DNA barcoding indicates multiple invasions of the freshwater snail Melanoides tuberculata sensu lato in Florida.

Melanoides tuberculata sensu lato (Thiaridae) are polymorphic female-clonal snails of Asian and African origins that have invaded freshwaters worldwide, including those in Florida. Although the snails have been documented in Florida for at least 70 years, no studies have investigated whether the observed distribution is due to a single introduction or multiple independent invasions. Here, cytochrome oxidase I was used to measure genetic diversity within and among sites in Florida and compare genetic diversity between Florida and other regions of the world. We also examined the relationship between shell morphology and haplotype diversity to determine if shell morphs can serve as a proxy for haplotypes. In total, we recovered 8 haplotypes randomly distributed across populations in Florida. Phylogenetic reconstruction supported the hypothesis of multiple invasions by diverse representatives of the M. tuberculata species complex. In contrast, shell morphology was not found to be a useful phylogeographic character, with divergent haplotypes represented by similar shell forms. These results suggest that the observed invasion patterns in Florida are best explained by serial introductions, and that shell morphology cannot be used to predict haplotypes or reconstruct invasion history of Melanoides tuberculata s.l. and that extensive taxonomic revisions are needed to investigate invasion dynamics.

Effects of Parasite Infection and Host Body Size on Habitat Associations of Invasive Aquatic Snails: Implications for Environmental Monitoring.

The Comal River, a spring-fed system in central Texas, was invaded in the 1960s by two Asian aquatic snails (Thiaridae: red-rimmed melania Melanoides tuberculata and quilted melania Tarebia granifera) and subsequently by three of their trematode parasites (the avian eye-fluke Philophthalmus gralli in the 1960s; the gill trematode Centrocestus formosanus in the 1990s; and the intestinal fluke Haplorchis pumilio in the 2000s). Previous snail collections (2001-2002) established that habitat conditions significantly affect the distribution of both snail species. However, the effects of snail size (known to influence infection prevalence) and habitat conditions (known to influence snail size) on trematode infection patterns in this system were not evaluated. In a re-evaluation of this data set, logistic regression analyses with individual snails showed that for both M. tuberculata and T. granifera populations, large snails were more likely to be infected than small snails, and habitat conditions were significantly related to infection in T. granifera. However, only snail size was significant in explaining the probability of infection in M. tuberculata. This result was confirmed by linear regression models, which showed that both infected and noninfected M. tuberculata used similar habitats, as large individuals in both infection categories were found in patches dominated by fine substrates and high levels of aquatic vegetation and detritus. For the large size-class of T. granifera, noninfected individuals were found primarily in habitats with silt/sand substrates and high vegetation and detritus cover, while infected individuals occurred among all available habitats. Using these results, we suggest that targeted sampling of large individuals of M. tuberculata in habitats with high detritus and vegetation and large individuals of T. granifera in any habitat can be used to efficiently ascertain parasite "hot spots" and to evaluate changes in parasite prevalence or detect the invasion of new parasites in these thiarid snails.

Validation of phylogenetic signals in amplified fragment length data: testing the utility and reliability in closely related taxa.

BACKGROUND: Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers. FINDINGS: We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates. CONCLUSION: For researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.