Conserv Genet (2012) 13:1677–1683 DOI 10.1007/s10592-012-0406-2 SHORT COMMUNICATION Testing for hybridization and assessing genetic diversity in Morelet’s crocodile (Crocodylus moreletii) populations from central Veracruz Ricardo González-Trujillo • David Rodriguez • Alberto González-Romero • Michael R. J. Forstner Llewellyn D. Densmore III • Vı́ctor Hugo Reynoso • Received: 6 March 2012 / Accepted: 25 August 2012 / Published online: 26 September 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Among the loss of genetic diversity due to population declines, population fragmentation and habitat loss, hybridization also stands as a threat to Morelet’s crocodile (Crocodylus moreletii) populations. Genetic surveys in Belize and the Yucatan Peninsula have detected evidence of hybridization with the American crocodile (C. acutus). Admixture between these two species is most likely driven by human-mediated translocations. Along the central gulf coast of Mexico, C. moreletii populations are presumed to be purebred. To test this, we use nine microsatellite loci and sequence data from the mitochondrial control region to detect if C. acutus alleles have introgressed into populations of C. moreletii from central Veracruz. In 2010, C. moreletii was transferred from Electronic supplementary material The online version of this article (doi:10.1007/s10592-012-0406-2) contains supplementary material, which is available to authorized users. R. González-Trujillo
A. González-Romero Red de Biologı́a y Conservación de Vertebrados, Instituto de Ecologı́a A.C., AP 63, 91070 Xalapa, Veracruz, Mexico D. Rodriguez Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853-2701, USA M. R. J. Forstner Department of Biology, Texas State University-San Marcos, San Marcos, TX 78666, USA L. D. Densmore III Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, USA V. H. Reynoso (&) Departamento de Zoologı́a, Instituto de Biologı́a, Universidad Nacional Autónoma de México, 04510 Mexico, DF, Mexico e-mail: vreynoso@ibiologia.unam.mx Appendix I to II of CITES based on a whole species demographic analysis, which indicated that populations had recovered across its range. Our study shows that populations in central Veracruz are purebred, although they exhibit low levels of genetic diversity most likely caused by inbreeding. Our data also suggest there is fragmentation among populations of C. moreletii, which may lead to further loss of genetic variation. Due the purity and low genetic diversity of C. moreletii populations from central Veracruz, we recommend increased protection and active management practices that take genetic data into account. Keywords Morelet’s crocodile
Microsatellites
mtDNA
Hybridization
Introgression
Genetic diversity
Mexico Introduction Human-mediated hybridization, loss of genetic diversity, small population sizes, and loss of gene flow because of decreased habitat connectivity, are major threats to Morelet’s crocodile populations (Cedeño-Vázquez et al. 2008; Rodriguez et al. 2008). Hybridization is a natural phenomenon that contributes to evolutionary processes such as speciation; however, in recent years, anthropogenic factors have disrupted natural breeding barriers facilitating contact among allopatric species (Olden et al. 2004; Shaddick et al. 2011). Species translocations, a common practice among wildlife managers, can threaten the purity of local populations (Rodriguez et al. 2008) by increasing the probability of hybridization and introgression. Translocations can produce hybrid swarms, which can lead to genetic extinction of the parental species (Rhymer and Simberloff 1996; Schlaepfer et al. 2005). Hybrids present a further threat 123 1678 because they can compete for the same ecological resources as the parental species (Chunco et al. 2012). When working with endangered species there is no official policy guideline for dealing with hybrids (Allendorf et al. 2004), and management strategies depend on whether the hybrids arose naturally or due to anthropogenic factors. For example, Rodriguez et al. (2011) suggested removing hybrids from the wild when they resulted from escaped heterospecific individuals. In practice, Mexican environmental agencies do not remove hybrids that have arisen through natural hybridization among sympatric species (Sánchez et al. 2011). They do relocate non-natural hybrids and confiscated crocodiles to Units of Management for Wild Life Conservation (UMAs) to prevent adverse effects on populations of native fauna (SEMARNAT 2012). This has resulted in the pooling of animals of mixed genetic ancestry coming from unknown sites. UMAs are not always secure and escapes have occurred in the past (Villegas and Reynoso 2010). Therefore, tests for hybridization should be a standard part of management plans for crocodile populations. Species within the genus Crocodylus are known to readily hybridize in captivity (Fitzsimmons et al. 2002; Weaver et al. 2008). Morelet’s crocodile (Crocodylus moreletii) and the American crocodile (C. acutus), which are sympatric in the Yucatan Peninsula and Belize, have been shown to hybridize in the wild despite preferring different habitats (Ray et al. 2004; Cedeño-Vázquez et al. 2008; Machkour-M’Rabet et al. 2009; Rodriguez et al. 2008). Hekkala (2004) suggested that the hybrid zone is localized in Belize, but other studies using mitochondrial DNA (Cedeño-Vázquez et al. 2008) and microsatellites (Rodriguez et al. 2008) reported that hybrids were also detected in the Yucatan Peninsula. Furthermore, it is possible that marine currents around the Yucatán Peninsula could also push C. acutus individuals to other areas where only C. moreletii is found, increasing the hybridization area between both species (Machkour-M’Rabet et al. 2009). Because hybridization and translocations are common in Mexico, we expect that the hybridization zone is larger than previously thought, but without detailed sampling along the western Gulf coast of Mexico, namely Veracruz and Tamaulipas, we cannot delineate the boundary of the natural hybridization zone. In this study, we use both nuclear (microsatellites) and mitochondrial (control region sequences) markers to test whether C. acutus alleles have introgressed into populations of C. moreletii in central Veracruz. In addition to hybridization, hunting and population fragmentation can reduce population sizes enough to decrease levels of genetic diversity in future generations and cause bottleneck effects, which may impede genetic recovery or result in local extinctions. Therefore, we also estimate genetic diversity among C. moreletii from central Veracruz to evaluate how hunting pressure and habitat 123 Conserv Genet (2012) 13:1677–1683 fragmentation have affected the genetic composition of crocodiles found there. Materials and methods Sample collection We captured 43 C. moreletii between October 2005 and December 2006 from five localities in central Veracruz, Mexico (Fig. 1). These were Casitas (CS), Laguna Chica (LC), La Mancha (LM), San Julián (SJ), and Colegio de Posgraduados (CO). We extracted blood from the post-cranial sinus with an ethylenediaminetetraacetic acid (EDTA)treated VacutainerÒ. From each blood sample, we extracted total genomic DNA using the PUREGENE isolation kit (Gentra Systems, Minneapolis, MN). We amplified a region of the mitochondrial genome that included tRNAThr, tRNAPro, tRNAPhe, and the D-loop. We generated sequence data and alignments following the methods of CedeñoVázquez et al. (2008). Following the methods of Rodriguez et al. (2008), we also genotyped each individual using a panel of nine microsatellite loci to produce a comparable microsatellite allele matrix (Table 1). We performed a power test with the program R (R Core Team 2012) using a Chi-square test to assess the quantity of genetic variability per locus detected in each genetic deme. Detection of hybrids To identify potential hybrids between C. moreletii and C. acutus, we diagnosed species-specific mitochondrial (mtDNA) haplotypes and compared our sequences to those previously reported by Cedeño-Vázquez et al. (2008). We also used microsatellite genotypes and STRUCTURE 2.3.3 (Pritchard et al. 2000) to obtain individual assignment probabilities (qi) of 43 individuals to C. moreletii or C. acutus (Rodriguez et al. 2008) by constraining the number of populations to two (K = 2) and running 1,000,000 iterations after a burn-in of 100,000 iterations. We used the admixture model of ancestry and averaged the results from ten independent runs using CLUMPP (Jakobsson and Rosenberg 2007). To determine whether individuals were admixed, we used previously established probability thresholds for C. acutus–C. moreletii hybrids in the Yucatan Peninsula (Rodriguez et al. 2008), where any individual exhibiting a qi value less than 0.95 or greater than 0.05 was considered to have admixed ancestry. Genetic diversity We used 24 individuals in the estimates of genetic diversity for central Veracruz, removing hatchlings (n = 20) from CO Conserv Genet (2012) 13:1677–1683 1679 Fig. 1 A Barplot of averaged individual assignment probabilities (qi) from microsatellite data analyzed in STRUCTURE when K = 3, individuals are grouped by locality: Casitas (CS), Laguna Chica (LC), La Mancha (LM), San Julián (SJ), and Colpos (CO). B Map of collection localities (closed circles) with pie charts proportional to sample size and mitochondrial haplotype frequency. Map inset depicts the natural range of C. moreletii (vertical lines) and C. acutus (diagonal lines) Table 1 Summary microsatellite diversity measures for C. moreletii from central Veracruz, Mexico (n = 24) Locus A/La HbO/HcE P vald C391e 6 0.35/0.61 0.04 e 7 0.46/0.67 0.07 Cj18e 1 0.00/0.00 – Cj20f 3 0.58/0.67 0.13 Cj109f 3 0.52/0.48 0.87 Cj119e 3 0.37/0.46 0.01 e 2 4 0.14/0.13 0.33/0.47 1.00 0.06 \0.01 Cj16 Cj131 Cu5123e Cuj131e 4 0.13/0.52 Mean 3.67 0.32/0.44 a Allelic richness (number of alleles per locus) b Heterozygosity observed c Heterozygosity expected (gene diversity corrected for sample size, Nei 1987) d e f Probability value of HWE exact test (heterozygote deficit) Fitzsimmons et al. (2001) Dever and Densmore (2001) (Fig. 1), as they were most likely half or full-siblings (data not shown). For mtDNA sequence data, we used DnaSP v5 (Librado and Rozas 2009) to analyze the number of segregating sites (S), nucleotide diversity (p), and haplotype diversity (h), which was estimated using Fisher’s exact test. We also performed Tajima’s neutrality test (D) (Tajima 1989) using MEGA v4 (Tamura et al. 2007). For microsatellite data, we estimated observed (HO) and expected heterozygosity (HE) using the program CERVUS 3.0.3 (Kalinowski et al. 2007) and calculated the number of alleles per locus (A/L) using GenePop 4.0.7 (Rousset 2008). We also used Genepop to test for Hardy–Weinberg equilibrium (HWE) using Fisher’s exact test (Guo and Thompson 1992) and a Markov chain method (Dememorization: 10,000, Batches: 20, Iterations per batch: 5,000) for each locus and between loci. We evaluated conformation to HWE using the heterozygote deficiency alternative hypothesis, because microsatellites tend to contain null alleles causing homozygote excess. This was previously observed in American alligator (Alligator mississippiensis) populations (Davis et al. 2002; Ryberg et al. 2002). To test for the presence of null alleles we used the maximum likelihood estimation of null allele frequency and the expectation–maximization algorithm (Dempster et al. 1977) using Micro-Checker 2.3.3 (Oosterhout et al. 2004). We tested for linkage disequilibrium (LD) by using Fisher’s exact test (Slatkin 1995) with GenePop. Population structure To test for population structure among C. moreletii from central Veracruz, we inputted the 24 genotypes used in the estimations of diversity into STRUCTURE. We ran 1,000,000 iterations after a burn-in of 100,000 iterations. We chose the most appropriate K by evaluating the value of DK, and the average log likelihood for values of K ranging from one to six. The results from ten independent runs were summarized using Structure Harvester (Earl and VonHoldt 2012) and averaged using CLUMPP. To test for a bottleneck, we applied a Wilcoxon sign-rank test on the microsatellite data using the stepwise mutation model (SMM) and two-phased mutation model (TPM) using Bottleneck 1.2.02, which assumed that all loci match a mutation-drift equilibrium model (Cornuet and Luikart 1996). Assuming a random mating strategy, the intrapopulation inbreeding estimator (RIS) was obtained for each locus using the SMM (Slatkin 1995; Balloux and Lugon-Moulin 2002) in the program SPAGeDi 1.3 (Hardy and Vekemans 2002). The mean value of RIS was used because of the lack of information for earlier generations (Hedrick 1986). 123 1680 Conserv Genet (2012) 13:1677–1683 Results Discussion We did not detect introgression of C. acutus mtDNA haplotypes or microsatellite alleles into Veracruz given that all of the individuals sampled carried C. moreletii haplotypes and had qi values greater than 0.95 to C. moreletii in modelbased tests of hybridization. In Veracruz, we detected two C. moreletii haplotypes with two segregating sites (Fig. 1), low haplotype diversity (h = 0.391, SD = 0.091) and low nucleotide diversity (p = 0.00164, SD = 0.00038). We detected haplotypes CmA (frequency = 0.82) and CmC (frequency = 0.18), which correspond to those previously reported by Cedeño-Vázquez et al. (2008). CmA is present in all sampled localities, but CmC was only found in CO (Fig. 1). In CO, two hatchlings carried haplotype CmC and 17 carried haplotype CmA. The power test result (1 - b = 0.87) suggests that our sample size was large enough to detect the allelic diversity in this population. Eight microsatellite loci were polymorphic, while locus Cj18 was monomorphic in this population (Table 1). Allelic richness (A/L) was 3.67 and mean expected heterozygosity was 0.44 (Table 1). Global tests for deviation from HWE were significant (P \ 0.0001). Individually, locus Cj119, Cj16 and Cuj131 were not in HWE most likely owing to the presence of null alleles (data not shown). An evolutionary force will usually affect all loci not just a few of them (Dempster et al. 1977), therefore the deficit of heterozygotes in this case is most likely the result of nonrandom mating due to inbreeding or population subdivision (Balloux and Lugon-Moulin 2002). Among all possible pairs of loci combinations seven pairs (19 %) showed significant LD (P = 0.0003–0.048), indicating recent and rapid population growth (Slatkin, 1995). In our tests for population structure using microsatellite genotypes, DK values weakly suggest three demes, and the mean log likelihood is maximal at K = 3 (Mean LnP(K) = -87; DK = 36). When considering K = 3 as a hypothesis, individuals clustered more or less into groups by locality (Fig. 1). The value of K is possibly overestimated when there is deviation from HWE and LD, however STRUCTURE is usually very robust when these violations are due to inbreeding or population division (Pritchard et al. 2000). Deficiency of heterozygotes under the SMM may be caused by a bottleneck that occurred in the sampled population (H deficiency: P = 0.01953, H excess: P = 0.98, H both: P = 0.04), however demographic records for C. moreletii (CITES 2010) and the fragmentation suggested in our analyses indicate that the low genetic diversity observed is due to inbreeding (RIS = 0.287). The observed low heterozygosity may be indicative of a Wahlund effect caused by population structure, which is also supported by our K and RIS results. Hybridization 123 Rodriguez et al. (2008) suggested that hybridization between C. acutus and C. moreletii is more widespread than suggested by Hekkala (2004); however, our data indicate that C. acutus alleles and haplotypes have not introgressed beyond the central portion of Veracruz. Translocation or natural dispersion of C. acutus or admixed individuals into central Veracruz is most likely a rare event. Therefore, it is possible that genetically purebred populations of C. moreletii may extend from Veracruz to the northern limit of its distribution in central Tamaulipas (Groombridge 1987). The lack of haplotype CmC from southern C. moreletii populations (Cedeño-Vázquez et al. 2008), weak population structure, and evidence of inbreeding indicate that these populations have not mixed with C. moreletii that escaped from UMAs. Although, to verify this it is necessary to test for hybridization in wild populations from southern Tamaulipas and Veracruz. Genetic diversity and population structure In this population, observed heterozygosity (HO = 0.32) is lower compared to that of other C. moreletii populations and other crocodylian species (Table 2). Low HO and the positive value of RIS (0.2772) suggest a deficit of heterozygotes (Balloux and Lugon-Moulin 2002) due to nonrandom mating. This result can be attributed to territorial behavior, specific habitat requirements, or lack of habitat connectivity, which could cause an inbreeding effect or population subdivision as suggested by the STRUCTURE analysis. The HO value appears relatively high considering the population size (Glenn et al. 1998), however the tendency for high heterozygosity has been found in small populations with separate sexes (Allendorf and Luikart 2007). Moreover, in long-lived species like crocodiles, life history traits (i.e. delayed sexual maturity, longevity, and the presence of multiple generations) modulate the decline of genetic variation even after a possible demographic bottleneck (Bishop et al. 2009). The number of alleles per locus in this population (A/L = 3.67; n = 24) is lower than in A. mississippiensis (A/L = 6.8; n = 28) (Glenn et al. 1998), C. acutus (A/L = 4.50; n = 19), and other C. moreletii populations from the Yucatan Peninsula (A/L = 4.11; n = 32) (Rodriguez et al. 2008), which can be attributed to a inbreeding caused by the loss of habitat and the effects of hunting. Dever et al. (2002) found high levels of genetic variation (Ho = 0.49; A/L = 8.0; n = 233) in C. moreletii populations from Belize, however, these results should be taken with caution because Conserv Genet (2012) 13:1677–1683 1681 Table 2 Genetic diversity parameters for three species of crocodylians using different genetic methods Species Microsat (Ho/HE) A. mississippiensis 0.466/0.484 Glenn et al. (1998) 0.694/ Davis et al. (2001) 0.594/0.579 Isberg et al. (2004) C. moreletii mtDNA (h) mtDNA (p) ISSR 0.31/0.39 Rodriguez et al. (2008) 0–0.795/0–0.737 McVay et al. (2008) 0.656–0.762/ 0.32/0.44 C. acutus Source Pacheco (2010) 0.391 0.00164 Our study 0.182 0.0003 Stafford et al. (2003) 0.251 0.00072 Ray et al. (2004) 0.51/0.59 Rodriguez et al. (2008) 0.065–0.24 0.45/0.38 C. acutus haplotypes were found in several of these populations (Ray et al. 2004). In that case, introgression may have artificially increased genetic variation. The presence of CmA haplotype across the studied region implies historical gene flow, but the lack of CmB haplotype (Cedeño et al. 2008) in all localities, the microsatellite data, and the division of the population into three demes as suggested by the STRUCTURE analysis indicate that gene flow among populations towards the northern portion of the distribution of C. moreletii was interrupted (Fig. 1), hence the central region along the eastern coast of the Gulf of Mexico appears to be a historical contact zone between northern and southern populations. Management implications Managers should focus on wild purebred populations especially when humans have facilitated hybridization (Allendorf et al. 2001). To outline areas where purebred populations exist, it is first necessary to identify contact zones between species (Fitzsimmons et al. 2002; Rodriguez et al. 2011) and to describe the extent of genetic introgression using molecular tools. Hybrids resulting from human-mediated factors should be removed from the environment when detected. Purebred crocodile populations that have been subjected to heavy hunting pressures and are affected by habitat fragmentation require management strategies focused on increasing genetic diversity. We suggest that managers genetically evaluate nearby populations, select purebreds, and then translocate reproductive individuals to promote genetic connectivity among populations. The decision to remove C. moreletii from Appendix I was based on a Population Viability Analysis (PVA) (CITES 2010) that assumed panmixia across its distributional range. This does not take into account genetic structure, low adult migration rates, and social structure. The PVA models treat Machkour-M0 Rabet et al. (2009) Rodriguez et al. (2011) organisms as independent individuals in a homogeneous environment without considering genetic isolation or recovery at the population level (Lacy and Miller 2002). Based on our evidence, which clearly shows that demographic recovery (CITES 2010) does not necessarily imply a concomitant genetic recovery, we suggest that management authorities should characterize the population genetic structure across the species range before PVA modeling to define areas of real interbreeding based on fine-scale genetic and ecological data. Acknowledgments We thank Blanca Martı́nez-de León, Miguel Munguı́a, Roger Guevara and Christopher Guibal for their helpful comments and suggestions on an earlier version of this manuscript. We thank Jeremy Weaver and Michael Vandewege for lab assistance. This research was supported with partial funding by a Consejo Nacional de Ciencia y Tecnologı́a (CONACYT) doctoral fellowship (No: 172256) (RGT) and Red de Biologı́a y Conservación de Vertebrados del Instituto de Ecologı́a, A.C. Field work was conducted under special collecting permit (SGPA/DGVS/02227 and SGPA/DGVS/ 05851/07 to AGR. 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