A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Genetically informed captive breeding of hybrids of an extinct species of Galapagos tortoise.

Hybridization poses a major challenge for species conservation because it threatens both genetic integrity and adaptive potential. Yet, hybridization can occasionally offer unprecedented opportunity for species recovery if the genome of an extinct taxon is present among living hybrids such that selective breeding could recapture it. We explored the design elements for establishing a captive-breeding program for Galapagos tortoises (Chelonoidis spp.) built around individuals with admixed ancestry involving an extinct species. The target individuals were hybrids between the extinct species from Floreana Island, C. niger, and an extant species, C. becki, which were recently found in the endemic range of C. becki, from Wolf Volcano on Isabela Island. We combined genotypic data from 35 tortoises with high ancestry from C. niger with forward-in-time simulations to explore captive breeding strategies that maximized overall genetic diversity and ancestry from C. niger while accommodating resource constraints, species biology, and the urgency to return tortoises to Floreana Island for facilitating ecosystem restoration. Overall genetic diversity was maximized when in the simulation tortoises were organized in relatively small breeding groups. Substantial amounts of the C. niger genome were captured despite limited resources available for selectively breeding tortoises in captivity. Genetic diversity was maximized when captive-bred offspring were released to the wild rather than being used as additional breeders. Our results provide genetic-based and practical guidance on the inclusion of hybrids with genomic representation from extinct taxa into species restoration programs and informs the ongoing debate on the value of hybrids in biodiversity conservation.

Reproducción en Cautiverio Informada Genéticamente de Híbridos de una Especie Extinta de Tortuga de las Galápagos Resumen La hibridación representa un obstáculo importante para la conservación de especies ya que amenaza tanto a la integridad genética como al potencial adaptativo. Aun así, la hibridación ocasionalmente puede ofrecer una oportunidad sin precedentes para la recuperación de una especie si el genoma de un taxón extinto está presente entre los híbridos vivientes de tal manera que la reproducción selectiva pudiera recuperarlo. Exploramos los elementos de diseño para el establecimiento de un programa de reproducción en cautiverio de la tortuga de las Galápagos (Chelonoidis spp.) construido en torno a los individuos con linajes mixtos que incluyeran una especie extinta. Los individuos fueron los híbridos de la especie extinta en la Isla Floreana, C. niger, y la especie viviente C. becki, encontrados recientemente en la distribución geográfica endémica de la segunda especie en el Volcán Wolf (Isla Isabela). Combinamos los datos genotípicos de 35 tortugas con un linaje cargado de C. niger usando simulaciones futuras de la descendencia generada por el programa para explorar las estrategias de reproducción en cautiverio que maximizaran en general la diversidad genética y el linaje de C. niger a la vez que se ajustaba a las restricciones de recursos, la biología de la especie y la urgencia por regresar las tortugas a la Isla Floreana para facilitar la restauración del ecosistema. En general, la diversidad genética se maximizó cuando en la simulación las tortugas estuvieron organizadas en grupos de reproducción relativamente pequeños y cuando cantidades sustanciales del genoma de C. niger fueron capturados con base en los recursos disponibles para reproducir selectivamente a las tortugas en cautiverio. La diversidad genética se vio especialmente maximizada cuando las crías reproducidas en cautiverio fueron liberadas en lugar de ser utilizadas como reproductoras adicionales. Nuestros resultados proporcionan una guía práctica y basada en la genética para la inclusión de híbridos con representación genómica de un taxón extinto en los programas de restauración de especies. Cuando incorporamos a los híbridos con diversidad genética que previamente se creía perdida en los programas con el propósito de la reintroducción de especies, nuestro estudio informa al debate continuo sobre el valor de los híbridos para la conservación de la biodiversidad.

Genetic Pedigree Analysis of the Pilot Breeding Program for the Rediscovered Galapagos Giant Tortoise from Floreana Island.

An aim of many captive breeding programs is to increase population sizes for reintroduction and establishment of self-sustaining wild populations. Genetic analyses play a critical role in these programs: monitoring genetic variation, identifying the origin of individuals, and assigning parentage to track family sizes. Here, we use genetic pedigree analyses to examine 3 seasons of a pilot breeding program for the Floreana island Galapagos giant tortoise, C. niger, that had been declared extinct for ~150 years until individuals with mixed ancestry were recently discovered. We determined that 8 of 9 founding individuals were assigned parentage to at least 1 of 130 offspring produced, though there was considerable reproductive skew. In addition, we observed that genetic diversity of the progeny was lower than that of the founders. Despite the observed reproductive skew, we did not see evidence for assortative mating based on relatedness, but there was a trend toward reduced fitness when more related individuals bred. Finally, we found that the majority of progeny had ancestry assigned to the Floreana species (mean ± SE = 0.51 ± 0.02), though individual estimates varied. The success of these pilot seasons bodes well for a larger breeding program to help restore the previously extinct tortoise from Floreana island. Future efforts should continue to monitor for reproductive skew and assortative mating to maintain allelic diversity. We would also recommend forming smaller breeding groups and rotating individuals among them to prevent long-term reproductive skew among pairs.

Genome-Wide Assessment of Diversity and Divergence Among Extant Galapagos Giant Tortoise Species.

Genome-wide assessments allow for fuller characterization of genetic diversity, finer-scale population delineation, and better detection of demographically significant units to guide conservation compared with those based on "traditional" markers. Galapagos giant tortoises (Chelonoidis spp.) have long provided a case study for how evolutionary genetics may be applied to advance species conservation. Ongoing efforts to bolster tortoise populations, which have declined by 90%, have been informed by analyses of mitochondrial DNA sequence and microsatellite genotypic data, but could benefit from genome-wide markers. Taking this next step, we used double-digest restriction-site associated DNA sequencing to collect genotypic data at >26000 single nucleotide polymorphisms (SNPs) for 117 individuals representing all recognized extant Galapagos giant tortoise species. We then quantified genetic diversity, population structure, and compared results to estimates from mitochondrial DNA and microsatellite loci. Our analyses detected 12 genetic lineages concordant with the 11 named species as well as previously described structure within one species, C. becki. Furthermore, the SNPs provided increased resolution, detecting admixture in 4 individuals. SNP-based estimates of diversity and differentiation were significantly correlated with those derived from nuclear microsatellite loci and mitochondrial DNA sequences. The SNP toolkit presented here will serve as a resource for advancing efforts to understand tortoise evolution, species radiations, and aid conservation of the Galapagos tortoise species complex.

Glacial survival of trophically linked boreal species in northern Europe.

Whether non-arctic species persisted in northern Europe during the Last Glacial Maximum (LGM) is highly debated. Until now, the debate has mostly focused on plants, with little consideration for other groups of organisms, e.g. the numerous plant-dependent insect species. Here, we study the late-Quaternary evolution of the European range of a boreo-montane leaf beetle, Gonioctena intermedia, which feeds exclusively on the boreal and temperate trees Prunus padus and Sorbus aucuparia Using species distribution models, we estimated the congruence between areas of past and present suitable climate for this beetle and its host plants. Then we derived historical hypotheses from the congruent range estimates, and evaluated their compatibility with observed DNA sequence variation at five independent loci. We investigated compatibility using computer simulations of population evolution under various coalescence models. We find strong evidence for range modifications in response to late-Quaternary climate changes, and support for the presence of populations of G. intermedia in northern Europe since the beginning of the last glaciation. The presence of a co-dependent insect in the region through the LGM provides new evidence supporting the glacial survival of cold-tolerant tree species in northern Europe.

Multi-locus DNA sequence variation in a complex of four leaf beetle species with parapatric distributions: mitochondrial and nuclear introgressions reveal recent hybridization.

While the importance for including multiple independent loci in phylogeographic studies is largely acknowledged, a majority of these still focus on a single species. We combine the study of multilocus DNA sequence variation (one mitochondrial and four unlinked nuclear fragments) at both the inter- and intraspecific levels to explore the evolutionary history of four closely related specialized herbivorous insects (Chrysomelidae, genus Gonioctena). Analyzing the sequences for samples collected across their entire European range allows us to (1) characterize the genetic boundaries among species, i.e. the degree of lineage sorting, (2) infer their phylogenetic relationships and (3) explore reproductive barriers among them in regions where their ranges overlap. For two sister species, we identify multiple independent cases of unidirectional transfer of genetic material (introgression) at both mitochondrial and nuclear loci, demonstrating recent hybridization between both species in the overlapping regions of their range. The highlighted pattern of genetic variation suggests that Gonioctena intermedia expanded its range into that of G. quinquepunctata, and that both species may experience mutual exclusion. Overall, this study illustrates that interpreting intraspecific genetic variation for the purpose of evolutionary inference without the broader context of the other closely related species could lead to erroneous conclusions.

Theory, practice, and conservation in the age of genomics: The Galápagos giant tortoise as a case study.

High-throughput DNA sequencing allows efficient discovery of thousands of single nucleotide polymorphisms (SNPs) in nonmodel species. Population genetic theory predicts that this large number of independent markers should provide detailed insights into population structure, even when only a few individuals are sampled. Still, sampling design can have a strong impact on such inferences. Here, we use simulations and empirical SNP data to investigate the impacts of sampling design on estimating genetic differentiation among populations that represent three species of Galápagos giant tortoises (Chelonoidis spp.). Though microsatellite and mitochondrial DNA analyses have supported the distinctiveness of these species, a recent study called into question how well these markers matched with data from genomic SNPs, thereby questioning decades of studies in nonmodel organisms. Using >20,000 genomewide SNPs from 30 individuals from three Galápagos giant tortoise species, we find distinct structure that matches the relationships described by the traditional genetic markers. Furthermore, we confirm that accurate estimates of genetic differentiation in highly structured natural populations can be obtained using thousands of SNPs and 2-5 individuals, or hundreds of SNPs and 10 individuals, but only if the units of analysis are delineated in a way that is consistent with evolutionary history. We show that the lack of structure in the recent SNP-based study was likely due to unnatural grouping of individuals and erroneous genotype filtering. Our study demonstrates that genomic data enable patterns of genetic differentiation among populations to be elucidated even with few samples per population, and underscores the importance of sampling design. These results have specific implications for studies of population structure in endangered species and subsequent management decisions.

Author Correction: Identification of Genetically Important Individuals of the Rediscovered Floreana Galápagos Giant Tortoise (Chelonoidis elephantopus) Provides Founders for Species Restoration Program.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Identification of Genetically Important Individuals of the Rediscovered Floreana Galápagos Giant Tortoise (Chelonoidis elephantopus) Provide Founders for Species Restoration Program.

Species are being lost at an unprecedented rate due to human-driven environmental changes. The cases in which species declared extinct can be revived are rare. However, here we report that a remote volcano in the Galápagos Islands hosts many giant tortoises with high ancestry from a species previously declared as extinct: Chelonoidis elephantopus or the Floreana tortoise. Of 150 individuals with distinctive morphology sampled from the volcano, genetic analyses revealed that 65 had C. elephantopus ancestry and thirty-two were translocated from the volcano's slopes to a captive breeding center. A genetically informed captive breeding program now being initiated will, over the next decades, return C. elephantopus tortoises to Floreana Island to serve as engineers of the island's ecosystems. Ironically, it was the haphazard translocations by mariners killing tortoises for food centuries ago that created the unique opportunity to revive this "lost" species today.