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Diversity, Phylogeny and Classification of Aquatic Fireflies

Jeng Ming-Luen1, Li-Wei Wu2, Ting-Wei Chen3 and R.J. Villanueva4

1: National Museum of Natural Science, Taichung City, Taiwan, ROC (mantis001@gmail.com)

2: Experimental Forest, National Taiwan University, Zhushan Township, Taiwan, ROC

3: Dept. of Life Sciences, National Taiwan Normal University, Taipei City, Taiwan, ROC

Abstract

New forms of aquatic firefly larvae were lately discovered in the Philippines. Their

adult incarnations have been successfully connected via DNA sequence

comparison. A three-gene molecular phylogeny of Pacific Asian Luciolinae was

constructed. The resulted Bayesian tree revealed five lineages of aquatic fireflies,

including two new Filipino groups which will be described as new genera.

Japanese Luciola cruciata-owadai is the sister group of the genus Aquatica in NE

and E Asia. Luciola substriata species complex may have terrestrial [Abscondita +

Pygoluciola] as its sister group.

Acknowledgement

We are grateful to H Cahilog, J Ronel Gil, HT Pham, NY Tsai, YT Wang, and TR Chen for various assistance

in the field trips to the Philippines and Vietnam.

Literature Cited

1. Bertrand HPI. 1972. Coléoptères aquatiques: Larves et Nymphes. JJ Symoens.

2. Jeng ML et al. 2003. Lampyridae. [Jäch MA, Ji L (eds)] Water Beetles of China 3. Wien. (review

of aquatic fireflies)

3. Fu X et al. 2010. Zootaxa 2530: 1-18. (genus Aquatica)

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)

6. McDermott FA. 1966. Coleopterum Catalogus, Supplementa. [Steel W (ed)] pars 9, Lampyridae. s-Gravenhage.

7. Ronquist F et al. 2012. Systematic Biology 61: 1-4. (MrBayes 3.2)

6. Vietnamese M05 (V genus sp Vtd) clustered with Filipino groups.

Adults found near creek, larvae unknown.

7. 2 spp. of Aquatica (M19, M129) and 1 sp. of L. substriata complex (M07)

newly discovered, expanding known distribution range of each group.

8. Distribution

4. Filipino aquatic fireflies: 2 groups in Clade 1, 1 sp in Clade 2.

Filipino grp 1: larvae slender, with soft cuticle (M03, 20, 21, 22).

Filipino grp 2: larvae robust, thickly cuticled with projections (M14).

Filipino species: M07 (adult), M15 (larva).

5. Synapomorphies:

Clade 1: tracheal gills (larva); incomplete V6 lanterns (male).

Clade 2: metapneustic (larva); V-shaped V6 lanterns (male).

Questions and Goals of the Study

Background

Material and Methods

Discussion

1. Evolution of aquatic fireflies with at least two origins:

Clade 1: likely having a single origin of aquatic larvae.

Clade 2: may have evolved from ancestor with terrestrial larvae.

2. Monophyly of [Luciola cruciata-owdai + Aquatica] is incongruent

with morphological phylogeny5. Better to be treated as separate

genera. Genus Aquatica may have higher diversity and greater

range in SE Asia.

3. The two Filipino groups will be described as new genera.

4. M05 is crucial for understanding the origin of Filipino groups and

requires more investigation.

5. Luciola substriata complex may have [Abscondita + Pygoluciola] as

its sister group, consistent with previous phylogenetics5.

6. Philippines has great firefly diversity and unique lucioline lineages.

7. The three-gene tree gives sufficient resolution to lucioline phylogeny.

Taxon sampling will be expanded.

Fig. 2. A phylogeny of Pacific Asian Luciolinae based on a combined dataset of DNA sequence of 2532 bps from three genes (mt

CO1, 16S and nuclear EF1α) of 88 specimens. The Bayesian tree was inferred using MrBayes 3.2 under the GTR+I+G model.

The tree was rooted on Elateridae, and showed two distant, well supported aquatic clades (light blue and green nodes). Five

aquatic lineages were specified and highlighted in different colors. Values at nodes represent posterior probability.

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

M07-M15 in Clade 2 are larvae and their adult incarnations. Values at nodes represent posterior probability.

Fig. 4. Distribution of five lineages of aquatic fireflies. Filipino group 2 is also found in Taiwan.

Luciola cruciata-owadai genus Aquatica Two Filipino groups Luciola substriata complex

Clade 1 Clade 2

Questions:

1. What are the adult incarnation of these larvae?

2. Which groups do they belong to?

3. How are they related to the other aquatic lineages?

Goals:

1. Using DNA sequence comparison to make larva-adult connection.

2. To build a phylogeny of Pacific Asian Luciolinae to reveal positions

of the Filipino groups and their relationship with other lineages.

3. A phylogenetic classification of aquatic fireflies.

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.

1) DNA extracted from thoracic muscle of beetles preserved in 95% ethanol alcohol.

2) 135 specimens of 5 lampyrid-related families used, including larvae in question

and possible adult incarnations collected simultaneously or from nearby locations.

3) There genes: mt CO1, 16S and nuclear EF1α amplified by PCR. Sequence of CO1 and

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).

4) 88 taxa (including 71 luciolines with sequence of all 3 genes + 2 spp. from GenBank

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

million generations.

Results

1. Monophyly of Luciolinae supported, four major clades recognized.

2. Aquatic fireflies clustered into well-supported Clade 1 and Clade 2.

3. Larva-adult connections in Filipino samples: M17-M122, M14-M18

in Clade 1, M07-M15 in Clade 2, M119-M123 in a terrestrial lineage.

The study was supported by the Ministry of Science and Technology, Taiwan, R.O.C.

NSC100-2313-B178-002-MY2 & 102-2313-B178-003-MY3.

Fig. 1. Three forms of aquatic

larvae found sympatrically

in the Philippines

4: Davao Medical School Foundation, Davao City, Mindanao, Republic of the Philippines