Abstract
Abstract DNA barcoding often fails to identify species despite its undisputed advantages. Hybridization, sample contamination, incomplete lineage sorting and nuclear copies of mitochondrial genes (NUMTs) are often put forward as explanations but have seldomly been tested. Here I used available RNA-sequencing data to explore this issue in four Chorthippus grasshopper species. I was able to exclude NUMTs, contamination and recent hybridization as probable causes of the low barcoding performance. Using a phylogenetic method, I estimated the nuclear and mitochondrial mutation rates as 1.31 × 10−9–2.27 × 10−9 and 8.1 × 10−9–1.4 × 10−8 mutations/site/year, respectively. These grasshoppers therefore did not exhibit a particularly low mitochondrial mutation rate compared to other insect species. Using coalescence simulation, I was able to show that two simple demographic scenarios, with a divergence period of 1–3 Myr, provided a good fit to the mitochondrial genealogies in three of the four target species. Interestingly, the mitochondrial genealogy of Chorthippus mollis was inconsistent with a neutral evolution pattern, suggesting that it had undergone adaptive selection.