Zoo animals as "proxy species" for threatened sister taxa: Defining a novel form of species surrogacy.

The vast number of species threatened with extinction, coupled with the limited resources to support them, results in the need to prioritize species for conservation action. Similarly, zoo collection managers must prioritize species for inclusion at their institutions, which are also limited by space and resources. While conservation status is one factor considered by zoos, weight is also given to qualitative features and practical considerations when evaluating the fit of different species. Resultantly, the species prioritized by zoos have limited overlap with those prioritized for conservation. Several recent studies have highlighted that the majority of species maintained in zoos are ranked globally as least concern. Given the centrality of conservation to the modern mission of zoos, there is value in identifying clear connections between non-threatened species in zoos and the roles they can play in conservation. Surrogate species approaches have been used in many instances to facilitate indirect conservation, and several distinctive types of surrogacy have been formally described. The aim of this study is to define a novel form of species surrogacy (i.e., "proxy species") that draws utility from non-threatened species maintained in zoos. A proxy species is here defined as a non-threatened species managed ex situ that can be used as a flagship for a related threatened species that is morphologically similar but not otherwise represented in zoos. The benefits of this approach and opportunities for its application are here reviewed. This concept will provide opportunities to enhance the value of pre-existing zoo collections, and ultimately, support conservation objectives.

Genotyping on the ark: A synthesis of genetic resources available for species in zoos.

Using molecular genetic information to guide population management can improve the sustainability of species in captivity. However, empirical population genetics has not been commonly applied to species management programs in zoos. One limitation may be the availability of genetic resources (e.g., markers, primers, etc.) for species held in zoos. To assess the extent to which species held in zoos have been studied using population genetics in the wild, we conducted a systematic literature review of close to 8,000 papers. We synthesized information on the availability and scale of population genetics studies across amphibian, bird, mammal, and reptile species held in zoos, and discussed their potential for informing ex situ management. We found that more than half of the species in zoos (52%) already have some genetic markers described in the literature specific for them, or a congeneric species, that could be further developed to aid the management of zoo populations, and the accumulation of these resources has been steady over the past decades. Furthermore, the proportion of species with genetic resources is even higher (62%) for species that are being managed through a formal breeding program in zoos. Our study provides encouraging results for captive program managers interested in integrating population genetics into ex situ management strategies.

De Novo Genome and Transcriptome Assembly of the Canadian Beaver (Castor canadensis).

The Canadian beaver (Castor canadensis) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon-gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology.

Delimiting shades of gray: phylogeography of the Northern Fulmar, Fulmarus glacialis.

The Northern Fulmar (Fulmarus glacialis) is a common tube-nosed seabird with a disjunct Holarctic range. Taxonomic divisions within the Northern Fulmar have historically been muddled by geographical variation notably including highly polymorphic plumage. Recent molecular analyses (i.e., DNA barcoding) have suggested that genetic divergence between Atlantic and Pacific populations could be on par with those typically observed between species. We employ a multigene phylogenetic analysis to better explore the level of genetic divergence between these populations and to test an old hypothesis on the origin of the modern distribution of color morphs across their range. Additionally, we test whether mutations in the melanocortin-1 receptor gene (MC1R) are associated with dark plumage in the Northern Fulmar. We confirmed that mitochondrial lineages in the Atlantic and Pacific populations are highly divergent, but nuclear markers revealed incomplete lineage sorting. Genetic divergence between these populations is consistent with that observed between many species of Procellariiformes and we recommend elevating these two forms to separate species. We also find that MC1R variation is not associated with color morph but rather is better explained by geographical divergence.

Frequency matrix approach demonstrates high sequence quality in avian BARCODEs and highlights cryptic pseudogenes.

The accuracy of DNA barcode databases is critical for research and practical applications. Here we apply a frequency matrix to assess sequencing errors in a very large set of avian BARCODEs. Using 11,000 sequences from 2,700 bird species, we show most avian cytochrome c oxidase I (COI) nucleotide and amino acid sequences vary within a narrow range. Except for third codon positions, nearly all (96%) sites were highly conserved or limited to two nucleotides or two amino acids. A large number of positions had very low frequency variants present in single individuals of a species; these were strongly concentrated at the ends of the barcode segment, consistent with sequencing error. In addition, a small fraction (0.1%) of BARCODEs had multiple very low frequency variants shared among individuals of a species; these were found to represent overlooked cryptic pseudogenes lacking stop codons. The calculated upper limit of sequencing error was 8 × 10(-5) errors/nucleotide, which was relatively high for direct Sanger sequencing of amplified DNA, but unlikely to compromise species identification. Our results confirm the high quality of the avian BARCODE database and demonstrate significant quality improvement in avian COI records deposited in GenBank over the past decade. This approach has potential application for genetic database quality control, discovery of cryptic pseudogenes, and studies of low-level genetic variation.

DNA barcoding birds: from field collection to data analysis.

As of February 2011, COI DNA barcode sequences (a 648-bp segment of the 5' end of the mitochondrial gene cytochrome c oxidase I, the standard DNA barcode for animals) have been collected from over 23,000 avian specimens representing 3,800 species, more than one-third of the world's avifauna. Here, we detail the methodology for obtaining DNA barcodes from birds, covering the entire process from field collection to data analysis. We emphasize key aspects of the process and describe in more detail those that are particularly relevant in the case of birds. We provide elemental information about collection of specimens, detailed protocols for DNA extraction and PCR, and basic aspects of sequencing methodology. In particular, we highlight the primer pairs and thermal cycling profiles associated with successful amplification and sequencing from a broad range of avian species. Finally, we succinctly review the methodology for data analysis, including the detection of errors (such as contamination, misidentifications, or amplification of pseudogenes), assessment of species resolution, detection of divergent intraspecific lineages, and identification of unknown specimens.

DNA barcode libraries provide insight into continental patterns of avian diversification.

BACKGROUND: The causes for the higher biodiversity in the Neotropics as compared to the Nearctic and the factors promoting species diversification in each region have been much debated. The refuge hypothesis posits that high tropical diversity reflects high speciation rates during the Pleistocene, but this conclusion has been challenged. The present study investigates this matter by examining continental patterns of avian diversification through the analysis of large-scale DNA barcode libraries. METHODOLOGY AND PRINCIPAL FINDINGS: Standardized COI datasets from the avifaunas of Argentina, the Nearctic, and the Palearctic were analyzed. Average genetic distances between closest congeners and sister species were higher in Argentina than in North America reflecting a much higher percentage of recently diverged species in the latter region. In the Palearctic genetic distances between closely related species appeared to be more similar to those of the southern Neotropics. Average intraspecific variation was similar in Argentina and North America, while the Palearctic fauna had a higher value due to a higher percentage of variable species. Geographic patterning of intraspecific structure was more complex in the southern Neotropics than in the Nearctic, while the Palearctic showed an intermediate level of complexity. CONCLUSIONS AND SIGNIFICANCE: DNA barcodes can reveal continental patterns of diversification. Our analysis suggests that avian species are older in Argentina than in the Nearctic, supporting the idea that the greater diversity of the Neotropical avifauna is not caused by higher recent speciation rates. Species in the Palearctic also appear to be older than those in the Nearctic. These results, combined with the patterns of geographic structuring found in each region, suggest a major impact of Pleistocene glaciations in the Nearctic, a lesser effect in the Palearctic and a mild effect in the southern Neotropics.

Searching for evidence of selection in avian DNA barcodes.

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species.

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae).

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.

DNA barcodes provide new evidence of a recent radiation in the genus Sporophila (Aves: Passeriformes).

The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.

Probing evolutionary patterns in neotropical birds through DNA barcodes.

BACKGROUND: The Neotropical avifauna is more diverse than that of any other biogeographic region, but our understanding of patterns of regional divergence is limited. Critical examination of this issue is currently constrained by the limited genetic information available. This study begins to address this gap by assembling a library of mitochondrial COI sequences, or DNA barcodes, for Argentinian birds and comparing their patterns of genetic diversity to those of North American birds. METHODOLOGY AND PRINCIPAL FINDINGS: Five hundred Argentinian species were examined, making this the first major examination of DNA barcodes for South American birds. Our results indicate that most southern Neotropical bird species show deep sequence divergence from their nearest-neighbour, corroborating that the high diversity of this fauna is not based on an elevated incidence of young species radiations. Although species ages appear similar in temperate North and South American avifaunas, patterns of regional divergence are more complex in the Neotropics, suggesting that the high diversity of the Neotropical avifauna has been fueled by greater opportunities for regional divergence. Deep genetic splits were observed in at least 21 species, though distribution patterns of these lineages were variable. The lack of shared polymorphisms in species, even in species with less than 0.5M years of reproductive isolation, further suggests that selective sweeps could regularly excise ancestral mitochondrial polymorphisms. CONCLUSIONS: These findings confirm the efficacy of species delimitation in birds via DNA barcodes, even when tested on a global scale. Further, they demonstrate how large libraries of a standardized gene region provide insight into evolutionary processes.

Diagnosing mitochondrial DNA diversity: applications of a sentinel gene approach.

Mitochondrial genomes show wide variation in their GC content. This study examines the correlations between mitochondrial genome-wide shifts in this feature and a fragment of the cytochrome c oxidase subunit I (COI) gene in animals, plants, and fungi. Because this approach utilizes COI as a sentinel, analyzing sequences from repositories such as GenBank and the Barcode of Life Data System (BOLD) can provide rapid insights into nucleotide usage. With this approach we probe nucleotide composition in a variety of taxonomic groups and establish the degree to which mitochondrial GC content varies among them. We then focus on two groups in particular, the classes Insecta and Aves, which possess the highest and lowest GC content, respectively. We establish that the sentinel approach provides strong indicators of mitochondrial GC content within divergent phyla (R values = 0.86-0.95, p < 0.001, in test cases) and provide evidence that selective pressures acting on GC content extend to noncoding regions of the plant and fungal mitochondrial genomes. We demonstrate that there is considerable variation in GC content of the mitochondrial genome within phyla and at each taxonomic level, leading to a substantial overlap zone in GC content between chordates and invertebrates. Our results provide a novel insight into the mitochondrial genome composition of animals, plants, and fungi and advocate this sentinel technique for the detection of rapid alterations in nucleotide usage as a measure of mitochondrial genome biodiversity.

Comprehensive DNA barcode coverage of North American birds.

DNA barcoding seeks to assemble a standardized reference library for DNA-based identification of eukaryotic species. The utility and limitations of this approach need to be tested on well-characterized taxonomic assemblages. Here we provide a comprehensive DNA barcode analysis for North American birds including 643 species representing 93% of the breeding and pelagic avifauna of the USA and Canada. Most (94%) species possess distinct barcode clusters, with average neighbour-joining bootstrap support of 98%. In the remaining 6%, barcode clusters correspond to small sets of closely related species, most of which hybridize regularly. Fifteen (2%) currently recognized species are comprised of two distinct barcode clusters, many of which may represent cryptic species. Intraspecific variation is weakly related to census population size and species age. This study confirms that DNA barcoding can be effectively applied across the geographical and taxonomic expanse of North American birds. The consistent finding of constrained intraspecific mitochondrial variation in this large assemblage of species supports the emerging view that selective sweeps limit mitochondrial diversity.