Luminescent characteristics and mitochondrial COI barcodes of nine cohabitated Taiwanese fireflies.

Background: Over 50 Taiwanese firefly species have been discovered, but scientists lack information regarding most of their genetics, bioluminescent features, and cohabitating phenomena. In this study, we focus on morphological species identification and phylogeny reconstructed by barcoding, as well as luminescent characteristics of cohabited Taiwanese firefly species to determine the key factors that influenced how distinct bioluminescent species evolved to coexist and proliferate within the same habitat.

Methods: In this study, 366 specimens from nine species were collected in northern Taiwan from April to August, 2016-2019. First, the species and sex of the specimens were morphologically and genetically identified. Then, their luminescent spectra and intensities were recorded using a spectrometer and a power meter, respectively. The habitat temperature, relative humidity, and environmental light intensity were also measured. The cytochrome oxidase I (COI) gene sequence was used as a DNA barcode to reveal the phylogenetic relationships of cohabitated species.

Results: Nine species-eight adult species (, , , , and ) and one larval -were morphologically identified. The nine species could be found in April-August. Six of the eight adult species shared an overlap occurrence period in May. Luminescent spectra analysis revealed that the of studied species ranged from 552-572 nm (yellow-green to orange-yellow). The average luminescent intensity range of these species was about 1.2-14 lux (182.1-2,048 nW/cm) for males and 0.8-5.8 lux (122.8-850 nW/cm) for females, and the maximum luminescent intensity of males was 1.01-7.26-fold higher than that of females. Compared with previous studies, this study demonstrates that different , species-specific flash patterns, microhabitat choices, nocturnal activity time, and/or an isolated mating season are key factors that may lead to the species-specific courtship of cohabitated fireflies. Moreover, we estimated that the fireflies start flashing or flying when the environmental light intensity decreased to 6.49-28.1 lux. Thus, based on a rough theoretical calculation, the sensing distance between male and female fireflies might be 1.8-2.7 m apart in the dark. In addition, the mitochondrial COI barcode identified species with high resolution and suggested that most of the studied species have been placed correctly with congeners in previous phylogenies. Several cryptic species were revealed by the COI barcode with 3.27%-12.3% variation. This study renews the idea that fireflies' luminescence color originated from the green color of a Lampyridae ancestor, then red-shifted to yellow-green in Luciolinae, and further changed to orange-yellow color in some derived species.