﻿Systematic review of the firefly genus Emeia Fu, Ballantyne & Lambkin, 2012 (Coleoptera, Lampyridae) from China

﻿Abstract The Luciolinae genus Emeia Fu, Ballantyne & Lambkin, 2012 is reviewed. Phylogenetic relationships based on cox1 DNA barcoding sequences from 42 fireflies and 2 outgroup species are reconstructed. The dataset included three main Lampyridae subfamilies: Luciolinae, Photurinae and Lampyrinae, and Emeia was recovered within Luciolinae. A new species, Emeiapulchra Zhu & Zhen sp. nov., is described from the wetland of Lishui, Zhejiang, China. Emeiapulchra is sister species to E.pseudosauteri from Sichuan, which is supported by morphological characters and a phylogeny based on DNA barcoding sequences. The two species are separated geographically as shown on the distribution map. A key to species of Emeia using males is provided.


Introduction
Emeia Fu, Ballantyne & Lambkin, 2012 (Luciolinae) was established as a monotypic genus (Fu et al. 2012) with Emeia pseudosauteri (Geisthardt 2004) as the type species. Emeia pseudosauteri was first described from Mount Emei, Sichuan, China by Michael Geisthardt in the genus Curtos Motschulsky, 1845 (Geisthardt 2004), and then transferred to Emeia based on morphological evidence (Fu et al. 2012). The genus Emeia Fu, Ballantyne & Lambkin had only one species (E. pseudosauteri) recorded in China before this study. The primary phenotypic feature of Emeia was the trilobite-like larva. The thoracic and abdominal terga of Emeia larvae are distinct. The lateral thoracic tergal margins are broad, similar to those of a trilobite "cephalon", while the abdomen is narrow and curls ventrad in the posterior part. At present, definition of the genus Emeia is based on the morphology of E. pseudosauteri, which makes it insufficient in light of the discovery of a second species.
In this study, based on specimens collected from Lishui, Zhejiang, China, we describe adults of Emeia pulchra Zhu & Zhen sp. nov. based on morphological and molecular data. We compare it with the previously described E. pseudosauteri. We also provide new information on the adult male hind wing venation of the type species E.pseudosauteri. With our detailed examination of both species, we present a systematic review of the genus Emeia and a key to species.

EL
elytral length; EW elytral width; PL pronotal length; BL body length (the sum of PL, EL and the length of the exposed portions of the head from the pronotum); BW body width (the greatest distance across the elytra, BW=2EW); T7, 8 abdominal tergite numbers; V6, 7 abdominal ventrite numbers.
Adult males of Emeia pulchra Zhu & Zhen sp. nov. were collected from Jiulong National Wetland Park, Lishui, Zhejiang Province in April, 2020. The holotype and paratypes of the new species are stored at School of Life Sciences, Westlake University, Hangzhou, Zhejiang. Samples of both male and female Emeia pseudosauteri were collected from Mt. Tian Tai, Sichuan Province in April, 2021.
Habitus images were taken using a Nikon D7500 camera. Images of genitalia were taken using a Nikon D7500 camera mounted on an SZ650 microscope (Chongqing Optec Instrument Co., Ltd.) under reflection or transmission light. Images were edited using Adobe Photoshop CS6. Morphological terminology and measurements follow those decribed in Douglas (2017). The body length (BL) is the sum of the pronotal length (PL) and elytral length (EL) plus the length of the exposed portions of the head from the pronotum. The abbreviations EW and BW (BW=2EW) denote elytral width and body width, respectively (Fig. 1A). The length and width of the aedeagus and aedeagal sheath were measured under the microscope using the OLYMPUS cellSens Dimension software (v 3.1.1) (Fig. 1B). The dissected aedeagus and aedeagal sheath structures are preserved in pure glycerol in small vials with the corresponding specimens.
We sequenced the cox1 gene barcode fragment from Emeia pseudosauteri and E. pulchra. Specifically, total DNA of the two Emeia species was isolated using the DNeasy Blood and Tissue Kit (Zhejiang Easy-Do Biotech CO., Ltd.), according to the manufacturer's protocol. The primers LCO 1490 and HCO 2198 (Folmer et al. 1994) were used to amplify the barcode fragments of the mitochondrial gene cytochrome c oxidase subunit I (cox1). We performed the PCR reaction in a 25 μL reaction mix containing 1× PCR buffer, 1 μL of each primer in a final concentration of 1 μM, 1 μL of template, 0.2 mM of each dNTP and 0.5 units of Taq polymerase (Takara Biomedical Technology CO., Ltd). The PCR thermal regime consisted of an initial denaturation at 95 °C for 3 min; 30 cycles of 30 s at 94 °C, 30 s at 48 °C and 30 s at 72 °C, followed by a 5 min final extension at 72 °C. PCR products were checked by electrophoresis in 1% agarose gel at 170 V for 20 min, and visualized under a UV transilluminator with nucleic acid dye (Cofitt Life Science, Hong Kong). The PCR products were cleaned using Easy Gel Extraction & Clean-up kit (Zhejiang Easy-Do Biotech CO., Ltd.). The cleaned products were sequenced with an ABI 3730XL sequencer (Applied Biosystems, California, USA) by Zhejiang Sunya Biotechnology Co., Ltd.

Phylogenetic analysis
The cox1 barcode sequences of E. pseudosauteri and E. pulchra share a 94% sequence identity over the 658 bp segment. The phylogeny constructed from cox1 of fireflies showed three main clades corresponding to Lampyrinae, Photurinae and Luciolinae (Fig. 2). Emeia pseudosauteri was recovered as sister to E. pulchra Zhu & Zhen sp. nov. within the subfamily Luciolinae, with strong support (100%).  (Fu et al. 2010). Emeia is distinguished from Curtos Motschulsky, 1845, as the species in Curtos have a distinctive longitudinal elytral humeral carina and parameres unequal in length (Fu et al. 2012). Emeia is closely related to Pygoluciola based on our cox1 phylogeny (Fig. 2), but the two genera can be distinguished by the shape of the pronotum, with median anterior margin gently rounded or slightly medianly emarginate in Pygoluciola (Ballantyne and Lambkin 2006) versus lateral margins of pronotum almost parallel in Emeia.
Description. Male: BL 10.0-10.4 mm; BW 3.5-3.7 mm (three individuals). Head. Antennae filiform, black, almost 2/3 as long as body length; antennomere 1 cone-shaped; 2 short and cylindrical; 3 to 10 compressed, not bifurcate; 11 th antennomere almost 1.5 times longer than 10 th , slightly dilated from base to apex. Concave between eyes dorsally in cross section, both eyes occupying about 2/3 width of whole head in ventral view. Eyes spherical, so that head cannot fully contract into pronotum. Mouthparts fully developed, clypeolabral suture flexible, outer edges of labrum reaching inner edges of closed mandibles.
Thorax. Scutellum black and slightly emarginate distally. Elytra elongated, dark brown to black, apices not deflexed in dorsal view, sides slightly convex. Hind wing well developed, r3 half the length of r4 (Fig. 4). Legs long and straight, without swelling on any part, dark brown to black, with dense white hairs.
Etymology. The specific name pulchra refers to the bright pronotum coloration.  There are many terrestrial snails and slugs in this habitat, which may be potential food for Emeia pulchra larvae. Combining descriptions from local people and our field observations, adult fireflies are usually observed mid-March. The protection of fireflies has been supported by the Lishui government and Jiulong National Wetland   Park management departments, and this area has been protected as Jiulong National Wetland Park (Fig. 7). Fan (2019) reported that the population size of E. pulchra has increased from 2014 to 2019 with the protection efforts.
Behavioral remarks. There are two obvious luminous bands at the terminal end of the adult male abdomen. The two bands both emit intermittent bright light during courtship. The male courtship behavior usually starts at 19:00 (approximately 1h after sunset), and peaks at about 20:30. Adult males rest on higher herbs and emit yellow and green flashing light. Males are reluctant flyers; the distance of each flight ranges from 0.5 to 5 m.

Key to species (adult males)
1 The elytral apices have a narrow orange stripe in both fresh and dried specimens; upper vein of MP 3+4 forked and reaching edge of hind wing (Fig. 10); phallus and parameres broad, 2 times as long as wide (Fig.12A)  The elytral apices are black in fresh and preserved specimens (Fig. 3A); upper vein of MP 3+4 reaching margin of hind wings, but without forks (Fig. 4); phallus and parameres slender, 3 times as long as wide (Fig. 6A)

Discussion
In this study, we summarized the diagnostic features of the genus Emeia. Emeia pulchra Zhu & Zhen, sp. nov. is morphologically similar to E. pseudosauteri Fu, Ballantyne & Lambkin, 2012 from Sichuan Province. However, we found differences in the antennal length and body size between the two species. The body size of a species may vary due to nutrition and environmental factors, so we did not include size in the diagnosis to the new species. The antenna of male E. pulchra (Fig. 3) is narrower than that of E. pseudosauteri (Fig. 8) in lateral view. Females of E. pseudosauteri have body length about 2/3 of that of the male and have normal elytra (Fig. 9), but their hind wings are small and shrunken, about 1/4 length of the male hind wings (Figs 10, 11). In the male, we found that the hind wing of E. pseudosauteri was relatively narrower and longer than that of E. pulchra. The elytral apice has a narrow orange stripe in both fresh and dried specimens of E. pseudosauteri, whereas it is black in E. pulchra (in three E. pulchra and six E. pseudosauteri examined). The observed body size of E. pseudosauteri (BL 6.6-7.2 mm; BW 2.7-2.9 mm; six individuals measured) was smaller than for E. pulchra (BL 10.0-10.4 mm; BW 3.5-3.7 mm; three individuals measured). In the male genitalia, the aedeagus of E. pulchra (Fig. 6A) is narrower than that of E. pseudosauteri (Fig. 12A), and the parameres are less curved (Figs 6B, 12B). In addition, the new species is only known from S. Zhejiang, whereas E. pseudosauteri is only found 1600 km westward, in the Sichuan Province (Fig. 13). The "barcode region" of cox1 is often used as an aid to new species' identification and distinction from close relatives in the Barcode of Life Data system (Ratnasingham and Hebert 2007;Lin et al. 2009). Currently, this method has been widely and successfully used to identify closely-related species and conspecific individuals. Our cox1 gene tree recovered the major subdivisions within Lampyridae, including Lampyrinae, Photurinae and Luciolinae. This tree is consistent with recent studies using 436 loci (Martin et al. 2019) or 15 mitochondrial genes (Chen et al. 2019), and supports that the placement of Emeia in Luciolinae (Fig. 2). Both the cox1 tree and morphology support E. pulchra as the closest sister species of E. pseudosauteri.