Sinopyrophorinae, a new subfamily of Elateridae (Coleoptera, Elateroidea) with the first record of a luminous click beetle in Asia and evidence for multiple origins of bioluminescence in Elateridae

Abstract The new subfamily Sinopyrophorinae within Elateridae is proposed to accommodate a bioluminescent species, Sinopyrophorusschimmeli Bi & Li, gen. et sp. nov., recently discovered in Yunnan, China. This lineage is morphologically distinguished from other click-beetle subfamilies by the strongly protruding frontoclypeal region, which is longitudinally carinate medially, the pretarsal claws without basal setae, the hind wing venation with a well-defined wedge cell, the abdomen with seven (male) or six (female) ventrites, the large luminous organ on the abdominal sternite II, and the male genitalia with median lobe much shorter than parameres, and parameres arcuate, with the inner margin near its apical third dentate. Molecular phylogeny based on the combined 14 mitochondrial and two nuclear genes supports the placement of this taxon far from other luminescent click-beetle groups, which provides additional evidence for the multiple origin of bioluminescence in Elateridae. Illustrations of habitus and main diagnostic features of S.schimmeli Bi & Li, gen. et sp. nov. are provided, as well as the brief description of its luminescent behavior.


Introduction
The cosmopolitan family Elateridae currently contains approximately 600 genera and almost 10,000 species (Costa et al. 2010, Kundrata andBocak 2011). Based on a synthesis of the work over the past several decades, Costa et al. (2010;and references therein) provided the main characteristics of adult and larval elaterid morphology and recognized 17 subfamilies. Thereafter, slight modifications of the above classification were proposed by Kundrata and Bocak (2011), Kundrata et al. (2016Kundrata et al. ( , 2018, Bocak et al. (2018), and Douglas et al. (2018) based on molecular analyses. Currently, 18 subfamilies and 37 tribes of Elateridae are recognized .
In 2017, during an expedition to the western Yunnan in China, in which the first author participated, a remarkable dusk-active bioluminescent click beetle with a single luminous organ on the abdomen was discovered. Since no bioluminescent representative of Elateridae has been recorded in Asia to date, morphological study and molecular phylogenetic analysis were undertaken simultaneously to clarify the identity and phylogenetic placement of the new taxon. He et al. (2019) published the mitochondrial genome of this species and provided a preliminary phylogenetic hypothesis for it based on the analysis of 13 protein-coding genes. Here, we formally describe this species in a new genus, which we propose to place into a new subfamily within Elateridae, and provide more robust phylogenetic hypothesis for this taxon.

Morphology
All specimens of the new species were collected from the vicinity of Longchuan County and Yingjiang County in western Yunnan, in subtropical evergreen broadleaf forests by searching for flashes or by setting light traps during the night. Specimens are deposited in the following collections: Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China (KIZ-CAS), The Insect Collection of Shanghai Normal University, Shanghai, China (SNUC), collection of Wen-Xuan Bi, Shanghai, China (CBWX), and the collection of Chang-Chin Chen, Tianjin, China (CCCC). Morphological terminology follows Calder (1996) and Costa et al. (2010), except for the wing venation, for which we follow Kukalova-Peck and Lawrence (1993,2004).

Phylogenetic analysis
To explore the phylogenetic position of the new Chinese luminescent click beetle, we newly generated 14 mitochondrial genes (13 protein-coding genes and 16S) and two nuclear rRNA genes (18S and 28S) for this species and merge them with the available four-gene Elateridae dataset (18S, 28S, 16S, cox1) by Kundrata et al. (2016Kundrata et al. ( , 2018 and Douglas et al. (2018) plus all publicly available Elateridae mitochondrial genomes downloaded from the GenBank (altogether 179 terminals representing 13 subfamilies). Four species of Phengodidae were used as outgroups following Kundrata et al. (2016Kundrata et al. ( , 2018) (Suppl. material 1, Table S1).
The mitogenome of the new species was obtained from GenBank (accession number MH065615) (He et al. 2019). Because we were not able to obtain high quality reference sequences of 18S and 28S from the Illumina reads previously used for assembling the mitogenome, we assembled 18S and 28S based on another batch of Illumina reads sequenced using fresh specimens collected from type locality later in June 13, 2018, and immediately frozen in liquid nitrogen and stored at -80 °C before use. Total genomic DNA (gDNA) was isolated from two male adults with Sodium Dodecyl Sulfonate method. Library (150-bp insert size) was prepared and sequenced on the Illumina HisSeq4000. Total 38 Gb clean reads were used for assembling 18S and 28S based on the following method: 1) reads were mapped to reference genes of rDNA using mrsFAST v3.3.0 with several iterations (Hach et al. 2014) (first iteration referred to genes downloaded from GenBank (NCBI Resource Coordinators 2016); later iterations referred to assembled contigs in the latest iteration); 2) mapped reads were then assembled using SPAdes v3.11 (Bankevich et al. 2012); 3) tandem contigs were filtered using mereps v2.6 because a huge number of repetitive reads greatly lowers assembly efficiency (Kolpakov et al. 2003); 4) steps one to three were automatically performed for 3-10 iterations until all genes were recovered.

Phylogenetic inference
Our phylogenetic analysis recovered similar topology ( Fig. 1) to that of , only Tetralobinae were placed in an unsupported terminal clade with Car-diophorinae + Negastriinae, and Dendrometrinae including Diplophoenicus Candèze (Morostomatinae) formed a separate clade. Similar to previously published phylogenies, we obtained low statistical support for the backbone of tree. All subfamilies were monophyletic, except for Dendrometrinae (due to the inclusion of Diplophoenicus) and Lissominae (due to the inclusion of Thylacosterninae). The newly sequenced luminescent species from China was found in an unsupported clade with Oestodinae and Hemiopinae as follows: Oestodes tenuicollis (Randall) + (Hemiops sp. + new luminescent taxon), far from other bioluminescent groups like Thylacosterninae and Agrypninae: Pyrophorini.   (Fig. 4) strongly protruding, longitudinally strongly carinate medially; antennomeres II and III short, subequal in length; clicking mechanism (i.e., prosternal process fitting into mesoventral cavity) fully developed; prosternal process straight in lateral view, pretarsal claw ( Description. Male. Body elongate, ~ 4.6 times as long as wide, weakly convex in lateral view. Vestiture of fine, suberect setae. Head with frontoclypeal region strongly protruding, inflexed at apex, medially longitudinally carinate; carina setose and apparently not joined to supra-antennal carinae, basally half as wide as frons, then narrowed and subparallel-sided, with cuticle between edges flat (Fig. 4). Antennal insertions concealed from above. Labrum free, transverse, anterior margin convex in dorsal view. Maxilla ( Fig. 7) with galea scooplike, anterior part covered with setae, denser on inner edge; lacinia elongate, densely pilose; palp with apical palpomere slightly expanded anteriorly. Labium ( Fig. 6) with prementum elongate, trilobed anteriorly. Mandibles ( Fig. 5) bidentate, apical tooth narrowly acute, subapical mesal tooth small. Antenna with 11 antennomeres, filiform; antennomeres II and III subequal in length (together 1/3 as long as antennomere IV), globular; remaining antennomeres at least five times longer than wide.
Etymology. The generic name is derived from the Latin prefix sino-, which means Chinese, and Pyrophorus, a bioluminescent click-beetle genus from Central and South America. Gender masculine.

Sinopyrophorus schimmeli
Head transverse, weakly convex, 0.75 times as long as wide, same width as pronotal anterior edge; sparsely and finely punctate. Frons rectangular, 1.4 times longer than width, 0.3 times as wide as head width across the eyes. Frontoclypeal region concave at sides beneath; with one small median depression. Eyes protuberant, median width of each eye ~ 0.7 times interocular distance in dorsal view. Mouthparts directed anteroventrally. Labrum (Fig. 5) 2.2 times wider than long. Antenna long, reaching second half of elytral length, ~ 0.7 times as long as body length; scape 2.3 times as long as  (14); not to scale (15, 16).
combined length of antennomeres II and III, and of approximately same length as antennomere 4, slightly curved; antennomeres 4-11 successively weakly lengthened, with fine and very long, distinct setae; setae almost as long as apical antennomere.
Prothorax (Fig. 8) slightly convex in lateral view, tallest anteriorly; ~ 1.2 times as long as wide in dorsal view, weakly narrowed anteriorly, slightly narrower than elytral humeral width; sides slightly sinuate; pronotal lateral carina complete; anterior angles short, subacute; hind angles narrowly acute, moderately produced posterolaterally, each with short carina; posterior edge straight from dorsal view, medially elevated; pronotal disk sparsely and finely punctate, with eight shallow depressions: single median and posteromedian, and three pairs at sides. Prosternum and hypomeron more coarsely punctate than pronotum. Elytra ~ 3.0 times as long as combined width, ~ 2.9 times as long as pronotum, parallel-sided; each elytron with three low and evenly spaced swellings, longitudinally arranged at basal half near suture; with nine punctate striae; apices conjointly rounded; epipleura short, abruptly narrowed near metacoxa. Legs long; tarsomeres I-III elongate, tarsomere I ~ 1.4 times as long as tarsomere II, tarsomere II as long as combined lengths of tarsomeres III and IV or as long as tarsomere V; tarsomeres III and IV ventrally lobate (Fig. 12).
Abdomen with each ventrite with paired depressions posterolaterally. Aedeagus (Fig. 20) robust, ~ 1.6 times as long as wide. Phallobase slightly longer than wide, narrowed dorsally, emarginate posteriorly. Median lobe approximately half as long as aedeagus, with basal struts 0.25 times total length of median lobe. Parameres ~ 1.5 times longer than median lobe, shorter than phallobase, with dorsal surface ~ 1.6 times longer than ventral one, partially fused basally in dorsal view; each paramere with dorsal surface angulate on inner margins at basal 2/5, strongly narrowed and curved mesad with dentate inner margin near apical 1/3 and rounded apex; bearing 20-30 fine setae near apex, longer on ventral surface. Female (Fig. 3). Body length 12.1-14.5 mm. Similar to male in its general appearance but with integument paler. Eyes smaller, median width of each eye ~ 0.4 times interocular distance in dorsal view. Antenna shorter, only reaching elytral humeri, ~ 0.3 times as long as body length. Pronotum relatively shorter with lateral margins more rounded, narrowed anteriorly, with rounded anterior angles. Elytra relatively longer, ~ 3.3 times as long as wide, ~ 3.0 times as long as pronotum. Legs relatively shorter. Abdominal luminescent organ smaller, occupying approximately one third of basal abdominal sternite width.
Immature stages. Unknown. Etymology. This species is named in honor of late Mr. Rainer Schimmel, a specialist in Elateridae, who kindly provided valuable comments at the beginning of this study.
Biological notes. All specimens of the new species were collected during the late May to June (i.e., the middle of the rainy season) from the mountain area in vicinity of Longchuan County or Yingjiang County, western Yunnan in subtropical evergreen broadleaf forests by searching for flashes or by light trapping during night. The adults of both sexes emitted a continuous yellowish green light from the abdominal luminous organs while in flight, or during a short time when preparing for flight or afterwards. During this process the luminous organ is exposed ventrally by raising and extending the abdomen from the metaventrite (Suppl. material 2, Movie S1). The reaction of the adults when disturbed during a flight is to retract their abdomen, hide their luminous organ, and show a death-feigning behavior (thanatosis). Thanatoid adults remained inactive for a long time, which was obviously different from the observation in Pyrophorini because the latter begin to emit light and are very active after being disturbed (Costa 1975). At least three lampyrid species (Luciola sp., Diaphanes sp., and Pyrocoelia sp.) occurred sympatrically with the new elaterid species and can be found simultaneously during night but can be easily distinguished by different flash patterns. Although some specimens of S. schimmeli were previously collected using light traps in 2016, this species was found to be luminescent only in 2017 when its bioluminescent behavior was observed in the field. This was caused by the abdominal luminescent organ being hidden when the beetle is not active.
Nomenclatural notes. He et al. (2019) reported the mitochondrial genome of S. schimmeli gen. et sp. nov. and used the genus and species name of the here described taxon in their study. The paper of He et al. (2019) (Fig. 4), which is medially distinctly longitudinally carinate, antennomeres II and III subequal in length and together less than half as long as antennomeres IV-XI, pronotal hind angles (Fig. 8) acute, produced posterolaterally, prosternal process (Fig. 8c) straight in lateral view, tarsomeres III and IV ( Fig. 12) with ventral lobes, abdomen with seven (male) or six (female) ventrites, with a luminous organ (Fig. 16) on sternite II, and aedeagus (Fig. 20) with a median lobe shorter than phallobase, and arcuate parameres.

Discussion
Phylogenetic placement and morphology of Sinopyrophorus schimmeli Bi & Li, gen. et sp. nov. He et al. (2019) provided the first preliminary phylogenetic hypothesis for S. schimmeli based on the analysis of 13 protein-coding mitochondrial genes. They found this species sister to a clade containing Dendrometrinae and Elaterinae; however, their sampling of the click-beetle lineages was limited and the position of S. schimmeli was not statistically supported.
Here, we used the most comprehensive dataset of Elateridae to date to elucidate the phylogenetic position of the first known Asian luminescent species. Our analysis (Fig. 1) placed S. schimmeli in a clade with Hemiops and Oestodes, which are both the type genera of the subfamilies Hemiopinae and Oestodinae, respectively, and which were not included in the study by He et al. (2019). However, the relationships among these lineages are not statistically supported and the long branches (see Kundrata et al. 2016) indicate a long-term independent evolution of these groups, which is supported by their distinct morphology. Sinopyrophorinae differ from both Hemiopinae and Oestodinae by having the strongly protruding frontoclypeal region with median longitudinal carina, the antennomeres II and III subequal in length and much shorter than remaining antennomeres (present only in the hemiopine genus Plectrosternus Lacordaire), the posterior margin of pronotum simple, without sublateral incisions or carinae, the prosternal process straight in lateral view, the posterior end of scutellar shield emarginate, the wide mesoventrite with an elongate mesoventral cavity which is only gradually narrowed posteriorly, the bisetose empodium (present in Oestodes, multisetose in Hemiopinae), the abdomen with six or seven ventrites and with a luminous organ, the male genitalia with a median lobe shorter than parameres, and each paramere strongly curved mesad with a dentate inner margin, the female genitalia with a more sclerotized ovipositor, longer paraprocts and styli attached subapically to gonocoxites (apically in Oestodes and Hemiopinae; A. Prosvirov, pers. comm.). These morphological differences in combination with the molecular phylogeny support the subfamilial rank of Sinopyrophorinae, especially when there are no obvious synapomorphies which would define the clade of Sinopyrophorinae + Oestodinae + Hemiopinae, or placement of Sinopyrophorus in any other subfamily. Since the backbone of the Elateridae phylogenetic tree is not or only weakly supported (Kundrata et al. 2016 this study), future genomic or transcriptomic data might help to resolve the relationships among the deep splits including Sinopyrophorinae.
Our molecular analysis in combination with the morphological investigation confirmed that S. schimmeli does not belong to any of described subfamilies containing bioluminescent species. The clade of Sinopyrophorinae, Oestodinae and Hemiopinae formed one of the basal radiations in Elateridae, far from the Agrypninae: Pyrophorini, which contains the majority of luminescent click beetles (Fig. 1). The position of S. schimmeli far from Pyrophorini is additionally supported by the morphological features such as the pretarsal claws without setae at base (present in Pyrophorini), hind wings with a well-defined wedge cell (absent in Pyrophorini), abdomen with six or seven ventrites (five in Pyrophorini) and the presence of styli on the gonocoxites of ovipositor (styli absent in Pyrophorini). Thylacosterninae, which include the luminescent Balgus schnusei, are regularly recovered inside Lissominae in recent DNA-based analyses (Kundrata et al. 2014(Kundrata et al. , 2016Bocak et al. 2018;this study). They differ from Sinopyrophorinae in having the flabellate antennae, deep antennal cavities lying beneath the hypomera, and membranous tarsal lobes (Costa 1984a, Costa et al. 2010. The members of the subfamily Campyloxeninae, which have not been included in any DNA-based study to date, differ from Sinopyrophorinae by much wider frontoclypeal region with a complete frontal carina (frontoclypeal region strongly protruding, relatively narrow and high, longitudinally carinate medially in S. schimmeli), narrowed apical portions of lateral lobes of mesoventrite (wide in S. schimmeli) ovalshaped mesoventral cavity (elongate, gradually narrowed posteriorly in S. schimmeli), abdomen with five ventrites and without a luminous organ (six or seven ventrites, and a large abdominal luminous organ in S. schimmeli), distinctly longer paraprocts, differently shaped aedeagus with the median lobe longer than parameres, and the paramere elongate, with a subapical hook, and apex oriented posteriorly (Costa 1975, Arias-Bohart 2015. Southern China including Yunnan is a world biodiversity hotspot (Myers et al. 2000) and hosts many endemic beetle species including Elateridae. Intensive entomological research in southern China and neighboring regions is necessary to find out if the newly established Sinopyrophorinae contains more species. Additionally, the discovery of a larva of S. schimmeli may help to better understand the morphological distinctiveness and systematic position of this interesting beetle lineage.

Evolution of bioluminescence in Elateridae
Bioluminescence has evolved independently many times in various organisms (Day et al. 2004). Within Coleoptera, bioluminescence is best known in Lampyridae (fireflies), but also present in Phengodidae, Rhagophthalmidae, and some Elateridae, all within the superfamily Elateroidea (e.g., Oba 2009). Additionally, it was reported also for the larvae of two species belonging to Staphylinidae (Costa et al. 1986, Rosa 2010), which are not closely related to Elateroidea (e.g., Zhang et al. 2018). Within the Elateroidea, bioluminescence independently evolved several times (Bocakova et al. 2007, Sagegami-Oba et al. 2007, Amaral et al. 2014, Fallon et al. 2018) but despite the recent progress in elucidating the phylogenetic relationships within this superfamily (Kundrata et al. 2014), the relationships between the luminescent lineages remain unresolved.
Within Elateridae, nearly all luminescent taxa belong to the agrypnine tribe Pyrophorini, and each of the remaining three smaller groups contain only a single bioluminescent species. All other bioluminescent subfamilies, except Sinopyrophorinae, also include non-luminescent species. Campyloxeninae were placed into Pyrophorini by Stibick (1979) but this was not accepted by later authors (see e.g., Costa et al. 2010). In fact, the members of Campyloxeninae differ diagnostically from Pyrophorini in e.g., the absent setae on tarsal claws, presence of the wedge cell in the hind wing venation and the ovispositor with styli (Costa 1975, Arias-Bohart 2015. Current molecular phylogeny suggests at least three independent origins of bioluminescence within Elateridae, i.e., in Agrypninae: Pyrophorini, Sinopyrophorinae and Thylacosterninae. The presence of a single luminous organ located on abdomen without any prothoracic bioluminescent organs, which is shared by S. schimmeli and the pyrophorine genus Hifo, is therefore an apparent homoplasy. Sequences of the fresh DNA-grade material of Campyloxeninae would help us to better understand the position of this group and if it represents a fourth origin of bioluminescence in Elateridae. All available phylogenetic analyses indicate that the ancestral state of Elateridae was nonluminescent and the luminescence was later obtained in several independent lineages (Bocakova et al. 2007, Sagegami-Oba et al. 2007, Douglas 2011, Kundrata and Bocak 2011, Kundrata et al. 2014.
In Lampyridae, bioluminescence was first gained by larvae as an aposematic warning display, and subsequently gained by adults and co-opted as a sexual signal; the overall trend of courtship is the use of pheromones in ancestral species, then pheromones used in conjunction with photic signals, then the sole use of photic signal Wenzel 2001, 2003;Ohba 2004;Stanger-Hall et al. 2018). Males of S. schimmeli have a large luminous organ on abdomen plus very long antennae (longer than in females, hypothesized to be for pheromone detection) and large eyes (larger than in females, and hypothesized to be for bioluminescence detection), which suggest the use of multiple communication channels for mate attraction (Stanger-Hall et al. 2018, and references therein). It is hypothesized that in elaterids, bioluminescence of the abdominal lantern is an optical signal for the intraspecific sexual communication, while the signals from the prothoracic lanterns (if present) serve to warn predators and may also provide illumination in flight (Lall et al. 2000(Lall et al. , 2010. Additionally, antennae are usually relatively longer in species which rely greatly on pheromones as sexual signals . Therefore, the external morphology of S. schimmeli suggests the combined sexual communication of both light signals and pheromones. The relatively larger sexual signal sensors of S. schimmeli males in comparison to conspecific females are in agreement with situation in many other insect groups, in which males are the more actively searching sex, with more sensitive sensors (Steinbrecht 1987. The discovery of S. schimmeli as the first record of a bioluminescent click beetle in Asia shed new light on the geographic distribution and evolution of luminescent click beetles. As a representative of a unique lineage, only distantly related to all other luminescent click beetles, S. schimmeli may serve as a new model taxon in the research of bioluminescence within Coleoptera. A project of de novo genome sequencing of S. schimmeli has already started and should help answer the questions related to the genome characteristics of this taxon (e.g., genome size, heterozygosity etc.), its genomic difference from those of luminescent pyrophorine click beetles, and the genomic basis of the origin of its bioluminescence.