1 2 3 4 Article type : Original Article 5 6 7 AID: 12307 8 9 ED: Karsten Schonrogge 10 AE: Gavin Broad 11 12 One generalist or several specialist species? Wide host range and diverse manipulations of 13 the hosts’ web building behaviour in the true spider parasitoid Zatypota kauros 14 (Hymenoptera: Ichneumonidae) 15 16 Running title: One generalist or several specialist species? 17 18 Stanislav Korenko*1, Tamara Spasojevic2,3, Stano Pekár4, Gimme. H. Walter5, Vlasta 19 Korenková6, Kateřina Hamouzová1, Michaela Kolářová1, Kristýna Kysilková1, Seraina 20 Klopfstein2,3 21 22 1 Department of Agroecology and Biometeorology, Faculty of Agrobiology, Food and Natural 23 Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6, Suchdol, 24 Czech Republic 25 2 Naturhistorisches Museum Bern, Abteilung Wirbellose Tiere, Bernastr. 15, CH-3005 Bern, 26 Switzerland 3 27 Institute of EcologyAuthor Manuscript and Evolution, University of Bern, Baltzerstr. 6, CH-3012 Bern, 28 Switzerland This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/icad.12307 This article is protected by copyright. All rights reserved 29 4 Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 30 611 37 Brno, Czech Republic 31 5 School of Biological Sciences, The University of Queensland, Brisbane QLD 4072, Australia 32 6 Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 33 20 Praha 4, Prague, Czech Republic 34 * Corresponding author: [email protected] 35 Abstract 36 1. Parasitoid wasps of the Polysphincta genus-group are highly specialised on their spider 37 hosts, and most of them are known to manipulate their hosts into building a special web in 38 which the parasitoid pupates. Trophic niche and the plasticity of host use were investigated 39 in the koinobiont parasitoid Zatypota kauros Gauld from Queensland, Australia. 40 2. We found that Z. kauros attacks spider hosts from different families, each differing widely 41 in their web-building behaviours, which makes it unique in the breadth of its host range. 42 Molecular analyses revealed that the taxon Zatypota kauros contains three divergent 43 mitochondrial lineages. Lineage A was associated exclusively with spiders of the genus 44 Anelosimus (Theridiidae), which builds tangle webs; lineage B was associated with the genus 45 Cyrtophora (Araneidae), which weaves tent webs; and lineage C was associated with a broad 46 range of hosts, including spiders of both the families Araneidae and Theridiidae. Unique host 47 manipulations could be observed in the web-building behaviours of the different host 48 groups. However, nuclear data from two ribosomal genes and three introns did not add any 49 support to the existence of different evolutionary lineages, nor did they coincide with the 50 different host groups. 51 3. The partial correspondence of mitochondrial lineage and host use, together with an 52 apparent mito-nuclear conflict might indicate maternal effects or very recent and/or 53 incomplete speciation in this taxon. Given their wide host range and intriguing interactions 54 with their hosts, the Z. kauros complex represents a promising system for studying parasitoid 55 specialization and its potential impact on speciation. 56 57 Keywords: behaviouralAuthor Manuscript manipulation – co-evolution – interactions – koinobionts – speciation 58 – silk– wasp – web architecture 59 60 Introduction This article is protected by copyright. All rights reserved 61 Understanding speciation is a major goal of evolutionary ecologists, but the processes 62 driving it remain poorly understood in most groups. This is also the case in insect parasitoids, 63 which have received only limited attention in speciation research (but see, e.g., Shaw, 2002; 64 Forbes et al., 2009; König et al., 2015), despite them constituting more than 5% of all 65 described species of multicellular organisms (Vié et al., 2009; Aguiar et al., 2013). Applying 66 ecological speciation theory (Schluter, 2009) to parasitoids, one would assume that the 67 major mechanisms associated with speciation are shifts in host-ranges and/or in the degree 68 of specialisation (e.g., Shaw, 2002). Unfortunately, detailed studies of the evolution of host 69 ranges in parasitoids are hampered by a lack of knowledge on the phylogeny of closely- 70 related species and on their host relations (even though there are notable exceptions, e.g., 71 (Shaw & Horstmann, 1997; Kankare et al., 2005; Tschopp et al., 2013). 72 The order Hymenoptera contains the most diversified parasitoids, both taxonomically 73 and ecologically (Eggleton & Belshaw, 1992). Some parasitoid species have been reported to 74 have wide host ranges which encompass insects or spiders from different families or even 75 orders, while other groups are much more specialized and have rather narrow host ranges 76 (Godfray, 1994, Yu et al., 2012). As a rule of thumb, the longer-lasting the association 77 between a parasitoid larva and its host, the higher the degree of specialization: species that 78 allow their hosts to continue their development, so-called koinobionts, tend to be more 79 specialized than idiobionts, which stop host development at the time of parasitization 80 (Althoff, 2003). 81 The Polysphincta genus-group (sensu Gauld & Dubois, 2006) (Ichneumonidae, 82 Pimplinae, Ephialtini) consists exclusively of koinobiont ectoparasitoids of spiders. Female 83 wasps of this group attach their eggs externally onto the opisthosoma or prosoma of spiders, 84 where the larvae manage to remain through several moult cycles of their hosts (Nielsen, 85 1923; Eberhard, 2000; Korenko et al., 2014). This strategy probably evolved from species 86 that feed, as larvae, on spider eggs, and the transition from egg mass scavengers to true 87 parasitoids of spiders probably occurred in a species that attacked the female of a hunter 88 spider species guarding its egg sac (Dubois et al., 2002; Gauld et al., 2002; Matsumoto, 89 2016). Author Manuscript 90 Polysphinctines are described as highly host-specific (e.g. Fitton et al., 1987; 91 Matsumoto, 2016) and exhibit a unique mode of host utilisation, including complex 92 behavioural repertoires (e.g. Eberhard, 2000; Matsumoto, 2009; Takasuka et al., 2017; This article is protected by copyright. All rights reserved 93 Messas et al. 2017; Korenko et al., 2018). Behavioural adaptations such as host manipulation 94 probably evolved through intimate interaction with the host behaviour and presumably 95 resulted in the restricted host ranges and high specificity of mutual interaction between 96 parasitoids and their spider hosts. Even though polysphinctines as a whole are associated 97 with spiders from different taxonomic groups utilizing different foraging techniques, the host 98 spectrum of a particular wasp species is usually restricted to a small group of taxonomically 99 closely-related species with similar behaviours. In all the species for which sufficient data are 100 available, the hosts always belong strictly to a single family (Fitton et al., 1987; Korenko et 101 al., 2011). The only exception is the recently studied Hymenoepimecis japi Loffredo & 102 Penteado-Dias, which is associated with orb-web building spiders from two families 103 (Araneidae and Tetragnathidae) (Messas et al., 2017). 104 The final instar larvae of polysphinctines have evolved an ability to manipulate the 105 web-spinning behaviour of the spider host shortly before the wasp pupates. They cause the 106 spider to construct a modified web structure enabling the wasp pupa to complete its 107 development in safe conditions (Kloss et al., 2016). Two different strategies can be 108 identified. In the first, the spider host is forced to build an additional or completely new, 109 three-dimensional silk barrier as a shelter for the wasp pupa (Matsumoto, 2009; Korenko & 110 Pekár, 2011; Korenko, et al., 2014). In the second, the manipulated spider reduces the 111 normal web to a simple and sparse, but strong silk construction (Eberhard, 2000; Eberhard, 112 2001; Korenko et al., 2015a; Kloss et al., 2016; Takasuka et al., 2015, 2017). These 113 modifications of the web structure into a more persistent cocoon web ascertain that the 114 wasp can pupate safely after the spider’s death (e.g. Kloss et al. 2016; Takasuka et al., 2015). 115 The type of behavioural manipulation in the spider host varies among groups and has to be 116 seen in connection to the spiders’ normal web building behaviour (e.g. Gonzaga et al., 2015). 117 Several studies have described behavioural manipulations of web-building spiders by 118 polysphinctine parasitoids from all over the world (Eberhard, 2000; Gonzaga et al., 2015; 119 Matsumoto, 2009; Korenko & Pekár, 2011; Korenko, 2016; Kloss et al., 2016; Takasuka et al., 120 2015, 2017). However, no such study was ever conducted in Australia, where 18 species of 121 the Polysphincta genusAuthor Manuscript -group have been reported to occur (Gauld, 1984). Certainly, this 122 number will increase given that several undescribed species are already known. Until now, 123 nothing was known about their ecology, including host association and behaviour. Here, we 124 describe for the first time host-parasitoid interactions in Zatypota kauros Gauld, an endemic This article is protected by copyright. All rights reserved 125 species in Australia. We focus on the host range of this parasitoid and on the behavioural 126 manipulations induced by the final instar larvae in their spider hosts, and find surprising 127 variability in both. By a combination of behavioural and molecular analyses, we investigate 128 whether the taxon currently known as Z. kauros represents a single, polyphagous and 129 behaviourally plastic species, or whether it constitutes a complex of more specialized 130 lineages. 131 132 Materials and methods 133 Field sampling 134 The host spectrum of Z.