Enhancing Biological Control against pests

André Filipe Fidalgo Casquilho Garcia

SCIENTIFIC ADVISORS:

Doutora Manuela Rodrigues Branco Simões

Doutor José Carlos Franco Santos Silva

THESIS PRESENTED TO OBTAIN THE DOCTOR DEGREE IN FORESTRY ENGINEERING AND NATURAL RESOURCES

2020

Enhancing Biological Control against Eucalyptus pests

André Filipe Fidalgo Casquilho Garcia

SCIENTIFIC ADVISORS: Doutora Manuela Rodrigues Branco Simões Doutor José Carlos Franco Santos Silva, Professor Auxiliar do Instituto Superior de Agronomia da Universidade de Lisboa

Jury

President: Doutora Maria Margarida Branco de Brito Tavares Tomé, Professora Catedrática do Instituto Superior de Agronomia da Universidade de Lisboa.

Members: Doutor Zvi Mendel, Full Professor aposentado do Agricultural Research Organization (ARO), Volcani Center, Israel; Doutora Maria Rosa Santos Paiva, Professora Catedrática da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa; Doutor António Maria Marques Mexia, Professor Catedrático do Instituto Superior de Agronomia da Universidade de Lisboa; Doutora Manuela Rodrigues Branco Simões, Professora Associada com Agregação do Instituto Superior de Agronomia da Universidade de Lisboa, orientadora; Doutora Elisabete Tavares Lacerda de Figueiredo Oliveira, Professora Auxiliar do Instituto Superior de Agronomia da Universidade de Lisboa.

THESIS PRESENTED TO OBTAIN THE DOCTOR DEGREE IN FORESTRY ENGINEERING AND NATURAL RESOURCES

Instituições Financiadoras e âmbito: Programa de doutoramento FCT (Sustainable Forests and Products, SUSFOR) - PD/BD/52693/2014

2020

Agradecimentos

Ao aproximar-se o fim de mais um capítulo da minha vida académica e pessoal, não poderia, nem posso deixar escapar a oportunidade de agradecer àqueles que de alguma forma me apoiaram, contribuíram e proporcionaram esta oportunidade. Sem o vosso apoio provavelmente não teria conseguido chegar até este momento. Ver o que vi, experenciar cenários novos e poder enfrentar os meus maiores medos. Agradeço à minha família, pai, mãe todo o apoio que me deram e têm dado ao longo da minha vida. Por me terem ajudado a superar aqueles momentos mais dificeis que vivi durante o período da tese. E, que apesar das emoções à flor da pele, provocadas pela distância ou pelo stress, sempre foram um dos meus pilares. À minha irmã e cunhado por todo o apoio que me deram quando mais precisei. E, por terem ficado perto a cuidar da família quando estive longe. Agradeço também, à minha namorada Cristina, que desde o ínicio desta etapa sempre me apoiou e incentivou a seguir atrás dos meus sonhos. E que mesmo sabendo dos diversos perigos, esteve sempre ao meu lado, dando-me apoio, conforto, e apaziguando aquela dor na alma. Provocada pela distância, por vezes, não só física mas também pelas experiencias menos positivas superadas. Foi sem dúvida, o meu porto de abrigo e o meu pilar! Sei que muitas vezes foste preterida, em determimento da tese. Mas o nosso amor mostrou ser mais forte! Obrigado por estares sempre a meu lado, dando o teu apoio. À minha colega e grande amiga Vera Zina por todo a companhia dada ao longo destes anos no ISA. Pela sua ajuda na construção de modelos para apresentações, devido ao seu espirito crítico e criativo. Mas acima de tudo pela amizada e partilha de ideias. Ao meu colega e amigo Gonçalo Duarte, pela arte dada a esta tese através das caricaturas de cada capítulo. E, pela partilha de ideias para vários delineamentos experimentais. Ao prof. Geoff Allen da Universidade da Tasmânia (UTAS), pelo acolhimento e apoio dado durante a minha estadia na Tasmânia. E, pela disponibilização do laboratório de entomologia para a manutenção das colónias. Ao Dr. David de Little, pelas indicações e avisos sobre os locais a prospectar. Ao Dr. Rolf Oberprieler e à Debbie Jennings por me terem recebido na ANIC – CSIRO. Pelo seu apoio logístico. E pelos ensinamentos do dr. Rolf Oberprieler na identificação dos “Gonipterus”. À Alice e Eduardo Gouveia, pelo suporte e apoio que me deram durante o período em que estive na Tasmânia. Aos meus colegas de casa, na Tasmânia, Henry e Mohammed. Ao Hugo Gonçalves, pela partilha de experiência, ensinamentos e apoio dado durante um período importante da tese. À Catarina Reis, à Joana Martins, à Ana Farinha e ao Pedro Nunes pelo apoio dado ao ouvirem os meus desabafos e pela ajuda dada na contagem dos insectos e na montagem dos ensaios de campo.

À Catarina Afonso, Artur Sarmento pelo cuidado que tiveram com os “Gonipterus” e por terem contabilizado os parasitóides. Quero agradecer também ao Dr. Carlos Valente, à Dra.Catarina Gonçalves, ao Dr. Nuno Borralho, do RAIZ (The Navigator Company) à Eng. Ana Reis, ao Eng. Luis Leal, da Altri Florestal, por terem apoiado este meu doutoramento desde a sua construção. Às empresas The Navigator Company, através do RAIZ, e à Altri Florestal por terem apoiado financeiramente o desenvolvimento desta tese, com a fantástica oportunidade de explorar as antípodas na busca por novos agentes de controlo biológico contra o “Gonipterus”. À CELPA pelo financiamento para manutenção do arboreto de eucaliptos e, em particular ao Eng. Armando Goes e à prof. Paula Soares. Ao programa doutoral Sustainable Forests and Products – SUSFOR (PD/BD/52693/2014; PD/BD/52693/2014), e projectos UID/AGR/00239/2013 e UID/AGR/00239/2019, financiados pela Fundação para a Ciência e Tecnologia (FCT). Ao projecto Europeu - HOlistic Management of Emerging forest pests and Diseases (HOMED – proj. nº 771271), pelo apoio financeiro durante parte da elaboração da tese. Ao professor José Carlos Franco, pela disponibilidade que sempre teve comigo quando precisei de conversar sobre o desenvolvimento prático dos ensaios de campo. Por mostrar a sua visão do objectivo a alcançar. E, pelo incentivo e calma transmitida naqueles momentos mais stressantes. Por fim, e independentemente de aparecer nesta posição dos agradecimentos, este é sem dúvida um dos maiores e mais calorosos agradecimentos. Esta pessoa é, aquela que ao longo dos últimos anos me acarinhou e moldou profissionalmente neste instituto. Deu- me “toda” a liberdade para delinear, gerir, explorar, descobir novas espécies de insectos em Portugal. Mas, ao mesmo tempo, tentava manter-me o foco por forma a concretizar os objectivos de cada tarefa. A sí, professora Manuela Branco, tenho de lhe agradecer por todas as oportunidades de crecimento pessoal e profissional. A sí, obrigado por todo o apoio dado na construção desta tese. A todos o meu muito obrigado!

Index

Abstract ...... I Resumo ...... II Resumo alargado ...... III Chapter 1 ...... 1 General introduction ...... 2 Control of invasive species ...... 4 Biological control ...... 7 Classical Biological Control ...... 7 Conservation biological control...... 12 Augmentative biological control ...... 13 Why Eucalyptus accumulated so many invasive species? ...... 14 Objectives ...... 19 References ...... 20 Chapter 2 ...... 33 First report of burwelli in Europe, a new invasive attacking eucalypts ...... 33 Abstract ...... 34 Case study ...... 34 Acknowledgments ...... 38 References ...... 39 Chapter 3 ...... 41 Biological control of Gonipterus: Uncovering the associations between eucalypts, weevils and parasitoids in their native range ...... 41 Abstract ...... 42 Introduction ...... 43 Material and methods ...... 47 Study area and surveys ...... 47 Site selection and field sampling ...... 47 Identification of Gonipterus specimens and egg parasitoids ...... 48 DNA extraction, sequencing and phylogenetic tree construction ...... 49 Parasitoid-Gonipterus-Eucalyptus associations ...... 51 Results and discussion ...... 52 Gonipterus species in Tasmania ...... 52

Phylogenetic relationships of Gonipterus species ...... 53 Egg parasitoids of Gonipterus species in Tasmania...... 54 Identified Eucalyptus-Gonipterus-parasitoid trophic associations ...... 56 Discussion ...... 61 Gonipterus species in Tasmania ...... 61 Phylogenetic relationships of Gonipterus species ...... 62 Egg parasitoids of Gonipterus species in Tasmania...... 62 Identified trophic associations: Eucalyptus - Gonipterus - egg parasitoid...... 64 Conclusion ...... 66 Acknowledgments ...... 67 References ...... 68 Supplementary material ...... 74 Chapter 4 ...... 107 Ophelimus sp., a new invasive gall wasp of in Europe, escapes the parasitism by chamaeleon due to an asynchronous life cycle...... 107 Abstract ...... 108 Introduction ...... 109 Materials and Methods ...... 110 Origin of Ophelimus spp. and Closterocerus chamaeleon ...... 110 Ophelimus sp...... 111 Closteroceus chamaeleon ...... 111 Data analysis ...... 113 Results ...... 114 Ophelimus sp...... 114 Closterocerus chamaeleon ...... 116 Discussion ...... 119 Ophelimus sp...... 119 Closterocerus chamaeleon ...... 119 Acknowledgements ...... 122 References ...... 123 Chapter 5 ...... 127 Novel prey boosts the expansion of host-plant range in a native predatory bug...... 127 Abstract ...... 128 Introduction ...... 129

Materials and methods ...... 130 Anthocoris nemoralis oviposition on Eucalyptus ...... 130 Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves .. 131 Results ...... 132 Survey of Anthocoris nemoralis on Eucalyptus trees ...... 132 Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves .. 132 Discussion ...... 134 Survey of Anthocoris nemoralis on Eucalyptus trees ...... 134 Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves .. 135 Acknowledgements ...... 137 References ...... 138 Chapter 6 ...... 141 Discussion and Conclusions ...... 142 Fortuitous biological control ...... 142 Classical biological control: the relevance of the trophic interactions in the native range ...... 143 Phenology and efficiency of a biocontrol agent ...... 144 Conservation biological control: can native predators be part of the solution? ...... 145 References ...... 147

Abstract

We investigated different biological control tactics against eucalypts pests, in four case studies: a) fortuitous introduction of a biocontrol agent; b) survey of new potential biocontrol agents for classical biological control; c) possible use of a parasitoid in the biological control of a new pest, which was previously introduced to cope with a congener pest species; and d) use of a native predator in conservation biological control. In the first study we report the presence of the gall wasp in Portugal (first record in Europe and Palearctic region), infesting leaves of , in four out of seven sampled locations, along Tagus river, between Lisbon and Castelo Branco. A new parasitoid species for Europe, Closterocerus sp. was found parasitizing E. burwelli. Classical biological control of Gonipterus platensis with Anaphes nitens proved to be insufficient in Portugal. A study on trophic interactions was carried out in Tasmania for the first time, for the selection of new potential biocontrol agents against G. platensis. Six Gonipterus species were identified. Five parasitoid species were collected from Gonipterus egg pods. No significant host specialization was observed between Gonipterus and egg parasitoids. Gonipterus platensis was mainly found on Eucalyptus ovata. In a third study, we demonstrated that Closterocerus chamaeleon, parasitoid of is able to complete development in Ophelimus sp., a gall wasp newly found in Southern Europe attacking E. globulus. We suggest that the lack of parasitism of Ophelimus sp. in field conditions is possibly due to life cycle asynchrony between the parasitoid and the gall wasp. The native predator Anthocoris nemoralis was observed feeding on nymphs of Glycaspis brimblecombei, an exotic psyllid pest of E. camaldulensis. We showed that A. nemoralis is able to lay eggs on the leaves of E. camaldulensis and to complete its development preying on G. brimblecombei nymphs. The predator significantly reduced psyllid population in mesocosm experiments. Altogether, our work shows constraints and opportunities on different aspects of the biological control of Eucalyptus pests in its invaded range.

Keywords: Eucalyptus; exotic pests; classical biological control; conservation biological control; fortuitous biological control

I

Resumo

Investigámos diferentes táticas de controlo biológico contra pragas exóticas dos eucaliptos, em quatro estudos de caso: a) introdução fortuita de um agente de controlo biológico; b) pesquisa de novas espécies de parasitóides para controlo biológico clássico; c) possível uso de um parasitóide no controlo biológico de uma nova praga, o qual foi previamente introduzido para controlar uma espécie do mesmo género; e d) utilização de um predador nativo em controlo biológico de conservação. No primeiro estudo Epichrysocharis burwelli foi detetado em Portugal (primeiro registo na Europa e na região Paleártica), em folhas de Corymbia citriodora, em quatro dos sete locais amostrados, ao longo do rio Tejo, entre Lisboa e Castelo Branco. Uma nova espécie de parasitóide, Closterocerus sp. foi observada parasitando E. burwelli. O controlo biológico clássico de Gonipterus platensis com Anaphes nitens provou ser insuficiente em Portugal. Um estudo de interações tróficas foi realizado pela primeira vez na Tasmânia, para seleção de novos agentes potenciais de controlo biológico de G. platensis. Foram identificadas seis espécies de Gonipterus e cinco espécies de parasitóides oófagos de Gonipterus. Não foi observada especialização significativa entre Gonipterus e os parasitóides identificados. Gonipterus platensis foi observado sobretudo associado a Eucalyptus ovata. Num terceiro estudo demonstrámos que Closterocerus chamaeleon, parasitóide de Ophelimus maskelli, é capaz de completar o desenvolvimento em Ophelimus sp., espécie galícola recém-encontrada no sul da Europa atacando E. globulus. A ausência de parasitismo de Ophelimus sp. em condições de campo é devida possivelmente à assincronia do ciclo de vida entre o parasitóide e Ophelimus sp. O predador nativo Anthocoris nemoralis foi observado alimentando-se de ninfas de Glycaspis brimblecombei, praga exótica de E. camaldulensis. Mostrámos que A. nemoralis é capaz de se reproduzir em folhas de E. camaldulensis e completar o desenvolvimento predando ninfas de G. brimblecombei. O predador reduziu significativamente a população do psilídeo em estudos realizados em mesocosmo. No seu conjunto esta tese demonstra limitações e oportunidades no uso de estratégias de controlo biológico para combater as pragas invasoras dos Eucaliptos.

Palavras-chave: eucaliptos; pragas exóticas; controlo biológico clássico; controlo biológico de conservação; controlo biológico fortuito.

II

Resumo alargado

Nesta tese, investigaram-se diferentes tácticas de controlo biológico contra pragas exóticas dos eucaliptos. A sua aplicação e optimização estão dependentes da existência de um agente de controlo biológico. Esta táctica é considerada com uma das mais sustentáveis em sistemas agro-florestais. Foram considerados quatro estudos de caso: a) introdução fortuita de um agente de controlo biológico; b) pesquisa de novas espécies de parasitóides para controlo biológico clássico de uma praga exótica; c) possível uso de um parasitóide no controlo biológico de uma nova praga, que foi introduzido para controlar uma espécie do mesmo género; e d) utilização de um predador nativo em controlo biológico de conservação de uma praga exótica. Muitas das espécies de insectos fitófagos dos eucaliptos fora da região nativa foram introduzidas acidentalmente. A sua descoberta ocorre por vezes através de prospecções, em plantações ou em espaço urbano. Em 2015, foi observada, na região de Lisboa, uma nova espécie cecidogénica, em árvores de Corymbia citriodora. Procedeu-se à prospecção deste insecto, em sete locais onde existia a planta hospedeira, para avaliar a sua distribuição geográfica, em Portugal. A sua presença foi registada através da observação de ramos, em quatro árvores, por local, determinando o nível de infestação e amostrando ramos infestados para identificação específica, em laboratório. A nova espécie galícola foi identificada como Epichrysocharis burwelli. Foi também observada a emergência de uma nova espécie de parasitóide do género Closterocerus, de origem não europeia. Trata-se do primeiro registo de E. burwelli e de Closterocerus sp. no continente Europeu. A presença deste parasitóide sugere um caso de controlo biológico fortuito, numa fase inicial de estabelecimento de uma praga exótica, que pode contribuir para limitar a expansão geográfica de E. burwelli. É recomendável que a distribuição de ambas as espécies continue a ser monitorizada e que se proceda à clarificação da identidade específica do parasitóide. O controlo biológico clássico de Gonipterus platensis, na Península Ibérica, através do parasitóide Anaphes nitens, mostrou ser insuficiente para reduzir eficazmente as populações desta praga do eucalipto, nomeadamente em regiões de maior altitude. Considerou-se, por isso, a hipótese de introduzir outros agentes de controlo biológico, que pudessem complementar a acção do referido parasitóide. Nesse sentido, realizaram- se pela primeira vez, na Tasmânia, entre 2016 e 2017, prospeções para, por um lado,

III

esclarecer as relações tróficas entre espécies de Eucalyptus e de Gonipterus, na região nativa, e, por outro, colher parasitóides oófagos de Gonipterus, tendo em vista a identificação e seleção de potenciais agentes de controlo biológico de G. platensis. Para tal, foram recolhidas ootecas de Gonipterus spp. em diferentes espécies de eucaliptos. A identificação das espécies de Gonipterus foi feita com recurso a análise molecular, mas apenas para os casos em que da ooteca eclodiram larvas de Gonipterus spp. e emergiu, pelo menos, uma espécie de parasitóide. Das 5313 e 1732 ootecas recolhidas, em 2016 e 2017, emergiram 962 e 376 parasitóides oófagos, respectivamente. Foram identificadas cinco espécies de parasitóides (Anaphes tasmaniae, A. inexpectatus, A. nitens, Cirrospilus sp., Euderus sp.) e cinco espécies de Gonipterus (G. platensis, G. pulverulentus, Gonipterus sp. 1, Gonipterus sp. 2, G. notographus). Não foi observada especialização significativa entre Gonipterus spp. e os parasitóides oófagos identificados. A espécie G. platensis foi encontrada principalmente em Eucalyptus ovata. A importância do conhecimento das relações tróficas entre hospedeiro – herbívoro – parasitóide, no seu ambiente nativo, é aqui demonstrada como sendo relevante, devendo ser incluída no desenvolvimento de programas de controlo biológico clássico. Por exemplo, o facto de 43% dos adultos de A. inexpectatus terem emergido de ootecas de G. notographus, recolhidas de eucaliptos do grupo dos ‘peppermints’ (E. amygdalina e E. pulchella), sugere a existência de uma eventual afinidade/preferência entre estas espécies. Ophelimus sp., é uma nova espécie galícola recentemente detectada no sul da Europa, associada a Eucalyptus da secção Maidenaria, nomeadamente E. globulus. Uma vez que Closterocerus chamaeleon é um parasitóide da espécie galícola O. maskelli, e com o objectivo de tentar controlar Ophelimus sp., praga de E. globulus em Portugal, foi avaliada a possibilidade de utilização deste parastóide como agente de controlo biológico de Ophelimus sp. Até à data não foi ainda identificado nenhum inimigo natural para esta espécie. Contudo, verificou-se que, em condições naturais, os adultos de C. chamaeleon não coexistem com o estado larvar de Ophelimus sp. Para este trabalho, prolongámos artificialmente a longevidade dos adultos do parasitóide, através da sua manutenção, em câmaras climatizadas, a 15±1ºC, com disponibilidade de alimento (água e mel). Nestas condições, foi possível prolongar a longevidade de C. chamaeleon até quatro meses. Demonstrou-se, em ensaios de laboratório e campo, que C. chamaeleon é capaz de reconhecer e parasitar as galhas de Ophelimus sp., bem como completar o desenvolvimento neste novo hospedeiro. A não observação de parasitismo de Ophelimus sp. por C. chamaeleon, em condições naturais, apesar da coexistência das duas espécies

IV

e da capacidade demonstrada do parasitóide reconhecer Ophelimus sp. como hospedeiro e ser capaz de o parasitar e completar o desenvolvimento, pode dever-se ao desfasamento entre os respectivos ciclos biológicos. Anthocoris nemoralis é uma espécie Paleártica, predadora nativa de psilídeos. Esta espécie foi observada a alimentar-se de ninfas de Glycaspis brimblecombei, em Eucalyptus camaldulensis. Em observações e ensaios de campo foi avaliada a hipótese de A. nemoralis poder reproduzir-se em E. camaldulensis. Uma vez que este antocorídeo faz a postura dos ovos nas folhas dos hospedeiros vegetais das suas presas e E. camaldulensis é uma espécie exótica em Portugal, esta observação documenta uma expansão do nicho ecológico usando novas espécies de plantas para ovipoisção. Foi também avaliada a capacidade do predador poder completar o desenvolvimento alimentando-se de ninfas de G. brimblecombei. Para tal, foram colocados, durante um mês, adultos de A. nemoralis em ramos de E. camaldulensis infestados com ovos e ninfas de G. brimblecombei e isolados através de mangas fechadas de Etamine. Este estudo foi efectuado em 2014 e 2015. Os resultados mostraram que as fêmeas do predador efetuaram a postura em folhas de E. camaldulensis. Foram contabilizados 139 ovos em 2014 e 61 em 2015. A presença de A. nemoralis nas mangas reduziu significativamente a população de psilídeos. Os resultados demonstraram que A. nemoralis tende a aumentar o seu leque presas e hospedeiros vegetais, sendo capaz de se reproduzir em E. camaldulensis e de predar, neste hospedeiro exótico, G. brimblecombei, praga recentemente introduzida em Portugal. A possibilidade de utilização de A. nemoralis como potencial agente de controlo biológico de G. brimblecombei através da promoção de tácticas de conservação ou largadas inundativas deve ser aprofundada.

Palavras-chave: eucaliptos; pragas exóticas; controlo biológico clássico; controlo biológico de conservação; controlo biológico fortuito.

V

Chapter 1

General introduction

1

Chapter 1

General introduction

Introductions of non-native species in a new geographical region are related with Human travels over long distances, since pre-historical times (Roques et al. 2010). Since the Neolithic, humans deliberately moved plants and from one region to another, for cultivation and food supply (Dyson 1964). This tendency sharply increased after the 15th century, with the discoveries and more recently with generalized global trade (Hulme 2009; Roques et al. 2010). After the industrial age, the number of records of new non- native species introduced worldwide has grown exponentially (Hulme 2009; Roques et al. 2010). This alarming trend is greatly explained by the increasing ability to transport goods and people across long distances, in a short time (Aukema et al. 2010; Hulme 2009; Paine et al. 2010; Roques et al. 2010). Luckily, not all the species that arrive into a new area get established (Tobin 2018; Williamson and Fitter 1996). After the arrival of an herbivore species in a new area, its establishment will primarily depend on the presence of suitable host plants (Keane and Crawley 2002; Lockwood et al. 2005). This is particularly relevant for highly specialized herbivore insect species. Some introduced non-native species feed exclusively, or to some extent, on a specific introduced exotic plants species (Roques et al. 2010). Consequently, the introduction of exotic plants, for agroforest or ornamental uses, has facilitated the establishment of new herbivore insect species (Roques et al. 2010). For example, more than 40 herbivore insect species originated from Australia, feeding on Eucalyptus, are currently established worldwide where Eucalyptus plantations are cultivated (Mansfield 2016). In fact, in most societies, agricultural, forest and urban landscapes are highly anthropized and often include high number of exotic plant species (Cadotte et al. 2017; Hoyle et al. 2017; Poland and McCullough 2006; Roques et al. 2010). Such modified ecosystems create suitable environments for invasive insect species to settle on (Poland and McCullough 2006; Roques et al. 2010). Biological invasions are one of the main causes of biodiversity loss on invaded regions, either through direct or indirect interactions among species (Cadotte et al. 2017; Kenis et al. 2017b). The ecolocigal impacts of species invasion will also affect local food-webs, and can culminate with species displacement or extintion (Sharma et al. 2018). Simultaneously, the impact of invasive species can also be evaluated and measured by the resulted economic impacts: yield losses in both agricultural and forest ecosystems; changes in market value for affected goods; associated treatment costs to reduce the yield loss; changes in land use, and Human welfare (Cadotte et al. 2017; Kenis et al. 2017b;

2

Chapter 1

Sharma et al. 2018). Some recent examples of invasive insect pests causing high economic damage include: the Emerald Ash Borer, Agrilus planipennis Fairmaire in United States of America, whose economic damages, due to treatment costs or tree removal, were estimated in about 10.7 billion dollars (Kovacs et al. 2010); the Eucalyptus weevil Gonipterus platensis Marell, which originated an economic impact in pulp industry, in Portugal, between 1996 to 2016, of about 7 billion euros (Valente et al. 2018); the establishment of the Chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu in Europe, which originated 50-70% yield reduction (EPPO 2005); the wood borers Anoplophora glabripennis (Motschulsky) and Anoplophora chinensis (Forster), for which the erradication costs in Europe already reached about 1500 million euros (Haack et al. 2010). Once established in the invaded range, herbivores may benefit from a predator/parasitoid- free environment (Keane and Crawley 2002). In their native region, herbivore species are normally kept at low density in consequence of bottom-up and top-down regulatory mechanisms (Pearson and Callaway 2005; Tobin 2015). A complex landscape of species interactions, involving competition with other herbivores, and several trophic levels of predators and parasitoids, created unique and complex communities, from which complex regulatory processes emerge (Polis 1994). Such mechanisms will keep populations at low density equilibrium or, when equilibria are disrupted, will pull populations again to new low equilibrium stages (e.g. Hunter et al. 1997). On the contrary, non-native herbivore may lack regulatory mechanisms in the invaded areas (Pearson and Callaway 2005). Nevertheless, native predators may adapt to the novel prey and exert some level of control (Carlsson et al. 2009; Garcia et al. 2019).

3

Chapter 1

Control of invasive species

Selection of control measures to cope with invasive species depends on the species establishment phase (Wittenberg and Cock 2005). Following a first detection, the first procedure should be the assessment of distributional range of the invasive species, to evaluate eradication possibility. Nevertheless, eradication is effective only when the gap between species arrival and first detection is relatively short (Pluess et al. 2012; Tobin 2018). When the species already covers a wide geographical area, the goal then is to use the most suitable control measure to keep the invasive species under acceptable economic threshold (Wittenberg and Cock 2005). The selection of control measures will have to take then into consideration species invasiveness, their biology and behavior, as well as available control tactics. Altogether, this requires a good knowledge of the target species and the control tactics. Table 1.1 summarizes major types of Integrated Pest Management (IPM) control measures, which can be used to control invasive herbivore insect species.

Table 1.1. – Integrated pest management control tactics against insect pests in agricultural and forest systems. Adapted from Amaro (2003).

Control tactic Type of impact Example

Use of semiochemicals for culling or disrupt Behavioral Direct population growth (e.g. mass trapping, mating disruption) Use of beneficial organisms (natural enemies) Biological Direct & Indirect that inflict harm and or feed on the target species Chemical Direct Use of insecticides Replacement of current tree species by non- Cultural Indirect host tree species; Host removal; Pruning Selection of less susceptible or resistant host Genetic Indirect plant genotypes; use genetically modified plants with resistant mechanisms Prohibition of importing plant material or food Legislation Indirect from country with presence of quarantine species Removal of pests from host trees (e.g. nest Mechanical Direct & Indirect removal; collar traps)

4

Chapter 1

Chemical control, with synthetic insecticides, has been one of the main and most direct control tactics used to cope with invasive species. However, the use of insecticides may have negative impacts on the environment and human health. Moreover, chemical control is not sustainable, since it has a short time effect over the target population, making necessary consecutives applications. This will be reflected in long-term high applications costs, as well as risk of insecticide resistance (Jetter and Paine 2004; Valente et al. 2018). Due to its negative impacts, chemical applications are being increasingly reduced, especially in forest ecosystems and urban areas (Zacharia 2011). Forests, in particular, support diverse number of plant species and provide habitat and resources for innumerous animals, which play relevant ecological services, such as pollination and biological control of pests (Aerts and Honnay 2011). Forests are also a major contributor to water drainage (FAO 2012; Barsoum et al. 2016). All these functions justify the high number of restrictions imposed by local authorities and certification committees to insecticide applications on natural or planted forests (FSC 2019). Similarly, urban forest areas are highly restrictive on the use of chemical control measures (Kenis et al. 2017a; Kristoffersen et al. 2007). Consequently, researchers and stakeholders have been giving increasing attention to other control tactics in forest protection, such as biological control. The main preventive tactic to cope with invasive species is through the application of restrictive legislation, as for transport of live plant material between different regions; and the requirement for a phytosanitary passport (Decreto-Lei n.º 92/2019; European Union 2014, 2019). That guarantees plant health, and that is pest free. By also imposing restrictions and requirements to wood packing material (e.g. types of wooden crates that may contain/transport wood borer species) it also increases security level regarding tree pests and diseases. The downside of this strategy will be the costs and market changes, such as the need for replacement of package material to meet the requirements of the country that imposed the restriction. One of the IPM tactics is the use of genetically improved plant species, which through certain processes can become more resilient or even less susceptible to a specific insect pest (Amaro 2003). These improvements can be obtained either by genetic transference between species, such as in the case of genetically modified organisms (GMO), for example Bt maize, or by development of resistant genotypes (Yang et al. 2016). The downside of this practices are the risks of genetic transfer from GMO’s to other plants in the wild and non-target effects of the toxins on other animals (e.g. bees while collecting pollen) (Van Wyk et al. 2010; Villanueva-Gutierrez et al. 2014). Yet, there are notorious

5

Chapter 1 benefits regarding the use of more resistant species, such as the reduction of pesticides required for the campaign; the improvement of environment quality and reduced costs of chemical applications for the stakeholders (Amaro 2003). Other directional and responsive action towards pest presence passes through the use of: behavioral tactics, such as the use of semiochemicals for mating disruption of insect pest or luring biocontrol agents from nearby areas to the target area (Branco et al. 2007; Franco et al. 2008). This behavioral tactic as a down side with high costs of the semiochemicals and the amount needed to increase efficiency. Cultural and physical tactics are considered preventive tactics, in which the stakeholder is the main control agent. Here, it is expected that the stakeholder will manage the pest species abundance through good management practices, such as: mulching; removal of susceptible host plant species; fertilization (Amaro 2003). Yet, these practices are highly time consuming and will mostly require more labor work, which will increase costs. In this thesis we will dealt with different studies related with biological control. Biological control is a management tactic to control either animals or plants, by resorting to other organisms. Biological control is considered one of the most sustainable and ecological friendly control tactics to cope with non-native species (Jetter and Paine 2004; Kenis et al. 2017a; Van Driesche et al. 2010). The promotion and practice of biological control methods has been increasing on visibility and is also generally well accepted by society (Jetter and Paine 2004). Jetter and Paine (2004), in a survey carried out in an urban area of Los Angeles, to local population, showed that citizens prefer biological control over chemical spray, to cope with non-native insect pests. Biological control tactics still requires scientific research to optimize results and adapt methods to different circumstances.

6

Chapter 1

Biological control

Biological control relies on the use or reinforce of organisms, which exert lethal activity on the target pest (Capinera 2008). In a broad sense, biological control includes the use of predators, parasitoids, phytophagous species and microorganisms (e.g. Bacillus thuringiensis) that can inflict damage to other living organisms (Capinera 2008; Eilenberg et al. 2001). Wittenberg and Cock (2005) classify biological control tactics in two major groups: a) Not self-sustaining, which may require some reinforcement (e.g. more than a single application or release, e.g. Bacillus thuringiensis that acts as insecticide); b) Self- sustaining, releases of biocontrol agents which are capable of reproduce and live freely and are capable of self-perpetuating in the released target region. Our work focuses on self-sustaining biological control tactics. These comprise at least three different approaches: classical biological control; augmentative releases; and conservation biological control. Below we will describe the postulates and applicability of these tactics.

Classical Biological Control Classical Biological Control (CBC) of invasive pest species is attained through the introduction of natural enemies from the same native range of the target pest (Cock et al. 2016; Wittenberg and Cock 2005). This practice involves searching of natural enemies on the native region of the pest; selecting best candidates for biological control; studying its biology and efficacy and, if considered safe and adequate, releasing these enemies on the target area (Hoddle 2005; Kenis et al. 2019). Classical Biological Control programs entails three main requirements that practitioners must deal with: 1) Efficiency of the selected natural enemy; 2) Adaptation to the new environmental conditions (e.g. climate); and 3) Risk to non-target species. In CBC programs, is of great interest to use highly specialized biocontrol agents, as they tend to fulfill the first and third requirements indicated above. Predators and parasitoids are predominantly used in CBC programs. Kenis et al. (2017a) refer that the establishment success of biocontrol agents in CBC programs was higher against insect pests of woody plants (37%), when compared to other species (30%). The same authors verified that 56% of released predators and parasitoids were used against woody plants insect pests. Ultimately, if the natural enemy species establishes and is efficient, it will permanently reduce the target pest population to sustainable levels. Consequently, the economic

7

Chapter 1 impact of the pest will be reduced sustainably, and no further actions are needed (Jetter and Paine 2004; Kenis et al. 2019). The first CBC program was developed in 1888-1889, in California (USA), targeting the cottony cushion scale, Icerya purchasi Maskell (Capinera 2008). This was seriously compromising the local citrus industry. Chemical control was, in this period, the major control measure. However, insecticide applications had becoming more and more demanding, due to resistance mechanism of the insect pest (Legner 2019). Following surveys conducted in Australia, the native region of the cottony cushion scale, the Vedalia , Rodolia cardinalis (Mulsant) was introduced in California for the biological control of I. purchasi (Capinera 2008). The success was outstanding. In a few years, due to effective biological control, I. purchasi damage on citrus industry was reduced to minimum levels, and chemical treatments were no longer needed (Caltagirone and Doutt 1989). Later, the Vedalia beetle was imported and released in other countries affected by I. purchasi, including Portugal, always with great biological control success (Amaro 2003; Capinera 2008; Hoddle 2013). Since the release of Vedalia beetle, more than 6175 attempts of CBC programs, involving about 2000 biocontrol agents, were carried out worldwide (Cock 2015; Kenis et al. 2017a; van Lenteren et al. 2006). Most of these biological control programs targeted invasive pests affecting woody plants (Kenis et al. 2017a). Globally, up to 2006, the leading promoters of CBC programs were the USA, Australia, New Zealand, South Africa, France, India and Israel (Cock 2015). Despite striking successes, several limitations and constraints hinder the success of CBC programs. It was estimated that only about 10 % of the CBC attempts resulted in a complete or partial success (Kenis et al. 2017a). The constraints to CBC success have been mainly associated with the biocontrol-agent efficiency, the development costs of CBC programs, and the impact of the released biocontrol agent to non-target organisms. Bellow, we refer the main aspects related with these constraints, which influence the development of a CBC program:

Efficiency - Efficiency of the CBC program is mostly associated with the biocontrol agent performance in the non-native range. Primarily, its efficacy relies on the relationship with its target host species. Therefore, a deep understanding of the biology and ecology of both species (the biological control agent and the target pest) is required to improve the success of the CBC program (Kenis et al. 2019).

8

Chapter 1

One of the constraints to the success of the CBC program is the correct identification of the target species in its native region (Rosen 1986). Inaccurate host-parasitoid, prey- predator relationships may result in the selection of poorly efficient or sub-optimal biological control agents. Errors result from the selection of unsuitable habitats, when collecting target insect pests and their natural enemies in the field; assignment of natural enemies to a wrong host or prey species; or contamination with non-host material (Bin et al. 2012). Frequently, there are several congeneric species of the target prey/host difficult to distinguish by morphological observations. An example is the case of Gonipterus scutellatus complex, which contains several species difficult or even impossible to distinguish morphologically (Mapondera et al. 2012). Difficulties in identification are particularly evident when the collector is searching for immature stages, eggs and larvae. The correct identification of the predator or parasitoid may further delay its use. Over the past few years, several improvements on species identification have occurred, mainly with molecular tools. These tools provide fast and precise species identification, and can contribute to solve misidentification problems related with cryptic species. A recent example is the identification of cryptic species and clarification of species misidentifications within the G. scutellatus complex (Mapondera et al. 2012). Using similar tools, on Chapter 3, we will test the hypothesis that parasitoid species may have different affinity towards different weevil species, within this complex. Inadequate environmental conditions on the non-native range may also hinder the efficiency of the biological control agent. In particular, climate plays a relevant role on the success of establishment and efficacy of the biocontrol agent. The use of species distribution models, such as Climex® (Hoddle 2005; Queiroz et al. 2013; Robertson et al. 2008), are of great use to predict the probability of establishment of the natural enemy in a new area with a given climate. This may be done by climate match of the native with the non-native region, or by mechanistic models using biological parameters of the studied species, such as temperature survival thresholds (Hoddle 2005; Robertson et al. 2008; Saavedra et al. 2015). Species distribution models can be used to narrow the area, within the native region of the target pest, where to search for biocontrol agents (Hoddle 2005; Robertson et al. 2008; Saavedra et al. 2015). Such area-oriented search also helps to reduce costs associated with species surveys in the field. Due to climatic constraints, the efficiency of the natural enemy may vary geographically, within the introduced range. For example, the psyllid parasitoid Psyllaephagous bliteus Riek, introduced in California, to control the eucalyptus red gum psyllid, Glycaspis brimblecombei Moore is more

9

Chapter 1 efficient in the more temperate littoral, than in the continental regions (Daane et al. 2012; Dahlsten et al. 2005). In Portugal, the parasitoid Anaphes nitens (Girault), introduced for the control of the eucalyptus snout beetle, G. platensis is efficient in the South and littoral areas, whereas the rate of parasitism sharply declines with elevation in mountain areas (Reis et al. 2012). This outcome was attributed mostly to the low winter temperatures at higher altitude.

Costs – Costs associated with CBC programs are difficult to estimate. They greatly vary with the chance of finding suitable material in the field and the facility of testing and studying the biocontrol agent, in the laboratory. Costs are also related with laboratory rearing, biological studies for those species poorly known, risk assessment assays and monitoring the successful establishment of the species on the target region (Kenis et al. 2019). Examples of biological assay and risk assessment studies conducted for the biological control of eucalyptus pests include the parasitoids A. inexpectatus Huber & Prinsloo, in Portugal (Valente et al. 2017a; Valente et al. 2017c), Closterocerus chamaeleon (Girault), in Israel (Protasov et al. 2007), P. bliteus, in USA (Dahlsten et al. 2005). Program costs may also include surveys for new biocontrol agents or for laboratory colony refreshment. For all these reasons, CBC programs require the existence of a stable funding source for its development and maintenance until successful establishment and self-perpetuation of the biocontrol agent (Legner 2019; Wittenberg and Cock 2005). Yet, the costs of CBC program may be greatly minimized, when the information on the biology of both the target pest and the biocontrol agent is already available (Kenis et al. 2019). When a biocontrol agent succeeds in one region, then its introduction in other regions is facilitated. In these cases, CBC will be managed at much lower costs. One example is the introduction of R. cardinalis in different continents as previously mentioned. The introduction of the egg parasitoid A. nitens, first released in South Africa, in 1926, for the control of G. scutellatus (s.l.), is another example (Tribe 2005). The success was high and in a few years the parasitism rates attained values up to 96 %, in the coastal areas (Tribe 2003). The parasitoid was then introduced from South Africa, to other African countries, California and Europe (Hanks et al. 2000; Pinet 1986; Santolamazza-Carbone and Cordero Rivera 2003). Although costs with CBC are quite high at the initial stages, at long term it offers high benefit/cost ratio, which may vary between 67 and 347 (Jetter and Paine 2004; Valente et al. 2018). Valente et al. (2018) showed that, in the case of releasing A. nitens in Portugal

10

Chapter 1 for the control of G. platensis, anticipating biological control by just one year could result in a benefit/cost ratio of 67. Nevertheless, in this case biological control was not completely successful, as parasitism rates were high in some areas within the release range (Reis et al. 2012).

Non-target effects - The release of non-native biocontrol agent(s) to control an exotic species in an invaded region may pose environmental risks (EPPO 2014; van Lenteren et al. 2006). The most frequent concern is that the natural enemy will prey or parasitize non- target species. This is particular relevant, if native species closely related to the target pest are present in the invaded area. Another possible risk will be interspecific competition with native predators and/or parasitoid species. In general, this is not expected, as the target species normally would not be controlled by other natural enemies. However, if a generalist predator / parasitoid is introduced, it might prey on non-target prey and by doing so also displace native local predators or parasitoids. This will cause a disruption of food webs and ultimately may originate extinction of native species. A major known example of an introduced biocontrol agent causing negative environmental impacts is the Asian ladybeetle, Harmonia axyridis Pallas, which was introduced in Europe for the control of aphids (Adriaens et al. 2003; Alhmedi et al. 2010; Kenis et al. 2010; Koch 2003; van Lenteren et al. 2007; Zhang et al. 2016). This ladybeetle species is very generalist, and has been observed preying on many different species, outcompeting its congeners on the released regions, and further affecting their populations by intra-guild predation (Adriaens et al. 2003; Koch 2003; van Lenteren et al. 2007). At least four ladybeetle species were shown to be affected by H. axyridis, in Europe (Kenis et al. 2010). A similar pattern was observed in North America, where the Asian ladybeetle was also intentionally introduced, between 1916 and 1981 (Koch 2003). In consequence of these types of risk, legislation has been produced to regulate CBC (an example is the Portuguse law Decreto-Lei n.º 92/2019), including the definition of native and exotic species; information about species introduction, as well has the risk- assessment studies needed (EPPO 2014; Decreto-Lei n.º 92/2019). Being impossible to test all potentially non-target prey/hosts, the selection of non-target organism for risk assessment should follow adequate criteria. One criterion is to select related non-target prey/hosts, with phylogenic proximity with the target species. A risk-assessment study on non-target prey was recently conducted by Valente et al. (2017a) for the release of A. inexpectatus, in Portugal, which considered: a) possible use of congeneric species; b)

11

Chapter 1 potential prey habiting the same habitat; c) similar egg structure and oviposition; d) economic value; and f) conservational status. Despite these concerns, we should note that biological control agents which have greatly affected non-target species occured in a very small number of cases (Simberloff and Stiling 1996). Conducting proper risk assessment studies will withdraw such possibility. This is one of the reasons why classical biological control requires intensive research.

Conservation biological control Conservation biological control is a self-sustainable biological control tactic in which the goal is to preserve and promote the targeted biocontrol agents (either native or exotic) within a delimited region (Eilenberg et al. 2001; Wittenberg and Cock 2005). This is usually carried out through habitat management, such as promoting plant diversity, by planting selected species or preserving semi-natural vegetation habitats, which will provide complementary food sources (e.g., nectar, pollen, honeydew), alternative prey/hosts, nesting habitats or refuges (Eilenberg et al. 2001). This will prevent the emigration of the target predators and/or parasitoids from the target area, while searching for the needed resources, and will favor the population growth of the biocontrol agents (Barbosa 1998). Conservational biological control approaches imply a deep understanding of the trophic relationships between host plant-pest and pest-predator, as well as of the ecological requirements of the predator for reproduction and survival (Chabaane et al. 2015). The habitat management can be conducted in a local scale, such as stand level, or at landscape level, by promoting land cover habitats needed for reproduction or shelter. Both parasitoids and predators use plant cues while searching for prey and these are key components for prey association by the biological control agents (Meiners and Hilker 1997; Scutareanu et al. 1996; Sigsgaard 2005). Plants specific volatiles, and herbivore- induced plant volatiles – HIPV may be used to retain the predators foraging in a given area. Several predators and parasitoids use also chemical cues emitted by the prey, such as their sexual pheromones. Similarly, these compounds may be used to retain the natural enemies in a given area, as demonstrated for the mealybugs´ parasitoid Anagyrus vladimiri (Triapitsyn) (=A. sp. near pseudococci) (Franco et al. 2011). Predators may require specific plant substrates for oviposition. The acceptance of a substrate to oviposit is crucial for the development of the predator offspring, and directly affects its performance on the target pest (Coll 1996). Although there are some studies approaching the selection substrate for oviposition by predators, these studies were

12

Chapter 1 mostly on native plants (e.g. Coll 1996). However, the acceptance of non-native host plant species for oviposition by native predators has been poorly studied. This aspect will be investigated in Chapter 5. These types of studies should be taken more into concern by researchers, once native generalist predators could be used as the first line of defense against unintentional arrival of a non-native species (Carlsson et al. 2009; Garcia et al. 2019).

Augmentative biological control Augmentative biological control is aimed at enhancing the efficiency of biological control by mass releasing biocontrol agents (Perez-Alvarez et al. 2019). Two types of augmentative releases may be considered, i.e. inundative or inoculative. Inundative releases are applied, when biological control is expected to be attained mostly by the released insects, whereas in inoculative releases the action is carried out by their progeny (Bellows and Fisher 1999). This biological control tactic is most used when the population density of the biocontrol agents is not enough to control the target pest or is not able to sustain a stable population, requiring increments on their number through mass rearing (Amaro 2003; Parrella et al. 1992). However, it is more common in agricultural ecosystems (where the crop lifecycle is relatively short, i.e., often less than one year) than in forests (Amaro 2003). Nevertheless, in both situations, it is possible to help the resident predators or parasitoids through augmentative releases and promote ecological friendly solutions to cope with insect pests (Perez-Alvarez et al. 2019). Augmentative biological control is usually dependent on the commercial availability of mass-reared selected biocontrol agents produced by local or international companies. In the case of Eucalyptus, an example of augmentative biological control is the local release of A. nitens, to increase the efficiency of this biological control agent against G. platensis (Santolamazza‐Carbone et al. 2019). Still, in this case the results were not satisfactory, which might be due to the season or the low number of individuals released (Santolamazza‐Carbone et al. 2019).

13

Chapter 1

Why Eucalyptus accumulated so many invasive species? Eucalypts are woody plants, of the Myrtaceae family, with approximately 800 described species (Coppen 2002). Native to Australia, Indonesia, Philippines, and New Guinea, many eucalypt species have been introduced in a wide variety of different regions, in all the continents (Coppen 2002; Doughty 2000). Still, only few species were selected and are widely cultivated (Coppen 2002). Species, such as Eucalyptus camaldulensis Dehnh., E. globulus Labill., E. grandis W.Hill ex Maiden and E. tereticornis Sm., dominate the major planted area of eucalypts worldwide (Doughty 2000). Some eucalypt hybrids are largely used in commercial plantations. Eucalypts are also planted as ornamental trees in urban areas, on landscape (e.g. as roadside trees) and as windbreaks (Doughty 2000; Paine et al. 2000). Eucalypts can play relevant ecosystem services (e.g. soil conservation, nectar source) and are important by their aesthetical and/or economic value both in urban and forest landscapes (Coppen 2002). For example, Zerga and Woldetsadik (2016) showed that Ethiopian farmers highlight eucalypts as their main or second main income source. Eucalypts have become a major source of timber and/or fibers, occupying large areas in several countries of both hemispheres (Wingfield et al. 2015). The first plantations of Eucalyptus, outside the native range, originated from seeds (Doughty 2000). This method had a great influence on reducing possible dissemination of eucalyptus pests (Doughty 2000). The high demands for fast growing woody species, especially for pulp industries major increased the trade of eucalypts seedlings and wood logs between countries (FAO 2007; 2009). Eucalypt plantations occupy currently an area of ca. 20 million ha, which corresponds to about 15 % of global planted forest (Wingfield et al. 2015). The planted area increased 14 million ha between 1985 and 2008 (Hurley et al. 2016). In parallel, an exponential increase of biological invasions was observed in eucalypts non-native range, since the 1980´s (Hurley et al. 2016). In Europe, until early 1990’s, only four insect pests of eucalypts, Ctenarytaina eucalypti (Maskell), G. platensis and Gonipterus sp. 2, that were previously identified as “Gonipterus scutellatus”, and semipunctata (Fabricius) were known to occur (Arzone and Meotto 1978; Azevedo and Figo 1979; Cadahia and Ruperez 1980; Mapondera et al. 2012). Since then, at least 17 eucalypts pests have been reported in the Mediterranean Basin (Garcia et al. unpublished data). Twelve of these species are present in Portugal (Table 1.2).

14

Chapter 1

Table 1.2. – Eucalyptus insect pests reported in the Mediterranean basin and known to occur in Portugal, with reference for its guild. Y – present in Portugal; N – not known to occur in Portugal

Presence in Species Guild Reference Portugal Blastopsylla occidentalis Taylor Sap-sucker Y Pérez Otero et al. 2011 Ctenarytaina eucalypti (Maskell) Sap-sucker Y Azevedo and Figo 1979 Ctenarytaina peregrina Sap-sucker N Roques et al. 2010 Hodkinson Ctenarytaina spatulata Taylor Sap-sucker Y Valente et al. 2004 Epichrysocharis burwelli Schauff Galler Y Franco et al. 2016 Glycaspis brimblecombei Moore Sap-sucker Y Valente and Hodkinson 2009 Gonipterus platensis Marelli Defoliator Y Mapondera et al. 2012

Gonipterus sp. nº 2 Defoliator N Mapondera et al. 2012 Leptocybe invasa Fisher & Galler Y Branco et al. 2006 LaSalle Ophelimus maskelli (Ashmead) Galler Y Branco et al. 2009 Ophelimus mediterraneus sp. n. Galler Y Borowiec et al. 2019 Phoracantha recurva Newman Wood-borer Y Valente and Ruiz 2002 Wood-borer Y Cadahia and Ruperez 1980 (Fabricius) Platyobria bienmani sp. nov. Sap-sucker N Burckhardt et al. 2014

Quadrastichodella nova Girault Galler N Doganlar and Doganlar 2008 Thaumastocoris peregrinus Sap-sucker Y Garcia et al. 2013 Carpintero & Dellapé sloanei (Blackburn) Defoliator N Sánchez et al. 2015

Eucalypts species accumulation has been also observed in other parts of the world (Hurley et al. 2016; Mansfield 2016). A possible explanation for such increments is the movement of goods and people worldwide (Paine et al. 2010). Yet, the economic importance of each insect pest varies among invaded regions. For example, the eucalyptus gall wasps Leptocybe invasa Fisher & La Salle in Brazil is more important than in Portugal. This is due to differences on the susceptibility of the most cultivated eucalyptus species in each country (ICNF 2013; Peris-Felipo et al. 2010; Schnell e Schühli et al. 2016). Due to contrasts between regions, implementation of biological control programs against invasive pest species also depends on the economic value of the host plant or its ecological importance. Classical biological control has been the most used control tactic against eucalyptus insect pests (Table 1.3). In most cases, biological control solved, or largely reduced damages caused by invasive eucalypts insect pests.

15

Chapter 1

Table 1.3. – List of Australian native Eucalyptus insect pests occurring outside their native region, to which Classical Biological Control program was developed or a predator or parasitoid was accidentally introduced, with mention to the guild of the biological control agent.

Pest species Biocontrol agent Guild References Phoracantha Avetianella longoi Siscaro; Parasitoid Hanks et al. 1996; semipunctata Jarra phoracantha Marsh & Austin; Lanfranco and Fabricius Syngaster lepidus Brullé; Megalyra Dungey 2001; Siscaro Phoracantha recurva fasciipennis Westwood 1992; Tribe 2003 Newman charybdis Cleobora mellyi Mulsant; Enoggera Predator and Murray et al. 2008; Stål nassaui (Girault); Neopolycystus Parasitoid Withers and Jones insectifurax Girault 2003 Enoggera reticulata Naumann Parasitoid Paine and Millar 2002 Blackburn

Trachymela Enoggera reticulata Naumann Parasitoid Tribe 2000 tincticollis (Blackburn) Gonipterus platensis Anaphes inexpectatus Huber and Parsitoid Sanches 2000; Valente (Marelli) Prinsloo; Anaphes nitens (Girault) et al. 2017b; Withers 2001 Gonipterus Anaphes nitens (Girault) Parasitoid Sanches 2000 pulverulentus Lea Gonipterus sp. 2 Anaphes nitens (Girault) Parasitoid Tooke 1955 Eriococcus coriaceus Rhyzobius ventralis (Erichson); Orchus Predator Morales and Bain Maskell chalybeus (Boisduval); Stathmopoda 1989; Withers 2001 melanochra Meyrick Anoeconeossa Psyllaephagus richardhenryi sp. nov., Parasitoid Berry 2007 communis Taylor fiscella Psyllaephagus gemitus Riek Parasitoid Withers 2001 Taylor Creiis lituratus Psyllaephagus richardhenryi sp. nov. Parasitoid Berry 2007 Froggatt Cryptoneossa Psyllaephagus perplexans Cockerell Parasitoid Jones et al. 2011 triangula Taylor Ctenarytaina eucalypti Psyllaephagus pilosus Noyes Parasitoid Chauzat et al. 2002; (Maskell) Santana and Burckhardt 2007; Withers 2001 Eucalyptolyma Psyllaephagus parvus Riek Parasitoid Jones et al. 2011 maideni Froggatt

Glycaspis Psyllaephagus bliteus Riek Parasitoid Boavida et al. 2016; brimblecombei Moore Bush et al. 2016; Dahlsten et al. 2005; Withers 2001

Glycaspis granulata Psyllaephagus bliteus Riek Parasitoid Withers 2001 (Froggatt) Thaumastocoris Cleruchoides noackae Lin and Huber Parasitoid Barbosa et al. 2017 peregrinus Carpintero & Dellapé

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Chapter 1

Leptocybe invasa Megastigmus lawsoni Doğanlar & Parasitoid Masson et al. 2017; Fisher & La-Salle Hassan; Megastigmus zvimendeli Mendel et al. 2017 Doğanlar & Hassan; Quadrastichus mendeli Kim & La Salle; Selitrichodes kryceri Kim & La Salle Ophelimus maskelli Closterocerus chamaeleon (Girault) Parasitoid Mendel et al. 2007; (Ashmead) Rizzo et al. 2006 Phylacteophaga Bracon phylacteophagus Austin Parasitoid Withers 2001 froggatti Riek lugens Walker urabae Austin and Allen Parasitoid Avila et al. 2013 Strepsicrates brevifacies (Hardy) Parasitoid Withers 2001 (=Stictea) macropetana Meyrick

Still, there is much to do regarding biological control of eucalyptus pests. In some cases, released biological control agents are not efficient enough, such as A. nitens used against G. plantensis in Iberian Peninsula (Reis et al. 2012; Valente et al. 2017c). Several eucalypts pest species are poorly known. Some were only described after being detect in the invaded areas, such as Leptocybe invasa (Mendel et al. 2004), Ophelimus maskelli (Withers 2001), and Ophelimus mediterraneus (Borowiec et al. 2019). In the case of O. mediterraneus, no parasitoid is known yet (Borowiec et al. 2019). For these cases, it is also harder to implement biological control strategies and search for suitable biocontrol agents, as their distribution in the native region and host range are unknown (Mendel et al. 2004). Moreover, accurate species and species biological traits are fundamental to fill up gaps about the target species, and host-parasitoid relationships, and may help promote a sustainable pest control. As an example, species sharing high number of morphological similarities can make taxonomical identifications difficult (e.g. see Mapondera et al. (2012) for Gonipterus spp.). Finally, the use of native predators as biocontrol agents against alien species, in the invaded region, has been scarcely explored (Carlsson et al. 2009). The knowledge on the relationship between exotic prey and native predators is scarce. Studies are even more limited when it is required to evaluate the interaction between the predator and the host plant recognition and acceptance as for oviposition. This is especially relevant when the host plant is an exotic species, such as in the case of Eucalyptus spp. In Chapter 5, we conducted a study to evaluate the response of a native generalist predator to an exotic psyllid prey species. We believe that biological control practitioners should spend more work on studying this type of relationships, once they

17

Chapter 1 can prove to be a good aid on decreasing the establishment probability of invasive species (Carlsson et al. 2009). Our thesis aims at resolving some questions related with biological control of eucalypts pests present in Portugal, and test hypothesis regarding its effectiveness either through the use of exotic biocontrol agents or native biocontrol agents. The main objectives of the thesis are indicated below.

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Chapter 1

Objectives

In this thesis, we studied four biological control case studies related with alien insect pests of Eucalyptus, including different approaches: a) a fortuitous introduction of a biocontrol agent; b) a survey of new potential biocontrol agents for classical biological control; c) the possible use in the biological control of a new insect pest of a parasitoid which was previously introduced to cope with a congener species; and d) the use of a native predator in conservation biological control. Our main objectives were: ➢ To survey the geographical distribution in Portugal of a recently established eucalypt insect pest, Epichrysocharis burwelli Schauff, and associated biocontrol agents, as possible case of fortuitous biological control (Chapter 2); ➢ To find biocontrol agents associated with Gonipterus platensis in Tasmania, and expand the knowledge on the Eucalyptus-Gonipterus-parasitoid associations aiming to improve future classical biological control programs against G. platensis and other invasive Gonipterus species (Chapter 3); ➢ To study the host-parasitoid interaction between Ophelimus sp. and Closterocerus chamaeleon, and testing the hypotheses that (i) Ophelimus sp. is not a suitable host for C. chamaeleon or, in alternative, (ii) is a suitable host but there is a seasonal asynchrony between C. chamaeleon and the gall-wasp phenology (Chapter 4); ➢ To evaluate the use of a native generalist predator, Anthocoris nemoralis, in an augmentative or conservation biological control approaches against the psyllid Glycaspis brimblecombei, to further reduce the psyllid population on highly susceptible eucalypts species (Chapter 5).

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References

Adriaens, T., Branquart, E., Maes, D., 2003. The Multicoloured Asian Ladybird Harmonia axyridis Pallas (Coleoptera: ), a threat for native aphid predators in Belgium?. Belgium J Zool 133:201-202 Aerts, R., Honnay, O., 2011. Forest restoration, biodiversity and ecosystem functioning. BMC Ecology 11:1-10 Alhmedi, A., Haubruge, É., Francis, F., 2010. Intraguild interactions implicating invasive species: Harmonia axyridis as a model species. Biotechnol Agron Soc Environ 14:187-201 Amaro, P., 2003. A Protecção Integrada. ISA Press Arzone, A., Meotto, F., 1978. Reperti biologici su Gonipterus scutellatus Gyll. (Col. ) infestante gli eucalipti della riviera. Ligure Redia 61:205-222 Aukema, J.E., Mccullough, D.G., Holle, B.V., Liebhold, A.M., Britton, K., Frankel, S.J., 2010. Historical accumuiation of nonindigenous forest pests in the continental United States. BioScience 60:886-897 Avila, G.A., Berndt, L.A., Holwell, G.I., 2013. Dispersal behavior of the parasitic wasp (: ): A recently introduced biocontrol agent for the control of (: ) in New Zealand. Biol Control 66:166-172 Azevedo, F., Figo, M.L., 1979. Ctenarytaina eucalypti Mask (Homoptera, ). Bol. San. Veg., Plagas 5:41-46 Barbosa, L.R., Rodrigues, Â.P., Soler. L.S., Fernandes, B.V., Castro, B.M.C., Wilcken, C.F., Zanuncio, J.C., 2017. Establishment in the field of Cleruchoides noackae (Hymenoptera: Mymaridae), an exotic egg parasitoid of Thaumastocoris peregrinus (: Thaumastocoridae). Fla Entomol 100:372-374 Barbosa, P., 1998. Conservation Biological Control. Elsevier Barsoum N, Gill, R., Henderson, L., Peace, A., Quine, C., Saraev, V., Valatin, G., 2016. Biodiversity and rotation length: economic models and ecological evidence. Forestry Commission, Forest Research 10 pp Bellows, T.S., Fisher, T.W., 1999. Handbook of Biological Control: Principles and Applications of Biological control. Academic Press Berry, J.A., 2007. Key to the New Zealand species of Psyllaephagus Ashmead (Hymenoptera: ) with descriptions of three new species and a new

20

Chapter 1

record of the psyllid hyperparasitoid Coccidoctonus psyllae Riek (Hymenoptera: Encyrtidae). Aust J Entomol 46:99-105 Bin, F., Roversi, P.F., van Lenteren, J.C., 2012. Erroneous host identification frustrates systematics and delays implementation of biological control. Redia 95:83-88 Boavida, C., Garcia, A., Branco, M., 2016. How effective is Psyllaephagus bliteus (Hymenoptera: Encyrtidae) in controlling Glycaspis brimblecombei (Hemiptera: )?. Biol Control 99:1-7 Borowiec, N., La Salle, J., Brancaccio, L., Thaon, M., Warot, S., Branco, M., Ris, N., Malausa, J.-C., Burks, R., 2019. Ophelimus mediterraneus sp. n. (Hymenoptera, ): a new Eucalyptus gall wasp in the Mediterranean region. B Entomol Res 109:678-694 Branco, M., Boavida, C., Durand, N., Franco, J.C., Mendel, Z., 2009. Presence of the Eucalyptus gall wasp Ophelimus maskelli and its parasitoid Closterocerus chamaeleon in Portugal: First record, geographic distribution and host preference. Phytoparasitica 37:51-54 Branco, M., Franco, J., Valente, C., Mendel, Z., 2006. Survey of eucalyptus gall wasps (Hymenoptera: Eulophide) in Portugal. Bol. San. Veg., Plagas 32:199-202 Branco, M., Franco, J.C., Dunkelblum, E., Assael, F., Protasov, A., Ofer, D., Mendel, Z., 2007. A common mode of attraction of larvae and adults of insect predators to the sex pheromone of their prey (Hemiptera: Matsucoccidae). B Entomol Res 96:179- 185 Burckhardt, D., Queiroz, D.L., Malenovský, I., 2014. First record of the Australian Platyobria Taylor, 1987 from Europe and P. biemani sp. nov. as a potential pest of Eucalyptus (Myrtaceae) (Hemiptera: Psylloidea). Entomologische Zeitschrift - Schwanfeld 124:109-112 Bush, S.J., Slippers, B., Neser, S., Harney, M., Dittrich-Schröder, G., Hurley, B.P., 2016. Six recently recorded australian insects associated with Eucalyptus in South Africa. Afr Entomol 24:539-544 Cadahia, D., Ruperez, A., 1980. Posible aparición de Phoracantha semipunctata (F) en España. Bol. San. Veg., Plagas 6:119-122 Cadotte, M.W., Yasui, S.L.E., Livingstone, S., MacIvor, J.S., 2017. Are urban systems beneficial, detrimental, or indifferent for biological invasion?. Biol Invasions 19:3489-3503

21

Chapter 1

Caltagirone, L.E., Doutt, R.L., 1989. The history of the vedalia beetle importation to california and its impact on the development of biological control. Annual Review of Entomology 34:1-16 Capinera, J.L., 2008. Encyclopedia of Entomology. vol 4. Springer-Verlag New York Inc, New York, United States Carlsson, N.O.L., Sarnelle, O., Strayer, D.L., 2009. Native predators and exotic prey –an acquired taste?. Front Ecol Environ 7:525-532 Chabaane, Y., Laplanche, D., Turlings, T.C.J., Desurmont, G.A., Phillips, R., 2015. Impact of exotic insect herbivores on native tritrophic interactions: a case study of the African cotton leafworm, Spodoptera littoralis and insects associated with the field mustard Brassica rapa. J Ecol 103:109-117 Chauzat, M.-P., Purvis, G., Dunne, R., 2002. Release and establishment of a biological control agent, Psyllaephagus pilosus for eucalyptus psyllid (Ctenarytaina eucalypti) in Ireland. Ann Appl Biol 141:293-304 Cock, M., 2015. The impacts of some classical biological control successes CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 10:1-58 Cock, M.J.W., Murphy, S.T., Kairo, M.T.K., Thompson, E., Murphy, R.J., Francis, A.W., 2016. Trends in the classical biological control of insect pests by insects: an update of the BIOCAT database. BioControl 61:349-363 Coll, M., 1996 Feeding and ovipositing on plants by an omnivorous insect predator. Oecologia 105:214-220 Coppen, J.J.W., 2002. Eucalyptus - The Genus Eucalyptus. Taylor & Francis, London Daane, K.M., Sime, K.R., Paine, T.D., 2012. Climate and the effectiveness of Psyllaephagus bliteus as a parasitoid of the red gum lerp psyllid. Biocontrol Sci Techn 22:1305-1320 Dahlsten, D.L., Daane, K. M., Paine, T. D., Sime, K. R., Lawson, S. A., Rowney, D. L., Roltsch, W. J., Andrews Jr. J. W., Kabashima, J. N., Shaw, D.A., Robb, K.L., Geisel, P. M., Chaney, W. E., Ingels, C. A., Varela, L. G., Bianchi, M. L., Taylor, G., 2005. Imported parasitic wasp helps control red gum lerp psyllid Calif Agr 59:229-234 Decreto-Lei n.º 92/2019 Diário da República n.º 130/2019, Série I Assegura a execução, na ordem jurídica nacional, do Regulamento (UE) n.º 1143/2014, estabelecendo

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Chapter 1

o regime jurídico aplicável ao controlo, à detenção, à introdução na natureza e ao repovoamento de espécies exóticas da flora e da fauna pp 3428-3442 Doganlar, O., Doganlar, M., 2008. First record of the Eucalyptus seed gall wasp, Quadrastichodella nova Girault, 1922, (Eulophidae: ) from Turkey. Turk J Zool 32:457-459 Doughty, R.W., 2000. The Eucalyptus: a natural and comercial history of the gum tree. The Johns Hopkins University, Baltimore. MD Dyson, R.H.J., 1964. On the Origins of the Neolithic Revolution Science 144:672-675 Eilenberg, J., Hajek, A., Lomer, C., 2001. Suggestions for unifying the terminology in biological control. BioControl 46:387-400 European Union (EU), 2019. Plant health & biosecurity - Trade in plants & plant products from non-EU countries. https://ec.europa.eu/food/plant/plant_health_biosecurity/non_eu_trade_en. Accessed on 14-10-2019 European Union (EU), 2014. Regulation (EU) No 1143/2014 of the European Parliament and of the Council of 22 October 2014 on the prevention and management of the introduction and spread of invasive alien species, L 317:35-55 https://eur-lex.europa.eu/legal- content/EN/TXT/PDF/?uri=CELEX:32014R1143&from=EN Accessed on 20-10-2019 European and Mediterranean Plant Protection Organization (EPPO), 2005. Data sheets on quarantine pests - Dryocosmus kuriphilus. Bulletin OEPP/EPPO Bulletin 35:422-424 European and Mediterranean Plant Protection Organization (EPPO), 2014. PM 6/2 (3) Import and release of non-indigenous biological control agents. OEPP/EPPO Bulletin 44:320-329 Food and Agriculture Organization of the United Nations (FAO), 2007. Item 5 - Global wood and wood products flow; trends and perspectives. FAO, China, Shanghai, June, 6 pp 13 Food and Agriculture Organization of the United Nations (FAO), 2009. Global demand for wood products. FAO, Rome Food and Agriculture Organization of the United Nations (FAO), 2012. State of the World's Forests. FAO, Rome

23

Chapter 1

Franco, J.C., Garcia, A., Branco, M., 2016. First report of Epichrysocharis burwelli in Europe, a new invasive gall wasp atacking eucalypts. Phytoparasitica 44:443-446 doi:10.1007/s12600-016-0539-9 Franco, J.C., Silva, E.B., Cortegano, E., Campos, L., Branco, M., Zada, A., Mendel, Z., 2008. Kairomonal response of the parasitoid Anagyrus spec. nov. near pseudococci to the sex pheromone of the vine mealybug Entomol Exp Appl 126:122-130 Franco, J.C. Silva, E. B., Fortuna, T., Cortegano, E., Branco, M., Suma, P., Torre, I. L., Russo, A., Elyahu, M., Protasov, A., Levi-Zada, A., Mendel, Z., 2011. Vine mealybug sex pheromone increases citrus mealybug parasitism by Anagyrus sp. near pseudococci (Girault) Biol Control 58:230-238 Forest Stewardship Council (FSC), 2019. FSC Pesticides Policy - FSC-POL-30-001 V3- 0 EN. FSC International Center, Garcia, A., Figueiredo, E., Valente, C., Monserrat, V.J., Branco, M., 2013. First record of Thaumastocoris peregrinus in Portugal and of the neotropical predator Hemerobius bolivari in Europe. B Insectol 66:251-256 Garcia, A., Franco, J.C., Branco, M., 2019. Novel prey boosts the expansion of host-plant range in a native predatory bug. BioControl 64: 677-683 Haack, R.A., Herard, F., Sun, J., Turgeon, J.J., 2010. Managing invasive populations of Asian longhorned beetle and citrus longhorned beetle: a worldwide perspective. Annu Rev Entomol 55:521-546 Hanks, L.M., Millar, J.G., Paine, T.D., Campbell, C.D., 2000. Classical biological control of the Australian weevil Gonipterus scutellatus (Coleoptera : Curculionidae) in California. Environ Entomol 29:369-375 Hanks, L.M., Paine, T.D., Millar, J.G., 1996. Tiny wasp helps protect eucalyptus from eualyptus longhorned borer. Calif Agr 50:14-16 Hoddle, M., 2013. Biological control of Icerya purchasi with Rodolia cardinalis in the Galapagos. UC Riverside, California. https://biocontrol.ucr.edu/rodolia/rodolia_icerya_biocontrol_galapagos.html. Accessed 20-07-2019 2019 Hoddle, M.S., Identifying the donor region within the home range of an invasive species: implications for classical biological control of pests In: Hoddle MS (ed) Second International Symposium on Biological Control of , Davos, Switzerland, 12-16 of September 2005. USDA Forest Service, pp 29-37

24

Chapter 1

Hoyle, H., Hitchmough, J., Jorgensen, A., 2017. Attractive, climate-adapted and sustainable? Public perception of non-native planting in the designed urban landscape. Landscape Urban Plan 164:49-63 Hulme, P.E., 2009. Trade, transport and trouble: managing invasive species pathways in an era of globalization. J Appl Ecol 46:10-18 Hunter, M.D., Varley, G.C., Gradwell, G.R., 1997. Estimating the relative roles of top- down and bottom-up forces on insect herbivore populations: a classic study revisited. PNAS 94:9176-9181 Hurley, B.P., Garnas, J., Wingfield, M.J., Branco, M., Richardson, D.M., Slippers, B., 2016. Increasing numbers and intercontinental spread of invasive insects on eucalypts. Biol Invasions 18:921-933 Instituto da Conservação da Natureza e das Florestas (ICNF), 2013. Inventário Florestal Nacional 6 (IFN6) - Áreas dos usos do solo e das espécies florestais de Portugal continental. Resultados preliminares. Instituto da Conservação da Natureza e das Florestas, Lisbon, Portugal Jetter, K., Paine, T.D., 2004. Consumer preferences and willingness to pay for biological control in the urban landscape. Biol Control 30:312-322 Jones, M.E., Daane, K.M., Paine, T.D., 2011. Establishment of Psyllaephagus parvus and P. perplexans as serendipitous biological control agents of Eucalyptus psyllids in southern California. BioControl 56:735-744 Keane, R.M., Crawley, M.J., 2002. Exotic plant invasions and the enemy release hypothesis. Trends Ecol Evol 17:164-170 Kenis, M.. Adriaens, T., Brown, P., Katsanis, A., Van Vlaenderen, J., Eschen, R., Golaz, L., Zindel, R., Martin y Gomez, G.S., Babendreier, D., Ware, R. 2010. Impact of Harmonia axyridis on European ladybirds: which species are most at risk? Bulletin IOBC/wprs 58:57-59 Kenis, M., Hurley, B., Colombari, F., Lawson, S., Sun, J., Wilcken, C., Weeks, R., Sathyapala, S., 2019. Guide to the classical biological control of insect pests in planted and natural forests. vol FAO Forestry Paper No. 182. Food and Agriculture Organization of the United Nations (FAO), Rome Kenis, M., Hurley, B.P., Hajek, A.E., Cock, M.J.W., 2017a. Classical biological control of insect pests of trees: facts and figures. Biol Invasions 19:3401-3417 Kenis, M., Roques, A., Santini, A., Liebhold, A.M., 2017b Impact of Non-native Invertebrates and Pathogens on Market Forest Tree Resources. In: Vilà M, Hulme

25

Chapter 1

PE (eds) Impact of Biological Invasions on Ecosystem Services, vol 12. Invading Nature - Springer Series in Invasion Ecology Springer, pp 103-117 Koch, R.L., 2003. The multicolored Asian lady beetle, Harmonia axyridis: A review of its biology, uses in biological control, and non-target impacts. J Insect Sci 3:1-16 Kovacs, K.F., Haight, R.G., McCullough, D.G., Mercader, R.J., Siegert, N.W., Liebhold, A.M., 2010. Cost of potential emerald ash borer damage in U.S. communities, 2009–2019. Ecol Econ 69:569-578 Kristoffersen, P., Rask, A. M., Grundy, A. C., Franzen, I., Kempenaar, C., Raisio, J., Schroeder, H., Spijker, J., Verschwele, A., Zarina, L., 2007. A review of pesticide policies and regulations for urban amenity areas in seven European countries. Weed Res 48:201-214 Lanfranco, D., Dungey, H.S., 2001. Insect damage in Eucalyptus: A review of plantations in Chile. Austral Ecol 26:477-481 Legner, E.F., 2019. Cottony-cushion scale. https://faculty.ucr.edu/~legneref/biotact/ch-35.htm Accessed on 09-08-2019 Lockwood, J.L., Cassey, P., Blackburn, T., 2005. The role of propagule pressure in explaining species invasions. Trends Ecol Evol 20:223-228 Mansfield, S., 2016. New Communities on Eucalypts Grown Outside Australia. Front Plant Sci 7:1-9 Mapondera, T.S., Burgess, T., Matsuki, M., Oberprieler, R.G., 2012. Identification and molecular phylogenetics of the cryptic species of the Gonipterus scutellatus complex (Coleoptera: Curculionidae: Gonipterini). Aust J Entomol 51:175-188 Masson, M.V., Tavares, W.S., Lopes, F.A., Souza, A.R., Ferreira-Filho, P.J., Barbosa, L.R., Wilcken, C.F., Zanuncio, J.C., 2017. Selitrichodes neseri (Hymenoptera: Eulophidae) recovered from Leptocybe invasa (Hymenoptera: Eulophidae) galls after initial release on Eucalyptus (Myrtaceae) in Brazil, and data on its biology. Fla Entomol 100:589-593 Meiners, T., Hilker, M., 1997. Host location in Oomyzus gallerucae (Hymenoptera: Eulophidae), an egg parasitoid of the elm Xanthogaleruca luteola (Coleoptera: Chrysomelidae). Oecologia 112:87-93 Mendel, Z., Protasov, A., Blumberg, D., Brand, D., Saphir, N., Madar, Z., La Salle, J., 2007. Release and recovery of parasitoids of the Eucalyptus gall wasp Ophelimus maskelli in Israel. Phytoparasitica 35:330-332

26

Chapter 1

Mendel, Z., Protasov, A., Fisher, N., La Salle, J., 2004. Taxonomy and biology of Leptocybe invasa gen. & sp. n. (Hymenoptera: Eulophidae), an invasive gall inducer on Eucalyptus. Aust J Entomol 43:101-113 Mendel, Z., Protasov, A., La Salle, J., Blumberg, D., Brand, D., Branco, M., 2017. Classical biological control of two Eucalyptus gall wasps; main outcome and conclusions. Biol Control 105:66-78 Morales, C.F., Bain, J., 1989. Eriococcus coriaceus Maskell, gum tree scale (Homoptera: ) vol Technical Communication No. 10. CAB International and DSIR, Oxon, UK Murray, T.J., Withers, T.M., Mansfield, S., Bain, J., 2008. Distribution and current status of natural enemies of in New Zealand. N Z Plant Protect 61:185-190 Paine, T.D., Dahlsten, D.L., Millar, J.G., Hoddle, M., Hanks, J.B., 2000. UC scientists apply IPM techniques to new Eucalyptus pests. Calif Agr 54:8-13 Paine, T.D., Millar, J.G. Biological control of introduced pests of Eucalyptus in California. In: 1st International Symposium on Biological Control of Arthropods, Honolulu, Hawaii, 2002. pp 66-71 Paine, T.D., Millar, J.G., Daane, K.M., 2010. Accumulation of pest insects on eucalyptus in california: Random process or smoking gun. J Econ Entomol 103:1943-1949 Parrella, M.P., Heinz, K.M., Nunney, L., 1992. Biological Control through Augmentative Releases of Natural Enemies: A strategy whose time has come. Am Entomol 38:172-180 Pearson, D.E., Callaway, R.M., 2005. Indirect nontarget effects of host-specific biological control agents: Implications for biological control. Biol Control 35:288-298 Perez-Alvarez, R., Nault, B.A., Poveda, K., 2019. Effectiveness of augmentative biological control depends on landscape context. Sci Rep 9:1-15 Pérez Otero, R., Borrajo, P., Mansilla, J.P., Ruiz, F., 2011. Primera cita en España de Psyllaephagus bliteus Riek (Hymenoptera, Encyrtidae), parasitoide de Glycaspis brimblecombei Moore (Hemiptera, Psyllidae). Bol San Veg, Plagas 37:37-44 Peris-Felipo, F.J., Bernués-Bañeres, A., Pérez-Laorga, E.A., Jiménez-Peydró, R., 2010. Nuevos datos sobre la distribución en España de Glycaspis brimblecombei Moore, 1964 (Hemiptera: Psyllidae), plaga de Eucalyptus camaldulensis. Bol Asoc española de Entomol 33:517-526

27

Chapter 1

Pinet, C., 1986. Patasson nitens, parasite spécifique de Gonipterus scutellatus en France. Bulletin OEPP/EPPO Bulletin 16:285-287 Pluess, T., Cannon, R., Jarošík, V., Pergl, J., Pyšek, P., Bacher, S., 2012. When are eradication campaigns successful? A test of common assumptions. Biol Invasions 14:1365-1378 Poland, T.M., McCullough, D.G., 2006. Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. Forestry 104:118-124 Polis, G.A., 1994. Food webs, trophic cascades and community structure. Aust J Ecol 19:121-136 Protasov, A., Blumberg, D., Brand, D., La Salle, J., Mendel, Z., 2007. Biological control of the eucalyptus gall wasp Ophelimus maskelli (Ashmead): taxonomy and biology of the parasitoid species Closterocerus chamaeleon (Girault), with information on its establishment in Israel. Biol Control 42:196-206 Queiroz, D.L., Majer, J., Burckhardt, D., Zanetti, R., Fernandez, J.I.R., Queiroz, E.C., Garrastazu, M., Fernandes, B. V., Anjos, N., 2013. Predicting the geographical distribution of Glycaspis brimblecombei (Hemiptera: Psylloidea) in Brazil. Aust J Entomol 52:20-30 Reis, A.R., Ferreira, L., Tomé, M., Araujo, C., Branco, M., 2012. Efficiency of biological control of Gonipterus platensis (Coleoptera: Curculionidae) by Anaphes nitens (Hymenoptera: Mymaridae) in cold areas of the Iberian Peninsula: Implications for defoliation and wood production in Eucalyptus globulus. Forest Ecol Manag 270:216-222 Rizzo, M.C., Lo Verde, G., Rizzo, R., Buccellato, V., Caleca, V., 2006. Introduzione di Closterocerus sp. in Sicilia per il controllo biologico di Ophelimus maskelli Ashmead (Hymenoptera Eulophidae) galligeno esotico sugli eucalipti. Boll Zool agr Bachic 38:237-248 Robertson, M.P., Kriticos, D.J., Zachariades, C., 2008. Climate matching techniques to narrow the search for biological control agents. Biol Control 46:442-452 Roques, A., Kenis, M., Lees, D., Lopez-Vaamonde, C., Rabitsch, W., Rasplus, J-Y., Roy, D., 2010. Alien terrestrial arthropods of Europe. Pensoft Publishers, Rosen, D., 1986. The role of taxonomy in effective biological control programs. Agr Ecosyst Environ 15:121-129

28

Chapter 1

Saavedra, M.C., Avila, G.A., Withers, T.M., Holwell, G.I., 2015. The potential global distribution of the Bronze bug Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae). Agr Forest Entomol 17:375-388 Sanches, M.A., 2000. Parasitism of eggs of Gonipterus scutellatus Gyllenhal, 1833 and Gonipterus gibberus Boisduval, 1835 (Coleoptera, Curculionidae) by the mymarid Anaphes nitens (Girault, 1928) (Hymenoptera, Mymaridae) in Colombo, PR. Brazil Arquivos do Instituto Biológico (São Paulo) 67:77-82 Sánchez, I., Amarillo, J.M., Molina, D., 2015. Primera cita de Trachymela sloanei (Blackburn, 1897) (Coleoptera, Chrysomelidae) en Europa. Revista gaditana de Entomología 6:127-130 Santana, D.L.Q., Burckhardt, D., 2007. Introduced Eucalyptus psyllids in Brazil. J For Res 12:337–344 Santolamazza-Carbone, S., Cordero-Rivera, A., 2003. Egg load and adaptive superparasitism in Anaphes nitens, an egg parasitoid of the Eucalyptus snout- beetle Gonipterus scutellatus. Entomol Exp Appl 106:127-134 Santolamazza‐Carbone, S., Pérez‐Rodríguez, A., García‐Fojo, R., Cordero‐Rivera, A., 2019. Local augmentation efficiency of Anaphes nitens (Hymenoptera, Mymaridae), the egg parasitoid of Gonipterus platensis (Coleoptera, Curculionidae). J Appl Entomol 143:574-583 Schnell e Schühli, G., Penteado, S.C., Barbosa, L.R., Filho, R.W., Iede, E.T., 2016. A review of the introduced forest pests in Brazil. Pesq Agropec Brasileira 51:397- 406 Scutareanu, P., Drukker, B., Bruin, J., Posthumus, M.A., Sabelis, M.W., 1996. Leaf volatiles and polyphenols in pear trees infested by Psylla pyricola. Evidence of simultaneously induced responses. Chemoecology 7:34-38 Sharma, K.R., Jaiswal, D.K., Babu, S.R., Saraoj, A.K., 2018. Impact of invasive insect pests species on agroecosystem in india and their management. IJAAS 4:19-26 Sigsgaard, L., 2005. Oviposition preference of Anthocoris nemoralis and A. nemorum (Heteroptera: Anthocoridae) on pear leaves affected by leaf damage, honeydew and prey. Biocontrol Sci Techn 15:139-151 Simberloff, D., Stiling, P., 1996. How risky is biological control?. Ecology 77:1965-1974 Siscaro, G., 1992. Avetianella longoi sp. n. (Hymenoptera Encyrtidae) egg parasitoid of Phoracantha semipunctata F.(Coleoptera Cerambycidae). Boll Zool agr Bachic 24:205-212

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Tobin, P.C., 2015. Ecological consequences of pathogen and insect invasions. Curr Forestry Rep 1:25-32 Tobin, P.C., 2018. Managing invasive species. F1000Res 7 Faculty Rev-1686 Tooke, F.G.C., 1955. The Eucalyptus Snout beetle, Gonipterus scutellatus Gyll. a study of its ecology and control by biological means. Entomology Memoirs. Department of Agriculture and Forestry, Union of South Africa South Africa 282 pp Tribe, G.D., 2000. Ecology, distribution and natural enemies of the Eucalyptus- defoliating tortoise beetle Trachymela tincticollis (Blackburn) (Chrysomelidae: : Paropsina) in southwestern Australia, with reference to its biological control in South Africa. Afr Entomol 8:23-45 Tribe, G.D., 2003. Biological control of defoliating, and phloem- or wood-feeding Insects in commercial forestry in Southern Africa. In: Neuenschwander P, Borgemeister C, Langewald J (eds) Biological control in IPM systems in Africa. CABI Publishing, Tribe, G.D., 2005. The present status of Anaphes nitens (Hymenoptera: Mymaridae), an egg parasitoid of the Eucalyptus snout beetle Gonipterus scutellatus, in the Western Cape Province of South Africa South. Afr. For. J. 203:49-54 Valente, C., Afonso, C., Gonçalves, C.I., Alonso-Zarazaga, M.A., Reis, A., Branco, M., 2017a. Environmental risk assessment of the egg parasitoid Anaphes inexpectatus for classical biological control of the Eucalyptus snout beetle, Gonipterus platensis. BioControl 62:457-468 Valente, C., Gonçalves, C., Afonso, C., Reis, A., Branco, M., 2017b. Controlo biológico clássico do gorgulho-do-eucalipto: situação atual e perspetivas futuras Pragas e doenças emergentes em sistemas florestais In: Instituto Superior de Agronomia, Lisbon, June, 8, 2017 Valente, C., Gonçalves, C.I., Monteiro, F., Gaspar, J., Silva, M., Sottomayor, M., Paiva, M.R., Branco, M., 2018. Economic outcome of classical biological control: A case study on the Eucalyptus snout beetle, Gonipterus platensis, and the parasitoid Anaphes nitens. Ecol Econ 149:40-47 Valente, C., Gonçalves, C.I., Reis, A., Branco, M., 2017c. Pre-selection and biological potential of the egg parasitoid Anaphes inexpectatus for the control of the Eucalyptus snout beetle, Gonipterus platensis. J Pest Sci 90:911-923 Valente, C., Hodkinson, I., 2009. First record of the red gum lerp psyllid, Glycaspis brimblecombei Moore (Hem.: Psyllidae), in Europe. J Appl Entomol 133:315-317

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Valente, C., Ruiz, F. Detecção de Phoracantha recurva Newman (Coleoptera, Cerambycidae) em Portugal. In: X Congresso Ibérico de Entomologia, Zamora, 16-20 September 2002. Valente, C., Vaz, A., Pina, J., Manta, A., Sequeira, A. Control strategy against the eucalyptus snout beetle, Gonipterus scutellatus Gyllenhal (Coleoptera, Curculionidae), by the portuguese cellulose industry. In: Borralho NMG, Pereira JS, Marques C, Coutinho J, Madeira M, Tomé M (eds) Eucalyptus in a Changing World, Aveiro, 2004. IUFRO, pp 622-627 Van Driesche, R.G., et al. 2010. Classical biological control for the protection of natural ecosystems. Biol Control 54:S2-S33 van Lenteren, J.C., Bale, J., Bigler, F., Hokkanen, H.M., Loomans, A.J., 2006. Assessing risks of releasing exotic biological control agents of arthropod pests. Annu Rev Entomol 51:609-634 van Lenteren, J.C., Loomans, A.J.M., Babendreier, D., Bigler, F., 2007. Harmonia axyridis: an environmental risk assessment for Northwest Europe. BioControl 53:37-54 Van Wyk, A., Brink, A., Van Huyssteen, G., Jansen, J., 2010. Tree farming guidelines for private growers - Parte 2, silviculture. Sappi Forests, South Africa 123 pp Villanueva-Gutierrez, R., Echazarreta-Gonzalez, C., Roubik, D.W., Moguel-Ordonez, Y.B., 2014. Transgenic soybean pollen (Glycine max L.) in honey from the Yucatan peninsula, Mexico. Sci reps 4:1-4 Williamson, M., Fitter, A. 1996. The varying success of invaders. Ecology 77:1661-1666 Wingfield, M.J., Brockerhoff, E.G., Wingfield, B.D., Slippers, B. 2015. Planted forest health: The need for a global strategy. Science 349:832-836 Withers, T.M., 2001. Colonization of eucalypts in New Zealand by Australian insects. Austral Ecol 26:467-176 Withers, T.M., Jones, D.C., 2003. The seasonal abundance of the newly established parasitoid complex of the eucalyptus tortoise beetle (Paropsis charybdis). N Z Plant Protect 56:51-55 Wittenberg, R., Cock, M.J.W., 2005. Best Practices for the prevention and management of invasive alien species. In: Mooney HA, Mack RN, McNeely JA, Neville LE, Schei PJ, Waage JK (eds) Invasive Alien Species: A New Synthesis. Island Press, Washington - Covelo - London

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Yang, F., Kerns, D. L., Brown, S., Kurtz, R., Dennehy, T., Braxton, B., Head, G., Huang, F., 2016. Performance and cross-crop resistance of Cry1F-maize selected Spodoptera frugiperda on transgenic Bt cotton: implications for resistance management. Sci rep 6:1-7 Zacharia, J.T., 2011. Ecological Effects of Pesticides. In: Stoytcheva DM (ed) Pesticides in the Modern World - Risks and Benefits. InTech, pp 129-142 Zerga, B., Woldetsadik, M., 2016. Contribution of Eucalyptus tree farming for rural livelihood in Eza Wereda, Ethiopia Palgo. J Agric 3:111-117 Zhang, G.F., Lovei, G.L., Wu, X., Wan, F.H., 2016. Presence of native prey does not divert predation on exotic pests by Harmonia axyridis in its indigenous range. PloS one 11:1-15

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First report of Epichrysocharis burwelli in Europe, a new invasive gall wasp attacking eucalypts

Published in Phytoparasitica

Franco, J.C., Garcia, A. & Branco, M., 2016. First report of Epichrysocharis burwelli in Europe, a new invasive gall wasp atacking eucalypts. Phytoparasitica 44, 443–446. https://doi.org/10.1007/s12600-016-0539-9

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Abstract

Epichrysocharis burwelli (Hymenoptera: Eulophidae) was detected in Portugal, infesting the leaves of lemon-scented gum, Corymbia citriodora (Myrtaceae), in four out of seven sampled locations, along Tagus river, between the region of Lisbon and Castelo Branco. This is the first record of this alien all wasp in Europe and in the Palaearctic region. An unidentified species of Closterocerus, which is not known from Europe, was found parasitizing the larvae of E. burwelli.

Keywords: Corymbia citriodora; Eucalypt; Gall wasp; Insect pest; Alien species; Portugal

Case study

Eucalypts are a diverse group of woody plants of the Myrtaceae family, including over 800 species, distributed among three genera, Eucalyptus, Angophora, and Corymbia, which are native to Australia, Indonesia, the Philippines, and New Guinea (Doughty 2000; Coppen 2002; Paine et al. 2011). Many eucalypt species have been introduced in different regions in all the continents. However, only a few species are widely cultivated, such as Eucalyptus camaldulensis, E. globulus, E. grandis and E. tereticornis, which are the dominant planted species worldwide (Doughty 2000). These species are major source of timber and/or fibre for pulp industry. Eucalypts also have been planted as ornamental, landscape and windbreak trees (Doughty 2000). Some eucalypt species are used in the commercial production of essential oils, as for example the lemon-scented gum, Corymbia (=Eucalyptus) citriodora (Hook.) K.D. Hill & L.A.S. Johnson (Doughty 2000; Coppen 2002). The area of eucalypts plantation in the world totals ca. 20,000,000 ha, which corresponds to about 15 % of global planted forest (IUFRO 2015). In Portugal, the area of eucalypts includes more than 800,000 ha, mainly with E. globulus (ICNF 2013). Corymbia citriodora is grown in different countries mostly as ornamental, but its oil, whose main component is citronellal, responsible for the characteristic lemon scent of its leafs, has commercial application in perfumery and as an insect repellent (Coppen 2002). In Portugal, C. citriodora has been planted occasionally as ornamental tree in gardens, urban parks and for oil extraction. Eucalypts are attacked by many herbivore insects, in their native range. However, very few insect pests followed them in the different regions where they were introduced, due to the fact that most of the introduced eucalypts originate from seeds (Doughty 2000). However, this situation dramatically changed within the last

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10–20 years. The number of alien insect pests of eucalypts introduced in Europe increased exponentially. Until early 1990’s, only three insect pests of eucalypts were known in Europe and Mediterranean. Nowadays, at least 14 species have been introduced in the region, including three leaf gallers (Hymenoptera: Eulophidae), Leptocybe invasa Fisher &. LaSalle, Ophelimus maskelli (Ashmead) and Ophelimus sp. (Paine et al. 2010; Hurley et al. 2016). Recently, during a field trip to an urban park in Almada, with planted stands of different eucalypt species, we detected for the first time the presence of galls in leafs of C. citriodora. Later on, we concluded that these galls were produced by Epichrysocharis burwelli Schauff (Hymenoptera: Eulophidae). Here we report the presence of this alien wasp in Portugal, which is the first record in Europe. After being detected in June 2015, a survey was conducted in the central region of Portugal, between January and March 2016, in order to access the extent of dispersal of E. burwelli and determine its possible pathway. A total of seven sites with planted trees of C. citriodora were checked for the presence of the gall wasp, mostly in the area of Almada and Lisbon, and up to Castelo Branco region along Tagus river, including gardens, parks and a commercial plantation for oil extraction (Table 2.1). In each site, trees were inspected for the presence of leaf galls. In those where leaf galls were

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Table 2.1. – Results of the survey carried out in plots of Corymbia citriodora to detect the presence of Epichrysocharis burwelli.

Nº galls per leaf Parasitoid Site Lat. Long. Infestation level (mean ± SE) detection Tapada da Ajuda, 38,714 -9,192 1.0 141.2 ± 16.3 No Lisbon Sete-Rios, 38,747 -9,175 1.5 267.3 ± 34.4 Yes Lisbon Almada 38,662 -9,169 1.5 542.1 ± 74.9 No Escaroupim 39,070 -8,743 0.5 76.2 ± 18.4 No Constância 39,471 -8,332 - - Tramagal 39,452 -8,252 - - Venda - 39,596 -7,863 - Nova Lat. Latitude, Long. Longitude detected, four trees were randomly selected and in each of them a sample of four branches (one per cardinal direction) was locally verified to characterizing the infestation level. The following notation was used to classify each sampled branch: 0 – no presence of leaf galls; 1 – galls present in 1–20 % of the leafs; 2 – galls present in 21–50 % of the leafs; and 3 – galls present in more than 50 % of the leafs. Whenever E. burwelli was detected, samples were collected to determine its rate of emergence, as well as to investigate the eventual presence of parasitoids and corresponding level of parasitism. With that purpose, a total of 12 leaves per tree were randomly selected from two infested trees and transported to the laboratory. Each leaf was kept isolated within 110 ml plastic containers, stopped up with parafilm, in laboratory conditions (17.7 ± 1 °C and 71 ± 8 % r.h.) until the emergence of adult wasps (about 25 days). The number of galls per leaf, as well as the number of emerged adults of E. burwelli, was determined. When present, parasitoids were also collected. Vouchers with adults of both E. burwelli and parasitoids were kept for taxonomic identification. The identification of E. burwelli was carried out based on Schauff and Garrison (2000). Epichrysocharis burwelli was observed in four out of the seven sampled sites (Fig. 1 and Table 2.1). In those sites, the mean infestation level varied between 0.5 and 1.5, and the mean number of galls per leaf between 76 and 542 (Table 2.1). A parasitoid emerged from galls induced by E. burwelli in one of the sampled sites

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(Fig. 2.1 and Table 2.1). It was identified by Dr. Christer Hansen (Museum of Biology, Lund, Sweden) as Closterocerus sp., an unknown species in Europe. The genus Epichrysocharis includes four species, E. aligherini (Girault), E. fusca (Girault), E. nigriventris (Girault), and E. burwelli (Schauff and Garrison 2000). Epichrysocharis burwelli is distinguished from the other species of the genus by its “brown and yellow head with a uniformly brown mesosoma and metasoma and relatively short ovipositor” (Schauff and Garrison 2000). The biology and ecology of E. burwelli is poorly known. Pereira (2010) studied the seasonal dynamics of leaf infestation, as well as the life history and life cycle of E. burwelli in Brazil. The economic impact of the wasp was also estimated. Depending on the infestation level on the leaves (number of galls per leaf), E. burwelli may negatively affect the essential-oil yield in plantations of C. citriodora (Pereira et al. 2012). Native to Australia, E. burwelli has dispersed to the Neotropical and Nearctic regions. It was reported from California (Schauff and Garrison 2000) and Brazil (Berti Filho et al. 2004) on C. citriodora.

Fig. 2.1 a: Leaf of Corymbia citriodora with galls induced by Epichrysocharis burwelli (the length of the white bar is 1 cm); b: Adult of E. burwelli (0.6 mm) ;c: Adult of Closterocerus sp.(0.55 mm)

Our report is the first record of E. burwelli in the Palaearctic region. It took about 16 year to reach Europe, since its detection in North America, the first record outside its native area, and 12 years since its introduction in South America. This dispersion trend is in line with the invasive bridgehead effect (Lombaert et al. 2010), according to which the probability of an invasive species further spreading, after becoming established outside its native range, increases as a result of an existing larger source population. In Portugal,

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E. burwelli is actually restricted to an area along Tagus river, between Lisbon and Escaroupim, about 50 Km northeast of Lisbon. Based on its actual distribution and on the fact that three out of four sites in which the wasp was detected are located in a region around Lisbon, also corresponding to the higher levels of infestation, we suggest that the area of introduction of this alien species was most probably Lisbon, which has international maritime port and airport. Considering that E. burwelli is a foliage-feeding insect, importation of live plant material and cut branches of eucalypts is its possible pathway of introduction (see Hurley et al. 2016). We suggest that the introduction of this wasp into Portugal might be related to the importation of C. citriodora plants for ornamental purposes or oil production. In fact, the importation of eucalypts has been registering a significant increase recently. For example, the importation of Eucalyptus wood in Portugal increased more than 50 % in the period 2010–2014 in relation to the previous 5 years (CELPA 2014). Most of the Eucalyptus wood is coming from South America. Hurley et al. (2016) showed that the number of nonnative pests of eucalypts outside Australia has doubled in less than three decades. Epichrysocharis burwelli is the fourth leaf-gall wasp invading the Mediterranean area in the last 16 years. Gall formers are the third most important guild of introduced pest insects of eucalypts, after sup- sucking taxa and defoliators (Hurley et al. 2016).

Acknowledgments

We would like to thank Dr. Christer Hansen (Museum of Biology, Lund, Sweden) for the identification of Closterocerus sp. Thanks are also due to Ivone Neves and Clara Araújo (Altri-Florestal), Francisco Delgado, Paula Soares, Pedro Nunes (ISA, ULisboa), Hugo Gonçalves (Universidade Estadual do Espírito Santo, Brazil), and Cristina Oliveira for their help in the field survey and lab work, as well as to reviewers for their comments and suggestions. This work was funded by Fundação para a Ciência e Tecnologia (FCT) through the Doctoral Programme SUSFOR - Sustainable Forests and Products PD/00157/2012 and Project UID/AGR/00239/2013.

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References

Berti Filho, E., Costa, V. A., & Lasalle, J. (2004). Primeiro registro da vespa de galha Epichrysocharis burwelli (Hymenoptera: Eulophidae) em Corymbia (Eucalyptus) citriodora (Myrtaceae) no Brasil. Revista de Agricultura, 79 (3), 363–364 (in Portuguese). CELPA (2014). Boletim estatístico 2014. CELPA Associação da Indústria Papeleira, 99 pp. Available at http://www.celpa.pt/wp-content/uploads/2015/09/Boletim- Estatístico-da-Celpa-de-2014.pdf (in Portuguese) Coppen, J. J. W. (2002). Eucalyptus: the genus Eucalyptus. London: Taylor & Francis. Doughty, R.W. (2000). The Eucalyptus, a natural and commercial history of the Gum tree. Baltimore, USA: The Johns Hopkins University Press. Hurley, B. P., Garnas, J., Wingfield, M. J., Branco, M., Richardson, D. M., & Slippers, B. (2016). Increasing numbers and intercontinental spread of invasive insects on eucalypts. Biological Invasions, 18, 921–933. doi:10.1007/s10530-016-1081-x. ICNF – Instituto da Conservação da Natureza e das Florestas. (2013). IFN6 – Áreas dos usos do solo e das espécies florestais de Portugal continental: Resultados preliminares (p. 34). Lisboa: Instituto da Conservação da Natureza e das Florestas. (in Portuguese) IUFRO - International Union Forestry Research Organization (2015). Scientific cultivation and green development to enhance the sustainability of eucalypt plantations. IUFRO Eucalypt Conference 2015, Zhanjiang, Guangdong, China, October 21–24: available at http://www.euciufro2015.com/en/ Lombaert, E., Guillemaud, T., Cornuet, J.-M., Malausa, T., Facon, B., & Estoup, A. (2010). Bridgehead effect in the worldwide invasion of the biocontrol Harlequin ladybird. PLoS ONE, 5, e9743. Paine, T. D., Millar, J. G., & Daane, K. M. (2010). Accumulation of pest insects on eucalyptus in California: random process or smoking gun. Journal of Economic Entomology, 103, 1943–1949. Paine, T. D., Steinbauer, M. J., & Lawson, S. A. (2011). Native and exotic pests of Eucalyptus: a worldwide perspective. Annual Review of Entomology, 56, 181–201. Pereira, R.A. (2010). Aspectos morfo-bioecológicos de Epichrysocharis burwelli (Eulophidae, Hymenoptera), vespa-das-galhas das folhas de Corymbia citriodora. Tese apresentada para obtenção do título de Doutor em Ciências, Área de

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concentração: Entomologia, Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz”, Piracicaba (in Portuguese) Pereira, R. A., Berti Filho, E., & Moura, R. G. (2012). Rendimento de óleo essencial de Corymbia citriodora Hill& Johnson sob diferentes níveis de infestação de galhas de Epichrysocharis burwelli Schauff (Hymenoptera, Eulophidae). Revista de Agricultura, 87(1), 10–17 (in Portuguese). Schauff, M. E., & Garrison, R. (2000). An introduced species of Epichrysocharis (Hymenoptera: Eulophidae) producing galls on Eucalyptus in California with notes on the described species and placement of the genus. Journal of Hymenoptera Research, 9(1), 179–180.

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Chapter 3 Biological control of Gonipterus: Uncovering the associations between eucalypts, weevils and parasitoids in their native range

Published in Forest Ecology and Management

Garcia, A., Allen, G. R., Oberprieler, R. G., Ramos, A. P., Valente, C., Reis, A., Franco, J. C., Branco, M., 2019. Biological control of Gonipterus: Uncovering the associations between eucalypts, weevils and parasitoids in their native range. Forest Ecology and Management, 443(1): 106-116 https://doi.org/10.1016/j.foreco.2019.04.004

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Abstract The study was initiated by the relative failure of the parasitoid Anaphes nitens in controlling the eucalypt weevil Gonipterus platensis on the Iberian Peninsula. Our aim was to gain insight into the community of Gonipterus egg parasitoids occurring in Tasmania. During surveys in 2016 and 2017, adult weevils and egg pods were collected from Eucalyptus trees in Tasmania. The weevils were identified using male genital structure and DNA extracted from hatched larvae. Parasitoids that emerged from the egg pods were identified, and trophic associations of egg parasitoids, weevils and host plants were analyzed. Five species of the Gonipterus scutellatus complex, to which G. platensis belongs, were found, including Gonipterus sp. 2, which is reported for the first time from Tasmania. Molecular analysis corroborated previous phylogenetic studies of this group of species. A sixth species, G. notographus, was also collected. Most species were found to overlap in distribution in Tasmania and, despite being oligophagous, to display selectivity among Eucalyptus species used as hosts: G. platensis and G. pulverulentus were mainly found on E. ovata, Gonipterus sp. 1 on E. nitens and E. globulus and G. notographus on ‘peppermint’ species (E. amygdalina and E. pulchella). Five egg parasitoid species were found associated with these Gonipterus species: Anaphes inexpectatus, A. nitens, A. tasmaniae, Cirrospilus sp. and Euderus sp., with no apparent host specialization. Anaphes nitens, Cirrospilus sp. and Euderus sp. were more frequently found on E. ovata, possibly associated with G. platensis and G. pulverulentus, which were dominant on this host species. Conversely, A. inexpectatus was dominantly found on peppermints (43%), suggesting a main association with G. notographus. Anaphes nitens was found at 23 locations out of 117 and in 2017 was the most abundant parasitoid obtained, with an average 20% parasitism rate, indicating that this species is undergoing a geographical and population expansion since its first report from Tasmania in 2012. These findings contribute to the understanding of the parasitoid-Gonipterus-Eucalyptus trophic relationship and stand to improve future classical biological control programs against G. platensis and other invasive Gonipterus species.

Keywords: Eucalyptus; Anaphes inexpectatus; A. nitens; A. tasmaniae; Gonipterus platensis; Host associations

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Introduction The genus Gonipterus Schoenherr (Coleoptera: Curculionidae: Gonipterini) comprises about 20 Australian species of weevils, all feeding on Eucalyptus leaves (Tooke, 1955, Clarke et al., 1998, Mapondera et al., 2012, Oberprieler et al., 2014). Gonipterus species are oligophagous, feeding on several Eucalyptus species (Newete et al., 2011, Gonçalves et al., in preparations). Three Gonipterus species (G. platensis (Marelli), G. pulverulentus Lea and Gonipterus sp. 2) are regarded as having attained pest status in Eucalyptus plantations worldwide (Mapondera et al., 2012, Hurley et al., 2016). So far, classical biological control (CBC) has been the main strategy to control these weevil species, mainly through the egg parasitoid Anaphes nitens (Girault) (Hymenoptera: Mymaridae). However, A. nitens does not successfully control Gonipterus in some regions in Europe, South America, Western Australia and South Africa where Eucalyptus has been planted (Tribe, 2003, Loch, 2008, Mayorga et al., 2013, Valente et al., 2017a). For this reason, new biological control agents that might complement the activity of A. nitens have to be identified. Gonipterus first appeared outside of Australia in New Zealand in 1890 (Broun, 1893, Kuschel, 1990), subsequently in South Africa in 1916 (Mally, 1924, Tooke, 1955) and South America in 1925 (Marelli, 1926) and more recently also in western Europe and parts of the U.S.A. (Mapondera et al., 2012, Barratt et al., 2018) (Table 3.1.). The identification of these invasive Gonipterus weevils was controversial from the beginning (Tooke, 1955, Barratt et al., 2018), and they were eventually all named as G. scutellatus Gyllenhal (Table 3.1.). However, in a combined morphological-molecular study, Mapondera et al. (2012) showed that the Gonipterus populations in these different countries are not conspecific but represent three different species, all belonging to a G. scutellatus complex. This study revealed that the species present in Africa, Italy and France is as yet undescribed species (referred to as Gonipterus sp. 2) and the species in New Zealand, South America, United States of America (U.S.A.), Spain and Portugal are G. platensis, which had not been described from Australia. Another Gonipterus species (G. pulverulentus) is present in South America, seemingly limited to Argentina, Brazil and Uruguay (Mapondera et al., 2012). Gonipterus scutellatus was found to be a rare species limited to Tasmania and not introduced anywhere (Mapondera et al., 2012).

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Table 3.1 – Invasive Gonipterus species in countries with major Eucalyptus industries, with their previous and currently accepted names and year of recording

Gonipterus Gonipterus Year of Country References former names current name recording New Broun, 1893; Kuschel, 1990; G. scutellatus G. platensis 1890 Zealand Mapondera et al., 2012 South Mally, 1924; Tooke, 1955; Mapondera G. scutellatus Gonipterus sp. 2 1916 Africa et al., 2012 Dacnirotatus Marelli, 1926; Rosado-Neto and platensis Marelli; G. platensis Argentina 1926 Marques, 1996; Mapondera et al., 2012 G. scutellatus Dacnirotatus bruchi Marelli; Marelli, 1926; Rosado-Neto and G. pulverulentus Argentina 1926 G. gibberus Marques, 1996; Mapondera et al., 2012 Boisduval

G. scutellatus G. platensis Uruguay 1943 Bosq, 1943; Mapondera et al., 2012;

Barbiellini, 1955; Kober, 1955; G. scutellatus G. platensis Brazil 1954 Mapondera et al., 2012 Sampo, 1976; Arzone and Meotto, G. scutellatus Gonipterus sp. 2 Italy 1976 1978; Mapondera et al., 2012 Rabasse and Perrin, 1979; Mapondera G. scutellatus Gonipterus sp. 2 France 1977 et al., 2012

G. scutellatus G. platensis Spain 1991 Mansilla, 1992; Mapondera et al., 2012

U.S.A. Cowles and Downer, 1995; Haines and G. scutellatus G. platensis (California 1994/2004 Samuelson, 2006; Mapondera et al., /Hawaii) 2012

G. scutellatus G. platensis Portugal 1995 Sousa and Ferreira, 1996

Lanfranco and Dungey, 2001; G. scutellatus G. platensis Chile 1997 Mapondera et al., 2012

After the establishment of Gonipterus in South Africa, chemical control was used for some years in an attempt to control its heavy attacks on Eucalyptus plantations and mitigate the resulting defoliation and consequent wood losses (Tooke, 1955). However, the application of insecticides led only to a temporary suppression of the weevil and was not efficient enough to avoid economic losses. Chemical control is also unsustainable due to the need for successive treatments (Valente et al., 2018a). As an alternative approach, the South African government implemented a CBC program (Tooke, 1955, Tribe, 2003,

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Tribe, 2005) by importing the egg-parasitic mymarid wasp A. nitens (Girault) from Australia in 1926 and releasing it as a biocontrol agent releasing it in the country from 1927 (Tooke, 1955, Tribe, 2005, Barratt et al., 2018). The results were exceptionally good. After five years, parasitism rates reached 96–98% and the problem was almost solved, except in very limited areas (Tribe, 2003). The high success of this CBC program prompted other countries experiencing Gonipterus damage to also introduce A. nitens (Rivera et al., 1999, Paine et al., 2000, Lanfranco and Dungey, 2001). The parasitoid was thus imported from South Africa into North and South America (Hanks et al., 2000, Lanfranco and Dungey, 2001), southern Europe (Italy and France) (Malausa et al., 2008) and the Iberian Peninsula, where it was released between 1994 and 2000 (Rivera et al., 1999, Valente et al., 2018a). Despite the general success of A. nitens in these countries, parasitism rates were not as high as expected in some regions and insufficient to avoid severe defoliation and wood losses (Tribe, 2003, Reis et al., 2012). This is the case in parts of the Iberian Peninsula, where E. globulus plantations occupy an area of about 1.4 million ha and generate high economic value (Ruiz and Lopez, 2010, ICNF, 2013). In Chile, G. platensis is also still a problem in areas with particular climatic conditions (Lanfranco and Dungey, 2001), as is the case in Western Australia, where G. platensis established in 1995 and causes significant damage in eucalypt plantations despite the presence of the parasitoid (Loch, 2008). Climate is probably an important factor in unsuccessful biocontrol, as low parasitism rates usually occur in regions with cold winters, leading to a delay in the development of the parasitoid in spring. In Portugal, parasitism rates by A. nitens during spring were found to decrease exponentially with elevation and low mean temperatures in the coldest months (Valente et al., 2004, Reis et al., 2012). Such an outcome is possibly related to the limited activity and reproduction of the parasitoid at low temperature (Santolamazza-Carbone et al., 2006). However, Mapondera et al. (2012) noted that A. nitens is native to south-eastern continental Australia, where it naturally parasitizes Gonipterus sp. 2, which is widespread in this region, whereas G. platensis originates from Tasmania, where A. nitens appears not to be native (Barratt et al., 2018). Due to the incomplete success of biocontrol by A. nitens, G. platensis continues to inflict serious economic losses in some of the affected regions. In Portugal, Valente et al. (2018b) estimated economic losses of about 648 million € caused by G. platensis between 1996 and 2016, despite the fact that A. nitens is controlling the weevil in many parts of the country where Eucalyptus is grown. However, if A. nitens had not been introduced,

45

Chapter 3 wood losses would have been four to eleven times higher than currently observed (Valente et al., 2018a). Insecticides are currently applied in those areas where biological control by A. nitens is ineffective. However, this is not considered to be a sustainable control strategy in the long term (Valente et al., 2018a). Therefore, looking for new candidates for an effective biocontrol program against G. platensis in those areas is a high priority. CBC is the most sustainable long-term tactic to cope with non-native insect pests. This is especially true when the pest status results from an enemy-free zone following populations becoming isolated from their native natural enemies (Keane and Crawley, 2002). When successful, CBC introduces a population regulation mechanism that keeps the target pest at low, stable and economically sustainable population levels without need for further actions (DiTomaso et al., 2017). In contrast, application of pesticide will cause an immediate suppressive effect on the target population but with a null resilient persistence, and populations will continue growing afterwards, implying the need for successive treatments. Furthermore, pesticide applications have negative impacts on human health, non-target species and several ecosystem services (e.g., beneficial insects and water quality) (Zacharia, 2011). Forests are characterized by their long-term rotation period, complexity of ecosystems and high biodiversity (Barsoum et al., 2016). Forest ecological functions and complexity therefore justify high restrictions on insecticide applications, as imposed by certification committees (e.g. Forest Stewardship Council), and CBC is frequently the major option for pest control in forestry environments (Kenis et al., 2017). Still, CBC has its own difficulties. The main challenge is to find good biocontrol agents that will be able to establish and successfully control the pest in a particular region, without non-target effects (Hoddle, 2005, Robertson et al., 2008). Kenis et al. (2017) showed that globally CBC has a success rate of almost 50%, whereas establishment rates are about 37%. Unsatisfactory results in the control of G. platensis by A. nitens in some regions justify the need to search for new and more suitable/specific biocontrol candidates in the weevil’s native region (Mayorga et al., 2013, Valente et al., 2017a). In surveys conducted in Tasmania between 2010 and 2012, Valente et al. (2017b) identified six egg-parasitoid wasp species, A. inexpectatus Huber & Prinsloo, A. nitens, A. tasmaniae Huber & Prinsloo (Hymenoptera: Mymaridae), Centrodora damoni (Girault) (Hymenoptera: Aphelinidae), Cirrospilus sp. and Euderus sp. (Hymenoptera: Eulophidae), as well as three larval parasitoids, Anagonia sp. (Diptera: ), Entedon magnificus (Girault & Dodd)

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(Hymenoptera: Eulophidae) and Oxyserphus sp. (Hymenoptera: Proctotrupidae). However, due to the difficulties in identifying the different Gonipterus species, Valente et al. (2017b) could not establish clear relationships between the Gonipterus species and the parasitoids. The main objective of this study was therefore to find biocontrol agents that could be specifically associated with G. platensis in Tasmania. We used morphological and molecular studies to identify the sampled weevil and parasitoid specimens. A second, broader objective was to expand the knowledge on the Eucalyptus- Gonipterus-parasitoid associations, for which our sampling and study was likely to contribute new data for other Gonipterus species as well. To this end we conducted a sampling regime of Gonipterus species across differing regions of Tasmania. Egg masses of Gonipterus were collected and their emerging parasitoids identified. With this work we expect to contribute to the improvement of future CBC programs against G. platensis and other invasive Gonipterus species.

Material and methods

Study area and surveys The study was conducted in Tasmania (Australia). Two surveys were carried out on Eucalyptus trees growing along road sides, the first between October 2016 and January 2017 (hereafter referred to as the 1st survey) and the second between November and December 2017 (the 2nd survey). A total of 117 sites was sampled in the 1st survey (of which 27 were visited more than once), and 19 sites were sampled in the 2nd survey. This survey covered all of Tasmania but for the remote west coast and south-west regions, with the latter differing in vegetation from the eucalypt woodlands present throughout the rest of Tasmania.

Site selection and field sampling The selection of sites for the survey was based on the presence of eucalypt trees with branches up to 3 m in height, to allow hand sampling. Except for Deddington, Grindwald, Hobart Park, Runnymede and The Glen Road, which were also surveyed based during previous visits (by Carlos Valente and David de Little, personal communication), all remaining sites were selected by the first author during travels in Tasmania, aiming to cover the maximum diversity of habitats in the region. At each site, eucalypt species were identified, whenever possible, using the field guide EucaFlip: Life-Sized Guide to the Eucalypts of Tasmania (Wiltshire and Potts, 2007).

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GPS coordinates and altitude was also registered using the GPS Essential App for Android®. Eucalyptus trees were screened for the presence of any Gonipterus individuals (egg, larvae or adults). Gonipterus specimens were collected from leaves or branches and stored in clear plastic boxes (22 × 14 × 7 cm) and transported to the Entomology Laboratory of the University of Tasmania (UTAS) in Hobart. Collected specimens of Gonipterus were sorted according to developmental stage. Egg pods showing emergence holes were discarded (considered to not contain viable eggs), whereas viable egg pods were cut out the leaves using a hole punch, counted and stored individually in clear gelatin capsules (size 0 Capsuline®). In the laboratory the egg pods were maintained under controlled conditions (22 ± 1 °C; 14 L:10 D photoperiod; 60% RH) and observed every 2–3 days until eclosion of the larvae. All collected egg pods were sent as soon as possible to RAIZ (Portugal), under consignment of Australian Government authorities, and there maintained in quarantine. Newly hatched Gonipterus larvae were stored in absolute alcohol in 1 mL Eppendorf tubes. Voucher specimens of Gonipterus adults were preserved in absolute alcohol in 5 mL plastic vials for identification and subsequently deposited in the Australian National Insect Collection (ANIC) in and in the School of Agriculture (ISA) - University of Lisbon, collection.

Identification of Gonipterus specimens and egg parasitoids Identification of Gonipterus adults was based on the structure of the male genitalia, which are diagnostically different between the species (Mapondera et al., 2012). Though previous attempts had been made to identify females, morphological differences in this sex are very subtle and inconsistent, making them unreliable for species identification (Mapondera et al., 2012). Males were dissected by removing the entire abdomen with fine tweezers. The abdomen was then placed in 10% KOH solution at 70 °C for about 20 min to macerate the muscle tissues (Porto et al., 2016) and, when cleared, was rinsed in a Petri dish with an 80% ethanol solution, in which the genitalia were also severed from the abdomen under a microscope. The separated genitalia were then placed in glycerol for further clearing, study and temporary storage. Identification was made under a Leica M205C stereo microscope at 45× magnification, specifically by comparison of the diagnostic internal sclerite of the penis (Mapondera et al., 2012). Remaining body parts (head, thorax, wings and legs) of dissected specimens were stored in absolute alcohol in Eppendorf tubes. One adult male from each of the identified Gonipterus species was used to confirm identification through molecular methods (see 2.4.). The Gonipterus sp. 2

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Chapter 3 specimen used for sequencing was from mainland Australia (Canberra). The sequences from these species were used to construct the phylogenetic tree. Gonipterus larvae hatched from egg pods from which parasitoids had hatched from sibling eggs as well as non-parasitized Gonipterus larvae were preserved. A total of 64 Gonipterus larvae, one from each egg pod from which parasitoids had emerged from sibling eggs, were identified through molecular methods and used for constructing a phylogenetic tree. These egg pods were distributed among 27 sampling sites (Supplementary Table 3.1.). Additionally, 44 Gonipterus larvae, hatched from non- parasitized egg pods, collected from 35 sites, were also genetically analyzed to broader the sample of host tree with Gonipterus species. The low number of pairs of larvae and parasitoids from sibling eggs reflects the difficulties of obtaining fresh larvae available for molecular work, such as the inviability of many egg pods, probably due to handling and transport. Anaphes species were identified using the key of Huber and Prinsloo (1990), and other parasitoid species were identified, up to genus level, by Catarina Gonçalves (RAIZ, Portugal), using reference specimens previously determined by the late Dr John LaSalle at CSIRO, Australia. Some of the collected specimens were not possible to be characterizing up to species level, once there is no suitable literature available. Voucher specimens were preserved in absolute alcohol and stored in the ISA collection for future reference. All parasitoids reared at the RAIZ facilities in Portugal were used for further biological studies. Only reliably identified specimens were used to generate the distribution maps of Gonipterus and the parasitoid wasp species, using ESRI ArcGis® 10.3 software. Data on climate, mean temperatures of the three coldest and hottest months and mean annual precipitation were retrieved using WorldClim Version2 (Fick and Hijmans, 2017), based on the geographical coordinates of the collection sites.

DNA extraction, sequencing and phylogenetic tree construction DNA was extracted from the hind legs of adult Gonipterus males, and the rest of the body was preserved for reference. With larvae the entire body was used for DNA extraction. Extractions were conducted using the DNeasy Blood and Tissue® kit (QIAGEN, Hilden, Germany). The protocol provided by the manufacturer was followed, with the following modifications: initial sample washed with ultra-pure sterilized water, followed by instant freeze in liquid nitrogen to improve cell lysis and addition of 4uL of Rnase A after the incubation period (approx. 2 h at 56 °C).

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Polymerase Chain Reaction (PCR) was conducted using the primers Jerry (5′- CAA CAT TTA TTT TGA TTT TTT GG-3′) and Pat (5′- TCC AAT GCA CTA ATC TGC CAT ATT A-3′) (Simon et al., 1994, Mapondera et al., 2012), which extracted a fragment of 800 bp of the cytochrome oxidase I (COI) region of the mitochondrial DNA (mtDNA). Each 25 µL PCR reaction tube contained 12.5 µL of DreamTaq™ MasterMix (2x) solution (Thermo Fisher Scientific, Lithuania). 9.5 µL of ultra-pure sterilized water, 1 µL of each Jerry and Pat primers (each with a concentration of 10 µM) and 1 µL of DNA (approx. 5 ng). The PCR cycle program was as follows: initial denaturation at 95 °C for two minutes, followed by 40 cycles of denaturation at 95 °C for half a minute, annealing at 46 °C for a minute, extension at 72 °C for one minute and a final extension at 75 °C for five minutes. PCR cycles were conducted on a S1000 Bio-Rad Thermal Cycler. Quality of the PCR reactions was verified on an agarose gel (0.5% TBE). DNA sequences were obtained through Sanger sequencing protocol. Amplification of COI fragments was made on an ABI 3730 XL sequencer at Stabvida (http://www.stabvida.com). Each sequence, without any modification, was Blasted against the sequences of Gonipterus species available in GenBank (www.blast.ncbi.nlm.nih.gov) for a species match. Following this, each COI sequence was individually analyzed to remove background noise, by trimming 50 bp from the 3′-5′ end and 100 bp from the 5′-3′ end. Base peaks of dubious nucleotides were verified by eye, using BioEdit version 7.0.9 (Hall, 1999), prior to alignment. For alignment a closely related genus of Gonipterini was used, Oxyops Schoenherr (GenBank specimen reference KF016235), with a frame length of 790 bp. Sequence alignment was made using the Clustal X (v2.1) software (Larkin et al., 2007). Aligned sequences output was then added to MEGA version 7.0 (Kumar et al., 2016), in which both frame ends were trimmed up (after verification check for stop codons) to the first complete base column amongst all aligned sequences. The obtained sequences, for each representative specimen, used for the construction of the phylogenetic tree, were deposited in GenBank, (MK674085– MK674154). Parsimony-informative and conservative sites and pairwise distances were calculated and a phylogenetic tree constructed using MEGA7. The tree is based on 64 sequences of larvae (identified as Gonipterus 1–64) and 6 sequences of adult Gonipterus males (identified as Reference Gonipterus). Each sequenced Gonipterus larva originated from a different egg pod. The adults had been previously identified morphologically and were used as a positive control. A maximum-likelihood tree was constructed based on the

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Tamura 3-parameter model with gamma (G) parameter. Determination of branch node support was obtained using 1000 bootstrap replicates.

Parasitoid-Gonipterus-Eucalyptus associations Parasitism was estimated at site level as the proportion of parasitized egg pods of the viable ones (i.e. from which larvae or parasitoids had hatched). At each site, egg pods were sampled from two to five trees, the number obtained per site varying depending on local abundance. Differences in egg parasitism between parasitoid species and years (2016 and 2017) were estimated based on the number of parasitized egg pods and the total number of viable egg pods. For this we used generalized linear models with Binomial Distribution and Log-link Function. The data are presented as mean parasitism ± standard error, per species and year. Frequencies of parasitized egg pods between parasitoid species and between the two years were analyzed using a Chi-square test. Frequencies of the presence of Gonipterus specimens (larvae and adult males) among Eucalyptus species were compared using Fisher’s exact test. Only Eucalyptus species with more than one occurrence were considered. The parasitoid distributions on the sampled eucalypt species were compared by Chi-square or by Fisher’s exact test (whenever the expected cell values were lower than five). Statistical tests and analysis were carried out with IBM® SPSS v25.

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Results and discussion Gonipterus species in Tasmania

A total of 489 adult weevils were collected during the 1st survey, of which 194 (39.67%) were males (Supplementary Table 3.1.). No adult weevils were collected in the 2nd survey. Six Gonipterus species were identified. Five of the species belong to the G. scutellatus complex, namely G. platensis, G. pulverulentus, G. scutellatus, Gonipterus sp. 1 and Gonipterus sp. 2 (for further detail, see Mapondera et al., 2012). The sixth species, G. notographus, does not belong to this complex (Mapondera et al., 2012). The distribution maps show that G. platensis, Gonipterus sp. 1 and G. notographus co- exist in the same large region (Fig. 3.1.A, C, F). In contrast, G. pulverulentus and G. scutellatus have much narrower ranges in the south-eastern part of the island (Fig. 3.1.E, F). Comparison of mean temperatures and precipitation of the locations where our specimens were collected (Table 3.2.) reveals that the six Gonipterus species highly overlap in their realized climatic niche.

Fig. 3.1. – Geographical distribution of the Gonipterus species collected in Tasmania in 2016 (based on adult males and sequenced larvae).

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Table 3.2. – Mean temperature interval for the three coldest and hottest months and mean annual precipitation, extracted for the locations of Gonipterus species shown on Figure 1 (source: Fick and Hijmans, 2017).

Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus platensis pulverulentus scutellatus sp.2 sp.1 notographus

5.9–8.6; 7.0–8.6; 6.9–7.9; 7.3–8.1; 6.1–8.6; 6.1–8.8, Temperature (ºC) 14.5–16.6 14.8–16.4 15.2–15.3 15.3–16.5 14.8–16.7 14.7–17.1

Precipitation (mm) 791 737 765 878 809 820

Gonipterus sp. 2 was collected from two distant locations in Tasmania (Fig. 3.1.B). Only four specimens were identified, two adults and one larva in the northern region and a single larva in the south-central region.

Phylogenetic relationships of Gonipterus species COI amplification was successful for 70 specimens (Fig. 3.2.; Supplementary Table 3.2.). The aligned data set consisted of 652 bp, of which 501 bp were considered to be conservative and 126 bp parsimony-informative. The phylogenetic tree (Fig. 3.2.) includes sequences of morphologically identified males (see 2.3.) as references and sequences of morphologically unidentified larvae. It shows the G. notographus specimens clustering as a monophyletic group (species) (with 99% support) and being clearly different from the specimens forming the G. scutellatus complex. In this complex, five strongly supported species were identified from the reference sequences as G. platensis, G. pulverulentus, G. scutellatus and the two undescribed species referred to as Gonipterus sp. 1 and Gonipterus sp. 2 (Mapondera et al., 2012). Gonipterus platensis and G. pulverulentus formed a closely relatedspecies pair (with 85% support) separated from the cluster containing G. scutellatus and the two undescribed species (with 89% support). In G.pulverulentus, our sequences (Gonipterus 37 to Gonipterus 44) also showed very low variation (0.025 ± 0.007) but formed a cluster separated from the reference sequence. Gonipterus sp. 1 and Gonipterus sp. 2, although distinctly separated, group together (with 84% support) and closer to the G. scutellatus reference than to G. platensis and G. pulverulentus, forming a species pair that is the sister-group of G. scutellatus. The intraspecific variation in Gonipterus sp. 1 that was sampled was found to be very low (0.003 ± 0.002).

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Fig. 3.2. – Maximum-likelihood cladogram of collected Gonipterus species based on cytochrome oxidase sub-unit one (COI). Numbers below each branch node represent the specimen separation probability (values below 70% not shown). All sequences identified as “Reference Gonipterus” apply to male specimens morphologically identified; the remaining “Gonipterus” specimens are sequences obtained from hatched larvae. The outgroup sequence was retrieved from GenBank (see M&M - DNA extraction, sequencing and phylogenetic tree construction).

Egg parasitoids of Gonipterus species in Tasmania Totals of 5313 and 1732 egg pods were collected during the 1st and 2nd surveys, respectively, of which 493 (9.3%) and 189 (10.9%) were parasitized and from which 962

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Chapter 3 and 376 egg parasitoids emerged, respectively (Supplementary Table 3.1.). Five egg parasitoid species were morphologically identified, representing three genera, Anaphes Haliday (Mymaridae) with three species (A. tasmaniae, A. inexpectatus and A. nitens), Cirrospilus Westwood (Eulophidae) with one unidentified species and Euderus Haliday (Eulophidae) with one unidentified species. Over the two years of the survey, A. tasmaniae represented 38% of the sampled parasitoids, followed by A. inexpectatus (18%). However, we found differences in the proportion of parasitoid species between the two surveys. Whereas A. tasmaniae was the most abundant species in 2016, A. nitens was the most abundant one in the following year. The proportion of egg masses parasitized by the different parasitoid species also differed significantly between the two years (χ2 = 154.44. p < 0.001). Parasitism rates varied between surveys and among parasitoid species, ranging between 1% and 25% (Fig. 3.3.). There was a significant interaction between year and parasitoid species in regard to parasitism rate (Wald Chi2 = 132.91, df = 4, p < 0.001). Whereas A. nitens, Cirrospilus sp. and Euderus sp. increased their parasitism rates from 2016 to 2017 (Fig. 3.3.), A. tasmaniae and A. inexpectatus followed an inverse trend (Fig. 3.3.).

30% b

b 25% a

20% a

a 15% a b

10% Parasitism Parasitism rate

5% b a a 0% 2016 2017 2016 2017 2016 2017 2016 2017 2016 2017 A.A. inexpectatusinexpectatus A.A. tasmaniaetasmaniae A.A. nitensnitens CirrospilusCirrospilus sp. sp. EuderusEuderus sp. sp.

Fig. 3.3. – Mean parasitism rates (±SE) of each egg parasitoid species during the two survey periods. Different letters represent significant differences in parasitism rate between years (α < 0.05).

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In 2016 we collected 63 A. nitens specimens from 17 different locations across Tasmania, whereas in 2017 151 specimens were collected from only 11 locations (Fig. 3.4.C). The 2017 survey increased the number of new locations from where A. nitens is now know to occur in Tasmania by six, and across both surveys the number of sites is 23 (Fig. 3.4.C). The three Anaphes species had very similar geographical distributional patterns (Fig. 3.4.A–C), but the two Eulophidae (Cirrospilus sp. and Euderus sp.), though present across the latitudinal range (north–south), were found in fewer locations than the other parasitoid species. In 2017 Cirrospilus was only found in the vicinities of Cygnet, Kingston and Hobart in the south, although the northern locations were also surveyed in that year (Fig. 3.4.D). A similar pattern is apparent for Euderus sp., which was the less frequent species (Fig. 3.4.E).

Fig. 3.4. – Distribution of egg parasitoid records in Tasmania in 2016 (white triangle), 2017 (gray triangles) and both years (black triangles).

Identified Eucalyptus-Gonipterus-parasitoid trophic associations Several Eucalyptus species were sampled throughout the surveys, the predominant ones being E. ovata (28%), E. amygdalina and E. pulchella (the ‘peppermints’) (19%) and E. globulus (13%) (Supplementary Table 3.1). Approximately 18% of the sampled Eucalyptus trees were not identified at species level due to the absence of morphological characters needed for the identification (e.g. seed capsules), and the remaining 22% were other species encountered only sporadically throughout the survey sites (Supplementary Table 3.1.). From the adult weevils identified and the parasitized egg pods with emergences of both Gonipterus larvae and parasitoids, we could analyze the associations

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Chapter 3 between Gonipterus species and their Eucalyptus hosts (Table 3.3.) and parasitoid species (Table 3.4.).

Table 3.3. – Associations of Gonipterus weevils and their Eucalyptus host as obtained during the 1st survey. - Eucalyptus sp.; - E. globulus; - E. nitens; - E. ovata; - E. amygdalina or E. pulchella; - E. viminalis; - E. dalrympleana; - E. pauciflora; - E. obliqua. “n” – corresponde to the number of males or larvae.

Gonipterus Gonipterus Gonipterus Gonipterus

platensis pulverulentus sp. nº1 notographus

Sampled

Gonipterus

males n = 17 n = 40 n = 64 n = 68

Sampled Gonipterus

larvae n = 26 n = 54 n = 14 n = 13

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Table 3.4.. – Trophic interactions between Gonipterus, egg parasitoid species and Eucalyptus hosts recorded during both surveys (n - number of parasitized egg pods; “n.a.” - not observed; - Eucalyptus sp.; - E. globulus; - E. nitens; - E. ovata; - E. amygadalina or E. pulchella; - E. viminalis; - E. dalrympleana; - E. pauciflora; - E. obliqua).

Gonipterus Gonipterus Gonipterus Gonipterus Unidentified platensis pulverulentus sp. nº1 notographus Gonipterus

A. tasmaniae n.a.

n = 29 n = 4 n = 11 n =213

A.

inexpectatus n = 7 n = 1 n = 2 n = 7 n = 108

A. nitens n.a. n.a.

n = 3 n = 1 n = 91

Cirrospilus n.a. n.a. sp.

n = 2 n = 2 n = 204

Euderus sp. n.a. n.a. n.a.

n = 1 n = 42

Gonipterus platensis and G. pulverulentus were mainly found on E. ovata, both as adults (47% and 75%, respectively) and larvae (70% and 79%, respectively). In much smaller frequencies, G. platensis larvae were also encountered on E. globulus (13%) and E. viminalis (7%). Overall, G. platensis and G. pulverulentus presented a similar pattern of host association (p = 0.091, Fisher’s exact test). In contrast, G. notographus has a very different pattern of host association (p < 0.001, Fisher’s exact test), with both adults and larvae mostly found on peppermints (68% and 69%, respectively). Adults of G. notographus were present at 18 out of 20 sites with peppermints; at the other two sites host trees were represented by E. ovata and E. obliqua. The host association pattern of Gonipterus sp. 1 differed significantly from those of all the other species (p < 0.001, Fisher’s exact test). This weevil was most abundant on E. nitens (52%), but this is skewed

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Chapter 3 because all males found feeding on this species were collected at a single location, where it occurred in high abundance. Adults were more frequently found on E. globulus (25% of the cases where it was present), followed by E. viminalis (16%). The host pattern of the larvae of this species is probably more accurate, having been found most frequently on E. globulus (44%), followed by E. nitens (22%) and E. obliqua (22%). Gonipterus sp. 2 was collected in very low numbers (4 specimens), precluding any analysis. From larvae identified, we found that in 11 out of 18 sites (61%) different Gonipterus species co-occurred. The specific combinations were G. platensis × G. notographus, G. platensis × Gonipterus sp. 1, G. platensis × Gonipterus sp. 2, G. platensis × G. pulverulentus, G. notographus × Gonipterus sp. 1 and G. pulverulentus × Gonipterus sp.1. In contrast to the evident pattern of host preferences in the weevils, we could not infer such pattern for the parasitoid species (Table 3.4.). More relevant quantitative information may be extracted from the plant-parasitoid associations (Table 3.4.). Although we could not detect any parasitoid specialization towards a particular Eucalyptus species from the 728 such associations we found, we could detect significant differences among species from the tritrophic parasitoid-weevil-plant associations (Table 3.4.). Anaphes tasmaniae was found as the most common parasitoid species and mainly associated with G. platensis (29 egg pods parasitized, about 78% of G. platensis egg pods), but we also found Gonipterus sp. 1 and Gonipterus sp. 2 egg pods to be parasitized by it. Anaphes tasmaniae was found associated with seven different eucalypt species (Table 3.4.), mainly with E. ovata but also with E. nitens and peppermints. In contrast, Anaphes inexpectatus was found associated with four Gonipterus species (Table 3.4.). It was reared in equal proportions from G. platensis and G. notographus egg pods (41%), but the parasitized eggs pods of G. notographus were exclusively collected on peppermints. This percentage is similar to that of A. inexpectatus collected from unidentified Gonipterus egg pods that were collected also from peppermints (49%). Although both these parasitoid species thus parasitized several Gonipterus species, they showed significant differences in their Eucalyptus associations (χ2 = 52.464; p < 0.001), with A. tasmaniae mainly collected on E. ovata (54%) and A. inexpectatus on peppermints. In our survey we found 95 Gonipterus egg pods to be parasitized by A. nitens, from at least five Eucalyptus species (Table 3.4.). Although this parasitoid species is evidently able to locate hosts in a broad range of habitats, it showed a preference for egg pods on

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E. ovata (65%) and E. globulus (21%), and its pattern of plant association differed significantly from that of A. inexpectatus (χ2 = 60.913; p < 0.001) and A. tasmaniae (χ2 = 32.574; p < 0.001). Of the 267 Cirrospilus specimens reared, 78% were from egg pods collected on E. ovata. Four larvae from these pods were identified as G. platensis and G. pulverulentus, and as these two weevil species also occurred mainly in E. ovata, it is evident that they are host species for this Cirrospilus sp. The Cirrospilus-Eucalyptus association differed from that of A. inexpectatus (χ2 = 133.06; p < 0.001) and A. tasmaniae (χ2 = 62.713; p < 0.001) but not from that of A. nitens (χ2 = 8.905; p = 0.064). The Euderus sp. was the parasitoid collected in lowest numbers (82 specimens from 43 egg pods). Unfortunately, from the egg pods found parasitized by Euderus only one Gonipterus larva could be genetically identified. The egg pod was collected on peppermints and the larva was found to belong to G. notographus. However, Euderus sp. was found mainly associated with Gonipterus species associated with E. ovata (64%), indicating that G. notographus is neither its exclusive nor main host. Under quarantine conditions in Portugal, we managed to establish colonies of Euderus sp. and Cirrospilus sp. on G. platensis egg pods (Reis, unpublished data), confirming the suitability of G. platensis as host for these two egg parasitoid species. The Euderus-Eucalyptus association was similar to those of A. nitens (p = 0.294, Fisher’s exact test) and Cirrospilus sp. (p < 0.102, Fisher’s exact test) but differed from those of A. inexpectatus (p < 0.001, Fisher’s exact test) and A. tasmaniae (Fisher exact test, p = 0.004).

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Discussion Gonipterus species in Tasmania During our field surveys we collected six Gonipterus species distributed across several sites in Tasmania. Three of these species, G. platensis, Gonipterus sp. 1 and G. notographus, were ubiquitous and widely represented. The large distribution here recorded for G. platensis across Tasmania adds several new localities to the range reported by Mapondera et al. (2012). The differences between the two studies may reflect seasonal differences in the weevil activity and population dynamics (Estay et al., 2002), as well as collecting intensity. In contrast, G. pulverulentus and G. scutellatus were found in very few locations, as also recorded by Mapondera et al. (2012). Still, more data will be needed to interpret these results and improve accuracy about the natural distribution range of these two species. Gonipterus sp. 2 was found in the present study for the first time in Tasmania. Only four specimens were identified, two in the northern and two in the south-central region. Prior to this study, this species was known to occur naturally only in the south-eastern continental Australia (Mapondera et al., 2012). Gonipterus sp. 2 was introduced in southern Africa in 1916 and in southern Europe in 1976 (Italy and France), though misidentified as G. scutellatus (Mapondera et al., 2012). The fact that Gonipterus sp. 2 is very common in south-eastern continental Australia and was not recorded from Tasmania before suggests a recent introduction of the weevil in this region. Based on the locations and considering the climatic similarity between Tasmania and parts of (Peel et al., 2007), it is possible that this species may spread in Tasmania in future, especially if palatable Eucalyptus species are widespread. In its invaded range, G. platensis represents a major problem mostly in cooler regions (Reis et al., 2012). Tasmania has a temperate climate (Köppen classification: Cfb) (Peel et al., 2007), with rain distributed throughout the year and mild and moist summers. Based on ours surveys, we found that in its native range G. platensis is experiencing average winter temperatures between 5.9 °C and 8.6 °C and mild summer temperatures between 14.5 °C and 16.6 °C (Table 3.2.). Yet, outside of Australia this species is now widely distributed and shows large climatic tolerance in the countries where it was introduced. It occurs in regions with warm winter and hot summer temperatures, as in the Lisbon area (11–13.3 °C and 22–25 °C, respectively, for the mean of the coldest and hottest months). Nevertheless, G. platensis causes greatest damage mostly at sites with average maximum winter temperatures below 10 °C (Reis et al., 2012). This is thought to be due to its oviposition in late winter, before the egg parasitoid starts its activity. Present field records

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Phylogenetic relationships of Gonipterus species Phylogenetic analysis of the sampled Gonipterus specimens shows the G. platensis larvae to form a cluster with 88% bootstrap support. The sequences contain up to 1% divergence among all specimens (Supplementary Table 3.3.). The lack of genetic diversity in this species was already reported by Mapondera et al. (2012), who considered that the low diversity of haplotypes they found in Western Australia (where the species is introduced) may reflect a founder effect, although they did not encounter the Western Australia haplotype in their Tasmanian specimens. We were able to match this haplotype with that of a larva we collected at Snug Falls Road, south of Hobart (data not provided), indicating that the population of G. platensis in Western Australia may originate from south-eastern Tasmania. However, additional studies, such as divergence data analysis, are required to further investigate the intraspecific variability of G. platensis. Regarding Gonipterus sp. 2, the difference between our reference sequence (a male from Canberra) and the specimens collected in Tasmania is about 2.5%, which is not unexpected given the Canberra, on the Australian mainland wide range and presumably genetic variation of the species on the mainland. Future molecular studies may help to identify the origin and pathway of introduction of this species into Tasmania and whether the specimens found at the two different locations in Tasmania represent a single or multiple introductions. Overall the results obtained corroborate those of Mapondera et al. (2012) that specimens assigned as Gonipterus sp. 1 and Gonipterus sp. 2 represent two well supported new species. Gonipterus sp. 3, identified as the sister species of Gonipterus sp. 2 by Mapondera et al. (2012), was not represented in our analysis as it does not occur in Tasmania.

Egg parasitoids of Gonipterus species in Tasmania Five egg parasitoid species were commonly found associated with Gonipterus, A. tasmaniae, A. inexpectatus, A. nitens, Cirrospilus sp. and Euderus sp.. All these species were previously reported from Tasmania by Valente et al. (2017a). Our work confirms that all of them may parasitize our target species, G. platensis. In the field, egg masses of G. platensis were found parasitized by all species except the Euderus sp., but in our laboratory colonies G. platensis was confirmed to be a suitable host for Euderus sp. as well. In fact, despite the low numbers of weevil-parasitoid associations we recorded, it is

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Chapter 3 evident that all identified parasitoid species are able to parasitize two or more Gonipterus species. It is possible that some degree of parasitoid preference towards a particular Gonipterus species exists, but further study is needed to investigate this. For example, A. inexpectatus was more frequently obtained from Gonipterus species associated with peppermint eucalypts (Table 3.4.), whereas the other parasitoid species were more frequently recorded from Gonipterus species associated with the Maidenaria section of Eucalyptus (E. globulus, E. ovata and E. viminialis). Anaphes tasmaniae was the most abundant parasitoid in our survey and the major one associated with G. platensis, and it was also the most abundant species collected in 2010 and 2012 (Valente et al., 2017b). However, it is remarkable that A. tasmaniae was outnumbered by A. nitens in our 2017 survey. Anaphes nitens was first detected in Tasmania in 2012, when it accounted for merely 0.3% of the total number of egg parasitoids (Valente, pers. comm.). In our 1st survey, in 2016, we registered a significant increment to about 6%, and in our 2nd survey, in 2017, its proportion increased to nearly 40%, making it the dominant egg parasitoid encountered. This suggests that A. nitens is not only consolidating its presence in Tasmania but also increasing its geographical range in this invaded region. This expansion suggests that the species is establishing itself as a new egg parasitoid of Gonipterus populations in Tasmania. In its native range, A. nitens parasitizes Gonipterus sp. 2, as it does in Africa, Italy and France (Mapondera et al., 2012), but it evidently has the ability to parasitize several Gonipterus species as we here report it from two native Tasmanian species, G. platensis (which it also parasitizes in western Europe and in North and South America) and G. notographus. From the competition between A. nitens and A. inexpectatus observed in the laboratory (Valente et al., 2018b), it seems likely that A. nitens may also impact on the populations of A. inexpectatus and A. tasmaniae in the field. However, further data are needed to test this hypothesis. Our work confirms the natural ability of both A. tasmaniae and A. inexpectatus to parasitize the two most widespread invasive Eucalyptus weevils in the world, G. platensis and Gonipterus sp. 2. Anaphes inexpectatus and A. tasmaniae were first described from specimens collected in Tasmania during a search for new parasitoids to enhance the biological control of Gonipterus sp. 2 in South Africa (Huber and Prinsloo, 1990, Tribe, 2003). Both Anaphes species were imported to and mass-reared in South Africa, but their release (in Lesotho) was derailed by bureaucratic procedures (Tribe, 2003). More recently A. tasmaniae was released in Chile (Mayorga et al., 2013) and A. inexpectatus in Portugal,

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Chapter 3 following laboratory risk-assessment studies (Valente et al., 2017a, Valente et al., 2017b). However, so far neither of these attempts has resulted in confirmed successful establishment of either of the two species and effective biological control of Gonipterus by them. Competition with the already well-established A. nitens may hinder the establishment of A. inexpectatus in Portugal and perhaps also of that of A. tasmaniae in Chile. We found large variability in egg parasitism between sites, species and years. These could be related to differences in time of sampling, to parasitoid and/or host seasonality or to population dynamics of the parasitoids leading to fluctuations between years (van Nouhuys and Lei, 2004). As parasitism rates depend, among other factors, on the abundance of the host, and we could not control for the abundance of the Gonipterus egg masses and sample size, we need to consider these results with caution. It may be further expected that climate variations can affect the trophic chain (host plant – weevil – egg parasitoid) and cause large variation in parasitism from one year to another (Jeffs and Lewis, 2013). The variability found in our 2016 and 2017 samplings is further evidence that multiple surveys need to be undertaken, at different times of the year and over several seasons, when searching for biological control agents in a new area. Our surveys were conducted in spring, but the population dynamics of the different Gonipterus species and their egg parasitoids over the entire season is yet unknown, and it is possible that the interactions between hosts and parasitoids may change over the remaining part of the season and also between years with different climatic conditions. Although probably not as promising as in spring, the search for potential additional biological control agents of Gonipterus should also be conducted in autumn.

Identified trophic associations: Eucalyptus - Gonipterus - egg parasitoid The marked and different host preferences of the Gonipterus species (Table 3.3) suggest some degree of niche differentiation. In particular, G. notographus was notably associated with the peppermint species, whereas G. platensis, G. pulverulentus and Gonipterus sp. 1 were mainly associated with E. ovata and other species of the Maidenaria section. These results are based on the presence of both adults and egg pods. A few males of Gonipterus sp. 2 and Gonipterus sp. 1 were also found on peppermints, but this does not mean that these tree species are suitable for feeding, oviposition or larval development of these weevil species. Perching and resting on non-suitable host plants is quite common in insects (Andersson, 2009), and these males could have flown onto the peppermints from nearby true host species. At the Snug Falls Road site, for example, small peppermints and

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E. ovata trees were growing adjacent to each other, facilitating movements of the weevils between tree species. The host pattern shown by G. platensis larvae and adults in Tasmania is an accordance with the host range studies of this species conducted in Europe by Rivera and Carbone, 2000, Gonçalves et al., in preparations, which found a preference of this species for E. bicostata Maiden, Blakely & Simmonds, E. globulus, E. ovata, E. smithii R.T. Baker and E. viminalis Labill. as well as other species of the Maidenaria section. Similarly, in field studies in South Africa Newete et al. (2011) recorded feeding and oviposition preferences of Gonipterus sp. 2 (as G. “scutellatus”) for E. grandis W. Hill, E. nitens Maiden, E. smithii, E. urophylla S.T. Blake and E. viminalis. Earlier investigations into the host specificity of Gonipterus species in their native range are compromised, because different weevil species (including G. platensis and Gonipterus sp. 2) were lumped into G. scutellatus, even species such as G. notographus that do not belong in the G. scutellatus complex. Thus, the oviposition preference of G. scutellatus for the peppermint species E. amygdalina, E. pulchella and E. tenuiramis reported by Clarke et al. (1998) in Tasmania actually applies to G. notographus (based on voucher specimens deposited in ANIC), which is in agreement with our observations. Gonipterus sp. 2 and G. scutellatus were collected in very low numbers in this study, precluding the deduction of any host preference. Despite the limited number of associations between Gonipterus larvae and egg parasitoids identified in our study, the associations recorded between the weevils and their hosts allow some deductions about the associations between the weevils and their parasitoids. Thus, because G. notographus has a strong host preference for peppermints (Table 3.3.) and most A. inexpectatus specimens we reared were from eggs pods collected on peppermints, it is extremely likely that these A. inexpectatus specimens were parasitizing eggs of G. notographus and further that this weevil species is a major host of A. inexpectatus. Similarly, as A. tasmaniae mostly emerged from egg pods collected on E. ovata and E. nitens (Table 3.4.), a major interaction of this parasitoid species with G. platensis, G. pulverulentus and Gonipterus sp. 1 might be inferred. Egg masses were collected in low number from E. globulus. This is due to the reduced area that this tree species occupies in Tasmania. Most of the A. tasmaniae - G. platensis pairs were collected from E. ovata, which has a much wider geographical distribution area in Tasmania than E. globulus (Williams and Potts, 1996).

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Conclusion This work provides the first attempt at comprehending the Gonipterus-Eucalyptus- parasitoid associations in part of their native region, in Tasmania. The work conducted by Mapondera et al. (2012) on clarifying the existence of different species in the G. scutellatus complex group was a crucial prerequisite for this study. Some previously unknown relationships are here reported for the first time, such as the parasitism of G. notographus by A. nitens, A. inexpectatus and Euderus sp. or the parasitism of G. pulverulentus and Gonipterus sp. 1 by A. tasmaniae and A. inexpectatus, Four out of five egg parasitoids species reared from Gonipterus egg pods were associated with G. platensis. Anaphes tasmaniae was the most frequently found parasitoid and could therefore be a good candidate for additional biocontrol of G. platensis. However, the success in rearing the Cirrospilus and Euderus species in the laboratory (unpublished data), compared with the difficulties in rearing A. tasmaniae, suggest that it may be wise to further study the efficacy of these two species against G. platensis. These species identities also needs to be studied. Another striking result of our study is the increased abundance of A. nitens encountered in 2017 in Tasmania, where it has apparently been introduced only recently. As this species is a strong competitor of other Anaphes species (Valente et al., 2018b), its distribution and population sizes in Tasmania and its impact on the native egg parasitoids should be further investigated. Although further surveys and studies of the Gonipterus-parasitoid interactions are required, especially in the remote western part of Tasmania, we expect this work to contribute to the improvement of future CBC programs against G. platensis and other invasive Gonipterus species in the world.

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Acknowledgments We sincerely thank Stephen Quarrell (UTAS, Hobart), David de Little, Debbie Jennings (CSIRO, ANIC), Ana Cabral (ISA), Vera Zina (ISA), Catarina Gonçalves (RAIZ), Catarina Afonso (RAIZ), Artur Sarmento (Altri), Tatiana Valada (ISA) and Filomena Nobrega (INIAV) for the assistance provided to the study and the preparation of the manuscript. We also acknowledge the contribution of three anonymous reviewers, which with their comments and suggestions helped us improving the manuscript. This work was financially supported by the FCT through the Doctoral Programme SUSFOR – Sustainable Forests and Products PD/00157/2012, PhD grant PD/BD/52693/2014 to the first author, and Project UID/AGR/00239/2013, as well as by Altri Florestal and Raiz (part of The Navigator Company).

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References Andersson, P., 2009. Quantitative aspects of plant-insect interactions in fragmented landscapes: the role of insect search behavior. Department of Botany. Stockholm University, Stockholm, pp. 37. Arzone, A., Meotto, F. 1978. Reperti biologici su Gonipterus scutellatus Gyll. (Col. Curculionidae) infestante gli eucalipti della riviera Ligure. Redia 61: 205–222. Barbiellini, A. A. 1955. Combate à praga do eucalipto no Sul. Chacaras e Quintais 91(2): 191–192. Barratt, B. I. P., Cock, M. J. W., Oberprieler, R. G. 2018. Weevils as targets for biological control, and the importance of taxonomy and phylogeny for efficacy and biosafety. Diversity. 10, 1–19. Barsoum, N., Gill, R., Henderson, L., Peace, A., Quine, C., V., S., Valatin, G., 2016. Biodiversity and rotation length: economic models and ecological evidence. Forestry Commission, Forest Research, 1-10. Bosq, J. M. 1943. Segunda lista de Coleópteros argentinos dañinos a la agricultura. Ministerio de Agricultura de la Nación, Dirección de Sanidad Vegetal, Buenos Aires, 80 pp. Broun, T. 1893. Remarks on the Carabidae of New Zealand. Transactions and Proceedings of the New Zealand Institute 25, 194–198. Clarke, A.R., Paterson, S., Pennington, P. 1998. Gonipterus scutellatus Gyllenhal (Coleoptera: Curculionidae) oviposition on seven naturally co-occurring Eucalyptus species. For. Ecol. Manag. 119, 89–99. Cowles, R.S., Downer, J.A., 1995. Eucalyptus snout beetle detected in California. Calif. Agric. 49, 38-40. DiTomaso, J.M., Van Steenwyk, R.A., Nowierski, R.M., Meyerson, L.A., Doering, O.C., Lane, E., Cowan, P.E., Zimmerman, K., Pitcairn, M.J., Dionigi, C.P., 2017. Addressing the needs for improving classical biological control programs in the USA. Biol. Control 106, 35-39. Estay, S., Araya, J.E., Guerrero, M.A., 2002. Biología de Gonipterus scutellatus Gyllenhal (Coleoptera: Curculionidae) en San Felipe, Chile. Bol. San. Veg., Plagas 28, 391-397. Fick, S.E., Hijmans, R.J., 2017. Worldclim 2: New 1-km spatial resolution climate surfaces for global land areas. Int. J. Climatol. 37, 4302-4315

68

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Gonçalves, C.I., Vilas-Boas, L., Branco, M., Rezende, G.D., Valente, C. Host susceptibility to Gonipterus platensis (Coleoptera: Curculionidae) of Eucalyptus species. In prep.. Haines, W.P., Samuelson, G.A. 2006. The Eucalyptus snout beetle Gonipterus scutellatus (Coleoptera: Curculionidae) recently established in the Hawaiian Islands. Bishop Museum Occasional Papers 88, 25–26. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95-98. Hanks, L.M., Millar, J.G., Paine, T.D., Campbell, C.D., 2000. Classical biological control of the Australian weevil Gonipterus scutellatus (Coleoptera: Curculionidae) in California. Environ. Entomol. 29, 369-375. Hoddle, M.S., 2005. Identifying the donor region within the home range of an invasive species: implications for classical biological control of arthropod pests In: Hoddle, M.S., (Ed.), Second International Symposium on Biological Control of Arthropods. USDA Forest Service, Davos, Switzerland, pp. 29-37. Huber, J.T., Prinsloo, G.L., 1990. Redescription of Anaphes nitens (Girault) and description of two new species of Anaphes Haliday (Hymenoptera: Mymaridae), parasites of Gonipterus scutellatus Gyllenhal (Coleoptera: Curculionidae) in Tasmania. J. Aust. Ent. Soc. 29, 333-341. Hurley, B.P., Garnas, J., Wingfield, M.J., Branco, M., Richardson, D.M., Slippers, B., 2016. Increasing numbers and intercontinental spread of invasive insects on eucalypts. Biol. Invasions 18, 921-933. Instituto da Conservação da Natureza e da Floresta (ICNF), 2013. Inventário Florestal Nacional 6 (IFN6) - Áreas dos usos do solo e das espécies florestais de Portugal continental. Resultados preliminares. Instituto da Conservação da Natureza e das Florestas, Lisbon, Portugal, pp. 35. Jeffs, C.T., Lewis, O.T., 2013. Effects of climate warming on host–parasitoid interactions. Ecol. Entomol. 38, 209-218. Keane, R.M., Crawley, M.J., 2002. Exotic plant invasions and the enemy release hypothesis. Trends Ecol. Evol. 17, 164-170. Kenis, M., Hurley, B.P., Hajek, A.E., Cock, M.J.W., 2017. Classical biological control of insect pests of trees: facts and figures. Biol. Invasions 19, 3401-3417.

69

Chapter 3

Kober, E. 1955. Observações preliminares da ação de diversos inseticidas orgânicos de síntese, no controle ao Gonipterus gibberus Boisd., praga do eucalipto. Agron. Sulriograndense 2(1): 30–40. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874. Kuschel, G. 1990. in a suburban environment: a New Zealand case study. The identity and status of Coleoptera in the natural and modified habitats of Lynfield, Auckland (1974–1989). DSIR Plant Protection Report No. 3, 118pp. Lanfranco, D., Dungey, H.S., 2001. Insect damage in Eucalyptus: A review of plantations in Chile. Austral Ecol. 26, 477-481. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947- 2948. Loch, A.D., 2008. Parasitism of the Eucalyptus weevil, Gonipterus scutellatus Gyllenhal, by the egg parasitoid, Anaphes nitens Girault, in Eucalyptus globulus plantations in southwestern Australia. Biol. Control 47, 1-7. Malausa, J.C., Rabasse, J.M., Kreiter, P., 2008. Les insectes entomophages d’intérêt agricole acclimatés en France métropolitaine depuis le début du 20 ème siècle. Bulletin OEPP/EPPO Bulletin 38, 136-146. Mally, C. W. 1924. The Eucalyptus Snout-beetle (Gonipterus scutellatus, Gyll.). Journal of the Department of Agriculture, Union of South Africa, 51: 1–30. Mansilla, J.P.V., 1992. Presencia sobre Eucalyptus globulus Labill de Gonipterus scutellatus Gyll. (Col. Curculionidae) en Galicia. Bol. San. Veg., Plagas, 18, 547- 554. Mapondera, T.S., Burgess, T., Matsuki, M., Oberprieler, R.G., 2012. Identification and molecular phylogenetics of the cryptic species of the Gonipterus scutellatus complex (Coleoptera: Curculionidae: Gonipterini). Aust. J. Entomol. 51, 175-188. Marelli, C. A. 1926. La plaga de los gorgojos de los eucaliptos. Revista de la Sociedad Entomológica Argentina 1(1): 14–22. Mayorga, S.I., Jaques, L., Peragallo, M., 2013. Anaphes tasmaniae, parasitoid of Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae) introduced in Chile. 4th International Symposium on Biological Control of Arthropods, Pucón, Chile, pp. 1.

70

Chapter 3

Newete, S.W., Oberprieler, R.G., Byrne, M.J., 2011. The host range of the Eucalyptus weevil, Gonipterus “scutellatus” Gyllenhal (Coleoptera: Curculionidae), in South Africa. Ann. For. Sci. 68, 1005-1013. Oberprieler, R. G., Caldara, R., Skuhrovec, J., 2014. Bagoini Thomson, 1859; Gonipterini Lacordaire, 1863; Hyperini Marseul, 1863. In: Leschen, R. A. B., Beutel, R. G. (Eds.), Handbook of Zoology. Arthropoda: Insecta. Coleoptera, Beetles. Volume 3: Morphology and Systematics (Phytophaga). Walter de Gruyter, Berlin/Boston. pp. 452–476. Paine, T.D., Dahlsten, D.L., Millar, J.G., Hoddle, M., Hanks, J.B., 2000. UC scientists apply IPM techniques to eucalyptus pests. California Agriculture, California, United States, pp. 8-13. Peel, M.C., Finlayson, B.L., McMahon, T.A., 2007. Updated world map of the Koppen- Geiger climate classification. Hydrol. Earth Syst. Sci. Discuss. 4, 439-473. Porto, D.S., Melo, G.A.R., Almeida, E.A.B., 2016. Clearing and dissecting insects for internal skeletal morphological research with particular reference to bees. Rev. Bras. Entomol. 60, 109-113. Rabasse, J. M., Perrin, H. 1979. Introduction en France du charançon de l’Eucalyptus, Gonipterus scutellatus Gyll. (Coleoptera Curculionidae). Annales de Zoologie et Ecologie Animaux 11(3): 337–345. Reis, A.R., Ferreira, L., Tomé, M., Araujo, C., Branco, M., 2012. Efficiency of biological control of Gonipterus platensis (Coleoptera: Curculionidae) by Anaphes nitens (Hymenoptera: Mymaridae) in cold areas of the Iberian Peninsula: Implications for defoliation and wood production in Eucalyptus globulus. Forest Ecol. Manag. 270, 216-222. Rivera, A.C., Carbone, S.S., 2000. The effect of three species of Eucalyptus on growth and fecundity of the Eucalyptus snout beetle (Gonipterus scutellatus). Forestry 73, 21-29. Rivera, A.C., Santolamazza Carbone, S., A. Andrés, J., 1999. Life cycle and biological control of the Eucalyptus snout beetle (Coleoptera, Curculionidae) by Anaphes nitens (Hymenoptera, Mymaridae) in north-west Spain. Agricultural and Forest Entomology 1, 103-109. Robertson, M.P., Kriticos, D.J., Zachariades, C., 2008. Climate matching techniques to narrow the search for biological control agents. Biol. Control 46, 442-452.

71

Chapter 3

Rosado-Neto, G. H., Marques, M.I. 1996. Características do adulto, genitália e formas imaturas de Gonipterus gibberus Boisduval and G. scutellatus Gyllenhal (Coleoptera, Curculionidae). Rev. Bras. Zool. 13(1): 77–90. Ruiz, F., Lopez, G., 2010. Review of cultivation history and uses of eucalyptus in Spain. Conference of Eucalytptus species management, history and trens in Ethiopia 15- 17 September, Addis Ababa, 1-15. Sampo, A. 1976. Un Curculionide Gonopterino australiano defogliatore dell’Eucalipto per la prima volta in Europa (Coleoptera Curculionidae). Il floriculture 13: 86–87. Santolamazza-Carbone, S., Rodriguez-Illamola, A., Cordero Rivera, A., 2006. Thermal requirements and phenology of the Eucalyptus snout beetle Gonipterus scutellatus Gyllenhal. J. Appl. Entomol. 130, 368-376. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Lu, H., Flook, P., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651-701. Sousa, E.M.R., Ferreira, L.J.C. 1996. Gonipterus scutellatus Gyll., uma nova praga do eucalipto em Portugal. Revista Florestal 9, 4–7. Tooke, F. G. C. 1955. The Eucalyptus Snout-beetle, Gonipterus scutellatus Gyll. A study of its ecology and control by biological means. Entomology Memoirs, Union of South Africa, 3: 1–282. Tribe, G.D., 2003. Biological control of defoliating, and phloem- or wood-feeding Insects in commercial forestry in Southern Africa. In: Neuenschwander, P., Borgemeister, C., Langewald, J., (Eds.), Biological control in IPM systems in Africa. CABI Publishing. pp. 113–129 Tribe, G.D., 2005. The present status of Anaphes nitens (Hymenoptera: Mymaridae), an egg parasitoid of the Eucalyptus snout beetle Gonipterus scutellatus, in the Western Cape Province of South Africa. Southern African Forestry Journal 203, 49-54. Valente, C., Vaz, A., Pina, J., Manta, A., Sequeira, A., 2004. Control strategy against the eucalyptus snout beetle, Gonipterus scutellatus Gyllenhal (Coleoptera, Curculionidae), by the portuguese cellulose industry. In: Borralho, N.M.G., Pereira, J.S., Marques, C., Coutinho, J., Madeira, M., Tomé, M., (Eds.), Eucalyptus in a Changing World. IUFRO, Aveiro, pp. 622-627.

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Valente, C., Gonçalves, C.I., Reis, A., Branco, M., 2017a. Pre-selection and biological potential of the egg parasitoid Anaphes inexpectatus for the control of the Eucalyptus snout beetle, Gonipterus platensis. J. Pest Sci. 90, 911-923. Valente, C., Afonso, C., Gonçalves, C.I., Alonso-Zarazaga, M.A., Reis, A., Branco, M., 2017b. Environmental risk assessment of the egg parasitoid Anaphes inexpectatus for classical biological control of the Eucalyptus snout beetle, Gonipterus platensis. BioControl. Valente, C., Gonçalves, C.I., Monteiro, F., Gaspar, J., Silva, M., Sottomayor, M., Paiva, M.R., Branco, M., 2018a. Economic outcome of classical biological control: A case study on the Eucalyptus Snout Beetle, Gonipterus platensis, and the parasitoid Anaphes nitens. Ecol. Econom. 149, 40-47. Valente, C., Afonso, C., Gonçalves, C.I., Branco, M., 2018b. Assessing the competitive interactions between two egg parasitoids of the Eucalyptus snout beetle, Gonipterus platensis, and their implications for biological control. Biol. Control. DOI: 10.1016/j.biocontrol.2018.10.002 van Nouhuys, S., Lei, G., 2004. Parasitoid–host metapopulation dynamics: the causes and consequences of phenological asynchrony. J. Anim. Ecol. 73, 526-535. Williams, K., Potts, B., 1996. The natural distribution of Eucalyptus species in Tasmania. In: Elliott, H., Jarman, J., Brown, M., Hinley, D., (Eds.), Tasforests. Forestry Tasmania, Tasmania, pp. 39-165. Wiltshire, R., Potts, B., 2007. EucaFlip: Life-Sized Guide to the Eucalypts of Tasmania. University of Tasmania, Hobart, Tasmania. Zacharia, J.T., 2011. Ecological Effects of Pesticides. In: Stoytcheva, D.M., (Ed.), Pesticides in the Modern World - Risks and Benefits. InTech, 129-142

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Supplementary material

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Chapter 3 - Supplementary Table 3.1. List of surveyed locations with information regarding host plant species surveyed, Gonipterus egg pods, adults and associated egg parasitoids. In brackets is indicated the number of specimens used for phylogenetic analysis; * – a single Gonipterus male specimen was used for genetic analysis and is deposit at ISA; 1 – Specimens are deposit at ANIC; 2 – Specimens are deposit at ISA; n.a. – no specimens were collected. Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods2 collected1 collected2 inexpectatus spp. spp. 21/10/2016 HobartUTAS.1 -42,905 147,324 82 Eucalyptus sp. 0 0 0 0 0 0 0 0 21/10/2016 HobartUTAS.2 -42,905 147,324 82 Eucalyptus sp. 73 5 4 0 5 0 0 0 21/10/2016 HobartUTAS.3 -42,905 147,324 82 Eucalyptus sp. 0 0 0 - - - - - 22/10/2016 HobartMtNelson.1 -42,912 147,323 234 E. ovata 520 12* 21 10 4 0 0 0 22/10/2016 HobartMtNelson.2 -42,912 147,323 237 E. globulus 0 0 0 - - - - - 25/10/2016 LaserField.1 -42,949 147,328 290 E. ovata 50 2 5 0 0 0 0 0 25/10/2016 LaserField.2 -42,949 147,328 290 E. obliqua 0 0 0 - - - - - 25/10/2016 Scouts.1 -42,945 147,313 103 E. ovata 178(1) 5 7 11 0 0 16 0 E. amygdalina 25/10/2016 Scouts.2 -42,945 147,313 103 0 0 0 - - - - - or E. pulchella 25/10/2016 Scouts.3 -42,945 147,313 103 E. obliqua 0 0 0 - - - - - 26/10/2016 KingstonLeslieRoad.1 -42,973 147,271 177 E. viminalis 7 0 1 0 0 0 0 0 KingstonRobaCourtRoa 26/10/2016 -42,969 147,276 136 Eucalyptus sp. 2 0 1 0 0 0 0 0 d.1 KingstonCadesDriveRoa 26/10/2016 -42,969 147,268 163 E. globulus 27 3 3 0 0 0 0 0 d.1 KingstonCadesDriveRoa 26/10/2016 -42,969 147,268 163 E. viminalis 8 0 0 0 0 0 0 0 d.2 26/10/2016 HounRoad.1 -42,974 147,170 365 E. ovata 0 0 0 - - - - - E. amygdalina 26/10/2016 DipRoad.1 -42,977 147,119 183 0 0 0 - - - - - or E. pulchella E. amygdalina 26/10/2016 DipRoad.2 -42,977 147,119 183 3 0 0 0 0 0 0 0 or E. pulchella E. amygdalina 26/10/2016 DipRoad.3 -42,977 147,119 183 12 0 0 0 0 0 0 0 or E. pulchella 26/10/2016 DipRoad.4 -42,977 147,119 183 E. obliqua 0 0 0 - - - - -

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 26/10/2016 MtRiverRoad.1 -42,931 147,136 198 E. ovata 2 0 0 0 0 0 0 0 26/10/2016 MtRiverRoad.2 -42,931 147,136 198 E. globulus 0 0 0 - - - - - 26/10/2016 AllensRivuletRoad.1 -43,002 147,200 172 E. ovata 1 0 0 0 0 0 0 0 26/10/2016 AllensRivuletRoad_2.1 -43,006 147,215 136 E. ovata 100 0 1 9 0 0 0 0 26/10/2016 TinderboxRoad.1 -43,025 147,314 122 E. ovata 2 0 0 0 0 0 0 0 28/10/2016 Runnymede.1 -42,636 147,565 246 E. nitens 512(2) 9 14 67 45 0 0 0 28/10/2016 Airport.1 -42,845 147,529 12 E. viminalis 16 6 8 0 3 0 0 0 31/10/2016 Margate.1 -43,023 147,259 12 E. ovata 0 0 0 - - - - - 31/10/2016 Margate_2.1.1 -43,017 147,250 110 E. ovata 11(1) 0 4 0 3 0 0 0 31/10/2016 VanMoreyRoad.1 -43,042 147,241 122 Eucalyptus sp. 1 0 0 0 0 0 0 0 31/10/2016 SnugFallsRoad.1 -43,071 147,242 78 E. ovata 42 0 2 0 2 0 1 0 E. amygdalina 31/10/2016 OldStationRoad.1 -43,084 147,264 124 0 0 0 - - - - - or E. pulchella 31/10/2016 OldStationRoad.2 -43,084 147,264 124 E. ovata 0 0 0 - - - - - 31/10/2016 Doughboy. 1 -43,113 147,246 114 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 Doughboy.2 -43,113 147,246 114 Eucalyptus sp. 0 1 0 - - - - - 31/10/2016 Doughboy.3 -43,113 147,246 114 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 Doughboy.4 -43,113 147,246 114 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 Doughboy.5 -43,113 147,246 114 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 Doughboy.6 -43,113 147,246 114 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 WatsonRoad.1 -43,117 147,238 76 E. ovata 7 0 0 0 0 0 0 0 31/10/2016 WatsonRoad.2 -43,117 147,238 76 Eucalyptus sp. 19 2 1 0 0 0 0 0 31/10/2016 BrunyIsland_1 -43,155 147,339 50 Eucalyptus sp. 24 0 1 0 6 0 0 0 31/10/2016 BrunyIsland_2.1 -43,337 147,241 54 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 BrunyIsland._2.2 -43,337 147,241 54 Eucalyptus sp. 0 0 0 - - - - -

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 31/10/2016 BrunyIsland_3 -43,426 147,249 19 Eucalyptus sp. 0 0 0 - - - - - 31/10/2016 BrunyIsland_4 -43,318 147,249 24 Eucalyptus sp. 0 0 0 - - - - - 02/11/2016 ChannelHighway_1 -43,146 147,243 46 E. obliqua 0 0 0 - - - - - 02/11/2016 ChannelHighway_2 -43,224 147,256 55 E. ovata 0 0 0 - - - - - 02/11/2016 ChannelHighway_3.1 -43,247 147,246 35 E. ovata 0 0 0 - - - - - 02/11/2016 ChannelHighway_3.2 -43,247 147,246 35 E. globulus 4 0 0 0 0 0 0 0 02/11/2016 ChannelHighway_4 -43,268 147,242 24 E. globulus 9 0 0 2 0 0 0 0 02/11/2016 ChannelHighway_5 -43,279 147,208 35 Eucalyptus sp. 0 0 0 - - - - - 02/11/2016 ChannelHighway_6 -43,245 147,150 15 E. ovata 0 0 0 - - - - - 02/11/2016 Cygnet_1.1 -43,166 147,080 12 E. ovata 0 0 0 - - - - - 02/11/2016 Cygnet_1.2 -43,166 147,080 12 Eucalyptus sp. 0 0 0 - - - - - 02/11/2016 Cygnet_2 -43,190 147,010 14 E. globulus 39 0 1 0 0 0 0 0 02/11/2016 HuonHighway.1 -43,158 146,974 21 E. viminalis 0 0 0 - - - - - 02/11/2016 HuonHighway.2 -43,158 146,974 21 Eucalyptus sp. 0 0 0 - - - - - 02/11/2016 EsperanceCoastRd.1 -43,300 147,075 72 E. obliqua 0 0 0 - - - - - 02/11/2016 Judbury -43,002 146,927 55 E. ovata 66(18) 2 3 89 0 7 0 0 04/11/2016 FrogLodge -42,733 146,925 44 E. viminalis 58 1 3 11 5 1 0 0 04/11/2016 GordonRiverRoad -42,675 146,789 109 E. ovata 0 0 0 - - - - - 04/11/2016 MtFields -42,685 146,722 171 Eucalyptus sp. 11(1) 0 0 1 0 2 0 0 04/11/2016 ForeshoreRoad_2 -42,820 147,268 22 E. globulus 2 0 0 0 0 0 0 0 04/11/2016 ForeshoreRoad_1 -42,820 147,268 22 Eucalyptus sp. 0 0 0 - - - - - 07/11/2016 EllendaleRoad_1 -42,640 146,757 265 E. obliqua 80(5) 4 6 14 2 1 0 10 07/11/2016 EllendaleRoad_2 -42,573 146,697 298 E. obliqua 1 2 2 0 0 0 0 0 07/11/2016 DawsonRoad.1 -42,520 146,685 143 Eucalyptus sp. 10(1) 0 0 0 2 0 0 0

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 07/11/2016 DawsonRoad.2 -42,520 146,685 143 Eucalyptus sp. 0 0 0 - - - - - 07/11/2016 DawsonRoad.3 -42,520 146,685 143 Eucalyptus sp. 14 0 0 0 0 0 0 0 07/11/2016 DawsonRoad.4 -42,520 146,685 143 Eucalyptus sp. 11 0 0 0 0 0 0 0 07/11/2016 DawsonRoad.5 -42,520 146,685 143 Eucalyptus sp. 13 1 1 0 0 0 0 0 E. amygdalina 07/11/2016 LakeRepulseRoad.1 -42,510 146,647 144 131(3) 2 3 4 15 3 0 7 or E. pulchella 07/11/2016 LakeRepulseRoad.2 -42,510 146,647 144 Eucalyptus sp. 12 0 0 1 0 0 0 0 E. amygdalina 07/11/2016 Bothwell_1 -42,400 146,871 359 0 0 0 - - - - - or E. pulchella 07/11/2016 Bothwell_2 -42,400 146,947 336 E. delegatensis 17 0 1 0 0 0 0 0 07/11/2016 Oatlands.1 -42,333 147,400 431 Eucalyptus sp. 0 0 0 - - - - - 07/11/2016 Oatlands.2 -42,333 147,400 431 Eucalyptus sp. 0 0 0 - - - - - 07/11/2016 Oatlands.3 -42,333 147,400 431 Eucalyptus sp. 0 0 0 - - - - - 07/11/2016 Nugent -42,667 147,730 327 E. obliqua 1 0 0 0 0 0 0 0 09/11/2016 Runnymede_1 -42,636 147,565 246 E. nitens 119(2) 17* 13 8 0 0 0 0 10/11/2016 Propriedade.1 -42,273 146,895 601 E. delegatensis 19 0 1 0 0 0 0 0 E. amygdalina 10/11/2016 Propriedade.2 -42,273 146,895 601 2 0 0 0 0 0 0 0 or E. pulchella 10/11/2016 Interlake_1 -42,113 146,907 802 E. delegatensis 0 0 0 - - - - - 10/11/2016 Interlake_2 -42,128 146,962 704 Eucalyptus sp. 0 0 0 - - - - - 10/11/2016 HighlandLakes -42,195 146,897 640 E. delegatensis 0 0 0 - - - - - 10/11/2016 Tunbridge_1 -42,118 147,326 272 E. ovata 0 0 0 - - - - - E. amygdalina 10/11/2016 NileRoad -41,736 147,467 249 155(3) 5 6 0 96 0 0 0 or E. pulchella 10/11/2016 Deddington.1 -41,627 147,397 233 E. globulus 31 1 0 0 0 0 0 0 10/11/2016 Deddington.2 -41,627 147,397 233 Eucalyptus sp. 0 0 0 - - - - -

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 11/11/2016 Grindwald -41,356 147,014 129 E. globulus 32 0 1 0 0 5 0 0 11/11/2016 Launceston -41,507 147,374 343 E. ovata 0 0 0 - - - - - E. amygdalina 11/11/2016 Upper Esk -41,453 147,736 489 8 0 2 0 0 0 0 0 or E. pulchella 11/11/2016 Mathinna.1 -41,472 147,824 302 E. delegatensis 23 2 2 6 0 0 0 6 11/11/2016 StMary.1 -41,591 148,200 285 E. viminalis 0 0 0 - - - - - E. amygdalina 11/11/2016 StMary.2 -41,591 148,200 285 102 6 5 3 14 0 3 0 or E. pulchella 11/11/2016 TasmanHighway_1 -41,672 148,280 44 E. ovata 0 0 0 - - - - - 11/11/2016 TasmanHighway_2 -41,749 148,278 35 E. globulus 1 0 0 0 0 0 0 0 11/11/2016 Triabunna -42,499 147,923 3 E. ovata 7 0 0 0 0 0 0 0 E. amygdalina 11/11/2016 Buckland.1 -42,619 147,637 332 17 0 0 0 0 0 0 0 or E. pulchella E. amygdalina 11/11/2016 Buckland.2 -42,619 147,637 332 0 0 0 - - - - - or E. pulchella 11/11/2016 Buckland.3 -42,619 147,637 332 E. obliqua 0 0 0 - - - - - 16/11/2016 Forcett_1 -42,818 147,677 138 E. obliqua 0 0 0 - - - - - 16/11/2016 Forcett_2 -42,822 147,687 118 E. obliqua 0 0 0 - - - - - 16/11/2016 Forcett._3.1 -42,817 147,758 73 E. ovata 0 0 0 - - - - - E. amygdalina 16/11/2016 Forcett_3.2 -42,817 147,758 73 0 0 0 - - - - - or E. pulchella 16/11/2016 Blowhole -43,037 147,938 17 E. globulus 0 0 3 - - - - - 16/11/2016 ArthurHighway_1.1 -42,815 147,785 83 E. ovata 18 0 0 0 0 0 0 0 E. amygdalina 16/11/2016 ArthurHighway_1.2 -42,815 147,785 83 0 0 0 - - - - - or E. pulchella 16/11/2016 ArthurHighway_2.1 -42,953 147,874 42 E. obliqua 0 0 0 - - - - - E. amygdalina 16/11/2016 ArthurHighway_2.2 -42,953 147,874 42 4 0 0 0 0 0 0 0 or E. pulchella

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 16/11/2016 NubeenaRoad.1 -43,140 147,818 198 E. globulus 15 0 1 0 0 0 0 0 16/11/2016 NubeenaRoad.2 -43,140 147,818 198 E. obliqua 0 0 0 - - - - - E. amygdalina 16/11/2016 NubeenaRoad.3 -43,140 147,818 198 0 0 0 - - - - - or E. pulchella 16/11/2016 NubeenaBackRoad -43,091 147,765 167 E. globulus 23 0 0 0 0 0 0 0 16/11/2016 ArthurHighway_3 -42,918 147,825 17 E. ovata 17 0 0 0 0 0 2 0 20/11/2016 Runnymede_2.1 -42,651 147,547 242 E. ovata 31 2 2 0 1 0 1 0 E. amygdalina 20/11/2016 Runnymede_.2.2 -42,651 147,547 242 0 0 0 - - - - - or E. pulchella 20/11/2016 Runnymede_3.1 -42,633 147,620 303 E. ovata 1 0 2 0 0 0 0 0 E. amygdalina 20/11/2016 Runnymede_3.2 -42,633 147,620 303 0 0 2 - - - - - or E. pulchella E. amygdalina 20/11/2016 Buckland.2.1.1 -42,589 147,705 104 14 1 1 5 4 0 0 0 or E. pulchella E. amygdalina 20/11/2016 Buckland.3.1.1 -42,509 147,642 347 3 1 1 0 0 0 0 0 or E. pulchella 20/11/2016 Tunbridge_2 -42,120 147,313 323 E. ovata 198(1) 1 4 5 0 3 0 0 21/11/2016 Grindwald -41,356 147,014 129 E. globulus 17(3) 2 2 0 6 8 0 0 E. amygdalina 21/11/2016 Exeter_1.1 -41,329 146,943 110 6 0 0 1 0 0 0 0 or E. pulchella

21/11/2016 Exeter_1.2 -41,329 146,943 110 E. ovata 23 0 0 0 0 0 0 0

21/11/2016 Exeter_2 -41,331 146,941 118 Eucalyptus sp. 21 0 0 4 0 0 0 0 21/11/2016 WillowGlen.1 -41,499 146,620 315 E. globulus 0 0 0 - - - - - E. amygdalina 21/11/2016 WillowGlen.2 -41,499 146,620 315 0 0 0 - - - - - or E. pulchella 21/11/2016 RailtonRoad -41,418 146,525 114 E. ovata 151(2) 0 1 17 4 7 0 0

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 21/11/2016 BellamyRoad -41,221 146,291 116 E. obliqua 3 0 3 0 0 0 0 0 21/11/2016 ForthRoad -41,190 146,304 91 Eucalyptus sp. 2 0 0 0 0 0 0 0 21/11/2016 Stonehill.1 -41,304 146,709 252 E. ovata 7 0 0 0 0 0 0 0 21/11/2016 Stonehill.2 -41,304 146,709 252 Eucalyptus sp. 0 0 0 - - - - - 21/11/2016 HolwellRoad -41,267 146,781 322 E. globulus 11 0 0 0 0 0 0 0 OldBangorTramRoad_.1 22/11/2016 -41,229 147,026 143 E. ovata 13 0 0 0 0 0 0 0 .1 OldBangorTramRoad_1. E. amygdalina 22/11/2016 -41,229 147,026 143 5 0 0 0 0 0 0 0 2 or E. pulchella 22/11/2016 OldBangorTramRoad_2 -41,209 147,110 125 E. ovata 6(2) 0 0 5 0 0 0 0 22/11/2016 TheGlenRoad_1.1 -41,188 147,103 179 E. ovata 15 0 1 3 0 0 0 0 22/11/2016 TheGlenRoad_1.2 -41,188 147,103 179 E. globulus 43 6 0 7 2 1 0 0 22/11/2016 TheGlenRoad_2.1 -41,170 147,059 127 E. ovata 36(1) 1 2 6 2 0 0 0 E. amygdalina 22/11/2016 TheGlenRoad_2.2 -41,170 147,059 127 101 3 4 8 0 0 1 0 or E. pulchella 22/11/2016 Deddington.1 -41,627 147,397 233 E. globulus 5(1) 0 1 0 3 0 0 0 22/11/2016 Deddington.2 -41,627 147,397 233 Eucalyptus sp. 0 0 1 - - - - - 30/11/2016 AllensRivuletRoad_2.1 -43,006 147,215 136 E. ovata 107 2 3 0 0 0 0 0 E. amygdalina 30/11/2016 AllensRivuletRoad_2.2 -43,006 147,215 136 13 1 2 0 0 0 1 0 or E. pulchella 30/11/2016 SandflyRoad.1 -43,023 147,257 34 E. ovata 13 0 0 0 9 0 0 0

30/11/2016 SandflyRoad.2 -43,023 147,257 34 E. viminalis 14 0 1 0 0 0 0 0

30/11/2016 SandflyRoad.3 -43,023 147,257 34 E. viminalis 5 0 1 0 3 0 0 0 30/11/2016 SnugFallsRoad.1 -43,071 147,242 78 E. ovata 49 7 0 0 0 0 2 0 E. amygdalina 30/11/2016 SnugFallsRoad.2 -43,071 147,242 78 125 0 8 11 0 0 2 0 or E. pulchella

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Emerged Nº of Gonipterus Gonipterus Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 30/11/2016 ChannelHighway_4 -43,268 147,242 24 E. globulus 11(1) 1* 1 2 0 0 0 0 30/11/2016 Cygnet_3 -43,163 147,083 17 E. ovata 63(1) 0 0 4 0 7 0 0 30/11/2016 Cygnet_2 -43,190 147,010 14 E. globulus 44 1 1 1 3 1 1 0 30/11/2016 Judbury -43,002 146,927 55 E. ovata 29 0 0 3 0 0 0 0 03/12/2016 Lebrina.1 -41,169 147,263 180 E. ovata 1 0 0 0 0 0 0 0 E. amygdalina 03/12/2016 Lebrina.2 -41,169 147,263 180 0 3 0 - - - - - or E. pulchella 03/12/2016 FernyHillRoad_1.1 -41,156 147,301 85 E. viminalis 15 1 0 2 0 1 1 0 E. amygdalina 03/12/2016 FernyHillRoad_1.2 -41,156 147,301 85 30 0 0 0 0 0 0 0 or E. pulchella 03/12/2016 FernyHillRoad_1.3 -41,156 147,301 85 E. obliqua 0 3 6 - - - - - E. amygdalina 03/12/2016 FernyHillRoad_2.1 -41,152 147,301 79 0 1 0 - - - - - or E. pulchella 03/12/2016 FernyHillRoad_2.2 -41,152 147,301 79 E. ovata 32 0 0 5 0 0 0 0 03/12/2016 FernyHillRoad._3 -41,078 147,309 69 Eucalyptus sp. 27 0 0 44 5 2 0 0 03/12/2016 PippersBrooks -41,051 147,191 114 E. globulus 17 0 1 1 0 0 0 0 E. amygdalina 04/12/2016 WaterHouseRoad -40,930 147,823 69 5 1 0 0 0 0 0 0 or E. pulchella 04/12/2016 NorthAtkinsonRoad.1 -40,949 148,029 61 Eucalyptus sp. 9 0 0 0 0 0 0 0 E. amygdalina 04/12/2016 NorthAtkinsonRoad.2 -40,949 148,029 61 2 0 0 0 0 0 0 0 or E. pulchella 04/12/2016 TasmanHighway_5 -41,308 148,220 81 E. ovata 79 7 9 10 0 0 1 1 04/12/2016 TasmanHighway_6 -41,406 147,282 341 E. ovata 97 1 5 8 5 0 0 0 05/12/2016 Mathinna.1 -41,472 147,824 302 E. delegatensis 20 0 0 0 0 0 0 0 05/12/2016 Mathinna.2 -41,472 147,824 302 E. ovata 1 0 0 0 0 0 0 0 05/12/2016 StMary.1 -41,591 148,200 285 E. viminalis 32 1 4 2 3 0 1 0

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. E. amygdalina 05/12/2016 StMary.2 -41,591 148,200 285 16 1 5 0 0 0 0 0 or E. pulchella 05/12/2016 TasmanHighway_7.1 -41,693 148,281 29 E. ovata 9 0 2 0 0 0 0 0 05/12/2016 TasmanHighway_7.2 -41,693 148,281 29 E. globulus 0 0 0 - - - - - 05/12/2016 TasmanHighway_7.3 -41,693 148,281 29 E. delegatensis 0 0 0 - - - - - 05/12/2016 TasmanHighway_7.4 -41,693 148,281 29 E. viminalis 0 0 0 - - - - - 08/12/2016 Cambridge -42,833 147,449 49 E. nitens 6(1) 0 0 1 4 1 0 0 09/12/2016 HobartPark.1 -42,885 147,300 247 E. ovata 1 0 0 0 0 0 0 0 09/12/2016 HobartPark.2 -42,884 147,300 252 E. globulus 20 0 1 5 14 0 1 0 09/12/2016 HobartPark.3 -42,883 147,301 164 E. globulus 8 1 1 0 0 0 0 0 09/12/2016 HobartPark.4 -42,883 147,301 143 E. viminalis 12(1) 6 6 2 6 0 1 0 17/12/2016 KingstonLeslieRoad.1 -42,973 147,271 177 E. viminalis 24(1) 0 1 0 0 4 11 0 17/12/2016 KingstonLeslieRoad.2 -42,973 147,271 177 E. viminalis 0 0 1 - - - - - 17/12/2016 Cygnet_2 -43,190 147,010 14 E. globulus 5 0 0 0 0 0 4 0 17/12/2016 Scouts.1 -42,945 147,313 103 E. ovata 49 0 1 0 0 1 18 0 17/12/2016 SnugFallsRoad.1 -43,071 147,242 78 E. ovata 34(1) 0 3 1 0 0 14 0 17/12/2016 ChannelHighway_4 -43,268 147,242 24 E. globulus 12 0 1 0 0 0 0 0 E. amygdalina 17/12/2016 SnugFallsRoad.2 -43,071 147,242 78 37 1 2 1 0 0 3 0 or E. pulchella KingstonCadesDriveRoa 17/12/2016 -42,969 147,268 163 E. globulus 6 0 0 0 0 0 0 0 d.1 KingstonCadesDriveRoa 17/12/2016 -42,969 147,268 163 E. viminalis 0 0 0 - - - - - d.2 18/12/2016 HobartMtNelson.1 -42,912 147,323 230 E. ovata 237(6) 2 5 3 8 2 52 1 18/12/2016 HobartMtNelson.2 -42,912 147,323 237 E. globulus 7 0 0 0 0 0 0 0 18/12/2016 HobartPark.3 -42,883 147,301 164 E. globulus 18 0 0 0 5 0 0 0 18/12/2016 HobartPark.4 -42,883 147,301 143 E. viminalis 39(1) 2* 2 1 6 0 1 0

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 17/12/2016 Cygnet_3 -43,163 147,083 17 E. ovata 11 0 0 0 0 0 0 0 23/01/2017 FrogLodge -42,733 146,925 44 E. viminalis 5 0 2 0 0 2 2 0 23/01/2017 LyellHighway.1 -42,680 146,937 79 E. globulus 0 1 1 - - - - - 23/01/2017 LyellHighway.2 -42,680 146,937 79 Eucalyptus sp. 0 0 0 - - - - - 23/01/2017 Tarraleah -42,298 146,448 597 Eucalyptus sp. 0 0 0 - - - - - 23/01/2017 MarlboroughHighway_1 -42,112 146,518 708 E. delegatensis 0 0 0 - - - - - 23/01/2017 MarlboroughHighway_2 -42,016 146,579 982 E. gunni 0 0 0 - - - - - 23/01/2017 RailtonRoad -41,418 146,525 114 E. ovata 3 0 0 0 0 0 0 0 24/01/2017 BassHighway_1 -40,949 145,517 176 E. globulus 0 0 0 - - - - - 24/01/2017 BassHighway_2 -40,854 145,380 21 E. obliqua 0 0 0 - - - - - 24/01/2017 Stanley -40,762 145,297 42 E. globulus 0 0 0 - - - - - 24/01/2017 BassHighway_3.1 -40,940 144,940 57 Eucalyptus sp. 0 0 0 - - - - - 24/01/2017 BassHighway_3.2 -40,940 144,940 57 Eucalyptus sp. 0 0 0 - - - - - 24/01/2017 BassHighway_3.3 -40,940 144,940 57 Eucalyptus sp. 0 0 0 - - - - - 24/01/2017 KauriTimber -40,862 145,142 32 Eucalyptus sp. 0 0 0 - - - - - 24/01/2017 BassHighway_4 -41,002 145,746 19 E. ovata 0 0 0 - - - - - E. amygdalina 24/01/2017 Exeter.1 -41,329 146,943 110 0 4 0 - - - - - or E. pulchella 24/01/2017 Exeter.2 -41,329 146,943 110 E. ovata 9 0 5 0 0 0 0 0 24/01/2017 Grindwald -41,356 147,014 129 E. globulus 2 2 0 0 0 0 0 0 25/01/2017 TheGlenRoad_1.1 -41,188 147,103 179 E. ovata 0 0 0 - - - - - 25/01/2017 TheGlenRoad_1.2 -41,188 147,103 179 E. globulus 1 0 10 0 0 0 0 0 E. amygdalina 25/01/2017 TheGlenRoad_1.3 -41,188 147,103 179 0 0 1 - - - - - or E. pulchella 25/01/2017 TheGlenRoad_2.1 -41,170 147,059 127 E. ovata 0 1 0 - - - - - E. amygdalina 25/01/2017 TheGlenRoad_2.2 -41,170 147,059 127 21 3 5 0 0 0 1 0 or E. pulchella

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 25/01/2017 TheGlenRoad_2.3 -41,170 147,059 127 Eucalyptus sp. 0 1 0 - - - - - 25/01/2017 OldBangorTramRoad.2 -41,209 147,110 125 E. ovata 1 0 1 0 0 0 0 0 25/01/2017 FernyHillRoad_1.1 -41,156 147,301 85 E. viminalis 0 0 0 - - - - - E. amygdalina 25/01/2017 FernyHillRoad_1.2 -41,156 147,301 85 2 0 0 0 0 0 0 0 or E. pulchella 25/01/2017 FernyHillRoad_3 -41,078 147,309 69 Eucalyptus sp. 0 0 0 - - - - - 25/01/2017 TasmanHighway_6 -41,406 147,282 341 E. ovata 66 0 2 0 0 0 0 0 25/01/2017 Deddington.1 -41,627 147,397 233 E. globulus 0 0 1 - - - - -

25/01/2017 Deddington.2 -41,627 147,397 233 Eucalyptus sp. 0 0 0 - - - - - E. amygdalina 25/01/2017 NileRoad -41,736 147,467 249 4 3 8 0 0 0 0 0 or E. pulchella 25/01/2017 ForeshoreRoad_2 -42,820 147,268 22 E. globulus 0 0 2 - - - - - E. amygdalina 26/01/2017 HobartPark.5 -42,883 147,301 164 2 5 0 0 0 0 0 0 or E. pulchella 26/01/2017 HobartPark.4 -42,883 147,301 143 E. viminalis 46 0 3 0 0 0 0 0 26/01/2017 HobartPark.3 -42,883 147,301 164 E. globulus 0 0 0 - - - - - 26/01/2017 HobartPark.6 -42,885 147,300 247 Eucalyptus sp. 3 0 1 0 0 0 0 0 E. amygdalina 28/01/2017 DipRoad.1 -42,977 147,119 183 4 1 2 0 0 0 0 0 or E. pulchella 28/01/2017 DipRoad.3 -42,977 147,119 183 E. obliqua 0 0 0 - - - - - 28/01/2017 KingstonLeslieRoad.2 -42,973 147,271 177 E. viminalis 0 0 1 - - - - - 28/01/2017 AllensRivuletRoad_2.1 -43,006 147,215 136 E. ovata 70 0 9 0 0 0 19 0 E. amygdalina 28/01/2017 AllensRivuletRoad_2.2 -43,006 147,215 136 3 0 3 0 0 0 0 0 or E. pulchella 28/01/2017 SandflyRoad.1 -43,023 147,257 34 E. ovata 9 0 0 0 0 0 0 0 28/01/2017 SandflyRoad.2 -43,023 147,257 34 E. viminalis 0 0 0 - - - - -

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 28/01/2017 SnugFallsRoad.1 -43,071 147,242 78 E. ovata 10 0 0 0 0 0 1 0 E. amygdalina 28/01/2017 SnugFallsRoad.2 -43,071 147,242 78 6 0 2 0 0 0 1 0 or E. pulchella 28/01/2017 ChannelHighway_4 -43,268 147,242 24 E. globulus 0 0 0 - - - - - 28/01/2017 Cygnet_3 -43,163 147,083 17 E. ovata 2 0 0 0 0 0 0 0 28/01/2017 Cygnet_2 -43,190 147,010 14 E. globulus 4 0 3 0 0 0 0 0 28/01/2017 Cygnet_4.1 -43,183 146,986 21 E. ovata 54 1 1 0 0 0 3 0 28/01/2017 Cygnet_4.2 -43,183 146,986 21 E. viminalis 1 2 0 0 0 0 0 0 28/01/2017 Judbury -43,002 146,927 55 E. ovata 45 0 5 2 0 4 0 0 29/01/2017 NubeenaRoad.1 -43,140 147,818 198 E. globulus 7 0 1 0 0 0 0 0 29/01/2017 NubeenaRoad.2 -43,140 147,818 198 E. obliqua 0 0 0 - - - - - E. amygdalina 29/01/2017 NubeenaRoad.3 -43,140 147,818 198 0 0 0 - - - - - or E. pulchella 29/01/2017 NubeenaBackRoad -43,091 147,765 167 E. globulus 0 0 0 - - - - - 29/01/2017 ArthurHighway_3 -42,918 147,825 17 E. ovata 29 0 1 0 0 0 1 0 30/01/2017 HobartMtNelson.1 -42,912 147,323 230 E. ovata 99 2 7 0 0 0 7 0 30/01/2017 HobartMtNelson.2 -42,912 147,323 230 E. globulus 1 1 0 0 0 0 0 0 30/01/2017 Scouts.1 -42,945 147,313 103 E. ovata 12 2 2 0 0 0 0 0 E. amygdalina 30/01/2017 Scouts.2 -42,945 147,313 103 0 5* 1 - - - - - or E. pulchella KingstonCadesDriveRoa 24/11/2017 -42,969 147,268 163 E. globulus 56 n.a. n.a. 0 0 9 0 0 d.1 24/11/2017 ChannelHighway_4 -43,268 147,242 24 E. globulus 35 n.a. n.a. 0 0 0 0 0 28/11/2017 Cygnet_4.1 -43,183 146,986 21 E. ovata 13 n.a. n.a. 0 0 0 6 1 28/11/2017 Cygnet_3 -43,163 147,083 17 E. ovata 267 n.a. n.a. 13 17 85 51 49 28/11/2017 Cygnet_2 -43,190 147,010 14 E. globulus 204(2) n.a. n.a. 5 5 5 22 6

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Nº of Gonipterus Gonipterus Emerged Date of Eucalyptus Place of Survey Latitude Longitude Altitude collected adults male females A. Cirrospilus Euderus Survey species A. tasmaniae A. nitens egg pods collected1 collected2 inexpectatus spp. spp. 23/11/2017 HobartPark.3 -42,883 147,301 164 E. globulus 27 n.a. n.a. 0 0 0 0 0 23/11/2017 HobartPark.4 -42,883 147,301 143 E. viminalis 28 n.a. n.a. 0 0 0 1 0 24/11/2017 KingstonLeslieRoad.2 -42,973 147,271 177 E. viminalis 33 n.a. n.a. 0 0 0 0 0 22/11/2017 HobartMtNelson.1 -42,912 147,323 230 E. ovata 115 n.a. n.a. 0 0 0 0 0 24/11/2017 Scouts.1 -42,945 147,313 103 E. ovata 72 n.a. n.a. 1 0 1 0 0 28/11/2017 SnugFallsRoad.1 -43,071 147,242 78 E. ovata 65 n.a. n.a. 2 0 7 11 0 28/11/2017 Cygnet_1 -43,166 147,080 12 E. ovata 50 n.a. n.a. 0 0 6 0 0 28/11/2017 MissingLinkRoad -43,169 147,036 236 Eucalyptus sp. 41 n.a. n.a. 0 0 1 3 0 E. amygdalina 04/12/2017 FernyHillRoad_1 -41,156 147,301 85 198 n.a. n.a. 4 0 6 0 0 or E. pulchella 04/12/2017 FernyHillRoad_2 -41,152 147,301 79 E. ovata 111 n.a. n.a. 10 0 9 0 0 04/12/2017 FernyHillRoad_3 -41,078 147,309 69 Eucalyptus sp. 105 n.a. n.a. 4 0 9 0 0 05/12/2017 Grindwald -41,356 147,014 129 E. globulus 13 n.a. n.a. 0 0 0 0 0 E. ovata/ E. 05/12/2017 TheGlenRoad_2.2 -41,170 147,059 127 amygdalina or 16(2) n.a. n.a. 3 10 0 0 1 E. pulchella 05/12/2017 TheGlenRoad_1.2 -41,188 147,103 179 E. globulus 271 n.a. n.a. 0 0 0 0 0 05/12/2017 Deddington.1 -41,627 147,397 233 E. globulus 12 n.a. n.a. 0 0 13 0 0

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Chapter 3 - Supplementary Table 3.2. List of sequenced Gonipterus specimens, used for phylogenetic analysis, with data of the collector, collection date, country of collection and GenBank accession numbers. Gonipterus specimen Sequence_ID Collected_by Collection_date Country GenBank Accession number Gonipterus 01 Seq01 André Garcia 02-Nov-16 Australia MK674085 Gonipterus 02 Seq02 André Garcia 02-Nov-16 Australia MK674086 Gonipterus 03 Seq03 André Garcia 02-Nov-16 Australia MK674087 Gonipterus 04 Seq04 André Garcia 02-Nov-16 Australia MK674088 Gonipterus 05 Seq05 André Garcia 02-Nov-16 Australia MK674089 Gonipterus 06 Seq06 André Garcia 02-Nov-16 Australia MK674090 Gonipterus 07 Seq07 André Garcia 02-Nov-16 Australia MK674091 Gonipterus 08 Seq08 André Garcia 02-Nov-16 Australia MK674092 Gonipterus 09 Seq09 André Garcia 02-Nov-16 Australia MK674093 Gonipterus 10 Seq10 André Garcia 02-Nov-16 Australia MK674094 Gonipterus 11 Seq11 André Garcia 28-Nov-17 Australia MK674095 Gonipterus 12 Seq12 André Garcia 07-Nov-16 Australia MK674096 Gonipterus 13 Seq13 André Garcia 02-Nov-16 Australia MK674097 Gonipterus 14 Seq14 André Garcia 18-Dec-16 Australia MK674098 Gonipterus 15 Seq15 André Garcia 17-Dec-16 Australia MK674099 Gonipterus 16 Seq16 André Garcia 04-Nov-16 Australia MK674100 Gonipterus 17 Seq17 André Garcia 05-Dec-17 Australia MK674101 Gonipterus 18 Seq18 André Garcia 22-Nov-16 Australia MK674102 Gonipterus 19 Seq19 André Garcia 21-Nov-16 Australia MK674103 Gonipterus 20 Seq20 André Garcia 22-Nov-16 Australia MK674104 Gonipterus 21 Seq21 André Garcia 22-Nov-16 Australia MK674105 Gonipterus 22 Seq22 André Garcia 21-Nov-16 Australia MK674106 Gonipterus 23 Seq23 André Garcia 17-Dec-16 Australia MK674107 Gonipterus 24 Seq24 André Garcia 21-Nov-16 Australia MK674108 Gonipterus 25 Seq25 André Garcia 02-Nov-16 Australia MK674109 Gonipterus 26 Seq26 André Garcia 18-Dec-16 Australia MK674110 Gonipterus 27 Seq27 André Garcia 09-Dec-16 Australia MK674111 Gonipterus 28 Seq28 André Garcia 02-Nov-16 Australia MK674112 Gonipterus 29 Seq29 André Garcia 02-Nov-16 Australia MK674113 Gonipterus 30 Seq30 André Garcia 02-Nov-16 Australia MK674114 Gonipterus 31 Seq31 André Garcia 02-Nov-16 Australia MK674115 Gonipterus 32 Seq32 André Garcia 02-Nov-16 Australia MK674116 Gonipterus 33 Seq33 André Garcia 30-Nov-16 Australia MK674117 Gonipterus 34 Seq34 André Garcia 22-Nov-16 Australia MK674118 Gonipterus 35 Seq35 André Garcia 20-Nov-16 Australia MK674119 Gonipterus 36 Seq36 André Garcia 08-Dec-16 Australia MK674120 Gonipterus 37 Seq37 André Garcia 28-Nov-17 Australia MK674121 Gonipterus 38 Seq38 André Garcia 18-Dec-16 Australia MK674122 Gonipterus 39 Seq39 André Garcia 31-Oct-16 Australia MK674123 Gonipterus 40 Seq40 André Garcia 25-Oct-16 Australia MK674124 Gonipterus 41 Seq41 André Garcia 18-Dec-16 Australia MK674125

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Gonipterus 42 Seq42 André Garcia 18-Dec-16 Australia MK674126 Gonipterus 43 Seq43 André Garcia 18-Dec-16 Australia MK674127 Gonipterus 44 Seq44 André Garcia 18-Dec-16 Australia MK674128 Gonipterus 45 Seq45 André Garcia 02-Nov-16 Australia MK674129 Gonipterus 46 Seq46 André Garcia 07-Nov-16 Australia MK674130 Gonipterus 47 Seq47 André Garcia 21-Nov-16 Australia MK674131 Gonipterus 48 Seq48 André Garcia 21-Nov-16 Australia MK674132 Gonipterus 49 Seq49 André Garcia 07-Nov-16 Australia MK674133 Gonipterus 50 Seq50 André Garcia 30-Nov-16 Australia MK674134 Gonipterus 51 Seq51 André Garcia 07-Nov-16 Australia MK674135 Gonipterus 52 Seq52 André Garcia 28-Oct-16 Australia MK674136 Gonipterus 53 Seq53 André Garcia 07-Nov-16 Australia MK674137 Gonipterus 54 Seq54 André Garcia 07-Nov-16 Australia MK674138 Gonipterus 55 Seq55 André Garcia 09-Nov-16 Australia MK674139 Gonipterus 56 Seq56 André Garcia 28-Nov-16 Australia MK674140 Gonipterus 57 Seq57 André Garcia 09-Nov-16 Australia MK674141 Gonipterus 58 Seq58 André Garcia 10-Nov-16 Australia MK674142 Gonipterus 59 Seq59 André Garcia 10-Nov-16 Australia MK674143 Gonipterus 60 Seq60 André Garcia 10-Nov-16 Australia MK674144 Gonipterus 61 Seq61 André Garcia 07-Nov-16 Australia MK674145 Gonipterus 62 Seq62 André Garcia 05-Dec-17 Australia MK674146 Gonipterus 63 Seq63 André Garcia 07-Nov-16 Australia MK674147 Gonipterus 64 Seq64 André Garcia 07-Nov-16 Australia MK674148 Gonipterus 65 Seq65 André Garcia 30-Nov-16 Australia MK674149 Gonipterus 66 Seq66 André Garcia 23-Oct-16 Australia MK674150 Gonipterus 67 Seq67 André Garcia 09-Nov-16 Australia MK674151 Gonipterus 68 Seq68 André Garcia 08-Feb-17 Australia MK674152 Gonipterus 69 Seq69 André Garcia 18-Dec-16 Australia MK674153 Gonipterus 70 Seq70 André Garcia 25-Oct-16 Australia MK674154

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Chapter 3 - Supplementary Table 3.3. Estimates of Evolutionary Divergence between Sequences. The number of base substitutions per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Maximum Composite Likelihood model. The analysis involved 71 nucleotide sequences. There were a total of 652 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Reference Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Specimen 63 64 62 61 59 60 58 notographus 17 18 19 20 21 16 15 11 10 Gonipterus 63 0,001 0,003 0,001 0,003 0,004 0,004 0,011 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,025 0,025 Gonipterus 64 0,002 0,003 0,002 0,003 0,004 0,003 0,010 0,026 0,026 0,026 0,026 0,026 0,025 0,026 0,025 0,025 Gonipterus 62 0,006 0,008 0,003 0,003 0,004 0,004 0,010 0,027 0,027 0,027 0,027 0,027 0,026 0,027 0,026 0,026 Gonipterus 61 0,002 0,003 0,005 0,002 0,003 0,003 0,010 0,026 0,026 0,026 0,026 0,026 0,025 0,026 0,025 0,025 Gonipterus 59 0,005 0,006 0,008 0,003 0,003 0,003 0,010 0,025 0,025 0,025 0,025 0,025 0,025 0,026 0,025 0,025 Gonipterus 60 0,008 0,009 0,011 0,006 0,006 0,004 0,011 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,024 0,024 Gonipterus 58 0,009 0,008 0,009 0,008 0,008 0,011 0,010 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,024 0,024 Reference Gonipterus notographus 0,047 0,045 0,045 0,045 0,045 0,049 0,040 0,027 0,027 0,027 0,027 0,027 0,028 0,028 0,027 0,027 Gonipterus 17 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,146 0,000 0,000 0,000 0,000 0,001 0,003 0,002 0,002 Gonipterus 18 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,146 0,000 0,000 0,000 0,000 0,001 0,003 0,002 0,002 Gonipterus 19 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,146 0,000 0,000 0,000 0,000 0,001 0,003 0,002 0,002 Gonipterus 20 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,146 0,000 0,000 0,000 0,000 0,001 0,003 0,002 0,002 Gonipterus 21 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,146 0,000 0,000 0,000 0,000 0,001 0,003 0,002 0,002 Gonipterus 16 0,135 0,133 0,140 0,133 0,133 0,131 0,129 0,149 0,002 0,002 0,002 0,002 0,002 0,002 0,001 0,001 Gonipterus 15 0,140 0,138 0,144 0,138 0,138 0,135 0,133 0,153 0,005 0,005 0,005 0,005 0,005 0,003 0,003 0,003 Gonipterus 11 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 Gonipterus 10 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 Gonipterus 09 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 08 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 12 0,135 0,133 0,140 0,133 0,133 0,131 0,129 0,149 0,005 0,005 0,005 0,005 0,005 0,003 0,006 0,002 0,002 Gonipterus 07 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 06 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000

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Gonipterus 05 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 04 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 03 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 01 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 02 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 13 0,133 0,131 0,138 0,131 0,131 0,129 0,127 0,147 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,000 0,000 Gonipterus 25 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 26 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 14 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,002 0,003 0,003 Reference Gonipterus platensis 0,138 0,135 0,142 0,135 0,135 0,133 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,002 0,003 0,003 Gonipterus 27 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 28 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 29 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 24 0,138 0,140 0,142 0,135 0,135 0,133 0,135 0,155 0,008 0,008 0,008 0,008 0,008 0,006 0,009 0,008 0,008 Gonipterus 30 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 31 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 32 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 23 0,135 0,138 0,140 0,133 0,133 0,131 0,133 0,149 0,002 0,002 0,002 0,002 0,002 0,003 0,006 0,005 0,005 Gonipterus 22 0,135 0,138 0,140 0,133 0,133 0,131 0,133 0,149 0,002 0,002 0,002 0,002 0,002 0,003 0,006 0,005 0,005 Gonipterus 33 0,133 0,135 0,138 0,131 0,131 0,129 0,131 0,151 0,003 0,003 0,003 0,003 0,003 0,002 0,005 0,003 0,003 Gonipterus 36 0,133 0,135 0,137 0,131 0,131 0,129 0,131 0,151 0,006 0,006 0,006 0,006 0,006 0,005 0,008 0,006 0,006 Gonipterus 35 0,135 0,137 0,140 0,133 0,133 0,131 0,133 0,153 0,006 0,006 0,006 0,006 0,006 0,005 0,008 0,006 0,006 Gonipterus 34 0,136 0,138 0,140 0,134 0,134 0,127 0,133 0,153 0,008 0,008 0,008 0,008 0,008 0,006 0,009 0,008 0,008 Gonipterus 41 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,052 0,052 0,052 0,052 0,052 0,054 0,057 0,055 0,055 Gonipterus 38 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,054 0,054 0,054 0,054 0,054 0,055 0,059 0,057 0,057 Gonipterus 37 0,149 0,146 0,149 0,146 0,151 0,149 0,144 0,160 0,049 0,049 0,049 0,049 0,049 0,050 0,054 0,052 0,052

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Gonipterus 42 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,054 0,054 0,054 0,054 0,054 0,055 0,059 0,057 0,057 Gonipterus 43 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,054 0,054 0,054 0,054 0,054 0,055 0,059 0,057 0,057 Gonipterus 44 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,054 0,054 0,054 0,054 0,054 0,055 0,059 0,057 0,057 Gonipterus 39 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,052 0,052 0,052 0,052 0,052 0,054 0,057 0,055 0,055 Gonipterus 40 0,155 0,153 0,155 0,153 0,158 0,155 0,151 0,167 0,052 0,052 0,052 0,052 0,052 0,054 0,057 0,055 0,055 Reference Gonipterus pulverulentus 0,146 0,144 0,151 0,144 0,144 0,142 0,138 0,151 0,042 0,042 0,042 0,042 0,042 0,044 0,047 0,045 0,045 Gonipterus 53 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Gonipterus 52 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Gonipterus 51 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Gonipterus 54 0,168 0,165 0,170 0,165 0,161 0,159 0,160 0,169 0,094 0,094 0,094 0,094 0,094 0,096 0,100 0,098 0,098 Gonipterus 55 0,168 0,165 0,170 0,165 0,161 0,159 0,160 0,169 0,094 0,094 0,094 0,094 0,094 0,096 0,100 0,098 0,098 Gonipterus 56 0,168 0,165 0,170 0,165 0,161 0,159 0,160 0,169 0,094 0,094 0,094 0,094 0,094 0,096 0,100 0,098 0,098 Gonipterus 57 0,168 0,165 0,170 0,165 0,161 0,159 0,160 0,169 0,094 0,094 0,094 0,094 0,094 0,096 0,100 0,098 0,098 Gonipterus 50 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Reference Gonipterus sp. 1 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Gonipterus 49 0,163 0,160 0,165 0,160 0,156 0,154 0,156 0,164 0,092 0,092 0,092 0,092 0,092 0,094 0,098 0,096 0,096 Gonipterus 48 0,167 0,165 0,169 0,165 0,160 0,158 0,160 0,169 0,090 0,090 0,090 0,090 0,090 0,092 0,096 0,094 0,094 Gonipterus 47 0,165 0,163 0,167 0,163 0,158 0,156 0,158 0,167 0,088 0,088 0,088 0,088 0,088 0,090 0,094 0,092 0,092 Gonipterus 46 0,163 0,160 0,165 0,160 0,156 0,154 0,156 0,164 0,086 0,086 0,086 0,086 0,086 0,088 0,092 0,090 0,090 Gonipterus 45 0,173 0,175 0,180 0,175 0,175 0,171 0,175 0,176 0,085 0,085 0,085 0,085 0,085 0,087 0,091 0,089 0,089 Reference Gonipterus sp. 2 0,174 0,177 0,177 0,172 0,172 0,167 0,172 0,168 0,083 0,083 0,083 0,083 0,083 0,085 0,088 0,087 0,087 Reference Gonipterus scutellatus 0,152 0,150 0,154 0,150 0,146 0,148 0,145 0,161 0,094 0,094 0,094 0,094 0,094 0,092 0,092 0,094 0,094 Oxyops sp. (KF016235) 0,193 0,195 0,195 0,190 0,190 0,188 0,193 0,198 0,185 0,185 0,185 0,185 0,185 0,182 0,180 0,185 0,185

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Reference Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Specimen 09 08 12 07 06 05 04 03 01 02 13 25 26 14 platensis 27 28 Gonipterus 63 0,025 0,025 0,026 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,026 0,026 0,025 0,025 Gonipterus 64 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,026 0,026 0,026 0,026 0,026 0,026 Gonipterus 62 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,027 0,027 0,026 0,026 Gonipterus 61 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,026 0,026 0,025 0,025 Gonipterus 59 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 Gonipterus 60 0,024 0,024 0,025 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,025 0,025 0,024 0,024 Gonipterus 58 0,024 0,024 0,025 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,024 0,025 0,025 0,025 0,025 0,025 0,025 Reference Gonipterus notographus 0,027 0,027 0,028 0,027 0,027 0,027 0,027 0,027 0,027 0,027 0,027 0,028 0,028 0,028 0,028 0,028 0,028 Gonipterus 17 0,002 0,002 0,003 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 18 0,002 0,002 0,003 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 19 0,002 0,002 0,003 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 20 0,002 0,002 0,003 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 21 0,002 0,002 0,003 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 16 0,001 0,001 0,002 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 Gonipterus 15 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,002 0,002 0,003 0,003 Gonipterus 11 0,000 0,000 0,001 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 10 0,000 0,000 0,001 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 09 0,000 0,001 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 08 0,000 0,001 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 12 0,002 0,002 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,001 0,003 0,003 0,003 0,003 0,003 0,003 Gonipterus 07 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 06 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 05 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 04 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002

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Gonipterus 03 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 01 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 02 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 13 0,000 0,000 0,002 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,002 0,002 Gonipterus 25 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,002 0,002 0,000 0,000 Gonipterus 26 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,002 0,002 0,000 0,000 Gonipterus 14 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,002 0,002 Reference Gonipterus platensis 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,002 0,002 Gonipterus 27 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 Gonipterus 28 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 Gonipterus 29 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 0,000 Gonipterus 24 0,008 0,008 0,009 0,008 0,008 0,008 0,008 0,008 0,008 0,008 0,008 0,005 0,005 0,008 0,008 0,005 0,005 Gonipterus 30 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 0,000 Gonipterus 31 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 0,000 Gonipterus 32 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 0,000 Gonipterus 23 0,005 0,005 0,006 0,005 0,005 0,005 0,005 0,005 0,005 0,005 0,005 0,002 0,002 0,005 0,005 0,002 0,002 Gonipterus 22 0,005 0,005 0,006 0,005 0,005 0,005 0,005 0,005 0,005 0,005 0,005 0,002 0,002 0,005 0,005 0,002 0,002 Gonipterus 33 0,003 0,003 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,000 0,000 0,003 0,003 0,000 0,000 Gonipterus 36 0,006 0,006 0,008 0,006 0,006 0,006 0,006 0,006 0,006 0,006 0,006 0,003 0,003 0,006 0,006 0,003 0,003 Gonipterus 35 0,006 0,006 0,008 0,006 0,006 0,006 0,006 0,006 0,006 0,006 0,006 0,003 0,003 0,006 0,006 0,003 0,003 Gonipterus 34 0,008 0,008 0,009 0,008 0,008 0,008 0,008 0,008 0,008 0,008 0,008 0,005 0,005 0,008 0,008 0,005 0,005 Gonipterus 41 0,055 0,055 0,054 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 Gonipterus 38 0,057 0,057 0,055 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 Gonipterus 37 0,052 0,052 0,054 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 0,052 Gonipterus 42 0,057 0,057 0,055 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 Gonipterus 43 0,057 0,057 0,055 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057

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Gonipterus 44 0,057 0,057 0,055 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 0,057 Gonipterus 39 0,055 0,055 0,054 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 Gonipterus 40 0,055 0,055 0,054 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 0,055 Reference Gonipterus pulverulentus 0,045 0,045 0,044 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 0,045 Gonipterus 53 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Gonipterus 52 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Gonipterus 51 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Gonipterus 54 0,098 0,098 0,100 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 Gonipterus 55 0,098 0,098 0,100 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 Gonipterus 56 0,098 0,098 0,100 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 Gonipterus 57 0,098 0,098 0,100 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 0,098 Gonipterus 50 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Reference Gonipterus sp. 1 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Gonipterus 49 0,096 0,096 0,098 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 0,096 Gonipterus 48 0,094 0,094 0,096 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 Gonipterus 47 0,092 0,092 0,094 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 0,092 Gonipterus 46 0,090 0,090 0,092 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 0,090 Gonipterus 45 0,089 0,089 0,091 0,089 0,089 0,089 0,089 0,089 0,089 0,089 0,089 0,085 0,085 0,089 0,089 0,085 0,085 Reference Gonipterus sp. 2 0,087 0,087 0,088 0,087 0,087 0,087 0,087 0,087 0,087 0,087 0,087 0,083 0,083 0,087 0,087 0,083 0,083 Reference Gonipterus scutellatus 0,094 0,094 0,096 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 0,094 Oxyops sp. (KF016235) 0,185 0,185 0,187 0,185 0,185 0,185 0,185 0,185 0,185 0,185 0,185 0,180 0,180 0,180 0,180 0,180 0,180

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Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Specimen 29 24 30 31 32 23 22 33 36 35 34 41 38 37 42 43 44 Gonipterus 63 0,025 0,026 0,025 0,025 0,025 0,026 0,026 0,025 0,025 0,026 0,026 0,029 0,029 0,028 0,029 0,029 0,029 Gonipterus 64 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,029 0,029 0,028 0,029 0,029 0,029 Gonipterus 62 0,026 0,027 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,026 0,027 0,030 0,030 0,028 0,030 0,030 0,030 Gonipterus 61 0,025 0,026 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,029 0,029 0,028 0,029 0,029 0,029 Gonipterus 59 0,025 0,026 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,030 0,030 0,029 0,030 0,030 0,030 Gonipterus 60 0,024 0,025 0,024 0,024 0,024 0,025 0,025 0,024 0,024 0,025 0,024 0,030 0,030 0,028 0,030 0,030 0,030 Gonipterus 58 0,025 0,026 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,025 0,029 0,029 0,028 0,029 0,029 0,029 Reference Gonipterus notographus 0,028 0,029 0,028 0,028 0,028 0,028 0,028 0,028 0,028 0,028 0,028 0,032 0,032 0,031 0,032 0,032 0,032 Gonipterus 17 0,002 0,003 0,002 0,002 0,002 0,001 0,001 0,002 0,003 0,003 0,003 0,011 0,011 0,011 0,011 0,011 0,011 Gonipterus 18 0,002 0,003 0,002 0,002 0,002 0,001 0,001 0,002 0,003 0,003 0,003 0,011 0,011 0,011 0,011 0,011 0,011 Gonipterus 19 0,002 0,003 0,002 0,002 0,002 0,001 0,001 0,002 0,003 0,003 0,003 0,011 0,011 0,011 0,011 0,011 0,011 Gonipterus 20 0,002 0,003 0,002 0,002 0,002 0,001 0,001 0,002 0,003 0,003 0,003 0,011 0,011 0,011 0,011 0,011 0,011 Gonipterus 21 0,002 0,003 0,002 0,002 0,002 0,001 0,001 0,002 0,003 0,003 0,003 0,011 0,011 0,011 0,011 0,011 0,011 Gonipterus 16 0,001 0,003 0,001 0,001 0,001 0,002 0,002 0,001 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 15 0,003 0,004 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,004 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 11 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 10 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 09 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 08 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012

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Gonipterus 12 0,003 0,004 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,004 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 07 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 06 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 05 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 04 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 03 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 01 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 02 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 13 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 25 0,000 0,003 0,000 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 26 0,000 0,003 0,000 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 14 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012 Reference Gonipterus platensis 0,002 0,003 0,002 0,002 0,002 0,003 0,003 0,002 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 27 0,000 0,003 0,000 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 28 0,000 0,003 0,000 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 29 0,003 0,000 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 24 0,005 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,003 0,004 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 30 0,000 0,005 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 31 0,000 0,005 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 32 0,000 0,005 0,000 0,000 0,001 0,001 0,000 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 23 0,002 0,006 0,002 0,002 0,002 0,000 0,001 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 22 0,002 0,006 0,002 0,002 0,002 0,000 0,001 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012

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Gonipterus 33 0,000 0,005 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,003 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 36 0,003 0,008 0,003 0,003 0,003 0,005 0,005 0,003 0,002 0,004 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 35 0,003 0,008 0,003 0,003 0,003 0,005 0,005 0,003 0,003 0,003 0,011 0,012 0,011 0,012 0,012 0,012 Gonipterus 34 0,005 0,009 0,005 0,005 0,005 0,006 0,006 0,005 0,008 0,008 0,012 0,012 0,011 0,012 0,012 0,012 Gonipterus 41 0,055 0,057 0,055 0,055 0,055 0,054 0,054 0,055 0,056 0,055 0,057 0,001 0,003 0,001 0,001 0,001 Gonipterus 38 0,057 0,059 0,057 0,057 0,057 0,055 0,055 0,057 0,057 0,057 0,059 0,002 0,003 0,000 0,000 0,000 Gonipterus 37 0,052 0,054 0,052 0,052 0,052 0,050 0,050 0,052 0,052 0,052 0,054 0,006 0,005 0,003 0,003 0,003 Gonipterus 42 0,057 0,059 0,057 0,057 0,057 0,055 0,055 0,057 0,057 0,057 0,059 0,002 0,000 0,005 0,000 0,000 Gonipterus 43 0,057 0,059 0,057 0,057 0,057 0,055 0,055 0,057 0,057 0,057 0,059 0,002 0,000 0,005 0,000 0,000 Gonipterus 44 0,057 0,059 0,057 0,057 0,057 0,055 0,055 0,057 0,057 0,057 0,059 0,002 0,000 0,005 0,000 0,000 Gonipterus 39 0,055 0,057 0,055 0,055 0,055 0,054 0,054 0,055 0,056 0,055 0,057 0,000 0,002 0,006 0,002 0,002 0,002 Gonipterus 40 0,055 0,057 0,055 0,055 0,055 0,054 0,054 0,055 0,056 0,055 0,057 0,000 0,002 0,006 0,002 0,002 0,002 Reference Gonipterus pulverulentus 0,045 0,049 0,045 0,045 0,045 0,044 0,044 0,045 0,045 0,045 0,047 0,025 0,024 0,025 0,024 0,024 0,024 Gonipterus 53 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,109 0,111 0,109 0,111 0,111 0,111 Gonipterus 52 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,109 0,111 0,109 0,111 0,111 0,111 Gonipterus 51 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,109 0,111 0,109 0,111 0,111 0,111 Gonipterus 54 0,098 0,098 0,098 0,098 0,098 0,096 0,096 0,098 0,098 0,098 0,096 0,112 0,114 0,112 0,114 0,114 0,114 Gonipterus 55 0,098 0,098 0,098 0,098 0,098 0,096 0,096 0,098 0,098 0,098 0,096 0,112 0,114 0,112 0,114 0,114 0,114 Gonipterus 56 0,098 0,098 0,098 0,098 0,098 0,096 0,096 0,098 0,098 0,098 0,096 0,112 0,114 0,112 0,114 0,114 0,114 Gonipterus 57 0,098 0,098 0,098 0,098 0,098 0,096 0,096 0,098 0,098 0,098 0,096 0,112 0,114 0,112 0,114 0,114 0,114 Gonipterus 50 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,109 0,111 0,109 0,111 0,111 0,111

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Reference Gonipterus sp. 1 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,109 0,111 0,109 0,111 0,111 0,111 Gonipterus 49 0,096 0,096 0,096 0,096 0,096 0,094 0,094 0,096 0,096 0,096 0,094 0,105 0,107 0,105 0,107 0,107 0,107 Gonipterus 48 0,094 0,094 0,094 0,094 0,094 0,092 0,092 0,094 0,094 0,094 0,092 0,107 0,109 0,107 0,109 0,109 0,109 Gonipterus 47 0,092 0,092 0,092 0,092 0,092 0,090 0,090 0,092 0,092 0,092 0,090 0,106 0,107 0,106 0,107 0,107 0,107 Gonipterus 46 0,090 0,090 0,090 0,090 0,090 0,088 0,088 0,090 0,090 0,090 0,088 0,104 0,106 0,104 0,106 0,106 0,106 Gonipterus 45 0,085 0,089 0,085 0,085 0,085 0,083 0,083 0,085 0,085 0,085 0,085 0,103 0,105 0,103 0,105 0,105 0,105 Reference Gonipterus sp. 2 0,083 0,083 0,083 0,083 0,083 0,081 0,081 0,083 0,083 0,083 0,083 0,108 0,110 0,108 0,110 0,110 0,110 Reference Gonipterus scutellatus 0,094 0,100 0,094 0,094 0,094 0,096 0,096 0,094 0,094 0,092 0,093 0,120 0,122 0,116 0,122 0,122 0,122 Oxyops sp. (KF016235) 0,180 0,185 0,180 0,180 0,180 0,182 0,182 0,180 0,175 0,175 0,180 0,202 0,200 0,202 0,200 0,200 0,200

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Reference Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Gonipterus Reference Gonipterus Gonipterus Gonipterus Specimen 39 40 pulverulentus 53 52 51 54 55 56 57 50 Gonipterus sp. 1 49 48 47 Gonipterus 63 0,029 0,029 0,028 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,030 0,031 0,031 Gonipterus 64 0,029 0,029 0,027 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,030 0,031 0,031 Gonipterus 62 0,030 0,030 0,029 0,031 0,031 0,031 0,032 0,032 0,032 0,032 0,031 0,031 0,031 0,032 0,032 Gonipterus 61 0,029 0,029 0,027 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,030 0,031 0,031 Gonipterus 59 0,030 0,030 0,027 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,029 0,030 0,030 Gonipterus 60 0,030 0,030 0,027 0,029 0,029 0,029 0,029 0,029 0,029 0,029 0,029 0,029 0,029 0,030 0,029 Gonipterus 58 0,029 0,029 0,026 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,030 0,029 0,030 0,030 Reference Gonipterus notographus 0,032 0,032 0,029 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,031 0,032 0,031 Gonipterus 17 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 0,017 Gonipterus 18 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 0,017 Gonipterus 19 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 0,017 Gonipterus 20 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 0,017 Gonipterus 21 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 0,017 Gonipterus 16 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 Gonipterus 15 0,012 0,012 0,011 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 11 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 10 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 09 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 08 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 12 0,011 0,011 0,010 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,018 0,018

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Gonipterus 07 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 06 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 05 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 04 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 03 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 01 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 02 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 13 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 25 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 26 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 14 0,011 0,011 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,018 0,018 0,018 Reference Gonipterus platensis 0,011 0,011 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,018 0,018 0,018 Gonipterus 27 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 28 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 29 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 24 0,012 0,012 0,011 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,018 0,018 0,018 Gonipterus 30 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 31 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 32 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 23 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 Gonipterus 22 0,011 0,011 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 Gonipterus 33 0,012 0,012 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,019 0,018 0,018 Gonipterus 36 0,011 0,011 0,010 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,019 0,018 0,018 Gonipterus 35 0,011 0,011 0,010 0,018 0,018 0,018 0,019 0,019 0,019 0,019 0,018 0,018 0,018 0,018 0,018 Gonipterus 34 0,012 0,012 0,010 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,018 0,017 Gonipterus 41 0,000 0,000 0,007 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,020 0,020 0,020

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Gonipterus 38 0,001 0,001 0,007 0,021 0,021 0,021 0,022 0,022 0,022 0,022 0,021 0,021 0,020 0,021 0,020 Gonipterus 37 0,003 0,003 0,007 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,020 0,020 0,020 Gonipterus 42 0,001 0,001 0,007 0,021 0,021 0,021 0,022 0,022 0,022 0,022 0,021 0,021 0,020 0,021 0,020 Gonipterus 43 0,001 0,001 0,007 0,021 0,021 0,021 0,022 0,022 0,022 0,022 0,021 0,021 0,020 0,021 0,020 Gonipterus 44 0,001 0,001 0,007 0,021 0,021 0,021 0,022 0,022 0,022 0,022 0,021 0,021 0,020 0,021 0,020 Gonipterus 39 0,000 0,007 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,020 0,020 0,020 Gonipterus 40 0,000 0,007 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,021 0,020 0,020 0,020 Reference Gonipterus pulverulentus 0,025 0,025 0,019 0,019 0,019 0,020 0,020 0,020 0,020 0,019 0,019 0,019 0,019 0,019 Gonipterus 53 0,109 0,109 0,103 0,000 0,000 0,001 0,001 0,001 0,001 0,000 0,000 0,002 0,003 0,002 Gonipterus 52 0,109 0,109 0,103 0,000 0,000 0,001 0,001 0,001 0,001 0,000 0,000 0,002 0,003 0,002 Gonipterus 51 0,109 0,109 0,103 0,000 0,000 0,001 0,001 0,001 0,001 0,000 0,000 0,002 0,003 0,002 Gonipterus 54 0,112 0,112 0,105 0,002 0,002 0,002 0,000 0,000 0,000 0,001 0,001 0,003 0,003 0,003 Gonipterus 55 0,112 0,112 0,105 0,002 0,002 0,002 0,000 0,000 0,000 0,001 0,001 0,003 0,003 0,003 Gonipterus 56 0,112 0,112 0,105 0,002 0,002 0,002 0,000 0,000 0,000 0,001 0,001 0,003 0,003 0,003 Gonipterus 57 0,112 0,112 0,105 0,002 0,002 0,002 0,000 0,000 0,000 0,001 0,001 0,003 0,003 0,003 Gonipterus 50 0,109 0,109 0,103 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,000 0,002 0,003 0,002 Reference Gonipterus sp. 1 0,109 0,109 0,103 0,000 0,000 0,000 0,002 0,002 0,002 0,002 0,000 0,002 0,003 0,002 Gonipterus 49 0,105 0,105 0,099 0,003 0,003 0,003 0,005 0,005 0,005 0,005 0,003 0,003 0,003 0,002 Gonipterus 48 0,107 0,107 0,101 0,005 0,005 0,005 0,006 0,006 0,006 0,006 0,005 0,005 0,005 0,001 Gonipterus 47 0,106 0,106 0,099 0,003 0,003 0,003 0,005 0,005 0,005 0,005 0,003 0,003 0,003 0,002 Gonipterus 46 0,104 0,104 0,097 0,005 0,005 0,005 0,006 0,006 0,006 0,006 0,005 0,005 0,005 0,003 0,002 Gonipterus 45 0,103 0,103 0,095 0,067 0,067 0,067 0,068 0,068 0,068 0,068 0,067 0,067 0,065 0,070 0,068 Reference Gonipterus sp. 2 0,108 0,108 0,096 0,056 0,056 0,056 0,058 0,058 0,058 0,058 0,056 0,056 0,057 0,056 0,054 Reference Gonipterus scutellatus 0,120 0,120 0,112 0,078 0,078 0,078 0,080 0,080 0,080 0,080 0,078 0,078 0,076 0,076 0,074 Oxyops sp. (KF016235) 0,202 0,202 0,191 0,197 0,197 0,197 0,194 0,194 0,194 0,194 0,197 0,197 0,194 0,199 0,197

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Reference Gonipterus Gonipterus Reference Gonipterus Oxyops sp. Specimen 46 45 Gonipterus sp. 2 scutellatus (KF016235) Gonipterus 63 0,031 0,034 0,034 0,029 0,037 Gonipterus 64 0,030 0,034 0,034 0,028 0,037 Gonipterus 62 0,031 0,035 0,035 0,029 0,037 Gonipterus 61 0,030 0,034 0,034 0,028 0,036 Gonipterus 59 0,029 0,034 0,033 0,028 0,036 Gonipterus 60 0,029 0,033 0,033 0,028 0,036 Gonipterus 58 0,029 0,034 0,034 0,028 0,037 Reference Gonipterus notographus 0,031 0,034 0,032 0,030 0,038 Gonipterus 17 0,017 0,017 0,017 0,018 0,036 Gonipterus 18 0,017 0,017 0,017 0,018 0,036 Gonipterus 19 0,017 0,017 0,017 0,018 0,036 Gonipterus 20 0,017 0,017 0,017 0,018 0,036 Gonipterus 21 0,017 0,017 0,017 0,018 0,036 Gonipterus 16 0,017 0,017 0,017 0,018 0,036 Gonipterus 15 0,018 0,018 0,017 0,018 0,035 Gonipterus 11 0,018 0,018 0,017 0,019 0,036 Gonipterus 10 0,018 0,018 0,017 0,019 0,036 Gonipterus 09 0,018 0,018 0,017 0,019 0,036 Gonipterus 08 0,018 0,018 0,017 0,019 0,036 Gonipterus 12 0,018 0,018 0,018 0,019 0,037 Gonipterus 07 0,018 0,018 0,017 0,019 0,036 Gonipterus 06 0,018 0,018 0,017 0,019 0,036 Gonipterus 05 0,018 0,018 0,017 0,019 0,036 Gonipterus 04 0,018 0,018 0,017 0,019 0,036

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Gonipterus 03 0,018 0,018 0,017 0,019 0,036 Gonipterus 01 0,018 0,018 0,017 0,019 0,036 Gonipterus 02 0,018 0,018 0,017 0,019 0,036 Gonipterus 13 0,018 0,018 0,017 0,019 0,036 Gonipterus 25 0,018 0,017 0,017 0,018 0,035 Gonipterus 26 0,018 0,017 0,017 0,018 0,035 Gonipterus 14 0,017 0,017 0,017 0,018 0,035 Reference Gonipterus platensis 0,017 0,017 0,017 0,018 0,035 Gonipterus 27 0,018 0,017 0,017 0,018 0,035 Gonipterus 28 0,018 0,017 0,017 0,018 0,035 Gonipterus 29 0,018 0,017 0,017 0,018 0,035 Gonipterus 24 0,018 0,018 0,016 0,019 0,036 Gonipterus 30 0,018 0,017 0,017 0,018 0,035 Gonipterus 31 0,018 0,017 0,017 0,018 0,035 Gonipterus 32 0,018 0,017 0,017 0,018 0,035 Gonipterus 23 0,017 0,017 0,016 0,019 0,036 Gonipterus 22 0,017 0,017 0,016 0,019 0,036 Gonipterus 33 0,018 0,017 0,017 0,018 0,035 Gonipterus 36 0,018 0,017 0,017 0,018 0,034 Gonipterus 35 0,017 0,017 0,016 0,018 0,034 Gonipterus 34 0,017 0,017 0,017 0,018 0,035 Gonipterus 41 0,020 0,020 0,021 0,022 0,039 Gonipterus 38 0,020 0,020 0,021 0,023 0,039 Gonipterus 37 0,020 0,020 0,021 0,022 0,039 Gonipterus 42 0,020 0,020 0,021 0,023 0,039 Gonipterus 43 0,020 0,020 0,021 0,023 0,039

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Gonipterus 44 0,020 0,020 0,021 0,023 0,039 Gonipterus 39 0,020 0,020 0,021 0,022 0,039 Gonipterus 40 0,020 0,020 0,021 0,022 0,039 Reference Gonipterus pulverulentus 0,018 0,018 0,019 0,021 0,038 Gonipterus 53 0,003 0,013 0,012 0,015 0,038 Gonipterus 52 0,003 0,013 0,012 0,015 0,038 Gonipterus 51 0,003 0,013 0,012 0,015 0,038 Gonipterus 54 0,003 0,013 0,012 0,015 0,038 Gonipterus 55 0,003 0,013 0,012 0,015 0,038 Gonipterus 56 0,003 0,013 0,012 0,015 0,038 Gonipterus 57 0,003 0,013 0,012 0,015 0,038 Gonipterus 50 0,003 0,013 0,012 0,015 0,038 Reference Gonipterus sp. 1 0,003 0,013 0,012 0,015 0,038 Gonipterus 49 0,003 0,013 0,012 0,015 0,038 Gonipterus 48 0,002 0,014 0,012 0,015 0,039 Gonipterus 47 0,001 0,014 0,011 0,014 0,038 Gonipterus 46 0,013 0,011 0,014 0,039 Gonipterus 45 0,067 0,007 0,019 0,112 Reference Gonipterus sp. 2 0,052 0,025 0,018 0,046 Reference Gonipterus scutellatus 0,072 0,106 0,096 0,040 Oxyops sp. (KF016235) 0,199 0,221 0,220 0,201

105

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106

Chapter 4

Ophelimus sp., a new invasive gall wasp of Eucalyptus globulus in Europe, escapes the parasitism by Closterocerus chamaeleon due to an asynchronous life cycle

Published in Biological Control

Garcia, A., Gonçalves, H., Borowiec, N., Franco, J.C., Branco, M., 2019. Ophelimus sp., a new invasive gall wasp of Eucalyptus globulus in Europe, escapes the parasitism by Closterocerus chamaeleon due to an asynchronous life cycle. Biological Control 131: 1-7 https://doi.org/10.1016/j.biocontrol.2018.12.006

107

Chapter 4 Abstract

Ophelimus sp. (Hym: Eulophidae) is an Australian gall wasp newly found in Southern Europe forming galls on Eucalyptus globulus. A congeneric gall wasp O. maskelli, also established in the Mediterranean Basin, is currently controlled by the introduced parasitoid Closterocerus chamaeleon. To date, no parasitism was observed on Ophelimus sp. by C. chamaeleon. Here we analyze a possible escape from parasitism through an asynchronous life cycle in this host-parasitoid system. The ability of C. chamaeleon to oviposit and complete development on Ophelimus sp. was determined, both in laboratory and field experiments. Ophelimus sp. showed to be univoltine, with winter larval development and possible summer egg diapause, contrasting with the multivoltine behavior of O. maskelli, which completes 3-4 generations per year. Concomitantly, C. chamaeleon is normally collected in the field from May to October. In laboratory, under low temperatures (15±1ºC), adults of the parasitoid could survive up to four months. Both old (86-89 days) and young (< 16 days) parasitoid females showed similar parasitism behavior. Naïve parasitoid females oviposited in both O. maskelli (on E. camaldulensis) and Ophelimus sp. (on E. globulus), with no apparent preference between the two host species. We observed parasitism, when we exposed adults of C. chamaeleon to bagged eucalypt leaves infested with Ophelimus sp. galls, in field conditions. Altogether, our results demonstrate that C. chamaeleon is able to oviposit and complete development in Ophelimus sp. However, in field conditions, the lack of parasitism is possibly due to life cycle asynchrony between the parasitoid and the gall wasp.

Keywords: Life cycle asynchrony; Eucalyptus; Eulophidae; Gall wasps; Parasitism; Phenology

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Introduction Native to Australia, the genus Ophelimus Haliday (Hymenoptera: Eulophidae) consists of typical gall-inducing wasps. Currently, it includes about 51 described species (Ciesla, 2011; Noyes, 2018), and possibly many others still unknown, considering that the diversity of gall-maker insects in Australia is not fully studied (Blanche, 2000). The systematics of the genus is poorly known and needs revision (Borowiec et al. 2012; Protasov et al. 2007a). Five Ophelimus species established outside their native range. Ophelimus eucalypti (Maid.) was the first eucalypt gall wasp recorded outside Australia, in New Zealand in 1921, affecting Eucalyptus globulus Labill. and other eucalyptus species of the section Maidenaria (Raman and Withers, 2003; Withers, 2001). Six decades later, O. eucalypti was also reported attacking eucalypts of the section Transversaria, in New Zealand. Nevertheless, it was suggested that this second record corresponded to a new Ophelimus species, although morphologically indistinguishable from O. eucalypti, due to the different plant host range, i.e., Eucalyptus species from two different sections, Maidenaria versus Transversaria (Raman and Withers, 2003; Withers, 2001). In the following thirty-years, three other Ophelimus species invaded different areas worldwide: O. maskelli Ashmead, and two undescribed species. Among these, O. maskelli is the most widely distributed (Mansfield, 2016), being present in the Mediterranean Basin and North America (Branco et al., 2009; Burks et al., 2015; Caleca, 2010; Dhahri et al., 2010; Protasov et al., 2007a). Eucalyptus camaldulensis and E. tereticornis, both belonging to section Exertaria, are their main host species (Branco et al., 2014; Hill, 2004; Protasov et al., 2007a). Recently, the other two undescribed species were found in non-native eucalyptus plantations, in South America and Europe: i) Ophelimus sp. nº1 was detected in 2003, in Chile, damaging leaves and twigs of E. globulus (Aquino et al. 2014, 2015; Carlos Valente, personal comm.); and ii) Ophelimus sp. nº2 was observed in 2010, in France, and later in Portugal and Italy, damaging leaves of E. globulus and other related eucalypt species, within Maidenaria section (Borowiec et al., 2013; Garcia et al., 2014). Ophelimus sp. nº2 can be differentiated from O. maskelli based on molecular, morphological, and ecological aspects, as well as in relation to voltinism and morphometry of the galls (Borowiec et al., 2019). A program for the biological control of O. maskelli started in 2004, in Israel, with the release of Closterocerus chamaeleon (Girault) (Hymenoptera: Eulophidae: ) (Mendel et al., 2007; Protasov et al., 2007b). This parasitoid was also released in Italy, imported from Israel (Caleca et al., 2009; Rizzo et al., 2006). It rapidly dispersed across the Mediterranean basin, including Portugal (Borrajo et al., 2008; Branco et al., 2009;

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Caleca, 2010; Doganlar and Mendel, 2007; Lo Verde et al., 2010), and established in the entire region, efficiently controlling O. maskelli (Branco et al., 2009; Caleca et al., 2011; Mendel et al., 2017). Considering that C. chamaeleon is a parasitoid of Ophelimus species, we would expect that it would be able to parasitize Ophelimus sp. nº2. However, no parasitism of Ophelimus sp. nº2 has been observed in Portugal (Garcia et al., 2014) or in France (Borowiec et al., 2013). Besides O. maskelli and O. eucalypti, no other host species are reported in the Universal Chalcidoidea Database for C. chamaeleon (Noyes, 2018). The lack of parasitism by C. chamaeleon in the alien populations of Ophelimus sp. nº2 may be explained by the following two hypotheses: i) Ophelimus sp. nº2 is not a suitable host for C. chamaeleon; ii) Ophelimus sp. nº2 is a suitable host but there is a seasonal asynchrony between C. chamaeleon and the gall-wasp phenology. In the present work, we aimed at investigating the host-parasitoid interaction between Ophelimus sp. nº2 and C. chamaeleon, and testing the two mentioned hypotheses, based on both field and laboratory experiments. For simplicity, Ophelimus sp. nº2 will be therein notated as Ophelimus sp.

Materials and Methods

Origin of Ophelimus spp. and Closterocerus chamaeleon All the individuals of both gall wasps and the parasitoid originated from material collected in the arboretum of Instituto Superior de Agronomia (ISA), University of Lisbon, at Tapada da Ajuda, Lisbon (Lat: 38.715014, Long: -9.192270). This arboretum is composed by 30 plots of different Eucalyptus species. Ophelimus sp. was obtained from E. globulus, E. cinerea, or E. cypellocarpa, whereas O. maskelli originated from E. camaldulensis. The individuals of C. chamaeleon emerged from leaves of E. camaldulensis with galls of O. maskelli, collected between September and December 2015. The leaves were maintained within plastic boxes, until the emergence of the parasitoids. A total of 5 936 adult wasps were obtained. Each wasp was used only once in the experiments.

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Ophelimus sp. Gall morphology Galls biometric data was collected for Ophelimus sp., in comparison with O. maskeli, based on samples collected from infested leaves of E. globulus and E. camaldulensis, respectively. Gall size of the two wasp species was estimated (area considered as an ellipse, A= 휋푋푌), by measuring for each wasp species the semi-major radius (X) and semi-minor radius (Y) of the gall in 30 fully developed galls. Gall morphology was also described for each species. All measurements were carried out using digital eyepiece for microscope – DEM130 and image analysis FIJI software ImageJ (Schindelin et al. 2012).

Phenology Samples of E. globulus leaves were collected monthly, between May 2013 and April 2014, at Tapada da Ajuda, Lisbon, to study the phenology of Ophelimus sp. Each sample consisted of leaves with galls, selected from branches at about 1.5m height. Thirty galls were selected per sample from the collected leaves and dissected under a stereo microscope (Optika SZM-LED1), using a scalp blade. The developmental stage of the wasp within each gall (1st, 2nd or 3rd instar larvae, and pupae) was registered, based on Protasov et al. (2007a).

Closteroceus chamaeleon Longevity The effect of temperature in the longevity of C. chamaeleon was studied, in order to determine the best temperature conditions to maintain the adults of the parasitoid long enough up to the setup of the parasitism experiments with Ophelimus sp., as the parasitoid was not naturally available in the field during the developing period of the gall wasp. The longevity was determined under laboratory conditions, at three constant temperatures (5°C±1ºC, 15°C±1ºC and 25°C±1ºC), 60% relative humidity, and 12L:12D photoperiod. Two diet regimes, i.e., no food (control), and honey + water (50/50), were considered for each temperature treatment, with three replicates. Three rearing chambers (ARALAB – Fitoclima S600 PL/PLH) were used, one per temperature treatment. Feeding solutions were offered on a small cotton ball and renovated every 2 or 3 days. Recently emerged adult wasps (up to 2 days-old) were placed inside clear plastic boxes (9 x 9 x 7 cm), with a respiration hole on the lid (covered with a net with a mesh lesser than 1 mm). The number of wasps used per box varied between 16 and 42, depending on the

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Parasitism laboratory tests The hability of C. chamaeleon to parasitize Ophelimus sp. was studied in no-choice and choice experiments. In no-choice tests, one leaf section (1cm2) of eucalypt, containing between 10 to 20 galls of Ophelimus sp. or O. maskelli, was placed in a gelatin capsule (24mm length and 7mm diameter). Then one single naïve female of C. chamaeleon was introduced within the gelatin capsule and allowed to oviposit. Two types of females were tested: 1) young females, up to 15 days-old; and 2) old females, 86-89 days-old. Galls of Ophelimus sp. were collected from E. cinerea (n=12), E. cypellocarpa (n=23) and E. globulus (n=16), whereas those of O. maskelli originated from E. camaldulensis (n=17). During each trial, the parasitoid behavior was observed, under a stereo microscope, for 30 minutes. Parasitism occurrence was registered whenever the wasp ovipositor was inserted in the gall for more that one minute (based on Rizzo et al., 2015). At the end of each trial, five randomly selected galls were dissected to assess the larval stage of the corresponding Ophelimus species as described in 2.2.2. All C. chamaeleon used in the trials were females and were previously fed with honey and water (50/50) solution. In the choice test, each C. chamaeleon female (16 to 21 days-old) was exposed, in a gelatin capsule, to two eucalypt leaf sections (≈ 0.5cm2 each), each holding 5 to 10 galls and allowed to choose between E. globulus leaf section with Ophelimus sp. galls and E. camaldulensis leaf section containing O. maskelli galls. Leaf placement within the gelatin capsule was randomized in each replication to avoid a positioning effect. We used 16 replicates in the test. Parasitoid behaviour was registered throughout an observation period of 30 minutes. At the end of each replicate, five random selected galls of both Eucalyptus sections were open and the development stage of the gall wasps was assessed. Closterocerus chamaeleon females were fed, with honey and water solution, prior to the trials.

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Parasitism field tests The ability of C. chamaeleon to parasitize and complete development in Ophelimus sp. was also evaluated in field conditions, between December 2015 and January 2016. Leaves of E. cypellocarpa and E. globulus naturally infested with Ophelimus sp. were exposed to the parasitoid within insect net bags (12x18cm, mesh size < 1mm). Each selected galled leaf was previously cleaned with a brush to remove other insects and debris, before being exposed to the parasitoid. The parasitoids used in the study were obtained from field samples collected between late October and early November 2015 (see 2.1). In average, we released about 10 adults of C. chamaeleon per bag: 149 in Ophelimus sp. - E. cypellocarpa bags (n=15), and 151 in Ophelimus sp. - E. globulus bags (n=15). Five other infested leaves (for each eucalypts species) were also bagged without insects and used as control. In March 2016, all bags were brought to the laboratory for individual inspection under a stereoscope microscope. The number of closed galls and those with emergence holes were counted per bag, as well as the number of live and dead individuals of Ophelimus sp. and C. chameleon. Each leaf was then kept within a Petri dish (9 cm diameter), at laboratory conditions, for ca. 1 month, to allow insect emergence and identification. We also collected in the same period leaves of E. cypellocarpa and E. globulus naturally infested with Ophelimus sp. (n=20) and stored them within clear plastic boxes (30x20x10 cm) at room temperature, and determined the number of emerged insects ca. 1 month later, as a reference. Parasitism rate was determined on the number of C. chamaeleon and Ophelimus emerged. This was calculated by dividing the number of C. chamaeleon, by the sum of all emerged insects per leaf.

Data analysis The influence of different temperatures regimes (5ºC; 15ºC and 25ºC) and different feeding modalities on the survival ability of C. chamaeleon was evaluated through survival analysis and Kaplan-Meier test. The Log Rank (Mantel-Cox) test was used to compare parasitoid survival between the feeding modalities and between the temperature regimes. Behavior analysis of C. chamaeleon on no-choice tests was analyzed using Generalized linear models (GLM), binary logistic, where the dependent variable was the occurrence of a parasitism event on each tested eucalypt species. Data is presented as the probability of the parasitoid to accept each Ophelimus species. Levels comparison was made by pairwise comparison and the results presented by Wald Chi2 and Omnibus statistical test. For the analysis of parasitoid behavior in choice and no-choice tests and the parasitoid age

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Chapter 4 influence on the parasitism, we conducted a Chi-square test for each trial. All statistical analyzes were performed using the software IBM SPSS® v.25.

Results Ophelimus sp. Gall morphology Galls of O. maskelli on E. camaldulensis, and of Ophelimus sp. on E. globulus may be distinguished by color, texture and gall expansion. Ophelimus sp. galls are asymmetric in that they expand mainly on one side of the leaf blade, while O. maskelli galls are symmetric, expanding evenly on both sides of the leaf blade. Texture and color of the galls differ between both species and this feature may be useful to help discriminating between them in the field (Figure 4.1). Ophelimus maskelli galls are smooth to touch and can have a green or a green-reddish coloration, whereas galls of Ophelimus sp. turn brown, becoming brown/greyish and rough to touch. Significant differences were observed in gall size between the two Ophelimus species (t-test=12.197; df =41.683; p<0.001; Table 4.1). The galls of Ophelimus sp. have approximately half of the area of those of O. maskelli.

Table 4.1. – Biometric data and gall size (mean±SE) obtained for Ophelimus maskelli and Ophelimus sp. Mean values with different letters are significant different within each column (p <0.05). Nº Gall area Host tree species Gall wasp species specimens (mm2)

Eucalyptus camaldulensis Ophelimus maskelli 30 1.04±0.04a

Eucalyptus globulus Ophelimus sp. 30 0.53±0.02b

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A B

Figure 4.1. – A: Leaf portion of E. camaldulensis with an open gall and several other galls of O. maskelli; and B: Leaf portion of E. globulus with Ophelimus sp. galls. Scale bars correspond to 1 000µm.

Phenology During our survey (May 2013 to April 2014), leaves with developing galls of Ophelimus sp. were observed only between November and March (Figure 4.2). Between May and October 2013, only old galls (developed on the previous year and already open or dried) were observed. The presence of 1st instar larvae was detected between November and January; 2nd instar larvae, between December and March; and 3rd instar larvae, between February and March. Pupae of Ophelimus sp. were observed in March (Figure 4.2).

100% 80% 60% 40% 20% 0% 06.11.2013 12.12.2013 15.01.2014 07.02.2014 10.03.2014 Observation dates Larvae 1ºinstar Larvae 2ºinstar Larvae 3ºinstar Pupae Figure 4.2. – Relative abundance (%) of Ophelimus sp. developmental stages observed on dissected galls from Eucalyptus globulus, collected monthly (n=30) between November 2013 and March 2014.

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Closterocerus chamaeleon Longevity Lifespan of unfed C. chamaeleon wasps was inversely related with temperature (Table 4.2; Figure 4.3). At 25ºC, all adult parasitoids on the control group died in just four days, whereas at 5ºC the wasps on the control survived up to 57 days. Still, 50% mortality was observed on the first day on this last condition. However, when fed with honey and water, mean longevity of C. chamaeleon was maximal at 15ºC, and minimal at 5ºC (Table 4.2; Figure 4.3). At 15ºC, approximately half of the adult parasitoids survived up to 15 days, and the maximal longevity was 127 days, whereas at 5ºC and 25ºC, half of the studied individuals died within the first four days. Fed adult wasps of C. chamaeleon showed a significant increase on life expectancy, for both 15ºC (χ2=43.706; p<0.001) and 25ºC (χ2=57.431; p<0.001), in comparison with control. No significant difference was observed between the two food regimes at 5ºC (χ2=0.513; p= 0.474).

Table 4.2 – Estimate mean life expectancy (±SE), using Kaplan-Meier survival test, for Closterocerus chamaeleon at different temperatures, with and without food supply. Log-rank test, for survival rate at different temperatures for the same feeding treatment. Mean life span (days) Pair-wise Treatment χ 2 p 5ºC 15ºC 25ºC comparison 5ºC vs 15ºC 4.153 0.042

Control 5ºC vs 25ºC 32.697 <0.001 5.1±0.8 3.8±0.2 1.9±0.2 (no food) 15ºC vs 25ºC 58.213 <0.001

5ºC vs 15ºC 43.630 <0.001

Honey and 5.7±0.8 31.9±3.9 9.5±1.2 5ºC vs 25ºC 7.647 0.006 water 15ºC vs 25ºC 26.295 <0.001

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1 0.9 A 0.8 0.7 0.6 0.5 Control at 5ºC 0.4 Honey and Water at 5ºC 0.3 0.2 0.1 0 1 0.9 B 0.8 0.7 0.6 0.5

0.4 Survival 0.3 0.2 0.1 0

1 0.9 0.8 C 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 120 140 Days

Figure 4.3 – Survival curves for C. chamaeleon, with and without honey as food, for different temperature regimes: A – for C. chamaeleon at 5ºC; B – at 15ºC; and C – at 25ºC.

Parasitism laboratory tests In no-choice tests, the females of C. chamaeleon parasitized both Ophelimus species, and the level of parasitism significantly differed among gall wasps and host plants (Wald Chi2=36.838, df=3, p<0.001; Table 4.3). By performing pairwise comparison we could observe that the level of parasitism was higher, when the parasitoid was exposed to the galls of O. maskelli on leaves of E. camadulensis, but did not significantly differ from that registered in Ophelimus sp. on E. globulus (p=0.163). No significant difference was

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Table 4.3. – Probability of Parasitism (± SE) estimated by GLM models with Binomial distribution of C. chamaeleon when exposed to Ophelimus maskelli and Ophelimus sp. on different eucalypt species, in no-choice tests. Probability of Host plant Gall wasp N parasitism

E. camaldulensis O. maskelli 17 0.88±0.078a

E. cinerea Ophelimus sp. 12 0.33±0.136b

E. cypellocarpa Ophelimus sp. 23 0.22±0.086b

E. globulus Ophelimus sp. 16 0.69±0.116a

In choice tests, three out of 16 C. chamaeleon females parasitized both Ophelimus sp. and O. maskelli, six females parasitized only Ophelimus sp., and one female parasitized only O. maskelli. The remaining six females of the parasitoid parasitized none of the two Ophelimus species. The number of parasitism attempts was higher on Ophelimus sp. (15), compared to O. maskelli (7). Nevertheless, the frequency distribution was not significantly different from that expected if the parasitoid had no preferences (χ2=2.909; df=1; p=0.088). Dissection of the tested galls showed that the main larval stages were 2nd instar on Ophelimus sp. and both 1st larval and 2nd instar larvae on O. maskelli.

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Parasitism field tests A total of 2 237 adults of the gall wasp emerged from 4 220 galls, as well as 23 adults of C. chamaeleon (descendants from parasitoids released in December), in the leaves of E. cypellocarpa and E. globulus infested with Ophelimus sp., in which we had released C. chamaeleon. From the control E. cypellocarpa and E. globulus leaves, we recorded the emergence of 1 893 adults of Ophelimus sp., from 3 209 galls. No specimens of C. chamaeleon or other parasitoid were collected in this case.

Discussion Ophelimus sp. Our data confirmed that Ophelimus sp. is clearly different from O. maskelli in terms of gall morphology and host range, as well as phenology. The phenological data indicate that Ophelimus sp. has an univoltine cycle (Table 4.4): the period of adult emergence was limited to March-April, the new galls were observed only in October-November and larval development was registered from November (1st instar) till February (3rd instar). These results suggest that oviposition in early spring is possibly followed by an egg diapause during spring and summer. Therefore, we hypothesise that the galls are induced after diapause termination, in connection with egg eclosion and the begin of larval development, as gall induction by insects is generally associated with stimuli produced by first larval instar when feeding (Fernandes et al., 2012). Further studies are nevertheless needed to confirm this hypothesis. The univoltine cycle of Ophelimus sp. is in contrast with that of O. maskelli, which completes 3-4 generations per year (Protasov et al., 2007a).

Closterocerus chamaeleon Adult feeding on honey and water increased the longevity of C. chamaeleon, for the studied temperatures, except for 5ºC, as observed for other parasitoids species (e.g. Jacob and Evans, 2000). Wasp longevity of was maximal at 15ºC. In these conditions, adult wasps of C. chamaeleon were kept alive up to 4 months. The fact that a few individuals survived up to two weeks at 5ºC indicates that the parasitoid may survive several days in the field, during the winter. Interestingly at the age of 86-89 days-old, the females kept at 15ºC were still able of host searching and parasitizing, and showed no differences on the parasitism performance, in comparison with young females. Here we showed for the first time, based both on laboratory and field experiments, that C. chamaeleon is able to parasitize and complete development in Ophelimus sp. Apparently, the parasitoid was able to discriminate among host plants, as the parasitism

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Chapter 4 rate of Ophelimus sp. in no-choice tests varied among eucalypt species, suggesting that C. chamaeleon uses host plant chemicals as host selection cues to locate and/or accept the gall wasp as a host (Vinson, 1998). We may hypothesize that some host plant traits, such as wax on leaf surface, or leaf volatile compounds could reduce the acceptance of Ophelimus sp. by C. chamaeleon when developing in E. cinerea and E. cypellocarpa. For example, it is known that the profile of volatile leaf oils can be used to distinguish many eucalypt species (Li et al., 1995, 1996; Santadino et al., 2017). However, the observed differences on parasitism rate could be also associated with differences in the host developmental stage among the studied eucalypt species, as parasitoids usually show different levels of host acceptance depending on the host stage (Badshah et al., 2016; Jervis and Kidd, 1996; Yazdani et al., 2015). In fact, the dominant larval developmental stage of Ophelimus sp. in the experiments was 1st and 3rd instar, respectively in E. cinerea and E. cypellocarpa, but 2nd instar in E. globulus. Surprisingly, C. chamaeleon showed no preference between the galls of O. maskelli on E. camaldulensis and those of Ophelimus sp. on E. globulus, in choice tests. There were also no significant differences in parasitism rate for the same two combinations “gall wasp species-host plant” in no-choice tests. Our results suggest that, at least, when exposed directly to Ophelimus sp. galls, the parasitoid C. chamaeleon seems to accept it as a host, and parasitize it in a similar way to O. maskelli. However, in field conditions the parasitoid should be able to locate the host habitat, which in the case of Ophelimus sp. is different from that of O. maskelli, as these two gall wasps have different host plants. Based on our experiments, we cannot answer this question. Further studies, including wind tunnel and olfactometer essays, are needed to clarify this issue. Although we managed to show that C. chamaeleon was able to parasitize and complete development in Ophelimus sp., under field conditions, the number of F1 descendants obtained were modest. These numbers may result from methodological difficulties, such as wasps escaping from the bags, leaf drying up within the bags, as well as maintaining alive until winter (up to 3-4 months) the necessary adult females of C. chamaeleon, to expose them to Ophelimus sp.. In this last case, the fecundity of the parasitoid females was possibly affected by the artificial increment of their longevity (Jervis and Kidd, 1996). However, climatic conditions in winter could be also inappropriate for the reproductive and development performance of the parasitoid. Altogether, our results support the hypothesis according to which the lack of parasitism in Ophelimus sp. is explained based on a seasonal asynchrony between C. chamaeleon and

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Chapter 4 the gall-wasp phenology. In fact, if a parasitoid and its potential host are not active at the same time or season, they may occur in the same geographical area but not interacting. Parasitism escape, totally or partially, by phenological asynchrony, has been reported on several host-parasitoid systems (e.g Godfray et al., 1994; Van Nouhuys and Lei, 2004). Climate (temperature in particular) affects the development of both C. chamaeleon and Ophelimus sp. and we assume that climate may influence the outcome of this host- parasitoid asynchrony, since temperature varies among years. Warmer autumns and mild winters may naturally increase the synchrony between the phenology of both species, being possible that some natural parasitism may occur in very favorable years. Climate warming may thus create a window of opportunity for C. chamaeleon to naturally parasitize Ophelimus sp. in the studied area, as suggested in other insect models (e.g. Singer and Parmesan, 2010). In fact, in 2015, we observed unusual emergences of the parasitoid in November and December (Garcia et al. unpublished data), whereas normally we cannot find C. chamaeleon adults in late fall. The end of autumn 2015 was particularly hot in Portugal, with monthly mean air temperatures of approximately 19ºC in October and 16ºC in November, about 0.5ºC warmer than the homologous period in 2014. We assume that such exceptionally high temperatures in fall allowed O. maskelli and the parasitoid to develop an additional autumnal generation.

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Acknowledgements The authors wish to thank to Joana Martins, Pedro Nunes and Vera Zina for the help in the laboratory. The comments and suggestions of two anonymous reviewers, which helped us improving an early version of the manuscript, are also acknowledge. This work was funded by Fundação para a Ciência e Tecnologia (FCT) through the: Doctoral Programme SUSFOR - Sustainable Forests and Products PD/00157/2012, PD/BD/52693/2014; and Project UID/AGR/00239/2013, and through the Capes Foundation Proc. nº. BEX 0285/15- 3.

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References Aquino, D.A., Hernández, C.M., Cuello, E.M., Andorno, A.V., Botto, E.N., 2014. Primera cita de la Argentina de Ophelimus maskelli (Ashmead) (Hymenoptera: Eulophidae) y su parasitoide, Closterocerus chamaeleon (Girault) (Hymenoptera: Eulophidae). Rev. Soc. Entomol. Arg. 73, 179-182. Aquino, D.A., Hernández, C.M., Cuello, E.M., Andorno, A.V., Botto, E.N., 2015. Errata: Primera cita de la Argentina de Ophelimus maskelli (Ashmead) (Hymenoptera: Eulophidae) y su parasitoide, Closterocerus chamaeleon (Girault) (Hymenoptera: Eulophidae). Rev. Soc. Entomol. Arg.74, 97. Badshah, H., Ullah, F., Calatayud, P.A., Crickmore, N., 2016. Host stage preference and parasitism behaviour of Aenasius bambawaleian encyrtid parasitoid of Phenacoccus solenopsis. Biocontrol Sci. Techn. 26, 1605-1616. Blanche, K.R., 2000. Diversity of insect-induced galls along a temperature–rainfall gradient in the tropical savannah region of the Northern Territory, Australia. Austral. Ecol. 25, 311-318. Borowiec N, La Salle J, Brancaccio L, Thaon M, Warot S, Branco M, Ris N, Malausa JC, Burks RA (2019). Ophelimus mediterraneus sp. n. (Hymenoptera, Eulophidae): a new Eucalyptus gall wasp in the Mediterranean Region. Bull. Entomo. Res. 109(5), 678-694. Borowiec, N., Brancaccio, L., Thaon, M., Warot, S., Branco, M., La Salle, J., Ris, N., Malausa, J.-C., 2013. Gall waps in France and neighbouring countries: a new Ophelimus (Hymenoptera, Eulophidae) treatens Eucalyptus plantations. 6th International Symposium on the Biology and Ecology of Gall Inducing Arthtopods and related Endophytes, Lamington National Park, Queensland, Australia. Borowiec, N., Thaon, M., Brancaccio, L., Warot, S., Ris, N., Malausa, J.-C., 2012. L'eucalyptus menacé par une nouvelle espèce d'Ophelimus en France. Phytoma, France, pp. 42-44. Borrajo, P., López, M.A., Ocete, R., López, G., Ruiz, F., 2008. Primera cita de Closterocerus chamaeleon Girault (Hymenoptera, Eulophidae), parasitoide de Ophelimus maskelli Ashmead (Hymenoptera, Eulophidae) en la provincia de Huelva (SO España). Bol. San. Veg. Plagas 34, 383-385. Branco, M., Boavida, C., Durand, N., Franco, J.C., Mendel, Z., 2009. Presence of the Eucalyptus gall wasp Ophelimus maskelli and its parasitoid Closterocerus chamaeleon in Portugal: First record, geographic distribution and host preference. Phytoparasitica 37, 51-54.

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Branco, M., Dhahri, S., Santos, M., Ben Jamaa, M.L., 2014. Biological control reduces herbivore’s host range. Biological Control 69, 59-64. Burks, R.A., Mottern, J.L., Waterworth, R., Paine, T.D., 2015. First report of the Eucalyptus gall wasp, Ophelimus maskelli (Hymenoptera: Eulophidae), an invasive pest on Eucalyptus, from the Western Hemisphere. Zootaxa 3926, 448-450. Caleca, V., 2010. First record in Algeria of two Eulophid wasps: Closterocerus chamaeleon (Girault) and its host, the eucalyptus gall wasp Ophelimus Maskelli (Ashmead) (Hymenoptera: Eulophidae). Naturalista sicil. 34, 201-206. Caleca, V., Rizzo, M.C., Lo Verde, G., Rizzo, R., Buccellato, V., Luciano, P., Cao, O., Palmeri, V., Grande, S.B., Campolo, O., 2009. Diffusione di Closterocerus chamaeleon (Girault) introdotto in Sicilia, Sardegna e Calabria per il controllo biologico di Ophelimus maskelli (Ashmead) (Hymenoptera, Eulophidae), galligeno esotico sugli eucalipti. Atti 3° Congr. Selvicoltura, 638-642. Caleca, V., Verde, G.L., Rizzo, M.C., Rizzo, R., 2011. Dispersal rate and parasitism by Closterocerus chamaeleon (Girault) after its release in Sicily to control Ophelimus maskelli (Ashmead) (Hymenoptera, Eulophidae). Biological Control 57, 66-73. Ciesla, W.M., 2011. Forest Entomology - A Global Perspective. Wiley-Blackwell. Dhahri, S., Ben Jamaa, M.L., Lo Verde, G., 2010. First record of Leptocybe invasa and Ophelimus maskelli Eucalyptus gall wasps in Tunisia. Tunisian Journal of Plant Protection 5, 229-234. Doganlar, M., Mendel, Z., 2007. First record of the Eucalyptus gall wasp Ophelimus maskelli and its parasitoid, Closterocerus chamaeleon, in Turkey. Phytoparasitica 35, 333-335. Fernandes, G.W., Carneiro, M.A., Isaias, R.M., 2012. Gall-inducing insects: from anatomy to biodiversity. Insect bioecology and nutrition for integrated pest management. In: Panizzi, A., Parra, J., (Eds.), Insect Bioecology and Nutrition for Integrated Pest Management. CRC Press, Boca Raton, Florida, USA, pp. 369-395. Garcia, A., Matos, S., Borowiec, N., Branco, M., 2014. A new gall wasp Ophelimus sp. affecting Eucalyptus globulus in Southwestern Europe. Xth European Congress of Entomology, York, United Kingdom. Godfray, H.C.J., Hassell, M.P., Holt, R.D., 1994. The population dynamic consequences of phenological asynchrony between parasitoids and their hosts. J. Anim. Ecol. 63, 1-10. Hill, D.S., 2004. EucaLink - A Web Guide to the Eucalypts.

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Jacob, H.S., Evans, E.W., 2000. Influence of carbohydrate foods and mating on longevity of the parasitoid Bathyplectes curculionis (Hymenoptera: Ichneumonidae). Environ. Entomol. 29, 1088-1095. Jervis, M., Kidd, N., 1996. Insect Natural Enemies. Springer, Netherlands. Li, H., Madden, J.L., Potts, B.M., 1995. Variation in volatile leaf oils of the Tasmanian Eucalyptus species—1. Subgenus Monocalyptus. Biochem. Syst. Ecol. 23, 299- 318. Li, H., Madden, J.L., Potts, B.M., 1996. Variation in volatile leaf oils of the Tasmanian Eucalyptus species II. Subgenus Symphyomyrtus. Biochem. Syst. Ecol. 24, 547- 569. Lo Verde, G., Dhahri, S., Jamaa, M.L.B., 2010. First record in Tunisia of Closterocerus chamaeleon (Girault) parasitoid of the eucalyptus gall wasp Ophelimus maskelli (Ashmead) (Hymenoptera: Eulophidae). Naturalista sicil. 34, 207-210. Mansfield, S., 2016. New communities on eucalypts grown outside Australia. Front Plant. Sci. 7, 1812. Mendel, Z., Protasov, A., Blumberg, D., Brand, D., Saphir, N., Madar, Z., La Salle, J., 2007. Release and recovery of parasitoids of the Eucalyptus gall wasp Ophelimus maskelli in Israel. Phytoparasitica 35, 330-332. Mendel, Z., Protasov, A., La Salle, J., Blumberg, D., Brand, D., Branco, M., 2017. Classical biological control of two Eucalyptus gall wasps; main outcome and conclusions. Biological Control 105, 66-78. Noyes, J.S., 2018. Universal Chalcidoidea Database. http://www.nhm.ac.uk/chalcidoids. (Accessed 20-03-2018). Protasov, A., La Salle, J., Blumberg, D., Brand, D., Saphir, N., Assael, F., Fisher, N., Mendel, Z., 2007a. Biology, revised taxonomy and impact on host plants of Ophelimus maskelli, an invasive gall inducer on Eucalyptus spp. in the Mediterranean area. Phytoparasitica 35, 50-76. Protasov, A., Blumberg, D., Brand, D., La Salle, J., Mendel, Z., 2007b. Biological control of the eucalyptus gall wasp Ophelimus maskelli (Ashmead): taxonomy and biology of the parasitoid species Closterocerus chamaeleon (Girault), with information on its establishment in Israel. Biological Control 42, 196-206. Raman, A., Withers, T.M., 2003. Oviposition by introduced Ophelimus eucalypti (Hymenoptera: Eulophidae) and morphogenesis of female-induced galls on Eucalyptus saligna (Myrtaceae) in New Zealand. Bull. Entomol. Res. 93, 55-63.

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Rizzo, M.C., Lo Verde, G., Rizzo, R., Buccellato, V., Caleca, V., 2006. Introduzione di Closterocerus sp. in Sicilia per il controllo biologico di Ophelimus maskelli Ashmead (Hymenoptera Eulophidae) galligeno esotico sugli eucalipti. Boll. Zool. agr. Bachic. 38, 237-248. Rizzo, M.C., Lo Verde, G., Rizzo, R., Caleca, V., 2015. Risk assessment of non-target effects of Closterocerus chamaeleon (Girault) parasitoid of the eucalypt gall maker Ophelimus maskelli (Ashmead) (Hymenoptera, Eulophidae). Phytoparasitica 43, 407-415. Santadino, M., Lucia, A., Duhour, A., Riquelme, M., Naspi, C., Masuh, H., Liljesthröm, G., Coviella, C., 2017. Feeding preference of Thaumastocoris peregrinus on several Eucalyptus species and the relationship with the profile of terpenes in their essential oils. Phytoparasitica, 395-406. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682. Singer, M.C., Parmesan, C., 2010. Phenological asynchrony between herbivorous insects and their hosts: signal of climate change or pre-existing adaptive strategy? Philos. T. R. SOC. B. 365, 3161-3176. Van Nouhuys, S., Lei, G., 2004. Parasitoid–host metapopulation dynamics: the causes and consequences of phenological asynchrony. J. Anim. Ecol. 73, 526-535. Vinson, S.B., 1998. The general host selection behavior of parasitoid hymenoptera and a comparison of initial strategies utilized by larvaphagous and oophagous species. Biological Control 11, 79-96. Withers, T.M., 2001. Colonization of eucalypts in New Zealand by Australian insects. Austral Ecol. 26, 467-476. Yazdani, M., Feng, Y., Glatz, R., Keller, M.A., 2015. Host stage preference of Dolichogenidea tasmanica (Cameron, 1912) (Hymenoptera: Braconidae), a parasitoid of Epiphyas postvittana (Walker, 1863) (Lepidoptera: ). Austral Entomology 54, 325-331.

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Novel prey boosts the expansion of host-plant range in a native predatory bug.

Published in BioControl

Garcia, A., Franco, J.C., Branco, M., 2019. Novel prey boosts the expansion of host-plant range in a native predatory bug. BioControl 64: 677-683. https://doi.org/10.1007/s10526-019-09965-x

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Abstract Anthocoris nemoralis (Fabricius) is a Palearctic predator associated with psyllids in their native region. We found it preying on the non-native psyllid Glycaspis brimblecombei Moore, on Eucalyptus camaldulensis Dehnh. Here, we demonstrate the predator acceptance of E. camaldulensis leaves as asubstrate for oviposition. We further tested the ability of A. nemoralis to complete its life cycle on E.camaldulensis trees, while feeding on G. brimblecombei. For this purpose, we enclosed infested branches in bags with adult predators, during one-month trials. At the end of the trials we found eggs, nymphs and newly born adults of A. nemoralis, confirming that a complete life cycle had occurred. The psyllid population growth rate was lower in the bags with predators than in the control. These results point out: (1) the ability of A. nemoralis to feed and complete a life cycle on exotic novel prey; (2) the acceptance of an exotic host plant; and (3) A. nemoralis as a potential candidate for the biological control against invasive psyllid species.

Keywords: Anthocoris nemoralis; Glycaspis brimblecombei; Eucalyptus camaldulensis; Conservation biological control

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Introduction Predatory bugs (Hemiptera: Heteroptera) are a diverse group of insects, foraging in many different habitats, including aquatic and terrestrial environments. Minute pirate bugs (Anthocoridae) are mostly small predators (1.5–5 mm length) which primarily prey upon small soft-body arthropods, including aphids, psyllids,thrips, scale insects, psocids, mites, and eggs or small larvae of Lepidoptera, Coleoptera, and Diptera (Horton 2008; Jerinic- Prodanovic and Protic 2013). Anthocorids include species with variable habitat and prey selectivity. Whereas some are highly eurytopic, foraging on many different host plants, such as Anthocoris nemoralis (Fabricius), other species show high specialization, as for example Elatophilus spp., which feed on Matsucoccus scale insects in pine trees (Mendel et al. 1995; Horton 2008). The substrates selected by anthocorid females for oviposition are variable, depending on the taxa and the length of their ovipositor. Anthocorinae females have developed ovipositors and insert their eggs, often isolated, into plant tissues. The eggs are generally oviposited in stems, petioles, leaf veins (e.g., Anthocoris), conifer needles (e.g., Elatophilus), pedicels, and floral organs (e.g., Orius) (Péricart1972; Sigsgaard 2005; Horton 2008; Pascua et al. 2018). Native of Europe, A. nemoralis is one of the most common anthocorid species used as a biocontrol agent in augmentative releases (Horton et al. 2004; Sigsgaard 2010; Jerinic-Prodanovic and Protic 2013). It was also introduced in North America for the biological control of pear psylla, Cacopsylla pyricola (Förster) (Horton et al. 2004). Anthocoris nemoralis is a eurytopic species, foraging on many different host plants, including species from about 39 genera and 28 families (Horton et al. 2004). However, this predatory bug prefers to oviposit in certain host plants, and this is apparently the main reason why its populations are much more abundant in pear than in apple orchards, for example (Sigsgaard 2004). Also, it shows a certain degree of habitat specialization, as most of the reported host plants are trees or shrubs (Lundgren 2011). Although A. nemoralis is considered a generalist predator, which may feed on psyllids, aphids, thrips, eggs and larvae of Lepidoptera, and on mites, it is often associated with trees and shrubs that host psyllids (Péricart1972; Horton et al. 2004; Sigsgaard 2004; Emami et al. 2014). This apparent preference for psyllids is supported by laboratory experiments. As a predator that lays its eggs within plant tissues, the host plants of the prey have a more critical role in the prey-searching strategy of A. nemoralis and in its impact on prey populations, compared to other predatory insects with a different reproduction mode. Besides the use of host-plant semiochemical cues in prey

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Chapter 5 habitat location and prey location (Dicke et al. 1990), host plant characteristics may also affect the development and survival of the predator eggs. The red gum lerp psyllid, Glycaspis brimblecombei Moore (Hemiptera: ), is an insect pest of Eucalyptus spp., native of Australia, which recently invaded Europe (Valente and Hodkinson 2009). Intense attacks of this psyllid may lead to tree defoliation (Brennan et al. 1999). One of the main host species is Eucalyptus camaldulensis Dehnh., which is widely used as an amenity tree in urban parks and road sides, as well as in forests for soil and water conservation in the Mediterranean area (Peris-Felipo et al. 2010). The parasitoid Psyllaephagus bliteus Riek was introduced in California in 2000 for the control of the red gum lerp psyllid (Daane et al. 2012), and was established in Europe as a fortuitous biological control agent (Caleca et al. 2011). However, the biological control provided by P. bliteus is not effective enough to prevent the outbreaks of G. brimblecombei during summer (Daane et al. 2012; Boavida et al. 2016; Margiotta et al. 2017). In this context, a potential role of native predators exerting some kind of control upon G. brimblecombei during outbreaks would be relevant to investigate. In previous field observations, we found A. nemoralis frequently preying on G. brimblecombei infesting E. camaldulensis. Therefore, we hypothesized that A. nemoralis could play a significant role in the biological control of G. brimblecombei. Although G. brimblecombei was reported before as a prey of A.nemoralis in California (USA) (Brennan et al. 1999; Horton et al. 2004), no information is available on the ability of the predator to reproduce on Eucalyptus, nor on their possible impact as a biocontrol agent against the invasive psyllid. In the present work, we aimed at: (1) analyzing the use of Eucalyptus as habitat for oviposition by A. nemoralis in field conditions; (2) quantifying the egg laying activity of A. nemoralis on Eucalyptus leaves and testing their ability to complete a life cycle on this host plant; and (3) assessing the impact of A. nemoralis on G. brimblecombei populations.

Materials and methods Anthocoris nemoralis oviposition on Eucalyptus Visual surveys were carried out in Tapada da Ajuda, Lisbon, between 2017 and 2018, on five randomly selected trees of E. camaldulensis infested with G.brimblecombei. From each selected eucalypt tree, we randomly collected 40 leaves around the tree crown, to evaluate the presence of A. nemoralis (eggs, nymphs or adults). Samplings were done once a month, from April to July in each year. The laboratory observation of the leaves

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Chapter 5 was conducted under a stereoscopic microscope (Optika SZM-LED1, 30 ×). The presence or absence of the predator (eggs, nymphs or adults) was registered. The eggs of A. nemoralis were described. Some Anthocoridae nymphs were fed with G. brimblecombei on fresh leaves of E. camaldulensis until adulthood. All adults were subsequently identified as A. nemoralis using the key provided by Péricart (1972).

Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves In order to test the ability of A. nemoralis to reproduce and complete their development on Eucalyptus leaves while feeding on G. brimblecombei, we conducted a field exclusion trial. We selected 30 branches of E. camaldulensis trees infested with G. brimblecombei, measuring ca. 40 cm long. Each branch was isolated within a polyester sleeve (50 cm length × 15 cm width) with a mesh size under 1 mm. The number of G. brimblecombei eggs and nymphs of first to third instars per branch was counted using a head mount magnifier glass (4.8×). Nymphs of fourth and fifth instars, as well as all other insects present on the leaves, were removed using a brush. The trials were conducted between July and August 2014 and 2015, and lasted 25 and 39 days, respectively. During the trial period, air temperature ranged between 16 and 26.5°C in 2014, and between 18 and 29°C in 2015. No precipitation was registered in this period in either year. Three treatments, with ten replicates per treatment, were considered and randomly allocated to the selected branches as follows: (1) isolated branches within sleeves in which three adult females and one male of A. nemoralis were introduced (WA); (2) isolated branches within sleeves, without A. nemoralis (WO); and (3) exposed branches, without A. nemoralis (WS). Two replicates of WA were excluded in 2015 due to predator escape. The individuals of A. nemoralis used in the trials were obtained from Agrobío—Control biológico de plagas y polinización natural (Almeria, Spain). The age and reproductive stage of the females was unknown. At the end of each trial, all branches were cut and transported to the laboratory for thorough inspection. All leaves were observed under the stereoscopic microscope (Optika SZM-LED1, 30 ×). The number of individuals of both psyllids (eggs and nymphs) and A. nemoralis (eggs, nymphs and adults) was counted. Differences in the psyllid growth rate between treatments and years were tested using generalized linearmodels (GLM), with normal distribution and identity link function. So as to account for the difference in the duration of the experimental periods between years, the psyllid growth rate was standardized to a monthly (30 days) growth rate, Rm, as follows: 푅푚 =

푃 30 ln( 푡+1)× 푒 푃푡 푡 where Pt+1 is the psyllid final population, Pt is the initial population, and t is

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Chapter 5 the number of days of the trial. Anthocoris nemoralis oviposition differences between the two years and treatments were tested using a GLM, with negative binomial distribution and log link function. Differences in initial psyllid population were analyzed using the same modelling analysis. In all cases, we used a model with two factors (year and treatment) and year × treatment interaction. However, the year × treatment interaction was never significant. The statistical analysis was made using SPSS® v25 software (IBM® New York, USA).

Results Survey of Anthocoris nemoralis on Eucalyptus trees The A. nemoralis activity was recorded between May and July and between June and July in 2017 and 2018, respectively. Several specimens of A. nemoralis (both nymphs and adults) were observed feeding on G. brimblecombei nymphs. Eggs of A. nemoralis were observed on both young and fully developed leaves of E. camaldulensis. We further observed specimens of A. nemoralis trying to reach the psyllid by lifting up G. brimblecombei lerp. We observed that A. nemoralis is able to prey on both moving and non-moving individuals of G. brimblecombei. Apparently, A.nemoralis adults only prey on young psyllid nymphs from first to third instars. They were not observed preying on eggs of G. brimblecombei nor on nymphs of fourth and fifth instars.The observed A. nemoralis eggs were elongated and showed a yellowish coloration. Females insert the eggs in the leaf parenchyma. Insertion is slightly oblique, almost parallel to the leaf surface, leaving only the operculum exposed.

Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves At the beginning of the trials, the psyllid population was homogeneous, with no statistical differences in the initial population size between treatments pooled over two years (χ2= 0.039; df = 2; p = 0.981) or between years (χ2= 0.128; df = 1; p = 0.720). The average number of eggs and nymphs per branch ranged from 750 to 910. During the trials, the psyllid population declined in all treatments with monthly growth rates Rm < 1 (Table 5.1). However, we observed a significant treatment effect over the two years (χ2 = 14.896; df = 2; p = 0.001). The population decline was higher in the bags with the predator (WA) in comparison with the bags without the predator (WO) (RWA-RWO= - 0.0794, p = 0.039) or with those without sleeves (WS) (RWA-RWS = - 0.1482, p < 0.001). The growth rate ± SE was also higher in 2015 (Rm= 0.27 ± 0.02) in comparison with 2014 (Rm= 0.21

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± 0.02) (χ2 = 4.28; df = 1; p = 0.039). The interaction year × treatment was not significant (χ2 = 5.78; df = 2; p = 0.056).

Table 5.1 - Average initial population and growth rate of both Glycaspis brimblecombei and Anthocoris nemoralis. The data refer to the average (±SE) number of eggs, nymphs and adults found at the end of the trial as well as the

Glycaspis brimblecombei Anthocoris nemoralis

Psyllid Initial N. of Treatments* monthly Eggs per Nymphs Adults per number per branches growth rate branch per branch branch branch with eggs (Rm) WA 816 ± 35.6a 0.158 ± 0.023a 40.9 ± 9.1a 4.4 ± 0.9 7.1 ± 1.7 20

WO 809 ± 37.2a 0.245 ± 0.029b 0.3 ± 0.3b 0 0 1 WS 859 ± 38.1a 0.314 ± 0.035b 0.5 ± 0.3b 0 0 4 number of sleeves with eggs. The data is pooled for both years of 2014 and 2015. *Treatments: WA – branches with sleeves with Anthocoris nemoralis (three females and one male); WO – branches with sleeves without Anthocoris nemoralis; WS – branches without sleeves and Anthocoris nemoralis (N=20 per treatment). In the same column, mean values with different letters are significantly different (p < 0.05).

In both years, A. nemoralis females laid eggs on E.camaldulensis leaves in the WA treatment, confirming their ability to use this new host plant as an oviposition substrate (Table 5.1). The number of oviposited eggs per sleeve in the WA treatment ranged from nine to 139 in 2014, and from one to 61 in 2015. The natural occurrence of A. nemoralis eggs was verified in both years on the exposed branches, WS treatment, but in low numbers (Table 5.1). In 2014, only one WS branch contained two eggs, whereas, in 2015, only three WS branches contained eggs of the predator, with one, one, and six eggs, respectively. In the closed sleeves with no predators (WO), no eggs were found in 2014, nor in nine of the ten sleeves in 2015. In 2015, six old hatched eggs of the anthocorid were found in a unique branch. The presence of A. nemoralis eggs in this WO treatment bag in 2015 was probably due to undetected hatched eggs which were already present on the eucalypt leaves during the preparation of the experiment. The rate of reproduction of A. nemoralis on the WA treatment was not significantly different between 2014 and 2015 (χ2 = 2.137; df = 1; p = 0.144). At the end of the experiments, nymphs and newborn adults were present on the WA sleeves, with up to 13 nymphs and 14 adults per sleeve (Table 5.1). In contrast, the WO and WS treatments had no nymphs or adults (Table 5.1). Altogether, these results show evidence that A.nemoralis was able to complete its life cycle and reach its adult stage in the WA sleeves. Our observations point out that most of the newborn nymphs of the predator might have escaped due to their tiny size, or they might have been subjected to cannibalism, since more hatched eggs than nymphs were found at the end of the trial.

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Discussion Survey of Anthocoris nemoralis on Eucalyptus trees Our data shows that A. nemoralis was able to respond to a novel psyllid prey, under field conditions, and more interestingly, accepting Eucalyptus species as host plants for oviposition. Although A. nemoralis was previously reported in association with G. brimblecombei, in USA and Europe, our study is the first to investigate the feeding behaviour of A. nemoralis on this psyllid species, as well as its ability to use Eucalyptus as host plants for reproduction. The association between A. nemoralis and G. brimblecombei in Portugal is relatively recent, as this alien psyllid was first detected in the country in 2007 (Valente and Hodkinson 2009). Still, our findings support the idea that A. nemoralis is able to expand its ecological range by adopting new prey and host plants which are alien in its native region (Horton et al. 2004). From the literature, A. nemoralis is majorly associated with psyllids, but it has been reported from many host plant species. Therefore, we may classify it as stenophagous at prey level and as eurytopic at hostplant level. These kinds of predator are expected to show an innate response to prey kairomones and to general cues from host plants, as well as a learned reaction to specific semiochemicals from the hostplant (Vet and Dicke1992). The available experimental data for A. nemoralis support the predictions of Vet and Dicke (1992). Sigsgaard (2005) observed that the predator’s oviposition is stimulated by C. pyri honeydew, a prey kairomone. Scutareanu et al. (1996) showed that A. nemoralis respond to (E,E)-alpha-farnesene and methyl-salicylate, plant volatiles induced by pear psylla feeding on pear trees. Based on olfactometer studies, Drukker et al. (2000) showed that the response of A. nemoralis to herbivore-induced plant volatiles (HIPV), such as methyl- salicylate, only occurred after experiencing HIPV with prey (rein-forcing stimulus), whereas naïve bugs did not respond. The authors argued that this influence of the reinforcing stimulus on the responses of A. nemoralis to HIPVsuggests associative learning. This probable capacity to learn to associate HIPV with the presence of prey may, at least in part, explain the ability of A. nemoralis to explore new prey species and novel host plants, as in the case of G. brimblecombei on Eucalyptus. For example, Eucalyptus species have been shown to produce HIPV in response to herbivory by different insect pests, such as the gall wasp Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) (Mohamed 2016), Thaumastocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae) (Martins and Zarbin 2013), and the psyllid Ctenarytaina

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Chapter 5 eucalypti (Maskell) (Hemiptera: Psyllidae) (Troncoso et al. 2012). Specific studies are needed to test the sensitivity of A. nemoralis to volatile emissions (HIPV) from Eucalyptus. The adult specimens used in our trials were naïve to the target prey. We may further hypothesize that adults from colonies reared on the target prey and host plant system might have higher ability to find, feed and reproduce on this specific prey. Further studies are needed to test this assumption. Also, more studies are required to compare the performance of feral individuals when feeding on native psyllids versus non-native prey species. On the other hand, A. nemoralis showed no interaction with two other alien psyllid species found on Eucalyptus, also present in the same studied area, namely C. eucalypti and Ctenarytaina spatulate Taylor (Nunes et al. pers. comm.). It would be interesting to investigate the reasons why A. nemoralis apparently ignores these two eucalypt psyllids as potential prey. Asynchronous predator–prey lifecycles might explain the non-association with the two Ctenarytaina species. Alternatively, we may consider that the new association between A. nemor-alis and G. brimblecombei results from the abundance of the prey. Still, during our field observations on eucalyptus infested by G. brimblecombei, we observed generally low numbers of A. nemoralis. This also explains the low abundance of predators and the poor predation efficiency in the WS treatment. It would be relevant to test whether conservation biological control or inundative release strategies could increase the abundance and efficacy of A. nemoralis to control G.brimblecombei.

Reproduction and development of Anthocoris nemoralis on Eucalyptus leaves On the Eucalyptus sleeved branches, A. nemoralis was able to complete all development stages until adult-hood feeding exclusively on G. brimblecombei. In this group of insects, food quality is crucial and obligatory through predation, while complementation with other food sources (e.g. pollens or nectars) provides little orno nutritive value (Anderson 1961). However, we donot exclude the hypothesis that A. nemoralis might have fed on eucalypt leaves to acquire some water. The variable number of A. nemoralis eggs found on WA, in both years, could be due to differences in the adults’ age or to other factors such as temperature. These factors should be further investigated in controlled laboratory trials. Although A. nemoralis has been reported in association with a large range of host plants, the use of these plant species as reproduction habitats is scarcely documented. If the host plant is not suitable for reproduction, we may have a functional response of the predator, but no numerical response. In such situations, no effective biological control will occur. The G. brimblecombei growth rate under predation by A. nemoralis (WA) was

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Chapter 5 lower than the observed growth rate in WO and WS treatments, evidencing the impact of the predator activity. Still, the growth rate was negative (Rm<1) in all treatments, reflecting apopulation decline regardless of predation activity. Actually, a decline of the psyllid population is normally observed in Mediterranean climate during summer and after spring outbreaks (Laudonia et al. 2014; Boavida et al. 2016). Native generalist predators may play an important role against non-native species (Carlsson et al. 2009). These predatory species may provide the first line of defense against alien pests. In the present work, we confirmed the ability of A. nemoralis to prey on G. brimblecombei nymphs, suggesting that it may be a good candidate for conservation and/or augmentative biological control, complementing the activity of P. bliteus in regulating the psyllid populations in eucalypt stands in countries where both studied psyllid andpredator are present.

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Acknowledgements The authors wish to thank Ana Silva, Pedro Nunes and Vera Zina for the help in the field and laboratory work. We also acknowledge the contribution of the editors and reviewers, which with their comments and suggestions helped us improve the manuscript.

Funding This work was funded by Fundação para a Ciência e Tecnologia (FCT) through the Doctoral Programme SUSFOR—Sustainable Forests and Products (PD/00157/2012) and PhDgrant to the first author (PD/BD/52693/2014). KKL-JNF Israel (2015–2017) also supported the study. CEF is a research unitfunded by FCT (UID/AGR/00239/2019).

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References Anderson NH (1961) Life-histories of some british anthocoris (Hemiptera: Heteroptera), with special reference to food requirements and feeding habits. Imperial College of Science and Technology — University of London Boavida C, Garcia A, Branco M (2016) How effective is Psyllaephagus bliteus (Hymenoptera: Encyrtidae) in controlling Glycaspis brimblecombei (Hemiptera: Psylloidea)? Biol Control 99:1–7 Brennan EB, Gill RJ, Hrusa GF, Weinbaum SA (1999) First record of Glycaspis brimblecombei (Moore) (Homoptera: Psyllidae) in North America: initial observations and predator associations of a potentially serious new pest of Eucalyptus in California. Pan-Pacific Entomol 75:55–57 Caleca V, Lo Verde G, Maltese M (2011) First record in Italy of Psyllaephagus bliteus Riek (Hymenoptera Encyrtidae) parasitoid of Glycaspis brimblecombei Moore (Hemiptera: Psyllidae). Naturalista sicil 3–4:435–444 Carlsson NOL, Sarnelle O, Strayer DL (2009) Native predator sand exotic prey — an acquired taste? Front Ecol Environ 7:525–532 Daane KM, Sime KR, Paine TD (2012) Climate and the effec-tiveness of Psyllaephagus bliteus as a parasitoid of the red gum lerp psyllid. Biocontrol Sci Technol 22:1305– 1320 Dicke M, Sabelis MW, Takabayashi J, Bruin J, Posthumus MA (1990) Plant strategies of manipulating predator-prey interactions through allelochemicals: prospects for appli-cation in pest control. J Chem Ecol 16:3091–3118 Drukker B, Bruin J, Sabelis MW (2000) Anthocorid predators learn to associate herbivore-induced plant volatiles with presence or absence of prey. Physiol Entomol 25:260–265 Emami MS, Shishehbor P, Karimzadeh J (2014) The influences of plant resistance on predation rate of Anthocoris nemoralis (Fabricius) on Cacopsylla pyricola (Förster). Arch Phytopathol 47:2043–2050 Horton DR (2008) Minute pirate bugs (Hemiptera: Anthocori-dae). In: Capinera JL (ed) Encyclopedia of entomology.Springer, Dordrecht, pp 2402–2412 Horton DR, Lewis TM, Broers DA (2004) Ecological and geographic range expansion of the introduced predator Anthocoris nemoralis (Heteroptera: Anthocoridae) in North America: potential for non target effects? Am Entomol 50:18–30

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Jerinic-Prodanovic D, Protic L (2013) True bugs (Hemiptera, Heteroptera) as psyllid predators (Hemiptera, Psylloidea). ZooKeys 319:169–189 Laudonia S, Margiotta M, Sasso R (2014) Seasonal occurrence and adaptation of the exotic Glycaspis brimblecombei Moore (Hemiptera: Aphalaridae) in Italy. J Nat Hist 48:675–689 Lundgren JG (2011) Reproductive ecology of predaceous Heteroptera. Biol Control 59:37–52 Margiotta M, Bella S, Buffa F, Caleca V, Floris I, Giorno V, LoVerde G, Rapisarda C, Sasso R, Suma P, Tortorici F,Laudonia S (2017) Modeling environmental influences in the Psyllaephagus bliteus (Hymenoptera: Encyrtidae) — Glycaspis brimblecombei (Hemiptera: Aphalaridae) parasitoid-host system. J Econ Entomol 110:491–501 Martins CB, Zarbin PH (2013) Volatile organic compounds of conspecific-damaged Eucalyptus benthami influence responses of mated females of Thaumastocoris peregrinus. J Chem Ecol 39:602–611 Mendel Z, Carmi-Gera E, Podoler H, Assael F (1995) Repro-ductive behavior of the specialist predator Elatophilus hebraicus (Hemiptera: Anthocoridae). Ann Entomol SocAm 88:856–861 Mohamed ME (2016) The interaction between the gall wasp Leptocybe invasa and Eucalyptus camaldulensis leaves: a study of phyto-volatile metabolites. J Pharmacogn Phytother 8:90–98 Pascua MS, Rocca M, De Clercq P, Greco NM (2018) Host plantuse for oviposition by the insidious flower bug (Hemiptera:Anthocoridae). J Econ Entomol 112:219–225 Péricart J (1972) Hémiptères—Anthocoridae, Cimicidae,Microphysidae de l’Ouest- Paléarctique. faune de L’europeet du Bassin Méditerranéen. Masson & Cie, Paris Peris-Felipo FJ, Bernués-Bañeres A, Pérez-Laorga EA, Jiménez-Peydró R (2010) Nuevos datos sobre la distribución en España de Glycaspis brimblecombei Moore, 1964 (Hemi-ptera: Psyllidae), plaga de Eucalyptus camaldulensis. Bol Asoc Esp Entomol 33:517–526 Scutareanu P, Drukker B, Bruin J, Posthumus MA, Sabelis MW(1996) Leaf volatiles and polyphenols in pear trees infestedby Psylla pyricola. Evidence of simultaneously induced responses. Chemoecology 7:34–38 Sigsgaard L (2004) Oviposition preference of Anthocoris nemorum and A. nemoralis for apple and pear. Entomol Exp Appl 111:215–223

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Sigsgaard L (2005) Oviposition preference of Anthocoris nemoralis and A. nemorum (Heteroptera: Anthocoridae) on pear leaves affected by leaf damage, honeydew and prey. Biocontrol Sci Technol 15:139–151 Sigsgaard L (2010) Habitat and prey preferences of the two predatory bugs Anthocoris nemorum (L.) and A. nemoralis (Fabricius) (Anthocoridae: Hemiptera- Heteroptera). BiolControl 53:46–54 Troncoso C, Becerra J, Perez C, Hernandez V, Martin AS,Sanchez-Olate M, Rios D (2012) Induction of defensive responses in Eucalyptus globulus (Labill) plants, against Ctenarytaina eucalypti (Maskell) (Hemiptera: Psyllidae). Am J Plant Sci 3:589–595 Valente C, Hodkinson I (2009) First record of the red gum lerp psyllid, Glycaspis brimblecombei Moore (Hem.: Psyllidae), in Europe. J Appl Entomol 133:315–317 Vet LEM, Dicke M (1992) Ecology of infochemical use by natural enemies in a tritrophic context. Annu Rev Entomol 37:141–172

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General discussion and conclusions

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Discussion and Conclusions

We have conducted several studies related with the success of biological control of invasive pests in Eucalyptus plantations. Ours studies included both classical biological control and conservational biological control approaches. In this chapter we summarize main achievements and discuss our results in light of current theories on biologicol control.

Fortuitous biological control In Chapter 2, we report the presence, for the first time in Europe, of a new gall wasp of eucalypts, Epichrysocharis burwelli (Franco et al. 2016). This gall wasp is specialized on Corymbia citriodora and was first found in urban trees in Lisbon and surrounding areas. During our surveys, we identified a parasitoid species, Closterocerus sp. parasitizing E. burwelli, suggesting a possible case of fortuitous biological control. Alien insect species frequently attain outbreak levels in invaded regions due to their enemy-free status, i.e. the absence of their native natural enemies (Keane and Crawley 2002). However, not so rarely, alien insect pests may arrive together with their natural enemies to the invaded area, either directly from their native distribution range or through natural dispersion from other regions where intentional releases were first done (Neuenschwander et al. 2003). Thus, screening for the presence of natural enemies fortuitously introduced should be part of the first assessments. This should be carried out before starting any other measures regarding biological control. Over the years, several parasitoid species of eucalyptus pests have been recorded in Portugal, which arrived by chance. In fact, most of the parasitoids of eucalypts pests in Portugal have been unintentionally introduced. The only exceptions, so far, are the cases of Anaphes nitens and A. inexpectatus, which were both introduced in Portugal for the control of G. platensis (Valente et al. 2017a). Other parasitoid species, such as Avetianella longoi, parasitoid of the long horn beetle, Phoracantha semipunctata (Hanks et al. 1995), C. chamaeleon, parasitoid of Ophelimus maskelli (Branco et al. 2009), and Psyllaephagus bliteus, parasitoid of G. brimblecombei (Dhahri et al. 2014), appeared in the Iberian Peninsula without intentional release. Altogether, these results demonstrate that fortuitous biological control can play a relevant role in the control of invasive insect pests worldwide. Closterocerus sp. that we observed parasitizng E. burwelli is a non-European species, most probably of Australian origin

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(Christer Hansen, pers. com.). The genus Closterocerus is known to occur over the world, and include parasitoid of Diptera, Lepidoptera and Hymenoptera species (Gumovsky 2001; Yefremova 2008). We hypothesized that this parasitoid species might be a good biocontrol agent against E. burwelli. However, further studies are needed to clarify the taxonomic position of Closterocerus sp., as well as to better describe its host-parasitoid relationship and assess its efficacy as a biocontrol agent. This knowledge will be useful to the biological control of E. burwelli in Portugal, as well as in other invaded regions, such as USA, Brazil and Mexico (Berti-Filho et al. 2004; Pujade-Villar et al. 2019; Schauff and Garrison 2000). In Brazil, another potential biocontrol agent of E. burwelli was recorded, Selitrichoids sp. (Pereira 2010).

Classical biological control: the relevance of the trophic interactions in the native range

Finding good candidate species for biological control, suitable for the target species, climate conditions and region, implies a good knowledge of the host-parasitoid communities in its native range. The control of Gonipterus spp. by A. nitens is one of the most studied cases of biological control of eucalypts pests. This egg parasitoid was first released in South Africa in 1926, being an example of a biological control program with enormous success (Tooke 1955). Thereafter, the same parasitoid species was introduced in many other regions in South America, North America and Europe (Hanks et al. 2000; Lanfranco and Dungey 2001). Yet, the success of the parasitoid was not equal in all regions. In particular, a low efficiency of A. nitens to control G. platensis was demonstrated in high altitude eucalypts stands, in Portugal (Reis et al. 2012). Difficulties in determining the taxonomic position of the target insect species is a major constraint for the assessment of a biocontrol agent. Following the finding that G. scutellatus consisted of a complex of species (Mapondera et al. 2012), it became most relevant to identify the major parasitoid species associated with G. platensis in its native range. A first survey was carried out in Tasmania, followed by the introduction in Portugal of A. inexpectatus, in 2012 (Valente et al. 2017a). Yet, in this first survey the association between Gonipterus species and their parasitoids was not studied. Our work (Chapter 3) was the first attempt to study the Gonipterus spp. - parasitoid communities in their native range. We identified four egg parasitoid species associated with G. platensis in the native range, i.e. A. inexpectatus, A. nitens, A. tasmaniae, and Cirrospilus sp. Anaphes tasmaniae was the most frequent species. We further described for the first time new trophic relationships between native Gonipterus species and native

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Chapter 6 and exotic egg parasitoids in Tasmania. In particular, we highlight the association between G. pulverulentus and Gonipterus sp. 1, between A. inexpectatus and A. tasmaniae, and between G. notographus and A. inexpectatus and Euderus sp. Furthermore, from the collection of Gonipterus egg pods, we verified that A. nitens has increased its distribution range in Tasmania, since its discovery in this region (Valente et al. 2017b). In Portugal, successful quarantine colonies were stablished for Cirrospilus sp. and Euderus sp. Further studies are needed to obtain a wider view of their potential as biocontrol agents against Gonipterus species. Also, climate threshold limits of these parasitoid species should be determined, as well as their taxonomic and molecular characterization. This was not possible to cover on the present work. Our work further points out an apparent host plant association for Gonipterus species. While G. notographus is most associated with eucalypts from the peppermints group, G. platensis, G. pulverulentus and Gonipterus sp. 1 were commonly found on eucalypt species belonging to Maidenaria section. We also confirmed the presence of Gonipterus sp. 2 for the first time in Tasmania. This species was known only from South Africa, France and Italy (Mapondera et al. 2012). We believe that our findings will help to design and develop future classical biological control programs against Gonipterus species.

Phenology and efficiency of a biocontrol agent In Chapter 4, we studied a new gall wasp species of eucalypts, Ophelimus sp., which was recently described as Ophelimus mediterraneus (Borowiec et al. 2019). This new species was found in Southern Europe (France, Italy and Portugal), in 2010-2011. In the invaded range, no parasitoids were found associated with it (Borowiec et al. 2019). Yet, we hypothesized that C. chamaeleon, a highly efficient larval parasitoid which was released in the Mediterranean Basin to control O. maskelli (Mendel et al. 2007; Rizzo et al. 2006), could also parasitize O. mediterraneus. Our study demonstrated the relevance of the phenology for the efficiency of a biocontrol agent. In Portugal, O. mediterraneus was shown to be univoltine, with winter larval development. Yet, in natural conditions C. chamaeleon is not active during the winter (it is possibly overwintering). Thus, the parasitism would not occur in nature due to a lack of synchronism. In laboratory and field manipulative experiments, we demonstrated that C. chamaeleon can recognize O. mediterraneus as suitable host and parasitize it. Furthermore, we also showed that C. chamaeleon could complete development parasitizing the larvae of O. mediterraneus. Thus, the asynchronous life cycles of C. chamaeleon and O. mediterraneus are the most likely explanation for the non-recorded parasitism of O. mediterraneus, in nature.

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More recently, another Ophelimus species, O. migdanorum was described from Chile and interestingly was found to be parasitized by C. chamaeleon (Molina-Mercader et al. 2019). This new host – parasitoid association reinforces our hypothesis that C. chamaeleon can use different Ophelimus species as hosts. We believe that the use of C. chamaeleon has a biological control agent against O. mediterraneus should be further studied. Furthermore, studies regarding temperature limit threshold and climate effects for the occurrence of parasitism in this host-parasitoid system should be evaluated.

Conservation biological control: can native predators be part of the solution?

Studies on the performance of native generalist predators as biocontrol agents of invasive species are scarce (Carlsson et al. 2009). Nevertheless, there is an increase attention for the possible relevance of conservation biological control for the management of invasive species (Escobar-Ramírez et al. 2019). Studies on the interaction between non-native pest species and native predators are needed to achieve this objective. Since native predators do not share a co-evolutionary history with the invasive pest species, we may hypothesize that the invasive species could be vulnerable to the predator due to lack of natural defenses. However, the invasive species may be also resistant to the native predator. This outcome could result from the low searching ability or low capture efficiency of the predator on the new prey or its low performance on the new food source (Pintor et al. 2015). In Chapter 5, we evaluated the feeding and reproductive behavior of the native predator Anthocoris nemoralis while feeding on the alien psyllid pest of eucalypts, Glycaspis brimblecombei. Although A. nemoralis was previously found occurring in trees infested by G. brimblecombei (Laudonia and Garonna 2010), this novel predator-prey interaction was never studied. We showed that A. nemoralis was able to feed and reproduce while feeding on the psyllid. This new association may be explained by the predator preference for psyllids in general (Horton et al. 2004). However, the most interesting finding was the ability of A. nemoralis females using Eucalyptus leaves as a substrate for oviposition. Our study further showed that A. nemoralis could complete one generation and reach adulthood, feeding exclusively on G. brimblecombei and using Eucalyptus leaves for oviposition. The influence of host-plant semiochemicals cues must not be excluded, as it can be decisive for anthocorid species to find their prey (Scutareanu et al. 1996; Sigsgaard 2005).

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Therefore, we suggest that A. nemoralis is able to recognize and be attracted by this new host plant habitat. Still, it is intriguing why A. nemoralis was never observed interacting with the other eucalypts psyllid species present in Europe, which deserve further studies. We also assume that the preference of A. nemoralis for Eucalyptus trees habitat might increase by learning response to host-plant chemical cues. Similarly, its efficiency while preying on G. brimblecombei might increase by learning, as demonstrated in other prey- predator systems. Further studies should be made to test these hypotheses. When choosing its prey, A. nemoralis might not discriminate between the ones that are parasitized from those that are not. Therefore, we also propose that the impact of the predator on the parasitoid P. bliteus population, through intraguild predation should be assessed. Our work is a first step, identifying the potential relevance of this native predator for the control of the non-native psyllid populations. Still, knowledge on the impact of A. nemoralis on the population dynamics of G. brimblecombei and how it may be incremented through conservation or augmentative biological control tactics is lacking.

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References Berti-Filho, E., Costa, V.A., La Salle, J., 2004. Primeiro registro da vespa-da-galha, Epichrysocharis burwelli (Hymenoptera: Eulophidae) em Corymbia (Eucalyptus) citriodora (Myrtaceae) no Brasil. Revista de Agricultura, Piracicaba 79:363 Borowiec, N., La Salle, J., Brancaccio, L., Thaon, M., Warot, S., Branco, M., Ris, N., Malausa, J.-C., Burks, R., 2019. Ophelimus mediterraneus sp. n. (Hymenoptera, Eulophidae): a new Eucalyptus gall wasp in the Mediterranean region. Bull Entomol Res 109:678-694. Branco, M., Boavida, C., Durand, N., Franco, J.C., Mendel, Z., 2009. Presence of the Eucalyptus gall wasp Ophelimus maskelli and its parasitoid Closterocerus chamaeleon in Portugal: First record, geographic distribution and host preference. Phytoparasitica 37:51-54 Carlsson, N.O.L., Sarnelle, O., Strayer, D.L., 2009. Native predators and exotic prey –an acquired taste?. Front Ecol Environ 7:525-532 Dhahri, S., Ben Jamaa, M.L., Garcia, A., Boavida, C., Branco, M., 2014. Presence of Glycaspis brimblecombei and its Parasitoid Psyllaephagus bliteus in Tunisia and Portugal. Silva Lusitana 22:99-115 Escobar-Ramírez, S., Grass, I., Armbrecht, I., Tscharntke, T., 2019. Biological control of the coffee berry borer: Main natural enemies, control success, and landscape influence. Biol Control 136 doi:10.1016/j.biocontrol.2019.05.011 Franco, J.C., Garcia, A., Branco, M., 2016. First report of Epichrysocharis burwelli in Europe, a new invasive gall wasp atacking eucalypts. Phytoparasitica 44:443-446 Gumovsky, A.V., 2001 The status of some genera allied to Chrysonotomyia and Closterocerus (Hymenoptera: Eulophidae, Entedoninae), with description of a new species from Dominican Amber. Phegea 29:125-141 Hanks, L.M., Gould, R.J., Paine, T.D., Millar, J.G., Wang, Q., 1995. Biology and host relations of Avetianella longoi (Hymenoptera: Encyrtidae), and egg parasitoid of the Eucalyptus Longhorned Borer (Coleóptera: Cerambycidae). Ann Entomol Soc Am 88:666-671 Hanks, L.M., Millar, J.G., Paine, T.D., Campbell, C.D., 2000. Classical biological control of the Australian weevil Gonipterus scutellatus (Coleoptera : Curculionidae) in California. Environ Entomol 29:369-375 Horton, D.R., Lewis, T.M., Broers, D.A., 2004. Ecological and geographic range expansion of the introduced predator: Anthocoris nemoralis (Heteroptera:

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Anthocoridae) in North America: potential for nontarget effects?. American Entomologist 50:18-30 Keane, R.M., Crawley, M.J., 2002. Exotic plant invasions and the enemy release hypothesis. Trends Ecol Evol 17:164-170 Lanfranco, D., Dungey, H.S., 2001. Insect damage in Eucalyptus: A review of plantations in Chile. Austral Ecology 26:477-481 Laudonia, S., Garonna, A.P., 2010. The red gum lerp psyllid, Glycaspis brimblecombei, a new exotic pest of Eucalyptus camaldulensis in Italy. B Insectol 63:233-236 Mapondera, T.S., Burgess, T., Matsuki, M., Oberprieler, R.G., 2012. Identification and molecular phylogenetics of the cryptic species of the Gonipterus scutellatus complex (Coleoptera: Curculionidae: Gonipterini). Aust J Entomol 51:175-188 Mendel, Z., Protasov, A., Blumberg, D., Brand, D., Saphir, N., Madar, Z., La Salle, J., 2007. Release and recovery of parasitoids of the Eucalyptus gall wasp Ophelimus maskelli in Israel. Phytoparasitica 35:330-332 Molina-Mercader, G., Angulo, A.O., Sanfuentes, E., Hasbún, R., Olivares, T., Castillo- Salazar, M., Goycoolea, C., 2019. Detection and distribution of Ophelimus migdanorum and its possible biocontroller Closterocerus chamaeleon in productive areas of Eucalyptus globulus in Chile. Chil J Agr Res 79:337-346 Neuenschwander, P., Borgemeister, C., Langewald, J., 2003. Biological control in IPM systems in Africa. CABI Publishing, Pereira, R.A., 2010. Aspectos morfo-bioecológicos de Epichrysocharis burwelli (Eulophidae, Hymenoptera), vespa-das-galhas das folhas de Corymbia citriodora. Universidade de São Paulo - Escola Superior de Agricultura "Luiz de Queiroz". PhD thesis Pintor, L.M., Byers, J.E., Anderson, M., 2015. Do native predators benefit from non- native prey?. Ecology letters 18:1174-1180 Pujade-Villar, J., Díaz-Ramos, S.G., Rodríguez-Rivas, A., Cibrián-Llanderal, V.D., Askew, D., 2019. First Record of Epichrysocharis burwelli from Mexico, a new invasive gall wasp attacking Corymbia (Myrtaceae). Southwest Entomol 44:323- 325 Reis, A.R., Ferreira, L., Tomé, M., Araujo, C., Branco, M., 2012. Efficiency of biological control of Gonipterus platensis (Coleoptera: Curculionidae) by Anaphes nitens (Hymenoptera: Mymaridae) in cold areas of the Iberian Peninsula: Implications

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for defoliation and wood production in Eucalyptus globulus. Forest Ecol Manag 270:216-222 Rizzo, M.C., Lo Verde, G., Rizzo, R., Buccellato, V., Caleca, V., 2006. Introduzione di Closterocerus sp. in Sicilia per il controllo biologico di Ophelimus maskelli Ashmead (Hymenoptera Eulophidae) galligeno esotico sugli eucalipti. Boll Zool agr Bachic 38:237-248 Schauff, M.E., Garrison, R., 2000. An introduced species of Epichrysocharis (Hymenoptera: Eulophidae) producing galls on Eucalyptus in California with notes on described species and placement of the genus. J Hymenopt Res 9:176- 181 Scutareanu, P., Drukker, B., Bruin, J., Posthumus, M.A., Sabelis, M.W., 1996. Leaf volatiles and polyphenols in pear trees infested by Psylla pyricola. Evidence of simultaneously induced responses. Chemoecology 7:34-38 Sigsgaard, L., 2005 Oviposition preference of Anthocoris nemoralis and A. nemorum (Heteroptera: Anthocoridae) on pear leaves affected by leaf damage, honeydew and prey. Biocontrol Sci Techn 15:139-151 Tooke, F.G.C., 1955. The Eucalyptus Snout beetle, Gonipterus scutellatus Gyll. a study of its ecology and control by biological means. Entomology Memoirs. Department of Agriculture and Forestry, Union of South Africa Valente, C., Gonçalves, C., Afonso, C., Reis, A., Branco, M., 2017a. Controlo biológico clássico do gorgulho-do-eucalipto: situação atual e perspetivas futuras. Pragas e doenças emergentes em sistemas florestais, School of Agriculture, June, 8th, 2017, Lisbon Valente, C., Gonçalves, C.I., Reis, A., Branco, M., 2017b. Pre-selection and biological potential of the egg parasitoid Anaphes inexpectatus for the control of the Eucalyptus snout beetle, Gonipterus platensis. J Pest Sci 90:911-923 Wittenberg, R., Cock, M.J.W., 2005. Best Practices for the prevention and management of invasive alien species. In: Mooney, H.A., Mack, R.N., McNeely, J.A., Neville, L.E., Schei, P.J., Waage, J.K. (eds) Invasive Alien Species: A New Synthesis. Island Press, Washington - Covelo - London Yefremova, Z., 2008. Order Hymenoptera, family Eulophidae. In: van Harten A (ed) Arthropod fauna of the United Arab Emirates, vol 1. Dar Al Ummah Printing, Abu Dhabi, United Arab Emirates, pp 345-360

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