JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 294:302–311 (2002)
Studies on the Molecular Evolution of the
Crocodylia: Footprints in the Sands of Time
HERBERT C. DESSAUER,1 TRAVIS C. GLENNn,2,3 and
LLEWELLYN D. DENSMORE4
1
Department of Biochemistry and Molecular Biology, Louisiana State University
Health Sciences Center, New Orleans, Louisiana 70119
2
Savannah River Ecology Laboratory, Aiken, South Carolina 29802
3
Department of Biological Sciences, University of South Carolina, Columbia,
South Carolina 29208
4
Department of Biological Sciences, Texas Tech University, Lubbock, Texas
79409-3131
ABSTRACT
A reasonably large number of studies focusing on the molecular evolution of
crocodilians have been completed during the past 100 years. Proteins were initially studied before
DNA was known to carry the genetic information of cells and organisms, and were subsequently
studied to infer changes at the DNA level. More recently, studies on the DNA itself have been
completed. We have had the pleasure of taking part in or facilitating many studies conducted over
the past 50 years, especially several of the earliest studies done using newly developed molecular
techniques. We provide a review of the molecular genetic studies on crocodilians, summarizing the
findings of these studies as well as the context in which they were undertaken. This review is a
personal look at the history of molecular studies on the evolutionary biology of crocodilians. Our
excuse for this focus is that our professors, our students and we have had the opportunity to be
among the first to apply many new techniques to studies of crocodilians since 1950, when one of us
(HCD) was a graduate student of Roland Coulson and Tom Hernandez. Although we will review
much of the material in this subject area, we do not claim that it is complete. Instead, we focus our
presentation on work in which we have participated or with which we are particularly familiar. We
especially focus on materials relevant to the research presented at the 2nd International Crocodilian
DNA Workshop, 7–9 November, 2001, at the San Diego Zoo. Thus, the following review also stands as
a tribute to our mentors, students, and colleagues. J. Exp. Zool. (Mol. Dev. Evol.) 294:302–311,
2002. r 2002 Wiley-Liss, Inc.
IN THE BEGINNING
Studies on the molecular evolution of crocodilians have a much longer history than most
current researchers appreciate. Rudolf Krass
(1897; cited in Boyden, ’51) discovered the
precipitin reaction, where clear antisera and
antigens react to form visible products, more than
a century ago. Nuttall (’01; Fig. 1a) first published
how this technique could be used in zoological
classification (i.e., systematics) 100 years ago, and
he subsequently published a comparative molecular study that included crocodilians (Nuttall, ’04).
These studies predated the experiments demonstrating DNA as the molecule carrying genetic
information by decades and the discovery of the
molecular structure of DNA by half a century. The
studies using these techniques have been summarized previously (Boyden, ’51).
r 2002 WILEY-LISS, INC.
In 1950 Roland Coulson and Tom Hernandez
(Fig. 1b) initiated work on alligator nutrition and
metabolism at the Louisiana State University
Medical School in New Orleans. This work is
summarized in two thorough compendiums on
these subjects (Coulson and Hernandez, ’64, ’83).
Their unique studies were recognized in a 1953
article in Newsweek and by the dedication of the
1989 Symposium on the Biology of the Crocodylia
to Coulson (Dessauer, ’89).
Work at the medical school with Rol Coulson
and Tom Hernandez was a valuable educational
Grant sponsor: U.S. Department of Energy; Grant number: DEFC09-96SR18546; Grant sponsor: National Science Foundation; Grant
number: BSR-8607420; Grant sponsor: National Geographic Society;
Grant numbers: 6529-99 and 7007-01.
n
Correspondence to: Travis C. Glenn, Savannah River Ecology Lab,
PO Drawer E, Aiken, SC 29802. E-mail: Travis.Glenn@sc.edu
Received 24 January 2002; Accepted 2 October 2002
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/jez.10208
MOLECULAR GENETIC STUDIES OF CROCODILIANS
303
Fig. 1. Pioneers in the molecular studies of s: a. G. H. F.
Nuttall (M. Schaffer photo); b. Tom Hernandez and Roland
Coulson (Newsweek 1953 photo); c. members of the ALPHA
HELIX EXPEDITION, standing, left to right: A. C. Wilson,
W. Z. Lidicker, A. H. Brush, T. Gobble, R. G. Zweifel, H. G.
Cogger, and V. M. Sarich; kneeling: R. Storez and H. C.
Dessauer (R. G. Zweifel photo).
experience for the many students who worked
with them. They instilled the pleasure of doing
detailed work on a problem, and the importance
of obtaining accurate data. One learned to
handle alligators, collect their body fluids and
maintain them in captivity. Often the day would
begin with an alligator roundup, following the
escape of our specimens from their cadaver tank
homes. One time we located a specimen in the
dean’s office.
Dean Frey was one of the few people in the
medical school who had respect for research with
species other than E. coli, dogs, rats or humans.
His view of the research was not shared by all,
however. One time in particular, the building
engineer, upset with us for using distilled water of
the central supply, shut off the valve on the
storage tank on weekends. Our response was to
open the valve. In frustration, he poured salt into
the tank. Fortunately, we immediately detected
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H.C. DESSAUER ET AL.
the contamination as we were doing chloride
analyses at the time.
The senior author (HCD) decided to work on the
biochemistry of Anolis carolinensis for his dissertation. This led to many years of collaboration
with Dr. Wade Fox. While collaboration on studies
of anoles continued, additional population and
systematic studies on garter snakes and many
other reptiles were undertaken. Working on such
problems, Dessauer became an expert on applying
immunological, peptide fingerprinting, and especially high resolution electrophoretic methods in
comparative studies using proteins (Dessauer,
’74).
POPULATION GENETIC STUDIES
OF CROCODILIANS
Proteins
Ted Joanen, who was largely responsible for
turning the alligator population in Louisiana away
from the path to extirpation (Joanen and
McNease, ’87, ’89), learned of the work of
Dessauer and Fox on genetic polymorphisms and
sought help in finding molecular markers for use
in solving alligator population problems. Don
Gartside, a postdoctoral fellow from Australia,
and Dessauer surveyed alligators from the Rockefeller Refuge in southwestern Louisiana for
protein polymorphisms (cf. Smithies, ’59). Only
two of the 50 loci tested, muscle lactate dehydrogenase and a peptidase, were polymorphic, suggesting that alligators had one of the lowest levels
of genetic diversity of any vertebrate (Gartside
et al., ’77). Subsequently, low levels of protein
polymorphisms have been observed in alligators
from the Everglades (Menzies et al., ’79) and
South Carolina (Adams et al., ’80), and in the Nile
crocodile from Zimbabwe (Lawson et al., ’89).
Subsequent tests also indicated low indices of
diversity with RAPD, M-13, and banded krait
DNA probes (see Glenn et al., ’98).
Although the total amount of variation revealed
by protein polymorphisms was low, it did indicate
significant genetic variation among populations
from the extremes of the species range (Adams
et al., ’80). Most of this variation was, however,
due to low frequency private alleles. The genetic
inheritance of the polymorphic proteins has subsequently been demonstrated in alligator families
from the Rockefeller population (Davis et al.,
2001b; HCD unpublished data), but these allozymes have little power to detect variation among
individuals within populations.
Microsatellites
In 1993, Dessauer decided that microsatellites,
shown to be highly polymophic in a variety of
mammals, might also serve as a source of genetic
markers for alligators. Mike Braun, director of the
Smithsonian Institution’s Laboratory of Molecular
Systematics and a former graduate student of
Dessauer and contemporary of Densmore, invited
Dessauer to visit his lab to learn how to characterize microsatellites. Braun introduced Dessauer to Travis Glenn, a graduate student, for lab
bench instruction. Glenn’s research focused on
vertebrates whose populations had undergone
extreme population reduction (i.e., bottlenecks)
during their evolution. Soon Glenn decided to
include the American alligator in his dissertation
research, and a productive collaboration between
Dessauer and Glenn was initiated. Glenn’s dissertation is not only a source of information on
microsatellites of whooping cranes and alligators,
but also includes methods for doing research on
microsatellites (Glenn, ’97). Methods development
has continued as a part of Glenn’s research and
subsequent methods developed for microsatellite
DNA loci are available electronically at http://
www.uga.edu/srel/DNA_Lab/index.htm.
Microsatellites provided the highly polymorphic
loci needed for solving problems in population
genetics of crocodilians. Glenn, Dessauer and
colleagues developed primer pairs for 15 microsatellite loci (most of these microsatellite loci
contained dinucleotide repeats), 11 of which were
polymorphic in alligators from the Rockefeller
Refuge and from the Florida Everglades (Glenn
et al., ’96, ’98). Heterozygosities at microsatellite
loci were almost 20 times higher than those
obtained with allozymes. Sixteen alleles were
detected at one polymorphic locus in our relatively
small sample of alligators (Glenn et al., ’98).
Composite genotypes at the 11 polymorphic loci
were unique for each individual, 19 specimens
from Louisiana and 14 from Florida (Glenn et al.,
’98).
Populations from across the range of American
alligators could be distinguished from each other
by unique alleles or by frequency differences at
microsatellite loci (Glenn et al., ’98; Davis et al.,
2001a). Davis et al. (2001a) observed strong
differences among populations that could be
divided best by the Mississippi River. Most of the
populations studied by Davis et al. (2001a),
however, were from coastal regions. Additional
population-level work on Texas populations of the
MOLECULAR GENETIC STUDIES OF CROCODILIANS
American alligator has recently been completed by
Wade Ryberg and his collaborators (Ryberg et al.,
2002), using the microsatellite loci characterized
by Glenn et al. (’98). Ryberg et al. (2002) have
studied alligators from several localities in Texas
that vary in ecology and geographic distances,
focusing on inland versus coastal populations.
Davis et al. (2002) have now analyzed American
alligators from 12 localities from throughout the
species range, using a subset of the originally
described loci and two newly developed loci with
tetranucleotide repeats, finding support for additional subdivision among populations.
Other crocodilian species have also been studied
at the population level. Jennifer Dever, working in
Densmore’s (LDD) lab, completed the first population genetics study of a New World Crocodylus,
Morelet’s crocodiles (C. moreletii) (Dever et al.,
2002). Nancy FitzSimmons and her colleagues are
currently working on the genetics of ‘‘salties’’
(Crocodylus porosus) and ‘‘freshies’’ (C. johnsoni )
in Australia (FitzSimmons et al., 2001). Jacob
Gratten and colleagues are studying population
level problems in New Guinea (C. novaeguineae)
and Philippine (C. mindorensis) crocodiles (J.
Gratten, personal communication). Other population studies underway include Evon Hekkala and
colleagues’ study of Nile crocodiles (C. niloticus),
U. Frederick Pontillas and colleagues’ study of
Philippine crocodiles (C. mindorensis), and N.
FitzSimmons and colleagues study to reintroduce
Asian crocodiles (C. siamensis) into Vietnam
(FitzSimmons et al., 2002).
Early analyses indicated that the primers
designed from alligator microsatellite loci were
significantly less likely to amplify orthologous loci
from more distantly related species (Glenn et al.,
’96). Although the loci amplified were sometimes
polymorphic within Caiman (Verdade et al., 2002),
they were monomorphic within species of Crocodylus (S. Davis and N. FitzSimmons personal
communication). Later, primers for 16 microsatellites were developed from species of Crocodylus
(FitzSimmons et al., 2001). Many of these primers
have proven useful for analyzing homologous
microsatellites in other species of true crocodiles;
Dever and Densmore (2001) showed that primers
developed for Crocodylus johnsoni (FitzSimmons
et al., 2001) could be successfully employed to
examine polymorphism and population structure
in C. moreletti (Dever et al., 2002). In addition,
Zucoloto et al. (2002) have recently identified and
characterized 13 microsatellite loci for the broadCaiman latirostris. Thus, microsatellite loci are
305
available as genetic markers for most species of
crocodilians (with Tomistoma, Gavialis, and Osteolamus as probable exceptions).
Mitochondrial DNA
Although mitochondrial DNA (mtDNA) has
been used extensively in studies of populations of
other taxonomic groups (Avise, ’94), and in
phylogenetic analyses (see below and Glenn et al.,
2002), few studies of variation within and among
crocodilian populations have been completed. The
complete mitochondrial genome has been sequenced for the American alligator (Janke and
Arnason, ’97; Mindell et al., ’99) and spectacled
caiman (Janke et al., 2001). Now that primers are
available to amplify the control region of crocodilians (Glenn et al., 2002; Ray and Densmore,
2002), it is possible to determine the level of
variation present in most species. Because the
substitution rate of protein coding genes is high
(similar to mammals; Janke and Arnason, ’97;
Janke et al., 2001), one would expect high levels of
intraspecific variation within most crocodilians.
However, the pattern appears to be strikingly
different for American alligators (Glenn et al.,
2002). Additionally, no mtDNA variation was
detected among Chinese alligators (X. Wu, personal communication), but this isn’t surprising
given the extreme population bottleneck that has
occurred in this species. Although not as extreme
as alligators, the pattern of low intraspecific
variation, even in domain I of the control region,
may be common to many crocodilians (N. FitzSimmons, J. Gratten, and D. Ray, personal
communication; but see Ray and Densmore, 2002
for results on domain III). Thus, mtDNA may be
better suited as a marker to identify species,
subspecies, or deep population divergences (R.
Godshalk, E. Hekkala, P. Shaw, personal communication).
Studies of population dynamics
In 1990 one of us (HCD) began examining
allozymes in offspring from alligator nest sites on
the Rockefeller Refuge to determine if multiple
paternity occurs in alligator clutches. Even with
data on only two loci, the evidence suggested that
multiple males had fathered some clutches, however the results were inconclusive. Lisa Davis, a
graduate student working with TCG and Roger
Sawyer at the University of South Carolina,
decided to see if microsatellites could answer the
question. She along with Dr. Ruth Elsey, from the
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Rockefeller Refuge, collected eggs at 22 nests and
sampled blood from the guarding females. The
eggs were incubated, and blood was taken from
the hatchlings. Lisa analyzed microsatellite alleles
at five highly polymorphic loci in offspring from an
individual clutch, along with their putative maternal parent. Multiple paternity was found in
seven of the 22 clutches. One clutch was fathered
by at least three males (Davis et al., 2001b). It is
not yet known whether multiple paternity in
alligators results from within-season multiple
matings or from sperm storage. Additional studies
using microsatellite loci to elucidate the mating
patterns of alligators and other crocodilians are
underway in several groups.
Studies of contaminants
Crocodilians, especially American alligators,
have also served as the subject of studies regarding
the effects of contaminants (Guillette et al., 2000).
In addition to elucidating specific genes involved
with contaminant response, molecular genetic
markers are being used in studies of toxicology
and genotoxicology. For example, Rotstein et al.
(2002) used microsatellite loci to determine the
fertilization status of alligator blastodisks that
failed to continue to develop. Additional studies
are underway to assess the effects of contaminants
on allele frequencies of crocodiles (J. Dever,
personal communication) and mutation rates of
microsatellite DNA loci in alligators (D. Strickland, personal communication).
MOLECULAR STUDIES OF CROCODILIAN
SYSTEMATICS
Nuttall (’04), a famous British microbiologist,
was the first scientist to publish a comparative
molecular study. Shortly after Krass discovered
the precipitin reaction, Nuttall, based on crude
precipitin reactions, predicted the relationships of
man and other primates that has passed the test of
time. He and his colleague Graham-Smith (’04)
also demonstrated the close affinities of American
and Chinese alligators and their distant affinities
to the Nile crocodile. Gorman et al. (’71), using
microcomplement fixation (MC’F), showed that
Alligator mississippiensis was more similar to
Caiman than to Crocodylus. Zoologists were
beginning to take notice that molecules could
answer tough questions in genetics and systematics.
In 1969, Charles Sibley organized the ALPHA
HELIX EXPEDITION to New Guinea. He re-
cruited both field and molecular biologists as
participants. This was the first expedition with
the primary task of collecting tissues for molecular
studies. Included among the scientists were
biochemists Alan Wilson, Alan Brush and HCD,
paleontologist Vince Sarich; and a group of highly
skilled field men, including herpetologists Dick
Zweifel and Harold Cogger; and the mammalogist
Bill Lidicker. Sibley and other ornithologists other
than Alan Brush were not about when the photo
(Fig. 1c) was taken. Thousands of specimens were
collected, and used in a variety of research
projects. Unused materials are preserved in frozen
tissue collections in California and Louisiana.
Eventually, evidence on relationsips among the
8 extant genera of the Crocodylia began to appear.
Lou Densmore (LDD) became a graduate student
in biochemistry at the LSU Medical Center in
1977. At that time there was little definitive
molecular evidence on relationships within the
order. Under the direction of Dessauer (HCD),
Densmore decided to examine crocodilian relationships for his dissertation. Unfortunately, tissues
were available only from the American alligator,
one species of caiman, and from a specimen of the
salt water crocodile, which Dessauer had obtained
while a participant on the ALPHA HELIX EXPEDITION. The principal source of material for
his study would have to come from zoos, wildlife
foundations and commercial animal farms. A
Doctoral Dissertation Research grant from the
National Science Foundation supplied Densmore
with funds to travel to such institutions to collect
blood from crocodilians (Densmore, ’83). He
contacted curators and agents of these institutions. His enthusiasm and persistence talked most
curators, after some "soul searching," into allowing
him to bleed precious, endangered crocodilians in
their care. As a result of these efforts, he obtained
blood samples from all named crocodilians except
one subspecies each of Osteolamus and Crocodylus. These blood samples were utilized in Lou’s
pioneering work and in many subsequent studies
by other investigators.
Dessauer was with Densmore when the latter
visited the Reptile Breeding Foundation in Picton,
Ontario, Canada, where two of the only accessible
true gharials in North America were housed. The
specimens were maintained in large oval tubs.
Densmore thanked the director of the Foundation
effusively, assuring him that bleeding from caudal
vessels would not harm these precious animals.
Then the highly charged Densmore climbed into
the tubs and seemingly proved just the opposite.
MOLECULAR GENETIC STUDIES OF CROCODILIANS
He was so excited, when confronted with these
valuable specimens and in the presence of his
major professor, that he failed to find the caudal
vessel. To save the day, Dessauer took over, and
(heroically) promptly obtained blood from each
animal.
Immunological methods, protein fingerprinting,
and high-resolution electrophoresis of proteins
were the only methods available to Densmore
when he undertook his dissertation research.
Major conclusions suggested by the results he
obtained with these relatively simple methods
were (Densmore, ’81, ’83; Densmore and Dessauer, ’84):
1. The Crocodylia consist of three major groups:
a Crocodile Lineage (genera Crocodylus and
Osteolamus), an Alligator Lineage (genera Alligator, Caiman, Melanosuchus and Paleosuchus),
and a Gharial Lineage (genera Gavialis and
Tomistoma).
2. Molecules from species of the genus Crocodylus are very similar, suggesting members of the
genus had resulted from a relatively recent
radiation.
3. Gavialis and Tomistoma are sister species, a
conclusion in apparent conflict with morphological
evidence.
Other molecular studies have substantiated and
enlarged on Densmore’s conclusions. Sequence
differences in the Alpha and Beta hemoglobin
chains support the relatively close affinites of
Caiman and Alligator and their more distant
affinities to Crocodylus (Leclercq et al., ’81; Perutz
et al., ’81). Immunological comparisons of albumins, using the sensitive, quantitative MC’F
technique, confirmed Densmore’s placements of
crocodilians and offered a "time frame" for their
evolution (Hass et al., ’92). Although the protein
clock is not a perfect chronometer, the MC’F data
suggest a number of reasonable estimates: the
divergence of the Crocodile and Alligator Lineages
occurred in the late Cretaceous or early Tertiary;
the divergence of the Gharial and Crocodile
lineages and the separation of alligators from the
caimans took place during the Eocene; the
separation of Gavialis from Tomistoma, and the
separation of the American and the Chinese
alligator date from the Oligocene; whereas the
radiation of species of genus Crocodylus was most
recent, probably dating from the Pliocene.
DNA comparisons are now beginning to add
additional data about several aspects of crocodilian
evolution. Unique mitochondrial gene order was
307
discovered in sequence analyses of several crocodilians by Quinn and Mindell (’96). Restriction
endonuclease fingerprints of crocodilian mitochondrial and ribosomal DNA’s were concordant with
other molecular findings (Densmore and Owen,
’89; Densmore and White, ’91; White, ’92; P.S.
White and L. Densmore, personal communication). Sequences of the 12S segment of mitochondrial DNA’s yielded comparable findings. Of
approximately 250 base positions, between 71
and 84 % were shared between Crocodylus, Caiman, Gavialis and Tomistoma whereas a maximum of 94 % were shared between Gavialis and
Tomostoma. The two gharials shared 22 unique
nucleotide sites, whereas Tomistoma and Crocodylus shared only four (Gatesy and Amato, ’92).
Hass and her colleagues (’92) repeated the Gatesy
and Amato (’92) study, adding Alligator and four
outgroup species to the earlier data set. Their
analyses, which were tested cladistically, supported the Gavialis/Tomostoma association, as
well as other relationships predicted by previous
work. However, recent work by David Ray and his
colleagues in Densmore’s lab (Ray et al., 2001)
confirms that there still is much to do in
crocodilian systematics. They report levels of
mtDNA sequence divergence within one purported subspecies of the African dwarf crocodile
(Osteolaemus tetraspis tetraspis), that are similar
or higher than interspecific (and in some cases
intergeneric) comparsions among other crocodilians. Relationships within the Crocodylia, while
perhaps better understood than ever, are far from
incontrovertible.
Conflict between morphological
and molecular data
The lack of consensus in crocodilian phylogeny
is most evident in the placement of Gavialis by
molecular biologists that is completely at odds
with conclusions of morphologists (Norell, ’89;
Tarsitano et al., ’89), including the most recent
analyses (Brochu, 2001). Among morphologists,
only Aoki (’76) ever linked Tomistoma with
Gavialis. Steven Poe (’96) in a comprehensive
analysis of all of the morphological and molecular
data available at the time, concluded that the
relationships that Densmore (’83) originally posited were still strongly supported, including the
placement of the true and false gharial. However,
a new breed of crocodilian morphologist, led by
Chris Brochu, still find characters that suggest
that Gavialis and Tomistoma are not sister-taxa
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H.C. DESSAUER ET AL.
(Brochu, 2001). Currently, both groups of scientists are attempting to resolve the conflict between
the two data sets (Brochu and Densmore, 2001).
Collaborative interactions between morphologists
and laboratory oriented scientists can be expected
to solve the problem, yielding a definitive phylogeny and a more comprehensive understanding of
the processes involved in crocodilian evolution
(Brochu and Densmore, 2001). Several explanations have been put forward. Buffetaut (’85) has
attempted to reconcile the conflict by reinterpreting morphological characters and history of the
tomostomine and gavialid lineages (however, see
Norell, ’89). Others attribute the problem in
resolution to a possible differential expression of
regulatory genes in Gavialis as compared to
Tomistoma and other crocodilians (Wilson et al.,
’77; Hass et al., ’92; Davidson, 2001). Perhaps,
analyses of nuclear sequences currently underway
in several labs (e.g., Mike Braun’s group at the
Smithsonian) will help address this difficult
problem.
Explanations of a relatively recent
radiation of genus Crocodylus
The relatively recent radiation of species of
Crocodylus and the circumtropical distribution of
the genus have caused zoogeographers to seek
explanations as to how this happened. The
presence of species of Crocodylus in four zoogeographical realms on all habitable continents
apparently cannot be attributed to continental
drift, land bridges, or to any other geological
phenomenon. Both the distances involved and the
size of crocodiles appear too great to postulate
rafting as a dispersal mechanism.
Transoceanic migration offers an intriguing but
controversial explanation (Taplin and Grigg, ’81a;
Mazzotti and Dunson, ’89). The hypothesis suggests that the distribution of Crocodylus results
from relatively recent colonizations by one or
more species of the genus. This implies long,
transoceanic migrations across highly saline
stretches of ocean. As improbable as that may
seem, there is strong evidence that it is physiologically possible. Crocodylus porosus has been
observed in Oceania some 300 km from the
nearest land (Schmidt, ’57). Dunson (’70) has
maintained Crocodylus acutus for five months in
seawater with no ill effects. Taplin and Grigg
(’81b) discovered functional salt glands in the
tongues of crocodiles, glands that can produce a
secretion two to three times the osmotic pressure
of their blood. Seemingly, these secretions could
maintain normal osmotic pressure of the blood in
an hyperosmotic environment, a critical requirement for an animal swimming across an ocean.
Crocodiles have other capabilities that may
facilitate colonization. If sperm storage is indeed
possible in Crocodylus, as has been suggested in
the caiman (Davenport, ’95), then a single migrating female can establish a colony. Additionally, if
multiple paternity occurs in Crocodylus, as in
alligators (Davis et al., 2001b), then a single clutch
of eggs deposited by a migrating female could
bring genetic diversity to the colony from herself
and multiple males. Because sex is determined by
temperature (Ferguson and Joanen, ’82; Deeming
and Ferguson, ’89; Webb and Cooper-Preston,
’89), clutches are often biased to one sex or the
other (Lang et al. ’89; Rhodes and Lang, ’96).
However, about half of all clutches are composed
of both male and female offspring (Lang et al. ’89;
Rhodes and Lang, ’96). Finally, the large numbers
of offspring allows ample opportunity for selection
of genes most suited to a new environment and/or
maintenance of diversity in the most heterozygous
individuals. Thus, crocodiles have several physiological mechanisms that allow long-range dispersal and promote the genetic diversity needed for
selection within a changing environment.
HOPEFUL FUTURE FOR ENDANGERED
CROCODILIANS
Current wildlife management studies suggest
that any endangered crocodilian can be saved from
extinction. The management program of the
Louisiana Department of Wildlife and Fisheries,
which has assured the survival of alligators in
Louisiana (Joanen and McNease, ’87, ’89), exemplifies these possibilities. The program at the
Rockefeller Refuge (in conjunction with the
LSUMC where scientists continue to come to
learn methods) is a comprehensive, long-range
effort based upon sensible wildlife management
and fundamental research. The latter involved
working with fellow scientists on problems in
population genetics (Gartside et al., ’77; Glenn
et al., ’98; Davis et al., 2001a,b), temperature
dependent sex determination (Ferguson and Joanen, ’82), reproductive endocrinology (Vance, ’89),
and biochemistry, growth and nutrition (Coulson
and Hernandez, ’64, ’83; Coulson RA et al., ’87,
’89; Coulson TD et al., ’73).
Studies at the LSUMC have shown that alligators grow rapidly in captivity. Newborn American
MOLECULAR GENETIC STUDIES OF CROCODILIANS
alligators, maintained in tanks at a constant
temperature of 311C and fed ad lib, grow steadily
and average about four feet long by the end of one
year in captivity (Coulson TD et al., ’73; Coulson
RA et al., ’89). Chinese alligators under the same
conditions grow well but their appetites and
growth rates are influenced by the photoperiod
(Herbert et al., 2002). Elsey and her colleagues
reported that alligators, raised in captivity for a
year or two and then released into the wild, grow
faster than wild alligators, and many of the
females nest before they are six or seven years
old (Elsey et al., 2001a,b).
The biological problems associated with the
conservation of endangered crocodilians can be
solved using knowledge of other crocodilians as a
solid foundation. Unfortunately, the economic and
social issues associated with crocodilian conservation can be more vexing (Thorbjarnarson, ’99;
Hutton et al., 2001). We hope that the information
gained during the successful recovery of American
alligators and the current popularity of crocodilians can inspire future workers to solve the
societal issues needed to conserve all crocodilian
species.
CONCLUSION
The future for crocodilian molecular studies is
brighter than it has ever been. Advances in
methodology such as analyses of single nucleotide
polymorphisms (SNPs) and automated analyses of
microsatellites and DNA sequencing mean that
many studies of highly endangered species such as
Alligator sinensis, Crocodylus cataphractus, C.
intermedius and C. mindorensis are now feasible.
Such work is beginning or already in progress in
established labs in Florida, Louisana, Maryland,
New Mexico and Texas, as well as in Australia and
the Peoples’ Republic of China. However, it is the
new generation of bright, young molecular evolutionary biologists like Lisa Davis, Jennifer Dever,
Nancy FitzSimmons, Jacob Gratten, Evon Hekkala, Fred Pontillas, David Ray, Wade Ryberg,
Luciano Verdade, and Rodrigo Zucoloto that will
be carrying the ‘‘torch’’ of crocodilian population
genetics and molecular systematics into the 21st
century. From where we stand, and as evidenced
by the Crocodilian DNA Workshop in San Diego,
that torch is in very good hands.
ACKNOWLEDGMENTS
We thank the many colleagues who have worked
with and tolerated us over the years. It is difficult
309
to limit the list of people who should be recognized, but we wish to especially recognize the
efforts of some individuals not sufficiently mentioned above: T. Joanen, L. McNease, R. Elsey and
staff at the Rockefeller Wildlife Refuge, I. L.
Brisbin Jr., W. Stephens and staff at the Savannah
River Site; W. Rhodes and the staff at Santee
Coastal Reserve, R. Sawyer at the University of
South Carolina, Rol and Tom Coulson and Tom
Hernandez at the LSU Medical School, the many
curators at zoos, as well as personnel at both
private farms and state wildlife refuges who
allowed us access to their facilities, and the
farmers, students, trappers, and volunteers who
have assisted us in collecting samples. Last, but
not least, we thank our families who have given us
so much support over the years, tolerating everything from temporary storage of blood samples in
refrigerators, to three week collecting trips in the
‘‘field,’’ to many long hours in the lab.
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