Proc. NatI. Acad. Sci. USA
Vol. 91, pp. 10601-10605, October 1994
Evolution
Generic affinities among crocodilians as revealed by DNA
fingerprinting with a Bkm-derived probe
(restriction fIgment length polymorphism/multlocus DNA probe/repetitive DNA probe/phenogram/genetic profile)
RAMESH K. AGGARWAL*, KSHITISH C. MAJUMDAR*, JEFFREY W. LANGt, AND LALJI SINGH*t
*Centre for Cellular and Molecular Biology, Hyderabad 500 007, India; and tDepartment of Biology, University of North Dakota, Grand Forks, ND 58202
Communicated by M. S. Swaminathan, July 23, 1993
and the caimans (Caiman, Melanosuchus, Paleosuchus) as the
nearest sister taxa ofAlligator, whereas opinion is divided on
the affinities of the two gharial genera (Gavialis and Tomistoma) to each other and to other crocodilians. Some favor a
close relationship of gavials with crocodylids (1, 6, 9, 11),
whereas others place them in a separate family/lineage (12).
The use of DNA fingerprinting (13) has recently been
shown to be useful in estimating relative genetic variability
and in reconstructing the evolutionary relationships of natural populations of genetically isolated mammals (14). In the
present study, we have used DNA fingerprinting, with the
Bkm-2(8) probe, to study phylogenetic relationships among
18 of the 21 living species belonging to seven of the eight
genera of crocodilians. The Bkm sequences were first identified and isolated as a minor satellite DNA from the genomic
DNA of the female Indian banded krait (Bungarusfasciatus).
Since then, it has been demonstrated that the major component of Bkm consists of tandem repeats ofthe tetranucleotide
GATA, which shows extensive restriction fragment length
polymorphism in various eukaryotes and can therefore be
used as an efficient probe for genetic fingerprinting (15-22).
Our results, based on quantitative as well as qualitative
differences in the genetic fingerprint profiles obtained by use
of the Bkm-2(8) probe, suggest that the seven crocodilian
genera studied belong to two distinct groups; the first group
includes Alligator, Paleosuchus, and Caiman, and the second
group includes Crocodylus, Osteolaemus, Tomistoma, and
Gavialis. The results also suggest that the two gharial genera,
Tomistoma and Gavialis, are closely related.
Genetic fingerprint profiles have been sucABSTRACT
cesfublly used for estabishing biological relationships, in linkage analysis, and in studies ofpopulato structure but have not
so far been used for ascertalning phylogenetic relationships
among related groups of species and genera. This Is largely
because these proffles are thought to evolve too rapidly to be
informative over large time intervals. However, we show here
that among the Crocodilla, whose phylogeny is a debated Issue,
these profiles can provide phylogenetically useful formation.
By using the probe Bkm-2(8), DNA gerprints with distinct
bands distributed in the size range 0.5-23.0 kb were obtained
for individuals of 18 species belonging to seven of the eight
genera of crocodiflans. These genetic profiles showed hidividual-, species-, and restriction enzyme-specific patterns. In
addition, string differences were observed in the copy number of Bkm-related sequences in genomes of different crocodiflan species. The qualitative data from DNA flngirpit
proffles, and quantitae data on copy number variation in
Bkm-related sequences, suggest that these genera belong to two
distinct groups, one of which includes Aligator, Paeosuchuis,
and Caiman; the other incudes Crocodylus, Osteolkemus, Tomistoma, and Gavialis. A close relationship between Tomistoma
and Gabals is also suggested by these results.
Crocodilians are the sole living reptilian representatives of
the subclass Archosauria, a highly successful group in the
Mesozoic era both in numbers and in diversity. At present,
only 8 of the 124 described genera have survived and all of
these belong to the same suborder, Eusuchia (1). According
to most systematists, there are only 21 extant species, 11 of
which belong to Crocodylus, which is by far the largest genus.
The natural affinities among living crocodilians have so far
been determined primarily on the basis of comparative morphology and paleontological records. However, the resolving
power of these approaches has not been adequate to solve
certain problematic and confusing relationships within the
order Crocodilia. The commonality in life-style of many of
the crocodilian taxa may have led to similar adaptative
strategies-e.g., convergent skull morphology and head
shape. Such convergence in characters, although considered
phylogenetically important, has made interpretation of the
systematic relationships in crocodilians difficult (2). This has
led to the use of other approaches such as cytogenetic
parameters (3, 4), analysis of coevolving crocodilian-parasite
lineages (5), biochemical and immunological studies of proteins (6-8), and Southern blot and DNA sequence analyses
of mitochondrial and nuclear ribosomal DNA (9-11) to
resolve the natural affinities and evolutionary history of the
living crocodilians.
On the basis of the approaches described above, there is
general agreement in aing Osteolaemus with Crocodylus
MATERIALS AND METHODS
amples. Blood samples were collected from the heart or
brain plexus of 203 individuals and stored at -700C (Table 1).
DNA Fingerprinting. DNA isolation, digestion, gel electrophoresis, Southern blotting, and filter hybridization were done
as described by Lang et al. (22) and by Aggarwal et al. (23).
Slot Bloting. Slot blots were prepared in duplicate for each
individual of different species with 60, 180, and 360 ng of
DNA onto a Hybond-N membrane, using a Minifold II
apparatus (Schleicher & Schuell). The membranes were then
hybridized with the 32P-labeled single-stranded Bkm-2(8)
probe. To confirm that the quantity of DNA loaded for
different individuals was the same, the hybridized blots were
melted and rehybridized with a nick-translated 32P-labeled
Xenopus rDNA probe.
Scoring and Analysis of DNA Fingerprints. Distinct bands
representing DNA fragments ranging in size from 1.3 to 23.0
kb were scored in each genetic profile. All bands showing
similar sizes and intensities were considered to be identical.
Molecular size markers and duplicate samples from the same
individual were run on either side of the gel to check for
mobility distortion. Samples of a set of individuals representing a genus/species were run in each gel, along with the
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*To whom reprint requests should be addressed.
10601
10602
Evolution: Aggarwal et A
Table 1. Number, source, and origin of crocodilians used for
DNA fingerprinting
Sex
Crocodilian genera/species (captive
?
locality)
M
F
Paleosuchus palpebrosus (a)
1
Paleosuchus trigonatus (a)
1
1
Caiman crocodilus yacare (a)
1
Caiman latirostris (a)
1
Caiman crocodilus crocodilus (a, b)
7
10
Alligator sinensis (a)*
1
Alligator mississippiensis (b, c,t d*)
3
2
Gavialis gangeticus (b)
1
2
Tomistoma schlegelii (b, e)
3
1
Osteolaemus tetraspsis (a, b)
3
2
Crocodylus palustris (b, f)§
46
84
Crocodylus porosus (b, f)
3
6
Crocodylus acutus (a, c)l
3
Crocodylus niloticus (a, b)
5
Crocodylus moreleti (b)
1
2
Crocodylus rhombifer (a)
1
Crocodylus siamensis (b, e)
2
4
Crocodylus cataphractus (e)
1
1
a, Ocala; b, Madras Crocodile Bank (India); c, Gatorama; d,
University of North Dakota; e, Miami Zoo; f, Nehru Zoological Park,
Hyderabad, India.
*Origin, China.
tOrigin, Florida.
tOrigin, Louisiana.
§Origin, different parts of India and captive bred animals at Madras
Crocodile Bank.
¶Origin, Jamaica.
samples to be compared, to facilitate the comparison of DNA
fingerprints obtained from different gels.
DNA fingerprints were scanned and the fragments were
calibrated for size by using a A HindIII/EcoRI double digest
as the molecular size marker on the Biotrac DNA fingerprinting system (Foster and Freeman, Worcestershire, U.K.)
using the BIOWORLD program. The inter- and intrageneric/
species variability was estimated by calculating the difference value, D, in all possible pairwise combinations. The
difference value (D) between any two DNA profiles was
calculated as the number of fragments that were different
divided by the total number of fragments present in the two
individuals (14). The degree of relatedness within the members of the same species/genera was calculated by subtracting D (average of all the D values for the species/genera in
question) from 1. The degree of divergence between any two
genera was arrived at by averaging all the D values between
individuals of the two genera. The latter values were used to
construct a phylogenetic tree using the UPGMA (unweighted
pair group method with arithmetic means) option in the
NEIGHBOR program (Phylip software, version 3.41) of J.
Felsenstein (University of Washington, Seattle).
RESULTS
Qualitative Differences in DNA Profiles. DNA fingerprints,
with distinct scorable bands distributed in the size range of
0.5-23.0 kb and showing individual-, species-, and restriction
enzyme-specific patterns, were obtained (Figs. 1 and 2).
The average number of total bands in Paleosuchus,
Caiman, Alligator, Gavialis, Tomistoma, Osteolaemus, and
Crocodylus was 48.0, 26.0, 28.2, 24.0, 20.0, 27.7, and 25.3 in
their Alu I profiles and 46.3, 42.7, 40.5, 27.0, 29.0, 32.7, and
27.8 in their HinfI profiles, respectively. The overall signal of
hybridization was stronger in Paleosuchus, Caiman, and, to
a lesser extent, Alligator, than in Gavialis, Tomistoma,
Osteolaemus, and Crocodylus. Gavialis and Tomistoma
showed a particularly poor signal. The Alu I-digested DNA
Proc. Natl. Acad. Sci. USA 91 (1994)
profiles of Paleosuchus, Caiman, and Alligator (Fig. 1A) and
Gavialis, Tomistoma, Osteolaemus, and Crocodylus (Fig. 1
B and C) showed a distinct fingerprint divergence among
themselves. The maximum number of bands was visible in
the DNA profiles of two species of Paleosuchus, which were
almost evenly distributed along the length of the DNA
fingerprint (Fig. 1A, lanes 1-3). Similar DNA profiles but
with significantly fewer bands were detected in the three
species of Caiman (Fig. 1A, lanes 4-9). On the other hand,
DNA profiles of the remaining five genera showed distinctly
fewer bands per fingerprint, most of them being <4 kb (Fig.
1). In Crocodylus and Osteolaemus, there were many major
(high intensity) bands interspersed with minor (low intensity)
bands (Fig. 1B, lanes 9-15; Fig. 1C, lanes 1-16), whereas in
Gavialis and Tomistoma such bands were relatively few (Fig.
1B, lanes 1-8). These differences in band distribution and
band intensities were much more apparent in the corresponding Hinfl-digested DNA fingerprints (Fig. 2). Hinfl profiles
of Caiman and Alligator showed many bands > 4 kb when
compared to their Alu I genetic profiles (Fig. 1A, lanes 4-15;
Fig. 2A, lanes 3-13); they closely resembled those of Paleosuchus (Fig. 2A, lanes 1 and 2) with respect to size distribution and hybridization intensities. In the remaining four
genera (Gavialis, Tomistoma, Osteolaemus, and Crocodylus), the HinfI profiles, although distinct, were similar to their
Alu I profiles. The HinfI profiles of Gavialis and Tomistoma
(data not shown) showed only a shift in the position of bands
relative to their Alu I profiles.
Analysis of the fingerprint data also demonstrated that
while most of the bands in the genetic profiles were individual
specific, there were certain bands that were highly conserved
and were probably specific to a species/genus. The HinfI
fingerprints of five individuals of A. mississippiensis, from
two different localities in the United States, were characterized by the presence of a species-specific doublet > 5 kb (Fig.
2A, arrowheads, lanes 9-13). No such elements were detected in the corresponding Alu I profiles (Fig. 1A, lanes
11-15). Conserved bands (small arrowheads) in the fingerprints of individuals belonging to geographically different
localities were also present in Osteolaemus (Fig. 2B, lanes
1-3), C. acutus (Fig. 2B, lanes 4-7), and C. siamensis (Fig.
1C, lanes 6-8; Fig. 2B, lanes 9-11). The geographically
unrelated individuals of Gavialis and Tomistoma also showed
species-specific distribution of high-intensity major bands in
their genetic profiles. Gavialis profiles showed a seemingly
conserved doublet of 3.5 kb and a band in the 15-kb range
(Fig. 1B, lanes 1-4). In Tomistoma there were five such
bands in the range 1.5-2.2 kb and one major band of 4.2 kb
(Fig. 1B, lanes 5-8).
When hybridized blots were washed at a higher stringency,
the number of bands and the intensity of hybridization was
greatly reduced in Gavialis, Tomistoma, Osteolaemus, and
Crocodylus. By contrast, in Paleosuchus, Caiman, and Alligator the higher stringency of washing had virtually no effect on
the overall number and the intensity of bands in the genetic
profiles obtained with both the restriction enzymes. The intensity of signal of hybridization, the number of bands obtained,
and the sustenance of the pattern of genetic profiles at high
stringency of washing in Paleosuchus, Caiman, and Alligator
samples suggested a quantitative difference in the genomic
content of Bkm-related sequences in the genera tested.
Quantitative Differences in Bkm-Related Sequences. The
quantitative differences in the genomic content of Bkmrelated sequences in different crocodilian genera were studied by preparing slot blots with known but equal quantities of
total undigested genomic DNA of one individual each of all
the species tested and hybridizing them with the labeled
Bkm-2(8) probe. After autoradiography, each slot was numbered, cut out, and assayed. The results were verified by
studying samples from additional individuals of each species
Evolution: Aggarwal et al.
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Proc. Natl. Acad. Sci. USA 91 (1994)
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FIG. 1. Ala I DNA profiles of different crocodilians developed after hybridization with Bkm-2(8) probe. (A) Lanes: 1, P. palpebrosus; 2 and
3, P. trigonatus; 4, C. yacare; 5, C. latirostris; 6-9, C. crocodilus; 10, A. sinensis; 11-15, A. mississippiensis. Note overall stronger signal in
Paleosuchus (PA), Caiman (CA), and Alligator (AL) compared to the remaining genera shown in B and C. (B) Lanes: 1-4, G. gangeticus; 5-8,
T. schlegelii; 9-11, 0. tetraspsis; 12, C. moreletii; 13, C. niloticus; 14, C. siamensis; 15, C. palustris. Note that there are many more major bands
interspersed with minor bands in size range >2 kb in Osteolaemus (OT) and Crocodylus (CR) compared to Gavialis (GA) and Tomistoma (TO).
(C) Lanes: 1, C. porosus (Cr.po.); 2 and 3, C. acutus (Cr.a.); 4 and 5, C. niloticus (Cr.n.); 6-8, C. siamensis (Cr.s); 9-11, C. moreletii (Cr.m.);
12, C. rhombifer (Cr.r.); 13 and 14, C. cataphractus (Cr.c.); 15 and 16, C. palustris (Cr.p.). Arrowheads indicate probable species-specific marker
bands.
wherever possible. In all the species tested, an increase in the
concentration of DNA resulted in a concomitant increase in
signal strength as indicated by both radioactivity and photodensity. For each of the three DNA concentrations tested,
the hybridization signal for Paleosuchus, Caiman, and Alligator species was invariably 3- to 8-fold higher than that for
Gavialis, Tomistoma, Osteolaemus, and Crocodylus (Fig. 3).
The slot blot results clearly indicated two major groups of
crocodilians with respect to the copy number of Bkm-related
sequences. In the first group, comprising Paleosuchus,
Caiman, and Alligator, the copy number of Bkm-related
sequences in their genome was 3-8 times higher than in the
second group consisting of Gavialis, Tomistoma, OsteolaeA
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copy number of Bkm-related sequences was seemingly the
highest in Paleosuchus, followed by Caiman and Alligator,
suggesting that Alligator lies at the lower boundary of this
group. Qualitative differences apparent in the overall band
patterns for these genera led to the same conclusions.
Generic Affinities in the Pakosechus-Caiman-Aigator
Group. The DNA profiles of Paleosuchus and Caiman were
very similar. In both cases, a large number of bands > 5 kb
were obtained with both HinfI and Alu I (Fig. 1A, lanes 1-9;
Fig. 2A, lanes 1-8). However, while Alligator HinfI profiles
closely resembled those of Caiman and Paleosuchus with
respect to number, size, and distribution of bands (Fig. 2A,
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FIG. 2. HinfI DNA profiles of different crocodiians-developed after hybridization with Bkm-2(8) probe. (A) Lanes: 1 and 2, P. trigonatus
(PA); 3-8 Caiman (CA), 3, C. c. yacare; 4, C. latirostris; 5-8, C. c. crocodilus; 9-13, A. mississippiensis (AL). (B) Lanes: 1-3, 0. tetraspsis
(OT); 4-19 Crocodylus (CR), 4-7, C. acutus (Cr.a.); 8, C. niloticus (Cr.n.); 9-11, C. siamensis (Cr.s.); 12, C. porosus (Cr.po.); 13-15, C. moreletu
(Cr.m.); 16 and 17, C. catapharactus (Cr.c.); 18 and 19, C. palustris (Cr.p.). Arrowheads indicate probable species-specific marker bands.
Evolution: Aggarwal et al.
10604
Proc. Natl. Acad. Sci. USA 91 (1994)
lanes 9-13), Alu I fingerprints differed considerably from
those of the other two and had significantly fewer bands
(mainly <4 kb; Fig. 1A, lanes 10-15). These differences in
band patterns suggest a closer affinity between Paleosuchus
and Caiman than between either of these and Alligator. This
conclusion is further substantiated by the similarity in copy
number of Bkm-related sequences between Paleosuchus and
Caiman and significant differences between these two genera
on one hand and Alligator on the other. The genomic content
of Bkm-related sequences in Alligator is 0.5-0.75 that of
Paleosuchus and Caiman (Fig. 3).
Generic Affinis in the Gaviafis-Tomistomt-OsteolaemusCrocodylus Group. On the basis of the broad characteristics
of the genetic profiles of these four genera, it is possible to
further divide them into two subgroups: subgroup I, Crocodylus and Osteolaemus; subgroup 2, Gavialis and Tomistoma. The fingerprints of Osteolaemus and all the species of
Crocodylus showed a similar pattern of many high-intensity
major bands interspersed with low-intensity minor ones (Fig.
1B, lanes 9-15; Fig. 1C, lanes 1-16; Fig. 2B, lanes 1-19),
mostly in the >3-kb size range. By contrast, in the two
monotypic genera Gavialis and Tomistoma, more bands were
found in the lower molecular weight range; the remaining
bands (>3 kb) were mostly low-intensity minor bands (Fig.
1B, lanes 1-8; compare these with lanes 9-15 for Osteolaemus and Crocodylus).
Phylogenetic Analysis. A phylogenetic tree showing relationships among the seven genera of crocodilians was generated based on coefficients of dissimilarity (data not shown).
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FIG. 3. Quantitative differences in Bkm-related sequences in
genomic DNA of different species/genera of crocodilians. (A) Slot
blot of genomic DNA hybrid with 32P-labeled Bkm-2(8) probe.
(B) cpm per slot of genomic DNA plotted for different species/genera
of crocodilians. PA, Paleosuchus; CA, Caiman; AL, Alligator; GA,
Gavialis; TO, Tomistoma; OT, Osteolaemus; CR, Crocodylus; Slots:
1, P. palpebrosus; 2 and 3, P. trigonatus; 4, C. c. yacare; 5 and 6,
C. c. crocodilus; 7, A. sinensis; 8 and 9, A. mississippiensis; 10 and
11, G. gangeticus; 12 and 13, T. schlegelii; 14 and 15, 0. tetraspsis;
16, C. cataphractus; 17, C. rhombifer; 18, C. acutus; 19, C. niloticus;
20, C. siamensis; 21, C. moreletii; 22, C. porosus; 23, C. palustris.
Note the 4-8 times higher signal in Paleosuchus, Caiman, and
Alligator compared to the other genera, suggesting that these genera
belong to two distinct groups.
The phenogram obtained clearly shows that seven crocodilian genera belong to two major groups and that the two
gharial genera are closely related to the crocodylids-i.e.,
Crocodylus and Osteolaemus (Fig. 4). It also illustrates the
degree of divergence between different generic groups and
shows that the genus Alligator is more widely separated from
Caiman (P = 0.351) than Paleosuchus (P = 0.328). Also, the
genus Gavialis is clearly distinguishable from Tomistoma (P
= 0.395), as is Osteolaemus from Crocodylus (P = 0.371). On
the whole, Alligator lineage shows the highest degree of
divergence from both the Gavialis and Crocodylus lineages (P
= 0.477), which, within themselves, show a divergence of
only 0.425. The reliability of the phenogram, notwithstanding
its deep nodes and smaller internodes, is brought out by the
fact that in each case the standard error value was significantly smaller than the respective estimate of pairwise distance between any given two nodes.
DISCUSSION
Genetc Profile and Bill Reaedn. Genetic or DNA
fingerprinting provides a method for identification of individuals, confirmation of biological relationships (13), human
genetic analysis (24), and demographic studies (14, 25-28).
However, it had not until now been used for phylogenetic
analysis because the profiles were thought to evolve too
rapidly to be informative over large time intervals. In the
present investigation, we have used the twin approach of
analyzing quantitative differences as well as similarities and
dissimilarities in fingerprint profiles to infer phylogenetic
relationships among the crocodilians, which as a group have
undergone relatively recent divergence compared to their
ancient progenitors-i.e., Archosauromorpha.
According to Norell (29), for groups like Crocodilia, which
have undergone relatively recent divergence, only those
molecular sequences will be phylogenetically informative
that behave like fast-clock molecules-i.e., the ones that
reflect relatively higher rates of sequence (marker) substitutions/modifications (30), although such sequences may be
uninformative regarding relationship of the group with its
outgroup taxa because of the possibility of the sequences
having progressed to the point of randomization. Mitochondrial DNA markers, which evolve 5-10 times faster than
nuclear genes, can be used to reconstruct the phylogenetic
history of populations, but they do not provide any information about the extent of nuclear gene flow or variability,
which is central to the evolution of the overall makeup of an
organism. By contrast, multilocus hypervariable minisatellite
probes reveal enormous genetic variability in the form of
restriction fragment length polymorphism spread over the
entire genome; they evolve rapidly over long time periods,
allowing estimation of the overall relative genetic variability
and providing a more amenable molecular tool for looking at
the phylogeny of closely related groups.
The results presented here demonstrate the potential of the
technique of DNA fingerprinting by using a Bkm-2(8) probe
in the study of phylogenetic relationships among relatively
recently diversified, closely related crocodiians. This study
shows that, based on band-sharing coefficients, the degree of
relatedness among different individuals can be determined. It
also shows that there are a few specific bands (for one or both
restriction enzymes) that are unique to a species/genus and
appear consistently in all its individuals, related or unrelated
by descent or geography. The presence of such elements
suggests that there are, perhaps, some species-specific allelic
conserved domains in the genome that might serve as potential diagnostic markers to identify a species.
We show here that the true (Crocodylus) and dwarf African
(Osteolaemus) crocodiles are closely related sister taxa,
whereas alligators and caimans form a loose assemblage,
although Alligator is distinct from the two caiman genera
Evolution:
Aggarwal et al.
Proc. Natl. Acad. Sci. USA 91 (1994)
studied. In addition, the present study favors a sister-group
relationship between Tomistoma and Gavialis; these two in
turn form a sister group to crocodylids-i.e., Osteolaemus
and Crocodylus. The above conclusions regarding grouping
of crocodilians are further substantiated by the phenogram
developed from the data on band sharing. The phenogram,
besides indicating the probable phylogenetic relatedness of
the species/genera involved, also offers a semiquantitative
estimate of the degree ofgenetic divergence. It shows that the
Alligator lineage is most widely separated from the Gavialis
and Crocodylus lineages (P = 0.447), which, within themselves, are closer to each other and relatively less diverged (P
= 0.425). These measures of relatedness may, however, be
slightly inflated because of inherent problems of DNA fingerprinting technique, such as fortuitous comigration of
fragments generated by alleles at different loci, as well as
limitation in resolving fragments of nearly similar sizes (31).
But the fact that fingerprint-based phylogenetic analysis
makes use of the variability present in the genotype of the
organism lends it more credibility over the findings of traditional approaches that make use of the phenotypic variability,
which is influenced greatly by the immediate environment of
the organism. Nevertheless, the grouping of crocodilians
based on the present study corroborates earlier findings
based on traditional disciplines, as well as the more recent
biochemical and immunological studies of proteins, restriction fragment length polymorphism, and sequence data of
mitochondrial and nuclear ribosomal DNA.
A comparison of the quantitative data pertaining to Bkmrelated sequences in the genomes of various crocodilians
(Fig. 3) with the available information on the distribution of
fossils and living crocodilians through time (32) reveals that
Paleosuchus and Caiman, which show the highest copy
number of Bkm-related sequences in their genomes, are also
the more recently evolved genera belonging to the Alligator
lineage. The copy number of Bkm-related sequences in
Alligator, although less than in the caimans, is distinctly 3-5
Tomi stoma
Gavialis
Osteolaemus
Crocodyl us
Caiman
Paleosuchus
Alligator
0.50
1
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.
0.30
1
0.20
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0.0
p
FIG. 4. uPGMA phenogram showing relationships among crocodilian genera based on Alu I fingerprinting data. P, probable degree
of divergence. The reliability of the phenogram was also tested by
generating the most parsimonious tree (results not shown) for a
subset of data for 13 individuals belonging to 10 species of four
genera, using both the bootstrapping and branch-and-bound options
contained in version 3.0 of the PAUP program of David Swofford
(Illinois Natural History Survey, Champaign).
10605
times more than in the remaining four genera. It seems that
in this lineage, evolution has involved a substantial increase
in the copy number of Bkm-like short repeat sequences,
involving processes such as amplification and insertion of
DNA into chromosomes. This sets apart the Alligator lineage
from the rest of the crocodilians and also rules out the
possibility of its being closely related to the Gavial lineage,
notwithstanding the stepwise nature of evolutionary changes
in the copy number of minisatellite alleles (33).
We would like to thank Mrs. Seema Bhaskar for her excellent
technical assistance and Dr. P. M. Bhargava and Prof. H. Sharat
Chandra for useful suggestions and for going through the manuscript.
The help of M. Wise at St. Augustine Alligator Farm (Florida); B.
Ziegler at Metro Zoo, Miami (Florida); H. Hunt at Zoo Atlanta
(Georgia); D. D. Thielein and C. Clemons at Gatorama, Palmdale
(Florida); P. Kumar at Nehru Zoological Park, Hyderabad; and R.
Whitaker and H. Andrews at Madras Crocodile Bank, Madras, in
providing crocodile samples for this study is gratefully acknowledged. We are particularly grateful to Dr. J. Felsenstein, University
of Washington, Seattle, for advice on the proper use of his program
and for his critical comments and suggestions. J.W.L. was supported
by the Smithsonian Institution, National Science Foundation, and
National Geographic Society.
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