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. Electronic supplementary material The online version of this article (10.1186/s12862-018-1183-4) contains supplementary material, which is available to authorized users.


Background
Cryptic species can show remarkable diversity in aspects of their ecology, behaviour, and at the level of the genome. They exist across metazoan taxa [1], including globally important arthropod pest taxa, such as whiteflies [2], disease-vectoring mosquitoes [3], fruit flies [4], thrips [5,6] and mites [7,8], some of which are characterised by cryptic species complexes. Discovering cryptic diversity has important consequences for estimates of global biodiversity, conservation planning, and the management of pests and diseases. Morphologically similar species can vary in pest status due to differences in genotypic and/or phenotypic traits that influence their host range and specificity, geographic distribution, the ability to vector diseases, or insecticide resistance [8][9][10]. Therefore, recognising cryptic species and the differences in their biology and ecology are essential for effective management, with important implications for public health, agriculture and trade.
The diamondback moth, Plutella xylostella, is the major pest of brassicaceous crops worldwide, costing an estimated US$4 to US$5 billion annually in direct losses and management costs [11,12]. Insecticide resistance is widespread in P. xylostella populations around the world, fuelling wide-ranging research to develop alternative management tactics [11,13]. Plutella xylostella was initially recorded in Australia in the late 1800s and rapidly became a widespread pest of Brassica vegetables, and then canola following its expanded production from the 1990s [14,15]. Recently, Landry and Hebert [16], through mtDNA barcoding, identified a cryptic lineage of Plutella in Australia not detected in previous molecular studies of P. xylostella [14,[17][18][19][20][21]. Although external morphology was indistinguishable from P. xylostella, deep mtDNA divergence (8.6%), differences in genital morphology and endemism in Australia led them to describe a new species, Plutella australiana Landry & Hebert. Plutella australiana was originally collected together with P. xylostella in light trap samples in eastern Australia, suggesting at least some ecological overlap [16], but its biology, ecology and pest status were unknown.
The management of P. xylostella in Australian Brassica crops has been a significant challenge for decades [15,22], but the discovery of P. australiana has made the relative abundance and pest status of both species in these crops uncertain. With rare exception, P. xylostella and allied species feed on plants in the order Brassicales, mainly within the family Brassicaceae [16,23,24], implying that the host range of P. australiana may include cultivated brassicas. Widespread resistance to pyrethroid and organophosphate insecticides has been attributed to Australian populations of P. xylostella from all vegetable and canola production regions, which has led to ineffective control during outbreaks [22,25]. Plutella xylostella is well known as a migratory insect with a high capacity for gene flow [11,13], facilitating the rapid spread of resistance alleles. Australian P. xylostella are thought to disperse frequently, based on indirect evidence from ecological and genetic studies [14,15,26]. Most studies have found a lack of genetic differentiation at microsatellite loci and low sequence variation in mitochondrial DNA markers among Australian and New Zealand populations of P. xylostella, consistent with high gene flow and/or recent ancestry [14,15,17,18]. While species identification was not in question in these studies, somewhat inconsistent findings in two studies from eastern Australia using allozymes or SSR markers [19,20] might reflect the confounding presence of P. australiana samples [16]. Given these considerations, future management of Plutella in Australian crops will require thorough understanding of the ecological requirements, genetic traits and pest status of the two Plutella species. In addition, reproductive isolation between these two species is unknown but has implications for evolutionary inference and the potential for gene flow. The capacity for hybridization and introgression could lead to the exchange of insecticide resistance or other adaptive alleles [27,28].
Although mtDNA markers are widely used in studies of species identity and population structure [29][30][31], mitochondrial variation within or between species can be influenced by direct and/or indirect selection, or introgressive hybridization [32,33]. One factor that can confound mtDNA-based inference is interaction with inherited bacterial symbionts [34,35]. Wolbachia is a widespread endosymbiont thought to infect at least half of arthropod [36] and 80% of lepidopteran [37] species. It is mainly transmitted vertically from infected females to their offspring through the egg cytoplasm, and inheritance is therefore linked with mtDNA. To facilitate its spread, Wolbachia manipulates host reproductive biology to favour the fitness of infected females by inducing host phenotypes that distort sex ratios (malefeminization, male-killing or induction of parthenogenesis) or cause sperm-egg cytoplasmic incompatibility (CI) [38,39]. In the simple case involving a single CI-inducing strain, crosses with infected females are fertile but crosses between uninfected females and infected males fail to produce offspring. If maternal transmission is efficient and infected females have a reproductive advantage, Wolbachia infection can spread rapidly through an insect population [40], driving a selective sweep of a single haplotype and reducing mtDNA diversity [41]. Limited surveys to date have identified Wolbachia strains infecting P. xylostella at low frequency in populations from North America, Africa, Asia and Europe [18,42,43]. Because symbionts can contribute to reproductive isolation and influence mtDNA diversity [34,44], assessing their role can provide important insights into host evolution and population structure [35,[45][46][47].
Here we investigated the biology, ecology and population genetic structure of two cryptic Plutella species by collecting Plutella from brassicaceous plants throughout Australia and screening individuals to identify mtDNA lineages and Wolbachia infections. For a subset of populations, we examined genetic diversity using thousands of nuclear SNPs from across the genome. In addition, we assessed reproductive compatibility in laboratory crosses and determined the susceptibility of each species to commercial insecticides.

Sample collection
Plutella larvae (rarely, eggs or pupae) were collected from canola crops, Brassica vegetable crops, forage brassicas and wild brassicas throughout Australia between March 2014 and December 2015 ( Table 1). The wild species included wild radish, Raphanus raphanistrum, turnip weed, Rapistrum rugosum, sea rocket, Cakile maritima, Ward's weed, Carrichtera annua, African mustard, Brassica tournefortii, and mixed stands of sand rocket, Diplotaxis tenuifolia, and wall rocket, D. muralis. At each location, at least 25 individuals were collected from randomly selected plants to achieve a representative sample. Insect samples were collected from Brassica vegetables by hand, from sea rocket by beating plants over a collection tray and from other hosts using a sweep net. Each population sample was separately reared in ventilated plastic containers on leaves of the original host material for 1-2 days and thereafter on cabbage leaves. Non-parasitised pupae or late-instar larvae were fresh frozen at − 80 • C.

DNA isolation and COI genotyping
For each population sample, we aimed to genotype a minimum of 16 individuals where possible after removing parasitized individuals. Individual pupae (but not larvae) were sexed under a dissecting microscope, then genomic DNA was isolated by homogenising whole individuals followed by two phenol and one chloroform extractions according to Zraket et al. [48]. DNA was treated with RNase A, then precipitated and re-suspended in TE buffer. Plutella lineages were distinguished using a PCR-RFLP assay [49]. A 707 bp COI region was amplified using a combination of two primer pairs: (i) PxCOIF (5 -TCAACAAATCATAAAGATATTGG-3 ) and PxCOIR (5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ), and (ii) PaCOIF (5 -TCAACAAATCATAAGGATATTGG-3 ) and PaCOIR (5 -TAAACCTCTGGATGGCCAAAAAA TCA-3 ). Ten microliter reactions were run with 2 μL of MyTaq 5x buffer, 0.2 μL of each primer (10mM stocks), 1 μL of DNA (approx. 5 ng) and 0.05 μL of MyTaq polymerase (Bioline). Samples were amplified at 95 • C for 2 min, then 35 cycles at 95 • C for 10 s, 52 • C for 20 s, 72 • C for 30 s followed by a 5 min final extension at 72 • C. PCR products were digested at 37 • C for 1 h with 1 unit of AccI (NEB) restriction enzyme with 2 μL Cutsmart Buffer in a 20 μL reaction. Following digestion, products were separated using agarose gel electrophoresis (1.5%). Plutella xylostella products are approximately 516 bp and 191 bp and P. australiana products are 348 bp and 359 bp [49]. To examine mtDNA haplotypes, sequencing of the 707 bp COI amplicon was performed for 44 P. xylostella and 37 P. australiana individuals at the Australian Genome Research Facility (AGRF). In addition, we downloaded sequence trace files from Landry and Hebert [16] (dx.doi.org/10.5883/DS-PLUT1) and re-analysed, aligned and trimmed all sequences in GENEIOUS version 10.0.6 [50]. Haplotype networks were constructed using R package pegas version 0.9 [51].

Wolbachia screening and phylogenetics
Wolbachia infection was detected using two separate PCR assays of the 16S rRNA gene (16S-2 and 16S-6) according to Simoes et al. [52]. To identify Wolbachia strains, the Wolbachia surface protein (wsp) gene was sequenced in a subset of individuals. Amplification was performed using wsp81F and wsp691R sequence primers [53]. Amplicons were sequenced using the reverse primer and aligned in GENEIOUS version 10.0.6 [50]. We used a 493 bp alignment to construct a maximum likelihood phylogeny in RAxML version 8.2.4 [54] using a general time reversal substitution model [55] with 1000 bootstrap replicates.

RADseq library preparation and sequencing
Libraries were prepared for restriction-site-associated DNA sequencing (RADseq) according to a protocol modified from Baird et al. [56]. Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 • C, then heat inactivated at 80 • C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG xxxxxxTGCA-3 , bottom strand 5 -[P]xxxxxxCTGTCTC TTATACACATCTGACGCTGCCGACGA-3 , x represents sites for MIDs) were then added using 0.5 μL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. Library pools were sheared using a Bioruptor sonicator (Diagenode), then DNA fragments end-repaired using a Quick Blunting Kit (NEB), adenine overhangs added then P2 adapters (top strand 5 -[P]CTGTCTCTTATA CACATCTCCAGAATAG-3 , bottom strand 5 -GTCTCG TGGGCTCGGAGATGTGTATAAGAGACAGT-3 ) ligated. DNA purification between steps was performed using a MinElute PCR purification kit (Qiagen). Libraries were amplified using KAPA HiFi Hotstart Readymix , and hard filtered to remove sites within repetitive regions and retain sites genotyped in at least 70% of individuals with an average site depth of 12-100. Sites from the mitochondrial genome were excluded from all datasets.

Genetic diversity and population structure
Genetic diversity was calculated for Plutella populations of both species from five locations. The R package hierfstat [64] was used to calculate observed heterozygosity, gene diversity and the inbreeding coefficient, F IS , according to Nei [65]. Population means for site depth and number of SNPs, indels and private sites were calculated using the --depth function and vcfstats module in VCFtools version 0.1.12a [63]. The number of heterozygous sites within individuals was determined from all confidentlycalled sites excluding indels using a custom python script parseVCF.py [66] and visualised using R [67].
To examine population structure in P. australiana, a global estimate of F ST [68] with bootstrapped 99% confidence intervals (10 4 bootstrap replicates) was calculated in R package diveRsity [69]. Pairwise F ST values for all population pairs were calculated and significance determined using exact G tests (10 4 mc burnins, 10 3 batches, and 10 4 iterations per batch) in GENEPOP version 4.6 [70] after Bonferroni correction for multiple comparisons. Separate analysis of population structure was performed using the Bayesian clustering program STRUCTURE version 2.3.4 [71], first for all individuals of co-occurring Plutella species, and second for P. australiana alone. For all runs, we used a burnin length of 5 × 10 5 followed by a run length of 10 6 MCMC iterations and performed ten independent runs for each K value from 1 to 10, where K is the number of genotypic clusters, using a different random seed for each run, assuming the locprior model with correlated allele frequencies and λ set to 1. The optimal value of K was determined using the delta K method [72] implemented in STRUCTURE HARVESTER [73] and inspection of the likelihood distribution for each model. Q-matrices were aligned across runs using CLUMPP version 1.1.2 [74] and visualised using DISTRUCT version 1.1 [75].

Laboratory cultures of Plutella species
Laboratory cultures of P. australiana and P. xylostella were established from field populations and used for crossing experiments and insecticide bioassays. Plutella adults were collected at light traps at Angle Vale and Urrbrae, South Australia, in October-November 2015. Females were isolated and allowed to lay eggs, then identified using PCR-RFLP and progeny pooled to produce separate cultures of each species. A laboratory culture of the Waite Susceptible P. xylostella strain (S) has been maintained on cabbage without insecticide exposure for approximately 24 years (≈ 310 generations) and was used as a bioassay reference strain. All cultures were maintained in laboratory cages at 26 ± 2.0 • C and a 14:10 (L:D) hour photoperiod at the South Australian Research and Development Institute, Waite Campus, Adelaide, South Australia. The P. australiana culture was maintained on sand rocket, Diplotaxis tenuifolia, and the P. xylostella culture was maintained on cabbage, Brassica oleracea var. capitata. The purity of cultures was assessed regularly using PCR-RFLP.

Crossing experiments
Plutella australiana and P. xylostella pupae were sexed under a stereo microscope, then placed into individual 5 mL clear polystyrene tubes with fine mesh lids and gender visually confirmed after eclosion. Enclosures used for crossing experiments were 850 mL polypropylene pots (Bonson Pty Ltd) modified with lateral holes covered with voile mesh for ventilation. Crosses of single mating pairs were performed on laboratory benches at 26 ± 2.0 • C and 14:10 (L:D) photoperiod using 3-week old D. tenuifolia seedlings as the host plant. After seven days, adults were collected into a 1.5 mL tube and fresh frozen at − 80 • C for species confirmation using PCR-RFLP. Seedlings were examined and eggs counted under a stereo microscope, then returned to enclosures to allow egg hatch. Larvae were provided with fresh 3-4 week old seedlings until pupation, then pupae were individually collected into 5 mL tubes. Hybrid F1xF1 crosses and back-crosses were then performed as above. The presence of egg and adult offspring was recorded for all replicates, and for the majority of replicates (> 80%), the numbers of offspring were counted and used to calculate a mean.

Insecticide bioassays
Insecticide susceptibility of field-collected Plutella strains was compared to the susceptible P. xylostella (S) reference in dose-response assays using four commercial insecticides: Dominex (100 g L −1 alpha-cypermethrin), Proclaim (44 g kg −1 emamectin benzoate), Coragen (200 g L −1 chlorantraniliprole) and Success Neo (120 g L −1 spinetoram). Bioassays were performed by placing 3 rd instar larvae onto inverted leaf discs embedded in 1% agar in 90 mm Petri dishes. Cabbage leaves, Brassica oleracea. var. capitata were used for P. xylostella and canola leaves, B. napus var. ' ATR Stingray' , were used for P. australiana. Eight concentrations and a water-only control were evaluated for each insecticide using four replicates of ten larvae. A 4 mL aliquot of test solution was applied directly to leaves using a Potter Spray Tower (Burkard Manufacturing Co. Ltd.) calibrated to deliver an aliquot of 3.52 ± 0.09 mg cm -1 . After application, each dish was placed in a controlled temperature room at 25 ± 0.5 • C, then mortality was assessed after 48 h (Dominex, Success Neo) or 72 h (Proclaim, Coragen). Dose-response analysis was performed using log-logistic regression in R package drc [76] and the fitted models were used to estimate the lethal concentration predicted to cause 50% (LC 50 ) and 99% (LC 99 ) mortality of the test population. Resistance ratios were calculated by dividing the LC 50 and LC 99 estimates for field strains by the corresponding LC estimates for the P. xylostella (S) reference strain.

Geographic distribution and host associations
Plutella larvae were collected from brassicaceous plants at 75 locations in Australia and 1477 individuals were genotyped at the COI locus using PCR-RFLP to identify species. Of these, 88% (n = 1300) were genotyped as P. xylostella, 10% (n = 147) as P. australiana and 2% (n = 30) were unresolved (Table 1). Plutella australiana was identified in around one quarter (n = 20/75) of collections distributed across southern Australia, while P. xylostella occurred at all locations except Cunnamulla, Queensland, in a collection from wild African mustard, Brassica tournefortii ( Table 1). The sex ratio was not different from 1:1 for P. xylostella (481 females, 517 males, χ 2 = 1.2986, p = 0.2545) or P. australiana (63 females, 55 males, χ 2 = 0.5424, p = 0.4615). The relative incidence and abundance of P. australiana was > 2-fold higher in the eastern state of New South Wales than in other states (Fig. 1). Plutella australiana larvae were detected in 29% (n = 5/17) of collections from wild brassicas and from species including wild radish, Raphanus raphanistrum, wild turnip, Rapistrum rugosum, African mustard, B. tournefortii, and mixed stands of sand rocket, D. tenuifolia and wall rocket, D. muralis (Table 2). Among cultivated crops, P. australiana larvae occurred in 36% (n = 14/39) of samples from canola, consisting of 11% of total Plutella individuals from those crops, but were not identified from commercial Brassica vegetable farms (Table 2). However, P. australiana eggs were collected from kale at one farm.

Wolbachia infections
Plutella individuals (n = 1447) were screened for Wolbachia infection using 16S rRNA PCR assays. Only 1.5% (n = 19/1300) of P. xylostella collected from eight different locations were infected (Table 1). In contrast, all 147 P. australiana individuals were infected with Wolbachia across the 20 locations where this species occurred. To identify Wolbachia strains, a Wolbachia surface protein (wsp) amplicon was sequenced from 14 P. xylostella and 30 P. australiana individuals. Each species was infected with a different strain. The wsp sequence for Australian P. xylostella showed 100% identity to a Wolbachia supergroup A isolate infecting P. xylostella from Malaysia, plutWA1 [18]. For P. australiana, the wsp sequence showed 100% identity to a Wolbachia supergroup B isolate infecting a mosquito, Culex pipiens, from Turkey and the winter moth, Operophtera brumata, from the Netherlands (Fig. 2).

Crossing experiments
Inter-species single pair mating experiments showed that hybridization between P. australiana and P. xylostella was possible, yet less successful than intra-species crosses. While most intra-species crosses produced adult offspring, the fecundity of P. xylostella was >2-fold higher than P. australiana (Table 3). Both reciprocal inter-species crosses produced F1 adult offspring, but success was asymmetric and notably higher in the pairs with P. australiana females. In this direction, there was a strong male bias in the F1 progeny: from 76 cross replicates, 16 collectively produced 9 female and 80 male adults, a ratio of 8.9. Hybrid F1xF1 crosses for both parental lines produced F2 adult offspring (Table 4). For the P. australiana maternal line, parental back-crosses using F1 hybrid males successfully produced offspring, while parental back-crosses with F1 hybrid females were sterile. For the P. xylostella

Mitochondrial haplotype diversity
Mitochondrial haplotype networks of Australian Plutella were constructed using a 613 bp COI alignment that included 81 sequences from this study and 108 from Landry and Hebert [16]. We found low haplotype diversity within Australian P. xylostella, consistent with previous reports [17,18,77]. Only five haplotypes were identified among 102 individuals, including three identified by Saw et al. [17] and three occurring in single individuals (Fig. 3a). The most common haplotype, PxCOI01, occurred at high frequency and differed by a single base   Table S1). Nine closely related haplotypes were identified in 87 P. australiana individuals with seven occurring in single individuals (Fig. 3b). The most common haplotype, PaCOI01, occurred at high frequency and differed by 1-2 base mutations from other haplotypes (Fig. 3b, Additional file 1: Table S2).

Nuclear diversity and population structure
At five collection locations, P. australiana co-occurred with P. xylostella in sufficient numbers to enable comparison of nuclear genomes, though the relative abundance of species varied between locations. To ensure representation from the south-west region of Australia, the 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 had 17 P. australiana individuals and was included. To generate nuclear SNP markers, we performed RADseq for a total of 52 P. australiana and 47 P. xylostella individuals. Illumina sequencing and demultiplexing using RADtools [57] yielded 276.8 million raw sequence reads. Following read quality filtering and mapping, genotypes were called for 99 individuals from the two species. Hard filtering retained 300,241 confidently-called variant and invariant nuclear sites at a mean depth > 36 per individual, and a subset of 689 widely-dispersed Estimates of nuclear genetic diversity across 300,241 variant and invariant sites revealed a striking contrast between Plutella species, with notably higher diversity within populations of P. australiana than co-occurring populations of P. xylostella ( Table 5). The mean observed heterozygosity within populations ranged from 0.013-0.016 for P. australiana and 0.009-0.010 for P. xylostella. Similarly, the average numbers of SNPs, indels and private alleles were considerably higher within P. australiana populations. As P. australiana may have fixed nucleotide differences relative to the P. xylostella reference genome that may affect population level statistics, we also removed indels from this dataset and directly compared the heterozygosity among individuals using 289,347 sites. Plutella australiana individuals had on average a 1.5-fold higher proportion of heterozygous sites than P. xylostella individuals (Fig. 4).
Genetic structure among co-occurring populations of Plutella species was investigated using 689 widelydispersed nuclear SNPs in the program STRUCTURE. The delta K method predicted a strong optimal at K = 2 genotypic clusters. Plutella australiana and P. xylostella individuals were clearly separated into distinct genotypic clusters in accordance to their species identified through mtDNA genotypes regardless of geographic location (Fig. 5, left panel). Five individuals across four locations showed greater than 1% admixture as shown by sharing of colored bars.
Assessing population structure from datasets with multiple species can mask heirachical structure [78]. To address this, genotypes were separately called for 52    (Table 6).

Insecticide susceptibility
Bioassays revealed highly contrasting responses to insecticide exposure in P. xylostella and P. australiana field strains (Fig. 6). Plutella australiana showed extremely high susceptibility to all four insecticides evaluated: resistance ratios at the LC 50 and LC 99 estimates were less than 1.0 and showed that this strain was 1.5-fold to 7.4-fold more susceptible than the laboratory P. xylostella (S) reference (Additional file 1: Table S3). In contrast, resistance ratios at the LC 50 for the field P. xylostella strain ranged from 2.9 for Success Neo to 41.4 for Dominex, indicating increased tolerance to all insecticides. Comparison of the LC 99 estimates with commercial field doses for each insecticide implies differences in field efficacy between species. The commercial field rate of Dominex was > 8fold lower than the LC 99 for P. xylostella, suggesting likely poor field control of this strain, but was > 17-fold higher a b  than the LC 99 for P. australiana (Fig. 6). Control mortality was similar for the field and reference strains, averaging 3.1 to 4.4% across all bioassays.

Discussion
Cryptic species arise when divergence does not lead to morphological change [79]. The recent discovery of a cryptic ally, P. australiana, to the diamondback moth, P. xylostella, was unexpected given the breadth of previous molecular studies of this insect. Several factors may have contributed to this discovery, including the use of light traps for specimen collection, rather than limiting Fig. 4 Boxplot showing the proportion of heterozygous sites across 289,347 confidently-called nuclear sites for individuals of P. xylostella (light grey boxes, n = 47) and P. australiana (dark grey boxes, n = 52) from five locations. Heterozygosity was consistently higher in P. australiana sampling to Brassica vegetable farms. Landry and Hebert [16] also isolated DNA from legs, keeping most of each specimen intact and providing a morphological reference for examining unexpected genotypes. It is also possible that P. australiana was previously overlooked from nuclear DNA studies due to biases in amplification of divergent alleles. Here, we sought to determine whether P. australiana is an agricultural pest, and to understand its ecological and genetic differences from P. xylostella. Extensive larval sampling from wild and cultivated brassicaceous plants revealed that P. australiana co-occurs 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 development 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 sampling focused on relatively few introduced brassicaceous species common in agricultural areas, yet the Australian Brassicales is represented by 11 plant families [80], including several non-Brassicaceae on which P. xylostella and its allies have been documented feeding, such as Capparaceae [24], Cleomaceae [16] and Tropaeolaceae Plutella australiana Plutella xylostella Plutella australiana C a lc a E s p e r a n c e G il g a n d r a E s p e r a n c e C a lc a G o u lb u r n G il g a n d r a B o o m i  [23]. The Australian Brassicaceae has records for 61 genera and 205 species [80], including many introduced species but also a diversity of native genera, such as Lepidium, Blennodium, and Arabidella, that occur over vast areas of Australia. Wider sampling of native Brassicales may identify other suitable hosts for P. australiana. Plutella australiana larvae were not identified among samples from sixteen commercial Brassica vegetable crops despite the high suitability of these crops for P. xylostella [81], however eggs were collected from kale. It is possible that extreme insecticide susceptibility prevents juvenile P. australiana populations from establishing, as commercial Brassica vegetable crops are typically sprayed multiple times per crop cycle [22]. Our data show that P. australiana is far more susceptible than P. xylostella to four commonly used insecticides. At commercial application rates, these insecticides are likely to provide high-level control of P. australiana in Australian Brassica crops, but some products may provide marginal or poor control against P. xylostella due to insecticide resistance ( Fig. 6) [22,25]. Alternatively, some vegetable cultivars may not be attractive for oviposition or suitable for larval survival in P. australiana. We noted that P. australiana cultures provided with cabbage seedlings failed to produce viable eggs over seven days, but after replacing cabbage with Diplotaxis seedlings, egg-laying then occurred within 24 h. Exposure to host plants stimulates reproductive behaviour in P. xylostella [82], but olfactory cues for host recognition or oviposition [83][84][85] may differ between these Plutella species. Host preference and performance studies are required to test these hypotheses. Insecticide bioassays have been conducted routinely on Australian P. xylostella to monitor levels of insecticide resistance in field populations [22,25]. This method appears unlikely to be affected by the presence of P. australiana under typical conditions, as a period of laboratory rearing is usually necessary to multiply individuals prior to screening. In our experience, laboratory rearing of the two Plutella species on cabbage plants selects against P. australiana individuals when competing with P. xylostella in cages, causing the complete loss of P. australiana within a few generations. The reasons for this are unknown but may include differences in host preference or development rate, or direct competition.
Crossing experiments revealed that hybridization can occur between P. australiana and P. xylostella under controlled conditions and is most likely to occur in crosses involving Wolbachia-infected P. australiana females. Hybridization occurs in around 10% of animal species, particularly in captivity [86], but asymmetric reproductive isolation is commonly observed in reciprocal crosses between taxa [87]. In our experiments, a strong male bias in the offspring of interspecific crosses and failure to back-cross hybrid females both follow Haldane's rule [88], which predicts greater hybrid inviability or sterility in the heterogametic sex (female, in Lepidoptera). This pattern can arise from epistatic interactions between sex-linked and/or autosomal genes that result in genetic incompatibilities [89,90]. Although the back-crosses with F1 hybrid females were sterile, the back-crosses with hybrid males (to both species) were viable, which could enable the 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 99 for the corresponding Plutella strain transfer of genes between hybrid and/or parental species. However, it is unclear whether hybridization occurs in the wild.
Although P. australiana and P. xylostella show deep divergence (8.6%) in mtDNA [16], the sole use of mtDNA can be unreliable for inference of evolutionary history and should be corroborated using evidence from nuclear markers [34]. Our analysis revealed striking differences in nuclear diversity across the genome between co-existing populations of each Plutella species collected at the same locations and times, and from the same host plant species. Plutella xylostella populations from Australia and New Zealand have low levels of genetic diversity compared with populations from other continents, thought to reflect the recent introduction of this species from a small founding population [14,17,77]. Consistent with this view, we found a remarkable 1.5-fold reduction in heterozygosity across > 300,000 sites in P. xylostella compared with sympatric P. australiana populations.
However, both species showed limited mtDNA diversity with a single predominant haplotype. While outgroups from other continents were not available, comparative analysis of these closely-related Australian Plutella species suggested that patterns of mitochondrial and nuclear diversity are concordant in P. xylostella and consistent with a demographic bottleneck [17,18], but discordant in P. australiana.
Sequence variation in mitochondrial DNA can be strongly influenced by Wolbachia infection [41]. Extensive Wolbachia screening showed that each Plutella species was infected with a different strain at contrasting frequencies, and fit a 'most-or-few' pattern whereby species infection rates are often very low (<10%) or very high (> 90%) [91]. Infection incidence in P. xylostella was lower in Australia (1%) than previously reported across global samples (5%) [18]. Our finding of a single supergroup A strain showing 100% sequence similarity to a strain reported in P. xylostella from Malaysia, plutWA1 [18], provides some support of an Asian origin for Australian P. xylostella [17], though does not preclude this strain also occurring elsewhere.
Fixation of infection in P. australiana suggests that Wolbachia manipulates the reproductive biology of this species. We found no evidence of sex-ratio distortion, which has been associated with a Wolbachia strain, plutWB1, in P. xylostella [18]. High infection can be driven by cytoplasmic incompatibility (CI) [40]. The high frequency (87%) of a single mtDNA haplotype among P. australiana individuals implies that the spread of Wolbachia infection has driven a selective sweep of coinherited mtDNA through the population, causing a loss of mtDNA diversity [41]. High nuclear diversity (relative to sympatric P. xylostella) supports this hypothesis, because a demographic bottleneck should reduce diversity across the entire genome [34].
Plutella australiana and P. xylostella have co-existed in Australia for at least 125 years ( 1300 generations), yet have strongly divergent mitochondrial and nuclear genomes, Wolbachia infections and insecticide susceptibility phenotypes. Our observations during laboratory rearing and crossing experiments also suggested that interspecific differences in host plant use may exist. What explains such strong divergence between the two Plutella species, given sympatry and the capacity to hybridize? Endemism of P. australiana [16] implies an ancient evolutionary history in Australia, and our data provide support for existing views that Australian P. xylostella were recently introduced from a small ancestral source population, possibly from Asia [17,18,77]. Therefore, the two Plutella species may have diverged in allopatry and recently come into secondary contact. Maintenance of divergence suggests strong continuing reproductive isolation, which can evolve as a side-effect of allopatric divergence [44]. All 99 individuals that were RAD sequenced showed concordance in nuclear multilocus genotypes and mtDNA genotypes identified through PCR-RFLP regardless of geographic location, as shown by STRUCTURE analysis. Cryptic species in sympatry provides strong evidence of limited genetic exchange [79]. A small degree of genotypic admixture evident for a few individuals in the STRUCTURE plots might be explained by ancestral polymorphism or introgressive hybridization [28], or alternatively, could be an artefact if our dataset is not representative of the entire genetic background [33]. The level of hybridization that may be occurring between these species is unknown. Isolation may not be uniform across the genome [92,93], and scans of larger genomic regions may be required to identify introgression and detect hybrids.
The factors leading to reproductive isolation between the two Plutella species in nature are unknown but could include a range of pre-or post-mating isolation mechanisms, such as assortive mating or hybrid fitness costs. Behavioural mating choices are often the main isolating factor in sympatric animals [86]. Does Wolbachia cause a reproductive barrier? The contrast in infection status creates the potential for cytoplasmic incompatibility between species [94]. Interspecific crosses showed a pattern of asymmetric isolation consistent with the expected effects of unidirectional CI, where 21% crosses involving infected P. australiana females produced viable offspring, while the reciprocal CI-cross direction (uninfected P. xylostella females crossed with infected P. australiana males) was nearly sterile. However, this pattern was not continued in the F1 generation: infected hybrid males (derived from the P. australiana maternal line) produced offspring at comparable rates when back-crossed to either uninfected P. xylostella or infected P. australiana female parents. The role of Wolbachia-induced postzygotic isolation between the two Plutella species requires further study, though our results suggest it could be more important in the F0 generation. Wolbachia can contribute to post-zygotic genetic isolation after speciation by complementing hybrid incompatibilities [94,95]. Symbiont infections could also influence mating behaviour and contribute to pre-mating isolation [96].

Conclusions
The discovery of cryptic pest species introduces complexities for their management and also exciting opportunities for understanding ecological traits. We found strong genomic and phenotypic divergence in two cryptic mitochondrial Plutella lineages co-existing in nature, supporting their status as distinct species [16] despite the capacity to hybridize. Reproductive isolation is likely to have evolved during allopatric speciation, and genomewide sequence data suggest it has been maintained following secondary contact. Variation in Wolbachia infections might be one factor reinforcing reproductive barriers.
Plutella australiana co-occurs with P. xylostella throughout agricultural regions of southern Australia, but made up only 10% of Plutella juveniles collected from cultivated and wild brassicaceous plants. A lack of population structure across neutral SNP markers suggests that P. australiana populations are linked by high levels of gene flow, and also that P. australiana is a highly mobile species, which is supported by light trap collections [16] and seasonal colonization of canola crops. Future molecular analysis of Australian Plutella should include a species identification step using a molecular diagnostic assay. For ecological studies, it may be possible to perform molecular species identification to confidently distinguish a representative sub-sample of individuals or pooled samples. Our study has shown that while P. australiana can attack canola crops, there is no evidence of pest status in commercial Brassica vegetables crops, and bioassays suggested that field populations should be easily controlled with insecticides. Though P. australiana is a potential pest of some Australian Brassica crops, it is of secondary importance to the diamondback moth, P. xylostella.

Additional file
Additional file 1: Table S1 The four variable nucleotide sites among the five P. xylostella 613 bp COI haplotypes identified in 102 individuals from Australia. Table S2 The eight variable nucleotide sites among the nine P. australiana 613 bp COI haplotypes identified in 87 individuals from Australia. Table S3 Log-logistic regression statistics for dose-response bioassays on P. australiana and P. xylostella field strains and the P. xylostella reference strain exposed to four commercial insecticides. (PDF 91 kb)