Hidden diversity in the Brazilian Atlantic rainforest: the discovery of Jurasaidae, a new beetle family (Coleoptera, Elateroidea) with neotenic females

Beetles are the most species-rich animal radiation and are among the historically most intensively studied insect groups. Consequently, the vast majority of their higher-level taxa had already been described about a century ago. In the 21st century, thus far, only three beetle families have been described de novo based on newly collected material. Here, we report the discovery of a completely new lineage of soft-bodied neotenic beetles from the Brazilian Atlantic rainforest, which is one of the most diverse and also most endangered biomes on the planet. We identified three species in two genera, which differ in morphology of all life stages and exhibit different degrees of neoteny in females. We provide a formal description of this lineage for which we propose the new family Jurasaidae. Molecular phylogeny recovered Jurasaidae within the basal grade in Elateroidea, sister to the well-sclerotized rare click beetles, Cerophytidae. This placement is supported by several larval characters including the modified mouthparts. The discovery of a new beetle family, which is due to the limited dispersal capability and cryptic lifestyle of its wingless females bound to long-term stable habitats, highlights the importance of the Brazilian Atlantic rainforest as a top priority area for nature conservation.


Results
Based on their divergent morphology (Figs. 2-5, S1-S12) and the results obtained from the molecular phylogeny (Figs. 6, S13), we describe here Jurasai digitusdei gen. et sp. nov., J. itajubense gen. et sp. nov. and Tujamita plenalatum gen. et sp. nov., for which we erect a new family Jurasaidae fam. nov. Below we provide remarks on their biology, ecology and behavior, as well as concise diagnostic descriptions for all new taxa based primarily on salient diagnostic characters. Detailed morphological diagnoses and descriptions are given in the Supplementary Text. observations of behavior of the immature stages and adults. Immature stages of two species (out of three) representing two different genera are known to date. Larvae and pupae of J. itajubense and T. plenalatum occur sympatrically in the Municipal Biological Reserve Serra dos Toledos. They are usually found in the  www.nature.com/scientificreports www.nature.com/scientificreports/ appearance, with only certain parts of the head adult-like (Jurasai, Fig. 2e) or with the head, prothorax and legs adult-like, though different in appearance from the corresponding parts in the male (Tujamita, Fig. 2f) (Table S1). About a week after eclosion, the male actively searches for a female and then copulates several times each for a short period of time (Fig. 4, Supplementary Video). More detailed information on field and laboratory observations are given in the Supplementary Text. Systematics. Jurasai gen. nov. (Figs. 1d, 2a,c-e, 3a,b, 4, 5a-d, S1-S4, S6a-i, S8a,b, S10a-i, S11a,b, S12a-h).
phylogenetic analyses. All phylogenetic analyses placed Jurasaidae in Elateroidea and showed an identical backbone topology for the superfamily, with the main clades branching off in the following order: Artematopodidae + Omethidae (incl. Telegeusinae), Throscidae, Eucnemidae, Cerophytidae + Jurasaidae, and the terminal clade of "higher elateroids" sensu Kundrata et al. 13 (i.e., Lycidae, Iberobaeniidae, Lampyridae, Cantharidae, Elateridae, Omalisidae, Phengodidae and Rhagophthalmidae). The maximum likelihood (ML) phylogenetic tree of 251 terminals with collapsed branches and Elateroidea families highlighted is given in Fig. 6; the full-resolution tree is in Fig. S13. Jurasaidae were always placed as a sister group of Cerophytum elateroides (Cerophytidae), with 90-95% bootstrap support in the ML analyses and 100% posterior probabilities in the Bayesian (BI) analyses. This clade was sister to the "higher elateroids" with robust statistical support in the BI analyses and weaker support in the ML analyses (Figs. 6, S13). Jurasaidae formed a maximally supported monophylum, with Tujamita sister to a clade formed by two species of Jurasai. We associated the different developmental stages and sexes using molecular markers to confirm the field and laboratory observations (Fig. S14). The uncorrected pairwise genetic distance between cox1 sequences for J. itajubense and T. plenalatum was 28.5% (cox1 was not available for J. digitusdei).

Discussion
The majority of the currently recognized families of Elateroidea was described more than a hundred years ago, and only recent fieldwork resulted in surprising discoveries of Iberobaeniidae in southern Spain 9 and the here reported Jurasaidae in southeastern Brazil. These families had not been discovered earlier most probably due to a minute, soft body in adult males, neotenic, wingless females with a presumably short lifespan (only hypothesized in Iberobaeniidae), and a cryptic lifestyle. Jurasaidae males superficially resemble other small soft-bodied elateroids, with which they share the weakly sclerotized cuticle, widely separated and protruding eyes, partially reduced ventral mouthparts, falcate mandibles, prosternum in front of coxae very short and with incomplete prosternal process, widely open procoxae, conspicuous prothoracic tubular spiracles, conical and projecting coxae, exposed pro-and mesotrochantins, absent elytral costae, relatively shortened elytra compared to a flexible abdomen, hind wings with reduced venation and folded only longitudinally, and the abdomen with free ventrites connected by visible membranes (Table S2;  www.nature.com/scientificreports www.nature.com/scientificreports/ each one with a supporting sclerotized plate (Fig. S3j,k), and the larvae are characterized by the following unique characters: fused ventral mouthparts (i.e., maxillae and labium), labial channels located dorsally, and the basal half of the mandibles retracted deeply into the head capsule and linked to an inner sclerotized rod that extends into the prothorax (Figs. S10b,d,e, S11, S12c-f,i-l). Apart from the above-mentioned unique morphological features, Jurasaidae differ from each of the other soft-bodied elateroid groups in the Neotropics, i.e., Phengodidae, Lampyridae, Cantharidae, Lycidae, and Omethidae: Telegeusinae, by a number of additional features (see the Supplementary Text).
Due to the parallel origins of soft-bodiedness, body miniaturization and the characters connected with prematurely terminated metamorphosis in Elateroidea 9,14,16,22,23,29 , a detailed phylogenetic placement of Jurasaidae based on morphology alone would be precarious. Indeed, a preliminary examination of J. itajubense led the discoverers of this species to place it tentatively in Penicillophorini, a small group of uncertain position, either in Phengodidae or Omethidae: Telegeusinae 30,31 . However, our detailed morphological investigation revealed the considerable differences between Jurasaidae and Penicillophorini (see the Supplementary Text) and the molecular phylogenetic analyses placed Jurasaidae unambiguously to basal elateroid splits as a sister group of Cerophytidae, far from Phengodidae, Telegeusinae and other soft-bodied lineages (Figs. 6, S13). Although Jurasaidae and Cerophytidae adults are superficially different (i.e., both sexes in Cerophytidae are completely metamorphosed, with a fully sclerotized body and functional clicking mechanism), their larvae share the cylindrical, white and grub-like body, very small wedge-shaped head without dorsal and ventral epicranial sutures, non-opposable, flattened and channeled mandibles, labium with channels to fit the mandibular apices, prosternum with a median sclerotized endocarinate rod, and short legs. Remarkably, many of these features are also shared with Eucnemidae and Throscidae, and were used to define a monophyletic group formed by Eucnemidae, Throscidae and Cerophytidae 32,33 . On the other hand, the labial channels which fit the mandibular apices are an unambiguous synapomorphy of Cerophytidae + Jurasaidae. Since the phylogenetic relationships among the basal elateroids including the above-mentioned families have not yet been satisfactorily resolved (and this is also beyond the scope of this paper), more effort should be made to test the monophyly of Eucnemidae, Throscidae, Cerophytidae and Jurasaidae using more robust datasets and analytical approaches [11][12][13]32,33 . This would be crucial for understanding the evolution of modified larval morphology associated with the adaptation to burrowing in soil or rotten wood and feeding on juices of fungal hyphae.
The discovery of Jurasaidae and their placement in the basal Elateroidea shed new light on the evolution of neoteny in this beetle superfamily. Neoteny in Elateroidea has long been a widely studied phenomenon 19,21 , and recent studies repeatedly confirmed that it originated several times not only within the superfamily but also within several distantly related families 14,20,22,23,29 . Elateroidea are divided into the "basal grade" with mostly hard-bodied groups with only Omethidae and Jurasaidae having a soft body, and the robustly supported clade of "higher elateroids" which contains predominantly fully sclerotized click-beetles and the vast majority of soft-bodied lineages 13 . It is hypothesized that soft-bodiedness in both adult sexes is the initial stage of ontogenetic modifications leading to development of highly modified larviform females 20 . Until now, the neotenic females with variously modified morphology were known almost exclusively within terminal higher elateroids, and Omethidae: Telegeusinae were the only basal lineage with unknown but supposedly neotenic females. Jurasaidae represent the second loss of sclerotization among basal elateroid lineages, and are the first proven case of highly modified neotenic females outside the terminal clade of higher elateroids. Interestingly, the females of both genera in Jurasaidae exhibit different degrees of neoteny, with Jurasai being almost completely larviform and Tujamita having the head, prothorax and legs partly adult-like, although more reduced compared to an adult male (Supplementary Text, Table S1). Similar cases of different levels of morphological modifications in females of different genera were reported also for Lampyridae 19 , Lycidae 20 , Rhagophthalmidae 34 and Elateridae 23 . Not only females, but also males in Jurasaidae, present interesting morphological modifications connected with soft-bodiedness and neoteny. In Elateroidea, divergence in the abdominal morphology represents a continuous scale from five ventrites of which all or at least some are connate (the majority of the well-sclerotized elateroids) through various intermediate stages to seven or eight ventrites, which are all free and connected by extensive intersegmental membranes (majority of the soft-bodied lineages) 29 . Jurasaidae males represent an intermediate form; their abdomen is soft and with five free ventrites (Figs. S1h, S5f). A similar condition is only known in the otherwise morphologically dissimilar Brachypsectridae. Moreover, similar to females, Jurasai males are morphologically more affected by ontogenetic modifications than Tujamita males, having a pronotum without strengthening structures (pronotum with lateral carinae in Tujamita), elytra with median edges divergent posteriorly and with separately tapered apices (elytra parallel-sided, with sutural edges contiguous to apex in Tujamita), and with the abdomen narrower and more exposed (abdomen wider and shorter, usually covered by elytra in Tujamita) (Figs. 5, S1h, S5f, S6e,k). Finally, the two species of Jurasai exhibit different degrees of morphological modifications, with J. itajubense being more neotenic than J. digitusdei, having a reduced number of maxillary palpomeres, less compact and relatively narrower elytra, and much more reduced hind wing venation (Figs. 5a-d, S1a,b,f,g, S6c,g, S8a,b). Our present findings therefore represent additional evidence for the complex and gradual morphological modifications caused by the various degrees of incomplete metamorphosis in elateroid beetles.
The neotenic elateroid beetles are known for their extremely low dispersal ability, limited geographic ranges, and strong dependence on long-term climatically stable habitats including the humid tropics 9,20,35,36 . These attributes make neotenics excellent indicators of the long, uninterrupted evolutionary history of tropical forests. The Brazilian Atlantic rainforest is a complex biome formed by a network of ecosystems without clear limits, covering approximately 3,000 km of the eastern coast of Brazil 37 . It hosts one of the world's most diverse tropical forest biotas with an exceptionally high level of species endemism 38,39 but due to human activities leading to the intense landscape transformation during the last centuries, it is considered one of the most endangered biomes and belongs among the "hottest biodiversity hotspots" on the planet 24 www.nature.com/scientificreports www.nature.com/scientificreports/ matrices, with only less than 10% of its original coverage 25,41,42 and only about a third of its total extent preserved by conservation units 24 . Nevertheless, even those remaining forest fragments still harbor a multitude of newly discovered animal taxa including both vertebrates [43][44][45] and invertebrates [46][47][48][49][50][51] . Currently, the largest and best preserved portions of the Brazilian Atlantic rainforest are situated near the southern mountain ranges including Serras do Mar and da Mantiqueira 42 , where all Jurasaidae known thus far were collected. Such a late discovery of a new beetle family, which is due to the cryptic lifestyle of its representatives bound to these ancient, stable habitats, further stresses the importance of the Brazilian Atlantic rainforest as a top priority area for nature conservation.

Material and Methods
taxon sampling and collecting sites. A total of 120 specimens (21 adult males, 8 adult females, 4 pupae, 87 larvae) from two localities in the Brazilian Atlantic forest ecoregion in southeastern Brazil were studied (Supplementary Text). Larvae, pupae, adult males and females of the first two species were collected in the soil or using Malaise traps in the Serra dos Toledos reserve, and adult males of the third species were collected using Malaise traps in the Serra dos Órgãos National Park. For the detailed numbers of individuals under each species, corresponding localities and information on the collecting and rearing of specimens see the Supplementary Text. The Serra dos Toledos reserve is situated in the municipality of Itajubá, Minas Gerais state in the Mantiqueira mountain range. It comprises an area of 10.7 km 2 with altitudes ranging from 1,100 to 1,800 m. The reserve was originally covered by dense ombrophilous and Araucaria forests, but nowadays it includes secondary forests and suffers from the deforestation caused by the surrounding agricultural and livestock activities 52 . The Serra dos Órgãos National Park is situated in the municipality of Teresópolis, Rio de Janeiro state in the Serra do Mar mountain range. It covers an area of 105.3 km 2 with altitudes ranging from 200 to 2,285 m. This conservation unit comprises one of the largest area of dense ombrophilous vegetation in Rio de Janeiro state, although approximately 45% are under anthropogenic land use, mainly pastures. Both Serra dos Toledos and Serra dos Órgãos are situated in the Atlantic Rainforest domain 48,53 . The collection, maintenance, and shipping of specimens were conducted in accordance with the Brazilian environmental laws (Sisbio permits 53842-1, 53842-2, 53842-3, 43943-1, SisGen Shipping Registration R1CE59F, and authorization of the Municipal Government of Itajubá).
Laboratory methods. Whole genomic DNA was extracted using the E.Z.N.A Tissue DNA Kit (Omega Bio-tek Inc. Norcross, USA) following standard protocol but with the overnight incubation and elution performed twice with 100 µl Elution buffer each. Amplifications were performed using Qiagen Multiplex PCR Plus Master Kit (Qiagen, Hilden, Germany) according to the manufacturer protocols. The PCR amplification conditions and list of primers used were reported in previous study 54 . We sequenced two nuclear and two mitochondrial markers which have been widely used for Coleoptera and Elateriformia phylogenies 9,13,22,55,56 , i.e., 18S rRNA (1842-1843 bp), 28S rRNA (627-629 bp), rrnL (505 bp), and cox1 mtDNA (723 bp). Selection of these markers, which are abundantly represented in GenBank, enabled us to test the position of the newly discovered lineage using extensive outgroups. The PCR products were purified using Exonuclease I and FastAP Thermosensitive Alkaline Phosphatase (Life Technologies, Darmstadt, Germany), and subsequently sent for sequencing to the commercial facility run by Macrogen Europe, Netherlands.
Dataset assembly and phylogenetic analyses. In addition to the field and laboratory observations, we associated the different developmental stages and sexes using the sequences from 10 specimens (three adult males, two adult females, one pupa, four larvae) representing three morphologically defined species from two localities (Table S3). We used the alignment method and the maximum likelihood analysis as described below.
To investigate the position of the newly discovered lineage, we merged the sequences of individuals from the three newly discovered species (one per species) with the 251-taxon dataset from the most comprehensive phylogenetic study of Elateriformia to date 56 . Scirtiformia were used as an outgroup, since they represent basal Polyphaga 11,12 . Only terminals for which all four markers were present were included, except for a few taxonomically important lineages for which some fragments were missing (Table S3). Forward and reverse sequences were edited and assembled using Geneious 7.1.7 57 and multiple sequence alignments were generated using MAFFT 7.157 58 at default parameters. Alignment of the length invariable protein-coding cox1 sequences was checked by amino acid translation. Basic statistics and the pairwise sequence divergences based on uncorrected p-distance were calculated using MEGA 6.06 59 . The resulting alignment included 4780 homologous positions (2338, 1128, 591 and 723 positions for 18 S, 28 S, rrnL and cox1, respectively), and contained 2134 conserved, 2408 variable, and 1872 parsimony informative characters.
Model and best-fit partitioning testing was carried out using a greedy algorithm in PartitionFinder 1.1.1 60 under the corrected Akaike information criterion. Subsequent phylogenetic trees were generated using maximum likelihood analysis (ML) and Bayesian inference (BI). ML was conducted using RAxML 8.2.9 61,62 with the default settings, partitioned by genes and codons, and bootstrapped with 1000 pseudoreplicates. BI was performed using MrBayes 3.2.6 63 with the GTR + I + G model for most partitions (SYM + I + G for 28 S rRNA) and partitioning by genes and codon positions as recommended by PartitionFinder. Four chains were run for 40 million generations using the Markov chain Monte Carlo method. Stationary phase and convergence were checked using Tracer 1.7.1 64 and the first 20% of generations were discarded as burn-in. The posterior probabilities (PP) were calculated from the remaining trees. All analyses were run on the CIPRES web server 65 .
We also evaluated the occurrence of substitution saturation in our data using Xia's nucleotide substitution saturation test 66 implemented in DAMBE 5.6.14 67 . We analyzed separately each non-coding gene and each position of the protein-coding cox1 mtDNA using 10,000 replicates on the fully resolved sites and with the empirical proportion of invariant sites estimated from the data. Only little saturation was detected in our data except for the 3rd codon positions of the cox1 fragment, which were substantially saturated (Table S4). Therefore, we also performed the above-described ML and BI analyses with discarded third codon positions. Additionally, to test