Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Post-release genetic assessment of two congeneric weed biological control agents Carson C. Keever a,b , Levent Gültekin c , Robert S. Bourchier d , Judith H. Myers e , Jenny S. Cory a, ⁎ a Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada b Department of Biology, Kwantlen Polytechnic University, 12666 72nd Avenue, Surrey, BC V3W 2MB, Canada c Biodiveristy Application and Research Center, Atatürk University, Erzurum 25240, Turkey d Agriculture and Agri-Food Canada, 5403-1 Avenue South, PO Box 3000, Lethbridge T1J 4B1, Alberta, Canada e Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC V6T 1Z4, Canada ARTICLE INFO Keywords: Population genetics Invasive species Larinus Curculionidae Hybridization Classical biological control Weed biocontrol Host range ABSTRACT Introductions of biological control agents are unique field experiments to examine the response of novel or- ganisms to new environments. Post-release monitoring is particularly challenging where closely related and morphologically similar biological control agents have been released. Two closely-related phytophagous wee- vils, Larinus minutus and L. obtusus, were introduced as biological control agents for two major rangeland weeds, diffuse knapweed, Centaurea diffusa and spotted knapweed, C. stoebe subsp. micranthos in North America. The release of the Larinus species coincided with a decline in C. diffusa abundance in many areas. However, it was not clear whether both species of Larinus had become established as they are morphologically very similar. We asked: (1) Could genetic markers be used to identify both the weevil species? (2) Do both weevil species attack both knapweed species in Canada? (3) Are the levels of genetic diversity of the introduced populations of the weevils similar to those of a European source population? (4) Is genetic mixing between the weevil species occurring? Both microsatellite and CO1 mtDNA markers distinguish between the two weevil species. Larinus obtusus was only found on spotted knapweed, while L. minutus was more widely distributed and attacked both weed species. The relatively large initial introductions of weevils to British Columbia (4,300 L. minutus and 5,500 L. obtusus) together with rapid population growth and frequent redistribution among sites has been sufficient to maintain levels of genetic diversity. Results from small samples showed that haplotype, nucleotide diversity and allelic richness of the introduced populations were comparable to those of a European population. We found no evidence for hybridization in the introduced populations. 1. Introduction The deliberate or accidental movement of organisms through trade, agriculture and other means has led to major global issues with invasive species that can have wide-ranging, negative economic and environ- mental impacts (Myers and Bazely, 2003; Lockwood et al., 2013). Un- derstanding the process and pathways of invasion, and the factors that promote establishment and spread of successful invaders are critical to predicting the potential distribution and impact of introduced species (Estoup and Guillemaud, 2010; Marsico et al., 2010; Fauvergue et al., 2012; Hopper et al., 2019). One approach is to study intentional in- vasions, such as biological control introductions (Roderick and Navajas, 2003; Van Driesche et al., 2008; Heimpel and Mills, 2017), in which it might be possible to separate the relative importance of genetic diversity (Myers and Sabath, 1980; Roderick and Navajas, 2003), re- lease sizes and numbers (Grevstad, 1999; Hufbauer et al., 2013), cli- matic factors (Robertson et al., 2008) and the impacts of co-invaders on successful establishment (Stephens et al., 2013). Classical (importation) biological control can be a very effective and environmentally sus- tainable approach to the long-term reduction of widespread invasive species and has been highly successful with many exotic weeds (e.g. McFadyen, 1998; Suckling, 2013; Myers and Cory, 2017; Hinz et al., 2019a, 2019b). While the success of biological control agents is evident from population declines of the target species, understanding the many complex factors leading to success (or failure (Myers, 2000)) is often less clear. Recent, but relatively rare, post-release studies have de- monstrated some of the complexity underlying successful classical biological control. This includes variation in the relative success of https://doi.org/10.1016/j.biocontrol.2020.104462 Received 27 April 2020; Received in revised form 14 October 2020; Accepted 16 October 2020 ⁎ Corresponding author. E-mail addresses: carson.c.keever@gmail.com (C.C. Keever), lgul@atauni.edu.tr (L. Gültekin), robert.bourchier@canada.ca (R.S. Bourchier), myers@zoology.ubc.ca (J.H. Myers), jsc21@sfu.ca (J.S. Cory). Biological Control 152 (2021) 104462 Available online 17 October 2020 1049-9644/ Crown Copyright © 2020 Published by Elsevier Inc. All rights reserved. T