Population genomics of the invasive Northern Giant Hornet Vespa mandarinia in North America and across its native range.

The northern giant hornet Vespa mandarinia (NGH) is a voracious predator of other insect species, including honey bees. NGH's native range spans subtropical and temperate regions across much of east and southeast Asia and, in 2019, exotic populations of the species were discovered in North America. Despite this broad range and invasive potential, investigation of the population genomic structure of NGH across its native and introduced ranges has thus far been limited to a small number of mitochondrial samples. Here, we present analyses of genomic data from NGH individuals collected across the species' native range and from exotic individuals collected in North America. We provide the first survey of whole-genome population variation for any hornet species, covering this species' native and invasive ranges, and in doing so confirm likely origins in Japan and South Korea for the two introductions. We additionally show that, while this introduced population exhibited strongly elevated levels of inbreeding, these signatures of inbreeding are also present in some long-standing native populations, which may indicate that inbreeding depression alone is insufficient to prevent the persistence of NGH populations. As well as highlighting the importance of ongoing monitoring and eradication efforts to limit the spread of this species outside of its natural range, our data will serve as a foundational database for future genomic studies into introduced hornet populations.

From genome to proteome: Comprehensive identification of venom toxins from the Chinese funnel-web spider (Macrothelidae: Macrothele yani).

Macrothelidae is a family of mygalomorph spiders containing the extant genera Macrothele and Vacrothele. China is an important center of diversity for Macrothele with 65 % of the known species occurring there. Previous work on Macrothele was able to uncover several important toxin compounds including Raventoxin which may have applications in biomedicine and agricultural chemistry. Despite the importance of Macrothele spiders, high-quality reference genomes are still lacking, which hinders our understanding and application of the toxin compounds. In this study, we assembled the genome of the Macrothele yani to help fill gaps in our understanding of toxin biology in this lineage of spiders to encourage the future study and applications of these compounds. The final assembled genome was 6.79 Gb in total length, had a contig N50 of 21.44 Mb, and scaffold N50 of 156.16 Mb. Hi-C scaffolding assigned 98.19 % of the genome to 46 pseudo-chromosomes with a BUSCO score of 95.7 % for the core eukaryotic gene set. The assembled genome was found to contain 75.62 % repetitive DNA and a total of 39,687 protein-coding genes were annotated making it the spider genome with highest number of genes. Through integrated analysis of venom gland transcriptomics and venom proteomics, a total of 194 venom toxins were identified, including 38 disulfide-rich peptide neurotoxins, among which 12 were ICK knottin peptides. In summary, we present the first high-quality genome assembly at the chromosomal level for any Macrothelidae spider, filling an important gap in our knowledge of these spiders. Such high-quality genomic data will be invaluable as a reference in resolving Araneae spider phylogenies and in screening different spider species for novel compounds applicable to numerous medical and agricultural applications.

The pan-plastome of Hemerocallis citrina reveals new insights into the genetic diversity and cultivation history of an economically important food plant.

BACKGROUND: Hemerocallis citrina Baroni (Huang hua cai in Chinese) is a perennial herbaceous plant grown for its flower buds that are eaten fresh or dried and is known as the vegetarian three treasures. The nuclear genome of H. citrina has been reported, but the intraspecific variation of the plastome (plastid genome) has not yet been studied. Therefore, the panplastome of this species collected from diverse locations is reported here for the first time. RESULTS: In this study, 65 H. citrina samples were resequenced, de novo assembled, and aligned with the published plastome of H. citrina to resolve the H. citrina panplastome. The sizes of the 65 newly assembled complete plastomes of H. citrina ranged from 156,048 bp to 156,263 bp, and the total GC content ranged from 37.31 to 37.34%. The structure of the complete plastomes showed a typical tetrameric structure, including a large single copy (LSC), a small single copy (SSC), and a pair of inverted repeat regions (IRA and IRB). Many nucleotide variants were identified between plastomes, among which the variants in the intergenic spacer region were the most abundant, with the highest number of variants concentrated in the LSC region. Based on the phylogenetic tree constructed using the ML method, population structure analysis, and principal component analysis (PCA), the panplastome data were subdivided into five genetic clusters. The C5 genetic cluster was mostly represented by samples from Qidong, Hunan Province, while samples from Shanxi and Shaanxi Provinces were classified into the C4 genetic cluster. The greatest genetic diversity was found in the C1 genetic cluster, and the greatest genetic distance between any two clusters was found between the C4 and C5 clusters. CONCLUSION: The resolution of the panplastome and the analysis of the population structure of H. citrina plastomes provide important data for future breeding projects and germplasm preservation.

Plant organellar genomes: much done, much more to do.

Plastids and mitochondria are the only organelles that possess genomes of endosymbiotic origin. In recent decades, advances in sequencing technologies have contributed to a meteoric rise in the number of published organellar genomes, and have revealed greatly divergent evolutionary trajectories. In this review, we quantify the abundance and distribution of sequenced plant organellar genomes across the plant tree of life. We compare numerous genomic features between the two organellar genomes, with an emphasis on evolutionary trajectories, transfers, the current state of organellar genome editing by transcriptional activator-like effector nucleases (TALENs), transcription activator-like effector (TALE)-mediated deaminase, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), as well as genetic transformation. Finally, we propose future research to understand these different evolutionary trajectories, and genome-editing strategies to promote functional studies and eventually improve organellar genomes.

Multi-integrated genomic data for Passiflora foetida provides insights into genome size evolution and floral development in Passiflora.

Passiflora is a plant genus known for its extremely distinctive and colorful flowers and a wide range of genome size variation. However, how genome characteristics are related to flower traits among Passiflora species remains poorly understood. Here, we assembled a chromosome-scale genome of P. foetida, which belongs to the same subgenus as the commercial passionfruit P. edulis. The genome of P. foetida is smaller (424.16 Mb) and contains fewer copies of long terminal repeat retrotransposons (LTR-RTs). The disparity in LTR-RTs is one of the main contributors to the differences in genome sizes between these two species and possibly in floral traits. Additionally, we observed variation in insertion times and copy numbers of LTR-RTs across different transposable element (TE) lineages. Then, by integrating transcriptomic data from 33 samples (eight floral organs and flower buds at three developmental stages) with phylogenomic and metabolomic data, we conducted an in-depth analysis of the expression, phylogeny, and copy number of MIKC-type MADS-box genes and identified essential biosynthetic genes responsible for flower color and scent from glandular bracts and other floral organs. Our study pinpoints LRT-RTs as an important player in genome size variation in Passiflora species and provides insights into future genetic improvement.

Viral Prevalence and Genomic Xenology in the Coevolution of HzNV-2 (Nudiviridae) with Host Helicoverpa zea (Lepidoptera: Noctuidae).

Insect viruses have been described from numerous lineages, yet patterns of genetic exchange and viral prevalence, which are essential to understanding host-virus coevolution, are rarely studied. In Helicoverpa zea, the virus HzNV-2 can cause deformity of male and female genitalia, resulting in sterility. Using ddPCR, we found that male H. zea with malformed genitalia (agonadal) contained high levels of HzNV-2 DNA, confirming previous work. HzNV-2 was found to be prevalent throughout the United States, at more than twice the rate of the baculovirus HaSNPV, and that it contained several host-acquired DNA sequences. HzNV-2 possesses four recently endogenized lepidopteran genes and several more distantly related genes, including one gene with a bacteria-like sequence found in both host and virus. Among the recently acquired genes is cytosolic serine hydroxymethyltransferase (cSHMT). In nearly all tested H. zea, cSHMT contained a 200 bp transposable element (TE) that was not found in cSHMT of the sister species H. armigera. No other virus has been found with host cSHMT, and the study of this shared copy, including possible interactions, may yield new insights into the function of this gene with possible applications to insect biological control, and gene editing.

Comparative analysis of organellar genomes between diploid and tetraploid Chrysanthemum indicum with its relatives.

Chrysanthemum indicum, a species native to Eastern Asia is well known as one of the progenitor species of the cultivated Chrysanthemum which is grown for its ornamental and medicinal value. Previous genomic studies on Chrysanthemum have largely ignored the dynamics of plastid genome (plastome) and mitochondria genome (mitogenome) evolution when analyzing this plant lineage. In this study, we sequenced and assembled the plastomes and mitogenomes of diploid and tetraploid C. indicum as well as the morphologically divergent variety C. indicum var. aromaticum. We used published data from 27 species with both plastome and mitogenome complete sequences to explore differences in sequence evolution between the organellar genomes. The size and structure of organellar genome between diploid and tetraploid C. indicum were generally similar but the tetraploid C. indicum and C. indicum var. aromaticum were found to contain unique sequences in the mitogenomes which also contained previously undescribed open reading frames (ORFs). Across Chrysanthemum mitogenome structure varied greatly but sequences transferred from plastomes in to the mitogenomes were conserved. Finally, differences observed between mitogenome and plastome gene trees may be the result of the difference in the rate of sequence evolution between genes in these two genomes. In total the findings presented here greatly expand the resources for studying Chrysanthemum organellar genome evolution with possible applications to conservation, breeding, and gene banking in the future.

Comparative Plastomes of Curcuma alismatifolia (Zingiberaceae) Reveal Diversified Patterns among 56 Different Cut-Flower Cultivars.

Curcuma alismatifolia (Zingiberaceae) is an ornamental species with high economic value due to its recent rise in popularity among floriculturists. Cultivars within this species have mixed genetic backgrounds from multiple hybridization events and can be difficult to distinguish via morphological and histological methods alone. Given the need to improve identification resources, we carried out the first systematic study using plastomic data wherein genomic evolution and phylogenetic relationships from 56 accessions of C. alismatifolia were analyzed. The newly assembled plastomes were highly conserved and ranged from 162,139 bp to 164,111 bp, including 79 genes that code for proteins, 30 tRNA genes, and 4 rRNA genes. The A/T motif was the most common of SSRs in the assembled genomes. The Ka/Ks values of most genes were less than 1, and only two genes had Ka/Ks values above 1, which were rps15 (1.15), and ndhl (1.13) with petA equal to 1. The sequence divergence between different varieties of C. alismatifolia was large, and the percentage of variation in coding regions was lower than that in the non-coding regions. Such data will improve cultivar identification, marker assisted breeding, and preservation of germplasm resources.

Transcriptome sequencing of wolf spider Lycosa sp. (Araneae: Lycosidae) venom glands provides insights into the evolution and diversity of disulfide-rich toxins.

Wolf spiders in the genus Lycosa are important pest predators in agroforestry ecosystems, capable of feeding on a wide range of pests through the use of complex venom which can to quickly immobilize and kill prey. Because of these characteristics the toxins in wolf spiders venom may prove to be natural sources for novel drug development and biopesticides. To better understand the toxins in Lycosa venom we sequenced the transcriptome from venom glands from an undescribed species of Lycosa and comparatively analyzed the data using known protein motifs. A series of 19 disulfide-rich peptide (DRP) toxin sequences were identified and categorized into seven groups based on the number and arrangement of cysteine residues. Notably, we identified three peptide sequences with low identity to any known toxin, which may be toxin peptides specific to this species of Lycosa. In addition, to further understand the evolutionary relationships of disulfide-rich peptide toxins in spider venom, we constructed phylogenetic trees of DRP toxins from three spiders species and found that the Lycosa sp. DRPs are comparatively diverse with previous research results. This study reveals the toxin diversity of wolf spiders (Lycosa sp.) at the transcriptomic level and provides initial insights into the evolution of DRP toxins in spiders, enriching our knowledge of toxin diversity and providing new compounds for functional studies.

Ultra-deep sequencing of 45S rDNA to discern intragenomic diversity in three Chrysodeixis species for molecular identification.

Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.

A high-quality Bougainvillea genome provides new insights into evolutionary history and pigment biosynthetic pathways in the Caryophyllales.

Bougainvillea is a perennial ornamental shrub that is highly regarded in ornamental horticulture around the world. However, the absence of genome data limits our understanding of the pathways involved in bract coloration and breeding. Here, we report a chromosome-level assembly of the giga-genome of Bougainvillea × buttiana 'Mrs Butt', a cultivar thought to be the origin of many other Bougainvillea cultivars. The assembled genome is ~5 Gb with a scaffold N50 of 151 756 278 bp and contains 86 572 genes which have undergone recent whole-genome duplication. We confirmed that multiple rounds of whole-genome multiplication have occurred in the evolutionary history of the Caryophyllales, reconstructed the relationship in the Caryophyllales at whole genome level, and found discordance between species and gene trees as the result of complex introgression events. We investigated betalain and anthocyanin biosynthetic pathways and found instances of independent evolutionary innovations in the nine different Caryophyllales species. To explore the potential formation mechanism of diverse bract colors in Bougainvillea, we analyzed the genes involved in betalain and anthocyanin biosynthesis and found extremely low expression of ANS and DFR genes in all cultivars, which may limit anthocyanin biosynthesis. Our findings indicate that the expression pattern of the betalain biosynthetic pathway did not directly correlate with bract color, and a higher expression level in the betalain biosynthetic pathway is required for colored bracts. This improved understanding of the correlation between gene expression and bract color allows plant breeding outcomes to be predicted with greater certainty.

Chromosome-level genome assemblies from two sandalwood species provide insights into the evolution of the Santalales.

Sandalwood is one of the most expensive woods in the world and is well known for its long-lasting and distinctive aroma. In our study, chromosome-level genome assemblies for two sandalwood species (Santalum album and Santalum yasi) were constructed by integrating NGS short reads, RNA-seq, and Hi-C libraries with PacBio HiFi long reads. The S. album and S. yasi genomes were both assembled into 10 pseudochromosomes with a length of 229.59 Mb and 232.64 Mb, containing 21,673 and 22,816 predicted genes and a repeat content of 28.93% and 29.54% of the total genomes, respectively. Further analyses resolved a Santalum-specific whole-genome triplication event after divergence from ancestors of the Santalales lineage Malania, yet due to dramatic differences in transposon content, the Santalum genomes were only one-sixth the size of the Malania oleifera genome. Examination of RNA-seq data revealed a suite of genes that are differentially expressed in haustoria and might be involved in host hemiparasite interactions. The two genomes presented here not only provide an important comparative dataset for studying genome evolution in early diverging eudicots and hemiparasitic plants but will also hasten the application of conservation genomics for a lineage of trees recovering from decades of overexploitation.

Transcriptome-based analyses reveal venom diversity in two araneomorph spiders, Psechrus triangulus and Hippasa lycosina.

The spiders Psechrus triangulus and Hippasa lycosina are widely distributed in Yunnan Province, China, and are important natural enemies of agricultural pests, yet studies regarding the composition of their venom are lacking. In this study, cDNA libraries were constructed from venom gland tissue of P. triangulus and H. lycosina and used for transcriptomic analysis. From the analysis, 39 and 31 toxin-like sequences were predicted for P. triangulus and H. lycosina, respectively. The predicted neurotoxin sequences were categorized according to cysteine sequence motifs, and the predicted neurotoxin sequences of P. triangulus and H. lycosina could be classified into 9 and 6 toxin families, respectively. In addition, potential acetylcholinesterase, hyaluronidase, and astaxanthin-like metalloproteinases were identified through annotation. In summary, transcriptomic techniques were invaluable in mining the gene expression information from these two spider species to explore the toxin composition of their venom and determine how they differ. Studies of this type provide essential baseline data for studying the evolution and physiological activities of spider toxins and for the potential development of medicinal compounds.

The pan-plastome of tartary buckwheat (fagopyrum tataricum): key insights into genetic diversity and the history of lineage divergence.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is an important food and medicine crop plant, which has been cultivated for 4000 years. A nuclear genome has been generated for this species, while an intraspecific pan-plastome has yet to be produced. As such a detailed understanding of the maternal genealogy of Tartary buckwheat has not been thoroughly investigated. RESULTS: In this study, we de novo assembled 513 complete plastomes of Fagopyrum and compared with 8 complete plastomes of Fagopyrum downloaded from the NCBI database to construct a pan-plastome for F. tartaricum and resolve genomic variation. The complete plastomes of the 513 newly assembled Fagopyrum plastome sizes ranged from 159,253 bp to 159,576 bp with total GC contents ranged from 37.76 to 37.97%. These plastomes all maintained the typical quadripartite structure, consisting of a pair of inverted repeat regions (IRA and IRB) separated by a large single copy region (LSC) and a small single copy region (SSC). Although the structure and gene content of the Fagopyrum plastomes are conserved, numerous nucleotide variations were detected from which population structure could be resolved. The nucleotide variants were most abundant in the non-coding regions of the genome and of those the intergenic regions had the most. Mutational hotspots were primarily found in the LSC regions. The complete 521 Fagopyrum plastomes were divided into five genetic clusters, among which 509 Tartary buckwheat plastomes were divided into three genetic clusters (Ft-I/Ft-II/Ft-III). The genetic diversity in the Tartary buckwheat genetic clusters was the greatest in Ft-III, and the genetic distance between Ft-I and Ft-II was the largest. Based on the results of population structure and genetic diversity analysis, Ft-III was further subdivided into three subgroups Ft-IIIa, Ft-IIIb, and Ft-IIIc. Divergence time estimation indicated that the genera Fagopyrum and Rheum (rhubarb) shared a common ancestor about 48 million years ago (mya) and that intraspecies divergence in Tartary buckwheat began around 0.42 mya. CONCLUSIONS: The resolution of pan-plastome diversity in Tartary buckwheat provides an important resource for future projects such as marker-assisted breeding and germplasm preservation.

A Genomic Quantitative Study on the Contribution of the Ancestral-State Bases Relative to Derived Bases in the Divergence and Local Adaptation of Populus davidiana.

Identifying alleles associated with adaptation to new environments will advance our understanding of evolution from the molecular level. Previous studies have found that the Populus davidiana southwest population in East Asia has differentiated from other populations in the range. We aimed to evaluate the contributions of the ancestral-state bases (ASBs) relative to derived bases (DBs) in the local adaptation of P. davidiana in the Yunnan-Guizhou Plateau from a quantitative perspective using whole-genome re-sequencing data from 90 P. davidiana samples from three regions across the species range. Our results showed that the uplift of the Qinghai-Tibet Plateau during the Neogene and associated climate fluctuations during the Middle Pleistocene were likely an important factor in the early divergence of P. davidiana. Highly differentiated genomic regions between populations were inferred to have undergone strong linked natural selection, and ASBs are the chief means by which populations of P. davidiana adapt to novel environmental conditions; however, when adapting to regions with high environmental differences relative to the ancestral range, the proportion of DBs was significantly higher than that of background regions, as ASBs are insufficient to cope with these environments. Finally, a number of genes were identified in the outlier region.

Variation in mitogenome structural conformation in wild and cultivated lineages of sorghum corresponds with domestication history and plastome evolution.

BACKGROUND: Mitochondria are organelles within eukaryotic cells that are central to the metabolic processes of cellular respiration and ATP production. However, the evolution of mitochondrial genomes (mitogenomes) in plants is virtually unknown compared to animal mitogenomes or plant plastids, due to complex structural variation and long stretches of repetitive DNA making accurate genome assembly more challenging. Comparing the structural and sequence differences of organellar genomes within and between sorghum species is an essential step in understanding evolutionary processes such as organellar sequence transfer to the nuclear genome as well as improving agronomic traits in sorghum related to cellular metabolism. RESULTS: Here, we assembled seven sorghum mitochondrial and plastid genomes and resolved reticulated mitogenome structures with multilinked relationships that could be grouped into three structural conformations that differ in the content of repeats and genes by contig. The grouping of these mitogenome structural types reflects the two domestication events for sorghum in east and west Africa. CONCLUSIONS: We report seven mitogenomes of sorghum from different cultivars and wild sources. The assembly method used here will be helpful in resolving complex genomic structures in other plant species. Our findings give new insights into the structure of sorghum mitogenomes that provides an important foundation for future research into the improvement of sorghum traits related to cellular respiration, cytonuclear incompatibly, and disease resistance.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

Insights into the prey of Vespa mandarinia (Hymenoptera: Vespidae) in Washington state, obtained from metabarcoding of larval feces.

The northern giant hornet, Vespa mandarinia (Hymenoptera: Vespidae), was detected for the first time in North America in 2019. Four nests have since been located and removed in northwestern Washington State as part of an extensive survey and eradication program. This recent introduction into North America has prompted new research on the biology and ecology of V. mandarinia to help inform management strategies. In its native range, V. mandarinia is known to prey on a variety of insects including the economically important honey bee species Apis cerana and Apis mellifera. Although A. cerana has developed defense mechanisms against attack by V. mandarinia, A. mellifera have no such defenses and an entire hive can be quickly destroyed by only a few hornets. In North America the hornet has been observed foraging on paper wasps (Polistes dominula) and honey bees, but little else is known about prey use in its novel range. To address this knowledge gap, we employed a DNA metabarcoding approach to characterize species detected in larval feces collected from 3 of the 4 Washington V. mandarinia nests found to date. Sequences were recovered for 56 species across fourteen orders, of which 36 species were likely prey items and 20 were suspected inquilines. The most frequently detected species were other social Hymenoptera, with Dolichovespula maculata, P. dominula, and A. mellifera present in most samples. All of the species detected, except for A. mellifera, represent new prey records for V. mandarinia, with eight families of insects newly associated with giant hornets. These results suggest that V. mandarinia in Washington preys on an assortment of insects similar to those documented in its native range, and that this new invader has readily incorporated novel species into its foraging and diet.

CO1 barcodes resolve an asymmetric biphyletic clade for Diabrotica undecimpunctata subspecies and provide nucleotide variants for differentiation from related lineages using real-time PCR.

Diabrotica undecimpunctata is a multivoltine polyphagous beetle species that has long been documented as a significant agricultural pest throughout its native range in North America. This beetle can vector bacterial and viral plant pathogens that result in major losses to crops such as cucumber and soybean. Many countries outside the Americas treat D. undecimpunctata as a species of quarantine importance, while in the USA only the subspecies D. u. duodecimnotata is subject to quarantine, to prevent introduction from Mexico. Identification of D. undecimpunctata on the basis of morphology alone can be complicated given the use of conflicting characters in the description of some subspecific taxa. To better understand relationships among D. undecimpunctata subspecies and other related species, we sequenced mitochondrial cytochrome oxidase 1 (CO1) and nuclear internal transcribed spacer 2 (ITS2) DNA from individuals in different subspecific taxa and across different parts of the species range using museum samples and interceptions. When our data were combined with publicly available Diabrotica data, no pattern of divergence consistent with the currently recognized subspecific designations was found. In addition, we compared phylogenetic patterns in CO1 data from the congener D. virgifera to demonstrate the utility of mitochondrial data in resolving subspecies. From the CO1 data, a diagnostic real-time PCR assay was developed that could successfully identify all haplotypes within the large D. undecimpunctata clade for use in surveys and identification at ports of entry. These findings underscore the need to resolve molecular and morphological datasets into cogent, lineage-based groupings. Such efforts will provide an evolutionary context for the study of agriculturally important attributes of Diabrotica such as host preferences, xenobiotic metabolism, and natural and anthropogenic patterns of dispersal.