The Geographic Mosaic of Wolbachia Infection in Melanitis Leda Butterfly Opulationsp
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City University of New York (CUNY) CUNY Academic Works Dissertations and Theses City College of New York 2018 The Geographic Mosaic of Wolbachia Infection in Melanitis leda Butterfly opulationsP Brandon E. Latorre CUNY City College How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/cc_etds_theses/729 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] The Geographic Mosaic of Wolbachia Infection in Melanitis leda Butterfly Populations By Brandon Latorre Department of Biology A Thesis Submitted to The City College of New York in Partial Fulfillment of the Requirements for the Degree: BS/MS of Science New York, New York Summer 2018 Master's Committee: David J. Lohman, Amy Berkov, Ana Carnaval Brandon Latorre Master's Thesis Contribution: I extracted DNA from 57 Melanitis leda butterflies. The following PCR reactions were conducted: 56 specimens in cytB; 43 specimens in COI (both a and b); 48 specimens in EF1a (1, 2, and 3); 42 specimens in wg; and all MLST loci. In addition, I screened all M. leda butterflies for Wolbachia using the wsp locus. After sequences were acquired from macrogen, I used Sequencher 5.1 to align forward/reverse sequences and edited every sample from the PCRs that I did. To determine which Wolbachia coinfect M. leda in Australia and Fiji, I created the input file for PHASE 2.1 (implemented on DnaSP 6.0), but did not conduct analyses. With reference to the PubMLST Database, I identified the Wolbachia sequence types (STs) that are infecting the M. leda in the study and subsequently searched for other organisms that were infected with these Wolbachia STs. In addition, I downloaded and aligned all Wolbachia MLST sequence data to make every Wolbachia phylogeny. I aligned every data matrix using SeaView and concatenated MLST, nDNA, mtDNA, and mtDNA+nDNA sequences using SequenceMatrix 1.8. I then inferred the best-fit model for every loci or combination of loci using jModelTest 2. I used TNT 1.5 to conduct parsimony analyses on each data matrix. I conducted all Bayesian phylogenetic analyses using MrBayes 3.2.6 via Cipres Science Gateway. I calculated all COI pairwise distances using MEGA 7. I conducted all phylogenetic associative analyses using BaTS (although I did not make input files). I created a heatmap using HeatMapper. I used Adobe Illustrator CS5 to create and edit all maps and phylogenies. I used Microsoft Excel to create all tables. I wrote and revised the manuscript, incorporating edits and learning from comments made by my PI. 1 The geographic mosaic of Wolbachia infection in Melanitis leda butterfly populations Brandon LaTorre1, David J. Lohman1,2,3 1Biology Department, City College of New York, City University of New York, New York, NY 10031, USA 2Ph.D. Program in Biology, Graduate Center, City University of New York, New York, NY 10016, USA 3Entomology Section, National Museum of the Philippines, Manila 1000, Philippines Correspondence: Brandon Latorre, [email protected] Keywords: co-evolution, host-parasite interactions, Indo-Australian Archipelago, intracellular symbiont, mito-nuclear discord, Papilionoidea, Wolbachia pipientis Abstract Insects are the most species-rich multicellular taxon on the planet, and it is estimated that more than half of all insect species are infected with the endosymbiont Wolbachia. This bacterium can manipulate the reproduction of its host and potentially affect host evolution. Most surveys investigating patterns of Wolbachia infection in nature sample limited numbers of individuals; population-level investigations into geographic differences in infection status that might affect host evolution are few. To investigate geographic variability among populations of a single species, we assayed 133 Melanitis leda butterflies (Lepidoptera: Nymphalidae: Satyrinae) for Wolbachia infection collected throughout its range in the Old World tropics and subtropics from Ghana to Fiji. Potential effects of the parasite on the evolution of its host were assessed by inferring phylogenies of the host with nuclear and mitochondrial markers, and by inferring relationships among the Wolbachia strain (sequence type, ST) that we detected in different populations. Geographic variability was apparent on two levels: prevalence of infection and ST of infection. Every M. leda individual on Java and all landmasses south and east of Wallace’s 2 Line were all infected with at least one of three different sequence types; individuals in Australia and Fiji hosted at least two different STs. Butterflies collected elsewhere were often not infected, but most that were had a fourth ST, except for a single individual in Uganda with a fifth ST. Infection status coincided with patterns of nuclear and mitochondrial variability. With the exception of the Ugandan individual, there was no genetic structure in populations collected throughout Africa, Madagascar, South Asia, mainland Asia, the Philippines, Borneo, and Sumatra; this homogeneity implies frequent gene flow between these areas or may be the result of recent M. leda colonization events. Specimens from Australia, New Guinea, and the archipelagos of Southeast Asia and Oceania had nuclear and mitochondrial structure. The topology of nuclear and mitochondrial trees conflicted with regard to the placement of the clades containing specimens from the South Pacific and Ghana. Although this result is consistent with Wolbachia- imposed selection through linkage disequilibria, other factors may play a role in this discord, including the lower effective population size & higher mutation rate of mitochondrial DNA, as well as genetic differences expected between different geographical regions. These results are consistent with the notion that marine barriers limit the dispersal of the host, which seems to permit different strains of Wolbachia to enter insular populations through horizontal transmission at different times and places before subsequent vertical transmission. Some of these STs seem to exert selection on the host, but many do not. Introduction Diverse bacteria and fungi live within terrestrial arthropods, where they are primarily transmitted vertically and can affect the nutrition, development, defense, and reproduction of their hosts (Duron et al. 2008; Gibson & Hunter 2010; Moran et al. 2008). The Alphaproteobacteria genus 3 Wolbachia is perhaps the most common, widely distributed, and best studied of these heritable endosymbionts (Russell et al. 2009; Weinert et al. 2015), and has been detected in nematodes and arthropods, including the majority of insect species surveyed (Hilgenboecker et al. 2008; Weinert et al. 2015; Werren et al. 2008). The Wolbachia lineage is estimated to be ~200 my old, and its diversification coincides with that of insects, which seem to comprise the most common hosts (Gerth & Bleidorn 2016). Wolbachia strains are not recognized as species per se, but are categorized by their lineage. The taxon is divided into at least sixteen supergroups that infect various arthropods and nematodes (designated by letters; Baldo & Werren 2007; Glowska et al. 2015). Supergroup designations are assigned to different clades of phylogenetic networks inferred with portions of four protein-coding genes (ftsZ, gltA, groEL, and coxA), and part of the 16S rRNA gene (Ramirez-Puebla et al. 2015; Ros et al. 2009). Supergroups are further divided into sequence types (STs) on the basis of sequence differences at five multi-locus sequence typing (MLST) loci (designated by numbers; Baldo et al. 2006a; Baldo et al. 2006b). Infection of an individual organism with two or more Wolbachia strains has been observed in many arthropod taxa (Baldo et al. 2006b; Mitsuhashi et al. 2011; Narita et al. 2007; Vavre et al. 1999). This coexistence makes possible selective exchange of genetic material, and comparative genomics of Wolbachia demonstrates rampant recombination among different lineages (Baldo et al. 2006a; Werren & Bartos 2001). Frequent recombination between clonal lineages thus necessitates identification using multiple markers (hence the MLST framework), but recent evidence suggests that the standard Wolbachia MLST loci might not adequately represent the genomic variability of different lineages (Bleidorn & Gerth 2018). Although this MLST procedure is an improvement on the former method of ST identification using only one locus, Bliedorn & Gerth (2017) suggest pursuing a whole genome approach in characterizing 4 Wolbachia. The existence of an established database dedicated to Wolbachia characterization using these five markers (pubMLST.org/Wolbachia), and budgetary constraints influenced our decision to pursue MLST using five markers in our study. Different STs can impact their hosts in different ways, ranging from mutualism and apparently benign commensalism to parasitic manipulation. In parasitic interactions, Wolbachia commandeers the reproductive biology of its host to favor production of females, since the bacterium is primarily transmitted in ova. Because Wolbachia and mitochondria are both uniparentally inherited in the host female line, and because Wolbachia can exert significant selection pressure on its host, non-recombining mitochondrial loci in Wolbachia-infected organisms can act as if they are under selection, and their evolutionary history can differ from nuclear markers (Kodandaramaiah et al. 2013; Smith et al. 2012). Moreover, rare interspecific