Contrary to patterns observed at the broader spatial scales of my study, I observed strong genetic structure of W. smithii at the finest spatial scale, among leaves within a plant. Patterns of significant genetic differentiation among leaves (but not at larger spatial scales) most likely reflect patterns of female oviposition in the current year. Significant genetic differentiation among pitchers of a plant would arise if the larvae within each leaf are related, for example if they are siblings, and if female mosquitoes are laying their eggs in clumps within individual pitchers rather than dispersing single eggs among multiple pitchers or plants. In support of this hypothesis, the mean pairwise relatedness values between individual larvae, calculated at various scales in each peatland, indicated that relatedness of pairs of individuals was highest at the leaf compared to the broader scales (Figures 3.5 and 3.6). This pattern, and the possibility of female W. smithii depositing multiple eggs (i.e., offspring related as siblings or half-siblings) in the same pitcher, is not consistent with the suggestion of Heard (1994b) that females typically lay their eggs singly within pitchers. Others, however, have reported placement of multiple eggs into a single pitcher by W. smithii females (Bradshaw 1983). Differences between my results and the observations of Heard (1994b) may relate to differing habitat characteristics and therefore female mosquito behaviour, in the peatlands of Algonquin Provincial Park versus those of Atlantic Canada, where his study was performed. The differences may also relate to the shorter time frame encompassed in Heard‟s (1994b) study, which involved experimental manipulation of pitcher quality and recording of female oviposition over 3 days, compared to my study, in which larvae were collected after several possible weeks of natural female oviposition in the field. I hypothesized that in response to low pitcher plant density, female W. smithii would aggregate more of their eggs into single leaves, in order to avoid moving long distances to oviposit, as observed in the midge M. knabi (Rasic & Keyghobadi 2012b). As predicted, I did
find a significant negative relationship between an index of aggregation, or „clumping‟ of related larvae within leaves, and pitcher plant density (Figure 3.7). However, this relationship was driven almost entirely by a single peatland, West Rose, which had both a much higher degree of aggregation of related larvae within leaves, and a much lower plant density than all other peatlands in my study (Figure 3.8). Therefore, my analysis of the effect of pitcher plant density on oviposition patterns of female W. smithii is inconclusive. Further study, using more peatlands with low plant density, is required to resolve the relationship.
Interestingly, West Rose peatland stood out in my study for a number of reasons. In addition to driving the relationship between relatedness of larvae within leaves and pitcher plant density, W. smithii samples from West Rose peatland also had higher genetic diversity, lower overall relatedness of individuals, and higher genetic differentiation from other peatlands. For M. knabi, samples from West Rose peatland were also highly differentiated from all other peatlands, even nearby ones (Rasic & Keyghobadi 2012b). West Rose peatland differs from all other study peatlands in that it is essentially an island mat of sphagnum surrounded by water, in addition to having very low plant density compared to the other peatlands. These factors may be influencing genetic structure of W. smithii as well as M. knabi. Further sampling of peatlands with similarly low pitcher plant densities and/or isolation by water would be helpful in determining whether these are indeed the factors contributing to the unusual genetic make-up of insect populations in West Rose peatland.
Summary
I elucidated the population genetic structure of the pitcher plant mosquito W. smithii using 117 AFLP markers derived from four selective primer combinations. Unexpectedly, there was very little structure and very low genetic differentiation among W. smithii samples at all scales, save for among leaves within plants. Although W. smithii is traditionally thought to be a poor disperser, my results indicate that there are likely high levels of gene flow among peatlands even up to 26 km apart. I inferred higher levels of movement and gene flow at larger scales in W. smithii than has been observed in the other pitcher plant insect inquilines, the midge M. knabi and the flesh fly F. fletcheri. Among these insects, it appears that body size is not a predictor of dispersal ability. High gene flow among peatlands could be a consequence of wind-assisted dispersal of W. smithii, a hypothesis that warrants further study.
In association with significant genetic differentiation among samples of W. smithii collected from different leaves of the same plant, the average degree of relatedness of pairs of individuals was greatest at the leaf scale, compared to the broader scales. These results suggest that female mosquitoes are laying their eggs in clumps within individual leaves instead of dispersing eggs singly among multiple pitchers. I observed a significant negative relationship between pitcher plant density and the degree of clumping of eggs within leaves (i.e., the degree relatedness of larvae within leaves relative to broader scales), but this was driven primarily by West Rose peatland, which had both a much lower pitcher plant density than all other peatlands in my study and showed much higher relatedness of larvae at the leaf scale compared to broader scales. West Rose peatland was also unusual in that W. smithii samples from that peatland displayed high genetic diversity, low relatedness of individuals, and high differentiation from other peatlands (FST values), which perhaps could be explained by the peatland‟s lower pitcher plant density and isolation by water. Therefore, the relationship between plant density and egg clumping is inconclusive; future studies should sample mosquitoes from peatlands with a similarly low plant density as West Rose peatland, in order to resolve this unclear relationship.
References
Adams RM, Smith GW (1977) SEM survey of 5 carnivorous pitcher plant genera. American Journal of Botany 64: 265-272.
Addicott JF (1974) Predation and prey community structure – experimental study of effect of mosquito larvae on protozoan communities of pitcher plants. Ecology 55: 475-492. Allan GJ, Max TL (2010) Molecular genetic techniques and markers for ecological research.
Nature Education Knowledge 1: 2.
Armbruster P, Bradshaw WE, Holzapfel CM (1998) Effects of postglacial range expansion on allozyme and quantitative genetic variation of the pitcher-plant mosquito, Wyeomyia smithii. Evolution 52: 1697-1704.
Bohonak AJ (1999) Gene flow, and population structure. The Quarterly Review of Biology 74: 21-45.
Bonin A, Bellemain E, Eidesen PB, Pompanon F, Brochmann C, Taberlet P (2004) How to track and assess genotyping errors in population genetics studies. Molecular Ecology 13: 3261-3273.
Bonin A, Taberlet P, Miaud C, Pompanon F (2006) Explorative genome scan to detect candidate loci for adaptation along a gradient of altitude in the common frog (Rana temporaria). Molecular Biology and Evolution 23: 773-783.
Bossart JL, Prowell DP (1998) Genetic estimates of population structure and gene flow: limitations, lessons and new directions. Trends in Ecology and Evolution 13: 202- 206.
Bowler DE, Benton TG (2005) Causes and consequences of animal dispersal strategies. Biological Reviews 80: 205-225.
Bradshaw WE (1983) Phytotelmata: Terrestrial Plants as Hosts for Aquatic Insect Communities. Plexus Publishing, Medford, NJ.
Bradshaw WE, Armbruster PA, Holzapfel CM (1998) Fitness consequences of hibernal diapause in the pitcher-plant mosquito, Wyeomyia smithii. Ecology 79:1458-1462. Bradshaw WE, Creelman RA (1984) Mutualism between the carnivorous purple pitcher plant
and its inhabitants. American Midland Naturalist 112: 294-304.
Bradshaw WE, Holzapfel CM (2001) Genetic shift in photoperiodic response correlated with global warming. Proceedings of the National Academy of Sciences of the United States of America 98: 14509-14511.
Bradshaw WE, Holzapfel CM (2008) Genetic response to rapid climate change: it's seasonal timing that matters. Molecular Ecology 17: 157-166.
Bradshaw WE, Lounibos LP (1977) Evolution of dormancy and its photoperiodic control in pitcher-plant mosquitoes. Evolution 31: 546-567.
Buckley HL, Burns JH, Kneitel JM, Walters EL, Munguía P, Miller TE (2004) Small-scale patterns in community structure of Sarracenia purpurea inquilines. Community Ecology 5: 181-188.
Bullock JM, Kenward RE, Hails RS (2002) Dispersal Ecology. Blackwell Science Ltd, Malden.
Byrne DN, Rathman RJ, Orum TV, Palumbo JC (1996) Localized migration and dispersal by the sweet potato whitefly, Bemisia tabaci. Oecologia 105: 320-328.
Cadotte MW, Fukami T (2005) Dispersal, spatial scale, and species diversity in a hierarchically structured experimental landscape. Ecology Letters 8: 548-557. Chapin CT, Pastor J (1995) Nutrient limitations in the northern pitcher plant Sarracenia-
purpurea. Canadian Journal of Botany 73: 728-734.
Chapman JW, Reynolds DR, Smith AD, Riley JR, Telfer MG, Woiwod IP (2005) Mass aerial migration in the carabid beetle Notiophilus biguttatus. Ecological Entomology 30: 264-272.
Charlesworth B (2009) Effective population size and patterns of molecular evolution and variation. Nature Reviews Genetics 10: 195-205.
Crawford LA, Desjardins S, Keyghobadi N (2011) Fine-scale genetic structure of an endangered population of the Mormon metalmark butterfly (Apodemia mormo) revealed using AFLPs. Conservation Genetics 12: 991-1001.
Croteau EK (2010) Causes and consequences of dispersal in plants and animals. Nature Education Knowledge 1: 12.
Dahlem GA, Naczi RFC (2006) Flesh flies (Diptera: Sarcophagidae) associated with North American pitcher plants (Sarraceniaceae), with descriptions of three new species. Annals of the Entomological Society of America 99: 218-240.
Ehrich D (2006) AFLPDAT: a collection of R functions for convenient handling of AFLP data. Molecular Ecology Notes 6: 603-604.
Emerson KJ, Merz CR, Catchen JM, Hohenlohe PA, Cresko WA, Bradshaw WE, Holzapfel CM (2010) Resolving postglacial phylogeography using high-throughput sequencing. Proceedings of the National Academy of Sciences of the United States of America 107: 16196-16200.
Fish D, Hall DW (1978) Succession and stratification of aquatic insects inhabiting leaves of insectivorous pitcher plant, Sarracenia-purpurea. American Midland Naturalist 99: 172-183.
Frankham R, Ballou JD, Briscoe DA (2010) Introduction to Conservation Genetics, 2nd ed. Cambridge University Press, Cambridge.
Freeland JR (2005) Molecular Ecology. Wiley, West Sussex.
Gallie DR, Chang SC (1997) Signal transduction in the carnivorous plant Sarracenia purpurea - Regulation of secretory hydrolase expression during development and in response to resources. Plant Physiology 115: 1461-1471.
Giberson DJ, Hardwick ML (1999) Pitcher plants (Sarracenia purpurea) in eastern Canadian peatlands: ecology and conservation of the invertebrate inquilines. In “Invertebrates in Freshwater Wetlands of North America: Ecology and Management”. pp. 401- 422. Batzer DP, Rader RB, Wissinger SA (eds.) John Wiley and Sons, Inc. New York.
Gotelli NJ, Smith AM, Ellison AM, Ballif BA (2011) Proteomic characterization of the major arthropod associates of the carnivorous pitcher plant Sarracenia purpurea. Proteomics 11: 2354-2358.
Goudet J (2005) Hierfstat, a package for R to compute and test hierarchical F-statistics. Molecular Ecology Notes 5: 184-186. Latest version available at
http://www.unil.ch/popgen/softwares/hierfstat.htm.
Hagler JR, Jackson CG (2001) Methods for marking insects: current techniques and future prospects. Annual Review of Entomology 46: 511-543.
Hamilton MB (2009) Population Genetics. John Wiley & Sons, Oxford.
Hamilton R, Duffield RM (2002) Novel observations of midge and mosquito larval
population dynamics in leaves of the northern pitcher plant, Sarracenia purpurea L. Hydrobiologia 482: 191-196.
Hanski I (1999) Metapopulation Ecology. Oxford University Press, Oxford.
Hardy OJ (2003) Estimation of pairwise relatedness between individuals and characterization of isolation-by-distance processes using dominant genetic markers. Molecular
Ecology 12: 1577-1588.
Hartl DL, Clark AG (2007) Principles of Population Genetics, 4th ed. Sinauer & Associates, Sunderland MA.
Harvey E, Miller TE (1996) Variance in composition of inquiline communities in leaves of Sarracenia purpurea L. on multiple spatial scales. Oecologia 108: 562-566.
Heard SB (1994a) Pitcher-plant midges and mosquitoes – a processing chain commensalism. Ecology 75: 1647-1660.
Heard SB (1994b) Imperfect oviposition decisions by the pitcher plant mosquito (Wyeomyia smithii). Evolutionary Ecology 8: 493-502.
Hendry AP, Taylor EB, McPhail JD (2007) Adaptive divergence and the balance between selection and gene flow: lake and stream stickleback in the misty system. Evolution 56: 1199-1216.
Hoekman D, Winston R, Mitchell N (2009) Top-down and bottom-up effects of a processing detritivore. Journal of the North American Benthological Society 28: 552-559.
Holsinger KE, Weir BS (2009) Genetics in geographically structured populations: defining, estimating and interpreting FST. Nature Reviews Genetics 10: 639- 650.
Huffaker CB (1958) Experimental studies on predation: dispersion factors and predator-prey oscillations. Hilgardia 27: 83.
Istock CA, Weisburg WG (1987) Strong habitat selection and the development of population structure in a mosquito. Evolutionary Ecology 1: 348-362.
Jenkins DG, Brescacin CR, Duxbury CV, Elliott JA, Evans JA, Grablow KR, Katherine R, Hillegass M, Lyon BN, Metzger GA, Olandese ML, Pepe D, Silvers GA, Suresch HN, Hiliary N, Thompson TN, Trexler CM, Williams GE, Williams NC, Williams SE (2007) Does size matter for dispersal distance? Global Ecology and Biogeography 16: 415-425.
JMP®, Version 8.0.1.SAS Institute Inc., Cary, NC, 1989-2007.
Juniper BE, Robins RJ, Joel DM (eds.) (1989) The Carnivorous Plants. Academic Press, London.
Kareiva P (1987) Habitat fragmentation and the stability of predator-prey interactions. Nature 326: 388-390.
Keddy PA (2010) Wetland Ecology: Principles and Conservation, Second Edition. Cambridge University Press, Cambridge.
Keyghobadi N, Roland J, Fownes S, Strobeck C (2003) Ink marks and molecular markers: examining effects of landscape on dispersal using both mark-recapture and molecular methods. In "Ecology and Evolution Taking Flight: Butterflies as Model Study Systems". pp. 169-183. University of Chicago Press.
Kingsolver JG (1979) Thermal and hydric aspects of environmental heterogeneity in the pitcher plant mosquito. Ecological Monographs 49: 357-376.
Kneitel JM, Miller TE (2002) Resource and top-predator regulation in the pitcher plant (Sarracenia purpurea) inquiline community. Ecology 83: 680-688.
Kneitel JM, Miller TE (2003) Dispersal rates affect species composition in metacommunities of Sarracenia purpurea inquilines. American Naturalist 162: 165-171.
Krawchuk MA, Taylor PD (2003) Changing importance of habitat structure across multiple spatial scales for three species of insects. Oikos 103: 153-161.
Levins R (1969) Some demographic and genetic consequences of environmental
heterogeneity for biological control. Bulletin of the Entomological Society of America 15: 237–240.
Lindsay SW, Armstrong JRM, Schellenberg JRMA, Zeiler HA, Daly RJ, Salum FM, Wilkins HA (1995) Exposure to Gambian children to Anopheles-gambiae malaria vectors in an irrigated rice production area. Medical and Veterinary Entomology 9: 50-58.
Lynch M, Milligan BG (1994) Analysis of population genetic-structure with RAPD markers. Molecular Ecology 3: 91-99.
Manel S, Schwartz MK, Luikart G, Taberlet P (2003) Landscape genetics: combining
landscape ecology and population genetics. Trends in Ecology and Evolution 18: 189- 197.
Mantel N (1967) Detection of disease clustering and a generalized regression approach. Cancer Research 27: 209-220.
Matter SF, Roland J (2010) Local extinction synchronizes population dynamics in spatial networks. Proceedings of the Royal Society of London, B 277: 729- 737.
Mech DL (1983) Handbook of Animal Radio-Tracking. University of Minnesota Press. Meglecz E, Anderson SJ, Bourguet D, Butcher R, Caldas A, Cassel-Lundhagen A, Coeur
d'Acier A, Dawson DA, Faure N, Fauvelot C, Franck P, Harper G, Keyghobadi N, Kluetsch C, Muthulakshmi M, Nagaraju J, Patt A, Petenian F, Silvain JF, Wilcock HR (2007) Microsatellite flanking region similarities among different loci within insect species. Insect Molecular Biology 16: 175-185.
Meudt HM, Clarke AC (2007) Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends in Plant Science 12: 106-117.
Miner JA, Taylor PD (2002) Effects of peatland size and exposure on two species of Diptera inhabiting the pitcher plant Sarracenia purpurea L. Ecoscience 9: 347- 354.
Mitsch WJ, Gosselink JG (2007) Wetlands, Fourth Edition. John Wiley & Sons, Inc., New Jersey.
Mueller UG, Wolfenbarger LL (1999) AFLP genotyping and fingerprinting. Trends in Ecology & Evolution 14: 389-394.
Nastase AJ, De La Rosa C, Newell SJ (1995) Abundance of pitcher-plant mosquitoes, Wyeomyia smithii (Coq.) (Diptera, Culicidae) and midges, Metriocnemus knabi Coq.(Diptera, Chironomidae), in relation to pitcher characteristics of Sarracenia purpurea L. American Midland Naturalist 133: 44-51.
Osborne JL, Clark SJ, Morris RJ, Williams IH, Riley JR, Smith AD, Reynolds DR, Edwards AS (1999) A landscape-scale study of bumble bee foraging range and constancy, using harmonic radar. Journal of Applied Ecology 36: 519-533.
Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288-295.
Peterson MA, Denno RF (1998) The influence of dispersal and diet breadth on patterns of genetic isolation by distance in phytophagous insects. American Naturalist 152: 428- 446.
Ragland GJ, Kingsolver JG (2008) Evolution of thermotolerance in seasonal environments: the effects of annual temperature variation and life-history timing in Wyeomyia smithii. Evolution 62: 1345-1357.
Rasic G. “A model system in landscape genetics – the insect inhabitants of pitcher plants.” Ph.D. dissertation, The University of Western Ontario, 2011.
Rasic G, Keyghobadi N (2012a) The pitcher plant flesh fly exhibits a mixture of patchy and metapopulation attributes. Journal of Heredity 103: 703-710.
Rasic G, Keyghobadi N (2012b) From broadscale patterns to fine-scale processes: habitat structure influences genetic differentiation in the pitcher plant midge across multiple spatial scales. Molecular Ecology 21: 223-236.
R Development Core Team (2012) http://www.r-project.org
Schaal BA, Hayworth DA, Olsen KM, Rauscher JT, Smith WA (1998) Phylogeographic studies in plants: problems and prospects. Molecular Ecology 7: 465-474.
Schlotterer C (2004) The evolution of molecular markers – just a matter of fashion? Nature Reviews Genetics 5: 63-69.
Selkoe KA, Toonen RJ (2006) Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters 9: 615-629.
Slatkin M (1985) Gene flow in natural populations. Annual Review of Ecology and Systematics 16: 393-430.
Spitzer K, Danks HV (2006) Insect biodiversity of boreal peat bogs. Annual Review of Entomology 51: 137-161.
Srivastava DS, Kolasa J, Bengtsson J, Gonzalez A, Lawler SP, Miller TE, Munguia P, Romanuk T, Schneider DC, Trzcinski MK (2004) Are natural microcosms useful model systems for ecology? Trends in Ecology & Evolution 19: 379-384.
Stenseth NC, Lidicker Jr WZ (1992) Animal Dispersal. Small mammals as a model. Chapman & Hall, New York.
Steward CC, McWade JW (1960) The mosquitoes of Ontario (Diptera: Culicidae) with keys to the species and notes on distribution. Proceedings of the Entomological Society of Ontario 91: 121-188.
Trzcinski MK, Walde SJ, Taylor PD (2003) Colonisation of pitcher plant leaves at several spatial scales. Ecological Entomology 28: 482-489.
Vekemans X, Beauwens T, Lemaire M, Roldan-Ruiz I (2002) Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size. Molecular Ecology 11: 139-151.
Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP – a new technique for DNA- fingerprinting. Nucleic Acids Research 23: 4407-4414.
Vucetich JA, Waite, TA, Nunney L (1997) Fluctuating population size and the ratio of effective to census population size. Evolution 51: 2017-2021.
Wallis RC, Frempong-Boadu J (1967) Colonization of Wyeomyia smithii (Coquillett) from Connecticut. Mosquito News 27: 9-11.
Wang RJ, Ovaskainen O, Cao YD, Chen HQ, Zhou Y, Xu CR, Hanski I (2011) Dispersal in the Glanville fritillary butterfly in fragmented versus continuous landscapes:
comparison between three methods. Ecological Entomology 36: 251-260.
Whitlock MC, McCauley DE (1999) Indirect measures of gene flow and migration: FST not equal 1/(4Nm+1). Heredity 82: 117-125.
Whitlock R, Hipperson H, Mannarelli M, Butlin RK, Burke T (2008) An objective, rapid, and reproducible method for scoring AFLP peak-height data that minimizes genotyping error. Molecular Ecology Resources 8: 725–735.
Wiens JA (1989) Spatial scaling in ecology. Functional Ecology 3: 385-397. Wolf PG, Doche B, Gielly L, Taberlet P (2004) Genetic structure of Rhododendron
Wright S (1931) Evolution in Mendelian populations. Genetics 16: 97-159.
Wright S (1946) Isolation by distance under diverse systems of mating. Genetics 31: 39-59. Wright S (1965) The interpretation of population structure by F-statistics with special regard
to systems of mating. Evolution 19: 395-420.
Zabeau M, Vos P (1993) Selective restriction amplification: a general method for DNA fingerprinting. European Patent Publication 92402629 (Publication No.
EP0534858A1).
Zani PA, Cohnstaedt LW , Corbin D, Bradshaw WE, Holzapfel CM (2005) Reproductive value in a complex life cycle: heat tolerance of the pitcher-plant mosquito, Wyeomyia smithii. Journal of Evolutionary Biology 18: 101-105.