Galapagos giant tortoises are endemic to the Galapagos Archipelago, where they are found in isolated populations. While these populations are widely considered distinguishable in morphology, behavior, and genetics, the recent divergence of these taxa has made their status as species controversial. Here, we apply modern methods of species delimitation to whole genome resequencing data from 38 tortoises across all 13 extant taxa to assess support for delimiting these taxa as species. We find strong evidence to reject the hypothesis that all Galapagos giant tortoises belong to a single species. Instead, a conservative interpretation of model-based and divergence-based results indicates that these taxa form a species complex consisting of a minimum of 5 species, with some analyses supporting as many as 13 species. There is mixed support for the species status of taxa living on the same island, with some methods delimiting them as separate species and others suggesting there is a single species per island. These results make clear that Galapagos giant tortoise taxa represent different stages in the process of speciation, with some taxa further along in that evolutionary process than others. A better understanding of the more complex parts of that process is urgently needed, given the threatened status of Galapagos giant tortoises.

Captive breeding programs benefit from genetic analyses that identify relatedness between individuals, assign parentage to offspring, and track levels of genetic diversity. Monitoring these parameters across breeding cycles is critical to the success of a captive breeding program as it allows conservation managers to iteratively evaluate and adjust program structure. However, in practice, genetic tracking of breeding outcomes is rarely conducted. Here, we examined the first three offspring cohorts (2017–2020) of the genetically-informed captive breeding program for the Floreana Island Galapagos giant tortoise, Chelonoidis niger. This captive breeding program is unique as the Floreana tortoise has been extinct since the 1800s, but its genome persists, in part, in the form of living hybrids with the extant Volcano Wolf tortoise, Chelonoidis becki. Breeding over the study period took place at the Galapagos National Park Directorate breeding facility in four corrals, each containing three females and two males. Using 17 microsatellite markers, we were able to assign parentage to 94 of the 98 offspring produced over the study period. We observe that despite the addition of more founders since the pilot breeding program, the effective population size remains low, and changes to the arrangements of breeding corrals may be necessary to encourage more equal reproductive output from the males. This study demonstrates the value of hybrids for species restoration and the importance of continually reassessing the outcomes of captive breeding.