Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Chromosomal variation and perinatal mortality in San Diego zoo Soemmerring's gazelles.

Chromosomal translocations play a fundamental role in the evolution and speciation of antelopes (Antilopinae, Bovidae), with several species exhibiting polymorphism for centric fusions. For the past 35 years, the San Diego Zoo Global (SDZG) captive population of Soemmerring's gazelles has revealed complex karyotypes resulting from chromosomal translocations with diploid numbers ranging from 34 to 39. Poor reproductive performance of this species in captivity and elevated mortality the first month of life (perinatal) has been attributed to this chromosomal dynamism. We have extended the studies of karyotypic variation in the SDZG Soemmerring's gazelle population and analyzed the effect of chromosomal and genetic variation upon perinatal mortality. Karyotypes from 149 captive Soemmerring's gazelles were evaluated revealing two unreported autosomal combinations, now constituting a total of 15 distinct karyotypes for the 3 Robertsonian centric fusions originally described for this population. Among SDZG founders, distinct chromosomal variation and nuclear and mitochondrial genetic structure were detected corresponding to the institution of origin of the founders. Low levels of genetic distance and nucleotide diversity among individuals, in addition to high relatedness values, suggested that outbreeding is less of a concern than inbreeding for maintaining a sustainable captive population. Finally, analysis of karyotypes of offspring born into the SDZG Soemmerring's gazelle herds, in conjunction with the maternal karyotype showed association of chromosomal makeup with perinatal mortality. This supports the importance of continuing cytogenetic screening efforts, particularly to evaluate the presence of deleterious chromosomal rearrangements in stillborns.

Induced pluripotent stem cells from highly endangered species.

For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent extinction. We report generation of induced pluripotent stem cells (iPSCs) from two endangered species: a primate, the drill, Mandrillus leucophaeus and the nearly extinct northern white rhinoceros, Ceratotherium simum cottoni. iPSCs may eventually facilitate reintroduction of genetic material into breeding populations.

Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds.

Molecular phylogeny and chromosomal evolution of Alcelaphini (Antilopinae).

Robertsonian (Rb) translocations, in particular centric fusions, are thought to play a primary role in evolution and speciation of the Bovidae family. However, Rb fusions are often polymorphic within species, being suggested as phylogenetically uninformative characters. This work studies chromosome variation in 72 captive individuals of 6 species of Alcelaphini (Antilopinae): The hartebeest (genus Alcelaphus), hirola (Beatragus), black and blue wildebeests (Connochaetes), and the topi and bontebok (Damaliscus). We infer the phylogenic relationships among Alcelaphini species and determine patterns of chromosomal evolution using G-banded karyotypes and complete mitochondrial genome sequences. The molecular phylogeny showed an early divergence of Connochaetes, followed by the split of Alcelaphus plus Beatragus + Damaliscus as sister taxa. Mitochondrial and chromosomal phylogenies only differed in the position of the critically endangered Beatragus, likely due to homoplasic chromosome characters. Patterns of chromosome evolution, reconstructed using a probabilistic approach, suggest that chromosome changes leading to speciation in Alcelaphini do not exclusively involve consecutive reduction of diploid number through centric fusion but also the losses and reversions of Rb translocations in Beatragus and Damaliscus lineages. Our results provide evidence that complex scenarios of chromosomal rearrangements can be detected in relatively recent-diverged bovids, as in this group of antelopes.

An ancient icon reveals new mysteries: mummy DNA resurrects a cryptic species within the Nile crocodile.

The Nile crocodile (Crocodylus niloticus) is an ancient icon of both cultural and scientific interest. The species is emblematic of the great civilizations of the Nile River valley and serves as a model for international wildlife conservation. Despite its familiarity, a centuries-long dispute over the taxonomic status of the Nile crocodile remains unresolved. This dispute not only confounds our understanding of the origins and biogeography of the 'true crocodiles' of the crown genus Crocodylus, but also complicates conservation and management of this commercially valuable species. We have taken a total evidence approach involving phylogenetic analysis of mitochondrial and nuclear markers, as well as karyotype analysis of chromosome number and structure, to assess the monophyletic status of the Nile crocodile. Samples were collected from throughout Africa, covering all major bioregions. We also utilized specimens from museum collections, including mummified crocodiles from the ancient Egyptian temples at Thebes and the Grottes de Samoun, to reconstruct the genetic profiles of extirpated populations. Our analyses reveal a cryptic evolutionary lineage within the Nile crocodile that elucidates the biogeographic history of the genus and clarifies long-standing arguments over the species' taxonomic identity and conservation status. An examination of crocodile mummy haplotypes indicates that the cryptic lineage corresponds to an earlier description of C. suchus and suggests that both African Crocodylus lineages historically inhabited the Nile River. Recent survey efforts indicate that C. suchus is declining or extirpated throughout much of its distribution. Without proper recognition of this cryptic species, current sustainable use-based management policies for the Nile crocodile may do more harm than good.

Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination.

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny-based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co-evolved with body size. Multiple independent times smaller, shorter-lived species changed to having longer telomeres and expressing telomerase. Trade-offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human-like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.