Genetic structure, admixture and invasion success in a Holarctic defoliator, the gypsy moth (Lymantria dispar, Lepidoptera: Erebidae).

Characterizing the current population structure of potentially invasive species provides a critical context for identifying source populations and for understanding why invasions are successful. Non-native populations inevitably lose genetic diversity during initial colonization events, but subsequent admixture among independently introduced lineages may increase both genetic variation and adaptive potential. Here we characterize the population structure of the gypsy moth (Lymantria dispar Linnaeus), one of the world's most destructive forest pests. Native to Eurasia and recently introduced to North America, the current distribution of gypsy moth includes forests throughout the temperate region of the northern hemisphere. Analyses of microsatellite loci and mitochondrial DNA sequences for 1738 individuals identified four genetic clusters within L. dispar. Three of these clusters correspond to the three named subspecies; North American populations represent a distinct fourth cluster, presumably a consequence of the population bottleneck and allele frequency change that accompanied introduction. We find no evidence that admixture has been an important catalyst of the successful invasion and range expansion in North America. However, we do find evidence of ongoing hybridization between subspecies and increased genetic variation in gypsy moth populations from Eastern Asia, populations that now pose a threat of further human-mediated introductions. Finally, we show that current patterns of variation can be explained in terms of climate and habitat changes during the Pleistocene, a time when temperate forests expanded and contracted. Deeply diverged matrilines in Europe imply that gypsy moths have been there for a long time and are not recent arrivals from Asia.

Concordant species delimitation from multiple independent evidence: A case study with the Pachytriton brevipes complex (Caudata: Salamandridae).

Mitochondrial DNA (mtDNA) sequence data are widely used to delimit species. However, owing to its strict maternal inheritance in most species, mtDNA tracks female dispersion and dispersal only. The accuracy of mtDNA-derived species delimitation is often not explicitly tested using other independent evidence, such as nuclear DNA (nDNA) data, morphological data, or ecological data. Because species are independent evolutionary lineages that can form testable hypotheses, we present a multi-evidence case study on species delimitation that combines statistical approaches with spatially explicit ecological analysis. Montane salamanders of the Pachytriton brevipes complex (Salamandridae) from southeastern China exhibit conservative morphology and variable color patterning that impede species diagnosis. Recent studies proposed splitting P. brevipes into four species based on deep mtDNA divergence but also found discordance between mtDNA and nDNA trees. In this study, we test evolutionary independence of these hypothesized species lineages using two coalescent-based Bayesian methods (Bayes factor and BP&P). Despite significant conflict between mtDNA gene tree and the species phylogeny, the results reinforce the inference of at least four species in the complex as opposed to the one species recognized for over 130 years. Correlative ecological niche modeling and statistical analysis of environmental data indicate that suitable habitats for each species are isolated by incompatible intervening lowland regions, so the likelihood of gene flow among species is very low, which means species lineages should maintain their evolutionary independence. We demonstrate that concordance among independent evidence confirms species status, which forms the basis for accurate assessment of regional biodiversity.

Significance of pre-Quaternary climate change for montane species diversity: insights from Asian salamanders (Salamandridae: Pachytriton).

Despite extensive focus on the genetic legacy of Pleistocene glaciation, impacts of earlier climatic change on biodiversity are poorly understood. Because amphibians are highly sensitive to variations in precipitation and temperature, we use a genus of Chinese montane salamanders (Salamandridae: Pachytriton) to study paleoclimatic change in East Asia, which experienced intensification of its monsoon circulation in the late Miocene associated with subsequent Pliocene warming. Using both nuclear and mitochondrial DNA sequences, we reconstruct the species tree under a coalescent model and demonstrate that all major lineages originated before the Quaternary. Initial speciation within the genus occurred after the summer monsoon entered a stage of substantial intensification. Heavy summer precipitation established temporary water connectivity through overflows between adjacent stream systems, which may facilitate geographic range expansion by aquatic species such as Pachytriton. Species were formed in allopatry likely through vicariant isolation during or after range expansion. To evaluate the influence of Pliocene warming on these cold-adapted salamanders, we construct a novel temperature buffer-zone model, which suggests widespread physiological stress or even extinction during the warming period. A significant deceleration of species accumulation rate is consistent with Pliocene range contraction, which affected P. granulosus and P. archospotus the most because they lack large temperature buffer zones. In contrast, demographic growth occurred in species for which refugia persist. The buffer-zone model reveals the Huangshan Mountain as a potential climatic refugium, which is similar to that found for other East Asian organisms. Our approach can incorporate future climatic data to evaluate the potential impact of ongoing global warming on montane species (particularly amphibians) and to predict possible population declines.

Range-wide population genomics of the spongy moth, Lymantria dispar (Erebidae): Implications for biosurveillance, subspecies classification and phylogeography of a destructive moth.

The spongy moth, Lymantria dispar, is an irruptive forest pest native to Eurasia where its range extends from coast to coast and overspills into northern Africa. Accidentally introduced from Europe in Massachusetts in 1868-1869, it is now established in North America where it is considered a highly destructive invasive pest. A fine-scale characterization of its population genetic structure would facilitate identification of source populations for specimens intercepted during ship inspections in North America and would enable mapping of introduction pathways to help prevent future incursions into novel environments. In addition, detailed knowledge of L. dispar's global population structure would provide new insight into the adequacy of its current subspecies classification system and its phylogeographic history. To address these issues, we generated >2000 genotyping-by-sequencing-derived SNPs from 1445 contemporary specimens sampled at 65 locations in 25 countries/3 continents. Using multiple analytical approaches, we identified eight subpopulations that could be further partitioned into 28 groups, achieving unprecedented resolution for this species' population structure. Although reconciliation between these groupings and the three currently recognized subspecies proved to be challenging, our genetic data confirmed circumscription of the japonica subspecies to Japan. However, the genetic cline observed across continental Eurasia, from L. dispar asiatica in East Asia to L. d. dispar in Western Europe, points to the absence of a sharp geographical boundary (e.g., the Ural Mountains) between these two subspecies, as suggested earlier. Importantly, moths from North America and the Caucasus/Middle East displayed high enough genetic distances from other populations to warrant their consideration as separate subspecies of L. dispar. Finally, in contrast with earlier mtDNA-based investigations that identified the Caucasus as L. dispar's place of origin, our analyses suggest continental East Asia as its evolutionary cradle, from where it spread to Central Asia and Europe, and to Japan through Korea.

Development of an array of molecular tools for the identification of khapra beetle (Trogoderma granarium), a destructive beetle of stored food products.

Trogoderma granarium Everts, the khapra beetle, native to the Indian subcontinent, is one of the world's most destructive pests of stored food products. Early detection of this pest facilitates prompt response towards the invasion and prevents the need for costly eradication efforts. Such detection requires proper identification of T. granarium, which morphologically resembles some more frequently encountered, non-quarantine congeners. All life stages of these species are difficult to distinguish using morphological characters. Additionally, biosurveillance trapping can result in the capture of large numbers of specimens awaiting identification. To address these issues, we aim to develop an array of molecular tools to rapidly and accurately identify T. granarium among non-target species. Our crude, cheap DNA extraction method performed well for Trogoderma spp. and is suitable for downstream analyses including sequencing and real-time PCR (qPCR). We developed a simple quick assay usingrestriction fragment length polymorphism to distinguish between T. granarium and the closely related, congeneric T. variabile Ballion and T. inclusum LeConte. Based on newly generated and published mitochondrial sequence data, we developed a new multiplex TaqMan qPCR assay for T. granarium with improved efficiency and sensitivity over existing qPCR assays. These new tools benefit regulatory agencies and the stored food products industry by providing cost- and time-effective solutions to enhance the identification of T. granarium from related species. They can be added to the existing pest detection toolbox. The selection of which method to use would depend on the intended application.

Direct competition and potential displacement involving managed Trogoderma stored product pests.

The establishment of an exotic pest may require displacing local species with a similar niche. The potential of Trogoderma granarium to displace Trogoderma inclusum was explored in a stored product setting. We performed direct competition experiments varying commodity and temperature over different durations. At nine weeks T. inclusum outproduced T. granarium on all commodities at any temperature. However the proportion of T. granarium versus T. inclusum was greater at 32 °C compared to 25 °C. The nine-week production of T. granarium was best on wheat, while rice was optimal for T. inclusum. After 25 weeks, when adults were used at the start of competition, T. inclusum maintained an advantage in the direct competition. If larvae were used to initiate the competition for 25 weeks, the two species coexisted well at 25 °C, but T. granarium nearly excluded T. inclusum at 32 °C. Thus T. inclusum performs better in competition over shorter intervals when resources are plentiful, but T. granarium can be more successful over longer time periods, particularly when late instar larvae are involved. The finding suggests a real threat of introductions of T. granarium larvae to establish populations within grain storage infrastructure where T. inclusum is common.

Cryptic genetic diversity and associated ecological differences of Anastatus orientalis, an egg parasitoid of the spotted lanternfly.

Anastatus orientalis, native to northern China, is an egg parasitoid wasp of the spotted lanternfly (Lycorma delicatula) and is being tested as a potential biological control agent for invasive L. delicatula in the United States. As a component of these evaluations, live A. orientalis collected from Beijing and Yantai in China were reared in containment in the U.S. These specimens showed different responses in diapause behaviors to rearing conditions used previously by other researchers. To understand the primary mechanism potentially driving discrepancies in important life history traits, we used molecular tools to examine the genetic composition of A. orientalis from China and from South Korea, where the parasitoid has been introduced to aid in the population management of invasive L. delicatula. Molecular analysis of mitochondrial DNA recovered six haplotype groups, which exhibit biased frequency of abundance between collection sites. Some haplotypes are widespread, and others only occur in certain locations. No apparent pattern is observed between wasps collected from different years or emergence seasons. Uncorrected genetic distances between haplotype groups range from 0.44% to 1.44% after controlling for within-group variation. Genetic variance of A. orientalis is characterized by high levels of local diversity that contrasts with a lack of a broad-scale population structure. The introduced Korean population exhibits lower genetic diversity compared to native populations. Additionally, we created iso-female lines for major haplotype groups through laboratory rearing. Differences in diapause behavior were correlated with mitochondrial haplotype. Our results indicate that the observed life history traits in A. orientalis have a genetic base.

Assessing the host range of Anastatus orientalis, an egg parasitoid of spotted lanternfly (Lycorma delicatula) using Eastern U.S. non-target species.

The spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae), an invasive planthopper discovered in Pennsylvania, U.S. in 2014, has spread to many surrounding states despite quarantines and control efforts, and further spread is anticipated. A classical (importation) biological control program would contribute to the long-term management of L. delicatula in the eastern U.S. In its native range of China, Anastatus orientalis (Hymenoptera: Eupelmidae), an egg parasitoid, causes significant mortality. Anastatus orientalis consists of multiple haplotypes that differ in important biological parameters. To delineate the physiological host range of A. orientalis Haplotype C, we completed no-choice and choice testing. No-choice testing of non-target eggs from 36 insect species spanning six orders and 18 families showed that physiologically this haplotype of A. orientalis can develop in a variety of host species eggs from the families Coreidae, Fulgoridae, Pentatomidae, and Saturniidae. Ten of the 16 species that were attacked in the no-choice tests were also attacked in the choice tests. The production of progeny on non-target egg masses was significantly lower than on the controls (L. delicatula egg masses run simultaneously) in the no-choice and choice tests. For the non-target species that were attacked and resulted in female wasp progeny, these females were able to produce their own progeny at the same rate as control females that were reared from the L. delicatula eggs. Larger host eggs corresponded to an increased female-biased sex ratio of the progeny, suggesting that gravid females select them for fertilized eggs. Results from these studies suggest that A. orientalis Haplotype C prefers to parasitize L. delicatula egg masses but is capable of developing in some non-target species.

Tracking global invasion pathways of the spongy moth (Lepidoptera: Erebidae) to the United States using stable isotopes as endogenous biomarkers.

The spread of invasive insect species causes enormous ecological damage and economic losses worldwide. A reliable method that tracks back an invaded insect's origin would be of great use to entomologists, phytopathologists, and pest managers. The spongy moth (Lymantria dispar, Linnaeus 1758) is a persistent invasive pest in the Northeastern United States and periodically causes major defoliations in temperate forests. We analyzed field-captured (Europe, Asia, United States) and laboratory-reared L. dispar specimens for their natal isotopic hydrogen and nitrogen signatures imprinted in their biological tissues (δ2H and δ15N) and compared these values to the long-term mean δ2H of regional precipitation (Global Network of Isotopes in Precipitation) and δ15N of regional plants at the capture site. We established the percentage of hydrogen-deuterium exchange for L. dispar tissue (Pex = 8.2%) using the comparative equilibration method and two-source mixing models, which allowed the extraction of the moth's natal δ2H value. We confirmed that the natal δ2H and δ15N values of our specimens are related to the environmental signatures at their geographic origins. With our regression models, we were able to isolate potentially invasive individuals and give estimations of their geographic origin. To enable the application of these methods on eggs, we established an egg-to-adult fraction factor for L. dispar (Δegg-adult = 16.3 ± 4.3‰). Our models suggested that around 25% of the field-captured spongy moths worldwide were not native in the investigated capture sites. East Asia was the most frequently identified location of probable origin. Furthermore, our data suggested that eggs found on cargo ships in the United States harbors in Alaska, California, and Louisiana most probably originated from Asian L. dispar in East Russia. These findings show that stable isotope biomarkers give a unique insight into invasive insect species pathways, and thus, can be an effective tool to monitor the spread of insect pest epidemics.

Comparative multi-locus assessment of modern Asian newts ( Cynops, Paramesotriton, and Pachytriton: Salamandridae) in southern China suggests a shared biogeographic history.

Evolutionary biologists are always interested in deciphering the geographic context of diversification, therefore they introduced the concept of comparative phylogeography, which helps to identify common mechanisms that contribute to shared genetic structures among organisms from the same region. Here, we used multi-locus genetic data along with environmental data to investigate shared phylogeographic patterns among three Asian-endemic newt genera, Cynops, Paramesotriton and Pachytriton, which occurred in montane/submontane streams or ponds in southern China. Our 222 samples from 78 localities covered the entire range of the three genera and represented the largest dataset of this group to date. We reconstructed matrilineal genealogies from two protein-coding, mitochondrial genes, and gene network from two nuclear genes. We also estimated divergence times of major cladogenetic events and used occurrence data to evaluate niche difference and similarity between lineages. Our results revealed a common basal split in all three genera that corresponds to the separation of two geographic terrains of southern China. Those ancient divergence occurred during middle to late Miocene and likely correlate with paleoclimatic fluctuations caused by the uplift of the Qinghai-Xizang (Tibet) Plateau (QTP). Particularly, the strengthening and weakening of Asian summer monsoons during the Miocene may have profoundly impacted southern China and led to repeatedly vicariance in those newts. However, despite differences in realized niches between lineages, there is no evidence for divergence of fundamental niches. Preservation of old newt matriline lineages in mountains of southern China suggests that the region acts as both museums and cradles of speciation. Based on those results, we advocate a multi-pronged protection strategy for newts in the three genera.

Genome-scale phylogeography resolves the native population structure of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky).

Human-assisted movement has allowed the Asian longhorned beetle (ALB, Anoplophora glabripennis (Motschulsky)) to spread beyond its native range and become a globally regulated invasive pest. Within its native range of China and the Korean peninsula, human-mediated dispersal has also caused cryptic translocation of insects, resulting in population structure complexity. Previous studies used genetic methods to detangle this complexity but were unable to clearly delimit native populations which is needed to develop downstream biosurveillance tools. We used genome-wide markers to define historical population structure in native ALB populations and contemporary movement between regions. We used genotyping-by-sequencing to generate 6102 single-nucleotide polymorphisms (SNPs) and amplicon sequencing to genotype 53 microsatellites. In total, we genotyped 712 individuals from ALB's native distribution. We observed six distinct population clusters among native ALB populations, with a clear delineation between northern and southern groups. Most of the individuals from South Korea were distinct from populations in China. Our results also indicate historical divergence among populations and suggest limited large-scale admixture, but we did identify a restricted number of cases of contemporary movement between regions. We identified SNPs under selection and describe a clinal allele frequency pattern in a missense variant associated with glycerol kinase, an important enzyme in the utilization of an insect cryoprotectant. We further demonstrate that small numbers of SNPs can assign individuals to geographic regions with high probability, paving the way for novel ALB biosurveillance tools.

Tracking invasions of a destructive defoliator, the gypsy moth (Erebidae: Lymantria dispar): Population structure, origin of intercepted specimens, and Asian introgression into North America.

Genetic data can help elucidate the dynamics of biological invasions, which are fueled by the constant expansion of international trade. The introduction of European gypsy moth (Lymantria dispar dispar) into North America is a classic example of human-aided invasion that has caused tremendous damage to North American temperate forests. Recently, the even more destructive Asian gypsy moth (mainly L. d. asiatica and L. d. japonica) has been intercepted in North America, mostly transported by cargo ships. To track invasion pathways, we developed a diagnostic panel of 60 DNA loci (55 nuclear and 5 mitochondrial) to characterize worldwide genetic differentiation within L. dispar and its sister species L. umbrosa. Hierarchical analyses supported strong differentiation and recovered five geographic groups that correspond to (1) North America, (2) Europe plus North Africa and Middle East, (3) the Urals, Central Asia, and Russian Siberia, (4) continental East Asia, and (5) the Japanese islands. Interestingly, L. umbrosa was grouped with L. d. japonica, and the introduced North American population exhibits remarkable distinctiveness from contemporary European counterparts. Each geographic group, except for North America, shows additional lower-level structures when analyzed individually, which provided the basis for inference of the origin of invasive specimens. Two assignment approaches consistently identified a coastal area of continental East Asia as the major source for Asian invasion during 2014-2015, with Japan being another source. By analyzing simulation and laboratory crosses, we further provided evidence for the occurrence of natural Asian-North American hybrids in the Pacific Northwest, raising concerns for introgression of Asian alleles that may accelerate range expansion of gypsy moth in North America. Our study demonstrates how genetic data contribute to bio-surveillance of invasive species with results that can inform regulatory management and reduce the frequency of trade-associated invasions.

Identification of Tree Genera Used in the Construction of Solid Wood-Packaging Materials That Arrived at U.S. Ports Infested With Live Wood-Boring Insects.

Although international regulations have been successfully implemented to reduce the introduction and spread of plant pests through wood packaging material (WPM), wood-boring insects continue to be intercepted in WPM at U.S. ports of entry. Both hardwoods and softwoods are used in the construction of WPM for international trade; however, it is not clear if some types of wood pose higher risks than others for harboring wood borers. This study documented the taxonomic diversity of infested wood genera intercepted as a result of targeted WPM inspection at U.S. ports, and identified many of the wood-boring insects transported within them. The results of this study reveal associations among packaging woods, commodities, and shipment origins. The wood genera most frequently infested were Pinus Linnaeus (Pinales: Pinaceae), Picea Miller (Pinales: Pinaceae), and Populus Linnaeus (Malpighiales: Salicaceae), which were heavily represented as packaging for commodities such as stone, metal, vehicles, and machinery. In addition to these results, we summarized preferences by the wood borers to develop in living, stressed, dying, or dead hosts, the pest status of intercepted wood borers in their native and non-native ranges, and potential host range of intercepted wood borers to gauge potential for these taxa to become pests in North America. New possible host associations are reported for eight wood borer taxa. Taxonomy of host wood is presented as a new factor for consideration in pathway-level risk analysis of WPM, and the findings further reinforce the need for enhanced compliance with ISPM 15 to reduce entry of non-native wood-boring insects.

The tenth member of stout newt (Amphibia: Salamandridae: Pachytriton): Description of a new species from Guangdong, southern China.

Field surveys of less-explored forests often lead to discoveries of new amphibian species. During a series of field surveys in early 2017 at Mt. Lianhua (the Lotus Mountain) of Lianhuashan-Baipenzhu Provincial Nature Reserve in southern China, we found an undocumented population of stout newt, genus Pachytriton. Based on its distinct morphological characters and large genetic divergence from congeners (uncorrected p-distance > 5% in either mitochondrial nd2 or cytb sequences), we describe this population as the tenth species of the genus: Pachytriton airobranchiatus sp. nov. It can be distinguished from other members of Pachytriton by a combination of the following morphological characters: prominently elevated distal end of the epibranchial bone showing as two conspicuous protruding bulges behind the head; dorsal coloration can be either spotted or unspotted; bright orange dots absent on dorsum; costal grooves absent between axilla and groin; fingers I and toe I slender, do not forms a volar pad; dorsal tips of fingers and toes brown; and tips of fore- and hind limbs could contact when limbs adpressed against body flank. Phylogenetic analysis grouped the new species with P. wuguanfui, P. xanthospilos, and P. changi. Currently, the new species is known from the type locality and an adjacent mountain peak at about 1000 m elevation. It inhabits small montane streams in broadleaf forests near the top of the mountain.

Identification of wood-boring beetles (Cerambycidae and Buprestidae) intercepted in trade-associated solid wood packaging material using DNA barcoding and morphology.

Global trade facilitates the inadvertent movement of insect pests and subsequent establishment of populations outside their native ranges. Despite phytosanitary measures, nonnative insects arrive at United States (U.S.) ports of entry as larvae in solid wood packaging material (SWPM). Identification of wood-boring larval insects is important for pest risk analysis and management, but is difficult beyond family level due to highly conserved morphology. Therefore, we integrated DNA barcoding and rearing of larvae to identify wood-boring insects in SWPM. From 2012 to 2015, we obtained larvae of 338 longhorned beetles (Cerambycidae) and 38 metallic wood boring beetles (Buprestidae) intercepted in SWPM associated with imported products at six U.S. ports. We identified 265 specimens to species or genus using DNA barcodes. Ninety-three larvae were reared to adults and identified morphologically. No conflict was found between the two approaches, which together identified 275 cerambycids (23 genera) and 16 buprestids (4 genera). Our integrated approach confirmed novel DNA barcodes for seven species (10 specimens) of woodborers not in public databases. This study demonstrates the utility of DNA barcoding as a tool for regulatory agencies. We provide important documentation of potential beetle pests that may cross country borders through the SWPM pathway.

A new species of the genus Paramesotriton (Caudata: Salamandridae) from Fujian, southeastern China.

New amphibian species have been constantly discovered throughout southern China, including from areas close to heavily populated cities that remain poorly surveyed for amphibian diversity. We describe a new species of the newt genus Paramesotriton from Fujian, a developed province on the southeastern coast of mainland China. The mitochondrial genealogy suggests that the new species is the sister taxon to Paramesotriton hongkongensis, separated by an uncorrected pairwise distances of 5.8% at the ND2 gene fragment analyzed. In addition to the genetic divergence, the new species can be readily differentiated from its congeners by having: a very rough skin; a continuous, orange, vertebral ridge; few warts on each side of the vertebral ridge; numerous small irregular orange-red or yellow spots on the chin, venter, underside of axillae, flanks, lateral side of the tail, base of limbs and cloaca; a small groove at the base of the vomerine tooth series; relatively long tail, relatively flat cloaca in females; normally developed eyes, and the absence of vestigial gills and gill filaments in adults.

A missing geographic link in the distribution of the genus Echinotriton (Caudata: Salamandridae) with description of a new species from southern China.

Disjunct geographic distribution of a species or a group of species is the product of long-term interaction between organisms and the environment. Filling the distributional gap by discovery of a new population or a species has significant biogeographic implications, because it suggests a much wider past distribution and provides evidence for the route of range expansion/contraction. The salamandrid genus Echinotriton (commonly known as spiny salamanders, spiny newts, or crocodile newts) has two species that are restricted to two widely separated areas, one in eastern Zhejiang province, China and the other in the Ryukyu Archipelago of Japan. It has been hypothesized that Echinotriton was once continuously distributed between the two areas through a historical land bridge that connected mainland China, Taiwan, and the archipelago. Finding fossils or relic populations along the postulated distribution are strong evidence for the hypothesis. Hundred-twenty-two years after the description of E. andersoni and eight-one years after that of E. chinhaiensis, we discover a third species of Echinotriton in southern China, which fills the distributional gap of the former two species. Species status of the new species is confirmed through molecular phylogenetic analysis and morphological comparison. Mitochondrial DNA indicates that the new species is sister to E. chinhaiensis, while nuclear DNA does not support this relationship. The new species has a very large quadrate projection, a single line of lateral warts pierced by distal rib extremities, normally developed 5th toes, and conical skin tubercles. Our discovery supports the hypothesis that there was a continuous distribution of Echinotriton from eastern coastal China to the Ryukyu Archipelago. We suggest that other species of this genus may also be found in Taiwan. Due to the rarity of this new species, we urge all hobbyists to refrain themselves from collecting this salamander or leaking locality information if encountered, and boycott any trading.