Molecular Ecology MtDNA metagenomics rev eals large -scale invasion of belowground arthropod communities by introduced species Journal:For Molecular Review Ecology Only Manuscript ID MEC-16-1035.R1 Manuscript Type: From the Cover Date Submitted by the Author: n/a Complete List of Authors: Cicconardi, Francesco; Universitat Innsbruck, Institute of ecology Borges, Paulo Strasberg, Dominique; UMR PVBMT, Université de La Réunion, Oromí, Pedro López, Heriberto Pérez-Delgado, Antonio; IPNA-CSIC, Ecology and Evolution Casquet, Juliane Caujapé-Castells, Juli; Jardin Botanico Canario "Viera y Clavijo", Dept. of Molecular Biodiversity & DNA bank Fernández-Palacios, José María; Universidad de La Laguna, Island Ecology and Biogeography Research Group Thébaud, Christophe; Université Paul Sabatier, Emerson, Brent; IPNA-CSIC, Ecology and Evolution; Keywords: mesofauna, soil, island biogeography, Invasive Species, Invertebrates Page 1 of 28 Molecular Ecology 1 1 2 MtDNA metagenomics reveals large-scale invasion of belowground 3 arthropod communities by introduced species 4 5 6 Francesco Cicconardi 1, Paulo A. V. Borges 2 , Dominique Strasberg 3, Pedro Oromí 4, 7 Heriberto López 5, Antonio J. Pérez-Delgado 5, Juliane Casquet 6, Juli Caujapé-Castells 7, José 8 María Fernández-Palacios 8, Christophe Thébaud 6 & Brent C. Emerson 5,9 9 10 1Institute of Ecology, University of Innsbruck, Technikerstrasse 25, a-6020 Innsbruck, Austria. 11 2CE3C – Centre for Ecology,For Evolution Review and Environmental Only Changes / Azorean 12 Biodiversity Group and Universidade dos Açores –Departamento de Ciências Agrárias, 13 Rua Capitão João d’Ávila s/n, 9700-042, Angra do Heroísmo, Açores, Portugal. 14 3UMR PVBMT, Peuplements Végétaux et Bio-agresseurs en Milieu Tropical, Université de La 15 Réunion, 15 avenue René Cassin, CS 93002, 97 744 Saint Denis, Cedex 9, Reunion Island, 16 France. 17 4Departamento de Biología Animal y Edafología y Geología, Universidad de La Laguna, C/ 18 Astrofísico Francisco Sánchez, 38206 La Laguna, Tenerife, Canary Islands, Spain. 19 5Island Ecology and Evolution Research Group, IPNA-CSIC, 38206 La Laguna, Tenerife, Canary 20 Islands, Spain. 21 6Laboratoire Evolution & Diversité Biologique, UMR 5174 CNRS-Université Paul Sabatier- 22 ENFA, 31062 Toulouse Cedex 9, France. 23 7Departamento de Biodiversidad Molecular y Banco de ADN, Jardín Botánico Canario 24 ‘Viera y Clavijo’ - Unidad Asociada CSIC, Cabildo de Gran Canaria, Camino del Palmeral 25 15 de Tafira Alta, 35017 Las Palmas de Gran Canaria, Spain. 26 8Island Ecology and Biogeography Research Group. Instituto de Enfermedades Tropicales 27 y Salud Pública de Canarias (IUETSPC), Universidad de La Laguna, Tenerife, Canary 28 Islands 38206, Spain. 29 9School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich 30 NR4 7TJ, UK. 31 32 33 34 Key words: mesofauna, soil, introduced species, island biogeography, invertebrate 35 36 Corresponding author: Brent Emerson, Island Ecology and Evolution Research Group, 37 Instituto de Productos Naturales y Agrobiología (IPNA-CSIC), C/ Astrofísico Francisco 38 Sánchez 3, 38206 - La Laguna, Santa Cruz de Tenerife - Canary Islands, Spain. Ph: +34 39 922 256 847 (ext. 283). Fax: +34 922 260 135. Email: [email protected] 40 41 Short title: Soil mesofauna assembly by species introductions 42 43 44 Molecular Ecology Page 2 of 28 2 45 Abstract 46 47 Using a series of standardised sampling plots within forest ecosystems in remote 48 oceanic islands, we reveal fundamental differences between the structuring of 49 aboveground and belowground arthropod biodiversity that are likely due to large-scale 50 species introductions by humans. Species of beetle and spider were sampled almost 51 exclusively from single islands, while soil dwelling Collembola exhibited more than 52 tenfold higher species sharing among islands. Comparison of Collembola mitochondrial 53 metagenomic dataFor to a database Review of more than 80,000 Only Collembola barcode sequences 54 revealed almost 30% of sampled island species are genetically identical, or near 55 identical, to individuals sampled from often very distant geographic regions of the 56 world. Patterns of mtDNA relatedness among Collembola implicate human-mediated 57 species introductions, with minimum estimates for the proportion of introduced species 58 on the sampled islands ranging from 45-88%. Our results call for more attention to soil 59 mesofauna to understand the global extent and ecological consequences of species 60 introductions. Page 3 of 28 Molecular Ecology 3 61 Introduction 62 63 To understand soil ecosystem functioning, with reference to phenomena that extend 64 beyond soil itself, such as potential cascading effects across trophic levels or the impact 65 of introduced and potentially invasive non-native species on ecosystem processes (e.g. 66 Ehrenfeld 2010; Wardle et al. 2004; Yang et al. 2009), advances are needed to bridge the 67 gap between belowground and aboveground terrestrial systems. However, such 68 advances are limited by the paucity of biodiversity data for soil, which has been referred 69 to as the “third bioticFor frontier”, Review along with tropical Onlyforest canopies and ocean abysses 70 (André et al. 1994). Forest soils are especially challenging, as a single square metre of 71 temperate forest soil may contain more than 1000 species of invertebrates, most of 72 which are less than 2mm in length (Schaefer & Schauermann 1990). Much of the 73 invertebrate species diversity of soil remains uncatalogued, meaning that there is 74 probably no soil where we are able to identify, or even quantify all resident 75 invertebrates (Decaëns 2010; Wall et al. 2005). This lack of primary data on species 76 identity complicates the study and measurement of soil invertebrate biodiversity, which 77 is critically needed as the taxonomic composition of an ecosystem determines the 78 diversity of forms and functions. This functional component of biodiversity, which acts 79 as a key driver of ecosystem functioning (Violle et al. 2015), and this is likely to be of 80 great importance in soil ecosystems (Bardgett & Van der Putten 2014; Dominati et al. 81 2010; Heemsbergen et al. 2004; Lavelle et al. 2006). 82 For more than a decade, it has been recognised that DNA sequence analysis can 83 provide some relief to the taxonomic impediment – the limitation to science imposed by 84 the difficulty in identifying living species, most of which remain undescribed (Gaston 85 1991; Lomolino et al. 2010). The original methods for DNA barcoding (Hebert et al. 86 2003) have been developed into powerful and effective metabarcoding protocols that 87 are particularly well-suited for analysing species-rich assemblages of taxa like 88 invertebrates (e.g. Ramírez-González et al. 2013; Yu et al. 2012). More recently, shotgun 89 metagenomic sequencing of mixed insect species templates, with a particular focus on 90 beetle phylogenetics, has yielded numerous reads corresponding to the mitochondrial 91 DNA (mtDNA) genome that can be assembled into full or partial mitogenomes (Andújar 92 et al. 2015; Crampton-Platt et al. 2015; Gillett et al. 2014; Gómez-Rodríguez et al. 2015; Molecular Ecology Page 4 of 28 4 93 Tang et al. 2014). It has been pointed out that, in addition to phylogenetic 94 reconstruction, such extensive mtDNA genome data also offer great potential for 95 understanding how community assembly and structure influence functioning in 96 ecosystems by providing a powerful way to reveal biodiversity that was previously 97 “invisible” (Andújar et al. 2015). 98 Here we employ mitochondrial metagenomics to compare soil-dwelling and 99 aboveground arthropod communities sampled across three remote oceanic 100 archipelagos, two of which are located in the northern Atlantic (Canary Islands and 101 Azores) and the thirdFor (Mascarene Review Islands) being locOnlyated in the southwestern Indian 102 Ocean. For soil-dwelling arthropods we focus on the ubiquitous and dominant soil 103 mesofaunal taxon Collembola. Species identification of Collembola is complicated by (i) 104 small adult size that can typically range between 0.2 - 2 mm (Decaëns 2010), (ii) 105 pervasive cryptic species (Emerson et al. 2011) that can result in underestimates of 106 morphologically-derived species richness by more than an order of magnitude 107 (Cicconardi et al. 2013; Cicconardi et al. 2010), and (iii) changes in adult morphology 108 attributable to ecomorphosis, epitoky and cyclomorphosis (Hopkin 1997). 109 Metabarcoding has previously been used to suggest that a substantial proportion 110 of the Collembola fauna of the Canary Island of Tenerife is of recent origin (Ramírez- 111 González et al. 2013). However, the limited mtDNA sequence length of 220bp obtained 112 by Ramírez-González et al. (2013) resulted in taxonomic uncertainty for many 113 sequences, rendering quantitative comparisons of taxonomic relatedness unreliable. We 114 address this limitation by using a mitochondrial metagenomic approach to first robustly 115 assign DNA sequences to the class Collembola by means of phylogenetic analysis, and 116 then evaluate their distribution limits beyond the island where they were sampled. We 117 sample soil Collembola communities from forest ecosystems on the islands of Tenerife 118 (Canary Islands), Terceira (Azores) and Réunion (Mascarene Islands). We first compare 119 sharing of Collembola species among islands, placing our results into context by also 120 sampling and comparing aboveground arthropod communities