Hoekstra HE, Coyne JA. The locus of evolution: Evo devo and the genetics of adaptation. Evolution 2007;61:995-1016.Abstract

An important tenet of evolutionary developmental biology ("evo devo") is that adaptive mutations affecting morphology are more likely to occur in the cis-regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis-regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis-regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis-regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis-regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis-regulatory and structural mutations, with a substantial contribution of the latter.

Mullen LM, Hirschmann RJ, Prince KL, Glenn TC, Dewey MJ, Hoekstra HE. Sixty polymorphic microsatellite markers for the oldfield mouse developed in Peromyscus polionotus and Peromyscus maniculatus. Molecular Ecology 2006;6:36-40.Abstract

We isolated and characterized 60 novel microsatellite markers from the closely related oldfield mouse (Peromyscus polionotus) and deer mouse (Peromyscus maniculatus) for studies of conservation, ecological, quantitative and population genetics. We assessed all 60 markers in a wild population of Peromyscus polionotus rhoadsi (N = 20) from central Florida and found an average of nine alleles per marker and an observed heterozygosity (HO) of 0.66 (range = 0.00–1.00). These polymorphic markers contribute to the growing number of genomic resources for Peromyscus, an emerging model system for ecological and evolutionary research.

Turner LM, Hoekstra HE. Adaptive evolution of fertilization proteins within a genus: Variation in ZP2 and ZP3 in deer mice (Peromyscus). Molecular Biology and Evolution 2006;23:1656-69.Abstract

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.

Hoekstra HE. Genetics, development and evolution of adaptive pigmentation in vertebrates. Heredity 2006;97:222-34.Abstract

The study of pigmentation has played an important role in the intersection of evolution, genetics, and developmental biology. Pigmentation's utility as a visible phenotypic marker has resulted in over 100 years of intense study of coat color mutations in laboratory mice, thereby creating an impressive list of candidate genes and an understanding of the developmental mechanisms responsible for the phenotypic effects. Variation in color and pigment patterning has also served as the focus of many classic studies of naturally occurring phenotypic variation in a wide variety of vertebrates, providing some of the most compelling cases for parallel and convergent evolution. Thus, the pigmentation model system holds much promise for understanding the nature of adaptation by linking genetic changes to variation in fitness-related traits. Here, I first discuss the historical role of pigmentation in genetics, development and evolutionary biology. I then discuss recent empirically based studies in vertebrates, which rely on these historical foundations to make connections between genotype and phenotype for ecologically important pigmentation traits. These studies provide insight into the evolutionary process by uncovering the genetic basis of adaptive traits and addressing such long-standing questions in evolutionary biology as (1) are adaptive changes predominantly caused by mutations in regulatory regions or coding regions? (2) is adaptation driven by the fixation of dominant mutations? and (3) to what extent are parallel phenotypic changes caused by similar genetic changes? It is clear that coloration has much to teach us about the molecular basis of organismal diversity, adaptation and the evolutionary process.

Hoekstra HE, Hirschmann RJ, Bundey RA, Insel PA, Crossland JP. A single amino acid mutation contributes to adaptive beach mouse color pattern. Science 2006;313:101-4.Abstract

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.

Hoekstra HE, Nachman MW. Coat-color variation in rock pocket mice (Chaetopidus intermedius): from phenotype to genotype. Zoology 2005;133:79-99.Abstract


In a series of classic studies in mammalian evolutionary biology, Sumner (1921), Benson (1933), and Dice and Blossom (1937) described striking coat color variation in the rock pocket mouse, Chaetodipus intermedius, in the deserts of Arizona and New Mexico. These authors showed that C. intermedius coat color typically matches the color of the rocks on which the mice live; the dorsal pelage varies from a light, sandy color for populations found on some granites to a dark, nearly black color for populations found on basalt lava flows. Dice and Blossom (1937) suggested that this crypsis is an adaptation to avoid predation. Motivated by the wealth of data on the genetics, biochemistry, and molecular biology of the pigmentation process, we have used a candidate-gene approach to identify the genetic basis of adaptive coat color variation in C. intermedius. We review our recent studies on this topic with emphasis on the following key results: the identification of a single gene (the melanocortin-1-receptor, Mc1r) in one population that appears to be largely responsible for color differences, the balance between selection and migration among neighboring melanic and light races, and the finding that melanism has evolved independently on different lava flows through changes at different genes. 



Hoekstra HE, Krenz JG, Nachman MW. Local adaptation in the rock pocket mouse (Chaetodipus intermedius): Natural selection and phylogenetic history of populations. Heredity 2005;94:217-28.Abstract

Elucidating the causes of population divergence is a central goal of evolutionary biology. Rock pocket mice, Chaeotdipus intermedius, are an ideal system in which to study intraspecific phenotypic divergence because of the extensive color variation observed within this species. Here, we investigate whether phenotypic variation in color is correlated with local environmental conditions or with phylogenetic history. First, we quantified variation in pelage color (n=107 mice) and habitat color (n=51 rocks) using a spectrophotometer, and showed that there was a correlation between pelage color and habitat color across 14 sampled populations (R2=0.43). Analyses of mtDNA sequences from these same individuals revealed strong population structure in this species across its range, where most variation (63%) was partitioned between five geographic regions. Using Mantel tests, we show that there is no correlation between color variation and mtDNA phylogeny, suggesting that pelage coloration has evolved rapidly. At a finer geographical scale, high levels of gene flow between neighboring melanic and light populations suggest the selection acting on color must be quite strong to maintain habitat-specific phenotypic distributions. Finally, we raise the possibility that, in some cases, migration between populations of pocket mice inhabiting different lava flows may be responsible for similar melanic phenotypes in different populations. Together, the results suggest that color variation can evolve very rapidly over small geographic scales and that gene flow can both hinder and promote local adaptation.

Rosenblum EB, Hoekstra HE, Nachman MW. Adaptive reptile color variation and the evolution of the Mc1r gene. Evolution 2004;58:1794-808.Abstract

The wealth of information on the genetics of pigmentation and the clear fitness consequences of many pigmentation phenotypes provide an opportunity to study the molecular basis of an ecologically important trait. The melanocortin-1 receptor (Mc1r) is responsible for intraspecific color variation in mammals and birds. Here, we study the molecular evolution of Mc1r and investigate its role in adaptive intraspecific color differences in reptiles. We sequenced the complete Mc1r locus in seven phylogenetically diverse squamate species with melanic or blanched forms associated with different colored substrates or thermal environments. We found that patterns of amino acid substitution across different regions of the receptor are similar to the patterns seen in mammals, suggesting comparable levels of constraint and probably a conserved function for Mc1r in mammals and reptiles. We also found high levels of silent-site heterozygosity in all species, consistent with a high mutation rate or large long-term effective population size. Mc1r polymorphisms were strongly associated with color differences in Holbrookia maculata and Aspidoscelis inornata. In A. inornata, several observations suggest that Mc1r mutations may contribute to differences in color: (1) a strong association is observed between one Mc1r amino acid substitution and dorsal color; (2) no significant population structure was detected among individuals from these populations at the mitochondrial ND4 gene; (3) the distribution of allele frequencies at Mc1r deviates from neutral expectations; and (4) patterns of linkage disequilibrium at Mc1r are consistent with recent selection. This study provides comparative data on a nuclear gene in reptiles and highlights the utility of a candidate-gene approach for understanding the evolution of genes involved in vertebrate adaptation.

Hoekstra HE, Drumm KE, Nachman MW. Ecological genetics of adaptive color polymorphism in pocket mice: geographic variation in selected and neutral genes. Evolution 2004;58:1329-41.Abstract

Patterns of geographic variation in phenotype or genotype may provide evidence for natural selection. Here, we compare phenotypic variation in color, allele frequencies of a pigmentation gene (the melanocortin-1 receptor, Mc1r), and patterns of neutral mitochondrial DNA (mtDNA) variation in rock pocket mice (Chaetodipus intermedius) across a habitat gradient in southern Arizona. Pocket mice inhabiting volcanic lava have dark coats with unbanded, uniformly melanic hairs, whereas mice from nearby light-colored granitic rocks have light coats with banded hairs. This color polymorphism is a presumed adaptation to avoid predation. Previous work has demonstrated that two Mc1r alleles, D and d, differ by four amino acids, and are responsible for the color polymorphism: DD and Dd genotypes are melanic whereas dd genotypes are light colored. To determine the frequency of the two Mc1r allelic classes across the dark-colored lava and neighboring light-colored granite, we sequenced the Mc1r gene in 175 individuals from a 35-km transect in the Pinacate lava region. We also sequenced two neutral mtDNA genes, COIII and ND3, in the same individuals. We found a strong correlation between Mc1r allele frequency and habitat color and no correlation between mtDNA markers and habitat color. Using estimates of migration from mtDNA haplotypes between dark- and light-colored sampling sites and Mc1r allele frequencies at each site, we estimated selection coefficients against mismatched Mc1r alleles, assuming a simple model of migration-selection balance. Habitat-dependent selection appears strong but asymmetric: selection is stronger against light mice on dark rock than against melanic mice on light rock. Together these results suggest that natural selection acts to match pocket mouse coat color to substrate color, despite high levels of gene flow between light and melanic populations.

Hoekstra HE, Price T. Parallel evolution is in the genes. Science 2004;303:1779-81. PDF
Hoekstra HE. Unequal transmission of mitochondrial haplotypes in natural populations of field mice with XY females (genus Akodon). The American Naturalist 2003;161(1):29-39.Abstract

In species with fertile XY females, such as South American field mice (genus Akodon), there are two types of mitochondrial DNA (mtDNA), one passing from XX females and one from XY females. The XX mothers pass their mtDNA to their XX daughters. The XY mothers, however, produce both XX and XY daughters. Because of this breeding scheme, the XY mtDNA remains isolated whereas the XX lineage is continuously invaded by XY mtDNA haplotypes. Using a set of recursion equations, I predicted that XY mtDNA haplotypes should rapidly spread through entire populations composed of both XX and XY females. I examined patterns of nucleotide polymorphism and divergence from the mtDNA control region as well as phylogenetic patterns for evidence of an mtDNA sweep. I compared patterns in two sister species, Akodon boliviensis and Akodon azarae, that are composed of 35% and 10% XY females, respectively. Akodon boliviensis XY females are found in all clades of a phylogenetic mtDNA tree consistent with the spread of mtDNA haplotypes. In addition, A. azarae mtDNA haplotypes showed no deviations from neutrality. These results, in combination with high levels of mtDNA nucleotide diversity in XY females, suggest an ancient origin (1104 generations) of XY females in both A. boliviensis and A. azarae.

Hoekstra HE, Nachman MW. Different genes underlie adaptive melanism in different populations of rock pocket mice. Mol Ecol 2003;12:1185-94.Abstract

Identifying the genes responsible for adaptation has been an elusive goal in evolutionary biology. Rock pocket mice (Chaetodipus intermedius) provide a useful system for studying the genetics of adaptation: most C. intermedius are light-coloured and live on light-coloured rocks, but in several different geographical regions, C. intermedius are melanic and live on dark-coloured basalt lava, presumably as an adaptation for crypsis. Previous work demonstrated that mutations at the melanocortin-1 receptor gene (Mc1r) are responsible for the dark/light difference in mice from one population in Arizona. Here, we investigate whether melanism has evolved independently in populations of dark C. intermedius from New Mexico, and whether the same or different genes underlie the dark phenotype in mice from these populations compared with the dark mice from Arizona. Seventy-six mice were collected from pairs of dark and light localities representing four different lava flows and adjacent light-coloured rocks; lava flows were separated by 70-750 km. Spectrophotometric analysis of mouse pelage and of rock samples revealed a strong positive association between coat colour and substrate colour. No significant differences were observed in the colour of rocks among the four lava flows, suggesting that mice in these separate populations have experienced similar selection for crypsis. Despite this similarity in environment, melanic mice from the three New Mexico populations were slightly, but significantly, darker than melanic mice from Arizona. The entire Mc1r gene was sequenced in all mice. The previously identified mutations responsible for the light/dark difference in mice from Arizona were absent in all melanic mice from three different populations in New Mexico. Five new Mc1r polymorphisms were observed among mice from New Mexico, but none showed any association with coat colour. These results indicate that adaptive melanism has arisen at least twice in C. intermedius and that these similar phenotypic changes have a different genetic basis.

Nachman MW, Hoekstra HE, D'Agostino SL. The genetic basis of adaptive melanism in pocket mice. Proceedings of the National Academy of Sciences of the United States of America 2003;100:5268-73.Abstract

Identifying the genes underlying adaptation is a major challenge in evolutionary biology. Here, we describe the molecular changes underlying adaptive coat color variation in a natural population of rock pocket mice, Chaetodipus intermedius. Rock pocket mice are generally light-colored and live on light-colored rocks. However, populations of dark (melanic) mice are found on dark lava, and this concealing coloration provides protection from avian and mammalian predators. We conducted association studies by using markers in candidate pigmentation genes and discovered four mutations in the melanocortin-1-receptor gene, Mc1r, that seem to be responsible for adaptive melanism in one population of lava-dwelling pocket mice. Interestingly, another melanic population of these mice on a different lava flow shows no association with Mc1r mutations, indicating that adaptive dark color has evolved independently in this species through changes at different genes.

Lingenfelter PA, Delbridge ML, Thomas S, Hoekstra HE, Mitchell MJ, Graves JA, Disteche CM. Expression and conservation of processed copies of the RBMX gene. Mammalian Genome 2001;12:538-45.Abstract

RBMX and RBMY are members of an ancient pair of genes located on the sex chromosomes that encode RNA-binding proteins involved in splicing. These genes have differentiated and evolved separately on the X and Y Chromosomes. RBMY has acquired a testis-specific function, whereas, as shown here, RBMX is ubiquitously expressed and is subject to X inactivation. We have also found that multiple processed copies of RBMX are present in the human genome. RBMX-like sequences (RBMXLs) located on human Chrs 1, 4, 6, 9 (9p13 and 9p24), 11, 20, and X lack introns and thus probably result from retroposition events. We found RBMXLs to be conserved in primates and great apes at corresponding chromosomal locations, indicating that they arose prior to the divergence of human. Some of the RBMXLs show insertions, deletions, and stop codons, which would probably result in nonfunctional proteins. The RBMXL on Chr 20 is deleted in some individuals. Two of the largely intact RBMXLs, located on Chrs 1 and 9p13, are expressed in different tissues and may encode novel proteins involved in splicing in a tissue-specific manner. The RBMXL located at 9p13 is specifically expressed in testis, and to a lesser extent in brain, and may therefore play a role in testis function. This autosomal, testis-specific copy of RBMX could potentially compensate for RBMX that is presumably inactivated in male germ cells, in a manner analogous to autosomal retroposed copies of other X-linked genes.

Hoekstra HE, Hoekstra JM, Berrigan D, Vignieri SN, Hoang A, Hill CE, Beerli P, Kingsolver JG. Strength and tempo of directional selection in the wild. Proceedings of the National Academy of Sciences of the United States of America 2001;98:9157-60.Abstract

Directional selection is a major force driving adaptation and evolutionary change. However, the distribution, strength, and tempo of phenotypic selection acting on quantitative traits in natural populations remain unclear across different study systems. We reviewed the literature (1984-1997) that reported the strength of directional selection as indexed by standardized linear selection gradients (beta). We asked how strong are viability and sexual selection, and whether strength of selection is correlated with the time scale over which it was measured. Estimates of the magnitude of directional selection (absolute value of beta) were exponentially distributed, with few estimates greater than 0.50 and most estimates less than 0.15. Sexual selection (measured by mating success) appeared stronger than viability selection (measured by survival). Viability selection that was measured over short periods (days) was typically stronger than selection measured over longer periods (months and years), but the strength of sexual selection did not vary with duration of selection episodes; as a result, sexual selection was stronger than viability selection over longer time scales (months and years), but not over short time scales (days).

Kingsolver JG, Hoekstra HE, Hoekstra JM, Berrigan D, Vignieri SN, Hill CE, Hoang A, Gibert P, Beerli P. The strength of phenotypic selection in natural populations. The American Naturalist 2001;157:245-61.Abstract

How strong is phenotypic selection on quantitative traits in the wild? We reviewed the literature from 1984 through 1997 for studies that estimated the strength of linear and quadratic selection in terms of standardized selection gradients or differentials on natural variation in quantitative traits for field populations. We tabulated 63 published studies of 62 species that reported over 2,500 estimates of linear or quadratic selection. More than 80% of the estimates were for morphological traits; there is very little data for behavioral or physiological traits. Most published selection studies were unreplicated and had sample sizes below 135 individuals, resulting in low statistical power to detect selection of the magnitude typically reported for natural populations. The absolute values of linear selection gradients |beta| were exponentially distributed with an overall median of 0.16, suggesting that strong directional selection was uncommon. The values of |beta| for selection on morphological and on life-history/phenological traits were significantly different: on average, selection on morphology was stronger than selection on phenology/life history. Similarly, the values of |beta| for selection via aspects of survival, fecundity, and mating success were significantly different: on average, selection on mating success was stronger than on survival. Comparisons of estimated linear selection gradients and differentials suggest that indirect components of phenotypic selection were usually modest relative to direct components. The absolute values of quadratic selection gradients |gamma| were exponentially distributed with an overall median of only 0.10, suggesting that quadratic selection is typically quite weak. The distribution of gamma values was symmetric about 0, providing no evidence that stabilizing selection is stronger or more common than disruptive selection in nature.

Hoekstra HE, Hoekstra JM. An unusual sex-determination system in South American field mice (genus Akodon): The role of mutation, selection, and meiotic drive in maintaining XY females. Evolution 2001;55:190-7.Abstract

The mechanism of sex determination in mammals appears highly conserved: the presence of a Y chromosome triggers the male developmental pathway, whereas the absence of a Y chromosome results in a default female phenotype. However, if the Y chromosome fails to initiate the male pathway (referred to as Y*), XY* females can result, as is the case in several species of South American field mice (genus Akodon). The breeding genetics in this system inherently select against the Y* chromosome such that the frequency of XY* females should decrease rapidly to very low frequencies. However, in natural populations of Akodon, XY* females persist at substantial frequencies; for example, 10% of females are XY* in A. azarae and 30% in A. boliviensis. We develop a mathematical model that considers the potential roles of three evolutionary forces in maintaining XY* females: Y-to-Y* chromosome transitions (mutation), chromosome segregation distortion (meiotic drive), and differential fecundity (selection). We then test the predictions of our model using data from breeding colonies of A. azarae. We conclude that any single force is inadequate to maintain XY* females. However, a combination of segregation bias of the male and female Y chromosomes during spermatogenesis/oogenesis and increased fecundity in XY* females could account for the observed frequencies of XY* females.

Hess CM, Gasper J, Hoekstra HE, Hill CE, Edwards SV. MHC class II pseudogene and genomic signature of a 32-kb cosmid in the house finch (Carpodacus mexicanus). Genome Research 2000;10(5):613-23.Abstract

Large-scale sequencing studies in vertebrates have thus far focused primarily on the genomes of a few model organisms. Birds are of interest to genomics because of their much smaller and highly streamlined genomes compared to mammals. However, large-scale genetic work has been confined almost exclusively to the chicken; we know little about general aspects of genomes in nongame birds. This study examines the organization of a genomic region containing an Mhc class II B gene in a representative of another important lineage of the avian tree, the songbirds (Passeriformes). We used a shotgun sequencing approach to determine the sequence of a 32-kb cosmid insert containing a strongly hybridizing Mhc fragment from house finches (Carpodacus mexicanus). There were a total of three genes found on the cosmid clone, about the gene density expected for the mammalian Mhc: a class II Mhc beta-chain gene (Came-DAB1), a serine-threonine kinase, and a zinc finger motif. Frameshift mutations in both the second and third exons of Came-DAB1 and the unalignability of the gene after the third exon suggest that it is a nonfunctional pseudogene. In addition, the identifiable introns of Came-DAB1 are more than twice as large as those of chickens. Nucleotide diversity in the peptide-binding region of Came-DAB1 (Pi = 0.03) was much lower than polymorphic chicken and other functional Mhc genes but higher than the expected diversity for a neutral locus in birds, perhaps because of hitchhiking on a selected Mhc locus close by. The serine-threonine kinase gene is likely functional, whereas the zinc finger motif is likely nonfunctional. A paucity of long simple-sequence repeats and retroelements is consistent with emerging rules of chicken genomics, and a pictorial analysis of the "genomic signature" of this sequence, the first of its kind for birds, bears strong similarity to mammalian signatures, suggesting common higher-order structures in these homeothermic genomes. The house finch sequence is among a very few of its kind from nonmodel vertebrates and provides insight into the evolution of the avian Mhc and of avian genomes generally.

Hoekstra HE, Edwards SV. Multiple origins of XY female mice (genus Akodon): Phylogenetic and chromosomal evidence. Proceedings of the Royal Society B: Biological Sciences 2000;267:1825-31.Abstract

Despite the diversity in sex determination across organisms, theory predicts that the evolution of XY females is rare in mammals due to fitness consequences associated with infertility or the loss of YY zygotes. We investigated this hypothesis from a phylogenetic perspective by examining the inter- and intraspecific distribution of Y chromosomes in males and females (XY females) in South American field mice (Akodon). We found that XY females occurred at appreciable frequencies (10-66%) in at least eight Akodon species, raising the possibility that this system of sex determination has arisen multiple times independently. To determine the number of origins of XY females in Akodon, we constructed a molecular phylogeny of 16 species of Akodon based on mitochondrial DNA control region sequences. Both parsimony and maximum-likelihood reconstruction of ancestral states suggest that multiple steps (gains or losses of XY females) best explain the evolution of XY females, but do not clearly differentiate between single and multiple origins. We then directly compared functional and non-functional Y chromosomes in six species by Southern blot analysis. We found that male and female Y chromosome restriction fragment length polymorphism patterns were identical within species, but always differed between species, providing evidence that XY females arose at least six times within the Akodon lineage. To our knowledge, this pattern in Akodon is the first documentation of a novel sex-determining system arising multiple times within a tight clade of mammals. In addition, this system provides a clear test of the accuracy of phylogenetic methods to reconstruct ancestral states.