DOI: 10.5061/dryad.mgqnk9962
title: Hybrid lizards with introgressed mtDNA show increased resistance to DNA damage from Reactive Oxygen Species

description: Hybrids can experience genetic incompatibilities that lead to cellular
 dysfunction and infertility. Previous analyses of mitochondrial function
 in the lizards Urosaurus graciosus and Urosaurus ornatus found that
 hybrids, which had introgressed mtDNA, experienced higher ATP production
 which increases reactive oxygen species (ROS) production, one of the main
 causes of damage to DNA. Hybrid populations of these lizards have
 persisted so we predicted they may have mechanisms for protecting their
 DNA from damage. To test this, we exposed liver cells of these lizards to
 hydrogen peroxide, a source of ROS, and compared DNA damage using
 single-cell gel electrophoresis. Hybrid cells showed significantly less
 DNA damage. We then examined potential mechanisms to explain this result
 by comparing melanin content of livers and expression levels of
 antioxidant genes. If functioning to deal with ROS, these should be
 expressed higher in hybrids. Melanin content of livers was highly variable
 among U. graciosus individuals and therefore not highest in hybrids as
 expected. Eighteen antioxidant genes showed significant differential
 expression but were not expressed consistently higher in hybrids. The
 overall gene expression profiles of hybrids and U. graciosus were very
 similar while that of the mtDNA donor species, U. ornatus, was highly
 divergent from both. Three antioxidant genes with different expression
 levels in hybrids and that warrant further study were SOD2, SOD3, and
 PRDX2. These results show how species can compensate for mtDNA
 introgression and highlight the complexity of genetic interactions
 encountered when closely related species exchange genetic material in
 zones of secondary contact.
description: <em>Urosaurus graciosus</em> and <em>U.
 ornatus</em> lizards were sampled from 2 different populations of
 each type of lizard at the following locations in Arizona during June 2020
 (AZ permit #SP403988, IACUC Protocol #2019-0001): <em>U. ornatus
 </em>morphology and mtDNA n = 4: Burro Creek (n = 2,
 -113<sup>o </sup>27.00W, 34<sup>o</sup>32.55N),
 Wickenburg Wash (n = 2, -112° 42' 39.13W, 33° 57' 2.12N);
 <em>U. graciosus </em>morphology with <em>U. ornatus
 </em>mtDNA “<em>Hybrid </em>” n = 5: Detritial Wash (n =
 3, -114<sup>o</sup>30.84W, 35<sup>o</sup>49.06N),
 Date Creek (n = 2, -113<sup>o</sup>9.966W,
 34<sup>o</sup>9.160N), <em>U. graciosus
 </em>morphology and mtDNA n = 5: Walnut Creek (n = 3, -
 114<sup>o</sup>9.619W, 35<sup>o</sup>2.17N),
 Hassayampa Wash (n = 2, -112° 40' 54.35"W, 33°
 44.35"N). MtDNA type was verified using a three
 primer species specific PCR protocol that amplifed either a 81 bp fragment
 from <em>U. ornatus</em>/hybrid type mtDNA from the ND1 gene
 or a 141 bp fragment from <em>U. graciosus</em> type mtDNA
 (see Haenel and Del Gaizo Moore, 2018 for details). The conserved reverse
 primer was 5’-GGTCGTACTGGTTCTTTTGTGAATA-3’, the <em>U.
 ornatus</em> and hybrid specific primer was
 5’-CCCAAATGTAGTTGGCCCT-3’, and the <em>U. graciosus</em>
 specific primer was 5’-CGTCGCCTTCCTTACCCTAC-3’. Liver
 tissue was dissected out immediately following euthanasia by rapid
 freezing in liquid nitrogen, separated into approximately 0.05 gram
 aliquots and stored in a -80ºC freezer. The liver was examined here
 because it is very metabolically active and previous work on mitochondrial
 function found evidence of dysfunction for mitochondria taken from liver
 cells in these lizards (Haenel and Del Gaizo Moore, 2018). Comet assays
 followed the alkaline protocol described in detail in Collins (Collins,
 2014) with minor modifications. Solutions can be found in Collins
 (Collins, 2014) except where noted below. Each sample was coded by a 4
 digit number such that the experimenters did not know which species the
 sample belonged to until it was categorized for statistical analyses.
 Liver cells were isolated by gently passing approximately 0.05 g liver
 tissue through a 100 µm cell strainer (CELLTREAT<sup>®</sup>
 Scientific Products) in 3 ml Merchant’s Medium (0.14M NaCl, 1.47mM
 KH<sub>2</sub>PO<sub>4</sub>, 2.7mM KCl, 8.1mM
 Na<sub>2</sub>HPO<sub>4</sub>, 10mM
 Na<sub>2</sub>EDTA, pH 7.4) on ice. Preliminary tests found
 this amount of tissue gave an appropriate number of cells per image.
 Isolated cells (15 µL) were added to 90 µL Merchants Medium then incubated
 at 37<sup>o</sup>C for 20 minutes. Hydrogen peroxide (0.0878M
 H<sub>2</sub>O<sub>2</sub>) was added to
 treatments as a source of ROS while controls did not have any
 H<sub>2</sub>O<sub>2 </sub>added. Following
 incubation, 15 µL of the cells were added to 90 µL 1% LMP agarose
 (R&D Systems) and 50 µL of this agarose mixture was pipetted onto
 microscope slides (CometSlide™ R&D Systems). Slides were incubated
 at 4<sup>o</sup>C for 10 minutes then transferred to lysis
 buffer (CometAssey<sup>®</sup> Lysis Solution, R&D
 Systems) and incubated for 1 hour to overnight at
 4<sup>o</sup>C. Slides were then incubated in electrophoresis
 buffer (0.3M NaOH, 1mM EDTA) at 4<sup>o</sup>C for 40
 minutes. Single cell electrophoresis was carried out
 for 1 hour at 500 mA per cm<sup>2 </sup>in the same
 electrophoresis buffer at 4<sup>o</sup>C. Following
 electrophoresis, slides were rinsed twice in distilled water for 5
 minutes, placed in 70% ETOH for 5 minutes then dried in a
 37<sup>o</sup>C incubator for 15 minutes. Slides were then
 stained with Ethidium Bromide for 30 minutes and rinsed twice with
 distilled water. Comets were viewed and photographed with an
 EVOS<sub>fi</sub> (ThermoFisher Scientific) inverted digital
 microscope using the 10x objective and RTF filter. Photographs were taken
 of at least 12 randomly selected fields of each treatment and control.
 Photos of comets were scored using OpenComet (Gyori et al., 2014), a
 plugin for ImageJ (Schneider et al., 2012). The default settings in
 OpenComet were used and all scorable nuclei were included from each field.
 Following the initial run with OpenComet, each image was viewed to find
 additional outliers that needed to be removed before statistical analyses.
 These were mainly where OpenComet scored two comets as one due to an
 overlap on the image. Absorption spectroscopy is a
 commonly used method to quantify melanin in cells and tissue (Chung et
 al., 2019) and was used here. Samples for melanin quantification were
 obtained from liver tissue homogenized for the comet assay. Samples ranged
 in color from light tan to dark brown<em>.</em> Melanin
 standards ranging from 2 to 75 mg/mL were made from melanin
 (Sigma-Aldrich) dissolved in mitochondrial isolation buffer (Moore and
 Haenel, 2022). Absorbance at 475 nm was measured for each standard using
 an Omega BMG Labteck spectrophotometer, and a standard curve was plotted
 by subtracting out the background of the buffer alone. Then, 100 µL of
 each sample was dissolved in a 1 M NaOH solution, and absorbance was
 measured at 475 nm.  
description: # Hybrid lizards with introgressed mtDNA show increased resistance to DNA
 damage from Reactive Oxygen Species
 [https://doi.org/10.5061/dryad.mgqnk9962](https://doi.org/10.5061/dryad.mgqnk9962) ## Description of the data and file structure File: **Olive_moment_data.csv** data table in csv format Olive_moment_data: This sheet contains the 100 (or fewer if less than 100 available) randomly selected olive moment data for each individual. Column names Species: lizard type Ug = Urosaurus graciosus, Uo = Urosaurus ornatus, Hyb = hybrids between the two species ID: individual identifier for each individual Olive_Moment: Olive moment value calculated from OpenComet relative to the average control for that individual. **Location of samples** Species ID long lat Hybrid 455 -114° 30.84W, 35° 49.06N Hybrid 1033 -114° 30.84W, 35° 49.06N Hybrid 1203 -114° 30.84W, 35° 49.06N Hybrid 1335 -113° 9.966W, 34° 9.160N Hybrid 2152 -113° 9.966W, 34° 9.160N Urosaurus graciosus 523 -114° 9.619W, 35° 2.17N Urosaurus graciosus 1212 -114° 9.619W, 35° 2.17N Urosaurus graciosus 1323 -114° 9.619W, 35° 2.17N Urosaurus graciosus 2135 -112° 40.35W, 33° 44.35N Urosaurus graciosus 3122 -112° 40.35W, 33° 44.35N Urosaurus ornatus 1312 -113° 27.00W, 34° 32.55N Urosaurus ornatus 1331 -113° 27.00W, 34° 32.55N Urosaurus ornatus 1344 -112° 39.13W, 33° 57.12N Urosaurus ornatus 2222 -112° 39.13W, 33° 57.12N File name: **Melanin_data.csv** Column names ID: individual lizard identifier Loc: collection site location name long: longitude of collection site lat: latitude of collection site Spp: lizard type, Ug - Urosurus graciosus; Uo - Urosaurus ornatus, hyb - hybrid between these two species OD: UV absorbance at 475nm g_tissue: amount of liver originally used mgMelgtissue: estimated milligrams melanin per gram of liver tissue relMel: amount of melanin in sample compared to mean of Uo samples. Readme for gel_mtDNA.pdf and gel2_mtDNA.pdf files **photos in pdf format** Lane numbers correspond to IDs above and locations are given above as well. File name: design_240213.csv This file gives the conditions and sample names for the DEseq2 analyses given in the R code posted below. ## Code/Software FIle name: DEseq2_Uo_Ug_Hyb.R The File DEseq2_Uo_Ug_Hyb.R gives the R code for the differential expression analyses. Data files for these analyses are located at NCBI GEO accession # GSE255990 Below provides the versions of the packages used. \&gt; sessionInfo() R version 4.3.3 (2024-02-29) Platform: x86_64-pc-linux-gnu (64-bit) Running under: Ubuntu 22.04.4 LTS Matrix products: default BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0 LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0 attached base packages: \[1] stats4 stats graphics grDevices utils datasets methods base other attached packages: \[1] magrittr_2.0.3 readr_2.1.5 ggrepel_0.9.5 \[4] ggplot2_3.4.4 RColorBrewer_1.1-3 dplyr_1.1.4 \[7] pheatmap_1.0.12 DESeq2_1.40.2 SummarizedExperiment_1.30.2 \[10] Biobase_2.60.0 MatrixGenerics_1.12.3 matrixStats_1.2.0 \[13] GenomicRanges_1.52.1 GenomeInfoDb_1.36.4 IRanges_2.34.1 \[16] S4Vectors_0.38.2 BiocGenerics_0.46.0 loaded via a namespace (and not attached): \[1] utf8_1.2.4 generics_0.1.3 bitops_1.0-7 lattice_0.22-5 \[5] hms_1.1.3 EnhancedVolcano_1.18.0 grid_4.3.3 Matrix_1.6-3 \[9] BiocManager_1.30.22 fansi_1.0.6 scales_1.3.0 codetools_0.2-19 \[13] abind_1.4-5 cli_3.6.2 rlang_1.1.3 crayon_1.5.2 \[17] XVector_0.40.0 munsell_0.5.0 withr_3.0.0 DelayedArray_0.26.7 \[21] S4Arrays_1.0.6 tools_4.3.3 parallel_4.3.3 tzdb_0.4.0 \[25] BiocParallel_1.34.2 colorspace_2.1-0 locfit_1.5-9.8 GenomeInfoDbData_1.2.10 \[29] vctrs_0.6.5 R6_2.5.1 lifecycle_1.0.4 zlibbioc_1.46.0 \[33] pkgconfig_2.0.3 pillar_1.9.0 gtable_0.3.4 glue_1.7.0 \[37] Rcpp_1.0.12 tibble_3.2.1 tidyselect_1.2.0 rstudioapi_0.15.0 \[41] compiler_4.3.3 RCurl_1.98-1.14