A phylogenetic test of sympatric speciation in the Hydrobatinae (Aves: Procellariiformes).

Phylogenetic relationships among species can provide insight into how new species arise. For example, careful consideration of both the phylogenetic and geographic distributions of species in a group can reveal the geographic models of speciation within the group. One such model, sympatric speciation, may be more common than previously thought. The Hydrobatinae (Aves: Procellariformes) is a diverse subfamily of Northern Hemisphere storm-petrels for which the taxonomy is unclear. Previous studies showed that Hydrobates (formally Oceanodroma) castro breeding in the Azores during the cool season is sister species to H. monteiroi, a hot season breeder at the same locations, which suggests sympatric speciation by allochrony. To test whether other species within the subfamily arose via sympatric speciation by allochrony, we sequenced the cytochrome b gene and five nuclear introns to estimate a phylogenetic tree using multispecies coalescent methods, and to test whether species breeding in the same geographic area are monophyletic. We found that speciation within the Hydrobatinae appears to have followed several geographic modes of divergence. Sympatric seasonal species in Japan likely did not arise through sympatric speciation, but allochrony may have played a role in the divergence of H. matsudairae, a cool season breeder, and H. monorhis, a hot season breeder. No other potential cases of sympatric speciation were discovered within the subfamily. Despite breeding in the same geographic area, hydrobatine storm-petrels breeding in Baja California (H. microsoma and H. melania) are each sister to a species breeding off the coast of Peru (H. tethys and H. markhami, respectively). In fact, antitropical sister species appear to have diverged at multiple times, suggesting allochronic divergence might be common. In addition, allopatry has likely played a role in divergence of H. furcata, a north Pacific breeder, and H. pelagius, a north Atlantic breeder. This study demonstrates that a variety of mechanisms of divergence have played a role in generating the diversity of the Hydrobatinae and supports the current taxonomy of the subfamily.

Mechanisms of global diversification in the brown booby (Sula leucogaster) revealed by uniting statistical phylogeographic and multilocus phylogenetic methods.

Recent theoretical and empirical research suggests that statistical models based on coalescent theory can improve both phylogeographic and phylogenetic inference. An approach that involves elements of both statistical phylogeography (e.g. Isolation with Migration analyses) and multilocus phylogenetic inference (e.g. *beast) may be particularly useful when applied to populations with relatively old divergence times. Here, we use such an approach in the globally distributed brown booby (Sula leucogaster). We sampled 215 individuals from all major breeding areas and genotyped them at eight microsatellite and three nuclear intron loci. We found that brown booby populations were highly differentiated and that colonies can be grouped into four major genetic populations (Caribbean Sea, Central Atlantic Ocean, Indo-Central Pacific and Eastern Pacific). These populations apparently diverged in the absence of gene flow and, with one exception, currently exchange few to no migrants. The Eastern Pacific population diverged from all other populations approximately one million years ago [90% highest posterior density: 330,000-2,000,000 years ago] and exhibits a distinct male plumage, relative to other populations. However, recent gene flow from the Indo-Central Pacific into the Eastern Pacific appears to have occurred, suggesting that approximately one million years of genetic isolation and divergence in male plumage colour are not sufficient to prevent interbreeding. Gene flow following secondary contact of the Indo-Central Pacific and Eastern Pacific populations was not detected in previous mitochondrial DNA (mtDNA) studies, and the contrast between the mtDNA results and our current results highlights the advantage of a multilocus phylogeographic approach.

A multilocus phylogeny of the Sulidae (Aves: Pelecaniformes).

Gene trees will often differ from the true species history, the species tree, as a result of processes such as incomplete lineage sorting. New methods such as Bayesian Estimation of the Species Tree (BEST) use the multispecies coalescent to model lineage sorting, and directly infer the species tree from multilocus DNA sequence data. The Sulidae (Aves: Pelecaniformes) is a family of ten booby and gannet species with a global distribution. We sequenced five nuclear intron loci and one mitochondrial locus to estimate a species tree for the Sulidae using both BEST and by concatenating nuclear loci. We also used fossil calibrated strict and relaxed molecular clocks in BEAST to estimate divergence times for major nodes in the sulid phylogeny. Individual gene trees showed little phylogenetic conflict but varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. On the other hand, both the BEST and concatenated species trees were highly resolved, strongly supported, and topologically consistent with each other. The three sulid genera (Morus, Sula, Papasula) were monophyletic and the relationships within genera were mostly consistent with both a previously estimated mtDNA gene tree and the mtDNA gene tree estimated here. However, our species trees conflicted with the mtDNA gene trees in the relationships among the three genera. Most notably, we find that the endemic and endangered Abbott's booby (Papasula abbotti) is likely basal to all other members of the Sulidae and diverged from them approximately 22 million years ago.

Comparative phylogeography of brown (Sula leucogaster) and red-footed boobies (S. sula): the influence of physical barriers and habitat preference on gene flow in pelagic seabirds.

To test the hypothesis that both physical and ecological barriers to gene flow drive population differentiation in tropical seabirds, we surveyed mitochondrial control region variation in 242 brown boobies (Sula leucogaster), which prefer inshore habitat, and 271 red-footed boobies (S. sula), which prefer pelagic habitat. To determine the relative influence of isolation and gene flow on population structure, we used both traditional methods and a recently developed statistical method based on coalescent theory and Bayesian inference (Isolation with Migration). We found that global population genetic structure was high in both species, and that female-mediated gene flow among ocean basins apparently has been restricted by major physical barriers including the Isthmus of Panama, and the periodic emergence of the Sunda and Sahul Shelves in Southeast Asia. In contrast, the evolutionary history of populations within ocean basins differed markedly between the two species. In brown boobies, we found high levels of population genetic differentiation and limited gene flow among colonies, even at spatial scales as small as 500km. Although red-footed booby colonies were also genetically differentiated within ocean basins, coalescent analyses indicated that populations have either diverged in the face of ongoing gene flow, or diverged without gene flow but recently made secondary contact. Regardless, gene flow among red-footed booby populations was higher than among brown booby populations. We suggest that these contrasting patterns of gene flow within ocean basins may be explained by the different habitat preferences of brown and red-footed boobies.

Population genetic structure in Atlantic and Pacific Ocean common murres (Uria aalge): natural replicate tests of post-Pleistocene evolution.

Understanding the factors that influence population differentiation in temperate taxa can be difficult because the signatures of both historic and contemporary demographics are often reflected in population genetic patterns. Fortunately, analyses based on coalescent theory can help untangle the relative influence of these historic and contemporary factors. Common murres (Uria aalge) are vagile seabirds that breed in the boreal and low arctic waters of the Northern Hemisphere. Previous analyses revealed that Atlantic and Pacific populations are genetically distinct; however, less is known about population genetic structure within ocean basins. We employed the mitochondrial control region, four microsatellite loci and four intron loci to investigate population genetic structure throughout the range of common murres. As in previous studies, we found that Atlantic and Pacific populations diverged during the Pleistocene and do not currently exchange migrants. Therefore, Atlantic and Pacific murre populations can be used as natural replicates to test mechanisms of population differentiation. While we found little population genetic structure within the Pacific, we detected significant east-west structuring among Atlantic colonies. The degree that population genetic structure reflected contemporary population demographics also differed between ocean basins. Specifically, while the low levels of population differentiation in the Pacific are at least partially due to high levels of contemporary gene flow, the east-west structuring of populations within the Atlantic appears to be the result of historic fragmentation of populations rather than restricted contemporary gene flow. The contrasting results in the Atlantic and Pacific Oceans highlight the necessity of carefully considering multilocus nonequilibrium population genetic approaches when reconstructing the demographic history of temperate Northern Hemisphere taxa.

Sympatric speciation by allochrony in a seabird.

The importance of sympatric speciation (the evolution of reproductive isolation between codistributed populations) in generating biodiversity is highly controversial. Whereas potential examples of sympatric speciation exist for plants, insects, and fishes, most theoretical models suggest that it requires conditions that are probably not common in nature, and only two possible cases have been described for tetrapods. One mechanism by which it could occur is through allochronic isolation-separation of populations by breeding time. Oceanodroma castro (the Madeiran or band-rumped storm-petrel) is a small seabird that nests on tropical and subtropical islands throughout the Atlantic and Pacific Oceans. In at least five archipelagos, different individuals breed on the same islands in different seasons. We compared variation in five microsatellite loci and the mitochondrial control region among 562 O. castro from throughout the species' range. We found that sympatric seasonal populations differ genetically within all five archipelagos and have ceased to exchange genes in two. Population and gene trees all indicate that seasonal populations within four of the archipelagos are more closely related to each other than to populations from the same season from other archipelagos; divergence of the fifth sympatric pair is too ancient for reliable inference. Thus, seasonal populations appear to have arisen sympatrically at least four times. This is the first evidence for sympatric speciation by allochrony in a tetrapod, and adds to growing indications that population differentiation and speciation can occur without geographic barriers to gene flow.

Complex hybridization dynamics between golden-winged and blue-winged warblers (Vermivora chrysoptera and Vermivora pinus) revealed by AFLP, microsatellite, intron and mtDNA markers.

Blue-winged (Vermivora pinus) and golden-winged warblers (Vermivora chrysoptera) have an extensive mosaic hybrid zone in eastern North America. Over the past century, the general trajectory has been a rapid replacement of chrysoptera by pinus in a broad, northwardly moving area of contact. Previous mtDNA-based studies on these species' hybridization dynamics have yielded variable results: asymmetric and rapid introgression from pinus into chrysoptera in some areas and bidirectional maternal gene flow in others. To further explore the hybridization genetics of this otherwise well-studied complex, we surveyed variation in three nuclear DNA marker types--microsatellites, introns, and a panel of amplified fragment length polymorphisms (AFLPs)--with the goal of generating a multilocus assay of hybrid introgression. All markers were first tested on birds from phenotypically and mitochondrially pure parental-type populations from outside the hybrid zone. Searches for private alleles and assignment test approaches found no combination of microsatellite or intron markers that could separate the parental populations, but seven AFLP characters exhibited significant frequency differences among them. We then used the AFLP markers to examine the extent and pattern of introgression in a population where pinus-phenotype individuals have recently invaded a region that previously supported only a chrysoptera-phenotype population. Despite the low frequency of phenotypic hybrids at this location, the AFLP data suggest that almost a third of the phenotypically pure chrysoptera have introgressed genotypes, indicating the presence of substantial cryptic hybridization in the history of this species. The evidence for extensive cryptic introgression, combined with the lack of differentiation at other nuclear loci, cautions against hybrid assessments based on single markers or on phenotypic traits that are likely to be determined by a small number of loci. Considered in concert, these results from four classes of molecular markers indicate that pinus and chrysoptera are surprisingly weakly differentiated and that far fewer genetically 'pure' populations of chrysoptera may exist than previously assumed, two findings with broad implications for the conservation of this rapidly declining taxon.

Differentiation of sympatric populations of the band-rumped storm-petrel in the Galapagos Islands: an examination of genetics, morphology, and vocalizations.

In each of at least two locations within the Galapagos Islands, breeding band-rumped storm-petrels (Oceanodroma castro) form two distinct populations that use the same colony site at separate times of the year for reproduction. Temporal segregation of these populations raises the possibility that they are reproductively isolated and represent cryptic species. We examined variation in mitochondrial DNA, morphology, and vocalizations of storm-petrel populations nesting 6 months apart on the islet of Plaza Norte in the Galapagos. Seasonal populations displayed low but significant levels of differentiation in the mitochondrial control region, five morphological variables, and one feature of male vocalizations. Breeding populations appear to have been separated for approximately 1700 years. Given the recent divergence date and relatively high effective population sizes (4000-5600 females each), seasonal populations are unlikely to be in genetic equilibrium. As a result, the low divergence estimate probably reflects historical association and not contemporary genetic exchange. These populations are not sufficiently differentiated to be considered cryptic species. However, they are probably in the early stages of divergence. Consequently, we recommend that cool- and hot season populations on Plaza Norte be recognized as separate management units.

Mechanisms of population differentiation in seabirds.

Despite recent advances in population genetic theory and empirical research, the extent of genetic differentiation among natural populations of animals remains difficult to predict. We reviewed studies of geographic variation in mitochondrial DNA in seabirds to test the importance of various factors in generating population genetic and phylogeographic structure. The extent of population genetic and phylogeographic structure varies extensively among species. Species fragmented by land or ice invariably exhibit population genetic structure and most also have phylogeographic structure. However, many populations (26 of 37) display genetic structure in the absence of land, suggesting that other barriers to gene flow exist. In these populations, the extent of genetic structure is best explained by nonbreeding distribution: almost all species with two or more population-specific nonbreeding areas (or seasons) have phylogeographic structure, and all species that are resident at or near breeding colonies year-round have population genetic structure. Geographic distance between colonies and foraging range appeared to have a weak influence on the extent of population genetic structure, but little evidence was found for an effect of colony dispersion or population bottlenecks. In two species (Galapagos petrel, Pterodroma phaeopygia, and Xantus's murrelet, Synthliboramphus hypoleucus), population genetic structure, and even phylogeographic structure, exist in the absence of any recognizable physical or nonphysical barrier, suggesting that other selective or behavioural processes such as philopatry may limit gene flow. Retained ancestral variation may be masking barriers to dispersal in some species, especially at high latitudes. Allopatric speciation undoubtedly occurs in this group, but reproductive isolation also appears to have evolved through founder-induced speciation, and there is strong evidence that parapatric and sympatric speciation occur. While many questions remain unanswered, results of the present review should aid conservation efforts by enabling managers to predict the extent of population differentiation in species that have not yet been studied using molecular markers, and, thus, enable the identification of management units and evolutionary significant units for conservation.

A test of founder effect speciation using multiple loci in the auklets (Aethia spp.).

Whether speciation results more frequently from the genetic consequences of founder events or from gradual genetic divergence of large populations is a matter of debate. In this study, multiple analyses were applied to data from three loci (cytochrome b, alpha-enolase intron VIII, and MHC class II B) to test for founder effects associated with speciation in Aethia (Aves: Alcidae), a genus of seabirds thought to have undergone a rapid founder-induced radiation. Effective population sizes (N(e)) were derived from estimators of based on allelic diversity and the coalescent and from data on trans-species polymorphism. Results indicated that N(e) has been on the order of 10(5)-10(6) individuals throughout the evolutionary histories of least and crested auklets (A. pusilla and A. cristatella, respectively) and that N(e) of the ancestral species was at least 16,000 individuals. Computer simulations of MHC evolution indicated that a single-generation bottleneck at speciation could not have involved <85 individuals for each species. More moderate simulation scenarios indicated that population size could not have dropped below 2000 individuals at the time of species founding. Demographic history appears to have been stable for the auklets throughout the past several million years, and a founder effect associated with their speciation is unlikely.

The Isthmus of Panama: a major physical barrier to gene flow in a highly mobile pantropical seabird.

To further test the hypothesis that the Isthmus of Panama is a major barrier to gene flow in pantropical seabirds, we applied phylogeographic methods to mitochondrial control sequence variation in masked booby (Sula dactylatra) populations on either side of the Isthmus of Panama and the southern tip of Africa. In accord with Steeves et al. (2003), we found that all Caribbean masked boobies with the 'secondary contact' cytochrome b haplotype (m-B) shared a control region haplotype (Sd_100), which grouped with Indian-Pacific haplotypes and not Caribbean-Atlantic haplotypes. In addition, Sd_100 was more closely related to control region haplotypes in the Indian Ocean than in the Pacific. We also found that the 'secondary contact' birds diverged more recently from extant populations in the Indian Ocean than in the Pacific. Thus, it appears that these masked boobies did not breach the Isthmus of Panama. Rather, birds likely dispersed around the southern tip of Africa during favourable oceanographic conditions in the Pleistocene.

Male-biased mutation rates and the overestimation of extrapair paternity: problem, solution, and illustration using thick-billed murres (Uria lomvia, Alcidae).

The widespread utility of hypervariable loci in genetic studies derives from the high mutation rate, and thus the high polymorphism, of these loci. Recent evidence suggests that mutation rates can be extremely high and may be male biased (occurring in the male germ-line). These two factors combined may result in erroneous overestimates of extrapair paternity, since legitimate offspring with novel alleles will have more mismatches with respect to the biological father than the biological mother. As mutations are male driven, increasing the number of hypervariable loci screened may simply increase the number of mismatches between fathers and their legitimate offspring. Here we describe a simple statistic, the probability of resemblance (PR), to distinguish between mismatches due to parental misassignment versus mutation in either sex or null alleles. We apply this method to parentage data on thick-billed murres (Uria lomvia), and demonstrate that, without considering either mutations or male-biased mutation rates, cases of extrapair paternity (7% in this study) would be grossly overestimated (14.5%-22%). The probability of resemblance can be utilized in parentage studies of any sexually reproducing species when allele or haplotype frequency data are available for putative parents and offspring. We suggest calculating this probability to correctly categorize legitimate offspring when mutations and null alleles may cause mismatches.

Glacial vicariance and historical biogeography of rock ptarmigan (Lagopus mutus) in the Bering region.

In this paper, we address alternative hypotheses for the evolution of subspecies of rock ptarmigan (Lagopus mutus) endemic to the Aleutian Archipelago. To do this we examined patterns of genetic differentiation among populations of rock ptarmigan in the Aleutian Islands and parts of both Alaska and Siberia. Variation in mitochondrial control region sequences of 105 rock ptarmigan from 10 subspecies within the Bering region revealed three major phylogenetic lineages, two of which are endemic to the Aleutian Islands. Accordingly, haplotype and nucleotide diversities of rock ptarmigan within the archipelago are much higher than within mainland Alaska or Siberia. For Aleutian rock ptarmigan, analyses of molecular variance indicated significant genetic structuring and low estimates of gene flow among populations, despite small interisland distances within the archipelago. However, isolation by distance did not describe the pattern of gene flow or differentiation at this scale. Our estimates of divergence times of lineages suggest that Aleutian rock ptarmigan became isolated prior to the most recent Pleistocene glaciation event (late Wisconsin Stade) and that current patterns of genetic variation reflect the postglacial redistribution of divergent lineages and subsequent limited gene flow. In addition, genetic divergence among lineages was concordant with the distribution of plumage types among subspecies. The patterns of genetic variation described here for rock ptarmigan provide evidence for the role of glacial vicariance in contributing to genetic diversity within this and other Bering region species.

Mechanisms of population differentiation in marbled murrelets: historical versus contemporary processes.

Mechanisms of population differentiation in highly vagile species such as seabirds are poorly understood. Previous studies of marbled murrelets (Brachyramphus marmoratus; Charadriiformes: Alcidae) found significant population genetic structure, but could not determine whether this structure is due to historical vicariance (e.g., due to Pleistocene glaciers), isolation by distance, drift or selection in peripheral populations, or nesting habitat selection. To discriminate among these possibilities, we analyzed sequence variation in nine nuclear introns from 120 marbled murrelets sampled from British Columbia to the western Aleutian Islands. Mismatch distributions indicated that murrelets underwent at least one population expansion during the Pleistocene and probably are not in genetic equilibrium. Maximum-likelihood analysis of allele frequencies suggested that murrelets from "mainland" sites (from the Alaskan Peninsula east) are genetically different from those in the Aleutians and that these two lineages diverged prior to the last glaciation. Analyses of molecular variance, as well as estimates of gene flow derived using coalescent theory, indicate that population genetic structure is best explained by peripheral isolation of murrelets in the Aleutian Islands, rather than by selection associated with different nesting habitats. No isolation-by-distance effects could be detected. Our results are consistent with a rapid expansion of murrelets from a single refugium during the early-mid Pleistocene, subsequent isolation and divergence in two or more refugia during the final Pleistocene glacial advance, and secondary contact following retreat of the ice sheets. Population genetic structure now appears to be maintained by distance effects combined with small populations and a highly fragmented habitat in the Aleutian Islands.

A TEST OF THE GLACIAL REFUGIUM HYPOTHESIS USING PATTERNS OF MITOCHONDRIAL AND NUCLEAR DNA SEQUENCE VARIATION IN ROCK PTARMIGAN (LAGOPUS MUTUS).

The glacial refugium hypothesis (GRH) proposes that glaciers promoted differentiation and generation of intraspecific diversity by isolating populations in ice-free refugia. We tested three predictions of this hypothesis for the evolutionary divergence of rock ptarmigan (Lagopus mutus) during the Wisconsin glaciation of the late Pleistocene. To do this, we examined subspecies distributions, population genetic structure, and phylogenetic relationships in 26 populations across North America and the Bering Sea region. First, we analyzed sequence variation in the mitochondrial control region, in a nuclear intron (Gapdh), and in an internal transcribed spacer (ITS1). Control region sequences of 154 rock ptarmigan revealed strong population and phylogeographic structure. Variation in intron sequences of 114 rock ptarmigan also revealed significant population structure compatible with results for the control region. Rock ptarmigan were invariant for ITS1. Second, we show that five known Nearctic refugia and an Icelandic refugium are concordant with the current distribution of morphologically distinct subspecies; five of these six refugia are geographically concordant with the distribution of closely related control region haplotypes. Third, our estimates of the time since phylogenetic lineages diverged predated the last glacial maximum for all but two lineages. In addition, all lines of evidence suggest that two unknown refugia in the Bering Sea region supported rock ptarmigan during the Wisconsin glaciation. Overall, our results are most consistent with the hypothesis that isolated populations of rock ptarmigan diverged in multiple refugia during the Wisconsin and that geographic variation reflects patterns of recolonization of the Nearctic after the ice receded. The GRH may therefore offer the most plausible explanation for similar biogeographic patterns in a variety of Nearctic vertebrates.

POLYTOMIES AND THE POWER OF PHYLOGENETIC INFERENCE.

Although phylogenetic hypotheses can provide insights into mechanisms of evolution, their utility is limited by our inability to differentiate simultaneous speciation events (hard polytomies) from rapid cladogenesis (soft polytomies). In the present paper, we tested the potential for statistical power analysis to differentiate between hard and soft polytomies in molecular phytogenies. Classical power analysis typically is used a priori to determine the sample size required to detect a particular effect size at a particular level of significance (a) with a certain power (1 - ß). A posteriori, power analysis is used to infer whether failure to reject a null hypothesis results from lack of an effect or from insufficient data (i.e., low power). We adapted this approach to molecular data to infer whether polytomies result from simultaneous branching events or from insufficient sequence information. We then used this approach to determine the amount of sequence data (sample size) required to detect a positive branch length (effect size). A worked example is provided based on the auklets (Charadriiformes: Alcidae), a group of seabirds among which relationships are represented by a polytomy, despite analyses of over 3000 bp of sequence data. We demonstrate the calculation of effect sizes and sample sizes from sequence data using a normal curve test for difference of a proportion from an expected value and a t-test for a difference of a mean from an expected value. Power analyses indicated that the data for the auklets should be sufficient to differentiate speciation events that occurred at least 100,000 yr apart (the duration of the shortest glacial and interglacial events of the Pleistocene), 2.6 million years ago.

Sequence variation in the guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog.

We describe sequence variation in the mitochondrial control region and its nuclear homolog in three species and seven subspecies of guillemots (Cepphus spp.). Nuclear homologs of the 5' end of the control region were found in all individuals. Nuclear sequences were approximately 50% divergent from their mitochondrial counterparts and formed a distinct phylogenetic clade; the mitochondrial-nuclear introgression event must have predated the radiation of Cepphus. As in other vertebrates, the guillemot control region has a relatively conserved central block flanked by hypervariable 5' and 3' ends. Mean pairwise interspecific divergence values among control regions were lower than those in other birds. All individuals were heteroplasmic for the number of simple tandem nucleotide repeats (A(n)C) at the 3' end of the control region. Phylogenetic analyses suggest that black guillemots are basal to pigeon and spectacled guillemots, but evolutionary relationships among subspecies remain unresolved, possibly due to incomplete lineage sorting. Describing molecular variation in nuclear homologs of mitochondrial genes is of general interest in phylogenetics because, if undetected, the homologs may confound interpretations of mitochondrial phylogenies.

Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms.

Combination of the targeted amplification of nuclear introns and the analysis of single-stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.