Take out the rubbish – Removing NUMTs and pseudogenes from theBemisia tabacicryptic species mtCOI database

Abstract

Identification ofBemisia tabacicryptic whitefly species complex currently relies on molecular characterisation of the mitochondrial DNA cytochrome oxidase subunit I (mtCOI) partial gene, however, nuclear mitochondrial sequences (NUMTs), PCR-derived pseudogenes and/or poor sequence editing have hindered this effort. To-date,ca.5,175 partial (≥ 300bp) mtCOI sequences for species identification purposes have been reported. We reviewedca.10% of sequences representing the standardB. tabacispecies complex mtCOI dataset. We found that 333 sequences (64.9%) were NUMTs, pseudogenes and/or affected by poor sequence quality. Amino acid pattern analyses of high throughput sequencing-derived mtCOI gene from 24 ‘tabaci’ and ‘non-tabaci’ species enabled differentiation between NUMTs/pseudogene-affected and likely real mtCOI sequences, and that the SSA4, SSA5/SSA8, AsiaII-2 and AsiaII_4 species were NUMTs/pseudogenes artefacts. Intra-specific uncorrected nucleotide distances (p-dist) from our up-dated dataset ranged from 0-1.98%, inter-specificp-dist within phylogenetic clades ranged betweenca.2.5 and 8%, and 8 and >19% for species between phylogenetic clades. Differentiating between closely related species could therefore utilise an ‘average’p-dist of 2.5%. Despite the smallerB. tabacimtCOI dataset, six putative new species were identified. Adoption of our standardised workflow and up-dated mtCOI clean dataset could facilitate better diagnostics ofB. tabaciand ‘non-tabaci’ cryptic species.

Genetic diversity of whitefly (Bemisia spp.) on crop and uncultivated plants in Uganda: implications for the control of this devastating pest species complex in Africa

“…Identification of NUMTs and PCR artefacts in the sequences obtained was as described by Vyskočilová et al (2018) and Kunz et al (2019). Briefly, Sanger sequences generated in this study were aligned with high-throughput sequencing (HTS)-derived full mitogenome sequences downloaded directly from GenBank in Geneious Prime® 2019.2.1 with the MUSCLE alignment option set to eight iterations.…”

“…Whitefly mtCO1 sequences obtained in this study were aligned together with equivalent reference whitefly sequences obtained from Kunz et al (2019) in Geneious Prime® 2019.2.1. The model of molecular evolution was determined using JModelTest version 2.1.10. and phylogenetic trees generated using MrBayes version 3.2.6 set with the following commands: lset nst = 6 rates = gamma.…”

“…In the absence of reliable morphological features, mtCO1 barcoding (Frohlich et al 1999;Dinsdale et al 2010), genome-wide SNP markers and whole genome sequencing approaches (Wosula et al 2017;Chen et al 2019;de Moya et al 2019;Elfekih et al 2019;Mugerwa et al 2020) have been used to generate an improved understanding of the systematics within this species complex. A partial region of the mtCO1 gene has been the molecular marker used most widely by the whitefly research community to classify B. tabaci species, with to date > 40 cryptic putative species proposed based on > 3-4% nucleotide divergence (Frohlich et al 1999;Dinsdale et al 2010;Mugerwa et al 2018;Vyskocilova et al 2018;Kunz et al 2019). In Africa, B. tabaci species (East Africa 1 (EA1), Indian Ocean (IO), Mediterranean (MED), Middle East-Asia Minor (MEAM1, MEAM2-Africa), Morocco, New World (NW1)-Sudan [EU760727] and sub-Saharan Africa (SSA) species SSA1-SSA13 have been reported on beans, cassava, cotton, eggplant, tomato, sweet potato or various uncultivated plants (Frohlich et al 1999;Legg et al 2002Legg et al , 2014bBerry et al 2004;Sseruwagi et al 2006;Boykin et al 2012;Mugerwa et al 2012Mugerwa et al , 2018Tahiri et al 2013;Esterhuizen et al 2013).…”

“…Although the 3′ mtCO1 sequence has been widely used to delimit cryptic species within the B. tabaci complex, studies are progressively revealing some classifications to be errors caused by nuclear mitochondrial DNA (NUMTs) or chimeric PCR products (Tay et al 2017;de Moya et al 2019;Vyskočilová et al 2018;Kunz et al, 2019), as well as species status not correlating consistently with divergence in the mtCO1 barcode region (Qin et al 2016;Vyskočilová et al 2018;Mugerwa et al 2020). For describing whitefly diversity in this study, we have nevertheless adopted it as a method to indicate species which should be considered as putative awaiting further biological and genetic studies to confirm their taxonomic status.…”

Within-Season Changes in Land-Use Impact Pest Abundance in Smallholder African Cassava Production Systems

“…Amplicon HTS runs were performed on Illumina MiSeq System, and sequence data processed using Trimmomatic [ 23 ] and analyzed as outlined in Tay et al [ 22 ] using the Geneious software version 10.0.8. All mtCOI sequences were trimmed to 657 bp for molecular diagnostics of Bemisia species using reference sequences for Sub-Saharan Africa (SSA)1, SSA2, MED, IO and for pseudogene detection [ 24 ]. Detection of HTS-generated partial mCOI sequences of Eretmocerus and Encarsia parasitoids were identified using the reference sequences (GenBank MN646919, MN646949), prior to species identification against curated sequences in GenBank.…”