Cold tolerance and overwintering survival of Aphelinus certus (Hymenoptera: Aphelinidae), a parasitoid of the soybean aphid (Hemiptera: Aphididae) in North America.

Broad-spectrum insecticides are the main control measure of the invasive and economically damaging soybean aphid (Aphis glycines) in North America, although biological control by resident natural enemies can also greatly diminish population levels. One such natural enemy is the accidentally introduced Eurasian parasitoid Aphelinus certus (Hymenoptera: Aphelinidae), though its impact appears to be limited by low rates of parasitism early in the growing season. We tested the hypothesis that A. certus might experience high overwintering mortality. In the laboratory, we used thermocouple thermometry to measure the supercooling points of diapausing parasitoids and assessed parasitoid survival after exposure to ecologically relevant durations of low temperature. We found A. certus to be freeze-intolerant with a median supercooling point of -28°C. When exposed to temperatures of 0°C for up to 7 months, adults emerged only after exposures of at least 60 days and survival decreased with durations beyond 150 days. We also conducted in-field studies at sites from northern Minnesota to southern Iowa to determine if diapausing A. certus could overwinter above and below the snowpack. Survival was negatively correlated with increasing latitude and was greater for parasitoids placed on the ground than 1 meter off the ground, likely due to the warmer and stabler temperatures of the subnivean microclimate. Our results suggest that A. certus is capable of overwintering in the region inhabited by soybean aphid but may experience substantial mortality even under ideal conditions. Climate change is predicted to bring warmer, drier winters to the North American Midwest, with decreased depth and duration of snow cover, which may further reduce overwintering survival.

The genetic architecture of ecological adaptation: intraspecific variation in host plant use by the lepidopteran crop pest Chloridea virescens.

Intraspecific variation in ecologically important traits is a cornerstone of Darwin's theory of evolution by natural selection. The evolution and maintenance of this variation depends on genetic architecture, which in turn determines responses to natural selection. Some models suggest that traits with complex architectures are less likely to respond to selection than those with simple architectures, yet rapid divergence has been observed in such traits. The simultaneous evolutionary lability and genetic complexity of host plant use in the Lepidopteran subfamily Heliothinae suggest that architecture may not constrain ecological adaptation in this group. Here we investigate the response of Chloridea virescens, a generalist that feeds on diverse plant species, to selection for performance on a novel host, Physalis angulata (Solanaceae). P. angulata is the preferred host of Chloridea subflexa, a narrow specialist on the genus Physalis. In previous experiments, we found that the performance of C. subflexa on P. angulata depends on many loci of small effect distributed throughout the genome, but whether the same architecture would be involved in the generalist's adoption of P. angulata was unknown. Here we report a rapid response to selection in C. virescens for performance on P. angulata, and establish that the genetic architecture of intraspecific variation is quite similar to that of the interspecific differences in terms of the number, distribution, and effect sizes of the QTL involved. We discuss the impact of genetic architecture on the ability of Heliothine moths to respond to varying ecological selection pressures.

Factors limiting the spread of the protective symbiont Hamiltonella defensa in Aphis craccivora Aphids.

Many insects are associated with heritable symbionts that mediate ecological interactions, including host protection against natural enemies. The cowpea aphid, Aphis craccivora, is a polyphagous pest that harbors Hamiltonella defensa, which defends against parasitic wasps. Despite this protective benefit, this symbiont occurs only at intermediate frequencies in field populations. To identify factors constraining H. defensa invasion in Ap. craccivora, we estimated symbiont transmission rates, performed fitness assays, and measured infection dynamics in population cages to evaluate effects of infection. Similar to results with the pea aphid, Acyrthosiphon pisum, we found no consistent costs to infection using component fitness assays, but we did identify clear costs to infection in population cages when no enemies were present. Maternal transmission rates of H. defensa in Ap. craccivora were high (ca. 99%) but not perfect. Transmission failures and infection costs likely limit the spread of protective H. defensa in Ap. craccivora. We also characterized several parameters of H. defensa infection potentially relevant to the protective phenotype. We confirmed the presence of H. defensa in aphid hemolymph, where it potentially interacts with endoparasites, and performed real-time quantitative PCR (qPCR) to estimate symbiont and phage abundance during aphid development. We also examined strain variation of H. defensa and its bacteriophage at multiple loci, and despite our lines being collected in different regions of North America, they were infected with a nearly identical strains of H. defensa and APSE4 phage. The limited strain diversity observed for these defensive elements may result in relatively static protection profile for this defensive symbiosis.

Worldwide populations of the aphid Aphis craccivora are infected with diverse facultative bacterial symbionts.

Facultative bacterial endosymbionts can play an important role in the evolutionary trajectory of their hosts. Aphids (Hemiptera: Aphididae) are infected with a wide variety of facultative endosymbionts that can confer ecologically relevant traits, which in turn may drive microevolutionary processes in a dynamic selective environment. However, relatively little is known about how symbiont diversity is structured in most aphid species. Here, we investigate facultative symbiont species richness and prevalence among world-wide populations of the cowpea aphid, Aphis craccivora Koch. We surveyed 44 populations of A. craccivora, and detected 11 strains of facultative symbiotic bacteria, representing six genera. There were two significant associations between facultative symbiont and aphid food plant: the symbiont Arsenophonus was found at high prevalence in A. craccivora populations collected from Robinia sp. (locust), whereas the symbiont Hamiltonella was almost exclusively found in A. craccivora populations from Medicago sativa (alfalfa). Aphids collected from these two food plants also had divergent mitochondrial haplotypes, potentially indicating the formation of specialized aphid lineages associated with food plant (host-associated differentiation). The role of facultative symbionts in this process remains to be determined. Overall, observed facultative symbiont prevalence in A. craccivora was lower than that of some other well-studied aphids (e.g., Aphis fabae and Acyrthosiphon pisum), possibly as a consequence of A. craccivora's almost purely parthenogenetic life history. Finally, most (70 %) of the surveyed populations were polymorphic for facultative symbiont infection, indicating that even when symbiont prevalence is relatively low, symbiont-associated phenotypic variation may allow population-level evolutionary responses to local selection.

Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here, we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 vs G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Reduced-representation libraries in insect genetics.

Genotyping-by-sequencing of reduced-representation libraries has ushered in an era where genome-wide data can be gotten for any species. Here, I review research on this topic during the last two years, report meta-analysis of the results, and discuss analysis methods and issues. Scanning the literature from 2021 to 2022 identified 21 papers, the majority of which were on population differences, including local adaptation and migration, but several papers were on genetic maps and their use in assembly scaffolding or analysis of quantitative trait loci, on the origin of incursions of pest insects, or on infection rates of a pathogen in a disease vector. The research reviewed includes 33 species from 25 families and 11 orders. Meta-analysis showed that less than 16%, and most often, less than 1% of the genome was implicated in local adaptation and that the number of adaptive loci correlated with genetic divergence among populations.

Assemblies of the genomes of parasitic wasps using meta-assembly and scaffolding with genetic linkage.

Safe, effective biological-control introductions against invasive pests depend on narrowly host-specific natural enemies with the ability to adapt to a changing environment. As part of a project on the genetic architectures of these traits, we assembled and annotated the genomes of two aphid parasitoids, Aphelinus atriplicis and Aphelinus certus. We report here several assemblies of A. atriplicis made with Illumina and PacBio data, which we combined into a meta-assembly. We scaffolded the meta-assembly with markers from a genetic map of hybrids between A. atriplicis and A. certus. We used this genetic-linkage scaffolded (GLS) assembly of A. atriplicis to scaffold a de novo assembly of A. certus. The de novo assemblies of A. atriplicis differed in contiguity, and the meta-assembly of these assemblies was more contiguous than the best de novo assembly. Scaffolding with genetic-linkage data allowed chromosomal-level assembly of the A. atriplicis genome and scaffolding a de novo assembly of A. certus with this GLS assembly, greatly increased the contiguity of the A. certus assembly to the point where it was also at the chromosomal-level. However, completeness of the A. atriplicis assembly, as measured by percent complete, single-copy BUSCO hymenopteran genes, varied little among de novo assemblies and was not increased by meta-assembly or genetic scaffolding. Furthermore, the greater contiguity of the meta-assembly and GLS assembly had little or no effect on the numbers of genes identified, the proportions with homologs or functional annotations. Increased contiguity of the A. certus assembly provided modest improvement in assembly completeness, as measured by percent complete, single-copy BUSCO hymenopteran genes. The total genic sequence increased, and while the number of genes declined, gene length increased, which together suggest greater accuracy of gene models. More contiguous assemblies provide uses other than gene annotation, for example, identifying the genes associated with quantitative trait loci and understanding of chromosomal rearrangements associated with speciation.

Response to selection for parasitism of a suboptimal, low-preference host in an aphid parasitoid.

Risks of postintroduction evolution in insects introduced to control invasive pests have been discussed for some time, but little is known about responses to selection or genetic architectures of host adaptation and thus about the likelihood or rapidity of evolutionary shifts. We report here results on the response to selection and genetic architecture of parasitism of a suboptimal, low-preference host species by an aphid parasitoid, Aphelinus rhamni, a candidate for introduction against the soy bean aphid, Aphis glycines. We selected A. rhamni for increased parasitism of Rhopalsiphum padi by rearing the parasitoid on this aphid for three generations. We measured parasitism of R. padi at generations 2 and 3, and at generation 3, we crossed and backcrossed parasitoids from the populations reared on R. padi with those from populations reared on Aphis glycines and compared parasitism of both R. padi and Aphis glycines among F 1 and backcross females. Aphelinus rhamni responded rapidly to selection for parasitism of R. padi. Selection for R. padi parasitism reduced parasitism of Aphis glycines, the original host of A. rhamni. However, parasitism of R. padi did not increase from generation 2 to generation 3 of selection, suggesting reduced variance available for selection, which was indeed found. We tested the associations between 184 single nucleotide polymorphisms (SNP) and increased parasitism of R. padi and found 28 SNP loci, some of which were associated with increased and others with decreased parasitism of R. padi. We assembled and annotated the A. rhamni genome, mapped all SNP loci to contigs and tested whether genes on contigs with SNP loci associated with parasitism were enriched for candidate genes or gene functions. We identified 80 genes on these contigs that mapped to 1.2 Mb of the 483 Mb genome of A. rhamni but found little enrichment of candidate genes or gene functions.

Immune Suppressive Extracellular Vesicle Proteins of Leptopilina heterotoma Are Encoded in the Wasp Genome.

Leptopilina heterotoma are obligate parasitoid wasps that develop in the body of their Drosophila hosts. During oviposition, female wasps introduce venom into the larval hosts' body cavity. The venom contains discrete, 300 nm-wide, mixed-strategy extracellular vesicles (MSEVs), until recently referred to as virus-like particles. While the crucial immune suppressive functions of L. heterotoma MSEVs have remained undisputed, their biotic nature and origin still remain controversial. In recent proteomics analyses of L. heterotoma MSEVs, we identified 161 proteins in three classes: conserved eukaryotic proteins, infection and immunity related proteins, and proteins without clear annotation. Here we report 246 additional proteins from the L. heterotoma MSEV proteome. An enrichment analysis of the entire proteome supports vesicular nature of these structures. Sequences for more than 90% of these proteins are present in the whole-body transcriptome. Sequencing and de novo assembly of the 460 Mb-sized L. heterotoma genome revealed 90% of MSEV proteins have coding regions within the genomic scaffolds. Altogether, these results explain the stable association of MSEVs with their wasps, and like other wasp structures, their vertical inheritance. While our results do not rule out a viral origin of MSEVs, they suggest that a similar strategy for co-opting cellular machinery for immune suppression may be shared by other wasps to gain advantage over their hosts. These results are relevant to our understanding of the evolution of figitid and related wasp species.

Multifaceted determinants of host specificity in an aphid parasitoid.

The host specificity of insect parasitoids and herbivores is thought to be shaped by a suite of traits that mediate host acceptance and host suitability. We conducted laboratory experiments to identify mechanisms shaping the host specificity of the aphid parasitoid Binodoxys communis. Twenty species of aphids were exposed to B. communis females in microcosms, and detailed observations and rearing studies of 15 of these species were done to determine whether patterns of host use resulted from variation in factors such as host acceptance or variation in host suitability. Six species of aphids exposed to B. communis showed no signs of parasitism. Four of these species were not recognized as hosts and two effectively defended themselves from attack by B. communis. Other aphid species into which parasitoids laid eggs had low suitability as hosts. Parasitoid mortality occurred in the egg or early larval stages for some of these hosts but for others it occurred in late larval stages. Two hypotheses explaining low suitability were investigated in separate experiments: the presence of endosymbiotic bacteria conferring resistance to parasitoids, and aphids feeding on toxic plants. An association between resistance and endosymbiont infection was found in one species (Aphis craccivora), and evidence for the toxic plant hypothesis was found for the milkweed aphids Aphis asclepiadis and Aphis nerii. This research highlights the multifaceted nature of factors determining host specificity in parasitoids.

Counties not countries: Variation in host specificity among populations of an aphid parasitoid.

Parasitic wasps are among the most species-rich groups on Earth. A major cause of this diversity may be local adaptation to host species. However, little is known about variation in host specificity among populations within parasitoid species. Not only is such knowledge important for understanding host-driven speciation, but because parasitoids often control pest insects and narrow host ranges are critical for the safety of biological control introductions, understanding variation in specificity and how it arises are crucial applications in evolutionary biology. Here, we report experiments on variation in host specificity among 16 populations of an aphid parasitoid, Aphelinus certus. We addressed several questions about local adaptation: Do parasitoid populations differ in host ranges or in levels of parasitism of aphid species within their host range? Are differences in parasitism among parasitoid populations related to geographical distance, suggesting clinal variation in abundances of aphid species? Or do nearby parasitoid populations differ in host use, as would be expected if differences in aphid abundances, and thus selection, were mosaic? Are differences in parasitism among parasitoid populations related to genetic distances among them? To answer these questions, we measured parasitism of a taxonomically diverse group of aphid species in laboratory experiments. Host range was the same for all the parasitoid populations, but levels of parasitism varied among aphid species, suggesting adaptation to locally abundant aphids. Differences in host specificity did not correlate with geographical distances among parasitoid populations, suggesting that local adaption is mosaic rather than clinal, with a spatial scale of less than 50 kilometers. We sequenced and assembled the genome of A. certus, made reduced-representation libraries for each population, analyzed for single nucleotide polymorphisms, and used these polymorphisms to estimate genetic differentiation among populations. Differences in host specificity correlated with genetic distances among the parasitoid populations.

Genomes of the Hymenoptera.

Hymenoptera is the second-most sequenced arthropod order, with 52 publically archived genomes (71 with ants, reviewed elsewhere), however these genomes do not capture the breadth of this very diverse order (Figure 1, Table 1). These sequenced genomes represent only 15 of the 97 extant families. Although at least 55 other genomes are in progress in an additional 11 families (see Table 2), stinging wasps represent 35 (67%) of the available and 42 (76%) of the in progress genomes. A more comprehensive catalog of hymenopteran genomes is needed for research into the evolutionary processes underlying the expansive diversity in terms of ecology, behavior, and physiological traits within this group. Additional sequencing is needed to generate an assembly for even 0.05% of the estimated 1 million hymenopteran species, and we recommend premier level assemblies for at least 0.1% of the >150,000 named species dispersed across the order. Given the haplodiploid sex determination in Hymenoptera, haploid male sequencing will help minimize genome assembly issues to enable higher quality genome assemblies.

Population dynamics of Aphis glycines (Homoptera: Aphididae) and impact of natural enemies in northern China.

Field surveys of soybean aphid, Aphis glycines Matsumura, and its natural enemies, as well as natural enemy exclosure experiments, were conducted during 2003 and 2004 in soybean fields near Langfang, China. In 2003, aphid density increased six-fold during 12 d in July from 66+/-12 per 10 plants to a seasonal peak of 401+/-79 per 10 plants. Aphid density remained high for another 10 d and declined during late July and early August. In 2004, aphid density increased 29-fold during 13 d in July from 14+/-2 per 10 plants to a seasonal peak of 375+/-30 per 10 plants. Unlike 2003, aphid density remained relatively high during late July and August, peaking again at 296+/-31 per 10 plants on 24 August. In both years, aphid density remained below economic injury level and seemed to be limited by natural enemies. Exclosure of natural enemies led to increases in A. glycines density in 2003 and 2004. In 2003, peak aphid densities in large- and medium-mesh cages were three- and seven-fold higher, respectively, than densities on uncaged plants. In 2004, peak aphid densities in large- and medium-mesh cages were 2-fold and 30-fold higher, respectively, than densities on uncaged plants in one experiment. In another experiment, peak aphid densities in large-, medium-, and small-mesh cages were 8-fold, 28-fold, and 68-fold higher, respectively, than densities on uncaged plants. Both predators and parasitoids were important in limiting aphid density. We compare our results with those from North America and discuss implications for biological control.

Variation in genome size and karyotype among closely related aphid parasitoids (Hymenoptera, Aphelinidae).

Genome sizes were measured and determined for the karyotypes of nine species of aphid parasitoids in the genus Aphelinus Dalman,1820. Large differences in genome size and karyotype were found between Aphelinus species, which is surprising given the similarity in their morphology and life history. Genome sizes estimated from flow cytometry were larger for species in the Aphelinus mali (Haldeman, 1851) complex than those for the species in the Aphelinus daucicola Kurdjumov, 1913 and Aphelinus varipes (Förster,1841) complexes. Haploid karyotypes of the Aphelinus daucicola and Aphelinus mali complexes comprised five metacentric chromosomes of similar size, whereas those of the Aphelinus varipes complex had four chromosomes, including a larger and a smaller metacentric chromosome and two small acrocentric chromosomes or a large metacentric and three smaller acrocentric chromosomes. Total lengths of female haploid chromosome sets correlated with genome sizes estimated from flow cytometry. Phylogenetic analysis of karyotypic variation revealed a chromosomal fusion together with pericentric inversions in the common ancestor of the Aphelinus varipes complex and further pericentric inversions in the clade comprising Aphelinus kurdjumovi Mercet, 1930 and Aphelinus hordei Kurdjumov, 1913. Fluorescence in situ hybridization with a 28S ribosomal DNA probe revealed a single site on chromosomes of the haploid karyotype of Aphelinus coreae Hopper & Woolley, 2012. The differences in genome size and total chromosome length between species complexes matched the phylogenetic divergence between them.

Phased Genotyping-by-Sequencing Enhances Analysis of Genetic Diversity and Reveals Divergent Copy Number Variants in Maize.

High-throughput sequencing (HTS) of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken from heterogeneous populations of heterozygous individuals. This requires that a number of issues encountered with GBS be considered, including the sequencing of nonoverlapping sets of loci across multiple GBS libraries, a common missing data problem that results in low call rates for markers per individual, and a tendency for applicability only in inbred line samples with sufficient linkage disequilibrium for accurate imputation. We addressed these issues while developing and validating a new, comprehensive platform for GBS. This study supports the notion that GBS can be tailored to particular aims, and using Zea mays our results indicate that large samples of unknown pedigree can be genotyped to obtain complete and accurate GBS data. Optimizing size selection to sequence a high proportion of shared loci among individuals in different libraries and using simple in silico filters, a GBS procedure was established that produces high call rates per marker (>85%) with accuracy exceeding 99.4%. Furthermore, by capitalizing on the sequence-read structure of GBS data (stacks of reads), a new tool for resolving local haplotypes and scoring phased genotypes was developed, a feature that is not available in many GBS pipelines. Using local haplotypes reduces the marker dimensionality of the genotype matrix while increasing the informativeness of the data. Phased GBS in maize also revealed the existence of reproducibly inaccurate (apparent accuracy) genotypes that were due to divergent copy number variants (CNVs) unobservable in the underlying single nucleotide polymorphism (SNP) data.

United States Department of Agriculture-Agricultural Research Service research on biological control of arthropods.

During 1999-2001, ARS scientists published over 100 papers on more than 30 species of insect pest and 60 species of predator and parasitoid. These papers address issues crucial to the three strategies of biological control: conservation, augmentation and introduction. Conservation biological control includes both conserving extant populations of natural enemies by using relatively non-toxic pesticides and increasing the abundance of natural enemies in crops by providing or improving refuges for population growth and dispersal into crops. ARS scientists have been very active in determining the effects of pesticides on beneficial arthropods and in studying movement of natural enemies from refuges into crops. Augmentation involves repeated releases of natural enemies in the field, which can be inoculative or inundative. Inoculative releases are used to initiate self-propagating populations at times or in places where they would be slow to colonize. ARS scientists have studied augmentative biological control of a variety of pest insects. The targets are mostly pests in annual crops or other ephemeral habitats, where self-reproducing populations of natural enemies are not sufficiently abundant early enough to keep pest populations in check. ARS research in augmentative biological control centers on methods for rearing large numbers of healthy, effective natural enemies and for releasing them where and when they are needed at a cost less than the value of the reduction in damage to the crop. ARS scientists have researched various aspects of introductions of exotic biological control agents against a diversity of pest insects. The major issues in biological control introductions are accurate identification and adequate systematics of both natural enemies and target pests, exploration for natural enemies, predicting the success of candidates for introduction and the likelihood of non-target impacts, quarantine and rearing methods, and post-introduction evaluation of establishment, control and non-target impacts. ARS scientists have published research on several general issues in biological control. Among the most important are the mechanisms affecting mate- and host-finding and host specificity.

The genetic architecture of a complex ecological trait: host plant use in the specialist moth, Heliothis subflexa.

We used genetic mapping to examine the genetic architecture of differences in host plant use between two species of noctuid moths, Heliothis subflexa, a specialist on Physalis spp., and its close relative, the broad generalist H. virescens. We introgressed H. subflexa chromosomes into the H. virescens background and analyzed 1462 backcross insects. The effects of H. subflexa-origin chromosomes were small when measured as the percent variation explained in backcross populations (0.2-5%), but were larger when considered in relation to the interspecific difference explained (1.5-165%). Most significant chromosomes had effects on more than one trait, and their effects varied between years, sexes, and genetic backgrounds. Different chromosomes could produce similar phenotypes, suggesting that the same trait might be controlled by different chromosomes in different backcross populations. It appears that many loci of small effect contribute to the use of Physalis by H. subflexa. We hypothesize that behavioral changes may have paved the way for physiological adaptation to Physalis by the generalist ancestor of H. subflexa and H. virescens.

Outcrossing and crossbreeding recovers deteriorated traits in laboratory cultured Steinernema carpocapsae nematodes.

The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and 'tail standing' (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and 'tail standing' did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth.

Molecular phylogenetics and reproductive incompatibility in a complex of cryptic species of aphid parasitoids.

We infer the phylogeny of a complex of cryptic species and populations of parasitic wasps and examine how reproductive incompatibility maps onto the molecular phylogeny. We used four nuclear (28S-D2, ITS1, ITS2, ArgK) and two mitochondrial (COI, COII) gene regions to analyze relationships among populations in the Aphelinus varipes species complex (Hymenoptera: Aphelinidae) from throughout Eurasia (France, Georgia, Israel, China, Korea and Japan) and from three aphid hosts (Aphis glycines, Diuraphis noxia and Rhopalosiphum padi; Hemiptera: Aphididae). A combined analysis of 21 genotypes of Aphelinus resulted in six most-parsimonious trees, and successive approximations character-weighting selected two of these as best supported by the data. All six gene regions were necessary to fully resolve the relationships among taxa. Four clades within the A. varipes complex were distinguished: (1) Aphelinus kurdjumovi, (2) Aphelinus hordei, (3) Aphelinus atriplicis, Aphelinus varipes, and Aphelinus albipodus, and (4) Aphelinus certus (populations from China, Korea, and Japan). Based on rates of nucleotide substitutions, these clades diverged between 78 and 526 thousand years ago during a period of repeated glaciations in Eurasia. In laboratory crosses, A. kurdjumovi, A. hordei, and A. varipes were reproductively incompatible with one another and all other populations. A. atriplicis was incompatible with these three species, but not with A. certus. The populations of A. certus from China, Japan, and Korea were reproductively compatible with one another but not with the other populations. Thus, with one exception, entities that were phylogenetically distinct were also reproductively incompatible with one another. Our evidence on molecular differentiation and reproductive incompatibility supports recognition of at least five cryptic species in the A. varipes complex. We discuss likely reasons for the high rate of speciation in this complex.