Diversity, Phylogeny and Classification of Aquatic Fireflies Jeng Ming-Luen1, Li-Wei Wu2, Ting-Wei Chen3 and R.J. Villanueva4 Abstract 4. Filipino aquatic fireflies: 2 groups in Clade 1, 1 sp in Clade 2. New forms of aquatic firefly larvae were lately discovered in the Philippines. Their Filipino grp 1: larvae slender, with soft cuticle (M03, 20, 21, 22). adult incarnations have been successfully connected via DNA sequence Filipino grp 2: larvae robust, thickly cuticled with projections (M14). comparison. A three-gene molecular phylogeny of Pacific Asian Luciolinae was Filipino species: M07 (adult), M15 (larva). 5. Synapomorphies: constructed. The resulted Bayesian tree revealed five lineages of aquatic fireflies, Clade 1: tracheal gills (larva); incomplete V6 lanterns (male). including two new Filipino groups which will be described as new genera. Clade 2: metapneustic (larva); V-shaped V6 lanterns (male). Japanese Luciola cruciata-owadai is the sister group of the genus Aquatica in NE 6. Vietnamese M05 (V genus sp Vtd) clustered with Filipino groups. and E Asia. Luciola substriata species complex may have terrestrial [Abscondita + Adults found near creek, larvae unknown. Pygoluciola] as its sister group. 7. 2 spp. of Aquatica (M19, M129) and 1 sp. of L. substriata complex (M07) newly discovered, expanding known distribution range of each group. Background 8. Distribution About 20 out of the 2300 species of Lampyridae have aquatic larvae, exclusively in Luciolinae1,2,3. Morphological phylogeny showed three aquatic lineages4,5: genus Aquatica, Luciola cruciata-owadai, and Luciola substriata complex. No aquatic firefly was known in the Philippines6 until we discovered three forms of aquatic larvae in 2012-2014. Questions and Goals of the Study Luciola cruciata-owadai genus Aquatica Two Filipino groups Luciola substriata complex Fig. 4. Distribution of five lineages of aquatic fireflies. Filipino group 2 is also found in Taiwan. Questions: 1. What are the adult incarnation of these larvae? Discussion 1. Evolution of aquatic fireflies with at least two origins: 2. Which groups do they belong to? Fig. 1. Three forms of aquatic Clade 1: likely having a single origin of aquatic larvae. 3. How are they related to the other aquatic lineages? larvae found sympatrically in the Philippines Clade 2: may have evolved from ancestor with terrestrial larvae. Goals: 2. Monophyly of [Luciola cruciata-owdai + Aquatica] is incongruent 1. Using DNA sequence comparison to make larva-adult connection. Fig. 2. A phylogeny of Pacific Asian Luciolinae based on a combined dataset of DNA sequence of 2532 bps from three genes (mt 5 with morphological phylogeny . Better to be treated as separate CO1, 16S and nuclear EF1α) of 88 specimens. The Bayesian tree was inferred using MrBayes 3.2 under the GTR+I+G model. 2. To build a phylogeny of Pacific Asian Luciolinae to reveal positions The tree was rooted on Elateridae, and showed two distant, well supported aquatic clades (light blue and green nodes). Five genera. Genus Aquatica may have higher diversity and greater aquatic lineages were specified and highlighted in different colors. Values at nodes represent posterior probability. of the Filipino groups and their relationship with other lineages. range in SE Asia. 3. A phylogenetic classification of aquatic fireflies. Clade 1 Clade 2 3. The two Filipino groups will be described as new genera. Material and Methods 4. M05 is crucial for understanding the origin of Filipino groups and 1) DNA extracted from thoracic muscle of beetles preserved in 95% ethanol alcohol. requires more investigation. 2) 135 specimens of 5 lampyrid-related families used, including larvae in question 5. Luciola substriata complex may have [Abscondita + Pygoluciola] as and possible adult incarnations collected simultaneously or from nearby locations. its sister group, consistent with previous phylogenetics5. 3) There genes: mt CO1, 16S and nuclear EF1α amplified by PCR. Sequence of CO1 and 6. Philippines has great firefly diversity and unique lucioline lineages. EF1α aligned by codon position, 16S rDNA by default settings of MUSCLE in MEGA 5. Total alignment length: 2532 bps (CO1, 1244; 16S, 541; EF1α, 747). 7. The three-gene tree gives sufficient resolution to lucioline phylogeny. 4) 88 taxa (including 71 luciolines with sequence of all 3 genes + 2 spp. from GenBank ? Taxon sampling will be expanded. with CO1+16S) selected to build trees. A elaterid species served as root. 5) Bayesian tree inferred using MrBayes 3.27 with GTR+I+G model, running for 5 Acknowledgement We are grateful to H Cahilog, J Ronel Gil, HT Pham, NY Tsai, YT Wang, and TR Chen for various assistance million generations. in the field trips to the Philippines and Vietnam. Results Literature Cited 1. Bertrand HPI. 1972. Coléoptères aquatiques: Larves et Nymphes. JJ Symoens. 1. Monophyly of Luciolinae supported, four major clades recognized. 2. Jeng ML et al. 2003. Lampyridae. [Jäch MA, Ji L (eds)] Water Beetles of China 3. Wien. (review of aquatic fireflies) 2. Aquatic fireflies clustered into well-supported Clade 1 and Clade 2. 3. Fu X et al. 2010. Zootaxa 2530: 1-18. (genus Aquatica) 3. Larva-adult connections in Filipino samples: M17-M122, M14-M18 4. Fu X et al. 2012. Zootaxa 3405: 1-34. (terrestrial and aquatic larvae of Luciolinae) 5. Ballantyne LA, Lambkin CL. 2013. Zootaxa 3653: 1-48. (morphological phylogeny of Luciolinae) in Clade 1, M07-M15 in Clade 2, M119-M123 in a terrestrial lineage. Fig. 3. Illustrative larva and male adult of each aquatic firefly lineage (not in the same scale). M17-M122 and M18-M14 in Clade 1 and 6. McDermott FA. 1966. Coleopterum Catalogus, Supplementa. [Steel W (ed)] pars 9, Lampyridae. s-Gravenhage. M07-M15 in Clade 2 are larvae and their adult incarnations. Values at nodes represent posterior probability. 7. Ronquist F et al. 2012. Systematic Biology 61: 1-4. (MrBayes 3.2) 1: National Museum of Natural Science, Taichung City, Taiwan, ROC ([email protected]) 4: Davao Medical School Foundation, Davao City, Mindanao, Republic of the Philippines The study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. 2: Experimental Forest, National Taiwan University, Zhushan Township, Taiwan, ROC NSC100-2313-B178-002-MY2 & 102-2313-B178-003-MY3. 3: Dept. of Life Sciences, National Taiwan Normal University, Taipei City, Taiwan, ROC .