Asymmetric hybridization between non-native winter moth, Operophtera brumata (Lepidoptera: Geometridae), and native Bruce spanworm, Operophtera bruceata, in the Northeastern United States, assessed with novel microsatellites and SNPs.

The European winter moth, Operophtera brumata, is a non-native pest in the Northeastern USA causing defoliation of forest trees and crops such as apples and blueberries. This species is known to hybridize with O. bruceata, the Bruce spanworm, a native species across North America, although it is not known if there are hybrid generations beyond F1. To study winter moth population genetics and hybridization with Bruce spanworm, we developed two sets of genetic markers, single nucleotide polymorphisms (SNPs) and microsatellites, using genomic approaches. Both types of markers were validated using samples from the two species and their hybrids. We identified 1216 SNPs and 24 variable microsatellite loci. From them we developed a subset of 95 species-diagnostic SNPs and ten microsatellite loci that could be used for hybrid identification. We further validated the ten microsatellite loci by screening field collected samples of both species and putative hybrids. In addition to confirming the presence of F1 hybrids reported in previous studies, we found evidence for multi-generation asymmetric hybridization, as suggested by the occurrence of hybrid backcrosses with the winter month, but not with the Bruce spanworm. Laboratory crosses between winter moth females and Bruce spanworm males resulted in a higher proportion of viable eggs than the reciprocal cross, supporting this pattern. We discuss the possible roles of population demographics, sex chromosome genetic incompatibility, and bacterial symbionts as causes of this asymmetrical hybridization and the utility of the developed markers for future studies.

Geographic population structure of the African malaria vector Anopheles gambiae suggests a role for the forest-savannah biome transition as a barrier to gene flow.

The primary Afrotropical malaria mosquito vector Anopheles gambiae sensu stricto has a complex population structure. In west Africa, this species is split into two molecular forms and displays local and regional variation in chromosomal arrangements and behaviors. To investigate patterns of macrogeographic population substructure, 25 An. gambiae samples from 12 African countries were genotyped at 13 microsatellite loci. This analysis detected the presence of additional population structuring, with the M-form being subdivided into distinct west, central, and southern African genetic clusters. These clusters are coincident with the central African rainforest belt and northern and southern savannah biomes, which suggests restrictions to gene flow associated with the transition between these biomes. By contrast, geographically patterned population substructure appears much weaker within the S-form.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

In situ population structure and ex situ representation of the endangered Amur tiger.

The Amur tiger (Panthera tigris altaica) is a critically endangered felid that suffered a severe demographic contraction in the 1940s. In this study, we sampled 95 individuals collected throughout their native range to investigate questions relative to population genetic structure and demographic history. Additionally, we sampled targeted individuals from the North American ex situ population to assess the genetic representation found in captivity. Population genetic and Bayesian structure analyses clearly identified two populations separated by a development corridor in Russia. Despite their well-documented 20th century decline, we failed to find evidence of a recent population bottleneck, although genetic signatures of a historical contraction were detected. This disparity in signal may be due to several reasons, including historical paucity in population genetic variation associated with postglacial colonization and potential gene flow from a now extirpated Chinese population. Despite conflicting signatures of a bottleneck, our estimates of effective population size (N(e) = 27-35) and N(e)/N ratio (0.07-0.054) were substantially lower than the only other values reported for a wild tiger population. Lastly, the extent and distribution of genetic variation in captive and wild populations were similar, yet gene variants persisted ex situ that were lost in situ. Overall, our results indicate the need to secure ecological connectivity between the two Russian populations to minimize loss of genetic diversity and overall susceptibility to stochastic events, and support a previous study suggesting that the captive population may be a reservoir of gene variants lost in situ.

High levels of hybridization between molecular forms of Anopheles gambiae from Guinea Bissau.

In the malaria vector Anopheles gambiae Giles sensu stricto, two molecular forms denoted M and S are considered units of incipient speciation within this species. Very low hybrid frequencies and significant genetic differentiation have been found in sympatric M- and S-form populations. We studied the molecular form composition and the degree of genetic differentiation at 15 microsatellites in two samples of An. gambiae collected in two consecutive years from Bissau, Guinea Bissau. High frequencies of M/S hybrids (19-24%) were found in this area. Coincidently, very low levels of genetic differentiation were detected between forms when analysis involved microsatellites mapped at chromosome-3 (mean Fst, 0.000-0.002). The single exception was the X-linked AGXH678, for which high differentiation was measured (Fst, 0.158-0.301). This locus maps near the centromere of chromosome X, a low recombination region in which selection is likely to promote divergence between M and S forms. These results strongly suggest that the degree of isolation between M and S forms, considered the units of incipient speciation within An. gambiae, is not homogenous throughout the species distribution range.

Polymorphic microsatellite markers for the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae), a vector of human African trypanosomiasis.

Our understanding of Glossina fuscipes fuscipes, a major vector of sleeping sickness, has been severely constrained by a lack of genetic markers for mapping and population genetic studies. Here we present 10 newly developed microsatellite loci for this tsetse species. Heterozygosity levels in Moyo, an Ugandan population, averaged 0.57, with only two loci showing very low heterozygosity. Five loci carried more than six alleles. Together with five recently published microsatellite loci, this brings the number of available microsatellite loci for this species to 15. Their availability will greatly facilitate future studies on the genetics of this important human disease vector.

Inbreeding linked to amphibian survival in the wild but not in the laboratory.

We examined the effects of inbreeding on the performance of wood frog (Rana sylvatica) larvae in the field and in the laboratory. We used microsatellite analysis to establish the parentage and degree of inbreeding of the larvae. Two different estimators of inbreeding were used. The first was based on average multilocus heterozygosity, and the second was based on a molecular relatedness estimator. The estimators were highly correlated, and both showed a significant negative relationship between inbreeding and survival in the wild. However, there was no evidence that inbreeding influenced growth or development in the wild. Neither was there any evidence that inbreeding affected survival, growth, or development in the laboratory. These results suggest that, for wood frogs, inbreeding has a bigger effect on fitness in the wild than in captivity and that measurements of survival are more sensitive than measures of growth or development.

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon.

Point mutations in the voltage-gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine-phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine-serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S-form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild-type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.

Kin distribution of amphibian larvae in the wild.

According to kin selection theory, the location of an individual with respect to its relatives can have important ramifications for its fitness. Perhaps more than any other vertebrate group, anuran amphibian larvae have been the subject of many experiments on this topic. Some anuran species have been shown in the laboratory to recognize and associate with their siblings and half-siblings. However, due to the difficulty of identifying sibships, no kinship studies with anuran larvae have been conducted in the wild. Here, we use microsatellite analysis to show that wood frog (Rana sylvatica) tadpoles were nonrandomly distributed in two ponds with respect to their relatives. In one pond, the tadpoles were significantly clumped with their siblings or half-siblings as expected from other published laboratory studies on this species. However, in another pond, the tadpoles were significantly nonrandomly dispersed from their siblings or half-siblings. This is the first example of kin repulsion of nonreproductive animals in the wild and the first time a species has been shown to display both aggregation and repulsion under different circumstances. These results suggest that kin distribution is context dependent and demonstrate the importance of testing kin selection hypotheses under natural conditions.

Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms.

We present the results of a geographical survey of genetic variation in Anopheles gambiae M and S molecular forms from ten African countries at Intron I of the voltage-gated sodium channel gene. We found two major haplotypes separated by a single mutational step, which cosegregate almost completely with the rDNA sites that identify M and S, consistent with previous estimates of strong reductions of gene flow between the two forms. We also report ten additional haplotypes stemming from the two major haplotypes, mostly present in single localities. The low levels of genetic variation found in this intronic region are discussed in light of a possible selective sweep. These findings offer additional elements to the ongoing debate on the amount of genetic differentiation and isolation between the two molecular forms and on their taxonomic status.

Phylogeography and molecular rates of subterranean aquatic Stenasellid Isopods with a peri-Tyrrhenian distribution.

The subterranean Isopods belonging to the genus Stenasellus have an interesting disjunct distribution in the peri-Tyrrhenian area with morphologically closely related taxa occurring in Sardinia, Corsica, Tuscany and in the Pyrenees phreatic and interstitial waters. Because the dispersal capacities of these organisms are limited, their distribution has been associated traditionally with the tectonic events leading to the separation of the Sardinia-Corsica microplate from the Pyrenees and its subsequent movement towards the Italian peninsula. We sequenced a fragment of the mtDNA cytochrome oxidase I gene (COI) for multiple populations of the S. racovitzai species-group (Corsica, Sardinia, Tuscany) and S. virei (Pyrenees). We found that multiple phylogenetic analyses always gave the same topology, which is consistent with the genetic relations found using allozyme data, and with the palaeogeography of the area. The molecular data suggest that a combination of vicariance and dispersal events explain most effectively the present distribution pattern of these organisms. We also calculated COI rates and calibrated them against absolute time, taking advantage of the availability of two geologically based time estimates. Rates on all substitutions are similar to those published for other crustaceans for the same COI fragment, including taxonomically and ecologically distant groups. Rates on third codon positions or on transversions are generally lower than those found in other crustaceans.

Speciation within Anopheles gambiae--the glass is half full.

Restrictions to gene flow among molecular forms of the mosquito Anopheles gambiae sensu stricto reveal an ongoing speciation process affecting the epidemiology of malaria in sub-Saharan Africa.

Molecular biogeography of cave life: a study using mitochondrial DNA from bathysciine beetles.

This study focuses on phylogenetic relationships in two distinct species assemblages of cave-dwelling beetles with similar disjunct distributions in the Pyrenees and Sardinia. One assemblage contains six species in the genera Ovobathysciola (four species) and Patriziella (two species) on Sardinia and one species of Anillochlamys in the Pyrenees. Species within the two Sardinian genera co-occur in the same karst area. Although, they are believed to be each others closest relative, they have very different body types (globular body with short appendages in Ovobathysciola; elongated body with long appendages in Patriziella), which are believed to reflect different degrees of adaptation to cave life. The other assemblage of Bathysciine beetles includes three species in the genus Speonomus in the Pyrenees and one on Sardinia. All the species are rare and many are endangered. One issue of particular interest was whether Ovobathysciola and Patriziella are reciprocally monophyletic or whether each of the Patriziella species evolved independently from the co-occurring Ovobathysciola species, as the similar morphology of the Patriziella species might be due to convergence rather than common descent. Based on DNA sequences of the cytochrome oxidase I (COI) region of the mtDNA, neither scenario was supported. Rather, the two Patriziella species are sister taxa embedded within the Ovobathysciola radiation. In addition, the well-dated geological history of this region allowed us to calibrate absolute rates of COI evolution, the first such estimates for any insect. Finally this study suggests that the evolutionary acquisition of typical cave adaptations (e.g., elongated body and appendages) may occur at about the same rate as loss of traits (e.g., eyes and pigmentation) associated with cave life.

Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s.

Analyses of inversions in polytene chromosomes indicate that, in West Africa, Anopheles gambiae (sensu stricto) may be a complex of more than a single taxonomic unit, and these units have been called chromosomal forms. In order to determine whether this genetic discontinuity extends to the rest of the genome, as would be expected if reproductive isolation exists, we have sequenced several regions of both the nuclear and mitochondrial genomes. With one exception, we were unable to identify any nucleotide sites that differentiate the chromosomal forms. The exception was the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). Three sites in this region distinguish Mopti chromosomal form from Savanna and Bamako in Mali and Burkina Faso. However, outside these two countries, the association between chromosomal form and rDNA type does not always hold. Together with the variants in the rDNA intergenic spacer (IGS) described in the accompanying papers (della Torre et al., 2001; Favia et al., 2001), we can recognize two major types of rDNA, Type I and Type II (corresponding to molecular forms S and M in della Torre et al., 2001). Type I is widespread in West Africa and is the only type found outside of West Africa (i.e. Tanzania and Madagascar). Type II is confined to West Africa. We were unable to detect any heterozygosity for the ITS types even in five collections containing both types. A sample from the island of São Tomé could not be classified into either Type I or Type II as the rDNA had characteristics of both. In general, our results confirm that An. gambiae is not a single pan-mictic unit, but exactly how to define any new taxa remains problematic. Finally, we have found minor variants of the major rDNA types fixed in local populations; contrary to most previous studies, this suggests restricted gene flow among populations of this species.

Origin and evolutionary relationships of giant Galápagos tortoises.

Perhaps the most enduring debate in reptile systematics has involved the giant Galápagos tortoises (Geochelone nigra), whose origins and systematic relationships captivated Charles Darwin and remain unresolved to this day. Here we report a phylogenetic reconstruction based on mitochondrial DNA sequences from Galápagos tortoises and Geochelone from mainland South America and Africa. The closest living relative to the Galápagos tortoise is not among the larger-bodied tortoises of South America but is the relatively small-bodied Geochelone chilensis, or Chaco tortoise. The split between G. chilensis and the Galápagos lineage probably occurred 6 to 12 million years ago, before the origin of the oldest extant Galápagos island. Our data suggest that the four named southern subspecies on the largest island, Isabela, are not distinct genetic units, whereas a genetically distinct northernmost Isabela subspecies is probably the result of a separate colonization. Most unexpectedly, the lone survivor of the abingdoni subspecies from Pinta Island ("Lonesome George") is very closely related to tortoises from San Cristobal and Espanola, the islands farthest from the island of Pinta. To rule out a possible recent transplant of Lonesome George, we sequenced DNA from three tortoises collected on Pinta in 1906. They have sequences identical to Lonesome George, consistent with his being the last survivor of his subspecies. This finding may provide guidance in finding a mate for Lonesome George, who so far has failed to reproduce.

A molecular phylogeny of four endangered Madagascar tortoises based on MtDNA sequences.

Four of the five tortoise species in Madagascar, Pyxis arachnoides, P. planicauda, Geochelone radiata, and G. yniphora, are endemic and on the verge of extinction. Their phylogenetic relationships remain controversial and unresolved. Here we address the phylogeny of this group using DNA sequences for the 12S and 16S rDNA and cyt b genes in mitochondrial DNA. As outgroups we used two species of Geochelone, pardalis (mainland Africa) and nigra (Galápagos), as well as a more distant North American tortoise, Gopherus polyphemus. We conclude that the two Pyxis species are sister taxa and are imbedded in the genus Geochelone, rendering this latter genus paraphyletic. There is moderate support for the sister status of the two Madagascar Geochelone and for the monophyletic origin of all four endemics, suggesting a single colonization of the island. The separation of Madagascar from other land masses (90-165 mya) predates the origin of the endemic tortoises (estimated to be 14-22 mya). This suggests founding by rafting, a process known to have occurred with other tortoises. The derived morphological divergence of the Pyxis species in a relatively short period of time (13-20 my) stands in contrast to the notoriously slow rate of morphological evolution in most lineages of Chelonia.

Characterization of the soluble guanylyl cyclase beta-subunit gene in the mosquito Anopheles gambiae.

Genomic DNA corresponding to the soluble guanylyl cyclase beta-subunit (GCSbeta) gene was cloned and sequenced from Anopheles gambiae. The sequence was 8103 bp long and presumably included the entire coding region. The deduced amino acid sequence was 71% and 62% similar to previously known Drosophila and vertebrate GCSbeta, while the C-terminus of A. gambiae GCSbeta was shorter. Because of the conserved characteristics in each functional domain, the high G+C% in the third codon positions compared to the introns, the lack of internal stop codons, and the fact that we identified the gene from a cDNA, we conclude that this A. gambiae gene is functional. This is the first detailed description of a guanylyl cyclase gene structure (e.g. intron-exon boundaries). Interestingly, within the fifth intron we found high similarity to the flanking regions of the Pegasus-27 transposable element and other noncoding regions of the A. gambiae genome.

Population structure, speciation, and introgression in the Anopheles gambiae complex.

We review here what is known about the population structure and evolutionary dynamics of members of the Anopheles gambiae complex with emphasis on the situation in West Africa. First, the importance of the 2nd chromosome inversion polymorphism is demonstrated especially in adaptation to levels of aridity, a major environmental variable in Africa. This affects the distribution of karyotypes on both a macro- and micro-geographic scale as well as temporally. Such differentiation leads to karyotypes being differentially effective transmitters of malaria and differentially susceptible to indoor residual spraying of insecticides. Second, we review the evidence that cryptic taxa, especially in An. gambiae s.s., exist. This observation stems from both karyotype studies and molecular studies. It is abundantly clear that West African populations of An. gambiae s.s. are often not panmictic units, with premating factors evidently acting to maintain distinct genetic forms. Third, we review phylogenetic studies that have revealed the presence of introgression between the two most important vectors, An. gambiae and An. arabiensis. This is most evident for the 2nd chromosome inversions. This interpretation of phylogenetic data is consistent with a direct laboratory study indicating inversions in this chromosome are stably maintained in back-crossed populations. All of this information has led to the view that members of the An. gambiae complex are highly variable with an abundance of adaptive genetic variation. This presents a significant challenge to vector control programs designed to reduce malaria in sub-Saharan Africa.

Multiple origins of cytologically identical chromosome inversions in the Anopheles gambiae complex.

For more than 60 years, evolutionary cytogeneticists have been using naturally occurring chromosomal inversions to infer phylogenetic histories, especially in insects with polytene chromosomes. The validity of this method is predicated on the assumption that inversions arise only once in the history of a lineage, so that sharing a particular inversion implies shared common ancestry. This assumption of monophyly has been generally validated by independent data. We present the first clear evidence that naturally occurring inversions, identical at the level of light microscopic examination of polytene chromosomes, may not always be monophyletic. The evidence comes from DNA sequence analyses of regions within or very near the breakpoints of an inversion called the 2La that is found in the Anopheles gambiae complex. Two species, A. merus and A. arabiensis, which are fixed for the "same" inversion, do not cluster with each other in a phylogenetic analysis of the DNA sequences within the 2La. Rather, A. merus 2La is most closely related to strains of A. gambiae homozygous for the 2L+. A. gambiae and A. merus are sister taxa, the immediate ancestor was evidently homozygous 2L+, and A. merus became fixed for an inversion cytologically identical to that in A. arabiensis. A. gambiae is polymorphic for 2La/2L+, and the 2La in this species is nearly identical at the DNA level to that in A. arabiensis, consistent with the growing evidence that introgression has or is occurring between these two most important vectors of malaria in the world. The parallel evolution of the "same" inversion may be promoted by the presence of selectively important genes within the breakpoints.

Mitochondrial DNA rates and biogeography in European newts (genus Euproctus).

Sequence divergence for segments of three mitochondrial DNA (mtDNA) genes encoding the 12S and 16S ribosomal RNA and cytochrome b was examined in newts belonging to the genus Euproctus (E. asper, E. montanus, E. platycephalus) and in three other species belonging to the same family (Salamandridae), Triturus carnifex, T. vulgaris, and Pleurodeles waltl. The three Euproctus species occur (one species each) in Corsica, Sardinia, and the Pyrenees. This vicariant distribution is believed to have been determined by the disjunction and rotation of the Sardinia-Corsica microplate from the Pyreneean region. Because time estimates are available for the tectonic events that led to the separation of the three landmasses, we used sequence data to estimate rates of evolution for the three gene fragments and investigated whether they conform to the rate-constancy hypothesis. By the Tajima (1993, Genetics 135:599-607) test, we could not detect rate heterogeneities for the ribosomal genes and for transversions in the cytochrome b gene. Assuming that these sites are evolving linearly over time and that cessation of gene flow occurred simultaneously with vicariant events, we compared the time of divergence estimated by molecular distances with the divergence times based on the geological estimates. Because we have two estimates of divergence time from the geological record, the split of Corsica/Sardinia from the Pyrenees and the split of Corsica from Sardinia, we could compare ratios of molecular divergence with the ratio of geological time divergence. The ratios are very similar, indicating that the molecular clock hypothesis cannot be rejected. These geological events also allowed us to calculate absolute rates of evolution for ribosomal and cytochrome b genes and compare them to rates for the same regions in other salamandrids and other vertebrates. Ribosomal mtDNA rates are comparable to those reported for other vertebrates, but cytochrome b rates are 3-7 times lower in salamanders than in other ectotherms. From a phylogenetic perspective, our data suggest that the cladogenic events leading to species formation in Euproctus and Triturus occurred very closely in time, indicating that the two genera may not be monophyletic. A duplication of the cytochrome b gene in T. carnifex was found, and the implications of this finding for mtDNA phylogenetic studies are discussed.