High levels of genetic variability in west African Dwarf Crocodiles Osteolaemus tetraspis getraspis DAVID A. RAY,LP. SCOTT WHITE,2 HUYEN V. DUONG,l T. CULLEN3 and LLEWELLYN D. DENSMORE1 The African Dwarf Crocodile (Osteolaemus) has been a long-standing problem for crocodilian systematists. Previously divided into separate genera, present forms are currently recognized as two subspecies within the single named species, 0. tetraspis. We sequenced a 350 bp region of mitochondrial DNA in an attempt to elucidate the relationships within one of these forms, 0. t. tetraspis. Results indicate at least two distinct and well-supported groups with nucleotide sequence divergence levels comparable to those found between species of other crocodilians. These data lay the groundwork for a comprehensive systematic and population study of the genus. Key words: Osteolaemus, Crocodylia, mtDNA, systematics. INTRODUCTION number of Osteolaemus tetraspis populations in Liberia has dropped significantly in recent and crocodilians represent the only BIRDS years (Kofron 1992). To complicate matters, extant descendants of the ancient group the taxonomic status of forms currently placed known as Archosaurs. Modern crocodilians in the genus has been a source of dis- first appeared about 80 million years ago and agreement among crocodilian systematists radiated into over 125 genera. Of those, only since the early 20th century. eight survive in the present day. These genera are commonly grouped into three families, The genus is identified today as consisting the Alligatoridae, the Crocodylidae, and the of a single species and two subspecies - Gavialidae. The taxonomy and phylogenetics 0. t. tetraspis and 0. t. osborni (Brazaitis 1973). of many of these groups have been well Unfortunately, no unequivocal 0. t. osborni are studied (Poe 1996; Brochu 1997). However, known to exist in collections in the United with the exception of the American alligator States and, at the time samples were collected A. mississibbiensis verv little work on crocodilian for this study, permits allowing the import 11 , population genetics has been done. This of bIood were not in our possession. We deficit is currently being remedied by a number were, therefore, forced to limit this study to of surveys of several members of genus 0. t. tetraspis. However, as revealed by several Crocodylus (C. acutus, C. rhombt$e;fel; C. moreletti, previous molecular analyses also involving C. johnstoni and C. porosus). Studies of several members of this subspecies (Densmore and species of caiman have also been proposed. Owen 1989; Densmore and White 1991), substantial intraspecific genetic variation The genus Osteolaemus, to which the dwarf African crocodile belongs, is generally appears to be the ~le.In a more recent study considered the sister taxon to the genus (White and Densmore, unpubl. data) based on sequence analysis of the ND6-tRNAgIu-Cyt b Crocodylus. Dwarf crocodiles are classified as region of crocodilian mtDNA, considerable endangered and virtually nothing is known nucleotide divergence (0.098) was noted about the status of wild populations across between two individuals of 0.t. tetraspis. their range (Fig. 1) (Kofron and Steiner 1994). The animals are used for food and the As an extension to the findings of White hides utilized by native people for some and Densmore (unpubl. data), the same 350 products (Ross 1998), but the effects of base pair region of mitochondrial DNA was hunting pressure on dwarf crocodile numbers sequenced for 10 individuals of 0. t. tetraspis is unknown. Deforestation may also be taking as well as for a single individual of Crocodylus a toll. One study has suggested that the rhombifer. This region was chosen because there 'Department of Biological Sciences. Texas Tech University, Lubbock. TX 79409 USA. 'Life Sciences Division, Los Namos National Laboratory. Los Namos. NM 87545 USA. 'Cullen Vivarium. P.O. Box 878. Milwaukee. WI 52301 USA. Pages 58-69 in CROCODILIAN BIOLOGY AND EVOLUTION ed by Gordon C. Crigg. Frank Seebacher and Craig E. Franklin. Surrey Beatiy & Sons. Chipping Norton. 2000. RAY ET AL.: GENETIC VARIABILITY IN WEST AFRICAN DWARF CROCODILES 5 9 Total DNA was isolated from the blood samples using the SDS-Urea method of White et al. (1998). MtDNA regions including a portion of ND6, the entire tRNAg'" gene, and a portion of cyt b were amplified using two rounds of PCR. The first round yielded a product of -2000 bp. A smaller fragment, -350 bp, was then amplified in the second round using the larger fragment as a template. In either case, a master mix containing 500pl ddH20, lOOpl 10 mM dNTP mix, 90p1 10X Tag buffer (Fisher), and 100 p1 25 mM MgC12 solution (Fisher) was made. To a 0.5 mi PCR reaction tube were added 48p1 of the master mix, 4 p1 of template (7-10pM) DNA, and 3.0 pl of a 20 pM mixture of primers (Table 1). Taq polymerase (2.5 units) was added directly to the reaction vessel just before spinning down the contents in a microcentrifuge. For both first and second round ampli- Fig. I. Geographic range of Osteolaemus tetraspis (Ross fication reactions, samples were overlain with 1998). mineral oil, followed by an initial denaturation step at 94°C for three minutes. First round appears to be sufficient sequence variability amplification was performed using the follow- among crocodilian species to be sensitive ing cycle parameters: 94°C for 1 min., 50°C enough for population level studies, while the for 1 min., and 72°C for 90 sec.; 35 cycles. conserved regions are suitable for outgroup The smaller size of the second round product comparison (White and Densmore, unpubl. and a desire to decrease reaction time and data). As stated earlier, no 0. t. osborni were increase primer fidelity prompted the follow- available for analysis. However, if large ing changes to cycle parameters for the amounts of nucleotide sequence variation exist second amplification: 94°C for 15 sec., 53°C within the subspecies examined, we can likely for 30 sec., and 72°C for 50 sec.; 35 cycles. assume that larger, more significant differences A Perkin Elmer-Cetus DNA thermal cycler probably exist between the two currently (Branchburg, New Jersey) was used for both recognized forms. sets of reactions. Amplification was verified on 0.8% agarose gels, after which first and MATERIALS AND METHODS second round products were purified using the Qiagen (Santa Clarita, California) gel Blood from 10 individuals of 0. t. tetraspis was collected in acid citrate dextrose-B purification protocol. (ACD-B) (Densmore and White 1991) at the Upon isolation of the -350 bp products, Cullen Vivarium (Y4, Y 10, Y 11, Y 13, Y16, automated sequencing was performed using Y19, Y20), the St. Augustine Alligator Farm an ABI PRISM Model 310 and Amplitaq (FT), and from the collection of Bruce DNA polymerase FS (Perkin Elmer) at the Schwedick (1A and 2A). Animals Y10 and core laboratory of the Texas Tech Institute Y19 are known to have been collected in the of Biochemistry. The primary sequencing wild from Gabon. Unfortunately, the original primers were ND6L and CytB2Hint (Table 1). collection locales for the remaining Osteolaemw All sequences were aligned using ClustalW samples are not known. One blood sample (Thompson et al. 1994) with manual adjust- from C. rhombifer was obtained from the St ments. We included known DNA sequence of Augustine Alligator Farm. A. mississippiensis from White 1992; White and Table 1. Primer sequences for first and second round amplification reactions (courtesy of White and Densmore (unpubl. data)). 1st Round Primers Primer sequence' CB2H 5'-CCCTCAGAATGATATITGTCCTCA-3' ND5L2 5'-GCCCTACTNCAYTCNAGCACAATAGT-3' 2nd Round Primers CBPHint 5'-TTTCATCATGCNGARATGTTKGATGGGGY KGRAGGTG3' NDGL 5'-TATTTRGGNGGNATGSTGGTNGTNTITG-3' ' Degenerate base codes: R = A,G; Y = C,T; S = C,G; W = A,T; K = G,T; M = A,C; H = A,C.T, B = C,G,T; V = A,C,G; D = A,G,T; N = A,C,G,T - ~- GKOCODILIAN BIOLOGY AND EVOLUTION Densmore (submitted) for use as an additional Nei (1984) in order to allow comparisons with outgroup taxon. PAUP* v. 4.0bl (Swofford genetic distances calculated for sequence data 1998) was used to generate genetic distances from the same region from other crocodilians for phenogram construction and to perform studied by White 1992; White and Densmore parsimony analyses for estimating phylogeny. (submitted). Pairwise genetic distances from this analysis are presented in Table 2. Using RESULTS the Tajima-Nei distance matrix, a neighbour- joining analysis was performed yielding one Using the NDGL primer, we were able to tree (Fig. 2). consistently produce sequences that were clear and repeatable; other sequencing primers In addition, a maximum parsimony (phylo- genetic) analysis was performed. Of the 297 proved less reliable. Aligned DNA sequences total characters, 36 were determined to be are presented in the appendix. phylogenetically informative. An exhaustive The aligned sequences were used to estimate search was performed and 17 equally several of the measures of genetic distance parsimonious trees (score = 123) resulted. A available through PAUP (Tajima-Nei, Jukes- strict consensus phylogram of these produced Cantor, Kimura 3-parameter, and Tamura- one unresolved polytomy. Bootstrap analysis Nei). All produced similar distance matrices. with 1 000 replications produced a single tree We chose to use the algorithm of Tajima and with the same topology (Fig. 3). Table 2. Genetic distance matrix for all sequences. Distances were calculated using the algorithm of Tajima and Nei (1984). All