Cryptic Plutella species show deep divergence despite the capacity to hybridize

Cryptic Plutella species show deep divergence despite the capacity to hybridize Background: Understanding genomic and phenotypic diversity among cryptic pest taxa has important implications for the management of pests and diseases. The diamondback moth, Plutella xylostella L., has been intensively studied due to its ability to evolve insecticide resistance and status as the world’s most destructive pest of brassicaceous crops. The surprise discovery of a cryptic species endemic to Australia, Plutella australiana Landry & Hebert, raised questions regarding the distribution, ecological traits and pest status of the two species, the capacity for gene flow and whether specific management was required. Here, we collected Plutella from wild and cultivated brassicaceous plants from 75 locations throughout Australia and screened 1447 individuals to identify mtDNA lineages and Wolbachia infections. We genotyped genome-wide SNP markers using RADseq in coexisting populations of each species. In addition, we assessed reproductive compatibility in crossing experiments and insecticide susceptibility phenotypes using bioassays. Results: The two Plutella species coexisted on wild brassicas and canola crops, but only 10% of Plutella individuals were P. australiana. This species was not found on commercial Brassica vegetable crops, which are routinely sprayed with insecticides. Bioassays found that P. australiana was 19-306 fold more susceptible to four commonly-used insecticides than P. xylostella. Laboratory crosses revealed that reproductive isolation was incomplete but directionally asymmetric between the species. However, genome-wide nuclear SNPs revealed striking differences in genetic diversity and strong population structure between coexisting wild populations of each species. Nuclear diversity was 1.5-fold higher in P. australiana, yet both species showed limited variation in mtDNA. Infection with a single Wolbachia subgroup B strain was fixed in P. australiana, suggesting that a selective sweep contributed to low mtDNA diversity, while a subgroup A strain infected just 1.5% of P. xylostella. Conclusions: Despite sympatric distributions and the capacity to hybridize, strong genomic and phenotypic divergence exists between these Plutella species that is consistent with contrasting colonization histories and reproductive isolation after secondary contact. Although P. australiana is a potential pest of brassicaceous crops, it is of secondary importance to P. xylostella. Keywords: Plutella australiana, Plutella xylostella, Lepidoptera, hybridization, sympatric, insecticide resistance, Wolbachia *Correspondence: kym.perry@sa.gov.au School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, 5005, Australia South Australian Research and Development Institute, Adelaide, 5001, Australia Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 2 of 17 Background control during outbreaks [22, 25]. Plutella xylostella is well Cryptic species can show remarkable diversity in aspects known as a migratory insect with a high capacity for gene of their ecology, behaviour, and at the level of the genome. flow [11, 13], facilitating the rapid spread of resistance They exist across metazoan taxa [1], including globally alleles. Australian P. xylostella are thought to disperse fre- important arthropod pest taxa, such as whiteflies [2], quently, based on indirect evidence from ecological and disease-vectoring mosquitoes [3], fruit flies [4], thrips genetic studies [14, 15, 26]. Most studies have found a [5, 6] and mites [7, 8], some of which are characterised lack of genetic differentiation at microsatellite loci and low by cryptic species complexes. Discovering cryptic diver- sequence variation in mitochondrial DNA markers among sity has important consequences for estimates of global Australian and New Zealand populations of P. xylostella, biodiversity, conservation planning, and the management consistent with high gene flow and/or recent ancestry of pests and diseases. Morphologically similar species [14, 15, 17, 18]. While species identification was not in can vary in pest status due to differences in genotypic question in these studies, somewhat inconsistent find- and/or phenotypic traits that influence their host range ings in two studies from eastern Australia using allozymes and specificity, geographic distribution, the ability to vec- or SSR markers [19, 20] might reflect the confounding tor diseases, or insecticide resistance [8–10]. Therefore, presence of P. australiana samples [16]. Given these con- recognising cryptic species and the differences in their siderations, future management of Plutella in Australian biology and ecology are essential for effective manage- crops will require thorough understanding of the ecolog- ment, with important implications for public health, agri- ical requirements, genetic traits and pest status of the culture and trade. two Plutella species. In addition, reproductive isolation The diamondback moth, Plutella xylostella,isthe major between these two species is unknown but has impli- pest of brassicaceous crops worldwide, costing an esti- cations for evolutionary inference and the potential for mated US$4 to US$5 billion annually in direct losses gene flow. The capacity for hybridization and introgres- and management costs [11, 12]. Insecticide resistance sion could lead to the exchange of insecticide resistance or is widespread in P. xylostella populations around the other adaptive alleles [27, 28]. world, fuelling wide-ranging research to develop alter- Although mtDNA markers are widely used in stud- native management tactics [11, 13]. Plutella xylostella ies of species identity and population structure [29–31], was initially recorded in Australia in the late 1800s and mitochondrial variation within or between species can rapidly became a widespread pest of Brassica vegetables, be influenced by direct and/or indirect selection, or and then canola following its expanded production from introgressive hybridization [32, 33]. One factor that can the 1990s [14, 15]. Recently, Landry and Hebert [16], confound mtDNA-based inference is interaction with through mtDNA barcoding, identified a cryptic lineage inherited bacterial symbionts [34, 35]. Wolbachia is of Plutella in Australia not detected in previous molec- a widespread endosymbiont thought to infect at least ular studies of P. xylostella [14, 17–21]. Although exter- half of arthropod [36] and 80% of lepidopteran [37] nal morphology was indistinguishable from P. xylostella, species. It is mainly transmitted vertically from infected deep mtDNA divergence (8.6%), differences in geni- females to their offspring through the egg cytoplasm, and tal morphology and endemism in Australia led them inheritance is therefore linked with mtDNA. To facili- to describe a new species, Plutella australiana Landry tate its spread, Wolbachia manipulates host reproduc- &Hebert. Plutella australiana was originally collected tive biology to favour the fitness of infected females by together with P. xylostella in light trap samples in east- inducing host phenotypes that distort sex ratios (male- ern Australia, suggesting at least some ecological over- feminization, male-killing or induction of parthenogene- lap [16], but its biology, ecology and pest status were sis) or cause sperm-egg cytoplasmic incompatibility (CI) unknown. [38, 39]. In the simple case involving a single CI-inducing The management of P. xylostella in Australian Brassica strain, crosses with infected females are fertile but crosses crops has been a significant challenge for decades between uninfected females and infected males fail to [15, 22], but the discovery of P. australiana has made produce offspring. If maternal transmission is efficient the relative abundance and pest status of both species in and infected females have a reproductive advantage, these crops uncertain. With rare exception, P. xylostella Wolbachia infection can spread rapidly through an insect and allied species feed on plants in the order Brassicales, population [40], driving a selective sweep of a single hap- mainly within the family Brassicaceae [16, 23, 24], imply- lotype and reducing mtDNA diversity [41]. Limited sur- ing that the host range of P. australiana may include culti- veys to date have identified Wolbachia strains infecting vated brassicas. Widespread resistance to pyrethroid and P. xylostella at low frequency in populations from North organophosphate insecticides has been attributed to Aus- America, Africa, Asia and Europe [18, 42, 43]. Because tralian populations of P. xylostella from all vegetable and symbionts can contribute to reproductive isolation and canola production regions, which has led to ineffective influence mtDNA diversity [34, 44], assessing their role Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 3 of 17 ◦ ◦ can provide important insights into host evolution and 2 min, then 35 cycles at 95 C for 10 s, 52 C for 20 s, ◦ ◦ population structure [35, 45–47]. 72 C for 30 s followed by a 5 min final extension at 72 C. Here we investigated the biology, ecology and popula- PCR products were digested at 37 Cfor 1hwith 1unit tion genetic structure of two cryptic Plutella species by of AccI (NEB) restriction enzyme with 2 μLCutsmart collecting Plutella from brassicaceous plants throughout Buffer in a 20 μL reaction. Following digestion, products Australia and screening individuals to identify mtDNA were separated using agarose gel electrophoresis (1.5%). lineages and Wolbachia infections. For a subset of pop- Plutella xylostella products are approximately 516 bp and ulations, we examined genetic diversity using thousands 191 bp and P. australiana products are 348 bp and 359 bp of nuclear SNPs from across the genome. In addition, we [49]. To examine mtDNA haplotypes, sequencing of the assessed reproductive compatibility in laboratory crosses 707 bp COI amplicon was performed for 44 P. xylostella and determined the susceptibility of each species to com- and 37 P. australiana individuals at the Australian mercial insecticides. Genome Research Facility (AGRF). In addition, we down- loaded sequence trace files from Landry and Hebert Methods [16] (dx.doi.org/10.5883/DS-PLUT1) and re-analysed, Sample collection aligned and trimmed all sequences in GENEIOUS version Plutella larvae (rarely, eggs or pupae) were collected 10.0.6 [50]. Haplotype networks were constructed using from canola crops, Brassica vegetable crops, forage bras- R package pegas version 0.9 [51]. sicas and wild brassicas throughout Australia between March 2014 and December 2015 (Table 1). The wild Wolbachia screening and phylogenetics species included wild radish, Raphanus raphanistrum, Wolbachia infection was detected using two separate PCR turnip weed, Rapistrum rugosum, sea rocket, Cakile mar- assays of the 16S rRNA gene (16S-2 and 16S-6) accord- itima, Ward’s weed, Carrichtera annua, African mustard, ing to Simoes et al. [52]. To identify Wolbachia strains, the Brassica tournefortii, and mixed stands of sand rocket, Wolbachia surface protein (wsp) gene was sequenced in a Diplotaxis tenuifolia, and wall rocket, D. muralis.Ateach subset of individuals. Amplification was performed using location, at least 25 individuals were collected from ran- wsp81F and wsp691R sequence primers [53]. Amplicons domly selected plants to achieve a representative sample. were sequenced using the reverse primer and aligned in Insect samples were collected from Brassica vegetables by GENEIOUS version 10.0.6 [50]. We used a 493 bp align- hand, from sea rocket by beating plants over a collection ment to construct a maximum likelihood phylogeny in tray and from other hosts using a sweep net. Each pop- RAxML version 8.2.4 [54] using a general time reversal ulation sample was separately reared in ventilated plastic substitution model [55] with 1000 bootstrap replicates. containers on leaves of the original host material for 1–2 days and thereafter on cabbage leaves. Non-parasitised RADseq library preparation and sequencing pupae or late-instar larvae were fresh frozen at − 80 C. Libraries were prepared for restriction-site-associated DNA sequencing (RADseq) according to a protocol mod- DNA isolation and COI genotyping ified from Baird et al. [56]. Genomic DNA was quantified For each population sample, we aimed to genotype a using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng minimum of 16 individuals where possible after removing digested with 10 units of high fidelity SbfI in Cutsmart parasitized individuals. Individual pupae (but not larvae) Buffer (NEB) for 1 h at 37 C, then heat inactivated at were sexed under a dissecting microscope, then genomic 80 C for 20 min. One microlitre of P1 adapter (100nM) DNA was isolated by homogenising whole individuals with a 6-base molecular identifier (MID) (top strand followed by two phenol and one chloroform extrac- 5 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG tionsaccordingtoZraketetal. [48]. DNA was treated xxxxxxTGCA-3 , bottom strand 5 -[P]xxxxxxCTGTCTC with RNase A, then precipitated and re-suspended in TTATACACATCTGACGCTGCCGACGA-3,xrepre- TE buffer. Plutella lineages were distinguished using a sents sites for MIDs) were then added using 0.5 μLT4 PCR-RFLP assay [49]. A 707 bp COI region was amplified DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. using a combination of two primer pairs: (i) PxCOIF (5 - Library pools were sheared using a Bioruptor sonicator TCAACAAATCATAAAGATATTGG-3 ) and PxCOIR (Diagenode), then DNA fragments end-repaired using (5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ), and a Quick Blunting Kit (NEB), adenine overhangs added (ii) PaCOIF (5 -TCAACAAATCATAAGGATATTGG-3 ) then P2 adapters (top strand 5 -[P]CTGTCTCTTATA and PaCOIR (5 -TAAACCTCTGGATGGCCAAAAAA CACATCTCCAGAATAG-3 , bottom strand 5 -GTCTCG TCA-3 ). Ten microliter reactions were run with 2 μLof TGGGCTCGGAGATGTGTATAAGAGACAGT-3)lig- MyTaq 5x buffer, 0.2 μL of each primer (10mM stocks), ated. DNA purification between steps was performed 1 μL of DNA (approx. 5 ng) and 0.05 μLofMyTaq poly- using a MinElute PCR purification kit (Qiagen). Libraries were amplified using KAPA HiFi Hotstart Readymix merase (Bioline). Samples were amplified at 95 Cfor Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 4 of 17 Table 1 Collection details showing the frequency (f)of Plutella species and Wolbachia infections among Plutella populations from Australia P. australiana P. xylostella Location Collection date Latitude Longitude Host No. genotyped No. (f)No.(f)No.(f ) wol-infected ◦ ◦ Boomi NSW Sep-2014 -28.76 149.81 Canola 25 15 (0.60) 10 (0.40) 0 (0.00) ◦ ◦ Gilgandra NSW Sep-2014 -31.67 148.72 Wild turnip 23 21 (0.91) 2 (0.09) 0 (0.00) ◦ ◦ Ginninderra NSW Sep-2014 -35.19 149.05 Canola 15 2 (0.13) 13 (0.87) 0 (0.00) ◦ ◦ Ginninderra NSW Oct-2015 -35.19 149.05 Canola 34 27 (0.79) 7 (0.21) 0 (0.00) ◦ ◦ Goulburn NSW Nov-2015 -34.84 149.67 Canola 32 25 (0.78) 7 (0.22) 0 (0.00) ◦ ◦ Henty NSW Oct-2014 -35.60 146.95 Canola 18 1 (0.06) 17 (0.94) 0 (0.00) ◦ ◦ Narromine NSW Sep-2014 -32.22 148.03 Canola 26 0 (0.00) 26 (1.00) 1 (0.04) ◦ ◦ Richmond NSW Oct-2015 -33.60 150.71 Cabbage 21 0 (0.00) 21 (1.00) 0 (0.00) ◦ ◦ Wagga Wagga NSW Sep-2014 -35.04 147.33 Canola 21 5 (0.24) 16 (0.76) 0 (0.00) ◦ ◦ Werombi NSW Nov-2014 -33.99 150.64 Vegetables 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werombi NSW Oct-2015 -34.00 150.56 Kale 13 4 (0.31) 9 (0.69) 0 (0.00) ◦ ◦ Bundaberg QLD Oct-2014 -24.80 152.26 Canola 14 1 (0.07) 13 (0.93) 0 (0.00) ◦ ◦ Bundaberg QLD Sep-2015 -24.80 152.26 Canola 30 0 (0.00) 30 (1.00) 0 (0.00) ◦ ◦ Cunnamulla QLD Sep-2015 -28.07 145.68 African mustard 17 17 (1.00) 0 (0.00) 0 – ◦ ◦ Dalby QLD Sep-2014 -27.28 151.13 Canola 30 0 (0.00) 30 (1.00) 0 (0.00) ◦ ◦ Gatton QLD Oct-2014 -27.54 152.33 Broccoli 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Gatton QLD Nov-2015 -27.54 152.33 Broccoli 15 0 (0.00) 15 (1.00) 0 (0.00) ◦ ◦ Warwick QLD Oct-2015 -28.21 152.11 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Calca SA Apr-2014 -33.02 134.28 Sand rocket, Wall rocket 13 8 (0.62) 5 (0.38) 0 (0.00) ◦ ◦ Cocata SA Sep-2014 -33.20 135.13 Canola 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Colebatch SA Feb-2015 -35.97 139.66 Forage brassica 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Coonalpyn SA Oct-2015 -35.62 139.91 Wild radish 11 0 (0.00) 11 (1.00) 0 (0.00) ◦ ◦ Cowell SA Sep-2014 -33.66 137.16 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Keith SA Oct-2014 -36.09 140.29 Canola 32 0 (0.00) 32 (1.00) 6 (0.19) ◦ ◦ Lameroo SA Sep-2014 -35.32 140.51 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Lameroo SA Oct-2015 -35.17 140.48 Canola 14 0 (0.00) 14 (1.00) 0 (0.00) ◦ ◦ Littlehampton SA Oct-2014 -35.06 138.90 Cabbage 34 0 (0.00) 34 (1.00) 6 (0.18) ◦ ◦ Littlehampton SA Sep-2015 -35.06 138.90 Brussels sprouts 8 0 (0.00) 8 (1.00) 0 (0.00) ◦ ◦ Loxton SA Sep-2014 -34.37 140.72 Canola 31 0 (0.00) 31 (1.00) 0 (0.00) ◦ ◦ Loxton SA Oct-2015 -34.50 140.80 Canola 14 1 (0.07) 13 (0.93) 0 (0.00) ◦ ◦ Mallala SA Sep-2015 -34.38 138.50 Canola 26 0 (0.00) 26 (1.00) 0 (0.00) ◦ ◦ Meribah SA Sep-2014 -34.74 140.82 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Millicent SA Apr-2015 -37.61 140.34 Canola 9 0 (0.00) 9 (1.00) 2 (0.22) ◦ ◦ Minnipa SA Oct-2015 -32.81 135.16 Canola 22 1 (0.05) 21 (0.95) 0 (0.00) ◦ ◦ Moonaree SA Aug-2014 -31.99 135.87 Ward’s weed 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Mt Hope SA Sep-2014 -34.14 135.33 Canola 29 0 (0.00) 29 (1.00) 0 (0.00) ◦ ◦ Mt Hope SA Sep-2015 -34.20 135.34 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Padthaway SA Oct-2015 -36.56 140.43 Canola 18 2 (0.11) 16 (0.89) 0 (0.00) ◦ ◦ Picnic Beach SA Apr-2014 -34.17 135.27 Sea rocket 2 0 (0.00) 2 (1.00) 0 (0.00) ◦ ◦ Picnic Beach SA Sep-2014 -34.17 135.27 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Redbanks SA Oct-2014 -34.49 138.59 Canola 38 0 (0.00) 38 (1.00) 1 (0.03) ◦ ◦ Sherwood SA Oct-2014 -36.05 140.64 Wild radish 8 0 (0.00) 8 (1.00) 0 (0.00) Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 5 of 17 Table 1 Collection details showing the frequency (f)of Plutella species and Wolbachia infections among Plutella populations from Australia (Continued) P. australiana P. xylostella Location Collection date Latitude Longitude Host No. genotyped No. (f)No.(f)No.(f ) wol-infected ◦ ◦ Southend SA Apr-2015 -37.57 140.12 Sea rocket 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Tintinara SA Oct-2015 -35.97 139.66 Forage Brassica 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Ucontichie SA Sep-2014 -33.22 135.31 Canola 3 0 (0.00) 3 (1.00) 0 (0.00) ◦ ◦ Virginia SA Oct-2014 -34.64 138.54 Broccoli 18 0 (0.00) 18 (1.00) 1 (0.06) ◦ ◦ Virginia SA Sep-2015 -34.64 138.54 Cabbage 23 0 (0.00) 23 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Sep-2014 -33.55 134.86 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Mar-2015 -33.55 134.86 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Sep-2015 -33.55 134.86 Sea rocket 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Wirrabara SA Oct-2014 -32.99 138.31 Canola 28 2 (0.07) 26 (0.93) 0 (0.00) ◦ ◦ Wokurna SA Sep-2015 -33.67 137.96 Wild radish 24 1 (0.04) 23 (0.96) 0 (0.00) ◦ ◦ Wurramunda SA Apr-2014 -34.30 135.56 Wild canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Deddington TAS Nov-2014 -41.59 147.44 Kale 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Launceston TAS Nov-2014 -41.47 147.14 Wild mustard 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Newstead TAS Nov-2015 -41.59 147.44 Cauliflower 22 0 (0.00) 22 (1.00) 0 (0.00) ◦ ◦ Cowangie VIC Oct-2015 -35.10 141.33 Canola 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Ouyen VIC Sep-2014 -35.00 142.31 Canola 28 1 (0.04) 27 (0.96) 0 (0.00) ◦ ◦ Robinvale VIC Sep-2014 -34.81 142.94 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werribee VIC Oct-2014 -37.94 144.73 Cauliflower 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werribee VIC Nov-2015 -37.94 144.73 Cauliflower 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Yanac VIC Sep-2014 -36.06 141.25 Canola 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Boyup Brook WA Sep-2014 -33.64 116.40 Canola 26 2 (0.08) 24 (0.92) 0 (0.00) ◦ ◦ Dalwallinu WA Sep-2015 -30.28 116.66 Canola 20 0 (0.00) 20 (1.00) 0 (0.00) ◦ ◦ Dalyup WA Oct-2015 -33.72 121.64 Wild radish 22 3 (0.14) 19 (0.86) 0 (0.00) ◦ ◦ Esperance WA Sep-2014 -33.29 121.76 Canola 23 8 (0.35) 15 (0.65) 1 (0.07) ◦ ◦ Esperance WA Oct-2015 -33.79 122.13 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Gingin WA Dec-2014 -31.28 115.65 Red cabbage 23 0 (0.00) 23 (1.00) 1 (0.04) ◦ ◦ Kalannie WA Sep-2015 -30.00 117.25 Canola 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Manjimup WA Dec-2014 -34.18 116.23 Chinese cabbage 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Manjimup WA Nov-2015 -34.18 116.23 Brassica vegetables 13 0 (0.00) 13 (1.00) 0 (0.00) ◦ ◦ Narrogin WA Oct-2015 -32.95 117.32 Wild radish, wild canola 15 0 (0.00) 15 (1.00) 0 (0.00) ◦ ◦ Narrogin WA Oct-2015 -32.96 117.33 Canola 32 0 (0.00) 32 (1.00) 0 (0.00) ◦ ◦ Walkaway WA Sep-2014 -28.94 114.83 Canola 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Walkaway WA Sep-2014 -28.16 114.63 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) Total 1447 147 (0.10) 1300 (0.90) 19 (0.01) Australian states: NSW = New South Wales, QLD = Queensland, SA = South Australia, TAS = Tasmania, VIC = Victoria, WA = Western Australia All P. australiana individuals were infected with Wolbachia (Kapa Biosystems) and Nextera i7 and i5 indexed primers Illumina paired-end sequencing was performed using ◦ ◦ with PCR conditions: 95 C for 3 min, two cycles of 98 C HiSeq2500 (100 bp) or NextSeq500 (75 bp) at the AGRF. ◦ ◦ for 20 s, 54 C for 15 s, 72 C for 1 min, then 15 cycles of ◦ ◦ ◦ 98 C for 20 s, 65 C for 15 s, 72 C for 1 min followed by Read filtering and variant calling a final extension of 72 C for 5 min. Libraries were size- Sequence reads were demultiplexed using RADtools selected (300-700 bp) on 1–1.5% agarose gel and purified version 1.2.4 [57] allowing one base MID mismatch, using a minElute Gel Extraction Kit (Qiagen), then then TRIMMOMATIC version 0.32 [58]was used Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 6 of 17 to remove restriction sites, adapter sequences and a population pairs were calculated and significance deter- 4 3 thymine base from reverse reads introduced by the mined using exact G tests (10 mc burnins, 10 batches, P2 adapter, and quality filter using the ILLUMINA- and 10 iterations per batch) in GENEPOP version 4.6 CLIP tool with parameters: TRAILING:10 SLIDING- [70] after Bonferroni correction for multiple comparisons. WINDOW:4:15 MINLEN:40. Paired reads were aligned Separate analysis of population structure was performed to the P. xylostella reference genome (accession num- using the Bayesian clustering program STRUCTURE ver- ber: GCF_000330985.1) using STAMPY version 1.0.21 sion 2.3.4 [71], first for all individuals of co-occurring [59]with --baq and --gatkcigarworkaround options and Plutella species, and second for P. australiana alone. For expected substitution rate set to 0.03 for P. xylostella all runs, we used a burnin length of 5 × 10 followed by and 0.05 for P. australiana to reflect expected levels a run length of 10 MCMC iterations and performed ten of sequence divergence relative to the P. xylostella ref- independent runs for each K value from 1 to 10, where K erence genome. Duplicate reads were removed using is the number of genotypic clusters, using a different ran- PICARD version 1.71 [60]. Genotypes were called using dom seed for each run, assuming the locprior model with the Genome Analysis Toolkit (GATK) version 3.3-0 [61, correlated allele frequencies and λ set to 1. The optimal 62] HaplotypeCaller tool. We determined that base quality value of K was determined using the delta K method score recalibration using bootstrapped SNP databases was [72] implemented in STRUCTURE HARVESTER [73]and inappropriate for this dataset as it globally reduced qual- inspection of the likelihood distribution for each model. ity scores. For downstream comparisons between species, Q-matrices were aligned across runs using CLUMPP we joint-genotyped P. australiana and P. xylostella indi- version 1.1.2 [74] and visualised using DISTRUCT viduals using the GATK GenotypeGVCFs workflow. To version 1.1 [75]. examine finer scale population structure, we also joint- genotyped the P. australiana individuals alone. All vari- Laboratory cultures of Plutella species ant callsets were hard-filtered with identical parameters Laboratory cultures of P. australiana and P. xylostella were using VCFtools version 0.1.12a [63]: We removed indels established from field populations and used for cross- and retained confidently-called biallelic SNPs (GQ30) ing experiments and insecticide bioassays. Plutella adults genotyped in at least 70% of individuals with a mini- were collected at light traps at Angle Vale and Urrbrae, mum genotype depth of 5, minQ400, average site depth South Australia, in October–November 2015. Females of 12–100, minimum minor allele frequency of 0.05, in were isolated and allowed to lay eggs, then identified Hardy-Weinberg equilibrium at an alpha level of 0.05. To using PCR-RFLP and progeny pooled to produce sep- avoid linked sites, we used the VCFtools --thin func- arate cultures of each species. A laboratory culture of tion to retain only SNPs separated by a minimum of 2000 the Waite Susceptible P. xylostella strain (S) has been bp. To estimate genetic diversity, we generated a set of maintained on cabbage without insecticide exposure for all confidently-called variant and invariant sites (GQ30), approximately 24 years (≈ 310 generations) and was used and hard filtered to remove sites within repetitive regions as a bioassay reference strain. All cultures were main- and retain sites genotyped in at least 70% of individu- tained in laboratory cages at 26 ± 2.0 C and a 14:10 (L:D) als with an average site depth of 12–100. Sites from the hour photoperiod at the South Australian Research and mitochondrial genome were excluded from all datasets. Development Institute, Waite Campus, Adelaide, South Australia. The P. australiana culture was maintained on Genetic diversity and population structure sand rocket, Diplotaxis tenuifolia,and the P. xylostella cul- Genetic diversity was calculated for Plutella populations ture was maintained on cabbage, Brassica oleracea var. of both species from five locations. The R package hierf- capitata. The purity of cultures was assessed regularly stat [64] was used to calculate observed heterozygosity, using PCR-RFLP. gene diversity and the inbreeding coefficient, F , accord- IS ing to Nei [65]. Population means for site depth and num- Crossing experiments ber of SNPs, indels and private sites were calculated using Plutella australiana and P. xylostella pupae were sexed the --depth function and vcfstats module in VCFtools under a stereo microscope, then placed into individual version 0.1.12a [63]. Thenumberofheterozygoussites 5 mL clear polystyrene tubes with fine mesh lids and within individuals was determined from all confidently- gender visually confirmed after eclosion. Enclosures used called sites excluding indels using a custom python script for crossing experiments were 850 mL polypropylene parseVCF.py [66]and visualisedusingR[67]. pots (Bonson Pty Ltd) modified with lateral holes cov- To examine population structure in P. australiana,a ered with voile mesh for ventilation. Crosses of single global estimate of F [68] with bootstrapped 99% confi- mating pairs were performed on laboratory benches at ST 4 ◦ dence intervals (10 bootstrap replicates) was calculated 26 ± 2.0 C and 14:10 (L:D) photoperiod using 3-week in R package diveRsity [69]. Pairwise F values for all old D. tenuifolia seedlings as the host plant. After seven ST Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 7 of 17 days, adults were collected into a 1.5 mL tube and fresh was not different from 1:1 for P. xylostella (481 females, ◦ 2 frozen at − 80 C for species confirmation using PCR- 517 males, χ = 1.2986, p = 0.2545) or P. australiana RFLP. Seedlings were examined and eggs counted under (63 females, 55 males, χ = 0.5424, p = 0.4615). The rel- a stereo microscope, then returned to enclosures to allow ative incidence and abundance of P. australiana was egg hatch. Larvae were provided with fresh 3–4 week > 2-fold higher in the eastern state of New South Wales old seedlings until pupation, then pupae were individu- than in other states (Fig. 1). Plutella australiana lar- ally collected into 5 mL tubes. Hybrid F1xF1 crosses and vae were detected in 29% (n = 5/17) of collections from back-crosses were then performed as above. The presence wild brassicas and from species including wild radish, of egg and adult offspring was recorded for all replicates, Raphanus raphanistrum,wildturnip, Rapistrum rugosum, and for the majority of replicates (> 80%), the numbers of African mustard, B. tournefortii, and mixed stands of sand offspring were counted and used to calculate a mean. rocket, D. tenuifolia and wall rocket, D. muralis (Table 2). Among cultivated crops, P. australiana larvae occurred Insecticide bioassays in 36% (n = 14/39) of samples from canola, consist- Insecticide susceptibility of field-collected Plutella strains ing of 11% of total Plutella individuals from those crops, was compared to the susceptible P. xylostella (S) reference but were not identified from commercial Brassica veg- in dose-response assays using four commercial insecti- etable farms (Table 2). However, P. australiana eggs were −1 cides: Dominex (100 g L alpha-cypermethrin), Proclaim collected from kale at one farm. −1 −1 (44 g kg emamectin benzoate), Coragen (200 g L −1 chlorantraniliprole) and Success Neo (120 g L spine- Wolbachia infections rd toram). Bioassays were performed by placing 3 instar Plutella individuals (n = 1447) were screened for larvae onto inverted leaf discs embedded in 1% agar Wolbachia infection using 16S rRNA PCR assays. Only in 90 mm Petri dishes. Cabbage leaves, Brassica oler- 1.5% (n = 19/1300) of P. xylostella collected from eight acea.var. capitata were used for P. xylostella and canola different locations were infected (Table 1). In contrast, leaves, B. napus var. ‘ATR Stingray’, were used for all 147 P. australiana individuals were infected with P. australiana. Eight concentrations and a water-only con- Wolbachia across the 20 locations where this species trol were evaluated for each insecticide using four repli- occurred. To identify Wolbachia strains, a Wolbachia cates of ten larvae. A 4 mL aliquot of test solution was surface protein (wsp) amplicon was sequenced from 14 P. applied directly to leaves using a Potter Spray Tower xylostella and 30 P. australiana individuals. Each species (Burkard Manufacturing Co. Ltd.) calibrated to deliver an was infected with a different strain. The wsp sequence -1 aliquot of 3.52 ± 0.09 mg cm . After application, each for Australian P. xylostella showed 100% identity to a dish was placed in a controlled temperature room at 25 ± Wolbachia supergroup A isolate infecting P. xylostella 0.5 C, then mortality was assessed after 48 h (Dominex, from Malaysia, plutWA1 [18]. For P. australiana,the wsp Success Neo) or 72 h (Proclaim, Coragen). Dose-response sequence showed 100% identity to a Wolbachia super- analysis was performed using log-logistic regression in group B isolate infecting a mosquito, Culex pipiens,from Rpackage drc[76] and the fitted models were used to Turkey and the winter moth, Operophtera brumata,from estimate the lethal concentration predicted to cause 50% the Netherlands (Fig. 2). (LC ) and 99% (LC ) mortality of the test population. 50 99 Resistance ratios were calculated by dividing the LC and Crossing experiments LC estimates for field strains by the corresponding LC Inter-species single pair mating experiments showed that estimates for the P. xylostella (S) reference strain. hybridization between P. australiana and P. xylostella was possible, yet less successful than intra-species crosses. Results While most intra-species crosses produced adult off- Geographic distribution and host associations spring, the fecundity of P. xylostella was >2-fold higher Plutella larvae were collected from brassicaceous plants than P. australiana (Table 3). Both reciprocal inter-species at 75 locations in Australia and 1477 individuals were crosses produced F1 adult offspring, but success was genotyped at the COI locus using PCR-RFLP to iden- asymmetric and notably higher in the pairs with P. aus- tify species. Of these, 88% (n = 1300) were genotyped traliana females. In this direction, there was a strong male as P. xylostella, 10% (n = 147) as P. australiana and 2% bias in the F1 progeny: from 76 cross replicates, 16 collec- (n = 30) were unresolved (Table 1). Plutella australiana tively produced 9 female and 80 male adults, a ratio of 8.9. was identified in around one quarter (n = 20/75) of Hybrid F1xF1 crosses for both parental lines produced F2 collections distributed across southern Australia, while adult offspring (Table 4). For the P. australiana maternal P. xylostella occurred at all locations except Cunna- line, parental back-crosses using F1 hybrid males suc- mulla, Queensland, in a collection from wild African cessfully produced offspring, while parental back-crosses with F1 hybrid females were sterile. For the P. xylostella mustard, Brassica tournefortii (Table 1). The sex ratio Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 8 of 17 Fig. 1 The geographic distribution of P. xylostella (light grey) and P. australiana individuals (black) in larval collections from brassicaceous plants in Australia during 2014 and 2015. Pie diagrams show the relative proportion of each species at each location. Overlapped pies represent locations with 100% P. xylostella. Green highlighted circles indicate five locations from which individuals of each species were RAD sequenced maternal line, low fitness allowed only a single parental mutation from other haplotypes (Fig. 3a, Additional file 1: back-cross replicate, which involved a hybrid female and Table S1). Nine closely related haplotypes were identified was sterile. in 87 P. australiana individuals with seven occurring in single individuals (Fig. 3b). The most common haplotype, Mitochondrial haplotype diversity PaCOI01, occurred at high frequency and differed by 1-2 Mitochondrial haplotype networks of Australian Plutella base mutations from other haplotypes (Fig. 3b, Additional were constructed using a 613 bp COI alignment that file 1:Table S2). included 81 sequences from this study and 108 from Landry and Hebert [16]. We found low haplotype diversity Nuclear diversity and population structure within Australian P. xylostella, consistent with previous At five collection locations, P. australiana co-occurred reports [17, 18, 77]. Only five haplotypes were identi- with P. xylostella in sufficient numbers to enable compar- fied among 102 individuals, including three identified by ison of nuclear genomes, though the relative abundance Saw et al. [17] and three occurring in single individ- of species varied between locations. To ensure repre- uals (Fig. 3a). The most common haplotype, PxCOI01, sentation from the south-west region of Australia, the occurred at high frequency and differed by a single base Esperance population (n = 5) was formed by including one P. australiana individual from Boyup Brook. Despite only two P. xylostella individuals at Gilgandra, this population Table 2 Frequency of P. australiana in Plutella collections from had 17 P. australiana individuals and was included. To different Brassica host types generate nuclear SNP markers, we performed RADseq for Host No. No. P.aus No. No. P.aus a total of 52 P. australiana and 47 P. xylostella individuals. locations locations genotyped Illumina sequencing and demultiplexing using Wild brassicas 17 5 (0.29) 268 50 (0.19) RADtools [57] yielded 276.8 million raw sequence reads. Canola crops 39 14 (0.36) 848 93 (0.11) Following read quality filtering and mapping, genotypes Vegetable crops 16 1 (0.06) 287 4 (0.01) were called for 99 individuals from the two species. Hard filtering retained 300,241 confidently-called vari- Forage brassicas 3 0 (0.00) 44 0 (0.00) ant and invariant nuclear sites at a mean depth > 36 Presented are the numbers and proportion in parentheses of P. australiana across per individual, and a subset of 689 widely-dispersed collection locations and individuals genotyped Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 9 of 17 Fig. 2 Maximum likelihood phylogeny of 493 bp of Wolbachia wsp amplicons for Plutella and other arthropods. The strain infecting P. australiana (wAus)was identicaltoa Wolbachia supergroup B strain reported from Culex pipiens and Operophtera brumata. The strain infecting Australian P. xylostella was identical to a supergroup A strain (plutWA1) reported from Malaysian P. xylostella. Labels include the Wolbachia strain,hostspecies and GenBank accession number. Labels in bold denote strains sequenced in this study. The scale bar shows the mean number of nucleotide substitutions per site nuclear SNP variants (to avoid linkage bias) at a mean heterozygosity among individuals using 289,347 sites. depth > 36 per individual, for comparative analyses of Plutella australiana individuals had on average a 1.5-fold genetic diversity and population structure. The dataset higher proportion of heterozygous sites than P. xylostella with all confidently-called sites was used to estimate individuals (Fig. 4). population-level genetic diversity. Genetic structure among co-occurring populations Estimates of nuclear genetic diversity across 300,241 of Plutella species was investigated using 689 widely- variant and invariant sites revealed a striking contrast dispersed nuclear SNPs in the program STRUCTURE. between Plutella species, with notably higher diversity The delta K method predicted a strong optimal at K = 2 within populations of P. australiana than co-occurring genotypic clusters. Plutella australiana and P. xylostella populations of P. xylostella (Table 5). The mean observed individuals were clearly separated into distinct genotypic heterozygosity within populations ranged from 0.013– clusters in accordance to their species identified through 0.016 for P. australiana and 0.009–0.010 for P. xylostella. mtDNA genotypes regardless of geographic location Similarly, the average numbers of SNPs, indels and pri- (Fig. 5, left panel). Five individuals across four locations vate alleles were considerably higher within P. australiana showed greater than 1% admixture as shown by sharing of populations. As P. australiana may have fixed nucleotide colored bars. differences relative to the P. xylostella reference genome Assessing population structure from datasets with mul- that may affect population level statistics, we also removed tiple species can mask heirachical structure [78]. To indels from this dataset and directly compared the address this, genotypes were separately called for 52 Table 3 Fecundity of intra-species and reciprocal inter-species single pair crosses of P. australiana (P.aus) and P. xylostella (P.x) Cross (♀ × ♂) No. replicates No. reps eggs No. reps adults Mean ± SEM no. eggs Mean ± SEM no. adults P.aus♀ × P.aus♂ 42 37 (0.881) 34 (0.81) 40.86 ± 5.33 9.66 ± 1.7 P.x♀ × P.x♂ 63 59 (0.937) 59 (0.937) 83.82 ± 10.61 24.28 ± 3.27 P.aus♀ × P.x♂ 76 49 (0.645) 16 (0.211) 18.43 ± 3.02 1.17 ± 0.33 P.x♀ × P.aus♂ 85 62 (0.729) 3 (0.035) 15.16 ± 2.37 0.06 ± 0.03 Presented are the number and proportion in parentheses of replicates (reps) that produced eggs and adult offspring, and the mean ± standard error of the mean number of eggs and adult offspring per replicate Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 10 of 17 Table 4 Fecundity of hybrid F1 crosses and back-crosses Cross (♀ × ♂) No. replicates No. reps eggs No. reps adults Mean ± SEM no. eggs Mean ± SEM no. adults F0 P.aus♀ source (P.aus × P.x♂)♀ × (P.aus × P.x♂)♂ 4 4 (1.000) 2 (0.500) 66.00 ± 60.00 – (P.aus × P.x♂)♀ × P.aus♂ 7 7 (1.000) 0 (0.000) 20.33 ± 11.86 0.00 ± 0.00 P.aus♀ × (P.aus × P.x♂)♂ 9 5 (0.556) 2 (0.222) 6.38 ± 3.54 0.22 ± 0.44 (P.aus × P.x♂)♀ × P.x♂ 4 4 (1.000) 0 (0.000) 39.00 ± 19.00 0.00 ± 0.00 P.x♀ × (P.aus × P.x♂)♂ 15 15 (1.000) 4 (0.267) 36.75 ± 3.21 0.33 ± 0.62 F0 P.x♀ source (P.x × P.aus♂)♀ × (P.x × P.aus♂)♂ 6 5 (0.833) 4 (0.667) 74.50 ± 22.79 6.17 ± 5.27 (P.x × P.aus♂)♀ × P.aus♂ 1 0 (0.000) 0 (0.000) 0.00 0.00 Presented are the number and proportion in parentheses of replicates (reps) producing eggs and adult offspring, and the mean ± standard error of the mean numbers of eggs and adults offspring per replicate. A dash denotes an absence of count data P. australiana individuals, and hard filtering retained a set Insecticide susceptibility of 974 widely-dispersed SNP variants at a mean depth > Bioassays revealed highly contrasting responses to insec- 33 per individual for examination of finer scale structure ticide exposure in P. xylostella and P. australiana field among five populations. The delta K method predicted strains (Fig. 6). Plutella australiana showed extremely a weak modal signal at K = 3, but the highest likelihood high susceptibility to all four insecticides evaluated: resis- value occurred at K = 1. Bar plots for K =3showeda tance ratios at the LC and LC estimates were less than 50 99 high degree of admixture among individuals across the 1.0 and showed that this strain was 1.5-fold to 7.4-fold five populations, consistent with high levels of gene flow more susceptible than the laboratory P. xylostella (S) ref- across Australia (Fig. 5,right panel).Pairwise F was then erence (Additional file 1: Table S3). In contrast, resistance ST calculated for the five P. australiana populations using ratios at the LC for the field P. xylostella strain ranged 974 SNPs. The global estimate of F was not significantly from 2.9 for Success Neo to 41.4 for Dominex, indicat- ST different from zero, indicating the populations are not ing increased tolerance to all insecticides. Comparison of differentiated (F = 0.0002, 99% CI = -0.0274–0.0387). the LC estimates with commercial field doses for each ST 99 Further, pairwise F values were low, ranging from insecticide implies differences in field efficacy between ST –0.0041 to 0.0038, suggesting substantial gene flow among species. The commercial field rate of Dominex was > 8- populations separated by distances of between 341 and fold lower than the LC for P. xylostella, suggesting likely 2700 kilometres (Table 6). poor field control of this strain, but was > 17-fold higher ab Fig. 3 Mitochondrial DNA haplotype network for a P. xylostella (n = 102, 44 from this study, 58 from [16]) and b P. australiana (n = 87, 37 from this study, 50 from [16]) individuals from Australia based on a 613 bp COI sequence alignment. Haplotypes shared by more than one individual are shown in circles with a grey border with the number of individuals indicated inside the circle. Haplotypes connected by a line differ by a single mutation Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 11 of 17 Table 5 Population statistics for variant and invariant sites for sympatric populations of P. australiana (P. aus) and P. xylostella (P. x) from five locations Population Species n Sites Site depth SNPs Indels Private sites H H F O S IS Boomi NSW P. aus 11.1 276939 40 7198 1112 212 0.013 0.015 0.089 P. x 9.4 282989 42 4316 549 30 0.009 0.010 0.039 Calca SA P. aus 8.7 261496 30 6629 989 210 0.014 0.015 0.059 P. x 8.2 274973 42 4126 538 40 0.009 0.010 0.050 Esperance WA P. aus 4.5 269268 28 6543 998 210 0.016 0.015 -0.032 P. x 11.0 275299 35 4046 520 23 0.010 0.010 0.019 Gilgandra NSW P. aus 15.7 277136 39 7154 1088 212 0.014 0.015 0.079 P. x 1.9 277846 42 4149 505 28 0.009 0.009 -0.056 Goulburn NSW P. aus 6.8 256343 29 6471 968 190 0.013 0.015 0.058 P. x 12.8 274700 36 4052 513 26 0.009 0.010 0.052 n, number of individuals genotyped per locus; H , observed heterozygosity; H , gene diversity; F , Nei’s inbreeding coefficient O S IS than the LC for P. australiana (Fig. 6). Control mortality sampling to Brassica vegetable farms. Landry and Hebert was similar for the field and reference strains, averaging [16] also isolated DNA from legs, keeping most of each 3.1 to 4.4% across all bioassays. specimen intact and providing a morphological reference for examining unexpected genotypes. It is also possi- Discussion ble that P. australiana was previously overlooked from Cryptic species arise when divergence does not lead to nuclear DNA studies due to biases in amplification of morphological change [79]. The recent discovery of a divergent alleles. Here, we sought to determine whether cryptic ally, P. australiana, to the diamondback moth, P. australiana is an agricultural pest, and to understand its P. xylostella, was unexpected given the breadth of previ- ecological and genetic differences from P. xylostella. ous molecular studies of this insect. Several factors may Extensive larval sampling from wild and cultivated bras- have contributed to this discovery, including the use of sicaceous plants revealed that P. australiana co-occurs light traps for specimen collection, rather than limiting widely with P. xylostella throughout southern Australia and utilizes some of the same host plants. The relative abundance of P. australiana was on average 9-fold lower than P. xylostella. We observed higher proportions of P. australiana in larval collections from the eastern state of New South Wales, similar to the light trap samples from Landry and Hebert [16], possibly reflecting habitat suitability. Although we did not detect P. australiana in limited sampling from the island state of Tasmania, the presence of brassicas in the region and evidence from light traps that wind currents can transport Plutella moths across Bass Strait (Lionel Hill, Pers. Comm.) suggest it is likely to occur there. Our study confirms that the host range of P. australiana includes canola crops and wild brassicaceous species. In laboratory rearing, P. australiana completed develop- ment on sand rocket, D. tenuifolia, and canola, B. napus, and was also collected from several other wild species, though without rearing to confirm host status. Our sam- pling focused on relatively few introduced brassicaceous species common in agricultural areas, yet the Australian Fig. 4 Boxplot showing the proportion of heterozygous sites across Brassicales is represented by 11 plant families [80], 289,347 confidently-called nuclear sites for individuals of P. xylostella including several non-Brassicaceae on which P. xylostella (light grey boxes, n = 47) and P. australiana (dark grey boxes, n = 52) from five locations. Heterozygosity was consistently higher in and its allies have been documented feeding, such as P. australiana Capparaceae [24], Cleomaceae [16] and Tropaeolaceae Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 12 of 17 Plutella australiana Plutella xylostella Plutella australiana K = 2 K = 3 Fig. 5 Proportional assignment of Plutella individuals to genotypic clusters, K, based on STRUCTURE analysis. Individuals are represented by vertical bars and genotypic clusters are represented by different colors. Left panel: Analysis at K = 2 for 52 P. australiana and 47 P. xylostella individuals sorted left-to right by proportion of cluster membership. The predominantly red bars correspond to P. australiana individuals and the predominantly blue bars correspond to P. xylostella individuals identified through mtDNA genotypes. Locations are labelled for five individuals showing > 1% genotypic admixture. Right panel: Analysis at K = 3 for 52 P. australiana individuals sorted left-to-right by proportion of cluster membership within geographic locations, showing a high degree of genotypic admixture among individuals across locations [23]. The Australian Brassicaceae has records for 61 replacing cabbage with Diplotaxis seedlings, egg-laying genera and 205 species [80], including many introduced then occurred within 24 h. Exposure to host plants stim- species but also a diversity of native genera, such as Lep- ulates reproductive behaviour in P. xylostella [82], but idium, Blennodium,and Arabidella, that occur over vast olfactory cues for host recognition or oviposition [83–85] areas of Australia. Wider sampling of native Brassicales may differ between these Plutella species. Host prefer- may identify other suitable hosts for P. australiana. ence and performance studies are required to test these Plutella australiana larvae were not identified among hypotheses. samples from sixteen commercial Brassica vegetable Insecticide bioassays have been conducted routinely crops despite the high suitability of these crops for P. on Australian P. xylostella to monitor levels of insec- xylostella [81], however eggs were collected from kale. ticide resistance in field populations [22, 25]. This It is possible that extreme insecticide susceptibility pre- method appears unlikely to be affected by the presence vents juvenile P. australiana populations from establish- of P. australiana under typical conditions, as a period ing, as commercial Brassica vegetable crops are typi- of laboratory rearing is usually necessary to multiply cally sprayed multiple times per crop cycle [22]. Our data individuals prior to screening. In our experience, lab- show that P. australiana is far more susceptible than P. oratory rearing of the two Plutella species on cabbage xylostella to four commonly used insecticides. At com- plants selects against P. australiana individuals when mercial application rates, these insecticides are likely to competing with P. xylostella in cages, causing the com- provide high-level control of P. australiana in Australian plete loss of P. australiana within a few generations. The Brassica crops, but some products may provide marginal reasons for this are unknown but may include differ- or poor control against P. xylostella due to insecticide ences in host preference or development rate, or direct resistance (Fig. 6)[22, 25]. Alternatively, some vegetable competition. cultivars may not be attractive for oviposition or suit- Crossing experiments revealed that hybridization can able for larval survival in P. australiana.Wenoted that occur between P. australiana and P. xylostella under con- P. australiana cultures provided with cabbage seedlings trolled conditions and is most likely to occur in crosses failed to produce viable eggs over seven days, but after involving Wolbachia-infected P. australiana females. Hybridization occurs in around 10% of animal species, particularly in captivity [86], but asymmetric reproduc- Table 6 Pairwise comparisons of Weir and Cockerham’s [68] F ST tive isolation is commonly observed in reciprocal crosses (below diagonal) and geographic distance in kilometres (above between taxa [87]. In our experiments, a strong male diagonal) among populations of P. australiana from five locations bias in the offspring of interspecific crosses and failure to Boomi Calca Esperance Gilgandra Goulburn back-cross hybrid females both follow Haldane’s rule [88], which predicts greater hybrid inviability or sterility in the Boomi – 1555 2714 341 677 heterogametic sex (female, in Lepidoptera). This pattern Calca -0.0041 – 1167 1365 1434 can arise from epistatic interactions between sex-linked Esperance 0.0038 0.0014 – 2531 2572 and/or autosomal genes that result in genetic incompati- Gilgandra 0.0000 0.0036 -0.0005 – 364 bilities [89, 90]. Although the back-crosses with F1 hybrid Goulburn -0.0015 -0.0014 0.0034 0.0005 – females were sterile, the back-crosses with hybrid males (to both species) were viable, which could enable the Exact G tests were non-significant for all population pairs (p > 0.05) Gilgandra Boomi Calca Esperance Esperance Calca Goulburn Gilgandra Boomi Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 13 of 17 Fig. 6 Insecticide bioassay dose-response curves for P. australiana (dotted line) and P. xylostella (dashed line) field strains collected from Angle Vale and Urrbrae, South Australia, and a susceptible P. xylostella (S) reference strain (solid line), exposed to four commercial insecticides: Dominex, Coragen, Proclaim and Success Neo. Points are the mean observed response across 4 bioassay replicates of 10 larvae each and lines are the fitted log-logistic response curves with 95% confidence intervals shown in grey shading. The vertical red line represents the approximate commercial field dose for each insecticide and vertical black lines represent the estimated LC for the corresponding Plutella strain transfer of genes between hybrid and/or parental species. However, both species showed limited mtDNA diversity However, it is unclear whether hybridization occurs in with a single predominant haplotype. While outgroups the wild. from other continents were not available, comparative Although P. australiana and P. xylostella show deep analysis of these closely-related Australian Plutella species divergence (8.6%) in mtDNA [16], the sole use of mtDNA suggested that patterns of mitochondrial and nuclear can be unreliable for inference of evolutionary history diversity are concordant in P. xylostella and consistent and should be corroborated using evidence from nuclear with a demographic bottleneck [17, 18], but discordant in markers [34]. Our analysis revealed striking differences in P. australiana. nuclear diversity across the genome between co-existing Sequence variation in mitochondrial DNA can be populations of each Plutella species collected at the strongly influenced by Wolbachia infection [41]. Extensive same locations and times, and from the same host plant Wolbachia screening showed that each Plutella species species. Plutella xylostella populations from Australia and was infected with a different strain at contrasting fre- New Zealand have low levels of genetic diversity com- quencies, and fit a ‘most-or-few’ pattern whereby species pared with populations from other continents, thought infection rates are often very low (<10%) or very high to reflect the recent introduction of this species from (> 90%) [91]. Infection incidence in P. xylostella was lower a small founding population [14, 17, 77]. Consistent in Australia (1%) than previously reported across global with this view, we found a remarkable 1.5-fold reduction samples (5%) [18]. Our finding of a single supergroup in heterozygosity across > 300,000 sites in P. xylostella A strain showing 100% sequence similarity to a strain compared with sympatric P. australiana populations. reported in P. xylostella from Malaysia, plutWA1 [18], Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 14 of 17 provides some support of an Asian origin for Australian mechanisms, such as assortive mating or hybrid fitness P. xylostella [17], though does not preclude this strain also costs. Behavioural mating choices are often the main iso- occurring elsewhere. lating factor in sympatric animals [86]. Does Wolbachia Fixation of infection in P. australiana suggests that cause a reproductive barrier? The contrast in infection Wolbachia manipulates the reproductive biology of this status creates the potential for cytoplasmic incompati- species. We found no evidence of sex-ratio distortion, bility between species [94]. Interspecific crosses showed which has been associated with a Wolbachia strain, a pattern of asymmetric isolation consistent with the plutWB1,in P. xylostella [18]. High infection can be expected effects of unidirectional CI, where 21% crosses driven by cytoplasmic incompatibility (CI) [40]. The high involving infected P. australiana females produced viable frequency (87%) of a single mtDNA haplotype among offspring, while the reciprocal CI-cross direction (unin- P. australiana individuals implies that the spread of Wol- fected P. xylostella females crossed with infected P. aus- bachia infection has driven a selective sweep of co- traliana males) wasnearlysterile.However,thispattern inherited mtDNA through the population, causing a loss was not continued in the F1 generation: infected hybrid of mtDNA diversity [41]. High nuclear diversity (rela- males (derived from the P. australiana maternal line) pro- tive to sympatric P. xylostella) supports this hypothesis, duced offspring at comparable rates when back-crossed to because a demographic bottleneck should reduce diversity either uninfected P. xylostella or infected P. australiana across the entire genome [34]. female parents. The role of Wolbachia-induced postzy- Plutella australiana and P. xylostella have co-existed gotic isolation between the two Plutella species requires in Australia for at least 125 years (1300 generations), further study, though our results suggest it could be more yet have strongly divergent mitochondrial and nuclear important in the F0 generation. Wolbachia can contribute genomes, Wolbachia infections and insecticide suscep- to post-zygotic genetic isolation after speciation by com- tibility phenotypes. Our observations during laboratory plementing hybrid incompatibilities [94, 95]. Symbiont rearing and crossing experiments also suggested that infections could also influence mating behaviour and con- interspecific differences in host plant use may exist. What tributetopre-matingisolation [96]. explains such strong divergence between the two Plutella species, given sympatry and the capacity to hybridize? Conclusions Endemism of P. australiana [16] implies an ancient evo- The discovery of cryptic pest species introduces com- lutionary history in Australia, and our data provide sup- plexities for their management and also exciting oppor- port for existing views that Australian P. xylostella were tunities for understanding ecological traits. We found recently introduced from a small ancestral source pop- strong genomic and phenotypic divergence in two cryp- ulation, possibly from Asia [17, 18, 77]. Therefore, the tic mitochondrial Plutella lineages co-existing in nature, two Plutella species may have diverged in allopatry and supporting their status as distinct species [16] despite the recently come into secondary contact. Maintenance of capacity to hybridize. Reproductive isolation is likely to divergence suggests strong continuing reproductive isola- have evolved during allopatric speciation, and genome- tion, which can evolve as a side-effect of allopatric diver- wide sequence data suggest it has been maintained follow- gence [44]. All 99 individuals that were RAD sequenced ing secondary contact. Variation in Wolbachia infections showed concordance in nuclear multilocus genotypes and might be one factor reinforcing reproductive barriers. mtDNA genotypes identified through PCR-RFLP regard- Plutella australiana co-occurs with P. xylostella less of geographic location, as shown by STRUCTURE throughout agricultural regions of southern Australia, analysis. Cryptic species in sympatry provides strong evi- but made up only 10% of Plutella juveniles collected dence of limited genetic exchange [79]. A small degree from cultivated and wild brassicaceous plants. A lack of genotypic admixture evident for a few individuals in of population structure across neutral SNP markers the STRUCTURE plots might be explained by ances- suggests that P. australiana populations are linked by tral polymorphism or introgressive hybridization [28], or high levels of gene flow, and also that P. australiana is a alternatively, could be an artefact if our dataset is not highly mobile species, which is supported by light trap representative of the entire genetic background [33]. The collections [16] and seasonal colonization of canola crops. level of hybridization that may be occurring between Future molecular analysis of Australian Plutella should these species is unknown. Isolation may not be uniform include a species identification step using a molecular across the genome [92, 93], and scans of larger genomic diagnostic assay. 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Cryptic Plutella species show deep divergence despite the capacity to hybridize

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Life Sciences; Evolutionary Biology; Animal Systematics/Taxonomy/Biogeography; Entomology; Genetics and Population Dynamics; Life Sciences, general
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Abstract

Background: Understanding genomic and phenotypic diversity among cryptic pest taxa has important implications for the management of pests and diseases. The diamondback moth, Plutella xylostella L., has been intensively studied due to its ability to evolve insecticide resistance and status as the world’s most destructive pest of brassicaceous crops. The surprise discovery of a cryptic species endemic to Australia, Plutella australiana Landry & Hebert, raised questions regarding the distribution, ecological traits and pest status of the two species, the capacity for gene flow and whether specific management was required. Here, we collected Plutella from wild and cultivated brassicaceous plants from 75 locations throughout Australia and screened 1447 individuals to identify mtDNA lineages and Wolbachia infections. We genotyped genome-wide SNP markers using RADseq in coexisting populations of each species. In addition, we assessed reproductive compatibility in crossing experiments and insecticide susceptibility phenotypes using bioassays. Results: The two Plutella species coexisted on wild brassicas and canola crops, but only 10% of Plutella individuals were P. australiana. This species was not found on commercial Brassica vegetable crops, which are routinely sprayed with insecticides. Bioassays found that P. australiana was 19-306 fold more susceptible to four commonly-used insecticides than P. xylostella. Laboratory crosses revealed that reproductive isolation was incomplete but directionally asymmetric between the species. However, genome-wide nuclear SNPs revealed striking differences in genetic diversity and strong population structure between coexisting wild populations of each species. Nuclear diversity was 1.5-fold higher in P. australiana, yet both species showed limited variation in mtDNA. Infection with a single Wolbachia subgroup B strain was fixed in P. australiana, suggesting that a selective sweep contributed to low mtDNA diversity, while a subgroup A strain infected just 1.5% of P. xylostella. Conclusions: Despite sympatric distributions and the capacity to hybridize, strong genomic and phenotypic divergence exists between these Plutella species that is consistent with contrasting colonization histories and reproductive isolation after secondary contact. Although P. australiana is a potential pest of brassicaceous crops, it is of secondary importance to P. xylostella. Keywords: Plutella australiana, Plutella xylostella, Lepidoptera, hybridization, sympatric, insecticide resistance, Wolbachia *Correspondence: kym.perry@sa.gov.au School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, 5005, Australia South Australian Research and Development Institute, Adelaide, 5001, Australia Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 2 of 17 Background control during outbreaks [22, 25]. Plutella xylostella is well Cryptic species can show remarkable diversity in aspects known as a migratory insect with a high capacity for gene of their ecology, behaviour, and at the level of the genome. flow [11, 13], facilitating the rapid spread of resistance They exist across metazoan taxa [1], including globally alleles. Australian P. xylostella are thought to disperse fre- important arthropod pest taxa, such as whiteflies [2], quently, based on indirect evidence from ecological and disease-vectoring mosquitoes [3], fruit flies [4], thrips genetic studies [14, 15, 26]. Most studies have found a [5, 6] and mites [7, 8], some of which are characterised lack of genetic differentiation at microsatellite loci and low by cryptic species complexes. Discovering cryptic diver- sequence variation in mitochondrial DNA markers among sity has important consequences for estimates of global Australian and New Zealand populations of P. xylostella, biodiversity, conservation planning, and the management consistent with high gene flow and/or recent ancestry of pests and diseases. Morphologically similar species [14, 15, 17, 18]. While species identification was not in can vary in pest status due to differences in genotypic question in these studies, somewhat inconsistent find- and/or phenotypic traits that influence their host range ings in two studies from eastern Australia using allozymes and specificity, geographic distribution, the ability to vec- or SSR markers [19, 20] might reflect the confounding tor diseases, or insecticide resistance [8–10]. Therefore, presence of P. australiana samples [16]. Given these con- recognising cryptic species and the differences in their siderations, future management of Plutella in Australian biology and ecology are essential for effective manage- crops will require thorough understanding of the ecolog- ment, with important implications for public health, agri- ical requirements, genetic traits and pest status of the culture and trade. two Plutella species. In addition, reproductive isolation The diamondback moth, Plutella xylostella,isthe major between these two species is unknown but has impli- pest of brassicaceous crops worldwide, costing an esti- cations for evolutionary inference and the potential for mated US$4 to US$5 billion annually in direct losses gene flow. The capacity for hybridization and introgres- and management costs [11, 12]. Insecticide resistance sion could lead to the exchange of insecticide resistance or is widespread in P. xylostella populations around the other adaptive alleles [27, 28]. world, fuelling wide-ranging research to develop alter- Although mtDNA markers are widely used in stud- native management tactics [11, 13]. Plutella xylostella ies of species identity and population structure [29–31], was initially recorded in Australia in the late 1800s and mitochondrial variation within or between species can rapidly became a widespread pest of Brassica vegetables, be influenced by direct and/or indirect selection, or and then canola following its expanded production from introgressive hybridization [32, 33]. One factor that can the 1990s [14, 15]. Recently, Landry and Hebert [16], confound mtDNA-based inference is interaction with through mtDNA barcoding, identified a cryptic lineage inherited bacterial symbionts [34, 35]. Wolbachia is of Plutella in Australia not detected in previous molec- a widespread endosymbiont thought to infect at least ular studies of P. xylostella [14, 17–21]. Although exter- half of arthropod [36] and 80% of lepidopteran [37] nal morphology was indistinguishable from P. xylostella, species. It is mainly transmitted vertically from infected deep mtDNA divergence (8.6%), differences in geni- females to their offspring through the egg cytoplasm, and tal morphology and endemism in Australia led them inheritance is therefore linked with mtDNA. To facili- to describe a new species, Plutella australiana Landry tate its spread, Wolbachia manipulates host reproduc- &Hebert. Plutella australiana was originally collected tive biology to favour the fitness of infected females by together with P. xylostella in light trap samples in east- inducing host phenotypes that distort sex ratios (male- ern Australia, suggesting at least some ecological over- feminization, male-killing or induction of parthenogene- lap [16], but its biology, ecology and pest status were sis) or cause sperm-egg cytoplasmic incompatibility (CI) unknown. [38, 39]. In the simple case involving a single CI-inducing The management of P. xylostella in Australian Brassica strain, crosses with infected females are fertile but crosses crops has been a significant challenge for decades between uninfected females and infected males fail to [15, 22], but the discovery of P. australiana has made produce offspring. If maternal transmission is efficient the relative abundance and pest status of both species in and infected females have a reproductive advantage, these crops uncertain. With rare exception, P. xylostella Wolbachia infection can spread rapidly through an insect and allied species feed on plants in the order Brassicales, population [40], driving a selective sweep of a single hap- mainly within the family Brassicaceae [16, 23, 24], imply- lotype and reducing mtDNA diversity [41]. Limited sur- ing that the host range of P. australiana may include culti- veys to date have identified Wolbachia strains infecting vated brassicas. Widespread resistance to pyrethroid and P. xylostella at low frequency in populations from North organophosphate insecticides has been attributed to Aus- America, Africa, Asia and Europe [18, 42, 43]. Because tralian populations of P. xylostella from all vegetable and symbionts can contribute to reproductive isolation and canola production regions, which has led to ineffective influence mtDNA diversity [34, 44], assessing their role Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 3 of 17 ◦ ◦ can provide important insights into host evolution and 2 min, then 35 cycles at 95 C for 10 s, 52 C for 20 s, ◦ ◦ population structure [35, 45–47]. 72 C for 30 s followed by a 5 min final extension at 72 C. Here we investigated the biology, ecology and popula- PCR products were digested at 37 Cfor 1hwith 1unit tion genetic structure of two cryptic Plutella species by of AccI (NEB) restriction enzyme with 2 μLCutsmart collecting Plutella from brassicaceous plants throughout Buffer in a 20 μL reaction. Following digestion, products Australia and screening individuals to identify mtDNA were separated using agarose gel electrophoresis (1.5%). lineages and Wolbachia infections. For a subset of pop- Plutella xylostella products are approximately 516 bp and ulations, we examined genetic diversity using thousands 191 bp and P. australiana products are 348 bp and 359 bp of nuclear SNPs from across the genome. In addition, we [49]. To examine mtDNA haplotypes, sequencing of the assessed reproductive compatibility in laboratory crosses 707 bp COI amplicon was performed for 44 P. xylostella and determined the susceptibility of each species to com- and 37 P. australiana individuals at the Australian mercial insecticides. Genome Research Facility (AGRF). In addition, we down- loaded sequence trace files from Landry and Hebert Methods [16] (dx.doi.org/10.5883/DS-PLUT1) and re-analysed, Sample collection aligned and trimmed all sequences in GENEIOUS version Plutella larvae (rarely, eggs or pupae) were collected 10.0.6 [50]. Haplotype networks were constructed using from canola crops, Brassica vegetable crops, forage bras- R package pegas version 0.9 [51]. sicas and wild brassicas throughout Australia between March 2014 and December 2015 (Table 1). The wild Wolbachia screening and phylogenetics species included wild radish, Raphanus raphanistrum, Wolbachia infection was detected using two separate PCR turnip weed, Rapistrum rugosum, sea rocket, Cakile mar- assays of the 16S rRNA gene (16S-2 and 16S-6) accord- itima, Ward’s weed, Carrichtera annua, African mustard, ing to Simoes et al. [52]. To identify Wolbachia strains, the Brassica tournefortii, and mixed stands of sand rocket, Wolbachia surface protein (wsp) gene was sequenced in a Diplotaxis tenuifolia, and wall rocket, D. muralis.Ateach subset of individuals. Amplification was performed using location, at least 25 individuals were collected from ran- wsp81F and wsp691R sequence primers [53]. Amplicons domly selected plants to achieve a representative sample. were sequenced using the reverse primer and aligned in Insect samples were collected from Brassica vegetables by GENEIOUS version 10.0.6 [50]. We used a 493 bp align- hand, from sea rocket by beating plants over a collection ment to construct a maximum likelihood phylogeny in tray and from other hosts using a sweep net. Each pop- RAxML version 8.2.4 [54] using a general time reversal ulation sample was separately reared in ventilated plastic substitution model [55] with 1000 bootstrap replicates. containers on leaves of the original host material for 1–2 days and thereafter on cabbage leaves. Non-parasitised RADseq library preparation and sequencing pupae or late-instar larvae were fresh frozen at − 80 C. Libraries were prepared for restriction-site-associated DNA sequencing (RADseq) according to a protocol mod- DNA isolation and COI genotyping ified from Baird et al. [56]. Genomic DNA was quantified For each population sample, we aimed to genotype a using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng minimum of 16 individuals where possible after removing digested with 10 units of high fidelity SbfI in Cutsmart parasitized individuals. Individual pupae (but not larvae) Buffer (NEB) for 1 h at 37 C, then heat inactivated at were sexed under a dissecting microscope, then genomic 80 C for 20 min. One microlitre of P1 adapter (100nM) DNA was isolated by homogenising whole individuals with a 6-base molecular identifier (MID) (top strand followed by two phenol and one chloroform extrac- 5 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG tionsaccordingtoZraketetal. [48]. DNA was treated xxxxxxTGCA-3 , bottom strand 5 -[P]xxxxxxCTGTCTC with RNase A, then precipitated and re-suspended in TTATACACATCTGACGCTGCCGACGA-3,xrepre- TE buffer. Plutella lineages were distinguished using a sents sites for MIDs) were then added using 0.5 μLT4 PCR-RFLP assay [49]. A 707 bp COI region was amplified DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. using a combination of two primer pairs: (i) PxCOIF (5 - Library pools were sheared using a Bioruptor sonicator TCAACAAATCATAAAGATATTGG-3 ) and PxCOIR (Diagenode), then DNA fragments end-repaired using (5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ), and a Quick Blunting Kit (NEB), adenine overhangs added (ii) PaCOIF (5 -TCAACAAATCATAAGGATATTGG-3 ) then P2 adapters (top strand 5 -[P]CTGTCTCTTATA and PaCOIR (5 -TAAACCTCTGGATGGCCAAAAAA CACATCTCCAGAATAG-3 , bottom strand 5 -GTCTCG TCA-3 ). Ten microliter reactions were run with 2 μLof TGGGCTCGGAGATGTGTATAAGAGACAGT-3)lig- MyTaq 5x buffer, 0.2 μL of each primer (10mM stocks), ated. DNA purification between steps was performed 1 μL of DNA (approx. 5 ng) and 0.05 μLofMyTaq poly- using a MinElute PCR purification kit (Qiagen). Libraries were amplified using KAPA HiFi Hotstart Readymix merase (Bioline). Samples were amplified at 95 Cfor Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 4 of 17 Table 1 Collection details showing the frequency (f)of Plutella species and Wolbachia infections among Plutella populations from Australia P. australiana P. xylostella Location Collection date Latitude Longitude Host No. genotyped No. (f)No.(f)No.(f ) wol-infected ◦ ◦ Boomi NSW Sep-2014 -28.76 149.81 Canola 25 15 (0.60) 10 (0.40) 0 (0.00) ◦ ◦ Gilgandra NSW Sep-2014 -31.67 148.72 Wild turnip 23 21 (0.91) 2 (0.09) 0 (0.00) ◦ ◦ Ginninderra NSW Sep-2014 -35.19 149.05 Canola 15 2 (0.13) 13 (0.87) 0 (0.00) ◦ ◦ Ginninderra NSW Oct-2015 -35.19 149.05 Canola 34 27 (0.79) 7 (0.21) 0 (0.00) ◦ ◦ Goulburn NSW Nov-2015 -34.84 149.67 Canola 32 25 (0.78) 7 (0.22) 0 (0.00) ◦ ◦ Henty NSW Oct-2014 -35.60 146.95 Canola 18 1 (0.06) 17 (0.94) 0 (0.00) ◦ ◦ Narromine NSW Sep-2014 -32.22 148.03 Canola 26 0 (0.00) 26 (1.00) 1 (0.04) ◦ ◦ Richmond NSW Oct-2015 -33.60 150.71 Cabbage 21 0 (0.00) 21 (1.00) 0 (0.00) ◦ ◦ Wagga Wagga NSW Sep-2014 -35.04 147.33 Canola 21 5 (0.24) 16 (0.76) 0 (0.00) ◦ ◦ Werombi NSW Nov-2014 -33.99 150.64 Vegetables 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werombi NSW Oct-2015 -34.00 150.56 Kale 13 4 (0.31) 9 (0.69) 0 (0.00) ◦ ◦ Bundaberg QLD Oct-2014 -24.80 152.26 Canola 14 1 (0.07) 13 (0.93) 0 (0.00) ◦ ◦ Bundaberg QLD Sep-2015 -24.80 152.26 Canola 30 0 (0.00) 30 (1.00) 0 (0.00) ◦ ◦ Cunnamulla QLD Sep-2015 -28.07 145.68 African mustard 17 17 (1.00) 0 (0.00) 0 – ◦ ◦ Dalby QLD Sep-2014 -27.28 151.13 Canola 30 0 (0.00) 30 (1.00) 0 (0.00) ◦ ◦ Gatton QLD Oct-2014 -27.54 152.33 Broccoli 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Gatton QLD Nov-2015 -27.54 152.33 Broccoli 15 0 (0.00) 15 (1.00) 0 (0.00) ◦ ◦ Warwick QLD Oct-2015 -28.21 152.11 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Calca SA Apr-2014 -33.02 134.28 Sand rocket, Wall rocket 13 8 (0.62) 5 (0.38) 0 (0.00) ◦ ◦ Cocata SA Sep-2014 -33.20 135.13 Canola 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Colebatch SA Feb-2015 -35.97 139.66 Forage brassica 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Coonalpyn SA Oct-2015 -35.62 139.91 Wild radish 11 0 (0.00) 11 (1.00) 0 (0.00) ◦ ◦ Cowell SA Sep-2014 -33.66 137.16 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Keith SA Oct-2014 -36.09 140.29 Canola 32 0 (0.00) 32 (1.00) 6 (0.19) ◦ ◦ Lameroo SA Sep-2014 -35.32 140.51 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Lameroo SA Oct-2015 -35.17 140.48 Canola 14 0 (0.00) 14 (1.00) 0 (0.00) ◦ ◦ Littlehampton SA Oct-2014 -35.06 138.90 Cabbage 34 0 (0.00) 34 (1.00) 6 (0.18) ◦ ◦ Littlehampton SA Sep-2015 -35.06 138.90 Brussels sprouts 8 0 (0.00) 8 (1.00) 0 (0.00) ◦ ◦ Loxton SA Sep-2014 -34.37 140.72 Canola 31 0 (0.00) 31 (1.00) 0 (0.00) ◦ ◦ Loxton SA Oct-2015 -34.50 140.80 Canola 14 1 (0.07) 13 (0.93) 0 (0.00) ◦ ◦ Mallala SA Sep-2015 -34.38 138.50 Canola 26 0 (0.00) 26 (1.00) 0 (0.00) ◦ ◦ Meribah SA Sep-2014 -34.74 140.82 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Millicent SA Apr-2015 -37.61 140.34 Canola 9 0 (0.00) 9 (1.00) 2 (0.22) ◦ ◦ Minnipa SA Oct-2015 -32.81 135.16 Canola 22 1 (0.05) 21 (0.95) 0 (0.00) ◦ ◦ Moonaree SA Aug-2014 -31.99 135.87 Ward’s weed 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Mt Hope SA Sep-2014 -34.14 135.33 Canola 29 0 (0.00) 29 (1.00) 0 (0.00) ◦ ◦ Mt Hope SA Sep-2015 -34.20 135.34 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Padthaway SA Oct-2015 -36.56 140.43 Canola 18 2 (0.11) 16 (0.89) 0 (0.00) ◦ ◦ Picnic Beach SA Apr-2014 -34.17 135.27 Sea rocket 2 0 (0.00) 2 (1.00) 0 (0.00) ◦ ◦ Picnic Beach SA Sep-2014 -34.17 135.27 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Redbanks SA Oct-2014 -34.49 138.59 Canola 38 0 (0.00) 38 (1.00) 1 (0.03) ◦ ◦ Sherwood SA Oct-2014 -36.05 140.64 Wild radish 8 0 (0.00) 8 (1.00) 0 (0.00) Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 5 of 17 Table 1 Collection details showing the frequency (f)of Plutella species and Wolbachia infections among Plutella populations from Australia (Continued) P. australiana P. xylostella Location Collection date Latitude Longitude Host No. genotyped No. (f)No.(f)No.(f ) wol-infected ◦ ◦ Southend SA Apr-2015 -37.57 140.12 Sea rocket 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Tintinara SA Oct-2015 -35.97 139.66 Forage Brassica 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Ucontichie SA Sep-2014 -33.22 135.31 Canola 3 0 (0.00) 3 (1.00) 0 (0.00) ◦ ◦ Virginia SA Oct-2014 -34.64 138.54 Broccoli 18 0 (0.00) 18 (1.00) 1 (0.06) ◦ ◦ Virginia SA Sep-2015 -34.64 138.54 Cabbage 23 0 (0.00) 23 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Sep-2014 -33.55 134.86 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Mar-2015 -33.55 134.86 Sea rocket 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Walkers Beach SA Sep-2015 -33.55 134.86 Sea rocket 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Wirrabara SA Oct-2014 -32.99 138.31 Canola 28 2 (0.07) 26 (0.93) 0 (0.00) ◦ ◦ Wokurna SA Sep-2015 -33.67 137.96 Wild radish 24 1 (0.04) 23 (0.96) 0 (0.00) ◦ ◦ Wurramunda SA Apr-2014 -34.30 135.56 Wild canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Deddington TAS Nov-2014 -41.59 147.44 Kale 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Launceston TAS Nov-2014 -41.47 147.14 Wild mustard 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Newstead TAS Nov-2015 -41.59 147.44 Cauliflower 22 0 (0.00) 22 (1.00) 0 (0.00) ◦ ◦ Cowangie VIC Oct-2015 -35.10 141.33 Canola 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Ouyen VIC Sep-2014 -35.00 142.31 Canola 28 1 (0.04) 27 (0.96) 0 (0.00) ◦ ◦ Robinvale VIC Sep-2014 -34.81 142.94 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werribee VIC Oct-2014 -37.94 144.73 Cauliflower 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Werribee VIC Nov-2015 -37.94 144.73 Cauliflower 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Yanac VIC Sep-2014 -36.06 141.25 Canola 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Boyup Brook WA Sep-2014 -33.64 116.40 Canola 26 2 (0.08) 24 (0.92) 0 (0.00) ◦ ◦ Dalwallinu WA Sep-2015 -30.28 116.66 Canola 20 0 (0.00) 20 (1.00) 0 (0.00) ◦ ◦ Dalyup WA Oct-2015 -33.72 121.64 Wild radish 22 3 (0.14) 19 (0.86) 0 (0.00) ◦ ◦ Esperance WA Sep-2014 -33.29 121.76 Canola 23 8 (0.35) 15 (0.65) 1 (0.07) ◦ ◦ Esperance WA Oct-2015 -33.79 122.13 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) ◦ ◦ Gingin WA Dec-2014 -31.28 115.65 Red cabbage 23 0 (0.00) 23 (1.00) 1 (0.04) ◦ ◦ Kalannie WA Sep-2015 -30.00 117.25 Canola 18 0 (0.00) 18 (1.00) 0 (0.00) ◦ ◦ Manjimup WA Dec-2014 -34.18 116.23 Chinese cabbage 17 0 (0.00) 17 (1.00) 0 (0.00) ◦ ◦ Manjimup WA Nov-2015 -34.18 116.23 Brassica vegetables 13 0 (0.00) 13 (1.00) 0 (0.00) ◦ ◦ Narrogin WA Oct-2015 -32.95 117.32 Wild radish, wild canola 15 0 (0.00) 15 (1.00) 0 (0.00) ◦ ◦ Narrogin WA Oct-2015 -32.96 117.33 Canola 32 0 (0.00) 32 (1.00) 0 (0.00) ◦ ◦ Walkaway WA Sep-2014 -28.94 114.83 Canola 19 0 (0.00) 19 (1.00) 0 (0.00) ◦ ◦ Walkaway WA Sep-2014 -28.16 114.63 Canola 16 0 (0.00) 16 (1.00) 0 (0.00) Total 1447 147 (0.10) 1300 (0.90) 19 (0.01) Australian states: NSW = New South Wales, QLD = Queensland, SA = South Australia, TAS = Tasmania, VIC = Victoria, WA = Western Australia All P. australiana individuals were infected with Wolbachia (Kapa Biosystems) and Nextera i7 and i5 indexed primers Illumina paired-end sequencing was performed using ◦ ◦ with PCR conditions: 95 C for 3 min, two cycles of 98 C HiSeq2500 (100 bp) or NextSeq500 (75 bp) at the AGRF. ◦ ◦ for 20 s, 54 C for 15 s, 72 C for 1 min, then 15 cycles of ◦ ◦ ◦ 98 C for 20 s, 65 C for 15 s, 72 C for 1 min followed by Read filtering and variant calling a final extension of 72 C for 5 min. Libraries were size- Sequence reads were demultiplexed using RADtools selected (300-700 bp) on 1–1.5% agarose gel and purified version 1.2.4 [57] allowing one base MID mismatch, using a minElute Gel Extraction Kit (Qiagen), then then TRIMMOMATIC version 0.32 [58]was used Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 6 of 17 to remove restriction sites, adapter sequences and a population pairs were calculated and significance deter- 4 3 thymine base from reverse reads introduced by the mined using exact G tests (10 mc burnins, 10 batches, P2 adapter, and quality filter using the ILLUMINA- and 10 iterations per batch) in GENEPOP version 4.6 CLIP tool with parameters: TRAILING:10 SLIDING- [70] after Bonferroni correction for multiple comparisons. WINDOW:4:15 MINLEN:40. Paired reads were aligned Separate analysis of population structure was performed to the P. xylostella reference genome (accession num- using the Bayesian clustering program STRUCTURE ver- ber: GCF_000330985.1) using STAMPY version 1.0.21 sion 2.3.4 [71], first for all individuals of co-occurring [59]with --baq and --gatkcigarworkaround options and Plutella species, and second for P. australiana alone. For expected substitution rate set to 0.03 for P. xylostella all runs, we used a burnin length of 5 × 10 followed by and 0.05 for P. australiana to reflect expected levels a run length of 10 MCMC iterations and performed ten of sequence divergence relative to the P. xylostella ref- independent runs for each K value from 1 to 10, where K erence genome. Duplicate reads were removed using is the number of genotypic clusters, using a different ran- PICARD version 1.71 [60]. Genotypes were called using dom seed for each run, assuming the locprior model with the Genome Analysis Toolkit (GATK) version 3.3-0 [61, correlated allele frequencies and λ set to 1. The optimal 62] HaplotypeCaller tool. We determined that base quality value of K was determined using the delta K method score recalibration using bootstrapped SNP databases was [72] implemented in STRUCTURE HARVESTER [73]and inappropriate for this dataset as it globally reduced qual- inspection of the likelihood distribution for each model. ity scores. For downstream comparisons between species, Q-matrices were aligned across runs using CLUMPP we joint-genotyped P. australiana and P. xylostella indi- version 1.1.2 [74] and visualised using DISTRUCT viduals using the GATK GenotypeGVCFs workflow. To version 1.1 [75]. examine finer scale population structure, we also joint- genotyped the P. australiana individuals alone. All vari- Laboratory cultures of Plutella species ant callsets were hard-filtered with identical parameters Laboratory cultures of P. australiana and P. xylostella were using VCFtools version 0.1.12a [63]: We removed indels established from field populations and used for cross- and retained confidently-called biallelic SNPs (GQ30) ing experiments and insecticide bioassays. Plutella adults genotyped in at least 70% of individuals with a mini- were collected at light traps at Angle Vale and Urrbrae, mum genotype depth of 5, minQ400, average site depth South Australia, in October–November 2015. Females of 12–100, minimum minor allele frequency of 0.05, in were isolated and allowed to lay eggs, then identified Hardy-Weinberg equilibrium at an alpha level of 0.05. To using PCR-RFLP and progeny pooled to produce sep- avoid linked sites, we used the VCFtools --thin func- arate cultures of each species. A laboratory culture of tion to retain only SNPs separated by a minimum of 2000 the Waite Susceptible P. xylostella strain (S) has been bp. To estimate genetic diversity, we generated a set of maintained on cabbage without insecticide exposure for all confidently-called variant and invariant sites (GQ30), approximately 24 years (≈ 310 generations) and was used and hard filtered to remove sites within repetitive regions as a bioassay reference strain. All cultures were main- and retain sites genotyped in at least 70% of individu- tained in laboratory cages at 26 ± 2.0 C and a 14:10 (L:D) als with an average site depth of 12–100. Sites from the hour photoperiod at the South Australian Research and mitochondrial genome were excluded from all datasets. Development Institute, Waite Campus, Adelaide, South Australia. The P. australiana culture was maintained on Genetic diversity and population structure sand rocket, Diplotaxis tenuifolia,and the P. xylostella cul- Genetic diversity was calculated for Plutella populations ture was maintained on cabbage, Brassica oleracea var. of both species from five locations. The R package hierf- capitata. The purity of cultures was assessed regularly stat [64] was used to calculate observed heterozygosity, using PCR-RFLP. gene diversity and the inbreeding coefficient, F , accord- IS ing to Nei [65]. Population means for site depth and num- Crossing experiments ber of SNPs, indels and private sites were calculated using Plutella australiana and P. xylostella pupae were sexed the --depth function and vcfstats module in VCFtools under a stereo microscope, then placed into individual version 0.1.12a [63]. Thenumberofheterozygoussites 5 mL clear polystyrene tubes with fine mesh lids and within individuals was determined from all confidently- gender visually confirmed after eclosion. Enclosures used called sites excluding indels using a custom python script for crossing experiments were 850 mL polypropylene parseVCF.py [66]and visualisedusingR[67]. pots (Bonson Pty Ltd) modified with lateral holes cov- To examine population structure in P. australiana,a ered with voile mesh for ventilation. Crosses of single global estimate of F [68] with bootstrapped 99% confi- mating pairs were performed on laboratory benches at ST 4 ◦ dence intervals (10 bootstrap replicates) was calculated 26 ± 2.0 C and 14:10 (L:D) photoperiod using 3-week in R package diveRsity [69]. Pairwise F values for all old D. tenuifolia seedlings as the host plant. After seven ST Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 7 of 17 days, adults were collected into a 1.5 mL tube and fresh was not different from 1:1 for P. xylostella (481 females, ◦ 2 frozen at − 80 C for species confirmation using PCR- 517 males, χ = 1.2986, p = 0.2545) or P. australiana RFLP. Seedlings were examined and eggs counted under (63 females, 55 males, χ = 0.5424, p = 0.4615). The rel- a stereo microscope, then returned to enclosures to allow ative incidence and abundance of P. australiana was egg hatch. Larvae were provided with fresh 3–4 week > 2-fold higher in the eastern state of New South Wales old seedlings until pupation, then pupae were individu- than in other states (Fig. 1). Plutella australiana lar- ally collected into 5 mL tubes. Hybrid F1xF1 crosses and vae were detected in 29% (n = 5/17) of collections from back-crosses were then performed as above. The presence wild brassicas and from species including wild radish, of egg and adult offspring was recorded for all replicates, Raphanus raphanistrum,wildturnip, Rapistrum rugosum, and for the majority of replicates (> 80%), the numbers of African mustard, B. tournefortii, and mixed stands of sand offspring were counted and used to calculate a mean. rocket, D. tenuifolia and wall rocket, D. muralis (Table 2). Among cultivated crops, P. australiana larvae occurred Insecticide bioassays in 36% (n = 14/39) of samples from canola, consist- Insecticide susceptibility of field-collected Plutella strains ing of 11% of total Plutella individuals from those crops, was compared to the susceptible P. xylostella (S) reference but were not identified from commercial Brassica veg- in dose-response assays using four commercial insecti- etable farms (Table 2). However, P. australiana eggs were −1 cides: Dominex (100 g L alpha-cypermethrin), Proclaim collected from kale at one farm. −1 −1 (44 g kg emamectin benzoate), Coragen (200 g L −1 chlorantraniliprole) and Success Neo (120 g L spine- Wolbachia infections rd toram). Bioassays were performed by placing 3 instar Plutella individuals (n = 1447) were screened for larvae onto inverted leaf discs embedded in 1% agar Wolbachia infection using 16S rRNA PCR assays. Only in 90 mm Petri dishes. Cabbage leaves, Brassica oler- 1.5% (n = 19/1300) of P. xylostella collected from eight acea.var. capitata were used for P. xylostella and canola different locations were infected (Table 1). In contrast, leaves, B. napus var. ‘ATR Stingray’, were used for all 147 P. australiana individuals were infected with P. australiana. Eight concentrations and a water-only con- Wolbachia across the 20 locations where this species trol were evaluated for each insecticide using four repli- occurred. To identify Wolbachia strains, a Wolbachia cates of ten larvae. A 4 mL aliquot of test solution was surface protein (wsp) amplicon was sequenced from 14 P. applied directly to leaves using a Potter Spray Tower xylostella and 30 P. australiana individuals. Each species (Burkard Manufacturing Co. Ltd.) calibrated to deliver an was infected with a different strain. The wsp sequence -1 aliquot of 3.52 ± 0.09 mg cm . After application, each for Australian P. xylostella showed 100% identity to a dish was placed in a controlled temperature room at 25 ± Wolbachia supergroup A isolate infecting P. xylostella 0.5 C, then mortality was assessed after 48 h (Dominex, from Malaysia, plutWA1 [18]. For P. australiana,the wsp Success Neo) or 72 h (Proclaim, Coragen). Dose-response sequence showed 100% identity to a Wolbachia super- analysis was performed using log-logistic regression in group B isolate infecting a mosquito, Culex pipiens,from Rpackage drc[76] and the fitted models were used to Turkey and the winter moth, Operophtera brumata,from estimate the lethal concentration predicted to cause 50% the Netherlands (Fig. 2). (LC ) and 99% (LC ) mortality of the test population. 50 99 Resistance ratios were calculated by dividing the LC and Crossing experiments LC estimates for field strains by the corresponding LC Inter-species single pair mating experiments showed that estimates for the P. xylostella (S) reference strain. hybridization between P. australiana and P. xylostella was possible, yet less successful than intra-species crosses. Results While most intra-species crosses produced adult off- Geographic distribution and host associations spring, the fecundity of P. xylostella was >2-fold higher Plutella larvae were collected from brassicaceous plants than P. australiana (Table 3). Both reciprocal inter-species at 75 locations in Australia and 1477 individuals were crosses produced F1 adult offspring, but success was genotyped at the COI locus using PCR-RFLP to iden- asymmetric and notably higher in the pairs with P. aus- tify species. Of these, 88% (n = 1300) were genotyped traliana females. In this direction, there was a strong male as P. xylostella, 10% (n = 147) as P. australiana and 2% bias in the F1 progeny: from 76 cross replicates, 16 collec- (n = 30) were unresolved (Table 1). Plutella australiana tively produced 9 female and 80 male adults, a ratio of 8.9. was identified in around one quarter (n = 20/75) of Hybrid F1xF1 crosses for both parental lines produced F2 collections distributed across southern Australia, while adult offspring (Table 4). For the P. australiana maternal P. xylostella occurred at all locations except Cunna- line, parental back-crosses using F1 hybrid males suc- mulla, Queensland, in a collection from wild African cessfully produced offspring, while parental back-crosses with F1 hybrid females were sterile. For the P. xylostella mustard, Brassica tournefortii (Table 1). The sex ratio Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 8 of 17 Fig. 1 The geographic distribution of P. xylostella (light grey) and P. australiana individuals (black) in larval collections from brassicaceous plants in Australia during 2014 and 2015. Pie diagrams show the relative proportion of each species at each location. Overlapped pies represent locations with 100% P. xylostella. Green highlighted circles indicate five locations from which individuals of each species were RAD sequenced maternal line, low fitness allowed only a single parental mutation from other haplotypes (Fig. 3a, Additional file 1: back-cross replicate, which involved a hybrid female and Table S1). Nine closely related haplotypes were identified was sterile. in 87 P. australiana individuals with seven occurring in single individuals (Fig. 3b). The most common haplotype, Mitochondrial haplotype diversity PaCOI01, occurred at high frequency and differed by 1-2 Mitochondrial haplotype networks of Australian Plutella base mutations from other haplotypes (Fig. 3b, Additional were constructed using a 613 bp COI alignment that file 1:Table S2). included 81 sequences from this study and 108 from Landry and Hebert [16]. We found low haplotype diversity Nuclear diversity and population structure within Australian P. xylostella, consistent with previous At five collection locations, P. australiana co-occurred reports [17, 18, 77]. Only five haplotypes were identi- with P. xylostella in sufficient numbers to enable compar- fied among 102 individuals, including three identified by ison of nuclear genomes, though the relative abundance Saw et al. [17] and three occurring in single individ- of species varied between locations. To ensure repre- uals (Fig. 3a). The most common haplotype, PxCOI01, sentation from the south-west region of Australia, the occurred at high frequency and differed by a single base Esperance population (n = 5) was formed by including one P. australiana individual from Boyup Brook. Despite only two P. xylostella individuals at Gilgandra, this population Table 2 Frequency of P. australiana in Plutella collections from had 17 P. australiana individuals and was included. To different Brassica host types generate nuclear SNP markers, we performed RADseq for Host No. No. P.aus No. No. P.aus a total of 52 P. australiana and 47 P. xylostella individuals. locations locations genotyped Illumina sequencing and demultiplexing using Wild brassicas 17 5 (0.29) 268 50 (0.19) RADtools [57] yielded 276.8 million raw sequence reads. Canola crops 39 14 (0.36) 848 93 (0.11) Following read quality filtering and mapping, genotypes Vegetable crops 16 1 (0.06) 287 4 (0.01) were called for 99 individuals from the two species. Hard filtering retained 300,241 confidently-called vari- Forage brassicas 3 0 (0.00) 44 0 (0.00) ant and invariant nuclear sites at a mean depth > 36 Presented are the numbers and proportion in parentheses of P. australiana across per individual, and a subset of 689 widely-dispersed collection locations and individuals genotyped Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 9 of 17 Fig. 2 Maximum likelihood phylogeny of 493 bp of Wolbachia wsp amplicons for Plutella and other arthropods. The strain infecting P. australiana (wAus)was identicaltoa Wolbachia supergroup B strain reported from Culex pipiens and Operophtera brumata. The strain infecting Australian P. xylostella was identical to a supergroup A strain (plutWA1) reported from Malaysian P. xylostella. Labels include the Wolbachia strain,hostspecies and GenBank accession number. Labels in bold denote strains sequenced in this study. The scale bar shows the mean number of nucleotide substitutions per site nuclear SNP variants (to avoid linkage bias) at a mean heterozygosity among individuals using 289,347 sites. depth > 36 per individual, for comparative analyses of Plutella australiana individuals had on average a 1.5-fold genetic diversity and population structure. The dataset higher proportion of heterozygous sites than P. xylostella with all confidently-called sites was used to estimate individuals (Fig. 4). population-level genetic diversity. Genetic structure among co-occurring populations Estimates of nuclear genetic diversity across 300,241 of Plutella species was investigated using 689 widely- variant and invariant sites revealed a striking contrast dispersed nuclear SNPs in the program STRUCTURE. between Plutella species, with notably higher diversity The delta K method predicted a strong optimal at K = 2 within populations of P. australiana than co-occurring genotypic clusters. Plutella australiana and P. xylostella populations of P. xylostella (Table 5). The mean observed individuals were clearly separated into distinct genotypic heterozygosity within populations ranged from 0.013– clusters in accordance to their species identified through 0.016 for P. australiana and 0.009–0.010 for P. xylostella. mtDNA genotypes regardless of geographic location Similarly, the average numbers of SNPs, indels and pri- (Fig. 5, left panel). Five individuals across four locations vate alleles were considerably higher within P. australiana showed greater than 1% admixture as shown by sharing of populations. As P. australiana may have fixed nucleotide colored bars. differences relative to the P. xylostella reference genome Assessing population structure from datasets with mul- that may affect population level statistics, we also removed tiple species can mask heirachical structure [78]. To indels from this dataset and directly compared the address this, genotypes were separately called for 52 Table 3 Fecundity of intra-species and reciprocal inter-species single pair crosses of P. australiana (P.aus) and P. xylostella (P.x) Cross (♀ × ♂) No. replicates No. reps eggs No. reps adults Mean ± SEM no. eggs Mean ± SEM no. adults P.aus♀ × P.aus♂ 42 37 (0.881) 34 (0.81) 40.86 ± 5.33 9.66 ± 1.7 P.x♀ × P.x♂ 63 59 (0.937) 59 (0.937) 83.82 ± 10.61 24.28 ± 3.27 P.aus♀ × P.x♂ 76 49 (0.645) 16 (0.211) 18.43 ± 3.02 1.17 ± 0.33 P.x♀ × P.aus♂ 85 62 (0.729) 3 (0.035) 15.16 ± 2.37 0.06 ± 0.03 Presented are the number and proportion in parentheses of replicates (reps) that produced eggs and adult offspring, and the mean ± standard error of the mean number of eggs and adult offspring per replicate Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 10 of 17 Table 4 Fecundity of hybrid F1 crosses and back-crosses Cross (♀ × ♂) No. replicates No. reps eggs No. reps adults Mean ± SEM no. eggs Mean ± SEM no. adults F0 P.aus♀ source (P.aus × P.x♂)♀ × (P.aus × P.x♂)♂ 4 4 (1.000) 2 (0.500) 66.00 ± 60.00 – (P.aus × P.x♂)♀ × P.aus♂ 7 7 (1.000) 0 (0.000) 20.33 ± 11.86 0.00 ± 0.00 P.aus♀ × (P.aus × P.x♂)♂ 9 5 (0.556) 2 (0.222) 6.38 ± 3.54 0.22 ± 0.44 (P.aus × P.x♂)♀ × P.x♂ 4 4 (1.000) 0 (0.000) 39.00 ± 19.00 0.00 ± 0.00 P.x♀ × (P.aus × P.x♂)♂ 15 15 (1.000) 4 (0.267) 36.75 ± 3.21 0.33 ± 0.62 F0 P.x♀ source (P.x × P.aus♂)♀ × (P.x × P.aus♂)♂ 6 5 (0.833) 4 (0.667) 74.50 ± 22.79 6.17 ± 5.27 (P.x × P.aus♂)♀ × P.aus♂ 1 0 (0.000) 0 (0.000) 0.00 0.00 Presented are the number and proportion in parentheses of replicates (reps) producing eggs and adult offspring, and the mean ± standard error of the mean numbers of eggs and adults offspring per replicate. A dash denotes an absence of count data P. australiana individuals, and hard filtering retained a set Insecticide susceptibility of 974 widely-dispersed SNP variants at a mean depth > Bioassays revealed highly contrasting responses to insec- 33 per individual for examination of finer scale structure ticide exposure in P. xylostella and P. australiana field among five populations. The delta K method predicted strains (Fig. 6). Plutella australiana showed extremely a weak modal signal at K = 3, but the highest likelihood high susceptibility to all four insecticides evaluated: resis- value occurred at K = 1. Bar plots for K =3showeda tance ratios at the LC and LC estimates were less than 50 99 high degree of admixture among individuals across the 1.0 and showed that this strain was 1.5-fold to 7.4-fold five populations, consistent with high levels of gene flow more susceptible than the laboratory P. xylostella (S) ref- across Australia (Fig. 5,right panel).Pairwise F was then erence (Additional file 1: Table S3). In contrast, resistance ST calculated for the five P. australiana populations using ratios at the LC for the field P. xylostella strain ranged 974 SNPs. The global estimate of F was not significantly from 2.9 for Success Neo to 41.4 for Dominex, indicat- ST different from zero, indicating the populations are not ing increased tolerance to all insecticides. Comparison of differentiated (F = 0.0002, 99% CI = -0.0274–0.0387). the LC estimates with commercial field doses for each ST 99 Further, pairwise F values were low, ranging from insecticide implies differences in field efficacy between ST –0.0041 to 0.0038, suggesting substantial gene flow among species. The commercial field rate of Dominex was > 8- populations separated by distances of between 341 and fold lower than the LC for P. xylostella, suggesting likely 2700 kilometres (Table 6). poor field control of this strain, but was > 17-fold higher ab Fig. 3 Mitochondrial DNA haplotype network for a P. xylostella (n = 102, 44 from this study, 58 from [16]) and b P. australiana (n = 87, 37 from this study, 50 from [16]) individuals from Australia based on a 613 bp COI sequence alignment. Haplotypes shared by more than one individual are shown in circles with a grey border with the number of individuals indicated inside the circle. Haplotypes connected by a line differ by a single mutation Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 11 of 17 Table 5 Population statistics for variant and invariant sites for sympatric populations of P. australiana (P. aus) and P. xylostella (P. x) from five locations Population Species n Sites Site depth SNPs Indels Private sites H H F O S IS Boomi NSW P. aus 11.1 276939 40 7198 1112 212 0.013 0.015 0.089 P. x 9.4 282989 42 4316 549 30 0.009 0.010 0.039 Calca SA P. aus 8.7 261496 30 6629 989 210 0.014 0.015 0.059 P. x 8.2 274973 42 4126 538 40 0.009 0.010 0.050 Esperance WA P. aus 4.5 269268 28 6543 998 210 0.016 0.015 -0.032 P. x 11.0 275299 35 4046 520 23 0.010 0.010 0.019 Gilgandra NSW P. aus 15.7 277136 39 7154 1088 212 0.014 0.015 0.079 P. x 1.9 277846 42 4149 505 28 0.009 0.009 -0.056 Goulburn NSW P. aus 6.8 256343 29 6471 968 190 0.013 0.015 0.058 P. x 12.8 274700 36 4052 513 26 0.009 0.010 0.052 n, number of individuals genotyped per locus; H , observed heterozygosity; H , gene diversity; F , Nei’s inbreeding coefficient O S IS than the LC for P. australiana (Fig. 6). Control mortality sampling to Brassica vegetable farms. Landry and Hebert was similar for the field and reference strains, averaging [16] also isolated DNA from legs, keeping most of each 3.1 to 4.4% across all bioassays. specimen intact and providing a morphological reference for examining unexpected genotypes. It is also possi- Discussion ble that P. australiana was previously overlooked from Cryptic species arise when divergence does not lead to nuclear DNA studies due to biases in amplification of morphological change [79]. The recent discovery of a divergent alleles. Here, we sought to determine whether cryptic ally, P. australiana, to the diamondback moth, P. australiana is an agricultural pest, and to understand its P. xylostella, was unexpected given the breadth of previ- ecological and genetic differences from P. xylostella. ous molecular studies of this insect. Several factors may Extensive larval sampling from wild and cultivated bras- have contributed to this discovery, including the use of sicaceous plants revealed that P. australiana co-occurs light traps for specimen collection, rather than limiting widely with P. xylostella throughout southern Australia and utilizes some of the same host plants. The relative abundance of P. australiana was on average 9-fold lower than P. xylostella. We observed higher proportions of P. australiana in larval collections from the eastern state of New South Wales, similar to the light trap samples from Landry and Hebert [16], possibly reflecting habitat suitability. Although we did not detect P. australiana in limited sampling from the island state of Tasmania, the presence of brassicas in the region and evidence from light traps that wind currents can transport Plutella moths across Bass Strait (Lionel Hill, Pers. Comm.) suggest it is likely to occur there. Our study confirms that the host range of P. australiana includes canola crops and wild brassicaceous species. In laboratory rearing, P. australiana completed develop- ment on sand rocket, D. tenuifolia, and canola, B. napus, and was also collected from several other wild species, though without rearing to confirm host status. Our sam- pling focused on relatively few introduced brassicaceous species common in agricultural areas, yet the Australian Fig. 4 Boxplot showing the proportion of heterozygous sites across Brassicales is represented by 11 plant families [80], 289,347 confidently-called nuclear sites for individuals of P. xylostella including several non-Brassicaceae on which P. xylostella (light grey boxes, n = 47) and P. australiana (dark grey boxes, n = 52) from five locations. Heterozygosity was consistently higher in and its allies have been documented feeding, such as P. australiana Capparaceae [24], Cleomaceae [16] and Tropaeolaceae Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 12 of 17 Plutella australiana Plutella xylostella Plutella australiana K = 2 K = 3 Fig. 5 Proportional assignment of Plutella individuals to genotypic clusters, K, based on STRUCTURE analysis. Individuals are represented by vertical bars and genotypic clusters are represented by different colors. Left panel: Analysis at K = 2 for 52 P. australiana and 47 P. xylostella individuals sorted left-to right by proportion of cluster membership. The predominantly red bars correspond to P. australiana individuals and the predominantly blue bars correspond to P. xylostella individuals identified through mtDNA genotypes. Locations are labelled for five individuals showing > 1% genotypic admixture. Right panel: Analysis at K = 3 for 52 P. australiana individuals sorted left-to-right by proportion of cluster membership within geographic locations, showing a high degree of genotypic admixture among individuals across locations [23]. The Australian Brassicaceae has records for 61 replacing cabbage with Diplotaxis seedlings, egg-laying genera and 205 species [80], including many introduced then occurred within 24 h. Exposure to host plants stim- species but also a diversity of native genera, such as Lep- ulates reproductive behaviour in P. xylostella [82], but idium, Blennodium,and Arabidella, that occur over vast olfactory cues for host recognition or oviposition [83–85] areas of Australia. Wider sampling of native Brassicales may differ between these Plutella species. Host prefer- may identify other suitable hosts for P. australiana. ence and performance studies are required to test these Plutella australiana larvae were not identified among hypotheses. samples from sixteen commercial Brassica vegetable Insecticide bioassays have been conducted routinely crops despite the high suitability of these crops for P. on Australian P. xylostella to monitor levels of insec- xylostella [81], however eggs were collected from kale. ticide resistance in field populations [22, 25]. This It is possible that extreme insecticide susceptibility pre- method appears unlikely to be affected by the presence vents juvenile P. australiana populations from establish- of P. australiana under typical conditions, as a period ing, as commercial Brassica vegetable crops are typi- of laboratory rearing is usually necessary to multiply cally sprayed multiple times per crop cycle [22]. Our data individuals prior to screening. In our experience, lab- show that P. australiana is far more susceptible than P. oratory rearing of the two Plutella species on cabbage xylostella to four commonly used insecticides. At com- plants selects against P. australiana individuals when mercial application rates, these insecticides are likely to competing with P. xylostella in cages, causing the com- provide high-level control of P. australiana in Australian plete loss of P. australiana within a few generations. The Brassica crops, but some products may provide marginal reasons for this are unknown but may include differ- or poor control against P. xylostella due to insecticide ences in host preference or development rate, or direct resistance (Fig. 6)[22, 25]. Alternatively, some vegetable competition. cultivars may not be attractive for oviposition or suit- Crossing experiments revealed that hybridization can able for larval survival in P. australiana.Wenoted that occur between P. australiana and P. xylostella under con- P. australiana cultures provided with cabbage seedlings trolled conditions and is most likely to occur in crosses failed to produce viable eggs over seven days, but after involving Wolbachia-infected P. australiana females. Hybridization occurs in around 10% of animal species, particularly in captivity [86], but asymmetric reproduc- Table 6 Pairwise comparisons of Weir and Cockerham’s [68] F ST tive isolation is commonly observed in reciprocal crosses (below diagonal) and geographic distance in kilometres (above between taxa [87]. In our experiments, a strong male diagonal) among populations of P. australiana from five locations bias in the offspring of interspecific crosses and failure to Boomi Calca Esperance Gilgandra Goulburn back-cross hybrid females both follow Haldane’s rule [88], which predicts greater hybrid inviability or sterility in the Boomi – 1555 2714 341 677 heterogametic sex (female, in Lepidoptera). This pattern Calca -0.0041 – 1167 1365 1434 can arise from epistatic interactions between sex-linked Esperance 0.0038 0.0014 – 2531 2572 and/or autosomal genes that result in genetic incompati- Gilgandra 0.0000 0.0036 -0.0005 – 364 bilities [89, 90]. Although the back-crosses with F1 hybrid Goulburn -0.0015 -0.0014 0.0034 0.0005 – females were sterile, the back-crosses with hybrid males (to both species) were viable, which could enable the Exact G tests were non-significant for all population pairs (p > 0.05) Gilgandra Boomi Calca Esperance Esperance Calca Goulburn Gilgandra Boomi Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 13 of 17 Fig. 6 Insecticide bioassay dose-response curves for P. australiana (dotted line) and P. xylostella (dashed line) field strains collected from Angle Vale and Urrbrae, South Australia, and a susceptible P. xylostella (S) reference strain (solid line), exposed to four commercial insecticides: Dominex, Coragen, Proclaim and Success Neo. Points are the mean observed response across 4 bioassay replicates of 10 larvae each and lines are the fitted log-logistic response curves with 95% confidence intervals shown in grey shading. The vertical red line represents the approximate commercial field dose for each insecticide and vertical black lines represent the estimated LC for the corresponding Plutella strain transfer of genes between hybrid and/or parental species. However, both species showed limited mtDNA diversity However, it is unclear whether hybridization occurs in with a single predominant haplotype. While outgroups the wild. from other continents were not available, comparative Although P. australiana and P. xylostella show deep analysis of these closely-related Australian Plutella species divergence (8.6%) in mtDNA [16], the sole use of mtDNA suggested that patterns of mitochondrial and nuclear can be unreliable for inference of evolutionary history diversity are concordant in P. xylostella and consistent and should be corroborated using evidence from nuclear with a demographic bottleneck [17, 18], but discordant in markers [34]. Our analysis revealed striking differences in P. australiana. nuclear diversity across the genome between co-existing Sequence variation in mitochondrial DNA can be populations of each Plutella species collected at the strongly influenced by Wolbachia infection [41]. Extensive same locations and times, and from the same host plant Wolbachia screening showed that each Plutella species species. Plutella xylostella populations from Australia and was infected with a different strain at contrasting fre- New Zealand have low levels of genetic diversity com- quencies, and fit a ‘most-or-few’ pattern whereby species pared with populations from other continents, thought infection rates are often very low (<10%) or very high to reflect the recent introduction of this species from (> 90%) [91]. Infection incidence in P. xylostella was lower a small founding population [14, 17, 77]. Consistent in Australia (1%) than previously reported across global with this view, we found a remarkable 1.5-fold reduction samples (5%) [18]. Our finding of a single supergroup in heterozygosity across > 300,000 sites in P. xylostella A strain showing 100% sequence similarity to a strain compared with sympatric P. australiana populations. reported in P. xylostella from Malaysia, plutWA1 [18], Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 14 of 17 provides some support of an Asian origin for Australian mechanisms, such as assortive mating or hybrid fitness P. xylostella [17], though does not preclude this strain also costs. Behavioural mating choices are often the main iso- occurring elsewhere. lating factor in sympatric animals [86]. Does Wolbachia Fixation of infection in P. australiana suggests that cause a reproductive barrier? The contrast in infection Wolbachia manipulates the reproductive biology of this status creates the potential for cytoplasmic incompati- species. We found no evidence of sex-ratio distortion, bility between species [94]. Interspecific crosses showed which has been associated with a Wolbachia strain, a pattern of asymmetric isolation consistent with the plutWB1,in P. xylostella [18]. High infection can be expected effects of unidirectional CI, where 21% crosses driven by cytoplasmic incompatibility (CI) [40]. The high involving infected P. australiana females produced viable frequency (87%) of a single mtDNA haplotype among offspring, while the reciprocal CI-cross direction (unin- P. australiana individuals implies that the spread of Wol- fected P. xylostella females crossed with infected P. aus- bachia infection has driven a selective sweep of co- traliana males) wasnearlysterile.However,thispattern inherited mtDNA through the population, causing a loss was not continued in the F1 generation: infected hybrid of mtDNA diversity [41]. High nuclear diversity (rela- males (derived from the P. australiana maternal line) pro- tive to sympatric P. xylostella) supports this hypothesis, duced offspring at comparable rates when back-crossed to because a demographic bottleneck should reduce diversity either uninfected P. xylostella or infected P. australiana across the entire genome [34]. female parents. The role of Wolbachia-induced postzy- Plutella australiana and P. xylostella have co-existed gotic isolation between the two Plutella species requires in Australia for at least 125 years (1300 generations), further study, though our results suggest it could be more yet have strongly divergent mitochondrial and nuclear important in the F0 generation. Wolbachia can contribute genomes, Wolbachia infections and insecticide suscep- to post-zygotic genetic isolation after speciation by com- tibility phenotypes. Our observations during laboratory plementing hybrid incompatibilities [94, 95]. Symbiont rearing and crossing experiments also suggested that infections could also influence mating behaviour and con- interspecific differences in host plant use may exist. What tributetopre-matingisolation [96]. explains such strong divergence between the two Plutella species, given sympatry and the capacity to hybridize? Conclusions Endemism of P. australiana [16] implies an ancient evo- The discovery of cryptic pest species introduces com- lutionary history in Australia, and our data provide sup- plexities for their management and also exciting oppor- port for existing views that Australian P. xylostella were tunities for understanding ecological traits. We found recently introduced from a small ancestral source pop- strong genomic and phenotypic divergence in two cryp- ulation, possibly from Asia [17, 18, 77]. Therefore, the tic mitochondrial Plutella lineages co-existing in nature, two Plutella species may have diverged in allopatry and supporting their status as distinct species [16] despite the recently come into secondary contact. Maintenance of capacity to hybridize. Reproductive isolation is likely to divergence suggests strong continuing reproductive isola- have evolved during allopatric speciation, and genome- tion, which can evolve as a side-effect of allopatric diver- wide sequence data suggest it has been maintained follow- gence [44]. All 99 individuals that were RAD sequenced ing secondary contact. Variation in Wolbachia infections showed concordance in nuclear multilocus genotypes and might be one factor reinforcing reproductive barriers. mtDNA genotypes identified through PCR-RFLP regard- Plutella australiana co-occurs with P. xylostella less of geographic location, as shown by STRUCTURE throughout agricultural regions of southern Australia, analysis. Cryptic species in sympatry provides strong evi- but made up only 10% of Plutella juveniles collected dence of limited genetic exchange [79]. A small degree from cultivated and wild brassicaceous plants. A lack of genotypic admixture evident for a few individuals in of population structure across neutral SNP markers the STRUCTURE plots might be explained by ances- suggests that P. australiana populations are linked by tral polymorphism or introgressive hybridization [28], or high levels of gene flow, and also that P. australiana is a alternatively, could be an artefact if our dataset is not highly mobile species, which is supported by light trap representative of the entire genetic background [33]. The collections [16] and seasonal colonization of canola crops. level of hybridization that may be occurring between Future molecular analysis of Australian Plutella should these species is unknown. Isolation may not be uniform include a species identification step using a molecular across the genome [92, 93], and scans of larger genomic diagnostic assay. For ecological studies, it may be possible regions may be required to identify introgression and to perform molecular species identification to confidently detect hybrids. distinguish a representative sub-sample of individuals The factors leading to reproductive isolation between or pooled samples. Our study has shown that while P. the two Plutella species in nature are unknown but australiana can attack canola crops, there is no evidence could include a range of pre- or post-mating isolation of pest status in commercial Brassica vegetables crops, Perry et al. BMC Evolutionary Biology (2018) 18:77 Page 15 of 17 and bioassays suggested that field populations should be References 1. Pfenninger M, Schwenk K. Cryptic animal species are homogeneously easily controlled with insecticides. Though P. australiana distributed among taxa and biogeographical regions. BMC Evol Biol. is a potential pest of some Australian Brassica crops, it 2007;7:121. https://doi.org/10.1186/1471-2148-7-121. 2. 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Li Z, Feng X, Liu SS, You M, Furlong MJ. Biology, ecology, and samples, and KDP and SWB performed RADseq, COI genotyping, data analysis management of the diamondback moth in China. Annu Rev Entomol. and wrote the manuscript. SWB and CMW genotyped Wolbachia and 2016;61:277–96. https://doi.org/10.1146/annurev-ento-010715-023622. sequenced wsp. KJP, JKK and GJB cultured Plutella strains and performed 14. Endersby NM, McKechnie SW, Ridland PM, Weeks AR. Microsatellites insecticide bioassays and crossing experiments. All authors read and approved reveal a lack of structure in Australian populations of the diamondback the final manuscript. moth, Plutella xylostella (L.) Mol Ecol. 2006;15(1):107–18. https://doi.org/ 10.1111/j.1365-294X.2005.02789.x. Ethics approval and consent to participate 15. Furlong MJ, Spafford H, Ridland PM, Endersby NM, Edwards OR, Not applicable. Baker GJ, Keller MA, Paull CA. Ecology of diamondback moth in Australian canola: Landscape perspectives and the implications for management. Competing interests Aust J Exp Agr. 2008;48(12):1494–505. https://doi.org/10.1071/EA07413. The authors declare that they have no competing interests. 16. Landry JF, Hebert PDN. Plutella australiana (Lepidoptera, Plutellidae), an Publisher’s Note overlooked diamondback moth revealed by DNA barcodes. ZooKeys. Springer Nature remains neutral with regard to jurisdictional claims in 2013;327:43–63. https://doi.org/10.3897/zookeys.327.5831. published maps and institutional affiliations. 17. Saw J, Endersby NM, McKechnie SW. Low mtDNA diversity among widespread Australian diamondback moth Plutella xylostella (L.) suggests Author details isolation and a founder effect. Insect Sci. 2006;13(5):365–373. School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, 18. Delgado AM, Cook JM. Effects of a sex-ratio distorting endosymbiont on 5005, Australia. 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