DOI: 10.5061/dryad.w0vt4b924
title: Pervasive heteroplasmy in an invasive ambrosia beetle (Scolytinae) in southern California

description: Heteroplasmy, the presence of multiple distinct mitochondrial genotypes
 (mitotypes) within an individual, has long been thought to be a rare
 aberrance that is quickly removed by selection or drift. However,
 heteroplasmy is being reported in natural populations of eukaryotes with
 increasing frequency, in part due to the development of better diagnostic
 molecular methods. Here, we report a seemingly stable heteroplasmic state
 in California populations of the polyphagous shothole borer (PSHB),
 Euwallacea fornicatus; an exotic invasive ambrosia beetle that is causing
 significant tree dieback. We develop and validate a qPCR assay that
 utilizes novel locked nucleic acid probes to detect different mitotypes,
 and qualitatively assess heteroplasmy in individual PSHB. We prove the
 utility of this assay by: a) mitotyping field-collected PSHB to document
 the prevalence of heteroplasmy across its invasive range in southern
 California; and, b) measuring relative titers of eac h mitotype across
 multiple generations of heteroplasmic PSHB colonies in the laboratory to
 assess the stability of transmission through the maternal germline. We
 rule out the possibility that heteroplasmy is due to a NUMT, and find that
 heteroplasmic individuals are common in Californian field populations of
 PSHB, and that heteroplasmy persists in a stable state for at least 10
 generations in experimental colonies. We also looked for evidence of the
 common occurrence of paternal leakage, but found none. In light of our
 results, we discuss competing hypotheses as to how heteroplasmy may have
 first arisen, and continues to perpetuate, in Californian PSHB
 populations.
description: Flurescence data collected via Rotor-Gene Q, from qPCR assays
 utilizing LNA probes to detect two competing mitotypes (alleles) in single
 individuals.
description: # Pervasive heteroplasmy in an invasive ambrosia beetle (Scolytinae) in
 southern California
 [https://doi.org/10.5061/dryad.w0vt4b924](https://doi.org/10.5061/dryad.w0vt4b924) ## Description of the data and file structure Study of heteroplasmy in California populations of the polyphagous shothole borer (PSHB), *Euwallacea fornicatus*. Data is presented in a single Excel workbook, across four sheets (tabs). All fluorescence data was collected using the green and yellow channels on a QIAGEN Rotor Gene Q. Sheet 1: Validation of LNA assay - DNA extractions from two colonies with differing but pure mitotypes (H33 and H35) were used to make a series of mixes in the following ratios (H33:H35); 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 5:95. These mixes were then used to test the ability of the LNA-probe assay to qualitatively discriminate different relative titers. Data is presented in 8 columns ``` A - RUN; the assay was validated in it's entirety three times B - Haplotype Mix ratio (H35:H33); artificially created "heteroplasmic" mix of differing relative quantities of two pure haplotypes H35 and H33. The pure haplotypes and "No template Controls" (NTC) are utilized for normalizing (rescaling) the fluoresence data, using the formulae: Normalized df [yellow]=((df [yellow]X – df [yellow]NTC))/((df [yellow]H33 – df [yellow]NTC)) Normalized df [green]=((df [green]X – df [green]NTC))/((df [green]H35 – df [green]NTC)) ...where X is the sample, H33 and H35 are pure haplotypes, and NTC is a TE buffer (negative) control. NOTE: data in each subsequent sheet is normalized (rescaled) by the same process! C - Mean change in fluorescence green (H35); calculated per run from four replicate reactions D - Mean change in fluorescence yellow (H33); calculated per run from four replicate reactions E - Rescaled mean change in fluorescence green (H35); see article for means of calculation F - Rescaled mean change in fluorescence yellow (H33); see article for means of calculation G - Estimated percent H35 in mix H - Estimated percent H33 in mix ``` Sheet 2: Assessment of heteroplasmy in fresh eggs and pinned museum specimens. DNA extracted from individual eggs or a single leg from a museum specimen, using a Chelex 100 method. LNA-probe assay used to detect heteroplasmy in the extracts - each extract was tested in duplicate reactions. Data is presented across 18 columns. ``` A-I - Raw measures J - empty K - Type = Egg. Pinned museum specimen, or control L - DNA extraction/sample identifier M - Mean change in fluorescence green (H35); calculated from two replicate reactions N - Mean change in fluorescence yellow (H33); calculated from two replicate reactions O - Rescaled mean change in fluorescence green (H35) P - Rescaled mean change in fluorescence yellow (H33) Q - Estimated percent H35 in extract R - Estimated percent H33 in extract ``` Sheet 3: Assessment of heteroplasmy across multiple generation in the lab. Temporal changes in the relative abundance of two mitotypes in individuals collected from heteroplasmic colonies maintained at differing temperatures. LNA-probe assay used to detect heteroplasmy in each individual - DNA from each individual was tested in duplicate reactions. Data is presented across 14 columns. ``` A - Rearing temperature = 29 degrees C or 23 degrees California B - Generation (time point) = 1, 6, or 10 (29 degrees only) C - Line; three separate lines were maintained at each temperature (designated B, C, & D at 29 degrees, and C, E, and F at 23 degrees) D - Individual specimen number E - Rescaled mean change in fluorescence green (H35); calculated from two replicate reactions F - Rescaled mean change in fluorescence yellow (H33); calculated from two replicate reactions G - Estimated percent H35 in extract H - Estimated percent H33 in extract I - empty J-N - calculated means used to produce Figure 5 ``` Sheet 4: Evidence that paternal leakage is not commonplace. Individual virgin males and females, from separate homoplasmic H33 and H35 colonies, were paired to create two ‘hybrid’ crosses (H33 female x H35 male, and H35 female x H33 male). Subsequent broods were reared in artificial diet and five adult F1 offspring were evetually tested for evidence of heteroplasmy. LNA-probe assay used to detect heteroplasmy in each individual - DNA from each individual was tested in duplicate reactions. Data is presented in 8 columns. ``` A - Cross; either H35 female x H33 male, or H33 female x H35 male B - Sample ID = pair ID and brood offspring number (e.g., 10A-1 refers to pair 10A and F1 offspring number 1) C - Mean change in fluorescence green (H35); calculated from two replicate reactions D - Mean change in fluorescence yellow (H33); calculated from two replicate reactions E - Rescaled mean change in fluorescence green (H35) F - Rescaled mean change in fluorescence yellow (H33) G - Estimated percent H35 in extract H - Estimated percent H33 in extract ``` ## Sharing/Access information All data was derived from the present study and is not shared elsewhere.