AFLP markers resolve intra-specific relationships and infer genetic structure among lineages of the canyon treefrog, Hyla arenicolor

The canyon treefrog, Hyla arenicolor, is a wide-ranging hylid found from southwestern US into southern Mexico. Recent studies have shown this species to have a complex evolutionary history, with several phylogeographically distinct lineages, a probable cryptic species, and multiple episodes of mitochondrial introgression with the sister group, the H. eximia complex. We aimed to use genome wide AFLP markers to better resolve relationships within this group. As in other studies, our inferred phylogeny not only provides evidence for repeated mitochondrial introgression between H. arenicolor lineages and H. eximia/H. wrightorum, but it also affords more resolution within the main H. arenicolor clade than was previously achieved with sequence data. However, as with a previous study, the placement of a lineage of H. arenicolor whose distribution is centered in the Balsas Basin of Mexico remains poorly resolved, perhaps due to past hybridization with the H. eximia complex. Furthermore, the AFLP data set shows no differentiation among lineages from the Grand Canyon and Colorado Plateau despite their large mitochondrial sequence divergence. Finally, our results infer a well-supported sister relationship between this combined Colorado Plateau/Grand Canyon lineage and the Sonoran Desert lineage, a relationship that strongly contradicts conclusions drawn from the mtDNA evidence. Our study provides a basis for further behavioral and ecological speciation studies of this system and highlights the importance of multi-taxon (species) sampling in phylogenetic and phylogeographic studies.     

The comparative phylogeography of neotropical mammals: patterns of intraspecific mitochondrial DNA variation among bats contrasted to nonvolant small mammals

The major aim of this study was to compare the phylogeographic patterns of codistributed bats and small nonvolant Neotropical mammals. Cytochrome b sequences (mitochondrial DNA) were obtained for a total of 275 bats representing 17 species. The tissue samples were collected in coastal Brazil, and were available from Mexico and the Guyana. The study concentrates on four species (Artibeus lituratus, Carollia perspicillata, Sturnira lilium and Glossophaga soricina) which were well represented. The other 13 species were sequenced to test the generality of the patterns observed. In general, sequence divergence values within species were low, with most bat species presenting less than 4% average sequence divergence, and usually between 1 and 2.5%. Clades of highly similar haplotypes enjoyed broad distribution on a continental scale. These clades were not usually geographically structured, and at a given locality the number of haplotypes was high (8-10). As distance increased, some moderately divergent clades were found, although the levels of divergence were low. This suggests a geographical effect that varied depending on species and scale. Small nonvolant mammals almost invariably have high levels of sequence divergence (> 10%) for cytochrome b over much shorter distances (< 1000 km). The grain of intraspecific variation found in small nonvolant mammals is much finer than in bats. Low levels of geographical structuring cannot be attributed to a slower evolutionary rate of bat DNA in relation to other mammalian taxa. The phylogeographic pattern of bats contrasts sharply with the pattern found for Neotropical rodents and marsupials.     

How many species of Hipposideros have occurred on Madagascar since the Late Pleistocene?

Populations of the Malagasy Hipposideros commersoni (family Hipposideridae) are threatened by deforestation and hunting. Maximum likelihood and Bayesian analysis of 148 cytochrome b sequences found this species to be paraphyletic and composed of three well‐supported monophyletic clades. Clades B and C form a monophyletic lineage that can be referred to H. commersoni; these two clades are separated by 6% sequence variation. Clade A represents a distinct evolutionary lineage separate (9–11% average sequence divergence) from H. commersoni (clades B and C) and is named herein as a new species, H ipposideros cryptovalorona sp. nov. In the phylogeny presented herein, this species is strongly associated with the outgroup taxa Hipposideros gigas and Hipposideros vittatus, both restricted to Africa. External, cranial and dental measurements taken from the same individuals used in the molecular study indicate no clear distinction in morphology amongst these three clades; this includes noseleaf structure and craniodental characteristics. Principal component analyses showed limited separation of the three clades. Comparison to a Quaternary fossil species from north‐west Madagascar, Hipposideros besaoka, found little morphological overlap between any of the three clades and this extinct species. Hence, at least three species of Hipposideros have occurred on Madagascar since the Late Pleistocene, two extant (H. commersoni s.s. and H. cryptovalorona sp. nov.) and one extinct (H. besaoka). © 2015 The Linnean Society of London     

SPECIATION BY POLYPLOIDY IN TREEFROGS: MULTIPLE ORIGINS OF THE TETRAPLOID,HYLA VERSICOLOR

Speciation by polyploidy is rare in animals, yet, in vertebrates, there is a disproportionate concentration of polyploid species in anuran amphibians. Sequences from the cytochrome b gene of the mitochondrial DNA (mtDNA) were used to determine phylogenetic relationships among 37 populations of the diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and Hyla versicolor. The diploid species, H. chrysoscelis, consists of an eastern and a western lineage that have 2.3% sequence divergence between them. The tetraploid species, H. versicolor, had at least three separate, independent origins. Two of the tetraploid lineages are more closely related to one or the other of the diploid lineages (0.18%–1.4% sequence divergence) than they are to each other (1.9%–3.4% sequence divergence). The maternal ancestor of the third tetraploid lineage is unknown. The phylogenetic relationships between the two species and among lineages within each species support the hypothesis of multiple origins of the tetraploid lineages.     

Genomic structure of the luciferase gene and phylogenetic analysis in the Hotaria-group fireflies

The luminescent fireflies have species specific flash patterns, being recognized as sexual communication. The luciferase gene is the sole enzyme responsible for bioluminescence. We describe here the complete nucleotide sequence and the exon-intron structure of the luciferase gene of the Hotaria-group fireflies, H. unmunsana, H. papariensis and H. tsushimana. The luciferase gene of the Hotaria-group firefly including the known H. parvula spans 1950 bp and consisted of six introns and seven exons coding for 548 amino acid residues, suggesting highly conserved structure among the Hotaria-group fireflies. Although only one luciferase gene was cloned from H. papariensis, each of the two sequences of the gene was found in H. unmunsana (U1 and Uc) and H. tsushimana (T1 and T2). The amino acid sequence divergence among H. unmunsana, H. papariensis, and H. tsushimana only ranged from zero to three amino acid residues, but H. parvula differed by 10-11 amino acid residues from the other Hotaria-group fireflies, suggesting a divergent relationship of this species. Phylogenetic analysis using the deduced amino acid sequences of the luciferase gene resulted in a monophyletic group in the Hotaria excluding H. parvula, suggesting a close relationship among H. unmunsana, H. papariensis and H. tsushimana. Additionally, we also analyzed the mitochondrial cytochrome oxidase I (COI) gene of the Hotaria-group fireflies. The deduced amino acid sequence of the COI gene of H. unmunsana was identical to that of H. papariensis and H. tsushimana, but different by three positions from H. parvula. In terms of nucleotide sequences of the COI gene, intraspecific sequence divergence was sometimes larger than interspecies level, and phylogenetic analysis placed the three species into monophyletic groups unresolved among them, but excluded H. parvula. In conclusion, our results suggest that H. unmunsana, H. papariensis and H. tsushimana are very closely related or might be an identical species, at least based on the luciferase and COI genes.     

COMPARATIVE GENOMIC AND POPULATION GENETIC ANALYSES INDICATE HIGHLY POROUS GENOMES AND HIGH LEVELS OF GENE FLOW BETWEEN DIVERGENT HELIANTHUS SPECIES

While speciation can be found in the presence of gene flow, it is not clear what impact this gene flow has on genome- and range-wide patterns of differentiation. Here we examine gene flow across the entire range of the common sunflower, H. annuus , its historically allopatric sister species H. argophyllus and a more distantly related, sympatric relative H. petiolaris . Analysis of genotypes at 26 microsatellite loci in 1015 individuals from across the range of the three species showed substantial introgression between geographically proximal populations of H. annuus and H. petiolaris , limited introgression between H. annuus and H. argophyllus , and essentially no gene flow between the allopatric pair, H. argophyllus and H. petiolaris. Analysis of sequence divergence levels among the three species in 1420 orthologs identified from EST databases identified a subset of loci showing extremely low divergence between H. annuus and H. petiolaris and extremely high divergence between the sister species H. annuus and H. argophyllus , consistent with introgression between H. annuus and H. petiolaris at these loci. Thus, at many loci, the allopatric sister species are more genetically divergent than the more distantly related sympatric species, which have exchanged genes across much of the genome while remaining morphologically and ecologically distinct.     

Haematoloechus danbrooksi n. sp. (Digenea: Plagiorchioidea) from Rana vaillanti from Los Tuxtlas,Veracruz, Mexico

Haematoloechus danbrooksi n. sp. from the lungs of Rana vaillanti in Veracruz state, Mexico, was found. The new species is most similar morphologically to H. medioplexus, H. parviplexus, and H. meridionalis in having a ventral sucker less than half the diameter of the oral sucker. It differs from these species by the extension of the vitellaria, which are shorter in the new species, and in the shape of the tegumental spines, which are blunt in the new species. It differs from all other known species of Haematoloechus in the distribution of the uterine loops that are arranged diagonally and that present several short, extracecal. longitudinal loops in the postacetabular region. The new species shows 1.2% sequence divergence in partial 28S sequence with respect to H. medioplexus, 1.1% with H. parviplexus. and 2.5% with H. meridionalis, sequence divergences complementing the morphological differences.     

Spawning substrata are important for breeding habitat selection but do not determine premating reproductive isolation in three sympatric Hexagrammos species

Habitat use and spawning substrata were surveyed to characterize the contribution of habitat divergence to reproductive isolation in greenling Hexagrammos species. The spawning substrata and microhabitat in breeding territories differed amongst the three Hexagrammos species studied: H. octogrammus, H. agrammus and H. otakii used small red algae, surfgrass and bryozoans, respectively, as spawning substrata, and breeding territories were established in areas where those substrata were abundant. In contrast, non-territorial individuals were observed in a comparatively wider range of habitats than conspecific territories. Consequently, the distributions of non-territorial individuals of the three species partially overlapped. Since hybrids have been frequently collected, the difference in spawning substrata and the subsequent microhabitat divergence in breeding territories do not prevent females from encountering males of other species. Thus, in addition to habitat divergence, other factors such as behavioural differentiation may be needed to complete premating reproductive isolation amongst these three Hexagrammos species.     

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.      

Molecular evolution of the nontandemly repeated genes of the histone 3 multigene family.

In some species, histone gene clusters consist of tandem arrays of each type of histone gene, whereas in other species the genes may be clustered but not arranged in tandem. In certain species, however, histone genes are found scattered across several different chromosomes. This study examines the evolution of histone 3 (H3) genes that are not arranged in large clusters of tandem repeats. Although H3 amino acid sequences are highly conserved both within and between species, we found that the nucleotide sequence divergence at synonymous sites is high, indicating that purifying selection is the major force for maintaining H3 amino acid sequence homogeneity over long-term evolution. In cases where synonymous-site divergence was low, recent gene duplication appeared to be a better explanation than gene conversion. These results, and other observations on gene inactivation, organization, and phylogeny, indicated that these H3 genes evolve according to a birth-and-death process under strong purifying selection. Thus, we found little evidence to support previous claims that all H3 proteins, regardless of their genome organization, undergo concerted evolution. Further analyses of the structure of H3 proteins revealed that the histones of higher eukaryotes might have evolved from a replication-independent-like H3 gene.     

Phylogenetic relationships of Hynobius naevius (Amphibia: Caudata) as revealed by mitochondrial 12S and 16S rRNA genes

Using mitochondrial 12S and 16S rRNA sequences, we investigated phylogenetic relationships among populations of the endemic Japanese salamander Hynobius naevius. Monophyly of this species was recovered only in the maximum parsimony tree and was unresolved in maximum likelihood and Bayesian trees. Instead the following four haplotype clades consistently emerged clearly: Clade 1 from northwestern Kyushu, Clade 2 from Chugoku and northeastern Kyushu, Clade 3 from western Shikoku and Kyushu, and Clade 4 from Chubu-Kinki and central-eastern Shikoku. Of these, Clades 1 and 2, and Clades 3 and 4, respectively, correspond to Groups A and B previously recognized from the analyses of allozyme data in this species, but monophyly of these groups was not strongly supported. Unlike the previous results, the western and eastern samples from Shikoku did not form a clade, and were grouped with Kyushu-B in Clade 3 and Chubu-Kinki in Clade 4, respectively. The reason for this conflict between mtDNA and allozyme results is unknown, but might be related to retention of ancestral mtDNA polymorphism in Shikoku populations. Nearly simultaneous divergence of as many as four lineages in wide-ranging H. naevius is inferred for the late Miocene-Pliocene history of this taxon.     

Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects

The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages.     

Multiple unrelated founding events for the long-distance Pleistocene dispersal of the Salangid, Neosalanx taihuensis: a general demographic model for inshore-orientated freshwater fish

The Salangid icefish Neosalanx taihuensis (Salangidae) originated from inshore of the East China seas and underwent adaptive freshwater radiation from the mid-Miocene to the early Pleistocene. The distribution of its genetic diversity presents a random pattern inconsistent with contemporary hydrological structure. In the present study, coalescent simulations were used to analyze its Pleistocene dispersal history. Population history simulation supported the hypothesis of long-distance dispersal during the Pleistocene based on multiple unrelated founding events. This analogous genetic pattern has been described for other inshore-orientated freshwater fish, and may represent a general history dispersal model for the phylogeography of these species. From network analysis, three subclades (Clades 1-3) grouped consistently with three probable ancestral haplotypes (H36, H27, and H33). Demographic analysis also revealed that the ancestral haplotype group (Clade 1) dispersed into freshwater during an interglacial age about 0.35Ma, while Clades 2 and 3 dispersed about 0.12 and 0.145Ma, respectively. The N. taihuensis population remained relatively small for a considerable amount of time during the Pleistocene ages, with population expansion events mainly occurring after the last glacial maximum (LGM).     

Myoglobin of the shark Heterodontus portusjacksoni: isolation and amino acid sequence

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.     

Evolutionary distances in hawaiian drosophila measured by DNA reassociation

Summary Comparisons of the sequence divergence of three species of Hawaiian Drosophila have been made by hybridization of single-copy tracer DNA of each of the species with driver DNA from each species, and measurement of the average melting temperature (Tma) in a chaotropic solvent (2.4 M tetraethylammonium chloride) which minimizes differences due to base composition. Correction was made for the length of hybrid duplex regions to obtain the reduction in thermal stability due to divergence.An accuracy of ± 0.2°C was achieved and the mean reduction in Tm for hybridization betweenD. heteroneura andD. silvestris (found only on the island of Hawaii) was 0.55°C and betweenD. picticornis, found only on the island of Kauai, and the other two species was 2.13°C. The rate of DNA change is estimated to be between 0.2 and 0.4%/My by assuming that theD. heteroneura-D. silvestris divergence occurred 0.8 My ago and the divergence between these species andD. picticornis between 4 and 6 My ago.The general single copy DNA sequence divergence appears to be very much greater than the minimal coding region sequence divergence previously estimated from allozyme studies.     

Mitochondrial DNA data unveil highly divergent populations within the genus <Emphasis Type="Italic">Hynobius</Emphasis> (Caudata: Hynobiidae) in South Korea

Korean salamanders of the genus Hynobius are currently classified into 3 species, H. leechii, H. quelpaertensis, and H. yangi. To investigate the phylogenetic relationship of these species, we analyzed the partial sequence of mitochondrial cytochrome b gene (907 bp) of 197 specimens from 43 regions in South Korea. Of these specimens, 93 were additionally examined with 12S rRNA (799 bp). Based on the partial sequence of the mitochondrial cytochrome b gene and 12S rRNA, 89 and 36 haplotypes were defined, respectively, consisting of six subclades (H. leechii, H. quelpaertensis, H. yangi, HC1, HC2, and HC3). Among these subclades, the three subclades (HC1, HC2, and HC3) were clearly separated from the 3 previously reported species in the genus Hynobius. Pairwise sequence divergence between the six subclades ranged from 6.3 to 11.2% in cytochrome b gene and 2.0 to 4.3% in 12S rRNA. These results indicate there may be more divergent populations than the three currently described. Moreover, the estimation of divergence time revealed that the Hynobius species in South Korea diverged during the Miocene epoch, approximately 9 — 5 MYA. In addition, we confirmed the distribution of the three known species (H. leechii, H. quelpaertensis, and H. yangi) and determined the distributions of new, distinct groups (or subclades; HC1, HC1, and HC3). To more accurately establish the taxonomic status and population structure, further genetic, morphological, and ecological studies will be needed.     

Molecular evolutionary analysis of a histone gene repeating unit from Drosophila simulans

A repeating unit of the histone gene cluster from Drosophila simulans containing the H1, H2A, H2B and H4 genes (the H3 gene region has already been analyzed) was cloned and analyzed. A nucleotide sequence of about 4.6 kbp was determined to study the nucleotide divergence and molecular evolution of the histone gene cluster. Comparison of the structure and nucleotide sequence with those of Drosophila melanogaster showed that the four histone genes were located at identical positions and in the same directions. The proportion of different nucleotide sites was 6.3% in total. The amino acid sequence of H1 was divergent, with a 5.1% difference. However, no amino acid change has been observed for the other three histone proteins. Analysis of the GC contents and the base substitution patterns in the two lineages, D. melanogaster and D. simulans, with a common ancestor showed the following. 1) A strong negative correlation was found between the GC content and the nucleotide divergence in the whole repeating unit. 2) The mode of molecular evolution previously found for the H3 gene was also observed for the whole repeating unit of histone genes; the nucleotide substitutions were stationary in the 3' and spacer regions, and there was a directional change of the codon usage to the AT-rich codons. 3) No distinct difference in the mode or pattern of molecular evolution was detected for the histone gene repeating unit in the D. melanogaster and D. simulans lineages. These results suggest that selectional pressure for the coding regions of histones, which eliminate A and T, is less effective in the D. melanogaster and D. simulans lineages than in the other GC-rich species.     

Phylogeny and biogeography of the alpine newt Mesotriton alpestris (Salamandridae, Caudata), inferred from mtDNA sequences

In this paper, we performed phylogenetic analyses of Mesotriton alpestris populations from the entire range of species distribution, using fragments of two mtDNA genes, cytochrome b (309bp) and 16S rRNA ( approximately 500bp). Sequence diversity patterns and phylogenetic analyses reveal the existence of a relict lineage (Clade A) of late Miocene origin, comprising populations from south-eastern Serbia. This lineage is proposed to be ancestor to a western and an eastern lineage, which diverged during the middle Pliocene. The western lineage is further divided in two clades (Clades B, C) of middle Pliocene origin that represent populations from Italy (B) and populations from central Europe and Iberia (C). Further subdivision, dated back to the middle-late Pliocene, was found within the eastern lineage, representing southern (Clade D) and central-northern (Clade E) Balkan populations, respectively. Extensive sequence divergence, implying greater isolation in multiple refugia, is found within eastern clades, while the western clades seem to have been involved in the colonization of central, western and north-eastern Europe from a hypothetical refugium in central Europe. The extent of divergence does not support the current taxonomy indicating cryptic speciation in the Balkans, while paedomorphic lineages were found to have been evolved during early-middle Pleistocene probably as a response to the ongoing dramatic climatic oscillations.     

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.     

Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.