Estimating Gene Flow between Refuges and Crops: A Case Study of the Biological Control of Eriosoma lanigerum by Aphelinus mali in Apple Orchards Blas Lavandero 1 *, Christian C. Figueroa 3 , Pierre Franck 2 , Angela Mendez 1 1 Laboratorio de Interacciones Insecto-Planta, Universidad de Talca, Talca, Chile, 2 Plantes et Syste ` mes de culture Horticoles, INRA, Avignon, France, 3 Facultad de Ciencias, Instituto de Ecologı ´a y Evolucio ´ n, Universidad Austral de Chile, Valdivia, Chile Abstract Parasitoid disturbance populations in agroecosystems can be maintained through the provision of habitat refuges with host resources. However, specialized herbivores that feed on different host plants have been shown to form host-specialized races. Parasitoids may subsequently specialize on these herbivore host races and therefore prefer parasitizing insects from the refuge, avoiding foraging on the crop. Evidence is therefore required that parasitoids are able to move between the refuge and the crop and that the refuge is a source of parasitoids, without being an important source of herbivore pests. A North-South transect trough the Chilean Central Valley was sampled, including apple orchards and surrounding Pyracantha coccinea (M. Roem) (Rosales: Rosacea) hedges that were host of Eriosoma lanigerum (Hemiptera: Aphididae), a globally important aphid pest of cultivated apples. At each orchard, aphid colonies were collected and taken back to the laboratory to sample the emerging hymenopteran parasitoid Aphelinus mali (Hymenoptera: Aphelinidae). Aphid and parasitoid individuals were genotyped using species-specific microsatellite loci and genetic variability was assessed. By studying genetic variation, natural geographic barriers of the aphid pest became evident and some evidence for incipient host-plant specialization was found. However, this had no effect on the population-genetic features of its most important parasitoid. In conclusion, the lack of genetic differentiation among the parasitoids suggests the existence of a single large and panmictic population, which could parasite aphids on apple orchards and on P. coccinea hedges. The latter could thus comprise a suitable and putative refuge for parasitoids, which could be used to increase the effectiveness of biological control. Moreover, the strong geographical differentiation of the aphid suggests local reinfestations occur mainly from other apple orchards with only low reinfestation from P. cocinnea hedges. Finally, we propose that the putative refuge could act as a source of parasitoids without being a major source of aphids. Citation: Lavandero B, Figueroa CC, Franck P, Mendez A (2011) Estimating Gene Flow between Refuges and Crops: A Case Study of the Biological Control of Eriosoma lanigerum by Aphelinus mali in Apple Orchards. PLoS ONE 6(11): e26694. doi:10.1371/journal.pone.0026694 Editor: Jeffrey A. Harvey, Netherlands Institute of Ecology, Netherlands Received May 19, 2011; Accepted October 3, 2011; Published November 2, 2011 Copyright: ß 2011 Lavandero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the International Foundation for Science Grant No C/4023-2 and FONDECYT (Project Number 11080013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: blavandero@utalca.cl Introduction Natural enemies of insect pests are constantly disturbed in agroecological systems, and classical management practices can severely reduce parasitoid populations. The use of habitat refuges, offering shelter and alternative hosts for these organisms, has been proposed for maintaining high density of parasitoids close to cultivated plants, acting as a constant source to control agricultural pests [1]. At larger scales, landscape heterogeneity has been proposed to have a positive effect on natural enemy populations and parasitism rates in general [2]. Nevertheless, one must have enough evidence that parasitoids do disperse between the refuges and the crop, and that they exert an effect on the herbivore populations. Ecological specialization of herbivore insects could affect their relationship with the third trophic level. Specialist herbivores that feed on different host plants have been shown to form host- specialized races, evidenced through reduced migration and gene flow [3]. The effect on the next trophic level (the natural enemies) can follow the specialization of their herbivore host, resulting in the formation of specialized parasitoid races, in a process termed sequential radiation [4]. In fact, as herbivorous insects and their parasitoids interact with their environment on a fine spatial and temporal scale, sequential radiation may be quite common [5]. Thus, parasitoids coming from a refuge may not readily forage on the crop or they may be totally isolated if gene flow between the refuge and the crop is absent, in which case the refuge would not constitute a real source of parasitoids for improving biocontrol. Genetic markers, particularly highly polymorphic ones such as microsatellites, have been widely used to study several aspects of insect ecology. These DNA markers provide the raw data to estimate genetic diversity and gene flow between insect popula- tions or to reconstruct migration routes and colonization history. Using appropriate bioinformatic tools to analyze DNA marker data, gene flow and genetic diversity within insect species can be quantified, which is critical for explaining population structure and dynamics in time and space (for a review see [6]). For instance, microsatellites in combination with powerful analytical tools [7] have proven to be useful for describing movement of insect pests between continents (for the western corn rootworm see [8]; for the PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26694