Molecular ecology of the green mirid Creontiades dilutus Stål (Hemiptera: Miridae) - movement and host plant interactions across agricultural and arid environments. James Peter Hereward BSc. (Hons) University of Manchester A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2012 School of Biological Sciences Abstract Creontiades dilutus , the green mirid, is endemic to Australia and widely distributed across the continent. These bugs have been recorded on a broad range of host-plants including native species, weeds and several crops, particularly cotton, lucerne, and soy. The economic relevance of green mirids to the Australian cotton industry increased in recent years in response to the uptake of transgenic cotton, which controls Lepidopteran pests but is ineffective against Hemiptera. In this thesis I combined several molecular and ecological approaches to develop a better understanding of the species status of this insect, its use of multiple hosts and its long distance movement. Creontiades dilutus had reputedly been recorded in the USA during 2006. With collaborators at the USDA, I used sequence data (Cytochrome Oxidase I (COI) and 28S ribosomal gene) to establish that the insects concerned were highly unlikely to be C. dilutus . Subsequent taxonomic work confirmed that the USA species was indeed a separate species, Creontiades signatus. Using C. signatus as an out-group, further phylogenetic analyses showed that C. dilutus and C. pacificus are well differentiated according to the sequence of both genes. The COI sequences also indicated low levels of genetic diversity in C. dilutus (Pi = 0.0006), especially in comparison to C. pacificus (Pi = 0.0026). The low COI diversity indicated that more variable markers would be required for further analyses of gene flow in this species, and consequently 12 microsatellites were developed by enrichment. To understand the use of multiple hosts by C. dilutus , all the available host plant data were analysed. Most of the putative host plants recorded prior to this thesis were crop species or introduced weeds. As C. dilutus has not been recorded outside of Australia it was evident that a more thorough investigation of potential native hosts was necessary. Over three seasons of field surveys in central and eastern Australia I added an additional 25 species to the list of potential hosts, 22 of which are native to Australia. The presence of nymphs indicates that C. dilutus is indeed able to feed and reproduce on 46 host plant species, most in the family Fabaceae. Quantitative sampling, however, revealed a strong association between C. dilutus and two plant species in the genus Cullen. These two species are thus likely the primary host plants for green mirids. To test whether green mirid individuals show a strong preference for Cullen under field conditions I amplified Chloroplast DNA from DNA extracted from whole insects. These diet analyses demonstrated that C. dilutus individuals do feed on alternative host plants to the one from which they were collected, even when that was a Cullen species, and multiple host use by individuals was not infrequent. Green mirids are found, sometimes in large numbers, in arid parts of central Australia, and Miles (1995) suggested that this might be the main source of the mirids invading cotton and other crops in sub coastal eastern Australia. Green mirid abundance is seasonally inverse between the central arid regions and eastern cropping areas, and they likely experience different selective pressures in each region. The population genetic consequences of these dynamics were assessed by sequencing a mitochondrial COI fragment from individuals collected over 24 years, and screening microsatellite variation for 32 populations across two ii seasons. A single COI haplotype predominated in samples from 2006/2007, but in the older collections (1983 and 1993) a different haplotype was most prevalent. This is consistent with successive population contractions and expansions, likely in response to alternate periods of drought and flood in the arid interior of Australia. The microsatellite data showed genetic differentiation between populations, evidence for movement between sites, and also genetic signatures of bottleneck events. The Simpson Desert, in central Australia was identified as a source of recent immigrants to populations in Biloela (m = 0.15, BAYESASS), eastern Australia, supporting the view that long distance migration is, indeed, a regular part of the ecology of this species. Together, these data highlight that since the advent of agriculture in Australia, green mirid dynamics are still shaped by its adaptations to arid, spatiotemporally variable environments. Previous ecological studies presented evidence that C. dilutus may be a complex of cryptic species, and that two such cryptic species may be associated with cotton and lucerne crop hosts. Further, C. dilutus has a reported preference for lucerne over cotton, leading to the proposal of lucerne as a trap crop in cotton production systems. To test this proposition I sampled C. dilutus individuals from adjacent cotton and lucerne crops at three geographically separate sites within a single season. Individual-based clustering analyses using microsatellite data showed that gene flow was high across these crop hosts. Further, gut content analysis indicated that a relatively high proportion of individuals collected from one crop host had fed on the alternate host and several individuals had fed on both. These data support the presence of one species associated with cotton and lucerne, but also show that green mirids will readily move between these two hosts despite their relative preference for lucerne. The findings outlined above are discussed in relation to ecological perceptions of generalist habits, the application of genetic techniques to the solution of ecological problems involving multiple host use, and the management and research implications arising from the data presented in this thesis. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968 . I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Peer Reviewed Papers Coleman RJ, Hereward JP, De Barro PJ, Frohlich DJ, Adamczyk JJ, Goolsby JA. (2008) Molecular comparison of Creontiades plant bugs from south Texas and Australia. Southwestern Entomologist 33 , 111-117. Characterisation of 12 polymorphic microsatellites in the green mirid, Creontiades dilutus Stål (Hemiptera: Miridae) Authors: James P. Hereward, Michael G. Gardner, Cynthia Riginos, Paul J. DeBarro, Andrew J. Lowe. Published as on-line PDF http://tomato.biol.trinity.edu/manuscripts/10- 6/mer-10-0251.pdf with summary published in: Andris M, Aradottir GI, Arnau G , et al. (2010) Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2010-31 July 2010. Molecular Ecology Resources 10 , 1106- 1108. Hereward JP, Walter GH. (2012) Molecular interrogation of the feeding behaviour of field captured individual insects for interpretation of multiple host plant use. PLoS One 7, e44435. The following two papers were produced while I was under part time candidature and were the result of paid work, the subject of each was relevant to my research, and their production contributed to the methods used in this thesis. Ridley AW, Hereward JP, Daglish GJ , Raghu S, Collins PJ, Walter GH . (2011) The spatiotemporal dynamics of Tribolium castaneum (Herbst): adult flight and gene flow. Molecular Ecology 20 , 1635-1646. Carter KD, Seddon JM, Carter JK, Goldizen AW, Hereward JP (2012) Development of 11 microsatellite markers for Giraffa camelopardalis through 454 pyrosequencing, with primer options for an additional 458 microsatellites. Conservation Genetics Resources 4, 943-945. v Publications included in this thesis Chapter 2.1 Characterisation of 12 polymorphic microsatellites in the green mirid, Creontiades dilutus Stål (Hemiptera: Miridae) Authors: James P. Hereward, Michael G. Gardner, Cynthia Riginos, Paul J. DeBarro, Andrew J. Lowe. Published as on-line PDF http://tomato.biol.trinity.edu/manuscripts/10- 6/mer-10-0251.pdf with summary