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Emerging infectious diseases (EIDs) have been reported as causes of morbidity and mortality in free-living animal populations, including turtles and tortoises, and they have even resulted in species extinctions, with human activities contributing to the spread of many of these diseases. In the Galapagos, giant tortoises are endangered due to habitat change, invasive species, and other human impacts; however, the impact of EIDs on Galapagos tortoise conservation remains understudied. To fill this gap, we conducted health assessments of five tortoise species from the islands of Santa Cruz, Isabela and Española. We performed health evaluations of 454 animals and PCR testing for pathogens known to be relevant in other tortoise species. We identified two novel sequences of adenoviruses and four of herpesviruses. Based on alignments of the DNA polymerase gene and maximum likelihood phylogenetic analyses, we found both novel adenoviruses to be most closely related to red footed tortoise adenovirus 2, by nucleotide sequence and red footed tortoise adenovirus 1, based on amino acid sequence. Three of the herpesvirus sequences translated into the same deduced amino acid sequence; therefore, they may be considered the same viral species, closely related to terrapene herpesvirus 2. The fourth herpesvirus sequence was highly divergent from any sequence previously detected and is related to an eagle owl herpesvirus based on nucleotide sequence and to loggerhead oro-cutaneous herpesvirus based on amino acids. These novel viruses may be pathogenic for giant tortoises under specific conditions (e.g., stress). Continued screening is crucial to determine if these viruses play a role in tortoise fitness, morbidity and survival. This information allows us to provide recommendations to the Galapagos National Park Directorate and other institutions to improve the management of these unique species both in Galapagos and globally, and for tortoise reintroduction plans throughout the archipelago.

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.杂交对物种保护提出了重大挑战, 因为它威胁着物种的遗传完整性和适应性潜力。然而, 如果一个已灭绝类群的基因组存在于现存的杂交种中, 那么杂交也可以为物种恢复提供前所未有的机遇, 通过选择性育种重新获得灭绝物种的基因组。本研究探讨了如何利用含有已灭绝物种基因组的混合血统个体, 为加拉帕戈斯陆龟 （Chelonoidis spp.） 建立圈养繁殖计划。我们的目标个体是弗洛里安娜岛灭绝物种 C. niger 与近期在其分布范围内伊莎贝拉岛沃尔夫火山发现的现存物种 C. becki 的杂交后代。我们将 35 只含有较高比例 C. niger 血统的乌龟的基因型数据与对该计划获得后代的前进式模拟相结合, 在满足总体遗传多样性和 C. niger 血统最大化, 且考虑资源限制、物种生物学特性和弗洛里安娜岛急需重引入乌龟来促进生态系统恢复的情况下, 分析了可能的圈养繁殖策略。在模拟实验中, 建立相对较小的乌龟繁殖群时的总体遗传多样性最高, 且根据已有资源对圈养乌龟进行选择性育种可以捕获大量的 C. niger 基因组。若将圈养繁殖的后代释放到野外而不是继续用于繁殖, 则遗传多样性会特别高。我们的研究结果为将携带已灭绝类群的代表性基因组的杂交种纳入物种恢复计划提供了基于遗传学的实践指导。我们的研究还表明, 可以在物种重引入项目中纳入携带了被认为已经丢失的遗传多样性的杂交种, 这也为目前杂交种在生物多样性保护中的价值的争论提供了信息。【翻译: 胡怡思; 审校: 聂永刚】.

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.

During January 1994, feces from a captive juvenile Galápagos tortoise, Chelonoidis sp., from the Dallas Zoo, Dallas County, Texas was examined for coccidia. The tortoise was found to harbor an eimerian which is described as new. Sporulated oocysts of Eimeria iversoni n. sp. were ovoidal with a smooth, single-layered wall (∼ 0.5-0.8) that measured (L × W) 13.5 × 10.3 μm, with a length/width (L/W) ratio of 1.3; micropyle, oocyst residuum, and polar granule(s) were all absent; 2 conical projections were present on 1 end of oocyst and measured 1.0-1.5. Sporocysts were elongate-ellipsoidal and measured 8.3 × 4.5 μm, with L/W of 1.8; a Stieda body (∼ 0.5 high) was present, but substieda and parastieda bodies were absent; a sporocyst residuum was composed of 2-5 granules in a compact mass between sporozoites; sporozoites were banana-shaped and measured 9.5 × 2.5 in situ, with an ellipsoidal posterior refractile body and a spheroidal anterior refractile body. This is only the second time an eimerian has been reported from Galápagos tortoises.

article i nfo The retention time of food in the digestive tract of animals has important implications for digestive physiology. Retention time impacts digestive efficiency and among herbivores affects plant-animal interactions including herbivory and seed dispersal. Poorly studied yet iconic Galapagos tortoises are large- bodied generalist herbivores and ecosystem engineers which migrate seasonally. Potentially variable digesta retention times due to strong seasonal and altitudinal temperature gradients may influence tortoise seed dispersal abilities and rates of herbivory. We fed captive adult tortoises living in semi-natural conditions on Galapagos with inert particles and seeds from locally available fruits to determine whether seed size and ambient temperature influenced retention time. Median retention time varied from 6 to 28 days, with a mode of 12 days. Seed size had no effect on any of our measures of retention time, but ambient temperature was inversely correlated with retention times. Long retention time facilitates long distance seed dispersal by Galapagos tortoises, which may improve effectiveness. The effect of temperature, which may double from hot lowlands to cold highlands through the seasonal cycle, on tortoise digesta retention time will strongly influence seed dispersal efficiency and may influence patterns of food selection and migration in this species.

Galapagos tortoises possess two main shell forms, domed and saddleback, that correlate with the biogeographic history of this species group. However, the lack of description of morphological shell variation within and among populations has prevented the under- standing of the contribution of evolutionary forces and the potential role of ontogeny in shaping morphological shell differences. Here, we analyze two lineages of Galapagos tortoises inhabiting Santa Cruz Island by applying geometric morphometrics in combination with a photogrammetry 3D reconstruction method on a set of tortoises of different ages (from juvenile to adult). The aim of this study is to describe morphological features on the carapace that could be used for taxonomic recog- nition by taking into account confounding factors, such as the morphological changes occurring during growth. Our results indicate that despite the shared similarities of growth patterns and of morphological changes observed during growth, the two lineages and the differ- ent sexes can be distinguished on the basis of distinct carapace features. Lineages differ by the shape of the vertebral (especially concerning their width) and pleural scutes, with one lineage having a more compressed carapace shape, whereas the other possesses a carapace that is more elongated and expanded toward the sides as well as an higher positioning of the first vertebral scute. Furthermore, females have a more elongated and wider carapace shape than males. Finally, carapace shape changes with growth, with vertebral scutes becoming narrower and pleural scutes becoming larger during late ontogeny. J. Morphol. 272:379-386, 2011. 2011 Wiley-Liss, Inc.

The Galápagos giant tortoise is an icon of the unique, endemic biodiversity of Galápagos, but little is known of its parasitic fauna. We assessed the diversity of parasitic nematode communities and their spatial distributions within four wild tortoise populations comprising three species across three Galápagos islands, and consider their implication for Galápagos tortoise conservation programmes. Coprological examinations revealed nematode eggs to be common, with more than 80% of tortoises infected within each wild population. Faecal samples from tortoises within captive breeding centres on Santa Cruz, Isabela and San Cristobal islands also were examined. Five different nematode egg types were identified: oxyuroid, ascarid, trichurid and two types of strongyle. Sequencing of the 18S small-subunit ribosomal RNA gene from adult nematodes passed with faeces identified novel sequences indicative of rhabditid and ascaridid species. In the wild, the composition of nematode communities varied according to tortoise species, which co-varied with island, but nematode diversity and abundance were reduced or altered in captive-reared animals. Evolutionary and ecological factors are likely responsible for the variation in nematode distributions in the wild. This possible species/island-parasite co-evolution has not been considered previously for Galápagos tortoises. We recommend that conservation efforts, such as the current Galá-pagos tortoise captive breeding/rearing and release programme, be managed with respect to parasite biogeography and host-parasite co-evolutionary processes in addition to the bio-geography of the host.

We examined the phylogeography and history of giant Galapagos tortoise populations based on mito- chondrial DNA sequence data from 161 individuals from 21 sampling sites representing the 11 currently recognized extant taxa. Molecular clock and geological considerations indicate a founding of the monophyletic Galapagos lineage around 2-3 million years ago, which would allow for all the diversification to have occurred on extant islands. Founding events generally occurred from geologically older to younger islands with some islands colonized more than once. Six of the 11 named taxa can be associated with monophyletic maternal lineages. One, Geochelone porteri on Santa Cruz Island, consists of two distinct populations connected by the deepest node in the archipelago-wide phylogeny, whereas tortoises in northwest Santa Cruz are closely related to those on adjacent Pinzon Island. Volcan Wolf, the northernmost volcano of Isabela Island, consists of both a unique set of maternal lineages and recent migrants from other islands, indicating multiple colonizations possibly due to human transport or multiple colonization and partial elimination through competition. These genetic findings are consistent with the mixed morphology of tortoises on this volcano. No clear genetic differentiation between two taxa on the two southernmost volcanoes of Isabela was evident. Extinction of crucial populations by human activities confounds whether domed versus saddleback carapaces of different populations are mono- or polyphyletic. Our findings revealed a complex phylogeography and history for this tortoise radiation within an insular environment and have implications for efforts to conserve these endangered biological treasures.

Restoration of extirpated species via captive breeding has typically relied on population viability as the primary criterion for evaluating success. This criterion is inadequate when species reintroduction is undertaken to restore ecological functions and interactions. Herein we report on the demographic and ecological outcomes of a five-decade-long population restoration program for a critically endangered species of "ecosystem engineer": the endemic Española giant Galapagos tortoise (Chelonoidis hoodensis). Our analysis of complementary datasets on tortoise demography and movement, tortoise-plant interactions and Española Island's vegetation history indicated that the repatriated tortoise population is secure from a strictly demographic perspective: about half of tortoises released on the island since 1975 were still alive in 2007, in situ reproduction is now significant, and future extinction risk is low with or without continued repatriation. Declining survival rates, somatic growth rates, and body condition of repatriates suggests, however, that resources for continued population growth are increasingly limited. Soil stable carbon isotope analyses indicated a pronounced shift toward woody plants in the recent history of the island's plant community, likely a legacy of changes in competitive relations between woody and herbaceous plants induced by now-eradicated feral goats and prolonged absence of tortoises. Woody plants are of concern because they block tortoise movement and hinder recruitment of cactus--a critical resource for tortoises. Tortoises restrict themselves to remnant cactus patches and areas of low woody plant density in the center of the island despite an apparent capacity to colonize a far greater range, likely because of a lack of cactus elsewhere on the island. We conclude that ecosystem-level criteria for success of species reintroduction efforts take much longer to achieve than population-level criteria; moreover, reinstatement of endangered species as fully functioning ecosystem engineers may often require large-scale habitat restoration efforts in concert with population restoration.

A new fungal genus and species, Aphanoascella galapagosensis, recovered from carapace keratitis in a Galapagos tortoise residing in a south Texas zoological collection, is characterized and described. The presence of a pale peridium composed of textura epidermoidea surrounded by scarce Hulle cell-like chlamydospores, and the characteristic reticulate ascospores with an equatorial rim separates it from other genera within the Onygenales. The phylogenetic tree inferred from the analysis of D1/D2 sequences demonstrates that this fungus represents a new lineage within that order. As D1/D2 and ITS sequence data also shows a further separation of Aphanoascus spp. into two monophyletic groups, we propose to retain the generic name Keratinophyton for species whose ascospores are pitted and display a conspicuous equatorial rim, and thereby propose new combinations in this genus for four Aphanoascus species.

Seasonal migration has evolved in many taxa as a response to predictable spatial and temporal variation in the environment. Individual traits, physiology and social state interact with environmental factors to increase the complexity of migratory systems. Despite a huge body of research, the ultimate causes of migration remain unclear. A relatively simple, tractable system - giant tortoises on Santa Cruz Island, Galapagos, was studied to elucidate the roles of environmental variation and individual traits in a partial migratory system. Specifically, we asked: (i) do Galapagos tortoises undergo long-distance seasonal migrations? (ii) is tortoise migration ultimately driven by gradients in forage quality or temperature; and (iii) how do sex and body size influence migration patterns? We recorded the daily locations of 17 GPS-tagged tortoises and walked a monthly survey along the altitudinal gradient to characterize the movements and distribution of tortoises of different sizes and sexes. Monthly temperature and rainfall data were obtained from weather stations deployed at various altitudes, and the Normalized Difference Vegetation Index was used as a proxy for forage quality. Analyses using net displacement or daily movement characteristics did not agree on assigning individuals as either migratory or non-migratory; however, both methods suggested that some individuals were migratory. Adult tortoises of both sexes move up and down an altitudinal gradient in response to changes in vegetation dynamics, not temperature. The largest tagged individuals all moved, whereas only some mid-sized individuals moved, and the smallest individuals never left lowland areas. The timing of movements varied with body size: large individuals moved upward (as lowland forage quality declined) earlier in the year than did mid-sized individuals, while the timing of downward movements was unrelated to body size and occurred as lowland vegetation productivity peaked. Giant tortoises are unlikely candidates for forage-driven migration as they are well buffered against environmental fluctuations by large body size and a slow metabolism. Notably the largest, and presumably most dominant, individuals were most likely to migrate. This characteristic and the lack of sex-based differences in movement behaviour distinguish Galapagos tortoise movement from previously described partial migratory systems. published

Galápagos tortoises represent the only surviving lineage of giant tortoises that exhibit two different types of shell morphology. The taxonomy of Galápagos tortoises was initially based mainly on diagnostic morphological characters of the shell, but has been clarified by molecular studies indicating that most islands harbor monophyletic lineages, with the exception of Isabela and Santa Cruz. On Santa Cruz there is strong genetic differentiation between the two tortoise populations (Cerro Fatal and La Reserva) exhibiting domed shell morphology. Here we integrate nuclear microsatellite and mitochondrial data with statistical analyses of shell shape morphology to evaluate whether the genetic distinction and variability of the two domed tortoise populations is paralleled by differences in shell shape. Based on our results, morphometric analyses support the genetic distinction of the two populations and also reveal that the level of genetic variation is associated with morphological shell shape variation in both populations. The Cerro Fatal population possesses lower levels of morphological and genetic variation compared to the La Reserva population. Because the turtle shell is a complex heritable trait, our results suggest that, for the Cerro Fatal population, non-neutral loci have probably experienced a parallel decrease in variability as that observed for the genetic data.

Giant tortoises, a prominent symbol of the Galápagos archipelago, illustrate the influence of geological history and natural selection on the diversification of organisms. Because of heavy human exploitation, 4 of the 15 known species (Geochelone spp.) have disappeared. Charles Darwin himself detailed the intense harvesting of one species, G. elephantopus, which once was endemic to the island of Floreana. This species was believed to have been exterminated within 15 years of Darwin's historic visit to the Galápagos in 1835. The application of modern DNA techniques to museum specimens combined with long-term study of a system creates new opportunities for identifying the living remnants of extinct taxa in the wild. Here, we use mitochondrial DNA and microsatellite data obtained from museum specimens to show that the population on Floreana was evolutionarily distinct from all other Galápagos tortoise populations. It was demonstrated that some living individuals on the nearby island of Isabela are genetically distinct from the rest of the island's inhabitants. Surprisingly, we found that these “non-native” tortoises from Isabela are of recent Floreana ancestry and closely match the genetic data provided by the museum specimens. Thus, we show that the genetic line of G. elephantopus has not been completely extinguished and still exists in an intermixed population on Isabela. With enough individuals to commence a serious captive breeding program, this finding may help reestablish a species that was thought to have gone extinct more than a century ago and illustrates the power of long-term genetic analysis and the critical role of museum specimens in conservation biology.

A well-differentiated cutaneous mast cell tumour was diagnosed in a subadult female giant Galapagos tortoise. The tumour was a pedunculated, verrucose mass located near the base of the neck. The histological features, which were diagnostic for a mast cell tumour, included abundant intracytoplasmic granules that were stained metachromatically with Giemsa and toluidine blue stains. Mast cell tumours are rare in reptiles, and this is the first description of a mast cell tumour in a chelonian. Se diagnosticó un mastocitoma cutáneo bien diferenciado en una hembra subadulta de tortuga gigante de Galápagos. El tumor era una masa verrucosa pedunculada situada cerca de la base del cuello. Las características histológicas que eran diagnósticas de un tumor de mastocitos, incluían abundantes gránulos intracitoplasmáticos que se tiñeron con tinción de Giemsa y azul de toluidina. Los tumores de mastocitos son raros en los reptiles, y ésta es la primera es la primera descripción de un mastocitoma en un quelonio. Universidad Nacional, Costa Rica. Escuela de Medicina Veterinaria

As once boldly stated, ‘bad taxonomy can kill’, highlighting the critical importance of accurate taxonomy for the conservation of endangered taxa. The concept continues to evolve almost 15 years later largely because most legal protections aimed at preserving biological diversity are based on formal taxonomic designations. In this paper we report unrecognized genetic divisions within the giant tortoises of the Galápagos. We found three distinct lineages among populations formerly considered a single taxon on the most populous and accessible island of Santa Cruz; their diagnosability, degree of genetic divergence and phylogenetic placement merit the recognition of at least one new taxon. These results demonstrate the fundamental importance of continuing taxonomic investigations to recognize biological diversity and designate units of conservation, even within long-studied organisms such as Galápagos tortoises, whose evolutionary heritage and contribution to human intellectual history warrant them special attention.

The Galapagos Islands are home to 11 subspecies of large terrestrial tortoises (Geochelone nigra). All Galapagos tortoises are considered endangered and approximately 12,000 animals still exist. Until now, the reproductive cycle of the Galapagos tortoise has been studied only in captive animals, and no data from free-ranging tortoises have been available. During a one-year period, blood samples were collected from male and femaleG. nigraliving under seminatural conditions on Santa Cruz Island, Galapagos. Plasma steroid hormones were measured by radioimmunoassays (RIAs). In males, plasma testosterone and corticosterone increased a few months before the onset of the mating season. Peak levels were observed while most copulations occurred and environmental temperatures were highest. Both testosterone and corticosterone showed low levels during the cold and dry nesting season and high levels during the hot and rainy mating season. In females, testosterone and corticosterone also rose during the hot and rainy mating season. Both hormones peaked during the second half of the mating season and decreased during the cooler dry season. Female estradiol levels increased at the onset of the mating season, reaching the highest level at the peak of the mating season, which coincided with the highest annual temperatures measured. Estradiol slowly decreased within the next months and rapidly dropped at the onset of the nesting season when temperatures decreased. Progesterone levels were high close to the time of ovulation and showed clearly elevated levels at the beginning of the nesting season after some females had laid their first clutch. Progesterone decreased during the nesting season, when ambient temperatures began to decrease, and reached minimal levels in the postbreeding period shortly before the onset of the next mating season. There were significant annual variations in plasma testosterone in both males and females. Plasma corticosterone was generally higher in males than in females and varied throughout the year in both sexes.

The Parisian comparative anatomist Claude Perrault, dissecting an Indian giant tortoise in 1676, was the first to observe that the urinary bladder is of an extraordinary size in terrestrial tortoises. In 1799, the English comparative physiologist Robert Townson suggested that the bladder functioned as a water reservoir, as he had shown previously for frogs and toads. However, these observations went unnoticed in subsequent reports on tortoise water economy that were made by travellers and naturalists visiting the Galapagos Archipelago and marvelling over the huge numbers of giant tortoises that inhabited these desert-like islands. The first such report was by an American naval officer, David Porter, who was a privateer in the 1812-15 war with England. In his journal he referred to the constant supply of water which the Galapagos tortoises carried with them. References to the location in the body, as well as the amounts and quality of the water stored, were, however, contradictory. The confusion concerning the anatomical identity of the water reservoir in the Galapagos tortoise, Geochelone elephantopus, persisted throughout the nineteenth century, and continued when studies of tortoise water economy and drinking behaviour in arid environments were taken up independently in the desert tortoise, Gopherus agassizii, which inhabits the desert regions in the south-western United States. In 1881 Cox found large sacs filled with clear water under the carapace, but it was half a century later that these sacs were identified as the large bilobed bladder; references to specific water sacs continued to appear in the literature until the 1960s. Since 1970, information on the water economy of desert tortoises has been obtained from extensive field studies. Rates of disappearance of tritiated water injected into the body have shown that during the drought periods of the summer, water turnover (intake) rates do not differ from the rates of metabolic water production. Under these conditions urine is not voided, but is stored in the large bladder. During a drought period the bladder urine increases from initially low osmolality finally to reach isosmolality with the blood plasma. Soluble K+ is the major cation of the urine, but large amounts of K+ are also present as precipitated urates. During a drought period the body is in negative water balance, but despite substantial losses of total body water, the plasma concentrations of Na+ and Cl- can remain constant for many months, indicating regulation of the extracellular fluid and water content of the body tissues by reabsorption of water from the urinary bladder. The bladder thus acts both as a store for nitrogenous waste and K+ and as a water reservoir during droughts. Following rain showers, there is a sharp decline in tritium activity correlated with copious drinking from temporary pools of rain water. The old bladder urine is voided and most of the water drunk is stored as a highly dilute urine. In 1676 Perrault observed that in a freshwater turtle, Emys orbicularis, but not in the giant tortoise, two other bladders opened into the cloaca. By the mid-twentieth century it had been established that these cloacal bladders typically were restricted to species of chelonians that led a semi-terrestrial or semi-aquatic life. The function of the bladders has been debated since Townson observed in 1799 that dehydrated freshwater turtles took up water by anal drinking, suggesting that anal drinking served in the water economy of semi-terrestrial turtles. Since then, the bladders have been ascribed hydrostatic and respiratory functions, but the recent literature mostly argues for a respiratory function. The possible role of the cloacal bladders as a water reservoir in amphibious turtles is still open. Terrestrial amphibians and tortoises are unique among vertebrates in possessing large urinary bladders that may function as water reservoirs in dry environments. (ABSTRACT TRUNCATED)

Document S1. Supplemental Experimental Procedures and Results, One Table, One Figure and Supplemental ReferencesxDownload (.21 MB ) Document S1. Supplemental Experimental Procedures and Results, One Table, One Figure and Supplemental References

Background The giant Galápagos tortoise, Chelonoidis nigra, is a large-sized terrestrial chelonian of high patrimonial interest. The species recently colonized a small continental archipelago, the Galápagos Islands, where it has been facing novel environmental conditions and limited resource availability. To explore the genomic consequences of this ecological shift, we analyze the transcriptomic variability of five individuals of C. nigra, and compare it to similar data obtained from several continental species of turtles. Results Having clarified the timing of divergence in the Chelonoidis genus, we report in C. nigra a very low level of genetic polymorphism, signatures of a weakened efficacy of purifying selection, and an elevated mutation load in coding and regulatory sequences. These results are consistent with the hypothesis of an extremely low long-term effective population size in this insular species. Functional evolutionary analyses reveal a reduced diversity of immunity genes in C. nigra, in line with the hypothesis of attenuated pathogen diversity in islands, and an increased selective pressure on genes involved in response to stress, potentially related to the climatic instability of its environment and its elongated lifespan. Finally, we detect no population structure or homozygosity excess in our five-individual sample. Conclusions These results enlighten the molecular evolution of an endangered taxon in a stressful environment and point to island endemic species as a promising model for the study of the deleterious effects on genome evolution of a reduced long-term population size.

Background Although not unusual to find captive relicts of species lost in the wild, rarely are presumed extinct species rediscovered outside of their native range. A recent study detected living descendents of an extinct Galapagos tortoise species (Chelonoidis elephantopus) once endemic to Floreana Island on the neighboring island of Isabela. This finding adds to the growing cryptic diversity detected among these species in the wild. There also exists a large number of Galapagos tortoises in captivity of ambiguous origin. The recently accumulated population-level haplotypic and genotypic data now available for C. elephantopus add a critical reference population to the existing database of 11 extant species for investigating the origin of captive individuals of unknown ancestry. Methodology/Findings We reanalyzed mitochondrial DNA control region haplotypes and microsatellite genotypes of 156 captive individuals using an expanded reference database that included all extant Galapagos tortoise species as well as the extinct species from Floreana. Nine individuals (six females and three males) exhibited strong signatures of Floreana ancestry and a high probability of assignment to C. elephantopus as detected by Bayesian assignment and clustering analyses of empirical and simulated data. One male with high assignment probability to C. elephantopus based on microsatellite genotypic data also possessed a “Floreana-like” mitochondrial DNA haplotype. Significance Historical DNA analysis of museum specimens has provided critical spatial and temporal components to ecological, evolutionary, taxonomic and conservation-related research, but rarely has it informed ex situ species recovery efforts. Here, the availability of population-level genotypic data from the extinct C. elephantopus enabled the identification of nine Galapagos tortoise individuals of substantial conservation value that were previously misassigned to extant species of varying conservation status. As all captive individuals of C. elephantopus ancestry currently reside at a centralized breeding facility on Santa Cruz, these findings permit breeding efforts to commence in support of the reestablishment of this extinct species to its native range.

1. 1. The molecular weights, S values and base composition of the rRNAs of the Galapagos tortoise ( Testudo elephantus ) and of the turtles Pseudemys scripta elegans and Chrysemys picta marginata have been determined. 2. 2. The mol. wt (0·7 × 10 6 ) of the smaller rRNA subunit of all species was identical. 3. 3. The mol. wt and S values of the larger rRNA subunit were 1·53 × 10 6 and 27·0 for the Galapagos tortoise; 1·44 × 10 6 and 26·3 for Chrysemys ; and 1·46 × 10 6 and 26·6 for Pseudemys . 4. 4. All the rRNAs had a G + C content of about 60%. 5. 5. The mitochondrial rRNAs of the Galapagos tortoise and Pseudemys sedimented at about 17 and 13 S.

Somatic cell hybrids were produced between cultured fibroblasts from the Galapagos tortoise and the golden hamster using combined thermal and chemical selection. Hybrids between such disparate species may be useful in studying regulation of cellular growth and gene expression in higher organisms.

Summary Genes from recently extinct species can live on in the genomes of extant individuals of mixed ancestry. Recently, genetic signatures of the giant Galapagos tortoise once endemic to Floreana Island ( Chelonoidis elephantopus ) were detected within eleven hybrid individuals of otherwise pure Chelonoidis becki on Volcano Wolf, Isabela Island [1]. Movement of tortoises between islands by pirate and whaling ships was not uncommon during the 1800s [2], representing a likely mechanism by which individuals from Floreana were translocated to northern Isabela, despite being presumed extinct soon after Charles Darwin's historic voyage to the Galapagos Islands in 1835. These eleven hybrid individuals with C. elephantopus ancestry were thought to be the last genetic vestiges of a unique evolutionary lineage in the wild. Here, we report that reproductively mature purebred tortoises of the recently ‘extinct' C. elephantopus from Floreana Island are very likely still alive today, as identified and tracked through the genetic footprints left in the genomes of very recent hybrid offspring on Volcano Wolf. If found, these purebred C. elephantopus individuals could constitute core founders of a captive breeding program directed towards resurrecting this species.