Emily V. Saarinen

When Technology Meets Conservation: Increased Microsatellite Marker Production Using 454 Genome Sequencing on the Endangered Okaloosa Darter (Etheostoma okaloosae)

High-throughput sequencing affords a cost and time effective means of obtaining large numbers of genetic markers for conservation studies. Here, we present thousands of novel microsatellite loci developed for the Okaloosa darter, Etheostoma okaloosae, an endangered percid fish. We sequenced more than 29 million bp using 454 whole genome shotgun sequencing and employed free user-friendly bioinformatics tools to screen for microsatellite loci and design appropriate primers. We tested 39 primer sets for polymorphism and ran population-level analyses on a population of Okaloosa darters. Of these, 30 markers were variable with an observed and expected heterozygosity of 0.382 and 0.430, respectively, and allele numbers ranging from 2 to 13. Comparisons against the zebra fish reference genome, Danio rerio, revealed that these loci represent an adequate chromosomal coverage of the darter genome, although total genomic coverage was only 2.4-3.3%. We also tested these loci on the brown darter, E. edwini, and identified loci that will be useful for hybridization studies between these taxa.

Miami blue butterfly larvae (Lepidoptera: Lycaenidae) and ants (Hymenoptera: Formicidae): new information on the symbionts of an endangered taxon

Abstract Historical, anecdotal records of the state-endangered Miami blue butterfly, Cyclargus thomasi bethunebakeri (Comstock & Huntington) (Lepidoptera), have mentioned larval associations with the Florida carpenter ant, Camponotus sp. Recent population studies confirm that C. t. bethunebakeri larvae associate with Camponotus floridanus (Buckley) as well as another member of the genus, Camponotus planatus (Roger). Additionally, caterpillars have been observed tended by Crematogaster ashmeadi (Emery), Forelius pruinosus (Roger), and Tapinoma melanocephalum (Fab.). Field surveys of remaining Miami blue habitat and recent butterfly reintroduction sites reveal other potential ant associates, Paratrechina longicornis (Latreille) and Paratrechina bourbonica (Forel), and a host of possible predaceous ant species. The corresponding conservation implications are discussed. Detailed information is also presented about larval ant-associated organs and their mediation of this facultative symbiosis.

Development and characterization of polymorphic microsatellite loci in the endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri).

The Miami blue butterfly (Cyclargus thomasi bethunebakeri) is a state‐endangered taxon in Florida and a candidate for federal listing. Here we report 12 polymorphic microsatellite loci appropriate for use in population and conservation studies. We genotyped 114 individuals sampled from a metapopulation in the lower Florida Keys over a 2‐year period (2005–2006). These results show 4–14 alleles per locus, and ranges of observed and expected heterozygosities are 0.02679–0.79630 and 0.06154–0.69565, respectively. Large deviations from Hardy‐Weinberg equilibrium (HWE) are observed across the whole sample set. When a single breeding population is analysed alone, seven of the loci are in HWE.

Local extinction event despite high levels of gene flow and genetic diversity in the federally-endangered Miami blue butterfly

A certain level of genetic diversity and connectivity between populations is necessary to allow small populations to persist over time. In this study, we investigate the last two populations of the highly endangered Miami blue butterfly, one of which was extirpated in 2010. We investigate whether an erosion of genetic diversity was the cause for the extirpation of the Bahia Honda State Park (BHSP) population. As the level of existing molecular diversity within and gene flow between the last two populations of the endangered Miami blue butterfly was unknown, we present data from twelve polymorphic microsatellite loci as an assessment. We show that greater genetic diversity (allelic richness and observed heterozygosity) than originally predicted existed in the BHSP population prior to its extirpation and values were even greater in the Key West National Wildlife Refuge population. Allelic frequencies show short-range gene flow between neighboring habitat patches and underline the importance of habitat connectivity. It does not appear that loss of genetic diversity was the cause for the loss of the BHSP population. Greater consideration of habitat connectivity should be considered for short and long-term conservation and management of this taxon.

Using museum specimens to assess historical distribution and genetic diversity in an endangered butterfly

The Miami blue butterfly, Cyclargus thomasi bethunebakeri, is a state-endangered taxon in Florida and a candidate for federal listing. This once common butterfly saw a dramatic decline in population number and abundance in the 1970s and 1980s, but significant collections of individuals prior to this decline are deposited in natural history museums. Using museum specimens, we quantified the genetic diversity in a historical population present in Key Largo, Florida in 1940, 1960, and 1980. Genetic diversity was consistently high within this historical population, but diversity was observed to decrease over the decades sampled. A comparison of historical diversity from the Key Largo population with the extant populations on Bahia Honda State Park (BHSP) and Key West National Wildlife Refuge (KWNWR) revealed differences in allelic frequencies, but only minor differences in the overall number of alleles. The historical distribution of butterflies throughout the Florida Keys further suggests a metapopulation structure. This structure involved partially-isolated populations of C. t. bethunebakeri that were loosely connected via gene flow and that underwent localized extinction and colonization events along the chain of suitable habitat in the Florida Keys. It appears that a “mini-metapopulation” currently exists on BHSP and KWNWR; structures that are similar to the historical metapopulation structure and distribution of populations on a larger scale. Knowledge of historical distribution helps to plan future reintroduction events with captive-bred butterflies. Additional populations of butterflies may represent undiscovered genetic diversity that, if appropriate, may be further incorporated into captive-breeding efforts.

Conservation genetics of an endangered grassland butterfly (Oarisma poweshiek) reveals historically high gene flow despite recent and rapid range loss

In poorly dispersing species gene flow can be facilitated when suitable habitat is widespread, allowing for increased dispersal between neighbouring locations. The Poweshiek skipperling [Oarisma poweshiek (Parker)], a federally endangered butterfly, has undergone a rapid, recent demographic decline following the loss of tallgrass prairie and fen habitats range wide. The loss of habitat, now restricted geographic range, and poor dispersal ability have left O. poweshiek at increased risk of extinction. We studied the population genetics of six remaining populations of O. poweshiek in order to test the hypothesis that gene flow was historically high despite limited long‐distance dispersal capability. Utilising nine microsatellite loci developed by PacBio sequencing, we tested for patterns of isolation by distance, low population genetic structure and alternative gene flow models. Populations from southern Manitoba, Canada to the Lower Peninsula of Michigan, USA are only weakly genetically differentiated despite having low diversity. We found no support for isolation by distance, and Bayesian estimates of historical gene flow support our hypothesis that high levels of gene flow previously connected populations from Michigan to Wisconsin. Prairie grasslands have been reduced tremendously over the past century, but the low mobility of O. poweshiek suggests that rapid loss of populations over the past decade cannot be simply explained by fragmentation of habitat. As a species at high risk of extinction, understanding historical processes of gene flow will allow for informed management decisions with respect to head‐starting individuals for population reintroductions and for conserving networks of habitat that will allow for high levels of gene flow.

A long-term survey of spring monarch butterflies in north-central Florida

Long-term springtime counts of immature and adult monarch butterflies and their Asclepias humistrata host plants in north-central Florida reveal a close relationship between the milkweed’s phenology and the butterfly’s spring remigration from Mexico. Remigrant adults arrive after most frosts occur and as the milkweeds are flourishing but before the plants begin to senesce. The peak of adult arrival is during the first few days in April; the eggs that are laid during this peak develop through April, leading to a second peak in adult abundance in early May. These newly emerged adults continue the migration northward. In addition to assessing the phenology of annual events, our long-term survey, with regular monitoring from 1994–2017, has enabled us to evaluate long-term trends. Both adults and immatures have declined in abundance from 1985 to 2017; since 2005, both have declined by around 80%. This decline has occurred concurrently with the decline in the number of monarchs at their Mexican overwintering sites.

Butterfly Conservation Genetics

Conservation programs are challenged with preventing biodiversity loss; losses that have typically been managed as preservation of individual species and groups. An increasing focus on different levels of biodiversity has promoted methods evaluating the genetic diversity in these species as a tool in preventing species loss. Therefore, most conservation practitioners today realize the need to evaluate, preserve, and manage population-level and range-wide genetic diversity as an essential component in the preservation of species. Recent methodological (e.g. non-lethal tissue sampling) and technological advances (e.g. high-throughput sequencing) have greatly increased our ability to evaluate genetic diversity in endangered and threatened butterflies. In this chapter, we provide a framework and guide for conducting genetic research and for including it in the management plans of endangered Lepidoptera. We discuss the variety of molecular markers and tools available to researchers as well as guidance in implementing them. We provide a step-by-step guide for conducting tissue sampling, evaluating statistical concerns, and provide advice and troubleshooting tips. We review several case studies that have successfully used molecular data and incorporated the results into management plans. The chapter concludes with a summary of techniques, future directions, and lessons learned from conservation genetics study of Lepidoptera of conservation concern.