Native Range Populations Research Articles

The seeds of invasion: a comparison of endophytic seed bacteria of Acacia longifolia between its native and invasive ranges

Acacia longifolia is an aggressive invader in Mediterranean-type ecosystems severely impacting biodiversity and ecosystem functions. The species’ invasiveness has been linked to its ability to thrive in nutrient poor soils, high seed production, and quick establishment after fire. In this study, we identify and compare the bacterial endophytes of A. longifolia seeds collected from populations in the species’ native (Australia) and invasive (Portugal) ranges. For this, we characterised the morphology (length, width, and weight) of seeds from two sites in each range and isolated and cultivated bacteria from seeds. DNA fingerprinting and cluster analyses revealed slightly higher, and distinct, bacterial diversity associated with seeds collected from native range populations in comparison to those collected from invasive populations. Sequencing of the 16S rDNA gene identified 119 bacterial isolates from 15 genera, with Curtobacterium strains being common in both ranges. Several differences in bacterial genera were found among ranges and sites: Dermacoccus, Frigoribacterium, Kocuria, Pantoea and Phyllobacterium taxa were each unique to seeds from the native populations, while Brevundimonas, Microbacterium, Rhizobium and Sphingomonas taxa were only found in the invasive seeds. The genus Paraburkholderia occurred in all invasive-range seeds but was not isolated from the native-range. Bacillus and Paenibacillus co-occurred in seeds collected from all invaded sites, but the simultaneous presence of both taxa was not found in native-range seeds. We propose that the bacterial endophytes present in invasive-range seeds may be important players for the invasiveness of A. longifolia, due to their role as plant growth promoters, providing extra capabilities helping acacia expansion.

Post-invasion selection acts on standing genetic variation despite a severe founding bottleneck

Invasive populations often have lower genetic diversity relative to the native-range populations from which they derive.1,2 Despite this, many biological invaders succeed in their new environments, in part due to rapid adaptation.3,4,5,6 Therefore, the role of genetic bottlenecks in constraining the adaptation of invaders is debated.7,8,9,10 Here, we use whole-genome resequencing of samples from a 10-year time-series dataset, representing the natural invasion of the Asian honey bee (Apis cerana) in Australia, to investigate natural selection occurring in the aftermath of a founding event. We find that Australia's A.cerana population was founded by as few as one colony, whose arrival was followed by a period of rapid population expansion associated with an increase of rare variants.11 The bottleneck resulted in a steep loss of overall genetic diversity, yet we nevertheless detected loci with signatures of positive selection during the first years post-invasion. When we investigated the origin of alleles under selection, we found that selection acted primarily on the variation introduced by founders and not on the variants that arose post-invasion by mutation. In all, our data highlight that selection on standing genetic variation can occur in the early years post-invasion, even where founding bottlenecks are severe.

Niche unfilling dominates the naturalization of species from intercontinentally disjunct genera

AbstractAimQuantifying niche shifts of non‐native species and their interactions with natives is vital in evaluating invasion risk yet remains difficult. Climate filtering and competition with native congeners are two important processes impacting niche changes and yielding contradictory predictions for the coexistence of non‐native and native species. Closely related species representing the Eastern Asia (EA)–Eastern North America (ENA) floristic disjunction provide a powerful yet unexplored system for assessing the relative roles of these two processes. Here, we tested three niche change scenarios (unfilling, expansion and stable) using EA species naturalized in ENA. We aim to answer: (1) which scenario is observed for naturalized species? and (2) how has niche overlap between EA and ENA congeners changed after naturalization?LocationENA and EA.Time PeriodPresent.Major Taxa StudiedAngiosperms.MethodsNiches were estimated using both occurrences and predictions generated by ecological niche models. Niche changes of naturalized species and their niche overlap with natives were measured using Schoener's D. Contributions of niche expansion and unfilling on niche changes of naturalized niches were evaluated using hierarchical partitioning and linear regressions. We further measured pairwise phylogenetic distances among EA and ENA congeners and evaluated the association with niche overlap.ResultsUnfilling‐dominated postintroduction niche changes of the naturalized species. EA species naturalized in ENA exhibited lower niche overlap with ENA native congeners compared with native range populations in EA. These results remain when considering time since introduction and potential physical dispersal limitations interpreted by projecting suitable niche space between EA and ENA. However, dispersal limitation may contribute to the degree of niche unfilling.Main ConclusionsUsing intercontinental disjunct species as a study system, we found that naturalization is shaped jointly by climate filtering and competition with native congeners, providing valuable insights for evaluating invasion risk underlying global floristic exchanges.

From Alien Species to Alien Communities: Host- and Habitat-Associated Microbiomes in an Alien Amphibian

Alien species can host diverse microbial communities. These associated microbiomes may be important in the invasion process and their analysis requires a holistic community-based approach. We analysed the skin and gut microbiome of Eleutherodactylus johnstonei from native range populations in St Lucia and exotic range populations in Guadeloupe, Colombia, and European greenhouses along with their respective environmental microbial reservoir through a 16S metabarcoding approach. We show that amphibian-associated and environmental microbial communities can be considered as meta-communities that interact in the assembly process. High proportions of bacteria can disperse between frogs and environment, while respective abundances are rather determined by niche effects driven by the microbial community source and spatial environmental properties. Environmental transmissions appeared to have higher relevance for skin than for gut microbiome composition and variation. We encourage further experimental studies to assess the implications of turnover in amphibian-associated microbial communities and potentially invasive microbiota in the context of invasion success and impacts. Within this novel framework of “nested invasions,” (meta-)community ecology thinking can complement and widen the traditional perspective on biological invasions.

Genetic relationships among laboratory lines of the egg parasitoid Trissolcus japonicus from native and adventive populations

Candidate biological control agents of invasive insect pests are increasingly being found in new geographic regions as a result of unintentional introductions. However, testing the degree of genetic differentiation among adventive and native-range populations of these agents is rarely done. We used reduced-representation sequencing of genomic DNA to investigate the relationships among laboratory lines of Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae), an egg parasitoid and biological control agent of the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera, Pentatomidae). We compared sequences from multiple adventive populations in North America (Canada, USA) and Europe (Switzerland) with populations sourced from part of its native range in China. We found considerably more genetic variation among lines sourced from adventive populations than among those within native populations. In the Pacific Northwest of North America (British Columbia, Canada and Washington State, USA), we found preliminary evidence of three distinct genetic clusters, two of which were highly dissimilar from all other lines we genotyped. In contrast, we found that other adventive lines with close geographic proximity (two from Ontario, Canada, three from Switzerland) had limited genetic variation. These findings provide a basis for testing biological differences among lines that will inform their use as biological control agents, and provide evidence to support a hypothesis of several independent introductions of T. japonicus in western North America from different source areas.

Genetic analyses reveal a complex introduction history of the globally invasive tree Acacia longifolia

Acacia longifolia (Sydney golden wattle) is considered one of the most problematic plant invaders in Mediterranean-type ecosystems. In this study, we investigate the species’ invasion history by comparing the genetic diversity and structure of native (Australia) and several invasive range (Brazil, Portugal, South Africa, Spain, and Uruguay) populations and by modelling different introduction scenarios using these data. We sampled 272 A. longifolia individuals – 126 from different invasive ranges and 146 from the native range – from 41 populations. We genotyped all individuals at four chloroplast and 12 nuclear microsatellite markers. From these data we calculated diversity metrics, identified chloroplast haplotypes, and estimated population genetic structure based on Bayesian assignment tests. We used Approximate Bayesian Computation (ABC) models to infer the likely introduction history into each invaded country. In Australia, population genetic structure of A. longifolia appears to be strongly shaped by the Bass Strait and we identified two genetic clusters largely corresponding to mainland Australian and Tasmanian populations. We found invasive populations to represent a mixture of these clusters. Similar levels of genetic diversity were present in native and invasive ranges, indicating that invasive populations did not go through a genetic bottleneck. Bayesian assignment tests and chloroplast haplotype frequencies further suggested a secondary introduction event between South Africa and Portugal. However, ABC analyses could not confidently identify the native source(s) of invasive populations in these two countries, probably due to the known high propagule pressure that accompanied these introductions. ABC analyses identified Tasmania as the likely source of invasive populations in Brazil and Uruguay. A definitive native source for Spanish populations could also not be identified. This study shows that tracing the introduction history of A. longifolia is difficult, most likely because of the complexity associated with the extensive movement of the species around the world. Our findings should be considered when planning management and control efforts, such as biological control, in some invaded regions.

Individual vocal signatures show reduced complexity following invasion

The manner in which vocal learning is used for social recognition may be sensitive to the social environment. Biological invaders capable of vocal learning are useful for testing this possibility, as invasion alters population size. Some vocal learning species use frequency modulation patterns of acoustic signals for individual recognition. In such species, frequency modulation patterns should be more complex in larger social groups, reflecting greater selection for individual distinctiveness. We used numbers of nests and nest densities as proxies of local population sizes of native range monk parakeets, Myiopsitta monachus , in Uruguay and invasive range populations in the United States. Flock sizes were obtained to estimate maximum social group sizes per range. Supervised machine learning and frequency contours were employed to compare contact call structure between native and invasive range populations, and the effect of urban habitats on call structure was also assessed. Invasive range populations exhibited fewer nests, lower nest densities and smaller maximum flock sizes, which is consistent with a reduction in population size following invasion. Parakeets at invasive range sites also produced contact calls with simpler frequency modulation patterns. Beecher's statistic (HS) revealed reduced individual identity content and fewer possible unique individual signatures in invasive range calls. Simpler individual signatures are consistent with relaxed selection on the complexity of learned calls likely used for individual vocal recognition in the smaller local populations that we identified post-invasion. Frequency modulation patterns were simpler in urban habitats in both ranges, indicating that urban habitats could also alter the social environment and in turn influence the complexity of learned individual signatures. These findings contribute to a growing literature on the use of vocal learning for individual recognition and indicate that vocal learning can be used to produce individual vocal signatures in a manner sensitive to local population size. • Local population and social group size estimates were smaller in the invasive range. • Invasive range parakeets produced calls with simpler individual signatures. • Urban calls had simpler frequency modulation patterns in the native and invasive ranges. • Monk parakeets may use vocal learning for individual vocal recognition.

Single-step genomic BLUP enables joint analysis of disconnected breeding programs: an example with Eucalyptus globulus Labill.

Single-step GBLUP (HBLUP) efficiently combines genomic, pedigree, and phenotypic information for holistic genetic analyses of disjunct breeding populations. We combined data from two independent multigenerational Eucalyptus globulus breeding populations to provide direct comparisons across the programs and indirect predictions in environments where pedigreed families had not been evaluated. Despite few known pedigree connections between the programs, genomic relationships provided the connectivity required to create a unified relationship matrix, H, which was used to compare pedigree-based and HBLUP models. Stem volume data from 48 sites spread across three regions of southern Australia and wood quality data across 20 sites provided comparisons of model accuracy. Genotyping proved valuable for correcting pedigree errors and HBLUP more precisely defines relationships within and among populations, with relationships among the genotyped individuals used to connect the pedigrees of the two programs. Cryptic relationships among the native range populations provided evidence of population structure and evidence of the origin of landrace populations. HBLUP across programs improved the prediction accuracy of parents and genotyped individuals and enabled breeding value predictions to be directly compared and inferred in regions where little to no testing has been undertaken. The impact of incorporating genetic groups in the estimation of H will further align traditional genetic evaluation pipelines with approaches that incorporate marker-derived relationships into prediction models.

Genomic data support multiple introductions and explosive demographic expansions in a highly invasive aquatic insect.

The study of the genetic makeup and demographic fate of alien species is essential to understand their capacity to recover from founder effects, adapt to new environmental conditions and, ultimately, become invasive and potentially damaging. Here, we employ genomic data to gain insights into key demographic processes that might help to explain the extraordinarily successful invasion of the Western Mediterranean region by the North American boatman Trichocorixa verticalis (Hemiptera: Corixidae). Our analyses revealed the genetic distinctiveness of populations from the main areas comprising the invasive range and coalescent-based simulations supported that they originated from independent introductions events probably involving different source populations. Testing of alternative demographic models indicated that all populations experienced a strong bottleneck followed by a recent and instantaneous demographic expansion that restored a large portion (>30%) of their ancestral effective population sizes shortly after introductions took place (<60years ago). Considerable genetic admixture of some populations suggest that hypothetical barriers to dispersal (i.e., land and sea water) are permeable to gene flow and/or that they originated from introductions involving multiple lineages. This study demonstrates the repeated arrival of propagules with different origins and short time lags between arrival and establishment, emphasizing the extraordinary capacity of the species to recover from founder effects and genetically admix in invaded areas. This can explain the demonstrated capacity of this aquatic insect to spread and outcompete native species once it colonizes new suitable regions. Future genomic analyses of native range populations could help to infer the genetic makeup of introduced populations and track invasion routes.

Highly diverse and highly successful: invasive Australian acacias have not experienced genetic bottlenecks globally.

Invasive species may undergo rapid evolution despite very limited standing genetic diversity. This so-called genetic paradox of biological invasions assumes that an invasive species has experienced (and survived) a genetic bottleneck and then underwent local adaptation in the new range. In this study, we test how often Australian acacias (genus Acacia), one of the world's most problematic invasive tree groups, have experienced genetic bottlenecks and inbreeding. We collated genetic data from 51 different genetic studies on Acacia species to compare genetic diversity between native and invasive populations. These studies analysed 37 different Acacia species, with genetic data from the invasive ranges of 11 species, and data from the native range for 36 species (14 of these 36 species are known to be invasive somewhere in the world, and the other 22 are not known to be invasive). Levels of genetic diversity are similar in native and invasive populations, and there is little evidence of invasive populations being extensively inbred. Levels of genetic diversity in native range populations also did not differ significantly between species that have and that do not have invasive populations. We attribute our findings to the impressive movement, introduction effort and human usage of Australian acacias around the world.

Population genetics of invasive and native Nymphaea mexicana Zuccarini: Taking the first steps to initiate a biological control programme in South Africa

Nymphaea mexicana Zuccarini (Nympheaceae) (Mexican waterlily) is a rooted floating-leaved aquatic plant native to southern USA and Mexico that has become a problematic invasive alien plant in South Africa. Biological control is considered a desirable management strategy for the plant in South Africa. A good understanding of the genetic structure of invasive populations has been useful in other biological control programmes because taxonomic uncertainty about the target plant can result in natural enemies that are not adapted to the invasive populations being considered as potential agents. For N. mexicana, hybrids exist in the wild and horticultural trade, but identification is difficult, so understanding the genetic structure of populations is required to ensure that potential agents are collected off plants similar to invasive populations in South Africa. ISSR (inter-simple sequence repeats) analysis was used to determine whether invasive N. mexicana populations from South Africa were genetically similar to native range populations from USA or whether they were hybrids. Results from these analyses were matched with the morphotypes of each population based on petal colour, shape, and size. The genotypes suggested by the ISSR analyses corroborated the presence of both hybrid and pure forms of N. mexicana in South Africa. Populations of N. mexicana in the invaded range that are genetically similar to native range populations are more likely to be suitable for biological control, while other populations are likely to be hybrids formed by crossing of parents from the native range or within the horticultural trade, which may present difficulties for management using biocontrol.

Founder effects on sex determination systems in invasive social insects.

Invasive populations are often established from a small number of individuals, and thus have low genetic diversity relative to native-range populations. Social ants, bees and wasps (social Hymenoptera) should be vulnerable to such founder effects on genetic diversity because sex in these species is determined genetically via Complementary Sex Determination (CSD). Under CSD, individuals homozygous at one or more critical sex loci are inviable or develop as infertile diploid males. Low diversity at sex loci leads to increased homozygosity and diploid male production, increasing the chance of colony death. In this review, we identify behavioral, social and reproductive traits that preserve allele richness at sex loci, allow colonies to cope with diploid male production, and eventually restore sex allele diversity in invasive populations of social Hymenoptera that experience founding bottlenecks.

Variation in size and shape of toxin glands among cane toads from native-range and invasive populations

If optimal investment in anti-predator defences depends on predation risk, invading new regions (and thus, encountering different predators) may favour shifts in that investment. Cane toads offer an ideal system to test this prediction: expensive anti-predator toxins are stored mainly in parotoid glands whose dimensions are easy to measure, and toad invasions have changed the suites of predators they encounter. Although plasticity may influence parotoid morphology, comparisons between parents and progeny revealed that gland dimensions were highly heritable. That heritability supports the plausibility of an evolved basis to variation in gland dimensions. Measurements of 3779 adult toads show that females have larger glands than males, invasive populations have larger glands than in the native-range, and that parotoid sexual size dimorphism varies strongly among invaded areas. Geographic variation in parotoid morphology may be driven by predation risk to both adult toads and offspring (provisioned with toxins by their mother), with toxins allocated to eggs exacerbating the risk of cannibalism but reducing the risk of interspecific predation. Investment into chemical defences has evolved rapidly during the cane toad’s international diaspora, consistent with the hypothesis that organisms flexibly adjust resource allocation to anti-predator tactics in response to novel challenges.

Population genomics of two invasive mosquitoes (Aedes aegypti and Aedes albopictus) from the Indo-Pacific.

The arbovirus vectors Aedes aegypti (yellow fever mosquito) and Ae. albopictus (Asian tiger mosquito) are both common throughout the Indo-Pacific region, where 70% of global dengue transmission occurs. For Ae. aegypti all Indo-Pacific populations are invasive, having spread from an initial native range of Africa, while for Ae. albopictus the Indo-Pacific includes invasive populations and those from the native range: putatively, India to Japan to Southeast Asia. This study analyses the population genomics of 480 of these mosquitoes sampled from 27 locations in the Indo-Pacific. We investigated patterns of genome-wide genetic differentiation to compare pathways of invasion and ongoing gene flow in both species, and to compare invasive and native-range populations of Ae. albopictus. We also tested landscape genomic hypotheses that genetic differentiation would increase with geographical distance and be lower between locations with high connectivity to human transportation routes, the primary means of dispersal at these scales. We found that genetic distances were generally higher in Ae. aegypti, with Pacific populations the most highly differentiated. The most differentiated Ae. albopictus populations were in Vanuatu, Indonesia and Sri Lanka, the latter two representing potential native-range populations and potential cryptic subspeciation respectively. Genetic distances in Ae. aegypti increased with geographical distance, while in Ae. albopictus they decreased with higher connectivity to human transportation routes. Contrary to the situation in Ae. aegypti, we found evidence of long-distance Ae. albopictus colonisation events, including colonisation of Mauritius from East Asia and of Fiji from Southeast Asia. These direct genomic comparisons indicate likely differences in dispersal ecology in these species, despite their broadly sympatric distributions and similar use of human transport to disperse. Our findings will assist biosecurity operations to trace the source of invasive material and for biocontrol operations that benefit from matching genetic backgrounds of released and local populations.

The Susceptibility of Juvenile American Shad to Rapid Decompression and Fluid Shear Exposure Associated with Simulated Hydroturbine Passage

Throughout many areas of their native range, American shad (Alosa sapidissima) and other Alosine populations are in decline. Though several conditions have influenced these declines, hydropower facilities have had significant negative effects on American shad populations. Hydropower facilities expose ocean-migrating American shad to physical stressors during passage through hydropower facilities, including strike, rapid decompression, and fluid shear. In this laboratory-based study, juvenile American shad were exposed separately to rapid decompression and fluid shear to determine their susceptibility to these stressors and develop dose–response models. These dose–response relationships can help guide the development and/or operation of hydropower turbines and facilities to reduce the negative effects to American shad. Relative to other species, juvenile American shad have a high susceptibility to both rapid decompression and fluid shear. Reducing or preventing exposure to these stressors at hydropower facilities may be a potential method to assist in the effort to restore American shad populations.

Functional shifts in leaves of woody invaders of deciduous forests between their home and away ranges.

Temperate forests are widely invaded by shade-tolerant shrubs and trees, including those of Eastern North America (ENA). However, it remains unknown whether these invaders are 'preadapted' for success in their new ranges due to unique aspects of their evolutionary history or whether selection due to enemy release or other postintroduction processes have driven rapid evolution in the invaded range. We sampled leaf traits of populations of woody understory invaders across light gradients in their native range in Japan and in their invaded ENA range to examine potential phenotypic shifts related to carbon gain and nitrogen use between ranges. We also measured leaf traits in three co-occurring ENA native shrub species. In their invaded range, invaders invested significantly less in leaf chlorophyll content (both per unit leaf mass and area) compared with native range populations of the same species, yet maintained similar rates of photosynthesis in low light. In addition, compared with ENA natives, ENA invaders displayed greater trait variation in response to increasing light availability (forest edges, gaps), giving them a potential advantage over ENA natives in a variety of light conditions. We conclude that, for this group of species, newly evolved phenotypes in the invaded range are more important than preadaptation for their success as shade-tolerant forest invaders.

Ghosts from the past: even comprehensive sampling of the native range may not be enough to unravel the introduction history of invasive species-the case of Acacia dealbata invasions in South Africa.

Knowledge about the introduction history (source(s), number and size of introduction events) of an invasive species is a crucial prerequisite to understand invasion success and to facilitate effective and sustainable management approaches, especially for effective biological control. We investigated the introduction history of the Australian legume tree Acacia dealbata in South Africa. Results of this study will not only provide critical information for the management of this species in South Africa, but will also broaden our overall knowledge on the invasion ecology of this globally important invasive tree. We used nuclear microsatellite markers to compare the genetic diversity and structure between 42 native Australian and 18 invasive South African populations and to test different and competing introduction scenarios using Approximate Bayesian Computation analyses. Australian populations were characterized by two distinct genetic clusters, while South African populations lacked any clear genetic structure and showed significantly lower levels of genetic diversity compared to native range populations. South African populations were also genetically divergent from native populations and the most likely introduction scenario indicated an unknown source population. Although we cannot definitely prove the cause of the observed genetic novelty/diversification in South African Acacia dealbata populations, it cannot be attributable to insufficient sampling of native populations. Our study highlights the complexity of unravelling the introduction histories of commercially important alien species.

Rapid establishment of a flowering cline in Medicago polymorpha after invasion of North America.

To establish and spread in a new location, an invasive species must be able to carry out its life cycle in novel environmental conditions. A key trait underlying fitness is the shift from vegetative to reproductive growth through floral development. In this study, we used a common garden experiment and genotyping-by-sequencing to test whether the latitudinal flowering cline of the North American invasive plant Medicago polymorpha was translocated from its European native range through multiple introductions, or whether the cline rapidly established due to evolution following a genetic bottleneck. Analysis of flowering time in 736 common garden plants showed a latitudinal flowering time cline in both the native and invaded ranges where genotypes from lower latitudes flowered earlier. Genotyping-by-sequencing of 9,658 SNPs in 446 individuals revealed two major subpopulations of M.polymorpha in the native range, only one of which is present in the invaded range. Additionally, native range populations have higher genetic diversity than invaded range populations, suggesting that a genetic bottleneck occurred during invasion. All invaded range individuals are closely related to plants collected from native range populations in Portugal and southern Spain, and population assignment tests assigned invaded range individuals to this same narrow source region. Taken together, our results suggest that latitudinal clinal variation in flowering time has rapidly evolved across the invaded range despite a genetic bottleneck following introduction.

Population genomics of the Asian tiger mosquito, Aedes albopictus: insights into the recent worldwide invasion.

Aedes albopictus, the “Asian tiger mosquito,” is an aggressive biting mosquito native to Asia that has colonized all continents except Antarctica during the last ~30–40 years. The species is of great public health concern as it can transmit at least 26 arboviruses, including dengue, chikungunya, and Zika viruses. In this study, using double‐digest Restriction site‐Associated DNA (ddRAD) sequencing, we developed a panel of ~58,000 single nucleotide polymorphisms (SNPs) based on 20 worldwide Ae. albopictus populations representing both the invasive and the native range. We used this genomic‐based approach to study the genetic structure and the differentiation of Ae. albopictus populations and to understand origin(s) and dynamics of the recent invasions. Our analyses indicated the existence of two major genetically differentiated population clusters, each one including both native and invasive populations. The detection of additional genetic structure within each major cluster supports that these SNPs can detect differentiation at a global and local scale, while the similar levels of genomic diversity between native and invasive range populations support the scenario of multiple invasions or colonization by a large number of propagules. Finally, our results revealed the possible source(s) of the recent invasion in Americas, Europe, and Africa, a finding with important implications for vector‐control strategies.

Genetic reconstruction of a bullfrog invasion to elucidate vectors of introduction and secondary spread

Reconstructing historical colonization pathways of an invasive species is critical for uncovering factors that determine invasion success and for designing management strategies. The American bullfrog (Lithobates catesbeianus) is endemic to eastern North America, but now has a global distribution and is considered to be one of the worst invaders in the world. In Montana, several introduced populations have been reported, but little is known of their sources and vectors of introduction and secondary spread. We evaluated the genetic composition of introduced populations at local (Yellowstone River floodplain) and regional (Montana and Wyoming) scales in contrast to native range populations. Our objectives were to (1) estimate the number of introductions, (2) identify probable native sources, (3) evaluate genetic variation relative to sources, and (4) characterize properties of local‐ and regional‐scale spread. We sequenced 937 bp of the mitochondrial cytochrome b locus in 395 tadpoles collected along 100 km of the Yellowstone River, from three additional sites in MT and a proximate site in WY. Pairwise ΦST revealed high divergence among nonnative populations, suggesting at least four independent introductions into MT from diverse sources. Three cyt b haplotypes were identical to native haplotypes distributed across the Midwest and Great Lakes regions, and AMOVA confirmed the western native region as a likely source. While haplotype (H d = 0.69) and nucleotide diversity (π = 0.005) were low in introduced bullfrogs, the levels of diversity did not differ significantly from source populations. In the Yellowstone, two identified haplotypes implied few introduction vectors and a significant relationship between genetic and river distance was found. Evidence for multiple invasions and lack of subsequent regional spread emphasizes the importance of enforcing legislation prohibiting bullfrog importation and the need for continuing public education to prevent transport of bullfrogs in MT. More broadly, this study demonstrates how genetic approaches can reveal key properties of a biological invasion to inform management strategies.