The complete mitochondrial genome of the strawberry aphid Chaetosiphon fragaefolii Cockerell, 1901 (Hemiptera: Aphididae) from California, USA.

The aphid Chaetosiphon fragaefolii Cockerell, 1901 is an agricultural pest and known vector of strawberry viruses. To better understand its biology and systematics, we performed a genomic analysis on C. fragaefolii collected from Quinalt strawberry plants from Pacific Grove, Monterey county, California, USA using Oxford Nanopore and Illumina sequencing. The resulting data were used to assemble the aphids complete mitogenome. The mitogenome of C. fragaefolii is 16,108 bp in length and contains 2 rRNA, 13 protein-coding, and 22 tRNA genes (GenBank accession number LC590896). The mitogenome is similar in content and organization to other Aphididae. Phylogenetic analysis of the C. fragaefolii mitogenome resolved it in a fully supported clade in the tribe Macrosiphini. Analysis of the cox1 barcode sequence of C. fragaefolii from California found exact and nearly identical sequences to C. fragaefolii and Chaetosiphon thomasi Hille Ris Lambers, 1953, suggesting the two species are conspecific.

The Aphididae consists of more than 4700 species of aphids that occur worldwide (Kim and Lee 2008). About half of the species in the family are classified to its most specious subfamily, the Aphidinae (Choi et al. 2018). One of these species is the strawberry aphid, Chaetosiphon fragaefolii (Cockerell 1901). C. fragaefolii was originally named from specimens from Jerome, Arizona, but has since been reported throughout North and South America, Europe, South Africa, New Zealand, and Australia (Dixon et al. 1987; Blackman and Eastop 2000; Rondon and Cantliffe 2004). It is an agricultural pest and has been shown to transmit several viruses to strawberry plants, including the economically devastating strawberry mild yellow edge virus (Lavandero et al. 2012). A large number of C. fragaefolii cox1 barcode gene sequences are deposited in GenBank (Foottit et al. 2008; Gwiazdowski et al. 2015; Hebert et al. 2016), however, the mitochondrial genome of C. fragaefolii has not been analyzed. Here, we performed Oxford Nanopore and Illumina genome sequencing on a specimen of C. fragaefolii from California, USA to determine its mitogenome structure and phylogenetic relationship to other aphids in the Aphididae.

DNA was extracted from C. fragaefolii (Voucher Specimen- Hartnell College #264, Dr. Jeffery R. Hughey, jhughey@hartnell.edu) collected on a Quinalt strawberry plant from Pacific Grove, Monterey county, California (36°37′06.1′′N, 121°54′41.1′′W) using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following the protocol of Hughey et al. (2019). The DNA extract was concentrated to 10 µL using the Microcon DNA Fast Flow Centrifugal Filter Unit Cat # MRCF0R100 (MilliporeSigma, Burlington, MA). The Oxford Nanopore library and sequencing was performed using the Rapid Sequencing Kit (SQK-RAD004) on a R9.4.1 flow cell and MinION device following the manufacturer’s instructions (Oxford Nanopore Technologies, Oxford, UK). The Nanopore sequencing generated 161,000 reads. The 150 bp paired-end Illumina library construction and sequencing were performed by myGenomics, LLC (Alpharetta, GA) and generated 20,128,502 reads. The mitogenome was assembled de novo using Illumina reads with the default settings in MEGAHIT (Li et al. 2015), and the gaps closed by mapping both the Oxford Nanopore and Illumina reads onto the de novo contigs using the default settings in Geneious Prime® 2020.1.2 (Biomatters Limited, Auckland, New Zealand). The annotation was completed with MITOS (Bernt et al. 2013) and NCBI ORF-finder (https://www.ncbi.nlm.nih.gov/orffinder/). The C. fragaefolii mitogenome was aligned to other mitogenomes with MAFFT (Katoh and Standley 2013) and the phylogenetic analysis was executed using RaxML in Trex-online (Boc et al. 2012) with the GTR + gamma model and 1,000 bootstraps. The tree was visualized with TreeDyn 198.3 at Phylogeny.fr (Dereeper et al. 2008).

The mitogenome of C. fragaefolii is 16,108 bp in length and is AT skewed with a base composition of 45.6% A, 38.3% T, 10.5% C, and 5.6% G. It contains 22 tRNA (trnL and trnS are duplicated), 2 rRNA (rnl, rns), and 13 electron transport and oxidative phosphorylation genes. Nine of the protein-coding genes and 15 tRNAs are coded on the forward strand, with the remaining 13 genes transcribed on the reverse strand. The start codon for the protein-coding genes cox2, cox3, nad2, and nad6 is ATA; nad1, nad4, nad4L, and nad5 is TTA; cox1, atp6, and nad3 is ATT; atp8 is ATC; and Cyt b is ATG. Most of the protein-coding genes terminate with TAA, but nad4 and nad4L terminate with CAT; cox1 with ATT; and nad1 with AAT. The mitogenome content and organization of C. fragaefolii is similar to other genera in the tribe Macrosiphini, including Cavariella salicicola (Wang et al. 2013), Diuraphis noxia (Zhang et al. 2014), Indomegoura indica (Hong et al. 2019), Myzus persicae (Voronova et al. 2020), and Sitobion avenae (Zhang et al. 2016). These taxa however differed from Acyrthosiphon pisum (International Aphid Genomics Consortium 2010) in the position of one of the trnS genes.

Phylogenetic analysis of C. fragaefolii fully resolved it in a clade with six other mitogenomes from the Macrosiphini (Figure 1). Comparison of the C. fragaefolii cox1 California sequence to published cox1 barcodes in GenBank found two identical sequences identified as C. thomasi from Manitoba and Saskatchewan, Canada. Chaetosiphon thomasi is a holocyclic rose-feeding species that specifically colonizes the rose plant Rosa rugosa (Blackman et al. 1987). The rose feeding populations of C. thomasi differ from C. fragaefolii in having a shorter rostrum and distinctive fundatrix morphology (Blackman et al. 1987). Thirty-one other sequences deposited in GenBank identified as C. thomasi, C. fragaefolii, and Chaetosiphon sp. from around the world differed by a single transition from adenine to guanine at position 246 of the cox1 gene. The mutation is silent and codes for a methionine at amino acid 82 of cox1. Based on this genetic evidence, C. fragaefolii and C. thomasi appear to be conspecific, however both require DNA sequencing of topotype material before proposing a final taxonomic conclusion.

Figure 1.

RaxML phylogram of Chaetosiphon fragaefolii and representative Aphididae mitogenomes. The subfamily Greenideinae served as the outgroup and the three other taxa listed to the right are tribes in the subfamily Aphidinae (Hormaphidini, Macrosiphini, and Aphidini). The * indicates 100% bootstrap support based on 1000 nreps. The legend below represents the scale for nucleotide substitutions.