JOURNAL OF EXPERIMENTAL ZOOLOGY 309A (2008) A Journal of Integrative Biology Evidence of Multiple Paternity in Morelet’s Crocodile (Crocodylus moreletii) in Belize, CA, Inferred From Microsatellite Markers JOHN D. MCVAY1, DAVID RODRIGUEZ1, THOMAS R. RAINWATER2, JENNIFER A. DEVER3, STEVEN G. PLATT4, SCOTT T. MCMURRY2, MICHAEL R.J. FORSTNER5, AND LLEWELLYN D. DENSMORE1 1 Department of Biological Sciences, Texas Tech University, Lubbock 2 The Institute for Environmental and Human Health, Texas Tech University, Lubbock 3 Department of Biology, University of San Francisco, San Francisco 4 Department of Biology, Sul Ross State University, Alpine 5 Department of Biology, Texas State University, San Marcos ABSTRACT Microsatellite data were generated from hatchlings collected from ten nests of Morelet’s Crocodile (Crocodylus moreletii) from New River Lagoon and Gold Button Lagoon in Belize to test for evidence of multiple paternity. Nine microsatellite loci were genotyped for 188 individuals from the 10 nests, alongside 42 nonhatchlings from Gold Button Lagoon. Then mitochondrial control region sequences were generated for the nonhatchlings and for one individual from each nest to test for presence of C. acutus-like haplotypes. Analyses of five of the nine microsatellite loci revealed evidence that progeny from five of the ten nests were sired by at least two males. These data suggest the presence of multiple paternity as a mating strategy in the true crocodiles. This information may be useful in the application of conservation and management techniques to the 12 species in this r 2008 Wiley-Liss, Inc. genus, most of which are threatened or endangered. J. Exp. Zool. 309A, 2008. How to cite this article: McVay JD, Rodriguez D, Rainwater TR, Dever JA, Platt SG, McMurry ST, Forstner MRJ, Densmore LD. 2008. Evidence of multiple paternity in Morelet’s Crocodile (Crocodylus moreletii) in Belize, CA, inferred from microsatellite markers. J. Exp. Zool. 309A:[page range]. Morelet’s Crocodile (Crocodylus moreletii) is a relatively small-bodied member of its genus, native to southern Mexico, Belize, and Guatemala (Britton, 2002). Owing to threat from habitat loss and hunting, C. moreletii is currently listed on the IUCN Red List as a lower risk, but conservationdependent species (IUCN, 2006). Because of its threatened status, extensive research has been published on this species’ population genetics (Dever and Densmore, 2001; Dever et al., 2002; Ray et al., 2004). An important contribution to the conservation of a species is knowledge of its breeding strategies. Multiple paternity has been shown theoretically to increase effective population size (Sugg and Chesser, ’94), thus potentially increasing the overall genetic diversity of a population, particularly those populations that have recently underr 2008 WILEY-LISS, INC. gone a genetic bottleneck. Evidence of multiple paternity in offspring has been detected in a wide variety of invertebrates (e.g., Fjerdingstad et al., ’98; Grant sponsor: National Geographic Society; Grant number: 652999; Grant sponsor: National Science Foundation BSR; Grant number: 0444133; Grant sponsor: Environmental Protection Agency; Grant number: R826310; Grant sponsors: Royal Geographic Society; ARCS Foundation, Lubbock, TX; The Wildlife Conservation Society; Texas State University Graduate School Pre-doctoral Summer Fellowship; Texas Tech University Graduate School Summer Dissertation Research Grant. Submitted for the special volume of the 3rd International Workshop on Crocodylian Genetics and Genomics in JEZ-A (Ecological Genetics and Physiology). Correspondence to: John McVay, LSU Department of Biological Sciences, 107 Life Science Building, Baton Rouge, LA 70803. E-mail: jmcvay1@lsu.edu Received 16 July 2007; Revised 3 July 2008; Accepted 18 August 2008 Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/jez.500 2 J D. MCVAY ET AL. Paterson et al., 2001; Good et al., 2006) and vertebrates (e.g., Valenzuela, 2000; Tennessen and Zamudio, 2003; Waser and De Woody, 2006) using multilocus microsatellite data. Within the order Crocodylia, multiple paternity has been detected in the American Alligator (Alligator mississippiensis) (Davis et al., 2001), but has not been reported within the true crocodiles, Crocodylidae. FitzSimmons et al. (2001)developed the first microsatellite library for Crocodylus in the Australian Freshwater Crocodile (C. johnstoni) and the American Crocodile (C. acutus). Dever and Densmore (2001) later found a higher level of polymorphism present in several loci in C. moreletii than in C. johnstoni, the species for which some of the markers were originally developed. This study incorporates multiple parentage analyses (Jones and Ardren, 2003) of these previously developed and characterized microsatellite loci to test for evidence of multiple paternity within two populations of Morelet’s Crocodile in Belize, Central America. Our goal is to increase the knowledge of mating strategies in this wild crocodilian and help to improve management options for this and possibly other species of Crocodylus, a number of which are critically endangered (IUCN, 2006). MATERIALS AND METHODS Sample collection: From 1998 to 2000, as part of ongoing studies of the status, ecology, and ecotoxicology of Morelet’s Crocodile (Platt, ’96; Platt and Thorbjarnarson, 2000; Platt et al., 2000, 2006; Rainwater, 2003; Wu et al., 2006; Rainwater et al., 2007, 2008), crocodile nests were located and monitored at two localities in northern Belize, Gold Button Lagoon (GBL), and New River Watershed (NRW). Gold Button Lagoon (171550 N, 881450 W) is a large man-made lagoon located on Gold Button Ranch, a 10,526 ha private cattle ranch approximately 25 km southwest of Orange Walk Town, Orange Walk District. New River Watershed (171420 N, 881380 W) comprises the New River, New River Lagoon (NRW), and associated tributaries in the Orange Walk and Corozal Districts. To reduce the loss of nests to flooding, nests were checked after each rain event to monitor the threat of rising water levels. If nests were in jeopardy of being flooded, entire clutches were transported to the Lamanai Field Research Center (Indian Church Village, Orange Walk District), placed in field incubators (Rhodes and Lang, ’95), and maintained in natural nest J. Exp. Zool. material until hatching. Upon hatching, each neonate was measured, marked, and maintained in captivity for 2–4 weeks, after which sex was determined by cloacal examination of the genitalia (Allsteadt and Lang, ’95). A blood sample (ca. 0.5 mL) was collected from the post-cranial sinus, transferred to an ethylenediaminetetraacetic acid (EDTA)-treated Vacutainers, and centrifuged at 2,000 rpm for 10 min. The plasma supernatant was transferred to a collection tube and both it and the remaining packed cells were frozen at 251C until shipment to Texas Tech University for storage at 801C until analysis. Following sample collection, each hatchling crocodile was released into dense aquatic vegetation at its respective nest site. Data collection: DNA was extracted from blood using the PUREGENEs genomic DNA purification kit (Gentra Systems, Minneapolis, MN). Nine microsatellite loci (Table 1) were amplified via PCR using the Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany) or MJ Research PTC-200 (MJ Research, Inc., Waltham, MA) for 188 individuals representing one nest from NRL and nine nests from GBL. PCR reactions were as follows: 5–20 ng template DNA, 1.25 pmol each primer, 0.625 nmol dNTPs, 1  PCR buffer, 0.31 U of Taq polymerase, and 9.32 mL of ddH2O in a 12.5 mL reaction. A standard thermocycling protocol of 35 cycles was used (see Table 1 for annealing temperatures). Products with WellRED-labeled forward primers were analyzed and peaks sizes were recorded using a CEQTM 8000 or 8800 Genetic Analysis System (Beckman Coulter, Inc., Fullerton, CA). Blood samples from 42 nonhatchlings from GBL collected during the course of the field work were also genotyped for the same markers to establish estimates of population allele frequencies. Because Ray et al. TABLE 1. Microsatellite loci used in the evaluation of multiple paternity in Crocodylus moreletii Primer C391 Cj16 Cj20 Cj109 Cj127 Cj128 Cj131 CU5-123 Cuj131 No. alleles Size range (bp) C1A Ho He 7 1 5 4 4 3 1 3 1 146–181 133 151–179 364–382 338–356 228–234 210 206–212 183 58 62 62 62 58 58 58 58 58 0.795 0 0.707 0.5 0.488 ? 0 0.405 0 0.737 0 0.662 0.421 0.532 ? 0 0.406 0 Size ranges are those detected in this study. All primers are from FitzSimmons (2001). C1A 5 annealing temperature of each primer pair; Ho 5 observed heterozygosity; He 5 expected heterozygosity. MULTIPLE PATERNITY IN MORELET’S CROCODILE (2004) found evidence of hybridization with the American Crocodile (C. acutus) within putative populations of C. moreletii in Belize, we searched for alleles that were found by Rodriguez et al. (this volume) to be exclusive to C. acutus. Additionally, a 540 bp region of the mitochondrial control region was amplified for a single individual from each nest, and for the nonhatchlings, to test for evidence of haplotypes containing base-pair changes unique to C. acutus. Reactions were 25 mL with the same reagent concentrations and conditions (581C annealing temperature) as for the microsatellite loci. PCR products were purified using QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA). Bidirectional cycle sequencing reactions were performed using Big Dye 3.1 (Applied Biosystems, Inc., Foster City, CA), and excess dye terminator was removed from the reactions using sephadex gel columns; also see Rodriguez et al. (this volume). Products were then sequenced on an ABI-Prism 3100-Avant Genetic Analyzer (Applied Biosystems Inc.). Sequences were edited and aligned using Sequencher 4.1 (Gene Codes, Ann Arbor, MI). Data analyses: Allele counts and genotypes were used to assess the presence or absence of more than two parents in each nest. Genepop 3.4 (Raymond and Rousset,’95) was used to test for (1) linkage disequilibrium among within the GBL population, using a single individual from each nest and the 42 nonhatchlings and (2) heterozygote deficiency for each locus within each nest to test for the presence of null alleles. We calculated observed and expected heterozygosity for each locus using the 42 nonhatchlings and one hatchling from each GBL nest. Potential parental genotypes were reconstructed and the minimum number of fathers was inferred for each nest using GERUD 2.0 (Jones, 2005). GERUDsim 2.0 (Jones, 2005) was utilized to test the ability of GERUD to infer the correct number of fathers. Because the allele frequencies of each nest were found to be highly significantly different from each other (data not shown), 10,000 simulated nests were generated for the allele frequencies and hatchling number of each nest. Each simulation was given the parameters of two fathers, with the proportion of contribution by each father taken from the minimum father solution from GERUD with the highest likelihood. For those nests determined to have been sired by one male, the contribution proportion was an average of the primary and secondary contributions of those nests with two fathers. We then performed simulations (1,000 3 iterations) with three fathers for each nest, at a ratio of 20:5:5 for the contribution the 1, 2, and 3 degree fathers. Average relatedness coefficients (Queller and Goodnight, ’89) within nests from GBL were calculated using SPAGeDi (Hardy and Vekemans, 2002), using the genotypes of 42 nonhatchlings from GBL as reference allele frequencies. Kinship (Goodnight, 2000) was then used to test for significance of half-sibling relationships (rm 5 0.5, rp 5 0) among individuals within nests, where the null hypothesis is a full sibling relationship (rm 5 0.5, rp 5 0.5). RESULTS Six of nine microsatellite loci amplified in this study were polymorphic. The three monomorphic loci (Cj16, Cj131, Cuj131) were uninformative and were thus not included in the analysis. One polymorphic locus, Cj128, was deemed unreliable owing to the presence of more than two peaks in a large number of individuals and was also excluded. Testing for linkage disequilibrium yielded no significantly linked loci after Bonferroni correction; likewise, heterozygote deficiency was not detected for any locus within any nest. GERUD inferred more than one father for six of the ten nests examined. However, one nest (GBL-8) contained a single individual whose alleles at a single locus (Cj20) were not consistent with the null hypothesis of a single sire for the clutch. Following FitzSimmons (’98), this genotype was attributed to a mutation, most likely a null allele, at that locus in that individual, and not evidence of multiple paternity. Among the nests with a minimum of two fathers, the ratio of contributions by the primary and secondary father was approximately 4:1; this number was used in the GERUDsim analyses for the single father nests. Results of the relatedness, Kinship, GERUD, GERUDsim analysis are shown in Table 2. A single control region haplotype was detected among all individuals, identical to a haplotype found by Ray et al. (2004). Likewise, microsatellite alleles designated by Rodriguez et al. (this volume) as being specific to C. acutus were not detected in any of the individuals. DISCUSSION Results of this study indicate a sizable presence of multiple paternity in Morelet’s Crocodile, being detected in half of the nests examined; a rate higher than that found by Davis et al. (2001) in Alligator. Moreover, given the low levels of J. Exp. Zool. 4 J D. MCVAY ET AL. TABLE 2. Tabulated results of the analyses of five microsatellite loci in Crocodylus moreletii Nest ID N R   Nm 11 L GBL-1-1998 GBL-1-2000 GBL-4 GBL-5 GBL-6 GBL-8 GBL-9 GBL-12 12 28 30 15 19 17 20 18 0.3137 0.31 0.3049 0.2275 0.2422 0.3719 0.2432 0.1076 0.02 0 0.12 0.07 0.08 0.04 0.05 0.28 0 0 0 0 0 0 0 0.02 2 1 1 1 2 2 1 2 7 – – – 15 16 – 16 C391 GBL-13 NRL-1 10 19 0.424 N/A 0.11 N/A 0 N/A 2 2 9 15 Cj20 Cj202 Cj20, Cj127 C391 Cj20, Cj109 N 5 number of hatchlings; R 5 relatedness coefficient (SPAGeDi); , 5 proportions of pairwise Kinship half-sibling likelihoods with significance of Po0.05 and Po0.001, respectively; Nm 5 minimum number of fathers inferred by GERUD. 11 5 number of offspring assigned to the primary father in the most likely parent pair from GERUD; L 5 microsatellite loci responsible for detection of multiple sires in GERUD. 2 Results from this nest consistent with single sire and null allele at single locus in one individual. polymorphism, multiple paternity may actually be present at an even higher rate in this species and simply not detected as a consequence of shared alleles among fathers. Results of the GERUDsim simulations suggest that the power of GERUD in this particular analysis is low (ability to correctly detect two fathers was less than half in nine of ten nests simulated; nests with three fathers were correctly designated very rarely) supporting the speculation that the actual incidence of multiple paternity is higher than we detected. Additionally, the low levels of relatedness within nests suggest a higher rate of multiple paternity; however, insufficient sampling of the underlying population allele frequencies may be as likely a cause for these results. And although the mitochondrial data suggest that these nonhatchlings may be related themselves, an accurate relatedness estimate is difficult to obtain without a better estimate of the underlying polymorphism in the population. The results of the likelihood analysis shown in Table 2 also show significant half-sibling relationships in eight of nine nests. However, we feel the multiple pairwise comparisons reduce the significance and results should be viewed cautiously. From a more conservative position, only one of nine nests showed significance at Po0.001. This does not lend support to the GERUD results, however, potential relatedness of these nonhatchlings may have affected the outcome of this analysis. J. Exp. Zool. Of the three polymorphic loci applied in both this study and Dever et al. (2002), allele counts and frequencies for loci Cj109 and Cj20 were very similar; our study detected two fewer alleles (both rare) in Cj127. The low overall variation among loci has potentially limited our ability to detect polyandry, and has prohibited a robust parentage analysis. The need for further population genetic studies in this species warrant the construction of a new microsatellite library specific to this taxon. These samples were collected for use in an ecotoxicological study (Rainwater, 2003), and thus collection schemes were not tailored for a population genetic study. Jones (2005) pointed out that GERUD 2.0 can estimate parental genotypes of half-sib progeny arrays with equal accuracy, with or without a given maternal genotype. Nonetheless, without the maternal genotype, it is impossible to rule out multiple females sharing nests, given the results of the current study. However, although the use of a single nest by multiple female crocodilians has been previously reported (reviewed by Platt et al., 2004), to our knowledge it has never been observed in C. moreletii (Platt et al., 2008). Indeed, in over eight years of research on the nesting ecology of C. moreletii at GBL and NRW, no evidence has been found to suggest that multiple females deposit eggs in the same nest (Platt et al., 2008; Rainwater, personal observation). Nest sharing by multiple female crocodiles would likely be discernable by unusually large numbers of eggs in the nest chamber as well as a bimodal distribution in egg width (i.e., egg width of a clutch correlates with the size of the nesting female; thus, clutches from different females can be identified by differences in mean egg width) (Platt and Thorbjarnarson, 2000; Platt et al., 2004, 2008). Although the latter has been observed in C. acutus in the coastal zone of Belize (Platt and Thorbjarnarson, 2000), neither has been observed in over 100 C. moreletii nests examined at GBL and NRW, including the nests from which hatchlings sampled in this study originated. The lack of diversity among the maternally inherited control region sequences from these populations eliminates the likelihood of finding genetic sequence evidence of multiple maternity. Although we are not surprised by the presence of polyandry in this species, the levels are higher that initially suspected, based on the results from Davis et al. (2001). It remains to be determined whether multiple paternity is the result of multiple matings within a single mating period, or MULTIPLE PATERNITY IN MORELET’S CROCODILE because of female sperm storage, a strategy discovered very recently in A. mississippiensis (April Bagwill, personal communication). Regardless, it can be concluded that multiple paternity is a significant characteristic of the breeding strategy of Morelet’s Crocodile, a characteristic also recently found in captive and wild populations of the Indo-Pacific Crocodile, C. porosus (FitzSimmons, personal communication; Lewis et al., this volume). Clearly, this strategy is shared across a wide variety of taxa (a literature search will produce more than 100 articles published after 2005 on this topic). It is yet to be determined whether multiple paternity is an ancestral strategy common to the extant Crocodylia, or has arisen independently in these taxa. This is an important topic of study, because multiple mating may increase a population’s recovery rate after a bottleneck or large loss of genetic diversity. More thorough characterizations of both the mating system and sperm storage in this species are necessary for proper implementation of conservation and management techniques: an important task, as these data may be a particularly useful tool in the captive management or wild population recovery efforts in the Crocodylus species currently critically endangered. ACKNOWLEDGMENT Mark and Monique Howells and their staff at the Lamanai Field Research Center provided invaluable logistical support during the collection of samples. S. San Francisco and C. Conkelton were particularly helpful with the laboratory portion of this study. Revisions were improved through discussions with Z. Cheviron and M. Carling. This study was funded in through the National Geographic Society ] 6529-99 (L. D. D.); National Science Foundation BSR 0444133 (L. D. D.); Environmental Protection Agency ]R826310 (S. T. M.); Royal Geographic Society (S. T. M.); ARCS Foundation, Lubbock, TX (T. R. R.); The Wildlife Conservation Society (S. G. P.); Texas State University Graduate School Pre-doctoral Summer Fellowship (D. R.), and a Texas Tech University Graduate School Summer Dissertation Research Grant (D. R.), and by the Department of Biology, Texas State University and the Department of Biological Sciences at Texas Tech University. Samples were collected under the following permits: Belize Export Permit CD/72/2/ 99/19; CITES Export Permit 001078; CITES Import Permit 99US812795/9 and 01US019090/9. 5 LITERATURE CITED Allsteadt J, Lang JW. 1995. Sexual dimorphism in the genital morphology of young American Alligators, Alligator mississippiensis. Herpetologica 51:314–325. Britton A. 2002. 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