Among crocodilians, Crocodylus rhombifer is one of the world's most endangered species with the smallest natural distribution. In Cuba, this endemic species coexists with the American crocodile (Crocodylus acutus). Hybridization between these two species is well known in captivity and might occur in the wild, but has never been demonstrated genetically. Here, we combined molecular data with environmental, geographic, and fossil data to infer the evolutionary history of Crocodylus in the Cuban Archipelago, and to evaluate genealogical support for species boundaries. We analyzed seven microsatellite loci plus DNA sequence data from nuclear (RAG-1) and mitochondria! (cytochrome b and cytochrome oxidase I) genes from 89 wild-caught individuals in Cuba, Grand Cayman Island, Jamaica, and Central America, and two samples from zoo collections. Microsatellites showed evidence of introgression, suggesting potential hybridization among Cuban groups. In Cuba, C. acutus contained one mitochondrial DNA (mtDNA) haplotype, whereas C. rhombifer contained two haplotypes. MtDNA data showed that C. acutus is paraphyletic with respect to C. rhombifer, revealing 1% sequence divergence between species within Cuba vs. 8% divergence between Cuban forms and mainland C. acutus. We suggest that hybridization has been a historical as well as a current phenomenon between C. acutus and C. rhombifer. These findings suggest that long-term conservation of crocodiles in Cuba will require identification of genetically pure and hybrid individuals, and a decrease in anthropogenic activities. We also recommend more extensive morphological and genetic analyses of Cuban population to establish clear boundaries of the hybrid zone between C. acutus and C. rhombifer. J. Exp. Zool. 315:358-375, 2011. © 2011 Grant Sponsors: Smithsonian Tropical Research Institute; NSF; Grant number: DEB04441333.Yoamel Milian-Garcia and Miryam Venegas-Anaya contributed equally to this work.^Deceased.Correspondence to: Georgina Espinosa-Lopez, University of Havana, Cuba. E-mail: georgina@fbio.uh.cu, georgina@cim.uh.cu Received 23 [358][359][360][361][362][363][364][365][366][367][368][369][370][371][372][373][374][375]2011 and Crocodylus acutus inferred from multilocus markers. J. Hybridization plays a dual role in evolutionary biology and conservation. Evolutionarily, hybridization and introgression may contribute to genetic variability and increase fitness in small populations or they may result in the melding of two previously distinct evolutionary lineages (Grant and Grant, '92; Clarke et al., '98;Zimmer, 2002;Coyne and Orr, 2004a). On the other hand, hybridization can result in decreased fitness of hybrids contributing to maintain distinctive genetic lineages. Some lineages may tolerate a substantial amount of hybridization without losing their morphological or genetic distincriveness, whereas in other cases morphological intermediates are formed (Mavarez et al., 2006). In the case of unidirectional hybridization or introgression, one lineage may cause th...

Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value.