potheses, defining well-resolved nodes of the phylogeny, and identifying unresolved relationships, thereby focusing our efforts more efficiently.Here, we employ a statistical approach, using both parsimony and likelihood analyses of molecular sequence data to test a suite of previously proposed hypotheses of phylogenetic relationships in marmots (Marmota). A sequential phylogenetic estimation procedure was used that culminated in a maximum likelihood analysis utilizing a model of sequence evolution with parameters estimated from the data.Marmots are large terrestrial rodents found today throughout much of northern Eurasia and North America, including the Bering Strait region of western Alaska and eastern Siberia (Fig. 1). Their ecology and ethology has been studied extensively (e.g., There is a growing movement in systematics from simply making estimates of phylogeny to hypothesis testing and reliability estimation (Huelsenbeck and Rannala, 1997). This statistical perspective allows more precise delineation of which relationships are well understood and which need additional investigation. It also promotes the generation of explicit evolutionary and biogeographic models while providing the tools to reject hypotheses. These developments promise to accelerate our understanding of evolution by improving our hy- Syst. Biol. 48(4):715-734, 1999 Abstract.-There are 14 species of marmots distributed across the Holarctic, and despite extensive systematic study, their phylogenetic relationships remain largely unresolved. In particular, comprehensive studies have been lacking. A well-supported phylogeny is needed to place the numerous ecological and behavioral studies on marmots in an evolutionary context. To address this situation, we obtained complete cytochrome (cyt) b sequences for 13 of the species and a partial sequence for the 14th. We applied a statistical approach to both phylogeny estimation and hypothesis testing, using parsimony and maximum likelihood-based methods. We conducted statistical tests on a suite of previously proposed hypotheses of phylogenetic relationships and biogeographic histories. The cyt b data strongly support the monophyly of Marmota and a western montane clade in the Nearctic. Although some other scenarios cannot be rejected, the results are consistent with an initial diversification in North America, followed by an invasion and subsequent rapid diversification in the Palearctic. These analyses reject the two major competing hypotheses of M. broweri's phylogenetic relationships-namely, that it is the sister species to M. camtschatica of eastern Siberia, and that it is related closely to M. caligata of the Nearctic. The Alaskan distribution of M. broweri is best explained as a reinvasion from the Palearctic, but a Nearctic origin can not be rejected. Several other conventionally recognized species groups can also be rejected. Social evolution has been homoplastic, with large colonial systems evolving in two groups convergently. The cyt b data do not provide unambiguous resolution of se...

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of 125I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.

Acute infection with equine infectious anemia virus (EIAV), a lentivirus of horses, results in a persistent high-level viremia in Arabian foals affected with severe combined immunodeficiency (SCID). This observation argues against the idea that the transient nature of acute lentiviral viremia is solely a function of viral population dynamics. To extend these studies, EIAV-specific immune reconstitution was attempted prior to EIAV challenge in 2 SCID foals, using adoptively transferred virus-stimulated lymphocytes derived from persistently EIAV-infected half sibling donors. Following transfer, lymphocyte engraftment occurred in 1 foal, and EIAV-specific cytotoxic T lymphocytes as well as neutralizing antibody activity developed. Following a brief period of plasma viremia in this foal, EIAV replication was controlled and plasma virus could not be detected by RT-PCR or culture. These results provide further direct evidence that a specific immune response is required for termination of plasma viremia in acute lentiviral infections.

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Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)‐PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I‐specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT‐PCR and 19 putative alleles were identified in multiple clones from the same RT‐PCR. The primer pairs did not amplify putative non‐classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)‐A haplotype as determined by serology. However, horses sharing ELA‐A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA‐A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT‐PCR with classical MHC class I‐specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.