Patterns Of mtDNA Variation Research Articles

A phylogenetic approach for haplotype analysis of sequence data from complex mitochondrial mixtures

Massively parallel (next-generation) sequencing provides a powerful method to analyze DNA from many different sources, including degraded and trace samples. A common challenge, however, is that many forensic samples are often known or suspected mixtures of DNA from multiple individuals. Haploid lineage markers, such as mitochondrial (mt) DNA, are useful for analysis of mixtures because, unlike nuclear genetic markers, each individual contributes a single sequence to the mixture. Deconvolution of these mixtures into the constituent mitochondrial haplotypes is challenging as typical sequence read lengths are too short to reconstruct the distinct haplotypes completely. We present a powerful computational approach for determining the constituent haplotypes in massively parallel sequencing data from potentially mixed samples. At the heart of our approach is an expectation maximization based algorithm that co-estimates the overall mixture proportions and the source haplogroup for each read individually. This approach, implemented in the software package mixemt, correctly identifies haplogroups from mixed samples across a range of mixture proportions. Furthermore, our method can separate fragments in a mixed sample by the most likely originating contributor and generate reconstructions of the constituent haplotypes based on known patterns of mtDNA diversity.

Concordance in evolutionary history of threatened plant and insect populations warrant unified conservation management approaches

Threatened organisms may act as host to a suite of dependent organisms, which are potentially cothreatened, yet management is rarely coordinated between host and dependent species. Here, we test the congruency of patterns of genetic structure between two critically endangered interacting taxa; the feather-leaf banksia (Banksia brownii R.Br.), and its host-specific herbivorous plant-louse Trioza barrettae Taylor & Moir, to establish whether conservation actions should be implemented jointly for both species. We also examine the role of host population size and fire history on the density of psyllids on host plants. We show that the patterns of mtDNA variation in T. barrettae and microsatellite variation in both species support the presence of at least two conservation units across each species, with the microsatellites also showing a high evolutionary congruency between plant and insect populations. The extinction of divergent B. brownii populations, therefore, is likely to have resulted in the extinction of divergent plant-louse populations. Larger populations of host plant (>150) and more recent fire history (<20years since fire) are important factors in maintaining T. barrettae densities. High molecular congruency indicates the importance of considering patterns of genetic diversity of source material from both host and dependent organisms for ex situ conservation, in situ supplementations and reintroductions. As dependents such as T. barrettae are often lost to extinction before their host, considering the conservation of dependent biota in the early stages of species management is paramount.

Effects of Pleistocene climatic and geomorphological changes on the population structure of the restricted-range catfish Trichogenes longipinnis (Siluriformes: Trichomycteridae)

Trichogenes longipinnis Britski & Ortega is a narrowly distributed endemic and phenotypically variable catfish from the coastal basins of the Serra do Mar range in southeast Brazil. We examined patterns of mtDNA variation of this species in coastal basins of the Serra do Mar to determine the influences of past climatic and geomorphological processes in connection with the currently isolated basins. Allozyme data were also used to test the hypothesis that the different spotting patterns in the different areas could be the result of cryptic speciation. Regardless of body pigmentation, T. longipinnis specimens from across the basins were found to belong to a single species, but the populations were structured in accordance with the current hydrological watersheds, in four management units across the three distinct basins of its geographic distribution. Thus, the current genetic distribution may be best explained by both marine regressions and orogeny. Based on the low levels of genetic variation and high population structure observed, we suggest that T. longipinnis should be classified as “vulnerable” in the Brazilian red list of threatened fauna. Furthermore, we propose that the headwaters of the Parati-Mirim River basin should be incorporated as an extension of the Bocaina National Park to protect its genetically differentiated lineages.

Revealing latitudinal patterns of mitochondrial DNA diversity in Chileans

The territory of Chile is particularly long and narrow, which combined with its mountainous terrain, makes it a unique scenario for human genetic studies. We obtained 995 control region mitochondrial DNA (mtDNA) sequences from Chileans representing populations living at different latitudes of the country from the North to the southernmost region. The majority of the mtDNA profiles are of Native American origin (∼88%). The remaining haplotypes are mostly of recent European origin (∼11%), and only a minor proportion is of recent African ancestry (∼1%). While these proportions are relatively uniform across the country, more structured patterns of diversity emerge when examining the variation from a phylogeographic perspective. For instance, haplogroup A2 reaches ∼9% in the North, and its frequency decreases gradually to ∼1% in the southernmost populations, while the frequency of haplogroup D (sub-haplogroups D1 and D4) follows the opposite pattern: 36% in the southernmost region, gradually decreasing to 21% in the North. Furthermore, there are remarkable signatures of founder effects in specific sub-clades of Native American (e.g. haplogroups D1j and D4p) and European (e.g. haplogroups T2b3 and K1a4a1a+195) ancestry. We conclude that the magnitude of the latitudinal differences observed in the patterns of mtDNA variation might be relevant in forensic casework.

Intraspecific genetic variation in the common midwife toad (Alytes obstetricans): subspecies assignment using mitochondrial and microsatellite markers

The common midwife toad (Alytes obstetricans), widely distributed in the northern half of the Iberian Peninsula and part of Western Europe, is currently subdivided into four subspecies: A. o. obstetricans, A. o. boscai, A. o. pertinax and A. o. almogavarii. However, the delimitation of these subspecies and their ranges are still under discussion because strong discordances have been found between morphological and molecular data, and especially among different genetic markers. Here, we screen a set of novel microsatellite loci and mtDNA sequences of A. obstetricans populations representative of all currently recognized subspecies to investigate the correspondence between genetic groupings inferred from clustering analysis of microsatellite genotypes and the described subspecies and test whether patterns of mtDNA variation are concordant with those genetic clusters. Our results confirm previous expectations of extremely high intraspecific diversity in A. obstetricans in Iberia. Analyses of microsatellite and mtDNA data were concordant in recovering five well-defined groups, of which three correspond to previously defined subspecies, while the two additional clusters correspond to populations of subspecies A. o. boscai separated by the Douro River. Our results suggest the occurrence of two distinct genetic units within A. o. boscai that likely result from a long independent evolutionary history, thus deserving special attention from a conservation point of view.

Paleoclimatic modeling and phylogeography of least killifish, Heterandria formosa: insights into Pleistocene expansion-contraction dynamics and evolutionary history of North American Coastal Plain freshwater biota

BackgroundClimatic and sea-level fluctuations throughout the last Pleistocene glacial cycle (~130-0 ka) profoundly influenced present-day distributions and genetic diversity of Northern Hemisphere biotas by forcing range contractions in many species during the glacial advance and allowing expansion following glacial retreat ('expansion-contraction’ model). Evidence for such range dynamics and refugia in the unglaciated Gulf-Atlantic Coastal Plain stems largely from terrestrial species, and aquatic species Pleistocene responses remain relatively uninvestigated. Heterandria formosa, a wide-ranging regional endemic, presents an ideal system to test the expansion-contraction model within this biota. By integrating ecological niche modeling and phylogeography, we infer the Pleistocene history of this livebearing fish (Poeciliidae) and test for several predicted distributional and genetic effects of the last glaciation.ResultsPaleoclimatic models predicted range contraction to a single southwest Florida peninsula refugium during the Last Glacial Maximum, followed by northward expansion. We inferred spatial-population subdivision into four groups that reflect genetic barriers outside this refuge. Several other features of the genetic data were consistent with predictions derived from an expansion-contraction model: limited intraspecific divergence (e.g. mean mtDNA p-distance = 0.66%); a pattern of mtDNA diversity (mean Hd = 0.934; mean π = 0.007) consistent with rapid, recent population expansion; a lack of mtDNA isolation-by-distance; and clinal variation in allozyme diversity with higher diversity at lower latitudes near the predicted refugium. Statistical tests of mismatch distributions and coalescent simulations of the gene tree lent greater support to a scenario of post-glacial expansion and diversification from a single refugium than to any other model examined (e.g. multiple-refugia scenarios).ConclusionsCongruent results from diverse data indicate H. formosa fits the classic Pleistocene expansion-contraction model, even as the genetic data suggest additional ecological influences on population structure. While evidence for Plio-Pleistocene Gulf Coast vicariance is well described for many freshwater species presently codistributed with H. formosa, this species demography and diversification departs notably from this pattern. Species-specific expansion-contraction dynamics may therefore have figured more prominently in shaping Coastal Plain evolutionary history than previously thought. Our findings bolster growing appreciation for the complexity of phylogeographical structuring within North America’s southern refugia, including responses of Coastal Plain freshwater biota to Pleistocene climatic fluctuations.

Regional patterns of mtDNA diversity in Styela plicata, an invasive ascidian, from Australian and New Zealand marinas

The ascidian Styela plicata is abundant in harbours and marinas worldwide and has likely reached this distribution via human-mediated dispersal. Previous worldwide surveys based on mitochondrial cytochrome oxidase one (COI) sequences have described two divergent clades, showing overlapping distributions and geographically widespread haplotypes. These patterns are consistent with recent mixing among genetically differentiated groups arising from multiple introductions from historically distinct sources. In contrast, a study of Australian S. plicata using nuclear markers found that population differentiation along the eastern coast related to geographic distance and no evidence for admixture between previously isolated genetic groups. We re-examined the genetic patterns of Australian S. plicata populations using mtDNA (CO1) to place their genetic patterns within a global context, and we examined New Zealand populations for the first time. We found that the haplotypic compositions of Australian and New Zealand populations are largely representative of other worldwide populations. The New Zealand populations, however, exhibited reduced diversity, being potentially indicative of a severely bottlenecked colonisation event. In contrast to results from nuclear markers, population differentiation of mtDNA among Australian S. plicata was unrelated to geographic distance. The discrepancy between markers is likely to be a consequence of non-equilibrium population genetic processes that typify non-indigenous species.

Deep mtDNA subdivision within Linnean species in an endemic radiation of tiger beetles from New Zealand (genus Neocicindela)

The invertebrate fauna of New Zealand is of great interest as a geologically tractable model for the study of species diversification, but direct comparisons with closely related lineages elsewhere are lacking. Integrating population-level analyses with studies of taxonomy and clade diversification, we performed mtDNA analysis on Neocicindela (Cicindelidae, tiger beetles) for a broad sample of populations from 11 of 12 known species and 161 specimens (three loci, 1883 nucleotides), revealing 123 distinct haplotypes. Phylogenetic reconstruction recovered two main lineages, each composed of 5–6 Linnean species whose origin was dated to 6.66 and 7.26 Mya, while the Neocicindela stem group was placed at 10.82 ± 0.48 Mya. Species delimitation implementing a character-based (diagnostic) species concept recognized 19 species-level groups that were in general agreement with Linnean species but split some of these into mostly allopatric subgroups. Tree-based methods of species delimitation using a mixed Yule-coalescence model were inconclusive, and recognized 32–51 entities (including singletons), splitting existing species into up to 8 partially sympatric groups. These findings were different from patterns in the Australian sister genus Rivacindela, where character-based and tree-based methods were previously shown to produce highly congruent groupings. In Neocicindela, the pattern of mtDNA variation was characterized by high intra-population and intra-species haplotype divergence, the coexistence of divergent haplotypes in sympatry, and a poor correlation of genetic and geographic distance. These observations combined suggest a scenario of phylogeographic divergence and secondary contact driven by orogenetic and climatic changes of the Pleistocene/Pliocene. The complex evolutionary history of most species of Neocicindela due to the relative instability of the New Zealand biota resulted in populations of mixed ancestry but not in a general loss of genetic variation.

Effects of Wolbachia on mtDNA variation and evolution in natural populations of Tetranychus urticae Koch

We investigated the effects of Wolbachia infection on mtDNA variation in spider mites by sequencing a portion of the mitochondrial cytochrome oxidase I (COI) gene from 198 individuals of known infection status. Four Wolbachia strains were described in the current study, namely wUrtOri1, wUrtOri2, wUrtOri3 and wUrtCon1. As predicted, the haplotype and nucleotide diversity were lower in infected individuals than in uninfected individuals. However, these mtDNA haplotype data are not entirely concordant with the surface protein of wolbachia (wsp) sequence data and both infected and uninfected individuals were found of the same haplotype. Although values of Tajima's D and Fu & Li's F were consistently less than zero for most infected groups, McDonald-Kreitman tests suggested that the patterns of variation were different from those expected under neutrality in only the uninfected group. Thus, the neutrality tests do not show a clear effect of Wolbachia infection on patterns of mtDNA variation and substitution in spider mites.

Erratum to: Patterns of mtDNA diversity in North Atlantic populations of the mussel Mytilus edulis

The post-colonization direction of gene flow in Fig. 5 was reversed. Although this mistake alters some conclusions regarding post-colonization gene flow (p. 405), IM-derived estimates of population splitting (s) and divergence times are correct as reported and these data form the basis for the major conclusions regarding routes of colonization. The recombinant haplotype from Mahone NS is 677 not 667.

The Commensal Model for Human Settlement of the Pacific 10 Years on—What Can We Say and Where to Now?

ABSTRACT In 1998 my colleagues and I published the first full analysis of the use of Rattus exulans mitochondrial DNA phylogenies as a proxy for tracing human colonization and migration in Polynesia. In the 10 years since that publication, we have extended this approach geographically beyond Polynesia and chronologically through the use of ancient DNA. We have also extended the approach to other commensal animals and plants. This paper will review some of the major discoveries of the last 10 years of commensal research, highlighting the similarities and differences we can now observe in the patterns of mtDNA variation in the various commensal species and discussing the implications of these for understanding the human settlement of the Pacific. I will then present and discuss some of the problems and potential solutions for the further development of the commensal approach and the use of ancient DNA for addressing questions regarding the human settlement of islands in general.

Effects of a sex-ratio distorting endosymbiont on mtDNA variation in a global insect pest

BackgroundPatterns of mtDNA variation within a species reflect long-term population structure, but may also be influenced by maternally inherited endosymbionts, such as Wolbachia. These bacteria often alter host reproductive biology and can drive particular mtDNA haplotypes through populations. We investigated the impacts of Wolbachia infection and geography on mtDNA variation in the diamondback moth, a major global pest whose geographic distribution reflects both natural processes and transport via human agricultural activities.ResultsThe mtDNA phylogeny of 95 individuals sampled from 10 countries on four continents revealed two major clades. One contained only Wolbachia-infected individuals from Malaysia and Kenya, while the other contained only uninfected individuals, from all countries including Malaysia and Kenya. Within the uninfected group was a further clade containing all individuals from Australasia and displaying very limited sequence variation. In contrast, a biparental nuclear gene phylogeny did not have infected and uninfected clades, supporting the notion that maternally-inherited Wolbachia are responsible for the mtDNA pattern. Only about 5% (15/306) of our global sample of individuals was infected with the plutWB1 isolate and even within infected local populations, many insects were uninfected. Comparisons of infected and uninfected isofemale lines revealed that plutWB1 is associated with sex ratio distortion. Uninfected lines have a 1:1 sex ratio, while infected ones show a 2:1 female bias.ConclusionThe main correlate of mtDNA variation in P. xylostella is presence or absence of the plutWB1 infection. This is associated with substantial sex ratio distortion and the underlying mechanisms deserve further study. In contrast, geographic origin is a poor predictor of moth mtDNA sequences, reflecting human activity in moving the insects around the globe. The exception is a clade of Australasian individuals, which may reflect a bottleneck during their recent introduction to this region.

Two cryptic species of the Hediste diversicolor group (Polychaeta, Nereididae) in the Baltic Sea, with mitochondrial signatures of different population histories

A presence of two cryptic biological species of Hediste diversicolor complex polychaetes was corroborated in a geographical survey of some 30 populations from the eastern and southern coasts of the Baltic Sea, with data from four completely diagnostic allozyme characters. Species A was dominant in the northernmost part of the Baltic Hediste range (Bothnian Sea), whereas Species B alone was found in the south (Poland, Germany, Denmark). In the intervening region, comprising the majority of the sites studied in southern Finland and Estonia, the two species were usually found together, with no evidence of recent hybridisation (i.e., no heterozygote genotypes). While mitochondrial DNA also distinguished the two taxa, it was not similarly completely diagnostic, but there were rare cases (ca 5%) of lineage mismatch indicating that some introgression has occurred in the past. Comparison with published data suggests that species A also inhabits the North Sea–NE Atlantic–Mediterranean coasts, and species B is also present in the North Sea and the NW Atlantic (Canada). Within the Baltic, the two species show distinctly different patterns of mtDNA diversity, plausibly related to different colonisation histories. Species A shows a generally high haplotype and nucleotide diversity, whereas in species B we found only four deeply diverged groups of closely related haplotypes. Hypothetically this could indicate a recent expansion of species B from a small number of colonising individuals. Moreover, species B showed marked intraspecific geographical structuring, with co-incident genetic changes along the N Estonian–S Finnish coasts both in mtDNA and an allozyme marker; this pattern suggests a contact between two genetically distinct invasion waves of different origins. In all, species A and B represent good, reproductively isolated and partly sympatric species which require to be recognised in ecological work. A formal taxonomical description is needed, but awaits better, range-wide distributional and ecological characterisation and working out of morphological differences that enable a practical identification.

Patterns of mtDNA diversity in North Atlantic populations of the mussel Mytilus edulis

Patterns of (female) mitochondrial DNA diversity were investigated in the blue mussel Mytilus edulis. Mytilus edulis is a ubiquitous member of contemporary North Atlantic hard-substrate communities and well represented in studies of this region. Mytilus edulis was surveyed in North America and Europe, as well as mid-Atlantic sites in Greenland, Iceland, and the Faroe Islands. Mitochondrial DNA sequences revealed considerable population structure but no monophyly of haplotypes between any major regions. Coalescent analyses suggest that migration across the Atlantic Ocean has prominently been from North American source populations and that Greenland was colonized recently and exclusively from North America. In North America, there was support for two regional groups along the North American coastline. Surprisingly, we also found evidence of recombination between some Mytilus edulis and Mytilus galloprovincialis female mtDNA sequences, particularly in northern Europe.

Migration and Social Structure among the Hopewell: Evidence from Ancient DNA

For more than a century, archaeologists have studied the cultural and skeletal remains of the prehistoric Native Americans known as the “Hopewell Moundbuilders.” While many aspects of the Hopewell phenomenon are now well understood, questions still remain about the genetic makeup, burial practices, and social structure of Hopewell communities. To help answer these questions, we extracted mitochondrial DNA (mtDNA) from the skeletal remains of 39 individuals buried at the Pete Klunk Mound Group in Illinois. The pattern of mtDNA variation at this site suggests that matrilineal relationships did not strongly influence burial practices. Because different forms of mortuary activity were not associated with distinct genetic lineages, this study provides no evidence of a maternally inherited or ascribed status system in this society. The genetic data collected here also help clarify another aspect of Illinois Hopewell social structure by suggesting a matrilocal system of post-marital residence. Finally, when these data were considered in conjunction with mtDNA data previously collected from the Hopewell Mound Group in Ohio (Mills 2003), they demonstrated that migration and gene flow did accompany the cultural exchange between Hopewell communities in the Illinois and Ohio Valleys.

Population structure of bottlenose dolphins (Tursiops aduncus) impacted by bycatch along the east coast of South Africa

Shark nets placed along the coast of KwaZulu-Natal to protect bathers from shark attacks result in an incidental by-catch of bottlenose dolphins (Tursiops aduncus) at twice the level suggested by the IWC as the maximum sustainable capture rate for a cetacean population. Observational data reported the presence of at least two putative populations, one a seasonal migratory population moving into the KwaZulu-Natal area following the movement of sardines, and the other a coastal, apparently resident population. Some observations indicated possible further subdivision of the coastal population. We analysed nine microsatellite loci and 599 bps of the mitochondrial control region and found small but significant differentiation within the putative coastal population (FST = 0.022) separated north and south of Ifafa. There was weaker evidence for differentiation between the southern coastal and migratory populations (FST = 0.012). Genetic diversity was low for both types of markers in all populations, and the pattern of mtDNA variation was consistent with a founder event. Considering the high capture rate, the low level of genetic diversity, and the evidence of possible population differentiation along the coast, we suggest that particular care should be taken in managing the take from shark nets especially in the north coastal area of KZN where the population showed the higher degree of differentiation.

The population genetic consequences of diapause in Eudiaptomus copepods

Prolonged diapause (extended dormancy) is thought to greatly influence evolution in freshwater invertebrates by lengthening generation time, promoting higher levels of dispersal among populations by wind or animal vectors, and increasing effective population size. However, empirical tests of these predictions are relatively rare. Comparative studies can be informative in this regard, if the comparisons involve sympatric, closely related species that differ only in the presence or absence of a dormant life history stage. We built upon a previous study by ZELLER et al. (2006), which used this approach to study patterns of microsatellite variation in Eudiaptomus copepods from northern Europe. E. graciloides possesses diapausing eggs and adults, whereas the closely related species E. gracilis is ecologically and trophically similar but lacks diapause. To separate further historical processes from recent anthropogenic influences, we examined mitochondrial DNA sequence variation in these species from three lakes in northern Germany where they are sympatric. Biotic and abiotic variation among contemporary and historical populations was minimized by focusing on hydro-logically linked lakes separated by less than 10km. E. gracilis and E. graciloides possess very different patterns of mtDNA variation. Both species possess significant population structure on small spatial scales, but E. graciloides has far fewer alleles despite a larger average divergence. Phylogeographic analyses and Bayesian skyline plots revealed evidence for historic population expansions in both species, with the growth phase beginning thousands of generations earlier in E. gracilis than in E. graciloides. Levels of genetic diversity suggest that effective population size may be an order of magnitude larger in E. gracilis than in E. graciloides, even though E. graciloides possesses a diapausing egg bank. Although this would seem to be an ideal system for quantifying the genetic role of diapause, we found no support for the assumption that freshwater invertebrates with diapause categorically possess larger effective population sizes and higher rates of gene flow than closely related species that lack diapause.

Molecular systematics of Hispaniolan pupfishes (Cyprinodontidae: Cyprinodon): Implications for the biogeography of insular Caribbean fishes

We used sequence variation in the mtDNA control-region and ND2 and cyt b genes to assess the systematics and biogeography of the five species of pupfish ( Cyprinodon) on Hispaniola. These include four endemics, the relatively large-bodied Cyprinodon bondi, Cyprinodon nichollsi, and Cyprinodon sp., each from a separate lake in southwestern Hispaniola, and Cyprinodon higuey from a coastal lake in eastern Hispaniola. The fifth species consists of coastal populations referable to Cyprinodon variegatus riverendi. The results indicate that Hispaniola has been invaded by at least two forms, first by a late Pliocene progenitor of Cyprinodon variegatus ovinus and the large-bodied Hispaniolan species, and, more recently, by one or more ancestral forms allied with Cyprinodon variegatus variegatus and C. v. riverendi. Levels of divergence indicate that large expanses of open sea have not acted as long-term barriers to inter-island dispersal of cyprinodontiform fishes. This study, together with the molecular systematics of other insular Caribbean fishes, indicates that most insular groups originated from late Neogene dispersal from the mainland. The patterns of mtDNA variation in Cyprinodon showed little congruence with the species/subspecies taxonomy.

Decreased Diversity but Increased Substitution Rate in Host mtDNA as a Consequence of Wolbachia Endosymbiont Infection

A substantial fraction of insects and other terrestrial arthropods are infected with parasitic, maternally transmitted endosymbiotic bacteria that manipulate host reproduction. In addition to imposing direct selection on the host to resist these effects, endosymbionts may also have indirect effects on the evolution of the mtDNA with which they are cotransmitted. Patterns of mtDNA diversity and evolution were examined in Drosophila recens, which is infected with the endosymbiont Wolbachia, and its uninfected sister species D. subquinaria. The level of mitochondrial, but not nuclear, DNA diversity is much lower in D. recens than in D. subquinaria, consistent with the hypothesized diversity-purging effects of an evolutionarily recent Wolbachia sweep. The d(N)/d(S) ratio in mtDNA is significantly greater in D. recens, suggesting that Muller's ratchet has brought about an increased rate of substitution of slightly deleterious mutations. The data also reveal elevated rates of synonymous substitutions in D. recens, suggesting that these sites may experience weak selection. These findings show that maternally transmitted endosymbionts can severely depress levels of mtDNA diversity within an infected host species, while accelerating the rate of divergence among mtDNA lineages in different species.

Insights from pattern of mtDNA variation into the genetic history of São Tomé e Prı́ncipe

Sequence data from the hypervariable segments I (HVS-I) and II (HVS-II) was obtained for 30 Angolares, 35 Forros and 38 Tongas, three self-reported ethnic groups from São Tomé e Prı́ncipe, an African archipelago (Gulf of Guinea) whose settlement begun in the late 15th century. The repertory of mtDNA lineages denoted a fully African maternal pool primarily arisen from a Central/Southwestern substratum. The absence of any lineages of putative European descent means that the European impact at the mitochondrial pool was virtually nil. Angolares showed a clear reduction of mtDNA diversity and a slight genetic differentiation relatively to Tongas or Forros, whereas the two last groups did not present any signs of genetic boundaries between each other. The data now obtained reinforce the depiction of genetic substructuring in São Tomé e Prı́ncipe previously derived from Y-chromosome STRs.