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High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1,2,3,4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences., We thank them for their permission to publish. A.R., S.K., B.P.W. and A.M.P. were supported by the Intramural Research Program of the NHGRI, NIH (1ZIAHG200398). A.R. was also supported by the Korea Health Technology R&D Project through KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI17C2098). S.A.M., I.B. and R.D. were supported by Wellcome Trust grant WT207492; W.C., M. Smith, Z.N., Y.S., J.C., S. Pelan, J.T., A.T., J.W. and Kerstin Howe by WT206194; L.H., F.M., Kevin Howe and P. Flicek by WT108749/Z/15/Z, WT218328/B/19/Z and the European Molecular Biology Laboratory. O.F. and E.D.J. were supported by Howard Hughes Medical Institute and Rockefeller University start-up funds for this project. J.D. and H.A.L. were supported by the Robert and Rosabel Osborne Endowment. M.U.-S. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (750747). F.T.-N., J. Hoffman, P. Masterson and K.C. were supported by the Intramural Research Program of the NLM, NIH. C.L., B.J.K., J. Kim and H.K. were supported by the Marine Biotechnology Program of KIMST, funded by the Ministry of Ocean and Fisheries, Republic of Korea (20180430). M.C. was supported by Sloan Research Fellowship (FG-2020-12932). S.C.V. was funded by a Max Planck Research Group award from the Max Planck Society, and a Human Frontiers Science Program (HFSP) Research grant (RGP0058/2016). T.M.L., W.E.J. and the Canada lynx genome were funded by the Maine Department of Inland Fisheries & Wildlife (F11AF01099), including when W.E.J. held a National Research Council Research Associateship Award at the Walter Reed Army Institute of Research (WRAIR). C.B. was supported by the NSF (1457541 and 1456612). D.B. was funded by The University of Queensland (HFSP - RGP0030/2015). D.I. was supported by Science Exchange Inc. (Palo Alto, CA). H.W.D. was supported by NSF grants (OPP-0132032 ICEFISH 2004 Cruise, PLR-1444167 and OPP-1955368) and the Marine Science Center at Northeastern University (416). G.J.P.N. and the thorny skate genome were funded by Lenfest Ocean Program (30884). M.P. was funded by the German Federal Ministry of Education and Research (01IS18026C). M. Malinsky was supported by an EMBO fellowship (ALTF 456-2016). The following authors’ contributions were supported by the NIH: S. Selvaraj (R44HG008118); C.V.M., S.R.F., P.V.L. (R21 DC014432/DC/NIDCD); K.D.M. (R01GM130691); H.C. (5U41HG002371-19); M.D. (U41HG007234); and B.P. (R01HG010485). D.G. was supported by the National Key Research and Development Program of China (2017YFC1201201, 2018YFC0910504 and 2017YFC0907503). F.O.A. was supported by Al-Gannas Qatari Society and The Cultural Village Foundation-Katara, Doha, State of Qatar and Monash University Malaysia. C.T. was supported by The Rockefeller University. M. Hiller was supported by the LOEWE-Centre for Translational Biodiversity Genomics (TBG) funded by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK). H.C. was supported by the NHGRI (5U41HG002371-19). R.H.S.K. was funded by the Max Planck Society with computational resources at the bwUniCluster and BinAC funded by the Ministry of Science, Research and the Arts Baden-Württemberg and the Universities of the State of Baden-Württemberg, Germany (bwHPC-C5). B.V. was supported by the Biomedical Research Council of A*STAR, Singapore. T.M.-B. was funded by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (864203), MINECO/FEDER, UE (BFU2017-86471-P), Unidad de Excelencia María de Maeztu, AEI (CEX2018-000792-M), a Howard Hughes International Early Career award, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C.T. was supported by the European Research Council (ERC-2012-StG311000) and an Irish Research Council Laureate Award. M.T.P.G. was supported by an ERC Consolidator Award 681396-Extinction Genomics, and a Danish National Research Foundation Center Grant (DNRF143). T.W. was supported by the NSF (1458652). J. M. Graves was supported by the Australian Research Council (CEO561477). E.W.M. was partially supported by the German Federal Ministry of Education and Research (01IS18026C). Complementary sequencing support for the Anna’s hummingbird and several genomes was provided by Pacific Biosciences, Bionano Genomics, Dovetail Genomics, Arima Genomics, Phase Genomics, 10X Genomics, NRGene, Oxford Nanopore Technologies, Illumina, and DNAnexus. All other sequencing and assembly were conducted at the Rockefeller University, Sanger Institute, and Max Planck Institute Dresden genome labs. Part of this work used the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov). We acknowledge funding from the Wellcome Trust (108749/Z/15/Z) and the European Molecular Biology Laboratory., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2018-000792-M).

High-quality reference genomes are fundamental tools for understanding population history, and can provide estimates of genetic and demographic parameters relevant to the conservation of biodiversity. The federally endangered Pacific pocket mouse (PPM), which persists in three small, isolated populations in southern California, is a promising model for studying how demographic history shapes genetic diversity, and how diversity in turn may influence extinction risk. To facilitate these studies in PPM, we combined PacBio HiFi long reads with Omni-C and Hi-C data to generate a de novo genome assembly, and annotated the genome using RNAseq. The assembly comprised 28 chromosome-length scaffolds (N50 = 72.6 MB) and the complete mitochondrial genome, and included a long heterochromatic region on chromosome 18 not represented in the previously available short-read assembly. Heterozygosity was highly variable across the genome of the reference individual, with 18% of windows falling in runs of homozygosity (ROH) >1 MB, and nearly 9% in tracts spanning >5 MB. Yet outside of ROH, heterozygosity was relatively high (0.0027), and historical Ne estimates were large. These patterns of genetic variation suggest recent inbreeding in a formerly large population. Currently the most contiguous assembly for a heteromyid rodent, this reference genome provides insight into the past and recent demographic history of the population, and will be a critical tool for management and future studies of outbreeding depression, inbreeding depression, and genetic load.

The vaquita is the most critically endangered marine mammal, with fewer than 19 remaining in the wild. First described in 1958, the vaquita has been in rapid decline for more than 20 years resulting from inadvertent deaths due to the increasing use of large‐mesh gillnets. To understand the evolutionary and demographic history of the vaquita, we used combined long‐read sequencing and long‐range scaffolding methods with long‐ and short‐read RNA sequencing to generate a near error‐free annotated reference genome assembly from cell lines derived from a female individual. The genome assembly consists of 99.92% of the assembled sequence contained in 21 nearly gapless chromosome‐length autosome scaffolds and the X‐chromosome scaffold, with a scaffold N50 of 115 Mb. Genome‐wide heterozygosity is the lowest (0.01%) of any mammalian species analysed to date, but heterozygosity is evenly distributed across the chromosomes, consistent with long‐term small population size at genetic equilibrium, rather than low diversity resulting from a recent population bottleneck or inbreeding. Historical demography of the vaquita indicates long‐term population stability at less than 5,000 (Ne) for over 200,000 years. Together, these analyses indicate that the vaquita genome has had ample opportunity to purge highly deleterious alleles and potentially maintain diversity necessary for population health., see also the Perspective by Annabel Whibley

Survival at high altitude requires adapting to extreme conditions such as environmental hypoxia. To understand high-altitude adaptations in a primate, we assembled the genome of the gelada (Theropithecus gelada), an endemic Ethiopian monkey, and complemented it with population resequencing, hematological, and morphometric data. Unexpectedly, we identified a novel karyotype that may contribute to reproductive isolation between gelada populations. We also identified genomic elements including protein-coding sequences and gene families that exhibit accelerated changes in geladas and may contribute to high-altitude adaptation. Our findings lend insight into mechanisms of speciation and adaptation while providing promising avenues for functional hypoxia research.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). We confirm that long-read sequencing technologies are essential for maximizing genome quality and that unresolved complex repeats and haplotype heterozygosity are major sources of error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

The bushbuck is the most widespread bovid species in Africa. Previous mitochondrial studies have revealed a polyphyletic pattern suggesting the possible existence of two distinct species. To assess this issue, we have sequenced 16 nuclear genes and one mitochondrial fragment (cytochrome b gene + control region) for most species of the tribe Tragelaphini, including seven bushbuck individuals belonging to the two divergent mtDNA haplogroups, Scriptus and Sylvaticus. Our phylogenetic analyses show that the Scriptus lineage is a sister-group of Sylvaticus in the nuclear tree, whereas it is related to Tragelaphus angasii in the mitochondrial tree. This mito-nuclear discordance indicates that the mitochondrial genome of Scriptus was acquired by introgression after one or several past events of hybridization between bushbuck and an extinct species closely related to T. angasii. The division into two bushbuck species is supported by the analyses of nuclear markers and by the karyotype here described for T. scriptus (2n = 57 M/58F), which is strikingly distinct from the one previously found for T. sylvaticus (2n = 33 M/34F). Molecular dating estimates suggest that the two species separated during the Early Pleistocene after an event of interspecific hybridization, which may have mediated massive chromosomal rearrangements in the common ancestor of T. scriptus.

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is the smallest and one of the most endangered rhinoceros species, with less than 100 individuals estimated to live in the wild. It was originally divided into three subspecies but only two have survived, D. sumatrensis sumatrensis (Sumatran subspecies), and D. s. harrissoni (Bornean). Questions regarding whether populations of the Sumatran rhinoceros should be treated as different management units to preserve genetic diversity have been raised, particularly in light of its severe decline in the wild and low breeding success in captivity. This work aims to characterize genetic differentiation between Sumatran rhinoceros subspecies using complete mitochondrial genomes, in order to unravel their maternal evolutionary history and evaluate their status as separate management units. We identified three major phylogenetic groups with moderate genetic differentiation: two distinct haplogroups comprising individuals from both the Malay Peninsula and Sumatra, and a third group from Borneo. Estimates of divergence time indicate that the most recent common ancestor of the Sumatran rhinoceros occurred approximately 360,000 years ago. The three mitochondrial haplogroups showed a common divergence time about 80,000 years ago corresponding with a major biogeographic event in the Sundaland region. Patterns of mitochondrial genetic differentiation may suggest considering Sumatran rhinoceros subspecies as different conservation units. However, the management of subspecies as part of a metapopulation may appear as the last resource to save this species from extinction, imposing a conservation dilemma.

The Zoonomia Project is investigating the genomics of shared and specialized traits in eutherian mammals. Here we provide genome assemblies for 131 species, of which all but 9 are previously uncharacterized, and describe a whole-genome alignment of 240 species of considerable phylogenetic diversity, comprising representatives from more than 80% of mammalian families. We find that regions of reduced genetic diversity are more abundant in species at a high risk of extinction, discern signals of evolutionary selection at high resolution and provide insights from individual reference genomes. By prioritizing phylogenetic diversity and making data available quickly and without restriction, the Zoonomia Project aims to support biological discovery, medical research and the conservation of biodiversity., This project was funded by NIH NHGRI R01HG008742 (E.K.K., B.B., D.P.G., R.S., J.T.-M., J.J., H.J.N., B.P. and J. Armstrong), Swedish Research Council Distinguished Professor Award (K.L.-T., V.D.M., E.M. and J.R.S.M.), Swedish Research Council grant 2018-05973 (K.L.-T.), Knut and Alice Wallenberg Foundation (K.L.-T., V.D.M., E.M. and J.R.S.M.), Uppsala University (K.L.-T., V.D.M., E.M., J.R.S.M., J.J., J. Alfoldi and L.G.), Broad Institute Next10 (L.G.), Gladstone Institutes (K.S.P.), NIH NHGRI 5R01HG002939 (A.F.A.S. and R.H.), NIH NHGRI 5U24HG010136 (A.F.A.S. and R.H.), NIH NHGRI 5R01HG010485 (B.P. and M.D.), NIH NHGRI 2U41HG007234 (B.P., M.D. and J. Armstrong), NIH NIA 5PO1AG047200 (V.N.G.), NIH NIA 1UH2AG064706 (V.N.G.), BFU2017-86471-P MINECO/FEDER, UE (T.M.-B.), Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya GRC 2017 SGR 880 (T.M.-B.), Howard Hughes International Early Career (T.M.-B.), European Research Council Horizon 2020 no. 864203 (T.M.-B.), Obra Social ‘La Caixa’ (T.M.-B.), BBSRC BBS/E/T/000PR9818, BBS/E/T/ 000PR9783 (W.H. and W.N.), BBSRC Core Strategic Programme Grant BB/P016774/1 (W.H., W.N. and F.D.), Sir Henry Dale Fellowship 200517/Z/16/Z jointly funded by the Wellcome Trust and the Royal Society (N.R.C.), FJCI-2016-29558 MICINN (D.J.), Prince Albert II Foundation of Monaco and Canada, Global Genome Initiative, Smithsonian Institution (M.N.), European Research Council Research Grant ERC-2012-StG311000 (E.C.T.), Irish Research Council Laureate Award (E.C.T.), UK Medical Research Council MR/P026028/1 (W.H. and W.N.), National Science Foundation DEB-1457735 (M.S.S.), National Science Foundation DEB-1753760 (W.J.M.), National Science Foundation IOS-2029774 (E.K.K. and D.P.G.), Robert and Rosabel Osborne Endowment (H.A.L. and J.D.), Swedish Research Council, FORMAS 221-2012-1531 (J.R.S.M.), NSF RoL: FELS: EAGER: DEB 1838283 (D.A.R.) and Academy of Finland grant to Center of Excellence in Tumor Genetics Research no. 312042 (T.K. and J.T.).

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. Zoo Biol. XX:XX–XX, 2015. © 2015 Wiley Periodicals, Inc.

A female Southern black rhinoceros (Diceros bicornis minor) calf died unexpectedly at less than 12 hr of age, after an uncomplicated birth and uneventful early postpartum period. Gross necropsy revealed a 15-cm full thickness cleft palate, a patent foramen ovale, and four septal defects ranging from 0.3 to 1 cm in diameter. Histologic findings did not reveal any significant abnormalities. Karyotyping did not indicate any significant numerical or structural chromosomal abnormalities.

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.

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.

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.

Controversial taxonomic relationships within Propithecus have consistently made conservation and management decisions difficult. We present a multidisciplinary phylogenetic analysis of Propithecus supporting the elevation of 4 subspecies to specific status: P. diadema perrieri → P. perrieri, P. diadema candidus → P. candidus, P. diadema edwardsi → P. edwardsi, and P. verreauxi coquereli→P. coquereli; leaving P. diadema diadema as P. diadema and P. verreauxi verreauxi as P. verreauxi.

We mapped the chromosomal homology of Pygathrix namaeus (douc) with human and other primates by in situ hybridization of human chromosome paints. The synteny of 3 human chromosomes (1, 2, 19) is fragmented in the douc karyotype and the 23 human probes (autosomes plus X) provided 26 signals. There are associations between human chromosomes 14/15, 21/22, and 1/19. Human chromosomes 1 and 19 are divided in two segments and associated on douc chromosomes 8 and 10. The fragmentation and association of human chromosomes 1 and 19 is best explained as the result of a reciprocal translocation, which occurs in all documented Asian colobines studied, but not in the African species Colobus guereza. However, the homologs to douc chromosome 10 in all other Asian documented colobines show an additional pericentric inversion. Our results indicate that Pygathrix nemeus is karyologically the most conservative colobine species yet studied and that this species probably diverged early after the separation of Asian and African Colobinae. The data reinforce the monophyly of the Colobinae and their division into an African and an Asian clade.

The genus Equus is unusual in that five of the ten extant species have documented centric fission (Robertsonian translocation) polymorphisms within their populations, namely E. hemionus onager, E. hemionus kulan, E. kiang, E. africanus somaliensis, and E. quagga burchelli. Here we report evidence that the polymorphism involves the same homologous chromosome segments in each species, and that these chromosome segments have homology to human chromosome 4 (HSA4). Bacterial artificial chromosome clones containing equine genes SMARCA5 (ECA2q21 homologue to HSA4q31. 21) and UCHL1 (ECA3q22 homologue to HSA4p13) were mapped to a single metacentric chromosome and two unpaired acrocentrics by FISH mapping for individuals possessing odd numbers of chromosomes. These data suggest that the polymorphism is either ancient and conserved within the genus or has occurred recently and independently within each species. Since these species are separated by 1–3 million years of evolution, this polymorphism is remarkable and worthy of further investigations.

Przewalski’s wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.

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.

For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent their extinction. Cellular reprogramming is a means to capture the genomes of individual animals as induced pluripotent stem cells (iPSCs), which may eventually facilitate reintroduction of genetic material into breeding populations. Here, we describe a method for generating iPSCs from fibroblasts of mammalian endangered species.

The California condor is the largest flying bird in North America and belongs to a group of New World vultures. Recovering from a near fatal population decline, and currently with only 197 extant individuals, the species remains listed as endangered. Very little genetic information exists for this species, although sexing methods employing chromosome analysis or W-chromosome specific amplification is routinely applied for the management of this monomorphic species. Keeping in mind that genetic conditions like chondrodystrophy have been identified, preliminary steps were undertaken in this study to understand the genome organization of the condor. This included an extensive cytogenetic analysis that provided (i) a chromosome number of 80 (with a likelihood of an extra pair of microchromosomes), and (ii) information on the centromeres, telomeres and nucleolus organizer regions. Further, a comparison between condor and chicken macrochromosomes was obtained by using individual chicken chromosome specific paints 1–9 and Z and W on condor metaphase spreads. Except for chromosomes 4 and Z, each of the chicken (GGA) macrochromosomes painted a single condor (GCA) macrochromosome. GGA4 paint detected complete homology with two condor chromosomes, viz., GCA4 and GCA9 providing additional proof that the latter are ancestral chromosomes in the birds. The chicken Z chromosome showed correspondence with both Z and W in the condor. The homology suggests that the condor sex chromosomes have not completely differentiated during evolution, which is unlike the majority of the non-ratites studied up till now. Overall, the study provides detailed cytogenetic and basic comparative information on condor chromosomes. These findings significantly advance the effort to study the chondrodystrophy that is responsible for over ten percent mortality in the condor.

Reprogramming to induced pluripotency of cells from the endangered silver-maned drill and the northern white rhinoceros is reported. Induced pluripotent stem cells from endangered species may prove useful for species preservation in the future. 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.

A 10-mo-old female red kangaroo (Macropus rufus) presented with a unilateral congenital corneal opacity OD. Complete ophthalmic examination revealed a shallow anterior chamber and a focal area of corneal edema with multiple persistent pupillary membranes extending from the iris colarette to the corneal endothelium adjacent to the edematous area of cornea. High-resolution B-scan ultrasound of the anterior segment showed an area consistent with thinning of Descemet's membrane in the area of corneal edema. Ophthalmic examination and ultrasound findings are consistent with a diagnosis of Peters anomaly, a form of anterior segment dysgenesis. An electroretinogram performed on the affected animal did not reveal any specific abnormalities. Karyotype analyses revealed a normal diploid number (2n = 20, -XX), with an abnormal pericentric inversion in the second largest chromosomal pair. The kangaroo exhibits mild compensated vision deficits in the affected eye. The maternal and paternal adult pairing has been discontinued in an effort to prevent future offspring anomalies.

A neonate male owl monkey (Aotus sp.) was identified cytogenetically as a hybrid after it failed to nurse and died. Phenotypically, the male parent possessed characteristics of the “gray-neck group,” and G-banded karyotypes identified him as Aotus lemurinus griseimembra (2n = 53), heterozygous for the centric fusion of chromosomes 13 and 14. The female parent belonged to the “red-neck group” and was identified cytogenetically as Aotus nancymaae (2n = 54). The neonate hybrid had 2n = 54 chromosomes with 13 homologous pairs of autosomes, 26 nonhomologous autosomes, and XY sex chromosomes. Thirteen of the nonhomologous chromosomes represented the paternal complement, and 13 were from the maternal complement. Chromosomal rearrangements occurring between the karyotypes of A. l. griseimembra and A. nancymaae were believed to include two paracentric inversions, a reciprocal translocation, and two complex rearrangements involving pericentric inversion, telocentromeric fusion, and centromeric adjustment. ...

A female bonobo (Pan paniscus) born at the San Diego Zoo exhibited inability to nurse and progressive weakness plus multiple congenital abnormalities including aural canal atresia and stenosis, malformed auricles, clenched hands, lordosis, agenesis of the caudal vertebra and cardiac abnormalities. Chromosome analysis identified the bonobo as being trisomic for chromosome 17, the homolog of human chromosome 18. Genotyping with human microsatellites suggested the extra chromosome was maternal in origin. In addition, a male lowland gorilla (Gorilla gorilla gorilla), also born at the zoo, exhibited postnatal growth retardation, facial dysmorphisms and small hands with short fingers. Karyotype analysis revealed the gorilla carried a deletion of the distal q arm of chromosome 3, the homolog of human chromosome 4. The phenotypic and karyotypic abnormalities found in the bonobo and gorilla were consistent with the characteristics of human trisomy 18 and human deletion 4q syndrome, respectively.

G- and C-banded karyotypes of the two extant species of the mammalian order Proboscidea are presented for the first time. Chromosome complements were 2n = 56 in both Loxodonta africana and Elephas maximus. Comparisons between the species demonstrated a high level of chromosome band homology, with 26 conserved autosomal pairs. The normal diploid karyotype of L. africana had 25 acrocentric/telocentric and two metacentric/submetacentric autosomal pairs. E. maximus differed by having one less acrocentric and one additional submetacentric pair due to either a heterochromatic arm addition or deletion involving autosomal pair 27. Several acrocentric autosomes of L. africana exhibited small short arms that were absent in homologous chromosomes of E. maximus. The X chromosomes in both species were large submetacentric elements and were homologous. However, the small acrocentric Y chromosomes differed; in E. maximus it was slightly larger and had more distinct G-bands than its counterpart in L. africana. Extant Elephantidae appear to be relatively conservative in their rates of chromosomal change compared to some other mammalian families. The high-quality banded karyotypes presented here should prove useful as references in future chromosome analyses of elephant populations and in comparative cytogenetic studies with other ungulate orders.

G- and C-banded karyotypes of four species of the genus Kobus were compared using the standard karyotype of Bos taurus. Chromosomal complements were 2n = 50–54 in K. ellipsiprymnus, 2n = 50 in K. kob, 2n = 48 in K. leche, and 2n = 52 in K. megaceros. The number of autosomal arms in all karyotypes was 58. Fifteen autosomal pairs were conserved among these four species, including the 1;19 and 2;25 centric fusions, and autosomal differences involved eight centric fusion rearrangements. Five centric fusions were each unique to a particular taxon: 3;10 (K. leche), 3;11 and 6;29 (K. kob), and 5;17 and 7;11 (K. ellipsiprymnus). The 4;7 fusion occurred in K. leche and K. megaceros, whereas the 5;13 fusion occurred in K. kob and K. leche; the 6;18 fusion was found in three species but was absent in K. kob. Differences between the X chromosomes of the four Kobus species were attributed to heterochromatic additions or deletions, and Y-chromosome differences may have been the result of pericentric inversion. G-banded karyotypes of putative K. l. leche and K. l. kafuensis appeared identical, as did C-banded karyotypes of the two subspecies. Karyotypes of K. e. ellipsiprymnus and K. e. defassa differed as a result of the 6;18 centric fusion, which was polymorphic in K. e. defassa, and the 7;11 centric fusion, which was polymorphic in K. e. ellipsiprymnus but absent in K. e. defassa. Several centric fusions were related by monobrachial chain-IV complexes; however, records of hybridization indicate that reproductive isolation between at least certain species of Kobus is incomplete. Karyotypic differences between K. ellipsiprymnus (including K. e. ellipsiprymnus and K. e. defassa), K. kob, K. leche, and K. megaceros support the validity of these taxa, as well as the need to manage them as separate populations.

G- and C-banded karyotypes of Damaliscus hunteri, D. lunatus and D. pygargus were compared using the standard karyotype of Bos taurus. Chromosomal complements were 2n = 36 in D. lunatus jimela, 2n = 38 in D. pygargus phillipsi and D. p. pygargus, and 2n = 44 in D. hunteri. The fundamental number in all karyotypes was 60. Among the three species of Damaliscus, seven autosomal pairs and the X chromosomes were conserved. Y-chromosome differences were attributed to heterochromatic additions or deletions. Banded karyotypes of the two subspecies of D. pygargus exhibited complete homology. Chromosomal complements of D. pygargus and D. lunatus differed by a simple centric fusion. However, karyotypes of D. pygargus and D. lunatus differed from D. hunteri by numerous centric fusions, several of which were related by monobrachial chain complexes. Between the karyotypes of D. hunteri and D. pygargus or D. lunatus, there were two chain complexes, one involving five chromosomes (chain V) and the other involving 12 in pygargus (chain XII) or 13 in lunatus (chain XIII). There were also two simple centric fusions between D. hunteri and D. lunatus/D. pygargus; acrocentric chromosomes 13, 15, 20 and 22 in D hunteri were fused as 13;15 and 20;22 in D. lunatus and D. pygargus.

Chevrotains are small forest-dwelling ruminants of the family Tragulidae. The chromosome number of the lesser Malay chevrotain was determined to be 2n = 32, NF = 64, G- and Q-banding allowed the identification of homologous chromosomes, and C-banding demonstrated the presence of pericentromeric, telomeric and interstitial constitutive heterochromatin. Q-band comparisons with domestic cattle revealed relatively few monobrachial chromosome band homologies. However, the smallest biarmed autosome of the chevrotain, chromosome 15, was determined to be cytogenetically homologus with the acrocentric chromosome 19 of cattle. A molecular cytogenetic analysis confirmed this putative chromosomal homology. In fact, molecular cytogenetic analyses indicate complete conservation of synteny among mouse deer chromosome 15, domestic cattle chromosome 19, domestic pig chromosome 12 and human chromosome 17. In the light of these molecular cytogenetic data and since mouse deer chromosome 15 is submetacentric and appears homologous in banding to submetacentric chromosome 12 of the domestic pig, these outgroup comparisons indicate that the acrocentric condition of cattle chromosome 19 has been derived by inversion. Since this derivative condition is present in the Antilocapridae, Bovidae, Cervidae and Giraffidae, it is a chromosomal synapomorphy that unites these advance ruminant families within the Artiodactyla.

How to conserve our planet’s rapidly disappearing biodiversity is one of the greatest challenges of our generation. Among terrestrial vertebrate taxa, amphibians are most at risk with 41% of all known species experiencing population declines and one-third threatened with extinction. Although many institutions have responded by establishing captive assurance colonies for several critically endangered amphibians, the resources provided by these conservation organizations will not be enough to save all species ‘at risk’ without a multi-pronged approach. Around the world, zoos, aquariums, governments, and conservation NGOs are beginning to establish amphibian gene banks to conserve, in perpetuity, the remaining extant genetic diversity for many of these critically endangered species. A suite of biomaterials has been targeted for cryoconservation including blood, cell cultures, tissues, spermatozoa, eggs, and embryos. Several international workshops on amphibian gene banking and assisted reproductive technologies have been held between 2010 and 2012, bringing together leading experts in the fields of amphibian ecology, physiology, and cryobiology to synthesize emerging trends for biobanking amphibian genetic resources, provide opportunities for collaboration, and discuss future research directions. The following review paper and summary will provide a synopsis of these international workshops, in particular the hopes, realities, and current challenges inherent to this applied research field.

The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual); three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota.

There are many methods for assessing chromosomal stability, including the classical cytogenetic approaches described here, SNP genotyping described in Chapter 14, and the SKY, and FISH methods described in the previous edition of this manual. These methods differ in resolution and the types of abnormalities they can detect. The normal resolution obtainable by classic cytogenetic methods is estimated to be in the megabase range, while single nucleotide polymorphism (SNP) can give 2 kb resolution (Chapter 14). However, more resolution is not necessarily better; for example, SNP genotyping cannot be used to detect balanced translocations or inversions. Here we describe a simple, straightforward method for classical karyotyping of hPSCs.

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.

Two-toed sloth species, Linnaeus's and Hoffmman's, are frequent residents of zoo collections in North America. However, species identification has always been problematic because of their large overlap in external morphology, which represents an obstacle to the captive breeding program. We describe here a PCR-based technique that allows species identification of two-toed sloths without requiring sequencing, by using a mitochondrial marker (COI gene) and restriction enzyme assay. We also report intra- and inter-specific patterns of chromosome variation in captive two-toed sloths. Molecularly, we identified 22 samples of Linnaeus's and Hoffmman's two-toed sloths corresponding to 14 and 8 individuals, respectively. One animal was identified as a hybrid using the nuclear gene Enam having alleles derived from both species. The chromosome number in Hoffman's two-toed sloths showed low variation ranging only between 50 and 51. In contrast, Linnaeus's two-toed sloths appeared to vary widely, with diploid numbers ranging from 53 to 67, suggesting distinct geographic groups. The species identification method presented here represents a low-cost easy-to-use tool that will help to improve management of the captive population of two-toed sloths.

Background: Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. \ud \ud Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. \ud \ud Results: Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. \ud \ud Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2(m) and 3(a) are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2(m) existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2(m) form, originally thought to be the derived condition, might actually be the ancestral one. \ud \ud Conclusion: Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease. \ud \ud In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

The order Perissodactyla, the group of odd-toed ungulates, includes three extant families: Equidae, Tapiridae, and Rhinocerotidae. The extremely rapid karyotypic diversification in perissodactyls has so far prevented the establishment of genome-wide homology maps between these three families by traditional cytogenetic approaches. Here we report the first genome-wide comparative chromosome maps of African rhinoceroses, four tapir species, four equine species, and humans. These maps were established by multidirectional chromosome painting, with paint probes derived from flow-sorted chromosomes of Equus grevyi, Tapirus indicus, and Ceratotherium simum as well as painting probes from horse and human. The Malayan tapir (Tapirus indicus), Baird's tapir (T. bairdii), mountain tapir (T. pinchaque), lowland tapir (T. terrestris), and onager (E. hemionus onager), were studied by cross-species chromosome painting for the first time. Our results, when integrated with previously published comparative chromosome maps of the other perissodactyl species, have enabled the reconstruction of perissodactyl, ceratomorph, and equid ancestral karyotypes, and the identification of the defining evolutionary chromosomal rearrangements along each lineage. Our results allow a more reliable estimate of the mode and tempo of evolutionary chromosomal rearrangements, revealing a striking switch between the slowly evolving ceratomorphs and extremely rapidly evolving equids.

Although much is known about genetic variation in human and African great ape (chimpanzee, bonobo, and gorilla) genomes, substantially less is known about variation in gene-expression profiles within and among these species. This information is necessary for defining transcriptional regulatory networks that contribute to complex phenotypes unique to humans or the African great apes. We took a systematic approach to this problem by investigating gene-expression profiles in well-defined cell populations from humans, bonobos, and gorillas. By comparing these profiles from 18 human and 21 African great ape primary fibroblast cell lines, we found that gene-expression patterns could predict the species, but not the age, of the fibroblast donor. Several differentially expressed genes among human and African great ape fibroblasts involved the extracellular matrix, metabolic pathways, signal transduction, stress responses, as well as inherited overgrowth and neurological disorders. These gene-expression patterns could represent molecular adaptations that influenced the development of species-specific traits in humans and the African great apes.

Fibroblast cultures of a skin biopsy from an adult intact male Siberian tiger (Panthera tigris altaica) revealed an abnormal standard and G-banded karyotype diploid chromosome number of 2n = 39 XXY due to an extra sex chromosome as opposed to the expected 2n = 38 XY. The tiger was euthanatized 1 yr later due to acute multifocal intervertebral disc disease. Histopathology of the reproductive tract demonstrated a paucity of seminiferous tubules and these were devoid of spermatagonia. An increase in fibrous connective tissue was noted in sections of the prostate and epididymis, and expansion of the fibrous interstitium was observed in the testes.