The adult phenotype of an organism is the result of its genotype, the environment, and the interaction between the two. Assessing the relative contribution of these factors to the final adult phenotype continues to occupy researchers. Studies have shown clutch effects early in development but few have investigated the persistence of clutch effects on a longer time scale. Five clutches of American alligators were reared for 1 year in a common environment then assessed for the presence of clutch effects as they related to morphological and physiological characteristics. After 1 year, significant clutch effects were evident in all size related variables despite open access to food. Additionally, lung and liver masses remained different between clutches after animal mass was taken into account. Although clutch had no effect on resting heart rate, it significantly contributed to mean arterial pressure. During swimming and exhaustive exercise, the resulting respiratory and metabolic acidoses were strongly dependent on clutch. Therefore, while the environment can have significant influences on the American alligator from hatching to death, the measureable contribution of genetics to the morphology and physiology of the organism remains evident, even after 1 year of common rearing conditions. It behooves researchers to acknowledge and control for clutch effects when designing experiments.

Molecular systematists generally rely on computer algorithms to establish the alignment of DNA sequences. However, when alignment regions are characterized by multiple insertions and deletions, these gap-filled stretches of DNA are often excised before phylogenetic reconstruction. This exclusion of systematic data is generally determined by subjective criteria. We explore a replicable methodology in which the comparison of several multiple sequence alignments can be used to eliminate regions of unstable sequence alignment. Using crocodilian and insect mitochondrial (mt) ribosomal (r) DNA as examples, we caution against the removal of sequence data prior to phylogenetic reconstruction.

The single-copy RAG-1 gene is found throughout higher vertebrates and consists of a single 3.1-kb exon without intervening introns. A 2.9-kb region of the RAG-1 locus was sequenced for 14 basal taxa of birds plus the crocodylian outgroups Alligator and Gavialis. Phylogenetic analysis of the sequences supported the hypothesis that the deepest evolutionary split in extant birds separates paleognaths from neognaths. A deep division among neognaths separates the chicken- and duck-like birds ("galloanserines") from a clade consisting of all other birds ("plethornithines"). The relationships of these three basal clades in Aves were supported by high bootstrap (98 to 100%) and large decay index values (above 14). Additionally, the plethornithine clade is characterized by a 15-bp (five-codon) synapomorphic deletion relative to all other birds. RAG-1 evolves slowly, with a number of properties favoring its phylogenetic utility, including rarity of indels, minimal saturation of transition changes at 3rd positions of codons, nearly constant base composition across taxa, and no asymmetry in directional patterns of reconstructed change. However, RAG-1 does not evolve in a clocklike manner, suggesting that this gene cannot easily be used for estimating ages of ancient lineages.

The classical phylogeny of living reptiles pairs crocodilians with birds, tuataras with squamates, and places turtles at the base of the tree. New evidence from two nuclear genes, and analyses of mitochondrial DNA and 22 additional nuclear genes, join crocodilians with turtles and place squamates at the base of the tree. Morphological and paleontological evidence for this molecular phylogeny is unclear. Molecular time estimates support a Triassic origin for the major groups of living reptiles.

Phylogenetic analyses of non-protein-coding nucleotide sequences such as ribosomal RNA genes, internal transcribed spacers, and introns are often impeded by regions of the alignments that are ambiguously aligned. These regions are characterized by the presence of gaps and their uncertain positions, no matter which optimization criteria are used. This problem is particularly acute in large-scale phylogenetic studies and when aligning highly diverged sequences. Accommodating these regions, where positional homology is likely to be violated, in phylogenetic analyses has been dealt with very differently by molecular systematists and evolutionists, ranging from the total exclusion of these regions to the inclusion of every position regardless of ambiguity in the alignment. We present a new method that allows the inclusion of ambiguously aligned regions without violating homology. In this three-step procedure, first homologous regions of the alignment containing ambiguously aligned sequences are delimited. Second, each ambiguously aligned region is unequivocally coded as a new character, replacing its respective ambiguous region. Third, each of the coded characters is subjected to a specific step matrix to account for the differential number of changes (summing substitutions and indels) needed to transform one sequence to another. The optimal number of steps included in the step matrix is the one derived from the pairwise alignment with the greatest similarity and the least number of steps. In addition to potentially enhancing phylogenetic resolution and support, by integrating previously nonaccessible characters without violating positional homology, this new approach can improve branch length estimations when using parsimony.

The complete mitochondrial genome of the alligator, Alligator mississippiensis, was sequenced. The size of the molecule is 16,642 nucleotides. Previously reported rearrangements of tRNAs in crocodile mitochondrial genomes were confirmed and, relative to mammals, no other deviations of gene order were observed. The analysis of protein-coding genes of the alligator showed an evolutionary rate that is roughly the same as in mammals. Thus, the evolutionary rate in the alligator is faster than that in birds as well as that in cold-blooded vertebrates. This contradicts hypotheses of constant body temperatures or high metabolic rate being correlated with elevated molecular evolutionary rates. It is commonly acknowledged that birds are the closest living relatives to crocodiles. Birds and crocodiles represent the only archosaurian survivors of the mass extinction at the Cretaceous/Tertiary boundary. On the basis of mitochondrial protein-coding genes, the Haemothermia hypothesis, which defines birds and mammals as sister groups and thus challenges the traditional view, could be rejected. Maximum-likelihood branch length data of amino acid sequences suggest that the divergence between the avian and crocodilian lineages took place at approximately equal to 254 MYA.

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)-tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and-loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.

The process of multiple sequence alignment provides homology statements for the phylogenetic analysis of molecular data. Unfortunately, multiple alignments are frequently nonunique. Two sources of these multiple alignments are analysis based on different sets of alignment parameter values (gap:change cost ratios) and nonunique equally costly alignments based on a single set of alignment parameters. By "eliding" these individual alignments into a single grand alignment, phylogeny that is weighted toward those positions that align more consistently can be reconstructed. Positions that show greater variation among alignments will be relatively downweighted. The technique results in a weighting procedure that is a posteriori and based on the evidence established from the original sequence alignments.

The complete mitochondrial genomes of two reptiles, the common iguana (Iguana iguana) and the caiman (Caiman crocodylus), were sequenced in order to investigate phylogenetic questions of tetrapod evolution. The addition of the two species allows analysis of reptilian relationships using data sets other than those including only fast-evolving species. The crocodilian mitochondrial genomes seem to have evolved generally at a higher rate than those of other vertebrates. Phylogenetic analyses of 2889 amino-acid sites from 35 mitochondrial genomes supported the bird-crocodile relationship, lending no support to the Haematotherma hypothesis (with birds and mammals representing sister groups). The analyses corroborated the view that turtles are at the base of the bird-crocodile branch. This position of the turtles makes Diapsida paraphyletic. The origin of the squamates was estimated at 294 million years (Myr) ago and that of the turtles at 278 Myr ago. Phylogenetic analysis of mammalian relationships using the additional outgroups corroborated the Marsupionta hypothesis, which joins the monotremes and the marsupials to the exclusion of the eutherians.

In mammals, birds and reptiles the morphological development of the gonads appear to be conserved. This conservation is evident despite the different sex determining switches employed by these vertebrate groups. Mammals exhibit chromosomal sex determination (CSD) where the key sex determining switch is the Y-linked gene, SRY. Although SRY is the trigger for testis determination in mammals, it is not conserved in other vertebrate groups. However, a gene closely related to SRY, the highly conserved transcription factor, SOX9, plays an important role in the testis pathway of mammals and birds. In contrast to the CSD mechanism evident in mammals and birds, many reptiles exhibit temperature dependent sex determination (TSD) where the egg incubation temperature triggers sex determination. Here we examine the expression of SOX9 during gonadogenesis in the American alligator,(Alligator mississippiensis), a reptile that exhibits TSD. Alligator SOX9 is expressed in the embryonic testis but not in the ovary. However, the timing of SOX9 upregulation in the developing testis is not consistent with a role for this gene in the early stages of alligator sex determination. Since SOX9 upregulation in male embryos coincides with the structural organisation of the testis, SOX9 may operate farther downstream in the vertebrate sex differentiation pathway than previously postulated.