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 304 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 306 H.C. DESSAUER ET AL. 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 308 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. LITERATURE CITED Adams SE, Smith MH, Baccus R. 1980. Biochemical variation in the American alligator. Herpetologica 36:289–296. Aoki R. 1976. On the generic status of Mecistops (Crocodylidae), and the origin of Tomistoma and Gavilis. Bull. Atagawa Inst. 6l–7:23–30 (In Japanese). Avise JC. 1994. Molecular Markers, Natural History and Evolution. New York:Chapman & Hall. Boyden A. 1951. 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