| Summary: | Phylogeographic studies have revealed spatial genetic structure and inferred geographical processes that may have generated genetic diversity and divergence. These study results have implications not only on the processes that generate intraspecific and interspecific diversity but also on the essential integrals for defining evolutionary entities (e.g., species). However, the resulting phylogeographic inferences might be impacted by the sampling design, i.e., the number of individuals per population and the number of geographic populations studied. The effect of sampling bias on phylogeographic inferences remains poorly explored. With a comprehensive sampling design (including 186 samples from 56 localities), we studied the phylogeographic history of a Taiwanese endemic damselfly, <i>Psolodesmus mandarinus</i>, with a specific focus on testing the impact of the sampling design on phylogeographic inference. We found a significant difference in the genetic structure of eastern and western populations separated by the Central Mountain Range (CMR) of Taiwan. However, isolation by the CMR did not lead to reciprocally monophyletic geographic populations. We further showed that, when only a subset of individuals was randomly included in the study, monophyletic geographic populations were obtained. Furthermore, historical demographic expansion could become undetectable when only a subset of samples was used in the analyses. Our results demonstrate the impact of sampling design on phylogeographic inferences. Future studies need to be cautious when inferring the effect of isolation by a physical barrier.
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