Molecular phylogeny and evolution of symbiosis in a clade of Indopacific nudibranchs

Previous efforts at understanding the evolution of the genus Phyllodesmium, based on morphological analyses, have been plagued by poorly supported phylogenies (Ortiz and Gosliner, 2008; Moore and Gosliner, 2009, in press). It has been suggested (Moore and Gosliner, 2009) that a molecular phylogeny might provide more insight into this history than can be easily discovered using morphological data. In this study, 658bp of the cytochrome c oxidase subunit I gene (CO1), 441bp of the mitochondrial large ribosomal subunit (16S) gene, and 328bp of a protein-coding nuclear gene (histone 3) were sequenced for 18 species of Phyllodesmium and six outgroup species. A total of 464 parsimony informative sites were used for parsimony, maximum likelihood, and Bayesian inference of phylogeny analyses. All three analyses produced similar topologies, with the exception of a single difference within the parsimony analysis. Bootstrap values and posterior probabilities provided strong support at many shallow nodes, and the monophyly of Phyllodesmium was supported in every case. Three distinct clades of Phyllodesmium are evident in this analysis. One of these represents the majority of asymbiotic taxa. Phyllodesmium poindimiei, an asymbiotic species, is clearly a member of a symbiotic clade and appears to have secondarily lost its symbiotic relationship with zooxanthellae. There was moderate support confirming similar topological trends seen in earlier morphological phylogenies, including the hypothesis that symbiotic species associating with zooxanthellae have evolved more recently than non-symbiotic species. Despite the inclusion of a presumably conservative nuclear locus, some deep nodes are still unresolved or are not well supported. Future inclusion of additional taxa and more slowly evolving loci will likely improve resolution of these deeper nodes. The subsequent phylogeny supports previous hypotheses by Rudman (1991), Kempf (1991) and Burghardt et al. (2008b) that evolution of more complex digestive gland structures is related to increased complexity of symbiosis with zooxanthellae and greater efficiency of photosynthetic activity. Our phylogeny also demonstrates that this symbiosis has evolved only once in Phyllodesmium and that azooxanthellate species are sister, rather than basal, to zooxanthellate species.     

Diversity of algal endosymbionts (zooxanthellae) in octocorals: the roles of geography and host relationships

The presence, genetic identity and diversity of algal endosymbionts (Symbiodinium) in 114 species from 69 genera (20 families) of octocorals from the Great Barrier Reef (GBR), the far eastern Pacific (EP) and the Caribbean was examined, and patterns of the octocoral-algal symbiosis were compared with patterns in the host phylogeny. Genetic analyses of the zooxanthellae were based on ribosomal DNA internal transcribed spacer 1 (ITS1) region. In the GBR samples, Symbiodinium clades A and G were encountered with A and G being rare. Clade B zooxanthellae have been previously reported from a GBR octocoral, but are also rare in octocorals from this region. Symbiodinium G has so far only been found in Foraminifera, but is rare in these organisms. In the Caribbean samples, only Symbiodinium clades B and C are present. Hence, Symbiodinium diversity at the level of phylogenetic clades is lower in octocorals from the Caribbean compared to those from the GBR. However, an unprecedented level of ITS1 diversity was observed within individual colonies of some Caribbean gorgonians, implying either that these simultaneously harbour multiple strains of clade B zooxanthellae, or that ITS1 heterogeneity exists within the genomes of some zooxanthellae. Intracladal diversity based on ITS should therefore be interpreted with caution, especially in cases where no independent evidence exists to support distinctiveness, such as ecological distribution or physiological characteristics. All samples from EP are azooxanthellate. Three unrelated GBR taxa that are described in the literature as azooxanthellate (Junceella fragilis, Euplexaura nuttingi and Stereonephthya sp. 1) contain clade G zooxanthellae, and their symbiotic association with zooxanthellae was confirmed by histology. These corals are pale in colour, whereas related azooxanthellate species are brightly coloured. The evolutionary loss or gain of zooxanthellae may have altered the light sensitivity of the host tissues, requiring the animals to adopt or reduce pigmentation. Finally, we superimposed patterns of the octocoral-algal symbiosis onto a molecular phylogeny of the host. The data show that many losses/gains of endosymbiosis have occurred during the evolution of octocorals. The ancestral state (azooxanthellate or zooxanthellate) in octocorals remains unclear, but the data suggest that on an evolutionary timescale octocorals can switch more easily between mixotrophy and heterotrophy compared to scleractinian corals, which coincides with a low reliance on photosynthetic carbon gain in the former group of organisms.     

Molecular evidence of Pleistocene bidirectional faunal exchange between Europe and the Near East: the case of the bicoloured shrew (Crocidura leucodon, Soricidae)

We sequenced 1077 bp of the mitochondrial cytochrome b gene and 511 bp of the nuclear Apolipoprotein B gene in bicoloured shrew (Crocidura leucodon, Soricidae) populations ranging from France to Georgia. The aims of the study were to identify the main genetic clades within this species and the influence of Pleistocene climatic variations on the respective clades. The mitochondrial analyses revealed a European clade distributed from France eastwards to north-western Turkey and a Near East clade distributed from Georgia to Romania; the two clades separated during the Middle Pleistocene. We clearly identified a population expansion after a bottleneck for the European clade based on mitochondrial and nuclear sequencing data; this expansion was not observed for the eastern clade. We hypothesize that the western population was confined to a small Italo-Balkanic refugium, whereas the eastern population subsisted in several refugia along the southern coast of the Black Sea.     

Molecular phylogeny of Eastern Pacific porcelain crabs, genera Petrolisthes and Pachycheles, based on the mtDNA 16S rDNA sequence: phylogeographic and systematic implications

Porcelain crabs, genera Petrolisthes and Pachycheles, are diverse and abundant members of the eastern Pacific near-shore decapod crustacean community. Morphology-based taxonomic analyses of these crabs have determined groupings of affiliated species, but phylogenetic relationships remain unknown. We used sequence data from the mitochondrial 16S rRNA gene of 46 species of eastern Pacific porcelain crabs to perform phylogenetic analyses by distance and parsimony methods. Our results are used to compare the taxonomic significance of morphological and molecular characters, to examine sequence divergence rates of crab 16S rRNA genes, and to analyze the phylogeographic history of these crabs. Our phylogenetic trees indicate that the genus Petrolisthes is divided into two main clades, reflecting morphological features. One clade contains primarily tropical species, and the other contains species from throughout the eastern Pacific, as well as species in the genera Allopetrolisthes and Liopetrolisthes. Phylogenetic trees of Pachycheles suggest an antitropical distribution; north and south temperate species form one clade and tropical species form a second clade. Sequence divergence rates of the 16S rRNA gene from three pairs of geminate species can be used to date divergence times, and we discuss porcelain crab phylogeographic patterns in relation to paleogeographic events.     

The genetic identity of dinoflagellate symbionts in Caribbean octocorals

Many cnidarians (e.g., corals, octocorals, sea anemones) maintain a symbiosis with dinoflagellates (zooxanthellae). Zooxanthellae are grouped into clades, with studies focusing on scleractinian corals. We characterized zooxanthellae in 35 species of Caribbean octocorals. Most Caribbean octocoral species (88.6%) hosted clade B zooxanthellae, 8.6% hosted clade C, and one species (2.9%) hosted clades B and C. Erythropodium caribaeorum harbored clade C and a unique RFLP pattern, which, when sequenced, fell within clade C. Five octocoral species displayed no zooxanthella cladal variation with depth. Nine of the ten octocoral species sampled throughout the Caribbean exhibited no regional zooxanthella cladal differences. The exception, Briareum asbestinum, had some colonies from the Dry Tortugas exhibiting the E. caribaeorum RFLP pattern while elsewhere hosting clade B. In the Caribbean, octocorals show more symbiont specificity at the cladal level than scleractinian corals. Both octocorals and scleractinian corals, however, exhibited taxonomic affinity between zooxanthella clade and host suborder.Communicated by R.C. Carpenter     

Mycosporine-like amino acid content in four species of sea anemones in the genus Anthopleura reflects phylogenetic but not environmental or symbiotic relationships

We examine the occurrence of UV-absorbing, mycosporine-like amino acids (MAAs) in four sympatric species of sea anemones in the genus Anthopleura, all collected from intertidal habitats on the Pacific Coast of temperate North America. We compare patterns of MAAs in A. elegantissima of several types: specimens having predominately zooxanthellae (dinoflagellates comprising at least two species) or zoochlorellae as symbionts; those containing algal endosymbionts of both kinds, and naturally occurring aposymbiotic specimens that lack the endosymbionts typically found in most specimens. We also compare MAAs in zooxanthellate specimens of A. sola and A. xanthogrammica, and specimens from the asymbiotic species A. artemisia. Our findings indicate that the complements of the four major MAAs in these species of Anthopleura (mycosporine-taurine, shinorine, porphyra-334, and mycosporine-2 glycine) broadly reflect phylogenetic differences among the anemones rather than the taxon of endosymbionts, presence or absence of symbionts, or environmental factors. An exception, however, occurs in A. elegantissima, where mycosporine-2 glycine increases in concentration with the density of zooxanthellae. Our evidence also shows that A. elegantissima can accumulate MAAs from its food, which may explain the occasional occurrence of minor MAAs in some individuals.     

Biogeography and microhabitat variation in temperate algal-invertebrate symbioses: zooxanthellae and zoochlorellae in two Pacific intertidal sea anemones, Anthopleura elegantissima and A. xanthogrammica

Abstract. Temperate sea anemones in the genus Anthopleura are unique among cnidarians in harboring two phylogenetically distinct symbiotic algae, zooxanthellae (golden-brown dinophytes, Symbiodinium ) and zoochlorellae (green chlorophytes). To determine whether their physiological differences generate patterns in anemone habitat and biogeographic distribution, we sampled symbiotic algae in the small clonal A. elegantissima and the large solitary A. xanthogrammica at 8 field sites (and the other large solitary Anthopleura species at one site) spanning 18° of latitude along 2500 km of the Pacific coast of North America. We found that zoochlorellae predominate in low intertidal habitats and northerly latitudes and in A. xanthogrammica , while zooxanthellae constitute the majority of symbionts in high intertidal habitats and more southerly latitudes and in A. elegantissima. These data are consistent with published predictions based on photosynthetic efficiency of the two algae under varied temperature and light regimes in the laboratory. This anemone-algal system provides a potential biological signal of benthic intertidal communities' responses to El Niño events and long-term climate changes in the Pacific.     

The evolutionary history of seahorses (Syngnathidae: Hippocampus): molecular data suggest a West Pacific origin and two invasions of the Atlantic Ocean

Sequence data derived from four markers (the nuclear RP1 and Aldolase and the mitochondrial 16S rRNA and cytochrome b genes) were used to determine the phylogenetic relationships among 32 species belonging to the genus Hippocampus. There were marked differences in the rate of evolution among these gene fragments, with Aldolase evolving the slowest and the mtDNA cytochrome b gene the fastest. The RP1 gene recovered the highest number of nodes supported by >70% bootstrap values from parsimony analysis and >95% posterior probabilities from Bayesian inference. The combined analysis based on 2317 nucleotides resulted in the most robust phylogeny. A distinct phylogenetic split was identified between the pygmy seahorse, Hippocampus bargibanti, and a clade including all other species. Three species from the western Pacific Ocean included in our study, namely H. bargibanti, H. breviceps, and H. abdominalis occupy basal positions in the phylogeny. This and the high species richness in the region suggests that the genus evolved somewhere in the West Pacific. There is also fairly strong molecular support for the remaining species being subdivided into three main evolutionary lineages: two West Pacific clades and a clade of species present in both the Indo-Pacific and the Atlantic Ocean. The phylogeny obtained herein suggests at least two independent colonization events of the Atlantic Ocean, once before the closure of the Tethyan seaway, and once afterwards.     

基于线粒体基因组全序列的鲟形目鱼类(Pisces: Acipenseriformes)的分子系统发育重建

为厘清鲟形目鱼类的系统发育, 研究新测定了中华鲟(Acipenser sinensis)、长江鲟(A. dabryanus)、短吻鲟(A. brevirostrum)、纳氏鲟(A. naccarii)、鳇(Huso dauricus)和匙吻鲟(Polyodon spathula)共6种鲟类的线粒体全基因组序列。联合已测的17种鲟类的线粒体基因组数据, 利用最大似然法和贝叶斯法重建了鲟形目鱼类的分子系统发育关系, 并采用似然值检验对不同的树拓扑结构进行了评价。结果表明, 6种新测鲟类的线粒体基因组大小为16521—16766 bp, 编码13个蛋白质编码基因、22个转运RNA基因和2个核糖体基因, 与大多数已测的鲟类的线粒体基因组结构高度相似。基于23种鲟形目鱼类线粒体基因组数据, 系统发育分析的结果表明: (1)鲟形目的两个科, 匙吻鲟科(Polyodontidae)和鲟科(Acipenseridae)均为单系; (2)鲟科的内部亲缘关系复杂, 鲟属和鳇属的物种均不构成单系群。鲟科鱼类按分子系统发育重建结果可以分为3个类群: 尖吻鲟类(A. sturio - A. oxyrinchus clade)、大西洋鲟类(Atlantic clade)和太平洋鲟类(Pacific clade)。树拓扑结构的检验结果表明, 鲟科的系统发育关系为(尖吻鲟类(太平洋鲟类, 大西洋鲟类))。铲鲟属(Scaphirhynchus)是大西洋鲟类的基部类群。研究也说明线粒体基因组数据在鲟形目鱼类系统与进化研究方面具有重要应用价值。     

Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

Genetic analysis of sympatric char populations in western Alaska: Arctic char (Salvelinus alpinus) and Dolly Varden (Salvelinus malma) are not two sides of the same coin

The North Pacific Ocean has been of great significance to understanding biogeography and speciation in temperate faunas, including for two species of char (Salmonidae: Salvelinus) whose evolutionary relationship has been controversial. We examined the morphology and genetics (microsatellite and mitochondrial DNA) of Arctic char (Salvelinus alpinus) and Dolly Varden char (Salvelinus malma) in lake systems in western Alaska, the eastern and western Arctic, and south of the Alaskan Peninsula. Morphologically, each lake system contained two forms: one (Arctic char) largely confined to lake habitats and characterized by greater numbers of pyloric caeca, gill rakers, and shallower bodies, and another (Dolly Varden) predominated in adjacent stream habitats and was characterized by fewer pyloric caeca, gill rakers, and deeper bodies. MtDNA partial (550 bp) d-loop sequences of both taxa were interspersed with each other within a single 'Bering' clade and demographic inferences suggested historical gene flow from Dolly Varden to Arctic char had occurred. By contrast, the taxa were strongly differentiated in sympatry across nine microsatellite loci in both lakes. Our data show that the two taxa are highly genetically distinct in sympatry, supporting their status as valid biological species, despite occasional hybridization. The interaction between these species highlights the importance of the North Pacific, and Beringia in particular, as an evolutionary wellspring of biodiversity.     

Mitochondrial DNA phylogeography of European hedgehogs

European hedgehog populations belonging to Erinaceus europaeus and E . concolor have been investigated by mitochondrial DNA analysis. A 383 bp fragment of the cytochrome b gene has been sequenced and maximum parsimony and neighbour-joining trees of Tamura–Nei genetic distance values have been constructed. Similar topologies have been produced by both methods, showing a deep divergence between E . europaeus and E . concolor and a further subdivision of each species into a western and an eastern clade. A comparison with previously published allozyme data is made, and concordant and discordant patterns are discussed. The influence of Pleistocene glaciations on the observed pattern of divergence is inferred.     

Y chromosome phylogeny for cutthroat trout (Oncorhynchus clarkii) subspecies is generally concordant with those of other markers

Sequence divergence was evaluated in the non-recombining, male-specific OmyY1 region of the Y chromosome among the subspecies of cutthroat trout (Oncorhynchus clarkii) in the western United States. This evaluation identified subspecies-discriminating OmyY1-haplotypes within a ～1200 bp region of the OmyY1 locus and localized the region to the end of the Y chromosome by FISH analysis. OmyY1 sequences were aligned and used to reconstruct a phylogeny of the cutthroat trout subspecies and related species via maximum-parsimony and Bayesian analyses. In the Y-haplotype phylogeny, clade distributions generally corresponded to the geographic distributions of the recognized subspecies. This phylogeny generally corresponded to a mitochondrial tree obtained for these subspecies in a previous study. Both support a clade of trout vs. Pacific salmon, of rainbow trout, and of a Yellowstone cutthroat group within the cutthroat trout. In our OmyY1 tree, however, the cutthroat “clade”, although present topologically, was not statistically significant. Some key differences were found between trees obtained from the paternally-inherited OmyY1 vs. maternally-inherited mitochondrial haplotypes in cutthroat trout compared to rainbow trout. Other findings are: The trout OmyY1 region evolves between 3 and 13 times slower than the trout mitochondrial regions that have been studied. The Lahontan cutthroat trout had a fixed OmyY1 sequence throughout ten separate populations, suggesting this subspecies underwent a severe population bottleneck prior to its current dispersal throughout the Great Basin during the pluvial phase of the last ice age. The Yellowstone group is the most derived among the cutthroat trout and consists of the Yellowstone, Bonneville, Colorado, Rio Grande and greenback subspecies. Identification of subspecies and sex with this Y-chromosome marker may prove useful in conservation efforts.     

EVOLUTIONARY HISTORY OF NORTHERN HEMISPHERE NUCELLA (GASTROPODA,MURICIDAE): MOLECULAR,MORPHOLOGICAL, ECOLOGICAL,AND PALEONTOLOGICAL EVIDENCE

By combining data from a variety of sources we explore patterns of evolution and speciation in Nucella, a widely studied genus of shallow-water marine neogastropods. We present a hypothesis of phylogenetic relationships for all of the currently recognized species of northern hemisphere Nucella, based on an analysis of 718 base pairs of nucleotide sequence from the mitochondrial cytochrome b gene. The order of appearance of species in the fossil record is congruent with this hypothesis. The topology of the inferred phylogeny of Nucella, coupled with ecological, morphological, and fossil evidence, was used to address three main questions: (1) At what time and by which route was the North Atlantic invaded from the North Pacific compared to prior studies of the trans-Arctic interchange? (2) Do patterns of molecular variation within species corroborate the importance of climatic cycles in driving speciation in north temperate marine animals? (3) Was radiation in the direction of increased or decreased ecological specialization, body size, or vulnerability to predation? Molecular evidence confirmed that the sole North Atlantic species, N. lapillus, arose from a North Pacific ancestor. Biogeographic and paleontological evidence supported the dispersal of Nucella, and perhaps other interchange species, via the Eurasian Arctic. Rather intriguingly, the linkage of N. lapillus to a western as opposed to eastern Pacific clade, and the biogeographic origins of the eastern Pacific species, parallel closely similar patterns observed in another genus of rocky-shore gastropods, Littorina. This congruence, in conjunction with information on the climatic and geographic histories of the region, as well as the geographic arrangement of mtDNA haplotypes within Nucella species, supports a model of speciation in Nucella driven by cycles of climatic amelioration and deterioration that began during the Miocene. Calibrations from the fossil record of Nucella suggest that third position transitions and transversions accrue at a rate of 3–4% and 0.5% respectively per million yr. This supports an early participation by Nucella in the trans-Arctic interchange, as suggested by paleobiogeographic studies. Consistent with the unstable taxonomic history of species of Nucella, we found few nonmolecular traits to be phylogenetically informative. Among North Pacific species, more recently derived species (N. canaliculata and the N. emarginata clade) were more ecologically specialized (narrower diet and habitat range). Consistent with extensive intraspecific variation, shell traits were quite labile evolutionarily: neither overall size nor development of antipredatory traits exhibited consistent evolutionary trends over the history of the genus. Nurse eggs (unfertilized eggs consumed by developing embryos) were an ancestral trait that was lost evolutionarily in the two clades that also exhibited increased body size, suggesting that these two life-history traits may be coupled. The reduced number of chromosomes in N. lapillus is clearly a derived state and is consistent with White's (1978) observations on chromosome evolution in other clades.     

从细胞色素b基因序列探讨笛鲷属的分子系统发生关系

测定了9种中国南海的笛鲷属鱼类的细胞色素b基因的部分序列,结合来自GenBank中1种分布于菲律宾和9种分布于美国大西洋的笛鲷属鱼类的相应同源序列,用邻接法和最大简约法构建分子系统树。结果显示:红鳍笛鲷(Lutjanuserythropterus)与红笛鲷(L.sanguineus)之间的同源序列碱基差异百分率只有0.32%,支持二者是同种异名的观点;中国南海的笛鲷属鱼类间的平均碱基差异要高于美国大西洋笛鲷属鱼类。在MP和NJ树中,美国大西洋笛鲷表现为亲缘关系较近,来源于中国南海的笛鲷鱼类相对集中在树的基部,分歧较大。这与所研究的笛鲷地理分布和地理隔离基本相一致,同时也说明中国南海笛鲷分化较早并且分歧较大。     

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

基于核基因c-mos的鸫亚科部分鸟类系统发生关系

采用分子系统学方法对鸫亚科Turdinae 11属21种鸟类的核基因c-mos进行了系统发生分析.所测序列经对位排列后共572个位点,其中核苷酸变异位点111个,简约信息位点71个.以太平鸟Bombycilla garrulus作外群,采用邻接法、最大简约法和最大似然法分别构建其系统发生树.重建的系统发生树显示:所研究鸫亚科21种鸟类分成2个支系,第1个支系包括鸫属Turdus和地鸫属Zoothera.第2个支系包括红尾鸲属Phoenicurus、矶鸫属Monticola、水鸲属Rhyacornis、鸲属Tarsiger、溪鸲属Chainarrornis、石即鸟属Saxicola、燕尾属Enivurus、歌鸲属Luscinia和鹊鸲属Copsychus.红尾鸲属为并系类群,水鸲属和溪鸲属聚到这一支系;歌鸲属与燕尾属互为姐妹群,再与鸲属聚合构成另一支系;宝兴歌鸫Turdus mupinensis独立于鸫属及地鸫属之外,形成单独一个分支.     

SOME PROPERTIES OF DNA FROM ZOOXANTHELLAE HARBORED BY AN ANEMONE ANTHOPLEURA ELEGANTISSIMA1

Native DNA from the zooxanthellae harbored by an anemone, Anthopleura elegantissima consists of 2 species resolvable by differences in bouyant density in CsCl. Nuclear DNA has a density of 1.695 g/ml. Chloroplast DNA bands at a density of 1.688 g/ml. A third species of DNA, also with a density of 1.695 g/ml in the native state, is presumably mitochondrial DNA. It was detected in heat-denatured preparations. Nuclear DNA contains, in addition to the 4 usual bases, some 5-methyl cytosine. The latter was absent from chloroplast fractions.     

Rangewide population genetic structure of the African malaria vector Anopheles funestus

Anopheles funestus is a primary vector of malaria in Africa south of the Sahara. We assessed its rangewide population genetic structure based on samples from 11 countries, using 10 physically mapped microsatellite loci, two per autosome arm and the X (N = 548), and 834 bp of the mitochondrial ND5 gene (N = 470). On the basis of microsatellite allele frequencies, we found three subdivisions: eastern (coastal Tanzania, Malawi, Mozambique and Madagascar), western (Burkina Faso, Mali, Nigeria and western Kenya), and central (Gabon, coastal Angola). A. funestus from the southwest of Uganda had affinities to all three subdivisions. Mitochondrial DNA (mtDNA) corroborated this structure, although mtDNA gene trees showed less resolution. The eastern subdivision had significantly lower diversity, similar to the pattern found in the codistributed malaria vector Anopheles gambiae. This suggests that both species have responded to common geographic and/or climatic constraints. The western division showed signatures of population expansion encompassing Kenya west of the Rift Valley through Burkina Faso and Mali. This pattern also bears similarity to A. gambiae, and may reflect a common response to expanding human populations following the development of agriculture. Due to the presumed recent population expansion, the correlation between genetic and geographic distance was weak. Mitochondrial DNA revealed further cryptic subdivision in A. funestus, not detected in the nuclear genome. Mozambique and Madagascar samples contained two mtDNA lineages, designated clade I and clade II, that were separated by two fixed differences and an average of 2% divergence, which implies that they have evolved independently for approximately 1 million years. Clade I was found in all 11 locations, whereas clade II was sampled only on Madagascar and Mozambique. We suggest that the latter clade may represent mtDNA capture by A. funestus, resulting from historical gene flow either among previously isolated and divergent populations or with a related species.