Scott K. Davis

EVOLUTION OF RIBOSOMAL DNA: FIFTY MILLION YEARS OF RECORDED HISTORY IN THE FROG GENUS RANA

Evolution of nuclear ribosomal DNA (rDNA) arrays of frogs of the genus Rana was examined among 32 species that last shared a common ancestor approximately 50 million years ago. Extensive variation in restriction sites exists within the transcribed and nontranscribed rDNA spacer regions among the species, whereas rDNA coding regions exhibit comparatively little interspecific variation in restriction sites. The most parsimonious phylogenetic hypothesis for the evolution of the group was constructed based on variation in restriction sites and internal spacer lengths among the 32 species of Rana and one species of Pyxicephalus (examined for outgroup comparison). This analysis suggests that R. sylvatica of North America is more closely related to the R. temporaria group of Eurasia than to other North American Rana. The hypothesized phylogeny also supports the monophyly of the R. boylii group, the R. catesbeiana group, the R. palmipes group, the R. tarahumarae group, and the R. pipiens complex. Furthermore, the restriction site data provide information about the evolution within and among these species groups. This demonstrates that restriction site mapping of rDNA arrays provides a useful molecular technique for the examination of historical evolutionary questions across considerable periods of time.

Mining the Mammalian Genome for Artiodactyl Systematics

A total of 7,806 nucleotide positions derived from one mitochondrial and eight nuclear DNA segments were used to provide a robust phylogeny for members of the order Artiodactyla. Twenty-four artiodactyl and two cetacean species were included, and the horse (order Perissodactyla) was used as the outgroup. Limited rate heterogeneity was observed among the nuclear genes. The partition homogeneity tests indicated no conflicting signal among the nuclear genes fragments, so the sequence data were analyzed together and as separate loci. Analyses based on the individual nuclear DNA fragments and on 34 unique indels all produced phylogenies largely congruent with the topology from the combined data set. In sharp contrast to the nuclear DNA data, the mtDNA cytochrome b sequence data showed high levels of homoplasy, failed to produce a robust phylogeny, and were remarkably sensitive to taxon sampling. The nuclear DNA data clearly support the paraphyletic nature of the Artiodactyla. Additionally, the family Suidae is diphyletic, and the nonruminating pigs and peccaries (Suiformes) were the most basal cetartiodactyl group. The morphologically derived Ruminantia was always monophyletic; within this group, all taxa with paired bony structures on their skulls clustered together. The nuclear DNA data suggest that the Antilocaprinae account for a unique evolutionary lineage, the Cervidae and Bovidae are sister taxa, and the Giraffidae are more primitive.

Artiodactyl IgD: The Missing Link

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig δ genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cδ gene appears to have been duplicated from the Cμ gene >300 million yr ago. The ruminant μCH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, ∼20 million yr ago, inserted upstream of the δ gene hinge regions, and later modified by gene conversion. A short Sδ (switch δ) sequence resulting from the second duplication, is located immediately upstream of the bovine Cδ gene and directs regular μ-δ class switch recombination in the cow. The presence of Cδ genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

CONSERVATION OF MICROSATELLITE LOCI ACROSS SPECIES OF ARTIODACTYLS: IMPLICATIONS FOR POPULATION STUDIES

DNA microsatellites have proven useful as markers in studies of gene mapping due to their high level of polymorphism and broad genomic distribution. These properties suggest that they also will be useful for studies of population structure. The limiting factor is the development of polymerase chain reaction (PCR) primer sets for each new species of interest, as this requires the construction and screening of libraries of genomic DNA, DNA sequencing, and design of primers. The locations and sequences of some microsatellites have been shown to be conserved across species of mammals. We tested these markers in an array of closely related species of bovids and cervids. PCR primer pairs designed for bovine, ovine, or caprine microsatellite loci successfully amplified microsatellites in many other species of artiodactyls. When 20 primer sets were tested on 10 species of bovids and six species of cervids, polymorphic products were obtained for seven or more of the 16 species with 12 of the primer pairs. This success in using heterologous PCR primers to amplify microsatellite loci in several different species eliminates the need to develop new sets of primers for each species and therefore facilitates the use of DNA microsatellites as markers in studies of population genetics. The primers tested here form a suite of useful markers for studies of artiodactyls that contains more polymorphic loci than any existing set of allozyme or RFLP loci.

RIBOSOMAL-DNA, MITOCHONDRIAL-DNA, CHROMOSOMAL, AND ALLOZYMIC STUDIES ON A CONTACT ZONE IN THE POCKET GOPHER, GEOMYS

We studied 75 individuals of the Plains pocket gopher, Geomys bursarius, from eastern New Mexico, where the subspecies major and knoxjonesi hybridize. Each individual was examined for chromosome number, ribosomal DNA, mitochondrial DNA, and three protein systems for which reference parental populations were fixed for alternative alleles. Twenty individuals were indistinguishable from parental major, 14 individuals were indistinguishable from parental knoxjonesi, and 41 individuals had genotypes composed of combinations of character states that distinguish the two parental types. The parental types appear to represent discrete genetic entities that have restricted introgression across a narrow hybrid zone (width approximately 3 km, using the 20/80 criterion). Parental types overlap in geographic distribution near the center of the zone, and changes in mitochondrial DNA and the five nuclear markers are concordant across the zone. It is probable that there is premating isolation between knoxjonesi males and major females. The frequencies of individuals with certain genotypic combinations within our sample imply differential reproductive success of certain genotypes. We propose that F1s and highly heterozygous males are sterile and that hybrid females are less fertile than parental females. These postmating factors, along with premating isolation for one of the reciprocal crosses, probably account for the restriction of gene flow across the contact zone. The structure of the zone can be explained by the “dynamic equilibrium” model.

Evaluating a Putative Bottleneck in a Population of Bowhead Whales from Patterns of Microsatellite Diversity and Genetic Disequilibria

Abstract. A size-selected Balaena mysticetus genomic library was screened for clones containing simple sequence repeat, or microsatellite, loci. A total of 11 novel loci was identified. These loci were combined with a set of 9 published loci, for a total of 20 markers, and were scored across a sample of 108 bowhead whales from the Bering–Chukchi–Beaufort Seas population of bowhead whales. Genetic variability was measured in terms of polymorphism information content values and unbiased heterozygosity. From the latter, estimates of long-term effective population size were obtained. In addition, gametic phase disequilibrium among loci was investigated. Moderate to high levels of polymorphism were found overall, and the long-term effective size estimates were large relative to total population size. Tests of heterozygosity excess (Cornuet and Luikart 1996) and allele frequency distribution (Luikart et al. 1998) indicated that the possibility of a recent genetic bottleneck in the Bering–Chukchi–Beaufort Seas population of bowhead whales is highly unlikely. However, the fact that five loci displayed a statistically significant heterozygote deficiency remains to be explained.

Evaluation of genetic variation in a domestic population ofPenaeus vannamei (Boone): a comparison with three natural populations

Abstract Three natural populations of Penaeus vannamei from Mexico, Panama and Ecuador were compared to a farm population which has been closed since 1983. These populations were screened for allozyme variation at 26 loci. The observed levels of variation and heterozygosity in domestic and wild populations were very low, with average heterozygosity of only 0.017. Comparison of allelic frequencies showed very low levels of differentiation among the wild populations and analysis of population structure revealed extremely low levels of population subdivision. The aquaculture population showed slightly lower levels of heterozygosity and possessed fewer rare alleles than the wild populations, but showed no evidence of inbreeding or reduction in effective breeding population size.

A molecular cytogenetic analysis of the tribe Bovini (Artiodactyla : Bovidae : Bovinae) with an emphasis on sex chromosome morphology and NOR distribution

Q-band comparisons were made among representative species of the four genera of the tribe Bovini (Bos, Bison, Bubalus, Syncerus) as well as to selected outgroup taxa representing the remaining two tribes of the subfamily Bovinae (nilgai, Boselaphini; eland, Tragelphini), the Bovidae subfamily Caprinae (domestic sheep) and the family Cervidae (sika deer and white- tailed deer). Extensive autosomal arm homologies were noted, but relatively few derivative character states were shared. Focus was then made on variation of the sex chromosomes and the chromosomal distribution of nucleolar organizer regions (NORs). Bovine BAC clones were used in molecular cytogenetic analyses to decipher rearrangements of the sex chromosomes, and a pocket gopher 28s ribosomal probe was used to map the chromosomal locations of nucleolar organizing regions (NORs). Some of the more noteworthy conclusions drawn from the comparative analysis were that: 1. The Bovidae ancestral X chromosome was probably acrocentric and similar to acrocentric X chromosomes of the Bovinae; 2. The domestic sheep acrocentric X is probably a deriative character state that unites non-Bovinae subfamilies; 3. Bos and Bison are united within the tribe Bovini by the presence of shared derivative submetacentric X chromosomes; 4. Sika and white- tailed deer X chromosomes differ by inversion from X chromosomes of the Bovinae; 5. The Bovini ancestral Y chromosome was probably a small acrocentric; 6. Bos taurus, B. gaurus and B. banteng share derivative metacentric Y chromosomes; 7. Syncerus and Bubalus are united by the acquisition of X-specific repetitive DNA sequence on their Y chromosomes; 8. Bovinae and Cervidae X chromosome centromere position varies without concomitant change in locus order. Preliminary data indicate that a knowledge of the chromosomal distribution of NORs among the Bovidae will prove to be phylogenetically informative.

Systematic and Taxonomic Implications of Karyotypic, Electrophoretic, and Mitochondrial-Dna Variation in Peromyscus from the Pacific Northwest

Chromosomes, allozymes, and mitochondrial DNA (mtDNA) were analyzed to examine the taxonomic and systematic relationships of coastal Peromyscus from northern Washington to southern Alaska. All three datasets indicate that Peromyscus from this region constitute two distinct groups. One group comprises P. oreas, P. sitkensis , and several currently recognized subspecies of P. maniculatus including P. m. algidus, P. m. hylaeus, P. m. keeni, P. m. macrorhinus , and P. m. prevostensis . The second group comprises only populations of P. m. austerus . The former group is differentiated from the latter by number of autosomal arms, allele-frequency differences, and mtDNA haplotypes. This dichotomy in karyotype, and the level of allozymic and mtDNA divergence between the groups suggest that the groups constitute distinct species. We recommend that P. oreas, P. sitkensis, P. maniculatus algidus, P. m. hylaeus, P. m. keeni, P. m. macrorhinus , and P. m. prevostensis be recognized under the specific epithet of Peromyscus keeni .

Phylogenetic Relationships in the Subfamily Bovinae (Mammalia: Artiodactyla) Based on Ribosomal DNA

Relationships in the subfamily Bovinae were examined using restriction-endonuclease site mapping of nuclear-ribosomal DNA. Phylogenetic analysis of the data supports the monophyly of the tribe Bovini and a sister-group relationship between the tribes Bovini and Boselaphini. Two lineages exist within the Bovini, the buffaloes ( Syncerus and Bubalus ) and the cattle ( Bos and Bison ). Data are presented to support recent studies that suggest the genus Bos is paraphyletic with respect to the genus Bison and that the genera should be combined. A comparison of chromosomal and ribosomal-DNA data indicates that 2n = 60 and FN = 58 is the most likely ancestral condition for the Bovinae. Numerous Robertsonian events have occurred during the radiation of the group, particularly in the buffaloes. Phylogenies based on ribosomal DNA, morphology, and chromosomes are combined to give a hypothesis of evolution in the subfamily.