Patterns of genetic diversity of the cryptogenic red alga Polysiphonia morrowii (Ceramiales,Rhodophyta) suggest multiple origins of the Atlantic populations

The red alga Polysiphonia morrowii, native to the North Pacific (Northeast Asia), has recently been reported worldwide. To determine the origin of the French and Argentine populations of this introduced species, we compared samples from these two areas with samples collected in Korea and at Hakodate, Japan, the type locality of the species. Combined analyses of chloroplastic (rbcL) and mitochondrial (cox1) DNA revealed that the French and Argentine populations are closely related and differ substantially from the Korean and Japanese populations. The genetic structure of P. morrowii populations from South Atlantic and North Atlantic, which showed high haplotype diversity compared with populations from the North Pacific, suggested the occurrence of multiple introduction events from areas outside of the so‐called native regions. Although similar, the French and Argentine populations are not genetically identical. Thus, the genetic structure of these two introduced areas may have been modified by cryptic and recurrent introduction events directly from Asia or from other introduced areas that act as introduction relays. In addition, the large number of private cytoplasmic types identified in the two introduced regions strongly suggests that local populations of P. morrowii existed before the recent detection of these invasions. Our results suggest that the most likely scenario is that the source population(s) of the French and Argentine populations was not located only in the North Pacific and/or that P. morrowii is a cryptogenic species.     

Global distribution of cryptic native,introduced and hybrid lineages in the widespread estuarine amphipod Ampithoe valida

Biological invasions can pose a severe threat to coastal ecosystems, but are difficult to track due to inaccurate species identifications and cryptic diversity. Here, we clarified the cryptic diversity and introduction history of the marine amphipod Ampithoe valida by sequencing a mtDNA locus from 683 individuals and genotyping 10,295 single-nucleotide polymorphisms (SNPs) for 349 individuals from Japan, North America and Argentina. The species complex consists of three cryptic lineages: two native Pacific and one native Atlantic mitochondrial lineage. It is likely that the complex originated in the North Pacific and dispersed to the north Atlantic via a trans-arctic exchange approximately 3 MYA. Non-native A. valida in Argentina have both Atlantic mitochondrial and nuclear genotypes, strongly indicating an introduction from eastern North America. In two eastern Pacific estuaries, San Francisco Bay and Humboldt Bay, California, genetic data indicate human-mediated hybridization of Atlantic and Pacific sources, and possible adaptive introgression of mitochondrial loci, nuclear loci, or both. The San Francisco Bay hybrid population periodically undergoes population outbreaks and profoundly damages eelgrass Zostera marina thalli via direct consumption, and these ecological impacts have not been documented elsewhere. We speculate that novel combinations of Atlantic and Pacific lineages could play a role in A. valida’s unique ecology in San Francisco Bay. Our results reinforce the notion that we can over-estimate the number of non-native invasions when there is cryptic native structure. Moreover, inference of demographic and evolutionary history from mitochondrial loci may be misleading without simultaneous survey of the nuclear genome.

     

Population genetics of Haminoea (Haloa) japonica Pilsbry, 1895, a widespread non-indigenous sea slug (Mollusca: Opisthobranchia) in North America and Europe

Haminoea japonica is an opisthobranch mollusk with a large non-indigenous range. This species is a vector for a parasite that causes the human skin disease cercarial dermatitis, and may have negative effects on populations of native species. Molecular evidence from the mitochondrial cytochrome c oxidase I gene and the histone 3 nuclear gene indicates that previously published morphology-based hypotheses on the spread of H. japonica out of Japan are correct. The most likely explanation for the current range of the species, which includes Japan, Korea, France, Spain, Italy, Canada and the USA is a recent, human-mediated dispersal from Japanese populations. The highest levels of nucleotide and haplotype diversity are found in Japan. Non-indigenous populations have low levels of genetic diversity (indicating bottlenecking). Haplotypes that were detected in the non-indigenous range of H. japonica have only been found in two localities in the native range; these two localities are in north-eastern Japan. In addition, the haplotype network structure and Spatial Analysis of Molecular Variance results confirm the origins of non-indigenous populations most likely trace to north-eastern Japan, which is where most Pacific oyster exports to North America also originated. Because there are no major shipping ports in north-eastern Japan, ballast water is less likely to be the mechanism of dispersal. The results of this study provide important data for the development of policies and regulations aimed to prevent further spread of this species in non-indigenous ranges.     

Phylogeographic analysis of the brown alga Cutleria multifida (Tilopteridales,Phaeophyceae) suggests a complicated introduction history

In depth genetic comparisons of populations of Cutleria multifida (Tilopteridales, Phaeophyceae) collected from Europe, the northwestern Pacific Ocean, Australia and New Zealand using the DNA sequences of four gene regions (the mitochondrial cox2 and cox3 genes, the intergeneric spacer region adjacent to cox3, and the open reading frame) suggested that the northwestern European and Japanese populations were considerably greater in terms of their genetic divergence than Mediterranean, Australian or New Zealand populations. The haplotypes of the populations in northwestern European (distribution range including the type locality, seven haplotypes) and Japanese populations (seven haplotypes) were unique except for one shared haplotype. There were weak but positive correlations between the geographical distance and the genetic divergence among northwestern European and Japanese populations. Moreover, both female and male gametophytes occurred in eight of the nine Japanese localities, suggesting Japanese populations showed normal sexual heteromorphic life history of the species. In light of these results, it appears that Japanese populations were native to the area despite earlier hypothesis. In contrast, Australian and New Zealand populations were composed of only one haplotype that is very close to those found in northwestern Europe and Japan, suggesting a recent introduction history from Europe (or from northeastern Asia via Europe) by ship transport to Australia and New Zealand. The Mediterranean populations included two haplotypes identical to those found in northwestern Europe and Japan, and it is suggestive of transoceanic introductions of some populations between Mediterranean and Japanese coasts.     

Extensive Introgression of Mitochondrial DNA Found between two Genetically Divergent Forms of Threespine Stickleback, Gasterosteus Aculeatus, around Japan

The genetic structure and phylogenetic relationships of Gasterosteus aculeatus around Japan were investigated for 15 populations of the Japan Sea and Pacific Ocean forms previously detected by allozyme analyses. PCR-RFLP analysis of mtDNA ND5/6 gene indicated two major divergent clades: (1) Alaskan population and (2) Russian and Japanese populations. Japanese populations were clearly subdivided into G. aculeatus populations and hariyo (G. aculeatus leiurus). However, the relationships among Japanese G. aculeatus populations did not support the genetic divergence between Japan Sea and Pacific Ocean forms shown by allozyme analyses. This suggests that mtDNA genetype similarity between the two forms could have resulted from complete replacement of mtDNA from one form to the other. MtDNA introgression is possible in two direction: from Japan Sea form to Pacific Ocean form and the reverse. The haplotype composition of each population observed in the present study suggests that the second direction is more likely. The migrant number of females per generation (Nm) between populations of the two forms was remarkablely large (9.083–infinite), suggesting that gene flow is taking place between the populations at the present time or might have occurred until recent years.     

Four mutually incompatible Argentine ant supercolonies in Japan: inferring invasion history of introduced Argentine ants from their social structure

In recent years, highly invasive ant species successively invaded warm regions of Asia. In Japan, the Argentine ant, Linepithema humile, has become established in several coastal regions. This species forms unusual social organizations called supercolonies consisting of numerous mutually non-aggressive nests. We studied the behavioral relationships, similarity of cuticular hydrocarbon profiles (nestmate recognition cue), and genetic relationships among the introduced Argentine ant populations of Japan. The Japanese populations were divided into four behaviorally, chemically, and genetically distinct supercolonies, which may have derived from independent source populations. The result represents the recent trend of increasing invasions of invasive ants to Asia. The discontinuous distribution of one supercolony throughout most of the Japanese range suggests rapid expansion of the supercolony via human-mediated jump dispersal. Meanwhile, localization of the other three supercolonies in Kobe Port provides the first evidence for multiple invasions of distinct supercolonies into a base for international trade.     

Tri-oceanic connectivity of the supposedly cosmopolitan polychaete,Harmothoe imbricata (Annelida: Polynoidae): insights from the COI marker

ABSTRACT

In this study, we investigated the global population genetic structure of the polychaete, Harmothoe imbricata, to determine connectivity patterns within the species. We sequenced the mtDNA marker, cytochrome c oxidase I (COI) from 29 specimens sampled across three sites from the New England coast of the United States. These were supplemented with 145 archived sequences from GenBank and the Barcode of Life Database, representing 16 global populations, which encompassed three broadly defined marine biogeographic regions: the Atlantic, Pacific and Arctic. The resulting haplotype network and pairwise AMOVA results showed marked structure across all the major biogeographic regions and also provides evidence for cryptic diversity in the species. Haplotypes from Arctic populations were more closely related to each other than those from the northwestern and northeastern Atlantic. Two evolutionary divergent lineages were recovered from Los Angeles, California and Manitoba, Canada. The highest genetic diversity was observed in the Arctic populations, providing evidence for an Arctic origin for H. imbricata. While human-mediated introductions may have likely contributed to some of the genetic patterns observed in this study, future work should incorporate a nuclear DNA component which could shed more light on contemporary movement of this species across large spatial scales.     

Historical,human, and environmental drivers of genetic diversity in the red swamp crayfish (Procambarus clarkii) invading the Iberian Peninsula

1. Patterns of genetic diversity in invasive populations can be modulated by a range of factors acting at different stages of the invasion process, including the genetic composition of the source population(s), the introduction history (e.g. propagule pressure), the environmental suitability of recipient areas, and the features of secondary introductions.
2. The North American red swamp crayfish, Procambarus clarkii, is one of the most widely introduced freshwater species worldwide. It was legally introduced into Spain twice, near the city of Badajoz in 1973 and in the Guadalquivir marshes in 1974. Thereafter the species rapidly colonised almost the entire Iberian Peninsula.
3. We used seven nuclear microsatellites to describe the genetic diversity and structure of 28 locations distributed across the Iberian Peninsula and to explain the expansion process of the red swamp crayfish. Additionally, we analysed the relationship between environmental suitability and genetic diversity of the studied locations.
4. The red swamp crayfish had a clear spatial genetic structure in the Iberian Peninsula, probably determined by the two independent introduction events in the 1970s, which produced two main clusters separated spatially, one of which was dominant in Portugal and the other in Spain.
5. The human-mediated dispersal process seemed to have involved invasion hubs, hosting highly genetically diverse areas and acting as sources for subsequent introductions. Genetic diversity also tended to be higher in more suitable environments across the Iberian Peninsula .
6. Our results showed that the complex and human-mediated expansion of the red swamp crayfish in the Iberian Peninsula has involved several long- and short-distance movements and that both ecological and anthropogenic factors have shaped the genetic diversity patterns resulting from this invasion process. Early detection of potential invasion hubs may help to halt multiple short-distance translocations and thus the rapid expansion of highly prolific invasive species over non-native areas.

     

Mitochondrial DNA reveals multiple Northern Hemisphere introductions of Caprella mutica (Crustacea, Amphipoda)

Caprella mutica (Crustacea, Amphipoda) has been widely introduced to non-native regions in the last 40 years. Its native habitat is sub-boreal northeast Asia, but in the Northern Hemisphere, it is now found on both coasts of North America, and North Atlantic coastlines of Europe. Direct sequencing of mitochondrial DNA (cytochrome c oxidase subunit I gene) was used to compare genetic variation in native and non-native populations of C. mutica . These data were used to investigate the invasion history of C. mutica and to test potential source populations in Japan. High diversity (31 haplotypes from 49 individuals), but no phylogeographical structure, was identified in four populations in the putative native range. In contrast, non-native populations showed reduced genetic diversity (7 haplotypes from 249 individuals) and informative phylogeographical structure. Grouping of C. mutica populations into native, east Pacific, and Atlantic groups explained the most among-region variation (59%). This indicates independent introduction pathways for C. mutica to the Pacific and Atlantic coasts of North America. Two dominant haplotypes were identified in eastern and western Atlantic coastal populations, indicating several dispersal routes within the Atlantic. The analysis indicated that several introductions from multiple sources were likely to be responsible for the observed global distribution of C. mutica , but the pathways were least well defined among the Atlantic populations. The four sampled populations of C. mutica in Japan could not be identified as the direct source of the non-native populations examined in this study. The high diversity within the Japan populations indicates that the native range needs to be assessed at a far greater scale, both within and among populations, to accurately assess the source of the global spread of C. mutica .     

Population genetics of Himalayan balsam (Impatiens glandulifera): comparison of native and introduced populations

Background: Invasive species can interfere in the structure and functioning of ecosystems. Better understanding of the evolution of such species will be useful when planning their management and eradication.

Aims: We aimed to compare patterns of genetic variability in Impatiens glandulifera in native and introduced regions.

Methods: We used native samples from India and Pakistan, and non-native samples from Canada, Finland and the UK. Genetic analyses included genotyping using 10 microsatellite markers and sequencing of the nuclear ITS region.

Results: Mean allele numbers from native and introduced samples were even, 8.8 and 8.5, respectively, while expected heterozygosities were higher in native samples (mean 0.738) than in non-native samples (mean 0.477). Hardy–Weinberg equilibrium testing indicated significant heterozygote deficiencies at 70% of the loci. Inbreeding coefficients were high in both native and introduced regions (range 0.201–0.726). STRUCTURE analyses showed that native samples from India and Pakistan possessed similar clustering patterns while non-native samples from the UK and Canada resembled each other. One of the four Finnish populations had a similar pattern with the UK and Canadian populations, while the rest showed similarly unique genetic compositions. ITS sequencing indicated in Pakistani samples two polymorphic sites not found in Indian samples but present in some samples from Canada, Finland and the UK.

Conclusions: Distinct population genetic patterns indicate that human-mediated dispersal is important in I. glandulifera.     

Phylogeography of Asparagopsis taxiformis revisited: Combined mtDNA data provide novel insights into population structure in Japan

Six intraspecific lineages (Lineages 1–6) of Asparagopsis taxiformis have been previously established based on mitochondrial cox2‐cox3 intergenic spacer and a partial cox1 sequences. ‘Lineage 2’ (L2) was suggested to be a recent introduction to the Mediterranean Sea, but its source population has not yet been identified. In order to clarify the nature of northwestern Pacific populations, we performed extensive sampling in Japan (60 individuals from 16 locations) and molecular phylogenetic analyses based on mitochondrial sequences. Sixteen additional individuals, collected from eight locations in the Indo‐Pacific, Caribbean, and Mediterranean regions, were also analyzed. Combined sequence analyses revealed that the Japanese populations only consisted of L2. Out of 19 combined haplotypes identified within L2, two are shared between Japan and the Mediterranean Sea and the Hawaiian Islands, and 12 were identified as endemic to Japan. Genetic analyses of population differentiation suggested that Japanese populations are genetically isolated from the Mediterranean and the Hawaiian populations. A genetic disjunction appears to separate two subpopulations within Japan: one between Toi and Kagoshima and the other between Ojikajima Island and Kagoshima in the Kyushu area.     

Pathways of spread of the introduced ascidian Styela clava (Tunicata) in Northern Europe, as revealed by microsatellite markers

Styela clava, a solitary ascidian native to the NW Pacific, has become a conspicuous member of fouling communities in NW European waters. As its natural dispersal appears to be limited, the wide distribution of S. clava along coasts within its introduced range may be attributed to secondary spread assisted by human activities. Here, we used six microsatellite loci to examine the genetic diversity and extent of gene flow among S. clava populations in its European introduced range. Samples were collected from 21 populations within Europe (N = 808), 4 populations within the USA and two populations within the native range (Japan). Large variation in genetic diversity was observed among the European populations but were not explained either by the geographic distance from the first introduction area (i.e. Plymouth, UK) nor by the time elapsed since the introduction. No founder effect was observed in the introduced populations, except possibly in Puget Sound (USA). At least two different introductions occurred in Europe, identified as distinct genetic clusters: northern Danish populations (resembling one Japanese population), and the rest of Europe; a sample from Shoreham (England) possibly represents a third introduction. In North America, the population from the Atlantic was genetically similar to the majority of European populations, suggesting a European origin for populations on this seaboard, while populations from the Pacific coast were genetically similar to the same Japanese population as the Danish populations.     

Genetic diversity and relatedness in aquaculture and marina populations of the invasive tunicate Didemnum vexillum in the British Isles

Introductions of invasive, non-native species in the marine environment are increasing as human activity within coastal areas rises. Genetic datasets are useful tools to identify source populations, track routes of invasions, and illuminate the role of genetic variation in the establishment and subsequent spread of novel introductions. Here, a microsatellite dataset is used to estimate the genetic diversity and population structure of 7 introduced Didemnum vexillum populations in Britain and Ireland, 4 of which are associated with aquaculture and 3 with marinas. Genetic differentiation observed between these populations indicates human-mediated transport as the main mechanism underlying the population structure of D. vexillum in Britain and Ireland. In addition to elucidating patterns of population structure we found that aquaculture sites showed significantly higher genetic diversity (measured as allelic richness) in comparison to the marina sites. We discuss these findings in relation to the history of each invasion, the complex life history of D. vexillum, and available evidence of the relative invasiveness of these populations. Our results show numerous interesting patterns which highlight further research avenues to elucidate the complex factors underlying the global spread of this successful invader.

     

The presence of a cryptic barrier in the West Pacific Ocean suggests the effect of glacial climate changes on a widespread sea‐dispersed plant,Vigna marina (Fabaceae)

Ocean currents are an important driver of evolution for sea‐dispersed plants, enabling them to maintain reciprocal gene flow via sea‐dispersed diaspores and obtain wide distribution ranges. Although geographic barriers are known to be the primary factors shaping present genetic structure of sea‐dispersed plants, cryptic barriers which form clear genetic structure within oceanic regions are poorly understood. To test the presence of a cryptic barrier, we conducted a phylogeographic study together with past demographic inference for a widespread sea‐dispersed plant, Vigna marina, using 308 individuals collected from the entire Indo‐West Pacific (IWP) region. Chloroplast DNA variation showed strong genetic structure that separated populations into three groups: North Pacific (NP), South Pacific (SP) and Indian Ocean (IN) (F′_CT among groups = 0.954–1.000). According to the Approximate Bayesian computation inference, splitting time between NP and SP was approximately 20,200 years (95%HPD, 4,530–95,400) before present. Moreover, a signal of recent population expansion was detected in the NP group. This study clearly showed the presence of a cryptic barrier in the West Pacific region of the distributional range of V. marina. The locations of the cryptic barrier observed in V. marina corresponded to the genetic breaks found in other plants, suggesting the presence of a common cryptic barrier for sea‐dispersed plants. Demographic inference suggested that genetic structure related to this cryptic barrier has been present since the last glacial maximum and may reflect patterns of past population expansion from refugia.     

Genetic population structure of the masked palm civet Paguma larvata, (Carnivora: Viverridae) in Japan, revealed from analysis of newly identified compound microsatellites

The masked palm civet Paguma larvata (Carnivora: Viverridae) in Japan has been phylogeographically considered an introduced species from Taiwan. To reveal the population structures and relationships among the P. larvata populations in Japan, seven compound microsatellite loci were isolated from the genome and genotyped for 287 individuals collected from the field. STRUCTURE analysis and factorial correspondence analysis of genotyping data revealed that animals from Japan were divided into four genetic clusters. Geographic distribution of the genetic clusters partly referred to sampling areas, indicating multiple introductions into distinct areas of Japan or independent founding events leading to the generation of different genetic clusters within introduced populations in Japan. The large genetic differentiation of populations in the Shikoku District from those in other areas within Japan suggests that there were at least two introduction routes into Japan, and a possibility that some founders from areas other than Taiwan were also involved in the introduction into Japan. The genetic variation within Japanese populations were not markedly reduced compared with that of Taiwan. The results indicated that the Japanese populations of P. larvata could have retained moderate genetic diversity during founding events, because of multiple introductions, or a large number or high genetic diversity of founders. Although some individuals in Japan showed a sign of admixture between different clusters, there is no evidence that such an admixture markedly increased the genetic diversity within Japanese populations.     

Genetic divergence in wild populations of Medaka,Oryzias latipes (Pisces: Oryziatidae) from Japan and China

Allozyme studies of the cyprinodont fish Oryzias latipes have revealed that the wild populations of this species have differentiated regionally, and that Japanese wild populations are composed of two distinct groups, the Northern Population and the Southern Population. In order to elucidate the genetic relationship and the origin of the two major populations, allozymic characters of the specimens from 21 localities in Japan and China were compared. Although Chinese populations could be distinguished from Japanese ones, they were more closely related to the Southern Population, and showed few allozymic traits of the Northern Population. It is likely that the latter is a relic population which has survived the glacial period along the western coast of north-eastern Japan.     

Unexpectedly complex gradation of coral population structure in the Nansei Islands,Japan

To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence‐based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.     

Endangered freshwater limpets in Japan are actually alien invasive species

Currently, many species are facing the risk of extinction, as it one of the most serious conservation issues. Many conservation programs have evolved to prevent species extinction; however, developing these strategies may prove to be difficult for the species itself, which includes different cryptic species. In this paper, we document the invasion of non-native cryptic species of ancylin limpets, Ferrissia californica, with a molecular phylogenetic analysis. Three species (F. nipponica, F. japonica, Gundlachia japonica) have been described as native, whereas the present analysis shows that most of them are actually non-native F. californica, having been introduced from North America and spreading throughout Japan. In addition, we found a few subpopulations of the cryptic native species of the ancylin limpets, though the native limpet species is very rare, and has limited distribution. The conservation status assigned to ancylin limpets on the Japanese red list is problematic, because F. japonica is listed as an endangered species. All individuals identified as F. japonica are of F. californica, so the current red list treats invasive non-native species as endangered. Reassessment of the conservation status of native species and an estimation of the impact of non-native species is required to better delineate these limpet species by genetic markers.     

Genetically monomorphic invasive populations of the rapa whelk,Rapana venosa

Rapana venosa is a predatory marine gastropod native to the coastal waters of China, Korea, and Japan. Since the 1940s, R. venosa has been transported around the globe and introduced populations now exist in the Black Sea, the Mediterranean Sea, the Adriatic and Aegean seas, off the coasts of France and the Netherlands, in Chesapeake Bay, Virginia, USA, and in the Rio de la Plata between Uruguay and Argentina. This study surveyed variation in two mitochondrial gene regions to investigate the invasion pathways of R. venosa, identify likely sources for introduced populations, and evaluate current hypotheses of potential transportation vectors. Sequence data were obtained for the mitochondrial cytochrome c oxidase I and NADH dehydrogenase subunit 2 gene regions of 178 individuals from eight native locations and 106 individuals from 12 introduced locations. Collections from within the native range displayed very high levels of genetic variation while collections from all introduced populations showed a complete lack of genetic diversity; a single haplotype was common to all introduced individuals. This finding is consistent with the hypothesis that R. venosa was initially introduced into the Black Sea, and this Black Sea population then served as a source for the other secondary invasions by various introduction vectors including ballast water transport. Although non‐native R. venosa populations currently appear to be thriving in their new environments, the lack of genetic variability raises questions regarding the evolutionary persistence of these populations.     

Haplotype diversity of mt<Emphasis Type="Italic">COI</Emphasis> in the fall webworm <Emphasis Type="Italic">Hyphantria cunea</Emphasis> (Lepidoptera: Arctiidae) in introduced regions in China,Iran, Japan,Korea, and its homeland,the United States

Hyphantria cunea (Drury) has colonized many countries outside its native range of North America and has become a model species for studies of the colonization and subsequent adaptation of agricultural pests. Molecular genetic analyses can clarify the origin and subsequent adaptations to non-native habitats. Using the mitochondrial COI gene, we examined the genetic relationships between invasive populations (China, Iran, Japan, and Korea) and native populations (i.e., the United States). The Jilin (China) and Guilan (Iran) populations showed nine previously unknown haplotypes that differed from those found in the south–central United States, suggesting multiple colonization events and different regions of invasion. A dominant mtDNA haplotype in populations in the United States was shared by all of the populations investigated, suggesting that H. cunea with that haplotype have successfully colonized China, Iran, Japan, and Korea.