Diet breadth of the aphid predator Chrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae).

The performance (development and reproduction) of generalist predators can vary greatly among the prey species that they use, and these differences can influence the ability of predatory insects to suppress pest populations. The aim of this study was to compare the performance of larvae of the green lacewing Chrysoperla rufilabris (Burmeister, 1839) by offering 16 species of aphids and by assessing the effects of each species on the survival, larval development time, prey consumption, pupal mass and egg load of adult Chr. rufilabris females taking aphid phylogeny into account. Chrysoperla rufilabris larvae preyed on individuals from all 16 aphid species, but complete development, adult emergence and egg load production were achieved only in seven species. As a general pattern, the best levels of performance were achieved for an aphid clade that includes the soybean aphid, Aphis glycines (Matsumara, 1917), and for a milkweed-feeding species, Myzocallis asclepiadis (Monell, 1879). We found significant phylogenetic clustering for most of the performance traits indicating the aspects of specialization in the diet breadth of Chr. rufilabris despite the fact that this species is considered a generalist aphid predator. These findings can help us to understand the interactions of this species in agroecological food webs, where it is commonly found, and provide insights into why natural, conservation biological control or augmentative releases may succeed or fail.

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

Molecular Data Confirm Interspecific Limits of Four Alloxysta and One Phaenoglyphis Species of Parasitic Wasps within the Subfamily Charipinae (Cynipoidea: Figitidae).

The hymenopteran subfamily Charipinae (Cynipoidea: Figitidae) consist of a group of parasitic wasps that are exclusive hyperparasitoids of Hemipteran. The species boundaries in Charipinae have historically been unclear. While diagnostic morphological features have been established for the stepwise separation of species, it is recommended to confirm those limits using molecular data. Here, we focus on the genera Alloxysta Förster, 1869 and Phaenoglyphis Förster, 1869, both of which contain species that are hyperparasitoids of aphids. We sequenced three genes (mitochondrial COI and 16S rDNA, and nuclear ITS2 rDNA) from specimens that were identified as belonging to five species: Alloxysta brevis (Thomson, 1862), A. castanea (Hartig, 1841), A. ramulifera (Thomson, 1862), A. victrix (Westwood, 1833), and Phaenoglyphis villosa (Hartig, 1841). The phylogeny resulting from concatenating these genes supported the species status of the five morphologically identified taxa, with P. villosa nested within Alloxysta. Our study thus indicates that these molecular markers can successfully distinguish charipine species, and also indicates that the genera Alloxysta and Phaenoglyphis may be more closely related than previously hypothesized. We also present the first estimates of genetic distances for these species. Future studies that include more species, loci, and/or genomic data will complement our research and help determine species relationships within the Charipinae subfamily.

Gene silencing for invasive paper wasp management: Synthesized dsRNA can modify gene expression but did not affect mortality.

Invasive paper wasps such as Polistes dominula are a major pest and problem for biodiversity around the globe. Safe and highly targeted methods for the control of these and other social wasp populations are needed. We attempted to identify potentially-lethal gene targets that could be used on adult paper wasps in a gene silencing or RNA interference (RNAi) approach. Double-stranded RNA (dsRNA) was designed to target genes for which silencing has proven lethal in other insects. dsRNA was provided either orally to foragers or directly injected into the wasps. We also provided the dsRNA unprotected or protected from degradation by gut nucleases in two different forms (lipofectamine and carbon quantum dots). The effects of oral delivery of 22 different gene targets to forager wasps was evaluated. The expression of five different genes was successfully reduced following dsRNA ingestion or injection. These gene targets included the FACT complex subunit spt16 (DRE4) and RNA-binding protein fusilli (FUSILLI), both of which have been previously shown to have potential as lethal targets for pest control in other insects. However, we found no evidence of significant increases in adult wasp mortality following ingestion or injection of dsRNA for these genes when compared with control treatments in our experiments. The methods we used to protect the dsRNA from digestive degradation altered gene expression but similarly did not influence wasp mortality. Our results indicate that while many of the same gene targets can be silenced and induce mortality in other insects, dsRNA and RNAi approaches may not be useful for paper wasp control.

Multilocus coalescent analysis of haemoglobin differentiation between low- and high-altitude populations of crested ducks (Lophonetta specularioides).

Hypoxia is a key factor determining survival, and haemoglobins are targets of selection in species native to high-altitude regions. We studied population genetic structure and evaluated evidence for local adaptation in the crested duck (Lophonetta specularioides). Differentiation, gene flow and time since divergence between highland and lowland populations were assessed for three haemoglobin genes (α(A) , α(D) , ß(A) ) and compared to seven reference loci (six autosomal introns and mtDNA). Four derived amino acid replacements were found in the globin genes that had elevated Φ(ST) values between the Andean highlands and Patagonian lowlands. A single ß(A) -globin polymorphism at a site known to influence O(2) affinity was fixed for different alleles in the two populations, whereas three α(A) - and α(D) -globin polymorphisms exhibited high heterozygosity in the highlands but not in the lowlands. Coalescent analyses supported restricted gene flow for haemoglobin alleles and mitochondrial DNA but nonzero gene flow for the introns. Simulating genetic data under a drift-migration model of selective neutrality, the ß(A) -globin fell outside the 95% confidence limit of simulated data, suggesting that directional selection is maintaining different variants in the contrasting elevational environments, thereby restricting migration of ß(A) -globin alleles. The α(A) - and α(D) -globins, by contrast, did not differ from the simulated values, suggesting that variants in these genes are either selectively neutral, or that the effects of selection could not be differentiated from background levels of population structure and linkage disequilibrium. This study illustrates the combined effects of selection and population history on inferring levels of population divergence for a species distributed across an altitudinal gradient in which selection for hypoxia resistance has likely played an important role.

Identity, Prevalence, and Pathogenicity of Entomopathogenic Fungi Infecting Invasive Polistes (Vespidae: Polistinae) Paper Wasps in New Zealand.

Two species of entomogenous fungi were discovered infecting the invasive paper wasp Polistes chinensis during an ecological study on Farewell Spit, New Zealand. We sequenced two nuclear ribosomal RDNA genes, the internal transcribed spacer (ITS) and the small ribosomal subunit 18S, and one protein-coding gene, the translation elongation factor 1-alpha (ef1 α). Combining sequence information with morphological examination, we identified these species as Beauveria malawiensis and Ophiocordyceps humbertii. We estimated that these fungi produce infection in approximately 3.3% of colonies in our study population. In bioassays, we successfully infected P. chinensis individuals from healthy colonies with B. malawiensis, with significant effects on adult mortality. This is the first record of both B. malawiensis and O. humbertii from Polistine hosts in New Zealand, and the first investigation into disease causality by these pathogens in P. chinensis. Our findings may contribute to the future development of biological control agents for paper wasps in New Zealand and elsewhere around the world.

Genomic analyses of fairy and fulmar prions (Procellariidae: Pachyptila spp.) reveals parallel evolution of bill morphology, and multiple species.

Prions are small petrels that are abundant around the Southern Ocean. Here we use mitochondrial DNA (COI and cytochrome b) and nuclear reduced representation sequencing (ddRADseq) to examine the relationships within and between fairy (Pachyptila turtur) and fulmar (P. crassirostris) prions from across their distributions. We found that neither species was recovered as monophyletic, and that at least three species were represented. Furthermore, we detected several genetic lineages that are also morphologically distinct occurring in near sympatry at two locations (Snares Islands and Chatham Islands). The factors that have driven diversification in the fairy/fulmar prion complex are unclear but may include philopatry, differences in foraging distribution during breeding, differences in non-breeding distributions and breeding habitat characteristics. The observed distribution of genetic variation in the fairy/fulmar prion complex is consistent with population expansion from ice-free Last Glacial Maximum refugia into previously glaciated areas.

Viral communities in the parasite Varroa destructor and in colonies of their honey bee host (Apis mellifera) in New Zealand.

The parasitic mite Varroa destructor is a leading cause of mortality for Western honey bee (Apis mellifera) colonies around the globe. We sought to confirm the presence and likely introduction of only one V. destructor haplotype in New Zealand, and describe the viral community within both V. destructor mites and the bees that they parasitise. A 1232 bp fragment from mitochondrial gene regions suggests the likely introduction of only one V. destructor haplotype to New Zealand. Seventeen viruses were found in bees. The most prevalent and abundant was the Deformed wing virus A (DWV-A) strain, which explained 95.0% of the variation in the viral community of bees. Black queen cell virus, Sacbrood virus, and Varroa destructor virus 2 (VDV-2) played secondary roles. DWV-B and the Israeli acute paralysis virus appeared absent from New Zealand. Ten viruses were observed in V. destructor, with > 99.9% of viral reads from DWV-A and VDV-2. Substantially more variation in viral loads was observed in bees compared to mites. Where high levels of VDV-2 occurred in mites, reduced DWV-A occurred in both the mites and the bees co-occurring within the same hive. Where there were high loads of DWV-A in mites, there were typically high viral loads in bees.

Persistence of the invasive bird-parasitic fly Philornis downsi over the host interbreeding period in the Galapagos Islands.

Many parasites of seasonally available hosts must persist through times of the year when hosts are unavailable. In tropical environments, host availability is often linked to rainfall, and adaptations of parasites to dry periods remain understudied. The bird-parasitic fly Philornis downsi has invaded the Galapagos Islands and is causing high mortality of Darwin's finches and other bird species, and the mechanisms by which it was able to invade the islands are of great interest to conservationists. In the dry lowlands, this fly persists over a seven-month cool season when availability of hosts is very limited. We tested the hypothesis that adult flies could survive from one bird-breeding season until the next by using a pterin-based age-grading method to estimate the age of P. downsi captured during and between bird-breeding seasons. This study showed that significantly older flies were present towards the end of the cool season, with ~ 5% of captured females exhibiting estimated ages greater than seven months. However, younger flies also occurred during the cool season suggesting that some fly reproduction occurs when host availability is low. We discuss the possible ecological mechanisms that could allow for such a mixed strategy.

A Diverse Viral Community from Predatory Wasps in Their Native and Invaded Range, with a New Virus Infectious to Honey Bees.

Wasps of the genus Vespula are social insects that have become major pests and predators in their introduced range. Viruses present in these wasps have been studied in the context of spillover from honey bees, yet we lack an understanding of the endogenous virome of wasps as potential reservoirs of novel emerging infectious diseases. We describe the characterization of 68 novel and nine previously identified virus sequences found in transcriptomes of Vespula vulgaris in colonies sampled from their native range (Belgium) and an invasive range (New Zealand). Many viruses present in the samples were from the Picorna-like virus family (38%). We identified one Luteo-like virus, Vespula vulgaris Luteo-like virus 1, present in the three life stages examined in all colonies from both locations, suggesting this virus is a highly prevalent and persistent infection in wasp colonies. Additionally, we identified a novel Iflavirus with similarity to a recently identified Moku virus, a known wasp and honey bee pathogen. Experimental infection of honey bees with this novel Vespula vulgaris Moku-like virus resulted in an active infection. The high viral diversity present in these invasive wasps is a likely indication that their polyphagous diet is a rich source of viral infections.

Gene flow in the face of countervailing selection: adaptation to high-altitude hypoxia in the betaA hemoglobin subunit of yellow-billed pintails in the Andes.

When populations become locally adapted to contrasting environments, alleles that have high fitness in only one environment may be quickly eliminated in populations adapted to other environments, such that gene flow is partly restricted. The stronger the selection, the more rapidly immigrant alleles of lower fitness will be eliminated from the population. However, gene flow may continue to occur at unlinked loci, and adaptive divergence can proceed in the face of countervailing gene flow if selection is strong relative to migration (s > m). We studied the population genetics of the major hemoglobin genes in yellow-billed pintails (Anas georgica) experiencing contrasting partial pressures of oxygen in the Andes of South America. High gene flow and weak population subdivision were evident at seven putatively neutral loci in different chromosomal linkage groups. In contrast, amino acid replacements (Ser-beta13, Ser-beta116, and Met-beta133) in the betaA hemoglobin subunit segregated by elevation between lowland and highland populations with significantly elevated F(ST). Migration rates for the betaA subunit alleles were approximately 17-24 times smaller than for five unlinked reference loci, the alphaA hemoglobin subunit (which lacks amino acid replacements) and the mitochondrial DNA control region. The betaA subunit alleles of yellow-billed pintails were half as likely to be transferred downslope, from the highlands to the lowlands, than in the opposite direction upslope. We hypothesize that migration between the lowlands and highlands is restricted by local adaptation, and the betaA hemoglobin subunit is a likely target of selection related to high-altitude hypoxia; however, gene flow may be sufficiently high to retard divergence at most unlinked loci. Individuals homozygous for lowland alleles may have relatively little difficulty dispersing to the highlands initially but may experience long-term fitness reduction. Individuals homozygous for highland genotypes, in contrast, would be predicted to have difficulty dispersing to the lowlands if their hemoglobin alleles confer high oxygen affinity, predicted to result in chronic erythrocytosis at low elevation. Heterozygous individuals may have a dispersal advantage if their hemoglobin has a wider range of function due to the presence of multiple protein isoforms with a mixture of different oxygen affinities.

Polistes versicolor (Hymenoptera: Vespidae), an Introduced Wasp in the Galapagos Islands: Its Life Cycle and Ecological Impact.

The yellow paper wasp, Polistes versicolor (Olivier) was first recorded in the Galapagos archipelago in 1988. Its life cycle and ecological impacts were studied on two islands 11 yr after it was first discovered. This invasive wasp adapted quickly and was found in most environments. Colony counts and adult wasp monitoring showed a strong preference for drier habitats. Nest activities were seasonally synchronized, nest building followed the rains in the hot season (typically January-May), when insect prey increases, and peaked as temperature and rains started to decline. Next, the number of adult wasps peaked during the cool season when there is barely any rain in the drier zones. In Galapagos, almost half of the prey loads of P. versicolor were lepidopteran larvae, but wasps also carried spiders, beetles, and flies back to the colonies. An estimated average of 329 mg of fresh insect prey was consumed per day for an average colony of 120-150 wasp larvae. The wasps preyed upon native and introduced insects, but likely also affect insectivorous vertebrates as competitors for food. Wasps may also compete with native pollinators as they regularly visited flowers to collect nectar, and have been recorded visiting at least 93 plant species in Galapagos, including 66 endemic and native plants. Colonies were attacked by a predatory moth, Taygete sphecophila (Meyrick) (Lepidoptera: Autostichidae), but colony development was not arrested. High wasp numbers also affect the activities of residents and tourists. A management program for this invasive species in the archipelago is essential.

The potential for a CRISPR gene drive to eradicate or suppress globally invasive social wasps.

CRISPR gene drives have potential for widespread and cost-efficient pest control, but are highly controversial. We examined a potential gene drive targeting spermatogenesis to control the invasive common wasp (Vespula vulgaris) in New Zealand. Vespula wasps are haplodiploid. Their life cycle makes gene drive production challenging, as nests are initiated by single fertilized queens in spring followed by several cohorts of sterile female workers and the production of reproductives in autumn. We show that different spermatogenesis genes have different levels of variation between introduced and native ranges, enabling a potential 'precision drive' that could target the reduced genetic diversity and genotypes within the invaded range. In vitro testing showed guide-RNA target specificity and efficacy that was dependent on the gene target within Vespula, but no cross-reactivity in other Hymenoptera. Mathematical modelling incorporating the genetic and life history traits of Vespula wasps identified characteristics for a male sterility drive to achieve population control. There was a trade-off between drive infiltration and impact: a drive causing complete male sterility would not spread, while partial sterility could be effective in limiting population size if the homing rate is high. Our results indicate that gene drives may offer viable suppression for wasps and other haplodiploid pests.

Sub-lethal effects of permethrin exposure on a passerine: implications for managing ectoparasites in wild bird nests.

Permethrin is increasingly used for parasite control in bird nests, including nests of threatened passerines. We present the first formal evaluation of the effects of continued permethrin exposure on the reproductive success and liver function of a passerine, the zebra finch (Taeniopygia guttata), for two generations. We experimentally treated all nest material with a 1% permethrin solution or a water control and provided the material to breeding finches for nest building. The success of two consecutive clutches produced by the parental generation and one clutch produced by first-generation birds were tracked. Finches in the first generation were able to reproduce and fledge offspring after permethrin exposure, ruling out infertility. Permethrin treatment had no statistically significant effect on the number of eggs laid, number of days from clutch initiation to hatching, egg hatch rate, fledgling mass or nestling sex ratio in either generation. However, treating nest material with permethrin significantly increased the number of hatchlings in the first generation and decreased fledgling success in the second generation. Body mass for hatchlings exposed to permethrin was lower than for control hatchlings in both generations, but only statistically significant for the second generation. For both generations, an interaction between permethrin treatment and age significantly affected nestling growth. Permethrin treatment had no effect on liver function for any generation. Permethrin was detected inside 6 of 21 exposed, non-embryonated eggs (28.5% incidence; range: 693-4781 ng of permethrin per gram of dry egg mass). Overall, results from exposing adults, eggs and nestlings across generations to permethrin-treated nest material suggest negative effects on finch breeding success, but not on liver function. For threatened bird conservation, the judicious application of this insecticide to control parasites in nests can result in lower nestling mortality compared to when no treatment is applied. Thus, permethrin treatment benefits may outweigh its sub-lethal effects.

Signatures of high-altitude adaptation in the major hemoglobin of five species of andean dabbling ducks.

Hypoxia is one of the most important factors affecting survival at high altitude, and the major hemoglobin protein is a likely target of selection. We compared population genetic structure in the alphaA and betaA hemoglobin subunits (HBA2 and HBB) of five paired lowland and highland populations of Andean dabbling ducks to unlinked reference loci. In the hemoglobin genes, parallel amino acid replacements were overrepresented in highland lineages, and one to five derived substitutions occurred at external solvent-accessible positions on the alpha and beta subunits, at alpha(1)beta(1) intersubunit contacts, or in close proximity to inositolpentaphosphate (IPP) binding sites. Coalescent analyses incorporating the stochasticity of drift and mutation indicated that hemoglobin alleles were less likely to be transferred between highland and lowland populations than unlinked alleles at five other loci. Amino acid replacements that were overrepresented in the highlands were rarely found within lowland populations, suggesting that alleles segregating at high frequency in the highlands may be maladaptive in the lowlands and vice versa. Most highland populations are probably nonmigratory and locally adapted to the Altiplano, but gene flow for several species may be sufficiently high to retard divergence at unlinked loci. Heterozygosity was elevated in the alphaA or betaA subunits of highland populations exhibiting high gene flow between the southern lowlands and the highlands and in highland species that disperse seasonally downslope to midelevation environments from the central Andean plateau. However, elevated heterozygosity occurred more frequently in the alphaA subunit but not simultaneously in both subunits, suggesting that selection may be more constrained by epistasis in the betaA subunit. Concordant patterns among multiple species with different evolutionary histories and depths of historical divergence and gene flow suggest that the major hemoglobin genes of these five dabbling duck species have evolved adaptively in response to high-altitude hypoxia in the Andes.

Genetic structuring of the coastal herb Arthropodium cirratum (Asparagaceae) is shaped by low gene flow, hybridization and prehistoric translocation.

We examined the genetic structuring of rengarenga (Arthropodium cirratum; Asparagaceae), an endemic New Zealand coastal herb, using nuclear microsatellite markers. This species was brought into cultivation by Maori within the last 700-800 years for its edible roots and was transplanted beyond its natural distribution as part of its cultivation. We found very high levels of genetic structuring in the natural populations (FST = 0.84), indicating low levels of gene flow. Reduced genetic diversity was found in the translocated populations, suggesting a large loss of genetic diversity early in the domestication process. The data indicates that rengarenga was brought into cultivation independently at least three times, with the sources of these introductions located within a narrow area encompassing about 250km of coastline. Hybridization was inferred between A. cirratum and the closely related A. bifurcatum, despite A. birfucatum not occurring in the vicinity.

The ectoparasites of hybrid ducks in New Zealand (Mallard x Grey Duck).

We studied the population genetics of one population sample of hybrid Mallard x Grey Ducks and their lice in New Zealand. We aimed to document the relationship between ectoparasite load and host phenotype, and test for an association between the mtDNA diversity of the lice and their hosts, which is predicted based on maternal care. We found three feather lice species previously described for these hosts: Anaticola crassicornis (wing louse), Anatoecus dentatus (head louse), and Trinoton querquedulae (body louse). No new or rare lice species were uncovered. Most ducks in our sample were more Mallard-like than Grey Duck-like hybrids for the five colour and plumage traits examined. We confirm that based solely on phenotypic characters it is difficult to distinguish between Mallards, hybrids and Grey Ducks. We detected no association between the number of lice and host phenotype for two of the three louse species (while controlling for bird size). However, the Grey Duck-like hybrids had fewer head lice (A. dentatus) than their Mallard-like counterparts. Only three of the 40 hosts had mtDNA haplotypes that characterise Grey Ducks. We present the first genetic data of Anaticola crassicornis, Anatoecus dentatus and Trinoton querquedulae from New Zealand waterfowl. We found that the lice mtDNA had greater sequence diversity than the homologous gene for the ducks. A mitochondrial phylogeny for A. crassicornis collected from hosts worldwide has been previously published, and we added our novel data to infer evolutionary relationships among worldwide populations of this louse. None of the three lice species showed a close association of parasite and host mtDNA lineage despite lack of paternal care in these duck species.

A metatranscriptomic analysis of diseased social wasps (Vespula vulgaris) for pathogens, with an experimental infection of larvae and nests.

Social wasps are a major pest in many countries around the world. Pathogens may influence wasp populations and could provide an option for population management via biological control. We investigated the pathology of nests of apparently healthy common wasps, Vespula vulgaris, with nests apparently suffering disease. First, next-generation sequencing and metatranscriptomic analysis were used to examine pathogen presence. The transcriptome of healthy and diseased V. vulgaris showed 27 known microbial phylotypes. Four of these were observed in diseased larvae alone (Aspergillus fumigatus, Moellerella wisconsensis, Moku virus, and the microsporidian Vavraia culicis). Kashmir Bee Virus (KBV) was found to be present in both healthy and diseased larvae. Moellerella wisconsensis is a human pathogen that was potentially misidentified in our wasps by the MEGAN analysis: it is more likely to be the related bacteria Hafnia alvei that is known to infect social insects. The closest identification to the putative pathogen identified as Vavraia culicis was likely to be another microsporidian Nosema vulgaris. PCR and subsequent Sanger sequencing using published or our own designed primers, confirmed the identity of Moellerella sp. (which may be Hafnia alvei), Aspergillus sp., KBV, Moku virus and Nosema. Secondly, we used an infection study by homogenising diseased wasp larvae and feeding them to entire nests of larvae in the laboratory. Three nests transinfected with diseased larvae all died within 19 days. No pathogen that we monitored, however, had a significantly higher prevalence in diseased than in healthy larvae. RT-qPCR analysis indicated that pathogen infections were significantly correlated, such as between KBV and Aspergillus sp. Social wasps clearly suffer from an array of pathogens, which may lead to the collapse of nests and larval death.

Isolation and characterization of microsatellite loci from Arthropodium cirratum (Asparagaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Arthropodium cirratum (Asparagaceae) to study population genetic structure and translocation of this species. These markers were tested for cross-amplification in two other Arthropodium species. METHODS AND RESULTS: Sixteen microsatellite markers were developed from a genomic library and tested in three populations of A. cirratum. The loci exhibited one to five alleles per locus, with private alleles present in each of the populations. Cross-amplification tests in the two other New Zealand Arthropodium species revealed that many of the loci amplify and demonstrate polymorphism in A. bifurcatum. CONCLUSIONS: These markers will be useful for determining genetic structure in A. cirratum and for determining the origins of translocated populations of this species.

Explaining large mitochondrial sequence differences within a population sample.

Mitochondrial DNA sequence is frequently used to infer species' boundaries, as divergence is relatively rapid when populations are reproductively isolated. However, the shared history of a non-recombining gene naturally leads to correlation of pairwise differences, resulting in mtDNA clusters that might be mistaken for evidence of multiple species. There are four distinct processes that can explain high levels of mtDNA sequence difference within a single sample. Here, we examine one case in detail as an exemplar to distinguish among competing hypotheses. Within our sample of tree weta (Hemideina crassidens; Orthoptera), we found multiple mtDNA haplotypes for a protein-coding region (cytb/ND1) that differed by a maximum of 7.9%. From sequencing the whole mitochondrial genome of two representative individuals, we found evidence of constraining selection. Heterozygotes were as common as expected under random mating at five nuclear loci. Morphological traits and nuclear markers did not resolve the mtDNA groupings of individuals. We concluded that the large differences found among our sample of mtDNA sequences were simply owing to a large population size over an extended period of time allowing an equilibrium between mutation and drift to retain a great deal of genetic diversity within a single species.