Estimating sample sizes for DNA barcoding.

Sample size has long been one of the basic issues since the start of the DNA barcoding initiative and the global biodiversity investigation. As a contribution to resolving this problem, we propose a simple resampling approach to estimate several key sampling sizes for a DNA barcoding project. We illustrate our approach using both structured populations simulated under coalescent and real species of skipper butterflies. We found that sample sizes widely used in DNA barcoding are insufficient to assess the genetic diversity of a species, population structure impacts the estimation of the sample sizes, and hence will bias the species identification potentially.

Breeding system, colony and population structure in the weaver ant Oecophylla smaragdina.

Weaver ants (Oecophylla smaragdina) are dominant ants in open forests from India, Australia, China and Southeast Asia, whose leaf nests are held together with larval silk. The species, together with its sole congener O. longinoda, has been important in research on biological control, communication, territoriality and colony integration. Over most of the range, only one queen has been found per colony, but the occurrence of several queens per nest has been reported for the Australian Northern Territory. The number of males mating with each queen is little known. Here we report on the colony structure of O. smaragdina using published and new microsatellite markers. Worker genotype arrays reflect the occurrence of habitual polygyny (more than one queen per colony) in 18 colonies from Darwin, Northern Australia, with up to five queens inferred per colony. Monogyny (one queen per colony) with occasional polygyny was inferred for 14 colonies from Queensland, Australia, and 20 colonies from Java, Indonesia. Direct genotyping of the sperm carried by 77 Queensland queens and worker genotypic arrays of established colonies yielded similar results, indicating that less than half of the queens mate only once and some mate up to five times. Worker genotype arrays indicated that queens from Java and the Northern Territory also often mate with more than one male, but less often than those from Queensland. A strong isolation-by-distance effect was found for Queensland samples. The variation uncovered means that O. smaragdina is a more versatile study system than previously supposed.

Relish regulates expression of antimicrobial peptide genes in the honeybee, Apis mellifera, shown by RNA interference.

Relationships of immune genes in adult honeybees (Apis mellifera) were investigated using RNA interference (RNAi). Quantitative RT-PCR was applied to estimate gene expression and the extent of gene silencing. Relish is a transcription factor and forms an important part of the IMD signalling pathway. The expression of the immune gene Relish was significantly reduced by RNAi (ca. 70%). The proposed regulation of antimicrobial peptide genes by Relish could be established for abaecin and hymenoptaecin. These two genes showed a reduction in gene expression to the same extent as Relish. However, the antimicrobial peptide gene defensin-1 was not affected which suggests defensin-1 is regulated by a different signalling pathway.

Dynamics of the evolution of Batesian mimicry: molecular phylogenetic analysis of ant-mimicking Myrmarachne (Araneae: Salticidae) species and their ant models.

Batesian mimicry is seen as an example of evolution by natural selection, with predation as the main driving force. The mimic is under selective pressure to resemble its model, whereas it is disadvantageous for the model to be associated with the palatable mimic. In consequence one might expect there to be an evolutionary arms race, similar to the one involving host-parasite coevolution. In this study, the evolutionary dynamics of a Batesian mimicry system of model ants and ant-mimicking salticids is investigated by comparing the phylogenies of the two groups. Although Batesian mimics are expected to coevolve with their models, we found the phylogenetic patterns of the models and the mimics to be indicative of adaptive radiation by the mimic rather than co-speciation between the mimic and the model. This shows that there is strong selection pressure on Myrmarachne, leading to a high degree of polymorphism. There is also evidence of sympatric speciation in Myrmarachne, the reproductive isolation possibly driven by female mate choice in polymorphic species.

No sympatric speciation here: multiple data sources show that the ant Myrmica microrubra is not a separate species but an alternate reproductive morph of Myrmica rubra.

No aspect of speciation is as controversial as the view that new species can evolve sympatrically, among populations in close physical contact. Social parasitism has been suggested to yield necessary disruptive selection for sympatric speciation. Recently, mitochondrial DNA phylogeography has shown that the ant Myrmica microrubra is closely related to its host, Myrmica rubra, leading to the suggestion that sympatric speciation has occurred. We investigated the relationships between the two ant forms using mitochondrial and nuclear DNA sequences, microsatellite genotyping and morphometrics. Molecular phylogenetic and population structure analyses showed that M. microrubra does not evolve separately to its host but rather shares a gene pool with it. Probability analysis showed that mitochondrial DNA data previously adduced in favour of sympatric speciation do not in fact do so. Morphometrically, M. microrubra is most readily interpreted as a miniature queen form of M. rubra, not a separate species. Myrmica microrubra is not an example of speciation. The large (typical M. rubra) and small (M. microrubra) queen forms are alternative reproductive strategies of the same species. Myrmica microrubraSeifert 1993 is consequently synonymized here with M. rubra Linnaeus, 1758.

On the origin of termite workers: weighing up the phylogenetic evidence.

Resolving the phylogenetic history of a 'true' worker caste in termites is essential to our understanding of termite eusocial evolution. Whether this caste is ancient and monophyletic or derived and polyphyletic will have a tremendous impact on our interpretation of termite eusocial history and remains an outstanding question in termite biology. Recent work has begun to re-examine this question in light of new phylogenetic information, but new questions have now arisen about how best to model character state changes in termite caste systems. In the present paper, we compare the models of Grandcolas and D'Haese [J. Evol. Biol. 15 (2002) 885] and Thompson et al. [J. Evol Biol. 13 (2000) 8691 and attempt to make explicit how these proposals differ with respect to the number of, and homology between, character states. We highlight the support each model has for the two principal, but competing, evolutionary hypotheses outlined above.

Patterns of population subdivision and gene flow in the ant Nothomyrmecia macrops reflected in microsatellite and mitochondrial DNA markers.

The Australian endemic ant Nothomyrmecia macrops is renowned for having retained a large proportion of 'primitive' morphological and behavioural characters. Another less studied peculiarity of this species is the production of short-winged (brachypterous) female sexuals, which presumably are poor dispersers. The males, in contrast, bear a full set of normally developed wings and thus may disperse widely. We investigated patterns of genetic differentiation within and among three distantly separated populations in South Australia using nine polymorphic microsatellite loci and four regions of mitochondrial DNA (COI, COII, Cytb, lrRNA). We sampled eight subpopulations, one in the Lake Gilles CP, two near Penong and five around Poochera where distances ranged from 360 km to sites separated by 2-10 km. Only little differentiation was found at the local scale (within the assumed dispersal distance of males) using nuclear markers, whereas the three distant locations were moderately differentiated (FST = 0.06). Mitochondrial DNA genetic structure was much more pronounced on all scales (phiST = 0.98), with regular differences in both haplotype composition and frequency even occurring among closely located sites. This lack of congruence between nuclear and mitochondrial markers strongly suggests limited female dispersal and male-biased gene flow among populations. As to the conservation status of the species there is no evidence for severe population reductions in the recent past, which would have left populations genetically depauperate.

Genetic control of the honey bee (Apis mellifera) dance language: segregating dance forms in a backcrossed colony.

We studied the genetic control of the dance dialects that exist in the different subspecies of honey bees (Apis mellifera) by observing the variation in dance form observed in a backcross between two lines that showed widely different dance dialects. To do this we generated the reciprocal of the cross performed by Rinderer and Beaman (1995), thus producing phenotypic segregation of dance forms within a single colony rather than between colonies. Our results are consistent with Rinderer and Beaman (1995) in that inheritance of the transition point from round dancing --> waggle dancing is consistent with control by a single locus with more than one allele. That is, we found one dance type to be dominant in the F(1), and observed a 1:1 segregation of dance in a backcross involving the F(1) and the recessive parent. However, we found some minor differences in dance dialect inheritance, with the most significant being an apparent reversal of dominance between our cross (for us "black" is the dominant dialect) and that of Rinderer and Beaman (1995) (they report "yellow" to be the dominant dialect). We also found that our black bees do not perform a distinct sickle dance, whereas the black bees used by Rinderer and Beaman (1995) did perform such a dance. However, our difference in dominance need not contradict the results of Rinderer and Beaman (1995), as there is no evidence that body color and dominance for dance dialect are linked.

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia.

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insect's biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.

Clines maintained by overdominant selection in hybrid zones.

Hybrid zone models often consider environment-independent selection to operate against all hybrids. However, empirical studies suggest that hybrids may be as fit or fitter than the hybridizing parental taxa in some environments. In this study we develop a novel mathematical model to explore the effects of one form of hybrid superiority on the genetic structure of hybrid zones. Our primary goals were to investigate the allele frequency clines at a locus experiencing overdominant selection and at a linked neutral or underdominant locus. Our results indicate that overdominant selection results in flat equilibrium allele frequency clines throughout the hybrid zone and an excess of heterozygosity relative to Hardy-Weinberg equilibrium. However, the genetic clines at linked neutral or underdominant loci tend not to reflect this overdominance even when the loci are tightly linked. Overall, we conclude that overdominance is unlikely to be detected in genetic surveys unless many loci are assayed.

Reproduction and recruitment in perennial colonies of the introduced wasp Vespula germanica.

We investigated the genetic structure of perennial colonies of the yellowjacket wasp (Vespula germanica) in its introduced range in Australia and New Zealand. The nuclear genotypes of 712 gynes from 21 colonies, 147 workers from 5 colonies, and 81 males from 4 colonies were assayed at three polymorphic microsatellite loci. The mitochondrial haplotypes of all wasps also were determined for a 450-bp region of the mtDNA using double-stranded conformational polymorphism (DSCP) analysis. We found that multiple reproductives were needed to explain the genotypes of gynes, workers, and males in 7 of 21, 2 of 5, and 2 of 4 colonies, respectively, and that nestmate relatedness of these three castes equaled 0.42, 0.16, and 0.22, respectively. The mitochondrial data revealed that all individuals shared the same mtDNA haplotype in 20 of the 21 colonies. However, in one colony, gynes and workers displayed multiple mtDNA haplotypes, indicating that nonnestmate recruitment had occurred. Overall the genetic structure within the majority of perennial colonies conformed to expectations based on the biology of V. germanica and kin selection theory for polygyne colonies; multiple reproductives successfully produced offspring and were recruited into their natal nests, thereby maintaining relatively high relatedness between interacting individuals.

Hierarchical genetic structure of the introduced wasp Vespula germanica in Australia.

The wasp Vespula germanica is a highly successful invasive pest. This study examined the population genetic structure of V. germanica in its introduced range in Australia. We sampled 1320 workers and 376 males from 141 nests obtained from three widely separated geographical areas on the Australian mainland and one on the island of Tasmania. The genotypes of all wasps were assayed at three polymorphic DNA microsatellite markers. Our analyses uncovered significant allelic differentiation among all four V. germanica populations. Pairwise estimates of genetic divergence between populations agreed with the results of a model-based clustering algorithm which indicated that the Tasmanian population was particularly distinct from the other populations. Within-population analyses revealed that genetic similarity declined with spatial distance, indicating that wasps from nests separated by more than approximately 25 km belonged to separate mating pools. We suggest that the observed genetic patterns resulted from frequent bottlenecks experienced by the V. germanica populations during their colonization of Australia.

Mating behaviour of Rhytidoponera sp. 12 ants inferred from microsatellite analysis.

In the queenless ponerine ant Rhytidoponera sp. 12, all workers have a spermatheca and functional ovaries and are potentially able to mate and reproduce. Within a colony gamergates may either be full sisters to each other (Type 1 colony), or they may not be full sisters but still be significantly related to each other (Type 2 colony) due to daughter gamergates reproducing in their natal colonies after mating. Despite many studies the mating behaviour of R. sp. 12 has been poorly understood. In this study, we used microsatellite markers to investigate intracolony relatednesses of male mates to the gamergates (bmq) and between male mates (bmm), and mating frequencies and mating patterns, using gamergate DNA and sperm DNA isolated from the spermathecae of gamergates from five colonies. Average bmm and bmq estimates for all five colonies studied were not significantly different from zero, suggesting that on average, within colonies, mating males were unrelated both to each other and to the gamergates. A low frequency (3%) of multiple mating by gamergates was detected. Multiple mating by individual males with sister gamergates within Type 1 colonies was also detected at 3% and could give rise to half-sister nestmate workers. Polygamy in R. sp. 12 might indicate local female-biased operational sex ratios despite the expectation of overall male biases. Our results concur with previous reports that gamergates mate within the colony or nearby, but indicate more diversity in mating patterns than previously indicated for this polygynous ponerine ant species.

A molecular framework for the phylogeny of the ant subfamily dolichoderinae.

Partial sequences are reported for the mitochondrial genes for cytochrome oxidase subunits 2 and 3 and for cytochrome b, and the entire sequence of the gene for tRNA(Leu)(UUR) for species from 14 genera of dolichoderine ants and from three outgroup genera. Considerable variation was observed between tRNA genes in the size of the TPsiC arm and the DHU and anticodon loops and whether or not the TPsiC stem possesses a GC pair. The outgroup taxa showed complete TAA CO1 stop codons, but dolichoderines have either TA or T. The outgroup taxa showed a noncoding gap between the CO1 and the tRNA(Leu)(UUR) genes. A phylogeny-independent compatibility test using the amino acid sequences showed differences between the genes consistent with variation in evolutionary rates, according with other studies. Base compositions proved heterogeneous between species, hence phylogenetic analysis was restricted to the protein sequences using maximum likelihood and the mtREV24 replacement matrix. A maximum-likelihood consensus tree has similarities to those from morphological studies with some exceptions such Leptomyrmex falling within the dolichoderine genera rather than basally, and the accretion of genera formerly included under Iridomyrmex. Features of the tRNA genes and the CO1 termination codons agree quite well with the molecular phylogeny.

Complex social organization reflects genetic structure and relatedness in the cooperatively breeding bell miner, Manorina melanophrys.

The cooperatively breeding bell miner, Manorina melanophrys, differs from most other cooperative breeding species in the complexity of its social system, where discrete social organization occurs on at least three levels. Microsatellite markers were used to investigate the degree of genetic structure underlying the social organization of M. melanophrys by comparing colonies, coteries and nest contingents. The genetic data confirmed behavioural observations of M. melanophrys living in male kin-based groups between which females disperse short distances to breed. Estimates of FST revealed restricted gene flow between eight colonies located within 30 km that was significantly associated with geographical distance when the two most distant colonies were included. Within a high density colony significant differences were found between coteries; analysis of the degree of relatedness between coterie members showed that this is due to related individuals associating preferentially with each other. Similarly, the contingent of individuals attending a nest were generally close relatives of the young they were aiding, supporting models invoking kin selection as the selective agency mediating helping.

Phylogenetic analysis and trait evolution in Australian lineages of drywood termites (Isoptera, Kalotermitidae).

A phylogenetic analysis of Australian drywood termites (Isoptera, Kalotermitidae) based on partial sequence from the cytochrome oxidase II (COII) and cytochrome b genes is presented. In addition to providing new information on the evolutionary relationships among 25 species from seven genera, we evaluate the relative likelihoods of alternative topological hypotheses, including those derived from morphology-based classifications. We also test the applicability of a molecular clock for estimating the age of the Kalotermitidae and infer the evolution of species-specific variation for habitat type and soldier caste phragmosis by mapping this information onto the independently derived phylogeny. Maximum-likelihood analysis of both nucleotide and protein sequences from a multigene data set jointly support a single topology, which is shown to be the best estimate of the true phylogeny among the alternatives tested. Our results support the monophyly of all genera but question the discrimination between Procryptotermes and Cryptotermes. A basal dichotomy among generic groups suggests two principle lines of divergence within the family. Intergeneric relationships show mixed congruence to previous proposals, resulting in one morphology-based classification being rejected. A molecular clock hypothesis is not supported due to significant among-lineage rate heterogeneity in the COII gene. Patterns revealed through trait mapping suggest that the most recently diverged taxa tend to occupy the driest habitats and that these same taxa reflect a defensive transition away from large mandibulate soldiers toward small phragmotic soldiers. The association between habitat and defensibility supports the hypothesis that these two characters have been tightly linked throughout the social diversification of termites.

Current intraspecific dynamics of sequence evolution differs from long-term trends and can account for the AT-richness of honeybee mitochondrial DNA.

The very high AT content of hymenopteran mtDNA has warranted speculation about nucleotide substitution processes in this group. Here we investigate the pattern of honeybee, Apis mellifera, mtDNA nucleotide polymorphisms inferred from phylogeny in terms of differences between the ATPase6, COI, COII, COIII, cytochrome b, and ND2 genes and strand asymmetry in mutation rates. The observed transition/transversion ratios and the distribution of nonsynonymous substitutions between regions differed significantly. The pattern of differences between genes leading to these heterogeneities (the ATPase6 and COIII genes group apart from the rest) differed markedly from that predicted on the basis of long-term evolutionary change and may indicate differences between current and long-term dynamics of sequence evolution. Also, there is strong strand asymmetry in substitutions, which probably results in a mutability of G and C sufficiently high to account for the AT-richness of honeybee mtDNA.