RESEARCH PLAN

Our primary objectives are to: (1) reveal the historical phylogeography of invasive species in both their native and introduced ranges and (2) understand possible genetic/genotypic and demographic changes that have occurred to make a species invasive in its introduced range.

Specific Objectives

A. Forensic Phylogeography

1. How do patterns of genetic diversity differ between native and introduced populations?
2. How many independent introductions were there?
3. From where in the native range did invasive species originate?
4. How have invasive species spread within the introduced range?

B. Evolutionary Change

1. What genotypic and demographic changes have occurred during invasion?
2. Is there evidence for genetic change (e.g., hybridization) in invasive species?

Experimental Approaches

The PI’s and Chinese senior personnel will focus efforts on characterizing a minimum of ten target species, including species native to China and invasive in the Southeast U.S. and species from the Southeast U.S. and invasive in China, over the five year grant period. We have chosen these species because they represent particularly acute conservation or agronomic problems. The species include two bacterial plant pathogens: citrus greening disease, Liberibacter asiaticus, and bacterial fruit blotch, Acidovorax avenae ssp. citrulli; four plant species native to China and invasive in the Southeast U.S. (the herbaceous grass, Nepalese browntop, Microstegium vimineum, the shrub, Chinese privet, Ligustrum sinense, the tree, Mimosa, Albizia julibrissin, the vine, kudzu, Pueraria montana); and 4 species from the Southeast and invasive in China (herbaceous Carolina geranium, Geranium carolinianum, rest prickly pear, Opuntia stricta, smooth cordgrass, Spartina alterniflora, and tall morning glory, Ipomoea purpurea).

In addition to these ten species, we include support for the research of graduate students to study additional invasive species in China and the southeastern U.S. The advantage of this wider support is that we would broaden the taxonomic reach of the collaboration. We have assembled a team of senior personnel with interest in China who study a broad range of invasive taxa, including plants, invertebrates, pathogens, and fish. We also expect that the funding of this proposal would give support to parallel funding efforts by our Chinese and Taiwanese collaborators. That funding would extend the number and breadth of taxa investigated.

Genetic Diversity and Genetic Structure of Plant Species

We will utilize several genetic markers to address specific objectives A1 – A4 and B2. Since several of our proposed study species have been the subject of previous allozyme studies, allozymes will be the markers of choice for analyses of nuclear genes. Allozymes often provide 25-30 loci (10-15 polymorphic loci) for analyses and continue to be the least expensive of the commonly used marker systems (? $0.20 per locus per individual). If allozymes do not prove to be feasible or provide too few polymorphic loci, we will develop microsatellite markers for these species. Using different marker systems for different species should not present a problem since the comparisons of interest will be the inter-country intraspecific comparisons.

Marker development

For those species that have not been analyzed previously we will first test whether we can resolve allozyme loci for them. This phase involves determining the optimum combination of extraction and electrophoretic buffers and specific enzyme stains. These analyses will be conducted at UGA during the first year of the project utilizing material collected from either native or invasive U. S. populations. If adequate numbers of allozyme loci cannot be resolved, or if there are too few polymorphic loci available, we will develop a minimum of eight microsatellite loci for each species.

Maternally inherited cpDNA haplotypes will also be developed for each species. This analysis will provide a different perspective on the genetic structure among populations and may provide insights into the invasive histories of these species. Total genomic DNA will be extracted from leaf tissue and will be used as a template in PCR reactions with sets of primer pairs for the tRNA genes T, L and F which flank less well-conserved noncoding regions. The amplified PCR product will be sequenced in both directions on an automated sequencer. Insertion, deletion and substitution differences will be recorded and different haplotypes will be treated as alleles for genetic diversity and structure analyses.

Genetic diversity analyses

For each species, we will sample 36 individuals from each of 20 populations from each country covering the geographic range of the species in both native and introduced habitats. We will estimate the proportion of polymorphic loci (P), the number of alleles per polymorphic locus (AP), and the effective number of alleles (Ae) and observed (Ho) and expected (He) heterozygosity. We will test whether each population is in Hardy-Weinberg equilibrium. These parameters will be calculated for each population and for data pooled across all the populations representing each country.

Total genetic diversity among the 40 populations at each locus will be estimated by calculating Nei’s GST or by AMOVA. By using a hierarchical analysis, we will partition the total genetic diversity among populations into between country and among populations within countries components.

Comparative analyses - nuclear genes

The genetic composition and structure of these species could vary in several ways between China and the USA. First, we will determine whether total levels of genetic diversity differ between China and the U.S. In particular, we will compare overall levels of genetic diversity and the total number of alleles observed in each country. If the species was introduced multiple times from multiple locations in its native range, we would expect that the two countries might not differ in total levels of genetic diversity but that the invasive populations might have a subset of the rare alleles found in the native populations. We will also test whether individual populations from the native and invasive ranges contain similar levels of genetic diversity, have different variances in He values and differ in the levels of genetic structure among populations (GST). Finally, we will examine whether pairwise GST values are significantly correlated with geographic distance between populations from the two countries. If there have been multiple introductions of the species, we might expect greater genetic diversity within the invasive populations, less variation in He among populations, lower GST values and less evidence of isolation by distance. Further insights into the histories of the introduced populations can be gained by testing for evidence of a recent bottleneck as a result of relatively recent introductions and/or colonization. We will use the program BOTTLENECK to examine the genotypic composition of each population (i.e., test for an excess of expected heterozygosity Cornuet and Luihart 1997; Luihart et al. 1997). We will also use the program STRUCTURE to infer whether the U.S. populations consist of a mixture of genetic diversity from specific Chinese populations. This approach uses Bayesian analyses to assign individuals to populations based on their genetic composition (Pritchard and Wen 2004).

Hybridization

Since hybridization has been associated with invasiveness, we are particularly interested in genetic evidence for intra- and inter-specific hybridization in the introduced range (Ellstrand & Schierenbeck 2000; Gaskin & Schaal 2002; Saltonstall 2002). Ellstrand and Schierenbeck (2000) have estimated that approximately 40% of the invasive species in North America are of hybrid origin. To determine whether invasive populations of our target species are hybrids, we will compare the allelic composition of the invasive populations with their native populations. If the invasive populations have alleles that do not appear in the native populations it would indicate that hybridization has occurred, particularly if the alleles that are novel to the invasive populations occur in fairly high frequencies. If such a result is observed we will sample populations of candidate congener species to determine which species were involved in the formation of the invasive taxon.

Comparative analyses - cpDNA haplotypes

Phylogeographic analyses offer a way to address historical and contemporary population processes that influence current plant distributions. Methods that employ networks of genetic relationships that take population level properties into account are most appropriate for intraspecific studies (Posada and Crandall 2001). We will use cpDNA sequence polymorphisms to identify haplotypes and generate networks of haplotype relationships. Inference of intraspecific phylogeography of an introduced species would be expected to differ from that seen for populations of native species. Patterns of relationships of a species in its native range should be primarily influenced by landscape level processes and time since divergence. Patterns of relationships within a relatively recently introduced species will be heavily influenced by introduction history and by subsequent human-mediated movement. Although phylogeographic relationships in invasive species may be blurred by such mixing, information concerning the number and location of introductions and subsequent colonization history in the introduced range may be potentially retained in the haplotype networks. If haplotypes are present in the invasive populations that are not found in the native range this constitutes additional evidence for the hybrid origin of the invasive populations.

Genetic Diversity and Genetic Structure of Phytopathogens

To assess the genetic structure of U.S. and Chinese populations of phytopathogens, we will use restriction fragment length polymorphisms (RFLP) via pulse field gel electrophoresis (PFGE) and amplified fragment length polymorphisms (AFLP).

While REP-PCR is more economical, our experience indicates that it is less robust than PFGE. Hence PFGE will be used in combination with AFLP to analyze genetic diversity of phytopathogens. Additionally, unlike PFGE, live cultures are not necessary, and if needed, DNA can be easily shipped between laboratories.

For this study, representative populations of phytopathogens from cultivated and wild host plant ranges will be collected in the U.S. and China. In particular, since it is suspected that planting materials might be involved in the spread and invasiveness of phytopathogens, regions where seeds, seedlings and planting stock are produced will be sampled. The identity of the collected strains will be determined by molecular, biochemical and pathogenicity tests and cultures will be catalogued and stored for future reference.

Collaborators will be trained to conduct PFGE and AFLP according to a standard protocol and strains will be processed in-country. In cases where it is necessary to share materials, all necessary permits will be requested from official government agencies. Additionally, only DNA rather than live cultures will be shared. AFLP and PFGE fingerprints will be generated according to established protocols and data will be analyzed as previously described. Additionally, host range pathogenicity studies will be conducted using representative subpopulations of the different PFGE and AFLP haplotypes or groups (Walcott et al. 2004). Subpopulations of the phytopathogens will be artificially inoculated onto replicates of five different members of the respective host plant families under controlled greenhouse conditions and evaluated for disease incidence and severity. Host range studies will only be conducted in the countries of origin.

Evolutionary and Demographic Changes

A variety of factors might be responsible for making a species invasive in an introduced habitat. Three evolutionary hypotheses have emerged as promising candidates for further study and we propose to investigate them. First, it has been noted that individuals in the introduced range show increased vigor when compared to the native range (e.g., Willis et al. 2000). This pattern might arise if ecological conditions in the introduced environment are different from the native habitat (e.g., absence of natural enemies; Wolfe 2002). Alternatively, this pattern could arise from genetic changes following invasion. For example, there might be reallocation of energy devoted to the synthesis of defensive chemicals to growth and reproduction, thereby facilitating invasiveness (Blossey & Nötzold 1995). Genetic characteristics of populations, such as the amount of additive genetic variance for “invasiveness traits,” may also indicate the capacity for range expansion (Tsutsui et al. 2000: Kolbe et al. 2004). Third, changes in reproduction might accompany invasion: for example, an increase in the rate of selfing (which would be revealed by examining the genotypic composition of our population samples).

Addressing objective B1 will encompass several approaches. We will establish permanent plots in 6-10 populations within each country in which we will record detailed demographic data over the last four years of the grant period (and hopefully beyond the tenure of this grant). These populations will be carefully selected to represent the range of habitats in which the species occurs in its native and invasive ranges. The age (stage) structure of each population will be determined. Each population will be monitored at least twice yearly (at the beginning and at the end of each growing season) for recruitment, mortality, size and reproductive effort. Transition matrices will be developed for each population. These analyses will allow us to determine if invasive species exhibit changes in population growth and age-specific life history traits when they become invasive. We will also have the opportunity to record potentially informative environmental correlates, such as herbivore load.

It is also possible that the populations in China and the U.S. may differ in traits that are correlated with invasion success (Blair and Wolfe 2004). For example, Loret et al. (2005) demonstrated that vegetative propagation, leaf size and seed dispersal were positively correlated with invasiveness on Mediterranean islands. To examine whether Chinese and U.S. populations differ significantly for such traits, we will establish common gardens for each species in each country. Using a subset of the populations examined for genetic diversity (e.g., 15 populations) we will collect seeds from a minimum of 15 maternal individuals per population. We will then randomly plant two seedlings per maternal individual into a randomized block design (i.e., 2 seedlings, x 15 maternal individuals x 15 populations = 450 total plants per garden). Populations will be chosen to represent the entire range of the species in each country. We will measure a number of morphological and plant growth traits but will concentrate our efforts on defensive and reproductive traits. Where feasible we will establish two or more replicates of the common gardens in different parts of the species’ native and introduced ranges. Finally, we leave open the possibility that we would perform reciprocal transplant experiments across countries in order to better understand genotypic changes. Such experiments would not be done with disease agents and would be established only with approval of government agencies and with great care to prevent the escape of additional diversity.

Mating System Analyses

One of the major changes observed between native and invasive populations are changes in a species’ reproductive system. In particular, invasive populations may have evolved traits to ensure reproduction (e.g., selfing) in the face of long-distance colonization events. We will test this possibility in our species by using a subset of our polymorphic genetic markers to estimate the proportions of selfing and outcrossing in populations representing each country. We will utilize the populations and maternal individuals used for our common garden studies (see above). We will analyze 12 progeny from each maternal individual to obtain estimates of the proportions of selfing and outcrossing for each population using the protocols of Ritland (2002). We will also test these progeny arrays to determine if there is evidence for apomitic (asexual) seed production. If we observe differences between invasive and native populations or significant variation among populations within countries, where feasible, we will repeat our analyses on progeny arrays produced by individuals within the common gardens to determine whether differences seen in wild populations have a genetic basis.