Alan R. Templeton

Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and the apes

Recombinant DNA technology provides evolutionary biologists with another tool for making phylogenetic inference through contrasts of restriction endounuclease cleavage site maps or DNA sequences between homologous DNA segments found in different groups. This paper is limited to the problem of making phylogenetic inference from restriction site maps. Several methods for making such inference have already been used or proposed (Avise et al., 1979a, 1979b;NeiandLi, 1979; Ferris et al., 1981), but all these methods depend upon the assumption that shared restriction sites reflect common evolutionary origins and are not the result of convergent evolution. Unfortunately, convergent evolution occurs with high probability for this type of data (Templeton, 1983). In addition, data from several different restriction enzymes are generally pooled in these analyses. Recently, Adams and Rothman (1982) have examined the distributions of cleavage sites and related sequences for 54 restriction endonucleases. They concluded 1) that cleavage sites and related sequences are distributed non-randomly in most DNA sequences, 2) that there is considerable heterogeneity between different restriction enzymes (even those with recognition sequences of the same length) with respect to the number and distribution of their respective cleavage sites and related sequences, and 3) that inference of phylogenetic relationship based on distances will be biased. In addition, Brown et al. (1982) sequenced 896 base pairs of the mitochondrial DNA from humans and apes and concluded that about 90% of the substitutions were transitions. The predominance of transitions over transversions increases the probability of convergence over that expected when all base substitutions are assumed to be equally likely (Templeton, 1983). Therefore, a need exists for an algorithm of phylogenetic inference that deals more directly with the problem of convergent evolution and statistical inhomogeneity between different restriction enzymes. In this paper, I propose such an algorithm. After discussing the problem of estimation of a phylogenetic tree, the task of statistical testing is then addressed. First, I present a non-parametric statistical framework for testing the fit of one hypothesized phylogeny versus an alternative phylogeny. Second, non-parametric statistical procedures are presented for testing hypotheses about relative rates of evolution among the various lineages.

Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history

Since the 1920s, population geneticists have had measures that describe how genetic variation is distributed spatially within a species’ geographical range. Modern genetic survey techniques frequently yield information on the evolutionary relationships among the alleles or haplotypes as well as information on allele frequencies and their spatial distributions. This evolutionary information is often expressed in the form of an estimated haplotype or allele tree. Traditional statistics of population structure, such as F statistics, do not make use of evolutionary genealogical information, so it is necessary to develop new statistical estimators and tests that explicitly incorporate information from the haplotype tree. One such technique is to use the haplotype tree to define a nested series of branches (clades), thereby allowing an evolutionary nested analysis of the spatial distribution of genetic variation. Such a nested analysis can be performed regarding the geographical sampling locations either as categorical or continuous variables (i.e. some measure of spatial distance). It is shown that such nested phylogeographical analyses have more power to detect geographical associations than traditional, nonhistorical analyses and, as a consequence, allow a broader range of gene‐flow parameters to be estimated in a precise fashion. More importantly, such nested analyses can discriminate between phylogeographical associations due to recurrent but restricted gene flow vs. historical events operating at the population level (e.g. past fragmentation, colonization, or range expansion events). Restricted gene flow and historical events can be intertwined, and the cladistic analyses can reconstruct their temporal juxtapositions, thereby yielding great insight into both the evolutionary history and population structure of the species. Examples are given that illustrate these properties, concentrating on the detection of range expansion events.

GeoDis : a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes

The central focus of population genetics is the study of the distribution of the genetic variation within and among populations. This endeavour has often been accomplished by the use of genealogies upon which geographical information is incorporated in the search of association among genetic variation and geographical distribution (see Avise 1998). However, a particular population genetic structure can be the result of distinct processes acting in different points through time and space and may reflect historical rather than ongoing population level processes (Gerber & Templeton 1996). Templeton (1993) and Templeton et al . (1995) describe a methodology (cladistic nested analysis) in which population structure can be separated from population history when it is assessed through rigorous and objective statistical tests upon an estimated nested cladogram (see Templeton et al . 1992). GeoDis is a computer program that implements the cladistic nested analysis. The simplest test for geographical association is to treat sample locations as categorical variables. An exact permutational contingency test is performed for any clade at each nesting level. A chi-square statistic is calculated from the contingency tables in which rows are genetic clades and columns are geographical locations (see also software Chiperm, available at http://bioag.byu.edu/zoology/crandall_lab/ programs.htm). A more elaborate analysis can also be carried out by using information on geographical distances. Using the geographical coordinates of each population two main statistics are calculated, the clade distance ( D c ), which measures the geographical spread of a clade, and the nested clade distance ( D n ), which measures how a clade is geographically distributed relative to other clades in the same higher-level nesting category. In the case of riparian or coastal species, or in the case of species with constrained dispersal routes, a matrix of pairwise distances among the different locations better describes their geographical distribution. The analogue statistics ( D cl and D nl ) are calculated as the average pairwise distances between members of the same focal clade and the average pairwise distances between members of the focal clade with all members of the nesting clade (including the focal clade). An interior-tip statistic (I-T) is also estimated within each nested category as the average interior distance minus the average tip distance. For the calculation of these averages, each clade distance is weighted by the number of copies in that focal clade relative to the total number of copies in the nesting clade. This tip vs. interior contrast corresponds to a young vs. old contrast and, to a lesser extent, rare vs. common (Crandall & Templeton 1993). If the haplotype tree is rooted, say by an out-group, the user can also specify which haplotype is the oldest by designating it as the ‘interior’, and regarding the younger haplotypes all as ‘tips’. When root probabilities or out-group weights for the cladogram are specified (Castelloe & Templeton 1994), the correlation of both distance measures with out-group weights within each nested category is also estimated. The significance of these statistics is estimated through a Monte Carlo procedure. Null distributions are constructed by randomizing the contingency data table for each clade and nesting level and estimating again the test statistics for each randomized data set. Matrix randomization is accomplished by using the algorithm of Roff & Bentzen (1989), which preserves the marginals of the table (clade frequencies and sample sizes), while permuting the individual cells. A minimum number of 1000 random permutations are recommended to make statistical inference at the 5% level of significance (Edgington 1986). The output of GeoDis consists of the calculated statistics and their associated permutational P -values. Templeton (1998) provides a key for the interpretation of these results in biological terms. GeoDis has been written both in C and Java and includes new features, as weighted I-T statistics, and the possibility of using user-defined distances. A previous version of the program written in VAX/VMS Basic exists (AR Templeton). The C program prompts the user for all the options needed to run the program. The Java program provides an interface where the user selects the input and output files, the number of permutations, the possibility of using out-group weights, decimal degrees, and/or user-defined distances. The input file consists of the population information plus the description of the nested cladogram. Details are given in the program documentation. The GeoDis package, containing executables for Macintosh, PC, and Unix machines, documentation, and source code in Java and C is available for free from http:// bioag.byu.edu/zoology/crandall_lab/programs.htm.

CORRELATION OF PAIRWISE GENETIC AND GEOGRAPHIC DISTANCE MEASURES: INFERRING THE RELATIVE INFLUENCES OF GENE FLOW AND DRIFT ON THE DISTRIBUTION OF GENETIC VARIABILITY

Attempts to relate estimates of regional FST to gene flow and drift via Wrights (1931) equation FST ≈ 1/ (4Nm + 1) are often inappropriate because most natural sets of populations probably are not at equilibrium (McCauley 1993), as assumed by the island model upon which the equation is based, or ineffective because the influences of gene flow and drift are confounded in the product Nm. Evaluations of the association between genetic (FST) and geographic distances separating all pairwise populations combinations in a region allows one to test for regional equilibrium, to evaluate the relative influences of gene flow and drift on population structure both within and between regions, and to visualize the behavior of the association across all degrees of geographic separation. Tests of the model using microsatellite data from 51 populations of eastern collared lizards (Crotaphytus collaris collaris) collected from four distinct geographical regions gave results highly consistent with predicted patterns of association based on regional differences in various historical and ecological factors that affect the amount of drift and gene flow. The model provides a prerequisite for and an alternative to regional FST analyses, which often simply assume regional equilibrium, thus potentially leading to erroneous and misleading inferences regarding regional population structure.

Statistical phylogeography: methods of evaluating and minimizing inference errors

Nested clade phylogeographical analysis (NCPA) has become a common tool in intraspecific phylogeography. To evaluate the validity of its inferences, NCPA was applied to actual data sets with 150 strong a priori expectations, the majority of which had not been analysed previously by NCPA. NCPA did well overall, but it sometimes failed to detect an expected event and less commonly resulted in a false positive. An examination of these errors suggested some alterations in the NCPA inference key, and these modifications reduce the incidence of false positives at the cost of a slight reduction in power. Moreover, NCPA does equally well in inferring events regardless of the presence or absence of other, unrelated events. A reanalysis of some recent computer simulations that are seemingly discordant with these results revealed that NCPA performed appropriately in these simulated samples and was not prone to a high rate of false positives under sampling assumptions that typify real data sets. NCPA makes a posteriori use of an explicit inference key for biological interpretation after statistical hypothesis testing. Alternatives to NCPA that claim that biological inference emerges directly from statistical testing are shown in fact to use an a priori inference key, albeit implicitly. It is argued that the a priori and a posteriori approaches to intraspecific phylogeography are complementary, not contradictory. Finally, cross‐validation using multiple DNA regions is shown to be a powerful method of minimizing inference errors. A likelihood ratio hypothesis testing framework has been developed that allows testing of phylogeographical hypotheses, extends NCPA to testing specific hypotheses not within the formal inference key (such as the out‐of‐Africa replacement hypothesis of recent human evolution) and integrates intra‐ and interspecific phylogeographical inference.

Out of Africa again and again

The publication of a haplotype tree of human mitochondrial DNA variation in 1987 provoked a controversy about the details of recent human evolution that continues to this day. Now many haplotype trees are available, and new analytical techniques exist for testing hypotheses about recent evolutionary history using haplotype trees. Here I present formal statistical analysis of human haplotype trees for mitochondrial DNA, Y-chromosomal DNA, two X-linked regions and six autosomal regions. A coherent picture of recent human evolution emerges with two major themes. First is the dominant role that Africa has played in shaping the modern human gene pool through at least two—not one—major expansions after the original range extension of Homo erectus out of Africa. Second is the ubiquity of genetic interchange between human populations, both in terms of recurrent gene flow constrained by geographical distance and of major population expansion events resulting in interbreeding, not replacement.

Evolutionary Consequences of Seed Pools

The annual habit in plants is often accompanied by specialized physiological mechanisms which permit seeds to remain dormant for a few years to decades. This results in the establishment of a seed pool from which germinating individuals are drawn. The seed pool causes an overlapping of generations in a population which would otherwise be subdivided into discrete generations. Others have examined seed pools per se as evolutionary strategies, but we are examining the constraints a seed pool imposes on the evolution of loci controlling seedling and adult characters not directly related to seed dormancy. Since a seed pool introduces genetic constraints for all loci simultaneously, there is not one evolutionary impact of seed pools upon an annual population, but many. For those loci that yield constant fitness effects, the presence of a seed pool retards the annual rate of allele frequency change, but does not alter the final equilibrium. However, for those loci interacting with variable features of the environment in producing their fitness effects, the seed pool can alter the ultimate genetic outcome. In a cyclical environment, the seed pool can dampen fitness cycles with a short period (with respect to the extent of the seed pool over past years) and accentuate long cycles. More importantly, the seed pool may serve as an evolutionary filter that causes only a few years from the cycle to have any true evolutionary impact and to effectively eliminate the selective impact of other years. This evolutionary filter also operates for those loci interacting with environmental elements that fluctuate randomly from year to year. Hence, seed pools can greatly reduce the fitness uncertainty generated by cyclical or random environments and free the plant population from having to respond genetically to the fitness conditions realized in every year. Moreover, the seed pool allows the absolute quality of a given years environment to influence allele frequency changes and not just the relative fitnesses of the genotypes at that locus. Finally, we examined the selectively analogous situation of seedling and juvenile selection in perennial plants lacking a seed pool. In this case, the standing crop replaces the seed pool as the agent causing an overlapping of generations. Most of the conclusions about the evolutionary effects of seed pools in annuals carry over to the evolutionary effects of standing crops on seedling and juvenile selection in perennials. However, the evolutionary filter caused by a standing crop is less effective in filtering random seedling and juvenile fitness fluctuations in perennials than the filter caused by a seed pool with respect to random fitness fluctuations in annuals.

Using phylogeographic analyses of gene trees to test species status and processes

A gene tree is an evolutionary reconstruction of the genealogical history of the genetic variation found in a sample of homologous genes or DNA regions that have experienced little or no recombination. Gene trees have the potential of straddling the interface between intra‐ and interspecific evolution. It is precisely at this interface that the process of speciation occurs, and gene trees can therefore be used as a powerful tool to probe this interface. One application is to infer species status. The cohesion species is defined as an evolutionary lineage or set of lineages with genetic exchangeability and/or ecological interchangeability. This species concept can be phrased in terms of null hypotheses that can be tested rigorously and objectively by using gene trees. First, an overlay of geography upon the gene tree is used to test the null hypothesis that the sample is from a single evolutionary lineage. This phase of testing can indicate that the sampled organisms are indeed from a single lineage and therefore a single cohesion species. In other cases, this null hypothesis is not rejected due to a lack of power or inadequate sampling. Alternatively, this null hypothesis can be rejected because two or more lineages are in the sample. The test can identify lineages even when hybridization and lineage sorting occur. Only when this null hypothesis is rejected is there the potential for more than one cohesion species. Although all cohesion species are evolutionary lineages, not all evolutionary lineages are cohesion species. Therefore, if the first null hypothesis is rejected, a second null hypothesis is tested that all lineages are genetically exchangeable and/or ecologically interchangeable. This second test is accomplished by direct contrasts of previously identified lineages or by overlaying reproductive and/or ecological data upon the gene tree and testing for significant transitions that are concordant with the previously identified lineages. Only when this second null hypothesis is rejected is a lineage elevated to the status of cohesion species. By using gene trees in this manner, species can be identified with objective, a priori criteria with an inference procedure that automatically yields much insight into the process of speciation. When one or more of the null hypotheses cannot be rejected, this procedure also provides specific guidance for future work that will be needed to judge species status.

Ancestral Asian Source(s) of New World Y-Chromosome Founder Haplotypes

Haplotypes constructed from Y-chromosome markers were used to trace the origins of Native Americans. Our sample consisted of 2,198 males from 60 global populations, including 19 Native American and 15 indigenous North Asian groups. A set of 12 biallelic polymorphisms gave rise to 14 unique Y-chromosome haplotypes that were unevenly distributed among the populations. Combining multiallelic variation at two Y-linked microsatellites (DYS19 and DXYS156Y) with the unique haplotypes results in a total of 95 combination haplotypes. Contra previous findings based on Y- chromosome data, our new results suggest the possibility of more than one Native American paternal founder haplotype. We postulate that, of the nine unique haplotypes found in Native Americans, haplotypes 1C and 1F are the best candidates for major New World founder haplotypes, whereas haplotypes 1B, 1I, and 1U may either be founder haplotypes and/or have arrived in the New World via recent admixture. Two of the other four haplotypes (YAP+ haplotypes 4 and 5) are probably present because of post-Columbian admixture, whereas haplotype 1G may have originated in the New World, and the Old World source of the final New World haplotype (1D) remains unresolved. The contrasting distribution patterns of the two major candidate founder haplotypes in Asia and the New World, as well as the results of a nested cladistic analysis, suggest the possibility of more than one paternal migration from the general region of Lake Baikal to the Americas.

Deep resequencing reveals excess rare recent variants consistent with explosive population growth

Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.