Andrew M. Shedlock

The genome of the green anole lizard and a comparative analysis with birds and mammals

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

SINE insertions: powerful tools for molecular systematics.

Short interspersed repetitive elements, or SINEs, are tRNA-derived retroposons that are dispersed throughout eukaryotic genomes and can be present in well over 10(4) total copies. The enormous volume of SINE amplifications per organism makes them important evolutionary agents for shaping the diversity of genomes, and the irreversible, independent nature of their insertion allows them to be used for diagnosing common ancestry among host taxa with extreme confidence. As such, they represent a powerful new tool for systematic biology that can be strategically integrated with other conventional phylogenetic characters, most notably morphology and DNA sequences. This review covers the basic aspects of SINE evolution that are especially relevant to their use as systematic characters and describes the practical methods of characterizing SINEs for cladogram construction. It also discusses the limits of their systematic utility, clarifies some recently published misunderstandings, and illustrates the effective application of SINEs for vertebrate phylogenetics with results from selected case studies. BioEssays 22:148-160, 2000.

Retroposon Analysis of Major Cetacean Lineages: The Monophyly of Toothed Whales and the Paraphyly Of River Dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28–33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Origin of avian genome size and structure in non-avian dinosaurs

Avian genomes are small and streamlined compared with those of other amniotes by virtue of having fewer repetitive elements and less non-coding DNA. This condition has been suggested to represent a key adaptation for flight in birds, by reducing the metabolic costs associated with having large genome and cell sizes. However, the evolution of genome architecture in birds, or any other lineage, is difficult to study because genomic information is often absent for long-extinct relatives. Here we use a novel bayesian comparative method to show that bone-cell size correlates well with genome size in extant vertebrates, and hence use this relationship to estimate the genome sizes of 31 species of extinct dinosaur, including several species of extinct birds. Our results indicate that the small genomes typically associated with avian flight evolved in the saurischian dinosaur lineage between 230 and 250 million years ago, long before this lineage gave rise to the first birds. By comparison, ornithischian dinosaurs are inferred to have had much larger genomes, which were probably typical for ancestral Dinosauria. Using comparative genomic data, we estimate that genome-wide interspersed mobile elements, a class of repetitive DNA, comprised 5–12% of the total genome size in the saurischian dinosaur lineage, but was 7–19% of total genome size in ornithischian dinosaurs, suggesting that repetitive elements became less active in the saurischian lineage. These genomic characteristics should be added to the list of attributes previously considered avian but now thought to have arisen in non-avian dinosaurs, such as feathers, pulmonary innovations, and parental care and nesting.

Evolution of the salmonid mitochondrial control region

To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.

Phylogenomics of nonavian reptiles and the structure of the ancestral amniote genome

We report results of a megabase-scale phylogenomic analysis of the Reptilia, the sister group of mammals. Large-scale end-sequence scanning of genomic clones of a turtle, alligator, and lizard reveals diverse, mammal-like landscapes of retroelements and simple sequence repeats (SSRs) not found in the chicken. Several global genomic traits, including distinctive phylogenetic lineages of CR1-like long interspersed elements (LINEs) and a paucity of A-T rich SSRs, characterize turtles and archosaur genomes, whereas higher frequencies of tandem repeats and a lower global GC content reveal mammal-like features in Anolis. Nonavian reptile genomes also possess a high frequency of diverse and novel 50-bp unit tandem duplications not found in chicken or mammals. The frequency distributions of ≈65,000 8-mer oligonucleotides suggest that rates of DNA-word frequency change are an order of magnitude slower in reptiles than in mammals. These results suggest a diverse array of interspersed and SSRs in the common ancestor of amniotes and a genomic conservatism and gradual loss of retroelements in reptiles that culminated in the minimalist chicken genome.

Phylogenetics of modern birds in the era of genomics

In the 14 years since the first higher-level bird phylogenies based on DNA sequence data, avian phylogenetics has witnessed the advent and maturation of the genomics era, the completion of the chicken genome and a suite of technologies that promise to add considerably to the agenda of avian phylogenetics. In this review, we summarize current approaches and data characteristics of recent higher-level bird studies and suggest a number of as yet untested molecular and analytical approaches for the unfolding tree of life for birds. A variety of comparative genomics strategies, including adoption of objective quality scores for sequence data, analysis of contiguous DNA sequences provided by large-insert genomic libraries, and the systematic use of retroposon insertions and other rare genomic changes all promise an integrated phylogenetics that is solidly grounded in genome evolution. The avian genome is an excellent testing ground for such approaches because of the more balanced representation of single-copy and repetitive DNA regions than in mammals. Although comparative genomics has a number of obvious uses in avian phylogenetics, its application to large numbers of taxa poses a number of methodological and infrastructural challenges, and can be greatly facilitated by a ‘community genomics’ approach in which the modest sequencing throughputs of single PI laboratories are pooled to produce larger, complementary datasets. Although the polymerase chain reaction era of avian phylogenetics is far from complete, the comparative genomics era—with its ability to vastly increase the number and type of molecular characters and to provide a genomic context for these characters—will usher in a host of new perspectives and opportunities for integrating genome evolution and avian phylogenetics.

Developing markers for multilocus phylogenetics in non-model organisms: A test case with turtles

We present a strategy for phylogenetic marker development in non-model systems. Rather than using the traditional approach of comparing distantly related taxa to develop conserved primers for unknown species, we explore an alternative strategy that builds primers directly from a single, relatively well characterized species and applies those primers to increasingly distantly related taxa. We develop and test our protocol with turtles. Using a single BAC end-sequence library consisting of 3461 sequences totaling 2.43 million base pairs of data, we outline a procedure to flag repeat elements, followed by a BLAST approach to categorize sequences into high, low, and no similarity compartments compared to GenBank sequences. We developed and tested a panel of 96 primer pairs with a set of turtle tissues that forms a series of increasingly distantly related taxa with respect to the BAC reference species. Finally, we sequenced 11 of these newly discovered markers across a diverse set of 18 turtle species that spans the 210 million years of chelonian crown-group history and that includes representatives of most of the major clades of extant turtles. Our results indicate that large numbers of new, phylogenetically informative markers can be developed quickly and inexpensively from a single BAC, EST, or similar genomic resource, and that those markers provide reliable phylogenetic information across both shallow and deep levels of phylogenetic history. Our results also highlight the importance of screening for and managing repetitive elements found in randomly sequenced DNA fragments. We presume that our strategy should work well across any similarly divergent clade, suggesting that many-marker datasets can be developed quickly and efficiently for phylogenetic analysis.

Genome Evolution in Reptilia, the Sister Group of Mammals

The genomes of birds and nonavian reptiles (Reptilia) are critical for understanding genome evolution in mammals and amniotes generally. Despite decades of study at the chromosomal and single-gene levels, and the evidence for great diversity in genome size, karyotype, and sex chromosome diversity, reptile genomes are virtually unknown in the comparative genomics era. The recent sequencing of the chicken and zebra finch genomes, in conjunction with genome scans and the online publication of the Anolis lizard genome, has begun to clarify the events leading from an ancestral amniote genome--predicted to be large and to possess a diverse repeat landscape on par with mammals and a birdlike sex chromosome system--to the small and highly streamlined genomes of birds. Reptilia exhibit a wide range of evolutionary rates of different subgenomes and, from isochores to mitochondrial DNA, provide a critical contrast to the genomic paradigms established in mammals.

Retroposon Mapping in Molecular Systematics

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.