Effects Of Mitochondrial Heteroplasmy On Species Identification And Biodiversity Assessment Using DNA Barcoding

The cox1 genes of fig wasp were amplified using universal primers,and the cox1 fragments of the same individual were simultaneously sequenced by Sanger sequencing and Illumina Miseq sequencing.Based on the sequencing results,we did the following analysis:(1)DNA barcoding analysis based on Sanger sequencing;(2)Mitochondrial heteroplasmy analysis based on Illumina Miseq sequencing;(3)Exploration of the mitochondrial heteroplasmy effect on DNA barcoding by comparing Sanger sequencing and Illumina Miseq amplicon sequencing;(4)Verification the authenticity of mitochondrial heteroplasmy in a variety of ways.The main results were as follows:1.DNA barcoding analysis based Sanger sequencing.Based on morphological identification,DNA barcodings of 154 fig wasps belonging to 28 species,10 genera and 5 subfamilies of 3 families were obtained by Sanger sequencing.By checking whether there were inframe stop codons,three DNA barcodings were considered to be Numts;By constructing NJ phylogenetic trees,five DNA barcodings were considered to be cross-contamination during the experiment.Finally,146 DNA barcodings from 28 species were used for further analysis.By calculating the DNA barcoding intraspecific/interspecific genetic distances,we found that the DNA barcodes intraspecific genetic differences were less than 2% in 20 species,but the DNA barcodes intraspecific genetic differences were more than 2% in the other 8 species.There was an obvious overlaps in the range of intraspecific/interspecific genetic differences: the minimal interspecific genetic differences was 5.8%(Philotrypesis pilosa(HIS)Vs.Philotrypesis sp.(HIS)),while the maximum intraspecific genetic distances among four species including Philotrypesis sp.,Platyscapa sp.and E.altissima were 7.5%,6.7% and 6.7%,respectively.Clustering with 2% similarity,the 146 DNA barcodings can be clustered into 38 m OTUs.2.Mitochondrial heteroplasmy analysis based on Illumina Miseq sequencing.A total of 4798199 paired-end reads were obtained from the 146 fig wasps.After quality trimmed,it was found that the amplified sequences of the two specimens might originate from crosscontamination and were excluded.Finally,a total of 1915416 cox1 sequences were obtained from 144 fig wasps,with an average sequencing depth of 13300×.With 100% similarity,the haplotypes composition of the 146 specimens were analyzed one by one.It was found that 123 specimens(85.42%)contained more than two haplotypes,and 17 specimens(11.64%)contained more than 10 haplotypes.54 specimens(37.50%)contained MIN^ and distributed among 16 species(57.14%).Among them,the heteroplasmy differences were as high as 9.20%(BEN.Sycobia.sp.M12.2,BEN.Walkerella.benjamini.Female.1,CUR.Platyscapa.sp.Male.6,& HIS.Philotrypesis.sp.Male.1);The highest proportion of MIN^ was found in BEN.Walkerella.benjamini.Female.1: fourteen MIN^ accounted for 78.44% of the total sequence.3.For 7 species that may contain cryptic species identified by DNA barcoding,we integrated the Sanger sequencing results of 7 species and the haplotype sequences obtained by high-throughput sequencing and construct NJ trees.We found that although the DNA barcodes obtained by Sanger sequencing formed distinct differentiation branches on NJ trees,the highthroughput sequencing haplotypes from single head specimens were not only distributed on "sister species",even formed new "sister species",suggesting that the conclusion that the cryptic species can be identified by DNA barcode may be due to the heteroplasmy.4.Using high-throughput sequencing to simulate DNA metabarcoding,the haplotypes of 90 samples can be clustered into a single m OTU,the haplotypes of the remaining 54 samples can be clustered into multiple m OTUs,and even the two samples of Walkerella benjamini(BEN)can be clustered into 14 m OTUs,while the haplotypes of 28 species can be clustered into 93 m OTUs,which is much larger than that the number of actual species.5.Multiple ways to verify the authenticity of mitochondrial heteroplasmy.By analyzing synonymous/non-synonymous mutations,haplotype-specific long fragment amplification and genome-wide BLAST search for cox1 homologous fragments,we confirm that the majority of Numts can be excluded by our analysis methods,and the resulting haplotypes can accurately reflect the mitochondrial heterogeneity of samples.6.Mitochondrial heteroplasmy distribution has no obvious branch specificity;some species have obvious male-specific mitochondrial heterogenous distribution.In summary,we confirmed that mitochondrial heteroplasmy directly affects DNA barcoding for the first time,and when cox1-based DNA barcoding is used in biodiversity exploration,heteroplasmy will lead to a serious overestimation of biodiversity.