A global Brassica pest and a sympatric cryptic ally, Plutella australiana (Lepidoptera: Plutellidae), show strong divergence despite the capacity to hybridize

The diamondback moth, Plutella xylostella, 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 ally, Plutella australiana Landry & Hebert, with apparent endemism to Australia, immediately raised questions regarding the extent of ecological and genetic diversity between these two species, whether gene flow could occur, and ultimately if specific management was required. Here, we show that despite sympatric distributions and the capacity to hybridize in controlled laboratory experiments, striking differences in genetic and phenotypic traits exist that are consistent with contrasting colonization histories and reproductive isolation after secondary contact. Almost 1500 Plutella individuals were collected from wild and cultivated brassicaceous plants at 75 locations throughout Australia. Plutella australiana was commonly found on all Brassica host types sampled except commercial vegetables, which are routinely sprayed with insecticide. Bioassays using four commonly-used insecticides found that P. australiana was 19-306 fold more susceptible than P. xylostella. Genome-wide SNPs derived from RADseq revealed substantially higher levels of genetic diversity across P. australiana compared with P. xylostella nuclear genomes, 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. Although P. australiana is a potential pest of brassica crops, it is of secondary importance to P. xylostella.

bachia strains, the Wolbachia surface protein (wsp) gene was sequenced for a subset of individuals. Amplification was performed using wsp81F and wsp691R sequence primers (Zhou, Rousset, 160 & O'Neill, 1998). Amplicons were sequenced using the reverse primer and aligned in geneious  195 We determined that base quality score recalibration using bootstrapped SNP databases was in-196 appropriate for this dataset as it globally reduced quality scores. For downstream comparisons 197 between species, we joint-genotyped P. australiana and P. xylostella individuals using the geno-198 typeGVCFs workflow. To examine finer scale population structure, we also joint-genotyped the 199 P. australiana individuals alone. All variant callsets were hard-filtered with identical parameters  Turkey and the winter moth, Operophtera brumata, from the Netherlands ( Figure 2).

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Inter-species single pair mating experiments showed that hybridization between P. australiana 313 and P. xylostella was possible, yet less successful than intra-species crosses. While most intra-314 species crosses produced adult o↵spring, the fecundity of P. xylostella was >2-fold higher than 315 for P. australiana (Table 6). Both reciprocal inter-species crosses produced F1 adult o↵spring, 316 but success was asymmetric and notably higher in the pairs with P. australiana females. In 317 this direction, there was a strong male bias in the F1 progeny: from 76 cross replicates, 16 318 collectively produced 9 female and 80 male adults, a ratio of 8.9. Hybrid F1xF1 crosses for 319 both parental lines produced F2 adult o↵spring. For the P. australiana maternal line, parental 320 back-crosses using F1 hybrid males successfully produced o↵spring, while parental back-crosses 321 with F1 hybrid females were sterile. For the P. xylostella maternal line, low fitness allowed only 322 a single parental back-cross replicate, which involved a hybrid female and was sterile.

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Mitochondrial haplotype networks of Australian Plutella were constructed using a 613 bp COI 325 alignment that included 81 sequences from this study and 108 from Landry and Hebert (2013). 326 We found low haplotype diversity within Australian P. xylostella, consistent with previous re-  Table   331 S1). Similarly, nine closely related haplotypes were identified in 87 P. australiana individuals, 332 with seven occurring in single individuals ( Figure 5B). The most common haplotype, PaCOI01, occurred at high frequency and di↵ered by 1-2 bases from other haplotypes ( Figure 5b, with notably higher diversity in populations of P. australiana than co-occurring populations of 351 P. xylostella (Table 3). The mean observed heterozygosity within populations ranged from 352 0.13-0.16 for P. australiana and 0.009-0.010 for P. xylostella. Similarly, the average numbers 353 of SNPs, indels and private alleles were considerably higher within P. australiana populations.

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As P. australiana may have fixed nucleotide di↵erences relative to the P. xylostella reference 355 genome that may a↵ect population level statistics, we also removed indels from this dataset and  (Table 4).

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Bioassays revealed highly contrasting responses to insecticide exposure in P. xylostella and P.

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australiana field strains ( Figure 6). Plutella australiana showed extremely high susceptibility 380 to all four insecticides evaluated: resistance ratios at the LC 50 and LC 99 estimates were less 381 than 1.0, indicating that this strain was 1.5-fold to 7.4-fold more susceptible even than the 382 laboratory P. xylostella (S) reference. In contrast, resistance ratios at the LC 50 for the field 383 P. xylostella strain ranged from 2.9 for Success Neo to 41.4 for Dominex (  Asia (Delgado & Cook, 2009;Juric et al., 2017;Saw et al., 2006). Therefore, the two Plutella The discovery of cryptic pest species introduces complexities for their management and also 543 exciting opportunities for understanding ecological traits. We found strong genomic and pheno-544 typic divergence in two cryptic Plutella lineages co-existing in nature, supporting their status 545 as distinct species (Landry & Hebert, 2013), despite their capacity to hybridize. Reproductive 546 isolation is likely to have evolved during allopatric speciation, and genome-wide sequence data 547 suggest it has been maintained following secondary contact. Variation in Wolbachia infections 548 might be one factor reinforcing reproductive barriers.

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Plutella australiana co-occurs with P. xylostella throughout agricultural regions of southern The strain infecting P. australiana (wAus) was identical to a 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, host species and GenBank accession number. Labels in bold denote strains sequenced in this study.

Dominex Coragen
Success Neo Proclaim Figure 6: Dose response curves for P. xylostella and P. australiana field strains collected from Angle Vale and Urrbrae, South Australia, and a susceptible P. xylostella (S) reference strain, exposed to four commercial insecticides: Dominex, Coragen, Proclaim and Success Neo. Points are the mean observed response across four bioassay replicates and lines are the fitted log-logistic response curves with 95% confidence intervals shown in grey shading.    Table 5: Log-logistic regression statistics for dose-response bioassays on P. australiana (P. aus) and P. xylostella (P.x ) field strains and the P. xylostella (S) reference strain exposed to four commercial insecticides. Statistics presented include the number of insects tested (n), LC 50 and LC 99 estimates with 95% confidence limits, resistance ratios (RR) at each LC level, and ratios of the commercial field doses to the LC 99 estimates (Field dose ratio).  Table 6: Fecundity of intra-species and reciprocal inter-species single pair crosses of P. australiana (P.aus) and P. xylostella (P.x ). Presented are the number and proportion in parentheses of replicates that produced eggs and adult o↵spring, and the mean ± standard error of the mean number of eggs and adult o↵spring per replicate.