60 °Cto
improve the quality of PCR products. The PCR products were sepa-
rated by electrophoresis in 1.5% agarose gels, then were puried by
AxyPrep™ PCR Cleanup Kit and AxyPrep™ DNA Gel Extraction kit
(AXYGEN Biotechnology (Hangzhou) Limited, Hangzhou, China)
and then sequenced with ABI 3730 (Shanghai Sangon Biotechnol-
ogy Co., Ltd., Shanghai, China). The whole mitochondrial genome
was read at least two times. Sequences were assembled using
the programs seqman (DNASTAR 2001), BioEdit (http://
www.mbio.ncsu.edu/BioEdit/Bioedit.html) and chromas 2.22
(http://www.technelysium.com.au/chromas.html), then checked
manually. The complete mtDNA sequences of C. acutus and C.
novaeguineae were obtained. The resulting information has been
deposited in GenBank with the Accession Nos. HM636894–
HM636896.
2.3. Sequence analysis
Compared with the corresponding complete mtDNA sequence
of C. niloticus, the thirteen protein-coding genes were identified
using DNAStar (Version 5.01) and SEQUIN (Version 9.5). All of
the transfer RNA (tRNA) genes were identified using software tRNA
Scan-SE 1.21 (http://lowelab.ucsc.edu/tRNA scan-SE) except for the
tRNA
Ser(AGC)
, and their cloverleaf secondary structure and antico-
don sequences were identified using RNA structure 4.2. After
checking the position of the tRNA
Ser(AGC)
and two rRNA genes
against other crocodilians mtDNA sequences, positions of the
remaining genes were revised. The number of variable sites of
the nucleotide sequences was calculated by Mega v.3.1 (Kumar
et al., 2001).
2.4. Data analysis and phylogenetic reconstruction
Besides the genomes of C. acutus and C. novaeguineae, we ob-
tained all other samples used in the phylogenetic analyses based
on the 12 H-strand encoded protein-coding gene sequences as fol-
lows: Struthio camelus (Y12025), Gallus gallus (X52392), Caiman
crocodilus (AJ404872), Alligator mississippiensis (Y13113), Alligator
sinensis (AF511507), Paleosuchus trigonatus (AM493869), Paleosu-
chus palpebrosus (AM493870), C. siamensis (EF581859), C. porosus
(DQ273698), C. niloticus (DQ273697), O. tetraspis (EF551001), M.
cataphractus (EF551000), G. gangeticus (AJ810454), T. schlegelii
(NC_011074), Crocodylus palustris (GU144286)and Crocodylus
mindorensis (GU144287). The NAD6 gene was excluded from the
analyses, because it was encoded on the L strand and had the
strand-specific base composition bias of mtDNAs differing signifi-
cantly from that of the 12 H-strand encoded genes, which
influenced replacement patterns at the amino acid sequence level
(Asakawa et al., 1991; Dong and Kumazawa, 2005; Roos et al.,
2007). Because many molecular data have identified birds as the
closest living relatives of crocodilians (Feng et al., 2010; Gatesy
et al., 2003; Iwabe et al., 2005; Janke and Arnason, 1997; Ji et al.,
2006; Kumar and Hedges, 1998; Roos et al., 2007), we selected
two birds as outgroups here.
Sequences were aligned using Clustal X (1.8) as implemented in
Mega v.3.1, (Kumar et al., 2001; Thompson et al., 1997) and only
including the first and second codon positions. Two different phy-
logenetic trees (ML, BI) were reconstructed using PAUP v. 4.0b10
(Swofford, 2002) and MrBayes v.3.04b (Huelsenbeck and Ronquist,
2001). In maximum-likelihood analyses, Modeltest v.3.7 (Posada
and Crandall, 1998) determined the most appropriate evolutionary
model to be GTR + I+G(I= 0.3086, Alpha = 0.8788) with base fre-
quencies estimated by PAUP (A= 0.3444, C= 0.3601, G= 0.0872,
T= 0.2082). Heuristic searches were made with 100 replicates of
random- addition sequences and TBR branch swapping. The confi-
dence value was evaluated by bootstrap analysis with 100 repli-
cates. The Shimodaira–Hasegawa (SH) test (Shimodaira and
Hasegawa, 1999) was used for comparison of alternative trees rel-
ative to the best ML tree.
With the model previously specified, Bayesian inference (BI) of
the phylogeny was performed using MrBayes v.3.04b (Huelsenbeck
and Ronquist, 2001). One cold and three heated chains were run for
1000,000 generations, with trees sampled every 100 generations.
Stationarity of the Markov chain was determined as the point
when sampled log-likelihood values plotted against generation
time reached a stable value (split frequencies = 0.000000) (Feng
et al., 2010; Willis, 2009); generations sampled before the chains
reached stationarity (2500) were discarded as burn-in. At least
two independent runs were performed for each dataset in the
Bayesian analysis. The resulting trees were used to generate a
majority consensus tree with posterior probability values.
3. Results
3.1. Genomes and gene arrangement
The length of the complete mtDNA nucleotide sequences of C.
acutus and C. novaeguineae are 16,883 and 16,894 bp, respectively.
As expected, the gene arrangements of the molecule conform to
those of other crocodilians, differing from the typical vertebrate
gene arrangement (Feng et al., 2010; Ji et al., 2006; Li et al.,
2007; Zhong et al., 2005; Zhang et al., 2010). Both of them contain
13 protein-coding genes, two ribosomal RNAs (12S rRNA and 16S
rRNA), 22 transfer RNAs and a putative control region. Most of
these genes are coded on the H-strand with the exception of one
protein-coding gene (NAD6) and some tRNA genes (tRNA
Gln
,
Z. Man et al. / Molecular Phylogenetics and Evolution 60 (2011) 62–67 63
tRNA
Ala
, tRNA
Asn
, tRNA
Cys
, tRNA
Tyr
, tRNA
Ser (UCN)
, tRNA
Glu
and tRNA-
Pro
). The rearrangement altered the positons of tRNA
Phe
and
tRNA
Ser (AGY)
. Location of tRNA
Phe
between tRNA
Pro
and the D-loop
produces a TPF cluster (tRNA
Thr
–tRNA
Pro
–tRNA
Phe
); location of
tRNA
Ser(AGY)
between NAD4 and tRNA
His
produces a SHL
(tRNA
Ser(AGY)
–tRNA
His
–tRNA
Leu
) cluster (Fig. 1). These two rear-
rangements are common in all crocodilians whose complete mtD-
NAs have been published so far (Feng et al., 2010; Janke and
Arnason, 1997; Janke et al., 2001, 2005; Ji et al., 2006; Li et al.,
2007; Roos et al., 2007; Wu et al., 2003; Zhang et al., 2010).
Fig. 1 shows the high conservative structure of crocodilians’
mtDNA.
For C. acutus and C. novaeguineae, the relative order of nucleo-
tide composition is A>C>T>G, conforming to other crocodilians.
In C. acutus, the overall base compositions is A: 32.37%, C: 27.91%,
T: 25.11%, G: 14.61%, and the base composition of C. novaeguineae
is A: 32.23%, C: 28.53%, T: 24.48%, G: 14.76%. The gene length and
start/stop codons for protein-coding genes of each genome are
shown in Table 1. As in other crocodilian mtDNAs (Feng et al.,
2010; Li et al., 2007; Wu et al., 2003), there are overlapping and
noncoding bases between some genes and also some of them are
long. Specifically, in C. acutus and C. novaeguineae, the ATP8 and
ATP6 genes all overlap by 22 bp and the NAD5 and NAD6 all over-
lap by 50 bp. Also, like other crocodilians, the noncoding region
(47 bp in C. acutus and 48 bp in C. novaeguineae) between Cyt b
and tRNA
Thr
is the longest.
3.2. Protein-coding genes
The total length of 13 protein-coding genes is 11,471 bp in C.
acutus and 11,468 bp in C. novaeguineae, which form 67.94% and
67.88% of the whole mt genome respectively. In both crocodiles,
the longest protein-coding gene is NAD5, and the shortest is
ATP8. In C. acutus, ATG is the start codon in 8 of the 13 protein-cod-
ing genes, but NAD1, NAD3, NAD5 start with ATA, while COI,
NAD4L begin with the nonstandard start codons GTG, ACC, respec-
tively. In C. novaeguineae, there are nine protein-coding genes that
start with ATG, and ATA is the start codon only in the NAD3 and
NAD5 genes, while COI and NAD4L begin with the nonstandard
start codons GTG and ACC respectively. Stop codons in C. acutus
and C. novaeguineae are the same. Standard termination codon
TAA occurs in most genes; however, AGG terminates the NAD1
and NAD6 genes, and TAG terminates the NAD3 gene. Incomplete
termination codon T occurs in the COIII and Cyt bgenes. These
incomplete termination codons are common in metazoan mtDNAs
and can be converted into complete ones (TAA) by polyadenylation
after transcription (Boore, 2001).
3.3. Ribosomal and transfer RNA genes
Like other crocodilians, in C. acutus and C. novaeguineae mt gen-
omes, 12S rRNA (984 bp and 983 bp, respectively) and 16S rRNA
(1593 bp and 1595 bp, respectively) genes are located between
the D-loop and tRNA
Leu(UUR)
and spaced by tRNA
Val
. As in some
other vertebrates, all tRNA genes can be folded into a canonical clo-
verleaf secondary structure with the exception of tRNA
Ser(AGY)
,
which loses the ‘‘DHU’’ arm (Anderson et al., 1981; Frazer-Abel
and Hagerman, 1999; Zhang et al., 2003). These 22 tRNAs range
in size from 66 to 75 nucleotides.
3.4. Noncoding regions
In C. acutus and C. novaeguineae, the control region is located be-
tween the tRNA
Phe
and 12S rRNA, with a total length of 1155 and
1183 bp respectively. Ray and Densmore (2002) analyzed the gen-
eral structure and the conserved sequences of the mitochondrial
control region in crocodilians. Consistent with other crocodilians,
three domains (Domain|, Domain||, Domain|||) also form the con-
trol region of these two crocodiles. Both of them contain a long
poly-A region and tandemly repeated sequences immediately fol-
lowed the poly-A tracts. In C. acutus, there is a 51 bp region 5
0
-TAG-
GCTAAAATAGGAAAAATTTTTTTAAAAAAAATTAAAAATTTATTAACC-
3
0
, repeated three times. In C. novaeguineae, the region 5
0
-TAGGC-
CAAAATAGGAAAAAATTTTAAAAAATTTTAAAAAAATTTTAAACAAAT-
TATTAACC-3
0
(61 bp) is repeated four times.
In vertebrates, the origin of L strand replication (O
L
)(Yoshinori
and Mutsumi, 1999) usually occurs in a cluster of tRNA
Trp
tRNA
Ala
–tRNA
Asn
–tRNA
Cys
–tRNA
Tyr
region (WANCY), but it is com-
mon to find that the O
L
is not obvious in all crocodilians. Also in C.
acutus and C. novaeguineae, the O
L
region between tRNA
Asn
and
tRNA
Cys
, which typically has the potential to fold into a stable
stem-loop secondary structure, all have only 6 bp (AATCTT).
Fig. 1. Complete mitochondrial genome organization of crocodiles. The tRNAs are
identified by the single-letter amino acid code.
Table 1
Main structural feature of mt genomes of Crocodylus acutus and Crocodylus
novaeguineae.
Feature Crocodylus acutus Crocodylus novaeguineae
Total length 16,883 16,894
Control region 1155 1183
12S rRNA 984 983
16S rRNA 1593 1595
NAD1 963 (ATA/AGG) 963 (ATG/AGG)
NAD2 1056 (ATG/TAA) 1056 (ATG/TAA)
COI 1557 (GTG/TAA) 1557 (GTG/TAA)
COII 684 (ATG/TAA) 684 (ATG/TAA)
ATP8 162 (ATG/TAA) 162 (ATG/TAA)
ATP6 696 (ATG/TAA) 696 (ATG/TAA)
COIII 784 (ATG/T) 784 (ATG/T)
NAD3 348 (ATA/TAG) 348 (ATA/TAG)
NAD4L 294 (ACC/TAA) 294 (ACC/TAA)
NAD4 1374 (ATG/TAA) 1374 (ATG/TAA)
NAD5 1860 (ATA/TAA) 1860 (ATA/TAA)
NAD6 (L) 528 (ATG/AGG) 525 (ATG/AGG)
Cyt b1165 (ATG/T) 1165 (ATG/T)
Length is expressed as bp, and start/stop codons showed within parentheses. ‘‘(L)’’
denotes gene encoded on the L strand.
64 Z. Man et al. / Molecular Phylogenetics and Evolution 60 (2011) 62–67
3.5. Phylogenetic analysis
The 12 protein-coding gene sequences of 16 crocodilians were
used to reconstruct the ML and BI trees for inspecting the phylog-
eny in crocodilians, especially within the genus Crocodylus. Both
ML and BI analyses recovered the same tree topologies, so here
we show only the BI tree (Fig. 2). From the tree, we can see notably
that the basal split is between Alligatoridae and a branch compris-
ing traditional Crocodylidae and G. gangeticus/T. schlegelii, consis-
tent with previous molecular analyses (Aggarwal et al., 1994;
Densmore, 1983; Densmore and White, 1991; Feng et al., 2010;
Gatesy et al., 1993; Gatesy and Amato, 2008; Harshman et al.,
2003; Janke et al., 2005; Ji et al., 2006; Li et al., 2007; Roos et al.,
2007; White and Densmore, 2001; Willis, 2009). We thus support
recognition of only two Crocodylia families: Alligatoridae and
Crocodylidae.
In Alligatoridae, Paleosuchus has a closer relation with Caiman
than with Alligator, and many morphological and molecular data
have supported this conclusion (Brochu, 2003; Feng et al., 2010;
Gatesy et al., 2003; Gatesy and Amato, 2008; Harshman et al.,
2003). Regarding some controversial phylogenetic issues, G. gan-
geticus groups with T. schlegelii, and M. cataphractus is most closely
related to O. tetraspis rather than to Crocodylus. Within the genus
Crocodylus, we support the two separate New World and Indopacif-
ic groups (Brochu, 2000; Meganathan et al., 2010). C. acutus groups
with C. niloticus, and together they form the sister taxon to other
true crocodiles. In the Indopacific assemblage, C. novaeguineae is
closest to C. mindorensis, and C. siamensis is closer to C. palustris
than to C. porosus.
4. Discussion
4.1. Gene features
The organization of the genomes is consistent with other
crocodylian mt genomes published previously (Fig. 1). In contrast
to the traditional vertebrate mitochondrial genomes, such
gene features as, TPF (tRNA
Thr
–tRNA
Pro
–tRNA
Phe
) and SHL (tRNA-
Ser(AGY)
–tRNA
His
–tRNA
Leu
) clusters, base composition, tRNA
structures, tandemly repeated sequences and the unconspicuous
O
L
are all common in crocodilian mt genomes. The same gene order
and gene rearrangement illustrate again that the mtDNA of crocod-
ilians is extremely conservative. There are differences in start/stop
codons: the start codon of NAD1 (ATG) in C. novaeguineae differs
from those of some other true crocodiles, such as C. porosus (Li
et al., 2007), C. niloticus (Ji et al., 2006), C. palustris (Feng et al.,
2010), C. mindorensis (Feng et al., 2010) and C. acutus (this study),
in which the start codons of NAD1 are all ATA. As to NAD4L, occur-
rence of multiple nonstandard codons (ACC, AGC, ACT, ACA, ATC) is
probably a result of its high evolutionary rate (Li et al., 2007).
4.2. Phylogenetic analysis
4.2.1. Intergeneric crocodilian relationships
The conflict on the phylogenetic position of G. gangeticus and T.
schlegelii is longstanding, and most surveys have focused on it. Tra-
ditional morphological evidence placed G. gangeticus in Gavialidae,
which was the sister taxon of all other living crocodilians (Brochu,
1997; Norell, 1989; Tarsitano et al., 1989). When many new phylo-
genetic data emerged, such as karyotype, biochemistry, immunol-
ogy, molecular biology etc., many authors grouped G. gangeticus
with T. schlegelii (Densmore, 1983; Densmore and Owen, 1989;
Densmore and White, 1991; Feng et al., 2010; Gatesy et al.,
2003; Gatesy and Amato, 2008; Hass et al., 1992; Harshman
et al., 2003; Janke et al., 2005; Ji et al., 2006; Li et al., 2007; Roos
et al., 2007; White and Densmore, 2001; Willis et al., 2007). How-
ever, their joint placement within crocodilian phylogeny remained
controversial. Some authors claimed that G. gangeticus and T. schle-
gelii formed the sister group to the traditional Crocodylidae and
then recognized within Crocodylia only two families, Alligatoridae
and Crocodylidae, with the sister taxa Gavialis and Tomistoma in-
cluded in the Crocodylidae (Feng et al., 2010; Janke et al., 2005;
Ji et al., 2006; Li et al., 2007; Roos et al., 2007). Alternatively, the
sister taxa Gavialis/Tomistoma were combined as Gavialidae
(Gatesy et al., 2003; Gatesy and Amato, 2008; Willis et al., 2007).
From our analysis, we support the former. We include G. gangeticus
in Crocodylidae and place Gavialis/Tomistoma as the sister group of
the traditional Crocodylidae. Thus, the analyses support the recog-
nition of only two Crocodylia families: Alligatoridae and
Crocodylidae.
Although M. cataphractus is traditionally placed in Crocodylus,
recent molecular datasets suggest a close affinity between M.
cataphractus and O. tetraspis (Feng et al., 2010; Gatesy et al.,
2003; Gatesy and Amato, 2008; White and Densmore, 2001; Willis,
2009) and meanwhile, some other authors’ work places M. cataph-
ractus as the sister taxon to all other members of Crocodylus (Bro-
chu, 2000; Densmore and White, 1991; Li et al., 2007). McAliley
et al. (2006) supported the hypothesis that M. cataphractus was
not a member of Crocodylus or Osteolaemus and resurrected it an
historic genus, Mecistops; later investigations based on many vari-
ous molecular data also supported this hypogehsis (Feng et al.,
2010; Gatesy and Amato, 2008; Li et al., 2007; Willis et al., 2007;
Fig. 2. Consensus Bayesian tree reconstructed using 12 H-strand mitochondrial protein-coding genes and based on the evolutionary model of GTR + I+G. Numbers represent
BI (out of round brackets) and ML (in round brackets) posterior probabilities values, respectively.
Z. Man et al. / Molecular Phylogenetics and Evolution 60 (2011) 62–67 65
Willis, 2009). Our results also sustain this hypothesis in both ML
and BI trees, and there is strong support (100% and 100%, respec-
tively) for the sister-group relationship of M. cataphractus and O.
tetraspis. In Crocodylidae, the intergeneric relationships can be
concluded as ((Crocodylus,(Osteolaemus,Mecistops)), (Gavialis,
Tomistoma)). The remaining intergeneric relationships in Croco-
dylidae recovered by our new data and analyses match those of
other recent molecular research (Feng et al., 2010; Gatesy et al.,
2003; Gatesy and Amato, 2008; Ji et al., 2006; Willis, 2009).
4.2.2. Intrageneric phylogenetic relationships within Crocodylus
There are 11 species in traditional Crocodylus (we support M.
cataphractus isolated from Crocodylus in this study), but only three
complete mitochondrial genomes (C. siamensis (EF581859), C. poro-
sus (DQ273698), C. niloticus (DQ273697)) available from GenBank
prior to this paper. Owing to lack of sufficient data, some authors
included only two or three species of Crocodylus in phylogenetic
analyses, focusing on the intergeneric relationships (Gatesy et al.,
2003; Janke et al., 2005; Roos et al., 2007). We found that using dif-
ferent data often generated different intrageneric relationships
(Brochu, 2000; Feng et al., 2010; Gatesy and Amato, 2008; Ji
et al., 2006; Li et al., 2007; McAliley et al., 2006; Meganathan
et al., 2010; Willis, 2009). Phylogenetic relationships within Croco-
dylus are far less stable than the intergeneric relationships.
Meganathan et al. (2010) first emphasized phylogenetic rela-
tionships among the members of Crocodylus. Here, we add the
complete mitochondrial genomes of C. palustris and C. mindorensis
(Feng et al., 2010) and C. acutus and C. novaeguineae generated in
this study to reconstruct the intrageneric phylogeny of Crocodylus
(Fig. 2). Our results support monophyly of Crocodylus and of the
Indopacific group (Brochu, 2000), focusing our discussion on rela-
tionships among the Indopacific species.
In our analysis, C. novaeguineae and C. mindorensis form a clade,
as do C. siamensis and C. palustris. The sister-group relationship be-
tween C. novaeguineae and C. mindorensis had been supported by
some molecular and morphological data (Gatesy and Amato,
2008; Poe, 1996) but we report considerably higher bootstrap val-
ues (100% in ML and 100% in BI analyses) than do earlier studies.
Although morphological data placed C. palustris as the sister taxon
to all other Indopacific crocodiles (Brochu, 2000; Sadleir and
Makovicky, 2008), many molecular data grouped the three Old
World true crocodiles (C. palustris,C. porosus, and C. siamensis) to-
gether (Feng et al., 2010; Gatesy and Amato, 2008; Li et al., 2007;
Willis, 2009), and we got the same result. However, the interspe-
cies relations among the three crocodiles have some differences.
Our grouping of C. palustris and C. siamensis as a clade matches
the outcome of Li et al. (2007) but contradicts some previous pre-
dictions. For instance, Gatesy and Amato (2008) and Willis (2009)
grouped C. palustris and C. porosus as a clade, whereas Feng et al.
(2010) and Meganathan et al. (2010) gave strong support to a clade
comprising C. porosus and C. siamensis.
Our phylogenetic results within Crocodylus can be concluded as
((C. acutus,C. niloticus), ((C. mindorensis,C. novaeguineae), (C. poro-
sus,(C. siamensis,C. palustris)))). We need more mitochondrial gen-
omes and other molecular datasets of the true crocodiles to
examine the phylogenetic placement of the remaining New World
and Australian species within the genus Crocodylus.
Acknowledgments
We greatly appreciate the crocodile samples collected by Dr.
George Amato from American Museum of Natural History. This
work was supported by the National Natural Science Found of
China (No. 30470244), Specialized Research Fund for the Doctoral
Program of Higher Education (20070370002), the special Fund
for Excellent Creative Research Team of Animal Biology in Anhui
Normal University, and Provincial Key Laboratory of Biotic Envi-
ronment and Ecological Safety in Anhui.
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... A multiple alignment was performed through a Clustal W implemented in MEGA X using the C. acutus sequence in GenBank as reference (GQ144571, (Eaton et al., 2010)). To evaluate the relationship between the sequences in this study with the ones previously reported from different species, seven GenBank sequences were used (KF273840, KF273836, KF273834, KF273841, KF273838 (Bloor et al., 2015)), belonging to C. acutus from Colombia; HM636894, (Man et al., 2011), belonging to C. acutus of unreported origin; HQ585889 (Meganathan et al., 2011), belonging to C. moreletti from Belize. ...
... In order to evaluate the relationship between the sequences in this study with the four New World Crocodylus species previously reported, nine GenBank sequences were used, where six belonged to C. acutus (KF273834, KF273838, KF273836, KF273840, KF273841 (Bloor et al., 2015); HM636894 (Man et al., 2011), one to C. moreletii (HQ585889; (Meganathan et al., 2011)), one to C. intermedius (JF502242, (Meredith et al., 2011)) and one to C. rhombifer (JF502247.1, (Meredith et al., 2011)); in order to obtaining an adequate tree topology, we include C. niloticus (JF502246, (Meredith et al., 2011)), C. porosus (DQ273698, (Li et al., 2007)) and C. johnsoni (HM488008, (Meganathan et al., 2011)) as external groups, considering the genus Crocodylus as a monophyletic group (Brochu, 2003). ...
... The CaI haplotype in this COX1 region, is equal to the colombian haplotype KF273840 and to GQ144571 and, according to Eaton et al. (2010), tissue from an organism collected in Oaxaca, Mexico (possibly in the Chacahua Lagoon) was used. This haplotype occurs on the coasts of the Pacific and in the Mexican Caribbean; another Colombian haplotype (KF273841, Cac08) also appears in the Mexican clade; in addition, the haplotypes Cac01, Cac3 and Cac05 that correspond to the sequences KF273834, KF273836, KF273838 reported for Colombia by Bloor et al. (2015) form another branch in the clade of C. acutus, as well as the sequence HM636894 reported by Man et al. (2011), which does not indicate the locality of sampling, form another branch in the clade of C. acutus and it is very likely that the crocodile that was sampled comes from Colombia. This supports what was mentioned by Bloor et al. (2015), who consider the possibility of two lineages: The North American haplogroup, where CaI is located and the Central American haplotypes (C. ...
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Introduction: There is low evidence of genetic diversity and hybridization processes within Crocodylus acutus and C. moreletii populations. Objetive: To evaluate genetic diversity and some phylogenetic relationships in wild and captive populations of C. acutus and C. moreletii using the Barcode of Life Data System (COX1, cytochrome C oxidase subunit 1 gene). Methods: 28 individuals phenotypically like C. acutus located in the state of Guerrero, Oaxaca and Quintana Roo were sampled, as well as animals belonging to C. moreletii located in the states of Tabasco, Campeche, and Quintana Roo. 641 base pairs of nucleotide sequence from COX1 were used to obtain the haplotype and nucleotide diversity per population, and a phylogenetic and network analysis was performed. Results: Evidence of hybridization was found by observing C. moreletti haplotypes in animals phenotypically determined as C. acutus, as well as C. acutus haplotypes in animals classified as C. moreletti. Low haplotypic diversity was observed for C. acutus (0.455 ± 0.123) and for C. moreletii (0.505 ± 0.158). A phylogenetic tree was obtained in which the sequences of C. acutus and C. moreletii were grouped into two well-defined clades. Organisms identified phenotypically as C. acutus but with C. moreletii genes were separated into a different clade within the clade of C. moreletii. Conclusions: There are reproductive individuals with haplotypes different from those of the species. This study provides a small but significant advance in the genetic knowledge of both crocodile species and the use of mitochondrial markers, which in this case, the COX1 gene allowed the detection of hybrid organisms in wild and captive populations. Conservation efforts for both species of crocodiles should prevent the crossing of both threatened species and should require the genetic identification of pure populations, to design effective conservation strategies considering the possibility of natural hybridization in areas of sympatry.
... Similarly, there has been a synchronous burst of molecular studies of Crocodylia, with phylogenetic analyses of several mitochondrial and nuclear genes (e.g. Harshman et al., 2003;Janke et al., 2005;McAliley et al., 2006;Ji et al., 2006;Roos, Aggarwal & Janke, 2007;Willis et al., 2007;Gatesy & Amato, 2008;Meganathan et al., 2010;Yan et al., 2010;Man et al., 2011;Meredith et al., 2011;Oaks, 2011;Bittencourt et al., 2019;Milián-García et al., 2020;Hekkala et al., 2021;Pan et al., 2021), as well as a whole genome analysis (Green et al., 2014). However, despite these developments, the Gavialis-Tomistoma morphology versus molecular dichotomy remains largely unresolved. ...
... The molecular topology has withstood numerous independent analyses, including the use of multiple gene loci in mitochondrial and nuclear DNA (e.g. Harshman et al., 2003;Man et al., 2011;Oaks, 2011). By contrast, although morphological character datasets have been augmented with new characters, morphological characters are typically reused in subsequent iterations of a dataset, meaning that there is little real independence between analyses. ...
... Brochu, 2000Brochu, , 2007aBrochu et al., 2010;Poe, 1997;McAliley et al., 2006;Li et al., 2007) or Osteolaemus (e.g. Gatesy et al., 2003;Schmitz et al., 2003;Willis, 2009;Oaks, 2011;Man et al., 2011;Lee & Yates, 2018;Pan et al., 2021;Hekkala et al., 2021); and (5) the species interrelationships of the crown genus Crocodylus, as well as the resulting biogeographic implications (e.g. Meganathan et al., 2010;Meredith et al., 2011;Oaks, 2011;Nicolaï & Matzke, 2019;Delfino et al., 2020Delfino et al., , 2021. ...
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The capter presents a population study of Morelet's crocodile (Crocodylus moreletii) in Sian Ka'an Biosphere Reserve, Mexico. The results allow to modeling different harvesting scenarios of a wild population. The model shows that ranching can be a virtuous strategy that helps sustain and recover recover populations, as long as it includes the reintroduction to wildlife of a portion of the extracted individuals. If ranching also involves local communities, it can incentivize them to protect crocodile habitat inside and outside protected areas.
... En los últimos años, los marcadores mitocondriales han cobrado gran importancia en genética de la conservación, ya que permiten dilucidar relaciones filogenéticas entre distintas especies; además, debido a la particularidad de ser transmitido únicamente por vía materna y no recombinarse, el adn mitocondrial (adnm) permite una detección más eficiente de hibridación entre especies o subespecies (Meganathan et al., 2011). No obstante, en el caso de los Crocodylia, este marcador ha sido utilizado también para la detección de anomalías en el material genético (Ray et al., 2005), así como para formular hipótesis acerca de los orígenes de los Crocodylia del nuevo mundo, en los cuales, gracias a diversas muestras de adnm de tres continentes, se observó que existe una clara relación entre los Crocodylia de África y América (Man et al., 2011;Meredith et al., 2011); sin embargo, los estudios de estructura poblacional y filogeografía en los Crocodylia aún son escasos (Ray et al., 2004). ...
... En los últimos años, los marcadores mitocondriales han cobrado gran importancia en genética de la conservación, ya que permiten dilucidar relaciones filogenéticas entre distintas especies; además, debido a la particularidad de ser transmitido únicamente por vía materna y no recombinarse, el adn mitocondrial (adnm) permite una detección más eficiente de hibridación entre especies o subespecies (Meganathan et al., 2011). No obstante, en el caso de los Crocodylia, este marcador ha sido utilizado también para la detección de anomalías en el material genético (Ray et al., 2005), así como para formular hipótesis acerca de los orígenes de los Crocodylia del nuevo mundo, en los cuales, gracias a diversas muestras de adnm de tres continentes, se observó que existe una clara relación entre los Crocodylia de África y América (Man et al., 2011;Meredith et al., 2011); sin embargo, los estudios de estructura poblacional y filogeografía en los Crocodylia aún son escasos (Ray et al., 2004). ...
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La presente obra es una compilación de temas básicos y fundamentales, de utilidad para los biólogos, profesionistas en conservación y personas dedicadas al estudio de los cocodrilos en México. Consideramos que, para estudiar los efectos de la interacción del ser humano con las poblaciones de cocodrilos, así como de las interacciones entre las poblaciones de cocodrilos con el ambiente, se debe llevar a cabo una serie de métodos que permitan obtener datos útiles que ayuden a la conservación de estas especies. En este libro mostramos los enfoques y análisis más usados para evaluar aspectos demográficos, ecológicos y genéticos en las poblaciones de cocodrilos, así como estrategias para conservar a estas especies. Para esta obra se contó con la participación de destacados investigadores adscritos a diferentes instituciones académicas, gubernamentales y de ong del país, así como de expertos en la materia que colaboraron para integrar este documento, que es sólo un breve acercamiento al estudio y conservación de los cocodrilos, tocando temas diversos que incluyen la investigación, el manejo, la docencia y la divulgación. El libro se divide en dos apartados, el primero lo integran cuatro temas en los que se aborda información sobre la taxonomía, biología, importancia ecológica y cultural de los cocodrilos, así como temas de legislación útiles para su protección y aprovechamiento. Estos temas permiten tener una visión general de estos aspectos básicos pero necesarios en el planteamiento de estudios y estrategias de conservación y aprovechamiento sustentable. El segundo apartado, contiene cinco temas enfocados principalmente al diseño y aplicación de las técnicas de estudio para evaluar aspectos de bioacústica, densidad poblacional y la dinámica de las poblaciones de cocodrilos, así como su estructura genética. También se abordan de manera general las técnicas de conservación ex situ, así como el manejo y aprovechamiento de cocodrilos en áreas naturales protegidas.
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Notosuchia is a group of mostly terrestrial crocodyliforms. The presence of a prominent crest over-hanging the acetabulum, slender straight-shafted long bones with muscular insertions close to the joints, and a stable knee joint suggests that they had an erect posture. This stance has been proposed to be linked to endo-thermy, because it is present in mammals and birds and contributes to the efficiency of their respiratory systems. However, a bone paleohistological study unexpectedly suggested that Notosuchia were ectothermic organisms. The thermophysiological status of Notosuchia deserves further analysis, because the methodology of the previous study can be improved. First, it was based on a relationship between red blood cell size and bone vascular canal diameter tested using 14 extant tetrapod species. Here we present evidence for this relationship using a more comprehensive sample of extant tetrapods (31 species). Moreover, contrary to previous results, bone cross-sectional area appears to be a significant explanatory variable (in addition to vascular canal diameter). Second, red blood cell size estimations were performed using phylogenetic eigen-vector maps, and this method excludes a fraction of the phylogenetic information. This is because it generates a high number of eigenvectors requiring a selection procedure to compile a subset of them to avoid model overfitting. Here we inferred the thermophysiology of Notosuchia using phylogenetic logistic regressions, a method that overcomes this problem by including all of the phylogenetic information and a sample of 46 tetrapods. These analyses suggest that Araripesuchus wegeneri, Armadillosuchus arrudai, Baurusuchus sp., Iber-osuchus macrodon, and Stratiotosuchus maxhechti were ectothermic organisms.
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During the late twentieth and early twenty-first centuries, there has been a revolution in evolutionary biology. Traditional methods that had been applied to understanding relationships and natural history for hundreds of years have been supplemented (and sometimes replaced) by biochemical and molecular techniques that now allow us to examine the entire genomes of non-model organisms. Herein we review the use of these new technologies as they apply to crocodylians in general and specifically to the New-World members of the Alligatoridae and Crocodylidae. While generally concordant with traditional analyses, in some cases they have permitted cryptic species to be recognized. In addition, they have allowed crocodylian biologists to detect hybridization events between species, both in captivity and in the wild, that would not have been possible before their use. Hybridization may lead to the formation of new species, but it may also allow a common species to “swamp out” a rarer one. Because there appears to be little hybrid dysgenesis between many of the potential hybridizing forms, hybridization is potentially a serious problem for several New-World species.
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Concurrent with the development of the polymerase chain reaction (PCR) in the late 1980s as an essential technique in the molecular biology laboratory, optimal design of PCR primers became an important part of the process. It was soon learned, for example, that inattention to secondary structure in synthetic primers could create primer-dimers and hairpins and could seriously reduce or eliminate the yield of the PCR product. The presence of priming sites in the template other than the intended target—so called false priming sites—were also found to interfere with PCR efficiency, by generating unwanted PCR products and background on the gel. Furthermore, the efficient PCR experiment required the proper concentrations of salt, buffer, and nucleic acid and the accurate determination of melting temperatures of the primers and the template. Cumbersome calculations were required to produce these values and their complexity often resulted in errors.
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Attempts to assess the natural affinities and evolution of living crocodilians have been difficult and largely contradictory (Kalin, 1955; Steel, 1973; Dowling and Duellman, 1974). Morphological character analysis has been misleading due to the overall conservatism of these reptiles and to the tendencies toward parallelism and convergence of traits that has occurred during their evolution (Langston, 1973). These complications, together with the lack of critical fossils, have made paleontological interpretations extremely difficult (Sill, 1968; Hecht and Malone, 1972; Langston, 1973; Buffetaut, 1979). Because of such problems and the small number of living species, morphoclines are rare, further complicating the efforts of the comparative morphologist (Hecht and Malone, 1972).
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Recent investigations into the evolution of the living Crocodilia, belonging to the suborder Eusuchia, have revealed that the genus Gavialis may be its most primitive living member. New morphological studies have shown that the braincase structure, neural pocket, air sinus systems, jaw adductor mechanisms, pelvic and hindlimb morphology and epaxial musculature of the caudal region of Gavialis gangeticus do not correspond to the rest of the living Eusuchia. Contrary to the morphological findings, recent biochemical studies suggest a sister group relationship between Gavialis gangeticus and Tomistoma schlegelii, another longirostrine eusuchian. Judged by its morphology, Tomistoma is merely another member of the genus Crocodylus within the Eusuchia. This conflict in data either means that not enough of the genome of both Gavialis and Tomistoma is known, the shared genome represents the primitive states for these genes or that similar genotypes can give rise to rather different morphologies. As Gavialis resembles in some ways a Mesozoic level of organization it is considered to be a surviving eusuchian relict.
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The relationships of the three living groups of crocodilians (crocodylids, alligatorids and gavialids) are poorly understood. Recent molecular results favor a sister group relationship between the crocodylid genus Tomistoma and Gavialis, with this as the sister group to the crocodylids (Densmore, 1983). Buffetaut (1985) has reinterpreted some morphologic evidence as supportive of this viewpoint. This morphologic evidence is examined here using outgroup analysis; it fails to support this hypothesis. Few if any morphological features unambiguously support a Gavialis + crocodylid or Gavialis + Tomistoma relationship. Instead the classic pre-Darwinian phylogeny of Duméril (1806) is corroborated by anatomical evidence. This phylogeny supports a monophyletic crocodylid + alligatorid clade as a monophyletic sister group to Gavialis.
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— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
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We present restriction fragment analyses of mitochondrial and ribosomal DNAs in an effort to resolve some critical systematic questions among extant members of the order Crocodilia. This paper concentrates on interspecific relationships among all the species of the circumtropical genus Crocodylus (the true crocodiles), the relationship of Crocodylus to Osteolaemus (the dwarf-African crocodile) and tests the hypothesis that the two gharial genera, Gavialis and Tomistoma, are more closely related to one another than to any other group of living crocodilians. This first extensive molecular phylogeny of the true crocodiles not only aligns some species that are found in the same hemisphere but also places organisms that currently have disjunct distributions in the same clade. We also include a method of analysis (compatible parsimony) for restriction fragment data that appears to combine compatibility and parsimony criteria.