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e avian tree of life at all levels stands to benefit dramatically
from the orders-of-magnitude more sequence data available from NGSMs. Many studies have shown that phylogenetic reso- lution and accuracy are improved by increasing the numbers of loci and taxa sampled (e.g., Zwickl and Hillis , Rokas et al.
, Prasad et al. ). Previously unresolved avian relation-
ships (e.g., placement of Cathartidae and Otididae; Hackett et al.
), particularly those that involve rapid evolutionary radia-
tions (e.g., Hawaiian honeycreepers; Fleischer et al. ), may be more readily resolved with the application of NGSMs to obtain massive amounts of sequence. Such “phylogenomic” analyses of rapid radiations are likely to provide insight into processes of lin- eage sorting, whereas studying adaptive radiations with large data sets can also provide information about functional or adaptive variation. Phylogenomics can also be applied to selected groups (e.g., ducks, warblers, and hummingbirds) to study processes of hybridization.
To date, only a few studies (all non-avian; e.g., Moore et al.
, Willerslev et al. ) have employed NGSMs for phylo-
genetic analysis, and none has utilized the tagging methods de- scribed above. In a study that addressed the close relationships among extinct and extant rhinoceroses, Willerslev et al. () used next-generation sequencing of whole mitochondrial genomes from the black, woolly, Javan, and Sumatran rhinoceroses. is data set included > kilobases (kbp) of mitochondrial data and provided strong support for sister relationships within the rhinoc- eros clade, something that studies based on smaller data sets had been unable to provide. However, higher-level relationships were not resolved with this data set, which suggests a hard polytomy in this rapid radiation that should be further investigated with data from nuclear loci.
As tagging methods become routine, next-generation se- quence data of whole mitochondrial genomes and many nuclear loci are likely to become the standard in phylogenetic studies, so we may be perched on the verge of a phylogenomics era in ornithology.
Molecular evolution and comparative Genomics
Patterns and processes of sequence evolution—including evolu- tionary rates of nucleotide and amino acid change, nucleotide and codon usage bias, and insertion-deletion patterns—can be investi- gated to a deeper level with the massive data sets made possible by NGSMs. Next-generation sequencing methods also provide a way to investigate the unique patterns of evolution in avian-specific microchromosomes and macrochromosomes on a large scale. With the availability of multiple complete avian genomes, we an- ticipate new insights into the distribution of genes and recombi- nation between the two chromosome types as well as evolution or generation of novel avian microchromosomes. Difficulties inher- ent to previous genome-sequencing technologies contributed to the incomplete status of the Red Jungle Fowl (i.e., chicken, Gallus gallus) genome. e missing sequence, predominantly on micro- chromosomes (%), is being targeted with NGSMs at the Genome Center at Washington University.
Comparative avian genomics, facilitated by the ease and re- duced cost of genome sequencing with NGSMs, has made great strides in recent years, leading to genomic resources for the
chicken, Wild Turkey (Meleagris gallopavo), Zebra Finch (Tae- niopygia guttata), California Condor (Gymnogyps californianus), and White-throated Sparrow (Zonotrichia albicollis) (Romanov et al. ). A comparative genomic approach is extraordinarily useful for identifying functional loci related to morphological, be- havioral, and physiological variation and thus enables us to better understand the process of avian evolution. For instance, sequenc- ing multiple genomes of diverse taxa from an adaptive radiation such as the Galapagos finches or Hawaiian honeycreepers may identify the genes responsible for particular bill morphologies and other phenotypic traits. Furthermore, genome sequences for threatened taxa can be useful in developing comprehensive con- servation plans that increase genetic resistance to known threats. For example, Romanov et al. () performed cDNA se- quencing of a fibroblast cell line in California Condors and found that % of the reads were homologous with chicken genes, map- ping to nearly all chicken chromosomes. Further analysis of this data set and additional transcriptomes is expected to identify the mutation(s) responsible for a heritable embryonic lethal condition (chondrodystrophy). Genomic data appear to be useful for manag- ing genetic diversity in the California Condor and the goal of es- tablishing a viable, self-sustaining population. Indeed, it is hard to overestimate the potential benefits of comparative avian genom- ics to species conservation and the study of avian evolution. With the advent of NGSMs, avian comparative genomics will likely cre- scendo to the forefront of studies of avian evolution.
conservation and Population Genetics
Next-generation sequencing methods can contribute broadly to conservation and population genetic studies of birds (Romanov et al. ). Although we believe that whole-genome sequencing will be used sparingly for such studies in the near future (mainly be- cause such studies usually require large sample sizes of individuals but do not usually have the large budgets associated with medical or agricultural applications), NGSMs can still be very useful for developing variable markers such as microsatellites (Allentoft et al. ) and single-nucleotide polymorphisms (SNPs; e.g., No- vaes et al. , Vera et al. ) for non-model organisms. ese variable markers can then be used for subsequent high-resolution analyses of population samples at lower cost.
Methods to obtain microsatellite sequences can include di- rect shotgun libraries or, preferably, enrichment procedures to in- crease the representation of microsatellite sequences within the pool of DNA fragments to be sequenced (Santana et al. ). e enrichment procedures followed by Santana et al. () produced
microsatellite loci for three species (a wasp, a nematode, and
a fungus) from a total of only . Mbp of sequence (–% of the contigs > bp contained a usable microsatellite locus). e amount of sequence they obtained is a bit more than can be se- quenced on one th of a plate on a single GS-FLX run or about one third of one th of a plate on a Titanium run (at a cost of less than $, at most core facilities). Allentoft et al. () were able to obtain microsatellite sequences from shot- gun (unenriched) sequence derived from DNA extracted from bone of a late Holocene moa, Pachyornis elephantopus. ey used the sequences to design primers for a microsatellite locus that was then successfully amplified from DNA extracted from bones representing three moa genera.