Multiple mitogenomes indicate Things Fall Apart with Out of Africa or Asia hypotheses for the phylogeographic evolution of Honey Bees (Apis mellifera).

Previous morpho-molecular studies of evolutionary relationships within the economically important genus of honey bees (Apis), including the Western Honey Bee (A. mellifera L.), have suggested Out of Africa or Asia origins and subsequent spread to Europe. I test these hypotheses by a meta-analysis of complete mitochondrial DNA coding regions (11.0 kbp) from 22 nominal subspecies represented by 78 individual sequences in A. mellifera. Parsimony, distance, and likelihood analyses identify six nested clades: Things Fall Apart with Out of Africa or Asia hypotheses. Molecular clock-calibrated phylogeographic analysis shows instead a basal origin of A. m. mellifera in Europe ~ 780 Kya, and expansion to Southeast Europe and Asia Minor ~ 720 Kya. Eurasian bees spread southward via a Levantine/Nilotic/Arabian corridor into Africa ~ 540 Kya. An African clade re-established in Iberia ~ 100 Kya spread thereafter to westerly Mediterranean islands and back into North Africa. Nominal subspecies within the Asia Minor and Mediterranean clades are less differentiated than are individuals within other subspecies. Names matter: paraphyletic anomalies are artefacts of mis-referral in GenBank of sequences to the wrong subspecies, or use of faulty sequences, which are clarified by inclusion of multiple sequences from available subspecies.

Evidence for the persistence of ancient Beothuk and Maritime Archaic mitochondrial DNA genome lineages among modern Native American peoples.

The Beothuk were a Native American people who formerly occupied the island of Newfoundland, and who are generally accepted to have become culturally extinct in 1829. The Beothuk succeeded the Maritime Archaic people on the island after a hiatus of ca. 1.4 ka, and were themselves succeeded by the extant Mi'kmaq within historic times. Genetic continuity between ancient and modern Native Americans remains of interest. Complete aDNA mitogenomes from ancient Beothuk and Maritime Archaic were compared with the most closely related modern mitogenomes as obtained by BLAST search of GenBank. Beothuk mitogenomes in five clades are in one case identical to and otherwise differ by minima of three to eight SNPs from the most closely related modern mitogenomes. Maritime Archaic mitogenomes in 12 clades are in one case identical to and otherwise differ by minima of one to nine SNPs from the most similar modern mitogenomes. The single available modern Mi'kmaq mitogenome differs from the most similar Beothuk and Maritime Archaic mitogenomes by 12 and 22 SNPs, respectively. Phylogenetic analysis and sequence similarities imply lineage extinction of most ancient clades, as well as continuity of two Beothuk and at least one Maritime Archaic lineages in modern Native Americans and their descendants.

On the persistence and detectability of ancient Beothuk mitochondrial DNA genomes in living First Nations peoples.

Claims have long been made as to the survival to the present day of descendants of the Newfoundland Beothuk, a group generally accepted to have become extinct with the death of the last known member, Shanawdithit, in 1829. Interest has recently been revived by the availability of commercial genetic testing, which some claim can assign living individuals to specific Native American groups. We compare complete mitogenome sequences (16569 bp) from aDNA of eight distinct Beothuk lineages, including Shanawdithit's uncle Nonosabasut and his wife Demasduit, with three Newfoundland Mi'kmaq lineages and 21 other living Native Americans drawn from GenBank. A Newfoundland Mi'kmaq lineage in Haplogroup A is more similar to three Native Americans (1-3 SNPs) than to the most closely related Beothuk (24 SNPs). Nonosabasut in Haplogroup X is identical to a non-Beothuk Native American. Demasduit in Haplogroup C differs from three other Native Americans by 1-4 substitutions. Within a 2168 bp region of the HVS sequences available from living Mi'kmaq of the Miawpukek First Nation in Newfoundland, lineages in Haplogroups C, X, and A differ by 1, 4, and 8 substitutions, from the most similar Beothuk, and are more similar to other Native Americans. MtDNA genome sequences in living persons identical or similar to those of Beothuk do not necessarily indicate Beothuk ancestry. Mi'kmaq lineages cannot at this time be associated with any Beothuk lineages more closely than those of other Native Americans.

Phylogeographic mitogenomics of Atlantic cod Gadus morhua: Variation in and among trans-Atlantic, trans-Laurentian, Northern cod, and landlocked fjord populations.

The historical phylogeography, biogeography, and ecology of Atlantic cod (Gadus morhua) have been impacted by cyclic Pleistocene glaciations, where drops in sea temperatures led to sequestering of water in ice sheets, emergence of continental shelves, and changes to ocean currents. High-resolution, whole-genome mitogenomic phylogeography can help to elucidate this history. We identified eight major haplogroups among 153 fish from 14 populations by Bayesian, parsimony, and distance methods, including one that extends the species coalescent back to ca. 330 kya. Fish from the Barents and Baltic Seas tend to occur in basal haplogroups versus more recent distribution of fish in the Northwest Atlantic. There was significant differentiation in the majority of trans-Atlantic comparisons (ΦST = .029-.180), but little or none in pairwise comparisons within the Northwest Atlantic of individual populations (ΦST = .000-.060) or defined management stocks (ΦST = .000-.023). Monte Carlo randomization tests of population phylogeography showed significantly nonrandom trans-Atlantic phylogeography versus absence of such structure within various partitions of trans-Laurentian, Northern cod (NAFO 2J3KL) and other management stocks, and Flemish Cap populations. A landlocked meromictic fjord on Baffin Island comprised multiple identical or near-identical mitogenomes in two major polyphyletic clades, and was significantly differentiated from all other populations (ΦST = .153-.340). The phylogeography supports a hypothesis of an eastern origin of genetic diversity ca. 200-250 kya, rapid expansion of a western superhaplogroup comprising four haplogroups ca. 150 kya, and recent postglacial founder populations.

Intraspecific mitogenomics of three marine species-at-risk: Atlantic, spotted, and northern wolffish (Anarhichas spp.).

High-resolution mitogenomics of within-species relationships can answer such phylogeographic questions as how species survived the most recent glaciation, as well as identify contemporary factors such as physical barriers, isolation, and gene flow. We examined the mitogenomic population structure of three at-risk species of wolffish: Atlantic (Anarhichas lupus), spotted (A. minor), and northern (A. denticulatus). These species are extensively sympatric across the North Atlantic but exhibit very different life history strategies, a combination that results in concordant and discordant patterns of genetic variation and structure. Wolffish haplogroups were not structured geographically: Atlantic and spotted wolffish each comprised three shallow clades, whereas northern wolffish comprised two deeper but unstructured lineages. We suggest that wolffish species survived in isolation in multiple glacial refugia, either refugia within refugia (Atlantic and spotted wolffish) or more distant refugia (northern wolffish), followed by secondary admixture upon post-glacial recolonisation of the North Atlantic.

Post-glacial recolonization of insular Newfoundland across the Strait of Belle Isle gave rise to an endemic subspecies of woodland caribou, Rangifer tarandus terranovae (Bangs, 1896): evidence from mtDNA haplotypes.

Post-glacial origins of woodland caribou (Rangifer tarandus subsp.) on the island of Newfoundland and their relationship to mainland populations have been uncertain. Sequence analysis of 2223 bp of the mitochondrial DNA control region and cytochrome b gene from 233 Newfoundland caribou identified 32 haplotypes in four major clades. Comparison with other Nearctic caribou confirms a closer affinity of the basal Clade A with animals from the mainland, and as an outgroup to Clades B, C, and D that are endemic to the island. This indicates re-entry of caribou to post-glacial Newfoundland across the Strait of Belle Isle from Labrador, rather than from southern coastal refugia. Newfoundland caribou are a distinct subspecies, Rangifer tarandus terranovae (Bangs, 1896). Hierarchical AMOVA shows significant clinal differentiation of the major clades from northwest to southeast across the island. The isolated Avalon Peninsula population in the extreme southeast is genetically depauperate. Founder effects are evident in herds introduced to previously unoccupied areas by wildlife managers over the past 40-50 years. Reindeer introduced in the early 20th century have not contributed to mtDNA diversity in Newfoundland caribou.

Conservation genetics of high-arctic Gull species at risk: I. Diversity in the mtDNA control region of circumpolar populations of the Endangered Ivory Gull (Pagophila eburnea).

The high-arctic Ivory Gull (Pagophila eburnea) has recently undergone a sharp decline in numbers, and in Canada it is listed as "Endangered" under the Species-At-Risk Act. To test for circumpolar genetic distinctiveness, we examined 264 bp of the mtDNA Control Region Domain I from 127 museum specimens collected during the breeding season from northern Canada, Greenland, and Norway, and during the non-breeding season from adjacent overwintering grounds in Canada, Greenland, and a disjunct area in Alaska adjacent to the Bering Sea. Partition of genetic variance according to various phylogeographic and breeding ground models indicates no strong population structure, except that Alaska birds are consistently differentiated from other locations, and there are significant temporal shifts in haplotype frequencies. The evidence suggests that Ivory Gulls in Canada, Greenland, and Norway are a single genetic entity, in contrast to Alaska birds, which may represent a distinctive Siberian population.

Conservation genetics of high-arctic Gull species at risk: II. Diversity in the mtDNA control region of Threatened Ross's Gull (Rhodostethia rosea).

Ross's Gull (Rhodostethia rosea) is the rarest of Canadian high-arctic gulls, and is listed as Threatened under Canada's Species-At-Risk Act. The large majority of birds breed in Siberia: the origins and affinities of four extremely small breeding colonies observed since 1978 in the Canadian high arctic are unknown. We compared a 515-bp region of the mtDNA Control Region amplified from material in museum collections taken from non-breeding birds in Canada (n = 8) and Alaska (n = 6), the latter passage migrants from the Siberian populations. The Alaskan birds all have distinct haplotypes that differ by as many as six SNPs: Canadian birds taken in the vicinity of the breeding colonies show only two of these. We hypothesize the origins of the Canadian breeding colonies as recent founder events by small numbers of passage migrants from Siberia via Alaska. Ross's Gull maintains a very tenuous breeding presence in the Canadian high Arctic.

Quantitative Phylogenomics of Within-Species Mitogenome Variation: Monte Carlo and Non-Parametric Analysis of Phylogeographic Structure among Discrete Transatlantic Breeding Areas of Harp Seals (Pagophilus groenlandicus).

Phylogenomic analysis of highly-resolved intraspecific phylogenies obtained from complete mitochondrial DNA genomes has had great success in clarifying relationships within and among human populations, but has found limited application in other wild species. Analytical challenges include assessment of random versus non-random phylogeographic distributions, and quantification of differences in tree topologies among populations. Harp Seals (Pagophilus groenlandicus Erxleben, 1777) have a biogeographic distribution based on four discrete trans-Atlantic breeding and whelping populations located on "fast ice" attached to land in the White Sea, Greenland Sea, the Labrador ice Front, and Southern Gulf of St Lawrence. This East to West distribution provides a set of a priori phylogeographic hypotheses. Outstanding biogeographic questions include the degree of genetic distinctiveness among these populations, in particular between the Greenland Sea and White Sea grounds. We obtained complete coding-region DNA sequences (15,825 bp) for 53 seals. Each seal has a unique mtDNA genome sequence, which differ by 6 ~ 107 substitutions. Six major clades / groups are detectable by parsimony, neighbor-joining, and Bayesian methods, all of which are found in breeding populations on either side of the Atlantic. The species coalescent is at 180 KYA; the most recent clade, which accounts for 66% of the diversity, reflects an expansion during the mid-Wisconsinan glaciation 40~60 KYA. FST is significant only between the White Sea and Greenland Sea or Ice Front populations. Hierarchal AMOVA of 2-, 3-, or 4-island models identifies small but significant ΦSC among populations within groups, but not among groups. A novel Monte-Carlo simulation indicates that the observed distribution of individuals within breeding populations over the phylogenetic tree requires significantly fewer dispersal events than random expectation, consistent with island or a priori East to West 2- or 3-stepping-stone biogeographic models, but not a simple 1-step trans-Atlantic model. Plots of the cumulative pairwise sequence difference curves among seals in each of the four populations provide continuous proxies for phylogenetic diversification within each. Non-parametric Kolmogorov-Smirnov (K-S) tests of maximum pairwise differences between these curves indicates that the Greenland Sea population has a markedly younger phylogenetic structure than either the White Sea population or the two Northwest Atlantic populations, which are of intermediate age and homogeneous structure. The Monte Carlo and K-S assessments provide sensitive quantitative tests of within-species mitogenomic phylogeography. This is the first study to indicate that the White Sea and Greenland Sea populations have different population genetic histories. The analysis supports the hypothesis that Harp Seals comprises three genetically distinguishable breeding populations, in the White Sea, Greenland Sea, and Northwest Atlantic. Implications for an ice-dependent species during ongoing climate change are discussed.

Near neutrality, rate heterogeneity, and linkage govern mitochondrial genome evolution in Atlantic cod (Gadus morhua) and other gadine fish.

The mitochondrial DNA (mtDNA) genome figures prominently in evolutionary investigations of vertebrate animals due to a suite of characteristics that include absence of Darwinian selection, high mutation rate, and inheritance as a single linkage group. Given complete linkage and selective neutrality, mtDNA gene trees are expected to correspond to intraspecific phylogenies, and mtDNA diversity will reflect population size. The validity of these assumptions is, however, rarely tested on a genome-wide scale. Here, we analyze rates and patterns of molecular evolution among 32 whole mitochondrial genomes of Atlantic Cod (Gadus morhua) as compared with its sister taxon, the walleye pollock (Gadus [Theragra] chalcogrammus), and genomes of seven other gadine codfish. We evaluate selection within G. morhua, between sister species, and among species and intraspecific measures of linkage disequilibrium and recombination within G. morhua. Strong rate heterogeneity occurs among sites and genes at all levels of hierarchical comparison, consistent with variation in mutation rates across the genome. Neutrality indices (dN/dS) are significantly greater than unity among G. morhua genomes and between sister species, which suggests that polymorphisms within species are slightly deleterious, as expected under the nearly neutral theory of molecular evolution. Among species of gadines, dN/dS ratios are heterogeneous among genes, consistent with purifying selection and variation in functional constraint among genes rather than positive selection. The dN/dS ratio for ND4L is anomalously high across all hierarchical levels. There is no evidence for recombination within G. morhua. These patterns contrast strongly with those reported for humans: genome-wide patterns in other vertebrates should be investigated to elucidate the complex patterns of mtDNA molecular evolution.

Intraspecific phylogeographic genomics from multiple complete mtDNA genomes in Atlantic cod (Gadus morhua): origins of the "codmother," transatlantic vicariance and midglacial population expansion.

On the basis of multiple complete mitochondrial DNA genome sequences, we describe the temporal phylogeography of Atlantic cod (Gadus morhua), a lineage that has undergone a complex pattern of vicariant evolution, postglacial demographic shifts, and historic sharp population declines due to fishing and/or environmental shifts. Each of 32 fish from four spawning aggregations from the northwest Atlantic and Norway has a unique mtDNA sequence, which differs by 6-60 substitutions. Phylogenetic analysis identifies six major haplogroups that range in age from 37 to 75 KYA. The widespread haplotype identified by previous single-locus analyses at the center of a "star phylogeny" is shown to be a paraphyletic assemblage of genome lineages. The coalescent that includes all cod occurs 162 KYA. The most basal clade comprises two fish from the western Atlantic. The most recent superclade that includes all fish examined from Norway, and which includes 84% of all fish examined, dates to 128 KYA at the Sangamon/Würm interglacial, when ocean depths on continental shelves would have favored transcontinental movement. The pairwise mismatch distribution dates population expansion of this superclade to the middle of the Wisconsinan/Weichsel glaciation 59 KYA, rather than to a postglacial emergence from a marine refugium 12 KYA, or to more recent historic events. We discuss alternative scenarios for the expansion and distribution of the descendants of the "codmother" in the North Atlantic. Mitochondrial phylogenomic analyses generate highly resolved trees that enable fine-scale tests of temporal hypotheses with an accuracy not possible with single-locus methods.

Phylogeographic analysis of complete mtDNA genomes from Walleye Pollock (Gadus chalcogrammus Pallas, 1811) shows an ancient origin of genetic biodiversity.

Ursvik et al. compared the complete mitochondrial DNA (mtDNA) genome sequences of Walleye Pollock (Gadus ( = Theragra) chalcogrammus) from the Pacific Ocean with a pair of fish from an isolated population of Norwegian pollock in the Barents Sea. They concluded that the Norwegian population was recently introduced from the Pacific. We test this hypothesis within a temporal framework provided by a phylogeographic analysis of complete genomes from the pollocks' sister species, Atlantic Cod (Gadus morhua), and their divergence 3.5 mya. Pollock have a coalescent ancestor 189 +/- 25 kya. The two Norwegian fish have a common ancestor 87 +/- 7 kya, which suggests an ancient origin rather than a recent human-mediated introduction. Mitochondrial genomic biodiversity in pollock antedates the most recent glacial cycle. The clade structure of the whole-genome tree indicates that previously described single-locus mtDNA haplotypes and haplogroups are typically paraphyletic.

Phylogeographic genomics of mitochondrial DNA: Highly-resolved patterns of intraspecific evolution and a multi-species, microarray-based DNA sequencing strategy for biodiversity studies.

Phylogeographic genomics, based on multiple complete mtDNA genome sequences from within individual vertebrate species, provides highly-resolved intraspecific trees for the detailed study of evolutionary biology. We describe new biogeographic and historical insights from our studies of the genomes of codfish, wolffish, and harp seal populations in the Northwest Atlantic, and from the descendants of the founding human population of Newfoundland. Population genomics by conventional sequencing methods remains laborious. A new biotechnology, iterative DNA "re-sequencing", uses a DNA microarray to recover 30-300 kb of contiguous DNA sequence in a single experiment. Experiments with a single-species mtDNA microarray show that the method is accurate and efficient, and sufficiently species-specific to discriminate mtDNA genomes of moderately-divergent taxa. Experiments with a multi-species DNA microarray (the "ArkChip") show that simultaneous sequencing of species in different orders and classes detects SNPs within each taxon with equal accuracy as single-species-specific experiments. Iterative DNA sequencing offers a practical method for high-throughput biodiversity genomics that will enable standardized, coordinated investigation of multiple species of interest to Species at Risk and conservation biologists.

Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip.

BACKGROUND: Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific iodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA) genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. RESULTS: In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands > or = 12b that are conserved between primates and fish. CONCLUSION: Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by more than a few percent. The data suggest that interspecific cross-hybridization will not interfere with the accurate recovery of species-specific data from multispecies microarrays, provided that the species' DNA sequences differ by > 20% (mean of 5b differences per 25b oligo). Recovery of DNA sequence data from multiple, distantly-related species on a single multiplex gene chip should be a practical, highly-parallel method for investigating genomic biodiversity.

Mitochondrial genomics of gadine fishes: implications for taxonomy and biogeographic origins from whole-genome data sets.

Phylogenetic analysis of 13 substantially complete mitochondrial DNA genome sequences (14,036 bp) from 10 taxa of gadine codfishes and pollock provides highly corroborated resolution of outstanding questions on their biogeographic evolution. Of 6 resolvable nodes among species, 4 were supported by >95% of bootstrap replications in parsimony, distance, likelihood, and similarly high posterior probabilities in bayesian analyses, one by 85%-95% according to the method of analysis, and one by 99% by one method and a majority of the other two. The endemic Pacific species, walleye pollock (Theragra chalcogramma), is more closely related to the endemic Atlantic species, Atlantic cod (Gadus macrocephalus), than either is to a second Pacific endemic, Pacific cod (Gadus macrocephalus). The walleye pollock should thus be referred to the genus Gadus as originally described (Gadus chalcogrammus Pallas 1811). Arcto-Atlantic Greenland cod, previously regarded as a distinct species (G. ogac), are a genomically distinguishable subspecies within pan-Pacific G. macrocephalus. Of the 2 endemic Arctic Ocean genera, Polar cod (Boreogadus) as the outgroup to Arctic cod (Arctogadus) and Gadus sensu lato is more strongly supported than a pairing of Boreogadus and Arctogadus as sister taxa. Taking into consideration historical patterns of hydrogeography, we outline a hypothesis of the origin of the 2 endemic Pacific species as independent but simultaneous invasions through the Bering Strait from an Arcto-Atlantic ancestral lineage. In contrast to the genome data, the complete proteome sequence (3830 amino acids) resolved only 3 nodes with >95% confidence, and placed Alaska pollock outside the Gadus clade owing to reversal mutations in the ND5 locus that restore ancestral, non-Gadus, amino acid residues in that species.

EVOLUTIONARY INFERENCES FROM RESTRICTION MAPS OF MITOCHONDRIAL DNA FROM NINE TAXA OF XENOPUS FROGS.

Restriction endonuclease cleavage maps were prepared by the double digestion method for mitochondrial DNAs (mtDNAs) purified from Xenopus borealis, X. clivii, X. fraseri, X. muelleri, X. ruwenzoriensis, X. vestitus, X. laevis victorianus, X. l. laevis, and a variant of X. laevis designated X. laevis "davis." An average of 21 cleavage sites per genome were mapped with 11 restriction endonucleases. Among the four invariant sites found are three conserved not only among the Xenopus mtDNAs tested but also among nearly all vertebrate mtDNAs examined to date. Two of these are Sac II sites in the 12S and 16S ribosomal RNA genes, and one is a Hpa I site in the gene for asparagine transfer RNA. These three sites permit the alignment and comparison of mtDNAs from different vertebrate classes. Although most of the differences observed among the Xenopus maps are attributable to point mutations causing gain or loss of restriction sites, the maps also differ by three large length mutations in or near the displacement loop. Phylogenetic analysis of 30 informative sites suggests that those members of the laevis species-group that have 36 chromosomes per somatic cell can be divided into three subgroups: 1) X. borealis, X. clivii, and perhaps X. fraseri (the "borealis" subgroup), 2) X. muelleri, and 3) the subspecies of X, laevis. The mtDNA of the hexaploid (2n = 108) species, X. ruwenzoriensis, is most similar to that of taxa in the latter two subgroups, which contrasts with the morphological similarity of this species to X. fraseri. X. ruwenzoriensis may be an allopolyploid with a mother (the contributor of the cytoplasmic mtDNA genome) on the X. laevis or X. muelleri lineage and a father on the X. fraseri lineage. We present a model showing how mtDNA and nuclear genomes can yield contrasting phytogenies for species-groups that have undergone several rounds of interspecific hybridization. Comparison of mitochondrial and nuclear sequence divergences suggests that Xenopus mtDNA, like that of mammals and birds, evolves faster than nuclear DNA. Genetic distances among mtDNAs of Xenopus species are very large, generally approaching or exceeding one substitution per nucleotide.